A broad-spectrum neutralizing antibody against novel coronavirus and its application

Abstract

This invention provides a human antibody with neutralizing ability against the JN.1 variant of SARS-CoV-2 and its application, belonging to the field of biomedical technology. The antibody comprises a heavy chain sequence as shown in SEQ ID NO:1 and a light chain sequence as shown in SEQ ID NO:2; or comprises a heavy chain sequence as shown in SEQ ID NO:3 and a light chain sequence as shown in SEQ ID NO:4. The antibody of this invention binds to the extracellular domain of the JN.1 variant spike protein via ECG. 50 The value was significantly lower than that of its parent antibody, with a binding capacity increase of 3 to 5 times or more. In the pseudovirus neutralization experiment, the antibody showed a half-maximal neutralizing concentration (IC50) against JN.1 pseudovirus. 50 The antibody also showed significantly better performance than the parent antibody, with a neutralizing activity increase of 3 to 24 times or more. Furthermore, the antibody's melting temperature exceeded 75°C, demonstrating good thermal stability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a human antibody with the neutralizing ability of JN.1 mutant strains and its application. Background Technology

[0002] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) poses an ongoing challenge to human health and global public health systems. The continuous evolution of the virus, particularly the constant mutation of the spike protein, has led to the emergence of new variants of concern (VOCs) with enhanced transmissibility and immune evasion capabilities. This has severely weakened the efficacy of monoclonal antibody therapies and vaccines developed early on based on the original strains.

[0003] Among numerous neutralizing antibodies against SARS-CoV, the S309 antibody (whose clinically derived antibody is Sotrovimab, VIR-7831) has attracted considerable attention due to its unique broad-spectrum activity. Isolated from recovered SARS-CoV-1 patients, S309 achieves cross-neutralization against multiple sarbecoviruses, including SARS-CoV-1, the original SARS-CoV-2 strains, and their early variants (such as Alpha and Delta), by targeting a highly conserved epitope containing glycosylation sites (e.g., N343) on the spike protein receptor-binding domain (RBD). However, with the widespread prevalence of the Omicron lineage and its subvariants, its clinical efficacy faces serious challenges. In particular, mutations such as G339D carried by the Omicron BA.1 subtype have significantly reduced its neutralizing activity, while the subsequent dominant JN.1 variant (lineage BA.2.86.1.1) has accumulated multiple escape mutations in the RBD region, including R346T, V445H, G446S, and the crucial L455S. The L455S mutation, located directly at the ACE2 receptor binding interface, not only enhances the affinity between the virus and host cells but also completely disrupts the binding of a number of antibodies targeting Class 1 / 2 epitopes, such as S309. Therefore, the neutralizing ability of the S309 antibody against JN.1 is severely insufficient, necessitating engineering modification to restore and enhance its efficacy against prevalent variants.

[0004] Currently, strategies for antibody affinity maturation or broadening of the neutralization spectrum mainly include: library screening based on random mutations, rational design based on structural information, and computationally assisted deep mutation scanning. However, these methods often face challenges when dealing with variants like JN.1 that have undergone multidirectional mutations in key epitope regions (involving both directly contacting residues and adjacent residues affecting local conformation): simple single-point reversion mutations are often insufficient to reconstruct potent binding; while introducing too many mutations may disrupt the structural stability of the antibody itself, increase the risk of immunogenicity, or lead to a decrease in expression yield. Therefore, developing a method to systematically and precisely enhance the neutralizing activity of antibodies against immune escape variants such as JN.1 while maintaining the core framework and excellent physicochemical properties has become an urgent need and technical challenge in the field of antibody drug development. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a modified antibody with neutralizing ability against JN.1 variant strains and its application, wherein the antibody has enhanced binding activity and neutralizing ability against both SARS-CoV-2 wild type (WT) and JN.1 variant strains, and maintains excellent thermal stability.

[0006] The present invention provides an antibody with neutralizing ability of JN.1 mutant strain, comprising a heavy chain sequence as shown in SEQ ID NO:1 and a light chain sequence as shown in SEQ ID NO:2; Alternatively, it may contain a heavy chain sequence as shown in SEQ ID NO:3 and a light chain sequence as shown in SEQ ID NO:4.

[0007] Preferably, the constant region of the antibody is the constant region of human IgG1.

[0008] This invention provides a nucleic acid molecule encoding the antibody.

[0009] This invention provides a recombinant expression vector comprising the aforementioned nucleic acid molecule and the initial vector pcDNA3.4.

[0010] The present invention provides a recombinant cell obtained by transferring the recombinant expression vector into a host cell; the host cell includes Expi293F cells.

[0011] This invention provides the use of the antibody, the nucleic acid molecule, the recombinant expression vector, or the recombinant cell in the preparation of a medicament for the prevention and / or treatment of SARS-CoV-2 infection.

