Anti-pacap antibody and use thereof

Abstract

Provided are an anti-PACAP antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region sequence VH. The heavy chain variable region sequence VH includes VHCDR1, VHCDR2, and VHCDR3, wherein the VHCDR1, VHCDR2, and VHCDR3 are the following amino acid sequences: 1) SEQ ID NOs: 1-3 or; 2) SEQ ID NOs: 7-9; or 3) SEQ ID NO: 7, SEQ ID NO: 35, and SEQ ID NO: 9. The PACAP antibody or antigen-binding fragment provided in the present invention is expected to be used for the prevention and treatment of migraine, or for the detection of PACAP antigens.

Description

Anti-PACAP antibodies and their applications

[0001] Cross-reference to related applications

[0002] This application claims priority to an earlier application filed on October 17, 2024, with patent application number 202411458349.X and entitled "Anti-PACAP Antibody and Its Application". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of protein engineering, and more particularly to an anti-PACAP antibody and its applications. Background Technology

[0004] Pituitary adenylate cyclase-activated polypeptide (PACAP) is a member of the secretin / vasoactive peptide (VIP) / growth hormone-releasing hormone (GHRH) family. PACAP is a multifunctional vasodilatory peptide existing in two α-amidated active forms: one with 38 amino acids (PACAP38) and the other with 27 amino acids (PACAP27). Both peptides share the same N-terminal 27 amino acids and are synthesized from the same precursor protein, PreproPACAP, with PACAP38 being more prevalent in mammals, accounting for over 90%. PACAP is widely distributed in the brain and peripheral organs and expressed throughout the nervous system. PACAP plays multiple roles in neurodevelopment, neuroprotection, neuromodulation, neurogenic inflammation, and nociception. VIP, a member of the VIP / secretin / glucagon superfamily, is structurally linked to PACAP and has multiple functions in vivo. Its primary function is vasodilation, occurring in the systemic circulation and specific tissues, including the respiratory system, gastrointestinal tract, central nervous system, and peripheral nervous system. However, it participates in regulating various physiological processes, such as smooth muscle contraction, glandular secretion, immune responses, and inflammation. In addition to its vasodilatory function, VIP also acts as a neurotransmitter and neuromodulator in perivascular parasympathetic nerve fibers and cranial parasympathetic ganglia of the brain. PACAP27 shares 68% sequence identity with VIP at the amino acid level.

[0005] The biological effects of PACAP / VIP are mediated by three different G protein-coupled receptors: PAC1R, vasoactive intestinal peptide receptor type 1 (VIPR1), and vasoactive intestinal peptide receptor type 2 (VIPR2). PAC1R / PACAP has approximately 1000 times the affinity of PAC1R / VIP, while VIPR1 / VIPR2 have equal affinity for PACAP / VIP. Upon binding to their receptors, these receptors all lead to the production of cyclic adenosine monophosphate (cAMP) and the activation of downstream signaling.

[0006] PACAP levels are elevated in both healthy individuals and migraine sufferers during migraine attacks, and direct infusion of PACAP can induce migraine-like headaches within the first two hours of administration. Its ability to induce cerebral artery vasodilation suggests that this neuropeptide plays a role in migraines. Similarly, VIP has also been found to have elevated expression levels in migraine sufferers, and VIP infusion induces headaches.

[0007] Migraine prevention therapy using CGRP / CGRPR antibodies has been proven effective, but the response rate is low in different populations, indicating that migraines in the vast majority of people are induced by other factors. Antibody blocking therapy targeting PACAP / VIP is expected to resolve migraine symptoms in some people who are not sensitive to CGRP therapy and can complement existing therapies. Summary of the Invention

[0008] Specific aspects of the present invention include:

[0009] This invention provides an anti-PACAP antibody or its antigen-binding fragment, comprising a heavy chain variable region sequence VH, wherein the heavy chain variable region VH comprises VHCDR1, VHCDR2, and VHCDR3, wherein VHCDR1, VHCDR2, and VHCDR3 have the following amino acid sequences:

[0010] 1) SEQ ID NO: 1-3 or;

[0011] 2) SEQ ID NO: 7-9 or:

[0012] 3) SEQ ID NO: 7, SEQ ID NO: 35, SEQ ID NO: 9.

[0013] In a specific embodiment of the present invention, the antibody or its antigen-binding fragment further comprises a light chain variable region VL, wherein the light chain variable region VL comprises VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1, VLCDR2, and VLCDR3 have the following amino acid sequence:

[0014] 3) SEQ ID NO: 4-6 or;

[0015] 4) SEQ ID NO: 10-12 or

[0016] 6) SEQ ID NO: 36, SEQ ID NO: 11, SEQ ID NO: 12.

[0017] In a specific embodiment of the present invention, the heavy chain variable region VH and the light chain variable region CDR are combined as follows, in the order of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3:

[0018] i) SEQ ID NO: 1-6, or;

[0019] ii) SEQ ID NO: 7-12, or

[0020] iii) SEQ ID NO: 7, SEQ ID NO: 35, SEQ ID NO: 9, SEQ ID NO: 36, SEQ ID NO: 11, SEQ ID NO: 12.

[0021] In a preferred aspect of the invention, the heavy chain variable region VH of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 13, 15, 17, 19, or 37.

[0022] In a preferred aspect of the invention, the light chain variable region VL of the antibody or its antigen-binding fragment comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 14, 16, 18, 20, or 38.

[0023] Furthermore, the antibody or its antigen-binding fragment comprises the following combinations of heavy chain variable region VH and light chain variable region VL: SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20 and SEQ ID NO: 37 and 38.

[0024] In a specific embodiment of the present invention, the antibody or its antigen-binding fragment satisfies at least one of the following two conditions:

[0025] (1) The antibody or its antigen-binding fragment is selected from full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, nanobody (single-domain antibody) or minimum recognition unit (MRU);

[0026] (2) The antibody is a rabbit-derived, chimeric, or humanized antibody or a fully human antibody.

[0027] Furthermore, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region (CH) and the light chain comprises a light chain constant region (CL), and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody or a primate antibody.

