Combination therapy

Abstract

The present invention relates to a composition comprising an epnuclizumab and LuAG09222, and to a method for preparing the same. The composition may be used to treat or prevent, for example, headache, pain, migraine, cluster headache or endometriosis.

Description

BACKGROUND

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 509,854, filed June 23, 2023, the contents of which are incorporated by reference in their entirety.

[0004] SEQUENCE LIST DISCLOSURE

[0005] The contents of the electronic sequence listing (1143257o014013.xml; size: 25,446 bytes; and date created: June 17, 2024) are incorporated herein by reference in their entirety.

[0006] Sequences not allowed in St.26 Xml file due to sequence length

[0007] Table A below lists sequences present in priority application U.S. Provisional Application No. 63 / 509,854 (identified above, which is incorporated by reference in its entirety herein), but these sequences could not be included in the 1143257o014013.xml file submitted herewith due to sequence length.

[0008] TECHNICAL FIELD

[0009] The present invention relates to a composition comprising two antibodies or fragments thereof, including Fab fragments, specific for different targets. One of these antibodies or antibody fragments specifically binds to human Calcitonin Gene-Related Peptide (hereinafter “CGRP”), the other antibody or antigen-binding fragment thereof specifically binds to Pituitary Adenylate Cyclase Activating Polypeptide (“PACAP”). BACKGROUND

[0010] Calcitonin Gene-Related Peptide (CGRP) is produced as a multifunctional neuropeptide of 37 amino acids in length. Two forms of CGRP, CGRP-alpha and CGRP-beta, exist in humans and have similar activities. CGRP-alpha and CGRP-beta differ by three amino acids in humans and are derived from different genes. CGRP is released from many tissues, such as the trigeminal nerve, which release meningeal innervation upon activation, mediating neurogenic inflammation characterized by vasodilation, vascular leakage, and mast cell degradation. Durham, P.L., New Eng. J. Med.[New England Journal of Medicine], 350 (11): 1073-75 (2004). The biological effects of CGRP are mediated through CGRP receptors (CGRP-R), which are composed of seven transmembrane components and receptor-activity modifying proteins (RAMPs). CGRP-R also requires the activity of a receptor component protein (RCP), which is important for efficient coupling of G proteins to adenylyl cyclase and production of cAMP. Doods, H., Curr. Op. Invest. Drugs [Current Opinion in Investigational Drugs], 2(9): 1261-68 (2001).

[0011] Several antibodies have been approved for the treatment of migraine. These antibodies bind to CGRP or CGRP receptors. FDA-approved antibodies include Aimovig (erenumab), Ajovi (fremanezumab), Emgality (galcanezumab), and Vyepti (eptinezumab). The present invention relates to Vyepti (erenumab or Ab6 as used herein), which is a drug approved for the preventive treatment of migraine. The recommended dose is 100 mg intravenous infusion every 3 months. Some patients can benefit from a dose of 300 mg administered every 3 months.

[0012] 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 vasodilatory peptide that exists in two alpha-amidated active forms, one with 38 amino acids (PACAP38) and the other with 27 amino acids (PACAP27). Both peptides have the same N-terminal 27 amino acids and are synthesized from the same precursor protein, preproPACAP (see, Moody et al., “PACAP: tissue effects and potential therapeutic applications,” Frontiers in Endocrinology, 9: 871, 2018). PACAP38 is the more prevalent active form, accounting for up to 90% of PACAP forms in mammalian tissues (see, Kaiser and Russo, “PACAP: from structure to function,” Regulatory Peptides, 174: 29-38, 2012). Curr. Opin. Endocrinol. Diabetes Obes. [Endocrinology, Diabetes and Obesity, Current Opinions], 18(1): 61-67, 2011). PACAP38 is the more prevalent active form, accounting for up to 90% of PACAP forms in mammalian tissues (see, Kaiser and Russo, “PACAP: from structure to function,” Regulatory Peptides, 174: 29-38, 2012). Neuropeptides [Neuropeptides], 47: 451-461, 2013). The sequence of PACAP38 is identical in all mammals and differs by only one amino acid from avian and amphibian orthologs (see, Vaudry et al., “PACAP and its receptors: from structure to function,” Pharmacological Reviews, 57(2): 92-114, 2005). Pharmacol. Rev.[Pharmacology Review], 52:269-324, 2000). The secretin / VIP / GHRH family includes mammalian peptide histidine methionine (“PHM”), secretin, glucagon, glucagon-like peptide-1 (“GLP1”), glucagon-like peptide-2 (“GLP2”), glucose-dependent insulinotropic peptide (“GIP”), and growth hormone-releasing factor (“GRF”). PACAP27 shares 68% sequence identity with VIP at the amino acid level (see, Vaudry et al., 2000).

[0013] The biological effects of PACAP are mediated by three different G protein-coupled receptors: PAC1-R, vasoactive intestinal peptide receptor type 1 (“VPAC1-R”), and vasoactive intestinal peptide receptor type 2 (“VPAC2-R”). These receptors are expressed in various tissues. PAC1-R is particularly abundant in the nervous system (e.g., olfactory bulb, thalamus, hypothalamus, cerebellum, and dorsal horn of the spinal cord), pituitary gland, and adrenal glands. In contrast, VPAC1-R and VPAC2-R are primarily expressed in the lungs, liver, and testes, although both receptors have also been detected in other tissues. VPAC1-R expression has been detected in the nervous system (e.g., cerebral cortex and hippocampus), smooth muscle cells of the lungs, liver, and intestines, megakaryocytes, and platelets. VPAC1-R binds to receptor-associated membrane proteins (“RAMP”, specifically RAMP2) (see Christopoulos et al., J. Biol. Chem. [Journal of Biochemistry], 278:3293-3297, 2002). VPAC2-R expression profiles include the nervous system (e.g., thalamus, hippocampus, brainstem, and dorsal root ganglia (“DRG”), cardiovascular system, gastrointestinal system, pancreas, and reproductive system) (see Usdin et al., 2002). Endocrin [Journal of Endocrinology], 135:2662-2680, 1994; Sheward et al. Neurosci. [Neuroscience], 67:409-418, 1995.

[0014] PACAP is presumed to play a role in a variety of diseases and disorders, including but not limited to migraine, headache, and pain. Migraine is thought to be associated with neurovascular factors. Approximately 10% of American adults suffer from migraine, and migraine is typically accompanied by severe headache. Approximately 20%–30% of migraine sufferers experience aura, including focal neurological phenomena that precede and / or accompany the event. Several observations suggest a role for PACAP in migraine: (1) During a migraine attack (sudden onset), plasma levels of PACAP are elevated compared to interictal levels (see Tuka et al., Cephalalgia(see, Schytz et al., Cephalalgia, 33(13): 1085-1095, 2013); (2) infusion of PACAP38 elicits headache in healthy subjects and headache, and subsequent migraine-like attacks in migraine patients (see, Schytz et al., Cephalalgia, 33(13): 1085-1095, 2013; and Olesen et al., Cephalalgia, 32(4):337-345, 2012, respectively); (3) PACAP-induced vasodilation can play a role in neurogenic inflammation (see, Kaiser and Russo, J. Neurosci. Res., 87: 2389-2400, 2009); (4) PACAP-induced migraine is associated with photophobia, phonophobia, nausea, and responds to triptans (see, Amin et al., Headache, 52: 1168-1181, 2012); and (5) PACAP elicits vasodilation, photophobia, and mast cell degranulation and neuronal activation (see, Markovics et al., J. Neurosci., 32: 140-149, 2012). Brain (see, Amin et al., Headache, 52: 1168-1181, 2012); (3) PACAP-induced vasodilation can play a role in neurogenic inflammation (see, Kaiser and Russo, J. Neurosci. Res., 87: 2389-2400, 2009); and (4) PACAP-induced migraine is associated with photophobia, phonophobia, nausea, and responds to triptans (see, Amin et al., Headache, 52: 1168-1181, 2012). Brain (see, Amin et al., Headache, 52: 1168-1181, 2012); (3) PACAP-induced vasodilation can play a role in neurogenic inflammation (see, Kaiser and Russo, J. Neurosci. Res., 87: 2389-2400, 2009); and (4) PACAP-induced migraine is associated with photophobia, phonophobia, nausea, and responds to triptans (see, Amin et al., Headache, 52: 1168-1181, 2012). Neuropeptides (see, Amin et al., Headache, 52: 1168-1181, 2012); (3) PACAP-induced vasodilation can play a role in neurogenic inflammation (see, Kaiser and Russo, J. Neurosci. Res., 87: 2389-2400, 2009); and (4) PACAP-induced migraine is associated with photophobia, phonophobia, nausea, and responds to triptans (see, Amin et al., Headache, 52: 1168-1181, 2012). Brain (see, Amin et al., Headache, 52: 1168-1181, 2012); (3) PACAP-induced vasodilation can play a role in neurogenic inflammation (see, Kaiser and Russo, J. Neurosci. Res., 87: 2389-2400, 2009); and (4) PACAP-induced migraine is associated with photophobia, phonophobia, nausea, and responds to triptans (see, Amin et al., Headache, 52: 1168-1181, 2012). Neurobiology of Disease (see, Amin et al., Headache, 52: 1168-1181, 2012); (3) PACAP-induced vasodilation can play a role in neurogenic inflammation (see, Kaiser and Russo, J. Neurosci. Res., 87: 2389-2400, 2009); and (4) PACAP-induced migraine is associated with photophobia, phonophobia, nausea, and responds to triptans (see, Amin et al., Headache, 52: 1168-1181, 2012). Cephalalgia (see, Amin et al., Headache, 52: 1168-1181, 2012); (3) PACAP-induced vasodilation can play a role in neurogenic inflammation (see, Kaiser and Russo, J. Neurosci. Res., 87: 2389-2400, 2009); and (4) PACAP-induced migraine is associated with photophobia, phonophobia, nausea, and responds to triptans (see, Amin et al., Headache, 52: 1168-1181, 2012). Pharmacology & Therapeutics (see, Amin et al., Headache, 52: 1168-1181, 2012); (3) PACAP-induced vasodilation can play a role in neurogenic inflammation (see, Kaiser and Russo, J. Neurosci. Res., 87: 2389-2400, 2009); and (4) PACAP-induced migraine is associated with photophobia, phonophobia, nausea, and responds to triptans (see, Amin et al., Headache, 52: 1168-1181, 2012).

[0015] PACAP can also be involved in diseases and disorders other than migraine, headache, and pain. For example, PACAP can be associated with or even causally involved in anxiety disorders (WO 2012 / 106407), thrombocytopenia (WO 2004 / 062684), and inflammatory skin diseases (WO 2010 / 007175). PACAP and PAC1-R polymorphisms are associated with post-traumatic stress syndrome ("PTSD"), major depressive disorder, and generalized anxiety disorder in women, suggesting a role for PACAP in these conditions. Additionally, supporting a role for PACAP in thrombocytopenia, patients with trisomy 18 have excess PACAP and exhibit defective megakaryocyte maturation (see, Schytz et al., 2010; and Moody et al., J. Clin. Invest., 121(1): 300-312, 2011). Curr. Opin. Endocrinol. Diabetes Obes. Contemp. Endocrinol., Diabetes, and Obesity, 18(1): 61-67, 2011).

[0016] Further, PACAP and other neuropeptides such as calcitonin gene-related peptide (“CGRP”), substance P, neurokinin A, bradykinin, and endothelin-1 are expressed in the lower urinary tract (“LUT”) (see Arms and Vizzard, Handbook Exp. Pharmacol. [Experimental Pharmacology Manual], 202:395-423, 2011), and are reported to potentially play a role in LUT dysfunction and urinary tract disorders such as urinary tract infections (“UTIs”), abnormal voiding, urinary urgency, nocturia, urinary incontinence, overactive bladder, and pain associated with such conditions.

[0017] PACAP and PACAP receptors are also thought to modulate inflammatory and neuropathic pain, and are associated with pro- and anti-nociception (see, Davis-Taber et al., J. Pain [Pain], 9(5):449-56, 2008). Spinal desensitization and induction of neuropathic pain have also been reported to require PACAP (see, Mabuchi et al., J. Neurosci . [Journal of Neuroscience], 24(33):7283-91, 2004). Additionally, altered morphine withdrawal behavior in PACAP receptor-deficient mice has been reported, further indicating a role for PACAP in morphine withdrawal anxiolytic responses (see, Martin et al., Mol. Brain Res. [Molecular Brain Research], 110(1):109-18, 2003).

[0018] The present invention relates to an anti-PACAP antibody named LuAG09222, Ab10.H3, or ALD1910 (all aliases of the same antibody) (Moldovan Loomis et al., J Pharmacol Exp Ther [Journal of Pharmacology and Experimental Therapeutics] 369:26-36, April 2019). The drug is being tested in various clinical trials for migraine at different doses, and has just shown significant results in a proof-of-concept study for migraine (clinicaltrials.gov number NCT05133323; and The Pharma Letter [Pharmaceutical News] April 20, 2023 “Lundbeck posts positive Phase IIa results with LuAG09222”).

[0019] This invention relates to the use of epronaizumab and LuAG09222 as a combination therapy or preferably in a composition comprising the two drugs. The combination therapy or composition can be used to treat medical conditions, optionally headache, episodic or chronic migraine, cluster headache, endometriosis, or pain. Summary of the Invention

[0020] This disclosure provides methods for treating headaches in patients, including administering to patients in need an effective amount of an anti-CGRP antibody (e.g., epronezumab) or an antibody fragment thereof, as disclosed herein, and an effective amount of an anti-PACAP antibody (e.g., LuAG09222) or an antibody fragment thereof.

[0021] According to one embodiment, the present invention relates to the treatment of migraine. The antibody treatment may be initiated between attacks (i.e., between migraine attacks) or during a sudden attack (i.e., during a migraine attack). The migraine may include, for example, chronic migraine or episodic migraine; in a particular aspect of the invention, the patient suffers from chronic migraine. In this invention, the anti-CGRP antibody is referred to as Ab6, eponezumab, or Vyepti, all of which are intended to refer to the same antibody. The anti-PACAP antibody is referred to as LuAG09222, ALD1910, or Ab10.H3, all of which are intended to refer to the same antibody.

[0022] The antibodies of the present invention can be produced in yeast or mammalian cells, such as Pichia pastoris or CHO cells. The antibodies of the present invention can be in a composition comprising histidine (10–50 mM), polysorbate 80, or poloxamer 188 (0.005–0.05% w / v) at a pH of 5.0–6.8. The composition may additionally contain a tensioning agent, optionally NaCl (10–150 mM), sorbitol (50–250 mM), and / or arginine (50–250 mM). The total amount of the two antibodies in the combination may be 150 mg / ml, for example, iprodione at 100 mg / ml and LuAG09222 at 50 mg / ml. The formulation can be administered to a patient subcutaneously or intravenously (iv). Further examples of the formulation and co-formulations are described herein and in the claims. As used in this invention, histidine further includes the L form of histidine (L-histidine), sorbitol includes the L-sorbitol form, and arginine includes the L-arginine form.

[0023] In another embodiment of the application, the combination therapy or composition can be used in a method of reducing, treating or preventing migraine (with or without aura), cancer or tumor, angiogenesis associated with cancer or tumor growth, angiogenesis associated with cancer or tumor survival, weight loss, pain, hemiplegic migraine, cluster headache, migraineous neuralgia, chronic headache, tension headache, general headache, hot flashes, chronic episodic migraine, secondary headache due to underlying structural problems in the head or neck, cranial neuralgias, sinus headache (e.g., like sinusitis related headache), and headache or migraine caused by allergy. The antibodies and antibody fragments of the application are particularly useful in treating, preventing, ameliorating, managing, or reducing the risk of one or more of the following conditions or diseases: overactive bladder and other urinary conditions, including bladder infection, pain; chronic pain; neurogenic inflammation and inflammatory pain; neuropathic pain; eye pain; tooth pain; post-surgical pain, trauma-related pain, burn-related pain, diabetes; non-insulin dependent diabetes and other inflammatory autoimmune disorders, vascular disorders; inflammation; arthritis; bronchial hyperreactivity, asthma; shock; sepsis; opiate withdrawal syndrome; morphine tolerance; hot flashes in men and women; allergic dermatitis; psoriasis; encephalitis; brain trauma; epilepsy; neurodegenerative diseases; skin diseases, including pruritis, neurogenic cutaneous redness, cutaneous rosaceous macules and erythema; inflammatory bowel disease, irritable bowel syndrome, cystitis; dysmenorrhea and other conditions that can be treated or prevented or symptoms that are ameliorated by antagonizing CGRP and / or PACAP signaling. Of particular importance is the acute or prophylactic treatment of headache, including migraine and cluster headache, and other pain-related conditions as well as endometriosis.

[0024] In another embodiment of the application, the combination therapy or composition can be used in a method of reducing, treating or preventing gastroesophageal reflux and / or visceral pain associated with gastroesophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstrual periods, labor, menopause, prostatitis, or pancreatitis.

[0025] In migraine, superactivation of the trigeminal nerve leads to increased release of calcitonin gene-related peptide (CGRP) and other peptides, resulting in the release of neurogenic proinflammatory mediators. Over time, these mediators further increase CGRP synthesis and release, corresponding to a typical migraine attack. CGRP mediates its effects via vasodilation of cerebral and arterioles, activating adenylyl cyclase in smooth muscle cells (mainly in the trigeminal vascular network), which in turn leads to abnormal activation of nociceptors and initiation of pain sensation.

[0026] Based on several lines of evidence, pituitary adenylate cyclase-activating polypeptide (PACAP) is thought to contribute to the pathophysiology of headache; studies have demonstrated that PACAP levels are elevated during an aura phase compared to an interictal phase in migraine patients. In addition, PACAP can be released during migraine attacks, and PACAP levels are elevated in chronic migraine patients.

[0027] Additional evidence comes from provocation studies, in which administration of both PACAP-38 and PACAP-27 resulted in migraine-like events in both healthy subjects and migraine patients. PACAP can also play a role in cluster headache. Similarly, studies have demonstrated that PACAP levels are also elevated during an aura phase of cluster events compared to interictal attacks.

[0028] CGRP and PACAP neuropeptides have overlapping locations and physiological similarities, but also show different biological effects.

[0029] This suggests that dual neutralization of CGRP and PACAP peptides will result in profound clinical efficacy in a group of patients in the migraine / headache space compared to single target neutralization.

[0030] Eptinezumab and LuAG09222 are both genetically engineered humanized antibodies against human CGRP and PACAP, respectively, that can bind these neuropeptides, thereby blocking their binding to the respective receptors and thus inhibiting downstream signaling that can lead to headache and pain sensation.

[0031] These antibodies are IgGl kappa immunoglobulins containing human constant regions. Their light and heavy chain variable regions consist of human sequences and humanized rabbit sequences.

[0032] Eptinezumab is a prescription drug for the preventive treatment of adult migraine. It is administered as a 100 or 300 mg intravenous infusion (iv) treatment over 30 minutes. Patients are given 4 doses per year (i.e., one dose every 3 months).

