WO2016173558A1 - 抗诺如病毒gii.4型鼠源单克隆抗体的制备和应用 - Google Patents

抗诺如病毒gii.4型鼠源单克隆抗体的制备和应用 Download PDF

Info

Publication number
WO2016173558A1
WO2016173558A1 PCT/CN2016/080793 CN2016080793W WO2016173558A1 WO 2016173558 A1 WO2016173558 A1 WO 2016173558A1 CN 2016080793 W CN2016080793 W CN 2016080793W WO 2016173558 A1 WO2016173558 A1 WO 2016173558A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
variable region
light chain
heavy chain
chain variable
Prior art date
Application number
PCT/CN2016/080793
Other languages
English (en)
French (fr)
Inventor
黄忠
王晓黎
Original Assignee
中国科学院上海巴斯德研究所
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 中国科学院上海巴斯德研究所 filed Critical 中国科学院上海巴斯德研究所
Publication of WO2016173558A1 publication Critical patent/WO2016173558A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biomedicine, in particular, the invention relates to the preparation and application of a murine monoclonal antibody against norovirus GII.4.
  • Norovirus is one of the major pathogens causing sporadic cases of acute gastroenteritis and large outbreaks, and Norovirus can infect people of all ages. Although the symptoms caused by Norovirus infection are generally mild and self-limiting, the course lasts for 1-3 days, but it can cause more serious symptoms and even death in children, the elderly and people with immune dysfunction. . According to the amino acid sequence of the VP1 capsid protein, Norovirus can be divided into 6 genomes (G1-GVI), but only GI, GII and GIV can infect humans. The infection of human Norovirus is mainly caused by Norovirus GII, and the large outbreak is mostly caused by Norovirus GII.4. Norovirus infection caused by variants of the GII.4 strain accounts for approximately 55%-85% of cases of gastroenteritis worldwide, and most of those cases that require serious hospitalization or even death are mostly Caused by virus GII.4.
  • Norovirus is popular in both developed and developing countries, causing serious economic losses to countries, and the health of children and the elderly poses a great threat. So far, there are no listed preventive vaccines and special therapeutic drugs. Norovirus lacks a cell culture model and no small animal model, which has greatly hindered the research of vaccines and antiviral drugs.
  • the object of the present invention is to provide a preparation and application of a monoclonal antibody against mouse Norovirus GII.4.
  • a heavy chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 6.
  • a heavy chain of an antibody having a heavy chain variable region and a heavy chain constant region according to the first aspect of the invention.
  • the heavy chain amino acid sequence of the antibody is set forth in SEQ ID NO.: 3.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 7.
  • a light chain of an antibody having a light chain variable region and a light chain constant region according to the third aspect of the invention.
  • the light chain amino acid sequence of the antibody is set forth in SEQ ID NO.: 5.
  • an antibody having:
  • the antibody has:
  • a heavy chain according to the second aspect of the invention and/or a light chain according to the fourth aspect of the invention.
  • a recombinant protein having:
  • sequence of the polynucleotide has the polynucleotide sequence set forth in SEQ ID NO.: 2 and/or SEQ ID NO.: 4.
  • a vector comprising the polynucleotide of the seventh aspect of the invention of the invention is provided.
  • a genetically engineered host cell comprising the polynucleotide of the seventh aspect of the present invention or the genome of the seventh aspect of the present invention is provided.
  • a kit comprising:
  • the kit is an enzyme-linked immunosorbent assay kit.
  • an immunoconjugate comprising:
  • a coupled moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, and radiation A nucleus, or enzyme.
  • a pharmaceutical composition comprising the antibody according to the fifth aspect of the invention, the recombinant protein of the sixth aspect of the invention, or the eleventh aspect of the invention Immunoconjugate;
  • a pharmaceutically acceptable carrier is selected from:
  • a method for preparing a recombinant polypeptide comprising:
  • Figure 1 shows the purification of anti-GII.4 monoclonal antibody by polyacrylamide gel electrophoresis.
  • the five purified antibodies were each treated with a loading buffer containing a reducing agent, loaded onto a 12% polyacrylamide gel for electrophoresis, and stained with Coomassie blue for protein bands.
  • M protein molecular weight standard; 1, D11 mAb; 2, G9 mAb; 3, 2D8 mAb; 4, 7D8 mAb; 5, 8E1 mAb
  • Figure 2 shows the enzyme-linked immunosorbent assay (Elisa) to identify the ability of monoclonal antibodies to bind to different antigens.
  • Elisa enzyme-linked immunosorbent assay
  • Each well was coated with 100 ng of GII.4 (A) or GI.1 (B) virus-like particles on each Elisa plate, and each well was incubated with different concentrations of purified monoclonal antibody for 2 hours at 37 ° C, followed by HRP-labeled anti- The mouse secondary antibody was incubated.
  • Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody was used as an unrelated control.
  • Each point in the graph shows the OD450nm mean and standard deviation determined for three replicate samples.
  • Figure 3 shows the Western blot analysis.
  • the GII.4 virus-like particles were electrophoresed in a 12% polyacrylamide gel, then transferred onto a PVDF membrane, and hybridized with a purified monoclonal antibody.
  • M protein molecular weight standard; 1, D11 mAb; 2, G9 mAb; 3, 2D8 mAb; 4, 7D8 mAb; 5, 8E1 mAb; 6, mouse anti-GII.4 virus-like particle cloning antibody.
  • Figure 4 shows the sandwich Elisa detection of GI.1 and GII.4 virus-like particles.
  • Each well of Elisa was coated with 50 ul of 1:5000 diluted rabbit anti-GII.4 (A) or rabbit anti-GI.1 (B), and each well was added with different concentrations of GII.4 virus-like particles (A) and GI.1 virus-like particles (B) were incubated at 37 °C for 2 hours, followed by the addition of 10 ng of purified monoclonal antibody per well, and finally incubated with HRP-labeled anti-mouse secondary antibody.
  • Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody was used as an unrelated control.
  • Figure 5 shows an alternative neutralization assay to detect the activity of purified mAbs to inhibit the action of GII.4 virus-like particles and PGMIII.
  • 50 ⁇ l of 10 ug/ml PGMII was coated per well on Elisa plates, and different concentrations of monoclonal antibodies were incubated with 0.5 ug/ml GII.4 virus-like particles for 1 hour at room temperature, then added to Elisa plates, followed by rabbit anti-GII.4. Finally, the HRP-labeled anti-rabbit secondary antibody was used for incubation.
  • Anti-hepatitis B surface antigen (HBsAg) monoclonal antibody was used as an unrelated control.
  • Figure 6 shows the identification of monoclonal antibodies for recombinant expression of genes. 100 ng of GII.4 virus-like particles were coated on each well of Elisa plate, and each well was incubated with different concentrations of purified monoclonal antibody at 37 °C. Hour was followed by incubation with an HRP-labeled anti-mouse secondary antibody. The culture supernatant of the cells not transfected with the plasmid served as a blank control. The bar graph in the graph shows the mean and standard deviation of OD450nm for three replicate samples.
  • the present inventors obtained a monoclonal antibody against mouse Norovirus GII.4 by extensive and intensive research, and the experimental results show that the monoclonal antibody has a very high neutralizing activity against Norovirus GII.4. Moreover, the antibody does not have a cross-reaction with GI.1 virus-like particles, and can specifically recognize the GII.4 virus.
  • the invention also provides the use of the above monoclonal antibodies.
  • a monoclonal antibody 8E1 capable of specifically recognizing GII.4 was prepared using GII.4 virus-like particles as an immunogen. Elisa and alternative neutralization experiments indicate that the antibody can be used to detect and analyze GII.4 sensitively, and more importantly, it also has strong neutralizing activity.
  • Norovirus GII.4 belongs to the GII type of Norovirus and is the main pathogen causing the outbreak of Norovirus. Norovirus is popular in both developed and developing countries, causing serious economic losses to countries, and the health of children and the elderly poses a great threat. So far, there are no listed preventive vaccines and special therapeutic drugs.
  • the present invention prepares a GII.4 monoclonal antibody by using recombinant GII.4 virus-like particles as an immunogen.
  • the antibodies prepared by the present invention can be used not only as a tool for laboratory testing, but also as a reliable candidate for the preparation of therapeutic humanized monoclonal antibodies and a useful reagent for developing diagnostic methods.
  • virus-like particles are prepared using Norovirus GII.4VP1, and the amino acid sequence thereof is:
  • the serine (Ser) at position 309 was mutated to asparagine (Asn).
  • virus-like particles are prepared using Norovirus GI.1 VP1, and the amino acid sequence thereof is:
  • antibody or "immunoglobulin” is an isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • the "light chain" of a vertebrate antibody can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region.
  • Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which may be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • the term "monoclonal antibody (mAb)” refers to an antibody obtained from a substantially homogeneous population, ie, the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefits of monoclonal antibodies are that they are It is synthesized by the tumor culture and will not be contaminated by other immunoglobulins. The modifier "monoclonal” indicates the identity of the antibody and is obtained from a substantially homogeneous population of antibodies, which should not be construed as requiring any particular method for producing the antibody.
  • the present invention also includes a monoclonal antibody having the corresponding amino acid sequence of the anti-GII.4 virus monoclonal antibody, a monoclonal antibody having the variable region chain of the anti-GII.4 virus monoclonal antibody, and having the chain Other protein or protein conjugates and fusion expression products.
  • the invention encompasses any protein or protein conjugate having a light chain and a heavy chain comprising a hypervariable region (complementarity determining region, CDR) and a fusion expression product (ie, an immunoconjugate and a fusion expression product), as long as The hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules with the anti-GII.4 virus monoclonal A conjugate formed by the binding of an antibody or fragment thereof.
  • the invention also encompasses cell surface markers or antigens that bind to the anti-GII.4 viral monoclonal antibodies or fragments thereof.
  • the present invention encompasses not only intact monoclonal antibodies, but also immunologically active antibody fragments such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • immunologically active antibody fragments such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • variable region are used interchangeably with “complementarity determining region (CDR).
  • the heavy chain variable region of the antibody comprises the following three complementarity determining region CDRs:
  • CDR1 the amino acid sequence of which is GYSFTDYYMH (SEQ ID NO: 8), which encodes a nucleotide sequence
  • amino acid sequence of the heavy chain variable region is:
