WO2022068847A1 - 一种治疗或预防新型冠状病毒SARS-CoV-2引起的疾病的方法 - Google Patents

一种治疗或预防新型冠状病毒SARS-CoV-2引起的疾病的方法 Download PDF

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WO2022068847A1
WO2022068847A1 PCT/CN2021/121556 CN2021121556W WO2022068847A1 WO 2022068847 A1 WO2022068847 A1 WO 2022068847A1 CN 2021121556 W CN2021121556 W CN 2021121556W WO 2022068847 A1 WO2022068847 A1 WO 2022068847A1
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antibody
antigen
binding fragment
cov
seq
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PCT/CN2021/121556
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English (en)
French (fr)
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甘天翊
刘平兰
孙敏
宋洪涛
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山东博安生物技术股份有限公司
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Priority claimed from PCT/CN2021/098077 external-priority patent/WO2021244601A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates to a method for treating or preventing diseases caused by the novel coronavirus SARS-CoV-2, comprising administering to a subject in need thereof an effective amount of an antibody or antigen that binds to the S protein of the novel coronavirus SARS-CoV-2 Combine fragments.
  • SARS-CoV-2 virus Diseases caused by the SARS-CoV-2 virus include COVID-19 pneumonia and other related complications.
  • the SARS-CoV-2 virus is the original strain of the new coronavirus (Wuhan-Hu-1 strain) first discovered in 2019 and the subsequent The collective term for new coronavirus mutant strains (B.1.1.7 strain, B.1.351 strain, p.1 strain, B.1.617.2 strain and C.37 strain, etc.) Public health security has posed a major threat. At present, there are no vaccines or specific antiviral drugs for the prevention and treatment of new coronavirus pneumonia.
  • the successful development and marketing of neutralizing antibodies based on antibody technology is particularly important for the prevention and treatment of COVID-19 pneumonia and other related complications.
  • Antibody molecules can kill and remove virus particles and infected cells by blocking the binding of virus particles to their receptors, activating immune cells such as macrophages, NK cells, and complement and other mechanisms.
  • the SARS-CoV-2 virus belongs to the genus ⁇ , has an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140 nm.
  • the main structural proteins encoded by its genome are: surface spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N).
  • S surface spike protein
  • M membrane protein
  • E envelope protein
  • N nucleocapsid protein
  • the spike protein is expressed on the viral envelope, which can promote the binding of the virus to the receptor on the surface of the host cell, mediate the fusion of the viral antigen protein and the cell membrane, and is an important pathogenic factor.
  • the main clinical manifestations of COVID-19 patients are fever, dry cough and fatigue.
  • the present invention provides the application of an antibody or antigen-binding fragment that binds to the S protein of the new coronavirus SARS-CoV-2 in the preparation of a medicine for treating or preventing diseases caused by the SARS-CoV-2 virus.
  • the use of an antibody or antigen-binding fragment of the S protein of CoV-2 in the treatment or prevention of diseases caused by the SARS-CoV-2 virus, or an antibody or antigen that utilizes an antibody or antigen that binds to the S protein of the novel coronavirus SARS-CoV-2 A method for treating or preventing a disease caused by a SARS-CoV-2 virus with a binding fragment, comprising administering to a subject in need thereof the antibody or antigen-binding fragment that binds to the S protein of the novel coronavirus SARS-CoV-2, or administering
  • the pharmaceutical composition containing the antibody or antigen-binding fragment can block the cytopathic effect caused by the SARS-CoV-2 virus or neutralize the SARS-CoV-2 virus
  • the main structural proteins encoded by the SARS-CoV-2 virus genome are: surface spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N), wherein the spike protein expresses On the viral envelope, it can promote the binding of the virus to the host cell surface receptor, mediate the fusion of the viral antigen protein and the cell membrane, and is an important pathogenic factor.
  • S surface spike protein
  • M membrane protein
  • E envelope protein
  • N nucleocapsid protein
  • the main clinical manifestations of COVID-19 patients are fever, dry cough and fatigue. A small number of patients have symptoms such as nasal congestion, runny nose, sore throat, myalgia, and diarrhea.
  • the diseases caused by the SARS-CoV-2 virus include COVID-19 pneumonia and other related complications.
  • SARS-CoV-2 virus is the collective term for the original strain of the new coronavirus first discovered in 2019 and the subsequent mutant strains of the new coronavirus.
  • the SARS-CoV-2 virus includes Wuhan-Hu-1 strain, B.1.1.7 strain (also known as Alpha strain), B.1.351 strain (also known as Beta) strain), p.1 strain (also known as Gamma strain), B.1.617.2 strain (also known as Delta strain) and C.37 strain (also known as Ram strain) one or more of the Lambda strain).
  • the antibody or antigen-binding fragment can block the cytopathic or cytopathic effects caused by the SARS-CoV-2 virus by binding to the S protein on the new coronavirus (ie, SARS-CoV-2, also known as 2019-nCoV). and SARS-CoV-2 virus.
  • SARS-CoV-2 also known as 2019-nCoV
  • SARS-CoV-2 virus a spike protein on the surface of the SARS-CoV-2 virus, and the S protein contains two subunits S1 and S2.
  • the fact that the antibody can bind to the S protein on the SARS-CoV-2 virus refers to one or more of the S1 and S2 subunits of the S protein, or the RBD protein that binds to the S1 subunit.
  • the antibody or antigen-binding fragment thereof binds to residues A475, E484, G485, N487, Y489, Q493, S494, Y449, Y453, L455, F456, F486 and F490 of the RBD of the SARS-CoV-2 virus.
  • the antibody or antigen-binding fragment can block the pathological changes of ACE2-expressing cells caused by SARS-CoV-2 virus, or block the infection of ACE2-expressing cells by SARS-CoV-2 virus, invasion, etc.
  • the cells include cells that naturally express ACE2 or cells that artificially express ACE2.
  • the cells are mammalian cells. Further, the mammals include humans, and non-human animals such as mice or monkeys.
  • the COVID-19 treatment or prevention regimen of the present invention comprises administering to a subject an effective amount of the antibody or antigen-binding fragment thereof, which may be, for example, a fully human monoclonal neutralizing antibody; or administering to the subject an effective amount of the antibody or antigen-binding fragment thereof.
  • a pharmaceutical composition comprising an amount of the neutralizing antibody or antigen-binding fragment thereof.
  • the effective amount is, for example, an effective amount of an antibody or antigen-binding fragment that satisfies the effect of treating or preventing COVID-19.
  • about 30 mg to about 2400 mg of said antibody or antigen-binding fragment is administered to a subject in need thereof. Further, it can be about 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg of the antibody or antigen-binding fragment, preferably, about 1200 mg or about 2400 mg of the antibody or antigen-binding fragment.
  • the dose of the antibody or antigen-binding fragment is the dose for humans, according to the "Guidelines for Estimation of the Maximum Recommended Initial Dose of Drugs for the First Clinical Trial of Healthy Adult Volunteers" issued by the Center for Drug Evaluation of the State Drug Administration in 20120215, based on animal experiments
  • the therapeutically or prophylactically effective dose is calculated and taken in conjunction with the maximum tolerated dose to determine the upper limit of the dose range for humans. This is understandable to those skilled in the art.
  • a pharmaceutical composition comprising from about 30 mg to about 2400 mg of said antibody or antigen-binding fragment.
  • it can be a pharmaceutical composition containing about 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg of the antibody or antigen-binding fragment.
  • the pharmaceutical composition is a unit formulation containing between 30 mg and 2400 mg (eg, 30 mg, about 150 mg, about 600 mg, about 1200 mg, or about 2400 mg) of the antibody or antigen-binding fragment thereof; More preferably, the pharmaceutical composition is an injection preparation.
  • the present invention before administering the antibody or antigen-binding fragment to the subject, or administering the pharmaceutical composition containing the antibody or antigen-binding fragment thereof, it further comprises detecting the subject
  • the steps of determining the type of novel coronavirus infected by the test subject can be performed using conventional methods in the art.
  • the subject is a subject infected with SARS-CoV-2 virus, an asymptomatic subject who is in contact with a subject infected with SARS-CoV-2 virus, a healthy subject or other subjects who are not suitable for vaccination (for example, subjects who are not suitable for vaccination due to various underlying diseases, chronic diseases or other reasons); preferably, the subjects infected with SARS-CoV-2 virus include asymptomatic subjects with SARS-CoV-2 virus infection or COVID-19 patients. More preferably, the COVID-19 patient is a mild, moderate, severe or critical COVID-19 patient.
  • the subjects infected with SARS-CoV-2 virus are mild and moderate COVID-19 patients, that is, the onset time is less than or equal to 7 days and within 72 hours according to the NIH (National Institutes of Health). Patients diagnosed with mild and moderate novel coronavirus pneumonia (COVID-19) according to the treatment guidelines (COVID-19) issued by the National Institutes of Health in April 2020.
  • NIH National Institutes of Health
  • the administered dose of the antibody or antigen-binding fragment for the treatment of the mild and moderate COVID-19 patients is about 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg; preferably , about 1200mg or about 2400mg.
  • the subject infected with SARS-CoV-2 virus is a patient with mild and common novel coronavirus pneumonia, that is, the onset time is less than or equal to 7 days and within 72 hours according to the "Diagnosis and Treatment of Novel Coronavirus Pneumonia" Patients with mild and common new type of coronavirus pneumonia diagnosed with the "Protocol (Trial Version 8 Revised Edition)".
  • the administered dose of the antibody or antigen-binding fragment for treating the mild and common COVID-19 patients is about 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg; preferably, About 1200 mg or about 2400 mg.
  • the subject infected with the SARS-CoV-2 virus is a severe or critical type of novel coronavirus pneumonia patient, that is, within 72 hours of the onset time, according to the "New Coronary Virus Pneumonia Diagnosis and Treatment Scheme (Trial No. 1)" Patients with severe and critical new coronavirus pneumonia diagnosed with
  • the administered dose of the antibody or antigen-binding fragment for treating the severe and critical COVID-19 patients is about 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg; preferably, About 1200 mg or about 2400 mg.
  • the subjects infected with SARS-CoV-2 virus are severe and critical COVID-19 patients, that is, laboratory tests (such as RT-PCR tests) within 72 hours of the onset time confirm that they are infected with 2019- nCoV, patients with severe and critically ill coronavirus pneumonia diagnosed according to the treatment guidelines (COVID-19) issued by the NIH in April 2020.
  • the dose of LY-CovMab to treat the severe and critical COVID-19 patients is about 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg; preferably, about 1200 mg or about 2400 mg.
  • the antibody or antigen-binding fragment is used to prevent COVID-19, and the subject of administration is an asymptomatic subject who has been in contact with a patient diagnosed with SARS-CoV-2 infection, or a healthy subject tester.
  • the dose of LY-CovMab used to prevent COVID-19 using the antibody or antigen-binding fragment is about 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg; preferably, about 1200 mg or about 2400mg.
  • the antibody or antigen-binding fragment thereof comprises three light chain complementarity determining regions and/or three heavy chain complementarity determining regions, wherein
  • the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and LCDR3 shown in SEQ ID NO:8, and/or
  • the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10 and HCDR3 shown in SEQ ID NO:11;
  • the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO: 12, LCDR2 shown in SEQ ID NO: 13 and LCDR3 shown in SEQ ID NO: 14, and/or
  • the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10 and HCDR3 shown in SEQ ID NO:11;
  • the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:15, and/or
  • the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:16 and HCDR3 shown in SEQ ID NO:17; or
  • the three light chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7 and LCDR3 shown in SEQ ID NO:21, and/or
  • the three heavy chain complementarity determining regions of the antibody or antigen-binding fragment thereof comprise HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10 and HCDR3 shown in SEQ ID NO:11.
  • the antibody or its antigen-binding fragment comprises the light chain variable region shown in SEQ ID NO:1, and/or the heavy chain variable region shown in SEQ ID NO:2;
  • the antibody or antigen-binding fragment thereof comprises the light chain variable region shown in SEQ ID NO:3, and/or the heavy chain variable region shown in SEQ ID NO:2;
  • the antibody or antigen-binding fragment thereof comprises the light chain variable region shown in SEQ ID NO:4, and/or the heavy chain variable region shown in SEQ ID NO:5; or
  • the antibody or antigen-binding fragment thereof comprises the light chain variable region shown in SEQ ID NO:20, and/or the heavy chain variable region shown in SEQ ID NO:2.
  • the antibody comprises the heavy chain constant region shown in SEQ ID NO:18, or the light chain constant region shown in SEQ ID NO:19.
  • the above-mentioned antibody or antigen-binding fragment thereof with the above-mentioned sequence is provided.
  • the CA521 antibody is in B.1.617.2 strain (also known as Delta strain) and Use in the treatment and prevention of diseases caused by one or more of the C.37 strains (also known as Lambda strains).
  • the use comprises administering to a subject in need thereof a pharmaceutical composition containing from about 30 mg to about 2400 mg of the antibody or antigen-binding fragment.
  • it can be a pharmaceutical composition containing about 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg, preferably, about 1200 mg or about 2400 mg of the antibody or antigen-binding fragment.
  • the pharmaceutical composition is a unit formulation containing between 30 mg and 2400 mg (eg 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg, preferably about 1200 mg or about 2400 mg) of all The antibody or its antigen-binding fragment; more preferably, the pharmaceutical composition is an injection preparation.
  • an antibody or antigen-binding fragment thereof with the above sequence is provided, further preferably, the CA521 antibody is in Wuhan-Hu-1 strain, B.1.1.7 strain (also known as Alpha ( Alpha strain), B.1.351 strain (also known as Beta strain), p.1 strain (also known as Gamma strain), B.1.617.2 strain (also known as Del Use in the treatment and prevention of diseases caused by one or more of the Delta strain) and the C.37 strain (also known as the Lambda strain).
  • the use comprises administering to a subject in need thereof a pharmaceutical composition containing from about 30 mg to about 2400 mg of the antibody or antigen-binding fragment.
  • the pharmaceutical composition can be a pharmaceutical composition containing about 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg, preferably, about 1200 mg or about 2400 mg of the antibody or antigen-binding fragment.
  • the pharmaceutical composition is a unit formulation containing between 30 mg and 2400 mg (eg 30 mg, about 150 mg, about 600 mg, about 1200 mg or about 2400 mg, preferably about 1200 mg or about 2400 mg) of all The antibody or its antigen-binding fragment; more preferably, the pharmaceutical composition is an injection preparation.
  • the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof, preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, preferably, the pharmaceutically acceptable carrier Carriers include one or more of the following: pharmaceutically acceptable solvents, surfactants, osmotic pressure regulators, pH regulators, dispersing agents, additives or plasticizers, and the like.
  • the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof and a buffer.
  • the buffer includes one or more of trehalose and polysorbate 80.
  • the pH of the pharmaceutical composition is 5.5-6.5.
  • the buffer also includes one or more of histidine hydrochloride and histidine. Further, based on the total volume of the pharmaceutical composition, the molar ratio of histidine hydrochloride and histidine is 10.5:9.5.
  • the buffer includes one or more of histidine hydrochloride, histidine, trehalose, and polysorbate 80.
  • the pharmaceutical composition comprises 0.04-0.1 g/mL trehalose, 0.0001-0.0003 g/mL polysorbate 80, and 10-30 mg/mL of the antibody or antigen-binding fragments thereof.
  • the pharmaceutical composition comprises 10.5 mM histidine hydrochloride, 9.5 mM histidine, 0.08 g/mL trehalose, 0.0002 g/mL polysorbate 80, and 20 ⁇ 2 mg/mL of the antibody or antigen-binding fragment thereof.
  • the pharmaceutical composition also includes water.
  • the pharmaceutical composition comprises 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002g/mL polysorbate 80, and 20 ⁇ 2 mg/mL of the antibody or antigen-binding fragment thereof. Further, the pharmaceutical composition also includes water.
  • the preparation method of the pharmaceutical composition includes 1) preparation of a mixture of auxiliary materials, which includes, for example, 10.5 mM histidine hydrochloride, 9.5 mM histidine, 0.08 g/mL seaweed sugar, 0.0002g/mL polysorbate 80; 2) adding the antibody or its antigen-binding fragment to the adjuvant mixture to obtain the pharmaceutical composition, and controlling the final obtained pharmaceutical composition as described in
  • the concentration of the antibody or antigen-binding fragment thereof is, for example, 20 ⁇ 2 mg/mL.
  • the antibody or its antigen-binding fragment also includes a virus-removing filtration process before being added to the adjuvant mixture, and a concentration process (for example, ensuring that the antibody protein concentration is above 27 mg/mL, which is in the art.
  • Technicians can first prepare the stock solution of excipients as needed, and then use dilution buffer (for example, 10.5mM His-HCl, 9.5mM His, pH5.80-6.20) to dilute each component in the antibody protein and excipient stock solution to the final setting. concentration).
  • dilution buffer for example, 10.5mM His-HCl, 9.5mM His, pH5.80-6.20
  • a sterile filtration process is also included.
  • Those skilled in the art can use conventional virus removal filtration, concentration, and sterilization filtration means to realize the above process, which is achievable for those skilled in the art.
  • trehalose acts as a stabilizer and adjusts osmotic pressure
  • polysorbate 80 as a surfactant, is also a stabilizer in biological product injection
  • histidine and histidine hydrochloride are pH buffer.
  • the dosage form of the pharmaceutical composition is an injection preparation.
  • the pharmaceutical composition can be administered by intravenous injection (eg, intravenous drip) or by intraperitoneal injection.
  • the pharmaceutical composition is a unit formulation (or a formulation in a single dosage form).
  • the unit preparation is a preparation that satisfies a single administration dose.
  • the pharmaceutical composition of the present invention is an injection, and its administration mode is intravenous drip.
  • the scheme of the present invention includes diluting the pharmaceutical composition containing an effective amount of the antibody or antigen-binding fragment and then intravenously instilling it into the subject; preferably, the pharmaceutical composition is diluted with physiological saline ; more preferably, the pharmaceutical composition is diluted to 100-250mL using physiological saline, and administered to the subject within 30-120 minutes; more preferably, the pharmaceutical composition is diluted to 250mL using physiological saline, and Administered to the subject within 90-120 minutes; more preferably, the pharmaceutical composition is diluted to 100 mL with physiological saline and administered to the subject within 30 minutes; more preferably, the physiological saline is 0.9% Sodium chloride solution.
  • the pharmaceutical composition containing an effective amount of the antibody or antigen-binding fragment is diluted and administered to the subject by intravenous drip; preferably, the pharmaceutical composition is diluted with a 0.9% sodium chloride solution; More preferably, the pharmaceutical composition is diluted to 100-250 mL with 0.9% sodium chloride solution, and administered to the subject within 30-120 minutes; more preferably, the The pharmaceutical composition is diluted to 250mL and administered to the subject within 90-120 minutes; more preferably, the pharmaceutical composition is diluted to 100mL with a 0.9% sodium chloride solution and administered to the subject within 30 minutes By.
  • an antibody having the above sequence or an antigen-binding fragment thereof in combination with another antibody or antigen-binding fragment in the treatment or prevention of diseases caused by SARS-CoV-2 virus said The application includes administering to a subject in need thereof the antibody or antigen-binding fragment thereof having the above-mentioned sequence, and another antibody or antigen-binding fragment that binds to the novel coronavirus SARS-CoV-2; preferably, the additional One antibody or antigen-binding fragment binds to the S protein of the novel coronavirus SARS-CoV-2; more preferably, another antibody or antigen-binding fragment binds to the S protein of the antibody or antigen-binding fragment thereof with the above sequence
  • the epitopes do not overlap or partially overlap.
  • the antibody or antigen-binding fragment thereof having the above sequence, and another antibody or antigen-binding fragment that binds to the novel coronavirus SARS-CoV-2 are administered to the subject at the same time or at different times.
  • Table 1-1 shows the names of the above-mentioned SARS-CoV-2 virus strains, where they appear and the main mutations.
  • the neutralizing antibody provided by the present invention can effectively treat or prevent the disease caused by the SARS-CoV-2 virus, block the infection of the subject by the SARS-CoV-2 virus or neutralize the SARS-CoV-2 virus;
  • the pharmaceutical composition containing the antibody or antigen-binding fragment has good stability and can be stored at 2-8° C. for at least 6-24 months.
  • Figure 1 shows the serum titers of 4 kinds of S protein immunized mice
  • Figures 2A-2C show the binding sensitivity of each candidate antibody to the Spike S1+S2 protein of SARS-CoV-2 virus (Wuhan-Hu-1 strain) detected by ELISA;
  • Figures 3A-3B show the binding sensitivity of ELISA detection candidate antibodies to S1 of SARS-CoV-2 virus (Wuhan-Hu-1 strain);
  • Figure 4 shows the binding sensitivity of ELISA detection candidate antibody to S2 of SARS-CoV-2 virus (Wuhan-Hu-1 strain);
  • Figure 5 shows ELISA detection of candidate antibodies blocking the binding of ACE2 to Spike RBD protein of SARS-CoV-2 virus (Wuhan-Hu-1 strain);
  • Figures 6A-6C show flow cytometry detection of antibody binding to CHO-K1-2019-nCoV Spike cells (cells expressing the Spike protein of Wuhan-Hu-1);
  • Figure 7 shows flow cytometry detection of antibody blocking the binding of RBD of SARS-CoV-2 virus (Wuhan-Hu-1 strain) to 293T-hACE2 cells;
  • Figure 8 shows flow cytometry detection of antibody blocking hACE2 of SARS-CoV-2 virus (Wuhan-Hu-1 strain) and 293F-SARS-CoV-2-Spike cells (Spike expressing Wuhan-Hu-1) protein) binding;
  • Figure 9 shows ELISA detection of antibody binding to human C1q receptor
  • FIGS. 10A-10B show that OctetRED 96 detects the binding of IgG4 wild-type control antibody and candidate antibody CA530 to human CD64, respectively;
  • FIG. 11 shows that CA521 has no apparent ADCC effect on CHO-K1-spike cells
  • FIG. 12A shows that CA521 has no CDC effect on Daudi cells
  • Figure 12B shows that CA521 has no CDC effect on CHO-K1-spike cells
  • Figure 13 shows the ability of candidate antibodies to block infection of cells by a pseudovirus of the SARS-CoV-2 virus (Wuhan-Hu-1 strain);
  • Figure 14 shows the neutralizing activity of CA521 neutralizing antibody against SARS-CoV-2 virus (strain BetaCoV/Beijing/IMEBJ01/2020) in vitro;
  • FIG. 15 shows that CA304, CA413 and CA521 block the infection of Vero cells by a true virus of the SARS-CoV-2 virus (strain BetaCoV/Beijing/IMEBJ01/2020);
  • Figures 16A-16B show viral (a mouse-adapted strain of SARS-CoV-2, MASCp6) loads in the lungs and trachea 3 days after administration of CA304, CA413 or CA521 antibody;
  • Figure 16C shows 3 days after administration of CA521 antibody Virus (the mouse-adapted strain MASCp6 of SARS-CoV-2) in lung and trachea in prophylactic and therapeutic animal models;
  • Figure 17A shows a 3D structural model of S-ECD and CA521 binding of SARS-CoV-2 virus (Wuhan-Hu-1 strain), and Figure 17B shows binding epitope analysis of S-ECD and CA521 binding;
  • Figure 18 shows the binding sites of CA521, ACE2, H014, CB6 and RBD.
  • Figure 19 shows that CA304 blocks the binding of Spike S1+S2 proteins of different strains to ACE2.
  • Figure 20 shows that CA555 blocks the binding of Spike S1+S2 proteins of different strains to ACE2.
  • Figure 21 shows the pseudovirus neutralizing activity of CA304 against different strains.
  • Figure 22 shows the pseudovirus neutralizing activity of CA555 against different strains.
  • the first and second immunizations were both 20 ⁇ g antigen protein; the third and booster doses were increased to 35 ⁇ g antigen protein.
  • the interval between the first immunity and the second immunity is 10 days; the interval between the second immunity and the third immunity is 8 days; the interval between the third immunity and the booster is 7 days; and the interval between the booster and the spleen is 2 days.
  • mice were sacrificed, and the spleen was removed by dissection.
  • the spleen was ground and crushed with a syringe rubber stopper and filtered through a filter.
  • the filtered spleen cells were frozen and prepared.
  • RNA was extracted, cDNA was obtained. : performed as described in the A laboratory manual (eg Chapters 8 and 9).
  • the library capacity data of the constructed library are shown in Table 2.
  • Table 2 The capacity of the phage library constructed by immunizing mice with each S protein
  • Antibody ID source library source mouse panning method CA13 Q03 Q03 Plate Screening CA14 Q03 Q03 Plate Screening CA108 Q03 Q03 Magnetic bead screening CA219 Q06 Q06 Plate Screening CA272 Q07 Q07 Plate Screening CA304 Q01 Q01 Magnetic bead screening CA310 Q01 Q01 Magnetic bead screening CA313 Q14/15 Q14/15 Magnetic bead screening CA413 Q1/2 Q1/2 Plate Screening CA417 Q1/2 Q1/2 Plate Screening CA521 Q2 Q2 Magnetic bead screening CA530 Q2 Q2 Magnetic bead screening CA536 Q1 Q1 Magnetic bead screening CA555 Q1/2 Q1/2 Magnetic bead screening
  • the positive clone CA13 ⁇ 14 ⁇ 108 ⁇ 304 ⁇ 310 ⁇ 413 ⁇ 417 ⁇ 521 ⁇ 530 ⁇ 536 ⁇ 555 ⁇ 219 ⁇ 272 ⁇ 313 was constructed and sequenced.
  • the amino acid sequence of the variable region of each antibody is shown in Table 4: (The CDR region is underlined , the analysis system is the IMGT system.)
  • the antibody variable region gene was amplified by conventional molecular biology technology PCR (2 ⁇ Phanta Max Master Mix manufacturer: Vazyme Item No.: P515-P1-AA Lot No.: 7E351H9), and the antibody heavy chain variable region genes were separated by homologous recombination.
  • the vector pCDNA3.4 (Life Technology) with the nucleic acid sequence of the antibody heavy chain constant region sequence SEQ ID NO: 18 was ligated, and the antibody light chain variable region gene was ligated into the antibody light chain constant region sequence SEQ ID NO: 19.
  • the nucleic acid sequence of the vector pCDNA3.4 The nucleic acid sequence of the vector pCDNA3.4.
  • variable region sequences of the candidate antibodies in the examples of this application are shown in Table 4, and the heavy chain constant region sequence is SEQ ID NO: 18 (containing S228P, F234A and L235A mutations (numbering system is Eu Numbering system), also known as FALA mutation) , the light chain constant region sequence is SEQ ID NO: 19.
  • the sequenced positive clones were extracted with plasmids and co-transfected into HEK293 cells and cultured in a shaker at 37°C ⁇ 8% CO 2 ⁇ 125rpm. After transient expression for 7 days, the supernatant was purified by Protein A affinity chromatography to obtain antibodies, which were purified by protein A affinity chromatography. The UV280 was combined with the theoretical extinction coefficient to determine the antibody concentration.
  • Dilute Spike RBD (Yiqiao Shenzhou, 40592-V05H) 0.4 ⁇ g/mL with pH 9.6 CBS, coat the microtiter plate, 100 ⁇ L/well, incubate at 4°C overnight; wash the plate and block with 3% nonfat milk powder. After washing the plate, 50 ⁇ l of PBST-diluted antibody or ACE2 control protein was added to each well, and the final concentration of antibody or ACE2 control protein (Novoprotein, C05Y) was (4 ⁇ g/mL, 1 ⁇ g/mL, 0.25 ⁇ g/mL, 0.0625 ⁇ g/mL).
