Fusion protein

Abstract

A fusion protein is provided that blocks both a calcitonin gene related peptide (CGRP) pathway and a pituitary adenylate cyclase activated peptide (PACAP) pathway, where 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 can block CGRP and PACAP pathways at the same time, and the response rate of drugs for treating migraine is improved.

Description

A fusion protein Technical Field

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

[0002] Migraine is a neurovascular disease that affects 1.1 billion people worldwide, and the number is growing. The current global prevalence is as high as 12-15%, with a severe, chronic incidence of 1.4-2.2%. It is one of the ten leading causes of disability worldwide. The peak age of migraine onset is 37 years old, with women three times more likely than men. Attacks last from three to 72 hours and, in addition to unilateral headache, are accompanied by symptoms such as photophobia, phonophobia, nausea, and vomiting. Chronic migraine sufferers often suffer from cardiovascular disease, mental disorders, depression, and sleep difficulties. 90% of patients experience disruption to their work, study, and social life; 30% of women experience migraines; and 10% of children experience migraines. Currently, the diagnosis and treatment of this disease are problematic, with only 5% of patients receiving appropriate diagnosis and treatment. Treatment options include CGRP-targeted drugs, 5-HTr agonists, calcium channel blockers, antiepileptic drugs, antidepressants, NSAIDs, nerve blockers / stimulators, and other nonspecific medications.

[0003] The cause of migraine is unclear, hence the term "primary headache." It's generally believed that certain central nervous system stimuli, such as CSD (cortical spreading depression), trigger 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 Aδ fibers, triggering migraines directly or indirectly.

[0004] Currently, at least two major signaling pathways involved in migraine are known: CGRP and PACAP. Both CGRP and PACAP are neurotransmitters distributed throughout the nervous system and other tissues throughout the body. Their receptors are class B GPCR neurotransmitters. They specifically bind to their receptors, increasing intracellular cAMP (cyclic adenosine monophosphate) concentrations and achieving signaling.

[0005] Many new drugs are being developed to block signaling in these two pathways. Antagonists of CGRP and its receptors have been successfully used to treat and prevent migraines. Currently, four monoclonal antibodies are available in the US and Europe: Eptinezumab (Lunbeck), Erenumab (Amgen), Fremanezumab (Teva), and Galcanezumab (Eli Lilly). Small molecules targeting CGRP receptors are also available in the US and Europe: Atogepant (AbbVie), Ubrogepent (AbbVie), and Rimegepent (Pfizer). In contrast, antagonists of PACAP and PAC1 are still under development. Clinical studies have shown that administering PACAP to healthy individuals can induce migraine-like headaches. Elevated levels of PACAP have also been found in the blood of chronic migraine patients compared to healthy controls. These studies demonstrate a close relationship between PACAP and migraine.

[0006] According to the latest statistics from the New England Journal of Medicine, the response rates of major migraine treatments are very low. Response rates for preventive CGRP monoclonal antibodies are only 20-24%, while small-molecule CGRP receptor antagonists are only around 10-14%. One reason for this low response rate is that migraines involve multiple signaling pathways. Therefore, the development of dual- or multifunctional drugs is needed to maximize therapeutic efficacy and provide effective treatment options for the vast majority of migraine patients, especially those with intractable migraines.

[0007] Summary of the Invention

[0008] The present invention provides a fusion protein that blocks CGRP and PACAP pathways simultaneously. The fusion protein can improve the response rate of drugs for treating migraine.

[0009] In one aspect, the present invention provides a single domain antibody that blocks the calcitonin gene-related peptide (CGRP) pathway, wherein the single domain antibody comprises a CDR1 as shown 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 shown 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 shown 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.

[0010] On the other hand, 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, wherein 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).

[0011] The fusion protein comprises a peptide chain having a structure in which a PAC1 ECD is connected 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;

[0012] Optionally, the fusion protein comprises two peptide chains, wherein the structure of the peptide chains is that a PAC1 ECD is connected 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 connected by a linker;

[0013] Optionally, the fusion protein comprises three peptide chains, the structure of the peptide chains being that a PAC1 ECD is connected 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 connected via a linker.

[0014] In another aspect, the present invention provides nucleic acids encoding the above-mentioned single-domain antibodies and fusion proteins.

[0015] In another aspect, the present invention provides a vector comprising the above nucleic acid.

[0016] In another aspect, the present invention provides a cell comprising the above nucleic acid or vector.

[0017] In another aspect, the present invention provides a pharmaceutical composition or kit comprising the above-mentioned single domain antibody, fusion protein and a pharmaceutically acceptable carrier, diluent or excipient.

[0018] On the other hand, the present invention provides a 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 method for treating diseases related to CGRP and PACAP pathway activation.

[0019] 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 a large number of migraine patients, especially those with refractory migraine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Other features, objects and advantages of the present invention will become more apparent from the detailed description of the non-limiting embodiments made with reference to the following drawings. The drawings are only for the purpose of illustrating specific embodiments and are not to be considered as limiting the present invention. In the drawings:

[0021] FIG1 shows the electrophoresis results of RC-Fc.

[0022] FIG2 shows that the reconstructed CGRP receptor extracellular domain of the present invention retains the ability to bind to its ligand CGRP, but does not bind to the unrelated polypeptide PACAP.

[0023] FIG3 shows CGRP-induced cAMP production under different SK-N-MC cell numbers.

[0024] FIG4 shows the electrophoresis results of 42wtFc, 42hv10Fc, 42hv11Fc, and 42hv12Fc.

[0025] FIG5 shows that VHH No. 42 retains the ability to inhibit cellular cAMP production after humanization and Fc fusion.

[0026] Figure 6 shows the gene structure of human PAC1, wherein vertical bars represent exons.

[0027] FIG7 shows three ligands of PAC1, including PACAP (PACAP38), PACAP27 and VIP.

[0028] FIG8 shows the electrophoresis results of PAC1 ECD-Fc and PACs1 ECD-Fc.

[0029] FIG9 shows a comparison of the ability of PAC1 ECD-Fc and PAC1s ECD-Fc to bind to PACAP in vitro.

[0030] FIG10 shows the structure of BY003.

[0031] Figure 11 shows the working principle of BY003.

[0032] Figure 12 shows the electrophoresis results of BY003, where from left to right are the electrophoresis results of 42hv10FcNA-1×PAC1s ECD, 42hv10FcNA-2×PAC1s ECD, and 42hv10FcNA-3×PAC1s ECD, corresponding to the cases of one, two, and three PAC1s ECDs, respectively.

[0033] FIG13 shows the CGRP standard curves of three CGRPr stable cell lines.

[0034] FIG. 14 shows that BY003 inhibits CGRP-induced cAMP production in CHO-hCGRPrC3 cells.

[0035] FIG15 shows PACAP standard curves of six PAC1 stable cell lines.

[0036] FIG. 16 shows that BY003 blocks PACAP signaling at the cellular level. DETAILED DESCRIPTION

[0037] I. Definition

[0038] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are standard procedures. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0039] 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 non-naturally occurring single amino acid sequence, for example, by chemical bonding or recombinant means. A fused amino acid sequence can be produced by the recombination of two genes encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombined polynucleotide into a host cell.

[0040] 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 multiple polymers of amino acid residues. This encompasses 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 is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Polypeptides can be monomeric or multimeric.

[0041] With respect to antibody chain polypeptide sequences, the phrase "substantially identical" is understood to mean antibody chains that exhibit 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 phrase is understood to mean nucleotide sequences that exhibit 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.

[0042] Sequence "identity" or "identity" has an art-recognized meaning, 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, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0043] The term "antibody," also referred to in the art as "immunoglobulin" (Ig), refers to a protein constructed from paired 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 folds into many different globular domains connected by more linear peptide sequences. For example, an immunoglobulin light chain folds into a variable (VL) and a constant (CL) domain, while a heavy chain folds into a variable (VH) and three constant (CH1, 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).

