WO2020239014A1 - 抗cgrp抗体及其应用 - Google Patents

抗cgrp抗体及其应用 Download PDF

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WO2020239014A1
WO2020239014A1 PCT/CN2020/092892 CN2020092892W WO2020239014A1 WO 2020239014 A1 WO2020239014 A1 WO 2020239014A1 CN 2020092892 W CN2020092892 W CN 2020092892W WO 2020239014 A1 WO2020239014 A1 WO 2020239014A1
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antibody
seq
cgrp
antigen
amino acid
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PCT/CN2020/092892
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English (en)
French (fr)
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宋德勇
刘秀
董创创
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山东博安生物技术有限公司
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Publication of WO2020239014A1 publication Critical patent/WO2020239014A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to an anti-CGRP antibody or an antigen-binding fragment thereof, and a preparation method, composition and application thereof.
  • Calcitonin gene-related peptide is a 37 amino acid neuropeptide secreted by nerve cells in the central and peripheral nervous systems. In the human body, CGRP exists in two forms: CGRP ⁇ and CGRP ⁇ , which differ by only 3 amino acids, and come from different genes (Doods, H., Curr. Op. Invest. Drugs, 2(9):1261-78 (2001) )). CGRP is a neurotransmitter released by various tissues (such as the trigeminal nerve).
  • CGRP-R CGRP receptor
  • RAMP receptor component protein
  • CGRP is an effective vasodilator associated with the pathology of other vasomotor symptoms, such as all forms of vascular headache, including migraine (with or without aura) and cluster headache.
  • the level of CGRP in the external jugular vein of patients increases during migraine (Goadsby et al., Ann. Neurol. 28:183-7 (1990)).
  • Intravenous administration of human ⁇ -CGRP induces headache and migraine in patients with migraine without aura, which suggests that CGRP has a causal role in migraine (Lassen et al., Cephalalgia 22:54-61, (2002)).
  • Fremanezumab trade name Developed by Teva and approved by the U.S. Food and Drug Administration (FDA), it is a humanized monoclonal antibody targeting calcitonin gene-related peptide (CGRP) and approved for the treatment of adult migraine (Urits). ,Ivan et al. Pain and therapy vol.9,1(2020):195-215.).
  • Eptinezumab trade name It is a humanized monoclonal antibody targeting calcitonin gene-related peptide (CGRP), which can block the binding of CGRP to its receptor, and is approved for the preventive treatment of adult migraine (Dhillon S. 2020; 80 (7):733-739.).
  • Galcanezumab trade name Developed by Eli Lilly, it was approved by the U.S. Food and Drug Administration (FDA) and then approved by the European Medicines Agency (EMA). It is a humanized monoclonal antibody that targets the calcitonin gene-related peptide (CGRP), approved for the treatment of migraine in adult patients (Martin, V., Samaan, KH, Aurora, S. et al. Adv Ther 37, 2034-2049 (2020)). According to the label of the above three marketed antibodies approved by the US FDA, both Epitinezumab and Galcanezumab are injected subcutaneously once a month, and Fremanezumab can be injected once a month or once every 3 months.
  • FDA U.S. Food and Drug Administration
  • EMA European Medicines Agency
  • the present invention provides antibodies or antigen-binding fragments thereof having a novel amino acid sequence.
  • the antibodies or antigen-binding fragments thereof include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', and F (ab')2, Fv, scFv or dsFv fragments, etc.
  • the antibody or antigen-binding fragment thereof provided by the present invention includes:
  • LCDR Three light chain complementarity determining regions
  • HCDR heavy chain complementarity determining regions
  • amino acid sequence of HCDR1 is shown in SEQ ID NO: 4
  • amino acid sequence of HCDR2 is shown in SEQ ID NO: 5
  • amino acid sequence of HCDR3 is shown in SEQ ID NO: 6 or SEQ. ID NO: 7 or SEQ ID NO: 8, preferably SEQ ID NO: 6; or
  • the above variants refer to the amino acids in any one or more LCDR or HCDR regions being changed, preferably by amino acid substitutions, and more preferably conservative substitutions; for example, the LCDR1, LCDR2 and/or LCDR3 regions of the light chain variable region contain at least one And no more than 10 amino acid changes; and/or the HCDR1, HCDR2 and/or HCDR3 regions of the heavy chain variable region contain at least one and no more than 10 amino acid changes.
  • the antibody or antigen-binding fragment thereof comprises a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 9, or is at least 80% with the amino acid sequence shown in SEQ ID NO: 9 %, at least 85%, or at least 90% identical amino acid sequence; further comprising a heavy chain variable region, the amino acid sequence of which is shown in any one of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or An amino acid sequence that is at least 80%, at least 85%, or at least 90% identical to SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
  • the antibody or antigen-binding fragment thereof comprises the following light and heavy chain variable region sequences:
  • a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9 and/or a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10, or
  • a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9 and/or a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 11, or
  • the antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region comprises a ⁇ -1, ⁇ -2, ⁇ -3 or ⁇ -4 human heavy chain constant region or the human variant of the heavy chain constant region, preferably having the sequence ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH
  • the antibody further comprises a light chain constant region, wherein the light chain constant region comprises a lambda or kappa human light chain constant region, preferably its sequence is TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGLSSPVTKSFNRGEC.
  • the binding antigen of the aforementioned antibody or antigen-binding fragment thereof is CGRP, preferably, the CGRP is human CGRP.
  • the present invention also provides a nucleic acid encoding any of the above antibodies or antigen-binding fragments thereof, and the binding antigen of the antibody or antigen-binding fragment thereof is CGRP, especially human CGRP.
  • the nucleic acid encodes an antibody or antigen-binding fragment thereof containing the following amino acid sequence:
  • LCDR light chain complementarity determining regions
  • HCDR heavy chain complementarity determining regions
  • the present invention relates to nucleic acids that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to nucleic acids that can hybridize to the nucleic acid of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%. Moreover, the polypeptide encoded by the hybridizable nucleic acid has the same biological function and activity as the mature polypeptide.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also provides a vector containing a nucleotide sequence encoding an antibody that binds to CGRP or an antigen-binding fragment thereof.
  • the vector is an expression vector.
  • the vector of the present invention includes, but is not limited to, a viral vector, such as an adenovirus. Vectors, retroviral vectors, adeno-associated virus vectors, etc.; non-viral vectors, such as plasmids, transposon vectors, etc., wherein the plasmid vector is preferably a pCDNA3.4 (Life Technology) vector. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the present invention also provides a cell for expressing an antibody or antigen-binding fragment thereof that binds to CGRP, which cell contains an expression vector encoding an antibody or antigen-binding fragment thereof that binds to CGRP or a nucleic acid that encodes an antibody or antigen-binding fragment thereof that binds to CGRP,
  • the cell is a host cell containing the above-mentioned expression vector.
  • host cells expressing CGRP-binding antibodies or antigen-binding fragments thereof include, but are not limited to, mammalian cells, insect cells, plant cells, fungal cells, and prokaryotic cells.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • the host cell provided by the present invention for expressing the antibody or antigen-binding fragment thereof that binds to CGRP is HEK293.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, transformation can also be performed by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the antibody encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant antibody in the above method can be expressed in the cell, on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic cleavage, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • a pharmaceutical composition which comprises any of the above-mentioned CGRP-binding antibodies or antigen-binding fragments thereof, nucleic acids, carriers or cells, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes one or more of the following: pharmaceutically acceptable solvents, dispersants, additives, plasticizers, and pharmaceutical excipients. Generally, these substances are non-toxic, inert and pharmaceutically acceptable carrier media.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumor, intraperitoneal, intravenous, or local administration (such as injection administration).
  • the present invention also relates to a kit, which comprises any of the above-mentioned antibodies or antibody fragments and nucleic acids that bind to the CGRP antigen.
