Anti-CGRP receptor / anti-PAC1 receptor bispecific antigen-binding protein

Abstract

The present invention relates to antagonist antibodies of the human calcitonin gene-related peptide (CGRP) receptor and bispecific antigen-binding proteins derived from anti-CGRP antibodies that bind to and inhibit both the human CGRP receptor and another target, such as the human pituitary adenylate cyclase-activating polypeptide type I (PAC1) receptor. Pharmaceutical compositions comprising the anti-CGRP receptor antibodies and bispecific antigen-binding proteins and methods for making them are also disclosed. Methods of using the anti-CGRP receptor antibodies and bispecific antigen-binding proteins to ameliorate, treat, or prevent conditions associated with CGRP and PAC1 receptors, such as chronic pain, migraine, and cluster headache, are also described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 868,557, filed June 28, 2019, which is incorporated herein by reference in its entirety.

[0002] Description of electronically submitted text files

[0001] This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the Sequence Listing in computer readable format, created on June 26, 2020, is titled A-2314-WO-PCT_SeqList_ST25, and is 734 kilobytes in size.

[0003] The present invention relates to the field of biopharmaceuticals. In particular, the present invention relates to antibodies that specifically bind to the human calcitonin gene-related peptide (CGRP) receptor and potently inhibit its biological activity. The present invention also includes bispecific antigen-binding proteins derived from anti-CGRP receptor antibodies that can specifically bind to and inhibit the human CGRP receptor and another target, such as the human pituitary adenylate cyclase-activating polypeptide type I (PAC1) receptor. The present invention also relates to pharmaceutical compositions comprising the anti-CGRP receptor antibodies and bispecific antigen-binding proteins, and methods of making and using such antibodies and bispecific antigen-binding proteins. [Background technology]

[0004] Migraine is a recurrent headache that can be accompanied by severe pain, often accompanied by nausea, vomiting, and extreme sensitivity to light (photophobia) and sound (phonophobia), and is sometimes preceded by sensory warning symptoms or signs (aura). Migraine is a highly prevalent disease worldwide, with approximately 12% of the European population and 18% of women and 6% of men in the United States suffering from migraine attacks (Lipton et al., Neurology, Vol. 68:343-349, 2007; Lipton et al., Headache, Vol. 41:646-657, 2001). A study evaluating the prevalence of migraine in the United States reported that nearly half of the migraine patient population suffered from three or more migraines per month (Lipton et al., Neurology, Vol. 68:343-349, 2007). Furthermore, migraine is associated with several psychiatric and medical comorbidities, such as depression and vascular disorders (Buse et al., J. Neurol. Neurosurg. Psychiatry, Vol. 81:428-432, 2010; Bigal et al., Neurology, Vol. 72:1864-1871, 2009). Most available migraine therapies are either poorly tolerated or ineffective (Loder et al., Headache, Vol. 52:930-945, 2012; Lipton et al., 2001); therefore, migraine remains an unmet medical need.

[0005] A key component of migraine pathogenesis involves activation of the trigeminovascular system. Release of trigeminal and parasympathetic neurotransmitters from perivascular nerve fibers (Sanchez-del-Rio and Reuter, Curr. Opin. Neurol., Vol. 17(3):289-93, 2004) has been suggested to result in vasodilation of cranial vessels and be associated with migraine onset (Edvinsson, Cephalagia, Vol. 33(13):1070-1072, 2013; Goadsby et al., New Engl J Med., Vol. 346(4):257-270, 2002).

[0006] Calcitonin gene-related peptide (CGRP) belongs to the calcitonin family of peptides, which also includes calcitonin, amylin, and adrenomedullin. CGRP is a 37-amino acid peptide expressed in both the central and peripheral nervous systems and has been implicated as a key mediator in the initiation and progression of migraine pain. In addition to its ability to act as a vasodilator, CGRP also acts as a neurotransmitter in the trigeminal ganglion and trigeminal subnucleus caudalis, facilitating synaptic transmission and pain responses (Durham et al., Curr Opin Investig Drugs, Vol. 5:731-735, 2004; Zimmermann et al., Brain Res., Vol. 724:238-245, 1996; Wang et al., Proc Natl Acad Sci USA., Vol. 92:11480-11484, 1995; Poyner, Pharmacol. Ther., Vol. 56:23-51, 1992).

[0007] The CGRP receptor is a complex composed of the G protein-coupled calcitonin-like receptor (CLR) and the single-transmembrane domain protein receptor activity-modifying protein 1 (RAMP1). The CGRP receptor complex is located in migraine-related areas, including the cerebral vasculature, the trigeminocervical complex in the brainstem, and the trigeminal ganglion (Zhang et al., J. Neurosci., Vol. 27: 2693-2703, 2007; Storer et al., Br J Pharmacol., Vol. 142: 1171-1181, 2004; Oliver et al., J Cereb Blood Flow Metab., Vol. 22: 620-629, 2002). Several lines of evidence indicate that CGRP is a potent vasodilator and nociceptive modulator associated with migraine pathophysiology: (1) it is expressed in the trigeminal nervous system, which has been implicated in the pathophysiology of migraine; (2) CGRP levels are elevated in migraine patients during attacks (Bellamy et al., Headache, Vol. 46:24-33, 2006; Ashina et al., Pain, Vol. 86:133-138, 2000; Gallai et al., Cephalalgia, Vol. 15:384-390, 1995; Goadsby et al., Ann Neurol., Vol. 28:183-187, 1990; Goadsby et al., Ann Neurol., Vol. 23:193-196, 1988); (3) acute migraine therapies such as triptans restore CGRP levels to normal after treatment (Juhasz et al., Cephalalgia, Vol. 25:179-183, 2005); (4) CGRP infusion induces migraine episodes in migraine patients (Petersen et al., Br J Pharmacol., Vol. 143:1074-1075, 2004; Lassen et al., Cephalalgia, Vol. 22:54-61, 2002); and (5) CGRP antagonists have demonstrated efficacy in reversing acute migraine (Connor et al., Neurology, Vol. 73:970-977, 2009; Hewitt et al., Abstract for the 14 thCongress of the International Headache Society,2009;LBOR3;Ho et al.,Lancet,Vol.372:2115-2123,2008a;Ho et al.,Neurology,Vol.70:1304-1312,2008b). Furthermore, antibody antagonists directed against CGRP ligands or CGRP receptors have demonstrated clinical efficacy in the preventive treatment of episodic and chronic migraine headaches (see, e.g., Tepper et al., Lancet Neurol., Vol. 16:425-434, 2017; Goadsby et al., New England Journal of Medicine, Vol. 377:2123-2132, 2017; Detke et al., Neurology, Vol. 91(24):e2211-e2221, 2018; Stauffer et al., JAMA Neurol., Vol. 75(9):1080-1088, 2018).

[0008] Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid (PACAP38) or 27-amino acid (PACAP27) peptide originally isolated from ovine hypothalamic extracts based on its ability to stimulate cyclic AMP (cAMP) formation in anterior pituitary cells (Miyata et al., Biochem Biophys Res Commun., Vol. 164:567-574, 1989; Miyata et al., Biochem Biophys Res Commun., Vol. 170:643-648, 1990). PACAP belongs to the VIP / secretin / glucagon superfamily. The sequence of PACAP27 corresponds to the 27 N-terminal amino acids of PACAP38 and shares 68% identity with vasoactive intestinal peptide (VIP) (Pantaloni et al., J. Biol. Chem., Vol. 271:22146-22151, 1996; Pisegna and Wank, Proc. Natl. Acad. Sci. USA, Vol. 90:6345-49, 1993; Campbell and Scanes, Growth Regul., Vol. 2:175-191, 1992). The predominant form of the PACAP peptide in the human body is PACAP38, and its pharmacology has not been shown to differ from that of PACAP27. Three PACAP receptors have been reported: one receptor (PAC1 receptor) that binds PACAP with high affinity and has much lower affinity for VIP, and two receptors (VPAC1 and VPAC2 receptors) that recognize PACAP and VIP equally well (Vaudry et al., Pharmacol Rev., Vol. 61:283-357, 2009).

[0009] Human experimental migraine models using PACAP as a trigger to induce migraine-like headache support the approach of antagonizing the PACAP / PAC1 signaling pathway as a therapeutic agent for migraine prevention. PACAP38 is elevated in plasma during spontaneous migraine attacks in migraine patients, and these elevated PACAP38 levels can be normalized with sumatriptan, an acute migraine therapy (Tuka et al., Cephalalgia, Vol. 33:1085-1095, 2013; Zagami et al., Ann. Clin. Transl. Neurol., Vol. 1:1036-1040, 2014). Infusion of PACAP38 induces headache in healthy subjects and migraine-like headache in migraine patients (Schytz et al., Brain, Vol. 132:16-25, 2009; Amin et al., Brain, Vol. 137:779-794, 2014; Guo et al., Cephalalgia, Vol. 37:125-135, 2017). However, in the same model, VIP does not induce migraine-like headache in migraine patients (Rahmann et al., Cephalalgia, Vol. 28:226-236, 2008). The lack of migraine-like headache induction by VIP infusion suggests that the effects of the PACAP38 peptide are mediated through the PAC1 receptor, rather than the VPAC1 or VPAC2 receptors, because VIP has a much higher affinity for the latter two receptors. This concept is further supported by animal studies in which PAC1 receptor antagonists inhibited nociceptive neuronal activity in the trigeminocervical complex in in vivo models of migraine (Akerman et al., Sci. Transl. Med., Vol. 7:308ra157, 2015; Hoffmann et al., Cephalalgia, Vol. 37(1S):3, Abstract OC-BA-004, 2017). Collectively, these data suggest that pharmacological agents that inhibit PACAP activation of the PAC1 receptor may have potential for treating migraine.

[0010] Although effective migraine-specific preventive therapies have recently been recognized and become available, there remains a need to develop additional migraine therapies with novel mechanisms of action to treat patients who do not adequately respond to existing therapies. In particular, therapeutic molecules with dual functionality, antagonizing both the CGRP / CGRP receptor and PACAP / PAC1 receptor pathways, would be particularly beneficial. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Lipton et al,Neurology,Vol.68:343-349,2007 [Non-patent document 2] Lipton et al.,Headache,Vol.41:646-657,2001 [Non-patent document 3] Buse et al.,J.Neurol.Neurosurg.Psychiatry,Vol.81:428-432,2010 [Non-patent document 4] Bigal et al.,Neurology,Vol.72:1864-1871,2009 [Non-patent document 5] Loder et al.,Headache,Vol.52:930-945,2012 [Non-patent document 6] Lipton et al., 2001 [Non-Patent Document 7] Sanchez-del-Rio and Reuter,Curr.Opin.Neurol.,Vol.17(3):289-93,2004 [Non-patent document 8] Edvinsson,Cephalagia,Vol.33(13):1070-1072,2013 [Non-Patent Document 9] Goadsby et al.,New Engl J Med.,Vol.346(4):257-270,2002

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[0012] The present invention provides antibodies and antigen-binding fragments that specifically bind to and potently inhibit the activity of human CGRP receptors. The antibodies and antigen-binding fragments of the present invention are 2- to 4-fold more potent inhibitors of human CGRP receptor activation than previously described anti-CGRP receptor antibodies. For example, in some embodiments, the anti-CGRP receptor antibodies and antigen-binding fragments inhibit CGRP-induced activation of human CGRP receptors with an IC50 of less than 500 pM as measured by a cell-based cAMP assay. In other embodiments, the anti-CGRP receptor antibodies and antigen-binding fragments inhibit CGRP-induced activation of human CGRP receptors with an IC50 of less than 200 pM as measured by a cell-based cAMP assay. In certain embodiments, the anti-CGRP receptor antibodies and antigen-binding fragments inhibit CGRP-induced activation of human CGRP receptors with an IC50 of between about 50 pM and about 400 pM as measured by a cell-based cAMP assay.

[0013] In certain embodiments, the anti-CGRP receptor antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising complementarity determining regions CDRH1, CDRH2, and CDRH3. In certain embodiments, the anti-CGRP receptor antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 47 with a mutation at one or more amino acid positions 28, 30, 31, 32, 54, 56, 57, 58, 59, 60, 102, 105, 107, 111, and / or 113. In such embodiments, the mutations may be selected from T28N, T28K, T28R, T28H, T28F, T28W, T28Y, S30G, S30D, S30M, S31N, S31K, S31R, S31H, S31T, F32Y, D54A, S56E, I57D, K58E, K58D, K58T, Y59H, S60Y, N102D, N102E, D105R, D105E, S107Y, S107F, H111G, K113H, or a combination thereof. In one such embodiment, the mutations are selected from T28N, T28K, T28R, T28H, S31N, S31K, S31R, S31H, N102D, N102E, or a combination thereof. In these and other embodiments, the anti-CGRP receptor antibody or antigen-binding fragment of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 23 or SEQ ID NO: 24 with a mutation at one or more amino acid positions 26, 31, 32, 33, 53, 54, 56, 57, 94, 95, 96, 97, 98, and / or 100. Such mutations may include S26F, S26R, S26Y, N31R, N31I, N31W, N32S, N32Y, N32R, N32K, N32W, Y33T, Y33S, Y33A, Y33P, N53R, N53M, K54W, K54F, K54Y, P56A, S57G, S57R, S57Q, S94Y, S94W, R95Q, R95A, R95W, L96W, L96M, L96T, L96H, L96R, S97K, S97Q, S97T, S97R, A98S, A98V, V100T, V100I, or a combination thereof. In some embodiments, the mutation is selected from S26R, S26Y, N31I, N31R, N32K, N32Y, Y33A, Y33S, or a combination thereof.

[0014] In certain embodiments, the anti-CGRP receptor antibody or antigen-binding fragment comprises a CDRH1 comprising the CDRH1 consensus sequence, a CDRH2 comprising the CDRH2 consensus sequence, and a CDRH3 comprising the CDRH3 consensus sequence. In one embodiment, the CDRH1 consensus sequence is X1FX2X3X4GMH (SEQ ID NO: 471), where X1 is N, K, R, H, F, W, or Y; X2 is S, G, D, or M; X3 is S, T, N, K, R, or H; and X4 is F or Y. In another embodiment, the CDRH1 consensus sequence is X1FSX2FGMH (SEQ ID NO: 472), where X1 is N, K, R, or H and X2 is S, T, N, K, R, or H. In related embodiments, the CDRH2 consensus sequence is VISFX1GX2X3X4X5X6VDSVKG (SEQ ID NO: 473), where X1 is D or A; X2 is S or E; X3 is I or D; X4 is K, E, T, or D; X5 is Y or H; and X6 is S or Y. In these and other embodiments, the CDRH3 consensus sequence may be DRLX1YYX2SX3GYYX4YX5YYGMAV (SEQ ID NO: 474), where X1 is N, D, or E; X2 is D, E, or R; X3 is S, Y, or F; X4 is G or H; and X5 is K or H. The CDRs in the light chain variable region of the anti-CGRP receptor antibody or antigen-binding fragment of the invention may also comprise consensus sequences. For example, in some embodiments, an anti-CGRP receptor antibody or antigen-binding fragment of the invention comprises a CDRL1 comprising the CDRL1 consensus sequence, a CDRL2 comprising the CDRL2 consensus sequence, and a CDRL3 comprising the CDRL3 consensus sequence. In one embodiment, the CDRL1 consensus sequence is SGSX1SNIGX2X3X4VS (SEQ ID NO: 475), where X1 is F, R, Y, or S; X2 is N, R, I, or W; X3 is N, S, Y, R, K, or W; and X4 is Y, T, S, A, or P. The CDRL2 consensus sequence may be DNX1X2RX3X4 (SEQ ID NO: 476), where X1 is N, R, or M; X2 is K, W, F, or Y; X3 is P or A; and X4 is S, G, R, or Q.In a related embodiment, the CDRL3 consensus sequence is GTWDX1X2X3X4X5VX6 (SEQ ID NO: 477), where X1 is S, Y, or W; X2 is R, Q, A, or W; X3 is L, W, M, T, H, or R; X4 is S, K, Q, T, or R; X5 is A, S, or V; and X6 is V, T, or I.

[0015] In some embodiments, the anti-CGRP receptor antibody or antigen-binding fragment of the invention comprises one or more CDRs or variable regions derived from any of the anti-CGRP receptor antibodies described herein. For example, in certain embodiments, the anti-CGRP receptor antibody or antigen-binding fragment comprises: a CDRL1 comprising a sequence selected from SEQ ID NOs: 5-12; a CDRL2 comprising a sequence selected from SEQ ID NOs: 13-16; a CDRL3 comprising a sequence selected from SEQ ID NOs: 17-22; a CDRH1 comprising a sequence selected from SEQ ID NOs: 35-38; a CDRH2 comprising a sequence selected from SEQ ID NOs: 39-42; and a CDRH3 comprising a sequence selected from SEQ ID NOs: 44-46. The anti-CGRP receptor antibody or antigen-binding fragment of the invention may comprise a light chain variable region comprising a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 23-34. In these and other embodiments, the anti-CGRP receptor antibody or antigen-binding fragment of the invention comprises a heavy chain variable region comprising a sequence at least 90% identical or at least 95% identical to a sequence selected from SEQ ID NOs: 48-53. In one embodiment, the anti-CGRP receptor antibody or antigen-binding fragment comprises a light chain variable region comprising a sequence selected from SEQ ID NOs: 23-34 and a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 48-53.

[0016] In any of the embodiments described herein, including those described above, the anti-CGRP receptor antibody or antigen-binding fragment of the invention is a monoclonal antibody or antigen-binding fragment thereof. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof. In other embodiments, the monoclonal antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. In still other embodiments, the monoclonal antibody or antigen-binding fragment thereof is a fully human antibody or antigen-binding fragment thereof. The monoclonal antibody can be of any isotype, such as human IgG1, IgG2, IgG3, or IgG4. In one particular embodiment, the monoclonal antibody is a human IgG1 antibody. In another particular embodiment, the monoclonal antibody is a human IgG2 antibody.

[0017] The present invention also includes bispecific antigen-binding proteins derived from the anti-CGRP receptor antibodies described herein. Such bispecific antigen-binding proteins are capable of specifically binding to and inhibiting the human CGRP receptor and another target. In certain embodiments, the present invention provides bispecific antigen-binding proteins comprising a first binding domain that specifically binds to the human CGRP receptor and a second binding domain that specifically binds to the human PAC1 receptor. In such an embodiment, the first binding domain comprises a first light chain immunoglobulin variable region (VL1) and a first heavy chain immunoglobulin variable region (VH1), and the second binding domain comprises a second light chain immunoglobulin variable region (VL2) and a second heavy chain immunoglobulin variable region (VH2), wherein VL1 comprises (i) a CDRL1 selected from SEQ ID NOs: 5-12, (ii) a CDRL2 selected from SEQ ID NOs: 13-16, and (iii) a CDRL3 selected from SEQ ID NOs: 17-22, and VH1 comprises (i) a CDRH1 selected from SEQ ID NOs: 35-38, (ii) a CDRH2 selected from SEQ ID NOs: 39-42, and (iii) a CDRH3 selected from SEQ ID NOs: 44-46. In these and other embodiments, VL2 may comprise (i) a CDRL1 selected from SEQ ID NOs: 130-140, (ii) a CDRL2 having the sequence of SEQ ID NO: 141, and (iii) a CDRL3 selected from SEQ ID NOs: 142-145, and VH2 may comprise (i) a CDRH1 selected from SEQ ID NOs: 157-163, (ii) a CDRH2 selected from SEQ ID NOs: 164-194, and (iii) a CDRH3 selected from SEQ ID NOs: 195-198.

[0018] In some embodiments, the bispecific antigen-binding protein is an antibody, such as a heterodimeric antibody. The heterodimeric antibody may comprise a first light chain and a first heavy chain derived from a first antibody that specifically binds to the human CGRP receptor, and a second light chain and a second heavy chain derived from a second antibody that specifically binds to the human PAC1 receptor. In certain embodiments, the first and second heavy chains comprise one or more charge-pair mutations in the constant regions (e.g., CH3 domains) that promote heterodimer formation. For example, in some embodiments, the first heavy chain or the second heavy chain comprises at least one amino acid substitution replacing a lysine with a negatively charged amino acid (e.g., glutamic acid or aspartic acid) at positions 360, 370, 392, 409, and / or 439 according to the EU numbering system, and the other heavy chain comprises an amino acid substitution replacing an aspartic acid with a positively charged amino acid (e.g., lysine) at position 399 according to the EU numbering system and at least one amino acid substitution replacing a glutamic acid with a positively charged amino acid (e.g., lysine) at positions 356 and / or 357 according to the EU numbering system.

[0019] In certain embodiments, the first light chain and first heavy chain (or second light chain and second heavy chain) of a heterodimeric antibody of the present invention may contain one or more charge pair mutations to promote correct light-heavy chain pairing. In such embodiments, the first heavy chain may contain an amino acid substitution that introduces a charged amino acid (e.g., glutamic acid) with the opposite charge of the amino acid (e.g., lysine) introduced into the first light chain, so that the first light chain and the first heavy chain can be bound to each other. The charged amino acid (e.g., glutamic acid) introduced into the second light chain will preferably have the same charge as the amino acid (e.g., glutamic acid) introduced into the first heavy chain, rather than the opposite charge of the amino acid (e.g., lysine) introduced into the second heavy chain, so that the second light chain will be bound to the second heavy chain but will be repelled from the first heavy chain. In one embodiment, the first heavy chain comprises an amino acid substitution at position 183 (according to the EU numbering system) to introduce a charged amino acid, and the first light chain comprises an amino acid substitution at position 176 (according to the Kabat numbering system) to introduce a charged amino acid, wherein the charged amino acid introduced into the first heavy chain has the opposite charge of the amino acid introduced into the first light chain. In a related embodiment, the second heavy chain comprises an amino acid substitution at position 183 (according to the EU numbering system) to introduce a charged amino acid, and the second light chain comprises an amino acid substitution at position 176 (according to the Kabat numbering system) to introduce a charged amino acid, wherein the charged amino acid introduced into the second heavy chain has the opposite charge of the amino acid introduced into the second light chain. In certain embodiments, the first heavy chain comprises a S183E mutation, the first light chain comprises a S176K mutation, the second heavy chain comprises a S183K mutation, and the second light chain comprises a S176E mutation.

[0020] In certain embodiments, the anti-CGRP receptor antibody or heterodimeric antibody of the present invention may contain one or more modifications that affect the glycosylation of the antibody. In some embodiments, the anti-CGRP receptor antibody or heterodimeric antibody contains one or more mutations that reduce or eliminate glycosylation. In such embodiments, the aglycosylated antibody may contain a mutation at amino acid position N297 (according to the EU numbering scheme), such as an N297G mutation, in one or both heavy chains. The aglycosylated antibody may further contain mutations that stabilize the antibody structure. Such mutations may include cysteine ​​substitution pairs such as A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C (amino acid positions according to the EU numbering scheme). In one embodiment, the aglycosylated antibody contains R292C and V302C mutations (according to the EU numbering scheme) in one or both heavy chains. In certain embodiments, the aglycosylated anti-CGRP receptor antibody or heterodimeric antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 65 or SEQ ID NO: 66. The anti-CGRP receptor antibody or heterodimeric antibody of the invention may further comprise mutations that modulate other characteristics of the antibody, such as pharmacokinetic properties. In one such embodiment, the anti-CGRP receptor antibody or heterodimeric antibody may comprise M252Y, S254T, and T256E mutations (positions according to the EU numbering scheme) in one or both heavy chains.

[0021] The present invention also includes one or more isolated polynucleotides and expression vectors encoding the anti-CGRP receptor antibodies, antigen-binding fragments, and bispecific antigen-binding proteins (e.g., heterodimeric antibodies) described herein, or any of their components, and host cells, such as CHO cells, containing the encoding polyribonucleotides and expression vectors. In certain embodiments, the present invention includes methods for making the anti-CGRP receptor antibodies, antigen-binding fragments, and bispecific antigen-binding proteins (e.g., heterodimeric antibodies) described herein. In one embodiment, the method comprises culturing a host cell containing an expression vector encoding the anti-CGRP receptor antibody or antigen-binding fragment under conditions allowing for expression of the antibody or antigen-binding fragment, and recovering the antibody or antigen-binding fragment from the culture medium or host cell. In another embodiment, the method comprises culturing a host cell containing an expression vector encoding the bispecific antigen-binding protein under conditions allowing for expression of the antigen-binding protein, and recovering the antigen-binding protein from the culture medium or host cell.

[0022] The anti-CGRP receptor antibodies, antigen-binding fragments, and bispecific antigen-binding proteins (e.g., heterodimeric antibodies) described herein can be used in the manufacture of pharmaceutical compositions or medicaments for the treatment of conditions associated with the biological activity of CGRP receptors and / or PAC1 receptors, such as headache, migraine, cluster headache, vasomotor symptoms, and chronic pain. Accordingly, the present invention also provides pharmaceutical compositions comprising the anti-CGRP receptor antibodies, antigen-binding fragments, or bispecific antigen-binding proteins (e.g., heterodimeric antibodies) described herein and a pharmaceutically acceptable excipient. The pharmaceutical compositions can be used in any of the methods described herein.

[0023] In certain embodiments, the present invention provides methods for treating or preventing a headache condition in a patient in need thereof, comprising administering to the patient an effective amount of an anti-CGRP receptor antibody, antigen-binding fragment, or bispecific antigen-binding protein (e.g., a heterodimeric antibody) described herein. In some embodiments, the headache condition to be treated or prevented by the methods of the present invention is migraine. The migraine can be episodic migraine or chronic migraine. In other embodiments, the headache condition to be treated or prevented by the methods of the present invention is cluster headache. In certain embodiments, the methods provide prophylactic treatment for these conditions.

[0024] In some embodiments, the present invention provides methods for treating chronic pain in a patient in need thereof, comprising administering to the patient an effective amount of an anti-CGRP receptor antibody, antigen-binding fragment, or bispecific antigen-binding protein (e.g., a heterodimeric antibody) described herein. Chronic pain syndromes to be treated according to the methods of the present invention may include neuropathic pain, arthritic pain, such as pain associated with osteoarthritis or rheumatoid arthritis, visceral pain, such as pain associated with irritable bowel syndrome, Crohn's disease, ulcerative colitis, and interstitial cystitis.

[0025] The use of anti-CGRP receptor antibodies, antigen-binding fragments, and bispecific antigen-binding proteins (e.g., heterodimeric antibodies) in any of the methods disclosed herein or for the preparation of medicaments for administration according to any of the methods disclosed herein is particularly contemplated. For example, the present invention includes anti-CGRP receptor antibodies or bispecific antigen-binding proteins (e.g., heterodimeric antibodies) for use in methods for treating or preventing conditions associated with the biological activity of CGRP receptors and / or PAC1 receptors in a patient in need thereof. The conditions may include headaches (e.g., migraines or cluster headaches) and chronic pain.