  • biotin-labeled ACE2 protein (final concentration 0.04 ⁇ g/mL) was added, 50 ⁇ L/well, and incubated at 37°C for 1 h; after washing the plate, STREP/HRP diluted in PBST was added, 100 ⁇ L/well, and incubated at 37°C for 1 h. After washing the plate, 100 ⁇ L of TMB was added to each well to develop color, and 10 min later, 50 ⁇ L of 2M H 2 SO 4 was added to each well to stop the color development, and the OD450 was read with a microplate reader. Antibodies were present in 0.01M PBS buffer pH 7.4 prior to dilution. The results showed that CA304, CA310, CA413, CA417, CA521, CA530, CA536 and CA555 could all block the binding of Spike RBD protein to ACE2 by antibodies as shown in Figure 5 and Table 8.
  • Anti-human IgG antibody was amino-conjugated to CM5 biosensor chip by GE anti-human IgG Fc amino-conjugation kit to obtain approximately 1000 response units (RU).
  • Spike S2 protein (Yiqiao Shenzhou, 40590-V08B) or Spike S1+S2 protein (Yiqiao Shenzhou, 40589-V08B1) was buffered with HBS-EP + 1 ⁇ (GE, BR-1006-69) 2-fold serial dilution, starting at 50 nM, 2-fold dilution with 4 concentration gradients, and set to 0 concentration. Startup 3 times.
  • Antibody 2 ⁇ g/ml, injection time 100 s, flow rate 10 ⁇ L/min; antigen protein: binding for 120 s, flow rate 30 ⁇ L/min, dissociation 600 s; regeneration: regeneration with 3M MgCl 2 buffer for 30 s, flow rate 30 ⁇ L/min.
  • Binding constants (ka) and dissociation constants (kd) were calculated using the 1:1 binding binding model or the Two state reaction binding model (BIAcore Evaluation Software version 3.2), and the equilibrium dissociation constant (KD) was calculated as the ratio kd/ka.
  • Antibodies were present in 0.01M PBS buffer pH 7.4 prior to dilution. The results show that each candidate antibody can bind to the corresponding antigen, and the affinity data are shown in Table 9.
  • the antibody was diluted to 4 ⁇ g/mL with PBST, and the protein Spike RBD protein (Beijing Yiqiao Shenzhou, catalog number: 40592-V05H) was sequentially diluted with PBST to 25nM, 12.5nM, 6.25nM, 3.13nM, and PBST was used as a blank control.
  • the antibody was bound to a FAB2G sensor (Fortebio, catalog number: 18-5125 ) with a binding height of 1 nm, followed by binding and dissociation detection, binding time 200 s, dissociation time 400 s, and KD, 1:1 mode was used to calculate KD, Kon, Koff.
  • the binding kinetics of antibodies to trimeric proteins were detected using an OctetRED 96 instrument.
  • the antibody was diluted to 4 ⁇ g/mL with PBST, and SCB-2019 was sequentially diluted to 25nM, 12.5nM, 6.25nM, 3.125nM, and 1.5625nM with PBST, with PBST as blank control.
  • the antibody was loaded on the FAB2G sensor (Fortebio, catalog number: 18-5125), the binding height was 1 nm, and then the binding and dissociation detection was performed, the binding time was 200 s, and the dissociation time was 400 s.
  • the buffer system of CA304 and CA413 is: 10mM CH 3 COONa ⁇ 3H 2 O, 30mM NaCl, 0.03% Tween-20, 5% sucrose, 20mg/mL of the antibody, pH 6.0;
  • the buffer system of CA521 was 10.5 mM histidine hydrochloride, 9.5 mM histidine, 0.08 g/mL trehalose, 0.0002 g/mL polysorbate 80, water, and 21.5 mg/mL of the antibody, pH 6.0.
  • the three antibodies CA304, CA413 and CA521 can bind to CHO-K1 cells overexpressing the Spike protein of Wuhan-Hu-1, and the binding activity is good.
  • Antibody CA521 or Isotype isotype control (an irrelevant antibody with the same constant region and different variable region as CA521) was mixed with 2 ⁇ g/mL hACE2-Biotin, co-incubated with 293F-SARS-CoV-2-Spike (NbBiolab) cells, and then Cells were stained with Streptavidin APC secondary antibody (Invitrogen, 17-4317-82) and analyzed by flow cytometry. The results are shown in Figure 8, CA521 can block the binding of hACE2 to 293F-SARS-CoV-2-Spike with IC50 of 0.86nM, while the Isotype isotype control has no blocking activity.
  • the Isotype isotype controls in the above-mentioned 3.4.1-3.4.3 are identical, and its heavy chain sequence (variable region+constant region) is SEQ ID NO: 22, and the light chain sequence (variable region+constant region) is SEQ ID NO: twenty three.
  • the candidate antibody was diluted to 1 ⁇ g/mL with pH9.6 CBS, coated with an enzyme-labeled plate (Suzhou beaver, catalog number: 40301), and then blocked with 2% BSA for 1 h; the diluted C1q protein was added with PBST (PBS+0.05% Tween20).
  • PBST PBS+0.05% Tween20
  • IgG1 wild-type control antibody its heavy chain sequence (variable region + constant region) is SEQ ID NO: 24, and the light chain sequence (variable region + constant region) is SEQ ID NO: 25.
  • IgG4 wild-type control antibody its heavy chain sequence (variable region + constant region) is SEQ ID NO: 26, and the light chain sequence (variable region + constant region) is SEQ ID NO: 27.
  • OctetRED 96 detects the binding of candidate antibody, IgG4 wild-type control antibody to human CD64:
  • the binding kinetics of the antibody to CD64 protein was detected using the OctetRED 96 instrument.
  • the antibody was diluted to 3 ⁇ g/mL with PBST, and protein S-RBD-Fc (ACROBiosystems, catalog number: FCA-H52H2) was sequentially diluted with PBST to 15.4nM, 7.7nM, 3.85nM, 1.93nM, and PBST was used as a blank control.
  • the antibody was bound to a FAB2G sensor (Fortebio, catalog number: 18-5125), and then binding and dissociation detection was performed, and KD, Kon, and Koff were calculated in 1:1 mode after the detection.
  • Antibodies were present in 0.01M PBS buffer pH 7.4 prior to dilution.
  • the affinity of the IgG4 wild-type control antibody to the human CD64 receptor is 3.41E-09M, while the engineered candidate antibody CA530 has no binding to CD64, which can greatly reduce the ADE of the candidate antibody effect.
  • the IgG4 wild-type control antibody is the same as in 3.5.1.
  • Biacore 8K detects the binding of candidate antibody CA521 and CA521-IgG1 wild-type control antibody to human Fc ⁇ R:
  • a CM5 chip (GE Healthcare, BR-1005-30) was amino coupled with His Capture Kit (GE Healthcare, 28-9950-56). Dilute Fc ⁇ RI (R&D, 1257-Fc) to 0.5 ⁇ g/mL with 1 ⁇ HBS-EP+ buffer, dilute Fc ⁇ RIIA R167 (R&D, 1330-CD/CF), Fc ⁇ RIIA H167 (Acro, CD1-H5223), Fc ⁇ RIIB/C ( R&D, 1875-CD) to 1 ⁇ g/mL and injected onto the chip at a rate of 10 ⁇ L/min with a binding time of 90 s.
  • CA521 and CA521-IgG1 wild-type antibodies were diluted 2-fold with the same buffer from different starting concentrations (see Table 11) into 5 concentration gradients, respectively. Inject and dissociate. The final binding dissociation curve uses the results after subtracting the reference channel and the buffer channel. The experimental data were fitted with a steady-state model to obtain affinity data.
  • the antibody buffer system was 10.5 mM histidine hydrochloride, 9.5 mM histidine, 0.08 g/mL trehalose, 0.0002 g/mL polysorbate 80, water, and 21.5 mg/mL of the antibody, pH 6.0 .
  • CA521-IgG1 wild-type control antibody its heavy chain sequence (variable region+constant region) is SEQ ID NO:28, and its light chain sequence (variable region+constant region) is SEQ ID NO:29.
  • ADCP antibody-dependent phagocytosis
  • CD14 + monocytes were recovered and stimulated cells were induced with 1640 complete medium containing GM-CSF, IFN- ⁇ and LPS.
  • CHO-K1-spike (GenScript, RD00819) cells were stained with CSFE (Invitrogen, 65-0850-85) and resuspended in serum-free 1640 medium; the antibody to be tested was diluted with 1640 serum-free medium to 10 ⁇ g/mL ; Incubate CHO-K1-spike cells, macrophages, and the antibody to be tested in a 96-well cell culture plate in a 37°C 5% CO2 incubator for 2 hours.
  • the buffer system of the antibody before dilution was 10.5 mM histidine hydrochloride, 9.5 mM histidine, 0.08 g/mL trehalose, 0.0002 g/mL polysorbate 80, water, and 21.5 mg/mL of the antibody, pH6.0.
  • the ADCP effect results of different isoforms of antibody CA521 are shown in Table 13.
  • CA521 and Isotype isotype control (different from CA521 variable region, irrelevant antibodies with the same constant region) have no ADCP effect, only about 3%, which is Experimental background.
  • the unmodified wild-type CA521-IgG1 showed a strong phagocytic effect, with a phagocytic rate of 12.74%. It can be seen that the biological functions mediated by the modified CA521Fc end and Fc receptors are inhibited, so that the ADE effect can be effectively eliminated.
  • the Isotype isotype control is the same as the Isotype isotype control in 3.4.1-3.4.3.
  • the CA521-IgG1 wild-type control antibody was the same as in 3.5.3.
  • Table 14 shows the test materials for the detection of candidate antibodies ADCC and CDC
  • CHO-K1-spike cells with high viral spike protein expression were selected as target cells to study the toxic effect of antibody CA521 on target cells.
  • CA521-IgG1 which has ADCC effect on CHO-K1-spike cells, was selected as a positive control to verify the feasibility of the ADCC method
  • Rituxan which has CDC effect on Daudi cells, was selected as a system control to verify the feasibility of the CDC method, and then the antibody CA521 Detection of ADCC and CDC effects in CHO-K1-spike cells.
  • ADCC detection principle Promega company transformed Jurkat-ADCC effector cells to express the high-affinity Fc ⁇ RIIIa (V158) receptor on the cell surface, which can better bind to the Fc end of the antibody; and at the same time Introduce NFAT (a transcription factor expressed in immune cells, which can regulate gene transcription) and luciferase Luciferase gene, so that when ADCC is activated, it can activate the expression of Luciferase through intracellular signal transduction.
  • the substrate in the color solution emits light after the action of the substrate, thereby reflecting the effect intensity of ADCC through the detection of the luminescence intensity.
  • ADCC detection method 1) Target cell preparation: collect CHO-K1-spike cells, count, add ADCC buffer to dilute to 1x 10 6 cells/mL; 2) CA521 antibody sample gradient dilution: take an appropriate amount of sample and add ADCC buffer to gradually dilute 100 ⁇ g/mL, and this concentration is the initial concentration; take 200uL of the initial concentration sample, add it to the 96-well dilution plate, draw 40uL and add it to the 160uL dilution solution, and successively dilute 5-fold gradient for a total of 8 points for subsequent use; 3) Effector cell preparation: Collect Jurkat-ADCC effector cell culture medium in a 50mL centrifuge tube, centrifuge at 1500rpm, remove the supernatant, add ADCC buffer and pipette repeatedly, resuspend cells, count cells, add ADCC buffer and dilute to 2x10 6 Cells/mL, for subsequent use; 4) Add target cells, CA521 antibody and effector cells: Add target cells to a white 96
  • CDC detection principle The antibody binds to the target on the cell, and after binding to the complement, the complement effect is activated, resulting in the killing of the target cell.
  • the number of live cells is detected by the AlarmBlue live cell detection solution, which indirectly indicates the number of cells killed by the CDC effect of the target cells. Therefore, the weaker the detected signal, the less the number of live cells, and the greater the CDC effect. powerful.
  • Target cell preparation collect Daudi cells, CHO-K1-spike cells, count, add ADCC buffer to dilute to 6x 10 5 cells/mL; 2) Sample gradient dilution: take 10uL sample and add appropriate amount of ADCC buffer Dilute to 1mg/mL, after mixing, take out 30uL and add 270uL buffer to prepare 100ug/mL, with this concentration as the initial concentration; take 200uL of the initial concentration sample, add it to the 96-well dilution plate, and add 40uL to the 160uL dilution solution 3)
  • Add antibody, target cells and complement Add target cells to a transparent 96-well plate: 25uL/well, i.e.
  • the CDC detection results of Daudi cells as target cells in Figure 12A show that as the concentration of the positive control Rituxan (ie Grp.1) increases, the signal gradually decreases, indicating that the number of viable cells gradually decreases, indicating that Rituxan has a significant CDC effect on Daudi cells , while the signal value of CA521 (ie sample S20100501, Grp.2) did not change significantly with the increase of sample concentration, indicating that CA521 has no CDC effect on Daudi cells;
  • Figure 12B shows the detection results of samples with CHO-K1-spike as the target cell, the negative control IgG4 (ie Grp.2), with the increase of the concentration, the signal value does not change significantly, and the negative control IgG4 has no effect on CHO-K1-spike cells CDC effect; CA521 signal does not show a concentration-dependent curve with increasing concentration, indicating that it has no CDC effect on CHO-K1-spike cells.
  • the sequence of the negative control IgG4 (heavy chain only) is SEQ ID NO:30.
  • a pseudovirus of the S protein of Wuhan-Hu-1 was packaged using HIV lentiviral vector. After the pseudovirus was incubated with the antibody to be tested, it infected 293T-ACE2 cells, the number of cells per well: 3E4 cells/well, the amount of virus: 50 ⁇ L/well, and finally, the luminescence value of Luciferase was detected by chemiluminescence method RLU, and the antibody to be tested was calculated according to the RLU reading. The pseudovirus suppression rate.
  • the buffer system of the antibody was pH 7.4, 0.01M PBS buffer.
  • CA304, CA310, CA413, CA417, CA521, CA530 and CA555 antibodies could effectively block pseudovirus infection of cells, among which CA304, CA413, CA521, CA530 and CA555 had the strongest blocking ability, at 0.20nM, 0.41 nM, 0.56 nM, 0.11 nM and 0.98 nM ( Figure 13 and Table 15).
  • the positive control is the positive control SARS-CoV/SARS-CoV-2 Spike antibody produced by Beijing Yiqiao Shenzhou, product number: 4015D-D001.
  • Antibody ID IC50(nM) Antibody ID IC50(nM)
  • CA304 0.20 CA521 0.56 CA310 1.28 CA530 0.11 CA413 0.41 CA536 4.87 CA417 1.42 CA555 0.98 positive control 1.58
  • Sample group Each serially diluted antibody was mixed with 100PFU SARS-CoV-2 true virus, incubated at 37 degrees for 1 hour, added to a 24-well plate plated with Vero cell layer, and incubated at 37 degrees for 1 hour.
  • Positive control Mix equal volumes of SARS-CoV-2 true virus and DMEM medium stock solution, incubate at 37°C for 1h, add to a 24-well plate plated with Vero cell layer, and incubate at 37°C for 1h.
  • Negative control DMEM medium stock solution was incubated at 37°C for 1 hour, added to a 24-well plate plated with Vero cell layer, incubated at 37°C for 1 hour, and all experiments were set in duplicate wells.
  • PRNT Plaque Reduction Neutralization Test
  • the CA521 antibody (100mg/5mL) was diluted 3-fold with cell maintenance solution, mixed with an equal volume of SARS-CoV-2 virus (virus strain BetaCoV/Beijing/IMEBJ01/2020), and incubated at 37°C for 1h; the virus- The antibody mixture (200 ⁇ L/well) was added to a 24-well culture plate containing a single layer of dense Vero cells, and incubated at 37°C for 1 h; the virus antibody mixture was discarded, and an appropriate volume of preheated nutrient agar was added to each well, at 37°C, 5%
  • the culture in a CO 2 incubator add an appropriate volume of fixative on the 2nd day after infection, fix at room temperature for 1 h, discard the fixative and nutrient agar cap, and wash once with the fixative; add an appropriate volume of 1.0% crystal violet solution, at room temperature After staining for 1 h, the crystal violet solution was discarded, washed once with fixative solution, the number of plaques was counted
  • the results are shown in Figure 14.
  • the PRNT 90 and PRNT 50 of CA521 at the Vero cell level were 1.36 ⁇ g/mL and 0.11 ⁇ g/mL, corresponding to 9.11 nM and 0.737 nM, respectively, for the 2019-nCoV infection, indicating that the neutralizing antibody has a clear Biological efficacy of protecting cells from novel coronavirus infection.
  • the antibody buffer system was 10.5 mM histidine hydrochloride, 9.5 mM histidine, 0.08 g/mL trehalose, 0.0002 g/mL polysorbate 80, water, and 21.5 mg/mL of the antibody, pH 5.5 -6.5.
  • CA304, CA413 and CA521 all have good neutralizing activities, with IC50s of 0.080, 0.089 and 0.132 ⁇ g/mL, respectively.
  • the viral load in the lung tissue of the Balb/C mice in the control group was 10 10.43 RNA copies/g on the 3rd day after infection.
  • the viral loads were 10 5.35 RNA copy number/g, 10 5.23 RNA copy number/g and 10 5.89 RNA copy number/g, respectively, which were decreased by 10 5.08 , 10 5.2 and 10 4.54 after administration of the three antibody drugs, respectively. It can also effectively reduce the viral load.
  • CA521 is provided in the form of a preparation, and the preparation is a pharmaceutical composition containing CA521 antibody.
  • the pharmaceutical composition includes 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002g/mL polysorbate Ester 80, water, and 21.5 mg/mL of the antibody, pH pH 6.0.
  • CA304 or CA413 is provided in the form of a pharmaceutical composition containing the antibody, and the pharmaceutical composition includes: 10 mM CH 3 COONa ⁇ 3H 2 O, 30 mM NaCl, 0.03% Tween-20, 5% sucrose, 20 mg/ mL of the antibody pH 6.0.
  • mice The protective effect of CA521 in mice was evaluated using the SARS-CoV-2 mouse-adapted strain model established by the P3 laboratory of the Institute of Microbial Epidemiology, Academy of Military Medical Sciences, Academy of Military Sciences.
  • BALB/c mice aged 6-8 weeks were randomly divided into 3 groups: antibody prevention group (LY-CovMab-P, mice were given 20 mg/kg CA521 by intraperitoneal injection 12h before virus infection), antibody treatment group (LY-CovMab-P) CovMab-T, mice were given 20 mg/kg CA521 by intraperitoneal injection 2 h after virus infection) and control group (mice were given the same volume of PBS as the drug 12 h before infection and 2 h after infection).
  • mice BALB/c mice were anesthetized before infection, and 2 ⁇ 10 4 PFU/mL virus (Academy of Military Medical Sciences, MASCp6) was inoculated into mice (30 ⁇ L/mice) by intranasal route, and the mice were intraperitoneally routed before or after infection, respectively.
  • RNA Copies/mL CT*(-0.2899)+14.875, and then calculate the viral RNA load in tissues and organs according to 0.1 g of lung tissue and 0.01 g of trachea in each mL of liquid Amount (RNA copy number/g).
  • the viral load in the lung tissue of the BALB/c mice in the control group was 10 10.22 RNA copies/g on the 3rd day after infection, the prevention group (LY-CovMab-P) and the treatment group (LY-CovMab-P) -CovMab-T) mice on the third day after infection, the viral loads in lung tissue were 10 7.26 RNA copies/g and 10 7.63 RNA copies/g, respectively, which were decreased by 10 2.96 and 10 2.59 times after administration (P ⁇ 0.01).
  • the viral load of mice in the control group was 10 9.16 RNA copies/g on the third day after infection.
  • the viral loads of the mice in the prevention group and the treatment group were 10 7.28 and 10 7.21 RNA copies/g on the third day after infection, respectively, which were decreased by 10 1.88 and 10 1.95 times after administration (P ⁇ 0.01).
  • the above results show that in the SARS-CoV-2 mouse-adapted strain model, 20 mg/kg CA521 can reduce the virus titers in the lung and trachea in both prophylactic and therapeutic models.
  • the CA521 antibody has a clear protective effect against novel coronavirus infection in the BALB/c mouse model, and can effectively reduce the viral load in the lungs and trachea.
  • the safety pharmacology study in rhesus monkeys was carried out with an expanded single-dose toxicity study in rhesus monkeys (see A2020131-T005-01 for details).
  • Rhesus monkeys were given physiological saline or 50, 200, 800 mg/kg of CA521 (in this experiment CA521 was provided in the form of a preparation, the preparation was a pharmaceutical composition containing CA521 antibody, and the pharmaceutical composition included 10.5 mM histidine hydrochloride.
  • the non-invasive physiological signal telemetry system for large animals was used to detect the electrocardiogram and respiratory rate of lead II, and the intelligent non-invasive sphygmomanometer was used to measure the arterial blood pressure.
  • LY-CovMab was administered intravenously in rhesus monkeys for a 4-week toxicity test to observe the nature, degree, dose-effect and time-effect relationship and reversibility of the possible toxic reaction caused by the test product, determine the toxic target organs or tissues, and examine the immunity of the drug.
  • the originality, immunotoxicity and effects on cardiovascular, respiratory system and injection site provide reference for the safety of clinical use.
  • the general condition of the monkeys was observed every day during the administration period and the recovery period, and the body weight and food intake were measured once a week; the body temperature was measured 3 to 4 hours after the second and fifth administrations and at the end of the recovery period, and the second and fifth administrations were performed.
  • IL-6, IL-10) detection anti-LY-CovMab antibody detection was performed once before administration, on the 15th and 29th days of the experiment and on the 27th day of the recovery period; once at the end of the administration and at the end of the recovery period Urine and ophthalmological examination; at the end of administration and recovery period, 6 and 4 monkeys (half male and half male) were selected from each group respectively, and bone marrow was collected and then euthanized.
  • the monkeys in each group of LY-CovMab were collected blood samples from veins before the 1st and 4th administration and 5 minutes, 1, 6, 24, 72, and 168 hours after administration, respectively.
  • test results showed that 50, 200, and 800 mg/kg of LY-CovMab were intravenously injected into rhesus monkeys once a week, and the no-toxicity dose (NOAEL) was 800 mg/kg (after the fourth administration at this dose).
  • NOAEL no-toxicity dose
  • the AUC 0-336h of female and male monkeys were 1710 and 1820h*mg/mL, respectively).
  • 18 rhesus monkeys (half male and half male) were divided into 3 groups, each group of monkeys were injected with 15, 60, 240 mg/kg of CA521 intravenously at 11.94 mL/kg respectively, and before administration and immediately after administration (1min) , 5 minutes, 1, 6 hours, 1(24h), 3(72h), 7(168h), 10(240h), 14(336h), 21(504h), 28(672h), 35(840h), 42 (1008h) and 56 (1344h) days, blood samples were collected, and the concentration of CA521 in serum was determined by ELISA. The standard curve ranged from 78.1 to 10000ng/mL, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin 6.4.
  • CA521 is provided in the form of injection, and the preparation is a pharmaceutical composition containing CA521 antibody.
  • the pharmaceutical composition includes 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002g/mL polysorbate Ester 80, water, and 21.5 mg/mL of the antibody, pH 5.5-6.5.
  • the peak concentration Cmax is 1300 ⁇ 86.9 ⁇ g/mL
  • the terminal elimination half-life t 1/2 is 275 ⁇ 95.9h
  • the exposure AUC 0- ⁇ is 354000 ⁇ 114000h* ⁇ g /mL
  • the clearance rate of Cl was 0.188 ⁇ 0.0709mL/h/kg
  • the steady-state apparent volume of distribution V ss was 71.4 ⁇ 7.76mL/kg
  • the residence time MRT 0- ⁇ was 417 ⁇ 129h.
  • Anti-drug antibodies were detected before administration and 14, 28, 42, and 56 days after administration by ELISA.
  • the ADA (anti-drug antibody) results of all animals before administration and in each group were negative; 0 (0/6), 1 (1/6), and 0 (0/6) animals were found in each group 14 days after administration. Animal ADA results were positive, 1 (1/6), 1 (1/6), 0 (0/6) animals in each group were positive for ADA 28 and 42 days after the end of administration, respectively. There were 2 (2/6), 1 (1/6), and 1 (1/6) animals in each group that were positive for ADA on each day.
  • CA521 (in this experiment CA521 is provided in the form of a preparation, the preparation is a pharmaceutical composition containing CA521 antibody, the pharmaceutical composition includes 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002 g/mL polysorbate 80, water, and 21.5 mg/mL of the antibody, pH 5.5-6.5) produced no ADE effect or very low risk.
  • CA521 has no cross-reactivity with normal human tissue and rhesus monkey tissue.
  • Extended single-dose toxicity test in rhesus monkeys 50, 200, 800 mg/kg of CA521 on general state, body weight, food intake, body temperature, lead II electrocardiogram, respiratory rate and blood pressure, ophthalmic examination, hematology, blood No obvious abnormality was found in biochemistry, urine, immune index, bone marrow smear, injection site, gross anatomical observation, organ weight and coefficient, and histopathological examination.
  • 50-800 mg/kg there was no significant gender difference in the exposure of rhesus monkey serum CA521, and the increase in exposure was basically proportional to the increase in dose.
  • 10mg/mL CA521 has no hemolysis and aggregation effect on rabbit erythrocytes.
  • CA521 has very good security.
  • Figure 17A shows the 3D structural model of the binding of S-ECD and CA521.
  • the three RBDs of the Spike protein are asymmetrically bound to the Fab of the antibody, indicating that the Spike protein bound to CA521 has only one conformation, one Spike protein
  • the three RBDs of the trimer whether in Up or Down conformation, bind to three Fabs, respectively.
  • Figure 17B performs identifiable electron cloud density analysis on the binding surface of Fab and RBD to determine the binding site of Spike and Fab.
  • RBM receptor binding module 438-506
  • CA521 can block viral binding to ACE2 by directly binding to RBM.
  • the binding sites of RBD to H014 (Shenzhou cells) and CB6 (Junshi Bio) are shown in Figure 18, and the binding epitopes of H014 (Shenzhou cells) are shown in Z.Lv et al., Science 10.1126/science.abc5881 (2020), CB6 (Junshi Bio) for the binding epitope, see Shi,R.et al.A human neutralizing antibody targets the receptor binding site of SARS-CoV-2.Nature.2020 Aug;584(7819):120-124 https:// doi.org/10.1038/s41586-020-2381-y (2020), the binding site of CA521 is obviously different from the two.
  • CA521 binds directly to RBM, one antibody can bind to two RBDs of one Spike trimer, and all three RBDs
  • CA521 antibody ie CA521 FALA
  • SARS-CoV-2 pseudovirus is the SARS-Cov-2 pseudovirus against Wuhan-Hu-1 strain
  • the pseudovirus of mutant B.1.1.7 is against B.1.1 .7 (hCoV-19/England/QEUH-F56F0F/2021) strain of SARS-Cov-2 pseudovirus.
  • the specific process of the experiment 450 mL of DMEM was added with 50 mL of FBS and mixed to obtain DMEM (10% FBS).
  • the antibody CA521 (in this experiment, CA521 was provided in the form of a preparation, the preparation was a drug containing CA521 antibody) with DMEM (10% FBS).
