A method to reduce the ADE effect of viruses

Abstract

A method for reducing the viral adverse reaction-deprivation (ADE) effect is provided, which achieves this by administering a molecule that reduces the viral ADE effect to a patient who is infected with the virus or at risk of viral infection. This is achieved by molecularly modifying the Fc fragment of an antibody to obtain an Fc fragment with reduced Fc receptor binding / complement binding properties. An antibody containing this Fc fragment can reduce the viral ADE effect. This antibody is preferably used for the prevention and / or treatment of acute respiratory infectious diseases caused by coronavirus infection.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202010598424.8, filed on June 28, 2020, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of cellular immunotherapy, providing a method for reducing viral adverse event-induced degradation (ADE) and a molecule for reducing ADE. Specifically, this invention provides a molecule containing an Fc fragment with reduced Fc receptor binding / complement binding, thereby achieving a reduction in viral ADE. This invention also provides humanized antibodies that reduce viral ADE by binding to and blocking the binding of the SARS-CoV-2 spike protein (S protein) to the ACE2 receptor, and that efficiently neutralize SARS-CoV-2 virus-infected cells; and humanized antibodies that reduce viral ADE by cross-blocking the binding of SARS-CoV-2 and SARS-CoV spike proteins (S proteins) to the ACE2 receptor, and that efficiently neutralize SARS-CoV-2 and SARS-CoV virus-infected cells. Background Technology

[0004] The neutralization of viruses by antibodies is an important mechanism for the host to resist viral infection. Antibodies can clear viruses through the effector functions of their Fc fragments, including complement-mediated lysis of viral particles, antibody-mediated cytotoxicity, and phagocytosis [1]. However, many viruses can use this property of antibodies to promote infection of host cells and regulate host cell signaling pathways, thereby inhibiting antiviral immune responses. This process is called antibody-dependent enhancement (ADE).

[0005] The ADE phenomenon was first discovered in dengue virus (DENV)[2], and subsequently, many virus types, including Ebola virus, Zika virus (ZIKV), Chikungunya virus, and severe acute respiratory syndrome virus (SARS), have reported ADE phenomena in in vitro experiments[3-5]. Although there is still controversy as to whether ADE is a major risk in clinical practice, there are many clues indicating that ADE may become one of the important factors that cannot be ignored in the development of antiviral drugs and vaccines.

[0006] A retrospective analysis of a clinical trial indicated that the earliest approved dengue virus vaccine, CYD-TDV, had its application strategy reconsidered due to its risk of exacerbating infection [6], suggesting that ADE may affect the effectiveness of vaccine use in clinical practice. Furthermore, in SARS-infected patients, higher titers of anti-SARS immunoglobulin were detected in the serum of severely ill patients than in those with mild symptoms [7]. This phenomenon has also been confirmed in several recent studies on the novel coronavirus (SARS-CoV-2): the severity of COVID-19 infection patients is positively correlated with the titer of total antibody IgG [8-10]. Therefore, ADE that reduces antibodies may improve safety during antibody-mediated COVID-19 treatment.

[0007] The impact of ADE on the immune system should not be underestimated. The reduction of antibody ADE has important guiding significance for vaccines, convalescent serum, and neutralizing antibodies in the prevention or treatment of novel coronavirus infection. Summary of the Invention

[0008] To avoid the impact of viral ADE on vaccine efficacy and pursue safer clinical outcomes, the inventors used molecular biology techniques to modify the corresponding Fc fragments, obtaining molecules with reduced Fc receptor binding / complement binding, thereby reducing viral ADE. Verification has shown that the modified Fc fragment molecules provided by this invention, when administered as therapeutic or prophylactic agents to virus-infected individuals or subjects at risk of viral infection, exhibit reduced binding to cells expressing FcγR, such as B cells, monocytes, macrophages, and dendritic cells, thus reducing viral infection. Furthermore, the inventors have invented a molecule for reducing viral ADE, comprising a) an antigen-binding fragment that recognizes and binds to the viral capsid protein or extracellular domain; and the aforementioned b) Fc receptor-binding / complement-binding-reduced Fc fragment. Invention Details

[0010] The purpose of this invention is to provide a method for reducing the ADE effect of viruses.

[0011] Another object of the present invention is to provide a molecule containing an Fc fragment with reduced Fc receptor binding / complement binding, thereby achieving a reduction in viral ADE.

[0012] Another object of the present invention is to provide a molecule that reduces the ADE effect of a virus, the molecule comprising a) an antigen-binding fragment that recognizes and binds to the viral capsid protein or extracellular domain; and the aforementioned b) Fc receptor binding / complement binding reducing Fc fragment.

[0013] Another object of the present invention is to provide a humanized antibody that reduces the viral ADE effect, blocks the binding of the SARS-CoV-2 spike protein (S protein) to the ACE2 receptor, and efficiently neutralizes SARS-CoV-2 virus-infected cells.

[0014] Another object of the present invention is to provide a humanized antibody that reduces the viral ADE effect, cross-blocks the binding of SARS-CoV-2 and SARS-CoV spike protein (S protein) to the ACE2 receptor, and efficiently neutralizes SARS-CoV-2 and SARS-CoV virus-infected cells.

[0015] Accordingly, the present invention provides polynucleotides capable of encoding molecules with reduced Fc receptor binding / complement binding properties.

[0016] The present invention also provides a recombinant vector containing the said polynucleotide.

[0017] The present invention also provides a host cell comprising the polynucleotide and / or the recombinant vector.

[0018] The present invention also provides antibodies formed from the polynucleotide expressed via a recombinant vector and / or host cells.

[0019] The Fc fragment with reduced Fc receptor binding / complement binding provided by the present invention can be further prepared into a vaccine, wherein the vaccine comprises one or more of the following as active ingredients: the above-mentioned Fc modified fragment molecule, the above-mentioned polynucleotide, the above-mentioned recombinant vector, the above-mentioned host cell, recombinant bacteria, adenovirus, lentivirus or virus particles.

[0020] In one possible implementation, the vaccine includes one or more of the following: inactivated vaccine, live attenuated vaccine, mRNA vaccine, DNA vaccine, adenovirus vector vaccine, other viral vector vaccine, subunit vaccine, or virus particle.

[0021] In one possible implementation of the aforementioned vaccine, the vaccine further includes any one or a combination of at least two of pharmaceutically acceptable mediators, diluents, adjuvants, or excipients.

[0022] The present invention also provides the use of the above-mentioned Fc fragment with reduced Fc receptor binding / complement binding, the above-mentioned antibody, the above-mentioned polynucleotide, recombinant vector, host cell, recombinant bacteria, adenovirus, lentivirus or viral particles in the preparation of vaccines for the prevention and / or treatment of novel coronavirus infection.

[0023] The present invention also provides a treatment method for preventing or treating diseases or symptoms caused by the novel coronavirus, the method comprising administering a molecule of the Fc modified fragment described in the present invention, the antibody described above, the polynucleotide described above, a recombinant vector, a transgenic cell line, a recombinant bacterium, adenovirus, lentivirus, or viral particles. Attached Figure Description

[0024] Figure 1 Flow cytometry identification of FcR expression in CHO-K1-CD32A, CHO-K1-CD32B, and CHO-K1-CD64 cells

[0025] Figure 2 Fd11 modification reduces the binding of CoV2-HB27-IgG1 antibody to Fc receptor protein.

[0026] Figure 3 Fd11 modification reduces the binding of CoV2-HB27-IgG1 antibody to complement C1q protein.

[0027] Figure 4 Fd11 modification reduces the ADCC effect of CoV2-HB27-IgG1 antibody.

[0028] Figure 5 Antibodies against CoV2-HB27 of IgG1 and Fd11-IgG4 subtypes have virtually no ADCP effect.

[0029] Figure 6 Antibodies against CoV2-HB27 of the IgG1 and Fd11-IgG4 subtypes have virtually no CDC effect.

[0030] Figure 7 Fd6 and Fd11 modification reduces the ADE effect of CoV2-HB27-IgG1 antibody on CHO-K1-CD32A cells.

[0031] Figure 8 Fd6 and Fd11 modification reduces the ADE effect of CoV2-HB27-IgG1 antibody on CHO-K1-CD32B cells.

[0032] Figure 9 Fd11 modification reduces the ADE effect of SARS-2-H014-IgG1 antibody in CHO-K1-CD64 cells.

[0033] Figure 10 Fd6 and Fd11 modification reduces the ADE effect of CoV2-HB27-IgG1 antibody on Raji cells.

[0034] Figure 11Fd11 modification reduces the ADE effect of SARS-2-H014-IgG1 antibody on THP-1 cells.

[0035] Figure 12 Fd11 modification reduces the ADE effect of SARS-2-H014-IgG1 antibody on U937 cells. Detailed Implementation

[0036] Reducing antibody ADE is an important task in the development of antiviral biopharmaceuticals and vaccines.

[0037] This invention establishes a method for reducing viral ADE effects. Specifically, the inventors modified the antibody Fc fragment to reduce its binding to the Fc receptor / complement binding, thereby achieving a reduction in viral ADE.

[0038] Viruses include, but are not limited to, coronaviruses, influenza viruses, common cold viruses, parainfluenza viruses, upper respiratory syncytial virus, dengue virus, West Nile virus, Marburg virus, Lassa hemorrhagic fever virus, HIV virus, Ebola virus, varicella-zoster virus, CMV virus, hepatitis viruses, human herpes simplex virus, cytomegalovirus, rotavirus, Epstein-Barr virus, measles virus, mumps virus, human papillomavirus, 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; and pseudoviruses and true viruses of hepatitis A, hepatitis B, hepatitis C, and hepatitis E.

[0039] Antibody-dependent enhancement (ADE), also known as immune enhancement or disease enhancement, refers to the phenomenon where, after a virus binds to non-neutralizing antibodies or to sub-neutralizing concentrations of antibodies, the Fc fragment of the antibody binds to cells expressing FcRs and mediates viral entry into these cells, thereby enhancing viral infectivity. This phenomenon leads to increased infectivity and virulence. ADE can modulate the immune response and cause persistent inflammation, lymphopenia, and / or a cytokine storm.

