BR112015023429B1 - ISOLATED ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, PHARMACEUTICAL COMPOSITION AND USE OF SAID ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF - Google Patents

Abstract

ISOLATED ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF, ISOLATED POLYNUCLEOTIDE, EXPRESSION VECTOR, CELL LINE, PHARMACEUTICAL COMPOSITION AND USE OF AN ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF. Antibodies and antigen-binding fragments thereof that bind to human PAC1 are provided. Nucleic acids encoding antibodies and antigen-binding fragments thereof, vectors, and cells encoding the same are also provided. Antibodies and antigen-binding fragments thereof can inhibit the binding of PAC1 to PACAP, and are useful in various PAC1-related disorders, including the treatment and/or prevention of headache disorders, including migraine and headache. saved.

Description

PRIORITY

[1] This application claims the benefit of priority to US Provisional Application Serial Number 61/792,678, filed March 15, 2013, the entirety of which is incorporated herein by reference.

REFERENCE FOR SEQUENCE LISTING

[2] This application contains a Sequence Listing that has been submitted electronically in ASCII format through and is incorporated herein by reference in its entirety. Said ASCII copy, created on March 12, 2014, is called A-1804-PCT_SL.txt and is 461,614 bytes in size.

BASIS

[3] There is a significant unmet need for effective therapies, particularly prophylaxis therapies, for migraine. Results from several studies indicate that ~14 million migraine sufferers in the United States could qualify for and benefit from safe and effective preventive therapy. Currently approved migraine prophylactic therapies are only partially effective and have considerable side effect profiles that significantly limit the acceptability of these medications. Despite these limitations, 4.5 million individuals with frequent migraines in the United States take prophylactic medication for their migraines.

[4] Pituitary adenylate cyclase-activating polypeptides (PACAP) are peptides of 38 amino acids (PACAP38), or 27 amino acids (PACAP27) that were first isolated from an ovine hypothalamic extract based on their ability to stimulate the formation of cAMP in anterior pituitary cells (Miyata, A., et al., “Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells.”, Biochem Biophys Res Commun. 1989;164:567-574; Miyata, A ., et al., “Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38.”), Biochem Biophys Res Commun. 1990; 170:643-648). PACAP peptides belong to the VIP-secretin hormone-releasing hormone (GHRH)-glucagon vasoactive intestinal polypeptide superfamily with the sequence of human PACAP27 sharing 68% identity with vasoactive intestinal polypeptide (VIP) (Campbell, R.M. and Scanes, C.G., “ Evolution of the growth hormone-releasing factor (GRF) family of peptides. Growth Regul.” 1992; 2:175-191). The main form of PACAP peptide in the human body is PACAP38 and the pharmacology of PACAP38 and PACAP27 has not been shown to be different from each other. Unless otherwise noted here, PACAP or PACAP38 will be used to represent PACAP38, and PACAP27 will be used to specify PACAP27.

[5] Three PACAP receptors have been reported: one that binds PACAP with high affinity and has a very low affinity for VIP (PAC1 receptor, or simply “PAC1”) and two that recognize PACAP and VIP essentially equally well (VPAC1 and VPAC2, or simply “VPAC1” or “VPAC2”, respectively) (Vaudry, D., et al. “Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery.”, Pharmacol Rev. 2009; Sep 61( 3):283-357).

[6] PACAP is able to bind to all three receptors (PAC1, VPAC1, VPAC2) with similar potency and is therefore not particularly selective. VIP, on the other hand, binds with significantly higher affinity to VPAC1 and VPAC2 compared to PAC1. Maxadilan, a 65 amino acid peptide originally isolated from the sandfly (Lerner, E.A., et al., “Isolation of maxadilan, a potent vasodilatory peptide from the salivary glands of the sand fly Lutzomyia longipalpis”, J Biol Chem. 1991 Jun 15; 266(17):11234-6; Lerner, E.A., et al., “Maxadilan, a PAC1 receptor agonist from sand flies”, Peptides. Sep 2007; 28(9): 1651-1654.), is exquisitely selective for PAC1 compared to VPAC1 or VPAC2 and therefore can be used as a selective PAC1 agonist.

SUMMARY

[7] In one aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising: (A) a light chain CDR1 comprising (i) an amino acid sequence selected from the group consisting of the CDR1 LC sequences set forth in Table 4A, (ii) an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the CDR1 LC sequences set forth in Table 4A, or (iii) an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the CDR1 LC sequences set forth in Table 4A; (B) a light chain CDR2 comprising (i) an amino acid sequence selected from the group consisting of the CDR2 LC sequences set forth in Table 4A, (ii) an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the CDR2 LC sequences set forth in Table 4A, or (iii) an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the CDR2 LC sequences set forth in Table 4A; and (C) a light chain CDR3 comprising (i) an amino acid sequence selected from the group consisting of the CDR3 LC sequences set forth in Table 4A, (ii) an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the CDR3 LC sequences set forth in Table 4A, or (iii) an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the CDR3 LC sequences set forth in Table 4A.

[8] In another aspect, the invention includes an antibody or antigen-binding fragment thereof, wherein (A) the antibody or antigen-binding fragment thereof competes for binding, with a Ki of 10 nM or less (e.g. example, 5 nM or 1 nM), human PAC1 with a reference antibody, said reference antibody selected from the group consisting of 01B, 14A, 14B, 26A, 26B, 28A, 29A, 29B, 39A, and 39B; and (B) the antibody or antigen-binding fragment thereof specifically binds to human PAC1.

[9] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising: (A) a heavy chain CDR1 comprising (i) an amino acid sequence selected from the group consisting of the CDR1 HC sequences set forth in Table 4B, (ii) an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the CDR1 HC sequences set forth in Table 4B, or (iii) an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the CDR1 HC sequences set forth in Table 4B; (B) a heavy chain CDR2 comprising (i) an amino acid sequence selected from the group consisting of the CDR2 HC sequences set forth in Table 4B, (ii) an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the CDR2 HC sequences set forth in Table 4B, or (iii) an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the CDR2 HC sequences set forth in Table 4B; and (C) a heavy chain CDR3 comprising (i) an amino acid sequence selected from the group consisting of the CDR3 HC sequences set forth in Table 4B, (ii) an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the CDR3 HC sequences set forth in Table 4B, or (iii) an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the CDR3 HC sequences set forth in Table 4B.

[10] In another aspect, the invention includes an antibody or antigen-binding fragment thereof, wherein (A) the CDRLs comprise (i) a CDRL1 selected from the group consisting of CDRL1-C1 and CDRL1-C2, (ii ) a CDRL2 selected from the group consisting of CDRL2-C1, CDRL2-C2, and CDRL2-C3, and (iii) a CDRL3 consisting of CDRL3-C1; (B) CDRHs comprise (i) a CDRH1 selected from the group consisting of CDRH1-C1 and CDRH1-C2; (ii) a CDRH2 selected from the group consisting of CDRH2-C1, CDRH2-C2, and CDRH2-C3; and (iii) a CDRH3 consisting of CDRH3-C1, and (C) the antibody or antigen-binding fragment thereof specifically binds to human PAC1.

[11] In an above embodiment, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C1, CDRL3-C1, CDRH1-C1, CDRH2-C1, and CDRH3-C1.

[12] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C1, CDRL3-C1, CDRH1-C1, CDRH2-C2, and CDRH3-C1.

[13] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C1, CDRL3-C1, CDRH1-C1, CDRH2-C3, and CDRH3-C1.

[14] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C1, CDRL3-C1, CDRH1-C2, CDRH2-C1, and CDRH3-C1.

[15] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C1, CDRL3-C1, CDRH1-C2, CDRH2-C2, and CDRH3-C1.

[16] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C1, CDRL3-C1, CDRH1-C2, CDRH2-C3, and CDRH3-C1.

[17] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C2, CDRL3-C1, CDRH1-C1, CDRH2-C1, and CDRH3-C1.

[18] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C2, CDRL3-C1, CDRH1-C1, CDRH2-C2, and CDRH3-C1.

[19] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C2, CDRL3-C1, CDRH1-C1, CDRH2-C3, and CDRH3-C1.

[20] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C2, CDRL3-C1, CDRH1-C2, CDRH2-C1, and CDRH3-C1.

[21] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C2, CDRL3-C1, CDRH1-C2, CDRH2-C2, and CDRH3-C1.

[22] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C2, CDRL3-C1, CDRH1-C2, CDRH2-C3, and CDRH3-C1.

[23] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C3, CDRL3-C1, CDRH1-C1, CDRH2-C1, and CDRH3-C1.

[24] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C3, CDRL3-C1, CDRH1-C1, CDRH2-C2, and CDRH3-C1.

[25] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C3, CDRL3-C1, CDRH1-C1, CDRH2-C3, and CDRH3-C1.

[26] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C3, CDRL3-C1, CDRH1-C2, CDRH2-C1, and CDRH3-C1.

[27] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C3, CDRL3-C1, CDRH1-C2, CDRH2-C2, and CDRH3-C1.

[28] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C1, CDRL2-C3, CDRL3-C1, CDRH1-C2, CDRH2-C3, and CDRH3-C1.

[29] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C1, CDRL3-C1, CDRH1-C1, CDRH2-C1, and CDRH3-C1.

[30] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C1, CDRL3-C1, CDRH1-C1, CDRH2-C2, and CDRH3-C1.

[31] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C1, CDRL3-C1, CDRH1-C1, CDRH2-C3, and CDRH3-C1.

[32] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C1, CDRL3-C1, CDRH1-C2, CDRH2-C1, and CDRH3-C1.

[33] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C1, CDRL3-C1, CDRH1-C2, CDRH2-C2, and CDRH3-C1.

[34] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C1, CDRL3-C1, CDRH1-C2, CDRH2-C3, and CDRH3-C1.

[35] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C2, CDRL3-C1, CDRH1-C1, CDRH2-C1, and CDRH3-C1.

[36] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C2, CDRL3-C1, CDRH1-C1, CDRH2-C2, and CDRH3-C1.

[37] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C2, CDRL3-C1, CDRH1-C1, CDRH2-C3, and CDRH3-C1.

[38] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C2, CDRL3-C1, CDRH1-C2, CDRH2-C1, and CDRH3-C1.

[39] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C2, CDRL3-C1, CDRH1-C2, CDRH2-C2, and CDRH3-C1.

[40] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C2, CDRL3-C1, CDRH1-C2, CDRH2-C3, and CDRH3-C1.

[41] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C3, CDRL3-C1, CDRH1-C1, CDRH2-C1, and CDRH3-C1.

[42] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C3, CDRL3-C1, CDRH1-C1, CDRH2-C2, and CDRH3-C1.

[43] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C3, CDRL3-C1, CDRH1-C1, CDRH2-C3, and CDRH3-C1.

[44] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C3, CDRL3-C1, CDRH1-C2, CDRH2-C1, and CDRH3-C1.

[45] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C3, CDRL3-C1, CDRH1-C2, CDRH2-C2, and CDRH3-C1.

[46] In another embodiment above, the antibody or antigen-binding fragment thereof comprises CDRL1-C2, CDRL2-C3, CDRL3-C1, CDRH1-C2, CDRH2-C3, and CDRH3-C1.

[47] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising (i) heavy chain CDRs CDR1, CDR2 and CDR3, each having a CDR sequence corresponding heavy chain CDRs as above, and (ii) light chain CDRs CDR1, CDR2 and CDR3, each having a sequence of the corresponding heavy chain CDR as above.

[48] In one embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-8, CDRH2-2 and CDRH3-3; and light chain CDRs: CDRL1-3, CDRL2-2, CDRL3-2.

[49] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-11, CDRH2-5 and CDRH3-9; and light chain CDRs: CDRL1-13, CDRL2-1, CDRL3-1.

[50] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-1, CDRH2-4 and CDRH3-6; and light chain CDRs: CDRL1-2, CDRL2-1, CDRL3-1.

[51] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-1, CDRH2-4 and CDRH3-6; and light chain CDRs: CDRL1-1, CDRL2-1, CDRL3-1.

[52] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-3, CDRH2-5 and CDRH3-5; and light chain CDRs: CDRL1-1, CDRL2-1, CDRL3-1.

[53] In another embodiment, the invention includes an antibody or antigen-binding fragment

[54] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-9, CDRH2-6 and CDRH3-1; and light chain CDRs: CDRL1-4, CDRL2-3, CDRL3-3.

[55] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-4, CDRH2-7 and CDRH3-8; and light chain CDRs: CDRL1-6, CDRL2-4, CDRL3-5.

[56] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-10, CDRH2-1 and CDRH3-7; and light chain CDRs: CDRL1-5, CDRL2-3, CDRL3-4.

[57] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-6, CDRH2-8 and CDRH3-2; and light chain CDRs: CDRL1-7, CDRL2-5, CDRL3-6.

[58] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-7, CDRH2-3 and CDRH3-4; and light chain CDRs: CDRL1-8, CDRL2-6, CDRL3-7.

[59] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-2, CDRH2-8 and CDRH3-2; and light chain CDRs: CDRL1-9, CDRL2-5, CDRL3-8.

[60] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-6, CDRH2-8 and CDRH3-2; and light chain CDRs: CDRL1-10, CDRL2-5, CDRL3-6.

[61] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-6, CDRH2-8 and CDRH3-2; and light chain CDRs: CDRL1-10, CDRL2-5, CDRL3-6.

[62] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-6, CDRH2-8 and CDRH3-2; and light chain CDRs: CDRL1-12, CDRL2-5, CDRL3-6.

[63] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising heavy chain CDRs: CDRH1-5, CDRH2-8 and CDRH3-2; and light chain CDRs: CDRL1-11, CDRL2-5, CDRL3-8.

[64] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a heavy chain CDR1 selected from the group consisting of CDRH1-1, CDRH1-2, CDRH1 -3, CDRH1-4, CDRH1-5, CDRH1-6, CDRH1-7, CDRH1-8, CDRH1-9, CDRH1-10, and CDRH1-11.

[65] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a heavy chain CDR2 selected from the group consisting of CDRH2-1, CDRH2-2, CDRH2 -3, CDRH2-4, CDRH2-5, CDRH2-6, CDRH2-7, and CDRH2-8.

[66] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a heavy chain CDR3 selected from the group consisting of CDRH3-1, CDRH3-2, CDRH3 -3, CDRH3-4, CDRH3-5, CDRH3-6, CDRH3-7, CDRH3-8 and CDRH3-9.

[67] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a light chain CDR1 selected from the group consisting of CDRL1-1, CDRL1-2, CDRL1 - 3, CDRL1-4, CDRL1-5, CDRL1-6, CDRL1-7, CDRL1-8, CDRL1-9, CDRL1- 10, CDRL1-11, CDRL1-12 and CDRL1-13.

[68] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a light chain CDR2 selected from the group consisting of CDRL2-1, CDRL2-2, CDRL2 - 3, CDRL2-4, CDRL2-5, and CDRL2-6.

[69] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a light chain CDR3 selected from the group consisting of CDRL3-1, CDRL3-2, CDRL3 - 3, CDRL3-4, CDRL3-5, CDRL3-6, CDRL3-7, and CDRL3-8.

[70] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that optionally specifically binds to human PAC1, comprising a heavy chain CDR1 as above, a heavy chain CDR2 as above, a CDR3 of heavy chain as above, a light chain CDR1 as above, a light chain CDR2 as above, and a light chain CDR3 as above.

[71] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that optionally specifically binds to human PAC1, comprising a light chain variable region comprising (i) an amino acid sequence selected from the group consisting in the AA VL sequences set forth in Table 3A, (ii) an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the AA VL sequences set forth in Table 3A, or (iii) an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of the AA VL sequences set forth in Table 3A.

[72] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a heavy chain variable region comprising (i) an amino acid sequence selected from the group consisting of the AA VH sequences set forth in Table 3B, (ii) an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of the AA VH sequences set forth in Table 3B, or (iii) an amino acid sequence at least 95 % identical to an amino acid sequence selected from the group consisting of the AA VH sequences set forth in Table 3B.

[73] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a light chain variable region amino acid sequence 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, LV-12, LV-13, LV-14 , LV-15, LV-16, LV-17, LV-18, LV-19, LV-20, and LV-21.

[74] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a heavy chain variable region amino acid sequence selected from the group consisting of HV-01, HV -02, HV-03, HV-04, HV-05, HV-06, HV-07, HV-08, HV-09, HV-10, HV-11, HV-12, HV-13, HV-14 , HV-15, HV-16, HV- 17, HV-18, HV-19, HV-20, HV-21, HV-22, HV-23, HV-24, HV-25, and HV- 26.

[75] In one embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-01 and HV-01.

[76] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-02 and HV-02.

[77] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-03 and HV-02.

[78] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-04 and HV-03.

[79] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-05 and HV-03.

[80] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-06 and HV-03.

[81] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-04 and HV-04.

[82] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-04 and HV-05.

[83] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-07 and HV-04.

[84] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-07 and HV-05.

[85] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-07 and HV-03.

[86] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-04 and HV-06.

[87] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-04 and HV-06.

[88] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-05 and HV-05.

[89] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-08 and HV-05.

[90] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-09 and HV-05.

[91] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-05 and HV-07.

[92] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-05 and HV-08.

[93] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-09 and HV-07.

[94] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-09 and HV-08.

[95] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-09 and HV-09.

[96] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-05 and HV-09.

[97] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-05 and HV-10.

[98] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-08 and HV-10.

[99] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-05 and HV-11.

[100] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-05 and HV-12.

[101] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-05 and HV-13.

[102] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-08 and HV-13.

[103] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-10 and HV-14.

[104] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-11 and HV-14.

[105] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-12 and HV-15.

[106] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-12 and HV-16.

[107] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-13 and HV-17.

[108] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-14 and HV-18.

[109] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-14 and HV-19.

[110] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-15 and HV-20.

[111] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-16 and HV-21.

[112] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-17 and HV-22.

[113] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-18 and HV-23.

[114] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-19 and HV-24.

[115] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-19 and HV-25.

[116] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-20 and HV-21.

[117] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LV-21 and HV-26.

[118] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising (i) a sequence selected from the group consisting of the sequences set forth in Table 2A, or ( ii) a sequence at least 90% identical to one of the sequences set out in Table 2A, or (iii) a sequence at least 95% identical to one of the sequences set out in Table 2A.

[119] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising (i) a sequence selected from the group consisting of the sequences set forth in Table 2B, or ( ii) a sequence at least 90% identical to one of the sequences set out in Table 2B, or (iii) a sequence at least 95% identical to one of the sequences set out in Table 2B.