  • a composition a pharmaceutical composition comprising 10.5 mM histidine hydrochloride, 9.5 mM histidine, 0.08 g/mL trehalose, 0.0002 g/mL polysorbate 80, water, and 21.5 mg/mL of said antibody, pH of 5.5-6.5) Dilute 8 different concentration gradients (0.061-133.4nM and 0.61-1334nM two dilution gradients), 100 ⁇ l gradient dilution antibody samples and 50 ⁇ L 2.0 ⁇ 10 4 TCID 50 /mL pseudovirus (Wuhan-Hu-1 strain) The pseudovirus, or the pseudovirus of the B.1.1.7 mutant strain) solution was mixed at 37°C and incubated for 1 hour.
  • Huh-7 cells were trypsinized, and the density of Huh-7 cells was changed with DMEM ( 10% FBS) to 4 ⁇ 10 5 cells/mL, take out the 96-well cell plate from the carbon dioxide incubator, add 100 ⁇ L of Huh-7 cell suspension to each well, and place it in a carbon dioxide incubator (37°C ⁇ 1°C, 5°C). % ⁇ 1% CO 2 ) for 24 hours. After the incubation, 150 ⁇ L of supernatant was removed from each well, 100 ⁇ L of Bright-Glo chromogenic solution was added, and the reaction was performed in the dark for 2-3 minutes. Luminescence detection.
  • CA521 in the range of 0.061-133.4nM, the maximum inhibition rate of the neutralizing activity of the pseudovirus of Wuhan-Hu-1 strain and the pseudovirus of B.1.1.7 strain can reach a level close to 100%.
  • the IC50 of CA521 antibody against the pseudovirus of Wuhan-Hu-1 strain was 0.180 nM, and the IC50 against the pseudovirus of B.1.1.7 (hCoV-19/England/QEUH-F56F0F/2021) strain was 0.456 nM.
  • CA521 not only has excellent neutralizing activity against pseudovirus of Wuhan-Hu-1 strain, but also has good neutralizing activity against pseudovirus of British mutant strain.
  • the experimental method is as follows: (1) Prepare CBS buffer, dilute the Spike S1+S2 protein of each strain to 0.5 ⁇ g/mL with CBS buffer, and coat 100 ⁇ L/well in a 96-well ELISA plate, overnight at 4°C. (2) Block with 3% nonfat milk powder, 300 ⁇ L/well, incubate at 37°C for 1 h and wash the plate twice. (3) Use PBST to dilute the antibody from 16 ⁇ g/mL four-fold gradient to 8 concentrations, add 50 ⁇ L/well to a 96-well ELISA plate, and add 50 ⁇ L/well of ACE2-FC-biotin (1:5) to make the final concentrations respectively.
  • Inhibition % (OD450 without antibody-OD450)/(OD450 without antibody-OD450 of highest concentration antibody)*100%.
  • the experiment was set up with 2 replicate wells, and the data were plotted as Mean ⁇ standard error. The experimental results are shown in Table 23 and FIG. 19 .
  • the experimental results show that the IC50 of CA304 antibody to block the binding of S1+S2 protein of Wuhan-Hu-1 strain to ACE2 is 0.176nM, and the IC50 of blocking the binding of S1+S2 protein of B.1.351 strain to ACE2 is 0.514nM, The IC50 for blocking the binding of the S1+S2 protein of the B.1.1.7 strain to ACE2 was 1.751 nM, and the IC50 for blocking the binding of the S1+S2 protein of the p.1 strain to ACE2 was 0.753 nM.
  • CA304 not only has excellent blocking ability to the binding of S1+S2 protein of Wuhan-Hu-1 strain to ACE2, but also has good blocking ability to the binding of S1+S2 protein of Brazil and South Africa mutants to ACE2.
  • the strain has a certain blocking ability.
  • the experimental method is the same as 3.16.2, and the experimental results are shown in Table 24 and Figure 20.
  • the experimental method is the same as the neutralizing activity of the CA521 antibody against the new coronavirus pseudovirus mutant in 3.15.
  • the IC50 experimental results of the pseudovirus neutralizing activity of CA304 against different strains are shown in Table 25 and Figure 21 below.
  • CA304 not only has excellent neutralizing activity against pseudoviruses of Wuhan-Hu-1 strain, but also has good neutralizing activity against pseudoviruses of British and Brazilian mutant strains, and has certain neutralizing activity against pseudoviruses of South African mutant strains.
  • the experimental method is the same as the neutralization activity of the CA521 antibody against the new coronavirus pseudovirus mutant in 3.15.
  • the IC50 experimental results of the pseudovirus neutralization activity of CA555 against different strains are shown in Table 26 and Figure 22 below.
  • CA304 not only has excellent neutralizing activity to pseudoviruses of Wuhan-Hu-1 strain, but also has good neutralizing activity to pseudoviruses of British and Brazilian mutant strains, and has certain neutralizing activity to pseudoviruses of South African mutant strains.
  • Antibody sample ie CA521, provided in the form of a preparation, the preparation is a pharmaceutical composition containing CA521 antibody, the pharmaceutical composition includes 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002g/mL Polysorbate 80, water, and 21.5 mg/mL of the antibody, pH 5.5-6.5) diluted in 8 different concentration gradients (for B.1.617.2 pseudovirus, the dilution gradient ranged from 0.61 to 1334 nM; for Wuhan- Hu-1 strain pseudovirus, dilution gradient range 0.0061 ⁇ 13.34nM), 100 ⁇ L of serially diluted antibody sample and 50 ⁇ L of 2.0 ⁇ 10 4 TCID50/mL pseudovirus (B.1.617.2 strain pseudovirus) solution were mixed at 37°C Incubate for 1 hour, and after 40 minutes of incubation, trypsinize Huh-7 cells, adjust the cell density to 4 ⁇ 10 5 cells/mL, take out the 96-well
  • CA521 not only has excellent neutralizing activity against the pseudovirus of Wuhan-Hu-1 strain, but also has certain neutralizing activity against the pseudovirus of B.1.617.2 strain.
  • Antibody sample ie CA521, provided in the form of a preparation, the preparation is a pharmaceutical composition containing CA521 antibody, the pharmaceutical composition includes 10.5mM histidine hydrochloride, 9.5mM histidine, 0.08g/mL trehalose, 0.0002g/mL Polysorbate 80, water, and 21.5 mg/mL of the antibody, pH 5.5-6.5), diluted in 11 different concentration gradients (for C.37 strain pseudovirus, the dilution gradient ranged from 0.001 to 66667 nM; for Wuhan- Hu-1 strain pseudovirus, dilution gradient range 0.001 ⁇ 66667nM), 100 ⁇ L of the serially diluted antibody sample and 50 ⁇ L of 2.7 ⁇ 10 4 TCID50/ml pseudovirus (C.37 strain pseudovirus) solution were mixed at 37°C and incubated for 1 After 40 minutes of incubation, trypsinize HEK293T-hACE2 cells, adjust the cell density to 4 ⁇ 10 5 cells/ml, take out the
  • CA521 not only has excellent neutralizing activity against the pseudovirus of Wuhan-Hu-1 strain, but also has certain neutralizing activity against the pseudovirus of C.37 strain.
  • the CA521 antibody solution after virus removal was concentrated to 80-100g/L through a 30KD ultrafiltration membrane bag, and then replaced with a dialysis buffer (10.5mM His-HCl, 9.5mM His, pH 5.80-6.20), and dialyzed.
  • the volume is 7-10 times, and the TMP is controlled at 0.8-1.5 bar in the whole process, and then the CA521 antibody solution is washed out from the ultrafiltration system to ensure that the CA521 antibody protein concentration is above 27g/L.
  • excipient stock solution (10.5mM His-HCl, 9.5mM His, 0.32g/mL trehalose, 0.0008g/mL polysorbate 80, pH 5.80-6.20)
  • dilution buffer 10.5mM His-HCl, 9.5mM His, pH 5.80-6.20
  • the antibody protein solution was diluted to 20 ⁇ 2mg/mL
  • the pharmaceutical composition was obtained after sterile filtration (10.5mM histidine hydrochloride, 9.5mM histidine) acid, 0.08 g/mL trehalose, 0.0002 g/mL polysorbate 80, and 20 ⁇ 2 mg/mL of the antibody or antigen-binding fragment thereof).
  • the reference substance is the pharmaceutical composition of the present application that has been prepared according to the preparation method of the composition of the present application and has passed various strict quality tests. It is used as a reference substance that can be used normally by the instrument during the testing process, or as a reference substance for comparison with samples. .
  • the reference substance and the pharmaceutical composition samples of the present application were diluted to 1 mg with mobile phase (25 mM Na 3 PO 4 , 150 mM NaCl, 50 mM L-arginine, 5% isopropanol, pH adjusted to 6.80 ⁇ 0.05 with 85% phosphoric acid) /mL solution, filtered and set aside. After equilibrating the high performance liquid chromatograph, according to the set method, the wavelength is 280 nm, the column temperature is 30 ⁇ 2 °C, the flow rate is 0.4 mL/min, and 10 ⁇ L is injected, and the isocratic elution is performed for 15 min, and the chromatogram is recorded.
  • mobile phase 25 mM Na 3 PO 4 , 150 mM NaCl, 50 mM L-arginine, 5% isopropanol, pH adjusted to 6.80 ⁇ 0.05 with 85% phosphoric acid
  • sample buffer of the pharmaceutical composition of the present application 8.8 mL of sodium dihydrogen phosphate solution (50 mM), 1.2 mL of disodium hydrogen phosphate solution (50 mM), and 2 mL of 10% SDS solution were mixed, adjusted to pH 6.5, and then used Milli-Q Make up to 20 mL with water and mix well.
  • CE-SDS sample buffer Take 60 ⁇ L of CE-SDS sample buffer, add an appropriate volume of the sample of the pharmaceutical composition of the present application and Milli-Q water to make the solution volume 95 ⁇ L, add 5 ⁇ L of iodoacetamide (non-reducing CE-SDS)/2-mercaptoethanol (reducing CE-SDS) -SDS), the theoretical final concentration of the pharmaceutical composition sample is 1.0 mg/mL.
  • Substitute Milli-Q water for the sample and prepare non-reducing and reducing blank solutions according to the above operations. Pipette 90 ⁇ L of the prepared pharmaceutical composition sample into the inner tube, put it into the sample bottle and the sample tray, and inject the sample for analysis.
  • the capillary electrophoresis instrument uses a PDA detector, the No. 2 detection window, the total length of the capillary tube is 30.2 cm, the effective distance is 20 cm, the capillary tube temperature is 25.0 °C, and the sample tray temperature: 15.0 °C.
  • Antigen-antibody binding activity detection method enzyme-linked immunosorbent assay, ELISA
  • RBD-mFc was coated on a high-adsorption 96-well plate, incubated overnight at 4°C, and 300 ⁇ L of PBST solution was added to each well. After washing the plate four times, 200 ⁇ L of blocking solution (1% BSA-PBST) was added to each well, and incubated at 37°C for 1.5 After h-2h, 300 ⁇ L of PBST solution was added to each well, and after washing the plate four times, the reference substance, the quality control substance (diluted reference substance of different concentrations) and the test substance (that is, the sample of the pharmaceutical composition of this application) were diluted 8 different Concentration gradient (0.0256ng/mL ⁇ 2000ng/mL), 100ul/well was added to high adsorption 96-well plate, after incubation at 37°C for 2h, 300 ⁇ L of PBST solution was added to each well, the plate was washed four times, and 100ul/well was added with enzyme-labeled secondary antibody After incubation at 37°C for 60min, add 300 ⁇
  • the reference substance and the test substance were diluted with 10 different concentration gradients (0.001-15 ⁇ g/ml), and 100 ⁇ l of the gradient-diluted pharmaceutical composition sample was mixed with 50 ⁇ l of 2.0-2.6 ⁇ 10 4 TCID 50 /ml sham.
  • the virus solution was mixed at 37°C and incubated for 1 hour. After 40 minutes of incubation, HEK293T-hACE2 cells were trypsinized, the cell density was adjusted to 3-5 ⁇ 105 cells/ml, and the 96-well cell plate was taken out from the carbon dioxide incubator.
  • the pharmaceutical composition of the present application containing the antibody or antigen-binding fragment has good stability and can be stored at 2-8° C. for at least 6 months.
  • a total of 5 dose groups were designed, 30mg/time, 150mg/time, 600mg/time, 1200mg/time, 2400mg/time, 30mg/time dose group with 2 cases, and other dose groups with 10 subjects in each group.
  • 2 subjects were enrolled, all of whom received the test drug, only for observation and evaluation of safety (including immunogenicity) and tolerability, and blood sample collection and evaluation during admission to the research center.
  • the safety and tolerability of the drug is up to D8 after administration.
  • the 10 eligible subjects of each subsequent dose group were randomized into the test group or placebo at a ratio of 4:1 for PK, safety (including immunogenicity) and tolerability observation and evaluation.
  • Each subject received only one corresponding dose as a single intravenous infusion.
  • D1 On the first day (D1), an intravenous infusion of diluted LY-CovMab (ie, CA521 antibody) injection or an equal volume of placebo was administered. During hospitalization, blood samples were collected for pharmacokinetic and immunogenic analysis, and the subjects' vital signs, physical examination, 12-lead electrocardiogram, laboratory tests, and adverse events were recorded. Subjects can only leave the research center after completing
  • the trial starts from a low dose and proceeds from low to high group by group. Only when the previous dose group completes the D8 safety and tolerability evaluation and confirms that the drug is safe and tolerated, the next dose group test can be conducted.
  • blood samples were collected before dosing, 0.5 hours after the start of the infusion (D1), immediately after the end of the infusion (D1), and at 4 hours (D1), 8 hours (D1), 24 hours (D2), 48 hours (D3), 72 hours (D4). 168 hours (D8), 336 hours (D15), 504 hours (D22), 672 hours (D29), 840 hours (D36), 1008 hours (D43), 1176 hours (D50), 1344 hours (D57), 1680 hours (D71) and after 2352 hours (D99). Two milliliters of blood samples were collected at each time point and serum concentrations of LY-CovMab (ie, the CA521 antibody) were measured.
  • LY-CovMab ie, the CA521 antibody
  • Blood samples were collected from subjects in all dose groups (30mg, 150mg, 600mg, 1200mg and 2400mg) and were collected at 336h (D15), 672h (D29), 1008h (D43), 1344h (D57), 1680h (D71) and 2352h (D99) collection. At 2352h (D99) after administration, blood samples were collected from all dose groups except the 30 mg group. A 4 ml blood sample was collected at each time point for the detection of drug-resistant antibodies (ADA). Samples that are confirmed to be positive for ADA will be tested for neutralizing antibodies (Nabs).
  • ADA drug-resistant antibodies
  • LY-CovMab ie, CA521 antibody injection (ie, the pharmaceutical composition); specification: 100 mg (5 mL)/bottle. Storage: Store at 2-8°C, protected from light.
  • the pharmaceutical composition was prepared as in Example 4.
  • Placebo LY-CovMab (ie CA521 antibody) injection mimic. Specifications: 5mL/bottle. Storage: Store at 2-8°C, protected from light.
  • the LY-CovMab injection simulant has the same composition as the LY-CovMab (ie CA521 antibody) injection except that it does not contain the active ingredient antibody.
  • Blood samples were collected from all subjects before and at 336h (D15), 672h (D29), 1008h (D43), 1344h (D57) and 1680h (D71) after dosing. 4 mL of whole blood was collected at each time point for drug-resistant antibody (ADA) detection.
  • ADA drug-resistant antibody
  • Serum concentrations of LY-CovMab ie, the CA521 antibody
  • ELISA enzyme-linked immunosorbent assay
  • Drug concentration data were then analyzed using Phoenix WinNonlin software V8.1 (Pharsight Corp., Mountain View, CA, USA), and PK parameters were obtained by non-compartmental analysis. Actual sampling time was used to calculate PK parameters, including maximum concentration (Cmax), time to Cmax (Tmax), area under the serum concentration-time curve from 0 to the last sampling time with a concentration value (AUC0-last), elimination Rate constant ( ⁇ z), area under the serum concentration-time curve from 0 to infinity (AUC0-inf), elimination half-life (t1/2), clearance (CL), volume of distribution (Vz), mean residence time ( MRT) etc.
  • Cmax maximum concentration
  • Tmax time to Cmax
  • AUC0-last concentration value
  • ⁇ z elimination Rate constant
  • AUC0-inf elimination half-life
  • t1/2 elimination half-life
  • CL clearance
  • Vz volume of distribution
  • MRT mean residence time
  • the vital signs, physical examination, laboratory examination, 12-lead electrocardiogram, chest CT, etc. have no abnormality or the abnormality has no clinical significance;
  • women of childbearing age (defined as: women who have not undergone surgical sterilization or who have been less than 1 year postmenopausal) have a negative blood pregnancy test result.
  • Male and female subjects of reproductive age agreed to use effective contraception for at least 2 weeks prior to screening and up to 6 months after the trial medication.
  • HBsAg hepatitis B surface antigen
  • HCV hepatitis C virus
  • HSV hepatitis C virus
  • Smoking is defined as: average daily smoking ⁇ 5 cigarettes within 3 months before screening;
  • SARS-CoV-2 novel coronavirus
  • SARS-CoV-2 neutralizing antibodies participated in any other clinical studies with drug intervention within 3 months before screening, or The drug is still in the elimination period (5 half-lives), whichever is longer; has been vaccinated within 12 weeks before screening, or is scheduled to be vaccinated during the study and within 12 weeks after the study;
  • the incidence of drug-related TEAEs for each dose group was as follows: 150 mg (28.6%, 2/7), 600 mg (25%, 2/8), 1200 mg (14.3%, 1/7), 2400 mg (50 %, 4/8) and placebo (37.5%, 3/8). All drug-related TEAEs were grade 1, and most of them were recovery/resolution or recovery/resolution without action.
  • LY-CovMab ie CA521 antibody
  • LY-CovMab ie CA521 antibody
  • a single dose of LY-CovMab was shown to be safe and well tolerated in healthy Chinese adults.
  • Example 6 A multicenter, randomized, double-blind, placebo-controlled Chinese Phase II clinical trial to evaluate the efficacy and safety of a single dose of LY-CovMab (ie CA521 antibody) injection in patients with mild and common novel coronavirus pneumonia Research
  • Test drug LY-CovMab (ie, CA521 antibody) injection (ie, the pharmaceutical composition); specification: 100 mg (5 mL)/bottle. Storage: Store at 2-8°C, protected from light.
  • the pharmaceutical composition was prepared as in Example 4.
  • Placebo LY-CovMab injection simulant, specification: 5ml/bottle, storage: 2-8 °C dark storage.
  • the LY-CovMab injection simulant has the same composition as the LY-CovMab (ie CA521 antibody) injection except that it does not contain the active ingredient antibody.
  • This study adopts a multi-center, randomized, double-blind, single-dose, placebo-controlled design to evaluate the efficacy, safety, pharmacokinetics and immunogens of LY-CovMab injection in the treatment of patients with mild and common novel coronavirus pneumonia sex.
  • Test period and visit time the screening period is 3 days, the efficacy observation period is 29 days, and the safety, pharmacokinetics and immunogenicity observation period is 99 days.
  • Visit time Screening period D-3 ⁇ -1, the day of administration, D2, D3, D4, D5, D6, D7, D8, D9, D11, D13, D15, D22, D29, D57 and D99 after administration.
  • women of reproductive age (defined as: women who have not undergone surgical sterilization or who have been less than 1 year postmenopausal) have a negative pregnancy test result.
  • Male and female subjects of childbearing age agree to take effective contraceptive measures within 6 months after the trial drug;
  • Allergic constitution that is, those who are allergic to two or more kinds of drugs or two or more kinds of food, or those who may or are clearly allergic to any component of the research drug as judged by the researcher;
  • Test group LY-CovMab injection, specification: 100mg (5ml)/bottle, the dosage is 1200mg/time or 2400mg/time, diluted with 0.9% sodium chloride to a total volume of 250mL, intravenously instilled, infusion time It is 90+30min, single dose, and clinical routine treatment is given at the same time;
  • Control group LY-CovMab injection simulant, specification: 5ml/bottle, diluted with 0.9% sodium chloride to a total volume of 250ml, intravenous infusion, instillation time of 90+30min, single administration, and clinically administered at the same time Conventional treatment.
  • Time-weighted mean change from baseline in viral load from D1 to D7, with D1 as baseline
  • Antipyretic time (within 29 days);
  • Anti-drug antibody (ADA) positive rate and neutralizing antibody (Nab) positive rate and the corresponding antibody titers.
  • PK parameters of LY-CovMab peak concentration (C max ), time to peak (T max ), area under the drug-time curve from 0 to the last quantifiable concentration time point (AUC 0-last ), extrapolated drug-
  • AUC_%Extrap percent area under the curve
  • if the data allow, will also calculate the area under the curve from 0 to infinity (AUC0 -inf ), clearance (CL), half-life (t 1/2 ) ), volume of distribution (Vd).
  • Example 7 Evaluation of the efficacy, safety, tolerability, pharmacokinetic profile, immunogenicity of LY-CovMab (i.e. CA521) in patients with mild to moderate novel coronavirus pneumonia (COVID-19) US 2a A randomized, double-blind, placebo-controlled clinical study
  • the study also aimed to evaluate the safety, tolerability, PK and immunogenicity of LY-CovMab in adult patients with mild to moderate COVID 19.
  • Approximately 150 eligible patients will be graded by severity of COVID-19 (mild or moderate) to balance treatment groups under comparison and will be randomized 1:1:1 to treatment groups (LY-CovMab 1200 mg, LY-CovMab 2400 mg, or placebo).
  • nasopharyngeal swabs will be collected at the study site and SARS-CoV-2 viral load will be determined by RT-qPCR .
  • PK blood draws will be collected for 10 patients in each treatment group.
  • Predetermined doses are administered on day 1, and on days 15, 29, 57 and 99, blood samples will be drawn for immunogenicity assessment.
  • the Sponsor or Designee will establish an independent Data Safety Monitoring Board (DSMB) to review the accumulated study data periodically (in accordance with the DSMB charter) throughout the study to ensure patient safety and to review the conduct of the entire study.
  • DSMB may recommend in writing to the sponsor whether to continue, modify or discontinue the clinical study based on safety considerations.
  • the start of the study will be the date the first patient provides informed consent, and the end of the study will be the last scheduled visit/assessment of the last patient.
  • the study time for each patient will include the following.
  • the total study duration (including screening) per patient was approximately 102 days.
  • Number of planned patients About 150 patients are planned to be recruited.
  • ICF Informed Consent Form
  • the patient is male or non-pregnant and non-lactating female ⁇ 18 years of age (or the legal age of consent where the study takes place) at the time of consent.
  • the patient has a positive SARS-CoV-2 test (RT-PCR or antigen test) in a local laboratory within 3 days prior to randomization.
  • the patient is being treated for COVID-19 as an outpatient.
  • a positive virological test i.e. nucleic acid amplification test or antigen test
  • COVID-19 have mild symptoms of COVID-19, which may include fever, cough, sore throat, malaise, headache, muscle pain, nausea, vomiting, diarrhea, loss of taste or smell, and no shortness of breath or difficulty breathing
  • virological test ie nucleic acid amplification test or antigen test.
  • Female patients of childbearing potential must have a negative pregnancy test result at screening and be willing to use at least one highly effective method of contraception for 99 days after receiving the study treatment dose.
  • the patient has serious systemic diseases, including but not limited to: uncontrolled liver disease, kidney disease, cardiovascular disease, neurological disease, immune system disease, blood disease, tumor or psychiatric disease, or any other disease that cannot be safely followed status of the program.
  • systemic diseases including but not limited to: uncontrolled liver disease, kidney disease, cardiovascular disease, neurological disease, immune system disease, blood disease, tumor or psychiatric disease, or any other disease that cannot be safely followed status of the program.
  • the patient has any of the following conditions: asthma requiring daily treatment; history of allergic/eosinophilic asthma; history of allergic reactions; history of cystic fibrosis; any other respiratory disease not related to COVID-19; respiratory bacteria Infections such as suppurative tonsillitis, acute tracheobronchitis unrelated to COVID-19, sinusitis and otitis media unrelated to COVID-19, or baseline lung disease such as severe interstitial lung disease and/or bronchiectasis.
  • the patient has a serious active bacterial, fungal or viral infection (except for COVID-19).
  • the patient has been vaccinated against COVID-19 with an investigational or emergency use/approved vaccine.
  • the patient has laboratory abnormality in any one of the following laboratory parameters (according to the reference range of the local laboratory) at the time of screening.
  • ALT Alanine aminotransferase
  • AST aspartate aminotransferase
  • UPN upper limit of normal
  • the patient has participated in clinical studies of other drugs (including any studies for the treatment or prevention of COVID-19) within the past 3 months.
  • the patient is or has received specific monoclonal/polyclonal antibodies against SARS-CoV-2, antiviral drugs against SARS-CoV-2, and/or has received plasma from recovered patients with COVID-19 (recovery) period plasma) treatment, either for investigational use or for emergency use authorization.
  • the patient was receiving greater than 20 mg of prednisone equivalents of systemic steroids, interleukin-1 inhibitors, interleukin-6 inhibitors, or intravenous immunoglobulin at the time of screening. These drugs were also not allowed during the study.
  • the patient has a known or suspected allergy to any of the ingredients used in the study treatment formulation.
  • the patient is positive for hepatitis B surface antigen (HBsAg).
  • HCV hepatitis C virus
  • a human immunodeficiency virus (HIV) positive patient A human immunodeficiency virus (HIV) positive patient.
  • HIV human immunodeficiency virus
  • the patient has donated whole blood or blood components or had massive bleeding (>400 mL) within 3 months prior to randomization.
  • the investigator determines that participation in the study is not in the best interests of the patient.
  • the selected population will include all individuals who have signed an informed consent form (ICF).
  • ICF informed consent form
  • the full analysis set will include all randomised patients, regardless of protocol deviation or premature discontinuation. Allocation to treatment groups will be assigned based on initial randomization. FAS will serve as the basis for efficacy analysis.
  • the safety analysis group will include all randomized patients who have received one dose of study drug. Treatment group assignment in this population will be defined by the actual treatment received. This group will be used for security analysis.
  • PKCS PK concentration analysis group
  • PKPS PK parameter analysis set
  • the primary efficacy analysis will be a comparison of the time-weighted mean change from baseline in viral load between each LY-CovMab dose group in FAS and the placebo group.
  • the primary endpoint will be analyzed using an analysis of covariance model (ANCOVA) with treatment group and stratification factors as fixed effects and baseline viral load as covariate.
  • ANCOVA analysis of covariance model
  • Least squares mean estimates of the time-weighted mean change in viral load from day 1 to day 7 for each treatment group, and the difference between each LY CovMab group and placebo, with associated 95% CIs , standard error and corresponding P value.
  • time-weighted mean change in SARS-CoV-2 viral load from baseline to days 5, 11, 15, 22, and 29 in nasopharyngeal swab samples by RT-qPCR will be descriptively summarized at each time point. It may be analysed using a model similar to the primary endpoint, as appropriate.
  • Example 8 Dose-finding clinical study to evaluate the efficacy and safety of LY-CovMab in patients with severe and critical novel coronavirus pneumonia
  • This study adopted a multicenter, randomized, double-blind, placebo-controlled, parallel design.
  • a total of 108 patients with severe and critical new coronavirus pneumonia are planned to be enrolled and randomly assigned to the LY-CovMab 1200mg/time dose group, the LY-CovMab 2400mg/time dose group or the placebo group according to the ratio of 1:1:1.
  • composition and dosing schedule of the LY-CovMab group and the placebo group were the same as in the above examples.
