A fully human monoclonal antibody and uses thereof

Abstract

The present disclosure provides a fully human therapeutic monoclonal antibody or fragment thereof, which is a fully human antibody specifically binding to the RBD region of SARS-CoV-2 S protein obtained from isolating a single B cell clone from a convalescent blood sample of a Covid-19 patient, which does not produce ADE effect on SARS-CoV-2 infection of THP-1 cells and K562 cells. The monoclonal antibody has high affinity to the RBD of SARS-CoV-2 S protein, with a KD value of 5.0 x 10 ‑9 M; and the monoclonal antibody has blocking activity to the binding of the RBD of SARS-CoV-2 S protein to ACEII, with an IC50 value less than 50 nM.

Description

Technical Field

[0001] This invention belongs to the field of antibody engineering, specifically relating to a monoclonal antibody against coronaviruses and its application, particularly to a human monoclonal antibody that binds to coronavirus RBD, its preparation method, and its application. Background Technology

[0002] Common signs of coronavirus infection include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. Currently, there is no specific treatment for the disease caused by the novel coronavirus; treatment must be based on the patient's clinical condition. The "Diagnosis and Treatment Protocol for Novel Coronavirus-Infected Pneumonia (Trial Version 5)" has been published, which includes convalescent plasma therapy as one of the treatment measures for severe and critically ill patients. On February 8, the first phase of COVID-19 convalescent plasma therapy was conducted on three critically ill patients at the First People's Hospital of Jiangxia District. Currently, including subsequent treatments at other hospitals, more than 10 critically ill patients have received this therapy. Clinical feedback indicates that 12 to 24 hours after treatment, key inflammatory markers in laboratory tests significantly decreased, lymphocyte ratio increased, and key indicators such as blood oxygen saturation and viral load showed comprehensive improvement, with significant improvement in clinical signs and symptoms. The guidelines further refine the indications, contraindications, and situations where convalescent plasma is not suitable for clinical use, focusing on the therapeutic goal of neutralizing the virus. Convalescent plasma is primarily used for COVID-19 patients with rapidly progressing, severe, or critical illness. In principle, the course of illness should not exceed 3 weeks; if the patient tests positive for COVID-19 nucleic acid or is clinically diagnosed with viremia, it should be used as early as possible during the acute progression phase of the disease. Although convalescent plasma therapy has achieved some clinical success, limitations exist due to the limited availability of plasma from infected patients, high safety risks associated with purified antibodies, and unstable titers of specific antibodies. Monoclonal antibodies with high titers, stable performance, and good safety profiles show promising application prospects for controlling the COVID-19 pandemic. Current literature has published or taught reports on the preparation of protective neutralizing monoclonal antibodies against the SARS-CoV-2 spike protein RBD. Examples include the use of the SARS-CoV-2 spike protein RBD to generate protective neutralizing antibodies against the SARS-CoV-2 virus (e.g., bioRxiv, “SARS-CoV-2 and SARS-CoV Spike-RBD Structure and Receptor Binding Comparison and Potential Implications on Neutralizing Antibody and Vaccine Development”, 20200220). The SARS spike protein RBD and the SARS-CoV-2 spike protein RBD have cross-neutralizing epitope peptides. The anti-SARS monoclonal antibody CR3022 can bind to the SARS-CoV-2 spike protein RBD (Emerging Microbes & Infections, 9(1):382-385, 20200217). Using homology modeling, the hotspots and key residues of the protein-protein interaction interface of the SARS-CoV-2 CTD1 / human ACE2 complex were identified. Candidate inhibitors targeting the CTD1 region and the ACE2 binding surface were screened to block the recognition and binding of the virus to the human ACE2 protein.

[0003] As research into SARS-CoV-2 vaccines and therapeutic antibodies continues, recent reports indicate that some therapeutic antibodies can produce antibody-dependent enhancement (ADE) when used to treat SARS-CoV-2. ADE refers to antibody-dependent enhancement, where certain viruses, with the assistance of specific antibodies, exhibit significantly enhanced replication or infectivity, leading to more severe pathological damage during infection. In simpler terms, the antibody fails to neutralize the virus; instead, it acts as a "Trojan horse," enhancing the virus's ability to infect immune cells, producing more progeny viruses, and causing severe symptoms. ADE was first discovered during dengue virus infection. Dengue virus binding to non-neutralizing antibodies can enter macrophages via a bypass pathway, proliferate, and cause a secondary infection. This is especially true when the secondary infection uses a different viral strain than the primary infection, resulting in more severe symptoms and an ADE effect. Regarding coronaviruses, early 1980s research on feline infectious peritonitis virus (FIPV) vaccines found that low-titer neutralizing antibodies against the S protein could exacerbate symptoms and lead to more severe death. Among coronaviruses that infect humans, studies on SARS and MERS viruses have found that low-affinity antibodies against the S protein induced by vaccination can mediate viral entry into immune cells. Based on the molecular mechanisms of coronavirus infection, if anti-SARS-CoV-2 antibodies also produce an adverse drug reaction (ADE) effect, it will severely impact the clinical application of SARS-CoV-2 vaccines and therapeutic antibodies. However, whether specific neutralizing antibodies against the SARS-CoV-2 S protein produce an ADE effect, the molecular mechanisms underlying this ADE effect, and countermeasures are currently unknown. Summary of the Invention

[0004] To address the aforementioned issues, this invention isolated coronavirus RBD-specific memory B cells from PBMCs of recovered COVID-19 patients, amplified the light and heavy chain variable region sequences of antibodies, and transiently expressed them. Detection revealed that at least 20 antibodies could specifically bind to the coronavirus RBD with high affinity, and at least 7 antibodies could block or inhibit the binding of the novel coronavirus RBD to the host receptor ACE2. Further research found that the 7 antibody molecules capable of blocking or inhibiting the binding of the novel coronavirus RBD to the host receptor ACE2 did not produce an adverse drug reaction (ADE) during SARS-CoV-2 infection of THP-1 and K562 cells. However, two antibodies, Corn-01 and Corn-05, promoted SARS-CoV-2 entry into Raji host cells, exhibiting an ADE effect. The mechanism of antibody ADE was analyzed by examining the types of membrane-bound Fc receptors in Raji host cells. Furthermore, mutations in the antibody Fc segment reduced or eliminated the binding of antibodies to host cell FcγRs receptors via Fc, thereby avoiding the ADE effect during SARS-CoV-2 infection of host cells. Specifically:

[0005] On the one hand, the present invention provides a SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or a fragment thereof, characterized in that: the monoclonal antibody is a fully human antibody specifically binding to the RBD region of the SARS-CoV-2S protein obtained by isolating a single B cell clone from a blood sample of a patient infected with the novel coronavirus during the recovery period, and it does not produce an ADE effect on SARS-CoV-2 infected THP-1 cells and K562 cells.

[0006] Preferably, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the monoclonal antibody does not produce an ADE effect on SARS-CoV-2 infected Raji cells.

[0007] Preferably, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the monoclonal antibody produces an ADE effect on SARS-CoV-2-infected Raji cells in a partial concentration range of 10-10000 ng.

[0008] Preferably, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the monoclonal antibody produces an ADE effect on SARS-CoV-2-infected Raji cells in a partial concentration range of 50-3000 ng.

[0009] Preferably, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the monoclonal antibody has high affinity for the SARS-CoV-2S protein RBD and a KD value of 5.0 × 10⁻⁶. -9 Below M.

[0010] Preferably, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the IC50 value of the monoclonal antibody blocking the binding of ACEII to the SARS-CoV-2S protein RBD is less than 50 nM, preferably less than 30 nM, 25 nM, 20 nM, 15 nM or 10 nM.

[0011] Preferably, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or its fragment described in this invention is characterized in that a point mutation in the Fc segment of the monoclonal antibody alters its binding to the receptor.

[0012] Preferably, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the point mutation of the Fc segment of the monoclonal antibody reduces or eliminates its binding to Fc γRs.

[0013] Preferably, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the point mutation of the Fc segment of the monoclonal antibody includes an amino acid substitution, deletion or insertion mutation at any one or two sites in the group consisting of positions 234 and 235.

[0014] Preferably, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the point mutations in the Fc segment of the monoclonal antibody include L234A and L235A mutations.

[0015] Preferably, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention is characterized in that the heavy chain constant region of the monoclonal antibody has the sequence shown in SEQ ID NO:44.