  • the kit includes an antibody or antigen-binding fragment thereof of any of the following groups of CDR amino acid sequences: LCDR1 shown in SEQ ID NO:1, LCDR2 shown in SEQ ID NO: 2, SEQ ID NO: 3 shown in LCDR3, SEQ ID NO: 4, HCDR1, SEQ ID NO: 5, HCDR2, SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 HCDR3.
  • the kit further includes a detection reagent, a negative control, and a positive control for detecting the CGRP antigen-antibody reaction.
  • the antibody or its antigen-binding fragment of the present invention has a wide range of biological application value and clinical application value, and its application relates to the diagnosis and treatment of diseases related to CGRP, basic medical research, biological research, detection and other fields.
  • a preferred application is for clinical diagnosis and targeted therapy for CGRP.
  • the present invention relates to the use of the antibody or antigen-binding fragment, nucleic acid, vector or cell of any one of the foregoing aspects in the preparation of a pharmaceutical composition for treating or preventing diseases.
  • the present invention relates to the application of the antibody or its antigen-binding fragment or nucleic acid of any one of the foregoing aspects in the preparation of diagnostic and detection kits.
  • a method for treating or preventing diseases comprising administering the antibody or antigen-binding fragment, nucleic acid, vector, cell or pharmaceutical composition of the present invention to a subject in need.
  • a method for diagnosis and detection which includes administering the antibody or antigen-binding fragment, nucleic acid or kit of the present invention to a subject or sample in need.
  • the use of the antibody or antigen-binding fragment, nucleic acid, carrier, cell or pharmaceutical composition of any one of the foregoing aspects for the treatment and prevention of diseases is provided.
  • the use of the antibody or antigen-binding fragment, nucleic acid, or kit of any one of the foregoing aspects for detection and diagnosis is provided.
  • the disease is preferably a CGRP-related disease, further preferably, the CGRP-related disease is headache, and more preferably migraine (migrane), including episodic migrane, that is, every 4-14 attacks per month) and chronic migraine (chronic migrane, that is, the number of attacks per month>14).
  • migraine migraine
  • chronic migrane chronic migraine
  • the inventors succeeded in obtaining a class of anti-CGRP antibodies.
  • the experimental results show that the CGRP antibodies obtained in the present invention can effectively block the interaction between CGRP and its receptor, which is surprising
  • the optimized CGRP antibody of the present invention can effectively block the binding between CGRP and its receptor.
  • the optimized antibody After identification, the optimized antibody has high affinity, low immunogenicity, long half-life in vivo, and inhibits vasodilation The effect is remarkable.
  • the present invention has been completed on this basis.
  • the antibody of the present invention is designed to have engineered CDRs and some parts (all or part of the framework, hinge region, and constant region) of an antibody of human origin, which are the same as or identical to those derived from the human genome sequence. Basically the same (basically human).
  • the whole human framework, hinge region and constant region are human germline sequences as well as sequences with naturally occurring somatic mutations and sequences with engineered mutations.
  • the antibody of the present invention may comprise a framework, hinge or constant region, which is derived from a fully human framework, hinge or constant region containing one or more amino acid substitutions, deletions or additions therein.
  • the antibodies of the present invention are preferably substantially non-immunogenic in humans.
  • the antibody of the present invention is an IgG type antibody and has a "heavy" chain and a "light” chain cross-linked via intra-chain and inter-chain disulfide bonds.
  • Each heavy chain contains an N-terminal HCVR (or VH) and a heavy chain constant region (“HCCR”).
  • Each light chain contains an LCVR (or VL) and a light chain constant region (“LCCR”).
  • the HCVR and LCVR regions can be further subdivided into hypervariable regions (referred to as complementarity determining regions ("CDR")), which are interspersed in more conserved regions (referred to as framework regions ("FR”)).
  • CDR complementarity determining regions
  • FR framework regions
  • Each HCVR and LCVR is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3" and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3".
  • the CDR contains most of the residues that interact with the antigen.
  • CGRP and “CGRP ⁇ ” in the present invention have the same semantics without special instructions, and refer to any CGRP molecule or mutant that the antibody of the present invention can bind.
  • the full name of the candidate antibody or its abbreviation herein has the same meaning, and both refer to the same candidate antibody with the same light chain variable region and heavy chain variable region, and can be used interchangeably.
  • the full name of the candidate antibody is named CGRPM5-BA51.23 or CGRP-BA51.23 or CGRP-BA51.23-IgG2, it can be represented by the abbreviation "BA51.23" in this patent.
  • nucleic acid encoded by the term “nucleic acid” may include encoding nucleic acid, or may also include additional encoding and/or non-encoding sequences.
  • the "fusion protein" of the antibody or its antigen-binding fragment of the present invention refers to a protein form formed by connecting the antibody or its antigen-binding fragment of the present invention with other proteins by any conventional means in the art.
  • Conventional methods in the art include, but are not limited to: adding protein genes to form a long open reading frame, thereby expressing the corresponding fusion protein through gene expression; it is also possible to express the protein or protein unit separately, and then combine these proteins in a specific manner.
  • the structural units are linked together, for example, by modifying the corresponding protein unit to include a partner with good covalent binding ability, such as biotin/avidin (biotin/avidin) can enable the protein to interact through non-covalent interactions Form a fusion protein.
  • the "conservative substitution” in the present invention refers to the way in which one amino acid in the structure of a protein or polypeptide is replaced by another amino acid with similar physical and chemical properties. Such changes to the protein or polypeptide usually do not affect the function of the protein. Obvious or small impact. It should be understood that any amino acid substitution with the above characteristics is within the meaning of "conservative substitution” in the present invention.
  • Figure 1 shows the serum titer of BALB/c mice after three immunizations, and the serum titer is 12,500 times diluted.
  • Figure 2 shows the serum titer of BoAn-hMab mice after seven immunization, and the serum titer is 2500-fold dilution.
  • Figure 3 shows the binding activity between the candidate antibody and CGRP ⁇ protein detected by Elisa
  • Figure 4 shows the sensitivity of Elisa to detect the binding of candidate antibodies to CGRP ⁇
  • Figure 5 shows the binding sensitivity of the optimized BA51.23 antibody to CGPP ⁇
  • Figure 6 shows the detection of the binding activity of the optimized BA51.23 antibody to human CGRP
  • Figures 7A-C show that the optimized BA51.23 antibody stimulates capsaicin for vasodilation after 48h (Figure 7A), 96h ( Figure 7B), and 168h (Figure 7C) administered vehicle/antibody subcutaneously at a dose of 10 mg/kg Graph of inhibition over time
  • Figure 8A-C shows that the optimized BA51.23 antibody stimulates vasodilation of capsaicin after subcutaneous injection of vehicle/antibody at a dose of 10 mg/kg for 48h (Figure 8A), 96h ( Figure 8B), 168h (Figure 8C) AUC chart of inhibition
  • Figure 9 shows the optimized BA51.23 antibody in cynomolgus monkey drug-time curve (5mg/kg)
  • FIG 10 shows the immunogenicity test of CGRPM5-BA51.23-IgG2-N105D antibody
  • mice Human CGRP polypeptide (synthesized by GenScript) is coupled to KLH protein, and emulsified with gold adjuvant to immunize BoAn-hMab transgenic mouse with full human antibody. A total of 10 mice were immunized this time, and a total of 8 immunizations this time. The serum titers of mice after immunization are shown in Figure 2.
  • variable regions of the heavy and light chains were obtained from the cDNA by PCR, and then the heavy and light The variable region is obtained by overlapping extension PCR method to obtain ScFv, and the scFv is digested and ligated with plasmid pCOMB3x, and then the ligation product is electrotransfected into E. coli TG1 competent cells. After TG1 is cultured, phage infection is added, and the culture is recovered Supernatant.
  • the phage library CGRPM05 established with the mouse numbered CGRPM05 has a storage capacity of 2.4 ⁇ 10 8 ; the phage library CGRPM08 established with the mouse numbered CGRPM08 has a storage capacity of 1.7 ⁇ 10 8 .
