Nanobody aSA3 against sars, covid-19 and its mutants

Abstract

This invention relates to a broad-spectrum nanobody aSA3 against SARS, SARS-CoV-2, and their mutant strains, comprising CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:3, and CDR3 as shown in SEQ ID NO:4. This nanobody can bind with high affinity to the receptor-binding domain (RBD) of SARS-CoV-2 and SARS-CoV-2 and potently neutralize SARS-CoV-2 and their mutant strains.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to nanobody sequences for use in the treatment and diagnosis of COVID-19 and SARS viruses. Background Technology

[0002] The novel coronavirus (SARS-CoV-2, hereinafter referred to as SARS2) and SARS-CoV (SARS-CoV, hereinafter referred to as SARS1) belong to the sabezier coronavirus family. Both SARS2 and SARS1 enter cells and complete infection by binding to angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells through their spike protein receptor-binding domain (RBD). Antibodies targeting the RBD have been proven to be effective in neutralizing the virus, but antibodies need to overcome the challenge of viral mutation. Developing antibodies that target conserved sites of sabezier virus will provide anti-mutation capabilities.

[0003] Besides traditional antibodies, nanobodies (also known as VHHs) have also attracted widespread attention in the treatment of COVID-19. Because they contain only heavy chain variable regions, nanobodies have a molecular weight of only 13-15 kDa. Their small size allows them to recognize epitopes that traditional antibodies cannot access. Their single-domain structure also makes them easier to engineer to further improve affinity and virus neutralization capabilities. Furthermore, nanobodies can be nebulized and delivered directly to the lungs, or even administered via the nasal cavity, which can alleviate strain on medical resources. Several anti-COVID-19 nanobodies have been reported both domestically and internationally, but nanobodies capable of binding to conserved epitopes of the SARS-CoV-2 virus have only been reported in a few cases abroad, such as VHH72, Fu2, and Nb95, which can simultaneously bind to SARS2 and SARS1 RBDs. However, these nanobodies exhibit low affinity for both SARS2 and SARS1 RBDs and limited virus neutralization activity.

[0004] This invention relates to a high-affinity nanobody targeting SARS2 and SARS1 RBD, named aSA3, which has an affinity K for binding to SARS2 and SARS1 RBD. D The values ​​are 5.19 x 10 -11 M and 3.61x10 -10M can block the binding of ACE2 to SARS2 and SARS1 RBDs. Its Fc fusion protein aSA3-Fc can efficiently neutralize SARS1, SARS2 and their Beta, Delta and Omicron BA.1 mutant strains in vitro, and can also exert anti-Omicron BA.1 efficacy in hamsters. aSA3 binds to the core region of the RBD. When aSA3-Fc is fused with another nanobody targeting the RBM epitope, aRBD-2, a dual epitope-specific antibody (aRBD2-aSA3-Fc) is formed. This dual epitope antibody exhibits higher binding affinity and stronger neutralizing activity for Omicron BA.1 and BA.2, and can prevent Omicron BA.1 infection in hamsters at low doses via intranasal administration. Summary of the Invention

[0005] This disclosure provides a variable region sequence (VHH) of an alpaca-derived heavy chain antibody that can bind to SARS2 and SARS1 RBD with high affinity. This variable region sequence is also known as a nanobody and can be used for the prevention, treatment and / or diagnosis of SARS2 and SARS1 infection.

[0006] The inventors immunized two alpacas three times with recombinantly expressed SARS2 RBD protein. Peripheral blood lymphocytes were then isolated, and total RNA was extracted and reverse transcribed into cDNA. Using this cDNA as a template, nanobody sequences were amplified with specific primers, and a phage display library was constructed. The library was then screened using SARS1 RBD, resulting in several nanobodies with bispecificity for both SARS2 and SARS1 RBD. The antibody with the highest affinity and strongest ACE2-RBD blocking ability was named aSA3, and its amino acid sequence is as follows:

[0007] QVQLVESGGGLVQPGGSLRLSCAA SGFTSDHYALA WFRQAPGK E REGVSCIDSDGNPF YADSVKGRFTGSRDNAKNTVYLQMNSLKLEDTA VYYCAA GLWYGRSLNSFDYD YWGQGTQVTVSS(SEQ ID NO:1)

[0008] The three antigen complementarity-determining regions (CDR1, CDR2, and CDR3) of the nanobody aSA3 are shown in the underlined portion above, specifically:

[0009] CDR1: SGFTSDHYALA(SEQ ID NO:2)

[0010] CDR2:EREGVSCIDSDGNPF(SEQ ID NO:3)

[0011] CDR3: GLWYGRSLNSFDYD (SEQ ID NO: 4).

