Anti-COVID-19 nanobodies, preparation methods and applications

Abstract

This invention discloses a broad-spectrum anti-SARS-CoV-2 nanobody, its preparation method, and its applications, belonging to the field of biomedical technology. The complementary determinant region of the anti-SARS-CoV-2 nanobody consists of CDR1 and CDR3, where CDR1 is the amino acid sequence shown in SEQ ID NO.1 and CDR3 is the amino acid sequence shown in SEQ ID NO.3, or CDR1 is the amino acid sequence shown in SEQ ID NO.2 and CDR3 is the amino acid sequence shown in SEQ ID NO.4. This invention discloses a broad-spectrum anti-SARS-CoV-2 nanobody, its preparation method, and its applications, which possess broad-spectrum binding ability, low production cost, good heat resistance, and can be stored at room temperature.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a broad-spectrum anti-COVID-19 nanobody, its preparation method and its application. Background Technology

[0002] The novel coronavirus SARS-CoV-2 has been constantly mutating. Currently emerging variants include Alpha, Beta, Delta, Omicron BA.1 / BA.2 / BA.1.12.1 / BA.3 / BA.4 / 5, etc. These new variants are constantly evading existing vaccines and treatments, seriously endangering people's health and posing a severe challenge to the global pandemic.

[0003] Most currently marketed antibody drugs for treating COVID-19 are ineffective or have significantly reduced neutralizing ability against the Omicron variant. Only two cocktail antibody drugs still show good neutralizing activity against the Omicron variant, and our previously developed anti-COVID-19 nanobody also exhibits varying degrees of reduced neutralizing activity against the Omicron variant. Therefore, developing antibody drugs with better neutralizing effects and a broader spectrum remains one of the top priorities. Furthermore, as the global pandemic gradually becomes normalized, there is an urgent need for a drug that can prevent COVID-19 infection for use in densely populated places.

[0004] Currently available antibodies are all traditional antibody drugs, which are expensive to produce and require cold chain storage, making them difficult to apply in routine epidemic prevention. Nanobodies, as a new type of antibody, have advantages such as small molecular weight, high affinity, strong thermal stability, and low production cost, and are mainly derived from sharks and camels.

[0005] In summary, providing a novel anti-SARS-CoV-2 nanobody with broad-spectrum neutralizing ability, low production cost, good heat resistance, and room temperature storage is of great significance for the prevention and control of SARS-CoV-2. Summary of the Invention

[0006] The purpose of this invention is to propose an anti-COVID-19 nanobody, its preparation method and its application, which has broad-spectrum binding ability, low production cost, good heat resistance, can be stored at room temperature and has high specificity.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] The present invention provides an anti-COVID-19 nanobody, wherein the complementarity-determining region of the anti-COVID-19 nanobody is composed of CDR1 and CDR3, wherein CDR1 is the amino acid sequence shown in SEQ ID NO.1 and CDR3 is the amino acid sequence shown in SEQ ID NO.3, or CDR1 is the amino acid sequence shown in SEQ ID NO.2 and CDR3 is the amino acid sequence shown in SEQ ID NO.4.

[0009] Preferably, it further includes hypervariable regions HV2 and HV4, where HV2 is the amino acid sequence shown in SEQ ID NO.5 or SEQ ID NO.6, and HV4 is the amino acid sequence shown in SEQ ID NO.7 or SEQ ID NO.8.

[0010] Preferably, it further includes backbone regions FR1, FR2 and FR3, where FR1 is the amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10, FR2 is the amino acid sequence shown in SEQ ID NO. 11 or SEQ ID NO. 12, and FR3 is the amino acid sequence shown in SEQ ID NO. 13 or SEQ ID NO. 14.

[0011] Preferably, the amino acid sequence of the anti-COVID-19 nanobody is shown in SEQ ID NO.15 or SEQ ID NO.16.

[0012] SEQ ID NO.1: GSSCTLSD.

[0013] SEQ ID NO.2: DSSCALAS.

[0014] SEQ ID NO.3: EAYSYYCPLYGMGY.

[0015] SEQ ID NO.4: KAYCPYTAGMTFSSV.

[0016] SEQ ID NO.5: ATKKANLS.

[0017] SEQ ID NO.6: ATNEESLS.

[0018] SEQ ID NO.7: NTASK.

[0019] SEQ ID NO.8: NKASK.