[0026] The invention also includes the use of an anti-CGRP receptor antibody or a bispecific antigen-binding protein (e.g., a heterodimeric antibody) in the preparation of a medicament for treating or preventing a condition associated with the biological activity of a CGRP receptor and / or a PAC1 receptor in a patient in need thereof. The condition may include headache (e.g., migraine or cluster headache) and chronic pain. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic representation of the selection process for improved binding variants from a yeast-displayed antibody Fab variant library. [Figure 2] Figure 1 shows a schematic representation of the four charge-pair mutation (CPM) formats used to generate anti-CGRP receptor / PAC1 receptor bispecific heteroimmunoglobulins. The Kabat-EU numbering scheme is used to indicate the position of the charge-pair mutation within each chain. This IgG-like bispecific molecule is a heterotetramer containing two different light chains and two different heavy chains. HC1 and LC1 refer to the heavy and light chains, respectively, of one Fab binding arm, and HC2 and LC2 refer to the heavy and light chains, respectively, of the second Fab binding arm. For example, in the schematic representation, HC1 and LC1 correspond to the anti-CGRP receptor binding arm, and HC2 and LC2 correspond to the anti-PAC1 binding arm. However, the two binding arms can be swapped so that HC1 and LC1 correspond to the anti-PAC1 arm, and HC2 and LC2 correspond to the anti-CGRP receptor binding arm. Mutations at the designated positions are indicated by specific charged amino acids in the schematic representation, such as mutations to glutamic acid or lysine residues. However, other similarly charged amino acids may be used, such as aspartic acid in place of glutamic acid (and vice versa) and arginine residues in place of lysine residues. [Figure 3A]

[0033] Figure 3A shows the serum concentration-time profiles for bispecific heteroimmunoglobulin molecules after a single subcutaneous dose of 1 mg / kg in mice. Figure 3A shows the overall serum concentration over time for molecules 5601, 5602, 5603, 5604, 5605, 5606, 5607, 5608, and 5609, while Figure 3B shows the serum concentration over time for the intact forms of the molecules (i.e., both binding arms intact). Figures 3C and 3D show the overall and intact serum concentration-time profiles for molecules 5605, 5606, and 5607, respectively. [Figure 3B]

[0033] Figure 3A shows the serum concentration-time profiles for bispecific heteroimmunoglobulin molecules after a single subcutaneous dose of 1 mg / kg in mice. Figure 3A shows the overall serum concentration over time for molecules 5601, 5602, 5603, 5604, 5605, 5606, 5607, 5608, and 5609, while Figure 3B shows the serum concentration over time for the intact forms of the molecules (i.e., both binding arms intact). Figures 3C and 3D show the overall and intact serum concentration-time profiles for molecules 5605, 5606, and 5607, respectively. [Figure 3C]

[0033] Figure 3A shows the serum concentration-time profiles for bispecific heteroimmunoglobulin molecules after a single subcutaneous dose of 1 mg / kg in mice. Figure 3A shows the overall serum concentration over time for molecules 5601, 5602, 5603, 5604, 5605, 5606, 5607, 5608, and 5609, while Figure 3B shows the serum concentration over time for the intact forms of the molecules (i.e., both binding arms intact). Figures 3C and 3D show the overall and intact serum concentration-time profiles for molecules 5605, 5606, and 5607, respectively. [Figure 3D]

[0033] Figure 3A shows the serum concentration-time profiles for bispecific heteroimmunoglobulin molecules after a single subcutaneous dose of 1 mg / kg in mice. Figure 3A shows the overall serum concentration over time for molecules 5601, 5602, 5603, 5604, 5605, 5606, 5607, 5608, and 5609, while Figure 3B shows the serum concentration over time for the intact forms of the molecules (i.e., both binding arms intact). Figures 3C and 3D show the overall and intact serum concentration-time profiles for molecules 5605, 5606, and 5607, respectively. [Figure 4A] This figure shows the dose-dependent effect of the bispecific hetero-immunoglobulin molecule (hetero-IgG) 5605 on Maxadilan-induced increases in skin blood flow in rats. Rats were administered hetero-IgG intravenously at one of four doses ranging from 0.1 mg / kg to 30 mg / kg 24 hours before challenge with 10 ng of Maxadilan (intradermal injection). Skin blood flow was assessed 30 minutes after Maxadilan challenge by laser Doppler imaging. *p<0.05, ****p<0.0001 compared to the vehicle group by one-way ANOVA followed by Dunnett's MCT. [Figure 4B] This figure shows the dose-dependent effect of bispecific hetero-immunoglobulin molecule (hetero-IgG) 5606 on Maxadilan-induced increases in skin blood flow in rats. Rats were administered hetero-IgG intravenously at one of four doses ranging from 0.1 mg / kg to 30 mg / kg 24 hours before challenge with 10 ng of Maxadilan (intradermal injection). Skin blood flow was assessed 30 minutes after Maxadilan challenge by laser Doppler imaging. ****p<0.0001 compared to the vehicle group by one-way ANOVA followed by Dunnett's MCT. [Figure 4C]This figure shows the dose-dependent effect of the bispecific hetero-immunoglobulin molecule (hetero-IgG) 5607 on Maxadilan-induced increases in skin blood flow in rats. Rats were administered hetero-IgG intravenously at one of four doses ranging from 0.1 mg / kg to 30 mg / kg 24 hours before challenge with 10 ng of Maxadilan (intradermal injection). Skin blood flow was assessed 30 minutes after Maxadilan challenge by laser Doppler imaging. **p<0.01 compared to the vehicle group by one-way ANOVA followed by Dunnett's MCT. [Figure 5A] 1 is a serum concentration-time profile for bispecific heteroimmunoglobulin molecules 5605, 5606, and 5607 after a single intravenous dose of 1 mg / kg in cynomolgus monkeys. [Figure 5B] 1 is a serum concentration-time profile for bispecific heteroimmunoglobulin molecules 5605, 5606, and 5607 after a single subcutaneous dose of 2 mg / kg in cynomolgus monkeys. [Figure 6A] Figure 1 shows the dose-dependent effect of bispecific hetero-immunoglobulin molecule (hetero-IgG) 5607 on capsaicin-induced increases in skin blood flow in cynomolgus monkeys. After pre-treatment measurements on day 0, the hetero-IgG was administered intravenously to cynomolgus monkeys at a single dose of 10 mg / kg. Skin blood flow was assessed 30 minutes after capsaicin challenge (1 mg in 20 μL, topical application) by laser Doppler imaging on days 2, 4 / 5, and 8 / 9 after administration of the hetero-IgG. Data are shown as mean ± SEM. **p<0.01, ****p<0.0001 compared to day 0 by one-way ANOVA followed by Bonferroni's MCT. [Figure 6B]Figure 1 shows the dose-dependent effect of bispecific hetero-immunoglobulin molecule (hetero-IgG) 5607 on maxadilan-induced increases in skin blood flow in cynomolgus monkeys. After pre-treatment measurements on day 0, hetero-IgG was administered intravenously to cynomolgus monkeys at a single dose of 10 mg / kg. Skin blood flow was assessed 30 minutes after maxadilan challenge (1 ng in 20 μL, intradermal injection) by laser Doppler imaging on days 2, 4 / 5, and 8 / 9 after hetero-IgG administration. Data are shown as mean ± SEM. **p<0.01, ****p<0.0001 compared to day 0 by one-way ANOVA followed by Bonferroni's MCT. [Figure 7A] Shown is a ternary complex of the 4E4 Fab fragment (ribbon structure representation with heavy chain (HC) on the left and light chain (LC) on the right) bound to the CRLR ECD polypeptide (light gray surface representation) and the RAMP1 ECD polypeptide (medium gray surface representation). The dashed box highlights the paratope-epitope interface. [Figure 7B] 7A is a magnified view of the paratope-epitope interface showing the interactions of each of the six CDRs in the 4E4 Fab fragment with the CRLR and RAMP1 polypeptide components of the CGRP receptor. This view shows the region delineated by the dashed box in FIG. 7A rotated 90° about the horizontal axis and 45° about the vertical axis. [Figure 8A] Figure 8 shows dose-response curves for wild-type 4E4 anti-CGRP receptor monoclonal antibody (WT) and single-point alanine mutation variant antibodies for inhibition of CGRP-induced activation of the human CGRP receptor. Percent of control (POC), where control is defined as the activity of the CGRP agonist in the assay, is plotted against the log concentration of antibody. Figure 8A shows dose-response curves for the WT antibody and the CDRH2 D54A antibody variant (H_D54A). Figure 8B shows dose-response curves for the WT antibody and CDRH3 antibody variants H_Y103A, H_Y104A, H_Y109A, H_Y110A, and H_K113A. Figure 8C shows dose-response curves for the WT antibody and light chain antibody variants L-Y33A, L_K67A, and L_R95A. [Figure 8B] Figure 8 shows dose-response curves for wild-type 4E4 anti-CGRP receptor monoclonal antibody (WT) and single-point alanine mutation variant antibodies for inhibition of CGRP-induced activation of the human CGRP receptor. Percent of control (POC), where control is defined as the activity of the CGRP agonist in the assay, is plotted against the log concentration of antibody. Figure 8A shows dose-response curves for the WT antibody and the CDRH2 D54A antibody variant (H_D54A). Figure 8B shows dose-response curves for the WT antibody and CDRH3 antibody variants H_Y103A, H_Y104A, H_Y109A, H_Y110A, and H_K113A. Figure 8C shows dose-response curves for the WT antibody and light chain antibody variants L-Y33A, L_K67A, and L_R95A. [Figure 8C] Figure 8 shows dose-response curves for wild-type 4E4 anti-CGRP receptor monoclonal antibody (WT) and single-point alanine mutation variant antibodies for inhibition of CGRP-induced activation of the human CGRP receptor. Percent of control (POC), where control is defined as the activity of the CGRP agonist in the assay, is plotted against the log concentration of antibody. Figure 8A shows dose-response curves for the WT antibody and the CDRH2 D54A antibody variant (H_D54A). Figure 8B shows dose-response curves for the WT antibody and CDRH3 antibody variants H_Y103A, H_Y104A, H_Y109A, H_Y110A, and H_K113A. Figure 8C shows dose-response curves for the WT antibody and light chain antibody variants L-Y33A, L_K67A, and L_R95A. [Figure 9] 1 is a representation of the interactions between selected amino acids in the CDRH3 of 4E4 Fab and the CRLR and RAMP1 polypeptide subunits of the CGRP receptor. Amino acids in the CDRH3 of the Fab are shown in ball-and-stick format, while amino acids in the CRLR and RAMP1 polypeptides are shown in ball-and-stick format within the molecular surface. [Figure 10A]Figure 10 shows the binding profiles of soluble CGRP receptor to wild-type 4E4 anti-CGRP receptor monoclonal antibody (WT) and single-point alanine mutation variant antibodies by surface plasmon resonance. Figure 10A shows the binding profiles for the WT antibody and the CDRH2 D54A antibody variant (H_D54A). Figure 10B shows the binding profiles for the WT antibody and CDRH3 antibody variants H_Y103A, H_Y104A, H_Y109A, H_Y110A, and H_K113A. Figure 10C shows the binding profiles for the WT antibody and the light chain antibody variants L-Y33A, L_K67A, and L_R95A. [Figure 10B] Figure 10 shows the binding profiles of soluble CGRP receptor to wild-type 4E4 anti-CGRP receptor monoclonal antibody (WT) and single-point alanine mutation variant antibodies by surface plasmon resonance. Figure 10A shows the binding profiles for the WT antibody and the CDRH2 D54A antibody variant (H_D54A). Figure 10B shows the binding profiles for the WT antibody and CDRH3 antibody variants H_Y103A, H_Y104A, H_Y109A, H_Y110A, and H_K113A. Figure 10C shows the binding profiles for the WT antibody and the light chain antibody variants L-Y33A, L_K67A, and L_R95A. [Figure 10C] Figure 10 shows the binding profiles of soluble CGRP receptor to wild-type 4E4 anti-CGRP receptor monoclonal antibody (WT) and single-point alanine mutation variant antibodies by surface plasmon resonance. Figure 10A shows the binding profiles for the WT antibody and the CDRH2 D54A antibody variant (H_D54A). Figure 10B shows the binding profiles for the WT antibody and CDRH3 antibody variants H_Y103A, H_Y104A, H_Y109A, H_Y110A, and H_K113A. Figure 10C shows the binding profiles for the WT antibody and the light chain antibody variants L-Y33A, L_K67A, and L_R95A. [Figure 11]FIG. 1 is a graph showing the correlation between in vitro potency (IC50) for anti-CGRP receptor antibodies that inhibit human CGRP receptor activation as measured by a cell-based cAMP assay and the number of amino acids in CDR3 of the antibody's heavy chain variable region. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention is based, in part, on the design and generation of high-affinity antibodies that specifically bind to and potently inhibit the human CGRP receptor. The antibodies of the present invention are 2-4 times more potent inhibitors of human CGRP receptor activation than previously described anti-CGRP receptor antibodies. The isolated antibodies and antigen-binding fragments thereof can be used to inhibit, interfere with, or modulate the biological activity of the human CGRP receptor, including inhibiting or reducing CGRP-induced activation of the CGRP receptor, inhibiting or reducing vasodilation, and ameliorating or treating the symptoms of migraine and other vascular headaches. The enhanced inhibitory potency of anti-CGRP receptor antibodies also enables the creation of bispecific antigen-binding proteins that can bind to and inhibit separate targets, such as the human CGRP receptor and the human PAC1 receptor. Such bispecific antigen-binding proteins constructed from the anti-CGRP receptor antibodies of the present invention have improved inhibitory activity against the CGRP receptor compared to bivalent monoclonal antibodies, thereby reducing the effective therapeutic dose.

[0029] Thus, the present invention provides isolated antibodies and antigen-binding fragments thereof that specifically bind to calcitonin gene-related peptide (CGRP) receptors, particularly human CGRP receptors. Human CGRP receptors are heterodimers containing human calcitonin receptor-like receptor (CRLR or CLR) polypeptides (GenBank accession number U17473.1) and human receptor activity-modifying protein 1 (RAMP1) polypeptides (GenBank accession number AJ001014). Human CGRP receptors are G protein-coupled receptors that are actively coupled to adenylate cyclase. Activation of the human CGRP receptor by CGRP results in an increase in intracellular cyclic AMP (cAMP). The amino acid sequences for the full-length human CRLR and RAMP1 polypeptides and the extracellular domains derived from both polypeptides are listed in Table 1 below.

[0030] [Table 1]

[0031] The present invention provides antibodies that specifically bind to the human CGRP receptor. An "antibody" is a protein that includes an antigen-binding fragment that specifically binds to an antigen and a scaffold or framework portion that enables the antigen-binding fragment to adopt a conformation that promotes antibody binding to the antigen. As used herein, the term "antibody" generally refers to a tetrameric immunoglobulin protein containing two light chain polypeptides (each approximately 25 kDa) and two heavy chain polypeptides (each approximately 50-70 kDa). The term "light chain" or "immunoglobulin light chain" refers to a polypeptide that, from the amino terminus to the carboxyl terminus, contains a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be a human kappa (κ) or human lambda (λ) constant domain. The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from amino to carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG-class and IgA-class antibodies are further divided into subclasses, namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). Immunoglobulin heavy chain constant domains can be derived from any immunoglobulin isotype, including subtypes. Antibody chains are linked via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the two antibody heavy chains.

[0032] The present invention also includes antigen-binding fragments of the anti-CGRP receptor antibodies described herein. An "antigen-binding fragment," which is used interchangeably herein with "binding fragment" or "fragment," is a portion of an antibody that lacks at least some of the amino acids present in the full-length heavy and / or light chains, yet is still capable of specifically binding to an antigen. Antigen-binding fragments include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., the VH domain of camelid heavy chain antibodies; VHH fragments; see Cortez-Retamozo et al., Cancer Research, Vol. 64:2853-57, 2004), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments, and may be derived from any mammalian source, such as human, mouse, rat, rabbit, or camel. Antigen-binding fragments may compete with intact antibodies for binding to target antigens, and fragments can be produced by modification of intact antibodies (e.g., enzymatic or chemical cleavage) or can be synthesized de novo using recombinant DNA techniques or peptide synthesis. In some embodiments, an antigen-binding fragment comprises at least one CDR from an antibody that binds to the antigen, such as the heavy chain CDR3 from an antibody that binds to the antigen. In other embodiments, an antigen-binding fragment comprises all three CDRs from the heavy chain of an antibody that binds to the antigen or all three CDRs from the light chain of an antibody that binds to the antigen. In yet other embodiments, an antigen-binding fragment comprises all six CDRs (three from the heavy chain and three from the light chain) from an antibody that binds to the antigen.

[0033] The term "isolated molecule" (where the molecule is, for example, a polypeptide, polynucleotide, antigen-binding protein, antibody, or antigen-binding fragment) refers to a molecule that, by virtue of its origin or source, (1) is not associated with naturally associated components that accompany it in its native state; (2) is substantially free of other molecules from the same species; (3) is expressed by cells from a different species; or (4) is not naturally occurring. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates is "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. The purity or homogeneity of a molecule can be assayed by many means well known in the art. For example, the purity of a polypeptide sample can be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution can be provided by using HPLC or other purification means well known in the art.

[0034] In certain embodiments of the present invention, an antibody or antigen-binding fragment thereof specifically binds to the human CGRP receptor. An antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof "specifically binds" to a target antigen if, under similar binding assay conditions, it has a significantly higher binding affinity compared to its affinity for other unrelated proteins, such that it can distinguish between the antigens. An antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof that specifically binds to an antigen has an equilibrium dissociation constant (K D )≦1×10 -6 The antibody, binding fragment, antigen-binding protein or binding domain thereof may have a K D is ≦1×10 -8 In one embodiment, the antibody or binding fragment of the invention specifically binds to an antigen with "high affinity" when M is ≦5×10 9 K of M DIn another embodiment, the antibody or binding fragment of the invention binds to the human CGRP receptor with a mAb of ≦1×10 -9 K of M D In yet another embodiment, the antibody or binding fragment of the invention binds to the human CGRP receptor with a binding affinity of ≦5×10 -10 K of M D In another embodiment, the antibody or binding fragment of the invention binds to the human CGRP receptor with a mAb of ≦1×10 -10 K of M D In certain embodiments, the antibody or binding fragment of the invention binds to the human CGRP receptor with a binding affinity of ≦5×10 -11 K of M D In other embodiments, the antibody or binding fragment of the invention binds to the human CGRP receptor with a binding affinity of ≦1×10 -11 K of M D In one particular embodiment, the antibody or binding fragment of the invention binds to the human CGRP receptor with a mAb concentration of ≦5×10 -12 K of M D In another specific embodiment, the antibody or binding fragment of the invention binds to the human CGRP receptor with a -12 K of M D It binds to the human CGRP receptor.

[0035] Affinity can be determined using a variety of techniques, one example being an affinity ELISA assay. In various embodiments, affinity is determined by a surface plasmon resonance assay (e.g., a BIAcore®-based assay). Using this methodology, the binding rate constant (k a Unit: M -1 s -1 ) and dissociation rate constant (k d Unit: s -1 ) can be measured. Then, the equilibrium dissociation constant (K D Unit: M) is the ratio of reaction rate constants (k d / k aIn some embodiments, affinity can be determined by a kinetic method, such as the equilibrium exclusion binding assay (KExA), as described in Rathanaswami et al., Analytical Biochemistry, Vol. 373:52-60, 2008. The KinExA assay can be used to determine the equilibrium dissociation constant (K D Unit: M) and binding rate constant (k a Unit: M -1 s -1 The dissociation rate constant (k d Unit: s -1 ) are the values ​​of these (K D ×k a In another embodiment, affinity is determined by biolayer interferometry, such as that described in Kumaraswamy et al., Methods Mol. Biol., Vol. 1278:165-82, 2015, and used in the Octet® system (Pall ForteBio). The rate constant (k a and k d ) and affinity constant (K D ) can be calculated in real time using biolayer interferometry. In some embodiments, the antibodies, binding fragments, antigen-binding proteins or binding domains thereof described herein have a densitometric value of about 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 s -1 The following human CGRP receptors d (dissociation rate constant) (lower values ​​indicate greater binding avidity), and / or about 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 K for human CGRP receptors below M DThe binding affinity of the ATP-binding protein is determined by the equilibrium dissociation constant (Eq. 1.2.1), where a smaller value indicates a higher binding affinity.

[0036] Preferably, antibodies, binding fragments, antigen-binding proteins, or binding domains thereof of the present invention do not significantly bind to or cross-react with other members of the calcitonin family of receptors, such as human adrenomedullin 1 (AM1), human adrenomedullin 2 (AM2), or human amylin (e.g., human AMY1 receptor) receptors. An antibody, binding fragment, antigen-binding protein, or binding domain thereof "does not significantly bind" a target antigen if, under similar binding assay conditions, it has binding affinity for that antigen that is comparable to its affinity for other unrelated proteins. Antibodies, binding fragments, antigen-binding proteins, or binding domains thereof that do not significantly bind a target antigen can also include proteins that do not generate a signal for the target antigen that is statistically different from a negative control in affinity assays such as those described herein. By way of example, an antibody that generates a signal value for determining binding to the human AM1 receptor in an ELISA or surface plasmon resonance-based assay (e.g., a BIAcore®-based assay) that is not statistically different from the signal value generated in a negative control (e.g., buffer solution without antibody) is considered not to significantly bind to the human AM1 receptor. An antibody, binding fragment, antigen-binding protein, or binding domain thereof that does not significantly bind to an antigen is considered to be less than 1×10 for that antigen. -6 More than M or 1 x 10 -5 More than M or 1 x 10 -4 More than M or 1 x 10 -3 The antibodies, binding fragments, antigen-binding proteins or binding domains thereof of the invention may have an equilibrium dissociation constant (KD) greater than M. Thus, in certain embodiments, the antibodies, binding fragments, antigen-binding proteins or binding domains thereof of the invention selectively bind to the human CGRP receptor compared to the human AM1, human AM2, and human amylin (e.g., human AMY1 receptor) receptors. In other words, the antibodies, binding fragments, antigen-binding proteins or binding domains thereof of the invention do not significantly bind to the human AM1, human AM2, or human amylin (e.g., human AMY1 receptor) receptors.

[0037] The antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the present invention may inhibit, interfere with, or modulate one or more biological activities of human CGRP receptor. Biological activities of human CGRP receptor include, but are not limited to, inducing the CGRP-mediated receptor signaling pathway, inducing vasodilation, and inhibiting vasoconstriction. In some embodiments, the antibodies, binding fragments, antigen-binding proteins, or binding domains thereof of the present invention inhibit the binding of CGRP to human CGRP receptor. "Inhibition of binding" occurs when excess antibody, binding fragment, or antigen-binding protein reduces the amount of human CGRP receptor bound to CGRP, or vice versa, by, for example, at least about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or more, as measured, for example, by an in vitro competitive binding assay. Inhibition constants (Ki), which indicate how potent the antibodies, antigen-binding fragments, and antigen-binding proteins of the invention are in preventing the binding of CGRP to the human CGRP receptor, can be calculated from such competitive binding assays. 125 The K i value is calculated using the formula K i = IC 50 / (1 + ([L] / K d )), where [L] is the radioligand (e.g., CGRP) used. 125where Kd is the concentration of radioactive CGRP (I-labeled CGRP), and Kd is the dissociation constant of the radioligand. See, for example, Keen M, MacDermot J (1993) Analysis of receptors by radioligand binding. In: Wharton J, Polak JM (eds) Receptor autoradiography, principles and practice. Oxford University Press, Oxford. The lower the K value for an antagonist, the more potent the antagonist. In some embodiments, antibodies, antigen-binding fragments, or antigen-binding proteins of the invention compete with radiolabeled CGRP ligands for binding to human CGRP receptors with a K of ≦1 nM. In other embodiments, antibodies, antigen-binding fragments, or antigen-binding proteins of the invention compete with radiolabeled CGRP ligands for binding to human CGRP receptors with a K of ≦500 pM. In still other embodiments, antibodies, antigen-binding fragments, or antigen-binding proteins of the invention compete with a radiolabeled CGRP ligand for binding to the human CGRP receptor with a K of ≦200 pM. In certain other embodiments, antibodies, antigen-binding fragments, or antigen-binding proteins of the invention compete with a radiolabeled CGRP ligand for binding to the human CGRP receptor with a K of ≦100 pM.

[0038] In certain embodiments, the antibodies, antigen-binding fragments, or antigen-binding proteins of the present invention inhibit ligand-induced activation of the human CGRP receptor. The ligand can be the primary endogenous ligand of the receptor, such as CGRP, or the ligand can be another known agonist of the receptor. Various assays for assessing CGRP receptor activation are known in the art, including cell-based assays that measure ligand-induced calcium mobilization and cAMP production. An exemplary cell-based cAMP assay is described in Example 1. Other suitable CGRP receptor activation assays are described in Aiyar et al., Molecular and Cellular Biochemistry, Vol. 197:179-185, 1999; Pin et al., European Journal of Pharmacology, Vol. 577:7-16, 2007; U.S. Patent No. 8,168,592; and WO 2010 / 075238, all of which are hereby incorporated by reference in their entireties.

[0039] The inhibitory activity of an antibody, antigen-binding fragment, or antigen-binding protein against CGRP receptor activation can be quantified by calculating the IC50 in any functional assay for the receptor, such as those described above. "IC50" is the dose / concentration required to achieve 50% inhibition of biological or biochemical function. In the case of a radioligand, IC50 is the concentration of a competing ligand that displaces 50% of the specific binding of the radioligand. The IC50 of any particular substance or antagonist can be determined by constructing a dose-response curve and testing the effect of different concentrations of the drug or antagonist on reversing agonist activity in a particular functional assay. By determining the concentration required to inhibit half of the maximal biological response of the agonist, the IC50 value can be calculated for a given antagonist or substance. Thus, the IC50 value of any anti-CGRP receptor antigen-binding protein, antibody, or binding fragment of the invention can be calculated by determining the concentration of antigen-binding protein, antibody, or binding fragment required to inhibit half-maximal biological response of a ligand (e.g., CGRP) in activating the human CGRP receptor in any functional assay, such as the cAMP assay described in the Examples. An anti-CGRP receptor antigen-binding protein, antibody, or binding fragment that inhibits ligand-induced (e.g., CGRP-induced) activation of the CGRP receptor is understood to be a neutralizing or antagonist antigen-binding protein, antibody, or binding fragment of the CGRP receptor.

[0040] In certain embodiments, the antigen-binding proteins, antibodies, or antigen-binding fragments of the invention inhibit CGRP-induced activation of human CGRP receptors. For example, the antigen-binding proteins, antibodies, or antigen-binding fragments may inhibit CGRP-induced activation of human CGRP receptors with an IC50 of less than about 5 nM, less than about 3 nM, less than about 1 nM, less than about 800 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, or less than about 150 pM, as measured by a cell-based calcium mobilization assay or cAMP assay. In one specific embodiment, the antigen-binding proteins, antibodies, or antigen-binding fragments of the invention inhibit CGRP-induced activation of human CGRP receptors with an IC50 of less than about 5 nM, as measured by a cell-based cAMP assay. In another specific embodiment, the antigen-binding proteins, antibodies, or antigen-binding fragments of the invention inhibit CGRP-induced activation of human CGRP receptors with an IC50 of less than about 1 nM, as measured by a cell-based cAMP assay. In yet another specific embodiment, the antigen binding proteins, antibodies, or antigen-binding fragments of the invention inhibit CGRP-induced activation of the human CGRP receptor with an IC50 of less than about 500 pM, as measured by a cell-based cAMP assay. In another embodiment, the antigen binding proteins, antibodies, or antigen-binding fragments of the invention inhibit CGRP-induced activation of the human CGRP receptor with an IC50 of less than about 400 pM, as measured by a cell-based cAMP assay. In another embodiment, the antigen binding proteins, antibodies, or antigen-binding fragments of the invention inhibit CGRP-induced activation of the human CGRP receptor with an IC50 of less than about 200 pM, as measured by a cell-based cAMP assay. In some embodiments, the antigen binding proteins, antibodies, or antigen-binding fragments of the invention inhibit CGRP-induced activation of the human CGRP receptor with an IC50 of between about 0.1 nM and about 1 nM, as measured by a cell-based cAMP assay. In other embodiments, the antigen binding proteins, antibodies, or antigen binding fragments of the invention inhibit CGRP-induced activation of the human CGRP receptor with an IC50 of between about 50 pM and about 400 pM as measured by a cell-based cAMP assay.In yet other embodiments, the antigen binding proteins, antibodies or antigen binding fragments of the invention inhibit CGRP-induced activation of the human CGRP receptor with an IC50 of between about 100 pM and about 350 pM as measured by a cell-based cAMP assay.

[0041] In some embodiments, the antigen-binding proteins, antibodies, or antigen-binding fragments of the present invention selectively inhibit the human CGRP receptor compared to human AM1, human AM2, and / or human amylin receptors (e.g., human AMY1 receptor). The human AM1 receptor is composed of a human CRLR polypeptide and a RAMP2 polypeptide, whereas the human AM2 receptor is composed of a human CRLR polypeptide and a RAMP3 polypeptide. Therefore, antibodies or other binding proteins that bind only to CRLR (but not RAMP1) would not be expected to selectively inhibit the CGRP receptor, since the CRLR polypeptide is also a component of the AM1 and AM2 receptors. The human amylin (AMY) receptor is composed of a human calcitonin receptor (CT) polypeptide and one of the RAMP1, RAMP2, or RAMP3 subunits. Specifically, the human AMY1 receptor is composed of a CT polypeptide and a RAMP1 polypeptide, the human AMY2 receptor is composed of a CT polypeptide and a RAMP2 polypeptide, and the human AMY3 receptor is composed of a CT polypeptide and a RAMP3 polypeptide. Thus, antibodies or other binding proteins that bind only to RAMP1 (and not CRLR) would not be expected to selectively inhibit the CGRP receptor because the RAMP1 polypeptide is also a component of the human AMY1 receptor. An antigen-binding protein, antibody, or antigen-binding fragment "selectively inhibits" a particular receptor compared to other receptors when the IC50 of the antigen-binding protein, antibody, or antigen-binding fragment in an inhibition assay for that particular receptor is at least 50-fold lower than the IC50 in an inhibition assay for another "reference" receptor, e.g., human AM1, human AM2, or human amylin receptor. As noted above, the IC50 value of any anti-CGRP receptor antigen-binding protein, antibody, or antigen-binding fragment can be calculated by determining the concentration of the antigen-binding protein, antibody, or antigen-binding fragment required to inhibit half of the maximal biological response of a CGRP ligand when activating the human CGRP receptor in any functional assay, such as the cAMP assay described in the Examples.