[0033] LuAG09222 binds human PACAP with high affinity. LuAG09222 blocks migraine-associated effects (photophobia, vasodilation, temperature increase, and lacrimation) and can reduce pain in preclinical mechanistic models. Data from clinical studies confirm the safety, tolerability, and PK of LuAG09222. Human provocation studies confirm that LuAG09222 prevents PACAP38-induced vasodilation and prevents PACAP38-induced facial flushing and heart rate increase, and a Phase 2 study of LuAG09222 in migraine (HOPE study) provides evidence of efficacy of targeting PACAP in migraine prevention. Data from the Phase 2 HOPE trial (NCT05133323) highlight the role of Lu AG09222 as a potential preventive agent for migraine. Overall, the trial met its primary endpoint, with a significant intergroup difference observed in the number of monthly migraine days (MMD) in patients receiving the high-dose treatment group over the 12-week double-blind period. The multinational, multi-site trial featured 237 individuals with episodic or chronic migraine who had failed 2-4 prior preventive agents and were randomized to 750 mg (n = 97) or 100 mg Lu AG09222 (n = 46) or placebo (n = 94) for a treatment period of 4 weeks, with a 12-week safety follow-up. At the end of the 4-week period, investigators observed a 2.0-day difference (95% CI, -3.5 to -0.6; P =.0106) in the reduction of monthly migraine days (MMD) in the high-dose treatment group and placebo group individuals.

[0034] Developing a composition for subcutaneous administration comprising two products (epratuzumab and LuAG09222) provides additional therapeutic benefits and offers different and / or superior treatment options in headache and non-headache pain disorders. Defined segments / phenotypes in migraine (patients with inadequate response to anti-CGRP or patients with parasympathetic symptoms, high frequency / chronic migraine, patients requiring a switch / adjunct to anti-CGRP) and episodic cluster headache include subjects with elevated circulating CGRP and PACAP levels.

[0035] In yet another embodiment of the present invention, the combination therapy or composition can be used to treat, ameliorate, or prevent chronic or episodic migraine, cluster headache, endometriosis, or pain.

[0036] In a particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222, wherein the ipilimumab has a VH region as defined in SEQ ID NO.: 4 and a VL as defined in SEQ ID NO.: 10, and the LU AG09222 has a VH region as defined in SEQ ID NO.: 15 and a VL region as defined in SEQ ID NO.: 20.

[0037] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, which is formulated to maintain the biological activity and / or storage stability of the ipilimumab and LuAG09222 antibodies therein.

[0038] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, which maintains the biological activity and / or storage stability of the ipilimumab and LuAG09222 antibodies therein for at least 1 month, at least 2 months, at least 3 months, at least 3-6 months, at least 6-9 months, at least 9-12 months, or at least one year.

[0039] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, which comprises or further comprises histidine and polysorbate 80 or poloxamer 188.

[0040] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, which comprises or further comprises one, two, or all of the following excipients: NaCl, sorbitol, and arginine.

[0041] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, which comprises ipilimumab and LuAG09222 at a total concentration of 100 mg / mL to 300 mg / mL.

[0042] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, which comprises ipilimumab and LuAG09222 at a total concentration of 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, or 300 mg / mL.

[0043] In another particular embodiment, the present application provides a pharmaceutical composition comprising epratuzumab and LuAG09222 according to any of the preceding items, having a ratio of epratuzumab to LuAG09222 of about 1 : 1, 1 : 2, or 2 : 2.

[0044] In another particular embodiment, the present application provides a pharmaceutical composition comprising epratuzumab and LuAG09222 according to any of the preceding items, wherein the composition comprises 100-300 mg / mL of epratuzumab and 50-100 mg / mL of LuAG09222.

[0045] In another particular embodiment, the present application provides a pharmaceutical composition comprising epratuzumab and LuAG09222 according to any of the preceding items, wherein the composition comprises 100 mg / mL of epratuzumab and 50 mg / mL of LuAG09222.

[0046] In another particular embodiment, the present application provides a pharmaceutical composition comprising epratuzumab and LuAG09222 according to any of the preceding items, wherein epratuzumab and LuAG0922 are the only active ingredients in the composition.

[0047] The pharmaceutical composition according to any of the preceding claims, wherein the concentration of histidine ranges between 10 - 50 mM, optionally 20-40 mM, the concentration of poloxamer P188 ranges between 0.0025-0.0120% w / v, and the concentration of polysorbate 80 ranges between 0.005-0.05% w / v, these ranges including the end values.

[0048] In another particular embodiment, the present application provides a pharmaceutical composition comprising epratuzumab and LuAG09222 according to any of the preceding items, wherein the concentration of NaCl is between 10-150 mM, optionally 30-70 mM, the concentration of sorbitol (e.g. L-sorbitol) is 50-250 mM, optionally 90-180 mM, and the concentration of arginine (e.g. L-arginine) ranges between 50-250 mM, these ranges including the end values.

[0049] In another particular embodiment, the present application provides a pharmaceutical composition comprising epratuzumab and LuAG09222 according to any of the preceding items, wherein the pH is between 5.0-6.8, this range including the end values.

[0050] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, wherein the pH is 5.0, 5.5, 5.9, 6.0, 6.5 or 6.8.

[0051] The pharmaceutical composition according to any of the preceding claims is suitable for intravenous administration or subcutaneous administration.

[0052] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, comprising ipilimumab and LuAG90222 in a ratio of 2 : 1, 1 : 1 or 1 : 2, and comprising 20-40 mM histidine buffer, 90-180 mM sorbitol, Poloxamer P188 0.0025-0.0120% w / v and 30-70 mM NaCl, and having a pH of about 6, optionally pH 5.9.

[0053] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, comprising ipilimumab and LuAG90222 in a ratio of 2 : 1, and comprising 25-35 mM histidine buffer, 165-175 mM sorbitol, Poloxamer P188 0.0025-0.010% w / v and 25-35 mM NaCl, and having a pH of about 6, optionally pH 5.9.

[0054] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, comprising ipilimumab and LuAG90222 in a ratio of 1 : 1, and comprising 30-40 mM histidine buffer, 130-140 mM sorbitol, Poloxamer P188 0.005-0.010% w / v and 45-55 mM NaCl, and having a pH of about 6, optionally pH 5.9.

[0055] In another particular embodiment, the present application provides a pharmaceutical composition comprising ipilimumab and LuAG09222 according to any of the preceding items, wherein the ratio of ipilimumab and LuAG90222 is 1 : 2, and the pharmaceutical composition comprises 35-45 mM histidine buffer, 90-100 mM sorbitol, Poloxamer P188 0.010-0.0120% w / v and 65-75 mM NaCl, and has a pH of about 6, optionally pH 5.9.

[0056] In another particular embodiment, the present application provides a pharmaceutical composition comprising eptinezumab and LuAG09222 for use as a medicament according to any of the preceding items.

[0057] In another particular embodiment, the present application provides a pharmaceutical composition comprising eptinezumab and LuAG09222 for use in the treatment or prevention of pain according to any of the preceding items.

[0058] In another particular embodiment, the present application provides a pharmaceutical composition comprising eptinezumab and LuAG09222 for use in the treatment or prevention of pain according to any of the preceding items.

[0059] In another particular embodiment, the present application provides a pharmaceutical composition comprising eptinezumab and LuAG09222 for use in the treatment or prevention of pain according to any of the preceding items.

[0060] In another particular embodiment, the present application provides a pharmaceutical composition comprising eptinezumab and LuAG09222 or the use thereof according to any of the preceding items, wherein eptinezumab has a heavy chain as defined in SEQ ID NO.: 5 or SEQ ID NO.: 6 and a light chain as defined in SEQ ID NO.: 11.

[0061] In another particular embodiment, the present application provides a pharmaceutical composition comprising eptinezumab and LuAG09222 or the use thereof according to any of the preceding items, wherein LUAG09222 has a heavy chain as defined in SEQ ID NO: 16 or SEQ ID NO.: 26 and a light chain as defined in SEQ ID NO.: 21.

[0062] In another particular embodiment, the present application provides a method of treating or preventing pain, optionally chronic or episodic migraine or cluster headache, comprising subcutaneously or intravenously administering a combination of eptinezumab and LuAG09222 antibodies, wherein eptinezumab has a VH region as defined in SEQ ID NO.: 4 and a VL as defined in SEQ ID NO.: 10, and LU AG09222 has a VH region as defined in SEQ ID NO.: 15 and a VL region as defined in SEQ ID NO.: 20, and optionally wherein administering said combination of antibodies has an additive or synergistic effect on inhibiting, reducing or preventing the number, duration and / or intensity of migraine attacks compared to subcutaneously or intravenously administering eptinezumab or LuAG09222 alone.

[0063] In another particular embodiment, the present application provides a method of treating or preventing pain by administering eptinezumab and LuAG09222, wherein eptinezumab has a VH region as defined in SEQ ID NO.: 4 and a VL as defined in SEQ ID NO.: 10, and LU AG09222 has a VH region as defined in SEQ ID NO.: 15 and a VL region as defined in SEQ ID NO.: 20, and optionally wherein 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.

[0064] In another particular embodiment, the present application provides a method of treating or preventing headache, optionally chronic or episodic migraine or cluster headache, by administering eptinezumab and LuAG09222, wherein eptinezumab and LuAG09222 antibodies are administered subcutaneously or intravenously using a pharmaceutical composition according to the foregoing.

[0065] In another particular embodiment, the present application provides a method of treating or preventing pain (e.g., acute pain, chronic pain, neuropathic pain, nociceptive pain, and / or radicular pain) by administering eptinezumab and LuAG09222, wherein eptinezumab and LuAG09222 antibodies are administered subcutaneously or intravenously using a pharmaceutical composition according to the foregoing. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 The number of subjects treated with Ab6 (treatment group) or placebo in the human clinical trial described in Example 1 who had a 50%, 75%, or 100% reduction in migraine at each monitoring point throughout the time period is shown. The right bar for each group corresponds to patients receiving 1000 mg Ab6, while the left bar for each group corresponds to matched placebo controls. In each response rate group, the response rate for patients receiving Ab6 was significantly higher than placebo treated controls, with p values for each group indicated as 0.0155, 0.0034, and 0.0006, respectively.

[0067] Figure 2 The % change from baseline in the median (±QR) number of migraine days per month over the 12 weeks post-treatment for placebo and Ab6 treatment groups is shown. (p = 0.0078). The upper and lower lines show the results for placebo treated controls and patients administered 1000 mg Ab6, respectively.

[0068] Figure 3The median (% change from baseline) in the number of monthly migraine attacks is shown for placebo and Ab6 treatment groups over the 12 weeks post-treatment. The top and bottom lines show the results for placebo-treated control group and patients administered 1000 mg Ab6, respectively.

[0069] Figure 4 The median (% change from baseline) in the number of monthly migraine hours is shown for placebo and Ab6 treatment groups over the 12 weeks post-treatment. The top and bottom lines show the results for placebo-treated control group and patients administered 1000 mg Ab6, respectively.

[0070] Figure 5 The screening of patients, assignment to treatment and control groups, and loss of patients through follow-up are summarized.

[0071] Figure 6 HIT-6 responder analyses for Ab6 treatment and placebo groups are compared at baseline, week 4 post-treatment, week 8 post-treatment, and week 12 post-treatment.

[0072] Figure 7 The percentage of patients with HIT-6 analyses indicating that the impact of headache was only "some" or "little / no" at baseline and after Ab6 administration is shown. At baseline, the majority of patients were experiencing "substantial" or "severe" impact of migraine. At each subsequent time point, the percentage of patients with only "some" or "little / no" HIT-6 impact was significantly higher in patients administered 1000 mg Ab6 (left column for each group, blue) compared to placebo controls (right column for each group, red).

[0073] Figure 8 Pharmacokinetic (PK) profiles for Ab6 administered as a single dose of 1000 mg intravenously are included.

[0074] Figure 9 Plasma-free pharmacokinetic (PK) parameters N (number of patients), mean, and standard deviation (SD) for Ab6 administered as a single intravenous dose of 1000 mg are included. 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).

[0075] Figure 10 The change in monthly migraine days (mean +- SEM) for Ab6 (1000 mg i.v.) relative to placebo for the single dose of the study described in Example 1 is shown relative to baseline.

[0076] Figure 11 The average number of migraine days (+ / - SD) over time is shown for the entire analyzed population in the study described in Example 1. The visit intervals (where electronic diaries (eDiary) were completed 21–27 days) were standardized by multiplying the observed frequencies by the inverse of the completion rate.

[0077] Figure 12 The distribution and variation of actual migraine days in the Ab6 treatment group during weeks 1–4 of the study described in Example 1 are shown.

[0078] Figure 13 The distribution and variation of actual migraine days in the placebo group during weeks 1–4 of the study described in Example 1 are shown.

[0079] Figure 14 The distribution and variation of actual migraine days in the Ab6 treatment group during weeks 5–8 of the study described in Example 1 are shown.

[0080] Figure 15 The distribution and variation of actual migraine days in the placebo group during weeks 5–8 of the study described in Example 1 are shown.

[0081] Figure 16 The distribution and variation of actual migraine days in the Ab6 treatment group during weeks 9–12 of the study described in Example 1 are shown.

[0082] Figure 17 The distribution and variation of actual migraine days in the placebo group during weeks 9–12 of the study described in Example 1 are shown.

[0083] Figure 18 The 50% responder rates for the Ab6 and placebo groups in the study described in Example 1 are shown. Subjects with a ≥ 50% reduction in migraine frequency were considered 50% responders. Visit intervals (where the electronic diary was completed 21–27 days prior) were standardized by multiplying the observed frequency by the reciprocal of the completion rate.

[0084] Figure 19 This shows the 75% responder rate in the Ab6 and placebo groups of the study described in Example 1. Subjects with a ≥75% reduction in migraine frequency were considered 75% responders. Figure 18 The above is to be standardized.

[0085] Figure 20 The 100% responder rate is shown for the Ab6 and placebo treatment groups in the study described in Example 1. Subjects who experienced a 100% reduction in migraine frequency were considered 100% responders. Figure 18 The above is to be standardized.

[0086] Figure 21The average migraine severity over time for the entire analysis population of the study described in Example 1 is shown. An average score of 3 on the scale used represents "moderate pain."

[0087] Figure 22 Changes from baseline in the measured attributes for the placebo and treatment groups in the study described in Example 1 are summarized.

[0088] Figure 23 The percentage of migraine patients in the 300 mg, 100 mg, and placebo treatment groups on days 1, 7, 14, 21, and 28 in the clinical trial described in Example 2 is shown. The top row shows the results for placebo, the bottom row shows the results for the 300 mg dose, and the middle row shows the results for the 100 mg dose.

[0089] Figure 24 The percentage of patients achieving a 50% reduction in migraine days in the 300 mg and 100 mg treatment groups in the clinical trial described in Example 2 at month 1, months 1-3 (after the first infusion), and months 4-5 (after the second infusion) is shown. In each figure, the data bars show the results for the 100 mg, 300 mg, and placebo groups from left to right. Statistical significance is indicated as follows. ++ indicates statistically significant difference from placebo; + indicates statistically significant difference from placebo (unadjusted); and § indicates statistically significant difference from placebo (post-hoc analysis).

[0090] Figure 25 The percentage of patients achieving a 75% reduction in migraine days in the 300 mg and 100 mg treatment groups in the clinical trial described in Example 2 at month 1, months 1-3 (after the first infusion), and months 4-5 (after the second infusion) is shown. The order of the data and the statistical significance labels are as shown. Figure 24

[0091] Figure 26 The percentage of patients achieving a 100% reduction in migraine days in the 300 mg and 100 mg treatment groups in the clinical trial described in Example 2 at month 1, months 1-3 (after the first infusion), and months 4-5 (after the second infusion) is shown. The order of the data and the statistical significance labels are as shown. Figure 24

[0092] Figure 27 The characteristics of the patients in each treatment group in the clinical trial described in Example 3 are summarized. ​​Change from baseline in mean monthly days (MMD) over 1-3 months by baseline subgroups of human clinical trials in patients with chronic migraine, compared to placebo. In the graph, data points are means and lines show 95% confidence intervals (CIs) for the change from placebo for the 100 mg (upper line) or 300 mg (lower line) treatment groups for each subgroup labeled on the far left.

[0093] Figure 28 . Change from baseline in mean monthly days (MMD) over 1-3 months by baseline subgroups of human clinical trials in patients with chronic migraine, compared to placebo. In the graph, data points are means and lines show 95% confidence intervals (CIs) for the change from placebo for the 100 mg (upper line) or 300 mg (lower line) treatment groups for each subgroup labeled on the far left.

[0094] Figure 29 . Change from baseline in mean monthly days (MMD) over 1-3 months by baseline subgroups of human clinical trials in patients with chronic migraine, compared to placebo. In the graph, data points are means and lines show 95% confidence intervals (CIs) for the change from placebo for the 100 mg (upper line) or 300 mg (lower line) treatment groups for each subgroup labeled on the far left.

[0095] Figure 30 . Change from baseline in mean monthly days (MMD) over 1-3 months by baseline subgroups of human clinical trials in patients with chronic migraine, compared to placebo. In the graph, data points are means and lines show 95% confidence intervals (CIs) for the change from placebo for the 100 mg (upper line) or 300 mg (lower line) treatment groups for each subgroup labeled on the far left.

[0096] Figure 31 . Change from baseline in mean monthly days (MMD) over 1-3 months by baseline subgroups of human clinical trials in patients with chronic migraine, compared to placebo. In the graph, data points are means and lines show 95% confidence intervals (CIs) for the change from placebo for the 100 mg (upper line) or 300 mg (lower line) treatment groups for each subgroup labeled on the far left.

[0097] Figure 32 . Change from baseline in mean monthly days (MMD) over 1-3 months by baseline subgroups of human clinical trials in patients with chronic migraine, compared to placebo. In the graph, data points are means and lines show 95% confidence intervals (CIs) for the change from placebo for the 100 mg (upper line) or 300 mg (lower line) treatment groups for each subgroup labeled on the far left.

[0098] Figure 33 . Change from baseline in mean monthly days (MMD) over 1-3 months by baseline subgroups of human clinical trials in patients with chronic migraine, compared to placebo. In the graph, data points are means and lines show 95% confidence intervals (CIs) for the change from placebo for the 100 mg (upper line) or 300 mg (lower line) treatment groups for each subgroup labeled on the far left.

[0099] Figure 34 . Change from baseline in mean migraine days (MMD) across 2 dose intervals in episodic migraine patients using acute medication at least 1 day per month at baseline. Triangles: placebo (n = 222). Circles: 100 mg Ab6 per dose (n = 221). Squares: 300 mg Ab6 per dose (n = 222).

[0100] Figure 35 . Mean number of acute medication days in episodic migraine patients using acute medication at baseline. Triangles: placebo (n = 222). Circles: 100 mg Ab6 per dose (n = 221). Squares: 300 mg Ab6 per dose (n = 222).

