Anti-PACAP Antibody Compositions and Methods

Abstract

The present disclosure relates generally to anti-PACAP antibodies, pharmaceutical compositions comprising such antibodies, and methods of making and using such antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 226,875, filed July 29, 2021, the disclosure of which is incorporated by reference in its entirety, including any drawings.

[0002] Incorporating sequence tables This application contains a sequence listing, the entire contents of which are incorporated herein by reference. The attached sequence listing text file (titled "035680-502001WO_SequenceListing_ST26.xml") was created on July 26, 2022 and is 69KB.

[0003] FIELD OF THEINVENTION The present disclosure relates generally to anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibodies, pharmaceutical compositions comprising such antibodies, and methods of producing and using such monoclonal antibodies. [Background technology]

[0004] Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide involved in a wide range of functions, including nociception and primary headache. PACAP is a member of the secretin / vasoactive intestinal peptide (VIP) / growth hormone-releasing hormone (GHRH) family. The PACAP / VIP receptors PAC1, VPAC1, and VPAC2 are present in sensory neurons and in vascular smooth muscle associated with the trigeminovascular system. The PAC1 receptor binds with high affinity to PACAP, but much lower affinity to VIP. The VPAC1 and VPAC2 receptors recognize PACAP and VIP equally well.

[0005] PACAP is a multifunctional vasodilatory peptide that exists in two α-amidated active forms, one with 38 amino acids and the other with 27 amino acids. PACAP38 is the more common active form and accounts for up to 90% of the PACAP forms in mammalian tissues.

[0006] To our knowledge today, PACAP, but not VIP, is involved in symptoms such as migraine, cluster headache, and post-traumatic stress disorder (PTSD). For example, findings from experimental models of trigeminal activation show that elevated plasma PACAP levels have been recorded in acute migraine attacks and cluster headaches. This suggests that activation of the trigeminal nervous system may increase venous PACAP levels, and this change may be reduced by treating headaches. Moreover, in patients suffering from these conditions, injection of PACAP induces migraine or cluster headache. Moreover, serum levels of PACAP are associated with PTSD diagnosis and symptom severity in female patients. Summary of the Invention [Problem to be solved by the invention]

[0007] Anti-PACAP antibodies may be useful for treating various conditions, such as migraine, cluster headache, anxiety, and PTSD. However, to date, no antibodies targeting PACAP have been approved for therapeutic use. Therefore, there is a need for anti-PACAP antibodies suitable for therapeutic use in humans. [Means for solving the problem]

[0008] The present disclosure provides anti-PACAP antibodies that solve the above problems and meet the needs of the field.

[0009] summary In at least one embodiment, the disclosure provides an anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibody, wherein the antibody comprises heavy chain variable region (VH)-CDR1, VH-CDR2, and VH-CDR3 sequences set forth in SEQ ID NOs: 9, 2, and 3, respectively, and light chain variable region (VL)-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 10, 5, and 6, respectively.

[0010] In at least one embodiment, the disclosure provides an anti-PACAP antibody, wherein the antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, and VH-CDR3 sequences selected from the group consisting of a) SEQ ID NOs: 1, 2, and 3, respectively; b) SEQ ID NOs: 7, 2, and 3, respectively; and c) a variant of a) to b) comprising 1, 2, or 3 conservative amino acid substitutions; and wherein the antibody or antigen-binding fragment thereof comprises VL-CDR1, VL-CDR2, and VL-CDR3 sequences selected from the group consisting of d) SEQ ID NOs: 4, 5, and 6, respectively; e) SEQ ID NOs: 8, 5, and 6, respectively; and f) a variant of d) to e) comprising 1, 2, or 3 conservative amino acid substitutions.

[0011] In at least one embodiment, the disclosure provides an anti-PACAP antibody, wherein: The antibody comprises a VH sequence derived from SEQ ID NO: 19, wherein the VH sequence comprises a valine (V) or leucine (L) at residue 32 according to the Kabat numbering, and wherein the antibody comprises a VL sequence derived from SEQ ID NO: 22, wherein the VL sequence comprises an alanine (A) at residue 27E according to the Kabat numbering, and a tryptophan (W) and an alanine (A) at residues 50 and 51, respectively, according to the Kabat numbering. In at least one embodiment, the antibody further comprises a cysteine-alanine-isoleucine (CAI) at residues 92-94 of VH according to the Kabat numbering.

[0012] In at least one embodiment, the antibodies each target the human gene IGHV1-69 * 01 and IGKV4-1* 01, as well as functional variants thereof.

[0013] In at least one embodiment, the antibody comprises heavy chain framework region (VHFR)-1, VHFR-2, VHFR-3, and VHFR-4 sequences set forth in SEQ ID NOs: 29 to 32, respectively, and light chain framework region (VLFR)-1, VLFR-2, VLFR-3, and VLFR-4 sequences set forth in SEQ ID NOs: 33 to 36, respectively.

[0014] In at least one embodiment, the antibody comprises a VH sequence that is about 90%, about 95%, or about 99% identical to a sequence selected from SEQ ID NOs: 11 and 12, and a VL sequence that is about 90%, about 95%, or about 99% identical to a sequence selected from SEQ ID NOs: 20 and 21.

[0015] In at least one embodiment, the antibody comprises a VH sequence selected from SEQ ID NOs:11 and 12, and a VL sequence selected from SEQ ID NOs:20 and 21.

[0016] In at least one embodiment, the disclosure provides an anti-PACAP antibody, wherein the antibody comprises VH-CDR1, VH-CDR2, and VH-CDR3 sequences, and VL-CDR1, VL-CDR2, and VL-CDR3 sequences selected from the group consisting of a) SEQ ID NOs: 1, 2, and 3, and SEQ ID NOs: 4, 5, and 6, respectively; b) SEQ ID NOs: 7, 2, and 3, and SEQ ID NOs: 8, 5, and 6, respectively; c) SEQ ID NOs: 1, 2, and 3, and SEQ ID NOs: 8, 5, and 6, respectively; and d) SEQ ID NOs: 7, 2, and 3, and SEQ ID NOs: 4, 5, and 6, respectively.

[0017] In at least one embodiment, the antibody comprises VH and VL sequences that are about 90%, about 95%, or about 99% identical to a sequence selected from the group consisting of: a) SEQ ID NOs: 11 and 20, respectively; b) SEQ ID NOs: 12 and 21, respectively; c) SEQ ID NOs: 11 and 21, respectively; and d) SEQ ID NOs: 12 and 20, respectively.

[0018] In at least one embodiment, the antibody comprises a VH sequence and a VL sequence selected from the group consisting of a) SEQ ID NOs: 11 and 20, respectively; b) SEQ ID NOs: 12 and 21, respectively; c) SEQ ID NOs: 11 and 21, respectively; and d) SEQ ID NOs: 12 and 20, respectively.

[0019] In at least one embodiment, the antibody comprises a VH sequence of SEQ ID NO:11 or SEQ ID NO:12, wherein said VH sequence has a glutamine (Q) instead of glutamic acid (E) at residue 1 of SEQ ID NO:11 or 12.

[0020] In at least one embodiment, the antibody comprises a heavy chain constant region sequence selected from the group consisting of SEQ ID NOs: 37-51 or SEQ ID NO: 76, and a light chain constant region sequence selected from SEQ ID NOs: 23 and 24.

[0021] In at least one embodiment, the antibody comprises a heavy chain constant region sequence set forth in SEQ ID NO:43 and a light chain constant region sequence set forth in SEQ ID NO:23.

[0022] In at least one embodiment, the antibody comprises a heavy chain constant region sequence set forth in SEQ ID NO:43 and a light chain constant region sequence set forth in SEQ ID NO:23.

[0023] In at least one embodiment, the antibody comprises the heavy chain constant region sequence set forth in SEQ ID NO:42 and the light chain constant region sequence set forth in SEQ ID NO:23.

[0024] In at least one embodiment, the antibody comprises a heavy chain constant region sequence set forth in SEQ ID NO:76 and a light chain constant region sequence set forth in SEQ ID NO:23.

[0025] In at least one embodiment, the antibody is a human or humanized antibody. In at least one embodiment, the antibody is a humanized antibody.

[0026] In at least one embodiment, the antibody has a low or no immunogenic profile.

[0027] In at least one embodiment, the antibody has a humanness score of about 89% or greater.

[0028] In at least one embodiment, the antibody has a titer of less than 5×10 as measured by SPR at 37° C. -11 It has a KD of less than 100 moles (M).

[0029] In at least one embodiment, the antibody has a titer of 3×10 as measured by SPR at 37° C. -11 It has a KD of less than 100 moles (M).

[0030] In at least one embodiment, the antibody is an antigen-binding fragment.

[0031] In at least one embodiment, the antibody is a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb 2 , (scFv)2, or scFv-Fc.

[0032] In at least one embodiment, the antibody is a full length antibody.

[0033] In at least one embodiment, the antibody constant region is an IgG constant region.

[0034] In at least one embodiment, the constant region is an IgG1 constant region.

[0035] In at least one embodiment, the constant region is an IgG4 constant region.

[0036] In at least one embodiment, the constant region is selected from the group consisting of the human IgG1 sequence set forth in SEQ ID NO: 37, the human IgG1 FAB TAG sequence set forth in SEQ ID NO: 38, the human IgG1 KiH Hole sequence set forth in SEQ ID NO: 39, the human IgG1 KiH Knob sequence set forth in SEQ ID NO: 40, the human IgG1 (L235A, G237A) sequence set forth in SEQ ID NO: 41, the human IgG1 YTE sequence set forth in SEQ ID NO: 42, the human IgG1 (L235A, G237A, YTE) sequence set forth in SEQ ID NO: 76, the human IgG2 DASS sequence set forth in SEQ ID NO: 43, the human IgG4 sequence set forth in SEQ ID NO: 44, the human IgG4 KiH Hole sequence set forth in SEQ ID NO: 45, the human IgG4 KiH Knob sequence set forth in SEQ ID NO: 46, the human IgG4 (L235A, G237A) sequence set forth in SEQ ID NO: 47, the human IgG4 (L235E) sequence set forth in SEQ ID NO: 48, the human IgG4 YTE sequence set forth in SEQ ID NO: 49, the human IgG4 YTE KiH Hole sequence set forth in SEQ ID NO: 50, and the human IgG4 YTE KiH Hole sequence set forth in SEQ ID NO: 51. Knob sequences.

[0037] In at least one embodiment, the antibody heavy chain is an IgG2 heavy chain. In at least one embodiment, the heavy chain comprises the IgG2DASS sequence set forth in SEQ ID NO:43.

[0038] In at least one embodiment, the antibody light chain is a human kappa light chain.

[0039] In at least one embodiment, the antibody light chain is a human lambda light chain.

[0040] In at least one embodiment, the antibody comprises a full-length heavy chain sequence and a full-length light chain sequence that are about 90%, about 95%, or about 99% identical to a sequence selected from the group consisting of: a) SEQ ID NOs: 70 and 71, respectively; b) SEQ ID NOs: 74 and 71, respectively; c) SEQ ID NOs: 75 and 71, respectively; d) SEQ ID NOs: 72 and 73, respectively; e) SEQ ID NOs: 70 and 73, respectively; and f) SEQ ID NOs: 72 and 71, respectively.

[0041] In at least one embodiment, the antibody comprises a full-length heavy chain sequence and a full-length light chain sequence selected from the group consisting of: a) SEQ ID NOs: 70 and 71, respectively, b) SEQ ID NOs: 74 and 71, respectively, c) SEQ ID NOs: 75 and 71, respectively, d) SEQ ID NOs: 72 and 73, respectively, e) SEQ ID NOs: 70 and 73, respectively, and f) SEQ ID NOs: 72 and 71, respectively. In at least one embodiment, the heavy chain sequences have a glutamine (Q) instead of glutamic acid (E) at residue 1 of SEQ ID NOs: 70, 72, 74, and 75.

[0042] In at least one embodiment, the antibody is an antagonist of PACAP.

[0043] In at least one embodiment, the antibody specifically binds to PACAP.

[0044] In at least one embodiment, the disclosure provides a nucleic acid encoding an antibody described herein.

[0045] In at least one embodiment, the disclosure provides a vector comprising a nucleic acid described herein.

[0046] In at least one embodiment, the disclosure provides an engineered cell comprising a vector described herein.

[0047] In at least one embodiment, the disclosure provides a method of producing an antibody comprising culturing an engineered cell of the disclosure under conditions sufficient for the cell to produce the antibody.

[0048] In at least one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody described herein and a pharma- ceutically acceptable carrier.

[0049] In at least one embodiment, the disclosure provides a method of treating or preventing a condition in an individual comprising administering to the individual a therapeutically effective amount of an antibody or pharmaceutical composition of the disclosure, wherein the condition is selected from the group consisting of headache (e.g., migraine, cluster headache, refractory migraine), anxiety, depression, PTSD, co-occurring conditions (e.g., anxiety / depression / PTSD) with headache (e.g., migraine, cluster headache, refractory migraine), co-occurring anxiety disorder with migraine, complex regional pain syndrome, rosacea. In at least one embodiment, the headache is selected from the group consisting of migraine with aura, migraine without aura, hemiplegic migraine, cluster headache, migrainous neuralgia, chronic headache, episodic migraine, chronic migraine, medication overuse headache, and tension headache.

[0050] In at least one embodiment, the disclosure provides a method of treating or preventing migraine in an individual comprising administering to the individual a therapeutically effective amount of an antibody or pharmaceutical composition of the disclosure.

[0051] In at least one embodiment, the disclosure provides a method of treating or preventing migraine in an individual comprising administering to the individual a therapeutically effective amount of an antibody or pharmaceutical composition of the disclosure, wherein the individual is unresponsive to two to four available prophylactic medications. In at least one embodiment, the individual is unresponsive to two to four available prophylactic medications selected from the group consisting of: divalproex, sodium valproate, valproate, valproic acid, topiramate, gabapentin, propranolol, timolol, atenolol, metoprolol, nadolol, bisopropol, flunarizine, amitriptyline, nortriptyline, doxepin, fluoxetine, and candesartan.

[0052] In at least one embodiment, the present disclosure provides a method for treating or preventing migraine in an individual, comprising administering to the individual a therapeutically effective amount of an antibody or pharmaceutical composition of the present disclosure, wherein the individual is unresponsive to two to four applicable classes of prophylactic drugs. In at least one embodiment, the class of prophylactic drugs is selected from the group consisting of: antiepileptic drugs, beta blockers, tricyclic antidepressants, calcium channel blockers, angiotensin II receptor antagonists, botulinum toxin, and CGRP pathway monoclonal antibodies. In at least one embodiment, the class of prophylactic drugs is selected from different clusters, where the clusters are defined as follows: Cluster A: antiepileptic drugs, Cluster B: beta blockers, Cluster C: tricyclic antidepressants, Cluster D: calcium channel blockers, Cluster E: angiotensin II receptor antagonists, Cluster F, botulinum toxin, and Cluster G: calcitonin gene-related peptide (CGRP) pathway monoclonal antibodies.

[0053] In at least one embodiment, the individual is non-responsive to 2-3, at least 2, at least 3, at least 4, more than 2, or more than 3 prophylactic agents or classes of prophylactic agents.

[0054] In at least one embodiment, the disclosure provides a method of treating or preventing migraine in an individual, the method comprising the steps of selecting an individual who is non-responsive to two to four available prophylactic agents or classes of prophylactic agents, and administering to the individual a therapeutically effective amount of an antibody or pharmaceutical composition of the disclosure.

[0055] In at least one embodiment, the present disclosure provides a method for treating or preventing migraine in an individual who is non-responsive to a CGRP pathway monoclonal antibody, comprising administering to said individual a therapeutically effective amount of an antibody or pharmaceutical composition of the present disclosure. In at least one embodiment, the CGRP pathway monoclonal antibody comprises an anti-CGRP antibody (i.e., an anti-CGRP ligand antibody), an anti-CGRP-R antibody (i.e., an anti-CGRP receptor antibody), or both. In at least one embodiment, the anti-CGRP antibody is selected from fremanezumab, galcanezumab, eptinezumab, or a combination thereof. In at least one embodiment, the anti-CGRP-R antibody is erenumab.

