Antibody targeting CGRP and use thereof

Abstract

An antibody targeting CGRP and the use thereof. The antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3. The light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3. The antibody can specifically bind to human and rat CGRP polypeptides and α / β subtype CGRP polypeptides with high affinity, block the binding of CGRP to a receptor CGRPR thereof, reduce pain perception, and achieve the treatment of painful diseases such as migraines caused by CGRP. Additionally, the antibody can alleviate or treat hot flushes.

Description

Antibodies targeting CGRP and uses thereof

[0001] This application claims priority to Chinese patent application 2024115969172 with a filing date of 2024 / 11 / 8. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application belongs to the field of antibodies, and particularly relates to an antibody targeting CGRP and uses thereof. BACKGROUND

[0003] CGRP (calcitonin gene-related peptide) is composed of 37 amino acids, and belongs to the calcitonin family together with calcitonin, adrenomedullin and amylin. There are mainly two subtypes of α and β in human body, and the expression of the two subtypes in tissues and cell types is different, but mainly in neurons and some endocrine cells. Among them, α-CGRP is encoded by CALCA (Calcitonin α) gene, mainly exists in peripheral sensory nerves and central nervous system, and serves as a kind of neuropeptide, which is closely related to the occurrence of headache. β-CGRP is encoded by CALCB (Calcitonin β) gene, and mainly exists in the intestine, central nervous system and blood vessels.

[0004] The CGRP receptor is composed of CRLR (Calcitonin receptor-like receptor) and RAMP1 (Receptor activity-modifying protein 1). RAMP1 is an accessory protein responsible for binding to CRLR and forming a functional CGRP receptor (CGRPR). Upon CGRP binding to CGRPR, a conformational change in CGRPR occurs, activating intracellular G proteins (mainly Gs proteins). The activated Gs protein a subunit binds GTP (guanosine triphosphate), leading to dissociation of the G protein trimer into G a subunit and G b y subunit, further activating Adenylyl Cyclase. Adenylyl Cyclase catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP), leading to an increase in intracellular cAMP levels. The increased cAMP levels activate protein kinase A (PKA), which is able to phosphorylate a variety of downstream target proteins, modulating their activities, including ion channels, transcription factors, and other enzymes. Specific phosphorylation targets can include calcium ion channels, affecting intracellular calcium ion concentration by modulating calcium ion channels, thereby modulating the contraction and relaxation of vascular smooth muscle. It can also phosphorylate cAMP Response Element-Binding Protein (CREB), making it an activated transcription factor, promoting changes in gene expression, which can affect cell growth, differentiation, and survival. It can also activate phospholipase C (PLC), initiating inositol triphosphate (IP3) and diacylglycerol (DAG) signaling pathways, further modulating calcium ion release and protein kinase C (PKC) activity.

[0005] Migraine is a type of primary headache and a common neurological disorder. Diagnosis relies primarily on medical history and symptom description; the International Headache Society (IHS) provides diagnostic criteria for migraine. Migraines typically present as moderate to severe headaches, often accompanied by other symptoms such as nausea, vomiting, and sensitivity to light and sound. Migraines are throbbing and worsen with activity. Most patients experience unilateral headaches, but some experience bilateral headaches, and some experience alternating pain between the left and right sides. Each attack lasts from 4 to 72 hours, with specific manifestations and severity varying from person to person. Some migraine sufferers experience aura symptoms before the headache, known as "migraine aura." Aura symptoms may include visual changes (such as visual field defects, flashes of light, jagged patterns), sensory changes (such as numbness or tingling), and speech difficulties; the aura usually lasts 5 to 60 minutes. Some migraine sufferers also experience a prodromal phase, known as migraine without aura. The exact pathogenesis of migraines is not fully understood, but the following factors are believed to play a role: genetic factors, neuronal overexcitation, vascular changes, changes in neurotransmitter levels, diet, mental stress or emotional fluctuations, sleep, environmental factors, and hormonal changes, among others. Migraines are a complex disease, and although there is currently no cure, most patients can effectively control their symptoms and reduce the frequency and severity of attacks through appropriate medication and lifestyle management.

[0006] CGRP plays a crucial role in the occurrence and development of migraines. Recent studies have revealed the importance of CGRP in the pathological mechanisms of migraines, accelerating the development of novel migraine treatments. CGRP is a potent vasodilator that relaxes vascular smooth muscle, leading to vasodilation. CGRP also plays an important role in pain transmission, particularly in pain perception following inflammation and nerve damage. CGRP is also involved in the regulation of the central and peripheral nervous systems, including neurotransmitter release and neuronal excitability. During a migraine attack, CGRP release in the trigeminal nerve increases, thereby activating its receptors (mainly CGRP receptors), leading to vasodilation and perivascular inflammation, which in turn causes headache. CGRP-induced vasodilation and increased blood flow are considered part of the mechanism of migraine pain. CGRP can promote the release of inflammatory mediators, leading to neurogenic inflammation, which further stimulates the trigeminal nerve, enhancing pain perception.

[0007] The application of antibody drugs in the treatment of migraines, particularly monoclonal antibodies targeting calcitonin gene-related peptide (CGRP) and its receptor, has become a major breakthrough in migraine treatment in recent years. These drugs offer the advantages of high specificity and targeting, effectively blocking CGRP-mediated migraine attacks without significantly affecting other physiological processes, thus reducing side effects. They also exhibit good tolerability; clinical trials and practical applications show that CGRP monoclonal antibodies are generally well-tolerated, with fewer side effects compared to traditional drugs, making them more suitable for long-term use. In terms of efficacy, antibody drugs have shown significant effects in preventing and reducing the frequency of migraine attacks. Many patients have experienced a significant reduction in the number of migraine attacks and a marked improvement in their quality of life after using antibody drugs. Many antibody drugs are administered at a low frequency, typically monthly or quarterly, and this long-acting approach not only improves patient adherence but also reduces the inconvenience of frequent medication. The development and application of CGRP antibody drugs represent a major scientific advancement in the field of migraine treatment. These drugs not only demonstrate the potential of modern biotechnology but also provide new insights into the treatment of other neurological diseases. Summary of the Invention

[0008] This invention provides a neutralizing antibody against human CGRP, capable of specifically binding to human α-CGRP and β-CGRP and competitively inhibiting CGRP-mediated downstream signaling pathways. The CGRP antibody or antigen-binding fragment provided by this invention holds promise for the treatment of headaches such as migraines.

[0009] A first aspect of the present invention provides an antibody or antigen-binding portion thereof targeting CGRP, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3.

[0010] The CDR-H1 comprises an amino acid sequence as shown in any one of SEQ ID NO:77-83, the CDR-H2 comprises an amino acid sequence as shown in any one of SEQ ID NO:84-95, and the CDR-H3 comprises an amino acid sequence as shown in any one of SEQ ID NO:96-108; or,

[0011] The CDR-L1 contains an amino acid sequence as shown in any one of SEQ ID NO:109-116, the CDR-L2 contains an amino acid sequence as shown in any one of SEQ ID NO:117-122, and the CDR-L3 contains an amino acid sequence as shown in any one of SEQ ID NO:123-130.

[0012] In some embodiments, the heavy chain variable region and the light chain variable region of the antibody are combined in the following ways:

[0013] The CDR-H1 has the amino acid sequence shown in SEQ ID NO:77; the amino acid sequence of the CDR-H2 has at least 80% homology with SEQ ID NO:85, preferably with amino acid residue differences at positions 5, 7, 9, and / or 10; the amino acid sequence of the CDR-H3 has at least 80% homology with SEQ ID NO:97, preferably with amino acid residue differences at positions 1, 2, 6, and / or 8; the amino acid sequence of the CDR-L1 has at least 80% homology with SEQ ID NO:109, preferably with amino acid residue differences at positions 5 and / or 9; the CDR-L2 has the amino acid sequence shown in SEQ ID NO:117; the amino acid sequence of the CDR-L3 has at least 90% homology with SEQ ID NO:123, preferably with amino acid residue differences at position 8; or,

[0014] The CDR-H1 contains an amino acid sequence as shown in any one of SEQ ID NO:78-83, the CDR-H2 contains an amino acid sequence as shown in any one of SEQ ID NO:90-95, and the CDR-H3 contains an amino acid sequence as shown in any one of SEQ ID NO:104-108; the CDR-L1 contains an amino acid sequence as shown in any one of SEQ ID NO:111-116, the CDR-L2 contains an amino acid sequence as shown in any one of SEQ ID NO:118-122, and the CDR-L3 contains an amino acid sequence as shown in any one of SEQ ID NO:125-130.

[0015] In this invention, the amino acid sequences of the CDRs listed above are all as shown in the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as based on the Kabat definition rules and the Chothia definition rules based on the position of the structural loop region (see JMol Biol 273:927-48, 1997). In this invention, the combined definition rules, which include both the Kabat and Chothia definitions, can also be used to determine the amino acid residues in the variable domain sequence. The combined definition rules combine the scopes of the Kabat and Chothia definitions, thereby taking a broader range. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., the variable region) should be understood to encompass the complementarity-determining region defined by any of the known schemes described above. Although the scope of protection claimed in this invention is based on the sequence shown in the Kabat definition rules, the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0016] In this invention, the term "antigen-binding portion" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parent antibody binding activity. Preferably, the antibody fragment retains at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% or more of the binding affinity of the parent antibody to the target.

[0017] In some embodiments, CDR-H1 has the amino acid sequence shown in SEQ ID NO:77; the amino acid sequence of CDR-H2 differs from SEQ ID NO:85 by three or fewer amino acid residues from K5N, G7N, T9I, and I10V; the amino acid sequence of CDR-H3 differs from SEQ ID NO:97 by two or fewer amino acid residues from L1I, V2I, F6Y, and F8S; the amino acid sequence of CDR-L1 differs from SEQ ID NO:109 by two or fewer amino acid residues from N5D and / or N9A; and the amino acid sequence of CDR-L3 differs from SEQ ID NO:123 by either not containing or containing the amino acid residue Y8L.

[0018] In some embodiments, CDR-H1 has an amino acid sequence as shown in SEQ ID NO:77, CDR-H2 has an amino acid sequence as shown in any one of SEQ ID NO:84-89, and CDR-H3 has an amino acid sequence as shown in any one of SEQ ID NO:96-103; CDR-L1 has an amino acid sequence as shown in any one of SEQ ID NO:109-110, CDR-L2 has an amino acid sequence as shown in SEQ ID NO:117, and CDR-L3 has an amino acid sequence as shown in any one of SEQ ID NO:123-124.

[0019] Amino acid residue differences refer to mutations in amino acids compared to the original amino acid sequence, including the insertion, deletion, or substitution of amino acid residues. As is known to those skilled in the art, amino acids exist as residues in an amino acid sequence; therefore, when referring to a single amino acid in this document, it often has the same meaning as an amino acid residue and can be used interchangeably.

[0020] In this invention, the mutation may include mutations known to those skilled in the art, such as mutations that may be performed on antibodies during production or application, such as mutations on potential post-translational modifications (PTMs) sites, particularly in the CDR region, including mutations related to antibody aggregation, asparagine deamidation sites (NG, NS, NH, etc.), aspartic acid isomerization (DG, DP, etc.) sensitive sites, N-glycosylation (N-{P}S / T) sensitive sites, and oxidation sensitive sites.

