CN113563475B - Bispecific antibody for resisting novel coronavirus and application thereof - Google Patents

Bispecific antibody for resisting novel coronavirus and application thereof Download PDF

Info

Publication number
CN113563475B
CN113563475B CN202110849422.6A CN202110849422A CN113563475B CN 113563475 B CN113563475 B CN 113563475B CN 202110849422 A CN202110849422 A CN 202110849422A CN 113563475 B CN113563475 B CN 113563475B
Authority
CN
China
Prior art keywords
ser
gly
val
leu
thr
Prior art date
Legal status (The legal status 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 status listed.)
Active
Application number
CN202110849422.6A
Other languages
Chinese (zh)
Other versions
CN113563475A (en
Inventor
高福
吴燕
李世华
黎朝晖
谭曙光
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Microbiology of CAS
Original Assignee
Institute of Microbiology of CAS
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 Institute of Microbiology of CAS filed Critical Institute of Microbiology of CAS
Publication of CN113563475A publication Critical patent/CN113563475A/en
Application granted granted Critical
Publication of CN113563475B publication Critical patent/CN113563475B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • Communicable Diseases (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention relates to a bispecific antibody for resisting novel coronavirus and application thereof. The bispecific antibody is obtained by modifying new coronavirus monoclonal antibodies H4 and B38 through a genetic engineering method, can simultaneously identify different sites of a new coronavirus S protein RBD, has far higher neutralizing activity on a new coronavirus pseudovirus than a maternal monoclonal antibody, and has higher inhibiting activity on a new coronavirus live virus than the maternal monoclonal antibody. The bispecific antibody of the invention improves the selectivity and the neutralization activity of the maternal monoclonal antibody, improves the safety and the effectiveness of the monoclonal antibody drug, can be used as a potential drug for diagnosing, preventing and treating diseases caused by novel coronavirus, and has great market value and good application prospect.

