Neutralizing antibodies against orthomyxoviruses and methods of making and using the same

Abstract

The application provides a neutralizing antibody against a sapovirus and a preparation method and application thereof. A heavy chain CDR1 of the neutralizing antibody comprises an amino acid fragment as shown in SEQ ID NO. 1, a heavy chain CDR2 comprises an amino acid fragment as shown in SEQ ID NO. 2, and a heavy chain CDR3 comprises an amino acid fragment as shown in SEQ ID NO. 3; a light chain CDR1 of the neutralizing antibody comprises an amino acid fragment as shown in SEQ ID NO. 4, a light chain CDR2 comprises an amino acid fragment as shown in SEQ ID NO. 5, and a light chain CDR3 comprises an amino acid fragment as shown in SEQ ID NO. 6. The neutralizing antibody can specifically bind to a conserved epitope on the RBD domain on the spike protein of the sapovirus, thereby preventing the infection of the sapovirus on cells in a broad spectrum, and finally achieving a protection effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a neutralizing antibody against Sabeivirus, a preparation method thereof, and an application thereof. Background Art

[0002] Sarbecoviruses include SARS-CoV and SARS-CoV-2, two highly pathogenic coronaviruses that have been reported to infect humans, as well as other coronaviruses from bats and pangolins that may have the ability to infect humans. Therefore, developing broad-spectrum antibodies against Sarbecoviruses is crucial to addressing the potential threat posed by Sarbecoviruses. Summary of the Invention

[0003] Based on this, one of the purposes of the present application includes providing a broad-spectrum neutralizing antibody against Sarbecovirus, which can specifically bind to the conserved epitope on the RBD domain on the spike protein of Sarbecovirus, thereby broadly preventing Sarbecovirus from infecting cells and ultimately achieving a protective effect.

[0004] The technical solutions for achieving the above-mentioned objectives of this application include:

[0005] In a first aspect of the present application, a neutralizing antibody against a Sabeivirus is provided, wherein the heavy chain CDR1 of the neutralizing antibody comprises the amino acid fragment shown in SEQ ID NO.1, the heavy chain CDR2 comprises the amino acid fragment shown in SEQ ID NO.2, and the heavy chain CDR3 comprises the amino acid fragment shown in SEQ ID NO.3;

[0006] The light chain CDR1 of the neutralizing antibody comprises the amino acid fragment shown in SEQ ID NO.4, the light chain CDR2 comprises the amino acid fragment shown in SEQ ID NO.5, and the light chain CDR3 comprises the amino acid fragment shown in SEQ ID NO.6.

[0007] In some embodiments of the present application, the neutralizing antibody satisfies one or more of the following conditions:

[0008] (1) the heavy chain variable region of the neutralizing antibody is shown in SEQ ID NO. 7; and,

[0009] (2) The light chain variable region of the neutralizing antibody is shown in SEQ ID NO.8.

[0010] In some embodiments of the present application, the neutralizing antibody satisfies one or more of the following conditions:

[0011] 1) the species origin of the constant region of the neutralizing antibody is human; and,

[0012] 2) the constant region of the neutralizing antibody is an IgGl constant region.

[0013] In some embodiments of the present application, the neutralizing antibody satisfies one or more of the following conditions:

[0014] (I) the light chain constant region of the neutralizing antibody is as set forth in SEQ ID NO. 9; and,

[0015] (II) the heavy chain constant region of the neutralizing antibody is as set forth in SEQ ID NO. 10.

[0016] In a second aspect of the present application, a recombinant protein is provided, which comprises one or more of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, light chain CDR1, the light chain CDR2, the light chain CDR3, the heavy chain variable region and the light chain variable region defined in the first aspect.

[0017] In a third aspect of the present application, a detection reagent, a detection kit or a medicament is provided, comprising the neutralizing antibody provided in the first aspect or the recombinant protein provided in the second aspect.

[0018] In a fourth aspect of the present application, a nucleic acid is provided, comprising a nucleic acid fragment for encoding the neutralizing antibody provided in the first aspect or the recombinant protein defined in the second aspect.