  • Method 1 CGRP (1-37) protein (synthesized by GenScript) is coated with 1 ⁇ g/well and placed overnight at 4°C. The next day, the plate is blocked by 3% skimmed milk powder for 1h, and the phage library is added ( 2x 10 12 ) incubate for 2h, wash 4-10 times and elute the CGRP-bound phage with Elution Buffer (pH 2.2); Method 2: Neutroavidin protein (Thermo, 31000) is coated with 1 ⁇ g/plate, placed overnight at 4°C, After two days, the plate was blocked with 3% skimmed milk powder for 1 hour, 3 ⁇ g/library Biotin-CGRP (1-37) (GenScript synthesis) was added to incubate for 1 hour, then the phage library (2x 10 12 ) was added to incubate for 2 hours, washed 4-10 times before use Elution Buffer (pH 2.2) elutes the phage bound by CGRP
  • Magnetic bead screening obtained clones CGRPM5-BA51-IgG2 and CGRPM1-BA219-IgG2 (hereinafter referred to as BA51, BA219), where CGRPM1 represents the first wild-type mouse immunized, and BA represents magnetic bead screening.
  • the clones BA51, BA219, and QS89-2.3 were sent to Invitrogen Biotechnology Co., Ltd. for sequencing.
  • the antibody amino acid sequence is shown in Table 1.
  • the positive clones after sequencing were extracted with plasmids and then co-transfected into HEK293 cells. They were cultured in a shaker at 37°C ⁇ 8%CO2 ⁇ 125rpm. After 7 days of transient expression, the supernatant was purified by Protein A affinity chromatography to obtain CGRP antibody. UV280 combined with theoretical extinction coefficient to determine the antibody concentration.
  • Control antibody production Determine the amino acid sequence of CGRP antibody Fremanezumab through IMGT database and patent CN200680042443. After the complete gene synthesis, insert the vector pCDNA3.4 to express in HEK293 cells, and the antibody produced is named CGRP-TEVA-IgG2.
  • CGRP antibody Eptinezumab The amino acid sequence of CGRP antibody Eptinezumab was determined by IMGT database and patent CN201280035852. After the complete gene synthesis, it was inserted into the vector pCDNA3.4 and expressed in HEK293 cells, and the produced antibody was named CGRP-Alder-IgG2.
  • CGRP-Lilly-IgG2 The amino acid sequence of CGRP antibody galcanezumab was determined by IMGT database and patent CN201180028611. After the complete gene synthesis, it was inserted into the vector pCDNA3.4 and expressed in HEK293 cells, and the produced antibody was named CGRP-Lilly-IgG2.
  • Elisa was used to detect the binding of the antibody to different concentrations of CGRP ⁇ , so as to obtain an antibody with strong binding ability to CGRP ⁇ .
  • the above antibody sequence is inserted into the corresponding vector by conventional molecular biology techniques such as amplification, the cells are transfected and cultured, and the obtained antibody is purified and tested.
  • the experimental content in section 1.5 of the invention refer to the experimental content in section 1.5 of the invention.
  • Elisa to detect the binding ability of candidate antibodies to different concentrations of CGRP ⁇ , it is expected to obtain antibodies with strong binding ability to CGRP ⁇ .
  • Table 4 Elisa detects the binding sensitivity of candidate antibodies to CGRP ⁇
  • the antibody binding kinetics was measured using an Octet RED 96 instrument based on Biolayer Interferometry (BLI). Coupling ⁇ -CGRP (0.1 ⁇ g/mL) to Streptavidin(SA)Dip and Read TM Biosensors, the loading height is 0.19nm, and the antibody (antibody is stored in 25mM Tris, 0.1M Gly, 20mM NaCl, pH6.5 In the buffer), perform a 2-fold serial dilution with PBST, starting at 50 mM, and setting the concentration to 0, the Association time is set to 300s, and the Dissociation time is set to 900s. After the detection, use 1:1 Model Curve Fitting to calculate the binding constant (kon) and dissociation constant (kdis), and the equilibrium dissociation constant (kD) is calculated as the ratio kd/ka. The results are shown in Table 5.
  • the heavy chain N 105 of CGRPM5-BA51.23-IgG2 was mutated to D 105 and L 105 respectively through molecular biology technology.
  • the light chain variable region sequence of CGRPM5-BA51.23-IgG2-N105D is shown in SEQ ID NO: 9 (LCDR1-3 is shown in SEQ ID NO: 1-3), and the heavy chain variable region is shown in SEQ ID NO: 10 (HCDR1-3 is shown in SEQ ID NO: 4-6);
  • CGRPM5-BA51.23-IgG2-N105L light chain variable region sequence is shown in SEQ ID NO: 9 (LCDR1-3 is shown in SEQ ID NO: 1-3), the heavy chain variable region is shown in SEQ ID NO: 11 (HCDR1-3 is shown in SEQ ID NO: 4/5/7).
  • the above antibody sequence is inserted into the corresponding vector by conventional molecular biology techniques such as amplification, the cells are transfected and cultured, and the obtained antibody is purified and tested. For specific steps
  • CGRP antibodies Using cell-based cAMP activation experiments and binding experiments, the in vitro function of CGRP antibodies was tested. Dilute the cells with Stimulation Buffer, 4x 10 5 Cells/mL, and add them to a white 384 shallow-well plate, 5 ⁇ L/well, that is, 2000 cells/well. Dilute the sample (stored in 25mM Tris, 0.1M Gly, 20mM NaCl, pH6.5 buffer) with Stimulation Buffer to 200 ⁇ g/mL, and then perform 2-fold dilutions in sequence. A total of 8 concentration gradients are added to the cell wells. Medium, 2.5 ⁇ L/well. Dilute CGRP to 2 ⁇ g/mL with Stimulation Buffer and add it to cell wells at 2.5 ⁇ L/well.
  • the SD rat capsaicin-stimulated vasodilation model was used to study the function of CGRP antibody.
  • SD rats (6 in each group) were deeply anesthetized by intraperitoneal injection of sodium pentobarbital (50-60 mg/kg) and placed on a constant temperature blanket.
  • the vehicle/antibody (vehicle components: 10mM CH 3 COONa 3H 2 O, 30 mM NaCl, 0.03% Tween-20, 5% sucrose, pH 6.0, antibody stored in the solvent) was given by subcutaneous injection at a dose of 10 mg/kg for 48h After 96h and 168h, the baseline blood flow of the back skin was measured. After subcutaneous injection of capsaicin, the blood flow was continued to be measured.
  • the monthly migraine days of CGRP-Alder-IgG2 can be reduced by more than 50%.
  • the rate can reach more than 60% at a dose of 300mg.
  • the in vivo efficacy is better than other control antibodies, so we choose CGRP-Alder-IgG2 As the control antibody for this experiment.
  • the cynomolgus monkeys (2 in each group) were given subcutaneous injection of different CGRP antibodies (antibodies were stored in 10mM CH 3 COONa 3H 2 O, 30mM NaCl, 0.03% Tween-20, 5% sucrose, pH6 at a dose of 5 mg/kg).
  • the interval between administration of CGRP-TEVA-IgG2 can reach three months, so we chose CGRP-TEVA-IgG2 as the control antibody for this experiment.
  • the results show that compared with CGRP-TEVA-IgG2, CGRPM5-BA51.23-IgG2-N105D has a longer half-life, indicating that CGRPM5-BA51.23-IgG2-N105D has a longer-lasting therapeutic effect.
  • CGRPM5-BA51.23-IgG2-N105D can prolong the administration period, reduce treatment costs, and greatly relieve the economic pressure of patients; at the same time, CGRPM5-BA51.23-IgG2-N105D is compared with CGRP-TEVA -IgG2 can increase AUC and has higher bioavailability.