[0016] In one specific embodiment, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof of the present invention has the following characteristics:

[0017] HCDR1 selected from SEQ ID NO:1, 7, 13, 19, 25, 31, 37;

[0018] HCDR2 selected from SEQ ID NO:2, 8, 14, 20, 26, 32, 38;

[0019] HCDR3 selected from SEQ ID NO:3, 9, 15, 21, 27, 33, 39;

[0020] LCDR1 selected from SEQ ID NO:4, 10, 16, 22, 28, 34, 40;

[0021] LCDR2 selected from SEQ ID NO:5, 11, 17, 23, 29, 35, 41;

[0022] LCDR3 selected from SEQ ID NO:6,12,18,24,30,36,42.

[0023] Furthermore, the SARS-CoV-2S protein-ACEII receptor binding blocking monoclonal antibody or fragment thereof described in this invention has the following characteristics:

[0024] HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6;

[0025] HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12;

[0026] HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:18;

[0027] HCDR1 shown in SEQ ID NO:19, HCDR2 shown in SEQ ID NO:20, HCDR3 shown in SEQ ID NO:21, LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24;

[0028] HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26, HCDR3 shown in SEQ ID NO:27, LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, and LCDR3 shown in SEQ ID NO:30;

[0029] HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32, HCDR3 shown in SEQ ID NO:33, LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36; or

[0030] HCDR1 shown in SEQ ID NO:37, HCDR2 shown in SEQ ID NO:38, HCDR3 shown in SEQ ID NO:39, LCDR1 shown in SEQ ID NO:40, LCDR2 shown in SEQ ID NO:41, and LCDR3 shown in SEQ ID NO:42.

[0031] Secondly, the present invention provides a polynucleotide encoding the aforementioned monoclonal antibody or a fragment thereof.

[0032] Thirdly, the present invention provides a nucleic acid construct comprising the aforementioned polynucleotides of the present invention.

[0033] Preferably, the nucleic acid construct of the present invention is used to express the aforementioned monoclonal antibody or fragment thereof.

[0034] Fourthly, the present invention provides a host cell comprising the aforementioned polynucleotides or the aforementioned nucleic acid constructs of the present invention.

[0035] Fifthly, the present invention provides a composition comprising one or more monoclonal antibodies or fragments thereof selected from the group consisting of the aforementioned monoclonal antibodies or fragments thereof, and optionally a pharmaceutically acceptable carrier.

[0036] Preferably, the composition of the present invention comprises any two, three, four, five, six or seven monoclonal antibodies or fragments thereof selected from the group consisting of any one of the monoclonal antibodies or fragments thereof as described in any one of claims 1 to 13.

[0037] Fifthly, the present invention provides the use of the monoclonal antibody or fragment thereof described in any of the foregoing claims of the present invention, the polynucleotide described in the foregoing claims of the present invention, the nucleic acid construct described in the foregoing claims of the present invention, the host cell described in the foregoing claims of the present invention, and the composition described in the foregoing claims of the present invention in the preparation of a drug for the prevention or treatment of SARS-CoV-2 infection.

[0038] Preferably, the application described in this invention is characterized in that the prevention or treatment of SARS-CoV-2 infection includes reducing or lowering the risk of SARS-CoV-2 infection, alleviating the symptoms of SARS-CoV-2 infection-related diseases (e.g., COVID-19), shortening the course of SARS-CoV-2 infection-related diseases (e.g., COVID-19), promoting recovery from SARS-CoV-2 infection-related diseases (e.g., COVID-19), and reducing deaths caused by SARS-CoV-2 infection.

[0039] In a sixth aspect, the present invention provides a method for eliminating the ADE effect of antibodies when SARS-CoV-2 infects host cells, wherein the antibody is a neutralizing antibody against SARS-CoV-2S protein, and the binding of the anti-SARS-CoV-2S protein antibody to its receptor is altered by point mutation of the Fc segment of the antibody.

[0040] Preferably, in the method for eliminating the ADE effect of antibodies when SARS-CoV-2 infects host cells, the point mutation of the Fc segment reduces or eliminates the binding of anti-SARS-CoV-2S protein antibodies to Fc γRs.

[0041] Preferably, in the method for eliminating the ADE effect of antibodies when SARS-CoV-2 infects host cells, the Fc segment point mutation of the present invention includes an amino acid substitution, deletion, or insertion mutation at any one or two sites in the group consisting of positions 234 and 235.

[0042] Preferably, the method for eliminating the ADE effect of antibodies when SARS-CoV-2 infects host cells according to the present invention is characterized in that the neutralizing antibody against SARS-CoV-2 S protein is selected from any of the monoclonal antibodies or fragments thereof described in the foregoing claims of the present invention.

[0043] In one specific embodiment, the present invention provides an antibody that specifically binds to the SARS-CoV-2S protein, wherein the antibody is based on a natural human anti-SARS-CoV-2S protein antibody with an Fc segment point mutation, thereby avoiding the ADE effect (antibody-dependent enhancement effect) when the natural human anti-SARS-CoV-2S protein antibody is used to prevent SARS-CoV-2 from infecting host cells.

[0044] The antibody that specifically binds to the SARS-CoV-2 S protein did not produce an adverse drug reaction (ADE) in SARS-CoV-2-infected THP-1 cells, Raji cells, and K562 cells within a concentration range of 10-10000 ng / mL.

[0045] Furthermore, the antibody of the present invention, wherein the natural human anti-SARS-CoV-2S protein antibody is capable of neutralizing SARS-CoV-2 infection of Vero E host cells.

[0046] Furthermore, the antibody of the present invention, wherein the natural human anti-SARS-CoV-2S protein antibody has an ADE effect on SARS-CoV-2 infected Raji cells at least in a partial concentration range of 10-10000 ng / mL.

[0047] Furthermore, the antibody of the present invention, wherein the human anti-SARS-CoV-2S protein antibody has an ADE effect on SARS-CoV-2-infected Raji cells at least in a partial concentration range of 50-3000 ng / mL.

[0048] Furthermore, the antibody of the present invention, wherein the natural human anti-SARS-CoV-2S protein antibody has:

[0049] HCDR1 selected from SEQ ID NO:1, 7, 13, 19, 25, 31, 37;

[0050] HCDR2 selected from SEQ ID NO:2, 8, 14, 20, 26, 32, 38;

[0051] HCDR3 selected from SEQ ID NO:3, 9, 15, 21, 27, 33, 39;

[0052] LCDR1 selected from SEQ ID NO:4, 10, 16, 22, 28, 34, 40;

[0053] LCDR2 selected from SEQ ID NO:5, 11, 17, 23, 29, 35, 41;

[0054] LCDR3 selected from SEQ ID NO:6,12,18,24,30,36,42.

[0055] Furthermore, the antibody of the present invention, wherein the natural human anti-SARS-CoV-2S protein antibody has...

[0056] HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6;

[0057] HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12;

[0058] HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14, HCDR3 shown in SEQ ID NO:15, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:18;

[0059] HCDR1 shown in SEQ ID NO:19, HCDR2 shown in SEQ ID NO:20, HCDR3 shown in SEQ ID NO:21, LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24;

[0060] HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26, HCDR3 shown in SEQ ID NO:27, LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29, and LCDR3 shown in SEQ ID NO:30;

[0061] HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32, HCDR3 shown in SEQ ID NO:33, LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35, and LCDR3 shown in SEQ ID NO:36; or

[0062] HCDR1 shown in SEQ ID NO:37, HCDR2 shown in SEQ ID NO:38, HCDR3 shown in SEQ ID NO:39, LCDR1 shown in SEQ ID NO:40, LCDR2 shown in SEQ ID NO:41, and LCDR3 shown in SEQ ID NO:42.

[0063] Furthermore, in the antibody of the present invention, the point mutation of the Fc segment alters the binding of the anti-SARS-CoV-2 S protein antibody to its receptor.

[0064] Furthermore, in the antibody of the present invention, the point mutation of the Fc segment reduces or eliminates the binding of the anti-SARS-CoV-2S protein antibody to FcγRs.

[0065] Furthermore, the antibody of the present invention does not produce an ADE effect on SARS-CoV-2-infected THP-1 cells, Raji cells, and K562 cells in a concentration range of 10-10000 ng / mL.