[120] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a light chain amino acid sequence selected from the group consisting of LC-01, LC-02 , LC-03, LC-04, LC-05, LC-06, LC-07, LC-08, LC-09, LC-10, LC-11, LC-12, LC-13, LC-14, LC -15, LC-16, LC-17, LC-18, LC-19, LC-20, and LC-21.

[121] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising a heavy chain amino acid sequence selected from the group consisting of HC-01, HC-02 , HC-03, HC-04, HC-05, HC-06, HC-07, HC-08, HC-09, HC-10, HC-11, HC-12, HC-13, HC-14, HC -15, HC-16, HC-17, HC-18, HC- 19, HC-20, HC-21, HC-22, HC-23, HC-24, HC-25, HC-26, HC-27 , HC- 28, HC-29, HC-30, HC-31, HC-32, HC-33, HC-34, HC-35, HC-36, HC- 37, HC-38, HC-39, HC -40, HC-41, HC-42, HC-43, HC-44, HC-45, HC-46, HC-47, HC-48, HC-49, HC-50, HC-51, HC-52 , and HC-53.

[122] In one embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-01 and HC-01 (01A).

[123] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-01 and HC-02 (01B).

[124] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-02 and HC-03 (02A).

[125] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-02 and HC-04 (02B).

[126] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-02 and HC-05 (02C).

[127] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-03 and HC-03 (03A).

[128] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-06 (04A).

[129] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-07 (04B).

[130] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-08 (04C).

[131] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-06 (05A).

[132] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-07 (05B).

[133] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-08 (05C).

[134] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-06 and HC-06 (06A).

[135] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-06 and HC-07 (06B).

[136] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-06 and HC-08 (06C).

[137] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-09 (07A).

[138] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-10 (07B).

[139] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-11 (07C).

[140] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-12 (08A).

[141] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-13 (08B).

[142] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-14 (08C).

[143] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-07 and HC-09 (09A).

[144] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-07 and HC-10 (09B).

[145] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-07 and HC-11 (09C).

[146] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-07 and HC-12 (10A).

[147] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-07 and HC-13 (10B).

[148] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-07 and HC-14 (10C).

[149] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-07 and HC-06 (11A).

[150] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-07 and HC-07 (11B).

[151] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-07 and HC-08 (11C).

[152] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-15 (12A).

[153] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-16 (12B).

[154] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-04 and HC-17 (12C).

[155] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-12 (13A).

[156] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-13 (13B).

[157] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-14 (13C).

[158] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-08 and HC-12 (14A).

[159] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-08 and HC-13 (14B).

[160] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-08 and HC-14 (14C).

[161] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-12 (15A).

[162] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-13 (15B).

[163] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-14 (15C).

[164] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-18 (16A).

[165] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-19 (16B).

[166] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-20 (16C).

[167] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-21 (17A).

[168] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-22 (17B).

[169] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-23 (17C).

[170] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-18 (18A).

[171] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-19 (18B).

[172] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-20 (18C).

[173] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-21 (19A).

[174] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-22 (19B).

[175] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-23 (19C).

[176] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-24 (20A).

[177] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-25 (20B).

[178] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-09 and HC-26 (20C).

[179] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-24 (21A).

[180] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-25 (21B).

[181] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-26 (21C).

[182] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-27 (22A).

[183] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-28 (22B).

[184] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-29 (22C).

[185] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-08 and HC-27 (23A).

[186] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-08 and HC-28 (23B).

[187] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-08 and HC-29 (23C).

[188] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-30 (24A).

[189] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-31 (24B).

[190] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-32 (24C).

[191] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-33 (25A).

[192] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-34 (25B).

[193] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-35 (25C).

[194] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-36 (26A).

[195] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-37 (26B).

[196] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-05 and HC-38 (26C).

[197] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-08 and HC-36 (27A).

[198] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-08 and HC-37 (27B).

[199] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-08 and HC-38 (27C).

[200] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-10 and HC-39 (28A).

[201] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-10 and HC-40 (28B).

[202] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-10 and HC-41 (28C).

[203] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-11 and HC-39 (29A).

[204] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-11 and HC-40 (29B).

[205] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-11 and HC-41 (29C).

[206] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-12 and HC-42 (30A).

[207] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-12 and HC-43 (31A).

[208] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-13 and HC-44 (32A).

[209] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-14 and HC-45 (33A).

[210] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-14 and HC-46 (34A).

[211] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-15 and HC-47 (35A).

[212] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-16 and HC-48 (36A).

[213] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-17 and HC-49 (37A).

[214] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-18 and HC-50 (38A).

[215] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-19 and HC-51 (39A).

[216] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-19 and HC-52 (39B).

[217] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-20 and HC-48 (40A).

[218] In another embodiment, the invention includes an antibody or antigen-binding fragment thereof comprising LC-21 and HC-53 (41A).

[219] In another aspect, the invention includes an isolated antibody or antigen-binding fragment thereof from any of the above, wherein the antibody is a polyclonal antibody.

[220] In another aspect, the invention includes the isolated antibody of any of the above, wherein the isolated antibody is selected from the group consisting of a monoclonal antibody, a Fab fragment, a Fab' fragment, an F(ab') fragment 2, an Fv fragment, a diabody, and a single-chain antibody.

[221] In another aspect, the invention includes the isolated antibody as above, wherein the isolated antibody is a monoclonal antibody selected from the group consisting of a fully human antibody, a humanized antibody and a chimeric antibody.

[222] In one embodiment above, the antibody isolated according to any one above is a fully human monoclonal antibody.

[223] In another aspect, the invention includes the antibody isolated as above, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 type antibody.

[224] In another aspect, the invention includes the antibody isolated as above, wherein the antibody is an aglycosylated IgG1 antibody.

[225] In another aspect, the invention includes the antibody isolated as above, wherein the aglycosylated IgG1 antibody comprises a mutation at position N297 in its heavy chain.

[226] In another aspect, the invention includes the antibody isolated as above, wherein the aglycosylated IgG1 antibody comprises the N297G substitution in its heavy chain.

[227] In another aspect, the invention includes the antibody isolated as above, wherein the aglycosylated IgG1 antibody comprises the substitutions N297G, R292C and V302C in its heavy chain.

[228] In another aspect, the invention includes the antibody isolated with any of the above, wherein the antibody selectively inhibits hPAC1 over hVPA1 and hVPAC2.

[229] In another aspect, the invention includes the antibody isolated as above, wherein the selectivity ratio is greater than 100:1.

[230] In another aspect, the invention includes the antibody isolated as above, wherein the selectivity ratio is greater than 1000:1.

[231] In another aspect, the invention includes an isolated polynucleotide that encodes an antibody like any of the above.

[232] In another aspect, the invention includes an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising (i) a sequence selected from the group consisting of the sequences set forth in Table 1A, or (ii) a sequence at least 90% identical to one of the sequences set out in Table 1A, or (iii) a sequence at least 95% identical to one of the sequences set out in Table 1A.

[233] In another aspect, the invention includes an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to human PAC1, comprising (i) a sequence selected from the group consisting of the sequences set forth in Table 1B, or (ii) a sequence at least 90% identical to one of the sequences set forth in Table 1B, or (iii) a sequence at least 95% identical to one of the sequences set forth in Table 1B.

[234] In another aspect, the invention includes an isolated polynucleotide having a nucleic acid sequence selected from the group consisting of the sequences set forth in Table 1A, (ii) a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of the sequences set forth in Table 1A, (iii) a nucleic acid sequence at least 90% identical to a nucleic acid sequence selected from the group consisting of the sequences set forth in Table 1A, or (iv) a sequence of nucleic acid at least 95% identical to a nucleic acid sequence selected from the group consisting of the sequences set forth in Table 1A.

[235] In another aspect, the invention includes an isolated polynucleotide having a nucleic acid sequence selected from the group consisting of the sequences set forth in Table 1B, (ii) a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of the sequences set forth in Table 1B, (iii) a nucleic acid sequence at least 90% identical to a nucleic acid sequence selected from the group consisting of the sequences set forth in Table 1B, or (iv) a sequence of nucleic acid at least 95% identical to a nucleic acid sequence selected from the group consisting of the sequences set forth in Table 1B.

[236] In another aspect, the invention includes an isolated polynucleotide having a nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding the variable regions set forth in Table 3A, (ii) a nucleic acid sequence of at least 80 % identical to a nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding the variable regions set forth in Table 3A, (iii) a nucleic acid sequence at least 90% identical to a nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding the variable regions set forth in Table 3A, or (iv) a nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding the variable regions set out in Table 3A. In another aspect, the invention includes an isolated polynucleotide having a nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding the variable regions set forth in Table 3B, (ii) a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding the variable regions set forth in Table 3B, (iii) a nucleic acid sequence at least 90% identical to a nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding the variable regions set forth in Table 3B, or (iv) a nucleic acid sequence at least 95% identical to a nucleic acid sequence selected from the group consisting of nucleic acid sequences encoding the established variable regions in Table 3A.

[237] In another aspect, the invention includes an expression vector comprising an isolated nucleic acid or polynucleotide as any of the above.

[238] In another aspect, the invention includes a cell line transformed with an expression vector as above.

[239] In another aspect, the invention includes a method for preparing an antibody or antigen-binding fragment thereof as any of the above, comprising preparing the antibody or antigen-binding fragment thereof from a host cell such as any above which secretes the antibody or antigen-binding fragment thereof.

[240] In another aspect, the invention includes a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as any one above, and a pharmaceutically acceptable excipient.

[241] In another aspect, the invention includes a method for treating a condition associated with PAC1 in a patient, comprising administering to a patient an effective amount of an antibody or antigen-binding fragment thereof as any of the above.

[242] In another aspect, the invention includes the method as above, wherein the condition is headache.

[243] In another aspect, the invention includes the method as above, wherein the condition is migraine.

[244] In another aspect, the invention includes the method as above, wherein the migraine is episodic migraine.

[245] In another aspect, the invention includes the method as above, wherein the migraine is chronic migraine.

[246] In another aspect, the invention includes the method as above, wherein the condition is cluster headache.

[247] In another aspect, the invention includes the method of any of the above, wherein the method comprises prophylactic treatment.

BRIEF DESCRIPTION OF FIGURES

[248] FIG. 1 shows selected anti-hPAC1 light chain CDR sequences and defined anti-hPAC1 light chain CDR consensus sequences CDRL1-C1, CDRL1-C2, CDRL2-C1, CDRL2-C2, CDRL2-C3, and CDRL3-C1.

[249] FIG. 2 shows selected anti-hPAC1 heavy chain CDR sequences and defined anti-hPAC1 heavy chain CDR consensus sequences CDRH1-C1, CDRH1-C2, CDRH2-C1, CDRH2-C2, CDRH2-C3, and CDRH3-C1.

DETAILED DESCRIPTION

[250] The section titles here are for organizational purposes only and should not be construed as limiting the subject matter described.

[251] Unless otherwise defined, scientific and technical terms used in connection with the present invention will have the meanings that are commonly understood by those skilled in the art. Unless otherwise required by the context, singular terms shall include pluralities and plural terms shall include the singular.

[252] Generally, the nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those known and commonly used in the art. The methods and techniques of the present invention are generally carried out in accordance with conventional methods known in the art and as described in various more general and specific references that are cited and discussed throughout this specification unless otherwise indicated. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990) which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, or as is commonly performed in the art or as described herein. The terminologies used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are those known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analysis, pharmaceutical preparation, formulation and release, and patient treatment.

[253] It should be understood that this invention is not limited to the methodology, protocols, and reagents etc. particulars described here, and as such may vary. The terminology used here is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be defined solely by the claims.

[254] Unlike the working examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about”. The term “about” when used in connection with percentages means ±10%.

Definitions

[255] The term “polynucleotide” or “nucleic acid” includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides or a modified form of any type of nucleotide.

[256] An “isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof, which is not associated with the whole or a part of a polynucleotide in which the polynucleotide isolated is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that “a nucleic acid molecule comprising” a particular nucleotide sequence does not include intact chromosomes. Isolated nucleic acid molecules "comprising" specified nucleic acid sequences may include, in addition to the specified sequences, sequences encoding up to ten or even up to twenty other proteins or parts thereof, or may include operably linked regulatory sequences that control expression of the region encoding the recited nucleic acid sequences, and/or may include vector sequences.

[257] Unless otherwise specified, the left end of any single-stranded polynucleotide sequence discussed here is the 5' end; the left direction of the double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the direction of transcription; Sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5' to the 5' end of the RNA transcript are referred to as “upstream sequences;” Sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3' to the 3' end of the RNA transcript are referred to as “downstream sequences.”

[258] The term “control sequence” refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is linked. The nature of these control sequences may depend on the host organism. In particular embodiments, control sequences for prokaryotes may include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, control sequences for eukaryotes may include promoters, comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, and termination sequences. “Control sequences” may include leader sequences and/or fusion partner sequences.

[259] The term “vector” means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein-coding information into a host cell.

[260] The term “expression vector” or “expression construct” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the cell host) the expression of one or more heterologous coding regions operably linked thereto. An expression construct may include, among others, sequences that affect or control transcription, translation and, if introns are present, affect RNA processing of a coding region operably linked thereto.

[261] As used herein, “operationally linked” means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions under suitable conditions. For example, a control sequence in a vector that is “operationally linked” to a protein-coding sequence is linked thereto so that expression of the protein-coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

[262] The term “host cell” means a cell has been transformed, or is capable of being transformed, with a nucleic acid sequence and thus expresses a gene of interest. The term includes the offspring of the parent cell, regardless of whether the offspring are identical in morphology or genetic makeup to the original parent cell while the gene of interest is present.

[263] The term “transduction” means the transfer of genes from one bacterium to another, usually by bacteriophage. “Transduction” also refers to the acquisition and transfer of eukaryotic cellular sequences by replication-defective retroviruses.

[264] The term “transfection” means the uptake of foreign or exogenous DNA by a cell, and a cell has been “transfected” when exogenous DNA has been introduced into the cell membrane. Various transfection techniques are well known and are disclosed herein. See, for example, Graham et al. , 1973, Virology 52:456; Sambrook et al. , 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. These techniques can be used to introduce one or more exogenous DNA fractions into suitable host cells.

[265] The term “transformation” refers to a change in the genetic characteristics of the cell, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed where it is genetically modified from its native state by introducing new genetic material through transfection, transduction or other techniques. After transfection or transduction, the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell, or it can be maintained transiently as an episomal element without being replicated, or it can replicate independently as a plasmid. A cell is considered to be “stably transformed” when the transforming DNA is replicated as the cell divides.

[266] The terms “polypeptide” or “protein” are used interchangeably here to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an analogue or mimetic of a corresponding naturally occurring amino acid, as well as polymers of naturally occurring amino acids. The terms may also include polymers of amino acids that have been modified, for example by the addition of carbohydrate residues to form glycoproteins, or phosphorylated. Polypeptides and proteins can be produced by a cell naturally occurring and non-recombinant; or are produced by a genetically modified or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions of, additions to and/or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically include antibodies, e.g., anti-PAC1 antibodies (also known as PAC1 antibodies), PAC1-binding proteins, antibodies or sequences that have deletions of, additions to, and/or substitutions of a or more amino acids of an antigen-binding protein. The term “polypeptide fragment” refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion compared to the entire protein. These fragments may also contain modified amino acids compared to the entire protein. In certain embodiments, fragments are about five to 500 amino acids in length. For example, fragments may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of a PAC1-binding antibody, useful fragments include, among others, a CDR region, a heavy or light chain variable domain, a part of an antibody chain or just its variable domain including two CDRs, and the like.

[267] The term “isolated protein” (e.g., isolated antibody), “isolated polypeptide,” or “isolated antibody” means that a protein, polypeptide, or antibody is free of most other proteins in which it would normally be found and has been separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which they are associated in nature. Typically, an “isolated protein,” “isolated polypeptide,” or “isolated antibody” constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. . Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode an isolated protein. Preferably, the isolated protein, polypeptide or antibody is substantially free of other proteins or other polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, or other use.

[268] The terms “human PAC1”, “human PAC1”, “hPAC1” and “hPAC1”, “human PAC1 receptor”, “human PAC1 receptor”, “hPAC1 receptor” and “hPAC1 receptor” are used interchangeably. interchangeable and refer to the human pituitary adenylate cyclase-activating polypeptide type I receptor. hPAC1 is a 468 amino acid protein designated as P41586 (PACR_HUMAN) in the UniProtKB/Swiss-Prot database and is encoded by the ADCYAP1R1 gene. PACAP-27 and PACAP-38 are the main endogenous PAC1 agonists. Unless otherwise specified or clear from the context in which the term is used, “PAC1” refers to human hPAC1.

[269] A “variant” of a polypeptide (e.g., an antigen-binding protein, or an antibody) comprises an amino acid sequence in which one or more amino acid residues are inserted, deleted, and/or substituted in the amino acid sequence relative to the other polypeptide sequence. Variants include fusion proteins.

[270] A “derivative” of a polypeptide is a polypeptide (e.g., an antigen-binding protein, or an antibody) that has been chemically modified in some manner distinct from insertion, deletion, or substitution variants, e.g., through conjugation of another chemical moiety.

[271] The term “naturally occurring” as used throughout the specification in connection with biological materials such as polypeptides, nucleic acids, host cells and the like, refers to materials that are found in nature.

[272] Antibody or antigen-binding fragment thereof is said to “specifically bind” to its target when the dissociation constant (KD) is <10-6 M. The antibody specifically binds to the target antigen with “high affinity ” when the KD is <1x 10-8 M. In one embodiment, antibodies or antigen-binding fragments thereof will bind to PAC1, or human PAC1 with a KD <5x 10-7; in another embodiment the antibodies or antigen binding fragments thereof will bind with a KD <1x 10-7; in another embodiment the antibodies or antigen binding fragments thereof will bind with a KD <5x 10-8; in another embodiment the antibodies or antigen binding fragments thereof will bind with a KD <1x 10-8; in another embodiment the antibodies or antigen binding fragments thereof will bind with a KD <5x 10-9; in another embodiment the antibodies or antigen binding fragments thereof will bind with a KD <1x 10-9; in another embodiment the antibodies or antigen binding fragments thereof will bind with a KD <5x 10-10; in another embodiment the antibodies or antigen binding fragments thereof will bind with a KD <1x 10-10.

