CN114249821B

CN114249821B - Alpaca-derived nanobody combined with SARS-CoV-2RBD

Info

Publication number: CN114249821B
Application number: CN202011038123.6A
Authority: CN
Inventors: 金腾川; 马欢; 曾威红
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2020-09-25
Filing date: 2020-09-25
Publication date: 2023-06-16
Anticipated expiration: 2040-09-25
Also published as: CN114249821A

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 nanobody combined with SARS-CoV-2RBD

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to a nanometer antibody sequence for resisting SARS-CoV-2RBD for treatment and diagnosis.

Background

SARS-CoV-2 belongs to the coronavirus, which causes pneumonia called COVID-19.SARS-CoV-2 enters the cell after binding to angiotensin converting enzyme 2 (ACE 2) on the surface of epithelial cells via its Receptor Binding Domain (RBD) of surface spike protein (spike), and completes invasion.

Fully human antibodies isolated from recovered patients proved to have good antiviral effects, but these were all conventional monoclonal antibodies consisting of 2 heavy and 2 light chains. Has the limitations of large molecular weight, complex production process, difficult processing and transformation, and the like.

In camelids there is an antibody naturally lacking the light chain, i.e. a heavy chain antibody, the variable region of which consists of only the heavy chain, abbreviated VHH, and the variable region protein is less than 10 nanometers in diameter and is therefore also referred to as nanobody. The nano antibody has the advantages of small molecular weight, strong penetrability, easy expression, easy genetic modification, easy combination of a plurality of epitopes and the like.

No alpaca-derived natural nanobody against SARS-CoV-2RBD is available for treating COVID19.

Disclosure of Invention

The present disclosure provides alpaca-derived heavy chain antibody variable region sequences (VHH) capable of binding with high affinity to the novel coronavirus (SARS-CoV-2) Receptor Binding Domain (RBD), also known as nanobodies, which can be used for the prevention, treatment and/or diagnosis of SARS-CoV-2 infection.

The inventor adopts SARS-CoV-2RBD protein expressed by recombination in vitro to immunize 2 head little alpaca for 3 times, then separates out peripheral blood lymphocyte and extracts total RNA of cell, then reverse transcribes into cDNA. And then using the cDNA as a template, and amplifying the nano antibody sequence by using a specific primer. We isolated 7 nanobodies. Designated as aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54, respectively, the amino acid sequences thereof are as follows:

amino acid sequence of aRBD-2:

QVQLVESGGGLVQAGGSLRLSCAASGRTYTMGWFRQAPGKEREFVAAMRWSDTDYADSLKGRFTISRDNANNAMYLQMNSLGPEDTAVYYCAAGEAWLARSTHHYDYWGQGTQVTVSS(SEQ ID NO：22)

amino acid sequence of aRBD-3:

QVQLVESGGGLVQPGGSLRLSCAASGLTLDYYAIGWFRQAPGKEREGVSCISHPGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASPLALFRLCVLPSPLPYDYWGQGTQVTVSS(SEQ ID NO：23)

amino acid sequence of aRBD-5:

QVQLVESGGGLVQPGGTLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISGSGGITNYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAPVSHTVVAGCAFEAWTDFGSWGQGTQVTVSS(SEQ ID NO：24)

amino acid sequence of aRBD-7:

QLQLVESGGGLVQAGGSLRLSCAASERTFSGGVMGWFRQRPGKEREFVAAIRWNGASTFYADSVKGRFTCSRDNAKNTGYLQMNSLTPEDTAVYYCARAVRTYASSDYYFQERTYDYWGQGTQVTVSS(SEQ ID NO：25)

amino acid sequence of aRBD-41:

QVQLVESGGGLVQPGGSLRLSCAASGFTSGHYAIGWFRQAPGKEREGVSCIGSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGLWYGRSLNSFDYDYWGQGTQVTVSS(SEQ ID NO：26)

amino acid sequence of aRBD-42:

QLQLVESGGGLVQAGGSLRLSCAASGRTFSSATMGWFRQAPGKEREFVAAISWSGLSRYYADSVKGRFTISRDNAENTVYLQMNSLKTEDTAVYYCAADSWGCSGLGCYDARQYDVWGQGTQVTVSS(SEQ ID NO：27)

amino acid sequence of aRBD-54:

QLQLVESGGGLVQPGGSLRLSCAASGRTFGSFMGWFRQAPGQERDFVAAITWSGGSTYYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAARISSAYYTRSSSYAYWGQGTQVTVSS(SEQ ID NO：28)

the 3 complementarity determining regions (CDR 1, CDR2, and CDR 3) of the 7-strain nanobody are shown in the underlined section, 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：GRTFSSATM(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 different epitopes, and aRBD-2 and aRBD-7 bind different epitopes, so that two corresponding double epitope-specific antibodies aRBD-2-5 and aRBD-2-7 were constructed by combining them, respectively.

The bi-epitope specific antibody as used herein refers to an antibody constructed by linking two nanobodies capable of binding to two independent epitopes on, for example, SARS-CoV-2RBD, respectively, with flexible polypeptide chains.

Specifically, the invention provides the following technical schemes:

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

as set forth in SEQ ID NO:1, a CDR1 as shown in figure 1,

as set forth in SEQ ID NO: CDR2 and 2

As set forth in SEQ ID NO:3, CDR3;

as set forth in SEQ ID NO:4, a CDR1 as shown in figure 4,

as set forth in SEQ ID NO: CDR2 and 5

As set forth in SEQ ID NO: CDR3 shown in fig. 6;

as set forth in SEQ ID NO: the CDR1 shown in figure 7,

as set forth in SEQ ID NO: CDR2 and 8

As set forth in SEQ ID NO: CDR3 as shown in 9;

as set forth in SEQ ID NO:10, a CDR1 as shown in figure 10,

as set forth in SEQ ID NO:11 and CDR2 and

as set forth in SEQ ID NO:12, CDR3;

as set forth in SEQ ID NO:13, a CDR1 is shown in figure 13,

as set forth in SEQ ID NO: CDR2 and 14

As set forth in SEQ ID NO:15, CDR3;

as set forth in SEQ ID NO:16, a CDR1 as shown in figure 16,

as set forth in SEQ ID NO:17 and CDR2 shown in fig. 17

As set forth in SEQ ID NO:18, CDR3; and/or

As set forth in SEQ ID NO:19, a CDR1 as shown in figure 19,

as set forth in SEQ ID NO:20 and CDR2 shown in FIG. 20

As set forth in SEQ ID NO:21, and CDR3 as shown.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VHH comprises:

as set forth in SEQ ID NO:22, and a sequence of the amino acids shown in the specification,

as set forth in SEQ ID NO:23, and an amino acid sequence shown in seq id no

As set forth in SEQ ID NO:24, and a nucleotide sequence shown in seq id no.

As set forth in SEQ ID NO:25, and a sequence of the amino acids shown in the formula II,

as set forth in SEQ ID NO:26, and a sequence of the amino acids shown in the specification,

as set forth in SEQ ID NO:27, and/or

As set forth in SEQ ID NO:28, and a polypeptide comprising the amino acid sequence shown in seq id no.

3. The antibody or antigen-binding fragment thereof of

item

1 or 2, which is a bi-epitope specific antibody comprising, in order (e.g., in order of N-terminus to C-terminus) SEQ ID NO:22 and SEQ ID NO:24, or SEQ ID NO:22 and SEQ ID NO:25, preferably wherein SEQ ID NO:22 and SEQ ID NO:24 or SEQ ID NO:22 and SEQ ID NO:25 are linked by a linker (e.g., a flexible polypeptide chain, such as a GS linker).