[0028] Furthermore, the antibody or its antigen-binding fragment is characterized in that both the heavy chain constant region and the light chain constant region of the antibody or antigen-binding fragment are derived from human IgG antibodies. Preferably, the heavy chain constant region is a constant region of human IgG1, IgG2, or IgG4 subtypes; more preferably, the heavy chain constant region is IgG4 S228P, IgG4 S228P / L235E (IgG4-PE), IgG2 H268Q / V309L / A330S / P331S (IgG2m4), IgG2 V234A / G237A / P238S / H268A / V309L / A330S / P331S (IgG2c4d), IgG1 L234A / L235A (LALA), or IgG1 L234A L235A P329G (LALA-PG); preferably, the light chain constant region is a human Kappa or lambda chain constant region.

[0029] Furthermore, the antibody or its antigen-binding fragment is characterized in that the heavy chain constant region of the antibody or antigen-binding fragment includes SEQ ID NO: 31, and the light chain constant region of the antibody or antigen-binding fragment includes SEQ ID NO: 32.

[0030] Furthermore, the heavy chain of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO: 21, 23 or 39; the light chain of the antibody or its antigen-binding fragment contains an amino acid sequence as shown in SEQ ID NO: 22, 24 or 40.

[0031] Furthermore, the combination of heavy and light chains of the antibody or its antigen-binding fragment is SEQ ID NO: 21-22, SEQ ID NO: 23-24 or SEQ ID NO: 39-40.

[0032] The present invention also provides a humanized anti-PACAP antibody, characterized in that the following sequence fragments are inserted into the frame region (FR) of the heavy chain variable region and the light chain variable region of human IgG in the order of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3: i) SEQ ID NO: 1-6, or ii) SEQ ID NO: 7-12.

[0033] In a specific embodiment of the present invention, the framework region of the variable region of the human IgG heavy chain is IGHV3-64*04, IGHV3-66*01, IGHV3-53*04, IGHV3-53*01, IGHV3-NL1*01, IGHV3-23*04, IGHV3-30*01, IGHV3-30*02, IGHV3-30*09, IGHV3-53*06, IGHV3-53*03, IGHV3-53*02, IGHV3-48*01, IGHV3-74*01, or IGHV3-23*01.

[0034] In a specific embodiment of the present invention, the framework region of the variable region of the human IgG light chain is IGKV1-5*01, IGKV1-5*02, IGKV1-39*01, IGKV1-9*01, IGKV1-NL1*01, IGKV1-17*01, IGKV1-12*01, IGKV1-6*01, IGKV1-17*02, IGKV1-13*02, or IGKV1-33*01.

[0035] In a preferred aspect of the invention, the anti-PACAP antibody and its antigen-binding fragment, or the humanized anti-PACAP antibody, binds to PACAP and / or VIP with a KD less than or equal to 5E-5M, 5E-6M, 5E-7M, 5E-8M, 5E-9M, 5E-10M, 5E-11M, or 5E-12M, wherein the KD value is determined using SPR. In another preferred aspect of the invention, in cell experiments, the anti-PACAP antibody and its antigen-binding fragment, or the humanized anti-PACAP antibody, neutralizes PACAP and / or VIP-induced PAC1R, VIPR1, and / or VIPR2 signals at a KD less than 20 nM, less than 10 nM, less than 1 nM, or less than 0.1 nM.

[0036] The present invention also provides a nucleic acid molecule, characterized in that the nucleic acid molecule encodes any of the antibodies or antigen-binding fragments described above.

[0037] The present invention also provides a carrier, characterized in that the carrier contains the above-mentioned nucleic acid molecules.

[0038] The present invention also provides a host cell, characterized in that the host cell contains the above-mentioned vector or chromosome integrated with the above-mentioned nucleic acid molecules or expressing any of the above-mentioned antibodies or antigen-binding fragments thereof.

[0039] The present invention also provides a pharmaceutical composition comprising any of the antibodies or antigen-binding portions thereof described above, and a pharmaceutically acceptable adjuvant.

[0040] The present invention also provides the use of any of the above-mentioned antibodies or antigen-binding fragments thereof, the above-mentioned nucleic acid molecules, the above-mentioned carriers, and the above-mentioned cells, characterized in that they are used to prepare drugs or preparations for the prevention and / or treatment of headaches or migraines.

[0041] In a preferred aspect of the invention, the anti-PACAP antibody and its antigen-binding fragment, or the humanized anti-PACAP antibody, antagonizes, inhibits, neutralizes, or blocks at least one biological effect associated with human PACAP / VIP. In some embodiments, the anti-PACAP antibody and its antigen-binding fragment inhibit or neutralize at least one biological effect induced by PACAP / VIP, wherein the PACAP includes PACAP27 and / or PACAP38. In other embodiments, the anti-PACAP antibody and its antigen-binding fragment neutralize or inhibit PACAP activation of at least one of PAC1R, VIPR1, and / or VIPR2; or inhibit PACAP-induced cAMP production; and / or, when administered to a subject, reduce PACAP-induced vasodilation, photophobia, mast cell degranulation, and / or neuronal activation.

[0042] The present invention also provides a method for treating headaches or migraines, comprising administering, to a subject in need, any of the antibody molecules or their binding fragments described above, or the pharmaceutical compositions described above, in an amount effective in treating headaches or migraines.

[0043] In a further embodiment of the invention, the method further includes administering, separately or co-administering, another analgesic, for example, selected from the following.

[0044] The present invention also provides a method for blocking the binding of PACAP / VIP to its receptor in a subject in need of it, the method comprising administering to the subject an effective amount of any of the antibody molecules or their binding fragments or the pharmaceutical compositions described above.

[0045] The present invention also provides antibody conjugates in which the anti-PACAP antibody and its antigen-binding fragment are conjugated to at least one effector moiety, and optionally include a chemical linker. In another embodiment, the anti-PACAP antibody and its antigen-binding fragment are conjugated to one or more detectable moieties, such as those comprising a fluorescent dye, enzyme, substrate, bioluminescent material, radioactive material, chemiluminescent moiety, or a mixture thereof.