[0056] In at least one embodiment, the present disclosure provides a composition for use in accordance with the present disclosure.

[0057] Each of the aspects and embodiments described herein can be used together unless expressly or specifically excluded from the context of the embodiment or aspect. [Brief description of the drawings]

[0058] [Figure 1] Figure 1 shows the alignment of human PACAP38, PACAP27, and VIP polypeptide sequences. "*" indicates completely conserved residues. "." indicates conservation between residues with weakly similar properties, and ":" indicates conservation between residues with strongly similar properties.

[0059] [Diagram 2] Figures 2A-2B show alignments of the VL (Figure 2A) and VH (Figure 2B) sequences of several exemplary antibodies provided herein. The CDRs are defined according to Kabat numbering, except for heavy chain CDR-1, which is defined by AbM in Figure 2B.

[0060] [Diagram 3]3A-3C show exemplary results of the binding affinity of anti-PACAP antibody 605C to PACAP38, PACAP27 and VIP as measured by SPR at 37°C.

[0061] [Figure 4] 1 shows exemplary SPR experimental data for binding of anti-PACAP antibody 890C to PACAP38, PACAP27, VIP, and other glucagon-secretin family peptides.

[0062] [Diagram 5] 1 shows exemplary experimental data on the selectivity of activity of anti-PACAP antibody 890C for PACAP38 versus VIP in a cell-based cyclic AMP induction assay.

[0063] [Figure 6] 1 shows the apparent molecular weights of several exemplary anti-PACAP antibodies provided herein, as well as other anti-PACAP antibodies of various isotype formats.

[0064] [Figure 7] 1 shows an overview of the predicted immunogenicity profiles of the variable regions of 608C, 609C, 627C, 890C, and other anti-PACAP antibodies.

[0065] [Figure 8] The amino acid sequence of anti-PACAP antibody 890C is shown. CDRs are underlined (all CDRs defined according to Kabat definition except for heavy chain CDR-1 as defined by AbM). Constant regions are underlined with dotted lines. A330S and P331S substitutions on the heavy chain constant region (EU Fc numbering) are double underlined. The C-terminal lysine deletion (Δ447K; EU Fc numbering) is marked with an asterisk.

[0066] [Figure 9]The amino acid sequence of anti-PACAP antibody 608C is shown. CDRs are underlined (all CDRs defined according to Kabat definition except for heavy chain CDR-1 as defined by AbM). Constant regions are underlined with dotted lines. A330S and P331S substitutions on the heavy chain constant region (EU Fc numbering) are double underlined. The C-terminal lysine deletion (Δ447K; EU Fc numbering) is marked with an asterisk.

[0067] [Figure 10] The amino acid sequence of anti-PACAP antibody 627C is shown. CDRs are underlined (all CDRs defined according to Kabat definition except for heavy chain CDR-1 as defined by AbM). Constant regions are underlined with dotted lines. A330S and P331S substitutions on the heavy chain constant region (EU Fc numbering) are double underlined. The C-terminal lysine deletion (Δ447K; EU Fc numbering) is marked with an asterisk.

[0068] [Figure 11] The amino acid sequence of anti-PACAP antibody 609C is shown. CDRs are underlined (all CDRs defined according to Kabat definition except for heavy chain CDR-1 as defined by AbM). Constant regions are underlined with dotted lines. A330S and P331S substitutions on the heavy chain constant region (EU Fc numbering) are double underlined. The C-terminal lysine deletion (Δ447K; EU Fc numbering) is marked with an asterisk. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Detailed Description The present disclosure relates to anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibodies, particularly those with high affinity and low immunogenicity for PACAP when administered to humans. In humans, PACAP is generated from a 176 amino acid precursor protein encoded by the ADCYAP1 gene. There are two naturally occurring isoforms of PACAP: a 38 amino acid peptide (PACAP38) and a 27 amino acid peptide (PACAP27). PACAP38 corresponds to amino acids 132-169 of the precursor protein and has the sequence HSDGIFTDSYSRYRKQMAVKKYLAAVLGKRYKQRVKNK (SEQ ID NO: 13). PACAP27 is the amino-terminal fragment of PACAP38 and corresponds to amino acids 132-158 of the precursor protein. The sequence of PACAP27 is HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 14). Both PACAP38 and PACAP27 show high sequence similarity to vasoactive intestinal peptide (VIP), the sequence of which is HSDAVFTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 15). See Figure 1. In some embodiments, the anti-PACAP antibodies provided herein can bind to both PACAP38 and PACAP27 with high affinity. In other embodiments, the antibodies provided herein show low or no binding to VIP.

[0070] The disclosure also provides compositions and methods useful for producing such antibodies, nucleic acids encoding same, cells genetically modified with same, as well as methods for the treatment or prevention of various conditions, such as migraine, refractory migraine, etc.

[0071] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols generally identify like components unless the context dictates otherwise. The illustrative options set forth in the detailed description, drawings, and claims are not meant to be limiting. Other options may be used, and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects generally described herein and illustrated in the figures can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are expressly contemplated and made a part of this application.

[0072] definition Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by those of ordinary skill in the art to which this disclosure pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. Many of the techniques and methods described or referenced herein are well understood and commonly employed by those of ordinary skill in the art using conventional methodology.

[0073] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen / target). Unless otherwise specified, "binding affinity" as used herein refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed as a dissociation constant (KD). Affinity can be measured by common methods known in the art, including the methods described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, one example of which is an affinity ELISA assay. Additionally, affinity can be determined by surface plasmon resonance assays (SPR, e.g., BIAcore®-based assays). Using this method, the association rate constant (M -1 s -1 ka at 0.05 and the dissociation rate constant (s -1 The equilibrium dissociation constant (KD at M) can be calculated from the ratio of the rate constants (kd / ka). Binding affinity can also be measured by kinetic methods such as kinetic exclusion assay (KinExA) as described in Rathanaswami et al. Analytical Biochemistry, Vol.373:52-60, 2008. The KinExA assay can be used to determine the equilibrium dissociation constant (KD at M) and the binding rate constant (M -1 s -1 The dissociation rate constant (s -1 The kd at 100 kD can be calculated from these values ​​(KD x ka). Binding affinity can also be measured by the equilibrium / solution method.

[0074] "Specifically binds" generally means that an antibody binds to an epitope through its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope through its antigen-binding domain, rather than to a random, unrelated epitope. The term "specificity" is used herein to appreciate the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have a higher specificity for a particular epitope than antibody "B," or antibody "A" may be considered to bind epitope "C" with a higher specificity than related epitope "D." With particular reference to the antibodies described herein, "specifically binds" means that the antibody binds to PACAP27 and / or PACAP38 more readily than it binds to VIP. In one embodiment, the antibody specifically binds to human PACAP. For example, the antibody binds to PACAP27 and PACAP38 more readily than it binds to VIP.

[0075] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in a form in which they occur in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0076] The term "derived from" as used herein with respect to a protein or polypeptide refers to an origin or source and may include naturally occurring recombinant unpurified or purified polypeptides obtained from or derived based on a source or original protein or polypeptide. Thus, a protein or polypeptide derived from an original protein or polypeptide may partially or entirely comprise the original protein or polypeptide and may be a fragment or variant of the original protein or polypeptide. In some cases, a polypeptide sequence or domain derived from a source or origin may be genetically or chemically modified.

[0077] As used herein, the terms "administer," "administration," "administering," and the like refer to the delivery of a composition, formulation, or drug, such as an anti-PACAP antibody disclosed herein, by a route including, but not limited to, intravenous, intraarterial, intracranial, intramuscular, intraperitoneal, subcutaneous, intramuscular, or a combination thereof. The term includes, but is not limited to, administration by a medical professional and self-administration.

[0078] As used herein, the terms "treatment," "treating," and "treating" refer to at least amelioration, if not necessarily amelioration, of one or more symptoms. For example, these terms refer to utilizing an approach to obtain a beneficial or desired clinical outcome, including, but not limited to, an approach to achieve such a beneficial or desired clinical outcome, where the clinical outcome may include therapeutic measures that improve, cure, slow, alleviate symptoms, and / or halt progression of a pathological condition or disorder. Individuals in need of treatment include those who have already been diagnosed with or are suspected of having the disorder. As used herein, unless otherwise specified, a "therapeutically effective amount" of a drug or agent, such as an anti-PACAP antibody disclosed herein, is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder in a subject, or to delay or minimize one or more symptoms associated with the disease. A therapeutically effective amount of a compound, agent or drug means an amount of a therapeutic agent that, alone or in combination with other therapeutic agents, provides a therapeutic benefit in the treatment or management of a disease. The term "therapeutically effective amount" can encompass an amount that improves the overall treatment of a disease, reduces or avoids the symptoms or causes of a disease, or enhances the therapeutic effect of another therapeutic agent. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom of a disease, also referred to as a "therapeutically effective amount." The exact amount of a composition that comprises a "therapeutically effective amount" depends on the purpose of the treatment and can be ascertained by one of skill in the art using known techniques.

[0079] As used herein, a "subject" or "individual" includes animals, such as humans (e.g., human individuals) and non-human animals. In some embodiments, a "subject" or "individual" is a patient receiving medical treatment from a physician. Thus, a subject may be a human patient who has, is at risk of, or is suspected of having one or more symptoms of a target disease (e.g., migraine, refractory migraine, etc.) and / or disease. A subject may also be an individual who has been diagnosed at the time of diagnosis or thereafter as being at risk for a target condition. For example, a subject may be further characterized as being at risk for developing a condition described herein or a condition that would benefit from reduced PACAP activity.

[0080] The terms "cell", "cell culture", and "cell line" refer not only to a particular subject cell, cell culture, or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, regardless of the number of transplants or passages of the culture. It should be understood that not all progeny are exactly identical to the parent cell. This is because some modifications may occur in subsequent generations due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications) such that the progeny are not in fact identical to the parent cell. However, so long as the progeny retain the same functionality as the original cell, cell culture, or cell line, they are still within the scope of the term as used herein.

[0081] As used herein, the term "operably linked" refers to a physical or functional linkage between two elements (e.g., polypeptide or polynucleotide sequences) that allows them to operate in an intended manner. For example, the term "operably linked" when used in the context of an orthogonal DNA target sequence described herein, or a promoter sequence in a nucleic acid construct or an engineered response element, means that the orthogonal DNA target sequence and the promoter are in frame and at an appropriate spatial and distance from the polynucleotide of interest encoding the protein or RNA, allowing binding of each by a transcription factor or RNA polymerase to affect transcription.

[0082] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells (including mixtures thereof). "A and / or B" is used herein to include all of the following options: "A," "B," "A or B," and "A and B."

[0083] Where a range of values ​​is provided, unless the context clearly dictates otherwise, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is understood to be encompassed in the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, provided that a limit may be specifically excluded within a stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0084] All ranges disclosed herein also encompass all possible subranges and combinations of those subranges. Any range recited may be recognized as fully descriptive and allowing for division of the same range into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein may be readily divided into a lower third, middle third, upper third, etc. Also, as will be understood by those of skill in the art, all terms such as "up to," "at least," "greater than," "less than," etc. refer to ranges that include the recited numerical values ​​and are subsequently divided into subranges as described above. Finally, as will be understood by those of skill in the art, ranges include individual members. Thus, for example, a group containing 1-3 items refers to a group containing 1, 2, or 3 items. Similarly, a group containing 1-5 items refers to a group containing 1, 2, 3, 4, or 5 items.

[0085] It is understood that certain features of the present disclosure that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described in the context of a single embodiment for brevity may also be provided individually or in any suitable subcombination. All combinations of the embodiments according to the present disclosure are specifically embraced in the present disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed herein. Moreover, all subcombinations of the various embodiments and elements thereof are specifically embraced in the present disclosure and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein.

[0086] composition The present disclosure provides, inter alia, anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibodies. The anti-PACAP antibodies provided herein can block PACAP signaling through the PACAP receptor (i.e., PAC1) and PACAP signaling through the VIP receptors VPAC1 and VPAC2. In certain embodiments, the anti-PACAP antibodies provided herein have been engineered to improve the proportion of human sequences through humanization. Furthermore, the CDR3 sequences of the anti-PACAP antibodies provided herein are engineered to improve affinity, potency, or both. In some embodiments, the anti-PACAP antibodies have been engineered to include a heavy chain variable region (VH)-CDR3 having a glutamine (Q) at residue 96 of SEQ ID NO: 3 according to the Kabat numbering. In some embodiments, the anti-PACAP antibodies include a light chain variable region (VL)-CDR3 having a tryptophan (W) at residue 93 and an aspartic acid (D) at residue 95 of SEQ ID NO: 6 according to the Kabat numbering. In other embodiments, the anti-PACAP antibodies provided herein can be engineered to eliminate immunogenicity and reduce manufacturing burden. Additionally, the anti-PACAP antibodies provided herein can be engineered to achieve potent inhibition of PACAP-38 and / or PACAP-27-induced cyclic adenosine monophosphate (cAMP) production, as described in Example 5 and previously described by Wang, Li et al. 2004.

[0087] As described in more detail below, the antibodies provided herein have low or no potential risk of immunogenicity when administered to a subject. As used herein, "low or no potential risk of immunogenicity" refers to the inability of the therapeutic antibody to induce the formation of anti-drug antibodies (ADAs) when administered to a subject in sufficient amounts. ADAs are antibodies against therapeutic drugs that are generated by the immune system, which can reduce the efficacy of the drug, and more importantly, ADAs can also cause a variety of side effects, ranging from injection site rashes to fatal systemic inflammatory reactions. In certain embodiments, the antibodies provided herein have low or no potential risk of immunogenicity when administered to humans. For administration to humans, the potential risk of immunogenicity can be made lower or eliminated, for example, by engineering the antibody to have a higher humanness score, as well as removing residues that have been found to have a high potential risk of immunogenicity by predictive in vitro techniques. As used herein, "humanness score" refers to the percent sequence identity of the antibody to the human germline.

[0088] Those skilled in the art will readily understand how to calculate sequence identity between an antibody and a human germline. For example, anti-PACAP antibodies have a high humanness score, for example, about 89% or more. In addition, anti-PACAP antibodies have a strong affinity for PACAP. For example, some anti-PACAP antibodies provided herein have a humanness score of about 5×10 as measured by SPR at 37° C. -11 In some embodiments, the anti-PACAP antibodies provided herein have a KD of less than about 3×10 as measured by SPR at 37° C. -11 has a submolar (M) KD. In some embodiments, the anti-PACAP antibody preferentially binds to PACAP, including PACAP-38 and PACAP-27.

[0089] antigen binding molecule Antibody, as used herein, has its common meaning in the art and refers to an immunoglobulin molecule that recognizes and specifically binds to a target epitope via at least one antigen-binding domain in the variable region of the immunoglobulin molecule. The target can be a peptide, for example, a PACAP peptide. The antibodies of the present disclosure include full-length antibodies (including full-length polyclonal antibodies and full-length monoclonal antibodies), antigen-binding fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) variants, multispecific antibodies such as bispecific antibodies generated from at least two full-length antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigenic determinant portion of an antibody, and any other modified immunoglobulin molecule antibodies containing an antigen recognition site, so long as the antibody exhibits the desired biological activity. The antibody may be any of the five major classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on their heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively. In some embodiments, the antibody of the present disclosure is an IgG antibody. In more specific embodiments, the antibody of the present disclosure is an IgG2 antibody. In more specific embodiments, the antibody of the present disclosure is a modified IgG2 containing the A330P331 to S330S331 mutation (amino acid numbering with reference to the wild-type IgG2 sequence, Eur. J. Immunol. (1999) 29:2613-2624). The different classes of immunoglobulins have different well-known subunit structures and three-dimensional structures.

[0090] The antibody of the present disclosure may comprise one or more variable regions. The variable region of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held together in close proximity by the FRs and contribute to the formation of the antigen-binding site of the antibody with the CDRs of the other chain. There are at least two techniques for determining CDRs: (1) approaches based on sequence variation between species (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) approaches based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883; Oxford Molecular's AbM antibody modelling software and North numbering convention (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256). (2011)). Moreover, a combination of these two approaches is sometimes used in the art to determine CDRs.

