CN117777281A - anti-COVID-19 virus murine neutralizing antibody, humanized modified antibody and application thereof - Google Patents

Abstract

The invention relates to preparation of an anti-COVID-19 virus neutralizing antibody and design modification and application of a humanized antibody h8A 8. The amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody provided by the invention are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 in sequence; the amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody are shown in SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6 in sequence. The antibody of the invention can specifically bind to the novel coronavirus RBD protein and neutralize the novel coronavirus (SARS-CoV-2) and partial mutant thereof. The antibody provided by the invention can be used for preventing and treating coronavirus infection, and has important biological and medical significance. The design and transformation of the antibody prepared based on the hybridoma technology and the humanized antibody provided by the invention also provide a new technical path for preventing and treating the sudden infectious diseases.

Description

anti-COVID-19 virus murine neutralizing antibody, humanized modified antibody and application thereof

Technical Field

The invention relates to the technical fields of biological medicine and synthetic biology, in particular to an anti-COVID-19 virus murine neutralizing antibody and humanized transformation and application thereof.

Background

The novel coronavirus SARS-CoV-2 belongs to the genus beta-coronavirus of the family Coronaviridae and is a kind of single-stranded positive strand RNA virus with envelope on the surface. SARS-CoV-2 structural protein Spike (abbreviated as S protein) can specifically bind to angiotensin converting enzyme 2 (ACE 2) receptor of host cell, and is a key protein for virus invasion of host susceptible cell. The full length 1273 amino acid of S protein consists of two subunits S1 and S2, wherein the receptor domain RBD of the S1 subunit can be directly combined with ACE2 receptor of host cell, and the S2 subunit mainly mediates fusion between virus and host cell membrane.

Animal experiments and clinical experiments prove that the neutralizing antibody of the Receptor Binding Domain (RBD) which is mainly targeted to S1 subunit of S protein and is separated from a patient has a certain effect of preventing and treating new coronavirus infection, wherein partial SARS-CoV-2 neutralizing antibody has been approved by FDA to be applied to the clinical treatment of COVID-19. Because of stronger mutation capability of S protein and multiple mutation, the novel coronavirus S protein vaccine has stronger escape capability for the novel coronavirus vaccine used in various countries at present, and the existing novel coronavirus neutralizing antibody is subjected to genetic engineering modification aiming at great danger caused by the epidemic of SARS-CoV-2 and variant strains thereof to human health and economic society development, and the efficient broad-spectrum neutralizing antibody aiming at novel coronavirus S protein RBD is developed to prevent the combination of S protein and human ACE2, thereby blocking virus infection. The hybridoma technology can be used for rapidly preparing the anti-SARS-CoV-2 and the RBD neutralizing antibody of the variant strain thereof, has important biological and medical significance for preventing and treating coronavirus infection, and also provides a new technical path for preventing and treating sudden infectious diseases.

Disclosure of Invention

Based on the above, the invention aims to provide a preparation method of neutralizing antibodies against novel coronaviruses, humanized transformation and application thereof, and the transformed h8A8 antibody has strong neutralizing activity and broad spectrum.

In a first aspect of the invention, there is provided a neutralizing antibody against a novel coronavirus.

A neutralizing antibody for resisting a novel coronavirus, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 regions in a heavy chain variable region of the neutralizing antibody are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; the amino acid sequences of LCDR1, LCDR2 and LCDR3 regions in the light chain variable region of the neutralizing antibody are respectively shown as SEQ ID NO.4, AAS and SEQ ID NO. 6.

In some of these embodiments, the neutralizing antibody further comprises a heavy chain constant region selected from IgG1 or IgG4 and a light chain constant region comprising a member selected from kappa or Lambda subtypes.

In some of these embodiments, the neutralizing antibody is a murine RBD monoclonal neutralizing antibody against a novel coronavirus, and the heavy chain variable region has the amino acid sequence of SEQ ID No.7, or a sequence having at least 90% identity with SEQ ID No.7 and unchanged activity.

In some of these embodiments, the amino acid sequence of the antibody light chain variable region is SEQ ID No.8, or a sequence having at least 90% identity to SEQ ID No.8 and unchanged activity.

In some embodiments, the neutralizing antibody is a humanized anti-novel coronavirus RBD neutralizing antibody obtained by humanized engineering a murine antibody, wherein the neutralizing antibody is obtained by implanting a complementarity determining region of a heavy chain variable region as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, a light chain variable region as shown in SEQ ID NO.4, AAS, SEQ ID NO.6 into a human germline template FR region sequence, and recovering the heavy chain by A97T, G27F, S30A, V37I, M48I, I70L, V68A mutation, and/or recovering the light chain by Y53F, S7T mutation; mutation back of the heavy chain amino acid includes, but is not limited to, the seven sites, and mutation back of the light chain amino acid includes, but is not limited to, the two sites.

In some embodiments, the amino acid sequence of the heavy chain variable region of the neutralizing antibody may be SEQ ID No.9 from position 1 to 119 from the N-terminus, or a sequence having at least 90% identity and unchanged activity to positions 1 to 119 of SEQ ID No.9 from the N-terminus; the amino acid sequence of the light chain variable region of the neutralizing antibody can be SEQ ID NO.10 from the 1 st to 111 th positions from the N end or a sequence which has at least 90% of identity with SEQ ID NO.10 from the 1 st to 111 th positions from the N end and has unchanged activity; the neutralizing antibodies further comprise a heavy chain constant region selected from the group consisting of IgG1 or IgG4 and a light chain constant region comprising a light chain constant region selected from the group consisting of kappa or Lambda subtypes.