  • the heavy chain of the antibody comprises the above-described heavy chain variable region and heavy chain constant region, and the heavy chain constant region may be of murine or human origin.
  • the heavy chain amino acid sequence of the antibody is:
  • V L light chain variable region
  • the light chain variable region of an antibody according to the invention has a complementarity determining region CDR selected from the group consisting of:
  • amino acid sequence of the light chain variable region is:
  • the light chain of the antibody comprises the above-described light chain variable region and light chain constant region, and the light chain constant region may be of murine or human origin.
  • the light chain amino acid sequence of the antibody is:
  • the terms "antibody of the invention”, “protein of the invention”, or “polypeptide of the invention” are used interchangeably and refer to an antibody that specifically binds to an anti-GII.4 virus, for example, having a heavy chain (eg, SEQ ID A protein or polypeptide of NO.: amino acid sequence of 3) and/or light chain (such as the amino acid sequence of SEQ ID NO.: 5). They may or may not contain an initial methionine.
  • a heavy chain eg, SEQ ID A protein or polypeptide of NO.: amino acid sequence of 3
  • light chain such as the amino acid sequence of SEQ ID NO.: 5
  • the antibody is a murine or human murine chimeric monoclonal antibody against the GII.4 virus
  • the heavy chain constant region and/or the light chain constant region may be a humanized heavy chain Constant region or light chain constant region.
  • the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or a light chain constant region of human IgGl, IgG2 or the like.
  • the invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the invention encompasses any protein or protein conjugate having a heavy chain and a light chain comprising a variable region and a fusion expression product (ie, an immunoconjugate and a fusion expression product), as long as the variable region is conjugated to an antibody of the invention
  • the variable regions of the heavy and light chains are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are divided into four framework regions (FR), four
  • FR framework regions
  • the amino acid sequence of FR is relatively conservative and is not directly involved in the binding reaction.
  • CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest since at least some of them are involved in binding antigen. Accordingly, the invention includes those molecules having a light chain and heavy chain variable region of a monoclonal antibody having a CDR, as long as the CDR thereof is 90% or more (preferably 95% or more) with the CDR identified herein. Optimally 98% or more) homology.
  • the present invention encompasses not only intact monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of an antibody of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed by the 6
  • the antibody of the present invention refers to a polypeptide comprising the above CDR regions having anti-GII.4 virus binding activity.
  • the term also encompasses variant forms of a polypeptide comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include, but are not limited to, one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • the function of the protein is generally not altered.
  • the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA capable of hybridizing to the DNA encoding the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained using an antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof.
  • the invention also includes fragments of the antibodies of the invention.
  • the fragment will have at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
  • “conservative variant of the antibody of the present invention” means having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3, compared to the amino acid sequence of the antibody of the present invention. Amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • the present invention also provides a polynucleotide molecule encoding the above antibody or a fragment thereof or a fusion protein thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO.: 2, or 4 or may be a degenerate variant.
  • a "degenerate variant” in the present invention refers to an amino acid sequence encoding the same as the polypeptide of the present invention, but with SEQ ID NO.: 3, 5, 6, 7, 8, 9, 10, The nucleic acid sequences differing in the coding region sequences shown in 14, 15, and 16.
  • Polynucleotides encoding mature polypeptides of the invention include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide represented by SEQ ID NO.: 12 and/or SEQ ID NO.: 22.
  • the full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • One possible method is to synthesize related sequences by artificial synthesis, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This pass It is often cloned into a vector, transferred to a cell, and the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • the biomolecule (nucleic acid, protein, etc.) to which the present invention relates includes biomolecules existing in an isolated form.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention may be used alone or in combination or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agents, or capable of producing detectable products Enzyme.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used for binding to GII.4 virus-like particles, and thus can be used for the prevention and treatment of Norovirus (NoVs) which causes acute gastroenteritis.
  • NoVs Norovirus
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-mentioned monoclonal antibody (or a conjugate thereof) of the present invention and pharmacy An acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the present invention also provides a hybridoma cell strain which can produce the monoclonal antibody against GII.4 virus of the present invention; preferably, the present invention provides a high titer hybridoma cell strain against the anti-GII.4 virus monoclonal antibody.
  • the structure of the antibody of the present invention (such as the heavy chain variable region and the light chain variable region of an antibody) can be easily obtained by those skilled in the art, and then the monoclonal antibody of the present invention can be produced by a recombinant method.
  • Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art.
  • an antigen of the invention can be administered to an animal to induce production of a monoclonal antibody.
  • monoclonal antibodies hybridoma technology can be used (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol. 6: 511, 1976; Kohler et al, Eur. J. Immunol .6: 292, 1976; Hammerling et al, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981) or may be prepared by recombinant DNA method (U.S. Patent No. 4,816,567).
  • myeloma cells are those which are efficiently fused, produce stable, high levels of production of cell-supporting antibodies by selected antibodies, and are sensitive to the culture medium (HAT medium matrix), including myeloma cell lines, such as rodents.
  • Myeloma cell line including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, California, USA) and SP-2, NZ0 or X63-Ag8 -653 cells (available from American Type Culture Collection, Rockville, Maryland, USA).
  • Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J.
  • the medium in which the hybridoma cells are grown is analyzed to detect a single having the desired specificity
  • the production of cloned antibodies for example, by in vitro binding assays, for example, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • the location of the antibody-expressing cells can be detected by FACS.
  • Hybridoma clones can then be subcloned by limiting dilution steps and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) 59- 103 pages).
  • Suitable media for use in this purpose include, for example, DMEM or RPMI-1640 medium.
  • hybridoma cells can be grown as ascites tumors in animals.
  • the monoclonal antibodies secreted by the subclones are appropriately isolated from the culture medium, ascites or serum by a conventional immunoglobulin purification process, for example, protein A-Sepharose, hydroxyphosphorus. Gray stone chromatography, gel electrophoresis, dialysis or affinity chromatography.
  • the present invention provides a monoclonal antibody against the GII.4 virus.
  • the monoclonal antibody is prepared by culturing a hybridoma cell method. The supernatant of the hybridoma cell culture was taken, crude IgG was extracted by saturated ammonium sulfate precipitation method, and the crude antibody was purified by affinity chromatography column (Protein G-Sephrose).
  • the monoclonal antibody is prepared by the method of producing monoclonal antibodies using Balb/C mouse ascites.
  • the hybridoma cells were inoculated into the peritoneal cavity of the sensitized mice, and the abdomen was significantly enlarged in about 10 days.
  • Ascites was taken and crudely extracted by saturated ammonium sulfate precipitation, and then the crude antibody was purified by affinity chromatography (Protein G-Sephrose).
  • the immunoglobulin carries a detectable label. More preferably, the label is selected from the group consisting of a colloidal gold label, a horseradish peroxidase label, a colored label or a fluorescent label.
  • the colloidal gold label can be carried out by methods known to those skilled in the art.
  • the monoclonal antibody against the GII.4 virus is labeled with colloidal gold to give a colloidal gold-labeled monoclonal antibody.
  • the anti-GII.4 virus monoclonal antibody of the invention has good specificity and high titer.
  • test board of the present invention can be made by using a conventional test plate preparation method by using a test plate material commonly used in the art.
  • the invention discloses an immunoassay plate for detecting GII.4 virus, comprising a test strip and a support plate supporting the test strip, such as a PVC polyester plate, etc.; the test strip is made of filter paper, chromatography material, nitrocellulose The film and the absorbent paper are sequentially laminated, and the overlapping portion can be fixedly connected by a conventional method such as tape; wherein: the chromatographic material is pre-coated with a colloidal gold-labeled or colored-labeled anti-GII.4 virus monoclonal antibody or polyclonal An antibody, preferably an anti-GII.4 virus monoclonal antibody labeled with colloidal gold, an adsorption detection line and a quality control line on the nitrocellulose membrane;
  • the colloidal gold-labeled anti-GII.4 virus monoclonal antibody is coated with a solution of anti-GII.4 virus monoclonal antibody labeled with colloidal gold at a concentration of 0.5-1.5 mg/ml.
  • the coating amount is 50 ⁇ l / cm 2 ; the preferred concentration is 0.5 or 1.5 mg / ml, 50 ⁇ l / cm 2 ;
  • test plate was placed flat, and the sample was dropped on the filter paper.
  • the sample was about 120 ⁇ l, and the chromatographic results were observed within 3 to 5 minutes. The result is judged based on the position of the streaks that appear.
  • Invalid There is no ribbon in the quality control area and detection area or no ribbon appears in the quality control area and a ribbon appears in the detection area, indicating that the detection method is wrong or the detection board is deteriorated or invalid, and the detection board should be replaced.
  • the present invention relates to a method for detecting Norovirus in a sample dissolved in cells and/or tissues.
  • the method steps are substantially as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of GII.4 virus in the dissolved sample.