[0020] SEQ ID NO.9: VEQTPTTTTKEAGESLTINCVLK.

[0021] SEQ ID NO. 10: LEQTPTTTTKEAGESLTINCVLR.

[0022] SEQ ID NO. 11: TYWYFTKKGATKKA.

[0023] SEQ ID NO. 12: THWYFTKKGATNEE.

[0024] SEQ ID NO. 13: NLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHC.

[0025] SEQ ID NO. 14: SLSNGGRYAETVNKASKSFSLRISDLRVEDAGTYHC.

[0026] SEQ ID NO.15:

[0027] MAPRVEQTPTTTTKEAGESLTINCVLKGSSCTLSDTYWYFTKKGATKKANLSTGGRYSDTKNTASKSFSLRISDLRVEDSGTYHCEAYSYYCPLYGMGYEGGGTIVTVK.

[0028] SEQ ID NO.16:

[0029] MAPRLEQTPTTTTKEAGESLTINCVLRDSSCALASTHWYFTKKGATNEESLSNGGRYAETVNKASKSFSLRISDLRVEDAGTYHCKAYCPYTAGMTFSSVGGGGTTLTVK.

[0030] The present invention also provides a nucleic acid molecule comprising a nucleic acid sequence encoding the above-mentioned anti-SARS-CoV-2 nanobody.

[0031] Preferably, the nucleic acid molecule includes the nucleic acid sequence shown in SEQ ID NO.17 or SEQ ID NO.18.

[0032] SEQ ID NO.17:

[0033] ATGGCCCCACGGGTTGAAAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAAAGGTTCCAGCTGTACATTGAGTGACACGTACTGGTATTTCACAAAAAAGGGCGCAACAAAAAAGGCGAACTTATCAACTGGCGG ACGATACTCGGACACAAAGAATACGGCATCAAAGTCCTTTTCCTTGCGAATTAGTGACCTAAGAGTTGAAGACAGTGGTACATATCACTGTGAAGCGTATAGTTATTACTGTCCCCTCTATGGGATGGGTTATGAAGGAGGCGGCACCATTGTGACTGTGAAA.

[0034] SEQ ID NO.18:

[0035] ATGGCCCCACGGCTTGAAAAACACCGACAACGACAACAAAGGAGGCAGGCGAATCACTGACCATCAATTGCGTCCTAAGAGATTCCAGCTGTGCATTGGCTAGCAGCACTGGTATTTCACAAAAAAGGGCGCAACAAACGAGGAGAGCTTATCAAATGGCGGA CGATACGCGGAAACAGTGAACAAGGCATCAAAGTCCTTTTCTTTCGAATTAGTGACCTAAGAGTTGAAGACGCTGGTACATATCACTGTAAAGCGTATTGCCCGTATACAGCTGGGATGACGTTTTCCAGTGTCGGAGGAGGCGGCACCACTCTGACTGTGAAA.

[0036] The present invention also provides a recombinant vector comprising the above-described nucleic acid molecules.

[0037] The present invention also provides recombinant cells, which include the above-described nucleic acid molecules or the above-described recombinant vectors.

[0038] The present invention also provides a method for preparing the above-mentioned anti-COVID-19 nanobody. The preparation method includes the following steps: (1) immunizing a striped bamboo shark with SARS-CoV-2S1 protein, isolating peripheral blood mononuclear cells of the immunized striped bamboo shark and extracting total RNA, and using the RNA as a template to reverse transcribe and prepare cDNA; (2) using the cDNA as a template, PCR amplifying the variable region of the nanobody and constructing a phage display library; (3) screening phage antibodies with SARS-CoV-2 antigen specificity in the phage display library and sequencing them to obtain the nucleic acid sequence of the antibody; (4) using the nucleic acid sequence of the antibody to construct an expression vector, and transfecting it into cells for expression and purification to obtain the anti-COVID-19 nanobody.

[0039] The present invention also provides a pharmaceutical composition comprising any one or a combination of at least two of the above-described anti-COVID-19 nanobody, the above-described nucleic acid molecule, the above-described recombinant vector, and the above-described recombinant cells.

[0040] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, including any one or a combination of at least two of a diluent, an adhesive, or a preservative.

[0041] The present invention also provides the use of the above-mentioned anti-SARS-CoV-2 nanobody, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector, the above-mentioned recombinant cell, and the above-mentioned pharmaceutical composition in the preparation of anti-SARS-CoV-2 drugs.