[0066] Furthermore, in the antibody of the present invention, the Fc segment point mutation includes an amino acid substitution, deletion, or insertion mutation at any one or two sites in the group consisting of positions 234 and 235.

[0067] Furthermore, in the antibody of the present invention, the Fc segment point mutation includes a single amino acid substitution, deletion, or insertion mutation at any one or two sites in the group consisting of positions 234 and 235.

[0068] Furthermore, in the antibody of the present invention, the Fc segment point mutation includes L234A and L235A mutations.

[0069] In another specific embodiment, the present invention provides the use of an antibody in the preparation of a medicament for treating SARS-CoV-2 infection, wherein the antibody is as described in the first aspect of the present invention.

[0070] In another specific embodiment, the present invention provides the use of an antibody in the preparation of a medicament for treating diseases caused by SARS-CoV-2 infection, wherein the antibody is as described in the first aspect of the present invention, and the disease includes COVID-19.

[0071] In another specific embodiment, the present invention provides a polynucleotide that encodes the antibody described in the first aspect of the present invention.

[0072] In another specific embodiment, the present invention provides a carrier comprising the polynucleotides described in the fourth aspect of the present invention.

[0073] In another specific embodiment, the present invention provides a host cell comprising the polynucleotide or the vector described in the fourth aspect of the present invention.

[0074] In another specific embodiment, the present invention provides a pharmaceutical composition comprising one or more antibodies selected from the group consisting of antibodies described in the first aspect of the present invention, and optionally a pharmaceutically acceptable carrier.

[0075] In another specific embodiment, the present invention provides a method for eliminating the ADE effect of antibodies when SARS-CoV-2 infects host cells, wherein the antibody is a neutralizing antibody against SARS-CoV-2S protein, and the binding of the anti-SARS-CoV-2S protein antibody to its receptor is altered by point mutation of the Fc segment of the antibody.

[0076] Furthermore, in the method for eliminating the ADE effect of antibodies when SARS-CoV-2 infects host cells, the point mutation of the Fc segment reduces or eliminates the binding of anti-SARS-CoV-2S protein antibodies to FcγRRs.

[0077] Furthermore, in the method for eliminating the ADE effect of antibodies when SARS-CoV-2 infects host cells, the Fc segment point mutation of the present invention includes substitution, deletion or insertion mutations of amino acids at any one or two sites in the group consisting of positions 234 and 235.

[0078] To better understand this invention, some terms are first defined. Other definitions are listed throughout the detailed description section.

[0079] The term "coronavirus" refers to members of the order Nidovirales, family Coronaviridae, and genus Coronavirus. The coronaviruses described in this invention primarily relate to coronaviruses that infect humans, including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2 (2019-nCoV), with a particular focus on SARS-CoV, MERS-CoV, and SARS-CoV-2 (2019-nCoV).

[0080] The term "specificity" refers to the determination of the presence of said protein in a population of proteins and / or other biological heterogeneities, such as the binding reaction of the monoclonal antibody of the present invention to the SARS-CoV-2 RBD protein. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0081] The term "antibody" in this article is intended to include full-length antibodies and any antigen-binding fragments (abbreviated as antibody fragments) or single chains. A full-length antibody is a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can also be divided into hypervariable regions called complementarity-determining regions (CDRs), which are separated by more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component (C1q) of the conventional complement system.

[0082] The terms "monoclonal antibody," "monoclonal antibody," or "monoclonal antibody composition" refer to antibody molecules that consist of a single molecule. A monoclonal antibody composition exhibits specific binding specificity and affinity for a particular epitope.

[0083] In this article, the term "antigen-binding fragment" (or simply antibody fragment) refers to one or more fragments of an antibody that retain the ability to specifically bind antigens. It has been demonstrated that the antigen-binding function of an antibody can be performed using fragments of a full-length antibody. Examples of binding fragments included in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1; (ii) F(ab′)2 fragments, bivalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH1; (iv) Fv fragments consisting of the antibody's single arm VL and VH; (v) dAb fragments consisting of VH (Ward et al., (1989) Nature 341: 544-546); (vi) separated complementarity-determining regions (CDRs); and (vii) nanobodies, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by different genes, they can be linked via a synthetic linker that makes them single-chain proteins through recombination, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fc (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). These single-chain antibodies are also intended to be included in the terminology. These antibody fragments can be obtained using techniques commonly known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

[0084] The antigen-binding fragments of the present invention include those capable of specifically binding to coronavirus RBD. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv (scFv) fragments, and single-domain fragments.

[0085] The Fab fragment contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that it contains the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. The Fab' fragment is generated by cleaving the disulfide bond at the hinge cysteine ​​residue in the F(ab')2 pepsin digestion product. Further chemical conjugation of antibody fragments is known to those skilled in the art. The Fab and F(ab')2 fragments lack the fragment crystallizable (Fc) region of the intact antibody, are cleared more rapidly from animal circulation, and may have less nonspecific tissue binding than the intact antibody (see, for example, Wahl et al., 1983, J. Nucl. Med. 24:316).

[0086] As is generally understood in the art, the "Fc" region is a crystallizable constant region of an antibody fragment that does not contain an antigen-specific binding region. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of the antibody. The IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4 domains) in each polypeptide chain.

[0087] The “Fv” fragment is the smallest fragment of an antibody containing a complete target recognition and binding site. This region consists of a dimer (VH-VL dimer) of a heavy chain and a light chain variable domain bound together in a tight, non-covalent manner. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or only half of the Fv containing the three CDRs for target specificity) can have the ability to recognize and bind to the target, although its affinity is lower than that of the entire binding site.

[0088] A "single-chain Fv" or "scFv" antibody-binding fragment contains the VH and VL domains of the antibody, which are located within a single polypeptide chain. Typically, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, which allows the scFv to form a structure conducive to target binding.

[0089] A “single-domain fragment” consists of a single VH or VL domain that shows sufficient affinity for the coronavirus RBD. In one specific implementation, the single-domain fragment is camelized (see, for example, Riechmann, 1999, Journal of Immunological Methods 231:25–38).

[0090] The antibodies against coronavirus RBD of the present invention include derivatized antibodies. For example, derivatized antibodies are typically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protective / blocking groups, proteolytic cleavage, or linkage to cellular ligands or other proteins. Any of a number of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivatives may contain one or more non-natural amino acids, for example, using the ambrx technology (see, for example, Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0091] "Human antibodies" include antibodies having the amino acid sequence of human immunoglobulins, and include antibodies isolated from a human immunoglobulin library or from animals that are transgenic for one or more human immunoglobulins and do not express endogenous immunoglobulins. Human antibodies can be prepared by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publications WO 98 / 46645; WO 98 / 50433; WO 98 / 24893; WO 98 / 16654; WO 96 / 34096; WO96 / 33735; and WO 91 / 10741. Human antibodies can also be generated using transgenic mice that do not express functional endogenous immunoglobulins but can express human immunoglobulin genes. See, for example, PCT disclosures WO 98 / 24893; WO92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Patents 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. Additionally, using similar techniques, companies such as LakePharma, Inc. (Belmont, CA) or Creative BioLabs (Shirley, NY) can engage in providing human antibodies against selected antigens. Fully human antibodies recognizing selected epitopes can be generated using a technique known as “guided selection.” In this method, a non-human monoclonal antibody, such as a mouse antibody, is selected to guide the selection of a fully human antibody that recognizes the same epitope (see Jespers et al., 1988, Biotechnology 12:899-903).

[0092] The terms “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that specifically binds to an antigen” in this document.

[0093] For IgG antibodies, the term "high affinity" refers to a KD of 1.0 × 10⁻⁶ for the antigen. -6 For sizes below M, 5.0 × 10 is preferred. -8 M or less, preferably 1.0 × 10 -8 Below M, 5.0×10 -9 M or less, preferably 1.0 × 10 -9 Below M. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding for the IgM subtype refers to a KD of 10. -6 For sizes below M, 10 is preferred.-7 M or less, preferably 10 -8 Below M.

[0094] The terms "Kassoc" or "Ka" refer to the binding rate of a specific antibody-antigen interaction, while the terms "Kdis" or "Kd" refer to the dissociation rate of a specific antibody-antigen interaction. The term "KD" refers to the dissociation constant, obtained from the ratio of Kd to Ka (Kd / Ka), and expressed as molar concentration (M). The KD value of an antibody can be determined by methods known in the art. A preferred method for determining the antibody KD is by measuring it using a surface plasmon resonance (SPR) instrument, preferably a biosensor system such as the Biacore™ system.

