Monoclonal antibody for resisting MERS virus as well as preparation method and application of monoclonal antibody

Abstract

The invention discloses an anti-MERS virus monoclonal antibody as well as a preparation method and application thereof, and belongs to the field of biomedicine. The monoclonal antibody or the antigen binding fragment thereof specifically binds to the Middle East Respiratory Syndrome Coronavirus S1 protein, and the monoclonal antibody or the antigen binding fragment thereof comprises: a) a heavy chain variable region comprising HCDR1 as shown in SEQ ID NO: 3, HCDR2 as shown in SEQ ID NO: 4 and HCDR3 as shown in SEQ ID NO: 5; and b) a light chain variable region comprising an LCDR1 as shown in SEQ ID NO: 10, an LCDR2 as shown in SEQ ID NO: 11 and an LCDR3 as shown in SEQ ID NO: 12.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to monoclonal antibodies against MERS virus, their preparation methods, and applications. Background Technology

[0002] Currently, domestic and international research on therapeutic antibodies against MERS-CoV mainly focuses on the receptor-binding domain (RBD) of the viral spike protein (S protein). For example, the m336 antibody (developed in collaboration with Fudan University and NIH): is a fully human monoclonal antibody with extremely high neutralizing activity against pseudoviruses (IC50). 50 With a concentration of approximately 0.005 μg / mL and an affinity at the picomolar level, it is one of the representative candidate drugs. Other drugs, such as REGN3048, have also shown some neutralizing activity, but most studies are still in the laboratory discovery and preclinical functional validation stage.

[0003] Current technologies primarily focus on the discovery of antibody molecules themselves, the optimization of affinity, and the enhancement of in vitro neutralizing activity. Furthermore, most high-performance antibodies lack supporting stable, high-expression cell lines and mature purification processes, making large-scale production difficult. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a monoclonal antibody against MERS virus, its preparation method, and its applications.

[0005] The objective of this invention can be achieved through the following technical solutions: A first aspect of the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the Middle East Respiratory Syndrome Coronavirus S1 protein, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises: a) A heavy chain variable region comprising HCDR1 as shown in SEQ ID NO: 3, HCDR2 as shown in SEQ ID NO: 4, and HCDR3 as shown in SEQ ID NO: 5; and b) Light chain variable region comprising LCDR1 as shown in SEQ ID NO: 10, LCDR2 as shown in SEQ ID NO: 11 and LCDR3 as shown in SEQ ID NO: 12.

[0006] Optionally, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2.

[0007] A second aspect of the invention relates to an isolated nucleic acid molecule that encodes the aforementioned monoclonal antibody or its antigen-binding fragment.

[0008] A third aspect of the invention relates to an expression vector comprising the aforementioned nucleic acid molecule.

[0009] A fourth aspect of the invention relates to a host cell comprising the expression vector described above.

[0010] Optionally, the host cell is a CHO cell.

[0011] A fifth aspect of the invention relates to a pharmaceutical composition comprising the above-described isolated monoclonal antibody or its antigen-binding fragment, and a pharmaceutically acceptable carrier.

[0012] A sixth aspect of the invention relates to the use of the above-described isolated monoclonal antibody or its antigen-binding fragment in the preparation of a medicament for the prevention and / or treatment of Middle East Respiratory Syndrome Coronavirus infection.

[0013] A seventh aspect of the present invention relates to a method for preparing the above-described monoclonal antibody or its antigen-binding fragment, the method comprising the following steps: The host cells described above are cultured under conditions suitable for expressing the monoclonal antibody or its antigen-binding fragment; and The monoclonal antibody or its antigen-binding fragment is recovered from the host cell culture.

[0014] Optionally, the method further includes a step of purifying the monoclonal antibody or its antigen-binding fragment recovered from the host cell culture, the purification step comprising the following steps: a) Clarify the host cell culture; b) Load the sample treated in step a) onto Protein A affinity chromatography medium to capture the monoclonal antibody or its antigen-binding fragment; c) Elute the monoclonal antibody or its antigen-binding fragment from the Protein A affinity chromatography medium to obtain an eluent; and d) Perform tangential flow ultrafiltration on the eluent obtained in step c) to concentrate the monoclonal antibody or its antigen-binding fragment and / or replace the buffer.

