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WO2022001803A1 - Méthode permettant de réduire un effet d'ade viral - Google Patents

Méthode permettant de réduire un effet d'ade viral Download PDF

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WO2022001803A1
WO2022001803A1 PCT/CN2021/101975 CN2021101975W WO2022001803A1 WO 2022001803 A1 WO2022001803 A1 WO 2022001803A1 CN 2021101975 W CN2021101975 W CN 2021101975W WO 2022001803 A1 WO2022001803 A1 WO 2022001803A1
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virus
molecule
antibody
seq
binding
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Chinese (zh)
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谢良志
孙春昀
陈龙
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Sinocelltech Ltd
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Sinocelltech Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of cellular immunity, and provides a method for reducing the ADE effect of a virus, and a molecule for reducing the ADE effect of the virus.
  • the present invention provides a molecule comprising an Fc fragment with reduced Fc receptor binding/complement binding, thereby achieving a reduction in viral ADE.
  • the present invention also provides a humanized antibody that reduces the ADE effect of the virus, binds and blocks the binding of the SARS-CoV-2 spike protein (S protein) to the ACE2 receptor, and efficiently neutralizes the cells infected by the SARS-CoV-2 virus.
  • S protein SARS-CoV-2 spike protein
  • SARS-CoV-2 and SARS-CoV spike protein (S protein) can cross-block the binding of SARS-CoV-2 and SARS-CoV spike protein (S protein) to the ACE2 receptor, and efficiently neutralize SARS-CoV-2 and SARS-CoV virus-infected cells.
  • S protein SARS-CoV spike protein
  • Antibodies can eliminate viruses through the effector functions of their Fc segments, including complement-mediated lysis of virus particles, antibody-mediated cytotoxicity, and phagocytosis. [1]. However, many viruses can use the feature of antibodies to promote the infection of host cells and regulate the signaling pathways of host cells, thereby inhibiting the antiviral immune response, a process called antibody-dependent enhancement (ADE).
  • ADE antibody-dependent enhancement
  • ADE Dengue virus
  • Ebola virus Ebola virus
  • ZIKV Zika virus
  • Chikungunya virus Chikungunya virus
  • SARS severe acute respiratory syndrome virus
  • the inventors modified the corresponding Fc fragment by means of molecular biology, and obtained Fc fragments with reduced Fc receptor binding/complement binding. molecules, thereby achieving a reduction in the ADE effect of the virus. It has been verified that the Fc modified fragment molecules provided by the present invention can express interaction with B cells, monocytes, macrophages, dendritic cells, etc. Fc ⁇ R-expressing cells, such as dendritic cells, have a reduced ability to bind and thus reduce viral infection.
  • the inventors invented a molecule that reduces the effect of viral ADE, the molecule comprising a) an antigen-binding fragment that recognizes and binds to the viral coat protein or extramembrane domain; and the aforementioned b) reduced Fc receptor binding/complement binding Fc fragment.
  • the purpose of the present invention is to provide a method for reducing the effect of viral ADE.
  • Another object of the present invention is to provide a molecule comprising an Fc fragment with reduced Fc receptor binding/complement binding, thereby reducing viral ADE.
  • Another object of the present invention is to provide a molecule for reducing the effect of viral ADE, the molecule comprising a) an antigen-binding fragment that recognizes and binds to the viral coat protein or extramembrane domain; and the aforementioned b) Fc receptor binding/complement Binding reduces Fc fragment.
  • Another object of the present invention is to provide a human source that can reduce the ADE effect of the virus, block the binding of the SARS-CoV-2 spike protein (S protein) to the ACE2 receptor, and efficiently neutralize the cells infected by the SARS-CoV-2 virus. Antibodies.
  • Another object of the present invention is to provide a cross-blocking SARS-CoV-2 and SARS-CoV spike protein (S protein) binding to the ACE2 receptor that reduces the ADE effect of the virus, and efficiently neutralizes SARS-CoV-2 and SARS-CoV-2. Humanized antibodies to SARS-CoV virus infecting cells.
  • S protein SARS-CoV spike protein
  • the present invention provides polynucleotides capable of encoding molecules with reduced Fc receptor binding/complement binding properties.
  • the present invention also provides a recombinant vector comprising the polynucleotide.
  • the present invention also provides host cells comprising the polynucleotide and/or the recombinant vector.
  • the present invention also provides antibodies formed from the polynucleotides expressed by recombinant vectors and/or host cells.
  • the molecules of the Fc fragments with reduced Fc receptor binding/complement binding provided by the present invention can be further prepared into vaccines, and the vaccines include the molecules of the modified Fc fragments, the polynucleotides, the recombinant vectors, the host cells, and the recombinant bacteria.
  • the vaccines include the molecules of the modified Fc fragments, the polynucleotides, the recombinant vectors, the host cells, and the recombinant bacteria.
  • One or more of , adenovirus, lentivirus, or viral particles as the active ingredient.
  • the vaccine includes one or more of inactivated vaccines, attenuated vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, other viral vector vaccines, subunit vaccines or virus particles .
  • the vaccine further includes any one or a combination of at least two of pharmaceutically acceptable vehicles, diluents, adjuvants or excipients.
