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WO2018212468A1 - Protéine de fusion ayant un domaine fc dérivé d'anticorps de souris connecté à une protéine vlrb dérivée de myxine dont le domaine de queue hydrophobe a été retiré, et son utilisation - Google Patents

Protéine de fusion ayant un domaine fc dérivé d'anticorps de souris connecté à une protéine vlrb dérivée de myxine dont le domaine de queue hydrophobe a été retiré, et son utilisation Download PDF

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WO2018212468A1
WO2018212468A1 PCT/KR2018/004465 KR2018004465W WO2018212468A1 WO 2018212468 A1 WO2018212468 A1 WO 2018212468A1 KR 2018004465 W KR2018004465 W KR 2018004465W WO 2018212468 A1 WO2018212468 A1 WO 2018212468A1
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derived
vlrb
protein
target antigen
fusion protein
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Korean (ko)
Inventor
정태성
김경동
이정석
임세평
김영림
김재성
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Gyeongsang National University GNU
Immucell Co Ltd
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Gyeongsang National University GNU
Immucell Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to a fusion protein in which a mouse antibody-derived Fc region is linked to a fish-derived VLRB protein from which a hydrophobic tail domain has been removed, and a use thereof.
  • VLRs variable lymphocyte receptors
  • VLRB protein governing the antibody role in mammals unlike the immunoglobulin (immunoglobulin, Ig) of the mammal and expressed at a much simpler structure a single poly-peptide of its diversity is arithmetically possible to approximately 10 over 14, and such as pH, temperature There is a much more stable advantage to changes in the surrounding environment. Therefore, using the acquired immune system of the eel, it is possible to produce a customized antibody to a specific antigen inexpensively, easily and quickly, and to be a strong competitive alternative antibody candidate that can face the existing market of various antibodies. do.
  • An important factor in protein detection techniques based on antigen-antibody binding is to maintain specific binding of the antibody protein to the target antigen with high binding stability.
  • the most frequently used mouse antibody for example, has a Y-shaped structure with two binding sites to the antigen.
  • monoclonal antibodies have a U-shaped horseshoe structure and do not act as antibodies in a single body.
  • the present inventors have confirmed that in the case of the VLRB antibody of the eel, through the previous studies, about 20 to 30 units have a specific spherical shape consisting of a spherical shape.
  • this spherical monoclonal antibody significantly reduces its expression rate due to its high hydrophobicity at the C-terminus of the wild-type VLRB protein when expressed in an artificial environment such as human cell line 293.
  • an artificial environment such as human cell line 293.
  • musk eel antibody unlike the spherical form of the naturally occurring musk eel antibody, there is a disadvantage that it is difficult to investigate the binding ability with the antigen by ELISA, immunoblotting and the like.
  • the present inventors removed the gene of the C-terminal region that determines the three-dimensional structure while maintaining the site capable of binding the antigen in the mudfish antibody VLRB and introduced the Fc peptide domain of the mouse antibody capable of dimer and tetramer formation. To express higher expression, higher antigen binding capacity and lower molecular weight.
  • Korean Patent No. 1632620 discloses an 'anti-VEGF single variable domain fused to an Fc domain
  • Korean Patent No. 1660336 discloses' FGFR-Fc fusion protein and uses', but the present invention is disclosed.
  • the present invention was derived from the above requirements, and the present inventors have prepared a fusion protein in which an immunoglobulin Fc domain, a linker, and an immunoglobulin J chain are sequentially linked to an eel-derived VLRB protein from which a hydrophobic tail domain has been removed.
  • the fusion protein prepared above was expressed in a 293-F cell line, it was confirmed that a multimer structure of a dimer or a tetramer was formed, and an increase in avidity for the target antigen was significantly increased compared to that of the monomer.
  • the present invention was completed by confirming that in the structure of the fusion protein, the linker is not included in the structure of the fusion protein, the binding force to the target antigen is not increased even when the dimer is formed.