Description

Bispecific antibody for resisting novel coronavirus and application thereof
Technical Field
The invention relates to the technical field of biotechnology and immunology, in particular to a bispecific antibody for resisting novel coronavirus, a preparation method and application thereof.
Background
Spike protein (Spike, S protein) on the surface of the novel coronavirus (2019-nCoV) is combined with angiotensin converting enzyme 2(ACE2) molecule which is a host cell receptor in the process of infecting a host, so that fusion of a virus membrane and a host cell membrane is started, and the host cell is infected with the virus. The S protein is divided into two parts, S1 and S2, and research has proved that the Receptor Binding Domain (RBD) of C Terminal (CTD) of S1 is combined with ACE2 to mediate the membrane fusion process.
To date, neutralizing antibodies have proven to be an effective method of treating viral diseases. Currently marketed drugs for the treatment and prevention of viral infections are palivizumab (Synagis) for the prevention of Respiratory Syncytial Virus (RSV) infection in children, abalizumab (Trogarzo) for the treatment of HIV infection, and Rabishield for the prevention after rabies virus exposure. Furthermore, there are a number of monoclonal antibodies against different viruses at different stages of clinical research (https:// clinicaltirials. gov /). Antibodies act primarily by two aspects. In one aspect, an antibody having neutralizing activity can block viral infection by binding to viral envelope proteins, blocking binding of the virus to cellular receptors. Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) recruit immune cells and immune molecules, such as macrophages or complement, to eliminate free virus and infected cells.
Bispecific antibodies (BsAb) are one of the major directions in the field of antibody drug research in recent years. Bispecific antibodies are artificial antibodies containing two molecules capable of specifically recognizing and binding to different antigens or different antigenic sites. If the two antigens are located on different cell surfaces, the bispecific antibody can bridge the two antigen molecules, thereby forming cross-linking between cells and mediating the cells to generate targeted effector functions. BsAb has wide application prospect in biomedicine, especially in the immunotherapy of tumor. Bispecific antibodies (immune diabodies) for immunotherapy are artificial antibodies containing 2 specific cell receptor antigen binding sites, which can bridge between diseased cells (target cells) and functional cells (immune cells) and stimulate a directed immune response. The BsAb mediated immune cells (such as T cells, NK cells and the like) to kill tumor cells is a hot spot of the current immunotherapy application research, and the action mechanism of the BsAb is that the BsAb can be simultaneously combined with tumor-associated antigens and target molecules on immune effector cells, and the BsAb can directly guide the immune effector cells to specifically kill the tumor cells while activating the immune cells. Currently, approved bispecific antibodies on the market include two, EpCAM/CD3 and CD19/CD3, more than one hundred of which are still in clinical research. For infectious pathogenic microorganisms, particularly RNA viruses which are easy to mutate, a targeted single-site antibody is used for infection treatment, and the virus is easy to mutate under the selective pressure of the antibody, so that the binding of the antibody is escaped, and the antibody which is not sensitive to antibody treatment is generated. Meanwhile, two different sites of the virus antigen are targeted, so that the probability of generation of escape mutation of the virus can be greatly reduced. Moreover, targeting different sites can obviously improve the virus neutralization effect, reduce the dosage of the antibody and improve the treatment effect. For example, in clinical treatment of ebola virus infection, three different antibodies that bind to ebola virus surface Glycoprotein (GP) can be used to significantly reduce patient mortality and have significant therapeutic effects on ebola virus infection.
Bispecific antibodies are available in a variety of ways, and are prepared mainly by: chemical coupling method, hybrid-hybridoma method and gene engineering antibody preparation method. The chemical coupling method is a bispecific monoclonal antibody prepared by linking 2 different monoclonal antibodies together by means of chemical coupling, which is the earliest bispecific monoclonal antibody. The hybrid-hybridoma method is to produce bispecific monoclonal antibodies by means of cell hybridization or triple hybridomas, which are obtained by fusion of established hybridomas or established hybridomas with mouse lymphocytes, and can be used only for production of bispecific antibodies of murine origin, and thus, its application is greatly limited. With the rapid development of molecular biology technology, various construction modes of genetic engineering humanized or fully human bispecific antibodies appear, mainly including four types of bispecific miniantibodies, diabodies, single-chain diabodies and multivalent bispecific antibodies. At present, several genetic engineering bispecific antibody medicaments enter a clinical test stage internationally and show good application prospects.
The advantages of bispecific antibodies compared to monoclonal antibodies are: the bispecific antibody can simultaneously recognize two molecules, thereby improving the selectivity and functionality of the antibody and improving the safety and effectiveness of medicaments. Bispecific antibody drugs reduce development and clinical trial costs compared to treatment with a combination of two monoclonal antibody drugs. For infectious pathogens, simultaneous action on two different sites of the pathogen makes the development of antibody resistance less likely during therapy and suppresses the development of escape mutations in the virus over a broad range of therapies. Therefore, the development of bispecific antibodies targeting different sites of the novel coronavirus is of great significance for improving the treatment effect of the novel coronary pneumonia, reducing the generation of escape mutation and the like.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention aims to provide a bispecific antibody which has a specific targeting effect, good affinity to a novel coronavirus and remarkable virus inhibition activity, and an application thereof.
To achieve the above objects, in a first aspect, the present invention provides a bispecific antibody, which comprises the following technical schemes: the invention creatively discovers that after the monoclonal antibody B38 and the monoclonal antibody H4 are subjected to the genetic modification, the specific binding capacity of the original maternal monoclonal antibody can be better reserved, and the monoclonal antibody has the biological functions of two monoclonal antibodies, and has obvious advantages in virus neutralization activity, safety, stability and the like compared with the maternal monoclonal antibody.
In the invention, the heavy chain variable region CDR1 of the monoclonal antibody B38 contains an amino acid sequence shown by SEQ ID NO.9, CDR2 contains an amino acid sequence shown by SEQ ID NO.10, and CDR3 contains an amino acid sequence shown by SEQ ID NO. 11; the light chain variable region CDR1 of the monoclonal antibody B38 contains an amino acid sequence shown by SEQ ID NO.12, the CDR2 contains an amino acid sequence shown by SEQ ID NO.13, and the CDR3 contains an amino acid sequence shown by SEQ ID NO. 14.
The heavy chain variable region CDR1 of the monoclonal antibody H4 contains an amino acid sequence shown by SEQ ID NO.15, CDR2 contains an amino acid sequence shown by SEQ ID NO.16, and CDR3 contains an amino acid sequence shown by SEQ ID NO. 17; the light chain variable region CDR1 of the monoclonal antibody H4 contains an amino acid sequence shown by SEQ ID NO.18, CDR2 contains an amino acid sequence shown by SEQ ID NO.19, and CDR3 contains an amino acid sequence shown by SEQ ID NO. 20.
Different from the design of traditional monoclonal antibodies, polyclonal antibodies, single chain antibodies and the like, the factors and the faced technical difficulties of the bispecific antibodies need to be considered during the design are mainly as follows: the bispecific antibody is a fusion protein formed between antibodies with different structures, and the antibodies with different structures respectively combine different antigens of two cells or different sites of the same antigen to exert different functions. In order to make each antibody in the bispecific antibody fully exert its own function, the adverse effects of the mutual influence of the respective antibodies in sequence and structure on the respective functions should be minimized in antibody design. And bispecific antibodies when targeting different antigens or different sites, the binding affinities of bispecific antibodies to different antigens or different sites of the same antigen need to be balanced, matched to each other, rather than simply pursuing high affinity for both targets, in order to maximize neutralization of viral titers.
The invention discovers that the bispecific antibody obtained by different modification methods has great difference in affinity, neutralization activity and stability in the process of selecting monoclonal antibody B38 and monoclonal antibody H4 to construct the bispecific antibody through genetic modification. Through repeated screening and exploration, the invention provides two bispecific antibodies with excellent effects, which are obtained by modification through a genetic engineering method and are as follows:
(1) the N end of the light chain variable region of the bispecific antibody BS-mAb-1, the light chain variable region sequence of which is B38 monoclonal antibody, is connected with the C end of the light chain variable region of H4 monoclonal antibody through a connecting peptide; the heavy chain variable region sequence is that the N end of the heavy chain variable region of the monoclonal antibody B38 is connected with the C end of the light chain variable region of the monoclonal antibody H4 through a connecting peptide; or
(2) The bispecific antibody BS-mAb-2 comprising a B38 monoclonal antibody unit and a H4 single chain antibody unit,
the H4 single-chain antibody unit comprises 2 single-chain antibodies, and the single-chain antibodies are fusion peptides formed by connecting the C end of the heavy chain variable region of the H4 monoclonal antibody with the N end of the light chain variable region of the H4 monoclonal antibody through connecting peptides; the C ends of the 2 single-chain antibodies are respectively connected with the N ends of the 2 heavy chains of the B38 monoclonal antibody through connecting peptides, or the N ends of the 2 single-chain antibodies are respectively connected with the C ends of the 2 heavy chains of the monoclonal antibody through connecting peptides.
Further, the amino acid sequence of the connecting peptide is (GGGGS) n, wherein n is a natural number of 1-4. Preferably, in the above (1), n of the linker peptide is 2; in the above (2), n of the linker peptide in the fusion peptide is 4, and n of the linker peptide between the single-chain antibody and the heavy chain of the monoclonal antibody is 1.
In one embodiment, the bispecific antibody of the present invention comprises the heavy chain variable region of mab B38, the light chain variable region of mab B38, the heavy chain variable region of mab H4, the light chain variable region of mab H4, wherein,
the heavy chain variable region of mab B38 comprises: CDR1 with an amino acid sequence shown as SEQ ID NO.9, CDR2 with an amino acid sequence shown as SEQ ID NO.10 and CDR3 with an amino acid sequence shown as SEQ ID NO. 11; the light chain variable region of mab B38 comprises: CDR1 with an amino acid sequence shown as SEQ ID NO.12, CDR2 with an amino acid sequence shown as SEQ ID NO.13, and CDR3 with an amino acid sequence shown as SEQ ID NO. 14;
the heavy chain variable region of mab H4 comprises: CDR1 with an amino acid sequence shown in SEQ ID NO.15, CDR2 with an amino acid sequence shown in SEQ ID NO.16 and CDR3 with an amino acid sequence shown in SEQ ID NO. 17; the light chain variable region of mab H4 comprises: CDR1 with an amino acid sequence shown in SEQ ID NO.18, CDR2 with an amino acid sequence shown in SEQ ID NO.19 and CDR3 with an amino acid sequence shown in SEQ ID NO. 20.
Preferably, the amino acid sequence of the heavy chain variable region of monoclonal antibody B38 is shown in SEQ ID NO.32, and the amino acid sequence of the light chain variable region of monoclonal antibody B38 is shown in SEQ ID NO. 33; the amino acid sequence of the heavy chain variable region of the monoclonal antibody H4 is shown as SEQ ID NO.34, and the amino acid sequence of the light chain variable region of the monoclonal antibody H4 is shown as SEQ ID NO. 35.
Further preferably, the constant region of monoclonal antibody B38 and/or the constant region of monoclonal antibody H4; the constant region can be any suitable constant region known in the art.
In a preferred embodiment, the bispecific antibody is BS-mAb-1, which has the following structure (from N-terminus to C-terminus):
heavy chain: VHH4-VHB38-CHB38，
Light chain: VLH4-VLB38-CLB38，
Wherein VHH4And VHB38By a linker peptide, VLH4And VLB38Linked by a linker peptide;
in another preferred embodiment, the bispecific antibody is BS-mAb-2, which has the following structure (from N-terminus to C-terminus):
heavy chain: VHB38-CHB38-VHH4-VLH4，
Light chain: VLB38-CLB38；
Wherein VHH4And VLH4Connecting through connecting peptide to form single-chain antibody; CH (CH)B38And VHH4Linked by a linker peptide.
Preferably, the bispecific antibody comprises two identical heavy chains and two identical light chains.
Wherein the amino acid sequence of the connecting peptide is (GGGGS) n, wherein n is a natural number of 1-4. Preferably, in the antibody BS-mAb-1, n of the linker peptide is 2; in antibody BS-mAb-2, the VHH4And VLH4N of the connecting peptide between is 4, CHB38And VHH4N of the connecting peptide therebetween is 1.
In the description above and elsewhere in this application, VHB38Represents the heavy chain variable region of mAb B38, which is synonymous with (VH-B38); CH (CH)B38Represents the heavy chain constant region of the monoclonal antibody B38, comprising CH1-CH 3; VHH4Represents the heavy chain variable region of mAb H4, which is synonymous with (VH-H4); VLH4The variable region of the light chain of mAb H4, which is synonymous with (VL-H4); VLB38Represents the light chain variable region of mAb B38, synonymous with (VL-B38); CLB38Represents monoclonal antibody B38, abbreviated as CL. In some embodiments, the heavy chain constant region of mab B38 has the same sequence as the heavy chain constant region of mab H4; the light chain constant region of mab B38 has the same sequence as the light chain constant region of mab H4.
Preferably, the bispecific antibody BS-mAb-1 has the amino acid sequence of the light chain as shown in SEQ ID NO.4 or the amino acid sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more amino acids, and has the amino acid sequence of the heavy chain as shown in SEQ ID NO.2 or the amino acid sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more amino acids.
The invention adopts the form of fusion peptide to express the single-chain antibody, and discovers that the stability of the antibody structure and the combination with a target point can be better improved by respectively adopting the specific single-chain antibody fusion peptide sequence when the connection modes of the single-chain antibody and the monoclonal antibody are different through specific antibody structure and sequence design.
Preferably, in the bispecific antibody BS-mAb-2, the amino acid sequence of the single-chain antibody is shown in SEQ ID NO. 21; further preferably, the bispecific antibody BS-mAb-2 has the amino acid sequence of the light chain as shown in SEQ ID NO.8 or the amino acid sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more amino acids, and has the amino acid sequence of the heavy chain as shown in SEQ ID NO.6 or the amino acid sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more amino acids.
The above-mentioned "amino acid sequence of a protein having the same function by substitution, deletion or insertion of one or more amino acids" refers to a sequence which differs from the indicated sequence at one or more amino acid residues but retains the biological activity of the resulting molecule, and may be modified by "conservatively modified variants" or by "conservative amino acid substitutions" which refer to amino acid substitutions known to those skilled in the art, which are made so as not to generally alter the biological activity of the resulting molecule. In general, one skilled in the art recognizes that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity. Exemplary substitutions are preferably made according to the substitutions shown below:
table 1 exemplary conservative amino acid assignments
Residue of a proenzyme Conservative substitutions Ortho residue Conservative substitutions Residue of a proenzyme Conservative substitutions
Ala(A) Gly,Ser Pro(P) Ala Gly(G) Ala
Arg(R) Lys，His Ser(S) Thr His(H) Asn Gln
Asn(N) Gln,His Thr(T) Ser Ile(I) Leu,Val
Asp(D) Glu,Asn Trp(W) Tyr,Phe Lys(K) Arg,His
Cys(C) Ser,Ala Tyr(Y) Trp,Phe Met(M) Leu,Ile,Tyr
Gln(Q) Asn Val(V) Ile,Leu Phe(F) Tyr,Met,Leu
Glu(E) Asp,Gln
In the present invention, the bispecific antibody may be a murine antibody, a humanized antibody, a chimeric antibody or a recombinant antibody.
The human or humanized antibody comprises one of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, and an IgG4 antibody.
In a second aspect, the present invention also provides a gene encoding the bispecific antibody based on the amino acid sequence of the bispecific antibody described above.
Based on the codon encoding rules and the degeneracy and preference of codons, one skilled in the art can design an encoding gene based on the amino acid sequence of the bispecific antibody described above.
As a preferred embodiment of the invention, the nucleotide sequence of the light chain of the bispecific antibody is shown in SEQ ID NO.3, and the nucleotide sequence of the heavy chain is shown in SEQ ID NO. 1; or
The nucleotide sequence of the bispecific antibody light chain is shown as SEQ ID NO.7, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 5.
Further, the present invention also provides a biomaterial comprising the gene.
In the present invention, the biological material includes recombinant DNA, expression cassette, vector, host cell, engineering bacterium or cell line.
In a third aspect, the present invention also provides a method of making the bispecific antibody comprising: respectively constructing recombinant expression vectors containing heavy chain and light chain genes of the bispecific antibody BS-mAb-1 or BS-mAb-2; introducing the recombinant expression vector into a host cell to obtain a host cell for stably expressing the bispecific antibody; culturing host cells, and separating and purifying to obtain the bispecific antibody.
