WO2021142672A1 - 抗Echo30的抗体，相应的核酸，载体，宿主细胞，组合物 - Google Patents

抗Echo30的抗体，相应的核酸，载体，宿主细胞，组合物 Download PDF

Info

Publication number
WO2021142672A1
WO2021142672A1 PCT/CN2020/072311 CN2020072311W WO2021142672A1 WO 2021142672 A1 WO2021142672 A1 WO 2021142672A1 CN 2020072311 W CN2020072311 W CN 2020072311W WO 2021142672 A1 WO2021142672 A1 WO 2021142672A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
amino acid
echo30
antigen
acid sequence
Prior art date
Application number
PCT/CN2020/072311
Other languages
English (en)
French (fr)
Inventor
张黎
王康
郑滨洋
朱玲
苏璇
张倩
陶焱炀
崔仑标
潘红星
葛以跃
吴涛
王祥喜
朱凤才
Original Assignee
江苏省疾病预防控制中心(江苏省公共卫生研究院)
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 江苏省疾病预防控制中心(江苏省公共卫生研究院) filed Critical 江苏省疾病预防控制中心(江苏省公共卫生研究院)
Priority to PCT/CN2020/072311 priority Critical patent/WO2021142672A1/zh
Publication of WO2021142672A1 publication Critical patent/WO2021142672A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • This application belongs to the field of biomedicine, and specifically relates to anti-Echo30 antibodies, corresponding nucleic acids, vectors, host cells, and compositions.
  • Echovirus belongs to enteroviruses (EVs). Enteroviruses are composed of 67 unique serotypes of viruses in the Picornaviridae family. Picornaviruses are positive-strand RNA viruses, including poliovirus, Coxsackie virus groups A and B, Echo virus, and enterovirus 68-71 types.
  • the Echo virus particles are very small and spherical in shape.
  • the virus particles are composed of simple capsids and single positive-stranded RNA. They are naked and have no membrane, and the diameter is less than 30nm.
  • the capsid of the virus contains 4 kinds of proteins, namely VP1, VP2, VP3, and VP4.
  • the viral RNA is a single molecule, about 7.44 kb in length, and is the template for viral translation and replication.
  • enterovirus There are 64 different serotypes of enterovirus that can cause human infections, among which 34 serotypes of Echovirus are known.
  • the clinical classification of viruses is mainly based on their genetic characteristics.
  • Echovirus belongs to the human enterovirus HEV B species. Like other enteroviruses, the optimal growth temperature of Echovirus is 35-37°C, and it can be cultured in monkey kidney, human amniotic membrane cells, human laryngeal carcinoma cells, rhabdomyosarcoma, human embryonic lung and other cells.
  • Echo30 Human Echovirus 30
  • the virus is a cell-free virus that can survive stably in harsh environments such as high salt and high pH. Enterovirus detoxification takes a long time, and patients can still excrete the virus from the respiratory tract or intestinal tract within a few weeks after infection, thus causing virus transmission, infection, and even outbreak or epidemic.
  • outbreaks or epidemics of viral encephalitis caused by Echo30 have been repeatedly reported in Taiwan, Zhejiang, Fujian, Henan and other places in my country.
  • the present invention provides an anti-Echo30 antibody or antigen-binding fragment thereof that specifically binds to Echo30.
  • the amino acid sequences of exemplary CDRs and the amino acid sequences of the VH and VL regions of the heavy and light chains of an exemplary anti-Echo30 antibody are provided in the following specific embodiments.
  • Anti-Echo30 antibodies may include modifications and/or mutations that alter the characteristics of the antibody, such as extending half-life, increasing or decreasing ADCC, etc., as known in the art.
  • nucleic acid comprising a nucleotide sequence encoding the anti-Echo30 antibody of the present invention, and a vector comprising the nucleic acid.
  • prokaryotic and eukaryotic host cells transformed with a vector containing the nucleotide sequence encoding the disclosed anti-Echo30 antibody, and eukaryotic (such as mammalian) hosts engineered to express the nucleotide sequence cell.
  • eukaryotic such as mammalian
  • the present invention provides a product for detecting Echo30, and the product includes the Echo30 antibody of the present invention or an antigen-binding fragment thereof.
  • the present invention provides a product for diagnosing Echo30 infection or a disease caused by it.
  • the product includes the Echo30 antibody or antigen-binding fragment thereof of the present invention.
  • the invention provides a composition comprising the anti-Echo30 antibody described herein.
  • the composition generally includes one or more anti-Echo30 antibodies and/or salts thereof as described herein, and one or more excipients, carriers or diluents.
  • the present invention provides a method for treating diseases caused by Echo virus infection diagnosed with Echo30 antibodies.
  • the method generally involves administering to the subject an amount of an anti-Echo30 antibody described herein that is effective to provide a therapeutic benefit.
  • the anti-Echo30 antibody is usually administered as an intravenous infusion at a dose ranging from about 0.001 mg/kg to about 4 mg/kg.
  • Anti-Echo30 is usually administered as an intravenous infusion twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, or once every eight weeks Of antibodies.
  • Anti-Echo30 antibodies can be administered as a single therapeutic agent (monotherapy) or other therapeutic agents that are adjunct to those commonly (but not necessarily) used to treat diseases caused by Echo virus infection, or together with these other therapeutic agents.
  • the therapeutic agent should usually be used in its approved dosage, route of administration, and frequency of administration, but it can be used in a lower dosage.
  • Anti-Echo30 antibodies can be administered via various routes or modes of administration, including but not limited to intravenous infusion and/or injection, and subcutaneous injection.
  • the amount administered will depend on the route of administration, the schedule of administration, the type of disease being treated, the stage of the disease being treated, and other parameters as well known in the art, such as the age and weight of the patient.
  • the present invention provides a method for detecting Echo30 with an anti-Echo30 antibody.
  • the method includes the following steps: a) contacting the sample with the anti-Echo30 antibody of the present invention; b) detecting the reaction between the antibody and Echo30.
  • the present invention provides a method for diagnosing Echo30 infection with an anti-Echo30 antibody.
  • the method includes the following steps: a) contacting the sample with the anti-Echo30 antibody of the present invention; b) detecting the reaction between the antibody and Echo30; c) or a positive reaction occurs, then it is determined that the subject has been infected by Echo30 or has Disease caused by Echo30 infection.
  • the present invention provides a method for diagnosing diseases caused by Echo30 infection by using anti-Echo30 antibodies. a) Contact the sample with the antibody of the present invention; b) Detect the reaction of the antibody or its antigen-binding fragment with Echo30; c) If a positive reaction occurs, it is judged that the subject has been infected by Echo30 or has Echo30 Disease caused by infection.
  • the present invention provides anti-Echo30 antibody detection, diagnosis, and treatment uses.
  • the uses include, but are not limited to, the following uses: applications in the preparation of products for detecting Echo30; applications in the preparation of products for diagnosing Echo30 infections or diseases caused by them; applications in the preparation of drugs for treating or preventing Echo30 infections; Application in preparing medicine for treating or preventing diseases caused by Echo30 infection.
  • the diseases caused by Echovirus type 30 infection disclosed in the present invention include but are not limited to meningitis, upper respiratory tract infection, myocarditis, skin rash, vomiting, fever, and headache.
  • the upper respiratory tract infection includes flu-like symptoms such as cough and sore throat.
  • the meningitis includes aseptic meningitis, and aseptic meningitis includes childhood aseptic meningitis.
  • the anti-Echo30 antibodies described herein will provide therapeutic benefits to subjects diagnosed with diseases caused by Echo virus 30 infection.
  • Figure 1 shows the SDS-PAGE chart of the antibody of the present invention
  • Figure 2 shows the affinity activity curve of the antibody of the present invention, where A: 4B10-IgG, B: 4B10-Fab, C: 6C5-IgG, and D: 6C5-Fab;
  • Figure 3 shows the binding affinity curve of the virus of the present invention to the receptor, where A: FcRn, B: CD55;
  • Figure 4 shows the inhibition curve of the antibody of the present invention on the binding of the virus to its receptor, where A: the 6C5 antibody is added first and then the receptor, B: the receptor is added first and then the 6C5 antibody is added; C: the 4B10 antibody is added first and then the receptor is added Body, D: Add the receptor first and then add the 4B10 antibody;
  • Figure 5 shows the specificity determination diagram of the affinity activity of the antibody of the present invention
  • Figure 6 shows a statistical diagram of the neutralization activity of the antibody of the present invention, where A: 6C5, B: 4B10;
  • Figure 7 shows the specificity determination diagram of the neutralizing activity of the antibody of the present invention.
  • Figure 8 shows the results of the effect of antibodies on virus activity before or after the virus and cells contact, where A: before contact, B: after contact;
  • Figure 9 shows a statistical graph of serum titer after immunized mice with inactivated E30 particles
  • Figure 10 shows a graph showing the effect of the binding of the antibody of the present invention to the virus on the epitope
  • the present invention relates to antibodies and/or antigen-binding fragments thereof that specifically bind to human Echo30.
  • the term "antibody” refers to an immunoglobulin molecule that specifically binds to a specific antigen (here, Echo30).
  • the anti-Echo30 antibody of the present invention contains complementarity determining regions (CDRs) in both the light chain variable domain and the heavy chain variable domain, which are also called hypervariable regions.
  • CDRs complementarity determining regions
  • the more highly conserved parts of variable domains are called the framework (FR).
  • FR framework
  • the amino acid positions/boundaries describing the hypervariable regions of antibodies can vary depending on the context and various definitions known in the art.
  • variable domains Some positions within the variable domain can be regarded as hybrid hypervariable positions because these positions can be considered to be in the hypervariable region according to a set of criteria and are considered to be outside the hypervariable region according to a set of different criteria. One or more of these locations can also be found in extended hypervariable regions.
  • the present invention provides antibodies comprising modifications in these hybrid hypervariable positions.
  • the variable domains of the natural heavy and light chains each contain four FR regions connected by three CDRs (mostly by adopting a ⁇ -sheet configuration), and these three CDRs form a connection (and in some cases form a ⁇ -sheet configuration). Part of the structure) ⁇ -sheet structure ring.
  • the CDRs in each chain are tightly bound together by the FR region, and together with the CDRs from the other chain, contribute to the formation of the antibody's target binding site.
  • Kabat et al. Sequences of Proteins of Immunological Interest [Sequence of a protein of immunological interest] (National Institute of Health, Bethesda, Md. 1987).
  • the numbering of immunoglobulin amino acid residues is based on the immunoglobulin amino acid residue numbering system of Kabat et al.
  • the antibodies of the present invention can be polyclonal, monoclonal, genetically engineered and/or otherwise modified in nature, including but not limited to murine antibodies, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, and the like.
  • the antibody comprises all or part of the constant region of the antibody.
  • the constant region is an isotype selected from: IgA (eg, IgA1 or IgA2), IgD, IgE, IgG (eg, IgG1, IgG2, IgG3, or IgG4), and IgM.
  • the "constant region" of an antibody includes natural constant regions, allotypes, or natural variants.
  • the light chain constant region of the anti-Echo30 antibody can be a kappa (kappa) light chain region or a lambda (lambda) region.
  • the lambda light chain region can be any of the known subtypes, for example, lambda 1, lambda 2, lambda 3, or lambda 4.
  • the anti-Echo30 antibody comprises a kappa (kappa) light chain region.
  • monoclonal antibody as used herein is not limited to antibodies produced via hybridoma technology. Monoclonal antibodies are obtained from a single clone by any means available or known in the art, including any eukaryotic, prokaryotic or phage clone. The monoclonal antibodies used in the present invention can be prepared using a wide variety of techniques known in the art, including the use of hybridoma technology, recombinant technology, and phage display technology or a combination thereof.
  • chimeric antibody refers to a human immunoglobulin constant with variable sequences derived from non-human immunoglobulins (such as rat or mouse antibodies) and usually selected from human immunoglobulin templates. Area of antibodies.
  • Methods for making chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science [Science] 229(4719): 1202-7; Oi et al., 1986, BioTechniques [Biotechnology] 4: 214-221; Gillies et al., 1985, J. Immunol. Methods [Immunization Journal of Scientific Methods] 125: 191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins containing very few sequences derived from non-human immunoglobulins.
  • a humanized antibody will contain substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are Those regions of the human immunoglobulin sequence.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), usually that portion of the common sequence of human immunoglobulins.
  • Fc immunoglobulin constant region
  • Human antibodies include antibodies having the amino acid sequence of human immunoglobulins, and include those isolated from a human immunoglobulin library or from animals that are transgenic for one or more human immunoglobulins and do not express endogenous immunoglobulins Of antibodies.
  • Human antibodies can be prepared using various methods known in the art (including phage display methods) using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; PCT Publication WO98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741.
  • Human antibodies can also be produced using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. See, for example, PCT Publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; US Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598.
  • companies such as LakePharma, Inc. (Belmont, CA) or Creative BioLabs (Shirley, NY) can participate to provide human antibodies against selected antigens using techniques similar to those described above.
  • Fully human antibodies that recognize selected epitopes can be produced using a technique called "directed selection".
  • the selected non-human monoclonal antibody e.g., mouse antibody
  • the selected non-human monoclonal antibody is used to guide the selection of fully human antibodies that recognize the same epitope (see Jespers et al., 1988, Biotechnology 12:899-903 ).
  • the present invention also discloses antigen-binding fragments of antibodies capable of specifically binding Echo30.
  • antigen-binding fragments include (by way of example and not limitation) Fab, Fab', F(ab')2, Fv fragments, single chain Fv fragments, and single domain fragments.
  • the Fab fragment contains the constant domain and variable domain of the light chain and the first constant domain (CH1) and variable domain of the heavy chain.
  • the difference between the Fab' fragment and the Fab fragment lies in the addition of several residues at the carboxy terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody.
  • the F(ab') fragment is produced by cleaving the disulfide bond at the hinge cysteine of the F(ab')2 pepsin digestion product. Other chemical coupling methods of antibody fragments are known to those of ordinary skill in the art.
  • Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, are cleared more rapidly from the circulation of animals, and have lower non-specific tissue binding properties than intact antibodies (see, for example, Wahl et al., 1983, J. Nucl . Med. [Journal of Nuclear Medicine] 24:316).
  • the "Fv” fragment is the smallest fragment of an antibody that contains complete target recognition and binding sites.
  • This region is composed of a heavy chain variable domain and a light chain variable domain dimer (VH-VL dimer) that are tightly non-covalently associated.
  • VH-VL dimer light chain variable domain dimer
  • the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer.
  • the six CDRs confer target binding specificity to the antibody.
  • even a single variable domain or half of an Fv containing only three CDRs specific to the target
  • Single chain Fv or “scFv” antibody binding fragments include the VH domain and VL domain of an antibody, where these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain, which allows the scFv to form the desired structure for target binding.
  • a “single domain fragment” is composed of a single VH domain or VL domain with sufficient affinity for Echo30.
  • the single domain fragment is a camelized fragment (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25-38).
  • the anti-Echo30 antibody of the present invention includes a derivative antibody.
  • derivatized antibodies are usually glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized with known protection/blocking groups, proteolytic cleavage, and linked to cellular ligands. Body or other proteins.
  • known techniques can be used to carry out any of many chemical modifications, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
  • the derivative may contain one or more unnatural amino acids, for example using the Ambryx technology (see, for example, Wolfson, 2006, Chem. Biol. 13(10): 1011-2).
  • the anti-Echo30 antibody or binding fragment may be an antibody or fragment whose sequence has been modified to modify at least one constant region-mediated biological effect function.
  • an anti-Echo30 antibody can be modified to reduce at least one constant region-mediated biological effect function relative to an unmodified antibody, for example, to reduce interaction with Fc receptors (Fc ⁇ R) (such as Fc ⁇ RI, Fc ⁇ RIIA). , FcyRIIB, FcyRIIIA and/or FcyRIIIB).
  • Fc ⁇ R binding can be reduced by mutating the immunoglobulin constant region segment of the antibody at a specific region necessary for Fc ⁇ R interaction (see, for example, Canfield and Morrison, 1991, J. Exp. Med.
  • Anti-Echo30 antibodies or binding fragments described herein include antibodies that have been modified to obtain or improve at least one constant region-mediated biological effect function, such as to enhance Fc ⁇ R interactions, relative to unmodified antibodies (see, e.g., U.S. Patent Application No. 2006/0134709).
  • the anti-Echo30 antibody of the present invention may have a constant region that binds FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA, and/or FcyRIIIB with greater affinity than the corresponding wild-type constant region.
  • the anti-Echo30 antibodies of the present invention have lower levels of trehalose or lack trehalose.
  • Antibodies lacking trehalose have been associated with enhanced ADCC activity, especially at lower doses of antibody. See Shields et al., 2002, J. Biol. Chem. [Journal of Biological Chemistry] 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. [Journal of Biological Chemistry] 278:3466-73.
  • the method of preparing lower trehalose antibodies includes growing in murine myeloma YB2/0 cells (ATCC CRL 1662). YB2/0 cells express lower levels of FUT8 mRNA encoding ⁇ -1,6-trehalosyltransferase (an enzyme necessary for the trehalosylation of polypeptides).
  • Anti-Echo30 antibodies with affinity for Echo30 may be required for therapeutic and diagnostic uses. Therefore, the present invention encompasses antibodies with binding affinity to Echo30.
  • the anti-Echo30 antibody that binds to Echo30 has an affinity of at least about 1000 nM but can exhibit a higher affinity, for example, at least about 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM, 25nM, 20nM, 15nM, 10nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.1nM, 0.01nM or even higher.
  • the affinity of an antibody against Echo30 to Echo30 can be determined using techniques well known in the art or described herein, such as, but not limited to, ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance, or fluorescence polarization measurement.
  • Anti-Echo30 antibodies usually include a heavy chain and a light chain, and the heavy chain includes three complementarity determining regions ("CDR") called (in N ⁇ C order) VH CDR1, VH CDR2, and VH CDR3 herein.
  • Variable region (VH) the light chain includes a variable region (VL) having three complementarity determining regions (in N ⁇ C order) referred to herein as VL CDR1, VL CDR2, and VL CDR3.
  • VH complementarity determining regions
  • VL variable region having three complementarity determining regions (in N ⁇ C order) referred to herein as VL CDR1, VL CDR2, and VL CDR3.
  • Provided herein are the amino acid sequences of exemplary CDRs and the amino acid sequences of the VH and VL regions of the heavy and light chains of an exemplary anti-Echo30.
  • Specific examples of anti-Echo30 antibodies include these exemplary CDR and/or VH and/or VL sequences.
  • the anti-Echo30 antibody of the present invention includes a VH with at least one CDR, and/or a VL with at least one CDR;
  • the CDR sequence of VH is selected from the amino acid sequence shown in SEQ ID NO: 1-3;
  • the CDR sequence of VH is selected from: amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 1-3;
  • the CDR sequence of VH is selected from: amino acid sequences that have at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1-3, and have the same or similar functions;
  • the CDR sequence of VL is selected from the amino acid sequence shown in SEQ ID NO: 5-7;
  • the CDR sequence of VL is selected from: amino acid sequence formed by substitution, deletion or addition of one or more amino acids based on the amino acid sequence shown in SEQ ID NO: 5-7;