[0042] In some embodiments, the anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the present invention may bind to specific regions or epitopes of the human CGRP receptor. For example, in certain embodiments, the anti-CGRP receptor antibodies or antigen-binding fragments specifically bind to a residue, sequence of residues, or region of both the human CRLR and human RAMP1 polypeptides. In one embodiment, the anti-CGRP receptor antibodies or antigen-binding fragments specifically bind to an epitope formed from amino acids in both the human CRLR and human RAMP1 polypeptides (e.g., SEQ ID NOS: 1 and 2, respectively). In another embodiment, the anti-CGRP receptor antibodies or antigen-binding fragments specifically bind to an epitope formed from amino acids in the extracellular domains of both the human CRLR and human RAMP1 polypeptides (e.g., SEQ ID NOS: 3 and 4, respectively). In some embodiments, the epitope formed from amino acids in both the human CRLR and human RAMP1 polypeptides contains one or more cleavage sites for the AspN protease, which cleaves the peptide after an aspartic acid residue and several glutamic acid residues at the amino terminus. As used herein, "epitope" refers to any determinant capable of being specifically bound by an antibody or antigen-binding fragment thereof. An epitope is the region of an antigen that is bound by or interacts with an antibody or binding fragment that targets that antigen, and, if the antigen is a protein, includes specific amino acids that directly contact or interact with the antibody or binding fragment. Epitopes can be formed by both contiguous amino acids juxtaposed by tertiary folding of a protein or by non-contiguous amino acids. A "linear epitope" is an epitope that includes an epitope recognized by a primary amino acid sequence. Linear epitopes typically contain at least 3 or 4 amino acids, more typically at least 5, at least 6, or at least 7 amino acids, e.g., about 8 to about 10 amino acids of a unique sequence.A "conformational epitope," in contrast to a linear epitope, is a discontinuous group of amino acids (e.g., in a polypeptide, amino acid residues that are not contiguous in the primary sequence of the polypeptide but are sufficiently close to each other in terms of the tertiary and quaternary structure of the polypeptide to be bound by an antibody or binding fragment thereof).

[0043] In certain embodiments, the anti-CGRP receptor antibody or antigen-binding fragment specifically binds to the extracellular domain of a human CRLR polypeptide comprising the amino acid sequence of SEQ ID NO: 3 and / or the extracellular domain of a human RAMP1 polypeptide comprising the amino acid sequence of SEQ ID NO: 4. As described in Example 5, the crystal structure of a complex of the human CRLR N-terminal extracellular domain (ECD), human RAMP1 N-terminal ECD, and the Fab region of an anti-CRLR antagonist antibody revealed key amino acids within the CRLR / RAMP1 heterodimer (i.e., the human CGRP receptor) that constituted the binding interface with the anti-CGRP receptor Fab. These core interface amino acids, all of which contained at least one non-hydrogen atom within 5.0 Å of a non-hydrogen atom in Fab, include E23, L24, E25, E26, E29, R38, I41, M42, D70, G71, W72, F92, D94, F95, K103, H114, A116, S117, R119, T120, W121, T122, Y124, N128, T131, H132, and E133 (amino acid position numbering relative to SEQ ID NO: 1) in the CRLR polypeptide and R67, A70, D71, W74, E78, C82, F83, W84, and P85 (amino acid position numbering relative to SEQ ID NO: 2) in the RAMP1 polypeptide. Thus, in some embodiments, the anti-CGRP receptor antibody or antigen-binding fragment of the invention binds to the human CGRP receptor at an epitope comprising one or more amino acids selected from E23, L24, E25, E26, E29, R38, 141, M42, D70, G71, W72, F92, D94, F95, K103, H114, A116, S117, R119, T120, W121, T122, Y124, N128, T131, H132, and E133 in the human CRLR polypeptide of SEQ ID NO: 1 and one or more amino acids selected from R67, A70, D71, W74, E78, C82, F83, W84, and P85 in the human RAMP1 polypeptide of SEQ ID NO: 2.In other embodiments, the anti-CGRP receptor antibody or antigen-binding fragment of the invention binds to the human CGRP receptor at an epitope comprising one or more amino acids selected from E23, L24, E25, E26, R38, 141, D70, W72, D94, H114, A116, S117, R119, T120, Y124, T131, H132, and E133 in the human CRLR polypeptide of SEQ ID NO: 1 and one or more amino acids selected from A70, D71, W74, E78, and W84 in the human RAMP1 polypeptide of SEQ ID NO: 2.

[0044] The crystal structure of the human CGRP ECD-Fab complex described in Example 5 also revealed key residues in the CDRs of the heavy and light chains of the Fab that interacted with amino acids in the human CRLR ECD and human RAMP1 ECD polypeptides, thereby identifying important amino acids in the paratope of the antibody. The "paratope" is the region of an antibody that recognizes and binds to a target antigen. Paratope residues within 5.0 Å of residues in the CRLR ECD and RAMP1 ECD polypeptides include S26, S27, G30, N31, N32, Y33, D51, N52, K67, S94, and R95 in the light chain variable region (SEQ ID NO: 23) and T28, S31, F53, D54, G55, S56, L101, N102, Y103, Y104, D105, S106, S107, G108, Y109, Y110, H111, K113, and Y115 in the heavy chain variable region (SEQ ID NO: 47). Specific mutations of some of these residues or neighboring residues in the paratope were designed to improve interactions with core interface residues (i.e., residues in the epitope) in the human CGRP receptor, thereby improving the binding affinity and / or inhibitory potency of the resulting variant anti-CGRP receptor antibodies. See Examples 1 and 5.

[0045] Analysis of the paratope / epitope interface described in Example 5 revealed that the heavy chain variable region, in particular CDRH3, plays an important role in the inhibitory function and selectivity of anti-CGRP receptor antibodies for the CGRP receptor, because paratope residues in the heavy chain interact with amino acids in both the CRLR and RAMP1 polypeptide components of the receptor. Thus, in certain embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a heavy chain variable region comprising complementarity-determining regions CDRH1, CDRH2, and CDRH3, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 47 having a mutation at one or more amino acid positions 28, 30, 31, 32, 54, 56, 57, 58, 59, 60, 102, 105, 107, 111, and / or 113. In such embodiments, the mutations may be selected from T28N, T28K, T28R, T28H, T28F, T28W, T28Y, S30G, S30D, S30M, S31N, S31K, S31R, S31H, S31T, F32Y, D54A, S56E, I57D, K58E, K58D, K58T, Y59H, S60Y, N102D, N102E, D105R, D105E, S107Y, S107F, H111G, K113H, or a combination thereof. In some embodiments, the mutations are selected from T28N, T28K, T28R, T28H, S31N, S31K, S31R, S31H, N102D, N102E, or a combination thereof. In these and other embodiments, the CDRH3 of the anti-CGRP receptor antibody or antigen-binding fragment is greater than 15 amino acids in length, e.g., about 18 to about 25 amino acids in length. As described in Example 5, the potency of anti-CGRP receptor antibodies directly correlates with the length of the CDRH3 region, with greater potency observed for antibodies with longer CDRH3 regions. Without being bound by theory, it is believed that a longer CDRH3 region allows the antibody to effectively bind to a hidden epitope deep within the CRLR / RAMP1 interface. See Figure 7B.

[0046] In certain related embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising complementarity determining regions CDRL1, CDRL2, and CDRL3, wherein the light chain variable region comprises the sequence of SEQ ID NO:23 or SEQ ID NO:24 with a mutation at one or more amino acid positions 26, 31, 32, 33, 53, 54, 56, 57, 94, 95, 96, 97, 98, and / or 100. In some embodiments, the mutations are selected from S26F, S26R, S26Y, N31R, N31I, N31W, N32S, N32Y, N32R, N32K, N32W, Y33T, Y33S, Y33A, Y33P, N53R, N53M, K54W, K54F, K54Y, P56A, S57G, S57R, S57Q, S94Y, S94W, R95Q, R95A, R95W, L96W, L96M, L96T, L96H, L96R, S97K, S97Q, S97T, S97R, A98S, A98V, V100T, V100I, or a combination thereof. In one particular embodiment, the mutation is selected from S26R, S26Y, N31I, N31R, N32K, N32Y, Y33A, Y33S, or a combination thereof.

[0047] The antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the present invention may comprise one or more complementarity-determining regions (CDRs) derived from the light chain variable region and heavy chain variable region of an antibody that specifically binds to the human CGRP receptor as described herein. The term "CDR" refers to a complementarity-determining region (also called a "minimal recognition unit" or "hypervariable region") within an antibody variable sequence. There are three heavy chain variable region CDRs (CDRH1, CDRH2, and CDRH3) and three light chain variable region CDRs (CDRL1, CDRL2, and CDRL3). As used herein, the term "CDR region" refers to a group of three CDRs (i.e., three light chain CDRs or three heavy chain CDRs) present in a single variable region. The CDRs in each of the two chains are typically aligned by framework regions (FRs) to form a structure that specifically binds to a specific epitope or domain on a target protein (e.g., human CGRP receptor). From N-terminus to C-terminus, naturally occurring light and heavy chain variable regions typically have these elements in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system has been devised to assign numbers to the amino acids occupying each position in these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity-determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using this system. Other numbering systems for amino acids in immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0048] In certain embodiments, an antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention that specifically binds to human CGRP receptor comprises at least one light chain variable region comprising CDRL1, CDRL2, and CDRL3, and at least one heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, derived from any of the anti-CGRP receptor antibodies described herein. The light and heavy chain variable regions and associated CDRs of exemplary human anti-CGRP receptor antibodies are set forth below in Tables 2A and 2B, respectively.

[0049] [Table 2]

[0050] [Table 3]

[0051] [Table 4]

[0052] Anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the present invention may comprise one or more of the light chain CDRs (i.e., CDRLs) and / or heavy chain CDRs (i.e., CDRHs) presented in Tables 2A and 2B, respectively. For example, in some embodiments, anti-CGRP receptors, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the present invention comprise CDRL1 comprising a sequence selected from SEQ ID NOs: 5-12; CDRL2 comprising a sequence selected from SEQ ID NOs: 13-16; CDRL3 comprising a sequence selected from SEQ ID NOs: 17-22; CDRH1 comprising a sequence selected from SEQ ID NOs: 35-38; CDRH2 comprising a sequence selected from SEQ ID NOs: 39-42; and CDRH3 comprising a sequence selected from SEQ ID NOs: 44-46.

[0053] In some embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof, of the present invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 6, 13, and 17, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 7, 13, and 17, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 8, 14, and 18, respectively; (e) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: (f) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 10, 13, and 17, respectively; (g) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 11, 15, and 19, respectively; (h) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 11, 16, and 20, respectively; (i) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 12, 13, and 17, respectively; (j) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 21, respectively; or (k) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 22, respectively.In these and other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof, of the invention comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein (a) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (b) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 45, respectively; (c) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 36, 39, and 44, respectively; (d) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 40, and 44, respectively; (e) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 37, 41, and 44, respectively; or (f) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 38, 42, and 46, respectively.

[0054] In certain embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3; (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 6, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 7, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 45, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 8, 14, and 18, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (e) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 9, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (f) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 36, 39, and 44, respectively; (g) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 10, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (h) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 40, and 44, respectively; (i) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 37, 41, and 44, respectively; (j) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 11, 15, and 19, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 38, 42, and 46, respectively; (k) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 11, 16, and 20, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (l) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 12, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (m) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 21, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; or (n) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 22, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively.

[0055] In one embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof, comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 6, 13, and 17, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively. In another embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof, comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 7, 13, and 17, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively. In yet another embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 45, respectively. In yet another embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 8, 14, and 18, respectively, and wherein CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively.In one specific embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof, comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 9, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively.

[0056] In some embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention may comprise CDRs having sequences according to consensus CDR sequences generated from sequence alignments of CDR sequences from anti-CGRP receptor antibodies with enhanced inhibitory potency (see Example 1) or predicted from analysis of the structure of the paratope / epitope interface (see Example 5). For example, in certain embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention comprise a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein CDRH1 comprises a sequence according to the CDRH1 consensus sequence, CDRH2 comprises a sequence according to the CDRH2 consensus sequence, and CDRH3 comprises a sequence according to the CDRH3 consensus sequence. In one embodiment, the CDRH1 consensus sequence is X1FX2X3X4GMH (SEQ ID NO: 471), where X1 is N, K, R, H, F, W, or Y; X2 is S, G, D, or M; X3 is S, T, N, K, R, or H; and X4 is F or Y. In another embodiment, the CDRH1 consensus sequence is X1FSX2FGMH (SEQ ID NO: 472), where X1 is N, K, R, or H and X2 is S, T, N, K, R, or H. In a related embodiment, the CDRH2 consensus sequence is VISFX1GX2X3X4X5X6VDSVKG (SEQ ID NO: 473), where X1 is D or A; X2 is S or E; X3 is I or D; X4 is K, E, T, or D; X5 is Y or H; and X6 is S or Y. In another related embodiment, the CDRH3 consensus sequence is DRLX1YYX2SX3GYYX4YX5YYGMAV (SEQ ID NO: 474), where X1 is N, D, or E; X2 is D, E, or R; X3 is S, Y, or F; X4 is G or H; and X5 is K or H.

[0057] In certain embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 comprises a sequence according to the CDRL1 consensus sequence, CDRL2 comprises a sequence according to the CDRL2 consensus sequence, and CDRL3 comprises a sequence according to the CDRL3 consensus sequence. In such embodiments, the CDRL1 consensus sequence may be SGSX1SNIGX2X3X4VS (SEQ ID NO: 475), where X1 is F, R, Y, or S; X2 is N, R, I, or W; X3 is N, S, Y, R, K, or W; and X4 is Y, T, S, A, or P. In a related embodiment, the CDRL2 consensus sequence is DNX1X2RX3X4 (SEQ ID NO:476), where X1 is N, R, or M; X2 is K, W, F, or Y; X3 is P or A; and X4 is S, G, R, or Q. In yet another related embodiment, the CDRL3 consensus sequence is GTWDX1X2X3X4X5VX6 (SEQ ID NO:477), where X1 is S, Y, or W; X2 is R, Q, A, or W; X3 is L, W, M, T, H, or R; X4 is S, K, Q, T, or R; X5 is A, S, or V; and X6 is V, T, or I.

[0058] In some embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the present invention comprises an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL) derived from an antibody that specifically binds to the human CGRP receptor, such as the antibodies described herein. The term "variable region," used interchangeably herein with "variable domain" (light chain variable region (VL) and heavy chain variable region (VH)), refers to the region in each of the light and heavy immunoglobulin chains that is directly involved in binding the antibody to the antigen. As described above, the variable light and variable heavy chain regions have the same general structure, and each region contains four framework (FR) regions, the sequences of which are widely conserved and connected by three CDRs. The framework regions adopt a β-sheet structure, and the CDRs may form loops connecting the β-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and, together with the CDRs of the other chain, form an antigen-binding site. Thus, in some embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the present invention may comprise a light chain variable region selected from LV-01 to LV-12 as shown in Table 2A, and / or a heavy chain variable region selected from HV-01 to HV-07 as shown in Table 2B, as well as binding fragments, derivatives, and variants of these light and heavy chain variable regions.

[0059] Each of the light chain variable regions listed in Table 2A may be combined with any of the heavy chain variable regions listed in Table 2B to form the anti-CGRP receptor binding domain of an anti-CGRP antibody or antigen-binding fragment thereof of the invention or a bispecific antigen-binding protein of the invention. Examples of such combinations include, but are not limited to, (i) LV-03 and HV-02; (ii) LV-04 and HV-02; (iii) LV-01 and HV-03; (iv) LV-02 and any one of HV-03, HV-04, HV-05, and HV-06; (v) LV-05 and HV-02; (vi) LV-06 and HV-02; (vii) LV-07 and HV-02; (viii) LV-08 and HV-07; (ix) LV-09 and HV-02; (x) LV-10 and HV-02; (xi) LV-11 and HV-02; and (xii) LV-12 and HV-02.

[0060] In certain embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention comprise a light chain variable region comprising the sequence of SEQ ID NO:25 and a heavy chain variable region comprising the sequence of SEQ ID NO:48. In some embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention comprise a light chain variable region comprising the sequence of SEQ ID NO:26 and a heavy chain variable region comprising the sequence of SEQ ID NO:48. In other embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention comprise a light chain variable region comprising the sequence of SEQ ID NO:23 and a heavy chain variable region comprising the sequence of SEQ ID NO:49. In yet other embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention comprise a light chain variable region comprising the sequence of SEQ ID NO:24 and a heavy chain variable region comprising the sequence of SEQ ID NO:49. In some embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention comprise a light chain variable region comprising the sequence of SEQ ID NO:27 and a heavy chain variable region comprising the sequence of SEQ ID NO:48. In certain embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:28 and a heavy chain variable region comprising the sequence of SEQ ID NO:48.

[0061] In one embodiment, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:24 and a heavy chain variable region comprising the sequence of SEQ ID NO:50. In another embodiment, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:29 and a heavy chain variable region comprising the sequence of SEQ ID NO:48. In yet another embodiment, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:24 and a heavy chain variable region comprising the sequence of SEQ ID NO:51. In yet another embodiment, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:24 and a heavy chain variable region comprising the sequence of SEQ ID NO:52. In a particular embodiment, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO:30 and a heavy chain variable region comprising the sequence of SEQ ID NO:53. In another specific embodiment, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 31 and a heavy chain variable region comprising the sequence of SEQ ID NO: 48. In certain embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 32 and a heavy chain variable region comprising the sequence of SEQ ID NO: 48. In some embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 33 and a heavy chain variable region comprising the sequence of SEQ ID NO: 48. In other embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain variable region comprising the sequence of SEQ ID NO: 34 and a heavy chain variable region comprising the sequence of SEQ ID NO: 48.

[0062] In some embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising a sequence of contiguous amino acids that differs by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from a light chain variable region sequence in Table 2A, i.e., a VL selected from LV-01 to LV-12, where each such sequence difference is independently a deletion, insertion, or substitution of a single amino acid, and where the deletion, insertion, and / or substitution results in 15 or fewer amino acid changes relative to the aforementioned variable domain sequence. The light chain variable region in some anti-CGRP receptor antibodies, binding fragments, antigen-binding proteins, or binding domains thereof comprises a sequence of amino acids that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 23-34 (i.e., the light chain variable region in Table 2A).

[0063] In one embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising a sequence at least 90% identical to a sequence selected from SEQ ID NOs: 23-34. In another embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising a sequence at least 95% identical to a sequence selected from SEQ ID NOs: 23-34. In yet another embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising a sequence selected from SEQ ID NOs: 23-34. In some embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising the sequence of SEQ ID NO: 25. In other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising the sequence of SEQ ID NO: 26. In yet other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising the sequence of SEQ ID NO: 24. In still other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising the sequence of SEQ ID NO: 27. In one particular embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising the sequence of SEQ ID NO: 28. In another particular embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain variable region comprising the sequence of SEQ ID NO: 23.

[0064] In these and other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof, comprises a heavy chain variable region comprising a sequence of contiguous amino acids that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from a heavy chain variable region sequence in Table 2B, i.e., a VH selected from HV-01 to HV-07, wherein each such sequence difference is independently a single amino acid deletion, insertion, or substitution, and wherein the deletion, insertion, and / or substitution results in no more than 15 amino acid changes relative to the aforementioned variable domain sequence. The heavy chain variable region of some anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 47 to 53 (i.e., the heavy chain variable regions in Table 2B).

[0065] In one embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a heavy chain variable region comprising a sequence at least 90% identical to a sequence selected from SEQ ID NOs: 48-53. In another embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a heavy chain variable region comprising a sequence at least 95% identical to a sequence selected from SEQ ID NOs: 48-53. In yet another embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a heavy chain variable region comprising a sequence selected from SEQ ID NOs: 48-53. In some embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 48. In other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 49. In yet other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 50. In still other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 51. In certain embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 52. In other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 53.

[0066] The term "identity," as used herein, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity," as used herein, means the percent of identical residues between the amino acids or nucleotides in the compared molecules, and is calculated based on the smallest size of the molecules being compared. For these calculations, gaps in the alignment, if any, must be addressed by a specific mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. For example, sequence identity can be determined by standard methods commonly used to compare the similarity of amino acid positions in two polypeptides.Using a computer program such as BLAST or FASTA, two polypeptide or two polynucleotide sequences are aligned to optimize their respective residue matches (along the entire length of one or both sequences, or along a predetermined portion of one or both sequences). The program provides a default start penalty and a default gap penalty, and a scoring matrix such as PAM 250 (Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3, 1978) or BLOSUM62 (Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) can be used with the computer program. Then, for example, the percent identity can be calculated as follows: the total number of perfect matches is multiplied by 100, and then divided by the sum of the length of the longer sequence in the matched range and the number of gaps introduced into the longer sequence to align the two sequences. In calculating the percent identity, the sequences to be compared are aligned in a way that maximizes the match between the sequences.

[0067] The GCG program package is a computer program that can be used to determine percent identity, and this package includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align two polypeptides or two polynucleotides for which percent sequence identity is to be determined. The sequences are aligned so that their respective amino acids or nucleotides are optimally matched (the "match range" determined by the algorithm). A gap opening penalty (calculated as 3 x average diagonal, where "average diagonal" is the average of the diagonals of the comparison matrix used; "diagonal" is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (usually 1 / 10 of the gap opening penalty), and a comparison matrix such as PAM 250 or BLOSUM 62 are used with the algorithm. In certain embodiments, standard comparison matrices (for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; for the BLOSUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919) are also used by the algorithm.

[0068] Recommended parameters for determining percent identity of polypeptide or nucleotide sequences using the GAP program include the following: Algorithm: Needleman et al. 1970, J. Mol. Biol. 48:443-453; Comparison matrix: BLOSUM 62 from Henikoff et al., 1992 (supra); Gap penalty: 12 (but no penalty for end gaps) Gap length penalty: 4 Similarity threshold: 0

[0069] A particular alignment scheme for aligning two amino acid sequences may result in matching only a short region of the two sequences, and this small aligned region may have very high sequence identity despite the lack of significant relatedness between the two full-length sequences. Therefore, the selected alignment method (GAP program) may be adjusted, if necessary, to produce an alignment spanning at least 50 consecutive amino acids of the target polypeptide.

[0070] The anti-CGRP receptor antibody or antigen-binding protein of the present invention can comprise any immunoglobulin constant region. The term "constant region," used interchangeably herein with "constant domain," refers to all domains of an antibody other than the variable region. The constant region is not directly involved in antigen binding but exhibits various effector functions. As described above, antibodies are divided into specific isotypes (IgA, IgD, IgE, IgG, and IgM) and subtypes (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) depending on the amino acid sequence of the constant region of their heavy chain. The light chain constant region can be, for example, a kappa or lambda light chain constant region, such as the human kappa or lambda light chain constant region found in all five antibody isotypes. Examples of human immunoglobulin light chain constant region amino acid sequences are shown in the table below.

[0071] [Table 5]

[0072] The heavy chain constant region of the anti-CGRP receptor antibody or antigen-binding protein of the invention can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, e.g., a human alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region. In some embodiments, the anti-CGRP receptor antibody or antigen-binding protein comprises a heavy chain constant region derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin, such as a human IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In one embodiment, the anti-CGRP receptor antibody or antigen-binding protein comprises a heavy chain constant region derived from a human IgG1 immunoglobulin. In such embodiments, the human IgG1 immunoglobulin constant region may comprise one or more mutations that prevent glycosylation of the antibody or antigen-binding protein, as described in more detail herein. In another embodiment, the anti-CGRP receptor antibody or antigen-binding protein comprises a heavy chain constant region derived from a human IgG2 immunoglobulin. In yet another embodiment, the anti-CGRP receptor antibody or antigen-binding protein comprises a heavy chain constant region derived from a human IgG4 immunoglobulin. Examples of human IgG1, IgG2, and IgG4 heavy chain constant region amino acid sequences are shown in Table 4 below.

[0073] [Table 6]

[0074] [Table 7]

[0075] Each of the light chain variable regions disclosed in Table 2A and each of the heavy chain variable regions disclosed in Table 2B can be joined to the above-described light chain constant regions (Table 3) and heavy chain constant regions (Table 4) to form complete antibody light and heavy chains, respectively. Furthermore, each of the heavy and light chain sequences thus generated can be combined to form complete antibody structures or bispecific antigen-binding proteins as described in more detail below. It should be understood that the heavy and light chain variable regions provided herein can also be joined to other constant domains having sequences different from the exemplary sequences listed above.

[0076] The anti-CGRP receptor antibody or antigen-binding fragment of the present invention can be any of the anti-CGRP receptor antibodies or antigen-binding fragments disclosed herein. For example, in certain embodiments, the anti-CGRP receptor antibody or antigen-binding fragment is an anti-CGRP receptor antibody or antigen-binding fragment selected from any of the antibodies or antigen-binding fragments thereof listed in Tables 12, 13, and 14. In some embodiments, the anti-CGRP receptor antibody or antigen-binding fragment of the present invention is selected from antibodies 01, 02, 03, 04, 05, 06, 07, 08, 09, 10, 11, 12, 13, 14, and 15 or antigen-binding fragments thereof, with variable region and CDR sequences set forth in Tables 2A and 2B. In some embodiments, the anti-CGRP receptor antibody is an antibody selected from antibodies 01, 02, 03, 04, 05, and 06. The full-length light chain and full-length heavy chain sequences of these exemplary human anti-CGRP receptor antibodies are set forth in Tables 5A and 5B, respectively, below.

[0077] [Table 8]

[0078] [Table 9]

[0079] [Table 10]

[0080]

Table 11

[0081]

Table 12

[0082]

Table 13

[0083]

Table 14

[0084]

Table 15

[0085] Table 16

[0086]

Table 17

[0087]

Table 18

[0088]

Table 19

[0089] Table 20

[0090] Table 21

[0091] Table 22

[0092] Table 23

[0093] Table 24

[0094] Table 25

[0095] Table 26

[0096] Table 27

[0097] Table 28

[0098] Table 29

[0099]

Table 30

[0100] In certain embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the present invention may comprise a light chain selected from LC-01 to LC-16 as shown in Table 5A and / or a heavy chain selected from HC-01 to HC-14 as shown in Table 5B, as well as variants of these light and heavy chains. Each of the light chains listed in Table 5A may be combined with any of the heavy chains listed in Table 5B to form an anti-CGRP receptor antibody or antigen-binding fragment thereof of the present invention, or an anti-CGRP receptor binding domain of a bispecific antigen-binding protein of the present invention. For example, in certain embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the present invention comprise a light chain comprising the sequence of LC-03 (SEQ ID NO:71) and a heavy chain comprising the sequence of HC-02 (SEQ ID NO:86). In some embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the present invention comprise a light chain comprising the sequence of LC-05 (SEQ ID NO:73) and a heavy chain comprising the sequence of HC-02 (SEQ ID NO:86). In other embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain comprising the sequence of LC-07 (SEQ ID NO:75) and a heavy chain comprising the sequence of HC-08 (SEQ ID NO:92). In yet other embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain comprising the sequence of LC-02 (SEQ ID NO:70) and a heavy chain comprising the sequence of HC-08 (SEQ ID NO:92). In some embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain comprising the sequence of LC-09 (SEQ ID NO:77) and a heavy chain comprising the sequence of HC-02 (SEQ ID NO:86). In certain embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain comprising the sequence of LC-10 (SEQ ID NO:78) and a heavy chain comprising the sequence of HC-02 (SEQ ID NO:86). In one embodiment, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain comprising the sequence of LC-02 (SEQ ID NO:70) and a heavy chain comprising the sequence of HC-11 (SEQ ID NO:95).In another embodiment, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain comprising the sequence of LC-11 (SEQ ID NO:79) and a heavy chain comprising the sequence of HC-02 (SEQ ID NO:86). In yet another embodiment, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain comprising the sequence of LC-02 (SEQ ID NO:70) and a heavy chain comprising the sequence of HC-12 (SEQ ID NO:96). In yet another embodiment, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain comprising the sequence of LC-02 (SEQ ID NO:70) and a heavy chain comprising the sequence of HC-13 (SEQ ID NO:97).

[0101] In certain other embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention comprise a light chain comprising the sequence of LC-12 (SEQ ID NO: 80) and a heavy chain comprising the sequence of HC-14 (SEQ ID NO: 98). In some embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention comprise a light chain comprising the sequence of LC-13 (SEQ ID NO: 81) and a heavy chain comprising the sequence of HC-02 (SEQ ID NO: 86). In other embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention comprise a light chain comprising the sequence of LC-14 (SEQ ID NO: 82) and a heavy chain comprising the sequence of HC-02 (SEQ ID NO: 86). In yet other embodiments, anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof of the invention comprise a light chain comprising the sequence of LC-15 (SEQ ID NO: 83) and a heavy chain comprising the sequence of HC-02 (SEQ ID NO: 86). In some embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof of the invention comprises a light chain comprising the sequence of LC-16 (SEQ ID NO: 84) and a heavy chain comprising the sequence of HC-02 (SEQ ID NO: 86).

[0102] In certain embodiments, an anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a light chain comprising a sequence of contiguous amino acids that differs from a light chain sequence in Table 5A, i.e., a light chain selected from LC-01 to LC-16, by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, each such sequence difference independently being a deletion, insertion, or substitution of a single amino acid, and each deletion, insertion, and / or substitution results in 15 or fewer amino acid changes relative to the aforementioned light chain sequence. The light chain in some anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof comprises a sequence of amino acids that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 69-84 (i.e., the light chain in Table 5A).

