WO2022110742A1 - Anticorps humanisé dirigé contre de nouveaux peptides antigéniques spécifiques du coronavirus, procédé de préparation et utilisation - Google Patents

Anticorps humanisé dirigé contre de nouveaux peptides antigéniques spécifiques du coronavirus, procédé de préparation et utilisation Download PDF

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WO2022110742A1
WO2022110742A1 PCT/CN2021/097706 CN2021097706W WO2022110742A1 WO 2022110742 A1 WO2022110742 A1 WO 2022110742A1 CN 2021097706 W CN2021097706 W CN 2021097706W WO 2022110742 A1 WO2022110742 A1 WO 2022110742A1
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amino acid
antibody
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杨衡
李利利
高美玲
王雅楠
段静
万定一
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苏州方科生物科技有限公司
苏州系统医学研究所
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
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    • 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
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    • 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
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present disclosure belongs to the field of biomedicine, and relates to a human antibody bound to a novel coronavirus-specific antigen peptide and uses thereof. Specifically, the present disclosure relates to a monoclonal antibody that specifically binds to the RBD domain of the SARS-CoV-2 virus, which is located in the S protein of the SARS-CoV-2 virus, and the aforementioned antigenic peptide thereof is prepared Use in COVID-19 vaccine, preparation of medicaments for prevention and treatment of COVID-19.
  • the novel coronavirus belongs to the genus betacoronavirus, a linear single-stranded RNA (ssRNA) virus. Its genome is about 29903 nucleotides in length and contains 10 genes. Since January 10, 2020, the first SARS-CoV-2 genome sequence data was released, and since then, the genome sequences of multiple new coronaviruses isolated from patients have been released. On January 22, 2020, the Genome Science Data Center officially released the 2019 Novel Coronavirus Resource Library.
  • the 2019 novel coronavirus (SARS-CoV-2) is 80% similar to the genome sequence of the SARS virus that broke out in 2003, which is similar to the Bat SARS-like coronavirus isolated bat collected from domestic bats in February 2017 -SL-CoVZC45 has the highest genome sequence similarity with 88% similarity.
  • SARS-CoV-2 the 2019 novel coronavirus
  • the novel coronavirus is an enveloped positive-strand RNA virus containing a 30kb genome and four structural proteins, namely spike protein (S), envelope protein (E), membrane protein (M). ) and nucleocapsid protein (N).
  • S spike protein
  • E envelope protein
  • M membrane protein
  • N nucleocapsid protein
  • the S protein regulates viral attachment to receptors on target host cells.
  • the function of the E protein is to assemble the virus and act as an ion channel; the M protein, together with the E protein, plays a role in virus assembly and participates in the biosynthesis of new virus particles; the N protein forms a ribonucleoprotein complex with viral RNA.
  • the surface spike glycoprotein (S protein) of the novel coronavirus is responsible for attachment to host cells through interactions with host cell surface receptors (ACE2).
  • the S protein exists in the form of homotrimers, each monomer containing more than 1200 amino acids.
  • a small domain containing residues 306-575 was identified as the receptor-binding domain (RBD), of which residues 439-508 were termed the receptor-binding motif (RBM)
  • RBD receptor-binding domain
  • the base directly mediates the interaction with ACE2.
  • the entry of coronaviruses into cells depends on the binding of viral spike proteins to cellular receptors and the initiation of S protein by host cell proteases. Elucidating which cytokines are utilized by 2019-nCoV may provide new ideas for the spread of the virus and the discovery of therapeutic targets.
  • SARS-CoV-2 Spike protein consists of S1 domain and S2 domain.
  • S1 contains a receptor binding domain (RBD) that can specifically bind to the receptor on target cells, angiotensin-converting enzyme 2 (ACE2), which is the most critical step in its infection process. Therefore, it is generally believed that SARS-CoV-2 Spike Protein (RBD) has potential value in the diagnosis of the virus.
  • Recombinant RBD protein vaccine is one of the important new crown vaccine options.
  • Phage display technology was originally developed by the British Medical Research Council (Medical Research Council) in 1990 by preparing a human antibody library (library) and expressing it on the surface of phage in the form of antibody fragments (Fab, ScFv), thereby Antibody cloning techniques for screening specific antigens. It has been proposed that almost all recombinant human monoclonal antibodies that react specifically with antigens can be screened from the single-pot antibody library system. Therefore, when phage-displayed antibody technology is used, it is possible to obtain in vivo diagnostic or therapeutic applications. Various antibody fragments (Fab or ScFv).
