WO2017121307A1 - 一种C5aR抗体及其制备方法和应用 - Google Patents

一种C5aR抗体及其制备方法和应用 Download PDF

Info

Publication number
WO2017121307A1
WO2017121307A1 PCT/CN2017/070713 CN2017070713W WO2017121307A1 WO 2017121307 A1 WO2017121307 A1 WO 2017121307A1 CN 2017070713 W CN2017070713 W CN 2017070713W WO 2017121307 A1 WO2017121307 A1 WO 2017121307A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
sequence listing
acid sequence
sequence
Prior art date
Application number
PCT/CN2017/070713
Other languages
English (en)
French (fr)
Inventor
杨翠青
段清
徐丽娜
顾红专
刘礼乐
Original Assignee
上海开拓者生物医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海开拓者生物医药有限公司 filed Critical 上海开拓者生物医药有限公司
Priority to CN202110368308.1A priority Critical patent/CN112851817B/zh
Priority to CN201780003251.7A priority patent/CN108026178B/zh
Priority to CN202110369308.3A priority patent/CN113150151B/zh
Priority to US16/965,209 priority patent/US20210070875A1/en
Publication of WO2017121307A1 publication Critical patent/WO2017121307A1/zh

Links

Images

Classifications

    • 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
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the invention relates to the field of antibodies, in particular to a C5aR antibody and a preparation method and application thereof.
  • the complement system is a protein reaction system consisting of more than 30 kinds of plasma proteins and membrane proteins, which are widely distributed in blood, tissue fluid and cell surface, and have precise regulation mechanism.
  • the main physiological function of complement is to promote the phagocytic ability of phagocytic cells and to lyse target cells, so it is an important part of the body's immune defense mechanism.
  • the ligand C5a of C5aR which is the activation product of the complement system, is an important mediator and chemokine of the inflammatory response.
  • the binding of C5aR to the ligand C5a induces an inflammatory response and affects the coagulation and fibrinolysis system, resulting in damage to normal tissue cells, which is involved in the pathological processes of various diseases.
  • C5a is the medium with the strongest anaphylatoxin activity in the complement cleavage fragment, and its anaphylatoxin action is 20 times and 2500 times that of C3a and C4a, respectively.
  • C5a can also increase the permeability of blood vessels and stimulate smooth muscle contraction.
  • C5a High concentrations of C5a are chemotactic agents for neutrophils, eosinophils, and monocytes that induce these cells to move in a concentration gradient.
  • C5a has a significant enhancement effect on immune response, which can induce the secretion of cytokines such as interleukin IL-1, IL-6, IL-8 and tumor necrosis factor alpha (TNF- ⁇ ) by monocytes, and promote antigen and alloantigen. Induced T cell proliferation and promotion of antibody production by B cells.
  • cytokines such as interleukin IL-1, IL-6, IL-8 and tumor necrosis factor alpha (TNF- ⁇ )
  • C5aR belongs to the family of seven transmembrane G-protein coupled receptors.
  • C5aR is a receptor with high affinity for C5a, wherein Kd is about 1 nM, which is located on different types of cells including white blood cells. The number of receptors per cell is very high, and each white cell can be as high as 200,000 sites.
  • C5a has another receptor called c5a like receptor (C5L2), but it is also called GPR77 because it does not contain G protein.
  • GPR77 belongs to the C5a receptor subfamily and has 35% homology with CD88 in amino acid sequence. Its biological function is still unclear.
  • C5aR is involved in the pathogenesis of various diseases including rheumatoid arthritis, psoriasis, sepsis, reperfusion injury, and adult respiratory distress syndrome (Gerard and Gerard, 1994; Murdoch and Finn, 2000). How to block the transmission of C5aR signaling to downstream signaling pathways, thereby reducing inflammation, has always been a hot issue in immunology research.
  • antagonists of C5aR and C5a are mainly classified into anti-C5a antibody, anti-C5aR antibody, small molecule antagonist, C5a antisense peptide, C5a mutant or bacterial-derived chemotactic inhibitory protein.
  • C5aR and its ligands there are currently no new antagonists for C5aR and its ligands.
  • the technical problem to be solved by the present invention is to provide a C5aR antibody with high affinity and high specificity, and a preparation method and application thereof, in order to overcome the deficiency of the current lack of C5aR antibody.
  • the C5aR antibody has high affinity with the human C5aR protein; it can efficiently inhibit or block the binding of C5aR to C5a, significantly reduce the calcium flow signal, thereby down-regulating or cutting off the corresponding signaling pathway, and stopping the C5a of neutrophils in vitro. Directed migration; and can significantly inhibit the chemotaxis of neutrophils; thus giving hope for the treatment of diseases of inflammation, blood vessels or nervous system.
  • the present inventors used the human C5aR protein as an immunogen, and cloned the antibody heavy chain variable region and the light chain variable region gene using an optimized hybridoma technique, and the variable region gene can be grafted to the human antibody constant region gene to form a human.
  • a murine chimeric antibody a leader antibody that obtains a C5aR antibody.
  • it has high affinity with proteins such as human C5aR protein; it can effectively inhibit or block the binding of C5aR and C5a, and significantly reduce the calcium flow signal;
  • the amino acid sequence of the heavy chain variable region of the C5aR antibody and the light chain variable region of the C5aR antibody is then sequenced by molecular biological methods.
  • the present invention provides an isolated protein comprising one or more of a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3 of a C5aR antibody, and/or a light chain CDR1, a light chain CDR2 and a light chain of a C5aR antibody
  • a heavy chain CDR1 is SEQ ID No. 2, SEQ ID No. 10, SEQ ID No. 18, SEQ ID No. 26, SEQ ID No. 34 in the sequence listing.
  • the amino acid sequence of the heavy chain CDR2 is SEQ ID No. 3, SEQ ID No. 11, SEQ ID No. 19, SEQ ID No. 27, SEQ ID No. 35, SEQ ID No.
  • amino acid sequence of the heavy chain CDR3 is SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 20 in the sequence listing, SEQ ID No. 28, SEQ ID No. 36, SEQ ID No. 44 or SEQ ID No. 52;
  • amino acid sequence of the light chain CDR1 is SEQ ID No. 6, SEQ ID No. 14, in the sequence listing, SEQ ID No. 22, SEQ ID No. 30, SEQ ID No. 38, SEQ ID No. 46 or SEQ ID No. 54;
  • amino acid sequence of the light chain CDR2 is SEQ ID No. 7, in the sequence listing, SEQ ID No. 15, SEQ ID No. 23, SEQ ID No. 31, SEQ ID No. 39, SEQ ID No.
  • amino acid sequence of the light chain CDR3 is SEQ ID No. 8, SEQ ID No. 16, SEQ in the sequence listing. ID No. 24, SEQ ID No. 32, SEQ ID No. 40, SEQ ID No. 48 or SEQ ID No. 56;
  • the amino acid sequence of the heavy chain CDR1 is as SEQ ID No. 2, SEQ ID No. 10, SEQ ID No. 18, SEQ ID No. 26, SEQ ID No. 34, SEQ ID No. 42 in the sequence listing.
  • the amino acid sequence of SEQ ID No. 50 having at least 80% sequence homology; the amino acid sequence of the heavy chain CDR2 and SEQ ID No. 3, SEQ ID No. 11 in the sequence listing SEQ ID No. 19, SEQ ID No. 27, SEQ ID No. 35, The amino acid sequence of SEQ ID No. 43 or SEQ ID No. 51 having at least 80% sequence homology; the amino acid sequence of the heavy chain CDR3 and SEQ ID No. 4 in the sequence listing, The amino acid sequence represented by SEQ ID No.
  • SEQ ID No. 20 SEQ ID No. 28, SEQ ID No. 36, SEQ ID No. 44 or SEQ ID No. 52 has at least 80% sequence homology.
  • the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 6, SEQ ID No. 14, SEQ ID No. 22, SEQ ID No. 30, SEQ ID No. 38, SEQ ID in the sequence listing.
  • the amino acid sequence of No. 46 or SEQ ID No. 54 having at least 80% sequence homology; the amino acid sequence of the light chain CDR2 and SEQ ID No. 7, SEQ ID of the sequence listing Amino acid sequence having at least 80% sequence homology of the amino acid sequence represented by No. 15, SEQ ID No. 23, SEQ ID No. 31, SEQ ID No. 39, SEQ ID No.
  • amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 8, SEQ ID No. 16, SEQ ID No. 24, SEQ ID No. 32, SEQ ID No. 40 of the sequence listing,
  • amino acid sequence represented by SEQ ID No. 48 or SEQ ID No. 56 is represented by an amino acid sequence having at least 80% sequence homology.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 2 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 3 of the Sequence Listing and the amino acid sequence of the heavy chain CDR3.
  • SEQ ID No. 4 the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 10 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No. 11 of the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 12 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No.
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 20 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 26 of the Sequence Listing, and the heavy chain CDR2
  • the amino acid sequence is shown in SEQ ID No. 27 of the Sequence Listing and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 28 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 34 of the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No.
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 6 of the Sequence Listing, the light chain
  • the amino acid sequence of CDR2 is shown in SEQ ID No. 7 of the Sequence Listing and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 8 of the Sequence Listing; the amino acid sequence of the CDR1 of the light chain is SEQ ID No. 14 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 15 of the Sequence Listing and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 16 of the Sequence Listing; ammonia of the light chain CDR1
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 23 of the Sequence Listing and the amino acid sequence of the light chain CDR3 is as shown in SEQ ID No. 24 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 30 of the sequence listing
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 31 of the sequence listing
  • the amino acid sequence of the light chain CDR3 is as follows.
  • the SEQ ID No. 32 is shown in the list; the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 38 of the Sequence Listing, and the amino acid sequence of the light chain CDR2 is as shown in SEQ ID No.
  • the present invention provides an isolated protein comprising a heavy chain variable region of a C5aR antibody and/or a light chain variable region of a C5aR antibody, the amino acid sequence of the heavy chain variable region being SEQ ID No. 1 in the Sequence Listing SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 33, SEQ ID No. 41 or SEQ ID No. 49; the amino acid sequence of the light chain variable region is as The sequence listing shows SEQ ID No. 5, SEQ ID No. 13, SEQ ID No. 21, SEQ ID No. 29, SEQ ID No. 37, SEQ ID No. 45 or SEQ ID No. 53.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1 of the Sequence Listing and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 5 of the Sequence Listing;
  • the amino acid sequence of the variable region is shown in SEQ ID No. 9 of the Sequence Listing and the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 13 of the Sequence Listing;
  • the amino acid sequence of the light chain variable region shown in SEQ ID No. 17 is shown in SEQ ID No. 21 of the Sequence Listing;
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 25 of the Sequence Listing.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID No.
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing and the light chain variable region
  • amino acid sequence is shown in SEQ ID No. 37 of the Sequence Listing
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 41 of the Sequence Listing and the amino acid sequence of the variable region of the light chain is SEQ ID:
  • amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 49 of the sequence listing and the light chain variable region
  • amino acid sequence shown in Sequence Listing SEQ ID No.53.
  • the number in Table 1 is the sequence number "SEQ ID No.”
  • amino acid sequence of the heavy chain protein variable region of 5F8E2C11 is SEQ ID No. 1 of the sequence listing
  • the heavy chain protein variable region of 5F8E2C11 is The amino acid sequence of the CDR1 domain is SEQ ID No. 2 of the Sequence Listing.
  • the protein further comprises an antibody heavy chain constant region and/or an antibody light chain constant region, which is conventional in the art, preferably a mouse-derived antibody heavy chain constant region.
  • the antibody light chain constant region is conventional in the art, preferably a mouse-derived light chain antibody constant region.
  • the protein is a protein conventional in the art, preferably a C5aR antibody, more preferably an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, or a single chain antibody (single chain)
  • a single chain antibody single chain
  • scFv antibody fragment
  • sdAb single domain antibody
  • signle-domain antibody single-domain antibody
  • monoclonal antibodies or polyclonal antibodies prepared by the above antibodies can be developed by a variety of pathways and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is the preparation of monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.
