Antibody molecule specifically binding to CGRP receptor, and use thereof

Abstract

The present invention provides an antibody molecule or antigen-binding fragment that specifically binds to a calcitonin gene-related peptide (CGRP) receptor, a fusion protein comprising two antibody molecules or antigen-binding fragments of the CGRP receptor, and a fusion protein further comprising a binding domain that specifically binds to a human HSA protein. The present invention also provides a nucleic acid encoding the antibody molecule or antigen-binding fragment and the fusion proteins thereof, a vector, a cell, a kit and pharmaceutical composition containing same, a use in the preparation of a drug for diseases related to CGRP over-activation, and the like.

Description

Antibody molecules that specifically bind to a crgp receptor and uses thereof

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. 202410630087.4, filed May 21, 2024, the contents of which are incorporated by reference in their entirety.

[0003] Statement as to Federally Sponsored Research

[0004] The sequence listing associated with this application is provided in text format and is incorporated by reference herein in its entirety. Computer readable format copy of the sequence listing: File name: BIYO_002_01WO_SeqList_ST25.xml, Date recorded: May 19, 2025, File size 140,596 bytes. TECHNICAL FIELD

[0005] The present invention belongs to the field of biotechnology, in particular to an antibody molecule that specifically binds to a CGRP receptor and uses thereof. BACKGROUND

[0006] Primary headache, especially migraine among them, is a neurovascular disease with a high global incidence rate, and the incidence rate of severe chronic patients is also relatively widespread. Migraine lasts for 3 to 72 hours when it occurs, and is accompanied by symptoms such as photophobia, phonophobia, nausea, vomiting, etc. in addition to unilateral headache. Chronic patients often have cardiovascular problems, mental disorders, depression, sleep difficulties, etc. Some female patients often have more severe attacks during menstruation, with a longer duration (>72 hours). These patients need better headache management measures and drugs.

[0007] The cause of migraine is not clear, hence the name primary headache. It is generally believed that some stimulus of the central nervous system, such as CSD (Cortical Depression Spreading), causes the C fiber endings of the trigeminal nerve to release neurotransmitters such as CGRP (Calcitonin Gene-Related Peptide), PACAP (Pituitary Adenylate Cyclase-activating Peptide), Substance C, etc. These neurotransmitters bind to receptors on Aδ fibers, triggering migraine through direct or indirect pathways.

[0008] There are at least two major signaling pathways involved in migraine: CGRP and PACAP. Both CGRP and PACAP are neurotransmitters, distributed in the nervous system and other tissues throughout the body. Their receptors are both class B G protein-coupled receptors (GPCRs). Neurotransmitters achieve signal transmission by specifically binding to receptors, leading to an increase in intracellular cAMP (cyclic adenosine monophosphate) concentration.

[0009] CGRP binds to and activates CGRP receptors, leading to an increase in cAMP. PACAP has similar affinity for its three receptors (PAC1, VPAC1, and VPAC2), so all three may be involved in migraine signal transmission.

[0010] Many new drugs are developed to block signal transmission in these two pathways. CGRP and its receptor antagonists have been successfully used for the treatment and prevention of migraine,

[0011] Current market drugs for the treatment and prevention of migraine include Cox-2 inhibitors, non-steroidal analgesics, beta blockers, antiepileptic drugs, antidepressants, ergotamine, neurotoxins, CGRP antagonists, 5-HT receptor agonists, etc. Among them, CGRP antagonists are the most popular, and there are four monoclonal antibodies on the market in Europe and the United States: Eptinezumab (Lunbeck), Erenumab (Amgen), Fremanezumab (Teva), and Galcanezumab (Eli Lilly). There are also small molecules that block CGRP receptors, such as oral tablets Atogepant (AbbVie) and Ubrogepent (AbbVie), nasal sprays Zavegepant, and oral disintegrating tablets Rimegepent (Pfizer) on the market in Europe and the United States. However, CGRP receptor blockers have many shortcomings. Their antibodies and small molecules block signal transmission by binding to an active epitope on the CGRP receptor. This single-epitope blocking method cannot achieve the maximum inhibition of signal transmission. SUMMARY

[0012] In one aspect, the present application relates to an antibody molecule and antigen-binding fragments and related molecules thereof that can bind to CGRP receptor with high affinity and block its signaling. In some embodiments, the antibody molecule, antigen-binding fragments and related molecules thereof have a unique way of binding to CGRP receptor, in which a first antigen-binding domain binds to a first epitope of CGRP receptor, a second antigen-binding domain binds to a second epitope of CGRP receptor, and a third antigen-binding domain binds to albumin for prolonging the half-life of the antibody molecule, antigen-binding fragments and related molecules thereof in blood circulation. In some embodiments, the antibody molecule, antigen-binding fragments and related molecules provided by the present application bind to CGRP receptor in a new, high-efficiency way, which improves the tissue penetration ability and the ability to inhibit cAMP production.

[0013] In one aspect, the present application provides an antibody molecule or antigen-binding fragment thereof that specifically binds to a Calcitonin Gene-Related Peptide (CGRP) receptor, comprising a first antigen-binding domain comprising a heavy chain variable region that binds to the CGRP receptor. In some embodiments, the heavy chain variable region comprises a HCDR1 as set forth in SEQ ID NO: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or any variant thereof, a HCDR2 as set forth in SEQ ID NO: 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, or any variant thereof, and a HCDR3 as set forth in SEQ ID NO: 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, or any variant thereof.

[0014] In some embodiments, the heavy chain variable region of the first antigen-binding domain described above comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of:

[0015] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0016] (2) SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17;

[0017] (3) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0018] (4) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0019] (5) SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32;

[0020] (6) SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37;

[0021] (7) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0022] (8) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47;

[0023] (9) SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52;

[0024] (10) SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57;

[0025] (11) SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62; or

[0026] (12) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0027] In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from the group consisting of SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, and 132-135.

[0028] In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 60, a HCDR2 as set forth in SEQ ID NO: 61, and a HCDR3 as set forth in SEQ ID NO: 62.

[0029] In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of:

[0030] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0031] (2) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0032] (3) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0033] (4) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0034] (5) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; and

[0035] (6) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0036] In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 9, 19, 24, 39, 44, 64, and 132-134.

[0037] In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 20, a HCDR2 as set forth in SEQ ID NO: 21, and a HCDR3 as set forth in SEQ ID NO: 22. In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 19 and 132-134. In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 132.

[0038] In some preferred embodiments, the first antigen binding domain described above is a single domain antibody.

[0039] In some preferred embodiments, the first antigen binding domain described above is humanized.

[0040] In some embodiments, the antibody molecule or antigen binding fragment thereof described above further comprises a second antigen binding domain that binds to a CGRP receptor.

[0041] In some embodiments, the antigenic epitope of the first antigen binding domain described above is different from the antigenic epitope of the second antigen binding domain described above. In some preferred embodiments, the two antigenic epitopes do not overlap.

[0042] In some embodiments, the second antigen binding domain described above comprises a heavy chain variable region that binds to a CGRP receptor, the heavy chain variable region comprising a HCDR1 as set forth in SEQ ID NO: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or any variant thereof, a HCDR2 as set forth in SEQ ID NO: 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, or any variant thereof, and a HCDR3 as set forth in SEQ ID NO: 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, or any variant thereof.

[0043] In some embodiments, the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1, a HCDR2 and a HCDR3 selected from the group consisting of:

[0044] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0045] (2) SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17;

[0046] (3) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0047] (4) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0048] (5) SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32;

[0049] (6) SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37;

[0050] (7) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0051] (8) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47;

[0052] (9) SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52;

[0053] (10) SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57;

[0054] (11) SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62; or

[0055] (12) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0056] In some preferred embodiments, the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of:

[0057] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0058] (2) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0059] (3) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0060] (4) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0061] (5) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; or

[0062] (6) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0063] In some more preferred embodiments, the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 20, a HCDR2 as set forth in SEQ ID NO: 21, and a HCDR3 as set forth in SEQ ID NO: 22.

[0064] In some more preferred embodiments, the heavy chain variable region of the second antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from the group consisting of SEQ ID NOs: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, and 132-135.

[0065] In some more preferred embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the first antigen binding domain described above and the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the second antigen binding domain described above are selected from one group of the embodiments of Table 11.

[0066] In some more preferred embodiments, the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the first antigen binding domain described above are different from the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the second antigen binding domain described above.

[0067] In some more preferred embodiments, the heavy chain variable region of the second antigen binding domain described above comprises an HCDR1 as set forth in SEQ ID NO: 60, an HCDR2 as set forth in SEQ ID NO: 61, and an HCDR3 as set forth in SEQ ID NO: 62. In some more preferred embodiments, the heavy chain variable region of the second antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 59. In some more preferred embodiments, the heavy chain variable region of the second antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 135.

[0068] In some more preferred embodiments, the second antigen binding domain described above is a single domain antibody.

[0069] In some more preferred embodiments, the second antigen binding domain described above is humanized.

[0070] In some embodiments, the antibody molecule or antigen binding fragment thereof described above further comprises a third antigen binding domain. In some embodiments, the third antigen binding domain described above is used to extend the half-life of the antibody molecule or antigen binding fragment thereof described above.

[0071] In some preferred embodiments, the third antigen binding domain described above specifically binds to human albumin.

[0072] In some embodiments, the third antigen binding domain described above comprises a heavy chain variable region comprising a HCDR1 as set forth in SEQ ID NO: 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or any variant thereof, a HCDR2 as set forth in SEQ ID NO: 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, or any variant thereof, and a HCDR3 as set forth in SEQ ID NO: 72, 77, 82, 87, 92, 97, 102, 107, 112, 117, 122, 127, or any variant thereof.

[0073] In some embodiments, the heavy chain variable region of the third antigen binding domain described above comprises a HCDR1, a HCDR2 and a HCDR3 selected from the group consisting of:

[0074] (1) SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72;

[0075] (2) SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77;

[0076] (3) SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82;

[0077] (4) SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87;

[0078] (5) SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92;

[0079] (6) SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97;

[0080] (7) SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102;

[0081] (8) SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107;

[0082] (9) SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112;

[0083] (10) SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117;

[0084] (11) SEQ ID NO: 120, SEQ ID NO: 121, and SEQ ID NO: 122;

[0085] (12) SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127; and

[0086] (13) SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 144.

[0087] In some preferred embodiments, the heavy chain variable region of the third antigen binding domain described above comprises HCDR1, HCDR2, and HCDR3 selected from the group consisting of:

[0088] (1) SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92; or

[0089] (2) SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102.

[0090] In some embodiments, the first and / or second antigen binding domain described above comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, or any variants thereof; and / or, the third antigen binding domain described above comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 69, 74, 79, 84, 89, 94, 99, 104, 109, 114, 119, 124, or any variants thereof.

[0091] In some preferred embodiments, the first antigen binding domain described above comprises an amino acid sequence set forth in SEQ ID NO: 59.

[0092] In some preferred embodiments, the second antigen binding domain described above comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 19, 24, 39, 44, and 64.

[0093] In some preferred embodiments, the third antigen binding domain described above comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 89 and 99.

[0094] In some preferred embodiments, said heavy chain variable region of the third antigen binding domain described above comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from the group consisting of SEQ ID NO: 69, 74, 79, 84, 89, 94, 99, 104, 109, 114, 119, and 124.

[0095] In some preferred embodiments, said heavy chain variable region of the third antigen binding domain described above comprises a HCDR1 as depicted in SEQ ID NO: 90, a HCDR2 as depicted in SEQ ID NO: 91, and a HCDR3 as depicted in SEQ ID NO: 92; preferably said heavy chain variable region of the third antigen binding domain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 89.

[0096] In some preferred embodiments, said heavy chain variable region of the third antigen binding domain described above comprises a HCDR1 as depicted in SEQ ID NO: 100, a HCDR2 as depicted in SEQ ID NO: 101, and a HCDR3 as depicted in SEQ ID NO: 102; preferably said heavy chain variable region of the third antigen binding domain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 99.

[0097] In some preferred embodiments, said heavy chain variable region of the third antigen binding domain described above comprises a HCDR1 as depicted in SEQ ID NO: 142, a HCDR2 as depicted in SEQ ID NO: 143, and a HCDR3 as depicted in SEQ ID NO: 144. In some preferred embodiments, said heavy chain variable region of the third antigen binding domain comprises an amino acid sequence which is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 141.

[0098] In some preferred embodiments, said third antigen binding domain described above is a single domain antibody.

[0099] In some preferred embodiments, said third antigen binding domain described above is humanized.

[0100] In some preferred embodiments, said third antigen binding domain described above can prolong the half-life of said antibody molecule or antigen binding fragment thereof.

[0101] In some preferred embodiments, the antibody molecule or antigen binding fragment or molecule thereof described herein has a half-life in the human body of at least 10 days, at least 15 days, or at least 20 days.

[0102] In some embodiments, the first, second and / or third antigen binding domains described above are connected sequentially, directly or indirectly.

[0103] In some preferred embodiments, the first, second and / or third antigen binding domains described above are connected sequentially via a linker.

[0104] In some more preferred embodiments, the linker described above comprises a flexible linker and a rigid linker.

[0105] In some preferred embodiments, the C-terminus of the first antigen binding domain described above is connected to the N-terminus of the second antigen binding domain via a flexible linker.

[0106] In some preferred embodiments, the C-terminus of the second antigen binding domain described above is connected to the N-terminus of the third antigen binding domain via a rigid linker.

[0107] In some preferred embodiments, the C-terminus of the second antigen binding domain described above is connected to the N-terminus of the first antigen binding domain via a flexible linker. In some preferred embodiments, the C-terminus of the first antigen binding domain described above is connected to the N-terminus of the third antigen binding domain via a rigid linker.

[0108] In some preferred embodiments, the flexible polypeptide linker described above is selected from (G4S)n, (SG4)n, or G4(SG4)n, wherein n is an integer between 1-5, more preferably, the flexible polypeptide linker described above is GGGGSGGGGS (SEQ ID NO: 129).

[0109] In some preferred embodiments, the rigid polypeptide linker described above is selected from (EAAAKA)n, wherein n is an integer between 1-5, more preferably, the rigid polypeptide linker described above is EAAAKAEAAAKA (SEQ ID NO: 130).

[0110] In some embodiments, the first, second and / or third antigen binding domains described above are recombinant antibodies, preferably, llama-derived antibodies, chimeric antibodies, or humanized antibodies.

[0111] In some more preferred embodiments, the recombinant antibody described above is a nanobody; further preferably, the recombinant antibody described above is a humanized camelid VHH.

[0112] In some more preferred embodiments, the above antibody molecule or antigen binding fragment thereof comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 131. In some more preferred embodiments, the above antibody molecule or antigen binding fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 131.

[0113] In another aspect, the present application provides a molecule that binds to a Calcitonin Gene-Related Peptide (CGRP) receptor, characterized in that it comprises an antibody molecule or antigen binding fragment thereof as described herein.

[0114] In some embodiments, the above antibody molecule or antigen binding fragment thereof or molecule does not bind to Pituitary Adenylate Cyclase Activating Peptide (PACAP), PACAP27, or VIP (Vasoactive Intestinal Polypeptide).

[0115] In some embodiments, the above antibody molecule or antigen binding fragment thereof or molecule does not block the Pituitary Adenylate Cyclase Activating Peptide (PACAP) pathway.

[0116] In some embodiments, the above antibody molecule or antigen binding fragment thereof or molecule does not comprise an extracellular domain fragment (ECD) of Pituitary Adenylate Cyclase 1 (PAC1).

[0117] In some embodiments, the above antibody molecule or antigen binding fragment thereof or molecule does not comprise an Fc fragment.

[0118] In some embodiments, the above antibody molecule or antigen binding fragment thereof or molecule does not comprise an immunoglobulin constant region.

[0119] In some embodiments, the above antibody molecule or antigen binding fragment thereof or molecule has one or more of the following properties: (a) binds to human CGRP receptor with a Kd of 1 x 10 -7 M or less; (b) inhibits the binding of CGRP receptor to CGRP; (c) inhibits CGRP-induced cAMP production in CHOK1 cells expressing CGRP receptor; and (d) does not bind to human Amylin 1 receptor.

[0120] In another aspect, the present application provides a nucleic acid encoding the above antibody molecule or antigen binding fragment thereof or molecule of any of the above aspects.

[0121] In some preferred embodiments, the above first antigen binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, or any variant thereof.

[0122] In some preferred embodiments, the above-mentioned second antigen binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, or any variants thereof.

[0123] In some preferred embodiments, the above-mentioned third antigen binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 73, 78, 83, 88, 93, 98, 103, 108, 113, 118, 123, 128, or any variants thereof.

[0124] In another aspect, the present application provides a vector comprising the above-mentioned nucleic acid.

[0125] In another aspect, the present application provides a cell comprising the above-mentioned nucleic acid or vector.

[0126] In another aspect, the present application provides a pharmaceutical composition or a kit comprising the antibody molecule or antigen-binding fragment thereof of any of the above aspects and a pharmaceutically acceptable carrier, diluent or excipient.

[0127] In another aspect, the present application provides a kit comprising the antibody molecule or antigen-binding fragment thereof, molecule, nucleic acid, vector, and / or cell of any of the above aspects.

[0128] In another aspect, the present application provides a pharmaceutical composition comprising (1) the antibody molecule or antigen-binding fragment thereof, molecule, nucleic acid, vector, and / or cell of any of the above aspects, and (2) a pharmaceutically acceptable carrier, diluent or excipient.

[0129] In another aspect, the present application provides the use of the antibody molecule or antigen-binding fragment thereof, nucleic acid, vector, cell and / or pharmaceutical composition of any of the above aspects in the manufacture of a medicament for treating and / or preventing a disease associated with CGRP over-activation.

[0130] In another aspect, the present application provides the use of the antibody molecule or antigen-binding fragment thereof, molecule, nucleic acid, vector, and / or cell, and / or pharmaceutical composition of any of the above aspects in the manufacture of a medicament for treating and / or preventing a disease associated with CGRP over-activation.

[0131] In another aspect, the present application provides a method for treating and / or preventing a disease associated with CGRP over-activation in a subject, the method comprising: administering to the subject a therapeutically effective amount of the antibody molecule or antigen-binding fragment thereof, molecule, nucleic acid, vector, and / or cell, and / or pharmaceutical composition of any of the above aspects.

[0132] In another aspect, the present application provides use of the antibody molecule or antigen binding fragment thereof, molecule, nucleic acid, vector, and / or cell, and / or pharmaceutical composition of any of the above aspects in the treatment and / or prevention of a disease associated with over-activation of CGRP.

[0133] In some preferred embodiments, the disease is migraine; more preferably, the disease comprises episodic migraine, menstrual migraine, chronic migraine. BRIEF DESCRIPTION OF DRAWINGS

[0134] The accompanying drawings, which are incorporated herein and constitute part of this specification, illustrate embodiments consistent with the application and, together with the description, serve to explain the principles of the application.

[0135] Figure 1A shows a schematic diagram of CGRP receptor; Figure IB shows a schematic diagram of Amylin 1 receptor.