  • a phage human antibody library is constructed from the PBMC of COVID-19 patients, and the antibody cloning technology for screening specific antigens by specifically binding to the RBD domain of the spike protein.
  • the present disclosure provides an anti-SARS-CoV-2 antibody or an antigen-binding fragment thereof that can specifically bind to the RBD domain of the SARS-CoV-2 virus.
  • the RBD domain is one of the key factors for the SARS-CoV-2 virus to invade cells. By specifically binding to RBD, it can block the invasion of the new coronavirus to cells, and realize the treatment, prevention or diagnosis of the new coronavirus.
  • an anti-SARS-CoV-2 antibody or an antigen-binding fragment thereof specifically binds to the SARS-CoV-2 epitope, wherein the SARS-CoV-2 epitope comprises as shown in SEQ ID NO: Sequence shown in 1.
  • the antibody or antigen-binding fragment thereof according to (1) comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising a VH complementarity determining region (CDR) 1, VH complementarity determining region (CDR) 2 and VH complementarity determining region (CDR) 3, and said VL comprises VLCDR1, VLCDR2 and VLCDR3, wherein,
  • VHCDR1 comprises the amino acid sequence shown in SEQ ID NO:39
  • VHCDR2 comprises the amino acid sequence shown in SEQ ID NO:40
  • VHCDR3 comprises the amino acid sequence shown in SEQ ID NO:41 ;
  • VLCDR1 comprises as described in any one of SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54 or SEQ ID NO:57
  • VLCDR2 comprises the amino acid shown in any one of SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55 or SEQ ID NO:58 sequence
  • VLCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, or SEQ ID NO:59.
  • VH comprises the amino acid sequence set forth in SEQ ID NO: 15, and VL comprises the amino acid sequence set forth in SEQ ID NO: 17;
  • VH comprises the amino acid sequence set forth in SEQ ID NO: 19
  • VL comprises the amino acid sequence set forth in SEQ ID NO: 21;
  • VH comprises the amino acid sequence set forth in SEQ ID NO:23
  • VL comprises the amino acid sequence set forth in SEQ ID NO:25;
  • VH comprises the amino acid sequence set forth in SEQ ID NO:27
  • VL comprises the amino acid sequence set forth in SEQ ID NO:29
  • VH comprises the amino acid sequence set forth in SEQ ID NO:31
  • VL comprises the amino acid sequence set forth in SEQ ID NO:33;
  • VH comprises the amino acid sequence set forth in SEQ ID NO:35
  • VL comprises the amino acid sequence set forth in SEQ ID NO:37;
  • (b) comprising, for example, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: : 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 or SEQ ID NO: 38
  • SEQ ID NO: 16 comprising, for example, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: : 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 or SEQ ID NO: 38
  • SEQ ID NO: 16 comprising, for example, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: : 30, SEQ ID NO: 32, SEQ ID NO: 34
  • nucleotide sequence shown in any one of (a)-(c) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequences of sequence identity.
  • nucleotide sequence shown in any one of (e)-(g) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequences of sequence identity.
  • nucleotide sequence shown in any one of (i)-(k) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequences of sequence identity.
  • a method for preparing a host cell stably expressing a target protein comprising the vector described in (10), the step of transforming an initial host cell; optionally, the host cell is a Chinese hamster ovary cell .
  • a method for producing a target protein comprising using the host cell described in (11) or by the method described in (12), producing the target protein.
  • a kit comprising the antibody or antigen-binding fragment thereof according to any one of (1)-(6) or (14).
  • a pharmaceutical composition or vaccine wherein the pharmaceutical composition or vaccine contains the antibody or antigen-binding fragment thereof according to any one of (1)-(6) or (14).
  • the present disclosure provides a human antibody that can specifically bind to the novel coronavirus.
  • the antibody By binding to the RBD domain of the novel coronavirus, the antibody can block the invasion of the novel coronavirus into cells, thereby realizing the protection against the novel coronavirus. Prevention or treatment of viruses.
  • the detection of the virus can also be realized for the clinical diagnosis of patients with new coronary pneumonia.
  • the present disclosure provides a polynucleotide encoding a human antibody that can specifically bind to a novel coronavirus, a vector comprising the polynucleotide, a host cell, and the like, which can realize the expression and preparation of monoclonal antibodies.
