WO2017092645A1 - 一种抗呼吸道合胞病毒的全人源抗体 - Google Patents

一种抗呼吸道合胞病毒的全人源抗体 Download PDF

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WO2017092645A1
WO2017092645A1 PCT/CN2016/107658 CN2016107658W WO2017092645A1 WO 2017092645 A1 WO2017092645 A1 WO 2017092645A1 CN 2016107658 W CN2016107658 W CN 2016107658W WO 2017092645 A1 WO2017092645 A1 WO 2017092645A1
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antibody
seq
rsv
antigen
binding fragment
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French (fr)
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赵雪莲
刘国华
王全英
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Eliteimmune Inc
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Eliteimmune Inc
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Priority to EP16869955.1A priority Critical patent/EP3385278A4/en
Priority to KR1020187017477A priority patent/KR102184151B1/ko
Priority to JP2018527811A priority patent/JP6925336B2/ja
Priority to AU2016363455A priority patent/AU2016363455B2/en
Publication of WO2017092645A1 publication Critical patent/WO2017092645A1/zh
Priority to US15/990,999 priority patent/US10149903B2/en
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    • 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
    • A61K39/12—Viral antigens
    • A61K39/295—Polyvalent viral antigens; Mixtures of viral and bacterial antigens
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C07K16/11—Paramyxoviridae (F); Pneumoviridae (F), e.g. respiratory syncytial virus [RSV]
    • C—CHEMISTRY; METALLURGY
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
    • G01N2333/135—Respiratory syncytial virus

Definitions

  • the present invention relates to anti-respiratory syncytial virus antibodies or antigen-binding fragments thereof, in particular to fully human anti-respiratory syncytial virus (RSV) monoclonal antibodies.
  • RSV anti-respiratory syncytial virus
  • a humanized antibody can be prepared by substituting a corresponding portion of a human antibody with a region of the mouse antibody that is not critical for antigen specificity.
  • the recombinant antibody thus obtained has an immune response (human anti-mouse reaction) in a patient when it is administered to a patient because it contains a residual murine sequence. Therefore, it is desirable to prepare fully human antibodies that do not contain non-human sequences.
  • Humanized antibodies have been reported, for example, using phage display technology to construct and screen human antibody libraries to obtain humanized antibodies, or to implant lymphocytes from immune human donors into severe combined immunodeficient mice (SCID). To obtain a humanized antibody, or to construct a transgenic mouse expressing a human immunoglobulin gene by genetic engineering techniques to obtain a humanized antibody.
  • a fully humanized antibody against a pathogenic antigen can also be obtained by separating a cord blood containing a natural component of IgM by extensive screening of cord blood (for example, see U.S. Patent No. 6,391,635).
  • these methods either obtain antibodies with low affinity or rely on human donors with specific immune responses.
  • Respiratory syncytial virus (RSV, also known as syncytial virus, also belonging to the paramyxoviridae) is the most common cause of viral pneumonia in children, which can cause interstitial pneumonia and bronchiolitis.
  • RSV Respiratory syncytial virus
  • syncytial virus also belonging to the paramyxoviridae
  • syncytial virus In Beijing, 48% of viral pneumonia and 58% of bronchiolitis are caused by syncytial virus (1980-1984); in Guangzhou, 31.4% of pediatric pneumonia and bronchiolitis are caused by syncytial virus (1973-1986) In the United States, 20% to 25% of infant pneumonia and 50% to 75% of bronchiolitis are caused by syncytial virus.
  • the incubation period for RSV infection is 2 to 8 days (mostly 4 to 6 days).
  • a typical observation of syncytial virus pneumonia is interstitial infiltration of monocytes. Mainly manifested as alveolar septum widening and monocyte-based interstitial exudation, including lymphocytes, plasma cells and macrophages.
  • the alveolar cavity is filled with edema fluid and a transparent membrane of the lung is formed.
  • lymphocytic infiltration of the bronchiole wall is also seen.
  • multinucleated fusion cells were seen in the alveolar space, and the morphology was similar to measles giant cells, but no nuclear inclusion bodies were found.
  • RSV has two major surface glycoproteins, F and G. Two glycoproteins (90KDa and 68KDa) were exposed to the surface of the virion. The 90 kDa highly glycosylated G protein is responsible for binding viral particles to target cells. The F protein of 68KDa mediates viral envelope and cell fusion and syncytia formation. The F and G surface glycoproteins are the main protective antigens, and the nuclear protein N and the envelope protein M2 have less protective activity. Monoclonal antibodies against G glycoprotein are less likely to neutralize the virus than monoclonal antibodies against F glycoprotein and do not have inhibitory activity. The amino acid sequence of the F glycoprotein is approximately 90% conserved between the RSV subgroups associated with human infection.
  • the only anti-RSV monoclonal antibody marketed is only approved for the prevention of RSV infection in premature infants. It is an anti-F protein antibody.
  • the name of this antibody is pelizumab Synagis (manufactured by MedImmune), which is a humanized mouse.
  • an anti-RSV antibody or antigen-binding fragment thereof, wherein the antibody comprises:
  • the antibody comprises:
  • Three light chain CDRs having the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16 are selected.
  • said antibody or antigen-binding fragment thereof comprising:
  • the antigen binding fragment of the invention is selected from the group consisting of Fab, Fab', F(ab) 2 , single chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments.
