Human monoclonal antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Abstract

The present disclosure relates to antibodies that bind to and neutralize the coronavirus designated SARS-CoV-2 and methods of use thereof.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application Nos. 63 / 000,299, filed March 26, 2020; 63 / 002,896, filed March 31, 2020; 63 / 003,716, filed April 1, 2020; 63 / 023,545, filed May 12, 2020; 63 / 024,204, filed May 13, 2020; 63 / 024,248, filed May 19, 2020; This application claims priority to US Pat. No. 63 / 027,173, filed June 11, 2020, US Pat. No. 63 / 037,984, filed June 17, 2020, US Pat. No. 63 / 040,224, filed June 17, 2020, US Pat. No. 63 / 040,246, filed June 17, 2020, US Pat. No. 63 / 142,196, filed January 27, 2021, and US Pat. No. 63 / 161,890, filed March 16, 2021, each of which is incorporated by reference in its entirety.

[0002] Federal Grant Disclosure This invention was made with government support under award HR0011-18-2-0001 by the Defense Advanced Research Projects Agency (DARPA) and HHS Contract 75N93019C00074 awarded by the National Institute of Allergy and Infectious Diseases / National Institutes of Health. The federal government has certain rights in this invention.

[0003] Reference to an electronically submitted sequence listing The contents of the electronically submitted Sequence Listing (Name: 4815-001PC0D_SL_ST25.txt; Size: 87,769 bytes; and Creation Date: March 24, 2021) are incorporated herein by reference in their entirety.

[0004] This disclosure relates generally to the fields of medicine, infectious diseases, and immunology. More particularly, this disclosure relates to human antibodies that bind to the novel coronavirus designated SARS-CoV-2 and methods of use thereof. [Background technology]

[0005] The novel coronavirus (SARS-CoV-2) infection originated in mainland China and has spread to 179 other countries and territories. The novel coronavirus was identified in Wuhan, the capital of Hubei Province, China, after which 41 people developed pneumonia without apparent cause. The virus, which causes an acute respiratory illness known as coronavirus disease 2019 (COVID-19), can spread from person to person. The incubation period (time from exposure to onset of symptoms) ranges from 0 to 24 days, with an average of 3 to 5 days, but individuals may be contagious during this period and after recovery. Symptoms include fever, cough, and difficulty breathing. The estimated mortality rate in February 2020 was 2% of confirmed cases, with a higher rate among those requiring hospitalization.

[0006] As of February 10, 2020, there were 40,627 confirmed cases (6,495 severe), including all provincial-level regions in China. Many more people may be infected but have not been detected (especially with milder cases). As of February 10, 2020, 910 deaths have been attributed to the virus since the first confirmed death on January 9, and 3,323 have recovered. The first local transmission outside of China occurred among a family in Vietnam, while the first non-family-related international transmission occurred in Germany on January 22. The first death outside of China occurred in the Philippines, where a man from Wuhan died on February 1. As of February 10, 2020, the number of deaths attributed to the virus exceeded that of the 2003 global SARS pandemic. Summary of the Invention [Problem to be solved by the invention]

[0007] As of early February 2020, there were no licensed vaccines or specific treatments, although several vaccine approaches and antiviral drugs are under investigation. This outbreak has led to the World Health Organization (WHO) declaring a Public Health Emergency of International Concern (PHEIC) based on the potential impact the virus could have if it spreads to countries with weak health systems. Therefore, there is an urgent need to investigate the biology and pathology of SARS-CoV-2 and the human immune response to this virus. [Means for solving the problem]

[0008] Thus, the present disclosure provides a method for detecting COVID-19 infection caused by SARS-CoV-2 in a subject, the method comprising: (a) contacting a sample from the subject with an antibody or antibody fragment having the clone-pair heavy and light chain CDR sequences of Tables 3 and 4, respectively; and (b) detecting SARS-CoV-2 in the sample by binding of the antibody or antibody fragment to a SARS-CoV-2 antigen in the sample. The sample may be a bodily fluid, such as blood, sputum, tears, saliva, mucus or serum, semen, cervical mucus or vaginal secretions, amniotic fluid, placental tissue, urine, exudate, transudate, tissue scraping, or feces. Detection may involve ELISA, RIA, lateral flow assay, or Western blot. The method may further comprise performing steps (a) and (b) twice and measuring changes in SARS-CoV-2 antigen levels compared to the first assay. The antibody or antibody fragment may be encoded by a clone-pair variable sequence as set forth in Table 1. The antibody or antibody fragment may be encoded by light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair variable sequences as set forth in Table 1, or light and heavy chain variable sequences having 100% identity to the clone pair sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to the clone pair sequences in Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair sequences in Table 2. The antibody or antibody fragment may bind to the SARS-CoV-2 surface spike protein. The antibody fragment may be a recombinant scFv (single-chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment.

[0009] In another embodiment, a method is provided for treating a subject infected with SARS-CoV-2 or reducing the likelihood of infection in a subject at risk for SARS-CoV-2, comprising delivering to the subject an antibody or antibody fragment having the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair variable sequences as set forth in Table 1, or light and heavy chain variable sequences having 100% identity to the clone pair sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to the clone pair sequences in Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair sequences in Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. The antibody may be a chimeric or bispecific antibody. The antibody may be an IgG or recombinant IgG antibody or antibody fragment containing an Fc portion mutated to alter (eliminate or enhance) FcR interaction to increase half-life and / or increase therapeutic efficacy, such as LALA, LALA PG, N297, GASD / ALIE, DHS, YTE, or LS mutations, or glycans modified to alter (eliminate or enhance) FcR interaction, such as by enzymatic or chemical addition, or expression in a cell line engineered with glycan removal or defined glycosylation patterns. The antibody or antibody fragment may bind to a SARS-CoV-2 antigen, such as the surface spike protein. The antibody or antibody fragment may be administered before or after infection. The subject may be 60 years of age or older, may be immunocompromised, or may suffer from respiratory and / or cardiovascular disorders. Delivery may include administration of the antibody or antibody fragment or gene delivery by RNA or DNA sequences or vectors encoding the antibody or antibody fragment.

[0010] In yet another embodiment, a monoclonal antibody is provided, wherein the antibody or antibody fragment is characterized by the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair variable sequences as set forth in Table 1, or light and heavy chain variable sequences having 100% identity to the clone pair sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to the clone pair sequences in Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair sequences in Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. The antibody may be a chimeric antibody, a bispecific antibody, or an intrabody. The antibody may be an IgG or recombinant IgG antibody or antibody fragment that contains an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or increase therapeutic efficacy, such as the LALA, LALA PG, N297, GASD / ALIE, DHS, YTE, or LS mutations, or glycans that have been modified to alter (eliminate or enhance) FcR interactions, such as by enzymatic or chemical addition, or expression in a cell line engineered with glycan removal or defined glycosylation patterns. The antibody or antibody fragment may bind to the SARS-CoV-2 surface spike protein.

[0011] A hybridoma or engineered cell encoding an antibody or antibody fragment, wherein the antibody or antibody fragment is characterized by the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair variable sequences as set forth in Table 1, or light and heavy chain variable sequences having 100% identity to the clone pair sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to the clone pair sequences of Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair sequences of Table 2. The antibody fragment may be a recombinant scFv (single-chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. The antibody may be a chimeric antibody, a bispecific antibody, or an intrabody. The antibody may be an IgG or recombinant IgG antibody or antibody fragment that contains an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or increase therapeutic efficacy, such as the LALA, LALA PG, N297, GASD / ALIE, DHS, YTE, or LS mutations, or glycans that have been modified to alter (eliminate or enhance) FcR interactions, such as by enzymatic or chemical addition, or expression in a cell line engineered with glycan removal or defined glycosylation patterns. The antibody or antibody fragment may bind to the SARS-CoV-2 surface spike protein.

[0012] In yet another embodiment, a vaccine formulation is provided comprising one or more antibodies or antibody fragments characterized by the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively. At least one of the antibodies or antibody fragments may be encoded by light and heavy chain variable sequences according to the clone pair sequences of Table 1, light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair sequences of Table 1, or light and heavy chain variable sequences having at least 95% identity to the clone pair sequences of Table 1. At least one of the antibodies or antibody fragments may comprise light and heavy chain variable sequences according to the clone pair sequences of Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair sequences of Table 2. At least one of the antibody fragments is a recombinant scFv (single-chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. At least one of the antibodies may be a chimeric antibody, a bispecific antibody, or an intrabody. The antibody may be an IgG or recombinant IgG antibody or antibody fragment that contains an Fc portion that has been mutated to alter (eliminate or enhance) FcR interactions to increase half-life and / or increase therapeutic efficacy, such as the LALA, LALA PG, N297, GASD / ALIE, DHS, YTE, or LS mutations, or glycans that have been modified to alter (eliminate or enhance) FcR interactions, such as by enzymatic or chemical addition, or expression in a cell line engineered with glycan removal or defined glycosylation patterns. The antibody or antibody fragment may bind to the surface spike protein of a SARS-CoV-2 antigen.

[0013] In a further embodiment, there is provided a vaccine formulation comprising one or more expression vectors encoding a first antibody or antibody fragment as described herein. The expression vector may be a Sindbis virus vector or a VEE vector. The vaccine may be formulated for delivery by needle injection, jet injection, or electroporation. The vaccine formulation may further comprise one or more expression vectors encoding a second antibody or antibody fragment, such as a distinct antibody or antibody fragment of claims 26-34.

[0014] In yet a further embodiment, there is provided a method of protecting the health of a subject aged 60 years or older, an immunocompromised subject, or a subject suffering from a respiratory and / or cardiovascular disorder who is infected with or at risk of infection with SARS-CoV-2, comprising delivering to the subject an antibody or antibody fragment having the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively. The antibody or antibody fragment may be encoded by light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair variable sequences as set forth in Table 1, or light and heavy chain variable sequences having 100% identity to the clone pair sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to the clone pair sequences in Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair sequences in Table 2. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. The antibody may be an IgG or recombinant IgG antibody or antibody fragment containing an Fc portion mutated to alter (eliminate or enhance) FcR interaction to increase half-life and / or increase therapeutic efficacy, such as LALA, LALA PG, N297, GASD / ALIE, DHS, YTE, or LS mutations, or glycans modified to alter (eliminate or enhance) FcR interaction, such as by enzymatic or chemical addition, or expression in a cell line engineered with glycan removal or defined glycosylation patterns. The antibody may be a chimeric or bispecific antibody. The antibody or antibody fragment may be administered before or after infection. The antibody or antibody fragment may bind to a SARS-CoV-2 antigen, such as the surface spike protein. Delivery may include administration of the antibody or antibody fragment or gene delivery by RNA or DNA sequences or vectors encoding the antibody or antibody fragment. The antibody or antibody fragment may improve a subject's breathing compared to untreated controls and / or reduce viral load compared to untreated controls.

[0015] In yet a further embodiment, a method for determining the antigenic integrity, correct conformation, and / or correct sequence of a SARS-CoV-2 surface spike protein is provided, comprising: (a) contacting a sample containing the antigen with a first antibody or antibody fragment having the clone-pair heavy and light chain CDR sequences of Tables 3 and 4, respectively; and (b) determining the antigenic integrity, correct conformation, and / or correct sequence of the antigen by detectable binding of the first antibody or antibody fragment to the antigen. The sample may comprise a recombinantly produced antigen or vaccine formulation or vaccine production batch. Detection may include ELISA, RIA, Western blot, biosensor or biolayer interferometry using surface plasmon resonance, or flow cytometry staining. The first antibody or antibody fragment may be encoded by a clone-pair variable sequence as set forth in Table 1, light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone-pair variable sequence as set forth in Table 1, or light and heavy chain variable sequences having at least 95% identity to the clone-pair sequence as set forth in Table 1. The first antibody or antibody fragment may comprise light and heavy chain variable sequences according to the clone pair sequences in Table 2, or may comprise light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair sequences in Table 2, or may comprise light and heavy chain variable sequences having at least 95% identity to the clone pair sequences in Table 2. The first antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. The method may further comprise performing steps (a) and (b) twice to determine the antigen stability of the antigen over time.

[0016] The method may further include (c) contacting a sample containing the antigen with a second antibody or antibody fragment having the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively, and (d) determining antigen integrity of the antigen by detectable binding of the second antibody or antibody fragment to the antigen. The second antibody or antibody fragment may be encoded by a clone pair variable sequence as set forth in Table 1, light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair variable sequences as set forth in Table 1, or light and heavy chain variable sequences having at least 95% identity to the clone pair sequences as set forth in Table 1. The second antibody or antibody fragment may comprise light and heavy chain variable sequences according to the clone pair sequences of Table 2, or may comprise light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair sequences of Table 2, or may comprise light and heavy chain variable sequences having at least 95% identity to the clone pair sequences of Table 2. The second first antibody fragment can be a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. The method can further include performing steps (c) and (d) twice to determine the antigen stability of the antigen over time.

[0017] Also provided is a human monoclonal antibody or antibody fragment, or a hybridoma or engineered cell producing the same, which binds to the surface spike protein of a SARS-CoV-2 antigen.

[0018] In one embodiment (A1) provided herein, a method for detecting COVID-19 infection caused by SARS-CoV-2 in a subject includes (a) contacting a sample from the subject with an antibody or antibody fragment having the clone-pair heavy and light chain CDR sequences of Tables 3 and 4, respectively, and (b) detecting SARS-CoV-2 in the sample by binding of the antibody or antibody fragment to a SARS-CoV-2 antigen in the sample. In one embodiment of A1 (A2), the sample is a bodily fluid. In one embodiment of A1 or A2 (A3), the sample is blood, sputum, tears, saliva, mucus or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissue, urine, exudate, transudate, tissue scraping, or feces. In one embodiment of any one of A1-A3 (A4), detecting includes ELISA, RIA, lateral flow assay, or Western blot. In one embodiment of any one of A1-A4 (A5), the method further comprises performing steps (a) and (b) twice and measuring the change in SARS-CoV-2 antigen levels compared to the first assay. In one embodiment of any one of A1-A5 (A6), the antibody or antibody fragment is encoded by a clone pair variable sequence as set forth in Table 1. In one embodiment of any one of A1-A5 (A7), the antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to a clone pair variable sequence as set forth in Table 1. In one embodiment of any one of A1-A5 (A8), the antibody or antibody fragment is encoded by light and heavy chain variable sequences having 100% identity to a clone pair sequence as set forth in Table 1. In one embodiment of any one of A1-A5 (A9), the antibody or antibody fragment comprises light and heavy chain variable sequences according to the clone pair sequences in Table 2. In one embodiment of any one of A1 to A5 (A10), the antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80%, 90% or 95% identity to the clone pair sequences in Table 2. In one embodiment of any one of A1 to A10 (A11), the antibody or antibody fragment binds to the SARS-CoV-2 surface spike protein.In one embodiment of any one of A1 to A11 (A12), the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment or an Fv fragment.

[0019] In one aspect provided herein (A13), a method of treating a subject infected with SARS-CoV-2 or reducing the likelihood of infection in a subject at risk for SARS-CoV-2 comprises delivering to the subject an antibody or antibody fragment having the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively. In an aspect of A13 (A14), the antibody or antibody fragment is encoded by clone pair light and heavy chain variable sequences as set forth in Table 1. In an aspect of A13 or A14 (A15), the antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair sequences of Table 1. In an aspect of A13 (A16), the antibody or antibody fragment comprises light and heavy chain variable sequences according to the clone pair sequences of Table 2. In one embodiment of A13 (A17), the antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair sequences in Table 2. In one embodiment of A13 (A18), the antibody or antibody fragment comprises light and heavy chain variable sequences having at least 95% identity to the clone pair sequences in Table 2. In one embodiment of any one of A13 to A18 (A19), the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. In one embodiment of any one of A13 to A19 (A20), the antibody is an IgG or recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interaction to increase half-life and / or increase therapeutic effect, such as a LALA, LALA PG, N297, GASD / ALIE, DHS, YTE, or LS mutation, or glycans modified to alter (eliminate or enhance) FcR interaction, such as by enzymatic or chemical addition, or expression in a cell line modified with glycan removal or defined glycosylation pattern. In one embodiment of any one of A13 to A18 (A21), the antibody is a chimeric or bispecific antibody. In one embodiment of any one of A13 to A21 (A22), the antibody or antibody fragment binds to the SARS-CoV-2 surface spike protein.In one embodiment of any one of A13 to A22 (A23), the antibody or antibody fragment is administered before or after infection. In one embodiment of any one of A13 to A23 (A24), the subject is 60 years of age or older, is immunocompromised, or suffers from a respiratory and / or cardiovascular disorder. In one embodiment of any one of A13 to A24 (A25), delivery includes administration of the antibody or antibody fragment, or gene delivery by an RNA sequence or DNA sequence or vector encoding the antibody or antibody fragment.

[0020] In one aspect (A26), there is provided herein a monoclonal antibody, wherein the antibody or antibody fragment is characterized by the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively. In an aspect of A26 (A27), the antibody or antibody fragment is encoded by light and heavy chain variable sequences according to the clone pair sequences of Table 1. In an aspect of A26 (A28), the antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair sequences of Table 1. In an aspect of A26 (A29), the antibody or antibody fragment comprises light and heavy chain variable sequences according to the clone pair sequences of Table 2. In an aspect of A26 (A30), the antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair sequences of Table 2. In one embodiment of any one of A26 to A30 (A31), the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In one embodiment of any one of A26 to A30 (A32), the antibody is a chimeric antibody or a bispecific antibody. In one embodiment of any one of A26 to A32 (A33), the antibody is an IgG or recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interaction to increase half-life and / or increase therapeutic effect, such as a LALA, LALA PG, N297, GASD / ALIE, DHS, YTE, or LS mutation, or glycans modified to alter (eliminate or enhance) FcR interaction, such as by enzymatic or chemical addition, or expression in a cell line engineered with glycan removal or defined glycosylation pattern. In one embodiment of any one of A26 to A33 (A34), the antibody or antibody fragment binds to a SARS-CoV-2 antigen, such as a surface spike protein. In one embodiment of any one of A26 to A34 (A35), the antibody is an intracellular antibody.

[0021] In one embodiment provided herein (A36), the hybridoma or engineered cell encodes an antibody or antibody fragment characterized by the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively. In one embodiment of A36 (A37), the antibody or antibody fragment is encoded by light and heavy chain variable sequences according to the clone pair sequences in Table 1. In one embodiment of A36 (A38), the antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair variable sequences of Table 1. In one embodiment of A36 (A39), the antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 95% identity to the clone pair variable sequences of Table 1. In one embodiment of A36 (A40), the antibody or antibody fragment comprises light and heavy chain variable sequences according to the clone pair sequences in Table 2. In one embodiment of A36 (A41), the antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair variable sequences of Table 2. In one embodiment of A36 (A42), the antibody or antibody fragment comprises light and heavy chain variable sequences having at least 95% identity to the clone pair sequences of Table 2. In one embodiment of any one of A36 to A42 (A43), the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In one embodiment of any one of A36 to A43 (A44), the antibody is a chimeric antibody, bispecific antibody, or intrabody. In one embodiment of any one of A36 to A43 (A45), the antibody is an IgG or recombinant IgG antibody or antibody fragment comprising an Fc portion that has been mutated to alter (eliminate or enhance) FcR interaction to increase half-life and / or increase therapeutic effect, such as a LALA, LALA PG, N297, GASD / ALIE, DHS, YTE or LS mutation, or glycans that have been modified to alter (eliminate or enhance) FcR interaction, such as by enzymatic or chemical addition, or by removal of glycans or expression in a cell line engineered with a defined glycosylation pattern.In one embodiment of any one of A36 to A45 (A46), the antibody or antibody fragment binds to the SARS-CoV-2 surface spike protein.

[0022] In one embodiment provided herein (A47), the vaccine formulation comprises one or more antibodies or antibody fragments characterized by the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively. In one embodiment of A47 (A48), at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences according to the clone pair sequences of Table 1. In one embodiment of A47 (A49), at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair sequences of Table 1. In one embodiment of A47 (A50), at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences having at least 95% identity to the clone pair sequences of Table 1. In one embodiment of A47 (A51), at least one of said antibodies or antibody fragments comprises light and heavy chain variable sequences according to the clone pair sequences of Table 2. In one embodiment of A47 (A52), at least one of the antibodies or antibody fragments comprises light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to the clone pair sequences in Table 2. In one embodiment of any one of A47 to A52 (A53), at least one of the antibody fragments is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In one embodiment of any one of A47 to A52 (A54), at least one of the antibodies is a chimeric antibody, a bispecific antibody, or an intrabody. In one embodiment of any one of A47 to A54 (A55), the antibody is an IgG or recombinant IgG antibody or antibody fragment comprising an Fc portion that has been mutated to alter (eliminate or enhance) FcR interaction to increase half-life and / or increase therapeutic effect, such as a LALA, LALA PG, N297, GASD / ALIE, DHS, YTE or LS mutation, or glycans that have been modified to alter (eliminate or enhance) FcR interaction, such as by enzymatic or chemical addition, or by removal of glycans or expression in a cell line engineered with a defined glycosylation pattern.In one embodiment of any one of A47 to A55 (A56), the antibody or antibody fragment binds to the SARS-CoV-2 surface spike protein.

[0023] In one aspect provided herein (A57), the vaccine formulation comprises one or more expression vectors encoding a first antibody or antibody fragment according to any one of A26-A34. In an aspect of A57 (A58), the expression vector is a Sindbis virus vector or a VEE vector. In an aspect of A57 or A58 (A59), the vaccine formulation is formulated for delivery by needle injection, jet injection, or electroporation. In an aspect of A57 (A60), the vaccine formulation further comprises one or more expression vectors encoding a second antibody or antibody fragment, such as a distinct antibody or antibody fragment of any one of A26-A34.

[0024] In one aspect provided herein (A61), a method of protecting the health of a subject aged 60 years or older, an immunocompromised subject, or a subject suffering from a respiratory and / or cardiovascular disorder who is infected or at risk of infection with SARS-CoV-2 comprises delivering to said subject an antibody or antibody fragment having the clone pair heavy and light chain CDR sequences of Tables 3 and 4, respectively. In one aspect of A61 (A62), the antibody or antibody fragment is encoded by a clone pair light and heavy chain variable sequences as set forth in Table 1. In one aspect of A61 or A62 (A63), the antibody or antibody fragment is encoded by a clone pair light and heavy chain variable sequences having at least 95% identity as set forth in Table 1. In one aspect of A61 or A62 (A64), the antibody or antibody fragment is encoded by a clone pair light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair sequences of Table 1. In one embodiment of A61 (A65), the antibody or antibody fragment comprises light and heavy chain variable sequences according to the clone pair sequences in Table 2. In one embodiment of A61 (A66), the antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair sequences in Table 2. In one embodiment of A61 (A67), the antibody or antibody fragment comprises light and heavy chain variable sequences having at least 95% identity to the clone pair sequences in Table 2. In one embodiment of any one of A61 to A67 (A68), the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment, or an Fv fragment. In one embodiment of any one of A61 to A68 (A69), the antibody is an IgG or recombinant IgG antibody or antibody fragment comprising an Fc portion that has been mutated to alter (eliminate or enhance) FcR interaction to increase half-life and / or increase therapeutic effect, such as a LALA, LALA PG, N297, GASD / ALIE, DHS, YTE or LS mutation, or glycans that have been modified to alter (eliminate or enhance) FcR interaction, such as by enzymatic or chemical addition, or by removal of glycans or expression in a cell line engineered with a defined glycosylation pattern.In one embodiment of any one of A61 to A67 (A70), the antibody is a chimeric antibody or a bispecific antibody. In one embodiment of any one of A61 to A70 (A71), the antibody or antibody fragment is administered pre- or post-infection. In one embodiment of any one of A61 to A71 (A72), the antibody or antibody fragment binds to the SARS-CoV-2 surface spike protein. In one embodiment of any one of A61 to A72 (A73), delivery comprises administration of the antibody or antibody fragment or gene delivery by an RNA sequence or DNA sequence or vector encoding the antibody or antibody fragment. In one embodiment of A61 (A74), the antibody or antibody fragment improves breathing in a subject compared to an untreated control. In one embodiment of A61 (A75), the antibody or antibody fragment reduces viral load compared to an untreated control.

[0025] In one embodiment provided herein (A76), a method for determining the antigenic integrity, correct conformation, and / or correct sequence of a SARS-CoV-2 surface spike protein comprises: (a) contacting a sample containing the antigen with a first antibody or antibody fragment having the clone-pair heavy and light chain CDR sequences of Tables 3 and 4, respectively; and (b) determining the antigenic integrity, correct conformation, and / or correct sequence of the antigen by detectable binding of the first antibody or antibody fragment to the antigen. In one embodiment of A76 (A77), the sample comprises a recombinantly produced antigen. In one embodiment of A76 (A78), the sample comprises a vaccine formulation or vaccine production batch. In one embodiment of A76-A78 (A79), detecting comprises ELISA, RIA, Western blot, biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometry staining. In one embodiment of A76 to A79 (A80), the first antibody or antibody fragment is encoded by a clone pair variable sequence as set forth in Table 1. In one embodiment of A76 to A79 (A81), the first antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to a clone pair variable sequence as set forth in Table 1. In one embodiment of any one of A76 to A79 (A82), the first antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 95% identity to a clone pair sequence as set forth in Table 1. In one embodiment of any one of A76 to A79 (A83), the first antibody or antibody fragment comprises light and heavy chain variable sequences according to the clone pair sequences in Table 2. In one embodiment of any one of A76 to A79 (A84), the first antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair sequences of Table 2. In one embodiment of any one of A76 to A79 (A85), the first antibody or antibody fragment comprises light and heavy chain variable sequences having at least 95% identity to the clone pair sequences of Table 2.In one embodiment of any one of A76 to A85 (A86), the first antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab')2 fragment, or Fv fragment. In one embodiment of any one of A76 to A86 (A87), the method further comprises performing steps (a) and (b) twice to determine the antigenic stability of the antigen over time. In one embodiment of any one of A76 to A87 (A88), the method further comprises (c) contacting a sample containing the antigen with a second antibody or antibody fragment having clone-pair heavy and light chain CDR sequences of Tables 3 and 4, respectively, and (d) determining the antigenic integrity of the antigen by detectable binding of the second antibody or antibody fragment to the antigen. In one embodiment of A88 (A89), the second antibody or antibody fragment is encoded by a clone-pair variable sequence as set forth in Table 1. In one embodiment of A89 (A90), the second antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair variable sequences as set forth in Table 1. In one embodiment of A89 (A91), the second antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 95% identity to the clone pair sequences as set forth in Table 1. In one embodiment of A89 (A92), the second antibody or antibody fragment comprises light and heavy chain variable sequences according to the clone pair sequences in Table 2. In one embodiment of A89 (A93), the second antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to the clone pair sequences in Table 2. In one embodiment of A89 (A94), the second antibody or antibody fragment comprises light and heavy chain variable sequences having at least 95% identity to the clone pair sequences in Table 2. In one embodiment of A89 (A95), the second antibody fragment is a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment or an Fv fragment. In one embodiment of A89 (A96), the method further comprises performing steps (c) and (d) twice to determine the antigen stability of the antigen over time.