  • the present invention also provides the above-mentioned Fc receptor-binding/complement-binding-reduced Fc fragment molecules, the above-mentioned antibodies, the above-mentioned polynucleotides, recombinant vectors, host cells, recombinant bacteria, adenoviruses, lentiviruses or virus particles in preparation for prevention and/or vaccines for the treatment of novel coronavirus infections.
  • the present invention also provides a therapeutic method for preventing or treating a disease or disorder caused by a novel coronavirus, the method comprising administering the molecule of the Fc modified fragment of the present invention, the above-mentioned antibody, the above-mentioned polynucleotide, recombinant Vectors, transgenic cell lines, recombinant bacteria, adenoviruses, lentiviruses or viral particles.
  • Figure 1 Flow cytometric identification of the corresponding FcR expression in CHO-K1-CD32A, CHO-K1-CD32B, CHO-K1-CD64 cells
  • FIG. 1 Fd11 engineering reduces binding of CoV2-HB27-IgG1 antibody to Fc receptor protein
  • FIG. 7 Fd6, Fd11 engineering reduces the ADE effect of CoV2-HB27-IgG1 antibody on CHO-K1-CD32A cells
  • FIG. 8 Fd6, Fd11 engineering reduces the ADE effect of CoV2-HB27-IgG1 antibody on CHO-K1-CD32B cells
  • FIG. 11 Fd11 engineering reduces the ADE effect of SARS-2-H014-IgG1 antibody on THP-1 cells
  • Antibody-reducing ADE is an important task in the development of antiviral biologics and vaccines.
  • the present invention establishes a method for reducing the effect of viral ADE. Specifically, the inventor achieves the reduction of viral ADE by modifying the Fc fragment of an antibody to reduce its binding to Fc receptors/complement binding.
  • 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, Lassa hemorrhagic fever virus, HIV virus, Ebola virus, Herpes zoster virus, CMV virus, hepatitis virus, human herpes simplex virus, cytomegalovirus, rotavirus, Epstein-Barr virus, measles virus, mumps virus, human papilloma virus, flavivirus or influenza virus; preferably SARS- CoV-2, SARS-CoV, MERS-CoV; influenza A virus (including H10N8, H7N9, H1N1, H5N1, etc.), influenza B virus; Ebola virus; False and true viruses.
  • Antibody-dependent enhancement effect refers to the binding of the virus to a non-neutralizing antibody or to a sub-neutralizing concentration of the antibody, the Fc segment of the antibody and the surface-expressed FcR cells bind and mediate the entry of the virus into these cells, thereby enhancing the infectivity of the virus. This phenomenon results in enhanced infectivity and toxicity, and ADEs modulate immune responses and can cause persistent inflammation, lymphopenia, and/or cytokine storms.
  • the main mechanism of ADE is that the Fc segment of the antibody in the antigen-antibody complex interacts with specific cells such as B cells, monocytes, macrophages, and dendritic cells under conditions of incomplete neutralization or non-neutralization of the virus by antibodies. After the binding of Fc ⁇ R-expressing cells, endocytosis into target cells is achieved [11,12]. In other words, the antibody helps the virus to enter the target cell leading to an increase in the number of infected cells.
  • This process referred to as exogenous ADE, is Fc ⁇ receptor (FcyR) dependent and occurs when the organism is secondary to infection with a heterologous serotype virus.
  • FcyR Fc ⁇ receptor
  • Circulating antibodies produced during primary viral infection recognize and bind to the secondary infected virus, enhancing viral infectivity rather than promoting virus neutralization by internalizing virus-antibody immune complexes in Fc ⁇ R-bearing cells. Once internalized, these immune complexes may modulate innate antiviral cellular responses, resulting in a dramatic increase in viral production per cell, a process known as endogenous ADE. Exogenous and endogenous ADEs together promote the massive release of inflammatory and vasoactive mediators, ultimately leading to disease aggravation [12].
  • receptor is a biochemical concept that refers to a class of molecules that can transduce extracellular signals and produce specific effects within cells. The resulting effects may only last for a short period of time, such as altering the metabolism of cells or the movement of cells. It may also be a long-term effect, such as up- or down-regulating the expression of one or more genes.
  • Fc receptor refers to a receptor that binds to the Fc region of an antibody.
  • the receptors that bind IgG antibodies are gamma receptors, which include FcyRI, FcyRII, and FcyRIII subtypes.
  • Human Fc ⁇ receptors mainly include Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), and Fc ⁇ RIII (CD16).
  • Fc ⁇ RI CD64
  • Fc ⁇ RII CD32
  • Fc ⁇ RIII CD16
  • Different virus types cause ADE-dependent Fc receptors with certain differences.
  • SARS and dengue fever mainly report that ADE is caused by CD32A[11,13]
  • MERS mainly triggers ADE through CD32 and CD64[14]
  • macrophages As an important cell type for phagocytosing antigen-antibody complexes, macrophages simultaneously express several receptors of CD16, CD32, and CD64.
  • anti-S protein antibodies can induce more immune cells (especially inflammatory macrophages) to infiltrate the lungs after SARS infection.