  • the Elisa method was used to detect the content of anti-drug antibody in monkey serum (monkey serum from Example 4.5) at different time points after subcutaneous administration of 5 mg/kg antibody.

Abstract

公开了一种新的抗体，尤其是一种结合CGRP抗原的抗体或其抗原结合片段，以及其制备方法、组合物和应用。本发明提供的抗体与CGRP抗原有较强的结合能力，免疫原性低，体内半衰期长，可用于治疗和/或预防CGRP相关疾病，如头痛；还可用于免疫学检测CGRP抗原。

Description

抗CGRP抗体及其应用 技术领域
本发明涉及生物医学或生物制药技术领域，更具体地涉及抗CGRP的抗体或其抗原结合片段，及其制备方法、组合物和应用。
背景技术
降钙素基因相关肽(CGRP)是由中枢和外周神经系统的神经细胞分泌的37个氨基酸的神经肽。人体中，CGRP存在两种形式：CGRPα和CGRPβ，二者仅相差3个氨基酸，且来自不同的基因(Doods,H.,Curr.Op.Invest.Drugs,2(9):1261-78(2001))。CGRP是由多种组织(例如三叉神经)释放的神经递质，当被活化时在脑膜中释放神经肽，从而介导神经原性炎症，特征为血管舒张、血管渗漏以及肥大细胞退化(Durham,P.L.,New Eng.J.Med.,350(11):1073-75(2004))。CGRP的生物作用是通过CGRP受体(CGRP-R)介导，CGRP受体是由七次跨膜成分结合受体相关蛋白(RAMP)组成，CGRP-R进一步需要受体成分蛋白(RCP)的活性，该受体成分蛋白(RCP)对于通过G蛋白与腺苷酸环化酶有效偶联以及cAMP的产生是必要的。CGRP是与其他血管运动症状的病理学相关的有效血管扩张剂，所述症状例如所有形式的血管性头痛，包括偏头痛(有先兆或无先兆)和丛集性头痛。患者偏头痛期间颈外静脉中的CGRP水平升高(Goadsby et al.,Ann.Neurol.28:183-7(1990))。静脉施用人α-CGRP在患有无先兆偏头痛的患者中诱导头痛和偏头痛，这提示CGRP在偏头痛中具有原因作用(Lassen et al.,Cephalalgia 22:54-61,(2002))。
目前，针对CGRP靶点，已有三个抗体产品获批上市。Fremanezumab，商品名为
Figure PCTCN2020092892-appb-000001
由Teva公司开发，获美国食品药品管理局(FDA)批准上市，是一种靶向于降钙素基因相关肽(CGRP)的人源化单克隆抗体，被批准用于治疗成人偏头痛(Urits,Ivan et al.Pain and therapy vol.9,1(2020):195-215.)。Eptinezumab，商品名
Figure PCTCN2020092892-appb-000002
是一种靶向降钙素基因相关肽(CGRP)的人源化单克隆抗体，可以阻断CGRP与其受体的结合，被批准用于成人偏头痛的预防性治疗(Dhillon S.2020；80(7):733‐739.)。Galcanezumab，商品名
Figure PCTCN2020092892-appb-000003
由礼来公司研发，获美国食品药品监督管理局(FDA)批准上市，后获欧洲药物管理局(EMA)批准上市，是一种人源化单克隆抗体，靶向于降钙素基因相关肽(CGRP)，被批准用于治疗成人患者偏头痛(Martin,V.,Samaan,K.H.,Aurora,S.et al.Adv Ther 37,2034–2049(2020))。根据美国FDA批准的以上三个上市抗体的药品说明书(Label)，Eptinezumab和Galcanezumab均每月一次皮下注射，Fremanezumab可每月一次或每3个月一次皮下注射。
然而，面对患者对于疾病治疗的药物需求，尤其是抗体药物的需求，仍然亟待有免疫原性更低、半衰期更长，药物效果更优的抗CGRP拮抗剂抗体。
发明内容
本发明提供了具有新的氨基酸序列的抗体或其抗原结合片段，所述抗体或其抗原结合片段为包括单克隆抗体、多克隆抗体、嵌合抗体、人源化抗体、Fab、Fab’、F(ab’)2、Fv、scFv或dsFv片段等。
本发明所提供的抗体或其抗原结合片段，包括：
1)3个轻链互补决定区(LCDR)，其中，LCDR1氨基酸序列如SEQ ID NO:1所示，LCDR2氨基酸序列如SEQ ID NO:2所示，LCDR3氨基酸序列如SEQ ID NO:3所示；和3个重链互 补决定区(HCDR)，其中，HCDR1氨基酸序列如SEQ ID NO:4所示，HCDR2氨基酸序列如SEQ ID NO:5所示，HCDR3氨基酸序列如SEQ ID NO:6或SEQ ID NO:7或SEQ ID NO:8所示，优选为SEQ ID NO:6；或者
2)相对于1)中的CDR序列组合的变体，其中所述变体在任意一个或多个CDR区上共有1-10个氨基酸被取代。
上述变体是指在任意一个或多个LCDR或HCDR区上的氨基酸被改变，优选被氨基酸取代，进一步优选保守取代；例如轻链可变区的LCDR1、LCDR2和/或LCDR3区共包含至少一个且不超过10个氨基酸改变；和/或重链可变区的HCDR1、HCDR2和/或HCDR3区共包含至少一个且不超过10个氨基酸改变。
在本发明的一个方面中，所述抗体或其抗原结合片段包含轻链可变区，其氨基酸序列如SEQ ID NO:9所示，或与SEQ ID NO:9所示的氨基酸序列具有至少80％、至少85％或至少90％同一性的氨基酸序列；还包含重链可变区，其氨基酸序列为SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12任一所示，或者与SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12具有至少80％、至少85％或至少90％同一性的氨基酸序列。优选地，所述抗体或其抗原结合片段包含以下轻、重链可变区序列：
1)包含SEQ ID NO:9所示的氨基酸序列轻链可变区和/或包含与SEQ ID NO:10所示的氨基酸序列的重链可变区，或者
2)包含SEQ ID NO:9所示的氨基酸序列的轻链可变区和/或SEQ ID NO:11所示的氨基酸序列的重链可变区，或者
3)包含SEQ ID NO:9所示的氨基酸序列的轻链可变区和/或包含与SEQ ID NO:12所示的氨基酸序列的重链可变区。
在一个方面，所述抗体或其抗原结合片段包含重链恒定区，其中所述重链恒定区包含γ-1、γ-2、γ-3或γ-4人重链恒定区或所述人重链恒定区的变体，优选地，其序列为ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。在一个方面，所述抗体还包含轻链恒定区，其中所述轻链恒定区包含λ或κ人轻链恒定区，优选地，其序列为TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。
在本发明的一个方面，上述抗体或其抗原结合片段的结合抗原是CGRP，优选地，所述CGRP是人CGRP。
本发明还提供了编码含有上述任一抗体或其抗原结合片段的核酸，所述抗体或其抗原结合片段的结合抗原是CGRP，尤其是指人CGRP。
在本发明的一个方面中，所述核酸编码含有以下氨基酸序列的抗体或其抗原结合片段：
1)包含3个轻链互补决定区(LCDR)，其中，LCDR1氨基酸序列如SEQ ID NO:1所示，LCDR2氨基酸序列如SEQ ID NO:2所示，LCDR3氨基酸序列如SEQ ID NO:3所示；和3个重链互补决定区(HCDR)，其中，HCDR1氨基酸序列如SEQ ID NO:4所示，HCDR2氨基酸序列如SEQ ID NO:5所示，HCDR3氨基酸序列如SEQ ID NO:6或SEQ ID NO:7或SEQ ID NO:8所示，优选为SEQ ID NO:6；或者
2)相对于1)中的CDR序列组合的变体，其中所述变体在任意一个或多个CDR区上共有1-10个氨基酸被取代。
