CN114106191A - Bispecific antibody for neutralizing coronavirus - Google Patents

Bispecific antibody for neutralizing coronavirus Download PDF

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CN114106191A
CN114106191A CN202111563268.2A CN202111563268A CN114106191A CN 114106191 A CN114106191 A CN 114106191A CN 202111563268 A CN202111563268 A CN 202111563268A CN 114106191 A CN114106191 A CN 114106191A
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黄竞荷
吴凡
王应丹
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Fudan University
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Abstract

The invention relates to a bispecific antibody for neutralizing coronavirus and homodimer thereof, a nucleic acid molecule for coding the bispecific antibody, a vector containing the nucleic acid molecule, a host cell containing the vector, and application of the bispecific antibody and the homodimer thereof in preparing a medicament for treating or preventing diseases caused by coronavirus and application in detecting products; the bispecific antibody for neutralizing the coronavirus has excellent broad-spectrum neutralizing capacity for the coronavirus, particularly has obvious neutralizing capacity for various SARS-CoV-2 coronavirus mutant strains including Alpha, Beta, Gamma, Delta, Lambda or Omicron, and has good clinical application prospect in the future.

Description

Bispecific antibody for neutralizing coronavirus
Technical Field
The invention relates to a bispecific antibody for neutralizing coronavirus and homodimer thereof, a nucleic acid molecule for coding the bispecific antibody, a vector containing the nucleic acid molecule, a host cell containing the vector, and application of the bispecific antibody and the homodimer thereof in preparing a medicament for treating or preventing diseases caused by coronavirus, and application in detecting products, belonging to the field of biomedicine.
Background
Since 12 months 2019, the new coronavirus pneumonia (COVID-19) epidemic caused by the new coronavirus (SARS-CoV-2) rapidly spreads all over the world, and poses serious challenges to human health and global public health safety.
SARS-CoV-2 belongs to coronavirus family, and belongs to the same genus of coronavirus as SARS coronavirus which is outbreak in 2003, and the amino acid homology is as high as 77.2%. The major envelope protein of SARS-CoV-2 virus is Spike protein (also called Spike protein, S protein for short). The spike protein is hydrolyzed into two parts of S1 and S2 by protease in cells during virus infection, wherein S2 is transmembrane protein, and S1 has a Receptor Binding Domain (RBD) for recognizing and Binding cell Receptor angiotensin converting enzyme-2 (ACE-2). The spike protein consisting of S1 and S2 is a viral protein that the SARS-CoV-2 virus specifically recognizes, binds to a target cell receptor, and mediates viral infection, and is therefore also a target for recognition by neutralizing antibodies.
Until now, the clinical treatment of COVID-19 mainly comprises symptomatic support treatment, the complexity of clinical symptoms of new coronary pneumonia brings great challenges to doctors, and the development of specific drugs for treating the new coronary pneumonia has become a hotspot and frontier in the current drug research field.
Many studies at home and abroad report neutralizing antibodies isolated from the body of a new coronary patient, and the neutralizing antibodies mainly target an RBD region or an NTD (N-terminal domain) region on the spike protein of SARS-CoV-2 and prevent viruses from entering cells to achieve a protective effect. In animal experiments and clinical experiments, many antibodies have also been demonstrated to have certain effects in preventing and treating new coronavirus infection, and some of the neutralizing antibodies against SARS-CoV-2 have been approved by the FDA for clinical treatment of COVID-19. For example, REGEN-COV (casirivimab and imdevimab) combination antibody therapy covi-19 from recycling dollars corporation can reduce hospitalization and death by 100%.
However, SARS-CoV-2 belongs to the RNA virus, and the viral genome sequence is susceptible to mutation during the spread of an epidemic; for example, the recently occurring Alpha UK mutants B.1.1.7, Beta mutants B.1.351, Gamma Brazil mutant P.1 and Lambda Peru mutant C.37, etc., all over the world; in particular, more contagious circulating strains have recently emerged, the Delta mutant B.1.617.2 and the Omicron (Ormcken) mutant B.1.1.529. It has been reported that there are as many as 32 mutations in the spike protein (S protein) of the omrkron mutant, and that many of these mutations result in greater resistance to the new crown vaccines currently used in various countries, and that antibodies clinically treating new crowns are essentially ineffective.
Therefore, the development of antibodies capable of broadly neutralizing a variety of new mutant strains of coronavirus, especially antibodies capable of neutralizing newly emerged mutant strains of new coronavirus, is a very urgent research topic for researchers in the field.
In order to improve the neutralization breadth and the neutralization capacity of a neutralizing antibody on a new coronavirus, researchers genetically engineer the existing new coronavirus neutralizing antibody, and hopefully construct an antibody which can target multiple epitopes of the virus (effectively inhibit escape phenomena) and has better broad-spectrum property and neutralization activity. Bispecific antibodies are a promising direction of research.
Bispecific antibodies (BsAb) are artificial antibodies that contain two specific antigen binding sites and are capable of targeting two different epitopes simultaneously. BsAb can target two epitopes of the same molecule to play a role in multi-site specific binding, and can target two epitopes of different target molecules to play a role in bridging between different target molecules, so that BsAb has great potential as a therapeutic drug.
Bispecific antibodies were originally prepared by fusing hybridomas of two different antibodies. Because each hybridoma can produce different immunoglobulins, the hybridoma or tetraploid tumor obtained by fusion can theoretically produce an antibody with the antigen specificity of the first parent hybridoma and the second parent hybridoma at the same time, however, the light and heavy chain pairing combination of the antibody produced by the method is complex, the correct pairing ratio is low, and the expected effect of drug production cannot be achieved. Scientists have also developed methods such as "knob-in-hole" (knob-in-hole) and single gene encoded bispecific antibody construction.
Therefore, the skilled person would like to develop new bispecific antibodies capable of neutralizing various coronaviruses, in particular various new emergent mutant coronaviruses, by the above-described methods.