[0095] The term "EC50," also known as the half-maximal effect concentration, refers to the antibody concentration that produces a 50% maximum effect.

[0096] The term "IC50" refers to the half-maximal inhibitory concentration of a measured antagonist. It indicates the amount of a drug or substance (inhibitor) that inhibits a certain biological process (or certain substances contained in this process, such as enzymes, cell receptors, or microorganisms) to half its capacity.

[0097] The term "epitope" refers to a site on an antigen where B and / or T cells respond. B cell epitopes can be formed from consecutive amino acids or from discontinuous amino acids arranged side-by-side due to the ternary folding of proteins. Epitopes formed from consecutive amino acids are typically preserved upon exposure to denaturing solvents, while epitopes formed by ternary folding are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least three, and more often at least five or eight to ten amino acids in a unique spatial conformation.

[0098] The term "composition" in this invention refers to compositions comprising antibodies or antigen-binding fragments thereof as described herein. Compositions according to the invention can be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, and similar properties. Many suitable formulations can be found in formulations known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTINTM), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbon waxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbon waxes. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998), *Journal of Pharmaceutical Science and Technology*, 52: 238-311.

[0099] The compositions described in this invention are preferably injectable formulations, which may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, intravenous infusion, etc. These injectable formulations can be prepared by methods known to the public. For example, injectable formulations can be prepared, for instance, by dissolving, suspending, or emulsifying the antibodies or their salts described above in a sterile aqueous or oily medium conventionally used for injection. As aqueous media for injection, examples include physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. As oily media, examples include sesame oil and soybean oil, which can be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Therefore, the prepared injection is preferably filled in a suitable ampoule.

[0100] The term "amino acid mutation" as used herein refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence, or an alteration to a portion of the protein that is chemically linked to it. For example, a mutation could be an alteration to the carbohydrate or PEG structure attached to a protein. For clarity, unless otherwise stated, amino acid mutations typically refer to amino acids encoded by DNA, such as the 20 amino acids that have codons in DNA and RNA.

[0101] "Amino acid substitution" or "replacement" as used herein refers to the replacement of an amino acid at a specific position in the parental polypeptide sequence with a different amino acid. More precisely, in some embodiments, substitution is for an amino acid that is not naturally present at a specific position; these amino acids are not naturally present in the organism or in any organism. For example, substitution E272Y refers to a variant polypeptide in which glutamic acid at position 272 is replaced by tyrosine, in this case, an Fc variant. For clarity, engineering a protein to alter its coding sequence without changing the starting amino acid (e.g., changing CGG (encoding arginine) to CGA (still encoding arginine) to increase expression levels in a host organism) is not "amino acid substitution"; that is, although a new gene encoding the same protein is generated, if that protein has the same amino acid at its starting specific position, it is not amino acid substitution.

[0102] As used herein, “amino acid insertion” or “insertion” means the addition of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, -233E or 233E indicates the insertion of glutamic acid after position 233 and before position 234. Similarly, -233ADE or A233ADE indicates the insertion of AlaAspGlu after position 233 and before position 234.

[0103] As used herein, "amino acid deletion" or "deletion" means the removal of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, G236-, G236#, or G236del indicates a glycine deletion at position 236. Additionally, EDA233- or EDA233# indicates a deletion of the sequence GluAspAla starting at position 233.

[0104] As used in this article, "residue" refers to a position in a protein and the identity of its associated amino acid. For example, asparagine 297 (also known as Asn297 or N297) is residue 297 in human antibody IgG1.

[0105] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0106] First, this invention employs single-lymphocyte cloning technology to amplify multiple specific antibodies from a large number of single B cells from patients recovering from SARS-CoV-2 infection. Through recombinant expression, specific binding ability screening, epitope competition analysis, and SARS-CoV-2 S protein-ACEII binding blocking activity analysis, seven monoclonal antibodies were obtained that can bind to the SARS-CoV-2 S protein RBD with high affinity and specificity, and can block SARS-CoV-2 binding to the host cell receptor ACEII. The amino acid sequences of their light and heavy chain CDRs regions were also provided. During the screening process, it was found that the antibody's activity in blocking SARS-CoV-2 binding to the host cell receptor ACEII was not positively correlated with its affinity for the SARS-CoV-2 S protein RBD. The monoclonal antibodies of this invention achieved good neutralization and blocking effects in both pseudovirus neutralization assays and SARS-CoV-2 virus particle neutralization assays.

[0107] Secondly, this invention performed ADE analysis on seven monoclonal antibodies with SARS-CoV-2S protein-ACEII binding blocking activity obtained through screening. None of these antibodies produced an ADE effect during SARS-CoV-2 infection of THP-1 and K562 cells; suggesting that the seven monoclonal antibodies screened in this invention have a low ADE effect in clinical applications. Furthermore, the seven monoclonal antibodies of this invention are not only naturally occurring fully human antibodies found in the serum of recovered patients, but also target multiple different natural epitopes of the SARS-CoV-2S protein RBD, making them particularly suitable for "cocktail antibody therapy."

[0108] Third, this invention has discovered that antibodies Corn-01 and Corn-05 at specific concentration ranges produce an adverse drug reaction (ADE) effect when SARS-CoV-2 virus infects Raji host cells. Based on existing research reports, the mechanism of the ADE effect produced by neutralizing antibodies against SARS-CoV-2 was explored. According to the different types of Fc receptors on Raji host cells compared to other host cells that do not produce an ADE effect, the mechanism of the ADE effect produced by neutralizing antibodies against SARS-CoV-2 may be due to the binding of the antibody to the FcγRI receptor on the host cell membrane. This allows those skilled in the art to reduce or eliminate the ADE effect by blocking the binding of neutralizing antibodies against SARS-CoV-2 to the FcγRIIb receptor on host cells, thus preserving the virus-blocking activity of the antibody while eliminating the potential ADE effect, thereby improving the safety of clinical applications in humans. Attached Figure Description

[0109] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0110] Figure 1 : Fusion expression of SARS-CoV-2S1 protein and His.

[0111] Figure 2 : Fusion expression of SARS-CoV-2S1 protein and mFc.

[0112] Figure 3 Fusion expression of SARS-CoV-2S1 protein RBD and His.

[0113] Figure 4 Fusion expression of SARS-CoV-2S1 protein RBD and mFc.

[0114] Figure 5 : The fusion expression of human ACE2 and human Fc.

[0115] Figure 6 The recombinant antibodies Corn-02, Corn-10, and Corn-01 demonstrate their ability to block the binding of S1RBD-mFc to ACE2. CR3022 is a monoclonal antibody against SARS-CoV RBD disclosed in US2010172917A1; Corn-10 is a monoclonal antibody specifically binding to S1RBD-mFc prepared in Example 4 of this invention.

[0116] Figure 7 The ability of recombinant antibodies Corn-02, Corn-10, and Corn-01 to block the binding of S1-mFc to ACE2.

[0117] Figure 8 : The curve of the blocking effect of recombinant antibody on the binding of S1RBD-mFc and ACEII-hFc.

[0118] Figure 9 : Curves showing the blocking effect of Corn-01 and Corn-07 on the binding of S1RBD-mFc and ACEII-hFc (competitive ELISA method).

[0119] Figure 10 : Blocking effect curves of Corn-01 and Corn-07 on the binding of S1RBD-His and ACEII-his (pseudovirus infection fluorescence detection method).

[0120] Figure 11The ADE effect of Corn-01 in the process of SARS-CoV-2 viral particle infection of different host cells

[0121] Figure 12 The ADE effect of Corn-01, Corn-04, Corn-05, and Corn-06 on Raji cells infected with SARS-CoV-2 viral particles.

[0122] Figure 13 The ADE effect of Corn-01, Corn-05, and Corn-07 on Raji cells infected with SARS-CoV-2 virus particles.

[0123] Figure 14 The ADE effect of Corn-01 and Corn-01-LALA on SARS-CoV-2 virus particles infecting Raji cells.

[0124] Figure 15 The ADE effect of Corn-05 and Corn-05-LALA on SARS-CoV-2 virus particles infecting Raji cells.