4. The antibody or antigen binding fragment thereof of any one of claims 1-3, further having an Fc domain, preferably an IgG1 Fc domain, more preferably a human IgG1 Fc domain, the sequence of which is set forth in, for example, SEQ ID NO:30, the nucleotide sequence of the coding gene of the sequence of the human IgG1 Fc domain is set forth, for example, in SEQ ID NO: shown at 31.

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

6. An expression vector, for example, employing an expression vector based on one or more promoters and host cells, comprising the polynucleotide of item 5.

7. A host cell comprising the expression vector of claim 6, wherein the host cell is a host cell for expressing a foreign protein, e.g., a bacterium, a yeast, an insect cell, a mammalian cell.

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

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

Advantages and positive effects of the present disclosure

The nano antibody (VHH) is derived from a natural alpaca heavy chain antibody, so that the nano antibody has the characteristics of high stability, high expression level and high affinity.

A round dichroism experiment shows that the half-dissolving temperature (Tm value) of the 7-strain nano antibody is above 70 ℃.

After the 7-strain nano antibody is fused with a human IgG1 Fc segment, cloning the fused Protein into a pTT5 vector, carrying out secretory expression by using a mammalian cell 293F, and purifying the fused Protein in a culture medium supernatant by using a Protein A column after 3 days of expression, wherein the yield of the 7-strain antibody is above 90 mg/L.

The 7 antibodies can combine SARS-CoV-2RBD with high affinity. ELISA experiments show that the Fc fusion proteins of other antibodies of the present disclosure bind to the extracellular portion of SARS-CoV-2 spike protein (S1+S2) with higher affinity than ACE2, except for aRBD-42. As a result of Surface Plasmon Resonance (SPR) experiments, it was revealed that the affinity dissociation constants (K) of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42, aRBD-54 and SARS-CoV-2RBD _D ) Values were 2.60, 3.33, 16.3, 3.31, 21.9, 113 and 5.49nM (nanomoles per liter), respectively.

In addition to aRBD-42, the other 6 nano antibodies of the present disclosure can well inhibit human ACE2 and SARS-CoV-2RBD after fusion with human IgG1 FcAnd (5) combining. The competitive ELISA experiments showed that the Fc fusion proteins of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41 and aRBD-54 each compete with 10nM ACE2-Fc for SARS-CoV-2RBD, the IC thereof ₅₀ 2.68, 2.59, 1.89, 1.42, 5.76 and 2.07nM, respectively.

The nano antibodies aRBD-2 and aRBD-5 of the disclosure bind different epitopes, and aRBD-2 and aRBD-7 bind different epitopes, thus constructing two bi-epitope specific antibodies aRBD-2-5 and aRBD-2-7, SPR shows that the affinity with SARS-CoV-2RBD is greatly enhanced, K _D The values were 59.2pM (picomolar per liter) and 0.25nM, respectively.

The Fc fusion proteins of the nanobodies aRBD-2, aRBD-5 and aRBD-7 can neutralize SARS-CoV-2 to infect Vero E6 cells in vitro. Fc fusion proteins of aRBD-2, aRBD-5 and aRBD-7 were used to neutralize 200PFU SARS-CoV-2 infection of Vero E6 at a concentration ND of 100. Mu.L system ₅₀ 0.092, 0.413 and 0.591 μg/mL, respectively. Fc fusion proteins of the bispecific antibodies aRBD-2-5 and aRBD-2-7 neutralize 200PFU SARS-CoV-2 infection Vero E6 at a concentration ND of 100 μL system ₅₀ 0.0104 and 0.0067. Mu.g/mL, respectively.

Drawings

Figure 1. Phage display screening results for 7 nanobodies of the disclosure. (a) is the phase count result of two rounds of panning; (B) results of ELISA using monoclonal phage.

FIG. 2 shows the SDS-PAGE gel of the nanobody Fc fusion protein (A) and nanobody (B). Lanes M are markers, lanes 1 to 7 are the aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54 fusion proteins (A) and their respective Fc-cleaving nanobody proteins (B) in this order.

Figure 3 is a graph of the results of round dichroism (CD) experiments to detect denaturation temperatures of 7 nanobodies of the disclosure. (A) - (G) results of the detection of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54 in this order.