[0101] Figure 36 . Change from baseline in acute medication days for subgroups of episodic migraine patients with different baseline acute medication days. Solid line: patients using acute medication 10 days or more per month at baseline. Dashed line: patients using acute medication at least 1 day and less than 10 days per month at baseline. Triangles: placebo. Circles: 100 mg Ab6 per dose. Squares: 300 mg Ab6 per dose.

[0102] Figure 37 . Summary of acute medication days for subgroups of episodic migraine patients with baseline acute medication use.

[0103] Figure 38 . Day -1 was included in the migraine data. Day 0 was defined as the day of infusion. Thus, data on day 0 indicates the treatment effect after infusion.

[0104] Figure 39 . Dose projections have been performed using R software (nlmixr) based on clinical data from the LuAG09222 (Ab 10.H3) trial, in particular from the phase II trial in migraine patients. Monthly dosing for 7 months, then discontinued. DETAILED DESCRIPTION

[0105] The population PK model combining eptinezumab and LuAG09222 allowed to establish a PKPD model to determine the ratio of eptinezumab and LuAG09222 needed to have both free targets (CGRP and PACAP) below 50%. For both compounds, simultaneous bolus administration was explored for dosing. Dosing frequency was restricted to once a month and administration route was subcutaneous administration.

[0106] Eptinezumab has a terminal elimination half-life of 27 days and is used in migraine at 100 or 300 mg, while LuAG09222 has a terminal elimination half-life of approximately 15 days.

[0107] If less than 50% free target is to be obtained at monthly s.c. dosing intervals, a 100 mg dose of eptinezumab and a 50 mg dose of LuAG09222 are estimated to be needed.

[0108] When administered as a single agent, no treatment-related deaths or adverse outcomes attributable to pharmacological activity of eptinezumab or LuAG09222 were observed during single-dose or repeat-dose studies in rats or cynomolgus monkeys. No effects on reproductive function or performance, fertility, or early embryonic development were observed in rats following administration of eptinezumab. No evidence of parental effects or embryotoxicity, fetal toxicity, or teratogenicity was observed in rats or rabbits for eptinezumab or LuAG09222 administration. There were no effects on survival, physical development, behavior, or reproductive performance of the Fl generation during pre- and postnatal development studies in rats with eptinezumab.

[0109] In all nonclinical studies, the NOEL / NOAEL for IV administration of eptinezumab or LuAG09222 as a single agent was the highest dose administered (up to 150 mg / kg / dose).

[0110] Since both eptinezumab and LuAG09222 are humanized monoclonal antibodies, it is expected that administration or co-administration of the combination product will not produce pharmacokinetic interactions.

[0111] Definitions

[0112] It should be understood that the present application is not limited to the particular methodology, protocols, cell lines, animal species or genera, and reagents described, as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application which will be limited solely by the appended claims. As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the protein" includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this application belongs unless clearly indicated otherwise.

[0113] As used herein, the term "chronic migraine" refers to a condition in which a patient exhibits an average of at least 15 headache days per month, where a subset of these headache days meet the ICHD-3 criteria for migraine with or without aura. The term "episodic migraine" refers to a condition in which a patient exhibits an average of less than 15 headache days per month, where typically 4-15 are migraine phenotypes meeting the ICHD-3 definition of migraine with or without aura.

[0114] As used herein, the term "diagnosed with chronic migraine" refers to a patient who meets the clinical criteria for chronic migraine, whether or not the patient has been formally diagnosed. The term "diagnosed with episodic migraine" refers to a patient who meets the clinical criteria for episodic migraine, whether or not the patient has been formally diagnosed.

[0115] As used herein, the term "intravenous administration" refers to an administration mode in which a substance, e.g., an antibody, is introduced directly into the circulation of a patient, most typically into the venous circulation. The substance can be introduced into a carrier fluid, optionally an aqueous solution, e.g., normal saline. The substance can be administered in a single formulation or in multiple formulations, so long as administration is completed within a short period of time (e.g., within 1 day, preferably within 12 hours, more preferably within 6 hours, and most preferably within 1-2 hours). The term "subcutaneous administration" (or s.c. administration) refers to an administration mode in which a substance, e.g., an antibody, is administered to a layer of the skin known as the dermis, just beneath the epidermis layer and the dermis layer. Subcutaneous administration can be to a variety of sites, including the outer region of the upper arm and the abdominal, anterior thigh, upper back, or upper buttock region behind the hip bone.

[0116] As used herein, the term "baseline migraine days" refers to the number of migraine days exhibited by a patient over a specified period of time, e.g., prior to treatment. For example, baseline migraine days can be determined over a period of one month or more, e.g., by recording each day whether a migraine occurred.

[0117] As used herein, the term "monthly migraine days" refers to the number of days per month in which a patient experiences a migraine, i.e., at any time during the day the patient has symptoms meeting the clinical definition of a migraine. Monthly migraine days can be determined by recording each day whether a migraine occurred.

[0118] As used herein, the term "monthly headache days" refers to the number of days per month in which a patient experiences a headache, i.e., at any time during the day the patient has symptoms meeting the clinical definition of a headache. Monthly headache days can be determined by recording each day whether a headache occurred.

[0119] Calcitonin gene-related peptide ( CGRP): As used herein, CGRP encompasses the following Homo sapiens CGRP-alpha and Homo sapiens CGRP-beta amino acid sequences, which are available from American Peptides (Sunnyvale CA) and Bachem (Torrance, CA).

[0120] CGRP-α : ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-NH2 (SEQ ID NO.: 22), with the terminal phenylalanine amidated; CGRP-β: ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-NH2 (SEQ ID NO.: 23), with the terminal phenylalanine amidated; also encompassing any membrane-bound forms of these CGRP amino acid sequences, as well as mutants, splice variants, isoforms, orthologs, homologs, and variants of the sequences.

[0121] Pituitary adenylate cyclase-activating polypeptide (PACAP) : As used herein, PACAP includes any mammalian form of PACAP, and particularly encompasses the following Homo sapiens PACAP27 and Homo sapiens PACAP38 amino acid sequences, unless otherwise indicated.

[0122] PACAP38 : HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO.: 24), also including any mutants, splice variants, isoforms, orthologs, homologs, and variants of the sequence.

[0123] PACAP27 : HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO.: 25), also including any mutants, splice variants, isoforms, orthologs, homologs, and variants of the sequence.

[0124] Expression vectorThese DNA vectors contain elements that facilitate manipulation of the foreign protein for expression in the target host cell (e.g., a yeast or mammalian cell, optionally Pichia pastoris or CHO cell). Conveniently, manipulation of the sequence and production of DNA for transformation is first performed in a bacterial host, such as E. coli, and typically, the vector will include sequences that facilitate such manipulation, including a bacterial origin of replication and appropriate bacterial selectable marker. The selectable marker encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients that are not present in the culture medium. Exemplary vectors and methods for transforming yeast are described in, e.g., 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.

[0125] Expression vectors for yeast or mammalian cells will typically further include yeast- or mammal-specific sequences, including selectable nutritional or drug markers for identifying transformed yeast strains or transformed mammalian cells. The drug marker can further be used to amplify the copy number of the vector in the host cell.

[0126] The polypeptide coding sequence of interest is operably linked to transcriptional and translational regulatory sequences that provide for expression of the polypeptide in a host cell, such as Pichia pastoris or CHO cell. These vector components can include, but are not limited to, one or more of the following: enhancer elements, promoters, and transcription termination sequences. Sequences for secretion of the polypeptide, such as a signal sequence, etc., can also be included. A replication origin for the yeast or mammal is optional, as the expression vector is typically integrated into the host cell genome. In one embodiment of the application, the polypeptide of interest is operably linked or fused to a sequence that provides optimal secretion of the polypeptide from the yeast diploid cell.

[0127] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a preprotein is operably linked to DNA for a polypeptide in which the preprotein is involved in secretion of the polypeptide; DNA for a signal sequence is operably linked with DNA for a polypeptide in which the signal sequence is involved in secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a promoter or enhancer is operably linked to a DNA sequence to be transcribed if it functions in the transcription of the sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is most often accomplished by ligation at convenient restriction sites, or by the use of an appropriate vector as described below. R Techniques; Invitrogen, Carlsbad, CA). If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0128] A promoter is the non-translated sequence (usually within about 100 bp to 1000 bp) located upstream (5') to the start codon of a structural gene that controls the transcription and translation of a specific nucleic acid sequence to which it is operably linked. Such promoters are classified into several categories: inducible, constitutive, and repressible promoters (increasing the level of transcription in response to the absence of a repressor). Inducible promoters initiate increased levels of transcription from the DNA under the control of the promoter in response to certain changes in the culture conditions (e.g., the presence or absence of a nutrient or a change in temperature).

[0129] Promoter fragments can also be used as sites for homologous recombination and integration of the expression vector into the same site in the host genome; alternatively, a selection marker is used as a site for homologous recombination. Examples of suitable promoters from P. pastoris include the AOX1 and AOX2 promoters (Cregg et al. (1989) Mol. Cell. Biol Mol. Cell. Biol. 9:1316-1323); the ICL1 promoter (Menendez et al. (2003) Yeast Yeast 20(13): 1097-108); the glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) (Waterham et al. (1997) Gene Gene 186(1): 37-44); and the FLD1 promoter (Shen et al. (1998) Gene Gene 216(1):93 102). GAP The promoters are strong constitutive promoters, while the AOX and FLD1 promoters are inducible.

[0130] Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4, PH03, PH05, Pyk, and chimeric promoters derived therefrom. In addition, non-yeast promoters can be used in the present application, optionally mammalian, insect, plant, reptilian, amphibian, viral, and avian promoters. Most typically, the promoter will comprise a mammalian promoter (which can be endogenous to the expressed gene) or will comprise a yeast or viral promoter that provides for efficient transcription in the yeast system.

[0131] Examples of mammalian promoters include cytomegalovirus (CMV)-derived promoters, chicken 3-actin (CBM)-derived promoters, adenomatous polyposis coli (APC)-derived promoters, leucine-rich repeat G protein-coupled receptor 5 (LGR5) promoters, CAG promoters, beta-actin promoters, elongation factor 1 (EF1) promoters, early growth response 1 (EGR-1) promoters, eukaryotic initiation factor 4A1 (EIF4A1) promoters, simian virus 40 (SV40) early promoters, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoters, MoMuLV promoters, avian leukosis virus promoters, Epstein-Barr virus immediate early promoters, Rous sarcoma virus promoters, and human gene promoters (optionally but not limited to actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters), among others. Combinations of two or more of the foregoing promoters can also be used. In addition, inducible promoters can be used. The use of inducible promoters provides a molecular switch that enables the initiation of expression of a polynucleotide sequence that is operably linked when such expression is desired, or turns off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0132] Antibodies of the present application can be produced recombinantly not only directly, but also as fusion polypeptides with heterologous polypeptides, e.g., signal sequences or other polypeptides that have specific cleavage sites at the N-terminus of the mature protein or polypeptide. Typically, the signal sequence can be a component of the vector, or it can be attached to the coding sequence of the polypeptide to be expressed by the vector. Preferably, the heterologous signal sequence selected is one that is recognized and processed by the standard secretory pathways of the host cell. The Saccharomyces cerevisiae alpha factor prepro- signal has been shown to efficiently direct the secretion of a variety of recombinant proteins from Pichia pastoris. Other yeast signal sequences include the alpha mating factor signal sequence, invertase signal sequence, and signal sequences derived from other secreted yeast polypeptides. Additionally, these signal peptide sequences can be engineered to provide enhanced secretion in diploid yeast expression systems. Secretory signals for mammalian as well as yeast cells include mammalian signal sequences, which can be either heterologous to the protein being secreted, or can be the native sequence of the protein being secreted. Signal sequences include pre-peptide sequences, and in some cases can include pro-peptide sequences. Many such signal sequences are known in the art, including those found on immunoglobulin chains, such as the K28 pre-toxin sequence, PHA-E, FACE, human MCP-1, human serum albumin signal sequence, human Ig heavy chain, human Ig light chain, and the like. See, e.g., Hashimoto et al. Protein Eng [Protein Engineering] 11(2) 75 (1998); and Kobayashi et al. Therapeutic Apheresis [Therapeutic dissociation] 2(4) 257 (1998).

[0133] Transcription can be increased by inserting an transcriptional activator sequence into the vector. These activators are cis-acting elements of DNA, usually between about 10 to 300 bp, which act on a promoter to increase its transcription. Transcription enhancers are relatively independent in orientation and location, both within introns and within the coding sequence itself, and have been found both 5' and 3' to the transcriptional unit. Enhancers can be spliced into the expression vector at a position 5' or 3' to the coding sequence, but are preferably located at a site 5' from the promoter.

[0134] Expression vectors that are useful in eukaryotic host cells can also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly found in the 3' untranslated region of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments that are transcribed into the poly A segment of the mRNA.

[0135] The construction of suitable vectors containing one or more of the above components employs standard ligation techniques or PCR / recombinant methods. Isolated plasmids or DNA fragments are cut, trimmed, and religated in the desired form used to generate the desired plasmid, or via recombination methods. To analyze and confirm the correct sequence in the constructed plasmid, the ligation mixture is used to transform host cells, and successful transformants are selected for antibiotic resistance (e.g., ampicillin or bleomycin) where appropriate. Plasmids from the transformants are prepared, analyzed, and / or sequenced by restriction endonuclease digestion.

[0136] As an alternative to fragment restriction enzyme digestion and ligation, DNA sequences can be inserted into vectors using recombination methods based on the att site and recombinase. Such methods are exemplified by those developed by Landy (1989). Ann. Rev. Biochem [Biochemistry Annals] 58 As described in 913-949 and known to those skilled in the art, such a method utilizes intermolecular DNA recombination mediated by a mixture of recombinant proteins encoded by λ and E. coli. Recombination occurs at specific attachments to interacting DNA molecules ( att Between sites. For a description of att sites, see Weisberg and Landy (1983) Site-Specific Recombination in Phage Lambda, in Lambda II [Site-Specific Recombination of Phage λ in λII], edited by Weisberg. (Cold Spring Harbor, New York: Cold Spring Harbor Press), pp. 211-250. Switching the DNA fragments flanking the recombination site results in recombination... att A locus is a heterozygous sequence composed of sequences contributed by each parent vector. Recombination can occur between DNAs of any topological structure.

[0137] att The site can be introduced into the target sequence by: ligating the target sequence into a suitable vector; or by using specific primers to generate a site containing... att PCR products at site B; generating cDNA libraries cloned into suitable vectors containing the att site; etc.

[0138] As used herein, folding refers to the three-dimensional structure of peptides and proteins, in which interactions between amino acid residues play a role in stabilizing the structure. Proper folding is typically the peptide arrangement that leads to optimal biological activity, and in the case of antibodies, it can be conveniently monitored by assays of activity (e.g., antigen binding).

[0139] The expression host can be further modified by the introduction of sequences encoding one or more enzymes that enhance folding and disulfide bond formation, i.e., foldases, chaperones, etc. Such sequences can be expressed constitutively or inducibly in the yeast host cell using vectors, markers, etc. as known in the art. Preferably, the sequences, including transcriptional regulatory elements sufficient to achieve the desired expression pattern, are stably integrated into the yeast genome by targeted methods.

[0140] For example, eukaryotic PDIs are not only efficient catalysts of protein cysteine oxidation and disulfide bond isomerization, but also exhibit chaperone activity. Co-expression of PDIs can facilitate production of active proteins with multiple disulfide bonds. Expression of BIP (immunoglobulin heavy chain binding protein), cyclophilins, etc. are also of interest. In one embodiment of the application, each haploid parent strain expresses a different foldase, e.g., one strain can express BIP while the other strain can express PDI or a combination thereof.

[0141] The terms "desired protein" or "desired antibody" are used interchangeably and generally refer to a parent antibody specific for a target, i.e., a CGRP, PACAP, or chimeric or humanized antibody or binding moiety derived therefrom as described herein. The term "antibody" is intended to include any polypeptide chain-containing molecular structure having a specific shape that is suitable for and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The prototypical antibody molecule is an immunoglobulin, particularly an IgG from all sources, e.g., human, rodent, rabbit, bovine, ovine, porcine, canine, other mammals, chicken, other avians, etc., is considered an "antibody." Numerous antibody-encoding sequences have been described; and others can be developed by methods well known in the art. Examples include chimeric antibodies, human antibodies and other non-human mammal antibodies, humanized antibodies, single chain antibodies (e.g., scFv), camelid antibodies, nanobodies, IgNAR (single chain antibody derived from sharks), small modular immunopharmaceuticals (SMIPs), and antibody fragments (e.g., Fab, Fab', F(ab')2, etc.). See Streltsov VA et al., Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype, Protein Sci[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 [ 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 [ Mol Immunol. 2001 Aug; 38(4): 313-26; Hamers-Casterman C et al., Naturally occurring antibodies devoid of light chains, Nature. [ Nature 1993 Jun 3; 363(6428): 446-8; Gill DS et al., Biopharmaceutical drug discovery using novel protein scaffolds, Curr Opin Biotechnol [ Curr Opin Biotechnol. 2006 Dec; 17(6): 653-8. Epub 2006 Oct 19.

[0142] For example, antibodies or antigen-binding fragments can be produced by genetic engineering. In this technique, as with other methods, cells that produce antibodies are sensitized to the desired antigen or immunogen. Messenger RNA isolated from the antibody-producing cells is used as a template to prepare cDNA by PCR amplification. A library of vectors is produced by inserting appropriate portions of the amplified immunoglobulin cDNA into expression vectors, each vector containing one heavy chain gene and one light chain gene that retains the original antigen specificity. A combinatorial library is constructed by combining the heavy chain gene library with the light chain gene library. This produces a library of clones that co-express heavy and light chains (similar to Fab fragments or antigen-binding fragments of antibody molecules). The vectors carrying these genes are co-transfected into host cells. When the antibody genes are induced to synthesize in the transfected host, the heavy and light chain proteins self-assemble to produce active antibodies, which can be detected by screening with the antigen or immunogen.

[0143] Antibody-encoding sequences of interest include those encoded by the native sequences, as well as those encoded by nucleic acids that differ from the disclosed nucleic acid sequences due to the degeneracy of the genetic code and variants thereof. Variant polypeptides can include amino acid (aa) substitutions, additions or deletions. Amino acid substitutions can be conservative amino acid substitutions or substitutions that eliminate non-essential amino acids, for example, to alter glycosylation sites, or to minimize misfolding by substituting or deleting one or more cysteine residues that are not essential for function. Variants can be designed to retain or enhance the biological activity of specific regions of the protein (e.g., functional domains, catalytic amino acid residues, etc.). Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and / or fragments corresponding to functional domains. In vitro mutagenesis techniques for cloning genes are known. The present application also includes polypeptides modified using ordinary molecular biology techniques to increase their resistance to proteolytic degradation or to optimize solubility or to make them more suitable as therapeutic agents.