[0136] Figure 2 shows a structural diagram of the trivalent antibody molecule of the present application.

[0137] Figure 3 shows a result of electrophoresis of RC-Fc after purification.

[0138] Figure 4 shows a result of verification of binding of RC-Fc to CGRP, in which the reconstructed extracellular region of CGRP receptor retains the ability to bind its ligand CGRP, but not the irrelevant polypeptide PACAP.

[0139] Figure 5 shows the number of SK-N-MC cells and the corresponding standard curve of CGRP.

[0140] Figures 6A, 6B and 6C show a schematic diagram of Binning ELISA experiment.

[0141] Figure 7 shows a result of protein electrophoresis of 42hv.

[0142] Figure 8 shows a result of cell cAMP inhibition ability of VHHhvFc after humanization.

[0143] Figure 9 shows a structure of the trivalent antibody molecule BY002.

[0144] Figure 10 shows a result of SDS-PAGE of BY002 after purification.

[0145] Figure 11 shows Binding EC of BY002. 50 Results.

[0146] Figure 12 shows a structure of BY002 and control protein 42hv10Fc.

[0147] Figure 13 shows the binding results of BY002 to human Amylin 1 receptor ECD, rat CGRP receptor, and human Calcitonin receptor ECD.

[0148] Figure 14 shows that CGRP induces cAMP production on CHOK1-CGRPr cells.

[0149] Figure 15A shows that Amylin 1 induces cAMP production on CHOK1-Amy1r cells; Figure 15B shows that the effect of BY002 on Amylin 1 -induced cAMP production is smaller than that of the reference antibody Erenumab.

[0150] Figures 16A and 16B show the results of blood circulation and half-life experiments of Nanobody with anti-HSA VHH in mice and rats.

[0151] Figure 17 shows the 30-day PK performance of BY002 in rats. DETAILED DESCRIPTION

[0152] I. DEFINITIONS

[0153] In the present application, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art, unless otherwise indicated. Also, the terms and procedures of protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and related scientific procedures used herein are those commonly used in the corresponding fields and are well known and commonly practiced in the art. Also, for better understanding of the present application, the definitions and explanations of the relevant terms are provided below.

[0154] The singular form of a term is intended to include the plural form as well, and the plural form of a term is intended to include the singular form, unless the context requires otherwise. More specifically, as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes mixtures of cells, and so forth. In this application, the use of "or" means "and / or" unless stated otherwise.

[0155] The use of the term “comprising” and other forms such as “comprise”, “comprises” and “comprised of” is not limiting. The term “comprising” as used in the present application is to be interpreted in the same way as the term “including”. When the term “comprising” is used in the present specification and claims, it is to be interpreted as meaning that the encompassing process, method, composition of matter, or other activity includes the recited steps or elements, or combinations of recited steps or elements, but does not exclude any other steps or elements, or combinations of steps or elements. For the purposes of the present application, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising”. The term “comprising” (and related terms such as “comprise” or “comprises” or “include” or “including”) is not intended to exclude that in other certain embodiments, the method or process or composition of matter, etc. described can “consist of’ or “consist essentially of’ the features described.

[0156] As used herein, the terms “about” and “approximately” are used as equivalents, unless otherwise indicated. Any numerical values recited in this application, with or without the use of about / approximately, are intended to encompass any normal fluctuation that would be expected by a person of ordinary skill in the relevant art to occur in the associated measurement. In some embodiments, the term “approximately” or “about” refers to a range of 10% in either direction (greater than or less than) of the recited reference value, unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of the possible value).

[0157] As used herein, ranges provided in the specification and the appended claims are inclusive of the recited endpoints and of all sub-ranges between the recited endpoints.

[0158] Provided herein is the term “fusion” or “fused” when used in reference to an amino acid sequence (e.g., a peptide, polypeptide, or protein) refers to the combination of two or more amino acid sequences into a single amino acid sequence that does not occur in nature, e.g., by chemical linkage or recombinant means. The fused amino acid sequence can be produced recombinantly by two genes encoding polynucleotide sequences, and can be expressed by methods of introducing a construct containing the recombined polynucleotide into a host cell.

[0159] The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, or a collection of polymer of amino acid residues. It encompasses natural or artificial proteins, protein fragments and polypeptide analogs having protein sequences. The terms apply to amino acid polymers in which one or more amino acid residues are synthetic chemical mimics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. The polypeptides can be monomeric or multimeric.

[0160] The phrase "substantially identical" with respect to an antibody chain polypeptide sequence can be understood as an antibody chain that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polypeptide sequence. With respect to nucleic acid sequences, the term can be understood as a nucleotide sequence that exhibits at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference nucleic acid sequence.

[0161] The sequence "identity" or "identity" has the art-recognized meaning and can be calculated as the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Pres, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are a number of methods for measuring sequence identity between two polynucleotides or polypeptides, the term "identity" is art-recognized (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)). In some preferred embodiments, the percent identity between two sequences can be derived using BLAST program version 2.11.0 and its default parameters.

[0162] The term "antibody", also referred to in the art as "immunoglobulin" (Ig), refers to a protein constructed from pairs of heavy and light polypeptide chains; there are various Ig isotypes, including IgA, IgD, IgE, IgG, and IgM. When an antibody is properly folded, each chain is folded into a number of distinct globular domains connected by more linear peptide sequences. For example, an immunoglobulin light chain is folded into variable (VL) and constant (CL) domains, while a heavy chain is folded into variable (VH) and three constant (CHI, CH2, CH3) domains. The interaction of the heavy and light chain variable domains (VH and VL) results in the formation of an antigen binding region (Fv). The term antibody also includes herein chimeric antibodies, single domain antibodies, and homologues, derivatives or fragments of single domain antibodies or antigen binding fragments that retain the same. In some embodiments, the antibodies described herein are humanized antibodies or fully human antibodies. The variable domains can be further subdivided into hypervariable regions (called complementarity determining regions (CDRs)) that are spaced apart by relatively conserved regions (called framework regions (FRs)). Generally, each variable domain consists of three CDRs and four FRs, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from N- to C-terminus. In some embodiments, the distribution of amino acids in the various regions or domains follows the definition of the international ImMunoGeneTics information system® (IMGT®) (the international ImMunoGeneTics information system® (the international ImMunoGeneTics information )).

[0163] The term "single-domain antibody" (sdAb) or "nanobody" or "VHH" or "VHH domain" refers to an antibody that comprises only a single antibody variable domain. Like conventional antibodies, single-domain antibodies are capable of selectively binding a particular antigen. Single-domain antibodies have a molecular weight of only 12-15 kDa, much smaller than ordinary antibodies (150-160 kDa) that consist of two heavy chains and two light chains. The term "heavy-chain antibody," "heavy-chain single-domain antibody," or "nanobody single-chain antibody" comprises a single variable domain VHH (variable domain of heavy chain of heavy-chain antibody) and two constant domains (CH2 and CH3). Importantly, cloned and isolated VHH domains are fully stable polypeptides with full antigen-binding capacity of the original heavy-chain antibody. This class of antibodies is naturally missing other antibody light chains. Such single-domain antibodies are found in camelids. Single-domain antibodies have the advantages of structural stability, small molecules, good solubility, tolerance to various adverse environments, and ease of humanization, and have been widely used in miniaturized genetically engineered antibody research, new drug development, and diagnosis and treatment of diseases. Examples of single-domain antibodies include single-domain antibodies derived from camelids (llama and camel) and cartilaginous fish (e.g., nurse shark) and single-domain antibodies derived from human and mouse antibodies from recombinant methods. As used herein, the term "single-domain antibody" includes those sdAbs isolated directly from a VH, VHH, VL, or VNAR library of any origin by phage display or other techniques, sdAbs derived from the foregoing sdAbs, sdAbs produced recombinantly, and those sdAbs produced by further modification of such sdAbs (by humanization, affinity maturation, stabilization, solubilization, camelization, or other methods of antibody engineering). The present invention also relates to homologs, derivatives, or fragments that retain the antigen-binding function and specificity of sdAbs.

[0164] The term "linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Linkers are known in the art or described herein. In some embodiments, the linker is a flexible polypeptide. The flexible polypeptide consists of flexible amino acids selected from at least one of Gly, Ser, Ala, and Thr. Suitable non-immunogenic linker peptides are, for example, (G4S)n, (SG4)n, or G4(SG4)n peptide linkers. In some embodiments, the linker is a rigid polypeptide linker having an (EAAAK)n sequence, with internal hydrogen bonds and a closely associated peptide backbone, rigid and stable, can serve as a rigid spacer between protein domains. The rigid linker exhibits a relatively rigid structure, can effectively separate the protein domains at both ends, by changing the size of n (exemplarily, an integer between 1-5), can adjust the optimal distance between the domains, so as to ensure the activity and biological function of the proteins at both ends.

[0165] The term "CDR" refers to complementarity-determining region, each heavy chain and light chain of an antibody molecule is known to have 3 CDRs. CDR is also called hypervariable region, and exists in the variable region of each heavy chain and light chain of the antibody, with very high variability sites in the primary structure of CDR. In this specification, the CDR of the heavy chain is represented by CDR1, CDR2, CDR3 from the amino terminal of the amino terminal sequence of the heavy chain, and the CDR of the light chain is represented by CDR1, CDR2, CDR3 from the amino terminal of the amino terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure, and determine the specificity of the antigen bound by the antibody.

[0166] "Specific binding" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein, and refer to the ability of an antibody or antigen-binding fragment to form one or more noncovalent bonds with the same antigen via noncovalent interactions between the antibody combining site of the antibody and the antigen. The antigen can be an isolated antigen or present on a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) an antigen is one that binds to the antigen with an affinity 7 M -1 or 1 x 10 8 M -1 or a larger affinity constant Ka (or 1 x 10 -7 M or 1 x 10 -8M or lower dissociation constant (Kd) to the antigen. Affinity and dissociation constants can be determined by standard kinetic methods of antibody reactions, e.g., immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11 :54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art; see also U.S. Patent No. 7,229,619 describing exemplary SPR and ITC methods for calculating binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335). In some embodiments, determination of affinity or dissociation constants is by SPR methods, measuring the ability of a test molecule (e.g., an antibody molecule or antigen-binding fragment thereof, or a corresponding molecule) to bind a corresponding ligand in a PBS solution environment, at an ambient temperature of 25 degrees Celsius, with the test molecule immobilized on a test chip. In some preferred embodiments, the "specifically binds" refers to the ability of an antibody or antigen-binding fragment thereof to bind a target antigen in a mixture without significantly binding other components or antigens present in the mixture. In some preferred embodiments, the "specifically binds" refers to an antibody or antigen-binding fragment thereof that binds a non-target antigen with a binding affinity that is less than (weaker than) 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the binding affinity to the target antigen. In some embodiments, "binding" as described herein is specific binding.

[0167] The term "binding affinity" or "affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity of a 1 : 1 interaction between members of a cognate pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can be generally represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high affinity antibodies generally bind antigen more quickly and tend to remain bound longer. Various methods of measuring binding affinity are known in the art.

[0168] The term "epitope" refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds. An "epitope" is also referred to as an "antigenic determinant." Epitopes or antigenic determinants generally consist of chemically active surface groupings of molecules such as amino acids, carbohydrate, or sugar side chains and generally have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique stereochemical conformation, which can be a "linear" or "conformational" epitope. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996). In a linear epitope, all of the interaction sites between the protein and the interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction sites are spanned across amino acid residues in the protein that are separated from one another. Antibodies can be screened for competition in binding the same epitope, as detected by routine techniques known to those of skill in the art. For example, competition or cross-competition studies can be performed to obtain antibodies that compete or cross-compete for binding to an antigen.

[0169] The term "inhibit" or "neutralize" as used with respect to a biological activity of an antibody or antigen-binding fragment thereof, fusion protein, etc. of the application refers to the ability of the polypeptide to substantially antagonize, prevent, forestall, limit, slow, disrupt, abrogate, stop, reduce, or reverse, for example, the progression or severity of that which is inhibited, including but not limited to, a biological activity.

[0170] As used herein, the ability to "inhibit binding," "block binding," or "compete for the same epitope" refers to the ability of an antibody or antigen-binding fragment thereof, fusion protein, etc. to inhibit the binding of two molecules (e.g., a ligand and a corresponding receptor) to any detectable extent. In some embodiments, the ability to block binding between two molecules refers to an antibody or antigen-binding fragment thereof, fusion protein, etc. that inhibits the binding interaction between two molecules by at least 50%. In some embodiments, the inhibition can be greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 97%, greater than 98%, or greater than 99%.

[0171] The terms "nucleic acid" and "nucleic acid molecule" refer to an oligo- or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) that are linked together, typically through phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, and can be cDNA.

[0172] The term "vector" includes a vehicle which enables the expression of DNA operably linked to regulatory sequences such as a promoter region which control transcription of such DNA fragments. Such additional fragments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Accordingly, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors which are replicable in eukaryotic and / or prokaryotic cells and expression vectors which remain episomal or integrate into the host cell genome.

[0173] The term "pharmaceutical composition" refers to a preparation which is in a form suitable for administration into a subject and which allows the biological activity of the active ingredients contained therein to be effective, and which does not contain additional ingredients which are unacceptable to the subject to which the preparation is administered.

[0174] The term "pharmaceutically acceptable carrier" refers to one or more non-toxic materials which are suitable for use in administering a therapeutic agent, including but not limited to buffers, preservatives, compatible carriers, diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), excipients, vehicles, and optionally other additives or encapsulating materials. Pharmaceutically acceptable carriers suitable for use in the present application can be conventional pharmaceutical agents excipients; and compositions and formulations suitable for delivery of the disclosed neutralizing antibodies. In general, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations generally include injectable liquids, which include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle for

[0175] The term "treatment" refers to the partial or total remission of symptoms of an individual having a disease or condition, or remaining unchanged after treatment. Thus, treatment includes prevention, therapy, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or progression of the disease. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided herein and compositions provided herein.

[0176] As used herein, the term "isolated" refers to a state that has been obtained by human intervention from the natural state. If a certain "isolated" material or component is naturally occurring, it can be because it has been naturally altered, or the material has been separated from nature, or both. For example, a certain polynucleotide or polypeptide that is not isolated naturally occurs within a certain living organism, and the same polynucleotide or polypeptide of high purity that is separated from this natural state is referred to as an isolated polynucleotide or polypeptide. The term "isolated" does not exclude admixture of artificial or synthetic materials, nor does it exclude other impurities that do not affect the activity of the isolated material. In some embodiments, the antibody molecules or antigen-binding fragments thereof, fusion proteins, nucleic acids, vectors, or other molecules, or cells described herein are isolated.

[0177] Also provided are "conservative sequence modifications" of the sequences described in the sequence listing herein, i.e., those that do not deprive the antibody of its binding to the antigen encoded by or containing the amino acid sequence. These conservative changes include conservative substitutions, additions and deletions to the nucleotide and amino acid sequences. For example, a modification can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions that do not deprive the antibody of its binding to the antigen. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes in one or more or all of the heavy chain CDRs. Preferably, the above amino acid changes are amino acid substitutions, preferably conservative substitutions. In some embodiments, the antibody variant has at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the parent antibody over the region of the antibody sequence of interest. Exemplary substitutions are shown in Table 8 below:

[0178] Table 8 Amino acid conservative substitutions

[0179] II. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0180] In one aspect, the present application provides an antibody molecule or an antigen binding fragment thereof that specifically binds to a Calcitonin Gene-Related Peptide (CGRP) receptor, comprising a first antigen binding domain, wherein the first antigen binding domain comprises a heavy chain variable region that binds to the CGRP receptor, the heavy chain variable region comprising a HCDR1 as set forth in SEQ ID NO: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or any variant thereof, a HCDR2 as set forth in SEQ ID NO: 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, or any variant thereof, and a HCDR3 as set forth in SEQ ID NO: 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, or any variant thereof.

[0181] In some embodiments, the heavy chain variable region of the first antigen binding domain comprises a HCDR1, a HCDR2 and a HCDR3 selected from the group consisting of:

[0182] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0183] (2) SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17;

[0184] (3) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0185] (4) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0186] (5) SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32;

[0187] (6) SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37;

[0188] (7) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0189] (8) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47;

[0190] (9) SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52;

[0191] (10) SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57;

[0192] (11) SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62; or

[0193] (12) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0194] In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from the group consisting of SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, and 132-135.

[0195] In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises HCDR1, HCDR2, and HCDR3 selected from the group consisting of:

[0196] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0197] (2) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0198] (3) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0199] (4) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0200] (5) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; and

[0201] (6) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67;

[0202] In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 9, 19, 24, 39, 44, 64, and 132-134.

[0203] In some preferred embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 20, a HCDR2 as set forth in SEQ ID NO: 21, and a HCDR3 as set forth in SEQ ID NO: 22. In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 19. In some embodiments, the heavy chain variable region of the first antigen binding domain described above differs from SEQ ID NO: 19 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservatively substituted amino acids. In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 132. In some embodiments, the heavy chain variable region of the first antigen binding domain described above differs from SEQ ID NO: 132 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservatively substituted amino acids. In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 133. In some embodiments, the heavy chain variable region of the first antigen binding domain described above differs from SEQ ID NO: 133 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservatively substituted amino acids. In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 134. In some embodiments, the heavy chain variable region of the first antigen binding domain described above differs from SEQ ID NO: 134 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservatively substituted amino acids.

[0204] In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 60, a HCDR2 as set forth in SEQ ID NO: 61, and a HCDR3 as set forth in SEQ ID NO: 62.

[0205] In some preferred embodiments, the first antigen binding domain described above is a single domain antibody. In some preferred embodiments, the first antigen binding domain described above is humanized.

[0206] In some embodiments, the antibody molecule or antigen binding fragment thereof described above further comprises a second antigen binding domain that binds to a CGRP receptor.

[0207] In some embodiments, the CGRP antigenic epitope bound by the first antigen binding domain is different from the antigenic epitope of CGRP bound by the second antigen binding domain. In some preferred embodiments, the CGRP antigenic epitope bound by the first antigen binding domain does not overlap with the antigenic epitope of CGRP bound by the second antigen binding domain. In some preferred embodiments, the first antigen binding domain and the second antigen binding domain can simultaneously bind to the same CGRP receptor.

[0208] In some preferred embodiments, the second antigen binding domain described above comprises a heavy chain variable region that binds to a CGRP receptor. In some preferred embodiments, the heavy chain variable region described above comprises a HCDR1 as set forth in SEQ ID NO: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or any variant thereof, a HCDR2 as set forth in SEQ ID NO: 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, or any variant thereof, and a HCDR3 as set forth in SEQ ID NO: 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, or any variant thereof.

[0209] In some embodiments, the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of:

[0210] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0211] (2) SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17;

[0212] (3) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0213] (4) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0214] (5) SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32;

[0215] (6) SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37;

[0216] (7) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0217] (8) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47;

[0218] (9) SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52;

[0219] (10) SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57;

[0220] (11) SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62; or

[0221] (12) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0222] In some preferred embodiments, the heavy chain variable region of the second antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from the group consisting of SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, and 132-135.

