CN116891531A - Fully human bispecific antibody for resisting novel coronavirus variant - Google Patents
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Abstract
The invention discloses a fully human bispecific neutralizing antibody capable of resisting a new coronavirus, which is formed by modifying two strains of fully human neutralizing antibodies of the new coronavirus. The antibodies are capable of effectively neutralizing currently popular Omicron subforms bf.7, ba.5, xbb.1.5, bq.1.1 and ch.1.1 with strong escape ability. Compared with the parent monoclonal antibody and cocktail strategy, the bispecific antibody disclosed by the invention has stronger antiviral activity and the capability of inhibiting escape mutation, is a feasible and effective strategy for treating and preventing severe new coronaviruses, and has potential to become a candidate drug for intervention of the new coronaviruses.
Description
Technical Field
The invention discloses a bispecific antibody, and belongs to the technical field of polypeptides.
Background
The novel coronavirus is a single-stranded positive sense RNA virus with a capsule, the genome of which can be mutated by various mechanisms during replication, and the evolution rate is extremely high. From 2019 outbreak to date, new variant strains are continuously appeared, so that epidemic situation is repeated, and the number of infected people is continuously increased. The Omicron variant strain which is spread on a large scale in the world at present has the characteristics of strong infectivity, high spreading speed, strong immune escape capability and the like, and the variant strain continuously evolves to evolve various sub-variants such as BA.5, XBB, XBB.1.5, BQ.1.1 and the like, and the spreading of the variant strains greatly increases the risks of reinfection and breakthrough infection, thus bringing great challenges to epidemic prevention work in the world.
In the epidemic prevention and control process, therapeutic antibody medicaments make an important contribution, and monoclonal antibody medicaments or monoclonal antibody combined cocktail therapies developed by large-scale pharmaceutical enterprises such as Gelanin, gift, gelanin Smith, and the like are all used for prevention and clinical treatment by the FDA emergency use authorization in the United states. However, the continued adaptive evolution of viruses has impaired the efficacy of existing vaccines and escaped almost all of the developed neutralizing monoclonal antibodies. There is thus a need to develop new generation neutralizing antibody drugs that provide broad spectrum protection.
Bispecific antibodies are a novel antibody drug model, integrating two specific antibodies into one molecule, exhibiting unique properties that differ from a single antibody. Compared with strategies such as single monoclonal antibody, antibody cocktail and the like, the bispecific antibody has the following advantages: (1) The cocktail therapy needs to produce two antibodies separately, while the bispecific antibody only needs to produce one, so that the production cost is low, and the pre-clinical and clinical development are more convenient; (2) IgG- (scFv) 2 The form of the diabody has tetravalent antigen binding capacity, has improved neutralizing activity on viruses, and particularly integrates two strains of neutralizing antibodies with synergistic effect, wherein the neutralizing activity has 1+1>2', a synergistic effect; (3) The different binding epitopes of the two monoclonal antibodies are combined, so that more virus antigen site mutations can be tolerated, and the probability of occurrence of virus escape mutations in the treatment process can be reduced, thus the method has important significance for clinical treatment. Thus, development of an economical and efficient bispecific antibody drug is urgent.
Bispecific antibodies are not present in nature and can only be prepared manually. Through many years of research and technological development, bispecific antibodies have emerged in many different design strategies in structure. The full-length diabodies containing the Fc region and the fragment diabodies not containing the Fc region are largely classified into 2 classes according to whether they contain the Fc functional region. Full length diabodies are characterized by the retention of the Fc region, which provides longer half-life and stability in vivo, and antibodies with the Fc region are more convenient to purify. In addition, the Fc region may mediate some immune-related functions, such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). Full length bispecific antibodies containing Fc regions are Knobs-into-holes, crossmab, DVD-Ig, igG- (scFv) 2 Etc. Compared with the traditional antibody, the fragment double antibody is smaller in size due to lack of Fc, generally only 50 kDa, better in tissue permeability and certain in solid tumor treatment. Currently, such bispecific antibodies are mainly bi-Nanobody, biTE, DART, tandAbs, etc.