  • the sample used in the method of the invention may be any sample comprising cells present in the cell preservation solution, as used in liquid-based cell assays.
  • the invention also provides a kit comprising an antibody (or a fragment thereof) of the invention or a detection plate of the invention, in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, buffer Agents, etc.
  • the invention further designs a detection kit for detecting the level of GII.4 virus, the kit comprising an antibody for recognizing an anti-GII.4 virus, a lysis medium for dissolving the sample, and a detection of a common reagent and a buffer, such as each Buffers, detection labels, detection substrates, and the like.
  • the test kit can be an in vitro diagnostic device.
  • the present invention further contemplates the development of a kit for the diagnostic evaluation of GII.4 virus infection related conditions from a solution sample, which kit can detect the GII.4 virus present in the sample solution, wherein the cell preservation solution for storing the sample can be Cell preservation solutions such as liquid-based cell assays.
  • the anti-GII.4 virus monoclonal antibody of the invention has the advantages of high affinity and high specificity, and can be widely used in the detection field of preparing GII.4 virus, such as detection reagent or preparation field of detection equipment, etc., in specificity and sensitivity. And detection rate and other aspects have significant advantages over traditional detection methods or detection reagents.
  • the monoclonal antibody 8E1 of the present invention is capable of specifically recognizing Norovirus-like particles
  • the monoclonal antibody 8E1 of the present invention can specifically bind to Norovirus GII.4, and has no cross reaction with Norovirus GI.1, thereby realizing the interaction of Norovirus GI.1 and Norovirus GII.4. Identification.
  • the monoclonal antibody 8E1 of the present invention has potent neutralizing activity against Norovirus.
  • Viral-like particles were prepared by expressing Norovirus GII.4VP1 using a baculovirus-insect expression system [1] . 10 ug of virus-like particles (50 ul volume) were mixed with an equal volume of aluminum adjuvant (500 ug), and 6 weeks old female Balb/c mice were immunized intraperitoneally, and immunized once at 0 weeks, 2 weeks, and 4 weeks. At week 6, mouse serum was taken to detect neutralization titers. At week 7, a mouse with the highest neutralizing titer boosted 15 ug of GII.4 virus-like particles through the tail vein. After 3 days, mouse spleens were taken for preparation of hybridoma cells.
  • GII.4 virus-like particles were coated in 96-well plates at 100 ng per well, coated at 4 °C overnight, blocked with PBST containing 5% skim milk, and incubated with 50 ul of hybridoma culture per well for 2 hours at 37 °C. Then, it was incubated with HRP-labeled secondary antibody (sigma) for 1 hour, and finally a color reaction was carried out to read the absorbance of OD450.
  • mice Female Balb/c mice were intraperitoneally injected with 500 ul of liquid paraffin oil. Two weeks later, each mouse was intraperitoneally injected with 300,000 hybridoma cells. After 7 days, the 12-gauge needle was collected for ascites, centrifuged at 10,000 rpm for 10 min, the upper layer of fat and the lower layer of the precipitate were removed, and clarified ascites was taken for antibody purification. Purified ascites was obtained using a HiTrap HiTrapTM Protein G affinity column according to the instructions to obtain antibodies.
  • Elisa plate was coated overnight with 100 ng of GI.1 or GII.4 virus-like particles per well at 4 ° C to identify the binding ability of the monoclonal antibody.
  • Elisa plates were blocked with 5% skim milk in PBST for 1 hour at 37 ° C, and different concentrations (5 ug/ml, 2.5 ug/ml, 1.25 ug/ml and 0.625 ug/ml) were added to the monoclonal antibody at 50 ul per well. Incubate for 2 hours at ° C, followed by incubation with HRP-labeled anti-mouse secondary antibody, and finally read the absorbance OD450.
  • the protein samples were mixed with SDS-PAG loading buffer, boiled for 10 min, and protein samples were separated by 12% polyacrylamide gel.
  • Western blot analysis was performed by Coomassie blue staining showing protein bands or transferring proteins to PVDF membranes.
  • Monoclonal antibodies were diluted to a final concentration of 1 ug/ml into PBST containing 1% skim milk.
  • Murine anti-GII.4 polyclonal antibody was used at 1:1000 dilution, followed by incubation with HPR-labeled murine secondary antibody (sigma) and finally recorded using a LAS-400 luminescence image analyzer.
  • GI.1 virus-like particles or GII.4 virus-like particles were fully emulsified in equal volume with Freund's complete adjuvant, and subcutaneously injected into healthy rabbits at 150 ug/n. 150ug of GI.1 virus-like particles after 3 and 6 weeks Or GII.4 virus-like particles are mixed with an equal amount of Freund's incomplete adjuvant adjuvant, and fully emulsified for booster immunization. Serum was collected 2 weeks after the last immunization and stored at -80 °C after storage.
  • the anti-GI.1 virus-like granules of rabbit anti-GI.1 virus-like granules (prepared as above) and rabbit anti-GII.4 virus-like granules (preparation method as above) 1:5000 dilution (50 ul / well) 4 ° C overnight
  • the 96-well Elisa plate was coated, and the Elisa plate was blocked with 5% skim milk in PBST for 2 hours at 37 ° C.
  • the virus-like particles were added to the Elisa plate, starting at 40 ng/50 ul/well, and diluting 12 concentrations twice.
  • 96-well Elisa plates were coated with 10 ug/ml of Porcine Gastric Mucin III (PGM) (Shanghai Yuanmu Biotechnology Co., Ltd.) (50 ul/well), and Elisa plates were blocked overnight at 4 ° C with 5% skim milk in PBST. After the backup.
  • PGM Porcine Gastric Mucin III
  • the GII.4 virus-like particle specific monoclonal antibody was started at 8 ug/ml, diluted 2 times, and incubated with an equal volume of 0.5 ug/ml of GII.4 virus-like particles for 1 hour at room temperature, and then added to the PGM-coated 96.
  • the cells of the hybridoma cell line were first extracted with total RNA using Trizol reagent, and then the heavy and light chain full-length genes were amplified according to the 5' RACE kit instructions.
  • the HindIII and EcoRI restriction sites were introduced at the 5' and 3' ends of the heavy and light chains, respectively, by PCR amplification, and the amplified heavy and light chain genes were cloned into pGEM-T, respectively.
  • the positive clones were screened for sequencing, and the clones with the correct sequence were digested with HindIII and EcoRI, and the target fragment was purified by agarose gel electrophoresis, and then used with the plasmid pcDNA3.1 (Promage).
  • the T4 DNA ligase was ligated and constructed into eukaryotic expression vectors pcDNA3.1-(m8E1H) and pcDNA3.1-(m8E1L).
  • the pcDNA3.1-(m8E1H) and pcDNA3.1-(m8E1L) were co-transfected into CHO cells by liposome method. After 72 hours, the culture supernatant was collected for analysis, and the expression of the antibody in the culture supernatant was determined by ELISA: Plates were plated with GI.1 virus-like particles, blocked with 5% milk in PBST for 1 hour at 37 ° C, and cultured supernatants of different dilutions were added for 2 hours at 37 ° C, followed by HRP-labeled anti-mouse IgG secondary antibody. The incubation was carried out and finally the absorbance value OD450 was read.
  • the samples used for the analysis were all 50 ul hybridoma cultured cells.
  • Figure 2 shows that G9, D11, 2D8 and 8E1 can specifically recognize GII.4 virus-like particles, there is no cross-reactivity with GI.1 virus-like particles, while some monoclonal antibodies (eg, 7D8), and GI.1 virus The granules bind to the GII.4 virus-like particles and are unable to specifically recognize the GII.4 virus.
  • Example 3 Based on the monoclonal antibody sandwich Elisa can specifically detect sensitive GI.1 and GII.4 virus-like particles
  • the minimum detection limit of the virus-like particles by the monoclonal antibody was determined by sandwich Elisa (positive when OD450nm > 0.15).
  • Figure 4 shows that G9, D11, 7D8 and 8E1 mAbs were able to specifically detect GII.4 virus-like particles with minimum detection limits of 0.3125 ng, 0.3125 ng, 0.3125 ng, and 0.625 ng, respectively.
  • Tissue blood group antigen is a saccharide present on mucosal tissues and red blood cells and is a receptor required for Norovirus infection.
  • HBGA binding inhibition assays are widely used as antibody-mediated alternative neutralization assays for Norovirus.
  • HBGA in pig gastric mucin III (PGM) has been validated for use in alternative neutralization assays [2] .
  • the potential neutralizing activity of the five monoclonal antibodies G9, D11, 2D8, 7D8 and 8E1 was tested by an alternative neutralization test.
  • Figure 5 shows that D11, G9 and 8E1 showed good neutralizing activity against GII.4, and their EC50s which prevented the binding of virus-like particles to PGM were: 0.0979 ug/ml, 0.6948 ug/ml and 0.04357 ug/ml, respectively.
  • the above results indicate that 8E1 has much better neutralizing activity against GII.4 than other antibody strains, and the neutralizing activity of 8E1 monoclonal antibody against GII.4 is more than twice that of the D11 antibody strain and about 16 times that of the G9 antibody strain.
  • the heavy and light chain sequences of the cloned 8E1 monoclonal antibody are as follows (wherein the single underlined portion is the signal peptide sequence, the italicized portion is the variable region sequence, and the dotted line is underlined as the constant region sequence):
  • the 8E1 mAb heavy chain variable region amino acids are as follows (underlined for the heavy chain CDR regions):
  • the above heavy chain variable region belongs to the IGHV1 subgroup.
  • the 8E1 mAb light chain variable region amino acids are as follows (underlined for the heavy chain CDR regions):
  • the above light chain variable region belongs to the IGKV9 subgroup.
  • the coding sequences of the heavy and light chains were inserted into pcDNA3.1, respectively, and the expression vectors pcDNA3.1-(m8E1H) and pcDNA3.1-(m8E1L) were constructed. Then co-transfected CHO cells and detected whether the cell supernatant specifically binds to GII.4 virus-like particles by ELISA. The presence of antibodies.
  • Figure 6 shows that the cell supernatant expressing the 8E1 mAb sequence has a high binding signal and is associated with the dilution factor of the supernatant; whereas the supernatant of the control cells not transfected with the relevant plasmid has no binding signal, whether diluted or not. This result demonstrates that the 8E1 mAb of the present invention can be successfully expressed in host cells.
  • the present invention obtains a monoclonal antibody 8E1 capable of specifically binding to GII.4 virus particles, which has strong potential neutralizing activity and can be used as a therapeutic monoclonal antibody drug or as a detection reagent after humanization.
  • a monoclonal antibody 8E1 capable of specifically binding to GII.4 virus particles, which has strong potential neutralizing activity and can be used as a therapeutic monoclonal antibody drug or as a detection reagent after humanization.
  • sandwich ELISA the minimum detection limit of monoclonal antibody 8E1 to GII.4 virus-like particles was 0.625 ng, which provided a favorable theoretical basis for the development of these monoclonal antibodies into Norovirus detection kits.
  • the EC50 of the monoclonal antibody 8E1 against GII.4 obtained by the present invention for GII.4 and PGM is 0.04357 ug/ml, which is far superior to other monoclonal antibodies.

