HUMAN MONOCLONAL ANTIBODIES TO SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-CoV-2)

Information

  • Patent Application
  • 20230122364
  • Publication Number
    20230122364
  • Date Filed
    March 26, 2021
    3 years ago
  • Date Published
    April 20, 2023
    a year ago
Abstract
The present disclosure is directed to antibodies binding to and neutralizing tire coronavirus designated SARS-CoV-2 and methods for use thereof.
Description
BACKGROUND
1. Field of the Disclosure

The present disclosure relates generally to the fields of medicine, infectious disease, and immunology. More particular, the disclosure relates to human antibodies binding to a novel coronavirus designated SARS-CoV-2 and methods of use therefor.


2. Background

An epidemic of a novel coronavirus (SARS-CoV-2) affected mainland China, along with cases in 179 other countries and territories. It was identified in Wuhan, the capital of China's Hubei province, after 41 people developed pneumonia without a clear cause. The virus, which causes acute respiratory disease designated coronavirus disease 2019 (COVID-19), is capable of spreading from person to person. The incubation period (time from exposure to onset of symptoms) ranges from 0 to 24 days, with a mean of 3-5 days, but it may be contagious during this period and after recovery. Symptoms include fever, coughing and breathing difficulties. An estimate of the death rate in February 200 was 2% of confirmed cases, higher among those who require admission to hospital.


As of 10 Feb. 2020, 40,627 cases have been confirmed (6,495 serious), including in every province-level division of China. A larger number of people may have been infected, but not detected (especially mild cases). As of 10 Feb. 2020, 910 deaths have been attributed to the virus since the first confirmed death on 9 January, with 3,323 recoveries. The first local transmission outside China occurred in Vietnam between family members, while the first international transmission not involving family occurred in Germany on 22 January. The first death outside China was in the Philippines, where a man from Wuhan died on 1 February. As of 10 Feb. 2020, the death toll from this virus had surpassed the global SARS outbreak in 2003.


As of early February 2020, there is no licensed vaccine and no specific treatment, although several vaccine approaches and antivirals are being investigated. The outbreak has been declared a Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO), based on the possible effects the virus could have if it spreads to countries with weaker healthcare systems. Thus, there is an urgent need to explore the biology and pathology of SARS-CoV-2 and well as the human immune response to this virus.


SUMMARY

Thus, in accordance with the present disclosure, there is provided a method of detecting COVID-19 infection with SARS-CoV-2 in a subject comprising (a) contacting a sample from said subject with an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (b) detecting SARS-CoV-2 in said sample by binding of said antibody or antibody fragment to a SARS-CoV-2 antigen in said sample. The sample may be a body fluid, such as blood, sputum, tears, saliva, mucous or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissues, urine, exudate, transudate, tissue scrapings or feces. Detection may comprise ELISA, RIA, lateral flow assay or western blot. The method may further comprise performing steps (a) and (b) a second time and determining a change in SARS-CoV-2 antigen levels as compared to the first assay. The antibody or antibody fragment may be encoded by clone-paired variable sequences 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 clone-paired variable sequences as set forth in Table 1, or by light and heavy chain variable sequences having 100% identity to clone-paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90% or 95% identity to clone-paired sequences from Table 2. The antibody or antibody fragment may bind to a SARS-CoV-2 surface spike protein. The antibody fragment may be a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.


In another embodiment, there is provided a method of treating a subject infected with SARS-CoV-2 or reducing the likelihood of infection of a subject at risk of contracting SARS-CoV-2, comprising delivering to said subject an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from 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 clone-paired variable sequences as set forth in Table 1, or by light and heavy chain variable sequences having 100% identity to clone-paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90% or 95% identity to clone-paired sequences from 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 or a bispecific antibody. The antibody may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody or antibody fragment may bind to a SARS-CoV-2 antigen such as a surface spike protein. The antibody or antibody fragment may be administered prior to infection or after infection. The subject may be of age 60 or older, may be immunocompromised, or may suffer from a respiratory and/or cardiovascular disorder. Delivering may comprise antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment.


In yet another embodiment, there is provided a monoclonal antibody, wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from 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 clone-paired variable sequences as set forth in Table 1, or by light and heavy chain variable sequences having 100% identity to clone-paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90% or 95% identity to clone-paired sequences from 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, is bispecific antibody, or is an intrabody. The antibody may be an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody or antibody fragment may bind to a SARS-CoV-2 surface spike protein.


A hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from 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 clone-paired variable sequences as set forth in Table 1, or by light and heavy chain variable sequences having 100% identity to clone-paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90% or 95% identity to clone-paired sequences from 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 bean IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody or antibody fragment may bind to a SARS-CoV-2 surface spike protein.


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


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(s) may be Sindbis virus or VEE vector(s). 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 for a second antibody or antibody fragment, such as a distinct antibody or antibody fragment as described herein.


In yet a further embodiment, there is provided a method of protecting the health of a subject of age 60 or older, an immunocompromised, subject or a subject suffering from a respiratory and/or cardiovascular disorder that is infected with or at risk of infection with SARS-CoV-2 comprising delivering to said subject an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from 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 clone-paired variable sequences as set forth in Table 1, or by light and heavy chain variable sequences having 100% identity to clone-paired sequences as set forth in Table 1. The antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, or light and heavy chain variable sequences having at least 70%, 80%, 90% or 95% identity to clone-paired sequences from 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 an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fe portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA PG. N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern. The antibody may be a chimeric antibody or a bispecific antibody. The said antibody or antibody fragment may be administered prior to infection or after infection. The antibody or antibody fragment may bind to a SARS-CoV-2 antigen such as a surface spike protein. Delivering may comprise antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment. The antibody or antibody fragment may improve the subject's respiration as compared to an untreated control and/or may reduce viral load as compared to an untreated control.


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


The method may further comprise (c) contacting a sample comprising said antigen with a second antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and (d) determining antigenic integrity of said antigen by detectable binding of said second antibody or antibody fragment to said antigen. The second antibody or antibody fragment may be encoded by clone-paired variable sequences as set forth in Table 1, by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1, or by light and heavy chain variable sequences having at least 95% identity to clone-paired sequences as set forth in Table 1. The second antibody or antibody fragment may comprise light and heavy chain variable sequences according to clone-paired sequences from Table 2, may comprise light and heavy chain variable sequences having at least 70%, 80% or 90% identity to clone-paired sequences from Table 2, or may comprise light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 2. The second first antibody fragment may be a recombinant scFv (single chain fragment variable) antibody. Fab fragment, F(ab′)2 fragment, or Fv fragment. The method may further comprise performing steps (c) and (d) a second time to determine the antigenic stability of the antigen over time.


Also provided is human monoclonal antibody or antibody fragment, or hybridoma or engineered cell producing the same, wherein said antibody binds to a SARS-CoV-2 antigen surface spike protein. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one.” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.


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 the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.





BRIEF DESCRIPTION OF THE DRAWINGS

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


The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The 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.



FIG. 1. IMGT/DomainGapAlign results of COV2-2196 heavy and light chains. Key interacting residues and their corresponding residues in germline genes are colored in red.



FIG. 2. Identification of critical residues for COV2-21% and COV2-2130 through deep mutational scanning coupled with resistant variant selection. Antibody neutralization as measured by FRNT against reference strains and SARS-CoV-2 variants of concern. Neutralization assays were performed in duplicate and repeated twice, with results shown from one experimental replicate. Error bars denote the range for each point. Mutations compared to the WA-1 reference strain are denoted. B.1.1.7-OXF contains 69-70 and 144-145 deletion and the following substitutions: N501Y, A570D, D614G, P681H, and T716I.



FIGS. 3A-B. Identification of putative public clonotype members genetically similar to COV2-21% in the antibody variable gene repertoires of virus-naïve individuals. Antibody variable gene sequences from healthy individuals with the same sequence features as COV2-2196 heavy chain and light chain are aligned. Sequences from three different donors as well as cord blood included sequences with the features of the public clonotype. The sequence features and contact residues used in COV2-2196 are highlighted in red boxes below each multiple sequence alignment. (SEQ ID NOS: 1050-1054)



FIGS. 4A-E. Functional characteristics of neutralizing SARS-CoV-2 mAbs. FIG. 4A. Heatmap of mAb neutralization activity, hACE2 blocking activity, and binding to either trimeric S2Pecto protein or monomeric SRBD. MAbs are ordered by neutralization potency (highest at the top, lowest at the bottom). Dashed lines indicate the 13 antibodies with a neutralization IC50 value lower than 150 ng/mL for wt virus. IC50 values are visualized for viral neutralization and hACE2 blocking, while 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 2 independent experiments, each performed in technical duplicate. No inhibition indicates an IC50 value of >10,000 ng/mL, while no binding indicates an EC50 value of >10,000 ng/mL. FIGS. 4B-D. Correlation of hACE2 blocking, S2Pecto trimer binding, or SRBD binding of mAbs with their neutralization activity. R2 values are shown for linear regression analysis of log-transformed values. Purple circles indicate mAbs with a neutralization IC50 value lower than 150 ng/mL. FIG. 4E. Correlation of hACE2 blocking and S2Pecto trimer binding. R2 values are shown for linear regression analysis of log-transformed values.



FIGS. 5A-B. Epitope mapping of mAbs by competition-binding analysis and synergistic neutralization by a pair of mAbs. FIG. 4A. Left: biolayer interferometry-based competition binding assay measuring the ability of mAbs to prevent binding of reference mAbs COV2-2196 and rCR3022 to RBD fused to mouse Fc (RBD-mFc) loaded onto anti-mouse Fc biosensors. Values in squares are % of binding of the reference mAb in the presence of the competing mAb relative to a mock-competition control. Black squares denote full competition (<33% of binding relative to no-competition control), while white squares denote no competition (>67% of binding relative to no-competition control). Right: biolayer interferometry-based competition binding assay measuring the ability of mAbs to prevent binding of hACE2. Values denote % binding of hACE2, normalized to hACE2 binding in the absence of competition. Red color denotes competition of mAb with hACE2. FIG. 4B. Competition of neutralizing mAb panel with reference mAbs COV2-2130, COV2-2196, or rCR3022. Reference mAbs were biotinylated and binding of reference mAbs to trimeric S2Pecto was measured in the presence of saturating amounts of each mAb in a competition ELISA. ELISA signal for each reference mAb was normalized to the signal in the presence of the non-binding anti-dengue mAb 2D22. Black denotes full competition (<25% binding of reference mAb), grey denotes partial competition (25-60% binding of reference mAb), and white denotes no competition (>60% binding of reference mAb).



FIG. 6. SARS-CoV-2 neutralization curves for mAb panel. Neutralization of authentic SARS-CoV-2 by human mAbs. Mean±SD of technical duplicates is shown. Data represent one of two or more independent experiments



FIG. 7. Inhibition curves for mAb inhibition of S2Pecto binding to hACE2. Blocking of hACE2 binding to S2Pecto by anti-SARS-CoV-2 neutralizing human mAbs. Mean±SD of triplicates of one experiment is shown. Antibodies CR3022 and 2D22 served as controls.



FIG. 8. ELISA binding of anti-SARS-CoV-2 neutralizing human mAbs to trimeric SRBD, S2Pecto, or SARS-CoV S2Pecto antigen. Mean±SD of triplicates and representative of two experiments are shown. Antibodies CR3022 and 2D22 served as controls.





DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As discussed above, SARS-CoV-2 is a major health concern with active cases increasing daily. Therefore, understanding the biology of this virus and the nature and extent of the human immune response to the virus is of paramount importance. The inventors have identified the sequences of human antibodies to SARS-CoV-2. Those sequences and uses for such antibodies are disclosed herein.


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


I. Coronavirus 2019 (SARS-CoV-2)

SARS-CoV-2 is a contagious virus that causes the acute respiratory disease designated coronavirus disease 2019 (COVID-19), a respiratory infection. It is the cause of the ongoing 2019-20 coronavirus outbreak, a global health emergency. Genomic sequencing has shown that it is a positive-sense, single-stranded RNA coronavirus.


During the ongoing outbreak, the virus has often been referred to in common parlance as “the coronavirus”, “the new coronavirus” and “the Wuhan coronavirus”, while the WHO recommends the designation “SARS-CoV-2”. The International Committee on Taxonomy of Viruses (ICTV) announced that the official name for the virus is SARS-CoV-2.


Many early cases were linked to a large seafood and animal market in the Chinese city of Wuhan, and the virus is thought to have a zoonotic origin. Comparisons of the genetic sequences of this virus and other virus samples have shown similarities to SARS-CoV (79.5%) and bat coronaviruses (96%). This finding makes an ultimate origin in bats likely, although an intermediate host, such as a pangolin, cannot be ruled out. The virus could be a recombinant virus formed from two or more coronaviruses.


Human-to-human transmission of the virus has been confirmed. Coronaviruses are primarily spread through close contact, in particular through respiratory droplets from coughs and sneezes within a range of about 6 feet (1.8 m). Viral RNA has also been found in stool samples from infected patients. It is possible that the virus can be infectious even during the incubation period.


Animals sold for food were originally suspected to be the reservoir or intermediary hosts of SARS-CoV-2 because many of the first individuals found to be infected by the virus were workers at the Huanan Seafood Market. A market selling live animals for food was also blamed in the SARS outbreak in 2003; such markets are considered to be incubators for novel pathogens. The outbreak has 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 be the original source of viral transmission to humans.


With a sufficient number of sequenced genomes, it is possible to reconstruct a phylogenetic tree of the mutation history of a family of viruses. Research into the origin of the 2003 SARS outbreak has resulted in the discovery of many SARS-like bat coronaviruses, most originating in the Rhinolophus genus of horseshoe bats. SARS-CoV-2 falls into this category of SARS-related coronaviruses. Two genome sequences from Rhinolophus sinicus published in 2015 and 2017 show a resemblance of 80% to SARS-CoV-2. A third virus genome from Rhinolophus affinis. “RaTG13” collected in Yunnan province, has a 96% resemblance to SARS-CoV-2.[28][29] For comparison, this amount of variation among viruses is similar to the amount of mutation observed over ten years in the H3N2 human influenza virus strain.


SARS-CoV-2 belongs to the broad family of viruses known as coronaviruses; “nCoV” is the standard term used to refer to novel coronaviruses until the choice of a more specific designation. It is a positive-sense single-stranded RNA (+ssRNA) virus. Other coronaviruses are capable of causing illnesses ranging from the common cold to more severe diseases such as Middle East respiratory syndrome (MERS) and Severe acute respiratory syndrome (SARS). It is the seventh known coronavirus to infect people, after 229E, NL63, OC43, HKU1, MERS-CoV, and SARS-CoV.


Like SARS-CoV, SARS-CoV-2 is a member of the subgenus Sarbecovirus (Beta-CoV lineage B). Its RNA sequence is approximately 30,000 bases in length. By 12 January, 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 increased to 28 by 26 January. Except for the earliest GenBank genome, the genomes are under an embargo at GISAID. A phylogenic analysis for the samples is available through Nextstrain.


Publication of the SARS-CoV-2 genome led to several protein modeling experiments on the receptor binding protein (RBD) of the spike (S) protein of the virus. Results suggest that the S protein retains sufficient affinity to the Angiotensin converting enzyme 2 (ACE2) receptor to use it as a mechanism of cell entry. On 22 January, a group in China working with the full virus and a group in the U.S. working with reverse genetics independently and experimentally demonstrated human ACE2 as the receptor for SARS-CoV-2.


To look for potential protease inhibitors, the viral 3C-like protease M(pro) from the ORF1a polyprotein has also been modeled for drug docking experiments. Innophore has produced two computational models based on SARS protease, and the Chinese Academy of Sciences has produced an unpublished experimental structure of a recombinant SARS-CoV-2 protease. In addition, researchers at the University of Michigan have modeled the structures of all mature peptides in the SARS-CoV-2 genome using I-TASSER.


The first known human infection occurred in early December 2019. An outbreak of SARS-CoV-2 was first detected in Wuhan, China, in mid-December 2019, likely originating from a single infected animal. The virus subsequently spread to all provinces of China and to more than two dozen other countries in Asia, Europe, North America, and Oceania. Human-to-human spread of the virus has been confirmed in all of these regions. On 30 Jan. 2020, SARS-CoV-2 was designated a global health emergency by the WHO.


As of 10 Feb. 2020 (17:15 UTC), there were 40.645 confirmed cases of infection, of which 40.196 were within mainland China. Initially, nearly all cases outside China occurred in people who either traveled from Wuhan, or were in direct contact with someone who traveled from the area. Later, spread from travelers to other countries resulted in transmission in many countries in the world. While the proportion of infections that result in confirmed infection or progress to diagnosable SARS-CoV-2 acute respiratory disease remains unclear, the total number of deaths attributed to the virus was over 19,000 as of 25 Mar. 2020.


The basic reproduction number (R-zero) of the virus has been estimated to be between 1.4 and 3.9. This means that, when unchecked, the virus typically results in 1.4 to 3.9 new cases per established infection. It has been established that the virus is able to transmit along a chain of at least four people.


In January 2020, multiple organizations and institutions began work on creating vaccines 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 a vaccine is under development there. Shanghai East Hospital is also developing a vaccine in partnership with the biotechnology company Stemirna Therapeutics.


Elsewhere, three vaccine projects are being supported by the Coalition for Epidemic Preparedness Innovations (CEPI), including projects by the biotechnology companies Moderna and Inovio Pharmaceuticals and another by the University of Queensland. The United States National Institutes of Health (NIH) is cooperating with Moderna to create an RNA vaccine matching a spike of the coronavirus surface; Phase I clinical trials began in March 2020. Inovio Pharmaceuticals is developing a DNA-based vaccination and collaborating with a Chinese firm in order to speed its acceptance by regulatory authorities in China, hoping to perform human trials of the vaccine in the summer of 2020. In Australia, the University of Queensland is investigating the potential of a molecular clamp vaccine that would genetically modify viral proteins to make them mimic the coronavirus and stimulate an immune reaction.


In an independent project, the Public Health Agency of Canada has granted permission to the International Vaccine Centre (VIDO-InterVac) at the University of Saskatchewan to begin work on a vaccine. VIDO-InterVac aims to start production and animal testing in March 2020, and human testing in 2021. The Imperial College Faculty of Medicine in London is now at the stage of testing a vaccine on animals.


COVID-19 acute respiratory disease is a viral respiratory disease caused by SARS-CoV-2. It was first detected during the 2019-20 Wuhan coronavirus outbreak. Symptoms may include fever, dry cough, and shortness of breath. There is no specific licensed treatment available as of March 2020, with efforts focused on lessening symptoms and supporting functioning.


Those infected may either be asymptomatic or have mild to severe symptoms, like fever, cough, shortness of breath. Diarrhoea or upper respiratory symptoms (e.g., sneezing, runny nose, sore throat) are less frequent. Cases of severe infection can progress to severe pneumonia, multi-organ failure, and death. The time from exposure to onset of symptoms is estimated at 2 to 10 days by the World Health Organization, and 2 to 14 days by the US Centers for Disease Control and Prevention (CDC).


Global health organizations have published preventive measures individuals can take to reduce the chances of SARS-CoV-2 infection. Recommendations are similar to those previously published for other coronaviruses and include: frequent washing of hands with soap and water; not touching the eyes, nose, or mouth with unwashed hands; and practicing good respiratory hygiene.


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


Research into potential treatments for the disease were initiated in January 2020. The Chinese Center for Disease Control and Prevention started testing existing pneumonia treatments in coronavirus-related pneumonia in late January. There has also been examination of the RNA polymerase inhibitor remdesivir, and interferon beta. In late January 2020, Chinese medical researchers expressed an intent to start clinical testing on remdesivir, chloroquine, and lopinavir/ritonavir, all of which seemed to have “fairly good inhibitory effects” on SARS-CoV-2 at the cellular level in exploratory research. On 5 Feb. 2020, China started patenting use of remdesivir for the disease.


Overall mortality and morbidity rates due to infection with SARS-CoV-2 are unknown, both because the case fatality rate may be changing over time in the current outbreak, and because the proportion of infections that progress to diagnosable disease remains unclear. However, preliminary research into SARS-CoV-2 acute respiratory disease has yielded case fatality rate numbers between 2% and 3%, and in January 2020 the WHO suggested that the case fatality rate was approximately 3%. An unreviewed Imperial College preprint study among 55 fatal cases noted that early estimates of mortality may be too high as asymptomatic infections are missed. They estimated a mean infection fatality ratio (the mortality among infected) ranging from 0.8% when including asymptomatic carriers to 18% when including only symptomatic cases from Hubei province.


Early data indicates that among the first 41 confirmed cases admitted to hospitals in Wuhan, 13 (32%) individuals required intensive care, and 6 (15%) individuals died. Of those who died, many were in unsound health to begin with, exhibiting conditions like hypertension, diabetes, or cardiovascular disease that impaired their immune systems. In early cases of SARS-CoV-2 acute respiratory disease that resulted in death, the median time of disease was found to be 14 days, with a total range from six to 41 days.


II. Monoclonal Antibodies and Production Thereof

An “isolated antibody” is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In particular embodiments, the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most particularly more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver stain. 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.


The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 basic heterotetramer units along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain. In the case of IgGs, the 4-chain unit is generally about 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 intrachain disulfide bridges. Each H chain has at the N-terminus, a variable region (VH) followed by three constant domains (CH) for each of the alpha and gamma chains and four CH domains for mu and isotypes. Each L chain has at the N-terminus, a variable region (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable regions. The pairing of a V1 and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba 1. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk. Conn., 1994, page 71, and Chapter 6.


The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on the amino acid sequences of their constant domains (C). Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. They gamma and alpha classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.


The term “variable” refers to the fact that certain segments of the V domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable regions of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, 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 involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent complement deposition (ADCD).


The term “hypervariable region” when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the VH when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda. Md. (1991)); and/or those residues from a “hypervariable loop” (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (H1), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); and/or those residues from a “hypervariable loop”/CDR (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1), 56-65 (H2) and 105-120 (H3) in the VH when numbered in accordance with 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)). Optionally the antibody has symmetrical insertions at one or more of the following points 28, 36 (L1), 63.74-75 (L2) and 123 (L3) in the VL, and 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) in the VsubH when numbered in accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol. 309:657-670 (2001)).


By “germline nucleic acid residue” is meant the nucleic acid residue that naturally occurs in a germline gene encoding a constant or variable region. “Germline gene” is the DNA found in a germ cell (i.e., a cell destined to become an egg or in the sperm). A “germline mutation” refers to a heritable change in a particular DNA that has occurred in a germ cell or the zygote at the single-cell stage, and when transmitted to offspring, such a mutation is incorporated in every cell of the body. A germline mutation is in contrast to a somatic mutation which is acquired in a single body cell. In some cases, nucleotides in a germline DNA sequence encoding for a variable region are mutated (i.e., a somatic mutation) and replaced with a different nucleotide.


The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., 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, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567) after single cell sorting of an antigen specific B cell, an antigen specific plasmablast responding to an infection or immunization, or capture of linked heavy and light chains from single cells in a bulk sorted antigen specific collection. The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.


A. General Methods

It will be understood that monoclonal antibodies binding to SARS-CoV-2 will have several applications. These include the production of diagnostic kits for use in detecting and diagnosing SARS-CoV-2 infection, as well as for treating the same. In these contexts, one may link such antibodies to diagnostic or therapeutic agents, use them as capture agents or competitors in competitive assays, or use them individually without additional agents being attached thereto. The antibodies may be mutated or modified, as discussed further 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).


The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. The first step for both these methods is immunization of an appropriate host or identification of subjects who are immune due to prior natural infection or vaccination with a licensed or experimental vaccine. As is well known in the art, a given composition for immunization may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may 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 a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde and bis-biazotized benzidine. As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants in animals include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant and in humans include alum, CpG, MFP59 and combinations of immunostimulatory molecules (“Adjuvant Systems”, such as AS01 or AS03). Additional experimental forms of inoculation to induce SARS-CoV-2-specific B cells is possible, including nanoparticle vaccines, or gene-encoded antigens delivered as DNA or RNA genes in a physical delivery system (such as lipid nanoparticle or on a gold biolistic bead), and delivered with needle, gene gun, transcutaneous electroporation device. The antigen gene also can be carried as encoded by a replication competent or defective viral vector such as adenovirus, adeno-associated virus, poxvirus, herpesvirus, or alphavirus replicon, or alternatively a virus like particle.


In the case of human antibodies against natural pathogens, a suitable approach is to identify subjects that have been exposed to the pathogens, such as those who have been diagnosed as having contracted the disease, or those 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 subject may then be obtained.


The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate MAbs.


Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, lymph nodes, tonsils or adenoids, bone marrow aspirates or biopsies, tissue biopsies from mucosal organs like lung or GI tract, or from circulating blood. The antibody-producing B lymphocytes from the immunized animal or immune human are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized or human or human/mouse chimeric cells. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). HMMA2.5 cells or MFP-2 cells are particularly useful examples of such cells.


Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. In some cases, transformation of human B cells with Epstein Barr virus (EBV) as an initial step increases the size of the B cells, enhancing fusion with the relatively large-sized myeloma cells. Transformation efficiency by EBV is enhanced by using CpG and a Chk2 inhibitor drug in the transforming medium. Alternatively, human B cells can be activated by co-culture with transfected cell lines 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 those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods also is appropriate (Goding, pp. 71-74, 1986) and there are processes for better efficiency (Yu et al., 2008). Fusion procedures usually produce viable hybrids at low frequencies, about 1×10−6 to 1×10−8, but with optimized procedures one can achieve fusion efficiencies close to 1 in 200 (Yu et al., 2008). However, relatively low efficiency of fusion does not pose a problem, as the viable, fused hybrids are differentiated from the parental, infused cells (particularly the infused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the medium is supplemented with hypoxanthine. Ouabain is added if the B cell source is an EBV-transformed human B cell line, in order to eliminate EBV-transformed lines that have not fused to the myeloma.


The preferred selection medium is HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a line of EBV-transformed B cells, as here, ouabain may also be used for drug selection of hybrids as EBV-transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant.


Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like. The selected hybridomas are then serially diluted or single-cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for MAb production 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 animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. When human hybridomas are used in this way, it is optimal to inject immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. Alternatively, human hybridoma cells lines can be used in vitro to produce immunoglobulins in cell supernatant. The cell lines can be adapted for growth in serum-free medium to optimize the ability to recover human monoclonal immunoglobulins of high purity.


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


It also is contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. Single B cells identified as responding to infection or vaccination because of plasmablast aor activated B cell markers, or memory B cells labelled with the antigen of interest, can be sorted physically using paramagnetic bead selection or flow cytometric sorting, then RNA can be isolated from the single cells and antibody genes amplified by RT-PCR. Various single-cell RNA-seq methods are available to obtain antibody variable genes from single cells. Alternatively, antigen-specific bulk sorted populations of cells can be segregated into microvesicles and the matched heavy and light chain variable genes recovered from single cells using physical linkage of heavy and light chain amplicons, or common barcoding of heavy and light chain genes from a vesicle. Matched heavy and light chain genes from single cells also can be obtained from populations of antigen specific B cells by treating cells with cell-penetrating nanoparticles bearing RT-PCR primers and barcodes for marking transcripts with one barcode per cell. The antibody variable genes also can be isolated by RNA extraction of a hybridoma line and the antibody genes obtained by RT-PCR and cloned into an immunoglobulin expression vector. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new specificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.


Other U.S. patents, each incorporated herein by reference, that teach the production of antibodies useful in the present disclosure include U.S. Pat. No. 5,565,332, which describes the production of chimeric antibodies using a combinatorial approach; U.S. Pat. No. 4,816,567 which describes recombinant immunoglobulin preparations; and U.S. Pat. No. 4,867,973 which describes antibody-therapeutic agent conjugates.


B. Antibodies of the Present Disclosure

Antibodies according to the present disclosure may be defined, in the first instance, by their binding specificity. Those of skill in the art, by assessing the binding specificity/affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims. For example, the epitope to which a given antibody bind may consist of a single contiguous sequence of 3 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 the antigen molecule (e.g., a linear epitope in a domain). Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within the antigen molecule (e.g., a conformational epitope).


Two main categories of SARS-CoV-2 antigens are the surface spike (S) protein and the internal proteins, especially the nucleocapsid (N) protein. Antibodies to the S protein will be useful for prophylaxis, or therapy, or diagnostics, or for characterizing vaccines. S protein antibodies will have additional binding specificity with that protein, with particular antibodies binding to the full-length ectodomain of the SARS-CoV-2 S protein (presented as a monomer or oligomer such as a timer; with our without conformation stabilizing mutations such as introduction of prolines at critical sites (“2P mutation”)) and (a) anti-S protein antibodies that binds to the receptor binding domain (RBD), (b) anti-S protein antibodies that bind to domains other than the RBD. Some of the subset that bind to domains other than the RBD bind to the N terminal domain (NTD), while others bind to an epitope 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 can cross-react with both SARS-CoV-2 S protein and the S protein of other coronaviruses such as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63 and/or HCoV-HKU 1, as well as cross-neutralize both SARS-CoV-2 and these other coronaviruses.


Another specificity will be antibodies that bind to N antibodies (or other internal targets) that will have primarily diagnostics uses. For example, antibodies to N or other internal proteins of SARS-CoV-2 that specifically recognize SARS-CoV-2 or that cross-reactively recognize SARS-CoV-2 and other coronaviruses such as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63 and/or HCoV-HKU1.


Various techniques known to persons of ordinary skill 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, for example, routine cross-blocking assays, such as that described in Antibodies. Harlow and Lane (Cold Spring Harbor Press. Cold Spring Harbor. N.Y.). Cross-blocking can be measured in various binding assays such as ELISA, biolayer interferometry, or surface plasmon resonance. Other methods include alanine scanning mutational analysis, peptide blot analysis (Reineke, Methods Mol. Biol. 248: 443-63, 2004), peptide cleavage analysis, high-resolution electron microscopy techniques using single particle reconstruction, cryoEM, 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 the 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 the protein of interest, followed by binding the antibody to the deuterium-labeled protein. Next, the protein/antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to-hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that am not part of the interface. As a result, amino acids that form part of the protein/antibody interface may retain deuterium and therefore exhibit relatively higher mass compared to amino acids not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry analysis, thereby revealing the deuterium-labeled residues which correspond 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 the antibody neutralizes SARS-CoV-2, antibody escape mutant variant organisms can be isolated by propagating SARS-CoV-2 in vitro or in animal models in the presence of high concentrations of the antibody. Sequence analysis of the SARS-CoV-2 gene encoding the antigen targeted by the antibody reveals the mutation(s) conferring antibody escape, indicating residues in the epitope or that affect the structure of the epitope allosterically.


The term “epitope” refers to a site on an antigen to which B and/or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.


Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers of monoclonal antibodies (mAbs) directed against the same antigen according to the similarities of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (see U.S. Patent Publication 2004/0101920, herein specifically incorporated by reference in its entirety). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope represented by another category. This technology allows rapid filtering of genetically identical antibodies, such that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening. MAP may facilitate identification of rare hybridoma clones that produce mAbs having the desired characteristics. MAP may be used to sort the antibodies of the disclosure into groups of antibodies binding different epitopes.


The present disclosure includes antibodies that may bind to the same epitope, or a portion of the epitope. Likewise, the present disclosure also includes antibodies that compete for binding to a target or a fragment thereof with any of the specific exemplary antibodies described herein. One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference, the reference antibody is allowed to bind to target under saturating conditions. Next, the ability of a test antibody to bind to the target molecule is assessed. If the test antibody is able to bind to the target molecule following saturation 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 is not able to bind to the target molecule following saturation binding with the reference antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference antibody.


To determine if an antibody competes for binding with a reference anti-SARS-CoV-2 antibody, the above-described binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to the SARS-CoV-2 antigen under saturating conditions followed by assessment of binding of the test antibody to the SARS-CoV-2 molecule. In a second orientation, the test antibody is allowed to bind to the SARS-CoV-2 antigen molecule under saturating conditions followed by assessment of binding of the reference antibody to the SARS-CoV-2 molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to SARS-CoV-2, then it is concluded that the test antibody and the reference antibody compete for binding to SARS-CoV-2. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.


Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits 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. 199W 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 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 reduce or eliminate binding of the other.


Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort 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 with EM or crystallography also can demonstrate whether or not two antibodies that compete for binding recognize the same epitope.


In another aspect, there are provided monoclonal antibodies having clone-paired CDRs from the heavy and light chains as illustrated in Tables 3 and 4, respectively. Such antibodies may be produced by the clones discussed below in the Examples section using methods described herein.


In another aspect, the antibodies may be defined by their variable sequence, which include additional “framework” regions. Furthermore, the antibodies sequences may vary from these sequences, optionally using methods discussed in greater detail below. For example, nucleic acid sequences may vary from those set out above in that (a) the variable regions may be segregated away from the constant domains of the light and heavy chains, (b) the nucleic acids may vary from those set out above while not affecting the residues encoded thereby, (c) the nucleic acids may vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, (d) the nucleic acids may vary from those set out above by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C., (e) the amino acids may vary from those set out 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 vary from those set out above by permitting conservative substitutions (discussed below). Each of the foregoing applies to the nucleic acid sequences and the amino acid sequences.


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


Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. 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, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy-—the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.: Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726-730.


Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by 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 by inspection.


One particular example of algorithms that are suitable for determining percent sequence identity and sequence similarity are 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. BLAST and BLAST 2.0 can be used, for example, with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The rearranged nature of an antibody sequence and the variable length of each gene requires multiple rounds of BLAST searches for a single antibody sequence. Also, manual assembly of different genes is difficult and error-prone. The sequence analysis tool IgBLAST (world-wide-web at ncbi.nlm.nih.gov/igblast/) identifies matches to the germline V, D and J genes, details at rearrangement junctions, the delineation of Ig V domain framework regions and complementarity determining regions. IgBLAST can analyze nucleotide or protein sequences and can process sequences in batches and allows searches against the germline gene databases and other sequence databases simultaneously to minimize the chance of missing possibly the best matching germline V gene.


In one illustrative example, cumulative scores can be calculated using, 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). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 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 wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.


For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.


In one approach, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.


Yet another way of defining an antibody is as a “derivative” of any of the below-described antibodies and their antigen-binding fragments. The term “derivative” refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen but which comprises, one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to a “parental” (or wild-type) molecule. Such amino acid substitutions or additions may introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. The term “derivative” encompasses, for example, as variants having altered CH1, hinge, CH2. CH3 or CH4 regions, so as to form, for example, antibodies, etc., having variant Fc regions that exhibit enhanced or impaired effector or binding characteristics. The term “derivative” additionally encompasses non-amino acid modifications, for example, amino acids that may be glycosylated (e.g., have altered mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc. content), acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytic cleavage, linked to a cellular ligand or other protein, etc. In some embodiments, the altered carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function. In a specific embodiment, the altered carbohydrate modifications enhance antibody mediated effector function relative to the antibody lacking the carbohydrate modification. Carbohydrate modifications that lead to altered antibody mediated effector function are well known in the art (for example, see Shields, R. L. et 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; 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 of altering carbohydrate contents are known to those skilled in the art, see, e.g., Wallick, S. C. 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, M. H. 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. (2003) “Enhancement Of Therapeutic Protein In Vivo Activities Through Glycoengineering,” Nature Biotechnol. 21:414-21: Shields. R. L. et 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).