  • Subjects will take throat swab virological tests 1 day before administration, 2 days, 5 days, 8 days, 11 days, 14 days, and 28 days after administration to observe the virus The time of negative conversion and the negative conversion rate of the virus on the 8th day.
  • the subjects measured body temperature every day, recorded clinical symptoms, and performed blood routine examinations on the 2nd, 5th, 8th, 11th, 14th, 21st, and 28th days.
  • physical examination, 12-lead electrocardiogram examination, laboratory examination and other safety examinations were carried out according to regulations, and the occurrence of adverse events was recorded throughout the trial period.
  • Age ⁇ 18 years old, gender is not limited;
  • the onset time is less than or equal to 12 days;
  • ALT or AST level > 5 times the upper limit of normal range (ULN); ALT or AST level > 3 times ULN and total bilirubin level > 2 times ULN;
  • Example 9 Clinical study to evaluate the preventive efficacy and safety of LY-CovMab injection in healthy subjects in contact with patients with novel coronavirus pneumonia
  • This study adopted a multicenter, randomized, double-blind, placebo-controlled, parallel design. It is planned to enroll 2000 healthy subjects who have been in contact with patients with novel coronavirus pneumonia and randomly assign them to the LY-CovMab 1200mg/time dose group, the LY-CovMab 2400mg/time dose group or the placebo group according to the ratio of 1:1.
  • the composition and dosing schedule of the LY-CovMab group and the placebo group were the same as in the above examples.
  • Subjects must give informed consent to this trial, and voluntarily sign a written informed consent form, be able to maintain good communication with investigators and comply with various requirements of the clinical trial (planned visits, laboratory examinations and other trial procedures) ;
  • Age ⁇ 18 years old, gender is not limited;
  • Asymptomatic family members who have been in contact with cases diagnosed with SARS-CoV-2 infection and who must be randomized within 96 hours of collection of diagnostic test samples for confirmed cases of SARS-COV-2.
  • the vital signs, physical examination, laboratory examination, 12-lead electrocardiogram, chest CT, etc. had no abnormality or the abnormality had no clinical significance; the clinical significance was judged by the investigator to be in the stable stage;
  • women of childbearing age (defined as: women who have not undergone surgical sterilization or who have been less than 1 year postmenopausal) have a negative blood pregnancy test result.
  • Male and female subjects of reproductive age agreed to use effective contraception for at least 2 weeks prior to screening and up to 6 months after the trial medication.
  • asthma requiring daily treatment, any other chronic respiratory diseases, bacterial infections of the respiratory system such as suppurative tonsillitis, acute tracheobronchitis, sinusitis, otitis media, etc.
  • Other respiratory tracts that affect the evaluation of clinical trials disease.
  • Patients with underlying lung diseases such as severe pulmonary interstitial lesions and bronchiectasis confirmed by chest CT;
  • HBV hepatitis B surface antigen
  • HCV hepatitis C virus
  • HCV hepatitis C virus

Abstract

本发明提供一种治疗或预防新型冠状病毒SARS-CoV-2引起的疾病的方法，包括向有此需要的受试者施用所述结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段，或施用含所述抗体或抗原结合片段的药物组合物，以阻断SARS-CoV-2病毒引起的细胞病变或者中和SARS-CoV-2病毒，实现对受试者的治疗或预防效果。

Description

一种治疗或预防新型冠状病毒SARS-CoV-2引起的疾病的方法
本申请要求获得2020年9月29日提交的发明名称为"2019-nCoV中和抗体及其应用"，申请号为202011053501.8的中国申请；2020年9月29日提交的发明名称为"利用SARS-CoV-2中和抗体治疗或预防COVID-19的方法"，申请号为202011053486.7的中国申请；2020年11月13日提交的发明名称为"2019-nCoV中和抗体及其应用"申请号为202011266998.1的中国申请；2021年4月30日提交的发明名称为"SARS-CoV-2病毒的中和抗体及其应用"，申请号为202110482731.4的中国申请，以及2021年6月3日提交的发明名称为"SARS-CoV-2病毒的中和抗体及其应用"，国际申请号为PCT/CN2021/098077的国际申请的优先权，其全部内容通过引用并入本文。
技术领域
本发明涉及治疗或预防新型冠状病毒SARS-CoV-2引起的疾病的方法，其包括向有此需要的受试者施用有效量的结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段。
背景技术
SARS-CoV-2病毒引起的疾病包括COVID-19肺炎及其他相关并发症，SARS-CoV-2病毒为2019年首次发现的新冠病毒原始毒株(Wuhan-Hu-1毒株)与后续出现的新冠病毒突变株(B.1.1.7毒株、B.1.351毒株、p.1毒株和B.1.617.2毒株和C.37毒株等)的统称，该病毒对人类健康和世界公共卫生安全已构成重大威胁。目前还没有疫苗和抗病毒特效药物用于新型冠状病毒肺炎的防治，基于抗体技术中和抗体的成功研发与上市，对于COVID-19肺炎及其他相关并发症的预防和治疗尤为重要。抗体分子能够通过阻断病毒颗粒与其受体结合，激活巨噬细胞、NK细胞等免疫细胞和补体等多种机制来杀伤、清除病毒颗粒以及受感染细胞。
SARS-CoV-2病毒属于β属，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60～140nm。其基因组编码的主要结构蛋白有：表面棘突蛋白(S)、膜蛋白(M)、包膜蛋白(E)及核衣壳蛋白(N)。其中棘突蛋白表达在病毒包膜上，可促进病毒与宿主细胞表面受体结合，介导病毒抗原蛋白和细胞膜融合，是重要致病因子。COVID-19患者以发热、干咳、乏力为主要临床表现。少数患者伴有鼻塞、流涕、咽痛、肌痛和腹泻等症状。以轻中症患者为主，约占81％。重症患者多在发病一周后出现呼吸困难和/或低氧血症，严重者可快速进展为急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍及多器官功能衰竭等。
目前还没有针对SARS-CoV-2的抗体药物及疫苗上市，现有药物临床疗效不确切、特异性差。中国仅“莲花清瘟胶囊”获批用于“新型冠状病毒性肺炎的常规治疗中，可用于轻型、普通型引起的发热、咳嗽、乏力”；目前，美国FDA尚未批准治疗或预防COVID-19的药物上市。随着病毒的全球蔓延，患病人数和死亡人数不断增加，临床上迫切需要安全、有效、靶向性强的治疗药物。由于中和抗体具备一箭双雕的作用，既可以作为治疗性药物，也可以作为短期预防性药物。
发明内容
本发明提供一种结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段在制备 治疗或预防SARS-CoV-2病毒引起的疾病的药物中的应用，一种结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段在治疗或预防SARS-CoV-2病毒引起的疾病中的应用，或者一种利用结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段治疗或预防SARS-CoV-2病毒引起的疾病的方法，包括向有此需要的受试者施用所述结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段，或施用含所述抗体或抗原结合片段的药物组合物，以阻断SARS-CoV-2病毒引起的细胞病变或者中和SARS-CoV-2病毒，实现对受试者的治疗或预防效果。
所述SARS-CoV-2病毒基因组编码的主要结构蛋白有：表面棘突蛋白(S)、膜蛋白(M)、包膜蛋白(E)及核衣壳蛋白(N)，其中棘突蛋白表达在病毒包膜上，可促进病毒与宿主细胞表面受体结合，介导病毒抗原蛋白和细胞膜融合，是重要致病因子。COVID-19患者以发热、干咳、乏力为主要临床表现。少数患者伴有鼻塞、流涕、咽痛、肌痛和腹泻等症状。
在本发明的方案中，所述SARS-CoV-2病毒引起的疾病包括COVID-19肺炎及其他相关并发症。SARS-CoV-2病毒为2019年首次发现的新冠病毒原始毒株与后续出现的新冠病毒突变株的统称。进一步的，所述SARS-CoV-2病毒包括Wuhan-Hu-1毒株、B.1.1.7毒株(又称阿尔法(Alpha)毒株)、B.1.351毒株(又称贝塔(Beta)毒株)，p.1毒株(又称伽玛(Gamma)毒株)，B.1.617.2毒株(又称德尔塔(Delta)毒株)和C.37毒株(又称拉姆达(Lambda)毒株)中的一种或多种。
本申请方案中，所述抗体或抗原结合片段通过结合新型冠状病毒(即SARS-CoV-2,又称2019-nCoV)上的S蛋白，阻断SARS-CoV-2病毒引起的细胞病变或者中和SARS-CoV-2病毒。进一步的，所述S蛋白为SARS-CoV-2病毒表面的S蛋白(spike protein)，所述S蛋白含有两个亚基(subunit)S1和S2。更进一步的，所述抗体能结合所述SARS-CoV-2病毒上的S蛋白指的是结合S蛋白的S1和S2亚基中的一个或多个，或结合S1亚基的RBD蛋白。更进一步的，所述抗体或其抗原结合片段结合SARS-CoV-2病毒的RBD的A475、E484、G485、N487、Y489、Q493、S494、Y449、Y453、L455、F456、F486和F490残基。
在本发明的具体实施方式中，所述抗体或抗原结合片段能阻断SARS-CoV-2病毒引起的表达ACE2细胞的病变，或阻断SARS-CoV-2病毒对表达ACE2的细胞的感染、入侵等。更进一步的，所述细胞包括天然表达ACE2的细胞或人工表达ACE2的细胞。更进一步的，所述细胞为哺乳动物细胞。更进一步的，所述哺乳动物包括人、以及非人动物如鼠或猴等。
本发明的COVID-19治疗或预防方案包括对受试者施用有效量的所述抗体或其抗原结合片段，所述抗体例如可以是全人单克隆中和抗体；或对受试者施用含有效量的所述中和抗体或其抗原结合片段的药物组合物。所述有效量例如为满足治疗或预防COVID-19效果的抗体或抗原结合片段的有效量。
在本发明的一个具体实施方式中，包括向有需要的受试者施用约30mg至约2400mg的所述抗体或抗原结合片段。进一步的，可以是约30mg、约150mg、约600mg、约1200mg或约2400mg的所述抗体或抗原结合片段，优选的，为约1200mg或约2400mg的所述抗体或抗原结合片段。
所述抗体或抗原结合片段的剂量为针对人的剂量，根据国家药品监督管理局药品评审中心20120215发布的“健康成年志愿者首次临床试验药物最大推荐起始剂量的估算指导原则”，基于动物实验治疗或预防的有效剂量计算获得，同时结合最大能耐受剂量确定针对人的剂量范围的上限。这对于本领域技术人员来说是可以理解的。
在本发明的另一个具体实施方式中，包括向有需要的受试者施用含约30mg至约2400mg的所述抗体或抗原结合片段的药物组合物。进一步的，可以是含约30mg、约150mg、约600mg、约1200mg或约2400mg的所述抗体或抗原结合片段的药物组合物。
更优选的，所述药物组合物为单位制剂，该单位制剂含有30毫克和2400毫克之间(例如30mg、约150mg、约600mg、约1200mg或约2400mg)的所述抗体或其抗原结合片段；更优选的，所述药物组合物为注射制剂。
在本发明的另一个具体实施方式中，在对所述受试者施用所述抗体或抗原结合片段，或施用含所述抗体或其抗原结合片段的药物组合物之前，还包括检测所述受试者感染的新冠病毒类型的步骤，可以使用本领域常规手段进行。
在本发明的方案中，所述受试者为感染SARS-CoV-2病毒的受试者、与感染SARS-CoV-2病毒的受试者有接触的无症状的受试者、健康受试者、或者其他不适合接种疫苗的受试者(例如因为各种基础病、慢性病或其他原因不适合接种疫苗的受试者)；优选的，所述感染SARS-CoV-2病毒的受试者包括无症状的SARS-CoV-2病毒感染的受试者或者COVID-19患者。更优选，所述COVID-19患者为轻度、中度、重度或危重COVID-19患者。
在本发明的一个方案中，所述感染SARS-CoV-2病毒的受试者为轻度及中度COVID-19患者，即发病时间≤7天且72小时之内按照NIH(美国国立卫生研究院National Institutes of Health)于2020年4月发布的(COVID-19)的治疗指南确诊的轻度及中度新型冠状病毒肺炎的患者。
在本发明的一个方案中，治疗所述轻度及中度COVID-19患者的所述抗体或抗原结合片段的给药剂量为约30mg、约150mg、约600mg、约1200mg或约2400mg；优选的，约1200mg或约2400mg。
在本发明的一个方案中，所述感染SARS-CoV-2病毒的受试者为轻型及普通型新型冠状病毒肺炎患者，即发病时间≤7天且72小时之内按照《新型冠状病毒肺炎诊疗方案(试行第八版修订版)》确诊的轻型及普通型新型冠状病毒肺炎患者。
在本发明的一个方案中，治疗所述轻型及普通型COVID-19患者的所述抗体或抗原结合片段的给药剂量为约30mg、约150mg、约600mg、约1200mg或约2400mg；优选的，约1200mg或约2400mg。
在本发明的一个方案中，所述感染SARS-CoV-2病毒的受试者为重型及危重型新型冠状病毒肺炎患者，即发病时间72小时之内按照《新型冠状病毒肺炎诊疗方案(试行第八版修订版)》确诊的重型及危重型新型冠状病毒肺炎患者。
在本发明的一个方案中，治疗所述重型及危重型COVID-19患者的所述抗体或抗原结合片段的给药剂量为约30mg、约150mg、约600mg、约1200mg或约2400mg；优选的，约1200mg或约2400mg。
在本发明的一个方案中，所述感染SARS-CoV-2病毒的受试者为重度及危重COVID-19患者，即发病时间72小时内实验室检查(如RT-PCR检查)确认感染2019-nCoV，按照NIH于2020年4月发布的(COVID-19)的治疗指南确诊的重度及危重新冠状病毒肺炎患者。
在本发明的一个方案中，治疗所述重度及危重COVID-19患者的LY-CovMab给药剂量为约30mg、约150mg、约600mg、约1200mg或约2400mg；优选的，约1200mg或约2400mg。
在本发明的一个方案中，使用所述抗体或抗原结合片段预防COVID-19，施用受试者为 与诊断为SARS-CoV-2感染的患者有接触的无症状的受试者，或健康受试者。
在本发明的一个方案中，使用所述抗体或抗原结合片段预防COVID-19的LY-CovMab给药剂量为约30mg、约150mg、约600mg、约1200mg或约2400mg；优选的，约1200mg或约2400mg。
在本发明的具体实施方式中，所述抗体或其抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，其中
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:8所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:11所示的HCDR3；
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:12所示的LCDR1、SEQ ID NO:13所示的LCDR2和SEQ ID NO:14所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:11所示的HCDR3；
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:15所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:16所示的HCDR2和SEQ ID NO:17所示的HCDR3；或者
所述抗体或其抗原结合片段的3个轻链互补决定区包含SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:21所示的LCDR3，和/或所述抗体或其抗原结合片段的3个重链互补决定区包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:11所示的HCDR3。
进一步的，所述抗体或其抗原结合片段包含SEQ ID NO:1所示的轻链可变区，和/或SEQ ID NO:2所示的重链可变区；
所述抗体或其抗原结合片段包含SEQ ID NO:3所示的轻链可变区，和/或SEQ ID NO:2所示的重链可变区；
所述抗体或其抗原结合片段包含SEQ ID NO:4所示的轻链可变区，和/或SEQ ID NO:5所示的重链可变区；或者
所述抗体或其抗原结合片段包含SEQ ID NO:20所示的轻链可变区，和/或SEQ ID NO:2所示的重链可变区。
更进一步的，所述抗体包含SEQ ID NO:18所示的重链恒定区，或者包含SEQ ID NO:19所示的轻链恒定区。
在本发明的一个具体实施方式中，提供了下述结合SARS-CoV-2病毒S蛋白的抗体或其抗原结合片段：
抗体ID 轻链可变区序列 重链可变区序列 轻链恒定区序列 重链恒定区序列
CA304 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:19 SEQ ID NO:18
CA521 SEQ ID NO:3 SEQ ID NO:2 SEQ ID NO:19 SEQ ID NO:18
CA530 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:19 SEQ ID NO:18
CA555 SEQ ID NO:20 SEQ ID NO:2 SEQ ID NO:19 SEQ ID NO:18
在本发明的一个具体实施方式中，提供了上述具有上述序列的抗体或其抗原结合片段，进 一步优选的，CA521抗体在B.1.617.2毒株(又称德尔塔(Delta)毒株)和C.37毒株(又称拉姆达(Lambda)毒株)的一种或多种引起的疾病的治疗和预防中的应用。进一步，所述应用包括向有需要的受试者施用含约30mg至约2400mg的所述抗体或抗原结合片段的药物组合物。进一步的，可以是含约30mg、约150mg、约600mg、约1200mg或约2400mg，优选的，约1200mg或约2400mg的所述抗体或抗原结合片段的药物组合物。优选的，所述药物组合物为单位制剂，该单位制剂含有30毫克和2400毫克之间(例如30mg、约150mg、约600mg、约1200mg或约2400mg，优选的，约1200mg或约2400mg)的所述抗体或其抗原结合片段；更优选的，所述药物组合物为注射制剂。
在本发明的一个具体实施方式中，提供了具有上述序列的抗体或其抗原结合片段，进一步优选的，CA521抗体在Wuhan-Hu-1毒株、B.1.1.7毒株(又称阿尔法(Alpha)毒株)、B.1.351毒株(又称贝塔(Beta)毒株)，p.1毒株(又称伽玛(Gamma)毒株)，B.1.617.2毒株(又称德尔塔(Delta)毒株)和C.37毒株(又称拉姆达(Lambda)毒株)中的一种或多种引起的疾病的治疗和预防中的应用。进一步的，所述应用包括向有需要的受试者施用含约30mg至约2400mg的所述抗体或抗原结合片段的药物组合物。进一步的，可以是含约30mg、约150mg、约600mg、约1200mg或约2400mg，优选的，约1200mg或约2400mg的所述抗体或抗原结合片段的药物组合物。优选的，所述药物组合物为单位制剂，该单位制剂含有30毫克和2400毫克之间(例如30mg、约150mg、约600mg、约1200mg或约2400mg，优选的，约1200mg或约2400mg)的所述抗体或其抗原结合片段；更优选的，所述药物组合物为注射制剂。
在本发明的方案中，所述药物组合物包含所述的抗体或其抗原结合片段，优选地，所述药物组合物还包含药学上可接受的载体，优选地，所述药学上可接受的载体包括以下中的一种或多种：药学上可接受的溶剂、表面活性剂、渗透压调节剂、pH调节剂、分散剂、附加剂或塑形剂等。
在本发明的具体实施方式中，所述药物组合物包含所述抗体或其抗原结合片段以及缓冲液。进一步的，所述缓冲液包括海藻糖和聚山梨酯80中的一种或多种。更进一步的，所述药物组合物pH为5.5-6.5。更进一步的，所述缓冲液还包括盐酸组氨酸和组氨酸中的一种或多种。更进一步的，基于所述药物组合物的总体积，所述盐酸组氨酸和组氨酸的摩尔比为10.5：9.5。
进一步的，所述缓冲液包括盐酸组氨酸、组氨酸、海藻糖、聚山梨酯80中的一种或多种。更进一步的，基于所述药物组合物的总体积，所述药物组合物包括0.04-0.1g/mL海藻糖,0.0001-0.0003g/mL聚山梨酯80，以及10-30mg/mL的所述抗体或其抗原结合片段。更进一步的，基于所述药物组合物的总体积，所述药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖,0.0002g/mL聚山梨酯80,以及20±2mg/mL的所述抗体或其抗原结合片段。更进一步的,所述药物组合物还包括水。
更进一步的,针对CA521，CA304，CA555或CA413抗体,所述药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，以及20±2mg/mL的所述抗体或其抗原结合片段。更进一步的,所述药物组合物还包括水。
在本发明的方案中，所述药物组合物的配制方法，包括1)配制辅料混合液，所述配制辅料混合液包括例如10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80；2)将所述抗体或其抗原结合片段添加到所述辅料混合液中以获得所述药物组合物，控制最终获得的所述药物组合物中所述抗体或其抗原结合片段的浓度例如为20±2mg/mL。
进一步的，在上述配制方法中，所述抗体或其抗原结合片段在添加到所述辅料混合液之前还包括除病毒过滤过程，以及浓缩过程(例如保证抗体蛋白浓度在27mg/mL以上，本领域技术人员可以根据需要先配制辅料的母液，然后用稀释缓冲液(例如10.5mM His-HCl,9.5mM His,pH5.80-6.20)将抗体蛋白和辅料母液中的各成分稀释到最终设定的浓度)。更进一步的，在获得所述药物组合物之后还包括除菌过滤过程。本领域技术人员可以采用常规的除病毒过滤、浓缩，以及除菌过滤手段实现上述过程，这对于本领域人员来说是可以实现的。
在本申请的药物组合物中，海藻糖起到稳定剂和调节渗透压的作用，聚山梨酯80作为表面活性剂也是生物制品注射液中的稳定剂；组氨酸和盐酸组氨酸是pH缓冲剂。
在本发明的方案中，所述药物组合物的剂型为注射制剂。更进一步的，所述药物组合物可以通过静脉注射(例如静脉滴注)或者通过腹腔注射进行给药。更进一步的，所述药物组合物为单位制剂(或单剂量形式的制剂)。在本申请说明书中，所述单位制剂为满足一次给药剂量的制剂。
在本发明的另一个方案中，本发明的所述药物组合物为注射液，其施用方式为静脉滴注。
进一步的，本发明的方案包括将含有效量的所述抗体或抗原结合片段的药物组合物稀释后静脉滴注给所述受试者；优选的，使用生理盐水对所述药物组合物进行稀释；更优选的，使用生理盐水将所述药物组合物稀释为100-250mL，在30-120分钟内施用给受试者；更优选的，使用生理盐水将所述药物组合物稀释为250mL，在90-120分钟内施用给受试者；更优选的，使用生理盐水将所述药物组合物稀释为100mL，在30分钟内施用给受试者；更优选的，所述生理盐水为0.9％的氯化钠溶液。
进一步的，将含有效量的所述抗体或抗原结合片段的药物组合物稀释后静脉滴注给所述受试者；优选的，使用0.9％氯化钠溶液对所述药物组合物进行稀释；更优选的，使用0.9％的氯化钠溶液将所述药物组合物稀释至100-250mL，在30-120分钟内施用给受试者；更优选的，使用0.9％的氯化钠溶液将所述药物组合物稀释为250mL，在90-120分钟内施用给受试者；更优选的，使用0.9％的氯化钠溶液将所述药物组合物稀释为100mL，在30分钟内施用给受试者。