[0026] In one aspect provided herein (A97), there is provided a human monoclonal antibody or antibody fragment, or a hybridoma or modified cell producing the same, wherein the antibody is a human monoclonal antibody or antibody fragment, or a hybridoma or modified cell producing the same, that binds to the SARS-CoV-2 surface spike protein.

[0027] The use of the word "a" or "an," when used in conjunction with the term "comprising" in the claims and / or this specification, can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." The word "about" means plus or minus 5% of the specified number.

[0028] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description.

[0029] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0031] [Figure 1]Dose-response matrix evaluating the synergistic neutralizing activity of the COV2-2196 + COV2-2130 cocktail using live BSL3 SARS-CoV-2 virus. At the highest tested concentration (250 ng / mL) of each individual mAb (between 0 ng / mL COV2-2196 + 250 ng / mL COV2-2130 and 250 ng / mL COV2-2196 + 0 ng / mL COV2-2130), there was qualitatively very little unneutralized virus, but a range of lower Ab concentrations in the combination resulted in complete neutralization (100%). The box for 15.6 ng / mL COV2-2196 + 63 ng / mL COV2-2130 indicates the area where the combination of mAb COV2-2196 at 15.6 ng / mL and mAb COV2-2130 at 63 ng / mL neutralized 96% of the virus, while the individual Abs only neutralized 6 or 0%, respectively (ovals for 0 ng / mL COV2-2196 + 63 ng / mL COV2-2130 and 15.6 ng / mL COV2-2196 + 0 ng / mL COV2-2130). Average values ​​of three technical replicates are shown. [Figure 2] Dose-response matrix evaluating the synergistic neutralizing activity of the cocktail of mAb COV2-2196 and mAb COV2-2130 using BSL3 live SARS-CoV-2 virus. The mean of three technical replicates is shown. Data were visualized and synergy assessed using SynergyFinder software. [Figure 3] Dose-response matrix evaluating the synergistic neutralizing activity of the cocktail of mAb COV2-2196 and mAb COV2-2130 using BSL3 live SARS-CoV-2 virus. Synergy score explanation: <-10: The interaction is likely antagonistic; -10 to 10: The interaction is likely additive; >10: The interaction is likely synergistic. [Figure 4]Therapeutic effect of constructed neutralizing human mAbs on SARS-CoV-2 infection. (Figure 4A) Mice were inoculated with 105 PFU of MA-SARS-CoV-2 via the intranasal route, and 12 hours later, the indicated antibody treatments were given via intraperitoneal injection. Two days after virus challenge, lung viral load was measured by plaque assay. Individual mouse measurements and median titers are shown, and each group was compared to the isotype control using Kruskal-Wallis ANOVA with Dunn's post-hoc test (*p<0.05). Data represent a single experiment. (Figure 4B) 10-11-week-old Balb / c mice (3-9 mice per group per experiment) were treated with anti-Ifnar1 mAb and transduced with AdV-hACE2 via the intranasal route one day later. Four days later, mice were inoculated intranasally with 105 FFU of standard SARS-CoV-2, and 12 hours later, the indicated mAb treatments were given intraperitoneally. Two days after challenge, lung viral loads were measured by plaque assay. Two controls for performing plaque neutralization assays included lung homogenates from individual isotype-treated mice mixed 1:1 (volume:volume) with lung homogenates from uninfected mice or mice treated with the mAb COV2-2196+COV2-2130 cocktail. Individual mouse measurements and median titers are shown, and each group was compared to the isotype control using Kruskal-Wallis ANOVA with Dunn's post-hoc test (**p<0.01). Data represent a single experiment (Figure 4C). Cytokine and chemokine gene expression was measured by qPCR analysis from lungs harvested as in Figure 4B. Individual mouse measurements and median titers are shown. Groups were compared using the Mann-Whitney U test (*p<0.05; **p<0.01). [Figure 5] Antibody pharmacokinetics after infusion of human mAb into macaques. [Figure 6] SARS-CoV-2 viral load (measured as subgenomic newly produced RNA) in bronchoalveolar lavage fluid samples from macaques after intranasal and intratracheal challenge with wild-type SARS-CoV-2 virus. [Figure 7]SARS-CoV-2 viral load (measured as subgenomic newly produced RNA) in nasal swab specimens from macaques after intranasal and intratracheal challenge with wild-type SARS-CoV-2 virus. [Figure 8]Crystal structure of the RBD of the S protein complexed with Fab COV2-2196. (Figure 8A) Schematic representation of COV2-2196 complexed with the RBD. The heavy chain of COV2-2196 is shown in cyan, the light chain in magenta, and the RBD in green. The colors can be seen in Figure 8A of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 8B) The structure of the COV2-2196-RBD complex is superimposed onto the structure of the RBD-human ACE2 complex (PDB ID: 6M0J) using the RBD structure as a reference. The color scheme of the COV2-2196-RBD complex is the same as in Figure 8A. The RBD in the RBD-ACE2 complex is colored light blue, and the human ACE2 peptidase domain is colored gray. The color can be seen in Figure 8B of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 8C) The structure of the COV2-2196-RBD complex is superimposed on the structure of the spike with a single RBD in the "up" conformation (PDB ID: 6XM4), using the RBD in the "up" conformation as a reference. The color scheme of the COV2-2196-RBD complex is the same as that in Figure 8A. The three subunits of the spike are colored gray, yellow, or light blue, respectively (the subunit containing the RBD in the "up" conformation is yellow). The color can be seen in Figure 8C of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 8D) Schematic representation of the RBD epitope recognized by COV2-2196. Epitope residues are colored in different shades of green and labeled in black. The colors can be seen in Figure 8D of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 8E) Antibody-antigen interaction between COV2-2196 and the RBD. The RBD is shown in the same surface representation and orientation as in Figure 8D. The paratope residues of COV2-2196 are shown in stick representation. The heavy chain is colored cyan, and the light chain is colored magenta. The colors can be seen in Figure 8E of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. [Figure 9]Crystal structure of the RBD of the S protein complexed with both the Fabs COV2-2196 and COV2-2130. (Figure 9A) Schematic representation of the crystal structure of the RBD of the S protein complexed with both the Fabs COV2-2196 and COV2-2130. The RBD is shown in green, the heavy chain of COV2-2196 in cyan, the light chain of COV2-2196 in magenta, the heavy chain of COV2-2130 in yellow, and the light chain of COV2-2130 in orange. The CDRs of COV2-2130 are labeled. The colors can be seen in Figure 9A of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 9B) The structure of the COV2-2130-RBD complex is superimposed onto the structure of the RBD-ACE2 complex (PDB ID: 6M0J) using the RBD structure as a reference. The color scheme of the COV2-2130-RBD complex is the same as that of Figure 9A. The RBD in the RBD-ACE2 complex is colored light blue, and the human ACE2 peptidase domain is colored gray. The colors can be seen in Figure 9B of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 9C) The structure of the COV2-2130-RBD complex is superimposed onto the structure of a spike (PDB ID: 6ZOY), which contains all RBDs in the "down" conformation, using the RBDs within one protomer as a reference. The color scheme of the COV2-2130-RBD complex is the same as that of Figure 9A. The three protomers of the spike are colored gray, light blue, or purple, respectively. The colors can be seen in Figure 9C of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 9D) The structure of the COV2-2196-2130-RBD complex is superimposed onto the structure of a spike containing one RBD in the "up" conformation (PDB ID: 7CAK), using the RBD in the "up" conformation as a reference. The color scheme of the COV2-2130-RBD complex is the same as that in Figure 9A. The three protomers of the spike are colored gray, light blue, or purple, respectively. The colors can be seen in Figure 9D of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety.(Figure 9E) Schematic representation of the epitope of the RBD recognized by COV2-2130. Epitope residues are shown in different colors and labeled in black. The colors can be seen in Figure 9E of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 9F) Interaction of the paratope residues of COV2-2130 with the epitope. The RBD is shown in the same surface representation and orientation as in Figure 9E. The paratope residues are shown in stick representation. The heavy chain is colored yellow and the light chain is colored orange. The colors can be seen in Figure 9F of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. [Figure 10] (Figure 10A) IMGT / DomainGapAlign results for the heavy and light chains of COV2-2196. Key interacting residues and their corresponding germline gene residues are boxed. The SEQ ID NOs for the sequences in Figure 10A are shown below. [Table 1] (Figure 10B) Binding curves of COV2-2196 point mutants. cDNAs encoding the boxed heavy chain point mutants above were designed and synthesized as DNA to produce recombinant IgG proteins and tested for binding activity to the spike protein. The D108 residue mutant is shown in the upper left graph, the predicted somatic mutation revertant to the germline sequence is shown in the upper right graph, the P99 mutant is shown in the lower left graph, and the mutant with the disulfide bond removed in HCDR3 is shown in the lower right graph. [Figure 11]Comprehensive mutational scanning combined with selection of resistant mutants identified residues critical for COV2-2196 and COV2-2130. (Figure 11A) Logo plot of the escape rate for all mutations in the RBD region associated with strong escape in COV2-2196 (left) or COV2-2130 (right). Taller letters indicate greater antibody binding escape. Mutations are colored based on the extent to which they reduce RBD binding to human ACE2. Data shown are the average of two independent escape selection experiments using two independent yeast libraries. Correlations are shown in Figures 18B-C. Interactive, expandable versions of these logo plots are available at jbloomlab.github.io / SARS-CoV-2-RBD_MAP_AZ_Abs / . We measured the escape rate as described in Methods, where 0 indicates the mutant always binds to the antibody and 1 indicates that it always escapes antibody binding. This is the estimated percentage of cells expressing a particular mutant that falls into an antibody escape bin. (Figure 11B) Logo plots of the mutational escape rates of COV2-2196 and COV2-2130 achievable by single-base substitutions derived from the Wuhan-Hu-1 reference strain used for escape selection (Figures 11E-F). The effect of each substitution on ACE2 binding is shown in Figure 11A. (Figure 11C) Left panel: Comprehensive mutational scanning escape mutants of COV2-2196 mapped to the RBD surface within the RBD-COV2-2196 structure. Mutations that inhibit COV2-2196 binding are displayed on the RBD structure using a heat map. Blue indicates the RBD site with the highest cumulative antibody escape, while white indicates no escape was detected. Gray indicates residues whose deleterious effects on RBD expression preclude evaluation of the mutation's impact on antibody binding. Right panel: A magnified version of the left panel, showing interacting residues surrounding the most potent escape site in the RBD. The heavy chain of COV2-2196 is colored cyan, and the light chain is colored magenta. Two iterations were performed with independent libraries as described in Figure 11A. The colors can be seen in Figure 11C of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety.(Figure 11D) Right panel: Comprehensive mutational scanning escape mutants of COV2-2130 mapped to the RBD surface within the RBD-COV2-2130 structure. Mutations that abrogate COV2-2130 binding were displayed on the RBD structure using a heat map, as in Figure 11C. Left panel: A close-up of the left panel, showing interacting residues around the most potent escape site of the RBD. The heavy chain of COV2-2130 is colored yellow, and the light chain is colored salmon pink. The colors can be seen in Figure 11D of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety. (Figure 11E) Table showing the results of escape selection experiments of VSV-SARS-CoV-2 with COV2-2196, COV2-2130, and their combination. The number of escape mutants selected and the total number of escape selection replicates performed are listed, as well as the residues identified by sequencing the escape mutant viruses. (Figure 11F) Table showing the results of passaging SARS-CoV-2 in the presence of subneutralizing concentrations of AZD8895 (based on COV2-2196), AZD1061 (based on COV2-2130), and AZD7442 (AZD8895 + AZD1061). Resistance-associated viral mutations identified by sequencing neutralization-resistant plaques are displayed. (Figure 11G) Scatter plot showing the DMS data from Figure 11A, with the x-axis representing the mutation escape rate and the y-axis representing the effect on ACE2 binding. × indicates a mutation accessible only by multiple base substitutions, while ○ indicates a mutation accessible by a single base substitution. Amino acid substitutions selected by COV2-2130 in VSV-SARS-CoV-2 (K444R, K444E) or standard SARS-CoV-2 (R346I) are displayed. (Figure 11H) Antibody neutralization against the reference strain and SARS-CoV-2 variants of interest, as measured by FRNT. Neutralization assays were performed in duplicate and repeated twice, and results from one experimental replicate are shown. Error bars represent the range of each point. Mutations relative to the WA-1 reference strain are indicated. B.1.1.7-OXF contains deletions 69-70 and 144-145 and the following substitutions: N501Y, A570D, D614G, P681H, and T716I. [Figure 12]Superposition of the RBD-COV2-2196 substructure within the RBD-COV2-2196-2130 complex with the crystal structure of RBD-COV2-2196. [Figure 13]Similar aromatic stacking and hydrophobic interaction patterns at F486, a shared RBD site between the RBD-COV2-2196 and spike-S2E12 complexes. (Figures 13A and B) Identical hydrogen bonding patterns surrounding residue F486 in the structures of the two complexes. (Figure 13C) Detailed interactions between COV2-2196 and the RBD. The COV2-2196 heavy chain is colored cyan, the light chain is colored magenta, and the RBD is colored green. Key interacting residues are shown in stick representation. Water molecules involved in Ab-Ag interactions are represented as pink spheres. Direct hydrogen bonds are indicated by orange dashed lines, and water-mediated hydrogen bonds are indicated by yellow dashed lines. Colors can be seen in Extended Data Figure 2C of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 13D) The cryo-EM structure of S2E12 / RBD is superimposed on the crystal structure of COV2-2196 / RBD, using the antibody variable domain as a reference. The heavy chain of COV2-2196 is blue-green, and the light chain is magenta. The heavy chain of S2E12 is pale blue-green, and the light chain is bright pink. The two corresponding RBD structures are colored green or yellow, respectively. The colors can be seen in Extended Data Figure 2D of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety. (Figure 13E) Detailed interactions between the heavy chain and RBD of COV2-2130. Paratope residues are shown as stick representations and colored yellow, and epitope residues are shown as green sticks. Hydrogen bonds or strong polar interactions are represented as dashed magenta lines. The colors can be seen in Extended Data Figure 2E of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety. (Figure 13F) Detailed interactions between the light chain and RBD of COV2-2130. Paratope residues are shown as stick representations and colored orange, and epitope residues are shown as green sticks. Hydrogen bonds are represented as dashed magenta lines. The colors can be seen in Extended Data Figure 2F of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety. [Figure 14]Common clonotypes of anti-RBD antibodies with identical binding mechanisms. (Figure 14A) Crystal structure of COV2-2196 / RBD. (Figure 14B) Cryo-EM structure of S2E12 / RBD. (Figure 14C) Homology model of COV2-2381 / RBD. COV2-2072 encodes an N-linked glycosylation sequon in HCDR3, indicated by a gray sphere. Colors can be seen in Extended Data Figure 3D of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety. (Figure 14D) Homology model of COV2-2072 / RBD. (Figure 14E) Superposition of the crystal structure of COV2-2196 / RBD (Figure 14A) with the cryo-EM structure of S2E12 / RBD (Figure 14B). [Figure 15]Identification of putative public clonotype members genetically similar to COV2-2196 in the antibody variable gene repertoire of virus-naive individuals. Alignment of antibody variable gene sequences from healthy individuals with sequence features identical to those of the heavy chain (Figure 15A) and light chain (Figures 15A and 15B) of COV2-2196. Sequences from three different donors and umbilical cord blood contained sequences with the public clonotype features. Sequence features and contact residues used by COV2-2196 are highlighted in boxes below each multiple sequence alignment (boxes are colored red in Extended Data Figure 4A of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety). The SEQ ID NOs for the heavy chain sequences in Figure 15A are as follows: HIP1: SEQ ID NO: 166, HIP2: SEQ ID NO: 167, HIP3: SEQ ID NO: 168, and CORD: SEQ ID NO: 169. The SEQ ID NOs for the light chain sequences in Figure 15A are as follows: HIP1: SEQ ID NO:170, HIP2: SEQ ID NO:171, and HIP3: SEQ ID NO:172. The SEQ ID NOs for the light chain sequences of HIP1 in Figure 15B are as follows (top to bottom): SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, and SEQ ID NO:177. The SEQ ID NOs for the light chain sequences of HIP2 in Figure 15B are as follows (top to bottom): SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, and SEQ ID NO:182. The SEQ ID NOs for the light chain sequences of HIP3 in Figure 15B are as follows: SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, and SEQ ID NO:187 (top to bottom). [Figure 16](Figure 16A) Detailed HCDR3 loop structure of COV2-2130. Short-range hydrogen bonds that stabilize the loop conformation are indicated by dashed lines (dashed lines are colored magenta in Extended Data Figure 5A of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety). (Figure 16B) Residues of the light chain of COV2-2130 form aromatic stacking interactions and hydrogen bonds with HCDR3, further stabilizing the HCDR3 loop. (Figure 16C) The long LCDR1, HCDR2, and HCDR3 form a binding surface complementary to the RBD epitope. The RBD is shown as a gray surface representation. The heavy chain of COV2-2130 is colored yellow, HCDR3 orange, the light chain salmon pink, and LCDR1 magenta. The colors can be seen in Extended Data Figure 3D of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety. (FIG. 16D) A 180° rotated view of FIG. 16C. [Figure 17] The interface between COV2-2196 and COV2-2130 in the crystal structures of the RBD complexed with COV2-2196 and COV2-2130. The heavy or light chain of COV2-2196 is shown schematically in cyan or magenta, respectively, and the heavy or light chain of COV2-2130 is shown in yellow or salmon pink, respectively. The RBD is colored green. The colors can be seen in Extended Data Figure 6 of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety. Interface residues are shown as stick representations. [Figure 18]Identification of mutations affecting antibody binding by comprehensive mutational scanning and selection of antibody-resistant mutants by VSV-SARS-CoV-2 virus. (Figure 18A) Top: Flow cytometry plots showing representative gating strategies for selecting single yeast cells using forward and side scatter (first three panels) and for selecting yeast cells expressing the RBD (right panel). Each plot is derived from the previous gate. Bottom: Flow cytometry plots showing gating for RBD+, antibody-negative yeast cells (i.e., cells expressing the RBD but with mutations inhibiting antibody binding). Selection experiments with COV2-2196 or COV2-2130 are shown, with two independent libraries shown. (Figure 18B) Correlation of observed sites of escape of antibody binding between yeast library selection experiments using COV2-2196, COV2-2130, or a 1:1 mixture of COV2-2196 and COV2-2130. The x-axis shows the cumulative percent escape of each site for Library 1, and the y-axis shows the cumulative percent escape of each site for Library 2. Correlation coefficients and n are displayed on each graph. (Figure 18C) Correlation of observed mutations that escape antibody binding between yeast library selection experiments using COV2-2196, COV2-2130, or a 1:1 mixture of COV2-2196 and COV2-2130. The x-axis shows the percent escape of each amino acid mutation for Library 1, and the y-axis shows the percent escape of each amino acid mutation for Library 2. Correlation coefficients and n are displayed on each graph. (Figures 18D-F) DMS results for COV2-2196 (Figure 18D), COV2-2130 (Figure 18E), or a 1:1 mixture of COV2-2196 and COV2-2130. (Figure 18E) Left panel: Escape sites across the RBD are indicated by peaks corresponding to the logo plots in the middle and right panels. Center panel: Logo plot of cumulative escape mutation rates for all RBD sites, including potent escape mutations in COV2-2196 or COV2-2130, or COV2-2196 + COV2-2130, as in Figure 11A. Mutations are colored based on the extent to which they inhibit RBD binding to human ACE2.Right panel: Again, logo plots show cumulative escape rates, but are colored based on the extent to which mutations result in RBD expression in the yeast display system. Interactive, expandable versions of these logo plots are available at jbloomlab.github.io / SARS-CoV-2-RBD_MAP_AZ_Abs / . Colors can be seen in Extended Data Figure 7F of U.S. Provisional Patent Application No. 63 / 161,890, incorporated herein by reference in its entirety. (Figure 18G) Representative RTCA sensorgrams showing viruses that escaped antibody neutralization. Cytopathic effect (CPE) of Vero E6 cells inoculated with virus was monitored kinetically in the presence of a saturating concentration (5 μg / mL) of antibody, COV2-2130. Representative examples of escape (magenta) or no detectable escape (blue) are shown. Uninfected cells (green) or cells inoculated with virus without antibody (red) served as controls. The magenta and blue curves represent a single representative well, and the red and green controls are the average of two technical replicates. Colors can be seen in Extended Data Figure 7G of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 18H) Representative RTCA sensorgram verifying that the mutant viruses selected by COV2-2130 in Figure 18G indeed escaped COV2-2130 (magenta) but were neutralized by COV2-2196 (light blue). Colors can be seen in Extended Data Figure 7H of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 18I) Exemplary sensorgrams from individual wells of a 96-well E-Plate assay for escape selection experiments with COV2-2196, COV2-2130, and a 1:1 mixture of COV2-2196 and COV2-2130. Demonstration of escape from COV2-2130 is shown, but no escape was detected in the presence of COV2-2196 or COV2-2196 + COV2-2130. Positive and negative controls are shown on the first plate. [Figure 19] Standard SARS-CoV-2 virus method for selecting antibody-resistant mutants. [Figure 20]Functional characterization of neutralizing SARS-CoV-2 mAbs. (Figure 20A) Heatmap of mAb neutralization activity, hACE2 blocking activity, and binding to trimeric S2Pecto protein or monomeric SRBD. mAbs are ranked by neutralization potency (highest at the top, lowest at the bottom). Dashed lines indicate 13 antibodies with neutralization IC50 values ​​below 150 ng / mL of wt virus. IC50 values ​​are visualized for virus neutralization and hACE2 inhibition, and EC50 values ​​are visualized for binding. A recombinant form of the cross-reactive SARS-CoV SRBD mAb, CR3022, is shown as a positive control, while the anti-dengue mAb, 2D22, is shown as a negative control. Data are representative of at least two independent experiments, each performed with two technical replicates. "No inhibition" indicates an IC50 value >10,000 ng / mL, while "No binding" indicates an EC50 value >10,000 ng / mL. (Figures 20B-E) Correlation of mAb hACE2 blockade, S2Pectotrimer binding, or SRBD binding with their neutralizing activity. R2 values ​​from linear regression analysis of log-transformed values ​​are shown. Dark circles (shown in purple in Figures 1B-E of Example 5 of U.S. Provisional Patent Application No. 63 / 161,890, incorporated herein by reference in its entirety) indicate mAbs with neutralizing IC50 values ​​below 150 ng / mL. (Figure 20E) Correlation of hACE2 blockade and S2Pectotrimer binding. R2 values ​​from linear regression analysis of log-transformed values ​​are shown. (Figure 20F) Neutralization curves of COV2-2196 and COV2-2130 in a standard SARS-CoV-2 virus neutralization assay. Calculated IC50 values ​​are depicted in the graph. Error bars represent the standard deviation of each point. Data are representative of at least two independent experiments, each performed with two technical replicates. (Figure 20G) Neutralization curves of COV2-2196 and COV2-2130 in a pseudovirus neutralization assay. Error bars represent the standard deviation of each point. Values ​​shown are for two technical replicates from a single experiment. IC50 values ​​calculated from a minimum of six experiments are depicted in the graph. (Figure 20H) hACE2 blocking curves of COV2-2196, COV2-2130, and the non-blocking SARS-CoV mAb, rCR3022, in an hACE2-blocking ELISA.Calculated IC50 values ​​are graphed. Error bars represent the standard deviation of each point. Values ​​shown are from three technical replicates from a representative experiment repeated twice. (Figure 20I) ELISA binding of COV2-2196, COV2-2130, and rCR3022 to trimeric S2Pecto. Calculated EC50 values ​​are graphed. Error bars represent the standard deviation of each point. Values ​​shown are from three technical replicates from a representative experiment repeated twice. [Figure 21]Epitope mapping of mAbs by competitive binding analysis and synergistic neutralization by a pair of mAbs. (Figure 21A) Left: Bio-layer interferometry-based competitive binding assay measuring the ability of mAbs to inhibit the binding of reference mAbs COV2-2196 and rCR3022 to RBD fused to mouse Fc (RBD-mFc) loaded onto an anti-mouse Fc biosensor. Values ​​within boxes are the percent binding of reference mAbs in the presence of competing mAbs relative to mock competition controls. Black boxes indicate complete competition (<33% binding relative to non-competitive control), while open boxes indicate no competition (<67% binding relative to non-competitive control). Right: Bio-layer interferometry-based competitive binding assay measuring the ability of mAbs to inhibit hACE2 binding. Values ​​represent the percent binding of hACE2 normalized to hACE2 binding in the absence of competition. Shading indicates mAb competition with hACE2. (Shading is shown in red in Figure 2A of Example 5 of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety.) (Figure 21B) Competition of a panel of neutralizing mAbs with reference mAbs COV2-2130, COV2-2196, or rCR3022. Reference mAbs were biotinylated, and binding of the reference mAbs to trimeric S2Pecto was measured in the presence of saturating amounts of each mAb in a competition ELISA. The ELISA signal of each reference mAb was normalized to the signal in the presence of unconjugated anti-dengue mAb 2D22. Black indicates complete competition (<25% binding of the reference mAb), gray indicates partial competition (25-60% binding of the reference mAb), and white indicates no competition (>60% binding of the reference mAb). (Figure 21C) Synergistic neutralization of wild-type SARS-CoV-2 by COV2-2196 and COV2-2130. Top: Neutralization matrix with serial dilutions of each mAb. Experiments were performed with three technical replicates. The number of representative experiments performed with three technical replicates is shown. The percent neutralization with each of the mAbs in combination is shown in each box. The white to black heat map represents 0% to 100% neutralization, respectively. (The heat map is shown in white to red in Figure 2C of Example 5 of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety.)) (Figure 21D) Synergy matrix based on neutralization of SARS-CoV-2 in Figure 21C. Dark colors (shown as red in Figure 2D of Example 5 of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety) represent areas where synergistic neutralization was observed, and black boxes represent areas of maximal synergy between the two mAbs. [Figure 22]Epitope identification and structural characterization of mAbs. (Figure 22A) Identification of critical contact residues by alanine and arginine mutagenesis. Top: Binding of COV2-2130 (gold), COV2-2165 (maroon), or COV2-2196 (dark purple) to wild-type (wt) or mutated SRBD constructs as measured by biolayer interferometry. The y-axis shows the response normalized to the signal observed for binding to wt SRBD. Bottom: Representative binding curves of COV2-2196 to SRBD constructs containing wt or mutated critical contact residues. Colors can be seen in Figure 3A of Example 5 of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 22B) Crystal structure of SARS-CoV-2 (blue) and hACE2 (green) (PDB (6M0J). The hACE2 recognition motif is colored orange. Contact residues important for COV2-2130 are shown as gold spheres, while contact residues important for COV2-2196 are shown as purple spheres. Colors can be seen in Figure 3B of Example 5 of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety. (Figure 22C) ELISA binding of mAbs to the 60 amino acid hACE2 recognition motif. r2D22 (anti-dengue mAb) is shown as a negative control. Bottom: Structure of the hACE2 recognition motif in orange, with the critical contact residues of COV2-2196 shown in purple. Colors are also shown in their entirety, which are incorporated by reference. This can be seen in Figure 3C of Example 5 of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference. (Figure 22D) S2Pecto trimeric complexes of single Fabs: COV2-2130 (gold), COV2-2165 (maroon), or COV2-2196 (dark purple), visualized by negative stain electron microscopy. The RBD is shown in blue, and the N-terminal domain (NTD) of S is shown in red. Electron density is shown in gray. The trimeric state (open or closed) is represented for each complex. A representative 2D class average for each complex is shown at the bottom (box size is 128 pixels). The colors can be seen in Figure 3D of Example 5 of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety.(Figure 22E) COV2-2130 and COV2-2196 Fabs complexed with S2Pecto trimers. Simultaneous binding of COV2-2130 (gold) and COV2-2196 (purple) Fabs to S2Pecto trimers. Electron density is shown in gray. Trimeric states (open and closed) are indicated. A representative 2D class average of the complex is shown at the bottom (box size 128 pixels). All images were generated in Chimera. Colors can be seen in Figure 3E of Example 5 of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated herein by reference in its entirety. (Figure 22F) Competitive binding analysis visualized on S2Pecto trimers. The crystal structure of CR3022 was docked to the dual Fab:S2Pecto trimer structure. CR3022 is shown in cyan. The colors can be seen in Figure 3F of Example 5 of U.S. Provisional Patent Application No. 63 / 161,890, which is incorporated by reference in its entirety. Bottom: A quantitative Venn diagram describes the number of mAbs in each competitor group and the overlap between groups. [Figure 23]Protective effect of neutralizing human mAbs against SARS-CoV-2 infection. (Figure 23A) SARS-CoV-2 challenge model. 10-11-week-old Balb / c mice (4-5 mice per group in two experiments) were treated with anti-Ifnar1 mAb and transduced with AdV-hACE2 via the in route one day later. Four days later, mice were treated with 200 μg of mAb CoV2-2196, CoV2-2130, or the combination (1:1 ratio) or isotype control mAb via the i.p. route. One day later, they were inoculated with SARS-CoV-2 via the in. route. Tissues were harvested 7 days after administration for analysis (Figures 23C and 23D). (Figure 23B) Body weight change of mice in panel a (two-way ordinary ANOVA with Tukey's post-hoc test: ****P<0.0001). (Figure 2C) Viral load in the lungs, spleen, and heart was measured by RT-qPCR: Kruskal-Wallis ANOVA with Dunn's post-hoc test (*, P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). The dotted line indicates the detection limit of the assay. (Figure 23D) Cytokine and chemokine gene expression was measured by qPCR analysis: Kruskal-Wallis ANOVA with Dunn's post-hoc test (*, P<0.05, **P<0.01, ***P<0.001). (Figure 23E) MA-SARS-CoV-2 challenge model. 12-week-old Balb / c mice (n=10) were inoculated with 10 PFU of MA-SARS-CoV-2 via the i.n. route. Mouse weight changes are shown. (Figure 23F) Lung viral load was measured by RT-qPCR (left) or plaque assay (right) at 2 dpi from (Figure 23E): Kruskal-Wallis ANOVA with Dunn's post-hoc test (***P<0.001, ****P<0.0001). [Figure 24] SARS-CoV-2 neutralization curves for a panel of mAbs. Standard SARS-CoV-2 neutralization by human mAbs. Shown are the mean values ​​± SD of two technical replicates. Data represent one of two or more independent experiments. [Figure 25]Inhibition curves of mAbs that inhibit the binding of S2Pecto to hACE2. Blockade of hACE2 binding to S2Pecto by anti-SARS-CoV-2 neutralizing human mAbs. Mean values ​​± SD from three replicates of one experiment are shown. Antibodies CR3022 and 2D22 served as controls. [Figure 26] ELISA binding of anti-SARS-CoV-2 neutralizing human mAbs to trimeric SRBD, S2Pecto, or the S2Pecto antigen of SARS-CoV. Mean values ​​± SD of three replicates and representative of two experiments are shown. Antibodies CR3022 and 2D22 served as controls. [Figure 27] Mapping of critical contact residues of mAbs by alanine and arginine mutagenesis and biolayer interferometry. (Figure 27A) Left: Response values ​​for mAb binding to wt or mutant SRBD constructs, normalized to wt. Asterisks denote residues where increased dissociation of the mAb was observed, indicating that the residue is likely proximal to the mAb's epitope. Right: Full response curves for association and dissociation of mAbs with wt or mutant SRBD constructs. (Figure 27B) Structure of the RBD highlighting critical contact residues for several mAbs and their location in the structure. DETAILED DESCRIPTION OF THE INVENTION