Abstract

本发明公开了一种抗诺如病毒GII.4型鼠源单克隆抗体的制备和应用。实验结果表明，所述单克隆抗体具有极高的对诺如病毒GII.4的中和活性，而且该抗体不存在与GI.1病毒样颗粒的交叉反应，能够特异性识别GII.4病毒样颗粒。

Description

抗诺如病毒GII.4型鼠源单克隆抗体的制备和应用 技术领域
本发明属于生物医药领域，具体地说，本发明涉及抗诺如病毒GII.4型鼠源单克隆抗体的制备和应用。
背景技术
诺如病毒(NoVs)是导致急性肠胃炎的散发病例和大暴发的主要的病原体之一，而且，诺如病毒可以感染所有年龄段的人。尽管，诺如病毒感染后引起的症状一般比较温和，有自限性，病程持续1-3天左右，但是在小孩、老人以及免疫功能不全的人中仍可引起较为严重的症状，甚至引起死亡。根据VP1衣壳蛋白的氨基酸序列，诺如病毒可以分为6个基因组(G1-GVI)，但只有GI，GII和GIV可以感染人类。人诺如病毒的感染主要由诺如病毒GII所引起，而大的暴发流行多由诺如病毒GII.4导致。GII.4病毒株的变异体所导致的诺如病毒的感染在全球的肠胃炎病例中占了大约55％-85％，而且那些病情比较严重需要住院治疗甚至导致死亡的病例也大多由诺如病毒GII.4引起。
诺如病毒在发达国家和发展中国家均有流行，给各国带来了严重的经济损失，儿童、老人的健康造成了很大的威胁。到目前为止，没有上市的预防性疫苗和特效的治疗性药物。诺如病毒缺少细胞培养模型，也没有小动物模型，这给疫苗和抗病毒药物的研究带来了很大的阻碍。
因此，本领域技术人员致力于开发具有良好临床应用前景的抗诺如病毒药物。
发明内容
本发明的目的在于提供一种抗诺如病毒GII.4鼠源单克隆抗体的制备和应用。
本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO:8所示的CDR1，
SEQ ID NO:9所示的CDR2，和
SEQ ID NO:10所示的CDR3；
优选地，所述重链可变区具有SEQ ID NO:6所示的氨基酸序列。
本发明的第二方面，提供了一种抗体的重链，所述的重链具有如本发明第一方面所述的重链可变区和重链恒定区。
在另一优选例中，所述抗体的重链氨基酸序列如SEQ ID NO.:3所示。
本发明的第三方面，提供了一种抗体的轻链可变区，所述轻链可变区具有选自下组的互补决定区CDR：
SEQ ID NO:14所示的CDR1'，
SEQ ID NO:15所示的CDR2'，和
SEQ ID NO:16所示的CDR3'；
优选地，所述的轻链可变区具有SEQ ID NO:7所示的氨基酸序列。
本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如本发明第三方面所述的轻链可变区和轻链恒定区。
在另一优选例中，所述抗体的轻链氨基酸序列如SEQ ID NO.:5所示。
本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明第一方面所述的重链可变区；和/或
(2)如本发明第三方面所述的轻链可变区；
或者，所述抗体具有：
如本发明第二方面所述的重链；和/或如本发明第四方面所述的轻链。
本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
本发明的第七方面，提供了一种多核苷酸，它编码选自下组的多肽：
(1)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体；或
(2)如本发明第六方面所述的重组蛋白。
在另一优选例中，所述多核苷酸的序列具有如SEQ ID NO.:2和/或SEQ ID NO.:4所示的多核苷酸序列。
本发明的第八方面，提供了一种载体，它含有本发明本发明第七方面所述的多核苷酸。
本发明的第九方面，提供了一种遗传工程化的宿主细胞，其特征在于，它含有本发明第八方面所述的载体或基因组中整合有本发明七方面所述的多核苷酸。
本发明的第十方面，提供了一种试剂盒，所述试剂盒中包括：
本发明第五方面所述的抗体。
在另一优选例中，所述试剂盒为酶联免疫检测试剂盒。
本发明的第十一方面，提供了一种免疫偶联物，该免疫偶联物含有：
(a)本发明第五方面所述的抗体或本发明第六方面所述的重组蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射 性核素、或酶。
本发明的第十二方面，提供了一种药物组合物，所述组合物包含本发明第五方面所述的抗体、本发明第六方面所述的重组蛋白、或本发明第十一方面所述的免疫偶联物；以及
药学上可以接受的载体。
本发明的第十三方面，提供了一种重组多肽的制备方法，该方法包含：
(a)在适合表达的条件下，培养本发明第九方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是本发明第五方面所述的抗体或本发明第六方面所述的重组蛋白。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了聚丙烯酰胺凝胶电泳分析纯化的抗GII.4单抗。5种纯化的抗体分别经含有还原剂的上样缓冲液处理后上样到12％的聚丙烯酰胺凝胶中进行电泳，并以考马斯亮蓝染色显示蛋白条带。M,蛋白分子量标准；1，D11单抗；2，G9单抗；3，2D8单抗；4，7D8单抗；5，8E1单抗
图2显示了酶联免疫吸附实验(Elisa)鉴定单抗与不同抗原的结合能力。在Elisa板上每孔分别包被100ngGII.4(A)或者GI.1(B)病毒样颗粒，每孔分别加不同浓度的纯化的单抗在37℃孵育2小时，接着用HRP标记的抗鼠二抗进行孵育。抗乙肝表面抗原(HBsAg)单抗被用来做无关对照。图中每个点显示了三个重复样品测定的OD450nm平均值和标准差。
图3显示了Westernblot分析。GII.4病毒样颗粒经处理后，在12％的聚丙烯酰胺凝胶中进行电泳，接着转印到PVDF膜上，用纯化的单抗进行杂交。M,蛋白分子量标准；1，D11单抗；2，G9单抗；3，2D8单抗；4，7D8单抗；5，8E1单抗；6，鼠抗GII.4病毒样颗粒克隆抗体。
图4显示了夹心Elisa检测GI.1和GII.4病毒样颗粒。在Elisa板上每孔分别包被50ul 1:5000稀释的兔抗GII.4(A)或者兔抗GI.1(B)，每孔分别加不同浓度的GII.4病毒样颗粒(A)和GI.1病毒样颗粒(B)在37℃孵育2小时，接着每孔加入10ng的纯化的单抗，最后用HRP标记的抗鼠二抗进行孵育。抗乙肝表面抗原(HBsAg)单抗被用来做无关对照。
图5显示了替代中和试验检测纯化后单抗抑制GII.4病毒样颗粒与PGMIII作用的活性。在Elisa板上每孔包被50ul 10ug/ml PGMII，将不同浓度的单抗与0.5ug/ml GII.4病毒样颗粒在室温孵育1小时后加入Elisa板中，接着加入兔抗GII.4，最后用HRP标记的抗兔二抗进行孵育。抗乙肝表面抗原(HBsAg)单抗被用来做无关对照。
图6显示了基因重组表达的单克隆抗体的鉴定。在Elisa板上每孔分别包被100ngGII.4病毒样颗粒，每孔分别加不同浓度的纯化的单抗在37℃孵育2 小时，接着用HRP标记的抗鼠二抗进行孵育。未转染质粒的细胞的培养上清作为空白对照。图中柱状图显示了三个重复样品测定的OD450nm平均值和标准差。
具体实施方式
本发明人通过广泛而深入的研究，获得一种抗诺如病毒GII.4鼠源单克隆抗体，实验结果表明，所述单克隆抗体具有极高的对诺如病毒GII.4的中和活性，而且该抗体不存在与GI.1病毒样颗粒的交叉反应，能够特异性识别GII.4病毒。本发明还提供了上述单克隆抗体的用途。
具体地，本研究以GII.4病毒样颗粒作为免疫原制备了能特异性识别GII.4的单克隆抗体8E1。Elisa和替代中和实验等方法说明该抗体可以用来灵敏检测和分析GII.4，更重要的是其还具有强大的中和活性。
在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
诺如病毒GII.4
诺如病毒GII.4属于诺如病毒属GII型，是引起诺如病毒暴发流行的主要病原体。诺如病毒在发达国家和发展中国家均有流行，给各国带来了严重的经济损失，儿童、老人的健康造成了很大的威胁。到目前为止，没有上市的预防性疫苗和特效的治疗性药物。
本发明利用重组GII.4病毒样颗粒作为免疫原制备了GII.4单克隆抗体。本发明制备的抗体不仅可以用作实验室检测用工具，而且是制备治疗性人源化单抗的可靠候选者和开发诊断方法的有用试剂。
本发明中使用诺如病毒GII.4VP1制备了病毒样颗粒，其氨基酸序列为：
Figure PCTCN2016080793-appb-000002
309位丝氨酸(Ser)突变为天 冬酰胺(Asn)。
本发明中使用诺如病毒GI.1VP1制备了病毒样颗粒，其氨基酸序列为：
Figure PCTCN2016080793-appb-000003
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA,IgD,IgE,IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1,IgG2,IgG3,IgG4,IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
如本文所用，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂 交瘤培养来合成的，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明还包括具有所述的抗GII.4病毒单克隆抗体的相应氨基酸序列的单克隆抗体、具有所述的抗GII.4病毒单克隆抗体可变区链的单克隆抗体，以及具有这些链的其他蛋白质或蛋白质偶联物及融合表达产物。具体地，本发明包括具有含超变区(互补决定区，CDR)的轻链和重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该超变区与本发明的轻链和重链的超变区相同或至少90％同源性，较佳地至少95％同源性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与所述的抗GII.4病毒单克隆抗体或其片段结合的而形成的偶联物。本发明还包括与所述的抗GII.4病毒单克隆抗体或其片段结合的细胞表面标记物或抗原。
本发明不仅包括完整的单克隆抗体，还包括具有免疫活性的抗体片段，如Fab或(Fab')2片段；抗体重链；抗体轻链。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括以下三个互补决定区CDR：
CDR1，其氨基酸序列为GYSFTDYYMH(SEQ ID NO:8)，其编码核苷酸序列为，
Figure PCTCN2016080793-appb-000004
CDR2，其氨基酸序列为
Figure PCTCN2016080793-appb-000005
其编码核苷酸序列为，
Figure PCTCN2016080793-appb-000006
Figure PCTCN2016080793-appb-000007
CDR3，其氨基酸序列为
Figure PCTCN2016080793-appb-000008
其编码核苷酸序列为，
Figure PCTCN2016080793-appb-000009
在另一优选例中，所述重链可变区的氨基酸序列为：
Figure PCTCN2016080793-appb-000010
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区，所述重链恒定区可以为鼠源或人源。