  • virus-induced macrophage ADE can induce Anti-inflammatory macrophage M2 produces more inflammatory factors such as IL-6, IL-8, MCP-1, etc., resulting in strong lung damage [15].
  • B cells have strong CD32 expression, and there are relatively few studies on the effect of virus on B cell function after infection by ADE. Antiserum produced by SARS-CoV vaccine can enhance virus infection of B cells [16]. Compared with normal people, the B cell composition of patients after dengue virus infection is quite different. The number of immature B cells, transitional B cells and Breg in severe patients is significantly lower than that in mild patients. Mature B cells cannot produce IL-10, activation markers and the expression of antigen-presenting molecules, so the acute infection phase of dengue has a serious impact on B cell function [17], which may be related to ADE.
  • the inventors modified the corresponding Fc fragment by means of molecular biology to obtain a molecule of Fc fragment with reduced Fc receptor binding/complement binding, thereby reducing the ADE effect of the virus. Further, the inventors invented a molecule for reducing the effect of viral ADE, the molecule comprising a) an antigen-binding fragment that recognizes and binds to the viral coat protein or extramembrane domain; and the aforementioned b) Fc receptor binding/complement binding Decreased Fc fragments.
  • a molecule comprising a) an antigen-binding fragment that recognizes and binds to a viral coat protein or extramembrane domain; and b) an Fc fragment, wherein b) has been engineered by molecular biological means to have reduced Fc receptor binding and/or complement binding.
  • the molecule When the molecule is administered as a therapeutic or prophylactic agent to a subject infected with a virus, or at risk of being infected with a virus, due to its reduced Fc receptor binding and/or complement binding, it interacts with specific cells such as B cells, monocytes, Fc ⁇ R-expressing cells such as cells, macrophages, dendritic cells, etc. have decreased binding, with the consequence of decreased endocytosis of target cells. Viral infection is thereby reduced.
  • the inventors engineered antibodies COV2-HB27 and SARS-2-H014.
  • CoV2-HB27 is a humanized antibody that can block the binding of SARS-CoV-2 spike protein (S protein) to the ACE2 receptor and efficiently neutralize SARS-CoV-2 virus-infected cells.
  • SARS-2-H014 is a human that can cross-block the binding of SARS-CoV-2 and SARS-CoV spike protein (S protein) to the ACE2 receptor, and efficiently neutralize SARS-CoV-2 and SARS-CoV virus-infected cells.
  • Antibody For details of its preparation, structure and properties, please refer to patent application 202010219867.1 and PCT/CN2021/082374 (incorporated into this specification by reference in its entirety).
  • virus-like particle refers to a polyprotein structure consisting of the corresponding native viral structural proteins, but lacking all or part of the viral genome, especially the replication and infectious components of the viral genome, and therefore not replicable and infectious.
  • the polyprotein structure highly mimics its corresponding natural virus particles in shape and size, and can be formed spontaneously after recombinant expression of viral structural proteins.
  • antibody means an immunoglobulin molecule and refers to any form of antibody that exhibits the desired biological activity. Including, but not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), and even antibody fragments.
  • a full-length antibody structure preferably comprises 4 polypeptide chains, usually 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds.
  • intact antibodies can be assigned to five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, of which IgG and IgA can be further divided into subclasses (isotypes), such as IgG1, IgG2 , IgG3, IgG4, IgA1 and IgA2. Accordingly, the heavy chains of the five classes of antibodies are classified as alpha, delta, epsilon, gamma, and mu chains, respectively.
  • the light chain of an antibody can be classified into kappa and lambda based on the amino acid sequence of its light chain constant region.
  • each heavy chain has a variable region (VH, variable heavy chain domain) followed by 3 constant domains (CH1, CH2 and CH3, also known as heavy chain) constant region).
  • VH variable heavy chain domain
  • CH1 constant domains
  • 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 parts" (or “Fab fragments”) and an "Fc part” (or “Fc fragments”).
  • the "Fab portion” of an antibody comprises the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain.
  • the "Fc portion" of an antibody comprising two heavy chains, CH2 and CH3, is not directly involved in the binding of the antibody to antigen, but exhibits various effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and "antibody-dependent enhancement (ADE)”.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADE antibody-dependent enhancement
  • 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 specified, "binding affinity” as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). "KD”, "binding rate constant k on” and “off rate constant k off” is generally used to describe a molecule (e.g. an antibody) and its binding partner (e.g. antigen) between the affinity, i.e., binding a particular protein ligand tight degree. Binding affinity is affected by non-covalent intermolecular interactions such as hydrogen bonding, electrostatic interactions, hydrophobic and van der Waals forces between two molecules. Additionally, 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.
  • the inventors modified the heavy chain IgG1 constant region of the CoV2-HB27 antibody by means of molecular biology to obtain IgG1 subtype humanized antibodies with reduced Fc function CoV2-HB27-Fd6-IgG1 and CoV2- HB27-Fd11-IgG4; CoV2-HB27-Fd6-IgG1 antibodies have almost no binding to CD32a and CD32b; only weak binding to CD64, and only weak binding to C1q (see patent application 202010349190.3 and PCT/CN2021/ 089748). CoV2-HB27-Fd11-IgG4 antibody has almost no binding to CD32A, CD32B and CD64. Both CoV2-HB27-Fd6-IgG1 and CoV2-HB27-Fd11-IgG4 exhibited reduced ADE effects on CHO-K1-CD32A cells, CHO-K1-CD32B cells and Raji cells.
