Antigen binding fragment and antibody for resisting arbecovirus coronavirus as well as preparation method and application of antigen binding fragment and antibody

Abstract

The invention discloses an antigen binding fragment for resisting a sarbecovirus coronavirus, the antigen binding fragment comprises a complementarity determining region, the complementarity determining region comprises a CDR3, and the amino acid sequence of the CDR3 is shown as SEQ ID NO.3. The invention also discloses an antigen binding fragment for resisting the sarbecovirus coronavirus. The invention also discloses an antibody which comprises the antigen binding fragment. The invention also discloses a biological material related to the antigen binding fragment or the antibody, a method for preparing a single-chain antibody, a detection or diagnosis reagent and a pharmaceutical composition. The antigen binding protein and the antibody provided by the invention are broad-spectrum antibodies aiming at coronavirus, not only can effectively neutralize various variants, but also have high affinity and lasting effectiveness, can improve the ability of resisting newly appearing sbecovirus coronavirus strains, and are beneficial to improving the treatment effect and reducing the severe case rate.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to antigen-binding fragments, antibodies, preparation methods, related biological materials, detection or diagnostic reagents and pharmaceutical compositions against sarbecovirus coronavirus. Background Art

[0002] Sarbecovirus is a class of coronaviruses, including SARS-CoV and SARS-CoV-2 (new coronavirus). They mainly infect humans and certain animals and may cause severe respiratory diseases. Sarbecovirus has strong variability, especially in receptor binding and immune escape. The new coronavirus continues to produce new mutants, especially omicron mutants, which show higher infection rates, growth advantages and immune escape characteristics. JN.1 and KP.3 mutants spread rapidly around the world and become new "mutants that need attention". Neutralizing antibody drugs are an effective means to deal with new coronavirus infections, but with the emergence of omicron series mutants, most of the neutralizing antibody drugs on the market have escaped. Although a number of monoclonal antibodies against omicron have entered clinical research, the high production cost of fully human monoclonal antibodies also limits their widespread use.

[0003] The mutation of the new coronavirus causes immune escape, rendering neutralizing antibody drugs ineffective. Nanoantibodies have shown good application prospects in multiple disease areas. Therefore, the development of antibodies that can broadly neutralize sarbecovirus coronaviruses is of great significance for the prevention and treatment of the new coronavirus. Summary of the invention

[0004] The object of the present invention is to provide an antigen-binding fragment and antibody against sarbecovirus coronavirus, so as to effectively neutralize a variety of sarbecovirus coronavirus mutations and improve the ability to resist virus strains.

[0005] To achieve the above-mentioned object, the present invention discloses an antigen-binding fragment against sarbecovirus coronavirus, comprising a complementary determining region, wherein the complementary determining region comprises CDR3, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.3.

[0006] Furthermore, the complementary determining region also includes CDR1, and the amino acid sequence of CDR1 is shown in SEQ ID NO.2.

[0007] Among them, it also includes a framework region, which includes FR1, FR2, FR3 and FR4; the amino acid sequence of FR1 is shown in SEQ ID NO.4; the amino acid sequence of FR2 is shown in SEQ ID NO.5; the amino acid sequence of FR3 is shown in SEQ ID NO.6; and the amino acid sequence of FR4 is shown in SEQ ID NO.7.

[0008] The amino acid sequence of the antigen-binding fragment is as shown in SEQ ID NO.1, or includes a sequence having at least 80% identity with SEQ ID NO.1.

[0009] The present invention also discloses an antibody, characterized in that it comprises the above-mentioned antigen-binding fragment and is a single-chain, bivalent, multivalent, bispecific, multispecific or chimeric antibody.

