fusion proteins

Abstract

A fusion protein is provided that simultaneously blocks the calcitonin gene-related peptide (CGRP) pathway and the pituitary adenylate cyclase-activating polypeptide (PACAP) pathway, and the fusion protein comprises at least one single-domain antibody, at least one Fc fragment, and at least one extracellular domain fragment of pituitary adenylate cyclase 1 (PAC1 ECD). The fusion protein simultaneously blocks the CGRP and PACAP pathways, thereby improving the response rate of drugs for the treatment of migraine.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and in particular to a fusion protein that simultaneously blocks the calcitonin gene-related peptide (CGRP) pathway and the pituitary adenylate cyclase-activating peptide (PACAP) pathway, and its applications. [Background technology]

[0002] Migraine is a vascular neurological disorder affecting 1.1 billion people worldwide, and the number of patients is still increasing. Currently, the global incidence rate is high at 12-15%, with the incidence rate of severe chronic cases at 1.4-2.2%. It is one of the top 10 most disabling diseases in the world. The peak age of onset for migraine is 37 years old, and it is three times more common in women than in men. Attacks last from 3 to 72 hours. In addition to the unilateral headache, symptoms include photophobia, phonophobia, nausea, and vomiting. Chronic migraine patients often have cardiovascular disease, psychiatric disorders, depression, sleep disorders, and other problems. Work, study, and social life are affected in 90% of patients, with 30% of women and 10% of children affected by migraine. Currently, both the diagnosis and treatment of this disease are problematic, with only 5% of patients receiving a correct diagnosis and treatment. Treatment methods include CGRP-targeted drugs, 5-HTr agonists, calcium channel blockers, antiepileptics, antidepressants, NSAIDs, nerve blockade / stimulation, and other non-specific medications.

[0003] Migraine is called a primary headache because its cause is unknown. It is generally believed that some type of stimulation of the central nervous system, such as cortical spreading depression (CSD), causes the release of neurotransmitters such as calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating peptide (PACAP), and substance C from the C-fiber terminals of the trigeminal nerve. These neurotransmitters bind to receptors on A-delta fibers and cause migraine headaches directly or indirectly.

[0004] Currently, it is known that there are at least two major signaling pathways involved in migraine: CGRP and PACAP. Both CGRP and PACAP are neurotransmitters distributed throughout the nervous system and other tissues throughout the body. Both receptors are class B GPCR neurotransmitters. They specifically bind to the receptor and increase the intracellular cAMP (cyclic adenosine monophosphate) concentration, thereby achieving the purpose of signal transduction.

[0005] Many new drugs are being developed to block signaling in these two pathways. Antagonists of CGRP and its receptors have been successfully used in the treatment and prevention of migraines. Currently, four monoclonal antibodies are available on the European and US markets: eptinezumab (Lunbeck), erenumab (Amgen), fremanezumab (Teva), and galcanezumab (Eli Lilly). Small molecules against the CGRP receptor, including atogepant (AbbVie), ubrogepant (AbbVie), and rimegepant (Pfizer), are also available on the European and US markets. In contrast, antagonists of PACAP and PAC1 are still under development. Clinical trials have shown that administration of PACAP to healthy individuals can induce migraine-like headache attacks. PACAP has also been found to be present at higher concentrations in the blood of chronic migraine patients than in healthy individuals. All these studies indicate that PACAP is closely related to migraine headache.

[0006] According to the latest statistics published in the New England Journal of Medicine, the response rates of the major drugs used to treat migraine are extremely low. The response rate to prophylactic CGRP monoclonal antibodies is only 20–24%, and the response rate to small molecule CGRP receptor antagonists is only approximately 10–14%. One reason for the low response rate to drugs is the involvement of multiple signaling pathways in the development of migraine. Therefore, the development of bifunctional or multifunctional drugs is necessary to achieve maximum therapeutic efficacy and provide effective treatment for many migraine patients, especially those with intractable migraine. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides a fusion protein that simultaneously blocks the CGRP and PACAP pathways, which can improve the response rate of drugs for treating migraine. [Means for solving the problem]

[0008] In one aspect, the present invention provides a single domain antibody for blocking the calcitonin gene-related peptide (CGRP) pathway, wherein the single domain antibody comprises a CDR1 set forth in the amino acid sequence of SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, or 57, or any variant thereof; a CDR2 set forth in the amino acid sequence of SEQ ID NO: 3, 8, 13, 18, 23, 28, 33, 38, 43, 48, 53, or 58, or any variant thereof; and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, or 59, or any variant thereof.

[0009] In another aspect, the present invention provides a fusion protein that simultaneously blocks the calcitonin gene-related peptide (CGRP) pathway and the pituitary adenylate cyclase-activating peptide (PACAP) pathway, the fusion protein comprising at least one single domain antibody, at least one Fc fragment, and at least one extracellular domain fragment of pituitary adenylate cyclase 1 (PAC1 ECD).

[0010] The fusion protein comprises one peptide chain, and its structure is such that one PAC1 ECD is linked to the C-terminus of an Fc fragment via a linker, and the N-terminus of the Fc fragment is fused to the C-terminus of a single-domain antibody; Optionally, the fusion protein comprises two peptide chains, in which one PAC1 ECD is linked to the C-terminus of an Fc fragment via a linker, and the N-terminus of the Fc fragment is fused to the C-terminus of a single domain antibody, and the two PAC1 ECDs of the two peptide chains are linked via a linker; Optionally, the fusion protein comprises three peptide chains in which one PAC1 ECD is linked to the C-terminus of an Fc fragment via a linker, the N-terminus of the Fc fragment is fused to the C-terminus of a single domain antibody, and the three PAC1 ECDs of the three peptide chains are linked to each other via linkers.

[0011] In another aspect, the invention provides nucleic acids encoding the above-described single domain antibodies and fusion proteins.

[0012] In another aspect, the present invention provides a vector comprising the above-described nucleic acid.

[0013] In another aspect, the present invention provides a cell comprising the above-described nucleic acid or vector.

[0014] In another aspect, the present invention provides a pharmaceutical composition or kit comprising the above-described single domain antibody, fusion protein, and a pharmaceutically acceptable vector, diluent, or excipient.

[0015] In another aspect, the present invention provides the use of the above-mentioned single domain antibody, fusion protein, nucleic acid, vector, and / or cell and / or pharmaceutical composition or kit in the preparation of a medicament for the treatment of a disease associated with activation of the CGRP and PACAP pathways.

[0016] The fusion protein provided by the present invention can simultaneously block two signal pathways, improve the response rate of migraine patients to drugs, and provide an effective treatment method for many migraine patients, especially those with intractable migraine.

[0017] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments, taken in conjunction with the accompanying drawings, which are intended for the purpose of illustrating particular embodiments only and are not intended to limit the invention. In the accompanying drawings: [Brief explanation of the drawings]

[0018] [Figure 1] The results of electrophoresis of RC-Fc are shown. [Figure 2] We demonstrate that the reconstituted CGRP receptor extracellular domain of the present invention retains the ability to bind to its ligand, CGRP, but not to the unrelated polypeptide, PACAP. [Figure 3] CGRP-induced cAMP production under different SK-N-MC cell numbers is shown. [Figure 4] The results of electrophoresis of 42wtFc, 42hv10Fc, 42hv11Fc, and 42hv12Fc are shown. [Figure 5] This shows that VHH No. 42 after humanization and Fc fusion retains the ability to inhibit cellular cAMP production. [Figure 6] The gene structure of human PAC1 is shown. The vertical bars represent exons. [Figure 7] Three ligands of PAC1 are shown, including PACAP (PACAP38), PACAP27, and VIP. [Figure 8] The results of electrophoresis of PAC1 ECD-Fc and PACs1 ECD-Fc are shown. [Figure 9] 1 shows a comparison of the ability of PAC1 ECD-Fc and PAC1s ECD-Fc to bind to PACAP in vitro. [Figure 10] The structure of BY003 is shown. [Figure 11] The operating principle of BY003 is shown below. [Figure 12] The results of electrophoresis of BY003 are shown. From left to right, the results of electrophoresis of 42hv10FeNA-1 × PAC1s ECD, 42hv10FcNA-2 × PAC1s ECD, and 42hv10FcNA-3 × PAC1s ECD are shown, corresponding to the cases of one, two, and three PAC1s ECDs, respectively. [Figure 13] CGRP standard curves for three CGRPr stable cell lines are shown. [Figure 14] 1 shows that BY003 inhibits CGRP-induced cAMP production in CHO-hCGRPrC3 cells. [Figure 15] PACAP standard curves for six PAC1 stable cell lines are shown. [Figure 16] We show that BY003 blocks PACAP signaling at the cellular level. DETAILED DESCRIPTION OF THE INVENTION

[0019] I. Definition In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and experimental procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and common procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0020] The term "fusion" or "fused" when applied to amino acid sequences (e.g., peptides, polypeptides, or proteins) refers to the combination of two or more amino acid sequences into a single, non-naturally occurring amino acid sequence, for example, by chemical conjugation or recombination. A fused amino acid sequence can be produced by the recombination of two gene encoding polynucleotide sequences and expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0021] The terms "polypeptide," "peptide," and "protein" are used interchangeably in this disclosure to refer to a polymer of amino acid residues or an aggregate of polymers of multiple amino acid residues. The terms encompass natural or artificial proteins, protein fragments, and polypeptide analogs having a protein sequence. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to natural and non-natural amino acid polymers. Polypeptides can be monomers or polymers.