[0032] As mentioned above, SARS-CoV-2 is a serious health problem with active cases increasing daily. Therefore, understanding the biology and properties of this virus and the extent of the human immune response to the virus is of utmost importance. The present inventors have identified human antibody sequences against SARS-CoV-2. These sequences and uses of such antibodies are disclosed herein.

[0033] Furthermore, by studying the interaction of one antibody (COV2-2196) with the RBD in detail, the inventors identify a molecular basis for selecting a public SARS-CoV-2 clonotype that operates through a complex structural configuration involving both its heavy and light chains. The shared structural features of this clonotype contribute to the formation of a paratope that includes residues from both the heavy and light chains, but surprisingly, does not rely on the HCDR3, which typically governs antigen-antibody interactions. The inventors show that this public clonotype is one of the more frequently shared types of potent neutralizing antibodies produced by humans against the RBD of the SARS-CoV-2 S protein. Detailed structural studies reveal that the paratope of commonly formed antibodies gives rise to an "aromatic cage" formed by five aromatic residues within the paratope that surround the interface between the heavy and light chains. This cage structure coordinates with aromatic residues on the SARS-CoV-2 S protein, explaining the high specificity and affinity of these antibodies. Notably, the formation of this public clonotype requires both heavy and light chains (thus defining the canonical IGHV, IGHJ, IGLV, and IGLJ genes in the clonotype), but HCDR3 minimally influences this interaction. Because these IGHV1-58-IGHJ3 heavy chain and IGKV3-20-IGKJ1 light chain recombinations are common in the pre-immune B cell repertoire, such clones are likely to be generated in many individuals in response to SARS-CoV-2 infection or vaccination. Given the frequency of B cell clones in the human population and the neutralizing and protective potencies of antibodies encoded by the variable gene segments, the antigenic sites recognized by the complex pre-assembled structure of this public clonotype are likely to be important components of a preventative vaccine for COVID-19.

[0034] These and other aspects of the disclosure are described in detail below.

[0035] I. Coronavirus 2019 (SARS-CoV-2) SARS-CoV-2 is a contagious virus that causes an acute respiratory illness, termed coronavirus disease 2019 (COVID-19). This virus is responsible for the ongoing 2019-2020 coronavirus pandemic, a global health emergency. Genome sequencing has revealed that the virus is a positive-sense, single-stranded RNA coronavirus.

[0036] During the ongoing outbreak, the virus is often referred to in common parlance as "coronavirus," "novel coronavirus," and "Wuhan coronavirus," although the WHO recommends the name "SARS-CoV-2." The International Committee on Taxonomy of Viruses (ICTV) has published the official name for the virus as SARS-CoV-2.

[0037] Many early cases were linked to a large seafood and animal market in the Chinese city of Wuhan, and the virus is thought to be of zoonotic origin. Genetic sequence comparison of this virus sample with other virus samples shows similarity to SARS-CoV (79.5%) and bat coronaviruses (96%). This finding makes bats a likely ultimate source of origin, although intermediate hosts such as pangolins cannot be ruled out. The virus may be a recombinant virus formed from two or more coronaviruses.

[0038] Human-to-human transmission of the virus has been confirmed. Coronaviruses are spread primarily through close contact, especially through respiratory droplets from coughs and sneezes within a range of approximately 6 feet (1.8 m). Viral RNA has also been found in fecal samples from infected patients. The virus may be transmissible even during the incubation period.

[0039] Because many of the first individuals found infected with the virus worked at the Huanan Seafood Market, animals sold for consumption were initially suspected of being the reservoir or intermediate host of SARS-CoV-2. Markets selling live animals for consumption were also blamed during the 2003 SARS outbreak, and such markets are considered to be breeding grounds for new pathogens. This outbreak prompted a temporary ban on the trade and consumption of wild animals in China. However, some researchers have suggested that the Huanan Seafood Market may not have been the source of the virus's transmission to humans.

[0040] With a sufficient number of sequenced genomes, it is possible to reconstruct a phylogenetic tree of the mutation history of a family of viruses. Studies into the origins of the 2003 SARS pandemic led to the discovery of many SARS-like bat coronaviruses, many of which originated from horseshoe bats of the Rhinolophus genus. SARS-CoV-2 falls into this category of SARS-related coronaviruses. Two genome sequences from the Chinese horseshoe bat (Rhinolophus sinicus), published in 2015 and 2017, have shown 80% similarity to SARS-CoV-2. A third viral genome, "RaTG13," from the Chinese horseshoe bat (Rhinolophus affinis), which shares 96% similarity with SARS-CoV-2, was identified in Yunnan Province.

[28]

[29] For comparison, this amount of variation within the virus is similar to the amount of mutations observed over a 10-year period in H3N2 human influenza virus strains.

[0041] SARS-CoV-2 belongs to a broad family of viruses known as coronaviruses; until a more specific name is chosen, "nCoV" is the standard term used to refer to this novel coronavirus. SARS-CoV-2 is a positive-sense, single-stranded RNA (+ssRNA) virus. Other coronaviruses can cause illnesses ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). SARS-CoV-2 is the seventh coronavirus known to infect humans, following 229E, NL63, OC43, HKU1, MERS-CoV, and SARS-CoV.

[0042] SARS-CoV, SARS-CoV-2, etc. are members of the Sarbecovirus subgenus (Beta-CoV lineage B). Their RNA sequences are approximately 30,000 bases long. By January 12, five genomes of SARS-CoV-2 had been isolated from Wuhan and reported by the Chinese Center for Disease Control and Prevention (CCDC) and other institutions. The number of genomes had increased to 28 by January 26. With the exception of the earliest Genbank genomes, genomes are embargoed at GISAID. Phylogenetic analysis of samples is available through Nextstrain.

[0043] The publication of the SARS-CoV-2 genome prompted experiments to model several proteins in the receptor-binding protein (RBD) of the viral spike (S) protein. Results suggest that the S protein retains sufficient affinity for the angiotensin-converting enzyme 2 (ACE2) receptor and uses this as a mechanism for cellular internalization. On January 22, a Chinese group working with the intact virus and a US group working with reverse genetics independently experimentally demonstrated that human ACE2 is the receptor for SARS-CoV-2.

[0044] To explore potential protease inhibitors, the viral 3C-like protease M(pro) derived from the ORF1a polyprotein was also modeled for drug docking experiments. Innophore created two computational models based on the SARS protease, and the Chinese Academy of Sciences created an unpublished experimental structure of the recombinant SARS-CoV-2 protease. Additionally, researchers at the University of Michigan used I-TASSER to model the structures of all mature peptides in the SARS-CoV-2 genome.

[0045] The first known human infections occurred in early December 2019. The SARS-CoV-2 outbreak was first detected in Wuhan, China, in mid-December 2019 and was thought to have originated from a single infected animal. The virus subsequently spread throughout all provinces of China and to over 20 other countries in Asia, Europe, North America, and Oceania. Human-to-human transmission of the virus has been confirmed in all of these regions. On January 30, 2020, SARS-CoV-2 was designated a global health emergency by the WHO.

[0046] As of February 10, 2020 (17:15 UTC), there were 40,645 confirmed cases of infection, of which 40,196 were in mainland China. Initially, almost all cases outside of China occurred in travelers from Wuhan or in people with direct contact with travelers from the region. Spread from travelers to other countries subsequently infected many countries worldwide. Although the proportion of infections resulting in confirmed infection or progression to diagnosable SARS-CoV-2 acute respiratory disease remained unknown, the total number of deaths attributed to the virus exceeded 19,000 as of March 25, 2020.

[0047] The basic reproduction number (R0) of the virus is estimated to be between 1.4 and 3.9. This means that, without testing, the virus will typically result in 1.4 to 3.9 new cases for each confirmed infection. The virus has been determined to be transmissible through a chain of at least four people.

[0048] In January 2020, several organizations and institutions began working on producing a vaccine for SARS-CoV-2 based on the published genome. In China, the Chinese Center for Disease Control and Prevention is developing a vaccine against the novel coronavirus. The University of Hong Kong has also announced that it is developing a vaccine there. Shanghai Oriental Hospital is also working with biotechnology company Stemirna Therapeutics to develop a vaccine.

[0049] Three other vaccine initiatives are being supported by the Coalition for Epidemic Preparedness Innovations (CEPI), including one by biotechnology companies Moderna and Inovio Pharmaceuticals, and another by the University of Queensland. The U.S. National Institutes of Health (NIH) is working with Moderna to create an RNA vaccine that matches the spikes on the surface of the coronavirus, which began phase I clinical trials in March 2020. Inovio Pharmaceuticals is developing a DNA-based vaccine and is collaborating with a Chinese company to expedite approval by Chinese regulators, with clinical trials of the vaccine expected in the summer of 2020. In Australia, the University of Queensland is investigating the potential of a molecular clamp vaccine, which genetically mutates viral proteins to mimic the coronavirus and stimulate an immune response.

[0050] In an independent effort, the Public Health Agency of Canada has given permission to the University of Saskatchewan's International Vaccine Centre (VIDO-InterVac) to begin work on a vaccine. VIDO-InterVac aims to begin production and animal testing in March 2020, with human trials beginning in 2021. Imperial College London's School of Medicine is currently in the process of testing the vaccine on animals.

[0051] COVID-19 acute respiratory disease is a viral respiratory illness caused by SARS-CoV-2. The disease was first detected during the 2019-20 Wuhan coronavirus outbreak. Symptoms can include fever, dry cough, and shortness of breath. As of March 2020, there are no specific approved treatments available, with efforts focused on alleviating symptoms and supporting function.

[0052] Infected individuals may be asymptomatic or have mild to severe symptoms, such as fever, cough, and shortness of breath. Diarrhea or symptoms of upper respiratory tract infection (e.g., sneezing, runny nose, sore throat) are less common. Severe cases of infection can progress to severe pneumonia, multiple organ failure, and death. The time from exposure to the onset of symptoms is estimated by the World Health Organization to be 2 to 10 days, and by the Centers for Disease Control and Prevention (CDC) to be 2 to 14 days.

[0053] Health organizations around the world have issued preventive measures that individuals can take to reduce their chances of SARS-CoV-2 infection. Recommendations are similar to those previously issued for other coronaviruses and include frequent handwashing with soap and water, avoiding touching your eyes, nose, or mouth with unclean hands, and practicing good respiratory hygiene.

[0054] The WHO has published several testing procedures for SARS-CoV-2. Testing uses real-time reverse transcription polymerase chain reaction (rRT-PCR). Testing can be done on respiratory or blood samples. Results are generally available within hours to days.

[0055] Research into potential treatments for the disease began in January 2020. The Chinese Center for Disease Control and Prevention began testing existing pneumonia treatments in coronavirus-associated pneumonia in late January. The RNA polymerase inhibitor remdesivir and interferon beta are also being investigated. In late January 2020, Chinese medical researchers indicated their intention to begin clinical trials of remdesivir, chloroquine, and lopinavir / ritonavir, all of which appeared to have "fairly good inhibitory effects" on SARS-CoV-2 at the cellular level in exploratory studies. On February 5, 2020, China began patenting remdesivir for the disease.

[0056] The overall mortality and morbidity rates attributable to infection with SARS-CoV-2 are unknown in the current pandemic because the case fatality rate may change over time and the proportion of infections that progress to diagnosable disease remains unclear. However, preliminary studies of SARS-CoV-2 acute respiratory disease have yielded case fatality figures ranging from 2% to 3%, and the WHO indicated that the case fatality rate was approximately 3% in January 2020. A preprint study from Imperial College, which included 55 fatal cases, noted that early mortality estimates may be too high due to overlooking asymptomatic infections. They estimated that the average infection fatality rate (the death rate among infected individuals) ranged from 0.8% when including asymptomatic carriers to 18% when including only symptomatic cases in Hubei Province.

[0057] Early data indicate that of the first 41 confirmed cases admitted to hospitals in Wuhan, 13 (32%) individuals required intensive care and 6 (15%) died. Many of the deceased individuals had underlying health conditions, such as hypertension, diabetes, or cardiovascular disease, which compromise the immune system. In early cases of SARS-CoV-2 acute respiratory disease resulting in death, the median time to illness was found to be 14 days, with an overall range of 6 to 41 days.

[0058] II. Monoclonal Antibodies and Their Production An "isolated antibody" is one that has been separated and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody has been purified (1) to greater than 95% antibody by weight, most particularly 99% or more antibody by weight, as determined by the Lowry method; (2) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions with Coomassie blue or silver staining. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[0059] The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basic heterotetrameric units together with an additional polypeptide called the J chain, and therefore contain 10 antigen-binding sites, while secretory IgA antibodies can polymerize to form multivalent assemblies consisting of two to five basic four-chain units with the J chain. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced interchain disulfide bridges. Each H chain contains a variable region (V) at its N-terminus. H ), followed by three constant domains (C H ) and the μ isotype has four C H Each L chain has a variable domain (V L ) followed by a constant domain (C L ) V L is V H It is lined with CL is the heavy chain (C H1 ) and the first constant domain of the heavy-chain variable region. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable regions. V H and V L and form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71, and Chapter 6.

[0060] The light chains of all vertebrate species contain their constant domains (C L Based on the amino acid sequence of their heavy chains (C), they can be assigned to one of two distinct types, called kappa and lambda. H Depending on the amino acid sequence of the constant domain of the immunoglobulins, immunoglobulins can be assigned to different classes, or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are divided into C H They are further divided into subclasses based on relatively minor differences in sequence and function, with humans expressing the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0061] The term "variable" refers to the fact that certain segments of the V domain vary widely in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, this variability is not evenly distributed across the entire 110-amino acid length of the variable region. Instead, the V region consists of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions," each 9-12 amino acids long. Native heavy and light chain variable regions each contain four FRs, which largely adopt a β-sheet structure and are connected by three hypervariable regions that form loops that connect and sometimes form part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by FRs with the hypervariable regions from the other chain and contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD).

[0062] The term "hypervariable region," as used herein, refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally consist of amino acid residues from the "complementarity-determining regions," or "CDRs," (e.g., V ...L Residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in H and / or those residues from the "hypervariable loops" (e.g., V, V1, V2, V3, V4, V5, V6, V7, V8, V9, V10, V11, V12, V13, V14, V15, V16, V17, V18, V19, V20, V21, V22, V23, V24, V25, V26, V27, V38, V39, V40, V41, V42, V43, V44, V45, V56, V57, V58, V69, V69, V70, V71, V72, V73, V74, V75, V76, V77, V78, V79, V80, V81, V82, V83, V84, V85, V86, V87, V88, V99, V91, V92, V93, V94, V95, V96, V97, V98, V99, V100, V111, V121, V132, V142, V153, V163, V174, V175, V185, V196, V197, V198, V199, V200, V211, V221, V232, V243, V254, V264, V275, V286, V297, V298, V399, V399, V41, V42, V43, V54, V55, V56, V67, V68, V79, V81, V95, V102, V113, V114, V121, V132, V143, V154, V164, V L Residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in H residues 26-32 (H1), 52-56 (H2), and 95-101 (H3) within the "Hypervariable Loops" / CDRs; and / or such residues from the "hypervariable loops" / CDRs (e.g., V when numbered according to the IMGT numbering system: Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res. 28:219-221 (2000)). L Residues 27-38 (L1), 56-65 (L2), and 105-120 (L3) in H Optionally, the antibody comprises residues 27-38 (H1), 56-65 (H2), and 105-120 (H3) within the following sequence, when numbered according to AHo: Honneger, A. and Plunkthun, AJ Mol. Biol. 309:657-670 (2001): V L 28, 36 (L1), 63, 74-75 (L2) and 123 (L3) and V sub They have symmetric insertions within H at one or more of 28, 36 (H1), 63, 74-75 (H2), and 123 (H3).

[0063] "Germline nucleic acid residue" refers to a nucleic acid residue that naturally occurs in a germline gene encoding a constant or variable region. A "germline gene" is DNA found in germ cells (i.e., cells that are committed to becoming eggs or sperm). A "germline mutation" refers to a genetic change in a particular DNA that occurs in a germ cell or single-cell zygote, and such a mutation is incorporated into every cell of the body when passed on to offspring. A germline mutation is in contrast to a somatic mutation, which is acquired in a single somatic cell. In some cases, a nucleotide in a germline DNA sequence encoding a variable region is mutated (i.e., a somatic mutation) and replaced with a different nucleotide.

[0064] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma technique first described by Kohler et al., Nature, 256:495 (1975), or can be made using recombinant DNA techniques in bacterial, animal, eukaryotic, or plant cells after single-cell sorting of antigen-specific B cells, antigen-specific plasmablasts in response to infection or immunization, or capture of heavy and light chains from associated single cells within a bulk-sorted antigen-specific collection (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0065] A. General method It will be appreciated that monoclonal antibodies that bind to SARS-CoV-2 have several uses. These include the production of diagnostic kits used to detect and diagnose as well as treat SARS-CoV-2 infection. In these contexts, such antibodies can be associated with diagnostic or therapeutic agents, used as capture or competitor agents in competitive assays, or used individually without any additional agents associated therewith. Antibodies can be mutated or modified as further described below. Methods for preparing and characterizing antibodies are well known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Pat. No. 4,196,265).

[0066] Methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step in both of these methods is to immunize a suitable host or to identify a subject previously immunized by natural infection or vaccination with an approved or experimental vaccine. As is well known in the art, a given immunization composition may vary in its immunogenicity. Therefore, it is often necessary to boost the host's immune system, which can be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins, such as ovalbumin, mouse serum albumin, or rabbit serum albumin, can also be used as carriers. Means for conjugating polypeptides to carrier proteins are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bisbiazotized benzidine. Additionally, as is known in the art, the immunogenicity of certain immunogenic compositions can be enhanced by the use of nonspecific stimulators of the immune response known as adjuvants. Exemplary and preferred adjuvants in animals include complete Freund's adjuvant (a nonspecific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant; in humans, they include alum, CpG, MFP59, and combinations of immunostimulatory molecules ("adjuvant systems" such as AS01 or AS03). Additional experimental vaccination formats for inducing SARS-CoV-2-specific B cells are contemplated, including nanoparticle vaccines in physical delivery systems or genetically encoded antigens delivered as DNA or RNA genes (such as lipid nanoparticles or gold biolistic beads), delivered by needle, gene gun, or transdermal electroporation device.The antigen gene can be delivered by being encoded by a replication-competent or defective viral vector or alternatively a virus-like particle, such as an adenovirus, adeno-associated virus, poxvirus, herpesvirus or alphavirus replicon.

[0067] For human antibodies against naturally occurring pathogens, a preferred approach is to identify subjects who have been exposed to the pathogen, such as those who have been diagnosed with the disease or who have been vaccinated to generate protective immunity against the pathogen or to test the safety or efficacy of an experimental vaccine. Circulating anti-pathogen antibodies can be detected, and antibody-encoding or producing B cells from the antibody-positive subjects can then be obtained.

[0068] The amount of immunogenic composition used to produce polyclonal antibodies varies depending on the nature of the immunogen and the animal used for immunization. Various routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). Polyclonal antibody production can be monitored by collecting blood from the immunized animal at various times after immunization. A second booster injection can also be given. This process of boosting and titering is repeated until a suitable titer is obtained. Once a desired level of immunogenicity is achieved, the immunized animal is bled, and the serum can be isolated and stored, and / or the animal can be used to generate MAbs.

[0069] Following immunization, somatic cells with the potential to produce antibodies, particularly B lymphocytes (B cells), are selected for use in the MAb generation procedure. These cells can be obtained from biopsied spleens, lymph nodes, tonsils, or adenoids, bone marrow aspirates or biopsies, tissue biopsies from mucosal organs such as the lungs or gastrointestinal tract, or circulating blood. Antibody-producing B lymphocytes from the immunized animal or immunized human are then fused with cells of an immortalized myeloma cell, generally of the same species as the immunized animal, or human or human / mouse chimeric cells. Myeloma cell lines suitable for use in the fusion procedure to generate hybridomas are preferably non-antibody-producing cells, have high fusion efficiency, and possess enzyme deficiencies that prevent growth in specific selective media that support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cell types known to those skilled in the art can be used (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). HMMA2.5 cells or MFP-2 cells are particularly useful examples of such cells.

[0070] Methods for generating hybrids between antibody-producing spleen or lymph node cells and myeloma cells typically involve mixing somatic cells with myeloma cells at a 2:1 ratio in the presence of an agent(s) that promotes cell membrane fusion (chemically or electrically), although this ratio can vary from approximately 20:1 to approximately 1:1. Sometimes, the first step involves transforming human B cells with Epstein-Barr virus (EBV) to increase their size, thereby promoting fusion with the relatively large myeloma cells. The efficiency of EBV transformation is improved by using CpG and aChk2 inhibitors in the transformation medium. Alternatively, human B cells can be activated by co-culturing them with a transfected cell line expressing CD40 ligand (CD154) in medium containing additional soluble factors, such as IL-21 and human B-cell activating factor (BAFF), a type II member of the TNF superfamily. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and fusion methods using polyethylene glycol (PEG), such as 37% (v / v) PEG, have been described by Gefter et al. (1977). Electrically induced fusion methods are also feasible (Goding, pp. 71-74, 1986), and processes exist for better efficiency (Yu et al., 2008). Fusion procedures typically involve approximately 1 × 10 -6 ~1×10 -8Although viable hybrids are produced at low frequencies, optimized procedures can achieve fusion efficiencies approaching 1:200 (Yu et al., 2008). However, the relatively low fusion efficiency is not a problem because viable fused hybrids differentiate from the parent injected cells (especially injected myeloma cells, which normally continue to divide indefinitely) by culturing in selective medium. Selective medium generally contains drugs that block de novo synthesis of nucleotides in tissue culture media. Exemplary and preferred drugs are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). When azaserine is used, the medium is supplemented with hypoxanthine. If the source of the B cells is an EBV-transformed human B cell line, ouabain is added to eliminate EBV-transformed lines that have not fused to myelomas.

[0071] Preferred selective media are HAT or ouabain-containing HAT. Only cells capable of functioning the nucleotide salvage pathway can survive in HAT medium. Myeloma cells lack key enzymes in the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and therefore cannot survive. B cells can function this pathway, but their lifespan in culture is limited, generally dying within about two weeks. Therefore, the only cells that can survive in selective media are hybrids formed from myeloma and B cells. When the source of B cells used for fusion is an EBV-transformed B cell lineage, as described herein, ouabain can also be used for drug selection of hybrids because EBV-transformed B cells are susceptible to drug killing, while the myeloma partner used is selected to be resistant to ouabain.