[0040] The main mechanism of ADE is that, under conditions of incomplete or non-neutralization of the virus by the antibody, the Fc fragment of the antibody in the antigen-antibody complex binds to specific cells such as B cells, monocytes, macrophages, dendritic cells, etc., which express FcγR, and then achieves endocytosis into the target cells [11,12]. In other words, the antibody helps the virus enter the target cells, leading to an increase in the number of infected cells. This process is called exogenous ADE, which is Fcγ receptor (FcγR) dependent and occurs in organisms that are secondary infected with heterologous serotype viruses. Circulating antibodies produced during the primary viral infection recognize and bind to the secondary virus, enhancing viral infectivity by internalizing virus-antibody immune complexes carried by FcγR cells, rather than promoting viral neutralization. Once internalized, these immune complexes may regulate the response of innate antiviral cells, causing a significant increase in the amount of virus produced in each cell, a process called endogenous ADE. Both exogenous and endogenous ADE together promote the massive release of inflammatory and vasoactive mediators, ultimately leading to disease exacerbation

[12] .

[0041] The term "receptor" is a biochemical concept referring to a class of molecules that can transduce extracellular signals and produce specific effects within the cell. These effects may last only a short time, such as altering cellular metabolism or cell motility, or they may be long-lasting, such as upregulating or downregulating the expression of one or more genes.

[0042] The term "Fc receptor" or "FcR" refers to the receptor that binds to the Fc region of an antibody. The receptor that binds to IgG antibodies is a γ receptor, which includes the subtypes FcγRI, FcγRII, and FcγRIII. The human Fcγ receptor mainly includes FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). Different virus types have some differences in the Fc receptors that induce ADE-dependent reactions. SARS and dengue fever are mainly reported to induce ADE through CD32A[11,13], while MERS mainly induces ADE through CD32 and CD64

[14] , and Chikungunya virus mainly induces ADE through CD32 and CD16[5].

[0043] Macrophages are an important cell type that phagocytose antigen-antibody complexes and express several receptors, including CD16, CD32, and CD64. In the rhesus monkey model, after SARS infection, antibodies against the S protein can induce more immune cells (especially inflammatory macrophages) to infiltrate the lungs. At the same time, the virus-induced macrophage ADE can induce anti-inflammatory macrophages M2 to produce more inflammatory factors such as IL-6, IL-8, and MCP-1, resulting in severe lung damage

[15] .

[0044] B cells have strong CD32 expression. There are relatively few studies on the effects of viral ADE on B cell function after infection. Antiserum produced by SARS-CoV vaccine can enhance viral infection of B cells

[16] . The composition of B cells in patients infected with dengue virus is significantly different from that in normal people. The number of immature B cells, transitional B cells and Breg cells in severe patients is significantly lower than that in mild patients. Immature B cells after stimulation cannot produce IL-10, activation markers and antigen-presenting molecules. Therefore, the acute infection period of dengue fever has a serious impact on B cell function

[17] , which may be related to ADE.

[0045] The inventors used molecular biology techniques to modify the corresponding Fc fragments, obtaining molecules with reduced Fc receptor binding / complement binding, thereby reducing the viral ADE effect. Further, the inventors invented a molecule that reduces the viral ADE effect, comprising a) an antigen-binding fragment that recognizes and binds to the viral capsid protein or extracellular domain; and the aforementioned b) an Fc fragment with reduced Fc receptor binding / complement binding. The molecule comprises a) an antigen-binding fragment that recognizes and binds to the viral capsid protein or extracellular domain; and b) an Fc fragment, wherein b) is modified using molecular biology techniques to reduce Fc receptor binding and / or complement binding. When this molecule is administered as a therapeutic or prophylactic agent to subjects infected with the virus or at risk of viral infection, the reduced Fc receptor binding and / or complement binding leads to decreased binding to specific cells expressing FcγR, such as B cells, monocytes, macrophages, and dendritic cells, resulting in reduced endocytosis of target cells. This reduces viral infection.

[0046] In one embodiment of the present invention, the inventors modified the antibodies COV2-HB27 and SARS-2-H014. 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 SARS-CoV-2 virus-infected cells. Its preparation, structure, and performance are detailed in patent application 202010349190.3 and PCT / CN2021 / 089748 (incorporated in full by reference). SARS-2-H014 is a humanized antibody that can cross-block the binding of SARS-CoV-2 and the SARS-CoV spike protein (S protein) to the ACE2 receptor and efficiently neutralize SARS-CoV-2 and SARS-CoV virus-infected cells. Its preparation, structure, and performance are detailed in patent application 202010219867.1 and PCT / CN2021 / 082374 (incorporated in full by reference).

[0047] The term "virus-like particle" (VLP) or "pseudovirus" refers to a multi-protein structure composed of corresponding natural viral structural proteins, but lacking all or part of the viral genome, particularly the replication and infectious components of the viral genome, and therefore lacking replication and infectivity. This multi-protein structure highly mimics its corresponding natural viral particle in morphology and size and can spontaneously form after recombinant expression of viral structural proteins.

[0048] The term "antibody" refers to an immunoglobulin molecule, which is any form of antibody that exhibits desired biological activity. This includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), and even antibody fragments. Typically, a full-length antibody structure preferably comprises four polypeptide chains, typically two heavy (H) chains and two light (L) chains linked together by disulfide bonds.

[0049] Based on the amino acid sequence of their heavy chain constant region, complete antibodies can be classified into five classes: IgA, IgD, IgE, IgG, and IgM. IgG and IgA can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Correspondingly, the heavy chains of these five antibody classes are classified as α, δ, ε, γ, and μ chains, respectively. Based on the amino acid sequence of their light chain constant region, the light chains of antibodies can be classified as κ and λ.

[0050] Taking IgG antibodies as an example, from its N-terminus to its C-terminus, each heavy chain has a variable region (VH, heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3, also known as heavy chain constant domains). Similarly, from its N-terminus to its 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 domain). IgG antibodies are cleaved by papain to form two "Fab moieties" (or "Fab fragments") and an "Fc moieties" (or "Fc fragments"). The "Fab moieties" of the antibody contain the variable and constant domains of the light chain, as well as the variable and first constant domain (CH1) of the heavy chain. They possess antigen-binding capabilities. The "Fc moieties" of the antibody, containing CH2 and CH3 of the two heavy chains, are not directly involved in antibody-antigen binding but exhibit various effector functions, such as antibody-dependent cytotoxicity (ADCC) and antibody-dependent enhancement (ADE).

[0051] 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 stated, when used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). "KD," "binding rate constant k," etc. on "and dissociation rate constant k" off"Affinity is commonly used to describe the affinity between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen), that is, how tightly the ligand binds to a specific protein. Binding affinity is influenced by non-covalent intermolecular interactions, such as hydrogen bonds, electrostatic interactions, hydrophobicity between the two molecules, and van der Waals forces. Additionally, the binding affinity between a ligand and its target molecule can be affected by the presence of other molecules. Affinity can be analyzed using conventional methods known in the art, including ELISA as described herein."

[0052] In one embodiment of the present invention, the inventors modified the heavy chain IgG1 constant region of the CoV2-HB27 antibody using molecular biology techniques to obtain humanized antibodies CoV2-HB27-Fd6-IgG1 and CoV2-HB27-Fd11-IgG4 with reduced Fc function IgG1 subtypes. The CoV2-HB27-Fd6-IgG1 antibody shows almost no binding to CD32a and CD32b; it binds only weakly to CD64 and C1q (see patent application 202010349190.3 and PCT / CN2021 / 089748 for details). The CoV2-HB27-Fd11-IgG4 antibody shows almost no binding to CD32A, CD32B, and CD64. Both CoV2-HB27-Fd6-IgG1 and CoV2-HB27-Fd11-IgG4 exhibited reduced ADE effects in CHO-K1-CD32A, CHO-K1-CD32B, and Raji cells.

[0053] In another embodiment of the invention, the inventors modified the heavy chain IgG4 constant region of the SARS-2-H014 antibody using molecular biology techniques. The SARS-2-H014-Fd11-IgG4 antibody does not bind to CD32a, CD32b, CD16a, and C1q complement proteins, exhibits very weak binding to CD64 under high concentration conditions, and binds to FcRn similar to that of the IgG1 subtype antibody at pH 6.0 (see patent applications 202010219867.1 and PCT / CN2021 / 082374 for details). The SARS-2-H014-Fd11-IgG4 antibody showed a reduced ADE effect in CHO-K1-CD64, THP-1, and U937 cells.

[0054] To express the molecules of the present invention, standard recombinant DNA expression methods can be used (see, for example, Goeddel; GeneExpression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, the nucleotide sequence encoding the desired molecule of the present invention can be inserted into an expression vector, which is then 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 should be understood that the design of the expression vector, including the selection of regulatory sequences, is influenced by a variety of factors, such as the choice of host cell, the desired protein expression level, and whether the expression is constitutive or inducible.

[0055] The molecules of this invention can be recovered and purified from recombinant cell cultures by 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, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (“HPLC”) can also be used for purification. See, for example, Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997–2001), e.g., Chapters 1, 4, 6, 8, 9, and 10, each incorporated herein by reference in its entirety.

[0056] The molecules of this invention include naturally purified products, products obtained through chemical synthesis, and products derived from prokaryotic and eukaryotic hosts via recombinant technologies, such as yeast, higher plants, insects, and mammalian cells. The molecules of this invention can be glycosylated or non-glycosylated. Such methods are described in many standard laboratory manuals, such as Sambrook above, Sections 17.37–17.42; and Ausubel above, Chapters 10, 12, 13, 16, 18, and 20.

[0057] Therefore, embodiments of the present invention also include a host cell containing the above-mentioned carrier or nucleic acid molecule, wherein the host cell may be a higher eukaryotic host cell such as mammalian and insect cells, a lower eukaryotic host cell such as yeast cells, or a prokaryotic cell such as bacterial cells.

[0058] use

[0059] The method of the present invention can be used to treat, prevent or detect diseases caused by viruses such as SARS-CoV-2 and SARS-CoV, such as acute respiratory infectious diseases caused by SARS-CoV-2 and SARS-CoV viruses.

[0060] Pharmaceutical Composition

[0061] The molecules, nucleic acids, and carriers of the present invention can be combined with at least one other chemical agent to prepare a pharmaceutical composition comprising the above-mentioned active ingredients and one or more pharmaceutically acceptable carriers, diluents, or excipients; optionally, it may also contain one or more other therapeutic agents.

[0062] Reagent test kit

[0063] The present invention also relates to pharmaceutical packaging and reagent kits comprising one or more containers containing the pharmaceutical compositions of the present invention mentioned above. Associated with such containers may be a form of notification prescribed by a government agency regulating the production, use, or sale of a drug or biological product, reflecting approval by the agency that produces, uses, or sells the product for human administration.

[0064] Preparation and storage

[0065] The pharmaceutical compositions of the present invention can be prepared in a manner known in the art, for example by conventional methods of mixing, dissolving, granulating, grinding, emulsifying, encapsulating, embedding, or lyophilizing.