[0022] With respect to antibody chain polypeptide sequences, the phrase "substantially identical" may be understood to refer to the antibody chain exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference polypeptide sequence. With respect to nucleic acid sequences, the term may be understood to mean that the nucleotide sequence exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference nucleic acid sequence.

[0023] Sequence "identity" or "identity" has its meaning generally recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide, or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods for determining identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0024] The term "antibody," also known in the art as "immunoglobulin" (Ig), refers to a protein constructed from pairs of heavy and light polypeptide chains, with various Ig isotypes occurring, including IgA, IgD, IgE, IgG, and IgM. When an antibody is properly folded, each chain folds into a number of distinct globular domains connected by more linear peptide sequences. For example, an immunoglobulin light chain folds into a variable (VL) domain and a constant (CL) domain, while a heavy chain folds into a variable (VH) domain and three constant (CH1, CH2, CH3) domains. Interactions between the heavy and light chain variable domains (VH and VL) form an antigen-binding region (Fv).

[0025] The term "single-domain antibody" (sdAb) refers to an antibody containing only a single antibody variable region (VH / VL). Like conventional antibodies, single-domain antibodies can selectively bind to specific antigens. Their molecular weight is only 12-15 kDa, much smaller than that of typical antibodies (150-160 kDa), which consist of two heavy chains and two light chains. The terms "heavy-chain antibody," "heavy-chain single-domain antibody," or "nano-single-chain antibody" refer to an antibody that contains a single variable domain (VHH) and two constant domains (CH2 and CH3). Importantly, cloned and isolated VHH domains are intact, stable polypeptides that retain the full antigen-binding capacity of the original heavy-chain antibody. Compared to other antibodies, this type of antibody naturally lacks light chains. This type of single-domain antibody has been found in camelids. Single-domain antibodies have the advantages of a stable structure, small molecule affinity, good solubility, resistance to various harmful environments, and ease of humanization. They are widely used in miniaturized, genetically engineered antibody research, drug development, and disease diagnosis and treatment. Examples of single domain antibodies include those derived from camelids (alpacas and camels) and cartilaginous fish (e.g., nurse sharks), as well as those derived from human and murine antibodies by recombinant methods. As used herein, the term "single domain antibody" includes sdAbs isolated directly from VH, VHH, VL, or VNAR libraries of any source by phage display or other techniques, sdAbs derived from the aforementioned sdAbs, recombinantly produced sdAbs, and sdAbs produced by further modification of such sdAbs (by humanization, affinity maturation, stabilization, solubilization, camelization, or other methods of antibody engineering). The present disclosure further relates to homologs, derivatives, or fragments that retain the antigen-binding function and specificity of sdAbs.

[0026] 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. Flexible polypeptides are composed of flexible amino acids, where the flexible amino acids are 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, i.e., the peptide is selected from the group consisting of GGGGS, GGGGSGGGGS, SGGGGSGGGG, GGGGGSGGGGSSGGGGGS, GGGGSGGGGSGGGG, GGGGSGGGGSGGGGGS, GGGGSGGGGSGGGGGS, GSGSGSGS, GGSGSGSG, GGSGSG, and GGSG.

[0027] The term "CDR" refers to a complementarity-determining region, and each heavy and light chain of a known antibody molecule has three CDRs. CDRs, also called hypervariable regions, are present in the variable regions of each heavy and light chain of an antibody, and the primary structure of the CDRs is accompanied by highly variable regions. Herein, a heavy chain CDR is represented by CDR1, CDR2, and CDR3 from the amino terminus of the amino-terminal sequence of the heavy chain, and a light chain CDR is represented by CDR1, CDR2, and CDR3 from the amino terminus of the amino-terminal sequence of the light chain. These regions are adjacent to each other in the tertiary structure and determine the specificity of the antibody for binding to an antigen.

[0028] "Specific binding" or "immunospecific binding," with respect to an antibody or antigen-binding fragment thereof, are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more non-covalent bonds with an alloantigen through non-covalent interactions between the antibody-binding site of the antibody and the antigen. The antigen may be an isolated antigen or may be present on tumor cells. Typically, an antibody that immunospecifically binds (or specifically binds) an antigen has a binding activity of about 1 x 10 7 M-1 or 1 x 108 It binds to the antigen with an affinity constant Ka of M or greater (or a dissociation constant (Kd) of 1×10 M or 1×10 M or less). The affinity constant can be determined by standard kinetic methods of antibody reactions, such as 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. Pat. No. 7,229,619 for a description of exemplary SPR and ITC methods for calculating antibody binding affinity. Instruments and methods for real-time detection and monitoring of binding kinetics are known and commercially available (BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; see Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0029] The term "binding affinity" or "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed 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 antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art.

[0030] The terms "inhibit" or "neutralize" as used with respect to a biological activity of a fusion protein of the present disclosure refer to, for example, the ability of a polypeptide to substantially antagonize, inhibit, prevent, limit, slow down, destroy, eliminate, stop, reduce, or reverse the progression or severity of the inhibited (including, but not limited to, biological activity).

[0031] The terms "nucleic acid" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleic acids or nucleic acid derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded and may be cDNA.

[0032] The term "vector" includes vectors capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from elements that may include plasmid or viral DNA, or both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that results in expression of cloned DNA when introduced into an appropriate host cell. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain episomal or that integrate into the host cell genome.

[0033] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredients contained therein is present in a form that is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered.

[0034] The term "pharmaceutically acceptable vector" refers to one or more non-toxic substances administered with a therapeutic agent that do not interfere with the biological activity of the active ingredient, including, but not limited to, buffers, preservatives, compatible vectors, diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), excipients, vehicles, and optionally other additives or encapsulating substances. Pharmaceutical vectors suitable for the present disclosure can be conventional pharmaceutical preparation excipients, as well as compositions and preparations suitable for delivering the disclosed neutralizing antibodies. Generally, the nature of the vector will depend on the particular mode of administration used. For example, parenteral preparations usually comprise injectable fluids containing pharmaceutically and physiologically acceptable liquids as vehicles, such as water, physiological saline, balanced salt solutions, glucose solutions, glycerol, or the like. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid vehicles can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral vehicle, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0035] The term "treating" refers to a subject suffering from a disease or condition in which the subject's symptoms are partially or completely alleviated or remain unchanged after treatment. Thus, treatment includes prophylaxis, therapy, and / or cure. Prevention means keeping the underlying disease at bay and / or preventing the worsening of symptoms or the onset of disease. Treatment further includes any pharmaceutical use of the provided antibodies or antigen-binding fragments thereof, and any of the compositions provided herein.

[0036] Also provided are "conservative sequence modifications" of the sequences set forth in the sequence listings herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding of an antibody encoded by the nucleotide sequence or containing the amino acid sequence to an antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listings set forth herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. 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), 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). Thus, a predicted non-essential amino acid residue in an anti-BCMA antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes are present in one or more or all three heavy chain CDRs.Preferably, the amino acid changes are amino acid substitutions, preferably conservative substitutions. In some embodiments, the antibody variant has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the parent antibody over the target antibody sequence region. Exemplary substitutions are shown in Table 1 below: [Table 1]

[0037] II. DETAILED DESCRIPTION OF THE INVENTION In one aspect, the present invention provides a single domain antibody for blocking the calcitonin gene-related peptide (CGRP) pathway, wherein the single domain antibody comprises a CDR1 set forth in the amino acid sequence of SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, or 57, or any variant thereof; a CDR2 set forth in the amino acid sequence of SEQ ID NO: 3, 8, 13, 18, 23, 28, 33, 38, 43, 48, 53, or 58, or any variant thereof; and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, or 59, or any variant thereof.

[0038] In some preferred embodiments, the above-mentioned single domain antibody comprises a CDR1 set forth in the amino acid sequence of SEQ ID NO: 12 or any variant thereof, a CDR2 set forth in the amino acid sequence of SEQ ID NO: 13 or any variant thereof, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 14 or any variant thereof.

[0039] In some embodiments, the single domain antibody comprises a variable region set forth in the amino acid sequence of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 62, or 63, or any variant thereof.

[0040] In some preferred embodiments, the single domain antibody comprises a variable region set forth in the amino acid sequence of SEQ ID NO: 61, 62, or 63, or any variant thereof.

[0041] In some preferred embodiments, the single domain antibody comprises a variable region set forth in the amino acid sequence of SEQ ID NO: 61 or any variant thereof.

[0042] In some preferred embodiments, the amino acids in the above single domain antibodies have conservative substitutions, and the number of conservatively substituted amino acids does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0043] In one aspect, the present invention provides a fusion protein that simultaneously blocks the calcitonin gene-related peptide (CGRP) pathway and the pituitary adenylate cyclase-activating peptide (PACAP) pathway, the fusion protein comprising at least one single domain antibody, at least one Fc fragment, and at least one extracellular domain fragment of pituitary adenylate cyclase 1 (PAC1 ECD).

[0044] In some embodiments, the fusion protein comprises one peptide chain in which one PAC1 ECD is linked via a linker to the C-terminus of an Fc fragment, and the N-terminus of the Fc fragment is fused to the C-terminus of a single domain antibody.

[0045] In some embodiments, the fusion protein comprises two peptide chains in which one PAC1 ECD is linked to the C-terminus of an Fc fragment via a linker, the N-terminus of the Fc fragment is fused to the C-terminus of a single-domain antibody, and the two PAC1 ECDs of the two peptide chains are linked via a linker.