[0021] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same bases or amino acid monomer subunits—for example, if a position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0022] In some embodiments, the heavy chain variable region and the light chain variable region of the antibody are selected from the following combinations:

[0023] 1) The CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 is selected from the amino acid sequences shown in any one of SEQ ID NO:85-88, and the CDR-H3 is selected from the amino acid sequences shown in any one of SEQ ID NO:97-101; the CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:123;

[0024] 2) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:84, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:96; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123.

[0025] 3) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:89, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:102; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123.

[0026] 4) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:84, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:103; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:110, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:124.

[0027] 5) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:78, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:90, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:104; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:111, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:118, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:125.

[0028] 6) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:79, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:91, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:105; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:112, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:118, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:126.

[0029] 7) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:80, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:92, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:105; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:113, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:119, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:127.

[0030] 8) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:81, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:93, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:106; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:114, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:120, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:128.

[0031] 9) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:82, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:94, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:107; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:115, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:121, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:129; and,

[0032] 10) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:83, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:95, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:108; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:116, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:122, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:130.

[0033] In some embodiments, the heavy chain variable region and the light chain variable region of the antibody are selected from the following combinations:

[0034] 1) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:85, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:97; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123;

[0035] 2) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:86, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:97; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123.

[0036] 3) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:87, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:97; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123.

[0037] 4) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:89, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:97; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123.

[0038] 5) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:85, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:98; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123.

[0039] 6) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:85, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:99; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123;

[0040] 7) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:85, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:100; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123.

[0041] 8) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:85, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:101; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123;

[0042] 9) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:89, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:102; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123.

[0043] 10) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:88, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:102; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:123.

[0044] In some embodiments, the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 10, 12, 14, and 15; the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 11, 13, and 16; or,

[0045] The heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 17, 19, 21, 23, 25 and 27; the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 18, 20, 22, 24, 26 and 28.

[0046] In some embodiments, the heavy chain variable region and the light chain variable region of the antibody are selected from the following combinations:

[0047] 1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:10; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:11;

[0048] 2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:12; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:13;

[0049] 3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:14; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:13;

[0050] 4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:16;

[0051] 5) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:18;

[0052] 6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:20;

[0053] 7) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:21; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:22;

[0054] 8) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:24;

[0055] 9) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:25; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:26; and,

[0056] 10) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:27; the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:28.

[0057] In some embodiments, the antibody or its antigen-binding moiety comprises a heavy chain and a light chain; wherein,

[0058] The heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 29-33 and 39-47; the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 34-38; or,

[0059] The heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO:48-52, 58-63, and 69-72; the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:53-57, 64-68, and 73-76.

[0060] In some embodiments, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:29; the light chain comprises the amino acid sequence shown in SEQ ID NO:34; or,

[0061] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:31; the light chain comprises the amino acid sequence shown in SEQ ID NO:36; or,

[0062] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:31; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0063] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:31; the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or,

[0064] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:32; the light chain comprises the amino acid sequence shown in SEQ ID NO:36; or,

[0065] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:32; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0066] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:32; the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or,

[0067] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:33; the light chain comprises the amino acid sequence shown in SEQ ID NO:36; or,

[0068] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:33; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0069] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:33; the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or,

[0070] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:39; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0071] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:40; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0072] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:41; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0073] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:42; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0074] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:43; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0075] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:44; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0076] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:45; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0077] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:46; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0078] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:47; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or,

[0079] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:48; the light chain comprises the amino acid sequence shown in SEQ ID NO:53; or,

[0080] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:50; the light chain comprises the amino acid sequence shown in SEQ ID NO:55; or,

[0081] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:50; the light chain comprises the amino acid sequence shown in SEQ ID NO:56; or,

[0082] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:50; the light chain comprises the amino acid sequence shown in SEQ ID NO:57; or,

[0083] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51; the light chain comprises the amino acid sequence shown in SEQ ID NO:55; or,

[0084] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51; the light chain comprises the amino acid sequence shown in SEQ ID NO:56; or,

[0085] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51; the light chain comprises the amino acid sequence shown in SEQ ID NO:57; or,

[0086] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:52; the light chain comprises the amino acid sequence shown in SEQ ID NO:55; or,

[0087] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:52; the light chain comprises the amino acid sequence shown in SEQ ID NO:56; or,

[0088] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:52; the light chain comprises the amino acid sequence shown in SEQ ID NO:57; or,

[0089] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:58; the light chain comprises the amino acid sequence shown in SEQ ID NO:64; or,

[0090] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:60; the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or,

[0091] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:60; the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or,

[0092] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:60; the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or,

[0093] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:61; the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or,

[0094] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:61; the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or,

[0095] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:61; the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or,

[0096] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:62; the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or,

[0097] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:62; the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or,

[0098] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:62; the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or,

[0099] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:63; the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or,

[0100] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:63; the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or,

[0101] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:63; the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or,

[0102] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:69; the light chain comprises the amino acid sequence shown in SEQ ID NO:73; or,

[0103] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:71; the light chain comprises the amino acid sequence shown in SEQ ID NO:75; or,

[0104] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:71; the light chain comprises the amino acid sequence shown in SEQ ID NO:76; or,

[0105] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:72; the light chain comprises the amino acid sequence shown in SEQ ID NO:75; or,

[0106] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:72; the light chain comprises the amino acid sequence shown in SEQ ID NO:76; or,

[0107] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:131; the light chain comprises the amino acid sequence shown in SEQ ID NO:37.

[0108] This invention utilizes chemically solid-phase synthesized α-CGRP and β-CGRP short peptides, or samples of these short peptides conjugated with KLH and BSA, as immunogens. Healthy, specific pathogen-free (SPF-grade) Balb / c mice and New Zealand white rabbits were selected as immunization animals. After immunization, blood was excised from the mice or rabbits, and serum was collected. The titer of the human α-CGRP peptide bound to the immune serum was detected by ELISA. Several animals with the highest titers were selected for immunization shock. Furthermore, candidate antibodies with high affinity and excellent functional assays were screened using hybridoma screening or flow cytometry B-cell screening. Chimeric antibody samples were then constructed and further confirmed in vitro. Finally, seven preferred monoclonal antibodies with good activity were obtained, named 4B1D5, 4B1A9, 4B3A8, 4B6A9, RF03H8, RF03E2, and RF03E212.

[0109] A second aspect of the present invention provides a CGRP conjugate comprising one, two or more antibodies or antigen-binding moieties as described in the first aspect of the present invention.

[0110] In some embodiments, the CGRP conjugate also has one or more of the following features:

[0111] 1) The CGRP conjugate is Fab, Fab', F(ab')2, scFab, Fv, scFv or a full-length antibody;

[0112] 2) The CGRP conjugate is an antibody fusion protein; for example, a Fab fusion protein or a single-chain fusion protein;

[0113] 3) The CGRP conjugate is a chimeric antigen receptor;

[0114] 4) The CGRP conjugate is a human, mouse, or rabbit antibody; and,

[0115] 5) The CGRP conjugate is IgG in the form of YTE.

[0116] Examples of antigen-binding fragments of the present invention include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, scFv, linear antibodies, single (heavy) chain antibodies, nanobodies, and domain antibodies. Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0117] The term "antibody" as used in this invention can refer to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0118] A single-chain antibody fragment (scFv) is an antibody composed of the variable regions of the heavy chain and the variable region of the light chain linked by a linker, which is a short peptide of several amino acids, such as 15 to 20 amino acids.

[0119] An intact antibody has a complete Ig molecule structure, usually IgG or IgM. The antibody as a whole consists of four polypeptide chains: two light chains (L chain) and two heavy chains (H chain) with the same structure. The four chains form a typical "Y"-shaped antibody structure.

[0120] Fusion antibodies, also known as antibody fusion proteins (Ig fusion proteins), are products obtained by fusing antibody fragments with other bioactive proteins using genetic engineering techniques. Due to the differences in fusion proteins, these antibody fusion proteins possess a variety of biological functions, and the expressed recombinant protein does not affect the antigen-binding ability of the single-chain antibody, nor does it affect the biological characteristics of the protein it is fused with.

[0121] As used herein, the term "specificity" with respect to antibodies refers to an antibody that recognizes a specific antigen but substantially does not recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, this interspecies cross-reactivity itself does not alter the antibody's classification according to specificity. In another instance, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, this cross-reactivity itself does not alter the antibody's classification according to specificity. In some cases, the terms "specificity" or "specific binding" may be used to refer to the interaction of an antibody, protein, or peptide with a second chemical substance, meaning that the interaction depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, an antibody generally recognizes and binds to a specific protein structure, rather than the protein itself. If an antibody is specific for epitope "A," then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0122] As used in this article, the term "chimeric antigen receptor" or "CAR" refers to an engineered transmembrane protein that combines the specificity of an antigen-specific antibody with the function of a T-cell receptor. Generally, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. Specifically, it includes an extracellular domain capable of binding antigens (extracellular binding domain), a hinge domain, a transmembrane domain (transmembrane region), and a polypeptide that transmits cytoplasmic signals to the hinge domain (i.e., the intracellular signaling domain). The hinge domain can be considered as part of the structure that provides flexibility to the extracellular antigen-binding region. The intracellular signaling domain refers to proteins that transmit information into the cell via defined signal transduction pathways by generating second messengers to regulate cellular activity, or proteins that act as effectors corresponding to such messengers, generating signals that can promote the immune effector function of CAR-containing cells (e.g., CAR-T cells). The intracellular signaling domain includes the signal transduction domain and may also include co-stimulatory intracellular domains derived from co-stimulatory molecules.

[0123] The term "human antibody" includes antibodies having variable and constant regions of human immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" does not include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human backbone sequence (i.e., "humanized antibody").

[0124] "YTE" form of IgG is an engineered immunoglobulin G (IgG) antibody whose Fc region is specifically mutated to enhance its binding to the neonatal Fc receptor (FcRn). This modification aims to prolong the half-life of the IgG antibody, thereby improving its efficacy and ease of administration. The most common mutations in YTE form of IgG are M252Y, S254T, and T256E. These substitutions refer to amino acid positions 252, 254, and 256, replacing the original amino acids with tyrosine (Y), threonine (T), and glutamic acid (E), respectively. However, the mutation sites are not fixed; the mutation sites of YTE antibodies can be adjusted according to specific applications and targets to optimize the overall performance of the antibody. These mutations are mainly located in the CH2 domain of the Fc region. In this invention, the YTE is M254Y / S256T / T258E. These mutations enhance the binding strength of Fc to FcRn, thereby prolonging the half-life of the IgG antibody without affecting its binding ability to specific antigens. This modified IgG antibody has various therapeutic applications, and has shown good results in treating diseases such as paroxysmal sleep disorder.