The invention aims to construct a novel bispecific antibody for resisting a novel coronavirus variant strain so as to cope with continuous viral immune escape, and provides candidate medicines for resisting the novel coronavirus variant strain which has strong escape capacity and possibly appears in the future and is popular at present.
Disclosure of Invention
Based on the above purpose, the invention is to construct a fully human bispecific antibody against novel coronavirus variants by taking the monoclonal antibodies H4D12 (Chinese patent application CN 202211732654.4) with strong neutralizing effect on the wild type and Delta, BA.2, BA.2.75, BA.2.76 and other variants of the novel coronavirus and the monoclonal antibodies H4B6 (Chinese patent application CN 202211734994.0) with strong neutralizing effect on recently popular Omicron sub-variants such as BA.5, BA.4.6 and BF.7 as parent monoclonal antibodies.
The invention firstly provides a fully human bispecific antibody of an anti-novel coronavirus variant, which consists of two polypeptide chains and two monoclonal antibody light chains, wherein the polypeptide chains are defined as being formed by connecting a monoclonal antibody heavy chain and a single chain antibody in series, and the monoclonal antibody heavy chain and the monoclonal antibody light chain are from the same maternal monoclonal antibody; in the bispecific antibody of the present invention, the monoclonal antibody heavy chain and the monoclonal antibody light chain derived from the same parent monoclonal antibody constitute the first antigen recognition region. The single chain antibody refers to a monovalent antibody form formed by connecting an antibody heavy chain variable region and a light chain variable region in series; in the bispecific antibody of the present invention, the single chain antibody heavy chain variable region and the single chain antibody light chain variable region constitute a second antigen recognition region; in the present invention, a complete bispecific antibody comprises two first antigen recognition regions and two second antigen recognition regions.
According to the technical scheme provided by the invention, the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the heavy chain of the monoclonal antibody are respectively shown as the amino acid sequences of the 26 th to 33 th, 51 th to 57 th and 96 th to 106 th of SEQ ID NO.1, the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the light chain of the monoclonal antibody are respectively shown as the amino acid sequences of the 27 th to 32 th, 50 th to 52 th and 89 th to 97 th of SEQ ID NO.3, the amino acid sequences of the CDR1, CDR2 and CDR3 of the heavy chain of the single chain antibody are respectively shown as the amino acid sequences of the 487 th to 494, 512 th to 519 th and 558 th to 575 th of SEQ ID NO.1, and the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the light chain of the single chain antibody are respectively shown as the amino acid sequences of the 627 th to 632 th, 650 th to 652 th and 689 th to 696 th of SEQ ID NO. 1.
In the design of the present invention, the heavy chain of the monoclonal antibody and the light chain of the monoclonal antibody are derived from the parent monoclonal antibody H4B6 and form the first antigen recognition region of the bispecific antibody of the present invention, so that in the present invention, the heavy chain of the monoclonal antibody refers to the heavy chain of the parent monoclonal antibody H4B6, and the light chain of the monoclonal antibody refers to the light chain of the parent monoclonal antibody H4B 6. The heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are derived from the parent monoclonal antibody H4D12 and form the second antigen recognition region of the bispecific antibody of the invention, so that the heavy chain of the single-chain antibody refers to the heavy chain of the parent monoclonal antibody H4D12 and the light chain of the single-chain antibody refers to the light chain of the parent monoclonal antibody H4D 12. Earlier studies showed that both H4B6 and H4D12 recognize RBD regions of the new coronavirus. As taught by the general knowledge in the art, the variable regions of the heavy and light chains of antibodies are responsible for binding to antigen, wherein the hypervariable regions of the variable regions (also called complementarity determining regions, CDRs) constitute the antigen binding sites of the antibody molecules, in the solution of the present invention the bispecific antibody is composed of the 4 sets of CDR regions described above, namely, the monoclonal antibody heavy chain variable regions CDR1, CDR2, CDR3 and monoclonal antibody light chain variable regions CDR1, CDR2, CDR3 from H4B6 constitute two first antigen recognition regions that recognize the RBD region of the novel coronavirus; the single chain antibody heavy chain variable region CDR1, CDR2, CDR3 and single chain antibody light chain variable region CDR1, CDR2 and CDR3 from H4D12 constitute two second antigen recognition regions that recognize the new coronavirus RBD region.