  • a fully human anti-RSV monoclonal antibody R66 is contemplated.
  • the nucleic acid sequences of the heavy chain and light chain (KAPPA) of monoclonal antibody R66 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the amino acid sequences of the corresponding heavy chain and light chain (KAPPA) are respectively SEQ ID. NO: 3 and SEQ ID NO: 4.
  • the present invention also relates to an antibody which is formed by insertion, substitution and/or deletion of the heavy chain and/or light chain amino acid sequence of the above R66 antibody and which has the same function.
  • the present invention also relates to an antigen-binding fragment of the above R66 antibody, particularly an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab) 2 , single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments .
  • an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab) 2 , single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments .
  • the invention also relates to a single domain antibody, chimeric antibody, antibody fusion protein antibody, antibody/antibody fragment-factor fusion protein or antibody/antibody fragment comprising a single heavy chain and/or a single light chain of the above antibody, preferably R66. Chemical conjugate.
  • the invention provides a nucleic acid encoding the above antibody, comprising:
  • nucleic acid of the invention it comprises:
  • an anti-RSV antibody preferably a fully human anti-RSV monoclonal antibody, comprising the steps of:
  • an expression vector comprising a DNA molecule encoding an anti-RSV antibody of the present invention, preferably a fully human anti-RSV monoclonal antibody, and an expression control sequence operably linked to the DNA molecule;
  • the anti-RSV antibody (preferably a fully human anti-RSV monoclonal antibody).
  • a vector comprising a nucleic acid sequence of an anti-RSV antibody of the invention, preferably a fully human anti-RSV monoclonal antibody, and a host cell comprising the nucleic acid sequence or vector are provided.
  • the vector may be a recombinant expression vector.
  • the host cell may be 293T cells, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human liver cancer cells, 549A cells, 3T3 cells, And many other cell lines.
  • CHO Chinese hamster ovary
  • NSO Chinese hamster ovary
  • SP2 cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human liver cancer cells 549A cells
  • 3T3 cells And many other cell lines.
  • an anti-RSV antibody preferably a fully human anti-RSV monoclonal antibody
  • nucleic acid preferably a fully human anti-RSV monoclonal antibody
  • vector or host cell of the present invention for the preparation of a therapeutic agent for preventing or treating a disease associated with RSV .
  • a pharmaceutical composition for treating an RSV-related disease comprising a therapeutically effective amount of an anti-RSV antibody of the present invention (preferably a fully human anti-RSV monoclonal antibody), a nucleic acid, The vector or host cell, and one or more pharmaceutically acceptable carriers or excipients.
  • Pharmaceutically acceptable carriers or excipients are well known to those skilled in the art and include physiologically compatible aqueous and nonaqueous vehicles, stabilizers, preservatives, solubilizers, antioxidants, solvents, dispersion media, outer layers, buffers Liquid, serum protein, etc.
  • the RSV-related disease is selected from the group consisting of viral pneumonia, interstitial pneumonia, and bronchiolitis.
  • an anti-RSV antibody preferably a fully human anti-RSV monoclonal antibody
  • nucleic acid preferably a fully human anti-RSV monoclonal antibody
  • vector or host cell of the invention in the preparation of a detection reagent for detecting RSV.
  • the anti-RSV antibodies (preferably anti-RSV monoclonal antibodies) provided by the present invention are preferably fully human-derived, and have greater immunogenicity due to species differences than other animal-derived (e.g., murine-derived) anti-RSV antibodies. Reduced, good specificity, high affinity, if used in clinical will greatly reduce side effects. Furthermore, since the anti-RSV antibody (preferably a monoclonal antibody) of the present invention can specifically bind to the RSV F antigen, it is also useful in the diagnosis and detection of RSV. When applied to humans, it does not produce side effects similar to those of murine antibodies.
  • Figure 1 is an electrophoresis pattern after purification of R66
  • Figure 2 is a ELISA assay to detect the binding of monoclonal antibody R66 to RSV-F (Strain A2);
  • Figure 3 is a ELISA assay to detect the binding of monoclonal antibody R66 to RSV-F (Strain RSS-2);
  • FIG 4 is the result of analysis of the CDR of monoclonal antibody R66, wherein R66_HC is the heavy chain of R66, R66_KC It is the light chain of R66.
  • the nucleic acid sequence of the light chain (KAPPA) variable region of the monoclonal antibody R66 represented by the sequence of the heavy chain variable region of the monoclonal antibody R66 as shown in SEQ ID NO: 1 is sent to GENEWIZ. The company is synthesizing.
  • the artificially synthesized R66 heavy chain variable region and the light chain variable region were used as templates, and Taq enzyme and dNTPs were separately added, and primers were used for PCR to obtain a PCR product.
  • the PCR product was recovered using a rapid DNA product purification kit (purchased from Kangwei Century) to obtain 40 ⁇ l of PCR product for use.
  • the restriction fragment of the heavy chain variable region and the light chain variable region of R66 were digested respectively.
  • the double digestion system was: 0.5 ⁇ l of NheI/NotI, 3 ⁇ l of 10*FastDigest Green Reaction Buffer, and 26 ⁇ l of PCR product.
  • the temperature was constant at 37 °C. 5h.