[0066] After a pharmaceutical composition comprising the compounds of the present invention formulated in an acceptable carrier has been prepared, it can be placed in a suitable container and labeled for the treatment of the indicated condition. Such labels will include the dosage, frequency, and method of administration.

[0067] Drug combination

[0068] The pharmaceutical compositions comprising the antibodies of the present invention may also be combined with one or more other therapeutic agents, wherein the resulting combinations do not cause unacceptable adverse effects.

[0069] The following examples are provided to illustrate the present invention, and are not intended to limit the scope of the invention.

[0070] Example

[0071] Example 1: Construction of a CHO-K1 cell line stably expressing FcR

[0072] 1.1 Transfection of CHO-K1 cells

[0073] pCMV3 vector (resistance: Hygromycin) containing CD32A, CD32B, CD64, and FcRγ-chain (source: Beijing Sinocare Technology Co., Ltd.) was transfected into CHO-K1 cells (source: ATCC) to prepare CHO-K1 cells expressing CD32A (transfected with CD32A vector alone), CD32B (transfected with CD32B vector alone), or CD64 (co-transfected with CD64 and FcRγ-chain vector). CHO-K1 cells were digested and counted one day in advance, and 3.5 x 10⁻⁶ cells were added to T25 culture flasks. 6 Cells were collected and culture medium (DMEM + 10% FBS + 69 μg / mL proline) was added to a final volume of 7 mL. The cells were then plated overnight in a 37°C, 5% CO2 incubator. The next day, 20 μg each of CD32A and CD32B plasmids, and 10 μg each of CD64 and FcRγ-chain plasmids (CD64 and FcRγ-chain plasmids were mixed) were taken and diluted to 1 mL using CHO-K1 medium. 25 μL of Sinofection Transfection Reagent (source: Beijing Sinocare Technology Co., Ltd.) was diluted to 1 mL using CHO-K1 medium. The diluted Sinofection Reagent was added to the diluted plasmids and mixed thoroughly. The mixture was incubated at room temperature for 10 min. The transfection mixture was added to each T25 culture flask and incubated in a 37°C, 5% CO2 incubator for 4 hours. The supernatant was removed, and 7 mL of fresh culture medium was added before incubating in a 37°C, 5% CO2 incubator.

[0074] 1.2 Cell screening and screening of high-expression monoclonal cell lines

[0075] Three days after transfection, hygromycin was added to the cells to screen for positive transfection. One week later, the cells were digested and diluted to 0.5 cells / mL with hygromycin-containing medium. 100 μL of cell suspension was added to each well of a 96-well flat-bottom cell culture plate. The plates were incubated at 37°C in a 5% CO2 incubator. After monoclonal colonies formed in the wells, the monoclonal cells were expanded. FcR expression on the cells was detected using CD32 and CD64 antibodies (source: BD). Monoclonal colonies with high expression levels of CHO-K1-CD32A, CHO-K1-CD32B, and CHO-K1-CD64 were selected for further expansion and subsequent experiments. The cell flow cytometry results are shown below. Figure 1 As shown.

[0076] Example 2: Preparation of fake viruses

[0077] 2.1 SARS-CoV-2 pseudovirus packaging

[0078] Pseudoviruses expressing the full-length SARS-CoV-2S protein were packaged using 293T (source: ATCC). The 293T was digested one day in advance, counted, and 3.5 x 10⁻⁶ cells were added to a T25 culture flask. 6 Cells were cultured and the medium (DMEM + 10% FBS) was added to 7 mL. The cells were then plated overnight in a 37°C, 5% CO2 incubator. The next day, 20 μg of the SARS-CoV-2 Spike plasmid (source: Beijing Sinocare Technology Co., Ltd.) was taken and diluted to 1 mL with 293T culture. 25 μL of Sinofection Transfection Reagent (source: Beijing Sinocare Technology Co., Ltd.) was also diluted to 1 mL with 293T culture. The diluted Sinofection Reagent was added to the diluted plasmid and mixed well. The cells were incubated at room temperature for 10 min. The cell culture plates were then incubated at 37°C, 5% CO2 for 6 h, followed by medium replacement. After 24 h, the SARS-CoV-2 Spike-transfected 293T cells were infected with VSV pseudovirus (VSV△G-Luc). One h later, the cells were washed three times with PBS, and 7 mL of fresh 293T medium was added. After 24 hours, the supernatant was collected, filtered through a 0.45 μm filter membrane to remove cell debris, and the pseudovirus solution was obtained and stored at -80℃.

[0079] 2.2 Detection of SARS-CoV-2 pseudovirus titer

[0080] The virus was serially diluted 10-fold using limiting dilution, resulting in six viral concentrations, each with six replicates. The inoculation density in the 96-well plate was 3 × 10⁻⁶. 4 Cell / mL VERO E6 (source: Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) suspension, 100 μL / well. Add 100 μL of serially diluted virus to each well, using cell culture medium as a negative control. Mix well and incubate statically at 37℃, 5% CO2 for 24 h. After incubation, discard the supernatant and add 100 μL / well of 1x passive lysis buffer (source: Promega) to lyse cells. Transfer 40 μL / well to a 96-well white plate to detect fluorescence signal and calculate TCID using the Karber method. 50 value.

[0081] Example 3: Antibody Preparation

[0082] 3.1 Antibody Sequence Source: 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 SARS-CoV-2 virus-infected cells. Its preparation, structure, and performance are detailed in patent applications 202010349190.3 and PCT / CN2021 / 089748 (which are incorporated herein by reference).

[0083] SARS-2-H014 is a humanized antibody 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. Its preparation, structure, and properties are detailed in patent applications 202010219867.1 and PCT / CN2021 / 082374 (which are incorporated herein by reference).

[0084] 3.2 Production of CoV2-HB27-IgG1 antibody

[0085] The nucleotide sequence of the variable region of the CoV2-HB27 heavy chain was obtained by whole-genome synthesis (SEQ ID NO:5). The expression vector of the CoV2-HB27 heavy chain (SEQ ID NO:1) was obtained by in-fusion insertion into the pSE vector containing the heavy chain signal peptide (SEQ ID NO:3) and the heavy chain IgG1 constant region (SEQ ID NO:7) digested with ScaI+NheI (source: Fermentas, the same below).

[0086] The nucleotide sequence of the variable region (SEQ ID NO:6) of the CoV2-HB27 light chain was obtained by whole-genome synthesis. The expression vector of the CoV2-HB27 light chain (SEQ ID NO:2) was obtained by in-fusion insertion into a pSE vector digested with ScaI+BsiWI (source: Fermentas) containing the nucleotide sequence of the light chain signal peptide (SEQ ID NO:4) and the light chain kappa constant region (SEQ ID NO:8).

[0087] After plasmid extraction, 293E cells (source: Invitrogen, the same below) were transfected and cultured for expression for 7 days. High-purity antibodies were obtained by purification using a protein A purification column.

[0088] Fully synthesized primers for the CoV2-HB27 heavy chain variable region:

[0089] 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

[0090] 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)

[0091] Primers for splicing CoV2-HB27-IgG1 heavy chain:

[0092]

[0093] Fully synthesized COV2-HB27 light chain variable region primers:

[0094] TGGGCCCTTGGTGCTTGCGCTGGACACTGTCACCAGGGTGCCTTGTCCCCA 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

[0095] 3.3 Production of CoV2-HB27-Fd6-IgG1 antibody

[0096] To reduce the immune function mediated by the antibody Fc fragment, nucleotide mutations were performed on the constant region of the IgG1 subtype according to the literature

[18] to obtain the 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 contains the heavy chain signal peptide nucleotide sequence (SEQ ID NO:3), the heavy chain variable region nucleotide sequence (SEQ ID NO:5) and the 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 it into the pSE vector digested with HindIII+XbaI by in-fusion method.

[0097] Primers for splicing CoV2-HB27-Fd6-IgG1 heavy chain:

[0098]

[0099] The CoV2-HB27-Fd6-IgG1 heavy chain (SEQ ID NO:10) expression vector and the CoV2-HB27 light chain (SEQ ID NO:2) expression vector plasmid were extracted, transfected into 293E cells and cultured for 7 days. The CoV2-HB27-Fd6-IgG1 antibody with reduced Fc function was obtained by purification using a protein A purification column.

[0100] 3.4 Production of CoV2-HB27-Fd11-IgG4 antibody

[0101] To reduce the immune function mediated by the antibody Fc fragment, the constant region of the IgG4 subtype was mutated according to the literature

[18] to obtain the 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 contains 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). The expression vector containing the CoV2-HB27-Fd11-IgG4 heavy chain (SEQ ID NO:12) was obtained by in-fusion insertion into the pSE vector digested with HindIII+XbaI.

[0102] Primers for splicing CoV2-HB27-Fd11-IgG4 heavy chain:

[0103]

[0104] The CoV2-HB27-Fd11-IgG4 heavy chain (SEQ ID NO:12) expression vector and the CoV2-HB27 light chain (SEQ ID NO:2) expression vector plasmid were extracted, transfected into 293E cells and cultured for 7 days. The CoV2-HB27-Fd11-IgG4 antibody with reduced Fc function was obtained by purification using a protein A purification column.

[0105] 3.5 Production of SARS-2-H014-IgG1 antibody

[0106] The nucleotide sequence of the SARS-2-H014 heavy chain variable region (SEQ ID NO:16) was obtained by whole-genome synthesis. The SARS-2-H014 heavy chain (SEQ ID NO:13) expression vector was obtained by in-fusion insertion into a pSE vector digested with ScaI+NheI (source: Fermentas) containing the heavy chain signal peptide (SEQ ID NO:15) and the heavy chain IgG1 constant region (SEQ ID NO:7).

[0107] The variable region of the SARS-2-H014 light chain (SEQ ID NO:17) was obtained through whole-genome synthesis. This region was then inserted into a pSE vector digested with ScaI+BsiWI (source: Fermentas) containing the light chain signal peptide (SEQ ID NO:4) and the light chain kappa constant region nucleotide sequence (SEQ ID NO:18), respectively, to obtain the SARS-2-H014 light chain (SEQ ID NO:14) expression vector. After plasmid extraction, the vector was transfected into 293E cells (source: Invitrogen) and cultured for 7 days. High-purity antibodies were obtained after purification using a protein A purification column.