[0019] In a fifth aspect of the present application, a recombinant expression vector is provided, comprising the nucleic acid provided in the fourth aspect.

[0020] Optionally, the recombinant expression vector is an antibody expression vector.

[0021] Further optionally, the vector is selected from the group consisting of a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus or a combination thereof.

[0022] Still further optionally, the recombinant expression vector is an AbVec2.0-IGHGl and / or AbVec1.1-IGKC expression vector.

[0023] In a sixth aspect of the present application, a host cell is provided, characterized in that it comprises the nucleic acid provided in the fourth aspect or the recombinant expression vector provided in the fifth aspect.

[0024] Optionally, the host cell is a CHO cell, a COS cell, a NSO cell, a HeLa cell, a BHK cell or a HEK293 cell.

[0025] In a seventh aspect of the present application, a method for preparing the neutralizing antibody provided in the first aspect or the recombinant protein provided in the second aspect is provided, the preparation method comprising the following steps:

[0026] The host cell provided in the sixth aspect is cultured, and the antibody or recombinant protein is isolated from the obtained culture.

[0027] In the eighth aspect of the present application, the neutralizing antibody described in the first aspect or the recombinant protein described in the second aspect is provided.

[0028] Application in the preparation of medicines for preventing and treating Sabeivirus infections.

[0029] Compared with conventional technologies, the above technical solutions provided by this application have at least the following beneficial effects:

[0030] The neutralizing antibodies provided by the present application comprise CDRs of specific sequences, which can specifically bind to the spike protein of the sarbecovirus, specifically to the conserved epitopes on the RBD domain on the spike protein of the sarbecovirus, thereby preventing the infection of cells by the sarbecovirus, and ultimately achieving a protective effect. In addition, the neutralizing antibodies provided by the present application have a broad spectrum and can be applied to the infection prevention and control of representative viruses in a variety of SARS-CoV-2 mutant strains and other sarbecoviruses. At the same time, the neutralizing antibodies provided by the present application have a high neutralizing activity. On the whole, the present application provides a new candidate for the prevention and treatment of sarbecovirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0032] Figure 1 This is a graph showing the binding activity test results of the monoclonal neutralizing antibody in Example 2 of the present application;

[0033] Figure 2This is a graph showing the results of the neutralization test of the monoclonal neutralizing antibody against the live virus of the mutant strain of SARS-CoV-2 in Example 2 of this application;

[0034] Figure 3 Graph showing the results of a pseudovirus neutralization test of a representative Sarbecovirus strain using the neutralizing monoclonal antibody in Example 2 of the present application;

[0035] Figure 4 This is an epitope competition experiment between the monoclonal neutralizing antibody in Example 2 of this application and existing antibodies that bind to the RBD domain (B38, REGN10933, P2B-2F6, REGN10987, CR3022 and S309). DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent forms obtained also fall within the protection scope of this application. In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing embodiments and examples and are not intended to limit the present invention.

[0038] the term

[0039] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0040] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0041] In the present invention, "plurality", "multiple", "multiple times", "multiple", etc., unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0042] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0043] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0044] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0045] In the present invention, “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present invention.

[0046] In the present invention, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no conflicts or constraints.

[0047] In the present invention, the terms "first," "second," "third," and "fourth," etc., in "the first aspect," "the second aspect," "the third aspect," and "the fourth aspect," etc., are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," "third," and "fourth," etc., serve only as non-exhaustive enumeration and description and should be understood not to constitute a closed-ended limitation on quantity.

[0048] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0049] In the present invention, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0050] The temperature parameters in the present invention, unless otherwise specified, may be either constant temperature or fluctuating within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the precision range of the instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0051] In the present invention, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0052] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this invention are cited with all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description in this application.

[0053] SARS-CoV and SARS-CoV-2 are currently reported Sarbecovirus viruses that can infect humans, and the new coronavirus pneumonia has brought a significant burden to the world. Neutralizing antibodies targeting SARS-CoV-2 have also been shown to be effective and safe in preventing and treating SARS-CoV-2 virus infection. Specifically, neutralizing antibodies block the binding of SARS-CoV-2 virus to receptors on susceptible cells, thereby preventing SARS-CoV-2 virus from infecting susceptible cells and ultimately achieving a protective effect. The development of neutralizing antibodies targeting conserved epitopes of Sarbecovirus will provide an effective response to potential Sarbecovirus threats.