[0072] Culture provides a population of hybridomas from which specific hybridomas are selected. Hybridoma selection is typically accomplished by culturing the cells by single-clone dilution in microtiter plates, followed by testing the supernatants of individual clones for the desired reaction (approximately 2–3 weeks later). Assays should be sensitive, simple, and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, or dot immunobinding assays. Selected hybridomas are then cloned into individual antibody-producing cell lines by serial dilution or flow cytometric sorting for single-cell sorting, and these clones can then be propagated indefinitely to yield mAbs. Cell lines can be utilized to generate MAbs in two basic ways: A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal (e.g., a mouse). Optionally, the animal is primed with a hydrocarbon, particularly an oil such as pristane (tetramethylpentadecane), prior to injection. When using human hybridomas in this method, it is best to inject them into immunocompromised mice, such as SCID mice, to avoid tumor rejection. The injected animals develop tumors secreting the specific monoclonal antibodies produced by the fused cell hybrids. The animal's body fluids, such as serum or ascites, can then be harvested to provide high concentrations of MAbs. Individual cell lines can also be cultured in vitro, where they naturally secrete MAbs into the culture medium, from which high concentrations can be rapidly obtained. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. Cell lines can be adapted for growth in serum-free medium to optimize the ability to recover highly pure human monoclonal immunoglobulins.

[0073] If desired, MAbs produced by either means can be further purified using filtration, centrifugation, and various chromatographic methods, such as FPLC or affinity chromatography. Fragments of the monoclonal antibodies of the present disclosure can be obtained from the purified monoclonal antibodies by methods including digestion with enzymes, such as pepsin or papain, and / or cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0074] It is also contemplated that molecular cloning techniques can be used to generate monoclonal antibodies. Single B cells identified as responding to infection or vaccination due to plasmablast or activated B cell markers, or memory B cells labeled with an antigen of interest, can be physically sorted using paramagnetic bead selection or flow cytometry sorting, followed by isolation of RNA from the single cells and amplified antibody genes by RT-PCR. Various single-cell RNA-seq methods are available for obtaining antibody variable genes from single cells. Alternatively, antigen-specific bulk-sorted populations of cells can be separated into microvesicles and matched to the heavy and light chain variable genes recovered from the single cells using physical binding of heavy and light chain amplicons or common barcoding of heavy and light chain genes from the vesicles. Furthermore, heavy and light chain genes from matched single cells can be obtained from a population of antigen-specific B cells by treating the cells with cell-permeable nanoparticles bearing RT-PCR primers and barcodes that mark transcripts with one barcode per cell. Antibody variable genes can also be isolated by RNA extraction from hybridoma lines and antibody genes obtained by RT-PCR and cloned into immunoglobulin expression vectors. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from cell lines, and phagemids expressing appropriate antibodies are selected by panning with viral antigens. The advantage of this approach over traditional hybridoma techniques is approximately 10 4The goal of this method is to produce twice as many antibodies and screen them in one round, and to generate new specificities by combining heavy and light chains, thereby further increasing the chances of finding a suitable antibody.

[0075] Other U.S. patents, each incorporated herein by reference, that teach the generation of antibodies useful in the present disclosure include U.S. Pat. No. 5,565,332, which describes the generation of chimeric antibodies using combinatorial techniques; U.S. Pat. No. 4,816,567, which describes the preparation of recombinant immunoglobulins; and U.S. Pat. No. 4,867,973, which describes antibody-therapeutic agent conjugates.

[0076] B. Antibodies of the Present Disclosure Antibodies according to the present disclosure can be defined primarily by their binding specificity. One skilled in the art can determine whether a given antibody falls within the scope of the present claims by assessing the binding specificity / affinity of such an antibody using techniques well known to those skilled in the art. For example, the epitope to which a given antibody binds may consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) amino acids located within an antigen molecule (e.g., a linear epitope within a domain). Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within an antigen molecule (e.g., a conformational epitope).

[0077] Two major classes of SARS-CoV-2 antigens are the surface spike (S) protein and the endogenous proteins, particularly the nucleocapsid (N) protein. Antibodies against the S protein are useful for disease prevention, treatment, diagnosis, or vaccine characterization. S protein antibodies have additional binding specificity for this protein and include specific antibodies that bind to the full-length ectodomain of the SARS-CoV-2 S protein (present as monomers or oligomers, such as timers, with or without stabilizing conformational mutations, such as the introduction of proline at critical sites ("2P mutations")), as well as (a) anti-S protein antibodies that bind to the receptor-binding domain (RBD) and (b) anti-S protein antibodies that bind to domains other than the RBD. While some of the subset that bind to domains other than the RBD bind to the N-terminal domain (NTD), other subsets bind to epitopes other than the NTD or RBD, and (c) S protein antibodies may further be found to neutralize SARS-CoV-2 by blocking binding of the SARS-CoV-2 S protein to its receptor, human angiotensin-converting enzyme 2 (hACE2), with others that neutralize but do not block receptor binding. Finally, antibodies may cross-react with both the SARS-CoV-2 S protein and the S proteins of other coronaviruses, such as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, and / or HCoV-HKU1, and similarly cross-neutralize both SARS-CoV-2 and these other coronaviruses.

[0078] Another specificity is an antibody that binds to the N antibody (or other endogenous target), which has primarily diagnostic applications. For example, antibodies against the N protein or other endogenous proteins of SARS-CoV-2 specifically recognize SARS-CoV-2 or cross-react with SARS-CoV-2 and other coronaviruses, such as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, and / or HCoV-HKU1.

[0079] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include conventional cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Cross-blocking can be measured by various binding assays, such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke, Methods Mol. Biol. 248:443-63, 2004), peptide truncation analysis, high-resolution electron microscopy techniques using single-particle reconstruction, cryo-EM or tomography, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer Prot. Sci. 9:487-496, 2000). Another method that can be used to identify amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium-labeling a protein of interest and then binding an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, where exchangeable protons in amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids not part of the interface. As a result, amino acids that form part of the protein / antibody interface retain deuterium and may therefore exhibit a relatively higher weight than amino acids not included in the interface. After dissociation of the antibody, protease cleavage and mass spectrometry of the target protein reveal deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, e.g., Ehring, Analytical Biochemistry 267:252-259 (1999); Engen and Smith, Anal. Chem. 73:256A-265A (2001).When antibodies neutralize SARS-CoV-2, antibody escape mutants can be isolated by growing SARS-CoV-2 in vitro or in animal models in the presence of high concentrations of antibodies. Sequence analysis of SARS-CoV-2 genes encoding antigens targeted by antibodies reveals mutations that confer antibody escape, which reveal residues in the epitope or residues that allosterically affect the structure of the epitope.

[0080] The term "epitope" refers to a site on an antigen that elicits a B cell and / or T cell response. B cell epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically contains at least three, more usually at least five or 8-10, amino acids in a unique spatial conformation.

[0081] Modification-assisted antibody profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying multiple monoclonal antibodies (mAbs) targeting the same antigen according to the similarities in their binding profiles to chemically or enzymatically modified antigen surfaces (see U.S. Patent Application Publication No. 2004 / 0101920, specifically incorporated herein by reference in its entirety). Each classification may reflect unique epitopes that are distinctly different or partially overlapping with those presented by other classifications. This technique allows for the rapid selection of genetically identical antibodies, thereby focusing on genetically distinct antibodies for characterization. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired properties. MAP can be used to classify the antibodies of the present disclosure into groups of antibodies that bind to different epitopes.

[0082] The present disclosure includes antibodies that can bind to the same epitope or a portion of an epitope. Similarly, the present disclosure also includes antibodies that compete for binding to a target or a fragment thereof, including any of the specific exemplary antibodies described herein. Whether an antibody binds to the same epitope as a reference antibody or competes for binding thereto can be easily determined by using routine methods known in the art. For example, to determine whether a test antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to the target under saturating conditions. The ability of the test antibody to bind to the target molecule is then evaluated. If the test antibody can bind to the target molecule after saturation of binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody cannot bind to the target molecule after saturation of binding with the reference antibody, the test antibody may bind to the same epitope as the epitope bound by the reference antibody.

[0083] To determine whether an antibody competes for binding with a reference anti-SARS-CoV-2 antibody, the binding approach described above is performed in two ways: first, the reference antibody is allowed to bind to SARS-CoV-2 antigen under saturating conditions, and then the binding of the test antibody to the SARS-CoV-2 molecule is assessed; second, the test antibody is allowed to bind to SARS-CoV-2 antigen molecules under saturating conditions, and then the binding of the reference antibody to the SARS-CoV-2 molecule is assessed. In either approach, if only the first (saturating) antibody is able to bind to SARS-CoV-2, it is concluded that the test and reference antibodies compete for binding to SARS-CoV-2. As will be understood by those skilled in the art, an antibody that competes for binding with a reference antibody does not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0084] Two antibodies bind to the same or overlapping epitopes if they competitively inhibit (block) each other's binding to the antigen. That is, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. In some embodiments, an antibody or antibody fragment that binds to the same or overlapping epitope as COV2-2196 is used in combination with an antibody or antibody fragment that binds to the same or overlapping epitope as COV2-2130.

[0085] Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is due to it actually binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. Structural studies by EM or crystallography can also establish whether two antibodies compete for binding that recognize the same epitope.

[0086] In another aspect, there is provided a monoclonal antibody having clone paired CDRs from the heavy and light chains, respectively, as illustrated in Tables 3 and 4. Such antibodies can be produced using the methods described herein and by the clones illustrated below in the Examples section.

[0087] In another aspect, antibodies can be defined by their variable sequences, including additional "framework" regions. Furthermore, antibody sequences can optionally be varied from these sequences using methods described in more detail below. For example, nucleic acid sequences can be varied from those described above in the following ways: (a) the variable regions can be separated from the light and heavy chain constant domains; (b) the nucleic acid can be varied from those described above but without affecting the residues encoded therein; (c) the nucleic acid can be varied from those described above by a given percentage of homology, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%; or (d) the nucleic acid can be varied from those described above by incubation at temperatures between about 50°C and about 70°C. The amino acids may be varied by their ability to hybridize under high stringency conditions, exemplified by low salt and / or high temperature conditions, such as those provided by about 0.02M to about 0.15M NaCl; (e) the amino acids may be varied from those set forth above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology; or (f) the amino acids may be varied from those set forth above by allowing for conservative substitutions (described below). Each of the foregoing applies to nucleic acid and amino acid sequences.

[0088] When comparing polynucleotide and polypeptide sequences, two sequences are said to be "identical" if the sequences of nucleotides or amino acids in the two sequences are identical when aligned for maximum correspondence, as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20, usually 30 to about 75, or 40 to about 50, contiguous positions. After two sequences are optimally aligned, the sequence can be compared to a reference sequence over the same number of contiguous positions.

[0089] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) using default parameters. This program incorporates several alignment schemes described in the following references: Dayhoff, MO (1978) A model of evolutionary change in proteins--Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogeny pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W. and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor. 11:105;Santou,N.Nes,M.(1987)Mol.Biol.Evol.4:406-425;Sneath,PHAand Sokal,RR(1973)Numerical Taxonomy--the Principles and Practice of Numerical Taxonomy,Freeman Press,San Francisco,Calif.;Wilbur,WJand Lipman, DJ (1983) Proc. Natl. Acad., Sci. USA 80:726-730.

[0090] Alternatively, optimal alignment of sequences for comparison may be performed by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or inspection.

[0091] One specific example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. For example, BLAST and BLAST 2.0 can be used with the parameters described herein to determine percent sequence identity for polynucleotides and polypeptides of the present disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The rearranged nature of antibody sequences and the variable length of each gene necessitates multiple rounds of BLAST searches of a single antibody sequence. Furthermore, manual assembly of different genes is difficult and prone to error. The sequence analysis tool lgBLAST (World Wide Web: ncbi.nlm.nih.gov / igblast / ) identifies matches to germline V, D, and J genes, details rearrangement junctions, and describes the framework regions and complementarity-determining regions of Ig V domains. lgBLAST can analyze nucleotide or protein sequences, can process sequences in batches, and can simultaneously search germline gene databases and other sequence databases to minimize the chance of missing the best possible germline V gene match.

[0092] In one exemplary embodiment, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always >0) can be used to calculate the cumulative score. Extension of word hits in each direction is stopped if the cumulative alignment score falls by an amount X from the maximum achieved; if the accumulation of one or more negatively scored residue alignments causes the cumulative score to fall below zero; or if the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11 and an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment, a B of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0093] For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of word hits in each direction is stopped when: the cumulative alignment score falls by an amount X from the maximum achieved; when one or more negatively scored residue alignments cause the cumulative score to fall below zero; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.

[0094] In one approach, "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the positions of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) of up to 20 percent, typically 5-15 percent or 10-12 percent, compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. This percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing this number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying this result by 100 to obtain the percent sequence identity.

[0095] Yet another way to define an antibody is as a "derivative" of any of the antibodies and antigen-binding fragments thereof described below. The term "derivative" refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen but contains one, two, three, four, five, or more amino acid substitutions, additions, deletions, or modifications compared to the "parent (or wild-type)" molecule. Such amino acid substitutions or additions can introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term "derivative" encompasses, for example, variants with altered CH1, hinge, CH2, CH3, or CH4 regions, such as to form antibodies with mutated Fc regions that exhibit enhanced or impaired effector or binding properties. The term "derivative" additionally encompasses non-amino acid modifications, such as amino acids that can be glycosylated (e.g., containing modified mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.), acetylated, pegylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytic cleavage, conjugated to cellular ligands or other proteins, etc. In some embodiments, the altered carbohydrate modification modulates one or more of the following: antibody solubilization, enhanced intracellular trafficking and secretion of the antibody, enhanced antibody assembly, conformational integrity, and antibody-mediated effector function. In certain embodiments, the altered carbohydrate modification enhances antibody-mediated effector function compared to an antibody lacking the carbohydrate modification.Carbohydrate modifications that result in altered antibody-mediated effector functions are well known in the art (see, e.g., Shields, R. Let al. (2002) "Lack of Fucose on Human IgG N-Linked Oligosaccharide Improves Binding to Human Fc gamma RIII and Antibody-Dependent Cellular Toxicity," J. Biol. Chem. 277(30):26733-26740; Davies J. et al. (2001) "Expression of GnTIII in a Recombinant Anti-CD20 CHO Production Cell Line: Expression of Antibodies with Altered Glycoforms Leads to an Increase in ADCC Through Higher Affinity for Fc gamma RIII," Biotechnology & Bioengineering 74(4);288-294).Methods for modifying carbohydrate content are known to those skilled in the art, and include, for example, Wallick, SC et al. (1988) "Glycosylation of a VH residue of a monoclonal antibody against alpha(1-6) dextran increases its affinity for antigen," J. Exp. Med. 168(3):1099-1109; Tao, MH et al. (1989) "Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region," J. Immunol. 143(8):2595-2601; Routledge, E. G. et al. (1995) "The effect of aglycosylation on the immunogenicity of a humanized therapeutic CD3 monoclonal antibody," Transplantation 60(8):847-53; Elliott, S. et al. al.(2003)“Enhancement Of Therapeutic Protein In Vivo Activities Through Glycoengineering,”Nature Biotechnol.21:414-21;Shields,RLet al.(2002)“Lack of Fucose on Human IgG N-Linked Oligosaccharide Improves Binding to Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity,” J. Biol. Chem. 277(30); 26733-26740).

[0096] Derivative antibodies or antibody fragments can be produced with modified sequences or glycosylation states to confer desired levels of activity in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), or antibody-dependent complement deposition (ADCD) function as measured by bead-based or cell-based assays or in vivo studies in animal models.

[0097] Derivative antibodies or antibody fragments may be modified by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In one embodiment, an antibody derivative possesses similar or identical function as the parent antibody. In another embodiment, an antibody derivative exhibits altered activity compared to the parent antibody. For example, a derivative antibody (or fragment thereof) may bind to its epitope more tightly than the parent antibody or may be more resistant to proteolysis.

[0098] C. Antibody Sequence Modifications In various embodiments, one may choose to modify the sequence of an identified antibody for a variety of reasons, such as improved expression, improved cross-reactivity, or reduced off-target binding. Modified antibodies can be made by any technique known to those of skill in the art, including expression by standard molecular biology techniques or chemical synthesis of polypeptides. Methods of recombinant expression are addressed elsewhere herein. The following is a general discussion of techniques of interest related to antibody modification.

[0099] Hybridomas are cultured, then the cells are lysed, and total RNA is extracted. Random hexamers are used at room temperature to generate cDNA copies of the RNA, followed by PCR using multiple mixtures of PCR primers expected to amplify all human variable gene sequences. PCR products are cloned into the pGEM-T Easy vector and then sequenced by automated DNA sequencing using standard vector primers. Binding and neutralization assays can be performed using antibodies recovered from hybridoma supernatants and purified by FPLC using a protein G column.

[0100] Recombinant full-length IgG antibodies can be produced by subcloning the heavy and light chain Fv DNA from the cloning vector into an IgG plasmid vector and transfecting it into 293 (e.g., Freestyle) or CHO cells, and the antibody can be recovered and purified from the 293 or CHO cell supernatant. Other suitable host cell systems include bacteria such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco with or without human-like glycan modifications), algae, or various non-human transgenic related organisms such as mouse, rat, goat, or cow.

[0101] Expression of antibody-encoding nucleic acids for both subsequent antibody purification and host immunization is also contemplated. The antibody-encoding sequence can be RNA, such as natural or modified RNA. Modified RNA refers to specific chemical modifications that confer improved stability and reduced immunogenicity to mRNA, thereby facilitating the expression of therapeutically important proteins. For example, N1-methyl-pseudouridine (N1mΨ) outperforms several other nucleoside modifications and their combinations in terms of translational potency. In addition to blocking immune / eIF2α phosphorylation-dependent inhibition of translation, the incorporated N1mΨ nucleotide dramatically alters the kinetics of the translation process by increasing ribosome pausing and its density on mRNA. The increased ribosome load of modified mRNA favors ribosome recycling or the recruitment of new ribosomes to the same mRNA, making their initiation more permissive. Such modifications can be used to enhance antibody expression in vivo after RNA inoculation. Whether natural or modified, the RNA can be delivered as naked RNA or within a delivery vehicle such as a lipid nanoparticle.

[0102] Alternatively, DNA encoding an antibody can be used for the same purpose. The DNA contains an expression cassette containing a promoter that is active in the designed host cell. Advantageously, the expression cassette is contained in a replicable vector such as a conventional plasmid or minivector. Examples of vectors include viral vectors such as poxvirus, adenovirus, herpesvirus, and adeno-associated virus, and lentiviruses are also contemplated. Replicons encoding antibody genes, such as alphavirus replicons based on VEE virus or Sindbis virus, are also contemplated. Such vectors can be delivered by needle via intramuscular, subcutaneous, or intradermal routes, or by transcutaneous electroporation if in vivo expression is desired.

[0103] The ability to rapidly obtain antibodies produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to shorten process development programs. Lonza has developed a general method using pooled transfectants grown in CDACF medium to rapidly produce small amounts (up to 50 g) of antibody in CHO cells. While slightly slower than true transient systems, its advantages include high product concentration and the use of the same host and process as the production cell line. As an example of the growth and productivity of a GS-CHO pool expressing a model antibody in a disposable bioreactor, a harvest antibody concentration of 2 g / L was achieved in a disposable bag bioreactor culture (5 L feed volume) operated in fed-batch mode within 9 weeks of transfection.

[0104] Antibody molecules include fragments (F(ab'), F(ab')2, etc.) generated, for example, by proteolytic cleavage of mAbs or single-chain immunoglobulins, which can be produced, for example, by recombinant means. F(ab')2 antibody derivatives are monovalent, while F(ab')2 antibody derivatives are divalent. In one embodiment, such fragments can be combined with each other or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Importantly, such chimeric molecules can contain substituents capable of binding to different epitopes of the same molecule.

[0105] In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the same CDR sequences as those of the disclosed antibodies (e.g., a chimeric antibody or a CDR-grafted antibody). Alternatively, it may be desirable to make modifications that introduce conservative changes into the antibody molecule. In making such modifications, the hydropathic amino acid index can be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydropathic properties of amino acids contribute to the secondary structure of a resulting protein and thus determine the interaction of that protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0106] It is also understood in the art that such amino acid substitutions can be effectively made based on hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, specifies that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its neighboring amino acids, correlates with the biological properties of the protein. As detailed in U.S. Pat. No. 4,554,101, amino acid residues are assigned the following hydrophilicity values: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (-0.5); acidic amino acids: aspartic acid (+3.0±1), glutamic acid (+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic non-ionic amino acids: serine (+0.3), asparagine (+0.5), and arginine (+0.5). (+0.2), glutamine (+0.2) and threonine (-0.4), sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); hydrophobic non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5) and glycine (0); hydrophobic aromatic amino acids: tryptophan (-3.4), phenylalanine (-2.5) and tyrosine (-2.3).

[0107] It is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity to produce a biologically or immunologically altered protein. Such changes involve substitution of amino acids whose hydrophilicity values ​​are within ±2, particularly preferred, those within ±1, and even more particularly preferred, those within ±0.5.

[0108] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration the various aforementioned characteristics are well known to those of skill in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0109] The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functions can be obtained. For example, changing to IgG1 can increase antibody-dependent cellular cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.

[0110] Alternatively, or in addition, it may be useful to combine an amino acid modification with one or more additional amino acid modifications that alter the C1q-binding and / or complement-dependent cytotoxicity (CDC) function of the Fc region of an IL-23p19-binding molecule. Particularly interesting binding polypeptides may be those that bind C1q and exhibit complement-dependent cytotoxicity. Polypeptides that have pre-existing C1q-binding activity, and optionally also have the ability to mediate CDC, may be modified to enhance one or both of these activities. Amino acid modifications that alter C1q and / or its complement-dependent cytotoxicity function are described, for example, in WO 0042072, incorporated herein by reference.

[0111] For example, by modifying C1q binding and / or FcγR binding, the Fc region of an antibody can be engineered with altered effector functions, thereby altering CDC and / or ADCC activity. An "effector function" is involved in activating or reducing a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to, C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors; BCRs), and the like. Such effector functions may require an Fc region in combination with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

[0112] For example, a variant Fc region of an antibody can be generated that has improved C1q binding and improved FcγRIII binding (e.g., both improved ADCC activity and improved CDC activity). Alternatively, if it is desired to reduce or eliminate effector function, the variant Fc region can be modified by reducing CDC activity and / or reducing ADCC activity. In other embodiments, only one of these activities can be improved, and optionally the other activity can be reduced (e.g., generating an Fc region variant with improved ADCC activity but reduced CDC activity, or vice versa).

[0113] FcRn binding. Fc mutations can also be introduced to alter the interaction with the neonatal Fc receptor (FcRn) and improve pharmacokinetic properties. A collection of human Fc variants with improved FcRn binding has been described (Shields et al., (2001)). High-resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR, (J. Biol. Chem. 276:6591-6604). Numerous methods are known that can result in increased half-life (Kuo and Aveson, (2011)). For example, amino acid modifications can be made by techniques including alanine scanning mutagenesis, random mutagenesis, and screening to evaluate neonatal Fc receptor (FcRn) binding and / or in vivo behavior. Computational strategies followed by mutagenesis can be used to select one of the amino acid mutations to mutate.

[0114] Thus, the present disclosure provides variants of antigen binding proteins with optimized FcRn binding. In certain embodiments, the variants of antigen binding proteins comprise at least one amino acid modification in the Fc region of the antigen binding protein, wherein the modification is at least one of 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291, 292, 294, 297, 298, 299, 301, 302, 303, 305, 307, 308, 309, 311, 315, 317, 320, 322, 325, 327, 330, 332, 334, 335, 338, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 300, 301, 302, 303, 305, 307, 42, 343, 345, 347, 350, 352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 380, 382, ​​384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 403, 404 4, 408, 411, 412, 414, 415, 416, 418, 419, 420, 421, 422, 424, 426, 428, 433, 434, 438, 439, 440, 443, 444, 445, 446 and 447, wherein the numbering of the amino acids in the Fc region is that of the EU index of Kabat. In a further aspect of the disclosure, the modifications are M252Y / S254T / T256E.

[0115] In addition, various publications describe methods for obtaining physiologically active molecules with altered half-lives by introducing FcRn-binding polypeptides into the molecules or fusing them to antibodies with preserved FcRn-binding affinity but significantly reduced affinity for other Fc receptors, or to the FcRn-binding domain of an antibody. See, e.g., Kontermann (2009).

[0116] Derivatized antibodies can be used to alter the half-life (e.g., serum half-life) of a parent antibody in a mammal, particularly a human. Such alterations can result in a half-life of greater than 15 days, preferably greater than 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 2 months, 3 months, 4 months, or 5 months. Increasing the half-life of an antibody or fragment thereof of the present disclosure in a mammal, preferably a human, can result in a higher serum titer of the antibody or antibody fragment in the mammal, thereby reducing the frequency of administration of the antibody or antibody fragment and / or reducing the concentration of the antibody or antibody fragment administered. Antibodies or fragments thereof with increased in vivo half-lives can be generated by techniques known to those skilled in the art. For example, antibodies or fragments thereof with increased in vivo half-lives can be generated by modifying (e.g., substituting, deleting, or adding) amino acid residues identified as being involved in the interaction between the Fc domain and the FcRn receptor.

[0117] Beltramello et al. (2010) previously reported the modification of a neutralizing mAb by substituting alanine residues at positions 1.3 and 1.2 of the CH2 domain (according to the unique numbering for the C domain of IMGT) to generate a variant that tends to enhance dengue virus infection. This modification, also known as a "LALA" mutation, abolishes antibody binding to FcγRI, FcγRII, and FcγRIIIa, as described by Hessell et al. (2007). The mutated and unmodified recombinant mAbs were compared for their ability to neutralize and enhance infection by four dengue virus serotypes. The LALA mutant retained the same neutralizing activity as the unmodified mAb but did not fully enhance the activity. Therefore, this type of LALA mutation is contemplated in the context of the antibodies disclosed herein.

[0118] Altered glycosylation. A particular embodiment of the present disclosure is an isolated monoclonal antibody or antigen-binding fragment thereof containing substantially uniform glycans without sialic acid, galactose, or fucose. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, both of which may be linked to a heavy chain constant region or a light chain constant region, respectively. The substantially uniform glycans may be covalently linked to the heavy chain constant region.

[0119] Another embodiment of the present disclosure includes a mAb with a novel Fc glycosylation pattern. The isolated monoclonal antibody or antigen-binding fragment thereof exists in a substantially homogeneous composition represented by either the GNGN or G1 / G2 glycoforms. Fc glycosylation plays an important role in the antiviral and anticancer properties of therapeutic mAbs. This disclosure is consistent with recent studies showing that fucose-free anti-HIV mAbs increased anti-lentiviral cell-mediated virus inhibition in vitro. This embodiment of the present disclosure, with homogeneous glycans lacking core fucose, demonstrated increased protection against certain viruses by a factor of more than two. Eliminating core fucose dramatically improved the ADCC activity of mAbs mediated by natural killer (NK) cells, while appearing to have the opposite effect on polymorphonuclear cell (PMN) ADCC activity.