In a preferred embodiment, the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody is shown in amino acid sequences 1-117 of SEQ ID NO.1, the amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown in amino acid sequences 1-108 of SEQ ID NO.3, the amino acid sequence of the variable region of the heavy chain of the single chain antibody is shown in amino acid sequences 462-586 of SEQ ID NO.1, and the amino acid sequence of the variable region of the light chain of the single chain antibody is shown in amino acid sequences 601-707 of SEQ ID NO. 1.
In a more preferred embodiment, the constant region amino acid sequence of the heavy chain of the monoclonal antibody is shown as amino acid sequences 118-447 of SEQ ID NO.1, and the constant region amino acid sequence of the light chain of the monoclonal antibody is shown as amino acid sequences 109-214 of SEQ ID NO. 3.
In a particularly preferred embodiment, the monoclonal antibody heavy chain and the single chain antibody, and/or the single chain antibody heavy chain variable region and the single chain antibody light chain variable region between flexible connecting peptides. The flexible connecting peptides of the invention generally comprise small amino acids, non-polar such as Gly, polar such as Ser. These smaller size amino acids provide flexibility in linking peptides, allowing some mobility of the two proteins being linked. And, the addition of Ser can make the connecting peptide and water molecule form hydrogen bond, and endow the connecting peptide with stability in aqueous solution, so as to reduce the interaction between the connecting peptide and the front and back proteins. The most predominant flexible connecting peptide today consists of Gly and Ser residues ("GS" linker). The most widely used flexible connecting peptide has the sequence (Gly-Gly-Gly-Gly-Ser) n. By adjusting the value of n (the number of repeats), the length of the GS-linked peptide can be altered so that the two linked proteins can be optimally separated or made to interact.
In a specific embodiment of the invention, the amino acid sequence of the flexible connecting peptide is as shown in amino acid sequence 448-461, i.e., GGGGSGGGGSGGGG.
In a specific embodiment of the invention, the amino acid sequence of the polypeptide chain is shown as SEQ ID NO.1, and the amino acid sequence of the monoclonal antibody light chain is shown as SEQ ID NO. 3. The bispecific antibody is defined herein as "4B6-H-4D12". The invention constructs an IgG- (scFv) 2 Bispecific antibodies in the form, i.e. single chain antibodies (scFv) linked to the Fc function of normal IgG antibody moleculesThe C-terminal end of the energy region is combined with the target molecule through CDR regions at two ends of the molecule to realize double functions.
In a further embodiment of the present invention, the polynucleotide encoding the bispecific antibody is shown in SEQ ID NO.2, and the polynucleotide encoding the light chain of the monoclonal antibody is shown in SEQ ID NO. 4. It is reasonable to expect by those skilled in the art that the coding sequence of the bispecific antibody can be appropriately optimized without changing the amino acid sequence to facilitate the expression and purification of the protein, and thus other coding sequences of the bispecific antibody can also implement the bispecific antibodies defined in the present invention.
Third, the present invention provides an expression vector containing the above polynucleotide. In a specific embodiment of the invention, the expression vector is pcDNA3.4. It is reasonable to expect by the skilled person that other eukaryotic or prokaryotic expression vectors of the prior art may also be used for the expression of the bispecific antibodies of the invention.
Fourth, the present invention provides a host cell containing the above expression vector. Expression of the bispecific antibody is performed using an Expi293 expression system in one particular embodiment of the invention. It is reasonable to expect by the skilled person that other engineering cells of the prior art, such as CHO cells etc. can also be used for the expression of the bispecific antibodies of the invention.
Finally, the invention provides the application of the bispecific antibody in preparing a novel coronavirus virus therapeutic drug.