  • the inventors modified the heavy chain IgG4 constant region of the SARS-2-H014 antibody by means of molecular biology, and the SARS-2-H014-Fd11-IgG4 antibody was associated with CD32a, CD32b, CD16a and C1q Complement protein has no binding, has a very weak binding level to CD64 under high concentration conditions, and binds to FcRn similar to IgG1 subtype antibody under pH 6.0 conditions (see patent application 202010219867.1 and PCT/CN2021/082374 for details). SARS-2-H014-Fd11-IgG4 antibody showed reduced ADE effect on CHO-K1-CD64, THP-1 and U937 cells.
  • a nucleotide sequence encoding the desired molecule of the invention can be inserted into an expression vector, which is then transfected into a suitable host cell.
  • suitable host cells are prokaryotic cells and eukaryotic cells.
  • 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 an expression vector including selection regulatory sequences is influenced by a variety of factors, such as the choice of host cell, the level of protein expression desired, and whether expression is constitutive or inducible.
  • Molecules of the invention can be recovered and purified from recombinant cell culture by known methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, protein A affinity chromatography, protein G affinity chromatography, anionic Or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography.
  • HPLC High performance liquid chromatography
  • Molecules of the present 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. Molecules of the present invention may be glycosylated, or may be non-glycosylated. Such methods are described in a number of standard laboratory manuals, eg, Sambrook, supra, sections 17.37-17.42; Ausubel, supra, chapters 10, 12, 13, 16, 18 and 20.
  • embodiments of the present invention are also host cells comprising the vectors or nucleic acid molecules described above, 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 For prokaryotic cells such as bacterial cells.
  • 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 For prokaryotic cells such as bacterial cells.
  • the method of the present invention can be used to treat, prevent or detect viruses such as SARS-CoV-2, diseases caused by SARS-CoV, such as acute respiratory infectious diseases caused by SARS-CoV-2 and SARS-CoV viruses.
  • viruses such as SARS-CoV-2, diseases caused by SARS-CoV, such as acute respiratory infectious diseases caused by SARS-CoV-2 and SARS-CoV viruses.
  • One or more of the molecules, nucleic acids, and vectors of the present invention, together with at least one other chemical agent, can be formulated into pharmaceutical compositions comprising the above-mentioned active ingredients and one or more pharmaceutically acceptable carriers, diluents, or excipients agent; optionally, one or more other therapeutic agents may also be included.
  • the present invention also relates to pharmaceutical packages and kits comprising one or more containers containing the above-mentioned pharmaceutical compositions of the present invention.
  • Associated with such a container may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of a drug or biological product, which reflects approval for human administration by the agency of manufacture, use or sale of the product.
  • compositions of the present invention can be prepared in a manner known in the art, eg, by conventional mixing, dissolving, granulating, grinding, emulsifying, encapsulating, entrapping, or lyophilizing methods.
  • compositions comprising a compound of the present invention formulated in an acceptable carrier
  • suitable container and labeled for treatment of the indicated condition.
  • labels would include the amount, frequency and method of administration.
  • compositions comprising the antibodies of the invention are also combined with one or more other therapeutic agents, wherein the resulting combination does not cause unacceptable adverse effects.
  • the pCMV3 vector containing CD32A, CD32B, CD64 and FcR ⁇ -chain was transfected into CHO-K1 cells (source: ATCC) to prepare CD32A expression respectively (transfected with CD32A vector alone), CD32B (transfected with CD32B vector alone) or CD64 (co-transfected with CD64 and FcR ⁇ -chain vector) CHO-K1 cells.
  • CHO-K1 cells one day in advance digestion, counted and incubated at 3.5x10 6 cells in T25 flask and make up the medium (DMEM + 10% FBS + 69 ⁇ g / mL proline) to 7 mL, placed in 37 °C, 5% CO 2 Plate overnight in the incubator.
  • DMEM + 10% FBS + 69 ⁇ g / mL proline 10% FBS + 69 ⁇ g / mL proline
  • hygromycin was added to the cells to select transfected positive cells.
  • the cells were digested, and the cells were diluted to 0.5 cells/mL with hygromycin-containing medium, and 100 ⁇ L of the cell suspension was added to each well of a 96-well flat-bottom cell culture plate. Incubate in a 37 °C, 5% CO 2 incubator.
  • the monoclonal cells were expanded and cultured, and the CD32 and CD64 antibodies (source: BD) were used to detect the expression of FcR on the cells.
  • -K1-CD32B and CHO-K1-CD64 monoclones were expanded and used for subsequent experiments, and the results of flow cytometry were shown in Figure 1.
  • Pseudovirus expressing the full-length protein of SARS-CoV-2S was packaged using 293T (source: ATCC). 293T was digested one day in advance, counted, and 3.5x10 6 cells were added to a T25 culture flask, supplemented with medium (DMEM+10% FBS) to 7 mL, and placed in a 37°C, 5% CO 2 incubator for overnight plating.