  • the recombinant expression vector (the expression vector is pcDNA3.1-Zeo(+) (Invitrogen), and the constant region of the human antibody heavy/light chain was previously transformed into the expression vector by GENEWIZ) for double digestion.
  • the double digestion system was: 0.5 ⁇ l each of NheI/NotI, 3 ⁇ l of 10*FastDigest Green Reaction Buffer, 1 ⁇ g of the vector, 30 ⁇ l with H 2 O, and thermostated at 37 ° C for 30 min. Then, 2 ⁇ l of alkaline phosphatase and 3.5 ⁇ l of 10*buffer (NEB) were added and mixed, and a constant temperature water bath at 37 ° C for 2 h.
  • NheI, NotI, 10*FastDigest Green Reaction buffer used in the above-described target fragment and vector double digestion system were purchased from Thermo Scientific.
  • the heavy chain variable region and the light chain variable region target fragment of the digested R66 were subjected to 1% agarose gel electrophoresis, and the results were observed by a UV instrument. Use a knife to cut the target strip into a weighing Ep tube and use it quickly. Each of the target fragments was recovered by a speed agarose gel DNA recovery kit (purchased from Kangwei Century).
  • Each of the target fragments was ligated to the vector, and the ligation system was: 2 ⁇ l of the vector, 15 ⁇ l of the target fragment, 1 ⁇ l of T4 DNA Ligase (purchased from NEB), 2 ⁇ l of a buffer solution, and mixed, and kept in a constant temperature water bath at 16 ° C for 2 h.
  • a single colony was picked and inoculated into 5 ml of LB liquid medium containing sodium ampicillin (100 ⁇ g/mL), and cultured at 37 ° C for 15 h with shaking.
  • the plasmid was extracted using a high-purity plasmid miniprep kit (purchased from Kangwei Century) to obtain a plasmid containing the heavy chain (RH66) of R66 and the light chain (RK66) of R66, respectively, and sent for sequencing.
  • the sequencing results showed that the nucleic acid sequences of the heavy chain variable region and the light chain (KAPPA) variable region of monoclonal antibody R66 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and their corresponding heavy chain variable regions, The amino acid sequences of the light chain (KAPPA) variable region are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the 293T cells were transfected with the plasmid containing the R66 heavy chain (RH66) obtained in Example 1 and the plasmid containing the light chain of R66 (RK66), respectively.
  • the plasmid and PEI were separately diluted with Opti-MeM (1X) buffer, then the PEI-Opti-MeM mixture was slowly added to the plasmid-Opti-MeM mixture tube, allowed to stand at room temperature for 20 minutes, and then the PEI and plasmid mixture were added. In the cell suspension.
  • the cell concentration at the time of transfection was 0.25 to 0.5 ⁇ 10 6 cells/ml, and transfection of each well was carried out using 2.5 ⁇ g of a plasmid containing R66 heavy chain + 2.5 ⁇ g of a plasmid containing R66 light chain + 10 ⁇ g of PEI. After the end of transfection, the supernatant was harvested after incubation at 37 ° C for 48 h, and the supernatant was detected by ELISA.
  • the expressed antibody protein was purified using rProtein A Sepharose Fast Flow (GE).
  • the supernatant of the 293T cell culture expressing R66 was separately collected, centrifuged at 10,000 rpm for 10 min at 4 ° C, and the supernatant was taken. The supernatant was added to the used equilibration buffer (20 mM phosphate buffer, 150 mM sodium chloride, pH 7.4).
  • Lane M is the standard protein
  • Lane R66 is a purified R66 antibody with two distinct bands, a heavy chain of about 50 KDa and a light chain of about 22 KDa. .
  • the reagents used include:
  • PBS (1X) Configured with PBS (10X) and deionized water.
  • PBST PBS (1X) plus Tween-20 to a final concentration of 0.05%.
  • Blocking solution PBS (1X) + 2% BSA + 2% newborn calf serum, ready to use.
  • Diluent PBST + 1% BSA, diluted antibody.
  • Stop solution 6 ml of 95%-98% concentrated sulfuric acid was slowly added to 180 ml of water, cooled and set aside.
  • the R66 antibody prepared in the present invention was diluted to a working concentration of 10 ⁇ g/ml.
  • RSV-F (Strain A2) and RSV-F (Strain RSS-2) (purchased from Sino Biological Inc.) were diluted with PBS (1X) (pH 7.4) buffer as coating antigen to make the end of the coating antigen solution.
  • the concentration was 2 ng/ ⁇ l, and 100 ⁇ l of the coated antigen solution was added to each well of a 96-well plate, coated at 2-8 ° C overnight, washed 5 times with PBST, then 200 ⁇ l of blocking solution was added to each well, and blocked at 37 ° C for 2 h, closed. After the end, it was washed 5 times with PBST for 1 minute each time.
  • the antibody was diluted 10-fold with antibody dilution (PBST + 1% BSA): 7 autoclaved 1.5 ml centrifuge tubes were added, 270 ⁇ l of antibody dilution was added to each tube, and 30 ⁇ l of primary antibody was taken from the working solution.