[0012] Specifically, the present invention provides a nanobody or its antigen-binding fragment having CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:3, and CDR3 as shown in SEQ ID NO:4. This nanobody can bind to the receptor-binding domain (RBD) of SARS-CoV-1 and SARS-CoV-2 with high affinity and can block the binding of angiotensin-converting enzyme 2 (ACE2) to the RBD. After fusion with human IgG1 Fc, this nanobody can bind to the RBD of SARS-CoV-2 mutant strains with high affinity, and can efficiently neutralize SARS-CoV-1, wild-type (WT) SARS-CoV-2, and Beta, Delta, Omicron BA.1, and Omicron BA.1.1 mutant strains in vitro. It also exhibits anti-Omicron BA.1 virus infection efficacy in hamsters. The SARS1-binding epitopes of this nanobody are mainly located in the core region of the RBD, and the binding sites include the L355, Y356, S358, F361, S362, T363, F364, K365, C366, V394, R395, I489, G490 and Y494 residues of the SARS1 RBD. A bieptope antibody was formed by fusing the N-terminus of the nanobody with the fusion protein of human IgG1 Fc and the C-terminus of another nanobody targeting the receptor RBD binding motif (RBM) via the (GGGGS)4 adaptor protein. This bieptope antibody can bind to the RBD of WT SARS2, Omicron BA.1, and BA.2 mutant strains with higher affinity and can more effectively neutralize WT SARS2, Beta, Delta, Omicron BA.1, Omicron BA.1.1, and Omicron BA.2 mutant strains. Intraperitoneal injection of this bieptope antibody also showed anti-Omicron BA.1 infection efficacy in hamsters. In addition, this bieptope antibody can also effectively protect hamsters from Omicron BA.1 infection by intranasal administration.

[0013] Specifically, the present invention provides the following embodiments:

[0014] 1. A nanobody or an antigen-binding fragment thereof, comprising CDR1 as shown in SEQ ID NO:2, CDR2 as shown in SEQ ID NO:3, and CDR3 as shown in SEQ ID NO:4.

[0015] 2. The nanobody or its antigen-binding fragment according to Project 1, comprising the amino acid sequence shown in SEQ ID NO:1.

[0016] 3. The nanobody or its antigen-binding fragment according to Project 1 or 2 further comprises an Fc domain, preferably an IgG1 Fc domain, more preferably a human IgG1 Fc domain, wherein the human IgG1 Fc amino acid sequence is shown in SEQ ID NO:5.

[0017] 4. A biepisode antibody (e.g., in N-terminal to C-terminal order) comprising nanobody aRBD-2 and any one of items 1-3, wherein the amino acid sequence of said aRBD-2 is as shown in SEQ ID NO:6;

[0018] Preferably, the nanobody aRBD-2 and the nanobody or antigen-binding fragment of any one of items 1-3 are connected by a linker (e.g., a flexible polypeptide chain, such as a GS linker, preferably a (GGGGS)4 linker).

[0019] 5. A polynucleotide encoding a nanobody or antigen-binding fragment thereof according to any one of items 1-3 or a biepisode antibody according to item 4.

[0020] 6. An expression vector comprising the polynucleotides described in item 5.

[0021] 7. A host cell comprising the expression vector described in item 6, wherein the host cell is a host cell for expressing a foreign protein, such as a bacterial, yeast, insect cell, or mammalian cell.

[0022] 8. A pharmaceutical composition comprising any one of items 1-3, a nanobody or an antigen-binding fragment thereof, or a biepisode antibody as described in item 4, and a pharmaceutical carrier.

[0023] 9. The use of any one of the nanobodies or antigen-binding fragments thereof described in Items 1-3 or the dual epitope antibody described in Item 4 in the preparation of kits or medicines for the prevention, treatment and / or diagnosis of SARS1 and / or SARS2 infection.

[0024] 10. As described in Item 9, the SARS2 includes WT SARS2, Alpha, Beta, Gamma, Kappa, Lambda, Delta, Delta plus, and Omicron mutant strains.

[0025] Advantages and positive effects of this disclosure

[0026] The nanobody aSA3 described in this disclosure can bind to WT SARS2 and SARS1 RBD with high affinity K. D The values ​​are 5.19 x 10 -11 M and 3.61x10 -10 M, aSA3 can block the binding between viral RBD and ACE2. Fusion of aSA3 with human IgG1 Fc increases affinity for WT SARS2 and SARS1 RBDs. D The values ​​are 7.64 x 10 -12 M and 8.54x10 -11 M.

[0027] The nanobody fused with IgG1 Fc described in this disclosure can potently neutralize SARS1, SARS2, and their mutant strains in vitro. The half-maximal inhibitory concentration (IC50) for neutralizing SARS1, WT SARS2, Beta, Delta, Omicron BA.1, and BA.1.1 mutant strains is [not specified in the original text]. 50 The concentrations were 6.78, 26.56, 40.22, 13.32, 127.2, and 97.87 ng / mL, respectively.

[0028] The nanobody aSA3 fused with Fc described in this disclosure, administered intraperitoneally at a single dose of 10 mg / kg, significantly reduced viral titers in the trachea and lungs of hamsters infected with the Omicron BA.1 mutant strain.

[0029] The nanobody aSA3 fused with Fc and then fused with another nanobody aRBD-2 targeting receptor RBD binding motif (RBM) as described in this disclosure forms a dual epitope antibody aRBD2-aSA3-Fc. aRBD2-aSA3-Fc can bind to the RBD of WT SARS2, Omicron BA.1, and BA.2 mutant strains with higher affinity, and can strongly neutralize WT SARS2, Beta, Delta, Omicron BA.1, Omicron BA.1.1, and Omicron BA.2 mutant strains, neutralizing the IC50 of these viruses. 50 The dose is as low as single digit nanograms per milliliter; intraperitoneal injection of aRBD2-aSA3-Fc can also be effective against Omicron BA.1 infection in hamsters. In addition, low-dose intranasal administration of aRBD2-aSA3-Fc can effectively protect hamsters from Omicron BA.1 infection. Attached Figure Description

[0030] Figure 1The present disclosure discloses the screening, preparation, and characterization results of the nanobody aSA3's ability to block ACE2 and bind to RBD. (A) Monoclonal phage ELISA detection of the binding of the nanobody aSA3 to SARS1 and WT SARS2 RBDD (left) and SDS-PAGE electrophoresis results of the nanobody aSA3 and its Fc fusion protein aSA3-Fc (right); (B) ELISA detection of the binding of aSA3-Fc to SARS1, WT SARS2, and their mutant RBDs; (C) Competitive ELISA detection of the blocking effect of aSA3 on ACE2 binding to SARS1 and WT SARS2 RBDs; (D) and (E) SPR detection of the affinity of aSA3 to WT SARS2 and SARS1 RBDs; (F), (G), (H), and (I) SPR detection of the affinity of aSA3-Fc to WT SARS2, SARS1, Omicron BA.1, and Omicron BA.2 RBDs.