In some preferred embodiments thereof, the heavy chain of the neutralizing antibody has the amino acid sequence of SEQ ID No.9, or a sequence having at least 90% identity with SEQ ID No.9 and unchanged activity; the amino acid sequence of the light chain of the antibody is SEQ ID No.10 or a sequence which has at least 90% of identity with SEQ ID No.10 and has unchanged activity.

In a second aspect of the invention there is provided a nucleic acid molecule encoding a neutralising antibody of any one of the preceding claims, said nucleic acid molecule comprising the nucleotide sequences of HCDR1, HCDR2 and HCDR3 in the murine heavy chain variable region of said neutralising antibody and the nucleotide sequences of LCDR1, LCDR2 and LHCDR3 in the murine light chain variable region of said neutralising antibody.

The nucleic acid molecules also include a heavy chain constant region encoding a polypeptide selected from the group consisting of IgG1 and IgG4 and a light chain constant region comprising a polypeptide selected from the group consisting of kappa and Lambda subtypes.

In some embodiments, the nucleotide sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region encoded by the nucleic acid molecules are shown in sequence at positions 76-99, 151-174 and 289-324 from the 5' end of SEQ ID No. 11; the nucleotide sequences of the light chain variable regions HCDR1, HCDR2 and HCDR3 coded by the nucleic acid molecules are sequentially shown as 79 th to 108 th, 160 th to 168 th and 277 th to 303 th positions from the 5' end of SEQ ID No. 12.

In some preferred embodiments thereof, the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule is SEQ ID No.11 or has at least 90% identity to SEQ ID No. 11; the nucleotide sequence encoding the light chain variable region of the neutralizing antibody is SEQ ID No.12 or a sequence having at least 90% identity with SEQ ID No.12 and unchanged activity.

In some embodiments, the nucleic acid molecule is humanized, and the nucleotide sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are shown in sequence at positions 76-99, 151-174 and 289-324 from the 5' end of SEQ ID No. 13; the nucleotide sequences of LCDR1, LCDR2 and LHCDR3 in the light chain variable region are sequentially shown as 79 th to 108 th, 160 th to 168 th and 277 th to 303 th positions from the 5' end of SEQ ID No. 14; the nucleic acid molecule further comprises a heavy chain constant region selected from the group consisting of IgG1 or IgG4 and a light chain constant region comprising a light chain selected from the group consisting of kappa or Lambda subtypes.

In some preferred embodiments thereof, the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule is SEQ ID No.13 from position 1 to 357 from the 5 'end or has at least 90% identity to positions 1 to 357 from the 5' end of SEQ ID No. 13; the nucleotide sequence of the light chain variable region of the nucleic acid molecule is 1-333 th from the 5 'end of SEQ ID No.14 or has at least 90% of identity with the 1-333 st from the 5' end of SEQ ID No. 14.

In some preferred embodiments thereof, the nucleotide sequence of the heavy chain of the nucleic acid molecule is SEQ ID No.13 or has at least 90% identity with SEQ ID No. 13. The nucleotide sequence encoding the light chain of the humanized antibody is SEQ ID No.14 or a sequence having at least 90% identity with SEQ ID No.14 and unchanged activity.

In a third aspect of the invention, there is provided an amino acid sequence or protein molecule expressed from the above nucleic acid molecule.

In some of these embodiments, the amino acid sequence is humanized engineered, and the heavy chain amino acid sequence is grafted to human antibody heavy chain germ line genes IGHV1-69 x 02 and IGHJ1 x 01, SEQ ID No.1, SEQ ID No.2, SEQ ID No. 3; the humanized antibody light chain amino acid sequence is obtained by transplanting SEQ ID NO.4, AAS and SEQ ID NO.6 onto human antibody light chain germ line genes IGKV4-1 x 01 and IGKJ4 x 01.

In a fourth aspect of the invention, there is provided an expression vector comprising a nucleic acid molecule of any one of the above, in the form of an expression vector including, but not limited to: plasmid, expression cassette, recombinant vector, recombinant cell or recombinant bacterium.

In some of these embodiments, the expression vector form is a plasmid.

In some preferred embodiments, the plasmid is obtained by cloning the sequence of SEQ ID NO.13 from the 5' end at positions 1-357 onto the pRVL-5 vector, and the resulting recombinant plasmid is the recombinant chain expression plasmid of antibody h8A 8.

In some preferred embodiments, the plasmid is a light chain expression plasmid of antibody h8A8 obtained by cloning the sequence of SEQ ID No.14 from the 5' end at positions 1-333 onto the pRVL-4 vector.

In some more preferred embodiments, the expression vector is a recombinant cell obtained by co-transfecting HEK293T cells with the two recombinant expression plasmids described above.

In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising a neutralizing antibody according to any one of the preceding claims together with a pharmaceutically acceptable excipient, diluent or carrier.