[0054] The first aspect of the present application

[0055] The present application provides a neutralizing antibody against Sabeivirus, wherein the heavy chain CDR1 of the neutralizing antibody comprises the amino acid fragment shown in SEQ ID NO.1, the heavy chain CDR2 comprises the amino acid fragment shown in SEQ ID NO.2, and the heavy chain CDR3 comprises the amino acid fragment shown in SEQ ID NO.3;

[0056] The light chain CDR1 of the neutralizing antibody comprises the amino acid fragment shown in SEQ ID NO.4, the light chain CDR2 comprises the amino acid fragment shown in SEQ ID NO.5, and the light chain CDR3 comprises the amino acid fragment shown in SEQ ID NO.6.

[0057] Antibodies and variable regions

[0058] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0059] In the present application, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0060] In the present application, the antibody of the present application also includes its conservative variants, which means that compared with the amino acid sequence of the antibody of the present application, at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.

[0061] The present application includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present application also includes fragments, derivatives and analogs of the antibodies.

[0062] In the present application, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, include human and non-human parts and can be obtained by standard recombinant DNA techniques, which are all useful antibodies. A chimeric antibody is a molecule in which different parts are derived from different animal species, such as a chimeric antibody having a variable region from a mouse monoclonal antibody and a constant region from a human immunoglobulin (see, for example, U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, which are incorporated herein by reference in their entirety). These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.

[0063] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0064] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes, called kappa and lambda, based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into five main classes based on the amino acid sequence of their heavy chain constant regions: IgA, IgD, IgE, IgG, and IgM. Some of these are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different immunoglobulin classes are well known in the art.

[0065] Generally, an antibody's antigen-binding properties are described by three specific regions located in the variable regions of the heavy and light chains, known as the variable regions (CDRs). These regions are divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0066] Antibody preparation

[0067] The fragments of the DNA molecules of the antibodies or antigen-binding fragments thereof of the present application can be obtained using conventional techniques, such as by methods such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and heavy chain can be fused together to form single-chain antibodies. Once the relevant sequence information is obtained, recombinant methods can be used to obtain the relevant sequence fragments in large quantities. This is usually done by cloning them into vectors, then transferring them into cells, and then isolating the relevant sequence fragments from the host cells after propagation by conventional methods.

[0068] In addition, artificial synthesis methods can also be used to synthesize relevant sequence fragments, especially when the fragment length is shorter. Usually, by first synthesizing multiple small fragments and then connecting them, very long fragments of sequence can be obtained.

[0069] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0070] The present application also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0071] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0072] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0073] The resulting monoclonal antibodies can be characterized using conventional methods. For example, the binding specificity of the monoclonal antibodies can be determined using immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, using the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0074] The antibodies of the present application can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic lysis, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

[0075] Optionally, the neutralizing antibody satisfies one or more of the following conditions:

[0076] (1) the heavy chain variable region of the neutralizing antibody is as set forth in SEQ ID NO. 7; and,

[0077] (2) the light chain variable region of the neutralizing antibody is as set forth in SEQ ID NO. 8.

[0078] Optionally, the neutralizing antibody satisfies one or more of the following conditions:

[0079] 1) the species origin of the constant region of the neutralizing antibody is human; and,

[0080] 2) the constant region of the neutralizing antibody is an IgGl constant region.

[0081] Optionally, the neutralizing antibody satisfies one or more of the following conditions:

[0082] (I) the light chain constant region of the neutralizing antibody is as set forth in SEQ ID NO. 9; and,

[0083] (II) the heavy chain constant region of the neutralizing antibody is as set forth in SEQ ID NO. 10.