[0108] Synthesize primers for the entire SARS-2-H014 heavy chain variable region:

[0109]

[0110] Synthesize primers for the entire light chain variable region of SARS-2-H014:

[0111]

[0112]

[0113] 3.6 Production of SARS-2-H014-Fd11-IgG4 antibody

[0114] Construction and production of humanized antibody SARS-2-H014 with reduced Fc functional IgG4 subtype

[0115] To reduce the immune function mediated by the antibody Fc fragment, the constant region of the IgG4 subtype was mutated according to the literature

[18] to obtain the 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 splice PCR, which contains the heavy chain signal peptide nucleotide sequence (SEQ ID NO:15), the SARS-2-H014 heavy chain variable region nucleotide sequence (SEQ ID NO:16), and the 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 in-fusion insertion into the pSE vector digested with HindIII+XbaI (source: Fermentas).

[0116] Primers for splicing SARS-2-H014-Fd11-IgG4 heavy chain:

[0117] F44(SEQ ID NO:117) GTCACCGTCCTGACACGAAGCTTGCCGCCACCATG R44(SEQ ID NO:118) TGGGCCCTTGGTGCTTGC F45(SEQ ID NO:119) GCAAGCACCAAGGGCCCA R45 (SEQ ID NO:120) ACTATAGAATAGGGCCCTCTAGA

[0118] The expression vectors for the SARS-2-H014-Fd11-IgG4 heavy chain (SEQ ID NO:19) and the SARS-2-H014 light chain (SEQ ID NO:14) were extracted and transfected into HEK-293 cells for expression for 7 days. The high-purity humanized SARS-2-H014 antibody with reduced Fc function, namely SARS-2-H014-Fd11-IgG4, was obtained by purification using a protein A purification column.

[0119] 3.7 Antibody Production and Purification

[0120] 293E cells were passaged in SCD4-4-TC2 medium (source: Beijing Sinocare Technology Co., Ltd.) to 200 mL / flask, with an initial seeding density of 0.3–0.4 × 10⁻⁶ cells / vial. 6 Cells were cultured at 1.5–3 × 10⁻³ cells / mL in a CO₂ shaker at 37°C and 175 rpm. 6 After reaching cell / mL concentration, add 100 μg of a 1:1 mixture of light and heavy chain plasmid DNA and 800 μL of TF2 transfection reagent (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.), and continue culturing in a shaker until day 7. Centrifuge the culture medium at 4000 rpm for 25 min, collect the supernatant, and add 1 / 5 volume of stock buffer (source: Shenzhou Cell Engineering Co., Ltd.). Equilibrate the protein A chromatography column (source: Shenzhou Cell Engineering Co., Ltd.) with 5–10 column volumes of PBS. Add the filtered culture supernatant to the chromatography column, equilibrate again with 5–10 column volumes, and then elute the sample with sodium acetate buffer (source: Shenzhou Cell Engineering Co., Ltd.). After elution, neutralize the sample with Tris buffer for later use.

[0121] Example 4: Fc function of COV2-HB27-Fd11-IgG4 antibody

[0122] 4.1 CD16a binding function of CoV2-HB27-Fd11-IgG4 antibody

[0123] Avidin protein at a concentration of 10 μg / mL (source: Thermo, hereinafter the same) was coated onto 96-well plates, 100 μL per well, and incubated overnight at 2-8℃. The next day, after washing the plates and blocking at room temperature for 1 h, 100 μL of biotin-labeled CD16a-AVI-His(V158)+BirA protein at a concentration of 5 μg / mL (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, and the plates were incubated at room temperature for 1 h, followed by washing. 100 μL of CoV2-HB27 antibodies of different Fc functional forms were added, with antibody concentrations of 5 μg / mL and 1 μg / mL, respectively. After incubation for 1 h, the plates were washed to remove unbound antibodies. Goat anti-human IgGF(ab)2 / HRP (source: Jackson Immuno Research, hereinafter the same) was added, and the plates were washed repeatedly. Finally, substrate chromogenic buffer was added for color development. After termination, the OD was read using a microplate reader. 450 .

[0124] The results are as follows Figure 2 As shown in Figure A, the Fd11-IgG4 antibody with reduced Fc function binds only very weakly to CD16a compared to the IgG1 form.

[0125] 4.2 CD32 binding function of CoV2-HB27-Fd11-IgG4 antibody

[0126] Avidin protein at a concentration of 10 μg / mL was coated onto 96-well plates (100 μL per well) and incubated overnight at 2-8°C. The next day, after washing the plates and blocking for 1 hour at room temperature, 100 μL of biotin-labeled CD32a-AVI-His(R131)+BirA protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) at a concentration of 5 μg / mL or CD32b-AVI-HIS+BirA protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added. The plates were incubated for 1 hour at room temperature and then washed. 100 μL of CoV2-HB27 antibodies of different Fc functional forms were added at concentrations of 5 μg / mL and 1 μg / mL. After incubation for 1 hour, the plates were washed to remove unbound antibodies. Goat anti-human IgG F(ab)2 / HRP was added, followed by repeated washing. Substrate chromogenic buffer was added for color development. After termination, the OD was read using a microplate reader. 450 .

[0127] The results are as follows Figure 2 As shown, the Fd11-IgG4 form antibody with reduced Fc function has almost no binding to CD32a and CD32b compared to the IgG1 form antibody. Figure 2 B, 2C).

[0128] 4.3 CD64 binding function of CoV2-HB27-Fd11-IgG4 antibody

[0129] Avidin protein at a concentration of 10 μg / mL was coated onto 96-well plates (100 μL per well) and incubated overnight at 2-8°C. The next day, after washing the plates and blocking at room temperature for 1 hour, 100 μL of biotin-labeled CD64-AVI-His+BirA protein at a concentration of 0.5 μg / mL (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, and the plates were incubated at room temperature for 1 hour, followed by washing. Then, 100 μL of CoV2-HB27 antibodies of different Fc functional forms (concentrations of 5 μg / mL and 1 μg / mL) were added. After incubation for 1 hour, the plates were washed to remove unbound antibodies. Goat anti-human IgG F(ab)2 / HRP was added, followed by repeated washing. Finally, substrate chromogenic buffer was added for color development. After termination, the OD was read using a microplate reader. 450 .

[0130] The results are as follows Figure 2 As shown in D, the Fd6-IgG1 antibody with reduced Fc function has only a weaker binding to CD64 compared to the IgG1 antibody.

[0131] 4.4 C1q binding function of CoV2-HB27-Fd11-IgG4 antibody

[0132] Different concentrations of CoV2-HB27 antibodies with different Fc functional forms were coated onto 96-well plates, 100 μL / well, and incubated overnight at 4°C. Antibody concentrations of 5 μg / mL and 1 μg / mL were also added. The plates were washed the following day, blocked at room temperature for 1 h, and then 5 μg / mL of C1q complement protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was added, 100 μg / well, and incubated for 1 h. After washing to remove unbound protein, 0.5 μg / mL anti-C1q / HRP (source: Abcam) was added for incubation, followed by repeated washing. Finally, substrate chromogenic buffer was added for color development, and OD was measured after termination. 450 .

[0133] The results are as follows Figure 3 As shown, the Fd11-IgG4 antibody with reduced Fc function binds only weakly to C1q.

[0134] 4.5 Antibody-mediated ADCC function of CoV2-HB27-Fd11-IgG4

[0135] Using 293FT cell line (293FT-SARS-CoV-2-S, source: Shenzhou Cell Engineering Co., Ltd., hereinafter the same) transiently transfected with the full-length SARS-CoV-2 protein as target cells, and Jurkat cells (Jurkat-NFAT / Luc2P-CD16AV) stably transfected with CD16AV and NFAT-Luc2P as effector cells, the ADCC function of humanized antibodies was detected by reporter gene assay.

[0136] In a 96-well plate, the inoculation density was 50 μL / well and 1×10⁻⁶ μL / well. 5 Target cells and effector cells of equal volume and density were prepared at a concentration of [cell / mL]. Then, 50 μL of CoV2-HB27 antibody and H7N9-R1 negative control antibody with different Fc functional forms were added. CoV2-HB27-IgG1, CoV2-HB27-Fd11-IgG4 antibodies and H7N9-R1 negative control antibodies were added at concentrations of 20 μg / mL, 1 μg / mL, and 0.05 μg / mL, respectively. After mixing, the cells were incubated at 37°C and 5% CO2 for 6 h. Finally, 30 μL of 5×passive lysis buffer was added per well, and the cells were lysed. 10 μL of cell sample per well was used to detect RLU values. The dose-response bar chart was analyzed using GraphPad Prism software, with the ordinate representing the RLU value. The bioluminescence intensity induction fold = RLU value of the sample group / RLU value of the negative control group.

[0137] The results are as follows Figure 4 As shown, the Fd11-IgG4 form antibody with reduced Fc function has no ADCC activity.

[0138] 4.6 Antibody-mediated ADCP function of CoV2-HB27-Fd11-IgG4

[0139] Using 293FT-SARS-CoV-2-S as the target cell and 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, the function of humanized antibody-mediated ADCP was detected using the reporter gene assay.

[0140] In a 96-well plate, the inoculation density was 50 μL / well and 1×10⁻⁶ μL / well. 5Target cells and effector cells of equal volume and density were prepared at a concentration of [cell / mL]. Then, 50 μL of CoV2-HB27 antibody and H7N9-R1 negative control antibody with different Fc functional forms were added. Jurkat-NFAT / Luc2P-CD32A, Jurkat-NFAT / Luc2P-CD32B, and Jurkat-NFAT / Luc2P-CD64 were used as effector cells, with antibody concentrations of 20 μg / mL, 1 μg / mL, and 0.05 μg / mL, respectively. After mixing, the cells were incubated at 37°C and 5% CO2 for 6 h. Finally, 30 μL of 5×passive lysis buffer was added per well, and the cells were lysed. 10 μL of cell sample per well was used to detect RLU values. GraphPad Prism software was used to analyze and plot a dose-response bar chart, with the ordinate representing the RLU value. Bioluminescence intensity induction fold = RLU value of sample group / RLU value of negative control group.

[0141] The results are as follows Figure 5 As shown, when Jurkat-NFAT / Luc2P-CD32A, Jurkat-NFAT / Luc2P-CD32B, and Jurkat-NFAT / Luc2P-CD64 are used as effector cells ( Figure 5 A, 5B and Figure 5 C) Neither the IgG1 nor the Fd11-IgG4 form of CoV2-HB27 antibody showed ADCP activity.

[0142] 4.7 CoV2-HB27-Fd11-IgG4 antibody-mediated CDC function

[0143] Using 293FT-SARS-CoV-2-S cells as target cells, the CDC function of humanized antibodies was detected by the WST-8 assay.

