CN115697392A - Method for reducing virus ADE effect - Google Patents

Abstract

A method of reducing the effects of viral ADE is provided by administering a molecule that reduces the effects of viral ADE to a subject infected by, or at risk of infection by, a virus. By molecular biological modification of the Fc fragment of an antibody, an Fc fragment with reduced Fc receptor binding/complement binding properties is obtained. Antibodies comprising the Fc fragment are capable of reducing the effects of viral ADE. The antibody can be preferably used for preventing and/or treating acute respiratory infectious diseases caused by coronavirus infection.

Description

Method for reducing virus ADE effect

Cross Reference to Related Applications

This application claims the benefit of chinese patent application 202010598424.8 filed on 28/06/2020, the contents of which are incorporated herein by reference.

Technical Field

The invention relates to the technical field of cellular immunity, and provides a method for reducing virus ADE effect and a molecule for reducing virus ADE effect. In particular, the invention provides a molecule comprising an Fc fragment with reduced Fc receptor binding/complement binding, thereby achieving a reduction in viral ADE. The invention also provides a humanized antibody which can reduce the effect of virus ADE, combine and seal the combination of SARS-CoV-2spike protein (S protein) and ACE2 receptor, efficiently neutralize the SARS-CoV-2 virus infected cell, and reduce the effect of virus ADE, can cross-seal the combination of SARS-CoV-2 and SARS-CoV spike protein (S protein) and ACE2 receptor, and efficiently neutralize the SARS-CoV-2 and SARS-CoV virus infected cell.

Background

Neutralization of viruses by antibodies is an important mechanism of defense of the host against viral infection, and antibodies can achieve viral clearance through effector functions of their Fc segments, including complement-mediated lysis of viral particles, antibody-mediated cytotoxicity, and phagocytosis [1]. However, many viruses can utilize the property of antibodies to promote infection of host cells, regulate the signaling pathways of host cells, and thus suppress the antiviral immune response, a process known as Antibody-dependent enhancement (ADE).

The ADE phenomenon was originally found in dengue virus (DENGuevirus, DENV) [2], and has been reported in vitro in a number of virus types including Ebola virus (Ebola virus), zika virus (ZIKV), chikungunya virus (Chikungunya virus), severe acute respiratory syndrome virus (SARS) [3-5]. Although it is debated whether ADE is a major risk in the clinic, there are several lines that suggest that ADE may be one of the important factors in antiviral drug and vaccine development that is not negligible.

A retrospective analysis of a clinical trial indicated that the earliest approved dengue virus vaccine CYD-TDV was renegotiated for an applicable vaccine strategy because it carries a risk of exacerbating infection [6], suggesting that ADE may affect the effectiveness of the vaccine in the clinic. In addition, among SARS-infected patients, severe patients detected higher titers of anti-SARS immunoglobulin than serum from mild patients [7]. This phenomenon has also been confirmed in several recent studies on a novel coronavirus (SARS-CoV-2): the severity of the new corona infected patients has a positive correlation with the titer of total antibody IgG [8-10]. Reducing the ADE of the antibody may therefore improve safety during treatment of neocoronary infections with neutralizing antibodies.

The effects of ADE on the immune system can vary widely, and ADE with reduced antibodies has important instructional significance for vaccines, serum from convalescent individuals, and neutralizing antibodies to prevent or treat new coronavirus infections.

Disclosure of Invention

In order to avoid the influence of the virus ADE effect on the using effect of the vaccine and pursue safer clinical effect, the inventor uses a means of molecular biology to modify the corresponding Fc fragment to obtain the Fc fragment molecule with reduced Fc receptor binding/complement binding, thereby realizing the reduction of the virus ADE effect. It was verified that the Fc engineered fragment molecules provided by the present invention, when administered as a therapeutic or prophylactic agent to a virus-infected person or a subject at risk of a virus infection, can exhibit reduced binding to Fc γ R-expressing cells such as B cells, monocytes, macrophages, dendritic cells, and thus can reduce a virus infection. Further, the inventors have invented a molecule that reduces the effects of viral ADE, the molecule comprising a) an antigen-binding fragment that recognizes and binds to the viral coat protein or ectodomain; and b) the aforementioned Fc receptor binding/complement binding reduces Fc fragments.

Detailed Description

The invention aims to provide a method for reducing the effect of virus ADE.

It is another object of the invention to provide a molecule comprising an Fc fragment with reduced Fc receptor binding/complement binding, thereby achieving a reduction in viral ADE.

It is another object of the invention to provide a molecule that reduces the effects of viral ADE, the molecule comprising a) an antigen-binding fragment that recognizes and binds to the viral coat protein or extracellular domain; and b) the aforementioned Fc receptor binding/complement binding reducing Fc fragment.

It is still another object of the present invention to provide humanized antibodies that reduce the effects of viral ADE, block the binding of SARS-CoV-2spike protein (S protein) to the ACE2 receptor, and neutralize SARS-CoV-2 virus infected cells with high efficiency.

It is still another object of the present invention to provide humanized antibodies that can cross-block the binding of SARS-CoV-2 and SARS-CoV spike protein (S protein) to the ACE2 receptor, and neutralize SARS-CoV-2 and SARS-CoV virus infected cells with high efficiency, reducing the effects of viral ADE.

Accordingly, the present invention provides polynucleotides capable of encoding molecules having reduced Fc receptor binding/complement binding properties.

The invention also provides a recombinant vector comprising the polynucleotide.

The invention also provides a host cell comprising the polynucleotide and/or the recombinant vector.

The invention also provides an antibody formed by the polynucleotide after being expressed by a recombinant vector and/or a host cell.

The Fc fragment molecules with reduced Fc receptor binding/complement binding provided by the present invention can be further prepared into vaccines comprising as an active ingredient one or more of the above-described Fc modified fragment molecules, the above-described polynucleotides, the above-described recombinant vectors, the above-described host cells, recombinant bacteria, adenoviruses, lentiviruses, or viral particles.

In one possible implementation, the vaccine includes one or more of an inactivated vaccine, an attenuated vaccine, an mRNA vaccine, a DNA vaccine, an adenoviral vector vaccine, other viral vector vaccines, subunit vaccines, or viral particles.

In one possible implementation of the above vaccine, the vaccine further comprises any one or a combination of at least two of a pharmaceutically acceptable vehicle, diluent, adjuvant or excipient.

The invention also provides the Fc fragment molecule with reduced Fc receptor binding/complement binding, the antibody, the polynucleotide, the recombinant vector, the host cell, the recombinant bacterium, the adenovirus, the lentivirus or the virus particle, and the application of the Fc fragment molecule, the antibody, the polynucleotide, the recombinant vector, the host cell, the recombinant bacterium, the adenovirus, the lentivirus or the virus particle in preparing the vaccine for preventing and/or treating the novel coronavirus infection.

The invention also provides a method of treatment for preventing or treating a disease or condition caused by a novel coronavirus comprising administering a molecule comprising an Fc-engineered fragment according to the invention, an antibody as described above, a polynucleotide as described above, a recombinant vector, a transgenic cell line, a recombinant bacterium, an adenovirus, a lentivirus or a viral particle.

Drawings

FIG. 1: flow identification of corresponding FcR expression of CHO-K1-CD32A, CHO-K1-CD32B and CHO-K1-CD64 cells

FIG. 2 is a schematic diagram: fd11 engineering to reduce binding of CoV2-HB27-IgG1 antibodies to Fc receptor proteins

FIG. 3: fd11 engineering to reduce binding of CoV2-HB27-IgG1 antibodies to complement C1q protein

FIG. 4 is a schematic view of: fd11 engineering to reduce the ADCC Effect of CoV2-HB27-IgG1 antibodies

FIG. 5 is a schematic view of: the CoV2-HB27 antibodies of the IgG1 subtype and the Fd11-IgG4 subtype have substantially no ADCP effect

FIG. 6

FIG. 7: fd6 and Fd11 modification for reducing ADE effect of CoV2-HB27-IgG1 antibody on CHO-K1-CD32A cells

FIG. 8: fd6 and Fd11 are modified to reduce ADE effect of CoV2-HB27-IgG1 antibody on CHO-K1-CD32B cells

FIG. 9: fd11 engineering to reduce the ADE Effect of SARS-2-H014-IgG1 antibody on CHO-K1-CD64 cells

FIG. 10: fd6 and Fd11 modification for reducing ADE effect of CoV2-HB27-IgG1 antibody on Raji cells

FIG. 11: fd11 engineering to reduce the ADE Effect of SARS-2-H014-IgG1 antibodies on THP-1 cells

FIG. 12: fd11 engineering to reduce the ADE Effect of SARS-2-H014-IgG1 antibodies on U937 cells

Detailed Description

Reducing the ADE of antibodies is an important task in the development of antiviral biopharmaceuticals and vaccines.