[0046] In some embodiments, the fusion protein comprises three peptide chains in which one PAC1 ECD is linked to the C-terminus of an Fc fragment via a linker, the N-terminus of the Fc fragment is fused to the C-terminus of a single-domain antibody, and the three PAC1 ECDs of the three peptide chains are linked to each other via linkers.

[0047] In some embodiments, the single domain antibody is a single domain antibody that binds to a CGRP receptor.

[0048] In some embodiments, the above-mentioned single domain antibodies comprise a CDR1 set forth in the amino acid sequence of SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, or 57, or any variant thereof; a CDR2 set forth in the amino acid sequence of SEQ ID NO: 3, 8, 13, 18, 23, 28, 33, 38, 43, 48, 53, or 58, or any variant thereof; and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, or 59, or any variant thereof.

[0049] In some embodiments, the above-mentioned single domain antibody comprises a CDR1 set forth in the amino acid sequence of SEQ ID NO: 12 or any variant thereof, a CDR2 set forth in the amino acid sequence of SEQ ID NO: 13 or any variant thereof, and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 14 or any variant thereof.

[0050] In some preferred embodiments, the amino acids in one or more or all three of the above CDRs have conservative substitutions, and the number of conservatively substituted amino acids does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0051] In some embodiments, the single domain antibody comprises a variable region set forth in the amino acid sequence of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 62, or 63, or any variant thereof.

[0052] In some preferred embodiments, the single domain antibody comprises a variable region set forth in the amino acid sequence of SEQ ID NO: 61, 62, or 63, or any variant thereof.

[0053] In some more preferred embodiments, the single domain antibody comprises a variable region set forth in the amino acid sequence of SEQ ID NO: 61, or any variant thereof.

[0054] In some preferred embodiments, the amino acids in the above single domain antibodies have conservative substitutions, and the number of conservatively substituted amino acids does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0055] In some embodiments, the PAC1 ECD is selected from the ECDs of the PAC isomers: PAC1, PAC1s, or PAC1vs.

[0056] In some preferred embodiments, the PAC1 ECD is a PAC1s ECD.

[0057] In some preferred embodiments, the PAC1 ECD comprises the amino acid sequence of SEQ ID NO: 64, and amino acid sequences having 80% or greater identity thereto, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity thereto, and more preferably 98% or 99% or greater identity thereto.

[0058] In some preferred embodiments, the PAC1s ECD comprises the amino acid sequence of SEQ ID NO: 66, and amino acid sequences having 80% or more identity thereto, preferably amino acid sequences having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and more preferably amino acid sequences having 98% or 99% or more identity thereto.

[0059] In some embodiments, the Fc fragment comprises the hinge region, constant region 2 (CH2), and constant region 3 (CH3) domains derived from a human immunoglobulin.

[0060] In some preferred embodiments, the Fc fragment is selected from human IgG1, IgG2, IgG3, and IgG4, and more preferably, the Fc fragment is selected to be human IgG1.

[0061] In some preferred embodiments, the Fc fragment has an amino acid substitution, more preferably the amino acid substitution comprises a substitution of asparagine N at position 297 of the Fc fragment with alanine A, and each amino acid position of the Fc fragment is numbered using the EU antibody numbering system.

[0062] In some preferred embodiments, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 68, and amino acid sequences having 80% or greater identity thereto, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity thereto, and more preferably 98% or 99% or greater identity thereto.

[0063] In some embodiments, the linker is a flexible polypeptide linker.

[0064] In some preferred embodiments, the flexible polypeptide linker is selected from GGGGS, GGGGSGGGGS, SGGGGSGGGG, GGGGGSGGGGSSGGGGS, GGGGSGGGGSGGGGGS, GGGGSGGGGSGGGG, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGSGGGG, GSGSGSGS, GGSGSGSG, GGSGSG, and GGSG.

[0065] In some more preferred embodiments, the flexible polypeptide linker is GGGGS (SEQ ID NO: 79).

[0066] In another aspect, the present invention provides a nucleic acid encoding the above-described fusion protein.

[0067] In another aspect, the present invention provides a vector comprising the above-described nucleic acid.

[0068] In another aspect, the invention provides a cell comprising the nucleic acid or vector.

[0069] In another aspect, the present invention provides a pharmaceutical composition or kit comprising the above-described fusion protein and a pharmaceutically acceptable vector, diluent, or excipient.

[0070] In another aspect, the present invention provides the use of the fusion protein, nucleic acid, vector, and / or cell and / or pharmaceutical composition or kit described above in the preparation of a medicament for the treatment of a disease associated with activation of the CGRP and PACAP pathways.

[0071] In some preferred embodiments, the disease is migraine.

[0072] The above description is merely a preferred embodiment of the present invention and is a description of the technical principles used. Those skilled in the art should understand that the scope of the disclosure related to the present invention is not limited to the technical solution formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the concept disclosed above, for example, by replacing the above features with (but not limited to) technical features having similar functions disclosed in the present invention. [Example]

[0073] Example 1: Synthesis of a single domain antibody against the CGRP receptor By immunizing llamas with the ECD of the CGRP receptor, the optimal sequence of a single domain antibody against the CGRP receptor, 42hv10, was obtained after multiple rounds of screening and humanization.

[0074] To obtain highly selective and high-affinity binding antibodies, we immunized llamas using a protein fused to the in vitro-expressed and purified CGRP ECD and the crystallizable fragment (Fc) of human immunoglobulin G1 (IgG1) (called RC-Fc) as an immunogen. Lymphocytes were then isolated from high-titer llama blood and their messenger ribonucleic acid (mRNA) was extracted. These mRNAs were used to construct a phage library, which was then screened using enzyme-linked immunosorbent assay (ELISA) for positive colonies containing single-domain antibodies capable of binding to RC-Fc and those containing single-domain antibodies capable of binding only to Fc. These colonies contained all single-domain antibodies that bound to CGRP ECD and Fc. Colonies that bound only to Fc were excluded, and the remaining CGRP ECD-positive and Fc-negative colonies were sent for sequencing. Sequence analysis and verification yielded a batch of single-domain antibodies capable of specifically binding to CGRP ECD.

[0075] 1. Preparation of RC-Fc 1.1 Design of RC-Fc RC-Fc is formed by the fusion of the CGRP receptor: human Ramp1 ECD, CLR ECD, and Fc. Ramp1 ECD is derived from Genbank ID NM_005855.4 nucleic acid sequence 132-404 (SEQ ID NO: 69). The nucleic acid sequence is as follows: [ka] The corresponding amino acid sequence is derived from NP_005846.1 27-117 (SEQ ID NO: 70). The amino acid sequence is as follows: [ka] The CLR ECD is derived from Genbank ID NM_001271751.2 nucleic acid sequence 407-782 (SEQ ID NO: 71). The nucleic acid sequence is as follows: [ka] The corresponding amino acid sequence is derived from NP_001258680.1 amino acid sequence 23-146 (SEQ ID NO: 72). The amino acid sequence is as follows: [ka] The Fc portion is derived from Genbank ID JX292763.1 nucleic acid sequence 661-1359 (SEQ ID NO: 73). The nucleic acid sequence for cloning is as follows: [ka] The corresponding amino acid sequence Genbank ID AFR78282.1 221-452 (SEQ ID NO: 74) is as follows: [ka] Alternatively, the Fc portion is derived from Genbank ID MW176619.1 nucleic acid sequence 663-1358 (SEQ ID NO: 91). The nucleic acid sequence for cloning is as follows: [ka] The corresponding amino acid sequence Genbank ID QRG33954 221-452 (SEQ ID NO: 92) is as follows: [ka]

[0076] Due to the different amino acid sequences of the Ramp1 ECD and CLR ECD, when fused to Fc, they must be paired by the knobs-in-holes (KiH) technique, with the C-terminus of the Ramp1 ECD linked to the N-terminus of an Fc with the T366W mutation to form the R chain, and the C-terminus of the CLR ECD fused to the N-terminus of an Fc with the T366S, L368A, and Y407V mutations to form the C chain.

[0077] The amino acid sequence of the RC-Fc Ramp1 ECD (RC-Fc R) chain (SEQ ID NO: 75) is as follows: [ka] wherein the Fc fused to the RC-Fc R chain has the T366W mutation shown below (SEQ ID NO: 76): [ka] The amino acid sequence of the RC-Fc CLR ECD (RC-Fc C) chain (SEQ ID NO: 77) is as follows: [ka] wherein the Fc fused to the RC-Fc C chain has the T366S, L368A, and Y407V mutations shown below (SEQ ID NO: 78): [ka]

[0078] 1.2 Gene synthesis, expression, and purification of RC-Fc Taizhou Biotechnology Co., Ltd. (Biointron) was commissioned to synthesize the RC-Fc plasmid. The synthesized gene fragment was digested with NotI (New England BioLabs Inc., NEB, R0189L) and XbaI (New England BioLabs Inc., NEB, R0145L) and recovered (Biointron), then ligated with pcDNA3.4 vector (ThermoFisher Scientific, EL0011). The ligation product was transformed into Top10 competent cells. Positive clones were selected for scale-up cultivation, and small amounts of plasmid were extracted and sequenced using a Biointron-prepared plasmid mini-extraction kit (Biointron). Clones with the correct sequence for scale-up cultivation were inoculated into LB medium (Biointron), and large amounts of plasmid were extracted using a Biointron-prepared kit (Biointron) and re-sequenced for verification.