本发明涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的核酸。本发明特别涉及在严格条件下与本发明所述核酸可杂交的核酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且,可杂交的核酸编码的多肽与成熟多肽有相同的生物学功能和活性。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还提供了含有编码结合CGRP的抗体或其抗原结合片段的核苷酸序列的载体，优选地，所述载体是表达载体，本发明所述载体包括但不限于，病毒载体，如腺病毒载体、逆转录病毒载体、腺相关病毒载体等；非病毒载体，如质粒、转座子载体等，其中质粒载体优选为pCDNA3.4(Life Technology)载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
本发明还提供了用于表达结合CGRP的抗体或其抗原结合片段的细胞，该细胞含有编码结合CGRP的抗体或其抗原结合片段的表达载体或编码结合CGRP的抗体或其抗原结合片段的核酸，优选地，所述细胞是含有上述表达载体的宿主细胞。在本发明的一个方面中，表达结合CGRP的抗体或其抗原结合片段的宿主细胞包括但不限于哺乳动物细胞、昆虫细胞、植物细胞、真菌细胞、原核细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。优选地，本发明提供用于表达结合CGRP的抗体或其抗原结合片段的宿主细胞是HEK293。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔，脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的抗体。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层 析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
根据本发明的另一方面，还提供一种药物组合物，其包含上文任一所述结合CGRP的抗体或其抗原结合片段、核酸、载体或细胞，以及药学上可接受的载体。所述药学上可接受的载体包括以下中的一种或多种：药学上可接受的溶剂、分散剂、附加剂、塑形剂、药物辅料。通常，这些物质是无毒的、惰性的和药学上可接受的载体介质。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或如局部给药(如注射给药)。
本发明还涉及一种试剂盒，其包含上文任一所述结合CGRP抗原的抗体或其抗体片段、核酸。在本发明的一个方面中,所述试剂盒包含以下任一组CDR氨基酸序列的抗体或其抗原结合片段：SEQ ID NO:1所示LCDR1，SEQ ID NO:2所示LCDR2，SEQ ID NO:3所示LCDR3，SEQ ID NO:4所示HCDR1，SEQ ID NO:5所示HCDR2，SEQ ID NO:6或SEQ ID NO:7或SEQ ID NO:8所示HCDR3。在本发明的一个方面中,所述试剂盒还包括用于对CGRP抗原抗体反应进行检测的检测试剂、阴性对照、阳性对照。
本发明的抗体或其抗原结合片段有广泛生物应用价值和临床应用价值，其应用涉及到与CGRP相关的疾病的诊断和治疗、基础医学研究、生物学研究、检测等多个领域。一个优选的应用是用于针对CGRP的临床诊断和靶向治疗。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、核酸、载体或细胞在制备治疗或预防疾病的药物组合物中的应用。
在另一方面，本发明涉及前述任一方面所述的抗体或其抗原结合片段、核酸在制备诊断、检测试剂盒中的应用。
在另一方面，提供一种治疗或预防疾病的方法，包括将本发明的抗体或抗原结合片段、核酸、载体、细胞或药物组合物给予有需要的受试者。
在另一方面，提供一种诊断、检测的方法，包括将本发明的抗体或抗原结合片段、核酸或试剂盒给予有需要的受试者或样本。
在另一方面，提供前述任一方面所述的抗体或其抗原结合片段、核酸、载体、细胞或药物组合物用于治疗、预防疾病的用途。
在另一方面，提供前述任一方面所述的抗体或其抗原结合片段、核酸、或试剂盒用于检测、诊断的用途。
在本发明的一个方面，所述疾病优选是CGRP相关疾病，进一步优选地，所述CGRP相关疾病是头痛，更近一步优选为偏头痛(migrane)，包括发作性偏头痛(episodic migrane，即每月发作4-14次)和慢性偏头痛(chronic migrane，即每月发作次数>14次)。
本发明人通过广泛而深入的研究，经过大量的筛选，成功获得一类抗CGRP抗体，实验结果表明，本发明获得的CGRP抗体能够有效阻断CGRP与其受体之间的相互作用，令人意外的是，经本发明优化后的CGRP抗体除能够有效阻断CGRP与其受体之间的结合外，经鉴定，优化后抗体亲和力高，免疫原性低，体内半衰期长，且对血管扩张的抑制作用显著。在此基础上完成了本发明。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理 解。
本发明的抗体被设计为具有工程改造的CDR且具有人类来源的抗体的一些部分(框架、铰链区和恒定区全部或部分)，所述部分与衍生自人类基因组序列的框架和恒定区相同或基本相同(基本是人类的)。全人类框架、铰链区和恒定区是人类种系序列以及具有天然存在的体细胞突变的序列和具有工程改造的突变的序列。本发明的抗体可包含框架、铰链或恒定区，其衍生自其中含有一个或多个氨基酸取代、缺失或添加的全人类框架、铰链或恒定区。此外，本发明的抗体优选为在人类中基本上为非免疫原性的。
本发明的抗体是IgG类型抗体且具有经由链内和链间二硫键交联的“重”链和“轻”链。每一重链包含N-末端HCVR(或VH)和重链恒定区(“HCCR”)。每一轻链包含LCVR(或VL)和轻链恒定区(“LCCR”)。
HCVR和LCVR区可进一步细分为超变区(被称为互补决定区(“CDR”))，其散布于更保守的区域(被称为框架区(“FR”))。每一HCVR和LCVR由三个CDR和四个FR组成，其从氨基末端至羧基末端以下列顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。本文中，重链的三个CDR被称为“HCDR1、HCDR2和HCDR3”且轻链的三个CDR被称为“LCDR1、LCDR2和LCDR3”。CDR含有大多数与抗原形成相互作用的残基。
本发明中的“CGRP”和“CGRPα”在无特殊说明下语义相同，指任何本发明所述抗体可结合的CGRP分子或突变体。
如无特别说明，本文中候选抗体全称或其简称具有相同含义，均指具有相同轻链可变区和重链可变区的相同候选抗体，可以互换使用。示例性地，如候选抗体全称以CGRPM5-BA51.23或CGRP-BA51.23或CGRP-BA51.23-IgG2命名，其在本专利中可使用简称“BA51.23”表示。
术语编码的“核酸”可以是包括编码的核酸，也可以是还包括附加编码和/或非编码序列的核酸。
本发明所述抗体或其抗原结合片段的“融合蛋白”，指通过任意本领域常规手段将本发明所述的抗体或其抗原结合片段与其他蛋白连接形成的蛋白形式，这部分所述的任意本领域常规手段包括但不限于：通过蛋白质基因加入以形成长的开放性阅读框，从而通过基因表达的方式表达相应融合蛋白；也可以通过单独表达蛋白质或蛋白单元，然后利用特定方式将这些蛋白质或结构单元连接在一起，例如通过在修饰相应蛋白单元使其包含具有良好共价结合能力的配偶体，比如生物素/抗生物素蛋白(biotin/avidin)可以使得蛋白质能够通过非共价相互作用形成融合蛋白。
本发明中的“保守取代”指将蛋白或多肽结构中一种氨基酸被另一种与其在物化性质上相近的氨基酸所替代的方式，这种对蛋白质或多肽的改变通常对蛋白质功能的不产生明显影响或影响很小。应当理解为任何具有上述特点的氨基酸取代均在本发明所述的“保守取代”含义内。
附图说明