[0125] Other forms of antigen-binding fragments, including but not limited to Fab, scFv, and chimeric antibodies, can be prepared using the variable regions of the aforementioned antibodies. Chimeric antibodies contain variable regions derived from mice or rabbits and constant regions derived from humans, and their preparation methods are known in the art. The chimeric antibody names corresponding to the seven monoclonal antibody samples mentioned above are 4B1D5-VHVL, 4B1A9-VHVL, 4B3A8-VHVL, 4B6A9-VHVL, RF03H8-VHVL, RF03E2-VHVL, and RF03E212-VHVL, respectively. To further reduce immunogenicity, the CDRs of the aforementioned mouse or rabbit antibodies are transplanted into suitable human frame regions to generate the desired humanized antibodies; methods for preparing humanized antibodies are known in the art. The humanization process is briefly described as follows: One or more human antibody frameworks highly homologous to the donor antibody sequence are selected. The donor antibody's CDR is transplanted into the human antibody framework. Based on the antibody's three-dimensional structure, key light and heavy chain amino acid residues are then selected for reversion mutation to maintain the affinity and biological activity of the humanized antibody. Finally, a humanized antibody sequence is obtained. This humanized antibody sequence is cloned into an expression vector and expressed in CHO cells. The resulting recombinant humanized anti-CGRP antibody molecule is obtained through one-step purification using a ProteinA affinity column. Affinity assays and cell function tests confirm that the obtained humanized antibody can specifically bind to human α-CGRP and β-CGRP, as well as rat α-CGRP and β-CGRP, further inhibiting the activation of CGRP-induced signaling pathways and achieving the therapeutic effect of migraine. 4B1D5-VHVL, 4B1A9-VHVL, 4B3A8-VHVL, and 4B6A9-VHVL share certain sequence similarities. Humanization of 4B1D5-VHVL yielded the humanized sequence 4B1D5-H1L2. Using 4B1D5-H1L2 as the starting sequence, further engineering modifications were performed with similar sequences to enhance the affinity and activity of the anti-CGRP antibody, resulting in 4B1D5-H1L2-M9, hereinafter referred to as SZC465. The preferred humanized sequences obtained from RF03H8-VHVL, RF03E2-VHVL, and RF03E212-VHVL are RF03H8-H1L1, RF03E2-H3L1, and RF03E212-H2L2, respectively.

[0126] A third aspect of the present invention provides an isolated nucleic acid that encodes an antibody or its antigen-binding portion as described in the first aspect of the present invention or a CGRP conjugate as described in the second aspect of the present invention.

[0127] A fourth aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid as described in the third aspect of the present invention.

[0128] In some implementations, the recombinant expression vector is a plasmid, bacteriophage, or viral vector.

[0129] In some embodiments, the viral vector is a retroviral vector, an adenovirus vector, or an adeno-associated virus vector, and the retroviral vector is, for example, a lentiviral vector.

[0130] The term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors disclosed herein are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Suitable vectors include those designed for amplification and expansion or for expression, or both of the above. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules, or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0131] A fifth aspect of the present invention provides a transformant, said transformant being a host cell containing isolated nucleic acids as described in the third aspect of the present invention, or a recombinant expression vector as described in the fourth aspect of the present invention.

[0132] In some implementations, the host cell is a prokaryotic cell or a eukaryotic cell.

[0133] In some embodiments, the host cell is selected from yeast cells or mammalian cells; the mammalian cells are, for example, HEK293 cells or CHO cells.

[0134] As used herein, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. Host cells may be eukaryotic cells, such as plants, animals, fungi, or algae; or host cells may be prokaryotic cells, such as bacteria or protozoa.

[0135] Expression vectors can be transfected or introduced into suitable host cells. Various techniques can achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system, or TALEN system), transposons (Sleeping Beauty or PiggyBAC), gene guns, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are cultured in a medium and screened for suitable activity. The methods and conditions used to culture the resulting transfected cells and to recover the generated antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector and host cells used. Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide protrophic, biocidal (e.g., antibiotic) or heavy metal (e.g., copper) resistance to auxotrophic hosts. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cells via co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include splicing signals, as well as transcription promoters, enhancers, and termination signals.

[0136] The sixth aspect of the present invention provides a method for preparing an antibody or antigen-binding portion thereof targeting CGRP, the method comprising culturing a transformant as described in the fifth aspect of the present invention, and obtaining an antibody or antigen-binding portion thereof targeting CGRP from the culture.

[0137] A seventh aspect of the present invention provides a pharmaceutical composition comprising one or more of the following: an antibody or antigen-binding portion thereof as described in the first aspect of the present invention; a CGRP conjugate as described in the second aspect of the present invention; an isolated nucleic acid as described in the third aspect of the present invention; a recombinant expression vector as described in the fourth aspect of the present invention; and a transformant as described in the fifth aspect of the present invention.

[0138] In some embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0139] An eighth aspect of the present invention provides a CGRP detection reagent, the CGRP detection reagent comprising an antibody or its antigen-binding portion as described in the first aspect of the present invention, and / or a CGRP conjugate as described in the second aspect of the present invention. Furthermore, the CGRP detection reagent may also include standards, enzyme-labeled reagents, substrates, washing buffers, sample diluents, stop solutions, biotin-labeled antibody diluents, horseradish peroxidase-labeled avidin diluents, sealing films, and sealing bags. These reagents or materials are those well known to those skilled in the art.

[0140] The ninth aspect of the present invention provides the use of one or more of the following in the preparation of diagnostic or detection agents: an antibody or antigen-binding portion thereof as described in the first aspect of the present invention, a CGRP conjugate as described in the second aspect of the present invention, an isolated nucleic acid as described in the third aspect of the present invention, a recombinant expression vector as described in the fourth aspect of the present invention, a transformant as described in the fifth aspect of the present invention, and a pharmaceutical composition as described in the seventh aspect of the present invention.

[0141] In some implementations, the diagnostic or detection agent is used to diagnose or detect diseases, conditions, or symptoms mediated by CGRP.

[0142] The tenth aspect of the present invention provides the use of one or more of the following in the preparation of a medicament for the prevention or treatment of CGRP-mediated diseases, conditions or symptoms: an antibody or antigen-binding portion thereof as described in the first aspect of the present invention, a CGRP conjugate as described in the second aspect of the present invention, an isolated nucleic acid as described in the third aspect of the present invention, a recombinant expression vector as described in the fourth aspect of the present invention, and a transformant as described in the fifth aspect of the present invention.

[0143] In some implementations, the disease is selected from at least one of the following:

[0144] Migraine, cluster headache, tension headache, and hot flashes.

[0145] The eleventh aspect of the present invention provides a method for detecting CGRP, using one or more of the following: an antibody or its antigen-binding portion as described in the first aspect of the present invention, a CGRP conjugate as described in the second aspect of the present invention, an isolated nucleic acid as described in the third aspect of the present invention, a recombinant expression vector as described in the fourth aspect of the present invention, a transformant as described in the fifth aspect of the present invention, or a CGRP detection agent as described in the eighth aspect of the present invention.

[0146] In some embodiments of the present invention, the method is for non-diagnostic and / or therapeutic purposes, such as detecting the presence or absence of CGRP in a laboratory; or competing with other CGRP-targeting antibodies to detect whether there is competition between antibodies, i.e., whether the antigenic epitopes are the same or similar, etc.

[0147] One aspect of the present invention provides a method for diagnosing, preventing, improving, or treating CGRP-mediated diseases, symptoms, or conditions, the method comprising administering to a subject in need an effective amount of an antibody or its antigen-binding portion as described in the first aspect of the present invention, a CGRP conjugate as described in the second aspect of the present invention, an isolated nucleic acid as described in the third aspect of the present invention, a recombinant expression vector as described in the fourth aspect of the present invention, a transformant as described in the fifth aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention.

[0148] In some implementations, the CGRP-mediated disease, condition, or symptom is selected from at least one of the following:

[0149] Migraine, cluster headache, tension headache, and hot flashes.

[0150] As used herein, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "therapeuticly effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0151] Combinations of drugs containing anti-CGRP antibodies can be used for the prevention and treatment of headaches such as migraines. For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology, etc.

[0152] The present invention provides, in one aspect, an antibody or antigen-binding portion thereof as described in the first aspect of the present invention, a CGRP conjugate as described in the second aspect of the present invention, an isolated nucleic acid as described in the third aspect of the present invention, a recombinant expression vector as described in the fourth aspect of the present invention, a transformant as described in the fifth aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention, for the diagnosis, prevention, improvement, or treatment of CGRP-mediated diseases, symptoms, or conditions.

[0153] In some implementations, the CGRP-mediated disease, condition, or symptom is selected from at least one of the following:

[0154] Migraine, cluster headache, tension headache, and hot flashes.

[0155] By using techniques such as molecular docking between antibodies and peptides, it can be determined that although the epitopes of the anti-CGRP monoclonal antibody in this invention are also located in the C-terminal region of the peptide, the complex conformation is inconsistent with that of existing Galcanezmab, Fremanezumab, and Eptinezumab, and the epitopes are still different. Therefore, from a structural perspective, it can be explained that the monoclonal antibody in this invention has excellent blocking activity.

[0156] As used in this invention, the terms “comprising” or “including” are intended to mean that a composition and method includes the stated elements but excludes other elements, but, depending on the context, also includes the case of “consisting of”.

[0157] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0158] The reagents and raw materials used in this invention are all commercially available.

[0159] The positive and progressive effects of this invention are as follows: the antibody of this invention can specifically bind to human / rat CGRP peptides with high affinity, and can also bind to human and rat α / β subtype CGRP peptides with high affinity, blocking the binding of CGRP to its receptor CGRPR, thereby inhibiting the activation of downstream adenylate cyclase, reducing cAMP production, thereby alleviating pain perception and achieving the treatment of pain diseases such as migraine caused by CGRP.

[0160] In in vitro experiments, the antibody of this invention exhibits comparable or even superior affinity and functional activity compared to Fremanezumab and Eptinezumab. The antibody also demonstrates a long half-life in rats and good stability in vivo.

[0161] The anti-CGRP monoclonal antibody of this invention can significantly reduce the increase in cerebral blood flow in rats induced by capsaicin stimulation. Therefore, the anti-CGRP monoclonal antibody has high potential in the treatment of migraine. Attached Figure Description

[0162] Figure 1 shows the inhibitory activity of the 4B1D5 humanized antibody against cAMP production in the human-α-CGRP-driven HEK293-1B10 overexpressing cell line.

[0163] Figure 2 shows the inhibitory activity of 4B1D5-H1L2, 4B1A9, 4B3A8, and 4B6A9 on cAMP production in the human-α-CGRP-driven HEK293-1B10 overexpressing cell line.

[0164] Figure 3 shows the binding of the 4B1D5-H1L2 modified antibody to human-α-CGRP in an ELISA.

[0165] Figure 4 shows the ELISA binding of the 4B1D5-H1L2 modified antibody to rat-α-CGRP.

[0166] Figure 5 shows the inhibitory activity of the 4B1D5-H1L2 modified antibody against cAMP production in the human-α-CGRP-driven HEK293-1B10 overexpressing cell line.

[0167] Figure 6 shows the inhibitory activity of the RF03E2 humanized antibody against cAMP production in the human-α-CGRP-driven HEK293-1B10 overexpressing cell line.