[0091] In some embodiments, the anti-PACAP antibodies provided herein are full-length antibodies. Full-length antibodies can include four polypeptide units consisting of two identical heavy chains and two identical light chains linked by disulfide bonds, as described in more detail below. The light chains are generally shorter and have a smaller molecular weight than the heavy chains. Each polypeptide chain has a constant region and a variable region. The variable region is specific to each particular antibody. The light chain variable region is referred to as VL, and the light chain constant region is referred to as CL. Similarly, the heavy chain variable region is referred to as VH, and the heavy chain constant region is referred to as CH, with CH1, CH2, and CH3 each representing a different portion of the constant region of the heavy chain. In some embodiments, carbohydrates can be typically attached to the CH2 domain of the heavy chain. In addition, full-length antibodies can also include a fragment crystallizable (Fc) region. The Fc region includes only the constant regions of the heavy chain (CH2 and CH3). In contrast, the antigen-binding region fragment (Fab) can include both the constant and variable domains of the heavy and light chains (VH, VL, CH1, and CL). The variable region fragment (Fv) contains only the two variable domains.

[0092] As mentioned above, the antibody of the present disclosure may comprise one or more constant regions. The "constant region" of an antibody is a term well known in the art and refers to the portion of an antibody whose amino acid sequence is relatively constant among different molecules. Typically, the heavy chain constant region is composed of three different regions, designated CH1, CH2, and CH3, numbered from the amino-terminus (N-terminus) to the carboxy-terminus (C-terminus). A typical light chain has only one constant region, designated CL. The constant region of an antibody determines its specific effector function. Those skilled in the art will readily understand the terminology and structural features of the constant region of an antibody.

[0093] Additionally, the anti-PACAP antibodies of the present disclosure also include antigen-binding fragments that specifically bind to PACAP. An antigen-binding fragment as used herein refers to a portion of a full-length antibody. For example, in some embodiments, an antigen-binding fragment of an antibody as used herein refers to the antigen-determining variable region of a full-length antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGA CH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb 2 , (scFv)2, or scFv-Fc.

[0094] In one embodiment, the anti-PACAP antibody of the present disclosure is a blocking antagonist antibody that inhibits or reduces the biological activity of PACAP. In some embodiments, the blocking or antagonist antibody substantially or completely inhibits the biological activity of PACAP. The biological activity of PACAP may be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100% compared to its native biological activity. The ability of the anti-PACAP antibody of the present disclosure to antagonize PACAP can be measured, for example, in a cell-based assay by monitoring ligand-induced cyclic adenosine monophosphate (cAMP) production. In some embodiments, the anti-PACAP antibody of the present disclosure antagonizes PACAP-induced activation of human PAC1, VPAC1, and / or VPAC2 receptors. Various assays for assessing activation of PAC1, VPAC1, and / or VPAC2 receptors are known in the art, including cell-based assays that measure ligand-induced calcium mobilization and cAMP production. An exemplary cell-based cAMP assay is described in Example 5 and previously described by Wang, Li et al. 2004.

[0095] The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes are formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation.

[0096] The antibodies provided herein may be monoclonal antibodies. A "monoclonal antibody" refers to a homogeneous antibody population that is involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to a polyclonal antibody, which typically contains different antibodies against different antigenic determinants. The term "monoclonal antibody" includes both full-length monoclonal antibodies, as well as antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins that include antibody portions, and any other modified immunoglobulin molecule that contains an antigen recognition site. Furthermore, "monoclonal antibody" refers to an antibody produced by any method, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0097] The antibodies encompassed by the present disclosure may be human, non-human, humanized, murine, chimeric, or resurfaced antibodies. In some embodiments, the antibodies of the present disclosure may be humanized antibodies. As used herein, a humanized antibody refers to an antibody derived from a monoclonal antibody originally produced in a non-human animal, such as a rodent or rabbit. Certain amino acid residues of the monoclonal antibody, typically from a non-antigen-recognizing portion of the antibody, have been modified to be homologous to the corresponding residues of a human antibody of the corresponding isotype. Humanization can be performed using a variety of methods, for example by substituting at least a portion of a rodent or rabbit variable region for the corresponding region of a human antibody (see, e.g., U.S. Pat. Nos. 5,585,089 and 5,693,762; Jones et al, 1986, Nature 321:522-525; Riechmann et al, 1988, Nature 332:323-27; and Verhoeyen et al, 1988, Science 239: 1534-1536).

[0098] In one embodiment, the antibody of the disclosure comprises a human gene VH:IGHV1-69, respectively, as shown below: * 01 / VK:IGKV4-1 * The antibody is engineered to contain heavy and light chain variable framework regions that are the product of or derived from .01. [Table 1]

[0099] Examples of the above human germline derived variants can have heavy and / or light chain variable framework regions that contain at least one amino acid modification from the corresponding heavy and / or light chain variable framework regions of the corresponding non-human antibody (e.g., a heavy chain variant having a "CAI" motif at residues 92-94 according to the Kabat numbering).

[0100] Exemplary anti-PACAP antibody sequences are shown in the table below. In some embodiments, the anti-PACAP antibodies of the disclosure comprise a combination of the VH and VL CDR sequences provided in Table 2. In some embodiments, all CDRs are defined according to Kabat numbering, except for VH-CDR1, which is defined by AbM. [Table 2]

[0101] In some embodiments, Xa comprises V or L and Xb comprises A or S.

[0102] In some embodiments, the anti-PACAP antibody of the present disclosure comprises heavy chain variable region (VH)-CDR1, VH-CDR2, and VH-CDR3 sequences set forth in SEQ ID NOs: 9, 2, and 3, respectively; and light chain variable region (VL)-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 10, 5, and 6, respectively.

[0103] In some embodiments, the disclosure provides anti-PACAP antibodies having the VH-CDR1, VH-CDR2, and VH-CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and variants containing 1, 2, or 3 conservative amino acid substitutions. In other embodiments, the disclosure provides anti-PACAP antibodies having the VH-CDR1, VH-CDR2, and VH-CDR3 sequences set forth in SEQ ID NOs: 7, 2, and 3, respectively, and variants containing 7, 2, or 3 conservative amino acid substitutions.

[0104] In some embodiments, the disclosure provides anti-PACAP antibodies or antigen-binding fragments thereof having the VL-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and variants containing 4, 5, or 6 conservative amino acid substitutions. In other embodiments, the disclosure provides anti-PACAP antibodies having the VL-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 8, 5, and 6, respectively, and variants containing 8, 5, or 6 conservative amino acid substitutions.

[0105] In some exemplary embodiments, the disclosure provides anti-PACAP antibodies having VH-CDR1, VH-CDR2, and VH-CDR3 sequences, and VL-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, and SEQ ID NOs: 4, 5, and 6, respectively.

[0106] In some embodiments, the anti-PACAP antibodies of the disclosure comprise the VH-CDR1, VH-CDR2, and VH-CDR3 sequences, and the VL-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 1, 2, 3, and 4, 5, 6, respectively, shown in Figure 8. For example, the CDRs are underlined in Figure 8 (all CDRs are defined according to the Kabat definition, except for heavy chain CDR-1, which is defined by AbM).

[0107] In other exemplary embodiments, the present disclosure provides anti-PACAP antibodies having VH-CDR1, VH-CDR2, and VH-CDR3 sequences, and VL-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 7, 2, and 3, and SEQ ID NOs: 8, 5, and 6, respectively.

[0108] In some embodiments, the anti-PACAP antibodies of the disclosure comprise the (VH)-CDR1, VH-CDR2, and VH-CDR3 sequences, and the (VL)-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 7, 2, 3, and 8, 5, 6, respectively, as shown in Figure 9. For example, the CDRs are underlined in Figure 9 (all CDRs are defined according to the Kabat definition, except for heavy chain CDR-1, which is defined by AbM).

[0109] In some exemplary embodiments, the disclosure provides anti-PACAP antibodies having VH-CDR1, VH-CDR2, and VH-CDR3 sequences, and VL-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, and SEQ ID NOs: 8, 5, and 6, respectively.

[0110] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise (VH)-CDR1, VH-CDR2, and VH-CDR3 sequences, and (VL)-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 1, 2, 3, and 8, 5, 6, respectively, as shown in Figure 10. For example, the CDRs are underlined in Figure 10 (all CDRs are defined according to the Kabat definition, except for heavy chain CDR-1, which is defined by AbM).

[0111] In yet other exemplary embodiments, the present disclosure provides anti-PACAP antibodies having VH-CDR1, VH-CDR2, and VH-CDR3 sequences, and VL-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 7, 2, and 3, and SEQ ID NOs: 4, 5, and 6, respectively.

[0112] In some embodiments, the anti-PACAP antibodies of the disclosure comprise the (VH)-CDR1, VH-CDR2, and VH-CDR3 sequences, and the (VL)-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 7, 2, 3, and 4, 5, 6, respectively, shown in Figure 11. For example, the CDRs are underlined in Figure 11 (all CDRs are defined according to the Kabat definition, except for heavy chain CDR-1, which is defined by AbM).

[0113] In some embodiments, the anti-PACAP antibodies of the disclosure comprise a combination of the VH, VL, and FR sequences provided in Tables 3-5 below. In some embodiments, the anti-PACAP antibodies of the disclosure comprise a VH sequence provided in Table 3, wherein the VH sequence has a glutamine (Q) instead of glutamic acid (E) at residue 1 of SEQ ID NOs: 11, 12, 18, and 19. [Table 3] [Table 4] [Table 5]

[0114] Additionally, some exemplary anti-PACAP antibody heavy and light chain constant region sequences are provided in Tables 6-7 below. The heavy chain of the anti-PACAP antibody of the present disclosure can include a constant region, such as the constant region described in Table 6. In some embodiments, the immunologically inert constant region is modified, for example, some modified constant regions are described in Table 6. For example, the modified constant region can be within the residues listed in Table 6 herein using EU numbering based on the IgG1 sequence (i.e., SEQ ID NO: 37). In some embodiments, the heavy chain constant region of the anti-PACAP antibody of the present disclosure can be a human heavy chain having a modified constant region of IgG1 described in Table 6. For example, the modified constant region of IgG1 can be any of the following: human IgG1 FAB TAG, human IgG1 KiH Hole, human IgG1 KiH Knob, human IgG1 (L235A, G237A), human IgG1 YTE, human IgG1 (L235A, G237A, YTE), and variants thereof. In some embodiments, the heavy chain constant region of the anti-PACAP antibody of the present disclosure can be a human heavy chain with a modified constant region of IgG2 as described in Table 6. For example, the modified constant region heavy chain can be a human heavy chain IgG2 constant region containing the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wild-type IgG2 sequence, Eur. J. Immunol. (1999) 29:2613-2624) as described in Table 6, SEQ ID NO: human IgG2DASS; SEQ ID NO: 43. In some embodiments, the heavy chain constant region of the anti-PACAP antibody of the present disclosure can be a human heavy chain with a modified constant region of IgG4 as described in Table 6. For example, the modified constant region of IgG4 can be any of the following: human IgG4 KiH Hole, human IgG4 KiH Knob, human IgG4(L235A, G237A), human IgG4(L235E), human IgG4 YTE, human IgG4 YTE KiH Hole, human IgG4 YTE KiH Knob, and variants thereof.

[0115] In yet other embodiments, the constant region can be aglycosylated for N-linked glycosylation. In some embodiments, the constant region can be aglycosylated for N-linked glycosylation by mutating the oligosaccharide binding residue (e.g., Asn297) and / or adjacent residues that are part of the N-glycosylation recognition sequence in the constant region. In some embodiments, the constant region can be aglycosylated for N-linked glycosylation. The constant region can be aglycosylated for N-linked glycosylation enzymatically or by expression in a glycosylation-deficient host cell.

[0116] In yet other embodiments, the antibodies of the disclosure comprise any of the above constant regions, such as those set forth in Table 6, wherein the constant region sequence may further comprise a C-terminal lysine (K) at position 447 according to EUFC numbering. [Table 6-1] [Table 6-2] [Table 7]

[0117] In some embodiments, the anti-PACAP antibody of the present disclosure comprises heavy chain framework region (VHFR)-1, VHFR-2, VHFR-3, and VHFR-4 sequences set forth in SEQ ID NOs: 29-32, respectively; and light chain framework region (VLFR)-1, VLFR-2, VLFR-3, and VLFR-4 sequences set forth in SEQ ID NOs: 33-36, respectively.

[0118] In some embodiments, an anti-PACAP antibody of the disclosure comprises a VH sequence that is about 80%, about 85%, about 90%, about 95%, or about 99% identical to a sequence selected from SEQ ID NOs: 11 and 12, and a VL sequence that is about 80%, about 85%, about 90%, about 95%, or about 99% identical to a sequence selected from SEQ ID NOs: 20 and 21. In some embodiments, an anti-PACAP antibody of the disclosure comprises a VH sequence selected from SEQ ID NOs: 11 and 12, and a VL sequence selected from SEQ ID NOs: 20 and 21.

[0119] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise VH and VL sequences that are about 80%, about 85%, about 90%, about 95%, or about 99% identical to the sequences set forth in SEQ ID NOs: 11 and 20, respectively. In other embodiments, the anti-PACAP antibodies of the present disclosure comprise VH and VL sequences that are about 80%, about 85%, about 90%, about 95%, or about 99% identical to the sequences set forth in SEQ ID NOs: 12 and 21, respectively. In some embodiments, the anti-PACAP antibodies of the present disclosure comprise VH and VL sequences that are about 80%, about 85%, about 90%, about 95%, or about 99% identical to the sequences set forth in SEQ ID NOs: 11 and 21, respectively. In other embodiments, the anti-PACAP antibodies of the present disclosure comprise VH and VL sequences that are about 80%, about 85%, about 90%, about 95%, or about 99% identical to the sequences set forth in SEQ ID NOs: 12 and 20, respectively.

[0120] In some exemplary embodiments, an anti-PACAP antibody of the disclosure comprises a VH sequence and a VL sequence set forth, respectively, in SEQ ID NOs: 11 and 20. In some exemplary embodiments, an anti-PACAP antibody of the disclosure comprises a VH sequence and a VL sequence set forth, respectively, in SEQ ID NOs: 11 and 20, wherein the VH sequence has a glutamine (Q) instead of a glutamic acid (E) at residue 1 of SEQ ID NO: 11.

[0121] In some embodiments, an anti-PACAP antibody of the disclosure comprises the VH and VL amino acid sequences provided in SEQ ID NO: 11 and SEQ ID NO: 20, respectively, as provided in Figure 8. For example, the VH and VL are shown in bold in Figure 8.

[0122] In other exemplary embodiments, an anti-PACAP antibody of the disclosure comprises the VH and VL sequences set forth, respectively, in SEQ ID NOs: 12 and 21. In other exemplary embodiments, an anti-PACAP antibody of the disclosure comprises the VH and VL sequences set forth, respectively, in SEQ ID NOs: 12 and 21, wherein the VH sequence has a glutamine (Q) instead of glutamic acid (E) at residue 1 of SEQ ID NO: 12.

[0123] In some embodiments, an anti-PACAP antibody of the disclosure comprises the VH and VL amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 21, respectively, as provided in Figure 9. For example, the VH and VL are shown in bold in Figure 9.

[0124] In some exemplary embodiments, an anti-PACAP antibody of the disclosure comprises a VH sequence and a VL sequence set forth, respectively, in SEQ ID NOs: 11 and 21. In some exemplary embodiments, an anti-PACAP antibody of the disclosure comprises a VH sequence and a VL sequence set forth, respectively, in SEQ ID NOs: 11 and 21, wherein the VH sequence has a glutamine (Q) instead of a glutamic acid (E) at residue 1 of SEQ ID NO: 11.

[0125] In some embodiments, an anti-PACAP antibody of the disclosure comprises the VH and VL amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:21, respectively, as provided in Figure 10. For example, the VH and VL are shown in bold in Figure 10.