  • 293T was digested one day in advance, counted, and 3.5x10 6 cells were added to a T25 culture flask, supplemented with medium (DMEM+10% FBS) to 7 mL, and placed in a 37°C, 5% CO 2 incubator for overnight plating.
  • medium DMEM+10% FBS
  • VSV pseudovirus (VSV ⁇ G-Luc) was used to infect 293T cells transfected with SARS-CoV-2 Spike, 1h later, washed three times with PBS, and supplemented with 7mL of fresh 293T medium. After 24 hours, the supernatant was collected, filtered with a 0.45 ⁇ m filter to remove cell debris, and a pseudovirus solution was obtained, which was stored at -80°C.
  • the virus was diluted 10 times by limiting dilution method, and a total of 6 virus concentrations were set, each with 6 duplicate wells.
  • the 96-well plate was seeded at a density of 3 ⁇ 10 4 cells/mL VERO E6 (source: Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences) suspension, 100 ⁇ L/well. Add 100 ⁇ L of serially diluted virus to each well, use the cell culture medium as a negative control, mix well and place in a 37°C, 5% CO 2 incubator for 24 h. After the incubation, discard the supernatant, add passive lysis buffer (source: Promega) diluted to 1x, 100 ⁇ L/well, and mix to lyse the cells. 40 ⁇ L/well was transferred to a 96-well white bottom plate to detect the fluorescent signal, and the TCID 50 value was calculated using the Karber method.
  • CoV2-HB27 is a humanized antibody that can block the binding of the SARS-CoV-2 spike protein (S protein) to the ACE2 receptor and efficiently neutralize cells infected by the SARS-CoV-2 virus. Details of its preparation, structure and properties are detailed in patent application 202010349190.3 and PCT/CN2021/089748 (herein incorporated for reference and made a part hereof).
  • SARS-2-H014 is a human that can cross-block the binding of SARS-CoV-2 and SARS-CoV spike protein (S protein) to the ACE2 receptor, and efficiently neutralize SARS-CoV-2 and SARS-CoV virus-infected cells.
  • S protein SARS-CoV spike protein
  • Antibody Its preparation, structure and properties are detailed in patent application 202010219867.1 and PCT/CN2021/082374 (herein incorporated for reference and made a part hereof).
  • the nucleotide sequence of the CoV2-HB27 heavy chain variable region (SEQ ID NO: 5) was obtained by the method of total gene synthesis. Inserted into pSE vector cleaved by ScaI+NheI (source: Fermentas, the same below) with heavy chain signal peptide (SEQ ID NO:3) and heavy chain IgG1 constant region (SEQ ID NO:7) by In-fusion method The CoV2-HB27 heavy chain (SEQ ID NO: 1) expression vector was obtained from .
  • the nucleotide sequences of the CoV2-HB27 light chain variable region (SEQ ID NO: 6) were obtained by the method of total gene synthesis. Inserted by In-fusion method into ScaI+BsiWI (source: Fermentas) digested pSE with light chain signal peptide (SEQ ID NO:4) and light chain kappa constant region nucleotide sequence (SEQ ID NO:8) The CoV2-HB27 light chain (SEQ ID NO: 2) expression vector was obtained from the vector.
  • the plasmid was extracted, it was transfected into 293E cells (source: Invitrogen, the same below), cultured and expressed for 7 days, and purified with a protein A purification column to obtain a high-purity antibody.
  • F1 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) CCTCTGGAGGCCTCCTACACCTACTACCCTGAC 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) TGGGCCCTTGGTG
  • 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) GCCCTGGTTGCTG
  • the constant region of the IgG1 subtype was subjected to nucleotide mutation with reference to the literature [18] to obtain a genetically engineered heavy chain IgG1 constant region nucleotide sequence (Fd6-IgG1, SEQ ID NO:9).
  • the CoV2-HB27-Fd6-IgG1 heavy chain sequence (SEQ ID NO: 10) was obtained by PCR, which comprises the heavy chain signal peptide nucleotide sequence (SEQ ID NO: 3), the heavy chain variable region nucleotide sequence (SEQ ID NO: 3) ID NO: 5) and Fd6-IgG1 constant region nucleotide sequence (SEQ ID NO: 9).
  • the expression vector containing CoV2-HB27-Fd6-IgG1 heavy chain was obtained by inserting into the pSE vector digested by HindIII+XbaI by In-fusion method.
  • CoV2-HB27-Fd6-IgG1 heavy chain (SEQ ID NO:10) expression vector and CoV2-HB27 light chain (SEQ ID NO:2) expression vector plasmids were extracted, transfected into 293E cells, cultured and expressed for 7 days, and purified with protein A
  • the CoV2-HB27-Fd6-IgG1 antibody with reduced Fc function was obtained by column purification.
  • nucleotide mutations were performed on the constant region of the IgG4 subtype with reference to the literature [18] to obtain a genetically engineered heavy chain IgG4 constant region nucleotide sequence (Fd11-IgG4, SEQ ID NO: 11).
