WO2022179561A1

WO2022179561A1 - Neutralizing antibodies against covid-19 and methods of use thereof

Info

Publication number: WO2022179561A1
Application number: PCT/CN2022/077669
Authority: WO
Inventors: Zhiwei Chen; Biao ZHOU; Runhong ZHOU; FuK Woo CHAN; Kwok Yung Yuen
Original assignee: The University Of Hong Kong; Centre For Virology, Vaccinology And Therapeutics Limited
Priority date: 2021-02-24
Filing date: 2022-02-24
Publication date: 2022-09-01

Abstract

Disclosed are compositions and methods using antibodies and antibody fragments that bind SARS-CoV-2 receptor binding domain (RBD). In particular, disclosed are antibodies and antigen binding fragments thereof comprising six complementarity determining regions (CDRs), wherein the CDRs comprise: (1) the three light chain CDRs of SEQ ID NO: 5 and the three heavy chain CDRs of SEQ ID NO: 1, (2) the three light chain CDRs of SEQ ID NO: 6 and the three heavy chain CDRs of SEQ ID NO: 2, (3) the three light chain CDRs of SEQ ID NO: 7 and the three heavy chain CDRs of SEQ ID NO: 3, or (4) the three light chain CDRs of SEQ ID NO: 8 and the three heavy chain CDRs of SEQ ID NO: 4and wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 RBD.

Description

NEUTRALIZING ANTIBODIES AGAINST COVID-19 AND METHODS OF USE THEREOF

This international patent application claims the benefit of U.S. Provisional Patent Application No.: 63/152,874 filed on February 24, 2021, the entire content of which is incorporated by reference for all purpose.

FIELD OF THE INVENTION

The disclosed invention is generally in the field of SARS-CoV-2 and specifically in the area of neutralizing antibodies against SARS-CoV-2 and COVID-19.

BACKGROUND OF THE INVENTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted globally in over 27 million infections with and nearly 0.9 million deaths by early September 2020 since the discovery of the disease outbreak in December 2019 (Chan et al., Lancet 395: 514-523 (2020) ; Zhu et al., N. Engl. J. Med. 382: 727-733 (2020) ) . The growing Coronavirus Disease 2019 (COVID-19) pandemic calls for urgent development of effective prophylaxis and treatment. While triple combination therapy (interferon-β1b, lopinavir/ritonavir, and ribavirin) , remdesivir, and dexamethasone have each shown some clinical benefits in selected patient groups (Goldman et al., N. Engl. J. Med., doi: 10.1056/NEJMoa2015301 (2020) ; Boulware et al., N Engl J Med 383: 517-525 (2020) ; Hung et al., Lancet 395: 1695-1704 (2020) ) , the discovery of specific anti-SARS-CoV-2 agents with higher efficacy, better safety profile, and bio-availability remain essential for improving the clinical outcome of COVID-19 patients. Besides some drugs identified in large-scale drug repurposing programs (Riva et al., Nature, 10.1038/s41586-41020-42577-41581 (2020) ) , direct cloning of human neutralizing antibodies (HuNAbs) against SARS-CoV-2 have also been reported recently (Shi et al., Nature 584: 120-124 (2020) ; Zost et al., Nature 584: 443-449 (2020) ; Liu et al., Nature 584: 450-456 (2020) ; Cao et al., Cell 182: 73-84 e16 (2020) ; Robbiani et al., Nature 584: 437-442 (2020) ; Sun et al., MAbs 12: 1778435 (2020) ; Wu et al., Science 368: 1274-1278 (2020) ; Wu et al., Cell Host Microbe 27: 891-898 e895 (2020) ) . However, as there has never been an approved vaccine for any human pathogenic coronaviruses, whether or not HuNAbs are safe and account for the immune correlate of protection remains to be determined for COVID-19.

Unlike SARS patients, who had peak upper respiratory tract viral load at day 10 after symptom onset (Peiris et al., Lancet 361: 1767-1772 (2003) ) , COVID-19 patients exhibited peak salivary or upper respiratory viral loads during the first week after symptom onset (which declines over time) , which could account for the fast-spreading nature of the pandemic (To et al., Lancet Infect Dis 20: 565-574 (2020) ; Hung et al., Lancet Infect. Dis. (2020) ) . Regarding the humoral response, SARS-CoV-2-specific IgG and neutralizing antibody responses were quickly detectable in adult and children patients just 6 days after symptom onset (Suthar et al., medRxiv, doi: 10.1101/2020.1105.1103.20084442 (2020) ; Zhou et al., Immunity 53: 1-14 (2020) ; Liu et al., Emerg. Microbes Infect. 9: 1254-1258 (2020) ) . However, COVID-19 patients with higher titers of anti-spike (S) and anti-nucleocapsid (NP) IgM and IgG tend to have poorer disease outcomes (Tan et al., bioRxiv, 2020.2003.2024.20042382 (2020) ; Jiang et al., bioRxiv, 2020.2003.2020.20039495 (2020) ) . It has been reported that COVID-19 patients with severe disease developed significantly more robust SARS-CoV-2-specific NAb responses (Wang et al., bioRxiv, 2020.2006.2013.150250 (2020) ; Wang et al., J. Clin. Invest., 10.1172/JCI138759 (2020) ; Liu et al., Emerging Microbes &Infections 9: 1664-1670 (2020) ) . Nevertheless, convalescent plasma with high Nab titers from recovered patients has been reported to be beneficial in the treatment of severe COVID-19 in small case cohorts (Duan et al., Proc. Natl. Acad. Sci. U S A 117: 9490-9496 (2020) ) . To replace convalescent plasma, which is not readily available in most countries, HuNAbs have been recently identified and showed promising results in preclinical studies (Shi et al., Nature 584: 120-124 (2020) ; Zost et al., Nature 584: 443-449 (2020) ; Liu et al., Nature 584: 450-456 (2020) ; Cao et al., Cell 182: 73-84 e16 (2020) ; Robbiani et al., Nature 584: 437-442 (2020) ; Sun et al., MAbs 12: 1778435 (2020) ; Wu et al., Science 368: 1274-1278 (2020) ; Wu et al., Cell Host Microbe 27: 891-898 e895 (2020) ) . However, the in vivo efficacy of anti-SARS-CoV-2 HuNAbs in protecting against upper respiratory tract infection in a physiologically relevant animal model remains incompletely investigated. Given this, there is a need for a specific drug to treat COVID-19 patients.

BRIEF SUMMARY OF THE INVENTION

Disclosed are compositions and methods using antibodies and antibody fragments that bind SARS-CoV-2 receptor binding domain (RBD) . In particular, disclosed are antibodies and antigen binding fragments thereof comprising six complementarity determining regions (CDRs) ,

wherein the CDRs comprise:

(1) the three light chain CDRs of SEQ ID NO: 5 and the three heavy chain CDRs of SEQ ID NO: 1,

(2) the three light chain CDRs of SEQ ID NO: 6 and the three heavy chain CDRs of SEQ ID NO: 2,

(3) the three light chain CDRs of SEQ ID NO: 7 and the three heavy chain CDRs of SEQ ID NO: 3, or

(4) the three light chain CDRs of SEQ ID NO: 8 and the three heavy chain CDRs of SEQ ID NO: 4, and

wherein the antibody or antigen binding fragment thereof binds to SARS-COV-2 RBD.

In some forms, the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-32 of SEQ ID NO: 5, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 5, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 5. In some forms, the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-33 of SEQ ID NO: 1, a second heavy chain CDR comprising amino acids 51-58 of SEQ ID NO: 1, and a third heavy chain CDR comprising amino acids 97-110 of SEQ ID NO: 1. In some forms, the antibody or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1.

In some forms, the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-33 of SEQ ID NO: 6, a second light chain CDR comprising amino acids 51-53 of SEQ ID NO: 6, and a third light chain CDR comprising amino acids 90-98 of SEQ ID NO: 6. In some forms, the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-33 of SEQ ID NO: 2, a second heavy chain CDR comprising amino acids 51-58 of SEQ ID NO: 2, and a third heavy chain CDR comprising amino acids 97-112 of SEQ ID NO: 2. In some forms, the antibody or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

In some forms, the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-32 of SEQ ID NO: 7, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 7, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 7. In some forms, the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-33 of SEQ ID NO: 3, a second heavy chain CDR comprising amino acids 51-58 of SEQ ID NO: 3, and a third heavy chain CDR comprising amino acids 97-113 of SEQ ID NO: 3. In some forms, the antibody or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3.

In some forms, the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-32 of SEQ ID NO: 8, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 8, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 8. In some forms, the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-32 of SEQ ID NO: 4, a second heavy chain CDR comprising amino acids 50-56 of SEQ ID NO: 4, and a third heavy chain CDR comprising amino acids 95-106 of SEQ ID NO: 4. In some forms, the antibody or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

In some forms, the antibody or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1. In some forms, the antibody or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In some forms, the antibody or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3. In some forms, the antibody or antigen binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

In some forms, the antibody or antigen binding fragment thereof attenuates the ability of a ligand of SARS-COV-2 RBD to bind to ACE2. In some forms, the antibody or antigen binding fragment thereof comprises one or more constant domains from an immunoglobulin constant region (Fc) . In some forms, the constant domains are human constant domains. In some forms, the human constant domains are IgA, IgD, IgE, IgG or IgM domains. In some forms, the human IgG constant domains are IgG1, IgG2, IgG3, or IgG4 domains.

In some forms, the antibody or antigen binding fragment thereof is detectably labeled or comprises a conjugated toxin, drug, receptor, enzyme, receptor ligand. In some forms, the antibody is a monoclonal antibody, a human antibody, a chimeric antibody or a humanized antibody. In some forms, the antibody is a bispecific, trispecific or multispecific antibody.

Also disclosed are humanized antibodies and antigen binding fragment thereof comprising one or more human IgG4 constant domains and

a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1,

a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2,

a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3, or

a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

Also disclosed are pharmaceutical compositions comprising an antibody or antigen binding fragment thereof as disclosed herein and a physiologically acceptable carrier or excipient. In some forms, the pharmaceutical composition is for use in a method of preventing or treating COVID-19 in a subject. In some forms, the subject has COVID-19. In some forms, the subject is at risk of developing COVID-19. In some forms, the pharmaceutical composition is for use in a method of treating COVID-19. In some forms, the pharmaceutical composition is for use in a method of preventing COVID-19.

Also disclosed is use of a disclosed antibody or antigen binding fragment thereof in manufacture of a medicament for preventing or treating COVID-19 in a subject.

Also disclosed is the use of a disclosed antibody or antigen binding fragment thereof in manufacture of a medicament for treating COVID-19 in a subject.

Also disclosed is the use of a disclosed antibody or antigen binding fragment thereof in manufacture of a medicament for preventing COVID-19 in a subject.

Also disclosed are methods of detection or diagnosis of SARS-CoV-2 infection, comprising: (a) assaying the presence of SARS-COV-2 RBD in a sample from a subject using the antibody or antigen binding fragment thereof of any one of paragraphs 1-30 and (b) comparing the level of the SARS-COV-2 RBD with a control level, wherein an increase in the assayed level of SARS-COV-2 RBD compared to the control level is indicative of SARS-CoV-2 infection. In some forms, the presence of SARS-COV-2 RBD is assayed by enzyme linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , or fluorescence-activated cell sorting (FACS) .