[0031] Figure 2 The in vitro neutralization titers of aSA3-Fc against SARS1, SARS2, and their mutant strains. (A), (B), (C), and (D) represent the neutralization results of aSA3-Fc against SARS1, BA.1, BA.1.1, and BA.2 pseudoviruses, respectively; (E), (F), (G), and (H) represent the neutralization results of aSA3-Fc against WT SARS2, Beta, Delta, and Omicron BA.1 authentic viruses, respectively; error bars represent the standard deviations from two independent replicates, and curves were obtained using Prism software fitting. (I) Virus neutralization IC50 50 Value summary. ND indicates no neutralizing activity detected, NT indicates no detection.

[0032] Figure 3 Animal experiments were conducted to detect the anti-Omicron BA.1 efficacy of aSA3-Fc and aRBD2-aSA3-Fc in hamsters. (A) Experimental protocol; (B) Viral RNA copy number in the respiratory tract and left and right lungs of hamsters infected with Omicron BA.1 4 days after infection in the control group, prophylactic intraperitoneal injection group, and therapeutic intraperitoneal injection group; (C) Viral titers in the respiratory tract and left and right lungs of hamsters infected with Omicron BA.1 4 days after infection in different groups. * indicates statistical significance (P < 0.05).

[0033] Figure 4Crystal structure and analysis results of the aSA3-SARS1 RBD complex. (A) Cartoon representation of the aSA3-SARS1 RBD complex structure; (B) Binding site of aSA3 to SARS1 RBD, with the residues above the circle indicating the binding sites of aSA3 to SARS1 RBD.

[0034] Figure 5 Affinity characterization and virus neutralization results of the biepisode antibody aRBD2-aSA3-Fc. (A), (B), and (C) show the affinity results of aRBD2-aSA3-Fc binding to WT SARS2, BA.1, and BA.2RBD, respectively, using SPR detection; (D) shows the affinity K. D Value summary; (E) Results of neutralization of SARS1, BA.1, BA.1.1 and BA.2 pseudoviruses by aRBD2-aSA3-Fc; (F) Results of neutralization of WT SARS2, Beta, Delta and BA.1 true viruses by aRBD2-aSA3-Fc; (G) Results of virus neutralization IC 50 Values ​​summary.

[0035] Figure 6 The preventive effect of .aRBD2-aSA3-Fc intranasal administration on Omicron BA.1 infection in hamsters. (A) Experimental design; (B) Hamster weight changes; (C) Viral RNA copy number in the respiratory tract and left and right lungs of hamsters infected with Omicron BA.1 3 days after infection in the control group and different dose intranasal administration groups; (D) Viral titer in the respiratory tract and left and right lungs of hamsters infected with Omicron BA.1 3 days after infection in the control group and different dose intranasal administration groups. * indicates a significant difference (P < 0.05), ** indicates a significant difference (P < 0.01), and *** indicates a significant difference (P < 0.001). Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0037] Screening, preparation, ACE2 blocking ability detection, and RBD binding affinity characterization of the nanobody aSA3 described in Example 1

[0038] 1) Construction of RBD immune library

[0039] SARS2 RBD (QKV42562.1, aa 321-591) purified and expressed in HEK293F cells (ATCC, CBP60437) was mixed with Freund's adjuvant and administered subcutaneously to immunize alpacas three times at two-week intervals. Two 6-month-old female alpacas were immunized. Two weeks after the third immunization, blood was collected intravenously and leukocytes were separated. Total RNA was extracted using an OmegaBioTek RNA extraction kit, and genomic DNA was removed using DNase. Takara's PrimeScript RNA extraction kit was used. TM II. The 1st Strand cDNA Synthesis Kit was used to reverse transcribe RNA into cDNA. Using alpaca VHH-specific primers, the cDNA was used as a template for PCR amplification to obtain the VHH coding gene fragment. The amplified VHH sequence was cloned into the NcoI and NotI sites of phageparticle pR2 (MRC Laboratory of Molecular Biology) using Gibson assembly. The resulting Gibson assembly product was the initial nanobody phageparticle library. *E. coli* TG1 (MRC Laboratory of Molecular Biology) competent cells were prepared using the 10% glycerol washing method. The Gibson assembly product was then electroporated into the TG1 competent cells and plated on five 150 mm² LB agar plates (containing 2% glucose and 100 μg / mL ampicillin) to amplify the phageparticle library. After scraping, an appropriate amount of bacterial culture was inoculated into 200 mL of 2TY (containing 2% glucose and 100 μg / mL ampicillin) and cultured to the logarithmic growth phase. 10 μg / mL ampicillin was added to the agar plate. 12 PFU-modified KM13 helper phage (MRC Laboratory of Molecular Biology) was inoculated at 37°C for 45 minutes. 100 mL of the bacterial culture was centrifuged, and the cells were resuspended in 200 mL of 2TY (containing 0.1% glucose, 100 μg / mL ampicillin, and 50 μg / mL kanamycin). The cells were then incubated at 25°C for 20 hours to amplify phages displaying nanobodies. The phages were concentrated using PEG precipitation, resuspended in PBS, and stored on ice.