[0044] The term "single-domain antibody" (sdAb) refers to an antibody that contains only a single antibody variable region (VH / VL). Like conventional antibodies, single-domain antibodies can selectively bind to specific antigens. The molecular weight of a single-domain antibody is only 12-15kDa, which is much smaller than an ordinary antibody composed of two heavy chains and two light chains (150-160kDa). The term "heavy chain antibody", "heavy chain single-domain antibody" or "nano single-chain antibody" contains a single variable domain VHH (variable domain of heavy chain of heavy-chainantibody) and two constant domains (CH2 and CH3). Importantly, the cloned and isolated VHH domain is a completely stable polypeptide with the full antigen binding ability of the original heavy chain antibody. This type of antibody is naturally lacking in light chains compared to other antibodies. This type of single-domain antibody was discovered in camelids. Single domain antibodies have the advantages of stable structure, small molecules, good solubility, tolerance to various adverse environments, and easy humanization, and have been widely used in miniaturized genetic engineering antibody research, new drug development, and the diagnosis and treatment of diseases. The example of single domain antibodies includes single domain antibodies derived from camelids (alpaca and camel) and cartilaginous fish (e.g., nurse shark) and single domain antibodies derived from recombinant methods from human and mouse antibodies. As used herein, the term "single domain antibody" includes those sdAbs separated directly from VH, VHH, VL or VNAR libraries of any source by phage display or other technologies, sdAbs derived from aforementioned sdAbs, sdAbs produced by recombinant production, and those sdAbs produced by further modification of such sdAbs (by humanization, affinity maturation, stabilization, solubilization, camelization (camelization) or other methods of antibody engineering). The disclosure also relates to homologues, derivatives, or fragments retaining the antigen-binding function and specificity of sdAbs.

[0045] 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 is composed 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, i.e., peptides selected from GGGGS, GGGGSGGGGS, SGGGGSGGGG, GGGGGSGGGGSSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GSGSGSGSGS, GGSGSGSG, GGSGSG.

[0046] The term "CDR" refers to the complementarity-determining region, and each heavy chain and light chain of a known antibody molecule has three CDRs. CDRs are also called hypervariable regions and are present in the variable regions of each heavy chain and light chain of an antibody, with very high variability sites in the primary structure of the CDRs. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 at the amino terminus of the amino terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 at the amino terminus 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 to which the antibody binds.

[0047] "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 non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen. The antigen may be an isolated antigen or present on a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present in an amount of about 1 x 10 7 M-1 or 1×10 8 The affinity constant may be determined by standard kinetic methods for 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. Patent No. 7,229,619 for exemplary SPR and ITC methods for calculating the 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).

[0048] 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 indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity of a 1:1 interaction between members of a reactive binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods for measuring binding affinity are known in the art.

[0049] The terms "inhibit" or "neutralize" as used with respect to a biological activity of a fusion protein of the present disclosure refer to the ability of a polypeptide to substantially antagonize, inhibit, prevent, limit, slow, destroy, eliminate, stop, reduce or reverse, for example, the progression or severity of that which is inhibited (including but not limited to, a biological activity).

[0050] 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. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0051] The term "vector" includes vectors capable of expressing DNA, and the DNA is operably connected to a regulatory sequence that can influence the expression of this type of DNA fragment, such as a promoter region. This type of extra fragment can include promoter and terminator sequences, and optionally can include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors generally derive from plasmids or viral DNA, or can comprise both elements. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate host cells, cause the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, and are included in reproducible expression vectors in eukaryotic cells and / or prokaryotic cells and keep free expression vectors or are integrated into the host cell genome.

[0052] The term "pharmaceutical composition" refers to a preparation that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.

[0053] The term "pharmaceutically acceptable carrier" refers to one or more non-toxic materials that do not interfere with the biological activity of the active ingredient when administered with 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. Pharmaceutical carriers suitable for use in the present disclosure can be conventional pharmaceutical formulation excipients; as well as compositions and formulations suitable for delivering the disclosed neutralizing antibodies. In general, the nature of the carrier depends on the specific mode of administration used. For example, parenteral formulations typically include injectable liquids that include pharmaceutically and physiologically acceptable liquids such as water, saline, balanced salt solutions, dextrose water, glycerol, etc. as vehicles. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the administered pharmaceutical compositions may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate.

[0054] The term "treating" refers to treating a subject suffering from a disease or condition, meaning that the subject's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing worsening of symptoms or progression of the disease. Treatment also includes any pharmaceutical use of any of the antibodies or antigen-binding fragments thereof, and compositions provided herein.

[0055] Also provided are "conservative sequence modifications" of the sequences described in the sequence listings described herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listings described herein by standard techniques known in the art (e.g., 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 with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, a predicted nonessential 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 (e.g., see 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 made 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 variants have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the parent antibody over the region of the antibody sequence of interest. Exemplary substitutions are shown in Table 1 below:

[0056] Table 1 Conservative amino acid substitutions

[0057] II. Detailed description of specific implementation plan

[0058] In one aspect, the present invention provides a single domain antibody that blocks the calcitonin gene-related peptide (CGRP) pathway, wherein the single domain antibody comprises a CDR1 as shown 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 shown 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 shown 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.

[0059] In some preferred embodiments, the above-mentioned single-domain antibody comprises CDR1 as shown in the amino acid sequence of SEQ ID NO: 12 or any variant thereof, CDR2 as shown in the amino acid sequence of SEQ ID NO: 13 or any variant thereof, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 14 or any variant thereof.

[0060] In some embodiments, the single-domain antibody comprises a variable region as shown 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.

[0061] In some preferred embodiments, the single-domain antibody comprises a variable region as shown in the amino acid sequence of SEQ ID NO: 61, 62 or 63 or any variant thereof.

[0062] In some preferred embodiments, the single-domain antibody comprises a variable region as shown in the amino acid sequence of SEQ ID NO: 61 or any variant thereof.

[0063] In some preferred embodiments, the amino acids in the above-mentioned 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.

[0064] 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, wherein 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).

[0065] In some embodiments, the fusion protein comprises a peptide chain having a structure in which a PAC1 ECD is connected 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.

[0066] In some embodiments, the fusion protein comprises two peptide chains, the structure of the peptide chains being that a PAC1 ECD is connected 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 connected via a linker.

[0067] In some embodiments, the fusion protein comprises three peptide chains, the structure of the peptide chains being that a PAC1 ECD is connected 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 connected via a linker.

[0068] In some embodiments, the single-domain antibody is a single-domain antibody that binds to the CGRP receptor.

[0069] In some embodiments, the single-domain antibody comprises a CDR1 as shown 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 shown 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 shown 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.

[0070] In some embodiments, the single-domain antibody comprises CDR1 as shown in the amino acid sequence of SEQ ID NO: 12 or any variant thereof, CDR2 as shown in the amino acid sequence of SEQ ID NO: 13 or any variant thereof, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 14 or any variant thereof.

[0071] 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.

[0072] In some embodiments, the single-domain antibody comprises a variable region as shown 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.

[0073] In some preferred embodiments, the single-domain antibody comprises a variable region as shown in the amino acid sequence of SEQ ID NO: 61, 62 or 63 or any variant thereof.

[0074] In some more preferred embodiments, the single-domain antibody comprises a variable region as shown in the amino acid sequence of SEQ ID NO: 61 or any variant thereof.

[0075] In some preferred embodiments, the amino acids in the above-mentioned 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.

[0076] In some embodiments, the PAC1 ECD is selected from the ECD of the PAC isomers: PAC1, PAC1s, or PAC1vs.

[0077] In some preferred embodiments, the PAC1 ECD is PAC1s ECD.

[0078] In some preferred embodiments, the PAC1 ECD comprises the amino acid sequence of SEQ ID NO: 64 and an amino acid sequence having 80% or greater identity thereto, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity thereto, and more preferably an amino acid sequence having 98% or greater identity thereto.

[0079] In some preferred embodiments, the PAC1s ECD 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.

[0080] 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.

[0081] In some preferred embodiments, the Fc fragment is selected from human IgG1, IgG2, IgG3 and IgG4; more preferably, the Fc fragment is human IgG1.

[0082] In some preferred embodiments, the Fc fragment has an amino acid substitution; more preferably, the amino acid substitution includes substitution of asparagine N at position 297 on the Fc fragment with alanine A, wherein each amino acid site on the Fc fragment is numbered using the EU antibody numbering system.