Also disclosed are pharmaceutical compositions for use in a method of treating a subject infected by or at risk for infection by SARS-CoV-2, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of paragraph 31 if the subject has a disease characterized by increased expression of SARS-COV-2 RBD. In some forms, the antibody or antigen binding fragment thereof is an antibody or antigen binding fragment thereof as disclosed herein.

Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

Throughout this specification the word “comprise, ” or variations such as “comprises” or “comprising, ” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

FIGs. 1A-1C are line graphs illustrating results from experiments characterizing the monoclonal antibodies isolated from single B cells of convalescent COVID-19 patients. FIG. 1A shows the RBD-specific binding activities of sera derived from 3 (P1-P3) convalescent and 1 (P4) acute COVID-19 patients as measured by ELISA. FIG. 1B shows the spike-specific binding activities of sera derived from four COVID-19 patients as measured by ELISA. FIG. 1C shows the neutralization activities of sera derived from four COVID-19 patients as measured by pseudotyped SARS-CoV-2 inhibition in 293T-ACE2 cells.

FIGs. 2A-2C illustrate the results from the isolation of SARS-CoV-2 specific antibodies from sorted memory B cells. FIG. 2A is a graphical representation of the gating strategy for isolation of SARS-CoV-2 RBD-specific memory B cells by flow cytometry. FIG. 2B are graphs showing the RBD double positive cell population was obtained from each subject. FIG. 2C is a table showing the RBD-binding response of individual monoclonal antibodies from 4 subjects by ELISA. The color scale indicated the absorbance value at OD 450 nm. FIG. 2D are pie charts showing the antibody gene repertoire analysis of reactive B cells derived from each patient. The number of cloned antibody genes from each patient is shown in the center of each pie chart for both the heavy (H) and light (L) chains. The colors represent specific variable gene family. Each fragment of the same color stands for one specific sub-family. FIGs. 2E-2L are violin plots showing the percentage of somatic hypermutation (SHM) compared to germline sequences and the CDR3 amino acid lengths of cloned antibody H and L gene sequences analyzed for each subject: Patient 1 (P1: FIG. 2E and FIG. 2F) ; Patient 2 (P2: FIG. 2G and FIG. 2H) ; Patient 3 (P3: FIG. 2I and FIG. 2J) ; and Patient 4 (P4: FIG. 2K and FIG. 2L) .

FIGs. 3A and 3B are line graphs showing the RBD (FIG. 3A) and spike (FIG. 3B) specific binding activities of five HuNAbs, including A6, B4, B7, B8 and C5, measured by ELISA. FIGs. 3C and 3D are line graphs showing the neutralization activities of 5 HuNAbs against pseudotyped (FIG. 3C) and authentic (FIG. 3D) SARS-CoV-2 were determined in HEK 293T-ACE2 and Vero-E6 cells, respectively. HIV-1 specific HuNAb VRC01 served as a negative control. Each assay was performed in duplicates and the mean of replicates is shown with the standard error of mean (SEM) . FIG. 3E is a line graph showing the neutralization activity determined for the screened antibodies against SARS-CoV-2 pseudovirus. The HuNAbs with high neutralizations were color-coded.

FIGs. 4A-4D are line graphs showing the competition of four HuNAbs, including B4 (FIG. 4A) , B7 (FIG. 4B) , B8 (FIG. 4C) and C5 (FIG. 4D) , with human soluble ACE2 for binding to SARS-CoV-2 RBD as measured by SPR. The curves show binding of ACE2 to SARS-CoV-2 RBD with (red) or without (black) pre-incubation with each HuNAb.

FIGs. 5A-5D are binding curves showing the binding dynamics of four most potent HuNAbs (B4 (FIG. 5A) , B7 (FIG. 5B) , B8 (FIG. 5C) and C4 (FIG. 5D) ) to SARS-CoV-2 Spike glycoprotein.

FIGs. 6A-6P are line graphs illustrating the competitive binding between the four HuNAbs to SARS-CoV-2 RBD. Orange curve: the baseline; Green curve: the binding of test antibody to RBD; Blue Curve: the binding of test antibody (Ab1) to RBD after pre-incubation with the competitor antibody (Ab2) .

FIGs. 7A-7F are line graphs showing the lack of synergistic effect between pairs of these four HuNAbs by neutralization assay against the SARS-CoV-2 pseudovirus. The combined antibodies were mixed at 1: 1 ratio.

FIG. 8A is a schematic illustrating the experimental schedule. Four groups of hamsters (G1-G4) received intraperitoneally a single dose of 1.5 mg/kg of B8-IgG1 at one day before infection (-1 dpi) for pre-exposure prophylaxis, and at day one (1 dpi) , two (2 dpi) and three (3 dpi) post-infection for early treatment, respectively. FIG. 8B is a bar graph showing the amount of infectious virus (PFU) as measured in animal lungs by the viral plaque assay in Vero-E6 cells. The PFU/ml concentration is shown in log-transformed units. FIG. 8C is a bar graph showing the relative viral RdRp RNA copies (normalized to β-actin) were determined by RT-PCR in animal lungs. FIG. 8D is a bar graph showing the amount of infectious virus (PFU) as measured in NT homogenates by the viral plaque assay as mentioned above. FIG. 8E is a bar graph showing the viral loads in NT homogenates of each group were determined by RT-PCR assay. The viral load data is shown in log-transformed units.

FIG. 9A is a line graph showing the RBD-specific binding activities of B8-mIgA1, B8-mIgA1, B8-dIgA1 and B8-dIgA2 as compared to B8-IgG1 measured by ELISA. FIG. 9B is a line graph showing the spike-specific binding activities of B8-mIgA1, B8-mIgA1, B8-dIgA1 and B8-dIgA2 as compared to B8-IgG1 measured by ELISA. FIG. 9C is a line graph showing the neutralization activities of B8-mIgA1, B8-mIgA1, B8-dIgA1 and B8-dIgA2 as compared to B8-IgG1 measured by decreased pseudotyped SARS-CoV-2 infection in HEK 293T-ACE2 cells. FIG. 9D is a line graph showing the neutralization activities of B8-mIgA1, B8-mIgA1, B8-dIgA1 and B8-dIgA2 as compared to B8-IgG1 measured by decreased authentic SARS-CoV-2 infection in Vero-E6 cells. All the assays above (FIGs. 9A-9D) were performed in duplicates and the mean of the duplicates was shown with SEM. The antibody concentration in the x-axis is shown in log-transformed units. FIG. 9E is a graph showing the purity of dimeric B8-dgA1 as was confirmed by size exclusion chromatography (SEC) . FIG. 9F is a graph showing the purity of dimeric B8-dgA2 was confirmed by SEC. FIGs. 9G-9J are binding curves showing the binding of ACE2 to SARS-CoV-2 RBD with (blue) or without (blue) pre-incubation with B8-mIgA1, B8-mIgA2, B8-dIgA1, and B8-dIgA2, respectively, as measured by SPR.

[Rectified under Rule 91, 21.03.2022]
FIG. 10A is a schematic of the experimental schedule. Five groups of hamsters (n=4 per group) received a single dose of 4.5 mg/kg of B8-IgG1, B8-mIgA1 or B8-mIgA2 one day before viral challenge for pre-exposure prophylaxis by the intranasal route (circles) or the intraperitoneal route (triangles) , respectively. Control hamsters (n=4) received PBS. On day 0, each hamster was intranasally challenged with a dose of 105 PFU of SARS-CoV-2. All hamsters were sacrificed on 4 dpi for analysis. FIG. 10B is a bar graph showing the viral RNA load, measured by relative RdRp RNA copy numbers (normalized to β-actin) was determined by RT-PCR in animal lung homogenates. FIG. 10C is a bar graph showing the relative sub-genomic nucleocapsid (sgNP) RNA copy numbers (normalized to β-actin) were determined by RT-PCR in animal lung homogenates. FIG. 10D is a bar graph showing the amount of infectious virus (PFU) was measured in animal lung homogenates by the viral plaque assay in Vero-E6 cells. FIG. 10E is a bar graph showing the relative viral RdRp RNA copy numbers (normalized to β-actin) were determined by RT-PCR in NT homogenates. FIG. 10F is a bar graph showing the relative sgNP RNA copy numbers (normalized to β-actin) were determined by RT-PCR in NT homogenates. FIG. 10G is a bar graph showing the amount of infectious virus (PFU) was measured in animal NT homogenates by the viral plaque assay in Vero-E6 cells. Log-transformed units are shown in (B) to (H) . Statistics were generated using one-way ANOVA tests. *p<0.05; **p<0.01.

FIG. 11A is a schematic of the experimental schedule. Four groups of hamsters (n=4 per group) were inoculated intranasally with B8-dIgA1 or B8-dIgA2, either at a low dose of 4.5 mg/kg or at a high dose of 13.5 mg/kg, respectively, 12 hours before intranasal viral challenge. Another group of hamsters (n=4) received PBS as control. On day 0, each hamster was intranasally challenged with a dose of 10 ⁵ PFU of SARS-CoV-2. All hamsters were sacrificed on 4 dpi for analysis. Data represent a presentative experiment from three independent experiments. FIG. 11B is a bar graph showing the relative viral RdRp RNA copy numbers (normalized to β-actin) were determined by RT-PCR in animal lung homogenates. FIG. 11C is a bar graph showing the relative viral sgNP RNA copy numbers (normalized to β-actin) were determined by RT-PCR in animal lung homogenates. FIG. 11D is a bar graph showing the amount of infectious virus (PFU) was measured in animal lung homogenates by the viral plaque assay in Vero-E6 cells. FIG. 11E is a bar graph showing the relative viral RdRp RNA copy numbers (normalized to β-actin) were determined by RT-PCR in NT homogenates. FIG. 11F is a bar graph showing the relative viral sgNP RNA copies (normalized to β-actin) were determined by RT-PCR in NT homogenates. FIG. 11G is a bar graph showing the amount of infectious virus (PFU) was measured in NT homogenates by the viral plaque assay in Vero-E6 cells. Log-transformed units are shown in (B) to (H) . Statistics were generated using one-way ANOVA tests. *p<0.05; **p<0.01.

FIG. 12A is a schematic of the experiment schedule. Two groups of hamsters (n=4 per group) were inoculated intranasally with control dIgA1 and control dIgA2 at a high dose of 13.5 mg/kg 12 hours before intranasal viral challenge, respectively. Another group of hamsters (n=4) received PBS as control. On day 0, each hamster was intranasally challenged with a dose of 105 PFU of SARS-CoV-2. All hamsters were sacrificed on 4 dpi for analysis. FIGs. 12B-12D are bar graphs showing the viral loads in lung tissue as determined by three assays. FIGs. 12E-12G are bar graphs showing the viral loads in NT as determined by three assays.

FIG. 13A are graphical representations showing the effects of B8-dIgA2 on SARS-CoV-2 infection in the MucilAir ^TM model, consisting of primary human nasal epithelial cells but no DCs. B8-mIgA2 or B8-dIgA2 were pre-incubated at doses of 10, 100, and 1000 ng/ml, respectively, in the apical compartment with or without mucus for 1 hour, before adding 104 PFU of SARS-CoV-2 (BetaCoV/France/IDF00372/2020) for 4 hours. The viral RNA loads were measured by RT-PCR in both the apical and basal compartments and are shown in log-transformed units. FIG. 13B is a bar graph showing the CD209 or CD299 overexpressed-HEK 293T cells pre-treated for 6 hours with 10 ng/ml of B8-dIgA1 or B8-dIgA2 or control dIgA1 or control dIgA2 or PBS, respectively, prior to SARS-CoV-2 infection (MOI: 0.05) . Two days after infection, SARS-CoV-2 NP expression was quantified by the mean fluorescence intensity (MFI) after anti-NP IF staining. Statistics were generated using student-t tests. *p<0.05; **p<0.01; ***p<0.001.