[0040] 2) Screening of nanobody aSA3

[0041] SARS1 RBD (YP_009724390.1, aa308-577) was expressed and purified using HEK293F cells (ATCC, CBP60437). The RBD was diluted to 0.1 mg / mL with PBS, and 100 μL was added to one well of a 96-well Nunc Maxsorp plate. The plate was incubated overnight at 4°C, with one control well containing no antigen. After washing three times with PBS, 300 μL of MPBS (PBS containing 5% skim milk) was added to each well, and the plate was blocked at room temperature for 2 hours. After washing three times with PBS, 1 x 10⁻⁶ MPBS was added to each well. 11 Phage libraries were prepared using PFU (dissolved in 100 μL MPBS) and incubated at room temperature for 1 hour at 80 rpm. The phages were washed 30 times with PBST (0.1% Tween 20). 100 μL of 0.5 mg / mL trypsin was added to each well, and the mixture was digested at room temperature for 1 hour. The phages bound to the wells were eluted; these were the first round of eluted phages. The eluted phages were then used to infect 3 mL of logarithmic-phase TG1 bacteria, incubated at 37°C for 45 minutes, and spread onto a 150 mm LB agar plate (containing 2% glucose and 100 μg / mL ampicillin) and incubated overnight at 37°C. Add 4 mL of 2TY to a 150 mm plate, scrape off the colonies, mix the bacterial solution, and inoculate 100 μL into 100 mL of 2TY (containing 2% glucose and 100 μg / mL ampicillin). Incubate to the logarithmic growth phase, then add KM13 for infection to complete the first round of elution and phage amplification. Subsequently, dilute SARS-1 RBD to 0.02 mg / mL with PBS, add 100 μL to one well of a 96-well immunoassay plate, and incubate overnight at 4°C. Include one antigen-free control well. Wash three times with PBS, adding 300 μL of MPBS (PBS containing 5% skim milk) to each well and blocking at room temperature for 2 hours. Wash three times with PBS, adding 1 x 10⁻⁶ MPBS to each well. 9 The first round of amplification using PFU resulted in the elution of phage (dissolved in 100 μL MPBS), followed by incubation at 80 rpm for 1 hour at room temperature. The phage was then washed 30 times with PBST (0.2% Tween 20). 100 μL of 0.5 mg / mL trypsin was added to each well, and digestion was performed at room temperature for 1 hour, eluting the phage bound to the wells, thus completing the second round of selection. The eluted phage was then used to infect TG1 cells and plated on LB agar plates (containing 2% glucose and 100 μg / mL ampicillin), and cultured for 12 hours to form clones.

[0042] Forty-seven single clones were randomly selected from the phage clones eluted in the two rounds of screening and inoculated into 96-well cell culture plates containing 100 μL of 2TY medium (containing 2% glucose and 100 μg / mL ampicillin) per well, one clone per well, and cultured at 37°C with shaking at 250 rpm for 12 hours. More than 5 μL of the bacterial culture was transferred to a new 96-well plate containing 200 μL of 2TY medium (containing 2% glucose and 100 μg / mL ampicillin) per well for further culture (the remaining bacterial culture was added to a final concentration of 15% glycerol and stored at -80°C), and cultured at 37°C with shaking at 250 rpm for 1.5 hours until the OD600 reached approximately 0.5. 100 μL of the bacterial culture was aspirated from each well. 50 μL of a solution containing 4 × 10⁴ phosphate groups was added to each well. 8 Mix PFU KM13 with 2TY, incubate at 37°C for 45 minutes. Centrifuge at 3500g for 10 minutes, discard the supernatant, and resuspend the pellet in 200 μL of 2TY containing 0.1% glucose, 100 μg / mL ampicillin, and 50 μg / mL kanamycin. Incubate at 25°C with shaking at 250 rpm for 20 hours. Centrifuge at 3500g for 10 minutes, transfer 75 μL of the supernatant to each well of a 96-well plate containing 225 μL of MPBS, mix well, and store temporarily at 4°C. The preparation of the monoclonal phage is now complete.

[0043] SARS2 RBD protein (QKV42562.1, aa 321-591) and SARS1 RBD protein (YP_009724390.1, aa 308-577) were diluted to 1 μg / mL with PBS. 100 μL of each protein was added to each well of a 96-well immunoassay plate. A blank control (PBS wells) was also included. The plates were incubated overnight at 4°C. After washing three times with PBS, 300 μL of MPBS was added to each well, and the plates were blocked at room temperature for 2 hours. 100 μL of the prepared phage-MPBS mixture was added to each well, and the plates were incubated at room temperature for 1 hour. The plates were washed four times with PBST. HRP-anti-M13 antibody (Sino-Pharmaceutical) was diluted appropriately with MPBS, and 100 μL was added to each well of the immunoassay plate. The plates were incubated at room temperature for 1 hour. The plates were washed four times with PBST. Add 100 μL of TMB substrate (Beyond) to each well, wrap with aluminum foil to protect from light, and react at room temperature for 5 minutes. Stop the reaction by adding 50 μL of 1M H₂SO₄ to each well and measure the OD. 450nm Value. All ODs 450nm Positive clones with a value greater than 1 were sent to the company for sequencing. The sequencing results were analyzed and compared to obtain the aSA3 sequence. The single-clone phage ELISA results are as follows: Figure 1 As shown in A (left).