[0083] In some preferred embodiments, the above-mentioned 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, and more preferably an amino acid sequence having 98% or 99% or more identity thereto.

[0084] In some embodiments, the linker is a flexible polypeptide linker.

[0085] In some preferred embodiments, the above-mentioned flexible polypeptide linker is selected from GGGGS, GGGGSGGGGS, SGGGGSGGGG, GGGGGSGGGGSSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, GSGSGSGSGS, GGSGSGSG, GGSGSG, and GGSG.

[0086] In some more preferred embodiments, the flexible polypeptide linker is GGGGS (SEQ ID NO: 79).

[0087] In another aspect, the present invention provides a nucleic acid encoding the above fusion protein.

[0088] In another aspect, the present invention provides a vector comprising the above nucleic acid.

[0089] In another aspect, the present invention provides a cell comprising the above nucleic acid or vector.

[0090] In another aspect, the present invention provides a pharmaceutical composition or kit comprising the above-mentioned fusion protein and a pharmaceutically acceptable carrier, diluent or excipient.

[0091] In another aspect, the present invention provides use of the above-mentioned fusion protein, nucleic acid, vector and / or cell and / or pharmaceutical composition or kit in the preparation of a pharmaceutical composition for treating diseases associated with activation of the CGRP and PACAP pathways.

[0092] In some preferred embodiments, the above disease is migraine.

[0093] The above description is merely a preferred embodiment of the present invention and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of disclosure involved in the present invention is not limited to the technical solutions formed by the specific combination of the above-mentioned technical features, but also includes other technical solutions formed by any combination of the above-mentioned technical features or their equivalents without departing from the above-mentioned disclosed concepts. For example, a technical solution formed by replacing the above-mentioned features with (but not limited to) technical features with similar functions disclosed in the present invention.

[0094] Example

[0095] Example 1: Synthesis of single domain antibodies against CGRP receptor

[0096] By immunizing llamas with the ECD of the CGRP receptor, the optimal sequence 42hv10 of the anti-CGRP receptor single domain antibody was obtained after multiple rounds of screening and humanization.

[0097] To obtain highly selective and high-affinity binding antibodies, a protein fused to the crystallizable fragment (Fc) of human immunoglobulin G1 (IgG1) expressed and purified in vitro (RC-Fc) was used as an immunogen to immunize llamas. Lymphocytes were then isolated from high-titer llama blood, and their messenger RNA (mRNA) was extracted. This mRNA was used to construct a phage library, and then an enzyme-linked immunosorbent assay (ELISA) was used to screen for colonies containing single-domain antibodies that bind to RC-Fc and those that bind only to the Fc. These colonies included all single-domain antibodies that bind to both the CGRP ECD and Fc. Colonies that only bind to the Fc were excluded, and the remaining CGRP ECD-positive and Fc-negative colonies were sent for sequencing. Through sequence analysis and verification, a group of single-domain antibodies that specifically bind to the CGRP ECD were obtained.

[0098] 1. Preparation of RC-Fc

[0099] 1.1 Design of RC-Fc

[0100] RC-Fc is a fusion of the CGRP receptor: human Ramp1 ECD, CLR ECD, and Fc. The Ramp1 ECD is derived from Genbank ID NM_005855.4, nucleotide sequence 132-404 (SEQ ID NO: 69). The nucleotide sequence is as follows:

[0101] The corresponding amino acid sequence is from NP_005846.1 27-117 (SEQ ID NO: 70). The amino acid sequence is as follows:

[0102] The CLR ECD is derived from Genbank ID NM_001271751.2 nucleotide sequence 407-782 (SEQ ID NO: 71). The nucleotide sequence is as follows:

[0103] The corresponding amino acid sequence is from amino acid sequence 23-146 of NP_001258680.1 (SEQ ID NO: 72). The amino acid sequence is as follows:

[0104] The Fc portion is derived from Genbank ID JX292763.1 nucleic acid sequence 661-1359 (SEQ ID NO: 73). The nucleic acid sequence used for cloning is as follows:

[0105] The corresponding amino acid sequence Genbank ID AFR78282.1 221-452 (SEQ ID NO: 74) is as follows:

[0106] Alternatively, the Fc portion is derived from Genbank ID MW176619.1 nucleic acid sequence 663-1358 (SEQ ID NO: 91). The nucleic acid sequence used for cloning is as follows:

[0107] The corresponding amino acid sequence Genbank ID QRG33954 221-452 (SEQ ID NO: 92) is as follows:

[0108] Because the amino acid series of Ramp1 ECD and CLR ECD are different, they need to be paired through Knob in Hole (KiH) technology when fused with Fc. The C-terminus of Ramp1 ECD is connected to the Fc N-terminus with T366W mutation to form the R chain; the C-terminus of CLR ECD is fused to the Fc N-terminus with T366S, L368A and Y407V mutations to form the C chain.

[0109] The amino acid sequence of the RC-Fc Ramp1 ECD (RC-Fc R) chain (SEQ ID NO: 75) is as follows:

[0110] The Fc fused to the RC-Fc R chain has a T366W mutation, as shown below (SEQ ID NO: 76):

[0111] The amino acid sequence of the RC-Fc CLR ECD (RC-Fc C) chain (SEQ ID NO: 77) is as follows:

[0112] The Fc fused to the RC-Fc C chain has T366S, L368A, and Y407V mutations, as shown below (SEQ ID NO: 78):

[0113] As shown:

[0114] 1.2 RC-Fc gene synthesis, expression, and purification

[0115] The RC-Fc plasmid was synthesized by Taizhou Biotechnology Co., Ltd. (Baiying Bio) using the following method: the synthesized gene fragment was ligated with the pcDNA3.4 vector (ThermoFisher Scientific, EL0011) after digestion with NotI (New England BioLabs Inc., NEB, R0189L) / XbaI (New England BioLabs Inc., NEB, R0145L) (Baiying Bio). The ligation product was transformed into Top10 competent cells, and positive clones were selected for expansion and sequencing using a plasmid miniprep kit (Baiying Bio). Correctly sequenced clones were inoculated into LB medium (Baiying Bio) for expansion, and the plasmid was extracted in large quantities using a kit (Baiying Bio) and sequenced again.

[0116] The expression plasmid was transfected into 293 cells using a transfection reagent (Bai Ying homemade). After culturing for 5 days in a constant temperature incubator (Jingqi, IS-RDS6C5), the cell culture was harvested. The cell culture was centrifuged and the supernatant was taken. The cell debris was filtered to remove the cell debris and the clear 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 the A280 method. The absorbance value at 280 nm was read using a NanoDrop One (ThermoFisher Scientific, ND-ONE-W) instrument. The protein was aspirated into a dialysis bag and dialyzed in a beaker containing 1XPBS. The purified product was identified by electrophoresis. The electrophoresis results are shown in Figure 1.

[0117] 2. Verification of RC-Fc Binding to CGRP

[0118] The expressed and purified RC-Fc fusion protein was used for animal immunization only after being verified to be able to bind to its ligand CGRP. The verification method was enzyme-linked immunosorbent assay (ELISA) developed by Chengdu Apec Biotechnology Co., Ltd., as shown below.

[0119] Streptavidin-coated 96-well ELISA plate (ThermoFisher Scientific TM After washing three times with PBST (1xPBS (Absin, abs9266), 0.1% Tween 20 (Solarbio, T8220), pH = 7.4), 1000 ng of Biotin-CGRP (Nanjing Source Peptide Biotechnology Co., Ltd., A03Biotin137, P200629-HS319172) was added to each well and incubated at room temperature for 2 hours. 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 plate incubated with 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 Huaan Biotechnology, HR1214) diluted 1:5000 in blocking buffer (PBST + 3% skim milk powder (Solarbio, D8340)) was added and incubated at 37°C for 1 hour.

[0120] 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 reaction was incubated at 37°C for 7 minutes. 100 μL of 1M HCL (Cologne Chemical) was added to each well to terminate the reaction. Finally, the OD value was read at a wavelength of 450 nm using a microplate reader (ThermoFisher Scientific, 51119080ET). The OD450 value was analyzed using the 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 the negative control peptide PACAP was 0.069, and the Kd was invalid. The results are shown in Figure 2, showing that the extracellular domain of the CGRP receptor reconstructed in this example retains the ability to bind to its ligand CGRP, but does not bind to the unrelated peptide PACAP.

