A nanobody against mers, a nanobody fusion protein and application thereof

Abstract

This invention provides an anti-MERS nanobody, a nanobody fusion protein, and their applications, belonging to the field of antibody drug technology. The anti-MERS nanobody comprises the following amino acid sequences in CDR1, CDR2, and CDR3 regions: CDR1: GSIFSRYT; CDR2: ITSEGNT; CDR3: NAGGGGAYFYHGAS. The nanobody of this invention is obtained through phage display technology and possesses unique CDR partitions, with its antigen recognition epitope located in the RBD region of the S1 protein. The anti-MERS nanobody fusion protein provided by this invention features high expression, high stability, high specificity, high affinity, low immunogenicity, and low-cost large-scale production, making it suitable for industrial production and potentially useful in addressing the MERS pandemic crisis, thus possessing significant application value.

Description

Technical Field

[0001] This invention belongs to the field of antibody drug technology, and particularly relates to an anti-MERS nanobody, recombinant antibody and their applications. Background Technology

[0002] Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus, and cases continue to be reported in some areas. After infection with the MERS virus in humans, clinical manifestations range from asymptomatic or mild respiratory symptoms to severe acute respiratory illness and death. Typical clinical symptoms include fever, cough, and shortness of breath. Pneumonia is also a common clinical manifestation.

[0003] The MERS-CoV spike protein (S protein) plays a crucial role in viral infection and receptor response. This protein consists of two functional subunits: the N-terminal S1 subunit forms a globular head, while the membrane-embedded C-terminal S2 region forms a stem structure. The S protein first interacts with the human cell receptor dipeptidyl peptidase 4 (hDPP4) via its receptor-binding domain (RBD) on the S1 subunit, and then mediates the fusion of the viral envelope with the host cell membrane via the S2 subunit, thereby initiating viral invasion of target cells. Therefore, blocking the interaction between the RBD and hDPP4 using neutralizing antibodies may be an effective therapeutic strategy for MERS-CoV infection. To date, more than 20 antibodies have been developed, most of which target the RBD region of the S protein, interfering with hDPP4 binding through steric hindrance. However, as of now, no vaccine or antiviral therapy is available for the prevention or treatment of MERS-CoV infection.

[0004] Nanobodies (Nbs), as single-domain fragments (named VHH domains) of camel heavy chain antibodies, have gradually gained clinical acceptance as antiviral agents due to their unique molecular size, high specificity, high affinity, and ease of genetic engineering. Antigen-specific nanobodies can be obtained through immune libraries. After obtaining the nanobody library, antibodies are displayed on the surface of bacteriophages using phage display technology, and nanobodies that specifically bind to target antigens are screened. Fusing nanobodies with the Fc fragment of a full-length antibody to construct VHH-Fc antibodies can prolong the half-life of nanobodies in the blood, improve their stability and neutralizing potency, and may become an effective strategy for addressing the potential crisis of the MERS-CoV epidemic. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an anti-MERS nanobody, a recombinant antibody and its application. The specific anti-MERS virus nanobody provided by the present invention has a unique CDR region and can effectively neutralize the MERS virus.

[0006] This invention provides an anti-MERS nanobody comprising the following amino acid sequences in CDR1, CDR2, and CDR3 regions:

[0007] CDR1: GSIFSRYT

[0008] CDR2: ITSEGNT

[0009] CDR3: NAGGGGAYFYHGAS.

[0010] Preferably, the nanobody further includes the following amino acid sequences in the FR1, FR2, FR3, and FR4 framework regions:

[0011] FR1: QVQLVESGGGLVQPGGSLRLSCAAS

[0012] FR2: MGWFRQAPGKQRDFVAR

[0013] FR3: TYSDSVKGRFTISRDNAKNTVYLQMTSLKPEDTAVYYC

[0014] FR4:WGQGTQVTVSS.

[0015] Preferably, the amino acid sequence of the nanobody is shown in SEQ ID No. 1.

[0016] The present invention provides a gene encoding the nanobody, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0017] This invention provides a recombinant antibody, comprising the sequence of the nanobody and the constant region sequence of a human heavy chain antibody.

[0018] The present invention provides a recombinant vector for expressing the nanobody, comprising an initial vector and the gene.