In a sixth aspect of the invention there is provided the use of a neutralising antibody or nucleic acid molecule or expression vector or pharmaceutical composition of any of the above, including but not limited to:

(A1) Use of any of the nucleic acid molecules or expression vectors described above for the preparation of any of the antibodies or any of the pharmaceutical compositions described above;

(A2) The use of any of the above neutralizing antibodies in the preparation of a d pharmaceutical composition or vaccine against the covd-19 virus;

(A3) Use of any of the above neutralizing antibodies or nucleic acid molecules or expression vectors or pharmaceutical compositions for the preparation of a product for the prevention and/or treatment of a disease caused by SARS-CoV-2 infection;

(A4) Use of any of the above neutralizing antibodies or expression vectors or pharmaceutical compositions for the preparation of a product for inhibiting SARS-CoV-2 infection;

(A5) Use of any of the above neutralizing antibodies or nucleic acid molecules or expression vectors or pharmaceutical compositions for the preparation of a product for the detection of SARS-CoV-2;

(A6) Use of any of the above neutralizing antibodies or nucleic acid molecules or expression vectors or pharmaceutical compositions for the preparation of a product for neutralizing SARS-CoV-2;

(A7) Use of any of the above neutralizing antibodies or nucleic acid molecules or expression vectors or pharmaceutical compositions for the preparation of a product for detecting the RBD protein of SARS-CoV-2;

(A8) Use of any of the above neutralizing antibodies or nucleic acid molecules or expression vectors or pharmaceutical compositions for the preparation of a product for binding the RBD protein of SARS-CoV-2.

In some embodiments, the product is a genetic drug or a vaccine.

Compared with the prior art, the invention has the following beneficial effects:

the invention screens an anti-COVID-19 virus neutralizing antibody m8A8 and a preparation method thereof, wherein the antibody can specifically bind to a novel coronavirus RBD protein and neutralize a novel coronavirus (SARS-CoV-2) and mutant strains thereof. Thus, the invention provides a humanized modified antibody based on the murine antibody capable of specifically binding to the novel coronavirus RBD protein, the humanized modified antibody and a preparation method thereof. The anti-COVID-19 virus neutralizing antibody provided by the invention has stronger binding capacity, and particularly the modified h8A8 antibody has stronger neutralizing activity and broad spectrum, can be used for preventing and treating novel coronavirus infection, and has important biological and medical significance.

Drawings

FIG. 1 shows the affinity results of murine monoclonal antibody m8A 8.

FIG. 2 shows the results of m8A8 antibody novel coronavirus pseudovirus neutralization experiments.

FIG. 3 is a comparison of the affinity results of m8A8 and h8A8 antibodies.

FIG. 4 shows the results of a h8A8 antibody novel coronavirus pseudovirus neutralization assay.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental methods, in which specific conditions are not noted in the examples below, are generally carried out according to conventional conditions, for example, green and Sambrook-s.A.fourth edition, molecular cloning, A.laboratory Manual (Molecular Cloning: ALaboratory Manual), published in 2013, or according to the conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

Wherein HCDR1, HCDR2 and HCDR3 are three complementarity determining regions in the heavy chain variable region, and LCDR1, LCDR2 and LCDR3 are three complementarity determining regions in the light chain variable region. The sequence of the complementarity determining region is defined according to the International immunogenetics information System (International Immunogenetics information system, IMGT, hereinafter referred to as IMGT).

The murine anti-COVID-19 RBD monoclonal antibody described herein is designated herein as "m8A8"; the antibody is in various forms such as murine or humanized full-length antibody, fab fragment, F (ab') 2 fragment or single-chain Fv fragment; the humanized engineered antibody is designated herein as "h8A8".

The sequence related to the invention comprises the following steps:

SEQ ID No.1: m8A8 murine antibody heavy chain variable region HCDR1 amino acid sequence

SEQ ID No.2: m8A8 murine antibody heavy chain variable region HCDR2 amino acid sequence

SEQ ID No.3: m8A8 murine antibody heavy chain variable region HCDR3 amino acid sequence

SEQ ID No.4: m8A8 murine antibody light chain variable region LCDR1 amino acid sequence

SEQ ID No.5: m8A8 murine antibody light chain variable region LCDR2 amino acid sequence

SEQ ID No.6: m8A8 murine antibody light chain variable region LCDR3 amino acid sequence

SEQ ID No.7: m8A8 murine antibody heavy chain variable region amino acid sequence

SEQ ID No.8: m8A8 murine antibody light chain variable region amino acid sequence

SEQ ID No.9: h8A8 humanized antibody heavy chain amino acid sequence

SEQ ID No.10: h8A8 humanized antibody light chain amino acid sequence

SEQ ID No.11: m8A8 murine antibody heavy chain variable region nucleotide sequence

SEQ ID No.12: m8A8 murine antibody light chain variable region nucleotide sequence

SEQ ID No.13: h8A8 humanized antibody heavy chain nucleotide sequence

SEQ ID No.14: h8A8 humanized antibody light chain nucleotide sequence

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The present invention will be described in further detail with reference to specific examples.

Example 1 discovery and preparation method of murine anti-RBD monoclonal antibody m8A8

1. Mouse immunization and acquisition of anti-RBD antibody hybridoma cells:

the immunization orbit is taken for blood collection from the Balb/C mice of 6-8 weeks of age. First immunization of recombinant protein RBD emulsified with freund's complete adjuvant (near shore protein, DRA72, supra), 100 μg RBD recombinant protein was injected subcutaneously per mouse. Boosting is carried out at intervals of 1 week, RBD recombinant proteins are emulsified by Freund's incomplete adjuvant, 100 mug of RBD recombinant proteins are injected subcutaneously into each mouse, the orbit is sampled before injection, impact immunization is changed to the third immunization, RBD recombinant proteins without adjuvant are used as immunogens, 50 mug of RBD recombinant proteins are injected intraperitoneally into each mouse, the mice are sacrificed 3 days after impact immunization, and spleen cells are collected. Spleen cells and SP2/0 cell suspensions were mixed in a ratio of 5:1 cell number, resuspended, cell fused with PEG4000, and plated. Limiting dilution is carried out on the tenth day of cell fusion, and hybridoma cells which can secrete monoclonal antibodies against RBD are obtained after several rounds of screening.