  • the CDR sequence of VL is selected from: amino acid sequences that have at least 80% homology with the amino acid sequence shown in SEQ ID NO: 5-7 and have the same or similar functions.
  • At least 80% homology means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% homology.
  • the CDR sequence of VH is selected from the amino acid sequence shown in SEQ ID NO: 1-3; the CDR sequence of VL is selected from the amino acid sequence shown in SEQ ID NO: 5-7.
  • the CDR1 sequence of VH has the amino acid sequence shown in SEQ ID NO: 1; the CDR2 sequence of VH has the amino acid sequence shown in SEQ ID NO: 2; the CDR3 sequence of VH has the amino acid sequence shown in SEQ ID NO: 3 Sequence:
  • the CDR1 sequence of VL has the amino acid sequence shown in SEQ ID NO: 5; the CDR2 sequence of VL has the amino acid sequence shown in SEQ ID NO: 6; the CDR3 sequence of VL has the amino acid sequence shown in SEQ ID NO: 7.
  • the antibody of the present invention includes VH and/or VL, wherein:
  • the VH has the amino acid sequence shown in SEQ ID NO: 4, or the VH has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 4; or The VH has an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 4 and has the same or similar function;
  • the VL has the amino acid sequence shown in SEQ ID NO: 8, or the VL has an amino acid sequence formed by substitution, deletion or addition of one or more amino acids on the basis of the amino acid sequence shown in SEQ ID NO: 8; or The VL has an amino acid sequence that has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 8 and has the same or similar function.
  • At least 80% homology means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% homology.
  • the VH has the amino acid sequence shown in SEQ ID NO: 4; the VL has the amino acid sequence shown in SEQ ID NO: 8.
  • the present invention covers nucleic acid molecules encoding anti-Echo30 immunoglobulin light chain genes and heavy chain genes, vectors containing such nucleic acids, and host cells capable of producing the anti-Echo30 antibodies of the present invention.
  • the nucleic acid encoding the heavy chain variable region CDR1 has the sequence shown in SEQ ID NO: 9; the nucleic acid encoding the heavy chain variable region CDR2 has the sequence shown in SEQ ID NO: 10; encoding The nucleic acid of the heavy chain variable region CDR3 has the sequence shown in SEQ ID NO: 11; the nucleic acid that encodes the light chain variable region CDR1 has the sequence shown in SEQ ID NO: 13; the nucleic acid that encodes the light chain variable region CDR2 has SEQ ID NO: 14; the nucleic acid encoding the light chain variable region CDR3 has the sequence shown in SEQ ID NO: 15.
  • the nucleic acid encoding the variable region of the heavy chain has the sequence shown in SEQ ID NO: 12; the nucleic acid encoding the variable region of the light chain has the sequence shown in SEQ ID NO: 16.
  • the anti-Echo30 antibody of the present invention can be prepared by recombinantly expressing immunoglobulin light chain genes and heavy chain genes in host cells.
  • the host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, so that the light and heavy chains can be expressed in the host cell without any effort.
  • the light and heavy chains can be expressed in the host cell without any effort.
  • Standard recombinant DNA methods are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells such as those described in Molecular Cloning; A Laboratory Manual [Molecular Cloning: Laboratory Manual], Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel, FM, etc.) People, ed., Greene Publishing Associates (1989) and those in U.S. Patent No. 4,816,397.
  • DNA fragments encoding the variable region of the light chain and the variable region of the heavy chain are obtained. These DNAs can be obtained by amplifying and modifying germline DNA or cDNA encoding the variable sequence of the light chain and the variable sequence of the heavy chain, for example, using polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the germline DNA sequences of the human heavy chain and light chain variable region genes are known in the art (see, for example, the "VBASE” human germline sequence database; see also Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest [Immunologically Important Protein Sequence], Fifth Edition, USDepartment of Health and Human Services [United States Department of Health and Human Services], NIH Publication No. 91-3242; Tomlinson et al., 1992, J.Mol.Biol. [Molecular Journal of Biology] 22T: 116-198; and Cox et al., 1994, Eur. J. Immunol. [European Journal of Immunology] 24: 827-836; the contents of each are incorporated herein by reference).
  • DNA fragments encoding the VH and VL segments related to the anti-Echo30 antibody are obtained, standard recombinant DNA techniques can be used to manipulate these DNA fragments, for example, to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv. Gene.
  • a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operably linked” means that two DNA fragments are joined such that the amino acid sequence encoded by the two DNA fragments remains in frame.
  • the isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, CH3, and optionally CH4).
  • the sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat, EA, et al., 1991, Sequences of Proteins of Immunological Interest), fifth edition, USDepartment of Health and Human Services [US Department of Health and Human Services], NIH Publication No. 91-3242) and DNA fragments covering these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but in certain embodiments is IgG1 or IgG4.
  • the DNA encoding VH can be operably linked to another DNA molecule encoding only the CH1 constant region of the heavy chain.
  • the isolated DNA encoding the VL region can be converted into a full-length light chain gene (and Fab light chain gene) by operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL.
  • the sequence of the human light chain constant region gene is known in the art (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest [Sequences of Proteins of Immunological Interest], Fifth Edition, USDepartment of Health and Human Services [United States Department of Health and Human Services], NIH Publication No. 91-3242) and DNA fragments covering these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, but in certain embodiments is a kappa constant region.
  • the DNA fragments encoding VH and VL are operably linked to another fragment encoding the flexible linker (for example, encoding the amino acid sequence (Gly4 ⁇ Ser)3 (SEQ ID NO: 200)), so that VH And VL sequences can be expressed as adjacent single-chain proteins, where the VL and VH regions are joined by flexible linkers (see, for example, Bird et al., 1988, Science [Science] 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 85: 5879-5883; McCafferty et al., 1990, Nature [Nature] 348: 552-554.
  • the partial or full-length light chain and heavy chain DNA encoding obtained as described above is inserted into an expression vector, so that the gene is operably linked to transcription and translation control sequences.
  • operably linked means that the antibody gene is joined to the vector so that the transcription and translation control sequences within the vector provide its predetermined function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequence are selected to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more commonly, the two genes can be inserted into the same expression vector.
  • the antibody gene is inserted into the expression vector using standard methods (for example, joining of antibody gene fragments and complementary restriction sites on the vector, or blunt end joining when restriction sites are not present).
  • the expression vector may already carry the antibody constant region sequence.
  • one way to convert the VH sequence and VL sequence related to the anti-Echo30 monoclonal antibody into a full-length antibody gene is to separately insert them into an expression vector that already encodes the heavy chain constant region and the light chain constant region, so that the VH region
  • the segment is operably connected to one or more CH segments in the carrier, and the VL segment is operably connected to the CL segment in the carrier.
  • the recombinant expression vector may encode a signal peptide that promotes secretion of the antibody chain from the host cell.
  • the antibody chain gene can be cloned into a vector so that the signal peptide is linked to the amino terminus of the antibody chain gene in frame.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).
  • the recombinant expression vector of the present invention carries regulatory sequences that control the expression of antibody chain genes in host cells.
  • the term "regulatory sequence” is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes.
  • Such regulatory sequences for example, are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (CA), 1990 middle.
  • CA Gene Expression Technology
  • the design of the expression vector may depend on factors such as the selection of the host cell to be transformed, the amount of expression of the desired protein, and the like.
  • Suitable regulatory sequences for expression in mammalian host cells include viral elements that direct higher protein expression in mammalian cells, such as those derived from cytomegalovirus (CMV) (such as CMV promoter/enhancer), monkey virus 40 (SV40) (such as SV40 promoter/enhancer), adenovirus (for example, adenovirus major late promoter (AdMLP)), and polyoma virus promoter and/or enhancer.
  • CMV cytomegalovirus
  • SV40 monkey virus 40
  • AdMLP adenovirus major late promoter
  • polyoma virus promoter and/or enhancer for a further description of viral regulatory elements and their sequences, see, for example, US Patent No. 5,168,062 to Stinski, US Patent No. 4,510,245 to Bell et al., and US Patent No. 4,968,615 to Schaffner et al.
  • the recombinant expression vector of the present invention may carry additional sequences, such as sequences that regulate the replication of the vector in host cells (for example, the origin of replication) and selectable marker genes.
  • Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all of Axel et al.).
  • the selectable marker gene usually confers resistance to drugs such as G418, hygromycin, or methotrexate.
  • Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for selection/amplification of DHFR-host cells with methotrexate) and neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • neo gene for G418 selection.
  • DHFR dihydrofolate reductase
  • For the expression of the light chain and the heavy chain one or more expression vectors encoding the heavy chain and the light chain are transfected into the host cell using standard techniques.
  • the various forms of the term "transfection" are intended to cover a wide variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-polydextrose transfection and the like.
  • eukaryotic host cells e.g., mammalian host cells
  • eukaryotic cells e.g., mammalian host cells
  • Exemplary mammalian host cells for expressing the recombinant antibodies of the present invention include Chinese Hamster Ovary (CHO cells) (including DHFR-CHO cells, which are described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA [United States Proceedings of the National Academy of Sciences] 77: 4216-4220, used with DHFR selectable markers, for example, as described in Kaufman and Sharp, 1982, Mol. Biol.
  • Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not necessary for binding to Echo30. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention.
  • host cells can be co-transfected with two expression vectors of the present invention, the first vector encodes a heavy chain derived from a polypeptide and the second vector encodes a light chain derived from a polypeptide.
  • the two vectors may contain the same selectable marker, or they may each contain independent selectable markers.
  • a single vector encoding both the heavy chain polypeptide and the light chain polypeptide can be used.
  • nucleic acid encodes one or more portions of the anti-Echo30 antibody
  • other changes or mutations can be introduced into the coding sequence, for example to produce antibodies that encode different CDR sequences, antibodies that have reduced affinity for Fc receptors, or different subgroups.
  • the nucleic acid of the antibody can be introduced into the coding sequence, for example to produce antibodies that encode different CDR sequences, antibodies that have reduced affinity for Fc receptors, or different subgroups.
  • nucleic acid sequence of the anti-Echo30 antibody involved in the present invention is shown in SEQ NO ID: 9-16.
  • the anti-Echo30 antibody of the present invention can also be chemically synthesized (for example, using the method described in Solid Phase Peptide Synthesis [solid phase peptide synthesis], 2nd edition, 1984 Pierce Chemical Co. (The Pierce Chemical Co.), Rockford ( Rockford), Illinois (Ill) method). Variant antibodies can also be produced using a cell-free platform (see, for example, Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et al., 2013, Currem Opinion in Chemical Biology [New Views on Chemical Biology], 17: 420-426.
  • the anti-Echo30 antibody of the present invention can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity chromatography). And size fractionation column chromatography), centrifugation, different solubility, or by any other standard techniques for protein purification.
  • the anti-Echo30 antibody of the present invention can be fused to a heterologous polypeptide sequence described herein or otherwise known in the art to facilitate purification.
  • the anti-Echo30 antibody can (if necessary), for example, by high-performance liquid chromatography (see, for example, Fisher, Laboratory Techniques In Biochemistry And Molecular Biology [Biochemistry and Molecular Biology Laboratory Technology], Work and Burdon, eds., Elsevier Publishing Company (Elsevier, 1980) or by SuperdexTM75 column gel filtration chromatography (Pharmacia Biotech AB, Uppsala, Sweden (Pharmacia Biotech AB, Uppsala, Sweden)) for further purification.
  • high-performance liquid chromatography see, for example, Fisher, Laboratory Techniques In Biochemistry And Molecular Biology [Biochemistry and Molecular Biology Laboratory Technology], Work and Burdon, eds., Elsevier Publishing Company (Elsevier, 1980) or by SuperdexTM75 column gel filtration chromatography (Pharmacia Biotech AB, Uppsala, Sweden (Pharmacia Biotech AB, Uppsala, Sweden) for further purification.
  • the anti-Echo30 antibodies described herein may be in the form of compositions comprising the antibody and one or more carriers, excipients and/or diluents.
  • the composition can be formulated for a specific use, such as for veterinary use or human pharmaceutical use.
  • the form of the composition (eg, dry powder, liquid formulation, etc.) and the excipients, diluents and/or carriers used will depend on the intended use of the ADC and the mode of administration for therapeutic use.
  • the composition may be provided as part of a sterile, pharmaceutical composition that includes a pharmaceutically acceptable carrier.
  • This composition may be in any suitable form (depending on the desired method of administering it to a subject (ie, patient), such as a human subject).
  • the pharmaceutical composition can be administered to a subject by various routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, surface, or topical. The most appropriate route of administration in any given situation will depend on the particular antibody, the subject, and the nature and severity of the disease, as well as the subject's physiological conditions.
  • the pharmaceutical composition should be administered intravenously or subcutaneously.
  • the pharmaceutical composition may suitably be in the form of a unit dosage containing a predetermined amount of the anti-Echo30 antibody described herein per dosage.
  • the amount of anti-Echo30 antibody included in the unit dose will depend on the disease being treated and other factors as well known in the art.
  • Such unit dose may be in the form of a lyophilized dry powder containing a certain amount of antibody suitable for single administration, or in liquid form.
  • the dry powder unit dosage form can be packaged in a kit with a syringe, an appropriate amount of diluent and/or other components that can be used for administration.
  • the unit dose in liquid form may be suitably provided in the form of a syringe pre-filled with a certain amount of anti-Echo30 antibody suitable for single administration.
  • the pharmaceutical composition may also be provided in the form of a block containing a certain amount of anti-Echo30 antibody suitable for multiple administrations.
  • carriers a pharmaceutically acceptable carrier, excipient or stabilizer commonly used in the art (all of the above are referred to herein as "carriers”).
  • carriers that is, buffers, stabilizers, preservatives, ionic isotonic agents, non-ionic detergents, antioxidants and other mixed additives are mixed to prepare pharmaceutical compositions for storage as freeze-dried formulations or aqueous solutions. See, Remington’s Pharmaceutical Sciences, 16th edition (Osol editor, 1980). Such additives should be non-toxic to the recipient at the dosage and concentration used.
  • Buffers help keep the pH in a range close to physiological conditions. It can be present in a wide variety of concentrations, but should generally be present in a concentration ranging from about 2mM to about 50mM.
  • Suitable buffers for use with the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture , Citric acid-monosodium citrate mixture, etc.), succinate buffer (for example, succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate Buffer (for example, tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffer (for example, fumaric acid-monosodium fumarate mixture, fum
  • fumarate buffers such as 2-amino-2-hydroxymethyl-propane-1,3-diol (ie, Tris, THAM or Tris( Hydroxymethyl)aminomethane).
  • Isotonic agents sometimes referred to as “stabilizers” may be added to ensure the isotonicity of the liquid composition of the present invention, and these isotonic agents include polyhydroxy sugar alcohols, such as trihydric alcohols or higher sugar alcohols, such as, Glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol.
  • Stabilizers refer to a wide range of excipients, which in terms of function range from swelling agents to solubilizing therapeutic agents or additives that help prevent denaturation or adhesion to the container wall.
  • Typical stabilizers can be polyhydroxy sugar alcohols (listed above); amino acids (such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L -Leucine, 2-phenylalanine, glutamic acid, threonine, etc.), organic sugars or sugar alcohols (such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribose) Alcohol, inositol, galactitol), glycerol, etc., including cyclic polyols (such as inositol); polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, lipoic acid, sulfur Sodium glycolate, thioglycerol, ⁇ -monothioglycerol, and sodium thiosulfate; low molecular weight
  • Non-ionic surfactants or detergents can be added to help dissolve glycoproteins and protect glycoproteins from agitation-induced aggregation, which also allows the formulation to be exposed to shear stress surfaces without causing Denaturation of protein.
  • Suitable nonionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), and pluronic polyols.
  • the nonionic surfactant may be present in the range of about 0.05 mg/mL to about 1.0 mg/mL.
  • aqueous compositions suitable for administration via intravenous infusion include 10 mg/mL of anti-Echo 30 antibody, 15 mM histidine buffer, pH 6.0, 8.0% (w/v) sucrose and 0.05% ( w/v) Polysorbate 80.
  • the composition may be in the form of a lyophilized powder, which is used in 2.0 mL of sterile water or other solutions suitable for injection or infusion (e.g., 0.9% saline, Ringer's solution, lactated Ringer's solution) After rehydration, the above aqueous composition is provided.
  • the composition or the composition of other embodiments may also be in the form of a syringe or other device suitable for injection and/or infusion prefilled with a certain amount of the composition suitable for single administration of the anti-Echo30 antibody.
  • the anti-Echo30 antibody of the present invention can be administered alone (monotherapy) or in addition to other therapies, or together with other drugs that can treat diseases caused by Echo30 infection. Regardless of whether it is administered as a monotherapy or in addition to other therapies or drugs, or administered together with other therapies or drugs, a certain amount of anti-Echo30 antibody is administered.
  • Anti-Echo30 antibodies can be used as a supplement or in combination with other drugs.
  • the anti-Echo30 antibody and one or more other drugs can be formulated together into a single combined drug formulation, or can be formulated and administered separately on a single coordinated dosing schedule or on different dosing schedules.
  • Drugs administered as adjunct or together with an anti-Echo30 antibody usually have complementary activities to the anti-Echo30 antibody, so that the antibody and other drugs do not adversely affect each other. Make the overall treatment plan provide therapeutic benefits.
  • the amount of anti-Echo30 antibody administered will depend on various factors, including but not limited to: the specific type of solid tumor being treated, the stage of the solid tumor being treated, the mode of administration, the frequency of administration, the desired therapeutic benefit, and others Parameters (such as the age, weight, and other characteristics of the patient, etc.).
  • the determination of a dose effective to provide therapeutic benefit for a particular mode and frequency of administration is within the abilities of those of ordinary skill in the art.
  • the dose effective to provide therapeutic benefit can first be estimated from in vivo animal models or clinically. Suitable animal models for a wide variety of diseases are known in the art.
  • the anti-Echo30 antibodies disclosed herein can be administered using any route suitable for the condition to be treated.
  • Anti-Echo30 antibodies should usually be administered parenterally, that is, infusion, subcutaneous, intramuscular, intravenous (IV), intradermal, intrathecal, bolus injection, or epidural administration ((Shire et al., 2004, J .Pharm.Sciences[Journal of Pharmaceutical Sciences]93(6):1390-1402)).