  • the present disclosure provides methods for making human antibodies capable of specifically binding to the novel coronavirus.
  • Figure 1 shows the PCR agarose gel electrophoresis image when constructing the VL and VH-VL libraries.
  • the library includes PBMCs of new crown patients and normal human PBMCs as a control group.
  • Figure 2 shows the quality report of the sequencing of the phage-displayed library, including the library constructed from PBMCs of patients with COVID-19 and normal human PBMCs.
  • Figure 3 shows the results of the recombinant plasmid RBD-PATX2 agarose gel electrophoresis verification, and the purified RBD SDS-PAGE electrophoresis to verify the purity, and the purity is greater than 90%.
  • Figure 4 shows the ELISA results at different dilution concentrations of the antibody.
  • Figures 5A-5H show the results of antibody neutralization experiments, and Figure 5A shows the inhibition rate of the screened antibody sequences against SARS-CoV-2 pseudovirus;
  • Figures 5B-5H show the antibodies RBD-R3P1-A12, RBD -R3P2-A2, RBD-R3P2-B5, RBD-R3P1-B6, RBD-R3P1-E4 and R3P2-G1 against SARS-CoV-2 euvirus ( Figures 5B-5H: SARS-CoV2-NP or SARS-CoV -2-NP) inhibition rate test results.
  • SARS-CoV-2 also known as “2019-nCoV”
  • 2019-nCoV means the 2019 novel coronavirus.
  • COVID-19 means Corona Virus Disease 2019, referred to as “COVID-19”, which refers to pneumonia caused by 2019 novel coronavirus (SARS-CoV-2) infection .
  • SARS-CoV-2 2019 novel coronavirus
  • Sequence identity and “percent identity” in the present disclosure refer to the percentage of nucleotides or amino acids that are identical (ie, identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides and The number of positions containing the same nucleotide or amino acid residue is scored and compared to the number of positions containing different nucleotide or amino acid residues in the aligned polynucleotides or polypeptides.
  • Polynucleotides can differ at a position, eg, by containing different nucleotides (ie, substitutions or mutations) or deletions of nucleotides (ie, insertions of nucleotides or deletions of nucleotides in one or both polynucleotides).
  • Polypeptides can differ at one position, for example, by containing different amino acids (ie, substitutions or mutations) or missing amino acids (ie, amino acid insertions or amino acid deletions in one or both polypeptides).
  • Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in a polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.
  • phage display technology in the present disclosure refers to inserting the DNA sequence of exogenous protein or polypeptide into the appropriate position of the phage coat protein structural gene, so that the exogenous gene is expressed along with the expression of the coat protein. Biotechnology for the reassembly of phages for display on the surface of phages.
  • antibody in this disclosure refers to immunoglobulins or fragments thereof or derivatives thereof, and includes any polypeptide that contains an antigen binding site, whether produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, Mutated, grafted antibodies.
  • antibody also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function. Typically, such fragments will include antigen-binding fragments.
  • single-chain antibody in the present disclosure is an antibody formed by linking the variable region of the heavy chain and the variable region of the light chain by a short peptide (also known as a linker) of limited amino acids. .
  • peripheral blood mononuclear cells are cells in peripheral blood that have a single nucleus, including lymphocytes and monocytes.
  • RBD refers to the receptor binding domain (RBD) of the coronavirus S protein that binds to angiotensin-converting enzyme 2 (ACE2) on the surface of the virus and host cells. , play an important role in the process of entering host cells.
  • RBD has good accuracy and specificity for the new coronavirus, and can be used for the detection of SARS-CoV-2; at the same time, RBD plays a role in the process of SARS-CoV-2 invading cells, recognizing and specifically binding to RBD Can be used for the treatment of diseases caused by SARS-CoV-2.
  • IMGT numbering scheme in this disclosure is the result of Lefranc et al. introducing a new standardized numbering system for all protein sequences of the immunoglobulin superfamily, including variable domains from antibody light and heavy chains and from different species T cell receptor chain.
  • the IMGT numbering method continuously counts residues based on a germ-line V sequence (germ-line V) alignment.
  • the antibody numbering scheme adopted for antibodies in the present disclosure is the IMGT numbering scheme.
  • the present disclosure relates to the stringency of hybridization conditions used to define the degree of complementarity of two polynucleotides.
  • the aforementioned polynucleotides may be selected from DNA.