Murine antibody variable region gene acquisition: and extracting total RNA from the screened monoclonal cells with good states, synthesizing cDNA by using the extracted total RNA as a template and using a first-strand cDNA synthesis kit, and respectively amplifying the cDNA as the template by using specific primers to obtain a heavy chain variable region and a light chain variable region of the antibody.

The extension primers were as follows:

heavy chain: vhRevU: GAG GTS MAR CTG CAG SAG TCW SEQ ID No.5:

GGVhForU：GACAGT GGATARACM GAT GG SEQ ID No.15：

light chain:

2mFK：GAYATTGTGMTSACMCARWCTMCA SEQ ID No.16：

2mRK：TGGGAAGATGGATACAGTT SEQ ID No.17：

2. preparation and purification method of murine anti-RBD monoclonal antibody m8A8

Hybridoma cells secreting monoclonal antibodies against RBD (2.5-5 x 10 ⁶ Individual cells/mL) were inoculated into the peritoneal cavity of liquid paraffin-pretreated mice, each of which was injected with 200 μl-1mL of the cell suspension, and peritoneal fluid was formed after 1-2 weeks. Ascites is extracted, centrifuged for 10min at 1500g and the supernatant is collected. The murine anti-RBD monoclonal antibody with purity of more than 90% is prepared by affinity purification. The purified antibody was subjected to dialysis treatment to replace the antibody solution, and the solution was sterilized by filtration through a 0.22 μm filter, sampled and assayed for protein concentration by BCA method and stored in aliquots at-20 ℃.

Purifying:

purifying:

2.1 packing of purification column (after packing of column there is delamination, washing off ethanol preservation solution with distilled water of 5-10 column bed volumes)

2.2 equilibration with 5-10 bed volumes of binding buffer

2.3 samples were diluted 10-fold with binding buffer and loaded after filtration through a 0.45 μm filter.

2.4, after the sample is loaded, the sample is balanced by using a binding buffer solution until no sundries flow out.

2.5 eluting the antibody with elution buffer, collecting the elution peak, and adjusting pH to neutral with neutralization buffer 1M Tris-HCl (pH 9.0)

2.6 regeneration washes with 3-5 bed volumes of elution buffer after each use

2.7 after purification, the column was preserved with 20% ethanol to prevent the growth of microorganisms.

2.8 Coomassie brilliant blue fast dye liquor (Solebao, P1300) staining identification

EXAMPLE 2 evaluation of murine anti-RBD monoclonal antibodies

1. Affinity assay of antibodies: the binding capacity of the prepared antibodies to recombinant RBD was tested by ELISA.

As a result, as shown in FIG. 1, among all the prepared antibodies, the monoclonal antibody with clone number m8A8 was effective, and its binding capacity to recombinant RBD was < 4ng/mL, which was an antibody with higher affinity.

ELISA assay for antibody binding to RBD:

1.1 coating: the RBD recombinant protein (offshore protein, DRA 72) stock was diluted with phosphate buffer, added to the ELISA plate at 100 ng/well, 100. Mu.L/well, and coated overnight at 4 ℃.

1.2 after the above steps are completed, the ELISA plate is taken, PBST plate is washed 3 times, and is sealed by 5% skimmed milk powder, and incubated for 2 hours at room temperature.

1.3 after the above steps were completed, the ELISA plate was taken, blocking solution was discarded, PBST plate was washed 3 times, the antibody obtained in example 1 was diluted, 100. Mu.L of the dilution solution was added per well, incubation was performed for 2 hours at 37℃and then PBST plate was washed 3 times.

1.4 after the above procedure, 50. Mu.L of 1:10000-fold diluted enzyme-labeled antibody-goat anti-mouse IgG-HRP (Friedel-crafts, FDM 007) was added to each well, incubated for 1h at room temperature, and then PBST was washed 3 times.

1.5 after the above steps are completed, taking the ELISA plate, adding 100 mu LTMB substrate color development liquid into each hole, and incubating for 15min at room temperature

1.6 after the above steps, the ELISA plate was taken and 100. Mu.L of 1M sulfuric acid solution was added to each well to terminate the ELISA reaction.

1.7. The optical density value is measured by an enzyme label instrument at 450 nm.

2. Neutralization capacity determination of antibodies: new coronavirus pseudovirus activity assay

2.1 dilution of antibodies: the antibody to be tested is diluted to 10ug/mL firstly, 100 mu LDMEM is added from the second row to the eighth row in the 96-well plate, then 50 mu L of the pre-diluted antibody solution is added into the second row, the dilution is carried out downwards in sequence until the eighth row, 7 dilutions are carried out in total, and 50 mu L of the solution is sucked after the last dilution is uniformly mixed.

2.2 adding 35 mu L of pseudovirus into each well, mixing uniformly, and placing into a 37 ℃ incubator for incubation for 1h.

2.3 incubation to 0.5h, digestion of ACE2-293T cells can begin and single cell suspension density is adjusted to 1.6X105/mL.