The invention establishes a method for reducing the effect of virus ADE, and particularly, the inventor modifies an antibody Fc fragment to reduce the binding of the antibody Fc fragment to Fc receptor/complement so as to reduce the virus ADE.

Viruses include, but are not limited to, coronavirus, influenza virus, cold virus, parainfluenza virus, upper respiratory syncytial virus, dengue virus, west nile virus, marburg virus, lasalosis virus, HIV virus, ebola virus, herpes zoster virus, CMV virus, hepatitis virus, human herpes simplex virus, cytomegalovirus, rotavirus, EB virus, measles virus, mumps virus, human papilloma virus, flavivirus or influenza virus; preferably SARS-CoV-2, SARS-CoV, MERS-CoV; influenza a viruses (including H10N8, H7N9, H1N1, H5N1, etc.), influenza B viruses; ebola virus; and pseudoviruses and true viruses of hepatitis A, B, C, and E.

By "antibody-dependent potentiation (ADE)", otherwise known as immune enhancement or disease enhancement, is meant that after binding of the virus to a non-neutralizing antibody, or to a sub-neutralizing concentration of antibody, the Fc portion of the antibody binds to FcR surface-expressing cells and mediates entry of the virus into those cells, thereby enhancing the infectivity of the virus. This phenomenon leads to increased infectivity and toxicity, and ADE modulates the immune response and can cause persistent inflammation, lymphopenia and/or cytokine storm.

The main mechanism of ADE is that under the conditions of incomplete neutralization or non-neutralization of the antibody against the virus, the Fc fragment of the antibody in the antigen-antibody complex is bound to cells expressing Fc γ R such as B cells, monocytes, macrophages, dendritic cells, etc., and then endocytosis to the target cell is achieved [11,12]. In other words, the antibody helps the virus to enter the target cell resulting in an increase in the number of infected cells. This process, referred to as exogenous ADE, is Fc γ receptor (Fc γ R) dependent, occurring in the context of secondary infection of organisms with heterologous serotype viruses. Circulating antibodies produced during primary infection virus recognize and bind to secondary infection virus, enhancing virus infectivity through intracellular virus-antibody immune complexes carrying fcyr, rather than promoting virus neutralization. Once internalized, these immune complexes may modulate the innate anti-viral cell response, causing a large increase in the amount of virus produced per cell, a process known as endogenous ADE. Exogenous and endogenous ADE together contribute to inflammation and the massive release of vasoactive mediators, ultimately leading to disease progression [12].

The term "receptor" is a biochemical concept and refers to a class of molecules that can transmit extracellular signals and produce specific effects within the cell. The resulting effect may last only for a short time, such as altering the metabolism of the cell or the movement of the cell. It may also be a long-lasting effect, such as up-or down-regulating the expression of a certain gene or genes.

The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. Receptors to which IgG antibodies bind are gamma receptors, which include Fc γ RI, fc γ RII, and Fc γ RIII subtypes. Human Fc γ receptors mainly include Fc γ RI (CD 64), fc γ RII (CD 32), fc γ RIII (CD 16). There are some differences in the ADE-dependent Fc receptor elicited by different virus types, SARS, dengue are mainly reported to elicit ADE via CD32A [11,13], MERS are mainly responsible for ADE via CD32, CD64 [14], chikungunya virus is mainly responsible for ADE via CD32, CD16 [5].

Macrophages are important cell types for phagocytosis of antigen-antibody complexes, and express several receptors, CD16, CD32, and CD 64. In rhesus monkey animal models, after SARS infection, antibodies can induce more immune cells (especially inflammatory macrophages) to infiltrate into the lung, and simultaneously, virus-induced macrophage ADE can induce anti-inflammatory macrophage M2 to produce more inflammatory factors IL-6, IL-8, MCP-1 and the like, thus forming strong lung injury [15].

B cells have strong CD32 expression, and the virus has relatively few studies on the effects on B cell functions after B cell infection by ADE, and antiserum generated by SARS-CoV vaccine can enhance the infection of B cells by virus [16]. The B cell composition of patients after dengue virus infection is significantly different from that of normal, the number of immature B, transitional B cells and Breg in severe patients is significantly lower than that of mild patients, and the stimulated immature B cells are unable to produce the expression of IL-10, activation markers and antigen presenting molecules, so that the acute infection phase of dengue has a severe impact on B cell function [17], which may be related to ADE.

The inventors have used molecular biological means to modify the corresponding Fc fragment to obtain Fc fragment molecules with reduced Fc receptor binding/complement binding, thereby achieving a reduction in the effects of viral ADE. Further, the inventors have invented a molecule that reduces the effects of viral ADE, the molecule comprising a) an antigen-binding fragment that recognizes and binds to the viral coat protein or ectodomain; and b) the aforementioned Fc receptor binding/complement binding reduces Fc fragments. Comprising a) an antigen-binding fragment that recognizes and binds to a viral coat protein or an extracellular domain; and b) an Fc fragment, wherein b) the Fc receptor binding and/or complement binding is reduced by molecular biological engineering. When the molecule is administered as a therapeutic or prophylactic agent to a subject infected with, or at risk of infection by, a virus, binding to specific cells, such as B cells, monocytes, macrophages, dendritic cells and the like, expressing Fc γ rs, is reduced due to reduced Fc receptor binding and/or complement binding, with the consequence that endocytosis of the target cell is reduced. Thereby reducing viral infection.

In one embodiment of the present invention, the inventors engineered antibodies COV2-HB27 and SARS-2-H014.CoV2-HB27 is a humanized antibody capable of blocking the combination of SARS-CoV-2spike protein (S protein) and ACE2 receptor and neutralizing SARS-CoV-2 virus infected cell with high efficiency. Details of their preparation, structure and properties are given in patent applications 202010349190.3 and PCT/CN2021/089748 (incorporated herein by reference in their entirety). SARS-2-H014 is humanized antibody capable of cross-blocking binding of SARS-CoV-2 and SARS-CoV spike protein (S protein) to ACE2 receptor, and neutralizing SARS-CoV-2 and SARS-CoV virus infected cells with high efficiency. Their preparation, structure and properties are described in detail in patent applications 202010219867.1 and PCT/CN2021/082374 (incorporated herein by reference in its entirety).

The term "virus-like particle" (VLP) or "pseudovirus" refers to a polyprotein structure composed of the corresponding native viral structural proteins, but lacking all or part of the viral genome, in particular the replicative and infectious components of the viral genome, and thus being non-replicative and infectious. The polyprotein structure mimics its corresponding native viral particle in morphology and size to a high degree, and can form spontaneously upon recombinant expression of the viral structural proteins.

The term "antibody" means an immunoglobulin molecule, and refers to any form of antibody that exhibits a desired biological activity. Including but not limited to monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), even including antibody fragments. Typically, a full-length antibody structure preferably comprises 4 polypeptide chains, 2 heavy (H) chains and 2 light (L) chains, usually interconnected by disulfide bonds.

Intact antibodies can be assigned to the five classes IgA, igD, igE, igG and IgM antibodies, depending on the amino acid sequence of their heavy chain constant region, where IgG and IgA can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1 and IgA2. Accordingly, the heavy chains of five classes of antibodies fall into the α, δ, ε, γ and μ chains, respectively. The light chain of an antibody can be classified into κ and λ according to the amino acid sequence of its light chain constant region.

In the case of IgG antibodies, each heavy chain has, from its N-terminus to its C-terminus, a variable region (VH, heavy chain variable domain) followed by 3 constant domains (CH 1, CH2 and CH3, also known as heavy chain constant regions). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL, light chain variable domain) followed by a constant region (CL, also known as light chain constant region). IgG antibodies are cleaved by papain to form two "Fab portions" (or "Fab fragments") and an "Fc portion" (or "Fc fragment"). The "Fab portion" of the antibody comprises the variable region and constant domain of the light chain and the variable region and first constant domain (CH 1) of the heavy chain. Has antigen binding ability. The "Fc portion" of an antibody, comprising the CH2 and CH3 of the two heavy chains, is not directly involved in binding of the antibody to an antigen, but exhibits a variety of effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and "antibody-dependent enhancement (ADE)".