[0168] Figure 7 shows the ELISA binding of different antibodies to human-α-CGRP.

[0169] Figure 8 shows the ELISA binding of different antibodies to human-β-CGRP.

[0170] Figure 9 shows the ELISA binding of different antibodies to rat-α-CGRP.

[0171] Figure 10 shows the ELISA binding of different antibodies to rat-β-CGRP.

[0172] Figure 11 shows the inhibitory activity of different antibodies on cAMP production in the human-α-CGRP-driven HEK293-1B10 overexpressing cell line.

[0173] Figure 12 shows the inhibitory activity of different antibodies on cAMP production in the human-α-CGRP-driven SK-N-MC cell line.

[0174] Figure 13 shows the inhibitory activity of different antibodies on cAMP production in human-α-CGRP-driven L6 cell lines.

[0175] Figure 14 shows the pharmacokinetic analysis of the antibody in rats.

[0176] Figure 15 shows the efficacy of the antibody in a rat blood flow model.

[0177] Figure 16 shows the area under the blood flow line (AUC). Detailed Implementation

[0178] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0179] Example 1: Preparation of peptide samples for anti-CGRP antibody preparation and testing

[0180] Keyhole limpet hemocyanin (KLH); α-calcium gene-related peptide (α-CGRP); bovine serum albumin (BSA). Mice or rabbits were immunized individually and cross-immunized with KLH-Human-α-CGRP, BSA-Human-α-CGRP, KLH-Rat-α-CGRP, BSA-Rat-α-CGRP, KLH-Human-α-CGRP (8-37), and Human-α-CGRP antigen, respectively. The screening agents used for anti-CGRP antibodies included His-Human-α-CGRP, Human-α-CGRP, Human-β-CGRP, Human-α-CGRP (K24FITC), KLH-Human-α-CGRP, BSA-Human-α-CGRP, KLH-Rat-α-CGRP, BSA-Rat-α-CGRP, KLH-Human-α-CGRP (8-37), KLH-Human-β-CGRP, BSA-Human-β-CGRP, KLH-Rat-β-CGRP, and BSA-Rat-β-CGRP. CGRP was conjugated to KLH or BSA at a 1:1 mass ratio. All peptide samples were prepared by Jier Biochemical.

[0181] The sequence information is as follows:

[0182] KLH-Human-α-CGRP:

[0183] KLH-CACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF(SEQ ID NO:1)-NH2

[0184] BSA-Human-α-CGRP:

[0185] BSA-CACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF(SEQ ID NO:1)-NH2

[0186] KLH-Human-α-CGRP(8-37):

[0187] KLH-CVTHRLAGLSRSGGVVKNNFVPTNVGSKAF(SEQ ID NO:2)-NH2

[0188] Human-α-CGRP:

[0189] ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF(SEQ ID NO:3)-NH2

[0190] His-Human-α-CGRP:

[0191] MGHHHHHHHHGGGSACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF(SEQ ID NO:4)-NH2

[0192] Human-α-CGRP(K24FITC):

[0193] ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF(SEQ ID NO:4)-NH2(K24FITC)

[0194] KLH-Human-β-CGRP:

[0195] KLH-CACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF(SEQ ID NO:5)-NH2

[0196] BSA-Human-β-CGRP:

[0197] BSA-CACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF(SEQ ID NO:5)-NH2

[0198] Human-β-CGRP:

[0199] CACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF(SEQ ID NO:5)-NH2

[0200] KLH-Rat-α-CGRP:

[0201] KLH-CSCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF(SEQ ID NO:6)-NH2

[0202] BSA-Rat-α-CGRP:

[0203] BSA-CSCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF(SEQ ID NO:6)-NH2

[0204] KLH-Rat-β-CGRP:

[0205] KLH-CSCNTATCVTHRLAGLLRRSGGVVKDNFVPTNVGSKAF(SEQ ID NO:7)-NH2

[0206] BSA-Rat-β-CGRP:

[0207] BSA-CSCNTATCVTHRLAGLLRRSGGVVKDNFVPTNVGSKAF(SEQ ID NO:7)-NH2

[0208] Example 2: Construction of cell lines overexpressing CRLR and RAMP1

[0209] To screen for antibodies that can block CGRP binding to the CRLR / RAMP1 receptor, a HEK293 cell line expressing both human CRLR and human RAMP1 receptors (CRLR / RAMP1) was constructed. The target gene CRLR / RAMP1 was cloned into the target cell line using non-liposome transfection to form a stable, high-expression cell line. First, the human CRLR and human RAMP1 genes were cloned into pCDNA3.1(+)-HygroB and pCDNA3.1(+)-Zeo plasmids, respectively. Then, using non-liposome transfection, human CRLR and RAMP1 were cloned into the HEK293 cell line using fugene 6 (Promega, Madison, WI, USA). The cells were then cultured for two weeks under a selection pressure of 400 μg / mL hygromycin B + 800 μg / mL bleomycin. Finally, HEK293 monoclonal cell line that simultaneously highly expresses human CRLR and RAMP1 was screened out by flow cytometry and named HEK293-hCGRPR-1B10.

[0210] The amino acid sequence of human CRLR protein (underlined regions are extracellular regions, double underlined regions are signal peptides).

[0211] The amino acid sequence of human RAMP1 protein (underlined areas are extracellular regions, double underlines are signal peptides).

[0212] Example 3: Screening of Anti-CGRP Monoclonal Mouse Hybridoma Cells

[0213] 3.1 Mouse Immunity and Hybridoma Fusion

[0214] Anti-human CGRP monoclonal antibodies were produced by immunizing mice. Balb / c mice, female, 6-8 weeks old (purchased from Hubei Provincial Experimental Animal Research Center), were used in the experiment. The mice that had adapted to the environment were immunized individually and cross-immunized with KLH-Human-α-CGRP, BSA-Human-α-CGRP, KLH-Rat-α-CGRP, BSA-Rat-α-CGRP, KLH-Human-α-CGRP (8-37), and Human-α-CGRP antigen. For the initial immunization, each mouse was subcutaneously injected with 50 μg of the fusion protein and Freund's complete adjuvant. Subsequent booster immunizations were performed every two weeks, with each mouse subcutaneously injected with 50 μg of the fusion protein and Freund's complete adjuvant. After the 3rd to 5th immunizations, mice with high and plateauing antibody titers in their serum were selected for a shock immunization, using the unadjuvanted fusion protein as the immunogen, with each mouse injected with 50 μg of the fusion protein. Three to four days later, the mice were sacrificed, and spleen cells were harvested and fused with myeloma cells at a 5:1 ratio. Using an optimized PEG-mediated fusion method, spleen lymphocytes were fused with myeloma Sp2 / 0 cells to obtain hybridoma cells. Hybridoma cells were seeded into 96-well plates at a density of 1E5 cells / well / 200 μL of culture medium and incubated statically at 37°C and 5% CO2.

[0215] 3.2 Hybridoma Screening

[0216] For initial screening, positive clones were identified using an ELISA binding assay targeting human CGRP. One well of an ELISA plate was coated overnight with 100 μL of 2 μg / mL CGRP protein. After blocking with BSA, 100 μL of hybridoma supernatant was added, and the plate was incubated at 37°C for 1 hour. Subsequent steps included washing, incubation with mouse IgG secondary antibody, and color development, following standard ELISA procedures. Finally, the absorbance at 450 nm was read using an ELISA reader, and the optimal OD value was determined. 450The top 10% of clones were subjected to cAMP release function rescreening. Functional rescreening employed a cAMP release function assay based on in vitro CGRP-mediated HEK293-hCGRPR-1B10 cells (constructed in Example 2) to screen for anti-CGRP specific monoclonal antibody hybridoma supernatant and determine antibody titers. Homogeneous time-resolved fluorescence (HTRF) technology (Revity, Waltham, MA) was used in the screening, employing the Revvity cAMP assay kit. In short, on the day of assay, α-CGRP was diluted to a specific concentration with medium containing 500 μM IBMX (Merck, Burlington, MA), and 30 μL was mixed with an equal volume of the collected hybridoma supernatant in a 96-well plate and incubated at 37°C for 1 h. HEK293-hCGRPR-1B10 cells were trypsinized and resuspended in the same medium. 5 μL of cells and 5 μL of the mixture were added to each well of a 384-well white microplate and incubated at room temperature for 20 min. Then, 5 μL each of Camp-d2 and Anti-cAMP-Cryptate from the kit were added to each well, and the cells were incubated at room temperature in the dark for 1 hour. The plates were then read using the TR-FRET built-in template on a multi-functional microplate reader (Molecular Device, San Jose, CA). Finally, high-activity, optimized monoclonal hybridoma cells were obtained.

[0217] Logarithmic growth phase hybridoma cells were collected, and RNA was extracted using NucleoZol (MN) and reverse transcribed (PrimeScript™ Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained from reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sent to a sequencing company for sequencing. The amino acid sequence of the variable region of the mouse antibody obtained by sequencing is as follows:

[0218] >4B1D5 mouse heavy chain variable region amino acid sequence (4B1D5-VH)

[0219] >4B1D5 mouse light chain variable region amino acid sequence (4B1D5-VL)

[0220] >4B1A9 mouse heavy chain variable region amino acid sequence (4B1A9-VH)

[0221] >4B1A9 mouse light chain variable region amino acid sequence (4B1A9-VL)

[0222] >4B3A8 mouse heavy chain variable region amino acid sequence (4B3A8-VH)

[0223] >4B3A8 mouse light chain variable region amino acid sequence (4B3A8-VL)

[0224] >4B6A9 mouse heavy chain variable region amino acid sequence (4B6A9-VH)

[0225] >4B6A9 mouse light chain variable region amino acid sequence (4B6A9-VL)

[0226] Example 4: Screening of Anti-CGRP Monoclonal Rabbit B Cells

[0227] 4.1 Rabbit Immunization

[0228] Anti-human CGRP monoclonal antibodies were produced by immunizing rabbits. Three-month-old (2-2.5 kg) female New Zealand White rabbits (purchased from the Hubei Provincial Experimental Animal Research Center) were used in the experiment. Rabbits that had adapted to the environment were immunized individually and cross-immunized with KLH-Human-α-CGRP, BSA-Human-α-CGRP, KLH-Rat-α-CGRP, BSA-Rat-α-CGRP, KLH-Human-α-CGRP (8-37), and Human-α-CGRP antigens. For the initial immunization, each rabbit received a subcutaneous injection of 200 μg of the peptide plus Freund's complete adjuvant. Subsequent booster immunizations were performed every two weeks, with each rabbit receiving a subcutaneous injection of 200 μg of the peptide plus Freund's complete adjuvant. After the 4th-6th immunizations, rabbits with high antibody titers in their serum that were approaching a plateau were selected for a pulse immunization, using the unadjuvanted peptide as the immunogen, with each rabbit receiving an injection of 200 μg of the peptide.

