ANTI-PACAP ANTIBODY

Abstract

Nucleic acids comprising sequences that encode antibodies to human pituitary adenylate cyclase-activating peptide for the manufacture of antibodies to human pituitary adenylate cyclase-activating peptide.

Claims

1. A nucleic acid comprising a sequence encoding SEQ ID NO: 9 or 10.

2. The nucleic acid of claim 1, wherein the sequence encodes SEQ ID NO: 9 and wherein residue 62 is aspartic acid residue 104 is asparagine.

3. The nucleic acid of claim 1, wherein the sequence encodes SEQ ID NO: 9 and wherein residue 62 is alanine and residue 104 is threonine.

4. The nucleic acid of claim 1, wherein the sequence encodes SEQ ID NO: 9 and wherein residue 62 is glutamic acid and residue 104 is threonine.

5. The nucleic acid of claim 1, wherein the sequence encodes SEQ ID NO: 9 and wherein residue 62 is glutamine and residue 104 is threonine.

6. The nucleic acid of claim 1, wherein the sequence encodes SEQ ID NO: 10 and wherein residue 30 is serine and residue 55 is leucine.

7. The nucleic acid of claim 1, wherein the sequence encodes SEQ ID NO: 10 and wherein residue 30 is tryptophan and residue 55 is phenylalanine.

8. A nucleic acid comprising a sequence encoding SEQ ID NO: 1 or 2.

9. The nucleic acid of claim 8, wherein the sequence encodes SEQ ID NO: 1 and wherein residue 62 is aspartic acid, residue 104 is asparagine, residue 231 is proline, residue 237 is alanine and residue 238 is alanine.

10. The nucleic acid of claim 8, wherein the sequence encodes SEQ ID NO: 1 and wherein residue 62 is alanine, residue 104 is threonine, residue 231 is proline, residue 237 is alanine and residue 238 is alanine.

11. The nucleic acid of claim 8, wherein the sequence encodes SEQ ID NO: 1 and wherein residue 62 is glutamic acid, residue 104 is threonine, residue 231 is proline, residue 237 is alanine and residue 238 is alanine.

12. The nucleic acid of claim 8, wherein the sequence encodes SEQ ID NO: 1 and wherein residue 62 is glutamine, residue 104 is threonine, residue 231 is proline, residue 237 is alanine and residue 238 is alanine.

13. The nucleic acid of claim 8, wherein the sequence encodes SEQ ID NO: 2 and wherein residue 30 is serine and residue 55 is leucine.

14. The nucleic acid of claim 8, wherein the sequence encodes SEQ ID NO: 2 and wherein residue 30 is tryptophan and residue 55 is phenylalanine.

15. A vector comprising: (a) a first nucleic acid sequence encoding SEQ ID NO: 9 and a second nucleic acid sequence encoding SEQ ID NO: 10; or (b) a first nucleic acid sequence encoding SEQ ID NO: 1 and a second nucleic acid sequence encoding SEQ ID NO: 2.

16. The vector of claim 15, wherein the first nucleic acid sequence encodes SEQ ID NO: 9 and the second nucleic acid sequence encodes SEQ ID NO: 10 and wherein, residue 62 of SEQ ID NO: 9 is aspartic acid, residue 104 of SEQ ID NO: 9 is asparagine, residue 30 of SEQ ID NO: 10 is serine and residue 55 of SEQ ID NO: 10 is leucine.

17. The vector of claim 15, wherein the first nucleic acid sequence encodes SEQ ID NO: 9 and the second nucleic acid sequence encodes SEQ ID NO: 10 and wherein, residue 62 of SEQ ID NO: 9 is alanine, residue 104 of SEQ ID NO: 9 is threonine, residue 30 of SEQ ID NO: 10 is tryptophan and residue 55 of SEQ ID NO: 10 is phenylalanine.

18. The vector of claim 15, wherein the first nucleic acid sequence encodes SEQ ID NO: 9 and the second nucleic acid sequence encodes SEQ ID NO: 10 and wherein, residue 62 of SEQ ID NO: 9 is glutamic acid, residue 104 of SEQ ID NO: 9 is threonine, residue 30 of SEQ ID NO: 10 is tryptophan and residue 55 of SEQ ID NO: 10 is phenylalanine.

19. The vector of claim 15, wherein the first nucleic acid sequence encodes SEQ ID NO: 9 and the second nucleic acid sequence encodes SEQ ID NO: 10 and wherein, residue 62 of SEQ ID NO: 9 is glutamine, residue 104 of SEQ ID NO: 9 is threonine, residue 30 of SEQ ID NO: 10 is tryptophan and residue 55 of SEQ ID NO: 10 is phenylalanine.

20. The vector of claim 15, wherein the first nucleic acid sequence encodes SEQ ID NO: 1 and the second nucleic acid sequence encodes SEQ ID NO: 2 and wherein, residue 62 of SEQ ID NO: 1 is aspartic acid, residue 104 of SEQ ID NO: 1 is asparagine, residue 231 of SEQ ID NO: 1 is proline, residue 237 of SEQ ID NO: 1 is alanine, residue 238 of SEQ ID NO: 1 is alanine, residue 30 of SEQ ID NO: 2 is serine and residue 55 of SEQ ID NO: 2 is leucine.