[0044] 3) Preparation of nanobody aSA3 and its Fc fusion protein

[0045] Primers were designed to fuse the N-terminus of the nanobody aSA3 gene sequence with an IFNα protein signal peptide to guide secretory expression, and the C-terminus with human IgG1 Fc. A TEV restriction site or a (GGGGS)3 adapter was introduced between these components. The resulting compound was then cloned into the mammalian expression vector pTT5 (NRC Biotechnology Research Institute). The constructed vector was transiently transfected into mammalian HEK293F (ATCC) cells using PEI. After 3 days of culture, the supernatant was collected, and the fusion protein was purified using a Protein A column and subjected to SDS-PAGE electrophoresis. The results are shown below. Figure 1 As shown in right A, we obtained high-purity aSA3-Fc fusion protein from the supernatant. The aSA3-Fc fusion protein with the TEV site was digested with TEV (with a 6His tag). The digestion products were then passed through Protein A and a nickel column to remove incompletely digested protein, Fc, and TEV enzyme, respectively. The collected flow-throughs were concentrated and subjected to SDS-PAGE electrophoresis. The results are shown in Figure A. Figure 1 As shown in A (right), we obtained high-purity aSA3 nanobody protein from the flow-through.

[0046] The binding of the Fc fusion protein of the nanobody aSA3 to SARS2 and SARS1 RBDs was preliminarily characterized using a non-competitive ELISA: SARS2 and SARS1 RBDs were diluted to 2 μg / mL with PBS, and 100 μL was added to each well for coating. After routine washing and blocking, serially diluted aSA3-Fc fusion protein and ACE2-Fc protein (secretory expression of human ACE2 aa19-615 fragment fused with human IgG1 Fc in HEK293F cells, followed by purification with Protein A) were added sequentially and incubated at room temperature for 1 hour. After washing, HRP-conjugated anti-IgG1 Fc antibody (Beijing Yiqiao Shenzhou) was added to detect the binding of aSA3-Fc and ACE2-Fc. The results are as follows: Figure 1 As shown in C, aSA3-Fc has a higher affinity for both SARS2 and SARS1 RBDs than ACE2-Fc. The EC50 of aSA3-Fc binding to SARS2 and SARS1 RBDs... 50 The values ​​are 0.69 and 2.24 nM, respectively.

[0047] 4) Blocking function and binding characterization of nanobody aSA3 and its Fc fusion protein

[0048] The binding of the Fc fusion protein of the nanobody aSA3 to different SARS1, SARS2, and their mutant RBDs was characterized using ELISA: RBD proteins of SARS1, SARS2, and their mutants (Beijing Yiqiao Shenzhou) were diluted to 2 μg / mL with PBS, and 100 μL was added to each well for coating. After washing and blocking, aSA3-Fc fusion protein solution diluted 1:3 was added, and the mixture was incubated at room temperature for 1 hour. After washing, HRP-conjugated anti-human IgG1 Fc secondary antibody (Beijing Yiqiao Shenzhou) was added. After incubation for 1 hour, followed by washing, TMB was added for color development, and sulfuric acid was added to stop the reaction. The OD450 value was measured, and the OD450 value and concentration were fitted and analyzed. The results are shown below. Figure 1 As shown in Figure B, aSA3-Fc can bind to SARS1 RBD and WT SARS2 RBD with high affinity, and bind to EC 50 The values ​​were 0.870 and 0.618 nM, respectively; the binding EC50 of aSA3-Fc to the RBD of Alpha, Beta, Gamma, Kappa, Lambda, Delta, and Delta plus mutant strains was 0.870 and 0.618 nM, respectively. 50 The value and its combination with WT RBD EC 50 The values ​​were similar, but the binding EC to RBD of the Omicron BA.1 mutant strain was different. 50 The increase to 1.312 nM suggests that the binding site of aSA3 to RBD may be located at the mutation site of Omicron BA.1.

[0049] The blocking function of the screened nanobodies was characterized using a competitive ELISA method. Tandem repeats of SARS2 RBD (i.e., SARS2 RBD-tr2) and SARS1 RBD (i.e., SARS1 RBD-tr2) (Anhui Zhifei Biotechnology) were diluted to 1 μg / mL with PBS, and 100 μL was added to each well for coating. After routine washing and blocking, ACE2-Fc was diluted to 10 nM, and then the Fc fusion protein of nanobody aSA3 was serially diluted 1:4 using this ACE2-Fc solution. 100 μL of each gradient mixture was added to the wells coated with the antigen and incubated at room temperature for 1 hour. After washing four times, HRP-conjugated anti-IgG1 Fc antibody (Beijing Yiqiao Shenzhou) was added to detect the bound ACE2-Fc. The results are as follows: Figure 1 As shown in Figure C, aSA3 can effectively block the binding of ACE2-Fc to SARS2 and SARS1 RBDs, IC 50 The values ​​are 2.567 and 0.433 nM, respectively.