Disclosure of Invention
To solve the above technical problems, the present invention provides, in one aspect, a bispecific antibody for neutralizing coronavirus comprising two single chain antibody fragments scFv-1 and scFv-2, wherein,
the light chain variable region VL-1 of the scFv-1 comprises the LCDR1-1 sequence of the light chain variable region shown as SEQ ID NO.1, the LCDR2-1 sequence of the light chain variable region shown as SEQ ID NO.2 and the LCDR3-1 sequence of the light chain variable region shown as SEQ ID NO. 3;
the VH-1 of the heavy chain variable region of the scFv-1 comprises the HCDR1-1 sequence of the heavy chain variable region shown as SEQ ID NO.4, the HCDR2-1 sequence of the heavy chain variable region shown as SEQ ID NO.5 and the HCDR3-1 sequence of the heavy chain variable region shown as SEQ ID NO. 6;
the light chain variable region VL-2 of the scFv-2 comprises the LCDR1-2 sequence of the light chain variable region shown as SEQ ID NO.7, the LCDR2-2 sequence of the light chain variable region shown as SEQ ID NO.8 and the LCDR3-2 sequence of the light chain variable region shown as SEQ ID NO. 9;
the heavy chain variable region VH-2 of the scFv-2 comprises the sequence of HCDR1-2 of the heavy chain variable region shown as SEQ ID NO.10, the sequence of HCDR2-2 of the heavy chain variable region shown as SEQ ID NO.11 and the sequence of HCDR3-2 of the heavy chain variable region shown as SEQ ID NO. 12.
Preferably, the sequence of the variable region VL-1 of the light chain of the scFv-1 is shown as SEQ ID NO.13, or the variable region VL-1 has more than 80% of sequence homology with the sequence shown as SEQ ID NO. 13;
the sequence of the heavy chain variable region VH-1 of the scFv-1 is shown in SEQ ID NO.14, or the VH-1 has more than 80 percent of sequence homology with the sequence shown in SEQ ID NO. 14;
the sequence of the variable region VL-2 of the light chain of the scFv-2 is shown as SEQ ID NO.15, or the variable region VL-2 has more than 80% of sequence homology with the sequence shown as SEQ ID NO. 15;
the sequence of the heavy chain variable region VH-2 of the scFv-2 is shown in SEQ ID NO.16, or the VH-2 has more than 80 percent of sequence homology with the sequence shown in SEQ ID NO. 16.
The percentage of "sequence homology" with respect to an amino acid sequence is the percentage of sequence homology generated by determining the number of amino acid residues present in both sequences to generate the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100.
In a specific embodiment of the present invention, the variable light chain region VL-1 of scFv-1 may be obtained by performing a deletion, insertion or amino acid mutation of a small number of amino acids based on the sequence shown in SEQ ID NO.13 to obtain an amino acid sequence having a homology of 80% or more. Minor amino acid substitutions (deletions or insertions, or amino acid mutations, or substitutions of similar amino acids), particularly conservative amino acid substitutions in framework regions, result in variants which have high homology (greater than 80%) to the sequence shown in SEQ ID NO.13 and which retain the original properties and functions of the light chain variable region, i.e., those of an antibody which specifically binds to coronaviruses, and are thus within the scope of the present invention. Similarly, the heavy chain variable region VH-1 of scFv-1 may be subjected to a few amino acid deletions, insertions or amino acid mutations based on the sequence shown in SEQ ID NO.14, particularly conservative amino acid substitutions in the framework region, to obtain a variant which retains the original properties and functions of the heavy chain variable region, i.e., the properties and functions of an antibody specifically binding to coronavirus, and such variants are also within the scope of the present invention. Similarly, the same applies to the light chain variable region VL-2 of scFv-2 and the heavy chain variable region VH-2 of scFv-2, which are not described in detail.
The framework region refers to an amino acid sequence located between CDRs, and includes a framework region of a heavy chain variable region and a framework region of a light chain variable region.
Preferably, the C-terminus of scFv-1 is linked to the N-terminus of scFv-2 via a first linker peptide, or the C-terminus of scFv-2 is linked to the N-terminus of scFv-1 via a first linker peptide.
Preferably, the scFv-1 comprises the light chain variable region VL-1, the second linker peptide and the heavy chain variable region VH-1 in that order from N-terminus to C-terminus; or, the scFv-1 comprises the heavy chain variable region VH-1, the second linker peptide and the light chain variable region VL-1 in sequence from N-terminus to C-terminus;
the scFv-2 comprises the light chain variable region VL-2, a third linker peptide and the heavy chain variable region VH-2 in that order from N-terminus to C-terminus; or, the scFv-2 comprises the heavy chain variable region VH-2, the third linker peptide and the light chain variable region VL-2 in sequence from N-terminus to C-terminus;
the sequence of the first linker peptide is GlySer (Gly)4Ser)4Mode, the sequence of the second linker peptide and the third linker peptide is (Gly)4Ser)3Mode(s).
Preferably, the bispecific antibody further comprises the Fc domain of human IgG 1.
Preferably, the C-terminal of scFv-1 is linked to the N-terminal of scFv-2 via a first linker peptide, and the C-terminal of scFv-2 is linked to the Fc domain of human IgG1 via a hinge peptide; alternatively, the first and second electrodes may be,
the C-terminal of the scFv-2 is connected with the N-terminal of the scFv-1 through a first linker peptide, and the C-terminal of the scFv-1 is connected with the Fc domain of human IgG1 through a hinge peptide.
Preferably, the Fc domain of human IgG1 comprises, in order from N-terminus to C-terminus, heavy chain constant region CH2 and heavy chain constant region CH 3; the sequence of the heavy chain constant region CH2 is shown as SEQ ID NO. 17;
the sequence of the heavy chain constant region CH3 is shown in SEQ ID NO. 18; the sequence of the hinge peptide is shown in SEQ ID NO. 19.
In another aspect, the present invention provides a homodimer of a bispecific antibody for neutralizing coronavirus, wherein the homodimer of the bispecific antibody for neutralizing coronavirus is: when the bispecific antibody is expressed in a host cell, the Fc domain of the human IgG1 undergoes homodimerization to form a homodimer.
In a further aspect, the invention provides a nucleic acid molecule, wherein said nucleic acid molecule encodes a bispecific antibody as described above.
Preferably, in the nucleic acid molecule, the nucleic acid sequence encoding the scFv-1 is shown as SEQ ID NO. 20; in the nucleic acid molecule, the nucleic acid sequence for coding the scFv-2 is shown as SEQ ID NO. 21.
Preferably, the nucleic acid sequence encoding the bispecific antibody is shown in SEQ ID NO. 22.
In a further aspect, the present invention provides a vector comprising the nucleic acid molecule described above.