[0079] The expression plasmid was transfected into 293 cells using a transfection reagent (Biointron), and the cell culture was harvested after 5 days in a constant temperature incubator (Crystal, IS-RDS6C5). The cell culture was centrifuged, the supernatant was collected, cell debris was removed by filtration, and the clarified liquid was collected. The target protein was captured using a Protein A affinity chromatography column. The target protein was filtered, and the final yield was determined by measuring A280 and the absorbance at 280 nm was read using a NanoDrop One (ThermoFisher Scientific, ND-ONE-W) instrument. The protein was transferred to a dialysis bag and dialyzed in a beaker containing 1x PBS. The purified product was identified by electrophoresis. The electrophoresis results are shown in Figure 1.

[0080] 2. Verification of RC-Fc binding to CGRP The expressed and purified RC-Fc fusion protein was used for animal immunization only after its ability to bind to its ligand CGRP was verified by enzyme-linked immunosorbent assay (ELISA) at Chengdu NB Biolab Co., Ltd., specifically as follows.

[0081] A streptavidin-coated 96-well ELISA plate (ThermoFisher Scientific™, 15126) was washed three times with PBST (1x PBS (Absin, abs9266), 0.1% Tween 20 (Solarbio, T8220), pH = 7.4). Then, 1000 ng of biotin-CGRP (Nanjing Yuan-Peptide Biotechnology Co., Ltd., A03Biotin137, P200629-HS319172) was added to each well and incubated at room temperature for 2 hours. The PBST was removed, and 100 μL of serially diluted RC-Fc fusion protein in PBST was added to each well and incubated at 37°C for 1 hour. The ELISA plates incubated with the samples were washed five times with PBST, and then 100 μL of the corresponding secondary antibody anti-human IgG-HRP (anti-human immunoglobulin G-horseradish peroxidase, Hangzhou HuaBio, HR1214) diluted 1:5000 in blocking solution (PBST + 3% non-fat milk powder (Solarbio, D8340)) was added and incubated at 37°C for 1 h.

[0082] The ELISA plate incubated with the secondary antibody was washed five times with PBST, and 100 μL of TMB (trimethylolpropane) single-component colorimetric solution (Solarbio, 20190402) was added to each well. The plate was incubated at 37°C for 7 minutes. 100 μL of 1 M HCl (Kelong Chemicals) was added to each well to stop the reaction, and the OD values ​​were read at 450 nm using an ELISA reader (ThermoFisher Scientific, 51119080ET). OD values ​​were analyzed using GraphPad Prism 9.3.1 in Nonlinear Regression One Site Specific Binding mode, revealing a Bmax of 5.198 and a Kd of 16.12 nM for RC-Fc binding to CGRP. The Bmax of RC-Fc binding to the negative control polypeptide PACAP was 0.069, with an ineffective Kd. The results are shown in Figure 2 and demonstrate that the reconstituted CGRP receptor extracellular domain of the present invention retains the ability to bind its ligand, CGRP, but not the unrelated polypeptide, PACAP.

[0083] 3. Immunization of llamas with RC-Fc The llama immunization and VHH screening process was entrusted to Chengdu NB Biolab Co., Ltd. (NB Biolab). Before immunization, 10 mL of blood was collected as a negative serum control. Then, 0.5 mg of RC-Fc was homogenously mixed with 1 mL of complete Freund's adjuvant (CFA, Sigma, F5881) and injected subcutaneously. On day 21, 0.25 mg of antigen was homogenously mixed with 1 mL of incomplete Freund's adjuvant (IFA, Sigma, F5506) and then injected subcutaneously. On day 28, 10 mL of blood was collected for serum isolation. On day 42, 0.25 mg of antigen was homogenously mixed with 1 mL of IFA and injected subcutaneously. On day 49, 50 mL of peripheral blood was collected, and lymphocytes and serum were isolated. On day 63, 0.25 mg of antigen was homogeneously mixed with 1 mL of IFA and injected subcutaneously. On day 70, 50 mL of peripheral blood was collected, and lymphocytes and serum were separated.

[0084] 4. Gradient Detection RC-Fc was diluted to 2000 ng / mL in coating solution (50 mM NaHCO3 (pH = 9.6)), and 100 μL was added to each well of a 96-well ELISA plate and left overnight at 4°C. The ELISA plate coated overnight at 4°C was washed five times with PBST, tapped dry, and 300 μL of blocking solution was added to each well.

[0085] Serum was serially diluted 1:2 with TBST, with the first gradient serum being 1:2000 and the last gradient serum being 1:128000. After washing the antigen plate three times with 300 μL / well of PBST, 100 μL of diluted serum was added to each well and incubated at 37°C for 1 hour. After washing three times with 300 μL / well of PBST, 100 μL of anti-M13 antibody (NB Biolab, 052-101-005) at 1:2000 was added to each well and incubated at 37°C for 1 hour. After washing six times with 300 μL / well of PBST, anti-alpaca-HRP (NEB, S001H) diluted 1:10000 was added for 30 minutes at 37°C. The ELISA plates incubated with the secondary antibody were washed five times with PBST, 100 μL of TMB single-component colorimetric solution was added to each well and incubated at 37°C for 7 minutes, 100 μL of 1 M HCl was added to each well to stop the reaction, and OD450 readings were taken.

[0086] 5. Phage Library Construction Peripheral blood was selected using a high-gradient method, and PBMCs were isolated according to the instructions for Lymphocyte Separation Medium (TBD Sciences, LTS1077). Total RNA was extracted using RNAiso Plus (Foregene, RE-03111) reagent, and 5 μg of RNA was transcribed into cDNA using the PrimeScript™ II First-Strand cDNA Synthesis Kit (Takara, 9109). The cDNA stock solution was diluted 5-fold for nested PCR amplification, and approximately 750 base pair (BP) single-domain antibody (VHH) fragments were recovered by gel excision for a second round of PCR amplification. The purified PCR amplified product was the target VHH fragment. The target VHH fragment was ligated into the vector pComb3XSS (NEB, P001) via the Sfil restriction site. Immediately after electroporation, 1 mL of 2YT (culture medium containing tryptone (Solarbio, T8490), yeast extract (Solarbio, Y8020), and sodium chloride (Kelong Chemicals), preheated to 37°C) was added to the electroporation cup for recovery. The electroporation product was aspirated, and the electroporation cup was washed with 2YT culture medium. A total of 100 mL of recovered product was obtained and recovered at 37°C, 180 rpm for 45 minutes. -3 and 10 -4 To determine the number of library transformants, 100 μL of the resulting solution was diluted to 0.05 mL and spread onto a 90 mm plate. The remainder was centrifuged, resuspended in 8 mL of 2YT, and spread onto eight 200 mm plates. The next day, bacterial clones harboring the target VHH fragments were plated to determine the number of library transformants.

[0087] 6. Packaging the Phage Library The bacterial library was inoculated into 2 × 300 mL of 2YT+A (100 μg / mL ampicillin (Solarbio, A1170)) + G (1% glucose (Solarbio, G8150)) medium to an initial OD of 0.1–0.2 and cultured at 37 °C and 230 rpm until the OD reached >0.8. Helper phage M13KO7 (NBbiolab, P006) and TG1 bacteria (NBbiolab, P008) were added according to the OD (helper phage:bacteria ratio: 20:1). M13KO7 was added, mixed uniformly, and left to stand at 37°C for 30 minutes, slowly shaken at 180 rpm for 30 minutes, and centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded, and the precipitate was resuspended in an equal volume of 2YT+A+K (kanamycin (Solarbio, K1030) 50 μg / ml) culture medium overnight at 30°C and 220 rpm.

[0088] The overnight culture was centrifuged at 10,000 rpm at 4°C for 20 minutes, the supernatant was collected, and the precipitate was discarded. The centrifuge tube was replaced and the mixture was centrifuged again at 10,000 rpm at 4°C for 20 minutes, and the supernatant was collected.

[0089] PEG8000 (Solarbio, P8260) / NaCl was added at 1 / 5 of the supernatant volume, mixed uniformly, and allowed to precipitate on ice for over 2 hours. The mixture was centrifuged at 10,000 rpm for 20 minutes, the supernatant was discarded, and all the supernatant was removed after one centrifugation. The precipitate was resuspended in 1 mL of 1x PBS, and 1 / 5 volume of PEG8000 / NaCl was added for 1 hour for secondary precipitation.

[0090] The pellet was centrifuged at 12,000 rpm for 10 minutes, the supernatant was discarded, and all of the supernatant was removed after one centrifugation. Depending on the amount of pellet, 1x PBS was added to resuspend the pellet, and 100% glycerol was added to a final concentration of 50%. The mixture was mixed uniformly, dispensed into 1.5 mL EP (Eppendorf) tubes, and stored at -80°C.

[0091] 7. Screening for positive colonies that bind to RC-Fc (positive screening) RC-Fc was diluted to a final concentration of 5 μg / mL using a coating solution at pH 9.6 and added to the enzyme-labeled wells at 100 μL / well to coat eight wells for each target molecule (four wells for the second round of screening and two wells for the third round of screening). Coating was performed overnight at 4°C. The coating solution was then discarded, the cells were washed three times with PBS, and 300 μL of 3% BSA-PBS blocking solution was added to each well, and the cells were blocked at 37°C for 1 hour. After washing three times with PBS, 100 μL of the phage library was added and incubated at 37°C for 1 hour. Unbound phages were aspirated, washed six times with PBST, washed twice with PBS, 100 μL of Gly-HCl elution solution was added, and incubated at 37°C for 8 minutes to elute specifically bound phages. The eluate was transferred to a 1.5 mL sterile centrifuge tube and quickly neutralized with 10 μL of Tris-HCl neutralization buffer. 10 μL was taken for gradient dilution to determine the titer and calculate the panning recovery rate.

