CN114249822B - Alpaca-derived nanobody combined with SARS-CoV-2RBD - Google Patents

Abstract

The present disclosure relates to alpaca-derived antibodies or antigen binding fragments thereof that bind to SARS-CoV-2RBD, in particular alpaca-derived nanobodies or antigen binding fragments thereof that are capable of binding to a novel coronavirus (SARS-CoV-2) Receptor Binding Domain (RBD) with high affinity, which can be used for the prevention, treatment and/or diagnosis of SARS-CoV-2 infection.

Description

Alpaca-derived Nanobodies Binding to SARS-CoV-2 RBD

technical field

The invention belongs to the field of biotechnology, and in particular relates to an anti-SARS-CoV-2 RBD nanobody sequence for treatment and diagnosis.

Background technique

SARS-CoV-2 is a coronavirus, and the pneumonia it causes is called COVID-19. SARS-CoV-2 enters cells through the receptor binding domain (RBD) of its surface spike protein (spike) and angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells, and completes the infection.

Fully human antibodies isolated from recovered patients have been proven to have good antiviral effects, but these are traditional monoclonal antibodies consisting of 2 heavy chains and 2 light chains. It has the limitations of large molecular weight, complex production process, and difficult processing and transformation.

In camelids, there is an antibody that naturally lacks light chains, that is, heavy chain antibodies. Its variable region is only composed of heavy chains. The variable region is abbreviated as VHH. The diameter of the variable region protein is less than 10 nanometers, so it is also called nanobodies. Nanobodies have the advantages of small molecular weight, strong penetrability, easy expression, easy genetic modification, and easy binding to multiple epitopes.

There are currently no alpaca-derived natural nanobodies against the SARS-CoV-2 RBD approved for the treatment of COVID-19.

Contents of the invention

The present disclosure provides an alpaca-derived heavy chain antibody variable region sequence (VHH) capable of binding the new coronavirus (SARS-CoV-2) receptor binding region (RBD) with high affinity, and the variable region sequence is also called a nanobody , which can be used to prevent, treat and/or diagnose SARS-CoV-2 infection.

The inventors used the SARS-CoV-2 RBD protein recombinantly expressed in vitro to immunize two llamas three times, then isolated peripheral blood lymphocytes and extracted the total RNA of the cells, which were then reverse-transcribed into cDNA. Then use the cDNA as a template to amplify the nanobody sequence with specific primers. We isolated and obtained 7 nanobody strains. Named as aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54 respectively, their amino acid sequences are as follows:

Amino acid sequence of aRBD-2:

Amino acid sequence of aRBD-3:

Amino acid sequence of aRBD-5:

Amino acid sequence of aRBD-7:

Amino acid sequence of aRBD-41:

Amino acid sequence of aRBD-42:

Amino acid sequence of aRBD-54:

The 3 complementarity determining regions (CDR1, CDR2 and CDR3) of the 7 strains of nanobodies are shown in the underlined part, specifically:

aRBD-2:

CDR1: GRTYTM (SEQ ID NO: 1)

CDR2: EFVAAMRWSDTD (SEQ ID NO: 2)

CDR3: AGEAWLARSTHHYDY (SEQ ID NO: 3)

aRBD-3:

CDR1: GLTLDYYAI (SEQ ID NO: 4)

CDR2: EGVSCISHPGGSTN (SEQ ID NO: 5)

CDR3: ASPLALFRLCVLPSPLPYDY (SEQ ID NO: 6)

aRBD-5:

CDR1: GFTLDYYAI (SEQ ID NO: 7)

CDR2: EGVSCISGSGGITN (SEQ ID NO: 8)

CDR3: PVSHTVVAGCAFEAWTDFGS (SEQ ID NO: 9)

aRBD-7:

CDR1: ERTFSGGVM (SEQ ID NO: 10)

CDR2: EFVAAIRWNGASTF (SEQ ID NO: 11)

CDR3: RAVRTYASSDYYFQERTYDY (SEQ ID NO: 12)

aRBD-41:

CDR1: GFTSGHYAI (SEQ ID NO: 13)

CDR2: EGVSCIGSSDGSTY (SEQ ID NO: 14)

CDR3: AGLWYGRSLNSFDYDY (SEQ ID NO: 15)

aRBD-42:

CDR1: GRTFSSA™ (SEQ ID NO: 16)

CDR2: EFVAAISWSGLSRY (SEQ ID NO: 17)

CDR3: DSWGCSGLGC (SEQ ID NO: 18)

aRBD-54:

CDR1: GRTFGSFM (SEQ ID NO: 19)

CDR2: DFVAAITWSGGSTY (SEQ ID NO: 20)

CDR3: ARISSAYYTRSSSYAY (SEQ ID NO: 21).

Then, the inventors found that Nanobodies aRBD-2 and aRBD-5 bind to different epitopes, and aRBD-2 and aRBD-7 bind to different epitopes, so they were combined to construct two corresponding bi-epitope-specific antibodies aRBD-2-5 and aRBD-2-7.

The bi-epitope-specific antibody as used herein refers to a nanobody capable of binding to the RBD constructed by linking two nanobodies that can respectively bind to two independent epitopes on the SARS-CoV-2 RBD with a flexible polypeptide chain. Antibodies to both epitopes.

Specifically, the present invention provides the following technical solutions:

1. An alpaca-derived antibody or antigen-binding fragment thereof that binds to the SARS-CoV-2 RBD has a VHH having a group selected from the group consisting of:

CDR1 as shown in SEQ ID NO: 1,

CDR2 as shown in SEO ID NO: 2 and

CDR3 as shown in SEQ ID NO:3;

CDR1 as shown in SEQ ID NO: 4,

CDR2 as shown in SEQ ID NO: 5 and

CDR3 as shown in SEQ ID NO: 6;

CDR1 as shown in SEQ ID NO: 7,

CDR2 as shown in SEQ ID NO: 8 and

CDR3 as shown in SEQ ID NO:9;

CDR1 as shown in SEQ ID NO: 10,

CDR2 as shown in SEQ ID NO: 11 and

CDR3 as shown in SEQ ID NO: 12;

CDR1 as shown in SEQ ID NO: 13,

CDR2 as shown in SEQ ID NO: 14 and

CDR3 as shown in SEQ ID NO: 15;

CDR1 as shown in SEQ ID NO: 16,

CDR2 as shown in SEQ ID NO: 17 and

CDR3 as shown in SEQ ID NO: 18; and/or

CDR1 as shown in SEQ ID NO: 19,

CDR2 as shown in SEQ ID NO: 20 and

CDR3 as shown in SEQ ID NO:21.

2. The antibody or antigen-binding fragment thereof according to item 1, wherein the VHH comprises:

Amino acid sequence as shown in SEQ ID NO: 22,

Amino acid sequence as shown in SEQ ID NO: 23

Amino acid sequence as shown in SEQ ID NO:24.

the amino acid sequence shown in SEQ ID NO: 25,

the amino acid sequence shown in SEQ ID NO: 26,

an amino acid sequence as shown in SEQ ID NO: 27, and/or

Amino acid sequence as shown in SEQ ID NO:28.

3. The antibody or antigen-binding fragment thereof according to item 1 or 2, which is a bi-epitope-specific antibody comprising SEQ ID NO : 22 and SEQ ID NO: 24, or SEQ ID NO: 22 and SEQ ID NO: 25, preferably, wherein between SEQ ID NO: 22 and SEQ ID NO: 24 or SEQ ID NO: 22 and SEQ ID NO: 25 use A linker (eg, a flexible polypeptide chain, such as a GS linker) is connected.

4. The antibody or antigen-binding fragment thereof according to any one of items 1-3, which further has an Fc domain, preferably an IgG1 Fc domain, more preferably a human IgG1 Fc domain, the human IgG1 Fc domain The sequence is, for example, shown in SEQ ID NO: 30, and the nucleotide sequence of the gene encoding the human IgG1 Fc domain sequence is, for example, shown in SEQ ID NO: 31.

5. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of items 1-4.

6. An expression vector, such as an expression vector based on one or more promoters and a host cell, comprising the polynucleotide described in Item 5.

7. A host cell comprising the expression vector described in item 6, said host cell is a host cell for expressing a foreign protein, such as bacteria, yeast, insect cells, mammalian cells.

8. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of items 1-4 and a pharmaceutically acceptable carrier.

9. Use of the antibody or antigen-binding fragment thereof according to any one of items 1-4 in the preparation of a kit or medicament for preventing, treating and/or diagnosing SARS-CoV-2 infection.

Advantages and positive effects of the present disclosure

Since the nanobody (VHH) described in the present disclosure is derived from a natural alpaca heavy chain antibody, it has the characteristics of high stability, high expression and high affinity.

Circular dichroism experiments showed that the half-melting temperatures (Tm values) of the above seven strains of Nanobodies were all above 70°C.

After the above 7 nanobodies were fused with the Fc segment of human IgG1, they were cloned into the pTT5 vector, and secretory expression was performed using mammalian cell 293F. After 3 days of expression, the fusion protein in the medium supernatant was purified by Protein A column and found that, The yields of the 7 antibody strains were all above 90 mg/L.

All seven antibodies can bind SARS-CoV-2 RBD with high affinity. ELISA experiments show that, except for aRBD-42, the affinity of the Fc fusion proteins of other antibodies of the present disclosure for binding to the extracellular segment of the SARS-CoV-2 spike protein (S1+S2) is higher than that of ACE2. Surface plasmon resonance (SPR) experiments showed that the affinity dissociation constants of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42, aRBD-54 and SARS-CoV-2 RBD ( K _D ) values were 2.60, 3.33, 16.3, 3.31, 21.9, 113 and 5.49 nM (nanomoles per liter), respectively.

Except for aRBD-42, the other 6 nanobodies of the present disclosure can well inhibit the binding of human ACE2 to SARS-CoV-2 RBD after being fused with human IgG1 Fc. Competitive ELISA experiments showed that the Fc fusion proteins of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41 and aRBD-54 could compete with 10 nM ACE2-Fc for SARS-CoV-2 RBD, which The _IC50s were 2.68, 2.59, 1.89, 1.42, 5.76 and 2.07 nM, respectively.

The Nanobodies aRBD-2 and aRBD-5 of the present disclosure bind to different epitopes, and aRBD-2 and aRBD-7 bind to different epitopes, so two bi-epitope-specific antibodies, aRBD-2-5 and aRBD- 2-7, SPR showed that its affinity with SARS-CoV-2 RBD was greatly enhanced, with _KD values of 59.2pM (picomoles per liter) and 0.25nM, respectively.

The Fc fusion proteins of the Nanobodies aRBD-2, aRBD-5 and aRBD-7 of the present disclosure can neutralize SARS-CoV-2 infecting Vero E6 cells in vitro. The ND ₅₀ concentrations of the Fc fusion proteins of aRBD-2, aRBD-5 and aRBD-7 neutralizing 200 PFU of SARS-CoV-2 infecting Vero E6 in a 100 μL system were 0.092, 0.413 and 0.591 μg/mL, respectively. The Fc fusion proteins of the biepitope-specific antibodies aRBD-2-5 and aRBD-2-7 neutralized 200 PFU of SARS-CoV-2 infecting VeroE6 in a 100 μL system with ND ₅₀ concentrations of 0.0104 and 0.0067 μg/mL, respectively.

Description of drawings

Figure 1. Results of phage display screening of seven Nanobodies of the present disclosure. (A) is the phage counting result of two rounds of panning; (B) is the result of monoclonal phage ELISA.

Figure 2. SDS-PAGE gel electrophoresis results of the Nanobody Fc fusion protein (A) and Nanobody (B). Lane M is marker, and lanes 1 to 7 are aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54 fusion proteins (A) and their Fc-cleaved nano Antibody protein (B).

