Compositions and methods relating to anti-PACAP IgG4 antibodies

Abstract

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide involved in a wide range of functions, including nociception and primary headaches. PACAP is a member of the secretin / vasoactive intestinal peptide (VIP) / growth hormone-releasing hormone (GHRH) family. The present disclosure generally relates to anti-PACAP antibodies, pharmaceutical compositions comprising such antibodies, and methods for producing and using such antibodies. For example, the present invention provides an anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibody, comprising the full-length heavy chain amino acid sequence set forth in SEQ ID NO:1 and the full-length light chain amino acid sequence set forth in SEQ ID NO:2.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 482,680, filed February 1, 2023, the entire disclosure of which, including any drawings, is incorporated herein by reference.

[0002] Field The present disclosure relates generally to anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibodies, pharmaceutical compositions containing such antibodies, and methods of producing and using such monoclonal antibodies.

[0003] Incorporation of sequence listings

[0001] This application contains a Sequence Listing. The material in the attached Sequence Listing is incorporated herein by reference. The attached Sequence Listing XML file, named "2024-01-25 Sequence_Listing_ST26 035680-504001WO.xml", was created on January 25, 2024, and is 18,407 bytes. [Background technology]

[0004] background 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. PACAP / VIP receptors, PAC1, VPAC1, and VPAC2, are present in sensory neurons and vascular smooth muscle associated with the trigeminovascular system. The PAC1 receptor binds PACAP with high affinity and VIP with much lower affinity. The VPAC1 and VPAC2 receptors recognize PACAP and VIP equally well.

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

[0006] To our current knowledge, PACAP, but not VIP, is involved in conditions such as migraine, cluster headache, and post-traumatic stress disorder (PTSD). For example, in accordance with findings in experimental models of trigeminal activation, elevated plasma levels of PACAP have been documented in acute migraine attacks and cluster headaches. This suggests that activation of the trigeminal nervous system may result in elevated intravenous levels of PACAP. This elevated intravenous level of PACAP can be reduced by treating headache. Furthermore, in patients suffering from migraine or cluster headache, injection of PACAP induces either migraine or cluster headache. Furthermore, serum levels of PACAP have been associated with PTSD diagnosis and symptom severity in female patients.

[0007] Anti-PACAP antibodies can be useful in the treatment of 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.

[0008] The present disclosure provides anti-PACAP antibodies that solve problems and meet needs in the art. Summary of the Invention [Means for solving the problem]

[0009] overview In at least one embodiment, the present disclosure provides an anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibody, the antibody comprising a full-length heavy chain amino acid sequence set forth in SEQ ID NO:1 and a full-length light chain amino acid sequence set forth in SEQ ID NO:2.

[0010] In at least one embodiment, the present disclosure provides an anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibody, the antibody comprising a full-length heavy chain amino acid sequence set forth in SEQ ID NO:3 and a full-length light chain amino acid sequence set forth in SEQ ID NO:2.

[0011] In at least one embodiment, the present disclosure provides an anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibody comprising the full-length heavy chain amino acid sequence set forth in SEQ ID NO:1 and the full-length light chain amino acid sequence set forth in SEQ ID NO:2, wherein the antibody is produced by a method comprising introducing into a host cell a vector comprising a nucleic acid encoding the full-length heavy chain amino acid sequence set forth in SEQ ID NO:1 and the full-length light chain amino acid sequence set forth in SEQ ID NO:2, culturing the host cell under conditions sufficient for the cell to produce the antibody, and purifying the antibody from the cell culture.

[0012] In at least one embodiment, the present disclosure provides an anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibody comprising the full-length heavy chain amino acid sequence set forth in SEQ ID NO:3 and the full-length light chain amino acid sequence set forth in SEQ ID NO:2, wherein the antibody is produced by a method comprising introducing into a host cell a vector comprising a nucleic acid encoding the full-length heavy chain amino acid sequence set forth in SEQ ID NO:3 and the full-length light chain amino acid sequence set forth in SEQ ID NO:2, culturing the host cell under conditions sufficient for the cell to produce the antibody, and purifying the antibody from the cell culture.

[0013] In at least one embodiment, the antibody is produced at a titer of at least 2 g / L.

[0014] In at least one embodiment, the antibody is a human antibody or a humanized antibody. In at least one embodiment, the antibody is a humanized antibody.

[0015] In at least one embodiment, the antibody is an antagonist of PACAP.

[0016] In at least one embodiment, the antibody specifically binds to PACAP.

[0017] In at least one embodiment, the present disclosure provides nucleic acids encoding the antibodies described herein.

[0018] In at least one embodiment, the present disclosure provides a vector comprising a nucleic acid described herein.

[0019] In at least one embodiment, the present disclosure provides a method for producing an anti-PACAP antibody having a full-length heavy chain amino acid sequence set forth in SEQ ID NO: 1 or 3 and a full-length light chain amino acid sequence set forth in SEQ ID NO: 2, the method comprising the steps of introducing into a host cell a vector containing a nucleic acid encoding the full-length heavy chain amino acid sequence set forth in SEQ ID NO: 1 or 3 and the full-length light chain amino acid sequence set forth in SEQ ID NO: 2, culturing the host cell under conditions sufficient for the cell to produce the antibody, and purifying the antibody from the cell culture.

[0020] In at least one embodiment, the antibody is produced at a titer of at least 2 g / L.

[0021] In at least one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody described herein and a pharmaceutically acceptable carrier.

[0022] In at least one embodiment, the present disclosure provides a liquid pharmaceutical composition comprising an anti-PACAP antibody having a full-length heavy chain amino acid sequence set forth in SEQ ID NO: 1 or 3 and a full-length light chain amino acid sequence set forth in SEQ ID NO: 2, a pharmaceutically acceptable carrier, and water for injection.

[0023] In at least one embodiment, the pH of the pharmaceutical composition is from about 6.5 to about 8.0.

[0024] In at least one embodiment, the present disclosure provides a pre-filled syringe or pre-filled autoinjector containing a liquid pharmaceutical composition comprising an anti-PACAP antibody having a full-length heavy chain amino acid sequence set forth in SEQ ID NO: 1 or 3 and a full-length light chain amino acid sequence set forth in SEQ ID NO: 2.

[0025] In at least one embodiment, the pH of the pharmaceutical composition is from about 6.5 to about 8.0.

[0026] In at least one embodiment, the present 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 present disclosure, wherein the condition is selected from the group consisting of headache (e.g., migraine, cluster headache, treatment-refractory migraine), anxiety, depression, PTSD, a condition (e.g., anxiety / depression / PTSD) coexisting with headache (e.g., migraine, cluster headache, treatment-refractory migraine), an anxiety disorder coexisting with migraine, complex regional pain syndrome, and 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, migraine-like neuralgia, chronic headache, episodic migraine, chronic migraine, medication-overuse headache, and tension-type headache.

[0027] In at least one embodiment, the present disclosure provides a method of treating or preventing migraine in an individual, the method comprising administering to the individual a therapeutically effective amount of an antibody or pharmaceutical composition of the present disclosure.

[0028] In at least one embodiment, the present 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 present disclosure, wherein the individual has failed to respond to two to four available preventive drugs. In at least one embodiment, the individual has failed to respond to two to four available preventive drugs 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.

[0029] In at least one embodiment, the present 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 present disclosure, wherein the individual has failed to respond to two to four available preventative drug classes. In at least one embodiment, the preventative drug classes are 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 preventative drug classes are selected from different clusters, 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.

[0030] In at least one embodiment, the individual fails to respond 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.

[0031] In at least one embodiment, the present disclosure provides a method of treating or preventing migraine in an individual, the method comprising the steps of selecting an individual who has failed to respond 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.

[0032] In at least one embodiment, the present disclosure provides a method for treating or preventing migraine in an individual who is unable to respond to a CGRP pathway monoclonal antibody, comprising administering to the 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.

[0033] In at least one embodiment, the present disclosure provides a composition for use in accordance with the present disclosure.

[0034] Each of the aspects and embodiments described herein can be used together unless expressly or explicitly excluded from the context of the embodiment or aspect. [Brief explanation of the drawings]

[0035] [Figure 1]Figure 1 shows an alignment of human PACAP38, PACAP27, and VIP polypeptide sequences. "*" indicates well-conserved residues. "." indicates conservation between residues with weakly similar properties, and ":" indicates conservation between residues with strongly similar properties.

[0036] [Figure 2] Figure 2 shows the amino acid sequences of the anti-PACAP antibody 890C heavy chain IgG4 and 890C light chain. CDRs are underlined (all CDRs are defined according to the Kabat definition, except for heavy chain CDR-1, which is defined by AbM). The constant regions are underlined with a dotted line. The C-terminal lysine deletion (Δ447K; EU Fc numbering) is marked with an asterisk.

[0037] [Figure 3] Figure 3 shows the amino acid sequences of the anti-PACAP antibody 890C heavy chain IgG4 YTE and 890C light chain. CDRs are underlined (all CDRs are defined according to the Kabat definition, except for heavy chain CDR-1, which is defined by AbM). The constant regions are underlined with a dotted line. The M252Y, S254T, and T256E substitutions (EU Fc numbering) in the heavy chain constant region are double-underlined. The C-terminal lysine deletion (Δ447K; EU Fc numbering) is marked with an asterisk.

[0038] [Figure 4A-B] 4A-4C show exemplary results of the binding affinity of anti-PACAP antibody 605C to PACAP38, PACAP27, and VIP as measured by SPR at 37°C. [Figure 4C] 4A-4C show exemplary results of the binding affinity of anti-PACAP antibody 605C to PACAP38, PACAP27, and VIP as measured by SPR at 37°C.

[0039] [Figure 5]FIG. 5 shows exemplary experimental SPR data for the binding of anti-PACAP antibody 890C to PACAP38, PACAP27, VIP and other glucagon-secretin family peptides.