[0223] In some preferred embodiments, the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1 as shown in SEQ ID NO: 60, a HCDR2 as shown in SEQ ID NO: 61, and a HCDR3 as shown in SEQ ID NO: 62. In some embodiments, the heavy chain variable region of the second antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 135. In some embodiments, the heavy chain variable region of the second antigen binding domain described above comprises only up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservatively substituted amino acids compared to SEQ ID NO: 135. In some embodiments, the heavy chain variable region of the second antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 59. In some embodiments, the heavy chain variable region of the second antigen binding domain described above comprises only up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservatively substituted amino acids compared to SEQ ID NO: 59.

[0224] In some embodiments, the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of:

[0225] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0226] (2) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0227] (3) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0228] (4) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0229] (5) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; or

[0230] (6) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0231] In some embodiments, the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1 as shown in SEQ ID NO: 20, a HCDR2 as shown in SEQ ID NO: 21, and a HCDR3 as shown in SEQ ID NO: 22.

[0232] In some preferred embodiments, the second antigen binding domain described above is a single domain antibody. In some preferred embodiments, the second antigen binding domain described above is humanized.

[0233] In some embodiments, the combination of the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the first antigen binding domain described above and the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the second antigen binding domain described above is selected from the group of embodiments of Table 11.

[0234] Table 11. Combination of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the first and second antigen binding domain

[0235] In some embodiments, the C-terminus of the first antigen binding domain selected from the embodiments of Table 11 is directly sequentially connected to the N-terminus of the second antigen binding domain. In some embodiments, the C-terminus of the first antigen binding domain selected from the embodiments of Table 11 is indirectly sequentially connected (e.g. through a linker) to the N-terminus of the second antigen binding domain.

[0236] In some embodiments, the C-terminus of the second antigen binding domain selected from the embodiments of Table 11 is directly sequentially connected to the N-terminus of the first antigen binding domain. In some embodiments, the C-terminus of the second antigen binding domain selected from the embodiments of Table 11 is indirectly sequentially connected (e.g. through a linker) to the N-terminus of the first antigen binding domain.

[0237] In some embodiments, the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the first antigen binding domain described above are different from the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region of the second antigen binding domain described above.

[0238] In some embodiments, the antigenic epitope bound by the heavy chain variable region of the first antigen binding domain described above is different from the antigenic epitope bound by the heavy chain variable region of the second antigen binding domain described above. In some embodiments, the antigenic epitope bound by the heavy chain variable region of the first antigen binding domain described above does not overlap with the antigenic epitope bound by the heavy chain variable region of the second antigen binding domain described above. In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 10, a HCDR2 as set forth in SEQ ID NO: 11, and a HCDR3 as set forth in SEQ ID NO: 12, and the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 60, a HCDR2 as set forth in SEQ ID NO: 61, and a HCDR3 as set forth in SEQ ID NO: 62. In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 20, a HCDR2 as set forth in SEQ ID NO: 21, and a HCDR3 as set forth in SEQ ID NO: 22, and the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 60, a HCDR2 as set forth in SEQ ID NO: 61, and a HCDR3 as set forth in SEQ ID NO: 62. In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 25, a HCDR2 as set forth in SEQ ID NO: 26, and a HCDR3 as set forth in SEQ ID NO: 27, and the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 60, a HCDR2 as set forth in SEQ ID NO: 61, and a HCDR3 as set forth in SEQ ID NO: 62. In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 40, a HCDR2 as set forth in SEQ ID NO: 41, and a HCDR3 as set forth in SEQ ID NO: 42, and the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 60, a HCDR2 as set forth in SEQ ID NO: 61, and a HCDR3 as set forth in SEQ ID NO: 62.In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO:45, a HCDR2 as set forth in SEQ ID NO:46, and a HCDR3 as set forth in SEQ ID NO:47, and the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO:60, a HCDR2 as set forth in SEQ ID NO:61, and a HCDR3 as set forth in SEQ ID NO:62. In some embodiments, the heavy chain variable region of the first antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO:65, a HCDR2 as set forth in SEQ ID NO:66, and a HCDR3 as set forth in SEQ ID NO:67, and the heavy chain variable region of the second antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO:60, a HCDR2 as set forth in SEQ ID NO:61, and a HCDR3 as set forth in SEQ ID NO:62.

[0239] In some embodiments, the heavy chain variable region that binds a CGRP receptor comprises a HCDR1 as set forth in SEQ ID NO: 20, a HCDR2 as set forth in SEQ ID NO: 21, and a HCDR3 as set forth in SEQ ID NO: 22. In some embodiments, the heavy chain variable region that binds a CGRP receptor comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 132. In some embodiments, the heavy chain variable region that binds a CGRP receptor has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservatively substituted amino acids compared to SEQ ID NO: 132. In some embodiments, the heavy chain variable region that binds a CGRP receptor comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 133. In some embodiments, the heavy chain variable region that binds a CGRP receptor has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservatively substituted amino acids compared to SEQ ID NO: 133. In some embodiments, the heavy chain variable region that binds a CGRP receptor comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 134. In some embodiments, the heavy chain variable region that binds a CGRP receptor has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservatively substituted amino acids compared to SEQ ID NO: 134. In some embodiments, the heavy chain variable region that binds a CGRP receptor is a heavy chain variable region of the first antigen binding domain described above.

[0240] In some embodiments, the heavy chain variable region that binds a CGRP receptor comprises a HCDR1 as set forth in SEQ ID NO: 60, a HCDR2 as set forth in SEQ ID NO: 61, and a HCDR3 as set forth in SEQ ID NO: 62. In some embodiments, the heavy chain variable region that binds a CGRP receptor comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 135. In some embodiments, the heavy chain variable region that binds a CGRP receptor has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservatively substituted amino acids compared to SEQ ID NO: 135. In some embodiments, the heavy chain variable region that binds a CGRP receptor is a heavy chain variable region of the second antigen binding domain described above.

[0241] In some embodiments, the antibody molecule or antigen-binding fragment thereof described above further comprises a third antigen binding domain, which is used to prolong the half-life of the antibody molecule or antigen-binding fragment thereof described above.

[0242] In some preferred embodiments, the third antigen binding domain specifically binds to human albumin.

[0243] In some embodiments, the third antigen binding domain comprises a heavy chain variable region comprising a HCDR1 as set forth in SEQ ID NO: 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 142, or any variant thereof, a HCDR2 as set forth in SEQ ID NO: 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 143, or any variant thereof, and a HCDR3 as set forth in SEQ ID NO: 72, 77, 82, 87, 92, 97, 102, 107, 112, 117, 122, 127, 144, or any variant thereof.

[0244] In some embodiments, the heavy chain variable region of the third antigen binding domain comprises a HCDR1, a HCDR2 and a HCDR3 selected from the group consisting of:

[0245] (1) SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72;

[0246] (2) SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77;

[0247] (3) SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82;

[0248] (4) SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87;

[0249] (5) SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92;

[0250] (6) SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97;

[0251] (7) SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102;

[0252] (8) SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107;

[0253] (9) SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112;

[0254] (10) SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117;

[0255] (11) SEQ ID NO: 120, SEQ ID NO: 121, and SEQ ID NO: 122;

[0256] (12) SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127; or

[0257] (13) SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 144.

[0258] In some preferred embodiments, the heavy chain variable region of the third antigen binding domain described above comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of:

[0259] (1) SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92; or

[0260] (2) SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102.

[0261] In some preferred embodiments, the heavy chain variable region of the third antigen binding domain described above comprises a HCDR1 as set forth in SEQ ID NO: 142, a HCDR2 as set forth in SEQ ID NO: 143, and a HCDR3 as set forth in SEQ ID NO: 144. In some embodiments, the heavy chain variable region of the third antigen binding domain described above comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 141. In some embodiments, the heavy chain variable region of the third antigen binding domain described above differs from SEQ ID NO: 141 by only up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substituted amino acids.

[0262] In some preferred embodiments, the VHH amino acid sequence of the third antigen binding domain described above is set forth below (SEQ ID NO: 141; individual HCDR1-3 regions are underlined, corresponding to SEQ ID NOs: 142-144):

[0263] In some preferred embodiments, the third antigen binding domain described above is a single domain antibody. In some preferred embodiments, the third antigen binding domain described above is humanized.

[0264] In some preferred embodiments, the third antigen binding domain described above can extend the half-life of the antibody molecule or antigen binding fragment thereof, or corresponding fusion protein or molecule. In some preferred embodiments, the half-life of the antibody molecule or antigen binding fragment thereof, or corresponding fusion protein or molecule, in a human body is at least 10 days, at least 15 days, or at least 20 days. In some preferred embodiments, the half-life of the antibody molecule or antigen binding fragment thereof, or corresponding fusion protein or molecule, in a human body is about 10 days to about 15 days, about 15 days to about 20 days, about 15 days to about 20 days, or about 20 days to about 30 days.

[0265] In some embodiments, the first and / or second antigen binding domain described above comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, or any variant thereof; and / or, the third antigen binding domain described above comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 69, 74, 79, 84, 89, 94, 99, 104, 109, 114, 119, 124, or any variant thereof.

[0266] In some preferred embodiments, the first antigen binding domain described above comprises an amino acid sequence set forth in SEQ ID NO: 59.

[0267] In some preferred embodiments, the second antigen binding domain described above comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 19, 24, 39, 44, and 64.

[0268] In some preferred embodiments, the third antigen binding domain described above comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 89 and 99. In some preferred embodiments, the third antigen binding domain described above comprises an amino acid sequence set forth in SEQ ID NO: 141.

[0269] In some embodiments, the first, second, and / or third antigen binding domain described above are directly or indirectly connected in sequence.

[0270] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein comprises, from N-terminus to C-terminus: a first antigen binding domain, a second antigen binding domain, sequentially connected.

[0271] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein comprises, from N-terminus to C-terminus: a second antigen binding domain, a first antigen binding domain, sequentially connected.

[0272] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein comprises, from N-terminus to C-terminus: a first antigen binding domain, a second antigen binding domain, a third antigen binding domain, sequentially connected.

[0273] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein comprises, from N-terminus to C-terminus: a first antigen binding domain, a third antigen binding domain, a second antigen binding domain, sequentially connected.

[0274] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein comprises, from N-terminus to C-terminus: a second antigen binding domain, a first antigen binding domain, a third antigen binding domain, sequentially connected.

[0275] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein comprises, from N-terminus to C-terminus: a second antigen binding domain, a third antigen binding domain, a first antigen binding domain, sequentially connected.

[0276] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein comprises, from N-terminus to C-terminus: a third antigen binding domain, a first antigen binding domain, a second antigen binding domain, sequentially connected.

[0277] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein comprises, from N-terminus to C-terminus: a third antigen binding domain, a second antigen binding domain, a first antigen binding domain, sequentially connected.

[0278] In some preferred embodiments, the above first antigen binding domain, second antigen binding domain, and / or third antigen binding domain are sequentially connected by a linker.

[0279] In some more preferred embodiments, the above linker comprises a flexible linker and a rigid linker.

[0280] In some embodiments, the C-terminus of the first antigen binding domain described above is connected to the N-terminus of the second antigen binding domain through a linker. In some embodiments, the C-terminus of the first antigen binding domain described above is connected to the N-terminus of the second antigen binding domain through a flexible linker. In some embodiments, the C-terminus of the first antigen binding domain described above is connected to the N-terminus of the second antigen binding domain through a rigid linker.

[0281] In some embodiments, the C-terminus of the second antigen binding domain described above is connected to the N-terminus of the first antigen binding domain through a linker. In some embodiments, the C-terminus of the second antigen binding domain described above is connected to the N-terminus of the first antigen binding domain through a flexible linker. In some embodiments, the C-terminus of the second antigen binding domain described above is connected to the N-terminus of the first antigen binding domain through a rigid linker.

[0282] In some embodiments, the C-terminus of the first antigen binding domain described above is connected to the N-terminus of the third antigen binding domain through a linker. In some embodiments, the C-terminus of the first antigen binding domain described above is connected to the N-terminus of the third antigen binding domain through a flexible linker. In some embodiments, the C-terminus of the first antigen binding domain described above is connected to the N-terminus of the third antigen binding domain through a rigid linker.

[0283] In some embodiments, the C-terminus of the third antigen binding domain described above is connected to the N-terminus of the first antigen binding domain through a linker. In some embodiments, the C-terminus of the third antigen binding domain described above is connected to the N-terminus of the first antigen binding domain through a flexible linker. In some embodiments, the C-terminus of the third antigen binding domain described above is connected to the N-terminus of the first antigen binding domain through a rigid linker.

[0284] In some embodiments, the C-terminus of the second antigen binding domain described above is connected to the N-terminus of the third antigen binding domain through a linker. In some embodiments, the C-terminus of the second antigen binding domain described above is connected to the N-terminus of the third antigen binding domain through a flexible linker. In some embodiments, the C-terminus of the second antigen binding domain described above is connected to the N-terminus of the third antigen binding domain through a rigid linker.

[0285] In some embodiments, the C-terminus of the third antigen binding domain described above is connected to the N-terminus of the second antigen binding domain through a linker. In some embodiments, the C-terminus of the third antigen binding domain described above is connected to the N-terminus of the second antigen binding domain through a flexible linker. In some embodiments, the C-terminus of the third antigen binding domain described above is connected to the N-terminus of the second antigen binding domain through a rigid linker.

[0286] In some preferred embodiments, the C-terminus of the second antigen binding domain described above is connected to the N-terminus of the first antigen binding domain via a flexible linker.

[0287] In some preferred embodiments, the C-terminus of the first antigen binding domain described above is connected to the N-terminus of the third antigen binding domain via a rigid linker.

[0288] In some preferred embodiments, the flexible polypeptide linker described above is selected from (G4S)n, (SG4)n, or G4(SG4)n, wherein n is an integer between 1 and 5. More preferably, the flexible polypeptide linker described above is GGGGSGGGGS (SEQ ID NO: 129).

[0289] In some preferred embodiments, the rigid polypeptide linker described above is selected from (EAAAKA)n, wherein n is an integer between 1 and 5. More preferably, the rigid polypeptide linker described above is EAAAKAEAAAKA (SEQ ID NO: 130).

[0290] In some embodiments, the first, second, and / or third antigen binding domain described above is a recombinant antibody, preferably a llama-derived antibody, a chimeric antibody, or a humanized antibody.

[0291] In some more preferred embodiments, the recombinant antibody described above is a nanobody; further preferably, the recombinant antibody described above is a humanized camelid VHH.

[0292] In some embodiments, the antigen binding domain (e.g., the first, second, and / or third antigen binding domain) is a single domain antibody. In some embodiments, the heavy chain variable region (e.g., the heavy chain variable region comprised by the first, second, and / or third antigen binding domain) is a single domain antibody.

[0293] In another aspect, the present application provides a molecule that binds to a Calcitonin Gene-Related Peptide (CGRP) receptor, the molecule comprising an antibody molecule or an antigen binding fragment thereof described herein. In some embodiments, the molecule comprises a fusion protein. In some embodiments, the molecule is a fusion protein. In some embodiments, the fusion protein comprises an antibody molecule or an antigen binding fragment thereof described herein. In some embodiments, the fusion protein further comprises a fusion partner.

[0294] In some preferred embodiments, the above antibody molecule or antigen-binding fragment, fusion protein, or molecule thereof comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 131. In some preferred embodiments, the above antibody molecule or antigen-binding fragment, fusion protein, or molecule thereof comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservatively substituted amino acids compared to SEQ ID NO: 131. In some more preferred embodiments, the above antibody molecule or antigen-binding fragment, fusion protein, or molecule thereof comprises the amino acid sequence as set forth in SEQ ID NO: 131.

[0295] In some embodiments, the antibody molecule or antigen-binding fragment, fusion protein, or molecule thereof described herein does not bind to pituitary adenylate cyclase-activating peptide (PACAP). In some embodiments, the antibody molecule or antigen-binding fragment, fusion protein, or molecule thereof described herein is unable to bind to PACAP with a Kd of less than 1 x 10 -6 M, less than 1 x 10 -5 M, less than 1 x 10 -4 M, less than 1 x 10 -3 M, or less than 1 x 10 -2 M. In some embodiments, the pituitary adenylate cyclase-activating peptide (PACAP) is a human-derived PACAP (PACAP38). In some embodiments, the pituitary adenylate cyclase-activating peptide (PACAP) comprises the amino acid sequence:

[0296] In some embodiments, the antibody molecule or antigen-binding fragment, fusion protein, or molecule thereof described herein does not bind to PACAP27. In some embodiments, the antibody molecule or antigen-binding fragment, fusion protein, or molecule thereof described herein is unable to bind to PACAP27 with a Kd of less than 1 x 10 -6 M, less than 1 x 10 -5 M, less than 1 x 10 -4 M, less than 1 x 10 -3 M, or less than 1 x 10 -2 M. In some embodiments, the PACAP27 is a human-derived PACAP27. In some embodiments, the PACAP27 comprises the amino acid sequence: HSDGIFTDSYSRYRKQMAVKKYLAAVL (SEQ ID NO: 137).

[0297] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not bind to VIP (Vasoactive Intestinal Polypeptide). In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to VIP with a Kd of less than 1 x 10 -6 M, less than 1 x 10 -5 M, less than 1 x 10 -4 M, less than 1 x 10 -3 M, or less than 1 x 10 -2 M. In some embodiments, the VIP is derived from a human. In some embodiments, the VIP comprises the amino acid sequence: HSDAVFTDNYTRLRKQMAVKKYLNSILNK (SEQ ID NO: 138).

[0298] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not bind to the extracellular domain fragment (ECD) of Pituitary Adenylate Cyclase 1 (PAC1). In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to the extracellular domain fragment (ECD) of PAC1 with a Kd of less than 1 x 10 -6 M, less than 1 x 10 -5 M, less than 1 x 10 -4 M, less than 1 x 10 -3 M, or less than 1 x 10 -2 M. In some embodiments, the extracellular domain fragment (ECD) of Pituitary Adenylate Cyclase 1 (PAC1) is derived from a human. In some embodiments, the extracellular domain fragment (ECD) of PAC1 comprises the amino acid sequence of SEQ ID NO: 139 or 140.

[0299] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not block the Pituitary Adenylate Cyclase Activating Peptide (PACAP) pathway.

[0300] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an extracellular domain fragment (ECD) of pituitary adenylate cyclase 1 (PAC1). In some embodiments, the PAC1 ECD is selected from the ECD of PAC isoforms: PAC1, PAC1s, or PAC1vs. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 30 contiguous amino acids of an extracellular domain fragment (ECD) of pituitary adenylate cyclase 1 (PAC1). In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 50 contiguous amino acids of an extracellular domain fragment (ECD) of pituitary adenylate cyclase 1 (PAC1). In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 100 contiguous amino acids of an extracellular domain fragment (ECD) of pituitary adenylate cyclase 1 (PAC1). In some embodiments, the PAC1 ECD is a PAC1s ECD. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 30 contiguous amino acids of a PAC1s ECD. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 50 contiguous amino acids of a PAC1s ECD. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 100 contiguous amino acids of a PAC1s ECD.