The term "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise indicated, "binding affinity" as used herein refers to an intrinsic binding affinity that reflects the interaction between members of a binding pair (e.g., antibody and antigen) of 1. "KD" and "binding Rate constant k _on "and" dissociation rate constant k _off "is generally used to describe the affinity between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen), i.e., how tightly a ligand binds to a particular protein. Binding affinity is affected by non-covalent intermolecular interactions, such as hydrogen bonding, electrostatic interactions, hydrophobic and van der waals forces between two molecules. In addition, the binding affinity between a ligand and its target molecule may be affected by the presence of other molecules. Affinity can be analyzed by conventional methods known in the art, including the ELISA described herein.

In one embodiment of the invention, the inventors engineered the heavy chain IgG1 constant region of the CoV2-HB27 antibody by molecular biological means to obtain humanized antibodies CoV2-HB27-Fd6-IgG1 and CoV2-HB27-Fd11-IgG4 of the IgG1 subtype with reduced Fc function; the CoV2-HB27-Fd6-IgG1 antibody had little binding to CD32a and CD32 b; it binds only weakly to CD64 and to C1q (see patent applications 202010349190.3 and PCT/CN2021/089748 for details). The CoV2-HB27-Fd11-IgG4 antibody had little binding to CD32A, CD32B and CD 64. Both CoV2-HB27-Fd6-IgG1 and CoV2-HB27-Fd11-IgG4 showed reduced ADE effects on CHO-K1-CD32A cells, CHO-K1-CD32B cells and Raji cells.

In another embodiment of the invention, the inventors engineered the heavy chain IgG4 constant region of SARS-2-H014 antibody by molecular biological means, with SARS-2-H014-Fd11-IgG4 antibody having no binding to CD32a, CD32b, CD16a and C1q complement proteins, very weak binding to CD64 at high concentrations, and similar FcRn binding to IgG1 subtype antibodies at pH6.0 (see for details patent applications 202010219867.1 and PCT/CN 2021/082374). The SARS-2-H014-Fd11-IgG4 antibody showed reduced ADE effects on CHO-K1-CD64, THP-1 and U937 cells.

For Expression of the molecules of the invention, standard recombinant DNA Expression methods can be used (see, e.g., goeddel; gene Expression technology, methods in Enzymology 185, academic Press, san Diego, calif. (1990)). For example, a nucleotide sequence encoding a desired molecule of the invention may be inserted into an expression vector, which is subsequently transfected into a suitable host cell. Suitable host cells are prokaryotic and eukaryotic cells. Examples of prokaryotic host cells are bacteria and examples of eukaryotic host cells are yeast, insect or mammalian cells. It will be appreciated that the design of the expression vector, including the choice of regulatory sequences, will be influenced by a variety of factors, such as the choice of host cell, the level of expression of the desired protein, and whether expression is constitutive or inducible.

The molecules of the invention can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein a affinity chromatography, protein G affinity chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, e.g., colligan, current Protocols in Immunology, or Current Protocols in Protein Science, john Wiley & Sons, NY, N.Y. (1997-2001), e.g., chapters 1, 4, 6, 8, 9, 10, each of which is incorporated herein by reference in its entirety.

The molecules of the invention include naturally purified products, products of chemical synthetic methods, and products produced by recombinant techniques from prokaryotic and eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells. The molecules of the invention may be glycosylated or may be non-glycosylated. Such methods are described in many standard laboratory manuals, e.g., sambrook, supra, sections 17.37-17.42; ausubel, supra, chapters 10, 12, 13, 16, 18, and 20.

Thus, embodiments of the invention are also host cells comprising the above-described vectors or nucleic acid molecules, wherein the host cells may be higher eukaryotic host cells such as mammalian and insect cells, lower eukaryotic host cells such as yeast cells, and may be prokaryotic cells such as bacterial cells.

Use of

The method of the present invention can be used for treating, preventing or detecting diseases caused by viruses such as SARS-CoV-2 and SARS-CoV, such as SARS-CoV-2 and acute respiratory infectious diseases caused by SARS-CoV virus.

Pharmaceutical composition

One or more of the molecules, nucleic acids, vectors of the invention may be formulated with at least one other chemical agent to form a pharmaceutical composition comprising the active ingredient as described above and one or more pharmaceutically acceptable carriers, diluents or excipients; optionally, one or more additional therapeutic agents may also be included.

Reagent kit

The invention also relates to a pharmaceutical pack and a kit comprising one or more containers containing the above-mentioned pharmaceutical composition of the invention. Associated with such containers may be a notice in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice is approved for human administration by the agency of the manufacture, use or sale of the products.

Preparation and storage

The pharmaceutical compositions of the present invention may be prepared in a manner known in the art, for example, by conventional mixing, dissolving, granulating, levigating, emulsifying, entrapping or lyophilizing processes.

After pharmaceutical compositions comprising the compounds of the present invention formulated in an acceptable carrier have been prepared, they may be placed in an appropriate container and labeled for treatment of the indicated condition. Such labels would include the amount, frequency and method of administration.

Pharmaceutical combination

The pharmaceutical compositions comprising the antibodies of the invention described above are also combined with one or more other therapeutic agents, wherein the resulting combination does not cause unacceptable adverse effects.

The following examples are intended to illustrate the invention without limiting it.

Examples

Example 1: construction of CHO-K1 cell line stably expressing FcR

1.1 transfection of CHO-K1 cells

pCMV3 vector (resistant: hygromycin) (source, beijing Ohio Kaisha technologies Co., ltd.) containing CD32A, CD32B, CD64 and FcR γ -chain was transfected into CHO-K1 cells (source: ATCC) to prepare CHO-K1 cells expressing CD32A (CD 32A vector alone), CD32B (CD 32B vector alone) or CD64 (CD 64 and FcR γ -chain vector together), respectively. CHO-K1 cells were digested one day in advance, counted, and 3.5x10 added to T25 flasks ⁶ And make up the medium (DMEM +10% FBS + 69. Mu.g/mL proline) to 7mL, put at 37 ℃,5% CO ₂ Plates were plated in the incubator overnight. The next day, 20. Mu.g of CD32A plasmid, 20. Mu.g of CD32B plasmid, 10. Mu.g of each of CD64 plasmid and FcR γ -chain plasmid (CD 64 plasmid and FcR γ -chain plasmid are mixed), the plasmids were diluted to 1mL using CHO-K1 medium, 25. Mu.L of Sinofection reaction (source: beijing, qianzhou technology Co., ltd.) was taken, diluted to 1mL using CHO-K1 medium, the diluted Sino Transfection reaction was added to the diluted plasmid and mixed well, and the mixture was incubated at room temperature for 10min. Adding the transfection mixture to each T25 flask, respectively, at 37 deg.C, 5% ₂ After 4h incubation in the incubator, the supernatant was removed, 7mL of fresh medium was added, the mixture was incubated at 37 ℃ with 5% CO ₂ Culturing in an incubator.

1.2 cell selection and high expression monoclonal cell line selection

After 3 days of transfection, hygromycin (Hygromycin) was added to the cells to select cells positive for transfection. One week later, the cells were digested and diluted to 0.5/mL with hygromycin containing medium in 96-well flat-bottom cell culture plates100 μ L of cell suspension was added to each well. Put at 37 ℃ and 5% CO ₂ Culturing in an incubator. After the monoclonal antibody is formed in the culture hole, the monoclonal cell is expanded and cultured, the FcR expression condition on the cell is detected by using CD32 and CD64 antibodies (source: BD), the CHO-K1-CD32A, CHO-K1-CD32B and CHO-K1-CD64 monoclonal antibodies with high expression level are selected for expansion and culture and used for subsequent experiments, and the cell flow detection result is shown in figure 1.

Example 2: pseudovirus preparation

2.1 SARS-CoV-2 pseudovirus package

A pseudovirus expressing the full-length SARS-CoV-2S protein was packaged using 293T (source: ATCC). The 293T was digested one day in advance, counted and 3.5x10 was added to the T25 flask ⁶ The cells of (1), and the medium (DMEM +10% FBS) is replenished to 7mL, the mixture is charged at 37 ℃,5% CO ₂ Plates were plated in the incubator overnight. The next day, 20. Mu.g of SARS-CoV-2Spike plasmid (origin: beijing Yiqiao Shenzhou Tech Co., ltd.) was taken, 293T culture was used to dilute the plasmid to 1mL, 25. Mu.L of Sinofection Transfection Reagent (origin: beijing Yiqiao Shenzhou Tech Co., ltd.) was taken, 293T culture was used to dilute to 1mL, the diluted Sino Transfection Reagent was added to the diluted plasmid and mixed well, and the mixture was incubated at room temperature for 10min. Placing the cell plate at 37 deg.C, 5% CO ₂ The culture box of (2) is cultured for 6h and then the liquid is changed. 293T cells transfected with SARS-CoV-2Spike were infected with VSV pseudovirus (VSV. DELTA.G-Luc) after 24h, washed three times with PBS after 1h, and supplemented with 7mL of fresh 293T medium. After 24h, the supernatant was collected and filtered through a 0.45 μm filter to remove cell debris, thus obtaining a pseudovirus solution, which was stored at-80 ℃.