FIG. 14 is a flow chart of the SARS-CoV-2 S-B8 complex cryo-EM data processing. Different map density of RBD-Fab portions are emphasized in red cycle.

FIG. 15A is a molecular model showing the structural comparison of RBDs between Spike-B8 3u (different colors) and Spike-B8 2u1d (gray) . FIG. 15B is a molecular model showing that the ACE2 (chocolate color, PDB: 6M0J) may clash with the heavy chain (blue) and light chain (gold) of the B8-Fab. ACE2 and the Fab share overlapping epitopes on the RBM (dotted black circle) , and the framework of the B8-VL appears to clash with ACE2 (dotted black frame) . The RBD core and RBM are shown in light sky blue and green, respectively. FIG. 15C is an atomic model of an RBD-B8 complex portion in cartoon mode, shown with the same color scheme as in (B) . FIG. 15D is a molecular model showing the residues involved in interactions between B8 and the RBM. The heavy and light chain of the B8-Fab are in blue and gold, respectively. The RBM is shown in green.

FIGs. 16A-16C illustrate the results from preliminary analysis of the human nasal cytology data. FIG. 16A is a UMAP representation of the analysis based data submitted under accession code GSE171488 (healthy donor nasal brushing) and GSE164547 (COVID-19 patient nasal brushing) . Among CD14 positive cells, the neutrophil and monocyte-differentiated DCs were mainly increased in the nasal cytology samples of COVID-19 patients. FIG. 16B are graphs showing the increased CD209 expression on nasal DCs of COVID-19 patients. FIG. 16C shows the proportion of nasal DCs increased 6.5-fold from 2%to 13%in nasal samples compared between health and COVID-19 subjects.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

SARS-CoV-2 is characterized by a burst in upper-respiratory portal for high transmissibility. SARS-CoV-2 infects upper respiratory tract despite potent systemic neutralizing antibodies. In the face of this new virus, it is important to discover SARS-CoV-2 specific drugs for prevention and therapy. The problem is that there is no specific drug to treat SARS-CoV-2 infections and COVID-19 patients. The disclosed compounds and compositions solve this problem by providing human neutralizing antibodies (HuNAbs) for entry protection against SARS-CoV-2.

It was realized that development of human neutralizing antibodies (HuNAbs) for entry protection against SARS-CoV-2 would be very useful for treating and forestalling infection by SARS-CoV-2 and development of COVID-19. As disclosed herein, four HuNAbs (B8, B7, B4, and C5) were generated that bind to conformational determinants of viral receptor binding domain (RBD) . The disclosed SARS-CoV-2 HuNAbs, each with a distinct sequence, are newly discovered from 4 patients.

The disclosed antibody drugs were demonstrated to effectively compete with human cellular receptor ACE2 for RBD binding. HuNAbs B8, B7, B4, and C5 prevented entry of pseudovirus with IC ₉₀ values of 0.046 μg/ml, 0.094 μg/ml, 0.136 μg/ml, and 0.083 μg/ml, respectively, and live virus with IC ₉₀ values of 0.032 μg/ml, 0.060 μg/ml, 0.134 μg/ml, and 0.044 μg/ml, respectively, by competing with human cellular receptor ACE2 for RBD binding.

It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Disclosed are antibodies or fragments thereof that comprise such antibodies or fragments, that immunospecifically bind to SARS-CoV-2 RBD and are capable of substantially blocking SARS-CoV-2 RBD’s interaction with ACE2 in vitro, or in a recipient subject or patient. As used herein, a molecule that is “capable of substantially blocking SARS-CoV-2 RBD’s interaction with ACE2” denotes that the provision of such molecule attenuates SARS-CoV-2 RBD-ACE2 interactions by more than 50%, more preferably by more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99%or most preferably completely attenuates such interaction, as measured by any of the assays disclosed herein. Such antibodies and antibody fragments have particular utility in attenuating cell entry of SARS-CoV-2 RBD.

The disclosed subject matter can also involve humanized antibodies and fragments or human antibodies and fragments. Most preferably, such molecules will possess sufficient affinity and avidity to be able to bind to SARS-CoV-2 RBD when present in a subject.

CDR sequences of the variable domains are shown in bold italic:

B8VH:

C5VH:

B7VH:

B4VH:

B8VK:

C5VK:

B7VK:

B4VK:

Analyses of the CDRs of the identified antibodies were conducted in order to identify consensus CDR sequences and likely variant CDR sequences that would provide similar binding attributes. Such variant CDRs were computed using Blosum62. iij analysis according to Table 1. Table 1 presents the Blosum62. iij substitution scores. The higher the score the more conservative the substitution and thus the more likely the substitution will not affect function.

The disclosed subject matter permits the formation of novel antibodies and antigen-binding fragments having 1, 2, 3, 4, 5 or 6 variant CDRs. The substitution scores of Table 1 provide a means for determining the identities of permitted substitutions in CDRs and other pats of the variable regions. For example, if a particular residue of a particular CDR is found to vary as R or S, then since R and S have a substitution score of -1, any substitution of R or S having a substitution score of -1 or greater are as likely as the observed variants (R or S) (or are more likely than R or S) to create a variant CDR having binding attributes that are sufficiently similar to those of the particular CDR to permit the variant CDR to be employed in lieu thereof so as to form a functional anti-SARS-CoV-2 RBD antibody or antigen-binding fragment. For each position, the selection of a residue having a higher substitution score is preferred over the selection of a residue having a lower substitution score.

In addition to antibodies and antigen-binding fragments thereof that possess the CDRs of the anti-SARS-CoV-2 RBD antibodies: B8, B7, B4, and C5, also disclosed are antibodies and antigen-binding fragments thereof that possess CDRs having the above-described light and/or heavy chain consensus sequences.

The disclosed subject matter encompasses antibodies or fragments thereof comprising an amino acid sequence of a variable heavy chain and/or variable light chain that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to the amino acid sequence of the variable heavy chain and/or light chain of the hamster monoclonal antibody produced by any of the above clones, and which exhibit immunospecific binding to SARS-CoV-2 RBD. The disclosed subject matter further encompasses antibodies or fragments thereof that comprise a CDR that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to the amino acid sequence of a CDR of the above-listed clones and which exhibit immunospecific binding to SARS-CoV-2 RBD. The determination of percent identity of two amino acid sequences can be determined by BLAST protein comparison.

In a preferred embodiment, the antibody is an immunoglobulin molecule (e.g., an antibody, diabody, fusion protein, etc. ) that comprises one, two or three light chain CDRs and one, two or three heavy chain CDRs (most preferably three light chain CDRs and three heavy chain CDRs) , wherein the light chain CDRs include:

(1) the light chain CDR1 of anti-SARS-CoV-2 RBD antibody B8, B7, B4, or C5;

(2) a light chain CDR2 of anti-SARS-CoV-2 RBD antibody B8, B7, B4, or C5;and

(3) the light chain CDR3 of anti-human SARS-CoV-2 RBD antibody B8, B7, B4, or C5.

In an alternative preferred embodiment, the immunoglobulin molecule comprises one, two, or three light chain CDRs and one, two, or three heavy chain CDRs (most preferably three light chain CDRs and three heavy chain CDRs) , wherein the heavy chain CDRs include:

(1) the heavy chain CDR1 of anti-SARS-CoV-2 RBD antibody B8, B7, B4, and C5;

(2) the heavy chain CDR2 of anti-SARS-CoV-2 RBD antibody B8, B7, B4, and C5; and

(2) the heavy chain CDR3 of anti-SARS-CoV-2 RBD antibody B8, B7, B4, and C5.

In some forms, the antibody or an antigen-binding fragment thereof can comprise one, two, three, four, five, or more preferably, all 6 CDRs of the above-described preferred antibodies and will exhibit the ability to bind to SARS-CoV-2 RBD.

The Fc portion of the antibody may be varied by isotype or subclass, may be a chimeric or hybrid, and/or may be modified, for example to improve effector functions, control of half-life, tissue accessibility, augment biophysical characteristics such as stability, and improve efficiency of production (and less costly) . Many modifications useful in construction of disclosed antibodies and methods for making them are known in the art, see for example Mueller, et al., Mol. Immun., 34 (6) : 441-452 (1997) , Swann, et al., Cur. Opin. Immun., 20: 493-499 (2008) , and Presta, Cur. Opin. Immun. 20: 460-470 (2008) . In some embodiments the Fc region is the native IgG1, IgG2, or IgG4 Fc region. In some embodiments the Fc region is a hybrid, for example a chimeric consisting of IgG2/IgG4 Fc constant regions. Medications to the Fc region include, but are not limited to, IgG4 modified to prevent binding to Fc gamma receptors and complement, IgG1 modified to improve binding to one or more Fc gamma receptors, IgG1 modified to minimize effector function (amino acid changes) , IgG1 with altered/no glycan (typically by changing expression host) , and IgG1 with altered pH-dependent binding to FcRn. The Fc region may include the entire hinge region, or less than the entire hinge region.

As used herein, the term “antibody” is intended to denote an immunoglobulin molecule that possesses a “variable region” antigen recognition site. The term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain) . The variable region comprises a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region comprises amino acid residues from a “Complementarity Determining Region” or “CDR” (i.e., typically at approximately residues 24-34 (L1) , 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (H1) , 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; 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” (i.e., residues 26-32 (L1) , 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1) , 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917) . “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. The term antibody includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (See e.g., Muyldermans et al., 2001, Trends Biochem. Sci. 26: 230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1: 253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231: 25; International Publication Nos. WO 94/04678 and WO 94/25591; U.S. Patent No. 6,005,079) , single-chain Fvs (scFv) (see, e.g., see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994) ) , single chain antibodies, disulfide-linked Fvs (sdFv) , intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to the disclosed SARS-CoV-2 RBD antibodies) . In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

As used herein, the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody’s Complementarity Determining Regions ( “CDRs” ) and optionally the framework residues that comprise the antibody’s “variable region” antigen recognition site and exhibit an ability to immunospecifically bind antigen. Such fragments include Fab', F (ab') 2, Fv, single chain (ScFv) , and mutants thereof, naturally occurring variants, and fusion proteins comprising the antibody’s “variable region” antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc. ) . As used herein, the term “fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

Human, chimeric or humanized derivatives of anti-human SARS-CoV-2 RBD antibodies are particularly preferred for in vivo use in humans, however, murine antibodies or antibodies of other species may be advantageously employed for many uses (for example, in vitro or in situ detection assays, acute in vivo use, etc. ) . A humanized antibody may comprise amino acid residue substitutions, deletions or additions in one or more non-human CDRs. The humanized antibody derivative may have substantially the same binding, stronger binding or weaker binding when compared to a non-derivative humanized antibody. In specific embodiments, one, two, three, four, or five amino acid residues of the CDR have been substituted, deleted or added (i.e., mutated) . Completely human antibodies are particularly desirable for therapeutic treatment of human subjects.

Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences (see U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741) . Human antibodies can be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized using conventional methodologies with a selected antigen, e.g., all or a portion of a SARS-CoV-2 RBD polypeptide. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology (see, e.g., U.S. Patent No. 5,916,771) . The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13: 65-93, which is incorporated herein by reference in its entirety) . For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., International Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Patent Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Medarex (Princeton, NJ) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

A “chimeric antibody” is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4: 214; Gillies et al., 1989, J. Immunol. Methods 125: 191-202; and U.S. Patent Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397. Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239, 400; International Publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089) , veneering or resurfacing (EP 592, 106; EP 519, 596; Padlan, 1991, Molecular Immunology 28 (4/5) : 489-498; Studnicka et al., 1994, Protein Engineering 7: 805; and Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91: 969) , and chain shuffling (U.S. Patent No. 5,565,332) .

The disclosed subject matter also concerns “humanized antibodies” (see, e.g., European Patent Nos. EP 239, 400, EP 592, 106, and EP 519, 596; International Publication Nos. WO 91/09967 and WO 93/17105; U.S. Patent Nos. 5,225,539, 5,530,101, 5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan, 1991, Molecular Immunology 28 (4/5) : 489-498; Studnicka et al., 1994, Protein Engineering 7 (6) : 805-814; Roguska et al., 1994, PNAS 91: 969-973; Tan et al., 2002, J. Immunol. 169: 1119-1125; Caldas et al., 2000, Protein Eng. 13: 353-360; Morea et al., 2000, Methods 20: 267-79; Baca et al., 1997, J. Biol. Chem. 272: 10678-10684; Roguska et al., 1996, Protein Eng. 9:895-904; Couto et al., 1995, Cancer Res. 55 (23 Supp) : 5973s-5977s; Couto et al., 1995, Cancer Res. 55: 1717-22; Sandhu, 1994, Gene 150: 409-10; Pedersen et al., 1994, J. Mol. Biol. 235: 959-973; Jones et al., 1986, Nature 321: 522-525; Reichmann et al., 1988, Nature 332: 323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2: 593-596) . As used herein, the term “humanized antibody” refers to an immunoglobulin comprising a human framework region and one or more CDR’s from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR's is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor. ” Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95%or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDR’s, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human. One says that the donor antibody has been “humanized, ” by the process of “humanization, ” because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDR’s. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or a non-human primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin that immunospecifically binds to an FcγRIIB polypeptide, that has been altered by the introduction of amino acid residue substitutions, deletions or additions (i.e., mutations) .

DNA sequences coding for preferred human acceptor framework sequences include but are not limited to FR segments from the human germline VH segment VH1-18 and JH6 and the human germline VL segment VK-A26 and JK4. In a specific embodiment, one or more of the CDRs are inserted within framework regions using routine recombinant DNA techniques. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., 1998, “Structural Determinants In The Sequences Of Immunoglobulin Variable Domain, ” J. Mol. Biol. 278: 457-479 for a listing of human framework regions) .

A humanized or chimeric SARS-CoV-2 RBD antibody can include substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. Preferably, a SARS-CoV-2 RBD antibody also includes at least a portion of an immunoglobulin constant region (Fc) , typically that of a human immunoglobulin. The constant domains of the SARS-CoV-2 RBD antibodies may be selected with respect to the proposed function of the antibody, in particular the effector function which may be required. In some embodiments, the constant domains of the SARS-CoV-2 RBD antibodies are (or comprise) human IgA, IgD, IgE, IgG or IgM domains. In a specific embodiment, human IgG constant domains, especially of the IgG1 and IgG3 isotypes are used, when the humanized SARS-CoV-2 RBD antibodies is intended for therapeutic uses and antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity are needed. In alternative embodiments, IgG2 and IgG4 isotypes are used when the SARS-CoV-2 RBD antibody is intended for therapeutic purposes and antibody effector function is not required. The disclosed subject matter also encompasses Fc constant domains comprising one or more amino acid modifications which alter antibody effector functions such as those disclosed in U.S. Patent Application Publication Nos. 2005/0037000 and 2005/0064514.

In some embodiments, the SARS-CoV-2 RBD antibody contains both the light chain as well as at least the variable domain of a heavy chain. In other embodiments, the SARS-CoV-2 RBD antibody may further include one or more of the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4. In some embodiments, the constant domain is a complement fixing constant domain where it is desired that the antibody exhibit cytotoxic activity, and the class is typically IgG1. In other embodiments, where such cytotoxic activity is not desirable, the constant domain may be of the IgG2 class. The SARS-CoV-2 RBD antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.

The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor CDR or the consensus framework may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the donor antibody. Such mutations, however, are preferably not extensive. Usually, at least 75%of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences, more often 90%, and most preferably greater than 95%. Humanized antibodies can be produced using variety of techniques known in the art, including, but not limited to, CDR-grafting (European Patent No. EP 239, 400; International Publication No. WO 91/09967; and U.S. Patent Nos. 5,225,539, 5,530,101, and5,585,089) , veneering or resurfacing (European Patent Nos. EP 592, 106 and EP 519, 596; Padlan, 1991, Molecular Immunology 28 (4/5) : 489-498; Studnicka et al., 1994, Protein Engineering 7 (6) : 805-814; and Roguska et al., 1994, Proc. Natl. Acad. Sci. 91: 969-973) , chain shuffling (U.S. Patent No. 5,565,332) , and techniques disclosed in, e.g., U.S. Patent Nos. 6,407,213, 5,766,886, 5,585,089, International Publication No. WO 9317105, Tan et al., 2002, J. Immunol. 169: 1119-25, Caldas et al., 2000, Protein Eng. 13: 353-60, Morea et al., 2000, Methods 20: 267-79, Baca et al., 1997, J. Biol. Chem. 272: 10678-84, Roguska et al., 1996, Protein Eng. 9: 895-904, Couto et al., 1995, Cancer Res. 55 (23 Supp) : 5973s-5977s, Couto et al., 1995, Cancer Res. 55: 1717-22, Sandhu, 1994, Gene 150: 409-10, Pedersen et al., 1994, J. Mol. Biol. 235: 959-73, Jones et al., 1986, Nature 321: 522-525, Riechmann et al., 1988, Nature 332: 323, and Presta, 1992, Curr. Op. Struct. Biol. 2: 593-596. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; U.S. Publication Nos. 2004/0049014 and 2003/0229208; U.S. Patent Nos. 6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 and Riechmann et al., 1988, Nature 332: 323) .

The disclosed antibodies can be monospecific. Also of interest are bispecific antibodies, trispecific antibodies or antibodies of greater multispecificity that exhibit specificity to different targets in addition to SARS-CoV-2 RBD, such as other molecules of the immune system. For example, such antibodies may bind to both SARS-CoV-2 RBD and to an antigen that is important for targeting the antibody to a particular cell type or tissue (for example, to an antigen associated with a cancer antigen of a tumor being treated) . In another embodiment, such multispecific antibody binds to molecules (receptors or ligands) involved in alternative or supplemental immunomodulatory pathways, such as CTLA4, TIM3, TIM4, OX40, CD40, GITR, 4-1-BB, CD27/CD70, ICOS, B7-H4, LIGHT, PD-1 or LAG3, in order to diminish further modulate the immunomodulatory effects. Furthermore, the multispecific antibody may bind to effecter molecules such as cytokines (e.g., IL-7, IL-15, IL-12, IL-4 TGF-beta, IL-10, IL-17, IFNg, Flt3, BLys) and chemokines (e.g., CCL21) , which may be particularly relevant for down-modulating both acute and chronic immune responses.

The disclosed antibodies can be produced by any method known in the art useful for the production of polypeptides, e.g., in vitro synthesis, recombinant DNA production, and the like. Preferably, the antibodies are produced by recombinant DNA technology. The SARS-CoV-2 RBD antibodies may be produced using recombinant immunoglobulin expression technology. The recombinant production of immunoglobulin molecules, including humanized antibodies are described in U.S. Patent No. 4,816,397 (Boss et al. ) , U.S. Patent Nos. 6,331,415 and 4,816,567 (both to Cabilly et al. ) , U.K. patent GB 2,188,638 (Winter et al. ) , and U.K. patent GB 2,209,757. Techniques for the recombinant expression of immunoglobulins, including humanized immunoglobulins, can also be found, in Goeddel et al., Gene Expression Technology Methods in Enzymology Vol. 185 Academic Press (1991) , and Borreback, Antibody Engineering, W. H.Freeman (1992) . Additional information concerning the generation, design and expression of recombinant antibodies can be found in Mayforth, Designing Antibodies, Academic Press, San Diego (1993) .

An exemplary process for the production of the recombinant chimeric SARS-CoV-2 RBD antibodies can include the following: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody heavy chain in which the CDRs and variable region of a murine anti-human SARS-CoV-2 RBD monoclonal antibody are fused to an Fc region derived from a human immunoglobulin, thereby producing a vector for the expression of a chimeric antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody light chain of the murine anti-human SARS-CoV-2 RBD monoclonal antibody, thereby producing a vector for the expression of chimeric antibody light chain; c) transferring the expression vectors to a host cell by conventional molecular biology methods to produce a transfected host cell for the expression of chimeric antibodies; and d) culturing the transfected cell by conventional cell culture techniques so as to produce chimeric antibodies.

An exemplary process for the production of the recombinant humanized SARS-CoV-2 RBD antibodies can include the following: a) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an anti-human SARS-CoV-2 RBD heavy chain in which the CDRs and a minimal portion of the variable region framework that are required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, such as a murine anti-human SARS-CoV-2 RBD monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for the expression of a humanized antibody heavy chain; b) constructing, by conventional molecular biology methods, an expression vector that encodes and expresses an antibody light chain in which the CDRs and a minimal portion of the variable region framework that are required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, such as a murine anti-human SARS-CoV-2 RBD monoclonal antibody, and the remainder of the antibody is derived from a human immunoglobulin, thereby producing a vector for the expression of humanized antibody light chain; c) transferring the expression vectors to a host cell by conventional molecular biology methods to produce a transfected host cell for the expression of humanized antibodies; and d) culturing the transfected cell by conventional cell culture techniques so as to produce humanized antibodies.

With respect to either exemplary method, host cells may be co-transfected with such expression vectors, which may contain different selectable markers but, with the exception of the heavy and light chain coding sequences, are preferably identical. This procedure provides for equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA or both. The host cell used to express the recombinant SARS-CoV-2 RBD antibody can be either a bacterial cell such as Escherichia coli, or more preferably a eukaryotic cell (e.g., a Chinese hamster ovary (CHO) cell or a HEK-293 cell) . The choice of expression vector is dependent upon the choice of host cell, and may be selected so as to have the desired expression and regulatory characteristics in the selected host cell. Other cell lines that may be used include, but are not limited to, CHO-K1, NSO, and PER. C6 (Crucell, Leiden, Netherlands) .

Any of the above-described antibodies can be used to generate anti-idiotype antibodies using techniques well known to those skilled in the art (see, e.g., Greenspan, N.S. et al. (1989) “Idiotypes: Structure And Immunogenicity, ” FASEB J. 7: 437-444; and Nisinoff, A. (1991) “Idiotypes: Concepts And Applications, ” J. Immunol. 147 (8) : 2429-2438) .