[0015] The beneficial effects of this invention are: The anti-MERS virus monoclonal antibody (306-1C1) provided by this invention exhibits excellent antigen-binding ability and virus neutralizing activity, showing significant potential for clinical application. Firstly, at the molecular level, this antibody can specifically recognize and bind to the S1 protein of the MERS coronavirus. Surface plasmon resonance (SPR) detection shows that its equilibrium dissociation constant (KD) is 18.59 nM, exhibiting nanomolar-level high affinity binding characteristics, which is the physical basis for its potent blocking effect.

[0016] Secondly, at the in vitro cellular level, this antibody exhibited extremely strong neutralizing ability against MERS virus. Experiments confirmed that, regardless of whether it was targeting pseudovirus or highly pathogenic live virus (EMC / 2012 strain), the antibody significantly inhibited viral infection in a dose-dependent manner. Furthermore, the antibody demonstrated excellent therapeutic protective effects in in vivo animal models. In mouse challenge experiments, intervention with this antibody significantly alleviated the trend of weight loss caused by viral infection and increased the survival rate of infected mice to 100% (observed up to day 14), confirming its effective ability to inhibit viral replication or clear the virus in vivo, demonstrating clear preventive and therapeutic value.

[0017] Finally, this antibody also possesses good drug-like properties and industrialization prospects. Through optimized preparation processes, the antibody's expression yield in stable cell lines can reach 1.19 g / L, which is at a relatively high level in the industry and helps reduce production costs. Simultaneously, the antibody exhibits good thermal and storage stability, meeting the requirements for industrial production, transportation, and clinical storage, making it suitable for further development into a biological drug for the prevention or treatment of Middle East Respiratory Syndrome (MERS). Attached Figure Description

[0018] The invention will now be further described with reference to the accompanying drawings.

[0019] Figure 1 Construction of the MERS antibody plasmid in the embodiments of this application; Figure 2 This is for screening monoclonal cell lines in the embodiments of this application; Figure 3 The cell bank stability test in this embodiment is shown in A and B, where A and B are the cell viability and antibody expression levels of each cell line as a function of passage number, respectively. Figure 4 This refers to the sterility test results in the embodiments of this application; Figure 5 The mycoplasma detection results in the embodiments of this application. Figure 6 Photographs of the cryopreserved cell lines in the embodiments of this application; Figure 7 This is a diagram of antibody purification in an embodiment of this application; Figure 8 The images show the SDS-PAGE and Western Blot results in the embodiments of this application; where A is the reduced and unreduced SDS-PAGE electrophoresis images of MERS antibody (306-1C1) and negative control (NC-hIgG1); and B is the reduced and unreduced Western Blot results of MERS antibody (306-1C1) and negative control (NC-hIgG1).

[0020] Figure 9 The ELISA method used in this application example is to detect the binding between the antibody and the MERS S1 protein. Figure 10 The SPR analysis in this application illustrates the binding affinity between the antibody and the MERS S1 protein. Figure 11 This is a comparison of the pseudovirus neutralization effect of the antibodies in the embodiments of this application; Figure 12 This is a comparison of the actual toxicity neutralization effects of the antibodies in the embodiments of this application; Figure 13 This is a comparison of the changes in mouse body weight in the embodiments of this application; Figure 14 This is a comparison of mouse survival curves in the embodiments of this application; Figure 15 This is a comparison of viral titers in mouse lung tissue in the embodiments of this application; Figure 16 This is a flowchart of the plasmid construction process for this application. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] In some embodiments of the present invention, the preparation and application of a monoclonal antibody against MERS virus are disclosed, the preparation and verification of which include the following steps: 1. Plasmid Construction and Validation: The pcDNA5 / FRT vector (both pcDNA5 / FRT vector and CHO-K1 cells were purchased from Invitrogen Life Technologies) was digested with enzymes to obtain two fragments, one for the light chain and one for the heavy chain. These two fragments were then ligated to the light and heavy chain sequences of the target fragment. After ligation, single clones were selected for plasmid mini-prep, and then digested with restriction enzymes of both light and heavy chains for identification. Two single bands of the light and heavy chains were obtained, both of the correct size (greater than 2000 bp). Sequencing results and BLAST alignment confirmed that the antibody light and heavy chain sequences were consistent with the design and no mutations occurred. The pcDNA5 / FRT-MERSAb expression vector (e.g., ...) was successfully constructed. Figure 16 (As shown).