  • the CoV2-HB27-Fd11-IgG4 heavy chain sequence (SEQ ID NO: 12) was obtained by PCR, which comprises the heavy chain signal peptide nucleotide sequence (SEQ ID NO: 3), the heavy chain variable region nucleotide sequence (SEQ ID NO: 3) ID NO: 5) and the Fd11-IgG4 constant region nucleotide sequence (SEQ ID NO: 11).
  • the expression vector containing CoV2-HB27-Fd11-IgG4 heavy chain was obtained by inserting into pSE vector digested by HindIII+XbaI by In-fusion method.
  • CoV2-HB27-Fd11-IgG4 heavy chain (SEQ ID NO:12) expression vector and CoV2-HB27 light chain (SEQ ID NO:2) expression vector plasmids were extracted, transfected into 293E cells, cultured and expressed for 7 days, and purified with protein A
  • the CoV2-HB27-Fd11-IgG4 antibody with reduced Fc function was obtained by column purification.
  • SARS-2-H014 heavy chain variable region SEQ ID NO: 16
  • SARS was obtained by inserting into pSE vector with heavy chain signal peptide (SEQ ID NO: 15) and heavy chain IgG1 constant region (SEQ ID NO: 7) digested by ScaI+NheI (source: Fermentas) by In-fusion method -2-H014 heavy chain (SEQ ID NO: 13) expression vector.
  • the SARS-2-H014 light chain variable region (SEQ ID NO: 17) was obtained by the method of total gene synthesis, and inserted into the light chain signal peptide (SEQ ID NO: 4) and the light chain kappa constant by the In-fusion method
  • the SARS-2-H014 light chain (SEQ ID NO: 14) expression vector was obtained from the pSE vector digested by ScaI+BsiWI (source: Fermentas) with the nucleotide sequence of the region (SEQ ID NO: 18). After the plasmid was extracted, it was transfected into 293E cells (source: Invitrogen) for culturing and expression for 7 days, and purified by protein A purification column to obtain high-purity antibody.
  • nucleotide mutations were performed on the constant region of the IgG4 subtype with reference to the literature [18] to obtain a genetically engineered heavy chain IgG4 constant region nucleotide sequence (Fd11-IgG4, SEQ ID NO: 11).
  • the SARS-2-H014-Fd11-IgG4 heavy chain sequence (SEQ ID NO: 19) was obtained by splicing PCR, which contained the heavy chain signal peptide nucleotide sequence (SEQ ID NO: 15), and the SARS-2-H014 heavy chain could be Variable region nucleotide sequence (SEQ ID NO: 16) and Fd11-IgG4 nucleotide sequence (SEQ ID NO: 11).
  • the SARS-2-H014-Fd11-IgG4 heavy chain (SEQ ID NO: 19) expression vector was obtained by inserting into the pSE vector digested by HindIII+XbaI (source: Fermentas) by the In-fusion method.
  • Splicing SARS-2-H014-Fd11-IgG4 heavy chain primers Splicing SARS-2-H014-Fd11-IgG4 heavy chain primers:
  • a protein A purification column was used to obtain a highly purified IgG4 subtype humanized SARS-2-H014 antibody with reduced Fc function, namely SARS-2-H014-Fd11-IgG4.
  • the 293E cells were passaged to 200mL/flask with SCD4-4-TC2 medium (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.), the initial seeding density was 0.3-0.4 ⁇ 10 6 cell/mL, and the rotation speed was 175 rpm at 37 °C CO 2 .
  • Cell culture was performed in a shaker. After the cell density reaches 1.5 ⁇ 3 ⁇ 10 6 cells/mL, add a total of 100 ⁇ g light and heavy chain plasmid DNA mixed at a ratio of 1:1 and 800 ⁇ L TF2 transfection reagent (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) Cultivation was continued in the bed until harvest on the 7th day.
  • the culture medium was centrifuged at 4000rpm for 25min, the supernatant was collected, and 1/5 of the supernatant volume was added to the stock buffer (source: Shenzhou Cell Engineering Co., Ltd.).
  • Buffer source: Shenzhou Cell Engineering Co., Ltd.
  • Avidin protein (source: Thermo, the same below) at a concentration of 10 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 2-8°C. The next day, the plate was washed, and after blocking at room temperature for 1 h, 100 ⁇ L of biotin-labeled CD16a-AVI-His(V158) + BirA protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added at a concentration of 5 ⁇ g/mL, and the plate was washed after incubating at room temperature for 1 h. .
  • CoV2-HB27 antibodies with different Fc functional forms were added at the concentration of 5 ⁇ g/mL and 1 ⁇ g/mL. After 1 h of incubation, wash the plate to remove unbound antibodies, add goat anti-human IgGF(ab)2/HRP (source: Jackson ImmunoResearch, the same below), and repeat the plate washing after incubation, and finally add the substrate chromogenic solution for color development. microplate reading OD 450.
  • the Fd11-IgG4 format antibody with reduced Fc function has only very weak binding to CD16a compared to the IgG1 format.
  • Avidin protein at a concentration of 10 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, overnight at 2-8°C. The next day, the plate was washed, and after blocking at room temperature for 1 h, 100 ⁇ L of biotin-labeled CD32a-AVI-His(R131)+BirA protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) or CD32b-AVI-HIS was added at a concentration of 5 ⁇ g/mL. +BirA (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) protein, incubated at room temperature for 1 h and washed the plate.