[0010] The present invention also discloses a biological material related to the above-mentioned antigen-binding fragment or the above-mentioned antibody, characterized in that the biological material is any one of the following: A1. A nucleic acid molecule encoding the above-mentioned antigen-binding fragment or the above-mentioned antibody. A2. An expression frame containing the nucleic acid molecule of the above-mentioned antigen-binding fragment or the above-mentioned antibody. A3. A recombinant vector containing the nucleic acid molecule of the above-mentioned antigen-binding fragment or the above-mentioned antibody. A4. A recombinant cell containing the nucleic acid molecule of the above-mentioned antigen-binding fragment or the above-mentioned antibody. A5. A recombinant cell containing the expression frame of the nucleic acid molecule of the above-mentioned antigen-binding fragment or the above-mentioned antibody. A6. A recombinant cell containing the recombinant vector of the nucleic acid molecule of the above-mentioned antigen-binding fragment or the above-mentioned antibody.

[0011] The present invention also discloses a method for preparing an antibody, comprising the following preparation steps: S1. Use the sarbecovirus coronavirus spike protein to immunize the striped bamboo shark, separate the peripheral blood mononuclear cells of the immunized striped bamboo shark and extract the total RNA, and use the RNA as a template for reverse transcription to prepare cDNA.

[0012] S2. Using the cDNA as a template, PCR amplify the variable region of the single-chain antibody and construct a phage display library.

[0013] S3. Screening the phage display library for phage antibodies specific to the sarbecovirus coronavirus spike protein antigen, and sequencing to obtain the nucleic acid sequence of the antibody.

[0014] S4. Use the nucleic acid sequence to construct an expression vector or expression frame, and transfer it into cells for expression and purification to obtain the single-chain antibody.

[0015] Wherein, the sarbecovirus coronavirus spike protein is SARS-CoV-2 S protein.

[0016] In some embodiments, the construction of the expression vector in step S4 includes construction of a prokaryotic expression vector or expression cassette, which is transformed into Escherichia coli for expression. Alternatively, in other embodiments, the construction of the expression vector includes construction of a eukaryotic expression vector or expression cassette, which is transformed into yeast cells for expression.

[0017] The present invention also discloses a detection or diagnosis reagent, comprising a conjugate and the above-mentioned antigen-binding fragment or the above-mentioned antibody; the conjugate is at least one of an enzyme-labeled conjugate, a fluorescent marker, a chemiluminescent conjugate, a biotin-labeled conjugate, a toxin conjugate, and a radioactive marker.

[0018] The present invention also discloses a pharmaceutical composition, characterized in that it comprises an antibody-drug conjugate and the above-mentioned antigen-binding fragment or the above-mentioned antibody.

[0019] Based on the above scheme, the present invention has the following beneficial effects: 1. The antigen-binding protein and antibody of the present invention are from the striped bamboo shark, with stable physical and chemical properties, acid and alkali resistance, high temperature resistance, and reduced transportation and storage costs; they can be expressed in prokaryotes or yeast, greatly reducing production costs. The antigen-binding protein and antibody of the present invention are broad-spectrum antibodies against coronaviruses, which can not only effectively neutralize multiple variants, but also have high affinity and lasting effectiveness, can improve resistance to emerging virus strains, help improve treatment effects, and reduce the rate of severe illness.

[0020] 2. The antibodies of the present invention can widely bind to SARS-CoV-2 mutants in vitro, including WT, Delta, BA.1, BA.2, BA.4, BF.7, BQ.1.1, XBB.1.5, BA.2.86, JN.1, KP.3, and SARS-CoV-1, and can be used to develop detection and diagnostic reagents against sarbecovirus coronavirus.

[0021] 3. The antibodies of the present invention have broad-spectrum neutralization ability against the currently popular XBB, BA.2.86, JN.1, KP.2, and KP.3, and can neutralize the sarbecovirus family coronaviruses. They can effectively prevent mice from being infected with the new coronavirus by nasal drops in vivo, and can be further developed into a preventive or therapeutic drug composition against sarbecovirus coronaviruses.

[0022] 4. The antibodies of the present invention can be efficiently expressed in prokaryotic systems, and the expression process is standardized and controllable, which can greatly reduce production costs; single-chain antibodies have small molecular weight, stable physical and chemical properties, and good heat resistance, which can greatly reduce the transportation cost of preventive and therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the protein electrophoresis diagram of the purified antibody obtained in Example 1.

