Nanometer antibody TnbE12 for broad-spectrum neutralization of sand shell virus and application of nanometer antibody TnbE12

Abstract

The invention discloses a nano antibody TnbE12 capable of neutralizing a sand shell virus in a broad spectrum and application of the nano antibody TnbE12. The invention provides a TnbE12 nano antibody. Three CDRs in a variable region of the TnbE12 nano antibody are sequentially shown as the 26 site to the 33 site, the 51 site to the 57 site and the 96 site to the 105 site in SEQ ID NO: 9. The invention also provides a TnbE12 humanized nano antibody, which comprises a TnbE12 nano antibody variable region and a human Fc region. The TnbE12 nano antibody and the TnbE12 humanized nano antibody can be used for inhibiting and / or neutralizing coronavirus, so that diseases caused by the coronavirus are prevented and / or treated. The invention has important application value for prevention and control of coronavirus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a nanobody TnbE12 for neutralizing a broad spectrum of sarbecoviruses and application thereof. BACKGROUND

[0002] The novel coronavirus (SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2) belongs to the Sarbecovirus subgenus in the beta genus of coronaviruses. With the spread process, SARS-CoV-2 has produced many mutations, and the main epidemic variants are Alpha, Beta, Delta and Omicron strains, etc. The transmission ability of new variants is continuously enhanced, and different degrees of immune escape are produced, resulting in different degrees of decline in the protective efficacy of vaccines, and many monoclonal antibodies also have weakened neutralizing activity or even completely lost neutralizing activity to the mutant strains.

[0003] In addition to SARS-CoV-2, the severe acute respiratory syndrome coronavirus (SARS-CoV-1, Severe Acute Respiratory Syndrome Coronavirus 1) also belongs to the Sarbecovirus subgenus. The Sarbecovirus subgenus often uses bats as animal hosts, and can be further divided into three branches: the first type, the SARS-CoV-1 virus infecting humans and bat-derived SARS-CoV-1 virus strains similar to its genome, such as Bat SARS-like WIV1, etc.; the second type, bat-derived SARS-CoV-1-like virus strains, such as Bat SARS-like CoV ZC45 and Bat SARS-like CoV ZXC21, etc.; the third type, the SARS-CoV-2 virus infecting humans and bat-derived and pangolin-derived virus strains similar to its genome, such as BatCoV RaTG13, etc. Since the twentieth century, sarbecoviruses have caused two human infections (SARS-CoV-1 and SARS-CoV-2) in the human world, and researchers have also continuously discovered more sarbecoviruses similar to SARS-CoV-1 from natural hosts. In order to cope with future variants and sarbecoviruses that may evolve from natural hosts to infect humans, it is also crucial to find universal drugs and universal vaccines for preventing and treating sarbecovirus infections to prevent more variants and possible new coronavirus outbreaks in the future.

[0004] Conventional immunoglobulin IgG antibodies are composed of two identical heavy chains and two identical light chains, such as monoclonal antibodies or humanized mouse antibodies isolated from human body. In addition to conventional antibodies, there are unique heavy chain antibodies (HCAbs) in camelids, which are composed of two identical heavy chains, each of which is composed of 1 heavy chain variable region (VHH) and two constant regions (CH2 and CH3). VHH is the smallest complete functional structure of heavy chain antibody, also known as Nanobody or single domain antibody. Nanobody has many advantages such as small volume, strong specificity, strong stability, easy production, strong penetration and low immunogenicity. Benefiting from the small volume and high specificity of Nanobody, many more hidden antibody epitopes on target proteins can be easily recognized by Nanobody, which is conducive to further distinguishing more neutralizing antibody epitopes and providing innovative ideas for antibody drug and vaccine research and development. SUMMARY

[0005] The purpose of the present application is to provide a Nanobody TnbE12 that can neutralize a wide range of Sars-like viruses and its application.

[0006] The present application provides a Nanobody (named TnbE12 Nanobody) against coronavirus, wherein the CDR1, CDR2 and CDR3 in the variable region are sequentially shown as SEQ ID NO: 9, 26-33 (GSISTLNV), 51-57 (ITLDGRP) and 96-105 (KLENGGFFYY).

[0007] Specifically, the variable region of the TnbE12 Nanobody comprises a framework region FR and a complementarity determining region CDR; the CDR1, CDR2 and CDR3 are sequentially shown as SEQ ID NO: 9, 26-33 (GSISTLNV), 51-57 (ITLDGRP) and 96-105 (KLENGGFFYY); the FR1, FR2, FR3 and FR4 are sequentially shown as SEQ ID NO: 9, 1-25, 34-50, 58-95 and 106-116.

[0008] Specifically, the variable region of the TnbE12 Nanobody is composed of a framework region FR and a complementarity determining region CDR.

[0009] Specifically, the variable region of the TnbE12 Nanobody is shown as SEQ ID NO: 9.

[0010] The application also provides a variable region of a nanobody against coronavirus (named as TnbE12 nanobody variable region), wherein CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 9 as follows: 26-33 (GSISTLNV), 51-57 (ITLDGRP) and 96-105 (KLENGGFFYY) respectively.