在本发明的另一个方案中，提供了具有上述序列的抗体或其抗原结合片段与另外一种抗体或抗原结合片段联合在治疗或预防SARS-CoV-2病毒引起的疾病中的应用，所述应用包括向有此需要的受试者施用具有上述序列的所述抗体或其抗原结合片段，以及结合新型冠状病毒SARS-CoV-2的另外一种抗体或抗原结合片段；优选的，所述另外一种抗体或抗原结合片段与新型冠状病毒SARS-CoV-2的S蛋白结合；更优选的，另外一种抗体或抗原结合片段与具有上述序列的中所述抗体或其抗原结合片段结合S蛋白的表位不重叠或部分重叠。
进一步的，向受试者同一时间或不同时间施用具有上述序列的所述抗体或其抗原结合片段，和结合新型冠状病毒SARS-CoV-2的另外一种抗体或抗原结合片段。
表1-1显示了上述SARS-CoV-2病毒各毒株的名称，出现地以及主要的突变情况。
表1-1
Figure PCTCN2021121556-appb-000001
Figure PCTCN2021121556-appb-000002
本发明提供的方案具有以下的一种或多种优势：
1、本发明提供的中和抗体能够有效治疗或预防SARS-CoV-2病毒引起的疾病，阻断SARS-CoV-2病毒对受试者的感染或者中和SARS-CoV-2病毒；
2、对健康受试者进行单次给药的结果显示，所有不良反应均为1级，未见2级及以上不良反应，未见严重不良反应，LY-Covmab(即CA521抗体)在健康成人中的药代动力学(PK)谱显示了典型的单克隆抗体分布和消除特征。LY-CovMab(即CA521抗体)显示了剂量比例性。LY-CovMab(即CA521抗体)的单剂量在中国健康成人中显示出安全和良好的耐受性。
3、重复给药结果显示，具有良好的安全性和耐受性。
4、含有所述抗体或抗原结合片段的所述药物组合物具有良好的稳定性，能在2-8℃储存至少6-24个月以上。
附图说明
图1示出了4种S蛋白免疫小鼠血清滴度；
图2A-图2C示出了ELISA检测各候选抗体对SARS-CoV-2病毒(Wuhan-Hu-1毒株)的Spike S1+S2蛋白的结合灵敏度；
图3A-3B示出了ELISA检测候选抗体对SARS-CoV-2病毒(Wuhan-Hu-1毒株)的S1的结合灵敏度；
图4示出了ELISA检测候选抗体对SARS-CoV-2病毒(Wuhan-Hu-1毒株)的S2的结合灵敏度；
图5示出了ELISA检测候选抗体阻断ACE2与对SARS-CoV-2病毒(Wuhan-Hu-1毒株)的Spike RBD蛋白的结合；
图6A-图6C示出了流式细胞术检测抗体与CHO-K1-2019-nCoV Spike cells(表达Wuhan-Hu-1的Spike蛋白的细胞)的结合；
图7示出了流式细胞术检测抗体阻断SARS-CoV-2病毒(Wuhan-Hu-1毒株)的RBD与293T-hACE2细胞的结合；
图8示出了流式细胞术检测抗体阻断SARS-CoV-2病毒(Wuhan-Hu-1毒株)的hACE2与293F-SARS-CoV-2-Spike cells(表达Wuhan-Hu-1的Spike蛋白的细胞)的结合；
图9示出了ELISA检测抗体与人C1q受体的结合；
图10A-图10B分别示出了OctetRED 96检测IgG4野生型对照抗体和候选抗体CA530与人CD64的结合；
图11示出了CA521对CHO-K1-spike细胞没有明显的ADCC效应；
图12A示出了CA521对Daudi细胞没有CDC效应，图12B示出了CA521对CHO-K1-spike细胞没有CDC效应；
图13示出了候选抗体阻断SARS-CoV-2病毒(Wuhan-Hu-1毒株)的假病毒对细胞的感染的能力；
图14示出了CA521中和抗体体外对SARS-CoV-2病毒(病毒株为BetaCoV/Beijing/IMEBJ01/2020)的中和活性；
图15示出了CA304、CA413和CA521阻断SARS-CoV-2病毒(病毒株为BetaCoV/Beijing/IMEBJ01/2020)的真病毒感染Vero细胞；
图16A-16B示出了CA304,CA413或CA521抗体给药3天后肺与气管中病毒(SARS-CoV-2的小鼠适应菌株,MASCp6)载量；图16C示出了CA521抗体给药3天后预防和治疗动物模型中肺与气管中病毒(SARS-CoV-2的小鼠适应菌株MASCp6)载量；
图17A示出了SARS-CoV-2病毒(Wuhan-Hu-1毒株)的S-ECD和CA521结合的3D结构模型，图17B示出了S-ECD和CA521结合的结合表位分析；
图18示出了CA521、ACE2、H014、CB6与RBD的结合位点。
图19示出了CA304阻断不同毒株Spike S1+S2蛋白与ACE2的结合。
图20示出了CA555阻断不同毒株Spike S1+S2蛋白与ACE2的结合。
图21示出了CA304对不同毒株的假病毒中和活性。
图22示出了CA555对不同毒株的假病毒中和活性。
具体实施方式
下面结合具体实施例，进一步阐述本发明。所描述的实施例是本发明一部分实施例，而不是全部的实施例。应理解，举出以下实施例是为了向本发明所属技术领域的一般专业人员就如何利用本发明之方法和组合物提供一个完整的公开和说明，并非用于限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1.抗2019-nCoV(或SARS-CoV-2)单克隆抗体的产生
1.1免疫方案 免疫所用Wuhan-Hu-1的各S蛋白及所用小鼠如表1所示。
表1各S蛋白来源及所用小鼠
Figure PCTCN2021121556-appb-000003
共有Wuhan-Hu-1的Spike S1+S2、Spike S1、Spike S2、Spike RBD四种抗原蛋白，分为以下四种免疫方式：(1)Spike S1、Spike S2、Spike RBD三抗原联合免疫(3种抗原免疫剂量相同，混匀后注射)。(2)Spike S1+S2免疫。(3)Spike S1、Spike RBD两抗原联合免疫(2种抗原免疫剂量相同，混匀后注射)。(4)Spike S2。免疫采用腹部皮下多点注射，首免、二免均为20μg抗原蛋白；三免及加强剂量增加到35μg抗原蛋白。首免-二免间隔10天；二免-三免间隔8天；三免-加强间隔7天；加强-取脾间隔2天。4种S蛋白免疫小鼠后，检测小鼠血清滴度结果如图1。
1.2噬菌体库的建立
处死小鼠，解剖取出脾脏，把脾脏用注射器胶塞研磨破碎并用滤网过滤，把滤过的脾细胞冷冻备，提取RNA后获得cDNA，噬菌体库的建立步骤参照Carlos F.Barbas III，Phage display：A laboratory manual中(例如第8和9章)记载的方法进行。构建库的库容数据如表2所示。
表2各S蛋白免疫小鼠构建噬菌体库库容
库名 Q1 Q2 Q3 Q6 Q7 Q14/15 Q18/21
库容(个) 7.5×10 9 6.5×10 9 7.08×10 9 7.8×10 9 7.5×10 9 5.1×10 9 7.4×10 9
1.3以两种方法进行筛选
1.3.1平板筛选，用Wuhan-Hu-1的Spike S1+S2(义翘神州，40589-V8B1)或Spike S1蛋白(义翘神州，40591-V02H)或Spike S2蛋白(义翘神州，40590-V08B)或Spike RBD蛋白(义翘神州，40592-V05H)包被平板。第二天加入噬菌体库孵育2h，洗涤4-10次后用洗脱缓冲液洗脱特异性结合的噬菌体。
1.3.2磁珠筛选，将Wuhan-Hu-1的Spike S1+S2或Spike S1蛋白或Spike S2蛋白或Spike RBD蛋白按照常规步骤进行生物素化，再与Thermo的磁珠结合，经BSA封闭后与噬菌体库孵育2h，洗涤4-10次后用Elution Buffer洗脱特异性结合的噬菌体。筛选获得抗体克隆及来源见表3。
表3筛选得到anti-2019-nCoV抗体来源表
抗体ID 来源库 来源小鼠 淘洗方法
CA13 Q03 Q03 平板筛选
CA14 Q03 Q03 平板筛选
CA108 Q03 Q03 磁珠筛选
CA219 Q06 Q06 平板筛选
CA272 Q07 Q07 平板筛选
CA304 Q01 Q01 磁珠筛选
CA310 Q01 Q01 磁珠筛选
CA313 Q14/15 Q14/15 磁珠筛选
CA413 Q1/2 Q1/2 平板筛选
CA417 Q1/2 Q1/2 平板筛选
CA521 Q2 Q2 磁珠筛选
CA530 Q2 Q2 磁珠筛选
CA536 Q1 Q1 磁珠筛选
CA555 Q1/2 Q1/2 磁珠筛选
实施例2.完整抗体的分子构建与生产
构建阳性克隆CA13\14\108\304\310\413\417\521\530\536\555\219\272\313并测序，各抗体 可变区氨基酸序列如下表4：(CDR区以下划线标识，分析系统为IMGT系统。)
表4.活性克隆氨基酸序列的可变区序列
Figure PCTCN2021121556-appb-000004
Figure PCTCN2021121556-appb-000005
通过常规的分子生物学技术PCR(2×Phanta Max Master Mix厂家：Vazyme货号：P515-P1-AA批号：7E351H9)扩增抗体可变区基因，通过同源重组分别将抗体重链可变区基因连接入带有抗体重链恒定区序列SEQ ID NO:18的核酸序列的载体pCDNA3.4(Life Technology)，将抗体轻链可变区基因连接入带有抗体轻链恒定区序列SEQ ID NO:19的核酸序列的载体pCDNA3.4。本申请实施例中各候选抗体的可变区序列参见表4，重链恒定区序列为SEQ ID NO:18(含有S228P，F234A和L235A突变(编号系统为Eu Numbering系统)，又称FALA突变)，轻链恒定区序列为SEQ ID NO:19。将测序后的阳性克隆提取质粒后共转染进入HEK293细胞在37℃\8％CO 2\125rpm摇床中培养，瞬时表达7天后上清通过Protein A亲和层析，纯化获得抗体，并通过UV280结合理论消光系数确定抗体浓度。
实施例3.Anti-2019-nCoV抗体分子的表征
3.1 Elisa检测抗体与Wuhan-Hu-1毒株的各S蛋白的结合
用pH 9.6 CBS稀释以下各S蛋白至0.3μg/mL：Spike S1+S2(SB,40589-V08B1)、Spike S1(SB,40591-V02H)和Spike S2(SB,40590-V08B)。包被酶标板，100μL/孔，4℃孵育过夜；洗板后用3％脱脂奶粉封闭；洗板后加入PBST稀释好的抗体100μL(2μg/mL五倍稀释4个梯度：0.4μg/mL，0.08μg/mL，0.016μg/mL，0.0032μg/mL),37℃孵育1h。洗板后加入山羊抗 人Fab，37℃孵育1h。洗板后每孔加入100μL TMB底物显色，10min后每孔加入2M H 2SO 4终止显色，用酶标仪读取OD450。稀释之前抗体存在于pH7.4，0.01M PBS缓冲液中。结果显示，随着各个中和抗体浓度的升高，均显示出了其结合Wuhan-Hu-1的Spike S1+S2蛋白的能力(图2A-图2C和表5)。CA304、CA310、CA313、CA413、CA417、CA521、CA530、CA536和CA555显示出了结合Wuhan-Hu-1的Spike S1蛋白的能力(图3A-图3B和表6)。CA13、CA14、CA108、CA219和CA272显示出了结合Wuhan-Hu-1的Spike S2蛋白的能力(图4和表7)。
表5 ELISA检测各候选抗体对Wuhan-Hu-1的Spike S1+S2蛋白的结合灵敏度
Figure PCTCN2021121556-appb-000006
表6 ELISA检测各候选抗体对Wuhan-Hu-1的Spike S1蛋白的结合灵敏度
Figure PCTCN2021121556-appb-000007
表7 ELISA检测各候选抗体对Wuhan-Hu-1的Spike S2蛋白的结合灵敏度
Figure PCTCN2021121556-appb-000008
3.2 ELISA检测抗体阻断Wuhan-Hu-1的Spike RBD蛋白与ACE2的结合
用pH 9.6 CBS稀释Spike RBD(义翘神州,40592-V05H)0.4μg/mL，包被酶标板,100μL/孔，4℃孵育过夜；洗板后用3％脱脂奶粉封闭。洗板后每孔加入PBST稀释好的抗体或ACE2对照蛋白50μl，抗体或ACE2对照蛋白(Novoprotein，C05Y)终浓度为(4μg/mL、1μg/mL、0.25μg/mL、0.0625μg/mL)。然后加入生物素标记的ACE2蛋白(终浓度0.04μg/mL)，50μL/孔，37℃孵育1h；洗板后加入PBST稀释的STREP/HRP，100μL/孔，37℃孵育1h。洗板后每孔加入100μL TMB显色，10min后每孔加入50μL 2M H 2SO 4终止显色，用酶标仪读取OD450。稀释之前抗体存在于pH7.4，0.01M PBS缓冲液中。结果显示CA304、CA310、CA413、CA417、CA521、CA530、CA536和CA555均能抗体阻断Spike RBD蛋白与ACE2的结合见图5和表8。
表8检测候选抗体以及ACE2对照蛋白对Wuhan-Hu-1的Spike RBD蛋白结合灵敏度
Figure PCTCN2021121556-appb-000009
3.3检测抗体与Wuhan-Hu-1的各S蛋白的亲和力
3.3.1抗体与Spike S1+S2蛋白和Spike S2蛋白的结合动力学使用基于表面等离振子共振(surface plasmon resonance，SRP)技术的BIAcore 8K仪器测量。
通过GE抗人IgG Fc氨基偶联试剂盒，将抗人IgG抗体氨基偶联到CM5生物传感器芯片上以获得大约1000应答单位(response units，RU)。对于动力学测量，将Spike S2蛋白(义翘神州，40590-V08B)或Spike S1+S2蛋白(义翘神州，40589-V08B1)用HBS-EP +1×(GE，BR-1006-69)缓冲液2倍连续稀释，50nM起始，2倍稀释4个浓度梯度，并设置0浓度。Startup 3次。抗体：2μg/ml，进样时间100s，流速10μL/min；抗原蛋白：结合120s，流速30μL/min，解离600s；再生：用3M MgCl 2缓冲液再生30s，流速30μL/min。使用1:1 binding结合模型或Two state reaction结合模型(BIAcore Evaluation Software version 3.2)计算结合常数(ka)和解离常数(kd)，平衡解离常数(KD)以比率kd/ka计算。稀释之前抗体存在于pH7.4，0.01M PBS缓冲液中。结果显示各候选抗体均能与相应抗原结合，亲和力数据见表9。
表9 Biacore 8K检测候选抗体亲和力
Figure PCTCN2021121556-appb-000010
3.3.2抗体与Spike RBD蛋白或与Spike三聚体蛋白的结合动力学使用OctetRED 96仪器检测。
用PBST把抗体稀释至4μg/mL，用PBST把蛋白Spike RBD蛋白(北京义翘神州，目录号：40592-V05H)依次稀释至25nM、12.5nM、6.25nM、3.13nM，以PBST作为空白对照。将抗体结合到FAB2G传感器(Fortebio，目录号：18-5125)上，结合高度为1nm，然后进行结合和解离检测，结合时间200s，解离时间400s，检测结束后用1:1模式计算KD、Kon、 Koff。
抗体与三聚体蛋白(三叶草生物制药，SCB-2019)的结合动力学使用OctetRED 96仪器检测。用PBST把抗体稀释至4μg/mL，用PBST把SCB-2019依次稀释至25nM、12.5nM、6.25nM、3.125nM、1.5625nM，以PBST作为空白对照。将抗体Load到FAB2G传感器(Fortebio，目录号：18-5125)上，结合高度为1nm，然后进行结合和解离检测，结合时间200S，解离时间400S，检测结束后用1：1模式拟合计算出KD、Kon、Koff。
结果显示CA417、CA304、CA521、CA530、CA413和CA555抗体均能与相应抗原结合，亲和力数据见表10。
表10 ForteBio Octet检测候选抗体Spike RBD以及Spike三聚体亲和力检测
Figure PCTCN2021121556-appb-000011
3.4检测抗体与细胞的结合与阻断
3.4.1通过流式细胞术检测抗体与表达Wuhan-Hu-1的Spike蛋白的细胞的结合活性。
0.74μg/mL抗体(CA304、CA413、CA521)或Isotype同型对照(与CA521可变区不同，恒定区相同的无关抗体)与过表达Wuhan-Hu-1的Spike蛋白的CHO-K1(GenScript，RD00819)细胞孵育后，用anti-human IgG Fc/FITC二抗(Biolegend，409310)染色，再用流式细胞仪检测分析(
Figure PCTCN2021121556-appb-000012
公司ACEA Biosciences.Inc.，型号2060R)。其中，CA304、CA413的缓冲液体系为：10mM CH 3COONa·3H 2O,30mM NaCl,0.03％Tween-20,5％蔗糖,20mg/mL的所述抗体，pH6.0；CA521的缓冲液体系为10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，水，以及21.5mg/mL的所述抗体，pH6.0。
结果如图6A-图6C显示，CA304、CA413、CA521三个抗体均可以与过表达Wuhan-Hu-1的Spike蛋白的CHO-K1细胞结合，结合活性较好。
3.4.2通过流式细胞术检测抗体的阻断活性。
抗体CA521或Isotype同型对照(与CA521可变区不同，恒定区相同的无关抗体)与3μg/mLWuhan-Hu-1的RBD-Biotin孵育后，再与293T-hACE2细胞(NbBiolab)共孵育1h。用Streptavidin APC二抗(Invitrogen，17-4317-82)染色后用流式细胞仪检测分析。某一抗体浓度下的抑制率＝(0浓度抗体MFI-某浓度抗体MFI)/0浓度抗体MFI*100％。结果显示如图7所示，CA521可以阻断Wuhan-Hu-1的RBD与293T-hACE2细胞的结合，IC50为23nM，而Isotype同型对照无阻断活性。
3.4.3通过流式细胞术检测抗体阻断hACE2与293F-SARS-CoV-2-Spike的结合。
抗体CA521或Isotype同型对照(与CA521可变区不同，恒定区相同的无关抗体)与2 μg/mL hACE2-Biotin混合，共同与293F-SARS-CoV-2-Spike(NbBiolab)细胞共孵育，然后用Streptavidin APC二抗(Invitrogen，17-4317-82)将细胞染色，再用流式细胞仪检测分析。结果如图8显示，CA521可以阻断hACE2与293F-SARS-CoV-2-Spike的结合，IC50为0.86nM，而Isotype同型对照无阻断活性。
上述3.4.1-3.4.3中的Isotype同型对照相同，其重链序列(可变区+恒定区)为SEQ ID NO:22,轻链序列(可变区+恒定区)为SEQ ID NO:23。
3.5抗体ADE效应的去除
在SARS和新冠肺炎患者中均观察到ADE(抗体依赖增强作用)迹象。本发明提供的抗体重链恒定区进行了突变改造可以避免ADE效应。
3.5.1 ELISA检测候选抗体、IgG1野生型对照抗体、IgG4野生型对照抗体与C1q蛋白的结合：
用pH9.6 CBS稀释候选抗体至1μg/mL，包被酶标版(苏州海狸，目录号：40301)，然后用2％BSA封闭1h；加入PBST(PBS+0.05％Tween20)稀释好的C1q蛋白孵育1h，加入Shp pAb to C1q-HRP(Abcam,ab46191)孵育，每孔加入100μL TMB(梅科万德，目录号1001)底物显色，10min后每孔加入50μL 2M H 2SO 4终止显色，用酶标仪读取OD450。稀释之前抗体存在于pH7.4，0.01M PBS缓冲液中。
结果如图9显示，相比IgG1野生型对照抗体，候选抗体CA304、CA530、CA521、CA555，与人C1q受体的结合大大降低，由此可以极大降低ADE效应，使抗体安全性更好。
IgG1野生型对照抗体，其重链序列(可变区+恒定区)为SEQ ID NO:24,轻链序列(可变区+恒定区)为SEQ ID NO:25。IgG4野生型对照抗体，其重链序列(可变区+恒定区)为SEQ ID NO:26,轻链序列(可变区+恒定区)为SEQ ID NO:27。
3.5.2 OctetRED 96检测候选抗体、IgG4野生型对照抗体与人CD64的结合：
抗体与CD64蛋白的结合动力学使用OctetRED 96仪器检测。用PBST把抗体稀释至3μg/mL，用PBST把蛋白S-RBD-Fc(ACROBiosystems，目录号：FCA-H52H2)依次稀释至15.4nM、7.7nM、3.85nM、1.93nM，以PBST作为空白对照。将抗体结合到FAB2G传感器(Fortebio，目录号：18-5125)上，然后进行结合和解离检测，检测结束后用1:1模式计算KD、Kon、Koff。稀释之前抗体存在于pH7.4，0.01M PBS缓冲液中。
结果如图10A-图10B显示，IgG4野生型对照抗体与人CD64受体的亲和力为3.41E-09M，而经过改造的候选抗体CA530与CD64无结合，由此可极大的降低候选抗体的ADE效应。IgG4野生型对照抗体与3.5.1中相同。
3.5.3 Biacore 8K检测候选抗体CA521、CA521-IgG1野生型对照抗体与人FcγR的结合：
CM5芯片(GE Healthcare，BR-1005-30)用His Capture Kit(GE Healthcare，28-9950-56)进行氨基偶联。用1×HBS-EP+缓冲液稀释FcγRI(R&D，1257-Fc)至0.5μg/mL、稀释FcγRIIA R167(R&D，1330-CD/CF)、FcγRIIA H167(Acro，CD1-H5223)、FcγRIIB/C(R&D，1875-CD)至1μg/mL，并以10μL/min的速度注射到芯片上，结合时间90s。将CA521和CA521-IgG1野生型抗体用相同缓冲液分别从不同起始浓度(见表11)2倍稀释成5个浓度梯度。进行进样和解离。最终结合解离曲线使用扣减参比通道和缓冲液通道后的结果。实验数据用稳态拟合模型得到亲和力数据。抗体的缓冲液体系为10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL 海藻糖，0.0002g/mL聚山梨酯80，水，以及21.5mg/mL的所述抗体，pH6.0。
表11 Biacore 8K检测抗体与不同Fc受体结合的稀释倍数
分析物起始浓度(nM) FcγRI FcγRIIA R167 FcγRIIA H167 FcγRIIB/C
CA521-IgG1野生型 40 5000 5000 20000
CA521 40 20000 20000 20000
抗体CA521、CA521-IgG1野生型与人FcγR的亲和力结果如下表12。
表12不同亚型抗体与人FcγR的亲和力结果
Figure PCTCN2021121556-appb-000013
从结果中可看出，相比于CA521-IgG1野生型，与重链恒定区具有突变(S228P+F234A+L235A)改造的CA521对四种Fc受体的亲和力都有明显的降低，由此可以消除ADE效应。CA521-IgG1野生型对照抗体，其重链序列(可变区+恒定区)为SEQ ID NO:28,轻链序列(可变区+恒定区)为SEQ ID NO:29。
3.5.4检测候选抗体ADCP效应
ADCP(抗体依赖的细胞吞噬)是衡量抗体Fc与Fc受体结合生物学活性的一个重要指标。
复苏CD14 +单核细胞，用含GM-CSF、IFN-γ和LPS的1640完全培养基诱导刺激细胞。将CHO-K1-spike(GenScript，RD00819)细胞用CSFE(Invitrogen，65-0850-85)染色后，用无血清1640培养基重悬；将待测抗体用1640无血清培养基稀释为10μg/mL；将CHO-K1-spike细胞、巨噬细胞、待测抗体于96孔细胞培养板中于37℃ 5％CO2培养箱中共同孵育2小时。巨噬细胞和靶细胞比例为1:1，抗体终浓度2μg/mL，反应缓冲液为无血清1640培养基。每孔加入2μL APC-anti-CD206染色后，用100μL DPBS重悬细胞，上机检测。以FITC-H为横坐标、APC-H为纵坐标作图，计算吞噬率＝(FITC ++APC +细胞比例)/(APC +细胞比例)*100％。稀释之前的抗体的缓冲液体系为10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，水，以及21.5mg/mL的所述抗体，pH6.0。抗体CA521不同亚型的ADCP效应结果见表13。
表13抗体CA521不同亚型的ADCP效应
样品 吞噬率％
CA521 3.33
CA521-IgG1野生型 12.74
Isotype同型对照 3.76
可以看出，经过重链恒定区S228P+F234A+L235A突变改造后，CA521和Isotype同型对照(与CA521可变区不同，恒定区相同的无关抗体)均无ADCP效应，仅为3％左右，为实验背景。而未改造的CA521-IgG1野生型表现出较强的吞噬效应，吞噬率达12.74％。由此可见，改造后的CA521Fc端与Fc受体介导的生物学功能受到了抑制，从而可以有效消除ADE效应。Isotype同型对照与3.4.1-3.4.3中的Isotype同型对照相同。CA521-IgG1野生型对照抗体与3.5.3中相同。
3.6检测候选抗体ADCC和CDC
表14显示了检测候选抗体ADCC和CDC试验材料
Figure PCTCN2021121556-appb-000014
本实施例选用病毒刺突蛋白表达量较高的CHO-K1-spike细胞作为靶细胞，研究抗体CA521对靶细胞的毒性作用。选择对CHO-K1-spike细胞具有ADCC作用的CA521-IgG1作为阳性对照，验证ADCC方法的可行性，选择对Daudi细胞具有CDC作用的Rituxan作为系统对照，验证CDC方法的可行性，然后进行抗体CA521对CHO-K1-spike细胞的ADCC和CDC作用的检测。
3.6.1.ADCC检测原理：普洛麦格(Promega)公司改造Jurkat-ADCC效应细胞，使其细胞表面表达高亲和力的FcγRIIIa(V158)受体，能够与抗体Fc端更好地结合；且同时引入NFAT(免疫细胞中表达的转录因子，能够调节基因的转录)与荧光素酶Luciferase基因，使其在ADCC作用激活时，通过细胞内的信号转导，激活Luciferase的表达，与Bio-Lite显色液中底物作用后发光，从而通过发光强度的检测来反映ADCC的效应强度。
ADCC检测方法：1)靶细胞准备：收集CHO-K1-spike细胞，计数，加入ADCC缓冲液稀释为1x 10 6细胞/mL；2)CA521抗体样品梯度稀释：取适量样品加入ADCC缓冲液逐步稀释为100μg/mL，以此浓度为起始浓度；取200uL起始浓度样品，加入96孔稀释板中，吸取40uL加入到160uL稀释液中，依次5倍梯度稀释共8个点，备后续使用；3)效应细胞准备：收集Jurkat-ADCC效应细胞培养液于50mL离心管中，1500rpm离心，去上清，加入ADCC缓冲液反复吹吸，重悬细胞，细胞计数，加入ADCC缓冲液稀释为2x10 6细胞/mL，备后续使用；4)加入靶细胞、CA521抗体和效应细胞：在白色96孔板中加入靶细胞：25uL/孔；在铺有靶细胞的孔中加入梯度稀释的抗体，25uL/孔；加入效应细胞：25uL/孔，使E:T比例为50000:25000细胞/孔；将96孔板放入细胞培养箱(37℃,5％CO 2)培养5h；取出96 孔板，室温放置，使其温度平衡到室温；5)显色液显色：加入Bio-Glo显色液：75uL/孔；反应15min；Tecan酶标仪读数(化学发光)。
结果如图11所示，CA521(即样品S20100501,Grp.1)随着样品浓度增加，信号值没有明显变化，表明CA521对CHO-K1-spike细胞没有明显的ADCC效应；CA521-IgG1阳性对照(即Grp.2)，随着样品浓度的增加，信号逐渐增强，表明其对CHO-K1-spike细胞有ADCC效应。CA521-IgG1阳性对照的序列为3.5.3中CA521-IgG1野生型对照抗体的序列。