[273] An antibody, antigen-binding fragment thereof, or antigen-binding protein “selectively inhibits” a specific receptor relative to other receptors when the IC50 of the antibody, antigen-binding fragment thereof, or antigen-binding protein antigen in a specific receptor inhibition assay is at least 50 times lower than the IC50 in an inhibition assay of another “reference” receptor, for example, an hVPAC1 or hVPAC2 receptor. The “selectivity ratio” is the IC50 of the reference receiver divided by the IC50 of the specific receiver. For example, if hPAC1 is the specific receptor and hVPAC1 is the reference receptor, and the IC50 of a particular anti-PAC1 antibody described herein against hPAC1 is 10 nm and against hVPACI is 10 μm, that particular antibody can be characterized as having a 1000:1 selectivity ratio for hPAC1 over hVPAC1. IC50 is the dose/concentration required to achieve 50% inhibition (of a biological or biochemical function). With radioactive ligands, IC50 is the concentration of a competing ligand that displaces 50% of the specific binding of the radioligands. EC50 is the concentration or dose required to induce a response of 50% of the maximum response.

[274] Ki data are typically generated in the context of competition binding experiments, which are typically done by binding a radioactive isotope (e.g., a 125I) to an “agonist”. The experiments measure agonist binding at increasing concentrations of an “antagonist.” Ki refers to the concentration of the “antagonist” or test compound, for example, an anti-PAC1 test antibody, that would occupy 50% of the receptors if there were no radioligand present. Ki can be calculated using the equation Ki=IC50/(1+([L]/Kd)), where [L] is the concentration of the radioligand used (e.g., a 125I-labeled PAC-1 antibody) and Kd is the radioligand dissociation constant. See, for example, Keen M, MacDermot J (1993) Analysis of receptors by radioligand binding. In: Wharton J, Polak JM (eds) Receiver autoradiography, principles and practice. OxfordUniversity Press, Oxford.

[275] “Antigen-binding region” means a protein, or a part of a protein, that specifically binds a specified antigen. For example, that part of an antibody that contains the amino acid residues that interact with an antigen and give the antibody its specificity and affinity for the antigen is referred to as the “antigen-binding region.” An antigen binding region typically includes one or more “complementary binding regions” (“CDRs”). Certain antigen-binding regions also include one or more “framework” regions. A “CDR” is an amino acid sequence that contributes to antigen binding affinity and specificity. “Framework” regions can help maintain the proper conformation of CDRs to promote binding between the antigen-binding region and an antigen.

[276] In certain aspects, recombinant antibodies or antigen-binding fragments thereof that bind to the PAC1 protein, or human PAC1, are provided. In this context, a "recombinant protein" is a protein prepared using recombinant techniques, that is, through the expression of a recombinant nucleic acid as described herein. Methods and techniques for producing recombinant proteins are well known.

[277] The term “antibody” refers to an intact immunoglobulin of any isotype, or an antigen-binding fragment thereof that can compete with the intact antibody for specific binding to the target antigen and includes, for example, chimeric, humanized antibodies , fully human, and bispecific or antigen-binding fragments thereof. An “antibody” as such is a kind of an antigen-binding protein. An intact antibody will generally comprise at least two full heavy chains and several full light chains, but in some cases may include fewer chains such as naturally occurring antibodies in camelids that may comprise only heavy chains. Antibodies or antigen-binding fragments thereof may be derived exclusively from a single source, or may be “chimeric”, that is, different parts of the antibody may be derived from two different antibodies as described further below. Antibodies or binding fragments thereof can be produced in hybridomas, by recombinant DNA techniques, or by chemical or enzymatic cleavage of intact antibodies. Unless otherwise indicated, the term “antibodies” includes, in addition to antibodies comprising two entire heavy chains and two entire light chains, derivatives, variants, fragments and mutations thereof, of which are described below.

[278] The term “light chain” includes an entire light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. An entire light chain includes a variable region domain, VL, and a constant region domain, CL. The light chain variable region domain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[279] The term “light chain” includes an entire light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. An entire heavy chain includes a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is at the amino terminus of the polypeptide, and the CH domains are at the carboxyl terminus, with the CH3 being closest to the carboxy terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including subtypes IgG1, IgG2, IgG3 and IgG4), IgA (including subtypes IgA1 and IgA2), IgM and IgE. In one embodiment, the heavy chain is aglycosylated IgG1, for example, an IgG1 HC with an N297G mutation.

[280] The term “signal sequence”, “leader sequence” or “signal peptide” refers to a short peptide chain (3-60 amino acids long) that directs the transport of a protein. Signal peptides can also be called target signals, signal sequences, transit peptides, or a localization signal. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported, so that the biologically active form of the protein (e.g., an antibody as described here) is the shorter, cleaved form. Therefore, terms such as “antibody comprising a heavy chain...”, “antibody comprising a light chain...”, etc., where the antibody is characterized as having a heavy and/or light chain with a particular identified sequence, is understood to include antibodies having the specific identified sequences, antibodies having the specific identified sequences, except that the signal sequences are replaced by different signal sequences, of antibodies, as well as antibodies having the identified sequences, minus any signal sequences.

[281] The term “antigen-binding fragment” (or simply “fragment”) of an antibody or immunoglobulin chain (light or heavy chain), as used herein, comprises a part (regardless of how that part is obtained or synthesized ) of an antibody lacking at least some of the amino acids present in an entire chain, but which is capable of specifically binding to an antigen. These fragments are biologically active as they specifically bind to the target antigen and can compete with other antibodies or antigen-binding fragments thereof for specific binding to a given epitope. In one aspect, such a fragment will maintain at least one CDR present in the entire light or heavy chain, and, in some embodiments, will comprise a single heavy chain and/or light chain or part thereof. These biologically active fragments can be produced by recombinant DNA techniques, or can be produced, for example, by chemical or enzymatic cleavage of intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies, and single chain antibodies, and may be derived from any mammalian source, including, but not limited to, human , mouse, rat, camelid or rabbit. It is further envisioned that a functional portion of the antibodies disclosed herein, e.g., one or more CDRs, could be covalently linked to a second protein or small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties. or having a prolonged serum half-life.

[282] A “Fab fragment” is composed of a light chain and the CH1 and variable regions of a heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[283] An “Fc” region contains two heavy chain fragments comprising the CH1 and CH2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.

[284] A “Fab' fragment contains a light chain and a part of a heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, so that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[285] An “F(ab’)2 fragment” contains two light chains and two heavy chains containing a part of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains. An F(ab')2 fragment, therefore, is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

[286] The “Fv region” comprises the variable regions of both heavy and light chains, but is devoid of constant regions.

[287] “Single-chain antibodies” are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and United States Patent 4,946,778 and No. 5,260,203, the disclosures of which are incorporated by reference.

[288] A “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some cases, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[289] A “bivalent antigen-binding protein” or “bivalent antibody” comprises two antigen-binding sites. In some cases, the two binding sites have the same antigen specificities. Bivalent antibodies can be bispecific, see, below.

[290] A “multispecific antigen-binding protein” or “multispecific antibody” is one targeting more than one antigen or epitope.

[291] A “bispecific,” “dual-specific,” or “bifunctional” antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody having two different antigen-binding sites. Bispecific antibodies are a kind of multispecific antigen-binding protein or multispecific antibody and can be produced by a variety of methods, including, but not limited to, hybridoma fusion or Fab' fragment ligation. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen-binding protein or antibody will bind to two different epitopes, which may reside on the same or different protein targets.

[292] The term “neutralizing antigen-binding protein” or “neutralizing antibody” refers to an antigen-binding protein or antibody, respectively, that binds to a ligand, prevents the ligand from binding to its partner. binding and disrupts the biological response that would otherwise result from the binding of the ligand to its binding partner. In evaluating the binding and specificity of an antigen-binding protein, e.g., an antibody or immunologically functional antigen-binding fragment thereof, an antibody or fragment will substantially inhibit the binding of a ligand to its binding partner when an excess of antibody reduce the amount of binding partner for the ligand by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (as measured in an in vitro competitive binding assay). In the case of a PAC1 binding protein, this neutralizing molecule will decrease the ability of PAC1 to bind to PACAP, for example, PACAP-27 or PACAP-38.

[293] The term “antigen” or “immunogen” refers to a molecule or a part of a molecule capable of being bound by a selective binding agent, such as an antigen-binding protein (including, for example, an antibody or antigen-binding fragment of the same immunological functional), and furthermore that it is capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen may have one or more epitopes that are capable of interacting with different antibodies or fragments thereof.

[294] The term “epitope” is the part of a molecule that is bound by an antigen-binding protein (e.g., an antibody). The term includes any determinant capable of specifically binding to an antigen-binding protein, such as an antibody or a T-cell receptor. An epitope may be contiguous or noncontiguous (e.g., amino acid residues that are not contiguous to each other). another in the polypeptide sequence, but which in the context of the molecule are linked by the antigen-binding protein). In certain embodiments, the epitopes may be mimetic in that they comprise a three-dimensional structure that is similar to an epitope used to generate the antibody, yet comprising none or only some of the amino acid residues found in the epitope used to generate the antibody. Most often, epitopes reside on proteins, but in some cases they can reside on other types of molecules, such as nucleic acids. Epitope determinants include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics, and/or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

[295] The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by alignment and comparison of the sequences. “Percent identity” means the percentage of identical residues among the amino acids or nucleotides in the molecules being compared and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in the alignments (if any) must be addressed by a particular mathematical model or computer program (i.e., an “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.

[296] In calculating percent identity, the sequences being compared are aligned in a way that gives the greatest correspondence between the sequences. The computer program used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The GAP computer algorithm is used to align the two polypeptides or polynucleotides for which percent sequence identity will be determined. Sequences are aligned for optimal matching of their respective amino acids or nucleotides (the “matched range,” as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, where the “average diagonal” is the average of the diagonal of the comparison matrix being used; “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is generally 1/10 times the gap opening penalty) as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:1091510919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

[297] Recommended parameters for determining percent identity for polypeptide or nucleotide sequences using the GAP program are as follows: Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; Comparison matrix: BLOSUM 62 by Henikoff et al., 1992, supra; Gap Penalty: 12 (but with no penalty for gaps at the edge) Gap Length Penalty: 4 Similarity Threshold: 0

[298] Certain alignment schemes 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, even if there is no significant relationship between the two sequences. entire. In this regard, the selected alignment method (GAP program) can be adjusted if desired to result in an alignment that extends at least 50 contiguous amino acids from the target polypeptide.

[299] As used herein, “substantially pure” means that the described species of the molecule is the predominant species present, that is, on a molar basis it is more abundant than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition in which the object species comprises at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise at least 80%, 85%, 90%, 95% or 99% of all macromolecular species present in the composition. In other embodiments, the object species is purified to essential homogeneity in that contaminating species cannot be detected in the composition by conventional detection methods and, therefore, the composition consists of a single detectable macromolecular species.

[300] The term “treat” refers to any of the signs of successful treatment or improvement of an injury, pathology or condition, including any objective or subjective parameter such as reduction, remission; decreasing symptoms or making the injury, pathology or condition more tolerable to the patient; decrease in the rate of degeneration or decline; making the end point of degeneration less debilitating; improving the patient's physical or mental well-being. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric examinations, and/or a psychiatric evaluation. For example, certain methods presented here successfully treat migraines either prophylactically or as acute treatment, decreasing the frequency of migraines, decreasing the severity of migraines, and/or improving a symptom associated with migraine.

[301] An “effective amount” is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate symptoms and/or the underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remedy the damage that results from or is associated with migraine. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” is an amount sufficient to correct a disease state (e.g., migraine) or symptoms, particularly a state or symptoms associated with the disease state, or prevent, hinder, delay, or otherwise reverse the progression of the disease state or any other undesirable symptom associated with the disease in any aspect. A “prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or recurrence) of migraine, or reducing the likelihood of the onset (or recurrence) of migraines or migraine symptoms. The full therapeutic or prophylactic effect does not necessarily occur through the administration of one dose, and may occur only after the administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations.

[302] “Amino acid” includes its normal meaning in the art. The twenty naturally occurring amino acids and their abbreviations follow conventional usage. See, Immunology-A Synthesis, 2nd Edition, (E. S. Golub and D. R. Green, eds.), Sinauer Associates: Sunderland, Mass. (1991), incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides and are included in the phrase “amino acid ”. Examples of unconventional amino acids include: 4-hydroxyproline, Y-carboxyglutamate, ε-N,N, N-trimethyl-lysine, ε-N-acetyl-lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine , 5-hydroxylysine, o-N-methylarginine and other similar amino acids and imino acids (e.g. 4-hydroxyproline). In the polypeptide notation used here, the left direction is the amino terminal direction and the right direction is the carboxyl terminal direction, in accordance with standard usage and convention.

PAC1 in disease

[303]Neuropeptides present in the perivascular space of cranial vessels are implicated as important mediators of nociceptive input during migraine attacks. Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in trigeminal sensory neurons and can modulate nociception at different levels of the nervous system. Experimental studies in humans have shown that PACAP38 induces headache and migraine-like attacks (Schytz, et al., 2009, “PACAP38 induces migraine-like attacks in patients with migraine without aura”, Brain:132 pp 16-25), supporting the idea that PAC1 receptor antagonists can be used in the prophylactic and/or acute treatment of migraine.

Antibodies

[304] Antibodies that bind to the PAC1 protein, including human PAC1 protein (hPAC1) are provided here. The antibodies provided are polypeptides in which one or more complementarity determining regions (CDRs), as described herein, are incorporated and/or joined. In some antibodies, the CDRs are incorporated into a “framework” region, which orients the CDRs so that the appropriate antigen-binding properties of the CDRs are achieved. In general, the antibodies that are provided can interfere with, block, reduce, or modulate the interaction between PAC1 and its ligands, e.g., PACAP, such as PACAP-38.

[305] In certain embodiments, antibodies include, among others, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to as “antibody mimetic”), chimeric antibodies, humanized antibodies, human antibodies, fusions of antibody (sometimes referred to as “antibody conjugates”) and fragments thereof. The various structures are further described below.

[306] The antibodies provided here have been shown to bind PAC1, in particular human PAC1 and cyno PAC1. As further described in the examples below, certain antibodies have been tested and shown to bind to epitopes other than those bound by various other antibodies directed against one or another of the PAC1 components. The antibodies that are provided prevent PACAP (e.g. PACAP-38) from binding to its receptor. As a consequence, the antibodies provided here are capable of inhibiting PAC1 activity. In particular, antibodies that bind to these epitopes may have one or more of the following activities: inhibiting, inter alia, inducing PAC1 signal transduction pathways, inhibiting vasodilation, causing vasoconstriction, decreasing inflammation, e.g., neurogenic inflammation, and other physiological effects induced by PAC1 upon PACAP ligation.

[307] The antibodies that are disclosed here have a variety of uses. Some of the antibodies, for example, are useful in specific binding assays, affinity purification of PAC1, in particular hPAC1 or its ligands, and in screening assays to identify other antagonists of PAC1 activity. Some of the antibodies are useful for inhibiting the binding of a PAC1 ligand (e.g., PACAP-38) to PAC1.

[308] Antibodies can be used in a variety of treatment applications, as explained here. For example, certain PAC1 antibodies are useful for treating conditions associated with PAC1-mediated signaling, such as reducing, relieving, or treating migraine frequency and/or severity, reducing, relieving, or treating cluster headaches, reducing, relieving, or treating chronic pain, relieving or treating diabetes mellitus (type II), reducing, relieving, or treating cardiovascular disorders, and reducing, relieving or treating hemodynamic derangements associated with endotoxemia and septicemia in a patient. Other uses for antibodies include, for example, diagnosis of diseases or conditions associated with PAC1 and screening assays to determine the presence or absence of PAC1. Some of the antibodies described herein are useful in treating consequences, symptoms, and/or pathology associated with PAC1 activity. These include, but are not limited to, various types of headaches, including migraines (e.g., chronic and/or episodic migraines).

[309] Some of the antibodies provided have the structure normally associated with naturally occurring antibodies. The structural units of these antibodies typically comprise one or more tetramers, each composed of two identical couplets of polypeptide chains, although some mammalian species also produce antibodies having only a single heavy chain. In a typical antibody, each pair or couplet includes an entire “light” chain (in certain embodiments, about 25 kDa) and an entire “heavy” chain (in certain embodiments, about 50-70 kDa). Each individual immunoglobulin chain is composed of several “immunoglobulin domains,” each consisting of approximately 90 to 110 amino acids and expressing a characteristic folding pattern. These domains are the basic units of which antibody polypeptides are composed. The amino-terminal portion of each chain typically includes a variable domain that is responsible for antigen recognition. The carboxy-terminal portion is more evolutionarily conserved than the other end of the chain and is referred to as the “constant region” or “C region”. Human light chains are generally classified as kappa and lambda light chains and each contains a variable domain and a constant domain. Heavy chains are typically classified as mu, delta, gamma, alpha or epsilon chains, and these define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. Subtypes of IgM include IgM and IgM2. IgA subtypes include IgA1 and IgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The C heavy chain region typically comprises one or more domains that may be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. IgG heavy chains, for example, each contain three C region domains known as CH1, CH2, and CH3. The antibodies that are provided can have any of these isotypes and subtypes. In certain embodiments, the PAC1 antibody is of the IgG1, IgG2, or IgG4 subtype.

[310] Within the entire light and heavy chains, the variable and constant regions are joined by a “J” region of about twelve or more amino acids, with the heavy chain also including a “D” region of about ten more amino acids. See, for example, Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989, New York: Raven Press (incorporated herein by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair normally form the antigen-binding site.

[311] The variable regions of immunoglobulin chains generally have the same general structure, comprising regions of relatively conserved structure (FR) joined by three hypervariable regions, more often called “complementarity determining regions” or CDRs. The CDRs of the two chains of each aforementioned heavy chain/light chain pair are typically aligned by the framework regions to form a structure that specifically binds with a specific epitope on the target protein (e.g., PAC1). From N-terminal to C-terminal, naturally occurring light and heavy chain variable regions typically conform to the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A numbering system was devised to assign numbers of amino acids that occupy positions in each of 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.

[312] The various heavy chain and light chain variable regions provided herein are shown in tables 3A and 3B. Each of these variable regions can be associated with the heavy and light chain constant regions to form a complete antibody heavy and light chain, respectively. Furthermore, each of the heavy and light chain sequences generated in this way can be combined to form a complete antibody structure. It should be understood that the heavy chain and light chain variable regions provided herein can also be linked to other constant domains having sequences other than the exemplary sequences listed above.