[0092] Twenty-four single clones were randomly selected from the eluate titration plate using a sterile toothpick and inoculated into 1 mL of 2xYT-A. The culture was incubated at 37°C for 8 hours with shaking at 220 rpm. To 200 μL of the culture, M13K07 phage was added at a phage:TG1 ratio of 20:1. The culture was incubated at 37°C for 15 minutes and then incubated at 220 rpm for 45 minutes with shaking. 800 μL of 2xYT-AK was added and the culture was incubated overnight at 30°C with vigorous shaking. The next day, the cells were centrifuged at 12,000 rpm for 2 minutes, and the supernatant was collected for monoclonal ELISA identification.

[0093] 8. Screening for positive clones that bind to Fc (negative screening) Fc protein (NBbiolab, 20200706) was diluted to a final concentration of 2 μg / mL in a pH 9.6 coating solution, and 100 μL per well was added to ELISA wells for overnight coating at 4°C. The coating solution was then discarded, and the wells were washed three times with PBST. 200 μL of 5% nonfat milk was added to each well, and the wells were blocked at 37°C for 1 hour. The wells were then washed three times with PBST, and 50 μL of phage culture supernatant and 50 μL of 5% nonfat milk were added to each well and incubated at 37°C for 1 hour. After washing six times with PBST, 100 μL of horseradish peroxidase-labeled anti-M13 antibody (diluted 1:10,000 in PBS) was added at 100 μL per well and incubated at 37°C for 1 hour. The plate was washed six times with PBST. For color development, 100 μL / well of TMB color development solution was added for 7 minutes at 37° C. To stop the reaction, 50 μL / well of stop solution was added, and the optical density was measured at 450 nm.

[0094] 9. Exclusion of Fc-binding positive clones The results of the positive and negative screening were comprehensively analyzed to retain positive clones from the positive screening and exclude positive clones from the negative screening. Finally, the positive clones from the positive screening and the negative clones from the negative screening were sent to Chengdu Tsingke Biotech Co., Ltd. for sequencing.

[0095] 10. Sequence analysis results Three llamas were immunized with RC-Fc, and peripheral blood lymphocyte mRNA from two of the animals was used to establish a phage library. After screening and sequencing, a total of 567 VHH sequences were obtained (311 from animal NB268 and 258 from animal NB269). After sequence comparison, 74 sequences (16 from NB268 and 58 from NB269) were determined to be unique. Finally, 12 VHH sequences with relatively high bacterial culture OD450 values ​​and no obvious hotspot amino acids (extra cystine, N-linked glycosylation sites, etc.) were selected from these 74 unique VHH sequences for cloning, expression, and purification. The sequences of these 12 VHHs are as follows: A10-268-5: Nucleotide sequence (SEQ ID NO: 5) [ka] Amino acid sequence (SEQ ID NO: 1) [ka] Nucleotide sequence (SEQ ID NO: 10) [ka] Amino acid sequence (SEQ ID NO: 6) [ka] Nucleotide sequence (SEQ ID NO: 15) [ka] Amino acid sequence (SEQ ID NO: 11) [ka] Nucleotide sequence (SEQ ID NO: 20) [ka] Amino acid sequence (SEQ ID NO: 16) [ka] Nucleotide sequence (SEQ ID NO: 25) [ka] Amino acid sequence (SEQ ID NO: 21) [ka] Nucleotide sequence (SEQ ID NO: 30) [ka] Amino acid sequence (SEQ ID NO: 26) [ka] Nucleotide sequence (SEQ ID NO: 35) [ka] Amino acid sequence (SEQ ID NO: 31) [ka] Nucleotide sequence (SEQ ID NO: 40) [ka] Amino acid sequence (SEQ ID NO: 36) [ka] Nucleotide sequence (SEQ ID NO: 45) [ka] Amino acid sequence (SEQ ID NO: 41) [ka] Nucleotide sequence (SEQ ID NO: 50) [ka] Amino acid sequence (SEQ ID NO: 46) [ka] Nucleotide sequence (SEQ ID NO: 55) [ka] Amino acid sequence (SEQ ID NO: 51) [ka] Nucleotide sequence (SEQ ID NO: 60) [ka] Amino acid sequence (SEQ ID NO: 56) [ka]

[0096] 11. Cloning, expression, and purification of A10-268 and A10-269 A10 is the target code for the CGRP receptor, and NB268 and NB269 are the numbers of the two llamas. Expression and purification were performed by Biointron, and six histidines (6xHis) were ligated to the C-terminus of the VHH sequence for purification. The synthesized gene fragment was ligated into pcDNA3.4 vector (ThermoFisher Scientific, EL0011) digested with NotI (NEB, R0189L) and XbaI (NEB, R0145L) (Biointron) and recovered. The ligation product was transformed into Top10 competent cells, and positive clones were selected for scale-up cultivation. Small amounts of plasmid were extracted and sequenced using a Biointron-prepared Plasmid Mini Extraction Kit (Biointron). Clones with the correct sequence were seeded in LB medium (Biointron) for scale-up cultivation, and large quantities of the plasmid were extracted using a kit prepared by Biointron (Biointron). They were then sequenced again for verification. The expression plasmids were transfected into 293 cells using a transfection reagent (Biointron). The cell cultures were harvested after 5 days in a constant temperature incubator (Crystal, IS-RDS6C5). The cell cultures were centrifuged, and the supernatant was collected. Cell debris was filtered off, and the clarified liquid was collected. The target protein was captured using a Ni2 affinity chromatography column. The target protein was filtered, and the final yield was determined by measuring A280 and the absorbance at 280 nm was read using a NanoDrop One instrument. The protein was transferred to a dialysis bag and dialyzed against a beaker containing 1X PBS. The purified product was identified by SDS-PAGE electrophoresis. The 12 expressed proteins are listed in Table 2. [Table 2]

[0097] Example 2: Validation of anti-RC-Fc VHH 1. ELISA Validation of Anti-RC-Fc VHH RC-Fc VHH binds to the immunizing antigen The immunizing antigen RC-Fc was diluted to 2000 ng mL in coating solution (50 mM NaHCO3 (pH = 9.6)), and 100 μL was used to coat a 96-well ELISA plate (ThermoFisher Scientific, 449824) and incubated overnight at 4 °C. The ELISA plate was washed five times with PBST overnight at 4 °C, tapped dry, and 200 μL of blocking solution was added, followed by blocking at 37 °C for 2 hours. The PBST was removed, and 100 μL of VHH protein serially diluted in PBST was added to each well and incubated at 37 °C for 1 hour. The ELISA plate incubated with the sample was washed five times with PBST, followed by the addition of 100 μL of the corresponding secondary antibody mouse anti-His-HRP (mouse anti-histidine-peroxidase, Nanjing GenScript, A00186) diluted 1:5000 in blocking solution and incubation at 37 °C for 1 hour.

[0098] The ELISA plate incubated with the secondary antibody was washed five times with PBST, and 100 μL of TMB single-component colorimetric solution (Solarbio, 20190402) was added to each well. The plate was incubated at 37°C for 7 minutes. 100 μL of 1 M HCl (Kelong Chemicals) was added to each well to stop the reaction. Finally, OD450 readings were performed using an ELISA reader. Results were analyzed using a nonlinear regression log(agonist) vs. response (3-parameter) model in GraphPad Prism 9.3.1 software. The resulting binding EC 50 The values ​​are shown in Table 3. [Table 3]

[0099] 2. Cellular Validation of Anti-RC-Fc VHH 2.1 SK-N-MC cell line and culture The primary neuroblastoma cell line SK-N-MC was obtained from ATCC (HTB-10, lot: 70036281). The culture medium was DMEM / F-12 (Gibco, C11330500BT) + 10% FBS, referred to as SK cell culture medium.

[0100] 2.2 cAMP concentration test in SK-N-MC cells The concentration of cAMP produced by SK-N-MC cells was tested using the Cisbio cAMP-GS Dynamic Kit (Cisbio, 62AM4PEB). Unless otherwise specified, the procedure was performed according to the kit's instructions. The test culture medium was DMEM / F1-12 + 10% FBS + 1 mM IBMX (Solarbio, I10010), and the test ELISA plate was a white 384-well plate.

[0101] 2.2.1 Determination of SK-N-MC cell number and obtaining a CGRP standard curve On the day of the experiment, cells were detached from the culture flasks using 0.25% trypsin-EDTA (Gibco, 25200-056), washed once with PBS, counted, and diluted to the appropriate concentrations in test medium: 10,000, 7,500, 5,000, and 2,500 cells were used per test well.

[0102] Five microliters of cell solution diluted with stimulation buffer (stimulation buffer + 500 μM IBMX) and 5 μL of CGRP (Nanjing Yuan-Peptide Biotechnology Co., Ltd., Catalog No.: A03-137, Batch No.: yuanpeptide-997792) were added sequentially to wells of a ProxiPlate-384 Plus test plate (PerkinElmer, 6008280). The final CGRP concentrations ranged from 0 nM or 0.001 to 50 nM.

[0103] 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 stop the reaction. The test plate was left at room temperature for 60 minutes and then read using a fluorescence microplate reader (BMG LabTech, model: PHERAstar FSX) equipped with a 665 / 620 filter. The obtained values ​​were used to calculate ratios in Microsoft Excel using the following formula:

number

[0104] The calculated ratios were analyzed using a nonlinear regression log(agonist) versus response (three-parameter) model in GraphPad Prism 9.3.1 software, and the results are shown in Figure 3 , showing numbers of cells ranging from 2500 to 10000 per well; therefore, 2500 cells per well were selected for subsequent experiments.