  • the full-length antibody protein is a conventional full-length antibody of the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and the light chain variable region of the protein may also constitute a full human antibody full length protein with a human heavy chain constant region and a human light chain constant region.
  • the full length protein of the antibody is IgG1, IgG2, IgG3 or IgG4.
  • the single-chain antibody is a conventional single-chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.
  • the antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment of the art comprising a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain constant region.
  • the antigen-antibody binding domain protein fragments are Fab and F(ab').
  • the single domain antibodies are conventional single domain antibodies in the art, including heavy chain variable regions and heavy chain constant regions.
  • the single region antibodies are conventional single region antibodies of the art which include only heavy chain variable regions.
  • the preparation method of the protein is a conventional preparation method in the art.
  • the preparation method is preferably: Obtained from an expression transformant recombinantly expressing the protein or obtained by artificially synthesizing a protein sequence.
  • the method for isolating the expression transformant which recombinantly expresses the protein is preferably obtained by cloning a nucleic acid molecule encoding the protein and having a point mutation into a recombinant vector, and transforming the resulting recombinant vector into a transformant to obtain a recombinant expression.
  • the transformant can be isolated and purified by culturing the resulting recombinant expression transformant to obtain the protein.
  • the invention also provides a nucleic acid encoding the protein described above.
  • the nucleic acid encoding the heavy chain variable region is SEQ ID No. 57, SEQ ID No. 59, SEQ ID No. 61, SEQ ID No. 63, and SEQ. ID No. 65, SEQ ID No. 67 of the Sequence Listing or SEQ ID No. 69 of the Sequence Listing; and/or the nucleotide sequence of the nucleic acid encoding the variable region of the light chain is SEQ ID No. 58 of the Sequence Listing , Sequence Listing SEQ ID No. 60, Sequence Listing SEQ ID No. 62, Sequence Listing SEQ ID No. 64, Sequence Listing SEQ ID No. 66, Sequence Listing SEQ ID No. 68 or Sequence Listing SEQ ID No. 70 .
  • nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 57 of the Sequence Listing and the nucleic acid encoding the light chain variable region is set forth in SEQ ID No. 58 of the Sequence Listing; encoding the heavy
  • the nucleic acid of the chain variable region is as shown in SEQ ID No. 59 of the Sequence Listing and the nucleic acid encoding the variable region of the light chain is shown in SEQ ID No. 60 of the Sequence Listing; the nucleic acid encoding the heavy chain variable region is as follows.
  • the nucleic acid encoding the light chain variable region shown in SEQ ID No. 61 is shown in SEQ ID No.
  • nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 63 of the Sequence Listing.
  • nucleic acid encoding the light chain variable region is as shown in SEQ ID No. 64 of the Sequence Listing; the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 65 of the Sequence Listing and encodes the light chain variable.
  • the nucleic acid of the region is shown in SEQ ID No. 66 of the Sequence Listing; the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 67 of the Sequence Listing and the nucleic acid encoding the variable region of the light chain is SEQ ID.
  • nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 69 of the Sequence Listing and the nucleic acid encoding the light chain variable region is SEQ ID No. .70 shows.
  • the number in Table 2 is the sequence number "SEQ ID No.”, such as the heavy chain protein encoding 5F8E2C11
  • the nucleotide sequence of the variable region is SEQ ID No. 33 of the Sequence Listing.
  • nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 5F8E2C11 is from positions 76 to 105 of SEQ ID No. 57 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 5F8E2C11 is 148th to 195th in SEQ ID No. 57 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 5F8E2C11 is position 292 to 321 in SEQ ID No. 57 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 5F8E2C11 is from position 70 to position 120 in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 5F8E2C11 is 166th to 186th in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 5F8E2C11 is 283th to 306th in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 42B5G7D1 is from positions 76 to 105 of SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 42B5G7D1 is 148th to 198th in SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 42B5G7D1 is from positions 295 to 336 of SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 42B5G7D1 is from positions 70 to 117 in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 42B5G7D1 is from position 163 to position 183 in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 42B5G7D1 is 280th to 306th in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 43E8F5B6 is from positions 76 to 105 of SEQ ID No. 61 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 43E8F5B6 is 148th to 198th in SEQ ID No. 61 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 43E8F5B6 is from positions 295 to 339 of SEQ ID No. 61 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 43E8F5B6 is the 70th to 105th positions in SEQ ID No. 62 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 43E8F5B6 is from positions 151 to 171 in SEQ ID No. 62 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 43E8F5B6 is from positions 268 to 294 in SEQ ID No. 62 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 46H2A11C7 is from positions 76 to 105 of SEQ ID No. 63 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region of 46H2A11C7 is 148th to 198th in SEQ ID No. 63 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 46H2A11C7 is 295th to 306th in SEQ ID No. 63 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 46H2A11C7 is from positions 70 to 117 in SEQ ID No. 64 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 46H2A11C7 is from position 163 to position 183 in SEQ ID No. 64 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 46H2A11C7 is from positions 280 to 306 in SEQ ID No. 64 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 2A12B2B2 is from positions 76 to 105 of SEQ ID No. 65 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 2A12B2B2 is from positions 148 to 198 of SEQ ID No. 65 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 2A12B2B2 is from positions 295 to 303 in SEQ ID No. 65 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 2A12B2B2 is from positions 70 to 102 of SEQ ID No. 66 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 2A12B2B2 is 148th to 168th in SEQ ID No. 66 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 2A12B2B2 is from position 265 to position 291 in SEQ ID No. 66 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 8B5D1A9 is in SEQ ID No. 67 of the Sequence Listing. 76th to 105th;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 8B5D1A9 is from positions 148 to 198 of SEQ ID No. 67 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 8B5D1A9 is from positions 295 to 324 of SEQ ID No. 67 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 8B5D1A9 is from position 70 to position 120 in SEQ ID No. 68 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 8B5D1A9 is from positions 166 to 189 in SEQ ID No. 68 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 8B5D1A9 is from positions 286 to 312 in SEQ ID No. 68 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 9D5A12G7 is from positions 76 to 105 of SEQ ID No. 69 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 9D5A12G7 is 148th to 195th in SEQ ID No. 69 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 9D5A12G7 is position 292 to 321 in SEQ ID No. 69 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 9D5A12G7 is from positions 70 to 120 of SEQ ID No. 70 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 9D5A12G7 is 166th to 186th in SEQ ID No. 70 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 9D5A12G7 is position 283 to 306 in SEQ ID No. 70 of the Sequence Listing.
  • the preparation method of the nucleic acid is a preparation method conventional in the art, and preferably includes the steps of obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial total sequence synthesis. .
  • the base sequence encoding the amino acid sequence of the above protein may be appropriately introduced with a substitution, deletion, alteration, insertion or addition to provide a homologue of a polynucleotide.
  • a homologue of a polynucleotide of the invention can be made by replacing, deleting or increasing one or more bases encoding a gene of the protein sequence within the range of activity of the antibody.
  • the invention also provides a recombinant expression vector comprising the nucleic acid.
  • the recombinant expression vector can be obtained by a conventional method in the art, that is, the nucleic acid molecule of the present invention is ligated to various expression vectors.
  • the expression vector is a variety of vectors conventional in the art as long as it can accommodate the aforementioned nucleic acid molecule.
  • the vector preferably includes: various plasmids, cosmids, phage or viral vectors, and the like.
  • the present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.
  • the preparation method of the recombinant expression transformant is a preparation method conventional in the art, and preferably, the recombinant expression vector is transformed into a host cell.
  • the host cell is a variety of host cells conventional in the art, as long as it satisfies the stable self-replication of the above recombinant expression vector, and the nucleic acid carried can be efficiently expressed.
  • the host cell is an E. coli TG1 or BL21 cell (expressing a single-chain antibody or a Fab antibody), or a CHO-K1 cell (expressing a full-length IgG antibody).
  • the recombinant expression plasmid of the present invention can be obtained by transforming the aforementioned recombinant expression plasmid into a host cell.
  • the conversion method is a conventional transformation method in the art, preferably a chemical conversion method, a heat shock method or an electrotransformation method.
  • the present invention provides a method for producing a C5aR antibody, which comprises the steps of culturing the above recombinant expression transformant and obtaining a C5aR antibody from the culture.
  • the present invention also provides a method for detecting cells overexpressing a C5aR protein, comprising the steps of: contacting the above-mentioned protein with a sample to be tested in vitro, and detecting the binding of the above protein to the sample to be tested.
  • overexpression is conventional in the art and refers to overexpression of RNA or protein of C5aR protein in a sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and protein degradation), and due to protein Local overexpression and increased functional activity due to changes in delivery mode (nuclear localization) (as in the case of increased enzymatic hydrolysis of the substrate).
  • overexpression also means that the expression level of RNA or protein of the C5aR protein is increased by 50%, 60%, 70%, 80%, 90% or higher compared to the control sample or normal cells.
  • the detection mode of the combination is a conventional detection method in the art, preferably FACS detection.
  • the present invention provides the use of the above proteins for the preparation of a medicament.
  • the medicament is a medicament for use in anti-inflammatory, vascular or neurological diseases.
  • the present invention also provides a pharmaceutical composition comprising the above-mentioned protein as an active ingredient.
  • the pharmaceutical composition is a pharmaceutical composition for use in anti-inflammatory, vascular or neurological diseases.
  • the administration route of the pharmaceutical composition of the present invention is preferably administered by injection or orally.
  • the administration by injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.
  • the pharmaceutical composition is various dosage forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule or a granule. , injection or infusion, etc.
  • the pharmaceutical compositions of the present invention further comprise one or more pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier is A conventional pharmaceutical carrier in the art, which may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, and preferably include pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises from 0.01 to 99.99% of the above protein and from 0.01 to 99.99% of a pharmaceutically acceptable carrier, the percentage being a percentage by mass of the pharmaceutical composition.
  • the pharmaceutical composition is administered in an amount effective to reduce or delay the progression of a disease, degenerative or damaging condition.
  • the effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the results sought.
  • One skilled in the art can determine the effective amount by using the above factors such as the individual basis and using no more than conventional experiments.
