Influenza antibody variant, application and transformation method

Abstract

The invention discloses an influenza antibody variant, application and a transformation method, and relates to the technical field of immune antibodies, and a heavy chain variable region and a light chain variable region of the influenza antibody variant are nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. According to the technical scheme provided by the invention, the antibody variant with stronger virus removal capability is obtained by performing point mutation on the antibody CDR, and compared with an unoptimized wild type antibody, the antibody variant has better antiviral activity.

Description

Technical Field

[0001] The present invention relates to the field of immune antibody technology, and in particular to an influenza antibody variant and uses thereof. Background Art

[0002] Influenza virus is a major pathogen that poses a long-term threat to human health. It is mainly transmitted through the respiratory tract and can cause seasonal influenza, pandemic influenza, severe complications, and even death after infection. However, due to the rapid mutation and spread of the virus, the development and production of vaccines often face the problems of lag and mismatch. Hemagglutinin (HA) is the most abundant protein on the surface of IV. It can recognize host sialic acid receptors to mediate the fusion process with the host membrane, thereby inhibiting viral infection and inducing host antibody response. Therefore, HA is an important target for influenza vaccine and antibody design. Neutralizing influenza antibodies are currently an effective means of preventing and treating influenza viruses. Direct injection of neutralizing antibodies can rapidly reduce viral loads, alleviate symptoms, and accelerate recovery. However, most existing neutralizing influenza antibodies suffer from long production cycles, poor affinity, high screening costs, and technical difficulties. Therefore, improving the antiviral activity of existing neutralizing influenza antibodies is urgent.

[0003] Existing antibody modification strategies primarily include humanization, affinity maturation, stability modification, glycosylation modification, and bispecific antibody development. These strategies are combined to maximize antibody R&D efficiency based on specific therapeutic needs. Despite significant progress in antibody modification strategies, practical applications still face numerous challenges. For example, balancing antibody affinity and specificity, and evaluating the structure and properties of variants remain unresolved. Diversified and precise antibody modification strategies are still needed to expand the scope of antibody drug development and application.

[0004] In summary, how to develop a modification strategy to improve the antiviral activity of influenza antibodies to prepare influenza neutralizing antibodies is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] The technical solution of the present invention to solve the above technical problems is to provide an influenza antibody variant, which is a human monoclonal antibody, and the heavy chain variable region is respectively the nucleotide sequence shown in SEQ ID NO: 1 and the CDR amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region is respectively the nucleotide sequence shown in SEQ ID NO: 2 and the CDR amino acid sequence shown in SEQ ID NO: 4.

[0006] To solve the above technical problems, the present invention also proposes the use of the influenza antibody variants described above in the manufacture of a medicament for preventing or treating influenza infection in a subject.

[0007] To solve the above technical problems, the present invention also proposes a method for modifying an influenza antibody variant, which is used to prepare the influenza antibody variant described above, comprising the following steps: Molecular dynamics technology was used to simulate the complex system of antibodies and HA, and the specific binding mode of amino acid residues was examined. Based on the principle of reducing steric hindrance and maintaining the complete conformation of the antibody, the key amino acid (32Y) was screened in the antibody CDR region.