[0125] Figure 16 Neutralizing activity of Corn-01 and Corn-05 against SARS-CoV-2 viral particles

[0126] Figure 17 Neutralizing activity analysis of Corn-01 and Corn-01-LALA against SARS-CoV-2 viral particles

[0127] Figure 18 Analysis of the neutralizing activity of Corn-05 and Corn-05-LALA against SARS-CoV-2 viral particles. Detailed Implementation

[0128] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0129] Example 1: Recombinant Expression of SARS-CoV-2 Antigen and Host Receptor

[0130] The fully synthesized gene S1RBD (Accession: QHD43416.1, 319-541aa) was cloned into eukaryotic transient expression vectors with either a His tag or an mFc tag at the C-terminus using enzyme digestion. The obtained expression plasmids were transformed into *E. coli* for amplification, and the S1RBD-his and S1RBD-mFc expression plasmids were isolated. Following the instructions for using the 293fectin transfection reagent (Cat: 12347019, Gibco), the plasmids were transfected into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected. The S1RBD-mFc protein was purified using a ProA affinity chromatography column to obtain the S1RBD-mFc protein (amino acid sequence shown in SEQ ID NO: 46). The S1RBD-his protein (amino acid sequence shown in SEQ ID NO:45) was purified from the expression supernatant using a HisTrapHP affinity chromatography column. The purity of the obtained recombinant protein was then determined by SDS-PAGE. Figure 3-4 ).

[0131] The S1 gene (Accession: QHD43416.1, 1-685aa) was cloned from a purchased full-length SARS-CoV-2 expression vector (Cat: VG40589-UT, Beijing Yiqiao Shenzhou) using PCR. The cloned gene was then cloned into eukaryotic transient expression vectors with either an mFc tag or a His tag at the C-terminus, respectively, using restriction enzyme digestion. The obtained expression plasmids were transformed into *E. coli* for amplification, and the S1-mFc and S1-His expression plasmids were isolated. Following the instructions of the 293fectin transfection reagent (Cat: 12347019, Gibco), the plasmids were transfected into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected, purified, and the S1-mFc protein (amino acid sequence as shown in SEQ ID NO: 48) and S1-His protein (amino acid sequence as shown in SEQ ID NO: 47) were obtained. The purity of the obtained recombinant proteins was determined by SDS-PAGE. Figure 1-2 ).

[0132] The extracellular region gene ACEII(1-615) (Accession: NP_068576.1, 1-615aa) of ACEII was cloned from a purchased full-length human ACEII expression vector (Cat:HG10108-ACR, Beijing Yiqiao Shenzhou) using PCR. The ACEII extracellular region gene was then cloned into a eukaryotic transient expression vector with an hFc tag at the C-terminus using enzyme digestion. The obtained expression plasmid was transformed into *E. coli* for amplification, and the ACEII (1-615)-hFc expression plasmid was isolated. Following the instructions of the 293fectin transfection reagent (Cat:12347019, Gibco), the plasmid was transfected into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column to obtain ACEII(1-615)-hFc protein (amino acid sequence shown in SEQ ID NO:49). The purity of the obtained recombinant protein was then determined by SDS-PAGE. Figure 5 ).

[0133] Example 2: Isolation of SARS-CoV-2S1 protein RBD-specific memory B cells

[0134] Specific memory B cells from recovered COVID-19 patients were detected and sorted. IgG and IgM antibodies in patient serum were detected using a COVID-19 antibody detection kit, and serum samples positive for IgG antibodies were selected. B cells were enriched using the RosetteSep kit (Cat:15064, STEMCELL). Based on this, FITC-labeled S1-RBD-his was used to capture memory B cells specifically bound to the COVID-19 RBD, and flow cytometry was used for single-cell sorting.

[0135] Example 3: Amplification of human anti-SARS-CoV-2 RBD antibody sequence

[0136] RNA was extracted from single B cells using RNA magnetic beads (Nanjing Novizan) and then reverse transcribed into cDNA. The specific method is as follows:

[0137] 1. Dispense 5 μl of Catch Buffer B (TCL + 1% β-ME) into each well and sort individual memory B cells.

[0138] 2. Apply film and centrifuge at 2000 rpm for 1 min.

[0139] 3. Add 10 μl H2O and 33 μl Beads to each well, mix by blowing and aspiration, and incubate at room temperature for 10 min.

[0140] 4. Place on a magnetic rack, at room temperature for 5 minutes, then discard the supernatant.

[0141] 5. Rinse the magnetic beads with 200 μl of freshly prepared 80% ethanol in nuclease-free water at room temperature for 30 seconds, then discard the supernatant.

[0142] 6. Rinse once more, discard the supernatant, and air dry for 3 minutes.

[0143] 7. Remove the magnetic rack, add 12 μl of Mix 1 to each well, blow and aspirate 5 times, and let it incubate at room temperature for 5 minutes.

[0144] 8. Place on a magnetic rack, incubate at room temperature for 2 minutes, transfer 10 μl to a new plate, centrifuge at 300g for 30 seconds, and run program 1.

[0145] 9. Add 10 μl of Mix 2 to each well, mix well, centrifuge, and run program 2.

[0146] 10. Perform PCR on the synthesized cDNA as soon as possible.

[0147] Mix 1: 310μl H2O+50μl dNTP+20μl Random 6+20μl Oligo_dT

[0148] Mix 2: 170μl H2O+160μl Buffer+40μl DTT+20μl RNase I+10μl RTase IV (Cat: EN0601 and 18090010, ThermoFisher)

[0149] Program 1: 65℃ for 5 min → 4℃ for ∞

[0150] Program 2: 23℃ for 10 min → 50℃ for 30 min → 80℃ for 10 min → 4℃ ∞

[0151] Two-step PCR was used to amplify the variable region genes of the antibody heavy chain and light chain (Kappa). Primer sequences were obtained from pages 114-117 of the book *Human Monoclonal Antibodies*. The specific method is as follows:

[0152] First round PCR (Ig-VH1, Ig-VK1), reaction system (20 μl):

[0153]

[0154] Run the program:

[0155] 94℃5min→(94℃30s→51℃30s→72℃55s)×15Cycles

[0156] →(94℃30s→56℃30s→72℃55s)×30 Cycles

[0157] →72℃ for 8 minutes

[0158] →4℃∞

[0159] Second round PCR (Ig-VH2, Ig-VK2), reaction system (20 μl):

[0160]

[0161] Run the program:

[0162] 94℃5min→(94℃30s→57℃30s→72℃45s)×50 Cycles

[0163] →72℃ for 10 minutes

[0164] →4℃∞

[0165] The PCR products were separated and purified by agarose gel electrophoresis, and the variable regions of the antibody light and heavy chains were sequenced.

[0166] Example 4: Expression and Specific Binding Screening of Human Anti-SARS-CoV-2 RBD Antibodies

[0167] The 122 pairs of sequences were analyzed, and 49 pairs of antibody light and heavy chain variable region genes were synthesized and cloned into the whole antibody transient expression vector for recombinant expression and specificity identification. The fully synthesized antibody heavy chain variable region was cloned into the upstream of the human IgG1 heavy chain constant region coding gene in the eukaryotic transient expression vector pKN041 by enzyme digestion, and the fully synthesized antibody light chain variable region was cloned into the upstream of the human light chain Cκ coding gene in the eukaryotic transient expression vector pKN019 by enzyme digestion. Light and heavy chain expression vectors were constructed, and light and heavy chain expression plasmids were obtained. These were transformed into E. coli for amplification, and the antibody light and heavy chain plasmids were isolated. The antibody light and heavy chain plasmids were then transformed into HEK293 cells for recombinant expression according to the instructions of the transfection reagent 293fectin (Cat:12347019, Gibco).

[0168] Twenty-four hours after cell transfection, the supernatant was collected, and the binding of the antibody to S1RBD was measured using an Octet QKe system instrument from Fortebio, employing an antibody capture antibody (AHC) bioprobe to capture the Fc fragment of the anti-human antibody. During the assay, the antibody supernatant and antibody were passed through the surface of an AHC probe (Cat: 18-0015, PALL) for 240 s. S1RBD-mFc (KN expression, lot: 20200217A) was used as the mobile phase, with a recombinant S1RBD-mFc protein concentration of 100 nM. The binding time was 300 s, and the dissociation time was 300 s. After the experiment, a 1:1 Langmuir binding mode was fitted using software to calculate the kinetic constants of antigen-antibody binding. A total of 39 antibodies were detected, of which 20 antibodies specifically bound to S1RBD-mFc (Table 1).

