Peptide NbM9 capable of recognizing and neutralizing MERS-CoV and its application

Abstract

The present invention relates to a polypeptide NbM9 that can recognize and neutralize MERS-CoV, comprising three complementary determining regions (CDR1-3), the sequences of which are shown in SEQ ID NOs: 1-3. The present invention develops nanoantibody drugs targeting MERS-CoV. By preparing the MERS-CoV S protein, immunizing Bactrian camels, and utilizing a phage library platform technology to display nanomonoclonal antibodies, a nanoantibody VHH that specifically binds to MERS-CoV was screened, its CDR sequence was identified, and a humanized VHH-huFc1 was constructed. Simultaneously, a pseudovirus neutralization experiment was used to evaluate the efficacy of NbM9 in treating MERS-CoV infection, providing a potential detection agent and therapeutic drug for the prevention and treatment of MERS-CoV infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine. More particularly, it relates to a polypeptide that can bind to MERS-CoV, and also to the use of the polypeptide in preparing therapeutic drugs and diagnostic agents for MERS-CoV infection. Background Art

[0002] Middle East Respiratory Syndrome (MERS) was first identified in a 60-year-old Saudi Arabian male patient who died in June 2012. The disease has an incubation period of 2 to 14 days and typically presents as an acute respiratory infection with an acute onset and high fever (39-40°C), which may be accompanied by chills, rigors, cough, chest pain, headache, generalized muscle and joint aches, fatigue, and loss of appetite. Currently, there is no available vaccine or specific treatment. Although the disease initially occurred in the Middle East, it has gradually spread to 27 countries across Europe, Africa, Asia, and North America, driven by trade, tourism, and religious activities. Therefore, the development of safe and effective neutralizing antibodies against the MERS pathogen has important public health implications for the prevention and control of this epidemic.

[0003] The pathogen that causes MERS-CoV is MERS-CoV, an enveloped, single-stranded, positive-sense RNA virus belonging to the genus Betacoronavirus (β-CoV). The single-stranded RNA genome of MERS-CoV is approximately 30 kb in size and contains 10 open reading frames (ORFs), encoding 16 nonstructural proteins (nsp1-16) and four structural proteins: spike (S), envelope (E), matrix (M), and nucleocapsid (N). These four structural proteins form spherical, crown-like viral particles, with S and N being highly immunogenic. The S protein is a trimeric type I transmembrane glycoprotein located on the surface of the viral membrane, mediating viral attachment to host cells and viral-cell membrane fusion, and is a major determinant of cellular targeting and pathogenesis. The S protein can induce the production of neutralizing antibodies, which play a key role in preventing MERS-CoV infection. Therefore, immunizing animals with the S protein and screening for neutralizing antibodies would be an effective strategy for treating MERS-CoV infection.

[0004] In 1993, a new type of natural antibody from the camelid family was discovered. This antibody naturally lacks light chains and is composed only of heavy chains. Its heavy chain contains two constant regions (CH2 and CH3), a hinge region, and a variable heavy chain domain (VHH, i.e., the antigen-binding site). The relative molecular mass of this heavy chain variable region is approximately 13 kDa, only 1 / 10 of that of a conventional antibody. Its molecular height and diameter are both at the nanometer level, making it the smallest functional antibody fragment currently available. Therefore, it is also called a nanobody (Nb). Due to the high stability (it will not degrade even at 90°C), high affinity, over 80% homology with human antibodies, and low toxicity and immunogenicity of nanobodies, they have recently been widely used in the development of immunodiagnostic kits, imaging research, and antibody drug development for tumors, inflammation, infectious diseases, and neurological diseases. Summary of the Invention

[0005] The present invention uses camels to immunize them with antigens to produce camel-derived nano-monoclonal antibodies for the detection and treatment of MERS-CoV infection. Based on these studies, the present invention provides a polypeptide that binds to MERS-CoV, comprising three complementary determining regions (CDRs) 1-3, the sequences of which are shown in SEQ ID NOs: 1-3.

[0006] In one embodiment, the polypeptide is a Nanobody.

[0007] In a specific embodiment, the polypeptide further comprises four framework regions FR1-4, and the FR1-4 and the CDR1-3 are staggered in sequence. For example, the FR1-4 sequence can be designed as shown in SEQ ID NO: 4-7 (alpaca source), but the scope of the present invention is not limited thereto. The specific recognition and binding ability of the antibody is mainly determined by the CDR region sequence, and the FR sequence has little effect and can be designed according to the species, which is well known in the art. The FR region sequence of human, mouse or camel origin can be designed to connect the above-mentioned CDR to obtain a nanobody that can bind to MERS-CoV.