在另一优选例中，所述抗体的重链氨基酸序列为：
Figure PCTCN2016080793-appb-000011
如本文所用，术语“轻链可变区”与“VL”可互换使用。
在本发明的一个优选的实施方式中，根据本发明的抗体的轻链可变区，具有选自下组的互补决定区CDR：
CDR1'，其氨基酸序列为
Figure PCTCN2016080793-appb-000012
其编码核苷酸序列为，
Figure PCTCN2016080793-appb-000013
CDR2'，其氨基酸序列为
Figure PCTCN2016080793-appb-000014
其编码核苷酸序列为，
Figure PCTCN2016080793-appb-000015
CDR3'，其氨基酸序列为
Figure PCTCN2016080793-appb-000016
其编码核苷酸序列为，
Figure PCTCN2016080793-appb-000017
在另一优选例中，所述的轻链可变区的氨基酸序列为：
Figure PCTCN2016080793-appb-000018
在本发明的一个优选的实施方式中，所述抗体的轻链包括上述轻链可变区和轻链恒定区，所述轻链恒定区可以为鼠源或人源。
在另一优选例中，所述抗体的轻链氨基酸序列为：
Figure PCTCN2016080793-appb-000019
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合抗GII.4病毒的抗体，例如具有重链(如SEQ ID NO.:3的氨基酸序列)和/或轻链(如SEQ ID NO.:5的氨基酸序列)的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
在另一优选例中，所述的抗体为抗抗GII.4病毒的鼠或人鼠嵌合单克隆抗体，它的重链恒定区和/或轻链恒定区可以是人源化的重链恒定区或轻链恒定区。更优选地，所述的人源化的重链恒定区或轻链恒定区为人IgG1、IgG2等的重链恒定区或轻链恒定区。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链和轻链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链和轻链的可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链和/或轻链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的单克隆抗体轻链和重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上， 最佳地98％以上)的同源性。
本发明不仅包括完整的单克隆抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有抗GII.4病毒结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含人抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
Figure PCTCN2016080793-appb-000020
Figure PCTCN2016080793-appb-000021
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO.:2、或4所示的编码区序列相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指编码具有与本发明的多肽相同的氨基酸序列，但与SEQ ID NO.:3、5、6、7、8、9、10、14、15、16所示的编码区序列有差别的核酸序列。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与SEQ ID NO.:12和/或SEQ ID NO.:22所示的成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通 常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合GII.4病毒样颗粒，因而可用于预防和治疗诺如病毒(NoVs)是导致急性肠胃炎。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
杂交瘤细胞株
本发明还提供了可生产本发明针对抗GII.4病毒单克隆抗体的杂交瘤细胞株；优选的，本发明提供了高效价的针对抗GII.4病毒单克隆抗体的杂交瘤细胞株。
在获得生产本发明的抗GII.4病毒单克隆抗体的杂交瘤之后，本领域技术人员可以方便地利用该杂交瘤细胞株制备抗体。此外，本领域技术人员还可很方便地获知本发明的抗体的结构(比如抗体的重链可变区和轻链可变区)，然后可通过重组方法来制备本发明的单克隆抗体。
单克隆抗体的制备
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。例如，本发明抗原，可被施用于动物以诱导单克隆抗体的产生。对于单克隆抗体，可利用杂交瘤技术来制备(见Kohler等人,Nature 256；495,1975；Kohler等人,Eur.J.Immunol.6:511,1976；Kohler等人,Eur.J.Immunol.6:292,1976；Hammerling等人,In Monoclonal Antibodies and T Cell Hybridomas,Elsevier,N.Y.,1981)或可用重组DNA法(美国专利号4,816,567)制备。
代表性的骨髓瘤细胞是有效融合、通过选择的抗体产生细胞支持抗体的稳定高水平产生、且对培养基(HAT培养基基质)敏感的那些骨髓瘤细胞，包括骨髓瘤细胞株，例如鼠类的骨髓瘤细胞株，包括衍生自MOPC-21和MPC-11小鼠肿瘤的骨髓瘤细胞株(可购自Salk Institute Cell Distribution Center，圣地亚哥，加利福尼亚，美国)以及SP-2、NZ0或X63-Ag8-653细胞(可购自American Type Culture Collection，洛克维尔，马里兰，美国)。人骨髓瘤和小鼠-人杂合骨髓瘤细胞株也已被描述用于产生人单克隆抗体[Kozbor，J.Immunol.，133：3001(1984)；Brodeur等，单克隆抗体的生产技术和应用(Monoclonal Antibodies Production Techniques and Applications)，51-63页(Marcel Dekker，Inc.，纽约，1987)]。
对杂交瘤细胞生长于其中的培养基进行分析以检测具有所需特异性的单 克隆抗体的产生，如，通过体外结合分析例如，酶联免疫吸附分析(ELISA)或放射免疫分析(RIA)。表达抗体的细胞的位置可用FACS进行检测。然后，可将杂交瘤克隆通过有限稀释步骤形成亚克隆(subcloned)，并通过标准方法生长(Goding，单克隆抗体(Monoclonal Antibodies)：原则和实践(Principles and Practice)，Academic Press(1986)59-103页)。为了达到这一目的而使用的适合的培养基包括，例如，DMEM或RPMI-1640培养基。此外，杂交瘤细胞可在动物体内作为腹水瘤生长。
由亚克隆分泌的单克隆抗体从培养基、腹水或血清中通过常规的免疫球蛋白纯化工艺适当地得到分离，这些纯化工艺为例如，蛋白A-琼脂糖法(protein A-Sepharose)、羟基磷灰石层析、凝胶电泳、透析或亲和层析。
本发明提供了一种针对GII.4病毒的单克隆抗体。在本发明的一个优选的方案中，单克隆抗体采用培养杂交瘤细胞方法制备。取杂交瘤细胞培养的上清液，经饱和硫酸铵沉淀法粗提出IgG,再将粗提的抗体经亲和层析柱(Protein G-Sephrose)纯化。
本发明的一个优选的方案中，单克隆抗体采用Balb/C小鼠腹水生产单克隆抗体的方法制备。将约杂交瘤细胞接种到致敏的小鼠腹腔内，10天左右可见腹部明显胀大。抽取腹水，经饱和硫酸铵沉淀法粗提后,再将粗提的抗体经亲和层析柱(Protein G-Sephrose)纯化。
标记的免疫球蛋白(抗体)
在本发明的一个优选例中，所述免疫球蛋白带有可检测标记物。更佳地，所述的标记物选自下组：胶体金标记物、辣根过氧化物酶标记、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，抗GII.4病毒的单克隆抗体用胶体金标记，得到胶体金标记的单克隆抗体。
本发明的抗GII.4病毒单克隆抗体具有很好的特异性，很高的效价。
检测板及其材料
本发明的检测板可采用本领域常用的检测板材料，采用常规的检测板制备方法制成。
本发明检测GII.4病毒的免疫检测板，包括测试条和支撑测试条的支撑板，如可采用PVC聚脂胶板等；所述的测试条由滤样纸、层析材料、硝酸纤维素膜和吸水纸依次搭接组成，搭接部位可以采用常规的方法，如胶带等固定连接；其中：层析材料预包被胶体金标记或有色标记的抗GII.4病毒单克隆抗体或多克隆抗体，优选被胶体金标记的抗GII.4病毒单克隆抗体，硝酸纤维素膜上吸附检测线和质控线；
在一个优选的方案中：层析材料上预包被胶体金标记的抗GII.4病毒单克隆抗体是采用浓度为0.5-1.5mg/ml胶体金标记的抗GII.4病毒单克隆抗体溶液进行预包被的，包被量为50μl/cm2；优选的浓度为0.5或1.5mg/ml，50μl/cm2；
检测方法与结果判定
平放检测板，将试样滴在滤样纸上，试样约120μl，3～5min内观察层析结果。根据出现的条纹位置来判断结果。
阴性：质控区、检测区均出现明显的色带，示为阴性；
阳性：只在质控区出现明显色带，而在检测区无色带，示为阳性；
无效：质控区、检测区无任何色带或在质控区未出现色带而在检测区出现色带，表明检测方法错误或检测板变质或失效，应重新换取检测板检测。
方法和样本
本发明涉及用于在以细胞和/或组织溶解的样本检测诺如病毒的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中GII.4病毒的水平。本发明方法所使用的样本可以是存在于细胞保存液中的包括细胞的任何样本，正如在液基细胞检测法中所使用的。
试剂盒
本发明还提供了一种指含有本发明的抗体(或其片段)或本发明的检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明进一步设计用于检测GII.4病毒水平的检测试剂盒，该试剂盒包括识别抗GII.4病毒的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
本发明进一步设计开发用于对来自溶液样本的GII.4病毒感染相关情况诊断评估的试剂盒，该试剂盒可以检测存在于样本溶液中的GII.4病毒，其中保存样本的细胞保存液可以是诸如液基细胞检测法中的细胞保存液。
本发明的抗GII.4病毒单克隆抗体具有高亲和力、高特异性等优点，可广泛应用在制备GII.4病毒的检测领域，如检测试剂或检测设备的制备领域等，在特异性、灵敏度和检测率等方面较之传统的检测方法或检测试剂具有显著的优势。
本发明的主要优点在于：
(1)本发明的单克隆抗体8E1能够特异性的识别诺如病毒样颗粒；
(2)本发明的单克隆抗体8E1能够特异性的结合诺如病毒GII.4，与诺如病毒GI.1无交叉反应，从而实现对诺如病毒GI.1和诺如病毒GII.4的鉴定。
(3)本发明的单克隆抗体8E1对诺如病毒具有强大的中和活性。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用 的实验材料和试剂如无特别说明均可从市售渠道获得。