  • “Stringency” as used in this disclosure refers to temperature and ionic strength conditions and the presence or absence of certain organic solvents during hybridization. The higher the stringency, the higher the degree of complementarity between the target nucleotide sequence and the labeled polynucleotide sequence.
  • Stringent conditions refer to temperature and ionic conditions under which only nucleotide sequences having a high frequency of complementary bases will hybridize.
  • hybridize under conditions of high or very high stringency describes the conditions used for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Current Protocols in Molec ⁇ Lar Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the specific hybridization conditions referred to in this disclosure are as follows: 1) High stringency hybridization conditions: in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by washing with 0.2X SSC, 0.1% SDS at 65°C One or more times; 2) Very high stringency hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes with 0.2X SSC, 1% SDS at 65°C.
  • SSC sodium chloride/sodium citrate
  • the SARS-CoV-2 Spike protein consists of the S1 domain and the S2 domain, and is one of the keys for the new coronavirus to infect and invade cells.
  • S1 contains a receptor binding domain (RBD) that can specifically bind to the receptor angiotensin-converting enzyme 2 (ACE2) on target cells, which is the most critical step in its infection process. Therefore, it is generally believed that SARS-CoV-2 Spike Protein (RBD) has potential value in the diagnosis of the virus.
  • Recombinant RBD protein vaccine is one of the important new crown vaccine options.
  • the amino acid sequence of RBD is shown in SEQ ID NO: 1, and the N-terminal of RBD contains the signal peptide sequence of "MPLLLLLPLLWAGALA", which can effectively improve the expression of RBD protein. After the RBD protein is expressed and undergoes post-translational modification, the signal peptide will be cleaved, which does not affect the screening of anti-SARS-CoV-2 antibodies.
  • the gene sequence of RBD is shown in SEQ ID NO:2. The gene sequence of RBD was artificially synthesized, and the RBD gene was recombined into the expression vector PATX2 to obtain the RBD-PATX2 expression vector, and the cloning site was EcoR1/Not1.
  • the RBD-PATX2 expression vector is transfected into HEK293F cell line for culture, and the supernatant is collected for nickel column purification to obtain RBD protein.
  • antibodies or antigen-binding fragments with high affinity to RBD proteins are screened using phage display technology.
  • antibodies or antigen-binding fragments with high affinity to RBD proteins include polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic , recombinant, hybrid, mutated, grafted antibodies, or fragments of antibodies such as Fab, F(ab')2, Fv, scFv, Fd, dAb and others that retain antigen binding function.
  • the present disclosure takes the PBMCs of patients with COVID-19, constructs a phage display library containing the variable heavy chain (VH) and the variable light chain (VL), and conducts biopanning with the RBD protein to screen A human antibody that can specifically bind to the new coronavirus has arrived.
  • VH variable heavy chain
  • VL variable light chain
  • V ⁇ 1 GGTCCTGGGCCCAGTCTGTGCTG (SEQ ID NO: 68)
  • V ⁇ 2 GGTCCTGGGCCCAGTCTGCCCCTG (SEQ ID NO: 69)
  • V ⁇ 3 GCTCTGTGACCTCCTATGAGCTG (SEQ ID NO: 70)
  • V ⁇ 6 GTTCTTGGGCCAATTTTATGCTG (SEQ ID NO: 72)
  • V ⁇ 7 GGTCCAATTCYCAGGCTGTGGTG (SEQ ID NO: 73)
  • pATA-scFv-2 vector Heavy chain/light chain variable region PCR product Vector or PCR product 25 ⁇ g 10 ⁇ g Fast Digest NheI 5 ⁇ l 2 ⁇ l Fast Digest NotI 5 ⁇ l 2 ⁇ l 10 ⁇ Fast Digest Buffer 25 ⁇ l 10 ⁇ l ddH 2 O A total of 250 ⁇ L was added to the reaction system A total of 100 ⁇ L was added to the reaction system
  • Positive clones were selected and sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing.
  • the RBD-PATX2 expression vector was transfected into HEK293F cell line for culture, and the supernatant was collected for nickel column purification to obtain RBD protein; the purified RBD also needs to be subjected to SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis) to verify its purity .
  • step 2.1 three times in a cycle, and each input phage library uses the eluted phage after the previous round of amplification.
  • Antigen group Dilute the RBD recombinant protein with PBS to 4 ⁇ g/ml, add 100ul per well to the ELISA plate, and coat overnight at 4°C.
  • Control group 100ul PBS was added to each well of the microtiter plate, and it was coated overnight at 4°C.