[0046] The present invention also provides a diagnostic reagent for detecting the target antigen PACAP, the reagent comprising the anti-PACAP antibody and its antigen-binding fragment as described in the present invention and / or the antibody conjugate thereof, wherein the anti-PACAP antibody or its antigen-binding fragment is conjugated to one or more detectable portions in the antibody conjugate.

[0047] Technical terms

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0049] The "antibody" described in this invention includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDR, VH region, and / or VL region. Examples of antibodies include (but are not limited to) monoclonal antibodies, antibodies produced in a recombinant manner, monospecific antibodies, mouse or rabbit anti-human antibodies, humanized antibodies, chimeric antibodies, tetrameric antibodies containing two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, antibody-drug conjugates or conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelified antibodies, and antigen-binding fragments of any of the above.

[0050] Preferred antibody forms of the present invention include Fab, Fab′, Fv, scFv, and (Fab′)2 fragments.

[0051] Fab refers to a portion of an antibody molecule containing a variable and constant region of a light chain and a variable and constant region of a heavy chain linked by disulfide bonds. The term Fab′ typically refers to a fragment different from Fab with the addition of a small number of residues (including one or more cysteine ​​residues from the antibody hinge region) to the carboxyl terminus of the CH1 domain of the heavy chain, and is generally obtained by the reduction of (Fab′)2.

[0052] Fv refers to the smallest antibody fragment containing the antibody heavy chain variable region, the light chain variable region, and all antigen-binding sites.

[0053] scFv refers to an engineered antibody composed of a light chain variable region and a heavy chain variable region directly linked or linked by a single peptide chain. In some cases, a disulfide bond may also exist between the VH and VL regions of the scFv. In some embodiments of the present invention, scFv can form di-scFv, which refers to two or more individual scFvs linked in series to form an antibody. In some embodiments of the present invention, scFv can form (scFv)2, which refers to two or more individual scFvs linked in parallel to form an antibody.

[0054] (Fab′)2 refers to the dimer of Fab′, which is obtained by treating the entire antibody molecule with pepsin without subsequent reduction.

[0055] The antibody forms of the present invention also include single-domain antibodies (nanobodies) or the smallest recognition unit.

[0056] The term "single-domain antibody," also known as "nanobody," refers to an antibody that contains only one heavy chain variable region, and is therefore also called a VHH antibody.

[0057] The smallest recognition unit (MRU) is the molecular recognition unit. Antibody-antigen binding primarily involves CDRs, with CDR3, especially the heavy chain CDR3, being the most crucial. Based on this fact, short peptides derived from CDRs have been used to mimic the specific binding activity of parental antibodies, confirming that CDRs can indeed be the smallest molecules specifically binding antigens, and thus are referred to as the smallest recognition unit or molecular recognition unit.

[0058] The present invention also includes antibodies of conserved sequence variants of the antibody amino acid sequence, the conserved amino acid sequence variants including modifications of the amino acid sequence that do not significantly alter the target-binding activity of the antibodies of the present invention, such as those derived from "conservative substitutions" well known in the art.

[0059] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art. Exemplary substitutions are shown in the table below:

[0060] The antibody described in this invention also includes sequences whose amino acid sequences in the antibody heavy chain variable region and the antibody light chain variable region have 80% or more, or 90% or more, or 95% or more, identity.

[0061] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be conveniently performed using, for example, computer programs such as the Align program (DNAstar, Inc.). The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the Needleman and Wunsch algorithm in the GAP program integrated into the GCG software package (available at www.gcg.com) can be used to determine the percentage identity between two amino acid sequences using a Blossum 62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0062] In this invention, the terms "VH region" and "VL region" refer to the variable regions of a single antibody heavy and light chains that contain FR (framework regions) 1, 2, 3 and 4 and CDR (complementarity-determining regions) 1, 2 and 3, respectively.

[0063] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system or the IMGT numbering system. For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art, as detailed in the table below:

[0064] When referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0065] The boundaries of the CDR of the antibody of the present invention can be determined artificially according to any method or combination thereof in the art. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers the CDR sequence determined in any of the foregoing methods.

[0066] In this invention, the terms "VL" and "VL region" refer to the variable region of the light chain of the antibody.

[0067] In this invention, the terms "VH" and "VH region" refer to the variable region of the heavy chain of the antibody.

[0068] The term "constant region" in this invention has its usual meaning in the art. The heavy chain constant region consists of three domains (CH1, CH2, and CH3), and the light chain constant region consists of one domain (CL). The constant region does not directly participate in antibody-antigen binding but can exhibit various effector functions, such as antibody portions interacting with Fc receptors (e.g., Fcγ receptors), for example, the carboxyl-terminal portions of the light and / or heavy chains. The heavy chain constant region of this invention is preferably a constant region or a variant of the human IgG1, IgG2, or IgG4 subtypes with weaker Fc-mediated effector function. For example, introducing LALA mutations (L234A, L235A) into the Fc region of the IgG1 subtype can reduce antibody binding to the Fcγ receptor and its complement without affecting pharmacokinetics in vivo.

[0069] In this invention, the term "chimeric antibody" refers to "human-rabbit chimeric antibody," which is a monoclonal antibody produced by inserting the variable regions of the light and heavy chains of a rabbit monoclonal antibody into an expression vector containing the constant region of a human antibody, and then transforming mammalian cells for expression.

[0070] In this invention, the term "monoclonal antibody" generally refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the antibodies in the cluster are identical except for a small number of possible natural mutations. Monoclonal antibodies typically exhibit high specificity against a single antigenic site. In this invention, the term "isolated nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in isolated form, isolated from its natural environment or synthesized artificially, or an analogue thereof.

[0071] In this invention, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid molecule linked to it and self-replicating in a suitable host. Vectors include any genetic element, such as plasmids, transposons, artificial chromosomes, viruses, etc., which, when combined with appropriate control elements, are capable of self-replication and transferring gene sequences to or between hosts.