[0019] Preferably, the initial vector is a eukaryotic expression plasmid pcDNA3.1-Fc with a human Fc tag.

[0020] The present invention provides a recombinant cell expressing the nanobody, which is obtained by transferring the recombinant vector into a host cell.

[0021] Preferably, the host cell is an Expi 293F cell or a CHO-S cell.

[0022] This invention provides the use of the nanobody, the gene, the recombinant antibody, the recombinant vector, and the recombinant cell in the preparation of drugs for the prevention and / or treatment of MERS infection.

[0023] Compared with existing technologies, the present invention has the following advantages: The anti-MERS nanobody provided by the present invention has a unique CDR partition, and its antigen recognition epitope is located in the RBD region of the S1 protein. The nanobody in the present invention is obtained by constructing an anti-MERS virus antibody phage library through genetic engineering methods using phage surface display technology, followed by screening; the nanobody fusion protein binds to the S-ECD protein of MERS at the EC... 50 The concentration was 0.5673 ng / mL, and the EC that bound to the RBD protein was... 50 The concentration was 0.7872 ng / mL, indicating that MERS-1 has good binding activity to both MERS S-ECD and RBD. The affinity for MERS RBD was 0.046 nM, indicating high affinity activity. In vitro neutralization activity evaluation results showed an IC50 of 0.7872 ng / mL against MERS pseudovirus. 50 It has a concentration of 4.225 ng / mL and exhibits good neutralizing activity. Attached Figure Description

[0024] Figure 1 The SDS-PAGE spectra of the affinity chromatography-purified nanobody fusion protein MERS-1.

[0025] Figure 2 The HPLC-SEC spectrum of the nanobody fusion protein MERS-1;

[0026] Figure 3 To detect the binding activity of the nanobody fusion protein MERS-1 with MERS S-ECD and RBD proteins using ELISA;

[0027] Figure 4 The affinity of the nanobody fusion protein MERS-1 for the MERS RBD protein;

[0028] Figure 5 To demonstrate the neutralizing activity of the nanobody fusion protein MERS-1 against MERS pseudoviruses. Detailed Implementation

[0029] This invention provides an anti-MERS nanobody comprising the following amino acid sequences in CDR1, CDR2, and CDR3 regions:

[0030] CDR1: GSIFSRYT (SEQ ID NO.4)

[0031] CDR2:ITSEGNT (SEQ ID NO.5)

[0032] CDR3:NAGGGGAYFYHGAS (SEQ ID NO. 6).

[0033] In this invention, the nanobody preferably further includes the following amino acid sequences of the FR1, FR2, FR3 and FR4 framework regions:

[0034] FR1: QVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO.7)

[0035] FR2: MGWFRQAPGKQRDFVAR (SEQ ID NO.8)

[0036] FR3: TYSDSVKGRFTISRDNAKNTVYLQMTSLKPEDTAVYYC (SEQ ID NO.9)

[0037] FR4: WGQGTQVTVSS (SEQ ID NO. 10).

[0038] Furthermore, the amino acid sequence of the nanobody is shown in SEQ ID No. 1, and is as follows:

[0039] QVQLVESGGGLVQPGGSLRLSCAASGSIFSRYTMGWFRQAPGKQRDFVARITSEGNTTYSDSVKGRFTISRDNAKNTVYLQMTSLKPEDTAVYYCNAGGGGAYFYHGASWGQGTQVTVSS; Sequence partitions are shown in Table 1.

[0040] Table 1. Sequence partitioning of nanobodies

[0041]

[0042] The present invention also provides a gene encoding the nanobody, the nucleotide sequence of which is shown in SEQ ID No. 2, specifically as follows: CAGGTGCAGCTGGTGGAGAGCGGCGGCGGACTGGTGCAGCCAGGAGGAAGCCTGAGACTGTCCTGCGCCGCCAGCGGCTCCATCTTCAGCAGATACACAATGGGCTGGTTTAGGCAGGCCCCTGGCAAGCAGAGAGATTTCGTGGCCAGAATCACAAGCGAGGGCAACACCACCTACTCCGACAGCGTGAAGGGCAGATTCACCATCTCCAGAGATAATGCCAAGAACACCGTGTACCTGCAGATGACAAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGTAACGCCGGCGGCGGCGGCGCCTACTTTTACCACGGCGCCTCCTGGGGCCAGGGCACACAAGTGACAGTGAGCAGC, and the sequence partitioning is shown in Table 2.