[0144] In a 96-well plate, the inoculation density was 50 μL / well and 2 × 10⁻⁶ μL / well. 6 Target cells were added at a concentration of 100 μg / mL. 50 μL of rabbit complement (from One Lambda) and CoV2-HB27 antibodies of different Fc functional forms were added, and blank wells (cell-free), positive control (cells only), and H7N9-R1 negative control antibody groups were set up. The antibody concentrations were 100 μg / mL, 20 μg / mL, 4 μg / mL, 0.8 μg / mL, 0.16 μg / mL, 0.032 μg / mL, 0.0064 μg / mL, and 0.00128 μg / mL. After mixing, the mixture was incubated at 37°C in a 5% CO2 incubator for 2 hours. After incubation, 10 μL / well of WST-8 chromogenic solution was added. The 96-well plate was incubated in a CO2 incubator. After colorimetric stability, the absorbance was measured at 450 nm and 630 nm using a microplate reader. The absorbance value (OD) was recorded as the final value.450 –OD 630 The CDC killing effect of the antibody is calculated by subtracting the reading from the blank wells. Kill rate % = (positive control OD value – sample OD value) / positive control OD value × 100%.

[0145] The results are as follows Figure 6 As shown, CoV2-HB27 antibodies with different Fc functional forms had no CDC effect on target cells expressing SARS-CoV-2S protein.

[0146] Example 5: Fc modification reduces the ADE effect of antibodies on CD32A-expressing cells.

[0147] 5.1 Modification of Fd6 and Fd11 antibodies reduces the ADE effect of CoV2-HB27-IgG1 antibody in CHO-K1-CD32A cells.

[0148] CHO-K1-CD32A was digested one day in advance, and the cell density was adjusted to 3 x 10⁻⁶ cells using culture medium. 5 Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate overnight at 37°C with 5% CO2. The next day, add 50 μL of antibody at different concentrations (starting at 500 μg / mL, with 4-fold serial dilutions for a total of 9 gradients) to another 96-well cell culture plate. Add 500 TCID50 to each well. 50 SARS-CoV-2 pseudovirus, 50 μL / well. A group with virus but no antibody was used as a positive control, and a group without virus and antibody was used as a negative control. After mixing, the mixture was incubated at 37°C in a 5% CO2 incubator for 1 h. After incubation, 100 μL / well was transferred into pre-coated CHO-K1-CD32A cell plates and incubated statically at 37°C in a 5% CO2 incubator for 24 h. After incubation, 30 μL / well of passive lysis 5x buffer (from Promega) was added and the cells were lysed. 10 μL / well was transferred into a 96-well white-background plate to measure the fluorescence intensity (RLU). The sample RLU / positive control RLU was calculated and plotted using GraphPad. The results are shown below. Figure 7 As shown, Fd6 modification and Fd11 modification can significantly reduce the ADE effect of CoV2-HB27-IgG1 antibody on CHO-K1-CD32A cells.

[0149] Example 6: Fc modification reduces the ADE effect of antibodies on CD32B-expressing cells.

[0150] 6.1 Modification of Fd6 and Fd11 antibodies reduces the ADE effect of CoV2-HB27-IgG1 antibody in CHO-K1-CD32B cells.

[0151] CHO-K1-CD32B was digested one day in advance, and the cell density was adjusted to 3 x 10⁻⁶ cells using culture medium. 5 Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate overnight at 37°C with 5% CO2. The next day, add 50 μL of antibody at different concentrations (starting at 500 μg / mL, with 4-fold serial dilutions for a total of 9 gradients) to another 96-well cell culture plate. Add 500 TCID50 to each well. 50 SARS-CoV-2 pseudovirus, 50 μL / well. A group with virus but no antibody was used as a positive control, and a group without virus and antibody was used as a negative control. After mixing, the mixture was incubated at 37°C in a 5% CO2 incubator for 1 h. After incubation, 100 μL / well was transferred into pre-coated CHO-K1-CD32B cell plates and incubated statically at 37°C in a 5% CO2 incubator for 24 h. After incubation, 30 μL / well of passive lysis 5x buffer was added and the cells were lysed. 10 μL / well was transferred into a 96-well white-background plate to measure the fluorescence signal (RLU). The sample RLU / positive control RLU was calculated and plotted using GraphPad. The results are shown below. Figure 8 As shown, Fd6 modification and Fd11 modification can significantly reduce the ADE effect of CoV2-HB27-IgG1 antibody on CHO-K1-CD32B cells.

[0152] Example 7: Fc modification reduces the ADE effect of antibodies on CD64-expressing cells.

[0153] 7.1 Modification of Fd11 antibody reduces the ADE effect of SARS-2-H014-IgG1 antibody in CHO-K1-CD64 cells.

[0154] CHO-K1-CD64 cells were digested one day in advance, and the cell density was adjusted to 3 x 10⁻⁶ cells using culture medium. 5 Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate overnight at 37°C with 5% CO2. The next day, add 50 μL of antibody at different concentrations (starting at 100 μg / mL, with 5-fold serial dilutions for a total of 9 gradients) to another 96-well cell culture plate. Add 500 TCID50 to each well. 50SARS-CoV-2 pseudovirus, 50 μL / well. A group with virus but no antibody was used as a positive control, and a group without virus and antibody was used as a negative control. After mixing, the mixture was incubated at 37°C in a 5% CO2 incubator for 1 h. After incubation, 100 μL / well was transferred into pre-coated CHO-K1-CD64 cell plates and incubated statically at 37°C in a 5% CO2 incubator for 24 h. After incubation, 30 μL / well of passive lysis 5x buffer was added and the cells were lysed. 10 μL / well was transferred into a 96-well white-background plate to measure the fluorescence intensity (RLU). The sample RLU / positive control RLU was calculated and plotted using GraphPad. The results are shown below. Figure 9 As shown, Fd11 modification can significantly reduce the ADE effect of SARS-2-H014-IgG1 antibody on CHO-K1-CD64 cells.

[0155] Example 8: Fc modification reduces the ADE effect of antibodies on cells expressing multiple FcRs

[0156] 8.1 Fd6 and Fd11 modification reduces the ADE effect of CoV2-HB27-IgG1 antibody on Raji cells.

[0157] Add 50 μL of antibody to each well of a 96-well cell culture plate (starting at a final concentration of 500 μg / mL, with 4-fold serial dilutions for a total of 9 gradients). Add 500 TCID to each well. 50 SARS-CoV-2 pseudovirus, 50 μL / well. The group with virus but no antibody was used as a positive control, and the group without virus and antibody was used as a negative control. After mixing, the mixture was incubated at 37°C in a 5% CO2 incubator for 1 hour. After incubation, 3 x 10⁻⁶ spores were added to each well. 5 100 μL of Raji cells were incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, 30 μL of passive lysis 5x buffer was added to each well to lyse the cells. 10 μL of the lysate was transferred to a 96-well white-background plate to measure the fluorescence intensity (RLU). The sample RLU / positive control RLU was calculated and plotted using GraphPad. The results are shown below. Figure 10 As shown, Fd6 and Fd11 modification can significantly reduce the ADE effect of CoV2-HB27-IgG1 antibody on Raji cells.

[0158] 8.2 Modification of Fd11 antibody reduces the ADE effect of SARS-2-H014-IgG1 antibody on THP-1 cells.

[0159] THP-1 cells were induced for 3 days with 2.5 μg / mL PMA during culture to evaluate the ADE effect of different SARS-2-H014 subtype antibodies. THP-1 cells were digested one day before induction, and the cell density was adjusted to 3 x 10⁻⁶ cells using culture medium. 5 Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate overnight at 37°C with 5% CO2. The next day, add 50 μL of antibody at different concentrations (starting at 80 μg / mL, with 5-fold serial dilutions for a total of 9 gradients) to another 96-well cell culture plate. Add 500 TCID50 to each well. 50 SARS-CoV-2 pseudovirus, 50 μL / well. A group with virus but no antibody was used as a positive control, and a group without virus and antibody was used as a negative control. After mixing, the mixture was incubated at 37°C in a 5% CO2 incubator for 1 h. After incubation, 100 μL / well was transferred into pre-coated THP-1 cell plates and incubated statically at 37°C in a 5% CO2 incubator for 24 h. After incubation, 30 μL / well of passive lysis 5× buffer was added and the cells were lysed. 10 μL / well was transferred into a 96-well white-background plate to measure the fluorescence signal (RLU). The sample RLU / positive control RLU was calculated and plotted using GraphPad. The results are shown below. Figure 11 As shown, Fd11 modification can significantly reduce the ADE effect of SARS-2-H014-IgG1 antibody on THP-1 cells.

[0160] 8.3 Modification of Fd11 antibody reduces the ADE effect of SARS-2-H014-IgG1 antibody on U937 cells

[0161] U937 cells were induced for 3 days with 2.5 μg / mL PMA during culture to evaluate the ADE effect of different SARS-2-H014 subtype antibodies. U937 cells were digested one day before induction, and the cell density was adjusted to 3 x 10⁻⁶ cells using culture medium. 5 Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate overnight at 37°C with 5% CO2. The next day, add 50 μL of antibody at different concentrations (starting at 10 μg / mL, with 5-fold serial dilutions for a total of 6 gradients) to another 96-well cell culture plate. Add 500 TCID50 to each well. 50SARS-CoV-2 pseudovirus, 50 μL / well. A group with virus but no antibody was used as a positive control, and a group without virus and antibody was used as a negative control. After mixing, the mixture was incubated at 37°C in a 5% CO2 incubator for 1 h. After incubation, 100 μL / well was transferred into pre-coated U937 cell plates and incubated statically at 37°C in a 5% CO2 incubator for 24 h. After incubation, 30 μL / well of passive lysis 5× buffer was added and the cells were lysed. 10 μL / well was transferred into a 96-well white-background plate to measure the fluorescence signal (RLU). The sample RLU / positive control RLU was calculated and plotted using GraphPad. The results are shown below. Figure 12 As shown, Fd11 modification can significantly reduce the ADE effect of SARS-2-H014-IgG1 antibody on U937 cells.

[0162] Sequence List

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173] References

[0174] 1.van Erp,EA;Luytjes,W.;Ferwerda,G.;van Kasteren,PBFc-MediatedAntibody Effector Functions During Respiratory Syncytial Virus Infection andDisease.Frontiers in immunology 2019,10,548-548,doi:10.3389 / fimmu.2019.00548.

[0175] 2.Halstead,S.B.;Nimmannitya,S.;Yamarat,C.;Russell,P.K.Hemorrhagicfever in Thailand;recent knowledge regarding etiology.Japanese journal ofmedical science&biology 1967,20 Suppl,96-103.