[0301] In some embodiments, the extracellular domain fragment (ECD) of pituitary adenylate cyclase 1 (PAC1) comprises the amino acid sequence:

[0302] MHSDCIFKKEQAMCLEKIQRANELMGFNDSSPGCPGMWDNITCWKPAHVGEMVLVSCPELFRIFNPDQVWETETIGESDFGDSNSLDLSDMGVVSRNCTEDGWSEPFPHYFDACGFDEYESETGDQDYYYLSVKA (SEQ ID NO: 139). In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 30 contiguous amino acids in SEQ ID NO: 139. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 50 contiguous amino acids in SEQ ID NO: 139. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 100 contiguous amino acids in SEQ ID NO: 139.

[0303] In some embodiments, the PAC1s ECD comprises the amino acid sequence:

[0304] MHSDCIFKKEQAMCLEKIQRANELMGFNDSSPGCPGMWDNITCWKPAHVGEMVLVSCPELFRIFNPDQDMGVVSRNCTEDGWSEPFPHYFDACGFDEYESETGDQDYYYLSVKA (SEQ ID NO: 140). In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 30 contiguous amino acids in SEQ ID NO: 140. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 50 contiguous amino acids in SEQ ID NO: 140. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to a fragment of any 100 contiguous amino acids in SEQ ID NO: 140.

[0305] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an Fc fragment. An "Fc fragment" refers to a polypeptide sequence corresponding to or derived from a portion of an antibody that is generally capable of binding to an Fc receptor on a cell and / or the Clq component of complement, thereby mediating effector functions of the antibody. Fc stands for "fragment, crystallizable," i.e., the fragment of an antibody that will readily form protein crystals. Different protein fragments, originally described by proteolytic digestion, can define the general overall structure of an immunoglobulin. Generally, the Fc region is a homodimeric protein comprising two polypeptides associated by disulfide bonds and each comprising a hinge region, a CH2 domain, and a CH3 domain. However, recently, the term has been applied to the single chain monomer component consisting of CH3, CH2, and at least a portion of the hinge, which is sufficient to form a disulfide-linked dimer with such a second chain. For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31 : 169-217, 1994. As used herein, the term Fc fragment includes variants of naturally occurring sequences. In some embodiments, the Fc fragment comprises a hinge region, a constant region 2 (CH2) and a constant region 3 (CH3) domain derived from a human immunoglobulin.

[0306] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise an immunoglobulin constant region. The term "immunoglobulin constant region" or "constant region" refers to a peptide or polypeptide sequence corresponding to or derived from all or a portion of one or more constant domains (e.g., CHI, CH2, CH3) of an immunoglobulin. Immunoglobulin constant regions include light chain constant regions as well as heavy chain constant regions. The term "immunoglobulin light chain constant region" (also referred to as "light chain constant region" or "CL") refers to the constant region from an antibody light chain. The term "immunoglobulin heavy chain constant region" (also referred to as "heavy chain constant region" or "CH") refers to the constant region from an antibody heavy chain. Depending on the antibody isotype, CH can be further divided into a CHI domain, a CH2 domain, and a CH3 domain (IgA, IgD, IgG), or a CHI domain, a CH2 domain, a CH3 domain, and a CH4 domain (IgE, IgM). In some embodiments, the constant domains that make up the constant region are human.

[0307] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise a CH1 domain, a CH2 domain, a CH3 domain, and / or a CH4 domain. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise a CH1 domain. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise a CH2 domain. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise a CH3 domain. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not comprise a CH4 domain.

[0308] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is capable of binding to a human CGRP receptor with a Kd of 1 x 10 -7 In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is capable of binding to a human CGRP receptor with a Kd of not more than 100 nM, not more than 10 nM, not more than 1 nM, not more than 100 pM, not more than 10 pM, or not more than 1 pM. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is capable of binding to a human CGRP receptor with a Kd of 30 nM to 100 nM, 10 nM to 30 nM, 3 nM to 10 nM, 1 nM to 3 nM, 300 pM to 1 nM, 100 pM to 300 pM, 30 pM to 100 pM, 10 pM to 30 pM, 3 pM to 10 pM, or 1 pM to 3 pM.

[0309] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is capable of inhibiting the binding of a CGRP receptor to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP with a Kd of less than 1 x 10 -5 In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is capable of inhibiting the binding of a CGRP receptor to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP with a Kd of less than 1 x 10 -4 In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is capable of inhibiting the binding of a CGRP receptor to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP with a Kd of less than 1 x 10 -3 In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is capable of inhibiting the binding of a CGRP receptor to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP with a Kd of less than 1 x 10 -2 In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is capable of inhibiting the binding of a CGRP receptor to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP with a Kd of less than 1 x 10 -1 In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is capable of inhibiting the binding of a CGRP receptor to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP. In some embodiments, a CGRP receptor bound by the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is unable to bind to CGRP with a Kd of less than 1 x 10

[0310] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is capable of inhibiting cAMP production by CHOK1-CGRPr cells. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein has at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold superior ability to inhibit CGRP-induced cAMP production at the CHOK1-CGRPr cell level than Erenumab mAb. In some embodiments, the comparison of the ability to inhibit is based on IC50 50 (eg, IC 50 of 0.1 nM is 10-fold superior to a molecule with an IC 50 of 1 nM).

[0311] In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein does not bind to the human Amylin 1 receptor. In some embodiments, the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein is not capable of binding to the human Amylin 1 receptor with a Kd of less than 1 x 10 -6 M, less than 1 x 10 -5 M, less than 1 x 10 -4 M, less than 1 x 10 -3 M, or less than 1 x 10 -2 M.

[0312] In another aspect, the present application provides a nucleic acid encoding the antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule of any of the above aspects.

[0313] In some preferred embodiments, the above first antigen binding domain is encoded by a nucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, or any variants thereof.

[0314] In some preferred embodiments, the above second antigen binding domain is encoded by a nucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, or any variants thereof.

[0315] In some preferred embodiments, the above third antigen binding domain is encoded by a nucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 73, 78, 83, 88, 93, 98, 103, 108, 113, 118, 123, 128, or any variants thereof.

[0316] In another aspect, the present application provides a vector comprising the above nucleic acid.

[0317] In another aspect, the present application provides a cell comprising the above nucleic acid or vector.

[0318] In another aspect, the present application provides a kit comprising the antibody molecule or antigen-binding fragment thereof, fusion protein, molecule, nucleic acid, vector, or cell of any of the above aspects.

[0319] In another aspect, the present application provides a pharmaceutical composition comprising the antibody molecule or antigen-binding fragment thereof, fusion protein, molecule, nucleic acid, vector, or cell of any of the above aspects. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0320] The pharmaceutical compositions of the present application can be administered to a subject in need thereof by a variety of routes including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal, or by implantation or inhalation. The compositions of the present application can be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. The appropriate formulation and administration route can be selected based on intended application and treatment regimen.

[0321] The term "subject" includes any human or non-human animal, preferably a mammal. In some preferred embodiments, the subject is a human.

[0322] In another aspect, the present application provides the use of the antibody molecule or antigen-binding fragment thereof, fusion protein, molecule, nucleic acid, vector, cell, and / or pharmaceutical composition of any of the above aspects in the manufacture of a medicament for the treatment and / or prevention of a disease associated with CGRP overactivity.

[0323] In another aspect, the present application provides a method of treating and / or preventing a disease associated with CGRP overactivity in a subject, the method comprising: administering to the subject a therapeutically effective amount of the antibody molecule or antigen-binding fragment thereof, fusion protein, molecule, nucleic acid, vector, cell, and / or pharmaceutical composition of any of the above aspects.

[0324] In another aspect, the present application provides the use of the antibody molecule or antigen-binding fragment thereof, fusion protein, molecule, nucleic acid, vector, cell, and / or pharmaceutical composition or kit of any of the above aspects in the treatment and / or prevention of a disease associated with CGRP overactivity.

[0325] In another aspect, the application provides a method of inhibiting or blocking the binding of a CGRP receptor on the surface of a cell to CGRP, the method comprising contacting the cell with an antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein. In some embodiments, the cell is in vitro (e.g., in vitro or ex vivo). In some embodiments, the cell is in vivo in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an antibody molecule or antigen-binding fragment thereof, fusion protein, or molecule described herein.

[0326] In some preferred embodiments, the disease described above is migraine. In some preferred embodiments, the disease described above comprises episodic migraine, menstrual migraine, chronic migraine.

[0327] For the purposes of clarity and brevity, features described herein as part of some embodiments that are the same or separate will be understood to encompass some embodiments having all or some combination of the features described.

[0328] Other numbered embodiments of the technology

[0329] Other numbered embodiments of the application are as follows:

[0330] Item 1. An antibody molecule or antigen-binding fragment thereof that specifically binds to a Calcitonin Gene-Related Peptide (CGRP) receptor, comprising a first antigen binding domain comprising a heavy chain variable region that binds to a CGRP receptor, the heavy chain variable region comprising a heavy chain complementarity determining region (HCDR)1 as set forth in SEQ ID NO: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or any variant thereof, a heavy chain CDR2 (HCDR2) as set forth in SEQ ID NO: 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, or any variant thereof, and a heavy chain CDR3 (HCDR3) as set forth in SEQ ID NO: 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, or any variant thereof.

[0331] Item 2. The antibody molecule or antigen-binding fragment thereof of item 1, wherein the heavy chain variable region of the first antigen binding domain comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of:

[0332] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0333] (2) SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17;

[0334] (3) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0335] (4) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0336] (5) SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32;

[0337] (6) SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37;

[0338] (7) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0339] (8) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47;

[0340] (9) SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52;

[0341] (10) SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57;

[0342] (11) SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62; and

[0343] (12) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0344] Item 3. The antibody molecule or antigen-binding fragment thereof of item 2, wherein the heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from the group consisting of SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, and 132-135.

[0345] Item 4. The antibody molecule or antigen-binding fragment thereof of any one of items 1-3, wherein the heavy chain variable region of the first antigen binding domain comprises HCDR1, HCDR2, and HCDR3 selected from the group consisting of:

[0346] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0347] (2) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0348] (3) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0349] (4) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0350] (5) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; and

[0351] (6) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67;

[0352] Item 5. The antibody molecule or antigen-binding fragment thereof of item 4, wherein the heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from SEQ ID NO: 9, 19, 24, 39, 44, 64, and 132-134.

[0353] Item 6. The antibody molecule or antigen-binding fragment thereof of items 1-5, wherein the heavy chain variable region of the first antigen binding domain comprises HCDR1 as set forth in SEQ ID NO: 20, HCDR2 as set forth in SEQ ID NO: 21, and HCDR3 as set forth in SEQ ID NO: 22.

[0354] Item 7. The antibody molecule or antigen-binding fragment thereof of item 6, wherein the heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from SEQ ID NO: 19 and 132-134.

[0355] Item 8. The antibody molecule or antigen-binding fragment thereof of item 6, wherein the heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 132.

[0356] Item 9. The antibody molecule or antigen-binding fragment thereof of any one of items 1-8, wherein the first antigen binding domain is a single domain antibody.

[0357] Item 10. The antibody molecule or antigen-binding fragment thereof of any one of items 1-9, wherein the first antigen binding domain is humanized.

[0358] Item 11. The antibody molecule or antigen-binding fragment thereof of any one of items 1-10, further comprising a second antigen binding domain that binds to a CGRP receptor.

[0359] Item 12. The antibody molecule or antigen-binding fragment thereof of item 11, wherein the antigenic epitope of the first antigen binding domain is different from the antigenic epitope of the second antigen binding domain; preferably, the two antigenic epitopes do not overlap.

[0360] Item 13. The antibody molecule or antigen-binding fragment thereof of any one of items 11-12, wherein the second antigen binding domain comprises a heavy chain variable region that binds to a CGRP receptor, the heavy chain variable region of the second antigen binding domain comprising a HCDR1 as set forth in SEQ ID NO: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or any variant thereof, a HCDR2 as set forth in SEQ ID NO: 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, or any variant thereof, and a HCDR3 as set forth in SEQ ID NO: 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, or any variant thereof.

[0361] Item 14. The antibody molecule or antigen-binding fragment thereof of item 13, wherein the heavy chain variable region of the second antigen binding domain comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of:

[0362] (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;

[0363] (2) SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17;

[0364] (3) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0365] (4) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27;

[0366] (5) SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32;

[0367] (6) SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37;

[0368] (7) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42;

[0369] (8) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47;

[0370] (9) SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52;

[0371] (10) SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57;

[0372] (11) SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62; and

[0373] (12) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 67.

[0374] Item 15. The antibody molecule or antigen-binding fragment thereof of item 14, wherein the heavy chain variable region of the second antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, and 132-135.

[0375] Item 16. The antibody molecule or antigen-binding fragment thereof of any one of items 11-15, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the first antigen binding domain and the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the second antigen binding domain are selected from a group in an embodiment of Table 11.

[0376] Item 17. The antibody molecule or antigen-binding fragment thereof of any one of items 11-16, wherein the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the first antigen binding domain are different from the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the second antigen binding domain.

[0377] Item 18. The antibody molecule or antigen-binding fragment thereof of any one of items 13-17, wherein the heavy chain variable region of the second antigen binding domain comprises a HCDR1 as set forth in SEQ ID NO: 60, a HCDR2 as set forth in SEQ ID NO: 61, and a HCDR3 as set forth in SEQ ID NO: 62.

[0378] Item 19. The antibody molecule or antigen-binding fragment thereof of item 18, wherein the heavy chain variable region of the second antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 135.

[0379] Item 20. The antibody molecule or antigen-binding fragment thereof of item 18, wherein the heavy chain variable region of the second antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 59.

[0380] Item 21. The antibody molecule or antigen-binding fragment thereof of any one of items 11-20, wherein the second antigen binding domain is a single domain antibody.

[0381] Item 22. The antibody molecule or antigen-binding fragment thereof of any one of items 11-21, wherein the second antigen binding domain is humanized.

[0382] Item 23. The antibody molecule or antigen-binding fragment thereof of any one of items 1-22, further comprising a third antigen binding domain that specifically binds human albumin.

[0383] Item 24. The antibody molecule or antigen-binding fragment thereof of item 23, wherein the third antigen binding domain comprises a heavy chain variable region comprising a HCDR1 as set forth in SEQ ID NO: 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 142, or any variant thereof, a HCDR2 as set forth in SEQ ID NO: 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 143, or any variant thereof, and a HCDR3 as set forth in SEQ ID NO: 72, 77, 82, 87, 92, 97, 102, 107, 112, 117, 122, 127, 144, or any variant thereof.

[0384] Item 25. The antibody molecule or antigen-binding fragment thereof of item 24, wherein the heavy chain variable region of the third antigen binding domain comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of:

[0385] (1) SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72;

[0386] (2) SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77;

[0387] (3) SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82;

[0388] (4) SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87;

[0389] (5) SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92;

[0390] (6) SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97;

[0391] (7) SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102;

[0392] (8) SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107;

[0393] (9) SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112;

[0394] (10) SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117;

[0395] (11) SEQ ID NO: 120, SEQ ID NO: 121, and SEQ ID NO: 122;

[0396] (12) SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127; and

[0397] (13) SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO: 144.

[0398] Item 26. The antibody molecule or antigen-binding fragment thereof of item 25, wherein the heavy chain variable region of the third antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 69, 74, 79, 84, 89, 94, 99, 104, 109, 114, 119, and 124.

[0399] Item 27. The antibody molecule or antigen-binding fragment thereof of any one of items 25-26, wherein the heavy chain variable region of the third antigen binding domain comprises a HCDR1 as set forth in SEQ ID NO: 90, a HCDR2 as set forth in SEQ ID NO: 91, and a HCDR3 as set forth in SEQ ID NO: 92; preferably the heavy chain variable region of the third antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 89.

[0400] Item 28. The antibody molecule or antigen-binding fragment thereof of any one of items 25-26, wherein the heavy chain variable region of the third antigen binding domain comprises a HCDR1 as set forth in SEQ ID NO: 100, a HCDR2 as set forth in SEQ ID NO: 101, and a HCDR3 as set forth in SEQ ID NO: 102; preferably the heavy chain variable region of the third antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 99.

[0401] Item 29. The antibody molecule or antigen-binding fragment thereof of any one of items 25-26, wherein the heavy chain variable region of the third antigen binding domain comprises a HCDR1 as set forth in SEQ ID NO: 142, a HCDR2 as set forth in SEQ ID NO: 143, and a HCDR3 as set forth in SEQ ID NO: 144.

[0402] Item 30. The antibody molecule or antigen-binding fragment thereof of item 29, wherein the heavy chain variable region of the third antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 141.

[0403] Item 31. The antibody molecule or antigen-binding fragment thereof of any one of items 23-30, wherein the third antigen binding domain is a single domain antibody.

[0404] Item 32. The antibody molecule or antigen-binding fragment thereof of any one of items 23-31, wherein the third antigen binding domain is humanized.

[0405] Item 33. The antibody molecule or antigen-binding fragment thereof of any one of items 23-32, wherein the third antigen binding domain prolongs the half-life of the antibody molecule or antigen-binding fragment thereof.

[0406] Item 34. The antibody molecule or antigen-binding fragment thereof of any one of items 23-33, which has a half-life in humans of at least 10 days, at least 15 days, or at least 20 days.

[0407] Item 35. The antibody molecule or antigen-binding fragment thereof of any one of items 1-34, wherein the first, second, and / or third antigen binding domains are directly or indirectly sequentially linked.

[0408] Item 35.1. The antibody molecule or antigen-binding fragment thereof of item 35, wherein the first and third antigen binding domains are directly or indirectly sequentially linked.

[0409] Item 35.2. The antibody molecule or antigen-binding fragment thereof of any one of items 35-35.1, wherein the second and first antigen binding domains are directly or indirectly sequentially linked.

[0410] Item 35.3. The antibody molecule or antigen-binding fragment thereof of any one of items 35-35.2, wherein the first, second, and / or third antigen binding domains are sequentially linked by a linker.

[0411] Item 35.4. The antibody molecule or antigen-binding fragment thereof according to item 35.3, wherein the linker comprises a rigid linker.

[0412] Item 35.5. The antibody molecule or antigen-binding fragment thereof according to any one of items 35.1-35.4, wherein the C-terminus of the first antigen binding domain is connected to the N-terminus of the third antigen binding domain via a rigid linker.

[0413] Item 35.6. The antibody molecule or antigen-binding fragment thereof according to any one of items 35.4-35.5, wherein the rigid linker is a rigid polypeptide linker selected from the group consisting of (EAAAKA)n, wherein n is an integer between 1-5; preferably the rigid polypeptide linker is EAAAKAEAAAKA (SEQ ID NO: 130).

[0414] Item 35.7. The antibody molecule or antigen-binding fragment thereof according to any one of items 35.3-35.6, wherein the linker comprises a flexible linker.