2.2 SARS-CoV-2 pseudovirus titer detection

The virus was diluted 10-fold in gradient by limiting dilution, with 6 virus concentrations in total, each 6 replicate wells. The inoculation density in 96-well plates was 3X10 ⁴ cell/mL VERO E6 (source: center for cell resources of institute of basic medicine, national academy of sciences) suspension, 100. Mu.L/well. Adding 100 μ L of virus diluted in gradient into each well, mixing with cell culture medium as negative control, and adjusting the concentration at 37 deg.C and 5% CO ₂ In (3) cultureAnd (5) standing and culturing for 24h in the box. After the culture was completed, the supernatant was discarded, and 100. Mu.L/well of passive lysis buffer (origin: promega) diluted to 1X was added to the mixture, and the cells were lysed by mixing. Transferring 40 mu L/well into a 96-well white bottom plate to detect a fluorescence signal, and calculating the TCID by using a Karber method ₅₀ The value is obtained.

Example 3: antibody preparation

3.1 sources of antibody sequences: coV2-HB27 is a humanized antibody capable of blocking the combination of SARS-CoV-2spike protein (S protein) and ACE2 receptor and neutralizing SARS-CoV-2 virus infected cell with high efficiency. Their preparation, structure and properties are described in detail in patent applications 202010349190.3 and PCT/CN2021/089748 (incorporated herein by reference for all purposes).

SARS-2-H014 is humanized antibody capable of cross-blocking binding of SARS-CoV-2 and SARS-CoV spike protein (S protein) to ACE2 receptor, and neutralizing SARS-CoV-2 and SARS-CoV virus infected cells with high efficiency. Their preparation, structure and properties are described in detail in patent applications 202010219867.1 and PCT/CN2021/082374 (incorporated herein by reference).

3.2 Production of CoV2-HB27-IgG1 antibody

The nucleotide sequence of the heavy chain variable region of CoV2-HB27 (SEQ ID NO: 5) was obtained by a method of whole gene synthesis. The expression vector of CoV2-HB27 heavy chain (SEQ ID NO: 1) was inserted into a ScaI + NheI (source: fermentas, hereinafter) digested pSE vector with a heavy chain signal peptide (SEQ ID NO: 3) and a heavy chain IgG1 constant region (SEQ ID NO: 7) by the In-fusion method.

The nucleotide sequences of the variable regions of the CoV2-HB27 light chain (SEQ ID NO: 6) were obtained by a whole-gene synthesis method, respectively. The CoV2-HB27 light chain (SEQ ID NO: 2) expression vector was obtained by inserting into a ScaI + BsiWI (source: fermentas) digested pSE vector having a light chain signal peptide (SEQ ID NO: 4) and a light chain kappa constant region nucleotide sequence (SEQ ID NO: 8) by the In-fusion method.

After plasmid extraction, 293E cells (source: invitrogen, the same applies hereinafter) were transfected and cultured for 7 days, and purified by a protein A purification column to obtain a high-purity antibody.

Total gene synthesis of CoV2-HB27 heavy chain variable region primer:

F1(SEQ ID NO:35)	GCTACCAGGGTGCTGAGTGAGGTGAAACTGGTGGAGTCTGGAGGAGGACTG
R1(SEQ ID NO:36)	CAGGGAGCCTCCAGGCTTCACCAGTCCTCCTCC
F2(SEQ ID NO:37)	CCTGGAGGCTCCCTGAGACTGTCCTGTGCTGCC
R2(SEQ ID NO:38)	GTTGCTGAAGGTGAAGCCAGAGGCAGCACAGGA
F3(SEQ ID NO:39)	TTCACCTTCAGCAACTATGGGATGAGTTGGGT
R3(SEQ ID NO:40)	CTCTTGCCAGGAGCCTGTCTCACCCAACTCATC
F4(SEQ ID NO:41)	GGCTCCTGGCAAGAGATTGGAGTGGGTGGCTG
R4(SEQ ID NO:42)	AGGAGCCTCCAGAGGAAATCTCAGCCACCCACT

F5(SEQ ID NO:43)	CCTCTGGAGGCTCCTACACCTACTACCCTGAC
R5(SEQ ID NO:44)	GGTGAACCTGCCTGTCACTGTGTCAGGGTAGTA
F6(SEQ ID NO:45)	ACAGGCAGGTTCACCATCAGCAGGGACAATGCC
R6(SEQ ID NO:46)	TTGGAGGTAGAGGGTGTTCTTGGCATTGTCCCT
F7(SEQ ID NO:47)	ACCCTCTACCTCCAAATGAACTCCCTGAGGGCT
R7(SEQ ID NO:48)	GTAGTAGACTGCTGTGTCCTCAGCCCTCAGGGA
F8(SEQ ID NO:49)	ACAGCAGTCTACTACTGTGCCAGGTTCAGATAT
R8(SEQ ID NO:50)	CACTGTGCCTCCTCCTCCATCATATCTGAACCT
F9(SEQ ID NO:51)	GGAGGAGGCACAGTGGACTACTGGGGACAAGGC
R9(SEQ ID NO:52)	TGGGCCCTTGGTGCTTGCGCTGGACACTGTCACCAGGGTGCCTTGTCCCCA

splicing CoV2-HB27-IgG1 heavy chain primer:

the COV2-HB27 light chain variable region primer is synthesized by the whole gene:

F12(SEQ ID NO:57)	GCCACAGGAGTGCATAGTGAGATTGTGCTGACCCAGAGCCCTGCCACCCTG
R12(SEQ ID NO:58)	CCTCTCTCCAGGGCTCAGGGACAGGGTGGCAGG
F13(SEQ ID NO:59)	AGCCCTGGAGAGAGGGCTACCCTGTCCTGTAGG
R13(SEQ ID NO:60)	GTTGTCCACAGACTCAGATGCCCTACAGGACAG
F14(SEQ ID NO:61)	GAGTCTGTGGACAACTATGGCATCTCC
R14(SEQ ID NO:62)	GGAACCAGTTCATAAAGGAGATGCCATA
F15(SEQ ID NO:63)	TTATGAACTGGTTCCAACAGAAGCCTG
R15(SEQ ID NO:64)	AGTCTTGGGGCTTGTCCAGGCTTCTGTT
F16(SEQ ID NO:65)	ACAAGCCCCAAGACTGCTGATTTATGC
R16(SEQ ID NO:66)	GCCCTGGTTGCTGGCAGCATAAATCAGC
F17(SEQ ID NO:67)	GCCAGCAACCAGGGCTCTGGAGTGCCTGCCAGG
R17(SEQ ID NO:68)	GCCAGAGCCAGAGCCAGAGAACCTGGCAGGCAC
F18(SEQ ID NO:69)	GGCTCTGGCTCTGGCACAGACTTCTCCCTGACC
R18(SEQ ID NO:70)	CTCAGGTTCCAAGGAGGAGATGGTCAGGGAGAA
F19(SEQ ID NO:71)	TCCTTGGAACCTGAGGACTTTGCTGTCTACTTC
R19(SEQ ID NO:72)	CACCTCCTTGCTCTGTTGACAGAAGTAGACAGC
F20(SEQ ID NO:73)	CAGAGCAAGGAGGTGCCAAGGACCTTTGGACAA
R20(SEQ ID NO:74)	TGGTGCAGCCACCGTACGCTTAATCTCCACCTTGGTGCCTTGTCCAAAGGT

3.3 Production of CoV2-HB27-Fd6-IgG1 antibody

To reduce antibody Fc fragment-mediated immune function, reference was made to nucleotide mutations in the IgG1 subtype constant region [18] to generate a genetically engineered heavy chain IgG1 constant region nucleotide sequence (Fd 6-IgG1, SEQ ID NO: 9). The CoV2-HB27-Fd6-IgG1 heavy chain sequence (SEQ ID NO: 10) was obtained by PCR and comprised of a heavy chain signal peptide nucleotide sequence (SEQ ID NO: 3), a heavy chain variable region nucleotide sequence (SEQ ID NO: 5) and an Fd6-IgG1 constant region nucleotide sequence (SEQ ID NO: 9). An expression vector containing the CoV2-HB27-Fd6-IgG1 heavy chain (SEQ ID NO: 10) was obtained by inserting into the HindIII + XbaI-digested pSE vector by the In-fusion method.