[0176] 3.Kuzmina,N.A.;Younan,P.;Gilchuk,P.;Santos,R.I.;Flyak,A.I.;Ilinykh,P.A.;Huang,K.;Lubaki,N.M.;Ramanathan,P.;Crowe,J.E.,Jr.,et al.Antibody-Dependent Enhancement of Ebola Virus Infection by Human Antibodies Isolatedfrom Survivors.Cell reports 2018,24,1802-1815.e1805,doi:10.1016 / j.celrep.2018.07.035.

[0177] 4.Li,M.;Zhao,L.;Zhang,C.;Wang,X.;Hong,W.;Sun,J.;Liu,R.;Yu,L.;Wang,J.;Zhang,F.,et al.Dengue immune sera enhance Zika virus infection in humanperipheral blood monocytes through Fc gamma receptors.PLOS ONE 2018,13,e0200478,doi:10.1371 / journal.pone.0200478.

[0178] 5.Lum,F.-M.;Couderc,T.;Chia,B.-S.;Ong,R.-Y.;Her,Z.;Chow,A.;Leo,Y.-S.;Kam,Y.-W.;Rénia,L.;Lecuit,M.,et al.Antibody-mediated enhancement aggravateschikungunya virus infection and disease severity.Scientific Reports 2018,8,1860,doi:10.1038 / s41598-018-20305-4.

[0179] 6.Wilder-Smith,A.;Hombach,J.;Ferguson,N.;Selgelid,M.;O'Brien,K.;Vannice,K.;Barrett,A.;Ferdinand,E.;Flasche,S.;Guzman,M.,et al.Deliberationsof the Strategic Advisory Group of Experts on Immunization on the use of CYD-TDV dengue vaccine.The Lancet Infectious Diseases 2019,19,e31-e38,doi:10.1016 / S1473-3099(18)30494-8.

[0180] 7.Lee,N.;Chan,P.K.;Ip,M.;Wong,E.;Ho,J.;Ho,C.;Cockram,C.S.;Hui,D.S.Anti-SARS-CoV IgG response in relation to disease severity of severeacute respiratory syndrome.Journal of clinical virology:the officialpublication of the Pan American Society for Clinical Virology 2006,35,179-184,doi:10.1016 / j.jcv.2005.07.005.

[0181] 8.Zhang,B.;Zhou,X.;Zhu,C.;Feng,F.;Qiu,Y.;Feng,J.;Jia,Q.;Song,Q.;Zhu,B.;Wang,J.Immune phenotyping based on neutrophil-to-lymphocyte ratio and IgGpredicts disease severity and outcome for patients with COVID-19.medRxiv2020,10.1101 / 2020.03.12.20035048,2020.2003.2012.20035048,doi:10.1101 / 2020.03.12.20035048.

[0182] 9.Zhao,J.;Yuan,Q.;Wang,H.;Liu,W.;Liao,X.;Su,Y.;Wang,X.;Yuan,J.;Li,T.;Li,J.,et al.Antibody responses to SARS-CoV-2 in patients of novel coronavirusdisease 2019.Clinical Infectious Diseases 2020,10.1093 / cid / ciaa344,doi:10.1093 / cid / ciaa344.

[0183] 10.Tan,W.;Lu,Y.;Zhang,J.;Wang,J.;Dan,Y.;Tan,Z.;He,X.;Qian,C.;Sun,Q.;Hu,Q.,et al.Viral Kinetics and Antibody Responses in Patients with COVID-19.medRxiv 2020,10.1101 / 2020.03.24.20042382,2020.2003.2024.20042382,doi:10.1101 / 2020.03.24.20042382.

[0184] 11.Wang,S.F.;Tseng,S.P.;Yen,C.H.;Yang,J.Y.;Tsao,C.H.;Shen,C.W.;Chen,K.H.;Liu,F.T.;Liu,W.T.;Chen,Y.M.,et al.Antibody-dependent SARS coronavirusinfection is mediated by antibodies against spike proteins.Biochem BiophysRes Commun 2014,451,208-214,doi:10.1016 / j.bbrc.2014.07.090.

[0185] 12.Taylor,A.;Foo,S.S.;Bruzzone,R.;Dinh,L.V.;King,N.J.;Mahalingam,S.Fcreceptors in antibody-dependent enhancement of viral infections.Immunologicalreviews 2015,268,340-364,doi:10.1111 / imr.12367.

[0186] 13.Moi,M.L.;Lim,C.K.;Kotaki,A.;Takasaki,T.;Kurane,I.Development of anantibody-dependent enhancement assay for dengue virus using stable BHK-21cell linesexpressing Fc gammaRIIA.J Virol Methods 2010,163,205-209,doi:10.1016 / j.jviromet.2009.09.018.

[0187] 14.Wan,Y.;Shang,J.;Sun,S.;Tai,W.;Chen,J.;Geng,Q.;He,L.;Chen,Y.;Wu,J.;Shi,Z.,et al.Molecular Mechanism for Antibody-Dependent Enhancement ofCoronavirus Entry.Journal of virology 2020,94,doi:10.1128 / JVI.02015-19.

[0188] 15.Liu,L.;Wei,Q.;Lin,Q.;Fang,J.;Wang,H.;Kwok,H.;Tang,H.;Nishiura,K.;Peng,J.;Tan,Z.,et al.Anti-spike IgG causes severe acute lung injury byskewing macrophage responses during acute SARS-CoV infection.JCI Insight2019,4,doi:10.1172 / jci.insight.123158.

[0189] 16.Kam,Y.W.;Kien,F.;Roberts,A.;Cheung,Y.C.;Lamirande,E.W.;Vogel,L.;Chu,S.L.;Tse,J.;Guarner,J.;Zaki,S.R.,et al.Antibodies against trimeric Sglycoprotein protect hamsters against SARS-CoV challenge despite theircapacity to mediate FcgammaRII-dependent entry into B cells in vitro.Vaccine2007,25,729-740,doi:10.1016 / j.vaccine.2006.08.011.

[0190] 17.Upasani,V.;Vo,H.T.M.;Ung,S.;Heng,S.;Laurent,D.;Choeung,R.;Duong,V.;Sorn,S.;Ly,S.;Rodenhuis-Zybert,I.A.,et al.Impaired Antibody-IndependentImmune Response of B Cells in Patients With Acute Dengue Infection.Frontiersin Immunology 2019,10,doi:10.3389 / fimmu.2019.02500.