FIG. 4 is a graph showing the results of ELISA detection of binding between the Fc fusion protein of the nanobody and the extracellular domain protein of SARS-CoV-2 spike protein (S1+S2).

FIG. 5 shows the detection of affinity between the nanobody and SARS-CoV-2RBD using SPR. (A) The kinetics curves of the binding between the nanobodies aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42, aRBD-54, aRBD-2-5 and aRBD-2-7 and SARS-CoV-2RBD protein are detected by adopting an SPR method in turn. Where the solid line is the kinetic curve monitored in real time and the dashed line is the curve fitted using biacore evaluation software. The kinetic curves of the different antibody concentration gradients correspond sequentially from top to bottom to the top-to-bottom concentrations identified on the right.

FIG. 6 is a graph showing the results of detection of the binding of ACE2 to SARS-CoV-2RBD by the Fc fusion protein of the nanobody using a competitive ELISA.

Figure 7 in vitro virus neutralization experiments verify the function of the antibodies of the present disclosure. The experimental data analysis results of the in vitro neutralization of SARS-CoV-2 virus infected Vero E6 cells by the Fc fusion proteins of nanobody aRBD-2, aRBD-5 and aRBD-7 and the Fc fusion proteins of the double epitope specific antibodies aRBD-2-5 and aRBD-2-7.

Detailed Description

The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.

EXAMPLE 1 immunization of alpaca with SARS-CoV-2RBD and screening nanobodies

1) HEK293F cells (ATCC, CBP 60437) were used to express purified SARS-CoV-2RBD (QKV 42562.1, aa 321-591) and mixed with Freund's adjuvant, and the alpaca was immunized by subcutaneous injection at a dose of 500. Mu.g/dose three times, 2 weeks apart, for a total immunization of 2 first 6 month-sized llamas.

2) After 2 weeks of the third immunization, blood was collected intravenously and leukocytes were isolated from the blood. Total RNA was extracted using the RNA extraction kit from omegabiotek company while genomic DNA was removed using DNase. PrimeScript by TAKARA Co ^TM II 1st Strand cDNA Synthesis Kit reverse transcribes RNA into cDNA.

3) Preparing a nanobody phagemid library: the designed alpaca VHH specific primer is adopted, the cDNA is used as a template to amplify and obtain a VHH coding gene fragment, the amplified VHH sequence is cloned into NcoI and NotI sites of phagemid pR2 by adopting a Gibson assembly method, and the obtained Gibson assembly product is the initial nanobody phagemid library.

4) Electrotransformation TG1 amplified nanobody phagemid library: coli TG1 competent cells were prepared using a 10% glycerol wash method, and then the above Gibson assembly product was electrotransformed into TG1 competent cells, and 5 blocks of 150mm LB (LB/2%G/Amp) plates containing 2% glucose and 100 μg/mL ampicillin were plated to amplify the phagemid library.

5) Amplifying nanobody phage library: after scraping, a proper amount of bacterial liquid is inoculated with 200mL of 2TY (containing 2% glucose and 100 mug/mL ampicillin) to be cultivated to logarithmic phase, and 10 percent of bacterial liquid is added ¹² pfu of KM13 helper phage (purchased from MRC Laboratory of Molecular Biology), infected at 37℃for 45min, 100mL of bacterial solution was centrifuged, the bacterial cells were resuspended in 200mL of 2TY (containing 0.1% glucose, 100. Mu.g/mL and 50. Mu.g/mL kanamycin), and incubated at 25℃for 20h to amplify the phage displaying nanobodies. Concentrating the phase by PEG precipitation, and finally re-suspending with PBS and preserving on ice.