[0415] Item 35.8. The antibody molecule or antigen-binding fragment thereof according to any one of items 35.2-35.7, wherein the C-terminus of the second antigen binding domain is connected to the N-terminus of the first antigen binding domain via a flexible linker.

[0416] Item 35.9. The antibody molecule or antigen-binding fragment thereof according to any one of items 35.7-35.8, wherein the flexible linker is a flexible polypeptide linker selected from the group consisting of (G4S)n, (SG4)n, or G4(SG4)n, wherein n is an integer between 1-5; preferably the flexible polypeptide linker is GGGGSGGGGS (SEQ ID NO: 129).

[0417] Item 36. The antibody molecule or antigen-binding fragment thereof according to any one of items 1-35.9, wherein the first, second and / or third antigen binding domain is a recombinant antibody.

[0418] Item 36.1. The antibody molecule or antigen-binding fragment thereof according to item 36, wherein the first, second and / or third antigen binding domain is a llama-derived antibody, a chimeric antibody, or a humanized antibody.

[0419] Item 36.2. The antibody molecule or antigen-binding fragment thereof according to any one of items 36-36.1, wherein the recombinant antibody is a nanobody.

[0420] Item 36.3. The antibody molecule or antigen-binding fragment thereof of any one of items 36-36.2, wherein the recombinant antibody is a humanized camelid VHH.

[0421] Item 37. The antibody molecule or antigen-binding fragment thereof of any one of items 1-36.3, comprising an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 131.

[0422] Item 38. A molecule that binds to a Calcitonin Gene-Related Peptide (CGRP) receptor, comprising the antibody molecule or antigen-binding fragment thereof of any one of items 1-37.

[0423] Item 39. The antibody molecule or antigen-binding fragment thereof of any one of items 1-37, or the molecule of item 38, which antibody molecule or antigen-binding fragment thereof or molecule does not bind to Pituitary Adenylate Cyclase Activating Peptide (PACAP), PACAP27, or VIP (Vasoactive Intestinal Polypeptide).

[0424] Item 40. The antibody molecule or antigen-binding fragment thereof or molecule of any one of items 1-39, which antibody molecule or antigen-binding fragment thereof or molecule does not block the Pituitary Adenylate Cyclase Activating Peptide (PACAP) pathway.

[0425] Item 41. The antibody molecule or antigen-binding fragment thereof or molecule of any one of items 1-40, which antibody molecule or antigen-binding fragment thereof or molecule does not comprise an extracellular domain fragment (ECD) of Pituitary Adenylate Cyclase 1 (PAC1).

[0426] Item 42. The antibody molecule or antigen-binding fragment thereof or molecule of any one of items 1-41, which antibody molecule or antigen-binding fragment thereof or molecule does not comprise an Fc fragment.

[0427] Item 43. The antibody molecule or antigen-binding fragment thereof or molecule of any one of items 1-42, which antibody molecule or antigen-binding fragment thereof or molecule does not comprise an immunoglobulin constant region.

[0428] Item 44. The antibody molecule or antigen-binding fragment thereof or molecule of any one of items 1-43, which has one or more of the following properties:

[0429] (a) binds to a CGRP receptor with a Kd of 1 x 10 -7binds human CGRP receptor with a Kd of M or less; (b) inhibits binding of CGRP receptor to CGRP; (c) inhibits CGRP-induced cAMP production in CHOK1 cells expressing CGRPr; and (d) does not bind to human Amylin 1 receptor.

[0430] Item 45. A nucleic acid encoding the antibody molecule or antigen-binding fragment thereof of any one of items 1-37 and 39-44, or the molecule of any one of items 38-44.

[0431] Item 46. The nucleic acid of item 45, wherein:

[0432] the first antigen binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, or any variant thereof;

[0433] the second antigen binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, or any variant thereof; or the third antigen binding domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 73, 78, 83, 88, 93, 98, 103, 108, 113, 118, 123, 128, or any variant thereof.

[0434] Item 47. A vector comprising the nucleic acid of any one of items 45-46.

[0435] Item 48. A cell comprising the nucleic acid of any one of items 45-46 or the vector of item 47.

[0436] Item 49. A kit comprising the antibody molecule or antigen-binding fragment thereof of any one of items 1-37 and 39-44, the molecule of any one of items 38-44, the nucleic acid of any one of items 45-46, the vector of item 47, or the cell of item 48.

[0437] Item 50. A pharmaceutical composition comprising (1) the antibody molecule or antigen-binding fragment thereof of any one of items 1-37 and 39-44, the molecule of any one of items 38-44, the nucleic acid of any one of items 45-46, the vector of item 47, or the cell of item 48, and (2) a pharmaceutically acceptable carrier, diluent, or excipient.

[0438] Item 51. Use of the antibody molecule or antigen-binding fragment thereof according to any one of items 1-37 and 39-44, the molecule according to any one of items 38-44, the nucleic acid according to any one of items 45-46, the vector according to item 47, and / or the cell according to item 48, and / or the pharmaceutical composition according to item 50, in the manufacture of a medicament for the treatment and / or prevention of a disease associated with CGRP over-activation.

[0439] Item 52. A method for the treatment and / or prevention of a disease associated with CGRP over-activation in a subject, the method comprising: administering to the subject a therapeutically effective amount of the antibody molecule or antigen-binding fragment thereof according to any one of items 1-37 and 39-44, the molecule according to any one of items 38-44, the nucleic acid according to any one of items 45-46, the vector according to item 47, and / or the cell according to item 48, and / or the pharmaceutical composition according to item 50.

[0440] Item 53. Use of the antibody molecule or antigen-binding fragment thereof according to any one of items 1-37 and 39-44, the molecule according to any one of items 38-44, the nucleic acid according to any one of items 45-46, the vector according to item 47, and / or the cell according to item 48, and / or the pharmaceutical composition according to item 14, for the treatment and / or prevention of a disease associated with CGRP over-activation.

[0441] Item 54. The use, method or application according to any one of items 51-53, wherein the disease is migraine; preferably the disease comprises episodic migraine, menstrual migraine, chronic migraine.

[0442] Examples

[0443] Example 1: Preparation of immunogens

[0444] Anti-CGRP receptor Nanobodies were obtained by immunizing llamas with the ECD (extracellular domain) of the CGRP receptor, followed by multiple rounds of screening and humanization. Anti-human albumin Nanobodies were obtained by immunizing llamas with human albumin, followed by multiple rounds of screening and humanization.

[0445] To obtain antibodies with high selectivity and high affinity to bind CGRP receptor, the present application uses in vitro expressed and purified CGRP ECD and human immunoglobulin G1 (IgG1) fragment crystallizable (Fc) fusion protein (RC-Fc for short) as immunogen to immunize llamas, then isolates lymphocytes from high titer llama blood and extracts their messenger ribonucleic acid (mRNA). The mRNAs are used to construct phage libraries, and positive colonies containing single-domain antibodies that can bind RC-Fc and only Fc are screened out respectively by enzyme-linked immunosorbent assay (ELISA). These colonies include all single-domain antibodies that bind CGRP ECD and Fc, and the colonies that can only bind Fc are excluded. The remaining CGRP ECD positive and Fc negative colonies are sequenced. Through sequence analysis and verification, a batch of single-domain antibodies that can specifically bind CGRP ECD is obtained.

[0446] To obtain antibodies with high selectivity and high affinity to bind human albumin, the present application uses in vitro expressed and purified albumin as immunogen to immunize llamas, then isolates lymphocytes from high titer llama blood and extracts their messenger ribonucleic acid (mRNA). The mRNAs are used to construct phage libraries, and positive colonies containing single-domain antibodies that can bind human albumin are screened out by enzyme-linked immunosorbent assay (ELISA). Through sequence analysis and verification, a batch of single-domain antibodies that can specifically bind human albumin is obtained.

[0447] 1. Preparation of RC-Fc, immunological screening and obtaining of anti-CGRP receptor single-domain antibodies

[0448] RC-Fc is fused from human Ramp1 ECD, CLR (Calcitonin-receptor-like receptor) ECD and Fc. Ramp1 ECD is from the nucleic acid sequence 132-404 of Genbank ID NM_005855.4. The nucleic acid sequence is as follows (SEQ ID NO: 1):

[0449] The corresponding amino acid sequence is from NP_005846.1 27-117. The amino acid sequence is as follows (SEQ ID NO: 2):

[0450] CLR ECD is from the nucleic acid sequence 407-782 of Genbank ID NM_001271751.2. The nucleic acid sequence is as follows (SEQ ID NO: 3):

[0451] The corresponding amino acid sequence is from NP_001258680.1 23-146. The amino acid sequence is as follows (SEQ ID NO: 4):

[0452] The Fc part is from Genbank ID AFR78282.1 nucleic acid sequence 661-1359, protein sequence 221-330. The cloning uses the nucleic acid sequence as follows (SEQ ID NO: 5):

[0453] The amino acid sequence is as follows (SEQ ID NO: 6):

[0454] Because the amino acid sequences of Ramp1 ECD and CLR ECD are different, when fused with Fc, they need to be paired by Knob in Hole (KiH) technology (14). The C-terminal of Ramp1 ECD is connected with the N-terminal of Fc with T366W mutation to form the R chain; the C-terminal of CLR ECD is fused with the N-terminal of Fc with T366S, L368A and Y407V mutations to form the C chain.

[0455] The amino acid sequence of RC-Fc Ramp1 ECD (RC-Fc R) chain is (SEQ ID NO: 7):

[0456] The amino acid sequence of RC-Fc CLR ECD (RC-Fc C) chain is (SEQ ID NO: 8):

[0457] 2. Gene synthesis, expression and purification of RC-Fc

[0458] The RC-Fc plasmid is synthesized as follows: the synthesized gene fragment is ligated with the pcDNA3.4 vector (ThermoFisher Scientific, EL0011) recovered after NotI (New England BioLabs Inc. (NEB, R0189L) / XbaI (NEB, R0145L) enzyme digestion. The ligation product is transformed into Top10 competent cells, and positive clones are selected for expansion culture. The plasmid is extracted in small quantities using a plasmid extraction kit (self-made) for sequencing. The correct clones are inoculated in LB medium (self-made) for expansion culture, and the plasmid is extracted in large quantities using a plasmid extraction kit (self-made) and re-sequenced for verification.

[0459] The 293 cells were transfected with the expression plasmid using transfection reagent (self-made), and then cultured in a constant temperature incubator (Jingqi, IS-RDS6C5) for 5 days. After centrifugation, the supernatant was collected, filtered to remove cell debris, and the clear liquid was collected. The Protein A affinity chromatography column was used to capture the target protein. The target protein was filtered and the final yield was determined by measuring A280. The NanoDrop One (ThermoFisher Scientific, ND-ONE-W) instrument was used to read the absorbance value at 280 nm. The protein was absorbed into a dialysis bag and dialyzed in a beaker containing 1XPBS. The purified product was identified by electrophoresis (the results are shown in Figure 3).

[0460] 3. Verification of RC-Fc binding to CGRP

[0461] The RC-Fc fusion protein after expression and purification was verified to be able to bind to its ligand CGRP before being used for animal immunization. The binding enzyme-linked (ELISA) method was used for verification as follows.

[0462] Streptavidin-coated 96-well enzyme-labeled plates (ThermoFisher Scientific TM , 15126) were washed 3 times with PBST (1xPBS (Absin, abs9266), 0.1% Tween 20 (Solarbio, T8220), pH=7.4), and then 1000 ng of Biotin-CGRP (Nanjing Yuanpeibio Technology Co., Ltd., A03Biotin137, P200629-HS319172) was added to each well. Incubate at room temperature for 2 hours. Remove PBST and add 100 μL of RC-Fc fusion protein diluted with PBST to each well, and incubate at 37°C for 1 hour. Wash the enzyme-labeled plate incubated with the sample 5 times with PBST, then add 100 μL of corresponding secondary antibody Anti-human IgG-HRP (anti-human immunoglobulin G-horseradish peroxidase, Hangzhou Huaan Biological, HR1214) diluted with blocking solution (PBST+3% skim milk powder (Solarbio, D8340)) 1:5000, and incubate at 37°C for 1 hour.

[0463] The enzyme-labeled plate incubated with the secondary antibody was washed with PBST for 5 times, 100 μL TMB (Trimethylolpropane) single-component color developing liquid (Solarbio, 20190402) was added to each well, and incubated at 37°C for 7 minutes. 100 μL 1M HCL (Kelong Chemical Industry) was added to each well to terminate the reaction, and finally the OD value was read at 450 nm wavelength by an enzyme-labeled instrument (ThermoFisher Scientific, 51119080ET). The OD OD450 value was analyzed by Nonlinear Regression One Site Specific Binding mode of GraphPad Prism 9.3.1, and the Bmax of RC-Fc binding to CGRP was 5.198, and the Kd was 16.12 nM. The Bmax of RC-Fc binding to negative control polypeptide PACAP was 0.069, and the Kd was invalid. The results showed that the reconstructed extracellular region of CGRP receptor retained the ability to bind its ligand CGRP, but did not bind to irrelevant polypeptide PACAP (see Figure 4 for results).

[0464] Example 2: Preparation of single-domain antibodies against CGRP receptor

[0465] 1. Immunize llamas with RC-Fc

[0466] Before immunizing the llamas, 10 mL of blood was taken as a negative serum control. Then 0.5 mg of RC-Fc was mixed with 1 ml of Complete Freund’s Adjuvant (CFA, Sigma, F5881) and injected subcutaneously. On day 21, 0.25 mg of antigen was mixed with 1 ml of Incomplete Freund’s Adjuvant (IFA, Sigma, F5506) and injected subcutaneously. On day 28, 10 mL of blood was taken to separate the serum. On day 42, 0.25 mg of antigen was mixed with 1 ml of IFA and injected subcutaneously. On day 49, 50 mL of peripheral blood was collected to separate lymphocytes and serum. On day 63, 0.25 mg of antigen was mixed with 1 ml of IFA and injected subcutaneously. On day 70, 50 mL of peripheral blood was collected to separate lymphocytes and serum.

[0467] 2. Gradient detection

[0468] RC-Fc was diluted to 2000 ng / ml with coating solution (50 mM NaHCO3 PH=9.6), 100 μL was added to each well of the 96-well enzyme-labeled plate, and incubated at 4°C overnight. The enzyme-labeled plate coated overnight at 4°C was washed with PBST for 5 times, and dried, 300 μL of blocking solution was added to each well.

[0469] Serially dilute the serum 1:2 with TBST, the first gradient serum is 1:2000, the last gradient serum is 1:128000. After washing the antigen plate with 300 μL / well PBST for three times, add 100ul diluted serum to each well, incubate at 37 degrees for 1 hour, wash with 300 μL / well PBST for three times, add 100 μL 1:2000 anti-M13 antibody (NB Biolab, 052-101-005) to each well, incubate at 37 degrees for 1 hour, wash with 300 μL / well PBST for six times, add 1:10000 diluted anti-Alpaca-HRP (NEB, S001H), incubate at 37 degrees for 30 minutes. After the secondary antibody incubated enzyme-labeled plate, wash with PBST for 5 times, add 100 μL TMB single-component color developing liquid to each well, incubate at 37 degrees for 7 minutes, add 100 μL 1M HCL to each well to stop the reaction, and read OD450.

[0470] 3. Construction of phage library

[0471] Select high gradient peripheral blood, separate PBMC according to the instructions of lymphocyte separation medium (Tianjin Haoyang Biological, LTS1077). Extract total RNA with RNAiso Plus (Fuji Biological, RE-03111) reagent, transcribe 5 μg RNA into cDNA with PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara, 9109). Dilute the cDNA stock solution 5 times for nest PCR amplification, recover the single domain antibody (VHH) fragment of about 750 base pairs (BP) from the gel for the second round of PCR amplification, and purify the PCR as VHH fragment. The VHH fragment is connected with the vector pComb3XSS (NEB, P001) through Sfil enzyme cutting point, and 1 mL 2YT (culture medium containing tryptone (Solarbio, T8490), yeast extract (Solarbio, Y8020) and sodium chloride (Kelong Chemical Industry)) is added to the shock cup immediately after shocking. The shock product is sucked out and the shock cup is washed with 2YT medium, a total of 100 ml of recovery product is obtained, 37°C, 180 rpm (rev / min), recovery for 45 minutes, 100 μL is diluted to 10-3 and 10-4 to determine the number of library transformants, and is coated on 90 mm plates. The rest is centrifuged, resuspended with 8 mL 2YT, and coated on 8 pieces of 200 mm plates. The next day, the plates for determining the number of library transformants are obtained with VHH bacterial clones.

[0472] 4. Phage library packaging

[0473] Bacterial library was inoculated into 2x300 mL 2YT+A (Ampicillin (Solarbio, A1170) 100 ug / ml) + G (1% Glucose (Solarbio, G8150) medium to its initial OD600 = 0.1-0.2, incubated at 37°C, 230 rpm until OD600 = 0.8 or more. According to OD600 value, helper phage M13KO7 (NB biolab, P006) and TG1 bacteria (NB biolab, P008) were added (helper phage: bacteria = 20: 1). After adding M13KO7, mix well, stand at 37°C for 30 minutes, shake at 180 rpm for 30 minutes, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, resuspend the precipitate with an equal volume of 2YT+A+K (Kanamycin (Solarbio, K1030) 50 ug / ml) medium, 30°C, 220 rpm overnight.

[0474] The overnight culture was centrifuged at 10000 rpm for 20 min at 4°C, the supernatant was collected and the precipitate was discarded. Replace the centrifuge tube, centrifuge at 10000 rpm for 20 min at 4°C, collect the supernatant.

[0475] Add PEG8000 (Solarbio, P8260) / NaCl at 1 / 5 of the volume of the supernatant, mix well, and precipitate in an ice bath for 2 hours or more. Centrifuge at 10000 rpm for 20 minutes, discard the supernatant, and empty the supernatant once. Resuspend the precipitate with 1 mL of 1xPBS, and precipitate again with 1 / 5 volume of PEG8000 / NaCl for 1 hour.

[0476] Centrifuge at 12000 rpm for 10 minutes, discard the supernatant, and empty the supernatant once. According to the amount of precipitate, resuspend the precipitate with 1xPBS, add 100% glycerol to a final concentration of 50%, mix well, and aliquot into 1.5 mL EP (Eppendorf) tubes, and store at -80°C.

[0477] 5. Screening of positive colonies binding RC-Fc (positive screening)

[0478] Coat the RC-Fc with the coating buffer (pH 9.6) at a final concentration of 5 μg / mL, 100 μL / well, 8 wells for each target molecule (4 wells for the second round of screening, 2 wells for the third round of screening), 4°C overnight. Discard the coating buffer, wash with PBS for 3 times, add 300 μL of 3% BSA-PBS per well, 37°C for 1 hour. Wash with PBS for 3 times, add 100 μL of the phage library, 37°C for 1 hour. Discard the unbound phage, wash with PBST for 6 times, wash with PBS for 2 times, add 100 μL of Gly-HCl elution buffer, 37°C for 8 minutes, elute the specific binding phage; transfer the elution to a 1.5 mL sterile centrifuge tube, quickly neutralize with 10 μL of Tris-HCl neutralization buffer, take 10 μL for gradient dilution, determine the titer, and calculate the recovery rate of the panning.