[0126] In other exemplary embodiments, an anti-PACAP antibody of the disclosure comprises the VH and VL sequences set forth, respectively, in SEQ ID NOs: 12 and 20. In other exemplary embodiments, an anti-PACAP antibody of the disclosure comprises the VH and VL sequences set forth, respectively, in SEQ ID NOs: 12 and 20, wherein the VH sequence has a glutamine (Q) instead of a glutamic acid (E) at residue 1 of SEQ ID NO: 12.

[0127] In some embodiments, an anti-PACAP antibody of the disclosure comprises the VH and VL amino acid sequences set forth in SEQ ID NO: 12 and SEQ ID NO: 20, respectively, as provided in Figure 11. For example, the VH and VL are shown in bold in Figure 11.

[0128] In some embodiments, an anti-PACAP antibody of the disclosure comprises a heavy chain constant region sequence selected from the group consisting of SEQ ID NOs: 37-51 and a light chain constant region sequence selected from SEQ ID NOs: 23 and 24. In certain exemplary embodiments, an anti-PACAP antibody of the disclosure comprises a heavy chain constant region sequence set forth in SEQ ID NO: 43 and a light chain constant region sequence set forth in SEQ ID NO: 23.

[0129] The Kabat numbering system is generally used when referring to residues within the variable region (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Numbering of amino acid positions as Kabat refers to the numbering system used for the heavy or light chain variable regions of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). According to this system, the heavy chain variable region may include a single amino acid insertion after residue 52 of H2 (e.g., residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc.). The Kabat numbering of residues for a given antibody can be determined by aligning the homologous regions of the antibody's sequence with the "standard" Kabat numbered sequence. Instead, Chothia refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia VH-CDR1 loop varies in the VH between positions 32 and 34 depending on the length of the loop (this is because the Kabat numbering scheme places the insert in the VH at positions 35A and 35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0130] The residue at position 32 (Kabat numbering) of VH CDR1 is important for having a predicted low immunogenicity profile. Thus, in some embodiments, the antibodies provided herein comprise a valine (V) or a leucine (L) at position 32 (Kabat numbering) of VH CDR1. In some embodiments, the antibodies provided herein do not have a serine (S) at position 32 (Kabat numbering) of VH CDR1.

[0131] Further, in some embodiments, the antibodies provided herein have an alanine-isoleucine (AI) at positions 93 and 94, respectively (Kabat numbering) of VHFR-3. In some embodiments, the antibodies provided herein do not have an alanine-arginine (AR) at positions 93 and 94, respectively (Kabat numbering) of VHFR-3.

[0132] Additionally, positions 50 and 51 (Kabat numbering) of the VL CDR2 are also important for the low immunogenicity profile of the antibodies provided herein. Thus, in some embodiments, the antibodies provided herein comprise tryptophan-alanine (WA) at positions 50 and 51 (Kabat numbering), respectively, of the VL CDR2.

[0133] In some embodiments, the disclosure also encompasses an anti-PACAP antibody comprising a VH sequence derived from SEQ ID NO: 19 and a VL sequence derived from SEQ ID NO: 22. In some embodiments, the anti-PACAP antibody comprises a VH sequence having a valine (V) or leucine (L) at residue 32 of SEQ ID NO: 19 according to the Kabat numbering, and a VL sequence having an alanine (A) at residue 27E of SEQ ID NO: 22 according to the Kabat numbering, and a tryptophan (W) and an alanine (A) at residues 50 and 51, respectively, of SEQ ID NO: 22 according to the Kabat numbering. In certain embodiments, the anti-PACAP antibody provided herein further comprises a cysteine-alanine-isoleucine (CAI) motif at residues 92-94 according to the Kabat numbering in the VH. See Figures 2A-2B.

[0134] For all antibodies provided herein, the numbering of the constant and / or variable regions can be according to IMGT® (IMGT®, the international ImMunoGeneTics information System®; Lefranc MP et al., Nucleic Acids Res, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc MP, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc MP, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc MP et al., Dev Comp Immunol, 29(3):185-203 (2005); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)).

[0135] For example, in some embodiments, the disclosure further includes anti-PACAP antibodies comprising a combination of the VH and VL CDR sequences provided below in Table 8. In some embodiments, the CDR sequences are based on the IMGT numbering. [Table 8]

[0136] In some embodiments, the anti-PACAP antibodies provided herein comprise a combination of the VH and VL CDR sequences provided in Table 8, wherein each CDR sequence may contain one, two, or three conservative amino acid substitutions.

[0137] In some embodiments, the anti-PACAP antibody of the present disclosure comprises heavy chain variable region (VH)-CDR1, VH-CDR2, and VH-CDR3 sequences set forth in SEQ ID NOs: 61, 52, and 55, respectively, and light chain variable region (VL)-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 62, 53, and 57, respectively, and variants containing one, two, or three conservative amino acid substitutions.

[0138] In some embodiments, the anti-PACAP antibody of the present disclosure comprises heavy chain variable region (VH)-CDR1, VH-CDR2, and VH-CDR3 sequences set forth in SEQ ID NOs: 28, 52, and 55, respectively, and light chain variable region (VL)-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 62, 53, and 57, respectively, and variants containing one, two, or three conservative amino acid substitutions.

[0139] Additionally, anti-PACAP antibodies of the present disclosure can also include the FR sequences provided in Table 9 herein, as well as variants described in more detail below. [Table 9]

[0140] For all antibodies provided herein, the numbering of the constant and / or variable domains may be according to the "EU numbering system" (Edelman GM et al., Proc Natl Acad Sci USA, 63(1):78-85 (1969)). The complete correspondence of the human CH1, hinge, CH2, and CH3 constant regions of IGHG1 can be found in the IMGT® database (IMGT®, the international ImMunoGeneTics information System®; Lefranc MP et al., Nucleic Acids Rev, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1):219-21 (2000); Lefranc MP, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc MP, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc MP et al., Dev Comp Immunol, 29(3):185-203 (2005)); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007).

[0141] For example, the numbering of the human kappa immunoglobulin light chain constant domain (IGKC) can be according to the "EU numbering system" (Edelman GM et al., Proc Natl Acad Sci USA, 63(1):78-85 (1969)). The complete correspondence of the human CK domain can be found in the IMGT database (IMGT®, the international ImMunoGeneTics information System®; Lefranc MP et al, Nucleic Acids Rev, 27(1):209-12 (1999); Ruiz M et al., Nucleic Acids Res, 28(1); 219-21 (2000); Lefranc MP, Nucleic Acids Res, 29(1):207-9 (2001); Lefranc MP, Nucleic Acids Res, 31(1):307-10 (2003); Lefranc MP et al., Dev Comp Immunol, 29(3):185-203 (2005)); Kaas Q et al., Briefings in Functional Genomics & Proteomics, 6(4):253-64 (2007)).

[0142] In some embodiments, the anti-PACAP antibodies of the disclosure comprise a combination of full-length heavy and light chain sequences as provided in Table 10 below. In some embodiments, the anti-PACAP antibodies of the disclosure comprise a full-length heavy chain sequence as provided in Table 10, wherein the heavy chain sequence has a glutamine (Q) instead of glutamic acid (E) at residue 1 of residues 70, 72, 74, and 75 of SEQ ID NO:. In yet other embodiments, the antibodies of the disclosure comprise a full-length heavy chain sequence as provided in Table 10, wherein the constant region sequence may further comprise a C-terminal lysine (K) at position 447 according to EU Fc numbering. [Table 10-1] [Table 10-2]

[0143] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise a full-length heavy chain sequence that is at least about 80%, about 85%, about 90%, about 95%, about 99% identical to the sequences set forth in SEQ ID NOs: 70, 72, 74, and 75. In some embodiments, the anti-PACAP antibodies of the present disclosure comprise a full-length light chain sequence that is at least about 80%, about 85%, about 90%, about 95%, about 99% identical to the sequences set forth in SEQ ID NOs: 71 and 73.

[0144] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise full-length heavy chain and full-length light chain sequences that are at least about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NOs: 70 and 71, respectively.

[0145] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise full-length heavy chain and full-length light chain sequences that are at least about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NOs: 74 and 71, respectively.

[0146] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise full-length heavy chain and full-length light chain sequences that are at least about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NOs: 75 and 71, respectively.

[0147] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise full-length heavy chain and full-length light chain sequences that are at least about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NOs: 72 and 73, respectively.

[0148] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise full-length heavy chain and full-length light chain sequences that are at least about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NOs: 70 and 73, respectively.

[0149] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise full-length heavy chain and full-length light chain sequences that are at least about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NOs: 72 and 71, respectively.

[0150] In some exemplary embodiments, an anti-PACAP antibody of the disclosure comprises the full-length heavy chain and full-length light chain sequences set forth, respectively, in SEQ ID NOs: 70 and 71. In some embodiments, an anti-PACAP antibody of the disclosure comprises the full-length heavy and light chain amino acid sequences provided in SEQ ID NO: 70 and SEQ ID NO: 71, respectively, in Figure 8. For example, the constant regions are underlined with a dotted line in Figure 8.

[0151] In other exemplary embodiments, an anti-PACAP antibody of the disclosure comprises the full-length heavy chain and full-length light chain sequences set forth, respectively, in SEQ ID NOs: 72 and 73. In some embodiments, an anti-PACAP antibody of the disclosure comprises the full-length heavy and light chain amino acid sequences provided in SEQ ID NO: 72 and SEQ ID NO: 73, respectively, in Figure 9. For example, the constant regions are underlined with a dotted line in Figure 9.

[0152] In some exemplary embodiments, an anti-PACAP antibody of the disclosure comprises the full-length heavy chain and full-length light chain sequences set forth, respectively, in SEQ ID NOs: 70 and 73. In some embodiments, an anti-PACAP antibody of the disclosure comprises the full-length heavy and light chain amino acid sequences provided in SEQ ID NO: 70 and SEQ ID NO: 73, respectively, in Figure 10. For example, the constant regions are underlined with a dotted line in Figure 10.

[0153] In yet other exemplary embodiments, an anti-PACAP antibody of the disclosure comprises the full-length heavy chain and full-length light chain sequences set forth, respectively, in SEQ ID NOs: 72 and 71. In some embodiments, an anti-PACAP antibody of the disclosure comprises the full-length heavy and light chain amino acid sequences provided in SEQ ID NO: 71 and SEQ ID NO: 72, respectively, in Figure 11. For example, the constant regions are underlined with a dotted line in Figure 11.

[0154] In some embodiments, the anti-PACAP antibody of the present disclosure comprises a full-length heavy chain sequence provided herein above, wherein the heavy chain sequence has a glutamine (Q) instead of glutamic acid (E) at residue 1 of SEQ ID NOs: 70, 72, 74, and 75.

[0155] As discussed above, the present disclosure encompasses variants of any of the antibodies or antigen-binding fragments disclosed herein. A "variant" of a polypeptide, such as an immunoglobulin chain (e.g., VH, VL, HC, or LC), refers to a polypeptide that comprises an amino acid sequence that is at least about 80-99.9% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a reference amino acid sequence set forth herein. When the comparison is performed by the BLAST algorithm, the parameters of the algorithm are selected to give the maximum match between the respective sequences over the entire length of each reference sequence.

[0156] The term "percent identity" as used herein in the context of two or more nucleic acids or proteins refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotides or amino acids (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9% or more identity when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm using default parameters described below or by manual alignment and visual inspection. See, for example, the NCBI website (ncbi.nlm.nih.gov / BLAST). Such sequences are then said to be "substantially identical". This definition may also refer to or apply to the complement of a sequence. This definition also includes sequences that contain deletions and / or additions, as well as sequences that contain substitutions. Sequence identity can be calculated over a range of amino acids. For example, sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990), and the like. Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, using its default parameters.

[0157] "Identity" per se has an art-recognized meaning and can be calculated using published techniques. See, e.g., COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, AM, ed., Oxford University Press, New York, (1988); BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, DW, ed., Academic Press, New York, (1993); COMPUTER ANALYSIS OF SEQUENCE DATA, PART I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, (1994); SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G., Academic Press, (1987); and SEQUENCE ANALYSIS PRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, (1991). There are many methods for measuring identity between two polynucleotide or polypeptide sequences, and the term "identity" is well known to those skilled in the art (Carillo, H., and Lipton, D., SIAM J. Applied Math. 48:1073 (1988)). Commonly used methods for determining identity or similarity between two sequences include, but are not limited to, those disclosed in "Guide to Huge Computers," Martin J. Bishop, ed., Academic Press, San Diego, (1994), and Carillo, H., and Lipton, D., SIAM J. Applied Math. 48:1073 (1988).Methods for aligning polynucleotides or polypeptides are codified in computer programs including the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J. Mol. Biol. 215:403 (1990)), the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711 (which uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482 489 (1981)).

[0158] A polypeptide having, for example, an amino acid sequence at least 95% "identical" to a query amino acid sequence of the present disclosure contemplates that the amino acid sequence of the subject polypeptide is identical to the query sequence, except that the subject polypeptide sequence may contain up to 5 amino acid modifications per every 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to the query amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be inserted, deleted, or substituted with another amino acid. These modifications of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence, or at any position between these terminal positions, either individually among residues in the reference sequence, or interspersed in one or more contiguous groups within the reference sequence. As a practical matter, whether a particular polypeptide is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9% identical to a polypeptide sequence of the present disclosure (e.g., an anti-PACAP antibody provided herein) can be determined using known computer programs.

[0159] As a practical matter, whether any particular polypeptide is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9% identical to, for example, any of the amino acid sequences shown in Tables 1-10 can be conventionally determined using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present disclosure) and a reference sequence (also called global sequence alignment) can also be determined using the FASTDB computer program mentioned above. In sequence alignment, both the query sequence and the reference sequence are amino acid sequences. The result of said global sequence alignment is expressed as a percent identity. In one embodiment of the present disclosure, the parameters used in a FASTDB alignment of amino acid sequences to calculate percent identity are: matrix=PAM0, k-tuple=2, mismatch penalty=1, joining penalty=20, randomization group length=0, cutoff score=1, window size=sequence length, gap penalty=5, gap size penalty=0.05, window size=500, or the length of the subject amino acid sequence, whichever is shorter.

[0160] If the reference sequence is shorter than the query sequence due to N- or C-terminal deletions rather than internal deletions, the results must be manually corrected. This is because the FASTDB program does not take into account the N- and C-terminal truncations of the reference sequence when calculating the overall percent identity. In the case of N- and C-terminally truncated reference sequences, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal to the reference sequence, as a percentage of the total bases of the query sequence, that are not matched / aligned with the corresponding target residues. Whether or not a residue is matched / aligned is determined by the results of the sequence alignment in FASTDB. This percentage is subtracted from the percent identity calculated by the FASTDB program above using the specified parameters to obtain a final percent identity score. This final percent identity score is the one that is used for the purposes of this disclosure. For the purposes of manually adjusting the percent identity score, only residues at the N- and C-termini of the reference sequence that are not matched / aligned with the query sequence are considered; that is, only query residue positions outside the furthest N- and C-terminal residues of the reference sequence.

[0161] For example, a 90 amino acid residue reference sequence is aligned with a 100 residue query sequence to determine percent identity. Since the deletion occurs at the N-terminus of the reference sequence, the FASTDB alignment does not show matching / alignment of the first 10 residues at the N-terminus. Since the 10 unpaired residues represent 10% of the sequence (number of residues at the N-terminus and C-terminus that are not matched / aligned with the query sequence / total number of residues in the query sequence), 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%. In another example, a 90 residue reference sequence is compared with a 100 residue query sequence. Since this deletion is an internal deletion, there are no residues at the N-terminus or C-terminus of the reference sequence that are not matched / aligned with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only residue positions outside the N-terminus and C-terminus of the reference sequence that are not matched / aligned with the query sequence, as displayed in the FASTDB alignment, are manually corrected.