图1示出了BALB/c小鼠三免后血清滴度，血清滴度为12500倍稀释。
图2示出了BoAn-hMab小鼠七免后血清滴度，血清滴度为2500倍稀释。
图3示出了Elisa检测候选抗体与CGRPα蛋白之间的结合活性
图4示出了Elisa检测候选抗体与CGRPα的结合灵敏度
图5示出了优化后的BA51.23抗体与CGPPα的结合灵敏度
图6示出了优化后的BA51.23抗体与human CGRP结合活性检测
图7A-C示出了按照10mg/kg剂量皮下注射给予溶媒/抗体48h(图7A)，96h(图7B)，168h(图7C)后，优化后的BA51.23抗体对辣椒素刺激血管扩张的抑制作用随时间变化图
图8A-C示出了按照10mg/kg剂量皮下注射给予溶媒/抗体48h(图8A)，96h(图8B)，168h(图8C)后，优化后的BA51.23抗体对辣椒素刺激血管扩张的抑制作用AUC图
图9示出了优化后的BA51.23抗体在食蟹猴体内药时曲线(5mg/kg)
图10示出了CGRPM5-BA51.23-IgG2-N105D抗体免疫原性检测
具体实施方式
参照以下实施例可以更好地理解本发明。但是，应理解，以下实施例仅用于举例说明目的，而不应被理解为以任何方式限制本发明的保护范围。
实施例1.抗CGRP单克隆抗体的产生
1.1免疫
野生型小鼠免疫：将人CGRP多肽(GenScript合成)( https://www.ncbi.nlm.nih.gov/prote in/1005250A？report＝genbank&log$＝protalign&blast_rank＝14&RID＝907JCDCA014#feature_100 5250A；NCBI ACCESSION：1005250A)偶联到KLH蛋白上，然后与弗氏佐剂乳化后免疫BALB/c小鼠。首次免疫使用弗氏完全佐剂，二免至三免使用弗氏不完全佐剂，本次共免疫5只小鼠。选取血清滴度较高的小鼠加强免疫，4天后处死小鼠取出脾脏用于后续实验。小鼠免疫后血清滴度如图1所示。
转基因小鼠免疫：将人CGRP多肽(GenScript合成)偶联到KLH蛋白上，与金佐剂乳化后免疫博安生物全人抗体转基因小鼠BoAn-hMab。本次共免疫10只小鼠，本次共免疫八次。小鼠免疫后血清滴度如图2所示。
1.2噬菌体库的建立
取免疫小鼠的脾脏细胞，加入Trizol(Thermo Scientific，目录货号15596-026)，待裂解充分后加入1/5体积的氯仿，充分混匀，室温放置20min后4℃12000rpm离心20min，取上层水溶液，并加入等体积的异丙醇，室温放置20min，4℃ 12000rpm离心20min，弃去上清水溶液，加入75％乙醇洗涤两次，4℃ 12000rpm离心5min，弃去水溶液，保留沉淀，室温风干后加入DEPC水重悬沉淀获得RNA，获得的RNA使用罗氏反转录试剂盒Transcriptor First Strand cDNA Synthesis Kit(Roche Applied Science，目录货号4897030001)按照其说明书将RNA反转录成cDNA。噬菌体库的建立步骤参照Carlos F.Barbas III，Phage display：A laboratory manual中记载的方法进行，用PCR的方法从cDNA中获得重链和轻链的可变区，再将重链和轻链的可变区通过重叠延伸PCR的方法获得ScFv，ScFv酶切后与质粒pCOMB3x连接，然后将连接产物电转染至大肠杆菌TG1感受态细胞中，TG1经培养后加入噬菌体侵染，然后回收培养物上清。以编号为CGRPM05的小鼠建立的噬菌体库CGRPM05，库容2.4×10 8；以编号为CGRPM08的小鼠建立的噬菌体库CGRPM08，库容1.7×10 8。
1.3以两种方法进行筛选
平板筛选用2种方法，方法一：CGRP(1-37)蛋白(GenScript合成)以1μg/孔包被平板，4℃放置过夜，第二天通过3％脱脂奶粉封闭平板1h，加入噬菌体库(2x 10 12)孵育2h，洗涤4-10次后用Elution Buffer(pH 2.2)洗脱CGRP结合的噬菌体；方法二：Neutroavidin 蛋白(Thermo，31000)以1μg/包被平板，4℃放置过夜，第二天通过3％脱脂奶粉封闭平板1h，加入3μg/库的Biotin-CGRP(1-37)(GenScript合成)孵育1h，再加入噬菌体库(2x 10 12)孵育2h，洗涤4-10次后用Elution Buffer(pH 2.2)洗脱CGRP结合的噬菌体。
磁珠筛选，将Biotin-CGRP(1-37)与磁珠(Invitrogen Dynabeads M-280 Streptavidin，00355871)结合1h后与噬菌体库孵育，洗涤4-10次后用Elution Buffer(pH 2.2)洗脱,从而获得CGRP结合的克隆。
磁珠筛选获得克隆CGRPM5-BA51-IgG2和CGRPM1-BA219-IgG2(以下简称BA51、BA219)，其中CGRPM1代表免疫的第1只野生型小鼠，BA代表磁珠筛选。
1.4杂交瘤筛选
取CGRPQ Q40/Q42小鼠解剖取脾获得B细胞悬液，CGRPQ40/Q42脾细胞各取1x10 7cells与1x10 7的SP2/0细胞电融合，次日半固体铺皿，10天后挑单克隆至96孔板，用有限稀释法将稀释后的阳性细胞加入到96孔板中，共四个梯度：8cells/well、4cells/well、2cells/well、1cells/well，于37℃5％CO2培养9天；每个克隆挑选8个亚克隆于96w中，细胞活性检测后的阳性亚克隆转6孔板，提取亚克隆的RNA，反转录后获得cDNA，通过PCR的方法获得各亚克隆的重链和轻链可变区，用于抗体分子的构建与生产。此方法筛选获得了CGRPQS89-2.3-抗体(以下简称QS89-2.3)。
1.5抗体分子的构建与生产
将克隆BA51、BA219、QS89-2.3送Invitrogen生物技术有限公司测序，抗体氨基酸序列如表1所示。
表1.活性克隆氨基酸序列
Figure PCTCN2020092892-appb-000004
通过常规的分子生物学技术PCR(2*Phanta Max Master Mix厂家：Vazyme货号：P515-AA批号：TE211G8)扩增抗体可变区基因，借助重叠延伸PCR将信号肽与可变区基因连接，通过同源重组(ClonExpress Ⅱ One Step Cloning Kit厂家：Vazyme货号：C112-01批号：TE211L8)分别将抗体重链可变区基因连接入带有抗体Fc(IgG2亚型)序列的载体pCDNA3.4(Life Technology)，将抗体轻链可变区基因连接入带有抗体轻链恒定区序列的载体pCDNA3.4，序 列如下所示。
轻链恒定区序列：
Figure PCTCN2020092892-appb-000005
重链恒定区序列：
Figure PCTCN2020092892-appb-000006
将测序后的阳性克隆提取质粒后共转染进入HEK293细胞在37℃\8％CO2\125rpm摇床中培养，瞬时表达7天后上清通过Protein A亲和层析，纯化获得CGRP抗体，并通过UV280结合理论消光系数确定抗体浓度。
对照抗体生产：通过IMGT数据库及专利CN200680042443确定CGRP抗体Fremanezumab的氨基酸序列。全基因合成后插入载体pCDNA3.4通过HEK293细胞表达，生产的抗体命名为CGRP-TEVA-IgG2。
通过IMGT数据库及专利CN201280035852确定CGRP抗体Eptinezumab的氨基酸序列。全基因合成后插入载体pCDNA3.4通过HEK293细胞表达，生产的抗体命名为CGRP-Alder-IgG2。
通过IMGT数据库及专利CN201180028611确定CGRP抗体galcanezumab的氨基酸序列。全基因合成后插入载体pCDNA3.4通过HEK293细胞表达，生产的抗体命名为CGRP-Lilly-IgG2。
表2对照抗体氨基酸序列
Figure PCTCN2020092892-appb-000007
1.6利用Elisa检测抗体与CGRPα的结合活性