The cross-binding activity identification of the invention shows that the bispecific antibody 4B6-H-4D12 can cross-bind to the S-ECD proteins of BA.5, BA.4.6, BF.7, XBB and XBB.1.5, and has wider binding activity compared with the parent monoclonal antibody 4B6-H-4D 12. IC of bispecific antibody 4B6-H-4D12 for neutralizing BA.5 pseudovirus 50 10 ng/mL, neutralizing BF.7 pseudovirus IC 50 7 ng/mL, XBB.1.5 pseudovirus neutralizing IC 50 11 ng/mL, neutralizing BQ.1.1 pseudovirus IC 50 IC for neutralizing CH.1.1 pseudovirus at 20 ng/mL 50 Is 62 ng/mL. The results show that the bispecific antibody 4B6-H-4D12 has a more broad spectrum efficient neutralizing activity against the currently prevailing major omacron subvariant pseudoviruses than the parent mab and cocktail strategy.
The bispecific antibody provided by the invention has good affinity with the S antigen of omacron sub-variant, so that the development of the bispecific antibody into a novel coronavirus specific drug is possible.
Drawings
FIG. 1 is a schematic diagram of the structure of a bispecific antibody of the present invention;
FIG. 2 is a SDS-PAGE profile of bispecific antibody purification;
FIG. 3 ELISA assay for cross-binding activity of 4B6-H-4D 12;
FIG. 4 is a graph of the binding kinetics of 4B6-H-4D12 to the BA.5S-ECD protein;
FIG. 5 is a graph of the binding kinetics of 4B6-H-4D12 to BF.7S-ECD protein;
FIG. 6 is a graph of the binding kinetics of 4B6-H-4D12 to XBB S-ECD protein;
FIG. 7 is a graph of the binding kinetics of 4B6-H-4D12 to XBB.1.5S-ECD protein;
FIG. 8 neutralizing activity of antibodies against BA.5 pseudoviruses;
FIG. 9 neutralizing activity of antibodies against BF.7 pseudoviruses;
FIG. 10 neutralizing activity of antibodies against XBB.1.5 pseudoviruses;
FIG. 11 neutralizing activity of antibodies against BQ.1.1 pseudoviruses;
FIG. 12 neutralizing activity of antibodies against CH.1.1 pseudoviruses.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are only exemplary and do not limit the scope of the invention in any way, which is defined by the claims.
EXAMPLE 1 construction of bispecific antibodies
Earlier we obtained fully human monoclonal antibodies H with excellent broad-spectrum neutralization activity from memory B cells of recombinant novel coronavirus vaccinators by flow sorting-single cell PCR technology4B6 and H4D12, which take RBD domain of S protein of SARS-CoV-2 as target point, wherein H4D12 has strong neutralization effect on new coronavirus wild type and Delta, BA.2, BA.2.75, BA.2.76 and other variants thereof, IC 50 All are lower than 10 ng/mL (see Chinese patent application CN 202211732654.4), H4B6 has strong neutralization effect on recently popular Omicron sub-variants such as BA.5, BA.4.6, BF.7 and the like, and IC 50 Are all lower than 100 ng/mL (see Chinese patent application CN 202211734994.0).
VH and VL linkages of H4D12 were used according to literature to construct scFv of H4D12, followed by the same linker to link scFv of H4D12 to the C-terminus of H4B6 heavy chain, a key map was constructed as follows: xba I cleavage site-GCCGCCACC-signal peptide-H4 B6VH+CH-linker-H4D 12 VH-linker-H4D 12 VL-stop codon-BamH I cleavage site, the sequence of interest was synthesized and ligated into pcDNA3.4 expression vector via Xba I and BamH I, the gene synthesis and the construction of the 4B6-H-4D12 heavy chain recombinant expression plasmid are completed by general biology (Anhui) company, and then the plasmid and the H4B6 light chain expression plasmid are transfected into an Expi293F cell together to express the 4B6-H-4D12 bispecific antibody. FIG. 1 shows a schematic diagram of the structure of a bispecific antibody of the present invention, which is an IgG- (scFv) 2 The form of the bispecific antibody, i.e., a single chain antibody (scFv) derived from the parent mab H4D12 is linked to the C-terminal end of the Fc-functional region of the parent mab H4B6 derived from the normal structure, and bispecific recognition is achieved by binding to the target molecule through CDR regions at both ends of the molecule.