  • the present invention is a polynucleotide encoding an immunoglobulin Fc domain, which is sequentially linked to the 3'-end of the gene encoding the eel-derived variable lymphocyte receptor B (VLRB) protein from which the hydrophobic tail domain is removed; Linker sequences; And a polynucleotide encoding an immunoglobulin J chain.
  • VLRB variable lymphocyte receptor B
  • the present invention also provides a host cell transformed with the recombinant expression vector.
  • the present invention provides a murine tail derived VLRB (variable lymphocyte receptor B) protein produced by the host cell; Immunoglobulin Fc domains; Linker; And fusion proteins with sequentially linked immunoglobulin J chains.
  • VLRB variable lymphocyte receptor B
  • the present invention also provides a multivalent antibody having increased binding ability to a target antigen, characterized in that the fusion protein is composed of a dimer or a tetramer multimer by self-assembly.
  • the present invention provides a method for producing a multivalent antibody having increased binding ability to a target antigen by transforming the host cell with the recombinant expression vector.
  • the present invention also provides a multivalent antibody having increased binding ability to the target antigen produced by the above method.
  • the present invention also provides a method for detecting a target antigen by treating the multivalent antibody with a target antigen-containing suspect sample.
  • the present invention also provides a composition for detecting a target antigen, containing the polyvalent antibody as an active ingredient.
  • Antibodies of the present invention are not just a monoclonal antibody but have two or four binding sites with antigens to increase antigen-antibody binding ability, and by removing hydrophobic tail domains, increase expression rate in host cells, and stabilize the antibody itself. Also increased.
  • the existing eel antibody should be treated with a VLRB recognition antibody when the ELISA or immunoblotting and the secondary antibody sequentially, but the fusion antibody of the present invention by treating only the secondary antibody that can recognize the mouse Fc region Since there is an advantage that can be easily detected, the eel antibody fusion antibody of the present invention will be useful in the future biomarker development and diagnostics.
  • Figure 1 shows the structure of the eel wild type VLRB protein.
  • SP signal peptide
  • LRRNT N-terminal capped LRR
  • LRR leucine-rich repeat
  • LRRVs variable LRR modules
  • CP connecting peptide
  • LRRCT C-terminal capped LRR
  • HC hydrophobic C-terminus.
  • FIG. 2 is a schematic diagram of a recombinant expression vector (pmlg vector) for the production of a fusion protein of the present invention.
  • FIG. 3 is a schematic diagram of the VLRB fusion protein of the present invention.
  • Fc Fc domain of mouse IgG; J, J chain of human IgM; J M , J chain of mouse IgM; ⁇ , cysteine residues.
  • Figure 4 is a Western blotting result confirming the expression of the VLRB fusion protein of the present invention, A shows a result in a non-reducing condition, B is a reducing condition.
  • VLRB fusion protein clones # 18 and # 94
  • VLRB protein from the eel immunized with avian influenza virus (AIV).
  • AIV avian influenza virus
  • VHSV viral hemorrhagic sepsis virus.
  • Figure 7 shows the results confirmed by ELISA binding ability of the selected fusion protein for each expression type.
  • Figure 8 shows the results confirmed by immunocytochemistry for antigen binding ability of each fusion protein clone to the ACK-infected MDCK cells.
  • the present invention relates to a poly-encoding immunoglobulin Fc domain, which is sequentially linked to the 3'-terminus of a gene encoding an eel-derived variable lymphocyte receptor B (VLRB) protein from which a hydrophobic tail domain has been removed. Nucleotides; Linker sequences; And a polynucleotide encoding an immunoglobulin J chain.
  • VLRB variable lymphocyte receptor B
  • the fish-derived VLRB protein from which the hydrophobic tail domain is removed is a signal peptide (SP) from the N-terminus to the C-terminus.
  • SP signal peptide
  • LRRNT N-terminal capped LRR
  • LRRVs leucine-rich repeat
  • LRRVs variable LRR modules
  • CP connecting peptide
  • LRRRCT C-terminal capped LRR
  • Stalk domains Stalk domains. It contains LRRVs and LRRCT domains, which are sites, and is a VLRB protein capable of binding to a target antigen.