[0072] In this invention, the term "K" D “K” D "KD" or "KD" can be used interchangeably and usually refers to the equilibrium dissociation constant of antibody-antigen interaction. In this invention, "KD" is the ratio of the dissociation rate constant (kdis, also known as "dissociation rate (koff)" or "kd") to the binding rate constant (kon, also known as "binding rate (kon)" or "ka").

[0073] The twenty common amino acids mentioned in this article are listed in accordance with conventional usage. In this invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably, both referring to amino acid structural units. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0074] As used herein, the term “pharmaceuticalally acceptable carrier and / or excipient” refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art.

[0075] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, relieving symptoms (whether partial or complete), alleviating or improving prognosis, reducing or inhibiting disease recurrence, whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0076] As used in this article, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

[0077] As used in this article, the term “approximately” means plus or minus 10% of the values ​​stated herein, such as plus or minus 5%.

[0078] As used in this article, the term "more" means ≥2.

[0079] Beneficial technical effects

[0080] The PACAP antibody or antigen-binding fragment provided by this invention is expected to be used for the prevention and treatment of migraines. Attached Figure Description

[0081] Figure 1: The binding activity of anti-PACAP antibody to the antigen protein PACAP38 was detected by ELISA.

[0082] Figure 2: The binding activity of anti-PACAP antibody to the antigen protein PACAP27 was detected by ELISA.

[0083] Figure 3: The binding activity of anti-PACAP antibody to the antigen protein VIP was detected by ELISA.

[0084] Figure 4: The ability of anti-PACAP antibodies to neutralize PACAP27-induced PAC1R signaling

[0085] Figure 5: Ability of anti-PACAP antibody to neutralize PACAP27-induced PAC1R signaling

[0086] Figure 6: The ability of anti-PACAP antibodies to neutralize PACAP38-induced PAC1R signaling

[0087] Figure 7: The ability of anti-PACAP antibodies to neutralize PACAP38-induced PAC1R signaling

[0088] Figure 8: The ability of anti-PACAP antibody to neutralize VIP-induced PAC1R signaling

[0089] Figure 9: The ability of anti-PACAP antibody to neutralize PACAP27-induced VIR1 signaling

[0090] Figure 10: The ability of anti-PACAP antibody to neutralize PACAP38-induced VIPR1 signaling

[0091] Figure 11: The ability of anti-PACAP antibody to neutralize VIP-induced VIPR1 signaling

[0092] Figure 12: The ability of anti-PACAP antibody to neutralize PACAP27-induced VIR2 signaling

[0093] Figure 13. The ability of anti-PACAP antibody to neutralize PACAP38-induced VIPR2 signaling.

[0094] Figure 14: The ability of anti-PACAP antibody to neutralize VIP-induced VIPR2 signaling

[0095] Figure 15: Anti-PACAP antibody inhibits PACAP38-induced cAMP production in SHSY5Y cells

[0096] Figure 16: Anti-PACAP antibody inhibits PACAP27-induced cAMP production in SHSY5Y cells Detailed Implementation

[0097] This invention provides a neutralizing antibody against PACAP, capable of binding to PACAP38 / 27 and VIP, and blocking their binding to three functional receptors. The PACAP antibody or antigen-binding fragment provided by this invention holds promise for the prevention and treatment of migraines. The amino acid sequence of the CDR region of the anti-PACAP antibody is as follows:

[0098] The variable region of the anti-PACAP antibody is as follows:

[0099] This invention is prepared by immunizing New Zealand rabbits with chemically prepared PACAP38 / 27 / VIP peptides conjugated with keyfora hemocyanin (KLH), the sequences of which are known.

[0100] >PACAP38 amino acid sequence

[0101] >PACAP27 amino acid sequence

[0102] >VIP amino acid sequence

[0103] Each rabbit underwent a primary immunization by subcutaneous injection of 800 μg of KLH-conjugated peptide in Freund's complete adjuvant, followed by booster immunizations every two weeks by subcutaneous injection of 400 μg of KLH-conjugated peptide in Freund's incomplete adjuvant. 7-10 days after the fourth booster immunization, rabbits were bled and serum was collected. The titer of PACAP38 binding in the immune serum was measured using ELISA. Rabbits with high titers were selected for intraperitoneal injection of 400 μg of KLH-conjugated peptide for a shock immunization. 48-72 hours after the shock immunization, rabbit PBMCs and spleens were collected, and RNA was extracted from each. This RNA was then reverse transcribed into cDNA, and the light and heavy chains of the antibodies were amplified using specific primers. A phage library was constructed from the amplified antibody sequences. Phages specifically binding to the peptide were screened using solid-phase and liquid-phase panning. After single-clone plating, clones specifically binding to PACAP38 were further confirmed. Positive clones were sequenced to obtain the sequences of rabbit-derived anti-PACAP antibodies.

[0104] Other forms of antigen-binding fragments, including but not limited to Fab, scFv, and chimeric antibodies, can be prepared using the variable region of the aforementioned rabbit antibody. Chimeric antibodies contain a variable region derived from rabbits and a constant region derived from humans, thus exhibiting a longer half-life and lower immunogenicity when administered to human subjects. The method for preparing chimeric antibodies is known in the art, involving linking the light and heavy chain variable regions of the aforementioned rabbit antibody with the light and heavy chain constant regions of a human antibody to form a chimeric antibody. To further reduce immunogenicity, the CDR of the aforementioned rabbit antibody can be transplanted into an arbitrarily selected human frame region to generate the desired humanized antibody. The method for preparing humanized antibodies is known in the art, and a specific scheme can be found in Example 4. In short, based on the homology between the variable regions of rabbit and human antibodies, a human Germline sequence was selected, and the CDR region in the human Germline sequence was replaced with a rabbit CDR sequence. Then, based on the three-dimensional structure of the rabbit antibody, in order to maintain the affinity and biological activity of the humanized antibody, key light and heavy chain amino acid residues were selected for reverse mutation, and human amino acid residues were mutated into rabbit amino acid residues, finally obtaining the humanized anti-PACAP antibody sequence.