Splicing CoV2-HB27-Fd6-IgG1 heavy chain primer:

extracting CoV2-HB27-Fd6-IgG1 heavy chain (SEQ ID NO: 10) expression vector and CoV2-HB27 light chain (SEQ ID NO: 2) expression vector plasmid, transfecting 293E cells, culturing and expressing for 7 days, and purifying by adopting a protein A purification column to obtain the CoV2-HB27-Fd6-IgG1 antibody with reduced Fc function.

3.4 Production of CoV2-HB27-Fd11-IgG4 antibody

To reduce antibody Fc fragment-mediated immune function, the reference literature carries out nucleotide mutations in the constant region of IgG4 subtype [18] to obtain a genetically engineered nucleotide sequence of the heavy chain IgG4 constant region (Fd 11-IgG4, SEQ ID NO: 11). The CoV2-HB27-Fd11-IgG4 heavy chain sequence (SEQ ID NO: 12) comprising the heavy chain signal peptide nucleotide sequence (SEQ ID NO: 3), the heavy chain variable region nucleotide sequence (SEQ ID NO: 5) and the Fd11-IgG4 constant region nucleotide sequence (SEQ ID NO: 11) was obtained by PCR. An expression vector containing the CoV2-HB27-Fd11-IgG4 heavy chain (SEQ ID NO: 12) was obtained by inserting into the HindIII + XbaI-digested pSE vector by the In-fusion method.

Splicing CoV2-HB27-Fd11-IgG4 heavy chain primer:

extracting CoV2-HB27-Fd11-IgG4 heavy chain (SEQ ID NO: 12) expression vector and CoV2-HB27 light chain (SEQ ID NO: 2) expression vector plasmid, transfecting 293E cells, culturing and expressing for 7 days, and purifying by adopting a protein A purification column to obtain the CoV2-HB27-Fd11-IgG4 antibody with reduced Fc function.

3.5 Production of SARS-2-H014-IgG1 antibody

The nucleotide sequences of SARS-2-H014 heavy chain variable region (SEQ ID NO: 16) were obtained by whole gene synthesis method, respectively. The SARS-2-H014 heavy chain (SEQ ID NO: 13) expression vector was obtained by inserting into a ScaI + NheI (source: fermentas) digested pSE vector with heavy chain signal peptide (SEQ ID NO: 15) and heavy chain IgG1 constant region (SEQ ID NO: 7) by In-fusion method.

SARS-2-H014 light chain variable regions (SEQ ID NO: 17) were obtained by whole gene synthesis, respectively, and inserted into ScaI + BsiWI (source: fermentas) digested pSE vectors having light chain signal peptide (SEQ ID NO: 4) and light chain kappa constant region nucleotide sequence (SEQ ID NO: 18) by In-fusion method to obtain SARS-2-H014 light chain (SEQ ID NO: 14) expression vectors, respectively. After plasmid extraction, 293E cells (source: invitrogen) were transfected and cultured for 7 days, and purified using a protein A purification column to obtain high-purity antibody.

The primer of SARS-2-H014 heavy chain variable region is synthesized by the whole gene:

the SARS-2-H014 light chain variable region primer is synthesized by whole gene:

3.6 Production of SARS-2-H014-Fd11-IgG4 antibody

Construction and production of IgG4 subtype humanized antibody SARS-2-H014 with reduced Fc function

To reduce antibody Fc fragment-mediated immune function, the reference literature carries out nucleotide mutations in the constant region of IgG4 subtype [18] to obtain a genetically engineered nucleotide sequence of the heavy chain IgG4 constant region (Fd 11-IgG4, SEQ ID NO: 11). SARS-2-H014-Fd11-IgG4 heavy chain sequence (SEQ ID NO: 19) comprising a heavy chain signal peptide nucleotide sequence (SEQ ID NO: 15), SARS-2-H014 heavy chain variable region nucleotide sequence (SEQ ID NO: 16) and Fd11-IgG4 nucleotide sequence (SEQ ID NO: 11) was obtained by splicing PCR. The SARS-2-H014-Fd11-IgG4 heavy chain (SEQ ID NO: 19) expression vector was obtained by inserting into the HindIII + XbaI (origin: fermentas) -digested pSE vector by the In-fusion method.

Splicing SARS-2-H014-Fd11-IgG4 heavy chain primer:

F44(SEQ ID NO:117)	GTCACCGTCCTGACACGAAGCTTGCCGCCACCATG
R44(SEQ ID NO:118)	TGGGCCCTTGGTGCTTGC
F45(SEQ ID NO:119)	GCAAGCACCAAGGGCCCA
R45(SEQ ID NO:120)	ACTATAGAATAGGGCCCTCTAGA

extracting SARS-2-H014-Fd11-IgG4 heavy chain (SEQ ID NO: 19) expression vector and SARS-2-H014 light chain (SEQ ID NO: 14) expression vector plasmid, transfecting HEK-293 cells, culturing and expressing for 7 days, and purifying by protein A purification column to obtain IgG4 subtype humanized SARS-2-H014 antibody with high purity and reduced Fc function, namely SARS-2-H014-Fd11-IgG4.

3.7 antibody production purification

The 293E cells were passaged to 200 mL/bottle using SCD4-4-TC2 medium (source: beijing Yiqiao Shenzhou Tech Co., ltd.) at an initial inoculation density of 0.3-0.4X 10 ⁶ cell/mL CO at 37 ℃ and 175rpm ₂ Cell culture was performed in a shaker. When the cell density reaches 1.5-3 multiplied by 10 ⁶ After cells/mL, a total of 100. Mu.g of light and heavy chain plasmid DNA mixed as described in 1 and 800. Mu.L of TF2 transfection reagent (source: beijing Yiqiao Shenzhou technologies, inc.) were added and the culture was continued in a shaker until harvest on day 7. The culture broth was centrifuged at 4000rpm for 25min, and the supernatant was collected and 1/5 of the supernatant volume of stock buffer (source: shenzhou cell engineering Co., ltd.) was added. The protein A column (origin: shenzhou cell engineering Co., ltd.) was equilibrated by 5 to 10 times the column volume with PBS, the filtered culture supernatant was added to the column, and after 5 to 10 times the column volume was equilibrated again, the sample was eluted with sodium acetate buffer (origin: shenzhou cell engineering Co., ltd.). After elution, the sample was neutralized to neutral with Tris buffer for use.

Example 4: fc function of COV2-HB27-Fd11-IgG4 antibody

4.1 CD16a binding function of CoV2-HB27-Fd11-IgG4 antibody

Avidin protein (source: thermo, infra) was coated at a concentration of 10. Mu.g/mL on 96-well plates, 100. Mu.L per well, overnight at 2-8 ℃. The plate is washed the next day, after being sealed for 1h at room temperature, 100 mul of biotin-labeled CD16a-AVI-His (V158) + BirA protein (source: beijing Yi Qian Shenzhou science and technology Co., ltd.) with the concentration of 5 mug/mL is added, and the plate is washed after being incubated for 1h at room temperature. Are added separatelymu.L of different Fc functional forms of CoV2-HB27 antibody were added at concentrations of 5. Mu.g/mL and 1. Mu.g/mL. Washing the plate after incubation for 1h to remove unbound antibody, adding goat anti-human IgGF (ab) 2/HRP (source: jackson Immunoresearch company, the same below) for incubation, repeatedly washing the plate, adding substrate developing solution for developing color, and reading OD by an enzyme reader after termination ₄₅₀ 。

As a result, as shown in FIG. 2A, the Fd11-IgG4 form antibody having reduced Fc function had only extremely weak binding to CD16a as compared with the IgG1 form.

4.2 CD32 binding function of CoV2-HB27-Fd11-IgG4 antibody

Avidin protein was coated at 10. Mu.g/mL in 96-well plates at 100. Mu.L per well overnight at 2-8 ℃. Washing the plate the next day, sealing at room temperature for 1h, adding 100 μ L of biotin-labeled CD32a-AVI-His (R131) + BirA protein (source: beijing Yinqiao Shenzhou science Co., ltd.) or CD32b-AVI-HIS + BirA protein (source: beijing Yinqiao Shenzhou science Co., ltd.) at a concentration of 5 μ g/mL, incubating at room temperature for 1h, and washing the plate. mu.L of CoV2-HB27 antibodies in different Fc functional forms were added at concentrations of 5. Mu.g/mL and 1. Mu.g/mL. Washing the plate after incubation for 1h to remove unbound antibody, adding goat anti-human IgG F (ab) 2/HRP, incubating, repeatedly washing the plate, adding substrate developing solution for developing, and reading OD by enzyme-labeling instrument after termination ₄₅₀ 。

As a result, as shown in FIG. 2, the Fd11-IgG4 form of antibody having reduced Fc function showed almost no binding to CD32a and CD32b, as compared with the IgG1 form of antibody (FIG. 2B, 2C).