  • the fish-derived VLRB protein from which the hydrophobic tail domain has been removed may be a signal peptide that is a murine immunoglobulin ⁇ chain leader sequence, but is not limited thereto. If the signal peptide or sequence that can further enhance the extracellular secretion capacity of the recombinant protein can be used without limitation in its kind.
  • the murine immunoglobulin ⁇ chain leader sequence of the present invention may be composed of the nucleotide sequence of SEQ ID NO: 1, but is not limited thereto.
  • the gene encoding the VLRB protein derived from an eel extracted from the hydrophobic tail domain may be a gene encoding a VLRB protein having the highest binding ability to a target antigen.
  • the linker sequence may be a cysteine coding sequence, but is not limited thereto.
  • the immunoglobulin Fc domain may be derived from a mouse IgG, preferably consisting of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.
  • the immunoglobulin J chain may be derived from human or mouse IgM, preferably consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, but is not limited thereto.
  • recombinant refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid.
  • Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form.
  • Recombinant cells can also express genes found in natural cells, but the genes are modified and reintroduced into cells by artificial means.
  • recombinant expression vector means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.
  • Expression vectors can be constructed by methods well known to those skilled in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to a suitable promoter in the expression vector to drive mRNA synthesis. Expression vectors may also include ribosomal binding sites and transcription terminators as translation initiation sites.
  • the recombinant expression vector may preferably include one or more selectable markers, but is not limited thereto.
  • the marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells.
  • the present invention also provides a host cell transformed with the recombinant expression vector.
  • the host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell can be used by any host cell known in the art, for example, Escherichia coli Rosetta, E. coli JM109, E. coli BL21, E. coli RR1, Escherichia coli LE392, Escherichia coli B, Escherichia coli X 1776, Escherichia coli D ⁇ , Escherichia coli W3110, Bacillus sp. Strains such as Bacillus subtilis , Bacillus thuringiensis , Salmonella typhimurium , Enterobacteria and strains such as Serratia marcescens and various Pseudomonas species.
  • yeast Saccharomyce
  • insect cells human cells and animal cells (e.g., human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO), W138, BHK, COS-7, HepG2, 3T3, RIN and MDCK cell lines) and plant cells This can be used.
  • human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO), W138, BHK, COS-7, HepG2, 3T3, RIN and MDCK cell lines e.g., HEK, human embryonic kidney (HEK) 293, Chinese hamster ovary (CHO), W138, BHK, COS-7, HepG2, 3T3, RIN and MDCK cell lines
  • the present invention also relates to an eel-derived variable lymphocyte receptor B (VLRB) protein from which a hydrophobic tail domain has been removed produced by the host cell; Immunoglobulin Fc domains; Linker; And fusion proteins with sequentially linked immunoglobulin J chains.
  • VLRB variable lymphocyte receptor B
  • the fusion protein according to the present invention is a fusion protein in which the F-c domain derived from mouse IgG, the J chain derived from human or mouse IgM are sequentially connected to the C-terminus of the stalk domain of the fish-derived VLRB protein, and the Fc domain and the J chain are cysteine. It is a fusion protein linked by a linker.
  • the present invention also provides a multivalent antibody having increased binding ability to a target antigen, characterized in that the fusion protein consists of a dimer or a tetramer multimer by self-assembly.
  • the polyvalent antibody of the present invention is a fusion protein in which an F-domain of an immunoglobulin and a J-chain derived from an immunoglobulin are sequentially linked and recombined to an E. VLRB protein from which a C-terminal hydrophobic tail domain has been removed while maintaining the binding ability to a target antigen. It is characterized by having a dimer or tetramer form of.
  • the multivalent antibody of the present invention has a binding ability to a target antigen when the multivalent form of the fusion protein necessarily includes a cysteine linker connecting the Fc domain and the J chain of the fusion protein.