[0023] 2. Stable cell line screening: The constructed plasmid was transformed into CHO-K1 cells and transfected by electroporation. After transfection, L-aminomethylmethionine (MSX) was used to selectively stimulate the amplification of the GS gene and the target protein gene. Recombinant cell lines were obtained through screening and can grow and proliferate in glutamine-free medium, which avoids or reduces the damage and inhibitory effects of ammonia accumulated from glutamine degradation during cell metabolism. The supernatant of cells after MSX-assisted screening was used for ELISA detection to screen for cell lines that express antibodies. Figure 2 ).

[0024] 3. Cell bank construction: First, the selected monoclonal cells were progressively expanded into shake flasks for culture. Cell supernatant samples were taken from different passages to detect antibody expression levels, and antibody yield was determined using ELISA. More than 20 monoclonal cell lines were obtained using the limiting dilution method. These were then passaged and screened for antibody expression and stability, ultimately yielding 6 cell lines in good condition. The results of these 6 cell lines showed that the passage viability of the selected cell lines was above 95%, antibody expression levels were greater than 1 g / L, and the yield decline rate was 24%, meeting the requirements for stable cell line yield. Figure 3 For the top 6 clones, a research cell bank was established. Cell 306-1C1 exhibited the best growth characteristics. The cloned cells were expanded and cryopreserved, and sterility and mycoplasma tests were performed. The sterility and mycoplasma test results showed that the cells were sterile and no mycoplasma was detected. Figure 4 , Figure 5 After stable expansion, the cells were cryopreserved, with more than 20 cells stored in liquid nitrogen. Figure 6 ).

[0025] 120 mL of cell culture supernatant expressing MERS antibodies was collected, and the antibodies were purified using affinity chromatography. Antibody collection was performed by monitoring changes in UV value. Figure 7 As shown, antibody collection began when the UV value started to rise and stopped when the UV value returned to its initial value, collecting a total of 10 ml of antibody. The collected antibody was then transferred to a 15 mL volume of PBS, filtered through a 0.25 μm filter, and subjected to BCA quantification analysis. The quantification result was 9.5 mg / mL. Further calculations determined the antibody expression level to be 9.5 mg / mL. 15 / 120 = 1.19 g / L.

[0026] In some embodiments of this application, the amino acid sequence information of the obtained MERS antibody (306-1C1) is as follows: The amino acid sequence of the heavy chain is SEQ ID NO.1: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWVGWINTHTGQPTYAEDFKGRFAFSLETSVLSAYLEINNLKDEDTATYFCARGTSLLVGNWYFDVWG AGTTVTVSPASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; The CDR1 amino acid sequence of the heavy chain, SEQ ID NO.3, is: GYTFTNYGMN; The CDR2 amino acid sequence of the heavy chain, SEQ ID NO.4, is: WINTHTGQPTYAEDFKG; The CDR3 amino acid sequence of the heavy chain, SEQ ID NO.5, is: GTSLLVGNWYFDV.

[0027] The amino acid sequences SEQ ID NOs 6-9 of FR1 to FR4 are as follows: QIQLVQSGPELKKPGETVKISCKAS; WVKQAPGKGLKWVG; RFAFSLETSVLSAYLEINNLKDEDTATYFCAR; WGAGTTVTVSPASTKGPSV.

[0028] The amino acid sequence of the light chain is SEQ ID NO.2: DIVLTQSPASLAVSLGQRATISCRTSESVDSFGNSFLHWYHQKPGQPPKLLIYRAFNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPWTFGGGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; The CDR1 amino acid sequence of the light chain, SEQ ID NO.10, is: RTSESVDSFGNSFLH; The CDR2 amino acid sequence of the light chain, SEQ ID NO. 11, is: RAFNLES; The CDR3 amino acid sequence of the light chain, SEQ ID NO.12, is: QQSNEDPWT.