[0043] Table 2. Sequence partitioning of nanobody encoding genes

[0044]

[0045] This invention provides a recombinant antibody, comprising the sequence of the nanobody and the constant region sequence of a human heavy chain antibody; in this invention, the constant region sequence of the human heavy chain antibody is as shown in SEQ ID NO.3, specifically as follows:

[0046] GSPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0047] The present invention provides a recombinant vector for expressing the nanobody, comprising an initial vector and the gene.

[0048] In this invention, the initial vector is preferably a eukaryotic cell expression vector, which contains GG, a human IgG1 CH2 domain, and a human IgG1 CH3 domain in sequence from the N-terminus to the C-terminus; more preferably, the initial vector is a eukaryotic expression plasmid pcDNA3.1-Fc with a human Fc tag. This invention does not impose any particular limitation on the construction method of the recombinant vector; conventional recombinant vector construction methods in the art can be used.

[0049] This invention provides a recombinant cell expressing the nanobody, obtained by transferring the recombinant vector into a host cell. In this invention, the host cell is preferably Expi 293F cells or CHO-S cells.

[0050] This invention provides the use of the nanobody, the gene, the recombinant antibody, the recombinant vector, and the recombinant cell in the preparation of drugs for the prevention and / or treatment of MERS infection. In this invention, the drug includes antibody drugs, antibody-drug conjugates, vaccines, pharmaceutical compositions, etc.

[0051] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with techniques described in the literature in this field or according to product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0053] Example 1

[0054] Obtaining the anti-MERS nanobody MERS-1

[0055] In this embodiment, the VHH phage display immune library obtained after MERS RBD immunization was panned using phage display technology and screened by ELISA to obtain high-affinity anti-MERS heavy chain antibodies, including the following steps:

[0056] The VHH library obtained after MERS RBD immunization was cloned into the M13 phage vector and transformed to extract plasmids, thus constructing a VHH phage display immunization library.

[0057] A solid-phase affinity panning method was used to coat MERS RBD proteins. Nanobodies targeting MERS were panned from the VHH phage display library. Phages were eluted with Gly-HCl (pH=2.2), neutralized with Tris-HCl (pH=9.6), and then infected with E. coli TG1 for amplification. The titers of the amplified products were determined. The amplified phages were diluted, and specific single positive clones were screened by ELISA, using 620 nm as a reference and detecting the absorbance at 450 nm. After sequencing, repetitive sequences were removed to obtain the final positive clones. The MERS-1 phage showed strong positivity, and this sequence (SEQ ID NO.1) was selected for further cloning and expression.

[0058] Example 2

[0059] Transient expression and affinity chromatography purification of anti-MERS nanobody fusion protein

[0060] The DNA sequence encoding the constant region of the human heavy chain antibody shown in SEQ NO.3 was optimized for human cell codon preference and cloned into the pcDNA3.1 vector to construct the eukaryotic expression plasmid pcDNA3.1-Fc with a human Fc tag. The MERS-1 nanobody sequence obtained in Example 1 was cloned into this recombinant eukaryotic expression plasmid pcDNA3.1-Fc, successfully constructing the antibody expression vector pcDNA3.1-MERS-1-Fc. Using the Expi293F expression system, 100 μg of pcDNA3.1-MERS-1-Fc was transfected into 100 mL of Expi293F cells, following the instructions.

[0061] Six days later, the culture medium was harvested, and approximately 100 mL of supernatant was centrifuged. A 5 mL pre-packed Protein A affinity chromatography column was used, equilibrated with 20 mM PBS before loading. The sample was injected after the conductivity reached the baseline. After loading, the column was washed with 20 mM PBS until the baseline stabilized. The target protein was eluted with 0.1 M pH 2.7 glycine buffer. When the UV280 reached the baseline, the collection was stopped. The column was washed with at least 3 column volumes of 20 mM PBS until the baseline stabilized, and then washed with 20% ethanol.