The binding properties of any of the above antibodies can, if desired, be further improved by screening for variants that exhibit such desired characteristics. For example, such antibodies can be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains, such as Fab and Fv or disulfide-bond stabilized Fv, expressed from a repertoire or combinatorial antibody library (e.g., human or murine) . Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage, including fd and M13. The antigen binding domains are expressed as a recombinantly fused protein to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the immunoglobulins, or fragments thereof, include those disclosed in Brinkman, U. et al. (1995) “Phage Display Of Disulfide-Stabilized Fv Fragments, ” J. Immunol. Methods, 182: 41-50, 1995; Ames, R.S. et al. (1995) “Conversion Of Murine Fabs Isolated From A Combinatorial Phage Display Library To Full Length Immunoglobulins, ” J. Immunol. Methods, 184: 177-186; Kettleborough, C.A. et al. (1994) “Isolation Of Tumor Cell-Specific Single-Chain Fv From Immunized Mice Using Phage-Antibody Libraries And The Re-Construction Of Whole Antibodies From These Antibody Fragments, ” Eur. J. Immunol., 24: 952-958, 1994; Persic, L. et al. (1997) “An Integrated Vector System For The Eukaryotic Expression Of Antibodies Or Their Fragments After Selection From Phage Display Libraries, ” Gene, 187: 9-18; Burton, D.R. et al. (1994) “Human Antibodies From Combinatorial Libraries, ” Adv. Immunol. 57: 191-280; PCT Publications WO 92/001047; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Patents Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including humanized antibodies, or any other desired fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab’ and F (ab’) 2 fragments can also be employed using methods known in the art (such as those disclosed in PCT Publication WO 92/22324; Mullinax, R.L. et al. (1992) “Expression Of A Heterodimeric Fab Antibody Protein In One Cloning Step, ” BioTechniques, 12 (6) : 864-869; and Sawai et al. (1995) “Direct Production Of The Fab Fragment Derived From The Sperm Immobilizing Antibody Using Polymerase Chain Reaction And cDNA Expression Vectors, ” Am. J. Reprod. Immunol. 34: 26-34; and Better, M. et al. (1988) “Escherichia coli Secretion Of An Active Chimeric Antibody Fragment, ” Science 240: 1041-1043) . Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston, J.S. et al. (1991) “Protein Engineering Of Single-Chain Fv Analogs And Fusion Proteins, ” Methods in Enzymology 203: 46-88; Shu, L. et al., “Secretion Of A Single-Gene-Encoded Immunoglobulin From Myeloma Cells, ” Proc. Natl. Acad. Sci. (USA) 90: 7995-7999; and Skerra. A. et al. (1988) “Assembly Of A Functional Immunoglobulin Fv Fragment In Escherichia coli, ” Science 240: 1038-1040.

Phage display technology can be used to increase the affinity of an antibody for SARS-CoV-2 RBD. This technique would be useful in obtaining high affinity antibodies that could be used in the disclosed combinatorial methods. This technology, referred to as affinity maturation, employs mutagenesis or CDR walking and re-selection using such receptors or ligands (or their extracellular domains) or an antigenic fragment thereof to identify antibodies that bind with higher affinity to the antigen when compared with the initial or parental antibody (See, e.g., Glaser, S.M. et al. (1992) “Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System, ” J. Immunol. 149: 3903-3913) . Mutagenizing entire codons rather than single nucleotides results in a semi-randomized repertoire of amino acid mutations. Libraries can be constructed consisting of a pool of variant clones each of which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue. Mutants with increased binding affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased avidity to the antigen (e.g., ELISA) (see, e.g., Wu, H. et al. (1998) “Stepwise In Vitro Affinity Maturation Of Vitaxin, An Alphav Beta3-Specific Humanized Mab, ” Proc. Natl. Acad. Sci. (USA) 95 (11) : 6037-6042; Yelton, D.E. et al. (1995) “Affinity Maturation Of The BR96 Anti-Carcinoma Antibody By Codon-Based Mutagenesis, ” J. Immunol. 155: 1994-2004) . CDR walking which randomizes the light chain may be used possible (see, Schier et al. (1996) “Isolation Of Picomolar Affinity Anti-C-Erbb-2 Single-Chain Fv By Molecular Evolution Of The Complementarity Determining Regions In The Center Of The Antibody Binding Site, ” J. Mol. Biol. 263: 551-567) .

Thus, the use of random mutagenesis to identify improved CDRs is also contemplated. Phage display technology can alternatively be used to increase (or decrease) CDR affinity. This technology, referred to as affinity maturation, employs mutagenesis or “CDR walking” and re-selection uses the target antigen or an antigenic fragment thereof to identify antibodies having CDRs that bind with higher (or lower) affinity to the antigen when compared with the initial or parental antibody (see, e.g., Glaser, S.M. et al. (1992) “Antibody Engineering By Codon-Based Mutagenesis In A Filamentous Phage Vector System, ” J. Immunol. 149: 3903-3913) . Mutagenizing entire codons rather than single nucleotides results in a semi-randomized repertoire of amino acid mutations. Libraries can be constructed consisting of a pool of variant clones each of which differs by a single amino acid alteration in a single CDR and which contain variants representing each possible amino acid substitution for each CDR residue. Mutants with increased (or decreased) binding affinity for the antigen can be screened by contacting the immobilized mutants with labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased (or decreased) avidity to the antigen (e.g., ELISA) (see, Wu, H. et al. (1998) “Stepwise In Vitro Affinity Maturation Of Vitaxin, An Alphav Beta3-Specific Humanized Mab, ” Proc. Natl. Acad. Sci. (USA) 95 (11) : 6037-6042; Yelton, D.E. et al. (1995) “Affinity Maturation Of The BR96 Anti-Carcinoma Antibody By Codon-Based Mutagenesis, ” J. Immunol. 155: 1994-2004) . CDR walking which randomizes the light chain may be used possible (see, Schier et al. (1996) “Isolation Of Picomolar Affinity Anti-C-Erbb-2 Single-Chain Fv By Molecular Evolution Of The Complementarity Determining Regions In The Center Of The Antibody Binding Site, ” J. Mol. Biol. 263: 551-567) .

Methods for accomplishing such affinity maturation are described for example in: Krause, J.C. et al. (2011) “An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function Of A Human Antibody, ” MBio. 2 (1) pii: e00345-10. doi: 10.1128/mBio. 00345-10; Kuan, C.T. et al. (2010) “Affinity-Matured Anti-Glycoprotein NMB Recombinant Immunotoxins Targeting Malignant Gliomas And Melanomas, ” Int. J. Cancer 10.1002/ijc. 25645; Hackel, B.J. et al. (2010) “Stability And CDR Composition Biases Enrich Binder Functionality Landscapes, ” J. Mol. Biol. 401 (1) : 84-96; Montgomery, D.L. et al. (2009) “Affinity Maturation And Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41, ” MAbs 1 (5) : 462-474; Gustchina, E. et al. (2009) “Affinity Maturation By Targeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab Derived From A Synthetic

Human Antibody Library And Directed Against The Internal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With Improved HIV-1 Neutralization Potency And Breadth, ” Virology 393 (1) : 112-119; Finlay, W.J. et al. (2009) “Affinity Maturation Of A Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside And Outside The Complementarity-Determining Regions, ” J. Mol. Biol. 388 (3) : 541-558; Bostrom, J. et al. (2009) “Improving Antibody Binding Affinity And Specificity For Therapeutic Development, ” Methods Mol. Biol. 525: 353-376; Steidl, S. et al. (2008) “In Vitro Affinity Maturation Of Human GM-CSF Antibodies By Targeted CDR-Diversification, ” Mol. Immunol. 46 (1) : 135-144; and Barderas, R. et al. (2008) “Affinity maturation of antibodies assisted by in silico modeling, ” Proc. Natl. Acad. Sci. (USA) 105 (26) : 9029-9034.

The production and use of “derivatives” of any of the above-described antibodies and their antigen-binding fragments is also contemplated. 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, chimeric or humanized variants of any of antibodies 1.3, 4.5 or 7.8, as well 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) .

In some embodiments, a humanized antibody is a derivative. Such a humanized antibody comprises amino acid residue substitutions, deletions or additions in one or more non-human CDRs. The humanized antibody derivative may have substantially the same binding, better binding, or worse binding when compared to a non-derivative humanized antibody. In specific embodiments, one, two, three, four, or five amino acid residues of the CDR have been substituted, deleted or added (i.e., mutated) .

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.

Derivatized antibodies may be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, preferably a human. Preferably such alteration will 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 disclosed humanized antibodies 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. The humanized SARS-CoV-2 RBD antibodies can be engineered to increase biological half-lives (see, e.g. U.S. Patent No. 6,277,375) . For example, humanized SARS-CoV-2 RBD antibodies can be engineered in the Fc-hinge domain to have increased in vivo or serum half-lives.

Antibodies or fragments thereof with increased in vivo half-lives can be generated by attaching to said antibodies or antibody fragments polymer molecules such as high molecular weight polyethyleneglycol (PEG) . PEG can be attached to said antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N–or C-terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography.

The SARS-CoV-2 RBD antibodies may also be modified by the methods and coupling agents described by Davis et al. (See U.S. Patent No. 4,179,337) in order to provide compositions that can be injected into the mammalian circulatory system with substantially no immunogenic response.

One embodiment encompasses modification of framework residues of the humanized SARS-CoV-2 RBD antibodies. Framework residues in the framework regions may be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., U.S. Patent No. 5,585,089; and Riechmann, L. et al. (1988) “Reshaping Human Antibodies For Therapy, ” Nature 332: 323-327) .

Yet another embodiment encompasses anti-human SARS-CoV-2 RBD antibodies (and more preferably, humanized antibodies) and antigen-binding fragments thereof that are recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a heterologous molecule (i.e., an unrelated molecule) . The fusion does not necessarily need to be direct, but may occur through linker sequences.

In one embodiment such heterologous molecules are polypeptides having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids. Such heterologous molecules may alternatively be enzymes, hormones, cell surface receptors, drug moieties, such as: toxins (such as abrin, ricin A, pseudomonas exotoxin (i.e., PE-40) , diphtheria toxin, ricin, gelonin, or pokeweed antiviral protein) , proteins (such as tumor necrosis factor, interferon (e.g., α-interferon, β-interferon) , nerve growth factor, platelet derived growth factor, tissue plasminogen activator, or an apoptotic agent (e.g., tumor necrosis factor-α, tumor necrosis factor-β) ) , biological response modifiers (such as, for example, a lymphokine (e.g., interleukin-1 ( “IL-1” ) , interleukin-2 ( “IL-2” ) , interleukin-6 ( “IL-6” ) ) , granulocyte macrophage colony stimulating factor ( “GM-CSF” ) , granulocyte colony stimulating factor ( “G-CSF” ) , or macrophage colony stimulating factor, ( “M-CSF” ) ) , or growth factors (e.g., growth hormone ( “GH” ) ) ) , cytotoxins (e.g., a cytostatic or cytocidal agent, such as paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof) , antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine) , alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,

(carmustine; BSNU) and lomustine (CCNU) , cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin) , anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin) , antibiotics (e.g., dactinomycin (formerly actinomycin) , bleomycin, mithramycin, and anthramycin (AMC) ) , or anti-mitotic agents (e.g., vincristine and vinblastine) .