4.3 CD64 binding function of CoV2-HB27-Fd11-IgG4 antibody

Avidin protein was coated at 10. Mu.g/mL in 96-well plates at 100. Mu.L per well and overnight at 2-8 ℃. The plate was washed the next day, and after 1 hour of blocking at room temperature, 100. Mu.L of biotin-labeled CD64-AVI-His + BirA protein (source: beijing Yi Qianzhou science and technology Co., ltd.) was added at a concentration of 0.5. Mu.g/mL, and after 1 hour of incubation at room temperature, the plate was washed. mu.L of CoV2-HB27 antibodies in different Fc functional forms were added at concentrations of 5. Mu.g/mL and 1. Mu.g/mL. Washing the plate after incubation for 1h to remove unbound antibody, adding goat anti-human IgG F (ab) 2/HRP, incubating, washing the plate repeatedly, adding substrate color developing solution for color development, and stoppingEnzyme-linked immunosorbent assay (OD) reading ₄₅₀ 。

As a result, as shown in FIG. 2D, the Fd6-IgG1 format antibody with reduced Fc function had only a weak binding to CD64 as compared with the IgG1 format antibody.

4.4 C1q binding function of CoV2-HB27-Fd11-IgG4 antibody

Different concentrations of CoV2-HB27 antibodies in different Fc functional forms were coated on 96-well plates at 100. Mu.L/well overnight at 4 ℃ and antibody addition concentrations of 5. Mu.g/mL and 1. Mu.g/mL, respectively. The plate was washed the next day, and after 1 hour of blocking at room temperature, 5. Mu.g/mL of C1q complement protein (source: beijing Yiqian Shenzhou science Co., ltd.) was added thereto at a concentration of 100. Mu.g/well, followed by incubation for 1 hour. Washing the plate to remove unbound protein, adding 0.5 μ g/mL anti-C1q/HRP (source: abcam), incubating, washing the plate repeatedly, adding substrate developing solution for developing color, and detecting OD after stopping ₄₅₀ 。

As a result, as shown in FIG. 3, the Fd11-IgG4 form antibody having reduced Fc function was only weakly bound to C1 q.

4.5 ADCC function mediated by CoV2-HB27-Fd11-IgG4 antibody

The 293FT cell strain (293 FT-SARS-CoV-2-S, source: shenzhou cell engineering Co., ltd., the same below) which transiently expresses SARS-CoV-2 full-length protein was used as a target cell, jurkat cells (Jurkat-NFAT/Luc 2P-CD16 AV) which stably transfected CD16AV and NFAT-Luc2P were used as effector cells, and the ADCC function of the humanized antibody was detected by a reporter gene method.

In a 96-well plate, the density of access was 1X 10 at 50. Mu.L/well ⁵ cell/mL target cells and equal volume of equal density effector cells. 50 μ L of CoV2-HB27 antibody in a different Fc functional form and H7N9-R1 negative control antibody were then added. CoV2-HB27-IgG1, coV2-HB27-Fd11-IgG4 antibody, and H7N9-R1 negative control antibody were added at concentrations of 20. Mu.g/mL, 1. Mu.g/mL, and 0.05. Mu.g/mL. After mixing at 37 ℃ and 5% CO ₂ Incubate in incubator for 6h. Finally, 5 Xpassive lysine buffer (30. Mu.L/well) was added and the cells were lysed by mixing well. RLU values were measured on 10. Mu.L/well cell samples. Dose effect histograms were analyzed and plotted using GraphPad Prism software with RLU values on the ordinate. Bioluminescence intensity induction fold = sample group RLU value/negative control group RLU value.

As a result, as shown in FIG. 4, the Fd11-IgG4 form antibody having reduced Fc function had no ADCC effect.

4.6 ADCP function mediated by CoV2-HB27-Fd11-IgG4 antibody

The reporter gene method was used to detect the ADCP function mediated by the humanized antibody using 293FT-SARS-CoV-2-S as target cells and Jurkat cells (Jurkat-NFAT/Luc 2P-CD32A, jurkat-NFAT/Luc2P-CD32B or Jurkat-NFAT/Luc2P-CD 64) stably transfected with CD32A, CD32B or CD64 and NFAT-Luc2P as effector cells.

In a 96-well plate, the density of access was 1X 10 at 50. Mu.L/well ⁵ cells/mL target cells and equal volume of effector cells at equal density. 50 μ L of different Fc functional forms of CoV2-HB27 antibody and H7N9-R1 negative control antibody were then added. Jurkat-NFAT/Luc2P-CD32A, jurkat-NFAT/Luc2P-CD32B and Jurkat-NFAT/Luc2P-CD64 were used as effector cells, and the antibody addition concentrations were 20. Mu.g/mL, 1. Mu.g/mL and 0.05. Mu.g/mL. Mixing, then 37 ℃ and 5% CO ₂ Incubate in incubator for 6h. Finally, 5 Xpassive lysine buffer (30. Mu.L/well) was added and the cells were lysed by mixing well. RLU values were measured on 10. Mu.L/well cell samples. Dose effect histograms were analyzed and plotted using GraphPad Prism software with RLU values on the ordinate. Bioluminescence intensity induction fold = sample group RLU value/negative control group RLU value.

As shown in FIG. 5, when Jurkat-NFAT/Luc2P-CD32A, jurkat-NFAT/Luc2P-CD32B and Jurkat-NFAT/Luc2P-CD64 were used as effector cells (FIGS. 5A,5B and 5C), neither the antibody CoV2-HB27 in the form of IgG1 nor Fd11-IgG4 had ADCP effect.

4.7 CDC function mediated by CoV2-HB27-Fd11-IgG4 antibody

The CDC function of the humanized antibody was detected by the WST-8 method using 293FT-SARS-CoV-2-S as a target cell.

In a 96-well plate, the density of access was 2X 10 at 50. Mu.L/well ⁶ cell/mL of target cells. mu.L of rabbit complement (source: one lambda) and different Fc functional forms of CoV2-HB27 antibody were added at concentrations of 100. Mu.g/mL, 20. Mu.g/mL, 4. Mu.g/mL, 0.8. Mu.g/mL, 0.16. Mu.g/mL, 0.032. Mu.g/mL, and a detection blank well (no cells), a positive control (cells only inoculated) control, and a H7N9-R1 negative control antibody group were setg/mL, 0.0064. Mu.g/mL, 0.00128. Mu.g/mL. After mixing at 37 ℃ and 5% CO ₂ Incubate in incubator for 2h. After the culture, WST-8 developing solution was added at 10. Mu.L/well. Placing a 96-well plate in CO ₂ Incubating in an incubator, and determining the absorbance at 450nm and 630nm on an enzyme-labeling instrument after color development is stable. In the absorbance value (OD) ₄₅₀ –OD ₆₃₀ ) And readings from blank wells were subtracted to calculate the killing effect of CDC of the antibody. Percent killing (= (positive control OD value-sample OD value)/positive control OD value × 100%).

As a result, as shown in FIG. 6, none of the antibodies to CoV2-HB27 in the different Fc functional forms had CDC effect on the target cells expressing SARS-CoV-2S protein.

Example 5: fc engineering reduces ADE effects of antibodies on CD 32A-expressing cells

5.1 Fd6 and Fd11 antibody modification for reducing ADE effect of CoV2-HB27-IgG1 antibody on CHO-K1-CD32A cells

One day prior digestion of CHO-K1-CD32A followed by adjustment of cell density to 3x10 using medium ⁵ PermL, 96-well cell culture plates 100. Mu.L of cell suspension per well, 37 ℃ and 5% CO ₂ Plates were plated in the incubator overnight. The next day, another 96-well cell culture plate was added with different concentrations (starting at a final concentration of 500. Mu.g/mL, 4-fold dilution gradient, total 9 gradients) of antibody at 50. Mu.L/well. Add 500TCID per well ₅₀ 50 μ L/well of SARS-CoV-2 pseudovirus (III). The group with the virus and the group without the antibody are used as positive controls, and the group without the virus and the antibody is used as a negative control. Mixing, and placing at 37 deg.C and 5% CO ₂ Incubate for 1h. After completion of incubation, the cells were transferred to a plated CHO-K1-CD32A cell plate at 100. Mu.L/well, incubated at 37 ℃ and 5% CO ₂ And (5) standing and culturing for 24 hours in an incubator. After the culture was completed, 30. Mu.L/well of passive lysine 5x buffer (source: promega) was added, and the cells were lysed by mixing. 10 μ L/well was transferred to a 96-well white bottom plate fluorescence signal value (RLU). Sample RLU/positive control RLU was calculated and plotted using GraphPad, and the results are shown in FIG. 7, where Fd6 and Fd11 modifications significantly reduced the ADE effect of CoV2-HB27-IgG1 antibodies on CHO-K1-CD32A cells.