[0121] 3. Immunization of Llamas with RC-Fc

[0122] Immunization of llamas and VHH screening were performed by Chengdu Apac Biotechnology Co., Ltd. (Aapac Biotechnology). Before immunization, 10 mL of blood was collected as a negative serum control. 0.5 mg of RC-Fc was then mixed with 1 mL of complete Freund's Adjuvant (CFA, Sigma, F5881) and injected subcutaneously. On day 21, 0.25 mg of the 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 collected for serum separation. On day 42, 0.25 mg of the antigen was mixed with 1 mL of IFA and injected subcutaneously. On day 49, 50 mL of peripheral blood was collected for lymphocyte and serum separation. On day 63, 0.25 mg of the antigen was mixed with 1 mL of IFA and injected subcutaneously. On day 70, 50 mL of peripheral blood was collected for lymphocyte and serum separation.

[0123] 4. Gradient Detection

[0124] Dilute RC-Fc to 2000 ng / mL in coating solution (50 mM NaHCO3, pH 9.6) and add 100 μL to each well of a 96-well ELISA plate. Incubate overnight at 4°C. Wash the plate five times with PBST, pat dry, and add 300 μL of blocking solution to each well.

[0125] Serum was serially diluted 1:2 in TBST, with the first serum dilution at 1:2000 and the final serum dilution at 1:128,000. After washing the plate three times with 300 μL / well PBST, 100 μL of diluted serum was added to each well and incubated at 37°C for 1 hour. The plate was then washed three times with 300 μL / well PBST. 100 μL of 1:2000 anti-M13 antibody (NB Biolab, 052-101-005) was added to each well and incubated at 37°C for 1 hour. The plate was washed six times with 300 μL / well PBST, and a 1:10,000 dilution of anti-Alpaca-HRP (NEB, S001H) was added and incubated at 37°C for 30 minutes. The plate, which had been incubated with the secondary antibody, was washed five times with PBST. 100 μL of TMB One-Component Colorimetric Buffer was added to each well and incubated at 37°C for 7 minutes. The reaction was terminated with 100 μL of 1 M HCl per well, and the OD450 reading was measured.

[0126] 5. Construction of phage library

[0127] Select high gradient peripheral blood and separate PBMC according to the instructions of lymphocyte separation medium (Tianjin Haoyang Biological, LTS1077). Total RNA was extracted with RNAiso Plus (Fuji Biological, RE-03111) reagent and PrimeScript TM5 μg of RNA was transcribed into cDNA using the II 1st Strand cDNA Synthesis Kit (Takara, 9109). The cDNA stock solution was diluted 5-fold for nested PCR amplification. A single-domain antibody (VHH) fragment of approximately 750 base pairs (bp) was recovered from the gel and subjected to a second round of PCR amplification. The purified PCR product was the target VHH fragment. The target VHH fragment was ligated to the vector pComb3XSS (NEB, P001) via the Sfil restriction enzyme site. Immediately after electroporation, 1 mL of 2YT (preheated culture medium containing tryptone (Solarbio, T8490), yeast extract (Solarbio, Y8020), and sodium chloride (Cologne Chemicals) at 37°C) was added to the cuvette for revival. The electroporation product was aspirated and the cuvette was washed with 2YT medium. A total of 100 mL of the revive product was obtained. The product was revived at 37°C, 180 rpm, for 45 minutes. 100 μL of the product was serially diluted to 10 -3 and 10 -4 Measure the number of transformants in the library and plate them on 90 mm plates. Centrifuge the remaining cells, resuspend them in 8 mL of 2YT, and plate them on eight 200 mm plates. The next day, isolate bacterial clones harboring the target VHH fragment from the plates where the transformants were measured.

[0128] 6. Phage Library Packaging

[0129] The bacterial library was inoculated into 2×300 mL of 2YT+A (100 μg / mL of Ampicillin (Solarbio, A1170))+G (1% glucose (Solarbio, G8150)) medium to an initial OD600 of 0.1-0.2 and cultured at 37°C, 230 rpm, until the OD600 reached 0.8 or above. Helper phage M13KO7 (NBbiolab, P006) and TG1 bacteria (NBbiolab, P008) were added according to the OD600 value (helper phage: bacteria = 20:1). After adding M13KO7, the cells were mixed, allowed to stand at 37°C for 30 minutes, gently shaken at 180 rpm for 30 minutes, and centrifuged at 5000 rpm for 10 minutes. The supernatant was discarded, and the pellet was resuspended in an equal volume of 2YT+A+K (50 μg / mL of Kanamycin (Solarbio, K1030)) medium and incubated at 30°C, 220 rpm, overnight.

[0130] Centrifuge the overnight culture at 4°C, 10,000 rpm for 20 minutes, collect the supernatant, and discard the precipitate. Replace the centrifuge tube and centrifuge again at 4°C, 10,000 rpm for 20 minutes, and collect the supernatant.

[0131] Add PEG8000 (Solarbio, P8260) / NaCl to 1 / 5 the volume of the supernatant, mix thoroughly, and precipitate on ice for at least 2 hours. Centrifuge at 10,000 rpm for 20 minutes, discard the supernatant, and evacuate once to remove all the supernatant. Resuspend the pellet in 1 mL of 1× PBS and add 1 / 5 the volume of PEG8000 / NaCl for a second precipitation for 1 hour.

[0132] Centrifuge at 12000 rpm for 10 minutes, discard the supernatant, and centrifuge once to remove all the supernatant. Depending on the amount of precipitate, resuspend the precipitate in 1× PBS, add 100% glycerol to a final concentration of 50%, mix well, and dispense into 1.5 mL Eppendorf tubes. Store at -80°C.

[0133] 7. Screening for positive colonies binding to RC-Fc (positive screening)

[0134] RC-Fc was diluted to a final concentration of 5 μg / mL using a coating buffer (pH 9.6). 100 μL / well was added to the enzyme-labeled wells, coating 8 wells per target molecule (4 wells for the second round of screening and 2 wells for the third round). Coating was incubated at 4°C overnight. The coating buffer was discarded, the wells were washed three times with PBS, and 300 μL of 3% BSA-PBS blocking buffer was added to each well and blocked at 37°C for 1 hour. The wells were then washed three times with PBS, and 100 μL of phage library was added and incubated at 37°C for 1 hour. Unbound phage were aspirated, washed six times with PBST, and twice with PBS. Specifically bound phage were eluted by adding 100 μL of Gly-HCl eluent and incubating at 37°C for 8 minutes. This eluate was transferred to a sterile 1.5 mL centrifuge tube and quickly neutralized with 10 μL of Tris-HCl neutralization buffer. 10 μL of the eluate was serially diluted, titered, and the panning recovery rate was calculated.

[0135] From the eluate titer plate, randomly select 24 single colonies using a sterile toothpick and inoculate them into 1 mL of 2×YT-A. Incubate at 37°C with shaking at 220 rpm for 8 hours. Add M13K07 phage to 200 μL of this culture at a ratio of phage:TG1 = 20:1. Incubate at 37°C for 15 minutes, then shake at 220 rpm for 45 minutes. Add 800 μL of 2×YT-AK and incubate at 30°C with vigorous shaking overnight. The next day, centrifuge at 12,000 rpm for 2 minutes, and remove the supernatant for single-clone ELISA analysis.

[0136] 8. Screening of positive clones binding to Fc (counter-screening)

[0137] Fc protein (NBbiolab, 20200706) was diluted with coating buffer (pH 9.6) to a final concentration of 2 μg / mL. 100 μL / well was added to the enzyme-labeled wells and coated overnight at 4°C. The coating buffer was then discarded and the plates were washed three times with PBST. 200 μL of 5% skim milk was added to each well and blocked at 37°C for 1 hour. The plates were then washed three times with PBST. 50 μL of phage culture supernatant and 50 μL of 5% skim milk were added to each well and incubated at 37°C for 1 hour. The plates were washed six times with PBST. Horseradish peroxidase-conjugated anti-M13 antibody (diluted 1:10,000 in PBS) was added at 100 μL / well and incubated at 37°C for 1 hour. The plates were washed six times with PBST. TMB colorimetric solution (100 μL / well) was added for development at 37°C for 7 minutes. The reaction was terminated with stop solution (50 μL / well). Optical density was measured at 450 nm.