[0040] [Figure 6] FIG. 6 shows exemplary experimental data on the selectivity of anti-PACAP antibody 890C's activity towards PACAP38 compared to VIP in a cell-based cyclic AMP induction assay.

[0041] [Figure 7] FIG. 7 shows the apparent molecular weights of some exemplary anti-PACAP antibodies provided herein and other anti-PACAP antibodies in various isotype formats.

[0042] [Figure 8] FIG. 8 shows a summary of the predicted immunogenicity profiles of the variable regions of 608C, 609C, 627C, 890C and other anti-PACAP antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0043] Detailed Description The present disclosure relates to anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibodies, particularly antibodies that have high affinity for PACAP and low immunogenicity when administered to humans. In humans, PACAP is produced from a 176-amino acid precursor protein encoded by the ADCYAP1 gene. Two isoforms of PACAP naturally exist: 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: 4). PACAP27 is an 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: 5). Both PACAP38 and PACAP27 show high sequence similarity to vasoactive intestinal peptide (VIP). The sequence of VIP is HSDAVFTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 6). 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 have low or no binding affinity to VIP.

[0044] The present disclosure also provides compositions and methods useful for producing such antibodies, nucleic acids encoding such antibodies, and methods for treating or preventing various conditions, such as, for example, migraine, refractory migraine, etc.

[0045] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols generally identify like components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not intended to be limiting. Other alternatives 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 drawings may 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. definition

[0046] Unless otherwise defined, all technical terms, notations, and other scientific terms or terminology used herein shall have the meaning commonly understood by those skilled in the art to which this disclosure belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood by those skilled in the art and are commonly utilized using conventional methodologies.

[0047] "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, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens more rapidly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, an example of which is an affinity ELISA assay. Furthermore, affinity can be determined by surface plasmon resonance assay (SPR, e.g., a BIAcore®-based assay). Using this methodology, the association rate constant (ka, M -1 s -1 units) and dissociation rate constant (kd, s -1 The equilibrium dissociation constant (KD, in M ​​units) can then be calculated from the ratio of the kinetic rate constants (kd / ka). Binding affinity can also be determined using kinetic methods such as the Kinetic Exclusion Assay (KinExA) described by Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008. When the KinExA assay is used, the equilibrium dissociation constant (KD, in M ​​units) and the association rate constant (ka, M -1 s -1 unit) 1 The dissociation rate constant (kd, s -1 The binding affinity (units) can be calculated from these values ​​(KD x ka). Binding affinity can also be determined by the equilibrium / solution method.

[0048] "Specifically binds" generally means that an antibody binds to an epitope via its antigen-binding domain and that binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody "specifically binds" to an epitope if it binds to that epitope more readily via its antigen-binding domain than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" can be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind epitope "C" with higher specificity than it does to a related epitope "D." With specific 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, antibodies that bind to PACAP27 and PACAP38 more readily than they bind to VIP.

[0049] 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 found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0050] The term "derived from," as used herein with respect to a protein or polypeptide, refers to origin or source and can include naturally occurring, recombinant, unpurified, or purified polypeptides obtained from or based on a source or original protein or polypeptide. Thus, a protein or polypeptide derived from an original protein or polypeptide may comprise the original protein or polypeptide in part or in its entirety, or 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.

[0051] The terms "administer," "administration," "administering," and the like, as used herein, refer to the delivery of a composition or formulation or drug, such as an anti-PACAP antibody disclosed herein, by a route of administration including, but not limited to, intravenous, intraarterial, intracranial, intramuscular, intraperitoneal, subcutaneous, intramuscular, or a combination thereof. This term encompasses, but is not limited to, administration by a medical professional and self-administration.

[0052] As used herein, "treatment," "treating," and "to treat" refer to improving at least one or more symptoms, even if not necessarily ameliorating. For example, these terms refer to utilizing an approach to obtain a beneficial or desired clinical result (including, but not limited to, an approach to achieve such a beneficial or desired clinical result), where clinical results may include therapeutic measures that improve, cure, slow, alleviate the symptoms, and / or halt the progression of a pathological condition or disorder. Those in need of treatment may include those already diagnosed with or suspected of having a disorder. As used herein, and unless otherwise specified, a "therapeutically effective amount" of an agent or drug, 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 refers to the amount of a therapeutic agent alone, or the amount of a therapeutic agent in combination with other therapeutic agents, that provides a therapeutic benefit in the treatment or management of a disease. The term "therapeutically effective amount" can encompass an amount that improves overall treatment of a disease, reduces or avoids the symptoms or causes of a disease, or enhances the therapeutic effectiveness 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 or symptoms of a disease, which may also be 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 skilled in the art using known techniques.

[0053] As used herein, "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 under the care of a physician. Thus, a subject may be a human patient who has, is at risk of, or is suspected of having a disease of interest (e.g., migraine, refractory migraine, etc.) and / or one or more symptoms of the disease. A subject may also be an individual who has been diagnosed at or after diagnosis as being at risk for a condition of interest. For example, a subject may be further characterized as being at risk for developing a condition described herein or a condition for which reducing PACAP activity would be beneficial.

[0054] 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 transfers or passages in culture. It should be understood that not all progeny will be exactly identical to the parent cell. This is because certain 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), and therefore the progeny may not actually be identical to the parent cell, but still be within the scope of the term as used herein, so long as the progeny retains the same functionality as the original cell, cell culture, or cell line.

[0055] The term "operably linked" as used herein refers to a physical or functional linkage between two or more elements, such as polypeptide sequences or polynucleotide sequences, that allows them to function in their intended manner. For example, 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, the term "operably linked" means that the orthogonal DNA target sequence and the promoter are in-frame and appropriately spaced and distanced relative to the polynucleotide of interest encoding the protein or RNA, so that binding of the transcription factor or RNA polymerase, respectively, can affect transcription.

[0056] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" encompasses one or more cells, including mixtures thereof. "A and / or B" is used herein to encompass all of the following alternatives: "A," "B," "A or B," and "A and B."

[0057] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, of that range, and any other stated value or intervening value within that stated range, is encompassed within 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, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0058] All ranges disclosed herein encompass any and all possible subranges and combinations of subranges. Any range described can be understood to fully describe and allow for division of the same range into at least 2, 3, 4, 5, 10, etc. divisions. As a non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. As will be understood by those skilled 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 can be subsequently divided into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to groups having 1, 2, 3, 4, or 5 items, and so on.

[0059] It is understood that certain features of the present disclosure, which are described for clarity in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are described for brevity in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments of the present disclosure are specifically embraced by the present disclosure, and each and every combination is herein disclosed as if individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure, and each and every such subcombination is herein disclosed as if individually and expressly disclosed. composition

[0060] 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 VIP receptors, VPAC1 and VPAC2. In certain embodiments, the anti-PACAP antibodies provided herein are engineered by humanization to improve the percentage of human sequence. Furthermore, the anti-PACAP antibodies provided herein have their CDR3 sequences engineered to improve affinity, efficacy, or both. In some embodiments, the anti-PACAP antibodies provided herein can be engineered to eliminate immunogenicity and reduce manufacturing inconvenience. Furthermore, the anti-PACAP antibodies provided herein are engineered to achieve potent inhibition of PACAP-38 and / or PACAP-27-induced cyclic adenosine monophosphate (cAMP) production, as previously described by Wang, Li et al. 2004.

[0061] As described in more detail below, the antibodies provided herein present low or no potential immunogenicity risk when administered to a subject. As used herein, "low or no potential immunogenicity risk" refers to the inability of a therapeutic antibody to induce the formation of anti-drug antibodies (ADAs) when administered to a subject in sufficient amounts. ADAs are antibodies produced by the immune system against therapeutic drugs, which can reduce the efficacy of the drug and, more importantly, can cause adverse effects ranging from injection site rashes to potentially fatal systemic inflammatory reactions. In certain embodiments, the antibodies provided herein present low or no potential immunogenicity risk when administered to humans. For administration to humans, low or no potential immunogenicity risk can be achieved, for example, by engineering the antibody to have a higher humanity score and by removing residues that have been found to have a higher potential immunogenicity risk using predictive in vitro techniques. As used herein, "humanity score" refers to the percent sequence identity of an antibody to human germline sequences.

[0062] Those skilled in the art will readily understand how to calculate the sequence identity between an antibody and a human germline. For example, an anti-PACAP antibody has a high humanity score, for example, a humanity score greater than or equal to about 89%. Furthermore, the anti-PACAP antibody has a strong affinity for PACAP. For example, some of the anti-PACAP antibodies provided herein have a humanity 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 x 10 as measured by SPR at 37°C. -11 has a KD that is less than or equal to the molar concentration (M). In some embodiments, the anti-PACAP antibody preferentially binds to PACAP, including PACAP-38 and PACAP-27. antigen binding molecule

[0063] As used herein, antibody has its general meaning in the art and refers to an immunoglobulin molecule that recognizes and specifically binds to a target epitope through at least one antigen-binding domain in its variable region.The target can be a peptide, for example, a PACAP peptide.The antibody of the present disclosure encompasses full-length antibodies (including full-length polyclonal antibodies and full-length monoclonal antibodies).The antibody of the present disclosure is an IgG4 antibody.

[0064] The antibodies 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 heavy and light chain variable regions each consist of four framework regions (FR) connected to 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, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. At least two techniques exist for determining CDRs: (1) methods based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda, Md.)); and (2) methods 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 modeling software and North numbering convention (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256). (2011)). Furthermore, a combination of these two techniques is sometimes used to determine CDRs in the art.

[0065] The anti-PACAP antibodies provided herein are full-length antibodies. Full-length antibodies may comprise four polypeptide units consisting of two identical heavy chains and two identical light chains, held together 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 heavy chain constant region. In some embodiments, carbohydrates may typically be attached to the CH2 domain of the heavy chain. In addition, full-length antibodies may also contain a fragment crystallizable (Fc) region. The Fc region contains only the constant region (CH2 and CH3) from the heavy chain. In contrast, the antigen-binding fragment (Fab) can contain both the constant and variable domains of both the heavy and light chains (VH, VL, CH1 and CL), whereas the variable fragment (Fv) contains only the two variable domains.