  • the anti-Echo30 antibody is provided as a lyophilized powder in a vial.
  • the vial may contain 21 mg of anti-Echo30 antibody.
  • the lyophilized powder is rehydrated with sterile water for injection (SWFI) or other suitable medium to obtain a solution containing 10 mg/mL anti-Echo30 antibody.
  • SWFI sterile water for injection
  • the resulting reconstituted solution is further diluted with saline or other suitable medium, and the treatment is performed twice every 7 days, once every 7 days, once every 14 days, once every 21 days, once every 28 days, and once every 35 days. , Once every 42 days, once every 49 days or once every 56 days by IV infusion.
  • the infusion is over 90 minutes. In some embodiments, subsequent infusions are over 60 minutes.
  • the obtained reconstituted water solution is further diluted with saline or other suitable medium, and the treatment is performed twice every 7 days, once every 7 days, once every 14 days, once every 21 days, once every 28 days, and once every 35 days. , Once every 42 days, once every 49 days, or once every 56 days IT injection for administration.
  • the anti-Echo30 antibody may be administered on the same schedule as the other drugs or on a different schedule.
  • the anti-Echo30 antibody can be administered before or after other drugs, or simultaneously with these other drugs.
  • the present invention provides a method for detecting Echo30 in a sample, which includes the following steps: (1) Combining the monoclonal antibody or antigen-binding fragment of the present invention with the virus in the sample to form an antibody virus or antibody fragment virus Complex; (2) Detect the sample complex to determine whether there is virus in the sample.
  • the present invention provides a method for detecting Echo30 in a sample, including the following steps: (1) Adsorb the first antibody onto a solid support; (2) Add Echo30 to the above support. (3) Add a second antibody with a label to the above-mentioned support; (4) detect the presence of the label to determine whether Echo30 exists.
  • the present invention provides a method for diagnosing Echo30 infection or the disease caused by it, including the following steps: (1) Combining the monoclonal antibody or antigen-binding fragment of the present invention with the virus in the sample to form an antibody virus or Antibody fragment virus complex; (2) Detect the sample complex to determine whether there is virus in the sample; (3) If there is virus, it is determined that the subject is infected with Echo30, or the suspected patient is diagnosed as having a disease caused by Echo30 infection .
  • the present invention provides a method for diagnosing Echo30 infection or the disease caused by it, including the following steps: (1) Adsorb the first antibody onto a solid support; (2) In the above support Add a suspicious sample to be tested that may contain Echo30; (3) Add a labeled secondary antibody to the above-mentioned support; (4) detect the presence of the label to determine whether Echo30 exists; (5) If there is a virus, It is judged that the subject is infected with Echo30, or the suspected patient is diagnosed as having a disease caused by Echo30 infection.
  • the first antibody or the second antibody is the monoclonal antibody or antigen-binding fragment thereof of the present invention.
  • the detection method can use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competition method and similar detection methods.
  • ELISA enzyme-linked immunosorbent assay
  • enzyme immunoassay enzyme immunoassay
  • chemiluminescence immunoassay chemiluminescence immunoassay
  • radioimmunoassay radioimmunoassay
  • fluorescence immunoassay immunochromatography
  • competition method Using the competition method or the sandwich method, the above detection method can be used to detect the target antigen or antibody.
  • the competition method is to compare the quantitative relationship between the antigen in the sample and a known amount of labeled antigen that compete to bind to the monoclonal antibody of the present invention.
  • the immunological detection based on the competition method is carried out by adding a sample containing an unknown number of target antigens to the monoclonal antibody of the present invention coated on a solid support by a known physical or chemical method in advance. At the same time, the pre-quantified labeled target antigen is added for reaction. After the incubation, the solid support is washed, and the activity of the label bound to the support is detected.
  • the target antigen in the sample is sandwiched between the coated monoclonal antibody and the labeled monoclonal antibody, and then a label such as an enzyme substrate is added, and the color of the substrate is changed to detect and determine the presence of the antigen.
  • a label such as an enzyme substrate
  • the color of the substrate is changed to detect and determine the presence of the antigen.
  • a sample containing an unknown amount of target antigen is first added to a solid support that is pre-coated with the monoclonal antibody of the present invention by physical or chemical methods for reaction.
  • the labeled monoclonal antibody of the present invention is added to react. After the incubation, the support is washed, and then the activity of the label bound to the support is detected.
  • Labels can be radioisotopes such as 125 iodine, enzymes, enzyme substrates, luminescent substances such as isoluminol and acridinium esters, fluorescent substances such as fluorescein and rhodamine, biotin and colored substances such as latex particles and colloidal gold, etc. .
  • the labeling enzyme may be peroxidase (such as horseradish peroxidase HRP), alkaline phosphatase, ⁇ -galactosidase, and glucose oxidase.
  • Suitable substrates for these reactions are 2,2'-azino-bis(3-ethylbenzothiapyrroline-6sulfonic acid), luminol-hydrogen peroxide, o-phenylenediamine-peroxide Hydrogen (for peroxidase), p-nitrophenyl phosphate, 4-methyl umbelliferone phosphate, 3-(2'-spiraladamantane)-4-methoxy-4-(3"-phosphoryl ) Phenyl-1,2-diethoxy alkane (for alkaline phosphatase), p-nitrobenzene- ⁇ -D-galactose and methylumbelliferone- ⁇ -D-galactose (for ⁇ -galactose) Glycosidase).
  • labels include quantum dot labels, chromophore labels, enzyme labels, affinity ligand labels, electromagnetic spin labels, heavy atom labels, probes labeled with nanoparticle light scattering labels or other nanoparticles, Fluorescein isothiocyanate (FITC), TRITC, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-Red, isophycoerythrin (APC), epitope tags such as FLAG or HA epitopes, and enzyme tags such as alkaline phosphatase, horseradish peroxidase, I2-galactosidase, alkaline phosphatase, ⁇ -galactosidase or acetylcholine Esterase and hapten conjugates such as digoxigenin or dinitrophenol, or binding pairs capable of forming complexes such as streptavidin/biotin, avidin/biotin or antigen/
  • detectable molecules include but Not limited to fluorophores and other known previously mentioned, such as Principles of Fluorescence Spectroscopy edited by Joseph R. Lakowicz (Editor), Plenum Pub Corp, second edition (July 1999) and Richard P. Hoagland’s sixth edition Version of the Molecular Probes Handbook.
  • the markers include semiconductor nanocrystals such as quantum dots (ie, Qdots), see USP 6,207,392. Qdots can be purchased from Quantum Dot Corporation.
  • Semiconductor nanocrystals used in the present invention include Group II-V semiconductor nanocrystals such as MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe and their mixtures and Group III-V semiconductor nanocrystals such as GaAs, InGaAs, InP, InAs and their mixtures.
  • Group IV semiconductors such as germanium or silicon, or the use of organic semiconductors, may be convenient and feasible under certain conditions.
  • Semiconductor nanocrystals may also include alloys containing two or more semiconductors selected from Group III-V compounds, Group II-VI compounds, Group IV elements, and combinations thereof.
  • the fluorescent energy acceptor is attached to the detection probe as a label.
  • the fluorescent energy acceptor can be formed by reacting a compound with singlet oxygen to form a fluorescent compound, or by reacting a compound with an auxiliary compound and converting it into a fluorescent compound. Such compounds can be included in the buffer in the device of the invention.
  • the fluorescent energy acceptor may be a part of a compound including a chemiluminescent agent or group.
  • the fluorescent energy acceptor may include metal complexes of rare earth metals such as europium, samarium, and tellurium.
  • lanthanide markers such as europium(III) can provide effective long-term signal emission, and are not easy to photobleach, so they can contain processed/reacted samples.
  • the test device can be placed for a longer period of time if needed.
  • long-lived fluorescent europium (III) complex nanoparticles have been used as markers. For example, see Huhtinen et al. Clin. Chem. 2004 Oct; 50(10): 1935-6. However, when these internally labeled nanoparticles are used with time-resolved fluorescence detection, the measurement performance can be improved.
  • the dynamic range of the assay at low concentrations can be extended; moreover, by using the highly specific active nanoparticle marker coated with the detection antibody instead of the conventionally labeled detection antibody, the dynamic characteristics of the assay can also be improved .
  • europium(III) nanoparticles are very effective donors for fluorescence resonance energy transfer, so that simple, fast and efficient screening can be performed.
  • the label such as the fluorescent label disclosed herein, includes a nanoparticle label coupled to a biomolecule. In other words, nanoparticles can be used as detection or capture probes.
  • europium(III)-labeled nanoparticles linked to monoclonal antibodies or streptavidin (SA) can be used to detect specific analytes in a sample, such as nanoparticle-based immunoassays.
  • Nanoparticles can be used as substrates for attaching specific binding agents that target the analyte and the detection (such as a label) or capture component. Examples of related markers can be found in U.S.P. 4,695,554; 4,863,875; 4,373,932; and 4,366,241.
  • U.S.P 4,313,734 and 4,373,932 disclose colloidal metals and dyed particles.
  • U.S.P 4,954,452 discloses how to prepare and use non-metallic colloids;
  • U.S.P 4,252,459 discloses organic polymer latex particles used as markers.
  • the method of binding the label to the antigen or antibody can be performed by the maleimide method (J. Biochem. (1976), 79, 233) and the biotin activation method (J. Am. Chem. Soc. (1978) , 100, 3585), hydrophobic binding method, ester activation method or isocyanate method ("Enzyme immunoassay techniques", published in 1987 by IgakuShoin).
  • the above-mentioned markers are radioisotopes, a good radiation-proof work surface or liquid protection equipment must be used. If the above-mentioned label is an enzyme, a substrate needs to be added, and the activity of the enzyme is measured by a colorimetric method or a fluorometer. If the above-mentioned label is a fluorescent substance, a luminescent substance or a colored substance, the determination method can be determined by a method well-known in the art accordingly.
  • the samples used to detect Echo30 include, but are not limited to, blood, plasma and serum of animals or patients.
  • the invention also relates to a product for detecting Echo30, in particular to a kit for detecting Echo30.
  • the present invention also relates to a product for diagnosing Echo30 infection or diseases caused by Echo30, in particular to a kit for diagnosing Echo30 infection or diseases caused by Echo30.
  • the kit of the present invention contains the monoclonal antibody of the present invention or an antigen-binding fragment thereof.
  • the kit of the present invention also contains a detection reagent suitable for detecting an antigen-antibody reaction.
  • the kit of the present invention also includes a solid-phase substrate for the attachment of monoclonal antibodies or antigen-binding fragments thereof.
  • the solid-phase substrates include, but are not limited to, microplates, magnetic particles, filter paper for immunochromatography, and polymers such as polyphenylene. Ethylene, glass beads, glass filters and other insoluble carriers.
  • the kit of the present invention also contains some other components, including but not limited to enzymes for labeling, corresponding substrates, radioisotopes, light-reflecting substances, fluorescent substances, colored substances, buffers and the like.
  • the monoclonal antibody or antigen used must be labeled with the above-mentioned markers in advance.
  • the kit of the present invention also includes control standards.
  • Each mouse is immunized 5 times, with 100 ⁇ l of antigen each time, and a total of 3-3.5 ml of virus is required.
  • Two adjuvants were used for immunization, modified Freund's adjuvant and aqueous adjuvant respectively. Two groups were immunized with virus, with 3 mice each as a group, and 6 mice were immunized with one adjuvant. All immunizations were performed in the SPF animal room.
  • mice Female mice aged 5-8 weeks.
  • the immunogen and adjuvant are mixed and then emulsified separately for immunization.
  • the first injection uses Freund's complete adjuvant, and the next 4 booster injections use Freund's adjuvant. Incomplete adjuvant, mix well with equal volume of antigen and then inject.
  • the immunization method is multi-point injection on the back.
  • the main immunization amount is 100 ⁇ l antigen/mouse
  • the booster injection is 100 ⁇ l antigen/mouse.
  • the immunization cycle is 6-8 weeks.
  • Antiserum detection The virus is coated with an enzyme-labeled plate, and the antiserum titer is detected by the indirect ELISA method. Serum titer is greater than 1:50K before proceeding to the next step of fusion. If there are multiple mice whose titer exceeds 1:50K, select the mouse with the highest titer for fusion.
  • Myeloma cell preparation One week before fusion, resuscitate SP2/0 cells and culture them to logarithmic phase normally.
  • Spleen cell preparation select the mice to be fused, sacrifice them by cervical dislocation on the day of fusion, take the spleen, collect and count the spleen cells in the standard procedure.
  • Cell fusion mix myeloma cells and spleen cells in a ratio of 1:3-1:10, perform cell fusion operations in standard procedures, and then culture them in HAT DMEM complete medium. Hybridoma cells can be seen 3 days after fusion. Change the 1/2HAT complete medium on the 7th day, and change the 1/2HT medium on the 8th day. The screening test began about 10 days after the fusion.
  • Fusion screening draw 100 ⁇ l/well of cell supernatant for indirect ELISA detection. According to the ELISA results, the positive wells are judged. Use a single-channel pipette to pick the positive wells detected in the entire plate, and perform a second re-examination to further confirm the positive wells.
  • Subcloning Perform two rounds of subcloning on the re-screened positive well cells. (Because the positive cell line obtained from the first subcloning is not stable and may contain multiple hybridoma cells, it is generally believed that the hybridoma cells are a single cell line after the second subcloning and are determined to be positive).
  • Virus neutralization experiment inoculate cells with antibody-containing culture supernatant (culture supernatant prepared after the first subcloning, all positive for antigen ELISA) and a certain concentration of antibody, and plaques are reduced or not occurred The hole is the neutralizing antibody positive hole.
  • the above-mentioned positive cells were expanded and cultured and injected into the abdominal cavity of Balb/C mice (sensitized by Freund's incomplete adjuvant). Generally, the bulge of the mouse abdomen was seen in 7-10 days, which represented the production of ascites. When the mouse has obvious ascites, draw the ascites in time.
  • the ascites of the above cells was purified with Protein A/G, and the purity of the antibody after purification was greater than 90%. After checking the purity of the concentration, adjust the concentration to 2mg/ml.
  • amino acid sequence and nucleotide sequence of the heavy chain variable region are shown in SEQ ID NO: 4 and SEQ ID NO: 12.
  • amino acid sequence and nucleotide sequence of the light chain variable region are shown in SEQ ID NO: 8 and SEQ ID NO: 16.
  • the SPR experiment was performed using BIAcore T100 (Biacore, GE Healthcare), and the buffer was PBS containing 0.05% Tween-20.
  • the NHS/EDC method to fix the purified E30 whole virus particles on the surface of the CM5 sensor chip until the RU value reaches 740.
  • gradient concentrations of IgG or Fab flow through the chip at a rate of 20 ⁇ l/min, and use 10 mM glycine-hydrochloric acid (pH 1.7) to regenerate the chip after each injection cycle.
  • the binding affinity was obtained by using the software BIAevaluation (version 4.1) to fit the curve globally.
  • the competitive SPR procedure is similar to the above-mentioned SPR, and the E30 virus particles are immobilized on the CM5 chip until the RU value reaches 740.
  • the receptor or antibody is added to the first stitch until the fitting curve is saturated, and then the competing antibody or receptor to be tested is added to the second stitch.
  • the binding competition relationship between the two substances before and after the E30 virus particles on the chip is reflected by the change trend of the fitted curve.
  • the ELISA experiment was used for the determination.
  • the purified virus particles (E30, E6, E3, E11 or CVB3) were coated on an ELISA plate (Costar, Corning, USA) at 30 ng/well, and then incubated overnight at 4°C.
  • the coated plate was blocked with 1% BSA-containing PBST solution (PBS plus 0.1% Tween20) at 37°C for 2 hours. After that, the plate was washed five times with PBST, and 4B10-IgG or 6C5-IgG was added as the primary antibody to each well at a dilution of 1:1000 at 37°C for 1 hour.
  • the plate was washed again with PBST five times, and HRP-conjugated goat anti-mouse IgG H&L (1:3000 dilution) (Sigma-Aldrich, St. Louis, USA) was added as a secondary antibody. Place at 37°C for 0.5 hour.
  • the plate was washed five times with PBST, and 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Biyuntian, Shanghai, China) was added to each well for 5 minutes at room temperature. Finally, 2M H 2 SO 4 was added to the plate to stop the reaction, and the absorbance value of each well at 450 nm was read.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • Digest the cells cultured overnight in a 12-well plate into an EP tube, wash 3 times, remove the supernatant, add virus solution (MOI 1) to resuspend the cells, incubate on ice for 30 min, wash 5 times according to the above steps, Remove the supernatant, add antibody solution (final concentration of 0 nM, 3 nM, 30 nM, 300 nM) to resuspend the cells, incubate on ice for 30 min, wash 5 times according to the above steps, remove the supernatant, and finally add 200 ⁇ l of lysis buffer for lysis.
  • antibody solution final concentration of 0 nM, 3 nM, 30 nM, 300 nM
  • QPCR step quantify E30 remaining on the surface of RD cells after 4B10/6C5 treatment by QPCR.
  • E30 is mixed with various concentrations of 6C5 at 4°C.
  • the cells were washed 3 times, and total RNA was extracted by RNeasy mini kit (Qiagen, Hilden, Germany), and SuperScrip III Platinum SYBR Green One was used on QuantStudio Dx Real-Time PCR Instrument (Applied Biosystems, Foster City, USA) -Step qRT-PCR Kit (Invitrogen, Carlsbad, USA) for QPCR.
  • 20 ⁇ L reaction system contains 0.2 ⁇ L SuperScript III RT/Platinum Taq Mix, 5 ⁇ L 2X SYBR Green Reaction Mix, 10 ⁇ m forward primer (5'-AACAGCAGCGTTGCCCGCGTCTA-3') and reverse primer (5'-ACCCTGTAGTTCCCTACATA-3') 0.2 ⁇ L each , 2 ⁇ L total RNA, 2.4 ⁇ L RNase-free H 2 O.
  • the QPCR amplification program is: 42°C, 5 minutes for reverse transcription, 95°C, 5 minutes for reverse transcription inactivation. Then it was denatured at 95°C for 15s, a total of 40 cycles, annealed at 60°C and extended for 30s.
  • the endogenous housekeeping gene ⁇ -actin (forward primer: 5'-GCCCTGAGGCACTCTTCCA-3', reverse primer: 5'-CGGATGTCCACGTCACACTT-3') was used as an internal control to normalize the sample. Different samples were analyzed relative levels E30RNA by 2 - ⁇ Ct method.
  • Antibodies (4B10 and 6C5) can effectively inhibit the activity of the virus before the virus contacts the cell ( Figure 8A) or after the contact ( Figure 8B), indicating that the antibody can competitively inhibit the recognition of the virus and the virus receptor
  • the virus that binds and has bound the receptor can also be competed by antibodies (4B10 and 6C5).
  • the cultured E30 virus was inactivated with formaldehyde, concentrated, and sucrose density gradient centrifugation. Hollow and solid virus particles with higher purity were obtained in different sucrose gradient layers; the hollow and solid particles were injected into the immunized mice according to the steps in Example 1. , And then take blood to get antiserum.
  • the purified E30 whole particles are first coated on the Elisa plate, and then the plate is blocked according to the above Elisa method. Then add serum against E30 solid particles adjuvanted with aluminum hydroxide (anti-F-par sera), serum against virus hollow particles adjuvanted with aluminum hydroxide (anti-E-par sera) or aluminum hydroxide adjuvant Serum (anti-adj sera). After incubation at 37°C for 0.5 hour, all wells were washed five times, and HRP-conjugated 4B10/6C5-IgG was added for further incubation at 37°C for 0.5 hour. The wells were then washed, TMB substrate, 2M H 2 SO 4 was added , and read at A450 as described above. The calculation formula of the inhibition percentage is (OD negative control-OD serum)/OD negative control ⁇ 100%.
  • the inactivated E30 solid particle and hollow particle antiserum can effectively neutralize the virus E30's infection of cells, with titers reaching 1:12 and 1:11 respectively (Figure 9), and the combination of the two and the virus can block off About 60-80% of 4B10 and 6C5 epitopes. It shows that the epitopes recognized by 4B10 and 6C5 are immunodominant epitopes ( Figure 10).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Food Science & Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)