21. The vector of claim 15, wherein the first nucleic acid sequence encodes SEQ ID NO: 1 and the second nucleic acid sequence encodes SEQ ID NO: 2 and wherein, residue 62 of SEQ ID NO: 1 is alanine, residue 104 of SEQ ID NO: 1 is threonine, residue 231 of SEQ ID NO: 1 is proline, residue 237 of SEQ ID NO: 1 is alanine, residue 238 of SEQ ID NO: 1 is alanine, residue 30 of SEQ ID NO: 2 is tryptophan and residue 55 of SEQ ID NO: 2 is phenylalanine.

22. The vector of claim 15, wherein the first nucleic acid sequence encodes SEQ ID NO: 1 and the second nucleic acid sequence encodes SEQ ID NO: 2 and wherein, residue 62 of SEQ ID NO: 1 is glutamic acid, residue 104 of SEQ ID NO: 1 is threonine, residue 231 of SEQ ID NO: 1 is proline, residue 237 of SEQ ID NO: 1 is alanine, residue 238 of SEQ ID NO: 1 is alanine, residue 30 of SEQ ID NO: 2 is tryptophan and residue 55 of SEQ ID NO: 2 is phenylalanine.

23. The vector of claim 15, wherein the first nucleic acid sequence encodes SEQ ID NO: 1 and the second nucleic acid sequence encodes SEQ ID NO: 2 and wherein, residue 62 of SEQ ID NO: 1 is glutamine, residue 104 of SEQ ID NO: 1 is threonine, residue 231 of SEQ ID NO: 1 is proline, residue 237 of SEQ ID NO: 1 is alanine, residue 238 of SEQ ID NO: 1 is alanine, residue 30 of SEQ ID NO: 2 is tryptophan and residue 55 of SEQ ID NO: 2 is phenylalanine.

Description

EXAMPLES

Example 1. Engineering and Expression of Exemplified Anti-PACAP Antibodies

[0025] Significant problems associated with chemical and physical stability were encountered when constructing an anti-PACAP antibody of the present invention. For example, problems encountered with initial humanized constructs include low binding affinity, unacceptable immunogenicity, variable region deamidation, oxidation, isomerization and low potency.

[0026] Chemical and physical modifications were therefore engineered to improve the binding affinity, eliminate or reduce HC dimerization, reduce immunogenicity, and improve chemical and physical stability of the antibodies of the present invention. Amino acid modifications were engineered throughout both the heavy and light chains. Extensive protein stability studies were also performed and the constructed antibodies were screened for expression and thermostability properties as well as other properties including binding affinity. The following antibodies, which include numerous modifications from original constructs, are identified as possessing high binding affinity, being chemically and physically stabile, possessing low immunogenicity and possessing pharmacokinetic properties consistent with monthly administration. None of the modifications comprising the antibodies of the present invention were identified in initially humanized constructs.

[0027] Exemplified anti-PACAP antibodies of the present invention are presented in Table 1. The exemplified antibodies include heavy chains of SEQ ID NO: 1 and light chains of SEQ ID NO: 2 as well as human HC framework 3-23 and human kappa LCs with framework O18. Additionally, engineered modifications within both the light and heavy chains which improve chemical and physical stability as well as functional properties of the antibodies are provided in Table 1. The relationship of the various regions of the exemplified anti-PACAP antibodies is as follows (numbering of amino acids applies linear numbering; assignment of amino acids to variable domains is based on the International Immunogenetics Information System® available at www.imgt.org; assignment of amino acids to CDR domains is based on the well-known North numbering convention, with the exception of HCDR2 which is based on the well-known Kabat numbering convention):

TABLE-US-00001 TABLE 1 Amino acid regions of exemplified anti-PACAP antibodies of the present invention. SEQ ID NO: 1 SEQ ID NO: 2 Region Positions Region Positions HCVR FRH1  1-22 LCVR FRL1  1-23 HCDR1 23-35 LCDR1 24-34 FRH2 36-49 FRL2 35-48 HCDR2 50-66 LCDR2 49-56 FRH3 67-96 FRL3 57-88 HCDR3  97-112 LCDR3 89-97 FRH4 113-123 FRL4  98-107 Constant CH 124-449 Constant CL 108-214 Exemplified 62D; 104N; 231P; Exemplified 30S; 55L Ab A 237A; 238A Ab A Exemplified 62A; 104T; 231P; Exemplified 30W; 55F Ab B 237A; 238A Ab B Exemplified 62E; 104T; 231P; Exemplified 30W; 55F Ab C 237A; 238A AbC Exemplified 62Q; 104T; 231P; Exemplified 30W; 55F Ab D 237A; 238A Ab D

[0028] The following Examples and assays demonstrate that the antibodies of the present invention are useful for treating pain including primary and secondary headache and migraine. It should be understood however, that the following Examples are set forth by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.