材料和方法
1抗原制备及小鼠免疫
利用杆状病毒-昆虫表达系统，通过表达诺如病毒GII.4VP1制备了病毒样颗粒[1]。将10ug的病毒样颗粒(50ul体积)与等体积的铝佐剂(500ug)混合后腹腔免疫6周雌性Balb/c小鼠，在0周、2周、4周各免疫一次。在第6周时，采取小鼠血清检测中和滴度。第7周时，中和滴度最高的一只小鼠通过尾静脉加强免疫15ugGII.4病毒样颗粒。3天后，取小鼠脾脏用于制备杂交瘤细胞。
2杂交瘤细胞株的制备和筛选
小鼠尾静脉加强免疫3天后，取小鼠脾脏细胞与骨髓瘤细胞SP2/0通过PEG1500融合，制备杂交瘤细胞。9天之后，通过酶联免疫吸附试验筛选特异性分泌针对GII.4病毒样颗粒的抗体。简言之，GII.4病毒样颗粒包被96孔板，每孔100ng，4℃包被过夜，用含有5％脱脂牛奶的PBST封闭，每孔加50ul杂交瘤培养液在37℃孵育2小时，接着用HRP标记的二抗(sigma)孵育1小时，最后进行显色反应，读取OD450的吸光值。
3腹水制备和抗体纯化
雌性Balb/c小鼠腹腔注射500ul液体石蜡油，两周后，每只小鼠腹腔注射30万个杂交瘤细胞。7天后，12号针头收集腹水，10,000rpm离心10min，去除上层油脂和下层沉淀，取澄清的腹水进行抗体纯化。根据说明书，利用HiTrap HiTrapTM Protein G亲和柱(GE health care)纯化腹水获得抗体。
4酶联免疫吸附实验鉴定单克隆抗体
用每孔100ngGI.1或GII.4病毒样颗粒4℃过夜包被96孔Elisa板，鉴定单抗的结合能力。Elisa板经含5％脱脂牛奶的PBST在37℃封闭1个小时后，按每孔50ul将不同浓度(5ug/ml、2.5ug/ml、1.25ug/ml和0.625ug/ml)加入单抗37℃孵育2小时，接着用HRP标记的抗鼠二抗进行孵育，最后读取吸光值OD450。
5聚丙烯酰胺凝胶电泳和western blot分析
蛋白样品与SDS-PAG上样缓冲液混合后，煮沸处理10min，经12％聚丙烯酰胺凝胶分离蛋白样品。通过考马斯亮蓝染色显示蛋白条带或者将蛋白转移到PVDF膜上进行western blot分析。单克隆抗体按最终浓度1ug/ml稀释到含1％脱脂牛奶的PBST中。鼠抗GII.4多克隆抗体1:1000稀释使用，接着用HPR标记的鼠二抗(sigma)进行孵育，最后用LAS-400发光图像分析仪进行记录。
6兔抗GI.1病毒样颗粒或GII.4病毒样颗粒多克隆抗体的制备
GI.1病毒样颗粒或GII.4病毒样颗粒与弗氏完全佐剂等体积混合充分乳化，皮下注射健康兔子150ug/只。3周和6周后将150ug的GI.1病毒样颗粒 或GII.4病毒样颗粒与等量弗氏不完全佐剂佐剂混合，充分乳化后进行加强免疫。最后一次免疫后2周收集血清，分装后-80℃保存备用。
7夹心Elisa检测GI.1和GII.4病毒样颗粒
分别用兔抗GI.1病毒样颗粒的多抗血清(制备方法同上)和兔抗GII.4病毒样颗粒的多抗血清(制备方法同上)1:5000稀释度(50ul/孔)4℃过夜包被96孔Elisa板，Elisa板经含5％脱脂牛奶的PBST在37℃封闭2个小时后，将病毒样颗粒加入Elisa板中，40ng/50ul/孔始起，2倍比稀释12个浓度，37℃孵育2个小时，然后将病毒样颗粒特异的单抗10ng/50ul/孔37℃孵育1小时，接着用HPR标记的鼠二抗进行孵育，最后读取吸光值OD450。
8体外替代中和实验
用10ug/ml的猪胃粘液素Ⅲ(PGM)(上海远慕生物科技有限公司)(50ul/孔)室温包被96孔Elisa板，Elisa板经含5％脱脂牛奶的PBST在4℃封闭过夜后备用。将GII.4病毒样颗粒特异性单抗8ug/ml始起，2倍比稀释，与等体积0.5ug/ml的GII.4病毒样颗粒室温孵育1个小时后加到包被有PGM的96孔Elisa板上，室温孵育1个小时，然后加入兔抗GII.4病毒样颗粒多克隆抗体(制备方法同上)1:1000稀释液37℃孵育1小时，接着用HPR标记的兔二抗(sigma)进行孵育，最后读取吸光值OD450。
9单克隆抗体的基因序列扩增及表达载体的构建
先将杂交瘤细胞株的细胞用Trizol试剂提取总RNA，然后按照5’RACE试剂盒说明书扩增出重链和轻链全长基因。利用PCR扩增的方法在重链和轻链的5’端和3’端分别引入HindIII和EcoRI酶切位点，并将扩增出来的重链和轻链全的基因分别克隆到pGEM-T(Promage)中，筛选出阳性克隆测序，然后将序列正确的克隆用HindIII和EcoRI双酶切，经琼脂糖凝胶电泳纯化出目的片段后，与同酶切的质粒pcDNA3.1(Promage)用T4DNA连接酶连接，构建成真核表达载体pcDNA3.1-(m8E1H)和pcDNA3.1-(m8E1L)。
10单克隆抗体基因的重组表达鉴定
利用脂质体的方法共转染pcDNA3.1-(m8E1H)和pcDNA3.1-(m8E1L)到CHO细胞，72小时后收取培养上清进行分析，采用ELISA确定培养上清中的抗体的表达：用GI.1病毒样颗粒包板，用含5％牛奶的PBST于37℃封闭1小时，加入不同稀释度的待测培养上清37℃孵育2小时，接着用HRP标记的抗鼠IgG二抗进行孵育，最后读取吸光值OD450。
实施例1分泌GII.4特异抗体的杂交瘤细胞的筛选
免疫了GII.4病毒样颗粒小鼠的脾脏细胞用来制备杂交瘤细胞。通过Elisa实验筛选杂交瘤细胞上清，从而获得能够分泌具有结合病毒能力的杂交瘤细胞株。最终，五株具有优异结合能力的单抗被筛选出来，他们都能够结合GII.4病 毒样颗粒。亚型鉴定显示，G9、D11、7D8和8E1属IgG1，2D8属IgG3。
表1.分泌单抗的杂交瘤细胞株鉴定
杂交瘤细胞株 重链 轻链 与GII.4VLP结合能力*
G9 IgG1 kappa +++
D11 IgG1 kappa +++
2D8 IgG3 kappa +++
7D8 IgG1 kappa +++
8E1 IgG1 kappa +++
用于分析的样品均为50ul杂交瘤培养细胞。
*,+：OD450nm＞0.15；++：OD450nm＞0.3；+++：OD450nm＞0.5。
实施例2抗GII.4单抗的特异性分析
首先通过SDS-PAGE鉴定从腹水中纯化的GII.4单抗的纯度和完整性。图1显示了五种单抗的重链和轻链分别为50KD和25KD左右。接着，通过Elisa方法检测了单抗与不同抗原的反应活性，包括GI.1病毒样颗粒和GII.4病毒样颗粒。图2显示G9、D11、2D8和8E1可以特异性识别GII.4病毒样颗粒，不存在与GI.1病毒样颗粒的交叉反应，而有些单抗(如，7D8)，同时与GI.1病毒样颗粒和GII.4病毒样颗粒结合，无法特异性识别GII.4病毒。
最后，通过Western blot分析单抗与GII.4的结合情况，图3显示，五种抗体中：D11、G9和8E1能够识别天然结构的GII.4病毒样颗粒，不能够识别变性后的GII.4病毒样颗粒，提示D11、G9和8E1识别的表位可能是构象表位。
实施例3基于单抗的夹心Elisa可以特异灵敏地检测到GI.1和GII.4病毒样颗粒
通过夹心Elisa测定单抗对病毒样颗粒的最低检出限度(当OD450nm＞0.15时，判为阳性)。图4显示了G9、D11、7D8和8E1单抗均可特异灵敏地检测到GII.4病毒样颗粒，最低检出限度分别为：0.3125ng、0.3125ng、0.3125ng、0.625ng。
实施例4单克隆抗体的潜在中和活性
组织血型抗原(HBGA)是存在于粘膜组织和红细胞上的糖类，是诺如病毒感染所需的受体。HBGA的结合抑制试验被广泛用作为抗体介导的诺如病毒的替代中和试验。猪胃粘液素Ⅲ(PGM)中含有HBGA，已经被验证可以用于替代中和试验[2]。通过替代中和试验分别对G9、D11、2D8、7D8和8E1五种单抗的潜在中和活性进行检测。图5显示，D11、G9和8E1对GII.4显示了良好的中和活性，它们阻止病毒样颗粒与PGM结合的EC50分别是：0.0979ug/ml、0.6948ug/ml和0.04357ug/ml。上述结果表明，8E1对GII.4中和活性要远优于其它的抗体株，8E1单抗对GII.4的中和活性是D11抗体株的两倍多，是G9抗体株的16倍左右。
实施例5单克隆抗体的基因序列分析
克隆出来的8E1的单抗的重链和轻链序列如下(其中，单下划线部分为信号肽序列，斜体部分为可变区序列，虚线下划线为恒定区序列)：
8E1单抗重链核苷酸序列：
Figure PCTCN2016080793-appb-000022
Figure PCTCN2016080793-appb-000023
8E1单抗重链氨基酸序列：
Figure PCTCN2016080793-appb-000024
8E1单抗轻链核苷酸序列：
Figure PCTCN2016080793-appb-000025
Figure PCTCN2016080793-appb-000026
8E1单抗轻链氨基酸序列：
Figure PCTCN2016080793-appb-000027
进一步分析8E1单抗重链可变区和轻链可变区序列，8E1单抗重链可变区氨基酸如下(下划线标注的为重链CDR区)：
Figure PCTCN2016080793-appb-000028
上述重链可变区属于IGHV1亚群。
8E1单抗轻链可变区氨基酸如下(下划线标注的为重链CDR区)：
Figure PCTCN2016080793-appb-000029
上述轻链可变区属于IGKV9亚群。
各CDR区氨基酸序列和核苷酸序列总结于表2。
表2
Figure PCTCN2016080793-appb-000030
实施例6单克隆抗体基因的重组表达及鉴定
为了验证所克隆出的8E1单抗的基因是否正确，将重链和轻链的编码序列分别插入到pcDNA3.1中，构建表达载体pcDNA3.1-(m8E1H)和pcDNA3.1-(m8E1L)，然后共转染CHO细胞，并通过ELISA检测细胞上清中是否有特异性结合GII.4病毒样颗粒 的抗体存在。图6显示，表达8E1单抗序列的细胞上清有很高的结合信号，而且与上清的稀释倍数相关；而没有转染相关质粒的对照细胞的上清不管是否稀释都没有结合信号。该结果说明了本发明的8E1单抗能够成功地在宿主细胞中表达。
讨论
本发明获得了能特异结合GII.4病毒颗粒的单克隆抗体8E1，其具有强大的潜在中和活性，可以经过人源化后用作治疗性单克隆抗体药物或用作检测试剂。通过夹心ELISA检测，单克隆抗体8E1对GII.4病毒样颗粒的最低检出限度为0.625ng，这为将这些单克隆抗体开发成诺如病毒检测试剂盒提供了有利的理论依据。而本发明所得到的针对GII.4的单克隆抗体8E1对GII.4和PGM的相互作用的EC50为：0.04357ug/ml，远优于其它的单克隆抗体。而且，Western显示8E1单抗识别的表位为构象表位，这使得8E1只能识别空间构象正确的病毒颗粒，而不能识别解聚的和变性的病毒颗粒，能够用于可靠地病毒颗粒定量。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
参考文献
1.Huhti, L., et al., A comparison of methods for purification and concentration of norovirus GII-4 capsid virus-like particles. Arch Virol, 2010. 155(11): p.1855-8.
2.Lindesmith, L.C., et al., Immunogenetic mechanisms driving norovirus GII.4antigenic variation. PLoS Pathog,2012.8(5):p.e1002705.