[0191] 18. Ye Xin; Sun Le; Xu Shaoyu; Hu Qiyue; Tao Weikang; Zhang Lianshan. Cd47 antibody, antigen-binding fragment and medical use thereof. Google Patents: 2018. SEQUENCE LISTING <110> Shenzhou Cell Engineering Co., Ltd. <120> A method to reduce the ADE effect of viruses <130> PCT70916SXB <160> 120 <170> PatentIn version 3.3 <210> 1 <211> 1413 <212> DNA <213> Homo sapiens <400> 1 atggagttgg gactgagctg gattttcctt ttggctattt taaaaggtgt ccagtgtgag 60 gtgaaactgg tggagtctgg aggaggactg gtgaagcctg gaggctccct gagactgtcc 120 tgtgctgcct ctggcttcac cttcagcaac tatggggatga gttgggtgag acaggctcct 180 ggcaagagat tggagtgggt ggctgagatt tcctctggag gctcctacac ctactaccct 240 gacacagtga caggcaggtt caccatcagc agggacaatg ccaagaacac cctctacctc 300 caaatgaact ccctgagggc tgaggacaca gcagtctact actgtgccag gttcagatat 360 gatggaggag gaggcacagt ggactactgg ggacaaggca ccctggtgac agtgtccagc 420 gcaagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 480 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 720 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840 gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 900 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1140 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctccccgtg 1260 ctggactccg acggctcctt cttctctctac agcaagctca ccgtggacaa gagcaggtgg 1320 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380 cagaagagcc tctccctgtc tccgggtaaaa tga 1413 <210> 2 <211> 714 <212> DNA <213> Homo sapiens <400> 2 atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt ccatagtgag 60 attgtgctga cccagagccc tgccaccctg tccctgagcc ctggagagag ggctaccctg 120 tcctgtaggg catctgagtc tgtggacaac tatggcatct cctttatgaa ctggttccaa 180 cagaagcctg gacaagcccc aagactgctg atttatgctg ccagcaacca gggctctgga 240 gtgcctgcca ggttctctgg ctctggctct ggcacagact tctccctgac catctcctcc 300 ttggaacctg aggactttgc tgtctacttc tgtcaacaga caaggaggt gccaaggacc 360 tttggacaag caccaaggt ggagattaag cgtacggtgg ctgcaccatc tgtcttcatc 420 ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 480 aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 540 aactcccagg agagtgtcac agagcaggac agcaaggaca cacctacag cctcagcagc 600 accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 660 catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttag 714 <210> 3 <211> 57 <212> DNA <213> Homo sapiens <400> 3 atggagttgg gactgagctg gattttcctt ttggctattt taaaaggtgt ccagtgt 57 <210> 4 <211> 57 <212> DNA <213> Homo sapiens <400> 4 atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt cgatagt 57 <210> 5 <211> 363 <212> DNA <213> Homo sapiens <400> 5 gaggtgaaac tggtggagtc tggaggagga ctggtgaagc ctggaggctc cctgagactg 60 tcctgtgctg cctctggctt caccttcagc aactatggga tgagttgggt gagacaggct 120 cctggcaaga gattggagtg ggtggctgag atttcctctg gaggctccta cacctactac 180 cctgacacag tgacaggcag gttcaccatc agcagggaca atgccaagaa caccctctac 240 ctccaaatga actccctgag ggctgaggac acagcagtct actactgtgc caggttcaga 300 tatgatggag gaggaggcac agtggactac tggggacaag gcaccctggt gacagtgtcc 360 agc 363 <210> 6 <211> 333 <212> DNA <213> Homo sapiens <400> 6 gagattgtgc tgacccagag ccctgccacc ctgtccctga gccctggaga gagggctacc 60 ctgtcctgta gggcatctga gtctgtggac aactatggca tctcctttat gaactggttc 120 caacagaagc ctggacaagc cccaagactg ctgatttatg ctgccagcaa ccagggctct 180 ggagtgcctg ccaggttctc tggctctggc tctggcacag acttctccct gaccatctcc 240 tccttggaac ctgaggactt tgctgtctac ttctgtcaac agagcaagga ggtgccaagg 300 acctttggac aaggcaccaa ggtggagatt aag 333 <210> 7 <211> 993 <212> DNA <213> Homo sapiens <400> 7 gcaagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa tga 993 <210> 8 <211> 324 <212> DNA <213> Homo sapiens <400> 8 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg ttag 324 <210> 9 <211> 993 <212> DNA <213> Homo sapiens <400> 9 gcaagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgccggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc agcactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa agcaggtgg cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa tga <210> 10 <211> 1413 <212> DNA <213> Homo sapiens <400> 10 atggagttgg gactgagctg gattttcctt ttggctattt taaaaggtgt ccagtgtgag gtgaaactgg tggagtctgg aggaggactg gtgaagcctg gaggctccct gagactgtcc tgtgctgcct ctggcttcac cttcagcaac tatgggatga gttgggtgag acaggctcct 180 ggcaagagat tggagtgggt ggctgagatt tcctctggag gctcctacac ctactaccct 240 gacacagtga caggcaggtt caccatcagc agggacaatg ccaagaacac cctctacctc 300 caaatgaact ccctgagggc tgaggacaca gcagtctact actgtgccag gttcagatat 360 gatggaggag gaggcacagt ggactactgg ggacaaggca ccctggtgac agtgtccagc 420 gcaagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 480 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 720 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaagc cgccggggga 780 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840 gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 900 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1140 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380 cagaagagcc tctccctgtc tccgggtaaa tga 1413 <210> 11 <211> 985 <212> DNA <213> Homo sapiens <400> 11 gcaagcacca agggcccatc ggtcttcccg ctggcgccct gctccaggag cacctccgag 60 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300 aaatatggtc ccccatgccc accctgccca gcacctgagg ccgccggggg accatcagtc 360 ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420 tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600 tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660 gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780 cgcctcccgt gctggactcc 840. cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 900 aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc ctctccctgt ctctgggtaa father <210> 12 <211> 1405 <212> DNA <213> Homo sapiens <400> 12 atggagttgg gactgagctg gattttcctt ttggctattt taaaaggtgt ccagtgtgag gtgaaactgg tggagtctgg aggaggactg gtgaagcctg gaggctccct gagactgtcc 180. tgtgctgcct ctggcttcac cttcagcaac tatgggatga gttgggtgag acaggctcct ggcaagagat tggagtgggt ggctgagatt tcctctggag gctcctacac ctactaccct 240 gacacagtga caggcaggtt caccatcagc agggacaatg ccaagaacac cctctacctc 360. caaatgaact ccctgagggc tgaggacaca gcagtctact actgtgccag gttcagatat gatggaggag gaggcacagt ggactactgg ggacaaggca ccctggtgac agtgtccagc 420 gcaagcacca agggcccatc ggtcttcccg ctggcgccct gctccaggag cacctccgag 480 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 660 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 720 aaatatggtc ccccatgccc accctgccca gcacctgagg ccgccggggg accatcagtc 780 ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 840 tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 900 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 960 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 1020 tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 1080 gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1140 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag cgcctcccgt gctggactcc 1260. cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc ctctccctgt ctctgggtaa 1405 <210> 13 <211> 1410 <212> DNA <213> Homo sapiens <400> 13 atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgagtgag gtccaacttg tccagtctgg agcagaggtg aagaagcctg gagccacagt gaagatttcc 180. tgtaaggtgt ctggctactc cttcaccaac tactacatcc actgggtgaa gcaggctcct ggcaagtcct tggagtggat tggctacatt gacccattca atggaggcac ctctgacaac ctgaagttca agggagcagc caccctgaca gcagacacca gcacagacac agcctatatg gaactgtcct ccctgaggtc tgaggacaca gcagtctact actgtgccag gtctgaatat gacccatact atgtgatgga ctactgggga caaggcacca cagtgacagt gtccagcgca 420 agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 720 tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840 gtcacgtgcg tggtggtgga cgtgagccac gaagaccccg aggtcaagtt caactggtac 900 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1140 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260 gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1320 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380 aagagcctct ccctgtctcc gggtaaatga 1410 <210> 14 <211> 702 <212> DNA <213> Homo sapiens <400> 14 atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt ccatagtgag 60 attgtgctga cccagagccc tgacttccag tctgtgacac caaggagaa ggtgaccatc 120 acttgtaggg caagccagag catctccagc aacctgcact ggtatcaaca gaagcctgac 180 cagagcccaa aactgctgat taaatatgcc agccagagca tctctggcat cccaagcagg 240 ttctctggct ctggctctgg cacagacttc accctgacca tcaactcctt ggaggctgag 300 gactttggca tctacttctg tcaacagacc aacttctggc catacacctt tggacaaggc 360 accaaattg agattaagcg tacggtggct gcaccatctg tcttcatctt cccgccatct 420 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660 agctcgcccg tcacaaagag cttcaacagg ggagagtgtt aa 702 <210> 15 <211> 57 <212> DNA <213> Homo sapiens <400> 15 atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgagt 57 <210> 16 <211> 360 <212> DNA <213> Homo sapiens <400> 16 gaggtccaac ttgtccagtc tggagcagag gtgaagaagc ctggagccac agtgaagatt 60 tcctgtaagg tgtctggcta ctccttcacc aactactaca tccactgggt gaagcaggct 120 cctggcaagt ccttggagtg gattggctac attgacccat tcaatggagg cacctctgac 180 aacctgaagt tcaagggagc agccaccctg acagcagaca ccagcacaga cacagcctat 240 atggaactgt cctccctgag gtctgaggac acagcagtct actactgtgc caggtctgaa 300 tatgacccat actatgtgat ggactactgg ggacaaggca ccacagtgac agtgtccagc 360 <210> 17 <211> 321 <212> DNA <213> Homo sapiens <400> 17 gagattgtgc tgacccagag ccctgacttc cagtctgtga caccaaagga gaaggtgacc 60 atcacttgta gggcaagcca gagcatctcc agcaacctgc actggtatca acagaagcct 120 gaccagagcc caaaactgct gattaaatat gccagccaga gcatctctgg catcccaagc 180 aggttctctg gctctggctc tggcacagac ttcaccctga ccatcaactc cttggaggct 240 gaggactttg gcatctactt ctgtcaacag accaacttct ggccatacac ctttggacaa 300 ggcaccaaat tggagattaa g 321 <210> 18 <211> 324 <212> DNA <213> Homo sapiens <400> 18 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg ttaa 324 <210> 19 <211> 1402 <212> DNA <213> Homo sapiens <400> 19 atgggctggt ccctgattct gctgttcctg gtggctgtgg ctaccagggt gctgagtgag 60 gtccaacttg tccagtctgg agcagaggtg aagaagcctg gagccacagt gaagatttcc 120 tgtaaggtgt ctggctactc cttcaccaac tactacatcc actgggtgaa gcaggctcct 180 ggcaagtcct tggagtggat tggctacatt gacccattca atggaggcac ctctgacaac 240 ctgaagttca agggagcagc caccctgaca gcagacacca gcacagacac agcctatatg 300 gaactgtcct ccctgaggtc tgaggacaca gcagtctact actgtgccag gtctgaatat 360 gacccatact atgtgatgga ctactgggga caaggcacca cagtgacagt gtccagcgca 420 agcaccaagg gcccatcggt cttcccgctg gcgccctgct ccaggagcac ctccgagagc 480 acagccgccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac gaagacctac 660 acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagagagt tgagtccaaa 720 tatggtcccc catgcccacc ctgcccagca cctgaggccg ccgggggacc atcagtcttc 780 ctgttccccc caaaacccaa ggacactctc atgatctccc ggacccctga ggtcacgtgc 840 gtggtggtgg acgtgagcca ggaagacccc gaggtccagt tcaactggta cgtggatggc 900 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctga acggcaagga gtacaagtgc 1020 aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa ccatctccaa agccaaaggg cagccccgag agccacaggt gtacaccctg cccccatccc aggaggat gaccaagaac 1140. caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 1200 gaaagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caggctaacc gtggacaaga gcaggtggca ggaggggat gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc 1380 tccctgtctc tgggtaata aa <210> 20 <211> 451 <212> PRT <213> Homo sapiens <400> 20 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Meth Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Arg Tyr Asp Gly Gly Gly Gly Thr Val Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 21 <211> 218 <212> PRT <213> Homo sapiens <400> 21 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 22 <211> 121 <212> PRT <213> Homo sapiens <400> 22 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Arg Tyr Asp Gly Gly Gly Gly Thr Val Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 23 <211> 111 <212> PRT <213> Homo sapiens <400> 23 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 24 <211> 330 <212> PRT <213> Homo sapiens <400> 24 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 25 <211> 107 <212> PRT <213> Homo sapiens <400> 25 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 26 <211> 330 <212> PRT <213> Homo sapiens <400> 26 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 27 <211> 451 <212> PRT <213> Homo sapiens <400> 27 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Arg Tyr Asp Gly Gly Gly Gly Thr Val Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 28 <211> 448 <212> PRT <213> Homo sapiens <400> 28 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Arg Tyr Asp Gly Gly Gly Gly Thr Val Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 29 <211> 327 <212> PRT <213> Homo sapiens <400> 29 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 30 <211> 450 <212> PRT <213> Homo sapiens <400> 30 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Ala Pro Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Phe Asn Gly Gly Thr Ser Asp Asn Leu Lys Phe 50 55 60 Lys Gly Ala Ala Thr Leu Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Tyr Asp Pro Tyr Tyr Val Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 31 <211> 214 <212> PRT <213> Homo sapiens <400> 31 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Phe Gly Ile Tyr Phe Cys Gln Gln Thr Asn Phe Trp Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 32 <211> 120 <212> PRT <213> Homo sapiens <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Ala Pro Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Phe Asn Gly Gly Thr Ser Asp Asn Leu Lys Phe 50 55 60 Lys Gly Ala Ala Thr Leu Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Tyr Asp Pro Tyr Tyr Val Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 33 <211> 107 <212> PRT <213> Homo sapiens <400> 33 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp Phe Gly Ile Tyr Phe Cys Gln Gln Thr Asn Phe Trp Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 34 <211> 447 <212> PRT <213> Homo sapiens <400> 34 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Ala Pro Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asp Pro Phe Asn Gly Gly Thr Ser Asp Asn Leu Lys Phe 50 55 60 Lys Gly Ala Ala Thr Leu Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Glu Tyr Asp Pro Tyr Tyr Val Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 35 <211> 51 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 35 gctaccaggg tgctgagtga ggtgaaactg gtggagtctg gaggaggact g 51 <210> 36 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 36 cagggagcct ccaggcttca ccagtcctcc tcc 33 <210> 37 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 37 cctggaggct ccctgagact gtcctgtgct gcc 33 <210> 38 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 38 gttgctgaag gtgaagccag aggcagcaca gga 33 <210> 39 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 39 ttcaccttca gcaactatgg gatgagttgg gt 32 <210> 40 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 40 ctcttgccag gagcctgtct cacccaactc atc 33 <210> 41 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 41 ggctcctggc aagagattgg agtgggtggc tg 32 <210> 42 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 42 aggagcctcc agaggaaatc tcagccaccc act 33 <210> 43 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 43 cctctggagg ctcctacacc tactaccctg ac 32 <210> 44 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 44 ggtgaacctg cctgtcactg tgtcagggta gta 33 <210> 45 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 45 acaggcaggt tcaccatcag cagggacaat gcc 33 <210> 46 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 46 ttggaggtag agggtgttct tggcattgtc cct 33 <210> 47 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 47 accctctacc tccaaatgaa ctccctgagg gct 33 <210> 48 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 48 gtagtagact gctgtgtcct cagccctcag gga 33 <210> 49 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 49 acagcagtct actactgtgc caggttcaga tat 33 <210> 50 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 50 cactgtgcct cctcctccat catatctgaa cct 33 <210> 51 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 51 ggaggaggca cagtggacta ctggggacaa ggc 33 <210> 52 <211> 51 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 52 tgggcccttg gtgcttgcgc tggacactgt caccagggtg ccttgtcccc a 51 <210> 53 <211> 40 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 53 gtcaccgtcc tgacacgaag cttgccgcca ccatggagtt 40 <210> 54 <211> 62 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 54 aagcttgccg ccaccatgga gttgggactg agctggattt tccttttggc tattttaaaa 60 gg 62 <210> 55 <211> 57 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 55 ttttcctttt ggctatttta aaaggtgtcc agtgtgaggt gaaactggtg gagtctg 57 <210> 56 <211> 23 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 56 actatagaat agggccctct aga 23 <210> 57 <211> 51 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 57 gccacaggag tgcatagtga gattgtgctg acccagagcc ctgccaccct g 51 <210> 58 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 58 cctctctcca gggctcaggg acagggtggc agg 33 <210> 59 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 59 agccctggag agagggctac cctgtcctgt agg 33 <210> 60 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 60 gttgtccaca gactcagatg ccctacagga cag 33 <210> 61 <211> 27 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 61 gagtctgtgg acaactatgg catctcc 27 <210> 62 <211> 28 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 62 ggaaccagtt cataaaggag atgccata 28 <210> 63 <211> 27 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 63 ttatgaactg gttccaacag aagcctg 27 <210> 64 <211> 28 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 64 agtcttgggg cttgtccagg cttctgtt 28 <210> 65 <211> 27 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 65 acaagcccca agactgctga tttatgc 27 <210> 66 <211> 28 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 66 gccctggttg ctggcagcat aaatcagc 28 <210> 67 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 67 gccagcaacc agggctctgg agtgcctgcc agg 33 <210> 68 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 68 gccagagcca gagccagaga acctggcagg cac 33 <210> 69 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 69 ggctctggct ctggcacaga cttctccctg acc 33 <210> 70 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 70 ctcaggttcc aaggaggaga tggtcaggga gaa 33 <210> 71 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 71 tccttggaac ctgaggactt tgctgtctac ttc 33 <210> 72 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 72 cacctccttg ctctgttgac agaagtagac agc 33 <210> 73 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 73 cagagcaagg aggtgccaag gacctttgga caa 33 <210> 74 <211> 51 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 74 tggtgcagcc accgtacgct taatctccac cttggtgcct tgtccaaagg t 51 <210> 75 <211> 40 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 75 gtcaccgtcc tgacacgaag cttgccgcca ccatggagtt 40 <210> 76 <211> 62 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 76 aagcttgccg ccaccatgga gttgggactg agctggattt tccttttggc tattttaaaa 60 gg 62 <210> 77 <211> 57 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 77 ttttcctttt ggctatttta aaaggtgtcc agtgtgaggt gaaactggtg gagtctg 57 <210> 78 <211> 23 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 78 actatagaat agggccctct aga 23 <210> 79 <211> 57 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 79 ttttcctttt ggctatttta aaaggtgtcc agtgtgaggt gaaactggtg gagtctg 57 <210> 80 <211> 62 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 80 aagcttgccg ccaccatgga gttgggactg agctggattt tccttttggc tattttaaaa 60 gg 62 <210> 81 <211> 40 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 81 gtcaccgtcc tgacacgaag cttgccgcca ccatggagtt 40 <210> 82 <211> 22 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 82 ctatagaata gggccctcta ga 22 <210> 83 <211> 51 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 83 gctaccaggg tgctgagtga ggtccaactt gtccagtctg gagcagaggt g 51 <210> 84 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 84 cactgtggct ccaggcttct tcacctctgc tcc 33 <210> 85 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 85 cctggagcca cagtgaagat ttcctgtaag gtg 33 <210> 86 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 86 gttggtgaag gagtagccag acaccttaca gga 33 <210> 87 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 87 tactccttca ccaactacta catccactgg gt 32 <210> 88 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 88 acttgccagg agcctgcttc acccagtgga tg 32 <210> 89 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 89 aggctcctgg caagtccttg gagtggattg gc 32 <210> 90 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 90 ccattgaatg ggtcaatgta gccaatccac tc 32 <210> 91 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 91 tgacccattc aatggaggca cctctgacaa cc 32 <210> 92 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 92 ggctgctccc ttgaacttca ggttgtcaga gg 32 <210> 93 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 93 ttcaagggag cagccaccct gacagcagac acc 33 <210> 94 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 94 catataggct gtgtctgtgc tggtgtctgc tgt 33 <210> 95 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 95 gacacagcct atatggaact gtcctccctg agg 33 <210> 96 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 96 gtagactgct gtgtcctcag acctcaggga gga 33 <210> 97 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 97 gacacagcag tctactactg tgccaggtct gaa 33 <210> 98 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 98 catcacatag tatgggtcat attcagacct ggc 33 <210> 99 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 99 ccatactatg tgatggacta ctggggacaa ggc 33 <210> 100 <211> 51 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 100 tgggcccttg gtgcttgcgc tggacactgt cactgtggtg ccttgtcccc a 51 <210> 101 <211> 51 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 101 gccacaggag tgcatagtga gattgtgctg acccagagcc ctgacttcca g 51 <210> 102 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 102 cttctccttt ggtgtcacag actggaagtc agg 33 <210> 103 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 103 acaccaaagg agaaggtgac catcacttgt agg 33 <210> 104 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 104 gctggagatg ctctggcttg ccctacaagt gat 33 <210> 105 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 105 cagagcatct ccagcaacct gcactggtat ca 32 <210> 106 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 106 ggctctggtc aggcttctgt tgataccagt gc 32 <210> 107 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 107 agcctgacca gagcccaaaa ctgctgatta aa 32 <210> 108 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 108 gagatgctct ggctggcata tttaatcagc ag 32 <210> 109 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 109 cagccagagc atctctggca tcccaagcag gt 32 <210> 110 <211> 32 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 110 gccagagcca gagccagaga acctgcttgg ga 32 <210> 111 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 111 ggctctggct ctggcacaga cttcaccctg acc 33 <210> 112 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 112 ctcagcctcc aaggagttga tggtcagggt gaa 33 <210> 113 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 113 tccttggagg ctgaggactt tggcatctac ttc 33 <210> 114 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 114 ccagaagttg gtctgttgac agaagtagat gcc 33 <210> 115 <211> 33 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 115 cagaccaact tctggccata cacctttgga caa 33 <210> 116 <211> 51 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 116 tggtgcagcc accgtacgct taatctccaa tttggtgcct tgtccaaagg t 51 <210> 117 <211> 35 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 117 gtcaccgtcc tgacacgaag cttgccgcca ccatg 35 <210> 118 <211> 18 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 118 tgggcccttg gtgcttgc 18 <210> 119 <211> 18 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 119 gcaagcacca agggccca 18 <210> 120 <211> 23 <212> DNA <213> Artificial <220> <223> The sequence is artifically synthesized. <400> 120 actatagaat agggccctct aga 23