  • the present invention provides the use of the above proteins in anti-inflammatory, vascular diseases or neurological diseases.
  • the present invention provides the use of the above pharmaceutical composition for anti-inflammatory, vascular disease or neurological diseases.
  • the reagents and starting materials used in the present invention are commercially available.
  • the positive progress of the present invention is that the protein of the present invention is a C5aR antibody which has high affinity with the C5aR protein (affinity KD ⁇ 1*10 -8 M) and can efficiently inhibit or block C5aR and C5a. Binding significantly reduces the calcium flow signal, thereby down-regulating or cutting off the corresponding signaling pathway, halting the directed migration of C5a in vitro neutrophils; and significantly inhibiting the chemotaxis of neutrophils. Therefore, the C5aR antibody can be used for the preparation of a drug such as inflammation, vascular disease or nervous system disease.
  • a drug such as inflammation, vascular disease or nervous system disease.
  • Figure 1 shows the results of FACS screening of HEK293 cells transfected with C5aR protein.
  • Figure 2 is a serum antibody titer of mice after immunization with C5aR polypeptide by ELISA.
  • Figure 3 is a ELISA showing the binding of a C5aR antibody to a specific epitope of C5aR.
  • Figure 4 is a FACS assay for the binding of C5aR antibody to CHOK1-hC5aR.
  • FIG. 5 shows FLIPR detection of C5aR antibodies blocking C5a-mediated calcium flux signals.
  • Figure 6 is a human neutrophil chemotaxis assay to detect C5aR antibodies blocking C5a-induced cell migration.
  • the room temperature described in the examples is room temperature conventional in the art, and is generally from 10 to 30 °C.
  • C5aR is a 7-transmembrane protein whose extracellular domain includes 1 N-terminus and 3 extracellular loops.
  • the following five polypeptides were designed for the N-terminus and three extracellular loops of the C5aR protein, respectively.
  • the specific sequences are shown in Table 3.
  • the polypeptides shown in Table 3 were synthesized by Jill Biochemical Shanghai Co., Ltd. Immunogen A was obtained by coupling these polypeptides to keyhole limpet hemocyanin (KLH). For the method of coupling, see “Establishment and Application of Indirect ELISA Method for PRRS Antibody” [J]. Veterinary Science and Technology of China, 2005(6).
  • the stable cell line used for immunogen B uses a lentivirus infection method.
  • the nucleotide sequence encoding the full-length amino acid sequence of human C5aR (as shown in SEQ ID No. 72 of the Sequence Listing) was cloned into the lentiviral vector pLVX-IRES vector (purchased from Clontech) and a plasmid was prepared.
  • the plasmid was transfected by lipofection (transfection using X-treme GENE HP DNA Transfection Reagent, available from Roche, Cat. No. Cat# 06 366 236 001, and operating according to the instructions) to the HEK293 cell line for viral packaging.
  • the viral suspension was collected for infection with HEK293 cell line and CHOK1 cell line (both purchased from Invitrogen).
  • the infection method was as follows: cells of HEK293 cell line and CHOK1 cell line were plated at a concentration of 1e 5 /well into a 6-well plate containing 10% (w/w) fetal bovine serum in complete medium, in which complete medium against HEK293 was used.
  • DMEM medium purchased from Gibco
  • the complete medium for CHOK1 was Ham's F-12 Nutrient Mixture medium (purchased from Gibco), placed in an incubator at 37 ° C, 5% (v/v) CO 2 overnight. to cultivate.
  • the medium was discarded the next day, and a 1 ml/well virus suspension was added, and the virus infection solution was discarded after overnight incubation.
  • the plates were subcloned and cultured at 37 ° C under 5% (v/v) CO 2 conditions. After approximately 2 weeks, a portion of the monoclonal wells were selected and expanded into 6-well plates.
  • the amplified clones were screened by flow cytometry using a known C5aR antibody (purchased from Abeam).
  • the cell line with good growth and high fluorescence intensity is selected, and the monoclonal cell line continues to expand and the liquid nitrogen is frozen, that is, the immunogen B is obtained.
  • the specific selection results are shown in Table 4 and Figure 1.
  • the positive cells (%) in Table 4 refer to the percentage of positive cells in the total number of cells.
  • Table 4 illustrates that a series of HEK293 cell lines positive for C5aR expression have been prepared, thus obtaining immunogen B.
  • the human C5aR full-length amino acid sequence cDNA (shown in SEQ ID No. 71 of the Sequence Listing) was cloned into the pCDNA3.1 vector (purchased from Invitrogen) and coated into a 1.0 ⁇ m gold colloidal bullet (purchased from Bio-rad) and used.
  • Helios gene gun immunization Helios Gene Gun System, Bio-rad, Cat. No. 165-2431
  • the method of coating to 1.0 ⁇ m gold colloidal bullet and immunization was carried out by referring to the Helios gene gun specification.
  • Immunogen A immunization was performed using Balb/c and SJL mice (purchased from Shanghai Slack Company) at 6-8 weeks old, and the mice were raised under SPF conditions. At the time of primary immunization, immunogen A was emulsified with Freund's complete adjuvant and intraperitoneally injected with 0.25 ml, that is, 50 ⁇ g of immunogen A was injected per mouse. In booster immunization, immunogen A was emulsified with Freund's incomplete adjuvant and intraperitoneally injected with 0.25 ml, ie 50 ⁇ g of immunogen A per mouse.
  • the first and second immunizations were separated by 2 weeks, and each subsequent immunization interval was 3 weeks.
  • blood was collected 1 week after each immunization, and antibody titer and specificity in serum were measured by FACS. After the second booster immunization, the FACS test serum antibody titer reached 1:1000 or more.
  • Immunogen C immunization Balb/c and SJL mice (purchased from Shanghai Slack Company) were used at 6-8 weeks old, and the mice were raised under SPF conditions. All mice were immunized 4 times with the Helios gene gun in the abdomen, 4 shots each time, 1.0 microgram of cDNA per shot. The interval between the initial immunization and the first booster immunization was 2 weeks, and each booster interval was 3 weeks later. Blood was collected 1 week after each boost, and antibody titer in serum was measured by ELISA or FACS. After the second booster immunization, the serum antibody titer of FACS was 1:1000 or more, and the ELISA titer was above 1:10000.
  • mice can achieve a FACS titer of 1:1000 or more after three immunizations.
  • mice were subjected to final booster immunization 3-5 days prior to cell fusion.
  • the mice immunized with the immunogen A were finally immunized with the corresponding polypeptides shown in Table 3; the mice immunized with the immunogen B and the immunogen C were finally immunized with a HEK293 stable cell line expressing human C5aR.
  • mice were sacrificed after 3-5 days, and spleen cells were collected. NH 4 OH was added to a final concentration of 1% (w/w), and erythrocytes were lysed in spleen cells to obtain a spleen cell suspension.
  • DMEM basal medium was used for 1000 rpm.
  • the cells were washed three times per minute, and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC) at a 5:1 ratio of viable cells, and cells were subjected to high-efficiency electrofusion (see METHODS IN ENZYMOLOGY, VOL.220). Fusion.
  • the fused cells were diluted into DMEM medium containing 20% fetal bovine serum and 1 ⁇ HAT in a percentage by mass. Then added to the 96-well cell culture plate at 1 ⁇ 10 5 /200 ⁇ l per well. In a 5% CO 2 , 37 ° C incubator, the percentage is a volume percent.
  • hybridoma cells with MFI values >50 in FACS experiments were selected as eligible positive clones.
  • Selected hybridoma cells were subcloned in a 96-well plate by limiting dilution in DMEM medium containing 10% (w/w) FBS (purchased from invitrogen) at 37 ° C, 5% (v/v) CO Culture under 2 conditions.
  • a preliminary screening was performed with Acumen 10 days after subcloning, and a single positive monoclonal was selected and amplified into 24-well plates for further culture.
  • Antigen binding positive was determined by FACS after 3 days and biological activity was assessed using the C5aR receptor ligand binding assay (evaluation criteria were MFI values >50 in FACS experiments).
  • the best clones were selected and cultured in DMEM medium containing 10% (w/w) FBS (purchased from invitrogen) at 37 ° C, 5% (v/v) CO 2 conditions.
  • the optimal clone is expanded and cultured, and the hybridoma cells of the present invention are obtained by cryopreservation of liquid nitrogen, and can be used for subsequent antibody production and purification.
  • Hybridoma cells produce antibodies at low concentrations, about 1-10 ⁇ g/ml, with large changes in concentration. Moreover, the various proteins produced in the culture of the medium and the fetal bovine serum components contained in the culture medium have different degrees of interference with many biological activity analysis methods, so that small-scale (1-5 mg) antibody production purification is required.
  • the hybridoma cells obtained in Example 1 were inoculated into a T-75 cell culture flask and domesticated for 3 passages using a production medium (Hybridoma serum free medium, available from Invitrogen). After the growth state is good, inoculate the cell culture spinner. 500 ml of production medium was added to each of 2 liters of culture spinner flasks, and the seed density was 1.0 ⁇ 10 5 /ml. The cap was capped and the roller was placed in a bottle shaker in a 37 ° C incubator at 3 rpm. After 14 days of continuous spin culture, the cell culture medium was collected, and the cells were removed by filtration, and filtered through a 0.45 ⁇ m filter to clarify the culture supernatant. The clarified culture supernatant can be purified immediately or frozen at -30 °C.
  • a production medium Hybridoma serum free medium, available from Invitrogen
  • the monoclonal antibody in the culture supernatant (300 mL) of the clarified hybridoma cells was purified using a 2 mL Protein G column (purchased from GE Healthcare).
  • the Protein G column was first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.2) and the clarified culture supernatant was then loaded onto a Protein G column with a flow rate of 3 mL/min. After loading, the protein G column was washed with equilibration buffer, and the volume of the equilibration buffer was 4 times the volume of the protein G column bed.
  • equilibration buffer PBS phosphate buffer, pH 7.2
  • the C5aR antibody bound to the Protein G column was eluted with an eluent (0.1 M glycine hydrochloride buffer, pH 2.5), and the elution condition (A280 ultraviolet absorption peak) was monitored with a UV detector.
  • the eluted antibody was collected, neutralized by adding 10% 1.0 M Tris-HCl buffer, and the percentage was a volume percentage, and then immediately dialyzed against PBS phosphate buffer overnight, and the solution was changed once a day and continued to permeate. Analysis for 3 hours.
  • the dialyzed C5aR antibody was collected, sterile-filtered with a 0.22 micron filter, and stored aseptically to obtain a purified C5aR antibody.
  • the purified C5aR antibody was assayed for protein concentration (A280/1.4), purity, and endotoxin (Lonza kit). The results are shown in Table 6. As a result, it was found that the endotoxin concentration of the antibody end product was within 1.0 EU/mg. A purified C5aR antibody was obtained.
  • A enzyme-linked immunosorbent assay (ELISA) to detect the binding of antibodies to C5aR protein.
  • the purified C5aR antibody obtained in Example 2 was cross-reactive with the five polypeptides of the C5aR extracellular domain, that is, the purified immunogen A obtained in Example 1.
  • the 5 polypeptides of the purified immunogen A obtained in Example 1 were each diluted with PBS to a final concentration of 1.0 ⁇ g/mL, and then added to a 96-well ELISA plate at 100 ⁇ l per well. Incubate overnight at 4 °C with plastic film, wash plate twice with wash solution [PBS containing 0.01% (v/v) Tween20], add blocking solution [containing 0.01% (v/v) Tween 20 and 1 % (v/v) BSA in PBS] was blocked at room temperature for 2 hours. The blocking solution was discarded, and 100 ⁇ l of the purified C5aR antibody obtained in Example 2 was added per well.
  • Table 7 shows that the binding sites of the purified antibody to C5aR are mainly at the N-terminus and the second. Extracellular loop (ECL#2).
  • the IgG control was human IgG and the data in the table is the OD 450 nm value.
  • the nucleotide sequence containing the full-length amino acid sequence encoding human C5aR in the step (2) of Example 1 was introduced into the CHOK1 cell line to obtain a CHOK1 stable cell line containing human C5aR (herein referred to as CHOK1-hC5aR stable cell line). Then, the culture was expanded to 90% confluence in a T-75 cell culture flask, the medium was exhausted, and washed twice with PBS buffer (Phosphate buffer saline, purchased from Invitrogen), followed by an enzyme-free cell dissociation solution (Versene). Solution, purchased from Life Technology, Inc., handles and collects cells.
  • PBS buffer Phosphate buffer saline, purchased from Invitrogen
  • Versene enzyme-free cell dissociation solution