[0138] 9. Exclude Fc-binding positive clones

[0139] The results of the positive and negative screening were analyzed together to retain positive clones and exclude positive clones. Finally, the positive and negative clones were sent to Chengdu Qingke Zixi Biotechnology Co., Ltd. for sequencing.

[0140] 10. Sequence Analysis of Results

[0141] Three llamas were immunized with RC-Fc, and a phage library was established using mRNA from peripheral blood lymphocytes of two of these animals. After screening and sequencing, a total of 567 VHH sequences were obtained (311 from animal NB268 and 258 from animal NB269). Sequence comparison determined that 74 sequences (16 from NB268 and 58 from NB269) were unique. Finally, from these 74 unique VHH sequences, 12 with high OD450 values ​​and no obvious hotspot amino acids (such as extra cystine or N-linked glycation sites) were selected for cloning, expression, and purification. The sequences of these 12 VHHs are as follows:

[0142] A10-268-5:

[0143] Nucleotide sequence (SEQ ID NO: 5)

[0144] Amino acid sequence (SEQ ID NO: 1)

[0145] A10-268-18:

[0146] Nucleotide sequence (SEQ ID NO: 10)

[0147] Amino acid sequence (SEQ ID NO: 6)

[0148] A10-268-42:

[0149] Nucleotide sequence (SEQ ID NO: 15)

[0150] Amino acid sequence (SEQ ID NO: 11)

[0151] A10-268-76:

[0152] Nucleotide sequence (SEQ ID NO: 20)

[0153] Amino acid sequence (SEQ ID NO: 16)

[0154] A10-268-103:

[0155] Nucleotide sequence (SEQ ID NO: 25)

[0156] Amino acid sequence (SEQ ID NO: 21)

[0157] A10-268-273:

[0158] Nucleotide sequence (SEQ ID NO: 30)

[0159] Amino acid sequence (SEQ ID NO: 26)

[0160] A10-269-56:

[0161] Nucleotide sequence (SEQ ID NO: 35)

[0162] Amino acid sequence (SEQ ID NO: 31)

[0163] A10-269-62:

[0164] Nucleotide sequence (SEQ ID NO:40)

[0165] Amino acid sequence (SEQ ID NO: 36)

[0166] A10-269-165:

[0167] Nucleotide sequence (SEQ ID NO:45)

[0168] Amino acid sequence (SEQ ID NO:41)

[0169] A10-269-172:

[0170] Nucleotide sequence (SEQ ID NO: 50)

[0171] Amino acid sequence (SEQ ID NO: 46)

[0172] A10-269-238:

[0173] Nucleotide sequence (SEQ ID NO: 55)

[0174] Amino acid sequence (SEQ ID NO: 51)

[0175] A10-269-385:

[0176] Nucleotide sequence (SEQ ID NO: 60)

[0177] Amino acid sequence (SEQ ID NO: 56)

[0178] 11. Cloning, expression and purification of A10-268 and A10-269

[0179] A10 is the target code for the CGRP receptor, and NB268 and NB269 are the designations for the two llamas. Expression and purification were performed by Bio-Bio. Six histidine residues (6xHis) were attached to the C-terminus of the VHH sequence for purification. The synthesized gene fragment was ligated into the pcDNA3.4 vector (ThermoFisher Scientific, EL0011) after digestion with NotI (NEB, R0189L) / XbaI (NEB, R0145L) (Bio-Bio). The ligation product was transformed into Top10 competent cells, and positive clones were selected for expansion. A small amount of plasmid was extracted using a plasmid miniprep kit (Bio-Bio) for sequencing. Correctly sequenced clones were inoculated into LB medium (Bio-Bio) for expansion, and the plasmid was extracted using a kit (Bio-Bio) for large-scale extraction and resequencing. 293 cells were transfected with the expression plasmid using a transfection reagent (Baiying, Inc.). After 5 days of incubation in a constant temperature incubator (Jingqi, IS-RDS6C5), the cell cultures were harvested, centrifuged, and the supernatant was removed by filtration to remove cell debris. The clear fluid 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 the A280 value. The absorbance at 280 nm was read using a NanoDrop One instrument. The protein was aspirated into a dialysis bag and dialyzed in a beaker containing 1X PBS. The purified product was identified by SDS-PAGE electrophoresis. The 12 expressed proteins are shown in Table 2.

[0180] Table 2 Twelve proteins expressed and their corresponding concentrations

[0181] Example 2: Validation of anti-RC-Fc VHH

[0182] 1. ELISA validation of anti-RC-Fc VHH

[0183] RC-Fc VHH binds to immune antigen

[0184] The immune antigen RC-Fc was diluted to 2000 ng / mL in coating buffer (50 mM NaHCO3, pH = 9.6). 100 μL of the RC-Fc was coated onto a 96-well microtiter plate (ThermoFisher Scientific, 449824) and incubated overnight at 4°C. The plate was washed five times with PBST, patted dry, and blocked with 200 μL of blocking buffer at 37°C for 2 hours. The PBST was removed, and 100 μL of serially diluted VHH protein in PBST was added to each well and incubated at 37°C for 1 hour. The plate was washed five times with PBST, and then 100 μL of the corresponding secondary antibody (Mouse Anti-His-HRP, Nanjing GenScript, A00186) diluted 1:5000 in blocking buffer was added and incubated at 37°C for 1 hour.

[0185] 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 reaction was incubated at 37°C for 7 minutes. 100 μL of 1 M HCL (Cologne Chemical) was added to each well to terminate the reaction. Finally, the OD450 reading was read using a microplate reader. The results were analyzed using the Nonlinear regression log (agonist) vs. response (three parameters) model in GraphPad Prism 9.3.1 software to obtain the Binding EC 50 See Table 3 for values.

[0186] Table 3 12 expressed proteins and their corresponding Binding EC 50 value

[0187] 2. Cell Validation of Anti-RC-Fc VHH

[0188] 2.1 SK-N-MC cell line and culture

[0189] 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.

[0190] 2.2 cAMP concentration test of SK-N-MC cells

[0191] The cAMP concentration produced by SK-N-MC cells was measured using the Cisbio cAMP-GS Dynamic Kit (Cisbio, 62AM4PEB). The assay was performed according to the kit instructions unless otherwise specified. The assay medium was DMEM / F1-12 + 10% FBS + 1 mM IBMX (Solarbio, I10010), and the assay was performed using a white 384-well microtiter plate.

[0192] 2.2.1 Determination of SK-N-MC Cell Number and Obtaining the CGRP Standard Curve

[0193] 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 to the appropriate concentration with test medium. 10,000, 7,500, 5,000, and 2,500 cells were tested per test well.

[0194] 5 μL of cell suspension and 5 μL of CGRP (Nanjing Yuanpeptide Biotechnology Co., Ltd., Catalog No. A03-137, Lot No. yuanpeptide-997792) diluted in Stimulation Buffer + 500 μM IBMX were added to the wells of a ProxiPlate-384 Plus assay plate (PerkinElmer, 6008280). The final CGRP concentration range was 0 nM or 0.001-50 nM.

[0195] After adding CGRP, the test plate was incubated at 37°C for 15 minutes. The reaction was then terminated by adding 5 μL of cAMP-d2 and 5 μL of Anti-cAMP-Cryptate. The plate was allowed to stand at room temperature for 60 minutes before reading using a fluorescence microplate reader (BMG LabTech, Model: PHERAstar FSX) with a 665 / 620 filter. The resulting values ​​were used to calculate the ratio (Ratio) using Microsoft Excel using the following formula:

[0196] The calculated ratios were analyzed using the nonlinear regression log (agonist) vs. response (three parameters) model in GraphPad Prism 9.3.1 software. The results are shown in Figure 3 , which shows that the number of cells per well ranged from 2500 to 10,000. Therefore, 2500 cells per well were selected for subsequent experiments.