[0105] 3. Evaluation of the ability of partial anti-CGRP receptor VHHs to inhibit cellular cAMP production A10-268-5, A10-268-18, A10-268-42, A10-268-76, A10-268-103, and A10-268-273 were used for cell function tests. On the day of the experiment, SK-NM cells were detached from the culture flask with 0.25% trypsin-EDTA, washed once with PBS, counted, and diluted with test medium to a cell solution of 5 million cells / mL and used within 30 minutes.

[0106] Five microliters of the cell solution and 5 microliters of anti-RC-Fc single domain antibody diluted five-fold in the stimulation solution (final concentration: 0 nM or 0.0001 to 100 nM) were added to the wells of the test plate and incubated in a 37°C incubator for 30 minutes.

[0107] Five microliters of CGRP diluted in the stimulus solution (final concentration 5 nM) was added, and the test plate was placed in a 37°C incubator for 15 minutes. The reaction was then stopped by adding 5 μL of cAMP-d2 and 5 μL of anti-cAMP-cryptate. The test plate was then left at room temperature for 60 minutes, after which it was read using a 665 / 620 filter on a fluorescence microplate reader. The resulting values ​​were used to calculate the 665 / 620 ratio, which was then analyzed using a nonlinear regression log(inhibitor) vs. response (3-parameter) model in GraphPad Prism 9.3.1 software. The results are shown in Table 4. The IC50 values ​​in the table show that the four VHHs have the ability to inhibit cAMP production in SK-N-MC cells, and their activity is as follows: A10-268-273>A10-268-42>A10-268-103>A10-268-18. A10-268-42 was selected as the lead VHH for the next evaluation step. [Table 4]

[0108] 4. Humanization, cloning, expression, and purification of A10-268-42 Using the IMGT sorting method, A10-268-42 (42wt) was divided into four framework regions and three CDR regions. The 3D structure of 42wt was predicted using AlphaFold2, and the cardinality of this structure was obtained using a self-developed method. Next, the 42wt sequence was compared with 100 human IGHV3 sequences using the IGBLAST application (https: / / www.nebi.nlm.nih.gov / igblast / ) to identify 14 non-human amino acids in the four framework regions of A10-268-42. These 14 amino acids were divided into 13 groups (42hv1–42hv13), and 1–10 amino acids in each group were converted to human amino acids. The structures of these 13 VHHs with different degrees of humanized amino acid combinations were predicted using AlphaFold2. These 3D structures were compared with the 42wt structure, and the three most similar combinations (42hv10, 42hv11, and 42hv12) were selected. Along with 42wt itself, the four VHHs mentioned above were fused to Fc, in which the 297th amino acid was mutated from N to A. Finally, the functions of these four proteins were compared via cellular cAMP experiments.

[0109] The C-terminus of 42wt, 42hv10, 42hv11, and 42hv12 were linked to the N-terminus of Fc, respectively, to form fusion proteins (42wtFc, 42hv10Fc, 42hv11Fc, and 42hv12Fc), and gene synthesis, expression, and purification were entrusted to Biointron using the same methods as above. Protein electropherograms are shown in Figure 4.

[0110] The sequences of 42hv10, 42hv11, and 42hv12 are as follows: [ka] Fc fragment with amino acid at position 297 mutated from N to A (SEQ ID NO: 68) [ka]

[0111] 5. Verification of the cellular cAMP inhibitory ability of humanized A10-268-42 (42hv10Fc) On the day of the experiment, neuroblastoma cells were detached from the culture flasks with 0.25% trypsin-EDTA, washed once with PBS, counted, and diluted with test medium to a cell solution of 5 million cells / mL and used within 30 minutes.

[0112] Five microliters of cell solution, 5 μL of 42wtFc, 42hv10Fc, 42hv11Fc, and 42hv12Fc proteins serially diluted five-fold in stimulation solution, and the control antibody erenumab (Biointron, A03EB) were added sequentially to wells of a ProxiPlate-384 Plus test plate at concentrations ranging from 0.0003 to 100 nM and incubated at 37°C for 30 minutes.

[0113] 5 μL of CGRP diluted with the stimulation solution (final concentration 5 nM) was added, and the test plate was placed in a 37°C incubator. After 15 minutes of incubation, 5 μL of cAMP-d2 and 5 μL of anti-cAMP-cryptate were added to stop the reaction. The test plate was then left at room temperature for 60 minutes, after which the readings were taken using a 665 / 620 filter on a fluorescence microplate reader. The obtained values ​​were used to calculate the 665 / 620 ratio, which was then analyzed using a nonlinear regression log(inhibitor) vs. response (3-parameter) model in GraphPad Prism 9.3.1 software. The results are shown in Figure 5. As can be seen from Figure 5, the humanization of all three combinations, 42hv10, 42hv11, and 42hv12, was successful, and their ability to inhibit cellular cAMP production was superior to that of the control antibody, erenumab. Based on the above results, the IC was two-fold better than the prototype. 50 42hv10, which has the following structure, was selected for further development.

[0114] Example 3: Synthesis of the extracellular domain of pituitary adenylate cyclase 1 (PAC1 ECD) 1. Gene synthesis, cloning, expression, and purification of PAC1 ECD and PAC1s ECD-Fc fusion proteins The official name of PAC1 is pituitary adenylate cyclase 1, and its Chinese name is pituitary adenylate cyclase 1, and its coding gene is ADCYAP1R1. PAC1 is a membrane protein belonging to the class B G protein-coupled receptor (class B GPCR). It is widely expressed in animals, but is mainly distributed in endocrine organs and the nervous system. PAC1 and its ligand, pituitary adenylate cyclase-activating polypeptide (PACAP, also known as PACAP38), constitute a signaling system that plays an important role in regulating growth, tissue repair, mood, metabolism, neuroprotection, and other functions.

[0115] ADCYAP1R1 has a total of 18 exons, 10 of which (exons 2, 3, 7-13, and 18) are constitutively expressed, while the others are expressed by alternative splicing, resulting in multiple isoforms, collectively referred to as isoforms. The amino-terminal (N-terminal) of the extracellular domain (ECD) of PAC1 is encoded by exons 2-6, the seven transmembrane domains (including the extracellular and intracellular portions) are encoded by exons 7-17, and the untranslated portion of the carboxyl-terminal (C-terminal) is encoded by exon 18.

[0116] In humans, the N-terminal extracellular domain (ECD) of PAC1 contains three isoforms: PAC1, PAC1s, and PAC1vs (Figure 6). When exons 2 to 6 are all expressed, it is PAC1; when exons 5 and 6 are truncated, it is PAC1s; and when exons 4, 5, and 6 are truncated, it is PAC1vs.

[0117] Ligands for PAC1 include PACAP (PACAP38), PACAP27, and VIP (vasoactive intestinal polypeptide) (Figure 7). The affinity of PAC1 and PACAP is in the single nanomolar (nM) range, but its affinity for VIP is 100-1000 times weaker. PAC1s lack the fifth and sixth exons but retain affinity for their ligand PACAP and increased affinity for VIP. The affinity of PAC1s for the ligands PACAP38 and PACAP27 is greatly reduced, but affinity for VIP is retained.

[0118] The PACAP trap is modified from PAC1 ECD. PAC1 ECD is the soluble extracellular domain of PAC1 that is released from the cell membrane. In the present invention, it has been found that this ECD also retains the ability to bind to PACAP in vitro. Furthermore, PAC1 ECD that does not contain exons 5 and 6, i.e., PAC1s ECD, has stronger binding ability to PACAP, and therefore, PAC1s ECD is used to construct the PACAP trap.

[0119] The PAC1 ECD is derived from Genbank ID NM_001199635.2, nucleic acid sequence 506 to 907 (SEQ ID NO: 65), amino acid sequence 24 to 155 (SEQ ID NO: 64). The nucleic acid sequence is: [ka] The amino acid sequence is: [ka]

[0120] The nucleic acid sequence used for cloning the PAC1s ECD was derived from Genbank ID NM_001199637.2 506-844 (SEQ ID NO: 67) and is as follows: [ka]

[0121]

[0039] The amino acid sequence (SEQ ID NO: 66) of PAC1s ECD (or additionally: from UniProt ID P41586-3 21-124), which is: [ka]

[0122] The C-terminus of PAC1 ECD and PAC1s ECD were fused to the N-terminus of Fc, and expressed as PAC1 ECD-Fc and PACs1 ECD-Fc. Gene synthesis and expression were entrusted to Biointron, and the same method as above was used. SDS-PAGE electrophoresis results are shown in Figure 8.

[0123] 2. ELISA evaluation of the ability of PAC1 ECD-Fc and PAC1s ECD-Fc to bind to PACAP After washing a streptavidin-coated 96-well ELISA plate (ThermoFisher Scientific™, 15126) three times with PBST (0.1% Tween 20, pH 7.4), 1000 ng of biotin-PACAP was added to each well and incubated at room temperature for 2 hours. The PBST was removed, and 100 μL of PAC1 ECD-Fe, PAC1s ECD-Fc, or negative control RC-Fc fusion protein serially diluted in PBST was added to each well and incubated at 37°C for 1 hour. The ELISA plate incubated with the samples was washed five times with PBST, and then 100 μL of the corresponding secondary antibody anti-human IgG-HRP (anti-human immunoglobulin G-horseradish peroxidase, Hangzhou HuaBio, HR1214) diluted 1:5000 in blocking solution was added and incubated at 37°C for 1 hour.