EXAMPLE 2 transient expression and affinity chromatography purification of bispecific antibodies
Using an Expi293 expression System, 100 mL of Expi293F cells were transfected after 50. Mu.g of heavy chain and 50. Mu.g of light chain were mixed, the culture broth was harvested after 5-6 days according to instructions (ThermoFisher Scientific, A14635), supernatant after centrifugation was about 30 mL, pre-loaded Protein G affinity chromatography column with volume of 5 mL was used, equilibrated with 20 mM PBS before loading, after conductivity to baseline, after loading was completed, the column was washed with 20 mM PBS to baseline plateau, the Protein of interest was eluted with 0.1M glycine buffer pH 2.7, after OD 280 After near baseline, collection was stopped.
Results: SDS-PAGE detects affinity chromatography purified bispecific antibody, see in particular FIG. 2, lanes 1, 2 and 3 are respectively the pre-column, post-column and eluent bands of 4B6-H-4D12, and protein samples can be reduced by mercaptoethanol into two fragments with the sizes of 75 kDa and 25 kDa, which correspond to the theoretical molecular weights of the heavy chain and the light chain of the antibody respectively, and are in line with expectations. Lane M is a molecular weight marker.
Example 3 identification of Cross-binding Activity of bispecific antibodies
1. Coating: 96-well ELISA plates were used on the day before the experiment, and the Omacron sub-variant BA.5S-ECD antigen (Acro, SPN-C5229), BA.4.6S-ECD antigen (Acro, SPN-C522 m), BF.7S-ECD antigen (Acro, SPN-C522 q), XBB S-ECD antigen (Acro, SPN-C5248) and XBB.1.5 antigen (Acro, SPN-C524 i) were diluted to 2. Mu.g/mL with a coating solution, and the ELISA plates were coated at 100. Mu.L per well overnight at 4 ℃.
2. Closing: plate washer (BIO-TEK, 405_LS) was used for 3 washes, 100 μl of blocking solution was added to each well and incubated at 37deg.C for 1 h.
3. Sample incubation: plates were washed 3 times, except for the first well, 100 μl of diluent was added to each well, the antibodies were diluted to 4 μg/mL for the first well, 4-fold gradient dilution, 100 μl/well, three duplicate wells were set per antibody, and incubated 1 h at 37 ℃.
4. Secondary antibody incubation: plates were washed 3 times, and HPR-labeled goat anti-human IgG secondary antibody (Abcam, ab 97225) was diluted 1:10000 with dilution, 100 μl per well was added to the ELISA plate corresponding well and incubated 1 h at 37 ℃.
5. Color development: washing the plate for 3 times, adding 100 mu L of TMB single-component color development liquid into each hole, developing for 6 min, keeping out of the sun at room temperature, and adding 50 mu L of termination liquid into each hole to terminate the reaction.
6. OD values at 450-630 nm were detected on a microplate reader, curves were drawn using Logistic four-parameter fitting, and the EC of the antibody was calculated 50 Values.
Results: the binding activity of the bispecific antibody to Omicron subtype S protein is shown in FIG. 3,4B6-H-4D12 and the S-ECD proteins of BA.5, BA.4.6, BF.7, XBB and XBB.1.5 can be combined effectively, and EC 50 56 ng/mL, 37 ng/mL, 47 ng/mL, 128 ng/mL, 85 ng/mL, respectively, while parent monoclonal antibody H4B6 is strongThe S-ECD proteins which bind to BA.5, BA.4.6, BF.7 are effective, but the binding activity to the subfractions XBB and XBB.1.5 is significantly reduced. The bispecific antibody 4B6-H-4D12 is shown to be capable of cross-binding to the S-ECD proteins of BA.5, BA.4.6, BF.7, XBB, XBB.1.5, and has a broader binding activity than the parent monoclonal antibody 4B6-H-4D 12.