Fig. 3. Circular dichroism (CD) experiment results of detecting the denaturation temperature of 7 Nanobodies of the present disclosure. (A)-(G) are the detection results of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54 in sequence.

Fig. 4. The result figure of detecting the binding between the Fc fusion protein of the nanobody and the extracellular segment protein of the SARS-CoV-2 spike protein (S1+S2) by ELISA.

Figure 5. Detection of affinity between the Nanobody and SARS-CoV-2 RBD by SPR. (A) to (I) adopt the method of SPR to detect Nanobody aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42, aRBD-54, aRBD-2-5 and Kinetic curves of binding between aRBD-2-7 and SARS-CoV-2 RBD protein. The solid line is the kinetic curve monitored in real time, and the dashed line is the curve fitted by biacore evaluation software. The kinetic curves of different antibody concentration gradients correspond from top to bottom with the concentrations marked on the right from top to bottom.

Figure 6. The results of detecting the binding of ACE2 to SARS-CoV-2 RBD by the Fc fusion protein of the nanobody detected by competitive ELISA.

Figure 7. In vitro virus neutralization experiments verify the function of the disclosed antibodies. Fc fusion proteins of nanobodies aRBD-2, aRBD-5 and aRBD-7 and biepitope-specific antibodies aRBD-2-5 and aRBD-2-7 and their Fc fusion proteins neutralize SARS-CoV-2 virus in vitro The experimental data analysis results of infecting Vero E6 cells.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

Example 1 Using SARS-CoV-2 RBD to Immunize Alpacas and Screen Nanobodies

1) The purified SARS-CoV-2 RBD (QKV42562.1, aa 321-591) expressed in HEK293F cells (ATCC, CBP60437) was mixed with Freund's adjuvant, and the alpaca was subcutaneously injected with 500 μg/time for three times, Two 6-month-old female alpacas were vaccinated at intervals of 2 weeks.

2) Two weeks after the third immunization, blood was collected from a vein and white blood cells in the blood were separated. Total RNA was extracted using the RNA extraction kit from Omegabiotek, and genomic DNA was removed using DNase. The RNA was reverse-transcribed using the PrimeScript ^TM II 1st Strand cDNA Synthesis Kit from TAKARA, and the RNA was reverse-transcribed into cDNA.

3) Preparation of nanobody phagemid library: use the alpaca VHH-specific primers we designed to amplify the coding gene fragment of VHH using the above cDNA as a template, and clone the amplified VHH sequence into a phage using the method of Gibson assembly In the NcoI and NotI sites of pR2, the resulting Gibson assembly product is the initial nanobody phagemid library.

4) Electroporation of TG1 to amplify the nanobody phagemid library: Prepare Escherichia coli TG1 competent cells by washing with 10% glycerol, then electrotransform the above Gibson assembly product into TG1 competent cells, and coat 5 pieces of 150mm containing 2 % glucose and 100 μg/mL ampicillin in LB (LB/2%G/Amp) plates to amplify the phagemid library.

5) Amplify the nanobody phage library: After scraping the plate, take an appropriate amount of bacterial liquid and inoculate 200mL 2TY (containing 2% glucose and 100μg/mL ampicillin) to the logarithmic growth phase, and add 10 ¹² pfu of KM13 helper phage (purchased from MRC Laboratory of Molecular Biology), infected at 37°C for 45min, centrifuged with 100mL of bacterial liquid, resuspended with 200mL of 2TY (containing 0.1% glucose, 100μg/mL and 50μg/mL kanamycin), and cultured at 25°C for 20h to expand Increase the phage of the displayed Nanobody. The phage was concentrated by PEG precipitation, finally resuspended in PBS, and stored on ice.