Claims (13)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:8所示的CDR1，
    SEQ ID NO:9所示的CDR2，和
    SEQ ID NO:10所示的CDR3；
    优选地，所述重链可变区具有SEQ ID NO:6所示的氨基酸序列。
  2. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区和重链恒定区。
  3. 一种抗体的轻链可变区，其特征在于，所述轻链可变区具有选自下组的互补决定区CDR：
    SEQ ID NO:14所示的CDR1'，
    SEQ ID NO:15所示的CDR2'，和
    SEQ ID NO:16所示的CDR3'；
    优选地，所述的轻链可变区具有SEQ ID NO:7所示的氨基酸序列。
  4. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区和轻链恒定区。
  5. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：
    如权利要求2所述的重链；和/或如权利要求4所述的轻链。
  6. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  7. 一种多核苷酸，其特征在于，它编码选自下组的多肽：
    (1)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的 抗体；或
    (2)如权利要求6所述的重组蛋白。
  8. 一种载体，其特征在于，它含有本发明权利要求7所述的多核苷酸。
  9. 一种遗传工程化的宿主细胞，其特征在于，它含有权利要求8所述的载体或基因组中整合有权利要求7所述的多核苷酸。
  10. 一种试剂盒，其特征在于，所述试剂盒中包括：
    权利要求5所述的抗体。
  11. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)权利要求5所述的单克隆抗体或权利要求6所述的重组蛋白；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  12. 一种药物组合物，其特在于，所述组合物包含权利要求5所述的抗体、权利要求6所述的重组蛋白、或权利要求12所述免疫偶联物；以及
    药学上可以接受的载体。
  13. 一种重组多肽的制备方法，其特征在于，该方法包含：
    (a)在适合表达的条件下，培养权利要求9所述的宿主细胞；
    (b)从培养物中分离出重组多肽，所述的重组多肽是权利要求5所述的抗体或权利要求6所述的重组蛋白。
PCT/CN2016/080793 2015-04-30 2016-04-29 抗诺如病毒gii.4型鼠源单克隆抗体的制备和应用 WO2016173558A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510216744.1A CN106188281B (zh) 2015-04-30 2015-04-30 抗诺如病毒gii.4型鼠源单克隆抗体的制备和应用
CN201510216744.1 2015-04-30