Example 4: affinity identification of bispecific antibodies
1. Preparing a buffer solution: running Buffer (PBS+0.02% Tween 20+0.2% BSA), regeneration Buffer (10 mM Gly,pH 1.75).
2. Sample preparation: the antibody was diluted to 50 nM (7.5. Mu.g/mL) with a Running Buffer, the antigen was diluted to 100, 50, 25, 12.5, 6.25, 3.125 nM, and 250. Mu.L each was pipetted into the corresponding position in the 96-well plate. The plate on which the sample is arranged is an A plate, and the corresponding sample is added according to the design layout and then is arranged on the inclined plate frame.
3. Opening a Gator instrument, clicking data acquisition software, and selecting a program module after the instrument completes self-checking. HFC (Anti-HIgG Fc) probes were carefully removed from the cassette with forceps and immersed in 96-well plates into which 200. Mu.L of buffer had been added. The 96-well plate was placed on a horizontal plate rack and the plate on which the probes were placed was a B plate.
4. Sample detection was performed by selecting the K Assay procedure: selecting K Assay in a main interface Assay Setup, and setting Time to 600 s,Shaker A Speed to 400 rpm,Shaker B Speed to 1000 rpm in Equilibration Setting in Basic Parameters; selecting a sample type in a Plate Set Up according to a base-Loading-base-Association experimental scheme, and inputting sample information in a 96-well Plate, wherein the sample information comprises the name and concentration of a sample; the Position, time, speed and Step Type of each Step are defined in the Assay Steps, step 1 is Baseline, time is 60 s, speed is 1000 rpm, step 2 is Loading, time is 100 s, speed is 400 rpm, step 3 is Baseline, time is 60 s, speed is 1000 rpm, step 4 is Association, time is 300 s, speed is 1000 rpm, step 5 is Dissociation, time is 300 s, speed is 1000 rpm; clicking Start in Preview starts running the program.
5. Analysis of results: clicking New K Analysis in a main interface Results & Analysis module according to the operation result, and selecting an experiment to be analyzed in Experiment Selection; setting Ref. Probe in Reference to define a control Probe in the experiment, selecting a non-control hole, clicking an edition Formula, selecting a multiple reduction operation mode, subtracting the Reference, clicking Processed display Processed data; in Binding Fitting, fitting in Parameters selects Global, click Binding Curve Fit to calculate a fitted curve; in Kinetic Analysis, after selection Binding Fitting Graph, click Calculate Kinetics, the binding kinetics data ka, KD of the bispecific antibody 4B6-H-4D12 to different antigens are calculated and displayed.
Results: FIGS. 4-7 are graphs of the affinity constants of 4B6-H-4D12 and BA.5, BF.7, XBB, XBB.1.5, S-ECD, respectively, showing affinity constants KD of 1.52, 1.63, 7.42, 1.40 and nM, respectively, measured R 2 The values were all 99% or more (see Table 1). The results show that the bispecific antibody has good affinity with the S antigen of omacron subtype, so that the bispecific antibody can be developed into a novel coronavirus specific drug.
TABLE 1 data on the kinetics of binding of bispecific antibody 4B6-H-4D12 to different sub-variant S proteins
EXAMPLE 5 identification of pseudovirus neutralizing Activity of bispecific antibodies
1. Purified bispecific antibody was serially diluted 3-fold from the initial concentration with dmem+10% FBS medium, added to 96-well plates, 3 multiplex wells were set, and the volume was 50 μl/well; then 50. Mu.L of pseudovirus suspension of the novel coronavirus variant (diluted to appropriate titer with DMEM+10% FBS) was added to each well, thoroughly mixed, and a survival control (without virus and antibody) and a death control (with virus only) were additionally provided, and the mixture was subjected to 5% CO at 37 ℃ 2 Cell incubator incubate 1 h.
2. HEK293T cells were digested with 0.25% pancreatin and diluted to 2.5X10% with medium (DMEM+10% FBS) 5 cell/mL concentration, inoculated into 96-well cell culture plate, inoculation volume 100 μL/well, placing 5% CO at 37 ℃in 2 The cell culture incubator was incubated overnight.
3.48 After h, 100. Mu.L of the cell culture supernatant was discarded, 100. Mu.L of chromogenic substrate was added and incubated for 2 min in the dark. Transfer 150 μl to 96-well white microwell plate and read Luciferase signal values using Tecan Spark multifunctional microwell plate detector.