  • the cells were washed twice with PBS buffer, and after cell counting, the cells were diluted with PBS buffer to 2 ⁇ 10 6 cells per ml, and 2% calf serum blocking solution was added, the percentage being mass percentage. Incubate for 15 minutes at room temperature and then wash twice with PBS buffer. The collected cells were suspended in FACS buffer (PBS containing 2% FBS, the percentage being a mass percentage) to 3 ⁇ 10 6 cells/mL. 100 ⁇ L per well was added to a 96-well FACS reaction plate, and the purified C5aR antibody sample obtained in Example 2 was added to 100 ⁇ l per well, and incubated at 4 ° C for 1 hour.
  • FACS buffer PBS containing 2% FBS, the percentage being a mass percentage
  • the cells were washed twice with FACS buffer, and 100 ⁇ l of a fluorescent (Alexa 488)-labeled secondary antibody (purchased from Invitrogen) per well was added and incubated at 4 ° C for 1 hour.
  • the cells were washed three times with FACS buffer, and 100 ⁇ l of fixative [4% (v/v) paraformaldehyde] suspension cells were added to each well, and after 10 minutes, the cells were washed twice with FACS buffer.
  • the cells were suspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACS Calibur, available from BD).
  • FACS Fluorescence intensity value of the cell population measured by MFI.
  • FLIPR calcium assay detects C5aR antibodies blocking C5a-C5aR-mediated downstream signaling pathway
  • FLIPR Calcium Detection Kit ( Calcium Assay Evaluation Kit, Product #R8172) was purchased from Molecular Devices and the specific experimental procedures were performed according to the kit instructions.
  • the CHOK1-hC5aR stable cell line obtained in the step (2) of the first embodiment was further modified to introduce the Ga15 protein (wherein the Ga15 protein was registered in the NCBI database as NM002068); and the transfection was performed using X-treme GENE HP DNA Transfection Reagent, Purchased from Roche, Cat. No. 6306362236001, and operated according to the instructions), the antibiotics were screened to obtain a stable cell line of CHOK1-Ga15-hC5aR.
  • CHOK1-Ga15-hC5aR cells were expanded to 90% confluence in T-75 cell culture flasks, the medium was aspirated, washed twice with PBS buffer, and then with enzyme-free cell dissociation (Versene solution: purchased from Life) Technology company) processes and collects cells. After cell counting, the cells were diluted to 2 ⁇ 10 5 cells per ml with F-12K medium containing 10% (w/w) calf serum, and added to a 384-well plate at 50 ⁇ L per well, placed at 5 Incubate overnight in a %CO 2 , 37 ° C incubator.
  • Example 2 Remove the 384-well plate the next day, discard the medium, and add 40 ⁇ l of fluorescent dye (purchased from Calcium Assay Evaluation Kit, Product #R8172) was placed in the dark at room temperature for 1 hour.
  • the purified C5aR antibody obtained in Example 2 was diluted to 150 ⁇ g/ml with a buffer [HBSS buffer containing 20 mM HEPES and 0.1% (w/w) BSA] to obtain an antibody dilution solution. Further, 10 ⁇ l of the antibody dilution solution was added to the 384-well plate, and then left at room temperature for 1 hour in the dark.
  • a 384-well plate was placed in a FLIPR instrument with 30 microliters of a 384-well plate diluted to 1 nM of C5a solution for each test.
  • the results are shown in Figures 5 and 9, which show that the antibody to be tested binds to the C5aR on the cell surface, thereby blocking the C5a-mediated calcium flux signal.
  • the mIgG control is murine IgG, and the data in the table is the average fluorescence intensity value of the cell population measured by MFI.
  • Human neutrophil chemotaxis assay detects C5aR antibodies blocking C5a-induced cell migration
  • the freshly obtained whole blood was diluted with a phosphate buffered saline PBS in a volume ratio of 1:1 to obtain diluted whole blood, and the diluted whole blood was gently flattened on a Ficoll liquid surface (purchased from GE Healthcare) using a sterile pipette.
  • the volume ratio of Ficoll to diluted whole blood is 3:4, avoiding shaking and mixing, and centrifuging at 400g for 20 minutes at room temperature 20°C.
  • the centrifuge tube after centrifugation is divided into four layers, the upper layer is plasma, and the middle white layer For mononuclear lymphocytes, the lower two layers are Ficoll and erythrocyte-neutrophil layers, respectively.
  • dextran Extran, molecular weight 500,000, purchased from Shanghai Source Ye Biotechnology Co., Ltd.
  • the human peripheral blood neutrophils obtained in the step (1) of Example 5 were plated to a 96-well cell culture plate at 100 ⁇ l per well of 2 ⁇ 10 6 cells.
  • the C5aR antibody was then dosed from 0.5 ⁇ g/mL to 10 ⁇ g/mL [The purified C5aR antibody obtained in Example 2 was diluted with chemotaxis buffer containing: 49% (v/v) RPMI 1640, 49 % (v/v) M199 and 0.02% (v/v) BSA] were added to the 96-well cell culture plate and incubated for 20 minutes at 37 ° C in a 5% (v/v) CO 2 incubator.
  • the incubated cells were then added to the upper chamber of a 3.0 ⁇ m pore size 24-well Transwell (purchased from Corning), and the upper chamber of Transwell was placed in the lower chamber containing 10 nM C5a protein at 37 ° C, 5% (v/ v) Incubate for 30 minutes in a CO 2 incubator during which cells migrate into the lower chamber.
  • the cells that migrated into the lower chamber were transferred to another 96-well plate and then measured by adding 100 ⁇ L of Celltiter-Glo (purchased from Promega).
  • the results are shown in Figure 6 and Table 10.
  • the data in Figure 6 demonstrates that the antibodies to be tested all have an activity of inhibiting human neutrophil migration.
  • Table 10 illustrates that the antibody to be tested blocks C5a-induced cell migration.
  • the IgG control was murine IgG, and the data in the table is the fluorescence intensity value measured by Celltiter-Glo.
  • Parallel sample 4 312.96 334.79 756.92 2037.87 Fluorescence intensity value 2A12B2B2 8B5D1A9 9D5A12G7 IgG control Parallel sample 1 669.59 655.03 174.68 5174.74 Parallel sample 2 596.81 713.26 211.07 5000.06 Parallel sample 3 232.90 705.98 574.97 2,576.45 Parallel sample 4 203.79 749.65 611.36 2641.96
  • RNA isolation The supernatant obtained from the subcloning culture of Example 1 was tested for antigen binding (ie, after the assays and activity assays of Examples 3 to 5), and 5 ⁇ 10 7 hybridoma cells were collected by centrifugation and added. 1 mL Trizol was mixed and transferred to a 1.5 mL centrifuge tube and allowed to stand at room temperature for 5 minutes. 0.2 mL of chloroform was added, shaken for 15 seconds, allowed to stand for 2 minutes, centrifuged at 12000 g for 5 minutes at 4 ° C, and the supernatant was transferred to a new 1.5 mL centrifuge tube. 0.5 mL of isopropanol was added, and the liquid in the tube was gently mixed.
  • Reverse transcription and PCR 1 ⁇ g of total RNA was taken, 20 ⁇ L of the system was placed, reverse transcriptase was added, and the reaction was carried out at 42 ° C for 60 minutes, and the reaction was terminated by reaction at 7 ° C for 10 minutes.
  • a 50 ⁇ L PCR system was configured, including 1 ⁇ L of cDNA, 25 pmol of each primer, 1 ⁇ L of DNA polymerase, and a matching buffer system, 250 ⁇ mol dNTPs.
  • the PCR program was set up, pre-denatured at 95 ° C for 3 minutes, denatured at 95 ° C for 30 seconds, annealed at 55 ° C for 30 seconds, extended at 72 ° C for 35 seconds, and 35 cycles and then extended at 72 ° C for 5 minutes to obtain a PCR product.
  • the kit for reverse transcription was PrimeScript RT Master Mix, purchased from Takara, catalog number RR036; the kit used for PCR was Q5 super-fidelity enzyme, purchased from NEB, catalog number M0492.
  • PCR product 5 ⁇ L was detected by agarose gel electrophoresis, and the positive samples were purified using a column recovery kit, wherein the recovery kit was Gel & PCR Clean-up, available from MACHEREY-NAGEL, Cat. No. 740609.
  • the ligation reaction was carried out: 50 ng of sample, 50 ng of T vector, 0.5 ⁇ L of ligase, 1 ⁇ L of buffer, 10 ⁇ L of reaction system, and the reaction product was ligated at 16 ° C for half an hour to obtain a ligation product, wherein the ligated kit was T4 DNA ligase, purchased from NEB, and the product number was M0402.
  • the number in Table 11 is the sequence number "SEQ ID No.”
  • amino acid sequence of the heavy chain protein variable region of 5F8E2C11 is SEQ ID No. 1 of the sequence listing
  • the heavy chain protein variable region of 5F8E2C11 is The amino acid sequence of the CDR1 domain is SEQ ID No. 2 of the Sequence Listing.
  • the number in Table 12 is the sequence number "SEQ ID No.”
  • the nucleotide sequence of the heavy chain protein variable region encoding 5F8E2C11 is SEQ ID No. 33 of the Sequence Listing.
  • nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 5F8E2C11 is SEQ ID of the sequence listing 76th to 105th in No.57;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 5F8E2C11 is 148th to 195th in SEQ ID No. 57 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 5F8E2C11 is position 292 to 321 in SEQ ID No. 57 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 5F8E2C11 is from position 70 to position 120 in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 5F8E2C11 is 166th to 186th in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 5F8E2C11 is 283th to 306th in SEQ ID No. 58 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 42B5G7D1 is from positions 76 to 105 of SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 42B5G7D1 is 148th to 198th in SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 42B5G7D1 is from positions 295 to 336 of SEQ ID No. 59 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 42B5G7D1 is from positions 70 to 117 in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 42B5G7D1 is from position 163 to position 183 in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 42B5G7D1 is 280th to 306th in SEQ ID No. 60 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 43E8F5B6 is from positions 76 to 105 of SEQ ID No. 61 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 43E8F5B6 is 148th to 198th in SEQ ID No. 61 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 43E8F5B6 is from positions 295 to 339 of SEQ ID No. 61 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 43E8F5B6 is the 70th to 105th positions in SEQ ID No. 62 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 43E8F5B6 is from positions 151 to 171 in SEQ ID No. 62 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 43E8F5B6 is from positions 268 to 294 in SEQ ID No. 62 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 46H2A11C7 is from positions 76 to 105 of SEQ ID No. 63 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region of 46H2A11C7 is 148th to 198th in SEQ ID No. 63 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region of 46H2A11C7 is 295th to 312th in SEQ ID No. 63 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 46H2A11C7 is from positions 70 to 117 in SEQ ID No. 64 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 46H2A11C7 is from position 163 to position 183 in SEQ ID No. 64 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 46H2A11C7 is from positions 280 to 306 in SEQ ID No. 64 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 2A12B2B2 is from positions 76 to 105 of SEQ ID No. 65 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 2A12B2B2 is from positions 148 to 198 of SEQ ID No. 65 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 2A12B2B2 is from positions 295 to 303 in SEQ ID No. 65 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 2A12B2B2 is from positions 70 to 102 of SEQ ID No. 66 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 2A12B2B2 is 148th to 168th in SEQ ID No. 66 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 2A12B2B2 is from position 265 to position 291 in SEQ ID No. 66 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 8B5D1A9 is from positions 76 to 105 of SEQ ID No. 67 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 8B5D1A9 is in SEQ ID No. 67 of the Sequence Listing. 148th to 198th;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 8B5D1A9 is from positions 295 to 324 of SEQ ID No. 67 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 8B5D1A9 is from position 70 to position 120 in SEQ ID No. 68 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 8B5D1A9 is from positions 166 to 189 in SEQ ID No. 68 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 8B5D1A9 is from positions 286 to 312 in SEQ ID No. 68 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 9D5A12G7 is from positions 76 to 105 of SEQ ID No. 69 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 9D5A12G7 is 148th to 195th in SEQ ID No. 69 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 9D5A12G7 is position 292 to 321 in SEQ ID No. 69 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 9D5A12G7 is from positions 70 to 120 of SEQ ID No. 70 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 9D5A12G7 is 166th to 186th in SEQ ID No. 70 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 9D5A12G7 is position 283 to 306 in SEQ ID No. 70 of the Sequence Listing.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了一种C5aR抗体及其制备方法和应用。该C5aR抗体包括C5aR抗体的重链可变区重链CDR1、重链CDR2和重链CDR3中的一种或多种，和C5aR抗体的轻链可变区轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种。该C5aR抗体对人源C5aR蛋白具有高亲和力，能阻止C5aR蛋白与C5a的结合，从而下调或切断相应的信号通路，中止体外中性粒细胞的C5a定向迁移；并抑制中性粒细胞的趋化作用。