[0024] Figure 2-1 to Figure 2-11 This is the binding capacity EC50 test result in Example 2.

[0025] Figure 3-1 to Figure 3-11 This is the affinity KD test result in Example 3.

[0026] Figure 4-1 to Figure 4-11 The neutralization ability IC50 test result of Example 4.

[0027] Figure 5 This is the in vivo protection ability test result of the antigen-binding fragment or antibody of the present invention in Example 5. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and Examples. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0029] Embodiment 1 The present invention discloses an antigen-binding fragment or antibody against sarbecovirus coronavirus, comprising a complementary determining region and a framework region. The complementary determining region comprises CDR1 and CDR3. In this embodiment, the framework region comprises FR1, FR2, FR3 and FR4. The amino acid sequence is shown in Table 1 below: Table 1 Amino acid sequences of antigen-binding fragments or antibodies

[0030] The "variable region" or "variable domain" of an antibody refers to the domain at the amino terminus of the heavy chain or light chain of the antibody that recognizes and binds to the antigen. The composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing the antigen. The heavy chain variable domain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain the antigen binding site. The variable regions of the heavy and light chains are each composed of 2 or 3 complementarity-determining regions (CDRs) (also called hypervariable regions) connected by 4 framework regions (FRs) (2 complementarity-determining regions are obtained in the present invention). The scope of the framework region and CDR has been precisely defined by, for example, IMGT, and any CDR determination method well known in the art, including a combination of methods, can identify the CDRs of the variable domain. The CDRs in each chain are held together closely by FRs to form the variable region. In the present invention, the variable regions VL / VH of the heavy and light chains can be obtained by arranging and connecting the CDRs and FRs with the following numbers in the following combinations: FR1-CDR1-FR2-FR3-CDR3-FR4.

[0031] In other embodiments, the antibody or antigen-binding fragment can be in various forms such as single chain, bivalent, multivalent, bispecific, multispecific and chimeric antibodies. The antigen-binding molecule can be a protein or polypeptide, including single-chain antibodies, single-domain antibodies, various fusions and conjugates, such as immunoconjugates, antibody-drug conjugates (ADCs), multi / bispecific antibodies, chimeric antigen receptors (CARs), etc.

[0032] The amino acid sequence of the antibody or antigen-binding fragment also includes an amino acid sequence in which one or more amino acids of the amino acid sequence of SEQ ID NO.1 are substituted, deleted, or added and which has the same or similar function as the protein shown in SEQ ID NO.1, or a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO.1. The framework region of the antibody, except the complementary determining regions of CDR1 and CDR3, can be replaced with the framework region of antibodies of other species. The term "sequence identity" (also referred to as "sequence identity") of the present invention refers to the amount of the degree of consistency between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Usually, before calculating the percentage of consistency between two amino acid or nucleotide sequences, a sequence alignment is performed and a gap (if any) is introduced. If at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be consistent or matched at that position; if the amino acid residues or bases in the two sequences are different, then it is considered that they are inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence consistency. In other algorithms, the number of gaps and / or the length of the gaps are also taken into account. For the purpose of the present invention, the publicly available alignment software BLAST (available on the webpage ncbi.nlm.nih.gov) can be used to obtain the best sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences by using the default settings. In some embodiments, the "at least 80% sequence identity" of the present invention includes, but is not limited to, at least 85%, at least 90%, or even 100% sequence identity.

[0033] The following is a description of the preparation method of the antigen-binding fragment or antibody of the present invention in conjunction with specific examples.