[0105] The above-mentioned humanized antibody sequence was constructed into an expression vector using conventional molecular biology methods. The recombinant humanized anti-PACAP antibody molecule was obtained by transient transfection in CHO cells and one-step purification with Protein A. This antibody can specifically bind to PACAP / VIP and block its binding to receptors PAC1R / VIPR1 / VIPR2, further inhibiting the activation of downstream signaling pathways.

[0106] Example

[0107] Example 1: Preparation of immunogen and animal immunization

[0108] PACAP38 / PACAP27 / VIP peptides were synthesized chemically based on known sequences. 3 mg of KLH (H8283, Sigma) was dissolved in 600 μL of PBS, and 0.5 mg of sulf-SMCC (M5525, Sigma) was dissolved in 100 μL of PBS. The dissolved SMCC solution was then slowly added dropwise to the KLH solution while gently mixing, and the reaction was allowed to proceed at room temperature for 2 hours. The solution was then transferred to a dialysis bag and dialyzed against PBS to remove excess SMCC overnight. The next day, after changing the dialysis buffer 1-2 times, 4 mg of the dissolved peptides were slowly added dropwise to the semi-conjugates while mixing, and the reaction was allowed to proceed at room temperature for 4 hours. After the reaction was complete, the peptides were aliquoted and stored at -80°C.

[0109] Each New Zealand rabbit was initially immunized by subcutaneous injection of 800 μg of KLH-conjugated peptide from Freund's complete adjuvant. Booster immunizations were then performed every two weeks by subcutaneous injection of 400 μg of KLH-conjugated peptide from Freund's incomplete adjuvant. 7-10 days after the fourth booster immunization, the rabbits were bled and serum was collected. The titer of PACAP38 binding in the immune serum was measured using ELISA. Rabbits with the highest titers were selected for intraperitoneal injection of 400 μg of KLH-conjugated peptide for pulse immunization.

[0110] Example 2: Construction of PACAP / VIP receptor overexpression stable cell lines

[0111] To screen for antibodies that can block the binding of PACAP / VIP to its receptor, a HEK293 cell line overexpressing PAC1R was constructed. The target gene was cloned into the HEK293 cell line using non-liposome transfection to form a stable high-expression cell line. The full-length human PAC1R gene was cloned into the pCDNA3.1(+)-CMV-MCS-NEO vector to construct the pCDNA3.1(+)-CMV-MCS-NEO-huPAC1R plasmid. Then, using non-liposome transfection, human PAC1R was cloned into the HEK293 cell line using fugene 6 (Promega, Madison, WI, USA). The cells were cultured for two weeks under a selection pressure of 1 mg / mL genimycin. Finally, flow cytometry was used to screen for monoclonal cell lines that highly expressed human PAC1R. HEK293 cell lines overexpressing human VIPR1 and VIPR2 were constructed using the same method. The sequences are as follows:

[0112] >Human PAC1R protein amino acid sequence

[0113] >Acid sequence of human VIR1 protein

[0114] >Human VIR2 protein amino acid sequence

[0115] Example 3: Screening of anti-PACAP antibodies

[0116] Whole blood was collected from rabbits subjected to shock immunization to isolate PBMCs. After sacrifice, the spleen was harvested, and RNA was extracted from both PBMCs and spleen. The RNA was then mixed and reverse transcribed into cDNA. Specific primers were used to amplify the light and heavy chains of the antibody separately. The amplified antibody library was ligated into a phage vector to construct a phage library. PACAP38 / VIP was coated onto a solid-phase vector, and the obtained phage library was subjected to positive panning. Positive clones G085 / G136 were sequenced and validated for recombinant expression.

[0117] Example 4: Preparation of humanized antibodies

[0118] 4.1 Humanized Antibody Design

[0119] The homology of the heavy and light chain variable regions of the G085 / G136 antibody with the human germline gene sequence (according to the Kabat numbering system) was compared. The germline sequence with the highest homology was selected as the template sequence. The CDR region of G085 / G136 was transplanted onto the template sequence to form a CDR transplanted antibody, labeled as G085-H0L0. Its amino acid sequence is as follows, where H0 is the heavy chain variable region sequence and L0 is the light chain variable region sequence.

[0120] >The amino acid sequence of the heavy chain variable region of the G085 CDR transplanted antibody (G085-H0)

[0121] >Amino acid sequence of the light chain variable region of the G085 CDR transplanted antibody (G085-L0)

[0122] >The amino acid sequence of the heavy chain variable region of the G136 CDR transplanted antibody (G136-H0)

[0123] >Amino acid sequence of the light chain variable region of the G136CDR transplanted antibody (G136-L0)

[0124] Based on the structural alignment with the variable region of the rabbit parent antibody, reversion mutant sequences were designed. Different combinations of heavy and light chains were employed, and the optimal combination (SEQ ID NO: 13-14 and 15-16) was selected according to activity and degree of humanization. These combinations were then further combined with the human IgG constant region to obtain a full-length humanized antibody. The human IgG heavy chain constant region can be selected from IgG1, IgG2, or IgG4 subtypes, and the light chain constant region can be selected from Kappa or lambda subtypes, or other constant regions known in the art. For example, the heavy chain constant region of G085 / G136 is selected from IgG4 S228P, and the light chain constant region is selected from the Kappa chain. The constant region sequences are as follows.

[0125] >Amino acid sequence of the S228P heavy chain constant region of human IgG4

[0126] >Amino acid sequence of the light chain constant region of human Kappa

[0127] Furthermore, a series of mutations were performed on the CDR region of the parental G136 humanized molecule, and a better mutant molecule G136-1 was obtained through structural docking. Similarly, it was combined with the constant region of human IgG to obtain a full-length humanized antibody.