Example 6: fc engineering reduces ADE effects of antibodies on CD32B expressing cells

6.1 Fd6 and Fd11 antibody modification for reducing ADE effect of CoV2-HB27-IgG1 antibody on CHO-K1-CD32B cells

After one day prior digestion of CHO-K1-CD32B, cell density was adjusted to 3x10 using medium ⁵ PermL, 96-well cell culture plates 100. Mu.L of cell suspension per well, 37 ℃ and 5% CO ₂ Plates were plated in the incubator overnight. The next day, another 96-well cell culture plate was added with different concentrations (starting at a final concentration of 500. Mu.g/mL, 4-fold dilution gradient, total 9 gradients) of antibody at 50. Mu.L/well. Add 500TCID per well ₅₀ 50 μ L/well of SARS-CoV-2 pseudovirus (III). The group with the virus and the group without the antibody are used as positive controls, and the group without the virus and the antibody is used as a negative control. Mixing, and placing at 37 deg.C and 5% CO ₂ Incubate for 1h. After completion of incubation, the cells were transferred to a plated CHO-K1-CD32B cell plate at 100. Mu.L/well, incubated at 37 ℃ and 5% CO ₂ And (5) performing static culture in an incubator for 24 hours. After the culture was completed, passive lysine 5xbuffer (30. Mu.L/well) was added, and the cells were lysed by mixing. 10 μ L/well was transferred to a 96-well white bottom plate fluorescence signal value (RLU). Sample RLU/positive control RLU was calculated and plotted using GraphPad, and the results are shown in FIG. 8, where Fd6 and Fd11 modifications significantly reduced the ADE effect of CoV2-HB27-IgG1 antibodies on CHO-K1-CD32B cells.

Example 7: fc engineering reduces ADE effects of antibodies on CD 64-expressing cells

7.1 Fd11 antibody engineering reduces the ADE effect of SARS-2-H014-IgG1 antibody on CHO-K1-CD64 cells

One day prior digestion of CHO-K1-CD64 followed by adjustment of cell density to 3x10 using medium ⁵ PermL, 96-well cell culture plates 100. Mu.L of cell suspension per well, 37 ℃ and 5% CO ₂ Plates were plated in the incubator overnight. The next day, another 96-well cell culture plate was loaded with 50 μ L/well of antibody at different concentrations (starting at a final concentration of 100 μ g/mL, 5-fold gradient dilution, for a total of 9 gradients). Add 500TCID per well ₅₀ 50 μ L/well of SARS-CoV-2 pseudovirus (III). The group with virus and without antibody is used as positive control, and the group without virus and antibody is used as negative control. Mixing uniformlyThen placed at 37 ℃ and 5% CO ₂ Incubate for 1h. After completion of incubation, the cells were transferred to a plated CHO-K1-CD64 cell plate at 100. Mu.L/well, incubated at 37 ℃ and 5% CO ₂ And (5) performing static culture in an incubator for 24 hours. After the culture, passive lysine 5xbuffer (30. Mu.L/well) was added and the cells were lysed by mixing. 10 μ L/well was transferred to a 96-well white bottom plate fluorescence signal value (RLU). Sample RLU/positive control RLU was calculated and plotted using GraphPad, and the results are shown in FIG. 9, where the Fd11 engineering significantly reduced the ADE effect of SARS-2-H014-IgG1 antibody on CHO-K1-CD64 cells.

Example 8: fc engineering reduces ADE effects of antibodies on cells expressing multiple FcRs

8.1 Fd6 and Fd11 engineering to reduce the ADE effect of CoV2-HB27-IgG1 antibodies on Raji cells

To a 96-well cell culture plate, 50. Mu.L/well of antibody at different concentrations (starting at a final concentration of 500. Mu.g/mL, diluted in 4-fold gradients, for a total of 9 gradients) was added. Add 500TCID per well ₅₀ 50 μ L/well of SARS-CoV-2 pseudovirus (III). The group with the virus and the group without the antibody are used as positive controls, and the group without the virus and the antibody is used as a negative control. Mixing, and placing at 37 deg.C and 5% CO ₂ Incubate for 1h. Add density 3x10 per well after incubation is complete ⁵ 100. Mu.L/mL of Raji cells, incubated at 37 ℃ and 5% CO ₂ And (5) performing static culture in an incubator for 24 hours. After the culture was completed, passive lysine 5xbuffer (30. Mu.L/well) was added, and the cells were lysed by mixing. 10. Mu.L/well was transferred to a 96-well white bottom plate fluorescence signal (RLU). Sample RLU/positive control RLU was calculated and plotted using GraphPad, and the results are shown in FIG. 10, where Fd6 and Fd11 engineering significantly reduced the ADE effect of the CoV2-HB27-IgG1 antibody on Raji cells.

8.2 Fd11 antibody engineering to reduce the ADE effect of SARS-2-H014-IgG1 antibody on THP-1 cells

The THP-1 cells were cultured with 2.5. Mu.g/mL PMA for 3 days and then used to evaluate the ADE effect of SARS-2-H014 antibody of different subtypes. One day prior to induction THP-1 cells were digested and the cell density adjusted to 3x10 using medium ⁵ mL, adding 100. Mu.L of cell suspension per well of a 96-well cell culture plate, then placing the plate at 37 ℃ and 5% ₂ CulturingPlates were plated in the box overnight. The next day, another 96-well cell culture plate was added with different concentrations (starting at a final concentration of 80. Mu.g/mL, 5-fold dilution gradient, total 9 gradients) of antibody at 50. Mu.L/well. 500TCID per well ₅₀ 50 μ L/well of SARS-CoV-2 pseudovirus (III). The group with virus and without antibody is used as positive control, and the group without virus and antibody is used as negative control. Mixing, and placing at 37 deg.C and 5% CO ₂ Incubate for 1h. After completion of incubation, transfer to plated THP-1 cell plates at 100. Mu.L/well, and 5% CO at 37 ℃ ₂ And (5) performing static culture in an incubator for 24 hours. After the culture was completed, passive lysine 5 Xbuffer (30. Mu.L/well) was added thereto, and the cells were lysed by mixing. 10. Mu.L/well was transferred to a 96-well white bottom plate fluorescence signal (RLU). Sample RLU/positive control RLU was calculated and plotted using GraphPad, and the results are shown in FIG. 11, where Fd11 engineering significantly reduced the ADE effect of SARS-2-H014-IgG1 antibody on THP-1 cells.

8.3 Fd11 antibody engineering reduces ADE effects of SARS-2-H014-IgG1 antibodies on U937 cells

The ADE effect of different subtypes of SARS-2-H014 antibodies was evaluated 3 days after the addition of 2.5. Mu.g/mL PMA during U937 cell culture. U937 cells were digested one day prior after induction, and cell density was adjusted to 3x10 using medium ⁵ PermL, 96-well cell culture plates 100. Mu.L of cell suspension per well, 37 ℃ and 5% CO ₂ Plates were plated in the incubator overnight. The next day, another 96-well cell culture plate was added with different concentrations (starting at a final concentration of 10. Mu.g/mL, 5-fold dilution gradient, total 6 gradients) of antibody at 50. Mu.L/well. Add 500TCID per well ₅₀ 50 μ L/well of SARS-CoV-2 pseudovirus (III). The group with the virus and the group without the antibody are used as positive controls, and the group without the virus and the antibody is used as a negative control. Mixing, and placing at 37 deg.C and 5% CO ₂ Incubate for 1h. Transferring to a plated U937 cell plate at 100. Mu.L/well after incubation, at 37 ℃ 5% ₂ And (5) standing and culturing for 24 hours in an incubator. After the culture was completed, passive lysine 5 Xbuffer (30. Mu.L/well) was added thereto, and the cells were lysed by mixing. 10. Mu.L/well was transferred to a 96-well white bottom plate fluorescence signal (RLU). Sample RLU/positive control RLU was calculated and plotted using GraphPad, with the results shown in FIG. 12, for Fd11Can obviously reduce the ADE effect of the SARS-2-H014-IgG1 antibody on U937 cells.