  • PBST + 1% BSA antibody dilution
  • 1:10 dilution After the solution was vortexed and mixed, mark it as 1:10 dilution, take 30 ⁇ l from the 1:10 diluted solution into the next tube, and so on, 1:10, 1:100, 1: 1000, 1:10000, 1:10000000, 1:1000000, 1:10000000, respectively, added to the corresponding wells, 100 ⁇ l per well, do two parallels, and set up two blank wells to replace the primary antibody with PBST, incubate at 37 °C 90 min, then washed 5 times with PBST.
  • the antibody was diluted 5000-fold with antibody dilution (PBST+1% BSA), 100 ⁇ l per well, incubated at 37 °C for 1 h, then washed 5 times with PBST, added TMB coloring solution, 100 ⁇ l/well, and incubated for 15 min at room temperature. After the end of the incubation, the reaction was terminated by the addition of H 2 SO 4 stop solution. Read the OD 450 reading on the microplate reader.
  • the R66 antibody binding RSV-F (Strain A2) EC 50 value 0.001791 ⁇ g / mL, were lower than the values of Synagis 50 EC (0.004881 ⁇ g / mL, ELISA was performed under the same conditions with Synagis And obtained by analysis with GraphPad Prism software).
  • EC 50 values of R66 binding of the antibody to RSV-F (Strain RSS-2 ) were 0.001463 ⁇ g / mL, 50 is also lower than the values of Synagis EC (0.001878 ⁇ g / mL, with the same Synagis
  • the conditions were subjected to ELISA and analyzed by GraphPad Prism software). This indicates that the R66 antibody of the present invention has higher sensitivity than Synagis.
  • the antibody variable region sequence information was introduced into the IgBLAST program (version 1.6.1) and compared with the human variable region original sequence library.
  • the variable region of the antibody was further divided into three framework regions (FRs) and two complementarities. Decision Areas (CDRs).
  • the antibody sequences were then introduced into the IMGT High V-Quest system for alignment to identify CDR3 and FR4 using the same library.
  • the digital calibration of all antibody sequences is based on the KABAT system. The results of the analysis comparison are shown in Figure 4.
  • amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 of the monoclonal antibody are SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10, respectively, and the corresponding nucleotide sequences are SEQ ID NO, respectively. : 5, SEQ ID NO: 7 and SEQ ID NO: 9; the amino acid sequences of the light chain CDR1, CDR2 and CDR3 are SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16, respectively, corresponding nucleosides