[0197] 3. Evaluating the Ability of Partially Anti-CGRP Receptor VHHs to Inhibit Cellular cAMP Production

[0198] A10-268-5, A10-268-18, A10-268-42, A10-268-76, A10-268-103, and A10-268-273 were used in cell function testing. On the day of the experiment, SK-NM cells were detached from the culture flask using 0.25% Trypsin-EDTA, washed once with PBS, counted, and diluted to 5 million cells / mL in assay medium. The cells were used within 30 minutes.

[0199] 5 μL of cell solution and 5 μL of anti-RC-Fc single domain antibody (final concentration 0 nM or 0.0001-100 nM) diluted 5-fold in stimulation solution were added to the wells of the test plate, and the plates were incubated in a 37° C. incubator for 30 minutes.

[0200] 5 μL of CGRP diluted in stimulation solution (final concentration 5 nM) was added and the test plate was incubated in a 37°C incubator for 15 minutes. 5 μL of cAMP-d2 and 5 μL of Anti-cAMP-Cryptate were then added to terminate the reaction. The test plate was left at room temperature for 60 minutes and read using a 665 / 620 filter on a fluorescence microplate reader. The 665 / 620 ratio was calculated and analyzed using the Nonlinear regression log (inhibitor) vs. response (three parameters) model in GraphPad Prism 9.3.1 software. The results are shown in Table 4. 50 The results showed that four VHHs had the ability to inhibit cAMP production in SK-N-MC cells, with the activity ranking as A10-268-273 > A10-268-42 > A10-268-103 > A10-268-18. A10-268-42 was selected as the lead VHH for the next step of evaluation.

[0201] Table 4 Six VHHs used in cell function tests and their corresponding ICs 50 value

[0202] 4. Humanization, cloning, expression and purification of A10-268-42

[0203] The IMGT sorting method was used to divide the four framework regions and three CDR regions of A10-268-42 (42wt). AlphaFold2 was used to predict the 3D structure of 42wt, and the base number of this structure was obtained using an independently developed method. The sequence of 42wt was then compared with the IGHV3 sequences of 100 people using the IGBLAST application (https: / / www.ncbi.nlm.nih.gov / igblast / ) to identify the 14 non-human amino acids in the four frameworks of A10-268-42. These 14 amino acids were divided into 13 groups (42hv1 to 42hv13), with 1-10 amino acids in each group becoming human amino acids. These 13 VHHs with different degrees of humanized amino acid combinations were all predicted by AlphaFold2. These 3D structures were compared with the 42wt structure, and the three combinations with the closest similarity (42hv10, 42hv11, and 42hv12) were selected. Together with 42wt itself, the above four VHHs were fused with Fc in which the 297th amino acid was mutated from N to A. Finally, the functions of these four proteins were compared through cellular cAMP experiments.

[0204] The C-termini of 42wt, 42hv10, 42hv11, and 42hv12 were connected to the N-terminus of Fc to form fusion proteins (42wtFc, 42hv10Fc, 42hv11Fc, and 42hv12Fc). Gene synthesis, expression, and purification were commissioned to Bio-Tech using the same methods as above. The protein electrophoresis diagram is shown in Figure 4.

[0205] The sequence of 42hv10, 42hv11, and 42hv12 is as follows:

[0206] 42hv10 (SEQ ID NO: 61):

[0207] 42hv11 (SEQ ID NO: 62):

[0208] 42hv12 (SEQ ID NO: 63):

[0209] Fc fragment in which amino acid position 297 is mutated from N to A (SEQ ID NO: 68)

[0210] 5. Verification of the cellular cAMP inhibitory ability of humanized A10-268-42 (42hv10Fc)

[0211] On the day of the experiment, neuroblastoma was isolated from the culture flask with 0.25% Trypsin-EDTA, washed once with PBS, counted, and diluted to 5 million cells / mL with test medium and used within 30 minutes.

[0212] 5 μL of cell fluid and 5 μL of 42wtFc, 42hv10Fc, 42hv11Fc, 42hv12Fc proteins diluted 5-fold in stimulation solution and control antibody Erenumab (Bio-Ying Bio, A03EB) were added to the wells of the ProxiPlate-384 Plus test plate in sequence, with concentrations ranging from 0.0003 to 100 nM, and incubated at 37°C for 30 minutes.

[0213] Add 5 μL of CGRP diluted with stimulation solution (final concentration is 5 nM), place the test plate in a 37°C incubator for 15 minutes, and then add 5 μL cAMP-d2 and 5 μL Anti-cAMP-Cryptate to terminate the reaction. After the test plate is placed at room temperature for 60 minutes, read the value using the 665 / 620 filter of the fluorescence microplate reader. After calculating the 665 / 620 ratio, the Nonlinear regression log (inhibitor) vs. response (three parameters) model in GraphPad Prism 9.3.1 software is used for analysis. The results are shown in Figure 5. As can be seen from Figure 5, the humanization of the three combinations of 42hv10, 42hv11, and 42hv12 has been successful, and their ability to inhibit cellular cAMP production is better than that of the control antibody Erenumab. Based on the above results, IC 50 The 42hv10, which is twice as good as the prototype, proceeds to the next stage of development.

[0214] Example 3: Synthesis of the extracellular domain of pituitary adenylate cyclase 1 (PAC1 ECD)

[0215] 1. Gene synthesis, cloning, expression, and purification of PAC1 ECD and PAC1s ECD-Fc fusion proteins

[0216] PAC1 stands for Pituitary Adenylate Cyclase 1 (PACAP), and its encoding gene is ADCYAP1R1. PAC1 is a membrane protein belonging to the Class B G protein-coupled receptor (GPCR). It is widely expressed in animals, but is primarily distributed in endocrine organs and the nervous system. PAC1 and its ligand, pituitary adenylate cyclase-activating polypeptide (PACAP, also known as PACAP38), form a signaling system that plays an important role in regulating growth, tissue repair, mood, metabolism, and neuroprotection.

[0217] ADCYAP1R1 has 18 exons, 10 of which (exons 2, 3, 7-13, and 18) are constitutively expressed. The others are expressed through alternative splicing, forming multiple homologous isoforms, which are collectively referred to as isoforms. The amino-terminus (N-terminus) of the extracellular domain (ECD) of PAC1 is encoded by exons 2-6, the seven transmembrane regions (including the extracellular and intracellular parts) are encoded by exons 7-17, and the untranslated portion of the carboxyl-terminus (C-terminus) is encoded by exon 18.

[0218] In humans, the N-terminal extracellular domain (ECD) of PAC1 consists of three isoforms: PAC1, PAC1s, and PAC1vs (Figure 6). If all exons 2-6 are expressed, it is PAC1; if exons 5 and 6 are cleaved, it is PAC1s; and if exons 4, 5, and 6 are cleaved, it is PAC1vs.

[0219] PAC1 ligands include PACAP (PACAP38), PACAP27, and VIP (Vasoactive Intestinal Polypeptide) (Figure 7). PAC1's affinity for PACAP is in the single nanomolar (nM) range, but its affinity for VIP is 100-1000-fold lower. Although PAC1s lacks exons 5 and 6, it retains affinity for its ligand PACAP and has an increased affinity for VIP. PAC1vs' affinity for its ligands PACAP38 and PACAP27 is significantly reduced, but it retains affinity for VIP.

[0220] The PACAP Trap is derived from the PAC1 ECD. The PAC1 ECD is the soluble extracellular domain of PAC1 that is free from the cell membrane. The present invention has shown that it retains the ability to bind PACAP in vitro. Furthermore, the PAC1 ECD lacking exons 5 and 6, known as the PAC1s ECD, exhibits stronger PACAP binding, thus enabling its use in the construction of the PACAP Trap.

[0221] The PAC1 ECD is derived from Genbank ID NM_001199635.2, nucleic acid sequence 506-907 (SEQ ID NO: 65), amino acid sequence 24-155 (SEQ ID NO: 64).