[0479] Plate the titer of the elution, randomly pick 24 single clones with a sterile toothpick, inoculate in 1 mL of 2×YT-A, 37°C, 220 rpm for 8 hours. Take 200 μL of the culture, add M13K07 phage at a ratio of phage: TG1 = 20: 1, 37°C for 15 minutes, then shake at 220 rpm for 45 minutes. Add 800 μL of 2×YT-AK, shake at 30°C overnight. Centrifuge at 12000 rpm for 2 minutes the next day, take the supernatant, and use it for single clone ELISA identification.

[0480] 6. Screening of positive clones binding Fc (counter-screening)

[0481] Dilute the Fc protein (NB biolab, 20200706) with the coating buffer (pH 9.6) to a final concentration of 2 μg / mL, add 100 μL / well to the enzyme-labeled wells, 4°C overnight, discard the coating buffer, wash with PBST for 3 times, add 200 μL of 5% skim milk per well, 37°C for 1 hour, wash with PBST for 3 times, add 50 μL of phage culture supernatant and 50 μL of 5% skim milk per well, 37°C for 1 hour. Wash with PBST for 6 times, add horseradish peroxidase-labeled anti-M13 antibody (diluted with PBS at 1:10000), 100 μL / well, 37°C for 1 hour. Wash the plate with PBST for 6 times. Add TMB color developing solution for color development, 100 μL / well, 37°C for 7 minutes, add stop solution to stop the reaction, 50 μL / well, and measure the optical density at 450 nm.

[0482] 7. Elimination of positive clones binding Fc

[0483] Analyze the results of the positive screening and the counter-screening, the purpose is to retain the positive clones of the positive screening and eliminate the positive clones of the counter-screening. Finally, sequence the clones that are positive in the positive screening and negative in the counter-screening.

[0484] 8. Results Sequence analysis

[0485] Three llamas were immunized with RC-Fc. Two of the animals, NB268 and NB269, were used to create phage libraries from their peripheral blood lymphocytes mRNA. After screening and sequencing, 567 VHH sequences were obtained (311 from NB268 and 258 from NB269). Sequence comparison determined that 74 sequences (16 from NB268 and 58 from NB269) were unique. From these 74 unique VHH sequences, 12 were selected for cloning, expression and purification because they had higher bacterial culture OD450 values and did not have obvious hot amino acids (such as extra cysteine, N-linked glycosylation sites).

[0486] The amino acid sequences of the 12 VHHs are as follows (each FR and CDR region is marked by <—->, and the CDR region is additionally marked by underlining):

[0487] The nucleic acid sequences of the 12 VHHs are as follows:

[0488] Table 9 summarizes the relevant sequence information of the 12 VHHs.

[0489] Table 9 Llama anti-CGRP receptor VHH related sequence information

[0490] 9. Cloning, expression and purification of A10-268 and A10-269

[0491] A10 is the target code of CGRP receptor, 268 and 269 are the numbers of two llamas. 6 histidines (6xHis) are connected at the C-terminal of VHH sequence for purification. The synthetic gene fragment is ligated with Notl (NEB, R0189L) / Xbal (NEB, R0145L) digested pcDNA3.4 vector (ThermoFisher Scientific, EL0011). The ligation product is transformed into Top10 competent cells, and positive clones are picked and cultured for plasmid extraction and sequencing. The correct clones are inoculated in LB medium (self-made) for expansion culture, and the plasmid is extracted and sequenced again. The expression plasmid is transfected into 293 cells using transfection reagent (self-made), and the cell culture is harvested after 5 days of incubation in a constant temperature incubator (Jingqi, IS-RDS6C5). The supernatant is obtained by centrifugation, and the cell debris is removed by filtration. The target protein is captured by using a Ni2 affinity column. The final yield of the target protein is determined by filtering and measuring A280, and the absorbance value at 280 nm is read by NanoDrop One instrument. The protein is absorbed into a dialysis bag and dialyzed in a beaker containing 1XPBS. The purified product is identified by SDS-PAGE electrophoresis. The 12 expressed proteins are shown in Table 1.

[0492] Table 1: 12 A10 VHHs expressed and purified and their protein concentrations

[0493] Example 3: ELISA verification of anti-RC-Fc VHH

[0494] The immunogen RC-Fc is diluted to 2000 ng / ml with coating solution (50 mM NaHCO3 pH = 9.6) and 100 μL is added to a 96-well enzyme-labeled plate (ThermoFisher Scientific, 449824) and incubated at 4°C overnight. The enzyme-labeled plate incubated at 4°C overnight is washed 5 times with PBST, tapped dry, and 200 μL of blocking solution is added and incubated at 37°C for 2 hours. Remove PBST and add 100 μL of VHH protein diluted with PBST to each well, and incubate at 37°C for 1 hour. Wash the enzyme-labeled plate incubated with the sample 5 times with PBST, then add 100 μL of corresponding secondary antibody Mouse Anti-His-HRP (mouse anti-histidine-peroxidase, Nanjing Kingsway, A00186) diluted 1:5000 with blocking solution, and incubate at 37°C for 1 hour.

[0495] The enzyme-labeled plate incubated with the secondary antibody was washed with PBST for 5 times, 100 μL TMB single-component color developing liquid (Solarbio, 20190402) was added to each well, and incubated at 37°C for 7 minutes, 100ul 1M HCL (Kelong Chemical Industry) was added to each well to terminate the reaction, finally OD450 reading was taken with the enzyme-labeled instrument, the results were analyzed by Nonlinear regression log (agonist) vs. response (three parameters) model in GraphPad Prism 9.3.1 software, and the Binding EC 50 values are shown in Table 2.

[0496] Table 2 12 A10 VHH and their Binding EC values 50 values

[0497] Example 4: Cell verification of anti-RC-Fc VHH

[0498] 1. SK-N-MC cell line and culture

[0499] The primary Neuroblastoma cell line SK-N-MC was from ATCC (HTB-10, lot: 70036281). The culture medium was DMEM / F-12 (Gibco, C11330500BT) + 10% FBS, abbreviated as SK cell culture medium.

[0500] 2. SK-N-MC cell cAMP concentration test

[0501] The cAMP concentration produced by SK-N-MC cells was tested by Cisbio's cAMP-GS Dynamic Kit (Cisbio, 62AM4PEB) kit, and the operation method was carried out according to the kit instructions unless otherwise specified. The test medium was DMEM / F1-12 + 10% FBS + 1mM IBMX (Solarbio, I10010), and the test enzyme-labeled plate was a white 384-well plate.

[0502] 3. Determination of the number of SK-N-MC cells and obtaining of the CCGRP standard curve

[0503] On the day of the experiment, cells were separated from the culture bottle with 0.25% Trypsin-EDTA (Gibco, 25200-056), washed once with PBS, counted, and diluted to the appropriate concentration with the test medium, and tested with 10000, 7500, 5000, 2500 cells per test well.

[0504] In the wells of a ProxiPlate-384 Plus assay plate (PerkinElmer, 6008280), 5 μL of cell suspension and 5 μL of CGRP (Nanjing Yuanpeptide Biotechnology Co., Ltd., Cat. No. A03-137, Batch No. yuanpeptide-997792) diluted with stimulation buffer + 500 uM IBMX were sequentially added. The final concentration range of CGRP was 0 and 0.001 to 50 nM.

[0505] After the addition of CGRP, the assay plate was incubated in a 37°C incubator for 15 minutes, and then 5 μL of cAMP-d2 and 5 μL of Anti-cAMP-Cryptate were added to terminate the reaction. After the assay plate was placed at room temperature for 60 minutes, the reading was performed using a fluorescence microplate reader (BMG LabTech, Model: PHERAstar FSX) with a 665 / 620 filter. The obtained values were used to calculate the ratio (Ratio) according to the following formula:

[0506] The calculated ratio was analyzed using the Nonlinear regression log (agonist) vs. response (three parameters) model in the GraphPad Prism 9.3.1 software, and the results are shown in Figure 5. The above experimental results show that the number of cells per well can be from 2500 to 10000, and in this example, 2500 cells per well were selected for subsequent experiments.

[0507] 4. Evaluation of the ability of anti-CGRP receptor VHH to inhibit the production of cAMP in cells

[0508] A10-268-5, A10-268-18, A10-268-42, A10-268-76, A10-268-103, A10-269-238, A10-268-273 were tested for cell function. On the day of the experiment, SK-N-M cells were detached from the culture flask using 0.25% Trypsin-EDTA, washed once with PBS, counted, and diluted to a cell suspension of 5 million cells / mL in test medium, and used within 30 minutes.

[0509] In the wells of the test plate, 5 μl of cell suspension and 5 μl of anti-RC-Fc single-domain antibody diluted 5-fold in series (final concentration 0 and 0.0001 to 100 nM) were sequentially added, and incubated in a 37°C incubator for 30 minutes.

[0510] Add 5 μL of CGRP diluted in stimulation buffer (final concentration is 5 nM), incubate the plate for 15 minutes at 37°C, then add 5 μL of d2 and 5 μL of Eu to stop the reaction. After adding CGRP, incubate the plate for 15 minutes at 37°C, then add 5 μL of cAMP-d2 and 5 μL of Anti-cAMP-Cryptate to stop the reaction. After incubating the plate for 60 minutes at room temperature, read the plate using the 665 / 620 filter of the fluorescence plate reader, and calculate the 665 / 620 ratio. The results are shown in Table 3. The IC 50 values in the table show that four VHHs have the ability to inhibit the production of cAMP by SK-N-MC cells, and the activities are in the order: A10-268-273 > A10-268-42 > A10-268-103 > A10-268-18.

[0511] Table 3. Anti-CGRP receptor VHHs and their corresponding IC 50 values

[0512] Example 5: Determining the binding sites of VHHs on RC-Fc

[0513] As can be seen from Table 3, in the CHO cell test system expressing human CGRP receptor, A10-268-18, A10-268-42, A10-268-103, and A10-268-273 have the ability to inhibit CGRP-induced cAMP production, and the others do not. Obviously, these VHHs bind to different epitopes on the CGRP receptor. In order to determine which VHHs bind to the same epitope on the CGRP receptor and which bind to different epitopes, a test called Binning ELISA (double VHH binding ELISA) was used to make the distinction.

[0514] In this experiment, A and B VHHs are added to the wells of the plate coated with antigen. If the two VHHs bind to the same epitope on the antigen, the signal of the Binning ELISA will not be much different from that obtained by adding either of the two VHHs alone, i.e., the signal intensity is equal to A or B (Figures 6A, 6B). But if the two VHHs bind to different epitopes, the signal of the Binning ELISA will increase significantly, i.e., the signal intensity is equal to A+B (see Figure 6C).

[0515] Antigen RC-Fc was diluted to 1081 ng / ml with coating buffer (50 mM NaHCO3 pH=9.6) and 100 μL was added to a 96-well enzyme-linked immunosorbent assay (ELISA) plate (ThermoFisher Scientific, 449824) and incubated at 4 °C overnight. The 4 °C overnight ELISA plate was washed 5 times with PBST, tapped dry, 200 μL blocking buffer was added and incubated at 37 °C for 2 hours. After removing the PBST, the ELISA plate was washed once with PBST and 50 μL of each protein solution was added to each well, each protein solution containing 50 μL of the VHH protein to be tested diluted to 3 nM with PBST, and incubated at 37 °C for 1 hour. The ELISA plate incubated with the samples was washed 5 times with PBST, and 100 μL of the corresponding secondary antibody Mouse Anti-His-HRP (Nanjing Kingsway, A00186) diluted 1:5000 with blocking buffer was added to each well and incubated at 37 °C for 1 hour.

[0516] The ELISA plate incubated with the secondary antibody was washed 5 times with PBST, 100 μL of TMB one-component color developing solution (Solarbio, 20190402) was added to each well, and incubated at 37 °C for 7 minutes. The reaction was stopped by adding 100 μL of 1 M HCL (Kelong Chemical) to each well, and finally the OD450 reading was taken with an enzyme-linked immunosorbent assay (ELISA) reader. The values obtained are shown in Table 4.

[0517] Table 4: Double VHH binding ELISA experiment results for 8 VHHs

[0518] The table shows the double VHH binding ELISA experiment results for 8 VHHs. Of the 8 VHHs, 1-7 are from Table 4, and the 8th VHH is a negative control that does not bind to the CGRP receptor ECD. The values in each cell of the table are the sum of the signals of the two VHHs, and the values in the rightmost column and the bottom row are the sum of the signals of each VHH and the negative control VHH together. Since the negative control VHH does not produce a signal, all of these signal values represent the signal of each VHH when it is present alone.

[0519] If the double VHH value in the table is comparable to or smaller than the signal value of either of the two VHHs when it is present alone, it is defined as the two VHHs binding to the same epitope. However, if the double VHH value in the table is greater than the signal value of either of the two VHHs when it is present alone, it is defined as the two VHHs binding to different epitopes.

[0520] From the table, we can see that the signal produced by two No. 1 VHHs together is 1.30, which is smaller than the signal 1.36 produced by No. 1 VHH and No. 8 (negative) VHH together, because two No. 1 VHHs bind to the same epitope, and the signal will not increase. The signal produced by No. 1 VHH and No. 2 VHH together is 2.47, which is smaller than 2.63 when No. 2 VHH is alone, so No. 1 and No. 2 VHHs bind to the same epitope. But the signal produced by No. 1 and No. 4 VHHs together is 2.28, which is larger than 1.36 when No. 1 VHH is alone, and also larger than 1.93 when No. 4 VHH is alone, so No. 1 VHH and No. 4 VHH bind to different epitopes. By interpreting the values in the table in this way, we can conclude that No. 1, No. 2, No. 3, No. 5, No. 6, and No. 7 VHHs bind to the same epitope, and No. 4 VHH binds to another epitope.

[0521] Example 6: Humanization of VHH and verification of cell cAMP inhibition ability

[0522] 1. Humanization of VHH

[0523] The 4 framework regions and 3 CDR regions of VHH are divided by IMGT sorting method. AlphaFold2 is used to predict the 3D structure, and the basis of this structure is obtained by using the self-developed evaluation method. Then through the IGBLAST application (https: / / www.ncbi.nlm.nih.gov / igblast / ), the sequence of VHH is compared with 100 human IGHV3 sequences to find out the non-human amino acids in the 4 Frameworks of VHH. These amino acids are divided into 10-13 groups (hv1 to hv13), and each group has 1-10 amino acids that are changed to human amino acids. The 13 VHHs with different combinations of humanized amino acids are subjected to AlphaFold2 prediction structure, and the 3D structures of these VHHs are compared with the 3D structure of the original VHH before modification. The three combinations with the highest degree of similarity are selected, and the original VHH itself is fused with Fc for expression. Finally, the functions of the four proteins (42wtFc, 42hv10Fc, 42hv11Fc, 42hv12Fc) are compared by cell cAMP experiment. A10-269-238 is humanized in the same way.

[0524] Among them, the VHH amino acid sequences of 42hv10, 42hv11, and 42hv12 are as follows (each CDR region is marked with an underline):

[0525] Among them, the VHH-238 amino acid sequence after humanization is as follows (each CDR region is marked with an underline):

[0526] 2. Cloning, expression, and purification after humanization

[0527] The C-terminus of the humanized VHH (VHHhv) was directly connected to the N-terminus of Fc to form a fusion protein VHHhvFc, which was synthesized, expressed and purified as described in Example 1. The protein electropherogram of 42hv is shown in Figure 7.

[0528] 3. Verification of the cell cAMP inhibition ability of humanized VHHhvFc

[0529] On the day of the experiment, Neuroblastoma cells were separated from the culture bottle with 0.25% Trypsin-EDTA, washed once with PBS, counted, and diluted to 5 million cells / mL with test medium. The cells were used within 30 minutes.

[0530] 5 μL of 42wtFc, 42hv10Fc, 42hv11Fc, 42hv12Fc protein and control antibody Erenumab (Biyuntong, A03EB) diluted 5-fold serially with stimulation solution were added to the wells of the ProxiPlate-384 Plus test plate, with a concentration range of 0.0003 to 100 nM, and incubated at 37°C for 30 minutes.

[0531] 5 μL of CGRP diluted with stimulation solution (final concentration 5 nM) was added, and the test plate was incubated in a 37°C incubator for 15 minutes, followed by the addition of 5 μL of d2 and 5 ul of Eu to terminate the reaction. After the addition of CGRP, the test plate was incubated in a 37°C incubator for 15 minutes, followed by the addition of 5 μL of cAMP-d2 and 5 μL of Anti-cAMP-Cryptate to terminate the reaction. After the test plate was incubated at room temperature for 60 minutes, the 665 / 620 filter of the fluorescence microplate reader was used to read the values, and the 665 / 620 ratio was calculated. The results were analyzed using the Nonlinear regression log(inhibitor) vs. response(three parameters) model in GraphPad Prism 9.3.1 software, and are shown in Figure 8.

[0532] As can be seen from Figure 8, the humanization of the three groups of 42hv10, 42hv11 and 42hv12 was successful, and their ability to inhibit the production of cell cAMP was better than that of the control antibody Erenumab. Based on the above results, the IC 50 The 42hv10, which was twice as good as the original prototype, was further developed.

[0533] Other VHHs were humanized using the same humanization method.

[0534] Example 7: Preparation of anti-HSA single-domain antibody

[0535] 1. Immunization of American camel with human albumin

[0536] Take 10 mL of blood before immunization of American camel, and keep it as negative serum control. Then mix 0.5 mg of human albumin (Chengdu Rongsheng Pharmaceutical Co., Ltd., S10940024) with 1 mL of Complete Freund's Adjuvant (CFA, Sigma, F5881) and inject subcutaneously. On day 21, mix 0.25 mg of antigen with 1 mL of Incomplete Freund's Adjuvant (IFA, Sigma, F5506) and inject subcutaneously. On day 28, take 10 mL of blood and separate the serum. On day 42, mix 0.25 mg of antigen with 1 mL of IFA and inject subcutaneously. On day 49, take 50 mL of peripheral blood to separate lymphocytes and serum. On day 63, mix 0.25 mg of antigen with 1 mL of IFA and inject subcutaneously. On day 70, take 50 mL of peripheral blood to separate lymphocytes and serum.

[0537] 2. Gradient detection

[0538] Coat the enzyme-labeled plate with albumin diluted to 2000 ng / ml with coating solution (50 mM NaHCO3, PH = 9.6), add 100 μL to each well of the 96-well enzyme-labeled plate, and incubate at 4°C overnight. Wash the enzyme-labeled plate coated at 4°C overnight with PBST 5 times, pat dry, and add 300 μL of blocking solution to each well.