[0029] The exemplified anti-PACAP antibodies of the present invention can be expressed and purified essentially as follows. A glutamine synthetase (GS) expression vector containing a DNA sequence encoding a HC amino acid sequence according to Table 1 (for example, a DNA sequence of SEQ ID NO:11 encoding a HC of Exemplified Antibody B presented in Table 1) and a DNA sequence encoding a LC amino acid sequence according to Table 1 (for example, a DNA sequence of SEQ ID NO:12 encoding a LC of Exemplified Antibody B presented in Table 1) is used to transfect a Chinese hamster ovary cell line (CHO) by electroporation. The expression vector encodes an SV Early (Simian Virus 40E) promoter and the gene for GS. Expression of GS allows for the biochemical synthesis of glutamine, an amino acid required by the CHO cells. Post-transfection, cells undergo bulk selection with 50 μM L-methionine sulfoximine (MSX). The inhibition of GS by MSX is utilized to increase the stringency of selection. Cells with integration of the expression vector cDNA into transcriptionally active regions of the host cell genome can be selected against CHO wild type cells, which express an endogenous level of GS. Transfected pools are plated at low density to allow for close-to-clonal outgrowth of stable expressing cells. The masterwells are screened for antibody expression and then scaled up in serum-free, suspension cultures to be used for production. Clarified medium, into which the antibody has been secreted, is applied to a Protein A affinity column that has been equilibrated with a compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed with 1M NaCl to remove nonspecific binding components. Bound antibody is eluted, for example, with sodium citrate at pH (approx.) 3.5 and fractions are neutralized with 1M Tris buffer. Antibody fractions are detected, such as by SDS-PAGE or analytical size-exclusion, and then are pooled. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. Exemplified anti-PACAP antibody of the present invention is concentrated and/or sterile filtered using common techniques. The purity of exemplified antibody after these chromatography steps is greater than 95%. Exemplified anti-PACAP antibody of the present invention may be immediately frozen at −70° C. or stored at 4° C. for several months.

Example 2. Binding Affinity

[0030] Binding affinity (K.sub.D) for each antibody-antigen complex is determined using a kinetic exclusion 96-well plate-based assay (adapted from Estep et al., mAbs, 2013, 5:270-278). Briefly, a 3-fold dilution series for each Exemplified Ab (set forth in Table 1) is prepared from a starting concentration of 7290 pM to 41 fM; each series including an antibody-blank control. Samples are prepared in 3% (w/v) Blocker A solution (MSD, #R93AA-1) and a fixed final antigen concentration of 30 pM of N-terminal biotinylated PACAP27 (custom synthesis, CPC Scientific) or N-terminal biotinylated PACAP38 (Anaspec, #23590) is added to each sample. A volume of 100 μl of each antigen-antibody sample is added to individual wells of a 96-well microtiter plate (Greiner, EK-20101) in duplicate. The plate is sealed with optical adhesive film (Thermo Fisher Scientific, #4311971) and incubated at 37° C. for 3 to 4 days to allow for sample equilibrium. On the day before analysis, each row of a 96-well MSD Standard plate (MSD, #L15XA) is coated with 30 μl of the corresponding antibody (as used in the titration series) at a concentration of 3 μg/ml in phosphate buffered saline (PBS). On the day of the experiment, the MSD Standard plate is washed once with 150 μl PBS and blocked with 150 μl of 3% Blocker A solution for 45 min at 30° C. with shaking at 300 rpm on a MaxQ 4450 benchtop shaker (Thermo Fisher Scientific). Following three washes with PBS, 50 μl of each antigen-antibody sample (prepared and incubated as described above) is added to the MSD Standard plate and incubated for 150 seconds at 30° C. with shaking at 300 rpm. Following a single wash with PBS, 50 μl of 1 μg/ml SULFO-TAG labeled Streptavidin (MSD, #R32AD-5) prepared in 1% (w/v) Blocker A solution is added and the MSD Standard plate and the plate is incubated at 30° C. for 3 minutes with shaking at 300 rpm. The plate is washed three more times with PBS followed by the addition of 150 μl/well of 1× Read buffer (MSD, #R92TC-2). The MSD Standard plate is read using a MESO Quickplex SQ 120/1300 instrument (Meso Scale Discovery). Data analysis is done using SigmaPlot (Version 12.5, Systat Software) and binding affinity (K.sub.D) is determined using SigmaPlot's integrated Four Parameter Logistic Curve Model. Results are provided in Table 2.

TABLE-US-00002 TABLE 2 Binding affinity (K.sub.D) of antibody-antigen complexes at 37° C. Binding Affinity, K.sub.D (pM, 37° C.) Antigen Antigen Antibody (PACAP38) (PACAP27) Exemplified Ab A not determined 37.0 Exemplified Ab B 12.8 11.6 Exemplified Ab C 14.6 14.5 Exemplified Ab D 9.0 10.5