Description

一种C5aR抗体及其制备方法和应用
本申请要求申请日为2016年1月12日的中国专利申请CN201610018727.1的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及抗体领域，具体涉及一种C5aR抗体及其制备方法和应用。
背景技术
补体系统是由30多种血浆蛋白和膜蛋白组成，广泛存在于血液、组织液和细胞表面的、具有精密调控机制的蛋白反应系统。补体的主要生理功能是促进吞噬细胞的吞噬能力和溶解靶细胞，因此是机体免疫防御机制的重要组成部分。
C5aR的配体C5a，是补体系统活化产物，是炎症反应的重要介质和趋化因子。C5aR与配体C5a结合后诱导炎症反应并影响凝血及纤溶系统，导致正常组织细胞的损伤，其参与多种疾病的病理过程。C5a是补体裂解片段中显示过敏毒素作用最强的介质，其过敏毒素作用分别为C3a和C4a作用的20倍和2500倍。此外，C5a还可以增加血管的通透性，刺激平滑肌收缩。高浓度的C5a是中性粒细胞、嗜酸粒细胞和单核细胞的趋化剂，可诱导这些细胞顺浓度梯度方向移动。C5a对免疫应答有明显增强作用，可诱导单核细胞分泌白介素IL-l、IL-6、IL-8及肿瘤坏死因子α(TNF-α)等细胞因子的表达，促进抗原及同种异体抗原诱导的T细胞增殖及促进B细胞产生抗体等。
C5aR属于七次跨膜的G-蛋白偶联受体家族。C5aR是对C5a具有高亲和力的受体，其中Kd约为1nM，其位于包括白细胞在内的不同类型的细胞上。每个细胞的受体数量都非常高，每个白细胞可高达200000个位点。C5a还有另外一个受体，称为C5a样受体(c5a like receptor，C5L2)，但由于其不含G蛋白，又称GPR77。GPR77属于C5a受体亚家族成员，在氨基酸序列上与CD88有35％的同源性，目前其生物学功能仍不明确。
C5aR参与包括风湿性关节炎、牛皮癣、败血症、再灌注损伤和成年呼吸窘迫综合症在内的各种疾病的发病机制(Gerard and Gerard，1994；Murdoch and Finn，2000)。如何阻断C5aR信号的向下游信号通路传导，从而减轻炎症反应一直是免疫学研究的热点问题。目前C5aR和C5a的拮抗剂主要分为抗C5a抗体、抗C5aR抗体、小分子拮抗剂、C5a反义肽、C5a突变体或细菌来源的趋化抑制蛋白等。但是目前仍未有上市的、针对C5aR及其配体的相关拮抗剂新药。
发明内容
本发明所要解决的技术问题是为了克服目前缺少C5aR抗体的不足，提供一种亲和力高、特异性强的C5aR抗体及其制备方法和应用。所述的C5aR抗体与人源C5aR蛋白具有高度亲和力；能够高效地抑制或阻断C5aR与C5a的结合，显著降低钙流信号，从而下调或切断相应的信号通路，中止体外中性粒细胞的C5a定向迁移；并能够显著抑制中性粒细胞的趋化作用；从而给炎症、血管或神经系统的疾病的治疗带来希望。
本发明人以人源C5aR蛋白作为免疫原，采用优化的杂交瘤技术，克隆抗体重链可变区和轻链可变区基因，可变区基因可以嫁接到人源抗体恒定区基因从而形成人鼠嵌合抗体，即获得C5aR抗体的先导抗体。再通过一系列的对先导抗体的初步生产、纯化和检定，获得与人源C5aR蛋白等蛋白具有高度亲和力；能够高效地抑制或阻断C5aR与C5a的结合，显著降低钙流信号；并且能够显著抑制中性粒细胞的趋化作用的C5aR抗体。然后通过分子生物学方法测序获知C5aR抗体的重链可变区和C5aR抗体的轻链可变区的氨基酸序列。
本发明提供一种分离的蛋白质，其包括C5aR抗体的重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或，C5aR抗体的轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种，所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42或SEQ ID No.50所示；所述重链CDR2的氨基酸序列如序列表SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43或SEQ ID No.51所示；所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44或SEQ ID No.52所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46或SEQ ID No.54所示；所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47或SEQ ID No.55所示；所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48或SEQ ID No.56所示；
或者，所述重链CDR1的氨基酸序列与如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42或SEQ ID No.50所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR2的氨基酸序列与如序列表中SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、 SEQ ID No.43或SEQ ID No.51所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR3的氨基酸序列与如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44或SEQ ID No.52所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR1的氨基酸序列与如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46或SEQ ID No.54所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR2的氨基酸序列与如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47或SEQ ID No.55所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR3的氨基酸序列与如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48或SEQ ID No.56所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示。
较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.3所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.4所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.11所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.12所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.19所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.20所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.26所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.27所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.28所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.34所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.35所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.36所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.42所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.43所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.44所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.50所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.51所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.52所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.7所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.8所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.15所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.16所示；所述轻链CDR1的氨 基酸序列如序列表SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.23所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.24所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.30所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.31所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.32所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.38所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.39所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.40所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.46所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.47所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.48所示；或，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.54所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.55所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.56所示。
本发明提供一种分离的蛋白质，其包括C5aR抗体的重链可变区和/或C5aR抗体的轻链可变区，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.33、SEQ ID No.41或SEQ ID No.49所示；所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.29、SEQ ID No.37、SEQ ID No.45或SEQ ID No.53所示。
较佳地，所述重链可变区的氨基酸序列如序列表SEQ ID No.1所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.5所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.9所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.13所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.17所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.21所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.25所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.29所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.33所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.37所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示；或，所述重链可变区的氨基酸序列如序列表SEQ ID No.49所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.53所示。
综上所述，上述氨基酸序列的编号如表1所示：
表1 C5aR抗体蛋白序列编号
Figure PCTCN2017070713-appb-000001
Figure PCTCN2017070713-appb-000002
其中，表1中的数字即为序列表“SEQ ID No.”编号，如5F8E2C11的重链蛋白可变区的氨基酸序列为序列表SEQ ID No.1，而5F8E2C11的重链蛋白可变区中CDR1域的氨基酸序列为序列表SEQ ID No.2。
较佳地，所述的蛋白质还包括抗体重链恒定区和/或抗体轻链恒定区，所述的抗体重链恒定区为本领域常规，较佳地为小鼠源抗体重链恒定区。所述的抗体轻链恒定区为本领域常规，较佳地为小鼠源轻链抗体恒定区。
所述的蛋白质为本领域常规的蛋白质，较佳地为C5aR抗体，更佳地为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区也可与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’)。
所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定区。
所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。
其中，所述蛋白质的制备方法为本领域常规的制备方法。所述制备方法较佳地为： 从重组表达该蛋白质的表达转化体中分离获得或者通过人工合成蛋白质序列获得。所述的从重组表达该蛋白质的表达转化体中分离获得优选如下方法：将编码所述蛋白质并且带有点突变的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述蛋白质。
本发明还提供一种核酸，其编码上述的蛋白质。
较佳地，编码所述重链可变区的核酸如序列表SEQ ID No.57、序列表SEQ ID No.59、序列表SEQ ID No.61、序列表SEQ ID No.63、序列表SEQ ID No.65、序列表SEQ ID No.67或序列表SEQ ID No.69所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.58、序列表SEQ ID No.60、序列表SEQ ID No.62、序列表SEQ ID No.64、序列表SEQ ID No.66、序列表SEQ ID No.68或序列表SEQ ID No.70所示。
更佳地，编码所述重链可变区的核酸如序列表SEQ ID No.57所示且编码所述轻链可变区的核酸如序列表SEQ ID No.58所示；编码所述重链可变区的核酸如序列表SEQ ID No.59所示且编码所述轻链可变区的核酸如序列表SEQ ID No.60所示；编码所述重链可变区的核酸如序列表SEQ ID No.61所示且编码所述轻链可变区的核酸如序列表SEQ ID No.62所示；编码所述重链可变区的核酸如序列表SEQ ID No.63所示且编码所述轻链可变区的核酸如序列表SEQ ID No.64所示；编码所述重链可变区的核酸如序列表SEQ ID No.65所示且编码所述轻链可变区的核酸如序列表SEQ ID No.66所示；编码所述重链可变区的核酸如序列表SEQ ID No.67所示且编码所述轻链可变区的核酸如序列表SEQ ID No.68所示；或，编码所述重链可变区的核酸如序列表SEQ ID No.69所示且编码所述轻链可变区的核酸如序列表SEQ ID No.70所示。
上述核苷酸序列的编号如表2所示：
表2 C5aR抗体基因序列编号
克隆号 重链蛋白可变区 轻链蛋白可变区
5F8E2C11 57 58
42B5G7D1 59 60
43E8F5B6 61 62
46H2A11C7 63 64
2A12B2B2 65 66
8B5D1A9 67 68
9D5A12G7 69 70
其中，表2中的数字即为序列表“SEQ ID No.”编号，如编码5F8E2C11的重链蛋白 可变区的核苷酸序列为序列表SEQ ID No.33。
其中，编码5F8E2C11的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.57中的第76位至第105位；
编码5F8E2C11的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.57中的第148位至第195位；
编码5F8E2C11的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.57中的第292位至第321位；
编码5F8E2C11的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.58中的第70位至第120位；
编码5F8E2C11的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.58中的第166位至第186位；
编码5F8E2C11的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.58中的第283位至第306位；
编码42B5G7D1的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.59中的第76位至第105位；
编码42B5G7D1的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.59中的第148位至第198位；
编码42B5G7D1的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.59中的第295位至第336位；
编码42B5G7D1的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.60中的第70位至第117位；
编码42B5G7D1的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.60中的第163位至第183位；
编码42B5G7D1的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.60中的第280位至第306位；
编码43E8F5B6的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.61中的第76位至第105位；
编码43E8F5B6的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.61中的第148位至第198位；
编码43E8F5B6的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.61中的第295位至第339位；
编码43E8F5B6的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.62中的第70位至第105位；
编码43E8F5B6的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.62中的第151位至第171位；
编码43E8F5B6的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.62中的第268位至第294位。
编码46H2A11C7的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.63中的第76位至第105位；
编码46H2A11C7的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.63中的第148位至第198位；
编码46H2A11C7的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.63中的第295位至第306位；
编码46H2A11C7的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.64中的第70位至第117位；
编码46H2A11C7的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.64中的第163位至第183位；
编码46H2A11C7的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.64中的第280位至第306位。
编码2A12B2B2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.65中的第76位至第105位；
编码2A12B2B2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.65中的第148位至第198位；
编码2A12B2B2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.65中的第295位至第303位；
编码2A12B2B2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.66中的第70位至第102位；
编码2A12B2B2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.66中的第148位至第168位；
编码2A12B2B2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.66中的第265位至第291位。
编码8B5D1A9的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.67中 的第76位至第105位；
编码8B5D1A9的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.67中的第148位至第198位；
编码8B5D1A9的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.67中的第295位至第324位；
编码8B5D1A9的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.68中的第70位至第120位；
编码8B5D1A9的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.68中的第166位至第189位；
编码8B5D1A9的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.68中的第286位至第312位。
编码9D5A12G7的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.69中的第76位至第105位；
编码9D5A12G7的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.69中的第148位至第195位；
编码9D5A12G7的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.69中的第292位至第321位；
编码9D5A12G7的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.70中的第70位至第120位；
编码9D5A12G7的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.70中的第166位至第186位；