[0144] Chimeric antibodies can be produced by recombinant means that combine variable light and heavy chain regions (V L and V H ) obtained from antibody-producing cells of one species with constant light and heavy chain regions from another species. Typically, chimeric antibodies utilize rodent or rabbit variable regions and human constant regions to produce antibodies having primarily human domains. Production of such chimeric antibodies is well known in the art and can be achieved by standard means (e.g., as described in U.S. Patent No. 5,624,659, which is incorporated herein by reference in its entirety). It is further contemplated that the human constant region of the chimeric antibodies of the present application can be selected from the group consisting of IgGl, IgG2, IgG3, and IgG4 constant regions.

[0145] Humanized antibodies are engineered to contain even more human-like immunoglobulin domains and incorporate only the complementarity determining regions of the animal-derived antibody. This is accomplished by carefully examining the sequences of the hypervariable loops of the variable region of the monoclonal antibody and fitting them to the structure of a human antibody chain. Although it appears complex, the process is straightforward in practice. See, e.g., U.S. Patent No. 6,187,287, which is incorporated by reference herein in its entirety.

[0146] In addition to intact immunoglobulins (or recombinant counterparts thereof), immunoglobulin fragments (e.g., Fab', F(ab')2, or other fragments) comprising an epitope binding site can also be synthesized. A "fragment" or minimal immunoglobulin can be designed using recombinant immunoglobulin technology. For example, an "Fv" immunoglobulin for use in the present application can be produced by synthesizing a fused variable light chain region and variable heavy chain region. Combinations of antibodies are also of interest, e.g., diabodies, which include two different Fv specificities. In another embodiment of the present application, immunoglobulin fragments encompass SMIPs (small molecule immunopharmaceuticals), camelid antibodies, nanobodies, and IgNARs.

[0147] Immunoglobulins and fragments thereof can be post-translationally modified, e.g., to add an effector moiety, e.g., a chemical linker, a detectable moiety, e.g., a fluorescent dye, an enzyme, a toxin, a substrate, a bioluminescent material, a radioactive material, a chemiluminescent moiety, etc., or a specific binding moiety, e.g., streptavidin, avidin, or biotin, etc., which can be used in the methods and compositions of the present application. Examples of additional effector molecules are provided below.

[0148] A polynucleotide sequence is "corresponding" to a polypeptide sequence if translation of the polynucleotide sequence according to the genetic code produces the polypeptide sequence (i.e., the polynucleotide sequence "encodes" the polypeptide sequence), and one polynucleotide sequence is "corresponding" to another polynucleotide sequence if the two polynucleotide sequences encode the same polypeptide sequence.

[0149] A "heterologous" region or domain of a DNA construct is a recognizable segment of DNA in a larger DNA molecule that is not associated with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene is typically flanked by DNA that is not flanked by mammalian genomic DNA in the genome of the source organism. Another example of a heterologous region is a construct in which the coding sequence itself (e.g., a cDNA in which the genomic coding sequence includes introns, or a synthetic sequence having codons different from the native gene) is not found in nature. Allelic variation or naturally occurring mutational events do not give rise to a heterologous region of DNA as defined herein.

[0150] A “coding sequence” is the in-frame sequence of a codon that (in terms of the genetic code) corresponds to or encodes a protein or peptide sequence. Two coding sequences correspond to each other if the sequence or its complement encodes 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 are typically located at the 3' end of the coding sequence. A “promoter sequence” is a regulatory region of DNA that binds to RNA polymerase in the cell and initiates transcription of the downstream (3' direction) coding sequence. A promoter sequence typically contains additional sites for binding to regulatory molecules (e.g., transcription factors) that influence the transcription of the coding sequence. When RNA polymerase binds to the promoter sequence in the cell and transcribes the coding sequence into mRNA (which is then translated into a protein encoded by the coding sequence), the coding sequence is “under the control” or “effectively linked” to the promoter.

[0151] Vectors are used to introduce foreign substances (such as DNA, RNA, or proteins) into an organism or host cell. Typical vectors include recombinant viruses (for polynucleotides) and liposomes (for peptides). A "DNA vector" is a replicon, such as a plasmid, bacteriophage, or granule, that attaches another polynucleotide fragment to it, causing the attached fragment to replicate. An "expression vector" is a DNA vector that contains regulatory sequences that will instruct the appropriate host cell to synthesize a peptide. This typically means a promoter that binds to RNA polymerase and initiates mRNA transcription, as well as a ribosome binding site and initiation signal to direct the conversion of mRNA into one or more peptides. The polynucleotide sequence is incorporated into the expression vector at the correct site and in the correct reading frame, and then the vector transforms into a suitable host cell, enabling the production of a peptide encoded by the polynucleotide sequence.

[0152] The "amplification" of polynucleotide sequences is the in vitro generation of multiple copies of a specific nucleic acid sequence. The amplified sequence is usually in DNA form. Van Brunt (1990, Bio / Technol A review article in [Biotechnology], 8(4):291-294, describes a variety of techniques used to perform such amplifications. Polymerase chain reaction, or PCR, is the prototype for nucleic acid amplification, and the use of PCR in this article should be considered as an example of other suitable amplification techniques.

[0153] The general structure of antibodies in vertebrates is now well understood (Edelman, GM, Ann. N.Y. Acad. Sci[Annals of the New York Academy of Sciences], 190: 5 (1971)). Antibodies are composed of two identical light chain polypeptides ("light chains") of molecular weight approximately 23,000 daltons and two identical heavy chains ("heavy chains") of molecular weight 53,000-70,000. The four chains are linked by disulfide bonds in a "Y" configuration, with the light chain looping around the heavy chain from the opening of the "Y" configuration. The "branch" portion of the "Y" configuration is called the F ab region; the stem portion of the "Y" configuration is called the F C region. The direction of the amino acid sequence is 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 for the antigen that elicited it, and the variable region is approximately 100 amino acids in length, with minor variations in length between light and heavy chains and between different antibodies.

[0154] The variable region is linked in each chain to a constant region that extends the remaining length of the chain, and the constant region in a particular class of antibody does not vary with the specificity (i.e., the antigen that elicited it) of the antibody. There are five known major classes of constant regions, which determine the class of the immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE correspond to gamma, mu, alpha, delta, and epsilon heavy chain constant regions). The constant region or class determines the subsequent effector functions of the antibody, including activation of complement (Kabat, E. A., Structural Concepts in Immunology and Immunochemistry, 2nd Ed., pp. 413-436, Holt, Rinehart, Winston (1976)) and other cellular responses (Andrews, D. W. et al., Clinical Immunobiology [Clin. Immunol. Immunopathol.], pp. 1-18, W. B. Sanders (1980); Kohl, S. et al., Immunology [Immunol.], 48: 187 (1983)); while the variable region determines the antigen with which it will react. Light chains are classified as kappa or lambda. Each heavy chain can be made with either kappa or lambda light chains. When immunoglobulins are produced by hybridomas or by B cells, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide bonds.

[0155] The expression "variable region" or "VR" refers to the domain within each pair of light and heavy chains in an antibody that is directly involved in the binding of the antibody to an antigen. Each heavy chain has a variable domain (V H ) at one end, followed by a number of constant domains. Each light chain has a variable domain (VL The other end has a constant structural domain; the constant structural domain of the light chain is aligned with the first constant structural domain of the heavy chain, and the variable structural domain of the light chain is aligned with the variable structural domain of the heavy chain.

[0156] The terms "complementarity-determining region," "hypervariant region," or "CDR" refer to one or more hypervariable or complementarity-determining regions (CDRs) found in the variable regions of the light or heavy chains of an antibody (see Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Maryland, (1987)). These terms include hypervariable regions or hypervariable loops in the three-dimensional structure of antibodies as defined by Kabat et al. ("Sequences of Proteins of Immunological Interest," Kabat E. et al., US Dept. of Health and Human Services, 1983) (Chothia and Lesk, 1983). J Mol. Biol. [Journal of Molecular Biology] 196 901-917 (1987). CDRs in each chain are tightly linked by framework regions and, together with CDRs in the other chain, promote the formation of antigen-binding sites. Within the CDRs, there are selective amino acids described as selectivity-determining regions (SDRs), which represent key contact residues used by the CDRs in antibody-antigen interactions (Kashmiri, S., Methods [Methods], 36:25-34 (2005). In this invention, when a specific antibody amino acid or nucleic acid residue is represented by a number, it usually refers to its position in a specific amino acid or nucleic acid sequence (i.e., a specific sequence identifier) ​​and / or according to the numbering by Kabat et al.

[0157] The expressions "framework region" or "FR" refer to one or more framework regions within the variable region of antibody light and heavy chains (see Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These expressions include those regions of the amino acid sequence of the variable region of antibody light and heavy chains that are interposed between the CDRs.

[0158] "Cmax" refers to the maximum (or peak) concentration of an antibody or other compound in a test region (e.g., serum or another compartment, such as cerebrospinal fluid) reached after administration of the drug. For example, serum Cmax can be measured from serum (prepared by collecting a blood sample, clotting it by centrifugation or other means, and separating the solid components to yield serum (blood that does not contain blood cells and clotting factors)) and then detecting the concentration of the analyte in the serum by ELISA or other means known in the art.

[0159] Unless otherwise indicated, "AUC" refers to the area under the concentration-time curve expressed in mg / mL hr (or, equivalently, mg hr / ml). "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 to infinity from time = 0.

[0160] "I max " refers to the maximal pharmacodynamic response elicited by a dose of an anti-CGRP antibody (preferably 350 mg or more, more typically at least 750 mg or 1000 mg) compared to the response elicited by a lower dose of the anti-CGRP antibody, for example where such response can be detected by inhibition of vasodilation following topical administration of capsaicin.

[0161] Antibody Ab6 (Eptinezumab) sequence

[0162] Antibody Ab6 (Eptinezumab) contains the variable light chain sequence QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQK PGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKR (SEQ ID NO.: 10) as listed below.

[0163] Antibody Ab6 (eptinezumab) contains the light chain sequence set forth below: QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.: 11).

[0164] Antibody Ab6 (eptinezumab) contains the variable heavy chain set forth below: EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS (SEQ ID NO.: 4).

[0165] Antibody Ab6 (eptinezumab) contains the heavy chain sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 6)

[0166] Alternatively, the heavy chain of Ab6 (etrolizumab) can lack the C-terminal lysine, i.e., a heavy chain sequence comprising the sequence listed below: EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO.: 5).

[0167] In one embodiment of the application described herein (infra), Fab fragments can be produced by enzymatic digestion (e.g., papain) of Ab6. In another embodiment of the application, Ab6 or a Fab fragment thereof can be produced by expression 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 pastoris) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[0168] The CDR regions of Ab6 (etrolizumab) are outlined below

[0169] Light chain CDR 1 QASQSVYHNTYLA (SEQ ID NO.: 7)

[0170] Light chain CDR 2 DASTLAS (SEQ ID NO.: 8)

[0171] Light chain CDR 3 LGSYDCTNGDCFV (SEQ ID NO.: 9)

[0172] Heavy chain CDR 1 GYYMN (SEQ ID NO.: 1)

[0173] Heavy chain CDR 2 VIGINGATYYASWAKG (SEQ ID NO.: 2)

[0174] Heavy chain CDR 3 GDI (SEQ ID NO.: 3)

[0175] Antibody LuAG09222

[0176] LuAG09222 contains a variable heavy chain sequence as set out below: EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMT WVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSS (SEQ ID NO.: 15) LuAG09222 contains a heavy chain sequence as set out below: EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMT WVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO.: 16).

[0177] According to some aspects of the application, LuAG009222 can have a terminal lysine in the heavy chain as set out below:

[0178] EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMTWVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 26)

[0179] LuAG09222 contains a variable heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMTWVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 26)

[0180] LuAG09222 contains a variable heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAASGIDLNSYYMTWVRQAPGKGLEWIGFIDAGGDAYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 26)

[0181] In one embodiment of the application described herein (below), Fab fragments can be produced by enzymatic digestion (e.g., papain) of Ab10.H3. In another embodiment of the application, LuAG09222 can be produced by expression in mammalian cells, such as CHO, NSO or HEK 293 cells, fungal, insect or microbial systems, such as yeast cells (e.g., diploid yeast, such as diploid Pichia pastoris) and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[0182] The CDR regions of LuAG09222 are outlined below

[0183] Light chain CDR 1 QSSESVYGNYLA (SEQ ID NO.: 17)

[0184] Light chain CDR 2 EASKLES (SEQ ID NO.: 18)

[0185] Light chain CDR 3 AGGDISEGVA (SEQ ID NO.: 19)

[0186] Heavy chain CDR 1 SYYMT (SEQ ID NO.: 12)

[0187] Heavy chain CDR 2 FIDAGGDAYYASWAKG (SEQ ID NO.: 13)

[0188] Heavy chain CDR 3 DLDL (SEQ ID NO.: 14)

[0189] As described herein, antibodies and fragments thereof can be post-translationally modified to add effector moieties, such as chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties, such as, for example, streptavidin, avidin, biotin, cytotoxins, cytotoxic agents, and radioactive materials.

[0190] Antibodies or fragments thereof can also be chemically modified to provide additional advantages, such as increased polypeptide solubility, stability, and circulation time (half-life in vivo) or decreased immunogenicity (see U.S. Patent No. 4,179,337). Chemical moieties for derivatization can be selected from water-soluble polymers, such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like. Antibodies and fragments thereof can be modified at random positions within the molecule or at predetermined positions within the molecule, and can include one, two, three, or more attached chemical moieties.

[0191] The polymer can have any molecular weight, and can be branched or unbranched. For polyethylene glycol, a preferred molecular weight is about 1 kDa to about 100 kDa (the term“about” indicates that in the preparation of polyethylene glycol, some molecules will weigh more than the stated molecular weight, and some less), to facilitate handling and manufacturing. Other sizes can be used, depending on the desired therapeutic properties (e.g., the duration of sustained release desired, the effect on biological activity, if any, ease of handling, degree or lack of antigenicity, and other known effects of polyethylene glycol on therapeutic proteins or analogs). For example, the average molecular weight of the polyethylene glycol can be about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa. Branched polyethylene glycol is described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al, Appl. Biochem. Biotechnol . [Applied Biochemistry and Biotechnology], 56:59-72 (1996); Vorobjev et al, Nucleosides and Nucleotides [ Nucleosides and Nucleotides], 18:2745-2750 (1999); and Caliceti et al, Bioconjug. Chem. [ Bioconjugate Chemistry], 10:638-646 (1999), the disclosure of each of which is incorporated herein by reference.

[0192] There are many attachment methods available to those skilled in the art, see, e.g., EP 0 401 384, which is incorporated herein by reference (coupling of PEG to G-CSF), see also Malik et al, Exp. Hematol[Experimental Hematology] 20: 1028-1035 (1992) (reporting pegylation of GM-CSF using trityl chloride). For example, the polyethylene glycol can be covalently bound via a reactive group such as a free amino or carboxyl group through an amino acid residue. Reactive groups are those to which an activated polyethylene glycol molecule can bind. Amino acid residues having a free amino group can include lysine residues and N-terminal amino acid residues; those having a free carboxyl group can include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues. Thiol groups can also be used as a reactive group for attaching a polyethylene glycol molecule. Attachment on an amino group, such as at the N-terminus or on a lysine group, is preferred for therapeutic purposes.

[0193] As noted above, polyethylene glycol can be attached to a protein by linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a polypeptide through a covalent bond to a lysine, histidine, aspartic acid, glutamic acid, or cysteine residue. One or more reaction chemistries can be employed to attach polyethylene glycol to a particular amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine, and combinations thereof).

[0194] Alternatively, the antibody or fragment thereof can have an increased half-life in vivo by fusion with albumin (including, but not limited to, recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued March 2, 1999, European Patent No. 0 413 622, and U.S. Patent No. 5,766,883, issued June 16, 1998, which are incorporated by reference herein in their entireties) or other circulating blood proteins such as transferrin or ferritin. In preferred embodiments, the polypeptides and / or antibodies (including fragments or variants thereof) of the present application are fused to a mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin, as shown in European Patent 0 322 094, which is incorporated by reference herein in its entirety). The present application also encompasses polynucleotides encoding the fusion proteins of the present application. Figure 1 and Figure 2 The present application also encompasses polynucleotides encoding the fusion proteins of the present application.

[0195] With respect to detectable moieties, additional exemplary enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase, and luciferase. Other exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Other exemplary chemiluminescent moieties include, but are not limited to, luminol. Other exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Other exemplary radioactive materials include, but are not limited to, iodine 125 (125I), technetium 99 (99Tc), indium 113m (113mIn), and thallium 201 (201T1). 125I), carbon 14 ( 14 C), Sulfur 35 ( 35 S), tritium ( 3 H) and phosphorus 32 ( 32 P).

[0196] Regarding the functional component, exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil decarbazine; alkylating agents such as nitrogen mustard, thiotepa, chlorambucil, melphalan, carmastine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatin. II) (DDP) cisplatin and carboplatin (paraplatin); anthracyclines include daunorubicin (formerly donorubicin), doxorubicin (adriamycin), detoxin, erythromycin, idarubicin, epirubicin, mitoxantrone, and bifenthrin; antibiotics include actinomycin (actinomycin D), bleomycin, calciferin, styracin, and anthramycin (AMC); and antimitotic drugs, such as vinca alkaloids, vincristine, and vinblastine. Other cytotoxic agents include paclitaxel (tacrolimus), ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B, bacitracin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, anthraquinone, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mytotane (O,P'-(DDD)), interferon, and mixtures of these cytotoxic agents.

[0197] Other cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calciferine, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, and bleomycin. Toxic enzymes derived from plants and bacteria (e.g., ricin, diphtheria toxin, and pseudomonadoxins) can be conjugated with humanized antibodies or chimeric antibodies or their binding fragments to produce cell type-specific killers (Youle et al.). Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 77:5483 (1980); Gilliland et al., Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 77:4539 (1980); Krolick et al. Proc. Nat'l Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 77:5419 (1980)).

[0198] Other cytotoxic agents include the cytotoxic ribonucleases described by Goldenberg in U.S. Patent No. 6,653,104. Embodiments of the present application also relate to radioimmunoconjugates in which a radionuclide emitting alpha or beta particles is stably coupled to an antibody or binding fragment thereof, with or without the use of a complex forming agent. Such radionuclides include beta-emitters such as phosphorus-32 (P), scandium-47 (Sc), copper-67 (Cu), gallium-67 (Ga), yttrium-88 (Y), yttrium-90 (Y), iodine-125 (I), iodine-131 (I), samarium-153 (Sm), lutetium-177 (Lu), rhenium-186 (Re) or rhenium-188 (Re), and alpha-emitters such as astatine-211 (At), lead-212 (Pb), bismuth-212 (Bi) or -213 (Bi) or actinium-225 (Ac). 32 47 67 67 88 90 125 131 153 177 186 188 211 212 212 213 225

[0199] Methods for conjugating antibodies or binding fragments thereof to detectable moieties, etc. are known in the art, such as those described in Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Methods, 40:219 (1981); and Nygren, J., Histochem. and Cytochem. 30:407 (1982). Nature Biochemistry J. Immunol. Meth Histochem. and Cytochem.