Example 3. In Vivo PACAP Degradation

[0031] Degradation of circulating PACAP38, in vivo, is assessed in rats. One of control IgG4 antibody (10 mg/kg) or Exemplified Antibody A (10 mg/kg) is injected into rats intravenously. Baseline PACAP38, in plasma, is assessed for each rat and then rats are injected, intravenously, with 6 μg/kg PACAP38. Following PACAP38 injection, plasma samples are collected over a 120 minute period and total levels of PACAP38 (bound or unbound to antibody) are determined via a modified ELISA format. Briefly, a 96 well MSD plate (MSD, catalog #: L15xA-1) is coated with 30 μl/well of 1 ug/ml anti-PACAP38 antibody (US Biological Cat #P1775-03C) in PBS and incubated overnight at 40° C. The plate is washed three times with 200 μl/well PBST followed by blocking with 150 μl/well 3% Blocker A (MSD Cat #R93BA-2) in PBS at room temperature for 1 hour with rotation at 650 rpm. Thereafter, the plate is washed three times with 200 μl/well PBST. Calibrator of PACAP38 (Bachem Cat #H430-0500) is diluted at 1000 pg/mL with Assay Buffer containing one part diluent 2 (MSD Cat #R51-BB-3) and thirty-nine parts diluent 3 (MSD Cat #R51BA-3) supplemented with 200 μg/ml HBR1 (Scantibodies Inc, Cat #3KC533) and 10 μg/ml of Biotin-Exemplified Antibody A. Calibrator standard curve is prepared by three-fold serial dilution of 1000 pg/mL calibrator with Assay Buffer. Plasma samples are diluted 40 fold in diluent 3 supplemented with 200 μg/mL HBR1 and 10 μg/mL of Biotin-Exemplified Antibody A. 25 μl of different concentrations of calibrator and diluted plasma samples are added to individual wells of the assay plate and incubated at room temperature for 2 hours with rotation at 650 rpm. After washing the plate 3 times with 200 μl/well PBST, 25 μl of 0.5 μg/ml Sulfo-tag labeled goat anti-human IgG (MSD Cat #R32AJ-1) in diluent 3 is added to each well and the plate is incubated at room temperature for 1 hour. Plates are then washed three times with 200 μl/well PBST and 150 μl of 2× read buffer T (MSD, Cat #R92TC-3) is added to each well and signal is read with a MSD plate reader (results, presented as detectable PACAP38 levels, mean±SEM (n=2-3)). Results are presented in Table 3.

TABLE-US-00003 TABLE 3 PACAP38 plasma levels PACAP38 Plasma Levels (ng/mL) at time points post PACAP38 injection (mean ± SEM (N)) Antibody baseline 5 mins. 30 mins. 60 mins. 90 mins. 120 mins. Exemplified Ab A 0.4 ± 0.0 200.0 ± 23.0  74.1 ± 11.8 37.8 ± 3.2  20.7 ± 2.9  11.7 ± 2.5  (2) (3) (3) (3) (3) (2) Control IgG4 Ab 0.5 ± 0.5 6.2 ± 2.5 2.3 ± 0.9 1.1 ± 0.3 0.5 ± 0.2 0.3 ± 0.1 (3) (3) (3) (3) (3) (3)

[0032] Results presented in Table 3 demonstrate Exemplified Antibody A prevents PACAP38 distribution upon intravenous injection but does not prevent PACAP38 in vivo degradation.

Example 4. Inhibition of PACAP27, PACAP38, or VIP-Induced cAMP

[0033] Neutralization of PACAP27, PACAP38, and VIP induced cAMP stimulation, by the antibodies of the present invention, is assessed in CHO-K1 cells (ATCC, catalog #CCL-61) transfected with vectors expressing either human PAC 1 (NP_001186564.1) or human VPAC2 (NP_003373.2). CHO-K1 cells are harvested with nonenzymatic Cell Dissociation Buffer (Gibco #313131-014), counted, centrifuged and resuspended in assay buffer to 10,000 cells/25 μl. Concentrated cells (10,000 in 25 μl) are added to wells of a 96-well plate.

[0034] Exemplified Antibodies (set forth in Table 1) are serial diluted in 100 μl assay buffer (HBSS with calcium and magnesium (Hyclone, ThermoScientific, SH30268), 0.1% BSA (Sigma-Aldrich, #A7888), 500 μM IBMX (Sigma-Aldrich, 15879)), to 4-times desired final concentration. Diluted Exemplified Antibodies are incubated with agonist (at 2× the final concentration) for 15 minutes at room temperature. Final concentrations of agonist: 100 pM for PACAP27 and PACAP38 in the human PAC1, 800 pM PACAP27 and PACAP38 in the human VPAC2, 800 pM VIP in the human VPAC2 assays. Thereafter, 25 μl of Exemplified Antibody-agonist solution is added to individual wells of the 96-well plate (containing cells) and incubated for 1 hour at room temperature. cAMP formation in each well is determined using cAMP femto 2 (Cisbio, 62AM5PEC). 25 μl cAMP-d2 working solution (Cisbio, 62AM5PEC) is added to each well followed by the addition of 25 μl Anti cAMP-Cryptate working solution and the plate is incubated for 1 hr at room temperature in the dark. Resulting HTRF signal is measured at 665 and 620 nm using an Envision 12 (Perkin Elmer, excitation 330 nm) instrument, and the ratio of 665/620 is plotted to quantify cAMP (data are plotted using Graphpad Prism 7 and IC50 values are determined using the integrated Four Parameter Logistic Curve Model fitting routines). Results are provided in Table 4 as mean±SEM.