[0011] The TnbE12 nanobody variable region comprises a framework region FR and a complementarity determining region CDR, wherein CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 9 as follows: 26-33 (GSISTLNV), 51-57 (ITLDGRP) and 96-105 (KLENGGFFYY) respectively, and FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 9 as follows: 1-25, 34-50, 58-95 and 106-116 respectively.

[0012] The FR1 can also be a polypeptide segment obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the polypeptide segment shown in SEQ ID NO: 9 and having the same function as the polypeptide segment. The FR2 can also be a polypeptide segment obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the polypeptide segment shown in SEQ ID NO: 9 and having the same function as the polypeptide segment. The FR3 can also be a polypeptide segment obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the polypeptide segment shown in SEQ ID NO: 9 and having the same function as the polypeptide segment. The FR4 can also be a polypeptide segment obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the polypeptide segment shown in SEQ ID NO: 9 and having the same function as the polypeptide segment.

[0013] Specifically, the TnbE12 nanobody variable region is composed of a framework region FR and a complementarity determining region CDR.

[0014] Specifically, the TnbE12 nanobody variable region is shown in SEQ ID NO: 9.

[0015] The application also provides a humanized nanobody against coronavirus (named as TnbE12 humanized nanobody), comprising two segments: a TnbE12 nanobody variable region and a human Fc segment.

[0016] Specifically, the TnbE12 humanized nanobody comprises the following segments from N-terminus to C-terminus: TnbE12 nanobody variable region, human Fc segment. Specifically, the TnbE12 humanized nanobody consists of the following two segments from N-terminus to C-terminus: TnbE12 nanobody variable region, human Fc segment. Specifically, the TnbE12 humanized nanobody consists of the following three segments from N-terminus to C-terminus: TnbE12 nanobody variable region, connecting peptide, and human Fc segment.

[0017] Specifically, the human Fc segment is as shown in SEQ ID NO: 12.

[0018] Specifically, the human Fc segment can be as follows (a) or (b):

[0019] (a) a protein obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the protein as shown in SEQ ID NO: 12 and having the same function as the protein;

[0020] (b) a protein having more than 95% identity with the protein as shown in SEQ ID NO: 12 and having the same function as the protein.

[0021] Specifically, the amino acid sequence of the connecting peptide is GGGGS.

[0022] Specifically, the connecting peptide can also be other connecting peptides commonly used by those skilled in the art.

[0023] A nucleic acid molecule encoding the TnbE12 nanobody also falls within the protection scope of the present application.

[0024] A nucleic acid molecule encoding the TnbE12 nanobody variable region also falls within the protection scope of the present application.

[0025] Specifically, the nucleic acid molecule is as shown in SEQ ID NO: 10.

[0026] A nucleic acid molecule encoding the TnbE12 humanized nanobody also falls within the protection scope of the present application.

[0027] Specifically, the nucleic acid molecule consists of the following three segments from upstream to downstream: the segment as shown in SEQ ID NO: 10, the segment as shown in SEQ ID NO: 11 at positions 962-976, and the segment as shown in SEQ ID NO: 11 at positions 977-1672.

[0028] The present application also protects the use of the TnbE12 nanobody or the TnbE12 nanobody variable region or the TnbE12 humanized nanobody in the preparation of a drug for inhibiting and / or neutralizing coronavirus.

[0029] The application also protects a medicine for inhibiting and / or neutralizing a coronavirus, the active ingredient of which is a TnbE12 nanobody or a TnbE12 nanobody variable region or a TnbE12 humanized nanobody.

[0030] The application also protects the use of a TnbE12 nanobody or a TnbE12 nanobody variable region or a TnbE12 humanized nanobody in the preparation of a medicine for preventing and / or treating a disease caused by a coronavirus.

[0031] The application also protects a medicine for preventing and / or treating a disease caused by a coronavirus, the active ingredient of which is a TnbE12 nanobody or a TnbE12 nanobody variable region or a TnbE12 humanized nanobody.

[0032] The naming system of the CDRs is IMGT.

[0033] Specifically, the coronavirus is a coronavirus of the beta genus.

[0034] Specifically, the coronavirus is a coronavirus of the sarbecovirus subgenus.

[0035] Specifically, the coronavirus is SARS-CoV-2. The SARS-CoV-2 can be a wild strain or a naturally mutated strain. The SARS-CoV-2 is a SARS-CoV-2 that infects humans.

[0036] Specifically, the coronavirus is SARS-CoV-1. The SARS-CoV-1 can be a wild strain or a naturally mutated strain. The SARS-CoV-1 can be a SARS-CoV-1 that infects humans.

[0037] Specifically, the coronavirus is a pangolin coronavirus (Pangolin CoV), such as Pangolin CoV GD, Pangolin CoV GX, etc.

[0038] Specifically, the coronavirus is a bat coronavirus (Bat CoV), such as Bat CoV RaTG13, Bat CoV WIV16, etc.