[0084] The second aspect of this application

[0085] The present application provides a recombinant protein comprising one or more of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, light chain CDR1, the light chain CDR2, the light chain CDR3, the heavy chain variable region, and the light chain variable region defined in the first aspect. Optionally, the recombinant protein further comprises a combination of the heavy chain constant region and the light chain constant region defined in the first aspect.

[0086] Optionally, the recombinant protein can further comprise a tag fragment that assists in the expression and / or purification of the recombinant protein, including but not limited to a 6His tag.

[0087] In the present application, the recombinant protein (or polypeptide) includes but is not limited to fusion protein.

[0088] In the present application, the recombinant protein can be monomer, dimer, or multimer.

[0089] The third aspect of this application

[0090] The present application provides a detection reagent, a detection kit or a medicament, comprising the neutralizing antibody provided in the first aspect or the recombinant protein provided in the second aspect.

[0091] In the present application, "medicament" refers to any compound having desired biological activity and having reactive functional groups for preparing the conjugate of the present application. The desired biological activity includes diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals. Therefore, the term "medicament" refers to compounds identified by the official compendia, such as the United States Pharmacopeia, and the official compendia of homoeopathic therapeutics, the National Formulary, or any supplement to them. Typical medicaments are listed in the Physicians' Desk Reference (PDR) and the Orange Book of the U.S. Food and Drug Administration (FDA). It should be understood that as new types of medicaments are discovered and developed, they should also be included in the "medicament" of the conjugate of the present application.

[0092] The fourth aspect of this application

[0093] The present application provides a nucleic acid comprising a nucleic acid fragment for encoding the neutralizing antibody provided in the first aspect or the recombinant protein defined in the second aspect.

[0094] The fifth aspect of this application

[0095] The present application provides a recombinant expression vector comprising the nucleic acid provided in the fourth aspect.

[0096] Alternatively, the recombinant expression vector is an antibody expression vector. The antibody expression vector of the present application is not particularly limited, including but not limited to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or a combination thereof. Further alternatively, the recombinant expression vector is AbVec2.0-IGHG1 and / or AbVec1.1-IGKC expression vector.

[0097] The sixth aspect of this application

[0098] The present application provides a host cell comprising the nucleic acid provided in the fourth aspect or the recombinant expression vector provided in the fifth aspect.

[0099] In some examples, the nucleic acid can be integrated into the genome of the host cell. The present application does not specifically limit the type of host cell. Examples include, but are not limited to, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, and HEK293 cells. For example, the host cell is a 293T cell or an Expi293F cell.

[0100] The seventh aspect of this application

[0101] The present application provides a method for preparing the neutralizing antibody provided in the first aspect or the recombinant protein provided in the second aspect, the preparation method comprising the following steps:

[0102] The host cell provided in the sixth aspect is cultured, and the antibody or recombinant protein is isolated from the obtained culture.

[0103] The eighth aspect of the present application

[0104] The present application provides the use of the neutralizing antibody described in the first aspect or the recombinant protein described in the second aspect in the preparation of a drug for preventing and treating Sabeivirus infection.

[0105] The definition of drug refers to the third aspect above.

[0106] In this application, prevention and treatment include prevention, control, auxiliary treatment, etc. Specific embodiments

[0108] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made according to experimental manuals or conventional conditions in the art, or according to conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0109] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0110] Example 1. Construction of an expression vector for monoclonal neutralizing antibodies against Sarbecovirus Construction, expression and purification

[0111] The monoclonal neutralizing antibody against Sarbecovirus prepared in this example comprises a heavy chain variable region comprising VHCDR1 as shown in SEQ ID NO.1, VHCDR2 as shown in SEQ ID NO.2, and VHCDR3 as shown in SEQ ID NO.3, and a light chain variable region comprising VLCDR1 as shown in SEQ ID NO.4, VLCDR2 as shown in SEQ ID NO.5, and VLCDR3 as shown in SEQ ID NO.6.

[0112] Specifically, its heavy chain variable region is shown in SEQ ID NO.7, its light chain variable region is shown in SEQ ID NO.8, its light chain constant region is shown in SEQ ID NO.9, and its heavy chain constant region is shown in SEQ ID NO.10.