[313] Nucleic acids encoding the antibodies described herein, or parts thereof, are also provided, including nucleic acids encoding one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, polynucleotides that encode heavy chain variable regions or just CDRs, polynucleotides sufficient for use as hybridization probes, PCR primers, or sequencing primers to identify, analyze, mutate, or amplify a polynucleotide encoding a polypeptide, antisense nucleic acids to inhibit expression of a polynucleotide, and sequences complementary to the previous ones. Nucleic acids can be of any length. They may have, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500 or more nucleotides in length, and/or may comprise one or more additional sequences, e.g., regulatory sequences, and/or be parts of a larger nucleic acid, e.g., a vector. Nucleic acids may be single-stranded or double-stranded and may comprise RNA and/or DNA nucleotides, and artificial variants thereof (e.g., peptide nucleic acids).

[314] Tables 1A and 1B show exemplary nucleic acid sequences. Any variable regions provided herein can be ligated to these constant regions to form complete heavy and light chain sequences. However, it should be understood that these constant region sequences are provided as specific examples only -- one skilled in the art may employ other constant regions, including IgG1 heavy chain constant region, IgG3 or IgG4 heavy chain constant regions, any one of seven lambda light chain constant regions, including hCL-1, hCL-2, hCL-3, and hCL-7; constant regions that have been modified for improved stability, expression, manufacturability, or other desired characteristics and the like. In some embodiments, the variable region sequences are joined to other constant region sequences that are known in the art.

[315] Specific examples of entire light and heavy chains of the antibodies that are provided and their corresponding nucleic acid and amino acid sequences are summarized in Tables 1A, 1B, 2A and 2B. Tables 1A and 2A show exemplary light chain sequences, and Tables 1B and 2B show exemplary heavy chain sequences, which are shown without respective signal sequences.

Antibody Variable Domains

[316] Also provided are antibodies (and corresponding nucleic acid sequences) that contain an antibody light chain variable region or an antibody heavy chain variable region, as shown in Tables 3A and 3B below, and immunologically functional fragments, derived , muteins and variants of these light chain and heavy chain variable regions.

[317] Also included are nucleic acid sequences encoding the variable regions shown in Tables 3A and 3B. Because these sequences are contained in the entire nucleic acid sequences of the corresponding antibodies shown in Tables 1A and 1B, they are not set out as separate sequences in a table or sequence listing, but can be easily verified by one skilled in the art. technique based on the variable regions provided in Tables 3A and 3B, together with the entire DNA sequences in Tables 1A and 1B.

[318] Antibodies of this type can generally be designated by the formula “VHx/VLy,” where “x” corresponds to the number of heavy chain variable regions and “y” corresponds to the number of light chain variable regions.

[319] Each of the heavy chain variable regions listed in Table 3B can be combined with any of the light chain variable regions shown in Table 3A to form an antibody.

CDRs

[320] The antibodies disclosed here are polypeptides into which one or more CDRs are grafted, inserted and/or joined. An antibody can have 1, 2, 3, 4, 5 or 6 CDRs. An antibody, therefore, may have, for example, a heavy chain CDR1 (“CDRH1”), and/or a heavy chain CDR2 (“CDRH2”), and/or a heavy chain CDR3 (“CDRH3”), and/or a light chain CDR1 (“CDRL1”), and/or a light chain CDR2 (“CDRL2”), and/or a light chain CDR3 (“CDRL3”). Some antibodies include a CDRH3 and a CDRL3. Specific heavy and light chain CDRs are identified in Tables 3A and 3B, respectively.

[321] Complementarity determining regions (CDRs) and framework regions (FR) of a given antibody can be identified using the system described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 913242, 1991. Certain antibodies that are disclosed herein include one or more amino acid sequences that are identical or have substantial sequence identity to the amino acid sequences of one or more of the CDRs set forth in Table 4A (CDRLs) and Table 4B (CDRHs). .

[322] The structure and properties of CDRs within a naturally occurring antibody have been described, supra. Briefly, in a traditional antibody, the CDRs are incorporated within a structure in the heavy and light chain variable region where they constitute the regions responsible for antigen binding and recognition. A variable region comprises at least three heavy or light chain CDRs, see, supra (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service N.I.H., Bethesda, MD; see also Chothia and Lesk, 1987, J . Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342: 877-883), within a framework region (designated framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991, supra; see also Chothia and Lesk, 1987, supra). The CDRs provided here, however, can not only be used to define the antigen-binding domain of a traditional antibody structure, but can also be incorporated into a variety of other polypeptide structures, as described here.

[323] Some of the antibodies disclosed herein share certain regions or sequences with other antibodies disclosed herein. These relationships are summarized in the tables above.

[324] In one aspect, the isolated antibodies provided herein may be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment of antibody of the same.

[325] In another embodiment, the antibody fragment of the isolated antibodies provided herein may be a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody, or a chain antibody molecule. only.

[326] In another embodiment, the isolated antibody provided here is a human antibody and may be of the IgG1, IgG2, IgG3 or IgG4 type. In another embodiment, the isolated antibody is one of the IgG1 or IgG2 type. In another embodiment, the isolated antibody is one of the IgG2 type. In another embodiment, the isolated antibody is one of the IgG1 type. In another embodiment, the IgG1-type antibody is an aglycosylated IgG1 antibody. In another embodiment, the aglycosylated IgG1 antibody has an N297G mutation.

[327] In another embodiment, the antibody consists solely of a light or heavy chain polypeptide as set forth in Tables 2A-2B. In some embodiments, the antibody consists only of a light chain variable or heavy chain variable domain such as those listed in Tables 3A and 3B. These antibodies can be pegylated with one or more PEG molecules.

[328] In yet another aspect, the isolated antibody provided herein can be coupled to a label group and can compete for binding to the extracellular portion of human PAC1 with an antibody from one of the isolated antibodies provided herein. In one embodiment, the isolated antibody provided herein can reduce monocyte chemotaxis, inhibit monocyte migration into tumors, or inhibit the accumulation and function of tumor-associated macrophages in a tumor when administered to a patient.

[329] As will be appreciated by those skilled in the art, for any antibody with more than one CDR of shown sequences, any combination of CDRs selected independently of the shown sequences is useful. Thus, antibodies with one, two, three, four, five or six of the independently selected CDRs can be generated. However, as will be appreciated by those skilled in the art, specific embodiments generally utilize combinations of CDRs that are non-repetitive, e.g., antibodies are not generally made with two CDRH2 regions, etc.

Monoclonal Antibodies

[330] Antibodies that are provided include monoclonal antibodies that bind to PAC1. Monoclonal antibodies can be produced using any known technique, for example, immortalizing spleen cells harvested from transgenic animals after completion of the immunization program. Spleen cells can be immortalized using any known technique, for example, fusing them with myeloma cells to produce hybridomas. Myeloma cells for use in fusion procedures for hybridoma production preferably are non-antibody producing, have high fusion efficiency, and enzyme deficiency that renders them incapable of growing in certain selective media that support the growth of only the desired fused cells (hybridomas). Examples of cell lines suitable for use in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC - 11, MPC11-X45-GTG 1.7 and S194/5XXO Bul; Examples of cell lines used in mouse fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[331] In some cases, a hybridoma cell line is produced by immunizing an animal (e.g., a transgenic animal having human immunoglobulin sequences) with a PAC1 immunogen; harvesting spleen cells from immunized animals; fusing harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; establishing hybridoma cell lines from the hybridoma cells, and identifying a hybridoma cell line that produces an antibody that binds to PAC1 (e.g., as described in Examples 1-3, below). Such hybridoma cell lines, and anti-PAC1 monoclonal antibodies produced thereby, are aspects of the present application.

[332] Monoclonal antibodies secreted by a hybridoma cell line can be purified using any known technique.

Chimeric and Humanized Antibodies

[333] Chimeric and humanized antibodies based on the above sequences are also provided. Monoclonal antibodies for use as therapeutic agents can be modified in several ways before use. An example is a chimeric antibody, which is an antibody composed of protein segments from different antibodies that are covalently joined to produce functional immunoglobulin light or heavy chains or immunologically functional parts thereof. Generally, a part of the heavy chain and/or light chain is identical to or homologous to a corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains is/are identical or homologous. to a corresponding sequence in antibodies derived from another species or belonging to another class or subclass of antibody. For methods relating to chimeric antibodies, see, for example, US Patent 4,816,567; and Morrison et al. , 1985, Proc. Natl. Academic. Sci. USA 81:6851-6855, which are incorporated herein by reference. CDR grafting is described, for example, in United States Patent Nos. 6,180,370, No. 5,693,762, No. 5,693,761, No. 5,585,089 and No. 5,530,101.

[334] Generally, the objective of preparing a chimeric antibody is to create a chimera in which the number of amino acids from the intended patient species is maximized. An example is the “CDR-grafted” antibody, in which the antibody comprises one or more complementarity-determining regions (CDRs) of a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chains are/ are identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another class or subclass of antibody. For use in humans, the variable region or CDRs selected from a rodent antibody are often grafted onto a human antibody, replacing the naturally occurring variable regions or CDRs of the human antibody.

[335] A useful type of chimeric antibody is a “humanized” antibody. Generally, a humanized antibody is produced from a monoclonal antibody initially created in a non-human animal. Certain amino acid residues in this monoclonal antibody, typically from non-antigen-recognizing portions of the antibody, are modified to be homologous to corresponding residues in a human antibody of corresponding isotype. Humanization can be accomplished, for example, using various methods by substituting at least a portion of a rodent variable region for the corresponding regions of a human antibody (see, e.g., U.S. Patent Nos. 5,585,089 and 5,693,762; Jones et al. ., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:32327; Verhoeyen et al., 1988, Science 239:1534-1536).

[336] In one aspect, the CDRs of the light and heavy chain variable regions of the antibodies provided here are grafted to the framework regions (FRs) of antibodies of the same or different phylogenetic species. For example, the heavy and light chain variable region CDRs described herein can be grafted onto the consensus human FRs. To create consensus human FRs, FRs from various human heavy chain or light chain amino acid sequences can be aligned to identify a consensus amino acid sequence. In other embodiments, the FRs of a heavy chain or light chain disclosed herein are replaced with the FRs of a different heavy chain or light chain. In one aspect, rare amino acids in the FRs of the anti-PAC1 antibody heavy and light chains are not replaced, while the rest of the FR amino acids are replaced. A “rare amino acid” is a specific amino acid that is in a position where this particular amino acid is not normally found in a FR. Alternatively, variable regions grafted from the heavy or light chain can be used with a constant region that is different from the constant region. of the particular heavy or light chain as disclosed herein. In other embodiments, the grafted variable regions are part of a single-chain Fv antibody.

[337] In certain embodiments, constant regions from non-human species can be used together with human variable regions to produce hybrid antibodies.

Fully Human Antibodies

[338] Fully human antibodies are also provided. Methods are available to prepare fully human antibodies specific to a given antigen without exposing humans to the antigen (“fully human antibodies”). One specific means provided for implementing 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 endogenous Ig genes have been inactivated is one means of producing fully human monoclonal antibodies (mAbs) in the mouse, an animal that can be immunized with any desirable antigen. The use of fully human antibodies can minimize the immunogenic and allergic responses that can sometimes be caused by the administration of mouse or mouse-derived mAbs to humans as therapeutic agents.

[339] Fully human antibodies can be produced by immunizing transgenic animals (usually mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose typically have six or more contiguous amino acids, and are optionally conjugated to a carrier, such as a hapten. See, for example, Jakobovits et al., 1993, Proc. Natl. Academic. 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 this method, transgenic animals are produced by incapacitating the endogenous mouse immunoglobulin loci that encode the mouse heavy and light immunoglobulin chains, and inserting into the mouse genome large fragments of DNA from the human genome containing loci that encode chain proteins. heavy and light human. Partially modified animals, which have less than the full complement of human immunoglobulin loci, are then crossed to obtain an animal having all the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies that are immunospecific for the immunogen but have human rather than murine amino acid sequences, including the variable regions. For further details of these methods, see, for example, WO96/33735 and WO94/02602. Additional methods relating to transgenic mice for preparing human antibodies are described in US Patent 5,545,807; 6,713,610; 6,673,986;6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299 and 5,545,806; in PCT publications WO91/10741, WO90/04036, and in EP 546073B1 and EP 546073A1.

[340] The transgenic mice described above, referred to herein as “HuMab” mice, contain a human immunoglobulin gene minilocus that encodes heavy ([mu] and [gamma]) and light [kappa] chain immunoglobulin sequences, along with target mutations that inactivate the endogenous [mu] and [kappa] chain loci (Lonberg et al., 1994, Nature 368:856-859). In this sense, mice show reduced expression of mouse IgM or [kappa] and in response to immunization, and the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgG [kappa] monoclonal antibodies (Lonberg et al., supra.; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13: 65-93; Harding and Lonberg, 1995, Ann. N.Y Acad. Sci. 764:536-546). The preparation of HuMab mice is described in detail in 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. 00:65-93; Harding and Lonberg, 1995, Ann. N.Y Acad. Sci. 764:536-546; Fishwild et al. , 1996, Nature Biotechnology 14:845-851; The aforementioned references are incorporated herein by reference in their entirety for all purposes. See, US Patent 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 US Patent 5,545,807; International Publication WO 93/1227; WO 92/22646; and WO 92/03918, the disclosures of which are incorporated herein by reference in their entireties for all purposes. Technologies used for the production of 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, HCo7 and HCo12 transgenic mouse strains can be used to generate anti-PAC1 antibodies. Further details on the production of human antibodies using transgenic mice are provided in the examples below.

[341] Using hybridoma technology, antigen-specific human mAbs with the desired specificity can be produced and selected from transgenic mice such as those described above. These antibodies can be cloned and expressed using a suitable vector and host cells, or the antibodies can be harvested from cultured hybridoma cells.

[342] Fully human antibodies can also be derived from phage-display libraries (as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 22 7:381; and Marks et al., 1991, J. Mol. Biol. 222:581). Phage display techniques mimic immune selection through the presentation of antibody repertoires on the surface of filamentous bacteriophages, and subsequent selection of the phage by binding to an antigen of choice. One such technique is described in PCT Publication WO 99/10494 (incorporated herein by reference), which describes the isolation of functional, high-affinity agonistic antibodies to MPL and msk receptors and using this approach.

Bispecific or Bifunctional Antibodies

[343] The antibodies that are provided also include bispecific and bifunctional antibodies that include one or more CDRs or one or more variable regions as described above. A bispecific or bifunctional antibody, in some cases, is a hybrid artificial antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including, but not limited to, hybridoma fusion or ligation of Fab' fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553.

Various Other Ways

[344] Some of the antibodies provided are variant forms of the antibodies disclosed above. For example, some of the antibodies have one or more conservative amino acid substitutions in one or more of the heavy or light chains, variable regions, or CDRs listed above.

[345] Naturally occurring amino acids can be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) acids: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatics: Trp, Tyr, Phe.

[346] Conservative amino acid substitutions may involve exchanging a member of one of these classes with another member of the same class. Conservative amino acid substitutions can include non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include peptidomimetics and other inverted forms of amino acid moieties.

[347] Non-conservative substitutions may involve exchanging a member of one of the aforementioned classes for a member of another class. These substituted residues can be introduced into regions of the antibody that are homologous with human antibodies, or into non-homologous regions of the molecule.

[348] When making these changes, according to certain embodiments, the hydropathic amino acid index may be considered. The hydropathic profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repeatedly averaging these values along the peptide chain. Each amino acid was assigned a hydropathic index depending on its hydrophobicity and charge characteristics. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[349] The importance of the hydropathic profile in conferring interactive biological function of a protein is understood in the art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids can be replaced by other amino acids having a similar hydropathic index or score and still retain similar biological activity. When making changes based on the hydropathic index, in certain embodiments, substitution of amino acids whose hydropathic indexes are within ±2 is included. In some aspects, those within ±1 are included, and in other aspects, those within ±0.5 are included.

[350] It is also understood in the art that the substitution of like amino acids can be made effectively based on hydrophilicity, particularly where the biologically functional protein or peptide thus created is intended for use in immunological modalities, as in the present case. In certain embodiments, the greater local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen binding or immunogenicity, that is, with a biological property of the protein.

[351] The following hydrophilicity values were assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+ 3.0±1); glutamate (+3.0±1); serine (+ 0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in certain embodiments, substitution of amino acids whose hydrophilicity values are within ±2 is included, in other embodiments, those that are within ±1 are included, and in still other embodiments , those within ±0.5 are included. In some cases, one can also identify epitopes from primary amino acid sequences based on hydrophilicity. These regions are also referred to as “epitope core regions.”

[352] Exemplary conservative amino acid substitutions are set out in Table 6.

[353] One skilled in the art will be able to determine suitable variants of polypeptides as set forth herein using well-known techniques. One skilled in the art can identify suitable areas of the molecule that can be altered without destroying activity by target regions not believed to be important for activity. The specialist will also be able to identify residues and portions of the molecules that are conserved among similar polypeptides. In other embodiments, even areas that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying biological activity or without adversely affecting the structure of the polypeptide.

[354] Additionally, one skilled in the art can review structure-function studies by identifying residues in similar polypeptides that are important for activity or structure. Given this comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may choose chemically similar amino acid substitutions for these predicted important amino acid residues.

[355] A person skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to this structure in similar polypeptides. Taking this information into account, one skilled in the art can predict the alignment of the amino acid residues of an antibody with respect to its three-dimensional structure. One skilled in the art may choose not to prepare radical changes for amino acid residues predicted to be on the surface of the protein, since these residues may be involved in important interactions with other molecules. Furthermore, one skilled in the art can generate test variants containing a unique amino acid substitution at each desired amino acid residue. These variants can then be screened using assays for PAC1 neutralizing activity, (see examples below) thus producing information regarding which amino acids can be changed and which should not be changed. In other words, based on information obtained in these routine experiments, one skilled in the art can easily determine the amino acid positions where further substitutions should be avoided alone or in combination with other mutations.

[356] Several scientific publications have focused on the prediction of secondary structure. See, Moult, 1996, Curr. Op. in Biotech. 7:422-427; Chou et al., 1974, Biochem. 13:222-245; Chou et al., 1974, Biochemistry 113:211-222; Chou et al., 1978, Adv. Enzymol. Report. Areas Mol. Biol. 47:45-148; Chou et al. , 1979, Ann. Rev. Biochem. 47:251-276; and Chou et al., 1979, Biophys. J. 26:367-384. Additionally, computer programs are currently available to assist with secondary structure prediction. One method for predicting secondary structure is based on homology modeling. For example, two polypeptides or proteins that have a sequence identity greater than 30%, or similarity greater than 40% may have similar structural topologies. The recent growth of the protein structural database (PDB) has provided increased predictability of secondary structure, including the potential number of folds within the structure of a polypeptide or protein. See, Holm et al., 1999, Nucl. Acid. Res. 27:244-247. It has been suggested (Brenner et al., 1997, Curr. Op. Struct. Biol. 7:369-376) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved , structural forecasting will become dramatically more accurate.