[0039] The inventors of the application used SARS-CoV-2 BA.4 / 5 S protein and SARS-CoV-1 S protein as bait to isolate nanobodies with broad-spectrum neutralizing ability from alpacas immunized with SARS-CoV-2 RBD protein, adenovirus vaccine AdC68-19S (immunized with chimpanzee adenovirus vector vaccine carrying novel coronavirus protein antigen) and SARS-CoV-2 S protein. Further, the inventors fused the variable region of the nanobody with human antibody Fc to obtain humanized nanobodies with broad-spectrum neutralizing ability.

[0040] The nanobody provided by the application has the effect of efficiently neutralizing various strains of coronavirus of the Sarbecovirus subgenus, and the discovery of the antibody has great application value for the prevention and control of coronavirus of the Sarbecovirus subgenus (such as SARS-CoV-2), and will have far-reaching social significance. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 When the protein liquid to be purified is the supernatant containing SARS-CoV-2 RBD protein, the gel filtration chromatogram.

[0042] Figure 2 When the protein liquid to be purified is the supernatant containing SARS-CoV-2 S protein, the gel filtration chromatogram.

[0043] Figure 3 When the protein liquid to be purified is the supernatant containing SARS-CoV-2 BA.4 / 5 S protein, the gel filtration chromatogram.

[0044] Figure 4 When the protein liquid to be purified is the supernatant containing SARS-CoV-1 S protein, the gel filtration chromatogram. DETAILED DESCRIPTION

[0045] The application will be further described in detail below in conjunction with the specific embodiments. The examples given are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0046] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified. In the following examples, quantitative tests were set up in triplicate, and the results were averaged, unless otherwise specified.

[0047] 293F cells: Thermo Fisher Scientific, product catalog number R79007. Plasmid pFastBac-dual: Gibco, product catalog number 10712024. Plasmid pcDNA3.1(+): Invitrogen, product catalog number V790-20. 293T cells: ATCC, product catalog number CRL-11268. Insect cell culture medium (Sf-900 II SFM): Gibco, product catalog number 10902-088. SMM 293-TII medium: Yiqiao Shenzhou, product catalog number M293TII. Adenovirus vaccine AdC68-19S, i.e., ChAdTS-COVID-19S virus liquid prepared in patent application 202010369075.2 (application publication number CN 113583978 A, application publication date November 2, 2021).

[0048] Example 1, screening of nanobodies

[0049] I. Preparation of proteins

[0050] 1. Preparation of SARS-CoV-2 RBD protein

[0051] The SARS-CoV-2 RBD fusion protein is shown in SEQ ID NO: 1. In SEQ ID NO: 1, the first to 33th amino acid residues constitute a signal peptide, the 34th to 256th amino acid residues constitute a SARS-CoV-2 RBD, the 257th to 264th amino acid residues constitute a strep tag, the 265th to 272th amino acid residues constitute a Flag tag, and the 273rd to 278th amino acid residues constitute a His6 tag. The SARS-CoV-2 RBD fusion protein exists in a dimeric form, and the expected molecular weight of the dimer is about 50 kDa.

[0052] The small fragment between the BamHI and HindIII enzyme cutting sites of the plasmid pcDNA3.1(+) was replaced with a double-stranded DNA molecule (encoding the protein shown in SEQ ID NO: 1) shown in SEQ ID NO: 2, to obtain a recombinant plasmid pcDNA3.1-SARS-CoV-2RBD. The recombinant plasmid pcDNA3.1-SARS-CoV-2RBD was transfected into 293F cells, which were cultured in SMM 293-TII medium for 72 h, then centrifuged at 4000 rpm for 30 min, and the supernatant containing the SARS-CoV-2 RBD protein was collected.

[0053] 2. Preparation of SARS-CoV-2 S protein

[0054] SARS-CoV-2 spike 2P extracellular region fusion protein (referred to as SARS-CoV-2 S protein) is shown in SEQ ID NO: 3. In SEQ ID NO: 3, the first to 13th amino acid residues constitute a signal peptide, the 14th to 1211th amino acid residues constitute a SARS-CoV-2 extracellular region, the 1212th to 1219th amino acid residues constitute a 3C enzyme cleavage site, the 1220th to 1227th amino acid residues constitute a linker peptide, the 1228th to 1255th amino acid residues constitute a trimer tag (functioning to facilitate the formation of a stable trimer), the 1256th to 1263rd amino acid residues constitute a strep tag, and the 1264th to 1269th amino acid residues constitute a His6 tag. The SARS-CoV-2 S protein exists in the form of a trimer, and the expected molecular weight of the trimer is 420 kDa. Compared with the corresponding protein in the wild-type novel coronavirus, the protein shown in SEQ ID NO: 3 is modified as follows: two mutations are introduced, i.e., the S1 / S2 enzyme cleavage site is mutated from “RRKR” to “GSAS”, and “KV” is mutated to “PP” to increase protein stability; a 3C enzyme cleavage site, a linker peptide, a trimer tag, a strep tag, and a His6 tag are introduced at the C-terminus.

[0055] The small fragment between the BamHI and HindIII enzyme cleavage sites of the plasmid pcDNA3.1(+) is replaced with the double-stranded DNA molecule shown in SEQ ID NO: 4 (encoding the protein shown in SEQ ID NO: 3) to obtain the recombinant plasmid pcDNA3.1-SARS-CoV-2 spike 2P. The recombinant plasmid pcDNA3.1-SARS-CoV-2 spike 2P is transfected into 293F cells, which are cultured for 72 h using SMM 293-TII medium, then centrifuged at 4000 rpm for 30 min, and the supernatant containing the SARS-CoV-2 S protein is collected.