[0066] As described above, the antibodies of the present disclosure comprise one or more constant regions. The term "constant region" of an antibody is a well-known term in the art and refers to a portion of an antibody whose amino acid sequence is relatively constant among different molecules. Generally, the heavy chain constant region is composed of three distinct 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. The terminology and structural features of the constant region of an antibody will be readily understood by those skilled in the art.

[0067] In certain embodiments, the anti-PACAP antibodies of the present disclosure are blocking antagonist antibodies and inhibit or reduce the biological activity of PACAP. In some embodiments, blocking or antagonist antibodies substantially or completely inhibit 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 antibodies of the present disclosure to antagonize PACAP can be measured, for example, in a cell-based assay that monitors ligand-induced cyclic adenosine monophosphate (cAMP) production. In some embodiments, the anti-PACAP antibodies of the present disclosure antagonize PACAP-induced activation of human PAC1, VPAC1, and / or VPAC2 receptors. Various assays for assessing the 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 Wang, Li et al. 2004.

[0068] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to a portion of an antigen that can be recognized and specifically bound by a specific antibody. When an antigen is a polypeptide, an epitope can be formed by either consecutive amino acids or non-consecutive amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by consecutive amino acids are generally retained even when the protein is denatured, while epitopes formed by tertiary folding are generally lost when the protein is denatured.

[0069] The antibody provided herein may be a monoclonal antibody. A "monoclonal antibody" refers to a homogeneous antibody population that is involved in highly specific recognition and binding to a single antigenic determinant, or epitope. This is in contrast to a polyclonal antibody, which generally contains different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses full-length monoclonal antibodies. Furthermore, "monoclonal antibody" refers to such antibodies produced in any number of ways, including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0070] The antibody encompassed by the present disclosure can be a human antibody, a non-human antibody, a humanized antibody, a mouse antibody, a chimeric antibody, or a resurfacing antibody. In some embodiments, the antibody of the present disclosure can be a humanized antibody. 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 this monoclonal antibody, generally amino acid residues from the non-antigen-recognizing portion of the antibody, are modified to be homologous to the corresponding residues in the human antibody of the corresponding isotype. Humanization can be performed, for example, by replacing at least a portion of a rodent or rabbit variable region of a human antibody using various methods (see, e.g., U.S. Pat. Nos. 5,585,089 and 5,693,762; Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239: 1534-1536).

[0071] The Kabat numbering system is commonly used when referring to residues in the variable region (approximately residues 1-107 of the light chain and residues 1-113 of 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 similar to that in Kabat refers to the numbering system used for the heavy or light chain variable regions of the antibody compilations 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) as well as residue insertions after residue 82 of the heavy chain FR (e.g., residues 82a, 82b, and 82c, etc.). The Kabat numbering of residues can be determined for a given antibody by aligning the antibody sequence in the region of homology with a "standard" Kabat numbered sequence. Chothia instead refers to the location of the structural loop (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 between positions 32 and 34 in the VH depending on the length of the loop (this is because the Kabat numbering scheme allows for insertions of positions 35A and 35B in the VH; if neither 35A nor 35B is present, the end of the loop is at position 32; if only 35A is present, the end of the loop is at position 33; and if both 35A and 35B are present, the end of the loop is at position 34).The AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software.

[0072] For all antibodies provided herein, the numbering of the constant and / or variable regions may be according to IMGT® (IMGT®, 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)).

[0073] For all antibodies provided herein, the numbering of constant and / or variable domains may also be according to the "EU numbering system" (Edelman GM et al., Proc Natl Acad Sci USA, 63(1):78-85 (1969)). The complete correspondence for the human CH1, hinge, CH2, and CH3 constant regions of IGHG1 can be found in the IMGT™ database (IMGT®, 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)).

[0074] For example, the numbering of the human kappa immunoglobulin light chain constant domain (IGKC) 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 for the human CK domain can be found in the IMGT database (IMGT®, 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)).

[0075] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise a combination of the full-length heavy and light chain sequences provided in Table 1 below. [Table 1]

[0076] In some embodiments, 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%, or about 99% identical to the sequence set forth in SEQ ID NOs: 1 and 3. In some embodiments, 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%, or about 99% identical to the sequence set forth in SEQ ID NO: 2.

[0077] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise full-length heavy chain and light chain sequences that are at least about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NOs: 1 and 2, respectively.

[0078] In some embodiments, the anti-PACAP antibodies of the present disclosure comprise full-length heavy chain and light chain sequences that are at least about 80%, about 85%, about 90%, about 95%, or about 99% identical to SEQ ID NOs: 3 and 2, respectively.

[0079] In some exemplary embodiments, anti-PACAP antibodies of the present disclosure comprise the full-length heavy and light chain sequences set forth in SEQ ID NOs: 1 and 2, respectively. In some embodiments, anti-PACAP antibodies of the present disclosure comprise the full-length heavy and light chain amino acid sequences provided in Figure 2, SEQ ID NO: 1 and SEQ ID NO: 2, respectively. For example, the constant regions are underlined with dotted lines in Figure 2.

[0080] In some exemplary embodiments, anti-PACAP antibodies of the present disclosure comprise the full-length heavy and light chain sequences set forth in SEQ ID NOs: 3 and 2, respectively. In some embodiments, anti-PACAP antibodies of the present disclosure comprise the full-length heavy and light chain amino acid sequences provided in Figure 3, SEQ ID NO: 3 and SEQ ID NO: 2, respectively. For example, the constant regions are underlined with dotted lines in Figure 3.

[0081] As noted above, the present disclosure encompasses variants of any of the antibodies disclosed herein. A "variant" of a polypeptide, such as an immunoglobulin chain (e.g., VH, VL, HC, or LC), refers to a polypeptide comprising an amino acid sequence that is at least about 80-99.9% identical or similar (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%) to a reference amino acid sequence described herein, when the comparison is performed using a BLAST algorithm with algorithm parameters selected to maximize match between the respective sequences over the entire length of each reference sequence.

[0082] The term "percent identity," when 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 specified 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 over a specified region when compared and aligned for maximum correspondence over a comparison window or specified region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with the 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 encompasses sequences with deletions and / or additions, as well as sequences with substitutions. Sequence identity can be calculated over a range of amino acids. For example, sequence identity can be calculated over a region at least about 20 amino acids or nucleotides in length, or over a region 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, and FASTA (Atschul et al., J Mol Biol 215:403, 1990).Sequence identity can be measured using sequence analysis software, for example, the Sequence Analysis Software Package by the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), using its default parameters.

[0083] "Identity" has its own art-recognized meaning and can be calculated using published techniques.See, for example, 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 several methods for measuring the identity between two polynucleotide or polypeptide sequences, while 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, S. F. et al., J. Mol. Biol. 215:403 (1990)), and 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)).

[0084] A polypeptide having an amino acid sequence that is at least, for example, 95% "identical" to a query amino acid sequence of the present disclosure is intended to mean 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 changes per 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence that is 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 changes to the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, either individually interspersed among residues in the reference sequence or in one or more contiguous groups within the reference sequence. Indeed, whether any 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.

[0085] In fact, 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%, or 99.9% identical to, for example, the amino acid sequence shown in Table 1 can be routinely determined using known computer programs. Determining the best overall match between a query sequence (the sequence of the present disclosure) and a reference sequence is also referred to as global sequence alignment, and a preferred method for this can 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 the global sequence alignment is expressed as percent identity. In one embodiment of the present disclosure, the parameters used for FASTDB alignment of amino acid sequences to calculate percent identity are as follows: Matrix=PAM 0, 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.

[0086] If the reference sequence is shorter than the query sequence due to N- or C-terminal deletion, not due to internal deletion, manual correction must be made to the result.This is because the FASTDB program does not consider the N- and C-terminal truncation of the reference sequence when calculating the global percent identity.For the reference sequence that is truncated at the N- and C-terminus, the percent identity is corrected by calculating the number of residues in the query sequence that are at the N- and C-terminus of the reference sequence that are not matched / matched with the corresponding subject residues relative to the query sequence as a percentage of the total number of bases in the query sequence.Whether a residue is matched / matched is determined by the result of FASTDB sequence alignment.This percentage is then subtracted from the percent identity calculated by the above-mentioned FASTDB program using specified parameters to obtain a final percent identity score.This final percent identity score is used for the purposes of the present disclosure. Only residues at the N- and C-termini of the reference sequence that are not matched / matched with the query sequence, i.e., only query residue positions outside the farthest N- and C-terminal residues of the reference sequence, are considered for the purposes of manual adjustment of the percent identity score.

[0087] For example, a 90-amino acid reference sequence is aligned with a 100-residue query sequence to determine percent identity. There is a deletion at the N-terminus of the reference sequence, so the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of unmatched N- and C-terminal residues / total number of residues in the query sequence), so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues are perfectly matched, the final percent identity will be 90%. In another example, a 90-residue reference sequence is compared with a 100-residue query sequence. In this case, the deletion is internal, so there are no residues at the N- or C-terminus of the reference sequence that do not match / align with the query. In this case, manual correction of the percent identity calculated by FASTDB is not performed. Again, only residue positions outside the N- and C-termini of the reference sequence, as indicated by the FASTDB alignment, that are not matched / aligned with the query sequence are manually corrected.