[0029] The amino acid sequences of FR1 to FR4, SEQ ID NOs 13 to 16, are as follows: DIVLTQSPASLAVSLGQRATISC; WYHQKPGQPPKLLIY; GIPARFSGSGSRTDFTLTINPVEADDVATYYC; FGGGTKLEIKRTVAAPSVFIFPPSDEQLK.

[0030] The results of SDS-PAGE and Western Blot are as follows: Figure 8 As shown in the figure. SDS-PAGE results showed that 306-1C1 exhibited a typical IgG molecular structure, with heavy chain (approximately 50 kDa) and light chain (approximately 25 kDa) bands appearing under reducing conditions, without any obvious extraneous bands. The negative control (NC-hIgG1) band position was consistent with that of typical human IgG. Western blot results showed that both 306-1C1 and the negative control were colored at their corresponding molecular weight positions, with good specificity and no non-specific bands.

[0031] The ELISA test data were plotted and analyzed, and the results are as follows: Figure 9As shown, the MERS antibody (306-1C1) exhibited concentration-dependent binding activity to the MERS S1 protein (purchased from Sinocare, catalog number 40069-V08H). According to the plotted dose-response curve, the OD450 value gradually decreased as the antibody concentration decreased from 0.12 μg / mL, indicating specific binding of the antibody to the S1 protein. Nonlinear regression analysis yielded an EC50 value of 3.59 ng / mL for the MERS antibody (306-1C1), indicating high binding affinity for the MERS S1 protein. In contrast, the negative control (NC-hIgG1) showed extremely low OD450 values ​​at all tested concentrations, confirming the high specificity of the binding between the MERS antibody and the S1 protein.

[0032] The SPR data were plotted and analyzed, and the results are as follows: Figure 10 As shown, the MERS antibody (306-1C1) exhibits highly specific binding to the MERS S1 protein immobilized on the chip surface. The binding response units (RU) increase with increasing antibody concentration, indicating that the binding reaction follows a concentration-dependent law. Kinetic parameter analysis shows that the binding rate constant (ka) between the MERS antibody (306-1C1) and the MERS S1 protein is 1.41 × 10⁻⁶. 5 M -1 s -1 The dissociation rate constant (kd) is 2.62 × 10⁻⁶. -3 s -1 The equilibrium dissociation constant (KD) was 18.59 nM. This KD value indicates that the MERS antibody (306-1C1) binds to the MERS S1 protein with nanomolar-level high affinity.

[0033] Based on the data from the pseudovirus neutralization test, the results are as follows: Figure 11 As shown, the MERS antibody (306-1C1) effectively neutralized MERS pseudovirus infection in HUH7 cells. With increasing antibody concentration, the detected chemiluminescence value (RLU) significantly decreased, indicating that the antibody inhibited viral infection in a concentration-dependent manner. Nonlinear regression analysis using GraphPad Prism software fitted the inhibition curve, calculating an IC50 value of 0.25 μg / mL for the MERS antibody (306-1C1), indicating good pseudovirus neutralizing activity. At the highest tested concentration (10 μg / mL), the MERS antibody (306-1C1) inhibited 99.84% of viral infection. In contrast, the negative control (NC-hIgG1) showed no significant neutralizing activity at any tested concentration, with RLU values ​​similar to the viral control group.

[0034] Results of the real poison neutralization test are as follows Figure 12As shown, the MERS antibody (306-1C1) exhibited a dose-dependent neutralizing effect in the live virus neutralization experiment. With increasing antibody concentration, the number of virus-infected cells decreased significantly, indicating that the antibody effectively inhibited the infection of cells by live MERS virus (EMC / 2012 strain). The ND50 value of the MERS antibody (306-1C1) was calculated to be 0.987 μg / mL using the Spearman-Kärber method. At the highest tested concentration, the antibody showed a significant viral inhibitory effect. In contrast, the negative control (NC-hIgG1) did not show significant neutralizing activity at any tested concentration, and its infection rate was comparable to that of the virus control group.