[0128] >G136-1 Heavy Chain Variable Region Amino Acid Sequence

[0129] >G136-1 light chain variable region amino acid sequence

[0130] 4.2 Expression and purification of humanized antibodies

[0131] The variable and constant regions of the selected combination were ligated to obtain the full-length antibody sequence. The amino acid sequence of the full-length antibody G085 / G136 / G136-1 is shown below. The sequence encoding this antibody was cloned into the pTT5 vector to construct an expression plasmid. Expi293F cells with a viability of 95% or higher were seeded in expression medium at a density of 0.8E6 / mL and cultured for 24 hours at 37°C, 130 rpm, and 6% CO2. The mass ratio of 1 mg / mL PEI transfection reagent to plasmid was 2:1. Taking the transfection of 200 mL of Expi293F cells as an example, 10 mL of Opti-MEM and 200 μg of plasmid were mixed and incubated for 5 min; another 10 mL of Opti-MEM and 400 μL of PEI were mixed and incubated for 5 min. The plasmid mixture and PEI mixture were then mixed and incubated at room temperature for 15 min. The plasmid and PEI mixture was slowly added to 200 mL of Expi293F cells and cultured in a shaker at 37°C and 130 rpm in 6% CO2. 18-24 h after transfection, 0.8% of the transfection volume of 250 mM sodium valproate solution was added. On days 1 and 3 after transfection, 5% of the culture medium was added. On day 5 after transfection, the cell supernatant was collected, centrifuged at high speed, and filtered to remove impurities for purification. The recombinant antibody expression supernatant was purified using a Protein A column. The column was washed with PBS buffer until the A280 reading dropped to baseline, and the target protein was eluted with 100 mM glycine (pH 3.0). Immediately after elution with 1 M Tris-HCl (pH 8.0), the collected purified antibody was replaced with PBS, and the absorbance at 280 nm was measured to calculate the antibody concentration.

[0132] >G085 heavy chain amino acid sequence

[0133] >G085 light chain amino acid sequence

[0134] >G136 heavy chain amino acid sequence

[0135] >G136 light chain amino acid sequence

[0136] >G136-1 heavy chain amino acid sequence

[0137] >G136-1 light chain amino acid sequence

[0138] Example 5: Determination of anti-PACAP antibody affinity

[0139] 5.1 ELISA-based PACAP27 / PACAP38 / VIP binding activity

[0140] The binding activity of the expressed humanized antibody to the antigen proteins PACAP27 / PACAP38 / VIP was detected by ELISA, as follows: Recombinant human PACAP27 / PACAP38 / VIP protein was diluted to 1 μg / mL with PBS buffer (pH 7.4), and 100 μL was coated onto a 96-well microplate (Costar, 42592) at 4°C overnight. The next day, the liquid in the wells was discarded, and the plate was washed three times with PBST (300 μL / well). Then, blocking buffer (PBST containing 3% BSA) was added to each well, and the plate was blocked at 37°C for 2 h. After blocking, discard the liquid in the wells, wash the plate three times with PBST (300 μL / well), then add 100 μL of serially diluted antibody sample (initial concentration 10 μg / mL), incubate at 37°C for 1 h, wash three times with PBST (300 μL / well), add 1:5000 diluted HRP-labeled Goat Anti-human IgG (H+L) secondary antibody, incubate at 37°C for 1 h, wash four times with PBST (300 μL / well), develop the color and read the absorbance at 450 nm on a microplate reader (see Figure 1-3), calculate the binding EC50 of PACAP antibody to PACAP27 / PACAP38 / VIP proteins. 50 The results (Table 1) show that the antibodies disclosed in this paper have good binding activity with both PACAP27 and PACAP38, and G085 also has good binding activity with VIP. Alder-Ab1H is the control antibody, and its sequence is as follows:

[0141] >Alder-Ab1H heavy chain amino acid sequence

[0142] Alder-Ab1H light chain amino acid sequence

[0143] Table 1. EC50 values ​​of PACAP antibody binding to PACAP27 / PACAP38 / VIP proteins

[0144] In the table, N / A indicates that in the ELISA method, the binding curve between the antibody and VIP cannot be fully plotted, so the EC50 value cannot be calculated.

[0145] 5.2 Determination of antibody affinity constant

[0146] The affinity constants of two humanized antibodies, G085 and G136, and the control antibody Alder Ab1H for PACAP38 / PACAP27 / VIP were determined by surface plasmon resonance (SPR) using a ProteinA chip (Cytiva, Cat. No.: 29127555) with a Biacore 8K instrument. Antibodies were injected at a flow rate of 10 μL / min for 12 s. Different concentrations of PACAP38 / PACAP27 / VIP protein were injected at a flow rate of 30 μL / min for 120 s, followed by monitoring for dissociation for 360 s. Finally, the chip was regenerated by flowing glycine (pH 1.7) at a flow rate of 30 μL / min for 30 s. Kinetic parameters were calculated using the 1:1 Binding model in the Bia-evaluation analysis software. The affinity constant, expressed as KD, was calculated from the kd / ka (dissociation rate / binding rate) ratio. The affinity assay results for the four antibodies G085, G136, G136-1, and Alder Ab1H are as follows: G085, G136, and G136-1 all have strong affinity for PACAP38 / PACAP27, and G085 also has a strong affinity for VIP.

[0147] Table 2. Affinity of PACAP antibodies to PACAP27 / PACAP38 / VIP proteins

[0148] Example 6. The ability of anti-PACAP antibody to neutralize PACAP27 / PACAP38 / VIP-induced cAMP production

[0149] 6.1 The ability of anti-PACAP antibody to neutralize PACAP27 / PACAP38 / VIP-induced PAC1R signaling was tested in a cell-based assay.

[0150] The antibody solution, serially diluted 3x, was incubated with PACAP38 (1 nM) / PACAP27 (1 nM) / VIP (10 nM) at a final concentration of 4x for 30 minutes. While incubating the antigen-antibody mixture, HEK293 cells overexpressing PAC1R were digested, washed, and resuspended in cell culture medium at 1x10⁶ cells / ml. Cells (5 μL) and the antigen-antibody complex (5 μL) were transferred to a 384-well plate and incubated with shaking at room temperature for 1 hour. After incubation, Eu³⁺-labeled mAb anti-cAMP (5 μL) and d²-labeled cAMP (5 μL) from a cAMP detection kit (62AM4PEC, Cisbio) were added, and the plate was incubated with shaking for 1 hour. After incubation, the plate was read (excitation 330 nm, emission 620 / 665 nm), and the ratio of 620:665 signals was determined (Figure 4-8). The IC50 value (nM) of the antibody neutralizing PACAP27 / PACAP38 / VIP-induced PAC1R signal was calculated. The results showed that G085 neutralized PACAP-induced PAC1R signal with similar activity to the control, while G136-1 exhibited superior blocking activity compared to G136.