为了确定获得的抗体与抗原的结合能力，利用Elisa检测抗体与不同浓度CGRPα的结合情况，从而获得与CGRPα具有较强结合能力的抗体。
包被不同浓度(0.2μg/mL、0.1μg/mL、0.05μg/mL、0.025μg/mL、0.0125μg/mL、0.00625μg/mL、0.003125μg/mL、0μg/mL)的抗原CGRPα(4013281-1000，Bachem)，100μL/孔4度过夜；用3％脱脂奶粉37℃封闭1h；每孔加入1μg/mL候选抗体各100ul(抗体保存在25mM Tris，0.1M Gly，20mM NaCl，pH6.5的缓冲液中)，37℃孵育1h；然后加入山羊抗人 IgG/HRP，37℃孵育1h，显色10min后，酶标仪上读取OD450。
实验结果如图3所示，抗体CGRPM5-BA51-IgG2、CGRPM1-BA219-IgG2和CGRP QS89-2.3-IgG2在实验所示浓度范围内均有与抗原较高的结合能力。
实施例2.人源化抗体的获得
2.1抗体人源化
借助Discovery Studio软件对抗体进行三维结构模拟，结合CDR graft对鼠源序列进行人源化改造，通过回复突变分析确定回复突变位点，最终获得抗体CGRPM5-BA51.23、BA51.27、CGRPM1-BA219.53、BA219.54(本发明中以下简称BA51.23、BA51.27、BA219.53、BA219.54)。BA51.23克隆经序列分析重链属于胚系基因IGHV1-2家族，轻链属于胚系基因IGKV1-33家族。序列如表3所示。
表3人源化抗体氨基酸序列
Figure PCTCN2020092892-appb-000008
以上抗体序列经扩增等常规的分子生物学技术插入相应载体，细胞转染并培养，所得抗体纯化并检测，具体步骤参考发明1.5节实验内容。
实施例3.候选抗体的表征
3.1 Elisa检测候选抗体与CGRPα蛋白的结合
利用Elisa检测候选抗体与不同浓度CGRPα的结合能力，期望获得与CGRPα具有较强结合能力的抗体。
包被不同浓度(0.2μg/mL、0.1μg/mL、0.05μg/mL、0.025μg/mL、0.0125μg/mL、0.00625μg/mL、0.003125μg/mL、0μg/mL)的抗原CGRPα(4013281-1000，Bachem)，100μL/ 孔4度过夜；用3％脱脂奶粉37℃封闭1h；每孔加入1μg/ml候选抗体各100μL(抗体保存在25mM Tris，0.1M Gly，20mM NaCl，pH6.5的缓冲液中)，37℃孵育1h；然后加入山羊抗人IgG/HRP，37℃孵育1h，显色10min后，酶标仪上读取OD450。结果见图4和表4。
结果显示CGRPM5-BA51.23-IgG2、CGRPM5-BA51.27-IgG2、与对照抗体CGRP-TEVA-IgG2、CGRP-LILY-IgG2具有接近的灵敏度。
表4 Elisa检测候选抗体与CGRPα结合灵敏度
样品 EC50(μg/mL)
CGRPM5-BA51.23-IgG2 0.036
CGRPM5-BA51.27-IgG2 0.034
CGRP-QS89-2.3-IgG2 0.083
CGRP-TEVA-IgG2 0.040
CGRP-ALD-IgG2 0.031
CGRP-LILY-IgG2 0.037
3.2 BLI检测候选抗体与CGRP蛋白的结合
抗体结合动力学使用基于生物膜干涉技术(Biolayer Interferometry BLI)Octet RED 96仪器测量。将α-CGRP(0.1μg/mL)偶联到Streptavidin(SA)Dip and Read TM Biosensors上，loading高度为0.19nm，将抗体(抗体保存在25mM Tris，0.1M Gly，20mM NaCl，pH6.5的缓冲液中)用PBST进行2倍连续稀释，50mM起始，并设置0浓度，Association时间设置为300s，Dissociation时间设置为900s。检测结束后使用1:1Model的Curve Fitting计算结合常数(kon)、解离常数(kdis)，平衡解离常数(kD)以比率kd/ka计算。结果见表5。
结果显示：抗体CGRPM5-BA51.23-IgG2、CGRPM5-BA51.27-IgG2与CGRP-TEVA-IgG2相比有着更弱的解离趋势，CGRPM5-BA51.23-IgG2、CGRPM5-BA51.27-IgG2的亲和力强于CGRP-TEVA-IgG2。
表5.Octet检测候选抗体结合动力学
抗体ID Kon(1/Ms) Kdis(1/s) KD(M)
CGRPM5-BA51.23-IgG2 1.22E+05 <1.0E-07 <1.0E-12
CGRPM5-BA51.27-IgG2 1.62E+05 <1.0E-07 <1.0E-12
CGRP-TEVA-IgG2 5.93E+05 1.60E-05 2.71E-11
CGRP-QS89-2.3-IgG2 2.02E+05 6.42E-05 3.18E-10
实施例4 BA51.23抗体优化
4.1.BA51.23抗体序列优化
为了进一步获得效果更优的CGRP抗体分子，通过分子生物学技术将CGRPM5-BA51.23-IgG2的重链N 105分别突变为D 105和L 105。CGRPM5-BA51.23-IgG2-N105D轻链可变区序列如SEQ ID NO:9所示(LCDR1-3如SEQ ID NO:1-3所示)，重链可变区如SEQ ID NO:10所示(HCDR1-3如SEQ ID NO:4-6所示)；CGRPM5-BA51.23-IgG2-N105L轻链可变区序列如SEQ ID NO:9所示(LCDR1-3如SEQ ID NO:1-3所示)，重链可变区如SEQ ID NO:11所示(HCDR1-3如SEQ ID NO:4/5/7所示)。以上抗体序列经扩增等常规的分子生物学技术插入相应载体，细胞转染并培养，所得抗体纯化并检测，具体步骤参考发明1.5节实验内容。
4.2利用Elisa检测优化后抗体与CGRPα的结合灵敏度
利用Elisa检测优化后抗体与不同浓度CGRPα的结合能力，期望获得与CGRPα具有较强结合能力的抗体。
包被不同浓度(0.2μg/mL、0.1μg/mL、0.05μg/mL、0.025μg/mL、0.0125μg/mL、0.00625μg/mL、0.003125μg/mL、0μg/mL)的抗原CGRPα(4013281-1000，Bachem)，100μL/孔4度过夜；用3％脱脂奶粉37℃封闭1h；每孔加入1μg/ml候选抗体(样品保存在25mM Tris，0.1M Gly，20mM NaCl，pH6.5的缓冲液中)各100μL，37℃孵育1h；然后加入山羊抗人IgG/HRP，37℃孵育1h，显色10min后，酶标仪上读取OD450。结果见图5及表6：
结果显示优化后抗体CGRPM5-BA51.23-IgG2-N105D和CGRPM5-BA51.23-IgG2-N105L比CGRPM5-BA51.23-IgG2具有强的结合能力。
表6优化后BA51.23与CGPPα的结合灵敏度EC50
ID EC50(ng/ml)
CGRPM5-BA51.23-IgG2-N105L 10.59
CGRPM5-BA51.23-IgG2-N105D 8.437
CGRPM5-BA51.23-IgG2 13.45
4.3优化后BA51.23抗体与CGRP结合活性检测
使用基于细胞的cAMP活化实验和结合实验，对CGRP抗体的体外功能进行检测。用Stimulation Buffer稀释细胞，4x 10 5Cells/mL，加入到白色384浅孔板中，5μL/孔，即2000个/孔。用Stimulation Buffer把样品(样品保存在25mM Tris，0.1M Gly，20mM NaCl，pH6.5的缓冲液中)稀释至200μg/mL,再依次进行2倍稀释，共8个浓度梯度，加入到细胞孔中，2.5μL/孔。用Stimulation Buffer把CGRP稀释至2μg/mL，加入到细胞孔中，2.5μL/孔。室温孵育1.5h。用Lysis buffer把cAMP-D2稀释5倍，加入到细胞孔中，5μL/孔。用Lysis buffer把Anti-cAMP-Cryptate稀释5倍，加入到细胞孔中，5μl/孔。室温避光孵育1h。用酶标仪HTRF模块读数。结果见图6和表7：