[357] Additional methods for predicting secondary structure include “segmentation” (Jones, 1997, Curr. Opin. Struct. Biol. 7:377-387; Sippl et al., 1996, Structure 4:15-19), “analysis profile” (Bowie et al., 1991, Science 253:164-170; Gribskov et al., 1990, Meth. Enzym. 183:146-159; Gribskov et al., 1987, Proc. Nat. Acad. Sci. 84:4355-4358), and “evolutionary linkage” (See, Holm, 1999, supra; and Brenner, 1997, supra).

[358] In some embodiments, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for the formation of protein complexes, (4 ) alter ligands or antigen binding affinities, and/or (4) confer or modify other physicochemical or functional properties on these polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made to the naturally occurring sequence. Substitutions can be made in the part of the antibody that lies outside the domains forming intermolecular contacts). In these embodiments, conservative amino acid substitutions may be used that do not substantially alter the structural characteristics of the parent sequence (e.g., one or more amino acid substitutions that do not disrupt the secondary structure that characterizes the parent or native antibody). Examples of polypeptide secondary and tertiary structures recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed.), 1984, W. H. New York: Freeman and Company; Introduction to Protein Structure (Branden and Tooze, eds.), 1991, New York: Garland Publishing; and Thornton et al., 1991, Nature 354:105, which are incorporated herein by reference.

[359] Additional preferred antibody variants include cysteine variants in which one or more cysteine residues in the native or parent amino acid sequence are deleted from or replaced with another amino acid (e.g., serine). Cysteine variants are useful, particularly when antibodies must be refolded into a biologically active conformation. Cysteine variants may have fewer cysteine residues than the native antibody, and generally have an even number to minimize interactions resulting from unpaired cysteines.

[360] The heavy and light chains, variable region domains and CDRs that are disclosed can be used to prepare polypeptides that contain an antigen-binding region that can specifically bind to PAC1. For example, one or more of the CDRs listed in Tables 4A and 4B can be incorporated into a molecule (e.g., a polypeptide) covalently or non-covalently to prepare an immunoadhesion. An immunoadhesion may incorporate the CDRs as part of a larger polypeptide chain, may covalently link the CDRs to another polypeptide chain, or may incorporate the CDRs noncovalently. CDRs allow immunoadhesion to specifically bind to a particular antigen of interest (e.g., PAC1 or epitope thereof).

[361] Mimetics (e.g., “peptide mimetics” or “peptidomimetics”) based on the variable region domains and CDRs that are described herein are also provided. These analogs may be peptides, non-peptides, or combinations of peptide and non-peptide regions. Fauchere, 1986, Adv. Drug Res. 15:29; Veber and Freidinger, 1985, TINS p. 392; and Evans et al., 1987, J. Med. Chem. 30:1229, which are incorporated herein by reference for any purpose. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce a similar therapeutic or prophylactic effect. These compounds are often developed with the aid of computerized molecular modeling. Generally, peptidomimetics are proteins that are structurally similar to an antibody exhibiting a desired biological activity, such as here the ability to specifically bind to PAC1, but have one or more peptide bonds optionally replaced by a bond selected from: -CH2NH-, -CH2S -, -CH2- CH2-, -CH-CH-(cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-, by methods well known in the art. Systematic replacement of one or more amino acids from a consensus sequence with D-amino acids of the same type (e.g., D-lysine in place of L-lysine) can be used in certain embodiments to generate more stable peptides. Furthermore, restricted peptides comprising a consensus sequence or a substantially identical consensus sequence variation can be generated by methods known in the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61:387), incorporated herein by reference. ), for example, by the addition of internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize the peptide.

[362] Derivatives of the antibodies that are described herein are also provided. The derivatized polypeptide may comprise any molecule or substance that imparts a desired property to the antibody or fragment, such as increased half-life in a particular use. The derivatized antibody may comprise, for example, a detectable moiety (or label) (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule, a detectable bead (such as a magnetic or electron-dense bead (e.g., gold)), or a molecule that binds to another molecule (e.g., biotin or streptavidin)), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antibody for a specific use particular (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses). Examples of molecules that can be used to derivatize an antibody include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-bound or PEGylated derivatives of antibodies can be prepared using well-known techniques. Certain antibodies include a pegylated single-chain polypeptide as described herein. In one embodiment, the antibody is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant may be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyrrolidone), polyethylene glycols, propylene glycol homopolymers, polypropylene oxide/propylene oxide copolymers, ethylene, polyoxyethylated polyols and polyvinyl alcohols.

[363] Other derivatives include covalent or aggregative conjugates of TIMP-3 binding proteins with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of PAC1 binding protein . For example, the conjugating peptide may be a heterologous signal (or leader) peptide, for example, the yeast factor-alpha leader, or a peptide such as an epitope tag. Fusion proteins containing PAC1 antibodies may comprise peptides added to facilitate purification or identification of PAC1 binding protein (e.g., poly-His). A PAC1 binding protein can also be linked to the FLAG peptide as described in Hopp et al., 1988, Bio/Technology 6:1204; and US Patent 5,011,912. The FLAG peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody (mAb), allowing rapid assay and easy purification of the expressed recombinant protein. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).

[364] Oligomers that contain one or more PAC1 binding proteins can be employed as PAC1 antagonists. Oligomers can be in the form of covalently linked or non-covalently linked dimers, trimers or higher oligomers. Oligomers comprising two or more PAC1 binding proteins are envisioned for use, with one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.

[365] One embodiment is directed to oligomers comprising multiple PAC1-binding polypeptides together through covalent or non-covalent interactions between peptide moieties fused to the PAC1-binding proteins. These peptides can be peptide ligands (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain antibody-derived polypeptides are among the peptides that can promote oligomerization of PAC1-binding proteins bound thereto, as described in more detail below.

[366] In particular embodiments, the oligomers comprise two to four PAC1 binding proteins. The PAC1 binding protein moieties of the oligomer may be in any of the forms described above, for example, variants or fragments. Preferably, the oligomers comprise PAC1 binding proteins that have PAC1 binding activity.

[367] In one embodiment, an oligomer is prepared using polypeptides derived from immunoglobulins. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various antibody-derived polypeptide moieties (including the Fc domain) has been described, for example, by Ashkenazi et al. , 1991, Proc. Natl. Academic. Sci. USA 88:10535; Byrn et al. , 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11.

[368] One embodiment is directed to a dimer comprising two fusion proteins created by fusing a PAC1 binding protein to the Fc region of an antibody. The dimer can be prepared, for example, by inserting a gene fusion encoding the fusion protein into a suitable expression vector, expressing the gene fusion in host cells transformed with the recombinant expression vector, and allowing the fusion protein to form. expresses a lot of very similar antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to produce the dimer.

[369] The term “Fc polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of these polypeptides containing the hinge region that promotes dimerization are also included. Fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of easy purification by affinity chromatography with protein A or protein G columns.

[370] A suitable Fc polypeptide, described in PCT application WO 93/10151 and US Patent 5,426,048 and No. 5,262,522, is a single-chain polypeptide extending from the N-terminal to the C-terminal hinge region. native terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in United States Patent No. 5,457,035 and in Baum et al., 1994, EMBO J. 13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 was changed from Leu to Ala, amino acid 20 was changed from Leu to Glu and amino acid 22 was changed from Gly to Ala. The mutein has reduced affinity for Fc receptors.

[371] In other embodiments, the variable part of the heavy and/or light chains of a PAC1 binding protein such as those disclosed herein can be replaced with the variable part of an antibody heavy and/or light chain.

[372] Alternatively, the oligomer is a fusion protein comprising several PAC1 binding proteins, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are described in US Patent 4,751,180 and No. 4,935,233.

[373] Another method for preparing oligomeric PAC1 binding protein derivatives involves the use of a leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., 1988, Science 240:1759), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, and the lung surfactant protein D (SPD)-derived leucine zipper described in Hoppe et al. , 1994, FEBS Letters 344:191, incorporated herein by reference. The use of a modified leucine zipper that allows stable trimerization of a heterologous protein fused to it is described in Fanslow et al. , 1994, Semin. Immunol. 6:267-278. In one approach, recombinant fusion proteins comprising a PAC1-binding protein fragment or derivative fused to a leucine zipper peptide are expressed in suitable host cells and the soluble oligomeric PAC1-binding protein fragments or derivatives that form are recovered from the culture supernatant.

[374] In certain embodiments, the antibody has a KD (equilibrium binding affinity) of less than 1 pM, 10 pM, 100 pM, 1 nM, 2 nM, 5 nM, 10 nM, 25 nM or 50 nM.

[375] Another aspect provides an antibody having a half-life of at least one day in vitro or in vivo (e.g., when administered to a human). In one embodiment, the antibody has a half-life of at least three days. In another embodiment, the antibody or part thereof has a half-life of four days or more. In another embodiment, the antibody or part thereof has a half-life of eight days or more. In another embodiment, the antibody or antigen-binding portion thereof is derivatized or modified so that it has a longer half-life compared to the underivatized or unmodified antibody. In another embodiment, the antibody contains point mutations to increase serum half-life, as described in WO 00/09560, published February 24, 2000, incorporated by reference.

Glycosylation

[376] The antibody may have a glycosylation pattern that is different or altered from that found in native species. As is known in the art, glycosylation patterns may depend both on the sequence of the protein (e.g., the presence or absence of particular glycosylation amino acid residues, discussed below), and on the host cell or organism in which the protein is produced. Particular expression systems are discussed below.

[377] Glycosylation of polypeptides is typically N-linked or O-linked. N-linked generally refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tri-peptide sequences asparagine-X-serine and asparagine-X threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic binding of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tri-peptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[378] The addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the tri-peptide sequences described above (for N-linked glycosylation sites). The change can also be made by adding, or substituting for, one or more serine or threonine residues to the starting sequence (for O-linked binding sites). To facilitate this, the amino acid sequence of the antibody can be altered by changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases so that codons are generated that will translate into desired amino acids.

[379] Another means to increase the number of carbohydrate moieties in the antibody is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require the production of the protein in a host cell that has glycosylation capacity for N- and O-linked glycosylation. Depending on the coupling mode used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those in cysteine, (d) free hydroxyl groups such as those in serine , threonine or hydroxyproline, (e) aromatic residues such as those in phenylalanine, tyrosine or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330 published September 11, 1987 and in Aplin and Wriston, 1981, CRC Crit.Rev, Biochem., pp. 259-306.

[380] The removal of carbohydrate moieties present in the initial antibody can be carried out chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all of the sugars except the linker sugar (N-acetylglucosamine or N-acetylgalactosamine), leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al. , 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties into polypeptides can be achieved by use of a variety of endo- and exo-glucosidases as described by Thotakura et al. , 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be prevented by the use of the compound tunicamycin, as described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation of protein-N-glycoside bonds.

[381] Therefore, aspects include antibody glycosylation variants in which the number and/or type of glycosylation site has been altered compared to the amino acid sequences of the parent polypeptide. In certain embodiments, antibody protein variants comprise a greater or lesser number of N-linked glycosylation sites than the native antibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue except proline. Substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence will prevent the addition of an N-linked carbohydrate chain present in the native polypeptide. For example, glycosylation can be reduced by deleting an Asn or replacing the Asn with a different amino acid. In other embodiments, one or more new N-linked sites are created. Antibodies typically have an N-linked glycosylation site in the Fc region.

[382] Human IgG1 is known to have a glycosylation site at N297 (EU numbering system) and glycosylation contributes to the effector function of IgG1 antibodies. Groups mutated N297 in an effort to prepare aglycosylated antibodies. The mutations focus on replacing N297 with amino acids that resemble asparagine in physicochemical nature, such as glutamine (N297Q) or with alanine (N297A) that mimics asparagines without polar groups.

[383] As used herein, “aglycosylated antibody” or “aglycosylated Fc” refers to the glycosylation state of the residue at position 297 of Fc. An antibody or other molecule may contain glycosylation at one or more sites, but may still be considered an aglycosylated antibody or aglycosylated Fc fusion protein.

[384] In an effort to prepare functionless IgG1 Fc molecules, it was discovered that mutation of amino acid N297 of human IgG1 to glycine, i.e., N297G, provides superior purification efficiency and biophysical properties over other amino acid substitutions at this residue. Furthermore, it was found that anti-PAC1 antibodies in an aglycosylated IgG1 structure prevented Fc gamma receptor-induced platelet depletion as assessed using a phagocytosis assay (see Example 5). Thus, in preferred embodiments, anti-PAC1 antibodies comprise antibodies with a “B” or “C” suffix, designating aglycosylated variants of IgG1. In certain embodiments, an antibody comprises the Fc having an N297G substitution.

[385] An Fc comprising a human IgG1 Fc having the N297G mutation can also comprise new insertions, deletions, and substitutions. In certain embodiments, the human IgG1 Fc comprises the N297G substitution and is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95 % identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to one or more of the amino acid sequences set forth in Table 2B (anti-hPAC1 Ab amino acid sequences).

[386] Molecules containing glycosylated IgG1 Fc may be less stable than molecules containing glycosylated IgG1 Fc. The Fc region can therefore be further engineered to increase the stability of the aglycosylated molecule. In some embodiments, one or more amino acids are substituted for cysteine to form disulfide bonds in the dimeric state. Residues corresponding to V259, A287, R292, V302, L306, V323 or I332 of an IgG1 amino acid heavy chain sequence can therefore be replaced with cysteine. In preferred embodiments, specific pairs of residues are substituted so that they preferentially form a disulfide bond with one another, thereby limiting or preventing scrambling of the disulfide bonds. Preferred pairs include, among others, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C.

[387] Specifically exemplified here are aglycosylated IgG1 Fc-containing heavy chains (N297G) in which one or both residues R292 and V302 are replaced with cysteine. Aglycosylated IgG1 (N297G) antibodies with a “C” suffix contain R292C and V302C substitutions in their heavy chains.

Markers and Effector Groups

[388] In some embodiments, the antigen binding comprises one or more markers. The term “marker group” or “marker” means any detectable marker. Examples of suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., zipper pair sequences leucine, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the antibody through spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used as seen for adjustment.

[389] The term “effector group” means any group coupled to an antibody that acts as a cytotoxic agent. Examples of suitable effector groups are radioisotopes or radionuclides (e.g. 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I). Other suitable groups include toxins, therapeutic groups, or chemotherapeutic groups. Examples of suitable groups include calicheamicin, auristatins, geldanamycin and maytansine. In some embodiments, the effector group is coupled to the antibody through spacer arms of various lengths to reduce potential steric hindrance.

[390] In general, markers fall into a variety of classes, depending on the assay in which they are being detected: a) isotopic markers, which can be radioactive or heavy isotopes; b) magnetic markers (e.g. magnetic particles); c) redox active fractions; d) optical dyes; enzymatic groups (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). In some embodiments, the labeling group is coupled to the antibody through spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art.

[391] Specific markers include optical dyes, including, but not limited to, chromophores, phosphors, and fluorophores, with the latter being specific in many cases. Fluorophores can be “small molecule” fluorine or protein fluorine.

[392] “Fluorescent label” means any molecule that can be detected through its inherent fluorescent properties. Suitable fluorescent markers include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl coumarins, pyrene, Malacite green, stilbenes, Lucifer yellow, BlueJ Cascade, Texas Red, IAEDANS, EDANS, FL BODIPY, LC Red 640, Cy 5 Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor Dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633 , Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), Blue Cascade, Yellow Cascade and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, Rhodamine and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in MOLECULAR PROBES HANDBOOK by Richard P. Haugland, expressly incorporated by reference.

[393] Suitable proteinaceous fluorescent markers also include, among others, green fluorescent protein, including Renilla, Ptilosarcus or Aequorea species of GFP (Chafie et al., 1994, Science 263:802-805), EGFP (Clontech Labs., Inc. , Genbank Accession Number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., Quebec, Canada; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178182) , enhanced yellow fluorescent protein (EYFP, Clontech Labs., Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA. 85:2603-2607) and Renilla (WO92/15673, WO95/07463, WO98/14605, WO98/26277, WO99/49019, US Patents 5292658, 5418155,5683888, 5741668, 577 7079, 5804387, 5874304 , 5876995, 5925558).

Nucleic acids

[394] A further aspect provides nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids comprising a nucleotide sequence listed in Tables 1A and 1B under particular hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. As defined here, a moderately stringent hybridization condition uses a prewash solution containing 5x sodium/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6x SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of 42°C) and washing conditions of 60°C, in 0.5x SSC, 0.1% SDS. A hybridization condition stringent hybridizes in 6x SSC at 45°C, followed by one or more washes in 0.1x SSC, 0.2% SDS at 68°C. Furthermore, one skilled in the art can manipulate hybridization and/or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids comprising nucleotide sequences that are at least 65%, 70%, 75%, 80% , 85%, 90%, 95%, 98% or 99% identical to each other usually remain hybridized to each other.

[395] The basic parameters that affect the choice of hybridization conditions and guidance for defining suitable conditions are established by Sambrook, Fritsch, and Maniatis (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor , N.Y., supra; 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 those skilled in the art based on , for example, length and/or composition of the nucleic acid.

[396] Changes can be introduced by mutation in a nucleic acid, thus leading to changes in the amino acid sequence of a polypeptide (for example, an antibody or antibody derivative) that it encodes. Mutations can be introduced using any known technique. In one embodiment, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered using, for example, a random mutagenesis protocol. However, a mutant polypeptide is produced that can be expressed and screened for a desired property.

[397] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, nucleotide substitutions can be prepared leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations may be introduced into a nucleic acid that selectively alters the biological activity of a polypeptide it encodes. For example, mutation can quantitatively or qualitatively alter biological activity. Examples of quantitative changes include increasing, reducing, or eliminating activity. Examples of qualitative changes include changing the antigen specificity of an antibody. In one embodiment, a nucleic acid encoding any antibody described herein can be mutated to alter the amino acid sequence using molecular biology techniques that are well established in the art.

[398] Another aspect provides nucleic acid molecules that are suitable for use as primers or hybridization probes for detecting nucleic acid sequences. A nucleic acid molecule may comprise only a portion of a nucleic acid sequence that encodes an entire polypeptide, e.g., a fragment that can be used as a probe or primer or a fragment that encodes an active moiety (e.g., a PAC1 binding) of a polypeptide.