[0034] 1. Construction of scFv phage library and screening of scFv 1. Antigen preparation The gene sequence encoding the novel coronavirus Omicron spike protein was optimized and synthesized by GenScript, and constructed into the pcDNA3.1 vector, transformed into E. coli clone TOP10, and the plasmid was extracted using an endotoxin removal kit (MeiGen, P1114). 1.5 mg of plasmid was dissolved in 25 ml of 293 culture medium (Sino Biological, M293TII), and 4.5 ml of PEI transfection reagent (1 mg / ml) was dissolved in 25 ml of 293 culture medium. PEI and plasmid were thoroughly mixed and allowed to stand at room temperature for 15 minutes. The plasmid transfection reagent mixture was added to a 4×10 6The expi293 cells were cultured at 37 degrees and 5% CO2 for 4-5 days. The supernatant was collected by centrifugation and filtered with a 0.45μm filter membrane. Affinity purification was performed using TED Ni filler (Tian Di Ren He, SA035100). The specific steps were referred to the filler instructions. The purified S protein was dialyzed in PBS buffer, the antigen purity was detected by SDS-PAGE, the antibody concentration was calculated by A280 determination of absorbance, and the protein was aliquoted and frozen at -80 degrees for later use.

[0035] 2. Shark immunization: Use SARS-CoV-2 S protein to immunize the striped bamboo shark, separate the peripheral blood mononuclear cells of the immunized striped bamboo shark and extract the total RNA, and use the RNA as a template for reverse transcription to prepare cDNA.

[0036] Take 150μg of SARS-CoV-2 S antigen and dilute it to 250μl with PBS, and fully shake and emulsify it in an equal volume of aluminum adjuvant. Inject bamboo sharks subcutaneously and intramuscularly at multiple points, and immunize them alternately with BA.2, BA.4, and BA.7 S antigens, with an interval of 2 weeks. After the last immunization, collect tail vein blood, centrifuge to separate plasma, and separate PBMCs with lymphocyte separation fluid.

[0037] 3. Construction of single-chain phage library: Using cDNA as template, PCR amplified the variable region of single-chain antibody to construct phage display library.

[0038] PBMCs were lysed with trizol, centrifuged at 12000rpm to remove the precipitate, and 200ul of chloroform was added to the supernatant, shaken for 15s, allowed to stand at room temperature for 15 minutes, and centrifuged at 12000rpm for 15min. Carefully aspirate the upper transparent layer, add an equal volume of isopropanol, and precipitate at room temperature for 10min. Centrifuge at 12000rpm for 10min, and wash the precipitate twice with 75% ethanol. Aspirate the ethanol and dissolve the RNA precipitate with DEPC water. cDNA was synthesized using the reverse transcription kit of bio-rad (bio-rad, Cat: 1708891). Using the cDNA as a template and specific primers, the amino acid sequence is as follows: Upstream primer: ATGGCCSMACGGSTTGAACAAACACC Downstream primer: WTTCACAGTCASARKGGTSCC The variable region of the single-chain antibody was amplified using upstream and downstream primers, constructed into the pcantab5e vector, and electroporated into the TG1 competent state. The plate was coated and a 10-fold gradient dilution was performed to calculate the library capacity. The plate was cultured overnight at 37°C, and the colonies on the plate were scraped with a coating rod, resuspended in the culture medium, and glycerol was added at a final concentration of 20%, and stored at -80°C for later use. 48 clones were selected for bacterial liquid PCR identification of the library positive conversion rate and sequencing to verify the antibody diversity. After identification, the positive conversion rate was 100% and the library diversity was 87.2%.

[0039] Take 100 μL of the single-chain antibody phage library prepared in Example 1, inoculate it in 50 mL of 2YT medium containing ampicillin antibiotics and 1% glucose, culture at 37°C to the logarithmic phase, add 20 times the number of helper phage M13K07, mix well, let stand at 37°C for 20 minutes, shake and culture for 30 minutes, centrifuge, discard the medium, add 50 mL of 2YT ampicillin kanamycin medium, shake and culture at 30°C overnight, centrifuge the next day, collect the supernatant, add 1 / 4 volume of PEG / NaCl, precipitate the recombinant phage at 4°C for 2 hours, collect the phage precipitate by centrifugation, dissolve it with 5 mL PBS, repeat the precipitation once, dissolve the phage with PBS, add glycerol with a final concentration of 15%, store it in aliquots at -80°C for use, take 10 μL for 10-fold gradient dilution, infect the logarithmic phase TG1, culture it overnight, and calculate the phage library titer.