2.4 100. Mu.LACE 2-293T cells were added to each well of the 96-well plate and incubated in incubator at 37℃for 2d.

2.5 the culture supernatant from the 96-well plates was discarded, washed 1 time with PBS, 100. Mu.L of 1X Passive Lysis Buffer (Promega, E194A) was added to each well, and the mixture was left to stand or shake at low speed for 30min, and 20. Mu.L of the lysate from each well was transferred to a white light-resistant 96-well plate.

2.6 detection of Single luciferase with Cystation 5 multifunctional cell imaging microplate Detector by Bertonian instruments, USA, 50. Mu. L LuciferaseAssay Substrate (Promega, E1501) was added per well; the IC50 was calculated by plotting with software GraphPadPrism 9.

As shown in FIG. 2, the m8A8 antibody was effective in neutralizing the novel coronavirus pseudovirus, with an IC50 of 4.19ng/mL for the WH1 (wild-type) strain pseudovirus, an IC50 of 3.57ng/mL for the B.1.617.2 strain pseudovirus, an IC50 of 8.61ng/mL for the B.1.529 strain pseudovirus, an IC50 of 134.30ng/mL for the BA.2 strain pseudovirus, an IC50 of 16.52ng/mL for the BA.3 strain pseudovirus, an IC50 of 19.26ng/mL for the BA.4& BA.5 strain pseudovirus, an IC50 of 44.41ng/mL for the BA.2.75 strain pseudovirus, and an IC50 of 149.7ng/mL for the BQ.1 strain pseudovirus. The above results indicate that: the m8A8 antibody has strong neutralization activity and broad spectrum.

The m8A8 murine antibody information was obtained by Sanger sequencing and comparing the sequencing results on IMGT, as follows:

m8A8 murine antibody heavy chain variable region CDR1 amino acid sequence SEQ ID NO.1:

GFTFAFYW

m8A8 murine antibody heavy chain variable region CDR2 amino acid sequence SEQ ID NO.2:

IYPGNGDT

m8A8 murine antibody heavy chain variable region CDR3 amino acid sequence SEQ ID NO.3:

TRAENYDGWFDY

m8A8 murine antibody light chain variable region CDR1 amino acid sequence SEQ ID NO.4:

QSVAYDGNNY

m8A8 murine antibody light chain variable region CDR2 amino acid sequence:

AAS

m8A8 murine antibody light chain variable region CDR3 amino acid sequence SEQ ID NO.6:

QQSNDYPYT

m8A8 murine antibody heavy chain variable region amino acid sequence SEQ ID NO.7:

QVQLQQSGAELARPGASVKLSCKASGFTFAFYWIQWIKQRPGQGLEWIGAIYPGNGDTKYSQKFRGKATLTADKSSMTAYMQLSGLASEDSAVYYCTRAENYDGWFDYWGQGTLVTVSA

m8A8 murine antibody light chain variable region amino acid sequence SEQ ID NO.8:

DIVLTQSPGSLTVSLGQRATISCKASQSVAYDGNNYMNWYQQKPGQPPKLLIFAASNLQSGVPTRFSGSGSGTDFTLSILPVEEEDAAIYYCQQSNDYPYTFGGGTKLEIK

m8A8 murine antibody heavy chain variable region nucleotide sequence SEQ ID No.11:

CAGGTTCAGCTCCAGCAGTCTGGGGCTGAGCTGGCAAGACCTGGGGCTTCCGTGAAGTTGTCCTGCAAGGCTTCTGGCTTCACCTTTGCTTTCTACTGGATACAGTGGATAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCTATTTATCCTGGAAATGGTGATACTAAATACAGTCAGAAGTTCAGGGGCAAGGCCACATTGACTGCAGATAAATCCTCCATGACAGCCTATATGCAACTCAGCGGCTTGGCATCTGAGGACTCTGCGGTCTATTACTGTACAAGAGCAGAAAACTATGATGGTTGGTTTGATTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

m8A8 murine antibody light chain variable region nucleotide sequence SEQ ID No.12:

GACATCGTGCTGACCCAGAGCCCCGGCAGCCTGACCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGCCTACGACGGCAACAACTACATGAACTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTTCGCCGCCAGCAACCTGCAGAGCGGCGTGCCCACCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAGCATCCTGCCCGTGGAGGAGGAGGACGCCGCCATCTACTACTGCCAGCAGAGCAACGACTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAG

EXAMPLE 3 humanized engineering of murine antibody m8A8, affinity, neutralization Activity detection

1. Humanized design of murine antibody m8A8

By comparing IMGT (http:// IMGT. Cines. FR) human antibody heavy and light chain variable region germline gene databases, heavy and light chain variable region germline genes with high homology with a murine antibody are respectively selected as templates, and CDRs of the murine antibody are respectively transplanted into corresponding human templates to form variable region sequences with the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. And (3) according to the requirement, back-mutating key amino acids in the framework sequence into amino acids corresponding to the murine antibody so as to ensure the original affinity, thus obtaining the humanized monoclonal antibody. The method comprises the following steps:

the humanized light chain template of the murine antibody m8A8 is a human antibody light chain germ line gene IGKV4-1 x 01 and IGKJ4 x 01, the humanized heavy chain template is a human antibody light chain germ line gene IGHV1-69 x 02 and IGHJ1 x 01, and the CDRs of the murine antibody are respectively transplanted into the human templates, so that the corresponding humanized antibody is obtained. And (3) carrying out back mutation on key amino acids in the FR region sequence of the humanized antibody to amino acids corresponding to the murine antibody according to the requirement so as to ensure the original affinity. Specific back mutations were designed as follows:

h8A8-H：Graft(IGHV1-69*02)+A97T,G27F,S30A,V37I,M48I,I70L,V68Ah8A8-L：Graft(IGKV4-1*01)+Y53F,S7T

note that: graft represents implantation of murine antibody CDRs into human germline template FR region sequences; a97T represents mutating a97 th position a of Graft to T, and so on. The numbering of the back mutated amino acids is the natural sequence numbering.