[0169] Table 1. Kinetic parameters of specific binding between candidate antibodies and S1RBD-mFc.

[0170]

[0171]

[0172]

[0173] As shown in Table 1, among the 39 successfully expressed antibody molecules, 20 antibodies that showed specific binding ability to S1RBD-mFc after initial screening were clone numbers 1, 2, 29, 5, 6, 31, 15, 22, 16, 17, 18, 36, 35, 33, 27, 28, 38, 32, 24, and 11. Clones 1, 17, 22, 5, 24, 32, 38, and 11 were selected from the initial screening for further study due to their high affinity. These antibody clones 5, 17, 11, 24, 32, 38, and 1 are also referred to below as Corn-01, Corn-02, Corn-03, Corn-04, Corn-05, Corn-06, and Corn-07.

[0174] The HCDR1-HCDR3 of the antibody Corn-01 are SEQ ID NO:1-3, and the LCDR1-LCDR3 are SEQ ID NO:4-6.

[0175] The HCDR1-HCDR3 of the antibody Corn-02 are SEQ ID NO:7-9, and the LCDR1-LCDR3 are SEQ ID NO:10-12.

[0176] The HCDR1-HCDR3 of the antibody Corn-03 are SEQ ID NO:13-15, and the LCDR1-LCDR3 are SEQ ID NO:16-18.

[0177] The HCDR1-HCDR3 of the antibody Corn-04 are SEQ ID NO:19-21, and the LCDR1-LCDR3 are SEQ ID NO:22-24.

[0178] The HCDR1-HCDR3 of the antibody Corn-05 are SEQ ID NO:25-27, and the LCDR1-LCDR3 are SEQ ID NO:28-30.

[0179] The HCDR1-HCDR3 of the antibody Corn-06 are SEQ ID NO:31-33, and the LCDR1-LCDR3 are SEQ ID NO:34-36.

[0180] The HCDR1-HCDR3 of the antibody Corn-07 are SEQ ID NO:37-39, and the LCDR1-LCDR3 are SEQ ID NO:40-42.

[0181] Example 5: ELISA blocking activity of the antibody

[0182] Clones with high affinity as detected by Fortebio in the supernatant were further subjected to ELISA for blocking activity assay. The specific method is as follows:

[0183] 1. Plate coating: Coat with human ACE2-hFc(1-615) (KN expression, lot: 20200213C) at a concentration of 0.75 ug / ml; 100 μl per well; 4℃ O / N;

[0184] 2. Blocking: 5% BSA in PBS, 37℃, 120 min, wash 4 times with PBST;

[0185] 3. Add primary antibody: 120ul 30ng / ml S1-RBD-mFc (KN expression, lot: 20200217A), add 120ul each of Corn-09, CR3022, Corn-08, Corn-02, Corn-10, and Corn-01 at a concentration of 10ug / ml and 5+17 (mixed 1:1, 5ug / ml), shake slightly to mix and let stand for 50min, take 2 100ul samples from each well and add human ACE2-hFc (1-615) to coat each well in parallel;

[0186] 4. Add secondary antibody: HRP-anti-mouse IgG (Cat:115-035-071, Jackson Immuno Research) (1:5000) 37℃, 45 min, wash plate 4 times with PBST;

[0187] 5. Color development: TMB (Cat:ME142, Beijing Taitianhe Biotechnology) color development, 37℃, 10min;

[0188] 6. Termination: The reaction is terminated with 2M HCl;

[0189] 7. Reading: Read and record the absorbance value of the well plate at a wavelength of 450 nm.

[0190] The results are as follows Figures 6-7 As shown, recombinant antibodies Corn-01 and Corn-02 exhibit significant blocking activity and a synergistic effect in the binding of S1RBD to ACEII and S1 to ACEII. Recombinant antibody Corn-10 cannot block the binding of S1RBD to ACE2, but it can inhibit the binding of S1 to ACE2. These results indicate that antibodies Corn-01 and Corn-02 share the same binding site with ACE2 on the SARS-CoV-2 S1 protein, and therefore can directly block the binding of S1RBD to ACE2.

[0191] Further, for antibodies Corn-01, Corn-02, Corn-03, Corn-04, Corn-05, Corn-06, and Corn-07, a serial dilution method was used for ELISA. In short, after coating and blocking according to the above method, in the step of adding primary antibody, 100 μL of each of the above-mentioned test antibody (initial concentration 40 μg / mL, 1.5-fold serial dilution, 12 gradients) and S1-RBD-mFc 70 ng / mL were mixed and incubated at 37°C for 50 min. Two 100 μL samples from each sample were then added in parallel to the ACE2-hFc coated wells; then secondary antibody was added, color development was performed, the assay was stopped, and the results were read. The results are as follows... Figures 8-9 As shown.

[0192] All six antibodies significantly inhibited the binding of ACEII-hFc to S1RBD-mFc, and their half-maximal inhibitory concentrations (IC50) are shown in Table 2.

[0193] Table 2: Half-maximal inhibitory concentrations of antibodies against the binding of ACEII-hFc to S1RBD-mFc

[0194]

[0195]

[0196] Example 6. Assay method for antibody neutralization of SARS-CoV-2 virus

[0197] 6.1 Pseudovirus Infection Test – Fluorescence Method

[0198] To estimate the neutralizing activity of Corn-01 and Corn-07 against SARS-CoV-2, the neutralizing activity assay of Huh7 cells infected with the novel coronavirus pseudovirus from the National Institutes for Food and Drug Control (NIFDC) was used. Different concentrations of the antibodies to be evaluated were neutralized with 750 TCID50 / well of pseudovirus particles (transfected with a luciferase reporter gene) at 37°C, and then 2 × 10⁶ cells were inoculated into each well. 4 / well huh7 cells were cultured in a CO2 incubator at 37℃ for 20-28 h. After 20-28 h, 100 μl of luciferase assay reagent was added to each well of the cell plate, and the reaction was carried out in the dark for 2 min. The fluorescence detector readings were taken, the neutralization inhibition rate was calculated, and the IC50 was calculated using the Reed-Muench method based on the neutralization inhibition rate results.

[0199] The results are as follows Figure 10 As shown, Corn-07 can inhibit pseudovirus particles from entering host cells in a dose-dependent manner. The IC50 value calculated by the Reed-Muench method is 62 ng / mL, which is higher than the neutralizing activity of Corn-01 (IC50, 281 ng / mL).

[0200] 6.2 SARS-CoV-2 Virus Infection Test – Cytopathic Effect Neutralization Titration Method

[0201] 1) Take Vero-E6 cells in good growth condition, digest them, and adjust the cell density to 1×10⁻⁶. 5 Inoculate 100 μL / well (i.e., 100 μL / well) into a 96-well plate. 4 (cells), placed in a 37℃, 5% CO2 incubator for 12-16 hours;

[0202] 2) After 12-16 hours, discard the culture medium in the wells. First, add 50 μl of the sample at different final concentrations (maximum concentration 100 μg / ml, 3-fold dilution 8 times). Then, add 10 μl of the sample to each well. 2 50 μl of SARS-CoV-2 virus with CCID50 was used. Cell controls and virus controls were also included.

[0203] 3) The cytopathic effect was observed and measured 72 hours after sample addition. The experimental results are shown in Table 3.

[0204] Table 3. Results of antibody activity assays against SARS-CoV-2 virus-infected host cells - EC50

[0205]

[0206]

[0207] Example 7: ADE effect of Corn-01 antibody on SARS-CoV-2 infected cells

[0208] Antibody ADE was detected using lymphocytes (THP-1, Raji, and K562) infected with SARS-CoV-2 particles from the National Institutes for Food and Drug Control (NIFDC). Different concentrations of the SARS-CoV-2 antibodies to be evaluated were neutralized with 750 TCID50 / well of pseudovirus particles (transfected with a luciferase reporter gene) at 37°C, and then inoculated into 1x10 wells. 5 / well host cells were incubated at 37°C in a 5% CO2 incubator for 20-28 hours. After 28-28 hours, 100 μL of luciferase assay reagent (G7940, Promega) was added to each well of the cell plate and incubated in the dark for 2 minutes. The fluorescence was then read by a fluorescence detector, and the strength of ADE was evaluated based on the fluorescence signal intensity.