[0103] In these and other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, antigen-binding protein, or binding domain thereof comprises a heavy chain comprising a sequence of contiguous amino acids that differs by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from a heavy chain sequence in Table 5B, i.e., a heavy chain selected from HC-01 through HC-14, where each such sequence difference is independently a deletion, insertion, or substitution of a single amino acid, and where the deletion, insertion, and / or substitution results in 15 or fewer amino acid changes relative to the aforementioned heavy chain sequence. The heavy chain in some anti-CGRP receptor antibodies, antigen-binding fragments, antigen-binding proteins, or binding domains thereof comprises a sequence of amino acids that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 85-98 (i.e., the heavy chain in Table 5B).

[0104] The anti-CGRP receptor antibody, antigen-binding fragment, or antigen-binding protein of the present invention can be a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment of any of the foregoing. In certain embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, or antigen-binding protein is a monoclonal antibody or an antigen-binding fragment thereof. In such embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, or antigen-binding protein can be a chimeric antibody, a humanized antibody, or a fully human antibody having human immunoglobulin constant domains, or an antigen-binding fragment of any of the foregoing. In these and other embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, or antigen-binding protein is a human IgG1, IgG2, IgG3, or IgG4 antibody or antigen-binding fragment thereof. Thus, the anti-CGRP receptor antibody, antigen-binding fragment, or antigen-binding protein may, in some embodiments, have a human IgG1, IgG2, IgG3, or IgG4 constant domain. In one embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, or antigen-binding protein is a monoclonal human IgG1 antibody or antigen-binding fragment thereof. In another embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, or antigen-binding protein is a monoclonal human IgG2 antibody or antigen-binding fragment thereof. In yet another embodiment, the anti-CGRP receptor antibody, antigen-binding fragment, or antigen-binding protein is a monoclonal human IgG4 antibody or antigen-binding fragment thereof.

[0105] The term "monoclonal antibody" (or "mAb"), as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific and are directed against an individual antigenic site or epitope, in contrast to polyclonal antibody preparations, which usually contain different antibodies directed against different epitopes. Monoclonal antibodies can be produced using any technique known in the art, for example, by immortalizing spleen cells harvested from an animal after completion of an immunization schedule. Spleen cells can be immortalized using any technique known in the art, for example, by fusing spleen cells with myeloma cells to generate hybridomas. See, e.g., Antibodies; Harlow and Lane, Cold Spring Harbor Laboratory Press, 1st Edition (e.g., from 1988) or 2nd Edition (e.g., from 2014). Myeloma cells for use in hybridoma-producing fusion procedures are preferably non-antibody-producing, have high fusion efficiency, and possess enzyme deficiencies that prevent their growth in specific selective media that support the growth of only the desired fused cells (hybridomas). Examples of cell lines suitable for use in fusion with mouse cells include, but are not limited to, Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag41, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG1.7, and S194 / 5XXO B1. Examples of cell lines suitable for use in fusion with rat cells include, but are not limited to, R210.RCY3, Y3-Ag1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0106] In some examples, hybridoma cell lines are produced by immunizing an animal (e.g., a rabbit, rat, mouse, or transgenic animal having a human immunoglobulin sequence) with a CGRP receptor immunogen (see, e.g., WO 2010 / 075238); harvesting spleen cells from the immunized animal; fusing the harvested spleen cells with a myeloma cell line to thereby produce hybridoma cells; establishing hybridoma cell lines from the hybridoma cells, and identifying hybridoma cell lines that produce antibodies that bind to CGRP receptors. Another useful method for producing monoclonal antibodies is the SLAM method described in Babcook et al., Proc. Natl. Acad. Sci. USA, Vol. 93: 7843-7848, 1996.

[0107] Monoclonal antibodies secreted by hybridoma cell lines can be purified using any technique known in the art, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Hybridoma supernatants or mAbs can be further screened to identify mAbs with specific properties, such as the ability to bind to a CGRP receptor (e.g., a human CGRP receptor, a cynomolgus monkey CGRP receptor, or a rat CGRP receptor); cross-reactivity with other calcitonin receptor family members (e.g., human adrenomedullin or human amylin receptor); the ability to block or interfere with the binding of a CGRP ligand to a CGRP receptor, or the ability to functionally block CGRP-induced activation of a CGRP receptor, for example, using a cAMP assay as described herein.

[0108] In some embodiments, the anti-CGRP receptor antibodies, antigen-binding fragments, or antigen-binding proteins of the present invention are chimeric or humanized antibodies or antigen-binding fragments thereof based on the CDR and variable region sequences of the antibodies described herein. Chimeric antibodies are antibodies composed of protein segments from different antibodies covalently linked to generate functional immunoglobulin light or heavy chains or binding fragments thereof. Generally, portions of such heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass. For methods relating to chimeric antibodies, see, e.g., U.S. Pat. No. 4,816,567 and Morrison et al., 1985, Proc. Natl. Acad. Sci. USA 81:6851-6855, both of which are incorporated herein by reference in their entireties.

[0109] Generally, the goal in creating chimeric antibodies is to create chimeras that maximize the number of amino acids from the intended species or germline genes. One example is a "CDR-grafted" antibody, in which the antibody contains one or more CDRs derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chain is identical or homologous to corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass. CDR-grafting is described, for example, in U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101. For use in humans, variable regions or selected CDRs from rodent or rabbit antibodies are often grafted into human antibodies to replace the naturally occurring variable regions or CDRs of the human antibody. In some embodiments, variable regions or selected CDRs from a human antibody can be grafted into another human antibody from a different antibody class or subclass.

[0110] One useful form of chimeric antibody is a "humanized" antibody. Generally, humanized antibodies are generated from monoclonal antibodies initially produced in non-human animals such as rodents or rabbits. Certain amino acid residues in the monoclonal antibody are modified to become homologous to corresponding residues in a human antibody of the corresponding isotype; these modified amino acid residues typically originate from non-antigen-recognizing portions of the antibody. Humanization can be achieved, for example, by substituting at least a portion of a rodent or rabbit variable region for the corresponding region of a human antibody using various methods (see, e.g., U.S. Pat. Nos. 5,585,089 and 5,693,762; Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-1536).

[0111] In one aspect, the CDRs of the light and heavy chain variable regions of the antibodies provided herein (see Tables 2A, 2B, 6A, and 6B) are grafted onto the framework regions (FRs) of an antibody from the same or a different phylogenetic species. For example, the CDRs of the heavy and light chain variable regions listed in Tables 2A, 2B, 6A, and 6B can be grafted onto consensus human FRs or FRs from other human germline genes. To create consensus human FRs, FRs from several human heavy or light chain amino acid sequences can be aligned to identify a consensus amino acid sequence. Alternatively, the grafted variable regions from one heavy or light chain can be used with a constant region that is different from the constant region of that particular heavy or light chain as disclosed herein. In other embodiments, the grafted variable regions are part of a single-chain Fv antibody.

[0112] In certain embodiments, the anti-CGRP receptor antibody, antigen-binding fragment, or antigen-binding protein of the present invention is a fully human antibody or antigen-binding fragment thereof. Fully human antibodies that specifically bind to human CGRP receptors can be produced using immunogens described in WO 2010 / 075238, such as a polypeptide consisting of any one of the sequences set forth in SEQ ID NOS: 1-4, or fragments thereof. A "fully human antibody" is an antibody comprising variable and constant regions derived from or representing human germline immunoglobulin sequences. One specific means provided for achieving the production of fully human antibodies is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated is one means for producing fully human monoclonal antibodies (mAbs) in mice, animals that can be immunized with any desired antigen. The use of fully human antibodies can minimize the immunogenic and allergic responses that can result from administering mouse mAbs or mouse-derived mAbs to humans as therapeutic agents.

[0113] Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that lack endogenous immunoglobulin production and have the capacity to produce a repertoire of human antibodies. Antigens of interest typically contain six or more consecutive amino acids and are optionally conjugated to a carrier such as a hapten. See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551-2555; Jakobovits et al., 1993, Nature 362:255-258; and Bruggermann et al., 1993, Year in Immunol. 7:33. In one example of such a method, transgenic animals are generated by disabling endogenous mouse immunoglobulin loci encoding mouse heavy and light immunoglobulin chains and inserting human genomic DNA containing loci encoding human heavy and light chain proteins into a large fragment of the mouse genome. Partially modified animals with less than the full complement of human immunoglobulin loci are then crossbred to obtain animals with all the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies immunospecific for the immunogen, but with human rather than mouse amino acid sequences comprising the variable regions. For further details of such methods, see, e.g., WO 96 / 33735 and WO 94 / 02602.Additional methods relating to transgenic mice for producing human antibodies are described in U.S. Patent Nos. 5,545,807, 6,713,610, 6,673,986, 6,162,963, 5,939,598, 5,545,807, 6,300,129, 6,255,458, 5,877,397, 5,610, and 5,713,610. ,874,299 and 5,545,806, PCT publications WO 91 / 10741, WO 90 / 04036, WO 94 / 02602, WO 96 / 30498, WO 98 / 24893, and European Patent No. 546073B1 and European Patent Application Publication No. 546073A1.

[0114] These transgenic mice, called "HuMab" mice, contain a human immunoglobulin gene minilocus encoding unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous mu and kappa chain loci (Lonberg et al., 1994, Nature 368:856-859). Thus, the mice exhibit reduced expression of mouse IgM and kappa proteins, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgG kappa monoclonal antibodies (Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995, Ann. NY Acad. Sci. 764:536-546). HuMab mice were created by Taylor et al.,1992, Nucleic Acids Research 20:6287-6295;Chen et al.,1993,International Immunology 5:647-656;Tuaillon et al.,1994,J.Immunol.152:2912-2920;Lonberg et al.,1994,Nature 368:856-859;Lonberg,1994,Handbook of Exp.Pharmacology 113:49-101;Taylor et al.,1994,International Immunology 6:579-591;Lonberg and Huszar,1995,Intern.Rev.Immunol.13:65-93;Harding and Lonberg, 1995, Ann.NY Acad. Sci. 764:536-546; Fishwild et al., 1996, Nature Biotechnology 14:845-851, which are incorporated herein by reference in their entireties.See also U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, as well as U.S. Patent No. 5,545,807, WO 93 / 1227, WO 92 / 22646, and WO 92 / 03918, the disclosures of all of which are incorporated herein by reference in their entireties. The techniques utilized for producing human antibodies in these transgenic mice are also disclosed in WO 98 / 24893 and Mendez et al., 1997, Nature Genetics 15:146-156, which are incorporated herein by reference. For example, the HCo7 and HCo12 transgenic mouse strains can be used to generate fully human anti-CGRP receptor antibodies. One particular transgenic mouse strain suitable for producing fully human anti-CGRP receptor antibodies is described in U.S. Pat. Nos. 6,114,598; 6,162,963; 6,833,268; 7,049,426; 7,064,244; Green et al., 1994, Nature Genetics 7:13-21; Mendez et al., 1997, Nature Genetics 15:146-156; Green and Jakobovitis, 1998, J. Ex. Med, 188:483-495; Green, 1999, Journal of Immunological Methods 231:11-23; Kellerman and Green, 2002, Current Opinion in Biotechnology 13,593-597, all of which are incorporated herein by reference in their entireties.

[0115] Human-derived antibodies can also be generated using phage display technology. Phage display is described, for example, in WO 91 / 17271 by Dower et al., WO 92 / 01047 by McCafferty et al., and Caton and Koprowski, 1990, Proc. Natl. Acad. Sci. USA, 87:6450-6454, each of which is incorporated herein by reference in its entirety. Antibodies generated by phage technology are typically produced in bacteria as antigen-binding fragments, such as Fv or Fab fragments, and therefore lack effector function. Effector function can be introduced by one of two strategies: the fragment can be engineered into a complete antibody expressed in mammalian cells, as needed, or into a bispecific antibody fragment with a second binding site capable of eliciting effector function. Typically, antibody Fd fragments (VH-CH1) and light chains (VL-CL) are cloned separately by PCR and randomly recombined in combinatorial phage display libraries, which can then be selected for specific antigen binding. Antibody fragments are expressed on the surface of phage, and selection of Fv or Fab fragments (and thus phage containing DNA encoding antibody fragments) based on antigen binding is achieved by several rounds of antigen binding and reamplification, a procedure called panning. Antigen-specific antibody fragments are enriched and ultimately isolated. Phage display technology can also be used in an approach for humanizing rodent monoclonal antibodies called "guided selection" (see Jespers, LS, et al., 1994, Bio / Technology 12, 899-903). For this purpose, Fd fragments of mouse monoclonal antibodies can be displayed in combination with a human light chain library, and the resulting hybrid Fab library can then be selected using the antigen. The mouse Fd fragments then provide a template to guide the selection. The selected human light chains are then combined with a human Fd fragment library, and selection of the resulting library yields fully human Fabs.

[0116] Once cells producing an anti-CGRP receptor antibody according to the invention have been obtained using any of the above immunization and other techniques, the specific antibody gene may be cloned by isolating and amplifying the DNA or mRNA therefrom according to standard procedures as described herein. The antibody produced may then be sequenced, the CDRs identified, and the DNA encoding the CDRs manipulated as described herein to generate other anti-CGRP receptor antibodies, antigen-binding fragments, or antigen-binding proteins according to the invention.

[0117] Any of the anti-CGRP receptor antibodies or antigen-binding fragments thereof described herein can be used to construct bispecific antigen-binding proteins that can bind to and inhibit separate targets, such as the human CGRP receptor and the human PAC1 receptor. As used herein, the term "antigen-binding protein" refers to a protein that specifically binds to one or more target antigens. Antigen-binding proteins can include antibodies and antigen-binding fragments thereof. Antigen-binding proteins can also include proteins that contain one or more antigen-binding fragments incorporated into a single polypeptide chain or multiple polypeptide chains. For example, antigen-binding proteins can be bispecific antibodies (see, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, Vol. 90:6444-6448, 1993); intrabodies; domain antibodies (a single VL or VH domain, or two or more VH domains joined by a peptide linker; see Ward et al., Nature, Vol. 341:544-546, 1989); maxibodies (two scFvs fused to an Fc region; Fredericks et al., Protein Engineering, Design & Selection, Vol. 17:95-106, 2004; and Powers et al., Journal of Immunological minibodies (scFv fused to a CH3 domain; see Olafsen et al., Protein Eng Des Sel., Vol. 17:315-23, 2004); peptibodies (one or more peptides attached to an Fc region or antibody; see WO 00 / 24782); linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with complementary light chain polypeptides; see Zapata et al., Protein Eng Des Sel., Vol. 251:123-135, 2001); triabodies; tetrabodies; Eng., Vol. 8:1057-1062, 1995); small modular immunopharmaceuticals (see U.S. Patent Publication No. 20030133939); and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0118] In certain embodiments, the antigen-binding proteins of the invention are "bispecific," meaning that they can specifically bind to two different antigens, separate target antigens, such as human CGRP receptor and human PAC1 receptor. In some embodiments of the invention, the antigen-binding proteins are multivalent. The valency of a binding protein indicates the number of individual antigen-binding domains within the binding protein. For example, the terms "monovalent," "bivalent," and "tetravalent" with respect to antigen-binding proteins of the invention refer to binding proteins having one, two, and four antigen-binding domains, respectively. Thus, a multivalent antigen-binding protein comprises two or more antigen-binding domains. In certain embodiments, the bispecific antigen-binding proteins of the invention are bivalent. Thus, such bispecific, bivalent antigen-binding proteins contain two antigen-binding domains: one antigen-binding domain that binds to human CGRP receptor and one antigen-binding domain that binds to another target antigen, such as human PAC1 receptor.

[0119] As used herein, the term "antigen-binding domain," which is used interchangeably with "binding domain," refers to a region of an antigen-binding protein that contains amino acid residues that interact with an antigen and confer specificity and affinity to the antigen-binding protein for that antigen. In certain embodiments, the binding domain of an antigen-binding protein of the invention may be derived from an antibody or an antigen-binding fragment thereof. For example, the binding domain of a bispecific antigen-binding protein of the invention may comprise one or more complementarity-determining regions (CDRs) derived from the light and heavy chain variable regions of an antibody that specifically binds to the human CGRP receptor or the human PAC1 receptor. In some embodiments, the anti-CGRP receptor-binding domain of a bispecific antigen-binding protein of the invention comprises all six CDRs of the heavy and light chain variable regions of an anti-CGRP receptor antibody described herein, and the anti-PAC1 receptor-binding domain of a bispecific antigen-binding protein of the invention comprises all six CDRs of the heavy and light chain variable regions of an anti-PAC1 receptor antibody described herein. In some embodiments, the binding domains (anti-CGRP receptor binding domain, anti-PAC1 receptor binding domain, or both) of the bispecific antigen-binding proteins of the invention comprise a Fab, a Fab', a F(ab')2, an Fv, a single-chain variable fragment (scFv), or a nanobody. In one embodiment, both binding domains are Fab fragments. In another embodiment, one binding domain is a Fab fragment and the other binding domain is an scFv. In yet another embodiment, both binding domains are scFvs.

[0120] Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, which contains all but the first domain of the immunoglobulin heavy chain constant region. The Fab fragment contains the variable domains from the light and heavy chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" is composed of one immunoglobulin light chain (light chain variable region (VL) and constant region (CL)) and the CH1 domain and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fd fragment" contains the VH and CH1 domains from an immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of a Fab fragment.

[0121] An "Fc fragment" or "Fc region" of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. In certain embodiments, the antigen-binding proteins of the present invention comprise an Fc region derived from an immunoglobulin. The Fc region may be an Fc region derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region comprises the CH2 and CH3 domains derived from a human IgG1 or human IgG2 immunoglobulin. The Fc region may retain effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc region may be modified to reduce or eliminate effector function, as described in more detail herein.

[0122] A "Fab' fragment" is a Fab fragment that has one or more cysteine ​​residues from the antibody hinge region at the C-terminus of the CH1 domain.

[0123] A "F(ab')2 fragment" is a bivalent fragment containing two Fab' fragments linked by inter-heavy chain disulfide bridges at the hinge region.

[0124] An "Fv" fragment is the minimum fragment containing a complete antigen-recognition and binding site derived from an antibody. This fragment consists of a dimer of one immunoglobulin heavy chain variable region (VH) and one immunoglobulin light chain variable region (VL) in tight, non-covalent association. In this configuration, the three CDRs of each variable region interact to define an antigen-binding site on the surface of the VH-VL dimer. A single light or heavy chain variable region (or half of an Fv fragment containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the complete binding site containing both VH and VL.

[0125] A "single-chain variable fragment" or "scFv fragment" comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain, and optionally contain a peptide linker between the VH and VL domains which enables the Fv to form the desired structure for antigen binding (see, e.g., Bird et al., Science, Vol. 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85:5879-5883, 1988).

[0126] A "nanobody" is the heavy chain variable region of a heavy chain antibody. Such a variable domain is the smallest fully functional antigen-binding fragment of such a heavy chain antibody, with a molecular mass of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy chain antibodies lacking light chains naturally occur in certain species of animals, such as nurse sharks, Alabarea dwarf sharks, and camelids, including camels, dromedaries, alpacas, and llamas. The antigen-binding site in these animals is reduced to a single domain, the VHH domain. These antibodies use only the heavy chain variable region to form the antigen-binding region, i.e., these functional antibodies are heavy chain homodimers with only the structure H2L2 (also called "heavy chain antibodies" or "HCAbs"). Camelized VHHs reportedly contain hinge, CH2, and CH3 domains and are recombined with IgG2 and IgG3 constant regions lacking the CH1 domain. Camelized VHH domains have been found to bind antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276:26285-90, 2001) and have high stability in solution (Ewert et al., Biochemistry, Vol. 41:3628-36, 2002). Methods for generating antibodies with camelized heavy chains are described, for example, in U.S. Patent Application Publication Nos. 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds may be made from human variable-like domains that more closely match the shark V-NAR scaffold and may provide a framework for long transmembrane loop structures.

[0127] In certain embodiments, the binding domain of the bispecific antigen-binding protein of the invention comprises the immunoglobulin heavy chain variable region (VH) and light chain variable region (VL) of an antibody or antibody fragment that specifically binds to the desired antigen. For example, the anti-CGRP receptor binding domain of the bispecific antigen-binding protein of the invention comprises the VH and VL regions derived from an anti-CGRP receptor antibody, such as any of the anti-CGRP receptor antibodies described herein, and the anti-PAC1 receptor binding domain comprises the VH and VL regions derived from an anti-PAC1 receptor antibody, such as any of the anti-PAC1 receptor antibodies described herein. Binding domains that specifically bind to human CGRP receptor or human PAC1 receptor may be derived from known antibodies against these antigens or novel antibodies or antibody fragments obtained by novel immunization methods using antigenic proteins or fragments thereof, phage display, or other methods described herein or known in the art. The antibody from which the binding domain for the bispecific antigen-binding protein is derived may be a monoclonal antibody, a recombinant antibody, a human antibody, or a humanized antibody. In certain embodiments, the antibody from which the binding domain is derived is a monoclonal antibody. In these and other embodiments, the antibody is a human or humanized antibody, and can be of the IgG1, IgG2, IgG3, or IgG4 type.

[0128] The bispecific antigen-binding proteins of the present invention comprise a binding domain that specifically binds to the human CGRP receptor. In certain embodiments, the anti-CGRP receptor binding domain of the bispecific antigen-binding proteins of the present invention comprises a VH region and / or a VL region or a CDR region derived from an anti-CGRP receptor antibody or an antigen-binding fragment thereof. Preferably, the anti-CGRP receptor antibody or antigen-binding fragment thereof specifically binds to the human CGRP receptor and prevents or reduces receptor binding to CGRP. In some embodiments, the anti-CGRP receptor antibody or antigen-binding fragment thereof from which the anti-CGRP receptor binding domain is derived specifically binds to a residue or sequence of residues, or a region in both the human CRLR and human RAMP1 polypeptides. In one embodiment, the anti-CGRP receptor antibody or antigen-binding fragment thereof specifically binds to an epitope formed from amino acids in both the human CRLR and human RAMP1 polypeptides (e.g., SEQ ID NOs: 1 and 2, respectively). In another embodiment, the anti-CGRP receptor antibody or antigen-binding fragment thereof specifically binds to an epitope formed from amino acids in the extracellular domains of both the human CRLR and human RAMP1 polypeptides (e.g., SEQ ID NOs: 3 and 4, respectively). In some embodiments, the epitope formed from amino acids in both the human CRLR and human RAMP1 polypeptides contains one or more cleavage sites for the AspN protease, which cleaves the peptide after an aspartic acid residue and several glutamic acid residues at the amino terminus. In certain embodiments, the anti-CGRP receptor antibody or antigen-binding fragment thereof from which the anti-CGRP receptor binding domain is derived specifically binds to the extracellular domain of a human CRLR polypeptide comprising the amino acid sequence of SEQ ID NO:3 and / or the extracellular domain of a human RAMP1 polypeptide comprising the amino acid sequence of SEQ ID NO:4.

[0129] The variable regions or CDR regions of any of the anti-CGRP receptor antibodies or antigen-binding fragments described herein can be used to construct the anti-CGRP receptor binding domain of the bispecific antigen-binding proteins of the invention. As described in the Examples, the anti-CGRP receptor antibodies of the invention have enhanced inhibitory potency compared to previously described anti-CGRP receptor antibodies, such as the antibodies described in WO 2010 / 075238. The light and heavy chain variable regions and associated CDRs of exemplary human anti-CGRP receptor antibodies from which the anti-CGRP receptor binding domain of the bispecific antigen-binding proteins of the invention may be derived or constructed are set forth in Tables 2A and 2B, respectively.

[0130] The anti-CGRP receptor binding domain of the bispecific antigen-binding protein may comprise one or more of the CDRs set forth in Table 2A (light chain CDRs; i.e., CDRLs) and Table 2B (heavy chain CDRs, i.e., CDRHs). For example, in certain embodiments, the anti-CGRP receptor binding domain comprises one or more light chain CDRs selected from (i) a CDRL1 selected from SEQ ID NOs: 5-12, (ii) a CDRL2 selected from SEQ ID NOs: 13-16, and (iii) a CDRL3 selected from SEQ ID NOs: 17-22. In these and other embodiments, the anti-CGRP receptor binding domain comprises one or more heavy chain CDRs selected from (i) a CDRH1 selected from SEQ ID NOs: 35-38, (ii) a CDRH2 selected from SEQ ID NOs: 39-42, and (iii) a CDRH3 selected from SEQ ID NOs: 44-46.

[0131] In certain embodiments, the anti-CGRP receptor binding domain of the bispecific antigen binding protein of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 6, 13, and 17, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 7, 13, and 17, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 8, 14, and 18, respectively; (e) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 9, 13, and 18, respectively. and 17; (f) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 10, 13, and 17, respectively; (g) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 11, 15, and 19, respectively; (h) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 11, 16, and 20, respectively; (i) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 12, 13, and 17, respectively; (j) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 21, respectively; or (k) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 22, respectively.

[0132] In other specific embodiments, the anti-CGRP receptor binding domain of the bispecific antigen binding protein of the invention comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein (a) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (b) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 45, respectively; (c) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 36, 39, and 44, respectively; (d) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 40, and 44, respectively; (e) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 37, 41, and 44, respectively; or (f) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 38, 42, and 46, respectively.

[0133] In certain embodiments, the anti-CGRP receptor binding domain of the bispecific antigen binding protein of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3 and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3; (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 6, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 7, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 45, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 8, 14, and 18, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (e) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 9, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (f) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 36, 39, and 44, respectively; (g) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 10, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (h) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 40, and 44, respectively; (i) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 37, 41, and 44, respectively; (j) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 11, 15, and 19, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 38, 42, and 46, respectively; (k) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 11, 16, and 20, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (l) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 12, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (m) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 21, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; or (n) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 22, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively.

[0134] In some embodiments, the anti-CGRP receptor binding domain of the bispecific antigen binding protein of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3 and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3; (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 6, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 7, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 5, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 45, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 8, 14, and 18, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively; or (e) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 9, 13, and 17, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 35, 39, and 44, respectively.

[0135] The anti-CGRP receptor binding domain of the bispecific antigen-binding proteins of the invention may comprise a light chain variable region selected from the group consisting of LV-01, LV-02, LV-03, LV-04, LV-05, LV-06, LV-07, LV-08, LV-09, LV-10, LV-11, and LV-12 as shown in Table 2A, and / or a heavy chain variable region selected from the group consisting of HV-01, HV-02, HV-03, HV-04, HV-05, HV-06, and HV-07 as shown in Table 2B, and antigen-binding fragments, derivatives, muteins, and variants of these light and heavy chain variable regions. Each of the light chain variable regions listed in Table 2A may be combined with any of the heavy chain variable regions shown in Table 2B to form an anti-CGRP receptor binding domain suitable for incorporation into a bispecific antigen-binding protein of the invention.For example, in certain embodiments, the anti-CGRP receptor binding domain comprises a light chain variable region and a heavy chain variable region, and (a) the light chain variable region comprises the sequence of SEQ ID NO:25, and the heavy chain variable region comprises the sequence of SEQ ID NO:48; (b) the light chain variable region comprises the sequence of SEQ ID NO:26, and the heavy chain variable region comprises the sequence of SEQ ID NO:48; (c) the light chain variable region comprises the sequence of SEQ ID NO:23, and the heavy chain variable region comprises the sequence of SEQ ID NO:49; (d) the light chain variable region comprises the sequence of SEQ ID NO:24, and the heavy chain variable region comprises the sequence of SEQ ID NO:49; (e) the light chain variable region comprises the sequence of SEQ ID NO:27, and the heavy chain variable region comprises the sequence of SEQ ID NO:48; (f) the light chain variable region comprises the sequence of SEQ ID NO:28, and the heavy chain variable region comprises the sequence of SEQ ID NO:48; or (g) the light chain variable region comprises the sequence of SEQ ID NO:24, and the heavy chain variable region comprises the sequence of SEQ ID NO:50. (h) the light chain variable region comprises the sequence of SEQ ID NO:29 and the heavy chain variable region comprises the sequence of SEQ ID NO:48; (i) the light chain variable region comprises the sequence of SEQ ID NO:24 and the heavy chain variable region comprises the sequence of SEQ ID NO:51; (j) the light chain variable region comprises the sequence of SEQ ID NO:24 and the heavy chain variable region comprises the sequence of SEQ ID NO:52; (k) the light chain variable region comprises the sequence of SEQ ID NO:30 and the heavy chain variable region comprises the sequence of SEQ ID NO:53; (l) the light chain variable region comprises the sequence of SEQ ID NO: 31 and the heavy chain variable region comprises the sequence of SEQ ID NO: 48; (m) the light chain variable region comprises the sequence of SEQ ID NO: 32 and the heavy chain variable region comprises the sequence of SEQ ID NO: 48; (n) the light chain variable region comprises the sequence of SEQ ID NO: 33 and the heavy chain variable region comprises the sequence of SEQ ID NO: 48; or (o) the light chain variable region comprises the sequence of SEQ ID NO: 34 and the heavy chain variable region comprises the sequence of SEQ ID NO: 48.