通过对细胞功能活性的测定，结果显示，优化后的抗体均具有良好的CGRP结合活性。
表7.优化后BA51.23与CGRP结合活性检测EC50
ID EC50(μg/mL)
CGRPM5-BA51.23-IgG2-N105L 44.85
CGRPM5-BA51.23-IgG2-N105D 32.00
4.4优化后BA51.23抗体在SD大鼠辣椒素刺激血管扩张模型中的药效学研究
用SD大鼠辣椒素刺激血管扩张模型进行CGRP抗体的功能研究。SD大鼠(每组6只)由戊巴比妥钠(50-60mg/kg)腹腔注射深度麻醉后，置于恒温毯。按照10mg/kg的剂量皮下注射给予溶媒/抗体(溶媒成分为：10mM CH 3COONa■3H 2O，30mM NaCl，0.03％Tween-20，5％蔗糖，pH6.0，抗体保存在溶媒中)48h，96h，168h后，进行背部皮肤血流基线水平测定，并于皮下注射辣椒素后，继续测定血流。血流测定用双面胶将激光多普勒皮肤血流测定探头牢固粘附于待测试皮肤，打开激光多普勒血流仪及记录软件，记录10min血流平稳基线后，在待测皮肤皮下注射辣椒素后，继而继续测定20～30min，以每分钟的血流平均值作为一个perfusion unit(PU)，结果见图7A-7C。通过时间×血流变化率得到曲线下面积AUC，反应血流抑制效果，结果见图7A-7C和图8A-8C。
根据FDA公开的药品说明书信息，CGRP-Alder-IgG2的月偏头痛天数减少50％以上比率 在300mg剂量下可达到60％以上，体内药效优于其他对照抗体，所以我们选择CGRP-Alder-IgG2作为本次实验的对照抗体。结果显示，给药96h后，CGRP-Alder-IgG2的血流检测的AUC为CGRPM5-BA51.23-IgG2-N105D的1.36倍；给药168h后，CGRP-Alder-IgG2的血流检测的AUC为CGRPM5-BA51.23-IgG2-N105D的1.82倍；说明CGRPM5-BA51.23-IgG2-N105D相较于CGRP-Alder-IgG2等其他抗体对辣椒素刺激血管扩张的抑制作用更明显，预示着CGRPM5-BA51.23-IgG2-N105D有更好的临床治疗效果。
4.5 CGRP抗体在食蟹猴PK上的研究
按照5mg/kg的给药剂量给予食蟹猴(每组2只)皮下注射不同CGRP抗体(抗体保存于10mM CH 3COONa■3H 2O，30mM NaCl，0.03％Tween-20，5％蔗糖，pH6.0的缓冲液中)，并于给药前(0h)及给药后1h、4h、10h、1d、2d、3d、4d、5d、7d、10d、14d、21d和28d经静脉取出全血样品，置血样收集管中，冰盒内让其自然凝固，于血样取出后8h内置离心机中，1000～3000g离心10min，分离血清，置样本保存管中，用Elisa的方法检测抗体在食蟹猴体内的代谢情况。结果见图9和表8。
CGRP-TEVA-IgG2给药间隔可达到三个月，所以我们选择CGRP-TEVA-IgG2作为本次实验的对照抗体。结果显示相比于CGRP-TEVA-IgG2，CGRPM5-BA51.23-IgG2-N105D有更长时间的半衰期，预示着CGRPM5-BA51.23-IgG2-N105D具有更持久的治疗效果，在Fremanezumab三个月给药一次的基础上，CGRPM5-BA51.23-IgG2-N105D可延长给药周期，降低治疗费用，大大缓解患者的经济压力；同时，CGRPM5-BA51.23-IgG2-N105D相比于CGRP-TEVA-IgG2可提高AUC，具有更高的生物利用度。
表8.候选抗体在食蟹猴体内代谢
Parameter Unit CGRP-TEVA-IgG2 CGRPM5-BA51.23-IgG2-N105D
t 1/2 h 138.6 195.7
C max μg/mL 56.1 52.6
T max h 36.0 36.0
AUC (0-t) μg/mL*h 11460.8 13081.0
AUC (0-∞) μg/mL*h 12017.3 15001.7
V z/F mL/kg 81.6 95.0
CL z/F mL/h/kg 0.42 0.388
MRT (0-t) h 153.1 204.0
4.6 CGRP抗体免疫原性检测
利用Elisa的方法检测皮下给药5mg/kg抗体后不同时间点猴血清(猴血清来自于实施例4.5))中抗药抗体的含量。以CBS包被液(pH9.6碳酸溶液)包被CGRP-TEVA 0.125μg/mL、CGRPM5-BA51.23-IgG2-N105D 0.0625μg/mL，100μL/孔4度过夜；用3％脱脂奶粉37℃封闭1h；每孔加入100X食蟹猴血清，100μL，37度孵育1h；然后加入CGRP-TEVA-biotin 0.125μg/mL、CGRPM5-BA51.23-IgG2-N105D-biotin 0.0625μg/mL、，37℃孵育1h，洗掉，继而加入链霉素/HRP，37℃孵育1h；TMB显色10min后，酶标仪上读取OD450。检测结果见图10。
结果显示CGRPM5-BA51.23-IgG2-N105D与CGRP-TEVA-IgG2均没有检测到免疫原性。

Claims (10)

  1. 一种抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包括：
    1)3个轻链互补决定区，其中，LCDR1氨基酸序列如SEQ ID NO:1所示，LCDR2氨基酸序列如SEQ ID NO:2所示，LCDR3氨基酸序列如SEQ ID NO:3所示；和
    3个重链互补决定区，其中，HCDR1氨基酸序列如SEQ ID NO:4所示，HCDR2氨基酸序列如SEQ ID NO:5所示，HCDR3氨基酸序列如SEQ ID NO:6或SEQ ID NO:7或SEQ ID NO:8所示，优选为SEQ ID NO:6；或者
    2)相对于1)中的CDR序列组合的变体，其中所述变体在任意一个或多个CDR区上共有1-10个氨基酸被取代。
  2. 一种抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含与SEQ ID NO:9所示的氨基酸序列具有至少80％、至少85％或至少90％同一性的氨基酸序列的轻链可变区，和/或包含与SEQ ID NO:10、SEQ ID NO:11或SEQ ID NO:12任一项所示的氨基酸序列具有至少80％、至少85％或至少90％同一性的氨基酸序列的重链可变区。
  3. 根据权利要求2所述的抗体或其抗原结合片段，其特征在于包括氨基酸序列为SEQ ID NO:9的轻链可变区；和氨基酸序列为SEQ ID NO:10或SEQ ID NO:11或SEQ ID NO:12的重链可变区，优选重链可变区氨基酸序列为SEQ ID NO:10。
  4. 根据权利要求1至3任一所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段结合抗原是CGRP。
  5. 一种核酸，其编码权利要求1-4任一项所述抗体或其抗原结合片段。
  6. 一种载体，其包含权利要求5的核酸；优选地，所述载体是表达载体。
  7. 一种细胞，其包含权利要求5的核酸或权利要求6的载体。
  8. 一种药物组合物，其特征在于含有权利要求1-4任一项所述抗体或其抗原结合片段，或权利要求5所述的核酸，或权利要求6所述的载体，或权利要求7所述的细胞，及药学可接受的载体。
  9. 一种试剂盒，含有权利要求1-4任一项所述抗体或其抗原结合片段，或权利要求5所述的核酸。
  10. 权利要求1-4任一项所述抗体或其抗原结合片段，或权利要求5所述的核酸用于预防、治疗、检测或诊断与CGRP相关的疾病的应用，优选地，所述CGRP相关疾病是头痛，更优选为偏头痛。
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