The term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell so that the genetic material elements it carries are expressed within the host cell. The vector may contain various elements for controlling expression, such as a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, a reporter gene, and the like. In addition, the vector may contain a replication initiation site. The vector may also include components which assist its entry into the cell, such as viral particles, liposomes or protein coats, but not exclusively. In an embodiment of the present invention, the carrier may be selected from, but is not limited to: plasmids, phagemids, cosmids, artificial chromosomes (e.g., yeast artificial chromosome YAC, bacterial artificial chromosome BAC, or artificial chromosome PAC of P1 origin), bacteriophages (e.g., lambda phage or M13 bacteriophage), and animal viruses used as vectors, for example, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV 40).
In a further aspect, the present invention provides a host cell comprising the vector described above.
With respect to "host cells," one can select, but is not limited to: prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast cells and Aspergillus, insect cells such as S2 Drosophila cells and Sf9, and animal cell models such as fibroblast, CHO cell, COS cell, NSO cell, HeLa cell, BHK cell, and HEK293 cell.
Preferably, the host cell is a HEK293 cell.
In a further aspect, the present invention provides a pharmaceutical composition comprising the bispecific antibody described above, or comprising the homodimer described above.
In a further aspect, the present invention provides the use of the bispecific antibody, the homodimer, or the pharmaceutical composition for the manufacture of a medicament for the treatment or prevention of a disease caused by a coronavirus.
In a preferred embodiment of the invention, the use refers to the use in the preparation of a medicament for the treatment or prevention of diseases caused by SARS-CoV-2 and its mutant strains, SARS-CoV or SARS-like coronavirus.
In a more preferred embodiment of the invention, the SARS-CoV-2 mutant is an Alpha, Beta, Gamma, Delta, Lambda or Omicron mutant.
In a further aspect, the invention provides an assay product, wherein the assay product comprises a bispecific antibody as described above, or a homodimer as described above.
The test product is useful for detecting the presence or level of a coronavirus in a sample.
In one embodiment of the present invention, the detection product includes, but is not limited to, a detection reagent, a detection kit, a detection chip or test paper, and the like.
The bispecific antibody of the present invention can be labeled by chemical or genetic engineering methods, and the labeled antibody or antigen-binding fragment thereof can be used for detection; the labeled antibody or antigen binding fragment thereof falls within the scope of the present invention.
The specific detection method can adopt the following steps of 1) providing a sample; 2) contacting said sample with the bispecific anti-coronavirus antibody of the invention described above; 3) detecting an immune reaction between the sample and the antibody or antigen-binding fragment thereof.
In a further aspect, the present invention provides a method for producing the bispecific anti-coronavirus antibody described above, wherein the bispecific anti-coronavirus antibody described above is produced by culturing a host cell containing a nucleic acid molecule encoding the bispecific anti-coronavirus antibody described above.
In a further aspect of the present invention, there is provided a method for producing the homodimer of the bispecific antibody, comprising culturing the host cell, wherein the Fc domain of human IgG1 undergoes homodimerization when the bispecific antibody is expressed in the host cell, thereby producing the homodimer of the bispecific antibody.
In yet another aspect, the present invention provides a method for treating or preventing a disease caused by a coronavirus by administering to a patient a therapeutically effective amount of the bispecific antibody described above, or a homodimer thereof; or administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of the bispecific antibody described above, or a homodimer thereof. Preferably, the disease caused by coronavirus is SARS-CoV-2 and its mutant, SARS-CoV or SARS-like coronavirus caused disease. More preferably, the SARS-CoV-2 mutant is an Alpha, Beta, Gamma, Delta, Lambda or Omicron mutant.
The invention relates to a bispecific antibody for neutralizing coronavirus and homodimer thereof, a nucleic acid molecule for coding the bispecific antibody, a vector containing the nucleic acid molecule, a host cell containing the vector, and application of the bispecific antibody and the homodimer thereof in preparing a medicament for treating or preventing diseases caused by coronavirus and application in detecting products; the bispecific antibody for neutralizing the coronavirus has excellent broad-spectrum neutralizing capacity for the coronavirus, particularly has obvious neutralizing capacity for various SARS-CoV-2 coronavirus mutant strains including Alpha, Beta, Gamma, Delta, Lambda or Omicron, and has good clinical application prospect in the future.
Drawings
FIG. 1 is a plasmid map of pcDNA3.4-Fc expression vector used in the preparation of bispecific antibody of example 1 of the present invention;
FIG. 2 is a map of an expression plasmid constructed in the preparation of the bispecific antibody of example 1 of the present invention;
FIG. 3 is an SDS-PAGE pattern of examples 1 and 2 of the present invention, and comparative examples 1 and 2.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments. These embodiments are not intended to limit the present invention, and structural, methodological, or functional changes made by those skilled in the art according to these embodiments are included in the scope of the present invention.
Example embodiments will now be described more fully. Example embodiments may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of example embodiments to those skilled in the art.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, science Press, translated by Huang Petang et al) or according to the product instructions.
Example 1
The coronavirus neutralizing bispecific antibody of example 1, comprising two single chain antibody fragments scFv-1 and scFv-2.
In a specific embodiment of the present invention, the light chain variable region VL-1 of scFv-1 comprises the LCDR1-1 sequence of the light chain variable region shown in SEQ ID NO.1, the LCDR2-1 sequence of the light chain variable region shown in SEQ ID NO.2, and the LCDR3-1 sequence of the light chain variable region shown in SEQ ID NO. 3; the heavy chain variable region VH-1 of scFv-1 comprises the sequence HCDR1-1 of the heavy chain variable region shown in SEQ ID NO.4, the sequence HCDR2-1 of the heavy chain variable region shown in SEQ ID NO.5, and the sequence HCDR3-1 of the heavy chain variable region shown in SEQ ID NO. 6.
In a preferred embodiment of the invention, the light chain variable region VL-1 of scFv-1 has the sequence shown in SEQ ID NO.13, or has more than 80% sequence homology with the sequence shown in SEQ ID NO. 13; the sequence of the heavy chain variable region VH-1 of the scFv-1 is shown in SEQ ID NO.14, or the VH-1 has more than 80 percent of sequence homology with the sequence shown in SEQ ID NO. 14.
Specifically, in this embodiment, the light chain variable region VL-1 of scFv-1 has the sequence shown in SEQ ID NO.13, and the heavy chain variable region VH-1 of scFv-1 has the sequence shown in SEQ ID NO. 14.