[0008] Analyze the effect of the mutant amino acid (Y32D) on the trajectory and number of hydrogen bonds in the complex; Combined with genetic engineering technology, an influenza antibody variant (8Y32D) was obtained. The influenza antibody variant (8Y32D) of the present invention has the nucleotide sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, in its heavy and light chain variable regions. By subjecting the antibody's CDR regions to point mutations, an antibody variant with enhanced virus clearance was obtained. Compared to the unoptimized wild-type antibody (8WT, MEDI8852), both its HA binding ability and virus neutralization activity were significantly enhanced. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0010] Figure 1 In the figure, A is the binding ability of 8WT and 8Y32D according to the present invention, and B is the binding ability of 8WT and 8Y32D with viruses according to the present invention; Figure 2 This is a thermal stability analysis diagram of 8WT and 8Y32D according to the present invention; Figure 3 Figure 1 shows the influenza virus prevention effects of 8WT and 8Y32D mice in mice. A1 and A2 are weight monitoring graphs of 8WT and 8Y32D mice, respectively. B1 and B2 are survival rate graphs of 8WT and 8Y32D mice, respectively. C1 and C2 show the viral loads in the lung tissues of 8WT and 8Y32D mice, respectively. Figure 4The effects of 8WT and 8Y32D on treating influenza virus in mice according to the present invention are shown in Figures A1 and A2, weight monitoring of 8WT and 8Y32D mice, respectively; B1 and B2, survival rate graphs of 8WT and 8Y32D mice, respectively; and C1 and C2, viral loads in lung tissues of 8WT and 8Y32D mice, respectively. Figure 5 The structural cluster and binding position analysis of 8WT and 8Y32D described in the present invention, wherein Figure 5 A is the distribution of variant structure clusters during simulation time, Figure 5 B is the ratio of variant structures in various structural clusters, Figure 5 C is the binding site of 8Y32D and HA stem region, Figure 5 D is the binding site of 8WT and the HA stem region; Figure 6 is the molecular dynamics simulation result of 8WT and 8Y32D described in the present invention, wherein Figure 6 A and Figure 6 B is the contact amino acid analysis between 8WT and 8Y32D and HA, Figure 6 C and Figure 6 D are the analysis of the number of hydrogen bonds between Arg56 and HA in the 8WT and 8Y32D complexes, Figure 6 E and Figure 6 F is the analysis of the number of hydrogen bonds between Ser57 and HA in the 8WT and 8Y32D complexes, respectively. DETAILED DESCRIPTION

[0011] The present invention provides an influenza antibody variant, its use and modification method, aiming to design an antibody variant with stronger virus clearance ability.

[0012] The influenza antibody variants, uses, and modification methods proposed in the present invention are described below in specific examples: Example 1: An influenza antibody variant, the influenza antibody variant (8Y32D) is a human monoclonal antibody, the heavy chain variable region has the nucleotide sequence shown in SEQ ID NO: 1 and the CDR amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region has the nucleotide sequence shown in SEQ ID NO: 2 and the CDR amino acid sequence shown in SEQ ID NO: 4, respectively.

[0013] Specifically, 8Y32D was obtained through genetic engineering technology, and the binding activity and virus neutralization activity were evaluated, which mainly included the following steps: (1) Binding activity detection: ELISA was used to detect the binding activity of the antibody to three different subtypes of HA protein. The specific steps are as follows: ELISA plates were coated with commercially available HA proteins of different subtypes (H1N1, H3N2, and H5N1) at a concentration of 0.2 μg / mL and incubated overnight at 4°C. After blocking with 5% BSA, the plates were incubated with antibody samples at 37°C for 2 hours. HRP-conjugated goat anti-human secondary antibody was then added and incubated at 37°C for 1 hour. 3,3',5,5'-tetramethylbenzidine (TMB) was added as a colorimetric solution, and the OD 450 (OD 450) was read using a microplate reader after stopping the color reaction.

[0014] The results are as follows Figure 1 As shown in A, the average binding activity results show that the variant (8Y32D) has a 4-fold increase in binding activity compared to 8WT.

[0015] (2) Virus neutralization activity detection: The neutralizing activity of the antibody against H1N1 (A / NY / 61 / LV16A) and H3N2 (A / 17 / HK / 2014 / 8296) influenza strains was tested. 8Y32D showed stronger neutralizing ability. The specific steps are as follows: Take 100 times TCID 50 The virus and the serially diluted antibodies were mixed in a 96-well plate, incubated at 37°C for 1 h, added to the MDCK monolayer cells, fixed with cold acetone, and the virus content in the cells was detected by ELISA to calculate the ID 50 , the results are as follows Figure 1 B. Thermal stability analysis of 8WT and 8Y32D is shown in Figure 2 shown.

[0016] The original 8Y32D heavy chain is: QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSDNAVWNWIRQSPSRGLEWLGRTYYRSGWYNDYAESVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARSGHITVFGVNVDAFDMWGQGTMVTVSS.

[0017] The original 8Y32D light chain is: DIQMTQSPSSSLSASVGDRVTITCRTSQSLSSYTHWYQQKPGKAPKLLIYAASSRGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSRTFGQGTKVEIK.

[0018] SEQ ID NO: 1 is: CAAGTGCAGCTGCAGCAGTCCGGCCCTGGCCTGGTGAAGCCTAGCCAGACCCTGAGCCTGACATGTGCCATCAGCGGAGATAGCGTATCCAGTGACAATGCCGTGTGGAACTGGATCAGACAGAGTCCTTCTCGCGGCCTGGAATGGCTGGGTAGAACATATTACCGCAGCGGATGGTACAACGACTACGCCGAGTCTGTGAAGAGCCGGATCACAATTAACCCTGATACCAGCAAAAACCAGTTCAGCTTGCAGCTGAACTCTGTTACCCCTGAGGATACAGCCGTGTACTATTGCGCCAGAAGCGGCCACATCACCGTGTTCGGCGTGAATGTGGATGCCTTTGATATGTGGGGCCAGGGCACAATGGTGACAGTGAGTAGCGCCAGCACAAAGGGCCCTTCTGTGTTCCCCCTGGCCCCTAGCAGCAAGAGCACCAGCGGCGGCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCAGAACCCGTGACCGTGAGCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTTCCTGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAGCTCTGTGGTGACCGTGCCCAGCAGCAGCCTGGGAACTCAGACATACATCTGCAATGTCAACCACAAGCCCAGCAACACCAAGGTGGACAAAAAGGTGGAGCCAAAGAGCTGC。

[0019] SEQ ID NO: 2 is as follows: .

[0020] SEQ ID NO: 3 is: CDRH1: SDNAVWN CDRH2: GRTYYRSGWYNDYAESVKS CDRH3: SGHITVFGVNVDAFDM SEQ ID NO: 4 is: CDRL1: RTSQSLSSYTH CDRL2: AASSRGS CDRL3: QQSRT Example 2: Use of 8Y32D according to Example 1 in the manufacture of a medicament for preventing or treating influenza infection in a subject.

[0021] (1) Analysis of the preventive effect of antibodies in mice: After analyzing the existing influenza antibody preventive dosing, 8WT and 8Y32D were intravenously injected at two different doses (1 mg / kg and 3 mg / kg). 24 hours later, H1N1 (A / NY / 61 / LV16A) and H3N2 (A / 17 / HK / 2014 / 8296) virus infections were performed intranasally. The survival rate and weight curve of the mice after vaccination were monitored, the virus prevention effect of the antibodies was analyzed, and the H1N1 and H3N2 virus loads in the mouse lungs were further detected. The results are as follows: Figure 3 As shown, the results showed that 8Y32D had a stronger antiviral effect than 8WT.

[0022] (2) Analysis of the therapeutic effect of antibodies in mice: After analyzing the existing influenza antibody treatment dosage, mice were infected with H1N1 (A / NY / 61 / LV16A) and H3N2 (A / 17 / HK / 2014 / 8296) viruses intranasally for 24 hours. 8WT and 8Y32D were then injected intravenously at a dose of 15 mg / kg. The survival rate and weight curve of the mice after virus infection and antibody vaccination were monitored to analyze the therapeutic effect of the antibodies. The H1N1 and H3N2 virus loads in the mouse lungs were further detected. The results are as follows: Figure 4 As shown, the results showed that 8Y32D had a stronger therapeutic effect on viruses than 8WT.

[0023] Dosage of 8WT: Vaccination with 3 mg / kg 8WT can achieve partial protection (70%) against H1N1 (A / NY / 61 / LV16A) and complete protection against H3N2 (A / 17 / HK / 2014 / 8296) influenza viruses. Vaccination with 15 mg / kg 8Y32D can treat 50% of mice infected with H1N1 (A / NY / 61 / LV16A) and 100% of mice infected with H3N2 (A / 17 / HK / 2014 / 8296) viruses.

[0024] Dosage of 8Y32D: Vaccination with 3 mg / kg 8Y32D can achieve complete protection against H1N1 (A / NY / 61 / LV16A) and H3N2 (A / 17 / HK / 2014 / 8296) influenza viruses, and vaccination with 15 mg / kg 8Y32D can cure 100% of mice infected with H1N1 (A / NY / 61 / LV16A) and H3N2 (A / 17 / HK / 2014 / 8296) viruses. Example 3: A method for modifying an influenza antibody variant, for preparing the influenza antibody variant described above, comprising the following steps: Molecular dynamics simulation of the antibody-HA complex system was performed using Gromacs 5.1.5 software. The specific parameters are as follows: During the simulation preprocessing phase, the pdb2gmx command was used to precondition the simulation system. The water molecule model was set to SPC (simple point charge). The simulation force field was set to the Gromos 53 A7 combined force field. The side chain protonation states of the titratable residues were specified based on Propka calculation results. The molecular dynamics simulation force field was set to the Gromos 54 A7 combined force field. The topology file and position restraint file required for the simulation were also generated. The editconf and genbox commands were used to place the complex system into a solvent-filled periodic box. The minimum distance between the complex and the solvent box was set to 0.8 nm. The first energy optimization was performed using grompp, and counterions were added to the simulation system using genion to achieve electroneutrality.