[0200] ​​​​​​​​​​​​​​​​​​​​​The embodiments described herein further include variants and equivalents that are substantially homologous to the antibody, antibody fragments, diabodies, SMIPs, camelid antibodies, nanobodies, IgNARs, polypeptides, variable regions, and CDRs listed herein. These can contain, for example, conservative substitution mutations (i.e., substitution of one or more amino acids with a similar amino acid). For example, a conservative substitution refers to the substitution of an amino acid with another amino acid in the same general class, e.g., the substitution of one acidic amino acid for another, one basic amino acid for another, or one neutral amino acid for another. The purpose of conservative amino acid substitution is well known in the art.

[0201] In another embodiment, the present application contemplates polypeptide sequences having at least 90% or greater sequence homology to any one or more of the polypeptide sequences of the antibody fragments, variable regions, and CDRs listed herein. More preferably, the present application contemplates polypeptide sequences having at least 95% or greater sequence homology, even more preferably at least 98% or greater sequence homology, and still more preferably at least 99% or greater sequence homology to any one or more of the polypeptide sequences of the antibody fragments, variable regions, and CDRs listed herein. Methods of determining homology between nucleic acid and amino acid sequences are well known to those of ordinary skill in the art.

[0202] Another embodiment of the present application contemplates incorporating these polynucleotides into expression vectors for expression in mammalian cells such as CHO, NSO, HEK-293, or in fungal, insect, or microbial systems such as yeast cells such as Pichia pastoris. Suitable Pichia species include, but are not limited to, P. pastoris. In one embodiment of the present application described herein (infra), Fab fragments can be produced by enzymatic digestion (e.g., papain) of Ab6 after expression of the full-length polynucleotide in a suitable host. In another embodiment of the present application, anti-CGRP antibodies, e.g., Ab6 or Fab fragments thereof, can be produced by expressing Ab6 polynucleotides in mammalian cells such as CHO, NSO, or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (e.g., diploid yeast, e.g., diploid Pichia pastoris) and other yeast strains. Suitable Pichia species include, but are not limited to, P. pastoris.

[0203] Host cells and vectors comprising the polynucleotides are also contemplated.

[0204] The present application further contemplates vectors comprising polynucleotide sequences encoding the variable heavy and light polypeptide sequences as well as the individual complementarity determining regions (CDRs or hypervariable regions) as listed herein, and host cells comprising the vector sequences. In one embodiment of the present application, the host cell is a yeast cell. In another embodiment of the present application, the yeast host cell belongs to the genus Pichia.

[0205] Methods of producing antibodies and fragments thereof

[0206] In another embodiment, the present application contemplates methods of producing anti-CGRP antibodies and fragments thereof. Methods of producing antibodies and fragments thereof secreted from a polyploid (preferably diploid or tetraploid) strain of mating-competent yeast are taught in, for example, U.S. Patent Application Publication No. US 2009 / 0022659 to Olson et al. and U.S. Patent No. 7,935,340 to Garcia-Martinez et al., the disclosure of each of which is incorporated by reference herein in its entirety. Methods of producing antibodies and fragments thereof in mammalian cells, such as CHO cells, are well known in the art.

[0207] Other methods of producing antibodies are also well known to those of ordinary skill in the art. For example, methods of producing chimeric antibodies are now well known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.; Morrison, N.B. et al., P.N.A.S. USA [Proc. Natl. Acad. Sci. USA], 81 :8651-55 (1984); Neuberger, M.S. et al., Nature [Nature], 314:268-270 (1985); Boulianne, G.L. et al., Nature [Nature], 312:643-46(1984), the disclosure of each of which is incorporated by reference herein in its entirety).

[0208] Likewise, other methods of producing humanized antibodies are now well known in the art (see, 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 No. 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, P.T. et al., Nature [Nature], 321 :522-525 (1986); Reichmann, L. et al., Nature [Nature], 332:323-327 (1988); Verhoeyen, M et al., Science [Science], 239:1534-36 (1988), the disclosure of each of which is incorporated by reference herein in its entirety).

[0209] Administration

[0210] A "pharmaceutical composition" refers to a chemical or biological composition suitable for administration to a mammal. Such compositions can be specially formulated for administration by one or more of a variety of routes including, but not limited to, intravenous or subcutaneous. Administration can be every 2 weeks, once a month (every 4 weeks), every other month, or every three months.

[0211] A "pharmaceutical excipient" or "pharmaceutically acceptable excipient" is a carrier with which the active therapeutic agent is formulated, typically a liquid. In one embodiment of the present application, the active therapeutic agent is a humanized antibody or one or more fragments thereof described herein. The excipient, while providing chemical and / or biological stability and release characteristics, generally does not provide any pharmacological activity to the formulation. Exemplary formulations can be found in, for example, Remington's Pharmaceutical Sciences, 19th Ed., Gennaro, A. Ed., 1995, which is incorporated by reference.

[0212] As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier can be suitable for intravenous, intraperitoneal, intramuscular, or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the application is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0213] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The present application contemplates pharmaceutical compositions in lyophilized form. The compositions can be formulated into solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The present application further contemplates the inclusion of stabilizers in the pharmaceutical compositions. For example, the proper fluidity can be maintained, for example, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0214] In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of an injectable composition can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, the basic polypeptide can be formulated as a timed release formulation, for example, as a composition that includes a slow release polymer. The active compounds can be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Numerous methods for preparing such formulations are known to those of skill in the art.

[0215] According to the present application, LuAG09222 and epratuzumab can be co-formulated according to the following examples.

[0216] Unless otherwise specified in the following examples, the total amount of epratuzumab and LuAG09222 in the pharmaceutical formulation is between 100 mg / mL and 300 mg / mL (optionally about 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL or 300 mg / mL), wherein the ratio of epratuzumab and LuAG00922 can vary. In one embodiment, the ratio of epratuzumab and LuAG09222 is 1 : 1, 1 : 2 and 2 : 1, which means that 100 mg / mL pharmaceutical formulation for example a ratio of 1 : 1 would contain 50 mg / mL epratuzumab and 50 mg / mL LuAG09222. The following gives examples of pharmaceutical compositions according to the present application.

[0217] According to one embodiment, the present application relates to a pharmaceutical composition comprising epratuzumab and LuAG90222 in a ratio selected from 2 : 1, 1 : 1 or 1 : 2, and further comprising 20-40 mM histidine buffer, 90-180 mM sorbitol, Poloxamer P188 0.0025-0.0120% w / v and 30-70 mM NaCl, and having a pH of about 6 (optionally between pH 5.5 and 6.4, optionally pH 5.9).

[0218] According to one embodiment, the present application is directed to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 2: 1, and comprising 25-35 mM histidine buffer, 165-175 mM sorbitol, Poloxamer P188 0.0025-0.010% w / v, and 25-35 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

[0219] According to one embodiment, the present application is directed to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 2: 1, and comprising 30 mM histidine buffer, 169 mM sorbitol, Poloxamer P188 0.005% w / v, and 30 mM NaCl, and having a pH of about 6 (optionally pH 5.9). The total combined protein concentration of ipilimumab and LuAG09222 in the composition can be 113 mg / mL.

[0220] According to one embodiment, the present application is directed to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 2: 1, and comprising 30-50 mM histidine buffer, 80-150 mM sorbitol, L-arginine hydrochloride 150-250 mM, and Poloxamer P188 0.02% w / v, and having a pH of about 6 (optionally pH 6.125). The total combined protein concentration of ipilimumab and LuAG09222 in the composition can be 140-150 mg / mL.

[0221] According to one embodiment, the present application is directed to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 2: 1, and comprising 30 mM histidine buffer, 80 mM sorbitol, L-arginine hydrochloride 150 mM, and Poloxamer P188 0.02% w / v, and having a pH of about 6 (optionally pH 6.125). The total combined protein concentration of ipilimumab and LuAG09222 can be about 150 mg / mL.

[0222] According to one embodiment, the present application is directed to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 2: 1, and comprising 50 mM histidine buffer, 150 mM sorbitol, L-arginine hydrochloride 250 mM, and Poloxamer P188 0.02%, and having a pH of about 6 (optionally pH 6.125). The total combined protein concentration of ipilimumab and LuAG09222 can be about 145 mg / mL.

[0223] According to one embodiment, the present application relates to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 1 : 1, and comprising 30-40 mM histidine buffer, 130-140 mM sorbitol, Poloxamer P188 0.005-0.010% w / v, and 45-55 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

[0224] According to one embodiment, the present application relates to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 1 : 1, and comprising 34 mM histidine buffer, 135 mM sorbitol, Poloxamer P188 0.008% w / v, and 48 mM NaCl, and having a pH of about 6 (optionally pH 5.9). The total protein concentration of ipilimumab and LuAG09222 can be 120 mg / mL.

[0225] According to one embodiment, the present application relates to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 1 : 1, and comprising 30 mM histidine buffer, 80 mM sorbitol, L-arginine 150 mM, and Poloxamer P188 0.02% w / v, and having a pH of about 6 (optionally pH 6.125). The total protein concentration of ipilimumab and LuAG09222 can be about 130 mg / mL.

[0226] According to one embodiment, the present application relates to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 1 : 1, and comprising 10 mM histidine buffer, 10 mM sorbitol, L-arginine hydrochloride 50 mM, and Poloxamer P188 0.02% w / v, and having a pH of about 6 (optionally pH 6.125). The total protein concentration of ipilimumab and LuAG09222 can be about 140 mg / mL.

[0227] According to one embodiment, the present application relates to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 1 : 2, and comprising 35-45 mM histidine buffer, 90-100 mM sorbitol, Poloxamer P188 0.010-0.0120% w / v, and 65-75 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

[0228] According to one embodiment, the present application relates to a pharmaceutical composition comprising epratuzumab and LuAG90222 in a ratio of 1 : 2, and comprising 38 mM histidine buffer, 96 mM sorbitol, Poloxamer P188 0.0114% w / v, and 69 mM NaCl, and having a pH of about 6 (optionally pH 5.9). The total protein concentration of epratuzumab and LuAG09222 can be 129 mg / mL.

[0229] In the above embodiments, arginine (optionally L-arginine) can be added at a concentration of 10-150 mM, optionally 10 mM, 50 mM, 80 mM, 100 mM, or 120 mM or 150 mM.

[0230] Additionally, in embodiments comprising arginine, the pharmaceutical formulation can comprise epratuzumab and LuAG90222 in a ratio selected from 1 : 2, 1 : 1, or 2: 1, and further comprising 30-50 mM histidine buffer, 50-250 mM sorbitol, and 10-150 mM NaCl, Poloxamer 188 0.005-0.05% w / v, 10-150 mM L-arginine hydrochloride, and having a pH of about 6 (optionally pH 5.9).

[0231] In another embodiment, the pharmaceutical formulation can comprise epratuzumab and LuAG90222 in a ratio selected from 1 : 2, 1 : 1, or 2: 1, and comprising 30-50 mM histidine buffer, 150-250 mM sorbitol, and 80-150 mM NaCl, 0.005-0.05% w / v Poloxamer P188, 80-150 mM L-arginine hydrochloride, and having a pH of about 6 (optionally pH 5.9). The total protein concentration of epratuzumab and LuAG09222 can be between 125-150 mg / mL.

[0232] In another embodiment, the pharmaceutical formulation can comprise epratuzumab and LuAG90222 in a ratio of 1 : 2, and comprising 10-50 mM histidine buffer, 50-250 mM sorbitol, and 10 -150 mM NaCl, 0.005-0.05% w / v Poloxamer P188, 10-150 mM L-arginine hydrochloride, and having a pH of about 6 (optionally pH 5.9). The total protein concentration of epratuzumab and LuAG09222 can be between 100-150 mg / mL.

[0233] In another embodiment, the pharmaceutical formulation can comprise epratuzumab and LuAG90222 in a ratio selected from 1 : 2, 2 : 1, and 1 : 1, and comprise 10-50 mM histidine buffer, 50-250 mM sorbitol, and 10 - 150 mM NaCl, 0.005-0.05% w / v poloxamer P188, 10-150 mM L-arginine hydrochloride, and have a pH of about 7 (optionally pH 6.75) or a pH of about 5 (optionally about 5.25). The total protein concentration of epratuzumab and LuAG09222 can be between 100-125 mg / mL.

[0234] According to one embodiment, the present application is directed to a pharmaceutical composition comprising epratuzumab and LuAG90222 in a ratio selected from 1 : 1, 1 :2, or 2 : 1, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, L-arginine hydrochloride 50-250 mM, and poloxamer P188 0.02% w / v, and having a pH of about 5 (optionally pH 5.5). The total protein concentration of epratuzumab and LuAG09222 can be between 140-155 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, or 155 mg / mL).

[0235] According to one embodiment, the present application is directed to a pharmaceutical composition comprising epratuzumab and LuAG90222 in a ratio selected from 1 : 1, 1 :2, or 2 : 1, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, L-arginine hydrochloride 50-250 mM, and poloxamer P188 0.02% w / v, and having a pH of about 5 (optionally pH 5.5). The total protein concentration of epratuzumab and LuAG09222 can be between 140-155 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, or 155 mg / mL).

[0236] According to one embodiment, the present application relates to a pharmaceutical composition comprising epratuzumab and LuAG90222 in a ratio selected from 1 : 1, 1 :2 or 2 : 1, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, L-arginine hydrochloride 50-250 mM and Poloxamer P188 0.02% w / v, and having a pH of about 5 (optionally pH 5.5). The total protein concentration of epratuzumab and LuAG09222 can be between 140-155 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL or 155 mg / mL).

[0237] According to one embodiment, the present application relates to a pharmaceutical composition comprising epratuzumab and LuAG90222 in a ratio selected from 1 : 1, 1 :2 or 2 : 1, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, L-arginine hydrochloride 50-250 mM and Poloxamer P188 0.02% w / v, and having a pH of about 7 (optionally pH 6.75). The total protein concentration of epratuzumab and LuAG09222 can be between 140-160 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, 155 mg / mL or 160 mg / mL).

[0238] According to one embodiment, the present application relates to a pharmaceutical composition comprising epratuzumab and LuAG90222 in a ratio selected from 1 : 1, 1 :2 or 2 : 1, and comprising 10-50 mM histidine buffer, 10-150 mM sorbitol, L-arginine hydrochloride 50-250 mM and Poloxamer P188 0.02% w / v, and having a pH of about 7 (optionally pH 6.75). The total protein concentration of epratuzumab and LuAG09222 can be between 140-160 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, 155 mg / mL or 160 mg / mL).

[0239] According to one embodiment, the present application relates to a pharmaceutical composition comprising ipilimumab and LuAG90222 in a ratio of 2: 1, and comprising 30-50 mM histidine buffer, 80-150 mM sorbitol, L-arginine hydrochloride 50-250 mM, and Poloxamer P188 0.02% w / v, and having a pH of about 7 (optionally pH 6.75). The total protein concentration of ipilimumab and LuAG09222 can be between 140-160 mg / mL (optionally 140 mg / mL, 145 mg / mL, 150 mg / mL, 155 mg / mL).

[0240] Further exemplary embodiments

[0241] Embodiment 1 (El) A composition comprising ipilimumab and LuAG09222.

[0242] E2. The composition of El, further comprising histidine and Poloxamer 188 or polysorbate 80.

[0243] E3. The composition of El and E2, further comprising one, two, or all of the following excipients: NaCl, sorbitol (e.g., L-sorbitol), and arginine (e.g., L-arginine).

[0244] E4. The pharmaceutical composition of any of the preceding embodiments, comprising ipilimumab and LuAG09222 in a total concentration of 100 mg / mL to 300 mg / mL.

[0245] E5. The pharmaceutical composition of any of the preceding embodiments, comprising ipilimumab and LuAG09222 in a total concentration of 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, or 300 mg / mL.

[0246] E6. The pharmaceutical composition of embodiments 4 or 5, having a ratio of ipilimumab to LuAG09222 of 1 : 1, 1 : 2, or 2 : 1.

[0247] E7. The pharmaceutical composition of any of the preceding embodiments, wherein the composition comprises 100-300 mg / mL of ipilimumab and 50-100 mg / mL of LuAG09222.

[0248] E8. The composition of any of the preceding embodiments, wherein the composition comprises 100, 150, 200, or 300 mg / ml of ipilimumab.

[0249] E9. The composition according to any of the preceding embodiments, wherein the composition comprises 50, 75, 100, or 150 mg / ml of LuAG09222.

[0250] E10. The composition according to any of the preceding embodiments, wherein the composition comprises 100 mg / ml of epratuzumab and 50 mg / ml of LuAG09222.

[0251] E11. The composition according to any of the preceding embodiments, wherein epratuzumab and LuAG09222 are the only active ingredients in the composition.

[0252] E12. The composition according to any of the preceding embodiments, wherein histidine is in the composition at a concentration between 10-50 mM, optionally 10 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM.

[0253] E13. The composition according to any of the preceding embodiments, wherein polysorbate 80 or poloxamer P188 is in the composition at 0.005-0.05% w / v.

[0254] E14. The composition according to any of the preceding embodiments, wherein one, two, or three of the following excipients are present in the composition: NaCl, sorbitol, and arginine.

[0255] E15. The composition according to any of the preceding embodiments, wherein the concentration of NaCl in the composition is between 10-150 mM, optionally 25 mM, 50 mM, 100 mM, or 150 mM.

[0256] E16. The composition according to any of the preceding embodiments, wherein sorbitol is present in the composition at a concentration between 50-250 mM, optionally 50 mM, 100 mM, 150 mM, 200 mM, or 250 mM.

[0257] E17. The composition according to any of the preceding embodiments, wherein arginine is present in the composition at a concentration between 50-250 mM, optionally 50 mM, 100 mM, 150 mM, 200 mM, or 250 mM.

[0258] E18. The composition according to any of the preceding embodiments, wherein the pH is between 5.0-6.8, optionally pH 5.0, pH 5.5, pH 5.9, pH 6.0, pH 6.5, or pH 6.8.

[0259] E19. The pharmaceutical composition according to any one of the preceding claims, wherein the concentration of histidine ranges between 10 - 50 mM, optionally 20-40 mM, the concentration of poloxamer P188 ranges between 0.0025 - 0.0120% w / v, and the concentration of polysorbate 80 ranges between 0.005-0.05% w / v, including the end values.

[0260] E20. The pharmaceutical composition according to any one of the preceding embodiments, wherein the concentration of NaCl is between 10-150 mM, optionally 30-70 mM, the concentration of sorbitol is 50-250 mM, optionally 90-180 mM, and the concentration of arginine (e.g. L-arginine) ranges between 50-250 mM, including the end values.