[0229] 4.2 Rabbit single B cell screening

[0230] One rabbit with good serum immunization and validation results was selected. Spleen and peripheral blood were collected for cell separation. Human-α-CGRP (K24FITC) was used as the selection molecule, and positive single B cells (memory B cells) were obtained by flow cytometry sorting. These cells were then cultured, and at least 960 single B cell clones (96-well * 10-well plates, after antigen enrichment) were selected. Finally, the cell supernatant was used to screen for positive clone B cells that specifically recognize the antigen using ELISA. Based on the ELISA results, up to 10 positive clone B cells were selected, and their antibody gene sequences were obtained through sequencing. The amino acid sequences are as follows:

[0231] >RF03G7 rabbit heavy chain variable region amino acid sequence (RF03G7-VH)

[0232] >RF03G7 rabbit light chain variable region amino acid sequence (RF03G7-VL)

[0233] >RF03H8 rabbit heavy chain variable region amino acid sequence (RF03H8-VH)

[0234] >RF03H8 rabbit light chain variable region amino acid sequence (RF03H8-VL)

[0235] >RF03E2 rabbit heavy chain variable region amino acid sequence (RF03E2-VH)

[0236] >RF03E2 rabbit light chain variable region amino acid sequence (RF03E2-VL)

[0237] >RF03E209 rabbit heavy chain variable region amino acid sequence (RF03E209-VH)

[0238] >RF03E209 rabbit light chain variable region amino acid sequence (RF03E209-VL)

[0239] >RF03E212 rabbit heavy chain variable region amino acid sequence (RF03E212-VH)

[0240] >RF03E212 rabbit light chain variable region amino acid sequence (RF03E212-VL)

[0241] >RF03G132 rabbit heavy chain variable region amino acid sequence (RF03G132-VH)

[0242] >RF03G132 rabbit light chain variable region amino acid sequence (RF03G132-VL)

[0243] The above-mentioned rabbit antibody was used to prepare an adult rabbit chimeric antibody. The heavy chain was constructed into a chimeric antibody of IgG4, and the light chain was constructed into the constant region of the IgG4 kappa chain. Functional experiments were performed on different batches to verify the antibody's properties. The functional experiments are detailed below: The detection method used a cAMP release function experiment based on in vitro CGRP-mediated HEK293-hCGRPR-1B10 cells (constructed in Example 2) to detect the prepared human-rabbit chimeric antibody. The detection was performed using homogeneous time-resolved fluorescence technology HTRF (Revvity, Waltham, MA) using the Revvity cAMP assay kit. In short, on the day of the assay, α-CGRP was diluted to a certain concentration with medium containing 500 μM IBMX (Merck, Burlington, MA), and the purified antibody was serially diluted with the same medium at a certain concentration ratio. 30 μL of α-CGRP dilution and an equal volume of purified antibody dilution were mixed in a 96-well plate and incubated at 37°C for 1 h. HEK293-hCGRPR-1B10 cells were digested with trypsin and resuspended in the above-mentioned culture medium. The purified antibody was serially diluted in the above-mentioned culture medium at specific concentration ratios. 5 μL of cells and 5 μL of the mixture were added to each well of a 384-well white microplate and incubated at room temperature for 20 min. Then, 5 μL each of cAMP-d2 and Anti-cAMP-Cryptate from the kit were added to each well and incubated at room temperature in the dark for 1 h. The cells were then read using a TR-FRET built-in template reader on a multifunctional microplate reader (Molecular Device, San Jose, CA). Cellular functional activity was measured. The cAMP release assay showed that the chimeric antibodies RF03E2, RF03H8, RF03G7, RF03E209, RF03E212, and RF03G132 exhibited excellent cell functional activity. Detailed data are shown in Table 1.

[0244] Table 1. Detection of cAMP release function of the antibody

[0245] Example 5: Humanization Design and Engineering of Anti-human CGRP Antibody

[0246] This patent relates to a method for antibody engineering, aiming to transform non-human antibodies into humanized antibodies, thereby improving their safety and efficacy in clinical applications. This method uses genetic engineering and protein engineering techniques to adjust the antibody structure, giving it better immune tolerance in the human body and enhancing its affinity and specificity. The antibodies involved are: 4B1D5, RF03H8, RF03E2, and RF03E212 antibodies, where 4B1D5 is mouse-derived, and RF03H8, RF03E2, and RF03E212 are rabbit-derived. The implementation method of this patent in the humanized antibody engineering method is as follows:

[0247] Selecting human antibody backbones: Screening and selecting appropriate human antibody backbones by comparing protein primary sequences and antibody structures;

[0248] CDR replacement: The non-human CDR regions of the 4B1D5, RF03H8, RF03E2, and RF03E212 antibodies are transplanted to replace the CDR regions of the selected human antibody backbone.

[0249] Based on the simulated three-dimensional structures of non-human antibodies 4B1D5, RF03H8, RF03E2, and RF03E212, some key residues of the replaced VH and VL were reverse-mutated. The final humanized molecule was obtained by cross-screening the VH and HL reverse-mutation sequences.

[0250] The constant region of the non-human antibody was replaced with the constant region of the human antibody. The heavy chain was constructed as a chimeric antibody of IgG4-S228P, and the light chain was constructed as the constant region of the kappa chain. Unless otherwise specified, the subtype of the humanized antibody is selected as IgG4-S228P / kappa.

[0251] 5.1 Humanization and Engineering of 4B1D5 Mouse Antibody

[0252] (1) Selection and reversion mutations in human FR regions

[0253] The heavy chain was selected from the IGHV1-46*01+IGHJ4*01 framework, and the light chain was selected from the IGKV1-16*01+IGKJ2*01 framework. The CDR region of the 4B1D5 murine antibody was transplanted into the above human template. H / L numbers indicate chimeric antibodies, H0 / L0 indicate framework region transplantation antibodies, and the others are designated as humanized designed antibodies. The obtained heavy / light chain sequences are as follows:

[0254] >4B1D5-H

[0255] In this invention, ** represents a stop codon.

[0256] >4B1D5-H0

[0257] >4B 1D5-H1

[0258] >4B 1D5-H2

[0259] >4B 1D5-H3

[0260] Light chain

[0261] >4B1D5-L

[0262] >4B 1D5-L0

[0263] >4B 1D5-L1

[0264] >4B 1D5-L2

[0265] >4B 1D5-L3

[0266] Combining the L and H sequences above yields a full-length antibody. Further techniques such as genetic engineering, protein engineering, cell culture, and protein purification are used to obtain antibody molecules with different combinations. Table 2 below lists the combinations used to prepare the antibodies.

[0267] Table 2. Humanized sample combinations of 4B1D5 antibody

[0268] The production steps for 4B1D5 chimeric and humanized antibodies are as follows: DNA sequences encoding the heavy and light chains of the antibody are synthesized, and a full-length IgG expression plasmid is constructed. Antibody expression is performed in 4 mL of CHO cell culture, and the supernatant is purified using a protein A affinity column. The purified antibody buffer is then exchanged for PBS using a PD-10 desalting column.

[0269] (2) cAMP function detection of 4B1D5 humanized antibody

[0270] All the aforementioned antibody molecules were detected using cell function experiments to verify their functional activities. The detection method employed a cAMP release assay based on in vitro CGRP-mediated cAMP release from HEK293-hCGRPR-1B10 cells (constructed in Example 2) to screen different humanized antibodies and determine their sequences. Homogeneous time-resolved fluorescence (HTRF) technology (Revity, Waltham, MA) was used in the assay, employing the Revvity cAMP assay kit. In short, on the day of the assay, α-CGRP was diluted to a specific concentration using medium containing 500 μM IBMX (Merck, Burlington, MA), and the purified antibody was serially diluted using the same medium at specific concentration ratios. 30 μL of α-CGRP dilution and an equal volume of purified antibody dilution were mixed in 96-well plates and incubated at 37°C for 1 h. HEK293-hCGRPR-1B10 cells were trypsinized and resuspended in the same medium. The purified antibodies were serially diluted using the above-mentioned culture medium at specific concentration ratios. 5 μL of cells and 5 μL of the mixture were added to each well of a 384-well white microplate and incubated at room temperature for 20 min. Then, 5 μL each of cAMP-d2 and Anti-cAMP-Cryptate from the kit were added to each well and incubated at room temperature in the dark for 1 h. The results were obtained using a TR-FRET built-in template reader (Molecular Device, San Jose, CA), as shown in Figure 1. The cAMP release assay showed that both the 4B1D5 chimeric antibody and its humanized antibody exhibited excellent activity. Among the humanized antibodies, 4B1D5-H1L2, 4B1D5-H2L2, and 4B1D5-H1L3 showed even better activity. Table 3 below shows the functional assay results of the above antibodies:

[0271] Table 3. Detection of cAMP release in HEK293-hCGRPR-1B10 cells, a humanized sample containing 4B1D5 antibody.

[0272] (3) Engineering modification of 4B1D5 humanized antibody

[0273] Antibody drugs, as important pharmaceutical and diagnostic reagents in the biomedical field, rely heavily on affinity for their biological function and efficacy. Therefore, improving the affinity of antibodies for target antigens is a crucial goal for enhancing antibody quality and efficacy. Comparing the preferred sequences 4B1D5, 4B1A9, 4B3A8, and 4B6A9 selected using hybridoma technology, these sequences exhibit similarity but show varying advantages and disadvantages in in vitro binding and function. To reduce the risk of point mutations (PTM) and improve antibody affinity, point mutations were performed on the 4B1D5 humanized sequence H1 / L2 combination. Before modification, the cellular functional activities of 4B1D5-H2L2, 4B1A9, 4B3A8, and 4B6A9 were measured; detailed data are shown in Figure 2 and Table 4. Modification was performed on the heavy chain portion of the 4B1D5-H1L2 antibody, while the light chain portion remained unchanged. A summary of the antibody engineering modifications is shown in Table 5. The binding strength of the modified antibody molecules to CGRP was detected by ELISA, and the cellular functional activity of the different antibody molecules was detected by cell function assays. Detailed data are shown in Figures 3, 4, and 5. cAMP release assays showed that antibodies 4B1A9, 4B3A8, 4B6A9, and 4B1D5-H1L2 exhibited excellent activity. The sample preparation process for the engineered molecules was the same as that for the humanized samples; specific mutations are shown in Table 5.

[0274] Table 4. ELISA binding and cAMP release function assays of the 4B1D5 modified antibody.

[0275] Table 5 Summary of Engineering Modification of 4B1D5-H1L2 Antibody

[0276] 4B1D5 Project Renovation Starting Sequence:

[0277] >4B1D5-H1

[0278] >4B 1D5-L2

[0279] Sequence information:

[0280] >4B1D5-H1-M1

[0281] >4B 1D5-H1-M2

[0282] >4B 1D5-H1-M3

[0283] >4B 1D5-H1-M4

[0284] >4B 1D5-H1-M5

[0285] >4B 1D5-H1-M6

[0286] >4B 1D5-H1-M7

[0287] >4B 1D5-H1-M8

[0288] >4B 1D5-H1-M9

[0289] (4) ELISA detection of the binding of 4B1D5 engineered antibody to human / rat-α-CGRP

[0290] The 4B1D5 engineered antibody used was selected for positive sequence identification using an ELISA binding assay targeting human / rat-α-CGRP. 100 μL / well of 2 μg / mL CGRP protein was coated onto an ELISA plate overnight. After blocking with BSA, 100 μL of antibody sample was added, and the plate was incubated at 37°C for 1 hour. The antibody concentration was 66.7 nmol / L as the maximum concentration, and the plate was serially diluted 3-fold. Subsequent steps included washing, IgG secondary antibody incubation, and color development according to standard ELISA procedures. Finally, the absorbance at 450 nm was read using an ELISA reader, and the data were processed using Graphpad Prism: OD values ​​for each concentration sample... 450 Using the average absorbance value as the Y-axis and the sample concentration value as the X-axis, a four-parameter fitting was performed to obtain EC. 50 The fitting constant R2 is shown in Figures 3 and 4 for detailed data.