[0088] Within the disclosed percent identity range, 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 alternative residues. In one aspect, the percent identity disclosed above is related to the entire sequence of the identified specific sequence, but should remain constant, and the amino acid residues that do not undergo variation are the amino acid residues of CDR, and the amino acid residues of framework undergo variation. For example, in one specific embodiment, where an anti-PACAP antibody of the present disclosure comprises at least one heavy chain comprising an amino acid sequence 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%, or 99.9% identical to the amino acid sequence of SEQ ID NO: 1, the CDR regions should remain constant and the framework regions are allowed to vary, provided that the overall percentage identity to SEQ ID NO: 1 falls within the scope of the embodiment. In one aspect, the variations are conservative substitutions in nature, although the present disclosure also encompasses non-conservative substitutions. Conservative substitutions, for purposes of the present disclosure, may be defined as set forth in Tables 2-4 below. Amino acids can be classified according to their physical properties and contribution to protein secondary and tertiary structure. A conservative substitution is recognized in the art as the substitution of one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are shown below. [Table 2]

[0089] Alternatively, conservative amino acids can be grouped as described in Lehninger (1975) Biochemistry, Second Edition; Worth Publishers, pp. 71-77, as follows: [Table 3]

[0090] Further alternative exemplary conservative substitutions are shown below. [Table 4] nucleic acid

[0091] As noted above, one aspect of the present disclosure pertains to recombinant nucleic acids comprising nucleic acid sequences encoding the antibodies of the present disclosure. In some embodiments, the recombinant nucleic acids of the present disclosure can be configured as expression cassettes or vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences, e.g., regulatory sequences, that allow for in vivo expression of the antibody in a host cell.

[0092] Nucleic acid molecules of the present disclosure can be of any length, including, for example, between about 1 Kb and about 50 Kb, e.g., between about 1.2 Kb and about 10 Kb, between about 2 Kb and about 15 Kb, between about 5 Kb and about 20 Kb, between about 10 Kb and about 20 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, e.g., between about 15 Kb and 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, between about 5 Kb and about 25 Kb, or between about 30 Kb and about 50 Kb.

[0093] Thus, in some embodiments, nucleic acid molecules comprising a nucleotide sequence encoding an antibody of the present disclosure are provided herein. In certain embodiments, the nucleic acid molecules provided herein comprise a nucleotide sequence encoding any of the polypeptide sequences disclosed herein, for example, those listed in Table 1. In some embodiments, the nucleotide sequence is incorporated into an expression cassette or expression vector. Those skilled in the art will understand that an expression cassette generally comprises a genetic material construct containing an antibody coding sequence and sufficient regulatory information to direct the appropriate transcription and / or translation of the coding sequence in recipient cells in vivo and / or ex vivo. Generally, the expression cassette can be inserted into a vector for targeting to a desired host cell and / or individual. Thus, in some embodiments, an expression cassette of the present disclosure comprises an antibody coding sequence operably linked to expression control elements, such as a promoter, and, if necessary, one or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0094] The expression cassette can be inserted into a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage as a linear or circular, single-stranded or double-stranded DNA or RNA polynucleotide molecule, which may be derived from any source and which is capable of genomic integration or autonomous replication, and which comprises one or more nucleic acid sequences linked in a functionally operable manner, e.g., operably linked nucleic acid molecule.

[0095] In some embodiments, the nucleic acid molecule of the present disclosure is incorporated into an expression vector. Those skilled in the art will understand that the term "vector" generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and can be used to transform host cells, for example, to introduce heterologous DNA into host cells. Thus, in some embodiments, a vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to cause replication of the inserted segment. In some embodiments, an expression vector can be an integrating vector.

[0096] 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 used broadly to refer to either a nucleic acid molecule (e.g., a transfer plasmid), which generally contains viral-derived nucleic acid elements that facilitate the transfer or integration of the nucleic acid molecule into a cellular genome, or a viral particle that mediates nucleic acid transfer. Viral particles generally contain various viral components and sometimes contain host cell components in addition to the nucleic acid(s). The term viral vector can refer to either a virus or viral particle capable of transferring a nucleic acid into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, or portions thereof, primarily derived from retroviruses. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements, including long terminal repeats, primarily derived from lentiviruses, a genus of retroviruses.

[0097] The nucleic acid sequence encoding the antibody disclosed herein can be optimized for expression in a target host cell.For example, the GC content of the sequence can be adjusted to the average level of a given cellular host, calculated by referring to the known gene expression in the host cell.Methods for optimizing codon usage are known in the art.The codon usage in the coding sequence of the antibody disclosed herein can be optimized to enhance expression in a host cell, and thus 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 specific host cell.

[0098] Also provided herein is a vector, plasmid, or virus that contains one or more nucleic acid molecules that encode any antibody disclosed herein.Nucleic acid molecules can be contained in a vector, and the vector can, for example, direct the expression of the nucleic acid molecules in the cells that are transformed / transduced with the vector.Vectors suitable for use in eukaryotic and prokaryotic cells are known in the art, are commercially available, or can be easily prepared by those skilled in the art. See, for example, 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 (both referred to herein as "Sambrook"); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (with additions 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).See 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, Ferre, 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 (with additions through 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV.

[0099] The introduction of DNA vector into cells, for example, eukaryotic cells, can be carried out by conventional transformation or transfection techniques.Suitable methods for the transformation or transfection of host cells can be found in Sambrook et al. (2012, above) and other standard molecular biology laboratory manuals, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, bullet introduction, nucleoporation, hydrodynamic impact and infection.

[0100] Viral vectors that can be used in the present disclosure include, for example, retroviral vectors, adenoviral vectors and adeno-associated viral vectors, lentiviral vectors, herpesvirus, simian virus 40 (SV40), and bovine papillomavirus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0101] For example, the antibodies disclosed herein can be produced in eukaryotic hosts, such as mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). The only important factor in selecting an expression system is that the components are compatible with each other. Those skilled in the art are able to make such a determination. Furthermore, those skilled in the art can consult P. Jones, "Vectors: Cloning Applications", John Wiley and Sons, New York, NY, 2009) for guidance in selecting an expression system.

[0102] The provided nucleic acid molecules may contain sequences that are naturally occurring or sequences that are different from naturally occurring sequences but encode the same polypeptide, for example, 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 a combination or modification of nucleotides within these types of nucleic acids.Furthermore, nucleic acid molecules may be double-stranded or single-stranded (e.g., either sense strand or antisense strand).

[0103] A nucleic acid molecule is not limited to a sequence encoding a polypeptide (e.g., an antibody), but may also include some or all of the non-coding sequences located upstream or downstream of the coding sequence (e.g., the coding sequence of an antibody). Those skilled in the art of molecular biology are familiar with conventional procedures for isolating nucleic acid molecules. If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription. Recombinant cells

[0104] The present disclosure provides methods for producing the antibodies described herein, which may include introducing into a host cell a vector containing nucleic acid encoding the full-length heavy chain amino acid sequence set forth in SEQ ID NO: 1 or 3 and the full-length light chain amino acid sequence set forth in SEQ ID NO: 2, culturing the host cell under conditions sufficient for the cell to produce the antibody, and purifying the antibody from the cell culture.

[0105] The nucleic acid of the present disclosure can be introduced into a host cell, for example, a Chinese hamster ovary (CHO) cell, to produce an engineered recombinant cell containing the nucleic acid molecule. The nucleic acid molecule (for example, DNA or RNA, including mRNA) or vector of the present disclosure can be introduced into a cell 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 for 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 molecule(s) in liposomes, lipid nanoparticle technology, biolistic injection, and direct microinjection of DNA into the nucleus. Furthermore, nucleic acid molecules can also 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 of the present disclosure can be introduced into a subject in the form of nucleic acids (e.g., DNA or RNA, including mRNA), so that the antibodies are produced by the subject's own cells. The present disclosure further provides modifications to the nucleotide sequences encoding the antibodies described herein that result in increased antibody expression, increased antibody stability, increased nucleic acid (e.g., mRNA) stability, or improved affinity or specificity of the antibody for PACAP.

[0106] Therefore, in some embodiments, nucleic acid molecules can be delivered by viral or non-viral delivery vehicles known in the art.For example, nucleic acid molecules can be stably integrated into the host genome, or can be episomal replicated, or can exist in recombinant host cells as a minicircle expression vector for transient expression.Therefore, in some embodiments, nucleic acid molecules are maintained and replicated as episomal units in recombinant host cells.In some embodiments, nucleic acid molecules are stably integrated into the genome of recombinant cells.Stable integration can be achieved by using classical random genome recombination techniques, or by using more sophisticated 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, nucleic acid molecules exist in recombinant host cells as a minicircle expression vector for transient expression.

[0107] Nucleic acid molecules can be encapsulated in viral capsids or lipid nanoparticles, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation.For example, nucleic acid can be introduced into cells by viral transduction.In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver nucleic acid to target cells by viral transduction.Several AAV serotypes have been described, and all known serotypes can infect cells from a variety of tissue types.AAV can be transduced in vivo into a wide range of species and tissues without evidence of toxicity, and also induces relatively mild innate and adaptive immune responses.

[0108] Lentivirus-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction.Lentivirus vectors offer several attractive properties as gene delivery vehicles, including: (i) sustained gene delivery due to stable integration of vector into host genome; (ii) ability to infect both dividing and non-dividing cells; (iii) wide tissue tropism, including important gene therapy and cell therapy target cell types; (iv) no viral protein expression after vector transduction; (v) ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) relatively easy system for vector manipulation and construction.

[0109] In some embodiments, host cells can be genetically engineered (e.g., transduced or transformed or transfected) with, for example, a vector construct of the present application, which can be, for example, a viral vector or vector for homologous recombination containing a nucleic acid sequence homologous to a portion of the host cell's genome, or an expression vector for expressing a polypeptide of interest. 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 set forth in SEQ ID NO: 1) and LC (e.g., the amino acid sequence set forth in SEQ ID NO: 2) can be cloned and engineered into expression vectors using known methods. The engineered immunoglobulin expression vector can then be stably transfected into the engineered cells.

[0110] In some embodiments, the engineered cell is a eukaryotic cell. In some embodiments, the engineered cell is an animal cell. In some embodiments, the animal cell is a vertebrate cell or an invertebrate cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the animal cell is a non-human animal cell. In some embodiments, the engineered cell is a non-human primate cell. 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 epidermoid larynx cells, human fibroblasts, 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 herein.