  • Blocking Add 300 ⁇ l of 5% skim milk (dissolved in PBS) to each well of the ELISA plate, and incubate at 30 degrees for 2 hours.
  • Phage incubation Dilute the eluted phage after each round of amplification to the required titer with 1% skim milk (dissolved in PBS). Add 100 ⁇ l per well to the ELISA plate and incubate with gentle shaking at room temperature for 2 hours.
  • Washing discard the liquid in the ELISA plate, and wash each well three times with 300 ⁇ l, 0.05% PBST.
  • Elution phage infection take a part of the diluted third round eluted phage and mix with 200ul of E. coli TG1 in log phase. The mixture was incubated at 37°C for 30 minutes and poured onto 2 ⁇ YT-A (Amp 100 ⁇ g/ml) solid medium. Incubate overnight at 37°C.
  • Antigen group Dilute the RBD recombinant protein with PBS to 4 ⁇ g/ml, add 100ul of each well to the ELISA plate, and coat overnight at 4°C.
  • Control group 100ul PBS was added to each well of the microtiter plate, and it was coated overnight at 4°C.
  • Blocking Add 300 ⁇ l of 5% skim milk (dissolved in PBS) to each well of the ELISA plate, and incubate at 30 degrees for 2 hours.
  • Phage incubation 100 ⁇ l of monoclonal culture supernatant per well was added to the ELISA plate, and incubated with gentle shaking at room temperature for 2 hours.
  • Washing discard the liquid in the ELISA plate, and wash each well three times with 300 ⁇ l, 0.05% PBST.
  • the clones in the antigen group greater than 3 times of the control group were designated as positive clones, and the positive clones were sent for sequencing. After eliminating wrong antibody sequences and repetitive antibody sequences, 6 high-affinity antibody sequences were finally obtained.
  • the sequences of the highly specific antibodies are as follows.
  • the obtained phage-positive clones were screened, and the full sequence was sequenced to obtain the corresponding antibody heavy chain and light chain, and the full sequence was as follows:
  • the amino acid sequence of the RBD-R3P1-A12 antibody is the sequence shown in SEQ ID NO: 3;
  • the nucleotide sequence of the RBD-R3P1-A12 antibody is the sequence shown in SEQ ID NO: 4;
  • the amino acid sequence of the RBD-R3P2-A2 antibody is the sequence shown in SEQ ID NO: 5;
  • the nucleotide sequence of the RBD-R3P2-A2 antibody is the sequence shown in SEQ ID NO: 6;
  • the amino acid sequence of the RBD-R3P2-B5 antibody is the sequence shown in SEQ ID NO: 7;
  • the nucleotide sequence of the RBD-R3P2-B5 antibody is the sequence shown in SEQ ID NO: 8;
  • the amino acid sequence of the RBD-R3P1-B6 antibody is the sequence shown in SEQ ID NO: 9;
  • the nucleotide sequence of the RBD-R3P1-B6 antibody is the sequence shown in SEQ ID NO: 10;
  • the amino acid sequence of the RBD-R3P1-E4 antibody is the sequence shown in SEQ ID NO: 11;
  • the nucleotide sequence of the RBD-R3P1-E4 antibody is the sequence shown in SEQ ID NO: 12;
  • the amino acid sequence of the RBD-R3P2-G1 antibody is the sequence shown in SEQ ID NO: 13;
  • the nucleotide sequence of the RBD-R3P2-G1 antibody is the sequence shown in SEQ ID NO: 14;
  • the amino acid sequence of the RBD-R3P1-A12 antibody heavy chain is the sequence shown in SEQ ID NO: 15;
  • the nucleotide sequence of the RBD-R3P1-A12 antibody heavy chain is the sequence shown in SEQ ID NO: 16;
  • the amino acid sequence of the RBD-R3P1-A12 antibody light chain is the sequence shown in SEQ ID NO: 17;
  • the nucleotide sequence of the RBD-R3P1-A12 antibody light chain is the sequence shown in SEQ ID NO: 18;
  • the amino acid sequence of the RBD-R3P2-A2 antibody heavy chain is the sequence shown in SEQ ID NO: 19;
  • the nucleotide sequence of the RBD-R3P2-A2 antibody heavy chain is the sequence shown in SEQ ID NO: 20;