Sequence List

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18. Yexin; sunle; xushao yoga; a mustache; a pottery Weikang; zhang lianshan, cd47 antibody, antibody-binding fragment and median use of therapeutics, google Patents, 2018.

Claims (32)

1. A method of reducing the effects of viral ADE, the method comprising administering to a subject infected with, or at risk of infection by, a virus:

   the molecule comprises

   a) An Fc fragment with reduced Fc receptor binding/complement binding; and preferably

   b) An antigen-binding fragment that recognizes and binds to the viral coat protein or extracellular domain.
2. The method of claim 1, wherein said pathogen is selected from the group consisting of:

   coronavirus, influenza virus, cold virus, parainfluenza virus, upper respiratory syncytial virus, dengue virus, west nile virus, marburg virus, lasa hemorrhagic fever virus, HIV virus, ebola virus, herpes zoster virus, CMV virus, hepatitis virus, human herpes simplex virus, cytomegalovirus, rotavirus, EB virus, measles virus, mumps virus, human papilloma virus, flavivirus or influenza virus; preferably SARS-CoV-2, SARS-CoV, MERS-CoV; influenza a viruses (including H10N8, H7N9, H1N1, H5N1, etc.), influenza B viruses; ebola virus; and hepatitis A, B, C, and E.
3. The method of any one of claims 1-2, wherein the Fc fragment is from the heavy chain constant region of a human antibody, murine antibody, rabbit antibody or other mammalian antibody.
4. The method according to claim 3, wherein the Fc fragment is from an antibody of the IgG, igM or IgA subtype of a human antibody, preferably from an antibody of the IgG1, igG2, igG3 or IgG4 subtype.
5. The method of any one of claims 1-4, wherein the Fc fragment has the following properties:

   reduced binding to one or more antibody Fc receptors; and/or

   (ii) does not bind or has insignificant binding to one or more complements;

   preferably, the Fc receptor is CD16a, CD16b, CD32a, CD32b, CD64, CD89, fcRn.
6. The method of claim 5, wherein the Fc fragment comprises the sequence of SEQ ID NO 26.
7. The method of claim 6, wherein the molecule is an IgG antibody comprising

   27, the heavy chain sequence of SEQ ID NO; and

   the light chain sequence of SEQ ID NO. 21.
8. The method of claim 5, wherein the Fc fragment comprises the sequence of SEQ ID NO 29.
9. The method of claim 8, wherein the molecule is an IgG antibody comprising

   (a) The heavy chain sequence of SEQ ID NO 28; and

   light chain sequence of SEQ ID NO 21 or

   (b) The heavy chain sequence of SEQ ID NO. 34; and

   31, SEQ ID NO.
10. The method of any of claims 1-9, wherein the molecule comprises

    One or more of a) an antigen binding fragment that recognizes and binds to the viral coat protein or extracellular domain; and

    one or more b) Fc mutant fragments with reduced Fc receptor binding/complement binding.
11. The method of any one of claims 1 to 10, wherein the molecule is conjugated to another macromolecule, preferably via a linker,

    preferably, the other macromolecule is a polysaccharide, a peptide/protein or PEG;

    preferably, the peptide/protein is modified or mutant engineered albumin (HSA).
12. A molecule for reducing the effects of viral ADE, the molecule comprising

    a) An Fc fragment with reduced Fc receptor binding/complement binding; and preferably

    b) An antigen binding fragment that recognizes and binds to the viral coat protein or extracellular domain.
13. The molecule of claim 12, wherein the pathogen is selected from the group consisting of:

    coronavirus, influenza virus, cold virus, parainfluenza virus, upper respiratory syncytial virus, dengue virus, west nile virus, marburg virus, lasa hemorrhagic fever virus, HIV virus, ebola virus, herpes zoster virus, CMV virus, hepatitis virus, human herpes simplex virus, cytomegalovirus, rotavirus, EB virus, measles virus, mumps virus, human papilloma virus, flavivirus or influenza virus; preferably SARS-CoV-2, SARS-CoV, MERS-CoV; influenza a viruses (including H10N8, H7N9, H1N1, H5N1, etc.), influenza B viruses; ebola virus; and hepatitis A, B, C, and E.
14. The molecule of any of claims 12-13, wherein the Fc fragment is from a heavy chain constant region of a human antibody, a murine antibody, a rabbit antibody, or other mammalian antibody.
15. The molecule of claim 14, wherein the Fc fragment is from an antibody of the IgG, igM or IgA subtype of a human antibody, preferably from an antibody of the IgG1, igG2, igG3 or IgG4 subtype.
16. The molecule of claim 15, wherein the Fc fragment

    Significantly reduced binding to one or more antibody Fc receptors; and/or

    (ii) does not bind or has insignificant binding to one or more complements;

    preferably, the Fc receptor is CD16a, CD16b, CD32a, CD32b, CD64, CD89, fcRn.
17. The molecule of claim 16, wherein the molecule comprises the sequence of SEQ ID No. 26.
18. The molecule of claim 16, wherein the molecule is an IgG antibody comprising

    The heavy chain sequence of SEQ ID NO 27; and

    the light chain sequence of SEQ ID NO 21.
19. The molecule of claim 15, wherein the molecule comprises the sequence of SEQ ID NO. 29.
20. The molecule of claim 16, wherein the molecule is an IgG antibody comprising

    (a) The heavy chain sequence of SEQ ID NO 28; and

    the light chain sequence of SEQ ID NO:21, or

    (b) The heavy chain sequence of SEQ ID NO. 34; and

    31, light chain sequence of SEQ ID NO.
21. The molecule of any one of claims 12-20, wherein the molecule comprises

    One or more of a) an Fc fragment with reduced Fc receptor binding/complement binding; and preferably

    One or more b) antigen binding fragments that recognize and bind to the viral coat protein or the extracellular domain.
22. A conjugate of the molecule of any one of claims 12-21 with another macromolecule,

    other macromolecules are polysaccharides, peptides/proteins or PEG;

    preferably, the peptide/protein is modified or mutant engineered albumin (HSA);

    preferably, the aforementioned molecules are conjugated to other macromolecules via linkers.
23. A nucleic acid encoding the molecule of any one of claims 12-21, which is mRNA and/or DNA.
24. An expression vector comprising the nucleic acid of claim 23.
25. A host cell comprising the nucleic acid of claim 23 or the expression vector of claim 24.
26. A process for the production of a molecule according to any one of claims 12 to 21, which comprises culturing a host cell according to claim 25 under conditions suitable for expression of the aforementioned protein molecule, and recovering the expressed product from the culture medium.
27. A pharmaceutical composition comprising

    a) The molecule of any one of claims 12 to 21, the conjugate of claim 22, the nucleic acid of claim 23, or the expression vector of claim 24; and/or

    c) A pharmaceutically acceptable carrier, excipient or stabilizer, preferably in the form of a lyophilized formulation or an aqueous solution.
28. The molecule of any one of claims 12 to 21, the conjugate of claim 22, the nucleic acid of claim 23 or the expression vector of claim 24, or the pharmaceutical composition of claim 27 for use in the prevention or treatment of a disease caused by a virus, preferably a coronavirus, more preferably SARS-CoV-2.
29. Use of the molecule of any one of claims 12 to 21, the conjugate of claim 22, the nucleic acid of claim 23 or the expression vector of claim 24, or the pharmaceutical composition of claim 27 for the preparation of a medicament for the prevention of a disease caused by a virus, preferably a coronavirus, more preferably SARS-CoV-2.
30. A pharmaceutical combination comprising

    The molecule of any one of claims 12-21, the conjugate of claim 22, the nucleic acid of claim 23 or the expression vector of claim 24, or the pharmaceutical composition of claim 27; and

    one or more additional therapeutic agents.
31. A kit comprising

    The molecule of any one of claims 12-21, the conjugate of claim 22, the nucleic acid of claim 23 or the expression vector of claim 24, or the pharmaceutical composition of claim 27;

    preferably, the first and second liquid crystal display panels are,

    still further comprising means for administering the drug.
32. A method of treating a disease caused by a virus, preferably a coronavirus, more preferably SARS-CoV-2, comprising administering to a subject the molecule of any one of claims 12 to 21, the conjugate of claim 22, the nucleic acid of claim 23 or the expression vector of claim 24, or the pharmaceutical composition of claim 27, the pharmaceutical combination of claim 30 or the kit of claim 31.

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