Abstract

提供一种抗Echo30的抗体，以及相应的核酸，载体，宿主细胞，组合物，该抗体的制备方法、检测方法和治疗用途。

Description

抗Echo30的抗体，相应的核酸，载体，宿主细胞，组合物 技术领域
本申请属于生物医学领域，具体涉及抗Echo30的抗体，相应的核酸，载体，宿主细胞，组合物。
背景技术
埃可病毒(Echovirus)属于肠道病毒(Enteroviruses，EVs)。肠道病毒由微小核糖核酸病毒科中的67种独特的血清型的病毒组成。小核糖核酸病毒是正链RNA病毒，包括脊髓灰质炎病毒、柯萨奇病毒A组和B组、埃可病毒、肠病毒68-71型。埃可病毒颗粒体积极小，呈球形，病毒颗粒由简单的衣壳和单正股RNA组成，裸露无膜，直径小于30nm。病毒的衣壳含4种蛋白质，分别为VP1、VP2、VP3、VP4。病毒的RNA为单一分子，长约7.44kb，是病毒翻译和复制的模板。有64种不同的血清型的肠道病毒可引起人类感染，其中已知埃可病毒有34个血清型。在临床上病毒的分型主要基于它们的基因特征，埃可病毒属于人肠道病毒HEV B种。与其它肠道病毒一样，埃可病毒的最适生长温度为35-37℃，能在猴肾、人羊膜细胞、人喉癌细胞、横纹肌肉瘤、人胚肺等细胞进行培养。
人类埃可病毒30(Echo30)是埃可病毒最常见的类型之一，是引起儿童和成人无菌性脑膜炎的主要病原体。在儿童病毒性脑炎病例中，由Echo30引起的病例占80％～93％。该病毒是一种无胞膜病毒，在高盐、高pH值等恶劣环境下均可稳定存活。肠道病毒排毒时间长，患者感染后的数周内仍能从呼吸道或肠道中排出病毒，从而引起病毒传播、感染甚至暴发或流行。近年来在我国台湾、浙江、福建和河南等地多次报道由Echo30引起病毒性脑炎的暴发或流行。
目前对Echo30相关研究比较少，更无特异性抗体的报道。本申请目的是制备针对Echo30的特异性抗体以便检测Echo30及其导致的相关疾病。
发明内容
本发明提供与Echo30特异性结合的抗Echo30的抗体或其抗原结合片段。例示性CDR的氨基酸序列以及例示性抗Echo30抗体的重链及轻链的VH区及VL区的氨基酸序列提供于下文具体实施方式中。
抗Echo30的抗体可包括更改抗体的特性的修饰和/或突变，这些特性诸如，延长半衰期、增加或减少ADCC等如本领域已知。
本文提供包含编码本发明的抗Echo30的抗体的核苷酸序列的核酸，以及包含核酸的载体。另外，本文提供用包含编码所披露的抗Echo30的抗体的核苷酸序列的载体转化的原核及真核宿主细胞，以及经工程化以表达核苷酸序列的真核(诸如，哺乳动物)宿主细胞。还提供通过培养宿主细胞及回收抗体来制造抗体的方法，且进一步在下文具体实施方式中论述。
本发明提供检测Echo30的产品，所述产品包括本发明的Echo30抗体或其抗原结合片段。本发明提供诊断Echo30感染或其导致的疾病的产品，所述产品包括本发明的Echo30抗体或其抗原结合片段。
在另一方面中，本发明提供包括本文所描述的抗Echo30的抗体的组合物。组合物通常包含一种或多种如本文所描述的抗Echo30的抗体和/或其盐、及一种或多种赋形剂、运载体或稀释剂。
本发明提供用抗Echo30的抗体治疗经诊断患有埃可病毒感染导致的疾病的方法。该方法通常涉及向受试者施用一定量的本文所描述的可有效地提供治疗益处的抗Echo30的抗体。通常以静脉内输注在介于约0.001mg/kg至约4mg/kg的范围内的剂量下来施用抗Echo30的抗体。通常以一周两次、一周一次、每两周一次、每三周一次、每四周一次、每五周一次、每六周一次、每七周一次或每八周一次静脉内输注来施用抗Echo30的抗体。
可施用抗Echo30的抗体作为单一治疗剂(单药疗法)或辅助通常(但非必需)用于治疗埃可病毒感染导致的疾病的那些的其他治疗剂，或与这些其他治疗剂一起施用。治疗剂通常应以其经批准的剂量、施用途径及施用频率来使用，但可以更低的剂量来使用。
抗Echo30的抗体可经由各种施用途径或模式来施用，这些施用途径或模式包括但不限于静脉内输注和/或注射、皮下注射。施用的量将取决于施用途径、 给药时间表、所治疗的疾病类型、所治疗的疾病所处的阶段、以及如本领域熟知的其他参数，例如患者的年龄和体重。
本发明提供用抗Echo30的抗体检测Echo30的方法。该方包括如下步骤：a)将样品与本发明的抗Echo30的抗体进行接触；b)检测抗体与Echo30的反应。
本发明提供用抗Echo30的抗体诊断Echo30感染的方法。该方法包括如下步骤：a)将样品与本发明的抗Echo30的抗体进行接触；b)检测抗体与Echo30的反应；c)或出现阳性反应，则判断该受试者已被Echo30感染或患有Echo30感染导致的疾病。
本发明提供用抗Echo30的抗体诊断Echo30感染导致的疾病的方法。a)将样品与本发明的抗体进行接触；b)检测所述的抗体或其抗原结合片段与Echo30的反应；c)或出现阳性反应，则判断该受试者已被Echo30感染或患有Echo30感染导致的疾病。
本发明提供抗Echo30抗体的检测、诊断、以及治疗用途。所述用途包括但不限于以下用途：在制备检测Echo30的产品中的应用；在制备诊断Echo30感染或其导致的疾病的产品中的应用；在制备治疗或预防Echo30感染的药物中的应用；在制备治疗或预防Echo30感染导致的疾病的药物中的应用。
本发明中所公开的埃可病毒30型感染导致的疾病包括但不限于脑膜炎，上呼吸道感染，心肌炎，皮疹，呕吐，发烧，头痛。
所述上呼吸道感染包括咳嗽、咽痛等流感样症状。
所述脑膜炎包括无菌性脑膜炎，无菌性脑膜炎又包括儿童无菌性脑膜炎。
基于本文提供的数据，预期本文所描述的抗Echo30的抗体将向诊断为埃可病毒30感染导致的疾病的受试者提供治疗益处。
附图说明
图1显示本发明抗体的SDS-PAGE图；
图2显示本发明抗体的亲和活性曲线，其中，A：4B10-IgG，B：4B10-Fab，C：6C5-IgG，D：6C5-Fab；
图3显示本发明病毒与受体结合的亲和力曲线，其中，A：FcRn，B：CD55；
图4显示本发明的抗体对病毒与其受体结合的抑制曲线，其中，A：先加入6C5 抗体后加入受体，B：先加入受体后加入6C5抗体；C：先加入4B10抗体后加入受体，D：先加入受体后加入4B10抗体；
图5显示本发明抗体的亲和活性专一性测定图；
图6显示本发明的抗体的中和活性统计图，其中，A：6C5，B：4B10；
图7显示本发明的抗体的中和活性专一性测定图；
图8显示病毒与细胞接触前或接触后抗体对病毒活性的影响结果图，其中，A：接触前，B：接触后；
图9显示灭活E30颗粒免疫小鼠后血清的效价统计图；
图10显示本发明的抗体与病毒结合对抗原表位的影响图；
图11显示本发明的抗体与病毒结合对病毒稳定性影响的结果图，其中：A：pH＝5.5的缓冲液，B：pH＝7.4的缓冲液，C：A导数值，D：B导数值。
具体实施方式
除非本文中另外定义，否则结合本发明使用的科学与技术术语应具有由本领域普通技术人员通常理解的含义。
抗Echo30的抗体及结合片段
在一个方面中，本发明是关于特异性结合人类Echo30的抗体和/或其抗原结合片段。
如本文所使用，术语“抗体”(Ab)是指与特定抗原(此处为Echo30)特异性结合的免疫球蛋白分子。本发明的抗Echo30的抗体包含轻链可变域及重链可变域两者中的互补决定区(CDR)，还称为高变区。可变域的更高度保守部分称为框架(FR)。如本领域中已知，描绘抗体的高变区的氨基酸位置/边界可取决于上下文及本领域中已知的各种定义而变化。可变域内的一些位置可视为杂交高变位置，原因在于这些位置可根据一组标准认为在高变区内而根据一组不同的标准认为在高变区外部。这些位置中的一个或多个还可在延伸的高变区中找到。本发明提供包含这些杂交高变位置中的修饰的抗体。天然重链及轻链的可变域各自包含由三个CDR连接的四个FR区(大部分通过采用β片层构形)，这三个CDR形成连接(且在一些情况下形成β片层结构的一部分)β片层结构的环。各链中的CDR 由FR区紧密结合在一起，且与来自另一链的CDR一起促使形成抗体的靶结合位点。参见Kabat等人，Sequences of Proteins of Immunological Interest[免疫学感兴趣的蛋白质的序列](美国国立卫生研究院，马里兰州贝塞斯达(National Institute of Health，Bethesda，Md.)1987)。如本文所使用，除非另外指明，否则免疫球蛋白氨基酸残基的编号是根据Kabat等人的免疫球蛋白氨基酸残基编号系统来进行。
本发明的抗体可为多克隆、单克隆、基因工程化和/或在本质上另外修饰的，包括但不限于鼠源抗体、嵌合抗体、人源化抗体、人类抗体、单链抗体等。在各种实施例中，抗体包含抗体的恒定区的全部或一部分。在一些实施例中，恒定区为选自以下各项的同种型：IgA(例如，IgA1或IgA2)、IgD、IgE、IgG(例如，IgG1、IgG2、IgG3或IgG4)及IgM。
如本文所使用，抗体的“恒定区”包括天然恒定区、同种异型或天然变体。
抗Echo30的抗体的轻链恒定区可为κ(kappa)轻链区或λ(lambda)区。λ轻链区可为已知亚型中的任一者，例如，λ1、λ2、λ3或λ4。在一些实施例中，抗Echo30的抗体包含κ(kappa)轻链区。
如本文所用的术语“单克隆抗体”不限于经由杂交瘤技术产生的抗体。单克隆抗体通过本领域中可用的或已知的任何方式自单一克隆获得，包括任何真核、原核或噬菌体克隆。用于本发明的单克隆抗体可使用本领域中已知的广泛多种技术来制备，包括杂交瘤技术、重组技术及噬菌体展示技术或其组合的使用。
如本文所使用，术语“嵌合”抗体是指具有源自非人类免疫球蛋白(诸如，大鼠或小鼠抗体)的可变序列及通常选自人类免疫球蛋白模板的人类免疫球蛋白恒定区的抗体。用于制造嵌合抗体的方法为本领域中已知的。参见例如，Morrison，1985，Science[科学]229(4719)：1202-7；Oi等人，1986，BioTechniques[生物技术]4：214-221；Gillies等人，1985，J.Immunol.Methods[免疫学方法期刊]125：191-202；美国专利号5,807,715；4,816,567；和4,816,397中。
非人类(例如，鼠类)抗体的“人源化”形式为含有源自非人类免疫球蛋白的极少序列的嵌合免疫球蛋白。通常，人源化抗体将包含实质上所有至少一个及典型地两个可变域，其中所有或实质上所有CDR区与非人类免疫球蛋白的那些区 相对应且所有或实质上所有FR区为人类免疫球蛋白序列的那些区。人源化抗体还可包含免疫球蛋白恒定区(Fc)的至少一部分，通常为人类免疫球蛋白共同序列的那部分。抗体人源化方法为本领域中已知。参见例如，Riechmann等人，1988，Nature[自然]332：323-7；授予Queen等人的美国专利号5,530,101、5,585,089、5,693,761、5,693,762、及6,180,370；EP239400；PCT公开WO 91/09967；美国专利号5,225,539；EP592106；EP519596；Padlan，1991，Mol.Immunol.[分子免疫学]，28：489-498；Studnicka等人，1994，Prot.Eng.[蛋白质工程]7：805-814；Roguska等人，1994，Proc.Natl.Acad.Sci.[美国国家科学院院刊]91：969-973；及美国专利号5,565,332。
“人类抗体”包括具有人类免疫球蛋白的氨基酸序列的抗体，且包括自人类免疫球蛋白库或自针对一种或多种人类免疫球蛋白转基因且并不表达内源性免疫球蛋白的动物分离的抗体。人类抗体可利用各种本领域中已知的方法(包括噬菌体展示方法)使用源自人类免疫球蛋白序列的抗体库来制备。参见美国专利号4,444,887及4,716,111；PCT公开WO98/46645；WO 98/50433；WO 98/24893；WO 98/16654；WO 96/34096；WO 96/33735；和WO 91/10741。人类抗体还可使用不能表达功能内源性免疫球蛋白但可表达人类免疫球蛋白基因的转基因小鼠来制造。参见例如，PCT公开WO 98/24893；WO 92/01047；WO 96/34096；WO 96/33735；美国专利号5,413,923；5,625,126；5,633,425；5,569,825；5,661,016；5,545,806；5,814,318；5,885,793；5,916,771；和5,939,598中。另外，诸如LakePharma，Inc.(Belmont，CA)或Creative BioLabs(Shirley，NY)的公司可参与以使用类似于上文所描述的技术提供针对所选择的抗原的人类抗体。识别所选择表位的全人类抗体可使用被称作“定向选择”的技术来产生。在此方法中，所选择非人类单克隆抗体(例如，小鼠抗体)用于引导识别相同表位的完全人类抗体的选择(参见Jespers等人，1988，Biotechnology[生物技术]12：899-903)。
本发明的还披露能够特异性地结合Echo30的抗体的抗原结合片段。抗原结合片段的实例包括(借助于实例且非限制的)Fab、Fab’、F(ab’)2、Fv片段、单链Fv片段及单一域片段。
Fab片段含有轻链的恒定域及可变域以及重链的第一恒定域(CH1)及可变域。Fab’片段与Fab片段的不同之处在于：在重链CH1域的羧基端处的几个残基的 添加，包括来自抗体铰链区的一个或多个半胱氨酸。F(ab’)片段是通过裂解在F(ab’)2胃蛋白酶消化产物的铰链半胱氨酸处的双硫键来制造。抗体片段的其他化学偶联法为本领域普通技术人员已知。Fab及F(ab’)2片段缺乏完整抗体的Fc片段，从动物的循环更快速清除，且具有比完整抗体更低的非特异性组织结合性(参见例如，Wahl等人，1983，J.Nucl.Med.[核医学杂志]24：316)。
“Fv”片段为含有完全靶识别及结合位点的抗体的最小片段。此区由紧密非共价缔合的一个重链可变域及一个轻链可变域的二聚体(VH-VL二聚体)构成。在此构型中，各可变域的三个CDR相互作用以限定VH-VL二聚体的表面上的靶结合位点。通常，六个CDR赋予抗体以靶结合特异性。然而，在一些情况下，即使单一可变域(或仅包含对标靶具有特异性的三个CDR的Fv的一半)可具有识别及结合标靶的能力，但其比完整结合位点具有更低的亲和力。
“单链Fv”或“scFv”抗体结合片段包含抗体的VH域及VL域，其中这些域存在于单一多肽链中。通常，Fv多肽进一步包含在VH域与VL域之间的多肽连接体，该多肽连接体使得scFv能够形成用于靶结合的所需结构。
“单一域片段”是由对Echo30具有足够亲和力的单一VH域或VL域构成。在一具体实施例中，单一域片段为驼峰化片段(参见例如，Riechmann，1999，Journal of Immunological Methods[免疫学方法杂志]231：25-38)。
本发明的抗Echo30的抗体包括衍生抗体。例如，但并非限制性的，衍生抗体通常通过糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、利用已知保护/阻断基团衍化、蛋白质分解性裂解、连接至细胞配体或其他蛋白质来修饰。可利用已知技术来进行诸多化学修饰中的任一者，这些技术包括但不限于特异性化学裂解、乙酰化、甲酰化、衣霉素(tunicamycin)的代谢合成等。另外，衍生物例如使用Ambryx技术(参见例如，Wolfson，2006，Chem.Biol[化学生物学].13(10)：1011-2)可含有一个或多个非天然氨基酸。
抗Echo30的抗体或结合片段可为其序列已经修饰以更改至少一个恒定区介导的生物效应功能的抗体或片段。例如，在一些实施例中，抗Echo30抗体可经修饰以相对于未经修饰的抗体减少至少一个恒定区介导的生物效应功能，例如，减少与Fc受体(FcγR)(诸如，FcγRI、FcγRIIA、FcγRIIB、FcγRIIIA和/或FcγRIIIB)中的一个或多个的结合。FcγR结合可通过使抗体在对于FcγR相互 作用必需的特定区处的免疫球蛋白恒定区区段突变来减少(参见例如，Canfield及Morrison，1991，J.Exp.Med.[实验医学杂志]173：1483-1491；及Lund等人，1991，J.Immunol[免疫学杂志].147：2657-2662)。抗体的FcγR结合能力的减少还可减少依赖于FcγR相互作用的其他效应功能，诸如，调理作用(opsonization)、吞噬及抗原依赖性细胞毒性(“ADCC”)。