Specifically, the light chain variable region VL-1 of the single-chain antibody fragment scFv-1 of example 1 of the present application uses the sequence of the light chain variable region of mab GW01, and the heavy chain variable region VH-1 uses the sequence of the heavy chain variable region of mab GW 01. For the technical content of monoclonal antibody GW01, see invention patent application publication No. CN111793129A (i.e., application No. 20201074031.9 filed by the applicant of the present application on year 2020, month 07 and 28); according to the published information, monoclonal antibody GW01 is a fully human antibody that specifically binds to the receptor binding region RBD of SARS-CoV and the S1 protein of SARS-CoV-2.
In a specific embodiment of the present invention, the light chain variable region VL-2 of scFv-2 comprises the LCDR1-2 sequence of the light chain variable region shown in SEQ ID NO.7, the LCDR2-2 sequence of the light chain variable region shown in SEQ ID NO.8 and the LCDR3-2 sequence of the light chain variable region shown in SEQ ID NO. 9; the heavy chain variable region VH-2 of the scFv-2 comprises the sequence of HCDR1-2 of the heavy chain variable region shown as SEQ ID NO.10, the sequence of HCDR2-2 of the heavy chain variable region shown as SEQ ID NO.11 and the sequence of HCDR3-2 of the heavy chain variable region shown as SEQ ID NO. 12.
In a preferred embodiment of the invention, the light chain variable region VL-2 of scFv-2 has the sequence shown in SEQ ID NO.15, or has more than 80% sequence homology with the sequence shown in SEQ ID NO. 15; the sequence of the heavy chain variable region VH-2 of the scFv-2 is shown in SEQ ID NO.16, or the VH-2 has more than 80 percent of sequence homology with the sequence shown in SEQ ID NO. 16.
In the embodiment, the sequence of the variable region VL-2 of the light chain of the scFv-2 is shown in SEQ ID NO. 15; the sequence of the heavy chain variable region VH-2 of the scFv-2 is shown as SEQ ID NO. 16.
Specifically, the light chain variable region VL-2 of the single-chain antibody fragment scFv-2 of example 1 of the present application was the sequence of the light chain variable region of mAb 16L9, and the heavy chain variable region VH-2 was the sequence of the heavy chain variable region of mAb 16L 9. For technical content of monoclonal antibody 16L9, see patent application publication No. CN112159469A (i.e., application No. 202011065506.2 filed by the applicant of the present application on Ser. No. 09/30/2020); according to the published information, monoclonal antibody 16L9 is a fully human antibody that specifically binds to the receptor binding region RBD of S1 protein of SARS-CoV-2.
In a particular embodiment of the present application, the C-terminus of scFv-1 is linked to the N-terminus of said scFv-2 via a first linker peptide. In another embodiment of the present application, the C-terminus of scFv-2 may also be linked to the N-terminus of said scFv-1 via a first linker peptide.
In this example, the C-terminus of scFv-1 is linked to the N-terminus of scFv-2 via a first Linker peptide (Linker-a), and scFv-1 comprises light chain variable region VL-1, a second Linker peptide (Linker-b), and heavy chain variable region VH-1 in that order from N-terminus to C-terminus, and scFv-2 comprises light chain variable region VL-2, a third Linker peptide (Linker-C), and heavy chain variable region VH-2 in that order from N-terminus to C-terminus.
That is, the bispecific antibody of example 1, in order from N-terminus to C-terminus: scFv-1-Linker-a-scFv-2; more specifically, the bispecific antibody of example 1, in order from N-terminus to C-terminus: VL-1-Linker-b-VH-1-Linker-a-VL-2-Linker-c-VH-2.
Of course, in other embodiments of the present application, the antibody may have the structure VH-1-Linker-b-VL-1-Linker-a-VL-2-Linker-c-VH-2, VL-1-Linker-b-VH-1-Linker-a-VH-2-Linker-c-VL-2, or VH-1-Linker-b-VL-1-Linker-a-VH-2-Linker-c-VL-2.
In other embodiments of the present application, the antibody may have the structure VL-2-Linker-c-VH-2-Linker-a-VL-1-Linker-b-VH-1, VH-2-Linker-c-VL-2-Linker-a-VL-1-Linker-b-VH-1, VL-2-Linker-c-VH-2-Linker-a-VH-1-Linker-b-VL-1, or VH-2-Linker-c-VL-2-Linker-a-VH-1-Linker-b-VL-1.
In this example, the first Linker peptide (Linker-a) between scFv-1 and scFv-2 was selected from GlySer (Gly)4Ser)4The mode is shown in SEQ ID NO. 23.
In this example, the sequences of the second Linker peptide (Linker-b) and the third Linker peptide (Linker-c) were (Gly)4Ser)3The mode is shown in SEQ ID NO. 24.
In a particular embodiment of the present application, the bispecific antibody further comprises an Fc domain of human IgG 1.
In a specific embodiment of the present application, the C-terminus of scFv-1 is linked to the N-terminus of scFv-2 via a first linker peptide, and the C-terminus of scFv-2 is linked to the Fc domain of human IgG1 via a Hinge peptide (Hinge). In another embodiment of the present application, the C-terminal of scFv-2 is linked to the N-terminal of scFv-1 via a first linker peptide, and the C-terminal of scFv-1 is linked to the Fc domain of human IgG1 via a Hinge peptide (Hinge).
In this example, the sequence of the bispecific antibody, from N-terminus to C-terminus, is: scFv-1-Linker-a-scFv-2-Hinge-Fc.
In a particular embodiment of the present application, the Fc domain of human IgG1 comprises, in order from N-terminus to C-terminus, heavy chain constant region CH2 and heavy chain constant region CH 3;
that is, the sequence of the bispecific antibody of example 1, from N-terminus to C-terminus, is:
VL-1—Linker-b—VH-1—Linker-a—VL-2—Linker-c—VH-2—Hinge—CH2—CH3。
wherein, the sequence of the heavy chain constant region CH2 is shown in SEQ ID NO. 17; the sequence of the heavy chain constant region CH3 is shown in SEQ ID NO. 18; the sequence of Hinge peptide Hinge is shown in SEQ ID NO. 19.