3.6.2.CDC检测原理：抗体与细胞上的靶点结合，在与补体结合后，激活补体效应，致使靶细胞被杀死。此实验通过AlarmBlue活细胞检测液检测活细胞的数量，从而间接的指示出靶细胞被CDC效应杀死的细胞数目，因此检测到的信号越弱，说明活细胞数目越少，从而表明CDC效应越强。
CDC检测方法：1)靶细胞准备：收集Daudi细胞,CHO-K1-spike细胞，计数，加入ADCC缓冲液稀释为6x 10 5细胞/mL；2)样品梯度稀释：取10uL样品加入适量ADCC缓冲液稀释为1mg/mL，混匀后取出30uL加入270uL缓冲液配制为100ug/mL，以此浓度为起始浓度；取200uL起始浓度样品，加入96孔稀释板中，吸取40uL加入到160uL稀释液中，依次5倍梯度稀释共8个点，备后续使用；3)加入抗体、靶细胞和补体：在透明96孔板中加入靶细胞：25uL/孔，即15000细胞/孔；加入梯度稀释的样品：12.5uL/孔；37度放置30min；加入补体：补体与ADCC缓冲液以1:1.5稀释，加补体12.5uL/孔，使补体终浓度为10％；37度二氧化碳培养箱培养2小时；显色液显色：加入AlarmaBlue活细胞染色液,5uL/孔，继续放入37度二氧化碳培养箱中培养2h；读数，以560nm激发光，590nm的发射光检测荧光信号。
图12A的Daudi细胞为靶细胞的CDC检测结果显示，阳性对照Rituxan(即Grp.1)随着样品浓度增加，信号逐渐降低，表明活细胞数目逐渐减少，表明Rituxan对Daudi细胞有明显的CDC效应，而CA521(即样品S20100501,Grp.2)随着样品浓度增加，信号值没有明显变化，说明CA521对Daudi细胞没有CDC效应；
图12B的CHO-K1-spike为靶细胞的样品检测结果显示，阴性对照IgG4(即Grp.2)，随着浓度的增加，信号值没有明显变化，阴性对照IgG4对CHO-K1-spike细胞没有CDC效应；CA521随着浓度的增加，信号不显示浓度依赖性曲线，表明其对CHO-K1-spike细胞没有CDC效应。阴性对照IgG4(只有重链)的序列为SEQ ID NO:30。
3.7抗体在假病毒上的中和活性
采用HIV慢病毒载体包装Wuhan-Hu-1的S蛋白的假病毒。假病毒与待检抗体孵育后侵染293T-ACE2细胞,每孔细胞数：3E4cells/孔，病毒量：50μL/孔，最后采用化学发光法检测Luciferase发光值RLU，根据RLU读值计算待检抗体的假病毒抑制率。抗体的缓冲液体系为pH7.4，0.01M PBS缓冲液。
结果显示CA304、CA310、CA413、CA417、CA521、CA530和CA555抗体均能有效阻断假病毒对细胞的感染，其中CA304、CA413、CA521、CA530和CA555的阻断能力最强，分别为0.20nM、0.41nM、0.56nM、0.11nM和0.98nM(图13和表15)。阳性对照为北京义翘神州自产的阳性对照SARS-CoV/SARS-CoV-2 Spike抗体，货号：4015D-D001。
表15候选抗体在假病毒上的中和活性
抗体ID IC50(nM) 抗体ID IC50(nM)
CA304 0.20 CA521 0.56
CA310 1.28 CA530 0.11
CA413 0.41 CA536 4.87
CA417 1.42 CA555 0.98
阳性对照 1.58    
3.8抗体阻断SARS-CoV-2真病毒引起的细胞病变
3.8.1实验材料
表16抗体阻断SARS-CoV-2真病毒引起的细胞病变的实验材料
器材名称 品牌或生产厂商 型号
SARS-CoV-2真病毒 / BetaCoV/Beijing/IMEBJ01/2020
Vero细胞 ATCC CCL-81
DMEM培养基 Gibco 11965092
FBS Gibco 10091155
3.8.2实验方法：
样品组：每种梯度稀释的抗体与100PFU SARS-CoV-2真病毒混合，37度孵育1小时，加至铺有Vero细胞层的24孔板中，37度孵育1h。阳性对照：SARS-CoV-2真病毒和DMEM培养基母液取等体积混合后，37度孵育1h，加至铺有Vero细胞层的24孔板中，37度孵育1h。阴性对照：DMEM培养基母液37度孵育1小时，加至铺有Vero细胞层的24孔板中，37度孵育1h，所有实验均设置两复孔。
移去抗体和病毒混合液，加入1mL 1.0％(w/v)的用含4％FBS(v/v)DMEM稀释的LMP凝胶，继续37度孵育2天，用溶于4％(v/v)甲醛的1％结晶紫(w/v)染色，通过蚀斑数统计抗体中和效果。所有实验均按经批准的三级生物安全操作程序进行。
结果显示候选抗体CA304、CA530、CA310、CA555在10nM浓度及以上时完全阻断病毒的侵染；CA521、CA417在6.25μg/mL浓度及以上，完全阻断病毒的侵染。抗体的缓冲液体系为pH7.4，0.01M PBS缓冲液。候选抗体阻断真病毒对细胞的感染结果如表17所示。
表17候选抗体阻断真病毒对细胞的感染
Figure PCTCN2021121556-appb-000015
-：此浓度抗体阻断了病毒引起的细胞病变
+：此浓度抗体未阻断病毒引起的细胞病变
3.9采用蚀斑减少中和试验法(Plaque Reduction Neutralization Test，PRNT)检测CA521对真病毒的中和活性。
3.9.1.CA521在SARS-CoV-2感染Vero细胞上的中和活性
用细胞维持液将CA521抗体(100mg/5mL)按3倍倍比稀释，与等体积SARS-CoV-2病毒(病毒株为BetaCoV/Beijing/IMEBJ01/2020)混合，37℃孵育1h；将病毒-抗体混合液(200μL/孔)加入含单层致密Vero细胞的24孔培养板中，37℃培养1h；弃病毒抗体混合液，每孔加入适当体积预热的营养琼盖，37℃、5％CO 2培养箱继续培养，在感染后第2天加入适当体积的固定液，室温固定1h，弃固定液和营养琼盖，用固定液清洗1次；加入适当体积的1.0％结晶紫溶液，室温染色1h，弃结晶紫溶液，用固定液清洗1次，计数出斑数，按照公式计算抑制率(抑制率＝(1-抗体组/对照组)×100％)，并采用Graphpad Prism 7软件进行统计分析。研究结果如图14所示，CA521在Vero细胞水平对新冠病毒感染的PRNT 90和PRNT 50分别为1.36μg/mL和0.11μg/mL，分别对应9.11nM和0.737nM，表明中和抗体具有明确的保护细胞免受新型冠状病毒感染的生物学药效。
3.9.2.CA304、CA413和CA521在SARS-CoV-2感染Vero细胞上的中和活性
将5倍连续稀释的单克隆抗体加入到大约100PFU的SARS-CoV-2(病毒株为BetaCoV/Beijing/IMEBJ01/2020)中，在37℃下孵育1h。然后，将混合物加入到24孔板中的Vero细胞单层中，37℃孵育1h。去除混合物，加入1mL含1.0％(w/v)LMP琼脂糖(Promega)和4％(v/v)胎牛血清的DMEM，覆盖于感染细胞上。37℃孵育2天后，用溶解在4％(v/v)甲醛中的1％(w/v)结晶紫染色，以观察斑块。所有实验都是按照经批准的生物安全三级设施的标准操作程序进行的。抗体的缓冲液体系为10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，水，以及21.5mg/mL的所述抗体，pH为5.5-6.5。
从结果图15中可看到，CA304、CA413和CA521均有较好的中和活性，IC50分别为0.080、0.089和0.132μg/mL。
3.10抗体动物体内药效实验
3.10.1 CA304,CA413或CA521在动物体内药效实验
无特异性致病性的6-8周雌性BALB/c小鼠被异氟醚轻麻醉，并将溶于30μL DMEM中的2×10 3PFU的SARS-CoV-2的小鼠适应菌株(军事医学科学院，MASCp6)经鼻滴入。小鼠在感染后2小时后给予20mg/kg的CA304(n＝4)或CA413(n＝4)或CA521(n＝4)PBS(n＝7)单次腹腔给药治疗。在注射3天后(3dpi)采集小鼠肺和气管组织，进行病毒滴度和器官检验。实验方案经中国AMMS实验动物中心动物实验委员会批准。实验结果如图16A-16B所示，对照组的Balb/C小鼠感染后第3天肺组织的病毒载量为10 10.43RNA拷贝数/g，抗体给药组感染后第3天肺组织的病毒载量分别为10 5.35RNA拷贝数/g、10 5.23RNA拷贝数/g和10 5.89RNA拷贝数/g，三个抗体药物给药后分别降低了10 5.08、10 5.2和10 4.54，在气管中同样可有效的降低病毒载量。
本实验中CA521以制剂的形式提供，制剂为含CA521抗体的药物组合物，药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，水，以及21.5mg/mL的所述抗体，pH为pH6.0。
CA304或CA413以制剂形式提供，所述制剂为含所述抗体的药物组合物，药物组合物包括：10mM CH 3COONa·3H 2O,30mM NaCl,0.03％Tween-20,5％蔗糖,20mg/mL的所述抗体 pH6.0。
检测结果如图16A-16B和表18所示，显示了抗体给药3天后肺与气管中病毒载量。
表18抗体给药3天后肺与气管中病毒载量
Figure PCTCN2021121556-appb-000016
3.10.2 CA521在预防和治疗动物模型中的药效实验
使用SARS-CoV-2小鼠适应株模型，腹腔注射20mg/kg LY-CovMab，在预防和治疗模型中均可显著降低肺和气管内病毒滴度。
采用军事科学院军事医学研究院微生物流行病研究所P3实验室建立的SARS-CoV-2小鼠适应株模型评价CA521在小鼠体内的保护效果。6-8周龄的BALB/c小鼠，随机分为3组：抗体预防组(LY-CovMab-P，小鼠在病毒感染前12h腹腔注射给予20mg/kg CA521)，抗体治疗组(LY-CovMab-T，小鼠在病毒感染后2h腹腔注射给予20mg/kg CA521)和对照组(小鼠在感染前12h和感染后2h给予与药物等体积的PBS)。感染前麻醉BALB/c小鼠，将2×10 4PFU/mL的病毒(军事医学科学院，MASCp6)以滴鼻方式接种小鼠(30μL/只)，并分别在感染前或感染后经腹腔途径注射CA521。每日观察小鼠状态，并在感染后第3天，解剖小鼠，取肺组织和气管装在研磨管中，加入1mL细胞维持液，用组织研磨仪研磨至均匀，8000rpm/min离心10min。取100μL上清，采用QIAGEN公司的QIAampViral RNA Mini Kit提取病毒核酸。采用Takara公司的One Step RT-PCR kit(RR064A)进行定量RT-PCR检测,检测病毒RNA。依据病毒RNA拷贝数和CT值的换算公式：RNA Copies/mL＝CT*(-0.2899)+14.875，然后按每mL液体中的0.1g肺组织和0.01g气管计算组织脏器中的病毒RNA载量(RNA拷贝数/g)。
检测结果如图16C所示，对照组的BALB/c小鼠感染后第3天肺组织的病毒载量为10 10.22RNA拷贝数/g，预防组(LY-CovMab-P)和治疗组(LY-CovMab-T)小鼠感染后第3天肺组织的病毒载量分别为10 7.26RNA拷贝数/g和10 7.63RNA拷贝数/g，给药后分别降低10 2.96与10 2.59倍(P<0.01)。对照组的小鼠感染后第3天气管的病毒载量为10 9.16RNA拷贝数/g。预防组和治疗组小鼠感染后第3天气管的病毒载量分别为10 7.28和10 7.21RNA拷贝数/g，给药后分别降低10 1.88与10 1.95倍(P<0.01)。上述结果表明，在SARS-CoV-2鼠适应株模型中，20mg/kg CA521，在预防和治疗模型下均可降低肺和气管内病毒滴度。CA521抗体在BALB/c小鼠模型中对新型冠状病毒感染具有明确的保护效果，可有效降低肺部和气管内病毒载量。
3.11安全药理学
3.11.1.单次给药毒性研究
恒河猴安全药理学试验伴随扩展的恒河猴单次给药毒性试验开展(详见A2020131-T005-01)。恒河猴单次静脉注射给予生理盐水或50、200、800mg/kg的CA521(本实验中CA521以制剂的形式提供，制剂为含CA521抗体的药物组合物，药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，水，以及21.5mg/mL的所述抗体，pH为5.5-6.5)，每天观察动物一般状态；给药前及给药后1～2、6～7h、第2、7、14d，采用大动物无创生理信号遥测系统检测II导联心电图、呼吸频率，采用智能无创血压计测定动脉血压。
实验期间，各组猴自主活动正常、精神状况良好，未见中枢神经系统异常行为表现。给药后各检测时间点，各剂量CA521对II导联心电图、呼吸频率、血压等各指标无明显改变。上述结果表明，50、200、800mg/kg的CA521对中枢神经系统、心血管系统和呼吸系统无影响。
3.11.2重复给药毒性研究
对LY-CovMab进行恒河猴静脉注射4周毒性试验，观察该供试品可能引起毒性反应的性质、程度、量效和时效关系及可逆性，判断毒性靶器官或靶组织，考查药物的免疫原性、免疫毒性及对心血管、呼吸系统、注射部位的影响，为临床使用的安全性提供参考。
本试验设4个组，分别为对照组及LY-CovMab 50、200、800mg/kg组，每组10只恒河猴，雌雄各半。各组猴均按40mL/kg的体积分别静脉注射对照品(0.9％氯化钠注射液)及相应浓度的LY-CovMab。每周1次、连续5次，停药恢复观察4周。首次给药当天定义为试验第1天。
给药期及恢复期每天观察猴一般状况，每周测定体重及摄食量1次；第2、5次给药后3～4小时及恢复期结束各测定1次体温，第2、5次给药后6～7小时及恢复期结束各进行1次II导联心电图、呼吸频率、血压检查；第2、5次给药后1天及恢复期结束各进行1次血液学、血生化、补体(C3、C4)、循环免疫复合物(CIC)、免疫球蛋白(IgA、IgG、IgM)、T淋巴细胞亚群(CD45 +CD20 -CD3 +、CD45 +CD20 -CD3 +CD4 +、CD45 +CD20 -CD3 +CD8 +、CD45 +CD20 -CD3 +CD4 +/CD45 +CD20 -CD3 +CD8 +)、B淋巴细胞(CD45 +CD3 -CD20 +)、NK细胞(CD45 +CD3 -CD20 -CD16 +CD56 +)、单核细胞(CD45 +CD3 -CD20 -CD14 +)检查；第2、5次给药后6、24小时及恢复期结束各测定1次细胞因子(IFN-γ、TNF-α、IL-2、IL-6、IL-10)检测；给药前、试验第15、29天及恢复期第27天各进行1次抗LY-CovMab抗体检测；给药结束及恢复期结束各进行1次尿液、眼科检查；给药结束及恢复期结束，各组分别取6、4只猴(雌雄各半)，采集骨髓后实施安乐死，进行大体解剖观察、脏器重量测定及组织病理学检查。LY-CovMab各组猴分别于第1、4次给药前及给药后5分钟、1、6、24、72、168小时从静脉采集血样，以ELISA法检测血药浓度并计算相关参数。
试验结果显示，恒河猴每周1次连续5次静脉注射50、200、800mg/kg的LY-CovMab，其无毒性反应剂量(NOAEL)为800mg/kg(该剂量下第4次给药后雌、雄猴的AUC 0-336h分别为1710、1820h*mg/mL)。
试验期间，各组猴一般状态、体重、摄食量、体温、II导联心电图、呼吸频率及血压、眼科检查、血液学、血生化、尿液、免疫指标、骨髓涂片、注射部位、大体解剖观察、脏器重量与系数及组织病理学检查均未见明显异常。
试验期间，LY-CovMab各组猴均无抗LY-CovMab抗体的产生。
在50～800mg/kg剂量范围内，恒河猴血清中LY-CovMab的暴露量无明显性别差异，暴露量增加与剂量增加基本成比例，连续给药4周未见明显蓄积。
3.12药代动力学
18只恒河猴(雌雄各半)分3组，各组猴按11.94mL/kg分别单次静脉注射15、60、240mg/kg的CA521，并于给药前及给药结束即刻(1min)，5分钟、1、6小时、1(24h)、3(72h)、7(168h)、10(240h)、14(336h)、21(504h)、28(672h)、35(840h)、42(1008h)、56(1344h)天采集血样，以ELISA法测定血清中CA521浓度，标准曲线范围为78.1～10000ng/mL，以Phoenix WinNonlin 6.4计算药动学参数。本实验中CA521以注射剂的形式提供，制剂为含CA521抗体的药物组合物，药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，水，以及21.5mg/mL的所述抗体，pH为5.5-6.5。
恒河猴分别单次静脉注射15、60、240mg/kg的CA521后，各剂量组主要药代动力学参数结果如下表19所示，以恒河猴单次静脉推注给予60mg/kg CA521注射液为例说明主要的药代动力学参数，达峰浓度C max为1300±86.9μg/mL，末端消除半衰期t 1/2为275±95.9h，暴露量AUC 0-∞为354000±114000h*μg/mL，清除率Cl为0.188±0.0709mL/h/kg，稳态表观分布容积V ss为71.4±7.76mL/kg，滞留时间MRT 0-∞为417±129h。
表19恒河猴单次静脉注射CA521注射液药代动力学参数
Figure PCTCN2021121556-appb-000017
以ELISA法检测给药前及给药结束后14、28、42、56天的抗药抗体。给药前及各组所有动物ADA(抗药抗体)结果均呈阴性；给药结束后14天各组分别有0(0/6)、1(1/6)、0(0/6)只动物ADA结果呈阳性，给药结束后28和42天各组分别有1(1/6)、1(1/6)、0(0/6)只动物ADA结果呈阳性，给药结束后56天各组分别有2(2/6)、1(1/6)、1(1/6)只动物ADA结果呈阳性。
综上，单次静脉注射15、60、240mg/kg CA521后各剂量组动物主要药代动力学参数无明显性别差异。在15～240mg/kg范围内，CA521在恒河猴体内暴露量增加比例与剂量增加比例基本一致。在研究剂量下，给药后56天内部分恒河猴有抗药抗体检出。
3.13毒理研究
经研究，CA521(本实验中CA521以制剂的形式提供，制剂为含CA521抗体的药物组合物，药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，水，以及21.5mg/mL的所述抗体，pH为5.5-6.5)不会产生ADE效应或风险极低。CA521与正常人体组织和恒河猴组织无交叉反应性。扩展的恒河猴单次给药毒性试验，50、200、800mg/kg的CA521对动物一般状态、体重、摄食量、体温、II导联心电图、呼吸频率及血压、眼科检查、血液学、血生化、尿液、免疫指标、骨髓涂片、注射部位、大体解剖观察、脏器重量与系数及组织病理学检查均未见明显异常。在50～800mg/kg剂量范围内，恒河猴血清中CA521的暴露量无明显性别差异，暴露量增加与剂量增加基本成比例。10mg/mL的CA521对兔红细胞无溶血和凝聚作用。在本试验条件下，恒河猴单次静脉注射50、200、800mg/kg的CA521，其无毒性反应剂量(NOAEL)为800mg/kg，该剂量下雌、雄猴的AUC0-336h均为1840000h*μg/mL。综上，CA521具有非常好的安全性。
3.14抗体晶体结构解析和结合表位分析
对候选抗体进行晶体结构解析和结合表位分析。2mg/mL纯化的Wuhan-Hu-1 Spike蛋白的胞外区(1-1208位氨基酸，聚合成三聚体，水木未来提供)(S-ECD)与6.6mg/mL CA521在冰上孵育1min后进行冷冻电镜分析。样品制备由Thermo Fisher Vitrobot Mark IV完成，3.5μL S-ECD和CA521(具有含FALA突变的SEQ ID NO:18重链恒定区，又称为CA521 FALA)的复合物置于包被有自制连续还原石墨烯氧化膜的辉光放电多孔碳膜上，在100％湿度、8℃条件下，将网格中多余的溶液吸干2.0s，然后将网格快速冻结在经液氮冷却的液态乙烷中。使用装备有Gatan K3直接电子计数摄影机的Thermo Fisher Titan Krios G3i电子显微镜进行数据采集。本实验中抗体存在的缓冲液体系为：10mM CH 3COONa·3H 2O,30mM NaCl,0.03％Tween-20,5％蔗糖,21.5mg/mL的所述抗体,pH6.0。
图17A显示S-ECD和CA521结合的3D结构模型，此模型中可看到，Spike蛋白的3个RBD均与抗体的Fab不对称结合，表明与CA521结合的Spike蛋白只有一种构象，一个Spike三聚体的3个RBD，无论其是Up还是Down构象，均分别与3个Fab结合。图17B对Fab和RBD的结合表面进行可识别电子云密度分析，确定了Spike与Fab的结合位点。RBD残基A475、E484、G485、N487、Y489、Q493和S494周围的氢键和盐桥以及Y449、Y453、L455、F456、F486和F490周围的疏水作用共同促成了RBD与CA521之间的相互作用。13个残基中有9个与ACE2结合位点重叠，包括Y449、Y453、L455、F456、A475、F486、N487、Y489和Q493，其他的4个氨基酸残基，如E484、G485、F490和S494，与这9个参与ACE2直接接触的残基距离不超过2个氨基酸。所有这些残基都位于与ACE2直接结合的受体结合组件(RBM)(438-506)中，因此CA521可以通过直接结合于RBM，阻断病毒与ACE2的结合。RBD与H014(神州细胞)和CB6(君实生物)结合的位点见图18，H014(神州细胞)的结合表位参见Z.Lv et al.,Science 10.1126/science.abc5881(2020)，CB6(君实生物)的结合表位参见Shi,R.et al.A human neutralizing antibody targets the receptor binding site of SARS-CoV-2.Nature.2020 Aug；584(7819):120-124 https://doi.org/10.1038/s41586-020-2381-y(2020),CA521的结合位点明显不同于二者。CA521与RBM直接结合，一个抗体可以结合一个Spike三聚体的两个RBD，并且全部3个RBD均被Fab占据，由此可以解释CA521优异的结合和阻断活性。
3.15 CA521抗体对新冠假病毒突变株的中和活性
采用抗体为CA521抗体(即CA521 FALA)；SARS-CoV-2假病毒为针对Wuhan-Hu-1株的SARS-Cov-2假病毒；突变株B.1.1.7的假病毒为针对B.1.1.7(hCoV-19/England/QEUH-F56F0F/2021)病毒株的SARS-Cov-2假病毒。
3.15.1试剂耗材见表20
表20 CA521抗体对新冠假病毒突变株的中和活性试剂耗材
Figure PCTCN2021121556-appb-000018
3.15.2.实验具体过程：450mLDMEM加入50mL FBS混匀得到DMEM(10％FBS)，用DMEM(10％FBS)将抗体CA521(本实验中CA521以制剂的形式提供，制剂为含CA521抗体的药物组合物，药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，水，以及21.5mg/mL的所述抗体，pH为5.5-6.5)稀释8个不同浓度梯度(0.061～133.4nM和0.61～1334nM两种稀释梯度)，100μl梯度稀释抗体样品与50μL2.0×10 4TCID 50/mL假病毒(Wuhan-Hu-1株的假病毒，或B.1.1.7突变株的假病毒)溶液在37℃下混合后孵育1小时，孵育40分钟后，胰蛋白酶消化Huh-7细胞，将Huh-7细胞的密度用DMEM(10％FBS)调整至4×10 5个/mL，从二氧化碳培养箱中取出96孔细胞板，每孔分别加入100μL Huh-7细胞悬液，放置到二氧化碳培养箱(37℃±1℃，5％±1％CO 2)内继续培养24小时，培养结束后，每孔吸掉150μL上清液，加入100μL显色液Bright-Glo，避光反应2-3分钟后，用酶标仪进行化学发光检测。
3.15.3.结果与分析
计算中和抑制率，使用四参数方程拟合回归模型，以浓度值为X轴，以抑制率为Y轴，使用四参数回归模型拟合生成抗体对不同假病毒突变株中和活性的曲线，利用使用四参数拟合法计算IC50。
根据CA521抗体浓度范围0.061～133.4nM得到的抑制率，拟合四参数曲线并计算IC50,结果如表21所示:
表21 CA521抗体的IC50和最大抑制率Imax(％)
Wuhan-Hu-1株的假病毒 B.1.1.7株的假病毒
Imax(％) 96.46 96.77
IC50(nM) 0.180 0.456
由实验结果可知，CA521在0.061～133.4nM范围，对Wuhan-Hu-1株的假病毒和B.1.1.7株的假病毒的中和活性的最大抑制率都可以达到接近100％的水平。CA521抗体对于Wuhan-Hu-1株的假病毒的IC50为0.180nM，对B.1.1.7(hCoV-19/England/QEUH-F56F0F/2021)株的假病毒的IC50为0.456nM。CA521不仅对Wuhan-Hu-1株的假病毒具有优异的中和活性，对英国突变株的假病毒也具有良好的中和活性。
3.16 ELISA检测CA304，CA555抗体阻断不同突变株S1+S2蛋白与ACE2的结合
3.16.1各毒株Spike S1+S2蛋白来源如下表22所示：
表22各毒株Spike S1+S2蛋白来源
Figure PCTCN2021121556-appb-000019
3.16.2.CA304阻断各毒株Spike S1+S2蛋白与ACE2的结合的IC50
实验方法如下：(1)配置CBS缓冲液，用CBS缓冲液将各毒株Spike S1+S2蛋白稀释至0.5μg/mL，按照100μL/孔包被于96孔ELISA板中，4℃过夜。(2)3％脱脂奶粉封闭，300μL/孔，37℃孵育1h后洗板2次。(3)用PBST将抗体从16μg/mL四倍梯度稀释8个浓度，按50μL/孔加入96孔ELISA板中，加入ACE2-FC-biotin(1:5)50μL/孔，使其终浓度分别为0.24μg/mL(Wuhan-Hu-1)、0.02μg/mL(B.1.351)、0.02μg/mL(p.1)、0.2μg/mL(B.1.1.7)。37℃孵育1h后，洗板2次。(4)加入STREP/HRP二抗(1:200稀释)100μL/孔，37℃孵育1h后，洗板5次。(5)每孔加入100μL TMB显色液，显色10min后，每孔加50μL 2M H 2SO 4终止反应。(6)多功能酶标仪读取OD450值。抑制率％＝(无抗体的OD450-OD450)/(无抗体OD450–最高浓度抗体OD450)*100％。实验设置2复孔，数据作图以Mean±standard error表示。实验结果如表23和图19所示。
表23 CA304阻断各毒株Spike S1+S2蛋白与ACE2的结合的IC50
Figure PCTCN2021121556-appb-000020
由实验结果可知，CA304抗体阻断Wuhan-Hu-1株的S1+S2蛋白与ACE2的结合的IC50为0.176nM，阻断B.1.351株S1+S2蛋白与ACE2的结合的IC50为0.514nM，阻断B.1.1.7株的S1+S2蛋白与ACE2的结合的IC50为1.751nM，阻断p.1株的S1+S2蛋白与ACE2的结合的IC50为0.753nM。CA304不仅对Wuhan-Hu-1株的S1+S2蛋白与ACE2的结合具有优异的阻断能力，对巴西、南非突变株的S1+S2蛋白与ACE2的结合也具有良好的阻断能力，对英国株具有一定的阻断能力。
3.16.3.CA555阻断各毒株Spike S1+S2蛋白与ACE2的结合的IC50
实验方法同3.16.2，实验结果如表24以及图20所示。
表24 CA555阻断各毒株Spike S1+S2蛋白与ACE2的结合的IC50
Figure PCTCN2021121556-appb-000021
由实验结果可知，CA555抗体阻断Wuhan-Hu-1株的S1+S2蛋白与ACE2的结合的IC50为0.187nM，阻断B.1.351株S1+S2蛋白与ACE2的结合的IC50为0.367nM，阻断p.1株的S1+S2蛋白与ACE2的结合的IC50为0.355nM。CA555不仅对Wuhan-Hu-1株的S1+S2蛋白与ACE2的结合具有优异的阻断能力，对巴西、南非突变株的S1+S2蛋白与ACE2的结合也具有良好的阻断能力。从图20可以看出,CA555对英国株也有良好的阻断效果。