[0050] The affinity between the nanobody aSA3 and its Fc fusion protein and SARS2 and SARS1 RBDs was characterized using SPR (Spatial Resonance Processing). SARS2 and SARS1 RBD proteins were dissolved in sodium acetate at pH 4.5 and coupled to one channel of a CM5 chip, with a control channel uncoupled from the protein. The chips were then blocked with ethanolamine. The nanobody and its Fc fusion protein were diluted 1:1 with PBS and then flowed through both channels at a rate of 30 μL / min, with the reactivity index (RU) measured. After one cycle, the bound antibody was washed away with 50 mM NaOH to regenerate the chip. All operations were performed on a Biacore system, and the results were analyzed using the Biacore evaluation program. Figure 1 As shown in DI, the affinity K for aSA3 to bind to SARS2 and SARS1 RBDs D The values ​​were 51.9 and 361 pM, respectively; the affinity K for the aSA3 Fc fusion protein to bind to SARS2 and SARS1 RBDs was 51.9 and 361 pM, respectively. D The values ​​were 7.64 and 85.4 pM, respectively; consistent with the ELISA results, the affinity of aSA3-Fc for Omicron BA.1RBD decreased to K. D The value was 1.88 nM, and the affinity for Omicron BA.2RBD decreased to K. D The value is 5.52 nM.

[0051] Example 2 characterizes the in vitro neutralizing efficacy of the Fc fusion protein of the nanobody aSA3 against SARS1, SARS2, and mutant strains.

[0052] The neutralizing efficacy of aSA3-Fc, the control antibody Sotrovimab (produced by GlaxoSmithKline, obtained from Beijing Institute of Biotechnology), and Nb21-Fc (a nanobody Nb21 expressed in tandem with human IgG1 Fc; the Nb21 antibody sequence can be found in the published paper: Science. 2020 Dec 18; 370(6523):1479-1484.) against SARS-1 was tested using a pseudovirus neutralization assay. ACE2-293T cells (Shenzhou Institute of Biomedical Technology) were used at 2 x 10⁻⁶ cells per well. 4Cells were seeded in 96-well plates and cultured overnight. aSA3-Fc cells were serially diluted with DMEM containing 10% FBS, followed by the addition of an equal volume of a solution containing SARS-1 pseudovirus (obtained from Beijing Institute of Biotechnology, with a viral load of 1,500,000 RLU). The cells were incubated at 37°C for 1 hour, the cell culture supernatant was removed, and 100 μL of a pseudovirus-antibody mixture was added. The cells were then cultured for 3 days. Cells were lysed and chemiluminescent buffer was added. The intensity of firefly fluorescence was detected using a microplate reader, the inhibition rate was calculated, and the results were fitted using Prism software to obtain the IC50. 50 Value. Result as follows Figure 2 As shown in A and I, aSA3-Fc can effectively neutralize SARS-1 pseudovirus, IC 50 The value was 6.78 ng / mL, with a neutralizing potency 3.9 times that of the control antibody Sotrovimab. Nb21-Fc, which cannot bind to the SARS1 RBD, did not show neutralizing activity. Using a similar method, aSA3-Fc showed potent neutralization of Omicron BA.1 and BA.1.1 pseudoviruses (obtained from the Beijing Institute of Biotechnology), with an IC50 value of 6.78 ng / mL, indicating a neutralizing effect 3.9 times greater than that of the control antibody Sotrovimab. Nb21-Fc, which cannot bind to the SARS1 RBD, did not show neutralizing activity. 50 The concentrations were 127.2 and 97.87 ng / mL, respectively. The neutralizing potency of aSA3-Fc against BA.1 was comparable to that of Sotrovimab, with the neutralizing potency against BA.1.1 being approximately twice that of Sotrovimab; however, aSA3-Fc exhibited lower neutralizing activity against BA.2 pseudoviruses, with an IC50 value of 127.2 and 97.87 ng / mL. 50 The concentration was approximately 5.7 μg / mL, with a neutralizing activity 7.7 times lower than that of Sotrovimab; Nb21-Fc lost its neutralizing activity against the Omicron mutant. Figure 2 BD and I).

[0053] The neutralizing efficacy of aSA3-Fc and the control antibody Nb21-Fc against SARS-2 and its Beta, Delta, and Omicron BA.1 variants was assessed using a plaque reduction neutralization assay. This experiment was conducted in a biosafety level 3 laboratory, using Vero E6 cells (ATCC) at 1.5 x 10⁻⁶ cells per well. 5Antibody-virus antibodies were inoculated into 24-well plates and cultured overnight. aSA3-Fc cells were serially diluted with DMEM containing 2.5% FBS. An equal volume of 600 PFU / ml SARS-2 and its variants (Wuhan Institute of Virology, Chinese Academy of Sciences) was then added, and the antibody-virus mixture was incubated at 37°C for 1 hour. The mixture was then added to the surface of Vero cells. Cells with only virus and no antibody served as a negative control. After incubation at 37°C for 1 hour, the antibody-virus mixture was removed, and DMEM containing 2.5% FBS and 0.9% carboxymethyl cellulose was added. After 3 days of culture, the plates were fixed with 8% paraformaldehyde, stained with 0.5% crystal violet, and the number of plaques was detected. The inhibition rate was calculated, and the results were fitted to obtain the IC50. 50 Value. Result as follows Figure 2 As shown in EI, the IC50 of aSA3-Fc neutralizes live viruses from the original SARS2, Beta, Delta, and Omicron BA.1 mutant strains. 50 The concentrations were 26.56, 40.22, 13.32, and 32.44 ng / mL, respectively. Although the control antibody Nb21-Fc was 5.9 and 5.4 times more potent than aSA3-Fc in neutralizing WT SARS2 and Delta, respectively, it could not neutralize Beta and Omicron BA.1 mutant strains.