Example 1 preparation of coronavirus-neutralizing bispecific antibody
Step 1) construction of antibody expression vector pcDNA3.4-Fc containing Fc gene fragment
The gene fragments of the fully human IgG1 signal peptide gene SP, Hinge peptide Hinge, heavy chain constant region CH2 and heavy chain constant region CH3, namely the SP-Fc gene, were synthesized by Kinry. AgeI and BamHI enzyme cutting sites are inserted between a signal peptide and a hinge peptide gene, and are separated by a GTACGC sequence, namely, an SP-AgeI-BamHI-Fc sequence is synthesized, the SP-AgeI-BamHI-Fc gene is connected to a pcDNA3.4 vector by a TA cloning mode, and finally, the pcDNA3.4-Fc expression vector is obtained, wherein the pcDNA3.4 vector is purchased from Seimer Feishel scientific and technical Co., Ltd, and the specific plasmid map of the vector is shown in figure 1.
Step 2) Synthesis of antibody Gene sequences
As described above, the light chain variable region VL-1 of the single-chain antibody fragment scFv-1 of example 1 uses the sequence of the light chain variable region of mAb GW01 (shown in SEQ ID NO. 13), and the heavy chain variable region VH-1 uses the sequence of the heavy chain variable region of mAb GW01 (shown in SEQ ID NO. 14); the second Linker peptide (Linker-b) between the two is shown in SEQ ID NO. 24.
Corresponding to the sequence, the nucleic acid sequence for coding scFv-1 is shown as SEQ ID NO. 20;
the light chain variable region VL-2 of the single-chain antibody fragment scFv-2 of example 1 used the sequence of the light chain variable region of mAb 16L9 (shown in SEQ ID NO. 15), and the heavy chain variable region VH-2 used the sequence of the heavy chain variable region of mAb 16L9 (shown in SEQ ID NO. 16); the third Linker peptide (Linker-c) between the two is shown in SEQ ID NO. 24.
Corresponding to the sequence, the nucleic acid sequence for coding scFv-2 is shown as SEQ ID NO. 21.
A nucleotide sequence corresponding to the single-chain antibody scFv-1-Linker-a-scFv-2 is synthesized by Nanjing Kingsler company according to a conventional method, and is shown as SEQ ID NO. 22.
Step 3) construction of expression vector of bispecific antibody gene
The N end and C of the nucleotide sequence of the single-chain antibody scFv-1-Linker-a-scFv-2 synthesized in the step 2) are cut by AgeI and BamHI respectively, the target fragment after recovery is purified by a connecting gel and is added into the pcDNA3.4-Fc expression vector constructed in the step 1), DH5 alpha competent cells are transformed to construct the expression plasmid of the final bispecific antibody, the expression plasmid is named as pcDNA3.4-GW01-16L9, and the plasmid map is shown in figure 2.
Step 4) expression of bispecific antibody in mammalian cell 293F
The expression plasmid for bispecific antibody was purified by plasmid purification kit (mayo) and expressed by HEK293F cells co-transfected with EZ Trans cell transfection reagent (lithami).
The specific transfection steps are as follows: 50ml of 293F cells at 1.2X 10 the day before transfection6The cells/mL are plated in 250mL cell culture shake flasks, the expression plasmid constructed in step 3) is fully mixed with the transfection reagent EZ-Trans on the day of transfection (mass to volume ratio of DNA: EZ-Trans ═ 1:3) and dissolved in serum-free OPM medium to obtain an EN-Trans mixture (i.e. 60. mu.g DNA and 180. mu.L EZ-Trans are dissolved in 4mL medium), the EZ-Trans-DNA mixture is uniformly added into HEK293F cells in the shape of raindrops after standing for 15 minutes, and cell culture supernatants are obtained by centrifugation six days after transfection for the subsequent extraction and purification steps of bispecific antibody. (Note: the Fc domain of human IgG1 undergoes homodimerization to form homodimers when the antibody is expressed in a host cell; identified after subsequent extraction and purification.)
Step 5) extraction and purification of antibodies
The cell supernatant collected in step 4) above was filtered through a 0.45 μ M filter, the supernatant was diluted with 1 × PBS binding buffer, and the bispecific antibody containing IgG1 Fc in the supernatant was purified using a protein-G column (tiandi human and biotechnology, japan) according to the instructions for the protein-G column. The bispecific antibody obtained was purified and named diabody GW01-16L 9.
The absorbance at 280nm was measured using a Nanodrop2000(ThermoFisher) and the antibody concentration was calculated. After affinity purification, the antibody was subjected to purity analysis and identification by SDS-PAGE, 5. mu.l of the purified sample was mixed with 20. mu.l of 5 Xloading buffer, the mixture was placed in a 100 ℃ metal water bath and heated for 10 minutes, 10. mu.l of the heated sample mixture was loaded on PAGE gels (Nanjing King Shirui Biotech Co., Ltd.) and separated by electrophoresis according to the molecular weight, the Gel of the separated sample was stained with Coomassie Brilliant blue R250 for 3 hours, and then decolorized with a decolorizing solution, and SDS-PAGE detection results expressing the purified bispecific antibody were obtained by photographing with a GelDoc Go Gel Imaging System (BIO-RAD), as shown in FIG. 3. SDS-PAGE of the antibodies of example 2, described below, and comparative examples 1 and 2 are shown in FIG. 3.
Example 2
The sequence structure of the antibody of example 2, from N-terminus to C-terminus, is:
VL-2—Linker-c—VH-2—Linker-a—VL-1—Linker-b—VH-1—Hinge—CH2—CH3。
the antibody preparation process of example 2 is substantially the same as that of example 1 except that step 2) is synthesized according to the respective antibody sequences, and details are not repeated.
The antibody of example 2, designated as dual anti-16L 9-GW 01.
Effect data
Production of SARS-CoV-2 and its mutant strain, SARS-CoV, bat SARS coronavirus (BtsL-CoV) WIV-1, Rs3367 pseudovirus
SARS-CoV-2 and its mutant, SARS-CoV, BtSL-CoV WIV-1, Rs3367, pseudovirus are non-replication defective retrovirus particles with their respective Spike membrane proteins (Spike, S) on the surface, carrying luciferase reporter gene, and can simulate the infection process of SARS-CoV-2 and its mutant, BtSL-CoV WIV-1, Rs3367, SARS-CoV virus to host cells (such as human liver cancer cell line Huh-7, 293T cell line 293T-ACE2 stably expressing human ACE2 receptor), and express luciferase reporter gene in infected cells. Since pseudoviral infection does not produce viral particles with infectious capacity, the relevant procedures can be safely performed in biosafety secondary laboratories.