[0040] 4. Single-chain antibody screening: Screen phage antibodies with SARS-CoV-2 antigen specificity in the phage display library and sequence them to obtain the nucleic acid sequence of the antibody.

[0041] S antigen was diluted with CBS buffer and coated in a 96-well ELISA plate, 1ug / well, coated at 4℃ for 12 hours, the antigen was discarded, blocking solution (5% skim milk dissolved in PBST) was added, 200 μL / well, blocked at 37℃ for 2 hours, and the plate was washed 4 times with 0.05% PBST. 100 times the storage capacity of recombinant phage was diluted in 100 μL blocking solution, added to the ELISA wells coated with RBD, incubated at room temperature for 2 hours, and the phage was discarded. Washed 10 times with 0.1% PBST, 2 minutes / time, eluted with 100 μL triethylamine for 10 minutes, the eluate was removed, and 1 M Tris-HCl (pH 7.4) was immediately added to neutralize the eluate. The neutralized eluate was added to 3 mL of logarithmic growth phase TG1, left to stand for 30 minutes, and shaken for 30 minutes. Take 10 μL for 10-fold gradient dilution, calculate the storage capacity, and centrifuge the rest. 300 μL of culture medium was reserved to resuspend the bacteria, and then spread on 2YT solid culture medium plates, and inverted and cultured at 37°C overnight. On the second day, the library capacity was calculated for the gradient dilution plates, and the bacteria were scraped off the bacterial library plates with a spreader, resuspended in 3 mL 2YT culture medium, and glycerol was added to a final concentration of 20%, and frozen at -80 for later use to obtain the first round of bacterial strain library. 100 μL of the first round of bacterial strain library was taken in 50 mL of culture medium, shaken and cultured at 37°C until the logarithmic phase, and the first round of recombinant phage library was prepared according to step 3.

[0042] The second and third rounds of screening were carried out according to the above steps. The number of washes was increased by 5 times in each round of screening, and the amount of antigen was reduced by 3 times. A single clone was picked in a 96-well plate and cultured overnight at 37°C. On the second day, 5 μL of bacterial solution was taken in a new 96-well plate (300 μL culture medium / well), cultured at 37°C to the logarithmic phase, and 20 times the amount of helper phage was added. The plate was allowed to stand at 37°C for 30 minutes, and then cultured for 30 minutes. The plate was centrifuged and the culture medium was removed. 300 μL of 2YT ampicillin culture medium was added, cultured overnight at 30°C, and 100 μL of supernatant was taken in an ELISA plate coated with S antigen (50 ng / well) and incubated at room temperature for 2 hours. At the same time, the recombinant phage library was used as a positive control, and the helper phage was used as a negative control. The plate was washed 4 times with PBST, and HRP-labeled anti-M13 antibody (purchased from Sino Biological) was added at 100 μL / well, and incubated at 37°C for 1 hour. The plate was washed 6 times with PBST, and TMB colorimetric substrate (purchased from millipore) was added at 100 μL / well, and incubated at 37°C in the dark for 15 minutes. 50 μL 1M H2SO4 was added to terminate the reaction, and the absorbance at 450 nm was detected. Positive clones were selected for sequencing, and antibodies with different sequences were selected for the next step of expression vector construction and purification.

[0043] 2. Single-chain Antibody Expression and Purification An expression vector or expression cassette is constructed using the nucleic acid sequence, and is transferred into cells for expression and purification to obtain an antigen-binding fragment or a single-chain antibody. Construction of an expression vector includes construction of a prokaryotic expression vector or expression cassette, which is transformed into Escherichia coli for expression. Alternatively, in other embodiments, construction of an expression vector includes construction of a eukaryotic expression vector or expression cassette, which is transformed into yeast cells for expression.

[0044] In this example, prokaryotic expression was used: the positive clones verified by phage elisa were sequenced to obtain antibodies with different sequences. Different antibody sequences were amplified by PCR, with homology arms at both ends. After the PCR products were recovered, they were constructed into the pET28a vector using the homologous recombination method, and the ligated products were added to the competent E. coli TOP 10 and cultured at 37°C overnight. Single clones were picked for colony PCR, and the antibody sequence was verified by sequencing.