[0111] In the methods described herein, host cells are cultured under conditions sufficient for the cells to produce an antibody of the disclosure. In some embodiments, the antibody is produced at a titer of at least 2 g / L.

[0112] The methods of producing the antibodies described herein further include purifying the produced antibodies from the recombinant cells and / or the medium in which the recombinant cells were cultured. Thus, antibodies produced by the methods disclosed herein also fall within the scope of the present disclosure. Pharmaceutical Composition

[0113] The anti-PACAP antibodies or nucleic acids of the disclosure can be incorporated into compositions, including pharmaceutical compositions.

[0114] In another aspect, the antibody or 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 include pharmaceutical compositions that generally include one or more of an antibody, a nucleic acid, and a pharmaceutically acceptable excipient, such as a carrier.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.

[0115] The pharmaceutical compositions provided herein may be in any form that allows the composition to be administered to an individual. In certain embodiments, the pharmaceutical composition is suitable for human administration. The scope of the present disclosure includes anti-PACAP antibodies (e.g., those in Table 1) or pharmaceutical compositions thereof that contain a pharmaceutically acceptable carrier, but that are substantially devoid of water, such as dried, e.g., lyophilized, compositions. As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more particularly in humans. A carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Physiological saline solution and aqueous dextrose and 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 E.W. Martin. In some embodiments, the pharmaceutical composition is sterilely formulated for administration to an individual or animal (some non-limiting examples include humans or mammals). In some embodiments, the individual is a human.

[0116] In some embodiments, the anti-PACAP antibody having the full-length heavy chain amino acid sequence set forth in SEQ ID NO: 1 and the full-length light chain amino acid sequence set forth in SEQ ID NO: 2 is in the form of a liquid pharmaceutical composition comprising a pharmaceutically acceptable carrier and water for injection. In some embodiments, the pH of the liquid pharmaceutical composition is about 6.5 to 8.0.

[0117] In some embodiments, the anti-PACAP antibody having the full-length heavy chain amino acid sequence set forth in SEQ ID NO: 3 and the full-length light chain amino acid sequence set forth in SEQ ID NO: 2 is in the form of a liquid pharmaceutical composition comprising a pharmaceutically acceptable carrier and water for injection. In some embodiments, the pH of the liquid pharmaceutical composition is about 6.5 to 8.0.

[0118] In some embodiments, the pharmaceutical composition of the present disclosure is formulated to be suitable for the intended administration route to an individual.For example, the pharmaceutical composition can be formulated to be suitable for parenteral administration, intraperitoneal administration, colorectal administration, intraperitoneal administration, and intratumoral administration.In some embodiments, the pharmaceutical composition can be formulated for oral administration, rectal administration, transmucosal administration, intestinal administration, parenteral administration; intramuscular administration, subcutaneous administration, intradermal administration, intrathecal administration, direct intraventricular administration, intravenous administration, intraperitoneal administration, intranasal administration, intraocular administration, inhalation administration, insufflation administration, topical administration, dermal administration, transdermal administration, or intraarterial administration.It will be understood by those skilled in the art that the formulation should be suitable for the administration mode.

[0119] 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. The composition can be stable 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 a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants, such as sodium dodecyl sulfate. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is common to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and / or sodium chloride in the composition. Agents that delay absorption, such as aluminum monostearate and gelatin, can be included in the composition to bring about sustained absorption of the injectable composition.

[0120] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound 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 containing a basic dispersion medium and the required other ingredients from those enumerated above. method

[0121] The present disclosure further provides, inter alia, methods for treating or preventing conditions such as those described herein in an individual. In some embodiments, the present invention may include methods for treating or preventing any aspect of PACAP-related conditions, such as headache, migraine, cluster headache, and / or refractory migraine, anxiety, depression, PTSD, conditions (e.g., anxiety / depression / PTSD) coexisting with headache (e.g., migraine, cluster headache, refractory migraine), anxiety disorders coexisting with migraine, complex regional pain syndrome, and rosacea. For example, in the context of headache or migraine treatment, treatment includes reducing the severity, pain intensity (e.g., headache, i.e., head pain), and other associated symptoms, reducing the frequency of recurrence, improving the quality of life of the headache sufferer, and reducing the dosage of other medications required to treat the headache. With regard to migraine, other associated symptoms include, but are not limited to, nausea, vomiting, and hypersensitivity to light, sound, and / or movement. For cluster headaches, other accompanying symptoms include, but are not limited to, swelling under or around the eyes, excessive tearing, red eyes, nasal discharge or congestion, and facial flushing.

[0122] Further provided herein are methods for reducing symptoms of conditions such as those described herein in an individual. "Reducing" symptoms means reducing the severity or frequency of the symptom(s), or eliminating the symptom(s). Thus, as used herein, the terms "reducing the incidence," "preventing," or "prevention" refer to either reducing the severity of a particular disease, condition, symptom, or disorder (the terms disease, condition, and disorder are used interchangeably throughout this application). Reducing severity includes reducing the need for, amount of, and / or exposure to drugs and / or treatments commonly used for the condition. Reducing severity also includes reducing the duration and / or frequency of a particular condition, symptom, or disorder (e.g., delaying recurrent attacks or increasing the time until the next recurrent attack in an individual).

[0123] Furthermore, the present disclosure provides a method for ameliorating a condition, such as those described herein, in an individual. Ameliorating one or more symptoms of a condition, e.g., headache or migraine, or other PACAP-related condition described herein, refers to a reduction or improvement in one or more symptoms of the condition, e.g., headache or migraine, compared to when the anti-PACAP antagonist antibody is not administered. Amelioration can also include a shortening or reduction in the duration of the symptoms.

[0124] In some embodiments, the present disclosure further provides a method for controlling a condition such as those 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) the severity or duration of one or more symptoms of a condition, such as headache or migraine, or the frequency of headache or migraine attacks in an individual. For example, in an individual, the duration or severity of head pain or the frequency of attacks is reduced by at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to pre-treatment levels. The reduction in the duration or severity of head pain or the frequency of attacks can last for any length of time, for example, 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.

[0125] As used herein, "delaying" the onset of a condition, such as a PACAP-related condition, such as a migraine or headache, refers to prolonging, preventing, slowing, retarding, stabilizing, and / or postponing the progression of the condition or disease. This delay can be of various lengths of time, depending on the condition or disease being treated and / or the individual's history. As will be apparent to those skilled in the art, a sufficient or significant delay can encompass prevention, in that the individual does not actually experience a headache (e.g., migraine). A method for "delaying" the onset of symptoms is a method that reduces the probability of symptoms occurring within a given time frame and / or reduces the severity of symptoms within a given time frame, compared to when the method is not used. Such comparisons are generally based on clinical trials using a statistically significant number of subjects.

[0126] The "onset" or "progression" of a condition, e.g., a PACAP-related condition described herein, refers to the initial manifestation and / or subsequent progression of a disorder or a symptom or side effect of such a disorder, e.g., photophobia or photoaversion. The onset of headache or migraine can be detected and assessed using standard clinical techniques well known in the art. However, onset also refers to progression, which may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes onset, recurrence, and onset. As used herein, the "onset" or "onset" of a condition, such as headache or migraine, encompasses initial onset and / or recurrence. A condition may be, for example, a primary or 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, general headache, paroxysmal hemicrania, and hemicrania continua. Secondary headaches include, for example, headaches caused by homeostatic disorders, such as autonomic dysreflexia, anxiety disorders coexisting with migraine, and headaches caused by complex regional pain syndrome.In addition, the subject may have migraine, headache, and pain associated with a disease or condition, such as a condition selected from the group consisting of cluster headache and / or refractory migraine, anxiety, depression, PTSD, conditions (e.g., anxiety / depression / PTSD) coexisting with headache (e.g., migraine, cluster headache, refractory migraine), anxiety disorders coexisting 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, migraine-like neuralgia, chronic headache, episodic migraine, chronic migraine, medication-overuse headache, and tension-type headache.

[0127] In other embodiments, the individual may have a condition not enumerated herein, but which is believed to require treatment with the antibodies or pharmaceutical compositions described herein.

[0128] Migraine is a chronic paroxysmal neurological disorder characterized by moderate or severe headache attacks and reversible neurological and generalized 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 can occur on one or both sides of the head, can be localized to a specific location, can radiate from a single point across the head, or can have a vice-like quality. Headaches can be sharp, throbbing, or dull. Headaches can develop gradually or suddenly and can last less than an hour or for several days.

[0129] The International Headache Society (IHS) defines migraine as a recurrent headache disorder manifesting as attacks lasting 4 to 72 hours. Typical headache characteristics include unilateral location, a pulsating nature, moderate or severe intensity, exacerbation 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 IHS Headache Classification Committee, there are two major types of migraine: 1) migraine without aura, which is a clinical syndrome characterized by distinctive headache and associated symptoms; and 2) migraine with aura, which is primarily characterized by transient focal neurological symptoms that usually precede or sometimes accompany the headache.

[0130] Migraine with aura, also known as classic migraine, includes symptoms such as recurrent attacks of unilateral, fully reversible visual, sensory, or other central nervous system symptoms, which usually develop gradually and are usually followed by headache and accompanying migraine symptoms. These attacks usually last several minutes. Migraine can be chronic. Furthermore, chronic migraine can also include episodic subtypes of migraine. Chronic migraine occurs 15 or more days per month for more than three months, with migraine headache features present at least 8 days per month.

[0131] Furthermore, the method provided herein can be used to treat individuals with cluster headache.The symptoms of cluster headache include, but are not limited to, attacks of severe, strictly unilateral pain in the orbit, supraorbital region, temporal region, or any combination thereof, lasting 15 to 180 minutes, occurring once every two days to eight times a day.The pain is associated with ipsilateral conjunctival congestion, lacrimation, nasal congestion, rhinorrhea, frontal and facial sweating, miosis, ptosis and / or eyelid edema, and / or restlessness or agitation.