[0136] In some embodiments, the anti-CGRP receptor binding domain comprises a light chain variable region comprising a sequence of contiguous amino acids that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from a light chain variable region sequence in Table 2A, i.e., a VL selected from LV-01, LV-02, LV-03, LV-04, LV-05, LV-06, LV-07, LV-08, LV-09, LV-10, LV-11, and LV-12, wherein each such sequence difference is independently a deletion, insertion, or substitution of a single amino acid, and wherein the deletion, insertion, and / or substitution results in no more than 15 amino acid changes relative to the aforementioned variable domain sequence. The light chain variable region in some CGRP receptor binding domains comprises a sequence of amino acids having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 23-34 (i.e., the light chain variable regions in Table 2A). In one embodiment, the anti-CGRP receptor binding domain comprises a light chain variable region comprising a sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 23-34. In another embodiment, the anti-CGRP receptor binding domain comprises a light chain variable region comprising a sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 23-34.

[0137] In these and other embodiments, the anti-CGRP receptor binding domain comprises a heavy chain variable region comprising a sequence of contiguous amino acids that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from a heavy chain variable region sequence in Table 2B, i.e., a VH selected from HV-01, HV-02, HV-03, HV-04, HV-05, HV-06, and HV-07, wherein each such sequence difference is independently a deletion, insertion, or substitution of a single amino acid, and wherein the deletion, insertion, and / or substitution results in no more than 15 amino acid changes relative to the aforementioned variable domain sequence. The heavy chain variable region in some anti-CGRP receptor binding domains comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 47-53 (i.e., the heavy chain variable region in Table 2B). In one embodiment, the anti-CGRP receptor binding domain comprises a heavy chain variable region comprising a sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 48-53. In another embodiment, the anti-CGRP receptor binding domain comprises a heavy chain variable region comprising a sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 48-53.

[0138] In certain embodiments, the bispecific antigen binding proteins of the invention comprise a binding domain that specifically binds to the human pituitary adenylate cyclase-activating polypeptide type I (PAC1) receptor. In such embodiments, the bispecific antigen binding protein comprises a first binding domain that specifically binds to the human CGRP receptor and a second binding domain that specifically binds to the human PAC1 receptor. Because both CGRP receptor and PAC1 receptor signaling have been implicated in the control of cerebrovascular tone, the bispecific binding proteins of the invention provide a means of simultaneously modulating both signaling cascades to ameliorate conditions associated with dysregulation of the cranial vasculature, such as cluster headache and migraine.

[0139] Human PAC1 is a 468 amino acid protein (NCBI Reference Sequence NP_001109.2) encoded by the ADCYAP1R1 gene on chromosome 7. The human PAC1 receptor is a G protein-coupled receptor that actively couples to adenylate cyclase. Activation of the human PAC1 receptor by endogenous ligands (e.g., PACAP38 or PACAP27) increases intracellular cyclic AMP (cAMP). The amino acid sequence for human PAC1 is shown below as SEQ ID NO: 129. [ka]

[0140] Amino acids 1-23 of the human PAC1 protein (SEQ ID NO: 129) constitute a signal peptide that is generally removed from the mature protein. The mature human PAC1 protein has the basic structure of a G protein-coupled receptor, consisting of seven transmembrane domains, an extracellular domain composed of an N-terminal region and three extracellular loops, three intracellular loops, and a C-terminal cytoplasmic domain. The N-terminal extracellular domain is located at approximately amino acids 24-153 of SEQ ID NO: 129, and the beginning of the seven transmembrane domain begins at amino acid 154 of SEQ ID NO: 129. The C-terminal cytoplasmic domain is located at approximately amino acids 397-468 of SEQ ID NO: 129. For the location of domains within the amino acid sequence, see Blechman and Levkowitz, Front. Endocrinol., Vol. 4(55):1-19, 2013. The terms "human PAC1," "human PAC1 receptor," "hPAC1," and "hPAC1 receptor" are used interchangeably and may refer to the polypeptide of SEQ ID NO: 129, the polypeptide of SEQ ID NO: 129 minus the signal peptide (amino acids 1-23), an allelic variant of the human PAC1 receptor, or a splice variant of the human PAC1 receptor.

[0141] In certain embodiments, the anti-PAC1 binding domain of the bispecific antigen-binding protein of the invention comprises a VH region and / or a VL region or a CDR region derived from an anti-PAC1 receptor antibody or antigen-binding fragment thereof. Preferably, the anti-PAC1 receptor antibody or antigen-binding fragment specifically binds to the human PAC1 receptor and prevents or reduces binding of the receptor to its ligands, such as PACAP-38 and / or PACAP-27. In some embodiments, the anti-PAC1 receptor antibody or antigen-binding fragment specifically binds to the extracellular region of the human PAC1 receptor. In one embodiment, the anti-PAC1 receptor antibody or antigen-binding fragment specifically binds to the amino-terminal extracellular domain of the PAC1 receptor (i.e., amino acids 24-153 of SEQ ID NO: 129). In certain embodiments, the anti-PAC1 antibody or antigen-binding fragment from which the anti-PAC1 binding domain of the bispecific antigen-binding protein of the invention is derived has a denaturing activity of ≦1×10 as measured by a surface plasmon resonance assay (e.g., a BIAcore®-based assay). -9 M, ≤ 1 × 10 -10 M, ≤ 1 × 10 -11 M or lower K D It binds to the human PAC1 receptor.

[0142] In some embodiments, the anti-PAC1 antibody or antigen-binding fragment from which the anti-PAC1 binding domain of the bispecific antigen-binding protein of the invention is derived selectively inhibits the human PAC1 receptor relative to the human VPAC1 and human VPAC2 receptors. As described above, the selective inhibition of any particular antibody, antigen-binding fragment, or antigen-binding protein can be determined by comparing the IC50 of the antibody, antigen-binding fragment, or antigen-binding protein in an inhibition assay for a particular receptor (e.g., the human PAC1 receptor) to the IC50 of the antibody, antigen-binding fragment, or antigen-binding protein in an inhibition assay for another "reference" receptor (e.g., the human VPAC1 or human VPAC2 receptor). The IC50 value for any anti-PAC1 antibody, antigen-binding fragment, or antigen-binding protein can be calculated as described herein, for example, by determining the concentration of the antibody, antigen-binding fragment, or antigen-binding protein required to inhibit half of the maximal biological response of the PACAP ligand (PACAP-27 or PACAP-38) in activating the human PAC1 receptor in any functional assay, such as the cAMP assay described in the Examples. An anti-PAC1 receptor antigen-binding protein, antibody, or binding fragment that inhibits ligand-induced (e.g., PACAP-induced) activation of the PAC1 receptor is understood to be a neutralizing or antagonist antigen-binding protein, antibody, or binding fragment of the PAC1 receptor.

[0143] The variable regions or CDR regions of any anti-PAC1 receptor antibody or antigen-binding fragment thereof, such as the antibodies and binding fragments described herein, can be used to construct the anti-PAC1 binding domain of the bispecific antigen-binding proteins of the invention. The light and heavy chain variable regions and associated CDRs of exemplary human anti-PAC1 receptor antibodies from which the anti-PAC1 binding domain of the bispecific antigen-binding proteins of the invention may be derived or constructed are set forth below in Tables 6A and 6B, respectively.

[0144] [Table 31]

[0145] [Table 32]

[0146] [Table 33]

[0147] [Table 34]

[0148] [Table 35]

[0149] [Table 36]

[0150] [Table 37]

[0151] The anti-PAC1 receptor binding domain of the bispecific antigen-binding protein may comprise one or more of the CDRs set forth in Table 6A (light chain CDRs; i.e., CDRLs) and Table 6B (heavy chain CDRs, i.e., CDRHs). For example, in certain embodiments, the anti-PAC1 receptor binding domain comprises one or more light chain CDRs selected from (i) a CDRL1 selected from SEQ ID NOs: 130-140, (ii) a CDRL2 having the sequence of SEQ ID NO: 141, and (iii) a CDRL3 selected from SEQ ID NOs: 142-145. In these and other embodiments, the anti-PAC1 receptor binding domain comprises one or more heavy chain CDRs selected from (i) a CDRH1 selected from SEQ ID NOs: 157-163, (ii) a CDRH2 selected from SEQ ID NOs: 164-194, and (iii) a CDRH3 selected from SEQ ID NOs: 195-198.

[0152] In some embodiments, the anti-PAC1 receptor binding domain of the bispecific antigen binding protein of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 133, 141, and 142, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 134, 141, and 142, respectively; (e) CDRL1, CDRL2 (f) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 136, 141, and 143, respectively; (g) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 137, 141, and 143, respectively; (h) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 138, 141, and 144, respectively; (i) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 139, 141, and 145, respectively; or (j) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 140, 141, and 142, respectively.

[0153] In other embodiments, the anti-PAC1 receptor binding domain of the bispecific antigen binding protein of the invention comprises a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3, wherein (a) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 165, and 195, respectively; (b) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 166, and 195, respectively; (c) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 167, and 195, respectively; (d) CDRH1, (e) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 168, and 195, respectively; (f) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 169, and 195, respectively; (g) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 171, and 195, respectively; (h) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 172, and 195, respectively. (i) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 158, 173, and 196, respectively; (j) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 174, and 195, respectively; (k) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 175, and 195, respectively; (l) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 176, and 195, respectively; (m) CDRH1, CDRH2, and CDR (n) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 159, 178, and 197, respectively; (o) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 160, 179, and 196, respectively; (p) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 180, and 195, respectively; (q) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 161, 181, and 198, respectively;(r) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 159, 182, and 196, respectively; (s) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 162, 183, and 196, respectively; (t) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 163, 177, and 198, respectively; (u) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 163, 177, and 198, respectively; (v) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 159, 185, and 195, respectively; (w) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 186, and 195, respectively; (x) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 187, and 195, respectively; (y) (z) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 188, and 195, respectively; (aa) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 190, and 195, respectively; (ab) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 191, and 195, respectively; (ac) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 192, and 195, respectively; (ad) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 193, and 195, respectively; or (ae) CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 194, and 195, respectively.

[0154] In certain embodiments, the anti-PAC1 receptor binding domain of the bispecific antigen binding protein of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3 and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3; (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 165, and 195, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 166, and 195, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 167, and 195, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 168, and 195, respectively; (e) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 169, and 195, respectively; (f) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 170, and 195, respectively; (g) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 171, and 195, respectively; (h) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 133, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 172, and 195, respectively; (i) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 158, 173, and 196, respectively; (j) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 174, and 195, respectively; (k) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 175, and 195, respectively; (l) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 134, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 176, and 195, respectively; (m) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 135, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 158, 177, and 196, respectively; (n) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 135, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 159, 178, and 197, respectively; (o) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 136, 141, and 143, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 160, 179, and 196, respectively; (p) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 180, and 195, respectively; (q) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 135, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 161, 181, and 198, respectively; (r) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 135, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 159, 182, and 196, respectively; (s) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 135, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 162, 183, and 196, respectively; (t) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 137, 141, and 143, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 163, 177, and 198, respectively; (u) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 138, 141, and 144, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 184, and 195, respectively; (v) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 139, 141, and 145, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 159, 185, and 195, respectively; (w) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 184, and 195, respectively; (x) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 186, and 195, respectively; (y) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 187, and 195, respectively; (z) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 188, and 195, respectively; (aa) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 189, and 195, respectively; (ab) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 140, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 190, and 195, respectively; (ac) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 140, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 191, and 195, respectively; (ad) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 140, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 192, and 195, respectively; (ae) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 193, and 195, respectively; or (af) CDRL1, CDRL2 and CDRL3 have the sequences of SEQ ID NOs: 131, 141 and 142, respectively, and CDRH1, CDRH2 and CDRH3 have the sequences of SEQ ID NOs: 157, 194 and 195, respectively.

[0155] In some embodiments, the anti-PAC1 receptor binding domain of the bispecific antigen binding protein of the invention comprises a light chain variable region comprising CDRL1, CDRL2, and CDRL3 and a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3; (a) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 165, and 195, respectively; (b) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 166, and 195, respectively; (c) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 184, and 195, respectively; (d) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 186, and 195, respectively; (e) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 175, and 195, respectively; (f) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 135, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 158, 177, and 196, respectively; (g) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 174, and 195, respectively; (h) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 133, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 172, and 195, respectively; (i) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 170, and 195, respectively; or (j) CDRL1, CDRL2, and CDRL3 have the sequences of SEQ ID NOs: 132, 141, and 142, respectively, and CDRH1, CDRH2, and CDRH3 have the sequences of SEQ ID NOs: 157, 168, and 195, respectively.

[0156] The anti-PAC1 receptor binding domain of the bispecific antigen-binding protein of the present invention comprises a light chain variable region selected from the group consisting of LV-101, LV-102, LV-103, LV-104, LV-105, LV-106, LV-107, LV-108, LV-109, LV-110, and LV-111 as shown in Table 6A, and / or HV-101, HV-102, HV-103, HV-104, HV-105, HV-106, HV-107, HV-108, HV-109, and HV-110 as shown in Table 6B. 9, HV-110, HV-111, HV-112, HV-113, HV-114, HV-115, HV-116, HV-117, HV-118, HV-119, HV-120, HV-121, HV-122, HV-123, HV-124, HV-125, HV-126, HV-127, HV-128, HV-129, HV-130, HV-131, and HV-132, as well as antigen-binding fragments, derivatives, muteins, and variants of these light and heavy chain variable regions. Each of the light chain variable regions listed in Table 6A may be combined with any of the heavy chain variable regions shown in Table 6B to form an anti-PAC1 receptor-binding domain suitable for incorporation into a bispecific antigen-binding protein of the invention. For example, in certain embodiments, the anti-PAC1 receptor binding domain comprises a light chain variable region and a heavy chain variable region, wherein (a) the light chain variable region comprises the sequence of SEQ ID NO: 147, and the heavy chain variable region comprises the sequence of SEQ ID NO: 200; (b) the light chain variable region comprises the sequence of SEQ ID NO: 147, and the heavy chain variable region comprises the sequence of SEQ ID NO: 201; (c) the light chain variable region comprises the sequence of SEQ ID NO: 148, and the heavy chain variable region comprises the sequence of SEQ ID NO: 202; (d) the light chain variable region comprises the sequence of SEQ ID NO: 148 and the heavy chain variable region comprises the sequence of SEQ ID NO: 203; (e) the light chain variable region comprises the sequence of SEQ ID NO: 148 and the heavy chain variable region comprises the sequence of SEQ ID NO: 204; (f) the light chain variable region comprises the sequence of SEQ ID NO: 148 and the heavy chain variable region comprises the sequence of SEQ ID NO: 205; (g) the light chain variable region comprises the sequence of SEQ ID NO: 148 and the heavy chain variable region comprises the sequence of SEQ ID NO: 206;(h) the light chain variable region comprises the sequence of SEQ ID NO: 149 and the heavy chain variable region comprises the sequence of SEQ ID NO: 207; (i) the light chain variable region comprises the sequence of SEQ ID NO: 148 and the heavy chain variable region comprises the sequence of SEQ ID NO: 208; (j) the light chain variable region comprises the sequence of SEQ ID NO: 148 and the heavy chain variable region comprises the sequence of SEQ ID NO: 209; (k) the light chain variable region comprises the sequence of SEQ ID NO: 148 and the heavy chain variable region comprises the sequence of SEQ ID NO: 210; (l) the light chain variable region comprises the sequence of SEQ ID NO: 150 and the heavy chain variable region comprises the sequence of SEQ ID NO: 211; (m) whether the light chain variable region comprises the sequence of SEQ ID NO: 151 and the heavy chain variable region comprises the sequence of SEQ ID NO: 212; (n) whether the light chain variable region comprises the sequence of SEQ ID NO: 151 and the heavy chain variable region comprises the sequence of SEQ ID NO: 213; (o) whether the light chain variable region comprises the sequence of SEQ ID NO: 152 and the heavy chain variable region comprises the sequence of SEQ ID NO: 214; (p) whether the light chain variable region comprises the sequence of SEQ ID NO: 148 and the heavy chain variable region comprises the sequence of SEQ ID NO: 215; (q) whether the light chain variable region comprises the sequence of SEQ ID NO: 151 and the heavy chain variable region comprises the sequence of SEQ ID NO: 216; (r) the light chain variable region comprises the sequence of SEQ ID NO: 151 and the heavy chain variable region comprises the sequence of SEQ ID NO: 217; (s) the light chain variable region comprises the sequence of SEQ ID NO: 151 and the heavy chain variable region comprises the sequence of SEQ ID NO: 218; (t) the light chain variable region comprises the sequence of SEQ ID NO: 153 and the heavy chain variable region comprises the sequence of SEQ ID NO: 219; (u) the light chain variable region comprises the sequence of SEQ ID NO: 154 and the heavy chain variable region comprises the sequence of SEQ ID NO: 220; (v) the light chain variable region comprises the sequence of SEQ ID NO: 155 and the heavy chain variable region comprises the sequence of SEQ ID NO: 221; (w) whether the light chain variable region comprises the sequence of SEQ ID NO: 147 and the heavy chain variable region comprises the sequence of SEQ ID NO: 220; (x) whether the light chain variable region comprises the sequence of SEQ ID NO: 147 and the heavy chain variable region comprises the sequence of SEQ ID NO: 222; (y) whether the light chain variable region comprises the sequence of SEQ ID NO: 147 and the heavy chain variable region comprises the sequence of SEQ ID NO: 223; (z) whether the light chain variable region comprises the sequence of SEQ ID NO: 147 and the heavy chain variable region comprises the sequence of SEQ ID NO: 224; (aa) whether the light chain variable region comprises the sequence of SEQ ID NO: 147 and the heavy chain variable region comprises the sequence of SEQ ID NO: 225;(ab) the light chain variable region comprises the sequence of SEQ ID NO: 156 and the heavy chain variable region comprises the sequence of SEQ ID NO: 226; (ac) the light chain variable region comprises the sequence of SEQ ID NO: 156 and the heavy chain variable region comprises the sequence of SEQ ID NO: 227; (ad) the light chain variable region comprises the sequence of SEQ ID NO: 156 and the heavy chain variable region comprises the sequence of SEQ ID NO: 228; (ae) the light chain variable region comprises the sequence of SEQ ID NO: 147 and the heavy chain variable region comprises the sequence of SEQ ID NO: 229; or (af) the light chain variable region comprises the sequence of SEQ ID NO: 147 and the heavy chain variable region comprises the sequence of SEQ ID NO: 230;

[0157] In some embodiments, the anti-PAC1 receptor binding domain comprises a light chain variable region comprising a sequence of contiguous amino acids that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues from a light chain variable region sequence in Table 6A, i.e., a VL selected from LV-101, LV-102, LV-103, LV-104, LV-105, LV-106, LV-107, LV-108, LV-109, LV-110, and LV-111, wherein each such sequence difference is independently a deletion, insertion, or substitution of a single amino acid, and wherein the deletion, insertion, and / or substitution results in no more than 15 amino acid changes relative to the aforementioned variable domain sequence. The light chain variable region in some PAC1 receptor-binding domains comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 147-156 (i.e., the light chain variable regions in Table 6A). In one embodiment, the anti-PAC1 receptor-binding domain comprises a light chain variable region comprising a sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 147-156. In another embodiment, the anti-PAC1 receptor-binding domain comprises a light chain variable region comprising a sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 147-156.

[0158] In these and other embodiments, the anti-PAC1 receptor binding domain comprises a heavy chain variable region sequence in Table 6B, i.e., HV-101, HV-102, HV-103, HV-104, HV-105, HV-106, HV-107, HV-108, HV-109, HV-110, HV-111, HV-112, HV-113, HV-114, HV-115, HV-116, HV-117, HV-118, HV-119, HV-120, HV-121, HV-122, HV-123, HV-124, HV-125, HV-126, HV-127, HV-128, HV-129, HV-200, HV-201, HV-202, HV-203, HV-204, HV-205, HV-206, HV-207, HV-208, HV-209, HV-301, HV-309, HV-401, HV-410, HV-411, HV-412, HV-413, HV-414, HV-415, HV-416, HV-417, HV-418, HV-419, HV-500, HV-510, HV-511, HV-512, HV-513, HV-514, HV-515, HV-516, HV-517, HV-518, HV-519, HV-601, HV-620, HV-621, HV-622, HV-623, HV and a VH selected from HV-125, HV-126, HV-127, HV-128, HV-129, HV-130, HV-131, and HV-132, and a heavy chain variable region comprising a sequence of consecutive amino acids that differs by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, where each such sequence difference is independently a deletion, insertion, or substitution of a single amino acid, and the deletion, insertion, and / or substitution results in 15 or fewer amino acid changes relative to the aforementioned variable domain sequence. The heavy chain variable region in some anti-PAC1 receptor-binding domains comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 200 to 230 (i.e., the heavy chain variable region in Table 6B). In one embodiment, the anti-PAC1 receptor binding domain comprises a heavy chain variable region comprising a sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 200 to 230. In another embodiment, the anti-PAC1 receptor binding domain comprises a heavy chain variable region comprising a sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 200 to 230.

[0159] In certain embodiments, the bispecific antigen-binding proteins of the invention are antibodies. In certain embodiments, the bispecific antigen-binding proteins of the invention are heterodimeric antibodies (used interchangeably herein as "heteroimmunoglobulins" or "hetero-Igs"), which refer to antibodies comprising two different light chains and two different heavy chains. For example, in some embodiments, the heterodimeric antibody comprises a light chain and a heavy chain derived from an anti-PAC1 receptor antibody and a light chain and a heavy chain derived from an anti-CGRP receptor antibody. See Figure 2. As described in Example 3, the heteroimmunoglobulin format for bispecific molecules with target specificity for the human CGRP receptor and the human PAC1 receptor has a more desirable pharmacokinetic profile than molecules with a bivalent bispecific format, such as an IgG-Fab format.

[0160] Heterodimeric antibodies can comprise any immunoglobulin constant region, such as the light and heavy chain constant regions shown in Tables 3 and 4, respectively. The heavy chain constant region of the heterodimeric antibody can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, e.g., a human alpha-, human delta-, human epsilon-, human gamma-, or human mu-type heavy chain constant region. In some embodiments, the heterodimeric antibody comprises a heavy chain constant region derived from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In one embodiment, the heterodimeric antibody comprises a heavy chain constant region derived from a human IgG1 immunoglobulin. In such embodiments, the human IgG1 immunoglobulin constant region can comprise one or more mutations that prevent glycosylation of the heterodimeric antibody, as described in more detail herein. In another embodiment, the heterodimeric antibody comprises a heavy chain constant region derived from a human IgG2 immunoglobulin. In yet another embodiment, the heterodimeric antibody comprises a heavy chain constant region derived from a human IgG4 immunoglobulin.

[0161] Each of the variable regions disclosed in Tables 2A, 2B, 6A, and 6B can be combined with the light and heavy chain constant regions in Tables 3 and 4 to form the light and heavy chains, respectively, of a complete antibody. Furthermore, each of the heavy and light chain polypeptides so generated can be combined to form a complete bispecific antibody structure, e.g., a heterodimeric antibody. It should be understood that the heavy and light chain variable regions provided herein can also be combined with other constant domains having sequences that differ from the exemplary sequences listed in Tables 3 and 4.

[0162] To promote assembly of the light and heavy chains derived from an anti-CGRP receptor antibody and the light and heavy chains derived from an anti-PAC1 receptor antibody into a bispecific heterodimeric antibody, the light and / or heavy chains from each antibody can be engineered to reduce the formation of mispaired molecules. For example, one approach to promoting heterodimerization over homodimerization is the so-called "knobs-into-hole" method, which involves introducing mutations into the CH3 domains of two different antibody heavy chains at the contact interface. Specifically, one or more bulky amino acids in one heavy chain are replaced with amino acids with short side chains (e.g., alanine or threonine) to create "holes," while one or more amino acids with large side chains (e.g., tyrosine or tryptophan) are introduced into the other heavy chain to create "knobs." When the engineered heavy chains are coexpressed, a greater percentage of heterodimers (knobs-holes) are formed compared to homodimers (hole-hole or knob-knob). The "knob-into-hole" methodology is described in detail in WO 96 / 027011; Ridgway et al., Protein Eng., Vol. 9:617-621, 1996; and Merchant et al., Nat. Biotechnol., Vol. 16:677-681, 1998, all of which are incorporated by reference in their entirety.

[0163] Another approach to eliminating homodimer formation and promoting heterodimer formation involves utilizing an electrostatic steering mechanism (see Gunasekaran et al., J. Biol. Chem., Vol. 285:19637-19646, 2010, incorporated herein by reference in its entirety). This approach involves introducing or utilizing charged residues in the CH3 domains of each heavy chain so that two different heavy chains associate via opposite charges that generate electrostatic attraction. Homodimerization of identical heavy chains is unfavorable because identical heavy chains have the same charge and are therefore repulsive. This same electrostatic steering technique can be used to prevent the incorrect pairing of light chains with non-cognate heavy chains by introducing oppositely charged residues at the appropriate light chain-heavy chain pairing at the binding interface. Electrostatic steering techniques and suitable charge pair mutations to promote heterodimerization and proper light chain / heavy chain pairing are described in WO2009089004 and WO2014081955, both of which are incorporated by reference in their entirety.

[0164] In embodiments in which the bispecific antigen-binding protein of the invention is a heterodimeric antibody comprising a first light chain (LC1) and a first heavy chain (HC1) derived from a first antibody that specifically binds to the human CGRP receptor, and a second light chain (LC2) and a second heavy chain (HC2) derived from a second antibody that specifically binds to the human PAC1 receptor, HC1 or HC2 may comprise one or more amino acid substitutions to replace a positively charged amino acid with a negatively charged amino acid. For example, in one embodiment, the CH3 domain of HC1 or the CH3 domain of HC2 comprises an amino acid sequence that differs from the wild-type human IgG amino acid sequence such that one or more positively charged amino acids (e.g., lysine, histidine, and arginine) in the wild-type human IgG amino acid sequence are replaced with one or more negatively charged amino acids (e.g., aspartic acid and glutamic acid) at the corresponding positions in the CH3 domain. In these and other embodiments, an amino acid (e.g., lysine) at one or more positions selected from 360, 370, 392, 409, and 439 according to the EU numbering system is replaced with a negatively charged amino acid (e.g., aspartic acid and glutamic acid). Unless otherwise indicated by reference to a particular sequence, throughout this specification and claims, numbering of residues in an immunoglobulin heavy or light chain follows the Kabat-EU numbering system as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health Publication No. 91-3242, Bethesda, MD (1991) and Edelman et al., Proc. Natl. Acad. USA, Vol. 63:78-85 (1969).

[0165] Amino acid substitutions in an amino acid sequence are generally indicated herein by a single-letter abbreviation of the amino acid residue at a particular position, followed by the number of the amino acid position relative to the original sequence of interest, followed by the single-letter code of the substituted amino acid residue. For example, "T30D" represents a substitution of a threonine residue with an aspartic acid residue at amino acid position 30 relative to the original sequence of interest. Another example, "S218G" represents a substitution of a serine residue with a glycine residue at amino acid position 218 relative to the original amino acid sequence of interest.

[0166] In certain embodiments, the HC1 or HC2 of a heterodimeric antibody may comprise one or more amino acid substitutions to replace a negatively charged amino acid with a positively charged amino acid. For example, in one embodiment, the CH3 domain of HC1 or the CH3 domain of HC2 comprises an amino acid sequence that differs from the wild-type human IgG amino acid sequence, such that one or more negatively charged amino acids in the wild-type human IgG amino acid sequence are replaced with one or more positively charged amino acids at corresponding positions in the CH3 domain. In these and other embodiments, an amino acid (e.g., aspartic acid or glutamic acid) at one or more positions selected from 356, 357, and 399 according to the EU numbering system in the CH3 domain is replaced with a positively charged amino acid (e.g., lysine, histidine, and arginine).

[0167] In certain embodiments, the heterodimeric antibody comprises a first heavy chain comprising negatively charged amino acids at positions 392 and 409 (e.g., K392D and K409D substitutions) and a second heavy chain comprising positively charged amino acids at positions 356 and 399 (e.g., E356K and D399K substitutions). In other certain embodiments, the heterodimeric antibody comprises a first heavy chain comprising negatively charged amino acids at positions 370, 392, and 409 (e.g., K370D, K392D, and K409D substitutions) and a second heavy chain comprising positively charged amino acids at positions 356, 357, and 399 (e.g., E356K, E357K, and D399K substitutions). In certain embodiments, the heterodimeric antibody comprises a first heavy chain comprising negatively charged amino acids at positions 392, 409, and 439 (e.g., K392D, K409D, and K439D substitutions) and a second heavy chain comprising positively charged amino acids at positions 356 and 399 (e.g., E356K and D399K substitutions). In other embodiments, the heterodimeric antibody comprises a first heavy chain comprising negatively charged amino acids at positions 360, 370, 392, and 409 (e.g., K360E, K370E, K392E, and K409D substitutions) and a second heavy chain comprising positively charged amino acids at positions 357 and 399 (e.g., E357K and D399K substitutions). In any of the foregoing embodiments, the first heavy chain may be from an anti-PAC1 receptor antibody and the second heavy chain may be from an anti-CGRP receptor antibody. Alternatively, in any of the foregoing embodiments, the first heavy chain may be from an anti-CGRP receptor antibody and the second heavy chain may be from an anti-PAC1 receptor antibody.