TABLE-US-00004 TABLE 4 In Vitro Neutralization of PACAP27 and PACAP38 Induced cAMP Increase Inhibition (IC50) (pM, mean ± SEM (N)) PACAP38 PACAP27 PACAP38 PACAP27 VIP (100 pM) (100 pM) (800 pM) (800 pM) (800 pM) Antibody in hPAC1 in hPAC1 in hVPAC2 in hVPAC2 in hVPAC2 Exemplified  554 ± 264 2250 714 ± 194 781 ± 340 >100,000 Ab A (N = 2) (N = 1) (N = 6) (N = 2) (N = 1) Exemplified 265 ± 62  270 ± 103 294 ± 60  605 ± 135 >100,000 Ab B (N = 3) (N = 2) (N = 3) (N = 2) (N = 1) Exemplified 303 ± 15 311 ± 29 325 ± 99  595 ± 219 >100,000 Ab C (N = 3) (N = 2) (N = 3) (N = 2) (N = 1) Exemplified 205 ± 35 212 ± 42 243 ± 112 515 ± 127 >100,000 Ab D (N = 3) (N = 2) (N = 3) (N = 2) (N = 1)

Example 5. Immunogenicity Analysis

[0035] Immunogenicity assessment, in silico, is performed with EpiMatrix T-cell epitope prediction software (within the Interactive Screening and Protein Reengineering Interface (ISPRI) web-based portal (Epivax, Inc.)) including Tregitope-adjusted Epimatrix Scores. Light chain variable region (LCVR) and heavy chain variable region (HCVR) protein sequences associated with each antibody are separately analyzed. According to Epivax, protein sequences with Tregitope-adjusted Epimatrix scores greater than zero have a higher overall immunogenic potential. EpiMatrix Cluster Immunogenicity assessment is also performed for each LCVR and HCVR to identify T-cell epitope clusters within the CDR of each antibody. The presence of T-cell epitope clusters, as defined by the presence of 2 or more adjacent EpiBars, is associated with a higher overall immunogenic potential. Using the North CDR definition, no CDR-related T-cell epitope clusters are identified in Exemplified Antibodies A, B, or C. A T-cell Epitope cluster of 4 adjacent EpiBars is identified in CDR2 of the LCVR of comparator antibody ALD 1.H (described as “Ab 1.H” in U.S. Patent Publication Number US 2016/0304604) indicating that the comparator antibody has higher immunogenic potential. Results are provided in Tables 5A, 5B and 5C.

TABLE-US-00005 TABLE 5A In Silico immunogenicity risk assessment via EpiMatrix T-cell Epitope Prediction Epimatrix Score Antibody HCVR LCVR HCVR + LCVR Exemplified 58.84 19.44 40.61 Ab A Exemplified 65.12 26.41 47.21 Ab B Exemplified 58.74 26.41 43.79 Ab C Exemplified 65.49 26.41 47.41 Ab D ALD 1.H 70.11 35.2 53.16

TABLE-US-00006 TABLE 5B Tregitope-Adjusted Epimatrix Assessment Tregitope-Adjusted Epimatrix Score Antibody HCVR LCVR HCVR + LCVR Exemplified −32.82 −34.22 −33.46 Ab A Exemplified −26.54 −27.24 −26.86 Ab B Exemplified −32.92 −27.24 −30.29 Ab C Exemplified −26.17 −27.24 −26.67 Ab D ALD 1.H −28.41 1.41 −13.93

TABLE-US-00007 TABLE 5C In Silico immunogenicity risk assessment via EpiMatrix Cluster Immunogenicity assessment Antibody CDR-related Epibar Clusters Exemplified Ab A 0 Exemplified Ab B 0 Exemplified Ab C 0 Exemplified Ab D 0 ALD 1.H 1

Example 6. Neutralization of Mast Cell Degranulation-Induced PACAP Release

[0036] Human mast cells are differentiated in culture from human cord blood stem cells using StemSpan Media (StemCell) and SCF/IL-6. On the day of the assay, mast cells are plated at 100,000 cells per 50 μl into wells of a 96-well tissue culture plate in Tyrode's buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 10 mM HEPES and 0.1% BSA, pH 7.4). Cells are treated with PACAP38 or PACAP27 in the presence or absence of an Exemplified Antibody as described below.

[0037] Single point tests are carried out by adding 25 μl of 4×-concentrated Exemplified Antibody per well (final concentration of 153 μM Exemplified Antibody). Dose curve tests are carried out by adding 25 μl of 4×-concentrated antibody per well for a final dose range of 0 to 153 μM (three-fold dilutions). 25 μl of 4×-concentrated PACAP38 or PACAP27 is added to each well to a final concentration 1 μM PACAP (38 or 27). Assay medium only is used as a no treatment control and IgG4 mAb is used as a negative control. Testing is done in triplicate. The 96-well plates are placed in an incubator (37° C.) for 30 minutes. Following incubation, 30 μl/well of supernatant are collected and tryptase activity is measured by commercial assay (Millipore). Percent (%) tryptase release in supernatant is calculated by the following equation: ((Experimental tryptase release−vehicle control tryptase release)/(total tryptase release−vehicle control tryptase release)×100, where total tryptase release is obtained by freeze/thawing mast cells). Results are provided in Tables 6A, 6B and 6C.