  • CoV2-HB27 antibodies of different Fc functional forms at concentrations of 5 ⁇ g/mL and 1 ⁇ g/mL. After incubation for 1 h, wash the plate to remove unbound antibodies, add goat anti-human IgG F(ab)2/HRP and incubate, repeat the plate washing, add substrate color development solution for color development, and read the OD 450 with a microplate reader after termination.
  • the Fd11-IgG4 format antibody with reduced Fc function showed almost no binding to CD32a and CD32b compared with the IgG1 format antibody (Figs. 2B and 2C).
  • Avidin protein at a concentration of 10 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, overnight at 2-8°C. The next day, the plate was washed, and after blocking at room temperature for 1 h, 100 ⁇ L of biotin-labeled CD64-AVI-His+BirA protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added at a concentration of 0.5 ⁇ g/mL, and the plate was washed after incubating at room temperature for 1 h. Add 100 ⁇ L of CoV2-HB27 antibodies of different Fc functional forms at concentrations of 5 ⁇ g/mL and 1 ⁇ g/mL.
  • the Fd6-IgG1 format antibody with reduced Fc function has only weaker binding to CD64 than the IgG1 format antibody.
  • CoV2-HB27 antibodies with different Fc functional forms were coated on a 96-well plate, 100 ⁇ L/well, overnight at 4°C, and the antibody concentration was 5 ⁇ g/mL and 1 ⁇ g/mL.
  • the plate was washed the next day, and after blocking at room temperature for 1 h, 5 ⁇ g/mL of C1q complement protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, 100 ⁇ g/well, and incubated for 1 h.
  • C1q complement protein source: Beijing Yiqiao Shenzhou Technology Co., Ltd.
  • the Fd11-IgG4 format antibody with reduced Fc function has only weak binding to C1q.
  • the 293FT cell line (293FT-SARS-CoV-2-S, source: Shenzhou Cell Engineering Co., Ltd., the same below) expressing SARS-CoV-2 full-length protein transiently was used as the target cell to stably transfect CD16AV and NFAT-
  • the Jurkat cells of Luc2P Jurkat-NFAT/Luc2P-CD16AV are effector cells, and the ADCC function of the humanized antibody is detected by the reporter gene method.
  • Target cells at a density of 1 ⁇ 10 5 cells/mL and an equal volume of equal density of effector cells were inoculated in 50 ⁇ L/well in a 96-well plate. Afterwards, 50 ⁇ L of CoV2-HB27 antibody and H7N9-R1 negative control antibody of different Fc functional forms were added. CoV2-HB27-IgG1, CoV2-HB27-Fd11-IgG4 antibody and H7N9-R1 negative control antibody were added at concentrations of 20 ⁇ g/mL, 1 ⁇ g/mL and 0.05 ⁇ g/mL. 37 °C, 5% CO 2 incubator for 6h after mixing.
  • the Fd11-IgG4 antibody with reduced Fc function had no ADCC effect.
  • Jurkat cells stably transfected with CD32A, CD32B or CD64 and NFAT-Luc2P (Jurkat-NFAT/Luc2P-CD32A, Jurkat-NFAT/Luc2P-CD32B or Jurkat-NFAT /Luc2P-CD64) as effector cells, and the humanized antibody-mediated ADCP function was detected by reporter gene method.
  • Target cells at a density of 1 ⁇ 10 5 cells/mL and an equal volume of equal density of effector cells were inoculated in 50 ⁇ L/well in a 96-well plate. Afterwards, 50 ⁇ L of CoV2-HB27 antibody and H7N9-R1 negative control antibody of different Fc functional forms were added.
  • Jurkat-NFAT/Luc2P-CD32A, Jurkat-NFAT/Luc2P-CD32B and Jurkat-NFAT/Luc2P-CD64 were used as effector cells, and antibodies were added at concentrations of 20 ⁇ g/mL, 1 ⁇ g/mL and 0.05 ⁇ g/mL. 37 °C, 5% CO 2 incubator for 6h after mixing.
  • Fig. 5A, 5B and Fig. 5C The results are shown in Fig. 5.
  • Fig. 5A, 5B and Fig. 5C IgG1 and Fd11-IgG4 None of the forms of CoV2-HB27 antibodies had ADCP effects.
  • Target cells at a density of 2 ⁇ 10 6 cells/mL were seeded at 50 ⁇ L/well in a 96-well plate.
  • the addition concentrations were 100 ⁇ g/mL, 20 ⁇ g/mL, 4 ⁇ g/mL, 0.8 ⁇ g/mL, 0.16 ⁇ g/mL, 0.032 ⁇ g/mL, 0.0064 ⁇ g/mL, and 0.00128 ⁇ g/mL.