6) Panning

A. The first round: Dilute the expressed and purified SARS-CoV-2 RBD in Example 1 to 0.1 mg/mL with PBS, take 100 μL and add it to one well of a 96-well immunoplate (Nunc maxsorp plate), and coat overnight at 4°C , and set a well without antigen control at the same time. Wash 3 times with PBS, add 300 μL MPBS (PBS containing 5% skimmed milk) to each well to block at room temperature for 2 hours. Wash 3 times with PBS, add 1 x 10 ¹¹ pfu to each well to prepare phage library phage (dissolved in 100 μL MPBS), and incubate at room temperature at 80 rpm for 1 h. Wash 30 times with PBST (0.1% Tween 20). Add 100 μL of trypsin at a concentration of 0.5 mg/mL to each well, digest at room temperature for 1 hour, and the phage bound in the wells is eluted. Take 10 μL of the eluted phage to infect 1 mL of TG1 bacteria in logarithmic growth phase, and bathe in water at 37°C for 45 minutes. 100 μL, 10 μL and 1 μL of coated LB/2%G/Amp plates were counted respectively. Infect 3 mL of TG1 bacteria in logarithmic growth phase with the remaining phage solution, bathe in 37°C water for 45 minutes, spread a 150mm LB/2%G/Amp plate, and culture overnight at 37°C.

B. The second round: Add 4mL 2TY to the above 150mm plate, scrape off the colony, mix the bacterial solution and inoculate 100μL to 100mL 2TY/2%G/Amp medium, cultivate to logarithmic growth phase and then add KM13 Infection to make phage for Nanobody display. Subsequently, the SARS-CoV-2 RBD was diluted to 0.02 mg/mL with PBS, 100 μL was added to one well of a 96-well immunoplate, and coated overnight at 4°C, and a well without antigen control was set at the same time. Wash 3 times with PBS, add 300 μL MPBS (PBS containing 5% skimmed milk) to each well to block at room temperature for 2 hours. Wash 3 times with PBS, add 1 x 10 ⁸ pfu of the first-round eluted phage (dissolved in 100 μL MPBS) to each well, and incubate at room temperature at 80 rpm for 1 h. Wash 30 times with PBST (0.2% Tween 20). Add 100 μL of trypsin at a concentration of 0.5 mg/mL to each well, digest at room temperature for 1 hour, and the phage bound in the wells is eluted. Take 10 μL of the eluted phage to infect 1 mL of TG1 bacteria in logarithmic growth phase, and bathe in water at 37°C for 45 minutes. 100 μL, 10 μL and 1 μL of coated LB/2%G/Amp plates were counted respectively.

C. Phage counts for two rounds of panning elution are shown in Figure 1A. Compared with the control wells, the number of phages eluted from the RBD-coated wells was significantly more. The number of phages eluted from the RBD-coated wells in the first round was more than 70 times that of the control wells, and the ratio was even higher in the second round. It shows that the bacteriophages specific for RBD were successfully isolated and enriched.

7) Phage ELISA screening of anti-SARS-CoV-2 RBD nanobody monoclonal.

A. Preparation of monoclonal phage: Pick 31 monoclonals from the plates counted after the above two rounds of screening and elution, and inoculate each well into 96 wells containing 100 μL of 2TY medium (containing 2% glucose and 100 μg/mL ampicillin) In a cell culture plate, one clone per well was cultured at 37° C. with shaking at 250 rpm for 12 hours. Transfer more than 5 μL of the bacterial solution to a new 96-well plate containing 200 μl of 2TY medium (containing 2% glucose and 100 μg/mL Ampicillin) in each well for cultivation (add the remaining bacterial solution to a final concentration of 15% glycerol and store at -80°C ), shake at 37°C and 250rpm for 1.5h until the OD600 is about 0.5, and remove 100μL of bacterial solution from each well. Add 50 μL of 2TY containing 4×10 ⁸ pfu KM13 phage to each well, mix well, and incubate at 37°C for 45 min. Centrifuge at 3500 g for 10 min, discard the supernatant, resuspend the pellet in 200 μL of 2TY containing 0.1% glucose, 100 μg/mL Ampicillin and 50 μg/mL Kanamycin, and incubate with shaking at 25 °C and 250 rpm for 20 h. Centrifuge at 3500g for 10 min, transfer 75 μL of supernatant to each well of a 96-well plate containing 225 μL of MPBS, mix well, and temporarily store at 4°C for later use, and the preparation of monoclonal phage is now complete.