[0025] After system preprocessing, the entire system was first minimized using the steepest descent method according to the specified energy optimization mdp file settings to eliminate unreasonable atomic collisions and obtain an energy-converged simulation system. After energy convergence, a two-step 100-ps equilibration simulation was performed (NVT: constant number, volume, temperature; NPT: constant number, pressure, temperature). During the equilibration process, the V-rescale and Parrinello-Rahman algorithms were used to maintain the simulation system temperature at 300 K. In the GROMACS dynamic simulation parameter settings, the default system population was fixed, the initial gas pressure was 1 bar, and the coupling time was set to 0.1 ps; the system pressure was maintained at 1.01×10 5 Pa, and the coupling time is 2.0 ps. At the same time, the simulated isothermal compressibility ratio of the control water is set to 4.5×10 -5 Pa -1 Long-range electrostatic interactions were calculated using the PME method, and atomic bonds were constrained using the LINCS and SETTLE algorithms, with water molecules treated as is. The grid width was set to 1.2 Å, the simulation time step was 2 fs, and coordinates were saved every 2 ps for data analysis.

[0026] The antibody structure stored in the simulation calculation compression trajectory file is classified into structural clusters to simulate the specific structural differences of the antibody and select the structural cluster with the largest proportion of sampling configuration. The structural cluster analysis results are as follows: Figure 5 As shown in A and B.

[0027] In order to further compare the binding differences between 8Y32D and HA, the binding position of the complex, the contact interface, the effect of the mutant amino acids on the trajectory of the complex and the number of hydrogen bonds were analyzed in detail. Figure 5As shown in the figure, the binding position direction of 8Y32D and HA is significantly different from that of 8WT. The binding position direction of 8Y32D on the HA stem is tilted upward compared with 8WT, which indicates that the contact interface between 8Y32D and HA has changed.

[0028] In order to comprehensively compare the differences between 8WT and 8Y32D in binding to HA, the binding interface details of the two systems were analyzed in detail. Figure 6 As shown in A and B, the results of hydrogen bond number analysis are as follows Figure 6 As shown in Figures C, D, E, and F, in 8WT, the number of hydrogen bonds between Arg56 and HA ranges from 1 to 2, while in 8Y32D, the number increases to 1 to 3. The number of hydrogen bonds between Ser57 and HA in both 8WT and 8Y32D remains within the range of 1 to 2, indicating that the 8Y32D complex exhibits good stability.

[0029] Combining genetic engineering technology, 8WT and 8Y32D antibody plasmids were obtained based on the original sequence of 8WT antibody, which were transiently transfected into Expi CHO cells. The antibodies were purified and their antiviral activity was tested to verify the improvement in the antiviral activity of 8Y32D compared with 8WT. The average binding activity of 8Y32D against HA was increased by 4 times, and the antiviral neutralization activity was significantly improved.

[0030] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. An influenza antibody variant, characterized in that For immune protection against H1N1 or H3N2, the influenza antibody variant is a human monoclonal antibody, and the heavy chain variable region and the light chain variable region have the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

2. Use of the influenza antibody variant according to claim 1 in the manufacture of a medicament for preventing or treating influenza infection in a subject.

3. A method for modifying an influenza antibody variant, for preparing the influenza antibody variant according to claim 1, characterized in that: The following steps are involved: Molecular dynamics technology was used to simulate the complex system of antibody and hemagglutinin, to examine the specific binding mode of amino acid residues, and to screen out key amino acids in the antibody CDR region. Analyze the effects of amino acid mutations on the trajectory and number of hydrogen bonds in the complex; Combined with genetic engineering technology, influenza antibody variants are obtained.

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