本文所描述的抗Echo30的抗体或结合片段包括已经修饰以相对于未经修饰的抗体获得或改良至少一个恒定区介导的生物效应功能例如以增强FcγR相互作用的抗体(参见例如，美国专利申请号2006/0134709)。例如，本发明的抗Echo30的抗体可具有以比对应野生型恒定区更大亲和力结合FcγRI、FcγRIIA、FcγRIIB、FcγRIIIA和/或FcγRIIIB的恒定区。
在一些实施例中，本发明的抗Echo30的抗体具有较低水平的海藻糖或缺乏海藻糖。缺乏海藻糖的抗体已与增强的ADCC活性相关，尤其在较低剂量的抗体下。参见Shields等人，2002，J.Biol.Chem.[生物化学杂志]277：26733-26740；Shinkawa等人，2003，J.Biol.Chem.[生物化学杂志]278：3466-73。制备较低海藻糖抗体的方法包括在鼠骨髓瘤YB2/0细胞(ATCC CRL 1662)中生长。YB2/0细胞表达较低水平的编码α-1，6-海藻糖基转移酶(一种对于多肽的海藻糖基化必需的酶)的FUT8mRNA。
具有针对Echo30的亲和力的抗Echo30的抗体可能为治疗性及诊断用途所需的。因此，本发明涵盖具有针对Echo30的结合亲和力的抗体。在具体实施例中，结合Echo30的抗Echo30的抗体具有至少约1000nM的亲和力但可呈现更高亲和力，例如，至少约900nM、800nM、700nM、600nM、500nM、400nM、300nM、250nM、200nM、150nM、100nM、90nM、80nM、70nM、60nM、50nM、40nM、30nM、25nM、20nM、15nM、10nM、7nM、6nM、5nM、4nM、3nM、2nM、1nM、0.1nM、0.01nM或甚至更高。
抗Echo30的抗体针对Echo30的的亲和力可使用本领域中熟知或本文所描述的技术来测定，诸如但非限制性的，ELISA、等温滴定热量测定(ITC)、表面等离子共振或荧光偏振测定。
抗Echo30的抗体通常包含重链及轻链，该重链包含具有在本文中被称为(以N→C次序)VH CDR1、VH CDR2及VH CDR3的三个互补决定区(“CDR”)的 可变区(VH)，该轻链包含具有在本文中被称为(以N→C次序)VL CDR1、VL CDR2及VL CDR3的三个互补决定区的可变区(VL)。本文提供例示性CDR的氨基酸序列以及例示性抗Echo30的重链及轻链的VH区及VL区的氨基酸序列。抗Echo30抗体的具体实施例包括这些例示性CDR和/或VH和/或VL序列。
在一些实施例中，本发明的抗Echo30的抗体，其包含至少一个CDR的VH，和/或，至少一个CDR的VL；
VH的CDR序列选自：SEQ ID NO：1-3所示的氨基酸序列；
或
VH的CDR序列选自：在SEQ ID NO：1-3所示的氨基酸序列基础上经取代、缺失或添加一个或多个氨基酸形成的氨基酸序列；
或
VH的CDR序列选自：与SEQ ID NO：1-3所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；
VL的CDR序列选自：SEQ ID NO：5-7所示的氨基酸序列；
或
VL的CDR序列选自：在SEQ ID NO：5-7所示的氨基酸序列基础上经取代、缺失或添加一个或多个氨基酸形成的氨基酸序列；
或
VL的CDR序列选自：与SEQ ID NO：5-7所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列。
至少有80％同源性是指有80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源性。
在本发明的具体实施方案中，VH的CDR序列选自：SEQ ID NO：1-3所示的氨基酸序列；VL的CDR序列选自：SEQ ID NO：5-7所示的氨基酸序列。
更具体第，VH的CDR1序列具有SEQ ID NO：1所示的氨基酸序列；VH的CDR2序列具有SEQ ID NO：2所示的氨基酸序列；VH的CDR3序列具有SEQ  ID NO：3所示的氨基酸序列；VL的CDR1序列具有SEQ ID NO：5所示的氨基酸序列；VL的CDR2序列具有SEQ ID NO：6所示的氨基酸序列；VL的CDR3序列具有SEQ ID NO：7所示的氨基酸序列。
进一步，本发明的所述抗体包括VH和/或VL，其中：
所述VH具有SEQ ID NO：4所示的氨基酸序列，或所述VH具有在SEQ ID NO：4所示的氨基酸序列基础上经取代、缺失或添加一个或多个氨基酸形成的氨基酸序列；或所述VH具有与SEQ ID NO：4所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；
所述VL具有SEQ ID NO：8所示的氨基酸序列，或所述VL具有在SEQ ID NO：8所示的氨基酸序列基础上经取代、缺失或添加一个或多个氨基酸形成的氨基酸序列；或所述VL具有与SEQ ID NO：8所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列。
至少有80％同源性是指有80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源性。
在本发明的具体实施方案中，所述VH具有SEQ ID NO：4所示的氨基酸序列；所述VL具有SEQ ID NO：8所示的氨基酸序列。
编码抗Echo30的抗体的聚核苷酸、制备抗体的表达系统及方法
本发明涵盖编码抗Echo30的免疫球蛋白轻链基因及重链基因的核酸分子、包含此类核酸的载体及能够制造本发明的抗Echo30的抗体的宿主细胞。
在本发明的具体实施方案中，编码重链可变区CDR1的核酸具有SEQ ID NO：9所示的序列；编码重链可变区CDR2的核酸具有SEQ ID NO：10所示的序列；编码重链可变区CDR3的核酸具有SEQ ID NO：11所示的序列；编码轻链可变区CDR1的核酸具有SEQ ID NO：13所示的序列；编码轻链可变区CDR2的核酸具有SEQ ID NO：14所示的序列；编码轻链可变区CDR3的核酸具有SEQ ID NO：15所示的序列。
在本发明的具体实施方案中，编码重链可变区的核酸具有SEQ ID NO：12所示的序列；编码轻链可变区的核酸具有SEQ ID NO：16所示的序列。
本发明的抗Echo30的抗体可通过在宿主细胞中重组表达免疫球蛋白轻链基因及重链基因来制备。为以重组方式表达抗体，用携带编码抗体的免疫球蛋白轻链及重链的DNA片段的一个或多个重组表达载体来转染宿主细胞，使得在宿主细胞中表达轻链及重链且任选地分泌至培养宿主细胞的培养基中，可从该培养基回收抗体。标准重组DNA方法用于获得抗体重链及轻链基因，将这些基因并入至重组表达载体中且将载体引入至宿主细胞中，这些宿主细胞诸如描述于Molecular Cloning；A Laboratory Manual[分子克隆：实验室手册]，第二版(Sambrook，Fritsch及Maniatis(编)，冷泉港实验室(Cold Spring Harbor)，N.Y.，1989)，Current Protocols in Molecular Biology[当前分子生物学方案](Ausubel，F.M.等人，编，格林出版联合公司(Greene Publishing Associates)，1989)中及美国专利号4,816,397中的那些。
为产生编码此类抗Echo30的抗体的核酸，首先获得编码轻链可变区及重链可变区的DNA片段。这些DNA可通过扩增及修饰编码轻链可变序列及重链可变序列的种系DNA或cDNA来获得，例如使用聚合酶链反应(PCR)。人类重链及轻链可变区基因的种系DNA序列为本领域中已知(参见例如，“VBASE”人类种系序列数据库；还参见Kabat，E.A.等人，1991，Sequences of Proteins of Immunological Interest[免疫学重要性蛋白质序列]，第五版，U.S.Department of Health and Human Services[美国健康与公共事业部]，NIH公开号91-3242；Tomlinson等人，1992，J.Mol.Biol.[分子生物学杂志]22T：116-198；及Cox等人，1994，Eur.J.Immunol.[欧洲免疫学杂志]24：827-836；其各自的内容通过引用并入本文中)。
一旦获得编码抗Echo30抗体相关的VH区段及VL区段的DNA片段，则可进一步利用标准重组DNA技术操纵这些DNA片段例如以将可变区基因转化成全长抗体链基因、Fab片段基因或scFv基因。在这些操纵中，编码VL或VH的DNA片段可操作地连接至编码另一蛋白质(诸如抗体恒定区或挠性连接体)的另一DNA片段。如用于此情况下，术语“可操作地连接”意指两个DNA片段接合使得由两个DNA片段编码的氨基酸序列保留于框内。
编码VH区的经分离DNA可通过将编码VH的DNA可操作地连接至编码重链恒定区(CH1、CH2、CH3及任选地CH4)的另一DNA分子来转化成全长重 链基因。人类重链恒定区基因的序列为本领域中已知(参见例如，Kabat，E.A.，等人，1991，Sequences of Proteins of Immunological Interest[免疫学感兴趣的蛋白质的序列]，第五版，U.S.Department of Health and Human Services[美国健康与公共事业部]，NIH公开号91-3242)且涵盖这些区的DNA片段可通过标准PCR扩增来获得。重链恒定区可为IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区，但在某些实施例中为IgG1或IgG4。对于Fab片段重链基因，编码VH的DNA可以可操作地连接到仅编码重链CH1恒定区的另一DNA分子。
编码VL区的经分离DNA可通过将编码VL的DNA可操作地连接于编码轻链恒定区CL的另一DNA分子来转化成全长轻链基因(以及Fab轻链基因)。人类轻链恒定区基因的序列为本领域中已知(参见例如，Kabat等人，1991，Sequences of Proteins of Immunological Interest[免疫学感兴趣的蛋白质的序列]，第五版，U.S.Department of Health and Human Services[美国健康与公共事业部]，NIH公开号91-3242)且涵盖这些区的DNA片段可通过标准PCR扩增来获得。轻链恒定区可为κ或λ恒定区，但在某些实施例中为κ恒定区。为建立scFv基因，将编码VH及VL的DNA片段可操作地连接于编码挠性连接体(例如，编码氨基酸序列(Gly4～Ser)3(SEQ ID NO：200))的另一片段，使得VH及VL序列可被表达为邻接单链蛋白质，其中VL区及VH区由挠性连接体接合(参见例如，Bird等人，1988，Science[科学]242：423-426；Huston等人，1988，Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]85：5879-5883；McCafferty等人，1990，Nature[自然]348：552-554。
为表达本发明的抗Echo30的抗体，将如上文所描述获得的编码部分或全长轻链及重链的DNA插入至表达载体中，使得基因可操作地连接于转录及翻译控制序列。在此上下文中，术语“可操作地连接”意指，将抗体基因接合至载体中使得在载体内的转录及翻译控制序列提供调整抗体基因的转录及翻译的其预定功能。选择表达载体及表达控制序列以与所用表达宿主细胞相容。可将抗体轻链基因及抗体重链基因插入至独立载体中，或更通常将两个基因插入至同一表达载体中。
利用标准方法(例如，抗体基因片段及载体上的互补限制位点的接合，或在不存在限制位点时平头端接合)将抗体基因插入至表达载体中。在插入抗Echo30 抗体相关的轻链或重链序列之前，表达载体可能已经携带抗体恒定区序列。例如，将抗Echo30单克隆抗体相关的VH序列及VL序列转化成全长抗体基因的一种方法是分别地将其插入至已经编码重链恒定区及轻链恒定区的表达载体中，使得VH区段可操作地连接于在载体内的一个或多个CH区段，且VL区段可操作地连接于在载体内的CL区段。另外或可替代地，重组表达载体可编码促进抗体链自宿主细胞分泌的信号肽。抗体链基因可克隆至载体中，使得信号肽在框内连接于抗体链基因的氨基端。信号肽可为免疫球蛋白信号肽或异源信号肽(即，来自非免疫球蛋白蛋白质的信号肽)。
除抗体链基因以外，本发明的重组表达载体携带控制抗体链基因在宿主细胞中表达的调节序列。术语“调节序列”意在包括启动子、强化子及控制抗体链基因的转录或翻译的其他表达控制元件(例如，聚腺苷酸化信号)。此类调节序列，例如，描述于Goeddel，Gene Expression Technology：Methods in Enzymology[基因表达技术：酶学方法]185，学术出版社(Academic Press)，圣地亚哥(San Diego)，加利福尼亚州(CA)，1990中。本领域普通技术人员应了解，表达载体的设计(包括调节序列的选择)可能视诸如待转化的宿主细胞的选择、所需蛋白质的表达量等因素而定。用于哺乳动物宿主细胞表达的合适调节序列包括在哺乳动物细胞中引导较高蛋白质表达量的病毒元件，诸如，源自巨细胞病毒(CMV)(诸如，CMV启动子/强化子)、猴病毒40(SV40)(诸如，SV40启动子/强化子)、腺病毒(例如，腺病毒主要晚期启动子(AdMLP))及多瘤病毒的启动子和/或强化子。对于病毒调节元件及其序列的进一步描述，参见例如，Stinski的美国专利号5,168,062、Bell等人的美国专利号4,510,245及Schaffner等人的美国专利号4,968,615。
除抗体链基因及调节序列以外，本发明的重组表达载体可携带额外序列，这些额外序列诸如调节载体在宿主细胞中的复制的序列(例如，复制起点)及可选标记基因。可选标记基因有助于已引入载体的宿主细胞的选择(参见例如，均为Axel等人的美国专利号4,399,216、4,634,665及5,179,017)。例如，在已引入载体的宿主细胞上，可选择标记基因通常赋予对诸如G418、潮霉素或甲氨蝶呤的药物的抗性。合适可选标记基因包括二氢叶酸还原酶(DHFR)基因(适用于用甲氨蝶呤选择/扩增DHFR-宿主细胞)及neo基因(用于G418选择)。对于轻链及重链的表达，利用标准技术将编码重链及轻链的一个或多个表达载体转染至宿主细胞中。 术语“转染”的各种形式意在涵盖通常用于将外源性DNA引入至原核或真核宿主细胞中的广泛多种技术，例如，电穿孔、脂质体转染、磷酸钙沉淀、DEAE-聚葡萄糖转染及类似者。
有可能在原核宿主细胞或真核宿主细胞中表达本发明的抗体。在某些实施例中，抗体的表达是在最佳分泌正确地折叠及免疫主动抗体的真核细胞(例如，哺乳动物宿主细胞)中进行。用于表达本发明的重组抗体的例示性哺乳动物宿主细胞包括中国仓鼠卵巢(CHO细胞)(包括DHFR-CHO细胞，其描述于Urlaub及Chasin，1980，Proc.Natl.Acad.Sci.USA[美国国家科学院院刊]77：4216-4220中，与DHFR可选标记一起使用，例如，如Kaufman及Sharp，1982，Mol.Biol.[分子生物学]159：601-621中所描述)、NSO骨髓瘤细胞、COS细胞及SP2细胞。当将编码抗体基因的重组表达载体引入至哺乳动物宿主细胞中时，抗体是通过培养宿主细胞持续足以允许抗体在宿主细胞中表达或将抗体分泌至生长宿主细胞的培养基中的时间段来制造。可使用标准蛋白质纯化方法自培养基回收抗体。宿主细胞还可用于制造完整抗体的部分，诸如，Fab片段或scFv分子。应了解，以上程序上的变化形式是在本发明的范畴内。例如，可能需要用编码本发明的抗Echo30抗体的轻链或重链(但并非两者)的DNA转染宿主细胞。
重组DNA技术还可用于将编码对于与Echo30结合不必需的轻链及重链中的任一者或两者的DNA的一些或所有移除。自此类截断DNA分子表达的分子还由本发明的抗体涵盖。
对于本发明的抗Echo30的抗体的重组表达，可用本发明的两种表达载体共转染宿主细胞，第一载体编码源自多肽的重链且第二载体编码源自多肽的轻链。两种载体可含有相同可选标记，或其可各自含有独立可选标记。可替代地，可使用编码重链多肽及轻链多肽两者的单一载体。
一旦核酸编码抗Echo30的抗体的一个或多个部分，则可将其他更改或突变引入至编码序列中，例如以产生编码具有不同CDR序列的抗体、具有针对Fc受体亲和力减少的抗体或不同亚类的抗体的核酸。
在本发明的具体实施方案中，本发明涉及的抗Echo30抗体的核酸序列如SEQ NO ID：9-16所示。