2. Recombinant humanized antibody h8A8 design and modification

The sequence of SEQ ID No.11 is cloned to pRVL-5 vector, and the obtained recombinant plasmid is named pRVL-5-H8A8-H after being verified to be correct by sequencing, and is a recombinant expression plasmid of antibody H8A 8. The sequence of SEQ ID No.12 is cloned to pRVL-4 vector, and the obtained recombinant plasmid is named pRVL-4-h8A8-L after being verified to be correct by sequencing, and is a light chain expression plasmid of antibody h8A 8. The amino acid sequence of the heavy chain of the humanized antibody h8A8 is shown as SEQ ID No.9, and the amino acid sequence of the light chain is shown as SEQ ID No. 10.

In SEQ ID No.9, the heavy chain variable region VH is composed of amino acid residues 1 to 119 from the N-terminus (wherein, amino acid residues 26 to 33 constitute HCDR1 (SEQ ID No. 1), amino acid residues 51 to 58 constitute HCDR2 (SEQ ID No. 2), amino acid residues 96 to 108 constitute HCDR3 (SEQ ID No. 3)), amino acid residues 120 to 212 constitute the heavy chain constant region CH1, amino acid residues 213 to 224 constitute the heavy chain Hinge region Hinge, amino acid residues 225 to 337 constitute the heavy chain constant region CH2, and amino acid residues 338 to 444 constitute the heavy chain constant region CH 3).

In SEQ ID No.10, the light chain variable region VL is composed of amino acid residues 1 to 111 from the N-terminus (wherein amino acid residues 27 to 36 constitute LCDR1 (SEQ ID No. 4), amino acid residues 54 to 56 constitute LCDR2 (SEQ ID No. 5), amino acid residues 93 to 101 constitute LCDR3 (SEQ ID No. 6)), and amino acid residues 112 to 216 constitute the light chain constant region CL.

The DNA molecule shown in SEQ ID No.13 encodes the polypeptide (heavy chain) shown in SEQ ID No. 9. In SEQ ID No.13, nucleotides 1 to 357 from the 5' end encode VH (wherein nucleotides 76 to 99 encode HCDR1, nucleotides 151 to 174 encode HCDR2, nucleotides 289 to 324 encode HCDR 3), nucleotides 358 to 636 encode CH1, nucleotides 637 to 672 encode finger, nucleotides 673 to 1011 encode CH2, nucleotides 1012 to 1332 encode CH3, and nucleotides 1333 to 1335 are stop codons.

The DNA molecule shown in SEQ ID No.14 encodes the polypeptide (light chain) shown in SEQ ID No. 10. In SEQ ID No.14, nucleotides 1 to 333 from the 5' end encode VL (wherein, nucleotides 79 to 108 encode LCDR1, nucleotides 160 to 168 encode LCDR2, nucleotides 277 to 303 encode LCDR 3), nucleotides 334 to 650 encode CL, and nucleotides 651 to 653 are stop codons.

Wherein the sequence of the complementarity determining region is defined according to IMGT. Antibody h8A8 was IgG1 and light chain type was kappa (kappa).

h8A8 humanized antibody heavy chain amino acid sequence SEQ ID No.9:

EVQLVQSGAEVKKPGSSVKVSCKASGFTFAFYWIQWIRQAPGQGLEWIGAIYPGNGDTKYSQKFRGRATLTADKSTSTAYMELSSLRSEDTAVYYCTRAENYDGWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

h8A8 humanized antibody light chain amino acid sequence SEQ ID No.10:

DIVMTQTPDSLAVSLGERATINCKASQSVAYDGNNYMNWYQQKPGQPPKLLIFAASNLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNDYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

h8A8 humanized antibody heavy chain nucleotide sequence SEQ ID No.13:

GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTTCACCTTCGCCTTCTACTGGATCCAGTGGATCAGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGCCATCTACCCCGGCAACGGCGACACCAAGTACAGCCAGAAGTTCAGGGGCAGGGCCACCCTGACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGCACCAGGGCCGAGAACTACGACGGCTGGTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGTCCAAGTGTATTCCCACTCGCTCCAAGCTCTAAATCCACGAGCGGTGGTACGGCAGCCCTCGGATGCCTCGTCAAAGATTATTTTCCAGAACCTGTTACAGTATCTTGGAATAGCGGCGCGTTGACCAGTGGCGTCCATACATTTCCCGCGGTATTGCAATCATCTGGCCTCTATAGTCTTTCATCAGTGGTCACCGTCCCGAGTTCTAGCCTGGGTACACAGACTTACATTTGCAATGTAAACCATAAGCCAAGCAATACTAAAGTTGACAAGAAGGTCGAGCCCAAAAGCTGTGACAAGACTCATACCTGCCCGCCCTGCCCAGCCCCCGAACTTCTTGGTGGACCGTCCGTCTTCCTCTTTCCTCCTAAGCCGAAGGACACGCTGATGATCTCAAGGACTCCCGAAGTTACATGTGTGGTCGTAGATGTCTCACACGAGGACCCCGAGGTTAAATTCAATTGGTACGTCGACGGAGTAGAAGTTCATAACGCCAAAACTAAGCCGAGGGAGGAGCAATACAATTCAACGTATCGGGTCGTAAGCGTTCTTACCGTCCTCCATCAGGATTGGTTGAACGGTAAAGAATACAAGTGCAAAGTTAGCAATAAAGCCCTCCCCGCCCCGATAGAAAAGACTATAAGTAAGGCGAAAGGACAACCAAGAGAACCCCAAGTTTACACTTTGCCCCCCTCACGGGATGAACTTACAAAAAATCAAGTCTCCCTTACATGCCTGGTGAAGGGTTTCTACCCATCAGATATCGCAGTGGAATGGGAATCTAACGGACAACCAGAAAATAATTACAAGACGACGCCGCCAGTCCTCGACTCTGATGGGTCCTTTTTTTTGTACTCAAAACTTACTGTGGATAAAAGTAGGTGGCAACAAGGCAATGTGTTCTCCTGCTCAGTGATGCATGAGGCCCTTCACAATCATTATACCCAGAAATCATTGAGCCTGAGCCCCGGCAAATGA

h8A8 humanized antibody light chain nucleotide sequence SEQ ID No.14:

GACATCGTGATGACCCAGACCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGAGGGCCACCATCAACTGCAAGGCCAGCCAGAGCGTGGCCTACGACGGCAACAACTACATGAACTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTTCGCCGCCAGCAACCTGCAGAGCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGCAGAGCAACGACTACCCCTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAAAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGCTGA

3. expression and purification of recombinant h8A8 antibodies

The constructed expression plasmids pRVL-5-H8A8-H and pRVL-4-H8A8-L are used for co-transfecting HEK293T cells by using a transfection reagent PEI. The method comprises the following steps: HEK293T cells with good growth state are selected and inoculated into a 10cm dish one day before transfection, the density is about 50% -80%, and the culture medium is replaced by serum-free DMEM culture medium. Transfection complex preparation: the light chain plasmid and the heavy chain plasmid are respectively 12 mug, 60 mug of PEI is added, the total system is 1mL, the residual volume is cultivated and supplemented by DMEM base, and the mixture is placed for 15min at room temperature after being uniformly mixed; then adding the mixed solution into the prepared HEK293T cells, and gently mixing; culturing at 37deg.C in 5% CO2 incubator for 8 hr, removing supernatant, and replacing DMEM complete medium; 48h later, the OPTI-MEM medium was changed, 96h later, the supernatant was collected, centrifuged at 4℃and 5000g, and the purification method was the same as in example 1. The purified and substituted antibody solution was subjected to filtration sterilization with a 0.22 μm filter membrane and dialysis treatment, the protein concentration was measured by BCA method, and the antibody protein stock solution was split-packed at-20 ℃.

The affinity assay of the h8A8 antibody was performed in the same manner as in example 2, while setting the parent antibody m8A8 as a control, and showed that h8A8 had a stronger binding capacity with recombinant RBD < 1ng/mL (FIG. 3).

The h8A8 antibody was used to neutralize the novel coronavirus pseudovirus by the same method as in example 2, the h8A8 antibody was effective to neutralize the novel coronavirus pseudovirus, the IC50 for the WH1 (wild-type) strain pseudovirus was 15ng/mL, the IC50 for the B.1.617.2 strain pseudovirus was 6ng/mL, the IC50 for the B.1.1.529 strain pseudovirus was 36ng/mL, the IC50 for the BA.2 strain pseudovirus was 831ng/mL, the IC50 for the BA.3 strain pseudovirus was 117ng/mL, the IC50 for the BA.4& BA.5 strain pseudovirus was 114ng/mL, the IC50 for the BA.2.75 strain pseudovirus was 1305ng/mL, and the IC50 for the BQ.1 strain pseudovirus was 1305 5ng/mL. The above results indicate that the h8A8 antibody has strong neutralizing activity and broad spectrum (FIG. 4).

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. An anti-covd-19 virus neutralizing antibody comprising a heavy chain and a light chain;

the amino acid sequences of HCDR1, HCDR2 and HCDR3 regions in the heavy chain variable region of the neutralizing antibody are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3;

the amino acid sequences of LCDR1, LCDR2 and LCDR3 regions in the light chain variable region of the neutralizing antibody are shown in SEQ ID NO.4, AAS and SEQ ID NO.6, respectively.

2. The neutralizing antibody of claim 1 wherein: the neutralizing antibody is a RBD monoclonal neutralizing antibody of a murine anti-COVID-19 virus, and the amino acid sequence of a heavy chain variable region of the neutralizing antibody is SEQ ID No.7 or a sequence with the sequence consistency of not less than 90% with SEQ ID No.7 and unchanged activity;

the amino acid sequence of the light chain variable region of the neutralizing antibody is SEQ ID NO.8, or a sequence which has at least 90% of identity with SEQ ID NO.8 and has unchanged activity.

3. The neutralizing antibody of claim 1 wherein: the neutralizing antibody is obtained by implanting a complementarity determining region of a heavy chain variable region shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, a light chain variable region shown as SEQ ID NO.4, AAS and SEQ ID NO.6 into a human germline template FR region sequence, and carrying out mutation recovery on a heavy chain through A97T, G27F, S30A, V37I, M48I, I70L and V68A and/or carrying out mutation recovery on a light chain through Y53F and S7T.