[0162] Within the disclosed percent identity ranges, the present disclosure also relates to substitution variants of the disclosed polypeptides of the present disclosure. Substitution variants include polypeptides in which one or more amino acid residues are removed and replaced with another residue. In one embodiment, the percent identity disclosed above relates to the overall sequence of the identified specific sequence, but the amino acid residues that remain constant and are not subject to change are the amino acid residues of the CDRs, and the amino acid residues of the framework will be subject to change. For example, in one particular embodiment, if the anti-PACAP antibody of the present disclosure comprises at least one VH comprising an amino acid sequence that is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9% identical to the amino acid sequence of SEQ ID NO: 11, the CDR regions of the VH are allowed to remain constant and the framework regions are allowed to be variable, as long as the overall percent identity of SEQ ID NO: 11 falls within the scope of the embodiment. In one embodiment, the change is essentially a conservative substitution. However, the present disclosure also encompasses non-conservative substitutions. Conservative substitutions for the purposes of the present disclosure may be defined as shown in Tables 11-13 below. Amino acids can be classified according to their physical properties and contribution to the secondary and tertiary structure of proteins. Conservative substitutions are recognized in the art as replacing one amino acid with another amino acid with similar properties. Exemplary conservative substitutions are shown below. [Table 11]

[0163] Alternatively, conservative amino acids can be grouped as described in Lehninger (1975) Biochemistry, Second Edition; Worth Publishers, pp. 71-77. [Table 12]

[0164] Yet other alternative, exemplary conservative substitutions are set forth below. [Table 13]

[0165] nucleic acid As discussed above, one aspect of the disclosure relates to recombinant nucleic acids comprising nucleic acid sequences encoding the antibodies of the disclosure. In some embodiments, the recombinant nucleic acids of the disclosure can be configured as expression cassettes or vectors that contain these nucleic acid molecules operably linked to heterologous nucleic acid sequences, such as regulatory sequences that allow in vivo expression of the antibodies in a host cell.

[0166] The nucleic acid molecules of the present disclosure may be of any length, including, for example, from about 1 Kb to about 50 Kb, such as, for example, from about 1.2 Kb to about 10 Kb, from about 2 Kb to about 15 Kb, from about 5 Kb to about 20 Kb, from about 10 Kb to about 20 Kb, from about 5 Kb to about 40 Kb, from about 5 Kb to about 30 Kb, from about 5 Kb to about 20 Kb, or from about 10 Kb to about 50 Kb, from about 15 Kb to about 30 Kb, from about 20 Kb to about 50 Kb, from about 20 Kb to about 40 Kb, from about 5 Kb to about 25 Kb, or from about 30 Kb to about 50 Kb.

[0167] Thus, in some embodiments, a nucleic acid molecule is provided herein that comprises a nucleotide sequence encoding an antibody of the present disclosure. In certain embodiments, a nucleic acid molecule provided herein comprises a nucleotide sequence encoding any of the polypeptide sequences disclosed herein, including, for example, those listed in Tables 1-10. In some embodiments, the nucleotide sequence is incorporated into an expression cassette or expression vector. It will be understood by those skilled in the art that an expression cassette generally comprises a construct of genetic material that includes a coding sequence for an antibody or antigen-binding fragment thereof and sufficient regulatory information to direct proper transcription and / or translation of the coding sequence in a recipient cell, in vivo and / or ex vivo. Generally, the expression cassette can be inserted into a vector that targets a desired host cell and / or individual. Thus, in some embodiments, an expression cassette of the present disclosure comprises a coding sequence for an antibody or antigen-binding fragment thereof operably linked to expression control elements, such as a promoter and, optionally, any or a combination of other nucleic acid sequences that affect transcription or translation of the coding sequence.

[0168] An expression cassette comprises a nucleic acid molecule in which one or more nucleic acid sequences are functionally operatively linked, e.g., operably linked, as a linear or circular, single-stranded or double-stranded DNA or RNA polynucleotide molecule from any source capable of genomic integration or autonomous replication and can be inserted into a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage.

[0169] In some embodiments, the nucleic acid molecule of the present disclosure is incorporated into an expression vector.The term "vector" generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and those skilled in the art will understand that it can be used for transformation purposes, such as for the introduction of heterologous DNA into host cells.Thus, in some embodiments, a vector can be a replicon, such as a plasmid, a phage, or a cosmid, into which another DNA segment can be inserted to cause the replication of the inserted segment.In some embodiments, an expression vector can be an integrating vector.

[0170] In some embodiments, the expression vector may be a viral vector. As will be understood by those skilled in the art, the term "viral vector" is typically used to refer to a nucleic acid molecule (e.g., a transfer plasmid) that contains a nucleic acid element derived from a virus that facilitates the transfer or integration of a nucleic acid molecule into the genome of a cell, or to refer to a viral particle that mediates the transfer of nucleic acid. A viral particle typically contains various viral components, and in some cases, host cell components, in addition to the nucleic acid. The term viral vector can refer to a virus or viral particle that can transfer a nucleic acid to a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof that are primarily derived from a retrovirus. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof, including LTRs, that are primarily derived from the retrovirus genus lentivirus.

[0171] The nucleic acid sequences encoding the antibodies and antigen-binding fragments disclosed herein can be optimized for expression in a host cell of interest. For example, the GC content of the sequence can be adjusted to the average level for a given cellular host, calculated with reference to known genes expressed in the host cell. Methods for optimizing codon usage are known in the art. The codon usage in the coding sequence of the antibodies and antigen-binding fragments disclosed herein can be optimized to enhance expression in a host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons in the coding sequence are optimized for expression in a particular host cell.

[0172] Also provided herein are vectors, plasmids, or viruses comprising one or more of the nucleic acid molecules encoding any of the antibodies or antigen-binding fragments thereof disclosed herein. The nucleic acid molecules can be included within a vector that can, for example, direct expression in a cell transformed / transduced with the vector. Vectors suitable for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by one of ordinary skill in the art. See, e.g., Sambrook, J., & Russell, DW (2012), Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to as "Sambrook"); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (with supplements through 2014); Bollag, DM et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, MG et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997).The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, KB, Ferree, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference. .

[0173] DNA vectors can be introduced into cells, e.g., eukaryotic cells, via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, and include, for example, calcium phosphate transfection, DEAE-dextran-mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape-loading, bombardment, nucleoporation, hydrodynamic bombardment, and infection.

[0174] Viral vectors that can be used in the present disclosure include, for example, retroviral vectors, adenoviral vectors, and adeno-associated viral vectors, lentiviral vectors, herpes viruses, simian virus 40 (SV40), and bovine papilloma virus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0175] For example, the antibodies or antigen-binding fragments thereof disclosed herein can be produced in eukaryotic hosts, such as mammalian cells (e.g., COS cells, NIH3T3 cells, or HeLa cells). These cells are available from a number of sources, including the American Type Culture Collection (Manassas, VA). When selecting an expression system, it is only important that the components are compatible with each other. Such a determination is within the capabilities of an engineer or skilled artisan. Additionally, if guidance is needed in selecting an expression system, the skilled artisan can consult P. Jones, "Vectors: Cloning Applications", John Wiley and Sons, New York, NY, 2009).

[0176] The nucleic acid molecules provided may include naturally occurring sequences or sequences that are different from those naturally occurring, but may encode the same polypeptide, e.g., an antibody, due to the degeneracy of the genetic code. These nucleic acid molecules may be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as those produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, the nucleic acid molecules may be double-stranded or single-stranded (e.g., either the sense strand or the antisense strand).

[0177] A nucleic acid molecule is not limited to a sequence that codes for a polypeptide (e.g., an antibody), but may also include some or all of the non-coding sequences upstream or downstream of the coding sequence (e.g., the coding sequence for an antibody). Those skilled in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. If the nucleic acid molecule is a ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0178] Recombinant Cells and Cell Cultures The nucleic acid of the present disclosure can be introduced into a host cell, such as a Chinese Hamster Ovary (CHO) cell, to produce an engineered or recombinant cell containing the nucleic acid molecule. The introduction of the nucleic acid molecule (e.g., DNA or RNA, including mRNA) or vector of the present disclosure into a cell can be achieved by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery. For example, methods of introducing heterologous nucleic acid molecules into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of nucleic acid molecules in liposomes, lipid nanoparticle technology, biolistic injection, and direct microinjection of DNA into the nucleus. Additionally, the nucleic acid molecules can be introduced into mammalian cells by viral vectors such as lentivirus or adeno-associated virus. As discussed in more detail below, in some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure can be introduced into a subject in a nucleic acid form (e.g., DNA or RNA, including mRNA), such that the subject's own cells produce the antibody. The present disclosure further provides modifications to the nucleotide sequences encoding the anti-CoV-S antibodies described herein, resulting in increased antibody expression, increased antibody stability, increased nucleic acid (e.g., mRNA) stability, or improved affinity or specificity of the antibody for the CoV spike protein.

[0179] Thus, in some embodiments, the nucleic acid molecule can be delivered by a viral or non-viral delivery vehicle known in the art. For example, the nucleic acid molecule can be stably integrated into the host genome, replicated episomally, or present in the recombinant host cell as a minicircle expression vector for transient expression. Thus, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genome recombination techniques, or more precise techniques, such as guide RNA-directed CRISPR / Cas genome editing, or DNA-guided endonuclease genome editing using NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nuclease). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a minicircle expression vector for transient expression.

[0180] Nucleic acid molecules can be encapsulated in viral capsids or lipid nanoparticles, or delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acid into cells can be achieved by viral transduction. In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver nucleic acid to target cells via viral transduction. Several AAV serotypes have been reported, and all known serotypes can infect cells of multiple and diverse tissue types. AAV can transduce a wide range of species and tissues in vivo without toxicity, and generates relatively mild innate and adaptive immune responses.

[0181] Lentivirus-derived vector systems are also useful for nucleic acid delivery and gene therapy by viral transduction. Lentiviral vectors offer several attractive properties as gene delivery vehicles, including: (i) sustained gene delivery through stable vector integration into the host genome; (ii) ability to infect both dividing and non-dividing cells; (iii) broad tissue tropism, including important gene and cell therapy target cell types; (iv) no expression of viral proteins after vector transduction; (v) ability to deliver complex genetic elements such as polycistronic or intron-containing sequences; (vi) potentially safe integration site profile; and (vii) a relatively simple system for vector engineering and production.

[0182] In some embodiments, the host cell can be genetically engineered (e.g., transduced or transformed or transfected) with, for example, a vector construct of the present application, such as a viral vector or a vector for homologous recombination, which can contain a nucleic acid sequence homologous to a portion of the genome of the host cell or can be an expression vector for expression of a polypeptide of interest. The antibodies of the present invention can be prepared and purified using known methods. For example, cDNA sequences encoding HC (e.g., the amino acid sequence given by SEQ ID NO: 70) and LC (e.g., the amino acid sequence given by SEQ ID NO: 71) can be cloned and engineered into an expression vector using known methods. The engineered immunoglobulin expression vector can then be stably transfected into the engineered cell.

[0183] In some embodiments, the engineered cells are eukaryotic cells. In some embodiments, the engineered cells are animal cells. In some embodiments, the animal cells are vertebrate cells or invertebrate cells. In some embodiments, the animal cells are mammalian cells. In some embodiments, the animal cells are human cells. In some embodiments, the animal cells are non-human animal cells. In some embodiments, the engineered cells are non-human primate cells. In some embodiments, the engineered cells are selected from the group consisting of baby hamster kidney (BHK) cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NS0 mouse myeloma cells, human epidermal laryngeal cells, human fibroblast cells, human HEK-293 cells, human HeLa cells, human HepG2 cells, human HUH-7 cells, human MRC-5 cells, human muscle cells, mouse 3T3 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells. In some embodiments, the engineered cells are Pichia pastoris cells or Saccharomyces cerevisiae cells, all of which are also suitable for producing the antibodies described in the present invention.

[0184] In another aspect, provided herein is a cell culture comprising at least one recombinant cell disclosed herein and a medium. In general, the medium can be any suitable medium for culturing the cells described herein. Techniques for transforming the wide variety of host cells and species mentioned above are known in the art and described in the technical and scientific literature. Thus, a cell culture comprising at least one recombinant cell disclosed herein is also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0185] In some embodiments, the disclosure provides a method of producing an antibody or antigen-binding fragment thereof described herein, which can include culturing an engineered or recombinant cell described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment thereof.

[0186] In another aspect, animals are also provided that include the recombinant nucleic acids or vectors disclosed herein. In some embodiments, the present disclosure provides transgenic animals that are non-human animals. In some embodiments, the transgenic animals produce the antibodies or antigen-binding fragments disclosed herein.

[0187] The transgenic non-human host animals of the present disclosure are prepared using standard methods known in the art for introducing exogenous nucleic acids into the genome of non-human animals. In some embodiments, the non-human animal of the present disclosure is a mouse. Other animal species suitable for the compositions and methods of the present disclosure include animals that are (i) suitable for gene transfer and (ii) capable of rearranging immunoglobulin gene segments to generate antibody responses. Examples of such species include, but are not limited to, rats, rabbits, chickens, goats, pigs, sheep, and cows. Approaches and methods for producing transgenic non-human animals are known in the art. Exemplary methods include pronuclear microinjection, DNA microinjection, lentiviral vector-mediated DNA transfer into early embryos, and sperm-mediated gene transfer, adenovirus-mediated DNA transfer into animal sperm (e.g., pigs), retroviral vectors (e.g., birds), somatic cell nuclear transfer (e.g., goats). The state of the art in producing transgenic livestock is reviewed in Niemann, H. et al. (2005) Rev. Sci. Tech. 24:285-298.

[0188] In some embodiments, the animal is a vertebrate or invertebrate. In some embodiments, the animal is a mammalian subject. In some embodiments, the mammalian animal is a non-human animal. In some embodiments, the transgenic animal of the present disclosure can be produced using classical random genome recombination techniques, or using more sophisticated techniques such as guide RNA-guided CRISPR / Cas genome editing, or DNA-guided endonuclease genome editing using NgAgo (Natronobacterium gregorii Argonaute), or TALEN genome editing (Transcription Activator-Like Effector Nuclease). In some embodiments, the transgenic animal of the present disclosure can be produced using transgenic microinjection techniques, which do not require the use of homologous recombination techniques, and therefore are believed to be easier to prepare and select than approaches using homologous recombination.

[0189] In another aspect, provided herein is a method for producing an antibody or antigen-binding fragment thereof, wherein the method comprises growing (i) a transgenic animal disclosed herein, or (ii) a recombinant cell disclosed herein, under conditions such that the antibody or antigen-binding fragment is produced.

[0190] In some embodiments, the method for producing an antibody or antigen-binding fragment thereof described herein further comprises isolating the produced antibody or antigen-binding fragment from (i) the transgenic animal, or (ii) the recombinant cell and / or the medium in which the recombinant cell was cultured. In some embodiments, the mammal is a non-human primate. Thus, the antibody or antigen-binding fragment produced by the method disclosed herein is also within the scope of the present disclosure.

[0191] Pharmaceutical Compositions The anti-PACAP antibodies, nucleic acids of the present disclosure can be incorporated into compositions, including pharmaceutical compositions.

[0192] In another aspect, the antibody, nucleic acid of the present disclosure can be incorporated into a composition, such as a pharmaceutical composition, suitable for various downstream applications.Exemplary compositions of the present disclosure generally include pharmaceutical compositions that include one or more antibodies, nucleic acids, and pharma- ceutically acceptable excipients, such as carriers.In some embodiments, the composition is a sterile composition.In some embodiments, the composition is formulated as a vaccine.In some embodiments, the composition further includes an adjuvant.

[0193] The pharmaceutical compositions provided herein can be in any form that allows the composition to be administered to an individual. In some particular embodiments, the pharmaceutical compositions are suitable for administration to humans. The scope of the present disclosure includes a dry, e.g., lyophilized, composition comprising an anti-CoV-S antigen-binding polypeptide, e.g., an antibody or antigen-binding fragment thereof (e.g., of Table 1), or a pharmaceutical composition thereof that includes a pharmaceutical acceptable carrier but is substantially free of water. As used herein, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more specifically, in humans. A carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline, aqueous dextrose, and aqueous glycerol solutions can also be used as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene glycol, water, ethanol, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. In some embodiments, the pharmaceutical composition is formulated aseptically for administration to an individual or animal (some non-limiting examples include a human or mammal). In some embodiments, the individual is a human.