[0113] SEQ ID NO.1: GFSFSDAW,

[0114] SEQ ID NO.2: VSSEIGGGTT,

[0115] SEQ ID NO.3: TTGVDIVVMMYADDAFDI,

[0116] SEQ ID NO.4: SLRSYY,

[0117] SEQ ID NO.5: GKN,

[0118] SEQ ID NO.6:TSRDSSGNHVI,

[0119] SEQ ID NO.7:

[0120] EVQLVESGGGLVKPGGSLRLSCAAS GFSFSDAW MSWVRQAPGKGLEWVGR VSSEIGGGTT DYAAP VKGRFTISRDDSKNTLFLQMSSLKTEDTAVYYC TTGVDIVVMMYADDAFDI WGQGTMVTVSS,

[0121] SEQ ID NO.8:

[0122] SSELTQDPAVSVALGQTVRITCQGD SLRSYY ASWYQQKPGQAPVLVIY GKN NRPSGIPDRFSGSSSGNTASLTITGPQAEDEADYYC TSRDSSGNHVI FGGGTKLTVL,

[0123] SEQ ID NO.9:

[0124] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,

[0125] SEQ ID NO.10:

[0126] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0127] The method for preparing the monoclonal neutralizing antibody against Sarbecovirus in this embodiment comprises the following steps:

[0128] 1. The nucleotide sequence fragments encoding the heavy chain variable region (SEQ ID NO. 7) and light chain variable region (SEQ ID NO. 8) of the monoclonal neutralizing antibody against Sarbecovirus were respectively integrated into AbVec2.0-IGHG1 and AbVec1.1-IGKC containing the heavy and light chain constant region sequence fragments of human IgG1 antibody (reference for vector use: Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. Tiller T, Meffre E, Yurasov S, Tsuiji M, Nussenzweig MC, Wardemann HJ Immunol Methods. 2008 Jan 1; 329(1-2): 112-24. Epub 2007 Oct 31.10.1016 / j.jim.2007.09.017 PubMed). 17996249), and obtained recombinant expression vectors capable of expressing the heavy chain and light chain of the target antibody respectively.

[0129] 2. Cell Transfection, Expression and Purification of Monoclonal Neutralizing Antibodies Against Sarbecovirus

[0130] 1. Cell transfection and expression of monoclonal neutralizing antibodies

[0131] The Gibco Expi293F expression system was used and transfection was performed according to the instructions. The steps are briefly described as follows:

[0132] 30 μg of DNA from two recombinant expression vectors expressing the heavy and light chains of the monoclonal neutralizing antibody were mixed with the transfection reagent ExpiFectamine TM Mix and let stand at room temperature for 20 minutes to form a stable complex;

[0133] Then add 25.5mL to adjust the concentration to 2.9×10 6 cells / mL in Expi 293F cell culture medium;

[0134] Incubate at 37°C, 8% (v / v) CO2, and 125 rpm in a shaker for 20 h;

[0135] Add transfection enhancer 1 and transfection enhancer 2 provided by the Expi293F expression system;

[0136] Continue to be placed in 37℃, 8% (v / v) CO2, 125 rpm shaker for 4 days.

[0137] 2、Purification

[0138] The culture prepared in step 1 was collected and centrifuged at 3000 rpm for 15 minutes to collect the cell culture supernatant, and the antibody was purified by Protein A magnetic beads from Genscript. The purification steps are briefly described as follows:

[0139] Mix the Protein A magnetic beads with the cell culture supernatant and combine on a shaker at room temperature for 4 hours;

[0140] Magnetic stand adsorbs magnetic beads, discards cell supernatant, and washes magnetic beads with 1x PBS containing 0.1% (v / v) Tween 20 at pH 7.0 for 5 times;

[0141] Eluted with Elution buffer of pH 2.0 0.1M glycine;

[0142] Equilibrated with pH 8.5 1M Tris buffer;

[0143] Take the equilibrated monoclonal neutralizing antibody for desalting and DPBS solvent replacement.

[0144] The purified neutralizing antibody was stored in a -80℃ refrigerator.