  • the acid sequences are SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, respectively.

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Abstract

针对RSV的抗体(优选为全人源抗体R66)、其编码核酸、包含它的载体和宿主细胞以及它的制备方法。上述针对RSV的抗体(优选为全人源抗体R66)在预防和治疗RSV相关疾病中的应用，以及它们在检测RSV中的应用。上述针对RSV的抗体优选为是全人源的单克隆抗体，与其它动物源性(如鼠源性)的抗RSV抗体相比，因物种差异导致的免疫原性大大减少，且特异性好、亲和力高，如果用于临床将大大降低副作用。

Description

一种抗呼吸道合胞病毒的全人源抗体 技术领域
本发明涉及抗呼吸道合胞病毒抗体或其抗原结合片段，特别是全人源的抗呼吸道合胞病毒(RSV)单克隆抗体。
背景技术
人源化抗体可以通过用人源抗体的相应部分，取代小鼠抗体对抗原特异性不重要的区域而制备得到。由此得到的重组抗体由于其含有残余的鼠类序列，因此在将其给予病人时，往往会在病人中引发免疫应答反应(人抗鼠反应)。因此，希望制备出不含非人类序列的全人源抗体。人源化抗体已经有过报道，例如利用噬菌体展示技术构建和筛选人源抗体文库，以获得人源化抗体，或者将来自免疫人类供体的淋巴细胞植入重度联合免疫缺陷小鼠(SCID)，以获得人源化抗体，或者利用基因工程技术构建表达人类免疫球蛋白基因的转基因小鼠，以获得人源化抗体。针对致病抗原的完全人源化抗体，也可以通过对脐带血的大量筛选，将包含IgM天然全组分的脐带血进行分离而得到(例如，参见美国专利No.6,391,635)。然而上述这些方法，或者得到的是亲和力低的抗体，或者需要依赖具有特定免疫应答的人类供体。
呼吸道合胞病毒(RSV，简称合胞病毒，也属副粘病毒科)是引起小儿病毒性肺炎最常见的病原，可引起间质性肺炎，及毛细支气管炎。在北京，48％的病毒性肺炎和58％的毛细支气管炎系由合胞病毒引起(1980～1984)；在广州，小儿肺炎及毛细支气管炎的31.4％由合胞病毒引起(1973～1986)；在美国，20％～25％的婴幼儿肺炎和50％～75％的毛细支气管炎由合胞病毒引起。
RSV感染的潜伏期为2～8天(多为4～6天)。合胞病毒肺炎的典型所见是单核细胞的间质浸润。主要表现为肺泡间隔增宽和以单核细胞为主的间质渗出，其中包括淋巴细胞、浆细胞和巨噬细胞。此外肺泡腔充满水肿液，并可见肺透明膜形成。在一些病例，亦可见细支气管壁的淋巴细胞浸润。在肺实质出现伴有坏死区的水肿，导致肺泡填塞、实变和萎 陷。少数病例在肺泡腔内可见多核融合细胞，形态与麻疹巨细胞相仿，但找不到核内包涵体。
RSV有两个主要的表面糖蛋白，F和G。两个糖蛋白(90KDa和68KDa)暴露于病毒粒子表面。90KDa的高度糖基化的G蛋白负责将病毒颗粒结合到靶细胞上。68KDa的F蛋白介导病毒被膜与细胞融合和合胞体形成。所述的F和G表面糖蛋白为主要的保护性抗原，核蛋白N和被膜蛋白M2具有较小的保护性活性。抗G糖蛋白的单克隆抗体比抗F糖蛋白的单克隆抗体不太可能中和病毒，且不具有抑制融合活性。F糖蛋白的氨基酸序列在与人感染有关的RSV亚组间有大约90％的保守性。
目前唯一上市的抗RSV单克隆抗体只批准用于预防早产儿感染RSV，是抗F蛋白的抗体，该抗体的名称是帕利珠单抗Synagis(MedImmune制造)，是一种人源化的鼠单克隆抗体，该药通过呼吸道合胞病毒融合蛋白阻止病毒向下呼吸道扩散。
发明内容
本发明的目的是提供抗RSV抗体或其抗原结合片段，优选为全人源抗RSV单克隆抗体或其抗原结合片段。
根据本发明的一个方面，涉及抗RSV抗体或其抗原结合片段，其中所述抗体包含：
至少一个重链CDR，其具有选自SEQ ID NO：6、SEQ ID NO：8和SEQ ID NO：10所示的氨基酸序列；和/或
至少一个轻链CDR，其具有选自SEQ ID NO：12、SEQ ID NO：14和SEQ ID NO：16所示的氨基酸序列。
进一步地，对于所述的抗体或其抗原结合片段，其中所述抗体包含：
选自具有如SEQ ID NO：6、SEQ ID NO：8和SEQ ID NO：10所示的氨基酸序列的三个重链CDR；和/或
选自具有如SEQ ID NO：12、SEQ ID NO：14和SEQ ID NO：16所示的氨基酸序列的三个轻链CDR。
优选地，对于所述的抗体或其抗原结合片段，其包含：
具有如SEQ ID NO：3所示的氨基酸序列的重链可变区和/或具有如SEQ ID NO：4所示的氨基酸序列的轻链可变区，所述抗体是全人源抗RSV单克隆抗体R66。
任选地，本发明所述的抗原结合片段选自Fab、Fab'、F(ab)2、单链Fv(scFv)、Fv、dsFv、 双抗体、Fd和Fd'片段。
根据本发明的另一个方面，涉及全人源抗RSV单克隆抗体R66。单克隆抗体R66的重链、轻链(KAPPA)的核酸序列分别如SEQ ID NO：1和SEQ ID NO：2所示，其相应的重链、轻链(KAPPA)的氨基酸序列分别如SEQ ID NO：3和SEQ ID NO：4所示。
本发明还涉及通过对上述R66抗体的重链和/或轻链氨基酸序列的插入、取代和/或缺失形成的并具有相同功能的抗体。
本发明还涉及上述R66抗体的抗原结合片段，特别是选自Fab、Fab'、F(ab)2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd'片段的抗原结合片段。
本发明还涉及含有上述抗体(优选为R66)的单个重链和/或单个轻链的单域抗体、嵌合抗体、抗体融合蛋白抗体、抗体/抗体片段-因子融合蛋白或抗体/抗体片段-化学偶联物。
本发明提供编码上述抗体的核酸，其包含：
至少一个编码重链CDR的核苷酸序列，其选自SEQ ID NO：5、SEQ ID NO：7和SEQ ID NO：9；和/或