[0062] Example 3

[0063] Purity detection of MERS-1 nanobody fusion protein

[0064] The MERS-1 nanobody fusion protein obtained by affinity chromatography purification in Example 2 was centrifuged using an ultrafiltration tube, concentrated, and transferred to 20 mM PBS. 10 μg was then subjected to SDS-PAGE electrophoresis to determine the protein molecular weight.

[0065] Adjust the protein concentration to 1 mg / mL, centrifuge 1 mL of protein at high speed and transfer it to a high performance liquid chromatography (HPLC) detection vial. Use a size exclusion chromatography (SEC) column with a pore size of 150 Å. Before loading the sample, rinse the system with pure organic phase at a low flow rate (0.5-1 mL / min) for 10 min to remove air bubbles. Switch to the initial proportioned mobile phase and run at the analytical method flow rate for 60 min until the baseline is stable. Load the sample and detect the absorbance signal at 280 nm.

[0066] The SDS-PAGE results of the MERS-1 nanobody fusion protein are shown in the figure. Figure 1 The protein sample size is as expected. The marker molecular weights from largest to smallest are 100, 70, 55, and 40 kDa.

[0067] The HPLC-SEC detection results of the MERS-1 nanobody fusion protein are shown in the figure. Figure 2 The protein sample showed a single peak shape, a very small amount of aggregates, a monomer content of 99.367%, high purity, and good uniformity.

[0068] Example 4

[0069] Evaluation of the binding activity of MERS-1 nanobody fusion protein

[0070] The binding activity of the purified antibody MERS-1 obtained in Example 2 to MERS S-ECD and RBD proteins was detected using the ELISA method. MERS S-ECD and RBD proteins were diluted to 2 μg / mL with coating buffer and coated onto ELISA plates, 100 μL per well, and incubated overnight at 4°C. The plates were washed three times with PBST, and 100 μL of 2% BSA was added to each well, followed by blocking at 37°C for 1 hour. After washing three times with PBST, the antibodies were diluted to 1 μg / mL with diluent, and serially diluted 4-fold. 100 μL of each antibody was added to the ELISA plates, with 3 replicates and 8 gradients, and incubated at 37°C for 1 hour. After washing three times with PBST, 100 μL of HPR-labeled goat anti-human IgG secondary antibody diluted 1:10000 was added, and the plates were incubated at 37°C for 1 hour. After washing three times with PBST, 100 μL of TMB chromogenic buffer was added, and the plates were incubated at room temperature in the dark for 6 minutes. The reaction was then stopped by adding 50 μL of stop solution. The absorbance at 450 nm was measured using a microplate reader with 630 nm as a reference.

[0071] The binding activity of MERS-1 to MERS S-ECD and RBD was detected, see details below. Figure 3 The present invention relates to the MERS-1 antibody binding to the S-ECD protein of MERS in EC2. 50The concentration was 0.5673 ng / mL, and the EC that bound to the RBD protein was... 50 The concentration was 0.7872 ng / mL. The results indicate that MERS-1 exhibits good binding activity to both MERS S-ECD and RBD.

[0072] Example 5

[0073] Affinity evaluation of MERS-1 nanobody fusion protein

[0074] The affinity of the purified antibody MERS-1 obtained in Example 2 for MERS RBD was determined using bio-layer interferometry (BLI). BLI is a label-free, real-time optical detection technique that can detect and track the entire process of binding and dissociation between molecules in solution and molecules immobilized on the surface of a biosensor. The results are recorded as a sensor map plotting the offset distance (in nm) of the interference spectrum curve and the reaction time, providing kinetic and affinity data.

[0075] During the assay, the purified antibody MERS-1 obtained in Example 2 was immobilized onto the surface of the Protein A biosensor. The mobile phase was a solution containing different concentrations of MERS RBD protein (12.5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 800 nM). The obtained signal curves were analyzed using GraphPad Prism 8 software to obtain the affinity detection graph of the neutralizing antibody and MERS RBD provided in this embodiment of the invention.

[0076] The results are as follows Figure 4 As shown, the affinity of MERS-1 for MERS RBD is 0.046 nM, indicating that the antibody MERS-1 provided in this embodiment of the invention has high affinity activity.