In the preparation of the bispecific antibody, the skilled in the art can select the host cell, expression vector, method for introducing the expression vector into the host cell, and method for separating and purifying the antibody, which are conventional in the art, as required.
In a fourth aspect, the present invention provides a pharmaceutical composition comprising the bispecific antibody of the present invention, on the basis of the bispecific antibody described above.
Preferably, the pharmaceutical composition further comprises other effective components or auxiliary materials allowed in the pharmaceutical field.
In a fifth aspect, the invention also provides a diagnostic reagent or kit comprising a bispecific antibody according to the invention. Preferably, it comprises BS-mAb-1 and/or BS-mAb-2.
In a sixth aspect, the present invention provides any one of the following uses of the bispecific antibody or the gene encoding the bispecific antibody or the biological material containing the encoding gene or the pharmaceutical composition or the kit:
(1) the application in preparing the medicine for preventing or treating the diseases caused by the novel coronavirus infection;
(2) the application in preparing a novel coronavirus diagnostic reagent or a diagnostic kit;
(3) the application in preparing novel coronavirus vaccines;
(4) the application in preventing or treating SARS-CoV-2 coronavirus caused diseases;
(5) the application in detecting novel coronavirus.
Further, the present invention provides a pharmaceutical composition in a single dose form, wherein the single dose form contains 180mg to 6000mg of the bispecific antibody of the present invention; preferably wherein the single dose form contains 180mg to 3000mg of a bispecific antibody according to the invention; more preferably, wherein the single dose form contains 500mg to 1800mg of the bispecific antibody according to the invention; further preferably wherein the single dose form contains 900mg to 1800mg of the bispecific antibody of the invention; still further preferred, wherein the single dose form contains 500mg-1000mg of the bispecific antibody according to the invention.
The pharmaceutical composition of the present invention in a single dosage form, wherein the pharmaceutical composition is formulated in a form suitable for intravenous administration.
Further, the present invention provides a method for preventing or treating a disease caused by a novel coronavirus infection, comprising administering 180mg to 6000mg of the bispecific antibody of the present invention to a human in need thereof; preferably, it comprises administering 180mg to 3000mg of a bispecific antibody of the invention to a human in need thereof; more preferably, it comprises administering 500mg-1800mg of a bispecific antibody of the invention to a human in need thereof; further preferably, it comprises administering 900mg to 1800mg of a bispecific antibody of the invention to a human in need thereof; still further preferably, it comprises administering 500mg-1000mg of the bispecific antibody of the present invention to a human in need thereof.
The invention provides a method for preventing or treating diseases caused by novel coronavirus infection, which comprises the steps of administering the bispecific antibody of the invention to a receptor in need, and administering for 1-2 times per week and continuously for 2-4 weeks; preferably, the administration is 2 times per week for 4 weeks.
Further, the present invention provides a method for diagnosing a novel coronavirus infection, comprising contacting a test sample with the bispecific antibody, pharmaceutical composition or formulation of the present invention.
In the present invention, the novel coronavirus is SARS-CoV-2 coronavirus.
The invention has the following beneficial effects: the invention utilizes gene engineering and antibody engineering methods to construct the anti-new coronavirus bispecific antibody containing a single-chain antibody and a complete monoclonal antibody structure, the bispecific antibody fusion protein retains the complete monoclonal antibody structure, has a highly stable symmetrical structure, and can not generate protein isomers with other structures when host expression is carried out, thereby greatly reducing the difficulty of extraction and purification processes, and having the advantages of simple preparation and high yield. The bispecific antibody of the invention better retains the biological functions of maternal monoclonal antibodies B38 and H4, realizes that one bispecific antibody molecule simultaneously has the biological functions of two monoclonal antibodies, simultaneously identifies different sites of a novel coronavirus S protein RBD, has much higher neutralization activity on a novel coronavirus pseudovirus than the maternal monoclonal antibodies, has higher inhibitory activity on a novel coronavirus live virus than the maternal monoclonal antibodies, and has low toxicity and good stability. The bispecific antibody of the invention improves the selectivity and the neutralization activity of the maternal monoclonal antibody, improves the safety and the effectiveness of a monoclonal antibody medicament, can be used as a potential candidate medicament for diagnosing, preventing and treating diseases caused by novel coronavirus, and has great market value and good application prospect.
Drawings
FIG. 1 is a schematic structural diagram of the bispecific antibodies BS-mAb-1(A) and BS-mAb-2(B) of the present invention.
FIG. 2 is a graph of the results of molecular sieve chromatography and SDS-PAGE for the bispecific antibodies BS-mAb-1(A) and BS-mAb-2 (B). Wherein "-" on the gel chart indicates that DTT (non-reducing SDS-PAGE) was not added; "+" indicates addition of DTT (reducing SDS-PAGE).
FIG. 3 is a schematic diagram of the detection of the two-site binding characteristics of BS-mAb-1(A) and BS-mAb-2(B) binding to RBD. Wherein, in panel A, H4-BS-mAb-1 indicates that the RBD-immobilized probe was first saturated with excess H4 antibody and then bound with BS-mAb-1, and the curve was significantly elevated after addition of the BS-mAb-1 antibody compared to the H4-H4 binding curve, indicating that it was able to bind to H4 saturated RBD. H4-H4 shows that the first phase first flows through the H4 antibody, and then the second phase flows through the H4 antibody again; H4-BS-mAb-1 is shown as flowing through the H4 antibody first in the first phase, and then flowing through the BS-mAb-1 antibody after the second phase; B38-BS-mAb-1 is shown as flowing through the B38 antibody after the first phase, and flowing through the BS-mAb-1 antibody after the second phase. In the B diagram, the reference A diagram is read.
FIG. 4 is a graph showing the results of kinetic curves for the binding of H4, B38, BS-mAb-1 and BS-mAb-2 antibodies to RBD protein.
FIG. 5 is a graph of the results of neutralizing activity of H4, B38, BS-mAb-1 and BS-mAb-2 antibodies against 2019-nCoV pseudovirus at different concentrations.
FIG. 6 is a graph showing the results of neutralization activities of the antibodies BS-mAb-1 and BS-mAb-2 against 2019-nCoV live virus at different concentrations.
FIG. 7 is a chart showing SDS-PAGE detection results of the anti-1 #, 6# and 12# antibodies.
Fig. 8 is a graph of the results of neutralization activity of 1#, 6# and 12# antibodies against 2019-nCoV live virus at different concentrations.
FIG. 9 is a graph showing the results of the viral load assay of rhesus monkey pharyngeal swab in example 10.
FIG. 10 is a graph showing the results of the rhesus nasal swab viral load assay of example 10.
FIG. 11 is a graph showing the results of the rhesus monkey anal swab viral load assay of example 10.
Detailed Description
The preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 bispecific antibody construction
In the construction of bispecific antibodies, the variable region arrangement of the two antibodies may have an effect on their binding ability to the antigen, while different constructions may lead to differences in antiviral activity. Thus, there remains a great uncertainty as to whether a bispecific antibody constructed based on two different site antibodies retains its dual site binding activity and can have improved antiviral activity. The bispecific antibody is a fusion protein formed between antibodies with different structures, and the antibodies with different structures are respectively combined with different antigens of two cells or different sites of the same antigen to play different functions. In order to make each antibody in the bispecific antibody fully exert its own function, this example designs a large number of alternative bispecific antibodies in the design of antibodies, in order to minimize the adverse effects of the mutual influence of the respective antibodies in sequence and structure on the respective functions.
Construction of BS-mAb-1
In the construction of BS-mAb-1, the V regions of B38 and H4 were constructed in tandem with a GS flexible hinge region as a junction (fig. 1A), and since the antigen binding sites are the shortest in relative distance, it is assumed that there may be some advantage in binding to adjacent epitopes, and the construction is characterized in that:
the light chain sequence is (VL-H4) - (GG)GGS)2-(VL-B38)-CL；
The heavy chain sequence is (VH-H4) - (GGGGS)2-(VH-B38)-CH1-CH2-CH3。
The light and heavy chain DNA sequences (shown in SEQ ID NO:3 and SEQ ID NO:1, respectively) were cloned into expression vectors pCAGGS (available from Addgene), respectively, to yield recombinant expression vectors encoding the antibody heavy and light chains, respectively. The construction scheme for the constructs expressing the heavy and light chains is as follows:
heavy chain coding sequence (5 '-3'): CMV promoter-EcoR I cleavage site-leader sequence Gene (sequence SEQ ID NO.22) (VH-H4) - (GGGGS)2- (VH-B38) -CH1-CH2-CH3 gene-Xho I cleavage site;
light chain (κ) coding sequence (5 '-3'): CMV promoter-Sac I enzyme cutting site-leader sequence gene (sequence SEQ ID NO.23) - (VL-H4) - (GGGGS)2- (VL-B38) -CL gene-Xho I cleavage site;
construction of BS-mAb-2
In the construction of BS-mAb-2, the VL and VH regions of the H4 antibody are connected by a GS flexible hinge region to construct a single chain antibody (scFv), and then the H4-scFv is constructed at the C-terminus of the heavy chain of the B38 antibody to form a bispecific antibody construction of B38 plus H4-scFv (fig. 1B), which has the construction structure:
the light chain sequence is (VL-B38) -CL
The heavy chain sequence is (VH-B38) -CH1-CH2-CH3-GGGS- (VH-H4) - (GGGGS)4-(VL-H4)
The light and heavy chain DNA sequences (shown in SEQ ID NO:7 and SEQ ID NO:5, respectively) were cloned into expression vectors pCAGGS (purchased from Addgene), respectively, to obtain recombinant expression vectors encoding the antibody heavy and light chains, respectively. The construction scheme of the constructs for expressing the heavy and light chains is as follows:
heavy chain coding sequence (5 '-3'): CMV promoter-EcoR I enzyme cleavage site-leader sequence gene (sequence SEQ ID NO.24) - (VH-B38) -CH1-CH2-CH3-GGGS- (VH-H4) - (GGGGS)4- (VL-H4) gene-Xho I cleavage site;
light chain (κ) coding sequence (5 '-3'): CMV promoter-Sac I cleavage site-leader gene (SEQ ID NO.25) - (VL-B38) -CL gene-Xho I cleavage site.
Example 2 construction of recombinant expression of bispecific antibody and purification of protein expression
In this example, two different bispecific antibodies, BS-mAb-1 and BS-mAb-2, were constructed based on two novel coronavirus S antigen-specific antibodies H4 and B38, the heavy chain amino acid sequences of which are shown in SEQ ID No.2 and SEQ ID No.6, and the light chain amino acid sequences of which are shown in SEQ ID No.4 and SEQ ID No.8, respectively.
HEK293T cells (ATCC CRL-3216) were cultured in DMEM with 10% FBS. HEK293T cells were co-transfected with recombinant expression vectors encoding the heavy and light chains of the antibodies for the BS-mAb-1 and BS-mAb-2 bi-specificities, respectively, described above. After 4-6 hours of transfection, the cell culture medium was replaced with serum-free DMEM, and the culture was continued for 3 days. The supernatant was collected, then DMEM was supplemented, the culture was continued for 4 days, and then the supernatant was collected again.
The collected supernatant was centrifuged at 5000rpm for 30min, then mixed with an equal volume of buffer containing 20mM sodium phosphate (pH 7.0), followed by filtration through a 0.22 μm filter, and then loaded onto a protein A pre-column (5mL, GE Healthcare). Proteins bound to the pre-packed column were eluted with 10mM glycine (pH 3.0). The eluted fractions were concentrated and then purified by molecular sieve chromatography. Subsequently, the purified target protein was detected by SDS-PAGE (reducing and non-reducing). The results are shown in FIG. 2. The results of FIG. 2 show that purified antibodies BS-mAb-1 and BS-mAb-2 were obtained by purification, the antibodies showed a single band in SDS-PAGE under non-reducing conditions, the disulfide bonds of the Fc region of the antibodies were opened in SDS-PAGE under reducing conditions, and thus two bands were shown, and the purity of the antibodies was over 95%.
EXAMPLE 3 assessment of the Dual-site binding Capacity of the BS-mAb-1 and BS-mAb-2 antibodies to the S protein RBD
In this example, the binding analysis of the purified antibody and the S protein RBD was performed by ForteBio Octet RED 96 biofilm layer surface interference technique, and whether the antibodies BS-mAb-1 and BS-mAb-2 have the double site binding ability was evaluated.
The histidine-tagged S protein RBD (science.2020Jun 12; 368(6496): 1274-. To evaluate the two-site binding ability of the BS-mAb-1 antibody, the RBD protein-immobilized probe was first bound to and saturated with H4 antibody (science.2020Jun 12; 368(6496): 1274-1278). The BS-mAb-1 was then flowed through the probes saturated with H4 antibody with an equal concentration of H4 antibody mixture. The results showed that RBD saturated with H4 antibody was still able to bind BS-mAb-1, indicating that it contains BS38 antibody that is not competitive with H4 antibody, in comparison to the level of binding to RBD by H4-H4 antibody (fig. 3 a). Similarly, the RBD protein-immobilized probe was first bound to B38 antibody and allowed to saturate. Then, a mixture of BS-mAb-1 and an equal concentration of B38 antibody (science.2020Jun 12; 368(6496):1274-1278) was passed through the probe saturated with B38 antibody. The results showed that RBD saturated with B38 antibody was still able to bind BS-mAb-1, indicating that it contains H4 antibody that is not competitive with B38 antibody, in comparison to the level of binding to RBD of B38-B38 antibody (a of fig. 3). The two-site binding ability of the BS-mAb-2 antibody was examined by similar experiments as described above, and the results showed that the BS-mAb-2 antibody also had two-site binding ability, indicating that both H4 and scFv of B38 antibody contained therein exerted antigen binding ability (B of FIG. 3).
Example 4 evaluation of the binding Capacity of BS-mAb-1 and BS-mAb-2 antibodies to RBD
First, an anti-human IgG antibody (available from GE Healthcare) was immobilized in a flow cell (Fc) of a CM5 chip in an amino-coupled manner. The fixed amount is controlled around 8,000 Response Units (RU). Then, in the form of antibody capture, respectively, combined with purified B38, H4, BS-mAb-1 or BS-mAb-2 antibody. In addition, RBD protein was diluted serially in 20mM HEPES, 150mM NaCl, pH 7.4 solution in multiple ratios. Serial dilutions of RBD protein (6.25 nM-100 nM) were then run sequentially through each channel (one by one starting from low concentration). Kinetic curves of each antibody binding to RBD protein were recorded and kinetic constants were calculated using BIAevaluation software 8K (Biacore, Inc.) software (fig. 4).
The results in FIG. 4 show that the affinity of the H4 and B38 antibodies to RBD is 8.25nM and 22.5nM, respectively, while the affinity of the bispecific antibodies BS-mAb-1 and BS-mAb-2 to RBD is 4.14nM and 21.7nM, respectively. This indicates that the affinity of the bispecific antibody for RBD is maintained at the same level as the B38 and H4 antibodies.
Example 5 evaluation of the ability of the BS-mAb-1 and BS-mAb-2 antibodies to neutralize 2019-nCoV pseudovirus
The full-length gene of hACE2 (UNIPRO: Q9BYF1) was constructed into Pcdna4.0 expression plasmid (purchased from Invitrogen), was down-transferred to Hela cells, and cultured under a selection pressure of 2. mu.g/ml puromycin (purchased from Gibco Co., Ltd.), and single cells were selected as monoclonal cells, and a Hela stably-transferred cell line (Hela-hACE2) stably expressing hACE2 gene was prepared for use.
Purified B38, H4, BS-mAb-1 or BS-mAb-2 antibodies were diluted from 200. mu.g/mL to a 10 th gradient and incubated with a pseudovirus expressing the new coronavirus S antigen (wild-type VSV-SARS-CoV-2 pseudovirus) (obtained from the Chinese food and drug assay institute) separately at 37 ℃ for 2 hours. After incubation, the virus was added to a 96-well plate previously inoculated with a Hela-transfected cell line expressing human ACE2 (Hela-hACE2) and incubated at 37 ℃ with 5% CO2After 24 hours of incubation in the incubator, the neutralizing titer of the B38, H4, BS-mAb-1 or BS-mAb-2 antibodies was calculated by measuring the level of luciferase substrate reaction activated after pseudovirus infection. The results are shown in FIG. 5. FIG. 5 shows the neutralizing activity of B38, H4, BS-mAb-1 or BS-mAb-2 antibodies against 2019-nCoV pseudovirus at different concentrations. The results show that the neutralizing titer (half-neutralization concentration, NC) of the BS-mAb-1 antibody against 2019-nCoV pseudovirus50) 0.119. mu.g/mL relative to H4 (NC)500.718 μ g/mL) and B38 (NC)500.681 μ g/mL) antibody increased 6-7 fold. The neutralizing titer of the BS-mAb-2 antibody to the 2019-nCoV pseudovirus was 0.048. mu.g/mL, which is a 14-15 fold improvement over the H4 and B38 antibodies. Therefore, bispecific antibodies BS-mAb-1 and BS-mAb-2 were constructed with higher neutralizing activity relative to the H4 and B38 monoclonal antibodies.
Example 6 evaluation of the ability of the BS-mAb-1 and BS-mAb-2 antibodies to neutralize 2019-nCoV live Virus
The purified BS-mAb-1 and BS-mAb-2 antibodies of example 1 were diluted from 100. mu.g/mL to a 10 th gradient and then separately dosed with half of the Tissue Culture Infectious Dose (TCID)50) BetacoV/Shenzhen/SZTH-003/2020 virus (GISAID number: EPI _ ISL \u)406594) was mixed and incubated at 37 degrees Celsius for 2 hours. After incubation, the virus was added to 96-well plates pre-seeded with Vero cells and incubated at 37 deg.C, 5% CO2After 4 days in the incubator, cytopathic effect (CPE) was observed and the neutralizing titers of the BS-mAb-1 and BS-mAb-2 antibodies were calculated. The results are shown in FIG. 6. FIG. 6 shows the neutralizing activity of the BS-mAb-1 and BS-mAb-2 antibodies against live 2019-nCoV virus at different concentrations. The results showed that the neutralizing titer (half inhibitory concentration, IC50) of the BS-mAb-1 antibody against the live 2019-nCoV virus was 1.725. mu.g/mL, and the neutralizing titer (half inhibitory concentration, IC50) of the BS-mAb-2 antibody against the live 2019-nCoV virus was 0.656. mu.g/mL, with good neutralizing activity.