编码9D5A12G7的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.70中的第283位至第306位。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明还提供一种包含所述核酸的重组表达载体。
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。
本发明还提供一种包含上述重组表达载体的重组表达转化体。
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或BL21细胞(表达单链抗体或Fab抗体)，或者CHO-K1细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
本发明提供一种C5aR抗体的制备方法，其包括如下步骤：培养上述的重组表达转化体，从培养物中获得C5aR抗体。
本发明还提供一种检测过表达C5aR蛋白的细胞的方法，包括如下的步骤：上述的蛋白质与待检样品在体外接触，检测上述的蛋白质与所述待检样品的结合即可。
所述的过表达的含义为本领域常规，指C5aR蛋白在待检样品中的RNA或蛋白质的过表达(由于转录增加、转录后加工、翻译、翻译后加工以及蛋白质降解改变)，以及由于蛋白质运送模式改变(核定位增加)而导致的局部过表达和功能活性提高(如在底物的酶水解作用增加的情况下)。较佳地，过表达也指相比对照样品或正常细胞，C5aR蛋白的RNA或蛋白质的表达水平提高50％、60％、70％、80％、90％或更高。
所述结合的检测方式是本领域常规的检测方式，较佳地为FACS检测。
本发明提供上述蛋白质在制备药物中的应用。
较佳地，所述的药物是用于抗炎症、血管疾病或神经系统疾病的药物。
本发明还提供一种药物组合物，其活性成分包括上述的蛋白质。
较佳地，所述的药物组合物是用于抗炎症、血管疾病或神经系统疾病的药物组合物。
本发明所述的药物组合物的给药途径较佳的为注射给药或口服给药。所述注射给药较佳的包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳的为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳的为片剂、胶囊、颗粒剂、注射剂或输注剂等。
较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为 本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。
本发明提供上述蛋白质在抗炎症、血管疾病或神经系统疾病中的应用。
本发明提供上述药物组合物在抗炎症、血管疾病或神经系统疾病中的应用。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明所述的蛋白质是一种C5aR抗体，其与C5aR蛋白具有高度亲和力(亲和力KD＜1*10-8M)，能够高效地抑制或阻断C5aR与C5a的结合，显著降低钙流信号，从而下调或切断相应的信号通路，中止体外中性粒细胞的C5a定向迁移；并能够显著抑制中性粒细胞的趋化作用。因此该C5aR抗体能够运用于治疗炎症、血管疾病或神经系统疾病等药物的制备中。
附图说明
图1为C5aR蛋白转染的HEK293细胞FACS筛选检测结果。
图2为ELISA检测C5aR多肽免疫后小鼠血清抗体效价。
图3为ELISA检测C5aR抗体与C5aR特定表位的结合。
图4为FACS检测C5aR抗体与CHOK1-hC5aR的结合反应。
图5为FLIPR检测C5aR抗体阻断C5a介导的钙流信号。
图6为人中性粒细胞趋化实验检测C5aR抗体阻断C5a诱导的细胞迁移。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例中所述的室温为本领域常规的室温，一般为10～30℃。
实施例1
C5aR抗体的制备
(一)、免疫原A的制备
C5aR是一个7次跨膜蛋白，它的胞外结构域包括1个N末端和3个胞外环。设计下列分别针对C5aR蛋白的N末端和3个胞外环的5条多肽，具体序列见表3。表3所示的多肽由吉尔生化上海有限公司合成。将这些多肽与钥孔血蓝蛋白(keyhole limpet hemocyanin，KLH)偶联后即得到免疫原A。偶联的方法参见《PRRS抗体间接ELISA检测方法的建立及应用》[J].中国兽医科技，2005(6)。
表3免疫原A的多肽序列
编号 胞外结构域 序列
C5aR多肽-01 C5aR胞外N末端-1 参见序列表SEQ ID No.43
C5aR多肽-02 C5aR胞外N末端-2 参见序列表SEQ ID No.44
C5aR多肽-03 C5aR胞外环-1 参见序列表SEQ ID No.45
C5aR多肽-04 C5aR胞外环-2 参见序列表SEQ ID No.46
C5aR多肽-05 C5aR胞外环-3 参见序列表SEQ ID No.47
(二)免疫原B的制备
免疫原B所用到的稳定细胞系采用慢病毒感染法。编码人源C5aR全长氨基酸序列(如序列表SEQ ID No.72所示)的核苷酸序列被克隆到慢病毒载体pLVX-IRES载体(购自Clontech)并制备质粒。采用脂质体转染法将质粒转染(转染使用X-treme GENE HP DNA Transfection Reagent，购自Roche公司，货号Cat#06 366 236 001，并按说明书操作)到HEK293细胞系中进行病毒包装，收集病毒悬液对HEK293细胞系和CHOK1细胞系(均购自Invitrogen)进行感染。感染方法如下：将HEK293细胞系和CHOK1细胞系的细胞以1e5/孔的浓度铺入含10％(w/w)胎牛血清的完全培养基的6孔板，其中针对HEK293的完全培养基为DMEM培养基(购自Gibco)，针对CHOK1的完全培养基为Ham's F-12Nutrient Mixture培养基(购自Gibco)，37℃，5％(v/v)CO2条件下置于培养箱中过夜培养。第二天弃去培养基，加入1ml/孔的病毒悬液，过夜孵育后弃去病毒感染液。再加入2ml含800μg/mL的潮霉素B(Hygromycin B)和10％(w/w)胎牛血清的DMEM培养基(购自Invitrogen)，选择性培养2周，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃，5％(v/v)CO2条件下培养，大约2周后选择部分单克隆孔扩增到 6孔板中。对扩增后的克隆用已知的C5aR抗体(购自Abcam)经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存，即获得免疫原B。具体选择结果如表4和图1所示，表4中阳性细胞(％)指阳性细胞占总细胞数目的百分比。表4说明，已经制得一系列C5aR阳性表达的HEK293细胞系，因此获得了免疫原B。
表4 C5aR蛋白转染的HEK293细胞FACS筛选检测结果
Figure PCTCN2017070713-appb-000003
(三)、免疫原C的制备
人源C5aR全长氨基酸序列cDNA(如序列表SEQ ID No.71所示)被克隆到pCDNA3.1载体(购自Invitrogen)并包被到1.0μm金胶体子弹(购自Bio-rad)，并用Helios基因枪免疫(Helios Gene Gun System，Bio-rad，货号165-2431)，即得免疫原C。其中，包被到1.0μm金胶体子弹和免疫的方法参见Helios基因枪说明书进行制定。
A、免疫原A免疫采用6～8周龄Balb/c和SJL小鼠(购自上海斯莱克公司)，小鼠在SPF条件下饲养。初次免疫时，免疫原A用弗氏完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A。加强免疫时，免疫原A用弗氏不完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A。初次免疫与第一次加强免疫之间间隔2周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA和FACS检测血清中免疫原A的抗体效价和特异性，结果如图2和表5所示。表5说明，经C5aR胞外区多肽免疫的小鼠的免疫后血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度为106左右。其中空白对照为1％(w/w)BSA，其中批次指第三次加强免疫后第七天的小鼠血清，表中的数据为OD450nm值。
表5 ELISA检测C5aR多肽免疫后小鼠血清抗体效价
Figure PCTCN2017070713-appb-000004
Figure PCTCN2017070713-appb-000005
B、免疫原B免疫采用6～8周龄Balb/c和SJL小鼠(购自上海斯莱克公司)，小鼠在SPF条件下饲养。含有编码人源C5aR全长氨基酸序列的核苷酸序列的pIRES质粒[参见实施例1步骤(二)]转染HEK293细胞系，得含人源C5aR的HEK293稳定细胞系(转染使用X-treme GENE HP DNA Transfection Reagent，购自Roche公司，货号Cat#06366236001，并按说明书操作)在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用DMEM基础培养基洗涤2次，然后用无酶细胞解离液(购自Invitrogen)37℃处理直至细胞从培养皿壁上可脱落，收集细胞。用DMEM基础培养基洗涤2次，进行细胞计数后将细胞用磷酸盐缓冲液(pH7.2)稀释至2×107细胞每毫升。每只小鼠每次免疫时腹腔注射0.5毫升细胞悬液。第一次与第二次免疫之间间隔2周，以后每次免疫间隔3周。除第一次免疫以外，每次免疫1周后采血，用FACS检测血清中抗体效价和特异性。在第二次加强免疫后，FACS检测血清抗体效价达到1:1000以上。
C、免疫原C免疫采用6～8周龄Balb/c和SJL小鼠(购自上海斯莱克公司)，小鼠在SPF条件下饲养。所有小鼠经腹部用Helios基因枪免疫4次，每次4枪，每枪1.0微克cDNA。初次免疫与第一次加强免疫之间隔2周，以后每次加强免疫间隔3周。每次加强免疫1周后采血，用ELISA或FACS检测血清中抗体效价。在第二次加强免疫后，FACS检测血清抗体效价达到1:1000以上，ELISA效价在1:10000以上。
通常以免疫原A～C进行免疫，大部分小鼠经3次免疫后FACS效价均可达到1:1000以上。
在细胞融合前3-5天，将所选择的小鼠进行最终加强免疫。其中用免疫原A进行免疫的小鼠用如表3所示相应的多肽进行最终免疫；用免疫原B和免疫原C进行免疫的小鼠用表达人C5aR的HEK293稳定细胞系进行最终免疫。(3-5天后处死小鼠，收集脾细胞。加入NH4OH至终浓度1％(w/w)，裂解脾细胞中参杂的红细胞，获得脾细胞悬液。用DMEM基础培养基1000转每分钟离心清洗细胞3次，然后按活细胞数目5:1比率与小鼠骨髓瘤细胞SP2/0(购自ATCC)混合，采用高效电融合方法(参见METHODS IN ENZYMOLOGY，VOL.220)进行细胞融合。融合后的细胞稀释到含20％胎牛血清、1×HAT的DMEM培养基中，所述百分比为质量百分比。然后按1×105/200微升每孔加入到96孔细胞培养板中，放入5％CO2、37℃培养箱中，所述百分比为体积百分比。14天后用Acumen(微孔板细胞检测法)筛选细胞融合板上清，将Acumen中MFI值>100的 阳性克隆扩增到24孔板，在含10％(w/w)HT胎牛血清的DMEM的培养基中，于37℃、5％(v/v)CO2条件下扩大培养。培养3天后取24孔板中扩大培养的培养液进行离心，收集上清液，对上清液进行抗体亚型分析，用FACS确定对C5aR阳性细胞的结合活性(结合活性的检测方法请分别参见实施例3A和实施例3B)。
根据24孔板筛选结果，挑选FACS实验中MFI值>50的杂交瘤细胞为符合条件的阳性克隆。选择符合条件的杂交瘤细胞用有限稀释法在96孔板进行亚克隆，在含10％(w/w)FBS的DMEM培养基中(购自invitrogen)37℃、5％(v/v)CO2条件下培养。亚克隆后10天用Acumen进行初步筛选，挑选单个阳性单克隆扩增到24孔板继续培养。3天后用FACS确定抗原结合阳性并用C5aR受体配体结合实验评估生物活性(评估标准为FACS实验中MFI值>50)。
根据24孔板样品检测结果，挑选出最优的克隆，并于含10％(w/w)FBS的DMEM培养基中(购自invitrogen)在37℃、5％(v/v)CO2条件下将该最优的克隆进行扩大培养，液氮冻存即得本发明杂交瘤细胞，并可用于后续的抗体生产和纯化。
实施例2
先导抗体的生产和纯化
杂交瘤细胞产生的抗体浓度较低，大约仅1-10μg/毫升，浓度变化较大。且培养基中细胞培养所产生的多种蛋白和培养基所含胎牛血清成分对很多生物活性分析方法都有不同程度的干扰，因此需要进行小规模(1-5毫克)抗体生产纯化。
将实施例1所得的杂交瘤细胞接种到T-75细胞培养瓶并用生产培养基(Hybridoma serum free medium，购自Invitrogen公司)驯化传代3代。待其生长状态良好，接种细胞培养转瓶。每个2升的培养转瓶中加入500毫升生产培养基，接种细胞密度为1.0×105/毫升。盖紧瓶盖，将转瓶置于37℃培养箱中的转瓶机上，转速3转/分钟。连续旋转培养14天后，收集细胞培养液，过滤去除细胞，并用0.45微米的滤膜过滤至培养上清液澄清。澄清的培养上清液可马上进行纯化或-30℃冻存。
澄清的杂交瘤细胞的培养上清液(300mL)中的单克隆抗体用2mL蛋白G柱(购自GE Healthcare)纯化。蛋白G柱先用平衡缓冲液(PBS磷酸缓冲液，pH7.2)平衡，然后将澄清的培养上清液上样到蛋白G柱，控制流速在3mL/分钟。上样完毕后用平衡缓冲液清洗蛋白G柱，平衡缓冲液的体积为4倍蛋白G柱柱床体积。用洗脱液(0.1M甘氨盐酸缓冲液，pH2.5)洗脱结合在蛋白G柱上的C5aR抗体，用紫外检测器监测洗脱情况(A280紫外吸收峰)。收集洗脱的抗体，加入10％1.0M Tris-HCl缓冲液中和pH，所述百分比为体积百分比，然后立即用PBS磷酸缓冲液透析过夜，第二天换液1次并继续透 析3小时。收集透析后的C5aR抗体，用0.22微米的滤器进行无菌过滤，无菌保存，即得纯化的C5aR抗体。
将纯化的C5aR抗体进行蛋白浓度(A280/1.4)、纯度、内毒(Lonza试剂盒)等检测分析，结果如表6所示，结果发现，抗体最终产品内毒素浓度在1.0EU/毫克以内，得到了纯化的C5aR抗体。
表6纯化的C5aR抗体检测分析
Figure PCTCN2017070713-appb-000006
实施例3
先导抗体的检定
A、酶联免疫吸附实验(ELISA)检测抗体与C5aR蛋白的结合。对实施例2所得的纯化的C5aR抗体与C5aR胞外结构域的5条多肽，即实施例1获得的纯化的免疫原A分别进行交叉反应。
将实施例1获得的纯化的免疫原A的5条多肽分别用PBS稀释至终浓度1.0μg/mL，然后以100μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用洗板液[含有0.01％(v/v)Tween20的PBS]洗板2次，加入封闭液[含有0.01％(v/v)Tween20和1％(v/v)BSA的PBS]室温封闭2小时。倒掉封闭液，加入实施例2所得的纯化的C5aR抗体100μl每孔。37℃孵育2小时后，用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma)，37℃孵育2小时后，用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次。加入TMB底物100μl每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μl每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A450nm数值，结果如图3和表7所示，表7说明，纯化后的抗体与C5aR的结合位点主要在N末端和第二个胞外环(ECL#2)。其中IgG对照为人 IgG，表中的数据为OD450nm值。
表7 ELISA检测C5aR抗体与C5aR特定表位的结合
Figure PCTCN2017070713-appb-000007
B、流式细胞实验(FACS)检测抗体与C5aR表达细胞的结合
将实施例1步骤(二)中所述含有编码人源C5aR全长氨基酸序列的核苷酸序列导入CHOK1细胞株得含人C5aR的CHOK1稳定细胞株(此处称为CHOK1-hC5aR稳定细胞株)，然后在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用PBS缓冲液(Phosphate buffer saline，购自Invitrogen)洗涤2次，然后用无酶细胞解离液(Versene solution，购自Life technology公司)处理和收集细胞。用PBS缓冲液洗涤细胞2次，进行细胞计数后将细胞用PBS缓冲液稀释至2×106细胞每毫升，加入2％小牛血清封闭液，所述百分比为质量百分比。室温孵育15分钟，然后用PBS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(含有2％FBS的PBS，所述百分比为质量百分比)悬浮至3×106细胞/mL。按每孔100微升加入到96孔FACS反应板中，加入实施例2所得的纯化的C5aR抗体待测样品每孔100微升，4℃孵育1小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗(购自Invitrogen)，4℃孵育1小时。用FACS缓冲液离心洗涤3次，加入每孔100微升固定液[4％(v/v)多聚甲醛]悬浮细胞，10分钟后用FACS缓冲液离心洗涤2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。结果如图4和表8所示，表8说明，待测抗体可结合细胞表面的C5aR。其中mIgG对照为鼠IgG，作为阴性对照。表8中的数据为MFI所测细胞群的平均荧光强度值。
表8 FACS检测C5aR抗体与CHOK1-hC5aR的结合反应
Figure PCTCN2017070713-appb-000008
Figure PCTCN2017070713-appb-000009
实施例4
FLIPR钙检测实验检测C5aR抗体阻断C5a-C5aR介导的下游信号通路
FLIPR钙检测试剂盒(
Figure PCTCN2017070713-appb-000010
Calcium Assay Evaluation Kit，Product#R8172)购自Molecular Devices，具体实验步骤按照试剂盒说明书进行。
将实施例1步骤(二)中得到的CHOK1-hC5aR稳定细胞株进一步改造，导入Ga15蛋白(其中，Ga15蛋白在NCBI数据库的登录号为NM002068)；转染使用X-treme GENE HP DNA Transfection Reagent，购自Roche公司，货号Cat#06366236001，并按说明书操作)，经过抗生素筛选得到CHOK1-Ga15-hC5aR稳定细胞株。
CHOK1-Ga15-hC5aR细胞在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用PBS缓冲液洗涤2次，然后用无酶细胞解离液(Versene solution:购自Life technology公司)处理和收集细胞。进行细胞计数后将细胞用含10％(w/w)小牛血清的F-12K培养基稀释至2×105细胞每毫升，按每孔50微升加入到384孔板中，放置在5％CO2、37℃ 培养箱中过夜培养。第二天取出384孔板，弃去培养基，加入40微升荧光染料(购自
Figure PCTCN2017070713-appb-000011
Calcium Assay Evaluation Kit，Product#R8172)后于室温避光放置1小时。将实施例2所得的纯化的C5aR抗体用缓冲液[含20mM HEPES和0.1％(w/w)BSA的HBSS缓冲液]稀释至150μg/ml，得抗体稀释溶液。再向384孔板中加入抗体稀释溶液每孔10微升，再于室温避光放置1小时。将384孔板和每孔装有30微升稀释至1nM的C5a溶液的384孔板一起放置在FLIPR仪器中进行检测。结果如图5和表9所示，表9说明，待测抗体可结合细胞表面的C5aR，从而阻断C5a介导的钙流信号。其中mIgG对照为鼠IgG，表中的数据为MFI所测细胞群的平均荧光强度值。
表9 FLIPR检测C5aR抗体阻断C5a介导的钙流信号
Figure PCTCN2017070713-appb-000012
实施例5
人中性粒细胞趋化实验检测C5aR抗体阻断C5a诱导的细胞迁移
(一)Ficoll分离全血获取外周血中性粒细胞PMNs。
将新鲜获取的全血用磷酸缓冲液PBS以1:1的体积比例稀释得稀释后的全血，用无菌吸管轻轻将稀释后的全血铺平在Ficoll液面(购自GE Healthcare)，Ficoll与稀释后的全血的体积比为3:4，避免震荡混匀，以400g转速室温20℃梯度离心30分钟，离心后的离心管分为四层，上层为血浆，中间白膜层为单核淋巴细胞，下面两层分别为Ficoll和红细胞-中性粒细胞层。弃掉血浆、白膜层、Ficoll，留红细胞-中性粒细胞层沉淀，用PBS稀释至25mL，再加入25mL 3％(w/w)葡聚糖(Dextran，分子量500，000，购自上海源叶生物科技有限公司)，室温静置20分钟进行红细胞沉降。然后取上清液，500g转速室温离心10分钟，弃上清，取细胞沉淀。将细胞沉淀用无菌水重悬至25mL，颠倒离心管使之充分混匀，反应28秒后加入等体积1.8％(w/w)氯化钠溶液(购自Sigma)终止红细胞裂解。500g转速室温离心5分钟，弃上清。将细胞沉淀用趋化缓冲液(49％RPMI1640、49％M199和2％透析的FBS的混合液，所述百分比为质量百分比)重悬至25mL，计数并离心，最后将中性粒细胞重悬至趋化缓冲液，细胞浓度调整为2×107细胞每毫升，得人外周血中性粒细胞。
(二)人外周血中性粒细胞趋化实验
将实施例5步骤(一)获得的人外周血中性粒细胞以2×106个细胞100微升每孔，铺至96孔细胞培养板。然后将浓度范围从0.5μg/mL到10μg/mL的C5aR抗体[将实施例2所得的纯化的C5aR抗体用趋化缓冲液稀释，趋化缓冲液包含：49％(v/v)RPMI1640、49％(v/v)M199和0.02％(v/v)BSA]加入到该96孔细胞培养板中，于37℃、5％(v/v)CO2培养箱孵育20分钟。然后将孵育后的细胞加入到3.0μm孔径的24孔Transwell(购自Corning)的上室中，再将Transwell的上室放置到含有10nM C5a蛋白的下室，在37℃、5％(v/v)CO2培养箱中孵育30分钟，期间有细胞迁移进入下室。将迁移进入下室的细胞转移到另一个96孔板，然后加入100μL Celltiter-Glo(购自Promega)进行测量。结果如图6和表10所示。图6的数据说明，待测抗体均具有抑制人中性粒细胞迁移的活性。表10说明，待测抗体可阻断C5a诱导的细胞迁移。其中IgG对照为鼠IgG，表中的数据为Celltiter-Glo所测的荧光强度值。
表10人中性粒细胞趋化实验检测C5aR抗体阻断C5a诱导的细胞迁移
荧光强度值 5F8E2C11 42B5G7D1 43E8F5B6 46H2A11C7
平行样1 574.97 1200.89 844.26 1455.62
平行样2 429.41 1477.46 807.87 1506.57
平行样3 400.30 298.40 705.98 1928.70
平行样4 312.96 334.79 756.92 2037.87
荧光强度值 2A12B2B2 8B5D1A9 9D5A12G7 IgG对照
平行样1 669.59 655.03 174.68 5174.74
平行样2 596.81 713.26 211.07 5000.06
平行样3 232.90 705.98 574.97 2576.45
平行样4 203.79 749.65 611.36 2641.96
实施例6
轻重链可变区氨基酸序列测定
总RNA分离：将实施例1亚克隆培养所得的上清液检验过抗原结合后(即经过实施例3～5的检定和活性测定后)，通过离心搜集5×107个杂交瘤细胞，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟。加0.2mL氯仿，振荡15秒，静置2分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中。加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清。加入1mL 75％乙醇(所述百分比为体积百分比)，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H2O溶解(55℃水浴促溶10分钟)，即得总RNA。
逆转录与PCR：取1μg总RNA，配置20μL体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μL PCR体系，包括1μL cDNA、每种引物25pmol、1μL DNA聚合酶以及相配的缓冲体系、250μmol dNTPs。设置PCR程序，预变性95℃3分钟，变性95℃30秒，退火55℃30秒，延伸72℃35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒为Q5超保真酶，购自NEB，货号M0492。