4. A neutralizing antibody according to claim 3 wherein the amino acid sequence of the heavy chain variable region of said neutralizing antibody comprises SEQ ID No.9 from position 1 to 119 from the N-terminus or a sequence having at least 90% identity with SEQ ID No.9 from position 1 to 119 from the N-terminus and having unchanged activity;

the amino acid sequence of the light chain variable region of the neutralizing antibody comprises SEQ ID NO.10 from the 1 st to 111 th positions from the N end or a sequence which has at least 90% of identity with SEQ ID NO.9 from the 1 st to 111 th positions from the N end and has unchanged activity;

preferably, it is:

the heavy chain amino acid sequence of the neutralizing antibody is shown as SEQ ID NO.9, or a sequence which has at least 90% of identity with SEQ ID NO.9 and has unchanged activity;

the light chain amino acid sequence of the neutralizing antibody is shown as SEQ ID NO.10 or a sequence which has at least 90% of identity with SEQ ID NO.10 and has unchanged activity.

5. A nucleic acid molecule encoding the neutralizing antibody or antigen binding portion thereof of any one of claims 1-4.

6. The nucleic acid molecule of claim 5, wherein the nucleotide sequence encoding the heavy chain variable region of the neutralizing antibody is SEQ ID No.11 or has at least 90% identity to SEQ ID No. 11; the nucleotide sequence encoding the light chain variable region of the neutralizing antibody is SEQ ID No.12 or has at least 90% identity to SEQ ID No. 12.

7. The nucleic acid molecule of claim 5, wherein,

the nucleotide sequences encoding HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are:

sequentially shown as 76 th to 99 th bits, 151 th to 174 th bits and 289 th to 324 th bits from the 5' end of SEQ ID No. 13;

the nucleotide sequences encoding LCDR1, LCDR2 and LHCDR3 in the light chain variable region are:

sequentially shown as 79 th to 108 th positions, 160 th to 168 th positions and 277 th to 303 th positions from the 5' end of SEQ ID No. 14;

preferably, it is:

the nucleotide sequence encoding the heavy chain variable region of the neutralizing antibody is SEQ ID No.13 having at least 90% identity from position 1 to 357 from the 5 'end or with SEQ ID No.13 from position 1 to 357 from the 5' end; the nucleotide sequence encoding the light chain variable region of the humanized antibody is 1-333 of SEQ ID No.14 from the 5 'end or has at least 90% identity with 1-333 of SEQ ID No.14 from the 5' end;

more preferably, the nucleotide sequence encoding the heavy chain of the neutralizing antibody is SEQ ID No.13 or has at least 90% identity to SEQ ID No. 13;

the nucleotide sequence encoding the light chain of the humanized antibody is SEQ ID No.14 or has at least 90% identity to SEQ ID No. 14.

8. An expression vector having a nucleic acid molecule according to any one of claims 5 to 7, wherein the expression vector includes, but is not limited to: plasmid, expression cassette, recombinant vector or recombinant plasmid, recombinant cell or recombinant bacterium.

9. A pharmaceutical composition or vaccine, characterized in that the active ingredient of the pharmaceutical composition comprises a neutralizing antibody according to any one of claims 1-4 and a pharmaceutically acceptable excipient, diluent or carrier.

10. The application is as follows:

(A1) Use of the expression vector of claim 8 for the preparation of a neutralizing antibody according to any one of claims 1-4 or a pharmaceutical composition or vaccine according to claim 9;

(A2) Use of a neutralizing antibody according to any one of claims 1-4 for the preparation of a pharmaceutical composition or vaccine against the covd-19 virus;

(A3) Use of a neutralizing antibody according to any one of claims 1-4 or a nucleic acid molecule according to any one of claims 5-7 or an expression vector according to claim 8 or a pharmaceutical composition or vaccine according to claim 9 for the preparation of a product for the prevention and/or treatment of a disease caused by SARS-CoV-2 infection;

(A4) Use of a neutralizing antibody according to any one of claims 1-4 or a nucleic acid molecule according to any one of claims 5-7 or an expression vector according to claim 8 or a pharmaceutical combination or vaccine according to claim 9 for the preparation of a product for inhibiting SARS-CoV-2 infection;

(A5) Use of a neutralizing antibody according to any one of claims 1-4 or a nucleic acid molecule according to any one of claims 5-7 or an expression vector according to claim 8 for the preparation of a product for the detection of SARS-CoV-2;

(A6) Use of a neutralizing antibody according to any one of claims 1-4 or a nucleic acid molecule according to any one of claims 5-7 or an expression vector according to claim 8 or a pharmaceutical composition or vaccine according to claim 9 for the preparation of a product for neutralizing SARS-CoV-2;

(A7) Use of a neutralizing antibody according to any one of claims 1-4 or a nucleic acid molecule according to any one of claims 5-7 or an expression vector according to claim 8 for the preparation of a product for detecting the RBD protein of SARS-CoV-2;

(A8) Use of a neutralizing antibody according to any one of claims 1-4 or a nucleic acid molecule according to any one of claims 5-7 or an expression vector according to claim 8 or a pharmaceutical composition or vaccine according to claim 9 for the preparation of a product for binding the RBD protein of SARS-CoV-2.