[0012] Preferably, the SARS-CoV-2 infection includes infection caused by the JN.1 variant.

[0013] This invention provides a method for preparing the antibody, comprising the following steps: 1) Recombinant cells were obtained by co-transfecting a heavy-chain expression vector expressing a heavy chain sequence and a recombinant expression vector expressing a light chain sequence into mammalian suspension cells; 2) Culture the recombinant cells, collect the culture supernatant containing the antibody, capture the antibody, and obtain the antibody.

[0014] Preferably, the capture in step 2) is performed using affinity chromatography; the medium for affinity chromatography is protein A or a functional analogue of protein A.

[0015] Compared with the prior art, the present invention has the following beneficial effects: Based on the broad-spectrum neutralizing antibody S309, this invention has rationally designed and screened two modified antibodies that have enhanced binding activity and neutralizing ability against both SARS-CoV-2 wild-type (WT) and JN.1 variants, while maintaining excellent thermal stability.

[0016] Specifically, the representative antibodies Mut-1, Mut-2, and the JN.1 mutant strain spike protein extracellular domain (S) in this invention ECD The combination of EC 50 The values ​​were significantly lower than those of the parental antibodies, with an increase of 3 to 5 times or more. In the pseudovirus neutralization experiment, the half-maximal neutralizing concentration (IC50) of the above antibodies against JN.1 pseudovirus was significantly lower. 50 The antibody described in this invention is also significantly superior to the parent antibody, with an improvement of 3 to 24 times or more. Furthermore, the melting temperature (T0) of the antibody described in this invention was determined by differential scanning fluorometry. m All of them exceeded 75℃, showing good thermal stability. Attached Figure Description

[0017] Figure 1 SDS-PAGE spectra of antibodies Mut-1, Mut-2, and parental antibodies; Figure 2 Antibodies Mut-1, Mut-2, and parental antibodies against SARS-CoV-2 wild-type strain S ECD Binding activity; Figure 3 Antibodies Mut-1, Mut-2, and parental antibodies against the JN.1 mutant strain S ECD Binding activity; Figure 4 The neutralizing activities of antibodies Mut-1, Mut-2, and parental antibodies against SARS-CoV-2 wild-type and JN.1 variant pseudoviruses were evaluated. Figure 5 Thermostability analysis of antibodies Mut-1, Mut-2 and parental antibodies. Detailed Implementation

[0018] This invention provides an antibody with neutralizing ability against JN.1 mutant strains, comprising a heavy chain sequence as shown in SEQ ID NO:1 and a light chain sequence as shown in SEQ ID NO:2, as detailed below: SEQ ID NO: 1 (Mut-1-H chain): QVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYCARDSTRGAWFGWSLIGGFDNWGQGTLVTVSS SEQ ID NO: 2 (Mut-1-K chain): EIVLTQSPGTLSLSPGERATLSCRASGTVSSASLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCIFHDTSLTFGGGTKVEIKR.

[0019] Or it may contain a heavy chain sequence as shown in SEQ ID NO:3 and a light chain sequence as shown in SEQ ID NO:4, as follows: SEQ ID NO: 3 (Mut-2-H chain) QVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYCARDYTRGAWFGWSLIGGFDNWGQGTLVTVSS SEQ ID NO: 4 (Mut-2-K chain) EIVLTQSPGTLSLSPGERATLSCRASQTVSASSLAWYQQKPGQAPRLLIYGFSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHDRSLTFGGGTKVEIKR.

[0020] In this invention, the constant region of the antibody is the constant region of human IgG1, specifically as follows: SEQ ID NO: 5 (Heavy chain constant region) GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAGGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCAGGTAAA。

[0021] SEQ ID NO: 6 (Light chain constant region)

[0022] ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGT GTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGTACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAATGA.

[0023] In this invention, the antibody targets the extracellular domain of the spike protein (S) of the SARS-CoV-2 JN.1 variant. ECD The binding affinity of EC 50 The value is below 0.2 μg / mL; the antibody against JN.1 S ECD The binding affinity of the antibody is 3-5 times higher than that of the parent antibody; the antibody can effectively neutralize pseudoviruses carrying the spike protein of the SARS-CoV-2 JN.1 variant, with a neutralizing half-maximal inhibitory concentration (IC50) of 100%. 50 The concentration was better than 6 μg / mL, representing a 3-24 fold increase compared to the parental antibody. The melting temperature (T0) of the antibody was determined using differential scanning fluorometry (DSF). m (Above 75℃)

[0024] The present invention also provides a nucleic acid molecule encoding the antibody. The present invention does not specify a particular method for preparing the nucleic acid molecule; it can be prepared artificially.

[0025] This invention provides a recombinant expression vector comprising the aforementioned nucleic acid molecule and the initial vector pcDNA3.4. The present invention does not specify a particular method for preparing the recombinant expression vector; conventional methods for preparing recombinant vectors in the art can be used.