本发明的抗Echo30的抗体还可利用化学合成法(例如，利用描述于Solid Pha se Peptide Synthesis[固相肽合成]，第2版，1984皮尔斯化学公司(The Pierce Chemical Co.)，罗克福德(Rockford)，伊利诺伊州(Ill)中的方法)来制造。变体抗体还可使用无细胞平台来产生(参见例如，Chu等人，Biochemia[生物化学]第2期，2001(罗氏分子生物公司(Roche Molecular Biologicals))及Murray等人，2013，Currem Opinion in Chemical Biology[化学生物学新见]，17：420-426。
一旦已利用重组表达制造本发明的抗Echo30的抗体，则其可通过本领域中已知的任何纯化免疫球蛋白分子的方法来纯化，例如，通过层析(例如，离子交换、亲和层析及尺寸分级柱层析)、离心、不同可溶性、或通过纯化蛋白质的任何其他标准技术。此外，可将本发明的抗Echo30的抗体融合至本文所描述或本领域中另外已知便于纯化的异源多肽序列。
一旦分离，则抗Echo30的抗体可(若需要)例如通过高效液相层析(参见例如，Fisher，Laboratory Techniques In Biochemistry AndMolecular Biology[生物化学和分子生物学实验室技术]，Work及Burdon，编，爱思唯尔出版公司(Else vier)，1980)或通过SuperdexTM75管柱凝胶过滤层析(法玛西亚生物技术公司(Pharmacia Biotech AB)，乌普萨拉(Uppsala)，瑞典(Pharmacia Biotech AB，Uppsala，Sweden))来进一步纯化。
药物组合物
本文所描述的抗Echo30的抗体可呈组合物形式，这些组合物包含抗体及一种或多种运载体、赋形剂和/或稀释剂。可以将组合物配制用于特定用途，例如用于兽医用途或人类的药物用途。组合物的形式(例如干粉、液体配制品等)和使用的赋形剂、稀释剂和/或运载体将取决于ADC的预期用途，以及针对治疗用途的给药方式。
对于治疗性用途，组合物可作为包括药学上可接受的运载体的无菌、药物组合物的一部分来提供。此组合物可呈任何合适的形式(视向例如人类受试者的受试者(即，患者)施用其的所需方法而定)。药物组合物可利用各种途径来向受试者施用，这些途径诸如，经口、经皮、皮下、鼻内、经静脉内、肌内、瘤内、鞘内、表面或局部。在任何给定情况下的最合适施用途径将视特定抗体、受试者、及疾病的性质与严重程度以及受试者的生理条件而定。通常，药物组合物应经静脉内 或皮下施用。
药物组合物可宜以每剂量含有预定量的本文所描述的抗Echo30的抗体的单位剂量形式存在。包括于单位剂量中的抗Echo30的抗体的数量将视所治疗的疾病以及如本领域中所熟知的其他因素而定。此类单位剂量可呈含有适合于单次施用的一定量的抗体的冻干干粉形式，或呈液体形式。干燥粉单位剂型可以与注射器、适量的稀释剂和/或其他可用于给予的组分包装在试剂盒中。呈液态形式的单位剂量可宜呈预填有适合于单次施用的一定量的抗Echo30的抗体的注射器形式来提供。
药物组合物还可呈含有适合于多次施用的一定量的抗Echo30的抗体的块体形式来提供。
可通过使具有所需纯度的抗体与任选地选用的本领域中通常采用的药学上可接受的运载体、赋形剂或稳定剂(以上所有在均本文中均被称为“运载体”)，即，缓冲剂、稳定剂、防腐剂、离子等张剂、非离子清洁剂、抗氧化剂及其他混杂添加剂混合来制备用于储存为冻干配制品或水溶液的药物组合物。参见，Remington’s Pharmaceutical Sciences[雷明顿药物科学]，第16版(Osol编辑，1980)。这样的添加剂在所用的剂量和浓度下应对接受者无毒。
缓冲剂有助于将pH保持在接近生理条件的范围内。其可在广泛多种浓度下存在，但应通常以介于约2mM至约50mM范围内的浓度存在。适用于与本披露一起使用的缓冲剂包括有机酸和无机酸两者及其盐，如柠檬酸盐缓冲液(例如，柠檬酸一钠-柠檬酸二钠混合物、柠檬酸-柠檬酸三钠混合物、柠檬酸-柠檬酸一钠混合物等)、琥珀酸盐缓冲液(例如，琥珀酸-琥珀酸一钠混合物、琥珀酸-氢氧化钠混合物、琥珀酸-琥珀酸二钠混合物等)、酒石酸盐缓冲液(例如，酒石酸-酒石酸钠混合物、酒石酸-酒石酸钾混合物、酒石酸-氢氧化钠混合物等)、富马酸盐缓冲液(例如，富马酸-富马酸一钠混合物、富马酸-富马酸二钠混合物、富马酸一钠-富马酸二钠混合物等)、葡糖酸盐缓冲液(例如，葡糖酸-葡糖酸钠混合物、葡糖酸-氢氧化钠混合物、葡糖酸-葡糖酸钾混合物等)、草酸盐缓冲液(例如，草酸-草酸钠混合物、草酸-氢氧化钠混合物、草酸-草酸钾混合物等)、乳酸盐缓冲液(例如，乳酸-乳酸钠混合物、乳酸-氢氧化钾混合物、乳酸-乳酸钾混合物等)以及乙酸盐缓冲液(例如，乙酸-乙酸钠混合物、乙酸-氢氧化钠混合物等)。另外，可使 用富马酸盐缓冲液、组氨酸缓冲液及三甲胺盐，诸如，2-氨基-2-羟甲基-丙-1，3-二醇(即，Tris、THAM或三(羟甲基)氨基甲烷)。
可添加有时被称为“稳定剂”的等张剂以确保本发明的液体组合物的等张性，且这些等张剂包括多羟基糖醇、例如三元醇或更高糖醇，诸如，丙三醇、赤藻糖醇、阿拉伯糖醇、木糖醇、山梨醇及甘露醇。稳定剂是指广泛类型的赋形剂，其就功能而言范围从膨胀剂到溶解治疗剂或者有助于防止变性或粘附到容器壁的添加剂。典型的稳定剂可以是多羟基糖醇(上面列举的)；氨基酸(如精氨酸、赖氨酸、甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、丙氨酸、鸟氨酸、L-亮氨酸、2-苯丙氨酸、谷氨酸、苏氨酸等)、有机糖或糖醇(如乳糖、海藻糖、水苏糖、甘露醇、山梨糖醇、木糖醇、核糖醇、肌醇、半乳糖醇)、甘油等，包括环多醇(如肌醇)；聚乙二醇；氨基酸聚合物；含硫还原剂，诸如，尿素、谷胱甘肽、硫辛酸、硫乙醇酸钠、硫代甘油、α-单硫代甘油及硫代硫酸钠；低分子量多肽(例如，具有10个残基或更少残基的肽)；亲水性聚合物，诸如，聚乙烯吡咯啶酮单糖，诸如木糖、甘露糖、果糖、葡萄糖；双糖，诸如，乳糖、麦芽糖、蔗糖及海藻糖；及三糖，诸如，棉子糖；以及多糖如右旋糖酐。稳定剂可以介于每重量的抗Echo30抗体0.5重量％至10重量％范围内的量存在。
可添加非离子表面活性剂或清洁剂(还称为“润湿剂”)以帮助溶解糖蛋白以及保护糖蛋白免于搅拌诱发的聚集，其还准许配制品暴露于剪切应力表面而不导致蛋白质的变性。合适非离子表面活性剂包括聚山梨醇酯(20、80等)、泊洛沙姆(poloxamer)(184、188等)及普洛尼克(pluronic)多元醇。非离子表面活性剂可以约0.05mg/mL至约1.0mg/mL的范围存在。
适合于经由静脉内输注施用的水性组合物的具体例示性实施例包含10mg/mL的抗Echo30的抗体，15mM组氨酸缓冲液，pH 6.0，8.0％(w/v)蔗糖及0.05％(w/v)聚山梨醇酯80。组合物可呈冻干粉末形式，该冻干粉末在用2.0mL无菌水或其他适合于注射或输注的溶液(例如，0.9％盐水、林格氏溶液(Ringer’s solution)、乳酸化林格氏溶液等)复水后，提供上文水性组合物。组合物或其他实施例的组合物还可呈预填有一定量的适合于单次施用抗Echo30的抗体的组合物的注射器或其他适合于注射和/或输注的器件形式。
本发明的抗Echo30的抗体可单独施用(单药疗法)或辅助其他疗法，或与其 他可以治疗Echo30感染导致的疾病的药物一起施用。无论是作为单药疗法施用或辅助其他疗法或药物，抑或与其他疗法或药物一起施用，施用一定量的抗Echo30的抗体。
辅助疗法
抗Echo30的抗体可用于辅助或并与具有其他药物一起使用。当辅助使用时，抗Echo30的抗体及一种或多种其他药物可共同配制成单一的组合药物配制品，或可在单一协同给药方案上或在不同给药方案上分开配制及施用。辅助或与抗Echo30的抗体一起施用的药物通常具有针对抗Echo30的抗体的互补活性，使得抗体与其他药物彼此没有不利影响。使得整体治疗方案提供治疗益处。
剂量及施用方案
所施用抗Echo30的抗体的量将视各种因素而定，这些因素包括但不限于：所治疗实体瘤的特定类型、所治疗实体瘤的阶段、施用模式、施用频率、所需治疗益处及其他参数(诸如，患者的年龄、体重及其他特征等)。针对特定模式及施用频率的有效提供治疗益处的剂量的测定是在本领域普通技术人员的能力内。
有效提供治疗益处的剂量可首先自活体内动物模型或临床来估计。用于广泛多种疾病的合适动物模型为本领域中已知。
本文披露的抗Echo30的抗体可利用适合于待治疗的病状的任何途径来施用。抗Echo30的抗体通常应非经肠施用，即，输注、皮下、肌内、静脉内(IV)、皮内、鞘内、推注、或硬膜外施用((Shire等人，2004，J.Pharm.Sciences[药物科学杂志]93(6)：1390-1402))。在一个实施例中，抗Echo30抗体作为小瓶中的冻干粉末提供。小瓶可含有21mg的抗Echo30抗体。在施用之前，用注射用无菌水(SWFI)或其他合适介质复水冻干粉末以得到含有10mg/mL抗Echo30抗体的溶液。在一些实施例中，所得复水溶液进一步用盐水或其他合适介质稀释，且经由每7天两次、每7天一次、每14天一次、每21天一次、每28天一次、每35天一次、每42天一次、每49天一次或每56天一次IV输注来施用。在一些实施例中，对于第一循环，输注历经90分钟进行。在一些实施例中，后续输注历经60分钟。在其他实施例中，所得复水溶液进一步用盐水或其他合适介质稀释，且经由每7天两次、每7天一次、每14天一次、每21天一次、每28天一次、每35天一次、 每42天一次、每49天一次或每56天一次IT注射来施用。
当辅助其他药物施用或与这些一种或多种其他药物一起施用时，抗Echo30的抗体可能以与其他药物相同的时间表或以不同时间表施用。当以相同时间表施用时，抗Echo30的抗体可在其他药物之前、之后施用，或与这些其他药物一起同时施用。
检测方法/诊断方法
一方面，本发明提供了检测样品中Echo30的方法，包括以下几个步骤：(1)将本发明的单克隆抗体或其抗原结合片段与上述样品中的病毒结合而成抗体病毒或抗体片段病毒复合物；(2)检测该样品复合物以确定样品中是否有病毒。
另一方面，本发明提供了一种检测样品中Echo30的方法，包括以下几个步骤：(1)将第一抗体吸附到固相支持物上；(2)在上述支持物中加入可能含有Echo30的可疑待测样品；(3)在上述支持物中加入带有标记物的第二抗体；(4)检测该标记物的存在从而判定Echo30是否存在。
一方面，本发明提供了诊断Echo30感染或其导致的疾病的方法，包括以下几个步骤：(1)将本发明的单克隆抗体或其抗原结合片段与样品中的病毒结合而成抗体病毒或抗体片段病毒复合物；(2)检测该样品复合物以确定样品中是否有病毒；(3)若有病毒，则判断该受试者感染了Echo30，或确诊疑似患者患有Echo30感染导致的疾病。
另一方面，本发明提供了一种诊断Echo30感染或其导致的疾病的方法，包括以下几个步骤：(1)将第一抗体吸附到固相支持物上；(2)在上述支持物中加入可能含有Echo30的可疑待测样品；(3)在上述支持物中加入带有标记物的第二抗体；(4)检测该标记物的存在从而判定Echo30是否存在；(5)若有病毒，则判断该受试者感染了Echo30，或确诊疑似患者患有Echo30感染导致的疾病。所述第一抗体或第二抗体都是本发明的所述的单克隆抗体或其抗原结合片段。
检测方法可以使用酶联免疫吸附(ELISA)、酶免疫检测、化学发光免疫检测、放射免疫检测、荧光免疫检测、免疫色谱法、竞争法及类似检测方法。利用竞争法或夹心法方式，上述检测方法可以用于检测目标抗原或抗体。
竞争法是比较样品中抗原和一种已知量的标记抗原竞争结合本发明所述单 克隆抗体的数量关系。开展基于竞争法的免疫学检测是将含有未知数量的目标抗原的样品加入到事先用已知的物理或化学方法把本发明所述单抗包被到固相支持物上而得以进行的。同时加入预先定量的标记后的目标抗原进行反应。孵育后，冲洗固相支持物，检测结合到该支持物上的标记物的活性。
在夹心法中，样品中的目标抗原被夹在包被单抗和标记单抗之间，然后再加入标记物比如酶的底物，通过底物颜色的变化检测并判定抗原的存在。开展基于夹心法的免疫学检测，例如，先把含有一种未知数量目标抗原的样品加入到用物理或化学方法预先包被了本发明中所述单克隆抗体的固相支持物上进行反应。然后，加入本发明所述的标记单抗进行反应。孵育后，冲洗该支持物，再对结合到该支持物上的标记物的活性进行检测。标记物可以是放射性同位素如125碘、酶、酶的底物、发光物质如异鲁米诺和吖啶酯、荧光物质如荧光素和罗丹明、生物素和有色物质如乳胶颗粒和胶体金等。标记用的酶可以是过氧化物酶(如辣根过氧化物酶HRP)、碱性磷酸酶、β半乳糖苷酶和葡萄糖氧化酶。对于这些反应中合适的底物有2，2＇-连氮基-双(3-乙基苯并噻吡咯啉-6磺酸)、鲁米诺-过氧化氢、邻苯二胺-过氧化氢(针对过氧化物酶)、对硝基苯磷酸盐、4-甲基磷酸伞型酮、3-(2＇-螺旋金刚烷)-4-甲氧基-4-(3＂-磷酰基)苯基-1，2-二乙氧基烷(针对碱性磷酸酶)、对硝基苯-β-D-半乳糖和甲基伞形酮-β-D-半乳糖(针对β半乳糖苷酶)。其它的标记包括量子点标记、生色团标记、酶标记、亲和配体标记、电磁自旋标记、重原子标记、标记有纳米微粒光散射标记或其它纳米微粒的探针、异硫氰酸荧光素(FITC)、TRITC、罗丹明、四甲基罗丹明、R-藻红蛋白、Cy-3、Cy-5、Cy-7、得克萨斯红、Phar-Red、异藻红蛋白(APC)、表位标记如FLAG或HA表位、以及酶标记如碱性磷酸酶、辣根过氧化物酶、I2-半乳糖苷酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶和半抗原偶联物如洋地黄毒苷或二硝基苯酚、或能够形成配合物的结合配对如链霉抗生物素蛋白/生物素、抗生物素蛋白/生物素或抗原/抗体配合物如包括兔IgG和抗-兔IgG；荧光基团如伞形三糖(umbelliferone)、荧光素、异硫氰酸荧光素、罗丹明、四甲基罗丹明、伊红、绿荧光蛋白、藻红、香豆素、甲基香豆素、芘、孔雀绿、二苯乙烯、荧光黄、Cascade蓝、二氯三嗪基荧光素、丹磺酰氯、藻红蛋白、荧光镧系络合物如包括铕和铽、Cy3、Cy5、分子信标(molecular beacons)和其荧光衍生物、发光材料如鲁米诺；光散射或细胞质基因组共振材料如金或银颗粒或量子斑(quantumdot)：或放射性材料如14C、 123I、124I、131I、Tc99m、35S或3H；或球珠(spherical shell)，以及标记有本领域已知的任何其它信号产生标记物的探针。例如，可检测的分子包括但不限于荧光基团以及前面所述其它已知的，如在Joseph R.Lakowicz(Editor)所编的Principles of Fluorescence Spectroscopy，Plenum Pub Corp，第二版(July 1999)和Richard P.Hoagland的第六版的Molecular Probes Handbook所描述的。在某些实施方式中，标记物包括半导体纳米微晶如量子斑(即Qdots)，参见U.S.P 6,207,392。Qdots可从Quantum Dot Corporation购得。用于本发明的半导体纳米微晶包括Group II-V半导体的纳米微晶如MgS、MgSe、MgTe、CaS、CaSe、CaTe、SrS、SrSe、SrTe、BaS、BaSe、BaTe、ZnS、ZnSe、ZnTe、CdS、CdSe、CdTe、HgS、HgSe、HgTe和其混合物以及Group III-V半导体的纳米微晶如GaAs、InGaAs、InP、InAs和其混合物。Group IV半导体如锗或硅的使用，或有机半导体的使用，在某些条件下可能是方便可行的。半导体纳米微晶也可以包括合金，其含有两种或多种选自于Group III-V化合物、Group II-VI化合物、Group IV元素和其组合物的半导体。