[0045] The clones with correct sequencing were inoculated into 10 mL LB kanamycin medium, cultured overnight with shaking, plasmids were extracted, transformed into E. coli expression bacteria Arctic express, and a single clone was picked. Cultured in LB medium until the logarithmic phase, 0.1 mM IPTG was added, and expression was induced at 16°C for 20 hours. The bacterial precipitate was collected, crushed under high pressure, centrifuged, and the supernatant was collected for Ni column affinity purification. The obtained crude extract was then subjected to ion exchange and molecular sieve to finally obtain the antigen-binding fragment or antibody (79C11) of the present invention. The electrophoresis diagram of the purified protein is as follows: Figure 1 As shown, the purity is >95%.

[0046] Embodiment 2 In this example, the antigen-binding fragment or antibody obtained in Example 1 is tested for binding ability.

[0047] The detection process is as follows: the S proteins of different strains (WT strain, early mutant Delta and different mutants of omiron) were diluted in CBS buffer, coated on the ELISA plate, 50 ng / 100 μL / well, incubated at 4°C for 12 hours, the antigen was discarded, and blocking solution (5% skim milk dissolved in PBST) was added, 200 μL / well, blocked at 37°C for 2 hours, washed with 0.05% PBST, 200 μL / well, washed 4 times, 2 minutes / time, discarded the liquid, and patted dry; the 79C11 antibody was diluted in PBST, starting from 100 μg / mL, and diluted 4 times, with a total of 11 dilutions, 100 Add μL / well to the ELISA plate, incubate at 37℃ for 2 hours, wash the plate 4 times with PBST, 2 minutes / time, add HRP-labeled anti-his secondary antibody, incubate at 37℃ for 1 hour, wash the plate 6 times with PBST, 2 minutes / time, shake off the liquid, pat dry, add TMB colorimetric substrate, 100 μL / well, incubate at 37℃ in the dark for 15 minutes, add 50 μL 1M H2SO4 to terminate, detect the absorbance at 450 nm, and calculate the EC50 of antigen-antibody binding.

[0048] Test results: As shown in Figure 2 ( Figure 2-1 to Figure 2-11 ), the results show that the antigen-binding fragment or antibody (79C11) of the present invention has a strong binding ability to different mutant strains.

[0049] Embodiment 3 In this example, the antigen-binding fragment or antibody obtained in Example 1 is subjected to affinity testing.

[0050] The detection process is as follows: the affinity of the antigen-binding fragment or antibody (79C11) obtained in Example 1 to the S protein of different mutant strains is detected by surface plasmon resonance technology (SPR). The experiment was carried out on a Biacore 8K surface plasmon resonance instrument. The CM5 chip was activated using a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The S mutant proteins were diluted in 10 mM sodium acetate buffer at pH 4.5 to 5.5 and coupled to the CM5 chip. The antigen-binding fragment or antibody (79C11) of the present invention was diluted 2-fold from 200 nM to 12.5 nM, the binding phase was 240 seconds, the dissociation phase was 400 seconds, the flow rate was constant at 30 μL / min, and the binding analysis was performed using a multi-cycle kinetic method. The KD data were fitted using the global fitting method using the Biacore Insight evaluation software.

[0051] Test results: As shown in Figure 3 ( Figure 3-1 to Figure 3-11 ), the affinity KD of the antigen binding fragment or antibody (79C11) to the S protein of different mutant strains is shown in Table 2 below. The results show that 79C11 has a strong affinity for different mutant strains.

[0052] Table 2 Affinity KD of antigen-binding fragments or antibodies (79C11) for S protein of different mutants

[0053] Embodiment 4 In this example, the neutralizing ability of the antigen-binding fragment or antibody obtained in Example 1 is tested.