TABLE-US-00008 TABLE 6A Single Point Inhibition of PACAP (27 and 38) Induced Tryptase Release in Human Mast Cells Percentage Tryptase Release upon addition of PACAP38 or 27 (%) Antibody PACAP38 PACAP27 Exemplified Ab B 4.9 ± 1.1 .sup. 21 ± 1.4 Exemplified Ab C 5.5 ± 1.4 14.2 ± 1.1 Exemplified Ab D 4.2 ± 0.7 15.1 ± 4.0 Assay Medium only −0.3 ± 0.3  −0.3 ± 0.3 IgG4 mAb 103.6 ± 0.5  109.7 ± 0.4 

TABLE-US-00009 TABLE 6B Dose Curve Inhibition of PACAP27 Induced Tryptase Release in Human Mast Cells Percentage Tryptase Release Dose upon addition of PACAP27 Antibody (uM) (%) Exemplified Ab B 153 10.3 ± 0.9 51 19.4 ± 6.4 17 11.6 ± 7.1 5.6  8.1 ± 2.9 1.8 11.7 ± 1.6 0.6 54.0 ± 2.0 0.2 78.1 ± 1.0 0 85.2 ± 3.4 Exemplified Ab C 153  9.7 ± 2.3 51 17.8 ± 2.3 17  8.0 ± 0.6 5.6 11.5 ± 4.9 1.8 10.4 ± 2.6 0.6 55.4 ± 6.0 0.2 85.2 ± 1.6 0 85.8 ± 4.8 Exemplified Ab D 153  9.4 ± 0.8 51 26.0 ± 6.3 17 19.8 ± 3.4 5.6  16.4 ± 10.8 1.8 20.3 ± 8.4 0.6 73.0 ± 2.1 0.2 86.5 ± 1.8 0 92.7 ± 1.5 Assay Medium only −0.2 ± 1.0 IgG4 mAb 153 94.8 ± 4.8

TABLE-US-00010 TABLE 6C Dose Curve Inhibition of PACAP38 Induced Tryptase Release in Human Mast Cells Percentage Tryptase Release Dose upon addition of PACAP38 Antibody (uM) (%) Exemplified Ab B 153  2.0 ± 0.3 51 14.6 ± 2.1 17 14.9 ± 2.6 5.6 22.0 ± 4.1 1.8 25.2 ± 1.4 0.6 52.4 ± 9.3 0.2 73.7 ± 1.3 0 77.2 ± 1.0 Exemplified Ab C 153  2.8 ± 0.3 51 12.1 ± 2.3 17 15.5 ± 0.9 5.6 21.9 ± 3.8 1.8 26.5 ± 4.3 0.6 66.6 ± 1.0 0.2 77.6 ± 0.3 0 80.3 ± 2.6 Exemplified Ab D 153  1.7 ± 0.1 51 14.7 ± 1.5 17 19.6 ± 3.6 5.6 25.9 ± 6.2 1.8 31.3 ± 3.8 0.6 64.5 ± 4.1 0.2 74.0 ± 1.8 0 76.5 ± 0.7 Assay Medium only −0.2 ± 1.0 IgG4 mAb 153 79.6 ± 4.1

Example 7. In Vivo PACAP-Induced cAMP Neutralization

[0038] Neutralization of PACAP-induced plasma cAMP increase, in vivo, is assessed in CD-1 murine model (Envigo). Briefly, male CD-1 mice are subcutaneously administered 10 mg/kg of one of: Control IgG4 antibody (N=6); Control IgG4 antibody plus PBS/rolipram only (N=5); Exemplified Antibody B (N=5); Exemplified Antibody C (N=5); or Exemplified Antibody D (N=6). Three days-post antibody treatment, mice are intravenously administered PACAP38 (13 nmols/kg) plus rolipram (100 μg/mL); some mice previously treated with IgG4 control antibody are intravenously administer PBS (containing 0.2% ethanol, 1% BSA, 5 ml/kg, and rolipram (100 μg/ml) only. Ten minutes post-PACAP38 treatment, blood is collected in EDTA containing tubes and plasma is separated by centrifugation. Plasma levels of cAMP are determined using commercially available cAMP ELISA (Cell Bios, Inc.) according to manufacturer instructions. Statistical analysis applies log transformed cAMP concentration in a one-way ANOVA (Graphpad Prism 7) followed by Dunnett's post hoc analysis. Results are set forth in Table 7 as mean±SE.

TABLE-US-00011 TABLE 7 PACAP-Induced Plasma cAMP Levels Plasma cAMP levels Antibody (nM, mean ± SEM) IgG4 Control Ab 238 ± 60 (N = 5) * (plus PBS/rolipram only) IgG4 Control Ab (plus PACAP) 4095 ± 318 (N = 6) Exemplified Ab B (plus PACAP) 715 ± 60 (N = 5) * Exemplified Ab C (plus PACAP) 666 ± 96 (N = 5) * Exemplified Ab D (plus PACAP) 560 ± 130 (N = 6) * * p < 0.0001 vs. IgG4 Control Ab (PACAP38)

Example 8. Neutralization of Trigeminal Ganglia Stimulation-Induced Dural Plasma Protein Extravasation In Vivo

[0039] The ability of anti-PACAP antibodies of the present invention to block, in vivo, plasma protein extravasation induced by release of PACAP following stimulation of the trigeminal ganglia is examined using a rat model. Briefly, Sprague-Dawley rats (Envigo, males, 250-350 g) are subcutaneously administered one of: 1-10 mg/kg of Exemplified Antibody A; 3-30 mg/kg of Exemplified Antibody B; or 3 or 10 mg/kg of Control IgG4 Ab. Seventy-two hours later, rats are anesthetized with Nembutal (65 mg/kg, ip.) and placed in a stereotaxic frame (David Kopf Instruments) with the incisor bar set at −2.5 mm. A mid-line sagittal scalp incision is made, followed by two pairs of bilateral holes drilled through the skull (3.2 mm posteriorly, 1.8 and 3.8 mm laterally, all coordinates referenced to bregma). Pairs of stainless steel stimulating electrodes (Rhodes Medical Systems, Inc.) are lowered through the holes in both hemispheres to a depth of 9.2 mm. from the dura. Two-minutes prior to trigeminal ganglion stimulation, fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) (20 mg/kg, iv.), a marker for protein extravasation, is injected into the femoral vein. The left trigeminal ganglion is stimulated for 5 minutes at a current intensity of 1.0 mA (5 Hz, 5 msec pulse duration) with a Model S48 Grass Instrument Stimulator with PSIU6 photoelectric isolation unit (Grass-Telefactor). Five minutes following stimulation, the rats are sacrificed by exsanguination with 40 ml of saline.