6) Panning (Panning)

A. First round: the SARS-CoV-2RBD purified in example 1 was diluted to 0.1mg/mL with PBS, 100. Mu.L was added to a well of a 96-well immunoplate (Nunc maxsorp plate), coated overnight at 4℃and a well of no antigen control was set. Washing with PBS 3 times, and adding 300 μl of MPBS (PBS containing 5% skimmed milk) to each well, and sealing at room temperature for 2 hr. Washing 3 times with PBS, 1X 10 was added to each well ¹¹ phage library phase (dissolved in 100. Mu.L MPBS) was prepared above pfu and incubated at 80rpm for 1h at room temperature. PBST (0.1% Tween 20) was used for 30 washes. 100. Mu.L trypsin at 0.5mg/mL was added to each well, and the mixture was digested at room temperature for 1h, and the phase bound in the wells was eluted. 10. Mu.L of eluted phage was used to infect 1mL of TG1 bacteria in logarithmic growth phase, and the mixture was subjected to a water bath at 37℃for 45min. 100. Mu.L, 10. Mu.L and 1. Mu.L of coated LB/2%G/Amp plates were counted, respectively. The remaining phage solution was all infected with 3mL of TG1 bacteria in logarithmic growth phase, water-bath at 37℃for 45min, coated with 1 block of 150mm LB/2%G/Amp plates, and incubated overnight at 37 ℃.

B. A second wheel: adding 4mL of 2TY to above 150mm plate, scraping colony, mixing bacterial liquid uniformly, inoculating 100 μL to 100mL of 2TYAnd (2) in a/2%G/Amp culture medium, adding KM13 infection after culturing to a logarithmic phase to prepare the phage displayed by the nano antibody. SARS-CoV-2RBD was then diluted to 0.02mg/mL with PBS, 100. Mu.L was added to one well of a 96-well immunoplate, coated overnight at 4℃and a well of no antigen control was set. Washing with PBS 3 times, and adding 300 μl of MPBS (PBS containing 5% skimmed milk) to each well, and sealing at room temperature for 2 hr. Washing 3 times with PBS, 1X 10 was added to each well ⁸ The first round of elution phase (in 100. Mu.L MPBS) amplified above pfu was incubated at room temperature for 1h at 80 rpm. PBST (0.2% Tween 20) was used for 30 washes. 100. Mu.L trypsin at 0.5mg/mL was added to each well, and the mixture was digested at room temperature for 1h, and the phase bound in the wells was eluted. 10. Mu.L of eluted phage was used to infect 1mL of TG1 bacteria in logarithmic growth phase, and the mixture was subjected to a water bath at 37℃for 45min. 100. Mu.L, 10. Mu.L and 1. Mu.L of coated LB/2%G/Amp plates were counted, respectively.

C. The phase counts of two rounds of channeling elution are shown in FIG. 1A. The RBD coated wells eluted with significantly more than the control wells, with the first round of RBD Kong Xituo coated with more than 70 times more than the control wells and the second round of RBD coated wells, with a higher ratio. Demonstrating that phages specific for RBD were successfully isolated and enriched.

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

A. Preparation of monoclonal phase: from the plates counted after the above 2 rounds of screening elution, 31 individual clones were picked and inoculated into 96-well cell culture plates containing 100. Mu.L of 2TY medium (containing 2% glucose and 100. Mu.g/mL ampicillin) per well, 1 clone was 1 well, and shake cultured at 37℃and 250rpm for 12 hours. Transfer more than 5. Mu.L of the broth to a fresh 96-well plate containing 200. Mu.L of 2TY medium (2% glucose and 100. Mu.g/mL Ampicillin) per well (the remaining broth was added to 15% glycerol at a final concentration, -80℃for storage), shake culture at 37℃at 250rpm for 1.5h to an OD600 of about 0.5, and aspirate 100. Mu.L of broth per well. 50. Mu.L of the solution containing 4X 10 was added to each well ⁸ pfu KM13 phage 2TY, mixed well and incubated at 37℃for 45min.3500g was centrifuged for 10min, the supernatant was discarded, and the pellet was resuspended in 200. Mu.L of 2TY containing 0.1% glucose, 100. Mu.g/mL Ampicillin and 50. Mu.g/mL Kanamycin, 25℃and 250rpm shaking culture for 20h. Centrifugation at 3500g for 10min, transfer 75. Mu.L of supernatant to 225. Mu.L per wellIn the 96-well plate holes of MPBS, uniformly mixing, and temporarily storing at 4 ℃ for standby, thus finishing the preparation of the monoclonal phage.