A derivative antibody or antibody fragment can be generated with an engineered sequence or glycosylation state to confer preferred 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) functions as measured by bead-based or cell-based assays or in vivo studies in animal models.


A derivative antibody or antibody fragment may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In one embodiment, an antibody derivative will possess a similar or identical function as the parental antibody. In another embodiment, an antibody derivative will exhibit an altered activity relative to the parental antibody. For example, a derivative antibody (or fragment thereof) can bind to its epitope more tightly or be more resistant to proteolysis than the parental antibody.


C. Engineering of Antibody Sequences

In various embodiments, one may choose to engineer sequences of the identified antibodies for a variety of reasons, such as improved expression, improved cross-reactivity or diminished off-target binding. Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides. Methods for recombinant expression are addressed elsewhere in this document. The following is a general discussion of relevant goals techniques for antibody engineering.


Hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Assay of binding and neutralization may be performed using antibodies collected from hybridoma supernatants and purified by FPLC, using Protein G columns.


Recombinant full-length IgG antibodies can be generated by subcloning heavy and light chain Fv DNAs from the cloning vector into an IgG plasmid vector, transfected into 293 (e.g., Freestyle) cells or CHO cells, and antibodies can be collected and purified from the 293 or CHO cell supernatant. Other appropriate host cells systems include bacteria, such as E. coli, insect cells (S2, Sf9, Sf29, High Five), plant cells (e.g., tobacco, with or without engineering for human-like glycans), algae, or in a variety of non-human transgenic contexts, such as mice, rats, goats or cows.


Expression of nucleic acids encoding antibodies, both for the purpose of subsequent antibody purification, and for immunization of a host, is also contemplated. Antibody coding sequences can be RNA, such as native RNA or modified RNA. Modified RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, N1-methyl-pseudouridine (N1mΨ) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. In addition to turning off the immune/eIF2α phosphorylation-dependent inhibition of translation, incorporated N1mΨ nucleotides dramatically alter the dynamics of the translation process by increasing ribosome pausing and density on the mRNA. Increased ribosome loading of modified mRNAs renders them more permissive for initiation by favoring either ribosome recycling on the same mRNA or de novo ribosome recruitment. Such modifications could be used to enhance antibody expression in vivo following inoculation with RNA. The RNA, whether native or modified, may be delivered as naked RNA or in a delivery vehicle, such as a lipid nanoparticle.


Alternatively, DNA encoding the antibody may be employed for the same purposes. The DNA is included in an expression cassette comprising a promoter active in the host cell for which it is designed. The expression cassette is advantageously included in a replicable vector, such as a conventional plasmid or minivector. Vectors include viral vectors, such as poxviruses, adenoviruses, herpesviruses, adeno-associated viruses, and lentiviruses are contemplated. Replicons encoding antibody genes such as alphavirus replicons based on VEE virus or Sindbis virus are also contemplated. Delivery of such vectors can be performed by needle through intramuscular, subcutaneous, or intradermal routes, or by transcutaneous electroporation when in vivo expression is desired.


The rapid availability of antibody produced in the same host cell and cell culture process as the final cGMP manufacturing process has the potential to reduce the duration of process development programs. Lonza has developed a generic method using pooled transfectants grown in CDACF medium, for the rapid production of small quantities (up to 50 g) of antibodies in CHO cells. Although slightly slower than a true transient system, the advantages include a higher product concentration and use of the same host and process as the production cell line. Example of growth and productivity of GS-CHO pools, expressing a model antibody, in a disposable bioreactor: in a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g/L was achieved within 9 weeks of transfection.


Antibody molecules will comprise fragments (such as F(ab′), F(ab′)2) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. F(ab′) antibody derivatives are monovalent, while F(ab′)2 antibody derivatives are bivalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form “chimeric” binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.


In related embodiments, the antibody is a derivative of the disclosed antibodies, e.g., an antibody comprising the CDR sequences identical to those in the disclosed antibodies (e.g., a chimeric, or CDR-grafted antibody). Alternatively, one may wish to make modifications, such as introducing conservative changes into an antibody molecule. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.


It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: basic amino acids: arginine (+3.0), lysine (+3.0), and histidine (−0.5); acidic amino acids: aspartate (+3.0±1), glutamate (+3.0±1), asparagine (+0.2), and glutamine (+0.2); hydrophilic, nonionic amino acids: serine (+0.3), asparagine (+0.2), glutamine (+0.2), and threonine (−0.4), sulfur containing amino acids: cysteine (−1.0) and methionine (−1.3); hydrophobic, nonaromatic 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).


It is understood that an amino acid can be substituted for another having a similar hydrophilicity and produce a biologically or immunologically modified protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.


As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.


The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, changing to IgG1 can increase antibody dependent cell cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.


Alternatively or additionally, it may be useful to combine amino acid modifications with one or more further amino acid modifications that alter C1q binding and/or the complement dependent cytotoxicity (CDC) function of the Fc region of an IL-23p19 binding molecule. The binding polypeptide of particular interest may be one that binds to C1q and displays complement dependent cytotoxicity. Polypeptides with pre-existing C1q binding activity, optionally further having the ability to mediate CDC may be modified such that one or both of these activities are enhanced. Amino acid modifications that alter C1q and/or modify its complement dependent cytotoxicity function are described, for example, in WO/0042072, which is hereby incorporated by reference.


One can design an Fc region of an antibody with altered effector function, e.g., by modifying C1q binding and/or FcγR binding and thereby changing CDC activity and/or ADCC activity. “Effector functions” are responsible for activating or diminishing 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; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fe region to be combined 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.).


For example, one can generate a variant Fe region of an antibody with improved C1q binding and improved FcγRIII binding (e.g., having both improved ADCC activity and improved CDC activity). Alternatively, if it is desired that effector function be reduced or ablated, a variant Fc region can be engineered with reduced CDC activity and/or reduced ADCC activity. In other embodiments, only one of these activities may be increased, and, optionally, also the other activity reduced (e.g., to generate an Fc region variant with improved ADCC activity, but reduced CDC activity and vice versa).


FcRn binding. Fc mutations can also be introduced and engineered to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties. A collection of human Fc variants with improved binding to the FcRn have 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). A number of methods are known that can result in increased half-life (Kuo and Aveson, (2011)), including amino acid modifications may be generated through techniques including alanine scanning mutagenesis, random mutagenesis and screening to assess the binding to the neonatal Fc receptor (FcRn) and/or the in vivo behavior. Computational strategies followed by mutagenesis may also be used to select one of amino acid mutations to mutate.


The present disclosure therefore provides a variant of an antigen binding protein with optimized binding to FcRn. In a particular embodiment, the said variant of an antigen binding protein comprises at least one amino acid modification in the Fc region of said antigen binding protein, wherein said modification is selected from the group consisting of 226, 227, 228, 230, 231, 233, 234, 239, 241, 243, 246, 250, 252, 256, 259, 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, 342, 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, 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 of the Fe region as compared to said parent polypeptide, wherein the numbering of the amino acids in the Fc region is that of the EU index in Kabat. In a further aspect of the disclosure the modifications are M252Y/S254T/T256E.


Additionally, various publications describe methods for obtaining physiologically active molecules whose half-lives are modified, see for example Kontermann (2009) either by introducing an FcRn-binding polypeptide into the molecules or by fusing the molecules with antibodies whose FcRn-binding affinities are preserved but affinities for other Fe receptors have been greatly reduced or fusing with FcRn binding domains of antibodies.


Derivatized antibodies may be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, particularly a human. Such alterations may result in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The increased half-lives of the antibodies of the present disclosure or fragments thereof in a mammal, preferably a human, results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus reduces the frequency of the administration of said antibodies or antibody fragments and/or reduces the concentration of said antibodies or antibody fragments to be administered. Antibodies or fragments thereof having increased in vivo half-lives can be generated by techniques known to those of skill 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 involved in the interaction between the Fc domain and the FcRn receptor.


Beltramello et al. (2010) previously reported the modification of neutralizing mAbs, due to their tendency to enhance dengue virus infection, by generating in which leucine residues at positions 1.3 and 1.2 of CH2 domain (according to the IMGT unique numbering for C-domain) were substituted with alanine residues. This modification, also known as “LALA” mutation, abolishes antibody binding to FcγRI, FcγRII and FcγRIIIa, as described by Hessell et al. (2007). The variant and unmodified recombinant mAbs were compared for their capacity to neutralize and enhance infection by the four dengue virus serotypes. LALA variants retained the same neutralizing activity as unmodified mAb but were completely devoid of enhancing activity. LALA mutations of this nature are therefore contemplated in the context of the presently disclosed antibodies.


Altered Glycosylation. A particular embodiment of the present disclosure is an isolated monoclonal antibody, or antigen binding fragment thereof, containing a substantially homogeneous glycan 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 attached to heavy chain or light chain constant regions respectively. The aforementioned substantially homogeneous glycan may be covalently attached to the heavy chain constant region.


Another embodiment of the present disclosure comprises a mAb with a novel Fc glycosylation pattern. The isolated monoclonal antibody, or antigen binding fragment thereof, is present in a substantially homogenous composition represented by the GNGN or G1/G2 glycoform. Fc glycosylation plays a significant role in anti-viral and anti-cancer properties of therapeutic mAbs. The disclosure is in line with a recent study that shows increased anti-lentivirus cell-mediated viral inhibition of a fucose free anti-HIV mAb in vitro. This embodiment of the present disclosure with homogenous glycans lacking a core fucose, showed increased protection against specific viruses by a factor greater than two-fold. Elimination of core fucose dramatically improves the ADCC activity of mAbs mediated by natural killer (NK) cells but appears to have the opposite effect on the ADCC activity of polymorphonuclear cells (PMNs).


The isolated monoclonal antibody, or antigen binding fragment thereof, comprising a substantially homogenous composition represented by the GNGN or G1/G2 glycoform exhibits increased binding affinity for Fc gamma RI and Fc gamma RIII compared to the same antibody without the substantially homogeneous GNGN glycoform and with G0, G1F, G2F, GNF, GNGNF or GNGNFX containing glycoforms. In one embodiment of the present disclosure, the antibody dissociates from Fc gamma RI with a Kd of 1×10−8 M or less and from Fc gamma RIII with a Kd of 1×10−7 M or less.


Glycosylation of an 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 may also be used. The recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequences 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.


The glycosylation pattern may be altered, for example, by deleting one or more glycosylation site(s) found in the polypeptide, and/or adding one or more glycosylation site(s) that are not present in the polypeptide. Addition of glycosylation sites to the Fe region of an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). An exemplary glycosylation variant has an amino acid substitution of residue Asn 297 of the heavy chain. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original polypeptide (for O-linked glycosylation sites). Additionally, a change of Asn 297 to Ala can remove one of the glycosylation sites.


In certain embodiments, the antibody is expressed in cells that express beta (1,4)-N-acetylglucosaminyltransferase III (GnT III), such that GnT III adds GlcNAc to the IL-23p19 antibody. Methods for producing antibodies in such a fashion are provided in WO/9954342, WO/03011878, patent publication 20030003097A1, and Umana et al., Nature Biotechnology, 17:176-180, February 1999. Cell lines can be altered to enhance or reduce or eliminate certain post-translational modifications, such as glycosylation, using genome editing technology such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). For example, CRISPR technology can be used to eliminate genes encoding glycosylating enzymes in 293 or CHO cells used to express recombinant monoclonal antibodies.


Elimination of monoclonal antibody protein sequence liabilities. It is possible to engineer the antibody variable gene sequences obtained from human B cells to enhance their manufacturability and safety. Potential protein sequence liabilities can be identified by searching for sequence motifs associated with sites containing:

    • 1) Unpaired Cys residues,
    • 2) N-linked glycosylation,
    • 3) Asn deamidation,
    • 4) Asp isomerization,
    • 5) SYE truncation,
    • 6) Met oxidation,
    • 7) Trp oxidation,
    • 8) N-terminal glutamate,
    • 9) Integrin binding,
    • 10) CD11c/CD18 binding, or
    • 11) Fragmentation


      Such motifs can be eliminated by altering the synthetic gene for the cDNA encoding recombinant antibodies.


Protein engineering efforts in the field of development of therapeutic antibodies clearly reveal that certain sequences or residues are associated with solubility differences (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 mutations in the literature indicate 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.


Stability. Antibodies can be engineered for enhanced biophysical properties. One can use elevated temperature to unfold antibodies to determine relative stability, using average apparent melting temperatures. Differential Scanning Calorimetry (DSC) measures the heat capacity, Cp, of a molecule (the heat required to warm it, per degree) as a function of temperature. One can use DSC to study the thermal stability of antibodies. DSC data for mAbs is particularly interesting because it sometimes resolves the unfolding of individual domains within the mAb structure, producing up to three peaks in the thermogram (from unfolding of the Fab. CH2, and CH3 domains). Typically unfolding of the Fab domain produces the strongest peak. The DSC profiles and relative stability of the Fe portion show characteristic differences for the human IgG1, IgG2, IgG3, and IgG4 subclasses (Garber and Demarest, Biochem. Biophys. Res. Commun. 355, 751-757, 2007). One also can determine average apparent melting temperature using circular dichroism (CD), performed with a CD spectrometer. Far-UV CD spectra will be measured for antibodies in the range of 200 to 260 nm at increments of 0.5 nm. The final spectra can be determined as averages of 20 accumulations. Residue ellipticity values can be calculated after background subtraction. Thermal unfolding of antibodies (0.1 mg/mL) can be monitored at 235 nm from 25-95° C. and a heating rate of 1° C./min. One can use dynamic light scattering (DLS) to assess for propensity for aggregation. DLS is used to characterize size of various particles including proteins. If the system is not disperse in size, the mean effective diameter of the particles can be determined. This measurement depends on the size of the particle core, the size of surface structures, and particle concentration. Since DLS essentially measures fluctuations in scattered light intensity due to particles, the diffusion coefficient of the particles can be determined. DLS software in commercial DLA instruments displays the particle population at different diameters. Stability studies can be done conveniently using DLS. DLS measurements of a sample can show whether the particles aggregate over time or with temperature variation by determining whether the hydrodynamic radius of the particle increases. If particles aggregate, one can see a larger population of particles with a larger radius. Stability depending on temperature can be analyzed by controlling the temperature in situ. Capillary electrophoresis (CE) techniques include proven methodologies for determining features of antibody stability. One can use an iCE approach to resolve antibody protein charge variants due to deamidation, C-terminal lysines, sialylation, oxidation, glycosylation, and any other change to the protein that can result in a change in pI of the protein. Each of the expressed antibody proteins can be evaluated by high throughput, free solution isoelectric focusing (IEF) in a capillary column (cIEF), using a Protein Simple Maurice instrument. Whole-column UV absorption detection can be performed every 30 seconds for real time monitoring of molecules focusing at the isoelectric points (pIs). This approach combines the high resolution of traditional gel IEF with the advantages of quantitation and automation found in column-based separations while eliminating the need for a mobilization step. The technique yields reproducible, quantitative analysis of identity, purity, and heterogeneity profiles for the expressed antibodies. The results identify charge heterogeneity and molecular sizing on the antibodies, with both absorbance and native fluorescence detection modes and with sensitivity of detection down to 0.7 μg/mL.


Solubility. One can determine the intrinsic solubility score of antibody sequences. The intrinsic solubility scores can be calculated using CamSol Intrinsic (Sormanni et al., J Mol Biol 427, 478-490, 2015). The amino acid sequences for residues 95-102 (Kabat numbering) in HCDR3 of each antibody fragment such as a scFv can be evaluated via the online program to calculate the solubility scores. One also can determine solubility using laboratory techniques. Various techniques exist, including addition of lyophilized protein to a solution until the solution becomes saturated and the solubility limit is reached, or concentration by ultrafiltration in a microconcentrator with a suitable molecular weight cut-off. The most straightforward method is induction of amorphous precipitation, which measures protein solubility using a method involving protein precipitation using ammonium sulfate (Trevino et al., J Mol Biol, 366: 449-460, 2007). Ammonium sulfate precipitation gives quick and accurate information on relative solubility values. Ammonium sulfate precipitation produces precipitated solutions with well-defined aqueous and solid phases and requires relatively small amounts of protein. Solubility measurements performed using induction of amorphous precipitation by ammonium sulfate also can be done easily at different pH values. Protein solubility is highly pH dependent, and pH is considered the most important extrinsic factor that affects solubility.


Autoreactivity. Generally, it is thought that autoreactive clones should be eliminated during ontogeny by negative selection, however it has become clear that many human naturally occurring antibodies with autoreactive properties persist in adult mature repertoires, and the autoreactivity may enhance the antiviral function of many antibodies to pathogens. It has been noted that HCDR3 loops in antibodies during early B cell development are often rich in positive charge and exhibit autoreactive patterns (Wardemann et al., Science 301, 1374-1377, 2003). One can test a given antibody for autoreactivity by assessing the level of binding to human origin cells in microscopy (using adherent HeLa or HEp-2 epithelial cells) and flow cytometric cell surface staining (using suspension Jurkat T cells and 293S human embryonic kidney cells). Autoreactivity also can be surveyed using assessment of binding to tissues in tissue arrays.


Preferred residues (“Human Likeness”). B cell repertoire deep sequencing of human B cells from blood donors is being performed on a wide scale in many recent studies. Sequence information about a significant portion of the human antibody repertoire facilitates statistical assessment of antibody sequence features common in healthy humans. With knowledge about the antibody sequence features in a human recombined antibody variable gene reference database, the position specific degree of “Human Likeness” (HL) of an antibody sequence can be estimated. HL has been shown to be useful for the development of antibodies in clinical use, like therapeutic antibodies or antibodies as vaccines. The goal is to increase the human likeness of antibodies to reduce potential adverse effects and anti-antibody immune responses that will lead to significantly decreased efficacy of the antibody drug or can induce serious health implications. One can assess antibody characteristics of the combined antibody repertoire of three healthy human blood donors of about 400 million sequences in total and created a novel “relative Human Likeness” (rHL) score that focuses on the hypervariable region of the antibody. The rHL score allows one to easily distinguish between human (positive score) and non-human sequences (negative score). Antibodies can be engineered to eliminate residues that are not common in human repertoires.


D. Single Chain Antibodies

A single chain variable fragment (scFv) is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma or B cell. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.


Flexible linkers generally are comprised of helix- and turn-promoting amino acid residues such as alanine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5×106 different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VHC terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers.


The recombinant antibodies of the present disclosure may also involve sequences or moieties that permit dimerization or multimerization of the receptors. Such sequences include those derived from IgA, which permit formation of multimers in conjunction with the J-chain. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chains may be modified with agents such as biotin/avidin, which permit the combination of two antibodies.


In a separate embodiment, a single-chain antibody can be created by joining receptor light and heavy chains using a non-peptide linker or chemical unit. Generally, the light and heavy chains will be produced in distinct cells, purified, and subsequently linked together in an appropriate fashion (i.e., the N-terminus of the heavy chain being attached to the C-terminus of the light chain via an appropriate chemical bridge).


Cross-linking reagents are used to form molecular bridges that tie functional groups of two different molecules, e.g., a stabilizing and coagulating agent. However, it is contemplated that dimers or multimers of the same analog or heteromeric complexes comprised of different analogs can be created. To link two different compounds in a step-wise manner, hetero-bifunctional cross-linkers can be used that eliminate unwanted homopolymer formation.


An exemplary hetero-bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the selective agent).


It is preferred that a cross-linker having reasonable stability in blood will be employed. Numerous types of disulfide-bond containing linkers are known that can be successfully employed to conjugate targeting and therapeutic/preventative agents. Linkers that contain a disulfide bond that is sterically hindered may prove to give greater stability in vivo, preventing release of the targeting peptide prior to reaching the site of action. These linkers are thus one group of linking agents.


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 methyl groups. It is believed that steric hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to the delivery of the attached agent to the target site.


The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3′-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.


In addition to hindered cross-linkers, non-hindered linkers also can be employed in accordance herewith. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood in the art. Another embodiment involves the use of flexible linkers.


U.S. Pat. No. 4,680,338, describes bifunctional linkers useful for producing conjugates of ligands with amine-containing polymers and/or proteins, especially for forming antibody conjugates with chelators, drugs, enzymes, detectable labels and the like. U.S. Pat. Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing a labile bond that is cleavable under a variety of mild conditions. This linker is particularly useful in that the agent of interest may be bonded directly to the linker, with cleavage resulting in release of the active agent. Particular uses include adding a free amino or free sulfhydryl group to a protein, such as an antibody, or a drug.


U.S. Pat. No. 5,856,456 provides peptide linkers for use in connecting polypeptide constituents to make fusion proteins, e.g., single chain antibodies. The linker is up to about 50 amino acids in length, contains at least one occurrence of a charged amino acid (preferably arginine or lysine) followed by a proline, and is characterized by greater stability and reduced aggregation. U.S. Pat. No. 5,880,270 discloses aminooxy-containing linkers useful in a variety of immunodiagnostic and separative techniques.


E. Multispecific Antibodies

In certain embodiments, antibodies of the present disclosure are bispecific or multispecific. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a single antigen. Other such antibodies may combine a first antigen binding site with a binding site for a second antigen. Alternatively, an anti-pathogen arm may be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and Fc gamma RIII (CD16), so as to focus and localize cellular defense mechanisms to the infected cell. Bispecific antibodies may also be used to localize cytotoxic agents to infected cells. These antibodies possess a pathogen-binding arm and an arm that binds the cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope 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. A bispecific anti-ErbB2/Fc alpha antibody is shown in WO98/02463. U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.


Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).


According to a different approach, antibody variable regions with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain bonding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant effect on the yield of the desired chain combination.


In a particular embodiment of this approach, the bispecific antibodies are 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. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986). 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 that are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.


Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.


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) describe a procedure wherein 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 Fab′ fragments generated 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 is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.


Techniques exist that facilitate the direct recovery of Fab′-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a humanized bispecific antibody F(ab)2 molecule. Each Fab′ fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.


Various techniques for making 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. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker that is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, 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).


In a particular embodiment, a bispecific or multispecific antibody may be formed as a DOCK-AND-LOCK™ (DNL™) complex (see. e.g., U.S. Pat. No. 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.) Generally, the technique takes advantage of the specific and high-affinity binding interactions that occur between a dimerization and docking domain (DDD) sequence of the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequence derived 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 may be attached to any protein, peptide or other molecule. Because the DDD sequences spontaneously dimerize and bind to the AD sequence, the technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences.


Antibodies with more than two 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). A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present disclosure can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fe region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable regions. For instance, the polypeptide chain(s) 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 an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or 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, for instance, comprise from about two to about eight light chain variable region polypeptides. The light chain variable region polypeptides contemplated here comprise a light chain variable region and, optionally, further comprise a CL domain.


Charge modifications are particularly useful in the context of a multispecific antibody, where amino acid substitutions in Fab molecules result in reducing the mispairing of light chains with non-matching heavy chains (Bence-Jones-type side products), which can occur in the production of Fab-based bi-/multispecific antigen binding molecules with a VH/VL exchange in one (or more, in case of molecules comprising more than two antigen-binding Fab molecules) of their binding arms (see also PCI publication no. WO 2015/150447, particularly the examples therein, incorporated herein by reference in its entirety).


Accordingly, in particular embodiments, an antibody comprised in the therapeutic agent comprises

    • (a) a first Fab molecule which specifically binds to a first antigen
    • (b) a second Fab molecule which specifically binds to a second antigen, and wherein the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced by each other,
    • wherein the first antigen is an activating 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 activating T cell antigen; and
    • wherein
    • 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 (numbering according to Kabat), and wherein 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 (numbering 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 (numbering according to Kabat), and wherein 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 (numbering according to Kabat EU index).


      The antibody may not comprise both 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., remain unexchanged).


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 substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)), 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 independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).


In a further embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).


In a particular embodiment, in the constant domain CL of the first Fab molecule under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)) and the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in one preferred embodiment independently by lysine (K) or arginine (R)), and in the constant domain CH1 of the first Fab molecule under a) the amino acid at position 147 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index) and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to Kabat EU index).


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 CH1 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).


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 lysine (K) (numbering according to Kabat) and the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat), and in the constant domain CH1 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).


F. Chimeric Antigen Receptors

Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs)) are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell, with transfer of their coding sequence facilitated by retroviral vectors. In this way, a large number of target-specific T cells can be generated for adoptive cell transfer. Phase I clinical studies of this approach show efficacy.


The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta transmembrane and endodomain. Such molecules result in the transmission of a zeta signal in response to recognition by the scFv of its target. An example of such a construct is 14g2a-Zeta, which is a fusion of a scFv derived from hybridoma 14g2a (which recognizes disialoganglioside GD2). When T cells express this molecule (usually achieved by oncoretroviral vector transduction), they recognize and kill target cells that express GD2 (e.g., neuroblastoma cells). To target malignant B cells, investigators have redirected the specificity of T cells using a chimeric immunoreceptor specific for the B-lineage molecule, CD19.


The variable portions of an immunoglobulin heavy and light chain are fused by a flexible linker to form a scFv. This scFv is preceded by a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent surface expression (this is cleaved). A flexible spacer allows to the scFv to orient in different directions to enable antigen binding. The transmembrane domain is a typical hydrophobic alpha helix usually derived from the original molecule of the signaling endodomain which protrudes into the cell and transmits the desired signal.


Type I proteins are in fact two protein domains linked by a transmembrane alpha helix in between. The cell membrane lipid bilayer, through which the transmembrane domain passes, acts to isolate the inside portion (endodomain) from the external portion (ectodomain). It is not so surprising that attaching an ectodomain from one protein to an endodomain of another protein results in a molecule that combines the recognition of the former to the signal of the latter.


Ectodomain. A signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if the receptor is to be glycosylated and anchored in the cell membrane. Any eukaryotic signal peptide sequence usually works fine. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g., in a scFv with orientation light chain-linker-heavy chain, the native signal of the light-chain is used


The antigen recognition domain is usually an scFv. There are however many alternatives. An antigen recognition domain from native T-cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g., CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor). In fact, almost anything that binds a given target with high affinity can be used as an antigen recognition region.


A spacer region links the antigen binding domain to the transmembrane domain. It should 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. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For most scFv based constructs, the IgG1 hinge suffices. However, the best spacer often has to be determined empirically.


Transmembrane domain. The transmembrane domain is a hydrophobic alpha helix that spans the membrane. Generally, the transmembrane domain from the most membrane proximal component of the endodomain is used. Interestingly, using the CD3-zeta transmembrane domain may result in incorporation of the artificial TCR into the native TCR a factor that is dependent on the presence of the native CD3-zeta transmembrane charged aspartic acid residue. Different transmembrane domains result in different receptor stability. The CD28 transmembrane domain results in a brightly expressed, stable receptor.


Endodomain. This is the “business-end” of the receptor. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used endodomain component is CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed.


“First-generation” CARs typically had the intracellular domain from the CD3 ξ-chain, which is the primary transmitter of signals from endogenous TCRs. “Second-generation” CARs add intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells. More recent, “third-generation” CARs combine multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to further augment potency.


G. ADCs

Antibody Drug Conjugates or ADCs are a new class of highly potent biopharmaceutical drugs designed as a targeted therapy for the treatment of people with infectious disease. ADCs are complex molecules composed of an antibody (a whole mAb or an antibody fragment such as a single-chain variable fragment, or scFv) linked, via a stable chemical linker with labile bonds, to a biological active cytotoxic/anti-viral payload or drug. Antibody Drug Conjugates are examples of bioconjugates and immunoconjugates.


By combining the unique targeting capabilities of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, antibody-drug conjugates allow sensitive discrimination between healthy and diseased tissue. This means that, in contrast to traditional systemic approaches, antibody-drug conjugates target and attack the infected cell so that healthy cells are less severely affected.


In the development ADC-based anti-tumor therapies, an anticancer drug (e.g., a cell toxin or cytotoxin) is coupled to an antibody that specifically targets a certain cell marker (e.g., a protein that, ideally, is only to be found in or on infected cells). Antibodies track these proteins down in the body and attach themselves to the surface of cancer cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or internalizes the antibody together with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released and kills the cell or impairs viral replication. Due to this targeting, ideally the drug has lower side effects and gives a wider therapeutic window than other agents.


A stable link between the antibody and cytotoxic/anti-viral agent is a crucial aspect of an ADC. Linkers are based on chemical motifs including disulfides, hydrazones or peptides (cleavable), or thioethers (noncleavable) and control the distribution and delivery of the cytotoxic agent to the target cell. Cleavable and noncleavable types of linkers have been proven to be safe in preclinical and clinical trials. Brentuximab vedotin includes an enzyme-sensitive cleavable linker that delivers the potent and highly toxic antimicrotubule agent Monomethyl auristatin E or MMAE, a synthetic antineoplastic agent, to human specific CD30-positive malignant cells. Because of its high toxicity MMAE, which inhibits cell division by blocking the polymerization of tubulin, cannot be used as a single-agent chemotherapeutic drug. However, the combination of MMAE linked to an anti-CD30 monoclonal antibody (cAC10, a cell membrane protein of the tumor necrosis factor or TNF receptor) proved to be stable in extracellular fluid, cleavable by cathepsin and safe for therapy. Trastuzumab emtansine, the other approved ADC, is a combination of the microtubule-formation inhibitor mertansine (DM-1), a derivative of the Maytansine, and antibody trastuzumab (Herceptin®/Genentech/Roche) attached by a stable, non-cleavable linker.


The availability of better and more stable linkers has changed the function of the chemical bond. The type of linker, cleavable or noncleavable, lends specific properties to the cytotoxic (anti-cancer) drug. For example, a non-cleavable linker keeps the drug within the cell. As a result, the entire antibody, linker and cytotoxic agent enter the targeted cancer cell where the antibody is degraded to the level of an amino acid. The resulting complex—amino acid, linker and cytotoxic agent—now becomes the active drug. In contrast, cleavable linkers are catalyzed by enzymes in the host cell where it releases the cytotoxic agent.


Another type of cleavable linker, currently in development, adds an extra molecule between the cytotoxic/anti-viral drug and the cleavage site. This linker technology allows researchers to create ADCs with more flexibility without worrying about changing cleavage kinetics. Researchers are also developing a new method of peptide cleavage based on Edman degradation, a method of sequencing amino acids in a peptide. Future direction in the development of ADCs also include the development of site-specific conjugation (TDCs) to further improve stability and therapeutic index and a emitting immunoconjugates and antibody-conjugated nanoparticles.


H. BiTES

Bi-specific T-cell engagers (BiTEs) are a class of artificial bispecific monoclonal antibodies that are investigated for the use as anti-cancer drugs. They direct a host's immune system, more specifically the T cells' cytotoxic activity, against infected cells. BiTE is a registered trademark of Micromet AG.


BiTEs are fusion proteins consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, and the other to an infected cell via a specific molecule.


Like other bispecific antibodies, and unlike ordinary monoclonal antibodies, BiTEs form a link between T cells and target cells. This causes T cells to exert cytotoxic/anti-viral activity on infected cells by producing proteins like perforin and granzymes, independently of the presence of MHC I or co-stimulatory molecules. These proteins enter infected cells and initiate the cell's apoptosis. This action mimics physiological processes observed during T cell attacks against infected cells.


I. Intrabodies

In a particular embodiment, the antibody is a recombinant antibody that is suitable for action inside of a cell—such antibodies are known as “intrabodies.” These antibodies may interfere with target function by a variety of mechanism, such as by altering intracellular protein trafficking, interfering with enzymatic function, and blocking protein-protein or protein-DNA interactions. In many ways, their structures mimic or parallel those of single chain and single domain antibodies, discussed above. Indeed, single-transcript/single-chain is an important feature that permits intracellular expression in a target cell, and also makes protein transit across cell membranes more feasible. However, additional features are required.


The two major issues impacting the implementation of intrabody therapeutic are delivery, including cell/tissue targeting, and stability. With respect to delivery, a variety of approaches have been employed, such as tissue-directed delivery, use of cell-type specific promoters, viral-based delivery and use of cell-permeability/membrane translocating peptides. With respect to the stability, the approach is generally to either screen by brute force, including methods that involve phage display and may include sequence maturation or development of consensus sequences, or more directed modifications such as insertion stabilizing sequences (e.g., Fc regions, chaperone protein sequences, leucine zippers) and disulfide replacement/modification.


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


By virtue of their ability to enter cells, intrabodies have additional uses that other types of antibodies may not achieve. In the case of the present antibodies, the ability to interact with the MUC1 cytoplasmic domain in a living cell may interfere with functions associated with the MUC1 CD, such as signaling functions (binding to other molecules) or oligomer formation. In particular, it is contemplated that such antibodies can be used to inhibit MUC1 dimer formation.


J. Purification

In certain embodiments, the antibodies of the present disclosure may be purified. The term “purified,” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.


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 to polypeptide and non-polypeptide fractions. Having separated the polypeptide 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 a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.


In purifying an antibody 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 may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.


Commonly, complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody. Alternatively, antigens may be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter or bead. The antibodies are bound to a support, contaminants removed (e.g., washed away), and the antibodies released by applying conditions (salt, heat, etc.).


Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides 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, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.


It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.


III. Active/Passive Immunization and Treatment/Prevention of SARS-CoV-2 Infection
A. Formulation and Administration

The present disclosure provides pharmaceutical compositions comprising anti-SARS-CoV-2 virus antibodies and antigens for generating the same. Such compositions comprise a prophylactically or therapeutically effective amount of an antibody or a fragment thereof, or a peptide immunogen, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the 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 solutions and aqueous dextrose and glycerol solutions can also be employed 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.


The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, 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 saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.


Active vaccines are also envisioned where antibodies like those disclosed are produced in vivo in a subject at risk of SARS-CoV-2 infection. Such vaccines can be formulated for parenteral administration. e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Administration by intradermal and intramuscular routes are contemplated. The vaccine could alternatively be administered by a topical route directly to the mucosa, for example, by nasal drops, inhalation, by nebulizer, or via intrarectal or vaginal delivery. Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.


Passive transfer of antibodies, known as artificially acquired passive immunity, generally will involve the use of intravenous or intramuscular injections. The forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (MAb). Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin. However, passive immunity provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e., sterile and syringeable.


Generally, the ingredients of compositions of the disclosure are supplied either 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 sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.


The compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.


2. ADCC


Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or fragments thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. By “antibody having increased/reduced antibody dependent cell-mediated cytotoxicity (ADCC)” is meant an antibody having increased/reduced ADCC as determined by any suitable method known to those of ordinary skill in the art.


As used herein, the term “increased/reduced ADCC” is defined as either an increase/reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and/or a reduction/increase in the concentration of antibody, in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The increase/reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example, the increase in ADCC mediated by an antibody produced by host cells engineered to have an altered pattern of glycosylation (e.g., to express the glycosyltransferase. GnTIII, or other glycosyltransferases) by the methods described herein, is relative to the ADCC mediated by the same antibody produced by the same type of non-engineered host cells.