SARS-CoV-2, BtSL-CoV WIV-1, Rs3367 and SARS-CoV pseudoviruses were obtained by co-transfection of 293T cells with respective S protein expression plasmids and HIV Env-deficient backbone plasmids with luciferase reporter genes (pNL4-3.Luc. R-E-).
The S gene sequences of SARS-CoV-2, SARS-CoV, BtSL-CoV WIV-1 and Rs3367 are designed according to NCBI GenBank sequences NC-045512, ABD72979.1, KC881007.1 and KC881006.1, the gene sequences are synthesized by Nanjing Kingsry company after being optimized by codons, and are connected to pcDNA3.1 eukaryotic expression vectors to construct SARS-CoV-2, SARS-CoV, BtSL-CoV WIV-1 and Rs3367S protein expression plasmids. Among them, the SARS-CoV-2 mutant pseudoviruses Alpha, Beta, Gamma, Delta, Lambda, and Omicron require corresponding point mutation and deletion mutation on the S protein expression plasmid. pNL4-3.Luc. R-E-backbone plasmid was derived from the U.S. NIH AIDS Reagent Program. All plasmids were amplified by transformation of DH 5. alpha. competent cells and purified using a plasmid purification kit from the production of the organism, the purification procedure being as per the kit instructions.
293T cells were cultured in DMEM medium containing 10% fetal bovine serum (Gibco) and plated onto 10cm cell plates prior to transfection. After 24 hours of culture, the backbone plasmid (pNL4-3.Luc. R-E-) was co-transfected into 293T cells at a ratio of 3:1 with plasmids expressing SARS-CoV, BtSL-CoV WIV-1, Rs3367, SARS-CoV-2 and mutant strains thereof using EZTrans cell transfection reagent (Liji organisms), see the instructions for the use of EZ Trans cell transfection reagent for details of transfection procedures. After 48 hours of transfection, the supernatant containing pseudovirus was collected, centrifuged at 2500 rpm for 10 minutes to remove cell debris, and then frozen in a freezer at-80 ℃ for detection of neutralizing antibodies.
Second, the detection of the neutralizing Activity of the bispecific antibodies (GW01-16L9 and 16L9-GW01) of examples 1 and 2 of the present application against the pseudoviruses of SARS-CoV-2 and its mutants (Alpha, Beta, Gamma, Delta, Lambda and Omicron), as well as various coronaviruses including SARS-CoV, Bat-type coronavirus BtSL-CoV WIV1, Rs3367
Different concentrations of bispecific antibody were tested on 96-well cell plates to inhibit pseudovirus infection of Huh-7 cells to test their neutralizing ability against SARS-CoV-2 and its mutant, SARS-CoV, BtSL-CoV WIV-1, and Rs3367 viruses.
The detection method is roughly as follows: 1) huh-7 cells were seeded in 96-well cell plates at 1X 10 cells per well437 ℃ and 5% CO2Culturing in a cell culture box for 24 hours; 2) the example and comparative antibodies were diluted in cell culture media to different concentrations, mixed with equal volumes of pseudovirus dilutions containing 100TCID50, and incubated at 37 ℃ for 1 hour; 3) discarding the cell culture solution, adding 50 μ l virus-antibody complex into each well, setting multiple wells, and setting no antibodyGroup, virus-free group and positive serum control group; 4) after 12 hours of culture, 150 mul of maintenance liquid is added into each hole, and the culture is continued for 48 hours at 37 ℃; 5) using a Luciferase Assay kit (Luciferase Assay System, Promega Cat. # E1500) to lyse cells and detect Luciferase activity of each well, wherein the specific detection method refers to the kit instructions; detecting the chemiluminescence RLU value of each hole by using a multifunctional microplate reader (Perkin Elmer); 6) percent neutralization inhibition of pseudovirus by different concentrations of antibody was calculated from the ratio of antibody to virus control RLU values, and half the inhibitory dose IC50 (in μ g/ml) of antibody against virus was calculated using PRISM7 software (GraphPad).
Experimental group 1: the bispecific antibody GW01-16L9 of example 1 of the present application was used;
experimental group 2: the bispecific antibody 16L9-GW01 of example 1 of the present application was used;
comparative group 1: adopting fully human novel coronavirus monoclonal antibody GW01 (comparative example 1);
comparative group 2: fully human novel coronavirus mab 16L9 (comparative example 2) was used.
The results are shown in table 1 below:
TABLE 1
Figure BDA0003421100850000141
Table 1 shows the neutralization IC50 results of the bispecific antibodies (GW01-16L9 and 16L9-GW01) of examples 1 and 2, and the control antibodies (mab GW01 and mab 16L9) against various novel coronavirus mutants and various other coronaviruses.
As can be seen from table 1:
1) the bispecific antibodies of examples 1 and 2 (GW01-16L9 and 16L9-GW01) have potent neutralizing ability against a variety of mutant strains of new coronavirus, including SARS-CoV-2Alpha, Beta, Gamma, Delta, Lambda and Omicron, as well as SARS-CoV, Bat coronavirus BtSL-CoV WIV1, Rs3367 pseudoviruses, demonstrating the broad spectrum of neutralizing coronavirus by the bispecific antibodies of examples 1 and 2 (GW01-16L9 and 16L9-GW01) and by the bispecific antibodies of examples 1 and 2 (GW01-16L9 and 16L9-GW 01).
2) It was surprisingly found that the IC50 values of comparative examples 1 and 2 (mAb GW01 and mAb 16L9) were both greater than 50. mu.g/ml for the newly emerging Omicron mutant, whereas the IC50 values of the bispecific antibodies (GW01-16L9 and 16L9-GW01) combined therewith were 0.0538. mu.g/ml and 0.5334. mu.g/ml, respectively. This indicates that monoclonal antibody GW01 and monoclonal antibody 16L9 have no neutralizing effect on the Omicron mutant strain respectively, but the bispecific antibodies (GW01-16L9 and 16L9-GW01) formed by combining the monoclonal antibodies unexpectedly show remarkable neutralizing capability, especially GW01-16L9 shows remarkable strong neutralizing capability.
The discovery also makes the inventor feel very unexpected and surprised, and hopes to disclose and popularize clinically in time, and contributes to the prevention and control of a new round of epidemic caused by the Omicron mutant strain.