[0124] The ELISA plate incubated with the secondary antibody was washed five times with PBST, and 100 μL of TMB (trimethylolpropane) single-component colorimetric solution (Solarbio, 20190402) was added to each well. The plate was incubated at 37°C for 7 minutes. 100 μL of 1 M HCl (Kelong Chemicals) was added to each well to stop the reaction. Finally, OD450 readings were performed using an ELISA reader. The results were analyzed using a nonlinear regression log(agonist) vs. response (3-parameter) model. The results are shown in Figure 9. As can be seen from this figure, the binding affinity of PAC1s ECD-Fc to PACAP was 40-fold higher than that of PAC1 ECD-Fc, while the extracellular domain of the CGRP receptor, RC-Fc, had no binding affinity.

[0125] Example 4: Construction of 42hv10FcNA-PAC1s ECD (BY003) bifunctional fusion protein 1. Cloning, expression, and purification of BY003 BY003 contains a fusion of three molecules: 42hv10, FcNA (in which amino acid 297 of Fc is mutated from N to A), and PAC1s ECD. The C-terminus of 42hv10 is linked to the N-terminus of FcNA, and the C-terminus of FcNA is linked to PAC1s ECD via the linker GGGGS (SEQ ID NO: 79). One, two, or three PAC1s ECDs can be present. In the case of multiple PAC1s ECDs, the PAC1s ECDs are linked to each other via GGGGS. The structure of BY003, which consists of two PAC1s ECDs, is shown in Figure 10, and the operating principle of BY003 is shown in Figure 11.

[0126] Gene synthesis was commissioned to Biointron, using the same method as above. SDS-PAGE electrophoresis results are shown in Figure 12.

[0127] 2. Cellular function of anti-42hv10 in BY003 (inhibition of cAMP production induced by CGRP in CHO-CGRPrC3 cells) In this example, the validated indicator of cellular function is the ability of BY003 to inhibit the production of cAMP.

[0128] 2.1 Establishment and culture of cell lines expressing CGRP receptors A CHO cell pool stably expressing human Ramp1 ECD and CLR ECD (referred to as CHO-hCGRPr or CHO-A10 cells) was obtained from Taizhou Biointron Biotechnology Co., Ltd. (catalog number CHOK1-C21738001). The culture medium was DMEM / F-12 + 10% FBS + 1x penicillin-streptomycin mixture (Solarbio, P1400) + 10 μg / mL puromycin (Solarbio, P8230) + 50 μg / mL hygromycin B (Solarbio, H8080), referred to as CHO-hCGRPr culture medium.

[0129] The CHO-hCGRPr cell pool was diluted to 5 cells / mL with CHO-hCGRPr culture medium. 100 μL of the diluted cell solution was then transferred to two wells of a 96-well cell culture plate (Thermo Scientific, 167008) and cultured in a CO2 incubator until individual cell colonies differentiated. Six colonies were transferred from the 96-well plate to a 6-well cell culture plate (Corning, 3516). After growing to 90% confluence, the cells were transferred to a T-75 (Corning, 430541U) cell culture flask. Once the cells reached full confluence, they could be used for functional testing and cryopreserved in liquid nitrogen.

[0130] The cellular cAMP concentration was tested using the Cisbio cAMP-GS Dynamic Kit (Cisbio, 62AM4PEB). Unless otherwise specified, the procedure was performed according to the kit's instructions. The test culture medium was DMEM / F1-12 + 10% FBS + 1 mM IBMX, and the test ELISA plate was a white 384-well plate.

[0131] 2.2 Obtaining a CGRP standard curve for monoclonal stable cell lines From the six single-cell clones, clones 1, 2, and 3 were selected to evaluate their ability to produce cAMP. The parental CHOK1 cell line (ECACC, 85051005) not transfected with CGRP receptor was added as a control. On the day of the experiment, cells were detached from the culture flask with 0.25% trypsin-EDTA (Gibco, 25200-056), washed once with PBS, counted, and diluted with test medium to a cell solution of 5 million cells / mL and used within 30 minutes.

[0132] Five μL of cell solution diluted with stimulation buffer (stimulation buffer + 500 μM IBMX) and 5 μL of CGRP (Nanjing Yuan-Peptide Biotechnology Co., Ltd., A03-137, batch number: yuanpeptide-997792) were added sequentially to wells of a ProxiPlate-384 Plus test plate. The final concentration of CGRP ranged from 0 nM or 0.000001 to 100 nM, and the CGRP was serially diluted 10-fold.

[0133] 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 stop the reaction. The test plate was left at room temperature for 60 minutes and then read using a fluorescence microplate reader (BMG LabTech, model: PHERAstar FSX) equipped with a 665 / 620 filter. The obtained values ​​were calculated using Microsoft Excel to obtain the 665 / 620 ratio, and finally analyzed using a nonlinear regression log(agonist) vs. response (3-parameter) model in GraphPad Prism 9.3.1 software. The results are shown in Figure 13. Based on the results, cell line 3 was selected for subsequent experiments and designated CHO-CGRPrC3.

[0134] 2.3 Evaluation of the ability of BY003 to inhibit CGPR-induced cAMP production in CHO-CGRPrC3 cells On the day of the experiment, CHO-hCGRPrC3 cells were detached from the culture flask with 0.25% trypsin-EDTA, washed once with PBS, counted, and diluted with test medium to a cell solution of 5 million cells / mL and used within 30 minutes.

[0135] Five microliters of cell solution and 5 μL of BY003 and control antibody erenumab (Biointron, A03EB) serially diluted five-fold in stimulation solution were added sequentially to wells of a ProxiPlate-384 Plus test plate at concentrations ranging from 0.0003 to 100 nM and incubated at 37°C for 30 minutes.

[0136] 5 μL of CGRP diluted in stimulation solution (final concentration 1.4 nM) was added, and the test plate was placed in a 37°C incubator. After 15 minutes of incubation, 5 μL of cAMP-d2 and 5 μL of anti-cAMP-cryptate were added to stop the reaction. The test plate was then left at room temperature for 60 minutes, after which it was read using a 665 / 620 filter on a fluorescence microplate reader. The resulting values ​​were used to calculate the 665 / 620 ratio, which was then analyzed using a nonlinear regression log(inhibitor) vs. response (3-parameter) model in GraphPad Prism 9.3.1 software. The results are shown in Figure 14. As can be seen, BY003's ability to inhibit cAMP production in CHO-hCGRPrC3 cells was 2.5-fold stronger than erenumab.

[0137] 3. Cellular function of anti-42hv10 in BY003 (inhibition of cAMP production induced by CGPR in CHO-hPAC1 cells) 3.1 Establishment and culture of stable cell lines expressing human PAC1 Cell pools stably expressing human PAC1 ECD and PAC1s ECD CHO (referred to as CHO-A05 cells and CHO-A05s cells) were obtained from Taizhou Biointron Biotechnology Co., Ltd. (catalog numbers CHOK1-A05 and CHOK1-A05s). The culture medium was DMEM / F-12 + 10% FBS + 1x penicillin-streptomycin mixture (Solarbio, P1400) + 10 μg / mL puromycin (Solarbio, P8230), referred to as CHO-PAC1 culture medium.

[0138] CHO-A05 and CHO-A05s cell pools were diluted to 5 cells / mL in CHO-PAC1 culture medium. 100 μL of the diluted cell solution was then transferred to the wells of two 96-well cell culture plates (Thermo Scientific, 167008) and cultured in a CO2 incubator until individual cell colonies differentiated. Six colonies were transferred from the 96-well plate to a 6-well cell culture plate (Corning, 3516). After growing to 90% confluence, the cells were transferred to a T-75 (Corning, 430541U) cell culture flask. Once the cells reached full confluence, they could be used for testing and cryopreserved in liquid nitrogen.

[0139] 3.2 Obtaining a cAMP standard curve for the monoclonal CHO-A05 cell line On the day of the experiment, six monoclonal CHO-A05 cells not transfected with PAC1 and the parental CHOK1 cell line (ECACC, 85051005) were isolated from culture flasks with 0.25% trypsin-EDTA, washed once with PBS, counted, and diluted to a cell solution of 5 million cells / mL in test medium and used within 30 minutes.

[0140] Five microliters of cell solution diluted with stimulation buffer (stimulation buffer + IBMX) and 5 μL of PACAP (Nanjing Yuan-Peptide Biotechnology Co., Ltd., A05-138, batch number: Yuanpeptide-194972) were added sequentially to wells of a ProxiPlate-384 Plus test plate. The final concentrations of PACAP ranged from 0.0001 to 100 nM.

[0141] After adding PACAP, 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 stop the reaction. The test plate was left at room temperature for 60 minutes and then read using a 665 / 620 filter on a fluorescence microplate reader (BMG LabTech, model: PHERAstar FSX). The resulting values ​​were calculated using Microsoft Excel to obtain the 665 / 620 ratio, and finally analyzed using a nonlinear regression log(agonist) vs. response (3-parameter) model in GraphPad Prism 9.3.1 software. The results are shown in Figure 15. Based on the results, cell line 4 was selected for subsequent experiments and designated CHO-hPAC1C4.