B. Phage ELISA detection: diluting SARS-CoV-2RBD protein with PBS to 1 μg/mL, respectively coating 96-well immunoplates with 100 μl/well, additionally providing blank (PBS wells, coating overnight at 4deg.C, washing with PBS for 3 times, adding 300 μl of MPBS to each well, sealing at room temperature for 2h, adding 100 μl of the above-prepared phage MPBS mixture to each well, incubating at room temperature for 1h, washing with PBST plate for 4 times, moderately diluting HRP-anti M13 antibody with MPBS (Yiqishenzhou), adding 100 μl to each well of above immunoplates, incubating at room temperature for 1h, washing with PBST plate for 4 times, adding 100 μl of TMB chromogenic substrate (Biyun) to each well, coating light-shielding with aluminum foil paper, reacting at room temperature for 5min, adding 50 μl of 1M H to each well ₂ SO ₄ Stop reaction and measure OD _450nm Values. The results are shown in FIG. 1B.

C. All OD's were taken _450nm Positive clones with a value greater than 1 were sent to the company for sequencing, and the sequencing results were analyzed and aligned to finally determine 7 positive monoclonal clones, named aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54, respectively, as described above.

EXAMPLE 2 expression purification of the resulting nanobody and Fc fusion protein thereof

1) Designing a primer, fusing IFN alpha protein signal peptide at the N end of the gene sequence of the nano antibody to guide secretion expression, fusing human IgG1 Fc at the C end of the gene sequence of the nano antibody, introducing a TEV enzyme cleavage site between the two, and then cloning the gene sequence into a mammalian expression vector pTT 5. The constructed vector is transiently transfected into a mammalian cell HEK293F by PEI, the supernatant is collected after 3 days of culture, fusion proteins in the supernatant are purified by a Protein A column, SDS-PAGE electrophoresis is carried out, and the result is shown in figure 2A, and from the supernatant, the high-purity nano antibody Fc fusion Protein is obtained.

2) The fusion Protein is digested by TEV, and then the digested products are respectively passed through a Protein G column and a nickel column, so that the incompletely digested proteins, fc and TEV enzymes are respectively removed, flow-through is collected, SDS-PAGE electrophoresis is carried out after concentration, and as a result, as shown in figure 2B, the high-purity nanobody Protein is obtained from the flow-through.

Example 3 characterization of the nanobody

1) Stability of nanobodies was characterized by Circular Dichroism (CD): the nano antibody solution of the example is respectively replaced by PBS and diluted to OD _280nm About 0.6, then detecting by a circular dichroscope, wherein the detection wavelength ranges from 280nm to 180nm, and the temperature ranges from 20 ℃ to 95 ℃. Each test was repeated twice. And processing the data by Prism software, selecting the change condition of a spectrum value at 205nm along with the temperature, and further fitting the Tm value. As a result, the Tm values of the aRBD-2-Fc, aRBD-3-Fc, aRBD-5-Fc, aRBD-7-Fc, aRBD-41-Fc, aRBD-42-Fc and aRBD-54-Fc were 72.33, 75.44, 73.37, 78.98, 71.26, 98.23 and 71.07 ℃respectively, as shown in FIG. 3.