[0035] In some embodiments of the present invention, a mouse experimental model is constructed to verify the therapeutic effect of the MERS antibody in the animal model, specifically including the following steps: 1. Animal preparation and grouping: 6-8 week old female hDPP4 transgenic mice were randomly divided into two groups: antibody treatment group (5 mice), which received MERS antibody (306-1C1) treatment; negative control group (5 mice), which received NC-hIgG1 antibody treatment.

[0036] 2. Viral infection: Mice were fixed after being lightly anesthetized and infected with the EMC-Mers strain by intranasal instillation, 200 FFU / mouse.

[0037] 3. Antibody therapy: 24 hours after challenge, the corresponding antibody was injected via the tail vein. Antibody therapy group: MERS antibody was injected, dose 100 μL / animal; negative control group: an equal dose of NC-hIgG1 antibody was injected.

[0038] 4. Observation and recording: Record the weight of mice on the day of infection (Day 0). Thereafter, weigh the two groups of mice at regular intervals every day and observe and record the mortality of mice. Continue weighing for 14 consecutive days.

[0039] To analyze weight changes, use Graphpad to create a weight change graph, such as... Figure 13 As shown.

[0040] The mortality of mice in both groups was monitored after infection. All mice in the treatment group survived for 14 days; in the control group, 2 mice died on Day 5, 2 mice died on Day 6, and all mice died on Day 7. The survival curves for both groups are shown below. Figure 14 The results showed that the MERS antibody (306-1C1) exhibited complete protection in the hDPP4 transgenic mouse model: effectively controlling weight loss caused by viral infection and significantly improving survival rate (P<0.01).

[0041] Live virus titers were detected in lung tissues of mice from both groups on day 5 post-infection. The results are as follows: Figure 15 The results showed that no live virus was detected in the lung tissue of the treatment group mice, while live virus was detected in the lung tissue of all mice in the control group. On day 14 post-infection, live virus titer was measured in the lung tissue of the treatment group mice, and no live virus was detected.

[0042] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0043] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. An isolated monoclonal antibody or its antigen-binding fragment thereof, which specifically binds to the Middle East Respiratory Syndrome Coronavirus S1 protein, wherein, The monoclonal antibody or its antigen-binding fragment comprises: a) A heavy chain variable region comprising HCDR1 as shown in SEQ ID NO: 3, HCDR2 as shown in SEQ ID NO: 4, and HCDR3 as shown in SEQ ID NO: 5; and b) A light chain variable region comprising LCDR1 as shown in SEQ ID NO: 10, LCDR2 as shown in SEQ ID NO: 11, and LCDR3 as shown in SEQ ID NO:

12.

2. The isolated monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO: 1, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:

2.

3. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof as described in claim 1 or 2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. The host cell according to claim 8, characterized in that, The host cell is a CHO cell.

7. A pharmaceutical composition comprising the isolated monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

8. Use of the isolated monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2 in the preparation of a medicament for the prevention and / or treatment of Middle East Respiratory Syndrome Coronavirus infection.

9. A method for preparing the monoclonal antibody or antigen-binding fragment thereof as described in claim 1 or 2, the method comprising the following steps: The host cells of claim 5 or 6 are cultured under conditions suitable for expressing the monoclonal antibody or its antigen-binding fragment; and The monoclonal antibody or its antigen-binding fragment is recovered from the host cell culture.

10. The method for preparing the monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2 according to claim 9, characterized in that, The method also includes the step of purifying the monoclonal antibody or its antigen-binding fragment recovered from the host cell culture, the purification step comprising the following steps: a) Clarify the host cell culture; b) Load the sample treated in step a) onto Protein A affinity chromatography medium to capture the monoclonal antibody or its antigen-binding fragment; c) Elute the monoclonal antibody or its antigen-binding fragment from the Protein A affinity chromatography medium to obtain an eluent; and d) Perform tangential flow ultrafiltration on the eluent obtained in step c) to concentrate the monoclonal antibody or its antigen-binding fragment and / or replace the buffer.

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