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CN-115594757-B - Neutralizing antibody against novel coronavirus, preparation method, preparation and application thereof

CN115594757BCN 115594757 BCN115594757 BCN 115594757BCN-115594757-B

Abstract

The invention relates to the technical field of biology, and discloses a neutralizing antibody for resisting novel coronaviruses, a preparation method, a preparation and application thereof. The nanobody comprises complementarity determining regions CDR1, CDR2 and CDR3 shown in SEQ ID NO. 2-4. The nano antibody and RBD have clear recognition and binding mechanism, can be combined with SARS-CoV-2 efficiently and specifically, has affinity reaching picomolar level (Kd <1x10 ‑12 M), has a recognition and binding mode different from ACE2, still has better neutralization biological activity (IC 50:16.37 nM), can obtain excellent effect in SARS-CoV-2 detection or SARS-CoV-2-induced diseases, and has wide application prospect in the biological and medical fields.

Inventors

  • TAN JINGQUAN
  • Li Dianfan
  • Dimitri Ravillet

Assignees

  • 南京晶准生物科技有限公司

Dates

Publication Date
20260508
Application Date
20210628

Claims (11)

  1. 1. A nano antibody for resisting SARS-CoV-2 of novel coronavirus is characterized by that the amino acid sequence of said nano antibody includes complementarity determining regions CDR1, CDR2 and CDR3, and the CDR1 is shown as SEQ ID NO.2, the CDR2 is shown as SEQ ID NO.3 and the CDR3 is shown as SEQ ID NO. 4.
  2. 2. The nanobody of claim 1, wherein the nanobody comprises one or more of the framework regions FR1, FR2, FR3 and FR4, FR1 as shown in SEQ ID No.5, FR2 as shown in SEQ ID No.6, FR3 as shown in SEQ ID No.7, and FR4 as shown in SEQ ID No. 8.
  3. 3. The nanobody according to claim 2, wherein the amino acid sequence of the nanobody is shown in SEQ ID No. 1.
  4. 4. An isolated polynucleotide encoding the nanobody of claim 1.
  5. 5. The polynucleotide according to claim 4, wherein the sequence of the polynucleotide is shown in SEQ ID NO. 9.
  6. 6. A construct, characterized in that, comprising the polynucleotide according to claim 4.
  7. 7. A nanobody expression system comprising the construct or genome of claim 6 integrated with an exogenous polynucleotide of claim 4 or 5.
  8. 8. A pharmaceutical composition comprising one or more nanobodies according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.
  9. 9. A detection reagent or a detection kit, characterized by comprising one or more nanobodies according to any one of claims 1 to 3.
  10. 10. Use of a nanobody according to any one of claims 1 to 3 or a polynucleotide according to claim 4 or 5 or a construct according to claim 6 or an expression system according to claim 7 or a pharmaceutical composition according to claim 8 for: I) The application in preparing the medicine for preventing and/or treating the disease caused by the novel coronavirus SARS-Cov-2; II) application in immunological examination and analysis for non-disease diagnosis and treatment purpose or in preparation of detection reagent or kit for novel coronavirus SARS-Cov-2.
  11. 11. A method for producing a nanobody according to any one of claims 1 to 3, comprising the step of culturing the nanobody expression system under a condition suitable for expression of the nanobody, thereby expressing the nanobody, and purifying and isolating the nanobody.