至少一个编码轻链CDR的核苷酸序列，其选自SEQ ID NO：11、SEQ ID NO：13和SEQ ID NO：15。
优选地，对于本发明所述的核酸，其包含：
如SEQ ID NO：1所示的编码重链可变区的核酸和/或如SEQ ID NO：2所示的编码轻链可变区的核酸。
根据本发明的另一个方面，提供制备抗RSV抗体(优选为全人源抗RSV单克隆抗体)的方法，包括如下步骤：
(1)提供表达载体，所述表达载体含有编码本发明的抗RSV抗体(优选为全人源抗RSV单克隆抗体)的DNA分子，以及与所述DNA分子可操作相连的表达调控序列；
(2)用所述表达载体转化宿主细胞；
(3)在适合所述抗RSV抗体(优选为全人源抗RSV单克隆抗体)表达的条件下培养所述宿主细胞；
(4)分离纯化获得所述的抗RSV抗体(优选为全人源抗RSV单克隆抗体)。
根据本发明的另一个方面，提供包含本发明的抗RSV抗体(优选为全人源抗RSV单克隆抗体)的核酸序列的载体以及包含所述核酸序列或载体的宿主细胞。
所述载体可以是重组表达载体。
所述宿主细胞可以是293T细胞、中国仓鼠卵巢(CHO)细胞、NS0、SP2细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝癌细胞、549A细胞、3T3细胞、以及许多其他细胞系。
根据本发明的另一个方面，提供本发明的抗RSV抗体(优选为全人源抗RSV单克隆抗体)、核酸、载体或宿主细胞在制备用于预防或治疗RSV相关疾病的治疗剂中的应用。
根据本发明的另一方面，提供用于治疗RSV相关疾病的药物组合物，药物组合物中包含治疗有效量的本发明的抗RSV抗体(优选为全人源抗RSV单克隆抗体)、核酸、载体或宿主细胞，和一种或多种药学上可接受的载体或赋形剂。药学上可接受的载体或赋形剂是本领域技术人员熟知的，包括生理相容的水性和非水性载体、稳定剂、防腐剂、增溶剂、抗氧化剂、溶剂、分散介质、外衣层、缓冲液、血清蛋白等。
所述RSV相关疾病选自由病毒性肺炎、间质性肺炎、毛细支气管炎组成的组。
根据本发明的另一方面，涉及本发明的抗RSV抗体(优选为全人源抗RSV单克隆抗体)、核酸、载体或宿主细胞在制备用于检测RSV的检测试剂中的应用。
本发明提供的抗RSV抗体(优选为抗RSV单克隆抗体)优选是全人源的，与其它动物源性(如鼠源性)的抗RSV抗体相比，因物种差异导致的免疫原性大大减少，且特异性好、亲和力高，如果用于临床将大大降低副作用。此外，由于本发明中的抗RSV抗体(优选为单克隆抗体)可以和RSV F抗原特异性结合，在RSV的诊断和检测中也有很好的应用。应用于人时不会产生类似鼠抗体所产生的副作用。
附图说明
图1是R66纯化后的电泳图；
图2是ELISA实验检测单克隆抗体R66与RSV-F(Strain A2)的结合；
图3是ELISA实验检测单克隆抗体R66与RSV-F(Strain RSS-2)的结合；
图4是单克隆抗体R66的CDR的分析结果，其中R66_HC为R66的重链，R66_KC 为R66的轻链。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明了，下面结合具体实施方式并参照附图，对本发明进一步详细说明。应该理解，这些描述只是示例性的，而并非要限制本发明的范围。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本发明的概念。
实施例1 全人源抗RSV抗体R66的制备
1、人工合成R66的重链可变区和轻链可变区
按照序列如SEQ ID NO：1所示的单克隆抗体R66的重链可变区、序列如SEQ ID NO：2所示的单克隆抗体R66的轻链(KAPPA)可变区的核酸序列送GENEWIZ公司人工合成。
将人工合成得到的R66重链可变区、轻链可变区作为模板，分别加入Taq酶和dNTPs，以及引物，进行PCR，得到PCR产物。
2、重组抗体的表达载体的构建
利用快速DNA产物纯化试剂盒(购自康为世纪)回收PCR产物，得到40μl PCR产物待用。
分别对R66的重链可变区、轻链可变区目的片段进行双酶切，双酶切体系为：NheⅠ/NotⅠ各0.5μl、10*FastDigest Green Reaction Buffer 3μl以及PCR产物26μl，37℃恒温5h。
对改造好的表达载体(表达载体为pcDNA3.1-Zeo(+)(Invitrogen公司)，由GENEWIZ公司预先将人抗体重链/轻链的恒定区改造到此表达载体中)进行双酶切，双酶切体系为：NheⅠ/NotⅠ各0.5μl、10*FastDigest Green Reaction Buffer 3μl、载体1μg、用H2O补齐30μl，37℃恒温30min。然后加入2μl碱性磷酸酶和3.5μl 10*buffer(NEB)混匀，37℃恒温水浴2h。
上述目的片段和载体的双酶切体系中所使用的NheⅠ、NotⅠ、10*FastDigest Green Reaction buffer均购自Thermo Scientific。
对将酶切后的R66的重链可变区、轻链可变区目的片段分别进行1％琼脂糖凝胶电泳，并通过紫外仪观察结果。用小刀将目的条带切下放入一个称量好重量的Ep管中，利用快 速琼脂糖凝胶DNA回收试剂盒(购自康为世纪)回收各个目的片段。
将各个目的片段分别和载体进行连接，连接体系为：载体2μl、目的片段15μl、T4DNA Ligase(购自NEB)1μl、缓冲液2μl，混匀后16℃恒温水浴保持2h。
将每个目的片段的所有连接产物加入到E.coli DH5α感受态细胞中，轻轻混匀，冰浴30min。42℃热激90s后，迅速置于冰浴5min。然后加入800μL培养基，37℃振荡(100r/min)温育1h。将培养菌液10000rpm离心15s，去除800ul上清，重悬沉淀，全部涂布于含氨苄青霉素钠(100μg/mL)的LB固体培养基上,正向放入37℃培养箱中培养1h，再倒置培养过夜至菌落清晰。挑取单菌落接种到5ml含氨苄青霉素钠(100μg/mL)的LB液体培养基中，37℃振荡培养15h。利用高纯度质粒小提试剂盒(购自康为世纪)提取质粒，获得分别含有R66的重链(RH66)、R66的轻链(RK66)的质粒，并送样测序。