[0194] In some embodiments, the pharmaceutical composition of the present disclosure is formulated to be suitable for the intended route of administration to an individual.For example, the pharmaceutical composition can be formulated to be suitable for parenteral, intraperitoneal, colorectal, intraperitoneal, and intratumoral administration.In some embodiments, the pharmaceutical composition can be formulated for oral, rectal, mucosal, enteral, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, injection, topical, dermal, transdermal, or intraarterial administration.Those skilled in the art will understand that the formulation should be suitable for the mode of administration.

[0195] For example, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In some embodiments, the composition should be sterile and fluid to the extent that easy syringability exists. It can be stabilized under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants, for example, sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is common to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, and / or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0196] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0197] method The present disclosure further provides a method for treating or preventing conditions as described herein, particularly in an individual. In some embodiments, the present invention can include a method for treating or preventing any aspect of PACAP-related symptoms, such as headache, migraine, cluster headache, and / or refractory migraine, anxiety, depression, PTSD, co-occurring conditions (e.g., anxiety / depression / PTSD) with headache (e.g., migraine, cluster headache, refractory migraine), co-occurring anxiety disorder with migraine, complex regional pain syndrome, and rosacea. For example, in the context of headache or migraine treatment, this includes reducing the severity, reducing the intensity of pain (e.g., headache, i.e., head pain) and other associated symptoms, reducing the frequency of recurrence, improving the quality of life of patients suffering from headache, and reducing the dose of other medications required to treat headache. In the case of migraine, other associated symptoms include, but are not limited to, nausea, vomiting, and hypersensitivity to light, sound, and / or movement. In the case of cluster headaches, other associated symptoms include, but are not limited to, swelling under or around the eyes, excessive watering, bloodshot eyes, runny or stuffy nose, facial redness, etc.

[0198] Further provided herein is a method for reducing symptoms of a condition in an individual, such as those conditions described herein. A "reduction" of a symptom refers to a reduction in the severity or frequency of the symptom, or the elimination of the symptom. Thus, the terms "reduction in incidence," "prophylaxis," or "prevention" as used herein refer to either a reduction in the severity of a particular disease, condition, symptom, or disorder (the terms disease, condition, and disorder are used interchangeably throughout this application). Reduction in severity includes reducing the drugs and / or therapies commonly used for the condition, for example, by reducing the need, amount, and / or exposure to the drug or therapy. Reduction in severity also includes reducing the duration and / or frequency of a particular condition, symptom, or disorder (including, for example, delaying or extending the time to the next episodic attack in an individual).

[0199] Further, the present disclosure provides a method for alleviating a condition as described herein in an individual. Ameliorating one or more symptoms of headache or migraine, or other PACAP-related condition as described herein, refers to a reduction or amelioration of one or more symptoms of a condition, e.g., headache or migraine, compared to not administering an anti-PACAP antagonist antibody. Amelioration also includes a shortening or reduction in the duration of the symptoms.

[0200] In some embodiments, the present disclosure further provides a method of controlling a condition as described herein in an individual. As used herein, "controlling headache" or "controlling migraine" or "controlling" another PACAP-related condition refers to maintaining or reducing (compared to pre-treatment levels) one or more symptoms of the condition in an individual, such as headache, migraine severity or duration, or headache or migraine attack frequency. For example, headache duration or severity or attack frequency is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in an individual compared to pre-treatment levels. The reduction in headache duration or severity, or attack frequency, may last for any period of time, such as 2 weeks, 4 weeks (1 month), 8 weeks (2 months), 12 weeks (3 months), 4 months, 5 months, 6 months, 9 months, 12 months, etc.

[0201] As used herein, "delaying" the onset of a condition, e.g., a PACAP-associated condition of migraine or headache, means to slow, hinder, slow, retard, stabilize, and / or postpone the progression of the condition or disease. This delay can be of various durations depending on the condition or disease and / or the medical history of the individual undergoing treatment. As will be appreciated by those skilled in the art, a sufficient or significant delay can in effect encompass prevention, in that the individual does not develop a headache (e.g., migraine). A method that "delays" the onset of a symptom is one that reduces the probability that the symptom will develop within a given time frame and / or reduces the severity of the symptom within a given time frame when compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of subjects.

[0202] "Onset" or "progression" of a condition, such as a PACAP-associated condition described herein, refers to the initial symptoms and / or subsequent progression of a disorder or symptom or side effect, such as photophobia or photoaversion. The onset of a headache or migraine can be detected and evaluated using standard clinical techniques known in the art. Onset, however, also refers to progression that may not be detectable. For purposes of this disclosure, onset or progression refers to the biological course of a condition. "Onset" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of a condition, such as a headache or migraine, includes the initial onset and / or recurrence. The condition may be, for example, a primary headache or a secondary headache. Primary headaches include, for example, migraine with aura, migraine without aura, hemiplegic migraine, episodic migraine, chronic migraine, abdominal migraine, cluster headache, tension-type headache, generalized headache, paroxysmal migraine, and persistent migraine. Secondary headaches include, for example, headaches caused by autonomic dysreflexia, anxiety disorders associated with migraine, and headaches caused by homeostatic disorders, such as complex regional pain syndrome.Furthermore, the subject may have a condition selected from the group consisting of migraine, headache, and pain associated with diseases or conditions, such as cluster headache and / or refractory migraine, anxiety, depression, PTSD, comorbid conditions (e.g., anxiety / depression / PTSD) associated with headache (e.g., migraine, cluster headache, refractory migraine), anxiety disorders associated with migraine, complex regional pain syndrome, and rosacea.In some embodiments, the headache may be selected from the group consisting of migraine with aura, migraine without aura, hemiplegic migraine, cluster headache, migrainous neuralgia, chronic headache, episodic migraine, chronic migraine, medication overuse headache, and tension-type headache.

[0203] In other embodiments, an individual may suffer from a condition not listed here, but is considered to be in need of treatment with the antibodies or pharmaceutical compositions described herein.

[0204] Migraine is a chronic paroxysmal neurological disorder characterized by attacks of moderate or severe headache and reversible neurological and general symptoms. The most characteristic symptoms associated with migraine include, but are not limited to, photophobia, phonophobia, and gastrointestinal symptoms such as nausea and vomiting. In contrast, headache generally refers to pain in any area of ​​the head. Headaches may occur on one or both sides of the head, may be localized to a specific location, or may radiate from a point to the entire head and may have a squeezing quality. Headaches may be sharp, throbbing, dull, etc. Headaches may begin gradually or suddenly and may last less than an hour or several days.

[0205] The International Headache Society (IHS) defines migraine as a recurrent headache disorder that manifests in attacks lasting 4 to 72 hours. Typical features of headache are unilateral location, pulsatile quality, moderate or severe intensity, aggravation by everyday physical activity, and association with nausea and / or photophobia and phonophobia. (Headache Classification Subcommittee of the International Headache Society (2018). The international classification of headache disorders 3rd edition. Cephalalgia 38:1-211). According to the Headache Classification Committee of the IHS, there are two main types of migraine: 1) migraine without aura, which is a clinical syndrome characterized by headache with specific features and associated symptoms; and 2) migraine with aura, which is mainly characterized by transient focal neurological symptoms that usually precede or occasionally accompany the headache.

[0206] Migraine with aura (also called classic migraine) includes symptoms such as repeated attacks of unilateral, fully reversible visual, sensory, or other central nervous system symptoms that usually develop gradually, followed by headache and associated migraine symptoms. These attacks usually last for a few minutes. Migraine may become chronic. In addition, chronic migraine may also include episodic subtypes of migraine. Chronic migraine occurs on 15 or more days per month, lasts for 3 months or more, and has migraine features for at least 8 days per month.

[0207] Additionally, the methods provided herein can be used to treat individuals suffering from cluster headaches. Symptoms of cluster headaches include, but are not limited to, attacks of severe, strictly unilateral pain in the orbit, supraorbit, temporal, or any combination of these sites, lasting 15-180 minutes and occurring once every other day to eight times a day. The pain is associated with ipsilateral conjunctival injection, lacrimation, nasal congestion, rhinorrhea, forehead and facial sweating, miosis, ptosis, and / or eyelid edema, and / or restlessness and agitation.

[0208] Cluster headaches can be episodic or chronic. Episodic cluster headaches are attacks lasting from 7 days to 1 year (separated by pain-free periods lasting at least 3 months). For example, episodic cluster headaches can present as attacks lasting from 7 days to 1 year (separated by pain-free periods lasting at least 3 months) separated by pain-free periods lasting at least 3 months. Furthermore, according to the International Classification of Headache Disorders, Third Edition, episodic cluster headaches can present as at least two cluster periods lasting from 7 days to 1 year (if untreated) separated by pain-free remission periods of 3 months or more. In contrast, chronic cluster headaches can be attacks that occur for more than a year without remission or remission periods of less than 3 months.

[0209] The term refractory migraine or resistant migraine has been used to describe persistent headaches that are difficult to treat or do not respond to standard and / or aggressive treatment. In some embodiments, refractory migraine as used herein requires prior unsuccessful treatment with 2-4 available prophylactic drugs or classes of prophylactic drugs. In certain embodiments, refractory migraine as used herein requires prior unsuccessful treatment with 2-3, at least 2, at least 3, at least 4, more than 2, or more than 3 available prophylactic drugs or classes of prophylactic drugs.

[0210] As used herein, "non-responsive" or "treatment failure" refers to the lack of effectiveness of a prophylactic agent or type of prophylactic agent in reducing the frequency, duration, and / or severity of migraine headaches in a patient after standard treatment of the drug or when treatment (depending on the prophylactic agent or class of prophylactic agents) must be discontinued because the patient cannot tolerate it due to adverse events or because the drug is contraindicated or not suitable for the patient.

[0211] Prophylactic drugs can be divided into several classes. For example, these classes include the following clusters: Cluster A, antiepileptic drugs; Cluster B, beta blockers; Cluster C, tricyclic antidepressants; Cluster D, calcium channel blockers; Cluster E, angiotensin II receptor antagonists; Cluster F, botulinum toxins; and Cluster G, calcitonin gene-related peptide (CGRP) pathway monoclonal antibodies. In some embodiments, refractory migraine as used herein requires prior unsuccessful treatment in any of 2-4 of the above clusters. In certain embodiments, refractory migraine as used herein requires prior unsuccessful treatment in any of 2-3, at least 2, at least 3, at least 4, more than 2, or more than 3 of the above clusters.

[0212] Examples of antiepileptic drugs include divalproex, sodium valproate, valproate, valproic acid, topiramate, and gabapentin. Examples of beta-blockers include propranolol, timolol, atenolol, metoprolol, nadolol, and bisopropol. Examples of tricyclic antidepressants include amitriptyline, nortriptyline, doxepin, and fluoxetine. Examples of calcium channel blockers include flunarizine. Examples of angiotensin II receptor antagonists include candesartan. Anti-CGRP pathway monoclonal antibodies are described below. For example, calcitonin gene-related peptide (CGRP) pathway monoclonal antibodies can include anti-CGRP antibodies, anti-CGRP receptor (CGRP-R) antibodies, or both. In an exemplary embodiment, the anti-CGRP antibody is fremanezumab. In another embodiment, the anti-CGRP antibody is galcanezumab. In one embodiment, the anti-CGRP antibody is eptinezumab. Examples of anti-CGRP-R antibodies include erenumab.

[0213] In certain embodiments, prophylactic drugs applicable to migraine headaches do not include acute treatments. In other embodiments, refractory migraine as encompassed by the present disclosure requires failure of treatment with 2-4, 3-4, at least 2, or at least 3 classes of prophylactic drugs as defined by the Refractory Headache Special Interest Section (RHSIS) of the American Headache Society (AHS). Under this definition, in some cases, an individual requires failure of 3 classes of prophylactic treatment.

[0214] Furthermore, a definition of pharmacologically intractable headache has been proposed by Silberstein SD, et al. (2010) (Defining the pharmacologically intractable headache for clinical trials and clinical practice. Headache 50(9):1499-1506). This definition builds on the AHS criteria and proposes a staging classification scheme of intractability to acute and preventive treatments, as well as an assessment of headache-related disability. Specifically, this definition proposes Class I (mild) refractory headache as failure of an adequate response to two different classes of non-specific acute treatments (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs), analgesic combinations); Class II (moderate) refractory headache as class I plus failure to respond to triptans or ergot derivatives (such as dihydroergotamine (DHE)); and Class III (severe) refractory headache as class I and II plus failure to respond to oral or parenteral opioids or corticosteroids in appropriate doses and in appropriate formulations, or parenteral dopamine antagonists.

[0215] Furthermore, the European Headache Federation (EHF) has published a consensus statement on the definition of chronic migraine (CM) in 2014. These criteria are limited to CM and require failure of three classes of preventive treatment. They also require appropriate treatment of psychiatric and other comorbid conditions, if possible, by a multidisciplinary team. In some embodiments, these criteria do not include acute treatment and degree of disability. Thus, when referring to refractory or resistant migraine, the present disclosure is intended to include all of the above definitions.

[0216] In other embodiments, the disclosure further provides a method of treating or preventing a headache in an individual. In some embodiments, the headache is a migraine. In some embodiments, the disclosure further provides a method of treating or preventing a migraine in an individual. In some embodiments, the disclosure further provides a method of treating or preventing episodic migraine. In some embodiments, the disclosure further provides a method of treating or preventing chronic migraine. In some embodiments, the headache is a cluster headache. In some embodiments, the headache is an episodic cluster headache. In some embodiments, the headache is a chronic cluster headache. In further embodiments, the disclosure further provides a method of treating or preventing refractory or resistant migraine in an individual. In additional embodiments, the disclosure provides a method of treating an individual diagnosed with migraine that has not responded to at least two, at least three, two to three, two to four, more than two, more than three, or up to four prior preventative therapies for migraine (i.e., refractory migraine).

[0217] In some embodiments, the therapeutic or prophylactic methods provided herein comprise administering to an individual a therapeutically effective amount of an anti-PACAP antibody or pharmaceutical composition described herein.

[0218] In certain embodiments, the condition to be treated or prevented is migraine or refractory migraine. In other embodiments, the condition to be treated or prevented is episodic migraine. In other embodiments, the condition to be treated or prevented is chronic migraine. In other embodiments, the method can also include administering to the individual a second agent, either simultaneously or sequentially with the anti-PACAP antibody. The second agent can be, for example, an acute migraine treatment agent. In yet other embodiments, the second agent can be a prophylactic treatment agent for migraine and / or refractory migraine.

[0219] Acute treatments for migraine headaches are known in the art and include nonsteroidal anti-inflammatory drugs (NSAIDs) and / or ergot alkaloids and / or triptans and / or 5 hydroxytryptamine 1F receptor agonists (i.e., ditanes), and gepants (i.e., calcitonin gene-related peptide receptor antagonists).

[0220] Non-limiting examples of NSAIDs that can be used in combination with anti-PACAP antibodies include aspirin, diclofenac, diflucinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, cyclooxygenase-2 (COX-2) inhibitors, celecoxib, rofecoxib, meloxicam, JTE-522, L-745, 337, NS398, or a pharma- ceutically acceptable salt thereof.

[0221] Non-limiting examples of triptans that can be used in combination with the anti-PACAP antibodies described herein include sumatriptan, zolmitriptan, naratriptan, rizatriptan, eletriptan, almotriptan, and afrovatriptan.

[0222] Non-limiting examples of ditanes that can be used in combination with the anti-PACAP antibodies of the present disclosure include lasmiditan.

[0223] Non-limiting examples of gepants that can be used in combination with the anti-PACAP antibodies of the present disclosure include ubrogepant, rimegepant, atogepant, and bazegepant.