  • the multivalent antibody of the present invention contains two or four VLRB proteins, which are blackfish antibodies, and thus is a multivalent antibody with significantly increased antigen binding ability as compared to VLRB monoclonal antibodies.
  • the present invention also provides
  • step (b) culturing the transformed host cell of step (a);
  • step (c) obtaining multimers of dimers or tetramers of fusion proteins from the host cell or the culture thereof cultured in step (b), wherein the multivalent antibody having increased binding capacity to the target antigen is It provides a manufacturing method.
  • the method for preparing the multivalent antibody of the present invention immunization by injecting an immunogen or antigen into the eel, to extract the blood from the immunized eel and to separate the lymphocytes, extract the total RNA of the lymphocytes based on the mature VLRBs After securing the mRNA, a polymerase chain reaction was carried out using the template to prepare a cDNA pool of VLRBs except for the hydrophobic region at the carboxy terminus.
  • Each clone of the cDNA prepared as described above was pmlg vector containing murine immunoglobulin ⁇ chain leader sequence, Fc domain of mouse IgG and polynucleotide encoding J chain of IgM (FIG. 2).
  • Cloning was performed to generate a recombinant expression vector capable of expressing a fusion protein comprising mudfish VLRBs with different antigen recognition sequences. Thereafter, 293-F cells were transfected with each recombinant expression vector, the transduced 293-F cells were cultured, and a culture solution was obtained to confirm reactivity with an immunogen or antigen by ELISA experiment. Finally, clones with excellent binding ability to the immunogen or antigen were selected, and the culture of the selected clones was Western blotting under non-reducing and reducing conditions to confirm that the fusion protein was formed into multimers.
  • the immunogen or antigen is not limited thereto, and may be a virus, a microorganism, or the like.
  • the present invention also provides a multivalent antibody having increased binding ability to a target antigen produced by the above method.
  • the hydrophobic tail domain is removed while maintaining the binding ability to the target antigen in the ingested VLRB protein, and the Fc domain derived from mouse IgG, the J chain derived from human or mouse IgM are sequentially located at the removed hydrophobic tail domain position.
  • the fusion protein linked to the protein consists of a dimeric or tetrameric multimeric form, and contains two or four mudfish VLRB proteins capable of binding to the target antigen. .
  • the present invention also provides a method for detecting a target antigen by treating the multivalent antibody with a target antigen-containing suspect sample.
  • the antigen is not limited thereto, but may be a virus or a microorganism.
  • the sample may be food, water, a solution containing a specific or unspecific microorganism, tissue, cells, blood, serum, plasma, saliva and the like, but is not limited thereto.
  • the target antigen detection method of the present invention can be carried out in an antigen-antibody reaction mode. In this case, it can be carried out using the multivalent antibody of the present invention that specifically binds to the target antigen to be detected.
  • the present invention can be used to detect the presence of a target antigen by carrying out according to conventional immunoassay methods. This immunoassay can be carried out according to various quantitative or qualitative immunoassay methods developed in the past.
  • Detection of the conjugate between the multivalent antibody and the target antigen of the present invention may be performed through an indirect direct enzyme linked immunosorbent assay (ELISA) or sandwich ELISA method, but is not limited thereto. Measurement of the final enzyme activity or signal in the indirect ELISA method and sandwich-ELISA method can be carried out according to various methods known in the art. Detection of such signals allows for qualitative or quantitative analysis of the multivalent antibodies of the invention.
  • ELISA indirect direct enzyme linked immunosorbent assay
  • the present invention also provides a composition for detecting a target antigen containing the multivalent antibody as an active ingredient, and a kit for detecting a target antigen comprising the composition as an active ingredient.
  • the hydrophobic tail domain is removed while maintaining the binding ability to the target antigen, and a Fc domain derived from mouse IgG, a J chain derived from human or mouse IgM are sequentially connected to the removed hydrophobic tail domain position.