[0222] The nucleic acid sequence is:

[0223] The amino acid sequence is:

[0224] The nucleic acid sequence used for cloning PAC1s ECD was derived from Genbank ID NM_001199637.2 506-844 (SEQ ID NO: 67):

[0225] The amino acid sequence of PAC1s ECD (or with addition: from UniProt ID P41586-3 21-124) (SEQ ID NO: 66) is:

[0226] The C-terminus of PAC1 ECD and PAC1s ECD was fused to the N-terminus of Fc to form PAC1 ECD-Fc and PACs1 ECD-Fc. Gene synthesis and expression were performed by Bio-Bio using the same methods as above. SDS-PAGE electrophoresis analysis is shown in Figure 8.

[0227] 2. ELISA Assessment of the Binding Ability of PAC1 ECD-Fc and PAC1s ECD-Fc to PACAP

[0228] Streptavidin-coated 96-well ELISA plate (ThermoFisher Scientific TM , 15126) were washed three times with PBST (0.1% Tween 20, pH = 7.4), and 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-Fc, 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 with 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 Huaan Biological, HR1214) diluted 1:5000 in blocking buffer was added and incubated at 37°C for 1 hour.

[0229] The ELISA plate incubated with the secondary antibody was washed five times with PBST. 100 μL of TMB (Trimethylolpropane) single-component colorimetric solution (Solarbio, 20190402) was added to each well and incubated at 37°C for 7 minutes. The reaction was terminated by adding 100 μL of 1 M HCl (Cologne Chemical) to each well. OD450 readings were taken on a microplate reader. The results were analyzed using the nonlinear regression log (agonist) vs. response (three parameters) model, as shown in Figure 9. The figure shows that the ability of PAC1s ECD-Fc to bind PACAP is 40-fold greater than that of PAC1 ECD-Fc, while the CGRP receptor extracellular domain RC-Fc has no binding ability.

[0230] Example 4: Construction of 42hv10FcNA-PAC1s ECD (BY003) bifunctional fusion protein

[0231] 1. Cloning, expression and purification of BY003

[0232] BY003 comprises a fusion of three molecules: 42hv10, FcNA (with amino acid 297 of the Fc modified from N to A), and PAC1s ECD. The C-terminus of 42hv10 is linked to the N-terminus of the FcNA, which in turn is connected to the PAC1s ECD via the linker GGGGS (SEQ ID NO:79). The number of PAC1s ECDs can be one, two, or three. In the case of multiple PAC1s ECDs, they are connected via GGGGS. The BY003 structure, consisting of two PAC1s ECDs, is shown in Figure 10, and the working principle of BY003 is illustrated in Figure 11.

[0233] Baiying Bio was commissioned to perform gene synthesis using the same method as above. The SDS-PAGE electrophoresis identification is shown in Figure 12.

[0234] 2. Cellular Function Verification of Anti-42hv10 in BY003 (Inhibition of CGPR-induced cAMP Production in CHO-CGRPrC3 Cells)

[0235] In this example, the verification indicator of cell function is the ability of BY003 to inhibit cAMP production.

[0236] 2.1 Establishment and culture of cell lines expressing CGRP receptors

[0237] A pool of CHO cells stably expressing human Ramp1 ECD and CLR ECD (referred to as CHO-hCGRPr or CHO-A10 cells) was obtained from Taizhou Baiying Biotechnology Co., Ltd., catalog number CHOK1-C21738001. The culture medium consisted of a mixture of DMEM / F-12, 10% FBS, 1x penicillin-streptomycin (Solarbio, P1400), 10 μg / mL puromycin (Solarbio, P8230), and 50 μg / mL hygromycin B (Solarbio, H8080), referred to as CHO-hCGRPr medium.

[0238] Dilute the CHO-hCGRPr cell pool to 5 cells / mL with CHO-hCGRPr medium. Transfer 100 μL of the diluted cell solution to the wells of two 96-well cell culture plates (Thermo Scientific, 167008) and culture in a CO2 incubator until individual cell colonies can be distinguished. Transfer six colonies from the 96-well plate to a 6-well cell culture plate (Corning, 3516). Once the cells reach 90% confluence, transfer them to a T-75 cell culture flask (Corning, 430541U). Once confluent, the cells can be used for functional testing and cryopreserved in liquid nitrogen.

[0239] Cellular cAMP concentration was measured using the Cisbio cAMP-GS Dynamic Kit (Cisbio, 62AM4PEB). Unless otherwise specified, procedures were performed according to the kit instructions. The assay medium was DMEM / F1-12 (10% FBS + 1 mM IBMX), and the assay was performed using a white 384-well microtiter plate.

[0240] 2.2 Obtaining the CGRP standard curve of monoclonal stable cell lines

[0241] From the six single-cell clones identified above, clones 1, 2, and 3 were selected to evaluate their cAMP production capacity. A non-CGRP receptor-transfected CHOK1 parent cell line (ECACC, 85051005) was also added as a control. On the day of the experiment, cells were detached from the culture flask using 0.25% Trypsin-EDTA (Gibco, 25200-056), washed once with PBS, counted, and diluted to 5 million cells / mL in assay medium. The cells were used within 30 minutes.

[0242] 5 μL of cell suspension and 5 μL of CGRP (Nanjing Yuanpeptide Biotechnology Co., Ltd., A03-137, Lot No.: yuanpeptide-997792) diluted in Stimulation Buffer + 500 μM IBMX were added to the wells of the ProxiPlate-384 Plus assay plate. The final CGRP concentration range was 0 nM or 0.000001-100 nM, with 10-fold serial dilutions.

[0243] After adding CGRP, the test plate was incubated in a 37°C incubator for 15 minutes. The reaction was then terminated by the addition of 5 μL of cAMP-d2 and 5 μL of Anti-cAMP-Cryptate. The plate was allowed to stand at room temperature for 60 minutes before reading using a fluorescence microplate reader (BMG LabTech, Model: PHERAstar FSX) using a 665 / 620 filter. The resulting values ​​were calculated using Microsoft Excel to calculate the 665 / 620 ratio, and finally analyzed using the nonlinear regression log (agonist) vs. response (three parameters) model in GraphPad Prism 9.3.1 software. The results are shown in Figure 13. Based on these results, cell line 3 was selected for subsequent experiments and designated CHO-CGRPrC3.

[0244] 2.3 Evaluation of BY003's ability to inhibit CGPR-induced cAMP production in CHO-CGRPrC3 cells

[0245] On the day of the experiment, CHO-hCGRPrC3 cells were isolated from the culture flask with 0.25% Trypsin-EDTA, washed once with PBS, counted, and diluted to 5 million cells / mL with assay medium and used within 30 minutes.

[0246] 5 μL of cell solution and 5 μL of BY003 and control antibody Erenumab (Bio-Ying Bio, A03EB) diluted 5-fold in stimulation solution were added to the wells of the ProxiPlate-384 Plus test plate, with concentrations ranging from 0.0003 to 100 nM, and incubated at 37°C for 30 minutes.

[0247] 5 μL of CGRP diluted in stimulation solution (final concentration 1.4 nM) was added, and the plate was incubated at 37°C for 15 minutes. The reaction was then terminated by the addition of 5 μL of cAMP-d2 and 5 μL of Anti-cAMP-Cryptate. The plate was then incubated at room temperature for 60 minutes using a fluorescence microplate reader with a 665 / 620 filter. The resulting values ​​were converted to 665 / 620 ratios and analyzed using the nonlinear regression log (inhibitor) vs. response (three parameters) model in GraphPad Prism 9.3.1 software. The results are shown in Figure 14. The figure shows that BY003 exhibits a 2.5-fold greater ability to inhibit cAMP production in CHO-hCGRPrC3 cells than Erenumab.

[0248] 3. Cellular Function Verification of Anti-42hv10 in BY003 (Inhibition of PACAP-induced cAMP Production in CHO-hPAC1 Cells)

[0249] 3.1 Establishment and culture of stable cell lines expressing human PAC1

[0250] CHO cell pools stably expressing human PAC1 ECD and PAC1s ECD (referred to as CHO-A05 and CHO-A05s cells) were obtained from Taizhou Baiying Biotechnology Co., Ltd., with catalog numbers CHOK1-A05 and CHOK1-A05s. The culture medium consisted of a DMEM / F-12 supplemented with 10% FBS, 1x penicillin-streptomycin (Solarbio, P1400), and 10 μg / mL puromycin (Solarbio, P8230), referred to as CHO-PAC1 medium.