[399] Probes based on nucleic acid sequence can be used to detect the nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide. The probe may comprise a label group, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. These probes can be used to identify a cell that expresses the polypeptide.

[400] Another aspect provides vectors comprising a nucleic acid encoding a polypeptide or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains). Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, e.g., recombinant expression vectors. Recombinant expression vectors may comprise a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Recombinant expression vectors include one or more regulatory sequences, selected based on the host cells to be used for expression, that is operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many host cell types (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter, and cytomegalovirus promoter), those that direct expression of the sequence of nucleotide only in certain host cells (e.g., tissue-specific regulatory sequences, see, Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science 236:1237, incorporated by reference herein in their entireties) and those that direct the inducible expression of a nucleotide sequence in response to particular treatment or condition (e.g., the metallothionin promoter in mammalian cells and the tet-responsive and/or streptomycin-responsive promoter in systems prokaryotes and eukaryotes (see, id.) It will be appreciated by those skilled in the art that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc. Expression vectors can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

[401] Another aspect provides host cells into which a recombinant expression vector has been introduced. A host cell can be any prokaryotic (e.g., E. coli) or eukaryotic (e.g., yeast, insect, or mammalian (e.g., CHO cells)) cell. Vector DNA can be introduced into prokaryotic or eukaryotic cells through conventional transfection or transformation techniques. For stable transfection in mammalian cells, it is known that, depending on the expression vector technique and transfection technique used, only a small fraction of cells can integrate foreign DNA into their genome. In order to identify and select these members, a gene encoding a selectable marker (e.g., for antibiotic resistance) is usually introduced into host cells along with the gene of interest. Preferred selectable markers include those that confer drug resistance, such as G418, hygromycin, and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while other cells die), among other methods.

Antibody Preparation

[402] Non-human antibodies that are provided may be, for example, derived from any antibody-producing animal, such as mouse, rat, rabbit, goat, donkey, or non-human primates (such as monkey, e.g., cynomolgus or rhesus monkey) or apes (e.g. chimpanzee)). Non-human antibodies may be used, for example, in in vitro cell culture and cell culture-based applications, or any other application where an immune response to the antibody does not occur or is negligible, can be avoided, is not a concern, or is desired. In certain embodiments, antibodies can be produced by immunizing animals using methods known in the art, as described above. Antibodies can be polyclonal, monoclonal, or can be synthesized in host cells expressing recombinant DNA. Fully human antibodies can be prepared as described above by immunizing transgenic animals containing human immunoglobulin loci or by selecting a phage display library that is expressing a repertoire of human antibodies.

[403] Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example, the standard somatic cell hybridization technique of Kohler and Milstein, 1975, Nature 256:495. Alternatively, other techniques for producing monoclonal antibodies can be employed, for example, viral or oncogenic transformation of B lymphocytes. A suitable animal system for preparing hybridomas is the murine system, which is a very well-established procedure. Immunization protocols and techniques for isolating fusion-immunized splenocytes are known and illustrative approaches are described in the Examples, below. For these procedures, B cells from immunized mice are typically fused with a suitable immortalized fusion partner, such as a murine myeloma cell line. If desired, rats or mammals other than this can be immunized instead of mice and B cells from these animals can be fused with the murine myeloma cell line to form hybridomas. Alternatively, a myeloma cell line from a source other than mice can be used. Fusion procedures for preparing hybridomas are also well known.

[404] The single-chain antibodies that are provided can be formed by linking heavy and light chain variable domain (Fv region) fragments through an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. These single-chain Fvs (scFvs) can be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (VL and VH). The resulting polypeptides may fold back on themselves to form antigen-binding monomers, or may form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et al. , 1997, Prot. Eng. 10:423; Kortt et al. , 2001, Biomol. Eng. 18:95-108). By combining different polypeptides comprising VL and VH, multimeric scFvs can be formed that bind to different epitopes (Kriangkum et al., 2001, Biomol. Eng. 18:31-40). Techniques developed for producing single-chain antibodies include those described in US Patent 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Academic. Sci. USA. 85:5879; Ward et al. , 1989, Nature 334:544, de Graaf et al., 2002, Methods Mol Biol. 178:379387. Single-chain antibodies derived from antibodies provided herein include, but are not limited to, scFvs comprising the variable domain combinations of the heavy and light chain variable regions shown in Tables 3A and 3B, or combinations of light and heavy chain variable domains that include CDRs shown in Tables 4A and 4B.

[405] The antibodies provided herein that are of one subclass can be changed to antibodies of a different subclass using subclass switching methods. Thus, IgG antibodies can be derived from an IgM antibody, for example, and vice versa. These techniques allow the preparation of new antibodies that have the antigen-binding properties of a particular antibody (the parent antibody) but also have biological properties associated with an antibody isotype or subclass different from the parent antibody. Recombinant DNA techniques can be employed. Cloned DNA encoding particular antibody polypeptides may be employed in these procedures, for example, DNA encoding the constant domain of an antibody of the desired isotype. See, for example, Lantto et al., 2002, Methods Mol. Biol. 178:303-316.

[406] Accordingly, antibodies that are provided include those comprising, for example, the variable domain combinations described, supra., having a desired isotype (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgE and IgD) , as well as Fab or F(ab')2 fragments thereof. Furthermore, if an IgG4 is desired, it may also be desired to introduce a point mutation (CPSCP->CPPCP) in the hinge region, as described in Bloom et al. , 1997, Protein Science 6:407, incorporated by reference here) to alleviate a tendency to form intra-H chain disulfide bonds that can lead to heterogeneity in IgG4 antibodies.

[407] Furthermore, techniques for deriving antibodies having different properties (i.e., different affinities for the antigen to which they bind) are also known. One such technique, known as strand shuffling, involves displaying immunoglobulin variable domain gene repertoires on the surface of filamentous bacteriophage, often referred to as phage display. Chain shuffling is used to prepare high affinity antibodies to the hapten 2-phenyloxazol-5-one, as described by Marks et al., 1992, BioTechnology 10:779.

[408] Conservative modifications can be prepared to the heavy and light chain variable regions described in Tables 3A and 3B, or the CDRs described in Tables 4A and 4B (and the corresponding modifications to the encoding nucleic acids) to produce a protein-binding protein. PAC1 having certain desirable functional and biochemical characteristics. Methods for achieving these modifications are described above.

[409] PAC1 antibodies can further be modified in several ways. For example, if used for therapeutic purposes, they can be conjugated with polyethylene glycol (pegylated) to prolong serum half-life or to enhance protein release. Alternatively, the V region of the subject's antibodies or fragments thereof may be fused to the Fc region of a different antibody molecule. The Fc region used for this purpose can be modified so that it does not bind complement, thereby reducing the likelihood of inducing cell lysis in the patient when the fusion protein is used as a therapeutic agent. Additionally, the subject's antibodies or functional fragments thereof can be conjugated to human serum albumin to enhance the serum half-life of the antibody or antigen-binding fragment thereof. Another useful fusion partner for antibodies or fragments thereof is transthyretin (TTR). TTR has the ability to form a tetramer, thus an antibody-TTR fusion protein can form a multivalent antibody that can increase its binding avidity.

[410] Alternatively, substantial modifications to the functional and/or biochemical characteristics of the antibodies described herein can be achieved by creating substitutions in the amino acid sequence of the heavy and light chains that differ significantly in their effect on maintaining (a) molecular structure in the area of substitution, for example, as a sheet or helical conformation, (b) charge or hydrophobicity of the molecule at the target site, or (c) volume of the side chain. A “conservative amino acid substitution” may involve a replacement of a native amino acid residue with a non-native residue that has little or no effect on the polarity or charge of the amino acid residue at that position. See, Table 6, supra. Furthermore, any native residue in the polypeptide can also be replaced with alanine, as has been previously described for alanine scanning mutagenesis.

[411] Amino acid substitutions (conservative or non-conservative) of antibodies can be implemented by those skilled in the art applying routine techniques. Amino acid substitutions can be used to identify important residues of the antibodies provided herein, or to increase or decrease the affinity of these antibodies for human PAC1 or to modify the binding affinity of other antibodies described herein.

Methods for Expressing Antibodies

[412] Expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes comprising at least one polynucleotide as described above are also provided herein, as well as host cells comprising such expression systems or constructs.

[413] The antibodies provided herein can be prepared by any of a number of conventional techniques. For example, PAC1 antibodies can be produced by recombinant expression systems using any known technique. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.) Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).

[414] Antibodies may be expressed in hybridoma cell lines (e.g., particular antibodies may be expressed in hybridomas) or in cell lines other than hybridomas. Expression constructs encoding antibodies can be used to transform a mammalian, insect, or microbial host cell. Transformation can be carried out using any known method for introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide into a virus or a bacteriophage and transduction into a host cell with the construct by transfection procedures known in the art, as exemplified in US Patent 4,399,216; 4,912,040; 4,740,461; 4,959,455. The ideal transformation procedure used will depend on what type of host cell is being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, among others, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotides in liposomes, mixing of nucleic acid with positively charged lipids, and direct microinjection of DNA into nuclei.

[415] Recombinant expression constructs typically comprise a nucleic acid molecule encoding a polypeptide comprising one or more of the following: one or more CDRs provided herein; a light chain constant region; a light chain variable region; a heavy chain constant region (e.g., CH1, CH2 and/or CH3); and/or other framework portion of a PAC1 antibody. These nucleic acid sequences are inserted into an appropriate expression vector using standard ligation techniques. In one embodiment, the heavy or light chain constant region is linked to the C-terminus of the anti-PAC1-specific heavy or light chain variable region and is ligated into an expression vector. The vector is generally selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery, allowing amplification and/or expression of the gene to occur). In some embodiments, vectors are used that employ protein fragment complementation assays using protein reporters, such as dihydrofolate reductase (see, for example, US Patent 6,270,964, which is incorporated herein by reference). Suitable expression vectors can be purchased, for example, from Invitrogen Life Technologies or BD Biosciences (formerly “Clontech”). Other useful vectors for cloning and expressing antibodies and fragments include those described in Bianchi and McGrew, 2003, Biotech. Biotechnol. Bioeng. 84:439-44, which is incorporated herein by reference. Additional suitable expression vectors are discussed, for example, in Methods Enzymol., vol. 185 (m. V. Goeddel, ed.), 1990, New York: Academic Press.

[416] Typically, expression vectors used in host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. These sequences, collectively referred to as “flanking sequences,” in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide into be expressed and a selectable marker element. Each of these sequences is discussed below.

[417] Optionally, the vector may contain a “tag” coding sequence, that is, an oligonucleotide molecule located at the 5' or 3' end of the PAC1 binding protein coding sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis), or another “tag” such as FLAG®, HA (influenza virus hemagglutinin) or myc, for which there are commercially available antibodies. This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification or detection of the host cell's PAC1-binding protein. Affinity purification can be carried out, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified PAC1 binding protein by various means, such as using certain peptidases for cleavage.

[418] Flanking sequences can be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a different species or strain of host cell), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic or native. As such, the source of a flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, as long as the flanking sequence is functional in, and can be activated by, the host cell machinery.

[419] Useful flanking sequences in the vectors can be obtained by any of several methods known in the art. Typically, flanking sequences useful here will be previously identified by mapping and/or restriction endonuclease digestion and therefore can be isolated from the appropriate tissue source using the appropriate restriction endonucleases. In some cases, the complete nucleotide sequence of a flanking sequence may be known. Here, the flanking sequence can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

[420] If all or only part of the flanking sequence is known, it can be obtained using the polymerase chain reaction (PCR) and/or by screening a genomic library with an appropriate probe such as an oligonucleotide and/or or flanking sequence fragment of the same or different species. Where the flanking sequence is not known, a fragment of DNA containing a flanking sequence can be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation can be carried out by restriction endonuclease digestion to produce the appropriate DNA fragment followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA) or other methods known to the person skilled in the art. The selection of appropriate enzymes to accomplish this purpose will be readily apparent to one skilled in the art.

[421] An origin of replication is typically a part of these commercially acquired prokaryotic expression vectors, and origin assists in amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, it can be chemically synthesized based on a known sequence linked to the vector. For example, the origin of replication of plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria, and several viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus 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 required for mammalian expression vectors (e.g., the SV40 origin is often used only because it also contains the virus's early promoter).

[422] A transcription termination sequence is normally located 3' to the end of a polypeptide coding region and serves to terminate transcription. Generally, a transcription termination sequence in prokaryotic cells is a G-C-rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library or even commercially acquired as part of a vector, it can also be easily synthesized using methods for nucleic acid synthesis such as those described here.

[423] A selectable marker gene encodes a protein necessary for the survival and growth of a host cell grown in selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin to prokaryotic host cells; (b) complement the cell's auxotrophic deficiencies; or (c) provide critical nutrients not available from the complex or defined medium. Specific selectable markers are the kanamycin resistance gene, ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, a neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells.

[424] Other selectable genes can be used to amplify the gene that will be expressed. Amplification is the process in which genes that are necessary for the production of a protein critical for cell growth or survival are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of selectable markers suitable for mammalian cells include the promoterless dihydrofolate reductase (DHFR) and thymidine kinase genes. Mammalian cell transformants are placed under selection pressure in which only the transformant is uniquely adapted to survive by virtue of the selectable gene present in the vector. Selection pressure is imposed by culturing transformed cells under conditions in which the concentration of the selection agent in the medium is successively increased, thus leading to amplification of the selectable gene and the DNA encoding another gene, such as an antibody that binds to PAC1. . As a result, increased amounts of a polypeptide such as an antibody are synthesized from the amplified DNA.

[425] A ribosome binding site is generally required for initiation of mRNA translation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is normally located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.

[426] In some cases, such as when glycosylation is desired in a eukaryotic host cell expression system, one can manipulate the various pre- or pro-sequences to improve glycosylation or yield. For example, one can alter the peptidase cleavage site of a particular signal peptide, or add prosequences, which can also affect glycosylation. The final protein product may have, at position - 1 (relative to the first amino acid of the mature protein) one or more additional amino acids incident to 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 enzymatic cleavage sites may result in a slightly truncated form of the desired polypeptide if the enzyme cuts in such an area within the mature polypeptide.

[427] Expression and cloning typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding a PAC1-binding protein. Promoters are non-transcribed sequences located upstream (i.e., 5') to the codon of a structural gene (usually within about 100 to 1000 bp) that control transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters can initiate increases in DNA transcription levels under their control in response to some changes in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on the other hand, uniformly transcribe the gene to which they are operationally linked, that is, with little or no control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to DNA encoding heavy chain or light chain comprising a PAC1 binding protein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector.

[428] Promoters suitable for use with yeast hosts are also known in the art. Yeast enhancers are advantageously used with yeast promoters. Promoters suitable for use with mammalian host cells are well known and include, among others, those obtained from the genome of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat-shock promoters and the actin promoter.

[429] Additional promoters that may be of interest include, among others: SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); 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); herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA. 78:1444-1445); metallothionin gene promoter and regulatory sequences (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA. 25:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA. 80:21-25). Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and are used in transgenic animals: control region of the elastase I gene that is active in the acinar cells of the pancreas (Swift et al., 1984, Cell 28:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); control region of the insulin gene that is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), control region of the immunoglobulin gene that is active in lymphoid cells (Grosschedl et al., 1984, Cell 38 :647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region that is active in the testis, breast, lymphoid cells and mast cells (Leder et al., Cell 1986:45 485-495), albumin gene control region that is active in the liver (Pinkert et al., 1987, Genes and Devel. 1 :268-276), the control region of the alpha-fetoprotein gene, which is active in the liver (Krumlauf et al., 1985, Mol.Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235 :53-58); control region of the alpha 1 antitrypsin gene which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); the beta-globin gene control region that is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene control region that is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 18:703-712); the myosin-2 light chain control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropic releasing hormone gene control region that is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[430] An enhancer sequence can be inserted into a vector to increase the transcription of DNA encoding light chain or heavy chain comprising human PAC1 binding protein by higher eukaryotes. Enhancers are cis-acting elements of DNA, generally about 10-300 base pairs in length, that act on a promoter to increase its transcription. Enhancers are relatively independent of orientation and position, being found at positions 5' and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein, and insulin). Typically, however, a virus enhancer is used. The SV40 enhancer, cytomegalovirus early promoter enhancer, the polyoma enhancer and adenovirus enhancers known in the art are exemplary enhancer elements for the activation of eukaryotic promoters. While an enhancer can be positioned in the vector 5' or 3' to a coding sequence, it is typically located at a site 5' of the promoter. A sequence encoding an appropriate native sequence or heterologous signal (leader sequence or signal peptide) can be incorporated into an expression vector to promote extracellular secretion of the antibody. The choice of signal or leader peptide depends on the type of host cells in which the antibody is to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence for interleukin-7 (IL-7) described in US Patent No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in EP Patent No. 0367 566; the interleukin-1 type I receptor signal peptide described in US Patent 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846.

[431] The expression vectors that are provided can be constructed from an initial vector such as a commercially available vector. These vectors may or may not contain all of the desired flanking sequences. When one or more of the flanking sequences described here are no longer present in the vector, they can be individually obtained and ligated into the vector. Methods used to obtain each of the flanking sequences are known to one skilled in the art.

[432] After the vector is constructed and a nucleic acid molecule encoding a light chain, a heavy chain, or a light chain and a heavy chain sequence comprising a PAC1 antigen binding sequence has been inserted into the site appropriate vector, the complete vector can be inserted into an appropriate host cell for polypeptide amplification and/or expression. Transformation of an expression vector for an antibody in a selected host cell can be carried out by known methods including transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection or other known techniques. The method selected will in part be a function of the type of host cell being used. These methods and other suitable methods are known to those skilled in the art and are set forth, for example, in Sambrook et al., 2001, supra.

[433] A host cell, when cultured under appropriate conditions, synthesizes an antibody that can be subsequently collected from the culture medium (if the host cell secretes into the medium) or directly from the host cell producing the same (if it is not secreted). Selection of an appropriate host cell will depend on several factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation), and ease of folding into a biologically active molecule.

[434] Mammalian cells available as a host for expression are well known in the art and include, among others, immortalized cell lines available from the American Type Culture Collection (ATCC), including, among others, Chinese Hamster Ovary (CHO) cells. , HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), and several other cell lines. In certain embodiments, cell lines can be selected by determining which cell lines have high levels of expression and constitutively produce antibodies with PAC1 binding properties. In another embodiment, a cell line of the B cell lineage that does not produce its own antibody, but has an ability to produce and secrete a heterologous antibody, can be selected.