[0042] The beneficial effects of this invention are as follows:

[0043] 1. Anti-COVID-19 nanobodies can effectively solve the problem of COVID-19 variants escaping, have broad-spectrum neutralizing ability, and can be efficiently expressed in prokaryotic and eukaryotic systems. They are low-cost to produce, have good heat resistance, can be stored at room temperature, and have high specificity. They have the potential to be developed into nasal sprays and have important application value in preventing COVID-19 infection.

[0044] 2. Anti-SARS-CoV-2 nanobodies have high affinity and broad-spectrum binding to viruses of the Sarbecovirus subgenus, including different SARS-CoV-2 variants. They can be efficiently expressed in eukaryotic systems with a standardized and controllable expression process, which can greatly reduce production costs. At the same time, they have small molecular weight and stable physicochemical properties. Attached Figure Description

[0045] Figure 1 This is an electrophoresis image of the purified protein of the anti-COVID-19 nanobody (41A4-Fc) of this invention.

[0046] Figure 2 This is an electrophoresis image of the purified protein of the anti-COVID-19 nanobody (1C12-Fc) of this invention.

[0047] Figure 3-14 The diagram shows the binding activity of the anti-SARS-CoV-2 nanobody (41A4-Fc) of this invention to the RBD protein of different mutant SARS-CoV-2 virus, as detected by ELISA.

[0048] Figure 15 The diagram shows the binding activity of the anti-SARS-CoV-2 nanobody (1C12-Fc) of this invention to the RBD protein of different mutant SARS-CoV-2 virus, as detected by ELISA. Detailed Implementation

[0049] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments.

[0050] Example 1:

[0051] This embodiment constructs a single-chain antibody phage library. The method for constructing a single-chain antibody phage library includes the following steps:

[0052] (1) Take 100 μg of SARS-CoV-2S1 protein (purchased from Beijing Yiqiao Shenzhou) and dissolve it in 250 μL of PBS. Then, mix it with an equal volume of aluminum adjuvant and shake it thoroughly to emulsify. Immunize the striped bamboo shark subcutaneously and intramuscularly at multiple points. The immunization was repeated 6 times, with an interval of 2 weeks between each immunization.

[0053] (2) Starting from the second immunization, 1 mL of blood was collected from the tail vein one week after each immunization. 0.5 mL of the blood was treated with anticoagulation to separate lymphocytes, lysed with trizol, and stored at -80℃ for later use. Another 0.5 mL of blood was taken and left to stand at room temperature for 1 hour. The serum was then centrifuged to separate the serum and the serum titer was detected. After the last immunization, blood was collected from the tail vein to detect the serum pseudovirus neutralization titer.

[0054] (3) Lymphocyte lysates from the 3rd, 4th, 5th and 6th immunizations were collected, and total RNA was extracted using chloroform. cDNA was synthesized using a reverse transcription kit from Bio-Rad (Bio-Rad, Cat: 1708891).

[0055] (4) Using the above cDNA as a template, specific primers were used;

[0056] Upstream primer: GCGAGGAGGAGGCCCAGCCGGCCATGGCCSMACGGSTTGAACAAACACC;

[0057] Downstream primer: ATAAGAATGCGGCCGCWTTCACAGTCASARKGGTSCC;

[0058] (Reference, doi:10.1016 / j.molimm.2006.07.299) Amplification of the variable region of single-chain antibody; the PCR reaction system includes:

[0059] The PCR amplification procedure is as follows:

[0060] Pre-denaturation at 98℃ for 30 seconds;

[0061] 98℃ denaturation for 10s, 61℃ annealing for 30s, 72℃ extension for 30s, 27 cycles;

[0062] Extend at 72℃ for 7 minutes;

[0063] (5) The PCR products were identified by 1% agarose gel electrophoresis and purified (Magen DNA Gel Extraction Kit) to obtain the single-chain antibody variable region PCR product. The single-chain antibody variable region PCR product and pcantab5e phage vector were digested with NotI and SfiI, respectively. The ligation was carried out overnight at 16℃ at a ratio of 150ng of vector and 50ng of fragment. After purification, the ligation product was electroporated into TG1 competent cells, plated, and serially diluted 10-fold to calculate the library volume. The cells were incubated overnight at 37℃. Colonies on the plates were scraped with a plating stick, resuspended in the culture medium, and glycerol was added to a final concentration of 20%. The cells were stored at -80℃ for later use. 96 clones were picked for bacterial PCR to identify the positive conversion rate of the library and sequencing to verify the antibody diversity. The library volume reached 2×10⁸, the positive conversion rate was 100%, and the library diversity was greater than 90%.