[0145] Example 2 Functional analysis of monoclonal neutralizing antibodies against Sarbecovirus

[0146] 1. Detection of the binding activity of the human-derived broad-spectrum monoclonal neutralizing antibody against Sarbecovirus prepared in Example 1 to antigens

[0147] The ability of the monoclonal neutralizing antibody to bind to SARS-CoV-2 spike protein S, SARS-CoV spike protein S, and RBD domains of SARS-CoV-2, SARS-CoV, Bat-CoV RaTG13, and Pangolin-CoV-2020 was determined by ELISA.

[0148] The steps are briefly described as follows:

[0149] (1) Coat 25 ng of SARS-CoV-2 spike protein S (SinoBiological, 40589-V08H25), SARS-CoV spike protein S, and SARS-CoV-2, SARS-CoV, Bat-CoV RaTG13, and Pangolin-CoV-2020 RBD proteins per well on an ELISA plate with DPBS (Dulbecco's Phosphate Buffered Saline) as the coating liquid at 4℃ overnight;

[0150] (2) 10% (v / v) calf serum in DPBS was used as a blocking solution and the cells were blocked at 37°C for 2 hours. The neutralizing antibody to be tested prepared in Example 1 after serial dilution was added and the cells were incubated at 37°C for 2 hours.

[0151] (3) Add HRP-conjugated Goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) diluted 1:40,000 as the secondary antibody and incubate at 37°C for 1 hour;

[0152] (4) After developing with TMB single-component colorimetric solution, the reaction was terminated with 2 M sulfuric acid and the absorbance A450 value was measured using a microplate reader.

[0153] result:

[0154] The results of the monoclonal neutralizing antibody binding activity test are shown in Figure 1 ,Depend on Figure 1 It can be seen that the binding activity of the monoclonal neutralizing antibody against SARS-CoV-2S protein is EC50 = 0.02 μg / mL, the binding activity against SARS-CoV S protein is EC50 = 0.07188 μg / ml, and the EC50 for SARS-CoV-2, SARS-CoV, Bat-CoV RaTG13 and Pangolin-CoV-2020RBD protein are 0.01832 μg / mL, 0.6423 μg / mL, 0.3532 μg / mL and 0.4605 μg / mL, respectively.

[0155] 2. Neutralization activity test of the broad-spectrum monoclonal neutralizing antibody against Sarbecovirus prepared in Example 1 against live SARS-CoV-2 mutant strains

[0156] The SARS-CoV-2 live virus neutralization experiment was performed using the SARS-CoV-2 live virus wild type strain (WT) SARS-CoV-2 / human / CHN / IQTC01 / 2020, Alpha mutant strain, Beta mutant strain, Eta mutant strain, Delta mutant strain and OmicronBA.1.1 mutant strain.

[0157] The steps are briefly described as follows:

[0158] The quantitative SARS-CoV-2 virus and serially diluted monoclonal neutralizing antibodies were mixed and incubated at 37°C for 1 hour, then added to a 96-well plate of pre-plated Vero E6 cells and cultured at 37°C for 24 hours.

[0159] The cell plates were fixed with 4% (v / v) paraformaldehyde for 2 hours before staining.

[0160] permeabilization with 0.2% (v / v) Triton X-100 at room temperature for 20 minutes;

[0161] Cross-reactive rabbit anti-SARS-CoV-N IgG (Sino Biological Inc) was used as the primary antibody and incubated at 37°C for 1 h to label viral antigens;

[0162] HRP-conjugated Goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch) was used as the secondary antibody and incubated at 37°C for 1 hour;

[0163] KPL TrueBlue Peroxidase substrates (SeraCare Inc) were used as the chromogenic substrate and the color was developed for 5 min;

[0164] The plate was scanned using a CTL ImmunoSpot S6 Ultra reader (Cellular Technology Ltd), and the number of stained viral antigen foci was counted. The virus neutralization activity (expressed as EC50) was also calculated.