[0168] To promote association of a particular heavy chain with its cognate light chain, both the heavy and light chains may contain complementary amino acid substitutions. As used herein, "complementary amino acid substitution" refers to a substitution of a negatively charged amino acid in one chain paired with a substitution of a positively charged amino acid in the other chain. For example, in some embodiments, a heavy chain contains at least one amino acid substitution to introduce a charged amino acid, and the corresponding light chain contains at least one amino acid substitution to introduce a charged amino acid, the charged amino acid introduced in the heavy chain having the opposite charge of the amino acid introduced in the light chain. In certain embodiments, one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into the first light chain (LC1), and one or more negatively charged residues (e.g., aspartic acid or glutamic acid) can be introduced into the heavy chain (HC1) that pairs at the LC1 / HC1 binding interface, while one or more negatively charged residues (e.g., aspartic acid or glutamic acid) can be introduced into the second light chain (LC2), and one or more positively charged residues (e.g., lysine, histidine, or arginine) can be introduced into the heavy chain (HC2) that pairs at the LC2 / HC2 binding interface. Electrostatic interactions will induce LC1 to pair with HC1 and LC2 to pair with HC2 due to the attraction of oppositely charged residues (polarity) at the interface. Heavy / light chain pairs with the same charged residues (polarity) at the interface (eg, LC1 / HC2 and LC2 / HC1) will repel each other, resulting in the suppression of unwanted HC / LC pairing.

[0169] In these and other embodiments, the CH1 domain of the heavy chain or the CL domain of the light chain comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more positively charged amino acids in the wild-type IgG amino acid sequence are replaced with one or more negatively charged amino acids. Alternatively, the CH1 domain of the heavy chain or the CL domain of the light chain comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more negatively charged amino acids in the wild-type IgG amino acid sequence are replaced with one or more positively charged amino acids. In some embodiments, one or more amino acids in the CH1 domain of the first and / or second heavy chain in the heterodimeric antibody at EU positions selected from F126, P127, L128, A141, L145, K147, D148, H168, F170, P171, V173, Q175, S176, S183, V185, and K213 are replaced with charged amino acids. In certain embodiments, a preferred residue for substitution with a negatively or positively charged amino acid is S183, where the amino acid position is according to the EU numbering system. In some embodiments, S183 is substituted with a positively charged amino acid. In alternative embodiments, S183 is substituted with a negatively charged amino acid. For example, in one embodiment, S183 is substituted with a negatively charged amino acid (e.g., S183E) in the first heavy chain, and S183 is substituted with a positively charged amino acid (e.g., S183K) in the second heavy chain.

[0170] In embodiments where the light chain is a kappa light chain, one or more amino acids in the CL domain of the first and / or second light chain in the heterodimeric antibody at a position according to EU and Kabat numbering in the kappa light chain selected from F116, F118, S121, D122, E123, Q124, S131, V133, L135, N137, N138, Q160, S162, T164, S174 and S176 are replaced with a charged amino acid. In embodiments where the light chain is a lambda light chain, one or more amino acids in the CL domain of the first and / or second light chain in the heterodimeric antibody at positions according to Kabat numbering in the lambda chain selected from T116, F118, S121, E123, E124, K129, T131, V133, L135, S137, E160, T162, S165, Q167, A174, S176, and Y178 are replaced with a charged amino acid. In some embodiments, a preferred residue for substitution with a negatively or positively charged amino acid is S176 (EU and Kabat numbering systems) of the CL domain of either a kappa or lambda light chain. In certain embodiments, S176 of the CL domain is replaced with a positively charged amino acid. In alternative embodiments, S176 of the CL domain is replaced with a negatively charged amino acid. In one embodiment, S176 is substituted with a positively charged amino acid (e.g., S176K) in the first light chain and S176 is substituted with a negatively charged amino acid (e.g., S176E) in the second light chain.

[0171] In addition to or instead of complementary amino acid substitutions in the CH1 and CL domains, the light and heavy chain variable regions of a heterodimeric antibody may contain one or more complementary amino acid substitutions to introduce charged amino acids. For example, in some embodiments, the heavy chain VH region or light chain VL region of a heterodimeric antibody comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more positively charged amino acids in the wild-type IgG amino acid sequence are replaced with one or more negatively charged amino acids. Alternatively, the heavy chain VH region or light chain VL region comprises an amino acid sequence that differs from the wild-type IgG amino acid sequence, such that one or more negatively charged amino acids in the wild-type IgG amino acid sequence are replaced with one or more positively charged amino acids.

[0172] V region interface residues in the VH region (i.e., amino acid residues that mediate assembly of the VH and VL regions) include Kabat positions 1, 3, 35, 37, 39, 43, 44, 45, 46, 47, 50, 59, 89, 91, and 93. One or more of these interface residues in the VH region may be substituted with a charged (positively or negatively charged) amino acid. In certain embodiments, the amino acid at Kabat position 39 in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid, e.g., lysine. In alternative embodiments, the amino acid at Kabat position 39 in the VH region of the first and / or second heavy chain is substituted with a negatively charged amino acid, e.g., glutamic acid. In some embodiments, the amino acid at Kabat position 39 in the VH region of the first heavy chain is substituted with a negatively charged amino acid (e.g., G39E) and the amino acid at Kabat position 39 in the VH region of the second heavy chain is substituted with a positively charged amino acid (e.g., G39K). In some embodiments, the amino acid at Kabat position 44 in the VH region of the first and / or second heavy chain is substituted with a positively charged amino acid, e.g., lysine. In alternative embodiments, the amino acid at Kabat position 44 in the VH region of the first and / or second heavy chain is substituted with a negatively charged amino acid, e.g., glutamic acid. In some embodiments, the amino acid at Kabat position 44 in the VH region of the first heavy chain is substituted with a negatively charged amino acid (e.g., G44E) and the amino acid at Kabat position 44 in the VH region of the second heavy chain is substituted with a positively charged amino acid (e.g., G44K).

[0173] V region interface residues in the VL region (i.e., amino acid residues that mediate assembly of the VH and VL regions) include Kabat positions 32, 34, 35, 36, 38, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 53, 54, 55, 56, 57, 58, 85, 87, 89, 90, 91, and 100. One or more interface residues in the VL region may be substituted with a charged amino acid, preferably an amino acid with the opposite charge to that introduced into the VH region of the cognate heavy chain. In some embodiments, the amino acid at Kabat position 100 in the VL region of the first and / or second light chain is substituted with a positively charged amino acid, e.g., lysine. In an alternative embodiment, the amino acid at Kabat position 100 in the VL region of the first and / or second light chain is substituted with a negatively charged amino acid, e.g., glutamic acid. In certain embodiments, the amino acid at Kabat position 100 in the VL region of the first light chain is substituted with a positively charged amino acid (e.g., G100K) and the amino acid at Kabat position 100 in the VL region of the second light chain is substituted with a negatively charged amino acid (e.g., G100E).

[0174] In certain embodiments, the heterodimeric antibody of the invention comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain, wherein the first heavy chain comprises amino acid substitutions at positions 44 (Kabat numbering), 183 (EU numbering), 392 (EU numbering), and 409 (EU numbering), the second heavy chain comprises amino acid substitutions at positions 44 (Kabat numbering), 183 (EU numbering), 356 (EU numbering), and 399 (EU numbering), and the first and second light chains comprise amino acid substitutions at positions 100 (Kabat numbering) and 176 (Kabat numbering), and the amino acid substitutions introduce charged amino acids at said positions. In a related embodiment, a glycine at position 44 (Kabat numbering) of the first heavy chain is replaced with glutamic acid, a glycine at position 44 (Kabat numbering) of the second heavy chain is replaced with lysine, a glycine at position 100 (Kabat numbering) of the first light chain is replaced with lysine, a glycine at position 100 (Kabat numbering) of the second light chain is replaced with glutamic acid, a serine at position 176 (Kabat numbering) of the first light chain is replaced with lysine, and a serine at position 176 (Kabat numbering) of the second light chain is replaced with glutamic acid. and a serine at position 183 (EU numbering) of the first heavy chain is replaced with glutamic acid, a lysine at position 392 (EU numbering) of the first heavy chain is replaced with aspartic acid, a lysine at position 409 (EU numbering) of the first heavy chain is replaced with aspartic acid, a serine at position 183 (EU numbering) of the second heavy chain is replaced with lysine, a glutamic acid at position 356 (EU numbering) of the second heavy chain is replaced with lysine, and / or an aspartic acid at position 399 (EU numbering) of the second heavy chain is replaced with lysine. Thus, in some embodiments, a heterodimeric antibody comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein (a) the first heavy chain comprises amino acid substitutions G44E, S183E, K392D, and K409D; (b) the first light chain comprises amino acid substitutions G100K and S176K; (c) the second heavy chain comprises amino acid substitutions G44K, S183K, E356K, and D399K; and (d) the second light chain comprises amino acid substitutions G100E and S176E.

[0175] In other embodiments, the heterodimeric antibody of the invention comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain, wherein the first heavy chain comprises amino acid substitutions at positions 183, 392, and 409 (all positions according to the EU numbering system), the second heavy chain comprises amino acid substitutions at positions 183, 356, and 399 (all positions according to the EU numbering system), and the first and second light chains comprise amino acid substitutions at position 176 (position according to the Kabat numbering system), and the amino acid substitutions introduce charged amino acids at said positions. In such embodiments, the serine at position 176 (according to Kabat numbering) of the first light chain is replaced with lysine; the serine at position 176 (according to Kabat numbering) of the second light chain is replaced with glutamic acid; the serine at position 183 (according to EU numbering) of the first heavy chain is replaced with glutamic acid, the lysine at position 392 (according to EU numbering) of the first heavy chain is replaced with aspartic acid, and the lysine at position 409 (according to EU numbering) of the first heavy chain is replaced with aspartic acid; the serine at position 183 (according to EU numbering) of the second heavy chain is replaced with lysine, the glutamic acid at position 356 (according to EU numbering) of the second heavy chain is replaced with lysine, and / or the aspartic acid at position 399 (according to EU numbering) of the second heavy chain is replaced with lysine. Thus, in some embodiments, a heterodimeric antibody comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein (a) the first heavy chain comprises amino acid substitutions S183E, K392D, and K409D; (b) the first light chain comprises amino acid substitution S176K; (c) the second heavy chain comprises amino acid substitutions S183K, E356K, and D399K; and (d) the second light chain comprises amino acid substitution S176E.

[0176] In yet another embodiment, the heterodimeric antibody of the invention comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain, wherein the first heavy chain comprises amino acid substitutions at positions 183, 370, 392, and 409 (all positions according to the EU numbering system), the second heavy chain comprises amino acid substitutions at positions 183, 356, 357, and 399 (all positions according to the EU numbering system), and the first and second light chains comprise amino acid substitutions at position 176 (position according to the Kabat numbering system), and the amino acid substitutions introduce charged amino acids at said positions. In such embodiments, the serine at position 176 (according to Kabat numbering) of the first light chain is replaced with a lysine; the serine at position 176 (according to Kabat numbering) of the second light chain is replaced with a glutamic acid; the serine at position 183 (according to EU numbering) of the first heavy chain is replaced with a glutamic acid, the lysine at position 370 (according to EU numbering) of the first heavy chain is replaced with an aspartic acid, and the lysine at position 392 (according to EU numbering) of the first heavy chain is replaced with an aspartic acid. a serine at position 183 (according to EU numbering) of the second heavy chain is replaced with lysine, a glutamic acid at position 356 (according to EU numbering) of the second heavy chain is replaced with lysine, a glutamic acid at position 357 (according to EU numbering) of the second heavy chain is replaced with lysine, and / or an aspartic acid at position 399 (according to EU numbering) of the second heavy chain is replaced with lysine. Thus, in some embodiments, a heterodimeric antibody comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein (a) the first heavy chain comprises amino acid substitutions S183E, K370D, K392D, and K409D; (b) the first light chain comprises amino acid substitution S176K; (c) the second heavy chain comprises amino acid substitutions S183K, E356K, E357K, and D399K; and (d) the second light chain comprises amino acid substitution S176E.

[0177] In certain embodiments, the heterodimeric antibody of the present invention comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain, wherein the first heavy chain comprises amino acid substitutions at positions 183, 392, 409, and 439 (all positions according to the EU numbering system), the second heavy chain comprises amino acid substitutions at positions 183, 356, and 399 (all positions according to the EU numbering system), and the first and second light chains comprise amino acid substitutions at position 176 (position according to the Kabat numbering system), and the amino acid substitutions introduce charged amino acids at said positions. In such embodiments, the serine at position 176 (according to Kabat numbering) of the first light chain is replaced with lysine; the serine at position 176 (according to Kabat numbering) of the second light chain is replaced with glutamic acid; the serine at position 183 (according to EU numbering) of the first heavy chain is replaced with glutamic acid, the lysine at position 392 (according to EU numbering) of the first heavy chain is replaced with aspartic acid, the lysine at position 409 (according to EU numbering) of the first heavy chain is replaced with aspartic acid, and the lysine at position 439 (according to EU numbering) of the first heavy chain is replaced with aspartic acid; the serine at position 183 (according to EU numbering) of the second heavy chain is replaced with lysine, the glutamic acid at position 356 (according to EU numbering) of the second heavy chain is replaced with lysine, and / or the aspartic acid at position 399 (according to EU numbering) of the second heavy chain is replaced with lysine. Thus, in some embodiments, a heterodimeric antibody comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein (a) the first heavy chain comprises amino acid substitutions S183E, K392D, K409D, and K439D; (b) the first light chain comprises amino acid substitution S176K; (c) the second heavy chain comprises amino acid substitutions S183K, E356K, and D399K; and (d) the second light chain comprises amino acid substitution S176E.

[0178] In some embodiments, the heterodimeric antibody of the invention comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain, wherein the first heavy chain comprises amino acid substitutions at positions 183, 360, 370, 392, and 409 (all positions according to the EU numbering system), the second heavy chain comprises amino acid substitutions at positions 183, 357, and 399 (all positions according to the EU numbering system), and the first and second light chains comprise amino acid substitutions at position 176 (position according to the Kabat numbering system), and the amino acid substitutions introduce charged amino acids at said positions. In such embodiments, the serine at position 176 (according to Kabat numbering) of the first light chain is replaced with a lysine; the serine at position 176 (according to Kabat numbering) of the second light chain is replaced with a glutamic acid; the serine at position 183 (according to EU numbering) of the first heavy chain is replaced with a glutamic acid, the lysine at position 360 (according to EU numbering) of the first heavy chain is replaced with a glutamic acid, and the lysine at position 370 (according to EU numbering) of the first heavy chain is replaced with a glutamic acid. a lysine at position 392 (according to EU numbering) of the first heavy chain is replaced with glutamic acid, a lysine at position 409 (according to EU numbering) of the first heavy chain is replaced with aspartic acid, a serine at position 183 (according to EU numbering) of the second heavy chain is replaced with lysine, a glutamic acid at position 357 (according to EU numbering) of the second heavy chain is replaced with lysine, and / or an aspartic acid at position 399 (according to EU numbering) of the second heavy chain is replaced with lysine. Thus, in some embodiments, a heterodimeric antibody comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein (a) the first heavy chain comprises amino acid substitutions S183E, K360E, K370E, K392E, and K409D; (b) the first light chain comprises amino acid substitution S176K; (c) the second heavy chain comprises amino acid substitutions S183K, E357K, and D399K; and (d) the second light chain comprises amino acid substitution S176E.

[0179] Any of the light and heavy chain constant domains, anti-PAC1 receptor variable regions, and anti-CGRP receptor variable regions described herein can be modified to contain one or more of the above-described charge pair mutations that promote proper assembly of heterodimeric antibodies. Exemplary full-length light chain and full-length heavy chain sequences derived from anti-CGRP receptor antibodies containing one or more charge pair mutations suitable for use in the heterodimeric antibodies of the invention are shown above in Tables 5A and 5B, respectively.

[0180] In some embodiments, the heterodimeric antibody of the invention comprises an anti-CGRP receptor antibody light chain from Table 5A and an anti-CGRP receptor antibody heavy chain from Table 5B. Exemplary pairs of anti-CGRP receptor antibody light and heavy chains that can be incorporated into the heterodimeric antibodies of the invention include LC-03 (SEQ ID NO:71) and HC-02 (SEQ ID NO:86); LC-04 (SEQ ID NO:72) and HC-03 (SEQ ID NO:87); LC-04 (SEQ ID NO:72) and HC-04 (SEQ ID NO:88); LC-04 (SEQ ID NO:72) and HC-05 (SEQ ID NO:89); LC-04 (SEQ ID NO:72) and HC-06 (SEQ ID NO:90); LC-04 (SEQ ID NO:72) and HC-07 (SEQ ID NO:91); LC-05 (SEQ ID NO:73) and HC-02 (SEQ ID NO:86); LC-06 (SEQ ID NO:74) and HC-03 (SEQ ID NO:87); LC-06 (SEQ ID NO:74) and HC-04 (SEQ ID NO:88); LC-07 (SEQ ID NO:75) and HC-08 (SEQ ID NO:92); LC-08 (SEQ ID NO:76) and HC-09 (SEQ ID NO:93); LC-08 (SEQ ID NO:76) and HC-09 (SEQ ID NO:93). These include, but are not limited to, C-10 (SEQ ID NO:94); LC-02 (SEQ ID NO:70) and HC-08 (SEQ ID NO:92); LC-09 (SEQ ID NO:77) and HC-02 (SEQ ID NO:86); LC-10 (SEQ ID NO:78) and HC-02 (SEQ ID NO:86); LC-02 (SEQ ID NO:70) and HC-11 (SEQ ID NO:95); LC-11 (SEQ ID NO:79) and HC-02 (SEQ ID NO:86); LC-02 (SEQ ID NO:70) and HC-12 (SEQ ID NO:96); LC-02 (SEQ ID NO:70) and HC-13 (SEQ ID NO:97); LC-12 (SEQ ID NO:80) and HC-14 (SEQ ID NO:98); LC-13 (SEQ ID NO:81) and HC-02 (SEQ ID NO:86); LC-14 (SEQ ID NO:82) and HC-02 (SEQ ID NO:86); LC-15 (SEQ ID NO:83) and HC-02 (SEQ ID NO:86); and LC-16 (SEQ ID NO:84) and HC-02 (SEQ ID NO:86). In certain embodiments, the heterodimeric antibody of the present invention comprises an anti-CGRP receptor antibody light chain comprising the sequence of SEQ ID NO: 72 and an anti-CGRP receptor antibody heavy chain comprising a sequence selected from SEQ ID NOs: 87-91.In other embodiments, the heterodimeric antibody of the invention comprises an anti-CGRP receptor antibody light chain comprising the sequence of SEQ ID NO: 74 and an anti-CGRP receptor antibody heavy chain comprising the sequence of SEQ ID NO: 87 or SEQ ID NO: 88. In yet other embodiments, the heterodimeric antibody of the invention comprises an anti-CGRP receptor antibody light chain comprising the sequence of SEQ ID NO: 76 and an anti-CGRP receptor antibody heavy chain comprising the sequence of SEQ ID NO: 93 or SEQ ID NO: 94.

[0181] The anti-CGRP receptor antibody light chain and / or heavy chain incorporated into the heterodimeric antibody of the present invention may comprise a sequence of consecutive amino acids that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid residues from the sequence of the light chain in Table 5A or the heavy chain in Table 5B, where each such sequence difference is independently a single amino acid deletion, insertion, or substitution. In some embodiments, the anti-CGRP receptor antibody light chain incorporated into the heterodimeric antibody of the present invention comprises a sequence of amino acids that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 69-84 (i.e., the anti-CGRP receptor antibody light chain in Table 5A). In one embodiment, the heterodimeric antibody of the present invention comprises an anti-CGRP receptor antibody light chain comprising a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 70-84. In another embodiment, the heterodimeric antibody of the present invention comprises an anti-CGRP receptor antibody light chain comprising a sequence at least 95% identical to a sequence selected from SEQ ID NOs: 70-84. In certain embodiments, the anti-CGRP receptor antibody heavy chain incorporated into the heterodimeric antibody of the present invention comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 85-98 (i.e., the anti-CGRP receptor antibody heavy chain in Table 5B). In one embodiment, the heterodimeric antibody of the present invention comprises an anti-CGRP receptor antibody heavy chain comprising a sequence at least 90% identical to a sequence selected from SEQ ID NOs: 86-98. In another embodiment, the heterodimeric antibody of the present invention comprises an anti-CGRP receptor antibody heavy chain comprising a sequence at least 95% identical to a sequence selected from SEQ ID NOs: 86-98.

[0182] Exemplary full-length light chain and full-length heavy chain sequences derived from anti-PAC1 receptor antibodies containing one or more charge pair mutations suitable for use in the heterodimeric antibodies of the invention are shown below in Tables 7A and 7B, respectively.

[0183] Table 38

[0184] Table 39

[0185] Table 40

[0186] Table 41

[0187] Table 42

[0188] Table 43

[0189] Table 44

[0190] Table 45

[0191] Table 46

[0192] Table 47

[0193] Table 48

[0194] Table 49

[0195]

Table 50

[0196] Table 51

[0197] Table 52

[0198] Table 53

[0199] Table 54

[0200] Table 55

[0201] Table 56

[0202] Table 57

[0203] Table 58

[0204] Table 59

[0205] Table 60

[0206] Table 61

[0207] Table 62

[0208] Table 63

[0209] Table 64

[0210] Table 65

[0211] Table 66

[0212] Table 67

[0213] Table 68

[0214] Table 69

[0215] Table 70

[0216] Table 71

[0217] Table 72

[0218] Table 73

[0219] Table 74

[0220] Table 75

[0221] Table 76

[0222] Table 77

[0223] Table 78

[0224] Table 79

[0225] Table 80

[0226] Table 81

[0227] Table 82

[0228] Table 83

[0229] Table 84

[0230] Table 85

[0231] Table 86

[0232] Table 87

[0233] Table 88

[0234] Table 89

[0235] Table 90

[0236]

Table 91

[0237] Table 92

[0238]

Table 93

[0239] Table 94

[0240]

Table 95

[0241] Table 96

[0242] Table 97

[0243] Table 98

[0244]

Table 99

[0245]

Table 100

[0246] Table 101

[0247] Table 102

[0248] Table 103

[0249] Table 104

[0250] Table 105

[0251] Table 106

[0252] Table 107

[0253] Table 108

[0254] Table 109

[0255] Table 110

[0256] Table 111

[0257] Table 112

[0258] Table 113

[0259] In some embodiments, heterodimeric antibodies of the invention comprise an anti-PAC1 receptor antibody light chain from Table 7A and an anti-PAC1 receptor antibody heavy chain from Table 7B. Exemplary pairs of anti-PAC1 receptor antibody light and heavy chains that can be incorporated into heterodimeric antibodies of the invention include LC-102 (SEQ ID NO:232) and HC-102 (SEQ ID NO:243); LC-102 (SEQ ID NO:232) and HC-103 (SEQ ID NO:244); LC-102 (SEQ ID NO:232) and HC-104 (SEQ ID NO:245); LC-102 (SEQ ID NO:232) and HC-105 (SEQ ID NO:246); LC-102 (SEQ ID NO:232) and HC-106 (SEQ ID NO:247); LC-102 (SEQ ID NO:232). and HC-107 (SEQ ID NO: 248); LC-102 (SEQ ID NO: 232) and HC-108 (SEQ ID NO: 249); LC-102 (SEQ ID NO: 232) and HC-109 (SEQ ID NO: 250); LC-102 (SEQ ID NO: 232) and HC-110 (SEQ ID NO: 251); LC-102 (SEQ ID NO: 232) and HC-111 (SEQ ID NO: 252); LC-102 (SEQ ID NO: 232) and HC-112 (SEQ ID NO: 253); LC-102 (SEQ ID NO: 232) and HC-149 (SEQ ID NO: 290); LC-102 (SEQ ID NO: 232) and HC-112 (SEQ ID NO: 253) No. 232) and HC-150 (SEQ ID NO: 291); LC-102 (SEQ ID NO: 232) and HC-153 (SEQ ID NO: 294); LC-102 (SEQ ID NO: 232) and HC-154 (SEQ ID NO: 295); LC-102 (SEQ ID NO: 232) and HC-155 (SEQ ID NO: 296); LC-102 (SEQ ID NO: 232) and HC-156 (SEQ ID NO: 297); LC-102 (SEQ ID NO: 232) and HC-157 (SEQ ID NO: 298); LC-102 (SEQ ID NO: 232) and HC-158 (SEQ ID NO: 299); LC-10 2 (SEQ ID NO: 232) and HC-159 (SEQ ID NO: 300); LC-102 (SEQ ID NO: 232) and HC-160 (SEQ ID NO: 301); LC-102 (SEQ ID NO: 232) and HC-167 (SEQ ID NO: 308); LC-102 (SEQ ID NO: 232) and HC-168 (SEQ ID NO: 309); LC-102 (SEQ ID NO: 232) and HC-169 (SEQ ID NO: 310); LC-102 (SEQ ID NO: 232) and HC-170 (SEQ ID NO: 311); LC-103 (SEQ ID NO: 233) and HC-113 (SEQ ID NO: 254);LC-103 (SEQ ID NO: 233) and HC-114 (SEQ ID NO: 255); LC-103 (SEQ ID NO: 233) and HC-115 (SEQ ID NO: 256); LC-103 (SEQ ID NO: 233) and HC-116 (SEQ ID NO: 257); LC-103 (SEQ ID NO: 233) and HC-117 (SEQ ID NO: 258); LC-103 (SEQ ID NO: 233) and HC-118 (SEQ ID NO: 259); LC-103 (SEQ ID NO: 233) and HC-119 (SEQ ID NO: 260); LC-103 (SEQ ID NO: 233) and HC-120 (SEQ ID NO: 261); LC-103 (SEQ ID NO: 233) and and HC-121 (SEQ ID NO: 262); LC-103 (SEQ ID NO: 233) and HC-122 (SEQ ID NO: 263); LC-103 (SEQ ID NO: 233) and HC-125 (SEQ ID NO: 266); LC-103 (SEQ ID NO: 233) and HC-126 (SEQ ID NO: 267); LC-103 (SEQ ID NO: 233) and HC-127 (SEQ ID NO: 268); LC-103 (SEQ ID NO: 233) and HC-128 (SEQ ID NO: 269); LC-103 (SEQ ID NO: 233) and HC-129 (SEQ ID NO: 270); LC-103 (SEQ ID NO: 233) and HC-130 (SEQ ID NO: 271); LC-103 (SEQ ID NO: 233) and HC-139 (SEQ ID NO: 280); LC-103 (SEQ ID NO: 233) and HC-140 (SEQ ID NO: 281); LC-104 (SEQ ID NO: 234) and HC-123 (SEQ ID NO: 264); LC-104 (SEQ ID NO: 234) and HC-124 (SEQ ID NO: 265); LC-105 (SEQ ID NO: 235) and HC-131 (SEQ ID NO: 272); LC-105 (SEQ ID NO: 235) and HC-132 (SEQ ID NO: 273); LC-106 (SEQ ID NO: 236) and HC-133 (SEQ ID NO: 274); LC-106 (SEQ ID NO: 236) and and HC-134 (SEQ ID NO: 275); LC-106 (SEQ ID NO: 236) and HC-135 (SEQ ID NO: 276); LC-106 (SEQ ID NO: 236) and HC-136 (SEQ ID NO: 277); LC-106 (SEQ ID NO: 236) and HC-141 (SEQ ID NO: 282); LC-106 (SEQ ID NO: 236) and HC-142 (SEQ ID NO: 283); LC-106 (SEQ ID NO: 236) and HC-143 (SEQ ID NO: 284); LC-106 (SEQ ID NO: 236) and HC-144 (SEQ ID NO: 285); LC-106 (SEQ ID NO: 236) and HC-145 (SEQ ID NO: 286);LC-106 (SEQ ID NO:236) and HC-146 (SEQ ID NO:287); LC-107 (SEQ ID NO:237) and HC-137 (SEQ ID NO:278); LC-107 (SEQ ID NO:237) and HC-138 (SEQ ID NO:279); LC-108 (SEQ ID NO:238) and HC-147 (SEQ ID NO:288); LC-108 (SEQ ID NO:238) and HC-148 (SEQ ID NO:289); LC-109 (SEQ ID NO:239) and HC-149 (SEQ ID NO:290); LC-109 (SEQ ID NO:239) and HC-150 (SEQ ID NO:291); LC-110 (SEQ ID NO:240) and HC-151 (SEQ ID NO:241) No. 292); LC-110 (SEQ ID NO: 240) and HC-152 (SEQ ID NO: 293); LC-111 (SEQ ID NO: 241) and HC-161 (SEQ ID NO: 302); LC-111 (SEQ ID NO: 241) and HC-162 (SEQ ID NO: 303); LC-111 (SEQ ID NO: 241) and HC-163 (SEQ ID NO: 304); LC-111 (SEQ ID NO: 241) and HC-164 (SEQ ID NO: 305); LC-111 (SEQ ID NO: 241) and HC-165 (SEQ ID NO: 306); and LC-111 (SEQ ID NO: 241) and HC-166 (SEQ ID NO: 307).