[0261] E21. The pharmaceutical composition according to any one of the preceding embodiments, comprising epratuzumab and LuAG90222 in a ratio of 2 : 1, 1 : 1 or 1 : 2, and comprising 20-40 mM histidine buffer, 90-180 mM sorbitol, poloxamer P188 0.0025-0.0120% w / v and 30-70 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

[0262] E22. The pharmaceutical composition according to any one of the preceding embodiments, comprising epratuzumab and LuAG90222 in a ratio of 2 : 1, and comprising 25-35 mM histidine buffer, 165-175 mM sorbitol, poloxamer P188 0.0025-0.010% w / v and 25-35 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

[0263] E23. The pharmaceutical composition according to any one of the preceding embodiments, comprising epratuzumab and LuAG90222 in a ratio of 1 : 1, and comprising 30-40 mM histidine buffer, 130-140 mM sorbitol, poloxamer P188 0.005-0.010% w / v and 45-55 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

[0264] E24. The pharmaceutical composition according to any of the preceding embodiments comprising eptinezumab and LuAG90222 in a ratio of 1 :2, and comprising 35-45 mM histidine buffer, 90-100 mM sorbitol, poloxamer P188 0.010-0.0120% w / v, and 65-75 mM NaCl, and having a pH of about 6 (optionally pH 5.9).

[0265] E25. The composition according to any of the preceding embodiments for use as a medicament.

[0266] E26. The composition according to any of the preceding embodiments for use in subcutaneous administration.

[0267] E27. The composition according to any of the preceding embodiments for use in the treatment or prevention of headache.

[0268] E28. The composition according to any of the preceding embodiments for use in the treatment or prevention of pain.

[0269] E29. The composition according to any of the preceding embodiments for use in the treatment or prevention of chronic or episodic migraine.

[0270] E30. The composition according to any of the preceding embodiments for use in the treatment or prevention of cluster headache.

[0271] E31. The composition according to any of the preceding embodiments for use in the treatment or prevention of endometriosis.

[0272] E32. The composition according to any of the preceding embodiments for use in the treatment or prevention of migraine (with or without aura), weight loss, cancer or tumour, angiogenesis associated with cancer or tumour growth, angiogenesis associated with cancer or tumour survival, hemiplegic migraine, cluster headache, migraineous neuralgia, chronic headache, tension headache, general headache, hot flashes, chronic-stroke migraine, secondary headache due to underlying structural problems of the head or neck, brain neuralgias, sinus headache (optionally e.g. rhinosinusitis-associated headache), headache or migraine caused by allergy, pain, inflammatory pain, postoperative incisional pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, pain resulting from burns, osteoporosis, gouty joint pain, pain associated with sickle cell crisis and other nociceptive pain, and hepatocellular carcinoma, breast cancer, cirrhosis, neurogenic pain, neuropathic pain, nociceptive pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovariopagia, reflex sympathetic dystrophy, neurogenic pain, osteoarthritis or rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, or visceral pain associated with gastroesophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstrual period, labour, menopause, prostatitis or pancreatitis.

[0273] E33. The composition according to any of the preceding embodiments for use in the treatment or prevention of chronic pain; neurogenic inflammation and inflammatory pain; neuropathic pain; ocular pain; dental pain; postoperative pain, trauma-related pain, diabetes; non-insulin dependent diabetes and other inflammatory autoimmune disorders, vascular disorders; inflammation; arthritis; sarcoidosis, bronchial hyperreactivity, asthma; shock; sepsis; opiate withdrawal syndrome; morphine tolerance; hot flashes in men and women; allergic dermatitis; psoriasis; encephalitis; brain trauma; epilepsy; neurodegenerative diseases; skin diseases, including pruritis, neurogenic cutaneous redness, cutaneous rosaceousness and erythema; inflammatory bowel disease, irritable bowel syndrome, cystitis; and dysmenorrhea.

[0274] E34. The composition according to any of the preceding embodiments for monthly or biweekly administration.

[0275] E35. The composition according to any of the preceding embodiments, wherein the composition comprises epratuzumab and a fragment of LuAG09222.

[0276] E36. The composition according to embodiment 26, wherein the fragment of epratuzumab and LuAG09222, respectively, comprises all 6 CDR regions of said antibody.

[0277] E37. The composition according to embodiment 26, wherein the fragment comprises or consists of the VH and VL regions of ipilimumab and LuAG09222.

[0278] E38. The composition according to any one of the preceding embodiments, wherein the composition comprises 50-150 mg / ml ipilimumab, optionally 50 mg / ml, 75 mg / ml, 100 mg / ml or 150 mg / ml.

[0279] E39. The composition according to any one of the preceding embodiments, wherein the composition comprises 25-100 mg / ml LuAG09222, optionally 25 mg / ml, 50 mg / ml, 75 mg / ml or 100 mg / ml.

[0280] E40. The composition according to any one of the preceding embodiments, for use in a method of subcutaneous administration.

[0281] E41. A method for treating or preventing headache, the method comprising administering to a patient in need thereof a composition according to any one of E1-E30.

[0282] E42. A method for treating or preventing pain, the method comprising administering to a patient in need thereof a composition according to any one of E1-E30.

[0283] E43. A method for treating or preventing chronic or episodic migraine, the method comprising administering to a patient in need thereof a composition according to any one of E1-E30.

[0284] E44. A method for treating or preventing cluster headache, the method comprising administering to a patient in need thereof a composition according to any one of E1-E30.

[0285] E45. A method for treating or preventing endometriosis, the method comprising administering to a patient in need thereof a composition according to any one of E1-E30.

[0286] E46. A method for treating or preventing migraine (with or without aura), weight loss, cancer or tumor, angiogenesis associated with cancer or tumor growth, angiogenesis associated with cancer or tumor survival, hemiplegic migraine, cluster headache, migraineous neuralgia, chronic headache, tension headache, general headache, hot flashes, chronic paroxysmal hemicrania, secondary headache due to underlying structural problems of the head or neck, brain neuralgias, sinus headache (optionally e.g. rhinosinusitis associated headache), headache or migraine caused by allergy, pain, inflammatory pain, postoperative incisional pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, pain resulting from burns, osteoporosis, gouty joint pain, pain associated with sickle cell crisis and other nociceptive pain, and hepatocellular carcinoma, breast cancer, cirrhosis, neuropathic pain, neurogenic pain, nociceptive pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovariopain, reflex sympathetic dystrophy, neurogenic pain, osteoarthritis or rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, or visceral pain associated with gastroesophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstrual period, labor, menopause, prostatitis, or pancreatitis, The method comprises administering to a patient in need thereof a composition according to any one of E1-E40.

[0287] E47. A method for treating or preventing chronic pain; neurogenic inflammation and inflammatory pain; neuropathic pain; ocular pain; dental pain; postoperative pain, trauma-related pain, diabetes; non-insulin dependent diabetes and other inflammatory autoimmune disorders, vascular disorders; inflammation; arthritis; sarcoidosis, bronchial hyperreactivity, asthma; shock; sepsis; opiate withdrawal syndrome; morphine tolerance; hot flashes in men and women; allergic dermatitis; psoriasis; encephalitis; brain trauma; epilepsy; neurodegenerative diseases; skin diseases, including pruritis, neurogenic cutaneous redness, cutaneous rosaceous macules and erythema; inflammatory bowel disease, irritable bowel syndrome, cystitis; and dysmenorrhea, The method comprises administering to a patient in need thereof a composition according to any one of E1-40.

[0288] E48. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of headache.

[0289] E49. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of pain.

[0290] E50. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of chronic or episodic migraine.

[0291] E51. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of cluster headache.

[0292] E52. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of endometriosis.

[0293] E53. Use of a 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 tumour, angiogenesis associated with cancer or tumour growth, angiogenesis associated with cancer or tumour survival, hemiplegic migraine, cluster headache, migraineous neuralgia, chronic headache, tension headache, general headache, hot flashes, chronic paroxysmal hemicrania, secondary headache due to underlying structural problems in the head or neck, brain neuralgias, sinus headache (optionally e.g. rhinosinusitis-associated headache), headache or migraine caused by allergy, pain, inflammatory pain, postoperative incisional pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, pain resulting from burns, osteoporosis, gouty joint pain, pain associated with sickle cell crisis and other nociceptive pain, and hepatocellular carcinoma, breast cancer, cirrhosis, neuropathic pain, neurogenic pain, nociceptive pain, trigeminal neuralgia, postherpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovariopain, reflex sympathetic dystrophy, neurogenic pain, osteoarthritic or rheumatoid arthritic pain, lower back pain, diabetic neuropathy, sciatica, or visceral pain associated with gastroesophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstruation, labor, menopause, prostatitis, or pancreatitis.

[0294] E54. Use of a composition according to E1-E40 for the manufacture of a medicament for the treatment or prevention of chronic pain; neurogenic inflammation and inflammatory pain; neuropathic pain; ocular pain; dental pain; postoperative pain, trauma-related pain, diabetes; non-insulin dependent diabetes and other inflammatory autoimmune disorders, vascular disorders; inflammation; arthritis; sarcoidosis, bronchial hyperreactivity, asthma; shock; sepsis; opiate withdrawal syndrome; morphine tolerance; hot flashes in men and women; allergic dermatitis; psoriasis; encephalitis; brain trauma; epilepsy; neurodegenerative diseases; skin diseases, including pruritis, neurogenic cutaneous redness, cutaneous rosaceousness and erythema; inflammatory bowel disease, irritable bowel syndrome, cystitis; and dysmenorrhea.

[0295] E55. The composition, method or use of any of the preceding embodiments, wherein the epratuzumab comprises all six CDR sequences: light chain CDR 1 SEQ ID NO.: 7, light chain CDR 2 SEQ ID NO.: 8, light chain CDR 3 SEQ ID NO.: 9, heavy chain CDR 1 SEQ ID NO.: 1, heavy chain CDR 2 SEQ ID NO.: 2, and heavy chain CDR 3 SEQ ID NO.: 3.

[0296] E56. The composition, method or use of any of the preceding embodiments, wherein the LU AG 09222 comprises all six CDR sequences: light chain CDR 1 SEQ ID NO.: 17, light chain CDR 2 SEQ ID NO.: 18, light chain CDR 3 SEQ ID NO.: 19, heavy chain CDR 1 SEQ ID NO.: 12, heavy chain CDR 2 SEQ ID NO.: 13, and heavy chain CDR 3 SEQ ID NO.: 14.

[0297] E57. The composition, method or use of any of the preceding embodiments, wherein the epratuzumab has a VH region as defined in SEQ ID NO.: 4 and a VL as defined in SEQ ID NO.: 10.

[0298] E58. The composition, method or use of any of the preceding embodiments, wherein the LU AG 09222 has a VH region as defined in SEQ ID NO.: 15 and a VL region as defined in SEQ ID NO.: 20.

[0299] E59. The composition, method or use of any of the preceding embodiments, wherein the epratuzumab has a heavy chain as defined in SEQ ID NO.: 5 or SEQ ID NO.: 6 and a light chain as defined in SEQ ID NO.: 11.

[0300] E60. The composition, method or use of any of the preceding embodiments, wherein the LU AG 09222 has a heavy chain as defined in SEQ ID NO.: 16 or SEQ ID No.: 26 and a light chain as defined in SEQ ID NO.: 21.

[0301] E61. The composition, method or use of any one of the preceding embodiments, wherein histidine is in the form of L-histidine, and / or sorbitol is in the form of L-sorbitol and arginine is in the form of L-arginine.

[0302] Further exemplary embodiments (EE)

[0303] Embodiment 1 (EE1 ) A method for treating a patient in need thereof by administering an effective amount of eptinezumab and LuAG09222.

[0304] EE2. The method of EE1, wherein eptinezumab and LuAG09222 are administered simultaneously or sequentially.

[0305] EE3. The method of any one of the preceding embodiments, wherein eptinezumab and LuAG09222 are administered sequentially within a time period of ½ - 2 hours, for example within ½ hour or 1 hour.

[0306] EE4. The method of any one of the preceding embodiments, wherein 50 - 150 mg / ml eptinezumab is administered, optionally 50 mg / ml, 75 mg / ml, 100 mg / ml or 150 mg / ml.

[0307] EE5. The method of any one of the preceding embodiments, wherein 25 - 100 mg / ml LuAG09222 is administered, optionally 25 mg / ml, 50 mg / ml, 75 mg / ml or 100 mg / ml.

[0308] EE6. The method of any one of the preceding embodiments, wherein eptinezumab is administered subcutaneously or intravenously, and LuAG09222 is administered subcutaneously or intravenously.

[0309] EE7. The method of any one of the preceding embodiments, for use in the treatment or prevention of headache.

[0310] EE8. The method of any one of the preceding embodiments, for use in the treatment or prevention of pain.

[0311] EE9. The method of any one of the preceding embodiments, for use in the treatment or prevention of chronic or episodic migraine.

[0312] EE10. The method of any one of the preceding embodiments, for use in the treatment or prevention of cluster headache.

[0313] EE11 The method of any one of the preceding embodiments, for use in the treatment or prevention of endometriosis.

[0314] EE12. The method of any of the preceding embodiments 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, migraineous neuralgia, chronic headache, tension headache, general headache, hot flashes, chronic-stroke migraine, secondary headache due to underlying structural problems in the head or neck, brain neuralgia, sinus headache (e.g., like sinusitis-related headache), headache or migraine caused by allergy, pain, inflammatory pain, post-surgical incision pain, complex regional pain syndrome, cancer pain, primary or metastatic bone cancer pain, fracture pain, osteoporotic fracture pain, pain resulting from burns, osteoporosis, gouty joint pain, pain associated with sickle cell crisis, and other nociceptive pain, and hepatocellular carcinoma, breast cancer, cirrhosis, neuropathic pain, neurogenic pain, nociceptive pain, trigeminal neuralgia, post-herpetic neuralgia, phantom limb pain, fibromyalgia, menstrual pain, ovarian pain, reflex sympathetic dystrophy, neurogenic pain, osteoarthritis or rheumatoid arthritis pain, lower back pain, diabetic neuropathy, sciatica, or visceral pain associated with gastroesophageal reflux, dyspepsia, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, ileitis, ulcerative colitis, renal colic, dysmenorrhea, cystitis, menstrual period, labor, menopause, prostatitis, or pancreatitis.

[0315] EE13. The method of any of the preceding embodiments for the treatment or prevention of chronic pain; neurogenic inflammation and inflammatory pain; neuropathic pain; ocular pain; dental pain; post-surgical pain, trauma-related pain, diabetes; non-insulin dependent diabetes and other inflammatory autoimmune disorders, vascular disorders; inflammation; arthritis; sarcoidosis, bronchial hyperreactivity, asthma; shock; sepsis; opiate withdrawal syndrome; morphine tolerance; hot flashes in men and women; allergic dermatitis; psoriasis; encephalitis; brain trauma; epilepsy; neurodegenerative diseases; skin diseases, including pruritis, neurogenic cutaneous redness, cutaneous rosaceous macules, and erythema; inflammatory bowel disease, irritable bowel syndrome, cystitis; and dysmenorrhea.

[0316] EE14. The method of any of the preceding embodiments, wherein the epratuzumab and LuAG09222 are administered monthly (every 4 weeks) or every 2 weeks.

[0317] EE15. The method of any of the preceding embodiments, wherein the epratuzumab comprises all six CDR sequences: light chain CDR 1 SEQ ID NO.: 7, light chain CDR 2 SEQ ID NO.: 8, light chain CDR 3 SEQ ID NO.: 9, heavy chain CDR 1 SEQ ID NO.: 1, heavy chain CDR 2 SEQ ID NO.: 2, and heavy chain CDR 3 SEQ ID NO.: 3.

[0318] EE16 The method according to any one of the preceding embodiments, wherein LUAG09222 comprises all six CDR sequences: light chain CDR 1 SEQ ID NO.: 17, light chain CDR 2 SEQ ID NO.: 18, light chain CDR 3 SEQ ID NO.: 19, heavy chain CDR 1 SEQ ID NO.: 12, heavy chain CDR 2 SEQ ID NO.: 13, and heavy chain CDR 3 SEQ ID NO.: 14.

[0319] EE17 The method according to any one of the preceding embodiments, wherein eteplumab has a VH region as defined in SEQ ID NO.: 4 and a VL as defined in SEQ ID NO.: 10.

[0320] EE18 The method according to any one of the preceding embodiments, wherein LUAG09222 has a VH region as defined in SEQ ID NO.: 15 and a VL region as defined in SEQ ID NO.: 20.

[0321] EE19 The method according to any one of the preceding embodiments, wherein the eteplumab has a heavy chain as defined in SEQ ID NO.: 5 or SEQ ID NO.: 6 and a light chain as defined in SEQ ID NO.: 11.

[0322] EE20 The method according to any one of the preceding embodiments, wherein the LUAG09222 has a heavy chain as defined in SEQ ID NO.: 16 or SEQ ID NO.: 26 and a light chain as defined in SEQ ID NO.: 21.

[0323] The above description of various illustrative embodiments of the application is not intended to be exhaustive or to limit the application to the precise form disclosed. While specific embodiments of, and examples for, the application are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the application, as those skilled in the relevant art will recognize. The teachings provided herein can be applied to other purposes, in addition to the examples presented herein.

[0324] These and other changes can be made to the application in light of the above Detailed Description. The terms used in the following claims should not be construed to limit the application to the specific embodiments disclosed in the specification and claims. Accordingly, the application is not limited by the disclosure, which has been set forth solely by way of illustration of the application.

[0325] The application can be practiced with the specific details set forth above and in the accompanying Examples. Numerous modifications and variations are possible in light of the above teachings and, therefore, the application is to be construed broadly within the scope of the appended claims.

[0326] The entire disclosure of each of the files cited in the Background of the Invention, the, the Examples, and the Abstract is hereby incorporated by reference.

[0327] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application, and are not intended to limit the scope of the application. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric pressure.

[0328] Examples

[0329] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application, and are not intended to limit the scope of the application. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric pressure.

[0330] Example 1

[0331] Human clinical study to assess safety and efficacy of anti-CGRP antibodies according to the present application

[0332] Clinical treatment regimen

[0333] The ability of the humanized anti-CGRP IgGl antibody identified herein as Ab6 (erenumab) to inhibit, reduce or prevent the number, duration and / or intensity of migraine attacks was assessed in human subjects.

[0334] Specifically, the clinical efficacy of the Ab6 antibody was tested in a placebo-controlled, double-blind, randomized study. All individuals in the study were selected according to specific criteria. In particular, all were diagnosed migraineurs (age < 50 years) (ICHD-II, 2004, section 1) and had a history of migraine > 12 months with > 5 and < 14 migraine days per 28-day period within the 3 months prior to screening.

[0335] In addition, all individuals in the study had completed a 28-day period of e-diary, used acute migraine medication < 14 days per 28-day period, and used triptans < 10 days per 28-day period within those days, within 3 months prior to screening and prior to randomization.