[0054] Example 3: In vivo characterization of the preventive and therapeutic effects of the Fc fusion protein of the nanobody aSA3 on hamsters infected with the Omicron mutant strain.

[0055] This experiment was conducted in a biosafety level 3 (P3) laboratory to evaluate the preventive and therapeutic effects of antibodies against COVID-19 infection in Syrian golden hamsters (procedure steps are described in [link to procedure]). Figure 3 A). To assess the preventive effect, the antibody (10 mg / kg dose, total volume 100 μL) was first injected into the peritoneal cavity of hamsters (Wuhan Institute of Biological Products). 24 hours later, the hamsters were anesthetized, and 1 x 102 antibodies were administered via nasal infection. 4 Hamsters were given PFU Omicron BA.1 live virus and then fed for 4 consecutive days before euthanasia and necropsy. Tracheal and left and right lung tissues were collected and analyzed for viral RNA copy number and live virus titer. Each treatment group contained 5 hamsters, and the untreated control group contained 6 hamsters.

[0056] Viral RNA copy number was detected using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. Viral titer was detected using a plaque assay. The supernatant from the centrifuged animal tissue was serially diluted 10-fold with DMEM containing 2.5% FBS, and cultured in 1.5 x 10⁻⁶ ppm solution. 5In 24-well plates containing Vero E6 cells (ATCC), cells were incubated at 37°C for 1 hour, then the supernatant was aspirated, and the cells were cultured in DMEM containing 2.5% FBS and 0.9% carboxymethyl cellulose for 3 days. Cells were fixed with 8% paraformaldehyde, stained with 0.5% crystal violet, and the number of plaques was counted to calculate the viral titer. Results are as follows: Figure 3 B and Figure 3 As shown in Figure C, the results indicated that prophylactic and therapeutic administration of 10 mg / kg aSA3-Fc (single-dose intraperitoneal injection) effectively reduced the viral RNA copy number in the respiratory tract and lung tissue of hamsters four days after infection with Omicron BA.1; more importantly, in the treatment group, live virus was no longer detectable in these tissues, while a large number of live virus remained in the untreated control group.

[0057] Structural analysis of the complex of nanobody aSA3 and SARS1 RBD described in Example 4

[0058] A complex was prepared by incubating aSA3 with SARS1 RBD-tr2 (i.e., tandemly repeated SARS1 RBD) (molar ratio 1.2:1). The complex was purified using a Superdex 200 molecular sieve and concentrated to 20 mg / mL using ultrafiltration. Crystallization conditions were then screened on a large scale using a crystal screening robot with a sitting drop method. High-quality single crystals were obtained using a vapor diffusion hanging drop method. The final crystallization solution was 0.2 M Li acetate, cacodylate, pH 6.5, and 20% PEG6000. The crystals were transferred to antifreeze and stored in liquid nitrogen. Diffraction of the complex crystals was performed at the Shanghai Synchrotron Radiation Facility, and data were collected. Data were processed using XDS and HKL2000. Using published RBD and nanobody structures with similar sequences as templates, the phase was resolved using molecular substitution in the CCP4 program. The complex structure was then refined using WinCoot and Phenix Refine. Based on the structural model, the binding interface was analyzed to reveal the interaction sites. The results are as follows: Figure 4 As shown in A and B, aSA3 mainly binds to the core region of the SARS1 RBD. The sites where SARS1 RBD interacts with aSA3 are L355, Y356, S358, F361, S362, T363, F364, K365, C366, V394, R395, I489, G490, and Y494.

[0059] Example 5 characterizes the affinity and in vitro viral neutralization titer of the biepisode antibody aRBD2-aSA3-Fc.

[0060] Based on the structural information, we fused the N-terminus of aSA3-Fc to the C-terminus of another nanobody, aRBD-2, targeting a different epitope, via a (GGGGS)4 linker, forming a biepisode antibody aRBD2-aSA3-Fc. The linker length allows both aSA3 and aRBD-2 to bind simultaneously to a single RBD. Expression was performed using the pTT5 vector (NRC Biotechnology Research Institute), and purification was achieved with Protein A. Subsequently, we again used SPR to detect the binding affinity of aRBD2-aSA3-Fc to WTSARS2, and the results are as follows. Figure 5 As shown in AD, the affinity K between aRBD2-aSA3-Fc and WT SARS2 RBD D The value was 0.358 pM, 21.3 times higher than that of aSA3-Fc; the affinity K of aRBD2-aSA3-Fc for Omicron BA.1 and BA.2 was... D The values ​​were 306 pM and 894 pM, respectively, which were 6.1 and 6.2 times higher than aSA3-Fc.

[0061] We also used spurious and real viruses to test the neutralizing activity of aRBD2-aSA3-Fc against SARS1 and SARS2 and their mutant strains, such as... Figure 5 As shown in E and G, the pseudovirus ICs of aRBD2-aSA3-Fc and Omicron BA.1, BA.1.1 and BA.2 are neutralized. 50 The concentrations were 4.00, 3.16, and 7.17 ng / mL, respectively, which were 31.8, 31.0, and 793.3 times stronger than aSA3-Fc, respectively. Figure 5 As shown in F and G, aRBD2-aSA3-Fc neutralizes WT SARS2, Beta, Delta, and Omicron BA.1 true viral IC. 50 The concentrations were 10.08, 4.59, 2.57 and 5.32 ng / mL, respectively, which were 2.6, 8.8, 5.2 and 6.1 times stronger than aSA3-Fc.