3.17 CA304和CA555对新冠突变株假病毒的中和活性
3.17.1.实验方法同3.15中CA521抗体对新冠假病毒突变株的中和活性，CA304对不同毒株的假病毒中和活性IC50实验结果如下表25以及图21所示。
表25 CA304对不同毒株的假病毒中和活性IC50实验结果
Figure PCTCN2021121556-appb-000022
由实验结果可知,CA304抗体对于Wuhan-Hu-1株的假病毒的IC50为0.821nM，对B.1.351株假病毒的IC50为12.18nM，对B.1.1.7株的假病毒的IC50为4.061nM，对p.1株假病毒的IC50为3.531nM。CA304不仅对Wuhan-Hu-1株的假病毒具有优异的中和活性，对英国、巴西突变株的假病毒也具有良好的中和活性，对南非突变株的假病毒具有一定中和活性。
3.17.2.实验方法同3.15中CA521抗体对新冠假病毒突变株的中和活性，CA555对不同毒株的假病毒中和活性IC50实验结果如下表26以及图22所示。
表26 CA555对不同毒株的假病毒中和活性IC50实验结果
Figure PCTCN2021121556-appb-000023
由实验结果可知，CA555抗体对于Wuhan-Hu-1株的假病毒的IC50为0.3284nM，对B.1.351株假病毒的IC50为13.89nM，对B.1.1.7株的假病毒的IC50为3.173nM，对p.1株假病毒的IC50为1.585nM。CA304不仅对Wuhan-Hu-1株的假病毒具有优异的中和活性，对英国、巴西突变株的假病毒也具有良好的中和活性，对南非突变株的假病毒具有一定的中和活 性。
3.18 CA521对新冠突变株假病毒的中和活性
表27 CA521抗体对新冠假病毒突变株的假病毒的中和活性试剂耗材
Figure PCTCN2021121556-appb-000024
3.18.1 CA521抗体对B.1.617.2株假病毒中和活性
抗体样品(即CA521，以制剂的形式提供，制剂为含CA521抗体的药物组合物，药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，水，以及21.5mg/mL的所述抗体，pH为5.5-6.5)稀释8个不同浓度梯度(对于B.1.617.2株假病毒，稀释梯度范围0.61～1334nM；对于Wuhan-Hu-1株假病毒，稀释梯度范围0.0061～13.34nM)，100μL梯度稀释的抗体样品与50μL 2.0×10 4TCID50/mL假病毒(B.1.617.2株假病毒)溶液在37℃下混合后孵育1小时，孵育40分钟后，胰酶消化Huh-7细胞，将细胞密度调整至4×10 5个/mL，从二氧化碳培养箱中取出96孔细胞板，每孔分别加入100μL细胞悬液，放置到二氧化碳培养箱(37℃±1℃，5％±1％CO 2)内继续培养24小时，培养结束后，每孔吸掉150μL上清液，加入100μL显色液Britelite plus，避光反应2-3分钟后，用酶标仪进行化学发光检测。计算中和抑制率，以浓度值为X轴，以抑制率为Y轴，使用四参数回归模型拟合生成抗体样品对B.1.617.2株假病毒的中和活性的曲线，使用四参数拟合法计算IC50。结果如表28所示:
表28 CA521对B.1.617.2株假病毒中和活性IC50实验结果
Figure PCTCN2021121556-appb-000025
由实验结果可知,CA521抗体对于Wuhan-Hu-1株的假病毒的IC50为0.13nM，对B.1.617.2株假病毒的IC50为29.38nM。CA521不仅对Wuhan-Hu-1株的假病毒具有优异的中和活性，对B.1.617.2株假病毒具有一定中和活性。
3.18.2 CA521抗体对C.37株假病毒中和活性
抗体样品(即CA521，以制剂的形式提供，制剂为含CA521抗体的药物组合物，药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80， 水，以及21.5mg/mL的所述抗体，pH为5.5-6.5)，稀释为11个不同浓度梯度(对于C.37株假病毒，稀释梯度范围0.001～66667nM；对于Wuhan-Hu-1株假病毒，稀释梯度范围0.001～66667nM)，100μL梯度稀释的抗体样品与50μL2.7×10 4TCID50/ml假病毒(C.37株假病毒)溶液在37℃下混合后孵育1小时，孵育40分钟后，胰酶消化HEK293T-hACE2细胞，将细胞密度调整至4×10 5个/ml，从二氧化碳培养箱中取出96孔细胞板，每孔分别加入100μL细胞悬液，放置到二氧化碳培养箱(37℃±1℃，5％±1％CO 2)内继续培养24小时，培养结束后，每孔吸掉150μL上清液，加入100μL显色液Britelite plus，避光反应2-3分钟后，用酶标仪进行化学发光检测。计算中和抑制率，以浓度值为X轴，以抑制率为Y轴，使用四参数回归模型拟合生成抗体样品对C.37株假病毒中和活性的曲线，使用四参数拟合法计算IC50。结果如表29所示:
表29 CA521对C.37株假病毒中和活性IC50实验结果
Figure PCTCN2021121556-appb-000026
由实验结果可知,CA521抗体对于Wuhan-Hu-1株的假病毒的IC50为0.274nM，对C.37株假病毒的IC50为132.4nM。CA521不仅对Wuhan-Hu-1株的假病毒具有优异的中和活性，对C.37株假病毒具有一定中和活性。
实施例4.本申请药物组合物
1.本申请药物组合物(注射液形式的)配制：
将除病毒过滤后CA521抗体溶液通过30KD超滤膜包浓缩至80-100g/L，然后用透析缓冲液(10.5mM His-HCl,9.5mM His,pH 5.80-6.20)进行缓冲液的置换，透析体积7-10倍，整个过程控制TMP在0.8-1.5bar，然后将CA521抗体溶液从超滤系统中冲洗出来，保证CA521抗体蛋白浓度在27g/L以上。然后向CA521抗体蛋白溶液中加入辅料母液(10.5mM His-HCl,9.5mM His,0.32g/mL海藻糖，0.0008g/mL聚山梨酯80,pH 5.80-6.20)，然后再用稀释缓冲液(10.5mM His-HCl,9.5mM His,pH5.80-6.20)将抗体蛋白溶液稀释至20±2mg/mL，除菌过滤后得到所述药物组合物(10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，以及20±2mg/mL的所述抗体或其抗原结合片段)。
2.药物组合物的稳定性检测：
参考品为根据本申请组合物制备方法制备出来的经各项严格质量检测合格的本申请的药物组合物，在检测过程作为仪器可以正常使用的参考品使用，或者作为与样品比较的参考品使用。
2.1电荷变异体检验(全柱成像毛细管等电聚焦电泳,icIEF)
将pI 7.05等电点标记物、pI 9.22等电点标记物、两性电解质(pH 3-10)、10M尿素和1％甲基纤维素按1:1:8:80:70的体积比混匀。取50μl参考品或本申请药物组合物样品于超滤离心管中，加入400μl超纯水，在离心机中10000rpm离心10min，取出超滤离心管内管进行涡旋混匀，取内管中溶液估测浓度，根据所测浓度进行如下稀释配制：取80μL预混液于1.5mL离心管中，加入一定体积的参考品或本申请药物组合物样品溶液以及Milli-Q水，使最终溶液体积为100μL，参考品或本申请药物组合物样品终浓度为0.2mg/mL，涡旋混匀，10000rpm离心10min(配制过程中的溶液体积可按照需要等比例缩放)。在全柱成像毛细管等 电聚焦电泳仪中设置进样时间：60s；聚焦第一阶段：1500V，1min；聚焦第二阶段：3000V，6min。使用iCE CFR Software软件计算等电点，并将数据导出，使用ChromPerfect软件对图谱进行分析，统计样品的等电点以及酸性峰、主峰、碱性峰的峰面积百分比，表27示出了三批本申请药物组合物注射液主峰含量检测结果。
表27三批本申请药物组合物注射液主峰含量检测结果
Figure PCTCN2021121556-appb-000027
2.2分子大小变异体检验(分子排阻色谱法，SEC-HPLC)
将参考品及本申请药物组合物样品用流动相(25mM Na 3PO 4，150mM NaCl，50mM L-精氨酸，5％异丙醇，用85％磷酸调节pH至6.80±0.05)稀释为1mg/mL的溶液，过滤，备用。将高效液相色谱仪平衡好后，按照设定的方法，波长280nm，柱温30±2℃，流速0.4mL/min进样10μL，等度洗脱15min，记录色谱图。
表28三批本申请药物组合物注射液抗体单体含量检测结果
Figure PCTCN2021121556-appb-000028
2.3分子大小变异体检验(毛细管凝胶电泳法，CE-SDS)
本申请药物组合物样品缓冲液配制：8.8mL磷酸二氢钠溶液(50mM)、1.2mL磷酸氢二钠溶液(50mM)、2mL 10％的SDS溶液混合，调整pH到6.5，再用Milli-Q水定容到20mL，混匀。取CE-SDS样品缓冲液60μL，加入适量体积本申请药物组合物样品和Milli-Q水使溶液体积为95μL，加入5μL的碘乙酰胺(非还原CE-SDS)/2-巯基乙醇(还原CE-SDS)，药物组合物样品理论终浓度为1.0mg/mL。涡旋混匀，短暂离心，在70℃的金属加热套下加热5min(非还原CE-SDS)/10min(还原CE-SDS)，冷却至室温，6000rpm离心10min。用Milli-Q水代替样品，照以上操作，配制成非还原与还原空白溶液。移取90μL制备好的药物组合物样品于内插管中，放入样品瓶和样品托盘中，进样分析。
毛细管电泳仪选用PDA检测器，2号检测窗口，毛细管总长30.2cm，有效距离20cm，毛细管温度25.0℃，样品盘温度：15.0℃。
表29三批本申请药物组合物注射液抗体分子大小变异体检测结果(还原CE-SDS)
Figure PCTCN2021121556-appb-000029
Figure PCTCN2021121556-appb-000030
表30三批本申请药物组合物注射液抗体分子大小变异体检测结果(非还原CE-SDS)
Figure PCTCN2021121556-appb-000031
2.4抗原抗体结合活性检测方法(酶联免疫吸附测定，ELISA)
将RBD-mFc包被于高吸附96孔板中，4℃孵育过夜，每孔加入300μL PBST溶液，洗板四次后，每孔加入200μL封闭液(1％BSA-PBST)，37℃孵育1.5h-2h后，每孔加入300μL PBST溶液，洗板四次后，参考品、质控品(稀释不同浓度的参考品)及供试品(即本申请药物组合物样品)溶液稀释8个不同浓度梯度(0.0256ng/mL～2000ng/mL)，100ul/孔加入高吸附96孔板中，37℃孵育2h后，每孔加入300μL PBST溶液，洗板四次，100ul/孔加入酶标二抗，37℃孵育60min后，每孔加入300μL PBST溶液，洗板四次，每孔加入100μL TMB溶液，37℃孵育10-20min后，每孔中加入100μL 1M磷酸终止。用酶标仪读板，450nm为检测波长，650nm为参比波长。以抗体浓度为X轴，OD 450nm-650nm平均值为Y轴，使用四参数方程拟合回归模型绘制标准曲线。
表31三批本申请药物组合物注射液抗原抗体结合活性检测结果
Figure PCTCN2021121556-appb-000032
2.5假病毒中和活性检验方法(细胞活性分析法)
参考品及供试品(即本申请药物组合物样品)溶液稀释10个不同浓度梯度(0.001～15μg/ml)，100μl梯度稀释药物组合物样品与50μl 2.0～2.6×10 4TCID 50/ml假病毒溶液在37℃下混合后孵育1小时，孵育40分钟后，胰酶消化HEK293T-hACE2细胞，将细胞密度调整至3～5×10 5个/ml，从二氧化碳培养箱中取出96孔细胞板，每孔分别加入100μl细胞悬液，放置到二氧化碳培养箱(37℃±1℃，5％±1％CO 2)内继续培养20～28小时，培养结束后，每孔吸掉150μl上清液，加入100μl显色液，避光反应2-3分钟后，用酶标仪进行化学发光检测。计算中和抑制率，使用四参数方程拟合回归模型，以浓度值为X轴，以抑制率为Y轴，使用四参数回归模型拟合生成参考品曲线与供试品曲线。
表32三批本申请药物组合物注射液假病毒中和活性检测结果
Figure PCTCN2021121556-appb-000033
可以看出，含有所述抗体或抗原结合片段的本申请药物组合物具有良好的稳定性，能在2-8℃储存至少6个月以上。
实施例5在健康受试者中评价LY-CovMab(即CA521抗体)注射液单次给药的安全性、耐受性、药代动力学特征及免疫原性临床研究
共设计5个剂量组，30mg/次、150mg/次、600mg/次、1200mg/次、2400mg/次，30mg/次剂量组入组2例，其他剂量组每组入组10例受试者。本研究第1个剂量组入组2例受试者，均接受试验药物，仅进行安全性(包括免疫原性)和耐受性观察和评价，在入住研究中心期间进行血液样本采集，并评估药物的安全性及耐受性至给药后D8，由研究者、申办者评估并共同确认其安全性后，可继续入组其他剂量组。后续各剂量组筛选合格的10例受试者以4:1比例随机进入试验组或安慰剂，进行PK、安全性(包括免疫原性)和耐受性观察和评价。每个受试者只接受一个相应剂量单次静脉滴注给药。
受试者在给药前14天内完成筛选工作，筛选合格的受试者于给药前一天(D-1)入住研究中心。在第一天(D1)给予静脉滴注稀释后的LY-CovMab(即CA521抗体)注射液或等体积的安慰剂。住院期间，采集血液样本用于药代动力学及免疫原性分析，并记录受试者生命体征、体格检查、12导联心电图、实验室检查以及不良事件发生情况。受试者在D8完成研究规定的试验流程和相关评估后，经研究者同意，方可离开研究中心。之后须按照方案规定的随访时间返回研究中心进行后续随访(150毫克、600毫克、1200毫克和2400毫克组的D15、D22、D29、D36、D43、D50、D57、D71和D99，30毫克组的D15、D29、D43、D50、D57、D71)，按照研究规定的试验流程采集血液样本用于药代动力学和免疫原性分析，并进行安全性评估。
试验从低剂量开始，由低到高逐组开展，只有在前一剂量组完成D8安全性和耐受性评价证实药物安全耐受的前提下，方可进行下一剂量组试验。
1)药代动力学样本采集：
对于150毫克、600毫克、1200毫克和2400毫克的剂量组(n＝40)，采集血样的时间为用药前、输液开始后0.5小时(D1)，输液结束后立即进行(D1)，以及在4小时(D1)、8小时(D1)、24小时(D2)、48小时(D3)、72小时(D4)。168小时(D8)，336小时(D15)，504小时(D22)，672小时(D29)，840小时(D36)，1008小时(D43)，1176小时(D50)，1344小时(D57)，1680小时(D71)和2352小时(D99)后。在每个时间点收集两毫升血样，并测量LY-CovMab(即CA521抗体)的血清浓度。
2)免疫原性样本采集：
采集所有剂量组(30mg、150mg、600mg、1200mg和2400mg)受试者的血样，并在用 药后336h(D15)、672h(D29)、1008h(D43)、1344h(D57)、1680h(D71)和2352h(D99)采集。在用药后2352h(D99)，从所有剂量组收集血样，30mg组除外。在每个时间点收集4毫升血样以检测抗药性抗体(ADA)。被确认为ADA阳性的样本将被检测为中和抗体(Nab)。
3)受试者的处置：
共有255名中国健康志愿者在知情同意书上签字，其中213人在筛选时没有通过。共有42人被随机分配到各组，其中2人在用药前退出。用药细节如下。2名受试者接受30毫克LY-CovMab(即CA521抗体)，7名受试者接受150毫克LY-CovMab(即CA521抗体)，8名受试者接受600毫克LY-CovMab(即CA521抗体)，7名受试者接受1200毫克LY-CovMab(即CA521抗体)，和8名受试者接受2400毫克LY-CovMab(即CA521抗体)，8名受试者接受安慰剂。42名参与者中共有37人完成了研究，5人退出了研究。
在42名随机参与者中，有30名男性(71.4％)和12名女性(28.6％)，平均年龄(平均值±SD)为27.5±6.94岁。在接受注射的40名成人中，平均身高(平均值±SD)为171.03±8.467厘米，平均体重(平均值±SD)为65.00±8.931公斤，平均BMI(平均值±SD)为22.16±1.932公斤/m2(表1)。
4)药物规格：
LY-CovMab(即CA521抗体)注射液(即所述药物组合物)；规格：100mg(5mL)/瓶。储存：2-8℃保存，避光。如实施例4所制备所得所述药物组合物。
剂量：30毫克，150毫克，600毫克，1200毫克，2400毫克。
给药方法：用0.9％的氯化钠稀释至总体积250毫升，然后在90分钟内(±3分钟)通过静脉滴注的方式进行单剂量注射。
5)安慰剂的规格、剂量和管理。
安慰剂：LY-CovMab(即CA521抗体)注射液模拟物。规格：5mL/瓶。储存：储存于2-8℃，避光保存。所述LY-CovMab注射液模拟物除了不含有活性成分抗体外，其他组成与LY-CovMab(即CA521抗体)注射液相同。
给药方法：用0.9％氯化钠稀释至总体积250毫升，然后在90分钟内(±3分钟)通过静脉滴注单剂量给药。
6)评价的标准
6-1)安全性评价
包括生命体征、体格检查、实验室检查(血液学、血液化学、尿液常规检查和凝血检查)、12导联心电图和不良事件。
6-2)免疫原性评估
在用药前和用药后336h(D15)、672h(D29)、1008h(D43)、1344h(D57)和1680h(D71)收集所有受试者的血样。在每个时间点收集4毫升的全血以检测抗药性抗体(ADA)。
LY-CovMab(即CA521抗体)的血清浓度是通过有效的酶联免疫吸附试验(ELISA)测定的。对ADA阳性的样本用有效的检测方法进一步检测是否存在中和抗体。还对ADA阳性样本的滴度进行了评估。
6-3)PK评价
血液样本在给药前、给药后0.5h(D1)、用药结束后立即(D1)、4h(D1)、8h(D1)、24h(D2)、48h(D3)、72h(D4)、168h(D8)、336h(D15)、504h(D22)、672h(D29)、 840h(D36)、1008h(D43)、1176h(D50)、1344h(D57)、1680h(D71)和2352h(D99)。在每个时间点收集2mL的全血，并测量血清中LY-CovMab(即CA521抗体)的浓度。
然后用Phoenix WinNonlin软件V8.1(Pharsight Corp.,Mountain View,CA,USA)对药物浓度数据进行分析，用非室间分析法获得PK参数。实际采样时间用于计算PK参数，包括最大浓度(Cmax)、达到Cmax的时间(Tmax)、从0到最后一个有浓度值的取样时间的血清浓度时间曲线下的面积(AUC0-last)、消除率常数(λz)、从0至无穷时间的血清浓度-时间曲线下的面积(AUC0-inf)、消除半衰期(t1/2)、清除率(CL)、分布容积(Vz)、平均停留时间(MRT)等。
7)入选标准
符合全部下列标准的受试者方可入选本研究：
1.对试验目的充分了解，对研究药物的药理作用及可能发生的不良反应基本了解；按赫尔辛基宣言精神，自愿签署书面知情同意书；
2.健康男性或女性，年龄18～45周岁(包括边界值)；
3.筛选时男性体重≥50kg，女性体重≥45kg，且体重指数(BMI)19.0≤BMI≤26.0kg/m2；
4.筛选期生命体征、体格检查、实验室检查、12导联心电图、胸部CT等无异常或异常无临床意义者；
5.筛选时，育龄女性(定义为：没有接受过手术绝育或绝经后不到1年的女性)的血妊娠试验结果为阴性。育龄男性和女性受试者同意至少在筛选前2周至试验用药后6个月内采取有效避孕措施。
8)排除标准
符合下列任一标准的受试者，不得入选本研究：
1.筛选前8周内做过手术或试验完成前有手术计划，且研究者与申办方确认后认为此类手术可能会给受试者带来不可接受的风险者；
2.给药前使用以下药物或治疗：
·给药前28天内使用任何处方药；
·给药前7天内使用任何非处方药，包括保健品类；
3.既往或目前患有呼吸系统、循环系统、消化系统、泌尿系统、生殖系统、神经系统、内分泌系统、免疫系统、血液学、精神病学、皮肤病学等任何临床的严重疾病或慢性疾病；或能干扰试验结果的任何疾病；
4.罹患新型冠状病毒肺炎或痊愈者；或者新型冠状病毒核酸，血清新冠病毒特异性抗体任一检测结果阳性；
5.已知对试验用药品的任何组分或类似药物有过敏史者，或为过敏体质者(既往对两种或两种以上食物或者药物过敏者)；
6.乙肝表面抗原(HBsAg)，丙型肝炎病毒(HCV)抗体、梅毒螺旋体抗体、人类免疫缺陷病毒(HIV)抗体任一检测结果阳性者；
7.嗜酒或嗜烟或酒精血液检测结果超过正常值范围上限者。嗜酒定义为：筛选前3个月内每周饮酒超过14单位酒精(1单位＝啤酒350mL，或白酒45mL，或葡萄酒150mL)。嗜烟定义为：筛选前3个月内平均每日吸烟量≥5支；
8.筛选时计划一年内生育者(包括其伴侣)，或妊娠期、哺乳期女性；
9.有吸毒或药物滥用史者，或者尿药筛检查结果阳性者；
10.筛选前3个月内献过全血、成分血或大量出血(>400ml)者；
11.既往接种过新型冠状病毒(SARS-CoV-2)疫苗或参与过SARS-CoV-2中和抗体临床试验；筛选前3个月内参加过任何其他有药物干预的临床研究，或筛选前该药物尚在消除期(5个半衰期)内，以时间长者为准；筛选前12周内接种过疫苗，或在研究期间和研究后12周内计划接种疫苗；
12.经询问，有晕血或晕针史者；
13.研究者认为不适宜参加本项临床试验或因其他原因不能完成本项研究者。
9)试验结果：
在42名随机参与者中，40人接受了注射，其中32人注射了LY-CovMab(即CA521抗体)，8人注射了安慰剂。静脉输液后，LY-CovMab(即CA521抗体)的血清暴露量(Cmax、AUC0-last、AUC0-inf)随着剂量从150mg增加到2400mg而以近似比例的方式增加。消除半衰期(t1/2)值在不同剂量组中没有差异，估计约为28.5天。12名受试者共报告了18例(30.0％)药物相关的治疗突发不良事件(TEAEs)，包括尿蛋白出现(25％，10/40)和血肌酐升高(7.5％，3/40)。每个剂量组的药物相关TEAE的发生率如下：150毫克(28.6％，2/7)，600毫克(25％，2/8)，1200毫克(14.3％，1/7)，2400毫克(50％，4/8)和安慰剂(37.5％，3/8)。所有与药物有关的TEAEs均为1级，其中大多数为恢复/解决或恢复/解决而不采取行动。
10)结论：
LY-Covmab(即CA521抗体)在健康成人中的药代动力学(PK)谱显示了典型的单克隆抗体分布和消除特征。LY-CovMab(即CA521抗体)显示了剂量比例性。LY-CovMab(即CA521抗体)的单剂量在中国健康成人中显示出安全和良好的耐受性。
实施例6在轻型及普通型新型冠状病毒肺炎患者中评价单次给予LY-CovMab(即CA521抗体)注射液有效性和安全性的多中心、随机、双盲、安慰剂对照的中国Ⅱ期临床研究
1)受试人群：轻型及普通型新型冠状病毒肺炎患者
2)受试者例数：每组30～50例，1200mg/次剂量组:2400mg/次剂量组:安慰剂组＝1:1:1
3)试验用药品：
试验药物：LY-CovMab(即CA521抗体)注射液(即所述药物组合物)；规格：100mg(5mL)/瓶。储存：2-8℃保存，避光。如实施例4所制备所得所述药物组合物。
安慰剂：LY-CovMab注射液模拟剂，规格：5ml/瓶，贮藏：2-8℃避光储存。所述LY-CovMab注射液模拟物除了不含有活性成分抗体外，其他组成与LY-CovMab(即CA521抗体)注射液相同。
4)总体设计：
本研究采用多中心、随机、双盲、单次给药、安慰剂对照设计，评价LY-CovMab注射液治疗轻型及普通型新型冠状病毒肺炎患者的疗效、安全性、药代动力学及免疫原性。
本研究共有3组，分别是1200mg/次剂量组、2400mg/次剂量组及安慰剂组。每组纳入30～50例受试者，共计90～150例。受试者在给药前3天内完成筛选工作，筛选合格的受试者按照1:1:1比例随机分至1200mg/次剂量组、2400mg/次剂量组及安慰剂组，临床疗效观察至29天，安全随访至99天。
此外，所有受试者在研究期间，根据方案规定的试验流程，采集血液样本用于药代动力学及免疫原性分析，根据方案规定的试验流程进行鼻咽拭子采集用于新型冠状病毒核酸定性 检测或新型冠状病毒病毒载量检测，记录受试者的临床症状和生命体征，进行体格检查、心电图检查、实验室检查，询问和记录不良事件和合并用药，用于疗效和安全性评价。
5)试验周期和访视时间：筛选期3天，有效性观察期为29天，安全性、药代动力学及免疫原性观察周期为99天。访视时间：筛选期D-3～-1，给药当天，给药后D2、D3、D4、D5、D6、D7、D8、D9、D11、D13、D15、D22、D29、D57及D99。
6)入选标准：符合全部下列标准的受试者方可入选本研究：
1.年龄18周岁及以上；
2.随机前发病时间≤7天且72小时之内按照《新型冠状病毒肺炎诊疗方案(试行第八版修订版)》确诊的轻型及普通型新型冠状病毒肺炎患者；
3.筛选时，育龄女性(定义为：没有接受过手术绝育或绝经后不到1年的女性)的妊娠试验结果为阴性。育龄男性和女性受试者同意在试验用药后6个月内采取有效避孕措施；
4.自愿签署书面知情同意书。
7)排除标准：符合下列任一标准的受试者，不得入选本研究：
1.呼吸频率≥30次/分或静息状态下，吸空气时指氧饱和度≤93％或动脉血氧分压(PaO2)/吸氧浓度(FiO2)≤300mmHg(1mmHg＝0.133kPa)；高海拔(海拔超过1000米)地区应根据以下公式对PaO2/FiO2进行校正：PaO2/FiO2×[760/大气压(mmHg)]，或临床症状进行性加重，肺部影像学显示24～48小时内病灶明显进展>50％者；
2.正在接受或既往接受过抗SARS-CoV-2的特异性单克隆/多克隆抗体、抗SARS-CoV-2的抗病毒药物和/或既往接受过COVID-19康复患者的血浆(恢复期血浆)治疗，无论是试验用还是紧急使用授权；
3.伴有严重的系统性疾病：包括但不限于肝脏疾病、肾脏疾病、心血管疾病、神经系统疾病、免疫系统疾病、血液疾病、肿瘤、精神疾病，经研究者判断不适合入组者；
4.既往接受过新冠疫苗，且筛选时的抗体水平超过临界值(阳性)；
5.有慢性呼吸道疾病，包括但不限于慢性阻塞性肺病、肺间质纤维化、支气管扩张、哮喘等，或伴有严重活动性细菌、真菌、病毒感染者(除COVID-19外)，或长期使用激素等免疫抑制剂的，经研究者判断不适合入组者；
6.筛选时，正在接受>20mg泼尼松等效剂量的全身性类固醇、白细胞介素-1抑制剂、白细胞介素-6抑制剂或静注免疫球蛋白治疗者；
7.筛选时出现以下任何实验室参数异常：
i.ALT或AST水平>2.5倍正常范围上限(ULN)
ii.总胆红素水平>1.5倍ULN；
iii.肌酐水平>1.5倍ULN；
8.在过去3个月内参加其它药物临床研究者(其中参加其他临床试验筛选失败者除外)；
9.过敏体质，即对两种或两种以上药物过敏者或者两种以上食物过敏者，或经研究者判断可能或明确对研究药物任一组分过敏者；
10.妊娠期、哺乳期女性；
11.人类免疫缺陷病毒(HIV)抗体检测阳性者；
12.研究者认为其他需要排除者。
8)给药方案：
试验组：LY-CovMab注射液，规格：100mg(5ml)/瓶，给药剂量为1200mg/次或2400mg/ 次，用0.9％氯化钠稀释至总体积250mL后，静脉滴注，滴注时间为90+30min，单次给药，同时给予临床常规治疗；
对照组：LY-CovMab注射液模拟剂，规格：5ml/瓶，用0.9％氯化钠稀释至总体积250ml后，静脉滴注，滴注时间为90+30min，单次给药，同时给予临床常规治疗。
9)生物样本采集：
药代动力学：
每组10例受试者于给药前，给药结束后即刻(D1)、给药结束后4h(D1)、给药开始后144h(D7)、336h(D15)、672h(D29)、1344h(D57)、2352h(D99)采集血液样本。每个时间点采集2mL全血，检测血清中LY-CovMab的浓度。
免疫原性：
所有受试者于给药前，给药开始后336h(D15)、672h(D29)、1344h(D57)、2352h(D99)采集血液样本。每个时间点采集4mL全血，检测血清中抗药抗体(ADA)，ADA确证为阳性的样本将进行滴度和中和抗体(Nab)检测。