[0056] 3. Preparation of SARS-CoV-2 BA.4 / 5 S protein

[0057] SARS-CoV-2 BA.4 / 5 spike 2P ectodomain fusion protein (referred to as SARS-CoV-2 BA.4 / 5 S protein) is shown in SEQ ID NO: 5. In SEQ ID NO: 5, the 1st-13th amino acid residues constitute a signal peptide, the 14th-1203rd amino acid residues constitute a SARS-CoV-2 ectodomain, the 1204th-1211th amino acid residues constitute a 3C enzyme cleavage site, the 1212th-1219th amino acid residues constitute a linker peptide, the 1220th-1247th amino acid residues constitute a trimer tag (functioning to facilitate the formation of a stable trimer), the 1248th-1255th amino acid residues constitute a strep tag, and the 1256th-1261th amino acid residues constitute a His6 tag. The SARS-CoV-2 BA.4 / 5 S protein exists in the form of a trimer, and the expected molecular weight of the trimer is 420 kDa.

[0058] The small fragment between the BamHI and HindIII enzyme cleavage sites of the plasmid pcDNA3.1(+) was replaced with a double-stranded DNA molecule (encoding the protein shown in SEQ ID NO: 5) shown in SEQ ID NO: 6, to obtain a recombinant plasmid pcDNA3.1-SARS-CoV-2 BA.4 / 5 spike 2P. The recombinant plasmid pcDNA3.1-SARS-CoV-2 BA.4 / 5 spike 2P was transfected into 293F cells, which were cultured for 72 h using SMM 293-TII medium, then centrifuged at 4000 rpm for 30 min, and the supernatant containing the SARS-CoV-2 BA.4 / 5 S protein was collected.

[0059] 4. Preparation of SARS-CoV-1 S protein

[0060] SARS-CoV-1 spike ectodomain fusion protein (referred to as SARS-CoV-1 S protein) is shown in SEQ ID NO: 7. In SEQ ID NO: 7, the 1st-13th amino acid residues constitute a signal peptide, the 14th-1195th amino acid residues constitute a SARS-CoV-1 ectodomain, the 1196th-1203rd amino acid residues constitute a 3C enzyme cleavage site, the 1204th-1211th amino acid residues constitute a linker peptide, the 1212th-1239th amino acid residues constitute a trimer tag (functioning to facilitate the formation of a stable trimer), the 1240th-1247th amino acid residues constitute a strep tag, and the 1248th-1253rd amino acid residues constitute a His6 tag. The SARS-CoV-1 S protein exists in the form of a trimer, and the expected molecular weight of the trimer is 420 kDa.

[0061] The supernatant containing SARS-CoV-1 S protein is prepared by using an insect baculovirus expression system, and the specific steps are as follows: the small fragment between the EcoRI and XbaI enzyme cutting sites of the plasmid pFastBac-dual is replaced with the double-stranded DNA molecule shown in SEQ ID NO: 8 (encoding the protein shown in SEQ ID NO: 7) to obtain the recombinant plasmid pFastBac-SARS-CoV-1 spike; the recombinant plasmid pFastBac-SARS-CoV-1 spike is introduced into E. coli DH10 Bac to obtain the recombinant Bacmid; the recombinant Bacmid is introduced into Sf9 cells to obtain the P1 generation virus liquid; the P1 generation virus liquid is used to infect Sf9 cells, and the insect cell culture medium is used for culture for 72 hours, then centrifuged at 4000 rpm for 15 min, and the supernatant is collected, which is the supernatant containing SARS-CoV-1 S protein.

[0062] II. Purification of protein

[0063] The protein liquid to be purified is: the supernatant containing SARS-CoV-2 RBD protein prepared in step 1, or the supernatant containing SARS-CoV-2 S protein prepared in step 2, or the supernatant containing SARS-CoV-2 BA.4 / 5 S protein prepared in step 3, or the supernatant containing SARS-CoV-1 S protein prepared in step 4.

[0064] 1. Affinity chromatography

[0065] The specifications of the chromatography column for affinity chromatography are: length 3 cm, inner diameter 1 cm.

[0066] The column filler for affinity chromatography is a nickel column bead (purchased from Qiagen Company, product catalog number 30230).

[0067] The following operation steps are performed in sequence: ① 500 mL of the protein liquid to be purified is loaded into the affinity chromatography column, and incubated at 4°C for 3 hours; ② the column is washed with 100 mL of HEPEs buffer (pH 7.2, 1M) containing 20 mM imidazole; ③ the target protein is eluted with 30 mL of HEPEs buffer (pH 7.2, 1M) containing 500 mM imidazole, and the solution after passing through the column is collected.

[0068] 2. The solution after passing through the column obtained after affinity chromatography is concentrated using a 10 kD concentration tube (purchased from Merck Company, product catalog number UFC800396) to obtain a concentrated solution with a volume of 1 mL.