Techniques for conjugating such therapeutic moieties to antibodies are well known; see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy” , in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Reisfeld et al. (eds. ) , 1985, pp. 243-56, Alan R. Liss, Inc. ) ; Hellstrom et al., “Antibodies For Drug Delivery” , in CONTROLLED DRUG DELIVERY (2nd Ed. ) , Robinson et al. (eds. ) , 1987, pp. 623-53, Marcel Dekker, Inc. ) ; Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review” , in MONOCLONAL ANTIBODIES ‘84: BIOLOGICAL AND CLINICAL APPLICATIONS, Pinchera et al. (eds. ) , 1985, pp. 475-506) ; “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy” , in MONOCLONAL ANTIBODIES FOR CANCER DETECTION AND THERAPY, Baldwin et al. (eds. ) , 1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, ” Immunol. Rev. 62: 119-158.

In one embodiment, the SARS-CoV-2 RBD antibodies or SARS-CoV-2 RBD fusion molecules include an Fc portion. The Fc portion of such molecules may be varied by isotype or subclass, may be a chimeric or hybrid, and/or may be modified, for example to improve effector functions, control of half-life, tissue accessibility, augment biophysical characteristics such as stability, and improve efficiency of production (and less costly) . Many modifications useful in construction of disclosed fusion proteins and methods for making them are known in the art, see for example Mueller, J.P. et al. (1997) “Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric IgG2/G4 Constant Regions Block Human Leukocyte Binding To Porcine Endothelial Cells, ” Mol. Immun. 34 (6) : 441-452, Swann, P.G. (2008) “Considerations For The Development Of Therapeutic Monoclonal Antibodies, ” Curr. Opin. Immun. 20: 493-499 (2008) , and Presta, L.G. (2008) “Molecular Engineering And Design Of Therapeutic Antibodies, ” Curr. Opin. Immun. 20: 460-470. In some embodiments the Fc region is the native IgG1, IgG2, or IgG4 Fc region. In some embodiments the Fc region is a hybrid, for example a chimeric consisting of IgG2/IgG4 Fc constant regions. Modifications to the Fc region include, but are not limited to, IgG4 modified to prevent binding to Fc gamma receptors and complement, IgG1 modified to improve binding to one or more Fc gamma receptors, IgG1 modified to minimize effector function (amino acid changes) , IgG1 with altered/no glycan (typically by changing expression host) , and IgG1 with altered pH-dependent binding to FcRn, and IgG4 with serine at amino acid resident #228 in the hinge region changed to proline (S228P) to enhance stability. The Fc region may include the entire hinge region, or less than the entire hinge region.

The therapeutic outcome in patients treated with rituximab (a chimeric mouse/human IgG1 monoclonal antibody against CD20) for non-Hodgkin’s lymphoma or Waldenstrom’s macroglobulinemia correlated with the individual’s expression of allelic variants of Fcγ receptors with distinct intrinsic affinities for the Fc domain of human IgG1. In particular, patients with high affinity alleles of the low affinity activating Fc receptor CD16A (FcγRIIIA) showed higher response rates and, in the cases of non-Hodgkin’s lymphoma, improved progression-free survival. In another embodiment, the Fc domain may contain one or more amino acid insertions, deletions or substitutions that reduce binding to the low affinity inhibitory Fc receptor CD32B (FcγRIIB) and retain wild-type levels of binding to or enhance binding to the low affinity activating Fc receptor CD16A (FcγRIIIA) .

Another embodiment includes IgG _2-4 hybrids and _IgG4 mutants that have reduce binding to FcR which increase their half-life. Representative IG _2-4 hybrids and IgG4 mutants are described in Angal, S. et al. (1993) “A Single Amino Acid Substitution Abolishes The Heterogeneity Of Chimeric Mouse/Human (Igg4) Antibody, ” Molec. Immunol. 30 (1) : 105-108; Mueller, J.P. et al. (1997) “Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric Igg2/G4 Constant Regions Block Human Leukocyte Binding To Porcine Endothelial Cells, ” Mol. Immun. 34 (6) : 441-452; and U.S. Patent No. 6,982,323. In some embodiments the IgG ₁ and/or IgG ₂ domain is deleted for example, Angal, s. et al. describe IgG ₁ and IgG ₂ having serine 241 replaced with a proline.

Substitutions, additions or deletions in the derivatized antibodies may be in the Fc region of the antibody and may thereby serve to modify the binding affinity of the antibody to one or more FcγR. Methods for modifying antibodies with modified binding to one or more FcγR are known in the art, see, e.g., PCT Publication Nos. WO 04/029207, WO 04/029092, WO 04/028564, WO 99/58572, WO 99/51642, WO 98/23289, WO 89/07142, WO 88/07089, and U.S. Patent Nos. 5,843,597 and 5,642,821. In one particular embodiment, the modification of the Fc region results in an antibody with an altered antibody-mediated effector function, an altered binding to other Fc receptors (e.g., Fc activation receptors) , an altered antibody-dependent cell-mediated cytotoxicity (ADCC) activity, an altered C1q binding activity, an altered complement-dependent cytotoxicity activity (CDC) , a phagocytic activity, or any combination thereof.

In some forms, the antibodies whose Fc region will have been modified so that the molecule will exhibit altered Fc receptor (FcR) binding activity, for example to exhibit decreased activity toward activating receptors such as FcγRIIA or FcγRIIIA, or increased activity toward inhibitory receptors such as FcγRIIB. Preferably, such antibodies will exhibit decreased antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) activities (relative to a wild-type Fc receptor) .

Modifications that affect Fc-mediated effector function are well known in the art (see U.S. Patent No. 6,194,551, and WO 00/42072; Stavenhagen, J.B. et al. (2007) “Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors, ” Cancer Res. 57 (18) : 8882-8890; Shields, R.L. 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 (9) : 6591-6604) . Exemplary variants of human IgG1 Fc domains with reduced binding to FcγRIIA or FcγRIIIA, but unchanged or enhanced binding to FcγRIIB, include S239A, H268A, S267G, E269A, E293A, E293D, Y296F, R301A, V303A, A327G, K322A, E333A, K334A, K338A, A339A, D376A. In some forms, the antibodies can be those whose Fc region will have been deleted (for example, an Fab or F (ab) ₂, etc. ) .

Any of the disclosed molecules can be fused to marker sequences, such as a peptide, to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I.A. et al. (1984) “The Structure Of An Antigenic Determinant In A Protein, ” Cell, 37: 767-778) and the “flag” tag (Knappik, A. et al. (1994) “An Improved Affinity Tag Based On The FLAG Peptide For The Detection And Purification Of Recombinant Antibody Fragments, ” Biotechniques 17 (4) : 754-761) .

The disclosed subject matter also encompasses antibodies or their antigen-binding fragments that are conjugated to a diagnostic or therapeutic agent or any other molecule for which serum half-life is desired to be increased. The antibodies can be used diagnostically (in vivo, in situ or in vitro) to, for example, monitor the development or progression of a disease, disorder or infection as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics. Such diagnosis and detection can be accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic group complexes such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent material such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive material such as, but not limited to, bismuth ( ²¹³Bi) , carbon ( ¹⁴C) , chromium ( ⁵¹Cr) , cobalt ( ⁵⁷Co) , fluorine ( ¹⁸F) , gadolinium ( ¹⁵³Gd, ¹⁵⁹Gd) , gallium ( ⁶⁸Ga, ⁶⁷Ga) , germanium ( ⁶⁸Ge) , holmium ( ¹⁶⁶Ho) , indium ( ¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In) , iodine ( ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I) , lanthanium ( ¹⁴⁰La) , lutetium ( ¹⁷⁷Lu) , manganese ( ⁵⁴Mn) , molybdenum ( ⁹⁹Mo) , palladium ( ¹⁰³Pd) , phosphorous ( ³²P) , praseodymium ( ¹⁴²Pr) , promethium ( ¹⁴⁹Pm) , rhenium ( ¹⁸⁶Re, ¹⁸⁸Re) , rhodium ( ¹⁰⁵Rh) , ruthemium ( ⁹⁷Ru) , samarium ( ¹⁵³Sm) , scandium ( ⁴⁷Sc) , selenium ( ⁷⁵Se) , strontium ( ⁸⁵Sr) , sulfur ( ³⁵S) , technetium ( ⁹⁹Tc) , thallium ( ²⁰¹Ti) , tin ( ¹¹³Sn, ¹¹⁷Sn) , tritium ( ³H) , xenon ( ¹³³Xe) , ytterbium ( ¹⁶⁹Yb, ¹⁷⁵Yb) , yttrium ( ⁹⁰Y) , zinc ( ⁶⁵Zn) ; positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.

The disclosed molecules can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980. Such heteroconjugate antibodies may additionally bind to haptens (such as fluorescein, etc. ) , or to cellular markers (e.g., PD-1, 4-1-BB, B7-H4, SARS-CoV-2 RBD, CD4, CD8, CD14, CD25, CD27, CD40, CD68, CD163, CTLA4, GITR, LAG-3, OX40, TIM3, TIM4, TLR2, LIGHT, etc. ) or to cytokines (e.g., IL-7, IL-15, IL-12, IL-4 TGF-beta, IL-10, IL-17, IFNg, Flt3, BLys) or chemokines (e.g., CCL21) , etc.

The disclosed molecules may be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen or of other molecules that are capable of binding to target antigen that has been immobilized to the support via binding to an antibody or antigen-binding fragment as disclosed. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

The disclosed subject matter additionally includes nucleic acid molecules (DNA or RNA) that encode any such antibodies or fragments, as well as vector molecules (such as plasmids) that are capable of transmitting or of replication such nucleic acid molecules and expressing such antibodies or fragments in a cell line. The nucleic acids can be single-stranded, double-stranded, may contain both single-stranded and double-stranded portions.

As used herein the term “modulate” relates to a capacity to alter an effect or result. In particular, the disclosed subject matter relates to polypeptides that comprise an anti-SARS-CoV-2 RBD antibody or any of its antigen-binding fragments that immunospecifically binds SARS-CoV-2 RBD.

As used herein, the terms “treat, ” “treating, ” “treatment” and “therapeutic use” refer to the elimination, reduction or amelioration of one or more symptoms of a disease or disorder that would benefit from an increased or decreased immune response. As used herein, a “therapeutically effective amount” refers to that amount of a therapeutic agent sufficient to mediate an altered immune response, and more preferably, a clinically relevant altered immune response, sufficient to mediate a reduction or amelioration of a symptom of a disease or condition. An effect is clinically relevant if its magnitude is sufficient to impact the health or prognosis of a recipient subject. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to reduce or minimize disease progression, e.g., delay or minimize an autoimmune response or an inflammatory response or a transplant rejection. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent or SARS-CoV-2 RBD antibody means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease, e.g., sufficient to enhance the therapeutic efficacy of a therapeutic antibody sufficient to treat or manage a disease.

As used herein, the term “prophylactic agent” refers to an agent that can be used in the prevention of a disorder or disease prior to the detection of any symptoms of such disorder or disease. A “prophylactically effective” amount is the amount of prophylactic agent sufficient to mediate such protection. A prophylactically effective amount may also refer to the amount of the prophylactic agent that provides a prophylactic benefit in the prevention of disease. Further, a prophylactically effective amount with respect to a prophylactic agent means that amount of prophylactic agent alone, or in combination with other agents, that provides a prophylactic benefit in the prevention of disease.