B. Phage phage ELISA detection: Dilute the SARS-CoV-2 RBD protein to 1 μg/mL with PBS, take 100 μL/well to coat the 96-well immunoplate, and set a blank control (PBS well, coat overnight at 4°C) Wash 3 times with PBS, add 300 μL MPBS to each well, and block at room temperature for 2 hours. Add 100 μL of the phage MPBS mixture prepared above to each well, and incubate at room temperature for 1 hour. Wash the plate 4 times with PBST. Dilute the HRP-anti M13 antibody appropriately with MPBS (sense Add 100 μL to each well of the above immune plate, incubate at room temperature for 1 h. Wash the plate 4 times with PBST. Add 100 μL of TMB chromogenic substrate (Biyuntian) to each well, wrap it in aluminum foil to avoid light, and store at room temperature React for 5 minutes. Add 50 μL of 1M H ₂ SO ₄ to each well to terminate the reaction, and measure the OD _450nm value. The results are shown in Figure 1B.

C. Send all positive clones with an OD _450nm value greater than 1 to the company for sequencing, analyze and compare the sequencing results, and finally determine 7 positive clones, named aRBD-2, aRBD-3, aRBD-5, and aRBD respectively as above -7, aRBD-41, aRBD-42 and aRBD-54.

Example 2 Expression and purification of nanobodies and their Fc fusion proteins

1) Design primers, fuse the N-terminus of the gene sequence of the Nanobody to IFNα protein signal peptide to guide secreted expression, fuse the C-terminus of the gene sequence of the Nanobody to human IgG1 Fc, and introduce a TEV restriction enzyme between them site, and then cloned into the mammalian expression vector pTT5. The constructed vector was transiently transfected into mammalian cells HEK293F with PEI, and the supernatant was collected after 3 days of culture. The fusion protein in the supernatant was purified by Protein A column, and subjected to SDS-PAGE electrophoresis. The results are shown in Figure 2A, from above In the serum, we obtained highly pure Nanobody Fc fusion protein.

2) Digest the fusion protein with TEV, then flow the digested products through Protein G column and nickel column respectively, so as to remove the undigested protein, Fc and TEV enzyme respectively, collect the flow-through, concentrate and carry out SDS-PAGE electrophoresis , the results are shown in Figure 2B, from the flow-through, we obtained highly pure Nanobody protein.

Example 3 Characterization of the Nanobodies

1) Use circular dichroism (CD) to characterize the stability of nanobodies: replace the nanobody solutions of the examples with PBS, dilute to a QD _280nm of about 0.6, and then detect with a circular dichroism instrument, and the detection wavelength range is 280nm-180nm , temperature from 20-95°C. Each assay was repeated twice. Prism software was used to process the data, and the variation of the spectral value at 205 nm with temperature was selected, and the Tm value was further fitted. The results are shown in Figure 3, the Tm The values are 72.33, 75.44, 73.37, 78.98, 71.26, 98.23 and 71.07°C, respectively.

2) Preliminary characterization of the binding of the Nanobody Fc fusion protein to the extracellular segment of the SARS-CoV-2 spike protein (S1+S2) by non-competitive ELISA: the SARS-CoV-2 SARS-CoV-2 spike protein (S1+S2) Extracellular segment (Val 16-Pro 1213, Beijing Yiqiao Shenzhou) was diluted to 2 μg/mL with PBS, and 100 μL was added to each well for coating. After routine washing and blocking, add 1: 2.5 Gradiently diluted nanobody Fc fusion protein and ACE2-Fc protein (the aa 19-615 segment of human ACE2 was fused with human IgG1 Fc, secreted by HEK293F cells, and then purified by Protein A) solution, incubated at room temperature for 1 Hour. After washing, HRP-coupled anti-IgG1 Fc antibody (Beijing Yiqiao Shenzhou) was added to detect the bound VHH-Fc and ACE2-Fc. The results are shown in Figure 4. Except for aRBD-42-Fc, the other 6 nanobody Fc The fusion proteins, namely aRBD-2-Fc, aRBD-3-Fc, aRBD-5-Fc, aRBD-7-Fc, aRBD-41-Fc and aRBD-54-Fc, all had higher affinity than ACE2-Fc, and their The _EC50s were 0.256, 0.098, 0.077, 0.105, 0.226, 0.164 nM, respectively.