[0539] Dilute the serum with TBST in a 1:2 series, with the first gradient serum being 1:2000 and the last gradient serum being 1:128000. After washing the antigen plate with 300 μL / well of PBST three times, add 100 ul of diluted serum to each well, incubate at 37°C for 1 hour, wash with 300 μL / well of PBST three times, add 100 μL of 1:2000 anti-M13 antibody (NB Biolab, 052-101-005) to each well, incubate at 37°C for 1 hour, wash with 300 μL / well of PBST six times, add 1:10000 diluted anti-Alpaca-HRP (NEB, S001H), and incubate at 37°C for 30 minutes. After incubating the enzyme-labeled plate with the secondary antibody, wash it with PBST 5 times, add 100 μL of TMB single-component color developing solution to each well, incubate at 37°C for 7 minutes, add 100 μL of 1M HCL to each well to stop the reaction, and read OD450.

[0540] 3. Construction of phage library

[0541] Select high-gradient peripheral blood, and separate PBMC according to the instructions for lymphocyte separation medium (Tianjin Haoyang Biological, LTS1077). Extract total RNA with RNAiso Plus (Fuji Biological, RE-03111) reagent, and use PrimeScriptTM II 1st Strand cDNA Synthesis Kit (Takara, 9109) to transcribe 5 pg RNA into cDNA. Dilute the cDNA stock 5 times for nest PCR amplification, cut the gel to recover the single domain antibody (VHH) fragments around 750 base pair (BP) for the second round of PCR amplification, and the purified PCR is the VHH fragments. The VHH fragments are ligated with the vector pComb3XSS (NEB, P001) through the Sfil enzyme cutting point. Immediately after electroporation, add 1 mL 2YT (a culture medium containing tryptone (Solarbio, T8490), yeast extract (Solarbio, Y8020) and sodium chloride (Kelong Chemical Industry), preheated at 37°C) to the electroporation cup, aspirate the electroporation product and wash the electroporation cup with 2YT medium, a total of 100 ml of recovery product, 37°C, 180 rpm (rev / min), recovery for 45 minutes, take 100 pL gradient dilution to 10-3 and 10-4 to determine the number of library transformants, and coat on 90 mm plates. The rest is centrifuged, resuspended with 8 mL 2YT, and coated on 8 pieces of 200 mm plates. The next day, the plates for determining the number of library transformants are obtained with VHH bacterial clones.

[0542] 4. Phage library packaging

[0543] The bacterial library is inoculated into 2 x 300 mL 2YT+A (ampicillin (Solarbio, A1170) 100 ug / ml) + G (1% glucose (Solarbio, G8150) medium to an initial OD600 = 0.1-0.2, 37°C, 230 rpm, and cultured to OD600 = 0.8 or more. According to the OD600 value, helper phage M13KO7 (NB biolab, P006) and TG1 bacteria (NB biolab, P008) are added (helper phage: bacteria = 20:1). After adding M13KO7, mix well, stand at 37°C for 30 minutes, shake at 180 rpm for 30 minutes, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, resuspend the precipitate with an equal volume of 2YT+A+K (kanamycin (Solarbio, K1030) 50 pg / ml) medium, and incubate at 30°C, 220 rpm overnight.

[0544] The overnight culture is centrifuged at 10000 rpm for 20 min at 4°C, the supernatant is collected, and the precipitate is discarded. Replace the centrifuge tube and centrifuge at 10000 rpm for 20 min at 4°C, collect the supernatant.

[0545] Add PEG8000 (Solarbio, P8260) / NaCl at 1 / 5 of the volume of supernatant, mix well, and precipitate in ice bath for more than 2 hours. Centrifuge at 10,000 rpm for 20 minutes, discard the supernatant, and empty the supernatant once. Resuspend the precipitate in 1 mL of 1x PBS, and precipitate again with 1 / 5 volume of PEG8000 / NaCl for 1 hour.

[0546] Centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, and empty the supernatant once. According to the amount of precipitate, resuspend the precipitate in 1x PBS, add 100% glycerol to a final concentration of 50%, mix well, and aliquot into 1.5 mL EP (Eppendorf) tubes, and store at -80°C.

[0547] 5. Screening of positive colonies that bind albumin

[0548] Dilute albumin to a final concentration of 5 μg / mL with coating solution at pH 9.6, add 100 μL / well to the enzyme-labeled wells, coat 8 wells for each target molecule (coat 4 wells in the second round of screening, and coat 2 wells in the third round of screening), and coat overnight at 4°C. Discard the coating solution, wash with PBS 3 times, add 300 μL of 3% BSA-PBS blocking solution per well, and block at 37°C for 1 hour. Wash with PBS 3 times, add 100 μL of phage library, and incubate at 37°C for 1 hour. Remove the unbound phage, wash with PBST 6 times, wash with PBS 2 times, add 100 μL of Gly-HCl elution solution, incubate at 37°C for 8 minutes, and elute the specifically bound phage; transfer the eluate to a 1.5 mL sterile centrifuge tube, quickly neutralize with 10 μL of Tris-HCl neutralization buffer, take 10 μL for gradient dilution, determine the titer, and calculate the recovery rate of the selection.

[0549] Drop the eluate on a plate, randomly pick 24 single clones with a sterile toothpick, inoculate in 1 mL of 2xYT-A, and incubate at 37°C with shaking at 220 rpm for 8 hours. Take 200 μL of the culture, add M13K07 phage at a ratio of phage: TG1 = 20:1, stand at 37°C for 15 minutes, and then incubate with shaking at 220 rpm for 45 minutes. Add 800 μL of 2xYT-AK, and incubate at 30°C with vigorous shaking overnight. Centrifuge at 12,000 rpm for 2 minutes the next day, take the supernatant, and use it for single clone ELISA identification.

[0550] 6. Sequence analysis of results

[0551] Three llamas were immunized with human albumin. The mRNA from the peripheral blood lymphocytes of the three animals were used to construct phage libraries. After screening and sequencing, 598 VHH sequences were obtained. Sequence comparison identified 50 unique sequences. From the 50 unique VHH sequences, 12 sequences were selected for cloning, expression and purification because they had high OD450 values and no obvious hot amino acids (such as extra cysteine, N-linked glycosylation sites).

[0552] The amino acid sequences of the 12 VHHs are as follows (each FR and CDR region is marked by <—->, and the CDR region is additionally marked by underlining):

[0553] (1) NB268-Anti-A11M-17 (A11-268-M17) (SEQ ID NO: 69)

[0554] (2) NB268-Anti-A11M-104 (A11-268-M104) (SEQ ID NO: 74)

[0555] (3) NB268-Anti-A11M-117 (A11-268-M117) (SEQ ID NO: 79)

[0556] (4) NB268-Anti-A11M-141 (A11-268-M141) (SEQ ID NO: 84)

[0557] (5) NB269-A11-H-16 (A11-269-H16) (SEQ ID NO: 89)

[0558] (6) NB269-A11-H-35 (A11-269-H35) (SEQ ID NO: 94)

[0559] (7) NB269-A11-H-88 (A11-269-H88) (SEQ ID NO: 99)

[0560] (8) NB269-A11-H-96 (A11-269-H96) (SEQ ID NO: 104)

[0561] (9) NB269-A11-M-144 (A11-269-H144) (SEQ ID NO: 109)

[0562] (10) NB269-A11-M-105 (A11-269-M105) (SEQ ID NO: 114)

[0563] (11) NB269-A11-M-131 (A11-269-M131) (SEQ ID NO: 119)

[0564] (12) NB269-A11-M-172 (A11-269-M172) (SEQ ID NO: 124)

[0565] The nucleic acid sequences of these 12 VHHs are as follows:

[0566] (1) NB268-Anti-A11M-17 (A11-268-M17) (SEQ ID NO: 73)

[0567] (2) NB268-Anti-A11M-104 (A11-268-M104) (SEQ ID NO: 78)

[0568] (3) NB268-Anti-A11M-117 (A11-268-M117) (SEQ ID NO: 83)

[0569] (4) NB268-Anti-A11M-141 (A11-268-M141) (SEQ ID NO: 88)

[0570] (5) NB269-A11-H-16 (A11-269-H16) (SEQ ID NO: 93)

[0571] (6) NB269-A11-H-35 (A11-269-H35) (SEQ ID NO: 98)

[0572] (7) NB269-A11-H-88 (A11-269-H88) (SEQ ID NO: 103)

[0573] (8) NB269-A11-H-96 (A11-269-H96) (SEQ ID NO: 108)

[0574] (9) NB269-A11-M-144 (A11-269-H144) (SEQ ID NO: 113)

[0575] (10) NB269-A11-M-105 (A11-269-M105) (SEQ ID NO: 118)

[0576] (11) NB269-A11-M-131 (A11-269-M131) (SEQ ID NO: 123)

[0577] (12) NB269-A11-M-172 (A11-269-M172) (SEQ ID NO: 128)

[0578] Table 10 summarizes the relevant sequence information for these 12 VHHs.

[0579] Table 10 llama anti-HSA VHH relevant sequence information

[0580] 7. Cloning, expression and purification of A11 VHH

[0581] A11 is the albumin target code. C-terminal of VHH sequence and 6 histidines (6xHis) fusion were used for purification. Synthetic gene fragment was ligated with Notl (NEB, R0189L) / Xbal (NEB, R0145L) digested pcDNA3.4 vector (self-made) (ThermoFisher Scientific, EL0011). The ligation product was transformed into Top10 competent cells, and positive clones were picked for expansion culture, and the plasmid was extracted with a small amount of plasmid extraction kit (self-made) for sequencing. The correct clone was inoculated in LB medium (self-made) for expansion culture, and the plasmid was extracted with a large amount of plasmid extraction kit (self-made) and re-sequenced for verification. The expression plasmid was transfected into 293 cells using transfection reagent (self-made), and the cell culture was harvested after 5 days of incubation in a constant temperature incubator (Jingqi, IS-RDS6C5). The supernatant was obtained after centrifugation of the cell culture, and the cell debris was removed by filtration, and the clear liquid was collected. The Ni2 affinity chromatography column was used to capture the target protein. The filtered protein was determined by A280 method to determine the final yield, and the 280 nm absorbance value was read by NanoDrop One instrument. The protein was absorbed into a dialysis bag and dialyzed in a 1XPBS beaker, and the purified product was identified by SDS-PAGE electrophoresis. Eight VHHs (A11-269-H16, A11-269-H35, A11-269-H88, A11-269-H96, A11-269-H144, A11-269-M105, A11-269-M131, A11-269-M172) obtained expression and purification products.

[0582] Example 8: ELISA verification of anti-albumin VHH

[0583] Human albumin, monkey albumin (Wuhan Mabco Biotech Co., Ltd., Cat. No. KT86371), mouse albumin (Wuhan Mabco Biotech Co., Ltd., Cat. No. KT210380) were diluted to 2000 ng / ml with coating solution (50 mM NaHCO3 pH = 9.6), and 100 μL was coated on a 96-well enzyme-labeled plate (ThermoFisher Scientific, 449824) at 4°C overnight. The enzyme-labeled plate incubated at 4°C overnight was washed 5 times with PBST, and dried, 200 μL blocking solution was added, and incubated at 37°C for 2 hours. Remove PBST, add 100 μL VHH protein diluted with PBST to each well, and incubate at 37°C for 1 hour. The enzyme-labeled plate incubated with the sample was washed 5 times with PBST, and then 100 μL of corresponding secondary antibody Mouse Anti-His-HRP (mouse anti-histidine-peroxidase, Nanjing KingsRiver, A00186) diluted 1:5000 with blocking solution was added, and incubated at 37°C for 1 hour.

[0584] The enzyme-labeled plate incubated with the second antibody was washed with PBST for 5 times, 100 μL TMB single-component color developing liquid (Solarbio, 20190402) was added to each well, and incubated at 37°C for 7 minutes, 100ul 1M HCL (Kelong Chemical Industry) was added to each well to terminate the reaction, finally OD450 reading was taken with microplate reader, the results were analyzed by Nonlinear regression log(agonist) vs.response(three parameters) model in GraphPad Prism 9.3.1 software, and the binding EC50 value was obtained as shown in Table 5.

[0585] Table 5 Binding EC of 8 anti-albumin VHH 50 value

[0586] Example 9: Humanization of anti-albumin VHH and ELISA verification

[0587] Humanization and verification were carried out according to the method of Example 6, and two VHHs capable of binding human, monkey and mouse albumin after humanization were obtained: A11-269-H16 and A11-269-H88. The names of the humanized VHHs were changed to H16hv3 and H88hv7, respectively. The verification data of the two humanized VHHs are shown in Table 6.

[0588] Table 6 Binding ELISA EC of two humanized VHHs 50 value

[0589] Example 10: Construction and verification of trivalent antibody molecules

[0590] 1. Construction of trivalent antibody molecule BY002

[0591] BY002 is connected by two linkers to form a tandem trivalent nanobody triad. Its structure is shown in Figure 9.

[0592] The gene synthesis, expression and purification method is referred to Example 2, and the C-terminal of the VHH sequence and 6 histidines (6xHis) are fused for purification. The results of SDS-PAGE of purified BY002 are shown in Figure 10.

[0593] The amino acid sequence of BY002 is shown in SEQ ID NO: 131:

[0594] 2. Activity verification of BY002

[0595] (1) High activity of BY002 in binding RC-Fc at low density of antigen

[0596] Antigens RC-Fc and 42hv10Fc were diluted to 30nM, 10nM, 1nM with coating buffer (50mM NaHC03 pH=9.6) and used to coat 96-well ELISA plates. ELISA was performed as described in Example 3. The binding EC 50 values are shown in Figure 11.

[0597] The control protein in the above experiment, 42hv10Fc, is a homodimer of VHH (Figure 12). The results show that at high concentration of antigen, BY002 and 42hv10Fc have the same binding capacity (Bmax and EC 50 50). At 10nM antigen, the Bmax of both are the same but the EC50 of BY002 is better than 42hv10Fc. At 1nM antigen, both the Bmax and EC 50 50 of BY002 are better than 42hv10Fc. This shows that the heterodimer design of BY002 is advantageous for maintaining better biological activity at low density of target.

[0598] (2) BY002 binds to human Amylin 1 receptor ECD but not to rat CGRP receptor and human Calcitonin receptor ECD

[0599] Antigens human CGRP receptor ECD (RC-Fc), human Amylin 1 receptor ECD (Beyotime, B22352901-CHO), human Calcitonin receptor ECD (Beyotime, B22352902-CHO) and rat CGRP receptor ECD (Beyotime, B21711601) were diluted to 30nM with coating buffer (50mM NaHC03 pH=9.6) and used to coat 96-well ELISA plates. ELISA was performed as described in Example 3. The results are shown in Figure 13. BY002 binds to human CGRP receptor and human Amylin 1 receptor ECD but not to rat and human Calcitonin receptor ECD.

[0600] (3) CGRP induces cAMP production in CHO-CGRPr cell line

[0601] CHO cell line expressing human CGRP receptor (CHOK1-CGRPr) was from Beyotime, Cat# CHOK1-C21738001. The culture medium is DMEM (Gibco, 11995065) + 10% FBS, abbreviated as CHOK1-CGRPr cell culture medium.

[0602] cAMP concentration was tested with Cisbio’s cAMP-GS Dynamic Kit (Cisbio, 62AM4PEB) kit, and the operation method was performed according to the kit instructions, except for special instructions. The test medium was DMEM + 10% FBS + 1 mM IBMX (Solarbio, I10010), and the test enzyme-labeled plate was white ProxiPlate-384 Plus test plate (PerkinElmer, 6008280).

[0603] On the day of the experiment, the cells were detached from the culture flask with 0.25% Trypsin-EDTA (Gibco, 25200-056), washed once with PBS, counted, and diluted to the appropriate concentration with the test medium, and 2500 cells per test well were tested.

[0604] 5 μL of cell solution and 5 μL of CGRP (Nanjing Yuanpeptide Biotechnology Co., Ltd., Item No.: A03-137, Batch No.: yuanpeptide-997792) diluted with stimulation solution (Stimulation Buffer + 500 uM IBMX) were added to the wells of the test plate in turn. The final concentration of CGRP ranged from 0 and 0.001 to 50 nM.

[0605] After adding CGRP, the test plate was incubated in a 37°C incubator for 15 minutes, and then 5 μL of cAMP-d2 and 5 μL of Anti-cAMP-Cryptate were added to terminate the reaction. After the test plate was placed at room temperature for 60 minutes, the reading was taken with the 665 / 620 filter of the fluorescence enzyme-labeled instrument (BMG LabTech, Model: PHERAstar FSX). The obtained value was calculated as the ratio (Ratio) using Microsoft Excel according to the following formula:

[0606] The calculated ratio was analyzed using the Nonlinear regression log (agonist) vs. response (three parameters) model in GraphPad Prism 9.3.1 software, and the results are shown in Figure 14.

[0607] The above experimental results show that CGRP can induce CHOK1-CGRPr to produce cAMP, and the EC 50The value is 0.104 nM. CGRP has no such function on CHOK1 cells. In addition, a polypeptide PACAP (Pituitary Adenylate Cyclase-activating Peptide, Nanjing Yuanpeptide, Catalog No. Yuanpeptide-194972) unrelated to CGRP cannot induce CHOK1-CGRPr to produce cAMP. This experiment verifies that CHOK1-CGRPr specifically expresses CGRP receptors and can be used to verify the function of BY002.

[0608] (4) BY002 inhibits CGRP-induced cAMP production in CHOK1-CGRPr cells

[0609] BY002 and the positive control antibody Erenumab (Bing Biosciences, B747201), and the negative control antibody Ctrl IgG were tested for cell function. On the day of the experiment, CHOK1-CGRPr cells were detached from the culture flask with 0.25% Trypsin-EDTA, washed once with PBS, counted, and diluted to 5 million cells / mL in test medium, and used within 30 minutes.

[0610] 5 μl of cells and 5 ul of BY002 (final concentrations of 0 and 0.0001 to 100 nM) diluted 5-fold in stimulation solution were added to the wells of the test plate in turn, and incubated at 37°C for 30 minutes.

[0611] 5 μL of CGRP diluted in stimulation solution (final concentration 5 nM) was added, and the test plate was incubated at 37°C for 15 minutes, then 5 μL d2 and 5 μL Eu were added to terminate the reaction. After the addition of CGRP (final concentration 1.4 nM), the test plate was incubated at 37°C for 15 minutes, then 5 μL cAMP-d2 and 5 μL Anti-cAMP-Cryptate were added to terminate the reaction. After the test plate was incubated at room temperature for 60 minutes, the 665 / 620 filter of the fluorescence microplate reader was used to read the values, and the 665 / 620 ratio was calculated. The results are shown in Table 7. Table 7 is the average of 4 independent experiments, and it can be seen that the IC50 of BY002 to inhibit the production of cAMP by CHOK1-CGRPr cells is 0.71 nM, which is 4 times that of the positive control antibody Erenumab; the negative control antibody Ctrl IgG has no inhibitory activity at 100 nM. 50

[0612] ​Table 7 IC of BY002 to inhibit cAMP production in CHOK1-CGRPr cells 50 Values

[0613] n = 4 experiments

[0614] (5) BY002 has no significant activity on CHOK1-Amy1r cells

[0615] To determine whether BY002 affects the activity of Amylin 1 receptor, CHOK1 (Biyang Bio, CHOK1) cells were transfected with human Amylin 1 receptor, and a stable cell pool expressing human Amylin 1 receptor (CHOK1-Amy1r) was obtained by neomycin selection. This cell pool can produce a concentration-dependent increase in cAMP in response to stimulation by human Amylin 1 (Nanjing Yuan Peptide, yuanpeptide-600021). In the presence of high concentrations of BY002, CHOK1-Amy1r cells still retain this activity, indicating that BY002 has no activity on Amylin 1 receptor, as shown in Figures 15A, B.