[0008] In a specific embodiment, the polypeptide is a camelid VHH or a humanized VHH.

[0009] The present invention also provides the use of the above polypeptide in preparing a detection agent for MERS-CoV or a detection agent for the S protein of MERS-CoV.

[0010] The present invention also provides the use of the above polypeptide in preparing a drug for treating MERS-CoV.

[0011] The present invention also provides a nucleic acid encoding the above polypeptide.

[0012] The present invention also provides the use of the above nucleic acid in preparing a drug for treating MERS-CoV.

[0013] The present invention develops nano-antibody drugs targeting MERS-CoV. By preparing MERS-CoV S protein, immunizing Bactrian camels, and utilizing a phage library to display nano-monoclonal antibodies, etc., a nano-antibody VHH that specifically binds to MERS-CoV was screened, its CDR sequence was identified, and a humanized VHH-huFc1 was constructed. Simultaneously, a pseudovirus neutralization experiment was used to evaluate the efficacy of NbM9 in treating MERS-CoV infection, providing potential detection agents and therapeutic drugs for the prevention and treatment of MERS-CoV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the antiserum titer detection curve one week after the alpaca was immunized with MERS-CoV-S protein for the fourth time.

[0015] Figure 2 The curves of the inhibition of MERS-CoV pseudovirus infection of ghost cells in vitro by antisera of different dilutions one week after the fourth immunization of camels, with the serum before immunization as the control.

[0016] Figure 3 The panning and identification of MERS-VHH phage antibody library, wherein A is the ELISA test statistical chart of the phage library after panning for MERS-CoV-S protein; B is the statistical chart of the first round (1 st ) Second round (2 nd ) and the third round (3 rd ) After panning, 24, 24 and 40 clones were selected from the phage antibody library for phage ELISA detection statistics.

[0017] Figure 4 Figure 3 is a statistical graph of ELISA detection of prokaryotically expressed VHH antibodies. Each point represents a clone. The vertical axis is the OD450 against the S protein / OD450 of the blank control. A ratio greater than 5.0 is defined as positive.

[0018] Figure 5 This is the SPR detection statistical graph of NbM9 antibody.

[0019] Figure 6 This is the OD450 statistical graph of the binding of NbM9 antibodies with different purified concentrations to MERS-S protein detected by ELISA.

[0020] Figure 7 This is the experimental curve of NbM9 antibody neutralizing MERS-CoV pseudovirus infection. DETAILED DESCRIPTION

[0021] 1. Alpaca immunization and acquisition of antiserum

[0022] Alpacas were initially immunized with an emulsified mixture of 250 μg of MERS-S protein and 250 μl of Freund's complete adjuvant, and boosted three times with MERS-S protein and 250 μl of Freund's incomplete adjuvant on days 14, 28, and 42. One week after the second and third immunizations, blood was collected to detect antiserum titers; one week after the fourth immunization, 200 ml of blood was collected for construction of a phage antibody library.

[0023] Antiserum titers were determined by ELISA. Plates were coated with 0.5 μg / ml MERS-S-his protein. 100 μl of serially diluted antiserum or purified antibody (pre-immune camel serum was used as a control) was added to each well. The plates were incubated at 37°C for 1.5 hours and washed twice. A 1:10,000 dilution of horseradish peroxidase-labeled goat anti-Llamma IgG (H+L) secondary antibody was added to each well. The plates were incubated at 37°C for 1 hour. After washing 4-6 times, 100 μl of TMB substrate was added and incubated at 37°C for 10 minutes. The reaction was terminated with 50 μl of 0.2 M H₂SO₄, and the OD₄450nm was measured. The serum titer for ELISA was defined as the highest dilution at which the OD₄450 was at least twice that of the blank control and greater than 0.2.

[0024] The results are as follows Figure 1 As shown, the titers of the antiserum from the four immune systems were 3.28×10 6 This shows that the antigen can induce camels to produce high-titer antiserum specific to MERS-S protein.