[0260] In certain embodiments, heterodimeric antibodies of the present invention comprise an anti-PAC1 receptor antibody light chain comprising the sequence of SEQ ID NO: 232 and an anti-PAC1 receptor antibody heavy chain comprising a sequence selected from SEQ ID NOs: 243-253, 290, 291, 294, and 295. In other embodiments, heterodimeric antibodies of the present invention comprise an anti-PAC1 receptor antibody light chain comprising the sequence of SEQ ID NO: 233 and an anti-PAC1 receptor antibody heavy chain comprising a sequence selected from SEQ ID NOs: 256, 257, 260, 261, and 268-271. In yet other embodiments, heterodimeric antibodies of the present invention comprise an anti-PAC1 receptor antibody light chain comprising the sequence of SEQ ID NO: 234 and an anti-PAC1 receptor antibody heavy chain comprising the sequence of SEQ ID NO: 264 or SEQ ID NO: 265. In yet other embodiments, heterodimeric antibodies of the present invention comprise an anti-PAC1 receptor antibody light chain comprising the sequence of SEQ ID NO: 236 and an anti-PAC1 receptor antibody heavy chain comprising the sequence of SEQ ID NO: 274 or SEQ ID NO: 275.

[0261] The anti-PAC1 receptor antibody light chain and / or heavy chain incorporated into the heterodimeric antibody of the present invention may comprise a sequence of consecutive amino acids that differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid residues from the sequence of the light chain in Table 7A or the heavy chain in Table 7B, where each such sequence difference is independently a deletion, insertion, or substitution of a single amino acid. In some embodiments, the anti-PAC1 receptor antibody light chain incorporated into the heterodimeric antibody of the present invention comprises a sequence of amino acids that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 231-241 (i.e., the anti-PAC1 receptor antibody light chain in Table 7A). In one embodiment, a heterodimeric antibody of the present invention comprises an anti-PAC1 receptor antibody light chain comprising a sequence at least 90% identical to a sequence selected from SEQ ID NOs: 232-241. In another embodiment, a heterodimeric antibody of the present invention comprises an anti-PAC1 receptor antibody light chain comprising a sequence at least 95% identical to a sequence selected from SEQ ID NOs: 232-241. In certain embodiments, an anti-PAC1 receptor antibody heavy chain incorporated into a heterodimeric antibody of the present invention comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs: 242-311 (i.e., the anti-PAC1 receptor antibody heavy chain in Table 7B). In one embodiment, a heterodimeric antibody of the present invention comprises an anti-PAC1 receptor antibody heavy chain comprising a sequence at least 90% identical to a sequence selected from SEQ ID NOs: 243-311. In another embodiment, the heterodimeric antibody of the invention comprises an anti-PAC1 receptor antibody heavy chain comprising a sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 243-311.

[0262] Any of the anti-CGRP receptor antibody light chains and heavy chains listed in Tables 5A and 5B can be combined with any of the anti-PAC1 receptor antibody light chains and heavy chains listed in Tables 7A and 7B to form bispecific heterodimeric antibodies of the invention. The structural features of exemplary bispecific heterodimeric antibodies of the invention (e.g., constituent anti-CGRP receptor antibody light chains and heavy chains and anti-PAC1 receptor antibody light chains and heavy chains) are set forth in Table 8 below. These antibodies contain one or more charge pairs as described herein to promote proper heavy and light chain pairing and heteromerization between the anti-CGRP receptor antibody heavy chain and the anti-PAC1 receptor heavy chain. Antibodies designated "A" or "E" contain mutations in the light and heavy chain constant regions according to the "v101" electrostatic steering strategy shown in Figure 2, while antibodies designated "B" contain mutations in the light and heavy chain constant regions according to the "v103" electrostatic steering strategy shown in Figure 2. Antibodies designated "C" or "F" contain mutations in the light and heavy chain constant regions according to the "v102" electrostatic steering strategy shown in Figure 2, while antibodies designated "D" or "G" contain mutations in the light and heavy chain constant regions according to the "v104" electrostatic steering strategy shown in Figure 2. Antibodies designated "E," "F," or "G" further contain M252Y, S254T, and T256E mutations in the CH2 domain of the heavy chain to improve circulating half-life by increasing the affinity of the molecule for the FcRn receptor. The variable light and heavy chain designations in Table 8 (e.g., LV-01, LV-02, LV-101, LV-102, HV-01, HV-02, HV-101, HV-102, etc.) are defined by the amino acid sequences in Tables 2A, 2B, 6A, and 6B and the nucleotide sequences in Tables 9 and 10. The light and heavy chain designations in Table 8 (e.g., LC-01, LC-02, LC-101, LC-102, HC-01, HC-02, HC-101, HC-102, etc.) are defined by the amino acid and nucleotide sequences in Tables 5A, 5B, 7A, and 7B. Thus, the complete sequence information for each of the four chains of the exemplary heterodimeric antibodies in Table 8 can be obtained by cross-reference to these tables.As an example, heterodimeric antibody iPS:454537 comprises an anti-CGRP receptor antibody light chain (LC-04) comprising the amino acid sequence of SEQ ID NO: 72, an anti-CGRP receptor antibody heavy chain (HC-03) comprising the amino acid sequence of SEQ ID NO: 87, an anti-PAC1 receptor antibody light chain (LC-106) comprising the amino acid sequence of SEQ ID NO: 236, and an anti-PAC1 receptor antibody heavy chain (HC-133) comprising the amino acid sequence of SEQ ID NO: 274.

[0263] [Table 114]

[0264] [Table 115]

[0265] [Table 116]

[0266] [Table 117]

[0267] [Table 118]

[0268] [Table 119]

[0269] [Table 120]

[0270] [Table 121]

[0271] Table 122

[0272] In certain embodiments, the bispecific antigen binding proteins of the invention are selected from the group consisting of iPS:454537, iPS:454539, iPS:454541, iPS:454543, iPS:454545, iPS:454547, iPS:454549, iPS:454551, iPS:454553, iPS:454555, iPS:454557(5601), iPS:454559, iPS:454561, iPS:454563, iPS:454565(5606), iPS:454567, iPS:454569, iPS: 454571, iPS:454573, iPS:454575, iPS:454577, iPS:454579, iPS:454581, iPS:454583, iPS:454585, iPS:454587, iPS:454589, iPS:454591, iPS:454 593, iPS:454595, iPS:454597, iPS:454599, iPS:454601, iPS:454603, iPS:454605, iPS:454607, iPS:454609, iPS:454611, iPS:454613, iPS:454615 , iPS:454617, iPS:454619, iPS:454621, iPS:454623, iPS:454625, iPS:454627, iPS:454629, iPS:454631, iPS:454633, iPS:454635, iPS:454637, iP S:454639, iPS:454641, iPS:454643, iPS:454645, iPS:454647, iPS:454649, iPS:454651, iPS:454653, iPS:454655, iPS:454657, iPS:454659, iPS:4 54661, iPS:454663, iPS:454665, iPS:454667, iPS:454669, iPS:454671, iPS:454673, iPS:454675, iPS:454677, iPS:454679, iPS:454681, iPS:4546 83, iPS:454685, iPS:454687, iPS:454689, iPS:454691, iPS:454693, iPS:454695, iPS:454697, iPS:454699, iPS:454701, iPS:454703, iPS:454705,iPS:454707、iPS:454709、iPS:454711、iPS:454713、iPS:454715、iPS:454717、iPS:454719、iPS:454721、iPS:454723、iPS:454725、iPS:454727、iPS: 454729、iPS:454731、iPS:454733、iPS:454735、iPS:454737、iPS:454739、iPS:454741、iPS:454743、iPS:454745、iPS:454747、iPS:454749、iPS:4547 51、iPS:454753、iPS:454755、iPS:454757、iPS:454759、iPS:454761、iPS: 454763、iPS:454765、iPS:454767、iPS:454769、iPS:454771、iPS:454773、i PS:454775、iPS:454777、iPS:454779、iPS:454781、iPS:454783、iPS:4547 85、iPS:454787、iPS:454789、iPS:454791、iPS:454793、iPS:454795、iPS:4 54797、iPS:454799、iPS:454801、iPS:454803、iPS:454805、iPS:454807、iPS:454809、iPS:454811、iPS:454813、iPS:454815、iPS:454817、iPS:45481 9、iPS:454821、iPS:454823、iPS:454825、iPS:454827、iPS:454829、iPS:4 54831、iPS:454833、iPS:454835、iPS:454837、iPS:454839、iPS:454841、iP S:454843、iPS:454845、iPS:454847、iPS:454849、iPS:454851、iPS:45485 3、iPS:454855、iPS:454857、iPS:454859、iPS:454861、iPS:454863、iPS:45 4865、iPS:454867、iPS:454869、iPS:454871、iPS:454873、iPS:454875、iPS:454877、iPS:454879、iPS:454881、iPS:454883、iPS:454885、iPS:454887、iPS:454889, iPS:454891, iPS:454893, iPS:454895, iPS:454897, iPS:454899, iPS:454901, iPS: 454903, iPS:454905, iPS:454907, iPS:454909, iPS:454911, iPS:454913, iPS:454915, iPS:4549 17, iPS:454919, iPS:571009(5602), iPS:571015(5603), iPS:571017(5604), iPS:571025(5605), iPS:571023(5607), iPS:571033(5608), and iPS:571824(5609). In some embodiments, the heterodimeric antibody is selected from the group consisting of iPS:454557 (5601), iPS:454565 (5606), iPS:454583, iPS:454585, iPS:454587, iPS:454729, iPS:454731, iPS:454733, iPS:454735, iPS:454737, iPS:454739, iPS:454741, iPS:454742, iPS:454743, iPS:454744, iPS:454745, iPS:454746, iPS:454747, iPS:454748, iPS:454749, iPS:454750, iPS:454751, iPS:454752, iPS:454753, iPS:454754, iPS:454755, iPS:454756, iPS:454757, iPS:454758, iPS:454759, iPS:454760, iPS:454761, iPS:454762, iPS:454763, iPS:454764, iPS:454765, iPS:454766, iPS:454767, iPS:454768, iPS:454769, iPS:454770, iPS:454771, iPS:454772, iPS:454773 PS:454741, iPS:454743, iPS:454745, iPS:454747, iPS:454749, iPS:454751, iPS:454753, iPS:4547 55, iPS:454757, iPS:454759, iPS:454761, iPS:454763, iPS:454765, iPS:454767, iPS:454769, iPS: 454771, iPS:454773, iPS:454775, iPS:454783, iPS:454785, iPS:454787, iPS:454789, iPS:454791, iPS:454793, iPS:454795, iPS:454797, iPS:454799, iPS:454801, iPS:454803, iPS:454805, iPS:454 807, iPS:454809, iPS:454811, iPS:454813, iPS:454815, iPS:454817, iPS:454819, iPS:454821, iPS :454823, iPS:454825, iPS:454827, iPS:454829, iPS:454831, iPS:454833, iPS:454835, iPS:454837,iPS:454839, iPS:454841, iPS:454843, iPS:454845, iPS:454847, iPS:454849, iPS:4548 51, iPS:454853, iPS:454855, iPS:454857, iPS:454859, iPS:454861, iPS:454863, iPS:4 54865, iPS:454867, iPS:454869, iPS:454871, iPS:454873, iPS:454875, iPS:454877, iP S:454879, iPS:454881, iPS:454883, iPS:454885, iPS:454887, iPS:454889, iPS:454891, iPS:454893, iPS:454895, iPS:454897, iPS:454899, iPS:454901, iPS:454903, iPS:4549 05, iPS:454907, iPS:454909, iPS:454911, iPS:454913, iPS:454915, iPS:454917, iPS:4 The antibody is selected from the group consisting of antibodies designated as iPS:54919, iPS:571009 (5602), iPS:571015 (5603), iPS:571017 (5604), iPS:571025 (5605), iPS:571023 (5607), iPS:571033 (5608), and iPS:571824 (5609). In certain embodiments, the heterodimeric antibody is selected from the group consisting of iPS:454557 (5601), iPS:454565 (5606), iPS:571009 (5602), iPS:571015 (5603), iPS:571017 (5604), iPS:571025 (5605), iPS:571023 (5607), iPS:571033 (5608), iPS:571824 (5609), iPS:454745, iPS:454757, iPS:454765, iPS:454777, iPS:454785, iPS:454791, iPS:454792, iPS:454793, iPS:454794, iPS:454795, iPS:454796, iPS:454797, iPS:454798, iPS:45479 ...9, iPS:454791, iPS:454791, iPS:454792, iPS:454793, iPS:454794, iPS:454795, 54749, iPS:454751, iPS:454753, iPS:454755, iPS:454757, iPS:454759, iPS:454761, iPS:454763, iPS:454765, iPS:4547 67, iPS:454769, iPS:454771, iPS:454775, iPS:454787, iPS:454789, iPS:454791, iPS:454793, iPS:454797, iPS:454799,The antibody is selected from the group consisting of antibodies designated as iPS:454815, iPS:454817, iPS:454819, iPS:454821, iPS:454823, iPS:454825, iPS:454827, iPS:454829, iPS:454831, iPS:454833, iPS:454835, iPS:454839, iPS:454841, iPS:454843, iPS:454845, and iPS:454851. In certain other embodiments, the heterodimeric antibody is an antibody selected from the antibodies designated as iPS:454557 (5601), iPS:571009 (5602), iPS:571015 (5603), iPS:571017 (5604), iPS:571025 (5605), iPS:454565 (5606), iPS:571023 (5607), iPS:571033 (5608), and iPS:571824 (5609), as described in Table 8. In one embodiment, the heterodimeric antibody is the antibody designated as iPS:571025 (5605), as described in Table 8. In another embodiment, the heterodimeric antibody is the antibody designated as iPS:454565 (5606) as described in Table 8. In yet another embodiment, the heterodimeric antibody is the antibody designated as iPS:571023 (5607) as described in Table 8.

[0273] Heterodimeric antibodies of the invention also include antibodies comprising the heavy and / or light chains described herein, wherein 1, 2, 3, 4, or 5 amino acid residues are missing from the N-terminus or C-terminus, or both, relative to any one of the heavy and light chains described in Tables 5A, 5B, 7A, and 7B, e.g., due to post-translational modifications resulting from the type of host cell the antibody is expressed in. For example, Chinese hamster ovary (CHO) cells often cleave the C-terminal lysine from antibody heavy chains.

[0274] Bispecific antigen-binding proteins of the invention, such as the heterodimers described herein, preferably inhibit activation of the human CGRP receptor and the human PAC1 receptor by their corresponding ligands. Methods for assessing ligand-induced activation of the CGRP receptor or ligand binding to the CGRP receptor are described above. Similar assays can be used to assess ligand-induced activation of the PAC1 receptor or ligand binding to the PAC1 receptor. For example, cell-based assays measuring ligand-induced calcium mobilization and cAMP production can be used to assess activation of the PAC1 receptor. The ligand can be an endogenous ligand of the receptor, such as PACAP38 or PACAP27, or the ligand can be another known agonist of the receptor, such as maxadilan. Maxadilan is a 65-amino acid peptide originally isolated from a sandfly that is highly selective for PAC1 compared to VPAC1 or VPAC2 and can therefore be used as a PAC1-selective agonist (Lerner et al., J Biol Chem., Vol. 266(17):11234-11236, 1991; Lerner et al., Peptides, Vol. 28(9):1651-1654, 2007). An exemplary cell-based cAMP assay for assessing PAC1 receptor activation is described in Example 2. Other suitable PAC1 receptor activation assays are described in Dickson et al., Ann. N.Y. Acad. Sci., Vol. 1070:239-42, 2006; Bourgault et al., J. Med. Chem., Vol. 52:3308-3316, 2009; and U.S. Patent Application Publication No. 2011 / 0229423 (all of which are incorporated by reference in their entirety).

[0275] In some embodiments, bispecific antigen-binding proteins (e.g., heterodimeric antibodies) of the invention inhibit PACAP (e.g., PACAP38 or PACAP27)-induced activation of human PAC1 receptor. For example, bispecific antigen-binding proteins (e.g., heterodimeric antibodies) may inhibit PACAP-induced activation of human PAC1 receptor with an IC50 of less than about 10 nM, less than about 8 nM, less than about 5 nM, less than about 3 nM, less than about 1 nM, less than about 800 pM, less than about 700 pM, or less than about 600 pM, as measured by cell-based cAMP or calcium mobilization assays. In one particular embodiment, bispecific antigen-binding proteins (e.g., heterodimeric antibodies) of the invention inhibit PACAP-induced activation of human PAC1 receptor with an IC50 of less than about 5 nM, as measured by cell-based cAMP assays. In another specific embodiment, a bispecific antigen-binding protein (e.g., a heterodimeric antibody) of the invention inhibits PACAP-induced activation of human PAC1 receptor with an IC50 of less than about 1 nM, as measured by a cell-based cAMP assay. In yet another specific embodiment, a bispecific antigen-binding protein (e.g., a heterodimeric antibody) of the invention inhibits PACAP-induced activation of human PAC1 receptor with an IC50 of less than about 800 pM, as measured by a cell-based cAMP assay. In some embodiments, a bispecific antigen-binding protein (e.g., a heterodimeric antibody) of the invention inhibits PACAP-induced activation of human PAC1 receptor with an IC50 of between about 0.5 nM and about 5 nM, as measured by a cell-based cAMP assay. In other embodiments, a bispecific antigen-binding protein (e.g., a heterodimeric antibody) of the invention inhibits PACAP-induced activation of human PAC1 receptor with an IC50 of between about 0.6 nM and about 3 nM, as measured by a cell-based cAMP assay. In yet other embodiments, the bispecific antigen-binding proteins (e.g., heterodimeric antibodies) of the invention inhibit PACAP-induced activation of the human PAC1 receptor with an IC50 of between about 0.5 nM and about 1 nM as measured by a cell-based cAMP assay.In certain embodiments, bispecific antigen-binding proteins (e.g., heterodimeric antibodies) of the invention inhibit CGRP-induced activation of the human CGRP receptor with an IC50 of less than about 1 nM, and inhibit PACAP-induced activation of the human PAC1 receptor with an IC50 of less than about 5 nM, both IC50 values ​​determined by a cell-based cAMP assay. In certain other embodiments, bispecific antigen-binding proteins (e.g., heterodimeric antibodies) of the invention inhibit CGRP-induced activation of the human CGRP receptor with an IC50 of less than about 500 pM, and inhibit PACAP-induced activation of the human PAC1 receptor with an IC50 of about 1 nM, both IC50 values ​​determined by a cell-based cAMP assay.

[0276] In certain embodiments, the anti-CGRP receptor antibodies and bispecific antigen-binding proteins of the present invention may contain one or more mutations or modifications to the constant region. For example, the heavy chain constant region or Fc region of an anti-CGRP receptor antibody or bispecific antigen-binding protein may contain one or more amino acid substitutions that affect the glycosylation, effector function, and / or Fcγ receptor binding of the antibody or antigen-binding protein. One of the functions of the Fc region of an immunoglobulin is to communicate with the immune system when the immunoglobulin binds to its target. This is commonly referred to as "effector function." This communication leads to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated through binding of the Fc region to Fc receptors on the surface of cells of the immune system. CDC is mediated through binding of Fc to proteins of the complement system, such as C1q.

[0277] In some embodiments, the anti-CGRP receptor antibodies and bispecific antigen-binding proteins of the invention comprise one or more amino acid substitutions in the constant region that enhance effector function, e.g., ADCC activity, CDC activity, ADCP activity, and / or the clearance or half-life of the antibody or antigen-binding protein. Exemplary amino acid substitutions (amino acid positions according to the EU numbering scheme) that can enhance effector function include E233L, L234I, L234Y, L235S, G236A, S239D, F243L, F243V, P247I, D280H, K290S, K290E, K290N, K290Y, R292P, E294L, Y296W, S298A, S298D, and S298V. , S298G, S298T, T299A, Y300L, V305I, Q311M, K326A, K326E, K326W, A330S, A330L, A330M, A330F, I332E, D333A, E333S, E333A, K334A, K334V, A339D, A339Q, P396L, or any combination thereof.

[0278] In other embodiments, the anti-CGRP receptor antibodies and bispecific antigen binding proteins of the invention comprise one or more amino acid substitutions in the constant region that reduce effector function. Exemplary amino acid substitutions (amino acid positions according to the EU numbering scheme) that can reduce effector function include, but are not limited to, C220S, C226S, C229S, E233P, L234A, L234V, V234A, L234F, L235A, L235E, G237A, P238S, S267E, H268Q, N297A, N297G, N297Q, V309L, E318A, L328F, A330S, A331S, P331S, or any combination thereof.

[0279] The anti-CGRP receptor antibodies and bispecific antigen-binding proteins of the invention may comprise one or more amino acid substitutions in their constant regions that modulate the pharmacokinetic properties of the antibodies and antigen-binding proteins. For example, in some embodiments, the anti-CGRP receptor antibodies and bispecific antigen-binding proteins of the invention comprise one or more amino acid substitutions in the constant region of one or both heavy chains that increase the affinity of the antibodies and antigen-binding proteins for the neonatal Fc receptor (FcRn receptor), thereby increasing the circulating half-life of the antibodies and antigen-binding proteins. Such amino acid substitutions (amino acid positions according to the EU numbering scheme) include L251R, M252Y, M252F, M252S, M252W, M252T, S254T, R255L, R255G, R255I, T256S, T256R, T256Q, T256E, T256D, T256A, T256N, V308T, L309P, Q311S, G385R, G385D, G385S, G385T, G385H, G385K, G385A, Q386T, Q386P, Q386R, Q386D, Q386P, Q386S, Q386R, Q386T, Q386P, Q386H ... In certain embodiments, the anti-CGRP receptor antibodies of the invention comprise M252Y, S254T, and T256E mutations (amino acid positions according to the EU numbering scheme) in one or both heavy chains. In certain other embodiments, bispecific antigen binding proteins of the invention, such as the heterodimeric antibodies described herein, comprise M252Y, S254T, and T256E mutations (amino acid positions according to the EU numbering scheme) in one or both heavy chains.

[0280] Other modifications of the anti-CGRP receptor antibodies or bispecific antigen-binding proteins of the invention that increase serum half-life may also be desirable, for example, by incorporating or adding a salvage receptor binding epitope (e.g., by mutating the appropriate region, or by incorporating the epitope into a peptide tag which is then fused to the antibody or antigen-binding protein at either end or in the middle, e.g., by DNA or peptide synthesis; see, e.g., WO 96 / 32478), or by the addition of a molecule such as PEG or other water-soluble polymer, e.g., a polysaccharide polymer. The salvage receptor binding epitope preferably constitutes a region in which any one or more amino acid residues from one or two loops of the Fc region are transferred to analogous positions in the antibody or antigen-binding protein. Even more preferably, three or more residues from one or two loops of the Fc region are transferred. Even more preferably, the epitope is taken from the CH2 domain of the Fc region (e.g., an IgG Fc region) and transferred to the CH1, CH3, or VH region, or two or more such regions, of the antibody or antigen-binding protein. Alternatively, the epitope is taken from the CH2 domain of the Fc region and transferred to the CL or VL region, or both, of the antigen binding protein. See WO 97 / 34631 and WO 96 / 32478 for a description of Fc variants and their interactions with salvage receptors. Other Fc modifications that increase the affinity of molecules for the FcRn receptor, thereby improving their serum half-life, are described in WO 2013 / 096221 and can be incorporated into the Fc region of an anti-CGRP receptor antibody or bispecific antigen-binding protein of the invention.

[0281] Glycosylation can contribute to the effector function of antibodies, particularly IgG1 antibodies. Thus, in some embodiments, anti-CGRP receptor antibodies and bispecific antigen-binding proteins (e.g., heterodimeric antibodies) of the present invention may contain one or more amino acid substitutions that affect the level or type of glycosylation of the antibody or antigen-binding protein. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linkage refers to the attachment of a sugar moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of a sugar moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of a sugar of N-acetylgalactosamine, galactose, or xylose to a single hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0282] In certain embodiments, glycosylation of the anti-CGRP receptor antibodies and bispecific antigen-binding proteins described herein may be increased by adding one or more glycosylation sites, for example, to the Fc region of the antibody or antigen-binding protein. Addition of glycosylation sites to an antibody or antigen-binding protein may conveniently be achieved by modifying the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Modifications may also be made by the addition to or substitution by one or more serine or threonine residues of the starting sequence (for O-linked glycosylation sites). To facilitate this, the amino acid sequence of an antibody or antigen-binding protein may be modified through alterations at the DNA level, in particular by mutating the DNA encoding the target polypeptide at preselected bases to generate codons that will be translated into the desired amino acids.

[0283] The present invention also encompasses the production of antibodies and antigen-binding proteins with modified glycosylation structures that result in altered effector activity, e.g., antibodies and antigen-binding proteins with absent or reduced fucosylation that exhibit improved ADCC activity. Various methods for reducing or eliminating fucosylation are known in the art. For example, ADCC effector activity is mediated by binding of antibody molecules to the FcγRIII receptor, which has been shown to depend on the glycosylation structure of the N-linked glycosylation at residue N297 in the CH2 domain. Nonfucosylated antibodies bind to this receptor with high affinity and elicit FcγRIII-mediated effector function more efficiently than naturally fucosylated antibodies. For example, recombinant production of nonfucosylated antibodies in CHO cells in which the alpha-1,6-fucosyltransferase enzyme has been knocked out results in antibodies with a 100-fold increase in ADCC activity (see Yamane-Ohnuki et al., Biotechnol Bioeng. 87(5):614-22, 2004). A similar effect can be achieved by reducing the activity of alpha-1,6-fucosyltransferase enzymes or other enzymes in the fucosylation pathway, for example, by siRNA or antisense RNA treatment, engineering cell lines to knock out the enzymes, or culturing with selective glycosylation inhibitors (see Rothman et al., Mol Immunol. 26(12):1113-23, 1989). Some host cell lines, such as the Lec13 or rat hybridoma YB2 / 0 cell lines, naturally produce antibodies with lower fucosylation levels (see Shields et al., J Biol Chem. 277(30):26733-40, 2002 and Shinkawa et al., J Biol Chem. 278(5):3466-73, 2003). Increasing the level of bisected carbohydrate chains, for example by recombinantly producing antibodies in cells overexpressing the GnTIII enzyme, has also been shown to increase ADCC activity (see Umana et al., Nat Biotechnol. 17(2):176-80, 1999).

[0284] In other embodiments, glycosylation of the anti-CGRP receptor antibodies and bispecific antigen-binding proteins described herein is reduced or eliminated by removing one or more glycosylation sites, for example, from the Fc region of the antibody or antigen-binding protein. N-linked glycosylation of an antigen-binding protein can be reduced or eliminated by amino acid substitutions that eliminate or alter N-linked glycosylation sites. In certain embodiments, the anti-CGRP receptor antibodies or bispecific antigen-binding proteins (e.g., heterodimeric antibodies) described herein comprise a mutation at amino acid position N297 (according to the EU numbering scheme), such as N297Q, N297A, or N297G, in one or both heavy chains. In some embodiments, the anti-CGRP receptor antibodies or bispecific antigen-binding proteins (e.g., heterodimeric antibodies) of the invention comprise an Fc region derived from a human IgG1 antibody with a mutation at amino acid position N297 (according to the EU numbering scheme) in one or both heavy chains. In one embodiment, an anti-CGRP receptor antibody or bispecific antigen-binding protein (e.g., a heterodimeric antibody) of the invention comprises an Fc region derived from a human IgG1 antibody with an N297G mutation in one or both heavy chains. For example, in some embodiments, an anti-CGRP receptor antibody or bispecific antigen-binding protein (e.g., a heterodimeric antibody) of the invention comprises a heavy chain comprising a heavy chain constant region comprising the sequence of SEQ ID NO:65.

[0285] To improve the stability of molecules containing the N297 mutation, the Fc region of an anti-CGRP receptor antibody or bispecific antigen-binding protein (e.g., a heterodimeric antibody) can be further engineered. For example, in some embodiments, one or more amino acids in the Fc region are substituted with cysteine ​​to promote disulfide bond formation in the dimeric state. Thus, residues corresponding to V259, A287, R292, V302, L306, V323, or I332 (according to the EU numbering scheme) in the IgG1 Fc region can be substituted with cysteine. Preferably, cysteine ​​substitutions are made so that specific pairs of residues preferentially form disulfide bonds with each other, thereby limiting or preventing scrambling of the disulfide bonds. Preferred pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. In certain embodiments, an anti-CGRP receptor antibody or bispecific antigen-binding protein (e.g., a heterodimeric antibody) described herein comprises an Fc region derived from a human IgG1 antibody with the mutations R292C and V302C in one or both heavy chains. In such embodiments, the Fc region may also comprise an N297 mutation, such as an N297G mutation, in one or both heavy chains. In some embodiments, an anti-CGRP receptor antibody or bispecific antigen-binding protein (e.g., a heterodimeric antibody) of the invention comprises a heavy chain comprising a heavy chain constant region comprising the sequence of SEQ ID NO: 66.