  • the amino acid sequence of the RBD-R3P2-A2 antibody light chain is the sequence shown in SEQ ID NO: 21;
  • the nucleotide sequence of the RBD-R3P2-A2 antibody light chain is the sequence shown in SEQ ID NO: 22;
  • the amino acid sequence of the RBD-R3P2-B5 antibody heavy chain is the sequence shown in SEQ ID NO: 23;
  • the nucleotide sequence of the RBD-R3P2-B5 antibody heavy chain is the sequence shown in SEQ ID NO: 24;
  • the amino acid sequence of the RBD-R3P2-B5 antibody light chain is the sequence shown in SEQ ID NO: 25;
  • the nucleotide sequence of the RBD-R3P2-B5 antibody light chain is the sequence shown in SEQ ID NO: 26;
  • the amino acid sequence of the RBD-R3P1-B6 antibody heavy chain is the sequence shown in SEQ ID NO: 27;
  • the nucleotide sequence of the RBD-R3P1-B6 antibody heavy chain is the sequence shown in SEQ ID NO: 28;
  • the amino acid sequence of the RBD-R3P1-B6 antibody light chain is the sequence shown in SEQ ID NO: 29;
  • the nucleotide sequence of the RBD-R3P1-B6 antibody light chain is the sequence shown in SEQ ID NO: 30;
  • the amino acid sequence of the RBD-R3P1-E4 antibody heavy chain is the sequence shown in SEQ ID NO: 31;
  • the nucleotide sequence of the RBD-R3P1-E4 antibody heavy chain is the sequence shown in SEQ ID NO: 32;
  • the amino acid sequence of the RBD-R3P1-E4 antibody light chain is the sequence shown in SEQ ID NO: 33;
  • the nucleotide sequence of the RBD-R3P1-E4 antibody light chain is the sequence shown in SEQ ID NO: 34;
  • the amino acid sequence of the RBD-R3P2-G1 antibody heavy chain is the sequence shown in SEQ ID NO: 35;
  • the nucleotide sequence of the RBD-R3P2-G1 antibody heavy chain is the sequence shown in SEQ ID NO: 36;
  • the amino acid sequence of the RBD-R3P2-G1 antibody light chain is the sequence shown in SEQ ID NO: 37;
  • the nucleotide sequence of the RBD-R3P2-G1 antibody light chain is the sequence shown in SEQ ID NO: 38;
  • amino acid sequence of CDR1 of the heavy chain of RBD-R3P1-A12, RBD-R3P2-A2, RBD-R3P2-B5, RBD-R3P1-B6, RBD-R3P1-E4 or RBD-R3P2-G1 antibody heavy chain is as shown in SEQ ID NO:39 the sequence shown;
  • amino acid sequence of the CDR2 of the heavy chain of the RBD-R3P1-A12, RBD-R3P2-A2, RBD-R3P2-B5, RBD-R3P1-B6, RBD-R3P1-E4 or RBD-R3P2-G1 antibody heavy chain is as shown in SEQ ID NO: 40 the sequence shown;
  • amino acid sequence of the CDR3 of the heavy chain of the RBD-R3P1-A12, RBD-R3P2-A2, RBD-R3P2-B5, RBD-R3P1-B6, RBD-R3P1-E4 or RBD-R3P2-G1 antibody heavy chain is as shown in SEQ ID NO: 41 the sequence shown;
  • the amino acid sequence of the CDR1 of the RBD-R3P1-A12 antibody light chain is the sequence shown in SEQ ID NO: 42;
  • the amino acid sequence of the CDR2 of the RBD-R3P1-A12 antibody light chain is the sequence shown in SEQ ID NO: 43;
  • amino acid sequence of the CDR3 of the RBD-R3P1-A12 antibody light chain is the sequence shown in SEQ ID NO: 44;
  • amino acid sequence of CDR1 of the RBD-R3P2-A2 antibody light chain is the sequence shown in SEQ ID NO: 45;
  • amino acid sequence of CDR2 of the RBD-R3P2-A2 antibody light chain is the sequence shown in SEQ ID NO: 46;
  • amino acid sequence of the CDR3 of the RBD-R3P2-A2 antibody light chain is the sequence shown in SEQ ID NO: 47;
  • amino acid sequence of CDR1 of the RBD-R3P2-B5 antibody light chain is the sequence shown in SEQ ID NO: 48;
  • amino acid sequence of CDR2 of the RBD-R3P2-B5 antibody light chain is the sequence shown in SEQ ID NO: 49;
  • amino acid sequence of the CDR3 of the RBD-R3P2-B5 antibody light chain is the sequence shown in SEQ ID NO: 50;
  • the amino acid sequence of the CDR1 of the RBD-R3P1-B6 antibody light chain is the sequence shown in SEQ ID NO: 51;