2) The binding of the nanobody Fc fusion protein to the extracellular segment of SARS-CoV-2 spike protein (S1+S2) was initially characterized by non-competitive ELISA: the extracellular fraction (Val 16-Pro 1213, beijing Yiqiao Shenzhou) of SARS-CoV-2SARS-CoV-2 spike Protein (S1+S2) was diluted to 2. Mu.g/mL with PBS, 100. Mu.L of each well was added for coating, after conventional washing and blocking, the nanobody Fc fusion Protein and ACE2-Fc Protein diluted in a 1:2.5 gradient were added in sequence (after aa 19-615 of human ACE2 was fused to human IgG1 Fc, secretory expression was performed using HEK293F cells, followed by purification using Protein A) solution, and incubated for 1 hour at room temperature. After washing, bound VHH-Fc and ACE2-Fc were detected with the addition of HRP-conjugated anti-IgG 1 Fc antibody (Beijing Yizhushen), and the results are shown in FIG. 4, in which the other 6 nanobody Fc fusion proteins, namely, aRBD-2-Fc, aRBD-3-Fc, aRBD-5-Fc, aRBD-7-Fc, aRBD-41-Fc and aRBD-54-Fc, have higher affinity than ACE2-Fc, in addition to aRBD-42-Fc, respectively ₅₀ 0.256, 0.098, 0.077, 0.105, 0.226, 0.164nM, respectively.

3) SPR was used to characterize the affinity between the nanobody and SARS-CoV-2 RBD: RBD protein is dissolved in sodium acetate with pH of 4.5, coupled to one channel of a CM5 chip, and a control channel without coupled protein is arranged and is blocked by ethanolamine. The 7 nanobodies were diluted 1:1 in PBS for 5 gradients, and then flowed through the above 2 channels at a rate of 30. Mu.L/min, respectively, while detecting signalsValue (RU). After one cycle was completed, the bound antibody was blotted off with 50mM NaOH to regenerate the chip. All operations are done on the Biacore T200 system. The results are shown in FIG. 5, and the results are analyzed using the Biacore evaluation procedure for the affinity K for RBD binding of aRBD-2, aRBD-3, aRBD-5, aRBD-7, aRBD-41, aRBD-42 and aRBD-54 _D Values were 2.60, 3.33, 16.3, 3.31, 21.9, 113 and 5.49nM, respectively. Meanwhile, according to competition experiments among antibodies, 2 bi-epitope specific antibodies aRBD-2-5 (the aRBD-2 and the aRBD-5 are connected end to end by using a GS connector with the sequence shown as SEQ ID NO:29 (GGGGSGGGGSGGGGS)) and aRBD-2-7 (the aRBD-2 and the aRBD-7 are connected end to end by using a GS connector with the sequence shown as SEQ ID NO:29 (GGGGSGGGGSGGGGS)) are designed, compared with a single body, the affinity of the bi-epitope specific antibodies is greatly improved, and the affinity K of the aRBD-2-5 and the aRBD-2-7 is greatly improved _D The values were 59.2pM and 0.25nM, respectively.

Example 4 characterization of the nanobody to inhibit the binding function of ACE2 to RBD

The blocking function of the nano antibody obtained by screening is characterized by adopting a competitive ELISA method. SARS-CoV-2RBD was diluted to 1. Mu.g/mL with PBS, 100. Mu.L of each well was used for coating, and subjected to conventional washing and blocking. Biotinylated ACE2-Fc was diluted to 10nM and then the nanobody Fc fusion protein was diluted with this ACE2-Fc solution in a 1:3 gradient in sequence, 100. Mu.L of each gradient mixture was added to antigen coated wells, respectively, and incubated for 1 hour at room temperature. After washing 4 times with PBST, bound biotinylated ACE2-Fc was detected by addition of HRP-conjugated strepitavidins (Biyun) and the results are shown in FIG. 6, wherein 6 nanobody Fc fusion proteins selected in addition to aRBD-42, aRBD-3-Fc, aRBD-5-Fc, aRBD-7-Fc, aRBD-41-Fc and aRBD-54-Fc each had the function of inhibiting the binding of ACE2-Fc to SARS-CoV-2RBD, and 10nM of ACE2-Fc was inhibited from binding to SARS-CoV-2RBD ₅₀ 2.68, 2.59, 1.89, 1.42, 5.76 and 2.07nM, respectively.