Use of Human PAC1 Antibodies for Diagnostic and Therapeutic Purposes

[435] Antibodies are useful for detecting PAC1 in biological samples and identifying cells or tissues that produce PAC1. For example, PAC1 antibodies can be used in diagnostic assays, e.g., binding assays, and/or quantifying PAC1 expressed in a tissue or cell. Antibodies that specifically bind to PAC1 can also be used to treat PAC1-related diseases in a patient in need of it. Furthermore, PAC1 antibodies can be used to inhibit PAC1 from forming a complex with its ligand PACAP (e.g., PACAP-38), thereby modulating the biological activity of PAC1 in a cell or tissue. Examples of activities that can be modulated include, among others, inhibition of vasodilation and/or reduction of neurogenic inflammation. Antibodies that bind to PAC1 can thus modulate and/or block the interaction with other binding compounds and as such may have therapeutic use in improving PAC1-related diseases.

Indications

[436] A disease or condition associated with human PAC1 includes any disease or condition whose onset in a patient is caused by, at least in part, the interaction of PAC1 with its ligand, PACAP (e.g., PACAP-38). The severity of the disease or condition can also be increased or decreased by the interaction of PAC1 with PACAP (e.g., PACAP-38). Examples of diseases and conditions that can be treated with the antibodies described herein include headaches, such as cluster headaches, migraines, including migraine headaches, chronic pain, type II diabetes mellitus, inflammation, e.g., neurogenic inflammation, disorders cardiovascular diseases, and hemodynamic derangement associated with endotoxemia and septicemia.

[437] In particular, antibodies described herein can be used to treat migraine, or as an acute treatment beginning after a migraine attack has begun, and/or as a prophylactic treatment, administered, for example, daily, weekly, biweekly, monthly, bimonthly, semi-annually, etc.) to prevent or reduce the frequency and/or severity of symptoms, e.g., pain symptoms, associated with migraine attacks.

[438] Infusion of the PAC1 receptor agonist PACAP causes migraine-like headache in migraine patients, suggesting that PAC1 blockade may be useful in treating migraine. Immunohistochemical studies in the cynomolgus monkey performed in support of the present invention have mapped the localization of PACAP and PAC1 to the parasympathetic pathway through the sphenopalatine ganglion (SPG; also known as the pterygopalatine ganglion), which also innervates the vasculature of the dura mater. The parasympathetic pathway is independent of and parallel to the sensory pathway that also controls the tone of the dural vasculature and therefore likely plays a role in the pathophysiology of migraine. Additional experiments performed in support of the present invention showed that a selective PAC1 Ab blocked electrically stimulated trigeminal cervical complex (TCC) activation, an electrophysiology model has been reported to correlate with clinical efficacy in migraine. Taken together, the evidence supports targeting PAC1 with a selective antagonist such as an anti-PAC1 antibody, as described here to treat migraine. The expected efficacy of an anti-PAC1 drug for the treatment of migraine in human patients is further supported by published articles, including, for example, Schytz, H.W., et al., “The PACAP receptor: a novel target for migraine treatment.” , Neurotherapeutics. 2010 Apr;7(2):191-6; Olesen J., et al., “Emerging migraine treatments and drug targets”, Trends Pharmacol Sci. 2011 Jun;32(6):352-9; and Schytz, H.W., et al., “PACAP38 induces migraine-like attacks in patients with migraine without aura,” Brain (2009) 132 (1): 16-25.

[439] Cluster headache is a condition that involves, as its most prominent feature, an immense, unilateral degree of pain. Some doctors and scientists have described the pain resulting from cluster headaches as the most intense pain a human can endure—worse than that from childbirth, burns, or broken bones. Cluster headaches usually occur periodically: spontaneous remission stops active periods of pain. The average age of onset of cluster headache is ~30-50 years. It is more prevalent in males with a male to female ratio ranging from 2.5:1 or 3.5:1. SPG stimulation is used for the treatment of cluster headache. More recently, Autonomic Technologies developed the ATI™ Neurostimulation System to deliver low-level (but high-frequency, physiologically locked) electrical stimulation to deliver acute SPG stimulation therapy with the intent of relieving acute debilitating headache pain in saved. Robust efficacy was demonstrated in their recent clinical trial (NCT01616511; Schoenen J, et al., “Stimulation of the sphenopalatine ganglion (SPG) for cluster headache treatment. Pathway CH-1: A randomized, sham-controlled study.”, Cephalalgia . . 2013;33(10):816-30. Taking into account the above and due to the fact that PACAP is one of the main neurotransmitters in SPG, a PAC1 receptor antagonist, such as an anti-PAC1 antibody described here, should have efficacy to treat cluster headache in humans.

Diagnostic Methods

[440] The antibodies described here can be used for diagnostic purposes to detect, diagnose or monitor diseases and/or conditions associated with PAC1. Also provided are methods for detecting the presence of PAC1 in a sample using classical immunohistological methods known to those skilled in the art (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, Vol 15 (Eds R.H. Burdon and P.H. van Knippenberg , Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; Jalkanen et al., 1987, J. Cell Biol. 105:3087-3096). PAC1 detection can be performed in vivo or in vitro.

[441] Diagnostic applications provided here include the use of antibodies to detect PAC1 expression and binding of ligands to PAC1. Examples of methods useful in detecting the presence of PAC1 include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).

[442] For diagnostic applications, antibodies will typically be labeled with a detectable labeling group. Examples of labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the antibody through spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used.

[443] In another aspect, an antibody can be used to identify a cell or cells that express PAC1. In a specific embodiment, the antibody is labeled with a labeling group and binding of the labeled antibody to PAC1 is detected. In a more specific embodiment, antibody binding to PAC1 is detected in vivo. In a more specific embodiment, the PAC1 antibody is isolated and measured using known techniques. See, for example, Harlow and Lane, 1988, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor (1991 ed. and periodic supplements); John E. Coligan, ed., 1993, Current Protocols In Immunology New York: John Wiley & Sons.

[444] Another aspect provides detection of the presence of a test molecule that competes for binding to PAC1 with the antibodies provided. An example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of PAC1 in the presence or absence of the test molecule. An increase in the amount of free antibody (i.e., antibody not bound to PAC1) indicates that the test molecule is able to compete for binding of PAC1 to the antibody. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence and absence of an antibody.

Treatment Methods: Pharmaceutical Formulations, Routes of Administration

[445] Methods for using the antibodies are also provided. In some methods, an antibody is given to a patient. The antibody inhibits the binding of PACAP to human PAC1.

[446] Pharmaceutical compositions comprising a therapeutically effective amount of one or a plurality of antibodies and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and/or adjuvant are also provided. Furthermore, methods of treating a patient, for example, for migraine, by administering such a pharmaceutical composition are included. The term “patient” includes human patients.

[447] Acceptable formulation materials are non-toxic to recipients at the dosages and concentrations used. In specific embodiments, pharmaceutical compositions comprising a therapeutically effective amount of antibodies to human PAC1 are provided.

[448] In certain embodiments, acceptable formulation materials preferably are non-toxic to recipients at the dosages and concentrations used. In certain embodiments, the pharmaceutical composition may contain formulation materials to modify, maintain or preserve, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration. of the composition. In these embodiments, suitable formulation materials include, among others, amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); thickening agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); dyes, flavorings and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity-increasing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); release vehicles; diluents; pharmaceutical excipients and/or adjuvants. See, REMINGTON’S PHARMACEUTICAL SCIENCES, 18” Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company.

[449] In certain embodiments, the ideal pharmaceutical composition will be determined by one skilled in the art depending, for example, on the intended route of administration, release format and desired dosage. See, for example, REMINGTON’S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, these compositions can influence the physical state, stability, in vivo release rate and in vivo clearance rate of the disclosed antibodies. In certain embodiments, the main vehicle or carrier in a pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles. In specific embodiments, pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may further include sorbitol or a suitable substitute. In certain embodiments of the invention, human PAC1 antibody compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulating agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra), in the form of a lyophilized cake or a solution. aqueous. Furthermore, in certain embodiments, the antibody to human PAC1 can be formulated as a lyophilisate using suitable excipients, such as sucrose.

[450] The pharmaceutical compositions of the invention can be selected for parenteral release. Alternatively, the compositions may be selected for inhalation or for release through the digestive tract, such as orally. The preparation of such compositions pharmaceutically is within the art.

[451] The formulation components are preferably present in concentrations that are acceptable for the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH, or at a slightly lower pH, typically within a pH range of about 5 to about 8.

[452] When parenteral administration is contemplated, therapeutic compositions can be provided in the form of a parenterally acceptable, pyrogen-free aqueous solution comprising human PAC1 binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the antibody to human PAC1 is formulated as a properly preserved, sterile, isotonic solution. In certain embodiments, the preparation may involve formulating the desired molecule with an agent, such as injectable microspheres, bioerosible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), granules, or liposomes, that can provide controlled or sustained release of the product that can be released through deposit injection. In certain modalities, hyaluronic acid can also be used, having the effect of promoting sustained duration in circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired antibody.

[453] Certain pharmaceutical compositions are formulated for inhalation. In some embodiments, antibodies to human PAC1 are formulated as a dry, inhalable powder. In specific embodiments, human PAC1 antibody inhalation solutions can also be formulated with a propellant for aerosol delivery. In certain embodiments, solutions may be nebulized. Pulmonary administration and methods for formulation, therefore, are further described in International Patent Application No. PCT/US94/001875, which is incorporated by reference and describes pulmonary delivery of chemically modified proteins. Some formulations can be administered orally. Antibodies to human PAC1 that are administered in this way can be formulated with or without carriers usually used in the composition of solid pharmaceutical forms, such as tablets and capsules. In certain embodiments, a capsule may be intended to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate absorption of the human PAC1 antibody. Diluents, flavorings, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

[454] Some pharmaceutical compositions comprise an effective amount of one or a plurality of antibodies to human PAC1 in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit dose form. Suitable excipients include, among others, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents such as starch, gelatin or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

[455] Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving antibodies to human PAC1 in sustained or controlled release formulations. Techniques for formulating a variety of other sustained or controlled release forms, such as liposome carriers, bioerosible microparticles or porous granules, and depot injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT/US93/00829, which is incorporated by reference and describes the controlled release of porous polymeric microparticles for releasing pharmaceutical compositions. Sustained release preparations may include semipermeable polymer matrices in the form of molded articles, e.g., films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactactates (as disclosed in US Patent No. 3,773,919 and European Patent Application Publication No. EP 058481, each of which is incorporated by reference), L-glutamic acid copolymers and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-inethacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167 -277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra) or poly-D(-)-3-hydroxybutyric acid (Application Publication European Patent No. EP133988). Sustained release compositions may also include liposomes which may be prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Academic. Sci. USA. 82:3688-3692; Publication of European Patent Application Nos. EP 036,676; EP 088,046 and EP 143,949, incorporated by reference.

[456] Pharmaceutical compositions used for in vivo administration are typically provided as sterile preparations. Sterilization can be easily accomplished by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method can be performed before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in a solution. Parenteral compositions are generally placed in a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[457] In certain embodiments, cells expressing a recombinant antibody, as disclosed herein, are encapsulated for release (see, Invest. Ophthalmol Vis Sci 43:3292-3298, 2002 and Proc. Natl. Acad. Sciences 103:3896-3901 , 2006).

[458] In certain formulations, an antibody has a concentration of at least 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml or 150 mg/ml. Some formulations contain a buffer, sucrose and polysorbate. An example of a formulation is one containing 50-100 mg/ml antibody, 5-20 mM sodium acetate, 5-10% w/v sucrose. and 0.002 - 0.008% w/v polysorbate. Certain formulations, for example, contain 65-75 mg/ml of an antibody in 9-11 mM sodium acetate buffer, 8-10% w/v sucrose, and 0.005-0.006% w/v polysorbate. The pH of certain formulations is in the range of 4.5-6. Other formulations have a pH of 5.0-5.5 (e.g. pH 5.0, 5.2 or 5.4).

[459] Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. These formulations can be stored in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration. Kits to produce a single dose administration unit are also provided. Certain kits contain a first container having a dry protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered prefilled syringes (e.g., liquid syringes and lyosyringes) are provided. The therapeutically effective amount of a pharmaceutical composition containing antibody to human PAC1 to be employed will depend, for example, on the therapeutic context and objectives. One skilled in the art will appreciate that appropriate dosage levels for treatment will vary depending, in part, on the molecule delivered, the indications for which the human PAC1 antibody is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain embodiments, clinicians can titrate the dosage and modify the route of administration to obtain the best therapeutic effect.

[460] A typical dose may range from about 1 μg/kg to about 30 mg/kg or more, depending on the above-mentioned factors. In specific embodiments, the dosage may range from 10 μg/kg to about 30 mg/kg, optionally from 0.1 mg/kg to about 30 mg/kg, alternatively from 0.3 mg/kg to about 20 mg /kg. In some applications, the dose is 0.5 mg/kg to 20 mg/kg. In some cases, an antibody is dosed at 0.3 mg/kg, 0.5 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, or 20 mg/kg. The dosage schedule in some treatment regimens is at a dose of 0.3 mg/kg qW, 0.5 mg/kg qW, 1 mg/kg qW, 3 mg/kg qW, 10 mg/kg qW, or 20 mg/kg kgqW.

[461] The dosing frequency will depend on the pharmacokinetic parameters of the particular human PAC1 antibody in the formulation used. Typically, a clinician administers the composition until a dosage is reached that achieves the desired effect. The composition, therefore, can be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion through an implanted device or catheter. Suitable dosages can be determined through the use of appropriate dose-response data. In certain embodiments, antibodies can be administered to patients over an extended period of time. Chronic administration of an antibody minimizes the adverse allergic or immune response commonly associated with antibodies that are not fully human, e.g., an antibody raised against a human antigen in a nonhuman animal, e.g., a non-fully human antibody or antibody non-human produced in a non-human species.

[462] The route of administration of the pharmaceutical composition is according to known methods, for example, orally, by injection intravenously, intraperitoneally, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intra-arterial, intraportal, or intralesional; by sustained release systems or by implantation devices. In certain embodiments, the compositions may be administered by bolus injection or continuously by infusion, or by implanted device.

[463] The composition can also be administered locally through the implantation of a membrane, sponge or other appropriate material, in which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implanted device is used, the device can be implanted in any suitable tissue or organ, and the release of the desired molecule can be through diffusion, time-released bolus, or continuous administration.

[464] It may also be desirable to use pharmaceutical antibody compositions for human PAC1 ex vivo. In these cases, cells, tissues or organs that have been removed from patients are exposed to pharmaceutical antibody compositions for human PAC1 after which the cells, tissues and/or organs are subsequently implanted back into the patient.

[465] In particular, antibodies to human PAC1 can be released by implanting certain cells that have been genetically modified, using methods such as those described here, to express and secrete the polypeptide. In certain embodiments, these cells may be human or animal cells, and may be autologous, heterologous, or xenogenic. In certain embodiments, cells can be immortalized. In other embodiments, in order to decrease the chances of an immune response, cells can be encapsulated to prevent infiltration of surrounding tissues. In other embodiments, the encapsulation materials are typically biocompatible, semipermeable polymeric shells or membranes that allow the release of the protein products but prevent destruction of the cells by the patient's immune system or other harmful factors in the surrounding tissues.

[466] The following examples, including the experiments performed and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

EXAMPLE 1 PAC1 ANTIBODIES A. Generation of anti-PAC1 antibodies

[467] Fully human antibodies were generated by immunizing XENOMOUSE animals with the PAC1 extracellular domain protein, DNA tagged with a T cell epitope tag, L1.2 cells expressing full-length human PAC1, and other hPAC1 antigens using standard methods. for example, substantially as detailed in US Patent Publication US 2010-0172895 A1.

B. Anti-PAC1 Antibody Screening

[468] Hybridoma Supernatants were screened for binding to PAC1 and also for functional antagonistic activity in an assay detecting their ability to block cAMP generation by activating PAC1 with PACAP (e.g., PACAP-27 or PACAP- 38) or a selective, exogenous peptide ligand (Maxadilan) and then counterscreened against the related receptors VPAC1 and VPAC2. Supernatants with desirable function and selectivity were sequenced and cloned, recombinantly expressed, purified, and retested for function and selectivity using standard methods, for example, substantially as detailed in US patent publication US 2010-0172895 A1, incorporated herein as reference.

C. Selectivity of PAC1 agonists

[469] Experiments were carried out to evaluate the activity and selectivity of the three PAC1 agonists (PACAP, VIP and Maxadilan) against the related receptors PAC1, VPAC1 and VPAC2. Data from these experiments are shown in Table 7 below, and demonstrate that PACAP has similar agonist activity at all three receptors, VIP has some activity at PAC1 but greater (~10-100x) activity at VPAC1 and VPAC2, and Maxadilan is highly selective. for PAC1 against VPAC1 and VPAC2.

EXAMPLE 2 ACTIVITY OF PAC1-SPECIFIC MONOCLONAL BLOCKING ANTIBODIES IN FUNCTIONAL ASSAY OF CAMP A. Anti-PAC1 Antibody Activity

[470] Selected hPAC1 antibodies as described herein were screened in an in vitro PAC1-mediated cAMP assay to determine intrinsic potency. The assay employed SH-SY-5Y, a human neuroblastoma cell line endogenously expressing hPAC1 (Biedler, J.L., et al., 1978, “Multiple neurotransmitter synthesis by human neuroblastoma cell lines and clones”. Cancer Res. 38 (11 Pt 1): 3751-7; Lutz, E.M., et al., “Characterization of novel splice variants of the PAC1 receptor in human neuroblastoma cells: consequences for signaling by VIP and PACAP.” Mol Cell Neurosci 2006;31:193-209) , and various cell lines expressing recombinant PAC1, VPAC1 & VPAC2 constructs having the following accession numbers: human PAC1 (hPAC1): NM_001118.4; short hPAC1 (hPAC1 with amino acid residues 89-109 deleted (range used: 1 - 88, 110 - 468)): NM_001199637.1; PAC1 cyno: XM_005549858.1; mouse PAC1: NM_133511.2; mouse PAC1: NM_001025372.2; human VPAC1 (hVPAC1): NM_004624.3; human VPAC2 (hVPAC2): NM_003382.4; mouse VPAC2: NM_017238.1; Mouse VPAC2: NM_009511.2.