[0120] An isolated monoclonal antibody or antigen-binding fragment thereof comprising a substantially homogeneous composition represented by GNGN or G1 / G2 glycoforms exhibits improved binding affinity to Fc gamma RI and Fc gamma RIII compared to the same antibody that is substantially free of homogeneous GNGN glycoforms and comprises G0, G1F, G2F, GNF, GNGNF, or GNGNFX-containing glycoforms. In one embodiment of the present disclosure, the antibody is at least 1x10 to 1x10 Fc gamma RI. -8 Dissociates with a Kd of 1 × 10 from Fc gamma RIII -7 It dissociates at the Kd of M.

[0121] Glycosylation of the Fc region is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to a peptide sequence of the asparagine side chain are asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. Thus, the presence of either of these peptide sequences in a polypeptide creates a potential glycosylation site.

[0122] For example, the glycosylation pattern can be altered by deleting one or more glycosylation sites found in the polypeptide and / or adding one or more glycosylation sites not present in the polypeptide. Addition of glycosylation sites to the Fc region of an antibody is conveniently accomplished by altering the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). An exemplary glycosylation variant is an amino acid substitution of residue Asn297 in the heavy chain. Alterations can also be made by adding or substituting one or more serine or threonine residues into the sequence of the original polypeptide (for O-linked glycosylation sites). Additionally, one glycosylation site can be removed by changing Asn297 to Ala.

[0123] In certain embodiments, the antibody is expressed in cells expressing β(1,4)-N-acetylglucosaminyltransferase III (GnT III), whereby GnT III adds GlcNAc to the IL-23p19 antibody. Methods for producing antibodies in this manner are described in WO 9954342, WO 03011878, JP 20030003097A1, and Umana et al., Nature Biotechnology, 17:176-180, February 1999. Genome editing techniques such as clustered regularly interspaced short palindromic repeats (CRISPR) can be used to engineer cell lines to enhance, reduce, or eliminate specific post-translational modifications, such as glycosylation. For example, CRISPR technology can be used to eliminate genes encoding glycosylation enzymes in 293 or CHO cells used to express recombinant monoclonal antibodies.

[0124] Elimination of sequence vulnerabilities in monoclonal antibody proteins. Antibody variable gene sequences obtained from human B cells can be modified to increase their manufacturability and safety. Potential protein sequence vulnerabilities can be identified by searching for sequence motifs related to sites containing: 1) unpaired Cys residue, 2) N-linked glycosylation, 3) Asn deamidation, 4) Asp isomerization, 5) Truncation of SYE, 6) oxidation of Met, 7) Oxidation of Trp 8) N-terminal glutamic acid, 9) integrin binding; 10) CD11c / CD18 binding, or 11) Fragmentation. Such motifs can be eliminated by modifying the synthetic gene of the cDNA encoding the recombinant antibody.

[0125] Protein engineering efforts in the field of therapeutic antibody development have demonstrated that specific sequences or residues are responsible for differences in solubility (Fernandez-Escamilla et al., Nature Biotech., 22(10), 1302-1306, 2004; Chennamsetty et al., PNAS, 106(29), 11937-11942, 2009; Voynov et al., Biocon. Chem., 21(2), 385-392, 2010). Evidence from solubility-altering mutants in the literature indicates that some hydrophilic residues, such as aspartic acid, glutamic acid, and serine, contribute significantly more favorably to protein solubility than other hydrophilic residues, such as asparagine, glutamine, threonine, lysine, and arginine.

[0126] Stability. Antibodies can be modified to improve their biophysical properties. Antibodies can be unfolded at high temperatures and the average apparent melting temperature used to determine relative stability. Differential scanning calorimetry (DSC) measures the heat capacity, C p DSC measures the heat required to warm a molecule per degree as a function of temperature. DSC can be used to test the thermal stability of antibodies. DSC data for mAbs can reveal the unfolding of individual domains within the mAb structure, which are often shown on the thermogram (Fab, C). H 2 and C HThis is of particular interest because it can produce up to three peaks (arising from the unfolding of three domains). Typically, unfolding of the Fab domain produces the most intense peak. The DSC profiles and relative stability of the Fc portion show distinct differences among human IgG1, IgG2, IgG3, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun. 355, 751-757, 2007). Circular dichroism (CD) performed with a CD spectrometer can also measure the average apparent melting temperature. Far-UV CD spectra of antibodies are measured in the range of 200-260 nm at 0.5 nm intervals. The final spectrum can be measured as the cumulative average of 20 runs. After background subtraction, residue ellipticity values ​​can be calculated. Thermal denaturation of antibodies (0.1 mg / mL) can be monitored at 235 nm from 25-95°C at a heating rate of 1°C / min. Dynamic light scattering (DLS) can be used to assess the tendency for aggregation. DLS is used to characterize the size of various particles, including proteins. When the system is not size-dispersed, the average effective diameter of the particles can be measured. This measurement depends on the particle core size, the size of the surface structures, and the particle concentration. DLS essentially measures the fluctuations in scattered light intensity by the particles, allowing the particle's diffusion coefficient to be determined. The DLS software in commercially available DLS instruments displays particle populations of different diameters. Stability testing can be easily performed using DLS. DLS measurements of a sample can indicate whether particles aggregate over time or with temperature changes by measuring whether the hydrodynamic radius of the particles increases. If particles aggregate, a population of larger particles with a larger radius can be observed. Temperature can be controlled in situ to analyze stability as a function of temperature. Capillary electrophoresis (CE) techniques include a proven method for characterizing antibody stability.The iCE approach can be used to separate antibody protein charge variants by deamidation, C-terminal lysine, sialylation, oxidation, glycosylation, and any other protein modifications that can alter the protein's pI. Each expressed antibody protein can be evaluated by high-throughput, free-solution capillary column isoelectric focusing (IEF, cIEF) using a Protein Simple Maurice instrument. UV absorbance detection of the entire column can be performed every 30 seconds to observe molecules focusing at their isoelectric point (pI) in real time. This approach combines the high-resolution properties of traditional gel IEF with the quantification and automation benefits of column-based separations, while eliminating the need for a flow step. This technique provides reproducible quantitative analysis of the identity, purity, and heterogeneity profile of expressed antibodies. The results identify charge heterogeneity and molecular sizing in antibodies in both absorbance and intrinsic fluorescence detection modes, with a detection sensitivity down to 0.7 μg / ml.

[0127] Solubility. The true solubility score of an antibody sequence can be measured. This can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol 427, 478-490, 2015). The amino acid sequence of residues 95-102 (Kabat numbering) within HCDR3 of each antibody fragment, such as an scFv, can be evaluated using an online program to calculate a solubility score. Solubility can also be measured using laboratory techniques. Various techniques exist, including adding lyophilized protein to a solution until the solution is saturated and the solubility limit is reached, or concentrating by ultrafiltration through a microconcentrator with a suitable molecular weight cutoff. The simplest method is to induce amorphous precipitation, which allows the solubility of the protein to be measured using a method for protein precipitation using ammonium sulfate (Trevino et al., J Mol Biol 366:449-460, 2007). Ammonium sulfate precipitation provides rapid and accurate information on relative solubility values. Ammonium sulfate precipitation produces a precipitated solution containing well-defined aqueous and solid phases, and relatively small amounts of protein are required. Solubility measurements, performed by inducing amorphous precipitation with ammonium sulfate, can also be easily performed at different pH values. Protein solubility is highly dependent on pH, and pH is considered to be the most important external factor affecting solubility.

[0128] Autoreactivity. It is generally believed that autoreactive clones must be eliminated during ontogeny by negative selection. However, it has become clear that many natural human antibodies with autoreactive properties remain in the mature repertoire of adults, and this autoreactivity may enhance the antiviral function of many antibodies against pathogens. The HCDR3 loops within antibodies during early B cell development are often rich in positive charges and are known to exhibit autoreactive patterns (Wardemann et al., Science 301, 1374-1377, 2003). The autoreactivity of a given antibody can be examined by assessing its level of binding to human-derived cells by microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometry cell surface staining (using suspended Jurkat T cells and 293S human embryonic kidney cells). Autoreactivity can also be investigated by assessing binding to tissues in tissue arrays.

[0129] Preferred residues ("human similarity"). Many recent studies have extensively performed deep sequencing of the B cell repertoire of human B cells from blood donors. Sequence information for a significant portion of the human antibody repertoire facilitates statistical evaluation of antibody sequence characteristics common to healthy humans. Knowledge of antibody sequence characteristics in reference databases of human recombinant antibody variable genes allows estimation of the degree of position-specific "human similarity" (HL) of antibody sequences. HL has been shown to be useful in the development of antibodies for clinical applications, such as therapeutic antibodies or antibodies as vaccines. The goal is to improve the human similarity of antibodies and reduce potential adverse reactions and anti-antibody immune responses that can significantly reduce the efficacy of antibody drugs or cause serious health consequences. The antibody characteristics of the combined antibody repertoire of approximately 400 million sequences from three healthy human blood donors were evaluated to generate a new "relative human similarity" (rHL) score that focuses on the hypervariable regions of antibodies. The rHL score allows for easy discrimination between human sequences (positive scores) and non-human sequences (negative scores). Antibodies can be engineered to eliminate residues that are uncommon in the human repertoire.

[0130] D. Single chain antibody Single-chain variable fragments (scFvs) are fusions of the variable regions of immunoglobulin heavy and light chains linked together by a short (usually serine or glycine) linker. These chimeric molecules retain the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of a linker peptide. This modification typically leaves specificity unchanged. Historically, these molecules were generated to facilitate phage display, which makes it extremely convenient to express antigen-binding domains as single peptides. Alternatively, scFvs can be generated directly from subcloned heavy and light chains from hybridomas or B cells. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules, and therefore require the use of common binding sites (e.g., protein A / G) to purify the antibody. These fragments can often be purified / immobilized using protein L, as protein L interacts with the variable region of the kappa light chain.

[0131] Flexible linkers are generally composed of amino acid residues that promote helices and turns, such as alanine, serine, and glycine; however, other residues can function as well. Tang et al. (1996) used phage display as a means to rapidly select linkers adapted for single-chain antibodies (scFv) from protein linker libraries. They constructed a random linker library in which genes for heavy and light chain variable domains were linked with segments encoding 18-amino acid polypeptides of variable composition. The scFv repertoire (approximately 5 × 10 6 The tethers (10 distinct members) were displayed on filamentous phage and subjected to affinity selection with haptens. The population of selected mutants showed greatly increased binding activity while retaining considerable sequence diversity. Subsequent screening of 1054 individual mutants yielded catalytically active scFvs that were efficiently produced in soluble form. Sequence analysis revealed the V as the only common feature of the selected tethers. HThese results reveal a conserved proline in the linker after the two C-terminal residues of α-glucan and abundant arginine and proline residues at other positions.

[0132] The recombinant antibodies of the present disclosure may also involve sequences or moieties that allow for receptor dimerization or multimerization. Such sequences include those derived from IgA, which in conjunction with the J chain allow for the formation of multimers. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin / avidin, allowing the two antibodies to be combined.

[0133] In a particular embodiment, single-chain antibodies can be produced by linking the receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains are produced and purified in separate cells and then linked together in an appropriate manner (i.e., the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a suitable chemical bridge).

[0134] Cross-linking reagents, such as stabilizers and coagulants, are used to form molecular bridges that connect the functional groups of two different molecules. However, it is contemplated that heterogeneous dimers or multimers composed of identical or different analogs can be produced. To link two different compounds in a stepwise manner, heterobifunctional cross-linkers can be used, which eliminate the formation of unwanted homopolymers.

[0135] Exemplary heterobifunctional crosslinkers contain two reactive groups: one that reacts with primary amine groups (e.g., N-hydroxysuccinimide) and the other that reacts with thiol groups (e.g., pyridyl disulfide, maleimide, halogen, etc.). The primary amine reactive group allows the crosslinker to react with lysine residues on one protein (e.g., the antibody or fragment of choice), and the thiol reactive group allows the crosslinker, already attached to the first protein, to react with cysteine ​​residues (containing no sulfhydryl groups) on the other protein (e.g., the agent of choice).

[0136] It is preferable to use a cross-linking agent that has reasonable stability in blood.Various types of disulfide bond-containing linkers are known that can be advantageously used to conjugate targeting agents and therapeutic / preventive agents.Linkers containing sterically hindered disulfide bonds have been shown to provide good stability in vivo, thereby preventing the release of targeting peptides before reaching the site of action.Therefore, these linkers are a group of linking agents.

[0137] Another cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and a methyl group. The steric hindrance of the disulfide bond is thought to serve to protect the bond from attack by thiolate anions, such as glutathione, that may be present in tissues and blood, thereby helping to prevent dissociation of the conjugate before the attached drug is delivered to the target site.

[0138] Like many other known cross-linking reagents, SMPT cross-linking reagents have the ability to cross-link functional groups such as the SH of cysteine ​​or primary amines (e.g., the ε-amino group of lysine). Another potential class of cross-linkers includes heterobifunctional photoreactive phenyl azides containing a cleavable disulfide bond, such as sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups, and the phenyl azide (upon photolysis) reacts nonselectively with any amino acid residue.

[0139] In addition to hindered crosslinkers, non-hindered linkers can also be used in accordance with the present invention. Other useful crosslinkers that are not considered to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such crosslinkers is well understood in the art. Another embodiment involves the use of flexible linkers.

[0140] U.S. Patent No. 4,680,338 describes bifunctional linkers useful for generating conjugates of ligands with amine-containing polymers and / or proteins, particularly for forming antibodies conjugated with chelators, drugs, enzymes, detectable labels, and the like. U.S. Patent Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing labile bonds that are cleavable under a variety of mild conditions. This linker is particularly useful in that the desired agent can be directly attached to the linker, and cleavage can result in release of the active agent. Specific applications include adding free amino groups or free sulfhydryl groups to proteins such as antibodies or drugs.

[0141] U.S. Patent No. 5,856,456 provides peptide linkers used to link polypeptide components to create fusion proteins, such as single-chain antibodies. The linkers are up to about 50 amino acids in length, contain at least one proline residue following a charged amino acid (preferably arginine or lysine), and are characterized by good stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses aminooxy-containing linkers useful in various immunodiagnostic and separation techniques.

[0142] E. Multispecific antibodies In certain embodiments, the antibodies of the present disclosure are bispecific or multispecific. Bispecific antibodies are antibodies with binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of a single antigen. In other such antibodies, a first antigen-binding site can be combined with a binding site for a second antigen. Alternatively, to target and target cellular defense mechanisms against infected cells, an anti-pathogen arm can be combined with an arm that binds to a trigger molecule on leukocytes, such as a T cell receptor molecule (e.g., CD3) or an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). Bispecific antibodies can also be used to localize cytotoxic drugs to infected cells. Such antibodies possess a pathogen-binding arm and an arm that binds a cytotoxic drug (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate, or radioisotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). WO 96 / 16673 describes a bispecific anti-ErbB2 / anti-Fc gamma RIII antibody, and U.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2 / anti-Fc gamma RI antibody. WO 98 / 02463 discloses a bispecific anti-ErbB2 / Fc α antibody. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2 / anti-CD3 antibody.

[0143] Methods for producing bispecific antibodies are known in the art. Traditionally, full-length bispecific antibodies are produced by coexpression of two immunoglobulin heavy-light chain pairs, each with a different specificity (Millstein et al., Nature, 305:537-539 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually achieved by affinity chromatography steps, which are quite cumbersome and result in low product yields. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0144] By different approaches, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusions comprise at least the hinge, C, H2 and C H3 The first heavy-chain constant region (C) containing the site necessary for light-chain binding, present in at least one of the fusions, comprises an Ig heavy-chain constant domain containing a portion of the Ig heavy-chain constant region (C) H1 ) DNA encoding the immunoglobulin heavy chain fusions and, optionally, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host cell. This provides greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide the optimal yield of the desired bispecific antibody. However, where high yields are obtained by expression of at least two polypeptide chains in equal ratios, or where this ratio does not significantly affect the yield of the desired chain combination, the coding sequences for two or all three polypeptide chains can be inserted into a single expression vector.

[0145] In a specific embodiment of this approach, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of only one-half of the immunoglobulin light chain in the bispecific molecule provides a facile separation method. This approach is disclosed in WO 94 / 04690. For further details on generating bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology, 121:210 (1986).

[0146] According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. A preferred interface is C H3 The antibody comprises at least a portion of a domain. In this method, one or more small amino acid side chains at the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine), a "cavity" of identical or similar size to the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over the yield of other unwanted end-products such as homodimers.

[0147] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be conjugated to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and to treat HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with various cross-linking techniques.

[0148] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229:81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The generated bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0149] Techniques exist that facilitate the direct recovery of Fab'-SH fragments from E. coli, which can then be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe the generation of F(ab')2 molecules of humanized bispecific antibodies. Each Fab' fragment was separately secreted from E. coli and subjected to in vitro-induced chemical coupling to form the bispecific antibody. The bispecific antibody thus formed was not only capable of binding to cells overexpressing the ErbB2 receptor and normal human T cells, but also of triggering the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0150] Various techniques for producing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described (Merchant et al., Nat. Biotechnol. 16, 677-681 (1998). doi:10.1038 / nbt0798-677pmid:9661204). For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol., 148(5):1547-1553, 1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers, which were then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), provides an alternative mechanism for making bispecific antibody fragments. The fragments are separated by a linker that is too short to allow pairing between the two domains on the same chain. L V coupled to H Therefore, V of one fragment H and V LThe domain is complementary to the V L and V H The Fv domains are forced to pair, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0151] In certain embodiments, bispecific or multispecific antibodies can be formed as DOCK-AND-LOCK™ (DNL™) complexes (see, e.g., U.S. Pat. Nos. 7,521,056, 7,527,787, 7,534,866, 7,550,143, and 7,666,400, the Examples section of each of which is incorporated herein by reference). In general, this technique utilizes the specific and high-affinity binding interactions that occur between the dimerization and docking domain (DDD) sequence of the regulatory (R) subunit of cAMP-dependent protein kinase (PKA) and the anchor domain (AD) sequence from any of a variety of AKAP proteins (Baillie et al., FEBS Letters. 2005;579:3264; Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004;5:959). The DDD and AD peptides can be conjugated to any protein, peptide, or other molecule, and because the DDD sequence spontaneously dimerizes and binds to the AD sequence, this technique allows the formation of complexes between any selected molecule capable of binding to the DDD or AD sequence.

[0152] Antibodies with three or more valencies are contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147:60, 1991; Xu et al., Science, 358(6359):85-90, 2017). Multivalent antibodies may be internalized (and / or catabolized) more quickly by cells expressing the antigen to which the antibody binds than bivalent antibodies. The antibodies of the present disclosure may be multivalent antibodies (e.g., tetravalent antibodies) having three or more antigen-binding sites, which can be readily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. Multivalent antibodies may comprise a dimerization domain and three or more antigen-binding sites. A preferred dimerization domain comprises (or consists of) an Fc region or hinge region. In this context, the antibody would comprise an Fc region and three or more antigen-binding sites amino-terminal to the Fc region. Preferred multivalent antibodies herein comprise (or consist of) three to about eight, but preferably four, antigen-binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), where the polypeptide chain comprises two or more variable regions. For example, the polypeptide chain may comprise VD1-(X1) n -VD2-(X2) n -Fc (wherein VD1 is a first variable region, VD2 is a second variable region, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1). For example, the polypeptide chain may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain; or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable region polypeptides. The multivalent antibody herein may comprise, for example, about two to about eight light chain variable region polypeptides. The light chain variable region polypeptide contemplated herein comprises a light chain variable region, and optionally a C L It further includes a domain.

[0153] Charge alteration is particularly useful in the context of multispecific antibodies, where amino acid substitutions within a Fab molecule can result in reduced mispairing of light chains with mismatched heavy chains (Bence Jones by-products), thereby allowing the generation of Fab-based bi / multispecific antigen-binding molecules with swapped VH / VL in one (or more, in the case of molecules comprising three or more antigen-binding Fab molecules) of their binding arms (see also PCT Application Publication No. WO 2015 / 150447, which is incorporated herein by reference in its entirety, and in particular the Examples therein).

[0154] Thus, in certain embodiments, the antibody included in the therapeutic agent is (a) a first Fab molecule that specifically binds to a first antigen; (b) a second Fab molecule that specifically binds to a second antigen, wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are interchanged; and wherein the first antigen is an activated T cell antigen and the second antigen is a target cell antigen, or the first antigen is a target cell antigen and the second antigen is an activated T cell antigen; i) in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by a positively charged amino acid (numbered according to Kabat) and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbered according to Kabat EU index); or ii) in the constant domain CL of the second Fab molecule under b) the amino acid at position 124 is substituted by a positively charged amino acid (numbered according to Kabat) and in the constant domain CH1 of the second Fab molecule under b) the amino acid at position 147 or the amino acid at position 213 is substituted by a negatively charged amino acid (numbered according to Kabat EU index). The antibody may not contain both the modifications mentioned under i) and ii): the constant domains CL and CH1 of the second Fab molecule are not replaced by each other (i.e., are maintained and not exchanged).

[0155] In another embodiment of the antibody, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbered according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)); and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 or the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbered according to Kabat EU index).

[0156] In a further embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is independently substituted by a lysine (K), an arginine (R) or a histidine (H) (numbering according to Kabat); and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is independently substituted by a glutamic acid (E) or an aspartic acid (D) (numbering according to Kabat EU index).

[0157] In a particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbered according to Kabat) (in one preferred embodiment, independently lysine (K) or arginine (R)), and the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbered according to Kabat) (in one preferred embodiment, independently lysine (K) or arginine (R)); and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbered according to Kabat EU index), and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbered according to Kabat EU index).

[0158] In a more particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by lysine (K) or arginine (R) (numbering according to Kabat); and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is substituted by glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by glutamic acid (E) (numbering according to Kabat EU index).

[0159] In an even more particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted by a lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by an arginine (R) (numbering according to Kabat); and in the constant domain CHI of the first Fab molecule under a) the amino acid at position 147 is substituted by a glutamic acid (E) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted by a glutamic acid (E) (numbering according to Kabat EU index).

[0160] F. Chimeric Antigen Receptor Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immune receptors, and chimeric antigen receptors (CARs)) are engineered receptors that transfer arbitrary specificities onto immune effector cells. Typically, these receptors are used to transfer the specificity of monoclonal antibodies into T cells, and the transfer of their coding sequences is facilitated by retroviral vectors. In this way, large numbers of target-specific T cells can be generated for adoptive cell transfer. Phase I clinical trials of this approach have demonstrated efficacy.

[0161] The most common form of these molecules is a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the CD3 zeta transmembrane and intramembrane domains. Such molecules transduce the zeta signal in response to recognition of their target by the scFv. An example of such a construct is 14g2a zeta, a fusion of an scFv derived from the hybridoma 14g2a (which recognizes the disialoganglioside GD2). When T cells express this molecule (usually achieved by transduction with an oncoretroviral vector), they recognize and kill target cells expressing GD2 (e.g., neuroblastoma cells). To target malignant B cells, researchers have redirected T cell specificity using chimeric immune receptors specific for the B-lineage molecule CD19.

[0162] The variable portions of the immunoglobulin heavy and light chains are fused via a flexible linker to form scFvs. This scFv is preceded by a signal peptide (which is cleaved off) to target the nascent protein to the endoplasmic reticulum for subsequent surface expression. The flexible spacer allows the scFv to be oriented in different directions to enable antigen binding. Typically, the transmembrane domain is a hydrophobic α-helix, usually derived from the original molecule's signaling endodomain, which protrudes into the cell and transmits the desired signal.

[0163] Type I proteins are actually two protein domains connected by a transmembrane alpha helix. The lipid bilayer of the cell membrane, through which the transmembrane domain runs, serves to separate the inner part (endodomain) from the outer part (ectodomain). It is not surprising that binding of the ectodomain of one protein to the endodomain of another protein would result in a molecule that combines the recognition of the former and the signal of the latter.

[0164] Ectodomain. The signal peptide directs the nascent protein to the endoplasmic reticulum, which is essential for the receptor to be glycosylated and anchored to the cell membrane. Generally, any eukaryotic signal peptide sequence works well. Generally, the signal peptide that naturally binds to the amino-terminal most component is used (e.g., in a light-chain-linker-heavy chain scFv, the natural signal of the light chain is used).

[0165] The antigen recognition domain is usually an scFv. However, many alternatives exist. Antigen recognition domains derived from natural T cell receptor (TCR) α and β single chains have been described, with simple ectodomains (e.g., the CD4 ectodomain, which recognizes HIV-infected cells) and more exogenous recognition components, such as bound cytokines (resulting in recognition of cells bearing cytokine receptors). In fact, almost anything that binds with high affinity to a given target can be used as an antigen recognition region.

[0166] The spacer region connects the antigen-binding domain to the transmembrane domain. The spacer region must be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. The simplest form is the hinge region from IgG1. Alternative forms include the CH2, CH3 regions of immunoglobulins and parts of CD3. For most scFv-based constructs, an IgG1 hinge is sufficient. However, the best spacer must often be determined experimentally.

[0167] Transmembrane domain. The transmembrane domain is a hydrophobic alpha helix that spans the membrane. Generally, a transmembrane domain derived from the most membrane-proximal component of the endodomain is used. Interestingly, the use of the CD3 zeta transmembrane domain can result in an artificial TCR incorporating a native TCR, a factor that depends on the presence of the native CD3 zeta transmembrane charged aspartic acid residue. Different transmembrane domains result in different receptor stabilities. The CD28 transmembrane domain apparently results in an expressed, stable receptor.

[0168] The endodomain. This domain is the "functional part" of the receptor. After antigen recognition, the receptor clusters and transmits a signal to the cell. The most commonly used endodomain component is CD3 zeta, which contains three ITAMs. This transmits an activation signal to T cells after antigen binding. However, CD3 zeta may not provide a fully competent activation signal, requiring additional costimulatory signaling.

[0169] "First-generation" CARs typically have an intracellular domain derived from the CD3 ξ chain, the primary transmitter of signals from endogenous TCRs. "Second-generation" CARs have added intracellular signaling domains derived from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to T cells. Preclinical studies have shown that second-generation CAR designs improve the antitumor activity of T cells. More recently, "third-generation" CARs have combined multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further increase potency.

[0170] G.ADC Antibody-drug conjugates (ADCs) are a new class of highly potent biologics designed as targeted therapies to treat people with infectious diseases. ADCs are conjugated molecules composed of an antibody (whole mAb or an antibody fragment such as a single-chain variable fragment, or scFv) linked to a biologically active cytotoxic / antiviral payload or drug via a stable chemical linker with a labile bond. Antibody-drug conjugates are examples of bioconjugates and immunoconjugates.