Publications (1)

Publication Number Publication Date
WO2016173558A1 true WO2016173558A1 (zh) 2016-11-03

Family

ID=57198181

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/080793 WO2016173558A1 (zh) 2015-04-30 2016-04-29 抗诺如病毒gii.4型鼠源单克隆抗体的制备和应用

Country Status (2)

Country Link
CN (1) CN106188281B (zh)
WO (1) WO2016173558A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109734801A (zh) * 2019-03-15 2019-05-10 南方医科大学 一种gii.4型诺如病毒广谱单克隆抗体的制备方法及用途
CN112159797A (zh) * 2020-09-18 2021-01-01 广东省微生物研究所（广东省微生物分析检测中心） 杂交瘤细胞株3g7 1b10、抗gii.4型诺如病毒p蛋白单克隆抗体和应用

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108727488B (zh) * 2017-04-13 2022-07-22 中国科学院上海巴斯德研究所 抗诺如病毒gii.17单克隆抗体的制备和应用
CN109081868B (zh) * 2017-06-14 2022-06-24 中国科学院上海巴斯德研究所 靶向寨卡病毒包膜蛋白保守表位的单克隆抗体及其应用
CN109957014B (zh) * 2017-12-25 2022-03-29 中国科学院上海巴斯德研究所 抗诺如病毒gii.3鼠源单克隆抗体的制备和应用
CN109265542B (zh) * 2018-09-27 2021-06-15 国药中生生物技术研究院有限公司 特异性结合诺如病毒gii.4基因型vp1蛋白或vlp的抗体及其制备方法和应用
CN110221080B (zh) * 2019-06-25 2021-09-17 上海交通大学 一种人源诺如病毒免疫胶体金试剂盒及细胞株
CN112175912B (zh) * 2020-09-18 2022-07-29 广东省微生物研究所（广东省微生物分析检测中心） 杂交瘤细胞株3g4 1d6、抗gii.4型诺如病毒p蛋白单克隆抗体和应用
EP4273155A1 (en) * 2020-12-30 2023-11-08 Grand Theravac Life Science (Nanjing) Co., Ltd. Norovirus virus-like particle, immune composition or kit, and use thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014126921A1 (en) * 2013-02-12 2014-08-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Monoclonal antibodies that neutralize norovirus

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104098698B (zh) * 2013-04-08 2019-04-05 中国人民解放军第二军医大学 一种抗cd3抗体及其制法和应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014126921A1 (en) * 2013-02-12 2014-08-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Monoclonal antibodies that neutralize norovirus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GUO, LI. ET AL.: "Preparation and characterization of monoclonal antibodies against Norovirus capsid protein.", CHINESE JOURNAL OF ZOONOSES, vol. 22, no. 5, 31 May 2006 (2006-05-31) *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109734801A (zh) * 2019-03-15 2019-05-10 南方医科大学 一种gii.4型诺如病毒广谱单克隆抗体的制备方法及用途
CN112159797A (zh) * 2020-09-18 2021-01-01 广东省微生物研究所（广东省微生物分析检测中心） 杂交瘤细胞株3g7 1b10、抗gii.4型诺如病毒p蛋白单克隆抗体和应用
CN112159797B (zh) * 2020-09-18 2022-08-05 广东省微生物研究所（广东省微生物分析检测中心） 杂交瘤细胞株3g7 1b10、抗gii.4型诺如病毒p蛋白单克隆抗体和应用

Also Published As

Publication number Publication date
CN106188281B (zh) 2019-10-08
CN106188281A (zh) 2016-12-07

Similar Documents

Publication Publication Date Title
CN111051347B (zh) Tigit抗体、其抗原结合片段及医药用途
WO2016173558A1 (zh) 抗诺如病毒gii.4型鼠源单克隆抗体的制备和应用
WO2018177393A1 (zh) B7-h3抗体、其抗原结合片段及其医药用途
CN110914304B (zh) Cd96抗体、其抗原结合片段及医药用途
WO2016112855A1 (zh) 抗cd19单克隆抗体及其制备方法
WO2016173559A1 (zh) 抗诺如病毒gi.1型鼠源单克隆抗体的制备和应用
CN111606993B (zh) 抗呼吸道合胞病毒的全人广谱中和抗体4f1及其应用
WO2018153366A1 (zh) Tim-3抗体、其抗原结合片段及医药用途
US20230138315A1 (en) Anti-angptl3 antibody and use thereof
KR101589135B1 (ko) 인간화 항-ｅｍａｐｉｉ 항체 및 이의 용도
CN108727488B (zh) 抗诺如病毒gii.17单克隆抗体的制备和应用
CN113817052A (zh) 抗SARS-CoV-2核衣壳蛋白单克隆抗体及其制备方法和用途
CN109879966B (zh) 基于鼠源cd19抗体的人源化设计及表达验证
CN111018984B (zh) 抗ck8单克隆抗体及其应用
CN109593131B (zh) 一种抗14-3-3η蛋白单克隆抗体及其用途
CN114685670A (zh) Cldn18.2抗体及其应用
CN109957014B (zh) 抗诺如病毒gii.3鼠源单克隆抗体的制备和应用
CN113321730B (zh) Cldn18.2抗体及其应用
CN116903739B (zh) 一种抗s100b蛋白的抗体及其应用
CN109897109B (zh) 抗肿瘤标志蛋白单克隆抗体及其用途
WO2022142272A1 (zh) Cldn18.2抗体及其应用
WO2023083327A1 (zh) 抗cldn18.2单克隆抗体及其应用
KR102662387B1 (ko) B7-h3 항체, 이의 항원-결합 단편 및 이의 의학적 용도
CN106608907B (zh) 抗ERPV-ATIp重组截短蛋白单克隆抗体的制备和应用
TW202222836A (zh) 特異性結合糖基化ceacam5的抗體

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: 16785986

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: 16785986

Country of ref document: EP

Kind code of ref document: A1