[0069] 3. Gel filtration chromatography

[0070] The specifications of the chromatography column for gel filtration chromatography are: length 24 cm, inner diameter 2 cm.

[0071] Column packing of gel filtration chromatography: superdex200 increase 10 / 300 GL (purchased from GE Healthcare Company, product catalog number 28-9909-44).

[0072] The following operation steps were performed: 0.5 mL of the concentrated solution obtained in step 2 was loaded, eluted with PBS buffer (pH 7.2, 10 mM) at a flow rate of 0.5 mL / min, and the post-column solution corresponding to the target peak was collected, which was the purified protein solution.

[0073] When the purified protein solution was the supernatant containing SARS-CoV-2 RBD protein, the gel filtration chromatogram was as shown in Figure 1 The retention volume corresponding to the target peak was 18.192 mL, and the purified protein solution obtained was named SARS-CoV-2 RBD protein solution.

[0074] When the purified protein solution was the supernatant containing SARS-CoV-2 S protein, the gel filtration chromatogram was as shown in Figure 2 The retention volume corresponding to the target peak was 8.633 mL, and the purified protein solution obtained was named SARS-CoV-2 S protein solution.

[0075] When the purified protein solution was the supernatant containing SARS-CoV-2 BA.4 / 5 S protein, the gel filtration chromatogram was as shown in Figure 3 The retention volume corresponding to the target peak was 8.989 mL, and the purified protein solution obtained was named SARS-CoV-2 BA.4 / 5 S protein solution.

[0076] When the purified protein solution was the supernatant containing SARS-CoV-1 S protein, the gel filtration chromatogram was as shown in Figure 4 The retention volume corresponding to the target peak was 11.627 mL, and the purified protein solution obtained was named SARS-CoV-1 S protein solution.

[0077] The related proteins used in the subsequent steps and subsequent examples were all provided by the purified protein solution.

[0078] III. Screening of immunized llama nanobody display library

[0079] 1. Immunization of llamas and establishment of nanobody display library

[0080] The llama immunization process was as follows:

[0081] First immunization: immunized with SARS-CoV-2 RBD protein, the protein immunization dose was 200 μg per animal (specifically, the protein solution was diluted to 1 mL, then mixed with 1 mL of complete Freund's adjuvant and used)

[0082] Second immunization: immunize SARS-CoV-2 RBD protein, the protein immunization dose is 200 μg per (specifically, the protein solution is diluted to 1 mL, and then mixed with 1 mL of incomplete Freund's adjuvant for use) ;

[0083] Third immunization: immunize SARS-CoV-2 RBD protein, the protein immunization dose is 200 μg per (specifically, the protein solution is diluted to 1 mL, and then mixed with 1 mL of incomplete Freund's adjuvant for use) ;

[0084] Fourth immunization: immunize adenovirus vaccine AdC68-19S, the immunization dose is 10 11 vp per;

[0085] Fifth immunization: immunize SARS-CoV-2 S protein, the protein immunization dose is 200 μg per (specifically, the protein solution is diluted to 1 mL, and then mixed with 1 mL of incomplete Freund's adjuvant for use) ;

[0086] Sixth immunization: immunize SARS-CoV-2 S protein, the protein immunization dose is 200 μg per (specifically, the protein solution is diluted to 1 mL, and then mixed with 1 mL of incomplete Freund's adjuvant for use).

[0087] The six immunizations are all in the form of neck subcutaneous immunization.

[0088] Seven days after the sixth immunization, 50 mL of blood is taken from the neck vein and sent to Chengdu Apark Biotechnology Co., Ltd. for the construction of a nanobody display yeast library, obtaining a yeast library with a diversity of 10 8 .

[0089] 2、First round of magnetic bead sorting

[0090] (1) 10 9 yeast cells are inoculated into 100 mL of SDCAA medium and cultured at 30°C, 250 rpm to OD 600nm value = about 7.

[0091] (2) After step (1) is completed, centrifuge at 4000 rpm for 5 minutes, discard the supernatant, and resuspend the precipitate in SGCAA medium (initial OD 600nm value = 0.5), 20°C, 250 rpm to OD 600nm value = about 4.

[0092] (3) After step (2) is completed, take 10 9Cells were collected by centrifugation at 3500 rpm for 5 min, and the precipitate was resuspended in 2 mL of buffer, bait protein (the bait protein was SARS-CoV-2 BA.4 / 5 S protein and SARS-CoV-1 S protein) was added to make the concentration in the system 100 nM, and the mixture was incubated at room temperature for 30 min and on ice for 10 min, then centrifuged at 3500 rpm for 5 min, and the precipitate was collected and washed with buffer.

[0093] (4) The precipitate obtained in step (3) was resuspended in 3 mL of buffer, 200 μL of Streptavidin MicroBeads (Miltenyi) was added, and the mixture was incubated on ice for 10 min (every 2 min, mix well), then 5 mL of buffer was added, mixed well, and centrifuged, the precipitate was resuspended in 10 mL of buffer, filtered through a 70 μm filter screen, and the filtrate was collected (the filtration was to remove cell clumps), which was the yeast magnetic bead suspension.