3) Use SPR to characterize the affinity between the nanobody and SARS-CoV-2 RBD: dissolve the RBD protein in sodium acetate at pH 4.5, couple it to a channel of the CM5 chip, and set an uncoupling protein at the same time The control channel was blocked with ethanolamine. The 7 Nanobodies were diluted 1:1 with PBS for 5 gradients, and then flowed through the above 2 channels respectively at a speed of 30 μL/min, and the signal value (RU) was detected simultaneously. After one cycle was completed, the chip was regenerated by aspirating off the bound antibody with 50 mM NaOH. All operations were performed on a Biacore T200 system. The results are shown in Figure 5. The Biacoreevaluation program was used to analyze the results, and the affinity K _D values of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54 combined with RBD 2.60, 3.33, 16.3, 3.31, 21.9, 113 and 5.49 nM, respectively. At the same time, we designed two bi-epitope-specific antibodies aRBD-2-5 based on the competition experiments between antibodies (aRBD-2 and aRBD-5 were linked head to tail with a GS linker whose sequence is shown in SEQ ID NO: 29 (GGGGSGGGGSGGGGS). ) and aRBD-2-7 (aRBD-2 and aRBD-7 are connected end-to-end with a GS linker with a sequence such as SEQID NO: 29 (GGGGSGGGGSGGGGS)), compared to monomers, the affinity of bi-epitope-specific antibodies is greatly The affinity K _D values of aRBD-2-5 and aRBD-2-7 were 59.2pM and 0.25nM, respectively.

Example 4 characterizes that the nanobody inhibits the binding function of ACE2 and RBD

The blocking function of the screened Nanobodies was characterized by competitive ELISA. Dilute SARS-CoV-2 RBD with PBS to 1 μg/mL, add 100 μL to each well for coating, and routinely wash and block. Dilute the biotinylated ACE2-Fc to 10nM, and then use the ACE2-Fc solution to sequentially dilute the Nanobody Fc fusion protein 1:3, and add 100 μL of each gradient mixture to the antigen-coated wells, and incubate at room temperature 1 hour. After washing with PBST for 4 times, HRP-coupled Streptavidin (Beiyuntian) was added to detect the bound biotinylated ACE2-Fc. The results are shown in Figure 6. Except for aRBD-42, the other 6 Nanobodies screened for Fc fusion The proteins aRBD-2-Fc, aRBD-3-Fc, aRBD-5-Fc, aRBD-7-Fc, aRBD-41-Fc and aRBD-54-Fc all have the ability to inhibit the binding of ACE2-Fc to SARS-CoV-2 RBD The IC ₅₀ for inhibiting the binding of 10 nM ACE2-Fc to SARS-CoV-2 RBD is 2.68, 2.59, 1.89, 1.42, 5.76 and 2.07 nM, respectively.

Example 5 characterizes the nanobody in vitro and SARS-CoV-2 invasion cell experiment

1) Inoculate Vero E6 cells (ATCC CBP60972) in a 96-well plate, culture medium is DMEM+10% FBS, and culture overnight at 37° C. and 5% CO ₂ . Dilute the Fc fusion protein of Nanobody aRBD-2 from 10 μg/mL to 0.041 μg/mL according to a 1:3 gradient, and dilute the Fc fusion proteins of aRBD-5 and aRBD-7 from 30 μg/mL according to a 1:3 gradient To 0.123μg/mL, the biepitope-specific antibodies aRBD-2-5 and aRBD-2-7 and their Fc fusion proteins were diluted from 1μg/mL to 0.0041μg/mL according to a 1:3 gradient, and the dilutions were DMEM+1%FBS, and then 50 μL were added to 96-well plates. Dilute SARS-CoV-2 (USA-WA1/2020 isolate) to 4000 PFU/mL, and the diluent is also DMEM+1% FBS, then take 50 μL of the SARS-CoV-2 dilution and add it to the antibody containing the gradient dilution At the same time, set a control without antibody, mix well, and incubate at 37°C for half an hour. Aspirate off the culture medium of Vero E6 cells, transfer the above 100 μL antibody and virus incubations to the wells inoculated with Vero E6 cells, and incubate for 1 h at 37°C and 5% CO ₂ . Aspirate the incubation, wash twice with PBS, add 100 μL DMEM (containing 10% FBS+0.5% methylcellulose) to each well, and incubate at 37° C., 5% CO2 for 48 hours. Each antibody concentration contained 2 replicate wells.