[0224] Preventive treatments for migraine and refractory migraine are known in the art. In one embodiment, the preventive treatment comprises topiramate. In another embodiment, the preventive treatment comprises onabotunamtoxin A. In one embodiment, the preventive treatment comprises a calcitonin gene-related peptide (CGRP) pathway monoclonal antibody. For example, the calcitonin gene-related peptide (CGRP) pathway monoclonal antibody can include an anti-CGRP antibody, an anti-CGRP receptor (CGRP-R) antibody, or both. In an exemplary embodiment, the anti-CGRP antibody is fremanezumab. In another embodiment, the anti-CGRP antibody is galcanezumab. In one embodiment, the anti-CGRP antibody is eptinezumab. Exemplary anti-CGRP-R antibodies include erenumab.

[0225] In certain embodiments, the anti-PACAP antibodies described herein are administered in combination with onabotunatoxinA.

[0226] In certain embodiments, the anti-PACAP antibodies described herein are administered in combination with any of the CGRP pathway monoclonal antibodies described above. In certain embodiments, the anti-PACAP antibodies described herein are administered in combination with fremanezumab.

[0227] In certain embodiments, the anti-PACAP antibodies described herein are administered in combination with galcanezumab.

[0228] In certain embodiments, the anti-PACAP antibodies described herein are administered in combination with eptinezumab.

[0229] In certain embodiments, the anti-PACAP antibodies described herein are administered in combination with erenumab.

[0230] In some embodiments, the anti-PACAP antibodies described herein can be used in combination with other anti-PACAP antibodies known in the art, such as those described in WO2017181031, WO2017181039, WO2017106578, and WO2019067293.

[0231] In some embodiments, the anti-PACAP antibodies described herein can be used in combination with the anti-PAC1 antibodies described in WO2019140216 and WO2014144632.

[0232] In some embodiments, the anti-PACAP antibodies described herein can be used in migraine patients in subpopulations identified by physiological measures of autonomic function. For example, subpopulations can be identified by (i) baseline ictal or interictal PACAP levels, plasma, tears, saliva, or other biological samples; (ii) baseline physiological measures of autonomic function, such as pupillary light reflex or galvanic skin response; and (iii) baseline ictal or interictal autonomic response to PACAP infusion.

[0233] In some embodiments, administration of a therapeutically effective amount of an anti-PACAP antibody or pharmaceutical composition described herein may result in a delay in the onset of a condition described herein, a shortened duration of a condition, or a reduced severity of a condition.

[0234] The administration of any one or more compositions described herein, such as anti-PACAP antibodies, can be incorporated into a therapeutic composition or combination therapy regimen for use in the treatment or prevention methods described herein. For example, a combination therapy of an anti-PACAP antibody with any of the above-mentioned CGRP pathway monoclonal antibodies (e.g., fremanezumab) can be incorporated into one therapeutic composition or into two separate therapeutic compositions for use in the treatment or prevention methods for PACAP-related conditions, such as headache, migraine, or any of the above-mentioned conditions. The anti-PACAP antibodies or pharmaceutical compositions of the present disclosure are typically administered as a solution or suspension formulation by injection or infusion. In an exemplary embodiment, the anti-PACAP antibody can be administered to an individual by direct injection. In another exemplary embodiment, the anti-PACAP antibody can be administered by systemic infusion. Some anti-PACAP antibodies of the present disclosure (e.g., 890C) are effective at a concentration equivalent to about 300 picomolar (pM) in cell-based assays. The cell-based assay may be one that follows cyclic adenosine monophosphate (cAMP) production in the presence of anti-PACAP antibodies (Wang, T., et al. (2004). Measurement of cAMP for G(αs)- and G(αi) Protein-Coupled Receptors (GPCRs). Assay Guidance Manual. S. Markossian, GS Sittampalam, A. Grossman et al. Bethesda (MD), Eli Lilly & Company and the National Center for Advancing Translational Sciences). cAMP is an important intracellular second messenger in GPCR signaling. Agonist activation of GPCRs that are coupled to G(αs) proteins results in increased production of intracellular cAMP levels, whereas activation of GPCRs that are coupled to G(αi) proteins results in decreased production of intracellular cAMP levels. Both of these changes in intracellular cAMP are mediated by modulation of adenylate cyclase activity. cAMP regulates the activity of cAMP-dependent protein kinase A (PKA), which plays an important role in various downstream cellular processes. There are a number of reagent kits available that can be used to measure intracellular cAMP levels. These include the HTRF cAMP kit from Cisbio, the LANCE cAMP kit from PerkinElmer, the HitHunter cAMP kit from DiscoverX, and the cAMP Direct Immunoassay kit from Abcam and BioVision. All of these assays are based on the use of antibodies that specifically recognize both intracellular cAMP and an exogenous labeled cAMP complex that serves as a competitor, after which the labeled cAMP conjugate is detected by various detection techniques, including fluorescence resonance energy transfer (FRET) or enzymatic reactions. Additionally, Promega's antibody-independent GloSensor cAMP assay uses a semi-split luciferase that reassembles upon binding to cAMP. Other anti-PACAP antibodies provided herein may be most effective at higher or lower concentrations, depending on their binding affinity to PACAP and the degree of expression of PACAP in the individual. In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are effective at concentrations equivalent to about 30 to about 90 pM in cell-based assays. In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are effective at concentrations equivalent to about 40 to about 80 pM in cell-based assays.In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are effective in a cell-based assay at a concentration equivalent to about 50 to about 70 pM, hi some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are effective in a cell-based assay at a concentration equivalent to about 60, 65, 67, 69, or 70 pM.

[0235] Systems and Kits Also provided herein are systems and kits that include the anti-PACAP antibodies, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided and described herein, as well as instructions for making and using the same. For example, in some embodiments, systems and / or kits are provided herein that include one or more of the anti-PACAP antibodies described herein, the recombinant nucleic acids described herein, the recombinant cells described herein, or the compositions described herein. In some embodiments, the systems and / or kits of the present disclosure further include one or more syringes (including pre-filled syringes) and / or catheters that are used to administer any one of the anti-PACAP antibodies, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided to an individual. In some embodiments, the kits can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, such as modulating the activity of cells in an individual in need thereof, inhibiting target cancer cells, or treating a disease.

[0236] Any of the above-mentioned systems and kits may further comprise one or more additional reagents, which may be selected from the following: a dilution buffer, a reconstitution solution, a wash buffer, a control reagent, a control expression vector, a negative control polypeptide, a positive control polypeptide, a bispecific binding agent, or a reagent for in vitro production of an engineered transmembrane protein.

[0237] In some embodiments, the system or kit may further include instructions for using the components of the kit to carry out the method. The instructions for carrying out the method are typically recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be present in the kit as a kit insert, on the label of the container of the kit or its components (i.e., on the packaging or subpackaging), etc. The instructions may be present as an electronic storage data file present on a suitable computer-readable storage medium, such as, for example, a CD-ROM, a diskette, a flash drive, etc. In some cases, the actual instructions may not be included in the kit, but a means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. As with the instructions, this means for obtaining the instructions may be recorded on a suitable substrate.

[0238] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0239] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. Although many sources of information, including scientific journal articles, patent documents, and textbooks, have been referenced herein, it will be expressly understood that this reference is not an admission that any of these documents form part of the common general knowledge in the art.

[0240] The discussion of the general methods described herein is for illustrative purposes only: other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are intended to be within the spirit and scope of this application. EXAMPLES

[0241] The practice of the present disclosure will employ, unless otherwise specified, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology well known to those skilled in the art. Such techniques are fully described in the above-cited documents. Additional embodiments are disclosed in more detail in the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure or claims in any way.

[0242] Example 1: Generation of anti-PACAP antibodies This example briefly describes the procedures for generating the antibodies provided in this disclosure.

[0243] Anti-PACAP monoclonal antibodies were isolated from immunized mice, and plasma cells were isolated from the mice based on their selectivity for PACAP. Single B cells were screened against PACAP-38 and VIP using known techniques, for example as described in: Winters et al. Rapid single B cell antibody discovery using nanopens and structured light. mAbs Volume 11, 2019 - Issue 6; Asensio et al. Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics. mAbs Volume 11, 2019 - Issue 5; Seah et al. Microfluidic single-cell technology in immunology and antibody screening. Mol Aspects Med. 2018;59:47-61; Proserpio et al. Single-cell technologies are revolutionizing the approach to rare cells. Immunol Cell Biol. 2016;94(3):225-229; El Debs et al. Functional single-cell hybridoma screening using droplet-based microfluidics. Proc Natl Acad Sci USA. 2012;109(29):11570-11575; and Theberge et al. Microdroplets in microfluidics: an evolving platform for discoveries in chemistry and biology. Angew Chem Int Ed Engl. 2010;49(34):5846-5868. Over 61,000 B cells were screened.Positive B cells secreting antigen-specific antibodies were isolated and sequenced.

[0244] Sequencing and cloning of 201 antibodies encoding immunoglobulin heavy and light chains were performed. Thirty-nine antibodies were identified with PACAP selective binding. Two clones were selected for further optimization. These antibodies showed high PACAP binding affinity but low or no VIP binding affinity. Furthermore, both showed high potency against PACAP but low or no potency against VIP.

[0245] Both antibodies showed high affinity and potency towards PACAP38 and PACAP27 as measured by SPR at 37° C. and a functional cell-based assay measuring PACAP27 / PACAP38-mediated cAMP accumulation using a human PAC1-expressing cell line. The optimization process generated over 660 humanized variants, generating high affinity, high potency antibody candidates while generating antibodies with low risk of potential immunogenicity as measured by the ProImmune REVEAL® (peptide MHC class II stability) assay and high humanness scores.

[0246] The humanized variant that retains titer is 100 million (10 8 The variants were further optimized through in vitro affinity maturation, which included the generation of a combinatorial library of over 1000 variants. Approximately 950 variants were further screened for potential manufacturability and immunogenicity while maintaining a potency profile.

[0247] Table 14 below shows exemplary humanized variants that were screened for binding affinity, potency, and predicted immunogenicity. [Table 14-1] [Table 14-2] Four humanized antibodies (890C, 608C, 627C, and 609C) demonstrated high affinity, high potency, and lower predicted immunogenicity.

[0248] Antibody developability criteria as described in Example 7 were used to identify variants with the best developability profile.

[0249] As can be seen from Table 14, the highest binding affinity does not necessarily correlate easily with the predicted immunogenicity. For example, variant 524C showed higher binding affinity than variants 890C, 608C, 627C, and 609C, but also showed a high predicted immunogenicity profile, whereas 890C, 608C, 627C, and 609C did not show the predicted immunogenicity profile at all. Furthermore, potency against PACAP is also not predictive of favorable immunogenicity and developability properties. As an example, variant 519C showed higher potency than variants 890C, 608C, 627C, and 609C, but also showed a high predicted immunogenicity profile, whereas 890C, 608C, 627C, and 609C did not show the predicted immunogenicity profile at all. As an example, the predicted immunogenicity profile was measured by the assay described in Example 3.

[0250] Example 2: Humanity of anti-PACAP antibodies This example illustrates the humanness of the antibodies provided in this disclosure.

[0251] Increasing the humanness of variable region sequences of monoclonal antibodies that are potential therapeutic candidates is an important approach to minimize potential immunogenicity. The anti-PACAP antibodies of the present disclosure were engineered to obtain a high humanness score without compromising the high affinity and high potency described herein. The humanness scores, i.e., similarity to human germline sequences, were obtained using various antibody analysis platforms, including IMGT and AbGenesis. Starting with a single input variable region antibody sequence, the closest human germline to that sequence was identified, and the percentage humanness of the variable heavy and variable light chains was calculated using AbGenesis software (release 4.1). The overall humanness of the antibody was calculated based on the average percent sequence identity values ​​of the heavy and light chains.

[0252] The humanness scores of several exemplary anti-PACAP antibodies of the present disclosure and several known anti-PACAP antibodies are shown in Table 15. [Table 15]

[0253] As shown above, the anti-PACAP antibodies (e.g., 608C, 627C, 609C, 604C, 605C, and 890C) all have at least 89% humanness, which is higher than the known anti-PACAP antibodies Ab1h described in WO2017181031 and antibody Ab10.H3 described in WO2017181039. Anti-PACAP antibodies AbC and D are described in WO2019067293. As explained in the examples below, sequences that are not identical to the germline are of particular interest for further immunogenicity prediction analysis.

[0254] Example 3: Predicted immunogenicity of anti-PACAP antibodies This example illustrates the predicted immunogenicity of the antibodies provided in this disclosure.

[0255] During the process of generating the antibodies described herein, antibodies predicted to be less immunogenic were further optimized, except for antibodies with epitopes predicted by peptide-MHC class II stability analysis.

[0256] Peptides synthesized from the heavy and light chain variable regions of the anti-PACAP antibodies provided herein, such as those described in Example 2 (Table 15), covering all non-germline residues, were incubated with recombinant MHC class II proteins. The ProImmune REVEAL® assay was used to assess the stability of peptide binding to MHC class II molecules. For example, synthetic peptides 15 residues long from the heavy and light chain sequences covering all non-germline residues were assessed for the antibodies tested.

[0257] The MHC class II alleles analyzed included all alleles with a frequency of at least 3% in the world population. Binding of peptides to MHC class II was detected by ELISA. These analyses identified predicted less immunogenic antibodies such as 608C, 609C, 627C, and 890C, for which all peptides tested showed no or less stable interactions with MHC II class proteins (Figure 7). In contrast, analyses of previously described anti-PACAP antibodies such as Ab1H, Ab10.H3, Ab B, Ab C, or Ab D all showed the predicted immunogenic epitopes.

[0258] Example 4: Kinetics of anti-PACAP antibodies This example illustrates the behavior of the antibodies provided in this disclosure.

[0259] Briefly, antibody-peptide interactions were measured using a Biacore S200 (GE Healthcare) as previously described (Andreu and Gomes 2002). Anti-human IgG Fc capture antibodies were immobilized on a CM5 biosensor chip via standard amine coupling. Anti-PACAP antibodies were injected and captured to 200-400 response units (RU). PACAP38, PACAP27, and VIP were diluted in HBS-EP+ buffer, pH 7.4, containing 0.1% w / v BSA (Bovostar, Cat. No. BSAS1.0) and 0.15 M NaCl (Sigma-Aldrich Cat. No. S7653). Antibody binding kinetics were determined by injecting two-fold serial dilutions of PACAP (12 nM-0 nM) and VIP (1200 nM-0 nM) in running buffer at 37 °C with a flow rate of 40 uL / min for 2 min, followed by a 10 min dissociation period. For subsequent cycles, the chip was regenerated with 0.85% phosphoric acid. Data were analyzed with Biacore S200 evaluation software (version 1.0). Association rate (ka) and dissociation rate (kd) constants were determined using a simple one-to-one Langmuir binding model, which was used to calculate the equilibrium dissociation constant (K D ) was calculated.

[0260] The same assay format was used to capture antibodies from the supernatant, and a single analyte injection of 33.3 nM PACAP was used to rank the off-rates of antibody variants. An exemplary Biacore-based off-rate ranking assay includes a single concentration of PACAP38 as shown in Table 16. [Table 16]

[0261] Figures 3A, 3B, and 3C show examples of binding affinity analysis of anti-PACAP antibody 605C to PACAP38, PACAP27, and VIP by SPR. Furthermore, Table 17 shows that anti-PACAP antibody 605C binds to PACAP38 and PACAP27 with high affinity and high selectivity. Since VIP has very fast on-rate and off-rate, the binding affinity at steady state was used. [Table 17]

[0262] Example 5: Anti-PACAP antibody titers This example illustrates the potency of the antibodies provided in this disclosure.

[0263] Cell-based assays for measuring PACAP-induced cAMP accumulation were performed according to previously described methods (Wang, Li et al. 2004). Specifically, inhibition of PACAP38 or PACAP27-induced signaling through PAC1, VPAC1, and VPAC2 receptors utilized CHO-K1 cell lines (Eurofins / DiscoverX) stably expressing either human PAC1, VPAC1, or VPAC2 receptors, and endpoints were determined using Promega cAMP-Glo™. Table 18 below shows the IC of several exemplary anti-PACAP antibodies provided herein. 50 To summarize: [Table 18]

[0264] Example 6: Selectivity of anti-PACAP antibodies This example illustrates the selectivity of the antibodies provided in this disclosure.