克隆与测序：取5μL PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2017070713-appb-000013
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μL，缓冲液1μL，反应体系10μL，于16℃反应半小时得连接产物，其中连接的试剂盒为T4DNA连接酶，购自NEB，货号M0402；取5μL连接产物加入100μL的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，冰浴5分钟。而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μL无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟，取出200μL涂布于含抗生素的LB固体培养基上于37℃孵箱过夜 培养。次日，使用T载体上引物M13F和M13R配置30μL PCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μL点于另一块含100nM氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μL进行琼脂糖凝胶电泳检测，将阳性样品进行测序。其中，测序的步骤参见Kabat，Sequences of Proteins of Immunological Interest，National Institutes of Health，Bethesda，Md.(1991)。
测序结果如表11～12所示：
表11 C5aR抗体蛋白序列编号
Figure PCTCN2017070713-appb-000014
其中，表11中的数字即为序列表“SEQ ID No.”编号，如5F8E2C11的重链蛋白可变区的氨基酸序列为序列表SEQ ID No.1，而5F8E2C11的重链蛋白可变区中CDR1域的氨基酸序列为序列表SEQ ID No.2。
表12 C5aR抗体基因序列编号
克隆号 重链蛋白可变区 轻链蛋白可变区
5F8E2C11 57 58
42B5G7D1 59 60
43E8F5B6 61 62
46H2A11C7 63 64
2A12B2B2 65 66
8B5D1A9 67 68
9D5A12G7 69 70
其中，表12中的数字即为序列表“SEQ ID No.”编号，如编码5F8E2C11的重链蛋白可变区的核苷酸序列为序列表SEQ ID No.33。
其中，编码5F8E2C11的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID  No.57中的第76位至第105位；
编码5F8E2C11的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.57中的第148位至第195位；
编码5F8E2C11的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.57中的第292位至第321位；
编码5F8E2C11的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.58中的第70位至第120位；
编码5F8E2C11的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.58中的第166位至第186位；
编码5F8E2C11的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.58中的第283位至第306位；
编码42B5G7D1的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.59中的第76位至第105位；
编码42B5G7D1的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.59中的第148位至第198位；
编码42B5G7D1的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.59中的第295位至第336位；
编码42B5G7D1的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.60中的第70位至第117位；
编码42B5G7D1的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.60中的第163位至第183位；
编码42B5G7D1的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.60中的第280位至第306位；
编码43E8F5B6的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.61中的第76位至第105位；
编码43E8F5B6的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.61中的第148位至第198位；
编码43E8F5B6的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.61中的第295位至第339位；
编码43E8F5B6的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.62中的第70位至第105位；
编码43E8F5B6的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.62中的第151位至第171位；
编码43E8F5B6的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.62中的第268位至第294位。
编码46H2A11C7的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.63中的第76位至第105位；
编码46H2A11C7的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.63中的第148位至第198位；
编码46H2A11C7的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.63中的第295位至第312位；
编码46H2A11C7的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.64中的第70位至第117位；
编码46H2A11C7的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.64中的第163位至第183位；
编码46H2A11C7的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.64中的第280位至第306位。
编码2A12B2B2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.65中的第76位至第105位；
编码2A12B2B2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.65中的第148位至第198位；
编码2A12B2B2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.65中的第295位至第303位；
编码2A12B2B2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.66中的第70位至第102位；
编码2A12B2B2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.66中的第148位至第168位；
编码2A12B2B2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.66中的第265位至第291位。
编码8B5D1A9的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.67中的第76位至第105位；
编码8B5D1A9的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.67中 的第148位至第198位；
编码8B5D1A9的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.67中的第295位至第324位；
编码8B5D1A9的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.68中的第70位至第120位；
编码8B5D1A9的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.68中的第166位至第189位；
编码8B5D1A9的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.68中的第286位至第312位。
编码9D5A12G7的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.69中的第76位至第105位；
编码9D5A12G7的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.69中的第148位至第195位；
编码9D5A12G7的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.69中的第292位至第321位；
编码9D5A12G7的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.70中的第70位至第120位；
编码9D5A12G7的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.70中的第166位至第186位；
编码9D5A12G7的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.70中的第283位至第306位。
应理解，在阅读了本发明的上述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种分离的蛋白质，其特征在于，其包括C5aR抗体的重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或，C5aR抗体的轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种，所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42或SEQ ID No.50所示；所述重链CDR2的氨基酸序列如序列表SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43或SEQ ID No.51所示；所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44或SEQ ID No.52所示；
    所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46或SEQ ID No.54所示；所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47或SEQ ID No.55所示；所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48或SEQ ID No.56所示；
    或者，所述重链CDR1的氨基酸序列与如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42或SEQ ID No.50所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR2的氨基酸序列与如序列表中SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43或SEQ ID No.51所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR3的氨基酸序列与如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44或SEQ ID No.52所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；
    所述轻链CDR1的氨基酸序列与如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46或SEQ ID No.54所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR2的氨基酸序列与如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47或SEQ ID No.55所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR3的氨基酸序列与如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48或SEQ ID No.56所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示。
  2. 如权利要求1所述的蛋白质，其特征在于，所述重链CDR1的氨基酸序列如序列表SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.3所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.4所示；
    所述重链CDR1的氨基酸序列如序列表SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.11所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.12所示；
    所述重链CDR1的氨基酸序列如序列表SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.19所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.20所示；
    所述重链CDR1的氨基酸序列如序列表SEQ ID No.26所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.27所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.28所示；
    所述重链CDR1的氨基酸序列如序列表SEQ ID No.34所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.35所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.36所示；
    所述重链CDR1的氨基酸序列如序列表SEQ ID No.42所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.43所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.44所示；
    所述重链CDR1的氨基酸序列如序列表SEQ ID No.50所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.51所示且所述重链CDR3的氨基酸序列如序列表SEQ ID No.52所示；
    所述轻链CDR1的氨基酸序列如序列表SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.7所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.8所示；
    所述轻链CDR1的氨基酸序列如序列表SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.15所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.16所示；
    所述轻链CDR1的氨基酸序列如序列表SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.23所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.24所示；
    所述轻链CDR1的氨基酸序列如序列表SEQ ID No.30所示，所述轻链CDR2的氨基 酸序列如序列表SEQ ID No.31所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.32所示；
    所述轻链CDR1的氨基酸序列如序列表SEQ ID No.38所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.39所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.40所示；
    所述轻链CDR1的氨基酸序列如序列表SEQ ID No.46所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.47所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.48所示；
    或，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.54所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.55所示且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.56所示。
  3. 一种分离的蛋白质，其特征在于，其包括C5aR抗体的重链可变区和/或C5aR抗体的轻链可变区，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.33、SEQ ID No.41或SEQ ID No.49所示；所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.29、SEQ ID No.37、SEQ ID No.45或SEQ ID No.53所示。
  4. 如权利要求3所述的蛋白质，其特征在于，所述重链可变区的氨基酸序列如序列表SEQ ID No.1所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.5所示；
    所述重链可变区的氨基酸序列如序列表SEQ ID No.9所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.13所示；
    所述重链可变区的氨基酸序列如序列表SEQ ID No.17所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.21所示；
    所述重链可变区的氨基酸序列如序列表SEQ ID No.25所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.29所示；
    所述重链可变区的氨基酸序列如序列表SEQ ID No.33所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.37所示；
    所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示；
    或，所述重链可变区的氨基酸序列如序列表SEQ ID No.49所示且所述轻链可变区的氨基酸序列如序列表SEQ ID No.53所示。
  5. 如权利要求1～4中任一项所述的蛋白质，其特征在于，所述的蛋白质还包括抗体 重链恒定区和/或抗体轻链恒定区。
  6. 如权利要求5所述的蛋白质，其特征在于，所述的抗体重链恒定区为小鼠源抗体重链恒定区；所述的抗体轻链恒定区为小鼠源抗体轻链恒定区。
  7. 如权利要求1或3所述的蛋白质，其特征在于，所述的蛋白质是C5aR抗体的单克隆抗体、抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体、单域抗体或单区抗体。
  8. 一种核酸，其特征在于，其编码如权利要求1～6中任一项所述的蛋白质。
  9. 如权利要求8所述的核酸，其特征在于，编码所述重链可变区的核酸如序列表SEQ ID No.57、序列表SEQ ID No.59、序列表SEQ ID No.61、序列表SEQ ID No.63、序列表SEQ ID No.65、序列表SEQ ID No.67或序列表SEQ ID No.69所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.58、序列表SEQ ID No.60、序列表SEQ ID No.62、序列表SEQ ID No.64、序列表SEQ ID No.66、序列表SEQ ID No.68或序列表SEQ ID No.70所示。
  10. 如权利要求9所述的核酸，其特征在于，编码所述重链可变区的核酸如序列表SEQ ID No.57所示且编码所述轻链可变区的核酸如序列表SEQ ID No.58所示；
    编码所述重链可变区的核酸如序列表SEQ ID No.59所示且编码所述轻链可变区的核酸如序列表SEQ ID No.60所示；
    编码所述重链可变区的核酸如序列表SEQ ID No.61所示且编码所述轻链可变区的核酸如序列表SEQ ID No.62所示；
    编码所述重链可变区的核酸如序列表SEQ ID No.63所示且编码所述轻链可变区的核酸如序列表SEQ ID No.64所示；
    编码所述重链可变区的核酸如序列表SEQ ID No.65所示且编码所述轻链可变区的核酸如序列表SEQ ID No.66所示；
    编码所述重链可变区的核酸如序列表SEQ ID No.67所示且编码所述轻链可变区的核酸如序列表SEQ ID No.68所示；
    或，编码所述重链可变区的核酸如序列表SEQ ID No.69所示且编码所述轻链可变区的核酸如序列表SEQ ID No.70所示。
  11. 一种包含如权利要求8～10中任一项所述的核酸的重组表达载体。
  12. 一种包含如权利要求11所述的重组表达载体的重组表达转化体。
  13. 一种C5aR抗体的制备方法，其包括如下步骤：培养如权利要求12所述的重组表达转化体，从培养物中获得C5aR抗体。
  14. 一种检测过表达C5aR蛋白的细胞的方法，其特征在于，包括如下的步骤：如权利要求1～6中任一项所述的蛋白质与待检样品在体外接触，检测如权利要求1～6中任一项所述的蛋白质与所述待检样品的结合即可。
PCT/CN2017/070713 2016-01-12 2017-01-10 一种C5aR抗体及其制备方法和应用 WO2017121307A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CN202110368308.1A CN112851817B (zh) 2016-01-12 2017-01-10 一种C5aR抗体及其制备方法和应用
CN201780003251.7A CN108026178B (zh) 2016-01-12 2017-01-10 一种C5aR抗体及其制备方法和应用
CN202110369308.3A CN113150151B (zh) 2016-01-12 2017-01-10 一种C5aR抗体及其制备方法和应用
US16/965,209 US20210070875A1 (en) 2016-01-12 2017-01-10 Anti-c5ar antibody and preparation method and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610018727.1 2016-01-12
CN201610018727.1A CN106957366B (zh) 2016-01-12 2016-01-12 一种C5aR抗体及其制备方法和应用