测序结果显示单克隆抗体R66的重链可变区、轻链(KAPPA)可变区的核酸序列分别如SEQ ID NO：1和SEQ ID NO：2所示，其相应的重链可变区、轻链(KAPPA)可变区的氨基酸序列分别如SEQ ID NO：3和SEQ ID NO：4所示。
实施例2 单克隆抗体R66的表达和纯化
用实施例1中获得的含有R66的重链(RH66)的质粒和含有R66的轻链(RK66)的质粒分别转染293T细胞。先用Opti-MeM(1X)buffer分别稀释质粒和PEI，然后将PEI-Opti-MeM混合物缓慢加入到质粒-Opti-MeM混合物管中，室温静置20分钟后，再将PEI和质粒混合物加入到细胞悬液中。转染时细胞浓度为0.25～0.5×106个细胞/ml，每孔细胞的转染使用2.5μg含有R66重链的质粒+2.5μg含有R66轻链的质粒+10μg PEI。转染结束后在37℃培养48h后收获上清，用ELISA检测上清。
使用rProtein A Sepharose Fast Flow(GE)纯化表达的抗体蛋白。分别收集表达R66的293T细胞培养物上清，10000rpm，4℃离心10min，取上清，将该上清加入到已用平衡缓冲液(20mM磷酸盐缓冲液，150mM氯化钠，pH7.4)平衡好的rProtein A Sepharose Fast Flow柱上，用同样的平衡缓冲液洗10个床体积，再用洗脱缓冲液(0.1M Gly-HCl缓冲液，pH2.5)流洗5个床体积，收集前3个床体积，向收集的洗脱液中加入1/10提及的中和液(1M Tris-HCl缓冲液，pH9.0)混匀，加入到Amicon Ultra-15Centrifugal Filters(Merck Millipore)中，5000G、4℃离心20min，浓缩蛋白，再向Amicon Ultra-15Centrifugal Filters中加入 上述平衡缓冲液，5000G、4℃离心20min，更换新的平衡缓冲液，重复3次，分别获得浓缩后的R66抗体蛋白，将其分别转到1.5ml离心管中，取样测定蛋白质含量，然后置于4℃保存。
分别取纯化的R66抗体进行电泳，结果如图1所示，其中泳道M是标准蛋白；泳道R66是纯化的R66抗体，有两个明显条带，分别是约50KDa的重链和约22KDa的轻链。
实施例3 R66抗体的ELISA检测
所用试剂包括：
PBS(1X)：用PBS(10X)和去离子水配置。
PBST：PBS(1X)加Tween-20至终浓度为0.05％。
封闭液：PBS(1X)+2％BSA+2％新生小牛血清，现用现配。
稀释液：PBST+1％BSA，稀释抗体用。
终止液：将6ml 95％-98％的浓硫酸慢慢加入180ml水中，冷却后备用。
一抗：本发明制备得到的R66抗体，稀释成工作浓度为10μg/ml。
二抗：Peroxidase-conjugated AffiniPure Goat Anti-Human IgG(H+L)(购自Jackson Immuno Research)，取1.5ml的无RNase水完全溶解后，备用。
用PBS(1X)(pH7.4)缓冲液稀释RSV-F(Strain A2)和RSV-F(Strain RSS-2)(购自Sino Biological Inc.)作为包被抗原，使包被抗原溶液的终浓度为2ng/μl，吸取100μl包被抗原溶液加入到96孔板的每孔中，2-8℃包被过夜，用PBST清洗5次，然后每孔加入200μl封闭液，37℃封闭2h，封闭结束后用PBST清洗5次，每次停留1分钟。
用抗体稀释液(PBST+1％BSA)将一抗做10倍稀释：即取7个高压灭菌的1.5ml离心管，每一管加入270μl的抗体稀释液，从工作溶液中取30μl一抗溶液，涡旋震荡混匀后，标记为1:10稀释，从1:10稀释的溶液中取30μl放入下一管中，以此类推，分别为：1:10，1:100，1:1000，1:10000，1:100000，1:1000000，1:10000000，分别加到相应的孔中，每孔100μl，做两个平行，并设立两个空白孔用PBST代替一抗，37℃孵育90min，然后用PBST清洗5遍。
用抗体稀释液(PBST+1％BSA)将二抗做5000倍稀释，每孔加入100μl，37℃孵育1h，然后用PBST清洗5遍，加入TMB显色液，100μl/孔，室温孵育15min，孵育结束后加入 H2SO4终止液终止反应。在酶标仪上读取OD450读数。
通过GraphPad Prism软件分析上述ELISA结果，获得R66抗体以及Synagis抗体的EC50值。
结果显示，表达和纯化的R66抗体与RSV-F(Strain A2)和RSV-F(Strain RSS-2)的结合具有剂量依赖性，说明R66抗体与RSV-F(Strain A2)和RSV-F(Strain RSS-2)的结合是特异性的。
从附图2可以看出，R66抗体与RSV-F(Strain A2)结合的EC50值为0.001791μg/mL，均低于Synagis的EC50值(0.004881μg/mL，用Synagis以相同条件进行ELISA并用GraphPad Prism软件分析获得)。从附图3可以看出，R66抗体与RSV-F(Strain RSS-2)结合的EC50值分别为0.001463μg/mL，也低于Synagis的EC50值(0.001878μg/mL，用Synagis以相同条件进行ELISA并用GraphPad Prism软件分析获得)。由此说明，本发明的R66抗体比Synagis具有更高的灵敏度。
实施例4 单克隆抗体R66的CDR的测定：测定单克隆抗体R66的重链CDR和轻链CDR