[0094] (5) The LS column (LS Columns, Miltenyi Biotec, 130-042-401) was placed in a magnetic stand, rinsed with 3 mL of cold buffer, and 8.5 mL of the yeast magnetic bead suspension obtained in step (4) was added. After the flow was completed, the LS column was immediately removed and placed back, 1 mL of buffer was added for rinsing, then the remaining yeast magnetic bead suspension was added, and after the flow was completed, the LS column was rinsed with 3 mL of buffer for 3 times, then the LS column was removed from the magnetic stand, and 6 mL of buffer was added for rinsing into a 15 mL centrifuge tube.

[0095] (6) The centrifuge tube obtained in step (5) was centrifuged at 3500 rpm for 5 min, and the precipitate (yeast magnetic beads) was collected, resuspended in 1 mL of SDCAA medium and cultured for 24 h, then centrifuged to collect the precipitate, resuspended in SGCAA medium and cultured for 36 h. During the culture process, the cells proliferated greatly and the number of magnetic beads remained unchanged, so the magnetic beads were diluted, and the precipitate collected later was mainly cells.

[0096] 3. Second round of flow sorting

[0097] (1) After step 2 was completed, the cells were collected by centrifugation, washed with buffer, and then 10 8 cells were resuspended in 500 μL of buffer, bait protein (the bait protein was SARS-CoV-2 BA.4 / 5 S protein and SARS-CoV-1 S protein) was added to make the concentration in the system 100 nM, and the mixture was incubated on ice for 30 min, then centrifuged at 3500 rpm for 5 min, and the cell precipitate was collected and washed with buffer.

[0098] (2) After step (1) is completed, resuspend the cells in 500 μL buffer, add 2.5 μL anti-HA-AF488 antibody (Cell Signaling) and 2.5 μL Streptavidin-PE antibody (eBioscience), incubate on ice for 30 minutes, wash 3 times with 1 mL buffer, resuspend in 4 mL buffer, pass through a 70 μm filter and collect the filtrate, which is the yeast cell suspension.

[0099] (3) The yeast cell suspension obtained in step (2) is loaded onto the Arial II flow sorter, and FITC-positive and PE-positive yeasts are sorted into a flow collection tube containing 2 mL of SDCAA medium.

[0100] (4) After step (3) is completed, 2 x 10 3 yeasts are taken from the collection tube and diluted in 200 μL of SDCAA medium, and plated on SDCAA plates, and incubated at 30°C overnight; the remaining positive cells are frozen.

[0101] 4. Identification of positive yeast monoclonal

[0102] (1) After step 3 is completed, pick the yeast monoclonal from the SDCAA plate into a 96-well deep well plate, add 400 μL of SDCAA medium to each well, and incubate at 30°C, 250 rpm overnight.

[0103] (2) After step (1) is completed, take 50 μL, centrifuge at 4000 rpm for 5 minutes, discard the supernatant, resuspend the precipitate in 400 μL of SGCAA medium, and incubate at 20°C, 250 rpm for 36 hours.

[0104] (3) After step (2) is completed, take 100 μL, centrifuge and discard the supernatant, wash the precipitate with buffer, then resuspend in 200 μL of buffer, add bait protein (the bait protein is SARS-CoV-2 BA.4 / 5 S protein and SARS-CoV-1 S protein) to make its concentration in the system 50 nM, incubate on ice for 30 minutes, then centrifuge at 3500 rpm for 5 minutes, collect the cell precipitate, and wash with buffer.

[0105] (4) Resuspend the cell precipitate obtained in step (3) in 200 μL of buffer, add 1 μL of anti-HA-AF488 antibody and 1 μL of Streptavidin-PE antibody, incubate on ice for 30 minutes, centrifuge to collect the cell precipitate, and resuspend in 200 μL of buffer after washing with buffer.

[0106] (5) The cell suspension obtained in step (4) is loaded onto the Fortessa flow analyzer, and the proportion of positive yeasts is detected, and a double-positive yeast monoclonal with a positive yeast proportion of more than 20% is selected.

[0107] (6) The yeast plasmid of the double-positive yeast monoclonal is extracted, sequenced, and the nanobody gene sequence is obtained.

[0108] A nanobody is obtained, which is named as TnbE12 nanobody. The amino acid sequence of the variable region of the TnbE12 nanobody is shown as SEQ ID NO: 9, and the encoding gene is shown as SEQ ID NO: 10.

[0109] Example 2, Preparation of TnbE12 humanized nanobody

[0110] The pMD18-T vector is a component of Takara No. 6011 pMD TM 18-T Vector Cloning Kit, https: / / www.takarabiomed.com.cn / ProductShow.aspx?m=20141220150817403017&productID=20141227130215047304.

[0111] I. Construction of recombinant expression vector

[0112] 1. The double-stranded DNA molecule shown in SEQ ID NO: 11 is inserted into the EcoRV enzyme cutting site of the pMD18-T vector to obtain a human Fc carrier. In SEQ ID NO: 11, the nucleotides 51-354 constitute a CMV Enhancer, the nucleotides 355-558 constitute a CMV Promoter, the nucleotides 890-892 constitute a start codon, the nucleotides 947-952 constitute a NheI enzyme cutting recognition sequence, the nucleotides 954-961 constitute a NotI enzyme cutting recognition sequence, the nucleotides 977-1672 encode a human Fc, the nucleotides 1673-1675 are a stop codon, and the nucleotides 1748-1869 constitute a SV40 Poly(A).