[0171] Antibody-drug conjugates combine the unique targeting capabilities of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs, allowing them to sensitively distinguish between healthy and diseased tissue. This means that, in contrast to traditional systematic approaches, antibody-drug conjugates target and attack infected cells while sparing healthy cells.

[0172] In developing ADC-based antitumor therapies, anticancer drugs (e.g., cytotoxins) are conjugated to antibodies that specifically target particular cellular markers (e.g., proteins ideally found only on or present on infected cells). The antibodies track these proteins throughout the body and attach themselves to the surface of cancer cells. A biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or internalizes the antibody along with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released, killing the cell or inhibiting viral replication. Because of this targeting, the drug ideally has fewer side effects and a broader therapeutic window than other agents.

[0173] A stable linkage between the antibody and the cytotoxic / antiviral drug is a critical aspect of ADCs. Linkers are based on chemical motifs, including disulfides, hydrazones, or peptides (cleavable) or thioethers (non-cleavable), to control the distribution and delivery of the cytotoxic drug to target cells. Both cleavable and non-cleavable linker types have been proven safe in preclinical and clinical studies. Brentuximab vedotin contains an enzyme-sensitive cleavable linker that delivers the synthetic anti-cancer agent monomethyl auristatin E (MMAE), a potent and highly toxic microtubule inhibitor, to human specific CD30-positive malignant cells. Due to its high toxicity, MMAE, which inhibits cell division by blocking tubulin polymerization, cannot be used as a single-agent chemotherapeutic agent. However, the combination of MMAE linked to an anti-CD30 monoclonal antibody (cAC10, a cell membrane protein for tumor necrosis factor, or TNF, receptor) has been proven to be stable in extracellular fluids, cleavable by cathepsins, and safe for treatment. Trastuzumab emtansine is another approved ADC, combining the microtubule inhibitor mertansine (DM-1), a derivative of maytansine, and the antibody trastuzumab (Herceptin® / Genentech / Roche), linked by a stable, non-cleavable linker.

[0174] The availability of better and more stable linkers changes the function of chemical bonds. The type of cleavable or non-cleavable linker confers specific properties to cytotoxic (anti-cancer) drugs. For example, a non-cleavable linker retains the drug intracellularly. As a result, the entire antibody, linker, and cytotoxic drug enters the target cancer cell, where the antibody is degraded to the amino acid level. The resulting complex (amino acids, linker, and cytotoxic drug) then becomes the active drug. In contrast, a cleavable linker is catalyzed by enzymes in the host cell, where it releases the cytotoxic drug.

[0175] Another type of cleavable linker, which adds an additional molecule between the cytotoxic / antiviral drug and the cleavage site, is currently under development. This linker technology allows researchers more flexibility in generating ADCs without worrying about altering cleavage kinetics. Researchers are also developing a new method for peptide cleavage based on Edman degradation, a method for sequencing amino acids within a peptide. Future directions in ADC development also include site-specific conjugation (TDC) to further improve stability and therapeutic index, as well as the development of alpha-radioimmunoconjugates and antibody-conjugated nanoparticles.

[0176] H.BiTE Bispecific T cell engagers (BiTEs) are a type of artificial, bispecific monoclonal antibody being investigated for use as anti-cancer drugs. BiTEs induce the cytotoxic activity of the host's immune system, more specifically, T cells, against infected cells. BiTE is a registered trademark of Micromet AG.

[0177] BiTEs are fusion proteins consisting of two single-chain variable fragments (scFv) of different antibodies or amino acid sequences from four different genes in a single peptide chain of approximately 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, while the other binds to infected cells via a specific molecule.

[0178] Like other bispecific antibodies and unlike conventional monoclonal antibodies, BiTEs form a link between T cells and target cells, which allows T cells to exert cytotoxic / viral activity against infected cells by producing proteins such as perforin and granzymes, independent of the presence of MHC I molecules or costimulatory molecules. These proteins enter infected cells and induce apoptosis of the cells, mimicking the physiological process observed during T cell attack on infected cells.

[0179] I. Intracellular antibodies In certain embodiments, the antibody is a recombinant antibody suitable for acting inside a cell; such antibodies are known as "intrabodies." These antibodies can interfere with target function through a variety of mechanisms, for example, by altering intracellular protein trafficking, interfering with enzyme function, or blocking protein-protein or protein-DNA interactions. In many respects, their structure mimics or resembles the structure of the single-chain and single-domain antibodies described above. Indeed, single transcripts / single chains are an important mechanism that allows intracellular expression in the target cell, as well as making protein translocation across the cell membrane more feasible. However, additional mechanisms are required.

[0180] Two major issues affecting the implementation of intracellular antibody therapy are delivery, including cell / tissue targeting, and stability. Regarding delivery, various approaches have been used, such as tissue-directed delivery, the use of cell-type specific promoters, viral delivery, and the use of cell-permeable / membrane-translocating peptides. Regarding stability, approaches generally involve either brute force screening, such as methods involving phage display and may include sequence maturation or consensus sequence development, or further targeted modifications, such as insertions and disulfide substitutions / modifications that stabilize the sequence (e.g., Fc regions, chaperone protein sequences, leucine zippers).

[0181] An additional mechanism that intracellular antibodies may require is a signal for intracellular targeting. Vectors that can target intracellular antibodies (or other proteins) to intracellular regions such as the cytoplasm, nucleus, mitochondria, and ER have been designed and are commercially available (Invitrogen Corp.; Persic et al., 1997).

[0182] Due to their ability to enter cells, intracellular antibodies have additional uses not available to other types of antibodies. In the case of the present antibodies, their ability to interact with the MUC1 cytoplasmic domain within live cells may result in inhibition of MUC1 CD-associated functions, such as signaling function (binding to other molecules) or oligomerization. In particular, it is contemplated that such antibodies may be used to inhibit MUC1 dimer formation.

[0183] J. Purification In certain embodiments, antibodies of the present disclosure may be purified. The term "purified," as used herein, is intended to refer to a composition that is isolatable from other components, and the protein may be purified to any degree relative to its naturally occurring state. A purified protein therefore also refers to a protein that has been freed from the environment in which it may naturally occur. When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms a major component of the composition, such as making up about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

[0184] Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu into polypeptide and non-polypeptide fractions. Once the polypeptide has been separated from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of pure peptides are ion exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibodies, etc., or heat denaturation followed by centrifugation; gel filtration; reverse-phase, hydroxylapatite, and affinity chromatography; and combinations of such and other techniques.

[0185] When purifying the antibodies of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide can be purified from other cellular components using an affinity column that binds to a tagged portion of the polypeptide. Generally, as is known in the art, the order in which the various purification steps are performed can be changed or certain steps can be omitted, and still result in a suitable method for preparing a substantially purified protein or peptide.

[0186] Generally, whole antibodies are fractionated using an agent that binds to the Fc portion of the antibody (i.e., Protein A). Alternatively, antigen can be used to simultaneously purify and select suitable antibodies. Such methods often use a selection agent bound to a support such as a column, filter, or beads. The antibody is bound to the support, contaminants are removed (e.g., washed away), and the antibody is released by application of conditions (salt, heat, etc.).

[0187] Various methods for quantifying the degree of purification of a protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, measuring the specific activity of an active fraction or assessing the amount of polypeptide within a fraction by SDS / PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction and compare it to the specific activity of the initial extract, thereby calculating the degree of purity. The actual units used to express the amount of activity will, of course, depend on the particular assay technique chosen and whether the expressed protein or peptide exhibits detectable activity following purification.

[0188] It is known that the migration of polypeptides can vary, sometimes significantly, under different SDS / PAGE conditions (Capaldi et al., 1977). Therefore, it will be understood that the molecular weight of purified or partially purified expression products may vary significantly under different electrophoretic conditions.

[0189] III. Active / Passive Immunization and Treatment / Prevention of SARS-CoV-2 Infection A. Formulation and Administration The present disclosure provides pharmaceutical compositions containing anti-SARS-CoV-2 virus antibodies and antigens for generating them. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or fragment thereof or a peptide immunogen and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more specifically, in humans. The term "carrier" refers to a diluent, excipient, or vehicle with which a therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0190] The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, as needed. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceuticals are described in "Remington's Pharmaceutical Sciences." Such compositions contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with an appropriate amount of carrier so as to provide a suitable dosage form for the patient. The formulation should be suitable for the method of administration, which can be oral, intravenous, intraarterial, buccal, nasal, aerosol, bronchial inhalation, rectal, vaginal, topical, or via artificial respiration.

[0191] Active vaccines are also contemplated when the disclosed antibodies are generated in vivo in subjects at risk for SARS-CoV-2 infection. Such vaccines can be formulated for parenteral administration, e.g., for injection via intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Intradermal and intramuscular routes of administration are contemplated. Alternatively, vaccines can be administered topically, e.g., via nasal sprays, inhalation with a nebulizer, or directly to the mucosa via rectal or vaginal delivery. Pharmaceutically acceptable salts include acid salts, e.g., those formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0192] Passive transfer of antibodies, known as artificially acquired passive immunity, generally involves the use of intravenous or intramuscular injections. Antibodies can be in the form of human or animal plasma or serum, as pooled intravenous human immune globulin (IVIG) or intramuscular human immune globulin (IG), as high-titer human IVIG or IG from immunized or disease-recovered donors, and as monoclonal antibodies (MAbs). Such immunity generally lasts only for a short period of time, and there are potential risks of hypersensitivity reactions and serum sickness, particularly with gamma globulins of non-human origin. However, passive immunization provides immediate protection. The antibodies are formulated in a carrier suitable for injection, i.e., sterile and injectable.

[0193] Generally, the components of the compositions of the present disclosure are provided separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by infusion, it can be administered using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0194] The compositions of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0195] 2. ADCC Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that results in the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which an Fc region-containing antibody or a fragment thereof specifically binds via a protein portion, typically at the N-terminus. An "antibody with increased / decreased antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to an antibody with increased / decreased ADCC as measured by any suitable method known to those skilled in the art.

[0196] As used herein, the term "increased / decreased ADCC" is defined as either an increase / decrease in the number of target cells lysed in a given time period at a given concentration of antibody in the medium surrounding the target cells by the ADCC mechanism defined above and / or an increase / decrease in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time period by the ADCC mechanism. The increase / decrease in ADCC is compared to the ADCC mediated by the same antibody produced by the same standard production, purification, formulation, and storage methods (known to those skilled in the art) but in unmodified host cells of the same type. For example, the increase in ADCC mediated by an antibody produced in a host cell modified by the methods described herein to have an altered glycosylation pattern (e.g., to express the glycosyltransferase GnTIII or other glycosyltransferase) is compared to the ADCC mediated by the same antibody produced in unmodified host cells of the same type.

[0197] 3.CDC Complement-dependent cytotoxicity (CDC) is a function of the complement system. It is a process of the immune system that kills pathogens by damaging their membranes, without the involvement of antibodies or cells of the immune system. There are three main processes. All three involve the insertion of one or more membrane attack complexes (MACs) into the pathogen, which causes lethal oncotic swelling, or CDC. This is one mechanism by which antibodies or antibody fragments have antiviral effects.

[0198] IV. Antibody Conjugates The antibodies of the present disclosure may be linked to at least one agent to form an antibody conjugate. To improve the effectiveness of antibody molecules as diagnostic or therapeutic agents, they are typically linked, covalently bonded, or complexed with at least one desired molecule or moiety. Such molecules or moieties may be, but are not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules conjugated to antibodies include toxins, antitumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelators, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any moiety that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or ligands, such as biotin.

[0199] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall into two categories: those for use in in vitro diagnostics, such as various immunoassays, and those for use in in vivo diagnostic protocols, commonly known as "antibody-directed imaging." Many suitable imaging agents are known in the art, as are methods for binding them to antibodies (see, for example, U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioisotopes, fluorescent dyes, NMR-detectable substances, and X-ray contrast agents.

[0200] In the case of paramagnetic ions, mention may be made, by way of example, of ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and / or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include, but are not limited to, lanthanum (III), gold (III), lead (II), and in particular bismuth (III).

[0201] In the case of radioisotopes for therapeutic and / or diagnostic use, astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, Copper 67 , 152 Eu, gallium 67 , 3 Hydrogen, iodine 123 , iodine 125 , iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, rhenium 186 ,rhenium 188 , 75 selenium, 35 Sulfur, Technetium 99m and / or yttrium 90 In many cases, the following may be mentioned for use in a particular embodiment: 125 I is preferred, and technetium is preferred because it has low energy and is suitable for wide-area detection. 99m and / or indium 111is also often preferred. Radioactively labeled monoclonal antibodies of the present disclosure can be produced according to methods well known in the art. For example, monoclonal antibodies can be iodinated by contact with sodium iodide and / or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite or an enzymatic oxidizing agent such as lactoperoxidase. Monoclonal antibodies according to the present disclosure can also be iodinated with technetium by a ligand exchange process, e.g., reducing pertechnate with a stannous solution, chelating the reduced technetium to a Sephadex column, and applying the antibody to the column. 99m Alternatively, direct labeling techniques can be used by incubating the antibody with a reducing agent such as pertechnate or SnCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediate functional groups often used to attach radioisotopes that exist as metal ions to antibodies are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0202] Among the fluorescent labels contemplated for use as conjugates are Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, rhodamine green, rhodamine red, renographin, ROX, TAMRA, TET, tetramethylrhodamine, and / or Texas Red.

[0203] A further class of antibodies contemplated by the present disclosure is intended primarily for in vitro use, in which the antibody is linked to an enzyme (enzyme tag) that generates a colored product upon contact with a secondary binding ligand and / or a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0204] Yet another known method for site-specifically binding a molecule to an antibody involves reacting the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with amino acids within the antigen-binding site, thereby disrupting this site and blocking specific antigen reaction. However, this may not be advantageous because it results in a loss of antigen binding by the antibody conjugate.

[0205] Molecules containing azide groups can also be used to form covalent bonds with proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). Specifically, 2- and 8-azido analogs of purine nucleotides have been used as site-specific photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 2- and 8-azido nucleotides have also been used to map nucleotide-binding domains in purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and can be used as antibody binding agents.

[0206] Several methods for linking or conjugating antibodies to their conjugate moieties are known in the art. One conjugation method involves the use of metal chelate complexes using organic chelators such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3α-6α-diphenylglycouril-3, bound to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies can also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reacting with isothiocyanates. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies, with a detectable imaging moiety attached to the antibody using a linker such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.

[0207] Another embodiment contemplates derivatizing immunoglobulins by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin using reaction conditions that do not alter the antibody binding site. Antibody conjugates produced according to this method have been disclosed to exhibit improved longevity, specificity, and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, in which the effector or reporter molecule is conjugated to a carbohydrate residue in the Fc region, has also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to generate antibodies with diagnostic and therapeutic potential and is currently undergoing clinical evaluation.

[0208] V. Immunodetection Methods In still further embodiments, the present disclosure relates to immunodetection methods for binding, purifying, removing, quantifying, or otherwise generally detecting SARS-CoV-2 and its associated antigens. While such methods can be applied in a conventional sense, another application is in the quality control and monitoring of vaccines and other viral stocks, where antibodies according to the present disclosure can be used to assess the quantity or integrity (i.e., long-term stability) of viral antigens. Alternatively, the methods can be used to screen various antibodies for appropriate / desired reaction characteristics.

[0209] Other immunodetection methods include specific assays for determining the presence of SARS-CoV-2 in a subject. A wide variety of assay formats are contemplated, particularly those used to detect SARS-CoV-2 in bodily fluids obtained from a subject, such as saliva, blood, plasma, sputum, semen, or urine. In particular, semen has been demonstrated as a viable sample for detecting SARS-CoV-2 (Purpura et al., 2016; Mansuy et al., 2016; Barzon et al., 2016; Gornet et al., 2016; Duffy et al., 2009; CDC, 2016; Halfon et al., 2010; Elder et al. 2005). Assays can be advantageously formatted for non-medical (home) use, including lateral flow assays (see below), similar to home pregnancy tests. These assays may be packaged in the form of a kit containing appropriate reagents and instructions for use by the subject's family.

[0210] Some immunodetection methods include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), immunoradiometric assays, fluorescent immunoassays, chemiluminescent assays, bioluminescent assays, and Western blots, to name a few. Competitive assays for detecting and quantifying SARS-CoV-2 antibodies targeting specific parasitic epitopes in a sample are also provided. The steps of various useful immunodetection methods are described in scientific literature, for example, in Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). Generally, immunoconjugation methods involve obtaining a sample suspected of containing SARS-CoV-2 and contacting the sample with a first antibody according to the present disclosure, optionally under conditions effective to allow the formation of an immune complex.

[0211] These methods include methods for purifying SARS-CoV-2 or related antigens from a sample. The antibody is preferably linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing SARS-CoV-2 or antigen components is applied to the immobilized antibody. Unwanted components are washed from the column, and the SARS-CoV-2 antigen is allowed to immunocomplex with the immobilized antibody, which is then recovered by removing the microorganism or antigen from the column.

[0212] Immunobinding methods include methods for detecting and quantifying the amount of SARS-CoV-2 or related components in a sample, as well as detecting and quantifying any immune complexes formed during the binding process. A sample suspected of containing SARS-CoV-2 or its antigen is obtained, contacted with an antibody that binds to SARS-CoV-2 or its component, and then the amount of immune complexes formed under specific conditions is detected and quantified. In terms of antigen detection, the biological sample analyzed can be any sample suspected of containing SARS-CoV-2 or a SARS-CoV-2 antigen, such as a tissue section or specimen, a homogenized tissue extract, a bodily fluid including blood or serum, or a secretion such as feces or urine.

[0213] Contacting a selected biological sample with an antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) generally involves simply adding the antibody composition to the sample and incubating the mixture long enough for the antibodies to form immune complexes, i.e., bind to any SARS-CoV-2 or antigens present. The sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, is then generally washed to remove any nonspecifically bound antibody species, thereby allowing the detection of only those antibodies specifically bound within the primary immune complexes.

[0214] In general, detecting the formation of immune complexes is known in the art and can be achieved by applying many techniques. These methods are generally based on detecting labels or markers, such as radioactive tags, fluorescent tags, biological tags, and enzyme tags. Patents relating to the use of such labels include U.S. Pat. Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Of course, as known in the art, further advantages can be found by using secondary binding ligands, such as a second antibody and / or a biotin / avidin ligand binding arrangement.

[0215] The antibody used for detection can itself be linked to a detectable label, allowing for detection of not only the label but also the amount of primary immune complexes in the composition. Alternatively, a first antibody that becomes bound within a primary immune complex can be detected by a second binding ligand that has binding affinity for the antibody. In such cases, the second binding ligand can be linked to a detectable label. The second binding ligand is often itself an antibody and is therefore sometimes referred to as a "secondary" antibody. The primary immune complexes are contacted with a labeled secondary binding ligand or antibody under effective conditions and for a time sufficient to allow secondary immune complexes to form. The secondary immune complexes are then generally washed to remove any nonspecifically bound labeled secondary antibodies or ligands, and the label remaining in the secondary immune complexes is then detected.

[0216] Another method involves detecting primary immune complexes using a two-step approach. A second binding ligand, such as an antibody, having binding affinity for the antibody is used to form secondary immune complexes as described above. After washing, the secondary immune complexes are again contacted with a third binding ligand or antibody having binding affinity for the second antibody under effective conditions and for a time sufficient to allow immune complexes (tertiary immune complexes) to form. The third ligand or antibody is linked to a detectable label, allowing the thus-formed tertiary immune complexes to be detected. This system can provide signal amplification, if desired.

[0217] One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, followed by a second antibody to detect biotin bound to the complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the first-step antibody. If the target antigen is present, some of the antibody binds to the antigen, forming a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by successive incubations in solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding an additional biotin moiety to the antibody / antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing a second-step antibody directed against biotin. This second-step antibody is labeled with an enzyme that can be used to detect the presence of the antibody / antigen complex, for example, by histoenzymatic chemistry using a chromogenic substrate. Suitable amplification can produce a conjugate visible to the naked eye.

[0218] Another known method of immunodetection is to use the immuno-PCR (polymerase chain reaction) technique. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA. However, instead of using multiple incubations of streptavidin and biotinylated DNA, the DNA / biotin / streptavidin / antibody complex is washed with a low pH or high salt buffer, which releases the antibody. The resulting wash solution is then used to carry out a PCR reaction using appropriate, well-controlled primers. At least in theory, the enormous amplification power and specificity of PCR can be exploited to detect a single antigen molecule.

[0219] A. ELISA Immunoassays, in their simplest and most direct sense, are binding assays. Particularly preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily understood that detection is not limited to such techniques, and Western blots, dot blots, FACS analysis, and the like can also be used.

[0220] In one exemplary ELISA, antibodies of the present disclosure are immobilized on a selected protein affinity surface, such as wells in a polystyrene microtiter plate. A test composition suspected of containing SARS-CoV-2 or a SARS-CoV-2 antigen is then added to the wells. After binding and washing to remove nonspecifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding another anti-SARS-CoV-2 antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA." Detection can also be achieved by adding a second anti-SARS-CoV-2 antibody followed by a third antibody linked to a detectable label that has binding affinity for the second antibody.

[0221] In another exemplary ELISA, a sample suspected of containing SARS-CoV-2 or a SARS-CoV-2 antigen is immobilized on a well surface and then contacted with an anti-SARS-CoV-2 antibody of the present disclosure. After binding and washing to remove nonspecifically bound immune complexes, the bound anti-SARS-CoV-2 antibody is detected. If the first anti-SARS-CoV-2 antibody is linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes can be detected using a second antibody linked to a detectable label that has binding affinity for the first anti-SARS-CoV-2 antibody.

[0222] Regardless of the format used, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detection of bound immune complexes, which are described below.

[0223] When coating a plate with either an antigen or an antibody, the wells of the plate containing the antigen or antibody solution are generally incubated overnight or for a specified period of time. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include bovine serum albumin (BSA), casein, or a powdered milk solution. Coating allows for blocking of nonspecific adsorption sites on the immobilized surface, thereby reducing background caused by nonspecific binding of the antiserum to this surface.

[0224] In ELISA, it is perhaps more customary to use secondary or tertiary detection means rather than direct procedures. Thus, after binding the protein or antibody to a well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilized surface is contacted with a biological sample for testing under conditions effective to allow immune complex (antigen / antibody) formation. Detection of the immune complex requires a labeled secondary binding ligand or antibody and a secondary binding ligand or antibody linked to a labeled tertiary antibody or third binding ligand.

[0225] "Under conditions effective to allow immune complex (antigen / antibody) formation" means that the conditions preferably include diluting the antigen and / or antibody with a solution such as BSA, bovine gamma globulin (BGG), or phosphate buffered saline (PBS) / Tween. These added agents also tend to aid in reducing nonspecific background.

[0226] Furthermore, "suitable" conditions mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. The incubation step may typically be about 1 to 2 to 4 hours at a temperature preferably on the order of 25°C to 27°C, or overnight at about 4°C.

[0227] After all incubation steps in an ELISA, the contact surface is washed to remove uncomplexed material. Preferred washing procedures include washing with a solution such as PBS / Tween or borate buffer. Specific immune complexes are formed between the test sample and the originally bound material, and after subsequent washing, even trace amounts of immune complexes can be measured.

[0228] To provide a means of detection, the second or third antibody has an associated label that allows it to be detected. Preferably, this label is an enzyme that generates color development upon incubation with a suitable chromogenic substrate. Thus, for example, it may be desirable to contact or incubate the first and second immune complexes with an antibody conjugated to urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase for a period of time under conditions that favor the development of further immune complex formation (e.g., incubation in a PBS-containing solution, such as PBS-Tween, for 2 hours at room temperature).

[0229] After incubation with the labeled antibody and subsequent washing to remove unbound material, the amount of label is quantified by incubation with a colorimetric substrate, such as urea, bromocresol purple, 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or HO, in the case of peroxidase as the enzyme label. Quantitation is then performed by measuring the degree of color development, for example, using a visible spectrum spectrophotometer.

[0230] In another embodiment, the present disclosure contemplates the use of a competitive format, which is particularly useful for detecting SARS-CoV-2 antibodies in a sample. In a competition-based assay, an unknown amount of analyte or antibody is measured by its ability to displace a known amount of labeled antibody or analyte. Thus, a quantifiable decrease in signal indicates the amount of unknown antibody or analyte in the sample.

[0231] Here, we propose to measure the amount of SARS-CoV-2 antibodies in a sample using a labeled SARS-CoV-2 monoclonal antibody. The basic format involves contacting a known amount of SARS-CoV-2 monoclonal antibody (linked to a detectable label) with a SARS-CoV-2 antigen or particle. The SARS-CoV-2 antigen or microorganism is preferably bound to a support. After the labeled monoclonal antibody is bound to the support, the sample is added and incubated under conditions that allow any unlabeled antibodies in the sample to compete with, and thus displace, the labeled monoclonal antibody. By measuring either the label lost or the label remaining (and subtracting from the original amount of bound label), one can determine how much unlabeled antibody is bound to the support and, therefore, how much antibody is present in the sample.

[0232] B. Western Blot Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. This technique uses gel electrophoresis to separate native or denatured proteins by polypeptide length (denatured) or 3D protein structure (native / non-denatured). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF) and probed (detected) with antibodies specific to the target protein.

[0233] Samples can be taken from whole tissues or cell cultures. Often, solid tissues are first mechanically disrupted using a blender (for relatively large sample volumes), a homogenizer (for relatively small volumes), or sonication. Cells can also be disrupted by one of the above mechanical methods. Note, however, that bacterial, viral, or environmental samples can be protein sources, and thus Western blots are not limited to cell studies alone. A combination of detergents, salts, and buffers can be used to promote cell lysis and solubilize proteins. Often, protease and phosphatase inhibitors are added to prevent the sample from digesting with its own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

[0234] Gel electrophoresis is used to separate proteins from a sample. Proteins can be separated by isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the sample treatment and the properties of the gel. This is a very useful method for determining proteins. Two-dimensional (2D) gels can also be used, where proteins from a single sample are spread across two dimensions. In the first dimension, proteins are separated according to their isoelectric point (the pH at which they have a neutral net charge), and in the second dimension, they are separated according to their molecular weight.

[0235] To make proteins available for antibody detection, they are transferred from the gel to a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on top of the gel, which is then layered with filter paper. The entire layer is placed in a buffer solution that moves to the top of the paper by capillary action, carrying the proteins with it. Another method for protein transfer, called electroblotting, uses an electric current to draw proteins from the gel to a PVDF or nitrocellulose membrane. The proteins move from the gel to the membrane while maintaining the organization that held them within the gel. This blotting process results in a thin surface layer of proteins exposed for detection (see below). Both types of membrane are chosen for their nonspecific protein-binding properties (i.e., they bind all proteins equally well). Protein binding is based on hydrophobic interactions and charge interactions between the membrane and the protein. Nitrocellulose membranes are less expensive than PVDF but are much more fragile and do not withstand repeated probing well. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be assessed by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once transferred, proteins are detected using labeled or unlabeled primary antibodies, followed by indirect detection using labeled Protein A or a secondary labeled antibody that binds to the Fc region of the primary antibody.