EXAMPLE 5 characterization of the nanobody in vitro neutralization of SARS-CoV-2 invasion cell experiments

1) Vero E6 cells (ATCC CBP 60972) were seeded in 96-well plates in DMEM+10% FBS,37℃and 5% CO ₂ Culturing overnight. The Fc fusion proteins of nanobodies aRBD-2 were diluted from 10. Mu.g/mL to 0.041. Mu.g/mL according to a gradient of 1:3, the Fc fusion proteins of aRBD-5 and aRBD-7 were diluted from 30. Mu.g/mL to 0.123. Mu.g/mL according to a gradient of 1:3, the double epitope-specific antibodies aRBD-2-5 and aRBD-2-7 and their Fc fusion proteins were diluted from 1. Mu.g/mL to 0.0041. Mu.g/mL according to a gradient of 1:3, and the dilutions were DMEM+1% FBS, followed by 50. Mu.L addition to 96-well plates, respectively. SARS-CoV-2 (USA-WA 1/2020 isolate) WAs diluted to 4000PFU/mL, the dilution WAs also DMEM+1% FBS, then 50. Mu.L of SARS-CoV-2 dilution WAs added to wells containing gradient diluted antibody, and control without antibody WAs set, mixed well and incubated at 37℃for half an hour. The culture medium of Vero E6 cells was aspirated, and 100. Mu.L of the above antibody and virus cultures were transferred to wells inoculated with Vero E6 cells, respectively, at 37℃and 5% CO ₂ Incubate for 1h. The incubations were aspirated, washed 2 times with PBS, and 100. Mu.L of DMEM (10% FBS+0.5% methylcellulose) was added to each well and incubated at 37℃under 5% CO2 for 48h. Each antibody concentration contained 2 duplicate wells.

2) The culture supernatant was aspirated, washed 2 times with PBS, 50. Mu.L of 4% paraformaldehyde in PBS was added to each well, and the mixture was fixed for 15 minutes and washed twice with PBS. Samples were incubated with PBS containing 0.1% Triton X-100 for 10 minutes, cell membranes were perforated, and washed 3 times with PBS. DMEM containing 10% fbs was added to block the non-specific binding sites and left at room temperature for 30min. PBS was washed 2 times, diluted anti-SARS-CoV-2N protein antibody (GeneTex, GTX 635679) to the appropriate concentration, 50. Mu.L per well was added and incubated for 1 hour at room temperature. PBST was washed 3 times. Diluted Alexa Fluor 488-conjugated secondary antibody (Thermo) was added to the appropriate concentration and 50. Mu.L per well was added and incubated for 1 hour at room temperature. Nuclei were stained with Hoechst 33342. The percentage of infected cells was calculated by taking a fluorescence image of the whole well with a 4-fold objective lens in cytoimager Cytation 5 (BioTek), and quantifying the total number of cells (as indicated by nuclear staining) and the total number of infected cells (as indicated by N protein staining) with a cell analysis module of Gen5 software (BioTek). Neutralization rate = 100× (percent 1-antibody well infected cells/percent no antibody well infected cells). Analysis of the data using Prism software, as shown in FIG. 7, the fit showed that aRBD-2-Fc, aRBD-5-Fc, aRBD-7-Fc, aRBD-2-5-Fc and aRBD-2-7-Fc neutralized SARND of S-CoV-2 infection Vero E6 cells ₅₀ (half-neutralized dose concentrations) are ND of 0.092, 0.413, 0.591, 0.0104 and 0.0067. Mu.g/mL, and aRBD-2-5 and aRBD-2-7, respectively ₅₀ Less than 0.004 μg/mL can be seen that the virus neutralization capacity of the bi-epitope specific antibodies is significantly better than that of a single nanobody. .

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims

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. 13,

CDR2 as shown in SEQ ID NO. 14, and

CDR3 as shown in SEQ ID NO. 15.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VHH comprises: the amino acid sequence shown in SEQ ID NO. 26.

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.