[0151] Table 3. IC50 values ​​of anti-PACAP antibodies neutralizing PACAP27 / PACAP38 / VIP-induced PAC1R signaling.

[0152] In the table, N / A indicates that the antibody is completely unable to neutralize the VIP-induced PAC1R signal, so the IC50 value cannot be calculated.

[0153] 6.2 The ability of anti-PACAP antibody to neutralize PACAP27 / PACAP38 / VIP-induced VIPR1 signaling was tested in a cell-based assay.

[0154] The antibody solution, serially diluted 3x, was incubated with PACAP38 (1 nM) / PACAP27 (0.5 nM) / VIP (0.5 nM) at a final concentration of 4x for 30 minutes. While incubating the antigen-antibody mixture, HEK293 cells overexpressing VIPR1 were digested, washed, and resuspended in cell culture medium at 1x10⁶ cells / ml. Cells (5 μL) and the antigen-antibody complex (5 μL) were transferred to 384-well plates and shaken at room temperature for 1 hour. After incubation, Eu from a cAMP assay kit (62AM4PEC, Cisbio) was added. 3+Labeled mAb anti-cAMP (5 μL) and d2-labeled cAMP (5 μL) were added, and the plate was incubated for 1 hour with shaking. After incubation, the plate was read (excitation 330 nm, emission 620 / 665 nm), and the 620:665 signal ratio was determined (Figure 9-11). The ICSO value (nM) of the VIPR1 signal induced by the antibody neutralization of PACAP27 / PACAP38 / VIP was calculated. The results showed that G085 antibody highly neutralized PACAP and VIP to induce VIPR1 signal, while G136 neutralized PACAP with high activity to induce VIPR1 signal and neutralized VIP with low activity to induce VIPR1 signal.

[0155] Table 4. IC50 values ​​of anti-PACAP antibodies neutralizing PACAP27 / PACAP38 / VIP-induced VIPR1 signaling.

[0156] In the table, N / A indicates that the antibody is completely unable to neutralize the VIPR1 signal induced by VIP, so the IC50 value cannot be calculated.

[0157] 6.3 The ability of anti-PACAP antibody to neutralize PACAP27 / PACAP38 / VIP-induced VIPR2 signaling was tested in a cell-based assay.

[0158] The antibody solution, serially diluted 3x, was incubated with PACAP38 (0.5 nM) / PACAP27 (0.75 nM) / VIP (0.5 nM) at a final concentration of 4x for 30 minutes. While incubating the antigen-antibody mixture, HEK293 cells overexpressing VIPR2 were digested, washed, and resuspended in cell culture medium at 1x10⁶ cells / ml. Cells (5 μL) and the antigen-antibody complex (5 μL) were transferred to 384-well plates and shaken at room temperature for 1 hour. After incubation, Eu of a cAMP assay kit (62AM4PEC, Cisbio) was added. 3+ Labeled mAb anti-cAMP (5 μL) and d2-labeled cAMP (5 μL) were added, and the plate was incubated for 1 hour with shaking. After incubation, the plate was read (excitation 330 nm, emission 620 / 665 nm), and the 620:665 signal ratio was determined (Figure 12-14). The IC50 value (nM) of the antibody neutralizing PACAP27 / PACAP38 / VIP-induced VIPR2 signal was calculated. The results showed that G085 antibody highly neutralized PACAP and VIP to induce VIPR2 signal, while G136 neutralized PACAP with high activity to induce VIPR2 signal and VIP with low activity to induce VIPR2 signal.

[0159] Table 5. IC50 values ​​of anti-PACAP antibodies neutralizing PACAP27 / PACAP38 / VIP-induced VIPR2 signaling.

[0160] In the table, N / A indicates that the antibody is completely unable to neutralize the VIP-induced VIPR2 signal, so the IC50 value cannot be calculated.

[0161] Example 7: Anti-PACAP antibody inhibits PACAP27 / PACAP38-induced cAMP production in SHSY5Y cells.

[0162] SHSY5Y cells are a human neuroblastoma cell line. These cells were identified as expressing the PAC1R protein, and PACAP stimulation activated downstream signaling pathways to produce cAMP, which could be used to evaluate the biological activity of anti-PACAP antibodies. 3x serially diluted antibody solutions were incubated with PACAP38 (1.5 nM) / PACAP27 (1.5 nM) at a final concentration of 4x for 30 minutes. While incubating with the antigen and antibody, SHSY5Y cells were digested, washed, and resuspended in cell culture medium at 2x10⁶ cells / ml. Cells (5 μL) and the antigen-antibody complex (5 μL) were transferred to 384-well plates and shaken at room temperature for 1 hour. After incubation, Eu3+-labeled mAb anti-cAMP (5 μL) and d2-labeled cAMP (5 μL) from a cAMP assay kit (62AM4PEC, Cisbio) were added, and the plate was incubated for 1 hour with shaking. After incubation, the plate was read (excitation 330 nm, emission 620 / 665 nm), and the ratio of 620:665 signal was determined (Figure 15-16). The IC50 value (nM) of antibody neutralization of cAMP generated in PACAP27 / PACAP38-induced SHSY5Y cells was calculated. G085 showed stronger blocking ability in SHSY5Y cells than the control antibody.

[0163] Table 6. IC50 values ​​of antibody G085 for neutralizing PACAP27 / PACAP38-induced cAMP production in SHSY5Y cells.

Claims

1. An anti-PACAP antibody or its antigen-binding fragment, comprising a heavy chain variable region sequence VH, wherein the heavy chain variable region sequence VH comprises VHCDR1, VHCDR2, and VHCDR3, wherein VHCDR1, VHCDR2, and VHCDR3 have the following amino acid sequence: 1) SEQ ID NO: 1-3 or; 2) SEQ ID NO: 7-9 or: 3) SEQ ID NO: 7, SEQ ID NO: 35, SEQ ID NO:

9.