[0132] Cluster headaches can be episodic or chronic. Episodic cluster headaches can be attacks occurring over a period of 7 days to 1 year, separated by pain-free periods lasting at least 3 months. For example, episodic cluster headaches can manifest as attacks occurring over a period of 7 days to 1 year, separated by pain-free periods lasting at least 3 months. Furthermore, episodic cluster headaches can also manifest as at least two cluster episodes lasting 7 days to 1 year (if untreated), separated by pain-free periods of remission of at least 3 months, according to the International Classification of Headache Disorders, Third Edition. In contrast, chronic cluster headaches can be attacks occurring over a period of 1 year or longer, without remission or with remission periods lasting less than 3 months.

[0133] The term "refractory migraine" or "resistant migraine" is used to describe persistent headaches that are difficult to treat or that do not respond to standard and / or aggressive treatments. In some embodiments, "refractory migraine," as used herein, requires failure of previous treatment with two to four available prophylactic drugs or classes of prophylactic drugs. In certain embodiments, "refractory migraine," as used herein, requires failure of previous treatment with two to three, at least two, at least three, at least four, more than two, or more than three available prophylactic drugs or classes of prophylactic drugs.

[0134] As used herein, "failure to respond" or "treatment failure" refers to the lack of effectiveness of a prophylactic agent or class of prophylactic agents in reducing the frequency, duration, and / or severity of migraine headaches in a patient after a standard treatment regimen with that drug, or the need to discontinue treatment (e.g., treatment with that prophylactic agent or class of prophylactic agents) due to adverse events that cause the patient to be unable to tolerate the treatment, or the drug being contraindicated or unsuitable for the patient.

[0135] Preventive 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 that a patient has failed previous treatment with any two to four of the above clusters. In certain embodiments, refractory migraine, as used herein, requires that a patient has failed previous treatment with any two to three, at least two, at least three, at least four, more than two, or more than three of the above clusters.

[0136] Exemplary antiepileptic drugs include divalproex, sodium valproate, valproate, valproic acid, topiramate, and gabapentin. Exemplary beta-blockers include propranolol, timolol, atenolol, metoprolol, nadolol, and bisoprolol. Exemplary tricyclic antidepressants include amitriptyline, nortriptyline, doxepin, and fluoxetine. Exemplary calcium channel blockers include flunarizine. Exemplary angiotensin II receptor antagonists include candesartan. Anti-CGRP pathway monoclonal antibodies are described below. For example, calcitonin gene-related peptide (CGRP) pathway monoclonal antibodies may 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. An exemplary anti-CGRP-R antibody is erenumab.

[0137] In certain embodiments, applicable prophylactic medications for migraine do not include acute treatments. In other embodiments, refractory migraine encompassed by the present disclosure requires failure of 2-4, 3-4, at least 2, or at least 3 classes of prophylactic medications as defined by the Refractory Headache Special Interest Section (RHSIS) of the American Headache Society (AHS). In some cases, under this definition, an individual must have failed 3 classes of prophylactic treatment.

[0138] 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 proposes a classification scheme based on the AHS criteria for grading headaches refractory to acute and preventive treatments, as well as a rating of headache-related disability. Specifically, this definition proposes class I (mild) refractory headaches that do not respond adequately to two different classes of nonspecific acute treatments (e.g., nonsteroidal anti-inflammatory drugs (NSAIDs) and combination analgesics); class II (moderate) refractory headaches that do not respond to triptans or ergot derivatives (e.g., dihydroergotamine (DHE)) in addition to class I headaches; and class III (severe) refractory headaches that do not respond to oral or parenteral opioids or corticosteroids or parenteral dopamine antagonists in sufficient doses and in appropriate formulations in addition to class I and II headaches.

[0139] Additionally, the European Headache Federation (EHF) provided 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 adequate treatment of psychiatric or other comorbid conditions by a multidisciplinary team, if available. In some embodiments, acute treatment and degree of disability are not included in these criteria. Therefore, when referring to intractable or resistant migraine, the present disclosure is intended to include all of the above definitions.

[0140] In other embodiments, the disclosure further provides a method of treating or preventing headache in an individual. In some embodiments, the headache is migraine. In some embodiments, the disclosure further provides a method of treating or preventing 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 cluster headache. In some embodiments, the headache is episodic cluster headache. In some embodiments, the headache is 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 previous preventative therapies for migraine (i.e., refractory migraine).

[0141] In some embodiments, the methods of treatment or prevention provided herein comprise administering to an individual a therapeutically effective amount of an anti-PACAP antibody or pharmaceutical composition described herein.

[0142] 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 may also include administering a second agent to the individual simultaneously or sequentially with the anti-PACAP antibody. The second agent may be, for example, an acute treatment for migraine. In yet other embodiments, the second agent may be a preventative treatment for migraine and / or refractory migraine.

[0143] Acute treatments for migraine 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., ditans), and gepants (i.e., calcitonin gene-related peptide receptor antagonists).

[0144] Non-limiting examples of NSAIDs that can be used in combination with anti-PACAP antibodies include aspirin, diclofenac, diflusinal, 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, rofecoxcib, meloxicam, JTE-522, L-745, 337, NS398, or a pharmaceutically acceptable salt thereof.

[0145] 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.

[0146] Non-limiting examples of ditanes that can be used in combination with the anti-PACAP antibodies of the present disclosure include lasmiditan.

[0147] 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 vazegepant.

[0148] Preventive treatments for migraine and / or refractory migraine are known in the art. In one embodiment, the preventive treatment includes topiramate. In another embodiment, the preventive treatment includes onabotulinumtoxinA. In one embodiment, the preventive treatment includes a calcitonin gene-related peptide (CGRP) pathway monoclonal antibody. For example, the calcitonin gene-related peptide (CGRP) pathway monoclonal antibody may 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. An exemplary anti-CGRP-R antibody includes erenumab.

[0149] In certain embodiments, the anti-PACAP antibodies described herein are administered in combination with onabotulinumtoxinA.

[0150] 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.

[0151] In certain embodiments, the anti-PACAP antibodies described herein are administered in combination with galcanezumab.

[0152] In certain embodiments, the anti-PACAP antibodies described herein are administered in combination with eptinezumab.

[0153] In certain embodiments, the anti-PACAP antibodies described herein are administered in combination with erenumab.

[0154] 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.

[0155] In some embodiments, the anti-PACAP antibodies described herein can be used in combination with the anti-PAC1 antibodies described in WO2019140216 and WO2014144632.

[0156] In some embodiments, the anti-PACAP antibodies described herein can be used for patients with migraine within a subpopulation identified by physiological measures of autonomic function. For example, the subpopulation can be identified by (i) baseline interictal or interictal PACAP levels in plasma, tears, saliva, or other biological samples; (ii) baseline physiological measurements of autonomic function (e.g., pupillary light reflex and galvanic skin response); and (iii) baseline interictal or interictal autonomic response to PACAP infusion.

[0157] 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 reduction in the duration of, or a reduction in the severity of, a condition discussed herein.

[0158] Administration of any one or more of the 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, 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 methods for treating or preventing 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 generally administered by injection or infusion as a solution or suspension formulation. In an exemplary embodiment, the anti-PACAP antibody can be administered directly to an individual by 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 in cell-based assays at concentrations equivalent to about 300 picomolar (pM). The cell-based assay can be an assay that monitors 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 G(αs)-coupled GPCRs results in increased levels of intracellular cAMP production, whereas activation of G(αi)-coupled GPCRs results in decreased levels of intracellular cAMP production.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. Several reagent kits that can be used to measure intracellular cAMP levels are available on the market. These include Cisbio's HTRF cAMP kit, PerkinElmer's LANCE cAMP kit, DiscoverX's HitHunter cAMP kit, and Abcam and BioVision's cAMP Direct Immunoassay kit. All of these assays are based on the use of antibodies that specifically recognize both intracellular cAMP and an exogenously labeled cAMP conjugate, which acts as a competitor, followed by detection of the labeled cAMP conjugate by various detection techniques, including fluorescence resonance energy transfer (FRET) or enzymatic reactions. Furthermore, 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 level of PACAP expression in an individual. In some embodiments, antibodies of the present disclosure are effective at concentrations equivalent to about 30 to about 90 pM in cell-based assays. In some embodiments, antibodies of the present disclosure are effective at concentrations equivalent to about 40 to about 80 pM in cell-based assays. In some embodiments, antibodies of the present disclosure are effective at concentrations equivalent to about 50 to about 70 pM in cell-based assays. In some embodiments, antibodies of the present disclosure are effective at concentrations equivalent to about 60 pM, 65 pM, 67 pM, 69 pM, or 70 pM in cell-based assays. Systems and Kits

[0159] Also provided herein are systems and kits that include the anti-PACAP antibodies, recombinant nucleic acids, or pharmaceutical compositions provided and described herein, as well as instructions for making and using the same. For example, in some embodiments, provided herein are systems and / or kits that include one or more of the anti-PACAP antibodies described herein, the recombinant nucleic acids described herein, or the pharmaceutical compositions described herein.

[0160] In some embodiments, provided herein is a pre-filled syringe or a pre-filled autoinjector containing a liquid pharmaceutical composition comprising an anti-PACAP antibody having a full-length heavy chain amino acid sequence set forth in SEQ ID NO: 1 and a full-length light chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, provided herein is a pre-filled syringe or a pre-filled autoinjector containing a liquid pharmaceutical composition comprising an anti-PACAP antibody having a full-length heavy chain amino acid sequence set forth in SEQ ID NO: 3 and a full-length light chain amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the liquid pharmaceutical composition has a pH of about 6.5 to 8.0. In some embodiments, the pre-filled syringe or pre-filled autoinjector contains at least 0.5 mL, 0.75 mL, 1 mL, 1.5 mL, 1.75 mL, or 2 mL of the liquid pharmaceutical composition. In some embodiments, the concentration of the anti-PACAP antibody in the liquid pharmaceutical composition in the pre-filled syringe or pre-filled autoinjector is about 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, 600 mg / mL, 700 mg / mL, 800 mg / mL, 900 mg / mL, or 1000 mg / mL.