[0165] result:

[0166] The results of the live virus neutralization test of monoclonal neutralizing antibodies are shown in Figure 2 ,Depend on Figure 2 It can be seen that this monoclonal neutralizing antibody can broadly neutralize a variety of SARS-CoV-2 virus mutants, and has good neutralizing activity (WT, 3.986μg / mL; Alpha, 1.594μg / mL; Beta, 3.815μg / mL; Delta, 1.801μg / mL; Eta, 3.877μg / mL; OmicronBA.1.1, 6.488μg / mL).

[0167] 3. Neutralization activity test of the human anti-Sarbecovirus broad-spectrum monoclonal neutralizing antibody prepared in Example 1 against pseudoviruses of representative Sarbecovirus strains

[0168] The antibody neutralizing activity was tested using pseudoviruses SARS-CoV, Pangolin-CoV-2020 and Bat-CoV WIV1, representative strains of the Sarbecovirus genus.

[0169] The steps are briefly described as follows:

[0170] The pCDNA3.1-SARS-CoV-S plasmid, pCDNA3.1-Pangolin-CoV-2020-S plasmid, and pCDNA3.1-Bat-CoV WIV1-S plasmid were transfected into 293T cells with 2 μg each of pSPAX2 and pLenti-GFP-luciferase, and the cell supernatant was collected 48 hours later to prepare pseudoviruses corresponding to SARS-CoV, Pangolin-CoV-2020, and Bat-CoV WIV1;

[0171] The pseudovirus and serially diluted monoclonal neutralizing antibodies were mixed and incubated at 37°C for 1 hour, then added to the digested 293T-ACE2 cells and cultured in 96-well white plates at 37°C for 48 hours;

[0172] After discarding the liquid from the cell plate, add the luciferase colorimetric substrate (Steady-Glo Luciferase asssy), incubate at room temperature for 3 minutes, and detect the fluorescence value;

[0173] The inhibition rate was calculated by comparing the readings with those of the wells with only pseudovirus added, and the pseudovirus neutralizing activity (expressed as EC50) was calculated.

[0174] result:

[0175] The results of the neutralization activity of monoclonal neutralizing antibodies against representative Sarbecovirus pseudovirus strains are shown in Figure 3 ,Depend on Figure 3 It can be seen that the neutralizing antibody can broadly neutralize a variety of Sarbecovirus viruses (Sarbecovirus), and has good neutralizing activity (SARS-CoV, 0.274μg / mL; Pangolin-CoV-2020, 0.2443μg / mL; Bat-CoV WIV1, 8.433μg / mL).

[0176] 4. Epitope competition experiment between the human anti-Sarbecovirus broad-spectrum monoclonal neutralizing antibody prepared in Example 1 and existing antibodies binding to the RBD domain (B38, REGN10933, P2B-2F6, REGN10987, CR3022 and S309)

[0177] A competitive ELISA experiment was used to compare the binding epitopes of existing antibodies that bind to the SARS-CoV-2 RBD domain (B38, REGN10933, P2B-2F6, REGN10987, CR3022 and S309) and the human anti-Sarbecovirus broad-spectrum monoclonal neutralizing antibody prepared in Example 1.

[0178] The steps are briefly described as follows:

[0179] (1) SARS-CoV-2 RBD protein was coated in a 96-well microtiter plate at a coating concentration of 2 μg / mL using Dulbecco's phosphate buffered saline (DPBS) as the coating solution at 4°C overnight.

[0180] (2) 10% (v / v) calf serum in DPBS was used as blocking solution and blocked at 37°C for 2 hours;

[0181] (3) The human anti-Sarbecovirus broad-spectrum monoclonal neutralizing antibody prepared in Example 1 and existing antibodies that bind to the SARS-CoV-2 RBD domain (B38, REGN10933, P2B-2F6, REGN10987, CR3022, and S309) were diluted to 20 μg / mL with DPBS (Dulbecco's phosphate buffer), added to the ELISA plate, and incubated at room temperature for 2 hours;

[0182] (4) The human anti-Sarbecovirus broad-spectrum monoclonal neutralizing antibody prepared in Example 1 and existing antibodies binding to the SARS-CoV-2 RBD domain (B38, REGN10933, P2B-2F6, REGN10987, CR3022, and S309) were biotinylated using Thermo Fisher's EZ-link NHS-PEG4-Biotin biotin labeling kit. 20 μg / mL of the biotinylated antibodies were added to the ELISA plate and incubated at room temperature for 2 hours.