[0209] The results show that ( Figure 11 Corn-01 in the IgG1 form showed significant ADE effects on Raji cells within a certain concentration range (50 ng / ml-3000 ng / ml); however, it did not produce ADE effects on THP-1 and K562 within the concentration range of 1-1000 ng / ml.

[0210] Example 8: Detection of the ADE effect of neutralizing antibodies during viral infection of susceptible host cells

[0211] Using the method of Example 7, Raji cells were used to detect the ADE effect of five fully human neutralizing antibodies against the RBD region of the SARS-CoV-2 S1 protein identified in previous screening. The results are as follows: Figure 12 , Figure 13 As shown.

[0212] Figure 12 , Figure 13 This indicates that different antibodies neutralizing the RBD region of the SARS-CoV-2S1 protein have varying abilities to induce adverse drug reaction (ADE) during viral infection of susceptible host cells. No significant ADE was observed in the IgG1 forms of Corn-04, Corn-06, and Corn-07 within a concentration range of 1-1000 ng / ml, while Corn-01 and Corn-05 produced ADE within a certain concentration range (50 ng / ml-3000 ng / ml).

[0213] The National Institutes for Food and Drug Control (NIFDC) used pseudovirus particles from the novel coronavirus to infect Raji cells for antibody ADE detection. Different concentrations of the SARS-CoV-2 antibody to be evaluated were neutralized with 750 TCID50 / well of pseudovirus particles (transfected with a luciferase reporter gene) at 37°C, and then seeded into Raji cells at 1x10⁵ / well. The cells were incubated in a CO₂ incubator at 37°C for 20–28 h. After 20–28 h, 100 μl of luciferase assay reagent was added to each well of the cell plate, and the reaction was carried out in the dark for 2 min. The fluorescence was then read using a fluorescence detector, and the strength of ADE was evaluated based on the fluorescence signal intensity.

[0214] Example 9: The effect of antibody Fc point mutation on host cell receptor affinity

[0215] Based on the types of Fc receptors on Raji host cells and other host cells that do not produce ADE effects, the location of Fc point mutations was determined, and the affinity of the mutated antibody for Fc receptors on host cells was tested. The Fc receptors of Corn-01 and Corn-05 were mutated into LALA (Fc-L234A, L235A) to construct Corn-01-LALA and Corn-05-LALA. The amino acid sequence of the wild-type heavy chain constant region before mutation is shown in SEQ ID NO:43, and the amino acid sequence of the mutant heavy chain constant region after mutation is shown in SEQ ID NO:44.

[0216] The affinity of Corn-05 (wtIgG1) and Corn-05-LALA for recombinant FcγRI (CD64) protein (10256-H08H, Sinopharm) and recombinant FcγRIIa (CD32A) protein (10374-H08H1, Sinopharm) was determined using the Fortebio Octet QKe system. Corn-05 and Corn-05-LALA antibodies were captured using an anti-human antibody Fc fragment capture antibody (AHC) bioprobe. 15 μg / ml of antibody was passed through the AHC probe (Cat:18-5060, PALL) for 120 s. 100 nM recombinant antigen was used as the mobile phase. The binding and dissociation times were 300 s. After the experiment, the blank control response values ​​were subtracted, and the kinetic constants of antigen-antibody binding were calculated using a 1:1 Langmuir binding model fitting. The results are shown in Table 4.

[0217] Table 4. Affinity constants of Corn-05 and Corn-05-LALA to CD64 and CD32A

[0218] Sample ID Loading Sample ID Response KD(M) kon(1 / Ms) kdis(1 / s) CD64-His Corn-05 0.1174 1.45E-08 1.20E+05 1.73E-03 CD64-His Corn-05-LALA 0.0344 3.66E-05 7.89E+02 2.89E-02 CD32A-His Corn-05 0.2302 5.60E-08 1.89E+06 1.06E-01 CD32A-His Corn-05-LALA 0.2254 6.62E-08 1.94E+06 1.29E-01

[0219] The results in Table 4 show that, compared with the wtIgG1 type Corn-05, the affinity of LALA mutation for each Fc γRs was altered.

[0220] Example 10: Point mutation of antibody Fc eliminates the ADE effect of antibody on host cells infected with SARS-CoV-2.

[0221] To verify whether reducing the affinity of neutralizing antibody Fc to host cell receptor FcγRI through point mutation could reduce or eliminate its ADE effect on SARS-CoV-2-infected host cells, the mutated Corn-01-LALA and Corn-05-LALA were compared with the unmutated Corn-01 and Corn-05, respectively. The method described in Example 7 was used to detect the ADE effect on SARS-CoV-2-infected host cells. The results are as follows: Figure 14 , Figure 15 As shown.

[0222] Figure 14 , Figure 15 This indicates that Corn-01-LALA and Corn-05-LALA eliminated the ADE phenomenon by introducing L234A and L235A mutations in the Fc region. Combined with the experimental results of Example 9, it can be inferred that the ADE effect induced by fully human neutralizing antibodies against the RBD region of the SARS-CoV-2 S1 protein during SARS-CoV-2 infection of the host may be mediated by the binding of the antibody Fc region to the FcrRI receptor on host cells. Therefore, by introducing L234A and L235A mutations in the Fc region, the binding of the antibody to the FcrRI receptor was greatly reduced, thereby preventing the occurrence of ADE.

[0223] Example 11: Point mutations in antibody Fc do not affect the neutralizing activity of the antibody against the novel coronavirus.

[0224] The neutralizing activity of antibodies was detected by infecting Huh-7 cells with SARS-CoV-2 particles from the National Institutes for Food and Drug Control (NIFDC). Different concentrations of the SARS-CoV-2 antibodies to be evaluated were neutralized with 750 TCID50 / well of pseudovirus particles (transfected with a luciferase reporter gene) at 37°C, and then 2 x 10n cells were seeded. 4 Huh-7 cells were cultured in each well at 37°C in a 5% CO2 incubator for 20-28 hours. After 28-28 hours, 100 μL of luciferase assay reagent (G7940, Promega) was added to each well of the cell plate, and the reaction was carried out in the dark for 2 minutes. The fluorescence was then read using a fluorescence detector, and the neutralizing activity of the antibody against the virus was evaluated based on the fluorescence signal intensity. The results are shown below. Figure 16 As shown. Figure 16 The results showed that the wtIgG1 forms of Corn-01 and Corn-05 exhibited good dose-dependent virus neutralizing activity.

[0225] The neutralizing activities of Corn-01 and Corn-05 LALA mutants (Fc-L234A, L235A) after ADE effects were eliminated were detected to assess the difference in neutralizing activity between wtIgG1 and LALA mutants (L234A, L235A). The results are as follows: Figure 17 and Figure 18 As shown.

[0226] Figure 17 and Figure 18 The results showed that, compared with Corn-01 and Corn-05, the neutralizing activity of the Fc segment LALA mutants Corn-01-LALA and Corn-05-LALA was not significantly altered. Therefore, the point mutation modification of the Fc segment of the anti-SARS-CoV-2 neutralizing antibody, as described above, can preserve its viral neutralizing activity.