[0291] (5) Detection of cAMP release function of 4B1D5 engineered antibody

[0292] The cAMP release function of the above-mentioned engineered 4B1D5 antibodies was detected using the same method as that of the humanized samples; detailed data are shown in Figure 5. The results indicate that both the humanized antibody 4B1D5-H1L2 and its engineered antibodies possess excellent activity, with the engineered antibodies 4B1D5-H1L2-M1, 4B1D5-H1L2-M4, 4B1D5-H1L2-M5, 4B1D5-H1L2-M8, and 4B1D5-H1L2-M9 exhibiting even better activity. Table 6 below shows the ELISA binding and release function detection results of the above antibodies:

[0293] Table 6. ELISA binding and cAMP release function assays of the 4B1D5 modified antibody.

[0294] 5.2 Humanization of RF03H8 rabbit antibody

[0295] (1) Selection and reversion mutations in human FR regions

[0296] The heavy chain was selected from the IGHV3-33*02+IGHJ2*01 framework, and the light chain was selected from the IGKV1-17*02+IGKJ4*01 framework. The CDR region of the RF03H8 rabbit antibody was transplanted into the above human template. H / L numbers indicate chimeric antibodies, H0 / L0 indicate framework region transplantation antibodies, and the others are designated as humanized design antibodies. The obtained heavy / light chain sequences are as follows:

[0297] >RF03H8-H

[0298] >RF03H8-H0

[0299] >RF03H8-H1

[0300] >RF03H8-H2

[0301] >RF03H8-H3

[0302] >RF03H8-L

[0303] >RF03H8-L0

[0304] >RF03H8-L1

[0305] >RF03H8-L2

[0306] >RF03H8-L3

[0307] Combining the L and H sequences above yields a full-length antibody. Further antibody molecules with different combinations are obtained using genetic engineering, protein engineering, cell culture, and protein purification techniques. The combinations used for antibody preparation are shown in Table 7.

[0308] Table 7. Humanized Sample Assemblies for RF03H8 Antibody

[0309] The production steps for RF03H8 chimeric and humanized antibodies are as follows: DNA sequences encoding the antibody heavy and light chains are synthesized, and a full-length IgG expression plasmid is constructed. Antibody expression is performed in 4 mL of CHO cell culture, and the supernatant is purified using a protein A affinity column. The purified antibody buffer is then exchanged for PBS using a PD-10 desalting column.

[0310] (2) SPR detection of RF03H8 humanized antibody

[0311] The detection data of individual concentration points for the RF03H8 humanized antibody were determined by surface plasmon resonance (SPR) to screen humanized samples. A Protein A chip was used in conjunction with Biacore. TM Detection was performed using an 8K SPR system. Antibodies were captured on the sensor chip using an Fc capture method, with Humanα-CGRP as the analyte. Biacore was used. TM 8K SPR system evaluation software obtains dissociation (K d ) and association (K a The rate constant data were analyzed. SPR results showed that both the chimeric antibody RF03H8-HL and its humanized antibody exhibited strong affinity. Among the humanized antibodies, RF03H8-H1L1 and RF03H8-H1L2 showed even stronger affinity and were therefore preferred sequences. The equilibrium dissociation constant (K03H8-HL) was also analyzed. D ) by K d With K a The ratio calculation and test data are shown in Table 8 below:

[0312] Table 8. Single-point SPR detection of RF03H8 antibody humanized samples.

[0313] 5.3 Humanization of RF03E2 rabbit antibody

[0314] (1) Selection and reversion mutations in human FR regions

[0315] The heavy chain was selected from the IGHV3-66*01+IGHJ1*01 framework, and the light chain was selected from the IGKV1-13*02+IGKJ4*01 framework. The CDR region of the RF03E2 rabbit antibody was transplanted into the above human template. H / L numbers indicate chimeric antibodies, H0 / L0 indicate framework region transplantation antibodies, and the others are designated as humanized design antibodies. The obtained heavy / light chain sequences are as follows:

[0316] >RF03E2-H

[0317] >RF03E2-H0

[0318] >RF03E2-H1

[0319] >RF03E2-H2

[0320] >RF03E2-H3

[0321] >RF03E2-H4

[0322] >RF03E2-L

[0323] >RF03E2-L0

[0324] >RF03E2-L1

[0325] >RF03E2-L2

[0326] >RF03E2-L3

[0327] Combining the L and H sequences above yields a full-length antibody. Further, different combinations of antibody molecules are obtained using genetic engineering, protein engineering, cell culture, and protein purification techniques. The combinations used for antibody preparation are shown in Table 9 below.

[0328] Table 9. Humanized Sample Assemblies of RF03E2 Antibody

[0329] The production steps for RF03E2 chimeric and humanized antibodies are as follows: DNA sequences encoding the heavy and light chains of the antibody are synthesized, and a full-length IgG expression plasmid is constructed. Antibody expression is performed in 4 mL of CHO cell culture, and the supernatant is purified using a protein A affinity column. The purified antibody buffer is then exchanged for PBS using a PD-10 desalting column.

[0330] (2) cAMP function detection of RF03E2 humanized antibody

[0331] All the above-mentioned antibody molecules were detected using cell function assays to verify their functional activities. Due to partial precipitation during the preparation of the humanized samples, only a portion of the samples were tested. The cAMP function detection method was consistent with that of the 4B1D5 humanized samples, as detailed in Figure 6. The cAMP release results showed that the humanized antibodies exhibited excellent activity. Table 10 below presents the functional detection results of the above antibodies:

[0332] Table 10. Detection of cAMP release in HEK293-hCGRPR-1B10 cells, a humanized sample containing RF03E2 antibody.

[0333] 5.4 Humanization of RF03E212 rabbit antibody

[0334] (1) Selection and reversion mutations in human FR regions

[0335] The heavy chain was selected from the IGHV3-66*01+IGHJ5*01 framework, and the light chain was selected from the IGKV1-9*01+IGKJ4*01 framework. The CDR region of the RF03E212 rabbit antibody was transplanted into the above human template. H / L numbers indicate chimeric antibodies, H0 / L0 indicate framework region transplantation antibodies, and the others are designated as humanized design antibodies. The obtained heavy / light chain sequences are as follows:

[0336] >RF03E212-H

[0337] >RF03E212-H0

[0338] >RF03E212-H1

[0339] >RF03E212-H2

[0340] >RF03E212-L

[0341] >RF03E212-L0

[0342] >RF03E212-L1

[0343] >RF03E212-L2

[0344] Combining the L and H sequences above yields a full-length antibody. Further, different combinations of antibody molecules are obtained using genetic engineering, protein engineering, cell culture, and protein purification techniques. The combinations used for antibody preparation are shown in Table 11 below.

[0345] Table 11 Humanized Sample Assemblies of RF03E212 Antibody

[0346] The production steps for RF03E212 chimeric and humanized antibodies are as follows: DNA sequences encoding the heavy and light chains of the antibody are synthesized, and a full-length IgG expression plasmid is constructed. Antibody expression is performed in 4 mL of CHO cell culture, and the supernatant is purified using a protein A affinity column. The purified antibody buffer is then exchanged for PBS using a PD-10 desalting column.

[0347] (2) SPR function detection of RF03E212 humanized antibody

[0348] The detection data of a single concentration point for the RF03E212 humanized antibody were determined by surface plasmon resonance (SPR) to screen humanized samples. A Protein A chip was used in conjunction with Biacore. TM Detection was performed using an 8K SPR system. Antibodies were captured on the sensor chip using an Fc capture method, with Humanα-CGRP as the analyte. Biacore was used. TM 8K SPR system evaluation software obtains dissociation (K d ) and association (K a Rate constant data. SPR results showed that both the chimeric antibody RF03E212-HL and its humanized antibody had strong affinity, with RF03E212-H2L2 showing stronger affinity in the humanized antibody, making it the preferred sequence. Equilibrium dissociation constant (K) D ) by K d With K a The ratio calculation and test data are shown in Table 12 below:

[0349] Table 12 Single-point SPR detection of RF03E212 antibody humanized samples

[0350] Example 6: Affinity detection of anti-human CGRP antibody binding to CGRP

[0351] 6.1 ELISA detection of the binding of anti-human CGRP antibody to human α-CGRP, human β-CGRP, rat α-CGRP, and rat β-CGRP

[0352] The anti-human CGRP antibody from Example 5 was subjected to species-cross ELISA detection of binding to human and rat α-CGRP and β-CGRP. Fremanezumab (Teva) and Eptinezumab (Lingbeck, Denmark) were selected as positive controls, and 4B1D5-H1L2-M9, RF03H8-H1L1, RF03E2-H3L1, and RF03E212-H2L2 were selected as preferred antibodies. Specifically, the concentrations of human α-CGRP, human β-CGRP, rat α-CGRP, and rat β-CGRP coated were 2 μg / mL. Dilute with PBS, 100 μL per well; seal with sealing film, incubate overnight at 2-8℃; remove sealing film, add 200 μL PBST to each well for washing, repeat 3 times; add 200 μL 3% BSA to each well, seal with sealing film, incubate at 37℃ for 2 h; remove sealing film, add 200 μL PBST to each well, repeat 3 times; initial sample concentration is 66.67 nmol / mL, serially diluted 3 times, 12 dilutions, 100 μL / well, diluent is PBS + 1% BSA; detect antigen-antibody binding; incubate at 37℃ for 1 h; remove sealing film, add 200 μL PBST to each well, repeat 3 times; HRP-IgG antibody (diluted with PBS + 1% BSA at 1:5000), 100 μL per well, incubate at 37℃ for 1 h; remove sealing film, add PBST to each well. 200 μL, repeated 4 times; add 100 μL of single-component TMB chromogenic solution to each well, seal with sealing film, and incubate at room temperature in the dark for 10 min; remove sealing film, add 100 μL of ELISA stop solution to each well; detect OD using a microplate reader. 450 Graphpad Prism data processing: OD values ​​for samples at various concentrations 450 The average absorbance was used as the Y-axis, and the sample concentration was used as the X-axis. Four-parameter fitting was performed to obtain EC50 and the fitting constant R². ELISA binding assay data showed that the selected antibodies exhibited strong binding affinity to both human and rat α / β-CGRP, and all showed strong cross-binding activity with rat α / β-CGRP. Detailed data are shown in Figures 7, 8, 9, and 10, and the fitted values ​​are shown in Table 13 below.