2. The anti-PACAP antibody or its antigen-binding fragment as described in claim 1, wherein the antibody or its antigen-binding fragment further comprises a light chain variable region VL, wherein the light chain variable region VL comprises VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1, VLCDR2, and VLCDR3 have the following amino acid sequence: 3) SEQ ID NO: 4-6 or; 4) SEQ ID NO: 10-12 or 6) SEQ ID NO: 36, SEQ ID NO: 11, SEQ ID NO:

12.

3. The anti-PACAP antibody or its antigen-binding fragment as described in claim 2, wherein the heavy chain variable region VH comprises VHCDR1, VHCDR2, and VHCDR3, and the light chain variable region VL comprises VLCDR1, VLCDR2, and VLCDR3; the CDR combinations of the heavy chain variable region VH and the light chain variable region are as follows, in the order of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, and VLCDR3: i) SEQ ID NO: 1-6, or; ii) SEQ ID NO: 7-12, or iii) SEQ ID NO: 7, SEQ ID NO: 35, SEQ ID NO: 9, SEQ ID NO: 36, SEQ ID NO: 11, SEQ ID NO:

12.

4. The anti-PACAP antibody or its antigen-binding fragment as described in claim 3, wherein the heavy chain variable region VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 13, 15, 17, 19, or 37.

5. The anti-PACAP antibody or its antigen-binding fragment as described in claim 3, wherein the light chain variable region VL comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity with SEQ ID NO: 14, 16, 18, 20, or 38.

6. The anti-PACAP antibody or its antigen-binding fragment according to any one of claims 3-5, comprising a combination of the following heavy chain variable region VH and light chain variable region VL: SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20 and SEQ ID NO: 37 and 38.

7. The anti-PACAP antibody or its antigen-binding fragment as described in any one of claims 1-6, wherein it satisfies at least one of the following two conditions: (1) The antibody or its antigen-binding fragment is selected from full-length antibody, Fab, Fab', F(ab')2, Fv, scFv, nanobody (single-domain antibody) or minimum recognition unit (MRU); (2) The antibody is a rabbit-derived, chimeric, or humanized antibody or a fully human antibody.

8. The anti-PACAP antibody or its antigen-binding fragment as claimed in any one of claims 1-7, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region (CH), the light chain comprises a light chain constant region (CL), and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody or a primate antibody. Preferably, said antibody or antigen binding fragment thereof is characterized in that, Both the heavy chain constant region and the light chain constant region of the antibody or antigen-binding fragment are derived from human IgG antibody. Preferably, the heavy chain constant region is a constant region of human IgG1, IgG2, or IgG4 subtypes. More preferably, the heavy chain constant region is IgG4 S228P, IgG4 S228P / L235E (IgG4-PE), IgG2 H268Q / V309L / A330S / P331S (IgG2m4), IgG2 V234A / G237A / P238S / H268A / V309L / A330S / P331S (IgG2c4d), IgG1 L234A / L235A (LALA), or IgG1 L234A L235A P329G (LALA-PG). Preferably, the light chain constant region is a human-derived Kappa or lambda chain constant region; Preferably, the heavy chain constant region of the antibody or antigen-binding fragment includes SEQ ID NO: 31, and the light chain constant region of the antibody or antigen-binding fragment includes SEQ ID NO:

32.

9. The anti-PACAP antibody or antigen-binding fragment thereof as claimed in claim 8, wherein the heavy chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 21, 23 or 39; and the light chain of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 22, 24 or 40; Preferably, the combination of heavy and light chains of the antibody or its antigen-binding fragment is SEQ ID NO: 21-22, SEQ ID NO: 23-24 or SEQ ID NO: 39-40.

10. A humanized anti-PACAP antibody, characterized in that The following sequence fragments are inserted into the frame regions (FRs) of the heavy chain variable region and the light chain variable region of human IgG in the order of VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3: i) SEQ ID NO: 1-6, or ii) SEQ ID NO: 7-12; Preferably, the framework region of the variable region of the human IgG heavy chain is IGHV3-64*04, IGHV3-66*01, IGHV3-53*04, IGHV3-53*01, IGHV3-NL1*01, IGHV3-23*04, IGHV3-30*01, IGHV3-30*02, IGHV3-30*09, IGHV3-53*06, IGHV3-53*03, IGHV3-53*02, IGHV3-48*01, IGHV3-74*01, or IGHV3-23*01; Preferably, the framework region of the variable region of the human IgG light chain is IGKV1-5*01, IGKV1-5*02, IGKV1-39*01, IGKV1-9*01, IGKV1-NL1*01, IGKV1-17*01, IGKV1-12*01, IGKV1-6*01, IGKV1-17*02, IGKV1-13*02, or IGKV1-33*01.

11. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or antigen-binding fragment of any one of claims 1-9 or the humanized anti-PACAP antibody of claim 10.

12. A vector, characterized in that, The carrier contains the nucleic acid molecule as described in claim 11.

13. A host cell, characterized in that, The host cell contains a vector of claim 12 or a chromosome in which the nucleic acid molecule of claim 11 is integrated, or expresses an antibody of any one of claims 1-9 or its antigen-binding fragment, or the humanized anti-PACAP antibody of claim 10.

14. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-9 or the humanized anti-PACAP antibody as described in claim 10, and a pharmaceutically acceptable adjuvant.

15. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the humanized anti-PACAP antibody according to claim 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, the cell according to claim 13, or the pharmaceutical composition according to claim 14, characterized in that, Used to prepare medicines or preparations for the prevention and / or treatment of headaches or migraines.

16. A diagnostic reagent for detecting the target antigen PACAP, said reagent comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-9 and / or a conjugate thereof, wherein the anti-PACAP antibody or antigen-binding fragment thereof is conjugated to one or more detectable moieties, said detectable moieties being selected from fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, chemiluminescent moieties, or mixtures thereof.

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