[0054] The detection process is as follows: the antigen-binding fragment or antibody (79C11) obtained in Example 1 is gradiently diluted, starting from 10 μg / mL and diluted 3 times, with a total of 6 dilution gradients, added to a 96-well plate, 60 μL / well, mixed with 650TCID50 pseudoviruses (WH-Hu-1, Beta, Delta, BA.1, BA.2), incubated at 37°C for 2 hours, 100 μL of the antibody-virus mixture is taken and cultured in Huh-7 cells cultured in a 96-well cell plate in advance, and cultured at 37°C 5% CO2 for 24 hours, and the IC50 of the antibody inhibiting viral infection is calculated by luciferase activity.

[0055] Inhibition rate %=(1-(average RLU of sample-average RLU of blank control) / (average RLU of negative control-average RLU of blank control))×100%.

[0056] The half-maximal inhibitory concentration (IC50) was calculated using 4-parameter logistic regression (GraphPad Prism).

[0057] Test results: As shown in Figure 4 ( Figure 4-1 to Figure 4-11 ), the IC50 of the antigen-binding fragment or antibody (79C11) is shown in Table 3. The results show that 79C11 has strong neutralizing ability against the original strain WT of the new coronavirus, the early mutant strain Delta and different mutant strains of omiron, and can neutralize the sarbecovirus family coronavirus SARS-CoV-1 and Pangolin CoV.

[0058] Table 3 IC50 of antigen-binding fragments or antibodies (79C11)

[0059] Embodiment 5 In this example, the antigen-binding fragment or antibody obtained in Example 1 was tested for its in vivo protective ability.

[0060] The detection process is as follows: the in vivo protection experiment was conducted in K18-ACE2 transgenic mice (purchased from Saiye). The antigen-binding fragment or antibody (79C11) was administered to mice by intranasal drops at a dose of 10 mg / kg, and a PBS control group was set up at the same time. One hour later, 5 × 104 FFU XBB.1 clinical strain of new coronavirus was administered by intranasal drops for virus attack. The mice were euthanized after three days of feeding, and lung tissues were collected. The FRNT method was used to detect the titer of new coronavirus in the lungs of mice, and the RT-qPCR method was used to detect the viral load in the lungs of mice. Lung tissue sections were sliced ​​and HE staining was performed for lung tissue pathological analysis. The virus attack experiment complies with animal welfare ethics and is conducted in a P3 laboratory.

[0061] Test results: Figure 5 As shown in A, the titer and load of coronavirus in the lungs of mice in the antigen-binding fragment or antibody (79C11) prevention group were significantly lower than those in the PBS control group. Figure 5 As shown in B, obvious pathological damage was observed in the lungs of mice in the PBS control group, while no obvious pathological damage was observed in the 79C11 prevention group. The results indicate that the antigen-binding fragment or antibody (79C11) of the present invention can be used for the emergency prevention and treatment of novel coronavirus infection.

[0062] Embodiment 6 This embodiment discloses a biomaterial related to an antigen-binding fragment or an antibody (79C11), wherein the biomaterial is any of the following: A1. Nucleic acid molecules encoding antigen-binding fragments or antibodies (79C11).

[0063] A2. An expression cassette containing the nucleic acid molecule described in A1.

[0064] A3. A recombinant vector containing the nucleic acid molecule described in A1.

[0065] A4. A recombinant cell containing the nucleic acid molecule described in A1.

[0066] A5. A recombinant cell containing the expression cassette described in A2.

[0067] A6. A recombinant cell containing the recombinant vector described in A3.

[0068] Embodiment 7 This embodiment discloses a detection or diagnostic reagent, including a conjugate and an antigen-binding fragment or antibody (79C11). The conjugate is at least one of an enzyme-labeled conjugate, a fluorescent marker, a chemiluminescent conjugate, a biotin-labeled conjugate, a toxin conjugate, and a radioactive marker.