[0040] Following sacrifice of the rats, the top of the skull is removed and dural membrane is collected. Membrane samples are removed from both hemispheres, rinsed with water, and spread flat on microscope slides. Slides are dried for 15 minutes on a slide warmer and tissues are cover-slipped with a 70% glycerol/water solution. A fluorescence microscope (Zeiss) equipped with a grating monochromator and a spectrophotometer are used to quantify FITC-BSA dye in each dural sample. The extravasation induced by electrical stimulation of the trigeminal ganglion is an ipsilateral effect (i.e. occurs only on the side of the dura in which the trigeminal ganglion is stimulated), thus allowing the unstimulated half of the dura to serve as a control. The ratio of extravasation in the dura from the stimulated side versus the unstimulated side is calculated. Results are provided in Table 8 (mean±SE).

TABLE-US-00012 TABLE 8 Trigeminal Ganglia Stimulation-Induced Dural Plasma Protein Extravasation Extravasation Ratio Antibody (mg/kg) (mean ± SEM) IgG4 Control Ab (10 mg/kg) (N = 3) 1.87 ± 0.04 Exemplified Ab A (1 mg/kg) (N = 3) 1.93 ± 0.04 Exemplified Ab A (3 mg/kg) (N = 3) 1.59 ± 0.07 Exemplified Ab A (10 mg/kg) (N = 3) 1.13 ± 0.05 IgG4 Control Ab (3 mg/kg) (N = 3) 1.82 ± 0.02 IgG4 Control Ab (10 mg/kg) (N = 3) 1.90 ± 0.04 IgG4 Control Ab (30 mg/kg) (N = 3) 1.95 ± 0.05 Exemplified Ab B (3 mg/kg) (N = 3) 1.83 ± 0.06 Exemplified Ab B (10 mg/kg) (N = 3) 1.21 ± 0.06 Exemplified Ab B (30 mg/kg) (N = 3) 1.07 ± 0.04

Example 9. Pharmacokinetics of Exemplified Anti-PACAP Antibodies

[0041] Serum pharmacokinetics of exemplified antibodies of the present invention are characterized in male cynomolgus monkeys (n=2) following a single SC administration by total IgG assay. Animals are injected subcutaneous with Exemplified Antibody B (10 mg/kg) and serum samples collected at 1, 3, 6, 24, 48, 72, 96, 120, 144, 168, 240, 336, 504 and 672 hours post injection. Total IgG assay is performed generally as described herein. Briefly, 100 uL/well of 1 ug/mL goat anti-human IgG F(ab′).sub.2 antibody (Jackson ImmunoResearch Laboratories, Inc., Catalog Number 109-006-097) is coated on an Immulon 4HBX plate. Wells are incubated with one of standards (a standard curve for Exemplified Antibody B is prepared from 15.63-1,000 ng/mL), control or serum samples, followed by 100 ul/well of 1:10,000 diluted mouse anti-human IgG Fc-horseradish peroxidase (HRP; SouthernBiotech, Catalog Number 9040-05) for detection. Unbound detection reagent is washed away. Thereafter, 100 ul/well TMB Microwell Peroxidase Substrate System is added to the wells. Color development is stopped by addition of 100 ul/well TMB Stop Solution and optical density is measured at 450 nm with wavelength correction set to 630 nm. Immunoreactivity is determined from known amounts of Exemplified Antibody B in 100% cynomolgus monkey serum, followed by a minimum required dilution of 1:10 in Blocker™ Casein in PBS using a 5-parameter algorithm (StatLia; version 3.2). Following procedures essentially as described above, apparent clearance (CL/F) of 0.49 mL/hr/kg, apparent volume of distribution (V/F) of 114.0 mL/kg, and terminal half-life of 171 hours calculated by non-compartmental analysis are calculated for Exemplified Antibody B. The pharmacokinetic results obtained are consistent with therapeutic antibodies capable of extended duration of action.