[0183] (5) Add HRP-labeled streptavidin from Beyotime to the ELISA plate and incubate at room temperature for 1 hour;

[0184] (6) After developing with TMB single-component colorimetric solution, the reaction was terminated with 2 M sulfuric acid, and the absorbance A450 value was measured using a microplate reader;

[0185] (7) The competition rate was calculated based on the A450 value of the competition ELISA absorbance, and an inhibition rate greater than 60% was defined as mutual competition.

[0186] result:

[0187] The results of competitive ELISA are shown in Figure 4 It can be seen that the competition rate of the broad-spectrum monoclonal neutralizing antibody against Sarbecovirus (marked with a black asterisk) prepared in Example 1 and the existing antibodies that bind to the SARS-CoV-2 RBD domain (B38, REGN10933, P2B-2F6, REGN10987, CR3022 and S309) is significantly less than 60%, and there is no epitope competition relationship. The epitope of the broad-spectrum monoclonal neutralizing antibody against Sarbecovirus prepared in Example 1 is specific.

[0188] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0189] The embodiments described above only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. In addition, it should be understood that after reading the above-mentioned teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A neutralizing antibody against the spike protein of a Sabeivirus, characterized in that: The amino acid sequence of the heavy chain CDR1 of the neutralizing antibody is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.3; The amino acid sequence of the light chain CDR1 of the neutralizing antibody is shown in SEQ ID NO.4, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.

6.

2. The neutralizing antibody against the spike protein of Sabeivirus according to claim 1, characterized in that The neutralizing antibody meets one or both of the following conditions: (1) the heavy chain variable region of the neutralizing antibody is shown in SEQ ID NO. 7; and, (2) The light chain variable region of the neutralizing antibody is shown in SEQ ID NO.

8.

3. The neutralizing antibody against the spike protein of a Sabeivirus according to claim 1 or 2, characterized in that The neutralizing antibody meets one or both of the following conditions: 1) The species origin of the constant region of the neutralizing antibody is human; and, 2) The constant region of the neutralizing antibody is an IgG1 constant region.

4. The neutralizing antibody against the spike protein of Sabeivirus according to claim 1 or 2, characterized in that The neutralizing antibody meets one or both of the following conditions: (I) the light chain constant region of the neutralizing antibody is shown in SEQ ID NO. 9; and, (II) The heavy chain constant region of the neutralizing antibody is shown in SEQ ID NO.

10.

5. A detection reagent, a detection kit or a drug, characterized in that: Comprising the neutralizing antibody according to any one of claims 1 to 4.

6. A nucleic acid, characterized in that It is used to encode the neutralizing antibody according to any one of claims 1 to 4.

7. A recombinant expression vector, characterized in that: Comprising the nucleic acid according to claim 6.

8. The recombinant expression vector according to claim 7, characterized in that The recombinant expression vector is selected from bacterial plasmid, bacteriophage, yeast plasmid, mammalian cell virus, retrovirus or a combination thereof.

9. The recombinant expression vector according to claim 8, characterized in that The mammalian cell virus includes adenovirus.

10. The recombinant expression vector according to claim 9, characterized in that The recombinant expression vector is AbVec2.0-IGHG1 and / or AbVec1.1-IGKC expression vector.

11. A host cell, characterized in that Comprising the nucleic acid according to claim 6 or the recombinant expression vector according to any one of claims 7 to 10.

12. The host cell according to claim 11, characterized in that The host cell is a CHO cell, a COS cell, a NSO cell, a HeLa cell, a BHK cell or a HEK293 cell.

13. The method for preparing the neutralizing antibody according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: The host cell according to any one of claims 11 to 12 is cultured, and the antibody is isolated from the obtained culture.

14. Use of the neutralizing antibody according to any one of claims 1 to 4 in the preparation of a medicament for treating Sabeivirus infection.

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