  • the fusion protein comprises a multivalent antibody in the form of a multimer as an active ingredient, and the kit of the present invention comprising the composition for detection is not limited thereto.
  • the kit is an immunoassay kit. May be direct-ELISA, indirect-ELISA, sandwich-ELISA, protein microarray, radioimmunoassay (RIA), and the like.
  • the wild type VLRB protein of the eel contains a strong hydrophobic region (HC) at the C-terminus (FIG. 1), and the expression rate and the secretion rate outside the cell are significantly reduced when expressed through the human cell line 293.
  • the hydrophobic region (HC) was deleted, and the Fc region (fragment crystallizable region) of the mouse antibody was connected to proceed with multimer fusion proteinization of VLRB.
  • the vector pmIg J was constructed by cloning into a vector for human cell expression so as to connect to the site.
  • a vector pmIg CJ prepared by adding cysteine (Cys) between the Fc region and the J chain region was completed.
  • the vector pmIg CJ M for tetramer production was produced by cloning the vector for human cell expression so as to be connected to the 3'-terminal region of the VLRB gene.
  • the produced vectors were transfected into host cells, and murine Ig ⁇ chains were used as signal peptides for the separation of various kinds of recombinant VLRB proteins produced from host cells to the outside of cells. (green fluorescent protein, GFP) was used as a reporter gene.
  • GFP green fluorescent protein
  • the monomer recombinant protein expressed from the expression vector pMono is in a form including LRRNT to LRRCT site and stalk site, which are known as antigen binding sites of VLRB derived from blackfish (FIG. 3).
  • MIg J a dimeric recombinant protein expressed from the expression vector pmIg J, includes the LRRNT to stalk portion of the VLRB from the eel, and the F-chain region of the mouse-derived IgG antibody and the J-chain region of the human-derived IgM antibody at the C-terminal portion of stalk. Are sequentially connected.
  • MIg CJ a dimeric recombinant protein expressed from the expression vector pmIg CJ, is a dimeric form that forms a stronger disulfide bond in the same dimeric protein, mIg J, in which cysteine is added between the Fc and J chain sites.
  • Expression vector pmIg CJ 4 incorporating the recombinant protein expressed from mIg CJ M M includes a stalk portion from the LRRNT VLRB, Fc portion of mouse IgG antibody derived from the C- terminal part of the stalk and mouse J chain-derived IgM, and It is expressed as a fusion protein in the form of cysteine added between the Fc region and the J chain region (FIG. 3).
  • Mono a monomeric recombinant protein, was identified as a monomer having a size of 40 to 43 kDa under both non-reducing and reducing conditions, and mIg J was found to have a band of 75 to 80 KDa, the expected molecular weight under reducing conditions (with ⁇ -ME). In non-reducing conditions, a band of about 200 kDa larger than the expected molecular weight of 150-160 kDa was identified. This is expected to increase the molecular weight due to glycosylation of the recombinant protein in the disulfide bond process for dimer formation. This phenomenon was also confirmed in mIg CJ and mIg CJ M.
  • avian influenza virus (AIV) specific antagonist VLRB clone # 94 and negative control clone # 18 validated in our previous studies
  • the mouse antibody-derived Fc region and human or mouse antibody-derived J chains were Linked VLRB fusion proteinization of the present invention proceeded.
  • Four fusion proteins of mono, mIg J, mIgCJ and mIg CJ M were produced using the two VLRB clones, and their reactivity with AIV was measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).
  • VHSV viral hemorrhagic sepsis virus
  • VDFB fusion proteins were treated on PVDF membranes coated with various concentrations of AIV antigen, and secondary antibodies were reacted to dot blotting. It was. As in the ELISA results, antigen-specific reactivity was not observed in mono 94 and dimer form mIg J 94, but the other dimer forms mIg CJ 94 and dimer and tetramer form mIg CJ M 94 were 100ng and 500ng. It showed strong reactivity against AIV antigen. In particular, the dimer / dimeric form of mIg CJ M 94 was strongly identified in response to 100 ng of antigen compared to other forms of VLRB fusion protein (FIG. 6).