Claims

1. A molecule that reduces the ADE effect of viruses, the molecule comprising Fc fragment containing the sequence of SEQ ID NO:26, the heavy chain sequence of SEQ ID NO:27, and the light chain sequence of SEQ ID NO:21; or Fc fragment containing the sequence of SEQ ID NO:29, the heavy chain sequence of SEQ ID NO:28, and the light chain sequence of SEQ ID NO:21; or The Fc fragment containing the sequence of SEQ ID NO:29, the heavy chain sequence of SEQ ID NO:34, and the light chain sequence of SEQ ID NO:

31.

2. The conjugate of the molecule of claim 1 with other macromolecules, wherein the other macromolecule is PEG.

3. The conjugate of claim 2, wherein the molecule is conjugated with other macromolecules via a linker.

4. A nucleic acid encoding the molecule of claim 1, wherein the molecule is mRNA and / or DNA.

5. An expression vector comprising the nucleic acid of claim 4.

6. A host cell comprising the nucleic acid of claim 4 or the expression vector of claim 5.

7. A method for producing the molecule of claim 1, comprising culturing the host cell of claim 6 under conditions suitable for expression of the molecule, and recovering the expressed product from the culture medium.

8. A pharmaceutical composition comprising... a) The molecule of claim 1, the conjugate of any one of claims 2-3, the nucleic acid of claim 4, or the expression vector of claim 5; and c) Pharmaceutically acceptable carriers.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable carrier is a pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution.

10. The pharmaceutical composition of claim 8 or 9, wherein the pharmaceutically acceptable carrier comprises an excipient or a stabilizer.

11. The molecule of claim 1, the conjugate of any one of claims 2-3, the nucleic acid of claim 4, the expression vector of claim 5, or the pharmaceutical composition of any one of claims 8-10, for use in the preparation of a medicament for the prevention of SARS-CoV-2 and / or diseases caused by SARS-CoV.

12. The application as described in claim 11, specifically its use in the preparation of a medicament for preventing disease caused by SARS-CoV-2.

13. A drug combination comprising... The molecule of claim 1, the conjugate of any one of claims 2-3, the nucleic acid of claim 4, or the expression vector of claim 5, or the pharmaceutical composition of any one of claims 8-10; and One or more other therapeutic agents.

14. A reagent kit comprising The molecule of claim 1, the conjugate of any one of claims 2-3, the nucleic acid of claim 4, the expression vector of claim 5, or the pharmaceutical composition of any one of claims 8-10.

15. The kit of claim 14, wherein, The kit further includes a device for administering the drug.

Images