3. CDC


Complement-dependent cytotoxicity (CDC) is a function of the complement system. It is the processes in the immune system that kill pathogens by damaging their membranes without the involvement of antibodies or cells of the immune system. There are three main processes. All three insert one or more membrane attack complexes (MAC) into the pathogen which cause lethal colloid-osmotic swelling, i.e., CDC. It is one of the mechanisms by which antibodies or antibody fragments have an anti-viral effect.


IV. Antibody Conjugates

Antibodies of the present disclosure may be linked to at least one agent to form an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radionuclides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or polynucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.


Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and those for use in vivo diagnostic protocols, generally known as “antibody-directed imaging.” Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236, 4,938,948, and 4,472,509). The imaging moieties used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.


In the case of paramagnetic ions, one might mention by way of example 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 especially bismuth (III).


In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine211, 14carbon, 51chromium, 36chlorine, 57cobalt, 58cobalt, copper67, 152Eu, gallium67, 3hydrogen, iodine123, iodine125, iodine131, indium111, 59iron, 2phosphorus, rhenium186, rheniumi188, 75selenium, 35sulphur, technicium99m and/or yttrium90. 125I is often being preferred for use in certain embodiments, and technicium99m and/or indium111 are also often preferred due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present disclosure may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium 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 disclosure may be labeled with technetium99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNCl2, a buffer solution such as sodium-potassium phthalate solution, and the antibody. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetracetic acid (EDTA).


Among the fluorescent labels contemplated for use as conjugates include 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.


Additional types of antibodies contemplated in the present disclosure are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with 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 of skill in the art and are described, for example, in 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.


Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.


Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989; Dholakia et al., 1989) and may be used as antibody binding agents.


Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S. Pat. Nos. 4,472,509 and 4,938,948). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.


In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are 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, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.


V. Immunodetection Methods

In still further embodiments, the present disclosure concerns immunodetection methods for binding, purifying, removing, quantifying and otherwise generally detecting SARS-CoV-2 and its associated antigens. While such methods can be applied in a traditional sense, another use will be in quality control and monitoring of vaccine and other virus stocks, where antibodies according to the present disclosure can be used to assess the amount or integrity (i.e., long term stability) of antigens in viruses. Alternatively, the methods may be used to screen various antibodies for appropriate/desired reactivity profiles.


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, but specifically those that would be used to detect SARS-CoV-2 in a fluid 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). The assays may be advantageously formatted for non-healthcare (home) use, including lateral flow assays (see below) analogous to home pregnancy tests. These assays may be packaged in the form of a kit with appropriate reagents and instructions to permit use by the subject of a family member.


Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. In particular, a competitive assay for the detection and quantitation of SARS-CoV-2 antibodies directed to specific parasite epitopes in samples also is provided. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). In general, the immunobinding methods include obtaining a sample suspected of containing SARS-CoV-2, and contacting the sample with a first antibody in accordance with the present disclosure, as the case may be, under conditions effective to allow the formation of immunocomplexes.


These methods include methods for purifying SARS-CoV-2 or related antigens from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the SARS-CoV-2 or antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the SARS-CoV-2 antigen immunocomplexed to the immobilized antibody, which is then collected by removing the organism or antigen from the column.


The immunobinding methods also include methods for detecting and quantifying the amount of SARS-CoV-2 or related components in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing SARS-CoV-2 or its antigens and contact the sample with an antibody that binds SARS-CoV-2 or components thereof, followed by detecting and quantifying the amount of immune complexes formed under the specific conditions. In terms of antigen detection, the biological sample analyzed may be any sample that is suspected of containing SARS-CoV-2 or SARS-CoV-2 antigen, such as a tissue section or specimen, a homogenized tissue extract, a biological fluid, including blood and serum, or a secretion, such as feces or urine.


Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to SARS-CoV-2 or antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.


In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. Patents concerning 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, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.


The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.


Further methods include the detection of primary immune complexes by a two-step approach. A second binding ligand, such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.


One method of immunodetection uses two different antibodies. A first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin. In that 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 to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites 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 the second step antibody against biotin. This second step antibody is labeled, for example, with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.


Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.


A. ELISAs

Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.


In one exemplary ELISA, the antibodies of the disclosure are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the SARS-CoV-2 or SARS-CoV-2 antigen is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-SARS-CoV-2 antibody that is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection may also be achieved by the addition of a second anti-SARS-CoV-2 antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.


In another exemplary ELISA, the samples suspected of containing the SARS-CoV-2 or SARS-CoV-2 antigen are immobilized onto the well surface and then contacted with the anti-SARS-CoV-2 antibodies of the disclosure. After binding and washing to remove non-specifically bound immune complexes, the bound anti-SARS-CoV-2 antibodies are detected. Where the initial anti-SARS-CoV-2 antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-SARS-CoV-2 antibody, with the second antibody being linked to a detectable label.


Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.


In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be 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 regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.


In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.


“Under conditions effective to allow immune complex (antigen/antibody) formation” means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.


The “suitable” conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25° C. to 27° C., or may be overnight at about 4° C. or so.


Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween. or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.


To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact or incubate the first and second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween). After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified. e.g., by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated. e.g., using a visible spectra spectrophotometer.


In another embodiment, the present disclosure contemplates the use of competitive formats. This is particularly useful in the detection of SARS-CoV-2 antibodies in sample. In competition-based assays, an unknown amount of analyte or antibody is determined by its ability to displace a known amount of labeled antibody or analyte. Thus, the quantifiable loss of a signal is an indication of the amount of unknown antibody or analyte in a sample. Here, the inventor proposes the use of labeled SARS-CoV-2 monoclonal antibodies to determine the amount of SARS-CoV-2 antibodies in a sample. The basic format would include contacting a known amount of SARS-CoV-2 monoclonal antibody (linked to a detectable label) with SARS-CoV-2 antigen or particle. The SARS-CoV-2 antigen or organism is preferably attached to a support. After binding of the labeled monoclonal antibody to the support, the sample is added and incubated under conditions permitting any unlabeled antibody in the sample to compete with, and hence displace, the labeled monoclonal antibody. By measuring either the lost label or the label remaining (and subtracting that from the original amount of bound label), one can determine how much non-labeled antibody is bound to the support, and thus how much antibody was present in the sample.


B. Western Blot

The Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.


Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.


The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.


In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies.


C. Lateral Flow Assays

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 sample (matrix) without the need for specialized and costly equipment, though many laboratory-based applications exist that are supported by reading equipment. Typically, these tests are used as low resources medical diagnostics, either for home testing, point of care testing, or laboratory use. A widely spread and well-known application is the home pregnancy test.


The technology is based on a series of capillary beds, such as pieces of porous paper or sintered polymer. Each of these elements has the capacity to transport fluid (e.g., urine) spontaneously. The first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) in which the manufacturer has stored the so-called conjugate, a dried format of bio-active particles (see below) in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the target molecule (e.g., an antigen) and its chemical partner (e.g., antibody) that has been immobilized on the particle's surface. While the sample fluid dissolves the salt-sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, the analyte binds to the particles while migrating further through the third capillary bed. This material has one or more areas (often called stripes) where a third molecule has been immobilized by the manufacturer. By the time the sample-conjugate mix reaches these strips, analyte has been bound on the particle and the third ‘capture’ molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color. Typically there are at least two stripes: one (the control) that captures any particle and thereby shows that reaction conditions and technology worked fine, the second contains a specific capture molecule and only captures those particles onto which an analyte molecule has been immobilized. After passing these reaction zones, the fluid enters the final porous material—the wick—that simply acts as a waste container. Lateral Flow Tests can operate as either competitive or sandwich assays. Lateral flow assays are disclosed in U.S. Pat. No. 6,485,982.


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 study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors and is well known to those of skill in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).


Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozen “pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in −70° C. isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections from the capsule. Alternatively, whole frozen tissue samples may be used for serial section cuttings.


Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden 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 may be substituted.


E. Immunodetection Kits

In still further embodiments, the present disclosure concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies may be used to detect SARS-CoV-2 or SARS-CoV-2 antigens, the antibodies may be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to SARS-CoV-2 or SARS-CoV-2 antigen, and optionally an immunodetection reagent.


In certain embodiments, the SARS-CoV-2 antibody may be pre-bound to a solid support, such as a column matrix and/or well of a microtiter plate. The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.


Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present disclosure.


The kits may further comprise a suitably aliquoted composition of the SARS-CoV-2 or SARS-CoV-2 antigens, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form.


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 are retained.


F. Vaccine and Antigen Quality Control Assays

The present disclosure also contemplates the use of antibodies and antibody fragments as described herein for use in assessing the antigenic integrity of a viral antigen in a sample. Biological medicinal products like vaccines differ from chemical drugs in that they cannot normally be characterized molecularly; antibodies are large molecules of significant complexity and have the capacity to vary widely from preparation to preparation. They are also administered to healthy individuals, including children at the start of their lives, and thus a strong emphasis must be placed on their quality to ensure, to the greatest extent possible, that they are efficacious in preventing or treating life-threatening disease, without themselves causing harm.


The increasing globalization in the production and distribution of vaccines has opened new possibilities to better manage public health concerns but has also raised questions about the equivalence and interchangeability of vaccines procured across a variety of sources. International standardization of starting materials, of production and quality control testing, and the setting of high expectations for regulatory oversight on the way these products are manufactured and used, have thus been the cornerstone for continued success. But it remains a field in constant change, and continuous technical advances in the field offer a promise of developing potent new weapons against the oldest public health threats, as well as new ones—malaria, pandemic influenza, and HIV, to name a few—but also put a great pressure on manufacturers, regulatory authorities, and the wider medical community to ensure that products continue to meet the highest standards of quality attainable.


Thus, one may obtain an antigen or vaccine from any source or at any point during a manufacturing process. The quality control processes may therefore begin with preparing a sample for an inmunoassay that identifies binding of an antibody or fragment disclosed herein to a viral antigen. Such immunoassays are disclosed elsewhere in this document, and any of these may be used to assess the structural/antigenic integrity of the antigen. Standards for finding the sample to contain acceptable amounts of antigenically correct and intact antigen may be established by regulatory agencies.


Another important embodiment where antigen integrity is assessed is in determining shelf-life and storage stability. Most medicines, including vaccines, can deteriorate over time. Therefore, it is critical to determine whether, over time, the degree to which an antigen, such as in a vaccine, degrades or destabilizes such that is it no longer antigenic and/or capable of generating an immune response when administered to a subject. Again, standards for finding the sample to contain acceptable amounts of antigenically intact antigen may be established by regulatory agencies.


In certain embodiments, viral antigens may contain more than one protective epitope. In these cases, it may prove useful to employ assays that look at the binding of more than one antibody, such as 2, 3, 4, 5 or even more antibodies. These antibodies bind to closely related epitopes, such that they are adjacent or even overlap each other. On the other hand, they may represent distinct epitopes from disparate parts of the antigen. By examining the integrity of multiple epitopes, a more complete picture of the antigen's overall integrity, and hence ability to generate a protective immune response, may be determined.


Antibodies and fragments thereof as described in the present disclosure may also be used in a kit for monitoring the efficacy of vaccination procedures by detecting the presence of protective SARS-CoV-2 antibodies. Antibodies, antibody fragment, or variants and derivatives thereof, as described in the present disclosure may also be used in a kit for monitoring vaccine manufacture with the desired immunogenicity.


G. Examples

The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate 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 disclosure.


Example 1—Materials and Methods for Example 2

Expression and purification of recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein. The DNA segments correspondent to the S protein RBD (residues 319-528) was sequence optimized for expression, synthesized, and cloned into the pTwist-CMV expression DNA plasmid downstream of the IL-2 signal peptide (MYRMQLLSCIALSLALVTNS) (Twist Bioscience). A three amino acid linker (GSG) and a His-tag were incorporated at the C-terminus of the expression constructs to facilitate protein purification. Expi293F cells were transfected transiently with the plasmid encoding RBD, and culture supernatants were harvested after 5 days. RBD was purified from the supernatants by nickel affinity chromatography with HisTrap Excel columns (GE Healthcare Life Sciences). For protein production used in crystallization trials, 5 μM kifunensine was included in the culture medium to produce RBD with high mannose glycans. The high mannose glycoproteins subsequently were treated with endoglycosidase F1 (Millipore) to obtain homogeneously deglycosylated RBD.


Isolation or generation of authentic SARS-CoV-2 viruses, including viruses with variant residues. The UK B.1.1.7-OXF isolate was obtained from a nasopharyngeal swab from an infected individual in Kent. England. The clinical studies to obtain specimens after written informed consent were approved by John Radcliffe Hospital in Oxford, U.K. The sample was diluted in DMEM with 2% FBS and passed through a 0.45 μm filter before adding to monolayers of Vero-hACE2-TMPRSS2 cells (a gift of A. Creanga and B. Graham). Two days later, supernatant was harvested to establish a passage zero (p0) stock. The 2019n-CoV/USA_WA1/2019 isolate of SARS-CoV-2 was obtained from the U.S. Centers for Disease Control (CDC) and passaged on Vero E6 cells. Individual point mutations in the spike gene (D614G and E484K/D614G) were introduced into an infectious cDNA clone of the 2019n-CoV/USA_WA1/2019 strain as described previously67. Nucleotide substitutions were introduced into a subclone puc57-CoV-2-F6 containing the spike gene of the SARS-CoV-2 wild-type infectious clone68. The full-length infectious cDNA clones of the variant SARS-CoV-2 viruses were assembled by in vitro ligation of seven contiguous cDNA fragments following the previously described protocol68. In vitro transcription then was performed to synthesize full-length genomic RNA. To recover the mutant viruses, the RNA transcripts were electroporated into Vero E6 cells. The viruses from the supernatant of cells were collected 40 h later and served as p0 stocks. All virus stocks were confirmed by sequencing.


Focus reduction neutralization test. Serial dilutions of mAbs or serum were incubated with 102 focus-forming units (FFU) of different strains or variants of SARS-CoV-2 for 1 h at 37° C. Antibody-virus complexes were added to Vero-hACE2-TMPRSS2 cell monolayer cultures in 96-well plates and incubated at 37° C. for 1 h. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. Plates were harvested 20 h later by removing overlays and fixed with 4% PFA in PBS for 20 min at room temperature. Plates were washed and sequentially incubated with an oligoclonal pool of anti-S mAbs and HRP-conjugated goat anti-human IgG in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies).


Multiple sequence alignments. The inventors searched for antibody variable gene sequences originating with the same features as those encoding COV2-2196 and retrieved the matching sequences from the repertoires of each individual examined. They searched for similar sequences in the publicly available large-scale antibody sequence repertoires for three healthy individuals and cord blood repertoires (deposited at SRP174305). The search parameters for the heavy chain were sequences with IGHV1-58 and IGHJ3 with the P99. D108, and F110 residues. Additionally, the search parameters for the light chain were sequences with Y92 and W98 residues. Sequences from a matching clonotype that belonged to each individual were aligned with either ClustalO (heavy chains) or with MUSCLE (light chains). Then, LOGOs plots of aligned sequences were generated using WebLogo.


Data and materials availability: The crystal structures reported in this paper have been deposited to the Protein Data Bank under the accession numbers 7L7D, 7L7E. Sequence Read Archive deposition for the aligned human antibody gene repertoire data set is deposited at the NCBI: PRJNA511481. All other data are available in the main text or the supplementary materials.


Example 2—Result and Discussion

Structural analysis of COV2-2196 in complex with RBD reveals how COV2-2196 recognizes the receptor-binding ridge on the RBD. One of the major contact residues, F486, situates at the center of the binding site, interacting extensively with the hydrophobic pocket (residue P99 of heavy chain and an “aromatic cage” formed by 5 aromatic side chains) between COV2-2196 heavy/light chains via a hydrophobic effect and van der Waals interactions. A hydrogen bond (H-bond) network, constructed with 4 direct Ab-Ag H-bonds and 16 water-mediated H-bonds, surround residue F486 and strengthen the Ab-Ag interaction. Importantly, for all residues except one (residue P99 of the heavy chain) that interact extensively with the epitope, they are encoded by germline sequences (IGHV1-58*01 and IGHJ3*02 for the heavy chain, IGKV3-20*01 and IGKJ1*01 for the light chain) (FIG. 1) or only their backbone atoms are involved in the Ab-Ag interactions, such as heavy chain N107 and G99 and light chain S94. The inventors noted another antibody in the literature, S2E12, that is encoded by the same IGHV/IGHJ and IGKV/IGKJ recombinations, with similar but most likely different IGHD genes to those of COV2-2196 (IGHD2-15 vs IGHD2-2)38. A comparison of the cryo-EM structure of S2E12 in complex with S protein (PDB 7K4N) suggests that the mAb S2E12 likely uses nearly identical Ab-Ag interactions as those of COV2-2196, although variations in conformations of interface residue side-chains can be seen. For example, for light chain residue Y92, the phenyl ring in the crystal structure is perpendicular to that ring in the EM structure as fitted.


The inventors searched genetic databases to determine if these structural features are present in additional SARS-CoV-2 mAbs isolated by others and found additional members of the clonotype (FIG. 1). Two other studies reported the same or a similar clonotype of antibodies isolated from multiple COVID-19 convalescent patients4,38, and one study found three antibodies with the same IGHV1-58 and IGKV3-20 pairing, without providing information on D or J gene usage39. All of these antibodies are reported to bind SARS-CoV-2 RBD avidly and to neutralize virus with high potency1,4,38,39. So far, there are only two atomic resolution structures of antibodies encoded by these VH(DH)JH and VK-JK recombinations available, the structure for COV2-2196 and that for S2E1238.


The inventors next determined whether they could identify potential precursors of this public clonotype in the antibody variable gene repertoires of circulating B cells from SARS-CoV-2-naïve individuals. The inventors searched for the V(D)J and VJ genes in previously described comprehensive repertoire datasets originating from 3 healthy human donors, without a history of SARS-CoV-2 infection, and in datasets from cord blood collected prior to the COVID-19 pandemico. A total of 386, 193, 47, or 7 heavy chain sequences for this SARS-CoV-2 reactive public clonotype was found in each donor or cord blood repertoire, respectively (FIG. 3A). Additionally, the inventors found 516,738 human antibody sequences with the same light chain V-J recombination (IGKV3-20-IGKJ1*01). A total of 103.534, 191,039, or 222,165 light chain sequences was found for this public clonotype in each donor respectively. Due to the large number of sequences, the top five abundant sequences were aligned from each donor. Multiple sequence alignments were generated for each donor's sequences using ClustalOmega, and logo plots were generated. The top 5 sequences with the same recombination event in each donor were identical, resulting in the same logo plots (FIGS. 3A-B).


The inventors noted that 8 of the 9 common residues important for binding in the antibody were encoded by germline gene sequences, and all were present all 14 members of the public clonotype listed here from four different antibody-discovery teams (FIG. 1).


Recently, viral variants with increased transmissibility and potential antigenic mutations have been reported in clinical isolates48-51. The inventors tested whether some of the variant residues in these rapidly emerging strains would abrogate the activity of these potently neutralizing antibodies. They tested a viral isolate from a nasal sample obtained at Oxford in the United Kingdom (a B.1.1.7 virus designated UK B.1.1.7-OXF), which contains B.1.1.7 lineage defining spike gene changes including the 69-70 and 144-145 deletions in the NTD, and substitutions at N501Y, A570D, D614G, and P681H49. The inventors also tested isogenic D614G and E484K variants in the WA-1 strain background (2019n-CoV/USA_WA1/2019, [WA-1]), all prepared as authentic SARS-CoV-2 viruses and used in focus reduction neutralization tests43. The E484K mutation was of special interest, since this residue is located within 4.5 Å of each of the mAbs in the complex of Fabs and RBD, albeit at the very periphery of the Fab footprints, is present in emerging lineages B.1.351 (501Y.V2)50 and P.1 (501Y.V3)51, and has been demonstrated to alter the binding of some monoclonal antibodies52,53 as well as human polyclonal serum antibodies54. Variants containing E484K also have been shown to be neutralized less efficiently by convalescent serum and plasma from SARS-CoV-2 survivors55,56. For COV2-2196, COV2-2130, and COV2-2050 (a third neutralizing antibody the inventors included for comparison as it interacts with the residue E484), they found virtually no impact of the D614G mutation or the suite of mutations present in the UK B.1.1.7-OXF strain; if anything, the inventors observed a trend toward slightly improved (2- to 3-fold reduction in IC50 values) against the latter circulating virus (FIG. 2). However, they did observe effects on neutralization with the D614G/E484K virus. COV2-2050 completely lost neutralization activity, consistent with our previous study defining E484K as a mutation abrogating COV2-2050 binding41.


Discussion. These structural analyses define the molecular basis for the frequent selection of a public clonotype of human antibodies sharing heavy chain V-D-J and light chain V-J recombinations that target the same region of the SARS-CoV-2 S RBD. Germline antibody gene-encoded residues in heavy and light chains play a vital role in antigen recognition, suggesting that few somatic mutations are required for antibody maturation of this clonotype. An IGHD2-gene-encoded disulfide bond provides additional restraint for the HCDR3 to adopt a conformation with shape and chemical complementarity to the antigenic site on RBD. It appears that three different IGHD2 genes (IGHD2-2, IGHD2-8, and IGHD2-15) encode portions of the HCDR3 that can function in the context of this clonotype. The inventors suggest that this occurrence of common germline gene-encoded antibodies with preconfigured structural features enabling high specificity and potent neutralizing activity is an unanticipated and beneficial feature of the primary human immune response to SARS-CoV-2. The selection of B cells from this public clonotype enabled rapid isolation of ultra-potent neutralizing antibodies that resist escape and possibly could account in part for the remarkable efficacy of S protein-based vaccines that is being observed in the clinic. One might envision an opportunity to elicit serum neutralizing antibody titers with even higher neutralization potency using domain- or motif-based vaccine designs for this antigenic site to prime human immune responses to elicit this clonotype.


The recent emergence of variant virus lineages with increased transmissibility and altered sequences in many known sites of neutralization is concerning for the capacity of SARS-CoV-2 to evade current antibody countermeasures in development and testing. The inventors tested the activity of the antibodies and the cocktail of both and found sustained activity against several important variants, including a virus containing the E484K mutation and a B.1.1.7 virus with multiple S gene variations. The genetic and structural basis for this broad activity is revealed in the crystal structures and DMS studies the inventors present here.


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Example 3—Materials and Methods for Example 4

Antibodies. The human antibodies studied in this paper were isolated from blood samples from two subjects in North America with previous laboratory-confirmed symptomatic SARS-CoV-2 infection that was acquired in China. The original clinical studies to obtain specimens after written informed consent were previously described1 and had been approved by the Institutional Review Board of Vanderbilt University Medical Center and the Research Ethics Board of the University of Toronto. The subjects (a 56-year-old male and a 56-year-old female) are a married couple and residents of Wuhan, China who traveled to Toronto, Canada, where PBMCs were obtained by leukopheresis 50 days after symptom onset. The antibodies were isolated using diverse tools for isolation and cloning of single antigen-specific B cells and the antibody variable genes encoding monoclonal antibodies1.


Cell culture. Vero E6 (CRL-1586, American Type Culture Collection (American Type Culture Collection, ATCC), Vero CCL81 (ATCC), HEK293 (ATCC), and HEK293T (ATCC) were maintained at 37° C. in 5% CO2 in Dulbecco's minimal essential medium (DMEM) containing 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1× non-essential amino acids, and 100 U/mL of penicillin-streptomycin. Vero-furin cells were obtained from T. Pierson (NIH) and have been described previously2. Expi293F cells (ThermoFisher Scientific. A1452) were maintained at 37° C. in 8% CO2 in Expi293F Expression Medium (ThermoFisher Scientific, A1435102). ExpiCHO cells (ThermoFisher Scientific, A29127) were maintained at 37° C. in 8% CO2 in ExpiCHO Expression Medium (ThermoFisher Scientific, A2910002). Mycoplasma testing of Expi293F and ExpiCHO cultures was performed on a monthly basis using a PCR-based mycoplasma detection kit (ATCC, 30-1012K).


Viruses. SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 was obtained from the Centers for Disease Control and Prevention (a gift from Natalie Thornburg). Virus was passaged in Vero CCL81 cells and titrated by plaque assay on Vero E6 cells. All work with infectious SARS-CoV-2 was approved by the Washington University School of Medicine or UNC-Chapel Hill Institutional Biosafety Committees and conducted in approved BSL3 facilities using appropriate powered air purifying respirators and personal protective equipment.


Recombinant antigens and proteins. A gene encoding the ectodomain of a prefusion conformation-stabilized SARS-CoV-2 spike (S2Pecto) protein was synthesized and cloned into a DNA plasmid expression vector for mammalian cells. A similarly designed S protein antigen with two prolines and removal of the furin cleavage site for stabilization of the prefusion form of S was reported previously3. Briefly, this gene includes the ectodomain of SARS-CoV-2 (to residue 1,208), a T4 fibritin trimerization domain, an AviTag site-specific biotinylation sequence, and a C-terminal 8×-His tag. To stabilize the construct in the prefusion conformation, the inventors included substitutions K986P and V987P and mutated the furin cleavage site at residues 682-685 from RRAR to ASVG. This recombinant spike 2P-stabilized protein (designated here as S2Pecto) was isolated by metal affinity chromatography on HisTrap Excel columns (GE Healthcare), and protein preparations were purified further by size-exclusion chromatography on a Superose 6 Increase 10/300 column (GE Healthcare). The presence of trimeric, prefusion conformation S protein was verified by negative-stain electron microscopy1. For electron microscopy with S and Fabs, the inventors expressed a variant of S2Pecto lacking an AviTag but containing a C-terminal Twin-Strep-tag, similar to that described previously3. Expressed protein was isolated by metal affinity chromatography on HisTrap Excel columns (GE Healthcare), followed by further purification on a StrepTrap HP column (GE Healthcare) and size-exclusion chromatography on TSKgel G4000SWXL (TOSOH). To express the SRBD subdomain of SARS-CoV-2 S protein, residues 319-541 were cloned into a mammalian expression vector downstream of an IL-2 signal peptide and upstream of a thrombin cleavage site, an AviTag, and a 6×-His tag. RBD protein fused to mouse IgG1 Fc domain (designated RBD-mFc), was purchased from Sino Biological (40592-V05H). For epitope mapping by alanine scanning, SARS-CoV-2 RBD (residues 334-526) or RBD single mutation variants were cloned with an N-terminal CD33 leader sequence and C-terminal GSSG linker, AviTag, GSSG linker, and 8×HisTag. Spike proteins were expressed in FreeStyle 293 cells (Thermo Fisher) and isolated by affinity chromatography using a HisTrap column (GE Healthcare), followed by size exclusion chromatography with a Superdex200 column (GE Healthcare). Purified proteins were analyzed by SDS-PAGE to ensure purity and appropriate molecular weights.


Electron microscopy (EM) stain grid preparation, imaging and processing of SARS-CoV-2 S2Pecto protein or S2Pecto/Fab complexes. To perform EM imaging, Fabs were produced by digesting recombinant chromatography-purified IgGs using resin-immobilized cysteine protease enzyme (FabALACTICA, Genovis). The digestion occurred in 100 mM sodium phosphate, 150 mM NaCl pH 7.2 for ˜16 hrs at RT. In order to remove cleaved Fe and intact IgG, the digestion mix was incubated with CaptureSelect Fc resin (Genovis) for 30 min at RT in PBS buffer. If needed, the Fab was buffer exchanged into Tris buffer by centrifugation with a Zeba spin column (Thermo Scientific).


For screening and imaging of negatively-stained (NS) SARS-CoV-2 S2Pecto protein in complex with human Fabs, the proteins were incubated for ˜1 hr and approximately 3 μL of the sample at concentrations of about 10 to 15 μg/mL was applied to a glow discharged grid with continuous carbon film on 400 square mesh copper EM grids (Electron Microscopy Sciences). The grids were stained with 0.75% uranyl formate (UF). Images were recorded on a Gatan US4000 4k×4k CCD camera using an FEI TF20 (TFS) transmission electron microscope operated at 200 keV and control with SerialEM5. All images were taken at 50,000× magnification with a pixel size of 2.18 Å/pix in low-dose mode at a defocus of 1.5 to 1.8 μm. Total dose for the micrographs was ˜25 to 38 e/Å2. Image processing was performed using the cryoSPARC software package6. Images were imported, and particles were CTF estimated. The images then were denoised and picked with Topaz7. The particles were extracted with a box size of 256 pixels and binned to 128 pixels. 2D class averages were performed and good classes selected for ab-initio model and refinement without symmetry. For EM model docking of SARS-CoV-2 S2Pecto protein, the closed model (PDB: 6VXX) was used in Chimera8 for docking to the EM map. All images were made with Chimera.


MAb production and purification. Sequences of mAbs that had been synthesized (Twist Bioscience) and cloned into an IgG1 monocistronic expression vector (designated as pTwist-mCis_G1) were used for mammalian cell culture mAb secretion. This vector contains an enhanced 2A sequence and GSG linker that allows simultaneous expression of mAb heavy and light chain genes from a single construct upon transfection9. The inventors previously described microscale expression of mAbs in 1 mL ExpiCHO cultures in 96-well plates1. For larger scale mAb expression, they performed transfection (1 to 300 mL per antibody) of CHO cell cultures using the Gibco™ ExpiCHO™ Expression System and protocol for 50 mL mini bioreactor tubes (Corning) as described by the vendor. Culture supernatants were purified using HiTrap MabSelect SuRe (Cytiva, formerly GE Healthcare Life Sciences) on a 24-column parallel protein chromatography system (Protein BioSolutions). 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.


ELISA binding assays. Wells of 96-well microtiter plates were coated with purified recombinant SARS-CoV-2 S protein or SARS-CoV-2 SRBD protein at 4° C. overnight. Plates were blocked with 2% non-fat dry milk and 2% normal goat serum in DPBS containing 0.05% Tween-20 (DPBS-T) for 1 hr. The bound antibodies were detected using goat anti-human IgG conjugated with HRP (horseradish peroxidase) (Southern Biotech) and TMB (3,3′,5,5′-tetramethylbenzidine) substrate (Thermo Fisher Scientific). Color development was monitored, 1N hydrochloric acid was added to stop the reaction, and the absorbance was measured at 450 nm using a spectrophotometer (Biotek). For dose-response assays, serial dilutions of purified mAbs were applied to the wells in triplicate, and mAb binding was detected as detailed above. Half-maximal effective concentration (EC50) values for binding were determined using Prism v8.0 software (GraphPad) after log transformation of mAb concentration using sigmoidal dose-response nonlinear regression analysis.


RBD minimal ACE2-binding motif peptide binding ELISA. Wells of 384-well microtiter plates were coated with 1 μg/mL streptavidin at 4° C. overnight. Plates were blocked with 0.5% BSA in DPBS containing 0.05% Tween-20 (DPBS-T) for 1 hr. Plates were washed 4× with 1×PBST and 2 μg/mL biotinylated-ACE2 binding motif peptide (cat. #LT5578, from LifeTein, LLC) was added to bind streptavidin for 1 hr at RT. Purified mAbs were diluted in blocking buffer, added to the wells, and incubated for 1 hr at RT. The bound antibodies were detected using goat anti-human IgG conjugated with HRP (horseradish peroxidase) (cat. #2014-05. Southern Biotech) and TMB (3,3′,5,5′-tetramethylbenzidine) substrate (ThermoFisher Scientific). Color development was monitored, 1N hydrochloric acid was added to stop the reaction, and the absorbance was measured at 450 nm using a spectrophotometer (Biotek). For dose-response assays, serial 3-fold dilutions starting at 10 μg/mL concentration of purified mAbs were applied to the wells in triplicate, and mAb binding was detected as detailed above.


Analysis of binding of antibodies to variant RBD proteins with alanine or arginine point mutations. Biolayer light interferometry (BLI) was performed using an Octet RED96 instrument (ForteBio; Pall Life Sciences) and wild-type RBD protein or a mutant RBD protein with a single amino acid change at defined positions to alanine or arginine. Binding of the RBD proteins were confirmed by first capturing octa-His-tagged RBD wild-type or mutant protein from a 10 μg/mL (≈200 nM) solution onto Penta-His biosensors for 300 sec. The biosensor tips then were submerged in binding buffer (PBS/0.2% Tween 20) for a 60 see wash, followed by immersion in a solution containing 150 nM of mAb for 180 sec (association), followed by a subsequent immersion in binding buffer for 180 sec (dissociation). Response for each RBD mutant protein was normalized to that of the wild-type RBD protein.


Focus reduction neutralization test (FRNT). Serial dilutions of mAbs were incubated with 102 FFU of SARS-CoV-2 for 1 hr at 37° C. The mAb-virus complexes were added to Vero E6 cell culture monolayers in 96-well plates for 1 hr at 37° C. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in Minimum Essential Medium (MEM) supplemented to contain 2% heat-inactivated FBS. Plates were fixed 30 hrs later by removing overlays and fixed with 4% PFA in PBS for 20 min at room temperature. The plates were incubated sequentially with 1 μg/mL of rCR3022 anti-S antibody10 and horseradish-peroxidase (HRP)-conjugated goat anti-human IgG in PBS supplemented with 0.1% (w/v) saponin (Sigma) and 0.1% bovine serum albumin (BSA). SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot 5.0.37 Macro Analyzer (Cellular Technologies). Data were processed using Prism software version 8.0 (GraphPad).


Generation of S protein pseudotyped lentivirus. Suspension 293 cells were seeded and transfected with a third-generation HIV-based lentiviral vector expressing luciferase along with packaging plasmids encoding for the following: SARS-CoV-2 spike protein with a C-terminal 19 amino acid deletion, Rev, and Gag-pol. Medium was changed 16 to 20 hrs after transfection, and the supernatant containing virus was harvested 24 hrs later. Cell debris was removed by low-speed centrifugation, and the supernatant was passed through a 0.45 μm filter unit. The pseudovirus was pelleted by ultracentrifugation and resuspended in PBS for a 100-fold concentrated stock.


Pseudovirus neutralization assay. Serial dilutions of mAbs were prepared in a 384-well microtiter plate and pre-incubated with pseudovirus for 30 minutes at 37° C., to which 293 cells that stably express human ACE2 were added. The plate was returned to the 37° C. incubator, and then 48 hrs later luciferase activity measured on an EnVision 2105 Multimode Plate Reader (Perkin Elmer) using the Bright-Glo™ Luciferase Assay System (Promega), according to manufacturer's recommendations. Percent inhibition was calculated relative to pseudovirus-alone control. IC50 values were determined by nonlinear regression using the Prism software version 8.1.0 (GraphPad). The average IC50 value for each antibody was determined from a minimum of 3 independent experiments.


MAb quantification. Quantification of purified mAbs was performed by UV spectrophotometry using a NanoDrop spectrophotometer and accounting for the extinction coefficient of human IgG.