Description

Neutralizing antibody against novel coronavirus, preparation method, preparation and application thereof Technical Field The invention relates to the field of biotechnology, relates to a neutralizing antibody for resisting novel coronavirus, a preparation method, a preparation and application thereof, in particular to a nano antibody for resisting novel coronavirus SARS-CoV-2, a preparation method, a preparation, a pharmaceutical composition, a detection kit and application thereof. Background Neutralizing antibodies are a class of antibodies that bind to epitopes of viral particles and reduce viral infectivity by blocking viral infection. The neutralizing antibody has wide sources, can be obtained from the blood plasma of a convalescence person, can also obtain the antibody for recognizing multiple epitopes by immunizing different virus antigen epitopes, has relatively mature preparation process and short research and development period, and therefore, research and development of the neutralizing antibody with high titer against viruses is also an effective means for preventing virus transmission and infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 is the causative agent of novel coronavirus infection (COVID-19) discovered at the end of 2019 and causing global transmission, the infection host relies on the recognition and combination of the receptor domain (RBD) of spike protein S to angiotensin converting enzyme 2 (ACE 2) of host epithelial cells, and then fusion with cell membranes, and the infection process of the virus is completed. The nano antibody has the advantages of small molecular weight, strong penetrability, easy expression, easy genetic modification, easy combination of a plurality of epitopes and the like. Thus, such antibodies can be used as candidates for effective neutralizing antibodies, but currently there is no marketed nanobody drug against SARS-CoV-2. Disclosure of Invention In order to overcome the defects in the prior art, the invention provides a nano antibody for resisting novel coronavirus SARS-CoV-2, a preparation method and application thereof, wherein the nano antibody has stronger specific binding capacity (Kd <1x10 -12 M) and clear binding epitope information with the novel coronavirus SARS-CoV-2, and the application of the nano antibody provides a wider means for diagnosis and treatment of the novel coronavirus SARS-CoV-2. The invention provides a nanometer antibody amino acid sequence (SC 4) of a novel coronavirus SARS-CoV-2S protein RBD structural Domain (Receptor-Binding Domain), wherein the nanometer antibody sequence comprises three complementarity determining regions CDR1, CDR2 and CDR3, and the amino acid sequence is as follows: Amino acid sequence of SC 4: QVQLQESGGGLVQAGGSLRLSCAASGSDFSSSTMGWYRQAPGKQREFVAISSEGSTSYAGSVKGRFTISRDNAKNTVYLQMNSLEPEDTAVYYCNVVDRWYDYWGQGTQVTVS（SEQ ID NO.1）. In the present invention, the nanobody includes three complementarity determining regions CDR1, CDR2 and CDR3, the amino acid sequences of which are as follows: CDR1 sequence GSDFSSST (SEQ ID NO. 2) CDR2 sequence ISSEGST (SEQ ID NO. 3) CDR3 sequence NVVDRWYDY (SEQ ID NO. 4). The nanobody of the invention may be an antibody comprising one or more of the above mentioned CDR1, CDR2 and CDR3, i.e. the nanobody may be a CDR1 comprising, or a CDR2 comprising, or a CDR3 comprising, or a CDR1 and CDR2 comprising, or a CDR1 and CDR3 comprising, or a CDR2 and CDR3 comprising, or a CDR1, CDR2 and CDR3 comprising. In a specific embodiment, the nanobody comprises at least CDR1. In a specific embodiment, the nanobody comprises at least CDR2. In a specific embodiment, the nanobody comprises at least CDR3. In the present invention, the further comprises a framework region, which is any framework region capable of achieving the nanobody function of the present invention, including human or murine. In the present invention, the nanobody may include one or more of the following framework regions: FR1 sequence QVQLQESGGGLVQAGGSLRLSCAAS (SEQ ID NO. 5); FR2 sequence MGWYRQAPGKQREFVA (SEQ ID NO. 6); FR3 sequence SYAGSVKGRFTISRDNAKNTVYLQMNSLEPEDTAVYYC (SEQ ID NO. 7); FR4 sequence WGQGTQVTVS (SEQ ID NO. 8). The nanobody of the present invention mainly recognizes a Receptor Binding Motif Region (RBM) that binds RBD. The invention further provides biological materials, derived antibodies, and the like, associated with the nanobodies. In one aspect, the invention provides an isolated polynucleotide encoding the anti-novel coronavirus SARS-CoV-2 antibody. The nucleic acid sequence is as follows: CAGGTGCAGCTGCAGGAGAGCGGCGGCGGACTGGTGCAGGCTGGAGGAAGCCTGAGACTGTCCTGTGCCGCCAGCGGCAGCGACTTCAGCTCCTCCACCATGGGCTGGTACAGACAGGCCCCCGGCAAGCAGAGAGAGTTCGTGGCCATCAGCTCCGAGGGCAGCACCTCCTACGCCGGCTCCGTGAAGGGCAGGTTCACAATCAGCAGAGATAACGCCAAGAACACAGTGTACCTGCAGATGAATAGCCTGGAGCCTGAGGACACCGCCGTGTACTACTGCAATGTGGTGGACAGATGGTACGACTACTGGGGCCAGGGCACCCAGGTGACAGTGAGC（SEQ ID NO.9）. in another aspect, the invention provides a construc