[0025] In order to further verify whether the high titer camel antiserum can effectively prevent MERS-CoV virus infection, a neutralization experiment of virus infection was performed. Antiserum and pre-immune serum of different dilution concentrations were incubated with MERS-CoV pseudovirus for 60 minutes, and then transferred to Vero cells. After 48 hours, the viral load was detected by Glo Max chemiluminescence microplate reader (Promega) and the neutralization effect was calculated. The results of the neutralization experiment showed that the antiserum induced by MERS-S protein inhibited 90% of MERS-CoV infection with an ID90 of more than 1000-fold dilution ( Figure 2 ). In summary, MERS-S protein induced high-titer antiserum, and the antiserum had the ability to effectively inhibit MERS-CoV pseudovirus infection.

[0026] 2. VHH phage library construction and panning

[0027] 200 ml of peripheral blood was collected from immunized camels and isolated using lymphocyte separation medium (GE Ficoll-Paque Plus). RNA was extracted according to the TRIzol operating manual and converted to cDNA using oligo(dT). The camel VHH gene was cloned into a phagemid plasmid through primer amplification and molecular cloning techniques. The VHH phage library was then transformed into TG1 bacteria.

[0028] In order to further identify whether the MERS-CoV-VHH phage library was successfully constructed, the VHH target gene of the camel immunized with the MERS-S protein was amplified by PCR. It can be seen that the target band is 450bp, and the size is in line with expectations, indicating that the MERS-CoV-VHH phage antibody library contains VHH genes. 25 clones were selected for sequencing, and 21 clones had target fragments inserted, with an insertion rate of approximately 84%. The sequencing results showed that these 21 clones did not have completely identical repeat sequences, and the library diversity was 100%. The alignment results showed that most of the differential sequences were in the CDR binding region. After testing, the capacity of the constructed CD4-VHH phage antibody library was 1.37×10 9 .

[0029] With the help of M13KO7 helper phage, the phage antibody library was revived using bacteria transformed with VHH-phagemid and precipitated with PEG / NaCl. The phage antibody library was enriched three times with 50 μg / ml of MERS-S-His protein. The enriched phage were eluted, transformed, plated, and single clones were picked for phage binding to CD4 protein ELISA. Clones with binding reads > 1.0 were sequenced and cloned into the expression vector pcDNA3.1. 293tt cells were transfected to express and produce nano-monoclonal antibodies.

[0030] The selected library was tested for binding to the MERS-S protein. The phage ELISA results showed that the binding readout of the CD4-VHH phage library to the CD4 protein before enrichment was 0.78, and the readings of the phage library after one, two, and three rounds of enrichment were 0.97, 2.59, and 3.34, respectively. Figure 3 A). To further verify the positive phage rate of MERS-CoV-VHH protein in the enriched library, 24, 24, and 40 clones were selected from the libraries after rounds 1, 2, and 3 of enrichment, respectively, for single phage ELISA testing. The results showed that 50% of the single phage clones in the second round of library were positive, and 75% of the phage clones in the third round of library were positive, and the Target / Blank ratio was >5 ( Figure 3B) The MERS-CoV-VHH phage library with high binding affinity was successfully enriched by MERS-S protein panning.

[0031] 3. Construction of VHH Prokaryotic Expression Library and VHH Expression

[0032] The 2nd-MERS-CoV-VHH and 3rd-MERS-CoV-VHH phage antibody libraries enriched after the second and third rounds of panning were amplified by PCR. All VHH gene fragments in the antibody library were obtained and purified, and the VHH gene fragments were cloned into a prokaryotic expression vector and transformed into the SS320 strain to construct a prokaryotic expression antibody library of VHH. The prokaryotic expression antibody library was spread on plates and cultured overnight. The next day, 182 monoclonal colonies were randomly selected and the antibody expression supernatant was induced using IPTG. The antibody supernatant was then tested for binding to the S protein by ELISA.

[0033] The results showed that 49 bacterial supernatants bound to the S protein and did not bind to the blank control. The S protein binding reading / blank control reading was greater than 5.0 ( Figure 4 Among them, the antibody NbM9 was screened out, the sequences of CDR1-3 are shown in SEQ ID NOs: 1-3, and the sequences of FR1-4 are shown in SEQ ID NOs: 4-7.

[0034] 4. VHH-huFc eukaryotic expression

[0035] Using molecular cloning techniques, the NbM9 gene was fused to the human Fc gene and inserted into the pCDNA3.4 eukaryotic expression vector to construct the NbM9-huFc-pCDNA3.4 expression plasmid. The constructed NbM9-huFc-pCDNA3.4 plasmid was transfected into 293tt cells to express NbM9-huFc (4NB). Cell supernatants were collected and analyzed by ELISA.