The dosage amounts and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophylactically effective. The dosage and frequency further will typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of cancer, the route of administration, as well as age, body weight, response, and the past medical history of the patient. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician’s Desk Reference (56 ^th Ed., 2002) .

Various delivery systems are known and can be used to administer the therapeutic or prophylactic compositions, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432) , construction of a nucleic acid as part of a retroviral or other vector, etc.

Methods of administering antibodies include, but are not limited to, pulmonary, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous) , epidural, and mucosal (e.g., intranasal and oral routes) . In a specific embodiment, the antibodies are administered by inhalation, intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by inhalation, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc. ) and may be administered together with other biologically active agents. Administration can be systemic or local. Pulmonary administration can be by, for example, use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., U.S. Patent Nos. 6,019,968; 5,985,20; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903. In a specific embodiment, it may be desirable to administer the pharmaceutical compositions locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering an antibody, care must be taken to use materials to which the antibody does not absorb.

In some embodiments, the antibodies are formulated in liposomes for targeted delivery of the antibodies. Liposomes are vesicles comprised of concentrically ordered phopsholipid bilayers which encapsulate an aqueous phase. Liposomes typically comprise various types of lipids, phospholipids, and/or surfactants. The components of liposomes are arranged in a bilayer configuration, similar to the lipid arrangement of biological membranes. Liposomes are particularly preferred delivery vehicles due, in part, to their biocompatibility, low immunogenicity, and low toxicity. Methods for preparation of liposomes are known in the art and are specifically contemplated, see, e.g., Epstein et al., 1985, Proc. Natl. Acad. Sci. USA, 82: 3688; Hwang et al., 1980 Proc. Natl. Acad. Sci. USA, 77: 4030-4; U.S. Patent Nos. 4,485,045 and 4,544,545.

Methods of preparing liposomes with a prolonged serum half-life, i.e., enhanced circulation time, such as those disclosed in U.S. Patent No. 5,013,556 can be used to make liposomes-antibody compositions. Preferred liposomes are not rapidly cleared from circulation, i.e., are not taken up into the mononuclear phagocyte system (MPS) . The disclosed subject matter also encompasses sterically stabilized liposomes which are prepared using common methods known to one skilled in the art. Although not intending to be bound by a particular mechanism of action, sterically stabilized liposomes contain lipid components with bulky and highly flexible hydrophilic moieties, which reduces the unwanted reaction of liposomes with serum proteins, reduces oposonization with serum components and reduces recognition by MPS. Sterically stabilized liposomes are preferably prepared using polyethylene glycol. For preparation of liposomes and sterically stabilized liposome, see, e.g., Bendas et al., 2001 BioDrugs, 15 (4) : 215-224; Allen et al., 1987 FEBS Lett. 223: 42-6; Klibanov et al., 1990 FEBS Lett., 268: 235-7; Blum et al., 1990, Biochim. Biophys. Acta., 1029: 91-7; Torchilin et al., 1996, J. Liposome Res. 6: 99-116; Litzinger et al., 1994, Biochim. Biophys. Acta, 1190: 99-107; Maruyama et al., 1991, Chem. Pharm. Bull., 39: 1620-2; Klibanov et al., 1991, Biochim Biophys Acta, 1062; 142-8; Allen et al., 1994, Adv. Drug Deliv. Rev, 13: 285-309. The disclosed subject matter also encompasses liposomes that are adapted for specific organ targeting, see, e.g., U.S. Patent No. 4,544,545, or specific cell targeting, see, e.g., U.S. Patent Application Publication No. 2005/0074403. Particularly useful liposomes for use in the disclosed compositions and methods can be generated by reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG derivatized phosphatidylethanolamine (PEG-PE) . Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. In some embodiments, a fragment of an antibody, e.g., F (ab’) , may be conjugated to the liposomes using previously described methods, see, e.g., Martin et al., 1982, J. Biol. Chem. 257: 286-288.

The SARS-CoV-2 RBD antibodies may also be formulated as immunoliposomes. Immunoliposomes refer to a liposomal composition, wherein an antibody or a fragment thereof is linked, covalently or non-covalently to the liposomal surface. The chemistry of linking an antibody to the liposomal surface is known in the art and are specifically contemplated, see, e.g., U.S. Patent No. 6,787,153; Allen et al., 1995, Stealth Liposomes, Boca Rotan: CRC Press, 233-44; Hansen et al., 1995, Biochim. Biophys. Acta, 1239: 133-144. In most preferred embodiments, immunoliposomes for use in the disclosed methods and compositions are further sterically stabilized. Preferably, the antibodies are linked covalently or non-covalently to a hydrophobic anchor, which is stably rooted in the lipid bilayer of the liposome. Examples of hydrophobic anchors include, but are not limited to, phospholipids, e.g., phosoatidylethanolamine (PE) , phospahtidylinositol (PI) . To achieve a covalent linkage between an antibody and a hydrophobic anchor, any of the known biochemical strategies in the art may be used, see, e.g., J. Thomas August, ed., 1997, Gene Therapy: Advances in Pharmacology, Volume 40, Academic Press, San Diego, CA, p. 399-435. For example, a functional group on an antibody molecule may react with an active group on a liposome associated hydrophobic anchor, e.g., an amino group of a lysine side chain on an antibody may be coupled to liposome associated N-glutaryl-phosphatidylethanolamine activated with water-soluble carbodiimide; or a thiol group of a reduced antibody can be coupled to liposomes via thiol reactive anchors, such as pyridylthiopropionylphosphatidylethanolamine. See, e.g., Dietrich et al., 1996, Biochemistry, 35: 1100-1105; Loughrey et al., 1987, Biochim. Biophys. Acta, 901: 157- 160; Martin et al., 1982, J. Biol. Chem. 257: 286-288; Martin et al., 1981, Biochemistry, 20: 4429-38. Although not intending to be bound by a particular mechanism of action, immunoliposomal formulations including an antibody are particularly effective as therapeutic agents, since they deliver the antibody to the cytoplasm of the target cell, i.e., the cell comprising the receptor to which the antibody binds. The immunoliposomes preferably have an increased half-life in blood, specifically target cells, and can be internalized into the cytoplasm of the target cells thereby avoiding loss of the therapeutic agent or degradation by the endolysosomal pathway.

The immunoliposomal compositions include one or more vesicle forming lipids, an antibody or a fragment or derivative thereof, and, optionally, a hydrophilic polymer. A vesicle forming lipid is preferably a lipid with two hydrocarbon chains, such as acyl chains and a polar head group. Examples of vesicle forming lipids include phospholipids, e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, sphingomyelin, and glycolipids, e.g., cerebrosides, gangliosides. Additional lipids useful in the formulations are known to one skilled in the art and are specifically contemplated. In some embodiments, the immunoliposomal compositions further comprise a hydrophilic polymer, e.g., polyethylene glycol, and ganglioside GM1, which increases the serum half-life of the liposome. Methods of conjugating hydrophilic polymers to liposomes are well known in the art and are specifically contemplated. For a review of immunoliposomes and methods of preparing them, see, e.g., U.S. Patent Application Publication No. 2003/0044407; PCT International Publication No. WO 97/38731, Vingerhoeads et al., 1994, Immunomethods, 4: 259-72; Maruyama, 2000, Biol. Pharm. Bull. 23 (7) : 791-799; Abra et al., 2002, Journal of Liposome Research, 12 (1&2) : 1-3; Park, 2002, Bioscience Reports, 22 (2) : 267-281; Bendas et al., 2001 BioDrugs, 14 (4) : 215-224, J. Thomas August, ed., 1997, Gene Therapy: Advances in Pharmacology, Volume 40, Academic Press, San Diego, CA, p. 399-435.

The antibodies can be packaged in a hermetically sealed container, such as an ampoule or sachette, indicating the quantity of antibody. In some forms, the antibodies are supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. Preferably, the antibodies are supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized antibodies should be stored at between 2 and 8℃ in their original container and the antibodies should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In some forms, antibodies are supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the antibody. Preferably, the liquid form of the antibodies are supplied in a hermetically sealed container at least 1 mg/ml, more preferably at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml of the antibodies.

The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each patient’s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For antibodies, the dosage administered to a patient is typically 0.0001 mg/kg to 100 mg/kg of the patient’s body weight. Preferably, the dosage administered to a patient is between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient’s body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies or fragments thereof may be reduced by enhancing uptake and tissue penetration of the antibodies by modifications such as, for example, lipidation.

In some forms, the compositions can be delivered in a controlled release or sustained release system. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies. See, e.g., U.S. Patent No. 4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698; Ning et al., 1996, “Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel, ” Radiotherapy &Oncology 39: 179-189, Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions, ” PDA Journal of Pharmaceutical Science &Technology 50: 372-397; Cleek et al., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application, ” Pro. Int’l. Symp. Control. Rel. Bioact. Mater. 24: 853-854; and Lam et al., 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery, ” Proc. Int’l. Symp. Control Rel. Bioact. Mater. 24: 759-760. In some forms, a pump may be used in a controlled release system (See Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 20; Buchwald et al., 1980, Surgery 88: 507; and Saudek et al., 1989, N. Engl. J. Med. 321: 574) . In some forms, polymeric materials can be used to achieve controlled release of antibodies (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds. ) , CRC Pres., Boca Raton, Florida (1974) ; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds. ) , Wiley, New York (1984) ; Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23: 61; See also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 7 1: 105) ; U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253) . Examples of polymers used in sustained release formulations include, but are not limited to, poly (2-hydroxy ethyl methacrylate) , poly (methyl methacrylate) , poly (acrylic acid) , poly (ethylene-co-vinyl acetate) , poly (methacrylic acid) , polyglycolides (PLG) , polyanhydrides, poly (N-vinyl pyrrolidone) , poly (vinyl alcohol) , polyacrylamide, poly (ethylene glycol) , polylactides (PLA) , poly (lactide-co-glycolides) (PLGA) , and polyorthoesters. In some forms, a controlled release system can be placed in proximity of the therapeutic target (e.g., the lungs) , thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984) ) . In some forms, polymeric compositions useful as controlled release implants are used according to Dunn et al. (See U.S. 5,945,155) . This particular method is based upon the therapeutic effect of the in situ controlled release of the bioactive material from the polymer system. The implantation can generally occur anywhere within the body of the patient in need of therapeutic treatment. In some forms, a non-polymeric sustained delivery system is used, whereby a non-polymeric implant in the body of the subject is used as a drug delivery system. Upon implantation in the body, the organic solvent of the implant will dissipate, disperse, or leach from the composition into surrounding tissue fluid, and the non-polymeric material will gradually coagulate or precipitate to form a solid, microporous matrix (See U.S. 5,888,533) . Controlled release systems are discussed in the review by Langer (1990, Science 249: 1527-1533) . Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more therapeutic agents, i.e., SARS-CoV-2 RBD antibodies. See, e.g., U.S. Patent No. 4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698; Ning et al., 1996, Radiotherapy &Oncology 39: 179-189; Song et al., 1995, PDA Journal of Pharmaceutical Science &Technology 50: 372-397; Cleek et al., 1997, Pro. Int’l. Symp. Control. Rel. Bioact. Mater. 24: 853-854; and Lam et al., 1997, Proc. Int’l. Symp. Control Rel. Bioact. Mater. 24: 759-760.