Competition-binding analysis through biolayer interferometry. Anti-mouse IgG Fc capture biosensors (FortéBio 18-5089) on an Octet HTX biolayer interferometry instrument (FortéBio) were soaked for 10 minutes in 1× kinetics buffer (Molecular Devices 18-1105), followed by a baseline signal measurement for 60 seconds. Recombinant SARS-CoV-2 RBD fused to mouse IgG1 (RBD-mFc, Sino Biological 40592-V05H) was immobilized onto the biosensor tips for 180 seconds. After a wash step in 1× kinetics buffer for 30 seconds, the reference antibody (5 μg/mL) was incubated with the antigen-containing biosensor for 600 seconds. Reference antibodies included the SARS-CoV human mAb CR3022 and COV2-2196. After a wash step in 1× kinetics buffer for 30 seconds, the biosensor tips then were immersed into the second antibody (5 μg/mL) for 300 seconds. Maximal binding of each antibody was normalized to a buffer-only control. Self-to-self blocking was subtracted. Comparison between the maximal signal of each antibody was used to determine the percent binding of each antibody. A reduction in maximum signal to <33% of un-competed signal was considered full competition of binding for the second antibody in the presence of the reference antibody. A reduction in maximum signal to between 33 to 67% of un-competed was considered intermediate competition of binding for the second antibody in the presence of the reference antibody. Percent binding of the maximum signal >67% was considered absence of competition of binding for the second antibody in the presence of the reference antibody.


High-throughput ACE-2 binding inhibition analysis. Wells of 384-well microtiter plates were coated with purified recombinant SARS-CoV-2 S2Pecto protein at 4° C. overnight. Plates were blocked with 2% non-fat dry milk and 2% normal goat serum in DPBS-T for 1 hr. Purified mAbs from microscale expression were diluted two-fold in blocking buffer starting from 10 μg/mL in triplicate, added to the wells (20 μL/well), and incubated for 1 hr at ambient temperature. Recombinant human ACE2 with a C-terminal FLAG tag protein was added to wells at 2 μg/mL in a 5 L/well volume (final 0.4 μg/mL concentration of ACE2) without washing of antibody and then incubated for 40 min at ambient temperature. Plates were washed, and bound ACE2 was detected using HRP-conjugated anti-FLAG antibody (Sigma) and TMB substrate. ACE2 binding without antibody served as a control. The signal obtained for binding of the ACE2 in the presence of each dilution of tested antibody was expressed as a percentage of the ACE2 binding without antibody after subtracting the background signal. Half-maximal inhibitory concentration (IC50) values for inhibition by mAb of S2Pecto, protein binding to ACE2 was determined after log transformation of antibody concentration using sigmoidal dose-response nonlinear regression analysis (Prism software, GraphPad Prism version 8.0). ACE2 blocking assay using biolayer interferometry biosensor. Anti-mouse IgG biosensors on an Octet HTX biolayer interferometry instrument (FortéBio) were soaked for 10 minutes in 1× kinetics buffer, followed by a baseline signal measurement for 60 seconds. Recombinant SARS-CoV-2 RBD fused to mouse IgG1 (RBD-mFc, Sino Biological 40592-V05H) was immobilized onto the biosensor tips for 180 seconds. After a wash step in Ix kinetics buffer for 30 seconds, the antibody (5 μg/mL) was incubated with the antigen-coated biosensor for 600 seconds. After a wash step in 1× kinetics buffer for 30 seconds, the biosensor tips then were immersed into the ACE2 receptor (20 μg/mL) (Sigma-Aldrich SAE0064) for 300 seconds. Maximal binding of ACE2 was normalized to a buffer-only control. Percent binding of ACE2 in the presence of antibody was compared to ACE2 maximal binding. A reduction in maximal signal to <30% was considered ACE2 blocking.


High-throughput competition-binding analysis. Wells of 384-well microtiter plates were coated with purified recombinant SARS-CoV-2 S protein at 4° C. overnight. Plates were blocked with 2% BSA in DPBS containing 0.05% Tween-20 (DPBS-T) for 1 hr. Micro-scale purified unlabeled mAbs were diluted ten-fold in blocking buffer, added to the wells (20 μL/well) in quadruplicates, and incubated for 1 hr at ambient temperature. A biotinylated preparation of a recombinant mAb based on the variable gene sequence of the previously described mAb CR302212 and also newly identified mAbs COV2-2096, -2130, and -2196 that recognized distinct antigenic regions of the SARS-CoV-2 S protein were added to each of four wells with the respective mAb at 2.5 μg/mL in a 5 μL/well volume (final 0.5 μg/mL concentration of biotinylated mAb) without washing of unlabeled antibody and then incubated for 1 hr at ambient temperature. Plates were washed, and bound antibodies were detected using HRP-conjugated avidin (Sigma) and TMB substrate. The signal obtained for binding of the biotin-labeled reference antibody in the presence of the unlabeled tested antibody was expressed as a percentage of the binding of the reference antibody alone after subtracting the background signal. Tested mAbs were considered competing if their presence reduced the reference antibody binding to less than 41% of its maximal binding and non-competing if the signal was greater than 71%. A level of 40-70% was considered intermediate competition.


Binding analysis of mAbs to alanine or arginine RBD mutants. Biolayer light interferometry was performed using an Octet RED96 instrument (FortéBio; Pall Life Sciences). Binding was confirmed by first capturing octa-His-tagged RBD mutants 10 μg/mL (≈200 nM) onto Penta-His biosensors for 300 s. The biosensors then were submerged in binding buffer (PBS/0.2% TWEEN 20) for a wash for 60 see followed by immersion in a solution containing 150 nM of mAbs for 180 sec (association), followed by a subsequent immersion in binding buffer for 180 see (dissociation). Response for each RBD mutant was normalized to that of wild-type RBD.


Mouse experiments using human hACE2-transduced mice. Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381-01). Virus inoculations were performed under anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering.


BALB/c mice were purchased from Jackson Laboratories (strain 000651). Female mice (10-11-week-old) were given a single intraperitoneal injection of 2 mg of anti-Ifnar1 mAb (MAR1-5A33, Leinco) one day before intranasal administration of 2.5×108 PFU of AdV-hACE2. Five days after AdV transduction, mice were inoculated with 4×105 PFU of SARS-CoV-2 by the intranasal route. Anti-SARS-CoV-2 human mAbs or isotype control mAbs were administered 24 hours prior to SARS-CoV-2 inoculation. Weights were monitored on a daily basis, and animals were sacrificed at days 5 or 7 post-infection, and tissues were harvested.


Measurement of viral burden. Tissues were weighed and homogenized with zirconia beads in a MagNA Lyser instrument (Roche Life Science) in 1 ml of DMEM media supplemented with 2% heat-inactivated FBS. Tissue homogenates were clarified by centrifugation at 10,000 rpm for 5 min and stored at −80° C. RNA was extracted using MagMax mirVana Total RNA isolation kit (Thermo Scientific) and a Kingfisher Flex 96 well extraction machine (Thermo Scientific). TaqMan primers were designed to target a conserved region of the N gene using SARS-CoV-2 (MN908947) sequence as a guide (L Primer: ATGCTGCAATCGTGCTACAA (SEQ ID NO: 1); R primer: GACTGCCGCCTCTGCTC (SEQ ID NO: 2); probe: /56-FAM/TCAAGGAAC/ZEN/AACATTGCCAA/3IABkFQ/). To establish an RNA standard curve, the inventors generated concatenated segments of the N gene in a gBlocks fragment (IDT) and cloned this into the PCR-II topo vector (Invitrogen). The vector was linearized, and in vitro T7-DNA-dependent RNA transcription was performed to generate materials for a quantitative standard curve.


Cytokine and chemokine mRNA measurements. RNA was isolated from lung homogenates at 7 dpi as described above. cDNA was synthesized from DNase-treated RNA using the High-Capacity cDNA Reverse Transcription kit (Thermo Scientific) with the addition of RNase inhibitor, following the manufacturer's protocol. Cytokine and chemokine expression was determined using TaqMan Fast Universal PCR master mix (Thermo Scientific) with commercial primers/probe sets specific for IFNγ (IDT: Mm.PT.58.41769240), IL-6 (Mm.PT.58.10005566), CXCL10 (Mm.PT.58.43575827), CCL2 (Mm.PT.58.42151692 and results were normalized to GAPDH (Mm.PT.39a.1) levels. Fold change was determined using the 2−ΔΔCt method comparing anti-SARS-CoV-2 specific or isotype control mAb-treated mice to naïve controls.


Mouse experiments using wild-type mice. Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the UNC Chapel Hill School of Medicine (NIH/PHS Animal Welfare Assurance Number is D16-00256 (A3410-01)). Virus inoculations were performed under anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering.


Mouse adapted SARS-CoV-2 (MA-SARS-CoV-2) virus. The virus was generated as described previously14. Virus was propagated in Vero E6 cells grown in DMEM with 10% Fetal Clone II and 1% Pen/Strep. Virus titer was determined by plaque assay. Briefly, virus was serial diluted and inoculated onto confluent monolayers of Vero E6 cells, followed by agarose overlay. Plaques were visualized on day 2 post-infection after staining with neutral red dye.


Wild-type mice. 12-month-old BALB/c mice from Envigo were used in experiments. Mice were acclimated in the BSL3 for at least 72 hours prior to start of experiments. At 6 hours prior to infection, mice were prophylactically treated with 200 μg of human monoclonal antibodies via intraperitoneal injection. The next day, mice were anesthetized with a mixture of ketamine and xylazine and intranasally infected with 10 PFU of MA-SARS-CoV-2 diluted in PBS. Daily weight loss was measured, and at two days post-infection mice were euthanized by isoflurane overdose prior to tissue harvest.


Plaque assay of lung tissue homogenates. The lower lobe of the right lung was homogenized in 1 mL PBS using a MagnaLyser (Roche). Serial dilutions of virus were titered on Vero E6 cell culture monolayers, and virus plaques were visualized by neutral red staining at two days after inoculation. The limit of detection for the assay is 100 PFU per lung.


Quantification and statistical analysis. The descriptive statistics mean±SEM or mean±SD were determined for continuous variables as noted. Technical and biological replicates are described in the figure legends. In the mouse studies, analysis of weight change and viral burden in vivo were determined by two-way ANOVA and Mann-Whitney tests, respectively. Statistical analyses were performed using Prism v8.0 (GraphPad).


REFERENCES FOR EXAMPLE 3



  • 1. Zost S J, G. P., Chen R E, Case J B, Reidy J X, Trivette A, Nargi R S, Sutton R E, Suryadevara N, Chen E C, Binshtein E, Shrihari S, Ostrowski M, Chu H Y, Didier J E, MacRenaris K W, Jones T, Day S, Myers L, Lee F E-H, Nguyen D C, Sanz I, Martinez D R, Baric R S, Thackray L B, Diamond M S, Carnahan R H, Crowe J E Jr., Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein. bioRxiv 2020.05.12.091462; doi: https://doi.org/10.1101/2020.05.12.091462 (2020)

  • 2. Mukherjee, S. et al. Enhancing dengue virus maturation using a stable furin overexpressing cell line. Virology 497, 33-40, doi:10.1016/j.virol.2016.06.022 (2016).

  • 3. Wrapp, D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260-1263, doi:10.1126/science.abb2507 (2020).

  • 4. Ohi, M., Li, Y., Cheng. Y. & Walz, T. Negative staining and image classification—Powerful tools in modern electron microscopy. Biol Proced Online 6, 23-34, doi:10.1251/bpo70 (2004).

  • 5. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152, 36-51, doi:10.1016/j.jsb.2005.07.007 (2005).

  • 6. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14, 290-296, doi:10.1038/nmeth.4169 (2017).

  • 7. Bepler, T., Noble, A. J., and Berger, B. Topaz-Denoise: general deep denoising models for cryoEM. bioRxiv. doi:10.1101/838920 (2019).

  • 8. Pettersen. E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25, 1605-1612, doi:10.1002/jcc.20084 (2004).

  • 9. Chng, J. et al. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells. MAbs 7, 403-412, doi:10.1080/19420862.2015.1008351 (2015).

  • 10. Yuan. M. et al. A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV. Science 368, 630-633, doi:10.1126/science.abb7269 (2020).

  • 11. Ianevski, A., He. L., Aittokallio, T. & Tang, J. SynergyFinder: a web application for analyzing drug combination dose-response matrix data. Bioinformatics 33, 2413-2415, doi:10.1093/bioinformatics/btx162 (2017).

  • 12. ter Meulen, J. et al. Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets. Lancet 363, 2139-2141, doi:10.1016/S0140-6736(04)16506-9 (2004).

  • 13. Sheehan, K. C. et al. Blocking monoclonal antibodies specific for mouse IFN-alpha/beta receptor subunit 1 (IFNAR-1) from mice immunized by in vivo hydrodynamic transfection. J Interferon Cytokine Res 26, 804-819 (2006).

  • 14. Dinnon K H, et al. A mouse-adapted SARS-CoV-2 model for the evaluation of COVID-19 medical countermeasures. bioRxiv 2020.05.06.081497; doi: https://doi.org/10.1101/2020.05.06.081497 (2020).



Example 4—Results

The S protein of SARS-CoV-2 is the molecular determinant of viral attachment, fusion, and entry into host cells3. The cryo-EM structure of a prefusion-stabilized trimeric S protein ectodomain (S2Pecto) for SARS-CoV-2 reveals similar features to that of the SARS-CoV S protein4. This type I integral membrane protein and class I fusion protein possesses an N-terminal subunit (S1) that mediates binding to receptor and a C-terminal subunit (S2) that mediates virus-cell membrane fusion. The S1 subunit contains an N-terminal domain (SNTD) and a receptor-binding domain (SRBD). SARS-CoV-2 and SARS-CoV, which share approximately 78% sequence identity in their genomes1 both use human angiotensin-converting enzyme 2 (hACE2) as an entry receptor5-7. Previous studies of human immunity to other high-pathogenicity zoonotic betacoronaviruses including SARS-CoV8-12 and Middle East respiratory syndrome (MERS)13-22 showed that Abs to the viral surface spike (S) glycoprotein mediate protective immunity. The most potent S protein-specific mAbs appear to neutralize betacoronaviruses by blocking attachment of virus to host cells by binding to the region on SRBD that directly mediates engagement of the receptor. It is likely that human Abs have promise for use in modifying disease during SARS-CoV-2 infection, when used for prophylaxis, post-exposure prophylaxis, or treatment of SARS-CoV-2 infection23. Many studies including randomized controlled trials evaluating convalescent plasma and one trial evaluating hyperimmune immunoglobulin are ongoing, but it is not yet clear whether such treatments can reduce morbidity or mortality.


The inventors isolated a large panel of SARS-CoV-2 S protein-reactive mAbs from the B cells of two individuals who were previously infected with SARS-CoV-2 in Wuhan China25. A subset of those antibodies bound to the receptor-binding domain of S (SRBD) and exhibited neutralizing activity in a rapid screening assay with authentic SARS-CoV-225. Here, the inventors defined the antigenic landscape of SARS-CoV-2 and determined which sites of SRBD are the target of protective mAbs. They tested a panel of 40 anti-S human mAbs previously pre-selected by a rapid neutralization screening assay in a quantitative focus reduction neutralization test (FRNT) with SARS-CoV-2 strain WA1/2020. These assays revealed the panel exhibited a range of half-maximal inhibitory concentration (IC50) values, from 15 to over 4,000 ng/mL (visualized as a heatmap in FIG. 4A, values shown in Table B. and full curves shown in FIG. 6). The inventors hypothesized that many of these SRBD-reactive mAbs neutralize virus infection by blocking SRBD binding to hACE2. Indeed, most neutralizing mAbs that were tested inhibited the interaction of hACE2 with trimeric S protein directly (FIG. 4A, FIG. 7). Consistent with these results, these mAbs also bound strongly to a trimeric S ectodomain (S2Pecto) protein or monomeric SRBD (FIG. 4A, FIG. 8). The inventors evaluated whether S2Pecto or SRBD binding or hACE2-blocking potency predicted binding neutralization potency independently, but none of these measurements correlated with neutralization potency (FIGS. 4B-D). However, each of the mAbs in the highest neutralizing potency tier (IC50<150 ng/mL) also revealed strongest blocking activity against hACE2 (IC50<150 ng/mL) and exceptional binding activity (EC50<2 ng/mL) to S2Pecto trimer and SRBD (FIG. 4E).


The inventors next defined the major antigenic sites on SRBD for neutralizing mAbs by competition-binding analysis. They first used a biolayer interferometry-based competition assay with a minimal SRBD domain to screen for mAbs that competed for binding with the potently neutralizing mAb COV2-2196 or a recombinant version of the previously described SARS-CoV mAb CR3022, which recognizes a conserved cryptic epitope10,26. The inventors identified three major groups of competing mAbs (FIG. 5A). The largest group of mAbs blocked COV2-2196 but not rCR3022, while some mAbs were blocked by rCR3022 but not COV2-2196. Two mAbs, including COV2-2130, were not blocked by either reference mAb. Most mAbs competed with hACE2 for binding, suggesting that they bound near the hACE2 binding site of the SRBD. The inventors used COV2-2196, COV2-2130, and rCR3022 in an ELISA-based competition-binding assay with trimeric S2Pecto protein and also found that SRBD contained three major antigenic sites, with some mAbs likely making contacts in more than one site (FIG. 5B). Most of the potently neutralizing mAbs directly competed for binding with COV2-2196.


Previous structural studies have defined the interaction between the SRBD and hACE228. Most of the interacting residues in the SRBD are contained within a 60-amino-acid linear peptide that defines the hACE2 recognition motif.


Here, the inventors defined the antigenic landscape for a large panel of highly potent mAbs against SARS-CoV-2. These detailed studies and the screening studies that identified this panel of mAbs from a larger panel of hundreds25 demonstrate that although diverse human neutralizing antibodies are elicited by natural infection with SARS-CoV-2, only a small subset of those mAbs are of high potency (IC50<50 ng/mL against live SARS-CoV-2 virus), and therefore, suitable for therapeutic development. Biochemical and structural analysis of these potent mAbs defined three principal antigenic sites of vulnerability to neutralization by human mAbs elicited by natural infection with SARS-CoV on the SRBD. Moreover, as SARS-CoV-2 continues to circulate, population immunity elicited by natural infection may start to select for antigenic variants that escape from the selective pressure of neutralizing antibodies, reinforcing the need to target multiple epitopes of S protein in vaccines or immunotherapeutics.


The common S gene variants across the globe reported to date are located at positions D614G, V483A, L5F, Q675H, H655Y and S939F30, far away from the amino acid variants at residues 486 or 487 identified in the inventors' mutation studies for the lead mAbs studied here. Rationally-selected therapeutic cocktails like the one described here might offer even greater resistance to SARS-CoV-2 escape. These studies set the stage for preclinical evaluation and development of the identified mAbs as candidates for use as COVID-19 immunotherapeutics in humans.


REFERENCES FOR EXAMPLE 4



  • 1. Zhou, P., et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273 (2020).

  • 2. Zhu, N., et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 382, 727-733 (2020).

  • 3. Pillay, T. S. Gene of the month: the 2019-nCoV/SARS-CoV-2 novel coronavirus spike protein. J Clin Pathol (2020).

  • 4. Wrapp, D., et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260-1263 (2020).

  • 5. Wan, Y., Shang, J., Graham, R., Baric, R. S. & Li, F. Receptor recognition by the novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus. J Virol 94(2020).

  • 6. Hoffmann, M., et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271-280 e278 (2020).

  • 7. Li, W., et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450-454 (2003).

  • 8. Sui, J., et al. Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association. Proc Natl Acad Sci USA 101, 2536-2541 (2004).

  • 9. ter Meulen, J., et al. Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets. Lancet 363, 2139-2141 (2004).

  • 10. ter Meulen, J., et al. Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants. PLoS Med 3, e237 (2006).

  • 11. Zhu, Z., et al. Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies. Proc Natl Acad Sci USA 104, 12123-12128 (2007).

  • 12. Rockx, B., et al. Structural basis for potent cross-neutralizing human monoclonal antibody protection against lethal human and zoonotic severe acute respiratory syndrome coronavirus challenge. J Virol 82, 3220-3235 (2008).

  • 13. Chen, Z., et al. Human neutralizing monoclonal antibody inhibition of Middle East respiratory syndrome coronavirus replication in the common marmoset. J Infect Dis 215, 1807-1815 (2017).

  • 14. Choi, J. H., et al. Characterization of a human monoclonal antibody generated from a B-cell specific for a prefusion-stabilized spike protein of Middle East respiratory syndrome coronavirus. PLoS One 15, e0232757 (2020).

  • 15. Niu, P., et al. Ultrapotent human neutralizing antibody repertoires against Middle East respiratory syndrome coronavirus from a recovered patient. J Infect Dis 218, 1249-1260 (2018).

  • 16. Wang, L., et al. Importance of neutralizing monoclonal antibodies targeting multiple antigenic sites on the Middle East respiratory syndrome coronavirus spike glycoprotein to avoid neutralization escape. J Virol 92(2018).

  • 17. Wang, N., et al. Structural definition of a neutralization-sensitive epitope on the MERS-CoV S1-NTD. Cell Rep 28, 3395-3405 e3396 (2019).

  • 18. Zhang, S., et al. Structural definition of a unique neutralization epitope on the receptor-binding domain of MERS-CoV spike glycoprotein. Cell Rep 24, 441-452 (2018).

  • 19. Corti, D., et al. Prophylactic and postexposure efficacy of a potent human monoclonal antibody against MERS coronavirus. Proc Natl Acad Sci USA 112, 10473-10478 (2015).

  • 20. Jiang, L., et al. Potent neutralization of MERS-CoV by human neutralizing monoclonal antibodies to the viral spike glycoprotein. Sci Transl Med 6, 234ra259 (2014).

  • 21. Tang, X. C., et al. Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution. Proc Natl Acad Sci USA 111, E2018-2026 (2014).

  • 22. Ying, T., et al. Exceptionally potent neutralization of Middle East respiratory syndrome coronavirus by human monoclonal antibodies. J Virol 88, 7796-7805 (2014).

  • 23. Jiang, S., Hillyer. C. & Du, L. Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses. Trends Immunol 41, 355-359 (2020).

  • 24. Valk, S. J., et al. Convalescent plasma or hyperimmune immunoglobulin for people with COVID-19: a rapid review. Cochrane Database Syst Rev 5, CD013600((2020).

  • 25. Zost, S. J., et al. Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein. bioRxiv, 2020.2005.2012.091462 (2020).

  • 26. Yuan. M., et al. A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV. Science 368.630-633 (2020).

  • 27. Ianevski, A., He, L., Aittokallio, T. & Tang, J. SynergyFinder: a web application for analyzing drug combination dose-response matrix data. Bioinformatics 33, 2413-2415 (2017).

  • 28. Lan, J., et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215-220 (2020).

  • 29. Dinnon, K. H., et al. A mouse-adapted SARS-CoV-2 model for the evaluation of COVID-19 medical countermeasures. bioRxiv. 2020.2005.2006.081497 (2020).

  • 30. Laha, S., et al. Characterizations of SARS-CoV-2 mutational profile, spike protein stability and viral transmission. bioRxiv, 2020.2005.2003.066266 (2020).










TABLE A







Activity Data










BINDING ASSAY RESULTS
NEUTRALIZATION ASSAY RESULTS



ELISA-Purified IgG
(Yes/No qualitative test, or IC50 value (ng/mL)













(OD 450 nm)

SARS-CoV-2

Nano-luciferase
















SARS-


SARS-

xCelligence neutralization

virus reduction



CoV-2
SARS-
SARS-
CoV

test (cell impedence)
SARS-CoV-
test

















Clone ID
Spike
CoV-2
CoV-2
Spike
hACE2

Estimated
reduction
SARS-



(COV2-xxxx)
trimer
RBD
NTD
trimer?
blocking
Qualitative
IC50
test 2 focus
CoV-2
SARS




















2050
4.3
4.3
0.12
0.11
Yes
Yes
nt
25
<50
nt


2046
4.3
4.2
0.09
0.09
Yes
Yes
nt
171
<100
nt


2113
4.4
4.4
0.4
0.1
Yes
Yes
nt
427
<300
nt


2132
4.4
4.4
0.15
0.09
Yes
Yes
nt
673
<300
nt


2098
4.3
4.2
0.15
0.09
Yes
Yes
nt
776
<100
nt


2068
4.3
4.3
0.1
0.1
Yes
Yes
nt
864
<300
nt


2082
4.3
4.3
0.11
3.9
Yes
Yes
nt
302
<200
>1,000


2103
4.3
4.3
0.12
0.53
Yes
Yes
nt
>1,000
nt
nt


2961
3.60
3.70
NT
0.10
Yes
Yes
nt
30
nt
nt
















TABLE B







Neutralization IC50, hACE2 blocking IC50, and EC50 values


for binding to S2Pecto or SRBD antigens for mAb panel

















SARS-CoV



Neutral-
hACE2
S2Pecto
SRBD
S2Pecto



ization
blocking
binding
binding
binding



IC50,
IC50,
EC50,
EC50,
EC50,


MAb
ng/mL
ng/mL
ng/mL
ng/mL
ng/mL















COV2-2015
892
68
2.2
5.1
 2.7


COV2-2050
80
63
1.7
1.6



COV2-2068
478
166
7.4
1.2



COV2-2082
204
43
1.1
0.6
19.9


COV2-2098
1,029
48
1.3
0.7



COV2-2103
1,969
79
2.9
1.1



COV2-2113
1,041
60
1.8
0.6



COV2-2258
989
76
2.5
1.6
11.2


COV2-2268
371
2,198
91.2
5.3



COV2-2308
394
75
2.7
1.9



COV2-2353
2,891
1,750
48.0
10.9 



COV2-2354
1,105
67
2.6
3.5



COV2-2489
4,378

13.0




COV2-2479
48
50
0.4
0.3



COV2-2499
27
57
1.4
1.1



COV2-2539
274
98
1.1
0.7



COV2-2562
348
154
1.0
0.6



COV2-2676
3,247

12.0




COV2-2677
1,618
107
2.4
0.7



COV2-2733
356
71
2.4
0.6



COV2-2752
349
53
1.9
0.7



COV2-2780
478
10,000
86.0
3.1



COV2-2807
907
1,753
13.0
55.1 



COV2-2812
1,020
1,387
45.4
2.1



COV2-2813
555
206
5.3
1.0



COV2-2819
114
60
0.9
0.8



COV2-2828
100
100
3.2
1.4
338  


COV2-2832
70
47
1.3
1.1



COV2-2835
190
91
3.4
1.2



COV2-2841
1,065
91
3.3
1.5



COV2-2919
1,091
1,000
153.0
90.3 



rCR3022


10.2
1.1
 5.2


r2D22





















TABLE 1







NUCLEOTIDE SEQUENCES FOR ANTIBODY VARIABLE


REGION










Clone
Seq ID
Chain
Variable Sequence Region













COV2-
3
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCC


2171


TGTGTAGCCTCTGGATTCACCTTTAGTTTCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGA





AAGGGGCTCGAGTGGGTGGCCAACATAAAGCAAGATGGAGGTGAGAAATACTATGTGGACTCT





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAC





AGCCTGAAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACTGTCTGCAGCAGCTGGGAC





TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



4
light
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACC





TGCTCTGGAGATAAATTGGGGGATAAATATGCTTGTTGGTACCAACAGAGGCCAGGCCAGTCC





CCTGTGCTGGTCATCTATCAAGATAGTAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGAC





TATTACTGTCAGGCGTGGGACAGCAGCACTGGAGTCTTCGGAACTGGGACCAAGGTCACCGTC





CTA





COV2-
5
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCC


2173


TGTGCAGCCTCTGGATTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCTGCTATTAATTGGAATGGTGGTAGCACAGGTTATGCAGACTCT





GTAAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACTCCCTGTATCTGCAAATGAAC





AGTCTGAGAGCCGAGGACACGGCCTTGTATCACTGTGCGAGACGGCGTAGCTCGTCCCGGTAT





AGCAGTGGCTGGTATATGTACTAGTACTACATGGACGTGTGGGGCAAAGGGACCACGGTCACC





GTCTCCTCA



6
light
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCCAGTCAGAGTGTTAGTACCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAACCTCCTGATCTATGAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGC





GGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCA





ACTTATTACTGCCAACAGTATAATACTTATTCGGGGACGTTCGGCCAAGGGACCAAGGTGGAA





ATCAAA





COV2-
7
heavy
CAGATCACCTTCAAGGAGTCTGGTCCTACGCTGGTGAAACCCACAGAGACCCTCACGCTGACC


2177


TGCACCTTCTCTGGGTTCTCAGTCAGCACTAGTGGAGAGGGTGTGGGCTGGATCCGTCAGCCC





CCAGGAAAGGCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGCCCA





TCTCTGAAGAGCAGGCTCACCATCACCAGGGACACCTCCAAAAACCAGGTGGTCCTTACAATG





ACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACACAGGCTTTGGTTCAGGGAT





GCTTTTGATATTTGGGGCCAAGGGACAACGGTCACCGTCTCCTCA



8
light
GACATCCAGATGACCCAGTCTCCATCGTCCCTGTCTGCATCTGTAGGAGACAGAGTCACAATC





ACTTGCCGGGCACGTCAGAGCATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCACAGTGGGGTCCCATCAAGGTTCAGT





GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCA





ACTTACTACTGTCAACAGACTTACAGTACCTTCTGGACGTTCGGCCAAGGGACCAACGTGGAA





ATCAAA





COV2-
9
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTAAGACTCTCC


2178


TGTGCAGCCTCTGGATTCACCTTTAGTACCTATTGGATGACCTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCT





GTTAAGTACCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAC





AGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTCGGGAGCAGCAGCTTTTAC





TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



10
light
TCCTATGAACTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACC





TGCTCTGGAGATAAATTGGGGGATAAATATGCTTGCTGGTATCAGCAGAAGCCAGGCCAGTCC





CCTGTGGTGGTCATCTATCAAGATAGCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGAC





TATTACTGTCAGGCGTGGGACAGCAGCACTGCGGTATTCGGCGGAGGGACCAAGCTGACCGTC





CTA





COV2-
11
heavy
CAGCTACAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACC


2179


TGCACTGTCTCTGGTGGCTCCATCAGCAGTGGAACTTACTACTGCGGCTGGATCCGCCAGCCC





CCAGGGAAGGGGCTGGAGTGGATTGGGAGTACATATTATGGTGGGAGCACCCTCTACAACCCG





TCCCTCAGGGGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTTTCCCTGAAGCTG





AGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTGCGAGACGGGGTAATTACTATGAT





AGTAAGAACTGGTTCGACCCCTTGGGGCCAGGGAACCCTTGGTCACCGTCTcCTCA



12
light
GAAATAGTGATGACGCAGTCTCCAGCCACCGTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGT





GCCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCA





GTTTATTACTGTCAGCAGTATAATAACTGGCCACCTATGTACACTTTTGGCCAGGGGACCAAG





GTGGAGATCAAA





COV2-
13
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGAGTCTCC


2181


TGTGCAGCGTCTGGATTCACCTTCAGTAGCCATGGCATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGTCAGTTATATGGTATGATGGAAGTAATAAATACTATGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTCTCTGCAAATGAAC





AGCCTGAGAGCCGAAGACACGGCTGTGTATTACTGTGCGAGAGAGAGCGCGGACATATCATCT





CGTCTTGACTACTGGGGCCGGGGAACCCTGGTCACCGTCTCCTCA



14
light
TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGCCCCCAGGACAAACGGCCAGGATCACC





TGCTCTGGAGATGCATTGCCAACAAAATATGCTTATTGGTACCAGCAGAAGTCAGGCCAGGCC





CCTGTGCTGGTCATCTATGACGACAGCAAACGACCCTCCGGGATCCCTGAGAGATTCTCTGGC





TCCAGCTCAGGGACAATGGCCACCTTGACTATCAGTGGGGCCCAGGTGGAGGATGAAGCTGAC





TACTACTGTTACTCAACAGACAGCAGTGGTAATGTCTTCGGAACTGGGACCAAGGTCACCGTC





CTA





COV2-
15
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGAGTCTCC


2183


TGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGGTATATCATATGATGGAAGCAATAAATACTACGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCGGACACTATGGTTCGGGGA





ACTTATTTTGAGTACTGGGGCCAGGGAACCCTGGTCACCGTCCCTCA



16
light
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCC





TGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCTTGGTACCAACAACACCCA





GGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGC





TTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGAC





GAGGCTGATTATTGCTGCAGCTCATATACAAGCAGCAGAGCTGTGCTATTCGGCGGAGGGACC





AAGCTGACCGTCCTA





COV2-
17
heavy
CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTAAAACCCACACAGACCCTCACGCTGACC


2184


TGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGCCTGGATCCGTCAGCCC





CCAGGAAAGGCCCTGGAGTGGCTTGCTCTCATTTATTGGGATGATGATAAGCGCTACAGCCCA





TCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATG





ACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGCACACAGACTTCCAACGCCCCAA





CTGCTACCATCTTTTGAAAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCC





TCA



18
light
CAGTCTGTGCTGACTCAGCCACCCTCGGTGTCTGAAGCCCCCAGGCAGAGGGTCACCATCTCC





TGTTCTGGAGGCAGCTCCAACATCGGAAATAATGCTGTAAACTGGTACCAGCAGCTCCCAGGA





AAGGCTCCCAAACTCCTCATCTATTATGATGATCTGCTGCCCTCAGGGGTCTCTGACCGATTC





TCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAAGATGAG





GCTGATTATTACTGTGCATCATGGGATGACAGCCTGATTGGTCCGGTATTCGGCGGAGGGACC





AAGCTGACCGTCCTA





COV-
19
heavy
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAGACTCTCC


2185


TGTGCAGTCTCTGGATTCACCTTCAGTAGCTACTGGATGCACTGGGTCCGCCAAGCTCCAGGG





AAGGGGCTGGTGTGGGTCTCACGTATTAATAGTGATGGGAGTAGCACAAGCTACGCGGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAAC





AGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAGAAGTGGAGCAGCTGGCTCAT





ATGGTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



20
light
TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACC





TGCTCTGGAGATGCATTGCCAAACCAATATGCTTATTGGTACCAGCAGAAGCCAGGCCAGGCC





CCTGTGCTGGTGATATATAAAGACAGTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAGCTCAGGGACAACAGTCACGTTGACCATCAGTGGAGTCCAGGCAGAAGACGAGGCTGAC





TATTACTGTCAATCAGCAGACAGCAGTGGTACATCTTGGGTGTTCGGCGGAGGGACCAAGCTG





ACCGTCCTA





COV2-
21
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2186


TGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAATCAATAAATACTACGCAGACGCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGGCCACGAAGTGGGAGCTACTAC





GCCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



22
light
GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGTCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTTTCAGCAGAAACCAGGGAAA





GCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTACAAAGTGGGGTCCCATCAAAGTTCAGC





GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCA





ACTTATTACTGCCAACAGTATAATAGTCACCCTCCCACTTTCGGCGGAGGGACCAAGGTGGAG





ATCAAA





COV2-
23
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2187


TGTGCAGCCTCTGGATTCACCTTCAGTTACTATCCTATGCACTGGCTCTGGGTCCGCCAGGCT





CCAGGCAAGGGGCTGGAGTGGGTGGCAGTTACATCATATGATGGAACCAATAAATACTACGCA





GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAA





ATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGAGCTACTAAC





TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



24
light
TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAACATTACC





TGTGGGGGAAACAACATTGGAAGAAAAAGTGTGCACTGGTACCAGCAGAAGTCAGGCCAGGCC





CCTGTGCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGAC





TATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATCCGGAGTGGGTGTTCGGCGGAGGGACC





AAGCTGACCGTCCTA





COV2-
25
heavy
GAAGCGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCC


2189


TGTGCAGCCTCTGGATTCACCTTTGATGATTCTGCCATGCACTGGGTCCGGCAAGCTCCAGGG





AAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAACGTAGGCTATGCGGACTCT





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAAC





AGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTACAAAAGCCTCTCGATATTGTAGTAGT





ACCATCTGCTATTGGAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



26
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAACTGGGGTCCCATCAAGGTTCAGT





GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGAAGTCTGCAACCTGAAGATTTTGCA





AGTTACTACTGTCAACAGAGTTACAGTACCCCCACTTTCGGCGGAGGGACCAAGGTGGAGATC





AAA





COV2-
27
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCC


2190


TGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGAAATGAACTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTGCCATATACTACGCAGACTCT





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAC





AGCCTGAGAGTCGAGGACACGGCTGTTTATTACTGTGCGAGAGAAGCGCGGTCACGATATTTT





GACTGGTTACCCTCGTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



28
light
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCC





TGCACTGGAACCAGCAGTGACATTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCA





GGCAAAGCCCCCAAACTCCTGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTACTCGC





TTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGAC





GAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCACCCATGTGGTGTTCGGCGGAGGG





ACCAAGCTGACCGTCCTA





COV2-
29
heavy
CAGGTGCAGGTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCC


2192


TGCAAGGCTTCTGGATACACCTTCACTACCTATGCTATGAATTGGGTGCGACAGGCCCCTGGA





CAAGGACTTGAGTGGATGGGATGGATCAACACCAACACTGGGAACCCAACGTATGCCCAGGGC





TTCACAGGACGGTTTGTCTTCTCCTTGGACACCTCTGTCAACACGGCATTTCTGCATATCGGC





AGCCTAAAGGCTGAGGACACTGCCGTGTATTACTGTGCGAGAGATCAGGACAGTGGCTACCCA





ACTTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTC



30
light
GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATC





TCCTGCAAGTCTAGTCAGAGCCTCCTGCATAGTGATGGAAAGACCTATTTGTATTGGTACCTG





CAGAAGCCAGGCCAGTCTCCACAGCTCCTGATCTATGAAGTTTCCAACCGGTTCTCTGGAGTG





CCAGATAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAG





GCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACAGCCTCCTCTCACTTTCGGCGGA





GGGACCAAGGTGGAGATCAAA





COV2-
31
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2193


TGTGCAGCCTCTGGATTCACCTTCAGTACCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGCGTGGGTGGCACTTATATCATATGATGGATATAATAAATACTACGCAGACTCC





GTGAGGGGCCGATTCACCATCTCCAGAATCAATTCCAAGAACACGCTGTCTCTGCAGATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTCTATTACTGTGCGAGAGGGTCAGCTGGAAACTACTAC





TACGGTATGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCA



32
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCAAGTCAGACCATTACCAACTATTTAAATTGGTATCAGCTGAAATCAGGGAGA





GCCCCCAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGT





GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCA





ACTTACTACTGTCAACAGAGTTACAGTACGCCGTACACTTTTGGCCAGGGGACCAAGCTGGAG





ATCAAA





COV2-
33
heavy
CAGGTGCATCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2195


TGTGCAGCCTCTGGATTCACCTTCAGTAACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATCTCAAATGATGAATTTAATAAATTCTATGCAAACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCGAAGAACACGGTGTATTTGCAATTGAAC





AGTCTGAGAACTGAGGACACGGCTCGATATTACTGTGCGAAAGGGGGGGATGGCAGTGGCTGG





GCCTGGGACGGTGATAACCCCCCAACGGACTACTGGGGCCAGGGAACCCTGGTCATCGTCTCT





TCA



34
light
GACATCGTGATGACCCAGTCTCCGGACTTCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATC





AGCTGCAAGTCCAGTCAGAGTGTTTTATACACCCCCAAGAATAAGAACTACTTAGCTTGGTAC





AAGCAGAAACCAGGACAGCCTCCTAAGGTGCTCATTTACTGGGCATCTACCCGGGAATCCGGG





GTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCATCATCAGCAGCCTG





CAGGCTGAGGATGCGGCAGTTTATTACTGTCAGCAATATTATACTGCTCCTCTCACTTTCGGC





GGTGGGACCAGGGTGGAGATCAA





COV2-
37
heavy
GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCC


2197


TGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGG





AAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCC





TTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGC





AGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACCCGACTATAGCAGTGGCTGG





TTTAGCTACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA



38
light
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTCTTAGCCTGGTACCAGCAGAAACCTGGC





CAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTC





AGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTT





GCAGTGTATTACTGCCAGCAGTATGGTCGCTCACCGATCACCTTCGGCCAAGGGACACGACTG





GAGATTAAA





COV2-
39
heavy
CAGGTCCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCC


2199


TGCAAGGTTTCCGGATACACCCTCACTGAACTATCCATACACTGGGTGCGACAGGCTCCTGGA





AAAGGGCTTGAGTGGATGGGAGGTTTTGATCCTGAAGATGCTGAAACAATCTACGCACAGCAG





TTCCAGGGCAGAGTCACCATGACCGAGGACACATCTACAGACACAGCCTACATGGAGCTGAGC





AGCCTGAAATCTGAGGACACGGCCCTGTATTACTGTGCAACAGGGTTCGCGGTGTTTGGGAGG





GCAGCAGTTCCCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



40
light
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACC





TGCTTTGGAGATAAATTGGGGGATAAATATGCTTGCTGGTTTCAGCAGAAGCCAGGCCAGTCC





CCTGTGCTGATCATCTATCAAGGTGCCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGAC





TATTACTGTCAGGCGTGGGACAGCAGCACTGTGGTTTTCGGCGGAGGGACCAAGCTGACCGTC





CTA





COV2-
41
light
CAGGTGCAGGTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2200


TGTGCAGCCTCTGGATTCACGTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATCTCACAATTGTAGTAATA





CCAGCTGCCCCAAATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



42
light
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCC





TGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCA





GGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGC





TTCTCTGGCTCCAGGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGAC





GAGGCTGATTATTATTGCAGGTCATATACAAGCAGCAGCACTCCTGTGGTGTTCGGCGGAGGG





ACCAAGCTGACCGTCCTA





COV2-
43
heavy
GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCC


2207


TGTAAGGGTTCTGGATACAGCTTTACCAGTCACTGGATCGGCTGGGTGCGCCAGATGCCCGGG





AAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATATAGCCCGTCC





TTCCAAGGGCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGC





AGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTGCCCTCAGAGAGCGAGGCGTA





CAGCTGTGGTCAGTTTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



44
light
CAGTCTGTGCTGACGCAGCCGCCGTCAGTGTCTGGGGGCCCAGGGCAGAGGGTCACCATCTCC





TGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCA





GGAACAGCCCCCAAACTCCTCATCTTTATTAACTCCAATCGGCCCTCAGGGGTCCCTGACCGC





TTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGAT





GAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGGGTGCCTTGTLCGGCGGAGGGACC





AAGCTGACCGTCCTA





COV2-
45
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2210


TGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACTCTTTATCTGCAAATGAAC





AGCCTGAGAGTTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCAAGAATGGTTCAGGGAG





TTATTCCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



46
light
GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTATGATGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGC





GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCA





ACTTACTATTGTCAACAGGCTAACAGTTTCCCTCCGACTTTCGGCCCTGGGACCAAAGTGGAT





ATCAAA





COV2-
47
heavy
CAGGTGCAGUTGGTGGAGTCCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2211


TGTGCAGCCTCTGGATTCACCTTCAGTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTATGTATTACTGTGCGAAAGATGGGAGTATAGCAGCAGCT





GACTACTGGGGCCAGGGAACCCTGGTCACCGTCCTC



48
light
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATC





AACTGCAAGTCCAGCCAGAGTGTCTTACACAGCTCCAACAACAAGGACTCCTTAGTTTGGTAC





CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTAGCCGGGAATCCGGG





GTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG





CAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCTTGGACGTTCGGC





CAAGGGACCAAGGTGGAAATCAAA





COV2-
49
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCC


2212


TGTGCAGCCTCTGGATTCACCTTCAGTAGTTATAGCATGAACTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCATCCATAAGTAATAGTAATAGTTTCATATACTACGCAGACTCA





ATGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAC





AGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGCGAGTTAACGGCAACTCAAACTGG





AACTTTGGGTCTTACTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTC





TCCTCA



50
light
GAAATTGTGTTGACACAGTCTCCAGCCATTTTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGTAGCTACTTAGCCTGGTACCAACAGAAGCCTGGCCAG





GCTCCCAGGCTCCTCATCTATGATACATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGACTTCACTCTCACTATCAGCAGCCTAGAGCCTGAAGATTTTGCA





TTTTATTACTGTCAGCAGCGTGGCAACTGGTGGACGTTCGCCCAAGGGACCAAGGTGGAAATC





AAA





COV2-
51
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCC


2215


TGTGCAGCCTCTGGATTCACTTTCAGTGGCTATAGCATGAACTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGTTACATATACTACGCAGACTCA





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAC





AGCCTGAGGGCCGAGGACACGGCTGTGTATTACTGTGCGAGATGGCTACAGTTAAGATCAGAC





TACTATTACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



52
light
GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAACAACTTAGCCTGGTACCAGCACAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTACAGTCTGAAGATTTTGCA





GTTTATTTCTGTCAGCAGTGTTATAACTGGCCTCCGTGGACGTTCGGCCAAGGGACCAAGGTG





GAAATCAAA





COV2-
53
heavy
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACC


2222


TGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGG





AAGGGACTGGAGTGGATTGGTTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTC





AAGAGTCGAGTCACCATATCAGTAGACATGTCCAAGAACCAGTTCTCCCTGAAGCTGAGGTCT





GTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGCCCCGAGGGAAAGGCTCCAATGG





GGGGAGTACTACTTCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



54
light
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCC





TGCACTGGAACCAGCAGTGATGTTGGGAGTTATAACCTTGTCTCCTGGTACCAACAGCATGCA





GGCAAAGCCCCCAAACTCATGATTTATGAGGTCATTAAGCGGCCCTCAGGGGTTTCTAATCGC





TTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACAATCTCTGGGCTCCAGGCTGAGGAC





GAGGCTGATTATTACTGCTGCTCATATGCAGTTAGTACCACTTATGTTATATTCGGCGGAGGG





ACCAAGCTGACCGTCCTA





COV2-
55
heavy
GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCC


2226


TGTAAGGGTTCTGGATACAGCTTTACCAACTCCTGGATCGGCTGGGTGCGCCAGATGCCCGGG





AAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCC





TTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGC





AGCCTGAAGGCCTCGGACACCGCCATCTATTACTGTGCGACACATCGTTGTAGTGGTGGTTTG





TGCTACTTAGCCTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



56
light
CAGCCTGTGCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTCGGTCACACTCACC





TGCACCCTGAGCAGCGGCTACAGTAATTATAAAGTGGACTGGTACCAGCAGAGACCAGGGAAG





GGCCCCCGGTTTGTGATGCGAGTGGGCACTGGTGGGATTGTGGGATCCAAGGGGGATGGCATC





CCTGATCGCTTCTCAGTCTTGGGCTCAGGCCTGAATCGGTACCTGACCATCAAGAACATCCAG





GAAGAGGATGAGAGTGACTACCACTGTGGGGCAGACCATGGCAGTGGGAGCAACTTCGTTTTT





GTGGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA





COV2-
57
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2227


TGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGTAAAAAAGACTATGCAGACTCC





GTGAAGGGCCGATTCACCATCrCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCAATCTCAGGGCGCTTAT





ATTTTGACTGGTTATAGGGGCTACGGTATGGACGTCTGGGGCCAGGGGACCACGGTCACCGTC





TCCTCA



58
light
GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATC





TCCTGCAGATCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACTTG





CAGAAGCCAGGGCAGTCTCCACAGTTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTC





CCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTATACTGAAAATCAGCAGAGTGGAG





GCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTTCAAACTCCATTCACTTTCGGCCCT





GGGACCAAAGTGGATATCAAA





COV2-
59
heavy
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACC


2228


TGCGCTGTCTATGGTGGGTCCTTCAGTGGTCACTACTGGAGCTGGATCCGCCAGCCCCCAGGG





AAGGGACTGGAGTGGATTGGGGAAATCAATCACAGTGGAAGCACCAACTACAACCCGTCCCTC





AAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCT





GTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGACCCCCCCAAGCAGCTCGTATTCAT





TACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA



60
light
GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCA





GTTTATTACTGTCAGCAGTATAATTACTGGCCTCCCCTCACTTTCGGCGGAGGGACCAAGGTG





GAGATCAAA





COV2-
61
heavy
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCC


2231


TGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGAACTGGGTCCGGCAACCTCCAGGG





AAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGATAGCATAGGCTATGCGGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAAC





AGTCTGAGAGCTGAGGACACGGCCATGTATTACTGTGCAAAAGGAAGGGGTGCTGGTTATACT





TCCTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA



62
light
TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACC





TGTGAGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCC





CCTGTGCTGGTCGTCTATGATGATAGCGGCCGGCCCTCAGGGATCCCTGAACGATTCTCTGGT





TCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGAC





TATTTCTGTCAGGTGTGGGATAGTAGTAGTGATCATCATGTGGTTTTCGGCGGAGGGACCAAG





CTGACCGTCCT





COV2-
63
heavy
CCTACACGACGCTCTTCCGATCTGGGGGTCACTGGATCGGCTGGGTGCGCCAGATGCCCGGGA


2233


AAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATATAGCCCGTCCT





TCCAAGGGCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCA





GCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGTGCCCTCAGAGAGCGAGGCGTAC





AGCTGTGGTCAGTTTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG



64
light
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCC





TGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCA





GGAACAGCCCCCAAACTCCTCATCTTTATTAACTCCAATCGGCCCTCAGGGGTCCCTGACCGC





TTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGAT





GAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGGGTGCCTTGTTCGGCGGAGGGACC





AAGCTGACCGTCCTA





COV2-
65
heavy
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCC


2046


TGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGG





AAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGCCTATACGGACTCT





GTGAAGGGCCGATTCACCATCTCCAGAGATAACGCCAAGAACTCCCTGTATCTGCAAATGAAC





AGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGCACACTCGACTGGACACCAA





TACTAGTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCCTCA



66
light
GACATCCAGATGACCCAGTCTCCATCCTCCTTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCAAGTCAGAGCATTAGCAGCTTTTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTATGCTGCATTCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGT





GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAATCTGAAGATTTTGCA





ACTTACTACTGTCAACAGAGTTACAATACCCCTTACACTTTTGGCCAGGGGACCAAGCTGGAG





ATCAAA





COV2-
67
hEavy
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCC


2047


TGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGG





AAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCT





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTATATCTGCAAATGAAC





AGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGTGTCGTCCATTACTAGCCTA





TTGGGATACTACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



68
light
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA





GTTTATTACTGTCAGCACCGTAGCAACTGGCCTCCGAGGCTCACTTTCGGCGGAGGGACCAAG





GTGGAGATCAAA





COV2-
69
heavy
CAGGTCCAGGTGGTTCAATCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2048


TGCAAGACTTCTGGAGACACCTCCAGCAGTTATACTGTCGGCTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAAGGATCATCCCTATCCTTGGTATAGCATACTCCGCACAGAAG





TTCCAGGGCAGACTCACGATTACCGCGGACAAATCCACGAGCACATCCTACATGGAGCTGAGC





AGCCTGAGATCTGAGGACACGGCCGTTTATTACTGTGCGAGAGGGGTGGAGCTGCTACTCCG





GGTTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



70
light
GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGTCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGT





GGCGGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCA





GTTTATTACTGTCAGCAGTATAATAACTTCCTCACTTTCGGCGGAGGGACCAAGGTAGAGATC





AAA





COV2-
71
heavy
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAGACTCTCC


2049


TGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGCACTGGGTCCGCCAAGTTCCAGGG





AAGGGGCTGGTGTGGGTCTCACGTATTAATAGTGATGGGAGTAGCACAAGCTACGCGGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGGAAATGAAC





AGTCTGAGAGCCCAGGACACGGCTGTTTATTACTGTGCAGGTTCCCCGTGGCTACGAGGCGAC





ATTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



72
light
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCC





TGCACCGGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGC





AGTGCCCCCACCACTGTGATCTATGAGGATAACCAAAGGCCCTCTGGGGTCCCTGATCGGTTC





TCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGGCTGATGACTGAG





GACGAGGCTGACTACTACTGTCAGTCTTATGATGGCAGCAATCATGCTGTGGTATTCGGCGGA





GGGACCAAGCTGACCGTCCTA





COV2-
73
heavy
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCC


2050


TGCAAGGCTTCTGGATACACCTTCACCGACTACTATATGCACTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTCGTGGCACAAACTATGCACAGAAG





TTTCAGGGCAGGGTCACTATGACCAGGGACACGTCCATCAGCACAGTCTACATGGAGCTGAGC





AGGCTGACATCTGACGACACGGCCGTCTATTACTGTGCGAGAGTGGTGGTCCTCGGCTATGGC





CGCCCAAACAATTACTATGATGGTAGGAATGTGTGGGACTACTGGGGCCAGGGAACCCTGGTC





ACCGTCTCCTCA



74
light
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCATCATCTCT





TGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTAAAGTGGTATCATCAGCTCCCAGGA





ACGGCCCCCAAACTCCTCATCTGTAGTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTC





TCTGGCTCCAAGTGTGACACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAG





GCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGCTTTGGTATTCGGCGGAGGGACC





AAGCTGACCGTCCTA





COV2-
75
heavy
CAGGTCACCTTGAGGGAGTCTGGTCCTGCGCTGGTGAAACCCACACAGACCCTCTCACTGACC


2051


TGCACCTTCTCTGGGTTCTCACTCGGCACTAGTGGAATGTGTGTGAGCTGGATCCGTCAGCCC





CCAGGGAAGGCCCTGGAGTGGCTTGCACGCATTGATTGGGATGATGATAAATACTACAGCACA





TCTCTGAAGACCAGGCTCACCATCTCCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATG





ACCAACATGGACCCTGTGGACACAGCCACGTATTACTGTGCACGGGGGGTGGTTACTTATGAC





TATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



76
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTGACCATC





ACTTGCCGGGCAAGTCAGAGCATTGCCGGCTATTTAAACTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTATGGTACAACCAGTTTGCAAAGTGGGGTCCCAGTAAGGTTCAGT





GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCA





ACTTACTACTGTCAACAGAGTTACAGTACCCCTGGCACCTTCGGCCAAGGGACACGACTGGAG





ATTAAA





COV2-
77
heavy
CAGGTCCAGCTGGTGCAATCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2054


TGCAAGGCTTCTGGAGACACCTTCAGCAGCTATACTATCAACTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAAGGATCATCCCTATCCTTGGTATACCAAACTACGCACAGAAA





TTCCAGGGCAGAGTCACCATTACCGCGGACAAATCCACGAGCACAGCCTTCATGGAGCTGAGC





AGCCTGAGATCTGAGGACACGGCCGTCTATTACTGTGCGAGAGGGAGGGGCTACAGTAACTAC





GGGGCCTCCTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA



78
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCAGGCGAGTCAGGACATTAACCACTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACCGGGGTCCCATCAAGGTTCAGT





GGAAGTGGATCTGGGACAGATTTTACTTTCACCATCACCAGCCTGCAGCCTGAAGATGTTGCA





ACATATTACTGTCAACAGTCTGATAATCTCCCCATGTACACTTTTGGCCAGGGGACCAAGCTG





GAGATCAAA





COV2-
79
heavy
GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCC


2055


TGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGAACTGGGTCCGGCAACCTCCAGGG





AAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGATAGCATAGGCTATGCGGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAAC





AGTCTGAGAGCTGAGGACACGGCCATGTATTACTGTGCAAAAGGAAGGGGTGCTGGTTATACT





TCCTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA



80
light
TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACC





TGTGAGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCC





CCTGTGCTGGTCGTCTATGATGATAGCGGCCGGCCCTCAGGGATCCCTGAACGATTCTCTGGT





TCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGAC





TATTTCTGTCAGGTGTGGGATAGTAGTAGTGATCATCATGTGGTTTTCGGCGGAGGGACCAAG





CTGACCGTCCT





COV2-
83
heavy
CAGGTGCAGCTGGCGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCC


2064


TGCAAGGCTGCTGGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACAGGCCACTGGA





CAAGGGCTTGAGTGGATGGGGTGGATGAACTCTAACAGTGGTAACGCAGGCTATGCACAGAAG





TTCCAGGGCAGAGTCACTATGACCAGGGACACCTCCACAAGTACAGCCTACATGGAGTTGAGC





AGCCTGACATCTGATGACACGGCCGTGTATTATTGTGCGAGAATGCGCACCGGCTGGCCCACA





CATGGCCGCCCGGATGACTTCTGGGGGCGGGGAACCCTGGTCACCGTCTCCTCA



84
light
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCT





TGTTCTGGAAGCAACTCCAACATCGGAAGTTATACTGTAAACTGGTACCAGCAGTTCCCAGGA





ACGGCCCCCAAACTCCTCATTTATGATAATAATCAGCGGACCTCAGGGGTCCCTGACCGATTC





TCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAG





GCTAATTATTACTGTTTAGTATGGGATGACAGCCTGAATGGCCTGGTATTCGGCGGAGGGACC





AAGCTGACCGTCCTA





COV2-
85
heavy
GAAGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAGACTCTCC


2068


TGTGCAGCCTCTGGGTTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCAGTTATTTATCCCGGTGGTAGCGCATTCTACGLAGACTCCGTG





AAGGGCCGATTCACCATCTCCAGACACAATTCCAACAACACACTGTGTCTTCAAATGAACAGC





CTGCGAACTGAGGACACGGCCGTGTATTATTGTGCGAGATCTTACGATATTTTGACTGGTTAT





AGAGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTC



86
light
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCC





TGCACTGGGAGCAGCTCCAACATCGGGTCAGGTTCTGATGTACACTGGTACCAGCAGCTTCCA





GGAACAGCCCCCAAACTCCTCATCTATGGCAACACCAATCGGCCCTCAGGGGTCCCTGACCGA





TTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGAT





GAGGCTGATTATTACTGCCAGTCCTATGACAGCAGGCTGAGTGGTTTTGTGGTATTCGGCGGA





GGGACCAAGCTGACCGTCCTA





COV2-
87
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCC


2069


TGTGCAGCCTCTGGACTCACCGTCAGTAGCAACTACATGAGTTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGCGTCTCAGTTATTTATGCCGGTGGTAATACATACTACGCAGACTCCGTG





AAGGGCAGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGC





CTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGCGATGGTGGTTATTACTCACCC





TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



88
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGGTCCTGATCTATGCTGCATCCACTATGCAAAGTGGGGTCCCATCAAGGTTCAGG





GGCAGTGGCTCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACTTGAGGATTTTGCA





ACTTACTACTGTCAACAGAGTTACAGTACCCCTCAGACGTTTGGCCAAGGGACCAAGGTGGAA





ATCAAA





COV2-
89
heavy
CAGGTGCAGTTACAGCAGTGGGGCGCAGGGCTGTTGAAGCCTTCGGAGACCCTGTCCCTCACC


2070


TGCGCTGTCTCTGGTGGGTCCTTCAGTGCTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGG





AAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTC





AGGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAATTCTCCCTGAAGCTGAGCTCT





GTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGTGGGTTATTCCCAAGGGTACTAC





TACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA



90
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCAAGTCAGAGCATTAGCAACTATTTAAATTGGTATCAGCAGAAACCGGGGAAA





GCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCCTCAAGGTTCAGT





GGCAGTGGATCTGGGACAGATTTCTCTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCA





ACTTACTCCTGTCAACAGAGTTACACTACCCTCCTCACTTTCGGCGGAGGGACCAAGGTGGAG





ATCAAA





COV2-
93
heavy
CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2078


TGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATAGTATCACCTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAAGGATCATCCCTGTCCTTGGTATAGCAAACTACGCACAGAAG





TTCCAGGACAGAGTCACGATTACGGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGC





AGCCTGAGATCTGAGGACACGGCCGTCTATTACTGTGCGAGAGTGGGCGTGAGTGGTTTTAAA





AGTGGCTCGAACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA



94
light
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCCTCTCC





TGCACTGGGAGCAACTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCA





GGAACAGCCCCCAAACTCCTCATCTATGGTAATAGCAATCGGCCCTCCGGGGTCCCTGACCGA





TTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGAT





GAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGATTCGGTGTTCGGCGGAGGG





ACCAAGGTGACCGTCCTA





COV2-
97
heavy
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCC


2081


TGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAATAATCAACCCTGGTGGTGGTAGTACAACCTACGCACAGAAG





TTCCAGGGCAGAGTCACCATGACCAGTGACACGTCCACGAGCACAGTCTACATGGAGCTGAGC





AGCCTGAGATCTGAGGACACGGCCATGTATTACTGTGCGAGAGGGGCAATTCCCCCAAATAGC





AGAGCCGAAATTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



98
light
GAAATAGTGATGACGCAGTGTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGTCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTGCCAGCAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCA





GTTTATTACTGTCAGCAATATTATAACTGGCCGCLCACTTTCGGCGGAGGGACCAAGGTGGAG





ATCAAA





COV2-
99
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCC


2082


TGTGCAGCCTCTGGATTCATCTTTGATGATTATGACATGACCTGGGTCCGCCAAGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCTGGTATTAGTTGGAATGGTGGTAACACAGGTTATGCAGACTCT





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAAC





AGTCTGAGAGCCGAGGACACGGCCTTGTATCACTGTGCAGTGATTATGTCTCCAATCCCCCGT





TATAGTGGCTACGATTGGGCGGGTGGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACC





GTCTCTTCA



100
light
TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACA





TGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGTC





CCTATACTTGTCATCTATGATAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGC





TCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGAC





TATTACTGTAACTCCCGGGACAGCAGTGGTAACGCCGTGGTGTTCGGCGGAGGGACCAAGCTG





ACCGTCCTA





COV2-
101
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2083


TGTGCAGCCTCTGGATTCACCTTCAGTAACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATGTCATATGATGGAAGTAATAAATACTATGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTATATTACTGTGCGAAAAATTTAGGACCCTATTGTAGT





GGTGGTACCTGCTATTCCTTAGTTGGTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCC





TCA



102
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCATCAAGGTTTCAGT





GGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCA





ACATATTACTGTCAACAGTATGCTAATCTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGAT





ATCAAA





COV2-
107
heavy
GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCC


2097


TGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCATTGGGTCCGGCAAGCTCCAGGG





AAGGGCCTAGAGTGGGTCTCAGGTATCAGTTGGAACAGTGGTACCATACGCTATGCGGACTCT





GTGAAGGGCCGATTCATCATCTCCAGAGACAACGCCAAGAACTCCTTGTATCTGCAAATGAAC





AGTCTGAGACCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATAATACGTCAGGGCGAA





GACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



108
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCAAGTCAGAACATTGCCAGCTATTTGAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGT





GGCAGTGGATCGGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGAGTTTGCA





ACTTACTACTGTCAACAGAGTTACAGTACCCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAA





ATCAAA





COV2-
109
heavy
GAGGTGCAACTGTTGGAGTCTGGGGGAGGCTTGATACAGCCTGGGGGGTCCCTGAGACTCTCC


2098


TGTGCAGCCTCTGGATTCACCTTTAGCAACTATGCCATGTCCTGGGTCCGCCAGGCCCCAGGG





AAGGGGCTGGAGTGGGTCTCAGGTATTATTAGTACCAGTGGTGGTGCCACATACAACGCAGAC





TCCGTGAGGGGCCGGTTCACCACCTCCAGGGACAATTCCAAGAACATACTGTATCTGCAAATG





AACAGCCTCAGAGGCGAGGACACGGCCGTTTATTACTGTGTGAAAGGTCTCTTTGACTGGTTC





CCGCTCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA



110
light
GACATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCATCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGAAGCAATTTAGCCTGGTACCAGCAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTCTGGTGCATCCACCAGGGCCACTGCTATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCACCAGCCTGCAGTCTGAAGATTGTGCA





GTTTATTACTGTCACCAGTATAATAACTGGCCTCAGACGTTCGGCCAAGGGACCAAGGTGGAA





ATCAAA





COV2-
111
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGTCCCTGAGACTCTCC


2103


TGTGCAGCCTCTGGATTCACCTTTAGTAGGCATTGGATGACCTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCT





GTGAAGGGCCGACTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAC





AGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACTGGGGTTCTATTACGGTGGA





GCCGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



112
light
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCC





TGCACCGGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGC





AGTGCCCCCACCACTGTGATCTCTGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTC





TCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAG





GACGAGGCTGACTACTACTGTCAGTCTTATGATGGCATCAATCGGGCATGGGTGTTCGGCGGA





GGGACCAAGCTGACCGTCCTA





COV2-
113
heavy
AACGTGCAATTAGTGGAGTCTGGGGGAGGCTTGGTTCAGCCTGGCGGGTCCCTGAGACTCTCC


2108


TGTGCAGCCTCTGGATTCACCTTTCATCATTATGCCATGCACTGGGTCCGACAAGCTCCAGGG





AAGGGCCTGGAGTGGGTCTCAGGTATTAGTGGGAGTAGTGATTACAGAGCCTATGCGGACTCT





CTGAAGGGCCGATTCACCATCTCCAGAGACTACGCCAAGAACTCCCTGTGGCTGCAAATGAAC





AGTCTGACATCTGAGGACACGGCCTTCTATTACTGTGCAAAGGGCGTTGACTATGGCGGCAAA





CTTGCCTACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



114
light
GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATC





TCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTCTTGGATACAACTCTTTGAGTTGGTACCTG





CAGAAGCCAGGGCAGTCTCCACACCTCCTGATCTATTTGGGCTCTAATCGGGCCTCCGGGGTC





CCTGACAGGTTCAGTGGCAGTGGATCAGCCACAGACTTTACACTGAAAATCAGCAGATTGGAG





GCTGAGGATGTTGGCGTTTATTACTGCATGCAAGCTCTACAAACTCCCCTCACCTTCGGCCAA





GGGACACGACTGGAGATTAAA





COV2-
115
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2110


TGTGCAGCCTCTGGATTCAGCTTCAGTAGCTATGTTATGAACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAGTAAATACTACGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACACTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGACATTGATAGTGGCTACGAT





CCTACCCCCGTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



116
light
GACATCCAGATGACCCACTCTCCATCCTCCCTGTCTGCATGTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GGCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGT





GGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCA





ACTTACTACTGTCAACAGAGTTACAGTTCCCTTTCGATCACCTTCGGCCAAGGGACACGACTG





GAGATTAAA





COV2-
117
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCC


2111


TGTGCAGCCTCTGGATTCACCTTCAGTAGCTACGACCTGCACTGGGTCCGCCAAGGTACAGGA





AAACGTCTGGAGTGGGTCTCAGCTATTGGTACTGCTGGTGACACATACTATCTAGGCTCCGTG





AAGGGCCGATTCACCATCTCCAGAGAAAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGC





CTGAGAGCCGGGGACACGGCTGTGTATTACTGTGCAAGAGTCCTCTATGATAGTAGTGGTTTT





TACAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



118
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATC





ACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGT





GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCA





ACTTACTACTGTCAACAGAGTTACGAAATCCCTCCGTGGACGTTCGGCCAAGGGACCAAGGTG





GAAATCAAA





COV2-
119
heavy
GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCC


2113


TGTGCAGCCTCTGAGGTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCACTTATTTATAGCGGTGGTACTACATACTACGCAGACTCCGTG





AAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACCCTGTATCTTCAAATGAACAGC





CTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGACTTTCTACGGTGGCACGACCTC





TGGGCCAGGGAACCCTGGCACCGTCTCCTCA



120
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCAGGCGAGTCAGGACATTAACAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATTAAGGTTCAGT





GGAAGTGGATCTGGGACAGATTTTATTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCA





ACATATTACTGTCAACAGTATGATAATCTCCCTCCAGTTTTCGGCGGAGGGACCAAGGTGGAG





ATCAAA





COV2-
121
heavy
CAGGTCCAGCTGGTGCAATCAGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2114


TGCAAGGCTTCTGGAGACACCTTCAGCAGCTATACTATCAACTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAAGGATCATCCCTATCCTTGGTATACCAAACTACGCACAGAAA





TTCCAGGGCAGAGTCACCATTACCGCGGACAAATCCACGAGCACAGCCTTCATGGAGCTGAGC





AGCCTGAGATCTGAGGACACGGCCGTCTATTACTGTGCGAGAGGGAGGGGCTACAGTAACTAC





GGGGCCTCCTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA



122
light
CAGTCTGTGCTGACGCAGCCGCCGTCAGTGTCTGGGGGCCCAGGGCAGAGGGTCACCATCTCC





TGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCA





GAAACAGCCCCCAAACTCCTCATCTATGCTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGA





TTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGAT





GAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGGTTCGGTATTCGGCGGAGGG





ACCAAGCTGACCGTCCTA





COV2-
123
heavy
CAGCTGCAGCTACAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTATCCCTCACC


2128


TGCACTGTCTCTGGTGGCTCCATCAGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCC





CCAGGGAACGGGCTGGAGTGGATTGGGAGTATCTATTATAGTGGGAGCACCTACTACAACCCG





TCCCTCAAGGGTCGAGTCTCCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTG





AGCTCTGTGACCGCCGCAGACACGGCTGTCTATTACTGTGCGAGAATCTTAGTAATTTTTACT





TTAAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



124
light
AATTTTATGGTGACTCAGCCCCAGTCTGTGTCGGAGTGTCCGGGGAAGACGGTAACCATCTCC





TGCACCGGCAGCAGTGGCAGCATTGCGAGCAACTATGTGCAGTGGTAGCAGCAGGGCCCGGGC





AGTGCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGGTTC





TCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATGTCTGGAGTGAAGACTGAG





GACGAGGCTGACTACTACTGTCAGTCTTATGATAGCGGCAATCCCATATTCGGCGGAGGGACC





AAGCTGACCGTCCTA





COV2-
127
heavy
GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCC


2132


TGTGCAGCCTCTGAGGTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCACTTATTTATAGCGGTGGTACTACATACTACGCAGACTCCGTG





AAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACCCTGTATCTTCAAATGAACAGC





CTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGACTTTCTACGGTGGCACGACCTC





TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



128
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCAGGCGAGTCAGGACATTAACAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATTAAGGTTCAGT





GGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCA





ACATATTACTGTCAACAGTATGATAATCTCCCTCCAGTTTTCGGCGGAGGGACCAAGGTGGAG





ATCAAA





COV2-
129
heavy
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACC


2137


TGCACTGTCTCTGGTGGCTCCGTCAGCAGTGGTAGTTACTACTGGAGCTGGATCCGGCAGCCC





CCAGGGAAGGGACTGGAGTGCATTGGGTATATCTATTACAGTGGGAGCTCCAACTACAACCCC





TCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGATG





AGCTCTGTGACCGCTGCGGACACGGCCGTATATTACTGTGCGGGGAGCCCTGTCCCTCCCACG





ATTGTGGGAGCTTCGTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



130
light
AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCTTCTCC





TGCACCGGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCAGCAGCGCCCGGGC





AGTGCCCCCACCACTGTGATCTATGAGGATAACCAAAGACCCTCTGGGGTCCCTGATCGATTC





TCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACTGAG





GACGAGGCTGACTACTACTGTCAGTCTTATGATGGCATCAATCGGTGGCTGGTATTCGGCGGA





GGGACCAAGCTGACCGTCCTA





COV2-
131
heavy
GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCC


2142


TGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGACTGGGTGCGCCAGATGCCCGGG





AAAGGCCTGGAGTGGATGGGGATCATTTATCCTGGTGACTCTGATACCAGATATAGCCCGTCC





TTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCACCAGCACCGCCTACCTGCAGTGGAGC





AGCTTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACGTGGAGAAGCAGCTGGTATT





TGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCA



132
light
CAGCCTGTGCTGACTCAGCCACCTTCTGCATCAGCCTCCCTGGGAGCCTCGGTCACACTCACC





TGCACCCTGAGCAGCGGCTACAGTAATTATAAAGTGGACTGGTACCAGCAGAGACCAGGGAAG





GGCCCCCGGTTTGTGATGCGAGTGGGCACTGGTGGGATTGTGGGATCCAAGGGGGATGGCATC





CCTGATCGCTTCTCAGTCTTGGGCTCAGGCCTGAATCGGTATCTGACAATCAAGAACATCCAA





GAAGAGGATGAGAGTGACTACCACTGTGGGGCAGACCATGGCAGTGGGAGCAACTTCGAGTAT





GTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA





COV2-
133
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCC


2143


TGTGCAGCCTCTGGATTCACCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCAGTTATTTATAGCGCTGGTAGCACATACTACGCAGACTCCGTG





AAGGGCAGATTCAGCATCTCCAGAGACAAGTCCAAGAACACGCTGTATCTTCAAATGAACAGC





CTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAAAGAAGGTGGATCGGGGAGCCTCCGC





TACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



134
light
CAGTCTGTGGTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCT





TGTTCTGGAAGCAGCTCCAACATCGGATATAATATTGTAAACTGGTACCAGCAGCTCCCAGGA





ACGGCCCCCAAACTCCTCATCTATAGTAATAATCAGAGGCCCTCAGGGGTCCCTGACCGATTC





TCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGTCCATCAGTGGGCTCCAGTCTGAGGATGAG





GCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTTATGTCTTCGGAACTGGGACC





AAGGTCACCGTCCTA





COV2-
135
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2145


TGTGCAGCCTCTGGATTCACCTTCAGTACGTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACACTATATCTGCAAATGATC





GuCCTGAGAGCTGAGGACACGGCTGTGTATTACLGTGCGAGAGATTGGGCACCTACGTACTAC





GATATGCCGAGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA



136
light
TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACC





TGTGGGGGAAACAACATTGGAAATAAAGGTGTGCATTGGTACCAGCAGAAGCCAGGCCAGGCC





CCTGTGCTGGTCGTCGATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAACTCTGGGAACACGGCCACCCTGATCATCAGCAGTGTCGAAGTCGGGGATGAGGCCGAC





TTTTACTGTCAGGTGTGGGATAGTAGTAGTGATCATCCGGGGGTGTTCGGCGGAGGGACCAAG





CTGACCGTCCTA





COV2-
137
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCC


2146


TGTGAAGCCTCTGGATTCACCTTCAGTAGTTCTGAAATCAACTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTTTCACACATTAGTAGTAGTGGTAGTATCATATACTACGCAGACTCT





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAC





AGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGGAGATCTTATAGAAGCAGCTGG





TACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



138
light
GACATCCAGTTGACCCAGTCTCCATCTTTCCTGTCTGCTTCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCCAGTCAGGGCATTAGCAGTTATTTAGCCTGGTATCAGCAAAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGC





GGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCA





ACTTATTACTGTCAACAGCTTAATAGTTACCCCGTGACGTTCGGCCAAGGGACCAAGGTGGAA





ATCAAA





COV2-
139
heavy
CAGGTGCAGCTGGCGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2147


TGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAATGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGrGCGAGAAGCACGAGTGGGAGCTACTAC





TACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



140
light
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCC





TGCACTGGAACCAGCAGTGACGTTGGTGATTATAACTATGTCTCCTGGTACCAACAACACCCA





GGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGC





TTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGAC





GAGGCTGAATATTACTGCAGCTCATATACAAGCAGCAGCACTCTACTTTATGTCTTCGGAACT





GGGACCAAGGTCACCGTCCTA





COV2-
141
heavy
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAGGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2151


TGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGGCAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAGGGATCATCCCTGTCTTTGGTACAGCAAACTACGCACAGAAG





TTCCAGGGCCGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTTCATGGAGCTGAAC





AGCCTGAGATCTGAGGACACGGCCGTCTATTACTGTGCGAGAATTGGGAGCTACCCTGAATAC





TTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA



142
light
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTTCTTAGCCTGGTACCAACAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA





GTTTATTACTGTCACTACCGTAGCAACTGGCCTCCGGTTCTCACTTTCGGCGGAGGGACCAAG





GTGGAGATC





COV2-
143
heavy
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2153


TGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCATCTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAACAAACTACGCACAGAAG





TTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACGTGGAACTGAGC





AGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGAATAGGCCATTTTGATAGTAGT





GGTTATTACTTAGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



144
light
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTTCTTAGCCTGGTACCAACAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGCCCACTGGCATCCCAGCCAGGTTCACT





GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA





GTTTATTACTGTCAGCACCGTACCAACTGGCCTCCCTTATTCACTTTCGGCCCTGGGACCAAA





GTGGATATCAAA





COV2-
145
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2155


TGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGCTCTGTTCTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATTTCATATGATGGAAATAATAAATACTACGCAGACTCC





GTGAGGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGACCTGAGGACACGGCTGTGTATTACTGTGCGAGACCATATACTGGGAGCTACAAG





AGCTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA



146
light
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATC





AACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTCCTTAGCTTGGTAC





CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGG





GTCCCTGACCGATTCAGTGGCAGUGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG





CAGGCTGAAGATGTGGCAGTATATTACTGTCAGCAATATTATAGTATTTCTTGGACGTTCGGC





CAAGGGACCAAGGTGGAAATCAAA





COV2-
147
heavy
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACC


2158


TGCACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGTTACTTCTGGAGCTGGATCCGCCAGCAC





CCAGGGAAGGGCCTGGAGTGGATTGGGTCCATCTATTACAGTGGGAGCACCTACTACAACCCG





TCCCTCAGGAGTCGAATTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTG





AGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGGGGGGGTTCGGGGAGTTAT





TCTCTTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



148
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCAGGCGAGTCAGGACATTACCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGT





GGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCA





ACATATTACTGTCAACAGTATGATAATCTCTACTCTGTACACTTTGGCCAGGGGACCAAGCTG





GAGATCAAA





COV2-
149
heavy
CAGGTGCAGCTGGTGGAGTCCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2161


TGTGCAGCCTCTGGATTCACCTTCAGTAGGCATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTATGCAGACTCC





GTGAAGGGCCGATTCGCCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGACCTGAGGACACGGCTGTATATTACTGTGCGAGAGATCCGAGCCCGTTAGTGCTT





ATTACTTCAATTGACTACTGGGGCCAGGGAACCCTGGTCACCCTCTCCTCA



150
light
TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACC





TGCTCTGGAGATGCATTGCCAAGGCAATATACTTATTGGTACCAGCAGAAGCCAGGCCAGGCC





CCTGTGTTGGTGATATATAAAGACAGTGAGAGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAGCTCAGGGACAACAGTCACGTTGACCATCAGTGGAGTCCAGGCAGAAGACGAGGCTGAC





TATTACTGTCAATCAGCAGACACCATTGGTACTTATTGGGTATTCGGCGGAGGGACCAAGCTG





ACCGTCCT





COV2-
151
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCC


2162


TGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGACCTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCT





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTCTCTGCAAATGAAC





AGCCTGAGAGTCGAGGACACGGCTGTGTATTATTGTGTGAGACTGGGGGTCAGCAGCTGGTAC





TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTGTCCTCA



152
light
TCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACC





TGCTCTGGAGATAAATTGGGGGATAAATATGCTTGCTGGTATCAGCAGAAGCCAGGCCAGTCC





CCTGTGCTGGTCATCTATCAAGATACCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGAC





TATTACTGTCAGGCGTGGGGCAGTAGCAGGGGAGTGTTCGGCGGAGGGACCAAGCTGACCGTC





CTA





COV2-
153
heavy
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2164


TGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTGCAGCAAACTACGCACAGAAC





TTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGGCTACATGCAACTGAGC





AGCCTGAGATTTGAGGACACGGCCGTGTATTACTGTGCGAGAACGTCTCACTATGATAGTAGT





GGTTCCTATTTTGAATACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



154
light
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGACCCTGAAGATTTTGCA





GTTTATTACTGTCACAAGCGTAGCAACTGGCCTCCTTCGCTCACTTTCGGCGGAGGGACCAAG





GTGGAGATCAA





COV2-
771
hEavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTGGTACAGCCTGGGGGGTCCGTGAGACTCTCC


2000


TGTGCAGCCTCTGGATTCACCTTTCGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCAGCTATTAGTGATAATGCTTATAGCACATACTACGCAGACTCC





GTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAAATGAAC





AGCCTGAGAGCCGAGGACACGGCCATATATTACTGTGCGAAGAATCTTTATAGTGGAAACTCC





CCATTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



772
light
NNNTNTGTGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACC





TGCTCTGGAGATAAATTGGGGGATAAATATGCTTGCTGGTATCAGCAGAAGCCAGGCCAGTCC





CCTGTGCTGGTCATCTATCAAGATAGCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGAC





TATTACTGTCAGGCGTGGGACAGCAGCACTGCCGTCTTCGGAACTGGGACCAAGGTCACCGTC





CT





COV2-
773
hEavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2001


TGTGCAGCGTCTGGATTCACCTTCAGTAGTTATGGCATGCATTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATGGCATGATGGAAGTAAGAAATACTATGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCAAGGTGGATATGATTAC





GTTTGGGGGAGTTATCGATATACATTTTACGTCTTTGACTACTGGGGCCAGGGAACCCTGGTC





ACCGTCTCCTCA



774
light
NANTNTGTGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACA





TGCCAAGGAGACAGCCTCAGAAACTATTATGCCAGCTGGTACCAGCAGAAGCCAGGACAGGCC





CCTGTAGTTGTCATGTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGAGTCTCTGGC





TCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGAC





TATTATTGTAACTCCCGGGACAGCAGTGGTAACCATCTGATATTCGGCGGAGGGACCAAGCTG





ACCGTCCT





COV2-
775
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCC


2002


TGTGCAGCCTCTGGATTCACCTTTAGTTTCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTGGCCAACATAAAGCAAGATGGAAGTGAGAAATACTATGTGGACTCT





GTGAAGGGCCGATTCAGCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAC





AGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGTTGGTAGCAGCAGCTGGTAT





TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



776
light
NNNNTTGTGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACC





TGCTCTGGAGATAAATTGGGGGATAAATATGCTTGCTGGTATCAGCAGAAGCCAGGCCAGTCC





CCTGTGCTGGTCATCTATCAAGATATCAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGAC





TATTACTGTCAGGCGTGGGACAGCAACACTGGAGTGTTCGGCGGAGGGACCAAGCTGACCGTC





CT





COV2-
777
heavy
GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCC


2003


TGCAAGGCTTCTGGATACACCCTCACCAGGTATGATATCCACTGGGTGCGACAGGCCACTGGA





CAAGGGCTTGAGTGGATGGGATGGTTGAACCCTAACGGTGGCAACACAGGCTATGCACAGAAG





TTCCAGGGCAGAGTCACCATGACCAGAAACACCGCCATAAGCACAGCCTACATGGAGCTGAGC





AGCCTGAGATCTGAGGACGCGGCCGTGTATTACTGTGCGAGGGGTCAGTGGGAACTAGACGCT





TGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCCTCA



778
light
CAGTNTGTGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACA





TGCCAAGGAGACAGCTTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCC





CCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGC





TCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGAC





TATTATTGTAACTCCCGGGACACCAGTGGTAACCATCTAGATGTGGTATTCGGCGGAGGGACC





AAGCTGACCGTCCT





COV2-
779
heavy
GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCACTGAAGGTCTCC


2004


TGCAAGGCTTCTGGATACACCTTCACCCGTTATGATATCAACTGGGTGCGACAGGCCACTGGA





CAAGGGCTTGAGTGGATGGGATGGATGAACCCTAACAGTGATAACACAGGCTATGCACAGAAG





TTCCAGGACAGAGTCACCATGACCAGGAACACCTCCATAAGCACAGTCTACATGGAGCTGAGC





AGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGGGGTCAGTGGGAGCTAGACGTT





TGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCCTCA



780
light
CAGNNTGTGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACACTCAGGATCACA





TGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCC





CCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGC





TCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGAC





TATTACTGTAACTCCCGGGACAGCAGCGGTCACCATCTAGATGTGGTATTCGGCGGAGGGACC





AAGCTGACCGTCCT





COV2-
781
heavy
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACC


2005


TGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGG





AAGGGACTGGAGTGGATTGGGTGTATCTATTACAGTGGGCGCACCAACTACAGCCCCTCCCTC





AAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCTGTTCTCCCTGAAGCTGAGCTCT





GTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGGAGGCCGCCCTGGGGCTGAAGGA





CCTTATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCA



782
light
CAGNNTGTGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACA





TGCCAAGGAGACAACCTCAGAAGGTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCC





CCTGTACTAGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGC





TCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGAC





TATTACTGTAACTCCCGGGACAGCAGTGGTAACCATGTGGTATTCGGCGGAGGGACCACGCTG





ACCGTCCT





COV2-
783
heavy
CAGGTGCAGCTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCC


2954


TGCAAGGCTTCTGGATACACCTTCAGTGACTATGCTATGAATTGGGTGCGACAGGCCCCTGGA





CAAGGACTTGAGTGGATGGGATGGATGAAGTCTAACAGTGGTAACACAGGCTATGCACAGAAG





TTCCAGGGCAGAGTCACTATGACCAGGAACACCTCCATAAGTACAGCCTACATGGAGTTGACC





AGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAATGCGCAGTGGCTGGCCCACA





CATGGCCGCCCGGATGACCACTGGGGCCGGGGAGCCCTGGTCACCGTCTCCTCAG



784
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTGGGAGACAGAGTCACCATC





ACTTGCCGGGCAAGTCAGAGCATTATCAGCTATTTAAATTGGTATCACCAGAAACCGGGGAAA





GCCCCTAAACTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGT





GGCAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCA





ACATATTACTGTCAACAGTCTGATAATCTCCCCATGTACACTTTTGGCCAGGGGACCAAGCTG





GAGATCAAAC





COV2-
785
heavy
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2956


TGTGCAGCCTCTGGATTCACCTTCGTTACCTCTGGCATACACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGGAGGGCCAAACAAGGAAGTA





CTATATTTCGGGGAGTTATTGGACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACC





GTCTCCTCA



786
light
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATC





AACTGCAAGTCCAGCCAGAGTGTTTTATACAGTTCCGAAATTAAGAACTACTTAGCTTGGTAT





CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATTCGGG





GTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG





CAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTGGTCCCCTGGACACTTTT





GGCCAGGGGACCAAGCTGGAGATCAAAC





COV2-
787
heavy
CAGGTCCAGCTTGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCC


2957


TGCAAGGTCTCTGGATACACCTTCACTGGCTATGTTGTACATTGGGTGCGCCAGGCCCCCGGA





CAAGACCTTGAGTGGATGGGATGGATCAACACTGGCTACGGCAACACAAAATATTCACAGAAG





TTCCAGGGCAGGGTCACTATTAGCTGGGACACATCCGCGACGACAGCCTACATGGAGCTGAGC





AACCTGAAATCTGAGGACAAGGCTGTTTATTATTGTGCGAGTATGAACCGGATGTCAGAGCAA





ACTTACTACGGAATGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCC



788
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGT





GGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCA





ACATATTACTGTCAACAGTATGATAAGCTCACLCTCGGCGGAGGGACCAAGGTGGAGATCAAA





C





COV2-
789
heavy
CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACC


2958


TGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGG





AAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAATCCGTCCCTC





AAGAGTCGAGTCACCATATCAGTGGACACGTCCAAGAACCACTTCTCCCTGAAAATGAACTCT





GTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGATGTCGCCAGATGGGGAACTTCTAC





TACTACTACATGGACGTCTGGGGCAAGGGGACCACGGTCACCGTCTCC



790
light
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGTCCCAGGGCAGAGGGTCACCGTCTCC





TGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTTTGATGTATATTGGTACCAGCAGTTTCTA





GGAACAGCCCCCAAACTCCTCATCTATGGCAACAACAATCGGCCCTCAGGCGTCCCTGACCGA





TTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAAGAT





GAGGCTGATTATTACTGCCAGTCCTTTGACATCGGCCGGGGTGGTTGGATTTTCGGCGGAGGG





ACCAAGCTGACCGTCCTAG





COV2-
791
heavy
CAGGTGCACCTACAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACC


2959


TGCACTGTCTCTGGTGGCTCCATCAATAATTATTACTGGAGCTGGATCCGGCAGCCCCCGGGG





AAGGGACTGGAGTGGATTGGGGAAATCCATTACAGTGGGAGCACCAGCTACAGCCCCTCCCTC





AAGAGTCGACTCAGCATATCAGTAGACAGGTCCAAGAACCAGTTCTCCCTGAAGCTGGCCTCT





GTGACCGCTGCAGACACGGCCGTGTATTACTGTGTGAGGGATAATTACTTTGATAATAGTGGT





CATCCTGTGTATCCGGTTCCCTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCC





TCAG



792
light
CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGTCCCAGGGCAGAGGGTCACCGTCTCC





TGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTTTGATGTATATTGGTACCAGCAGTTTCTA





GGAACAGCCCCCAAACTCCTCATCTATGGCAACAACAATCGGCCCTCAGGCGTCCCTGACCGA





TTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAAGAT





GAGGCTGATTATTACTGCCAGTCCTTTGACATCGGCCGGGGTGGTTGGATTTTCGGCGGAGGG





ACCAAGCTCACCGTCC





COV2-
793
heavy
CAGGTTCAGGTGGTGCAGTGTGGAGCTGAGGTAAAGAAGCCTGGGGCCTCAGTGAAGGTCTCC


2960


TGCAAGGCTTCTGGTTACACCTTTAAGAACTATGGGATCAGTTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACACAGGAAACACAAACTATGCACAGAAG





TTCCAGGGCAGAATGACCATGACCACAGACACATCCACGGGAACAGGTTATATGGAACTGAGG





AGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGTACAACGACGTCGACTTGAC





TACTGGGGCCAGGGAACCCTGGTCATCGTCTCGTGAG



794
light
GATATTGTGGTGACCCAGACTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATC





TCCTGCAAGTCTAGTGAGACCCTCCTGCATAGTGATGGAAAGACCTATTTGTCTTGGTATCTG





CAGAAGCCAGGCCAGCCTCCACAGCTCCTGATCTATGAAGTTTCCAACCGGTTCTCTGGAGTG





CCAGACAGGTTCAGTGGCAGCGGGTCAGGAACAGATTTCACACTTAAAATCGGCCGGGTGGAG





GCTGAGGATGTTGGGCTTTATTACTGCATGCAAAGTATACAGCTCGCCTTCGGCCAAGGGACA





CGACTGGAAATTGAAC





COV2-
795
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2150


TGTGCAGCCTCTGGATTCACCTTCAGTACGTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACACTATATCTGCAAATGATC





GGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTGGGCACCTACGTACTAC





GATATGCCGAGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA



796
light
TCCTATGAGCTGACACAGCCACCCTCGGTGTCAGTGGCCCCAGGAAAGACGGCCAGGATTACC





TGTGGGGGAAACAACATTGGAAATAAAGGTGTGCATTGGTACCAGCAGAAGCCAGGCCAGGCC





CCTGTGCTGGTCGTCGATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAACTCTGGGAACACGGCCACCCTGATCATCAGCAGTGTCGAAGTCGGGGATGAGGCCGAC





TTTTACTGTCAGGTGTGGGATAGTAGTAGTGATCATCCGGGGGTGTTCGGCGGAGGGACCAAG





CTGACCGTCCTA





COV2-
797
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2159


TGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAATGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAAGCACGAGTGGGAGCTACTAC





TACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



798
light
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCC





TGCACTGGAACCAGCAGTGACGTTGGTGATTATAACTATGTCTCCTGGTACCAACAACACCCA





GGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGC





TTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGAC





GAGGCTGAATATTACTGCAGCTCATATACAAGCAGCAGCACTCTACTTTATGTCTTCGGAACT





GGGACCAAGGTCACCGTCCTA





COV2-
799
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2160


TGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAATGGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAAGCACGAGTGGGAGCTACTAC





TACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA



800
light
CAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCC





TGCACTGGAACCAGCAGTGACGTTGGTGATTATAACTATGTCTCCTGGTACCAACAACACCCA





GGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGC





TTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGAC





GAGGCTGAATATTACTGCAGCTCATATACAAGCAGCAGCACTCTACTTTATGTCTTCGGAACT





GGGACCAAGGTCACCGTCCTA





COV2-
801
heavy
GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2166


TGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCATCTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAACAAACTACGCACAGAAG





TTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACGTGGAACTGAGC





AGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGAATAGGCCATTTTGATAGTAGT





GGTTATTACTTAGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



802
light
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTTCTTAGCCTGGTACCAACAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGCCCACTGGCATCCCAGCCAGGTTCACT





GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA





GTTTATTACTGTCAGCACCGTACCAACTGGCCTCCCTTATTCACTTTCGGCCCTGGGACCAAA





GTGGATATCAAA





COV2-
803
heavy
GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2169


TGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTGCAGCAAACTACGCACAGAAC





TTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGGCTACATGCAACTGAGC





AGCCTGAGATTTGAGGACACGGCCGTGTATTACTGTGCGAGAACGTCTCACTATGATAGTAGT





GGTTCCTATTTTGAATACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



804
light
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGACCCTGAAGATTTTGCA





GTTTATTACTGTCACAAGCGTAGCAACTGGCCTCCTTCGCTCACTTTCGGCGGAGGGACCAAG





GTGGAAATCAAA





COV2-
805
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCC


2175


TGTGTAGCCTCTGGATTCACCTTTAGTTTCTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGA





AAGGGGCTCGAGTGGGTGGCCAACATAAAGCAAGATGGAGGTGAGAAATACTATGTGGACTCT





GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAAC





AGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACTGTCTGGCAGCAGCTGGGAC





TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



806
light
TCCTATGAGCTGACACAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGCATCACC





TGCTCTGGAGATAAATTGGGGGATAAATATGCTTGTTGGTACCAACAGAGGCCAGGCCAGTCC





CCTGTGCTGGTCATCTATCAAGATAGTAAGCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGC





TCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGAC





TATTACTGTCAGGCGTGGGACAGCAGCACTGGAGTCTTCGGAAGTGGGACCAAGGTCACCGTC





CTA





COV2-
807
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCC


2191


TGTGCAGCCTCTGGATTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCTGCTATTAATTGGAATGGTGGTAGCACAGGTTATGCAGACTCT





GTAAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACTCCCTGTATCTGCAAATGAAC





AGTCTGAGAGCCGAGGACACGGCCTTGTATCACTGTGCGAGACGGCGTAGCTCGTCCCGGTAT





AGCAGTGGCTGGTATATGTACTACTACTACATGGACGTCTGGGGCAAAGGGACCACGGTCACC





GTCTCCTCA



808
light
GACATCCAGATGACCCAGTUTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCCAGTCAGAGTGTTAGTACCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAACCTCCTGATCTATGAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGC





GGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCA





ACTTATTACTGCCAACAGTATAATACTTATTCGGGGACGTTCGGCCAAGGGACCAAGGTGGAA





ATCAAA





COV2-
809
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCC


2194


TGTGCAGCCTCTGGATTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCTGCTATTAATTGGAATGGTGGTAGCACAGGTTATGCAGACTCT





GTAAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACTCCCTGTATCTGCAAATGAAC





AGTCTGAGAGCCGAGGACACGGCCTTGTATCACTGTGCGAGACGGCGTAGCTCGTCCCGGTAT





AGCAGTGGCTGGTATATGTACTACTACTACATGGACGTCTGGGGCABAGGGACCACGGTCACC





GTCTCCTCA



810
light
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCCAGTCAGAGTGTTAGTACCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAACCTCCTGATCTATGAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGC





GGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCA





ACTTATTACTGCCAACAGTATAATACTTATTCGGGGACGTTCGGCCAAGGGACCAAGGTGGAA





ATCAAA





COV2-
811
heavy
GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2198


TGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTGCAGCAAACTACGCACAGAAC





TTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGGCTACATGCAACTGAGC





AGCCTGAGATTTGAGGACACGGCCGTGTATTACTGTGCGAGAACGTCTCACTATGATAGTAGT





GGTTCCTATTTTGAATACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



812
light
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGACCCTGAAGATTTTGCA





GTTTATTACTGTCACAAGCGTAGCAACTGGCCTCCTTCGCTCACTTTCGGCGGAGGGACCAAG





GTGGAGATCAAA





COV2-
813
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2203


TGTGCAGCCTCTGGATTCACCTTCAGTACCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGCGTGGGTGGCACTTATATCATATGATGGATATAATAAATACTACGCAGACTCC





GTGAGGGGCCGATTCACCATCTCCAGAATCAATTCCAAGAACACGCTGTCTCTGCAGATGAAC





AGCCTGAGAGCTGAGGACACGGCTGTCTATTACTGTGCGAGAGGGTCAGCTGGAAACTACTAC





TACGGTATGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCA



814
light
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCAAGTCAGACCATTACCAACTATTTAAATTGGTATCAGCTGAAATCAGGGAGA





GCCCCCAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGT





GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCA





ACTTACTACTGTCAACAGAGTTACAGTACGCCGTACACTTTTGGCCAGGGGACCAAGGTGGAG





ATCAAA





COV2-
815
heavy
GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2214


TGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCATCTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAACAAACTACGCACAGAAG





TTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACGTGGAACTGAGC





AGCCTGAGATCTGAGGACACGGCCGTGTATTATTGTGCGAGAATAGGCCATTTTGATAGTAGT





GGTTATTACTTAGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



816
light
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGAGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTTCTTAGCCTGGTACCAACAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGCCCACTGGCATCCCAGCCAGGTTCACT





GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA





GTTTATTACTGTCAGCACCGTACCAACTGGCCTCCCTTATTCACTTTCGGCCCTGGGACCAAA





GTGGATATCAAA





COV2-
817
heavy
GAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCC


2216


TGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGA





CAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTGCAGCAAACTACGCACAGAAC





TTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGGCTACATGCAACTGAGC





AGCCTGAGATTTGAGGACACGGCCGTGTATTACTGTGCGAGAACGTCTCACTATGATAGTAGT





GGTTCCTATTTTGAATACTGGGGCCAGGGAACCCTGGGAGGGTCTCCTCA



818
light
GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAG





GCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGT





GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGACCCTGAAGATTTTGCA





GTTTATTACTGTCACAAGCGTAGCAACTGGCCTCCTTCGCTCACTTTCGGCGGAGGGACCAAG





GTGGAAATCAAA





COV2-
819
heavy
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2218


TGTGCAGCCTCTGGATTCACCTTCAGTAGTTATGCTCTGTTCTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATTTCATATGATGGAAATAATAAATACTACGCAGACTCC





GTGAGGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAAC





AGCCTGAGACCTGAGGACACGGCTGTGTATTACTGTGCGAGACCATATACTGGGAGCTACAAG





AGCTACATGGACGTCTGGGGCAAAGGGACCACGGTCACCGTCTCCTCA



820
light
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATC





AACTGCAAGTCCAGCCAGAGTGTTTTATACAGCTCCAACAATAAGAACTCCTTAGCTTGGTAC





CAGCAGAAACCAGGACAGCCTCCTAAGCTGCPCATTJACTGGGCATCTACCCGGGAATCCGGG





GTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG





CAGGCTGAAGATGTGGCAGTATATTACTGTCAGCAATATTATAGTATTTCTTGGACGTTCGGC





CAAGGGACCAAGGTGGAAATCAAA





COV2-
821
heavy
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC


2224


TGTGCAGCCTCTGGATTCACCTTCAGTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGC





AAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCC





GTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAAC





AGCCTGAGAGCTGAGGACACGGCTATGTATTACTGTGCGAAAGATGGGAGTATAGCAGCAGCT





GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA



822
light
GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATC





AACTGCAAGTCCAGCCAGAGTGTCTTACACAGCTCCAACAACAAGGACTCCTTAGTTTGGTAC





CAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTAGCCGGGAATCCGGG





GTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTG





CAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAGTACTCCTTGGACGTTCGGC





CAAGGGACCAAGGTGGAAATCAAA





COV2-
823
heavy
GAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCC


2235


TGTGCAGCCTCTGGGTTCATCGTCAGTAGCAACTACATGAGCTGGGTCCGCCAGGCTCCAGGG





AAGGGGCTGGAGTGGGTCTCAGTTATTTATAGCGGTGGTAGCACATACTACGCAGACTCCGTG





AAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGC





CTGAGAGCCGAGGACACGGCCGTATATTACTCTGCGAGAGAGAGCACGCAATGGGGCCAGGGA





ACCCTGGTCACCGTCTCCTCA



824
light
GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATC





ACTTGCCGGGCCAGTCACAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAA





GCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTCCCATCAAGGTTCAGC





GGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCA





ACTTATTACTGCCAACAGTATAATACTTATTCTCAAACGTTCGGCCAAGGGACCAAGGTGGAG





ATCAAA





COV2-
825
heavy
GAGGTGCAGCTGGTGCAGTCTGGGCCTGAGGTGAAGAAGCCTGGGACCTCAGTGAAGGTCTCC


2961


TGCAAGGCTTCTGGATTCACCTTTATGAGCTCTGCTGTGCAGTGGGTGCGACAGGCTCGTGGA





CAACGCCTTGAGTGGATAGGATGGATCGTCATTGGCAGTGGTAACACAAACTACGCACAGAAG





TTCCAGGAAAGAGTCACCATTACCAGGGACATGTCCACAAGCACAGCCTACATGGAGCTGAGC





AGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGGCCCCATATTGTAGTAGTATCAGC





TGCAATGATGGTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA



826
light
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAGAGAGCCACCCTC





TCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGC





CAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTC





AGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTT





GCAGTGTATTACTGTCAGCACTATGGTAGCTCACGGGGTTGGACGTTCGGCCAAGGGACCAAG





GTGGAAATCAAA
















TABLE 2







PROTEIN SEQUENCES FOR ANTIBODY


VARIABLE REGION










Clone
Seq ID
Chain
Variable Sequence Region





COV2-
157
heavy
EVQLVESGGGLVQPGGSLRLSCVASGFTFSFYWMSWVRQAPGKG


2171


LEWVANIKQDGGEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRA





EDTAVYYCARLSGSSWDFDYWGQGTLVTVSS



158
light
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQRPGQSPV





LVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQ





AWDSSTGVFGTGTKVTVL





COV2-
159
heavy
EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKG


2173


LEWVSAINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRA





EDTALYHCARRRSSSRYSSGWYMYYYYMDVWGKGTTVTV



160
light
DIQMTQSPSTLSASVGDRVTITCRASQSVSTWLAWYQQKPGKAP





NLLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYC





QQYNTYSGTFGQGTKVEIK





COV2-
161
heavy
QITFKESGPTLVKPTETLTLTCTFSGFSVSTSGEGVGWIRQPPG


2177


KALEWLAVIYWDDDKRYSPSLKSRLTITRDTSKNQVVLTMTNMD





PVDTATYYCAHRLWFRDAFDIWGQGTTVTVSS



162
light
DIQMTQSPSSLSASVGDRVTITCRARQSISNYLNWYQQKPGKAP





KLLIYAASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC





QQTYSTFWTFGQGTNVEIK





COV2-
163
heavy
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYWMTWVRQAPGKG


2178


LEWVANIKQDGSEKYYVDSVKYRFTISRDNAKNSLYLQMNSLRA





EDTAVYYCARVGSSSWYFDYWGQGTLVTVSS



164
light
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPV





VVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQ





AWDSSTAVFGGGTKLTVL





COV2-
161
heavy
QLQLQESGPGLVKPSETLSLTCTVSGGSISSGTYYCGWIRQPPG


2179


KGLEWIGSTYYGGSTLYNPSLRGRVTISVDTSKNQFSLKLSSVT





AADTAVYYCARRGNYYDSKNWFDPWGQGTLVTVSS



166
light
EIVMTQSPATVSVSPGERATLSCRASQSVSSNLAWYQQKPGQAP





RLLIYGASTRATGIPARFSASGSGTEFTLTISSLQSEDFAVYYC





QQYNNWPPMYTFGQGTKVEIK





COV2-
167
heavy
QVQLVESGGGVVQPGRSLRVSCAASGFTFSSHGMHWVRQAPGKG


2181


LEWVSVIWYDGSNKYYADSVKGRFTISRDNSKNTLSLQMNSLRA





EDTAVYYCARESADISSRLDYWGRGTLVTVSS



168
light
SYELTQPPSVSVSPGQTARITCSGDALPTKYAYWYQQKSGQAPV





LVIYDDSKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCY





STDSSGNVFGTGTKVTVL





COV2-
169
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKG


2183


LEWVAGISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA





EDTAVYYCARADTMVRGTYFEYWGQGTLVTVSS



170
light
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGK





APKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADY





CCSSYTSSRAVLFGGGTKLTVL





COV2-
171
heavy
QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVAWIRQPPG


2184


KALEWLALIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMD





PVDTATYYCAHRLPTPQLLPSFENWFDPWGQGTLVTVSS



172
light
QSVLTQPPSVSEAPRQRVTISCSGGSSNIGNNAVNWYQQLPGKA





PKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYY





CASWDDSLIGPVFGGGTKLTVL





COV2-
173
heavy
EVQLVESGGGLVQPGGSLRLSCAVSGFTFSSYWMHWVRQAPGKG


2285


LVWVSRINSDGSSTSYADSVKGRFTISRDNAKNTLYLQMNSLRA





EDTAVYYCAREVEQLAHMVDYWGQGTLVTVSS



174
light
SYELTQPPSVSVSPGQTARITCSGDALPNQYAYWYQQKPGQAPV





LVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQ





SADSSGTSWVFGGGTKLTVL





COV2-
175
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKG


2186


LEWVAVISYDGINKYYADAVKGRFTISRDNSKNTLYLQMNSLRA





EDTAVYYCARPRSGSYYAYFDYWGQGTLVTVSS



176
light
DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAP





KSLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDVATYYC





QQYNSHPPTFGGGTKVEIK





COV2-
177
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSYYPMHWLWVRQAPG


2187


KGLEWVAVTSYDGTNKYYADSVKGRFTISRDNSKNTLYLQMNSL





RAEDTAVYYCARGGATNFDYWGQGTLVTVSS



178
light
SYVLTQPPSVSVAPGKTANITCGGNNIGRKSVHWYQQKSGQAPV





LVVYDDSDRPSGIPSRFSGSNSGNTATLTISRVEAGDEADYYCQ





VWDSSSDHPEWVFGGGTKLTVL





COV2-
179
heavy
EAQLVESGGGLVQPGRSLRLSCAASGFTFDDSAMHWVRQAPGKG


2189


LEWVSGISWNSGNVGYADSVKGRFTTSRDNAKNSLYLQMNSLRA





EDTALYYCTKASRYCSSTICYWNWFDPWGQGTLVTVSS



180
light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP





KLLIYGASSLQTGVPSRFSGSGSGTDFTLTIRSLQPEDFASYYC





QQSYSTPTFGGGTKVEIK





COV2-
181
heavy
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKG


2190


LEWVSYISSSGSAIYYADSVKGRFTISRDNAKNSLYLQMNSLRV





EDTAVYYCAREARSRYFDWLPSYYFDYWGQGTLVTVSS



182
light
QSALTQPASVSGSPGQSITISCTGTSSDIGGYNYVSWYQQHPGK





APKLLIYDVSNRPSGVSTRFSGSKSGNTASLTISGLQAEDEADY





YCSSYTSSSTHVVFGGGTKLTVL





COV2-
183
heavy
QVQLVQSGSELKKPGASVKVSCKASGYTFTTYAMNWVRQAPGQG


2192


LEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVNTAFLHIGSLKA





EDTAVYYCARDQDSGYPTYYYYYMDVWGKGTTVTVSS



184
light
DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQK





PGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDV





GVYYCMQSIQPPLTFGGGTKVEIK





COV2-
185
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMHWVRQAPGKG


2193


LAWVALISYDGYNKYYADSVRGRFTTSRINSKNTLSLQMNSLRA





EDTAVYYCARGSAGNYYYGMDVWGQGTTVTVSS



186
light
DIQMTQSPSSLSASVGDRVTITCRASQTITNYLNWYQLKSGRAP





KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC





QQSYSTPYTFGQGTKLEIK





COV2-
187
heavy
QVHLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKG


2395


LEWVAVISNDEFNKFYANSVKGRFTISRDNSKNTVYLQLNSLRT





EDTARYYCAKGGDGSGWAWDGDNPPTDYWGQGTLVIVSS



188
light
DIVMTQSPDFLAVSLGERATISCKSSQSVLYTPKNKNYLAWYKQ





KPGQPPKVLIYWASTRESGVPDRESGSGSGTDFTLTISSLQAED





AAVYYCQQYYTAPLTFGGGTRVEI





COV2-
191
heavy
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKG


2197


LEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKA





SDTAMYYCARPDYSSGWPSYWYFDLWGRGTLVTVSS



192
light
EIVLTQSPGTLSLSPGERATLSCRASQSVSSNFLAWYQQKPGQA





PRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY





CQQYGRSPITFGQGTRLEIK





COV2-
193
heavy
QVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSIHWVRQAPGKG


2199


LEWMGGFDPEDAETIYAQQFQGRVTMTEDTSTDTAYMELSSLKS





EDTALYYCATGFAVFGRAAVPYWGQGTLVTVSS



194
light
SYELTQPPSVSVSPGQTASITCFGDKLGDKYACWFQQKPGQSPV





LIIYQGAKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQ





AWDSSTVVFGGGTKLTVI





COV2-
195
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG


2200


LEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA





EDTAVYYCAKDLTIVVIPAAPNFDYWGQGTLVTVSS



196
light
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGK





APKLMIYDVSNRPSGVSNRFSGSRSGNTASLTISGLQAEDEADY





YCSSYTSSSTPVVFGGGTKLTVL





COV2-
197
heavy
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSHWIGWVRQMPGKG


2207


LEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKA





SDTAMYYCASALRERGVQLWSVWGQGTLVTVSS



198
light
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGT





APKLLIFINSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADY





YCQSYDSSLGALFGGGTKLTVL





COV2-
199
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKG


2210


LEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRV





EDTAVYYCARDQEWFRELFLFDYWGQGTLVTVSS



200
light
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAP





KLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC





QQANSFPPTFGPGTKVDIK





COV2-
201
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKG


2211


LEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA





EDTAMYYCAKDGSIAAADYWGQGTLVTVSS



202
light
DIVMTQSPDSLAVSLGERATINCKSSQSVLHSSNNKDSLVWYQQ





KPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAED





VAVYYCQQYYSTPWTFGQGTKVEIK





COV2-
203
heavy
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKG


2212


LEWVSSISNSNSFTYYADSMKGRFTTSRDNAKNSLYLQMNSLRA





EDTAVYYCARVNGNSNWNFGSYYYYYMDVWGKGTTVTVSS



204
light
EIVLTQSPAILSLSPGERATLSCRASQSVSSYLAWYQQKPGQAP





RLLIYDTSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAFYYC





QQRGNWWTFAQGTKVEIK





COV2-
205
heavy
EVQLVESGGGLVKPGGSLRLSCAASGFTFSGYSMNWVRQAPGKG


2215


LEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRA





EDTAVYYCARWLQLRSDYYYFGMDVWGQGTTVTVSS



206
light
EIVMTQSPATLSVSPGERATLSCRASQSVSNNLAWYQHKPGQAP





RLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYPC





QQCYNWPPWTFGQGTKVEIK





COV2-
207
heavy
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWTRQPPGKG


2222


LEWIGYIYYSGSTNYNPSLKSRVTISVDMSKNQFSLKLRSVTAA





DTAVYYCARAPRERLQWGEYYPDYWGQGTLVTVSS



208
light
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHAGK





APKLMIYEVIKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADY





YCCSYAVSTTYVIFGGGTKLTVL





COV2-
209
heavy
EVQLVQSGAEVKKPGESLKISCKGSGYSFTNSWIGWVRQMPGKG


2226


LEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKA





SDTAIYYCATHRCSGGFCYLAYWGQGTLVTVSS



210
light
QPVLTQPPSASASLGASVTLTCTLSSGYSNYKVDWYQQRPGKGP





RPVMRVGTGGIVGSKGDGIPDRFSVLGSGLNRYLTIKNIQEEDE





SDYHCGADHGSGSNFVFVVFGGGTKLTVL





COV2-
211
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG


2227


LEWVAVIWYDGSKKDYADSVKGRFTISRDNSKNTLYLQMNSLRA





EDTAVYYCARDQSQGAYILTGYRGYGMDVWGQGTTVTVSS



212
light
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQK





PGQSPQFLIYLGSNRASGVPDRFSGSGSGTDFILKISRVEAEDV





GVYYCMQALQTPFTFGPGTKVDIK





COV2-
213
heavy
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGHYWSWTRQPPGKG


2228


LEWIGETNHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAA





DTAVYYCARPPQAARIHYYYYMDVWGKGTTVTVSS



214
light
ETVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAP





RLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC





QQYNYWPPLTFGGGTKVEIK





COV2-
215
heavy
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMNWVRQPPGKG


2231


LEWVSGISWNSDSIGYADSVKGRFTISRDNAKNSLYLQMNSLRA





EDTAMYYCAKGRGAGYTSYMDVWGKGTTVTVSS



216
light
SYVLTQPPSVSVAPGKTARITCEGNNIGSKSVHWYQQKPGQAPV





LVVYDDSGRPSGIPERFSGSNSGNTATLTISRVEAGDEADYFCQ





VWDSSSDHHVVFGGGTKLTVL





COV2-
217
heavy
YTTLFRSGGHWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQG


2233


QVTISADKSISTAYLQWSSLKASDTAMYYCASALRERGVQLWSV





WGQGTLVTVSS



218
light
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGT





APKLLIFINSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADY





YCQSYDSSLGALFGGGTKLTVL





COV2-
219
heavy
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKG


2046


LEWVSGISWNSGSIAYTDSVKGRFTISRDNAKNSLYLQMNSLRA





EDTALYYCAKAHSTGHQYYYGMDVWGQGTTVTVSS



220
light
DIQMTQSPSSLSASVGDRVTITCRASQSISSFLNWYQQKPGKAP





KLLIYAAFNLQSGVPSRFSGSGSGTDFTLTISSLQSEDFATYYC





QQSYNTPYTFGQGTKLEIK





COV2-
221
heavy
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKG


2047


LEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRA





EDTALYYCAKVSSITSLLGYYFDSWGQGTLVTVSS



222
light
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAP





RLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC





QHRSNWPPRLTFGGGTKVEIK





COV2-
223
heavy
QVQLVQSGAEVKKPGSSVKVSCKTSGDISSSYTVGWVRQAPGQG


2048


LEWMGRIIPILGIAYSAQKFQGRLTITADKSTSTSYMELSSLRS





EDTAVYYCARGVVAATPGWFDPWGQGTLVTVSS



224
light
ETVMTQSPATLSVSPGERVTLSCRASQSVSSNLAWYQQKPGQAP





RLLIYGASTRATGIPARFSGGGSGTEFTLTISSLQSEDFAVYYC





QQYNNFLTFGGGTKVEIK





COV2-
225
heavy
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQVPGKG


2049


LVWVSRINSDGSSTSYADSVKGRFTISRDNAKNTLYLEMNSLRA





QDTAVYYCAGSPWLRGDIDYWGQGTLVTVSS



226
light
NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSA





PTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLMTEDEAD





YYCQSYDGSNHAVVPGGGTKLTVL





COV2-
227
heavy
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQG


2050


LEWMGWINPNSRGTNYAQKFQGRVTMTRDTSISTVYMELSRLTS





DDTAVYYCARVVVLGYGRPNNYYDGRNVWDYWGQGTLVTVSS



228
light
QSVLTQPPSASGTPGQRVIISCSGSSSNIGSNTVKWYHQLPGTA





PKLLICSNNQRPSGVPDRFSGSKSDTSASLAISGLQSEDEADYY





CAAWDDSLNALVHGGGTKLTVL





COV2-
229
heavy
QVTLRESGPALVKPTQTLSLTCTPSGFSLGTSGMCVSWIRQPPG


2051


KALEWLARIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMD





PVDTATYYCARGVVTYDYWGQGTLVTVSS



230
light
DIQMTQSPSSLSASVGDRVTITCRASQSIAGYLNWYQQKPGKAP





KLLIYGTTSLQSGVPVRFSGSGSGTDFTLTISSLQPEDFATYYC





QQSYSTPGTFGQGTRLEIK





COV2-
231
heavy
QVQLVQSGAEVKKPGSSVKVSCKASGDTFSSYTINWVRQAPGQG


2054


LEWMGRIIPILGIPNYAQKFQGRVTITADKSTSTAFMELSSLRS





EDTAVYYCARGRGYSNYGASYYMDVWGKGTTVTVSS



232
light
DIQMTQSPSSLSASVGDRVTITCQASQDINHYLNWYQQKPGKAP





KLLIYDASNLETGVPSRFSGSGSGTDFTFTITSLQPEDVATYYC





QQSDNLPMYTFGQGTKLEIK





COV2-
233
heavy
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMNWVRQPPGKG


2055


LEWVSGISWNSDSIGYADSVKGRFTISRDNAKNSLYLQMNSLRA





EDTAMYYCAKGRGAGYTSYMDVWGKGTTVTVSS



234
light
SYVLTQPPSVSVAPGKTARITCEGNNIGSKSVHWYQQKPGQAPV





LVVYDDSGRPSGIPERFSGSNSGNTATLTISRVEAGDEADYFCQ





VWDSSSDHHVVFGGGTKLTVL





COV2-
237
heavy
QVQLAQSGAEVKKPGASVKVSCKAAGYTFTSYDINWVRQATGQG


2064


LEWMGWMNSNSGNAGYAQKFQGRVTMTRDTSTSTAYMELSSLTS





DDTAVYYCARMRTGWPTHGRPDDFWGRGTLVTVSS



238
light
QSVLTQPPSASGTPGQRVTISCSGSNSNIGSYTVNWYQQPPGTA





PKLLIYDNNQRTSGVPDRFSGSKSGTSASLAISGLQSEDEANYY





CLVWDDSLNGLVFGGGTKLTVL





COV2-
239
heavy
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKG


2068


LEWVSVIYPGGSAFYADSVKGRFTISRHNSNNTLCLQMNSLRTE





DTAVYYCARSYDILTGYRDAFDIWGQGTMVTVSS



240
light
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGSGSDVHWYQQLPGT





APKLLIYGNTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADY





YCQSYDSRLSGPVVFGGGTKLTVL





COV2-
241
heavy
EVQLVESGGGLVQPGGSLRLSCAASGLTVSSNYMSWVRQAPGKG


2069


LECVSVIYAGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE





DTAVYYCARGDGGYYSPPDYWGQGTLVTVSS



242
light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP





KVLIYAASTMQSGVPSRFRGSGSGTDFTLTISSLQLEDFATYYC





QQSYSTPQTFGQGTKVEIK





COV2-
243
heavy
QVQLQQWGAGLLKPSETLSLTCAVSGGSFSAYYWSWIRQPPGKG


2070


LEWIGEINHSGSTNYNPSLRSRVTISVDTSKNQFSLKLSSVTAA





DTAVYYCARVGYSQGYYYYYMDVWGKGTTVTVSS



244
light
DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAP





KLLIYAASSLQSGVPSRFSGSGSGTDFSLTISSLQPEDFATYSC





QQSYTTLLTFGGGTKVEIK





COV2-
247
heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYSITWVRQAPGQG


2078


LEWMGRIIPVLGIANYAQKFQDRVTITADKSTSTAYMELSSLRS





EDTAVYYCARVGVSGFKSGSNWYFDLWGRGTLVTVSS



248
light
QSVLTQPPSVSGAPGQRVTLSCTGSNSNIGAGYDVHWYQQLPGT





APKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADY





YCQSYDSSLSDSVFGGGTKVTVL





COV2-
251
heavy
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQG


2081


LEWMGIINPGGGSTTYAQKFQGRVTMTSDTSTSTVYMELSSLRS





EDTAMYYCARGAIPPNSRAEIDYWGQGTLVTVSS



252
light
ETVMTQSPATLSVSPGERVTLSCRASQSVSSNLAWCQQKPGQAP





RLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYC





QQYYNWPLTFGGGTKVEIK





COV2-
253
heavy
EVQLVESGGGVVRPGGSLRLSCAASGFIFDDYDMTWVRQAPGKG


2082


LEWVSGISWNGGNTGYADSVKGRFTISRDNAKNSLYLQMNSLRA





EDTALYHCAVIMSPIPRYSGYDWAGGAFDIWGQGTMVTVSS



254
light
SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQVPI





LVIYDKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCN





SRDSSGNAVVFGGGTKLTVL





COV2-
265
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKG


2083


LEWVAVMSYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA





EDTAVYYCAKNLGPYCSGGTCYSLVGDYWGQGTLVTVSS



256
light
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP





KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC





QQYANLPFTFGPGTKVDIK





COV2-
261
heavy
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKG


2097


LEWVSGISWNSGTIGYADSVKGRFITSRDNAKNSLYLQMNSLRP





EDTALYYCAKDIIRQGEDGMDVWGQGTTVTVSS



262
light
DIQMTQSPSSLSASVGDRVTITCRASQNIASYLNWYQQKPGKAP





KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEEFATYYC





QQSYSTPWTFGQGTKVEIK





COV2-
263
heavy
EVQLLESGGGLIQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKG


2098


LEWVSGIISTSGGATYNADSVRGRFTTSRDNSKNILYLQMNSLR





GEDTAVYYCVKGLFDWFPLWGQGTMVTVSS



264
light
DIVMTQSPATLSVSPGERAILSCRASQSVRSNLAWYQQKPGQAP





RLLISGASTRATAIPARFSGSGSGTEFTLTITSLQSEDCAVYYC





HQYNNWPQTFGQGTKVEIK





COV2-
265
heavy
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRHWMTWVRQAPGKG


2103


EEWVANIKQDGSEKYYVDSVKGRLTISRDNAKNSLYLQMNSLRA





TAVYYCARLGFYYGGADYWGQGTLVTVSS



266
light
NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSA





PTTVISEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAD





YYCQSYDGINRAWVFGGGTKLTVL





COV2-
267
heavy
NVQLVESGGGLVQPGGSLRLSCAASGFTFHHYAMHWVRQAPGKG


2108


LEWVSGISGSSDYRAYADSLKGRFTISRDYAKNSLWLQMNSLTS





EDTAFYYCAKGVDYGGKLAYFDSWGQGTLVTVSS



268
light
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSLGYNSLSWYLQK





PGQSPHLLIYLGSNRASGVPDRFSGSGSATDPTLKISRLEAEDV





GVYYCMQALQTPLTFGQGTRLEIK





COV2-
269
heavy
EVQLVESGGGVVQPGRSLRLSCAASGFSFSSYVMNWVRQAPGKG


2110


EWVAVISYDGSSKYYADSVKGRFTISRDNSKNTLYLQMNSLRA





DTAVYYCARDIDSGYDPTPVFDYWGQGTLVTVSS



270
light
DIQMTQSPSSLSACVGDRVTITCRASQSISSYLNWYQQKPGKGP





KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC





QQSYSSLSITFGQGTRLEIK





COV2-
271
heavy
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDLHWVRQGTGKR


2111


LEWVSAIGTAGDTYYLGSVKGRFTISRENAKNSLYLQMNSLRAG





DTAVYYCARVLYDSSGFYNWFDPWGQGTLVTVSS



272
light
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAP





KLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC





QQSYEIPPWTFGQGTKVEIK





COV2-
273
heavy
EVQLVESGGGLIQPGGSLRLSCAASEVTVSSNYMSWVRQAPGKG


2113


LEWVSLIYSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE





DTAVYYCARDFLRWHDLWGQGTLVTVSS



274
light
DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAP





KLLIYDASNLETGVPLRFSGSGSGTDFIFTISSLQPEDIATYYC





QQYDNLPPVFGGGTKVEIK





COV2-
275
heavy
QVQLVQSGAEVKKPGSSVKVSCKASGDTFSSYTINWVRQAPGQG


2114


LEWMGRIIPILGIPNYAQKFQGRVTITADKSTSTAFMELSSLRS





DTAVYYCARGRGYSNYGASYYMDVWGKGTTVTVSS



276
light
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPET





APKLLIYANSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADY





YCQSYDSSLSGSVFGGGTKLTVL





COV2-
277
heavy
QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPG


2128


NSLEWTGSIYYSGSTYYNPSLKGRVSISVDTSKNQPSLKLSSVT





ADTAVYYCARILVIFTLNWFDPWGQGTLVTVSS



278
light
NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSA





PTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAD





YCQSYDSGNPIFGGGTKLTVL





COV2-
281
heavy
EVQLVESGGGLIQPGGSLRLSCAASEVTVSSNYMSWVRQAPGKG


2132


LEWVSLIYSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE





DTAVYYCARDFLRWHDLWGQGTLVTVSS



282
light
DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAP





KLLIYDASNLETGVPLRFSGSGSGTDFTFTISSLQPEDIATYYC





QQYDNLPPVPGGGTKVEIK





COV2-
283
heavy
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPG


2237


KGLECIGYIYYSGSSNYNPSLKSRVTISVDTSKNQFSLKMSSVT





AADTAVYYCAGSPVPPTIVGASYWGQGTLVTVSS



284
light
NFMLTQPHSVSESPGKTVTFSCTGSSGSIASNYVQWYQQRPGSA





PTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAD





YYCQSYDGINRWLVFGGGTKLTVL





COV2-
285
heavy
EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIDWVRQMPGKG


2142


LEWMGIIYPGDSDTRYSPSFQGQVTISADKSTSTAYLQWSSLKA





SDTAMYYCARRGEAAGIWYFDLWGRGTLVTVSS



286
light
QPVLTQPPSASASLGASVTLTCTLSSGYSNYKVDWYQQRPGKGP





RPVMRVGTGGIVGSKGDGIPDRFSVLGSGLNRYLTIKNIQEEDE





SDYHCGADHGSGSNFEYVVFGGGTKLTVL





COV2-
287
heavy
EVQLVESGGGLVQPGGSLRLSCAASGFTVSSNYMSWVRQAPGKG


2143


LEWVSVIYSAGSTYYADSVKGRFSISRDKSKNTLYLQMNSLRAE





DTAVYYCAKEGGSGSLRYYYYGMDVWGQGTTVTVSS



288
light
QSVVTQPPSASGTPGQRVTISCSGSSSNIGYNIVNWYQQLPGTA





PKLLIYSNNQRPSGVPDRFSGSKSGTSASLSISGLQSEDEADYY





CAAWDDSLNGYVFGTGTKVTVL





COV2-
289
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMHWVRQAPGKG


2145


LEWVAVISYDGSNKYYADSVKGRFTTSRDNSKNTLYLQMIGLRA





EDTAVYYCARDWAPTYYDMPSAFDIWGQGTMVTVSS



290
light
SYVLTQPPSVSVAPGKTARITCGGNNIGNKGVHWYQQKPGQAPV





LVVDDDSDRPSGIPERFSGSNSGNTATLIISSVEVGDEADFYCQ





VWDSSSDHPGVFGGGTKITVL





COV2-
291
heavy
EVQLVESGGGLVQPGGSLRLSCEASGFTFSSSEINWVRQAPGKG


2146


LEWVSHISSSGSIIYYADSVKGRFTISRDNAKNSLYLQMNSLRA





EDTAVYYCARRSYRSSWYYYYGMDVWGQGTTVTVSS



292
light
DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAP





KLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYC





QQLNSYPVTFGQGTKVEIK





COV2-
293
heavy
QVQLAESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKG


2147


LEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRA





EDTAVYYCARSTSGSYYYGMDVWGQGTTVTVSS



294
light
QSALTQPASVSGSPGQSITISCTGTSSDVGDYNYVSWYQQHPGK





APKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEAEY





YCSSYTSSSTLLYVFGTGTKVTVL





COV2-
295
heavy
QVQLVQSGAEVRKPGSSVKVSCKASGGTFSSYAISWVRQAPGQG


2151


LEWMGGIIPVFGTANYAQKFQGRVTITADKSTSTAFMELNSLRS





EDTAVYYCARIGSYPEYPQHWGQGTLVTVSS



296
light
ETVLTQSPATLSLSPGERATLSCRASQSVSSFLAWYQQKPGQAP





RLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC





HYRSNWPPVLTFGGGTKVEI





COV2-
297
heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIIWVRQAPGQG


2153


LEWMGGIIPIFGTTNYAQKFQGRVTITADESTSTAYVELSSLRS





EDTAVYYCARIGHFDSSGYYLDYWGQGTLVTVSS



298
light
EIVLTQSPATLSLSPGERATLSCRASQSVSSFLAWYQQKPGQAP





RLLIYDASNRPTGIPARFTGSGSGTDFTLTISSLEPEDFAVYYC





QHRTNWPPLPTFGPGTKVDIK





COV2-
299
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYALFWVRQAPGKG


2155


LEWVAVISYDGNNKYYADSVRGRFTISRDNSKNTLYLQMNSLRP





EDTAVYYCARPYTGSYKSYMDVWGKGTTVTVSS



300
light
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNSLAWYQQ





KPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAED





VAVYYCQQYYSISWTFGQGTKVEIK





COV2-
301
heavy
QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYFWSWIRQHPG


2158


KGLEWIGSIYYSGSTYYNPSLRSRITISVDTSKNQFSLKLSSVT





AADTAVYYCARGGSGSYSLFDYWGQGTLVTVSS



302
light
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKPGKAP





KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDFATYYC





QQYDNLYSVHFGQGTKLEIK





COV2-
303
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFSRHAMHWVRQAPGKG


2161


LEWVAVISYDGSNKYYADSVKGRFAISRDNSKNTLYLQMNSLRP





EDTAVYYCARDPSPLVLITSIDYWGQGTLVTVSS



304
light
SYELTQPPSVSVSPGQTARITCSGDALPRQYTYWYQQKPGQAPV





LVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQ





SADTIGTYWVFGGGTKLTVL





COV2-
305
heavy
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVRQAPGKG


2162


LEWVANTRQDGSERYYVDSVKGRFTTSRDNAKNSLSLQMNSLRV





EDTAVYYCVRLGVSSWYPDYWGQGTLVTVSS




light
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPV





LVIYQDTKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQ





AWGSSRGVFGGGTKLTVL





COV2-
307
heavy
QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQG


2164


LEWMGGIIPIFGAANYAQNFQGRVTITADESTSTGYMQLSSLRF





EDTAVYYCARTSHYDSSGSYFEYWGQGTLVTVSS



308
light
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAP





RLLIYDASNRATGIPARFSGSGSGTDFTLTISSLDPEDFAVYYC





HKRSNWPPSLTFGGGTKVEI





COV2-
827
heavy
EVQLVESGGDLVQPGGSLRLSCAASGFTFRSYAMSWVRQAPGKG


2000


LEWVSAISDNAYSTYYADSVKGRFTISRDNSKNTLYLQMNSLRA





EDTAIYYCAKNLYSGNSPFDYWGQGTLVTVSS



828
light
XXVLTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPV





LVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQ





AWDSSTAVFGTGTKVTVI





COV2-
829
heavy
EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKG


2001


LEWVAVIWHDGSKKYYADSVKGRFTISRDNSKNTLYLQMNSLRA





EDTAVYYCARDQGGYDYVWGSYRYTFYVFDYWGQGTLVTVSS



830
light
XXVLTQDPAVSVALGQTVRITCQGDSLRNYYASWYQQKPGQAPV





VVMYGKNNRPSGIPDRVSGSSSGNTASLTITGAQAEDEADYYCN





SRDSSGNHLIFGGGTKLTVL





COV2-
831
heavy
EVQLVESGGGLVQPGGSLRLSCAASGFTFSFYWMSWVRQAPGKG


2002


LEWVANTRQDGSERYYVDSVKGRFSTSRDNAKNSLYLQMNSLRA





EDTAVYYCARVGSSSWYPDYWGQGTLVTVSS



832
light
XXVLTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQKPGQSPV





LVIYQDIKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQ





AWDSNTGVFGGGTKLTVL





COV2-
833
heavy
EVQLVQSGAEVKKPGASVKVSCKASGYTLTRYDIHWVRQATGQG


2003


LEWMGWLNPNGGNTGYAQKFQGRVTMTRNTAISTAYMELSSLRS





EDAAVYYCARGQWELDAWYFDLWGRGTLVTVSS



834
light
QXVLTQDPAVSVALGQTVRITCQGDSFRSYYASWYQQKPGQAPV





LVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCN





SRDISGNHLDVVFGGGTKLTVL





COV2-
835
heavy
EVQLVQSGAEVKKPGASLKVSCKASGYTFTRYDINWVRQATGQG


2004


LEWMGWMNPNSDNTGYAQKFQDRVTMTRNTSISTVYMELSSLRS





EDTAVYYCARGQWELDVWYFDLWGRGTLVTVSS



836
light
QXVLTQDPAVSVALGQTLRITCQGDSLRSYYASWYQQKPGQAPV





LVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCN





SRDSSGHHLDVVFGGGTKLTVL





COV2-
837
heavy
QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKG


2005


LEWIGCIYYSGRTNYSPSLKSRVTISVDTSKNLFSLKLSSVTAA





DTAVYYCARGGRPGAEGPYDAFDIWGQGTMVTVSS



838
light
QXVLTQDPAVSVALGQTVRITCQGDNLRRYYASWYQQKPGQAPV





LVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCN





SRDSSGNHVVFGGGTTLTVL





COV2-
839
heavy
QVQLVQSGSELKKPGASVKVSCKASGYTFSDYAMNWVRQAPGQG


2954


LEWMGWMKSNSGNTGYAQKFQGRVTMTRNTSISTAYMELTSLRS





EDTAVYYCARMRSGWPTHGRPDDHWGRGALVTVSS



840
light
DIQMTQSPSSLSASVGDRVTITCRASQSIISYLNWYHQKPGKAP





KLLIYAASSLQSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC





QQSDNLPMYTFGQGTKLEIK





COV2-
841
heavy
QVQLVESGGGVVQPGRSLRLSCAASGFTFVTSGIHWVRQAPGKG


2956


LEWVAVISYDGSNKYYADSVKGRPTISRDNSKNTLYLQMNSLRA





EDTAVYYCAKGGPNKEVLYFGELLDYGMDVWGQGTTVTVSS



842
light
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSEIKNYLAWYQQ





KPGQPPKLLIYWASTREFGVPDRFSGSGSGTDFTLTISSLQAED





VAVYYCQQYYSGPLDTFGQGTKLEIK





COV2-
843
heavy
QVQLVQSGAEVKKPGASVKVSCKVSGYTFTGYVVHWVRQAPGQD


2957


LEWMGWINTGYGNTKYSQKFQGRVTISWDTSATTAYMELSNLKS





EDKAVYYCASMNRMSEQTYYGMDVWGQGTTVTVS



844
light
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAP





KLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC





QQYDKLTLGGGTKVEIK





COV2-
845
heavy
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKG


2958


LEWTGEINHSGSTNYNPSLKSRVTTSVDTSKNHFSLKMNSVTAA





DTAVYYCARCRQMGNFYYYYMDVWGKGTTVTVS



846
light
QSVLTQPPSVSGVPGQRVTVSCTGSSSNIGAGFDVYWYQQFLGT





APKLLIYGNNNRPSGVPDRFSASKSGTSASLAITGLQAEDEADY





YCQSFDIGRGGWIFGGGTKLTVL





COV2-
847
heavy
QVHLQESGPGLVKPSETLSLTCTVSGGSINNYYWSWIRQPPGKG


2959


LEWIGEIHYSGSTSYSPSLKSRLSISVDRSKNQPSLKLASVTAA





DTAVYYCVRDNYFDNSGHPVYPVPWFDPWGQGTLVTVSS



848
light
QSVLTQPPSVSGVPGQRVTVSCTGSSSNIGAGFDVYWYQQFLGT





APKLLIYGNNNRPSGVPDRFSASKSGTSASLAITGLQAEDEADY





YCQSFDLGRGGWIFGGGTKLTV





COV2-
849
heavy
QVQVVQSGAEVKKPGASVKVSCKASGYTFKNYGISWVRQAPGQG


2960


LEWMGWISAYTGNTNYAQKFQGRMTMTTDTSTGTGYMELRSLRS





DDTAVYYCARVQRRRLDYWGQGTLVIVSS



850
light
DIVVTQTPLSLSVTPGQPASISCKSSETLLHSDGKTYLSWYLQK





PGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKIGRVEAEDV





GLYYCMQSIQLAFGQGTRLEIE





COV2-
851
heavy
EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMHWVRQAPGKGL


2150


EWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMIGLRAED





TAVYYCARDWAPTYYDMPSAFDIWGQGTMVTVSS



852
light
SYELTQPPSVSVAPGKTARITCGGNNIGNKGVHWYQQKPGQAPVI





VVDDDSDRPSGIPERFSGSNSGNTATLIISSVEVGDEADFYCQVW





DSSSDHPGVFGGGTKLTVI





COV2-
853
heavy
EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGI


2159


EWVAVISYDGSNKYYADSVKGRFTTSRDNSKNTLYLQMNSLRAED





TAVYYCARSTSGSYYYGMDVWGQGTTVTVSS



854
light
QSALTQPASVSGSPGQSITISCTGTSSDVGDYNYVSWYQQHPGKA





PKLMIYDVSNRPSGVSNRFSGSRSGNTASLTISGLQAEDEAEYYC





SSYTSSSTLLYVPGTGTKVTVL





COV2-
855
heavy
EVQLVESGGGVVQPGRSLRLSCAASGHTPSSYAMHWVRQAPGKGL


2160


EWVAVISYDGSNKYYADSVKGRFTTSRDNSKNTLYLQMNSLRAED





TAVYYCARSTSGSYYYGMDVWGQGTTVTVSS



856
light
QSALTQPASVSGSPGQSITISCTGTSSDVGDYNYVSWYQQHPGKA





PKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEAEYYC





SSYTSSSTLLYVFGTGTKVTVL





COV2-
857
heavy
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIIWVRQAPGQGL


2166


EWMGGIIPIFGTTNYAQKFQGRVTITADESTSTAYVELSSLRSED





TAVYYCARIGHFDSSGYYLDYWGQGTLVTVSS



858
light
EIVLTQSPATLSLSPGERATLSCRASQSVSSFLAWYQQKPGQAPR





LLIYDASNRPTGIPARFTGSGSGTDFTLTISSLEPEDFAVYYCQH





RTNWPPLPTFGPGTKVDIK





COV2-
859
heavy
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGL


2169


EWMGGIIPIFGAANYAQNFQGRVTITADESTSTGYMQLSSLRPED





TAVYYCARTSHYDSSGSYFEYWGQGTLVTVSS



860
light
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR





LLIYDASNRATGIPARFSGSGSGTDPTLTISSLDPEDFAVYYCHK





RSNWPPSLTPGGGTKVEIK





COV2-
861
heavy
EVQLVESGGGLVQPGGSLRLSCVASGFTFSPYWMSWVRQAPGKGL


2175


EWVANIKQDGGEKYYVDSVKGRPTISRDNAKNSLYLQMNSLRAED





TAVYYCARLSGSSWDFDYWGQGTLVTVSS



862
light
SYELTQPPSVSVSPGQTASITCSGDKLGDKYACWYQQRPGQSPVL





VIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAW





DSSTGVFGTGTKVTVL





COV2-
863
heavy
EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGL


2191


EWVSAINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAED





TALYHCARRRSSSRYSSGWYMYYYYMDVWGKGTTVTVSS



864
light
DIQMTQSPSTLSASVGDRVTITCRASQSVSTWLAWYQQKPGKAPN





LLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQ





YNTYSGTFGQGTKVEIK





COV2-
865
heavy
EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGL


2194


EWVSAINWNGGSTGYADSVKGRFTTSRDNAKNSLYLQMNSLRAED





TALYHCARRRSSSRYSSGWYMYYYYMDVWGKGTTVTVSS



866
light
DIQMTQSPSTLSASVGDRVTITCRASQSVSTWLAWYQQKPGKAPN





LLIYEASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQ





YNTYSGTFGQGTKVEIK





COV2-
867
heavy
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGI


2198
868

EWMGGIIPIFGAANYAQNFQGRVTITADESTSTGYMQLSSLRFED





TAVYYCARTSHYDSSGSYFEYWGQGTLVTVSS




light
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR





LLIYDASNRATGIPARFSGSGSGTDFTLTISSLDPEDFAVYYCHK





RSNWPPSLTFGGGTKVEIK





COV2-  
869
heavy
EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYAMRWVRQAPGKGL


2203


AWVALISYDGYNKYYADSVRGRFTISRINSKNTLSLQMNSLRAED





TAVYYCARGSAGNYYYGMDVWGQGTTVTVSS



870
light
DIQMTQSPSSLSASVGDRVTITCRASQTITNYLNWYQLKSGRAPK





LLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ





SYSTPYTPGQGTKVEIK





COV2-
871
heavy
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIIWVRQAPGQGL


2214


EWMGGIIPIFGTTNYAQKFQGRVTITADESTSTAYVELSSLRSED





TAVYYCARIGHFDSSGYYLDYWGQGTLVTVSS



872
light
EIVLTQSPATLSLSPGERATLSCRASQSVSSFLAWYQQKPGQAPR





LLIYDASNRPTGIPARFTGSGSGTDFTLTISSLEPEDFAVYYCQH





RTNWPPLFTFGPGTKVDIK





COV2-
873
heavy
EVQLVQSGAEVKKPGSSVRVSCKASGGTFSSYAISWVRQAPGQGI


2216


EWMGGIIPIFGAANYAQNFQGRVTITADESTSTGYMQLSSLRFED





TAVYYCARTSHYDSSGSYFEYWGQGTEVTVSS



874
light
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPR





LLIYDASNRATGIPARFSGSGSGTDFTLTISSLDPEDFAVYYCHK





RSNWPPSLTFGGGTKVEIK





COV2-
875
heavy
EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYALFWVRQAPGKGL


2218


EWVAVISYDGNNKYYADSVRGRFTISRDNSKNTLYLQMNSLRPED





TAVYYCARPYTGSYKSYMDVWGKGTTVTVSS



876
light
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNSLAWYQQK





PGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVA





VYYCQQYYSISWTFGQGTKVEIK





COV2-
877
heavy
EVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGL


2224


EWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAED





TAMYYCAKDGSIAAADYWGQGTLVTVSS



878
light
DIVMTQSPDSLAVSLGERATINCKSSQSVLHSSNNKDSLVWYQQK





PGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTTSSLQAEDVA





VYYCQQYYSTPWTFGQGTKVEIK





COV2-  
879
heavy
EVQLVESGGGLIQPGGSLRLSCAASGFIVSSNYMSWVRQAPGKGL


2235


EWVSVIYSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT





AVYYCARESTQWGQGTLVTVSS



880
light
DIQMTQSPSTLSASVGDRVTITCRASHSISSWLAWYQQKPGKAPK





LLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDEFATYYCQQ





YNTYSQTFGQGTKVEIK





COV2-
881
heavy
EVQLVQSGPEVKKPGTSVKVSCKASGFTFMSSAVQWVRQARGQRL


2961


EWIGWIVIGSGNINYAQKFQERVTITRDMSTSTAYMELSSLRSED





TAVYYCAAPYCSSISCNDGPDIWGQGTMVTVSS



882
light
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAP





RLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQ





HYGSSRGWTFGQGTKVEIK
















TABLE 3







HEAVY CHAIN SEQUENCES











CDRH1
CDRH2
CDRH3


Clone
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:





COV2-2171
GFTFSFYW
IKQDGGSK
ARLSGSSWDFDY



312
313
314





COV2-2173
GFTFDDYG
INWNGGST
ARRRSSSRYSSGWYMYYYYMDV



315
316
317





COV2-2177
GFSVSTSGEG
IYWDDDK
AHRLWFRDAFDI



318
319
320





COV2-2178
GFTFSTYW
IKQDGSEK
ARVGSSSWYFDY



321
322
323





COV2-2179
GGSISSGTYY
TYYGGST
ARRGNYYDSKNWFDR



324
323
326





COV2-2181
GFTFSSHG
IWYDGSNK
ARESADISSRLDY



327
328
329





COV2-2183
GFTFSSYA
ISYDGSNK
ARADTMVRGTYFEY



330
331
332





COV2-2184
GFSLSTSGVG
IYWDDDK
AHRLPTPQLLPSPENWFDP



333
334
335





COV2-2185
GFTFSSYW
INSDGSST
AREVEQLAHMVDY



336
337
338





COV2-2186
GFTFSSYA
ISYDGINK
ARPRSGSYYAYFDY



337
340
341





COV2-2187
GFTFSYYP
TSYDGTNK
ARGGATNFDY



342
343
344





COV2-2189
GFTFDDSA
ISWNSGNV
TKASRYCSSTICYWNWFDP



345
346
347





COV2-2190
GFTFSSYE
ISSSGSAI
AREARSRYPDWLPSYYFDY



348
349
350





COV2-2192
GYTFTTYA
INTNTGNP
ARDQDSGYPTYYYYYMDV



351
352
353





COV2-2193
GFTFSTYA
ISYDGYNK
ARGSAGNYYYGMDV



354
355
356





COV2-2195
GFTFSNYG
ISNDEFNK
AKGGDGSGWAWDGDNPPTDY



357
358
359





COV2-2197
GYSFTSYW
IYPGDSDT
ARPDYSSGWFSYWYPDL



363
364
365





COV2-2199
GYTLTELS
FDPEDAET
ATGFAVFGRAAVPY



366
367
368





COV2-2200
GFTFSSYG
ISYDGSNK
AKDLTIVVIPAAPNFDY



369
370
371





COV2-2207
GYSFTSHW
IYPGDSDT
ASALRERGVQLWSV



372
373
374





COV2-2210
GFTFSSYA
ISYDGSNK
ARDQEWFRELFLFDY



375
376
377





COV2-2211
GFTFSTYG
ISYDGSNK
AKDGSIAAADY



378
379
380





COV2-2212
GFTFSSYS
ISNSNSFI
ARVNGNSNWNFGSYYYYYMDV



381
382
383





COV2-2215
GFTFSGYS
ISSSSSYI
ARWLQLRSDYYYFGMDV



384
385
386





COV2-2222
GGSISSYY
IYYSGST
ARAPRERLQWGEYYFDY



387
388
389





COV2-2226
GYSFTNSW
IYPGDSDT
ATHRCSGGFCYLAY



390
391
392





COV2-2227
GFTFSSYG
IWYDGSKK
ARDQSQGAYILTGYRGYGMDV



393
394
395





COV2-2228
GGSFSGHY
INHSGST
ARPPQAARIHYYYYMDV



396
397
398





COV2-2231
GFTFDDYA
ISWNSDSI
ARGKGAGYTSYMDV



399
400
401





COV2-2233
LFRSGGHW
IYPGDSDT
ASALRERGVQLWSV



402
403
404





COV2-2046
GFTFDDYA
ISWNSGSI
AKAHSTGHQYYYGMDV



405
406
407





COV2-2047
GFTFDDYA
ISWNSGSI
AKVSSITSLLGYYFDS



408
409
410





COV2-2048
GDTSSSYT
IIPILGIA
ARGVVAATPGWFDP



411
412
413





COV2-2049
GFTFSSYW
INSDGSST
AGSPWLRGDIDY



414
415
416





COV2-2050
GYTFTDYY
INPNSRGT
ARVVVIGYGRPNNYYDGRNVWDY



417
418
419





COV2-2051
GFSLGTSGMC
IDWDDDK
ARGVVTYDY



420
421
422





COV2-2054
GDTFSSYT
IIPILGIP
ARGRGYSNYGASYYMDV



423
424
425





COV2-2055
GFTFDDYA
ISWNSDSI
AKGRGAGYTSYMDV



426
427
428





COV2-2064
GYTFTSYD
MNSNSGNA
ARMRTGWPTHGRPDDE



432
433
434





COV2-2068
GFTVSSNY
IYPGGSA
ARSYDILTGYRDAFDT



435
436
437





COV2-2069
GLTVSSNY
IYAGGNT
ARGDGGYYSPFDY



438
439
440





COV2-2070
GGSFSAYY
INHSGST
ARVGYSQGYYYYYMDV



441
442
443





COV2-2078
GGTFSSYS
IIPVLGIA
ARVGVSGFKSGSNWYFDL



447
448
449





COV2-2081
GYTFTSYY
INPGGGST
ARGAIPPNSRAEIDY



453
454
455





COV2-2082
GFIFDDYD
ISWNGGNT
AVIMSPIPRYSGYDWAGGAFDI



456
457
458





COV2-2083
GFTFSNYG
MSYDGSNK
AKNLGPYCSGGTCYSLVGDY



459
460
461





COV2-2097
GFTFDDYA
ISWNSGTI
AKDIIRQGEDGMDV



468
469
470





COV2-2098
GFTFSNYA
IISTSGGAT
VKGLFDWFPL



471
472
473





COV2-2103
GFTFSRHW
IKQDGSEK
ARLGFYYGGADY



474
475
476





COV2-2108
GFTFHHYA
ISGSSDYR
AKGVDYGGKLAYFDS



477
478
479





COV2-2110
GFSFSSYV
ISYDGSSK
ARDIDSGYDPIPVFDY



480
481
482





COV2-2111
GFTFSSYD
IGTAGDT
ARVLYDSSGPYNWFDP



483
484
485





COV2-2113
EVTVSSNY
IYSGGTT
ARDFLRWHDL



486
487
488





COV2-2114
GDTFSSYT
IIPILGIP
ARGRGYSNYGASYYMDV



489
490
491





COV2-2128
GGSISSSSYY
IYYSGST
ARILVIFTLNWFDP



492
493
494





COV2-2132
EVTVSSNY
IYSGGTT
ARDFLRWHDL



498
499
500





COV2-2137
GGSVSSGSYY
IYYSGSS
AGSPVPPTIVGASY



501
502
503





COV2-2142
GYSFTSYW
IYPGDSDT
ARRGEAAGIWYFDL



504
505
506





COV2-2143
GFTVSSNY
IYSAGST
AKEGGSGSLRYYYYGMDV



507
508
509





COV2-2145
GFTFSTYA
ISYDGSNK
ARDWAPTYYDMPSAFDI



510
511
512





COV2-2146
GFTFSSSE
ISSSGSII
ARRSYRSSWYYYYGMDV



513
514
515





COV2-2147
GFTFSSYA
ISYDGSNK
ARSTSGSYYYGMDV



516
517
518





COV2-2151
GGTFSSYA
IIPVFGTA
ARIGSYPEYFQH



519
520
521





COV2-2153
GGTFSSYA
IIPIFGTT
ARIGHFDSSGYYLDY



522
523
524





COV2-2155
GFTFSSYA
ISYDGNNK
ARPYTGSYRSYMDV



525
526
527





COV2-2158
GGSISSGGYF
IYYSGST
ARGGSGSYSLFDY



528
529
530





COV2-2161
GFTFSRHA
ISYDGSNK
ARDPSPLVLITSIDY



531
532
533





COV2-2162
GFTFSSYW
IRQDGSEK
VRLGVSSWYFDY



534
535
536





COV2-2164
GGTFSSYA
IIPIFGAA
ARTSHYDSSGSYFEY



537
538
539





COV2-2000
GFTFRSYA
ISDNAYST
AKNLYSGNSPFDY



833
884
885





COV2-2001
GFTPSSYG
IWHDGSKK
ARDQGGYDYVWGSYRYTFYVFDY



886
887
888





COV2-2002
GFTFSFYW
IKQDGSEK
ARVGSSSWYFDY



889
890
891





COV2-2003
GYTLTRYD
LNPNGGNT
ARGQWELDAWYFDI



892
893
894





COV2-2004
GYTFTRYD
MNPNSDNT
ARGQWELDVWYFDI



895
896
897





COV2-2005
GGSISSYY
IYYSGRT
ARGGRPGAEGPYDAFDI



898
899
900





COV2-2954
GYTFSDYA
MKSNSGNT
ARMRSGWPTHGRPDDH



901
902
903





COV2-2956
GFTFVTSG
ISYDGSNK
AKGGPNKEVLYFGELLDYGMDV



904
905
906





COV2-2957
GYTFTGYV
INTGYGNT
ASMNRMSE0TYYGMDV



907
908
909





COV2-2958
GGSFSGYY
INHSGST
ARCRQMGNEYYYYMDV



910
911
912





COV2-2959
GGSINNYY
IHYSGST
VRDNYFDNSGHPVYPVPWFDP



913
914
915





COV2-2960
GYTFKNYG
ISAYTGNT
ARVQRRRLDY



916
917
918





COV2-2150
GFTFSTYA
ISYDGSNK
ARDWAPTYYDMPSAFDI



919
920
921





COV2-2159
GFTFSSYA
ISYDGSNK
ARSTSGSYYYGMDV



922
923
924





COV2-2160
GFTFSSYA
ISYDGSNK
ARSTSGSYYYGMDV



925
926
927





COV2-2166
GGTFSSYA
IIPIFGTT
ARIGHFDSSGYYLDY



928
929
930





COV2-2169
GGTFSSYA
IIPIFGAA
ARTSHYDSSGSYFEY



931
932
933





COV2-2175
GFTFSFYW
IKQDGGEK
ARLSGSSWDFDY



934
935
936





COV2-2191
GFTFDDYG
INWNGGST
ARRRSSSRYSSGWYMYYYYMDV



937
938
939





COV2-2194
GFTFDDYG
INWNGGST
ARRRSSSRYSSGWYMYYYYMDV



940
941
942





COV2-2198
GGTFSSYA
IIPIFGAA
ARTSHYDSSGSYFEY



943
944
945





COV2-2203
GPTFSTYA
ISYDGYNK
ARGSAGNYYYGMDV



946
947
948





COV2-2214
GGTFSSYA
IIPIPGTT
ARIGHFDSSGYYLDY



949
950
951





COV2-2216
GGTFSSYA
IIPIPGAA
ARTSHYDSSGSYFEY



952
953
954





COV2-2218
GFTFSSYA
ISYDGNNK
ARPYTGSYKSYMDV



955
956
957





COV2-2224
GFTFSTYG
ISYDGSNK
AKDGSIAAADY



958
959
960





COV2-2235
GFIVSSNY
IYSGGST
ARESTQ



961
962
963





COV2-2961
GFTFMSSA
IVIGSGNT
AAPYCSSISCNDGFDI



964
965
966
















TABLE 4







LIGHT CHAIN SEQUENCES











CDRL1
CDRL2
CDRL3


Clone
SEQ ID NO:
SEQ ID NO:
SEQ ID NO:





COV2-2171
KLGDKY
QDS
QAWDSSTGV



543
544
545





COV2-2173
QSVSTW
EAS
QQYNTYSGT



546
547
548





COV2-2177
QSISNY
AAS
QQTYSTFWT



549
550
551





COV2-2178
KLGDKY
QDS
QAWDSSTAV



5b2
553
554





COV2-2179
QSVSSN
GAS
QQYNNWPPMYT



555
556
557





COV2-2181
ALPTKY
DDS
YSTDSSGNV



558
559
560





COV2-2183
SSDVGGYNY
DVS
SSYTSSRAVL



561
562
563





COV2-2184
SSNIGNNA
YDD
ASWDDSLIGPV



564
565
566





COV2-2185
ALPNQY
KDS
QSADSSGTSWV



567
568
569





COV2-2186
QGISNY
AAS
QQYNSHPPT



570
571
572





COV2-2187
NIGRKS
DDS
QVWDSSSDHPEWV



573
574
575





COV2-2189
QSISSY
GAS
QQSYSTPT



576
577
578





COV2-2190
SSDIGGYNY
DVS
SSYTSSSTHVV



579
580
581





COV2-2192
QSLLHSDGKTY
EVS
MQSIQPPLT



582
583
584





COV2-2193
QTITNY
AAS
QQSYSTPYT



585
586
587





COV2-2195
QSVLYTPRNKNY
WAS
QQYYTAPLT



588
589
590





COV2-2191
QSVSSNF
GAS
QQYGRSPIT



594
595
596





COV2-2199
KLGDKY
QGA
QAWDSSTVV



597
598
599





COV2-2200
SSDVGGYNY
DVS
SSYTSSSTPVV



600
601
602





COV2-2207
SSNIGAGYD
INS
QSYDSSLGAL



603
604
605





COV2-2210
QGISSW
DAS
QQANSFPPY



606
607
608





COV2-2211
QSVLHSSNNKDS
WAS
QQYYSTPWT



609
610
611





COV2-2212
QSVSSY
DTS
QQRGNWWT



612
613
614





COV2-2215
QSVSNN
GAS
QQCYNWPPWT



615
616
617





COV2-2222
SSDVGSYNL
EVI
CSYAVSTTYVI



618
619
620





COV2-2226
SGYSNYK
VGTGGIVG
GADHGSGSNFVEW



621
622
623





COV2-2227
QSLLHSNGYNY
LGS
MQALQTPPT



624
625
626





COV2-2228
QSVSSN
GAS
QQYNYWPPLT



627
628
629





COV2-2231
NIGSKS
DDS
QVWDSSSDHHVV



630
631
632





COV2-2233
SSNIGAGYD
INS
QSYDSSLGAL



633
634
635





COV2-2046
QSISSF
AAF
QQSYNTPYT



636
637
638





COV2-2047
QSVSSY
DAS
QHRSNWPPRLT



639
640
641





COV2-2048
QSVSSN
GAS
QQYNNFLT



642
643
644





COV2-2049
SGSIASNY
SDN
QSYDGSNHAVV



645
646
644





COV2-2050
SSNIGSNT
SNN
AAWDDSLNALV



648
649
650





COV2-2051
QSIAGY
GTT
QQSYSTDGT



651
652
653





COV2-2054
QDINHY
DAS
QQSDNLPMYT



654
655
656





COV2-2055
NIGSKS
DDS
QVWDSSSDHHVV



657
658
659





COV2-2064
NSNTGSYT
DNN
LVWDDSLNGLV



663
664
665





COV2-2068
SSNIGSGSD
GNT
QSYDSRLSGFVV



666
667
668





COV2-2069
QSISSY
AAS
QQSYSTPQT



669
670
671





COV2-2070
QSISNY
AAS
QQSYTTLLT



672
673
674





COV2-2078
NSNIGAGYD
GNS
QSYDSSLSDSV



678
679
680





COV2-2081
QSVSSN
GAS
QQYYNWPLT



684
685
686





COV2-2082
SLRSYY
DKN
NSRDSSGNAVV



687
688
689





COV2-2083
QDISNY
DAS
QQYANLPFT



690
691
692





COV2-2097
QNIASY
AAS
QQSYSTPWT



699
700
701





COV2-2098
QSVRSN
GAS
HQYNNWPQT



702
703
704





COV2-2103
SGSIASNY
EDN
QSYDGINRAWV



705
706
707





COV2-2108
QSLLHSLGYNS
LGS
MQALQTPLT



108
709
710





COV2-2110
QSISSY
AAS
QQSYSSLSIT



711
712
713





COV2-2111
QSISSY
AAS
QQSYEIPPWT



714
715
716





COV2-2113
QDINNY
DAS
QQYDNLPPV



717
718
719





COV2-2114
SSNIGAGYD
ANS
QSYDSSLSGSV



720
721
722





COV2-2128
SGSIASNY
EDN
QSYDSGNPI



723
724
725





COV2-2132
QDINNY
DAS
QQYDNLPPV



729
730
731





COV2-2137
SGSIASNY
EDN
QSYDGINRWLV



732
733
734





COV2-2142
SGYSNYK
VGTGGIVG
GADHGSGSNFEYVV



735
736
737





COV2-2143
SSNIGYNI
SNN
AAWDDSLNGYV



738
730
740





COV2-2195
NIGNKG
DDS
QVWDSSSDHPGV



741
742
743





COV2-2146
QGISSY
AAS
QQLNSYPVI



744
745
746





COV2-2147
SSDVGDYNY
DVS
SSYTSSSTLLYV



747
748
749





COV2-2151
QSVSSF
DAS
HYRSNWPPVIT



750
751
752





COV2-2153
QSVSSF
DAS
QHRTNWPPLFT



753
754
755





COV2-2155
QSVLYSSNNKNS
WAS
QQYYSISWT



756
757
758





COV2-2158
QDITNY
DAS
QQYDNLYSVH



759
760
761





COV2-2161
ALPRQY
Kns
QSADTIGTYWV



762
763
764





COV2-2162
KLGDKY
QDT
QAWGSSRGV



765
766
767





COV2-2164
QSVSSY
DAS
HKRSNWPPSLT



768
769
770





COV2-2000
KLGDKY
QDS
QAWLSSIAV



967
968
969





COV2-2001
SLRNYY
GKN
NSRDSSGNHLI



970
971
972





COV2-2002
KLGDKY
QDI
QAWDSNTGV



973
974
975





COV2-2003
SFRSYY
GKN
NSRDTSGNHLDVV



976
977
978





COV2-2004
SLRSYY
GKN
NSRDSSGHHLDVV



979
980
981





COV2-2005
NLRRYY
GKN
NSRDSSGNHVV



982
983
984





COV2-2954
QSIISY
AAS
QQSDNLPMYT



985
986
987





COV2-2306
QSVLYSSEIKNY
WAS
QQYYSGPLDT



988
989
990





COV2-2957
QDISNY
DAS
QQYDKLT



991
992
993





COV2-2958
SSNIGAGFD
GNN
QSFDIGRGGWI



994
995
996





COV2-2959
SSNIGAGFD
GNN
QSFDIGRGGWI



997
998
999





COV2-2960
ETLLHSDGKTY
EVS
MQSIQLA



1000
1001
1002





COV2-2150
NIGNKG
DDS
QVWDSSSDHPGV



1003
1004
1005





COV2-2159
SSDVGDYNY
DVS
SSYTSSSTLLYV



1006
1007
1008





COV2-2160
SSDVGDYNY
DVS
SSYTSSSTLLYV



1009
1010
1011





COV2-2166
QSVSSF
DAS
QHRTNWPPLFT



1012
1013
1014





COV2-2169
QSVSSY
DAS
HKRSNWPPSLT



1015
1016
1017





COV2-2175
KLGDKY
QDS
QAWDSSTGV



1018
1019
1020





COV2-2191
QSVSTW
EAS
QQYNTYSGI



1021
1022
1023





COV2-2194
QSVSTW
EAS
QQYNTYSGI



1024
1025
1026





COV2-2198
QSVSSY
DAS
HKRSNWPPSLT



540
1027
1028





COV2-2203
QTITNY
AAS
QQSYSTPYT



1029
1030
1031





COV2-2214
QSVSSF
DAS
QHRTNWPPLFT



1032
1033
1034





COV2-2216
QSVSSY
DAS
HKRSNWPPSLT



1035
1036
1037





COV2-2218
QSVLYSSNNKNS
WAS
QQYYSISWT



1038
1039
1040





COV2-2224
QSVLHSSNNKDS
WAS
QQYYSTPWT



1041
1042
1043





COV2-2235
HSISSW
KAS
QQYNTYSQT



1044
1045
1046





COV2-2961
QSVSSSY
GAS
QHYGSSRGWT



1047
1048
1049









All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.


VII. References

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims
  • 1. A method of detecting COVID-19 infection with SARS-CoV-2 in a subject comprising: (a) contacting a sample from said subject with an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and(b) detecting SARS-CoV-2 in said sample by binding of said antibody or antibody fragment to a SARS-CoV-2 antigen in said sample.
  • 2. The method of claim 1, wherein said sample is a body fluid.
  • 3. The method of claim 1, wherein said sample is blood, sputum, tears, saliva, mucous or serum, semen, cervical or vaginal secretions, amniotic fluid, placental tissues, urine, exudate, transudate, tissue scrapings or feces.
  • 4. The method of claim 1, wherein detection comprises ELISA, RIA, lateral flow assay or western blot.
  • 5. The method of claim 1, further comprising performing steps (a) and (b) a second time and determining a change in SARS-CoV-2 antigen levels as compared to the first assay.
  • 6. The method of claim 1, wherein the antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.
  • 7. The method of claim 1, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, 90% or 95% identity to clone-paired variable sequences as set forth in Table 1.
  • 8. The method of claim 1, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having 100% identity to clone-paired sequences as set forth in Table 1.
  • 9. The method of claim 1, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.
  • 10. The method of claim 1, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80%, 90% or 95% identity to clone-paired sequences from Table 2.
  • 11. The method of claim 1, wherein said antibody or antibody fragment binds to a SARS-CoV-2 surface spike protein.
  • 12. The method of claim 1, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.
  • 13. A method of treating a subject infected with SARS-CoV-2 or reducing the likelihood of infection of a subject at risk of contracting SARS-CoV-2, comprising delivering to said subject an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.
  • 14. The method of claim 13, the antibody or antibody fragment is encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1.
  • 15. The method of claim 13, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, 90% or 95% identity to clone-paired sequences from Table 1.
  • 16. The method of claim 13, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.
  • 17. The method of claim 13, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80% or 90% identity to clone-paired sequences from Table 2.
  • 18. The method of claim 13, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 2.
  • 19. The method of claim 13, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.
  • 20. The method of claim 13, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.
  • 21. The method of claim 13, wherein said antibody is a chimeric antibody or a bispecific antibody.
  • 22. The method of claim 13, wherein said antibody or antibody fragment binds to a SARS-CoV-2 surface spike protein.
  • 23. The method of claim 13, wherein said antibody or antibody fragment is administered prior to infection or after infection.
  • 24. The method of claim 13, wherein said subject is of age 60 or older, is immunocompromised, or suffers from a respiratory and/or cardiovascular disorder.
  • 25. The method of claim 13, wherein delivering comprises antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment.
  • 26. A monoclonal antibody, wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.
  • 27. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences according to clone-paired sequences from Table 1.
  • 28. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to clone-paired sequences from Table 1.
  • 29. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.
  • 30. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80%, 90%, or 95% identity to clone-paired sequences from Table 2.
  • 31. The monoclonal antibody of claim 26, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.
  • 32. The monoclonal antibody of claim 26, wherein said antibody is a chimeric antibody, or is a bispecific antibody.
  • 33. The monoclonal antibody of claim 26, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.
  • 34. The monoclonal antibody of claim 26, wherein said antibody or antibody fragment binds to a SARS-CoV-2 antigen such as a surface spike protein.
  • 35. The monoclonal antibody of claim 26, wherein said antibody is an intrabody.
  • 36. A hybridoma or engineered cell encoding an antibody or antibody fragment wherein the antibody or antibody fragment is characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.
  • 37. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences according to clone-paired sequences from Table 1.
  • 38. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences from Table 1.
  • 39. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 95% identity to clone-paired variable sequences from Table 1.
  • 40. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.
  • 41. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences from Table 2.
  • 42. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 2.
  • 43. The hybridoma or engineered cell of claim 36, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.
  • 44. The hybridoma or engineered cell of claim 36, wherein said antibody is a chimeric antibody, a bispecific antibody, or an intrabody.
  • 45. The hybridoma or engineered cell of claim 36, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.
  • 46. The hybridoma or engineered cell of claim 36, wherein said antibody or antibody fragment binds to a SARS-CoV-2 surface spike protein.
  • 47. A vaccine formulation comprising one or more antibodies or antibody fragments characterized by clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.
  • 48. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences according to clone-paired sequences from Table 1.
  • 49. The vaccine formulation of claim 47, wherein 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 clone-paired sequences from Table 1.
  • 50. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments is encoded by light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 1.
  • 51. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.
  • 52. The vaccine formulation of claim 47, wherein at least one of said antibodies or antibody fragments comprises light and heavy chain variable sequences having at least 70%, 80%, 90% or 95% identity to clone-paired sequences from Table 2.
  • 53. The vaccine formulation of claim 47, wherein at least one of said antibody fragments is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.
  • 54. The vaccine formulation of claim 47, wherein at least one of said antibodies is a chimeric antibody, is bispecific antibody or an intrabody.
  • 55. The vaccine formulation of claim 47, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA PG, N297, GASD/ALIE, DHS YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.
  • 56. The vaccine formulation of claim 47, wherein said antibody or antibody fragment binds to a SARS-CoV-2 surface spike protein.
  • 57. A vaccine formulation comprising one or more expression vectors encoding a first antibody or antibody fragment according to claim 26.
  • 58. The vaccine formulation of claim 57, wherein said expression vector(s) is/are Sindbis virus or VEE vector(s).
  • 59. The vaccine formulation of claim 57, formulated for delivery by needle injection, jet injection, or electroporation.
  • 60. The vaccine formulation of claim 57, further comprising one or more expression vectors encoding for a second antibody or antibody fragment.
  • 61. A method of protecting the health of a subject of age 60 or older, an immunocompromised, subject or a subject suffering from a respiratory and/or cardiovascular disorder that is infected with or at risk of infection with SARS-CoV-2 comprising delivering to said subject an antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively.
  • 62. The method of claim 61, the antibody or antibody fragment is encoded by clone-paired light and heavy chain variable sequences as set forth in Table 1.
  • 63. The method of claim 61, the antibody or antibody fragment is encoded by clone-paired light and heavy chain variable sequences having at least 95% identity to as set forth in Table 1.
  • 64. The method of claim 61, wherein said antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired sequences from Table 1.
  • 65. The method of claim 61, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.
  • 66. The method of claim 61, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80% or 90% identity to clone-paired sequences from Table 2.
  • 67. The method of claim 61, wherein said antibody or antibody fragment comprises light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 2
  • 68. The method of claim 61, wherein the antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.
  • 69. The method of claim 61, wherein said antibody is an IgG, or a recombinant IgG antibody or antibody fragment comprising an Fc portion mutated to alter (eliminate or enhance) FcR interactions, to increase half-life and/or increase therapeutic efficacy, such as a LALA, LALA PG, N297, GASD/ALIE, DHS, YTE or LS mutation or glycan modified to alter (eliminate or enhance) FcR interactions such as enzymatic or chemical addition or removal of glycans or expression in a cell line engineered with a defined glycosylating pattern.
  • 70. The method of claim 61, wherein said antibody is a chimeric antibody or a bispecific antibody.
  • 71. The method of claim 61, wherein said antibody or antibody fragment is administered prior to infection or after infection.
  • 72. The method of claim 61, wherein said antibody or antibody fragment binds to a SARS-CoV-2 surface spike protein.
  • 73. The method of claim 61, wherein delivering comprises antibody or antibody fragment administration, or genetic delivery with an RNA or DNA sequence or vector encoding the antibody or antibody fragment.
  • 74. The method of claim 61, wherein the antibody or antibody fragment improves the subject's respiration as compared to an untreated control.
  • 75. The method of claim 61, wherein the antibody or antibody fragment reduces viral load as compared to an untreated control.
  • 76. A method of determining the antigenic integrity, correct conformation and/or correct sequence of a SARS-CoV-2 surface spike protein comprising: (a) contacting a sample comprising said antigen with a first antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and(b) determining antigenic integrity, correct conformation and/or correct sequence of said antigen by detectable binding of said first antibody or antibody fragment to said antigen.
  • 77. The method of claim 76, wherein said sample comprises recombinantly produced antigen.
  • 78. The method of claim 76, wherein said sample comprises a vaccine formulation or vaccine production batch.
  • 79. The method of claim 76, wherein detection comprises ELISA, RIA, western blot, a biosensor using surface plasmon resonance or biolayer interferometry, or flow cytometric staining.
  • 80. The method of claim 76, wherein the first antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.
  • 81. The method of claim 76, wherein said first antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1.
  • 82. The method of claim 76, wherein said first antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 95% identity to clone-paired sequences as set forth in Table 1.
  • 83. The method of claim 76, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.
  • 84. The method of claim 76, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80% or 90% identity to clone-paired sequences from Table 2.
  • 85. The method of claim 76, wherein said first antibody or antibody fragment comprises light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 2.
  • 86. The method of claim 76, wherein the first antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.
  • 87. The method of claim 76, further comprising performing steps (a) and (b) a second time to determine the antigenic stability of the antigen over time.
  • 88. The method of claim 76, further comprising: (c) contacting a sample comprising said antigen with a second antibody or antibody fragment having clone-paired heavy and light chain CDR sequences from Tables 3 and 4, respectively; and(d) determining antigenic integrity of said antigen by detectable binding of said second antibody or antibody fragment to said antigen.
  • 89. The method of claim 88, wherein the second antibody or antibody fragment is encoded by clone-paired variable sequences as set forth in Table 1.
  • 90. The method of claim 89, wherein said second antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 70%, 80%, or 90% identity to clone-paired variable sequences as set forth in Table 1.
  • 91. The method of claim 89, wherein said second antibody or antibody fragment is encoded by light and heavy chain variable sequences having at least 95% identity to clone-paired sequences as set forth in Table 1.
  • 92. The method of claim 89, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences according to clone-paired sequences from Table 2.
  • 93. The method of claim 89, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences having at least 70%, 80% or 90% identity to clone-paired sequences from Table 2.
  • 94. The method of claim 89, wherein said second antibody or antibody fragment comprises light and heavy chain variable sequences having at least 95% identity to clone-paired sequences from Table 2.
  • 95. The method of claim 89, wherein the second antibody fragment is a recombinant scFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.
  • 96. The method of claim 89, further comprising performing steps (c) and (d) a second time to determine the antigenic stability of the antigen over time.
  • 97. A human monoclonal antibody or antibody fragment, or hybridoma or engineered cell producing the same, wherein said antibody binds to a SARS-CoV-2 surface spike protein.
PRIORITY CLAIM

This application claims benefit of priority to the following applications, each of which are hereby incorporated by reference in their entirety: U.S. Provisional Application Ser. Nos. 63/000,735 and 63/024,214, filed Mar. 27, 2020, and May 13, 2020, respectively; and U.S. Provisional Application Ser. Nos. 63/002,896, 63/024,248, 63/027,173, 63/037,984, 63/040,246, and 63/142,196, filed Mar. 31, 2020, May 13, 2020. May 19, 2020, Jun. 11, 2020, Jun. 17, 2020, and Jan. 27, 2021, respectively; and U.S. Provisional Application Ser. No. 63/023,545, filed May 12, 2020.

FEDERAL FUNDING DISCLOSURE

This invention was made with government support under HR0011-18-2-0001 awarded by the Defense Advanced Research Projects Agency (DARPA) and HHS Contract 75N93019C00074 awarded by the National Institutes of Allergy and Infection Disease/National Institutes of Health. The government has certain rights in the invention.

PCT Information
Filing Document Filing Date Country Kind
PCT/US21/24341 3/26/2021 WO
Provisional Applications (9)
Number Date Country
63000735 Mar 2020 US
63002896 Mar 2020 US
63023545 May 2020 US
63024214 May 2020 US
63024248 May 2020 US
63027173 May 2020 US
63037984 Jun 2020 US
63040246 Jun 2020 US
63142196 Jan 2021 US