  • the fusion protein of the invention was further prepared using other AIV specific VLRB clones validated through our previous studies, including A94 specific VLRB clone # 94.
  • A94 specific VLRB clone # 94 a total of six clones, including # 94 clones # 261, # 343, # 279, # 383, and # 662, are produced as four types of fusion proteins (mono, mIg J, mIgCJ, mIg CJ M ) Reactivity with the antigen AIV was measured using ELISA.
  • the # 94, # 261, and # 343 VLRB clones showed no specific reactivity in the monomeric mono and dimer form mIg J, but in the other dimer form mIg CJ and dimer / 4 dimer form.
  • the mIg CJ M showed strong reactivity, especially the dimer / dimer form mIg CJ M showed the strongest reactivity.
  • the fusion protein using the VLRB clones of # 279, # 383 and # 662 did not show specific reactivity in the monomeric mono and dimer form mIg J as described above, but the other dimer forms mIg CJ and 2
  • the mIg CJ M in the merged / conjugated form showed weaker reactivity to the AIV antigen, though weaker than the # 94, # 261 and # 343 VLRB clones.
  • the inventors also found that the mIg CJ M 94, mIg CJ M 261, mIg CJ M 343 proteins exhibiting strong reactivity to AIV through the ELISA results, and the mIg CJ M 18 protein having little reactivity to AIV (negative control). Immunohistochemistry was carried out using to analyze the binding ability to the antigen. Infecting AIV with MDCK (Madin-Darby canine kidney) cells, reacting the mIg CJ M form of VLRB dimer / 4-mer fusion protein, and reacting mouse Fc-binding antibody with Fluorescein isothiocyanate (FITC) to fluorescence staining Proceeded.
  • MDCK Medin-Darby canine kidney
  • the overall cell distribution was confirmed by nuclear staining using DAPI (4 ', 6-diamidino-2-phenylindole).
  • MIg CJ M 18 used as a negative control did not show any reactivity against MDIV cells infected with AIV, but all of the experimental groups treated with mIg CJ M 94, mIg CJ M 261 and mIg CJ M 343 showed strong fluorescence (Fig. 8).
  • the dimer and tetrameric fusion proteins of mIg CJ M 94, mIg CJ M 261 and mIg CJ M 343 using AIV specific clones # 94, # 261 and # 343 VLRB have high binding capacity to the target antigen. It could be confirmed that it has.

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Abstract

La présente invention concerne un anticorps multivalent ayant une force de liaison accrue par rapport à un antigène cible. L'anticorps multivalent a une protéine de fusion formée en un multimère dimérique ou tétramérique, la protéine de fusion ayant un récepteur B des lymphocytes variable dérivé de myxine (VLRB) dont un domaine de queue hydrophobe a été retiré, un domaine Fc d'immunoglobuline, un lieur et une chaîne J d'immunoglobuline connectés séquentiellement.
PCT/KR2018/004465 2017-05-18 2018-04-18 Protéine de fusion ayant un domaine fc dérivé d'anticorps de souris connecté à une protéine vlrb dérivée de myxine dont le domaine de queue hydrophobe a été retiré, et son utilisation Ceased WO2018212468A1 (fr)

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KR1020170061578A KR101934578B1 (ko) 2017-05-18 2017-05-18 소수성 테일 도메인이 제거된 먹장어 유래 VLRB 단백질에 마우스 항체 유래 Fc 도메인이 연결된 융합 단백질 및 이의 용도
KR10-2017-0061578 2017-05-18

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WO2023010060A3 (fr) * 2021-07-27 2023-07-06 Novab, Inc. Anticorps anti-vlrb génétiquement modifiés présentant des fonctions effectrices immunitaires
CN118979017A (zh) * 2024-06-06 2024-11-19 华中农业大学 一种高亲和力鼠抗乌鳢IgM单克隆抗体、杂交瘤细胞株及应用

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