[0336] Table 1 summarizes the demographic characteristics of the study population.

[0337]

[0338] Throughout the study, all individuals were required to use an electronic diary (e-diary) to record their migraine status on a daily basis. In the e-diary, study participants were required to record migraine days / month, migraine attacks / month, migraine hours / month, migraine severity, and any abortive medication use (e.g., triptans).

[0339] In addition, study participants were required to record their migraine status using the e-diary for a 28-day period prior to treatment with either the antibody or placebo to establish a baseline of migraine days / hours / attacks per month. In addition, this allowed study participants to become familiar with the use of the e-diary.

[0340] After the 28-day run-in, study participants were divided into two groups, each comprising 80 participants (n = 80) Figure 5 ). In the first group, the antibody treatment group (n = 80), each participant in the group was administered a single 1000 mg dose of Ab6 intravenously. In the second group (n = 80), the placebo group, each participant was given an intravenous injection containing only the aqueous solution of the antibody vehicle.

[0341] Individuals in the treatment and placebo groups were evaluated for 24 weeks after dose administration. Initially, a 12-week interim analysis was performed. Following the 12-week interim analysis, a refinement analysis was performed. This refinement analysis can include, for example, the addition or deletion of patient data according to the study protocol, for example, updating data that has not yet been fully loaded from the e-diary. This refinement resulted in slight changes, but did not change the overall conclusions.

[0342] The efficacy of the antibody compared to placebo was evaluated based on data recorded in the e-diary entries. For example, the analysis included comparing the recorded migraine days / month, migraine attacks / month, migraine hours / month of subjects between the treatment group compared to the placebo group. The percentage of responders (i.e., subjects with a 50%, 75%, and 100% reduction in migraine days) in each of the two groups was also compared.

[0343] In addition, the response of subjects treated with Ab6 and placebo in both groups to the MSQ and HIT-6 questionnaires will be assessed and compared. The MSQ is a commonly used disease-specific tool to assess the impact of migraine on health-related quality of life (HRQL). The MSQ contains 16 items of the Migraine-Specific Quality of Life Questionnaire (Version 1.0), which was developed by Glaxo Wellcome Inc. It is hypothesized that the MSQ can measure 3 parameters: (i) Role Function - Restrictive; (ii) Role Function - Preventive; and (iii) Emotional Function.

[0344] The HIT-6 or Functional Impact (also known as the Headache Impact Test or HIT-6) is similarly a well-known tool for assessing the intensity of migraine. This test uses six questions to illustrate the impact of headaches and their treatment on an individual's functional health and well-being.

[0345] Clinical Outcomes and Analysis

[0346] The results of this human clinical trial and analysis through Week 12 in treated subjects are summarized in Table 2 below.

[0347] Table 2. Responder Analysis of Migraine Days

[0348] In addition, the clinical study results were compared based on the number of responders in the treatment and placebo groups. As shown in Table 3 below, the number of subjects in the treatment and placebo groups who experienced a 50%, 75%, or 100% reduction in migraine days per month at the midpoint of the study was compared. Figure 1 As shown in the table, 60% of subjects in the Ab6 treatment group experienced at least a 50% reduction in headache days, 31% of subjects in the Ab6 treatment group experienced at least a 75% reduction in headache days, and 15% of subjects in the Ab6 treatment group experienced a 100% reduction in headache days.

[0349] In contrast, 33% of subjects in the placebo treatment group experienced at least a 50% reduction in headache days, 9% of subjects in the placebo treatment group experienced at least a 75% reduction in headache days, and 0% (none) of subjects in the placebo treatment group experienced a 100% reduction in headache days.

[0350] These results clearly indicate that there was a greater reduction in the number of migraine days in the Ab6 treatment group. However, the difference between these numbers would be even more pronounced for a significant placebo effect. (A heightened placebo effect is not unusual for migraine and other neurological drugs).

[0351] In addition, the % change in the number of migraine days per month compared to baseline was compared between the placebo and Ab6 treatment groups. As shown in Table 4 below, the % change in the number of migraine days per month compared to baseline was compared between the placebo and Ab6 treatment groups. Figure 2The median (% of QR) change from baseline in the number of monthly migraine days for the placebo and Ab6 treatment groups over the 12 weeks post-treatment was compared. These statistically significant (p = 0.0078) results clearly indicate that the Ab6 treatment group experienced a greater reduction in the number of monthly migraine days compared to the placebo treatment group compared to baseline.

[0352] In addition, the change from baseline in the number of monthly migraine attacks for the placebo and Ab6 treatment groups was compared. As shown in Figure 6, the median (% of QR) change from baseline in the number of monthly migraine attacks for the placebo and Ab6 treatment groups over the 12 weeks post-treatment was compared. These results indicate that the Ab6 treatment group experienced a significantly greater reduction in the number of monthly migraine attacks compared to the placebo treatment group compared to baseline. Figure 3 In addition, the change from baseline in the number of monthly migraine attacks for the placebo and Ab6 treatment groups was compared. As shown in Figure 6, the median (% of QR) change from baseline in the number of monthly migraine attacks for the placebo and Ab6 treatment groups over the 12 weeks post-treatment was compared. These results indicate that the Ab6 treatment group experienced a significantly greater reduction in the number of monthly migraine attacks compared to the placebo treatment group compared to baseline.

[0353] In addition, the change from baseline in the number of monthly migraine attacks for the placebo and Ab6 treatment groups was compared. As shown in Figure 6, the median (% of QR) change from baseline in the number of monthly migraine attacks for the placebo and Ab6 treatment groups over the 12 weeks post-treatment was compared. These results indicate that the Ab6 treatment group experienced a significantly greater reduction in the number of monthly migraine attacks compared to the placebo treatment group compared to baseline. Figure 4 In addition, the change from baseline in the number of monthly migraine attacks for the placebo and Ab6 treatment groups was compared. As shown in Figure 6, the median (% of QR) change from baseline in the number of monthly migraine attacks for the placebo and Ab6 treatment groups over the 12 weeks post-treatment was compared. These results indicate that the Ab6 treatment group experienced a significantly greater reduction in the number of monthly migraine attacks compared to the placebo treatment group compared to baseline.

[0354] In addition, the HIT-6 results for both groups were compared. As previously described, this questionnaire has received widespread acceptance for use in assessing the migraine condition of individuals with frequent / chronic migraine. Figure 6 A HIT-6 responder analysis for the Ab6 treatment group and placebo group was compared at baseline, post-treatment week 4, post-treatment week 8, and post-treatment week 12. The results at each time point showed a statistically significant improvement in the HIT-6 score for the Ab6 treatment group compared to the placebo group, i.e., 54.4% for the Ab6 treatment group compared to 30% for the placebo group at week 4 (p = 0.0023), 51.3% for the Ab6 treatment group compared to 38.0% for the placebo group at week 8 (p = 0.1094), and 61.1% for the Ab6 treatment group compared to 33.3% for the placebo group at week 12 (p = 0.0007). Figure 7 The percentage of patients in the placebo and Ab6 treatment groups who had some or little / no HIG-6 score over time is shown (showing statistical significance).

[0355] In addition, Figure 8 Pharmacokinetic (PK) profiles over a 24-week period post Ab6 administration for Ab6 administered as a single dose intravenously at 1000 mg (mg / mL).

[0356] Figure 9Plasma pharmacokinetic (PK) parameters N (number of patients), mean, and standard deviation (SD) for Ab6 including single intravenous dose of 1000 mg. 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).

[0357] Further analysis of patient data was performed from 12 weeks to 24 weeks. The treatment group continued to show a reduction in migraine days compared to the control group, however, the magnitude of the difference decreased over time. In addition, the control group showed fewer migraine days per month than baseline. This is believed to be due, at least in part, to "diary fatigue" in which patients can not report migraine on the day it actually occurs, avoiding the time and effort to answer more questions about migraine, which can be because they gave a positive answer to the question of whether they had a migraine on a given day.

[0358] Further analysis of the study results is shown in Figures 10 to 21 . These results include an analysis of the change from baseline in mean migraine days per month + / - SEM ( Figure 22 ) for Ab6 (1000 mg i.v.) versus placebo, the change in mean migraine days over time + / - SD ( Figure 23 ) for the entire analysis population. In addition, shown are the distribution and change in actual migraine days for the Ab6 treatment group during weeks 1-4 ( Figure 12 ), the distribution and change in actual migraine days for the placebo group during weeks 1-4 ( Figure 13 ), the distribution and change in actual migraine days for the Ab6 treatment group during weeks 5-8 ( Figure 14 ), the distribution and change in actual migraine days for the placebo group during weeks 5-8 ( Figure 15 ), the distribution and change in actual migraine days for the Ab6 treatment group during weeks 9-12 ( Figure 16 ), and the distribution and change in actual migraine days for the placebo group during weeks 9-12 ( Figure 17 ).

[0359] Responder rate analysis was also performed ( Figures 18 to 20 ). These figures show the 50%, 75%, and 100% responder rates for the Ab6 and placebo treatment groups, respectively. Subjects with a reduction in migraine frequency of > 50% were considered 50% responders. Subjects with a reduction in migraine frequency of > 75% were considered 75% responders. Likewise, subjects with a reduction in migraine frequency of 100% were considered 100% responders.

[0360] In Figure 10 and Figures 18 to 20 the inter-visit interval (where e-diary completion was 21-27 days) was standardized by multiplying the observed frequency by the inverse of the completion rate.

[0361] Migraine severity was also analyzed. Figure 21 Mean migraine severity over time is shown for the entire analysis population. An average score of 3 on the scale used represents "moderate pain."

[0362] Figure 22 Changes from baseline in migraine days, migraine attacks, migraine hours, mean migraine severity, headache frequency, and outcome measures including HIT-6 score, MSQ (migraine-specific quality of life questionnaire) RFP (role function-preventive), MSQ RFR (role function-restrictive), and MSQ EF (emotional function) were summarized.

[0363] Example 2

[0364] Human clinical study to assess safety and efficacy of an anti-CGRP antibody in patients with chronic migraine

[0365] This example describes a randomized, double-blind, placebo-controlled clinical trial to assess the safety and efficacy of Ab6 (erenumab) in the prevention of chronic migraine. In the study, 1,072 patients were randomized to receive Ab6 (300 mg or 100 mg) or placebo administered by infusion every 12 weeks. To be eligible for the trial, patients must have experienced at least 15 headache days per month, of which at least eight met criteria for migraine. Patients enrolled in the trial had an average of 16.1 migraine days per month at baseline. Endpoints included mean change from baseline in monthly migraine days, reduction in migraine attack rates on days 1 and 1-28, and at least 50%, 75%, and 100% reduction in mean monthly migraine days from baseline, change from baseline in mean monthly acute migraine-specific medication days, reduction in patient-reported impact scores on the Headache Impact Test (HIT-6).

[0366] Patient characteristics are summarized in Figure 27 with separate columns for patients receiving placebo, 100 mg antibody, or 300 mg antibody. The average number of years since diagnosis of migraine for patients was 17.0 to 19.0, the average duration of chronic migraine was 11.5 to 12.4 years, and the percentage of patients using at least one preventive medication was 44.3% to 45.2%. At baseline, the average number of migraine days per month was 16.1 for the two antibody treatment groups, and 16.2 for the placebo group.

[0367] A specified percentage (50%, 75%, or 100%) reduction in mean monthly migraine days from baseline refers to the number or percentage of patients in a treatment group who exhibited a given percentage reduction in monthly migraine days. For example, a patient who exhibited 16 monthly migraine days at baseline would be a 75% responder if monthly migraine days were reduced by at least 12 days per month over a specified time period.

[0368] Results are shown in Figures 23 to 27 Figure 23 The percentage of migraine patients in the 300 mg, 100 mg, and placebo treatment groups is shown on days 1, 7, 14, 21, and 28. The top row shows the results for placebo, the bottom row shows the results for the 300 mg dose, and the middle row shows the results for the 100 mg dose.

[0369] As shown in Figure 23 On day 1, the percentage reduction in migraine prevalence was 52% for the 300 mg dose, 50% for the 100 mg dose, and 27% for placebo. The reductions shown were statistically significant in the 100 mg and 300 mg treatment groups compared to the placebo group.

[0370] Figures 24 to 26 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, in the first month, in the first 3 months (after the first infusion), and in the 4th-5th months (after the second infusion) 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 shown as follows. ++ indicates a statistically significant difference from placebo; + indicates a statistically significant difference from placebo (unadjusted); and § indicates a statistically significant difference from placebo (post-hoc analysis).

[0371] Example 3

[0372] Baseline subgroup analysis of a human clinical study to assess the safety and efficacy of an anti-CGRP antibody in patients with chronic or episodic migraine

[0373] In the study of chronic migraine described in Example 3, each patient was assessed for potential medication overuse headache (MOH) at intake. MOH occurred in 39.9% (139 patients) in the 100 mg treatment group, 42.0% (147 patients) in the 300 mg treatment group, and 39.6% (145 patients) in the placebo group. Evaluation of treatment outcomes in this patient subgroup indicated that treatment with the anti-CGRP antibody was effective in patients with MOH ( Figure 29 ​). Specifically, in the 100 mg treatment group, patients with MOH at baseline changed their mean monthly migraine days by -3.0 days (95% CI, -4.56 to -1.52 days) compared to MOH patients receiving placebo. Similarly, in the 300 mg treatment group, patients with MOH at baseline changed their mean monthly migraine days by -3.2 days (95% CI, -4.66 to -1.78 days) compared to MOH patients receiving placebo. In contrast, for patients without MOH at baseline, the mean monthly migraine days changed by -1.3 days (95% CI, -2.43 to -0.16 days) in the 100 mg treatment group compared to patients without MOH at baseline receiving placebo. Likewise, for patients without MOH at baseline, the mean monthly migraine days changed by -2.1 days (95% CI, -3.24 to -0.88 days) in the 300 mg treatment group compared to patients without MOH at baseline receiving placebo. Efficacy was also shown for other subgroups, including for patients with mean migraine days (MMD) frequency of less than 17 days or greater than or equal to 17 days, patients with age at diagnosis of less than or equal to 21 years or greater than 21 years, patients with migraine duration of less than or equal to 15 years or greater than 15 years, patients with aura or without aura, patients using prior prophylactic medications or not, patients using concomitant prophylactic medications or not, and patients using triptans greater than or equal to 33% of days or less than 33% of days. In each case, efficacy was shown for each subgroup (Table 3). Figure 29 .

[0374] In another human clinical trial of episodic migraine patients, in a double-blind, parallel study, patients were randomized to receive Ab6 100 mg (n = 221), 300 mg (n = 222), or placebo (n = 222) intravenously every 3 months for a total of 4 infusions (Q3Mx4) over a 28-day screening period. Figure 28 Efficacy was shown for both the 100 mg and 300 mg treatment groups in months 1-3, with a mean change in migraine days of -3.9 days for the 100 mg treatment group and -4.3 days for the 300 mg treatment group, compared to -3.2 days for the placebo group. Efficacy was also shown for subgroups of patients, including for patients with mean migraine days (MMD) frequency of less than or equal to 9 days or greater than 9 days, patients with age at diagnosis of less than or equal to 21 years or greater than 21 years, patients with migraine duration of less than or equal to 15 years or greater than 15 years, and patients with aura or without aura.

[0375] Example 4

[0376] The impact of Ab6 treatment on medication use in patients with chronic and episodic migraines.

[0377] In the studies of patients with chronic migraine described in Example 3 and patients with episodic migraine described in Example 4, patients also recorded their use of acute medications in daily electronic diaries and were allowed to decide for themselves when to use them. Acute medications for migraines include ergot, triptans, and analgesics (such as NSAIDs, opioids, and caffeine-containing combination analgesics).

[0378] For further analysis, patients were stratified based on the number of days of acute medication use during the 28-day screening period (days 1–9 or ≥ 10; "baseline"). Acute medication days were calculated for each type of acute medication, as well as combinations thereof (meaning that if two or more types of medication were used on the same calendar day, they were counted as separate medication use days). For example, if a patient took both an opioid and a triptan on the same day, it was considered 2 days of acute medication use. These analyses included patients with at least one day of acute medication use during the 28-day baseline screening period.

[0379] In patients with chronic and episodic migraines who used acute medication during the 28-day baseline period, Ab6 treatment resulted in a greater mean reduction in monthly migraine days and acute medication days than placebo as early as the first month after administration, with similar results across two dose intervals over 6 months.

[0380] In chronic migraine patients who used acute medication for ≥ 1 day during the baseline period, Ab6 consistently showed a greater reduction in mean monthly migraine days compared to placebo over 6 months of treatment. Figure 30 Chronic migraine patients who used acute medication at least one day per month during the baseline period showed a greater reduction in acute medication use compared to placebo, as early as the first month after treatment and throughout the entire 6-month treatment period. Figure 31 In a subgroup of chronic migraine patients who took acute medication for 1–9 days at baseline, the change in the number of days of acute medication use compared to baseline was greater in the 300 mg Ab6 group than in the placebo group during the 6-month treatment period. Figure 32 Throughout the 6-month period, compared with placebo, a significant reduction in the number of days of medication per month was observed in patients who used the medication for at least 10 days per month at baseline in both Ab6 treatment groups. Figure 33 This study shows the variation in the number of days of medication use at month 1 and month 6 in subgroups of chronic migraine patients who used acute medication for ≥ 1 day, 1–9 days, and ≥ 10 days at baseline. In patients who used medication for 1–9 days / month at baseline, Ab6 showed a greater therapeutic effect than placebo in reducing acute medication use at month 6, even without 100 mg Ab6.

[0381] Similarly, patients with episodic migraine who used acute medication one or more days during the baseline period experienced a greater reduction in mean monthly migraine days over 6 months across 2 dose intervals with Ab6 than with placebo ( Figure 34 ). Patients with episodic migraine who used acute medication at least one day per month during the baseline period showed a greater reduction in acute medication use compared to placebo as early as month 1 post-treatment and throughout the 6-month treatment period ( Figure 35 ). In a subgroup of episodic migraine patients who took acute medication 1-9 days during the baseline period, the change in days of acute medication use over the 6-month treatment period with Ab6 was greater than with placebo ( Figure 36 ). A similar pattern was observed in a subgroup of patients who took acute medication > 10 days during the baseline period, although the smaller sample size over time can have resulted in a less consistent pattern. Figure 37 The change in days of medication use at month 1 and month 6 is shown for subgroups of episodic migraine patients who used acute medication > 1 day, 1-9 days, and > 10 days at baseline. Patients who used > 10 days / month at baseline experienced a greater reduction in acute medication use with Ab6 than placebo, with the exception of Ab6 100 mg at month 6.

[0382] Results indicate that both episodic migraine and chronic migraine patients, who are at risk for medication overuse headache (use of acute medication > 10 days / month), showed the greatest reduction in acute medication use, with Ab6 treatment generally resulting in a greater reduction in days of medication use than placebo.