[0142] 3.3 Evaluation of the ability of anti-BY003 to inhibit cAMP production in CHO-hPAC1C4 cells The cellular cAMP concentration was tested using a cAMP kit from Cisbio. Unless otherwise specified, the procedure was performed according to the kit's instructions. The test culture medium was DMEM / F1-12 + 10% FBS + 1 mM IBMX, and the test ELISA plate was a white 384-well plate.

[0143] Because BY003 is a bifunctional protein that acts on both the CGRP receptor and PACAP, CHO-hCGRPrC3 and CHO-hPAC1C4 cells were added to experimental test plates. One day before the experiment, the two types of cells were detached from the culture flasks with 0.25% trypsin-EDTA, washed once with PBS, counted, and diluted with test culture medium to a cell solution of 4 million cells / mL. After mixing the two cell solutions, 10 μL of the mixture was transferred to a 384-well cell culture plate (Corning, 3765) and placed in a CO2 incubator at 37°C overnight.

[0144] On the day of the experiment, the culture medium was removed, and 5 μL of BY003 single domain antibody serially diluted in stimulation solution (final concentrations were 0 nM or 0.1–1000 nM) was added to the wells of the test plate and incubated for 30 min in a 37°C incubator.

[0145] Five microliters of PACAP diluted in the stimulation solution (final concentration: 0.7 nM) was added. After the addition of PACAP, the test plate was placed in a 37°C incubator and incubated for 15 minutes. The reaction was then stopped by adding 5 μL of cAMP-d2 and 5 μL of anti-cAMP-cryptate. The test plate was left at room temperature for 60 minutes and then read using a fluorescence microplate reader (BMG LabTech, model: PHERAstar FSX) equipped with a 665 / 620 filter. The resulting values ​​were calculated using Microsoft Excel to obtain the 665 / 620 ratio, which was then analyzed using a nonlinear regression log(agonist) vs. response (3-parameter) model in GraphPad Prism 9.3.1 software. The results are shown in Figure 16.

[0146] In response to unmet clinical needs in the current field of migraine treatment and prevention, the present invention provides a fusion protein that simultaneously blocks the CGRP and PACAP pathways. By using a method of fusing a single-domain VHH to the CGRP receptor with a natural PAC1 soluble EDC, the CGRP and PACAP signaling pathways, both of which are closely related to migraine, are simultaneously blocked. The present inventors have successfully constructed the BY003 molecule and demonstrated that BY003 can block cAMP production induced by CGRP and PACAP at the cellular level.

Claims

1. 1. A single domain antibody for blocking the calcitonin gene-related peptide (CGRP) pathway, the single domain antibody comprising a CDR1 set forth in the amino acid sequence of SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, or 57, or any variant thereof; a CDR2 set forth in the amino acid sequence of SEQ ID NO: 3, 8, 13, 18, 23, 28, 33, 38, 43, 48, 53, or 58, or any variant thereof; and a CDR3 set forth in the amino acid sequence of SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, or 59, or any variant thereof; Preferably, the single domain antibody comprises a CDR1 as set forth in the amino acid sequence of SEQ ID NO: 12 or any variant thereof, a CDR2 as set forth in the amino acid sequence of SEQ ID NO: 13 or any variant thereof, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 14 or any variant thereof.

2. the single domain antibody comprises a variable region as set forth in the amino acid sequence of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 62, or 63, or any variant thereof; Preferably, the single domain antibody comprises a variable region as set forth in the amino acid sequence of SEQ ID NO: 61, 62, or 63, or any variant thereof; More preferably, said single domain antibody comprises a variable region as set forth in the amino acid sequence of SEQ ID NO: 61 or any variant thereof, 2. The single domain antibody of claim 1, wherein the amino acids of said single domain antibody preferably have conservative substitutions, and the number of conservatively substituted amino acids does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

3. A fusion protein that simultaneously blocks the calcitonin gene-related peptide (CGRP) pathway and the pituitary adenylate cyclase-activating peptide (PACAP) pathway, the fusion protein comprising at least one single domain antibody, at least one Fc fragment, and at least one extracellular domain fragment of pituitary adenylate cyclase 1 (PAC1 ECD).

4. the fusion protein comprises one peptide chain, and the structure of the peptide chain is such that one PAC1 ECD is linked to the C-terminus of an Fc fragment via a linker, and the N-terminus of the Fc fragment is fused to the C-terminus of a single-domain antibody; Optionally, the fusion protein comprises two peptide chains, the structure of the peptide chains being such that one PAC1 ECD is linked to the C-terminus of an Fc fragment via a linker, the N-terminus of the Fc fragment is fused to the C-terminus of a single domain antibody, and the two PAC1 ECDs of the two peptide chains are linked to each other via a linker; Optionally, the fusion protein comprises three peptide chains, the structure of which is such that one PAC1 ECD is linked to the C-terminus of an Fc fragment via a linker, the N-terminus of the Fc fragment is fused to the C-terminus of a single-domain antibody, and the three PAC1 ECDs of the three peptide chains are linked via linkers.

5. the single domain antibody is a single domain antibody that binds to a CGRP receptor; Preferably, said single domain antibody comprises a CDR1 as set forth in the amino acid sequence of SEQ ID NO: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, or 57 or any variant thereof, a CDR2 as set forth in the amino acid sequence of SEQ ID NO: 3, 8, 13, 18, 23, 28, 33, 38, 43, 48, 53, or 58 or any variant thereof, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, or 59 or any variant thereof, More preferably, said single domain antibody comprises a CDR1 as set forth in the amino acid sequence of SEQ ID NO: 12 or any variant thereof, a CDR2 as set forth in the amino acid sequence of SEQ ID NO: 13 or any variant thereof, and a CDR3 as set forth in the amino acid sequence of SEQ ID NO: 14 or any variant thereof, 5. The fusion protein of claim 3 or 4, wherein preferably, one or more or all three of the CDRs have conservative substitutions, and the number of conservatively substituted amino acids does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

6. the single domain antibody comprises a variable region as set forth in the amino acid sequence of SEQ ID NO: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 62, or 63, or any variant thereof; Preferably, the single domain antibody comprises a variable region as set forth in the amino acid sequence of SEQ ID NO: 61, 62, or 63, or any variant thereof; More preferably, said single domain antibody comprises a variable region as set forth in the amino acid sequence of SEQ ID NO: 61 or any variant thereof, 6. The fusion protein of any one of claims 3 to 5, wherein the amino acids of said single domain antibody have conservative substitutions, and the number of conservatively substituted amino acids does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

7. the PAC1 ECD is selected from the ECDs of the PAC isoforms: PAC1, PAC1s, or PAC1vs; Preferably, the PAC1 ECD is a PAC1s ECD; Preferably, the PAC1 ECD comprises the amino acid sequence of SEQ ID NO: 64, and amino acid sequences having 80% or greater identity thereto, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity thereto, more preferably 98% or 99% or greater identity thereto; The fusion protein of any one of claims 3 to 6, wherein the PAC1 ECD preferably comprises the amino acid sequence of SEQ ID NO: 66, and an amino acid sequence having 80% or more identity thereto, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and more preferably an amino acid sequence having 98% or 99% or more identity thereto.

8. the Fc fragment comprises a hinge region and constant region 2 (CH2) and constant region 3 (CH3) domains derived from a human immunoglobulin; Preferably, the Fc fragment is selected from human IgG1, IgG2, IgG3, and IgG4, more preferably, the Fc fragment is selected to be human IgG1; Preferably, the Fc fragment comprises an amino acid substitution, more preferably, the amino acid substitution comprises a substitution of asparagine N at position 297 of the Fc fragment with alanine A, and each amino acid position of the Fc fragment is numbered using the EU antibody numbering system; More preferably, the Fc fragment comprises the amino acid sequence of SEQ ID NO: 68, and an amino acid sequence having 80% or more identity thereto, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity thereto, more preferably an amino acid sequence having 98% or 99% or more identity thereto.

9. the linker is a flexible polypeptide linker; Preferably, said flexible polypeptide linker is selected from GGGGS (SEQ ID NO:79), GGGGSGGGGS (SEQ ID NO:80), SGGGGSGGGGG (SEQ ID NO:81), GGGGGSGGGGGSSGGGGGS (SEQ ID NO:82), GGGGSGGGGSGGGGGS (SEQ ID NO:83), GGGGSGGGGSGGGG (SEQ ID NO:84), GGGGSGGGGSGGGGGSGGGGGS (SEQ ID NO:85), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:86), GSGSGSGS (SEQ ID NO:87), GGSGSGSG (SEQ ID NO:88), GGSGSG (SEQ ID NO:89), and GGSG (SEQ ID NO:90); More preferably, the fusion protein according to any one of claims 3 to 8, wherein the flexible polypeptide linker is GGGGS (SEQ ID NO: 79).

10. A nucleic acid encoding a single domain antibody according to claim 1 or 2 and a fusion protein according to any one of claims 3 to 9.

11. A vector comprising the nucleic acid of claim 10.

12. A cell comprising the nucleic acid of claim 10 or the vector of claim 11.

13. A pharmaceutical composition or kit comprising a single domain antibody according to claim 1 or 2 and / or a fusion protein according to any one of claims 3 to 9, and a pharmaceutically acceptable vector, diluent or excipient.

14. 14. Use of a single domain antibody according to claim 1 or 2, a fusion protein according to any one of claims 3 to 9, a nucleic acid according to claim 10, a vector according to claim 11, and / or a cell according to claim 12 and / or a pharmaceutical composition or kit according to claim 13 in the preparation of a medicament for treating a disease associated with activation of the CGRP and PACAP pathway, Preferably, the disease is migraine.

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