在某些实施方式中，荧光能量受体连接到检测探针作为标记物。在一实施方式中，荧光能量受体可以通过化合物与单线态氧反应形成荧光化合物而形成，或通过化合物与一辅助化合物反应并将其转化为荧光化合物而形成。这类化合物可以包括于本发明装置中的缓冲液中。在其它的实施方式中，荧光能量受体可以是包括化学发光剂或基团的化合物的一部分，例如，荧光能量受体可以包括稀土金属如铕、钐、碲等金属络合物。这些材料因其具有尖锐的发光谱带而特别具有吸引力；而且，镧系标记物如铕(III)可以提供有效的长时间的信号发射，同时不易光漂白，因而可以使得含有处理/反应样品的测试装置在需要的情况下可以放置较长一段时间。在各种异源和同源免疫测定法中，已经使用长寿命的荧光铕(III)络合物纳米微粒作为标记物，例如可以参见Huhtinen et al.Clin.Chem.2004Oct；50(10)：1935-6。但这些内部标记的纳米微粒与时间分辨荧光检测一起使用时，测定性能可以改善。在异源测定中，低浓度下测定的动态范围可以扩展；而且，通过使用检测抗体涂敷的高特异活性纳米微粒标记物，而不是使用常规标记的检测抗体，测定的动力学特性也可以改善。在同源测定中，对于荧光共振能量转移来说，铕(III)纳米微粒是很有效的供体，从而可以进行简单、快速和高效的筛选。在一实施方式中，标记物如此处披露的荧光标记物，包括与生物分子偶联的纳米 微粒标记物。换句话说，纳米微粒可以用作检测或捕捉探针。例如，本发明中，可以利用连接到单抗或链霉抗生物素蛋白(SA)的铕(III)-标记的纳米微粒来检测样品中特定的分析物，如纳米微粒基的免疫测定。纳米微粒可以作为附着特定结合剂的底物，这些特定结合剂是针对分析物和检测(如标记物)或捕捉成分的。有关标记物的实例可以参见U.S.P 4,695,554；4,863,875；4,373,932；和4,366,241。U.S.P 4,313,734和4,373,932中则披露了胶体金属和染色颗粒。而U.S.P 4,954,452中则披露了如何制备和使用非金属的胶体；U.S.P 4,252,459中披露了用作标记物的有机聚合物乳胶颗粒。
把标记物结合到抗原或抗体上的方法，可以通过顺丁烯二酰亚胺法(J.Biochem.(1976)，79,233)、生物素活化法(J.Am.Chem.Soc.(1978)，100，3585)、疏水结合法、酯活化法或异氰酸酯法(＂Enzyme免疫测定techniques＂，published in 1987 by IgakuShoin)。
如果上述标记物为放射性同位素，则需使用好的防辐射的工作台面或液体防护设备。如果上述标记物为酶，需加入底物，酶的活性通过比色法或荧光计测定。如果上述标记物为荧光物质、发光物质或有色物质，测定方法可以相应的使用本领域熟知的方法进行测定。
本发明中，用于检测Echo30的样品包括但不限于动物或病人的血液、血浆血清等。
产品或试剂盒
本发明还涉及一种检测Echo30的产品，尤其涉及检测Echo30的试剂盒。本发明还涉及一种诊断Echo30感染或其导致的疾病的产品，尤其涉及诊断Echo30感染或其导致的疾病的试剂盒。
本发明所述试剂盒包含本发明所述的单克隆抗体或其抗原结合片段。本发明的试剂盒还包含适合检测抗原-抗体反应的检测试剂。
本发明的试剂盒还包括供单克隆抗体或其抗原结合片段附着的固相底物，所述固相底物包括但不限于微孔板、磁微粒、免疫色谱用滤纸、聚合体例如聚苯乙烯、玻璃珠、玻璃滤器和其它不溶的载体。
本发明的试剂盒还包含一些其它成分，包括但不限于标记用的酶、相应底物、 放射性同位素、反光物质、荧光物质、有色物质、缓冲液等。
本发明的试剂盒中，所使用的单克隆抗体或抗原必须事先用上述提到的标记物标记。
本发明的试剂盒还包括控制标准。
具体实施方式
以下实施例旨在举例说明本发明，而不限制其范围。
实施例1 单克隆抗体制备
1、免疫原(抗原)：Echo30。
每只小鼠免疫5次，每次100μl抗原，病毒共需3-3.5ml。
2、动物免疫
使用两种佐剂进行免疫，分别为改良的弗氏佐剂及水性佐剂，病毒免疫两组，每3只小鼠为一组，使用一种佐剂，病毒共免疫6只小鼠。所有免疫均在SPF动物房中进行。
选取5-8周龄Balb/c小鼠6只(雌鼠)，免疫原与佐剂混匀后分别乳化进行免疫，第一次注射使用弗氏完全佐剂，以后4次加强注射使用弗氏不完全佐剂，均与等体积抗原充分混匀后注射。
免疫方法为背部多点注射，免疫量主注射100μl抗原/只实验鼠，加强注射100μl抗原/只实验鼠，免疫周期6-8周。
3、单克隆抗体制备流程
抗血清检测：将病毒包板酶标板，间接ELISA方法检测抗血清效价。血清效价大于1:50K，方可进行下一步融合。如有多个小鼠效价超过1：50K，选择效价最高的小鼠进行融合。
骨髓瘤细胞制备：融合前一周，复苏SP2/0细胞，正常培养至对数期。
脾细胞制备：选定要融合的小鼠，融合当天用颈椎脱臼法处死，取脾，标准流程收集脾细胞并计数。
细胞融合：按1:3-1：10的比例混合骨髓瘤细胞和脾细胞，标准流程进行细胞 融合操作，随后用HAT DMEM完全培养基培养，融合后3天即可以看到杂交瘤细胞，第7天换1/2HAT完全培养基，第8天换1/2HT培养基。融合后10天左右开始进行筛选检测。
细胞融合结果：融合后用HAT选择性培养基培养，显微镜下观察，看到多个生长的杂交瘤细胞，证明融合操作成功。
融合筛选：吸取细胞上清100μl/孔进行间接ELISA检测。根据ELISA结果，判断阳性孔。用单道移液器挑检整板检测出的阳性孔，进行第二次复检，进一步确认阳性孔。
亚克隆：对复筛的阳性孔细胞做两轮亚克隆。(因为第一次亚克隆得到的阳性孔细胞株尚不稳定，有可能包含多个杂交瘤细胞，普遍认为第二次亚克隆后杂交瘤细胞为单个细胞株，并确定为阳性)。
4、病毒中和实验：将含有抗体的培养液上清(第一次亚克隆后制备的培养上清，均为对抗原ELISA阳性)与一定浓度的抗体一起接种细胞，噬斑减少或者没有发生的孔，即为中和抗体阳性孔。
5、腹水制备及抗体纯化
5.1腹水制备
将上述阳性细胞扩大培养并注射至Balb/C小鼠(经弗氏不完全佐剂至敏)的腹腔，一般7-10日可见小鼠腹部隆起即代表有腹水产生。当小鼠有明显腹水产生时及时抽取腹水。
5.2腹水纯化
将上述细胞的腹水，用Protein A/G进行纯化，纯化后抗体纯度大于90％。检测浓度纯度后，调整浓度至2mg/ml。
6、抗体鉴定
利用SDS-PAGE检验抗体的表达及纯化情况，结果见图1，证实得到较纯蛋白，可清晰观察到解链后的抗体轻、重链，注：图中M所在泳道代表的是蛋白Marker。
7、抗体序列测定
经测序，鉴定出的命名为6C5的单克隆抗体的氨基酸序列和核苷酸序列如下及表1所示。
重链可变区的氨基酸序列和核苷酸序列如SEQ ID NO：4和SEQ ID NO：12所示。
Figure PCTCN2020072311-appb-000001
轻链可变区的氨基酸序列和核苷酸序列如SEQ ID NO：8和SEQ ID NO：16所示。
Figure PCTCN2020072311-appb-000002
表1抗体序列
Figure PCTCN2020072311-appb-000003
实施例2 抗体功能
1、抗原抗体亲和力测定
1.1表面等离子共振法(SPR)
1.1.1步骤：
利用Pierce Fab Preparation Kit(Thermo)对4B10-IgG(全长抗体)、6C5-IgG(全长抗体)进行切割以及纯化得到4B10-Fab(抗体的Fab片段)、6C5-Fab(抗 体的Fab片段)。
使用BIAcore T100(Biacore，GE Healthcare)进行SPR实验，缓冲液为含有0.05％Tween-20的PBS。使用NHS/EDC方法将纯化的E30全病毒颗粒固定在CM5传感器芯片表面上，直到RU值达到740。然后，梯度浓度的IgG或Fab以20μl/min的速度流经芯片，并在每个注射周期后使用10mM甘氨酸-盐酸(pH 1.7)再生芯片。通过使用软件BIAevaluation(版本4.1)全局拟合曲线来获得结合亲和力。
1.1.2结果
结果如图2所示，4B10-IgG(全长抗体)，4B10-Fab(抗体的Fab片段)，6C5-IgG(全长抗体)，6C5-Fab(抗体的Fab片段)与E30的亲和力分别达到：2.88nM、67.70nM、1.51nM、3.68nM。
1.2竞争性SPR
1.2.1步骤
竞争性SPR步骤与上述SPR类似，将E30病毒颗粒固定在CM5芯片上直至RU值达到740。第一针加入受体或抗体直至拟合曲线饱和，然后第二针再进待测竞争性抗体或受体。通过拟合曲线的变化趋势反应前后两种物质针对芯片上E30病毒颗粒的结合竞争关系。
1.2.2结果
结果显示：病毒与受体(FcRn和CD55)的亲和力分别达到：2.38μM(图3A)和2.11μM(图3B)，远小于病毒与抗体的亲和力，并且相比于对照组(第一针加入1A1，一个针对E6病毒的特异性抗体)，加入E30抗体，能够强力抑制FcRn与CD55和E30的结合(对照成立，ICAM5是EVD68特异性的细胞受体)(图4A和图4C)。而相反地，先加入细胞受体并不能有效抑制抗体与病毒的结合(图4B和图4D)。
2、抗原抗体结合特异性测定
使用ELISA实验进行测定。
2.1步骤
将纯化的病毒颗粒(E30，E6，E3，E11或CVB3)以30ng/孔包被到ELISA 板(Costar，Corning，USA)上，然后在4℃下孵育过夜。在37℃下用含有1％BSA的PBST溶液(PBS加0.1％Tween20)封闭包被板2小时。此后，将板用PBST洗涤五次，并在37℃下以1：1000的稀释度将4B10-IgG或6C5-IgG作为第一抗体添加到每个孔中作用1小时。再次用PBST洗涤板五次，并添加HRP缀合的山羊抗小鼠IgG H&L(1:3000稀释)(Sigma-Aldrich,St.Louis,USA)作为二抗。在37℃下放置0.5小时。将板用PBST洗涤五次，并且在室温下将3,3'，5,5'-四甲基联苯胺(TMB)底物(碧云天，中国上海)添加到每个孔中作用5分钟。最后，将2M H 2SO 4加入板中以终止反应，并读取每个孔在450nm的吸光度值。
2.2结果
结果如图5所示，4B10和6C5均能特异性地识别E30，而与E3，E6，E11和CVB3不发生反应。说明二者是E30特异性的。
3、抗体中和活性检测
3.1减少斑块的中和试验(PRNT)
3.1.1步骤
将4B10/6C5-IgG，4B10/6C5-Fab或小鼠血清在DMEM培养基中稀释以达到2倍系列稀释，最高浓度分别为128nM，128nM，384nM或64nM。此外建立了一个没有任何抗体，Fab或血清的对照组。将相同数量的E30病毒(PFU范围从50到100)与每种稀释液混合，在37℃下孵育1小时，然后添加到接种在6孔板中的RD细胞的汇合单层中。将板放回细胞培养箱培养1小时，每20分钟轻轻震荡一下，之后用DMEM(pH 7.4)冲洗3次。用添加2％FBS的琼脂糖覆盖物(1％(质量体积比)AGAROSE Ⅱ(Amresco)，溶剂为双蒸水)(2mL/孔)覆盖，并在5％CO 2细胞培养箱中于37℃孵育3天。然后用2.5％的结晶紫染色使噬菌斑可视化，抑制百分比计算为(Ncontrol-Ntest)/Ncontrol x 100％，其中Ncontrol和Ntest分别代表对照组和测试组中观察到的噬菌斑计数的平均值。所有实验均重复三次。
3.1.2结果
结果如图6所示，4B10-IgG，4B10-Fab，6C5-IgG，6C5-Fab对E30的Neut50分别达到：0.3nM，25nM，1.2nM和6.8nM。
3.2交叉中和实验
3.2.1步骤
将50nM或500nM的4B10或6C5与一定量的E30(PFU＝～50)在37℃下混合1小时，然后添加到RD细胞铺满的单层中。在温育和漂洗后，将细胞用琼脂糖覆盖物覆盖并在37℃下再温育三天，然后用结晶紫染色，抑制率计算公式为：(Ncontrol-Ntest)/Ncontrol x 100％，其中Ncontrol和Ntest分别代表对照组和测试组中观察到的噬菌斑计数的平均值。所有实验均重复三次。
3.2.2结果
结果如图7所示，50nM和500nM的4B10-IgG和6C5-IgG均能够100％中和E30的感染活性，而等量的这两种抗体却对E3、E6、E11和CVB3起不到任何的中和作用，说明二者对E30的中和活性具有极高的专一性。
3.3 Pre/post attachment实验
3.3.1步骤
Pre attachment
将抗体溶液(终浓度为0nM、3nM、30nM、300nM)和病毒(MOI＝1)于冰上作用30min，将接种12孔板中过夜培养的细胞消化至EP管中，离心1000rpm，5min，移除上清，加入1ml DMEM，将细胞悬起，再次离心，重复3次，移除上清，加入抗体和病毒作用后的液体将细胞悬起，于冰上作用30min，之后如上清洗5次，移除上清，最后加入200μl裂解液裂解。
Post attachment：
将接种12孔板中过夜培养的细胞消化至EP管中，清洗3次，移除上清，加入病毒液(MOI＝1)将细胞重悬，冰上孵育30min，按照上述步骤洗5次，移除上清，加入抗体溶液(终浓度为0nM、3nM、30nM、300nM)将细胞重悬，冰上孵育30min，按照上述步骤清洗5次，移除上清，最后加入200μl裂解液进行裂解。
将Pre/post attachment得到的裂解液提取核酸，并进行QPCR定量分析。
QPCR步骤：通过QPCR对4B10/6C5处理后残留在RD细胞表面的E30进行定量。简而言之，在病毒附着于细胞的MOI为1的前后，E30在4℃下与各种浓度的 6C5混合。然后将细胞洗涤3次，并通过RNeasy mini试剂盒(Qiagen，Hilden，Germany)提取总RNA，并在QuantStudio Dx Real-Time PCR Instrument(Applied Biosystems,Foster City,USA)上使用SuperScrip III Platinum SYBR Green One-Step qRT-PCR Kit(Invitrogen,Carlsbad,USA)进行QPCR。20μL反应体系包含0.2μL SuperScript III RT/Platinum Taq Mix，5μL 2X SYBR Green Reaction Mix，10Μm正向引物(5'-AACAGCAGCGTTGCCCGCGTCTA-3')和反向引物(5'-ACCCTGTAGTTCCCTACATA-3')各0.2μL，2μL总RNA，2.4μL无RNase的H 2O。QPCR扩增程序为：42℃、5分钟用于逆转录，95℃、5分钟用于逆转录灭活。随后95℃变性15s，共40个循环，60℃退火并延伸30s。内源性管家基因β-肌动蛋白(正向引物：5'-GCCCTGAGGCACTCTTCCA-3'，反向引物：5'-CGGATGTCCACGTCACACTT-3')被用作内部对照以标准化样品。通过2 -ΔΔCt法进行不同样品中E30RNA的相对水平的分析。
3.3.2结果
结果如图8所示：病毒与细胞接触前(图8A)或接触后(图8B)抗体(4B10和6C5)均能够有效抑制病毒的活性，表明抗体可以竞争性抑制病毒与病毒受体的识别与结合，并且已经结合受体的病毒也可以被抗体(4B10和6C5)竞争下来。
4、抗体效价和抗体结合表位的免疫优势测定
将培养的E30病毒用甲醛进行灭活、浓缩、蔗糖密度梯度离心，在不同蔗糖梯度层得到纯度较高的空心和实心病毒颗粒；将空心和实心颗粒按照实施例1的步骤分别注射免疫小鼠，然后取血得到抗血清。
利用表位竞争测定
步骤：
为了测试与抗体4B10或6C5结合的表位是否具有免疫优势，首先将纯化的E30全颗粒包被在Elisa平板上，然后按照上述Elisa方法将平板封闭。然后加入针对氢氧化铝佐剂化的E30实心颗粒的血清(anti-F-par sera)，氢氧化铝佐剂化的病毒空心颗粒的血清(anti-E-par sera)或氢氧化铝佐剂的血清(anti-adj sera)。在37℃下孵育0.5小时后，将所有孔洗涤五次，并添加HRP缀合的4B10/6C5-IgG，以在37℃下进一步孵育0.5小时。然后洗涤孔，添加TMB底物，2M H 2SO 4，并 如上所述在A450读取。抑制百分比计算公式为(OD阴性对照-OD血清)/OD阴性对照×100％。
结果：
灭活的E30实心颗粒和空心颗粒的抗血清能够有效中和病毒E30对细胞的感染，效价分别达到1：12和1：11(图9)，并且它们二者与病毒的结合能够block掉约60-80％的4B10和6C5的抗原表位。表明4B10和6C5所识别的抗原表位是免疫优势表位(图10)。
6、抗体对病毒稳定性的影响
Thermofluor实验步骤：
使用MX3005 qPCR仪(Agilent，Santa Clara，USA)，用SYTO9和SYPROred(Invitrogen，Carlsbad，USA)作为荧光探针检测RNA和蛋白质的疏水性残基。建立50μl反应体系，该体系包含2μg目标蛋白，5μM SYTO9和3X SYPROred，并将温度从25℃升至99℃。以1℃的间隔一式三份记录荧光。
结果：4B10-IgG与6C5-IgG与E30的结合均能够微弱破坏E30的稳定性，提示二者的结结合区间可能与病毒的与受体的结合区域相关(图11)，图中E30F-particle。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。