Example 7 construction and validation of additional double antibodies
To demonstrate that the variable region arrangement of bispecific antibodies or a linker peptide linking two mab moieties can have an effect on their ability to bind to antigen, applicants have constructed additional bispecific antibodies based on mab B38 and H4. Other bispecific antibodies are: 1#, 6# and 12#, the structures are respectively:
1#：
light chain (VL-B38) -GQPKAAP- (VL-H4) - (TKQPS) -CL, the amino acid sequence is shown in SEQ ID NO. 26;
the heavy chain (VH-H4) -S- (VH-B38) -RT-CH1-CH2-CH3, and the amino acid sequence is shown as SEQ ID NO. 27;
6#：
light chain (VL-B38) - (G)10- (VL-H4) -CL having the amino acid sequence shown in SEQ ID NO. 28;
heavy chain (VH-H4) - (G)7- (VH-B38) -S-CH1-CH2-CH3, and the amino acid sequence is shown as SEQ ID NO. 29;
12#：
the light chain sequence is (VL-H4) - (G)10- (VL-B38) -CL having the amino acid sequence shown in SEQ ID NO. 30;
the heavy chain sequence is (VH-B38) - (G)7- (VH-H4) -S-CH1-CH2-CH3, and the amino acid sequence is shown as SEQ ID NO. 31.
(I) antibody construction
These 3 antibodies were prepared according to the construction of BS-mAb-1 in example 1, purified and checked for purity as described in example 2.
The results of fig. 7 show that purified antibodies # 1, # 6 and # 12 were obtained, the disulfide bonds of the Fc region of the antibodies were opened in SDS-PAGE under reducing conditions, thus showing two bands, and the antibody purity was more than 95%.
(II) evaluation of 2019-nCoV Living Virus neutralization Capacity
The purified antibodies 1#, 6# and 12# were diluted from 100. mu.g/mL to 10 th gradient and then mixed with half of the Tissue Culture Infectious Dose (TCID)50) BetacoV/Shenzhen/SZTH-003/2020 virus (GISAID number: EPI _ ISL _406594) was incubated at 37 ℃ for 2 hours with mixing. After incubation, the virus was added to 96-well plates pre-seeded with Vero cells and incubated at 37 ℃ in 5% CO2After 4 days in the incubator, cytopathic effect (CPE) was observed and the neutralizing titer of antibodies # 1, # 6 and # 12 was calculated. The results are shown in fig. 8, which shows the neutralizing activity of 1#, 6# and 12# antibodies against live 2019-nCoV virus at different concentrations. The results showed that the neutralizing titer (half inhibitory concentration, IC50) of the 1# antibody against the live 2019-nCoV virus was 5004. mu.g/mL, the neutralizing titer (half inhibitory concentration, IC50) of the 6# antibody against the live 2019-nCoV virus was 54304. mu.g/mL, and the neutralizing titer (half inhibitory concentration, IC50) of the 12# antibody against the live 2019-nCoV virus was 102.6. mu.g/mL.
Example 8: experiment of in vitro neutralizing activity of bispecific antibody against new coronavirus wild strain/variant pseudovirus
This experiment confirmed the neutralizing activity of the BS-mAb-2 antibody constructed in example 2 against 40 new coronavirus pseudovirion strains.
1) Material
Huh7 cells (JCRB, Cat #0403), Hospital SARS-Cov-2 pseudovirus strain 31, DMEM high-sugar medium, firefly luciferase assay reagent, sterile PBS (pH7.2), fetal bovine serum, 1% (m/v) double antibody (penicillin/streptomycin antibiotic), 0.25% (m/v) pancreatin-EDTA.
The details of the novel coronavirus pseudovirus constructed by 31 strains of Chinese food and drug research institute are shown in Table 2 below.
Table 2: 31 pseudoviruses
Figure BDA0003181840800000111
Figure BDA0003181840800000121
Figure BDA0003181840800000131
2) A dilution method comprises the following steps: diluting the stock solution (30.7g/L) by 30 times; then, as shown in Table 3 below, 96 well plates were prepared, and 150. mu.l/well of DMEM complete medium (1% double antibody, 25mM HEPES, 10% FBS) was added to column 2 (CC: cell control, cell and medium only, see Table 3), 100. mu.l/well of DMEM complete medium (pseudo-virus controls VC1, VC2, VC3 in the order of column 3, column 6, and column 9, and so on for the next 96 well plate; VC: pseudo-virus control, pseudo-virus, cell and medium only) was added to C4-G5, C7-G8, and C10-G11 wells, and 100. mu.l/well of DMEM complete medium was added to B4-B5, B7-B8, and B10-B11 wells, and 142.5. mu.l/well of DMEM complete medium was added to B10-B11 wells.
Sample adding: adding the antibodies to be detected into the holes B4-B5, B7-B8 and B10-B11: 7.5. mu.l/well (30-fold initial dilution of working solution) … … and so on.
3) Sample dilution: and adjusting a multi-channel pipette to 50 mu l, softly and repeatedly blowing and sucking the liquid in the wells B4-B5, B7-B8 and B10-B11 for 6-8 times, fully and uniformly mixing, then transferring 50 mu l of liquid to the corresponding wells C4-C5, C7-C8 and C10-C11, softly and repeatedly blowing and sucking for 6-8 times, transferring to the wells D4-D5, D7-D8 and D10-D11, and so on, and finally sucking and discarding 50 mu l of liquid from the wells G4-G5, G7-G8 and G10-G11, wherein the loading position of the 96-well cell culture plate refers to Table 3. And after the sample is added, the sample is put back.
4) Each pseudovirus strain was diluted to 1.3X 10 in DMEM complete medium4(1×104～2×104)TCID50Perml (diluted according to the provided dilution factor), adding pseudovirus 1 into the 3 rd to 5 th rows, adding pseudovirus 2 into the 6 th to 8 th rows, and adding into the 9 th to 11 th rowsPseudovirus 3, and so on, 50. mu.l was added to each well, so that the amount of pseudovirus was 650(500- & 1000)/well. After completion of the virus addition, the above 96-well plate was placed in a cell incubator (37 ℃, 5% CO)2) Incubate for 1 hour.
Table 3: 96-well plate sample adding position table
Figure BDA0003181840800000132
When the incubation time is half an hour, taking out the cells prepared in advance in the incubator (the confluence rate reaches 80% -90%), sucking and removing the culture medium in the bottle, adding 5ml of PBS buffer solution to clean the cells, pouring off the PBS, adding 3ml of 0.25% pancreatin-EDTA to immerse the cells for digestion for 1 minute, pouring off the pancreatin, placing the cells in the cell incubator for digestion for 5 minutes, slightly beating the side wall of the culture bottle to enable the cells to fall off, adding 10ml of culture medium to neutralize the pancreatin, after blowing and beating for several times, counting the cells, diluting the cells to 2 multiplied by 10 by using DMEM complete culture medium5One per ml. Incubate for 1 hour, add 100. mu.l cells per well in 96-well plate to make cells 2X 10 per well4And (4) respectively. Gently shaking the 96-well plate to disperse the cells uniformly in the wells, placing the 96-well plate in a cell culture box at 37 deg.C and 5% CO2Culturing for 20-28 hours. The 96-well plate was removed from the cell incubator, 150. mu.l of the supernatant was aspirated from each loading well using a multi-channel pipette, and then 100. mu.l of the luciferase assay reagent was added and reacted at room temperature for 2min in the dark. After the reaction is finished, repeatedly blowing and sucking the liquid in the reaction holes for 6-8 times by using a multi-channel pipettor to fully lyse the cells, sucking 150 mu l of liquid from each hole, adding the liquid into a corresponding 96-hole chemiluminescence detection plate, and placing the plate in a multifunctional microplate detector to read the luminescence value.
5) Calculating the neutralization inhibition rate: the inhibition rate is [ 1- (mean value of luminescence intensity of sample group-CC mean value of blank control)/(mean value of luminescence intensity of negative group-CC mean value of blank control value)]X 100%. According to the result of the neutralization inhibition rate, the IC is calculated by adopting a Reed-Muench method50The value is obtained. Wherein, the negative group comprises normal human serum without anti-new coronavirus antibody, pseudovirus, cells and culture medium.
Repeat the test: detection of IC of antibody and pseudovirus variants determined twice and three more times per variant by the above method50Mean is the final IC of the antibody against a variant50The value is obtained.
6) Results
The antibody concentration was diluted to 100. mu.g/mL for detection, and the results are shown in Table 4.
Table 4: results of detection of neutralizing Activity of antibodies of the present invention against different Virus strains
Figure BDA0003181840800000141
Figure BDA0003181840800000151
Therefore, the antibody can neutralize and inhibit various variant pseudovirus strains in vitro to different degrees, and the antibody has the advantages of effective effect on the new coronavirus and various variant strains thereof and wide application range. Among them, the b.1.617-1 strain, which is first found in india, is a delta strain, which is one of the most infectious respiratory viruses known at present and rapidly spread all over the world, and the neutralizing activity of the antibody of the present invention is extremely high.
Example 9: toxicity test of bispecific antibody
(one) repeated dosing experiments
This experiment evaluated the potential toxic response, toxic target organs, pharmacokinetic properties and immunogenicity of the BS-mAb-2 antibody administered to cynomolgus monkeys by repeated intravenous infusion 2 times per week for 4 weeks after continuous administration, and the recovery of toxic response or potential delayed toxic response 4 weeks after the last administration.
Experimental animals and groups: 40 cynomolgus monkeys (20/sex) (origin: Guangxi Xiongsen primate culture and development Limited; laboratory animal production license number: SCXK (Gui) 2016-. 2 doses per week for 4 weeks for a total of 9 doses (D1, D5, D8, D12, D15, D19, D22, D26, and D29). The injection pump is used for subcutaneous intravenous infusion administration of hind limbs of the cynomolgus monkey, the administration volume is 10mL/kg, and the administration speed is about 0.5 mL/kg/min. The day of first administration for males and females was defined as test D1 for animals of that sex, respectively.
Detection indexes are as follows: during the experiment, animals were subjected to clinical observations and their body weight, food intake, body temperature, electrocardiogram (vest telemetry), respiratory function (vest telemetry), electrocardiogram (limb lead II ECG), blood pressure, ophthalmic examination, blood cell count, coagulation function, blood biochemistry and urine analysis were monitored periodically. Blood concentration measurements and pharmacokinetic analyses were performed before and after D1 and D26 dosing, and before D15, D19 and D22 dosing. To evaluate the immune response, a subpopulation of lymphocytes (CD 3) was periodically tested+、CD3+CD4+、CD3+CD8+、CD20+、CD3+CD4+/CD3+CD8+) Cytokines (TNF-alpha, IFN-gamma, IL-2, IL-4, IL-5, IL-6), immunoglobulins (IgG, IgM, IgA), complements (C3, C4) and BS-mAb-2 antibodies. After the dosing period, the first 3/sex/group animals in groups 1-4 were euthanized as scheduled after 4 weeks of dosing (D30), and the remaining 2/sex/group animals in groups 1-4 were euthanized as scheduled at the end of the 4-week recovery period (D57). The animals in groups 1 to 4 were systematically dissected and examined for organ weight, gross anatomy, and histopathology.
As a result:
1) during the experiment, all animals in groups 1-4 were not shown to be dead or dying. No abnormal changes associated with the test article were observed in the clinical observations of the 25, 50 and 150mg/kg dose groups of animals.
2) During the experiment, the animals in the test article 25, 50 and 150mg/kg dose groups showed no changes in weight and weight gain, food intake, body temperature, electrocardiogram (vest telemetry), respiratory function (vest telemetry), electrocardiogram (limb lead II ECG), blood pressure, eye examination, blood cell count, blood coagulation function, blood biochemistry, urine analysis, lymphocyte subpopulation, cytokines, immunoglobulins and complement related to the test article or having toxicological significance.
3) After repeated intravenous infusion of the BS-mAb-2 antibody at 25, 50, and 150mg/kg doses to cynomolgus monkeys, anti-drug antibodies (ADA) were produced at 29 days after administration to 1 animal in each of the 50 and 150mg/kg dose groups, the ADA titers were 1:160 and 1:1280, respectively, the total positive rate of the test sample group was 1.4%, and the total positive rate of the individual was 6.7%. The time, incidence rate and titer of the antibody generation have no obvious sex difference and dose correlation; this indicates that cynomolgus monkeys are less immunogenic for the BS-mAb-2 antibody.
4) The results of animal pharmacokinetic parameters for each test group after repeated intravenous infusion of the test samples to cynomolgus monkeys are shown in table 5:
table 5: animal pharmacokinetic parameters results for antibody BS-mAb-2:
Figure BDA0003181840800000161
Figure BDA0003181840800000171
remarking: n is the number of animals; accumulation factor AF ═ AUClast,8th/AUClast,1st
After repeated intravenous infusion of the test article to cynomolgus monkeys, only C of low dose group D26maxAnd AUClastThe occurrence of male-female differences (p)<0.05, but with a slight difference, the male/female ratio being 1.24 and 1.37, respectively), and the pharmacokinetic parameters of the remaining male and female animals were not statistically different (p)>0.05) indicating that the parameters of different sexes of each group of animals are basically consistent. After the 8 th (D26) administration, the test sample groups showed accumulation factor (AF ═ AUC) in male and female animalslast,8th/AUClast,1st) Between 1.69 and 2.28, no significant drug accumulation was seen.
5) The test sample BS-mAb-2 antibody is repeatedly infused intravenously into cynomolgus monkeys in a dosage range of 25-150 mg/kg, the time variation trend of serum drug concentration among animals with different sexes is basically consistent, and the peak concentration of the serum drug and the exposure amount of the serum drug are positively correlated with the administration dosage.
6) The animals of the test article 25, 50 and 150mg/kg dose groups were euthanized at the end of the dosing (D30) and at the end of the observation period (D57), and no pathological changes associated with the test article were observed in the organ weights, gross anatomical examination and histopathological examination.
7) No abnormal changes associated with the test article were observed in the visual inspection, gross anatomy examination and histopathology examination of the administered part (i.e., injection part) of the test article 25, 50 and 150mg/kg dose groups.
And (4) conclusion: in summary, in the present experimental conditions, BS-mAb-2 antibody was administered to cynomolgus monkeys by repeated intravenous infusion at doses of 25, 50 and 150mg/kg 2 times per week for 4 weeks (9 doses total) with a recovery period of 4 weeks. No systemic toxicity reaction and toxic target organs are observed in animals of each dose group. The dose level at which no clinical adverse effects were observed in this experiment (NOAEL) was 150 mg/kg. C of post-drug Male at 8 th time (D26) of this dosemaxAnd AUClast4460ng/mL and 234 h-mg/mL, respectively, female animal CmaxAnd AUClast4480ng/mL and 218 h. mg/mL, respectively.
(II) Single dose experiment
This experiment evaluated the possible toxic reactions, toxic target organs and in vivo metabolic characteristics of cynomolgus monkeys after a single intravenous infusion of the BS-mAb-2 antibody, and the observed toxic reactions 14 days after the end of the dosing period.
6 cynomolgus monkeys, randomized into 3 groups (1/sex/group), group 1 was a negative control (sodium chloride injection, 0mg/kg), and groups 2-3 were given 50 and 300mg/kg of the BS-mAb-2 antibody, respectively. Animals were dosed on day 1 (D1) with a single dose of 12mL/kg by subcutaneous intravenous infusion using a syringe pump at a rate of about 0.5 mL/kg/min. During the experiment, the animals were observed continuously for about 4 hours after administration, and examined periodically for clinical observation, body weight, body temperature, electrocardiogram, blood cell count, blood coagulation function, blood biochemistry, T lymphocyte subpopulation, cytokine and pharmacokinetic index.
During the experiment, no death or dying phenomenon is seen in each group of animals. No clinical abnormal phenomena related to the test sample are found in the 50mg/kg dose group and the 300mg/kg dose group of the BS-mAb-2 antibody, and no abnormal changes related to the test sample are found in the weight, the weight gain, the body temperature, the electrocardiogram index, the blood cell count, the blood coagulation function, the blood biochemistry and the T lymphocyte subpopulation. At the end of the observation period (D15), the gross observations of the BS-mAb-2 antibody 50 and 300mg/kg dose groups were not abnormal and were not histopathologically examined. The results of toxin tests show that the change trends of the serum drug concentration among different sexes of animals in each group are basically consistent, and the serum drug concentration is positively correlated with the administration dose.
In conclusion, under the present experimental conditions, no clear toxic response associated with the BS-mAb-2 antibody was seen after a single intravenous infusion of the BS-mAb-2 antibody at the doses of 50 and 300mg/kg, with a level of no visible adverse effect (NOEL) of 300 mg/kg.
Example 10: antibody therapeutic experiments
In this example, the therapeutic effect of the BS-mAb-2 antibody in rhesus monkeys was evaluated by infecting a rhesus monkey model with SARS-CoV-2 virus.
1) Experimental materials:
(1) SARS-CoV-2 strain: the virus is separated from BSL-3 laboratory (the laboratory passes through the national CNAS approval and can be used for SARS-CoV-2 research after the national health committee approval) at the medical biology research institute of the Chinese academy of medicine of Yunnan province, and is subjected to Vero cell adaptation to complete the identification, sequencing and preservation of the virus seeds (SARS-CoV-2-KMS1/2020/GenBank access number: MT 226610.1).
(2) Cell: african green monkey kidney cell line (Vero) purchased from ATCC (Manassas, VA, USA) and cultured in MEM containing 100U/mL penicillin, 100. mu.g/mL streptomycin, 10% newborn bovine serum based on 5% CO2And culturing at 37 ℃.