[0353] Table 13 Detection of binding of different CGRP antibodies to α / β-CGRP in humans / rats using ELISA

[0354] 6.2 Detection of SPR binding between anti-human CGRP antibody and human α-CGRP

[0355] The affinity kinetics of the anti-human CGRP antibody in Example 5 with human-α-CGRP were determined. Surface plasmon resonance (SPR) was used, with five concentration points measured for each antibody using a Protein A chip in conjunction with Biacore.TM Detection was performed using an 8K SPR system. Antibodies were captured on a sensor chip using an Fc capture method, with human-α-CGRP as the analyte. Biacore was used. TM 8K SPR system evaluation software obtains dissociation (K d ) and association (K a Data on the rate constant. SPR binding assays showed that the selected preferred antibodies and positive antibodies all exhibited strong binding affinity to human-α-CGRP. Among them, 4B1D5-H1L2-M9, due to its extremely weak dissociation, bound even stronger to human-α-CGRP and is expected to have a greater advantage than other preferred antibodies and positive antibodies in in vivo experiments. Equilibrium dissociation constant (K0) D ) by K d With K a The ratio was calculated. The fitted values ​​are shown in Table 14 below:

[0356] Table 14. Affinity detection of different CGRP antibodies with human α-CGRP SPR

[0357] The inability to fit the values ​​of *4B1D5-H1L2-M9 is presumably due to excessively high affinity.

[0358] Example 7: Functional detection of anti-CGRP antibody blocking CGRP-induced cAMP release

[0359] The anti-human CGRP antibody in Example 5 was tested for its ability to block CGRP-induced cAMP release. The cell lines used for in vitro cAMP assays in this example included the human neuroblastoma cell line (SK-N-MC), the HEK293-hCGRPR-1B10 cell line obtained in the cell construction example, and rat L6 cells. The cAMP release function of the anti-human CGRP antibody in Example 5 was detected using the same method as the humanized samples in Example 5. Figure 11 shows the detection of cAMP release function of the anti-CGRP antibody blocking the HEK293-hCGRPR-1B10 overexpressing cell line; Figure 12 shows the detection of cAMP release function of the anti-CGRP antibody blocking the SK-N-MC cell line; and Figure 13 shows the detection of cAMP release function of the anti-CGRP antibody blocking the rat L6 cell line. The cAMP release experiment data from different cell lines showed that the selected preferred antibodies all exhibited strong activity, with 4B1D5-H1L2-M9 showing the best performance, demonstrating superiority over other preferred antibodies and positive antibodies. Table 15 below summarizes the data from functional assays of the above antibodies with different cell lines:

[0360] Table 15 Anti-CGRP antibodies block cAMP release in different cell lines induced by CGRP. 50 value

[0361] Example 8: Pharmacokinetic Detection of Anti-CGRP Antibody

[0362] This embodiment aims to study the pharmacokinetic characteristics of 4B1D5-H1L2-M9 and RF03E2-H3L1 in rats. It also evaluates the pharmacokinetics of the YTE mutant of 4B1D5-H1L2-M10 (IgG1 antibody) (M254Y / S256T / T258E, composed of heavy chain 4B1D5-H1-M10 YTE and light chain 4B1D5-L2, the amino acid sequence of 4B1D5-H1-M10 YTE is shown in SEQ ID NO:131) (unless otherwise specified, all antibodies in this patent are prepared in IgG4-s228p form). The pharmacokinetic assay is used to evaluate the metabolism and bioavailability of different monoclonal antibodies.

[0363] Eighteen healthy male adult SD rats aged 7-8 weeks (approximately 250g each) were selected as experimental animals. They were divided into 6 groups of 3 rats each, based on drug administration (see Table 16 for grouping details). Blood samples were collected from rats at different time points after drug administration (before administration, 0.5h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 168h, 240h, and 336h). The concentration of anti-CGRP monoclonal antibody in the blood was detected using ELISA. Detailed experimental results are shown in Figure 14. Pharmacokinetic curves were plotted based on the data to analyze the pharmacokinetic characteristics of different monoclonal antibodies. The pharmacokinetic curves showed that 4B1D5-H1L2-M9 and RF03E2-H3L1 had relatively long half-lives in rats, consistent with the pharmacokinetic characteristics of conventional antibodies. Furthermore, the YTE form of 4B1D5-H1L2-M10 also exhibited excellent pharmacokinetic performance.

[0364] Table 16 Rat PK Grouping Information

[0365] Example 9: Detection of the efficacy of anti-CGRP antibodies in a capsaicin-stimulated blood flow model

[0366] This embodiment aims to evaluate the efficacy of the anti-CGRP monoclonal antibody 4B1D5-H1L2-M9 in a simulated migraine pharmacological model, specifically through efficacy verification using a rat capsaicin blood flow dilation model. Capsaicin activates the transient receptor potential vanillic acid type 1 (TRPV1). When capsaicin binds to the TRPV1 receptor, it triggers the release of a series of bioactive substances, including the pro-inflammatory neuropeptide CGRP (calcitonin gene-related peptide). By uniformly applying capsaicin to the skin of the rat's skull, vasodilation can be observed. Blood flow is measured using laser Doppler imaging (LDI) to establish a skin blood flow model. This model is non-invasive, simple to operate, rapid, objective, and highly reproducible.

[0367] Twenty-five healthy adult male SD rats aged 7-8 weeks (approximately 250g each) were selected as experimental animals. All rats underwent a one-week acclimatization period before the experiment to ensure stable physiological conditions. They were randomly divided into five groups (n=5 per group) based on body weight: a model control group (Vehicle, saline), a 4B1D5-H1L2-M9-0.1mg / kg group, a 4B1D5-H1L2-M9-0.3mg / kg group, a 4B1D5-H1L2-M9-1.0mg / kg group, and a 4B1D5-H1L2-M9-3.0mg / kg group. All samples were administered intravenously. Capsaicin was dissolved in 100% ethanol at a concentration of 100mg / mL, and 20μL (2mg capsaicin) was applied to each rat. Blood flow rate was measured four minutes before each capsaicin application (1min, 2min, 3min, 4min), and the average value was taken as the baseline blood flow rate. Immediately after capsaicin application, the blood flow rate of the rats was measured, recording data at 19 points per minute. A blood flow rate change curve was obtained, and the blood flow rate change rate was calculated as: (single-point blood flow rate - average blood flow rate in the 4 minutes before capsaicin application) / single-point blood flow rate * 100%. Data were measured for each treatment group before administration and at 2, 4, 9, and 14 days after administration. Detailed results are shown in Figures 15 and 16.

[0368] Comparison of data from the 4B1D5-H1L2-M9-0.1 mg / kg, 4B1D5-H1L2-M9-0.3 mg / kg, 4B1D5-H1L2-M9-1.0 mg / kg, and 4B1D5-H1L2-M9-3.0 mg / kg groups showed that 4B1D5-H1L2-M9 exhibited some dose dependence. Within 14 days of administration, the 4B1D5-H1L2-M9-1.0 mg / kg and 4B1D5-H1L2-M9-3.0 mg / kg groups demonstrated excellent efficacy.

[0369] This embodiment uses a rat capsaicin stimulation model to simulate the effects of migraine medications and evaluates the efficacy of the aforementioned anti-CGRP monoclonal antibody. Experimental results show that the anti-CGRP monoclonal antibody can significantly reduce the increase in cerebral blood flow in rats induced by capsaicin stimulation, demonstrating excellent efficacy. Therefore, it has high potential in the treatment of migraines.

[0370] The CDR sequence involved in this invention is as follows:

[0371] CDR-H1-1:

[0372] CDR-H1-2:

[0373] CDR-H1-3:

[0374] CDR-H1-4:

[0375] CDR-H1-5:

[0376] CDR-H1-6:

[0377] CDR-H1-7:

[0378] CDR-H2-1:

[0379] CDR-H2-2:

[0380] CDR-H2-3:

[0381] CDR-H2-4:

[0382] CDR-H2-5:

[0383] CDR-H2-6:

[0384] CDR-H2-7:

[0385] CDR-H2-8:

[0386] CDR-H2-9:

[0387] CDR-H2-10:

[0388] CDR-H2-11:

[0389] CDR-H2-12:

[0390] CDR-H3-1:

[0391] CDR-H3-2:

[0392] CDR-H3-3:

[0393] CDR-H3-4:

[0394] CDR-H3-5:

[0395] CDR-H3-6:

[0396] CDR-H3-7:

[0397] CDR-H3-8:

[0398] CDR-H3-9:

[0399] CDR-H3-10:

[0400] CDR-H3-11:

[0401] CDR-H3-12:

[0402] CDR-H3-13:

[0403] CDR-L1-1:

[0404] CDR-L1-2:

[0405] CDR-L1-3:

[0406] CDR-L1-4:

[0407] CDR-L1-5:

[0408] CDR-L1-6:

[0409] CDR-L1-7:

[0410] CDR-L1-8:

[0411] CDR-L2-1:

[0412] CDR-L2-2:

[0413] CDR-L2-3:

[0414] CDR-L2-4:

[0415] CDR-L2-5:

[0416] CDR-L2-6:

[0417] CDR-L3-1:

[0418] CDR-L3-2:

[0419] CDR-L3-3:

[0420] CDR-L3-4:

[0421] CDR-L3-5:

[0422] CDR-L3-6:

[0423] CDR-L3-7:

[0424] CDR-L3-8:

[0425] 4B 1D5-H1-M10 YTE:

[0426] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An antibody targeting CGRP or its antigen-binding moiety, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3, characterized in that, The CDR-H1 comprises an amino acid sequence as shown in any one of SEQ ID NO:77-83, the CDR-H2 comprises an amino acid sequence as shown in any one of SEQ ID NO:84-95, and the CDR-H3 comprises an amino acid sequence as shown in any one of SEQ ID NO:96-108; or, The CDR-L1 contains an amino acid sequence as shown in any one of SEQ ID NO:109-116, the CDR-L2 contains an amino acid sequence as shown in any one of SEQ ID NO:117-122, and the CDR-L3 contains an amino acid sequence as shown in any one of SEQ ID NO:123-130.