4. With [1- (sample-surviving control signal)/(death control signal-surviving control signal)]Antibody neutralization was calculated at 100%, and antibody IC was calculated by fitting a curve to GraphPad Prism 8 50 Values.
Results: FIGS. 8-12 are graphs of pseudovirus neutralization activity of 4B6-H-4D12 with BA.5, BF.7, XBB.1.5, BQ.1.1 and CH.1.1, respectively, as a function of concentration, IC for the bispecific antibody 4B6-H-4D12 to neutralize BA.5 50 10 ng/mL, neutralizing BF.7 pseudovirus IC 50 7 ng/mL, XBB.1.5 pseudovirus neutralizing IC 50 11 ng/mL, neutralizing BQ.1.1 pseudovirus IC 50 IC for neutralizing CH.1.1 pseudovirus at 20 ng/mL 50 Is 62 ng/mL. The results show that the bispecific antibody 4B6-H-4D12 has a more broad spectrum efficient neutralizing activity against the currently prevailing major omacron subvariant pseudoviruses than the parent mab and cocktail strategy.
Claims (10)
1. The fully human bispecific antibody of the novel coronavirus variant is characterized in that the bispecific antibody consists of two polypeptide chains and two monoclonal antibody light chains, wherein each polypeptide chain is formed by connecting a monoclonal antibody heavy chain and a single chain antibody in series, the monoclonal antibody heavy chain and the monoclonal antibody light chain are derived from the same parent monoclonal antibody, the amino acid sequences of variable regions CDR1, CDR2 and CDR3 of the monoclonal antibody heavy chain are respectively shown as 26-33, 51-57 and 96-106 of SEQ ID NO.1, the amino acid sequences of variable regions CDR1, CDR2 and CDR3 of the monoclonal antibody light chain are respectively shown as 27-32, 50-52 and 89-97 of SEQ ID NO.3, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the single chain antibody are respectively shown as 487-494-558-519 of SEQ ID NO.1, and amino acid sequences of CDR1 are respectively shown as 27-32, 50-52 and 89-97 of SEQ ID NO.3, and the amino acid sequences of the variable regions CDR1 and light chain variable region of the monoclonal antibody light chain are respectively shown as amino acid sequences of SEQ ID NO. 1-632-519.
2. The specific antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in amino acid sequences 1-117 of SEQ ID NO.1, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in amino acid sequences 1-108 of SEQ ID NO.3, the amino acid sequence of the heavy chain variable region of the single chain antibody is shown in amino acid sequences 462-586 of SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the single chain antibody is shown in amino acid sequences 601-707 of SEQ ID NO. 1.
3. The bispecific antibody of claim 1, wherein the constant region amino acid sequence of the monoclonal antibody heavy chain is shown in amino acid sequences 118-447 of SEQ ID No.1, and the constant region amino acid sequence of the monoclonal antibody light chain is shown in amino acid sequences 109-214 of SEQ ID No. 3.
4. The bispecific antibody of claim 3, wherein the heavy chain of the monoclonal antibody and the single chain antibody and/or the heavy chain variable region of the single chain antibody and the light chain variable region of the single chain antibody are linked by a flexible linker peptide.
5. The bispecific antibody of claim 5, wherein the amino acid sequence of the flexible linker peptide is as depicted at amino acid positions 448-461.
6. The bispecific antibody of claim 5, wherein the amino acid sequence of the polypeptide chain is shown in SEQ ID No.1 and the amino acid sequence of the monoclonal antibody light chain is shown in SEQ ID No. 3.
7. A polynucleotide encoding the bispecific antibody of claim 6, wherein the polynucleotide of the polypeptide chain has the sequence shown in SEQ ID No.2 and the polynucleotide of the monoclonal antibody light chain has the sequence shown in SEQ ID No. 4.
8. An expression vector comprising the polynucleotide of claim 7.
9. A host cell comprising the expression vector of claim 8.
10. Use of the bispecific antibody of any one of claims 1-6 in the manufacture of a novel medicament for the treatment of coronavirus pneumonia.
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