(3) Experimental animals: rhesus monkeys, 1.5-1.7 years old, 1.5-2.5kg body weight, 16 male, were provided by the institute of medicinal and biological sciences, the institute of medical and biological, china medical sciences, central laboratory animals (laboratory animal production permit number: SCXK (yunnan) K2020-0005). All the experimental animals were used according to the regulations of the Committee for the administration of laboratory animals in Yunnan province and the institute of laboratory animals, and approved by the institutional animal ethics Committee of medical and biological research of Chinese academy of sciences (approval No. DWSP 202104010). All animal experiments were performed under biosafety third-class laboratory (ABSL-3) conditions certified by the China National acceptance Service for compliance Association (CNAS), and approved by the National health and wellness Commission for the laboratory work on SARS-CoV-2 virus.
2) Virus titer detection
Half Cell culture infectious dose method (50% Cell culture infections dos, CCID 50): vero cells were plated at 1X 10 the day before assay4～5×104The concentration of each cell/well was added to a 96-well cell culture plate in 100. mu.L total in MEM containing 10% newborn bovine serum, and the stock culture was poured out the next day and washed once with 200. mu.L PBS to remove the remaining serum and dead cells. Before the experiment, 100. mu.L of MEM complete medium containing 3% newborn calf serum was added to each well, and cells were cultured at 37 ℃ for further use. Simultaneously, the virus solution to be tested is taken out from minus 80 ℃, melted at normal temperature or 37 ℃, mixed evenly in a vortex manner, and diluted by 10 times (10 times) by using serum-free MEM culture solution-1、10-2、10-3、…、10-8). When diluting virus, the pipette tip for sucking virus liquid is suspended to squeeze virus liquid into the diluent (without extending the tip into the diluent), and after fully and uniformly mixing the virus liquid and the diluent in a vortex manner, the pipette tip is continuously diluted for one step, and the pipette tip is replaced once every time the virus liquid is diluted. And sequentially adding the diluted virus solution into Vero cells of a 96-well plate, wherein each dilution is 8 parallel wells, and a virus stock solution positive control well and an MEM culture solution negative control well are arranged. Then the cells were incubated at 37 ℃ with 5% CO2Cultured in the environment, and cytopathic effect (CPE) is observed up to day 6-7. The results were calculated by the Karber method, i.e., lgCCID50 ═ lg lowest dilution factor) - (dilutedGroup interval) × (sum of positive lesion aperture ratio-0.5) CCID50 ═ 10lgCCID50/0.1 mL.
3) Fluorescent PCR identification of SARS-CoV-2
Primers, probes and reagents: all primers were synthesized by Huada Gene Co, dissolved in double distilled water to a working solution concentration of 10. mu. mol/. mu.l, and stored at-20 ℃ for further use.
Table 6: SARS-CoV-2 fluorescent PCR identification primer sequence
Figure BDA0003181840800000191
4) Design of experiments
After the experimental monkeys are subjected to conventional quarantine, SARS-CoV-2 antibody detection negative is selected in the experiment. A control group, a low dose group, a high dose group and a preventive administration group were set, and 4 were included in each group.
The experimental scheme is as follows:
(1) counteracting toxic substances: the toxic dose of each experimental group is 5.0log CCID 50/nasal drop infection, 100 mu L;
(2) grouping:
prevention group: injecting antibody in an amount of 50mg/kg body weight by intravenous injection 12 hours before challenge;
low dose group: injecting the antibody for the first time 12 hours after the toxin is attacked, wherein the injection is intravenous injection with the weight of 25 mg/kg; a second injection of antibody was made 72 hours after the first injection, at the same dose as the first.
High dose group: injecting the antibody for the first time 12 hours after the toxin is attacked, wherein the injection is intravenous injection with the weight of 50 mg/kg; a second injection of antibody was made 72 hours after the first injection, at the same dose as the first.
Blank control group: no treatment is done.
(3) Observing the clinical performance of animals, measuring the body temperature and the body weight, collecting nasal swab samples, pharyngeal swab samples and anal swab samples every day for virus qPCR detection, and carrying out virus qPCR, subgenomic RNA and CCID50 detection on the nasal swab samples, the pharyngeal swab samples and the anal swab samples on days 1, 3, 5 and 7; on day 7, the experimental animals were sacrificed after anesthesia, and the main tissues were collected for pathology and viral load detection and analysis.
Detecting the content:
(1) post-infection detoxification and in-tissue viral load analysis
And (3) performing q-PCR after extracting RNA from the sample from throat swab, nose swab, anus swab and tissue, and calculating the viral load of the sample by using a standard curve and a Ct value.
(2) Histopathological examination
Animal tissue samples were fixed using 4% formaldehyde solution and embedded in paraffin.
(3) Statistical analysis
Data statistics were expressed as mean, or mean ± SD (neutralizing antibody as GMT or GMT ± SD) using Graphad prism8.0 software.
5) The experimental results are as follows:
5.1) clinical presentation:
(1) all experimental monkeys have no obvious changes in spirit and diet within 1-7 days; no animal death occurred; in the control group, 1 experimental monkey showed mild diarrhea from day 2, and the diet did not change significantly.
(2) Body temperature: except for the comparative obvious reduction trend of the body temperature of the experimental monkeys of the control group and the prevention group, the body temperature of all experimental monkeys has no obvious body temperature rise or reduction condition within 1-7 days, and the body temperature of all experimental monkeys is within the normal reference range value.
(3) Weight: no significant increase or decrease in body weight was observed in all experimental monkeys from day 1 to day 7.
5.2) Virus detection
Challenge test used 5.0log CCID50 virus dose per mouse, 100. mu.l nasal infection. Collecting throat, nose and anus swabs on days 1-7, and detecting the copy number of the virus. Viral load results for pharyngeal, nasal and anal swabs are shown below. (1) Throat swab results
The pharynx of the control group showed toxin expulsion from day 3, and obvious toxin expulsion occurred from 4-5 days, wherein the highest toxin expulsion occurred at day 4 and reaches 4.98log (copy/100 μ L), and no toxin expulsion occurred from day 6; the prevention group and the treatment group with the BS-mAb-2 antibody showed that the mean values of the test monkeys at 1-7 days were below the detection threshold, except that the individual monkeys detected viral load on the specified days, as shown in fig. 9.
(2) Nasal swab results
The virus detection results of the nasal swabs show that the control group has toxin expelling from day 1 to day 7, and obvious toxin expelling peaks appear in 4-5 days, wherein the highest toxin expelling peak appears in 5.52log (copy/100 muL) in day 4, and the toxin expelling peaks appear in 4.32log (copy/100 muL), 5.07log (copy/100 muL) and 4.89log (copy/100 muL) in day 1 in the prevention group, the BS-mAb-2 antibody low dose group and the BS-mAb-2 antibody high dose group, respectively, wherein the virus load is reduced by more than 2log values in days 4-7 of the prevention group and the BS-mAb-2 antibody low dose group compared with the control group; the high dose group showed a greater than 2log reduction in viral load on days 6-7 compared to the control group, as shown in detail in figure 10.
(3) Anal swab results
No fecal detoxification was detected in all experimental monkeys on days 1-7, as shown in FIG. 11.
5.3) gross detection
(1) Gross results
The general pathological observation results show that the lung volumes of the experimental monkeys in the antibody prevention group and the high-low dose group are basically normal, the lung colors are changed in a gray-red staggered mode at different degrees, and the lung surfaces are scattered at bleeding points, so that severe pulmonary bleeding or nodule conditions do not occur.
(2) Tissue viral load results
The tissue virus load detection results show that the virus load detection values of nasal mucosa, trachea and lung tissues of all experimental monkeys in the prevention group are lower than the detection threshold value, and the virus load is not detected in neck lymph nodes and lung lymph nodes. In the low dose group and the high dose group of the BS-mAb-2 antibody, SARS-CoV-2 virus was not detected in any tissues except for a small amount of viral nucleic acid detected in individual lung, lung stranguria, cervical stranguria, trachea, and nasal mucosa tissues of monkeys.
The above experimental results show that, when rhesus monkeys are subjected to SARS-CoV-2 virus challenge after preventive and therapeutic administration of the BS-mAb-2 antibody, virus proliferation and detoxification in early and middle stages of experimental monkeys in the low dose group, the high dose group, and the preventive group can be inhibited to some extent.
According to the above experimentIt can be known that the effective dose of the rhesus monkey is 25mg/kg when the rhesus monkey is infected by 100 mul drops of 5.0log CCID50 per dog and is injected with BS-mAb-225 mg/kg after being infected by virus by single administration for 12 hours, compared with a control group, the rhesus monkey has the obvious effect of reducing the virus gene copy of nasal and pharyngeal swabs and the virus infection capacity. According to the guidance principle of estimating the maximum recommended initial dose of the first clinical trial medicine of healthy adult volunteers issued by NMPA, the dosage is not in mg/m between species2Other cases of dose scaling: the protein with molecular weight more than 100000 dalton for intravascular administration should be converted according to mg/kg, and Human Equivalent Dose (HED) is calculated to be 8.6 mg/kg. The estimated effective dose of the human body is as follows according to the weight of 60kg of an adult: 500 mg. Considering the safety, 1/2-1/3 of the dosage is selected as the dosage for the first human administration, and the effective dosage range of the human is 180-250 mg.
According to a dose exploration test given to the cynomolgus monkey by single intravenous infusion, the level of no visible harmful effect (NOEL) is 300mg/kg, the corresponding Human Equivalent Dose (HED) is 100mg/kg, and the corresponding human dose is 6000mg calculated according to the weight of 60kg of a human body; according to the results of 4 weeks' toxicology studies with repeated administration to cynomolgus monkeys, no clinical adverse effects were observed at a dose level (NOAEL) of 150mg/kg and a corresponding Human Equivalent Dose (HED) of 50mg/kg, corresponding to a human dose of 3000mg, calculated on the basis of 60kg body weight of the human. The maximum human dose currently supported by preclinical studies is 6000 mg. The single administration dosage is 180-6000 mg, such as 180, 250, 500, 900, 1000, 1800mg, 3000mg or 6000mg, or the above numerical value interval. The mode of administration is single/multiple administration.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> institute for microbiology of Chinese academy of sciences
<120> bispecific antibody for resisting novel coronavirus and application thereof
<130> DSP1F211340JW
<150> 202010763140.X
<151> 2020-08-01
<160> 35
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1743
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
caggttcagt tagttcagag tggagcagaa gtgaagaagc ccggtgcctc tgtgaaagtg 60
tcatgcaaag catcaggata cacatttaca ggatactaca tgcactgggt taggcaggcg 120
cccggtcaag gcctggagtg gatgggcaga atcaacccta actctggcgg gacgaattat 180
gcacagaagt tccagggaag agtgacaatg acaagagata catcaatctc aacagcatac 240
atggaacttt caagacttag atcagatgac actgctgtct actactgcgc acgagtcccg 300
tactgctcat caacatcatg ccacagagat tggtactttg atctttgggg gagaggtaca 360
cttgtgacag tctcatcggg tggaggcgga tcagggggtg gtggctccga agtccagctc 420
gtcgagagtg gtggaggact ggttcaacct ggaggatcac ttagactttc atgcgcagca 480
tcaggattta tcgtgtcatc aaactacatg tcatgggtac gccaggctcc cgggaagggt 540
ctggagtggg tctcggtgat ctactcagga ggatcaacat actacgcaga ttcagtgaaa 600
ggaagattta caatctcaag acacaactca aagaatactc tttaccttca aatgaactca 660
cttagagcag aagacactgc ggtttactac tgcgcaagag aagcgtacgg aatggatgtg 720
tggggccagg gtacaacagt tactgtttcg tctgcgtcca ctaaaggccc ttccgtcttc 780
ccgctggcac cttcatcaaa gtctacttct ggtggaacgg cggctcttgg atgtctggtt 840
aaggactatt tcccggagcc tgtaactgta tcgtggaact caggagcact tacatcagga 900
gtgcacacat ttcctgcagt gcttcaatca tcaggacttt actcactttc atcagtggtg 960
acagtgcctt catcatcact tggaacacaa acatacatct gcaacgtgaa ccacaaacct 1020
tcaaacacaa aggttgacaa gaaggtcgag cctaaatcat gcgataagac tcatacatgc 1080
cctccttgcc ctgcacctga acttcttgga ggcccgtcgg tgtttttatt cccgccaaaa 1140
ccgaaggata cacttatgat ctcaagaaca cctgaagtga catgcgtggt ggtggatgtg 1200
tcacacgaag atcctgaagt gaaatttaac tggtacgtgg atggagtgga agtgcacaac 1260
gcaaagacta agcctagaga agaacaatac aactcaacat acagagtggt gtcagtgctt 1320
acagtgcttc accaagattg gcttaacgga aaggagtata aatgcaaagt gtcaaacaaa 1380
gcacttcctg cacctatcga gaagactata tcaaaagcaa aaggacaacc tagagaacct 1440
caagtgtaca cacttcctcc ttcaagagat gaacttacaa agaatcaggt gtcacttaca 1500
tgtttagtaa agggcttcta cccatctgac atcgcagtgg aatgggaatc aaacggacaa 1560
cctgagaata attataagac cactcctcct gtacttgatt cagatggatc attcttcctc 1620
tattcaaagt taaccgtgga taaatcaaga tggcaacaag gaaacgtgtt ctcctgttca 1680
gtgatgcacg aagcacttca caaccactac acacagaaga gtctatcact ttcacctgga 1740
tga 1743
<210> 2
<211> 580
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr
100 105 110
Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly
130 135 140
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
145 150 155 160
Ser Gly Phe Ile Val Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala
165 170 175
Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser Gly Gly Ser
180 185 190
Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg His
195 200 205
Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
210 215 220
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Ala Tyr Gly Met Asp Val
225 230 235 240
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
245 250 255
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
260 265 270
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
275 280 285
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
290 295 300
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
305 310 315 320
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
325 330 335
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
340 345 350
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
355 360 365
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
370 375 380
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
385 390 395 400
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
405 410 415
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
420 425 430
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
435 440 445
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
450 455 460
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
465 470 475 480
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
485 490 495
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
500 505 510
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
515 520 525
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
530 535 540
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
545 550 555 560
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
565 570 575
Leu Ser Pro Gly
580
<210> 3
<211> 1017
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gatatccaaa tgacccagag cccattgagc cttcctgtga cacctggaga acctgcatca 60
atctcatgca gatcatcaca atcacttctt gattcagatg atggaaacac ataccttgat 120
tggtaccttc agaagcccgg tcagtctcct cagctactaa tttatactct atcatacaga 180
gcatcgggcg taccggaccg cttctccgga tcagggtcag ggactgattt caccctaaag 240
atatccagag tggaagcaga agatgtggga gtgtactact gcatgcaaag aatcgaattt 300
cctcttacat ttggaggagg aacaaaggta gagatcaaag gtggaggagg atccggagga 360
ggtggatccg atatcgtgat gacgcagagc cctagctttc tttctgctag tgtaggcgac 420
agagtgacaa tcacatgcag agcatcacaa ggaatctcat cataccttgc atggtaccaa 480
cagaagccgg gaaaggcgcc caagctgctc atatatgctg cctcaactct gcagagtggc 540
gtgccgtctc gcttcagcgg tagcggatcg ggaacagagt ttacacttac aatctcatca 600
cttcaacctg aagatttcgc cacgtactac tgccaacaac ttaactcata ccctccttac 660
acatttggac aaggaacaaa gttagagatc aagcgaaccg tggcagcacc ttcagtgttt 720
atctttcctc cttcagatga acaacttaaa tcaggaactg cgtctgtagt ttgtcttctt 780
aacaacttct atccaagaga agcaaaggta cagtggaaag tggataacgc cctgcagtct 840
ggtaatagcc aagaatcagt gacagaacaa gattcaaagg acagtacata ctcactttca 900
tcaacactta cactttcaaa ggcggactac gagaagcata aggtgtacgc atgcgaagtg 960
acacaccaag gactttcatc acctgtgaca aagtccttca acagaggaga atgctga 1017
<210> 4
<211> 338
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser
20 25 30
Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
35 40 45
Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
65 70 75 80
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
85 90 95
Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr
115 120 125
Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
130 135 140
Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala Trp Tyr Gln
145 150 155 160
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr
165 170 175
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
180 185 190
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr
195 200 205
Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Pro Tyr Thr Phe Gly Gln
210 215 220
Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe
225 230 235 240
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
245 250 255
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
260 265 270
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
275 280 285
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr
290 295 300
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
305 310 315 320
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly
325 330 335
Glu Cys
<210> 5
<211> 2121
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaggtccagc tcgttgagtc cggtggggga ttggtgcaac ctggaggatc acttagactt 60
tcatgcgcag catcaggatt tatcgtgtca tcaaactaca tgtcatgggt acggcaggcg 120
ccagggaaag gcctagagtg ggtttcggtg atctactcag gaggatcaac atactacgca 180
gattcagtga aaggaagatt tacaatctca agacacaact caaagaatac tctttacctt 240
caaatgaact cacttagagc agaagacact gcggtgtact actgcgcacg ggaggcgtac 300
ggaatggatg tgtgggggca gggaacaaca gtcacggtct ctagcgccag caccaaaggc 360
ccgtctgtct ttccactcgc accttcatca aagagcacgt cgggagggac ggctgcgctt 420
ggatgtttag ttaaggacta tttcccagag ccggtcacag ttagctggaa ctcaggagca 480
cttacctcgg gcgttcacac ctttcctgca gtgcttcaat catcaggact ttactcactt 540
tcatcagtgg tgacagtgcc ttcatcatca cttggaacac aaacatacat ctgcaacgtg 600
aaccacaaac cttcaaacac aaaggtagac aagcgagtag aacctaaatc atgcgataag 660
acccatacat gccctccttg ccctgcacct gaacttcttg gaggtccatc ggtattctta 720
tttccgccaa agccgaagga tacacttatg atctcaagaa cacctgaagt gacatgcgtg 780