  • amino acid sequence of the CDR2 of the RBD-R3P1-B6 antibody light chain is the sequence shown in SEQ ID NO: 52;
  • the amino acid sequence of the CDR3 of the RBD-R3P1-B6 antibody light chain is the sequence shown in SEQ ID NO: 53;
  • amino acid sequence of CDR1 of the RBD-R3P1-E4 antibody light chain is the sequence shown in SEQ ID NO: 54;
  • amino acid sequence of the CDR2 of the RBD-R3P1-E4 antibody light chain is the sequence shown in SEQ ID NO: 55;
  • amino acid sequence of the CDR3 of the RBD-R3P1-E4 antibody light chain is the sequence shown in SEQ ID NO: 56;
  • the amino acid sequence of CDR1 of the RBD-R3P2-G1 antibody light chain is the sequence shown in SEQ ID NO: 57;
  • amino acid sequence of the CDR2 of the RBD-R3P2-G1 antibody light chain is the sequence shown in SEQ ID NO: 58;
  • amino acid sequence of the CDR3 of the RBD-R3P2-G1 antibody light chain is the sequence shown in SEQ ID NO: 59;
  • the linker amino acid sequence is the sequence shown in SEQ ID NO:60.
  • Embodiment 3 ELISA detects the OD value of antibody under different dilution concentration conditions
  • the RBD protein was diluted 1:500 with blocking solution, 100 ⁇ l of diluted serum was added to each well, and the reaction was carried out at room temperature for 1 hour.
  • the operation is performed by trained experimental operators. Before the experimental operation, change clothes in the clean area (put on disposable sterile clothing, change work shoes, wear masks, hats, disposable medical latex gloves) before entering the experimental area. Inside, perform experimental operations.
  • inhibition rate [1-(mean luminescence intensity of sample group-mean value of blank control CC)/(mean luminescence intensity VC of negative group-mean value of blank control CC)]*100%.
  • FIG. 5 shows a graph of the results of the neutralization experiment.
  • Figure 5A shows the inhibition rate of the screened antibody sequences against SARS-CoV-2 pseudovirus
  • Figures 5B-5H show the antibodies RBD-R3P1-A12 (Figure 5B: A12), RBD-R3P2-A2 ( Figure 5D, 5G: A2), RBD-R3P2-B5 ( Figure 5C: B5), RBD-R3P1-B6 ( Figure 5E: B6), RBD-R3P1-E4 ( Figure 5B, 5H: E4) and R3P2-G1 ( Figure 5F: G1) detection results of the inhibition rate of SARS-CoV-2 true virus (in Figures 5B-5H: SARS-CoV2-NP or SARS-CoV-2-NP), where antibody concentrations -1, -2, - 3 represents the concentration of 63ng, 250ng, and 1u

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Abstract

La présente divulgation concerne un anticorps humanisé dirigé contre de nouveaux peptides antigéniques spécifiques du coronavirus, un procédé de préparation et une utilisation. En particulier, la présente divulgation concerne un anticorps anti-SARS-CoV-2 ou un fragment de liaison à l'antigène de celui-ci, et son utilisation dans le diagnostic de maladies, la préparation d'un vaccin anti-COVID-19, et la préparation d'un médicament pour la prévention et le traitement de la COVID-19. L'anticorps anti-SARS-CoV-2 ou le fragment de liaison à l'antigène de celui-ci peut se lier au domaine RBD du nouveau coronavirus et bloquer l'invasion de cellules par le virus, ayant une importance clinique importante pour la prévention, le traitement ou la détection du nouveau coronavirus.
PCT/CN2021/097706 2020-11-25 2021-06-01 Anticorps humanisé dirigé contre de nouveaux peptides antigéniques spécifiques du coronavirus, procédé de préparation et utilisation WO2022110742A1 (fr)

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WO2022240887A1 (fr) * 2021-05-10 2022-11-17 Icahn School Of Medicine At Mount Sinai Procédés pour la détection et la graduation des réponses immunitaires virales cellulaires
CN113880947B (zh) * 2021-07-26 2023-07-04 中国人民解放军军事科学院军事医学研究院 小分子抗体及其编码基因和制备方法及应用和药物组合物
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