[0251] CHO-A05 and CHO-A05s cell pools were diluted to 5 cells / mL in CHO-PAC1 medium. 100 μL of the diluted cell suspension was then transferred to the wells of two 96-well cell culture plates (Thermo Scientific, 167008) and cultured in a CO2 incubator until individual colonies could be distinguished. Six colonies were transferred from the 96-well plates to a 6-well cell culture plate (Corning, 3516). Once the cells reached 90% confluence, they were transferred to T-75 (Corning, 430541U) cell culture flasks. Once confluent, the cells were ready for testing and cryopreservation in liquid nitrogen.

[0252] 3.2 Obtaining the cAMP standard curve of the monoclonal CHO-A05 cell line

[0253] On the day of the experiment, six monoclonal CHO-A05 cells and a CHOK1 parent cell line not transfected with PAC1 (ECACC, 85051005) were isolated from the culture flask using 0.25% Trypsin-EDTA, washed once with PBS, counted, and diluted to 5 million cells / mL with assay medium before use within 30 minutes.

[0254] 5 μL of cell suspension and 5 μL of PACAP (Nanjing Yuanpeptide Biotechnology Co., Ltd., A05-138, batch number: Yuanpeptide-194972) diluted with stimulation buffer (Stimulation Buffer + IBMX) were added to the wells of the ProxiPlate-384 Plus test plate. The final concentration of PACAP ranged from 0.0001 to 100 nM.

[0255] After adding PACAP, the test plate was incubated in a 37°C incubator for 15 minutes. The reaction was then terminated by adding 5 μL of cAMP-d2 and 5 μL of Anti-cAMP-Cryptate. The plate was incubated at room temperature for 60 minutes and then read using a fluorescence microplate reader (BMG LabTech, Model: PHERAstar FSX) using a 665 / 620 filter. The resulting values ​​were calculated as 665 / 620 ratios using Microsoft Excel and analyzed using nonlinear regression log (agonist) vs. response (three parameters) in GraphPad Prism 9.3.1 software. The results are shown in Figure 15. Based on these results, cell line No. 4 was selected for future experiments and designated CHO-hPAC1C4.

[0256] 3.3 Evaluation of the ability of anti-BY003 to inhibit cAMP production in CHO-hPAC1C4 cells

[0257] Cellular cAMP concentration was measured using the Cisbio cAMP Kit. Unless otherwise specified, the procedure was performed according to the kit instructions. The assay medium was DMEM / F1-12 (10% FBS + 1 mM IBMX), and the assay was performed using a white 384-well microtiter plate.

[0258] Because BY003 is a bifunctional protein, acting on both the CGRP receptor and PACAP, CHO-hCGRPrC3 and CHO-hPAC1C4 cells were added to the assay plates. The day before the experiment, both cells were detached from the culture flasks using 0.25% Trypsin-EDTA, washed once with PBS, counted, and diluted to a cell density of 4 million / mL in assay medium. After mixing the two cell cultures, 10 μL of the mixture was transferred to a 384-well cell culture plate (Corning, 3765) and incubated overnight at 37°C in a CO2 incubator.

[0259] On the day of the experiment, the culture medium was removed and 5 μL of BY003 single domain antibody serially diluted with stimulation solution (final concentration was 0 nM or 0.1-1000 nM) was added to the wells of the test plate and incubated in a 37°C incubator for 30 minutes.

[0260] 5 μL of PACAP diluted in stimulation solution (final concentration 0.7 nM) was added. After the addition of PACAP, the test plate was incubated in a 37°C incubator for 15 minutes. The reaction was then terminated by the addition of 5 μL of cAMP-d2 and 5 μL of Anti-cAMP-Cryptate. The test plate was incubated at room temperature for 60 minutes and then read using a fluorescence microplate reader (BMG LabTech, Model: PHERAstar FSX) using a 665 / 620 filter. The resulting values ​​were calculated using Microsoft Excel to calculate the 665 / 620 ratio, and finally analyzed using nonlinear regression log (inhibitor) vs. response (three parameters) in GraphPad Prism 9.3.1 software. The results are shown in Figure 16.

[0261] To address the unmet clinical needs in migraine treatment and prevention, this invention provides a fusion protein that simultaneously blocks both the CGRP and PACAP pathways. By fusing a single-domain VHH targeting the CGRP receptor with a native PAC1 soluble EDC, this protein simultaneously blocks both CGRP and PACAP signaling pathways, two pathways closely associated with migraine. The inventors successfully constructed the BY003 molecule and demonstrated its ability to block CGRP- and PACAP-induced cAMP production at the cellular level.

Claims

1. A single domain antibody that blocks the calcitonin gene-related peptide (CGRP) pathway, wherein: The single domain antibody comprises a CDR1 as shown 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 shown 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 shown 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 CDR1 as shown in the amino acid sequence SEQ ID NO: 12 or any variant thereof, CDR2 as shown in the amino acid sequence SEQ ID NO: 13 or any variant thereof and CDR3 as shown in the amino acid sequence SEQ ID NO: 14 or any variant thereof.

2. The single domain antibody according to claim 1, wherein The single-domain antibody comprises a variable region as shown 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 shown in the amino acid sequence of SEQ ID NO: 61, 62 or 63 or any variant thereof; More preferably, the single domain antibody comprises a variable region as shown in the amino acid sequence of SEQ ID NO: 61 or any variant thereof; Preferably, the amino acids in the 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.

3. A fusion protein that simultaneously blocks the calcitonin gene-related peptide (CGRP) pathway and the pituitary adenylate cyclase activating peptide (PACAP) pathway, wherein: 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).

4. The fusion protein according to claim 3, wherein The fusion protein comprises a peptide chain, wherein the structure of the peptide chain is that a PAC1 ECD is connected 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 is that a PAC1 ECD is connected 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 connected via a linker; Optionally, the fusion protein comprises three peptide chains, the structure of the peptide chains is that a PAC1 ECD is connected 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 connected via a linker.

5. The fusion protein according to claim 3 or 4, wherein The single domain antibody is a single domain antibody that binds to the CGRP receptor; Preferably, the single domain antibody comprises a CDR1 as shown 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 shown 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 shown 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, the single-domain antibody comprises CDR1 as shown in the amino acid sequence of SEQ ID NO: 12 or any variant thereof, CDR2 as shown in the amino acid sequence of SEQ ID NO: 13 or any variant thereof, and CDR3 as shown in the amino acid sequence of SEQ ID NO: 14 or any variant thereof; Preferably, the amino acids in one or more or all three of the CDRs have conservative substitutions, the conservatively substituted amino acids The number of acids does not exceed 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

6. The fusion protein according to any one of claims 3 to 5, wherein The single-domain antibody comprises a variable region as shown 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 shown in the amino acid sequence of SEQ ID NO: 61, 62 or 63 or any variant thereof; More preferably, the single domain antibody comprises a variable region as shown in the amino acid sequence of SEQ ID NO: 61 or any variant thereof; Preferably, the amino acids in the 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 fusion protein according to any one of claims 3 to 6, wherein The PAC1 ECD is selected from the ECD of PAC isomers: PAC1, PAC1s or PAC1vs; Preferably, the PAC1 ECD is PAC1s ECD; Preferably, the PAC1 ECD comprises the amino acid sequence of SEQ ID NO: 64 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 more identity thereto; Preferably, the PAC1s ECD 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 more identity thereto.

8. The fusion protein according to any one of claims 3 to 7, wherein The Fc fragment comprises a hinge region, a constant region 2 (CH2) and a constant region 3 (CH3) domain 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 from human IgG1; Preferably, the Fc fragment has an amino acid substitution; more preferably, the amino acid substitution comprises substitution of asparagine N at position 297 of the Fc fragment with alanine A, wherein each amino acid position on 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, and more preferably an amino acid sequence having 98% or 99% or more identity thereto.

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

10. A nucleic acid encoding the single domain antibody according to claim 1 or 2, or the 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 the single domain antibody according to claim 1 or 2 and / or the fusion protein according to any one of claims 3 to 9 and a pharmaceutically acceptable carrier, diluent or excipient.

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

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