gtggtggatg tgtcacacga agatcctgaa gtgaaattta actggtacgt ggatggagtg 840
gaagtgcaca acgcaaagac gaagcctaga gaagaacaat acaactcaac atacagagtg 900
gtgtcagtgc ttacagtgct tcaccaagat tggcttaacg gaaaggagta taaatgcaaa 960
gtgtcaaaca aagcacttcc tgcacctatc gagaagacga tatcaaaagc aaaaggacaa 1020
cctagagaac ctcaagtgta cacacttcct ccttcaagag atgaacttac aaagaatcag 1080
gtgtcactta catgtttagt caagggcttc tacccttcag atatcgcagt ggaatgggaa 1140
tcaaacggac aacctgagaa taattataag actacaccac cggtgttgga cagtgacggg 1200
agcttcttcc tgtattcaaa gctcactgtg gataaatcaa gatggcaaca aggaaacgtg 1260
ttctcgtgtt cagtgatgca cgaagcactt cacaaccact acacacagaa gtcgctgtca 1320
ctttcacctg gtggtggaag tcaagttcag ttggtacaga gcggagcaga agtgaagaag 1380
cccggtgcga gtgtgaaagt gtcatgcaaa gcatcaggat acacatttac aggatactac 1440
atgcactggg tacgacaggc tccgggtcaa ggtttagagt ggatgggcag aatcaaccct 1500
aactctggag gcaccaacta cgcacagaag ttccagggaa gagtgacaat gacaagagat 1560
acatcaatct caacagcata catggaactt tcaagactta gatcagatga cactgcggtc 1620
tattattgtg cacgggtacc ttactgctca tcaacatcat gccacagaga ttggtacttt 1680
gatctttggg gaagaggaac acttgtcact gtgtcgtcag gaggcggagg atcagggggt 1740
gggggcagcg gcggtggcgg ttctggtgga ggtggcagcg acatccaaat gacacaatca 1800
cctctttcac ttcctgtgac acctggagaa cctgcatcaa tctcatgcag atcatcacaa 1860
tcacttctgg actcggacga cggcaacaca taccttgatt ggtaccttca gaagccgggt 1920
caatcacctc aacttcttat ctacacactt tcatacagag cgtccggagt cccggacaga 1980
ttcagcggtt caggatcagg aacagatttc acgttgaaga tttctagagt ggaagcagaa 2040
gatgtgggag tgtactactg catgcaaaga atcgaatttc ctcttacatt tggaggtggc 2100
acgaaagtgg agatcaaatg a 2121
<210> 6
<211> 706
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Val Ser Ser Asn
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Glu Ala Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
115 120 125
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
130 135 140
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
165 170 175
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205
Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
210 215 220
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
225 230 235 240
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
260 265 270
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
275 280 285
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
290 295 300
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
340 345 350
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
355 360 365
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
385 390 395 400
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
405 410 415
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
420 425 430
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Ser Gln
435 440 445
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
450 455 460
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr
465 470 475 480
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
485 490 495
Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln
500 505 510
Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met
515 520 525
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala
530 535 540
Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr Phe
545 550 555 560
Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
565 570 575
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
580 585 590
Ser Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro
595 600 605
Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp
610 615 620
Ser Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
625 630 635 640
Gln Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly
645 650 655
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
660 665 670
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met
675 680 685
Gln Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu
690 695 700
Ile Lys
705
<210> 7
<211> 648
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gacatcgtga tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggccagtca gggcattagc agttatttag cctggtatca gcaaaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagag ttcactctca caatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtcaacag cttaatagtt accctccgta cacttttggc 300
caggggacca agctggagat caaacgaact gtggctgcac caagcgtgtt tatcttccct 360
cccagcgacg agcagctgaa gagcggcacc gccagcgtgg tctgtctcct gaacaacttc 420
tatcccaggg aggccaaggt ccagtggaaa gtggacaacg ccctgcaaag cggcaatagc 480
caggagtccg tcacagagca ggacagcaag gacagcacct acagcctgtc cagcaccctg 540
accctcagca aggccgacta cgagaagcac aaggtgtacg cttgcgaggt gacccatcag 600
ggcctgtcca gccccgtgac caagtccttc aacaggggcg aatgcagc 648
<210> 8
<211> 216
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Pro
85 90 95
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys Ser
210 215
<210> 9
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Gly Phe Ile Val Ser Ser Asn Tyr
1 5
<210> 10
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Ile Tyr Ser Gly Gly Ser Thr
1 5
<210> 11
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
Ala Arg Glu Ala Tyr Gly Met Asp Val
1 5
<210> 12
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
Gln Gly Ile Ser Ser Tyr
1 5
<210> 13
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
Ala Ala Ser
1
<210> 14
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
Gln Gln Leu Asn Ser Tyr Pro Pro Tyr Thr
1 5 10
<210> 15
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Gly Tyr Thr Phe Thr Gly Tyr Tyr
1 5
<210> 16
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Ile Asn Pro Asn Ser Gly Gly Thr
1 5
<210> 17
<211> 19
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr
1 5 10 15
Phe Asp Leu
<210> 18
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr
1 5 10
<210> 19
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Thr Leu Ser
1
<210> 20
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Met Gln Arg Ile Glu Phe Pro Leu Thr
1 5
<210> 21
<211> 259
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr
100 105 110
Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr
145 150 155 160
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu
165 170 175
Asp Ser Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro
180 185 190
Gly Gln Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser
195 200 205
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
210 215 220
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
225 230 235 240
Met Gln Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val
245 250 255
Glu Ile Lys
<210> 22
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
atgggatggt catgcatcat cctattccta gtagcaacag caacaggagt gcactca 57
<210> 23
<211> 66
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
atggatatga gagtgcctgc acaacttctt ggacttcttc ttctttggct tcctggagca 60
agatgc 66
<210> 24
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
atgggatggt catgcatcat cctcttccta gtagcaacag caacaggagt gcactca 57
<210> 25
<211> 63
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
atggagacgg atacgctgct cctgtgggtt ttgctgctgt gggttccagg ttccactggt 60
gac 63
<210> 26
<211> 362
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Gly Ala Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Phe
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Gly Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
Leu Asn Ser Tyr Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
115 120 125
Ile Lys Gly Gln Pro Lys Ala Ala Pro Asp Ile Gln Met Thr Gln Ser
130 135 140
Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys
145 150 155 160
Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr Leu
165 170 175
Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr
180 185 190
Thr Leu Ser Tyr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
195 200 205
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu
210 215 220
Asp Val Gly Val Tyr Tyr Cys Met Gln Arg Ile Glu Phe Pro Leu Thr
225 230 235 240
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Lys Gln Pro Ser Arg
245 250 255
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
260 265 270
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
275 280 285
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
290 295 300
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
305 310 315 320
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
325 330 335
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
340 345 350
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
355 360
<210> 27
<211> 592
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
50 55 60
Glu Trp Met Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg
115 120 125
Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser
130 135 140
Ser Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
145 150 155 160
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Val Ser
165 170 175
Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
180 185 190
Trp Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
195 200 205
Val Lys Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu
210 215 220
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
225 230 235 240
Cys Ala Arg Glu Ala Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
245 250 255
Val Thr Val Ser Ser Arg Thr Ala Ser Thr Lys Gly Pro Ser Val Phe
260 265 270
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
275 280 285
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
290 295 300
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
305 310 315 320
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
325 330 335
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
340 345 350
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
355 360 365
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
370 375 380
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
385 390 395 400
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
405 410 415
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
420 425 430
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
435 440 445
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
450 455 460
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
465 470 475 480
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
485 490 495
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
500 505 510
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
515 520 525
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
530 535 540
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
545 550 555 560
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
565 570 575
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
580 585 590
<210> 28
<211> 360
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Gly Ala Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Phe
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Gly Ile Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
Leu Asn Ser Tyr Pro Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
115 120 125
Ile Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asp Ile Gln Met
130 135 140
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser
145 150 155 160
Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn
165 170 175
Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu
180 185 190
Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val Pro Asp Arg Phe
195 200 205
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val
210 215 220
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Arg Ile Glu Phe
225 230 235 240
Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val
245 250 255
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
260 265 270
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
275 280 285
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
290 295 300
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
305 310 315 320
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
325 330 335
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
340 345 350
Lys Ser Phe Asn Arg Gly Glu Cys
355 360
<210> 29
<211> 598
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
20 25 30
Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Gly Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
50 55 60
Glu Trp Met Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala
65 70 75 80
Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser
85 90 95
Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg
115 120 125
Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser
130 135 140
Ser Gly Gly Gly Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly
145 150 155 160
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
165 170 175
Ser Gly Phe Ile Val Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala
180 185 190
Pro Gly Lys Gly Leu Glu Trp Val Ser Val Ile Tyr Ser Gly Gly Ser
195 200 205
Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg His
210 215 220
Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
225 230 235 240
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Ala Tyr Gly Met Asp Val
245 250 255
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ser Leu Ala Ser Thr
260 265 270
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
275 280 285
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
290 295 300
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
305 310 315 320
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
325 330 335
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
340 345 350
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
355 360 365
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
370 375 380
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
385 390 395 400
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
405 410 415
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
420 425 430
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
435 440 445
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
450 455 460
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
465 470 475 480
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
485 490 495
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
500 505 510
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
515 520 525
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
530 535 540
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
545 550 555 560
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
565 570 575
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
580 585 590
Leu Ser Leu Ser Pro Gly
595
<210> 30
<211> 360
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Leu Ser
20 25 30
Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
35 40 45
Gln Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr
50 55 60
Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser
65 70 75 80
Tyr Arg Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
85 90 95
Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly
100 105 110
Val Tyr Tyr Cys Met Gln Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly
115 120 125
Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly
130 135 140
Gly Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val
145 150 155 160
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser
165 170 175
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
180 185 190
Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
195 200 205
Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
210 215 220
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro
225 230 235 240
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
245 250 255
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
260 265 270
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
275 280 285
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
290 295 300
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
305 310 315 320
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
325 330 335
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
340 345 350
Lys Ser Phe Asn Arg Gly Glu Cys
355 360
<210> 31
<211> 596
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Val
35 40 45
Ser Ser Asn Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Val Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
65 70 75 80
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr
85 90 95
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
100 105 110
Tyr Cys Ala Arg Glu Ala Tyr Gly Met Asp Val Trp Gly Gln Gly Thr
115 120 125
Thr Val Thr Val Ser Ser Gly Gly Gly Gly Gly Gly Gly Gln Val Gln
130 135 140
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys
145 150 155 160
Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr Met His
165 170 175
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile
180 185 190
Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln Gly Arg
195 200 205
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu Leu
210 215 220
Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val
225 230 235 240
Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr Phe Asp Leu
245 250 255
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
260 265 270
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
275 280 285
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
290 295 300
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
305 310 315 320
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
325 330 335
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
340 345 350
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
355 360 365
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
370 375 380
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
385 390 395 400
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
405 410 415
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
420 425 430
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
435 440 445
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
450 455 460
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
465 470 475 480
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
485 490 495
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
500 505 510
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
515 520 525
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
530 535 540
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
545 550 555 560
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
565 570 575
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
580 585 590
Leu Ser Pro Gly
595
<210> 32
<211> 115
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Val Ser Ser Asn
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Ile Tyr Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Glu Ala Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr
100 105 110
Val Ser Ser
115
<210> 33
<211> 108
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Pro
85 90 95
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 34
<211> 126
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Pro Tyr Cys Ser Ser Thr Ser Cys His Arg Asp Trp Tyr
100 105 110
Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 35
<211> 113
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser
20 25 30
Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln
35 40 45
Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
65 70 75 80
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln
85 90 95
Arg Ile Glu Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
100 105 110
Lys