[0227] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims. sequence list <110> Maiwei (Shanghai) Biotechnology Co., Ltd. <120> A fully human monoclonal antibody and its application <141> 2020-10-22 <160> 49 <170> SIPOSequenceListing 1.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <400> 1 Gly Gly Val Phe Ser Ser Phe 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <400> 2 Ile Pro Val Leu Gly Ile 1 5 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <400> 3 Asp Arg Phe Val Glu Pro Ala Thr Asp Ala Tyr Phe Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <400> 4 Arg Ala Ser Gln Ser Gly Ser Ser Asn Leu Ala 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <400> 5 Gly Ala Ser Thr Arg Ala Thr 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <400> 6 Gln Gln Tyr Ser Asn Trp Leu Thr 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <400> 7 Gly Phe Thr Phe Ser Ser Tyr 1 5 <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <400> 8 Ser Ser Thr Ser Ser Phe 1 5 <210> 9 <211> 14 <212> PRT <213> Artificial Sequence <400> 9 Glu Val His Val Asp Thr Ala Met Asp Ala Tyr Phe Asp Tyr 1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <400> 10 Arg Ala Ser Gln Thr Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <400> 11 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <400> 12 Gln Gln Ser Tyr Ser Asn Pro Pro Leu Thr 1 5 10 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <400> 13 Gly Ile Thr Val Ser Lys Asn 1 5 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <400> 14 Tyr Ser Ala Gly Ser Thr 1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <400> 15 Gly Tyr Gly Asp Tyr Tyr Phe Asp Tyr 1 5 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <400> 16 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <400> 17 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <400> 18 Gln Gln Ala Asn Ser Phe Leu 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <400> 19 Gly Leu Thr Val Ser Ser Asn 1 5 <210> 20 <211> 6 <212> PRT <213> Artificial Sequence <400> 20 Tyr Ser Gly Gly Ser Thr 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial Sequence <400> twenty one Asp Val Ala Glu Ala Phe Asp Ile 1 5 <210> twenty two <211> 11 <212> PRT <213> Artificial Sequence <400> twenty two Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala 1 5 10 <210> twenty three <211> 7 <212> PRT <213> Artificial Sequence <400> twenty three Ala Ala Ser Thr Leu Gln Ser 1 5 <210> twenty four <211> 11 <212> PRT <213> Artificial Sequence <400> twenty four Gln Gln Ile Asn Ser Tyr Pro Pro Val Asn Thr 1 5 10 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <400> 25 Gly Gly Thr Phe Ser Ser Tyr 1 5 <210> 26 <211> 6 <212> PRT <213> Artificial Sequence <400> 26 Ile Pro Ile Phe Gly Ser 1 5 <210> 27 <211> 16 <212> PRT <213> Artificial Sequence <400> 27 Ser Pro Leu Gly Gly Gly Ser Gly Tyr Ser Val Ser Trp Phe Asp Pro 1 5 10 15 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <400> 28 Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 10 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <400> 29 Gly Ala Ser Thr Arg Ala Thr 1 5 <210> 30 <211> 10 <212> PRT <213> Artificial Sequence <400> 30 Gln Gln Tyr Ser Asn Trp Pro Pro Trp Thr 1 5 10 <210> 31 <211> 7 <212> PRT <213> Artificial Sequence <400> 31 Gly Phe Thr Phe Ser Ser Tyr 1 5 <210> 32 <211> 6 <212> PRT <213> Artificial Sequence <400> 32 Ser Gly Ser Gly Gly Ser 1 5 <210> 33 <211> 18 <212> PRT <213> Artificial Sequence <400> 33 Gly Tyr Thr Tyr Asp Ser Ser Gly Tyr Tyr Phe Arg Glu Asn Ala Phe 1 5 10 15 Asp Ile <210> 34 <211> 11 <212> PRT <213> Artificial Sequence <400> 34 Arg Ala Ser Gln Gly Ile Ser Asn Tyr Leu Ala 1 5 10 <210> 35 <211> 7 <212> PRT <213> Artificial Sequence <400> 35 Ala Ala Ser Thr Leu Gln Ser 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <400> 36 Leu Gln His Asn Ser Tyr Pro Tyr Thr 1 5 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <400> 37 Gly Phe Thr Phe Ser Ser Tyr 1 5 <210> 38 <211> 6 <212> PRT <213> Artificial Sequence <400> 38 Lys Gln Asp Ala Ser Glu 1 5 <210> 39 <211> 11 <212> PRT <213> Artificial Sequence <400> 39 Asp Leu Gly Ile Leu Trp Phe Gly Asp Tyr Pro 1 5 10 <210> 40 <211> 11 <212> PRT <213> Artificial Sequence <400> 40 Arg Ala Ser Gln Gly Ile Ser Asn Ser Leu Ala 1 5 10 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <400> 41 Ala Ala Ser Thr Leu Glu Ser 1 5 <210> 42 <211> 9 <212> PRT <213> Artificial Sequence <400> 42 Gln Gln Phe Tyr Ser Thr Pro Arg Thr 1 5 <210> 43 <211> 330 <212> PRT <213> Artificial Sequence <400> 43 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> 44 <211> 330 <212> PRT <213> Artificial Sequence <400> 44 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> 45 <211> 249 <212> PRT <213> Artificial Sequence <400> 45 Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala 1 5 10 15 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 20 25 30 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 35 40 45 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 50 55 60 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 65 70 75 80 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 85 90 95 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 100 105 110 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 115 120 125 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 130 135 140 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 145 150 155 160 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 165 170 175 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 180 185 190 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 195 200 205 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 210 215 220 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala 225 230 235 240 Ser Gly Ser His His His His His His 245 <210> 46 <211> 468 <212> PRT <213> Artificial Sequence <400> 46 Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala 1 5 10 15 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 20 25 30 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 35 40 45 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 50 55 60 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 65 70 75 80 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 85 90 95 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 100 105 110 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 115 120 125 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 130 135 140 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 145 150 155 160 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 165 170 175 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 180 185 190 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 195 200 205 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 210 215 220 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala 225 230 235 240 Ser Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro 245 250 255 Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu 260 265 270 Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser 275 280 285 Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu 290 295 300 Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr 305 310 315 320 Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln 355 360 365 Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val 370 375 380 Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val 385 390 395 400 Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln 405 410 415 Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 420 425 430 Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 435 440 445 Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 450 455 460 Ser Pro Gly Lys 465 <210> 47 <211> 695 <212> PRT <213> Artificial Sequence <400> 47 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ala Ser Gly 675 680 685 Ser His His His His His His 690 695 <210> 48 <211> 914 <212> PRT <213> Artificial Sequence <400> 48 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ala Ser Val 675 680 685 Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val 690 695 700 Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile 705 710 715 720 Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp 725 730 735 Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His 740 745 750 Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg 755 760 765 Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys 770 775 780 Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu 785 790 795 800 Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr 805 810 815 Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu 820 825 830 Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp 835 840 845 Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile 850 855 860 Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln 865 870 875 880 Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His 885 890 895 Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro 900 905 910 Gly Lys <210> 49 <211> 649 <212> PRT <213> Artificial Sequence <400> 49 Met Ser Ser Ser Ser Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala 1 5 10 15 Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe 20 25 30 Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp 35 40 45 Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn 50 55 60 Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala 65 70 75 80 Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln 85 90 95 Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys 100 105 110 Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser 115 120 125 Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu 130 135 140 Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu 145 150 155 160 Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu 165 170 175 Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg 180 185 190 Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu 195 200 205 Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu 210 215 220 Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu 225 230 235 240 His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile 245 250 255 Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly 260 265 270 Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys 275 280 285 Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala 290 295 300 Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu 305 310 315 320 Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro 325 330 335 Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly 340 345 350 Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp 355 360 365 Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala 370 375 380 Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 400 His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Ala 405 410 415 Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 420 425 430 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 435 440 445 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 450 455 460 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 465 470 475 480 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 485 490 495 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 500 505 510 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 515 520 525 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 530 535 540 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 545 550 555 560 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 565 570 575 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 580 585 590 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 595 600 605 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 610 615 620 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 625 630 635 640 Lys Ser Leu Ser Leu Ser Pro Gly Lys 645

Claims

1. A SARS-CoV-2 S protein-ACEII receptor binding blocking monoclonal antibody or antigen binding fragment thereof, characterized in that: The monoclonal antibody or its antigen-binding fragment has HCDR1 as shown in SEQ ID NO:25, HCDR2 as shown in SEQ ID NO:26, HCDR3 as shown in SEQ ID NO:27, LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:29, and LCDR3 as shown in SEQ ID NO:30, and the heavy chain constant region of the monoclonal antibody or its antigen-binding fragment has the sequence shown in SEQ ID NO:

44.

2. A polynucleotide encoding the monoclonal antibody of claim 1 or an antigen-binding fragment thereof.

3. A nucleic acid construct comprising the polynucleotide of claim 2.

4. A host cell comprising the polynucleotide of claim 2 or the nucleic acid construct of claim 3.

5. A composition comprising the monoclonal antibody of claim 1 or an antigen-binding fragment thereof, and optionally a pharmaceutically acceptable carrier.

6. The use of the monoclonal antibody of claim 1 or its antigen-binding fragment, the polynucleotide of claim 2, the nucleic acid construct of claim 3, the host cell of claim 4, and the composition of claim 5 in the preparation of a drug for the prevention or treatment of SARS-CoV-2 infection.

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