[0265] To determine the presence and extent of off-target binding, anti-PACAP antibodies, e.g., 890C, were tested against related peptides of the secretin-glucagon family (Table 19). The nine bioactive peptides of the glucagon superfamily are: PACAP, VIP, glucagon, glucagon-like peptides (GLP-1, GLP-2), growth hormone releasing factor (GRF or GHRF), peptide histidine methionine (PHM), secretin, and gastric inhibitory polypeptide (GIP). After antibody capture as described in Example 4, antibody-peptide interactions were measured by injecting 37.5 nM of each peptide diluted in 37° C. buffer at a flow rate of 50 mL / min for 2 min, followed by a dissociation phase of 4-5 min. Samples were injected in a multi-cycle manner over the freshly captured anti-PACAP antibody by regenerating the capture surface with two injections of 0.85% phosphoric acid at a flow rate of 30 μL / min. [Table 19]

[0266] As shown in Figure 4, at the stability level measured by Biacore (relative binding after 15 seconds dissociation), antibody 890C is highly selective for PACAP and does not or only minimally binds other glucagon-secretin family peptides. The affinity of 890C for PACAP38, PACAP27, and VIP, measured by SPR and expressed in pM, is 54,218 and 390,000, respectively. The selectivity of antibody 890C was assessed by comparing the inhibition of PACAP38 signaling to the inhibition of VIP signaling in a cyclic AMP (cAMP) measuring cell-based functional assay. As shown in Figure 5, antibody 890C showed >1000-fold selectivity for inhibiting PACAP38-induced cAMP production in CHO-K1 cells stably expressing the PAC1 receptor versus the inhibition of VIP-induced cAMP production in CHO-K1 cells stably expressing the VPAC1 receptor.

[0267] Example 7: Potential assessment using HPLC-SEC analysis The developability of antibody molecules can be evaluated using biophysical techniques that measure the non-specific interactions of antibody molecules. High performance liquid chromatography (HPLC) analysis of antibodies using silica-based columns or dextran-based size-exclusion columns has shown that the retention time of an antibody sample is related to its colloidal stability, with antibodies prone to precipitation or aggregation being retained longer on the column, likely due to non-specific interactions between the antibody and the column matrix (Kohli et al., (2015) mAbs, 7:4, 752-758, DOI: 10.1080 / 19420862.2015.1048410). The apparent molecular weight of an antibody can be calculated using molecular weight standards. A longer retention time is displayed as a smaller apparent molecular weight than that calculated based on the sequence.

[0268] As shown in Figure 6, the typical molecular weight of antibodies with acceptable IgG is approximately 155 kDa. Antibodies 609C and 890C eluted normally with an apparent molecular weight window of 155 kDa to 165 kDa, whereas Ab C and Ab D showed significantly elevated retention times with apparent molecular weights of approximately 95 to 105 kDa depending on the IgG isoform. These data indicate that antibodies 609C and 890C are expected to have better developability due to their appropriate colloidal stability reflected in their retention times on the column.

[0269] Example 8: Single-dose study of anti-PACAP monoclonal antibodies in cynomolgus monkeys Four biologic-naive male cynomolgus monkeys were administered a single subcutaneous dose of 10 mg / kg of anti-PACAP antibodies with the following CDRs (VH-CDR1, VH-CDR2, and VH-CDR3 sequences, and VL-CDR1, VL-CDR2, and VL-CDR3 sequences, as set forth in SEQ ID NOs: 1, 2, and 3, and SEQ ID NOs: 4, 5, and 6, respectively). Sampling was performed for pharmacokinetic and pharmacodynamic analysis, hematology, and clinical chemistry evaluation. Pharmacokinetic analysis was performed by ex vivo assays, e.g., by comparing serum samples from injected animals with pre-dose samples from the same animals. No persistent antibody-related changes in hematology, clinical chemistry, or body weight were observed. The time profile of the serum concentration of the antibody is comparable to a typical monoclonal antibody profile in cynomolgus monkeys.

[0270] While specific alternative examples of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. Accordingly, there is no intention to be limited to the precise abstract and disclosure presented herein. [Table 20-1] [Table 20-2] [Table 20-3] [Table 20-4] [Table 20-5] [Table 20-6]

Claims

1. An anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibody, comprising heavy chain variable region (VH)-CDR1, VH-CDR2, and VH-CDR3 sequences set forth in SEQ ID NOs: 9, 2, and 3, respectively, and light chain variable region (VL)-CDR1, VL-CDR2, and VL-CDR3 sequences set forth in SEQ ID NOs: 10, 5, and 6, respectively.

2. An anti-PACAP antibody, wherein the antibody or antigen-binding fragment thereof is a) SEQ ID NOs: 1, 2, and 3, respectively; b) SEQ ID NOs: 7, 2, and 3, respectively; and c) variants of a)-b) containing one, two, or three conservative amino acid substitutions; VH-CDR1, VH-CDR2, and VH-CDR3 sequences selected from the group consisting of: d) SEQ ID NOs: 4, 5, and 6, respectively; e) SEQ ID NOs: 8, 5, and 6, respectively; and f) variants of d) to e) containing one, two, or three conservative amino acid substitutions; and VL-CDR1, VL-CDR2, and VL-CDR3 sequences selected from the group consisting of:

3. An anti-PACAP antibody, wherein the antibody comprises a VH sequence derived from SEQ ID NO: 19, wherein the VH sequence comprises a valine (V) or leucine (L) at residue 32 according to Kabat numbering, and the antibody comprises a VL sequence derived from SEQ ID NO: 22, wherein the VL sequence comprises an alanine (A) at residue 27E according to Kabat numbering, and a tryptophan (W) and an alanine (A) at residues 50 and 51 according to Kabat numbering.

4. The antibody of claim 3, further comprising a cysteine-alanine-isoleucine (CAI) in VH at residues 92-94 according to Kabat numbering.

5. Human gene IGHV1-69, respectively ＊ 01 and IGKV4-1 ＊ The antibody of any one of claims 1 to 4, comprising heavy and light chain framework region (FR) sequences derived from 01, and functional variants thereof.

6. 5. The antibody of claim 1, comprising heavy chain framework region (VHFR)-1, VHFR-2, VHFR-3, and VHFR-4 sequences set forth in SEQ ID NOs: 29 to 32, respectively, and light chain framework region (VLFR)-1, VLFR-2, VLFR-3, and VLFR-4 sequences set forth in SEQ ID NOs: 33 to 36, respectively.

7. 5. The antibody of claim 1, comprising a VH sequence that is about 90%, about 95%, about 99%, or 100% identical to a sequence selected from SEQ ID NOs: 11 and 12, and a VL sequence that is about 90%, about 95%, about 99%, or 100% identical to a sequence selected from SEQ ID NOs: 20 and 21.

8. a) SEQ ID NOs: 1, 2, and 3, and SEQ ID NOs: 4, 5, and 6, respectively; b) SEQ ID NOs: 7, 2, and 3, and SEQ ID NOs: 8, 5, and 6, respectively; c) SEQ ID NOs: 1, 2, and 3, and SEQ ID NOs: 8, 5, and 6, respectively; and d) SEQ ID NOs: 7, 2, and 3, and SEQ ID NOs: 4, 5, and 6, respectively; an anti-PACAP antibody comprising VH-CDR1, VH-CDR2, and VH-CDR3 sequences, and VL-CDR1, VL-CDR2, and VL-CDR3 sequences selected from the group consisting of:

9. a) SEQ ID NOs: 11 and 20, respectively; b) SEQ ID NOs: 12 and 21, respectively; c) SEQ ID NOs: 11 and 21, respectively; and d) SEQ ID NOs: 12 and 20, respectively; 9. The antibody of any one of claims 1 to 4 and 8, comprising a VH sequence and a VL sequence that are about 90%, about 95%, about 99%, or 100% identical to a sequence selected from the group consisting of:

10. 9. The antibody of any one of claims 1 to 4 and 8, comprising a VH sequence of SEQ ID NO: 11 or SEQ ID NO: 12, wherein the VH sequence has a glutamine (Q) instead of glutamic acid (E) at residue 1 of SEQ ID NO: 11 or 12.

11. The antibody of any one of claims 1 to 4 and 8, comprising a heavy chain constant region sequence selected from the group consisting of SEQ ID NOs: 37 to 51 or 76, and a light chain constant region sequence selected from SEQ ID NOs: 23 and 24.

12. The antibody of any one of claims 1 to 4 and 8, wherein the antibody is human or humanized.

13. The antibody according to any one of claims 1 to 4 and 8, wherein the antibody is a humanized antibody.

14. The antibody of any one of claims 1 to 4 and 8, which has a low or no immunogenicity profile.

15. 9. The antibody of any one of claims 1 to 4 and 8, having a humanness score of about 89% or greater.

16. Approximately 5 × 10 as measured by SPR at 37 °C －１１ The antibody of any one of claims 1 to 4 and 8, having a KD of 1 molar (M) or less.

17. 9. The antibody according to any one of claims 1 to 4 and 8, wherein the antibody or antigen-binding fragment thereof has a chromatin density of about 3 x 10 when measured by SPR at 37°C. －１１ The antibody has a KD of 1 molar (M) or less.

18. The antibody of any one of claims 1 to 4 and 8, which is an antigen-binding fragment or a full-length antibody.

19. Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab') ３ , tetrabodies, triabodies, diabodies, single domain antibodies, DVD-Ig, Fcab, mAb ２ 9. The antibody of any one of claims 1 to 4 and 8, comprising (scFv)2, (scFv)3, or scFv-Fc.

20. The antibody according to any one of claims 1 to 4 and 8, wherein the constant region is an IgG constant region.

21. The antibody of claim 20, wherein the IgG constant region is an IgG1 constant region or an IgG4 constant region.

22. 22. The antibody of claim 21, wherein the constant region comprises a human IgG1 sequence set forth in SEQ ID NO: 37, a human IgG1 FAB TAG sequence set forth in SEQ ID NO: 38, a human IgG1 KiH Hole sequence set forth in SEQ ID NO: 39, a human IgG1 KiH Knob sequence set forth in SEQ ID NO: 40, a human IgG1 (L235A, G237A) sequence set forth in SEQ ID NO: 41, a human IgG1 YTE sequence set forth in SEQ ID NO: 42, a human IgG1 (L235A, G237A, YTE) sequence set forth in SEQ ID NO: 76, a human IgG2 DASS sequence set forth in SEQ ID NO: 43, a human IgG4 sequence set forth in SEQ ID NO: 44, a human IgG4 KiH Hole sequence set forth in SEQ ID NO: 45, or a human IgG4 KiH Knob sequence set forth in SEQ ID NO:

46. the antibody, comprising a sequence selected from the group consisting of a human IgG4 (L235A, G237A) sequence set forth in SEQ ID NO: 47, a human IgG4 (L235E) sequence set forth in SEQ ID NO: 48, a human IgG4 YTE sequence set forth in SEQ ID NO: 49, a human IgG4 YTE KiH Hole sequence set forth in SEQ ID NO: 50, and a human IgG4 YTE KiH Knob sequence set forth in SEQ ID NO:

51.

23. The antibody according to any one of claims 1 to 4 and 8, wherein the heavy chain is an IgG2 heavy chain.

24. The antibody of any one of claims 1 to 4 and 8, wherein the heavy chain comprises the IgG2DASS sequence set forth in SEQ ID NO:

43.

25. The antibody of any one of claims 1 to 4 and 8, wherein the light chain is a human kappa light chain or a human lambda light chain.

26. The antibody according to any one of claims 1 to 4 and 8, a) SEQ ID NOs: 70 and 71, respectively; b) SEQ ID NOs: 74 and 71, respectively; c) SEQ ID NOs: 75 and 71, respectively; d) SEQ ID NOs: 72 and 73, respectively; e) SEQ ID NOs: 70 and 73, respectively; and f) SEQ ID NOs: 72 and 71, respectively; The antibody described above, comprising a full-length heavy chain sequence and a full-length light chain sequence that are about 90%, about 95%, about 99%, or 100% identical to a sequence selected from the group consisting of:

27. The antibody of any one of claims 1 to 4 and 8, wherein the heavy chain sequence has a glutamine (Q) instead of a glutamic acid (E) at residue 1 of SEQ ID NOs: 70, 72, 74, and 75.

28. The antibody according to any one of claims 1 to 4 and 8, wherein the antibody is an antagonist of PACAP.

29. The antibody according to any one of claims 1 to 4 and 8, wherein the antibody specifically binds to PACAP.

30. A nucleic acid encoding the antibody according to any one of claims 1 to 4 and 8.

31. A vector comprising the nucleic acid of claim 30.

32. 32. An engineered cell comprising the vector of claim 31.

33. 33. A method of producing an antibody, comprising culturing the engineered cell of claim 32 under conditions sufficient for the cell to produce the antibody.

34. A pharmaceutical composition comprising the antibody of any one of claims 1 to 4 and 8 and a pharmaceutically acceptable carrier.

35. 10. A pharmaceutical composition for treating or preventing a condition in an individual, comprising the antibody of any one of claims 1 to 4 and 8, wherein the condition is selected from the group consisting of headache (e.g., migraine, cluster headache, treatment-refractory migraine), anxiety, depression, PTSD, a co-occurring condition (e.g., anxiety / depression / PTSD) with headache (e.g., migraine, cluster headache, treatment-refractory migraine), an anxiety disorder co-occurring with migraine, complex regional pain syndrome, and rosacea.

36. 36. The pharmaceutical composition of claim 35, wherein the headache is selected from the group consisting of migraine with aura, migraine without aura, hemiplegic migraine, cluster headache, migrainous neuralgia, chronic headache, episodic migraine, chronic migraine, medication overuse headache, and tension-type headache.

37. 10. A pharmaceutical composition for treating or preventing migraine in an individual, comprising the antibody of any one of claims 1 to 4 and 8, wherein the individual is unresponsive to two to four available prophylactic medications.

38. 38. The pharmaceutical composition of claim 37, wherein the individual is unresponsive to two to four available prophylactic medications selected from the group consisting of divalproex, sodium valproate, valproate, valproic acid, topiramate, gabapentin, propranolol, timolol, atenolol, metoprolol, nadolol, bisopropol, flunarizine, amitriptyline, nortriptyline, doxepin, fluoxetine, and candesartan.

39. 10. A pharmaceutical composition for treating or preventing migraine in an individual, comprising the antibody of any one of claims 1 to 4 and 8, wherein the individual is unresponsive to two to four available prophylactic drug classes.

40. 40. The pharmaceutical composition of claim 39, wherein the class of prophylactic agent is selected from the group consisting of antiepileptics, beta-blockers, tricyclic antidepressants, calcium channel blockers, angiotensin II receptor antagonists, botulinum toxin, and CGRP pathway monoclonal antibodies.

41. the classes of prophylactic agents are selected from different clusters, and the clusters are Cluster A: Antiepileptic drugs Cluster B: Beta-blockers Cluster C: Tricyclic antidepressants Cluster D: Calcium channel blockers Cluster E: Angiotensin II receptor antagonists Cluster F, botulinum toxin, and 40. The pharmaceutical composition of claim 39, defined as follows: Cluster G: calcitonin gene-related peptide (CGRP) pathway monoclonal antibodies.

42. A pharmaceutical composition for use in a method for treating or preventing migraine in an individual, comprising an antibody according to any one of claims 1 to 4 and 8, said method comprising the steps of selecting an individual who is unresponsive to two to four applicable prophylactic agents or classes of prophylactic agents, and administering said pharmaceutical composition to said individual.

43. 10. A pharmaceutical composition for treating or preventing migraine in an individual who is unresponsive to a CGRP pathway monoclonal antibody, the pharmaceutical composition comprising an antibody according to any one of claims 1 to 4 and 8.

44. 44. The pharmaceutical composition of claim 43, wherein the CGRP pathway monoclonal antibody comprises an anti-CGRP antibody, an anti-CGRP-R antibody, or both.

45. 44. The pharmaceutical composition of claim 43, wherein the anti-CGRP antibody is selected from fremanezumab, galcanezumab, eptinezumab, or a combination thereof.

46. A composition comprising the antibody according to any one of claims 1 to 4 and 8.

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