[0113] 2. Replace the small fragment between Nhe I and Not I restriction enzyme recognition sequences of the human Fc carrier (containing Nhe I restriction enzyme recognition sequence and Not I restriction enzyme recognition sequence, i.e. GCTAGCCGCGGCCGC) with the double-stranded DNA molecule shown in SEQ ID NO: 10 to obtain the recombinant plasmid TnbE12-Fc. The recombinant plasmid TnbE12-Fc expresses a TnbE12 humanized nanobody. The TnbE12 humanized nanobody consists of the following segments in order from N-terminus to C-terminus: a signal peptide, a protein segment shown in SEQ ID NO: 9 (variable region of the TnbE12 nanobody), a linker peptide (GGGGS), and a protein segment shown in SEQ ID NO: 12 (human Fc fragment). The signal peptide is cleaved in the cell, and the remaining active protein consists of the following segments: a protein segment shown in SEQ ID NO: 9 (variable region of the TnbE12 nanobody), a linker peptide (GGGGS), and a protein segment shown in SEQ ID NO: 12 (human Fc fragment).

[0114] II. Preparation of the TnbE12 humanized nanobody

[0115] 1. Transfect the recombinant plasmid TnbE12-Fc prepared in Step 1 into 293F cells, then culture in SMM 293-TII medium for 72 h, then centrifuge at 4°C, 4000 rpm for 30 min, and collect the supernatant.

[0116] 2. Affinity chromatography

[0117] Chromatography column specifications for affinity chromatography: length 3 cm, inner diameter 1 cm;

[0118] Column packing for affinity chromatography: protein A beads (Thermo, product catalog number 10006D);

[0119] Perform the following steps in order: ① Load 300 mL of the supernatant obtained in Step 1 into the affinity chromatography column, and incubate at 4°C for 16 hours; ② Wash the column with 60 mL of PBS buffer (pH 7.2, 10 mM); ③ Elute the target protein with 30 mL of elution buffer, and collect the solution after passing through the column.

[0120] Elution buffer: Take 7.5 g of glycine, dissolve in water, and use water to bring the volume to 500 mL. Adjust the pH to 3.0 with hydrochloric acid.

[0121] 3. Take the solution after passing through the column obtained in Step 2, concentrate it with an ultrafiltration concentration tube, and replace the system with PBS buffer (pH 7.2, 10 mM) to obtain an antibody solution with an antibody concentration of about 2 mg / mL, which is named TnbE12-Fc antibody solution.

[0122] Example 3, Detection of neutralization activity of TnbE12 humanized nanobody against SARS-CoV-2 and other sarbecoviruses I. Preparation of pseudovirus

[0123] The plasmid expressing viral membrane protein and the backbone plasmid pNL4-3R-E-luciferase were co-transfected into 293T cells, and after incubation, the pseudovirus with infectivity but without replication ability could be obtained, and its infectivity was similar to that of live virus. The backbone plasmid pNL4-3R-E-luciferase, i.e., the backbone plasmid pNL4-3R-E containing Luciferase (i.e., the vector with the luciferase gene containing backbone pNL4-3R-E in the literature): Wang Q, Liu L, Ren W, Gettie A, Wang H, Liang Q, Shi X, Montefiori DC, Zhou T, Zhang L. Cell Rep. 2019.

[0124] The coding gene of the coronavirus membrane protein was inserted between the BamHI and EcoRI enzyme digestion sites of the pcDNA3.1(+) vector to obtain a plasmid expressing the coronavirus membrane protein. The plasmid expressing the coronavirus membrane protein and the backbone plasmid pNL4-3R-E-luciferase were co-transfected into 293T cells, and incubated at 37°C (DMEM medium containing 10% fetal bovine serum was used), and the cell culture supernatant was collected after 60 hours of transfection, which was the virus liquid containing the coronavirus pseudovirus.

[0125] Virus liquids of 25 kinds of coronavirus strain pseudo-viruses were prepared, i.e. virus liquids of 20 kinds of new coronavirus strain pseudo-viruses, 1 kind of SARS-CoV-1 strain pseudo-virus, 2 kinds of bat coronavirus strain pseudo-viruses, and 2 kinds of pangolin coronavirus strain pseudo-viruses. The 20 kinds of new coronavirus strains refer to: new coronavirus wild strain, new coronavirus Beta strain, new coronavirus Delta strain, new coronavirus Omicron BA.1 strain, new coronavirus Omicron BA.2 strain, new coronavirus Omicron BA.4 / 5 strain, new coronavirus Omicron BF.7 strain, new coronavirus Omicron BQ.1 strain, new coronavirus Omicron BQ.1.1 strain, new coronavirus Omicron XBB strain, new coronavirus Omicron XBB.1 strain, new coronavirus Omicron XBB.1.5 strain, new coronavirus Omicron XBB.1.1.6 strain, new coronavirus Omicron CH.1.1 strain, new coronavirus Omicron EG.5.1 strain, new coronavirus Omicron BA.2.86 strain, new coronavirus Omicron HK.3 strain, new coronavirus Omicron HV.1 strain, new coronavirus Omicron JD.1.1 strain, new coronavirus Omicron JN.1 strain. The 2 kinds of bat coronavirus strains refer to Bat CoV WIV16 strain and Bat CoV RaTG13 strain. The 2 kinds of pangolin coronavirus strains refer to Pangolin CoV GD strain and Pangolin CoV GX strain. The membrane proteins of each coronavirus and the encoding genes of the membrane proteins are shown in Table 1.