[0616] (6) BY002 enters the blood circulation quickly and has a long half-life

[0617] BY002 is composed of three VHHs, not only has a small molecular weight and strong tissue penetration ability, but also has a long half-life in the blood circulation. To verify this effect, first, VHH 42hv10 and VHH H16 were connected by a 2xGGGGS linker to express a diabody (42hv10@H16), and VHH 42hv10 and a VHH (A10-269-56) that does not bind to HSA were connected in the same way to express a diabody (42hv10@56).

[0618] The mouse experiment plan was reviewed and approved by the animal ethics committee before implementation.

[0619] 42hv10@H16 (HSA-binding VHH diabody), 42hv10@1 (non-HSA-binding VHH diabody), and Ctrl IgG (control monoclonal antibody) were subcutaneously injected into two C57BL / 6J mice at a dose of 6 mg / kg, respectively, and then blood was drawn at 30, 60, 180, 300, and 1440 minutes to obtain plasma, and the protein concentration was detected by ELISA method, as shown in Figures 16A-16B.

[0620] Figure 16A shows that 42hv10@H16 with anti-HSA VHH enters blood circulation faster than control IgG, reaching 100 nM concentration at 30 minutes after subcutaneous injection, and its concentration is increasing within 24 hours. Monoclonal antibody reaches around 50 nM at 5 hours. Diabody 42hv10@56 without anti-HSA VH enters blood circulation even faster, reaching 700 nM at 30 minutes after subcutaneous injection, but then drops rapidly, almost to 0 nM at 24 hours.

[0621] The above experiments demonstrate that nanobody with anti-HSA VHH enters blood circulation fast after subcutaneous injection in mice, and has long half-life. This is an important advantage. BY002 is a trivalent nanobody with one more VHH. To demonstrate that BY002 also has the characteristics of fast entering blood circulation and long half-life after subcutaneous injection, two SD rats were injected subcutaneously with 6 mg / kg of BY002, and blood was drawn at 5, 30, 60, 120 and 240 minutes after injection to obtain plasma, and the concentration of BY002 in plasma was determined by ELISA. The results are shown in Figure 16B.

[0622] Figure 16B is the result of rat PK experiment, showing that BY002 maintains the characteristics of fast entering blood circulation and long half-life. The concentration of BY002 in plasma reaches 32 nM at 5 minutes after subcutaneous injection, which is 45 times the cellular IC50 (0.71 nM), and within 24 hours, the plasma concentration is increasing. The rat PK data support the use of BY002 for acute treatment of episodic migraine.

[0623] Figure 17 is the 30-day PK performance of BY002 in rats, with a Beta half-life of 3.8 days (estimated to be 15-20 days in humans), and detectable concentration of BY002 at 30 days. This PK result shows that BY002 has the potential to be used as a prophylactic drug once every two weeks or once a month.

[0624] In summary, the present application enhances the blocking activity of BY002 on the CGRP signaling pathway by using a way of binding two different epitopes on the CGRP receptor. The ability of BY002 to inhibit CGRP-induced cAMP production at the cellular level is 5 times superior to Erenumab, a marketed mAb with similar mechanism and use. By linking the nanobody into a trivalent nanobody with anti-HSA VHH, the half-life of BY002 of 40 kD is greatly extended. Therefore, BY002 has the potential of superior drug efficacy, fast onset and long half-life in the treatment and prevention of migraine. These advantages are concentrated in the same molecule, which is currently not achieved by small molecule and mAb CGRP antagonists on the market. BY002 can be used to treat moderate to severe, headache for several days long migraine (such as menstrual migraine) at one time, and can also be used to prevent episodic or chronic migraine according to the treatment regimen. BY002 fills the gap of the current market small molecule drugs with unsatisfactory drug efficacy and short half-life, and also solves the problem of slow onset of mAb, providing more precise drug treatment options for patients with migraine.

Claims

1. An antibody molecule or antigen-binding fragment thereof that specifically binds to a Calcitonin Gene-Related Peptide (CGRP) receptor, comprising a first antigen binding domain, the first antigen binding domain comprising a heavy chain variable region that binds to a CGRP receptor, the heavy chain variable region comprising a heavy chain complementarity determining region (HCDR)1 as set forth in SEQ ID NO: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or any variant thereof, a heavy chain CDR2 (HCDR2) as set forth in SEQ ID NO: 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, or any variant thereof, and a heavy chain CDR3 (HCDR3) as set forth in SEQ ID NO: 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, or any variant thereof.

2. The antibody molecule or antigen binding fragment thereof of claim 1, wherein, the heavy chain variable region of the first antigen binding domain comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of: (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; (2) SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17; (3) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; (4) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; (5) SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32; (6) SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37; (7) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42; (8) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; (9) SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52; (10) SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57; (11) SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62; and (12) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO:

67.

3. The antibody molecule or antigen-binding fragment thereof of claim 2, wherein, the heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, and 132-135.

4. The antibody molecule or antigen binding fragment thereof of any of claims 1-3, wherein, The heavy chain variable region of the first antigen binding domain comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of: (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; (2) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; (3) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; (4) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42; (5) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; and (6) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO:

67.

5. The antibody molecule or antigen-binding fragment thereof of claim 4, wherein, The heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 9, 19, 24, 39, 44, 64, and 132-134.

6. The antibody molecule or antigen-binding fragment thereof of claims 1-5, wherein, The heavy chain variable region of the first antigen binding domain comprises a HCDR1 as set forth in SEQ ID NO: 20, a HCDR2 as set forth in SEQ ID NO: 21, and a HCDR3 as set forth in SEQ ID NO:

22.

7. The antibody molecule or antigen-binding fragment thereof of claim 6, wherein, The heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 19 and 132-134.

8. The antibody molecule or antigen-binding fragment thereof of claim 6, wherein, The heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

132.

9. The antibody molecule or antigen-binding fragment thereof of any of claims 1-8, wherein, The first antigen binding domain is a single domain antibody.

10. The antibody molecule or antigen-binding fragment thereof of any of claims 1-9, wherein, The first antigen binding domain is humanized.

11. The antibody molecule or antigen binding fragment thereof according to any one of claims 1-10, further comprising a second antigen binding domain that binds to a CGRP receptor.

12. The antibody molecule or antigen-binding fragment thereof of claim 11, wherein, The antigenic epitope of the first antigen binding domain is different from the antigenic epitope of the second antigen binding domain; preferably, the two antigenic epitopes do not overlap. The heavy chain variable region of the first antigen binding domain comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of: (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; (2) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; (3) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; (4) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42; (5) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; and (6) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO:

67. The heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 9, 19, 24, 39, 44, 64, and 132-134. The heavy chain variable region of the first antigen binding domain comprises a HCDR1 as set forth in SEQ ID NO: 20, a HCDR2 as set forth in SEQ ID NO: 21, and a HCDR3 as set forth in SEQ ID NO:

22. The heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 19 and 132-134. The heavy chain variable region of the first antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

132. The first antigen binding domain is a single domain antibody. The first antigen binding domain is humanized.

11. The antibody molecule or antigen binding fragment thereof according to any one of claims 1-10, further comprising a second antigen binding domain that binds to a CGRP receptor. The antigenic epitope of the first antigen binding domain is different from the antigenic epitope of the second antigen binding domain; preferably, the two antigenic epitopes do not overlap.

13. The antibody molecule or antigen-binding fragment thereof of any one of claims 11-12, wherein, The second antigen binding domain comprises a heavy chain variable region that binds a CGRP receptor, the heavy chain variable region of the second antigen binding domain comprises a HCDR1 as set forth in SEQ ID NO: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or any variant thereof, a HCDR2 as set forth in SEQ ID NO: 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, or any variant thereof, and a HCDR3 as set forth in SEQ ID NO: 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, 67, or any variant thereof.

14. The antibody molecule or antigen-binding fragment thereof of claim 13, wherein, The heavy chain variable region of the second antigen binding domain comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of: (1) SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; (2) SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17; (3) SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22; (4) SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27; (5) SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32; (6) SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37; (7) SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42; (8) SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47; (9) SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52; (10) SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57; (11) SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62; and (12) SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO:

67.

15. The antibody molecule or antigen-binding fragment thereof of claim 14, wherein, The heavy chain variable region of the second antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, and 132-135.

16. The antibody molecule or antigen binding fragment thereof of any one of claims 11-15, wherein, The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the first antigen binding domain are different from the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the second antigen binding domain.

17. The antibody molecule or antigen binding fragment thereof of any one of claims 11-16, wherein, The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the first antigen binding domain are different from the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the second antigen binding domain.

18. The antibody molecule or antigen binding fragment thereof of any one of claims 13-17, wherein, The heavy chain variable region of the second antigen binding domain comprises a HCDR1 as set forth in SEQ ID NO: 60, a HCDR2 as set forth in SEQ ID NO: 61, and a HCDR3 as set forth in SEQ ID NO:

62.

19. The antibody molecule or antigen-binding fragment thereof of claim 18, wherein, The heavy chain variable region of the second antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

135.

20. The antibody molecule or antigen-binding fragment thereof of claim 18, wherein, The heavy chain variable region of the second antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

59.

21. The antibody molecule or antigen binding fragment thereof of any one of claims 11-20, wherein, The second antigen binding domain is a single domain antibody.

22. The antibody molecule or antigen binding fragment thereof of any one of claims 11-21, wherein, The second antigen binding domain is humanized.

23. The antibody molecule or antigen binding fragment thereof of any of claims 1-22, further comprising a third antigen binding domain that specifically binds to human albumin.

24. The antibody molecule or antigen binding fragment thereof of claim 23, wherein, The third antigen binding domain comprises a heavy chain variable region comprising a HCDR1 as set forth in SEQ ID NO: 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 142, or any variant thereof, a HCDR2 as set forth in SEQ ID NO: 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 143, or any variant thereof, and a HCDR3 as set forth in SEQ ID NO: 72, 77, 82, 87, 92, 97, 102, 107, 112, 117, 122, 127, 144, or any variant thereof.

25. The antibody molecule or antigen-binding fragment thereof of claim 24, wherein, The heavy chain variable region of the third antigen binding domain comprises a HCDR1, a HCDR2, and a HCDR3 selected from the group consisting of: (1) SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72; (2) SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77; (3) SEQ ID NO: 80, SEQ ID NO: 81, and SEQ ID NO: 82; (4) SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87; (5) SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92; (6) SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97; (7) SEQ ID NO: 100, SEQ ID NO: 101, and SEQ ID NO: 102; (8) SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; (9) SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112; (10) SEQ ID NO: 115, SEQ ID NO: 116, and SEQ ID NO: 117; (11) SEQ ID NO: 120, SEQ ID NO: 121, and SEQ ID NO: 122; (12) SEQ ID NO: 125, SEQ ID NO: 126, and SEQ ID NO: 127; and (13) SEQ ID NO: 142, SEQ ID NO: 143, and SEQ ID NO:

144.

26. The antibody molecule or antigen-binding fragment thereof of claim 25, wherein, the heavy chain variable region of the third antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from the group consisting of SEQ ID NO: 69, 74, 79, 84, 89, 94, 99, 104, 109, 114, 119, and 124.

27. The antibody molecule or antigen-binding fragment thereof of any one of claims 25-26, wherein, the heavy chain variable region of the third antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from the group consisting of SEQ ID NO: 69, 74, 79, 84, 89, 94, 99, 104, 109, 114, 119, and 124.

28. The antibody molecule or antigen binding fragment thereof of any one of claims 25-26, wherein, the heavy chain variable region of the third antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from the group consisting of SEQ ID NO: 69, 74, 79, 84, 89, 94, 99, 104, 109, 114, 119, and 124.

29. The antibody molecule or antigen-binding fragment thereof of any one of claims 25- 26, wherein, the heavy chain variable region of the third antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to one sequence selected from the group consisting of SEQ ID NO: 69, 74, 79, 84, 89, 94, 99, 104, 109, 114, 119, and 124.

30. The antibody molecule or antigen-binding fragment thereof of claim 29, wherein, the heavy chain variable region of the third antigen binding domain comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

141.

31. The antibody molecule or antigen-binding fragment thereof of any of claims 23-30, wherein, the third antigen binding domain is a single domain antibody.

32. The antibody molecule or antigen-binding fragment thereof of any of claims 23-31, wherein, the third antigen binding domain is humanized.

33. The antibody molecule or antigen-binding fragment thereof of any of claims 23-32, wherein, the third antigen binding domain can prolong the half-life of the antibody molecule or antigen binding fragment thereof.

34. The antibody molecule or antigen binding fragment thereof according to any one of claims 23-33, which has a half-life in humans of at least 10 days, at least 15 days, or at least 20 days.

35. The antibody molecule or antigen-binding fragment thereof of any of claims 1-34, wherein, the first, second and / or third antigen binding domain are directly or indirectly sequentially linked; Preferably, the first, second and / or third antigen binding domain are sequentially linked by a linker; More preferably, the linker comprises a flexible linker and a rigid linker; Preferably, the C-terminus of the second antigen binding domain is linked to the N-terminus of the first antigen binding domain by a flexible linker; Preferably, the C-terminus of the first antigen binding domain is linked to the N-terminus of the third antigen binding domain by a rigid linker; Preferably, the flexible polypeptide linker is selected from (G4S)n, (SG4)n, or G4(SG4)n, wherein n is an integer between 1-5; more preferably, the flexible polypeptide linker is GGGGSGGGGS (SEQ ID NO: 129); Preferably, the rigid polypeptide linker is selected from (EAAAKA)n, wherein n is an integer between 1-5; more preferably, the rigid polypeptide linker is EAAAKAEAAAKA (SEQ ID NO: 130).

36. The antibody molecule or antigen-binding fragment thereof of any of claims 1-35, wherein, the first, second and / or third antigen binding domain is a recombinant antibody, preferably a llama-derived antibody, a chimeric antibody, or a humanized antibody; More preferably, the recombinant antibody is a nanobody; further preferably, the recombinant antibody is a humanized camelid VHH.

37. The antibody molecule or antigen binding fragment thereof according to any one of claims 1-36, which comprises an amino acid sequence that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

131.

38. A molecule that binds to a calcitonin gene-related peptide (CGRP) receptor, characterized in that, comprising the antibody molecule or antigen binding fragment thereof according to any one of claims 1-37.

39. The antibody molecule or antigen binding fragment thereof according to any one of claims 1-37, or the molecule according to claim 38, which antibody molecule or antigen binding fragment thereof or molecule does not bind to Pituitary Adenylate Cyclase Activating Peptide (PACAP), PACAP27, or VIP (Vasoactive Intestinal Polypeptide).

40. The antibody molecule, or antigen-binding fragment or molecule thereof, of any of claims 1-39, which does not block the pituitary adenylate cyclase-activating peptide (PACAP) pathway.

41. The antibody molecule, or antigen-binding fragment or molecule thereof, of any of claims 1-40, which does not comprise an extracellular domain fragment (ECD) of pituitary adenylate cyclase 1 (PAC1).

42. The antibody molecule, or antigen-binding fragment or molecule thereof, of any of claims 1-41, which does not comprise an Fc fragment.

43. The antibody molecule, or antigen-binding fragment or molecule thereof, of any of claims 1-42, which does not comprise an immunoglobulin constant region.

44. The antibody molecule, or antigen-binding fragment or molecule thereof, of any of claims 1-43, which has one or more of the following properties: (a) binds human CGRP receptor with a Kd of 1 x 10 -7 M or less; (b) inhibits binding of CGRP receptor to CGRP; (c) inhibits CGRP-induced cAMP production in CHO K1 cells expressing CGRP receptor; and (d) does not bind to human Amylin 1 receptor.

45. A nucleic acid encoding the antibody molecule, or antigen-binding fragment thereof, of any of claims 1-37 and 39-44, or the molecule of any of claims 38-44.

46. The nucleic acid of claim 45, wherein: the first antigen binding domain is encoded by a nucleotide sequence selected from SEQ ID NO: 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, or any variant thereof; the second antigen binding domain is encoded by a nucleotide sequence selected from SEQ ID NO: 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, 68, or any variant thereof; or the third antigen binding domain is encoded by a nucleotide sequence selected from SEQ ID NO: 73, 78, 83, 88, 93, 98, 103, 108, 113, 118, 123, 128, or any variant thereof.

47. A vector comprising the nucleic acid of any of claims 45-46.

48. A cell comprising the nucleic acid of any of claims 45-46 or the vector of claim 47.

49. A kit comprising the antibody molecule, or antigen-binding fragment thereof, of any of claims 1-37 and 39-44, the molecule of any of claims 38-44, the nucleic acid of any of claims 45-46, the vector of claim 47, or the cell of claim 48.

50. A pharmaceutical composition comprising (1) the antibody molecule, or antigen-binding fragment thereof, of any of claims 1-37 and 39-44, the molecule of any of claims 38-44, the nucleic acid of any of claims 45-46, the vector of claim 47, or the cell of claim 48, and (2) a pharmaceutically acceptable carrier, diluent, or excipient.

51. Use of an antibody molecule or antigen-binding fragment thereof according to any one of claims 1-37 and 39-44, a molecule according to any one of claims 38-44, a nucleic acid according to any one of claims 45-46, a vector according to claim 47, and / or a cell according to claim 48, and / or a pharmaceutical composition according to claim 50, for the manufacture of a medicament for the treatment and / or prevention of a disease associated with over-activation of CGRP.

52. A method for treating and / or preventing a disease associated with over-activation of CGRP in a subject, the method comprising: administering to the subject a therapeutically effective amount of an antibody molecule or antigen-binding fragment thereof according to any one of claims 1-37 and 39-44, a molecule according to any one of claims 38-44, a nucleic acid according to any one of claims 45-46, a vector according to claim 47, and / or a cell according to claim 48, and / or a pharmaceutical composition according to claim 50.

53. Use of an antibody molecule or antigen-binding fragment thereof according to any one of claims 1-37 and 39-44, a molecule according to any one of claims 38-44, a nucleic acid according to any one of claims 45-46, a vector according to claim 47, and / or a cell according to claim 48, and / or a pharmaceutical composition according to claim 14, for the treatment and / or prevention of a disease associated with over-activation of CGRP.

54. The use, method or application of any one of claims 51-53, wherein, the disease is migraine; more preferably, the disease comprises episodic migraine, menstrual migraine, chronic migraine.

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