[0064] Example 2:

[0065] This embodiment describes the screening and expression of SARS-CoV-2 single-chain antibodies, including the following steps:

[0066] (1) Take 100 μL of the single-chain antibody phage library prepared in Example 1, inoculate it into 50 mL of 2YT medium containing ampicillin antibiotic and 1% glucose, and culture at 37°C to the logarithmic phase. Add 20 times the number of helper phage M13KO7, mix well, and let stand at 37°C for 20 minutes, then shake and culture for 30 minutes. Centrifuge, discard the medium, add 50 mL of 2YT ampicillin-kanamycin medium, and shake and culture overnight at 30°C. Centrifuge the next day, collect the supernatant, add 1 / 4 volume of PEG / NaCl, precipitate the recombinant phage at 4°C for 2 hours, centrifuge and collect the phage precipitate, dissolve it with 5 mL of PBS, repeat the precipitation once, dissolve the phage in PBS, add glycerol to a final concentration of 15%, aliquot and store at -80°C for later use. At the same time, take 10 μL to make a 10-fold serial dilution, infect the logarithmic phase TG1, culture overnight, and calculate the titer.

[0067] (2) Dilute Omicron RBD or S antigen to 10 μg / mL with CBS buffer, mix thoroughly, and coat 100 μL / well in a 96-well microplate. Incubate at 4°C for 12-16 hours, discard the supernatant, add blocking buffer (5% skim milk dissolved in PBST), block at 37°C for 2 hours, wash 4 times with 0.05% PBST for 2 minutes each time, and finally discard the liquid for later use.

[0068] (3) Screening: Take 100 times the volume of recombinant phage and dilute it in 100 μL of blocking solution. Add it to the enzyme-labeled wells coated with RBD or S antigen. After incubating at room temperature for 2 hours, wash with 0.1% PBST 5-10 times, 2 minutes each time. Finally, elute the phage bound to the plate with 100 μL of triethylamine and immediately add 1M Tris-HCl (pH 7.4) to neutralize the elution solution. Mix it with 3 mL of logarithmic growth phase TG1, let stand for 30 minutes, shake and incubate for 30 minutes, plate it, use one part for serial dilution to calculate the volume, and use the other part as a strain library. Incubate at 37℃ upside down overnight.

[0069] (4) Repeat the screening 2-4 times. The specific steps are as described in step (3). Based on step (3), change the conditions, increase the number of washing times by 15-30 times, reduce the amount of coating antigen by 1-3 times, select a single clone from the library obtained in the last round of screening, and perform phage ELISA verification.

[0070] (5) Phage ELISA verification: Select a single clone into a 96-well plate, incubate overnight at 37°C with shaking, and inoculate into a new 96-well plate the next day (300 μL of medium / well), incubate at 37°C with shaking until the logarithmic phase, add 20 times the amount of helper phage, incubate at 37°C for 30 minutes, then incubate with shaking for 30 minutes, replace the medium with 2YT ampicillin medium, and incubate overnight at 30°C with shaking. Take 100 μL of supernatant into an ELISA plate coated with RBD or S antigen (50 ng / well) and incubate at room temperature for 2 hours. Use a recombinant phage library as a positive control and an auxiliary phage as a negative control. Wash the plate 4 times with PBST, add 100 μL of HRP-labeled anti-M13 antibody (purchased from Chengdu Apak), incubate at 37°C for 1 hour, wash the plate 6 times with PBST, add 100 μL of TMB chromogenic substrate (purchased from Millipore), incubate at 37°C in the dark for 15 minutes, add 50 μL of 1M H2SO4 to stop the reaction, and measure the absorbance at 450 nm.

[0071] (6) HEK293 Expression: Positive clones verified by phage ELISA were sequenced to obtain antibodies with different sequences. Different antibody sequences were amplified by PCR and constructed into the pCMV-IgG1 vector. Plasmids with the correct sequences were transfected into HEK293 cells using PEI for expression for 3-7 days. The expression supernatant was collected, and after centrifugation to filter cell debris, Protein A affinity purification was performed to finally obtain the desired expression. Figure 1 and Figure 2 The purified anti-COVID-19 nanobodies shown are 41A4-Fc (SEQ ID NO.17) and 1C12-Fc (SEQ ID NO.18), with a purity >93%.