[0126] Table 1

[0127]

[0128]

[0129] II. Detection of neutralization activity of monoclonal antibodies

[0130] The test antibody was a TnbE12-Fc antibody solution prepared in Example 2.

[0131] HEK-293T-hACE2 cells are described in Ju B, Fan Q, Liu C, Shen S, Wang M, Guo H, Zhou B, Ge X, Zhang Z. Omicron BQ.1.1 and XBB.1 unprecedentedly escape broadly neutralizing antibodies elicited by prototype vaccination. Cell Rep. 2023 Jun 27;42(6):112532.

[0132] 1. The test antibody was gradiently diluted with DMEM medium containing 10% FBS to obtain dilutions of each dilution.

[0133] 2. Mix 100 μl of the dilution obtained in step 1 with 50 μl of the virus solution prepared in step 1 (virus content is 100 TCID50), incubate at 37°C for 1 hour. Set up a blank control with 100 μl of DMEM medium containing 10% FBS instead of 100 μl of dilution.

[0134] 3. After completing step 2, add 50 μl of HEK-293T-hACE2 cell suspension (about 2 x 10 4 cells), incubate at 37°C for 48 hours.

[0135] 4. After completing step 3, add 100 μl of PBS buffer and 50 μl of cell lysis solution (Bright-Glo TM Luciferase Assay System, Promega, E2650), stand for 2 min, then detect luciferase activity with a chemiluminescence instrument.

[0136] Each treatment was set up in triplicate, and the results were averaged.

[0137] Neutralization activity = (fluorescence intensity of blank control group - fluorescence intensity of experimental group with dilution) / fluorescence intensity of blank control group x 100%.

[0138] The concentration of the antibody corresponding to a neutralization activity of 50% is the IC50 value.

[0139] The IC50 value results are shown in Table 2. The TnbE12-Fc antibody has strong neutralizing ability against wild-type novel coronavirus and natural mutant strains, and the TnbE12-Fc antibody has strong neutralizing ability against the other 5 Sars viruses.

[0140] Table 2

[0141]

[0142] The application has been described in detail. Those skilled in the art will understand that they can make modifications and alterations to this application without departing from the spirit and scope of the application. Although this application presents specific examples, it is to be understood that further modifications can be made. In general, the principles of the application are intended to be included in any alteration, modification or improvement to the application, including changes made outside the scope of the disclosure herein, using conventional techniques known in the art. Some of the essential features can be applied within the scope of the following claims.

Claims

1. A Nanobody against coronavirus, wherein CDR1, CDR2 and CDR3 in the variable region are shown in SEQ ID NO: 9 in order of positions 26-33, 51-57 and 96-105.

2. The Nanobody of claim 1, wherein: The variable region comprises a framework region FR and a complementarity determining region CDR; CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 9 in order of positions 26-33, 51-57 and 96-105; FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 9 in order of positions 1-25, 34-50, 58-95 and 106-116. 3.A variable region of a Nanobody against coronavirus, wherein CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 9 in order of positions 26-33, 51-57 and 96-105.

4. The variable region of a Nanobody according to claim 3, characterized in that: The variable region comprises a framework region FR and a complementarity determining region CDR; CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 9 in order of positions 26-33, 51-57 and 96-105; FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 9 in order of positions 1-25, 34-50, 58-95 and 106-116. 5.A humanized Nanobody against coronavirus, comprising two segments: a Nanobody variable region, a human Fc segment; the variable region of the Nanobody is as claimed in claim 3 or 4. 6.A nucleic acid molecule encoding the Nanobody of claim 1 or 2, or encoding the variable region of the Nanobody of claim 3 or 4, or encoding the humanized Nanobody of claim 5. 7.Use of the Nanobody of claim 1 or 2, or the variable region of the Nanobody of claim 3 or 4, or the humanized Nanobody of claim 5 in the preparation of a medicament for inhibiting and / or neutralizing coronavirus. 8.A medicament for inhibiting and / or neutralizing coronavirus, wherein the active ingredient is the Nanobody of claim 1 or 2, or the variable region of the Nanobody of claim 3 or 4, or the humanized Nanobody of claim 5. 9.Use of the Nanobody of claim 1 or 2, or the variable region of the Nanobody of claim 3 or 4, or the humanized Nanobody of claim 5 in the preparation of a medicament for preventing and / or treating a disease caused by coronavirus. 10.A medicament for preventing and / or treating a disease caused by coronavirus, wherein the active ingredient is the Nanobody of claim 1 or 2, or the variable region of the Nanobody of claim 3 or 4, or the humanized Nanobody of claim 5.

Images