[0036] Affinity test of antibody NbM9 and MERS-S protein was conducted. Affinity was tested using the Fortebio biomolecular interaction platform. The antibody was immobilized onto the Anti-human IgG Fc Capture Biosensors (AHC) probe for 400 seconds, then bound to the antigen CD4-his protein for 180 seconds and 180 seconds, and the binding and dissociation of the antibody and antigen were observed. The instrument fitted the curve and the data were derived. The affinity test results are shown in Table 1. The affinity of most antibodies can reach 10 -12 (picomole level), the binding and dissociation curves are as follows Figure 5 As shown. It can be seen that we have obtained antibodies with high affinity.

[0037] Table 1 NbM9 affinity data

[0038] Clone ID Ka(1 / MS) Kd(1 / S) KD(M) Response(nm) NbM9 3.88E+05 ＜1E-07 ＜1E-12 0.477

[0039] 5. Antibody Serial Dilution ELISA

[0040] The detection plate was coated with 0.5 μg / ml MERS-S protein, 100 μl per well, incubated at 37°C for 2 hours, washed 2-4 times, blocked with 4% bovine serum, 250 μl per well, incubated at 37°C for 1 hour, washed 2-4 times, 100 μl of gradient diluted purified antibody was added to each well, incubated at 37°C for 1.5 hours, washed twice, 100 μl of 1:10000 diluted horseradish peroxidase-labeled anti-human antibody was added to each well, incubated at 37°C for 1 hour, washed 4-6 times, 100 μl of TMB substrate was added, incubated at 37°C for 10 minutes, 50 μl of 0.2M H2SO4 was used to stop the reaction, and OD450nm was measured. The results are as follows. Figure 6 As shown, when the concentration of antibody NbM9 was as low as 0.00188 μg / ml, the ratio of OD450 of its binding to MERS-S protein / OD450 of the blank control was still greater than 2.

[0041] 5. NbM9 neutralizes MERS-CoV pseudovirus

[0042] MERS-CoV pseudovirus was generated by co-transfecting 293T cells (ATCC) with expression vectors expressing firefly luciferase (pNL43R-E-luciferase) and pcDNA3.1 (Invitrogen). Viral supernatant was collected 48 hours later. Viral titer was determined by relative light units of luciferase activity (Bright-Glo Luciferase Assay Vector System, Promega Biosciences). The control monoclonal antibody was the anti-SFTSV antibody SNB02 (1 mg / ml), and in vitro neutralization experiments were performed on NbM9. The antibody was serially diluted to different concentrations and incubated with MERS-CoV pseudovirus at 5% CO2 and 37°C for 1 hour. 1×10 4 Vero cells were cultured in a 5% CO2 incubator at 37°C for 48 hours, and the half-maximal inhibitory concentration (IC50) of the monoclonal antibody was evaluated by detecting the luciferase activity.

[0043] The results are as follows Figure 7 As shown, NbM9 has good neutralizing activity, and the inhibition rate can reach 90% when the antibody concentration is 0.1376 μg / ml.

[0044] From the above experimental results, it can be seen that the antibody NbM9 of the present invention and its humanized form can specifically recognize and bind to MERS-CoV and its S protein, and can neutralize MERS-CoV and block its infection, thereby being used to treat MERS.

[0045] The above description is only a preferred embodiment of the present invention and is 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 in the scope of protection of the present invention.

Claims

1. A nanobody that can bind to MERS-CoV, characterized in that It includes three complementary determining regions CDR1-3, the CDR1 sequence is shown in SEQ ID NO: 1, the CDR2 sequence is shown in SEQ ID NO: 2, and the CDR3 sequence is shown in SEQ ID NO:

3.

2. The Nanobody according to claim 1, characterized in that It also includes four framework regions FR1-4, and the FR1-4 and the CDR1-3 are arranged in a staggered order.

3. The Nanobody according to claim 2, characterized in that The nanobody is a camel-derived VHH or a VHH linked to a human Fc region.

4. Use of the Nanobody according to any one of claims 1 to 3 in the preparation of a detection agent for MERS-CoV or a detection agent for the S protein of MERS-CoV.

5. Use of the Nanobody according to any one of claims 1 to 3 in the preparation of a therapeutic drug for MERS-CoV infection.

6. A nucleic acid, characterized in that Encoding the Nanobody of any one of claims 1 to 3.

7. Use of the nucleic acid according to claim 6 in the preparation of a therapeutic drug for MERS-CoV infection.

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