[471] SHSY-5Y cells were grown in a 1:1 mixture of Minimum Essential Medium (MEM) (Invitrogen 11095-072) and Ham's F12 nutrient mixture (Invitrogen 11765-047), 10% Fetal Bovine Serum (Invitrogen 10099 -141), 1X Penicillin-Streptomycin-Glutamine (Invitrogen 10378016) and 1X MEM Non-Essential Amino Acids (Invitrogen 11140-050). PAC1-CHO, rat PAC1, and PAC1 Cyno cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco Cat# 11965), 10% dialyzed FBS (Invitrogen 26400044), 1% MEM Non-Essential Amino Acids (Invitrogen 11140- 050), 1% sodium pyruvate (Invitrogen 11360070), 1% glutamine/pen/strep (Invitrogen 10378016). CHO-Trex-rat mouse VPAC2R stable cell lines were grown in Basal Medium: F12 (Invitrogen 11765-047), P/S/G: 1X (Invitrogen 10378016), Heat-Inactivated FBS: 10% (Invitrogen 10100147) , Blasticidin: 5 ug/ml (Invitrogen R21001) and Hygromycin: 400 ug/ml (Invitrogen 10687010). Cells were induced with Tetracycline (Sigma 87128): 3.5 ug/ml, add 24 hours before assay. hPAC1-CHO-K1 cells (Perkin Elmer; ES-272-A) were grown in Ham's F12 (Invitrogen 11765-047), 10% FBS (Invitrogen 10099-141), Pen-strep: 1X (Invitrogen 15140122), 400ug /ml G418 (Stock: 50mg/ml=>8ml/L medium) (Invitrogen 10131027), 250ug/ml Zeocin (Stock:100mg/ml=>2.5ml/L medium) (Invitrogen R25005). VPAC1/CHO-CRE-BLA cells (Invitrogen; Agonist:VIP) were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco Cat# 11965), Dialyzed FBS: 10% (Invitrogen 26400044), NEAA: 1X (Invitrogen 11140 -050), Pen/Strep: 1X (Invitrogen 15140122), HEPES (pH 7.3): 25 mM (Invitrogen 15630130). VPAC2/CHO-CRE-BLA cells (Invitrogen; Agonist:VIP) were grown as described above for VPAC1 cells, with the addition of Blasticidin: 5ug/ml (Invitrogen R21001) and Geneticin: 500 ug/ml (G418) (Invitrogen 10131027). SK-N-MC cells were grown in MEM (Invitrogen 11095-072), 10% FBS (Invitrogen 10099-141), 1X NEAA (Invitrogen 11140-050), 1X Sodium Pyruvate (Invitrogen 11360070), 1X glutamine/pen /strep (Invitrogen 10378016). L6 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco Cat# 11965), 10% Fetal Bovine Serum (Gibco Cat# 10099-141), 1X Glutamine/Pen/Strep (Gibco Cat# 10378-016).

[472]Cells were grown under the conditions described above and harvested at 80-90% confluency. Cells were dissociated with VERSENE (1:5000, Invitrogen, 15040-060) for SHSY5Y cells; with 0.05% Trypsin-EDTA (1x), Phenol Red (Invitrogen, 25300054) and then centrifuged in a Beckman GS-6R unit at 1000 rpm for 5 minutes at 4°C. Pellets were resuspended with cell freezing medium (Invitrogen, 12648010). Cells were then counted using a Z1 Coulter Particle Counter and adjusted to the desired concentration (5E6/ml or 2E6/ml) and aliquots were stored at -80°C or in liquid nitrogen until use. Concentrations of PAC1, hVPAC1/2 CHO cells were 4K/10 μl; the others at 2K/10 μl; for SKNMC and L6, the concentration was 1K/10 μl.

[473] The LANCE Ultra cAMP Assay Kit (PerkinElmer, Boston, MA) was used for screening. Assays were performed in triplicate in white Optiplates half-area 96-well plates (Costar 3642) in a total volume of 40 μl. Briefly, vials of frozen cells were thawed by incubation at 37°C in a water bath. Thawed cells were washed once with assay buffer (F12, 0.1% BSA, 1mM IBMX) and cell concentration was adjusted to 2K/10 μl. The cell suspension was distributed in 10-μl aliquots onto the 96 half-area white plates. Five microliters of antagonist (e.g., an anti-PAC1 antibody, as described here) was then added, and the samples were incubated for 30 minutes at room temperature with gentle shaking. Five microliters of agonist (e.g., PACAP38) was then added, and the samples were incubated for an additional 15 minutes, again at room temperature, with gentle shaking. Ten microliters of 4X Eu-cAMP working solution and ten microliters of 4X ULight-anti-cAMP working solution then added and the samples were incubated for an additional 45 minutes at room temperature.

[474] The samples were analyzed on an EnVision instrument at Em665/615nM. Data were processed by GraphPad Prism software version 5/6 (GraphPad Inc., La Jolla, CA) to show POC (percent control) as a function of antagonist anti-PAC1 antibody concentration tested, and were fitted with regression curves. standard nonlinear to produce IC50 values. POC was calculated as follows:

[475] All of the antibodies described here and tested in the assay had IC50 activity between about 0.1 nM and about 200 nM; most had activity between 0.1 nM and 100 nM. Similar experiments were performed using recombinant cells expressing cynomolgus PAC1 and mouse cells expressing rat PAC1 as described above. IC50 obtained using human and cynomolgus PAC1s were similar, while the antibodies tested did not appear to uniformly cross-react well with rat PAC1. Furthermore, cells expressing related receptors hVPAC1 (CRE-Bla-CHO-K1/ Invitrogen) and hVPAC2 (-Bla- CHO-K1/ Invitrogen) were used to evaluate the selectivity of the tested antibodies. None of the antibodies tested had significant inhibitory activity against hVPAC1 or hVPAC2 in the range tested (IC50 was >10,000 nM in all cases). Exemplary data is shown in Table 8, below.

[476] The differences in IC50 between human PAC1 and human VPAC1 and VPAC2 receptors illustrate the high selectivity of these antibodies for PAC1 over related receptors.

EXAMPLE 3 RADIOLIGAND BINDING ASSAYS FOR DETERMINING Ki OF RECEPTOR BLOCKING ANTIBODIES

[477] 125I-labeled PAC-1 antibodies (for PAC1 binding assay) or 125I-labeled PEG-PACAP38 antibodies (for PACAP38 binding assay), both obtained from Perkin Elmer (Billerica, MA), and cell membranes prepared from from hPAC-1 AequoScreen CHO (PerkinElmer Life and Analytical Sciences, Boston, Massachusetts) were used for radioligand binding experiments in the presence of different concentrations of test antibodies to determine the corresponding Ki values.

Cell Membrane Preparation

[478] Cells were grown to confluency in 40xT225 flasks in Ham's F12, 10%FBS, 100 μg/ml streptomycin, 400 μ/ml G418 (receptor expression selection) and Zeocin (Aequorin expression selection). The flasks were washed 1x with Ca/Mg-free PBS. PBS was removed completely. 5ml VERSENE was added to each vial and waited for about 5 min. The flasks were turned on their side and all cells were dislodged. VERSENE/cell suspension from all flasks were transferred to 50 ml conical tubes and placed on ice. To collect the remaining cells, the flasks were washed with ice-cold, Ca/Mg-free PBS with protease inhibitors and solution was added to the cells in tubes. The tubes were rotated at 1000 rpm for 10 min. Pellets were stored at -80°C.

[479]On the day of membrane preparation, the cell pellets were weighed and dissolved on ice. The pellets were resuspended in (10ml/gram of pellet) 10mM TrisHCl, pH7.4, 1mM EDTA, Roche protease inhibitors (1 tablet/50ml) and homogenized with 30 movements with the motorized dounce homogenizer. Cell homogenates were centrifuged at 2500 rpm(~1000g) for 10 minutes at 4°C. Supernatants were transferred to a Beckman centrifuge tube and kept on ice. The pellets were homogenized again with 15 ml of buffer and the rotation step was repeated. Both supernatants were combined and centrifuged at 48,400 g for 30 min. The pellets were resuspended in 15 ml buffer and cut using an 18G syringe to break the pellet. Pellets were homogenized with 20 strokes and centrifuged at 48,400 g for 30 min. Pellets were resuspended in ~6ml of PACAP Binding buffer without BSA (20mM Tris HCl, 5mM MgSO4+ Protease Inhibitors). The pellets were resuspended using an 18G syringe, dounce homogenized, aliquoted and stored at -70°C. Total membrane protein concentration was determined with a Microplate BCA Protein Assay (Pierce).

Binding Assay

[480] The PAC1 binding assay was set up at room temperature in plates containing 96 wells: 120 μl binding buffer (20 mM Tris-HCl, pH 7.5, 5.0 mM MgSO4, 150 mM NaCl, 0.1% BSA (Sigma), 1 tablet of CompleteTM buffer/50 ml (a protease inhibitor)); 10 μl test compound (20X); 50 μl human PAC1 CHO membrane suspension (0.2 μg per well); and 20 μl of 125I-PAC-1 antibody (10X; for PAC1 binding assay) or 125I-PEG-PACAP 38 (10X; for PACAP38 assay buffer).

[481] Non-specific binding was determined by adding 10 μl of (20X) 20μ M PACAP 6-38 into designated plates. The plates were incubated at room temperature for 2 hours with shaking at 60 rpm, and then the contents of each well were filtered through GF/C 96-well filter plates treated with 0.5% polyethyleneimine (PEI) (at least two hours ). The GF/C filter plates were washed five times with ice-cold 50 mM Tris, pH 7.5 and dried in an oven at +55 °C for 1 hour. The bottom parts of the GF/C plates were then sealed. 40 μl 20 MicroscintTM was added to each well, the tops of the GF/C plates were sealed with TopSealTM-A (a press-on adhesive sealing film), and the GF/C plates were counted with TopCount NXT (Packard). Data were analyzed using Prizm (GraphPad Software Inc.)

[482] Exemplary data showing Ki values obtained as described above are shown in Table 9, below. PAC1 binding assay data is shown with 125I-labeled PAC-1 antibodies indicated in different columns, and test antibodies as shown in different rows. Samples from the different production batches were run with some of the test antibodies as shown (01A, 26A, 39A). The results indicate that the tested anti-PAC1 antibodies that have biological activity in the cAMP assay described in Example 2 compete with the binding of a PAC1 agonist (125I-labeled PEG-PACAP38) and also cross-compete with another for the same epitope. or similar in hPAC1.

EXAMPLE 4 BINDING KD MEASURED BY KINEXA A. Experiments using whole cells expressing hPAC1.

[483] Anti-Pac1 antibody binding to CHOK1/huPac1 cells was tested in KinExA. Briefly, UltraLink Biosupport (Pierce cat# 53110) was pre-coated with goat anti-huFc (Jackson Immuno Research cat# 109-005-098) and blocked with BSA. 10 pM, 30 pM, 100 pM, and 300 pM of anti-Pac1 antibody was incubated with various densities (1.7x101 - 9x106 cells/mL) of CHOK1 cells expressing huPac1 in F12 medium containing 1% FBS and 0.05% azide of sodium. Samples containing anti-Pac1 antibody and whole cells were incubated with rotation at room temperature for 4 hours before 220-g centrifugation for 5 minutes at 4oC to separate whole cells and anti-Pac1 antibody-cell complexes from the anti-Pac1 antibody. Pac1 not bound. Supernatants containing free anti-Pac1 antibody were filtered through a 0.22-μm filter before loading onto goat anti-huFc-coated beads in KinExA. The amount of anti-Pac1 antibody bound to the bead was quantified by fluorescently labeled (Alexa Fluor 647) anti-huIgG (H+L) antibody (Jackson Immuno Research cat# 109-605-088). The binding signal is proportional to the concentration of free anti-Pac1 antibody in solution at each cell density. The equilibrium dissociation constant (KD) was estimated by n-curve analysis in KinExATM Pro software (Sapidyne Instruments, Inc., Boise, ID).

B. Experiments using hPAC1 extracellular domain (ECD; hPAC1 1-135).

[484]Binding of anti-Pac1 Abs to huPac1(1-135)::6xHis was tested in KinExA. Briefly, the UltraLink Biosupport (Thermo Fisher Scientific cat# 53110) was pre-coated with huPac1 (1- 135)::6xHis and blocked with BSA as described above. 30, 100, and 300 pM Ab were incubated with different concentrations of huPac1 (1-135)::6xHis at room temperature for at least 8 hours before running through the huPac1 (1-135)::6xHis-coated beads. The amount of Ab bound to the bead was quantified by fluorescently labeled goat anti-huIgG (H+L) antibody (DyLight 649) (Jackson Immuno Research cat# 109-495-088). The sign of binding is proportional to the free Ab concentration at the binding equilibrium. The equilibrium dissociation constant (KD) was obtained by nonlinear regression of the competition curves using a one-site homogeneous binding model (KinExATM Pro software).

[485] A summary of the equilibrium dissociation constants for binding of the samples tested with CHOK1/huPac1 and huPac1 (1-135)::6xHis cell is shown in Table 10 below (average followed by (range)).

EXAMPLE 5 Platelet phagocytosis phagocytosis

[486] To address any potential Fc gamma receptor-induced platelet depletion, a subset of the antibodies described here were tested in a Phagocytosis Assay.

A. Preparation of Peripheral Blood Leukocytes (PBL) and Platelets

[487] Human and cynomolgus platelets were prepared as previously described (Semple, J.W., et al., Blood (2007) 109: 4803-4805). Briefly, whole blood samples from healthy cynomolgus donors were centrifuged at 170 x g for 15 minutes without braking. Platelet-rich plasma (PRP) was collected from the top layer. The bottom layers of the sample were centrifuged at 2000 x g for 10 minutes to obtain the platelet-poor plasma (PPP) layer and buffy coat layer. Platelets in the PRP fraction were labeled with 20 μM Cell Tracker Green CMFDA for 30 minutes at room temperature. Labeled platelets were washed with PBS, resuspended in RPMI-1640 with 10% FBS, and adjusted to a concentration of 1 x 108 cells/mL. PBL were prepared from the buffy coat obtained from the same donor. RBCs were lysed with BD Pharm-lyse following the protocol provided by the vendor. After lysis, PBL were washed twice with RPMI-1640 with 10% FBS and the final concentration was adjusted to 5 x 106 cells/ml.

B. Phagocytosis Assay

[488] The phagocytosis reaction was initiated by incubating 100 μL each of CMFDA-labeled platelets and PBMC with test articles in the dark. After six hours of incubation, the reaction was stopped by placing the plates on ice. Extracellular fluorescence was stopped by incubating for 2 minutes with 0.1% trypan blue. After washing twice with ice-cold PBS, cells were incubated with propidium iodide and anti-CD14 for 30 min at 4°C. After washing, the cells were analyzed by a BD LSRII Flow Cytometer (Figure 2). Based on previous experiments, platelet-phagocytosing monocytes have median CMFDA fluorescence intensity values at least 2-fold or higher than negative controls (a-SA or a-DNP) (2).

[489] Exemplary data is shown in Table 11, below. Results show that none of the platelets activated by aglycosylated IgG1 variants (antibodies ending with “B” or “C”, although only “B” was tested in this study) analyzed in human or cynomolgus whole blood when tested at antibody concentrations up to 14 mg/mL. Likewise, no effect was seen on platelet phagocytosis in vitro up to and including the highest concentration tested (3mg/ml). In vitro neutrophil activation was observed at any concentration tested.

[490] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in these publications that may be used in connection with the subject matter disclosed herein. These publications are provided solely for dissemination prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors do not have the right to backdate this disclosure by virtue of prior invention or for any other reason. Any statements as to the date or representation as to the contents of these documents are based on information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

Claims (20)

1. Isolated antibody or antigen-binding fragment thereof that specifically binds to the human pituitary adenylate cyclase-activating polypeptide (PAC1) type I receptor, characterized by comprising 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: 153 and the heavy chain variable region comprises the sequence of SEQ ID NO: 174; (b) 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: 174; or (c) 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: 182. 2. Isolated antibody or antigen-binding fragment thereof, according to claim 1, characterized in that 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: 182. 3. Isolated antibody or antigen-binding fragment thereof, according to claim 1, characterized in that 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: 174. 4. Isolated antibody or antigen-binding fragment thereof, according to claim 1, characterized in that 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: 174. 5. Isolated antibody or antigen-binding fragment thereof, according to claim 1, characterized in that the antibody comprises: (a) a light chain comprising the sequence of SEQ ID NO: 79 and a heavy chain comprising the selected sequence of SEQ ID NOs: 107-109 and 131-133; or (b) a light chain comprising the sequence of SEQ ID NO: 82 and a heavy chain comprising the selected sequence of SEQ ID NOs: 107-109. 6. Isolated antibody or antigen-binding fragment thereof, according to claim 5, characterized in that the antibody comprises a light chain comprising the sequence of SEQ ID NO: 79 and a heavy chain comprising the sequence of SEQ ID NO: 131. 7. Isolated antibody or antigen-binding fragment thereof, according to claim 5, characterized in that the antibody comprises a light chain comprising the sequence of SEQ ID NO: 79 and a heavy chain comprising the sequence of SEQ ID NO: 132. 8. Isolated antibody or antigen-binding fragment thereof, according to claim 5, characterized in that the antibody comprises a light chain comprising the sequence of SEQ ID NO: 79 and a heavy chain comprising the sequence of SEQ ID NO: 133. 9. Isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, characterized in that the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody, or a single chain antibody. 10. Isolated antibody or antigen-binding fragment thereof, according to claim 9, characterized in that the monoclonal antibody is a fully human antibody. 11. Isolated antibody or antigen-binding fragment thereof, according to claim 9, characterized in that the antibody is an IgG1, IgG2, IgG3 or IgG4 type antibody. 12. Isolated antibody or antigen-binding fragment thereof, according to claim 11, characterized in that the antibody is an aglycosylated IgG1 antibody. 13. Isolated antibody or antigen-binding fragment thereof, according to claim 12, characterized in that the antibody comprises a mutation at position N297 in its heavy chain. 14. Isolated antibody or antigen-binding fragment thereof, according to claim 13, characterized in that the antibody comprises an N297G mutation in its heavy chain. 15. Isolated antibody or antigen-binding fragment thereof, according to claim 13, characterized in that the antibody further comprises R292C and V302C mutations in its heavy chain. 16. Pharmaceutical composition characterized by comprising an antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 15, and a pharmaceutically acceptable excipient. 17. Use of the antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 15, characterized in that it is in the preparation of a medicament for treating headache in a patient. 18. Use according to claim 17, characterized in that the headache is a migraine or cluster headache. 19. Use according to claim 18, characterized by the fact that the migraine is episodic migraine or chronic migraine. 20. Use according to any one of claims 17 to 19, characterized in that the treatment comprises prophylactic treatment.