将抗体可变区域序列信息导入IgBLAST程序(version 1.6.1)中与人类可变区域原始序列文库进行对比分析，抗体的可变区域被进一步划分出3个构架区(FRs)和2个互补性决定区(CDRs)。然后将抗体序列导入IMGT High V-Quest系统中使用之前相同的文库进行比对确定出CDR3和FR4。所有抗体序列的数字标定都是基于KABAT系统。分析比对结果如图4所示。
结果表明，该单克隆抗体的重链CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO：6、SEQ ID NO：8和SEQ ID NO：10，其对应的核苷酸序列分别为SEQ ID NO：5、SEQ ID NO：7和SEQ ID NO：9；轻链CDR1、CDR2和CDR3的氨基酸序列分别为SEQ ID NO：12、SEQ ID NO：14和SEQ ID NO：16，其对应的核苷酸序列分别为SEQ ID NO：11、SEQ ID NO：13和SEQ ID NO：15。
应当理解的是，本发明的上述具体实施方式仅仅用于示例性说明或解释本发明的原理，而不构成对本发明的限制。因此，在不偏离本发明的精神和范围的情况下所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。此外，本发明所附权利要求旨在涵盖落入所附权利要求范围和边界、或者这种范围和边界的等同形式内的全部变化 和修改例。
Figure PCTCN2016107658-appb-000001
Figure PCTCN2016107658-appb-000002
Figure PCTCN2016107658-appb-000003
Figure PCTCN2016107658-appb-000004
Figure PCTCN2016107658-appb-000005

Claims (17)

  1. 抗RSV的抗体或其抗原结合片段，其中所述抗体包含：
    至少一个重链CDR，其具有选自SEQ ID NO：6、SEQ ID NO：8和SEQ ID NO：10所示的氨基酸序列；和/或
    至少一个轻链CDR，其具有选自SEQ ID NO：12、SEQ ID NO：14和SEQ ID NO：16所示的氨基酸序列。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其中所述抗体包含：
    选自具有如SEQ ID NO：6、SEQ ID NO：8和SEQ ID NO：10所示的氨基酸序列的三个重链CDR；和/或
    选自具有如SEQ ID NO：12、SEQ ID NO：14和SEQ ID NO：16所示的氨基酸序列的三个轻链CDR。
  3. 根据权利要求2所述的抗体或其抗原结合片段，其中所述抗体包含：
    具有如SEQ ID NO：3所示的氨基酸序列的重链可变区和/或具有如SEQ ID NO：4所示的氨基酸序列的轻链可变区。
  4. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，其中所述抗原结合片段选自Fab、Fab'、F(ab)2、scFv、Fv、dsFv、双抗体、Fd和Fd'片段。
  5. 包含权利要求1-4中任一项所述的的抗体的单个重链、单个轻链或其抗原结合片段的单域抗体、嵌合抗体、抗体融合蛋白抗体、抗体/抗体片段-因子融合蛋白或抗体/抗体片段-化学偶联物。
  6. 编码权利要求1-4任一项所述的抗体或其抗原结合片段的核酸。
  7. 根据权利要求6所述的核酸，其包含：
    至少一个编码重链CDR的核苷酸序列，其选自SEQ ID NO：5、SEQ ID NO：7和SEQ ID NO：9；和/或
    至少一个编码轻链CDR的核苷酸序列，其选自SEQ ID NO：11、SEQ ID NO：13和SEQ ID NO：15。
  8. 根据权利要求7所述的核酸，其包含：
    如SEQ ID NO：1所示的编码重链可变区的核酸和/或如SEQ ID NO：2所示的编码轻链可变区的核酸。
  9. 含有权利要求6-8任一项所述的核酸的载体。
  10. 含有权利要求6-8任一项所述的核酸或权利要求9所述的载体的宿主细胞。
  11. 根据权利要求10所述的宿主细胞，其是用权利要求9所述的载体转化的宿主细胞。
  12. 权利要求1-4任一项所述的抗体或其抗原结合片段、权利要求5所述的抗体或融合蛋白或偶联物、权利要求6-8任一项所述的核酸、权利要求9所述的载体或权利要求10-11任一项所述的宿主细胞在制备用于预防或治疗RSV相关疾病的治疗剂中的应用。
  13. 根据权利要求12所述的应用，其中所述RSV相关疾病选自由病毒性肺炎、间质性肺炎、毛细支气管炎组成的组。
  14. 权利要求1-4任一项所述的抗体或其抗原结合片段、权利要求6-8任一项所述的核酸、权利要求9所述的载体或权利要求10-11任一项所述的宿主细胞在制备用于检测RSV的检测试剂中的应用。
  15. 用于治疗RSV相关疾病的药物组合物，包含治疗有效量的权利要求1-4任一项所述的抗体或其抗原结合片段、权利要求6-8任一项所述的核酸、权利要求9所述的载体或权利要求10-11任一项所述的宿主细胞，和一种或多种药学上可接受的载体或赋形剂。
  16. 制备抗RSV抗体或其抗原结合片段的方法，包括如下步骤：
    (1)提供表达载体，所述表达载体含有编码权利要求1-4任一项所述的抗体或其抗原结合片段的DNA分子，以及与所述DNA分子可操作相连的表达调控序列；
    (2)用所述表达载体转化宿主细胞；
    (3)在适合所述抗RSV抗体或其抗原结合片段表达的条件下培养所述宿主细胞；
    (4)分离纯化获得所述的抗RSV抗体或其抗原结合片段。
  17. 根据权利要求16所述的方法，其中所述抗RSV抗体是全人源抗RSV单克隆抗体。
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