TABLE-US-00013 Sequences Exemplified HC (SEQ ID NO. 1) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKGLEWVSA ISLSGGSTYYAXSHKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVREV GASXHNYYGMDVWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TKTYTCNVDHKPSNTKVDKRVESKYGPPCPXCPAPEXXGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG wherein, X at residue 62 is one of D, A, E and Q; X at residue 104 is one of N and T; X at residue 231 is one of P and S; X at residue 237 is one of A and F; and X at residue 238 is one of A and L. Exemplified LC (SEQ ID NO. 2) DIQMTQSPSSLSASVGDRVTITCRASQSIXRWLAWYQQKPGKAPKLLIHD ASQLXEGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQFDLLPLTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC wherein X at residue 30 is one of S and W; and X at residue 55 is one of L and F. Exemplified HCDR1 (SEQ ID NO. 3) AASGFTFSSYYMS Exemplified HCDR2 (SEQ ID NO. 4) AISLSGGSTYYAXSHKG wherein X at residue 13 is one of D, A, E and Q. Exemplified HCDR3 (SEQ ID NO. 5) VREVGASXHNYYGMDV wherein X at residue 8 is one of N and T. Exemplified LCDR1 (SEQ ID NO. 6) RASQSIXRWLA wherein X at residue 7 is one of S and W. Exemplified LCDR2 (SEQ ID NO. 7) HDASQLXE wherein X at residue 7 is one of L and F. Exemplified LCDR3 (SEQ ID NO. 8) QQFDLLPLT Exemplified HCVR (SEQ ID NO. 9) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKGLEWVSA ISLSGGSTYYAXSHKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVREV GASXHNYYGMDVWGQGTMVTVSS wherein, X at residue 62 is one of D, A, E and Q; and X at residue 104 is one of N and T. Exemplified LCVR (SEQ ID NO. 10) DIQMTQSPSSLSASVGDRVTITCRASQSIXRWLAWYQQKPGKAPKLLIHD ASQLXEGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQFDLLPLTFGG GTKVEIK wherein X at residue 30 is one of S and W; and X at residue 55 is one of L and F. Nucleotide Sequence Encoding HC of Exemplified Ab B (SEQ ID NO. 11) gaggtgcagctgttggagtctgggggaggc ttggtacagcctggggggtccctgagactc tcctgtgcagcctctggattcacctttagc agctattacatgagctgggtccgccaggct ccagggaaggggctggagtgggtctcagct attagtctgagtggtggtagcacatactac gcagcgtcccacaagggccggttcaccatc tccagagacaattccaagaacacgctgtat ctgcaaatgaacagcctgagagccgaggac acggccgtatattactgtgtccgggaggtg ggagctagcactcacaactactacggtatg gacgtctggggccaagggaccatggtcacc gtctcttcagcttctaccaagggcccatcg gtcttcccgctagcgccctgctccaggagc acctccgagagcacagccgccctgggctgc ctggtcaaggactacttccccgaaccggtg acggtgtcgtggaactcaggcgccctgacc agcggcgtgcacaccttcccggctgtccta cagtcctcaggactctactccctcagcagc gtggtgaccgtgccctccagcagcttgggc acgaagacctacacctgcaacgtagatcac aagcccagcaacaccaaggtggacaagaga gttgagtccaaatatggtcccccatgccca ccctgcccagcacctgaggccgccggggga ccatcagtcttcctgttccccccaaaaccc aaggacactctcatgatctcccggacccct gaggtcacgtgcgtggtggtggacgtgagc caggaagaccccgaggtccagttcaactgg tacgtggatggcgtggaggtgcataatgcc aagacaaagccgcgggaggagcagttcaac agcacgtaccgtgtggtcagcgtcctcacc gtcctgcaccaggactggctgaacggcaag gagtacaagtgcaaggtctccaacaaaggc ctcccgtcctccatcgagaaaaccatctcc aaagccaaagggcagccccgagagccacag gtgtacaccctgcccccatcccaggaggag atgaccaagaaccaggtcagcctgacctgc ctggtcaaaggcttctaccccagcgacatc gccgtggagtgggaaagcaatgggcagccg gagaacaactacaagaccacgcctcccgtg ctggactccgacggctccttcttcctctac agcaggctaaccgtggacaagagcaggtgg caggaggggaatgtcttctcatgctccgtg atgcatgaggctctgcacaaccactacaca cagaagagcctctccctgtctctgggt Nucleotide Sequence Encoding LC of Exemplified Ab B (SEQ ID NO. 12) gacatccagatgacccagtctccatcctcc ctgtctgcatctgtaggagacagagtcacc atcacttgccgggcgagtcagagtatttgg aggtggttggcctggtatcagcagaaacca gggaaagcccctaagctcctgatccacgat gcatcccaattgttcgaaggggtcccatca aggttcagtggaagtggatctgggacagat tttactttcaccatcagcagcctgcagcct gaagatattgcaacatattactgtcaacag tttgatttgctccctctcactttcggcgga gggaccaaggtggagatcaaacggaccgtg gctgcaccatctgtcttcatcttcccgcca tctgatgagcagttgaaatctggaactgcc tctgttgtgtgcctgctgaataacttctat cccagagaggccaaagtacagtggaaggtg gataacgccctccaatcgggtaactcccag gagagtgtcacagagcaggacagcaaggac agcacctacagcctcagcagcaccctgacg ctgagcaaagcagactacgagaaacacaaa gtctacgcctgcgaagtcacccatcagggc ctgagctcgcccgtcacaaagagatcaac aggggagagtgc Amino Acid Sequence of Recombinant Human PACAP 38 (SEQ ID NO. 13) HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK wherein K at residue 38 is post-translationally modified by C-terminal amidation Amino Acid Sequence of Recombinant Human PACAP 27 (SEQ ID NO. 14) HSDGIFTDSYSRYRKQMAVKKYLAAVL wherein L at residue 27 is post-translationally modified by C-terminal amidation