[0026] The present invention provides a recombinant cell obtained by transferring the recombinant expression vector into a host cell; the host cell includes Expi293F cells.

[0027] This invention provides the use of the antibody, the nucleic acid molecule, the recombinant expression vector, or the recombinant cell in the preparation of a medicament for the prevention and / or treatment of SARS-CoV-2 infection.

[0028] In this invention, the SARS-CoV-2 infection includes infection caused by the JN.1 variant; the drug also includes pharmaceutically acceptable excipients; the excipients include carriers or excipients.

[0029] This invention provides a method for preparing the antibody, comprising the following steps: 1) Recombinant cells were obtained by co-transfecting a heavy-chain expression vector expressing a heavy chain sequence and a recombinant expression vector expressing a light chain sequence into mammalian suspension cells; 2) Culture the recombinant cells, collect the culture supernatant containing the antibody, capture the antibody, and obtain the antibody.

[0030] In this invention, the capture is performed using affinity chromatography; the affinity chromatography medium is protein A or its functional analogue, wherein the functional analogue of protein A is Monofinity A resin.

[0031] 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.

[0032] Example 1

[0033] Antibody expression and purification

[0034] To ensure the accuracy and comparability of functional evaluations, standardized recombinant expression and high-purity preparation procedures were employed for all candidate antibody molecules. A professional company was commissioned to synthesize the variable regions encoding two antibodies (V...). H / V L Gene sequences of the heavy and light chains (SEQ ID NO:1 / SEQ ID NO:2 and SEQ ID NO:3 / SEQ ID NO:4) and the human IgG1 constant region (SEQ ID NO:5 and SEQ ID NO:6) were cloned into the mammalian expression vector pcDNA3.4 to obtain heavy and light chain expression plasmid pairs.

[0035] Using ExpiFectamine 293 transfection reagent (Thermo Fisher), equal amounts of heavy and light chain plasmids were co-transfected into Expi293F suspension cells in logarithmic growth phase. After transfection, the cells were cultured in a shaker at 37°C, 8% CO2, and 125 rpm. ExpiFectamine 293 Transfection Enhancer was added on day 2 of culture, and the cells were cultured until day 6.

[0036] Cell culture supernatant was harvested by centrifugation and clarified by filtration through a 0.22 μm filter. The clarified supernatant was loaded onto a Monofinity A affinity chromatography column (Cyitva) pre-equilibrated with equilibration buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4). After thorough washing with equilibration buffer, the target antibody was eluted with low-pH elution buffer (0.1 M glycine-HCl, pH 3.0) and immediately adjusted to pH 7.0 with neutralization buffer (1 M Tris-HCl, pH 9.0). The purified antibody solution was then transferred to PBS buffer (pH 7.4), and its purity and molecular size were verified by SDS-PAGE.

[0037] The results are as follows Figure 1 As shown, lanes 1, 2, and 3 are non-reducing SDS-PAGE sequences of Mut-1, Mut-2, and the parental antibody, respectively. The protein sample is located at 170 kDa, which is consistent with the common migration phenomenon of antibodies (theoretical molecular weight 150 kDa) in non-reducing electrophoresis, indicating that the molecular weight of the antibody is correct and the structure is intact. Lanes 4, 5, and 6 are non-reducing SDS-PAGE sequences of Mut-1, Mut-2, and the parental antibody, respectively. The protein sample can be reduced by mercaptoethanol to two fragments of 50 kDa and 25 kDa, which correspond to the theoretical molecular weights of the antibody heavy and light chains, respectively, which is as expected. Lane M is a molecular weight marker.

[0038] Example 2

[0039] ELISA combined with activity verification

[0040] To quantitatively assess the affinity and cross-reactivity of the modified antibody to antigens of different mutant strains, an ELISA experiment was performed: the secretory extracellular domains (S) of wild-type SARS-CoV-2 (WT) and JN.1 mutant strains were coated with carbonate-coated buffer (pH 9.6). ECDThe protein was diluted to 1 μg / mL and added to a 96-well ELISA plate, 100 μL per well, and incubated overnight at 4°C. The coating solution was discarded, and the plate was washed three times with PBST (PBS containing 0.2% Tween-20). 200 μL of PBST blocking buffer containing 2% BSA was added to each well, and the plate was incubated at 37°C for 1 hour. After washing, serially diluted antibody samples (quadruple dilution, starting concentration 1 μg / mL, 7 concentration points in total) were added, 100 μL per well, and the plate was incubated at 37°C for 1 hour. After washing, 100 μL of HRP-labeled goat anti-human IgG secondary antibody diluted 1:10000 was added to each well, and the plate was incubated at 37°C for 1 hour. After washing, TMB substrate solution was added for color development, and the reaction was stopped by adding an equal volume of 2 M H2SO4 after a certain time. Absorbance values ​​were read at 450 nm (detection wavelength) and 630 nm (reference wavelength) using a microplate reader; dose-response curves were fitted using GraphPad Prism software, and the half-maximal effective concentration (EC50) was calculated. 50 ).