Claims (19)

1. A bispecific antibody against a novel coronavirus, which comprises the heavy chain variable region of mAb B38, the light chain variable region of mAb B38, the heavy chain variable region of mAb H4, and the light chain variable region of mAb H4, wherein,
the heavy chain variable region of mab B38 comprises: CDR1 with an amino acid sequence shown as SEQ ID NO.9, CDR2 with an amino acid sequence shown as SEQ ID NO.10 and CDR3 with an amino acid sequence shown as SEQ ID NO. 11; the light chain variable region of mab B38 comprises: CDR1 with an amino acid sequence shown in SEQ ID NO.12, CDR2 with an amino acid sequence shown in SEQ ID NO.13 and CDR3 with an amino acid sequence shown in SEQ ID NO. 14;
the heavy chain variable region of mab H4 comprises: CDR1 with an amino acid sequence shown as SEQ ID NO.15, CDR2 with an amino acid sequence shown as SEQ ID NO.16, and CDR3 with an amino acid sequence shown as SEQ ID NO. 17; the light chain variable region of mab H4 comprises: CDR1 with an amino acid sequence shown in SEQ ID NO.18, CDR2 with an amino acid sequence shown in SEQ ID NO.19 and CDR3 with an amino acid sequence shown in SEQ ID NO. 20;
wherein the bispecific antibody is BS-mAb-1 having the following structure from N-terminus to C-terminus:
heavy chain: VHH4-VHB38-CHB38，
Light chain: VLH4-VLB38-CLB38，
Wherein VHH4And VHB38By a linker peptide, VLH4And VLB38Linked by a linker peptide; or,
the bispecific antibody is BS-mAb-2, which has the following structure from N-terminus to C-terminus:
heavy chain: VHB38-CHB38-VHH4-VLH4，
Light chain: VLB38-CLB38；
Wherein VHH4And VLH4Connecting through connecting peptide to form single-chain antibody; CH (CH)B38And VHH4Linked by a linker peptide;
the amino acid sequence of the connecting peptide is (GGGGS) n, wherein n is a natural number of 1-4.
2. The bispecific antibody of claim 1, wherein in antibody BS-mAb-1, n of the linking peptide is 2; in antibody BS-mAb-2, the VHH4And VLH4N of the connecting peptide between is 4, CHB38And VHH4N of the connecting peptide therebetween is 1.
3. The bispecific antibody of claim 2, wherein the amino acid sequence of the heavy chain variable region of mab B38 is set forth in SEQ ID No.32, and the amino acid sequence of the light chain variable region of mab B38 is set forth in SEQ ID No. 33; the amino acid sequence of the heavy chain variable region of the monoclonal antibody H4 is shown as SEQ ID NO.34, and the amino acid sequence of the light chain variable region of the monoclonal antibody H4 is shown as SEQ ID NO. 35.
4. The bispecific antibody of claim 3, wherein the bispecific antibody BS-mAb-1 has the amino acid sequence of its light chain as shown in SEQ ID No.4 and the amino acid sequence of its heavy chain as shown in SEQ ID No. 2; or,
the amino acid sequence of the light chain of the bispecific antibody BS-mAb-2 is shown in SEQ ID NO.8, and the amino acid sequence of the heavy chain of the bispecific antibody BS-mAb-2 is shown in SEQ ID NO. 6.
5. The bispecific antibody of any one of claims 1 to 4, wherein said bispecific antibody is a murine antibody, a humanized antibody, a chimeric antibody or a recombinant antibody.
6. The bispecific antibody of any one of claims 1-4, wherein the antibody is one of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody.
7. A gene encoding the bispecific antibody of any one of claims 1 to 6.
8. The gene of claim 7, wherein the nucleotide sequence of the light chain of the bispecific antibody is shown as SEQ ID No.3, and the nucleotide sequence of the heavy chain is shown as SEQ ID No. 1; or
The nucleotide sequence of the bispecific antibody light chain is shown as SEQ ID NO.7, and the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 5.
9. A biological material comprising the gene of claim 7 or 8, which is a recombinant DNA, an expression cassette, a vector, a host cell, an engineered bacterium or a cell line.
10. A process for the preparation of a bispecific antibody according to any one of claims 1 to 6, comprising: respectively constructing recombinant expression vectors containing heavy chain and light chain genes of the bispecific antibody BS-mAb-1 or BS-mAb-2; introducing the recombinant expression vector into a host cell to obtain a host cell for stably expressing the bispecific antibody; culturing host cells, and separating and purifying to obtain the bispecific antibody.
11. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 6.
12. A kit comprising the bispecific antibody according to any one of claims 1 to 6.
13. Use of the bispecific antibody of any one of claims 1 to 6 or the gene of claim 7 or 8 or the biological material of claim 9 or the pharmaceutical composition of claim 11 or the kit of claim 12 for any one of the following:
(1) the application in preparing the medicine for preventing or treating the diseases caused by the novel coronavirus infection;
(2) the application in preparing a novel coronavirus diagnostic reagent or a diagnostic kit.
14. A pharmaceutical composition in single dose form, wherein the single dose form contains 180mg-6000mg of the bispecific antibody of any one of claims 1 to 6.
15. The pharmaceutical composition of claim 14, wherein the single dose form contains 180mg to 3000mg of the bispecific antibody of any one of claims 1 to 6.
16. The pharmaceutical composition of claim 15, wherein the single dose form contains 500mg to 1800mg of the bispecific antibody of any one of claims 1 to 6.
17. The pharmaceutical composition of claim 16, wherein the single dose form contains 900mg to 1800mg of the bispecific antibody of any one of claims 1 to 6.
18. The pharmaceutical composition of claim 14, wherein the single dose form contains 500mg to 1000mg of the bispecific antibody of any one of claims 1 to 6.
19. The pharmaceutical composition of any one of claims 14 to 18, wherein the pharmaceutical composition is formulated in a form suitable for intravenous administration.
CN202110849422.6A 2020-07-31 2021-07-27 Bispecific antibody for resisting novel coronavirus and application thereof Active CN113563475B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010763140X 2020-07-31
CN202010763140 2020-07-31