[0383] The most frequently reported acute headache medications in > 10% of subjects included: Thomapyrin N (44.5%) (combination of acetaminophen, aspirin, and caffeine), ibuprofen (40.6%), sumatriptan (33.6%), acetaminophen (Tylenol) (20.3%), and naproxen sodium (10.2%). The most frequently reported prophylactic headache medication in > 10% of subjects was topiramate (12.5%).

[0384] Example 5

[0385] Efficacy of anti-CGRP antibodies in subjects experiencing acute attacks of migraine

[0386] This example describes a randomized, double-blind, placebo-controlled clinical trial to assess the safety and efficacy of Ab6 for acute treatment of migraine. In this study, approximately 450 patients are randomized to receive 100 mg Ab6 or placebo in a 1 : 1 ratio. During the screening period (about 1-8 weeks), patients are assessed for migraine frequency and medication use frequency. Eligible patients have a migraine attack frequency of about 4-15 migraine days per month in the 3 months prior to screening. Historically, a typical migraine attack in a subject, if left untreated, would be associated with moderate to severe headache and the most bothersome symptoms of nausea, photophobia, or phonophobia. Subjects must be free of headache for at least 24 hours prior to a qualifying migraine attack to participate in the trial. On the day of treatment, patients will visit the study site and receive an intravenous infusion of 100 mg Ab6 or placebo starting about 1-6 hours from the onset of the attack. Patients will not receive any other monoclonal antibodies (e.g., any CGRP-antagonistic antibodies) in the 6-month period prior to screening.

[0387] The primary endpoints are time to freedom from headache and time to freedom from most bothersome symptoms. Key secondary endpoints are freedom from headache at 2 hours and freedom from most bothersome symptoms at 2 hours. Secondary endpoints are time to headache relief, freedom from headache for 24 and 48 hours, use of rescue medication before 24 hours and before 48 hours, absence of photophobia at 2 hours, absence of phonophobia at 2 hours, absence of nausea at 2 hours, change from baseline in Headache Impact Test 6 (HIT 6) at week 4, and change from baseline in Migraine Treatment Optimized Questionnaire 6 (mTOQ-6) at week 4. Exploratory endpoints are freedom from headache at all time points beyond 2 hours, absence of photophobia at all time points beyond 2 hours, absence of phonophobia at all time points beyond 2 hours, absence of nausea at all time points beyond 2 hours, recurrence of pain when the subject is free from headache at 2 hours, Patient Global Change Impression (PGIC) at week 4, and time to next migraine. Headache is collected on a 4-point scale, with 3 being severe, 2 being moderate, 1 being mild, and 0 being no pain. Freedom from pain without rescue medication is no pain (0) (note that, in the trial, rescue medication is not permitted within 2 hours after completion of the infusion to distinguish the effect of the antibody from rescue medication, but, in normal use, rescue medication can optionally be used; use of any rescue medication is collected as data).

[0388] Statistical analyses are performed to determine the significance of differences in endpoints between patients receiving Ab6 or placebo, including time to freedom from pain and time to freedom from most bothersome symptoms, and each of the other endpoints described above.

[0389] Use of rescue medication refers to any intervention (medical or device) provided to the subject to alleviate the migraine. In this study, rescue medication should not be provided within 2 hours of completing study drug administration to distinguish the effect of the antibody from the effect of the rescue medication, however, the use of rescue medication is not prohibited. The study summarizes the proportion of subjects who required the use of rescue medication. Acute rescue medication includes any medication that treats migraine or migraine-associated symptoms, such as triptans, analgesics (e.g., non-opioid or opioid / narcotic), acetaminophen, NSAIDs, combination drugs (e.g., EXCEDRIN ® MIGRAINE ® ), antiemetics, ergotamine, ergot derivatives, etc.

[0390] The absence of migraine-associated symptoms (photophobia, phonophobia, and nausea) as reported by the subject refers to the absence or presence of the above migraine-associated symptoms. The study summarizes the proportion of subjects who had no symptoms without the administration of rescue medication.

[0391] Headache Impact Test (HIT-6) was assessed as change from baseline in total score and summarized in the study and compared between treatment groups.

[0392] Migraine Treatment Optimization Questionnaire 6 (mTOQ-6) was assessed as change from baseline in total score and summarized in the study and compared between treatment groups.

[0393] Time to headache relief was assessed as the first time point after completion of infusion that the subject reported relief of pain, meaning that their headache had gone from moderate or severe (2 or 3) to mild or no pain (1 or 0) without the administration of rescue medication.

[0394] Pain recurrence was assessed as the occurrence of any severity of headache within 48 hours of drug administration for patients who were headache free (0) at 2 hours. The study summarizes the proportion of patients who had a recurrence of headache of any severity.

[0395] The study demonstrates that Ab6 is effective and safe for the treatment of acute migraine.

[0396] Example 6

[0397] In the pivotal clinical study, patients received a 100 mg or 300 mg dose of Ab6, as described in Example 2. When evaluating the treatment effect, inclusion of Day -1 (post-Ab6 infusion) in the statistical analysis demonstrated a clear treatment effect immediately after infusion (p<0.0001) that was sustained through Day 2 (p<0.0001) and Day 7 (p<0.0001) (Figure 6). Figure 38). In the figure, day 0 is defined as the day of infusion, while day -1 data represent pre-infusion status. From day -1 (baseline, day before infusion) to day 0, the percentage of migraine was significantly reduced. Moreover, the effect magnitude was greater for the 300 mg dose than for the 100 mg dose, and both showed a greater effect than the placebo group.

[0398] Example 7

[0399] This example relates to the antibody Ab10.H3 (LuAG09222). Ab10.H3 has been studied in several clinical trials, including a phase I trial to determine safety and tolerability of increasing doses, and more recently a phase II study (referred to as the HOPE study, described earlier herein), showing an effect in the prevention of migraine. Thus, Ab10.H3 has proven to be safe and useful in the treatment of patients with migraine.

[0400] Ab10.H3 is described in patent application WO 2017181039, the content of which (including the sequence listing) is incorporated herein by reference in its entirety. Ab10.H3 is a humanized version of the antibody Ab10 described in WO 2017181039. WO 2017181039 describes that Ab10 and AB10.H3 have an increased inhibitory specificity for the PACAP1 receptor pathway over the VPAC1 or VPAC2 pathway: in the case of Ab10, humanization of Ab10 decreased the IC50 (pM) of PACAP38-induced PCA1-R-mediated cAMP increase, while humanization increased the IC50 (pM) of PACAP38-induced VPAC1-R-mediated and VPAC2-mediated cAMP increase, which overall indicates an increased inhibitory specificity for the PACAP1 receptor. Given the abundant expression of VCAP1 and VCAP2 receptors outside the nervous system (see paragraph in WO 2017181039), the decreased relative specificity for VCAP1 and VCAP2 receptors can reduce unintended effects, i.e. interactions with undesired target cells when the antibody is used as a therapeutic or prophylactic agent, which is a desirable property.

[0401] Thus, the humanized antibody provides an additional technical feature, i.e. an increased inhibitory specificity for the PACAP1 receptor pathway over the VPAC1 or VPAC2 pathway, which further complements the technical differences described above. The table below summarizes these points from WO 2017181039. Table 2 was generated by WO 2017181039 based on the cited table from WO 2017181039.

[0402]

[0403] Ab10 and AB10.H3 antibodies provide in vivo neural effects: As shown in Example 11 of WO 2017181039, Ab10.H and Ab10.H3 reduce light sensitivity in a PACAP-induced photophobia mouse model (see, e.g., paragraph of WO 2017181039). Photophobia, an extreme light sensitivity, is a symptom commonly experienced by patients with neurological disorders such as migraine, and thus the presently claimed antibodies are antibodies that can be used to treat or prevent such neurological disorders or symptoms. In Example 13 of WO 2017181039, Ab10.H3 reduced trigeminal parasympathetic reflexes in a rat model of cluster headache, trigeminal neuralgia and possible vascular dysfunction of migraine, as measured by lacrimation and nose temperature after intranasal administration of xylometazoline (see paragraphs - and Figure 34 and Figure 35 ). Furthermore, Example 8 WO2017181039 shows that Ab10 inhibits PACAP-induced cutaneous vasodilation in rabbits. Although cutaneous vascular effects were studied in this experiment, the neural mechanisms of vasoconstriction / vasodilation are essentially the same in the brain, and vasodilation is one of the pathogenic mechanisms of various types of headaches. Taken together, the antibodies can be used to treat or prevent neurological disorders or symptoms such as headaches.

[0404] Ab10 and AB10.H3 recognize unique epitopes within PACAP: As shown in Example 12 of WO 2017181039, Ab10 and Ab10.H3 recognize residues 19, 22, 23 and 27 of PACAP. The humanized versions of Ab10 presently claimed are also expected to recognize the same residues, as they share the same 6 CDR sequences as their parent Ab10. Binding to this particular epitope, particularly residues 23 and 27, appears to provide the unique and beneficial features described therein, namely the inability to bind to PACAP to cells expressing the PAC1 receptor.

[0405] Affinity: Humanization of Ab10 increased affinity for PACAP38 from 7.5E-11 to 2.9E-11 (Ab10.H), 2.2E-11 (Ab10.H2), 2.2E-11 (Ab10.H3) or 1.9E-11 (Ab10.H4) (Table 4 of WO 2017181039).

[0406] IC50: Humanization of Ab10 reduced the IC50 (pM) of 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), 22.7 (Ab10.H5) pM (see Table 2 and Table 3 of WO 2017181039).

[0407] Example 7

[0408] The high concentration co-formulation of Eculizumab-LuAG00922 requires a delicate balance of formulation components and pH. The correct balance of components and pH will provide low viscosity, which enables subcutaneous administration, and in addition, can enable a stable formulation with a long shelf life. For a typical monoclonal antibody (mAb) drug product formulation, the conformational and colloidal stability will be optimized based on the unique biophysical properties of the specific antibody. For a co-formulation of two mAbs, the formulation development becomes challenging when the biophysical properties of each mAb are significantly different from each other. In this case, the isoelectric point of Eculizumab is approximately 8.1, while the isoelectric point of LuAG00922 is approximately 6.9. It is generally desirable to formulate mAbs in compositions that are several pH units away from the isoelectric point to improve colloidal stability and solubility, which is key to stability at high concentrations. In addition, the behavior of LuAG00922 as the only antibody component in the high concentration formulation exhibits a unique pH dependent viscosity; at pH 5.5, the LuAG00922 formulation has a relatively high viscosity of > 70 cP when the concentration of LuAG00922 is higher than 158 mg / mL. As the pH is increased from 5.5 to 6.0 and further to 6.5, the viscosity of LuAG00922 shows a pH dependent charge patch change from a protonated positively charged patch at pH 5.5 to a deprotonated neutral charged patch at 6.0 and 6.5, resulting in a decrease in viscosity as the pH is increased.

[0409] Design of Experiment (DOE)

[0410] A formulation design space was created by varying the pH and the amount of L-histidine, sorbitol, L-arginine hydrochloride, poloxamer 188, and protein concentration. Since the use of 10 to 50 mM histidine buffer achieves the desired pH range, a pH range of 5.25 to 6.75 was investigated, which is expected to provide buffering capacity for a mAb subcutaneous formulation. NaCl and L-arginine hydrochloride excipient levels were investigated at 3 levels of 10, 80, 150 mM and are expected to act as potential stabilizers, viscosity reducing agents, and tonicity modifiers. L-sorbitol excipient levels were chosen at 50, 150, and 250 mM and are expected to act as potential stabilizers, cryoprotectants, and tonicity modifiers. Poloxamer 188 was investigated at 0.005%, 0.0275%, and 0.05% (w / v) and is expected to act as a surfactant to prevent protein aggregation and particle formation. Total antibody concentration levels were investigated at 100, 125, and 150 mg / mL to provide a high enough concentration to enable subcutaneous administration of the formulation. A deterministic screening experimental design (DOE) was created using JMP statistical software that used 7 factors, each with 3 levels, resulting in 18 formulations. This DOE was applied to epratuzumab-LuAG00922 co-formulations at ratios of 1 : 1, 1 : 2, and 2 : 1, resulting in a total of 54 formulations.

[0411] Formulation composition and buffer exchange

[0412] Reagents used to prepare the formulations are in Table 3 below. Working stock solutions of each formulation component were prepared by: 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 in Table 4 were generated by diluting the appropriate amount of each working stock solution of each formulation component into distilled, deionized water (Milli-Q water). The final pH of each formulation buffer was adjusted using 5 M hydrochloric acid, then the formulation was filtered with a 0.22 pm PES filter.

[0413] Table 3: Reagents used in the formulations.

[0414]

[0415] Eighteen formulation buffers containing L-histidine, L-sorbitol, and L-arginine hydrochloride were prepared by mixing together appropriate volumes of working stock solutions and diluting with distilled deionized water (Milli-Q water) to obtain the compositions in the table below. Three ratios of ipilimumab: LuAG00922 (1 : 2, 1 : 1, 2: 1) were chosen and each buffer was exchanged into the eighteen formulations using Big Tuna (Unchained Labs) to produce a total of 54 protein formulations.

[0416] Table 4: Eighteen formulation buffer compositions for ratios of 1 : 2, 1 : 1, and 2: 1 ipilimumab: LuAG00922.

[0417]

[0418] The stability of the 54 formulations was evaluated using size exclusion columns with ultra-high pressure liquid chromatography at the following conditions and time points.

[0419] 5°C (0, 1, 4, 7 months) 25°C (1, 4, 7 months) 40°C (1 month) Methods Protein concentration measurements: After buffer exchange into the desired co-formulation, protein concentration was measured using Solo VPE and Agilent Cary 60 UV-Vis spectrophotometer or Lunatic spectrophotometer (Unchained Labs) and known A280 extinction coefficients for ipilimumab and LuAG00922.

[0420] High throughput assays: Size exclusion chromatography with ultra-performance liquid chromatography (SE-UPLC) was performed using a Waters Acquity H-Class UPLC with a TUV detector connected to a Waters BEH SEC column (P / N 186005225). SE-UPLC analysis was performed at T = 0 and every time point at 5°C, 25°C, 30°C, and 40°C to assess the percentage of high molecular weight species (HMW%). The initial HMW% for ipilimumab was 0.32% and the initial HMW% for LuAG00922 was 0.94%. The total HMW% results at T = 0 for each of the 54 co-formulations after buffer exchange ranged from 0.39% to 1.40%. JMP DOE model analysis showed that changes in protein concentration, ratio of ipilimumab : LuAG00922, histidine concentration, L-arginine hydrochloride concentration, pH, and sorbitol concentration had a significant effect on the HMW% response. Sodium chloride concentration and poloxamer (P188) concentration did not show a significant change in the HMW% response.

[0421] High-throughput viscosity measurements were performed using a bead-based method based on Uncle (Nonlinear Laboratories Inc.) dynamic light scattering (DLS) (He et al. 2010, Analytical Biochemistry, Vol. 399, No. 1, pp. 141-143, High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions). The protein formulations were spiked with 100 nm diameter polystyrene beads (Thermo Scientific: P / N 3100A).

[0422]

[0423] Table 11. Co-formulations of ipilimumab : LuAG00922 at a ratio of 1 : 1 .

Claims

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

20.

2. The pharmaceutical composition of claim 1, formulated to maintain the biological activity and / or storage stability of the ipilimumab and LuAG09222 antibodies therein.

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

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

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

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

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

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

2.

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

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

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

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

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

14. The pharmaceutical composition of any one of the preceding claims, wherein the pH is between about 5.0-6.8, including the end values.

15. The pharmaceutical composition of any one of the preceding claims, wherein the pH is about 5.0, about 5.5, about 5.9, about 6.0, about 6.5, or about 6.

8.

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

17. The pharmaceutical composition of any one of the preceding claims, which comprises epratuzumab and LuAG90222 in a ratio of about 2 : 1, about 1 : 1, or about 1 : 2, and comprises about 20-40 mM histidine buffer, about 90-180 mM sorbitol, 0.0025-0.0120% w / v poloxamer P188, and 30-70 mM NaCl, and has a pH of about 6, optionally about pH 5.

9.

18. The pharmaceutical composition of any one of the preceding claims, which comprises epratuzumab and LuAG90222 in a ratio of 2 : 1, and further comprises 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 has a pH of about 6, optionally about pH 5.

9.

19. The pharmaceutical composition of any one of the preceding claims, which comprises epratuzumab and LuAG90222 in a ratio of 1 : 1, and further comprises 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 has a pH of about 6, optionally pH 5.

9.

20. The pharmaceutical composition according to any one of the preceding claims, comprising eptinezumab and LuAG90222 in a ratio of 1 :2, and further comprising about 35-45 mM histidine buffer, about 90-100 mM sorbitol, about 0.010-0.0120% w / v poloxamer P188, and about 65-75 mM NaCl, and having a pH of about 6, optionally pH 5.

9.

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

22. The pharmaceutical composition according to any one of the preceding claims, for use in the treatment or prevention of headache, optionally chronic or episodic migraine or cluster headache, further optionally wherein administration of the pharmaceutical composition comprising the antibody combination has an additive or synergistic effect on inhibiting, reducing or preventing the number, duration and / or intensity of migraine attacks compared to subcutaneous or intravenous administration of a pharmaceutical composition comprising the same dose of eptinezumab or LuAG09222 alone.

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

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

25. The pharmaceutical composition according to any one of the preceding claims, wherein eptinezumab comprises a heavy chain as defined in SEQ ID NO.: 5 or SEQ ID NO.: 6 and a light chain as defined in SEQ ID NO.:

11.

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

21.

27. A method of treating or preventing a headache, optionally chronic or episodic migraine or cluster headache, comprising or consisting of subcutaneously or intravenously administering a combination of eptinezumab and the LuAG09222 antibody, wherein eptinezumab comprises a VH region as defined in SEQ ID NO.: 4 and a VL as defined in SEQ ID NO.: 10, and LUAG09222 comprises a VH region as defined in SEQ ID NO.: 15 and a VL region as defined in SEQ ID NO.: 20, and optionally wherein administration of the antibody combination has an additive or synergistic effect on inhibiting, reducing or preventing the number, duration and / or intensity of migraine attacks compared to subcutaneously or intravenously administering the same dose of eptinezumab or LuAG09222 alone.

28. A method of treating or preventing pain, optionally any of acute pain, chronic pain, neuropathic pain, nociceptive pain and / or radicular pain, comprising or consisting of subcutaneously or intravenously administering a combination of eptinezumab and the LuAG09222 antibody, wherein eptinezumab comprises a VH region as defined in SEQ ID NO.: 4 and a VL as defined in SEQ ID NO.: 10, and LUAG09222 comprises a VH region as defined in SEQ ID NO.: 15 and a VL region as defined in SEQ ID NO.: 20, and optionally wherein administration of the antibody combination has an additive or synergistic effect on inhibiting or reducing pain compared to subcutaneously or intravenously administering the same dose of eptinezumab or LuAG09222 alone.

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

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