[0062] Example 6 characterizes the antiviral activity of the biepisode antibody aRBD2-aSA3-Fc in hamsters.

[0063] We first evaluated the preventive and therapeutic protective effects of aRBD2-aSA3-Fc administered via intraperitoneal injection against OmicronBA.1 infection in hamsters. The results are as follows: Figure 3As shown in B and C, intraperitoneal injection of 10 mg / kg aRBD2-aSA3-Fc effectively protected and treated hamsters infected with BA.1. Four days after infection, the viral RNA copy number in the respiratory tract and lung tissue of the treated hamsters decreased, and live virus was undetectable in these tissues, while the untreated control group still had a large amount of live virus. We also examined the preventive effect of aRBD2-aSA3-Fc on Omicron BA.1 infection in hamsters via nasal administration. To assess the preventive effect, four different doses of the antibody (5, 2, 1, and 0.5 mg / kg, 50 μL) were injected into the nasal cavity of anesthetized hamsters (Wuhan Institute of Biological Products). Three hours later, 1 x 102 hamsters were infected via nasal administration. 4 PFU Omicron BA.1 live virus was administered twice, at 24 and 48 hours post-infection. Hamsters were euthanized and necropsy was performed 3 days after infection. Tracheal and left / right lung tissues were collected for viral RNA copy number and live virus titer analysis (Figure 6A). Each dosage group and control group contained 5 hamsters. Results showed that the growth rate of hamsters in all four dosage groups was significantly faster than that in the control group. Figure 6 B), and the viral RNA levels in the respiratory tract and lung tissue of hamsters in all four dosage groups were significantly lower than those in the control group. Figure 6 C), and no live virus was detected in the respiratory tract and lung tissue of hamsters in all four dosage groups. Figure 6 (D). This indicates that even at a dose of 0.5 mg / kg, three intranasal administrations can protect hamsters from infection with Omicron BA.1.

[0064] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Nanobody or antigen-binding fragment thereof that binds to SARS2 and SARS1 RBD, comprising a CDR1 of an amino acid sequence as set forth in SEQ ID NO: 2, a CDR2 of an amino acid sequence as set forth in SEQ ID NO: 3, and a CDR3 of an amino acid sequence as set forth in SEQ ID NO:

4.

2. The Nanobody or antigen-binding fragment thereof of claim 1, having an amino acid sequence comprising an amino acid sequence as set forth in SEQ ID NO:

1.

3. The Nanobody or antigen-binding fragment thereof of claim 1 or 2, further comprising an Fc domain.

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

5. The Nanobody or antigen-binding fragment thereof of claim 3, wherein the Fc domain is a human IgG1 Fc domain, and wherein the human IgG1 Fc amino acid sequence is as set forth in SEQ ID NO:

5.

6. The Nanobody or antigen-binding fragment thereof of claim 4, wherein the Fc domain is a human IgG1 Fc domain, and wherein the human IgG1 Fc amino acid sequence is as set forth in SEQ ID NO:

5.

7. A biparatopic antibody comprising a Nanobody aRBD-2 and the Nanobody or antigen-binding fragment thereof of any one of claims 1-6, wherein the amino acid sequence of the aRBD-2 is as set forth in SEQ ID NO:

6.

8. A biparatopic antibody comprising a Nanobody aRBD-2 and the Nanobody or antigen-binding fragment thereof of any one of claims 1-6 in the order N-terminal to C-terminal, wherein the amino acid sequence of the aRBD-2 is as set forth in SEQ ID NO:

6.

9. The biparatopic antibody of claim 7 or 8, wherein the Nanobody aRBD-2 and the Nanobody or antigen-binding fragment thereof of any one of claims 1-6 are connected by a linker.

10. The biparatopic antibody of claim 9, wherein the linker is a flexible polypeptide chain.

11. The biparatopic antibody of claim 9, wherein the linker is a GS linker.

12. The biparatopic antibody of claim 10, wherein the linker is a GS linker.

13. The biparatopic antibody of claim 9, wherein the linker is a (GGGGS)4 linker.

14. The biparatopic antibody of any one of claims 10-12, wherein the linker is a (GGGGS)4 linker.

15. A polynucleotide encoding the Nanobody or antigen-binding fragment thereof of any one of claims 1-6 or the biparatopic antibody of any one of claims 7-14.

16. An expression vector comprising the polynucleotide of claim 15.

17. A host cell comprising the expression vector of claim 16, wherein the host cell is a host cell for expressing a foreign protein.

18. The host cell of claim 17, wherein the host cell is a bacterial, yeast, insect cell, or mammalian cell.

19. A pharmaceutical composition comprising the Nanobody or antigen-binding fragment thereof of any one of claims 1-6 or the biparatopic antibody of any one of claims 7-14, and a pharmaceutically acceptable carrier.

20. Use of the Nanobody or antigen-binding fragment thereof of any one of claims 1-6 or the biparatopic antibody of any one of claims 7-14 in the manufacture of a kit or medicament for the prevention, treatment and / or diagnosis of SARS1 and / or SARS2 infection.

21. The use according to claim 20, wherein the SARS2 comprises WT SARS2, Alpha, Beta, Gamma, Kappa, Lambda, Delta, Delta plus and Omicron variants.

Images