Publications (1)

Publication Number Publication Date
WO2017121307A1 true WO2017121307A1 (zh) 2017-07-20

Family

ID=59310823

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/070713 WO2017121307A1 (zh) 2016-01-12 2017-01-10 一种C5aR抗体及其制备方法和应用

Country Status (3)

Country Link
US (1) US20210070875A1 (zh)
CN (4) CN106957366B (zh)
WO (1) WO2017121307A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3632932A4 (en) * 2017-06-01 2021-08-18 Jiangsu Hengrui Medicine Co. Ltd. ANTI-CD40 ANTIBODIES, ANTIGEN BINDING FRAGMENT OF THIS ANTIGEN AND ASSOCIATED MEDICAL USE
EP3892634A4 (en) * 2018-11-30 2022-11-16 Jiangsu Hengrui Pharmaceuticals Co., Ltd. ANTI-CD40 ANTIBODIES, ANTIGEN BINDING FRAGMENT AND PHARMACEUTICAL USE THEREOF
WO2023056473A1 (en) * 2021-09-30 2023-04-06 The Regents Of The University Of California Anti-human astrovirus antibodies and related compositions and methods

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106957366B (zh) * 2016-01-12 2022-02-01 上海昀怡健康科技发展有限公司 一种C5aR抗体及其制备方法和应用
CA3127556A1 (en) * 2019-01-28 2020-08-06 Tuojie Biotech (Shanghai) Co., Ltd. Anti-cd79b antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
TW202246331A (zh) 2021-01-13 2022-12-01 美商威特拉公司 人源化補體５ａ受體１抗體及其使用方法
CN113512106B (zh) * 2021-04-28 2022-05-13 湖南农业大学 一种草鱼C5aR蛋白合成肽及其多克隆抗体

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010229122A (ja) * 2009-03-06 2010-10-14 Kumamoto Univ 抗癌剤、医薬、及び癌疾患の検査薬
CN103596982A (zh) * 2011-06-06 2014-02-19 诺沃—诺迪斯克有限公司 治疗性抗体
CN104059149A (zh) * 2006-08-22 2014-09-24 G2炎症私人有限公司 具有改进性能的抗-C5aR抗体
WO2014180961A1 (en) * 2013-05-08 2014-11-13 Novo Nordisk A/S Use of c5ar antagonists

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5449099A (en) * 1998-09-03 2000-03-27 Asahi Kasei Kabushiki Kaisha Novel receptor protein and method for diagnosing inflammatory diseases by using the same
EP1476469B1 (en) * 2002-01-25 2015-11-18 Novo Nordisk A/S Monoclonal antibodies against extracellular loops of c5ar
EP2826791A3 (en) * 2008-02-20 2015-07-29 Novo Nordisk A/S Humanized anti-C5aR antibodies
CN102597005A (zh) * 2009-06-23 2012-07-18 阿雷克森制药公司 与补体蛋白质结合的双特异性抗体
EP2327725A1 (en) * 2009-11-26 2011-06-01 InflaRx GmbH Anti-C5a binding moieties with high blocking activity
CN106957366B (zh) * 2016-01-12 2022-02-01 上海昀怡健康科技发展有限公司 一种C5aR抗体及其制备方法和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104059149A (zh) * 2006-08-22 2014-09-24 G2炎症私人有限公司 具有改进性能的抗-C5aR抗体
JP2010229122A (ja) * 2009-03-06 2010-10-14 Kumamoto Univ 抗癌剤、医薬、及び癌疾患の検査薬
CN103596982A (zh) * 2011-06-06 2014-02-19 诺沃—诺迪斯克有限公司 治疗性抗体
WO2014180961A1 (en) * 2013-05-08 2014-11-13 Novo Nordisk A/S Use of c5ar antagonists

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HUANG, LILI ET AL.: "Discovery of Human Antibodies against the C5aR Target Using Phage Display Technology", JOURNAL OF MOLECULAR RECOGNITION, vol. 4, no. 18, 31 December 2005 (2005-12-31), pages 327 - 333, ISSN: 0950-3499 *
WANG, HUIHUI ET AL.: "Complement C5a,C5a Receptor and their Antagonists: Research Advances", JOURNAL OF INTERNATIONAL PHARMACEUTICAL RESEARCH, vol. 37, no. 3, 30 June 2010 (2010-06-30), pages 181 - 186, XP055400548, ISSN: 1674-0440 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3632932A4 (en) * 2017-06-01 2021-08-18 Jiangsu Hengrui Medicine Co. Ltd. ANTI-CD40 ANTIBODIES, ANTIGEN BINDING FRAGMENT OF THIS ANTIGEN AND ASSOCIATED MEDICAL USE
US11525005B2 (en) 2017-06-01 2022-12-13 Jiangsu Hengrui Medicine Co., Ltd. Anti-CD40 antibody, antigen binding fragment thereof and medical use thereof
EP3892634A4 (en) * 2018-11-30 2022-11-16 Jiangsu Hengrui Pharmaceuticals Co., Ltd. ANTI-CD40 ANTIBODIES, ANTIGEN BINDING FRAGMENT AND PHARMACEUTICAL USE THEREOF
WO2023056473A1 (en) * 2021-09-30 2023-04-06 The Regents Of The University Of California Anti-human astrovirus antibodies and related compositions and methods

Also Published As

Publication number Publication date
CN106957366A (zh) 2017-07-18
CN108026178B (zh) 2021-04-23
CN112851817A (zh) 2021-05-28
CN113150151B (zh) 2022-06-21
US20210070875A1 (en) 2021-03-11
CN108026178A (zh) 2018-05-11
CN113150151A (zh) 2021-07-23
CN106957366B (zh) 2022-02-01
CN112851817B (zh) 2022-06-21

Similar Documents

Publication Publication Date Title
WO2017121307A1 (zh) 一种C5aR抗体及其制备方法和应用
CN111051347B (zh) Tigit抗体、其抗原结合片段及医药用途
CN106939047B (zh) 一种pd-l1抗体及其制备方法
JP7215759B2 (ja) ４－１ｂｂ抗体およびその製造方法と使用
CN110272490B (zh) 靶向ctla-4抗体、其制备方法和用途
CN112876564B (zh) 一种tslp相关病症治疗剂的开发和应用
CN110914304A (zh) Cd96抗体、其抗原结合片段及医药用途
WO2020239134A1 (zh) 抗体白介素4受体的抗体及其应用
CN111378043A (zh) 一种具有中和作用的人鼠嵌合抗Siglec-15全分子IgG及其制备方法和应用
CN113508139A (zh) 结合人lag-3的抗体、其制备方法和用途
WO2021218874A1 (zh) 一种靶向人claudin和人PDL1蛋白的双特异抗体及其应用
WO2017198148A1 (zh) 一种il-13抗体及其制备方法和应用
WO2020038404A1 (zh) 抗人claudin 18.2单克隆抗体及其应用
CN109021103B (zh) 抗人血管内皮生长因子的抗体及其制备方法和应用
WO2019114793A1 (zh) 一种egfr抗体及其制备方法和应用
CN107964045B (zh) 一种人鼠嵌合抗CXCR2全分子IgG及其应用
CN110343178B (zh) 抗人lag-3单克隆抗体及其应用
WO2023173258A1 (zh) 一种抗cd3抗体及其制备方法和用途
WO2018006824A1 (zh) 一种BLyS抗体及其制备方法和应用
CN109021104B (zh) 一种抗人血小板衍生生长因子β受体的抗体及其应用
CN115611976B (zh) 一种人MICA/Bα3区水解相关的抗原表位肽及其应用
EP2552958B1 (en) Monoclonal antibody directed against the p17 protein of hiv, capable of neutralising the binding of p17 to the p17 receptor (p17r)
JP7346576B2 (ja) 抗ｏｘ４０モノクローナル抗体とその応用
JP2023553205A (ja) Ｓｔｒｅｐ－ＴａｇＩＩタグに特異的に結合する抗体およびその使用
CN115521380A (zh) 靶向gpc3的抗原结合蛋白

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17738121

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17738121

Country of ref document: EP

Kind code of ref document: A1