[0286] The present invention includes one or more isolated polynucleotides or isolated nucleic acids encoding the anti-CGRP receptor antibodies or bispecific antigen-binding proteins and components thereof described herein. Additionally, the present invention encompasses vectors containing the nucleic acids, host cells or cell lines containing the nucleic acids, and methods for making the anti-CGRP receptor antibodies and bispecific antigen-binding proteins of the present invention. Nucleic acids include, for example, polynucleotides encoding all or a portion of an antibody, antigen-binding fragment, or bispecific antigen-binding protein, e.g., one or both chains of an anti-CGRP receptor antibody or heterodimeric antibody of the present invention, or a fragment, derivative, or variant thereof; polynucleotides sufficient for use as hybridization probes, PCR primers, or sequencing primers to identify, analyze, mutate, or amplify polynucleotides encoding polypeptides; antisense oligonucleotides for inhibiting expression of polynucleotides; and complementary sequences to the above. Nucleic acids may be of any length suitable for the desired use or function, may include one or more additional sequences, e.g., regulatory sequences, and / or may be part of a larger nucleic acid, e.g., a vector. Nucleic acid molecules of the present invention include DNA and RNA in both single- and double-stranded forms, as well as corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Nucleic acid molecules of the present invention include full-length genes or cDNA molecules, as well as combinations of fragments thereof. Nucleic acids of the present invention can be derived from human sources and non-human species.

[0287] The related amino acid sequence from an immunoglobulin or region thereof (e.g., variable region, Fc region, etc.) or polypeptide of interest may be determined by direct protein sequencing, and suitable encoding nucleotide sequences may be designed according to a universal codon table. Alternatively, genomic or cDNA encoding a monoclonal antibody from which a monoclonal antibody of the invention or binding fragment thereof or the binding domain of a bispecific antigen-binding protein of the invention may be derived can be isolated and sequenced from cells producing such an antibody using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody).

[0288] An "isolated nucleic acid," as used interchangeably herein with an "isolated polynucleotide," is a nucleic acid that, in the case of a nucleic acid isolated from a naturally occurring source, is separated from adjacent gene sequences present in the genome of the organism from which the nucleic acid is isolated. For example, in the case of a nucleic acid that is enzymatically or chemically synthesized from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid resulting from such a process is understood to be an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In a preferred embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecule is preferably derived from DNA or RNA that has been isolated at least once, in a substantially pure form and in an amount or concentration that allows for identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (e.g., those reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and / or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, typically present in eukaryotic genes. Non-translated DNA sequences can be present 5' or 3' from the open reading frame, provided that they do not interfere with manipulation or expression of the coding region. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' production of a nascent RNA transcript is referred to as the transcription direction, with the region of the DNA strand that is 5' to the 5' end of the RNA transcript and has the same sequence as the RNA transcript being referred to as the "upstream sequence," and the region of the DNA strand that is 3' to the 3' end of the RNA transcript and has the same sequence as the RNA transcript being referred to as the "downstream sequence."

[0289] The present invention also encompasses nucleic acids that hybridize to nucleic acids encoding polypeptides as described herein under moderately stringent conditions, more preferably under highly stringent conditions. Basic parameters influencing the selection of hybridization conditions and guidance for devising suitable conditions are provided by Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4) and can be readily determined by one of skill in the art based, for example, on the length and / or base composition of the DNA. One method of achieving moderately stringent conditions involves the use of a pre-wash solution comprising a hybridization buffer of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), about 50% formamide, 6×SSC, and a hybridization temperature of about 55° C. (or other similar hybridization solutions, such as those containing about 50% formamide with a hybridization temperature of about 42° C.), and wash conditions in 0.5×SSC, 0.1% SDS at about 60° C. Generally, highly stringent conditions are defined as hybridization conditions as above, except with a wash at about 68° C., 0.2×SSC, 0.1% SDS. SSPE (1x SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1x SSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization buffer and wash buffer, and after hybridization is complete, a 15 minute wash is performed.It should be understood that wash temperatures and wash salt concentrations can be adjusted as necessary to achieve the desired degree of stringency by applying basic principles governing hybridization reactions and duplex stability, as known to those of skill in the art and further described below (see, e.g., Sambrook et al., 1989).

[0290] When hybridizing a nucleic acid to a target nucleic acid of unknown sequence, the hybrid length is assumed to be the length of the hybridizing nucleic acid. When hybridizing nucleic acids of known sequence, the hybrid length can be determined by aligning the nucleic acid sequences and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids expected to be less than 50 base pairs in length should be 5-10°C lower than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following formula: For hybrids less than 18 base pairs in length, Tm (°C) = 2 (number of A+T bases) + 4 (number of G+C bases). For hybrids greater than 18 base pairs in length, Tm(°C) = 81.5 + 16.6(log10[Na+]+) + 0.41(%G+C) - (600 / N), where N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC = 0.165M). Preferably, each such hybridizing nucleic acid is at least 15 nucleotides (or more preferably at least 18 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, or at least 40 nucleotides, or most preferably at least 50 nucleotides) or at least 25 of the length of the nucleic acid of the invention to which it hybridizes. % (more preferably, at least 50%, or at least 60%, or at least 70% and most preferably at least 80%) and has at least 60% sequence identity (more preferably, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% and most preferably at least 99.5%) with the nucleic acid of the invention to which it hybridizes.Here, sequence identity is determined by comparing the sequences of hybridizing nucleic acids when aligned to maximize overlap and identity while minimizing sequence gaps, as described in more detail above.

[0291] Variants of the anti-CGRP receptor antibodies and antigen-binding proteins described herein can be prepared by site-directed mutagenesis of nucleotides in the DNA encoding the polypeptide, using cassette or PCR mutagenesis or other techniques well known in the art, such as those described in Example 1, to generate DNA encoding the variant, followed by expressing the recombinant DNA in cell culture as outlined herein. Antibodies and antigen-binding proteins containing variant CDRs having up to about 100-150 residues can also be prepared by in vitro synthesis using established techniques. Variants typically exhibit the same qualitative biological activity, e.g., antigen binding, as the naturally occurring analog. Such variants include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody or antigen-binding protein. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct retains the desired characteristics. Amino acid changes may also alter post-translational processing of the antibody or antigen-binding protein, such as by changing the number or position of glycosylation sites. In certain embodiments, variants of anti-CGRP receptor antibodies and antigen-binding proteins are generated with the intention of modifying those amino acid residues directly involved in epitope binding. In other embodiments, modifications of residues not directly involved in epitope binding or not involved in epitope binding at all are desirable for the purposes discussed herein. Mutagenesis in either the CDR and / or framework regions is contemplated. Covariance analysis techniques can be utilized by those skilled in the art to design useful modifications in the amino acid sequence of an antibody or antigen-binding protein.See, e.g., Chourier, et al., Proteins 41:475-484, 2000; Demarest et al., J. Mol. Biol. 335:41-48, 2004; Hugo et al., Protein Engineering 16(5):381-86, 2003; U.S. Patent Application Publication No. 2008 / 0318207; U.S. Patent Application Publication No. 2009 / 0048122; WO 2008 / 110348; and WO 2009 / 000099. Such modifications, as determined by covariance analysis, can improve the potency, pharmacokinetics, pharmacodynamics, and / or manufacturability properties of the antibody or antigen-binding protein.

[0292] Table 9 shows exemplary nucleic acids encoding the light and heavy chain variable regions of anti-CGRP receptor antibodies, and Table 10 shows exemplary nucleic acid sequences encoding the light and heavy chain variable regions of anti-PAC1 receptor antibodies. Polynucleotides encoding anti-CGRP receptor variable regions can be used, optionally in conjunction with nucleic acids encoding the light and heavy chain constant regions listed in Tables 3 and 4, respectively, to construct anti-CGRP receptor antibodies and antigen-binding fragments of the invention. Polynucleotides encoding anti-CGRP receptor and anti-PAC1 receptor variable regions can also be used to construct the anti-CGRP receptor and anti-PAC1 receptor binding domains, respectively, of the bispecific antigen-binding proteins (e.g., heterodimeric antibodies) described herein. Tables 5A and 5B show exemplary nucleic acid sequences encoding the complete light and heavy chains, respectively, of the anti-CGRP receptor antibodies described herein. Tables 7A and 7B show exemplary nucleic acid sequences encoding the complete light and heavy chains, respectively, of the anti-PAC1 receptor antibodies described herein. Polynucleotides encoding the anti-CGRP receptor antibody light and heavy chains can be co-expressed with polynucleotides encoding the anti-PAC1 receptor antibody light and heavy chains to produce bispecific antigen-binding proteins, such as heterodimeric antibodies of the invention.

[0293] [Table 123]

[0294] Table 124

[0295] Table 125

[0296] Table 126

[0297] Table 127

[0298] Table 128

[0299] Table 129

[0300] Table 130

[0301] Table 131

[0302] Table 132

[0303] Table 133

[0304] [Table 134]

[0305] [Table 135]

[0306] An isolated nucleic acid encoding an anti-CGRP receptor antibody, antigen-binding fragment, or anti-CGRP receptor binding domain of a bispecific antigen-binding protein of the invention may comprise a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to any of the nucleotide sequences listed in Tables 5A, 5B, and 9. In some embodiments, an isolated nucleic acid encoding an anti-CGRP receptor antibody light chain variable region comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 393-404. In certain embodiments, an isolated nucleic acid encoding an anti-CGRP receptor antibody light chain variable region comprises a sequence selected from SEQ ID NOs: 393-404. In related embodiments, an isolated nucleic acid encoding an anti-CGRP receptor antibody heavy chain variable region comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 405-411. In other related embodiments, the isolated nucleic acid encoding the anti-CGRP receptor antibody heavy chain variable region comprises a sequence selected from SEQ ID NOs: 405-411.

[0307] An isolated nucleic acid encoding an anti-PAC1 receptor binding domain of a bispecific antigen-binding protein of the invention may comprise a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to any of the nucleotide sequences listed in Tables 7A, 7B, and 10. In some embodiments, an isolated nucleic acid encoding an anti-PAC1 receptor antibody light chain variable region comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 412-422. In certain embodiments, an isolated nucleic acid encoding an anti-PAC1 receptor antibody light chain variable region comprises a sequence selected from SEQ ID NOs: 412-422. In related embodiments, an isolated nucleic acid encoding an anti-PAC1 receptor antibody heavy chain variable region comprises a sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a sequence selected from SEQ ID NOs: 423-454. In other related embodiments, the isolated nucleic acid encoding the anti-PAC1 receptor antibody heavy chain variable region comprises a sequence selected from SEQ ID NOs: 423-454.

[0308] In certain embodiments of an anti-CGRP receptor antibody of the invention or an embodiment in which a bispecific antigen-binding protein of the invention is a heterodimeric antibody, an isolated nucleic acid encoding an anti-CGRP receptor antibody light chain may comprise a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to any of the nucleotide sequences listed in Table 5A (e.g., SEQ ID NOS: 99-114). In some embodiments, an isolated nucleic acid encoding an anti-CGRP receptor antibody light chain of an anti-CGRP receptor antibody or heterodimeric antibody of the invention comprises a sequence selected from SEQ ID NOS: 99-114. In these and other embodiments, an isolated nucleic acid encoding an anti-CGRP receptor antibody heavy chain may comprise a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to any of the nucleotide sequences listed in Table 5B (e.g., SEQ ID NOS: 115-128). In some embodiments, an isolated nucleic acid encoding an anti-CGRP receptor antibody heavy chain of an anti-CGRP receptor antibody or heterodimeric antibody of the invention comprises a sequence selected from SEQ ID NOS: 115-128.

[0309] In some embodiments where the bispecific antigen-binding protein of the invention is a heterodimeric antibody, an isolated nucleic acid encoding an anti-PAC1 receptor antibody light chain may comprise a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to any of the nucleotide sequences listed in Table 7A (e.g., SEQ ID NOs: 312-322). In certain embodiments, an isolated nucleic acid encoding an anti-PAC1 receptor antibody light chain of a heterodimeric antibody of the invention comprises a sequence selected from SEQ ID NOs: 312-322. In these and other embodiments, an isolated nucleic acid encoding an anti-PAC1 receptor antibody heavy chain may comprise a nucleotide sequence that is at least 80% identical, at least 90% identical, at least 95% identical, or at least 98% identical to any of the nucleotide sequences listed in Table 7B (e.g., SEQ ID NOs: 323-392). In some embodiments, an isolated nucleic acid encoding an anti-PAC1 receptor antibody heavy chain of a heterodimeric antibody of the invention comprises a sequence selected from SEQ ID NOs: 323-392.

[0310] The nucleic acid sequences provided in Tables 5A, 5B, 7A, 7B, 9, and 10 are exemplary only. As will be understood by those of skill in the art, due to the degeneracy of the genetic code, a variety of nucleic acids may be generated, all of which encode the CDRs, variable regions, and heavy and light chains or other components of the antibodies and antigen binding proteins described herein. Thus, having identified a particular amino acid sequence, one of skill in the art could generate any number of different nucleic acids by simply altering the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein.

[0311] The present invention also includes vectors comprising one or more nucleic acids encoding one or more components (e.g., variable region, light chain, and heavy chain) of the anti-CGRP receptor antibodies, antigen-binding fragments, bispecific antigen-binding proteins, or binding domains thereof of the present invention. The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information to a host cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, such as recombinant expression vectors. The term "expression vector" or "expression construct," as used herein, refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid control sequences necessary for the expression of an operably linked coding sequence in a particular host cell. Expression vectors may include, but are not limited to, sequences that affect or control transcription, translation, and, if present, introns, affecting RNA splicing of the operably linked coding region. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

[0312] A secretory signal peptide sequence operably linked to the coding sequence of interest may also optionally be encoded by the expression vector, such that the expressed polypeptide may be secreted by the recombinant host cell for easier isolation of the polypeptide of interest from the cell, if desired. For example, in some embodiments, a signal peptide sequence may be added / fused to the amino terminus of any of the variable region polypeptide sequences listed in Tables 2A, 2B, 6A, and 6B, or any of the complete chain polypeptide sequences listed in Tables 5A, 5B, 7A, and 7B. In certain embodiments, a signal peptide having the amino acid sequence MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO:455) is fused to the amino terminus of any of the variable region polypeptide sequences listed in Tables 2A, 2B, 6A, and 6B, or any of the complete chain polypeptide sequences listed in Tables 5A, 5B, 7A, and 7B. In other embodiments, a signal peptide having the amino acid sequence of MAWALLLLTLLTQGTGSWA (SEQ ID NO:456) is fused to the amino terminus of any of the variable region polypeptide sequences listed in Tables 2A, 2B, 6A, and 6B, or any of the complete chain polypeptide sequences listed in Tables 5A, 5B, 7A, and 7B. In yet other embodiments, a signal peptide having the amino acid sequence of MTCSPLLLTLLIHCTGSWA (SEQ ID NO:457) is fused to the amino terminus of any of the variable region polypeptide sequences listed in Tables 2A, 2B, 6A, and 6B, or any of the complete chain polypeptide sequences listed in Tables 5A, 5B, 7A, and 7B.Other suitable signal peptide sequences that may be fused to the amino terminus of the variable region polypeptide sequences or complete chain polypeptide sequences described herein include MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 458), MEWTWRVLFLVAAATGAHS (SEQ ID NO: 459), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 460), METPAQLLFLLLLWLPDTTG (SEQ ID NO: 461), MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 462), MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 463), and the like.

[00130] Other signal peptides include the following: MDIRAPTQLLGLLLLWLPGAKC (SEQ ID NO: 463), MDIRAPTQLLGLLLLWLPGAKC (SEQ ID NO: 464), MDIRAPTQLLGLLLLWLPGARC (SEQ ID NO: 465), MDTRAPTQLLGLLLLWLPGATF (SEQ ID NO: 466), MDTRAPTQLLGLLLLWLPGARC (SEQ ID NO: 467), METGLRWLLLVAVLKGVQC (SEQ ID NO: 468), METGLRWLLLVAVLKGVQCQE (SEQ ID NO: 469), and MDMRAPTQLLGLLLLWLPGARC (SEQ ID NO: 470). Other signal peptides will be known to those of skill in the art and can be fused to any of the variable region polypeptide chains listed in Tables 2A, 2B, 6A, and 6B, or to the complete polypeptide chains listed in Tables 5A, 5B, 7A, and 7B, for example, to facilitate or optimize expression in a particular host cell.

[0313] Expression vectors used in host cells to produce the anti-CGRP receptor antibodies, antigen-binding fragments, and bispecific antigen proteins (e.g., heterodimeric antibodies) of the invention will typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences encoding components of the antibodies, antigen-binding fragments, and bispecific antigen-binding proteins. In certain embodiments, such sequences, collectively referred to as "flanking sequences," typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

[0314] Optionally, the vector may contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the polypeptide coding sequence, which oligonucleotide tag sequence encodes polyHis (such as hexaHis), FLAG, HA (influenza virus hemagglutinin), myc, or another "tag" molecule for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression and can serve as a means for affinity purification or detection of the polypeptide from host cells. Affinity purification can be achieved, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can then be removed from the purified polypeptide by various means, such as using specific cleavage peptidases.

[0315] The flanking sequences may be homologous (i.e., derived from the same species and / or strain as the host cell), heterologous (i.e., derived from a species other than the species or strain of the host cell), hybrid (i.e., a combination of flanking sequences from multiple sources), synthetic, or naturally occurring. Thus, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences are functional in, and can be activated by, the host cell machinery.

[0316] Flanking sequences useful in the vectors of the present invention can be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein will be previously identified by mapping and / or restriction endonuclease digestion, and can therefore be isolated from a suitable tissue source using appropriate restriction endonucleases. In some cases, the complete nucleotide sequence of the flanking sequence may be known. In this case, the flanking sequence may be synthesized using conventional methods for nucleic acid synthesis or cloning.

[0317] Whether all or only a portion of the flanking sequence is known, it may be obtained using the polymerase chain reaction (PCR) and / or by screening a genomic library with appropriate probes, such as oligonucleotides and / or flanking sequence fragments from the same or another species. If the flanking sequence is unknown, fragments of DNA containing the flanking sequence can be isolated from a larger fragment of DNA that may contain, for example, a coding sequence or one or more additional genes. Isolation can be achieved by generating appropriate DNA fragments by restriction endonuclease digestion, followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to those of skill in the art. The selection of appropriate enzymes to accomplish this purpose will be readily apparent to those of skill in the art.

[0318] Origins of replication are typically part of commercially purchased prokaryotic expression vectors; the origin aids in the amplification of the vector in host cells. If the vector of choice does not contain an origin of replication site, one can be chemically synthesized based on a known sequence, or it can be ligated into the vector. For example, the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (e.g., the SV40 origin is often used simply because it also contains the viral early promoter).

[0319] A transcription termination sequence is usually located 3' to the end of a polypeptide coding region and serves to terminate transcription. Typically, a transcription termination sequence in prokaryotic cells is a GC-rich fragment followed by a poly-T sequence. This sequence is easily cloned from a library or purchased commercially as part of a vector, but can also be easily synthesized using known methods for nucleic acid synthesis.

[0320] A selectable marker gene encodes a protein necessary for the survival and growth of host cells grown in selective culture media. Typical selectable marker genes (a) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, on prokaryotic host cells; (b) complement an auxotrophic deficiency of the cells; or (c) encode a protein that supplies a critical nutrient unavailable from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, the neomycin resistance gene can be used for selection in both prokaryotic and eukaryotic host cells.

[0321] Other selection genes can be used to amplify the gene to be expressed. Amplification is the process by which genes required for the production of proteins important for growth or cell survival are tandemly repeated in the chromosomes of successive generations of recombinant cells. Examples of suitable selection markers for mammalian cells include dihydrofolate reductase (DHFR) and promoter-less thymidine kinase genes. Mammalian cell transformants are placed under selection pressure that is specifically adapted so that only these transformants survive due to the selectable gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively increased, thereby resulting in the amplification of both the selectable gene and DNA encoding another gene, such as one or more components of the antibody, antigen-binding fragment, or bispecific antigen-binding protein described herein. As a result, large amounts of polypeptide are synthesized from the amplified DNA.

[0322] A ribosome binding site is usually required for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is usually located 3' of the promoter and 5' of the coding sequence of the polypeptide to be expressed. In certain embodiments, one or more coding regions may be operably linked to an internal ribosome binding site (IRES), allowing translation of two open reading frames from a single RNA transcript.

[0323] In some cases, such as when glycosylation is desired in eukaryotic host cell expression systems, various pre- or pro-sequences may be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide may be modified or a pro-sequence may be added, which may also affect glycosylation. The final protein product may have one or more additional amino acids at position -1 (relative to the first amino acid of the mature protein) accompanying expression, which may not have been completely removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site attached to the amino terminus. Alternatively, the use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide if the enzyme cleaves at such a region within the mature polypeptide.

[0324] Expression and cloning vectors of the invention typically contain a promoter that is recognized by the host organism and operably linked to a molecule encoding a polypeptide. As used herein, the term "operably linked" refers to the joining of two or more nucleic acid sequences in such a way as to produce a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule. For example, a control sequence in a vector "operably linked" to a protein-coding sequence is ligated to the protein-coding sequence such that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequence. More specifically, a promoter and / or enhancer sequence (including any combination of cis-acting transcriptional control elements) is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0325] A promoter is a non-transcribed sequence (generally within about 100-1000 bp) located upstream (i.e., 5') of the start codon of a structural gene and controls the transcription of the structural gene. Promoters are typically classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from polynucleotides under their control in response to some change in culture conditions, such as the presence or absence of nutrients or a change in temperature. Constitutive promoters, on the other hand, transcribe genes to which they are operably linked uniformly, i.e., with little or no control over gene expression. Numerous promoters recognized by a variety of potential host cells are well known. A suitable promoter is operably linked to a polynucleotide encoding, for example, the heavy chain, light chain, or other components of the antibodies, antigen-binding fragments, and bispecific antigen-binding proteins of the present invention by removing the promoter from the original nucleic acid by restriction enzyme digestion and inserting the desired promoter sequence into a vector.

[0326] Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably, simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0327] Additional promoters of interest include the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); ...

Claims

1. 1. A bispecific antigen-binding protein comprising a first binding domain that specifically binds to the human calcitonin gene-related peptide (CGRP) receptor and a second binding domain that specifically binds to the human pituitary adenylate cyclase-activating polypeptide type I (PAC1) receptor, the first binding domain comprises a first light chain immunoglobulin variable region (VL1) and a first heavy chain immunoglobulin variable region (VH1), and the second binding domain comprises a second light chain immunoglobulin variable region (VL2) and a second heavy chain immunoglobulin variable region (VH2); and VL1 comprises CDRL1, CDRL2, and CDRL3 having the sequences of SEQ ID NOs: 6, 13, and 17, respectively; VH1 comprises CDRH1, CDRH2, and CDRH3 having the sequences of SEQ ID NOs: 35, 39, and 44, respectively; and a bispecific antigen binding protein, wherein VL2 comprises CDRL1, CDRL2, and CDRL3 having the sequences of SEQ ID NOs: 131, 141, and 142, respectively, and VH2 comprises (i) CDRH1, CDRH2, and CDRH3 having the sequences of SEQ ID NOs: 157, 165, and 195, respectively, or (ii) CDRH1, CDRH2, and CDRH3 having the sequences of SEQ ID NOs: 157, 166, and 195, respectively.

2. 2. The bispecific antigen-binding protein of claim 1, wherein VL1 comprises the sequence of SEQ ID NO: 25 and VH1 comprises the sequence of SEQ ID NO:

48.

3. (a) VL2 comprises the sequence of SEQ ID NO: 147 and VH2 comprises the sequence of SEQ ID NO: 200; or (b) VL2 comprises the sequence of SEQ ID NO: 147 and VH2 comprises the sequence of SEQ ID NO:

201.

4. 4. The bispecific antigen-binding protein of any one of claims 1 to 3, wherein the binding protein is an antibody comprising a first light chain (LC1) and a first heavy chain (HC1) derived from a first antibody that specifically binds to a human CGRP receptor, and a second light chain (LC2) and a second heavy chain (HC2) derived from a second antibody that specifically binds to a human PAC1 receptor.

5. 5. The bispecific antigen-binding protein of claim 4, wherein LC1 comprises a sequence which is at least 90% identical to the sequence of SEQ ID NO: 72 and HC1 comprises a sequence which is at least 90% identical to a sequence selected from SEQ ID NOs: 87-91.

6. 6. The bispecific binding protein of claim 4, wherein LC1 comprises the sequence of SEQ ID NO: 72 and HC1 comprises a sequence selected from SEQ ID NOs: 87-91.

7. 7. The bispecific binding protein of any one of claims 4 to 6, wherein LC2 comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 232, and HC2 comprises a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 243 to 253.

8. 8. The bispecific binding protein of claim 4, wherein LC2 comprises the sequence of SEQ ID NO: 232 and HC2 comprises a sequence selected from SEQ ID NOs: 243-253.

9. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO: 72, HC1 comprises the sequence of SEQ ID NO: 87, LC2 comprises the sequence of SEQ ID NO: 232, and HC2 comprises the sequence of SEQ ID NO:

250.

10. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO:72, HC1 comprises the sequence of SEQ ID NO:87, LC2 comprises the sequence of SEQ ID NO:232, and HC2 comprises the sequence of SEQ ID NO:

243.

11. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO:72, HC1 comprises the sequence of SEQ ID NO:88, LC2 comprises the sequence of SEQ ID NO:232, and HC2 comprises the sequence of SEQ ID NO:

251.

12. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO:72, HC1 comprises the sequence of SEQ ID NO:88, LC2 comprises the sequence of SEQ ID NO:232, and HC2 comprises the sequence of SEQ ID NO:

244.

13. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO:72, HC1 comprises the sequence of SEQ ID NO:87, LC2 comprises the sequence of SEQ ID NO:232, and HC2 comprises the sequence of SEQ ID NO:

252.

14. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO:72, HC1 comprises the sequence of SEQ ID NO:89, LC2 comprises the sequence of SEQ ID NO:232, and HC2 comprises the sequence of SEQ ID NO:

253.

15. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO: 72, HC1 comprises the sequence of SEQ ID NO: 90, LC2 comprises the sequence of SEQ ID NO: 232, and HC2 comprises the sequence of SEQ ID NO:

247.

16. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO:72, HC1 comprises the sequence of SEQ ID NO:90, LC2 comprises the sequence of SEQ ID NO:232, and HC2 comprises the sequence of SEQ ID NO:

248.

17. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO:72, HC1 comprises the sequence of SEQ ID NO:87, LC2 comprises the sequence of SEQ ID NO:232, and HC2 comprises the sequence of SEQ ID NO:

245.

18. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO:72, HC1 comprises the sequence of SEQ ID NO:89, LC2 comprises the sequence of SEQ ID NO:232, and HC2 comprises the sequence of SEQ ID NO:

246.

19. The bispecific binding protein of any one of claims 4 to 8, wherein LC1 comprises the sequence of SEQ ID NO:72, HC1 comprises the sequence of SEQ ID NO:91, LC2 comprises the sequence of SEQ ID NO:232, and HC2 comprises the sequence of SEQ ID NO:

249.

20. 20. The bispecific antigen-binding protein of any one of claims 1 to 19, wherein the bispecific antigen-binding protein inhibits activation of the human CGRP receptor and the human PAC1 receptor.

21. 21. The bispecific antigen-binding protein of claim 20, wherein the bispecific antigen-binding protein inhibits CGRP-induced activation of the human CGRP receptor with an IC50 of less than 1 nM as measured by a cell-based cAMP assay.

22. 22. The bispecific antigen-binding protein of claim 20 or 21, wherein said bispecific antigen-binding protein inhibits PACAP-induced activation of the human PAC1 receptor with an IC50 of less than 1 nM as measured by a cell-based cAMP assay.

23. 20. One or more isolated polynucleotides encoding the bispecific antigen-binding protein of any one of claims 1 to 19.

24. 24. An expression vector comprising one or more isolated polynucleotides of claim 23.

25. A host cell comprising the vector of claim 24.

26. 26. A method for making a bispecific antigen binding protein that specifically binds to the human CGRP receptor and the human PAC1 receptor, the method comprising culturing the host cell of claim 25 under conditions that allow expression of said antigen binding protein; and recovering said antigen binding protein from the culture medium or the host cell.

27. 20. A pharmaceutical composition comprising the bispecific antigen-binding protein of any one of claims 1 to 19 and a pharmaceutically acceptable excipient.

28. 20. A pharmaceutical composition for treating or preventing a headache condition in a patient in need thereof, the pharmaceutical composition comprising a bispecific antigen-binding protein according to any one of claims 1 to 19.

29. 29. The pharmaceutical composition of claim 28, wherein the headache condition is migraine.

30. 29. The pharmaceutical composition of claim 28, wherein the headache condition is cluster headache.

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