[0069] Embodiment 8 This embodiment discloses a pharmaceutical composition, including an antibody-drug conjugate and an antigen-binding fragment or an antibody (79C11). A drug conjugate refers to a substance obtained by connecting a bioactive compound fragment (drug molecule) to an antibody or its antigen-binding fragment. In some embodiments of the present invention, the bioactive compound fragment is connected to the targeting portion through a linker. The linker can be broken in a specific environment or under a specific action (such as the action of a lysosomal protease), thereby separating the bioactive compound fragment from the targeting portion or the antibody or its antigen-binding fragment. In some embodiments of the present invention, the linker contains a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present invention, the bioactive compound fragment is directly connected to the targeting portion or the antibody or its antigen-binding fragment through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.

[0070] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. An antigen-binding fragment of an anti-sarbecovirus coronavirus, characterized in that: It comprises a complementary determining region, wherein the complementary determining region comprises CDR3, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.

3.

2. The anti-sarbecovirus antigen-binding fragment of claim 1, characterized in that: The complementary determining region also includes CDR1, and the amino acid sequence of CDR1 is shown in SEQ ID NO.

2.

3. The anti-sarbecovirus coronavirus antigen-binding fragment according to claim 1 or 2, characterized in that: Also includes a framework region, the framework region includes FR1, FR2, FR3 and FR4; The amino acid sequence of FR1 is shown in SEQ ID NO.4; The amino acid sequence of FR2 is shown in SEQ ID NO.5; The amino acid sequence of FR3 is shown in SEQ ID NO.6; The amino acid sequence of FR4 is shown in SEQ ID NO.

7.

4. The anti-sarbecovirus coronavirus antigen-binding fragment of claim 3, characterized in that: The amino acid sequence of the antigen-binding fragment is shown in SEQ ID NO.1, or includes a sequence having at least 80% identity with SEQ ID NO.

1.

5. An antibody, characterized in that: The invention comprises the antigen-binding fragment according to any one of claims 1 to 4, wherein the antibody is a single-chain, bivalent, multivalent, bispecific, multispecific or chimeric antibody.

6. A biomaterial related to the antigen-binding fragment according to any one of claims 1 to 4 or the antibody according to claim 5, characterized in that: The biological material is any of the following: A1. A nucleic acid molecule encoding the antigen-binding fragment of any one of claims 1 to 4, or the antibody of claim 5; A2. An expression cassette containing the nucleic acid molecule described in A1; A3. A recombinant vector containing the nucleic acid molecule described in A1; A4. A recombinant cell containing the nucleic acid molecule described in A1; A5. A recombinant cell containing the expression cassette described in A2; A6. A recombinant cell containing the recombinant vector described in A3.

7. A method for preparing an antibody, characterized in that: The method comprises the following preparation steps: S1. Immunize the bamboo shark with sarbecovirus coronavirus spike protein, separate the peripheral blood mononuclear cells of the bamboo shark after immunization and extract total RNA, and prepare cDNA by reverse transcription using the RNA as a template; S2. Using the cDNA as a template, PCR amplified the variable region of the single-chain antibody to construct a phage display library; S3. Screening the phage display library for phage antibodies specific to the sarbecovirus coronavirus spike protein antigen, and sequencing to obtain the nucleic acid sequence of the antibody; S4. Use the nucleic acid sequence to construct an expression vector or expression frame, and transfer it into cells for expression and purification to obtain the single-chain antibody.

8. The method for preparing an antibody according to claim 7, wherein: The sarbecovirus coronavirus spike protein is SARS-CoV-2 S protein; The constructing of the expression vector in step S4 includes constructing a prokaryotic expression vector or an expression cassette, which is transformed into Escherichia coli for expression; or the constructing of the expression vector includes constructing a eukaryotic expression vector or an expression cassette, which is transformed into yeast cells for expression.

9. A detection or diagnostic reagent, characterized in that: The invention comprises a conjugate and the antigen-binding fragment according to any one of claims 1 to 4 or the antibody according to claim 5; the conjugate is at least one of an enzyme-labeled conjugate, a fluorescent marker, a chemiluminescent conjugate, a biotin-labeled conjugate, a toxin conjugate and a radioactive marker.

10. A pharmaceutical composition, characterized in that: It comprises an antibody-drug conjugate and the antigen-binding fragment according to any one of claims 1 to 4 or the antibody according to claim 5.

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