In summary, the bispecific antibodies of examples 1 and 2 of the present application (GW01-16L9 and 16L9-GW01) have potent neutralizing ability against various new coronavirus mutants including SARS-CoV-2Alpha, Beta, Gamma, Delta, Lambda and Omicron, and various coronaviruses including SARS-CoV-2, SARS-CoV, BtSL-CoV WIV-1 and Rs3367, show excellent broad spectrum in neutralizing coronavirus, and are significantly superior to the antibodies casirivimab and imdevimab, etc., which are currently approved by the FDA for clinical treatment of COVID-19; in particular, the bispecific antibodies of examples 1 and 2 of the present application also surprisingly show a significant neutralizing capacity for the just recently emerging Omicron mutants.
Based on the effect data of the bispecific antibodies of examples 1 and 2 in the present application, the bispecific antibodies (GW01-16L9 and 16L9-GW01) composed of the combination of monoclonal antibodies GW01 and 16L9 have been demonstrated to have excellent broad spectrum in neutralizing coronavirus, and particularly have strong neutralizing ability for various new mutant strains of coronavirus.
Based on the disclosure and spirit of the present application, one skilled in the art can make simple adjustments based on the diabody sequence of example 1, for example, by exchanging the sequence of light chain and heavy chain based on the diabody sequence of example 1, to obtain antibodies such as VH-1-Linker-b-VL-1-Linker-a-VL-2-Linker-c-VH-2, VL-1-Linker-b-VH-1-Linker-a-VH-2-Linker-c-VL-2, or VH-1-Linker-b-VL-1-Linker-a-VH-2-Linker-c-VL-2. Alternatively, the sequence of the light chain and the heavy chain can be reversed based on the double antibody sequence of example 2 to obtain an antibody such as VH-2-Linker-c-VL-2-Linker-a-VL-1-Linker-b-VH-1, VL-2-Linker-c-VH-2-Linker-a-VH-1-Linker-b-VL-1 or VH-2-Linker-c-VL-2-Linker-a-VH-1-Linker-b-VL-1; it can be reasonably speculated that they all have similar effects to the double antibody of examples 1 and 2; such equivalents are intended to be encompassed by the present application.
The person skilled in the art can also make routine equivalent substitutions for linker peptide sequences, Fc domain sequences, hinge peptide sequences, etc., based on the double antibody sequences of examples 1 and 2 of the present application; the skilled person can also make amino acid insertion, substitution or deletion treatment on the basis of the double-antibody sequence of the present invention, which does not affect the overall effect of the antibody; such equivalents are intended to be encompassed by the present application.
As can be seen from the above, the bispecific antibody for neutralizing coronavirus and its homodimer of the present invention have excellent broad-spectrum, potent neutralizing ability against coronavirus, and therefore, those skilled in the art can develop a pharmaceutical composition comprising the bispecific antibody or its homodimer of the present invention on the basis of the knowledge of this technical disclosure; and, the use of the bispecific antibody or homodimer thereof of the present invention, or a pharmaceutical composition comprising the same, for the manufacture of a medicament for the treatment or prevention of a disease caused by a coronavirus.
In addition, the skilled person, knowing the above technical content, develops a detection product comprising the bispecific antibody of the present invention or its homodimer for detecting the presence or level of coronavirus in a sample.
Application example
This application example describes the use of the bispecific antibody of example 1 of the present application to treat diseases caused by coronaviruses including SARS-CoV-2.
While specific methods, dosages, and modes of administration are provided, it will be understood by those skilled in the art that variations may be made without materially affecting the treatment. Based on the guidance disclosed herein, coronavirus infection can be treated or prevented by administering a therapeutically effective amount of a bispecific antibody described herein, thereby reducing or eliminating coronavirus infection.
The specific application method is as follows:
1) pretreatment of the subject: in particular embodiments, the subject is treated prior to administration of a therapeutic agent comprising one or more antiviral drug therapies known to those skilled in the art. However, such pre-treatment is not always required and may be determined by a skilled clinician.
2) Administration of therapeutic compositions
After screening the subject, a therapeutically effective dose of the bispecific antibody of example 1 of the present application as described above is administered to the subject (e.g., an adult or newborn infant at risk of or known to be infected with SARS-CoV-2 coronavirus). Additional drugs, such as antiviral agents, can be administered to the subject simultaneously with, prior to, or subsequent to the administration of the disclosed agents. Administration is accomplished by any method known in the art, such as oral administration, inhalation, intravenous, intramuscular, intraperitoneal, or subcutaneous. The amount of the composition administered to prevent, reduce, inhibit and/or treat the condition of the subject depends on the subject being treated, the severity of the condition, and the mode of administration of the subject. Desirably, a therapeutically effective amount of an agent is an amount sufficient to prevent, reduce, and/or inhibit, and/or treat a condition in a subject without causing a substantial cytotoxic effect in the subject. An effective amount can be readily determined by one skilled in the art, for example, using routine experimentation to establish a dose response curve. Likewise, these compositions may be formulated with an inert diluent or a pharmaceutically acceptable carrier. In one specific example, the antibody is administered at 5mg per kg every two weeks or 10mg per kg every two weeks, depending on the particular stage of SARS-CoV-2 virus infection. In one example, the antibody is administered continuously. In another example, the antibody is administered at 50 μ g per kg twice weekly for 2-3 weeks. The therapeutic composition may be administered for an extended period of time (e.g., for a period of months or years).
3) Evaluation of
Monitoring a subject infected with SARS-CoV-2 for a reduction in the level of SARS-CoV-2 virus, or a reduction in one or more clinical symptoms associated with a new coronary pneumonia disease, following administration of one or more therapies. In a particular example, the subject is analyzed one or more times beginning 2 days after treatment. The object is monitored using any method known in the art. For example, biological samples from subjects, including pharyngeal swabs, can be obtained and evaluated for changes in SARS-CoV-2 virus levels.
4) Additional treatment
In particular embodiments, if the subject is stable or has a small, mixed or partial response to treatment, additional treatments can be performed after re-evaluation with the same protocol and substance formulation that they previously received for the desired time.