[0072] Example 3:

[0073] This embodiment tests the binding ability of the anti-COVID-19 nanobody prepared in Example 2, including the following steps:

[0074] (1) RBD (332-527) proteins (WH-Hu-1, Alpha, Beta, Delta, Delta plus, Kappa, Lambda, Omicron, SARS-CoV, Pangolin GD1, Bat Cov RaTG13) and MERS RBD were diluted in CBS buffer and coated with ELISA plates at 50 ng / 100 μL / well. The plates were incubated at 4°C for 12 hours. The antigens were discarded, and blocking buffer (5% skim milk dissolved in PBST) was added at 200 μL / well. The plates were blocked at 37°C for 2 hours. The plates were washed with 0.05% PBST at 200 μL / well, 4 times for 2 minutes each time. The liquid was then shaken off and the plates were patted dry.

[0075] (2) Dilute the 41A4-Fc and 1C12-Fc antibodies separately in PBST, starting from 100 μg / mL, and perform a semi-logarithmic dilution for a total of 12 dilutions. Add 100 μL / well to the microplate and incubate at 37°C for 2 hours. Wash the plate 4 times with PBST for 2 minutes each time. Add HRP-labeled anti-his secondary antibody and incubate at 37°C for 1 hour. Wash the plate 6 times with PBST for 2 minutes each time. Discard the liquid, pat dry, add 100 μL / well of TMB chromogenic substrate, and incubate at 37°C in the dark for 15 minutes. Add 50 μL of 1M H2SO4 to stop the reaction. Measure the absorbance at 450 nm and calculate the antigen-antibody binding EC50. The results are as follows: Figure 3 and Figure 15As shown, 41A4-Fc and 1C12-Fc bind well to the RBDs of different SARS-CoV-2 variants, SARS-CoV, Pangolin, and Bat RBDs, but do not bind to the MERS RBDs of other coronavirus genera. This indicates that 41A4-Fc and 1C12-Fc have the ability to bind to a broad spectrum of beta coronaviruses (Sarbecovirus subgenus) with high specificity.

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A shark nanobody against SARS-CoV-2, characterized in that: the CDR1 has an amino acid sequence as set forth in SEQ ID NO. 1, the CDR3 has an amino acid sequence as set forth in SEQ ID NO. 3, the HV2 has an amino acid sequence as set forth in SEQ ID NO. 5, and the HV4 has an amino acid sequence as set forth in SEQ ID NO. 7; or the CDR1 has an amino acid sequence as set forth in SEQ ID NO. 2, the CDR3 has an amino acid sequence as set forth in SEQ ID NO. 4, the HV2 has an amino acid sequence as set forth in SEQ ID NO. 6, and the HV4 has an amino acid sequence as set forth in SEQ ID NO.

8. 2.The shark nanobody against SARS-CoV-2 according to claim 1, characterized in that: the shark nanobody against SARS-CoV-2 has an amino acid sequence as set forth in SEQ ID NO. 15 or SEQ ID NO.

16. 3.A nucleic acid molecule, characterized in that: the nucleic acid molecule comprises a nucleic acid sequence encoding the shark nanobody against SARS-CoV-2 according to any one of claims 1-2; or the nucleic acid molecule comprises a nucleic acid sequence as set forth in SEQ ID NO. 17 or SEQ ID NO.

18. 4.A recombinant vector, characterized in that: the recombinant vector comprises the nucleic acid molecule according to claim 3. 5.A recombinant cell, characterized in that: the recombinant cell comprises the nucleic acid molecule according to claim 3 or the recombinant vector according to claim 4. 6.A pharmaceutical composition, characterized in that: the pharmaceutical composition comprises any one of the shark nanobody against SARS-CoV-2 according to any one of claims 1-2, the nucleic acid molecule according to claim 3, the recombinant vector according to claim 4, and the recombinant cell according to claim 5, or a combination of at least two thereof; and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. 7.Use of the shark nanobody against SARS-CoV-2 according to any one of claims 1-2, the nucleic acid molecule according to claim 3, the recombinant vector according to claim 4, the recombinant cell according to claim 5, or the pharmaceutical composition according to claim 6 in the preparation of an anti-SARS-CoV-2 drug. ​ ​ ​ ​

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