Claims (43)

  1. 一种抗Echo30的抗体或其抗原结合片段，其包含至少一个CDR的VH，和/或，至少一个CDR的VL；
    VH的CDR序列选自：SEQ ID NO：1-3所示的氨基酸序列；
    或
    VH的CDR序列选自：在SEQ ID NO：1-3所示的氨基酸序列基础上经取代、缺失或添加一个或多个氨基酸形成的氨基酸序列；
    或
    VH的CDR序列选自：与SEQ ID NO：1-3所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；
    VL的CDR序列选自：SEQ ID NO：5-7所示的氨基酸序列；
    或
    VL的CDR序列选自：在SEQ ID NO：5-7所示的氨基酸序列基础上经取代、缺失或添加一个或多个氨基酸形成的氨基酸序列；
    或
    VL的CDR序列选自：与SEQ ID NO：5-7所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列。
  2. 权利要求1所述的抗体或其抗原结合片段，其特征在于，至少有80％同源性是指有80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源性。
  3. 根据权利要求1或2所述的抗体或其抗原结合片段，其特征在于，VH的CDR序列选自：SEQ ID NO：1-3所示的氨基酸序列；VL的CDR序列选自：SEQ ID NO：5-7所示的氨基酸序列。
  4. 根据权利要求3所述的抗体或其抗原结合片段，其特征在于，VH的CDR1序列具有SEQ ID NO：1所示的氨基酸序列；VH的CDR2序列具有SEQ ID NO： 2所示的氨基酸序列；VH的CDR3序列具有SEQ ID NO：3所示的氨基酸序列；
    VL的CDR1序列具有SEQ ID NO：5所示的氨基酸序列；VL的CDR2序列具有SEQ ID NO：6所示的氨基酸序列；VL的CDR3序列具有SEQ ID NO：7所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体包括VH和/或VL，其中：
    所述VH具有SEQ ID NO：4所示的氨基酸序列，
    或
    所述VH具有在SEQ ID NO：4所示的氨基酸序列基础上经取代、缺失或添加一个或多个氨基酸形成的氨基酸序列；
    或
    所述VH具有与SEQ ID NO：4所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列；
    所述VL具有SEQ ID NO：8所示的氨基酸序列，
    或
    所述VL具有在SEQ ID NO：8所示的氨基酸序列基础上经取代、缺失或添加一个或多个氨基酸形成的氨基酸序列；
    或
    所述VL具有与SEQ ID NO：8所示的氨基酸序列至少有80％同源性的、且功能相同或相似的氨基酸序列。
  6. 根据权利要求5所述的抗体或其抗原结合片段，其特征在于，至少有80％同源性是指有80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％同源性。
  7. 根据权利要求5或6所述的抗体或其抗原结合片段，其特征在于，所述VH具有SEQ ID NO：4所示的氨基酸序列；所述VL具有SEQ ID NO：8所示的氨基酸序列。
  8. 根据权利要求1-7任一项所述的抗体或其抗原结合片段，其特征在于，所述抗原结合片段包括所述抗体的Fab片段、F(ab’)2片段、单链Fv片段。
  9. 根据权利要求1-8任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体包含κ轻链恒定区。
  10. 一种组合物，其包含权利要求1-9中任一项所述的抗体或其抗原结合片段。
  11. 权利要求10所述的组合物，其特征在于，所述组合物包括药物组合物，所述药物组合物包括权利要求1-9中任一项所述的抗体或其抗原结合片段以及药学上可接受的运载体。
  12. 一种预防或治疗Echo30感染的方法，其包括向有需要的患者施用如权利要求1-9中任一项所述的抗体或其抗原结合片段或如11所述的药物组合物。
  13. 一种预防或治疗Echo30感染导致的疾病的方法，其包括向有需要的患者施用如权利要求1-9中任一项所述的抗体或其抗原结合片段或如权利要求11所述的药物组合物。
  14. 根据权利要求13所述的方法，其特征在于，所述疾病包括脑膜炎，上呼吸道感染，心肌炎，皮疹，呕吐，发烧，头痛。
  15. 一种核酸，其包含编码如权利要求1-9中任一项所述的抗体或其抗原结合片段的核苷酸序列。
  16. 根据权利要求15所述的核酸，其特征在于，所述核苷酸序列如SEQ NO ID：9-16所示。
  17. 一种载体，其包含如权利要求15或16所述的核酸。
  18. 一种原核宿主细胞，其用如权利要求17所述的载体转化。
  19. 一种真核宿主细胞，其用如权利要求17所述的载体转化。
  20. 一种真核宿主细胞，其经工程化以表达如权利要求15或16所述的核酸。
  21. 根据权利要求19或20所述的真核宿主细胞，其为哺乳动物宿主细胞。
  22. 一种产生抗体或其抗原结合片段的方法，该方法包括：(a)培养如权利要求19或20所述的宿主细胞及(b)回收该抗体或其抗原结合片段。
  23. 检测样品中Echo30的产品，所述产品包括权利要求1-9中任一项所述的抗体或其抗原结合片段。
  24. 诊断Echo30感染或其导致的疾病的产品，其特征在于，所述产品包括权利要求1-9中任一项所述的抗体或其抗原结合片段。
  25. 根据权利要求23或24所述的产品，其特征在于，所述产品包括试剂盒，所述试剂盒包括附着于固相底物上的权利要求1-9中任一项所述的抗体或其抗原结合片段以及可检测的、被标记的第二抗体。
  26. 根据权利要求25所述的产品，其特征在于，所述第二抗体能特异性地结合Echo30。
  27. 根据权利要求23-26任一项所述的产品，其特征在于，所述试剂盒还包括控制标准。
  28. 根据权利要求24所述的产品，其特征在于，所述疾病包括脑膜炎，上呼吸道感染，心肌炎，皮疹，呕吐，发烧，头痛。
  29. 一种检测样品中Echo30的方法，其包括如下步骤：
    a)将样品与权利要求1-9中任一项所述的抗体或其抗原结合片段进行接触；
    b)检测所述抗体或其抗原结合片段与Echo30的反应。
  30. 根据权利要求31所述的方法，其中，所述抗体或其抗原结合片段附着在固相底物上。
  31. 根据权利要求32所述的方法，其中，所述的固相底物选自如下一组:微滴定板、磁颗粒、胶乳颗粒和硝化纤维素膜。
  32. 根据权利要求31所述的方法，其中，所述反应是通过酶显色进行测定的。
  33. 根据权利要求31所述的方法，其中，所述反应是通过荧光进行测定的。
  34. 根据权利要求31所述的方法，其中，所述反应是通过化学发光进行测定的。
  35. 一种诊断Echo30感染或其导致的疾病的方法，其包括如下步骤：
    a)将样品与权利要求1-9中任一项所述的抗体或其抗原结合片段进行接触；
    b)检测所述抗体或其抗原结合片段与Echo30的反应；
    c)或出现阳性反应，则判断该受试者已被Echo30感染或患有Echo30感染导致的疾病。
  36. 根据权利要求37所述的方法，其特征在于，所述疾病包括脑膜炎，上呼吸道感染，心肌炎，皮疹，呕吐，发烧，头痛。
  37. 根据权利要求37所述的方法，其中，所述抗体或其抗原结合片段附着在固相底物上。
  38. 根据权利要求39所述的方法，其中，所述的固相底物选自如下一组:微滴定板、磁颗粒、胶乳颗粒和硝化纤维素膜。
  39. 根据权利要求37所述的方法，其中，所述反应是通过酶显色进行测定的。
  40. 根据权利要求37所述的方法，其中，所述反应是通过荧光进行测定的。
  41. 根据权利要求37所述的方法，其中，所述反应是通过化学发光进行测定的。
  42. 权利要求1-9中任一项所述的抗体或其抗原结合片段的应用，其包括以下任一项应用：
    (1)在制备检测Echo30的产品中的应用；
    (2)在制备诊断Echo30感染或其导致的疾病的产品中的应用；
    (3)在制备治疗或预防Echo30感染的药物中的应用；
    (4)在制备治疗或预防Echo30感染导致的疾病的药物中的应用。
  43. 根据权利要求44所述的应用，其特征在于，所述疾病包括脑膜炎，上呼吸道感染，心肌炎，皮疹，呕吐，发烧，头痛。
PCT/CN2020/072311 2020-01-15 2020-01-15 抗Echo30的抗体，相应的核酸，载体，宿主细胞，组合物 WO2021142672A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/072311 WO2021142672A1 (zh) 2020-01-15 2020-01-15 抗Echo30的抗体，相应的核酸，载体，宿主细胞，组合物

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/072311 WO2021142672A1 (zh) 2020-01-15 2020-01-15 抗Echo30的抗体，相应的核酸，载体，宿主细胞，组合物

Publications (1)

Publication Number Publication Date
WO2021142672A1 true WO2021142672A1 (zh) 2021-07-22

Family

ID=76863497

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/072311 WO2021142672A1 (zh) 2020-01-15 2020-01-15 抗Echo30的抗体，相应的核酸，载体，宿主细胞，组合物

Country Status (1)

Country Link
WO (1) WO2021142672A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1872874A (zh) * 2006-06-14 2006-12-06 浙江大学 埃可病毒30型衣壳蛋白(vp)的制备方法
CN105348391A (zh) * 2015-11-03 2016-02-24 李越希 埃可病毒6型vp1蛋白特异性抗原表位及其融合蛋白的制备、应用
CN105906716A (zh) * 2016-04-27 2016-08-31 李越希 埃可病毒9型vp1蛋白特异性抗原表位及其融合蛋白的制备、应用
CN105949320A (zh) * 2016-04-27 2016-09-21 李越希 埃可病毒1型vp1蛋白特异性抗原表位及其融合蛋白的制备、应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1872874A (zh) * 2006-06-14 2006-12-06 浙江大学 埃可病毒30型衣壳蛋白(vp)的制备方法
CN105348391A (zh) * 2015-11-03 2016-02-24 李越希 埃可病毒6型vp1蛋白特异性抗原表位及其融合蛋白的制备、应用
CN105906716A (zh) * 2016-04-27 2016-08-31 李越希 埃可病毒9型vp1蛋白特异性抗原表位及其融合蛋白的制备、应用
CN105949320A (zh) * 2016-04-27 2016-09-21 李越希 埃可病毒1型vp1蛋白特异性抗原表位及其融合蛋白的制备、应用

Similar Documents

Publication Publication Date Title
JP7262440B2 (ja) 抗ｃｄ４７抗体及びその使用
CN112135626B (zh) 抗tigit抗体及其用途
US10584172B2 (en) Humanized monoclonal antibodies and methods of use
TWI409276B (zh) 抗－海帕西啶（ｈｅｐｃｉｄｉｎ）抗體及其用途
JP5814790B2 (ja) デングウイルス中和抗体およびその使用
US8609817B2 (en) Anti-hepcidin-25 selective antibodies and uses thereof
JP2017512209A (ja) デングウイルスに対する抗体分子およびその使用
CN113330036B (zh) 结合pd-l1和ox40的双特异性抗体
JP7463366B2 (ja) 新規の抗ジカウイルス抗体及びその使用
WO2021063352A1 (zh) 一种抗pd-l1抗原结合蛋白及其应用
US20130084301A1 (en) Cluster of Neutralizing Antibodies to Hepatitis C Virus
WO2012155819A1 (zh) Hbv特异性抗体
WO2021195385A1 (en) HUMAN MONOCLONAL ANTIBODIES TO SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-GoV-2)
WO2021142672A1 (zh) 抗Echo30的抗体，相应的核酸，载体，宿主细胞，组合物
WO2023154824A1 (en) Human monoclonal antibodies that broadly target coronaviruses
TW201620931A (zh) 具有抗ｃ型肝炎病毒的感染抑制活性的抗體
WO2022187050A1 (en) Anti-cd117 agents and compositions and methods for making and using the same
CN107286237B (zh) 一种抗丙型肝炎病毒抗体的获取以及应用
EP3820909B1 (en) Monoclonal antibody for the detection of the antiretroviral drug emtricitabine (ftc, 2',3'-dideoxy-5-fluoro-3'-thiacytidine)
KR20230042301A (ko) 샘플에서 sars-cov-2 단백질을 검출하기 위한 개선된 방법 및 키트
WO2021142671A1 (zh) 埃可病毒30型的单克隆抗体
CN114106162A (zh) 一种新型冠状病毒Spike蛋白抗体及其用途
CN111153989B (zh) Echo30单克隆抗体的制备及其用途
TW201915016A (zh) 單株抗體或其抗原結合片段及其用途
CN111153990B (zh) 针对Echo30的单克隆抗体、制备方法及其应用

Legal Events

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

Ref document number: 20913902

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20913902

Country of ref document: EP

Kind code of ref document: A1