[0161] In some embodiments, provided herein is a pre-filled syringe or a pre-filled autoinjector containing a liquid pharmaceutical composition comprising an anti-PACAP antibody having a full-length heavy chain amino acid sequence set forth in SEQ ID NO: 1 and a full-length light chain amino acid sequence set forth in SEQ ID NO: 2, and a pharmaceutically acceptable carrier. In some embodiments, provided herein is a pre-filled syringe or a pre-filled autoinjector containing a liquid pharmaceutical composition comprising an anti-PACAP antibody having a full-length heavy chain amino acid sequence set forth in SEQ ID NO: 3 and a full-length light chain amino acid sequence set forth in SEQ ID NO: 2, and a pharmaceutically acceptable carrier. In some embodiments, the pH of the liquid pharmaceutical composition is about 6.5 to 8.0. In some embodiments, the pre-filled syringe or pre-filled autoinjector contains at least 0.5 mL, 0.75 mL, 1 mL, 1.5 mL, 1.75 mL, or 2 mL of the liquid pharmaceutical composition. In some embodiments, the concentration of the anti-PACAP antibody in the liquid pharmaceutical composition in the pre-filled syringe or pre-filled autoinjector is about 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, 500 mg / mL, 600 mg / mL, 700 mg / mL, 800 mg / mL, 900 mg / mL, or 1000 mg / mL.

[0162] In some embodiments, the kit may have one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, e.g., to modulate cellular activity or treat a disease in an individual in need thereof.

[0163] Any of the above systems and kits may further comprise one or more additional reagents, which may be selected from 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 the in vitro production of an engineered transmembrane protein.

[0164] In some embodiments, the system or kit may further include instructions for practicing the method using the components of the kit. The instructions for practicing the method are generally 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 package insert, on a label on the container of the kit or its components (i.e., associated with the packaging or inner packaging), and similar forms. The instructions may be present as an electronic storage data file residing on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, etc. In some cases, the actual instructions are not present 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. Like the instructions, the means for obtaining the instructions may also be recorded on a suitable substrate.

[0165] 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.

[0166] No admission is made that any reference cited herein constitutes prior art. The discussion of references states what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. Although several sources of information, including scientific journal articles, patent documents, and textbooks, are referenced herein, it will be expressly understood that such reference does not constitute an admission that these documents form part of the general knowledge in the art.

[0167] The discussion of general methods presented herein is intended to be exemplary only: other alternative methods and substitutes will be apparent to those skilled in the art upon review of this disclosure, and such alternative methods and substitutes are to be included within the spirit and scope of this application. [Example]

[0168] 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 that are well known to those skilled in the art. Such techniques are fully explained in the literature cited above. 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 present disclosure or the claims in any way. Example 1 Generation of anti-PACAP antibodies

[0169] This example briefly describes the procedure for generating the antibodies provided in this disclosure.

[0170] 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 can be isolated, for example, by techniques known in the literature, such as 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. More than 61,000 B cells were screened against PACAP-38 and VIP.Positive B cells secreting antigen-specific antibodies were isolated and sequenced.

[0171] We sequenced and cloned 201 antibodies encoding immunoglobulin heavy and light chains. Thirty-nine antibodies with selective binding to PACAP were identified. Two clones were selected for further optimization. These antibodies exhibited high binding affinity to PACAP and low or no binding affinity to VIP. Furthermore, both antibodies exhibited high potency for PACAP and low or no potency for VIP.

[0172] Both antibodies showed high affinity and potency for 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 to generate candidate antibodies with high affinity and potency, and at the same time, reduced risk of potential immunogenicity, as measured by the ProImmune REVEAL® (peptide-MHC class II stability) assay and high humanity scores.

[0173] The potency-retaining humanized variant was 8 The antibody was further optimized by in vitro affinity maturation, which involved generating a combinatorial library of variants across species. Approximately 950 additional variants were screened to maintain the potency profile while addressing potential manufacturability and immunogenicity.

[0174] Table 5 below shows exemplary humanized variants that were screened for binding affinity, potency, and predicted immunogenicity. [Table 5-1] [Table 5-2] Four humanized antibodies (890C, 608C, 627C, and 609C) demonstrated high affinity, high potency, and low predicted immunogenicity.

[0175] Antibody developability criteria such as those described in Example 7 were used to identify variants with the best developability profile.

[0176] As can be seen from Table 5, the highest binding affinity does not necessarily correlate easily with predicted immunogenicity. For example, variant 524C exhibited higher binding affinity than variants 890C, 608C, 627C, and 609C, but also exhibited a predicted immunogenic profile, whereas 890C, 608C, 627C, and 609C had a predicted immunogenic profile that was not observed. Furthermore, potency against PACAP does not predict favorable qualities for immunogenicity and developability. For example, variant 519C exhibited higher potency than variants 890C, 608C, 627C, and 609C, but also exhibited a predicted immunogenic profile, whereas 890C, 608C, 627C, and 609C had a predicted immunogenic profile that was not observed. For example, predicted immunogenicity profiles were measured by the assay described in Example 3. Example 2 Humanity of anti-PACAP antibodies

[0177] This example describes the human nature of the antibodies provided in this disclosure.

[0178] Increasing the humanity of the variable region sequences of monoclonal antibodies that are potential therapeutic candidates is an important approach to minimize potential immunogenicity. The anti-PACAP antibody of the present disclosure was engineered to achieve a high humanity score without compromising the high affinity and high potency described herein. The humanity score, i.e., similarity to human germline sequences, was obtained using various antibody analysis platforms, including IMGT and AbGenesis. Starting from a single input variable region antibody sequence, the closest human germline sequence was identified, and the percentage of humanity of the variable heavy and light chains was computed using AbGenesis software (Release 4.1). The overall humanity of the antibody was calculated based on the average percent sequence identity values ​​of the heavy and light chains.

[0179] The humanity scores of some exemplary anti-PACAP antibodies of the present disclosure and some known anti-PACAP antibodies are shown in Table 6. [Table 6]

[0180] As described above, all of the anti-PACAP antibodies (e.g., 608C, 627C, 609C, 604C, 605C, and 890C) have at least 89% humanity, which is higher than the known anti-PACAP antibody Ab1h, described in WO2017181031, and antibody Ab10.H3, described in WO2017181039. Anti-PACAP antibodies Ab C and D are described in WO2019067293. Sequences that are not germline identical are of particular interest for further immunogenicity prediction analysis, as described in the Examples below. Example 3 Predicted immunogenicity of anti-PACAP antibodies

[0181] This example describes the predicted immunogenicity of the antibodies provided in this disclosure.

[0182] During the process of generating the antibodies described herein, antibodies with predicted low immunogenicity were taken forward for further optimization, excluding those with epitopes predicted by peptide-MHC class II stability analysis.

[0183] Peptides synthesized from the heavy and light chain variable regions of the anti-PACAP antibodies provided herein, including all non-germline residues, such as those described in Example 2 (Table 6), 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, 15-residue synthetic peptides derived from the heavy and light chain sequences, including all non-germline residues, were assessed for the tested antibodies.

[0184] The MHC class II alleles analyzed included all alleles with a frequency of at least 3% in the global population. Binding of peptides to MHC class II was detected by ELISA. These analyses identified predicted low-immunogenic antibodies, e.g., 608C, 609C, 627C, and 890C, for which all peptides tested showed no or low-stability interactions with MHC class II proteins (Figure 8). In contrast, analysis of previously described anti-PACAP antibodies, e.g., Ab1H, Ab10.H3, Ab B, Ab C, or Ab D, all showed predicted immunogenic epitopes. Example 4 Dynamics of anti-PACAP antibodies

[0185] This example describes the behavior of the antibodies provided in this disclosure.

[0186] Briefly, antibody-peptide interactions were determined on 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 by 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, catalog no. BSAS1.0) and 0.15 M NaCl (Sigma-Aldrich, catalog no. S7653). Antibody binding kinetics were determined by injecting two-fold serial dilutions of PACAP (12 nM to 0 nM) in running buffer and two-fold serial dilutions of VIP (1200 nM to 0 nM) in running buffer at a flow rate of 40 μL per minute at 37°C for 2 minutes, followed by a 10-minute dissociation period. The chip was regenerated using 0.85% phosphoric acid for subsequent cycles. Data were analyzed using Biacore S200 Evaluation Software (ver 1.0). The association rate constant (k a ) and dissociation rate constant (k d ) were determined using a simple one-to-one Langmuir binding model and used to calculate the equilibrium dissociation constant (K D ) was calculated.

[0187] The same assay format was used for supernatant-derived capture antibodies, using a single analyte injection of 33.3 nM PACAP for off-rate ranking of antibody variants. An exemplary Biacore-based off-rate ranking assay with a single concentration of PACAP38 is shown in Table 7. [Table 7-1] [Table 7-2]

[0188] 4A, 4B, and 4C show examples of SPR binding affinity analysis of anti-PACAP antibody 605C to PACAP38, PACAP27, and VIP. Furthermore, Table 8 shows that anti-PACAP antibody 605C binds to PACAP38 and PACAP27 with high affinity and selectivity. Steady-state binding affinity was used for VIP because of its very fast on-rate and off-rate. [Table 8] Example 5 Efficacy of anti-PACAP antibodies

[0189] This example describes the efficacy of the antibodies provided in this disclosure.

[0190] A cell-based assay for measuring PACAP-induced cAMP accumulation was performed according to a previously described method (Wang, Li et al. 2004). Specifically, a CHO-K1 cell line (Eurofins / Discover X) stably expressing either the human PAC1, VPAC1, or VPAC2 receptor was utilized to inhibit PACAP38- or PACAP27-induced signaling via the PAC1, VPAC1, and VPAC2 receptors, and endpoints were determined using Promega cAMP-Glo™. Table 9 below lists the IC values ​​of several exemplary anti-PACAP antibodies provided herein. 50 To summarize: [Table 9] Example 6 Selectivity of anti-PACAP antibodies

[0191] This example describes the selectivity of the antibodies provided in this disclosure.