2) Aspirate the medium supernatant, wash with PBS twice, add 50 μL of PBS containing 4% paraformaldehyde to each well, fix for 15 minutes, and wash twice with PBS. Incubate the sample with PBS containing 0.1% Triton X-100 for 10 minutes to perforate the cell membrane and wash 3 times with PBS. Add DMEM containing 10% FBS to block non-specific binding sites, and place at room temperature for 30 min. Wash twice with PBS, use diluted anti-SARS-CoV-2N protein antibody (GeneTex, GTX635679) to an appropriate concentration, add 50 μL to each well, and incubate at room temperature for 1 hour. Wash 3 times with PBST. Add diluted Alexa Fluor 488-conjugated secondary antibody (Thermo) to an appropriate concentration, add 50 μL to each well, and incubate at room temperature for 1 hour. Nuclei were stained with Hoechst 33342. Fluorescent images of the entire well were acquired with a 4x objective in a Cytation 5 (BioTek) cell imager, and the total number of cells (as indicated by nuclear staining) and the total number of infected cells (as indicated by N protein) were quantified using the Cell Analysis module of Gen5 software (BioTek). staining), and the percentage of infected cells was calculated. Neutralization rate=100×(1-percentage of infected cells in wells with antibody/percentage of infected cells in wells without antibody). Using Prism software to analyze the data results, as shown in Figure 7, the fitting shows that in aRBD-2-Fc, aRBD-5-Fc, aRBD-7-Fc, aRBD-2-5-Fc and aRBD-2-7-Fc The ND ₅₀ (half-neutralizing dose concentration) of Vero E6 cells infected with SARS-CoV-2 was 0.092, 0.413, 0.591, 0.0104 and 0.0067 μg/mL, while the ND of aRBD-2-5 and aRBD-2-7 ₅₀ is less than 0.004 μg/mL. It can be seen that the virus neutralization ability of bi-epitope-specific antibodies is significantly better than that of single nanobodies. .

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.

Claims (12)

1. Alpaca-derived antibodies or antigen binding fragments thereof that bind to SARS-CoV-2RBD, having a VHH with

CDR1 as shown in SEQ ID NO. 19,

CDR2 as shown in SEQ ID NO. 20, and

CDR3 as shown in SEQ ID NO. 21.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VHH comprises: the amino acid sequence shown in SEQ ID NO. 28.

3. The antibody or antigen binding fragment thereof of claim 1 or 2, further having an Fc domain.

4. The antibody or antigen binding fragment thereof of claim 3, wherein the Fc domain is an IgG1 Fc domain.

5. The antibody or antigen binding fragment thereof of claim 3, wherein the Fc domain is a human IgG1 Fc domain.

6. The antibody or antigen-binding fragment thereof of claim 5, wherein the amino acid sequence of the human IgG1 Fc domain is set forth in SEQ ID No. 30.

7. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-6.

8. An expression vector comprising the polynucleotide of claim 7.

9. A host cell comprising the expression vector of claim 8, said host cell being a host cell for expressing a foreign protein.

10. The host cell of claim 9, wherein the host cell is a bacterial, yeast, insect cell, or mammalian cell.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6 and a pharmaceutically acceptable carrier.

12. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6 in the manufacture of a kit or medicament for the treatment and/or diagnosis of SARS-CoV-2 infection.

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