[0236] C. Lateral Flow Assay Lateral flow assays, also known as lateral flow immunochromatographic assays, are simple devices intended to detect the presence (or absence) of a target analyte in a sample (matrix) without the need for dedicated and expensive equipment, although there are many laboratory-based applications supported by a reader. Typically, these tests are used as low-resource medical diagnostics, either for home testing, point-of-care testing, or laboratory applications. A widespread and well-known application is the home pregnancy test.

[0237] This technology is based on a series of capillary beds, such as small pieces of porous paper or sintered polymer. Each of these elements has the ability to spontaneously transport fluid (e.g., urine). The first element (sample pad) acts as a sponge, retaining excess sample fluid. Upon immersion, the fluid migrates to the second element (conjugate pad), where the manufacturer stores so-called conjugates, which are dried forms of bioactive particles (see below) in a salt-sugar matrix, all contained to ensure an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., an antibody) immobilized on the particle surface. The sample fluid dissolves the salt-sugar matrix while also dissolving the particles, and in a combined transport action, dissolves the sample and conjugate mixture as it flows through the porous structure. Thus, the analyte binds to the particles as it travels further through the third capillary bed. This material has one or more regions (often called strips) where the manufacturer immobilizes a third molecule. By the time the sample-conjugate mixture reaches these strips, the analyte has bound to the particle, and a third "capture" molecule has bound to the complex. After a while, as more fluid passes through the strip, particles accumulate, causing the strip area to change color. Typically, there are at least two strips: one (control) that captures any particles, indicating that the reaction conditions and technique worked well, and a second that contains a specific capture molecule and captures only particles with immobilized analyte molecules. After passing through these reaction areas, the fluid enters a final porous material, the wick, which simply serves as a waste container. Lateral flow tests can function as competitive or sandwich assays. Lateral flow assays are disclosed in U.S. Pat. No. 6,485,982.

[0238] D. Immunohistochemistry The antibodies of the present disclosure may also be used in conjunction with both fresh-frozen and / or formalin-fixed, paraffin-embedded tissue blocks prepared for immunohistochemical (IHC) studies. Methods for preparing tissue blocks from these particulate specimens have been successfully used in previous IHC studies of various prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0239] Briefly, frozen sections can be prepared by rehydrating 50 ng of frozen "ground" tissue in phosphate-buffered saline (PBS) in small plastic capsules at room temperature; pelleting the particles by centrifugation; resuspending them in viscous embedding medium (OCT); inverting the capsule and / or re-pelleting by centrifugation; flash-freezing in isopentane at -70°C; cutting the plastic capsule and / or removing the frozen cylinder of tissue; securing the cylinder of tissue on the chuck of a cryostat microtome; and / or cutting 25-50 serial sections from the capsule. Alternatively, frozen whole tissue specimens can be used to cut serial sections.

[0240] Permanent sections can be prepared by a similar method, including rehydrating a 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours to fix; washing / pelleting; resuspending in warm 2.5% agar; pelleting; chilling in ice water to set the agar; removing the tissue / agar block from the tube; infiltrating and / or embedding the block in paraffin; and / or cutting up to 50 serial permanent sections. Again, whole tissue samples can be used instead.

[0241] E. Immunodetection Kit In still further embodiments, the present disclosure relates to immunodetection kits for use with the immunodetection methods described above. Because antibodies can be used to detect SARS-CoV-2 or a SARS-CoV-2 antigen, the antibodies can be included in the kit. The immunodetection kit thus comprises, in suitable container means, a first antibody that binds to SARS-CoV-2 or a SARS-CoV-2 antigen, and optionally an immunodetection reagent.

[0242] In certain embodiments, SARS-CoV-2 antibodies may be pre-bound to a solid support, such as a column matrix and / or the wells of a microtiter plate. The immunodetection reagents of the kit can take any one of a variety of forms, including, for example, a detectable label associated with or linked to a given antibody. Detectable labels associated with or bound to a secondary binding ligand are also contemplated. An exemplary secondary ligand is a ligand of a secondary antibody that has binding affinity for the first antibody.

[0243] Further suitable immunodetection reagents for use in the present kits include two-component reagents comprising a second antibody having binding affinity for the first antibody, along with a third antibody having binding affinity for the second antibody, linked to a detectable label. As noted above, many exemplary labels are known in the art, and all such labels can be used in connection with the present disclosure.

[0244] The kit may further include an appropriately aliquoted composition of SARS-CoV-2 or a SARS-CoV-2 antigen, labeled or unlabeled, that can be used to generate a standard curve for the detection assay. The kit may contain the antibody-label conjugate either in fully conjugated form, in the form of an intermediate, or as separate moieties to be conjugated by the user of the kit. The kit components may be packaged in either aqueous medium or lyophilized form.

[0245] The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody may be placed, or preferably, suitably aliquoted. The kits of the present disclosure will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection- or blow-molded plastic containers into which the desired vials may be retained.

[0246] F. Vaccine and Antigen Quality Control Assays The present disclosure also contemplates the use of antibodies and antibody fragments as described herein to assess the antigenic integrity of viral antigens in a sample. Biological pharmaceuticals, such as vaccines, differ from chemical drugs in that they typically cannot be molecularly characterized. Antibodies are highly complex macromolecules with capabilities that vary widely from preparation to preparation. They are also administered to healthy individuals, including newborn children, and therefore their quality must be emphasized to the greatest extent possible to ensure they are effective in preventing or treating life-threatening diseases without causing harm themselves.

[0247] The increasing globalization of vaccine production and distribution has opened new possibilities for better managing public health issues, but it has also raised questions about the comparability and interchangeability of vaccines procured across various sources. International standardization of starting materials, manufacturing, and quality control testing, as well as high expectations for regulatory oversight throughout the course of these products' manufacture and use, have therefore been fundamental to their continued success. However, the field is constantly changing, and ongoing technological advances in the field, which promise to develop powerful new countermeasures against the oldest public health threats as well as new ones—malaria, pandemic influenza, and HIV, to name a few—put enormous pressure on manufacturers, regulators, and the broader medical community to ensure that products continue to meet the highest attainable standards of quality.

[0248] Thus, antigens or vaccines can be obtained from any source or at any point in the manufacturing process. Therefore, the quality control process begins with preparing samples for immunoassays to identify binding of the antibodies or fragments disclosed herein to viral antigens. Such immunoassays are described elsewhere herein, any of which may be used to assess the structural / antigen integrity of the antigen. Criteria for finding a sample containing an acceptable amount of antigenically correct and intact antigen may be established by regulatory authorities.

[0249] Another important aspect in which antigen integrity is assessed is to determine shelf life and storage stability. Most pharmaceuticals, including vaccines, can deteriorate over time. Therefore, it is important to determine whether an antigen, such as in a vaccine, deteriorates or becomes unstable over time, such that it is no longer antigenic when administered to a subject and / or is unable to elicit an immune response. Again, regulatory authorities may establish criteria for finding a sample containing an acceptable amount of antigenically intact.

[0250] In certain embodiments, a viral antigen may contain two or more protective epitopes. In these cases, it may prove useful to use an assay that examines the binding of two or more antibodies, for example, two, three, four, five, or more antibodies. These antibodies may bind to closely related epitopes, such as adjacent or even overlapping epitopes. Alternatively, they may represent distinct epitopes derived from different parts of the antigen. By examining the integrity of multiple epitopes, the overall integrity of the antigen can be more fully depicted, and thus the ability to generate a protective immune response can be measured.

[0251] Antibodies and fragments thereof as described in this disclosure can also be used in kits to monitor the effectiveness of vaccination treatments by detecting the presence of protective SARS-CoV-2 antibodies. Antibodies, antibody fragments, or variants and derivatives thereof as described in this disclosure can also be used in kits to monitor vaccine production with desired immunogenicity. For example, the present disclosure provides the following embodiments. [1] 1. An isolated antibody or antibody fragment that binds to a SARS-CoV-2 surface spike protein, (a) CDRH1 comprising the amino acid sequence of SEQ ID NO: 59, CDRH2 comprising the amino acid sequence of SEQ ID NO: 60, CDRH3 comprising the amino acid sequence of SEQ ID NO: 61, CDRL1 comprising the amino acid sequence of SEQ ID NO: 89, CDRH2 comprising the amino acid sequence of SEQ ID NO: 90, CDRH3 comprising the amino acid sequence of SEQ ID NO: 91; (b) CDRH1 comprising the amino acid sequence of SEQ ID NO: 68, CDRH2 comprising the amino acid sequence of SEQ ID NO: 69, CDRH3 comprising the amino acid sequence of SEQ ID NO: 70, CDRL1 comprising the amino acid sequence of SEQ ID NO: 98, CDRH2 comprising the amino acid sequence of SEQ ID NO: 99, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 100; (c) CDRH1 comprising the amino acid sequence of SEQ ID NO: 41, CDRH2 comprising the amino acid sequence of SEQ ID NO: 42, CDRH3 comprising the amino acid sequence of SEQ ID NO: 43, CDRL1 comprising the amino acid sequence of SEQ ID NO: 71, CDRH2 comprising the amino acid sequence of SEQ ID NO: 72, CDRH3 comprising the amino acid sequence of SEQ ID NO: 73; (d) CDRH1 comprising the amino acid sequence of SEQ ID NO: 44, CDRH2 comprising the amino acid sequence of SEQ ID NO: 45, CDRH3 comprising the amino acid sequence of SEQ ID NO: 46, CDRL1 comprising the amino acid sequence of SEQ ID NO: 74, CDRH2 comprising the amino acid sequence of SEQ ID NO: 75, CDRH3 comprising the amino acid sequence of SEQ ID NO: 76; (e) CDRH1 comprising the amino acid sequence of SEQ ID NO: 47, CDRH2 comprising the amino acid sequence of SEQ ID NO: 48, CDRH3 comprising the amino acid sequence of SEQ ID NO: 49, CDRL1 comprising the amino acid sequence of SEQ ID NO: 77, CDRH2 comprising the amino acid sequence of SEQ ID NO: 78, CDRH3 comprising the amino acid sequence of SEQ ID NO: 79; (f) CDRH1 comprising the amino acid sequence of SEQ ID NO: 50, CDRH2 comprising the amino acid sequence of SEQ ID NO: 51, CDRH3 comprising the amino acid sequence of SEQ ID NO: 52, CDRL1 comprising the amino acid sequence of SEQ ID NO: 80, CDRH2 comprising the amino acid sequence of SEQ ID NO: 81, CDRH3 comprising the amino acid sequence of SEQ ID NO: 82; (g) CDRH1 comprising the amino acid sequence of SEQ ID NO: 53, CDRH2 comprising the amino acid sequence of SEQ ID NO: 54, CDRH3 comprising the amino acid sequence of SEQ ID NO: 55, CDRL1 comprising the amino acid sequence of SEQ ID NO: 83, CDRH2 comprising the amino acid sequence of SEQ ID NO: 84, CDRH3 comprising the amino acid sequence of SEQ ID NO: 85; (h) CDRH1 comprising the amino acid sequence of SEQ ID NO: 56, CDRH2 comprising the amino acid sequence of SEQ ID NO: 57, CDRH3 comprising the amino acid sequence of SEQ ID NO: 58, CDRL1 comprising the amino acid sequence of SEQ ID NO: 86, CDRH2 comprising the amino acid sequence of SEQ ID NO: 87, CDRH3 comprising the amino acid sequence of SEQ ID NO: 88; (i) a CDRH1 comprising the amino acid sequence of SEQ ID NO: 62, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 63, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 64, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 65, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 66, and a CDRH3 comprising the amino acid sequence of SEQ ID NO: 67; or (j) CDRH1 comprising the amino acid sequence of SEQ ID NO: 65, CDRH2 comprising the amino acid sequence of SEQ ID NO: 66, CDRH3 comprising the amino acid sequence of SEQ ID NO: 67, CDRL1 comprising the amino acid sequence of SEQ ID NO: 95, CDRH2 comprising the amino acid sequence of SEQ ID NO: 96, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 97 1. An isolated antibody or antibody fragment comprising: [2] (a) the amino acid sequence of SEQ ID NO: 33 and the amino acid sequence of SEQ ID NO: 34; (b) the amino acid sequence of SEQ ID NO: 39 and the amino acid sequence of SEQ ID NO: 40; (c) the amino acid sequence of SEQ ID NO: 21 and the amino acid sequence of SEQ ID NO: 22; (d) the amino acid sequence of SEQ ID NO: 23 and the amino acid sequence of SEQ ID NO: 24; (e) the amino acid sequence of SEQ ID NO: 25 and the amino acid sequence of SEQ ID NO: 26; (f) the amino acid sequence of SEQ ID NO: 27 and the amino acid sequence of SEQ ID NO: 28; (g) the amino acid sequence of SEQ ID NO: 29 and the amino acid sequence of SEQ ID NO: 30; (h) the amino acid sequence of SEQ ID NO: 31 and the amino acid sequence of SEQ ID NO: 32; (i) the amino acid sequence of SEQ ID NO: 35 and the amino acid sequence of SEQ ID NO: 36; or (j) the amino acid sequence of SEQ ID NO: 37 and the amino acid sequence of SEQ ID NO: 38 2. The antibody or antibody fragment according to claim 1, comprising heavy and light chain variable sequences having at least 70%, 80%, 90% or 95% identity to [3] (a) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 33 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 34; (b) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 39 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 40; (c) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 21 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 22; (d) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 23 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 24; (e) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 25 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 26; (f) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 27 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 28; (g) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 29 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 30; (h) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 31 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 32; (i) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 35 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 36; or (j) a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 37 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 38 3. The antibody or antibody fragment according to 1 or 2 above, comprising: [4] 4. The antibody or antibody fragment according to any one of 1 to 3 above, which is monoclonal. [5] 5. The antibody or antibody fragment according to any one of 1 to 4 above, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, a Fab fragment, a F(ab')2 fragment or an Fv fragment. [6] 6. The antibody or antibody fragment according to any one of 1 to 5 above, which comprises a YTE mutation. [7] 7. The antibody or antibody fragment of any one of claims 1 to 6, which is an IgG or recombinant IgG antibody or antibody fragment comprising an Fc portion that has been mutated to alter (eliminate or enhance) FcR interaction to increase half-life and / or increase therapeutic effect, such as a LALA, LALA PG, N297, GASD / ALIE, DHS, YTE or LS mutation, or glycans that have been modified to alter (eliminate or enhance) FcR interaction, such as by enzymatic or chemical addition, or by expression in a cell line modified with glycan removal or defined glycosylation pattern. [8] 8. The antibody or antibody fragment of any one of claims 1 to 7, which is capable of neutralizing live BSL3 SARS-CoV-2 virus in a focus reduction neutralization test (FRNT) assay using a Vero-E2 cell culture monolayer, and optionally is capable of neutralizing 96% of the live BSL3 SARS-CoV-2 virus at a concentration of 250 ng / mL. [9] 9. The antibody or antibody fragment of any one of claims 1 to 8, which blocks binding of the receptor binding domain (RBD) to the human receptor angiotensin-converting enzyme 2 (ACE2), and optionally blocks activity against hACE2 with an IC50 of <150 ng / mL.

[10] 10. The antibody or antibody fragment of any one of claims 1 to 9, which is capable of neutralizing a SARS-CoV-2 virus comprising a spike protein comprising a D614G substitution, optionally wherein the spike protein does not comprise an E484K substitution.

[11] 11. The antibody or antibody fragment according to any one of 1 to 10, which is capable of binding to the RBD in an "up" conformation.

[12] 12. The antibody or antibody fragment according to any one of 1 to 11, which is capable of binding to the RBD in both the "up" and "down" conformations.

[13] 12. The antibody or antibody fragment according to any one of 1 to 11, which is unable to bind to the RBD in a "down" conformation.

[14] 14. The antibody or antibody fragment of any one of claims 1 to 13, which is capable of binding to the ectodomain of a trimeric spike protein and to the RBD of a monomeric spike protein, optionally with an EC50 of <2 ng / mL for the binding to the ectodomain of the trimeric spike protein and / or the binding to the RBD of the monomeric spike protein.

[15] 15. The antibody or antibody fragment according to any one of 1 to 14 above, further comprising a detectable label.

[16] 16. A method for treating a subject infected with SARS-CoV-2 or reducing the likelihood of infection in a subject at risk of contracting SARS-CoV-2, comprising delivering to the subject the first antibody or antibody fragment described in any one of 1 to 15.

[17] 16. A method of protecting the health of a subject aged 60 years or older, an immunocompromised subject, or a subject suffering from a respiratory and / or cardiovascular disorder who is infected or at risk of infection with SARS-CoV-2, comprising delivering to the subject the first antibody or antibody fragment of any one of claims 1 to 15.

[18] 18. The method of claim 16 or 17, further comprising delivering to the subject a second antibody or antibody fragment, optionally wherein the second antibody or antibody fragment is the antibody or antibody fragment of any one of claims 1 to 15.

[19] 1. A method for treating a subject infected with SARS-CoV-2 or reducing the likelihood of infection in a subject at risk of contracting SARS-CoV-2, comprising delivering to the subject a first antibody or antibody fragment and a second antibody or antibody fragment, wherein the first and second antibodies or antibody fragments are synergistic in neutralizing SARS-CoV-2.

[20] 20. The method of claim 18 or 19, wherein the first antibody or antibody fragment and the second antibody or antibody fragment have a synergy score of 17.4. [twenty one] 21. The method of any one of 18 to 20, wherein the dose of the first antibody or antibody fragment and the second antibody or antibody fragment can be reduced by more than three times the dose of the first antibody or antibody fragment or the second antibody or antibody fragment alone to achieve the same efficacy in virus neutralization. [twenty two] 22. The method of any one of 18 to 21, wherein the first antibody or antibody fragment is capable of binding to RBD in an "up" conformation and is incapable of binding to RBD in a "down" conformation. [twenty three] 23. The method according to any one of 18 to 22, wherein the second antibody or antibody fragment is capable of binding to the RBD in both the "up" and "down" conformations. [twenty four] 24. The method according to any one of 18 to 23, wherein the first antibody or antibody fragment and the second antibody or antibody fragment do not compete for binding to RBD. [twenty five] 25. The method according to any one of 18 to 24, wherein the first antibody or antibody fragment comprises CDRH1 comprising the amino acid sequence of SEQ ID NO: 59, CDRH2 comprising the amino acid sequence of SEQ ID NO: 60, CDRH3 comprising the amino acid sequence of SEQ ID NO: 61, CDRL1 comprising the amino acid sequence of SEQ ID NO: 89, CDRH2 comprising the amino acid sequence of SEQ ID NO: 90, or CDRH3 comprising the amino acid sequence of SEQ ID NO: 91.

[26] 26. The method according to any one of 18 to 25, wherein the second antibody or antibody fragment comprises CDRH1 comprising the amino acid sequence of SEQ ID NO: 68, CDRH2 comprising the amino acid sequence of SEQ ID NO: 69, CDRH3 comprising the amino acid sequence of SEQ ID NO: 70, CDRL1 comprising the amino acid sequence of SEQ ID NO: 98, CDRH2 comprising the amino acid sequence of SEQ ID NO: 99, or CDRH3 comprising the amino acid sequence of SEQ ID NO: 100.

[27] 27. The method according to any one of 18 to 26, wherein the first antibody or antibody fragment comprises a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 33 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 34.

[28] 28. The method according to any one of 18 to 27, wherein the second antibody or antibody fragment comprises a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 39 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 40.

[29] 29. The method according to any one of 16 to 28, wherein the delivery reduces expression of INF-γ, IL-6, CXCL10 and CCL2 in the subject.

[30] 30. The method according to any one of 16 to 29, wherein the delivery is intravenous.

[31] 31. The method according to any one of 16 to 30, wherein the subject is 60 years of age or older, is immunocompromised, or suffers from a respiratory disorder and / or a cardiovascular disorder.

[32] 32. The method of any one of 16 to 31, wherein the delivery improves breathing in the subject compared to an untreated control.

[33] 33. The method of any one of 16 to 32, wherein the delivery reduces viral load compared to an untreated control.

[34] 34. The method according to any one of 17 to 33, wherein the delivery is before infection.

[35] 34. The method according to any one of 17 to 33, wherein the delivery is after infection.

[36] 16. A vaccine formulation comprising one or more antibodies or antibody fragments according to any one of 1 to 15 above.

[37] 37. The vaccine formulation of claim 36, further comprising a second antibody or antibody fragment that binds to the SARS-CoV-2 surface spike protein, optionally wherein the second antibody or antibody fragment is the antibody or antibody fragment of any one of claims 1 to 15.

[38] 16. A vaccine formulation comprising one or more expression vectors encoding the first antibody or antibody fragment according to any one of 1 to 15 above.

[39] 39. The vaccine formulation according to claim 38, wherein the expression vector is a Sindbis virus vector or a VEE vector.

[40] 40. The vaccine formulation of claim 38 or 39, which is formulated for delivery by needle injection, jet injection or electroporation.

[41] 41. The vaccine formulation of claim 40, further comprising one or more expression vectors encoding a second antibody or antibody fragment that binds to the SARS-CoV-2 surface spike protein, optionally wherein the second antibody or antibody fragment is an antibody or antibody fragment described in any one of claims 1 to 15.

[42] 1. A method for detecting COVID-19 infection caused by SARS-CoV-2 in a subject, comprising: (a) contacting a sample derived from the subject with the antibody or fragment thereof described in any one of 1 to 15; (b) detecting SARS-CoV-2 in the sample by binding of the antibody or antibody fragment to a SARS-CoV-2 antigen in the sample; A method comprising:

[43] 43. The method of claim 42, wherein the sample is a body fluid.

[44] 44. The method of claim 42 or 43, wherein the sample is blood, sputum, tears, saliva, mucus or serum, semen, cervical mucus or vaginal secretions, amniotic fluid, placental tissue, urine, exudate, transudate, tissue scraping or stool.

[45] 45. The method according to any one of 42 to 44, wherein the detection comprises ELISA, RIA, lateral flow assay, or Western blot.

[46] 1. A method for determining the antigenic integrity, correct conformation and / or correct sequence of a SARS-CoV-2 surface spike protein, comprising: (a) contacting a sample containing the antigen with the antibody or fragment thereof described in any one of 1 to 15; (b) determining the antigenic integrity, correct conformation, and / or correct sequence of the antigen by detectable binding of the antibody or antibody fragment to the antigen; A method comprising:

[47] 47. The method of claim 46, wherein the sample comprises a recombinantly produced antigen.

[48] 47. The method of claim 46, wherein the sample comprises a vaccine formulation or a vaccine production batch.

[49] 49. The method according to any one of 46 to 48, wherein the detection comprises ELISA, RIA, Western blot, a biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometry staining.

[50] 50. The method according to any one of 46 to 49, further comprising carrying out steps (a) and (b) twice to determine the antigen stability of the antigen over time.

[51] 16. A hybridoma or modified cell comprising a polynucleotide encoding the antibody or antibody fragment according to any one of 1 to 15 above.

[52] An isolated human monoclonal antibody or antibody fragment, or a hybridoma or modified cell producing the same, wherein the antibody binds to the SARS-CoV-2 surface spike protein.

[53] 16. An isolated polynucleotide comprising a nucleic acid sequence encoding a heavy chain variable region of the antibody or antibody fragment described in any one of 1 to 15 and / or a nucleic acid sequence encoding a light chain variable region of the antibody or antibody fragment.

[54] 54. The polynucleotide according to 53 above, comprising any one of the sequences of SEQ ID NOs: 1 to 20.

[55] A vector comprising the polynucleotide described in 54.

[56] A host cell comprising a first vector comprising the polynucleotide described in 53 or 54, the vector described in 55, or a nucleic acid molecule encoding the heavy chain variable region of the antibody or antibody fragment thereof described in any one of 1 to 15, and a second vector comprising a nucleic acid molecule encoding the light chain variable region of the antibody or antibody fragment thereof.

[57] A method for producing an antibody or antibody fragment, comprising: (a) culturing the cells described in 56; and (b) isolating the antibody or antibody fragment thereof from the cultured cells.

[58] A composition comprising a first antibody or antibody fragment that binds to the SARS-CoV-2 surface spike protein and a second antibody or antibody fragment that binds to the SARS-CoV-2 surface spike protein, wherein the composition is optionally a pharmaceutically acceptable composition.

[59] A kit comprising a first antibody or antibody fragment that binds to the SARS-CoV-2 surface spike protein and a second antibody or antibody fragment that binds to the SARS-CoV-2 surface spike protein, optionally further comprising instructions for using the first antibody or antibody fragment and the second antibody or antibody fragment to treat a subject infected with SARS-CoV-2 or to reduce the likelihood of infection in a subject at risk of contracting SARS-CoV-2.

[60] 60. The composition or kit of claim 58 or 59, wherein the first antibody or antibody fragment and the second antibody or antibody fragment have a synergy score of 17.4.

[61] 61. The composition or kit of any one of 58 to 60, wherein the dose of the first antibody or antibody fragment and the second antibody or antibody fragment can be reduced by more than three times the dose of the first antibody or antibody fragment or the second antibody or antibody fragment alone to achieve the same efficacy in virus neutralization.

[62] 62. The composition or kit of any one of 58 to 61, wherein the first antibody or antibody fragment is capable of binding to RBD in an "up" conformation and is incapable of binding to RBD in a "down" conformation.

[63] 63. The composition or kit according to any one of 58 to 62, wherein the second antibody or antibody fragment is capable of binding to the RBD in both the "up" and "down" conformations.

[64] 64. The composition or kit according to any one of 58 to 63, wherein the first antibody or antibody fragment and the second antibody or antibody fragment do not compete for binding to RBD.

[65] The composition or kit according to any one of items 58 to 64, wherein the first antibody or antibody fragment comprises CDRH1 having the amino acid sequence of SEQ ID NO: 59, CDRH2 having the amino acid sequence of SEQ ID NO: 60, CDRH3 having the amino acid sequence of SEQ ID NO: 61, CDRL1 having the amino acid sequence of SEQ ID NO: 89, CDRH2 having the amino acid sequence of SEQ ID NO: 90, and CDRH3 having the amino acid sequence of SEQ ID NO: 91.

[66] The composition or kit according to any one of items 58 to 65, wherein the second antibody or antibody fragment comprises CDRH1 having the amino acid sequence of SEQ ID NO: 68, CDRH2 having the amino acid sequence of SEQ ID NO: 69, CDRH3 having the amino acid sequence of SEQ ID NO: 70, CDRL1 having the amino acid sequence of SEQ ID NO: 98, CDRH2 having the amino acid sequence of SEQ ID NO: 99, and CDRH3 having the amino acid sequence of SEQ ID NO: 100.

[67] 67. The composition or kit according to any one of 58 to 66, wherein the first antibody or antibody fragment comprises a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 33 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 34.

[68] 68. The composition or kit according to any one of items 58 to 67, wherein the second antibody or antibody fragment comprises a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 39 and / or a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 40.