[0077] Example 6

[0078] Evaluation of the in vitro neutralizing activity of the MERS-1 nanobody fusion protein

[0079] The in vitro neutralizing activity of the purified antibody MERS-1 against MERS pseudovirus in Example 2 was detected using a pseudovirus neutralization assay. The antibody was serially diluted three-fold from its initial concentration of 1 μg / mL using DMEM medium with 10% FBS, resulting in nine dilutions: 1 μg / mL, 0.33 μg / mL, 0.11 μg / mL, 0.037 μg / mL, 0.0123 μg / mL, 0.0041 μg / mL, 0.00137 μg / mL, 0.000457 μg / mL, and 0.00015 μg / mL. Each dilution was added to a 96-well cell culture plate with three replicates and a volume of 50 μL / well. Immediately afterward, 50 μL of MERS pseudovirus (Vazyme, DD1802) diluted to the appropriate titer with DMEM medium with 10% FBS was added to each well and thoroughly mixed. A survival control (without virus and antibody) and a death control (with virus only) were also included. The plates were incubated at 37°C in a 5% CO2 cell culture incubator for 1 h.

[0080] Vero E6 cells were digested with 0.25% trypsin and then diluted to 5 × 10⁶ cells / mL with DMEM medium + 10% FBS. 5 At a concentration of cells / mL, 100 μL was inoculated into each well of the antibody-virus mixture and incubated overnight at 37°C in a 5% CO2 cell culture incubator.

[0081] After 48 h, remove the cell culture plate, discard 100 μL of cell culture supernatant, add 100 μL of Luciferase chromogenic substrate, and incubate in the dark for 2 min. Transfer 150 μL of the mixture to a 96-well white microplate, and read the Luciferase signal value using a Tecan Spark multi-functional microplate reader. Calculate cell viability using (Luc sample wells - Luc death control) / (Luc survival control - Luc death control), and calculate the antibody IC50 using a GraphPad Prism 8 curve fit. 50 value.

[0082] To detect the neutralizing activity of the MERS-1 nanobody fusion protein against MERS pseudoviruses, see [link to details]. Figure 5 The MERS-1 nanobody fusion protein provided by this invention has an IC50 value against MERS pseudovirus. 50 It has a concentration of 4.225 ng / mL and exhibits good neutralizing activity.

[0083] As can be seen from the above embodiments, the MERS-1 nanobody fusion protein against MERS provided by the present invention has the characteristics of high expression, high stability, high specificity, high affinity, low immunogenicity, and low cost for large-scale production, making it suitable for industrial production and having significant application value.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nanobody against MERS, characterized in that, The amino acid sequences of the CDR1, CDR2, and CDR3 regions are as follows: CDR1: GSIFSRYT CDR2: ITSEGNT CDR3: NAGGGGAYFYHGAS.

2. The nanobody according to claim 1, characterized in that, The nanobody also includes the following amino acid sequences in the FR1, FR2, FR3, and FR4 framework regions: FR1: QVQLVESGGGLVQPGGSLRLSCAAS FR2: MGWFRQAPGKQRDFVAR FR3: TYSDSVKGRFTISRDNAKNTVYLQMTSLKPEDTAVYYC FR4: WGQGTQVTVSS.

3. The nanobody according to claim 2, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID No.

1.

4. A gene encoding the nanobody according to any one of claims 1 to 3, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No.

2.

5. A recombinant antibody, characterized in that, Includes the sequence of the nanobody according to any one of claims 1 to 3 and the constant region sequence of the human heavy chain antibody.

6. A recombinant vector expressing the nanobody according to any one of claims 1 to 3, characterized in that, Includes the initial vector and the gene as described in claim 4.

7. The recombinant vector according to claim 6, characterized in that, The initial vector was the eukaryotic expression plasmid pcDNA3.1-Fc with a human Fc tag.

8. A recombinant cell expressing the nanobody according to any one of claims 1 to 3, characterized in that, Obtained by transferring the recombinant vector of claim 6 or 7 into host cells.

9. The recombinant cell according to claim 8, characterized in that, The host cells are Expi 293F cells or CHO-S cells.

10. The use of the nanobody according to any one of claims 1 to 3, the gene according to claim 4, the recombinant antibody according to claim 5, the recombinant vector according to claim 6 or 7, and the recombinant cell according to claim 8 or 9 in the preparation of a medicament for treating MERS infection.

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