Publications (2)

Publication Number Publication Date
CN113563475A CN113563475A (en) 2021-10-29
CN113563475B true CN113563475B (en) 2022-07-22

Family

ID=78167869

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110849422.6A Active CN113563475B (en) 2020-07-31 2021-07-27 Bispecific antibody for resisting novel coronavirus and application thereof

Country Status (3)

Country Link
CN (1) CN113563475B (en)
TW (1) TW202208424A (en)
WO (1) WO2022022666A1 (en)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114149509B (en) * 2021-12-23 2023-04-28 中国人民解放军军事科学院军事医学研究院 Bispecific neutralizing antibody for resisting coronavirus and application thereof
CN113980135B (en) * 2021-12-27 2022-04-19 三优生物医药(上海)有限公司 Anti-drug antibody combined with coronavirus bispecific antibody, preparation method and application thereof
WO2023131262A1 (en) * 2022-01-10 2023-07-13 杰库(上海)生物医药研究有限公司 Antigen-binding protein specifically bound to sars-cov-2
CN116617388A (en) * 2022-01-26 2023-08-22 百奥泰生物制药股份有限公司 Use of antibodies targeting coronaviruses for preventing, treating or ameliorating covd-19
CN114605555B (en) * 2022-03-28 2023-09-05 中国医学科学院病原生物学研究所 Bispecific neutralizing antibody for resisting novel coronavirus SARS-CoV-2 and application thereof
CN115057939B (en) * 2022-06-28 2023-01-03 广东菲鹏制药股份有限公司 Bispecific binding proteins, medicaments and methods for binding novel coronaviruses

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017186950A1 (en) * 2016-04-28 2017-11-02 Biomunex Pharmaceuticals Bispecific antibodies targeting egfr and her2
CN111196855B (en) * 2018-11-19 2022-11-15 三生国健药业（上海）股份有限公司 anti-EGFR/PD-1 bispecific antibodies
CN111378048A (en) * 2018-12-28 2020-07-07 复旦大学 Antibody-polypeptide bispecific immunotherapeutics against middle east respiratory syndrome coronavirus
CN111333722A (en) * 2020-03-03 2020-06-26 江苏省疾病预防控制中心(江苏省公共卫生研究院) SARS-CoV-2 inhibitor and its application
CN111303280B (en) * 2020-03-22 2022-01-07 中国人民解放军军事科学院军事医学研究院 High-neutralization-activity anti-SARS-CoV-2 fully human monoclonal antibody and application
CN111423508A (en) * 2020-03-31 2020-07-17 江苏省疾病预防控制中心(江苏省公共卫生研究院) Separated SARS-CoV-2 protein binding molecule for resisting virus infection

Also Published As

Publication number Publication date
WO2022022666A1 (en) 2022-02-03
TW202208424A (en) 2022-03-01
CN113563475A (en) 2021-10-29

Similar Documents

Publication Publication Date Title
CN113563475B (en) Bispecific antibody for resisting novel coronavirus and application thereof
CN106519034A (en) Anti-PD-1 (Programmed Death-1) antibody and application thereof
US10030069B2 (en) High affinity human antibodies to human cytomegalovirus (CMV) gB protein
CN113444169B (en) Human monoclonal antibodies to novel coronaviruses and uses thereof
TW200927760A (en) Monoclonal antibodies that bind to hGM-CSF and medical compositions comprising same
CN112574300B (en) anti-SAR-COV-2 fully human monoclonal antibody and preparation method and application thereof
WO2022143816A1 (en) Full-human broad-spectrum cross-neutralizing antibody for sars-cov-2 and sars-cov and application thereof
CN108690134A (en) For treating hepatitis B infected and relevant disease antibody
WO2023046097A1 (en) Anti-tigit humanized antibody or antigen-binding fragment thereof and application thereof
TW202241955A (en) Garp protein antibody and application thereof
CN115087667A (en) Antigen binding proteins that specifically bind to SARS-CoV-2
CN113912706A (en) Antibody binding to hepatitis B virus surface antigen and application thereof
CN111450248A (en) Antibody medicine for preventing and treating PRRSV infection
CN115073593B (en) Novel coronavirus antibodies and uses thereof
WO2022148374A1 (en) Fully human broad-spectrum neutralizing antibody 76e1 against coronavirus, and use thereof
CN116419970B (en) Low toxicity anti-OX 40 antibodies, pharmaceutical compositions and uses thereof
CN114591428B (en) Anti-Dsg 1 antibodies and uses thereof
WO2023131262A1 (en) Antigen-binding protein specifically bound to sars-cov-2
WO2023103788A1 (en) Bispecific antibody that specifically binds to klebsiella pneumoniae o2 and o1 antigens, and composition
WO2022095045A1 (en) Sars-cov-2 antibody and application thereof
CN116178530A (en) Use of antibodies in the treatment of SARS-CoV-2 infection
CN117510621A (en) Broad-spectrum nano-antibodies N103 and N235 targeting novel coronavirus NTD, constructs and application thereof
CN116143910A (en) Nano antibody for targeting SARS-CoV-2 spike protein and application thereof
CN116574189A (en) Multiple antibodies against human IL-36R and/or human IL-1R3 and uses thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40055435

Country of ref document: HK

GR01 Patent grant
GR01 Patent grant