[0192] Anti-PACAP antibodies, such as 890C, were tested against related peptides from the secretin-glucagon family (Table 10) to determine the presence and extent of any off-target binding. The nine bioactive peptides in 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 buffer at 37°C for 2 minutes at a flow rate of 50 mL per minute, followed by a 4-5 minute dissociation phase. Samples were injected over the newly captured anti-PACAP antibody in a multi-cycle fashion by regenerating the capture surface with two injections of 0.85% phosphoric acid at a flow rate of 30 μL per minute. [Table 10]

[0193] As shown in Figure 5, at the stability level (relative binding after 15 seconds of dissociation) determined by Biacore, antibody 890C is highly selective for PACAP and exhibits minimal or no binding to 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-induced cAMP signaling with that of VIP-induced cAMP signaling in a cell-based functional assay measuring cyclic AMP (cAMP). As shown in Figure 6, antibody 890C demonstrated greater than 1000-fold selectivity for inhibiting PACAP38-induced cAMP production in CHO-K1 cells stably expressing the PAC1 receptor compared to VIP-induced cAMP production in CHO-K1 cells stably expressing the VPAC1 receptor. Example 7 Assessing developability using HPLC-SEC analysis

[0194] The developability of antibody molecules can be assessed using biophysical techniques that measure the nonspecific interactions of antibody molecules. High-performance liquid chromatography (HPLC) analysis of antibodies using either silica-based or dextran-based size-exclusion columns has shown that the retention time of antibody samples correlates with their colloidal stability. Antibodies that tend to precipitate or aggregate are retained longer on the column, likely due to nonspecific 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. Longer retention times indicate a lower apparent molecular weight than calculated based on the sequence.

[0195] As shown in Figure 7, the typical molecular weight of antibodies with acceptable IgG is approximately 155 kDa. Antibodies 609C and 890C eluted normally, with apparent molecular weights ranging from 155 kDa to 165 kDa, whereas Ab C and Ab D showed significantly increased retention times with apparent molecular weights ranging from approximately 95 to 105 kDa depending on the IgG isoform. These data demonstrate that antibodies 609C and 890C have adequate colloidal stability, as reflected by their retention times on the column, and are therefore predicted to have better developability. Example 8 Single-dose study of anti-PACAP monoclonal antibody in cynomolgus monkeys.

[0196] Four biologically naive male cynomolgus monkeys were subcutaneously administered a single dose of 10 mg / kg of anti-PACAP antibodies having the CDR sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Samples were collected for pharmacokinetic and pharmacodynamic analyses, hematology, and clinical biochemistry evaluations. Pharmacodynamic analyses were 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 were observed in hematology, clinical biochemistry, or body weight. The serum concentration-time profile of the antibody was comparable to that of a typical monoclonal antibody in cynomolgus monkeys.

[0197] While certain alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and contemplated within the true spirit and scope of the appended claims. Accordingly, there is no intention to be limited to the precise summary and disclosure presented herein.

Claims

1. An anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibody, comprising a full-length heavy chain amino acid sequence shown in SEQ ID NO: 1 and a full-length light chain amino acid sequence shown in SEQ ID NO:

2.

2. An anti-pituitary adenylate cyclase-activating polypeptide (PACAP) antibody, comprising a full-length heavy chain amino acid sequence shown in SEQ ID NO:3 and a full-length light chain amino acid sequence shown in SEQ ID NO:

2.

3. An anti-PACAP antibody comprising the full-length heavy chain amino acid sequence shown in SEQ ID NO: 1 and the full-length light chain amino acid sequence shown in SEQ ID NO: 2, wherein the antibody is produced by a method comprising the steps of introducing a vector comprising nucleic acid encoding the full-length heavy chain amino acid sequence shown in SEQ ID NO: 1 and the full-length light chain amino acid sequence shown in SEQ ID NO: 2 into a host cell, culturing the host cell under conditions sufficient for the cell to produce the antibody, and purifying the antibody from the cell culture.

4. An anti-PACAP antibody comprising the full-length heavy chain amino acid sequence shown in SEQ ID NO: 3 and the full-length light chain amino acid sequence shown in SEQ ID NO: 2, wherein the antibody is produced by a method comprising the steps of introducing a vector comprising a nucleic acid encoding the full-length heavy chain amino acid sequence shown in SEQ ID NO: 3 and the full-length light chain amino acid sequence shown in SEQ ID NO: 2 into a host cell, culturing the host cell under conditions sufficient for the cell to produce the antibody, and purifying the antibody from the cell culture.

5. 5. The antibody of claim 3 or 4, produced at a titer of at least 2 g / L.

6. The antibody of any one of the preceding claims, which is a human antibody or a humanized antibody.

7. The antibody of any one of the preceding claims, which is a humanized antibody.

8. The antibody of any one of the preceding claims, having a low or non-immunogenic profile.

9. The antibody of any one of the preceding claims, which is an antagonist of PACAP.

10. An antibody described in any one of the preceding claims, which specifically binds to PACAP.

11. A nucleic acid encoding an antibody according to any one of the preceding claims.

12. A vector comprising the nucleic acid of claim 11.

13. A method for producing an anti-PACAP antibody having a full-length heavy chain amino acid sequence shown in SEQ ID NO: 1 or 3 and a full-length light chain amino acid sequence shown in SEQ ID NO: 2, comprising the steps of introducing a vector containing nucleic acid encoding the full-length heavy chain amino acid sequence shown in SEQ ID NO: 1 or 3 and the full-length light chain amino acid sequence shown in SEQ ID NO: 2 into a host cell, culturing the host cell under conditions sufficient for the cell to produce the antibody, and purifying the antibody from the cell culture.

14. 14. The method of claim 13, wherein the antibody is produced at a titer of at least 2 g / L.

15. A pharmaceutical composition comprising an antibody according to any one of the preceding claims and a pharmaceutically acceptable carrier.

16. A liquid pharmaceutical composition comprising an anti-PACAP antibody having a full-length heavy chain amino acid sequence shown in SEQ ID NO: 1 or 3 and a full-length light chain amino acid sequence shown in SEQ ID NO: 2, a pharmaceutically acceptable carrier, and water for injection.

17. 17. The liquid pharmaceutical composition of claim 16, wherein the pH is from about 6.5 to about 8.

0.

18. A pre-filled syringe or pre-filled autoinjector containing a liquid pharmaceutical composition comprising an anti-PACAP antibody having a full-length heavy chain amino acid sequence shown in SEQ ID NO: 1 or 3 and a full-length light chain amino acid sequence shown in SEQ ID NO:

2.

19. 19. The pre-filled syringe or pre-filled autoinjector of claim 18, wherein the pH of the composition is from about 6.5 to about 8.

0.

20. A method of treating or preventing a condition in an individual, comprising the step of administering to the individual a therapeutically effective amount of the antibody or pharmaceutical composition of any one of the preceding claims, wherein the condition is selected from the group consisting of headache (e.g., migraine, cluster headache, treatment-refractory migraine), anxiety, depression, PTSD, conditions (e.g., anxiety / depression / PTSD) coexisting with headache (e.g., migraine, cluster headache, treatment-refractory migraine), anxiety disorders coexisting with migraine, complex regional pain syndrome, and rosacea.

21. 21. The method of claim 20, wherein the headache is selected from the group consisting of migraine with aura, migraine without aura, hemiplegic migraine, cluster headache, migraine-like neuralgia, chronic headache, episodic migraine, chronic migraine, medication overuse headache, and tension-type headache.

22. 10. A method of treating or preventing migraine in an individual, comprising administering to said individual a therapeutically effective amount of the antibody or pharmaceutical composition of any one of the preceding claims.

23. A method of treating or preventing migraine in an individual, comprising the step of administering to said individual a therapeutically effective amount of the antibody or pharmaceutical composition of any one of the preceding claims, wherein said individual has failed to respond to two to four available prophylactic agents.

24. 24. The method of claim 23, wherein the individual fails to respond to two to four available prophylactic agents selected from the group consisting of divalproex, sodium valproate, valproate, valproic acid, topiramate, gabapentin, propranolol, timolol, atenolol, metoprolol, nadolol, bisoprolol, flunarizine, amitriptyline, nortriptyline, doxepin, fluoxetine, and candesartan.

25. A method of treating or preventing migraine in an individual, comprising the step of administering to said individual a therapeutically effective amount of the antibody or pharmaceutical composition of any one of the preceding claims, wherein said individual has failed to respond to two to four available prophylactic drug classes.

26. 26. The method of claim 25, wherein the class of preventative drug 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.

27. The classes of prophylactic agents are selected from different clusters, wherein the clusters are selected from the following: 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.

26. The method of claim 25, wherein the method is defined as follows:

28. 28. The method of claims 23-27, wherein the individual fails to respond to two to three, at least two, at least three, at least four, more than two, or more than three prophylactic agents or classes of prophylactic agents.

29. 10. A method of treating or preventing migraine in an individual, comprising the steps of selecting an individual who has failed to respond to two to four available prophylactic agents or classes of prophylactic agents, and administering to said individual a therapeutically effective amount of the antibody or pharmaceutical composition of any one of the preceding claims.

30. 10. A method of treating or preventing migraine in an individual who is unable to respond to a CGRP pathway monoclonal antibody, comprising administering to said individual a therapeutically effective amount of the antibody or pharmaceutical composition of any one of the preceding claims.

31. 31. The method of claim 30, wherein the CGRP pathway monoclonal antibody comprises an anti-CGRP antibody, an anti-CGRP-R antibody, or both.

32. 31. The method of claim 30, wherein the anti-CGRP antibody is selected from fremanezumab, galcanezumab, eptinezumab, or a combination thereof.

33. 31. The method of claim 30, wherein the anti-CGRP-R antibody is erenumab.

34. A composition for use according to any of the preceding claims.

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