[69] 69. The kit according to any one of 59 to 68, wherein the first antibody or antibody fragment and the second antibody or antibody fragment are in separate containers. [Example]

[0252] The following examples are included to demonstrate preferred embodiments. It will be understood by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to work well in practicing the embodiments, and as such can be considered to constitute preferred ways of practicing the same. However, in light of the present disclosure, one of skill in the art will understand that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0253] Example 1 – Antibody Synergy Synergy is defined herein as the increased neutralizing activity mediated by a cocktail of two mAbs compared to that mediated by each individual mAb at the same total antibody concentration (in vitro) or dose (in vivo). To assess whether two mAbs in a cocktail act synergistically to neutralize SARS-CoV-2, we quantified synergy using a previously reported method (Ianevski A, He L, Aittokallio T, Tang J. Bioinformatics. 33, 2413-2415, 2017). To assess the significance of the beneficial effect of combining mAbs, we compared the observed combination response (dose-response matrix) with the expected response calculated by a synergy scoring model (Ianevski A, He L, Aittokallio T, Tang J. Bioinformatics. 33, 2413-2415, 2017). Viral neutralization was measured using a conventional focus reduction neutralization test (FRNT) assay using wild-type SARS-CoV-2 and Vero-E2 cell culture monolayers. Individual mAbs, COV2-2196 and COV2-2130, were mixed at different concentrations to assess the neutralizing activity of different mAb ratios in the cocktail. Specifically, 7-fold dilutions of mAb COV2-2130 (starting at 500 ng / mL) were mixed with 9-fold dilutions of mAb COV2-2196 (starting at 500 ng / mL) in a total volume of 50 μL for each condition. The mixture was then incubated with 50 μL of live SARS-CoV-2 in cell culture medium (RPMI-1640 medium supplemented with 2% FBS) and subsequently added to confluent Vero-E2 cells grown in 96-well plates. The control values ​​included dose-response values ​​for neutralizing activity measured separately for each individual mAb, COV2-2196 and COV2-2130, which were evaluated at the same doses as in the cocktail (see Figures 1-3). Each measurement was performed in duplicate. Next, the inventors calculated the virus neutralization rate for each condition and then calculated a synergy score value, which defines the interaction between these two mAbs in the cocktail as synergistic (synergy score = 17.4).Note that a synergy score below -10 indicates antagonism, a score between -10 and 10 indicates additive effects, and a score above 10 indicates synergy. The example in Figures 1-3 shows a dose-response matrix demonstrating that a 79 ng / mL dose of combined mAbs in the cocktail (16 ng / mL COV2-2196 and 63 ng / mL COV2-2130) had the same activity as 250 ng / mL of each individual mAb. This finding indicates that the dose can be reduced by more than three-fold in the cocktail to achieve the same efficacy in virus neutralization.

[0254] To evaluate the therapeutic efficacy of the treatments, we first tested mAb COV2-2196 or COV2-2130, or their 1:1 combination, using a MA-SARS-CoV-2 challenge model. All treatments reduced infectious virus in the lungs, as measured by plaque titers in lung tissue 2 days after virus inoculation. Lung burdens were reduced by up to 3 × 10 4 The cocktail treatment delivered at a dose of 400 μg per mouse (approximately 20 mg / kg) was the most effective, resulting in a significant 2-fold reduction in viral load, with four out of five animals in this treatment group already free of infectious virus in their lungs (Figure 4A). Similarly, treatment of mice with lungs transduced with recombinant adenovirus to express the SARS-CoV-2 receptor, human ACE2 (AdV-hACE2), with 400 μg of the mAb cocktail per mouse 12 hours after standard SARS-CoV-2 virus challenge completely neutralized infectious virus in the lungs in vivo (Figure 4B). Gene expression of the cytokines and chemokines INF-γ, IL-6, CXCL10, and CCL2, indicators of inflammation, was also reduced in the lungs of mAb cocktail-treated mice compared with the lungs of isotype control-treated mice (Figure 4C). Together, these results suggest the efficacy of post-exposure treatment mediated by the COV2-2196 + COV2-2130 cocktail in a SARS-CoV-2 challenge mouse model.

[0255] Example 2 – Non-human primate challenge study MAb production and purification. mAb sequences were synthesized (Twist Bioscience) and cloned into an IgG1 monocistronic expression vector (designated pTwist-mCis_G1) and used for mAb secretion by mammalian cell culture. This vector contains an enhanced 2A sequence and a GSG linker, allowing simultaneous expression of mAb heavy and light chain genes from a single construct upon transfection. 1 Microscale expression of mAbs in 1 mL ExpiCHO cultures in 96-well plates has been previously described by the inventors. 2 To express mAbs on a larger scale, we transfected CHO cell cultures (1–300 mL per antibody) using the Gibco™ ExpiCHO™ Expression System and 50 mL miniature bioreactor tube (Corning) protocol as described by the vendor. Culture supernatants were purified on a 24-column parallel protein chromatography system (Protein BioSolutions) using HiTrap MabSelect SuRe (Cytiva, formerly GE Healthcare Life Sciences). Purified mAbs were buffer-exchanged into PBS, concentrated using Amicon® Ultra-4 50 KDa centrifugal filter units (Millipore Sigma), and stored at 4°C until use. Purified mAbs were routinely tested for endotoxin levels and found to be <1 EU / mg IgG in NHP testing. Endotoxin testing was performed using PTS201F cartridges (Charles River) and an Endosafe Nexgen-MCS instrument (Charles River), which have a sensitivity range of 10 to 0.1 EU / mL.

[0256] We tested the protective efficacy of mAbs using a recently described SARS-CoV-2 non-human primate (NHP) challenge model. 3,4In this model, we tested COV2-2196, a neutralizing mAb encoded by the same variable gene segments as COV2-2196 but taking advantage of many significant amino acid differences in HCDR3 and LCDR3, as monotherapy. Animals received a 50 mg / kg dose of either mAb COV2-2196 or an isotype control mAb intravenously on day -3, followed by 1.1 x 10 4 Patients were challenged intranasally and intratracheally with a dose of 1000 PFU of SARS-CoV-2. After challenge, we assessed viral RNA by RT-qPCR in bronchoalveolar lavage (BAL) and nasal swabs. High levels of subgenomic viral RNA were observed in NHPs treated with the isotype control mAb, with a mean peak in nasal swabs of 7.53 (range 5.37-8.23) log 10 The mean peak RNA copies / swab in BAL was 4.97 (range 3.81–5.24) log 10 Subgenomic viral RNA was not detected in samples from the mAb-treated group (LOD = 50 [1.7 log 10 ]RNA copies / swab or RNA copies per mL), indicating protection. Pharmacokinetic analysis revealed stable concentrations of human mAb in the circulation of NHPs.

[0257] NHP Challenge Study. NHP research studies adhered to the principles set forth in the Guide for the Care and Use of Laboratory Animals, 8th Edition. The facility in which this study was conducted (Bioqual Inc., Rockville, MD) is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and licensed by the Office of Laboratory Animal Welfare. NHP studies were conducted in compliance with all relevant local, state, and federal regulations and were approved by the relevant Institutional Animal Care and Use Committee (IACUC).

[0258] Eight healthy adult rhesus macaques (Macaca mulatta) from India (weight 5-15 kg) were studied. Animals were randomly assigned to an anti-SARS-CoV-2 mAb treatment group (n=4 per group) and one control (isotype treatment) group (n=4 per group). Animals received either mAb COV2-2196 or an isotype control mAb intravenously at a dose of 50 mg / kg on day -3, followed by 1.1 x 10 4 Patients were challenged with PFU of SARS-CoV-2 for 3 days, administered 1 mL by the intravenous route and 1 mL by the intratracheal route. Viral RNA was assessed by bronchoalveolar lavage fluid examination and RT-qPCR in nasal swabs at multiple time points, as described. 5,6 All animals underwent a physical examination. In addition, all animals were observed daily with an internal scoring protocol approved by the Institutional Animal Care and Use Committee. These studies were unblinded.

[0259] Detection of circulating human mAbs in NHP serum. ELISA plates were coated with 1 μg / mL goat anti-human IgG (H+L) secondary antibody (monkey adsorbed) (Novus Biological) overnight at 4°C and then blocked for 2 hours. Serum samples were assayed in 3-fold dilutions starting at a 1:3 dilution in Blocker Casein (ThermoFisher) diluent in PBS. Samples were incubated for 1 hour at ambient temperature, then removed and the plate washed. Wells were then incubated for 1 hour with HRP-conjugated goat anti-human IgG (monkey adsorbed) (Southern Biotech) at a dilution of 1:4,000. Wells were washed and then incubated with SureBlue Reserve TMB Microwell Peroxidase Substrate (Seracare) (100 μL / well) for 3 minutes, followed by quenching with TMB Stop Solution (Seracare) (100 μL / well). Microplates were read at 450 nm. Concentrations of human mAbs were interpolated from the linear range of purified human IgG (Sigma) standard curves using version 8.0 of Prism software (GraphPad).

[0260] Materials and Methods for Examples 3-4 Expression and purification of the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. A DNA segment corresponding to the RBD (residues 319–528) of the S protein was synthesized and optimized for expression and cloned into the pTwist-CMV expression DNA plasmid downstream of the IL-2 signal peptide (MYRMQLLSCIALSLALVTNS) (Twist Bioscience). To facilitate protein purification, a three-amino acid linker (GSG) and a His tag were incorporated into the C-terminus of the expression construct. Expi293F cells were transiently transfected with the RBD-encoding plasmid, and culture supernatants were harvested 5 days later. The RBD was purified from the supernatant by nickel affinity chromatography using a HisTrap Excel column (GE Healthcare Life Sciences). To produce the protein used in crystallization experiments, 5 μM kifunensine was included in the medium to generate RBD with high-mannose glycans. This high-mannose glycoprotein was subsequently treated with endoglycosidase F1 (Millipore) to obtain a homogeneously deglycosylated RBD.

[0261] Expression and purification of recombinant COV2-2196 and COV2-2130 Fabs. DNA fragments corresponding to the heavy chain variable domains of COV2-2196 and COV2-2130, along with the CH1 domain and light chain variable domain of human IgG1 containing the human kappa chain constant domain, were synthesized and cloned into the pTwist vector (Twist Bioscience). This vector contains the heavy chain of each Fab, followed by a GGGGS linker, a furin cleavage site, a T2A ribosomal cleavage site, and the light chain of each Fab. Expression of the heavy and light chains is driven by the same CMV promoter. COV2-2196 and COV2-2130 Fabs were expressed in ExpiCHO cells by transient transfection with the expression plasmids. Recombinant Fabs were purified from the culture supernatant using an anti-CH1 Capture Select column (Thermo Fisher Scientific). For the RBD / COV2-2196 complex, the wild-type COV2-2196 sequence was used for expression. For the RBD / COV2-2196 / COV2-2130 complex, a modified version of the Fab of COV2-2196 was used, in which the first two amino acids of the variable region were mutated from QM to EV.

[0262] Crystallization and structure determination of the antibody-antigen complex. Purified COV2-2196 Fab was mixed with deglycosylated RBD at a molar ratio of 1:1.5, and the mixture was further purified by size-exclusion chromatography on a Superdex-200 Increase column (GE Healthcare Life Sciences) to obtain the antibody-antigen complex. To obtain the RBD / COV2-2196 / COV2-2130 ternary complex, purified deglycosylated RBD was mixed with both COV2-2196 and COV2-2130 Fab at a molar ratio of 1:1.5:1.5, and the ternary complex was purified on a Superdex-200 Increase column. The complex was concentrated to approximately 10 mg / mL and subjected to crystallization experiments. The RBD / COV2-2196 complex was crystallized in 16%-18% PEG3350, 0.2% Tris-HCl (pH 8.0-8.5), and the RBD / COV2-2196 / COV2-2130 complex was crystallized in 5% (w / v) PEG1000, 100 mM disodium phosphate / citric acid (pH 4.2), and 40% (v / v) reagent alcohol. For crystals of both complexes, a cryoprotectant solution was prepared by mixing the crystallization solution with 100% glycerol in a 20:7 volume ratio. Protein crystals were rapidly immersed in the cryoprotectant solution and then flash-frozen in liquid nitrogen. Diffraction data were collected at the Advanced Photon Source on beamline 21-ID-F for the RBD / COV2-2196 complex and on beamline 21-ID-G for the RBD / COV2-2196 / COV2-2130 complex. 58 and CCP4 Suite 59 The program Phaser 60 The crystal structures were solved by molecular replacement using the structures of the RBD complexed with Fab CC12.1 (PDB ID 6XC2) and the Fab structure of MR78 (PDB ID 5JRP) using Phenix. 61 or Coot 62 The structure was manually refined and rebuilt using the PyMOL software. This model has been deposited in the Protein Data Bank. All structural diagrams were generated using the PyMOL software. 63was used.

[0263] Hydrogen-deuterium mass spectrometry (HDX-MS) experiments were performed using an automated sample handling robot (LEAP technologies, Fort Lauderdale, FL, USA) coupled to an M-Class Acquity LC system and HDX Manager (Waters Ltd., Wilmslow, UK). 7.6 μL of 4.3 μM SARS-CoV-2 S2P spike trimer, either alone or in a 1:1.12 molar ratio with AZD1061 or AZD8895, was added to 52.4 μL of labeling buffer (50 mM potassium phosphate, pH 6.2) and incubated for 1 min at 20°C. After incubation, 50 μL of this sample was added to 50 μL of quench solution (50 mM potassium phosphate, 200 mM TCEP, 2 M Gdn-HCl, pH 2.3) at 1°C. Fifty microliters of the quenched sample was passed through an immobilized BEH pepsin column (Waters Ltd., Wilmslow, UK) at 100 μL / min (approximately 4,000 psi) for 4 min at 20 °C, and the resulting peptides were then trapped using a Vanguard precolumn Acquity UPLC BEH C18 trap column (1.7 μm, 2.1 mm × 5 mm, Waters Ltd., Wilmslow, UK). After valve switching, peptides were isolated using an Acquity UPLC BEH C18 analytical column (1.7 μm, 1 mm × 100 mm) with a gradient elution of 0–35% MeCN (0.2% v / v formic acid) in HO (0.2% v / v formic acid) at 40 μL / min over 6 min at 1 °C. Peptides were analyzed using an Orbitrap Fusion mass spectrometer (Thermo Fisher, Bremen, Germany) operating in either Orbitrap detection mode (deuterated samples) or Orbitrap-ion trap DDA mode (t = 0 samples) at a resolution of 120 K. Peptide MS / MS data used to identify peptides were analyzed using BioPharma Finder v3.0 (Thermo Fisher, Bremen, Germany), and deuterium incorporation was quantified using HDExaminer v2.0 (Sierra Analytics).CSV exports of peptide pool results and uptake tables from HDExaminer were reformatted using in-house R scripts to generate uptake plots and structural heatmaps using PAVED (University of Leeds). 64 .

[0264] ELISA binding of the COV2-2196 mutant. Wells of a 384-well microtiter plate were coated with purified recombinant SARS-CoV-2 S 6P protein overnight at 4°C. Plates were blocked for 1 hour with 2% nonfat dry milk and 2% normal goat serum in DPBS containing 0.05% Tween-20 (DPBS-T). Antibodies were diluted to 10 μg / mL and added to the wells in 23 two-fold titrations in DPBS-T, followed by incubation at room temperature for 1 hour. Bound antibodies were detected using goat anti-human IgG conjugated with horseradish peroxidase (Southern Biotech) and TMB substrate (Thermo Fischer Scientific). The reaction was quenched with 1 N hydrochloric acid, and absorbance was measured at 450 nm using a spectrophotometer (Biotek).

[0265] Mapping of all mutants that escape antibody binding. All mutants that escape antibody binding were identified using the DMS method. 41 The present inventors mapped the RBD mutant library by yeast display described above. 41,42 Briefly, a replicate mutation library was constructed with the spike receptor-binding domain (RBD) from SARS-CoV-2 (isolated Wuhan-Hu-1, Genbank accession number MN908947, residues N331-T531) containing 3,804 of 3,819 possible amino acid mutations, with >95% present as single mutations. To facilitate downstream sequencing, each RBD mutant was linked to a unique 16-nucleotide barcode sequence. As described above, the library was screened for RBD expression and binding to ACE2 to identify mutants that were either completely misfolded or nonfunctional (i.e., lacking adequate ACE2 binding affinity).41 ) RBD mutants were excluded.

[0266] Antibody escape mapping experiments were performed in biological replicates using two independent mutant RBD libraries as described above with minor modifications. 41 Briefly, mutant yeast libraries induced to express RBD were washed and incubated with 400 ng / mL of antibody for 1 hour at room temperature with gentle agitation. After antibody incubation, the library was secondary labeled with 1:100 FITC-conjugated anti-MYC antibody (Immunology Consultants Lab, CYMC-45F) to label for RBD expression and 1:200 PE-conjugated goat anti-human IgG (Jackson ImmunoResearch 109-115-098) to label for binding antibodies. Flow cytometry sorting was used to enrich for cells expressing RBD mutants with reduced antibody binding through a selection gate targeted at capturing unmutated SARS-CoV-2 cells labeled with an antibody concentration of 1% of the library sample. For each sample, approximately 10 million RBD+ cells were processed in a hemocytometer. Antibody-evaded cells were grown overnight in SD-CAA (6.7 g / L yeast nitrogen base, 5.0 g / L casamino acids, 1.065 g / L MES acid, and 2% w / v dextrose) to expand the cells prior to plasmid extraction.

[0267] Plasmid samples were prepared from antibody-free cell preselection and overnight cultures as described above (Zymoprep Yeast Plasmid Miniprep II). 41 16-nucleotide barcode sequences identifying each RBD variant were amplified by PCR and sequenced on an Illumina HiSeq 2500 with 50-bp single-end reads as described. 41、42 .

[0268] As noted, we calculated the evasion percentage with minor modifications as described below. 41We used the dms_variants package (jbloomlab.github.io / dms_variants / , version 0.8.2) to parse Illumina sequences using the previously described 65 A barcode / RBD lookup table was used to count each barcode RBD variant within each pre-selected population and antibody escape population.

[0269] For each antibody selection, we calculated the "escape fraction" of each barcode variant using the deep sequencing counts of each variant in the original population and the antibody escape population, using the aforementioned formula 41 We calculated the total fraction of the library that escaped antibody binding by . These escape fractions represent the estimated fraction of cells expressing a particular mutant that falls into an antibody escape bin, where a value of 0 means that the mutant always binds serum and a value of 1 means that it always escapes antibody binding. We then applied computational filters to remove mutants with highly deleterious mutations whose antibody escape may be caused simply by low sequencing counts or insufficient expression of a properly folded RBD on the yeast cell surface. 41、42 Specifically, as mentioned above, 42 Using exhaustive mutation scanning scores at the mutation and mutation levels, variants with (or containing) an ACE2 binding score <-2.35 or an expression score <-1 were removed. These filter criteria are slightly more stringent than those previously used to map panels of human antibodies. 41 , mAb 65 and polyclonal serum 54 Note that these are the same criteria used in a recent study to define RBD residues that affect binding of .

[0270] Next, we performed the following steps, as detailed and described in (jbloomlab.github.io / dms_variants / dms_variants.globalepistasis.html) 41 , an exhaustive epitaxy model implemented in the dms_variants package 66 Mutation-level escape scores were deconvolved into single-mutation escape fraction estimates using [translate]. The reported document-wide escape fractions are averaged across the entire library (correlations are shown in Figures 18A-B); these scores are also presented in Table B. For emphasis in the logo plots, strongly escaped sites from each antibody were heuristically determined as sites whose sum of mutational escape scores was at least 10-fold greater than the average site-wise sum of the selections and within 10-fold of the site-wise sum of the most strongly selected sites. A complete description of the computational analysis is available at github.com / jbloomlab / SARS-CoV-2-RBD_MAP_AZ_Abs. These results are also available in interactive form at https: / / jbloomlab.github.io / SARS-CoV-2-RBD_MAP_AZ_Abs / .

[0271] VSV-SARS-CoV-2 antibody escape selection experiments. For escape selection experiments with COV2-2196 and COV2-2130, we used replication-competent recombinant VSV viruses encoding spike proteins from SARS-CoV-2 with a C-terminal 21 amino acid deletion. 43 As previously described, spike-expressing VSV viruses were propagated in MA104 cells (African green monkey, ATCC CRL-2378.1). 43Virus stocks were titrated onto Vero E6 cell monolayer cultures. Plaques were visualized using neutral red staining. To screen escape mutants selected in the presence of COV2-2196, COV2-2130, or a cocktail consisting of a 1:1 mixture of COV2-2196 and COV2-2130, we used a real-time cell analysis assay (RTCA) and an xCELLigence RTCA MP analyzer (ACEA Biosciences Inc.) and the escape selection scheme described above. 41Briefly, 50 μL of cell culture medium (DMEM supplemented with 2% FBS) was added to each well of a 96-well E-plate to obtain a background reading. Eighteen thousand (18,000) Vero E6 cells were seeded per well in 50 μL of cell culture medium, and the plate was placed on the analyzer. Measurements were taken automatically every 15 minutes, and sensorgrams were visualized using RTCA software version 2.1.0 (ACEA Biosciences Inc.). VSV-SARS-CoV-2 virus (5e3 plaque-forming units (PFU) per well, approximately 0.3 MOI) was mixed with saturating neutralizing concentrations of COV2-2196, COV2-2130, or a 1:1 mixture of COV2-2196 and COV2-2130 antibodies (5 μg / ml total antibody concentration) in a total volume of 100 μL and incubated at 37°C for 1 hour. Sixteen to 20 hours after seeding, the virus-antibody mixture was added to the cell monolayer. Each plate contained controls, including wells containing virus alone in the absence of antibody and wells containing Vero E6 cells in medium. Plates were continuously measured (every 15 minutes) for 72 hours. Escape mutants were identified by monitoring the cell index for a decline in cell viability. To verify escape from antibody selection, wells in which cytopathic effects were observed in the presence of COV2-2130 were evaluated in subsequent RTCA experiments in the presence of 10 μg / mL of COV2-2130 or COV2-2196. After confirming the resistance of selected viruses to neutralization by COV2-2130, viral isolates were propagated in Vero E6 cells in the presence of 10 μg / mL of COV2-2130. Viral RNA was isolated using the QiAmp Viral RNA Extraction Kit (QIAGEN) according to the manufacturer's protocol, and the SARS-CoV-2 spike gene was reverse transcribed and amplified with the SuperScript IV One-Step RT-PCR Kit (ThermoFisher Scientific) using primers flanking the S gene. The amplified PCR products were purified using SPRI magnetic beads (Beckman Coulter) in a 1:1 ratio and sequenced by the Sanger method using primers that confer forward and reverse reads of the RBD.

[0272] Serial passage and testing of SARS-CoV-2 to select mAb-resistant mutations. The SARS-CoV-2 strain, USA-WA1...

Claims

1. A pharmaceutically acceptable composition comprising a first antibody or antibody fragment that binds to the SARS-CoV-2 surface spike protein and a second antibody or antibody fragment that binds to the SARS-CoV-2 surface spike protein, the first antibody or antibody fragment comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO:59, a CDRH2 comprising the amino acid sequence of SEQ ID NO:60, a CDRH3 comprising the amino acid sequence of SEQ ID NO:61, a CDRL1 comprising the amino acid sequence of SEQ ID NO:89, a CDRL2 comprising the amino acid sequence of SEQ ID NO:90, and a CDRL3 comprising the amino acid sequence of SEQ ID NO:91; the second antibody or antibody fragment comprises a CDRH1 comprising the amino acid sequence of SEQ ID NO: 68, a CDRH2 comprising the amino acid sequence of SEQ ID NO: 69, a CDRH3 comprising the amino acid sequence of SEQ ID NO: 70, a CDRL1 comprising the amino acid sequence of SEQ ID NO: 98, a CDRL2 comprising the amino acid sequence of SEQ ID NO: 99, and a CDRL3 comprising the amino acid sequence of SEQ ID NO: 100; composition.

2. the first antibody or antibody fragment comprises a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 34; the second antibody or antibody fragment comprises a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 40; 10. The pharmaceutically acceptable composition of claim 1.

3. 3. The pharmaceutically acceptable composition of claim 1 or 2, wherein the first antibody or antibody fragment comprises a YTE mutation and the second antibody or antibody fragment comprises a YTE mutation.

4. 4. The pharmaceutically acceptable composition of claim 1, wherein the first antibody or antibody fragment is an IgG1 antibody and the second antibody or antibody fragment is an IgG1 antibody.

5. A pharmaceutically acceptable composition according to any one of claims 1 to 4 for reducing the likelihood of infection in a subject at risk of contracting SARS-CoV-2.

6. A pharmaceutically acceptable composition according to any one of claims 1 to 4 for treating a subject infected with SARS-CoV-2.

7. An isolated antibody or antibody fragment that binds to the SARS-CoV-2 surface spike protein, comprising: a CDRH1 comprising the amino acid sequence of SEQ ID NO:59; a CDRH2 comprising the amino acid sequence of SEQ ID NO:60; a CDRH3 comprising the amino acid sequence of SEQ ID NO:61; a CDRL1 comprising the amino acid sequence of SEQ ID NO:89; a CDRL2 comprising the amino acid sequence of SEQ ID NO:90; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:

91.

8. 8. The antibody or antibody fragment of claim 7, comprising a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable sequence comprising the amino acid sequence of SEQ ID NO:

34.

9. 9. The antibody or antibody fragment of claim 7 or 8, comprising a YTE mutation.

10. The antibody or antibody fragment of any one of claims 7 to 9, which is an IgG1 antibody.

11. An isolated antibody or antibody fragment that binds to the SARS-CoV-2 surface spike protein, comprising: a CDRH1 comprising the amino acid sequence of SEQ ID NO:68; a CDRH2 comprising the amino acid sequence of SEQ ID NO:69; a CDRH3 comprising the amino acid sequence of SEQ ID NO:70; a CDRL1 comprising the amino acid sequence of SEQ ID NO:98; a CDRL2 comprising the amino acid sequence of SEQ ID NO:99; and a CDRL3 comprising the amino acid sequence of SEQ ID NO:

100.

12. 12. The antibody or antibody fragment of claim 11, comprising a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 39 and a light chain variable sequence comprising the amino acid sequence of SEQ ID NO:

40.

13. 13. The antibody or antibody fragment of claim 11 or 12, comprising a YTE mutation.

14. The antibody or antibody fragment of any one of claims 11 to 13, which is an IgG1 antibody.

15. The antibody or antibody fragment of any one of claims 7 to 14, which is monoclonal.

16. The antibody or antibody fragment of any one of claims 7 to 15, further comprising a detectable label.

17. A composition comprising the antibody or antibody fragment of any one of claims 7 to 16.

18. 1. A method for detecting COVID-19 infection by SARS-CoV-2 in a subject, comprising: (a) contacting a sample from the subject with the antibody or fragment thereof according to any one of claims 7 to 16; (b) detecting SARS-CoV-2 in the sample by binding of the antibody or antibody fragment to a SARS-CoV-2 antigen in the sample; and A method comprising:

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