[0041] The results are as follows Figure 2 and Figure 3 As shown, Mut-1 and Mut-2 are related to JN.1 S ECD EC 50 The concentrations were 0.0871 μg / mL and 0.1286 μg / mL, respectively, compared to the parental antibody (EC). 50 = 0.4854 μg / mL), the binding capacity was increased by approximately 5.6-fold and 3.8-fold, respectively. In contrast, another modified antibody of the same parent antibody, Mut-7, bound EC... 50 The concentration was 59.52 μg / mL, which showed no improvement in binding ability compared to the parent antibody.

[0042] Table 1. Representative antibody binding activities

[0043] Example 3

[0044] fake virus neutralization experiment

[0045] To verify whether the increased binding activity translates into enhanced biological function, a pseudovirus neutralization experiment was performed: A pseudovirus (Vazyme) carrying the SARS-CoV-2 WT or JN.1 spike protein was pre-incubated with serially triple-diluted antibodies (starting concentration 100 μg / mL) at 37°C for 1 hour. The pre-incubation mixture was added to 96-well plates pre-seeded with 293T cells. The plates were incubated at 37°C in a 5% CO2 incubator for 48 hours. Cells were lysed, and reporter gene activity was measured using a luciferase assay kit. The half-maximal inhibitory concentration (IC50) was determined by calculating relative luciferase activity and fitting a dose-response curve. 50 ).

[0046] The results are as follows Figure 4 As shown, Mut-1 and Mut-2 have IC values ​​against the JN.1 pseudovirus. 50 The concentrations were 0.8632 μg / mL and 5.423 μg / mL, respectively, compared to the parental antibody (IC50). 50 = 20.83 μg / mL), and the neutralizing activities were increased by approximately 24-fold and 3.8-fold, respectively.

[0047] Example 4

[0048] Antibody thermal stability analysis

[0049] The thermodynamic stability of the antibody was assessed using differential scanning fluorometry: the purified antibody was diluted to 0.5 mg / mL with PBS, and the sample was placed in a dedicated capillary tube of the Uncle protein stability analysis platform. The temperature was programmed to increase from 25 °C to 95 °C at a rate of 1 °C / min, while the fluorescence signal was monitored in real time. The melting temperature (T0) of the antibody was determined by analyzing the inflection point of the fluorescence intensity change with temperature. m ).

[0050] The results are as follows Figure 5 As shown, T of Mut-1 and Mut-2 m The temperature value exceeds 75℃, indicating good thermal stability, which is beneficial for its formulation development and long-term storage.

[0051] As can be seen from the above embodiments, the antibody provided by the present invention has the ability to neutralize JN.1 mutant strains. The antibody has enhanced binding activity and neutralizing ability against both SARS-CoV-2 wild type (WT) and JN.1 mutant strains, and maintains excellent thermal stability.

[0052] 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 modified antibody with neutralizing ability of JN.1 mutant strains, characterized in that, It includes the heavy chain sequence as shown in SEQ ID NO:1 and the light chain sequence as shown in SEQ ID NO:2; Alternatively, it may contain a heavy chain sequence as shown in SEQ ID NO:3 and a light chain sequence as shown in SEQ ID NO:

4.

2. The antibody according to claim 1, characterized in that, The constant region of the antibody is the constant region of human IgG1.

3. A nucleic acid molecule encoding the antibody of claim 1 or 2.

4. A recombinant expression vector, characterized in that, It comprises the nucleic acid molecule and the initial vector pcDNA3.4 as described in claim 3.

5. A recombinant cell, characterized in that, The recombinant expression vector of claim 4 is obtained by transferring it into a host cell; the host cell includes Expi293F cells.

6. The use of the antibody of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4, or the recombinant cell of claim 5 in the preparation of a medicament for the prevention and / or treatment of SARS-CoV-2 infection.

7. The application according to claim 6, characterized in that, The SARS-CoV-2 infections mentioned include those caused by the JN.1 variant.

8. The method for preparing the antibody according to claim 1 or 2, characterized in that, Includes the following steps: 1) Recombinant cells were obtained by co-transfecting recombinant expression vectors expressing heavy chain sequences and light chain sequences into mammalian suspension cells; 2) Culture the recombinant cells, collect the culture supernatant containing the antibody, capture the antibody, and obtain the antibody.

9. The preparation method according to claim 8, characterized in that, Step 2) The capture is performed using affinity chromatography; the medium for affinity chromatography is protein A or a functional analogue of protein A.

Images