10)评价指标：
主要终点指标：
病毒载量较基线的时间加权平均变化值(从D1至D7，D1为基线)；
次要终点指标：
1.发展为重型或危重型的患者比例(按照《新型冠状病毒肺炎诊疗方案(试行第八版修订版)》评估，29天内)；
2.新型冠状病毒核酸检测转阴时间(29天内)；
3.第3、5、7、11、15、22、29天病毒载量较基线的变化情况；
4.病毒载量较基线的时间加权平均变化值(D5、D11、D15、D22、D29)；
5.第7，15天新型冠状病毒核酸检测转阴率；
6.症状改善的患者比例(D7、D11、D15、D22、D29)；
7.症状恢复的患者比例(D7、D11、D15、D22、D29)；
8.症状恢复时间(29天内)；
9.退热时间(29天内)；
10.需要高流量氧疗或无创通气或有创机械通气的患者比例(29天内)；
11.全因死亡率(29天内及99天内)。
探索性指标
突变毒株感染患者症状改善的比例(D7、D11、D15、D22、D29)；
突变毒株感染患者症状恢复的比例(D7、D11、D15、D22、D29)。
安全性评价
生命体征；
体格检查；
实验室检查(血常规、血生化、尿常规、凝血功能)；
12导联心电图；
不良事件。
免疫原性评价
抗药抗体(ADA)的阳性率和中和抗体(Nab)的阳性率，以及相应的抗体滴度。
药代动力学评价
LY-CovMab的PK参数：峰浓度(C max)、达峰时间(T max)、从0到最后可定量浓度时间点间的药时曲线下面积(AUC 0-last)、外推的药-时曲线下面积百分比(AUC _％Extrap)，如数据允许，还将计算从0时至无限时间的药时曲线下面积(AUC 0-inf)、清除率(CL)、半衰期(t 1/2)、分布容积(Vd)。
实施例7在轻度至中度新型冠状病毒肺炎(COVID-19)患者中评价LY-CovMab(即CA521)的疗效、安全性、耐受性、药代动力学特征、免疫原性的美国2a期随机、双盲、安慰剂对照的临床研究
1)研究目标和终点
Figure PCTCN2021121556-appb-000034
Figure PCTCN2021121556-appb-000035
采用随机、双盲、安慰剂对照、平行剂量探索的2a期研究，旨在评估LY-CovMab与安慰剂在轻度至中度COVID 19成人门诊患者中的疗效。该研究还旨在评估LY-CovMab在轻度至中度COVID 19成年患者中的安全性、耐受性、PK和免疫原性。
筛选评估将在研究治疗的3天内完成。所有患者在基线访问的3天内必须有SARS-CoV-2的本地实验室阳性结果。
约150名符合条件的患者将按COVID-19的严重程度(轻度或中度)进行分级，以平衡比较下的治疗组，并按1:1:1随机分为治疗组(LY-CovMab 1200毫克，LY-CovMab 2400毫克，或安慰剂)。
-LY-CovMab 1200毫克：约50名患者
-LY-CovMab 2400毫克：约50名患者
-安慰剂：约50名患者
在第1天，患者将接受一次LY-CovMab或安慰剂的静脉(IV)注射。在治疗后29天内，将对患者进行临床疗效观察，在治疗后99天内，将对患者进行安全性观察。研究访问将在第3、5、7、11、15、22、29、57和99天进行。研究访问期间将收集以下样本。
-在第1天用药预定剂量，以及第3、5、7、11、15、22和第29天，将在研究地点收集鼻咽拭子，通过RT-qPCR测定SARS-CoV-2病毒载量。
-在第1天用药预定剂量，输液结束后立即，以及输液结束后的0.5、1和4小时，将抽取血样进行PK分析。在第7、15、29、57和99天将进行额外的PK抽血。将为每个治疗组的10名患者收集PK抽血。
-在第1天用药预定剂量，以及第15、29、57和99天，将抽取血样进行免疫原性评估。
从第1天到第29天的整个临床疗效观察期，将监测疾病严重程度和COVID-19症状。对 所有患者的生存状态/全因死亡率的评估将记录到第99天。所有患者的安全性将被观察到第99天。
安全性和耐受性将通过TEAEs、SAEs、临床实验室检查(血液学、化学、凝血学和尿液分析)、12导联心电图、生命体征(包括收缩压和舒张压、心率、呼吸率、氧饱和度[SpO 2]和体温)、和体检来评估，按照评估时间表。不良事件将根据美国国家癌症研究所不良事件通用术语标准(NCI-CTCAE 5.0版)进行严重程度分级。
主办方或指定方将成立一个独立的数据安全监测委员会(DSMB)，在整个研究过程中定期(根据DSMB章程)审查积累的研究数据，以确保患者的安全并审查整个研究的进行。DSMB可以根据安全方面的考虑，以书面形式向主办方建议是否继续、修改或停止临床研究。
2)研究期限：
研究的开始将是第一个病人提供知情同意书的日期，而研究的结束将是最后一个病人的最后一次预定访问/评估。
每个病人的研究时间将包括以下内容。
-筛选期:第-3天至第-1天
-临床疗效观察期:第1天至第29天
-安全性、PK、免疫原性和生存状态/全因死亡率期:第1天至第99天
每位患者的总研究时间(包括筛选)大约为102天。
3)计划中的病人数量：计划招募约150名患者。
4)入选标准
符合全部下列标准的受试者方可入选本研究：
1.由病人或其合法授权代表签署的知情同意书(ICF)。
2.患者在同意时是男性或非怀孕和非哺乳期的女性≥18岁(或研究发生地的法定同意年龄)。
3.患者在随机化前3天内，当地实验室SARS-CoV-2检测(RT-PCR或抗原检测)结果为阳性。
4.患者作为门诊病人接受COVID-19的治疗。
5.患者的COVID-19发病时间在随机化前≤7天。
6.患者的COVID-19严重程度为轻度至中度，符合FDA筛选时的严重程度分类。
轻度:
a)病毒学试验(即核酸扩增试验或抗原试验)检测结果呈阳性
b)患有COVID-19的轻度疾病症状，可能包括发烧、咳嗽、喉咙痛、身体不适、头痛、肌肉疼痛、恶心、呕吐、腹泻、味觉或嗅觉丧失，没有呼吸急促或呼吸困难
c)没有表明中度、重度或危重COVID-19的临床症状
中度:
a)通过病毒学检测(即核酸扩增检测或抗原检测)呈阳性。
b)患有COVID-19的中度疾病的症状，这可能包括任何轻度疾病的症状或劳累时的呼吸急促
c)暗示患有COVID-19的中度疾病的临床症状，如呼吸频率≥20次/分钟，心率≥90次/分钟，海平面室内空气中SpO 2>93％或海拔高于4000英尺时>90％。
d)没有表明严重或危重疾病严重程度的临床症状
7.有生育能力的女性患者在筛查时必须有妊娠试验的阴性结果，并愿意在接受研究治疗剂量后99天内使用至少一种高效的避孕方法。
8.男性患者必须同意在接受研究治疗剂量后的99天内使用屏障式避孕方法。
5)排除标准
符合下列任一标准的受试者，不得入选本研究：
1.患者有严重的全身性疾病，包括但不限于：无法控制的肝脏疾病、肾脏疾病、心血管疾病、神经系统疾病、免疫系统疾病、血液疾病、肿瘤或精神疾病，或任何其他无法安全遵循方案的状况。
2.病人有以下任何一种情况：需要每天治疗的哮喘病；过敏性/嗜酸性哮喘病史；过敏性反应病史；囊性纤维化病史；与COVID-19无关的任何其他呼吸道疾病；呼吸系统细菌感染，如化脓性扁桃体炎、与COVID-19无关的急性气管支气管炎、与COVID-19无关的鼻窦炎和中耳炎，或基线肺部疾病，如严重肺间质病变和/或支气管扩张症。
3.患者有严重的细菌、真菌或病毒的活动性感染(COVID-19除外)。
4.有SARS-CoV-2血清学阳性史。
5.在作为本研究入选标准的SARS-CoV-2的PCR或抗原检测之前，有阳性病史。
6.患者已经用研究性或紧急使用/批准的疫苗接种过COVID-19疫苗。
7.计划在研究过程中接受任何疫苗。在研究过程中，包括筛选期间，不允许接种疫苗，但流感疫苗除外。如果患者接受流感疫苗，它必须不是减毒活疫苗，而且必须在第29天访问后至少21天内进行接种。
8.患者在筛查时进行的以下任何一项实验室参数(根据当地实验室的参考范围)出现实验室异常。
a)丙氨酸氨基转移酶(ALT)或天门冬氨酸氨基转移酶(AST)>2.5×正常上限(ULN)值
b)总胆红素水平>1.5×ULN值
c)肌酐清除率<60mL/min/1.73m 2
9.患者在过去3个月内参加过其他药物的临床研究(包括任何治疗或预防COVID-19的研究)。
10.患者正在或曾经接受过针对SARS-CoV-2的特异性单克隆/多克隆抗体、针对SARS-CoV-2的抗病毒药物，和/或曾经接受过COVID-19康复患者的血浆(康复期血浆)治疗，无论是研究性使用还是紧急使用授权。
11.患者在筛查时正在接受大于20毫克泼尼松当量的系统性类固醇、白细胞介素-1抑制剂、白细胞介素-6抑制剂或静脉免疫球蛋白。在研究期间也不允许使用这些药物。
12.患者已知或怀疑对研究治疗配方中使用的任何成分过敏。
13.女性患者计划在服用研究药物的99天内怀孕或计划开始辅助生殖，如体外受精。
14.男性患者计划在研究过程中或在服用研究药物的99天内捐赠精子。
15.患者乙型肝炎表面抗原(HBsAg)阳性。
16.患者丙型肝炎病毒(HCV)感染阳性。HCV抗体反应，但确认未检测到HCV RNA的患者，如果这种情况以前被认为是稳定的，不需要治疗，或在完成适当的治疗后，肝功能正常，可以入选。
17.人类免疫缺陷病毒(HIV)阳性的患者。
18.尿液药物筛查呈阳性的患者，由研究者酌情排除。
19.患者在随机化前3个月内捐献过全血或血液成分或有大量出血(>400毫升)。
20.研究者确定参与研究不符合患者的最佳利益。
6)研究性产品：
名称：LY-CovMab
剂量：1200毫克或2400毫克
给药方式：静脉输液，90至120分钟
7)安慰剂产品：
名称：安慰剂
剂量：不适用
给药方式：静脉输液，90至120分钟
8)统计学方法：
样本量：
计划以1:1:1的随机比例招募约150名患者(每个治疗组n＝50名患者)进行研究。确定样本量是为了反映本研究的探索性质。
分析人群：
入选人群
入选人群将包括所有签署知情同意书(ICF)的个人。
完整分析组(Full Analysis Set)
完整的分析集(FAS)将包括所有被随机选中的患者，无论是否偏离方案或过早中止。治疗组的分配将根据最初的随机分配来指定。FAS将作为疗效分析的基础。
按方案设置(Per protocol Se)
没有严重违反协议的FAS的子集。
安全分析组(Safety Analysis Set)
安全性分析组将包括所有接受过一剂研究药物的随机患者。这个群体中的治疗组分配将由实际接受的治疗来定义。这个群体将被用于安全分析。
PK浓度分析组
PK浓度分析组(PKCS)被定义为所有接受LY-CovMab并且至少有一个可评估的血清浓度结果的患者。PKCS也将用于免疫原性的分析。
PK参数分析组
PK参数分析集(PKPS)是指所有接受LY-CovMab的患者，他们有足够的血清浓度数据来获得PK参数的估计，并且没有重大的协议偏差或其他对PK数据有影响的事件。
疗效分析
本研究是探索性的。数据将按观察结果使用，不使用归因方法。报告的P值将被视为名义值。
主要疗效分析
主要疗效分析将是比较FAS中每个LY-CovMab剂量组和安慰剂组之间病毒载量从基线的时间加权平均变化。
主要终点将采用协方差分析模型(ANCOVA)进行分析，治疗组和分层因素为固定效应(fixed effects)，基线病毒载量为协变量(covariate)。
将报告每个治疗组从第1天到第7天病毒载量的时间加权平均变化的最小二乘法平均估计值，以及每个LY CovMab组与安慰剂之间的差异，以及相关的95％CI、标准误差和相应的P值。
次要疗效分析
连续终点分析
通过RT-qPCR检测鼻咽拭子样本，SARS-CoV-2病毒载量从基线到第5、11、15、22和29天的时间加权平均变化，将在每个时间点进行描述性总结。它可能会酌情使用与主要终点类似的模型进行分析。
以下两个连续疗效变量将按访问进行描述性的总结。从基线开始的变化将在每个基线后的访问中计算和总结。可以酌情使用重复测量的混合模型(MMRM)来分析随时间的变化，以治疗组、分层因素(如果适用)、访问和治疗-访问交互作用作为固定效应，以基线值作为协变量。
-从基线到第3、5、7、11、15、22和29天的SARS-CoV-2病毒载量的变化
-第7、11、15和29天，世界卫生组织(WHO)8分制的基线变化。
事件终点的时间分析
事件发生的时间终点。
-通过RT-qPCR检测SARS-CoV-2为阴性的时间，随后没有出现RT-qPCR阳性。
-根据美国食品和药物管理局(FDA)推荐的COVID-19常见症状评估，到第29天症状改善的时间
-根据美国食品和药物管理局推荐的COVID-19常见症状评估，截至第29天的症状缓解时间
上述事件终点的时间分布将用Kaplan-Meier方法估计。每个治疗组的事件时间中位数及其95％CI将被报告，结果将按治疗组以图表形式呈现。此外，治疗效果危险比(HR)及其95％CI可能会酌情使用Cox回归模型来估计，治疗组和分层因素(如果适用)为固定效应。
二元端点分析
二元端点。
-第7天RT-qPCR检测SARS-COV-2阴性的患者比例
-在第7、11、15和29天，通过FDA的症状调查表显示症状改善的患者比例
-在第7天、第11天、第15天和第29天，通过FDA的症状调查表显示症状消失的患者比例
-在第29天因COVID-19恶化而需要住院治疗的患者比例。
实施例8在重度及危重新型冠状病毒肺炎患者中评价LY-CovMab的疗效和安全性的剂量探索临床研究
本研究采用多中心、随机、双盲、安慰剂对照、平行设计。计划入组108例重度及危重新型冠状病毒肺炎患者，按照1:1:1的比例随机分配到LY-CovMab 1200mg/次剂量组、LY-CovMab 2400mg/次剂量组或安慰剂组。
LY-CovMab组和安慰剂组的成分和给药方案与以上实施例相同。
受试者在给药前3天内完成筛选，经筛选合格的受试者随机进入相应试验组接受试验药物或安慰剂治疗，单次给药，临床观察周期为28天。
受试者将于给药前1天，给药后第2天、第5天、第8天、第11天、第14天、、第28天分别进行咽拭子病毒学检测，以观察病毒的转阴时间及第8天病毒转阴率。受试者每天测量体温，记录临床症状，并于第2天、第5天、第8天、第11天、第14天、第21天、第28天进行血常规检查。给药后按照规定进行体格检查、12导联心电图检查、实验室检查等安全性检查，在整个试验期间，记录不良事件发生情况。
入选标准
符合全部下列标准的受试者方可入选本研究：
1.年龄≥18岁，性别不限；
2.随机前72小时内实验室检查(RT-PCR)确认感染2019-nCoV，按NIH指南确诊的重度及危重新型冠状病毒肺炎患者；
3.距离发病时间≤12天；
4.育龄女性受试者筛选时妊娠试验阴性，且受试者及其伴侣在试验期间无妊娠计划并自愿采取有效避孕措施，避免使自己或伴侣怀孕；
5.必须对本试验知情同意，并自愿签署书面的知情同意书，能够遵守临床试验的各种要求(计划内访视、实验室检查和其他试验程序等)。
排除标准
符合下列任一标准的受试者，不得入选本研究：
1.预计48小时内死亡者；
2.计划一年内生育，或妊娠期、哺乳期女性，或育龄期女性血妊娠筛查呈阳性；
3.过敏体质(对两种或两种以上的药物或食物过敏)，或经研究者判断可能或明确对研究药物任一组分过敏者；
4.有以下严重呼吸系统疾病病史者：如哮喘，支气管扩张、慢性阻塞性肺部疾病、肺间质病等影响临床试验评估的呼吸系统疾病；
5.伴有其他严重疾病或疾病史者，包括但不限于未控制已经转移不能切除的恶性肿瘤、血液病、恶液质、活动性出血、已知严重肾功能不全(估计肾小球滤过率≤30mL/min/1.73m2)或接受连续性肾脏替代治疗，血液透析，腹膜透析的患者、严重营养不良、精神疾病等；
6.筛选时出现以下任何实验室参数异常(根据当地实验室参考范围)：
a.ALT或AST水平>5倍正常范围上限(ULN)；ALT或AST水平>3倍ULN且总胆红素水平>2倍ULN；
b.抗HIV检测阳性；
7.在过去3个月内参与了其它药物的临床研究(包括新型冠状病毒肺炎治疗的研究，如给药前30天内接受了SARS-CoV-2预防治疗；既往接受过SARS-CoV-2特异性单克隆抗体的治疗；既往曾接受过COVID-19康复者血浆治疗者)；
8.研究者认为参加本项研究不符合受试者最大利益，或可能存在的潜在的违背试验依从性或者影响安全性和有效性评价的其他任何情况者(包括将在72小时内转移到不是研究地点的另一家医院)。
实施例9在与新型冠状病毒肺炎患者接触的健康受试者中评价LY-CovMab注射液的预防疗效和安全性的临床研究
本研究采用多中心、随机、双盲、安慰剂对照、平行设计。计划入组2000例与新型冠状 病毒肺炎患者接触的健康受试者，按照1:1的比例随机分配到LY-CovMab 1200mg/次剂量组、LY-CovMab 2400mg/次剂量组或安慰剂组。LY-CovMab组和安慰剂组的成分和给药方案与以上实施例相同。
受试者在给药前3天内完成筛选，经筛选合格的受试者随机进入相应试验组接受试验药物或安慰剂治疗，单次给药，临床观察周期为3个月。
入选标准
符合全部下列标准的受试者方可入选本研究：
1.受试者必须对本试验知情同意，并自愿签署书面的知情同意书，能够和研究者保持良好的沟通并且遵守临床试验的各种要求(计划内访视、实验室检查和其他试验程序)；
2.年龄≥18岁，性别不限；
3.与诊断为SARS-CoV-2感染的病例有接触的无症状的家庭成员，且必须在收集确诊病例SARS-COV-2的诊断试验样本的96小时内进行随机化。筛选期生命体征、体格检查、实验室检查、12导联心电图、胸部CT等无异常或异常无临床意义者；有临床意义经研究者判断处于稳定期者；
4.筛选时，育龄女性(定义为：没有接受过手术绝育或绝经后不到1年的女性)的血妊娠试验结果为阴性。育龄男性和女性受试者同意至少在筛选前2周至试验用药后6个月内采取有效避孕措施。
排除标准
符合下列任一标准的受试者，不得入选本研究：
1.罹患新型冠状病毒肺炎或痊愈者；或者新型冠状病毒核酸，血清新冠病毒特异性抗体任一检测结果阳性；
2.与曾有SARS-CoV-2感染患者生活史者；伴有严重的系统性疾病：包括但不限于肝脏疾病、肾脏疾病、心血管疾病、神经系统疾病、免疫系统疾病、血液疾病、肿瘤、精神疾病，经研究者判断不适合入组者；
3.有以下状况的受试者：需每日治疗的哮喘，任何其他慢性呼吸道疾病，呼吸系统细菌感染如化脓性扁桃体炎，急性气管支气管炎，鼻窦炎，中耳炎等其他影响临床试验评估的呼吸道疾病。胸部CT证实存在严重的肺间质病变、支气管扩张等基础性肺部疾病患者；
4.既往参与过新型冠状病毒肺炎预防和治疗药物研究(如既往接受过SARS-CoV-2特异性单克隆抗体的治疗；既往接受过COVID-19康复者血浆治疗者；或既往接受过新型冠状病毒肺炎疫苗者等)在过去3个月内参与了其它药物的临床试验研究。；
5.过敏体质(对两种或两种以上的药物或食物过敏)，或经研究者判断可能或明确对研究药物任一组分过敏者；
6.妊娠期、哺乳期女性
7.乙肝表面抗原(HBsAg)检测阳性、丙型肝炎病毒(HCV)抗体、梅毒螺旋体抗体、人类免疫缺陷病毒(HIV)抗体阳性；
8.研究者认为其他需要排除者。

Claims (14)

  1. 一种结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段在制备治疗或预防SARS-CoV-2病毒引起的疾病的药物中的应用,
    包括向有此需要的受试者施用所述结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段；
    所述SARS-CoV-2病毒包括Wuhan-Hu-1毒株、B.1.1.7毒株、B.1.351毒株、p.1毒株、B.1.617.2毒株和C.37毒株中的一种或多种，所述疾病包括COVID-19肺炎及其他相关并发症；
    优选的，所述抗体或抗原结合片段结合SARS-CoV-2病毒的RBD的A475、E484、G485、N487、Y489、Q493、S494、Y449、Y453、L455、F456、F486和F490残基。
  2. 一种结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段在治疗或预防SARS-CoV-2病毒引起的疾病中的应用，
    包括向有此需要的受试者施用所述结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段；
    所述SARS-CoV-2病毒包括Wuhan-Hu-1毒株、B.1.1.7毒株、B.1.351毒株、p.1毒株、B.1.617.2毒株和C.37毒株中的一种或多种，所述疾病包括COVID-19肺炎及其他相关并发症；
    优选的，所述抗体或抗原结合片段结合SARS-CoV-2病毒的RBD的A475、E484、G485、N487、Y489、Q493、S494、Y449、Y453、L455、F456、F486和F490残基。
  3. 一种利用结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段治疗或预防SARS-CoV-2病毒引起的疾病方法，
    包括向有此需要的受试者施用所述结合新型冠状病毒SARS-CoV-2的S蛋白的抗体或抗原结合片段；
    所述SARS-CoV-2病毒包括Wuhan-Hu-1毒株、B.1.1.7毒株、B.1.351毒株、p.1毒株、B.1.617.2毒株和C.37毒株中的一种或多种，所述疾病包括COVID-19肺炎及其他相关并发症；
    优选的，所述抗体或抗原结合片段结合SARS-CoV-2病毒的RBD的A475、E484、G485、N487、Y489、Q493、S494、Y449、Y453、L455、F456、F486和F490残基。
  4. 根据权利要求1-3任一项所述的应用或方法，所述抗体或抗原结合片段包含3个轻链互补决定区和/或3个重链互补决定区，其中
    所述抗体或抗原结合片段的3个轻链互补决定区包含SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:8所示的LCDR3，和/或所述抗体或抗原结合片段的3个重链互补决定区包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:11所示的HCDR3；
    所述抗体或抗原结合片段的3个轻链互补决定区包含SEQ ID NO:12所示的LCDR1、SEQ ID NO:13所示的LCDR2和SEQ ID NO:14所示的LCDR3，和/或所述抗体或抗原结合片段的3个重链互补决定区包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:11所示的HCDR3；
    所述抗体或抗原结合片段的3个轻链互补决定区包含SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:15所示的LCDR3，和/或所述抗体或抗原结合片段的3个重链互补决定区包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:16所示的HCDR2和SEQ ID NO:17所示的HCDR3；或者
    所述抗体或抗原结合片段的3个轻链互补决定区包含SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:21所示的LCDR3，和/或所述抗体或抗原结合片段的 3个重链互补决定区包含SEQ ID NO:9所示的HCDR1、SEQ ID NO:10所示的HCDR2和SEQ ID NO:11所示的HCDR3。
  5. 根据权利要求1-4任一项所述的应用或方法，其特征在于，
    所述抗体或抗原结合片段包含SEQ ID NO:1所示的轻链可变区，和/或SEQ ID NO:2所示的重链可变区；
    所述抗体或抗原结合片段包含SEQ ID NO:3所示的轻链可变区，和/或SEQ ID NO:2所示的重链可变区；
    所述抗体或抗原结合片段包含SEQ ID NO:4所示的轻链可变区，和/或SEQ ID NO:5所示的重链可变区；或者
    所述抗体或抗原结合片段包含SEQ ID NO:20所示的轻链可变区，和/或SEQ ID NO:2所示的重链可变区。
  6. 根据权利要求1至5任一所述的应用或方法，其特征在于，所述抗体包含SEQ ID NO:18所示的重链恒定区，或者包含SEQ ID NO:19所示的轻链恒定区。
  7. 根据权利要求1至6任一所述的应用或方法，其中所述抗体或抗原结合片段以药物组合物的形式施用，所述药物组合物含有所述抗体或抗原结合片段以及缓冲液；优选的，所述缓冲液包括海藻糖和聚山梨酯80中的一种或多种；更优选的，所述药物组合物pH为5.5-6.5；更优选的，所述缓冲液还包括盐酸组氨酸和组氨酸中的一种或多种；更优选的，所述盐酸组氨酸和组氨酸的摩尔比为10.5：9.5；更优选的，基于所述药物组合物的总体积，所述药物组合物包括0.04-0.1g/mL海藻糖,0.0001-0.0003g/mL聚山梨酯80，以及10-30mg/mL的所述抗体或抗原结合片段；更优选的，基于所述药物组合物的总体积，所述药物组合物包括10.5mM盐酸组氨酸,9.5mM组氨酸,0.08g/mL海藻糖，0.0002g/mL聚山梨酯80，以及20±2mg/mL的所述抗体或抗原结合片段。
  8. 根据权利要求1至7任一所述的应用或方法，所述受试者为感染SARS-CoV-2病毒的受试者、与感染SARS-CoV-2病毒的受试者有接触的无症状的受试者、健康受试者、或者其他不适合接种疫苗的受试者；优选的，所述感染SARS-CoV-2病毒的受试者包括无症状的SARS-CoV-2病毒感染的受试者或者COVID-19患者；更优选，所述COVID-19患者为轻度、中度、重度或危重COVID-19患者。
  9. 根据权利要求1至8任一所述的应用或方法，其中所述感染SARS-CoV-2病毒的受试者为轻度及中度COVID-19患者；优选的，所述COVID-19患者为发病时间≤7天且为在72小时之内按照NIH指南确诊的轻度及中度COVID-19患者；
    所述感染SARS-CoV-2病毒的受试者为轻型及普通型新型冠状病毒肺炎患者；优选的，所述COVID-19患者为发病时间≤7天且72小时之内按照《新型冠状病毒肺炎诊疗方案(试行第八版修订版)》确诊的轻型及普通型新型冠状病毒肺炎患者；或者
    所述感染SARS-CoV-2病毒的受试者为重度及危重COVID-19患者；优选的，所述COVID-19患者为72小时内实验室检查(如RT-PCR检查)确认感染SARS-CoV-2，且按NIH指南确诊的重度及危重COVID-19的患者。
  10. 根据权利要求1至9任一所述的应用或方法，包括向有此需要的受试者施用约30mg至约2400mg，优选的，约1200mg或约2400mg的权利要求1至6中任一所述抗体或抗原结合片段；优选的，向有此需要的受试者施用含有效量的权利要求1至6中任一所述抗体或抗原结合片段的药物组合物；更优选的，所述药物组合物为单位制剂，该单位制剂含有约30mg 至约2400mg，优选的，约1200mg或约2400mg的权利要求1至6中任一所述抗体或抗原结合片段；更优选的，所述药物组合物为注射制剂。
  11. 根据权利要求1至10任一所述的应用或方法，将含有效量的所述抗体或抗原结合片段的药物组合物稀释后静脉滴注给所述受试者；优选的，将所述药物组合物稀释至100-250mL，在30-120分钟内施用给受试者；更优选的，将所述药物组合物稀释为250mL，在90-120分钟内施用给受试者；更优选的，将所述药物组合物稀释为100mL，在30分钟内施用给受试者；更优选的，使用0.9％氯化钠溶液对所述药物组合物进行稀释。
  12. 根据权利要求1至11任一所述的应用或方法，包括向有此需要的受试者施用权利要求1至6中任一所述抗体或抗原结合片段，以及结合新型冠状病毒SARS-CoV-2的另外一种抗体或抗原结合片段；优选的，所述另外一种抗体或抗原结合片段与新型冠状病毒SARS-CoV-2的S蛋白结合；更优选的，另外一种抗体或抗原结合片段与权利要求1至6中任一所述抗体或抗原结合片段结合S蛋白的表位不重叠或部分重叠。
  13. 根据权利要求1至12任一所述的应用或方法，在对所述受试者施用所述抗体或抗原结合片段，或施用含所述抗体或抗原结合片段的药物组合物之前，还包括检测所述受试者感染的新冠病毒类型的步骤。
  14. 根据权利要求12所述的应用或方法，向受试者同一时间或不同时间施用权利要求1至6中任一所述抗体或抗原结合片段，以及结合新型冠状病毒SARS-CoV-2的另外一种抗体或抗原结合片段。
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