It should be understood that although the present description refers to embodiments, not every embodiment contains only a single technical solution, and such description is for clarity only, and those skilled in the art should make the description as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments understood by those skilled in the art.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Figure IDA0003421100900000011
Figure IDA0003421100900000021
Figure IDA0003421100900000031
Figure IDA0003421100900000041
Figure IDA0003421100900000051
Figure IDA0003421100900000061
Figure IDA0003421100900000071
Figure IDA0003421100900000081
Figure IDA0003421100900000091
Figure IDA0003421100900000101
Figure IDA0003421100900000111
Figure IDA0003421100900000121
Figure IDA0003421100900000131
Figure IDA0003421100900000141
Figure IDA0003421100900000151
Figure IDA0003421100900000161
Figure IDA0003421100900000171
Figure IDA0003421100900000181
Figure IDA0003421100900000191

Claims (15)

1. A bispecific antibody that neutralizes coronaviruses, characterized in that:
the bispecific antibody comprises two single chain antibody fragments scFv-1 and scFv-2, wherein,
the light chain variable region VL-1 of the scFv-1 comprises the LCDR1-1 sequence of the light chain variable region shown as SEQ ID NO.1, the LCDR2-1 sequence of the light chain variable region shown as SEQ ID NO.2 and the LCDR3-1 sequence of the light chain variable region shown as SEQ ID NO. 3;
the VH-1 of the heavy chain variable region of the scFv-1 comprises the HCDR1-1 sequence of the heavy chain variable region shown as SEQ ID NO.4, the HCDR2-1 sequence of the heavy chain variable region shown as SEQ ID NO.5 and the HCDR3-1 sequence of the heavy chain variable region shown as SEQ ID NO. 6;
the light chain variable region VL-2 of the scFv-2 comprises the LCDR1-2 sequence of the light chain variable region shown as SEQ ID NO.7, the LCDR2-2 sequence of the light chain variable region shown as SEQ ID NO.8 and the LCDR3-2 sequence of the light chain variable region shown as SEQ ID NO. 9;
the heavy chain variable region VH-2 of the scFv-2 comprises the sequence of HCDR1-2 of the heavy chain variable region shown as SEQ ID NO.10, the sequence of HCDR2-2 of the heavy chain variable region shown as SEQ ID NO.11 and the sequence of HCDR3-2 of the heavy chain variable region shown as SEQ ID NO. 12.
2. The bispecific antibody of claim 1, wherein:
the sequence of the variable region VL-1 of the light chain of the scFv-1 is shown as SEQ ID NO.13, or the variable region VL-1 has more than 80% of sequence homology with the sequence shown as SEQ ID NO. 13;
the sequence of the heavy chain variable region VH-1 of the scFv-1 is shown in SEQ ID NO.14, or the VH-1 has more than 80 percent of sequence homology with the sequence shown in SEQ ID NO. 14;
the sequence of the variable region VL-2 of the light chain of the scFv-2 is shown as SEQ ID NO.15, or the variable region VL-2 has more than 80% of sequence homology with the sequence shown as SEQ ID NO. 15;
the sequence of the heavy chain variable region VH-2 of the scFv-2 is shown in SEQ ID NO.16, or the VH-2 has more than 80 percent of sequence homology with the sequence shown in SEQ ID NO. 16.
3. The bispecific antibody of claim 2, wherein:
the C-terminal of the scFv-1 is linked to the N-terminal of the scFv-2 via a first linker peptide, or the C-terminal of the scFv-2 is linked to the N-terminal of the scFv-1 via a first linker peptide.
4. The bispecific antibody of claim 3, wherein:
the scFv-1 comprises the light chain variable region VL-1, a second linker peptide and the heavy chain variable region VH-1 in sequence from N-terminus to C-terminus; or, the scFv-1 comprises the heavy chain variable region VH-1, the second linker peptide and the light chain variable region VL-1 in sequence from N-terminus to C-terminus;
the scFv-2 comprises the light chain variable region VL-2, a third linker peptide and the heavy chain variable region VH-2 in that order from N-terminus to C-terminus; or, the scFv-2 comprises the heavy chain variable region VH-2, the third linker peptide and the light chain variable region VL-2 in sequence from N-terminus to C-terminus;
the sequence of the first linker peptide is GlySer (Gly)4Ser)4Mode, the sequence of the second linker peptide and the third linker peptide is (Gly)4Ser)3Mode(s).
5. The bispecific antibody of any one of claims 1 to 4, characterized in that:
the bispecific antibody also includes the Fc domain of human IgG 1.
6. The bispecific antibody of claim 5, wherein:
the C-terminus of the scFv-1 is linked to the N-terminus of the scFv-2 via a first linker peptide, the C-terminus of the scFv-2 is linked to the Fc domain of human IgG1 via a hinge peptide, or,
the C-terminal of the scFv-2 is connected with the N-terminal of the scFv-1 through a first linker peptide, and the C-terminal of the scFv-1 is connected with the Fc domain of human IgG1 through a hinge peptide.
7. The bispecific antibody of claim 6, wherein:
the Fc domain of the human IgG1 comprises a heavy chain constant region CH2 and a heavy chain constant region CH3 from N end to C end;
the sequence of the heavy chain constant region CH2 is shown as SEQ ID NO. 17;
the sequence of the heavy chain constant region CH3 is shown in SEQ ID NO. 18;
the sequence of the hinge peptide is shown in SEQ ID NO. 19.
8. A homodimer of a bispecific antibody that neutralizes coronaviruses, characterized in that:
the homodimer of the coronavirus neutralizing bispecific antibody is: when the bispecific antibody of claim 5 is expressed in a host cell, the Fc domain of human IgG1 undergoes homodimerization to form a homodimer.
9. A nucleic acid molecule, characterized in that: the nucleic acid molecule encodes a bispecific antibody according to any one of claims 1 to 7.
10. The nucleic acid molecule of claim 9, wherein:
in the nucleic acid molecule, the nucleic acid sequence for coding the scFv-1 is shown as SEQ ID NO. 20;
in the nucleic acid molecule, the nucleic acid sequence for coding the scFv-2 is shown as SEQ ID NO. 21.
11. A vector comprising the nucleic acid molecule as claimed in claim 9.
12. A host cell comprising the vector of claim 11.
13. A pharmaceutical composition characterized by: the pharmaceutical composition comprises a bispecific antibody according to any one of claims 1 to 7, or a homodimer according to claim 8.
14. Use of a bispecific antibody according to any one of claims 1 to 7, a homodimer according to claim 8, or a pharmaceutical composition according to claim 13 for the preparation of a medicament for the treatment or prevention of a disease caused by a coronavirus.
15. An assay product characterized by: the assay product comprises a bispecific antibody according to any one of claims 1 to 7, or a homodimer according to claim 8.
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