  • Example 5 Fc engineering reduces the ADE effect of antibodies on CD32A expressing cells
  • CHO-K1-CD32A was digested one day in advance, the cell density was adjusted to 3x10 5 /mL with medium, and 100 ⁇ L of cell suspension was added to each well of a 96-well cell culture plate, and then placed in a 37°C, 5% CO 2 incubator for overnight plating. The next day, another 96-well cell culture plate was added with different concentrations (final concentration of 500 ⁇ g/mL starting, 4-fold gradient dilution, 9 gradients in total), 50 ⁇ L/well. Add 500 TCID 50 of SARS-CoV-2 pseudovirus to each well, 50 ⁇ L/well. The group with virus and no antibody was used as a positive control, and the group without virus and antibody was used as a negative control.
  • Example 6 Fc engineering reduces the ADE effect of antibodies on CD32B expressing cells
  • CHO-K1-CD32B was digested one day in advance, the cell density was adjusted to 3x10 5 /mL with medium, 100 ⁇ L of cell suspension was added to each well of a 96-well cell culture plate, and then placed in a 37°C, 5% CO 2 incubator for overnight plating. The next day, another 96-well cell culture plate was added with different concentrations (final concentration of 500 ⁇ g/mL starting, 4-fold gradient dilution, 9 gradients in total), 50 ⁇ L/well. Add 500 TCID 50 of SARS-CoV-2 pseudovirus to each well, 50 ⁇ L/well. The group with virus and no antibody was used as a positive control, and the group without virus and antibody was used as a negative control.
  • Example 7 Fc engineering reduces the ADE effect of antibodies on CD64 expressing cells
  • CHO-K1-CD64 was digested one day in advance, the cell density was adjusted to 3x10 5 /mL with medium, and 100 ⁇ L of cell suspension was added to each well of a 96-well cell culture plate, and then placed in a 37°C, 5% CO 2 incubator for overnight plating. The next day, another 96-well cell culture plate was added with antibodies of different concentrations (final concentration 100 ⁇ g/mL starting, 5-fold gradient dilution, 9 gradients in total), 50 ⁇ L/well. Add 500 TCID 50 of SARS-CoV-2 pseudovirus to each well, 50 ⁇ L/well. The group with virus and no antibody was used as a positive control, and the group without virus and antibody was used as a negative control.
  • Example 8 Fc engineering reduces the ADE effect of antibodies on cells expressing various FcRs
  • Antibodies of different concentrations were added to the 96-well cell culture plate, 50 ⁇ L/well.
  • the group with virus and no antibody was used as a positive control, and the group without virus and antibody was used as a negative control.
  • 100 ⁇ L of Raji cells with a density of 3 ⁇ 10 5 /mL were added to each well, and the cells were placed in a 37° C., 5% CO 2 incubator for static culture for 24 h.
  • THP-1 cells were cultured with 2.5 ⁇ g/mL PMA and induced for 3 days to evaluate the ADE effect of antibodies of different subtypes of SARS-2-H014.
  • THP-1 cells were digested one day before induction, and the cell density was adjusted to 3x10 5 /mL with medium. 100 ⁇ L of cell suspension was added to each well of a 96-well cell culture plate and then placed in a 37°C, 5% CO 2 incubator for overnight plating. The next day, another 96-well cell culture plate was added with different concentrations (final concentration of 80 ⁇ g/mL starting, 5-fold gradient dilution, 9 gradients in total), 50 ⁇ L/well.
  • U937 cells were cultured with 2.5 ⁇ g/mL PMA and induced for 3 days to evaluate the ADE effect of antibodies of different subtypes of SARS-2-H014.
  • U937 cells were digested one day in advance after induction, and the cell density was adjusted to 3x10 5 /mL with medium. 100 ⁇ L of cell suspension was added to each well of a 96-well cell culture plate and then placed in a 37°C, 5% CO 2 incubator for overnight plating. The next day, another 96-well cell culture plate was added with different concentrations (final concentration 10 ⁇ g/mL starting, 5-fold gradient dilution, 6 gradients in total), 50 ⁇ L/well.

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Abstract

L'invention concerne une méthode permettant de réduire un effet d'ADE viral. La méthode atteint son objectif par l'administration de molécules qui réduisent l'effet d'ADE viral à un sujet qui est infecté par un virus ou qui est susceptible d'être infecté par un virus. Par la réalisation d'une modification de biologie moléculaire sur un fragment Fc d'un anticorps, un fragment Fc présentant des performances de liaison au récepteur Fc/au complément réduites est obtenu. L'anticorps contenant le fragment Fc peut réduire l'effet d'ADE viral. L'anticorps est de préférence utilisé pour prévenir et/ou traiter des infections respiratoires aiguës provoquées par une infection à coronavirus.
PCT/CN2021/101975 2020-06-28 2021-06-24 Méthode permettant de réduire un effet d'ade viral Ceased WO2022001803A1 (fr)

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CN119265271A (zh) * 2024-12-09 2025-01-07 上海益诺思生物技术股份有限公司 一种评价ade效应的系统及其应用

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WO2018011283A1 (fr) * 2016-07-13 2018-01-18 Humabs Biomed Sa Nouveaux anticorps se liant spécifiquement aux épitopes du virus du zika et leurs utilisations
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CN119265271A (zh) * 2024-12-09 2025-01-07 上海益诺思生物技术股份有限公司 一种评价ade效应的系统及其应用

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