2. The antibody or its antigen-binding portion as described in claim 1, characterized in that, The heavy chain variable region and light chain variable region of the antibody are selected from the following combinations: The CDR-H1 has the amino acid sequence shown in SEQ ID NO:77; the amino acid sequence of the CDR-H2 has at least 80% homology with SEQ ID NO:85, preferably with amino acid residue differences at positions 5, 7, 9, and / or 10; the amino acid sequence of the CDR-H3 has at least 60% homology with SEQ ID NO:97, preferably with amino acid residue differences at positions 1, 2, 6, and / or 8; the amino acid sequence of the CDR-L1 has at least 80% homology with SEQ ID NO:109, preferably with amino acid residue differences at positions 5 and / or 9; the CDR-L2 has the amino acid sequence shown in SEQ ID NO:117; the amino acid sequence of the CDR-L3 has at least 90% homology with SEQ ID NO:123, preferably with amino acid residue differences at position 8; or, The CDR-H1 contains the amino acid sequence shown in any one of SEQ ID NO:78-83, the CDR-H2 contains the amino acid sequence shown in any one of SEQ ID NO:90-95, and the CDR-H3 contains the amino acid sequence shown in any one of SEQ ID NO:104-108; the CDR-L1 contains the amino acid sequence shown in any one of SEQ ID NO:111-116, the CDR-L2 contains the amino acid sequence shown in any one of SEQ ID NO:118-122, and the CDR-L3 contains the amino acid sequence shown in any one of SEQ ID NO:125-130; Preferably, CDR-H1 has the amino acid sequence shown in SEQ ID NO:77; the amino acid sequence of CDR-H2 differs from SEQ ID NO:85 by three or fewer amino acid residues from K5N, G7N, T9I, and I10V; the amino acid sequence of CDR-H3 differs from SEQ ID NO:97 by two or fewer amino acid residues from L1I, V2I, F6Y, and F8S; the amino acid sequence of CDR-L1 differs from SEQ ID NO:109 by two or fewer amino acid residues from N5D and / or N9A; CDR-L2 has the amino acid sequence shown in SEQ ID NO:117; and the amino acid sequence of CDR-L3 differs from SEQ ID NO:123 by either not containing or containing the amino acid residue Y8L. More preferably, CDR-H1 has the amino acid sequence shown in SEQ ID NO:77, CDR-H2 has the amino acid sequence shown in any one of SEQ ID NO:84-89, and CDR-H3 has the amino acid sequence shown in any one of SEQ ID NO:96-103; CDR-L1 has the amino acid sequence shown in any one of SEQ ID NO:109-110, CDR-L2 has the amino acid sequence shown in SEQ ID NO:117, and CDR-L3 has the amino acid sequence shown in any one of SEQ ID NO:123-124.

3. The antibody or its antigen-binding portion as described in claim 1 or 2, characterized in that, The CDRs of the heavy chain variable region and the light chain variable region of the antibody are selected from the following combinations: 1) The CDR-H1 comprises the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 is selected from the amino acid sequences shown in any one of SEQ ID NO:85-89, and the CDR-H3 is selected from the amino acid sequences shown in any one of SEQ ID NO:97-102; the CDR-L1 comprises the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 comprises the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 comprises the amino acid sequence shown in SEQ ID NO:123; 2) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:84, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:96; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:

123. 3) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:89, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:102; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:109, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:

123. 4) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:77, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:84, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:103; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:110, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:117, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:

124. 5) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:78, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:90, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:104; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:111, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:118, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:

125. 6) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:79, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:91, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:105; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:112, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:118, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:

126. 7) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:80, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:92, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:105; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:113, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:119, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:

127. 8) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:81, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:93, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:106; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:114, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:120, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:

128. 9) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:82, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:94, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:107; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:115, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:121, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:129; and, 10) The CDR-H1 contains the amino acid sequence shown in SEQ ID NO:83, the CDR-H2 contains the amino acid sequence shown in SEQ ID NO:95, and the CDR-H3 contains the amino acid sequence shown in SEQ ID NO:108; the CDR-L1 contains the amino acid sequence shown in SEQ ID NO:116, the CDR-L2 contains the amino acid sequence shown in SEQ ID NO:122, and the CDR-L3 contains the amino acid sequence shown in SEQ ID NO:

130.

4. The antibody or its antigen-binding moiety according to any one of claims 1-3, characterized in that, The heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 10, 12, 14, and 15; the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 11, 13, and 16; or, The heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 17, 19, 21, 23, 25 and 27; the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 18, 20, 22, 24, 26 and 28; Preferably, the heavy chain variable region and the light chain variable region of the antibody are selected from the following combinations: 1) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:10; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:11; 2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:12; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:13; 3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:14; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:13; 4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:16; 5) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:17; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:18; 6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19; the light chain variable region contains the amino acid sequence shown in SEQ ID NO:20; 7) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:21; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:22; 8) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:23; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:24; 9) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:25; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:26; and, 10) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:27; the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

28.

5. The antibody or its antigen-binding moiety according to any one of claims 1-4, characterized in that, The antibody or its antigen-binding moiety comprises a heavy chain and a light chain; wherein... The heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 29-33 and 39-47; the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 34-38; or, The heavy chain comprises an amino acid sequence as shown in any one of SEQ ID NO:48-52, 58-63, and 69-72; the light chain comprises an amino acid sequence as shown in any one of SEQ ID NO:53-57, 64-68, and 73-76. Best location: The heavy chain comprises the amino acid sequence shown in SEQ ID NO:29; the light chain comprises the amino acid sequence shown in SEQ ID NO:34; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:31; the light chain comprises the amino acid sequence shown in SEQ ID NO:36; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:31; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:31; the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:32; the light chain comprises the amino acid sequence shown in SEQ ID NO:36; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:32; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:32; the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:33; the light chain comprises the amino acid sequence shown in SEQ ID NO:36; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:33; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:33; the light chain comprises the amino acid sequence shown in SEQ ID NO:38; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:39; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:40; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:41; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:42; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:43; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:44; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:45; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:46; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:47; the light chain comprises the amino acid sequence shown in SEQ ID NO:37; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:48; the light chain comprises the amino acid sequence shown in SEQ ID NO:53; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:50; the light chain comprises the amino acid sequence shown in SEQ ID NO:55; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:50; the light chain comprises the amino acid sequence shown in SEQ ID NO:56; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:50; the light chain comprises the amino acid sequence shown in SEQ ID NO:57; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51; the light chain comprises the amino acid sequence shown in SEQ ID NO:55; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51; the light chain comprises the amino acid sequence shown in SEQ ID NO:56; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:51; the light chain comprises the amino acid sequence shown in SEQ ID NO:57; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:52; the light chain comprises the amino acid sequence shown in SEQ ID NO:55; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:52; the light chain comprises the amino acid sequence shown in SEQ ID NO:56; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:52; the light chain comprises the amino acid sequence shown in SEQ ID NO:57; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:58; the light chain comprises the amino acid sequence shown in SEQ ID NO:64; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:60; the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:60; the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:60; the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:61; the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:61; the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:61; the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:62; the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:62; the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:62; the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:63; the light chain comprises the amino acid sequence shown in SEQ ID NO:66; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:63; the light chain comprises the amino acid sequence shown in SEQ ID NO:67; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:63; the light chain comprises the amino acid sequence shown in SEQ ID NO:68; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:69; the light chain comprises the amino acid sequence shown in SEQ ID NO:73; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:71; the light chain comprises the amino acid sequence shown in SEQ ID NO:75; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:71; the light chain comprises the amino acid sequence shown in SEQ ID NO:76; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:72; the light chain comprises the amino acid sequence shown in SEQ ID NO:75; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:72; the light chain comprises the amino acid sequence shown in SEQ ID NO:76; or, The heavy chain comprises the amino acid sequence shown in SEQ ID NO:131; the light chain comprises the amino acid sequence shown in SEQ ID NO:

37.

6. A CGRP conjugate, characterized in that, The CGRP conjugate comprises one, two, or more antibodies or antigen-binding moieties as described in any one of claims 1-5. Preferably, the CGRP conjugate also has one or more of the following characteristics: 1) The CGRP conjugate is Fab, Fab', F(ab')2, scFab, Fv, scFv or a full-length antibody; 2) The CGRP conjugate is an antibody fusion protein; for example, a Fab fusion protein or a single-chain fusion protein; 3) The CGRP conjugate is a chimeric antigen receptor; 4) The CGRP conjugate is a human, mouse, or rabbit antibody; and, 5) The CGRP conjugate is IgG in the form of YTE.

7. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the antibody or its antigen-binding portion as described in any one of claims 1-5 or the CGRP conjugate as described in claim 6.

8. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 7; Preferably, the recombinant expression vector is a plasmid, bacteriophage, or viral vector; More preferably, the viral vector is a retroviral vector, an adenovirus vector, or an adeno-associated virus vector, and the retroviral vector is, for example, a lentiviral vector.

9. A transformant, characterized in that, The transformant is a host cell containing the isolated nucleic acid as described in claim 7, or the recombinant expression vector as described in claim 8; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; More preferably, the host cell is selected from yeast cells or mammalian cells; the mammalian cell is, for example, HEK293 cells.

10. A method for preparing an antibody or its antigen-binding moiety targeting CGRP, characterized in that, The method includes culturing the transformant as described in claim 9, and obtaining an antibody or antigen-binding moiety targeting CGRP from the culture.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises one or more of the antibody or its antigen-binding portion as described in any one of claims 1-5, the CGRP conjugate as described in claim 6, the isolated nucleic acid as described in claim 7, the recombinant expression vector as described in claim 8, and the transformant as described in claim 9; Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

12. A CGRP detection reagent, characterized in that, The CGRP detection reagent includes an antibody or its antigen-binding portion as described in any one of claims 1-5, and / or a CGRP conjugate as described in claim 6.

13. The use of one or more of the antibody or antigen-binding portion thereof as described in any one of claims 1-5, the CGRP conjugate as described in claim 6, the isolated nucleic acid as described in claim 7, the recombinant expression vector as described in claim 8, the transformant as described in claim 9, and the pharmaceutical composition as described in claim 11 in the preparation of diagnostic or detection reagents; Preferably, the diagnostic agent or detection agent is used to diagnose or detect diseases, symptoms, or conditions mediated by CGRP.

14. The use of one or more of the antibody or antigen-binding portion thereof as claimed in any one of claims 1-5, the CGRP conjugate as claimed in claim 6, the isolated nucleic acid as claimed in claim 7, the recombinant expression vector as claimed in claim 8, and the transformant as claimed in claim 9 in the preparation of a medicament for the prevention or treatment of CGRP-mediated diseases, conditions, or symptoms; Preferably, the CGRP-mediated disease, condition, or symptom is selected from at least one of the following: Migraine, cluster headache, tension headache, and hot flashes.

15. A method for detecting CGRP, characterized in that, CGRP can be detected using one or more of the following: the antibody or its antigen-binding portion as described in any one of claims 1-5; the CGRP conjugate as described in claim 6; the isolated nucleic acid as described in claim 7; the recombinant expression vector as described in claim 8; the transformant as described in claim 9; and the CGRP detection agent as described in claim 12; preferably, the method is for non-diagnostic purposes.

16. A method for diagnosing, preventing, improving, or treating a CGRP-mediated disease, condition, or symptom, the method comprising administering to a subject in need an effective amount of an antibody or antigen-binding portion thereof as described in any one of claims 1-5, a CGRP conjugate as described in claim 6, an isolated nucleic acid as described in claim 7, a recombinant expression vector as described in claim 8, a transformant as described in claim 9, or a pharmaceutical composition as described in claim 11; Preferably, the CGRP-mediated disease, condition, or symptom is selected from at least one of the following: Migraine, cluster headache, tension headache, and hot flashes.

17. The antibody or antigen-binding portion thereof as described in any one of claims 1-5, the CGRP conjugate as described in claim 6, the isolated nucleic acid as described in claim 7, the recombinant expression vector as described in claim 8, the transformant as described in claim 9, or the pharmaceutical composition as described in claim 11, for the diagnosis, prevention, improvement, or treatment of CGRP-mediated diseases, conditions, or symptoms; Preferably, the CGRP-mediated disease, condition, or symptom is selected from at least one of the following: Migraine, cluster headache, tension headache, and hot flashes.

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