In some forms, such as where the therapeutic or prophylactic composition is a nucleic acid encoding a SARS-CoV-2 RBD antibody or an antigen-binding fragment thereof, the nucleic acid can be administered in vivo to promote expression of its encoded antibody, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (See U.S. Patent No. 4,980,286) , or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont) , or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (See e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868) , etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.

Treatment of a subject with a therapeutically or prophylactically effective amount of antibody can include a single treatment or, preferably, can include a series of treatments.

The compositions include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically effective amount of a prophylactic and/or therapeutic agent disclosed herein or a combination of those agents and a pharmaceutically acceptable carrier. Preferably, the disclosed compositions include a prophylactically or therapeutically effective amount of antibody and a pharmaceutically acceptable carrier.

In some forms, 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, adjuvant (e.g., Freund’s adjuvant (complete and incomplete) , 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 preferred 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. 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.

Generally, the ingredients of compositions 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 can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, 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.

A. Inhalation Means

The dosage formulations are typically loaded in capsules or reservoirs, which are loaded into inhalers. The dosage formulations may be used with various inhaler types, such as dry powder inhalers, pressurized metered-dose inhalers, soft-mist inhalers, and medical nebulizers (Rubokas et al., Med Princ Pract, 25 (suppl 2) : 60–72 (2016) ) . Preferably, the dosage formulations are used with the dry powder inhalers.

1. Dry Powder Inhalers

DPIs are breath actuated, thus the problem of coordinated inspiration with actuation, as in the case of pMDIs, is avoided. The delivery of antibodies using DPIs can occur with a range of drying technologies such as spray drying, freeze drying, spray freeze drying or air jet micronization. For example, the spray drying of drugs in antibody formulations has been shown to be appropriate for manufacturing particles with a small aerodynamic size.

The dry powder inhaler types may carry one or more units, each unit containing capsules with one or more doses. The dry powder inhalers may contain a reservoir with multiple doses dose metering means. Exemplary dry powder inhaler types include single unit capsule dose in an inhaler, single unit disposable dose in the inhaler, multiple unit dose with pre-metered units in a replaceable set in an inhaler, and multiple dose in a reservoir in an inhaler. Exemplary commercially available dry powder inhalers include

(Novartis Ag Corporation Switzerland, Basel, Switzerland) ,

(Boehringer Ingelheim Pharma KG, Ingelheim am Rhein, Fed Rep Germany)

(Novartis Ag Corporation Switzerland, Basel, Switzerland) , DIRECT

(Direct-Haler A/S Corp Denmark, Odense Sv Denmark) ,

(Glaxo Group Limited Corp, Brentford, Middlesex United Kingdom) ,

(Astra Aktiebolag Corp., Sodertalie Sweden) ,

(Orion Corporation, Espoo Finland) , and Nexthaler (Lavorini et al. Multidisciplinary Respiratory Medicine, 12: 11 (2017) ) .

2. Pressurized Metered-Dose Inhalers

pMDIs are robust canisters enclosing a drug dissolved or dispersed in liquefied propellants. Actuation of the device with coordinated inspiration results in the release of a precise dose. The propellant rapidly evaporates owing to its high vapor pressure, leaving an accurate dose of the aerosolized drug particles to be inhaled by the patient. pMDI devices have traditionally been used in the treatment of asthma since the 1950s.

3. Soft Mist Inhalers

SMIs are hand-held propellant-free metered dose inhalation devices that generate slow-moving aqueous aerosols for deep-lung deposition. An example is the

(Aradigm Corp., Novo Nordisk, Hayward, Calif., USA) , an SMI that is able to deliver liposome-DNA complexes in respirable aerosols.

4. Medical Nebulizers

Compared to other inhalation devices, nebulizers can generate large volumes of “respirable” aerosol, with no need to perform drying procedures, as in the case of DPIs, or involve propellants, as in case of pMDIs. There are three types of nebulizer: air jet, ultrasonic and vibrating mesh. The air jet nebuliser employs compressed gas passing through a narrow “venturi” nozzle at the bottom of the device to convert the liquid medication into “respirable” aerosol droplets. By contrast, the ultrasonic nebuliser utilizes ultrasound waves generated via a piezoelectric crystal vibrating at a high frequency to convert the liquid into aerosols. However, the vibrating mesh nebulizer operates using a different principle, by utilizing a vibrational element that transmits the vibrations to a perforated plate with multiple micro-sized apertures to push the medication fluid through and generate slow-moving aerosol droplets with a narrow size distribution.

The disclosed compositions and methods can be further understood through the following numbered paragraphs.

1. An antibody or antigen binding fragment thereof comprising six complementarity determining regions (CDRs) ,

wherein the CDRs comprise:

2. The antibody or antigen binding fragment thereof of paragraph 1, wherein the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-32 of SEQ ID NO: 5, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 5, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 5.

3. The antibody or antigen binding fragment thereof of one of

paragraphs

1 or 2, wherein the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-33 of SEQ ID NO: 1, a second heavy chain CDR comprising amino acids 51-58 of SEQ ID NO: 1, and a third heavy chain CDR comprising amino acids 97-110 of SEQ ID NO: 1.

4. The antibody or antigen binding fragment thereof of one of

paragraphs

1, 2, or 3 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5.

5. The antibody or antigen binding fragment thereof of one of paragraphs 1 or 2-4 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1.

6. The antibody or antigen binding fragment thereof of paragraph 1, wherein the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-33 of SEQ ID NO: 6, a second light chain CDR comprising amino acids 51-53 of SEQ ID NO: 6, and a third light chain CDR comprising amino acids 90-98 of SEQ ID NO: 6.

7. The antibody or antigen binding fragment thereof of one of

paragraphs

1 or 6, wherein the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-33 of SEQ ID NO: 2, a second heavy chain CDR comprising amino acids 51-58 of SEQ ID NO: 2, and a third heavy chain CDR comprising amino acids 97-112 of SEQ ID NO: 2.

8. The antibody or antigen binding fragment thereof of one of

paragraphs

1, 6, or 7 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6.

9. The antibody or antigen binding fragment thereof of one of paragraphs 1 or 6-8 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

10. The antibody or antigen binding fragment thereof of paragraph 1, wherein the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-32 of SEQ ID NO: 7, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 7, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 7.

11. The antibody or antigen binding fragment thereof of one of

paragraphs

1 or 10, wherein the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-33 of SEQ ID NO: 3, a second heavy chain CDR comprising amino acids 51-58 of SEQ ID NO: 3, and a third heavy chain CDR comprising amino acids 97-113 of SEQ ID NO: 3.

12. The antibody or antigen binding fragment thereof of one of

paragraphs

1, 10, or 11 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7.

13. The antibody or antigen binding fragment thereof of one of paragraphs 1 or 10-12 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3.

14. The antibody or antigen binding fragment thereof of paragraph 1, wherein the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-32 of SEQ ID NO: 8, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 8, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 8.

15. The antibody or antigen binding fragment thereof of one of

paragraphs

1 or 14, wherein the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-32 of SEQ ID NO: 4, a second heavy chain CDR comprising amino acids 50-56 of SEQ ID NO: 4, and a third heavy chain CDR comprising amino acids 95-106 of SEQ ID NO: 4.

16. The antibody or antigen binding fragment thereof of one of

paragraphs

1, 14, or 15 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

17. The antibody or antigen binding fragment thereof of one of paragraphs 1 or 14-16 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

18. The antibody or antigen binding fragment thereof of paragraph 1 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1.

19. The antibody or antigen binding fragment thereof of paragraph 1 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

20. The antibody or antigen binding fragment thereof of paragraph 1 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3.

21. The antibody or antigen binding fragment thereof of paragraph 1 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

22. The antibody or antigen binding fragment thereof of any one of paragraphs 1-21, wherein the antibody or antigen binding fragment thereof attenuates the ability of a ligand of SARS-CoV-2 RBD to bind to ACE2.

23. The antibody or antigen binding fragment thereof of any one of paragraphs 1-22 comprising one or more constant domains from an immunoglobulin constant region (Fc) .

24. The antibody or antigen binding fragment thereof of paragraph 23, wherein the constant domains are human constant domains.

25. The antibody or antigen binding fragment thereof of paragraph 24, wherein the human constant domains are IgA, IgD, IgE, IgG or IgM domains.

26. The antibody or antigen binding fragment thereof of paragraph 25, wherein human IgG constant domains are IgG1, IgG2, IgG3, or IgG4 domains.

27. The antibody or antigen binding fragment thereof of any one of paragraphs 1-26, wherein the antibody or antigen binding fragment thereof is detectably labeled or comprises a conjugated toxin, drug, receptor, enzyme, receptor ligand.

28. The antibody or antigen binding fragment thereof of any one of paragraph 1-27, wherein the antibody is a monoclonal antibody, a human antibody, a chimeric antibody or a humanized antibody.

29. The antibody or antigen binding fragment thereof of any one of paragraphs 1-28, wherein the antibody is a bispecific, trispecific or multispecific antibody.

30. A humanized antibody or antigen binding fragment thereof comprising one or more human IgG4 constant domains and

31. A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of paragraphs 1-30 and a physiologically acceptable carrier or excipient.

32. The pharmaceutical composition of paragraph 31 for use in a method of preventing or treating COVID-19 in a subject.

33. The pharmaceutical composition for use of paragraph 32 wherein the subject has COVID-19.

34. The pharmaceutical composition for use of paragraph 32 wherein the subject is at risk of developing COVID-19.

35. The pharmaceutical composition of paragraph 31 for use in a method of treating COVID-19.

36. The pharmaceutical composition of paragraph 31 for use in a method of preventing COVID-19.

37. Use of the antibody or antigen binding fragment thereof of any of paragraphs 1-30 in manufacture of a medicament for preventing or treating COVID-19 in a subject.

38. Use of the antibody or antigen binding fragment thereof of any of paragraphs 1-30 in manufacture of a medicament for treating COVID-19 in a subject.

39. Use of the antibody or antigen binding fragment thereof of any of paragraphs 1-30 in manufacture of a medicament for preventing COVID-19 in a subject.

40. A method of detection or diagnosis of SARS-CoV-2 infection, comprising: (a) assaying the presence of SARS-CoV-2 RBD in a sample from a subject using the antibody or antigen binding fragment thereof of any one of paragraphs 1-30 and (b) comparing the level of the SARS-CoV-2 RBD with a control level, wherein an increase in the assayed level of SARS-CoV-2 RBD compared to the control level is indicative of SARS-CoV-2 infection.

41. The use of paragraph 38, wherein the presence of SARS-CoV-2 RBD is assayed by enzyme linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , or fluorescence-activated cell sorting (FACS) .

42. A pharmaceutical composition for use in a method of treating a subject infected by or at risk for infection by SARS-CoV-2, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of paragraph 31 if the subject has a disease characterized by increased expression of SARS-CoV-2 RBD.

43. The method of paragraph 42 wherein the antibody or antigen binding fragment thereof is the antibody or antigen binding fragment thereof of any one of paragraphs 1-30.

Examples

Example 1: SARS-CoV-2 hijacks neutralizing dimeric IgA for enhanced nasal infection and injury.

Robust severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in nasal turbinate (NT) accounts for high viral transmissibility, yet whether neutralizing IgA antibodies can control it remains unknown. In this non-limiting example, the efficacy of the receptor binding domain (RBD) -specific monomeric B8-mIgA1 and B8-mIgA2, and dimeric B8-dIgA1 and B8-dIgA2 against intranasal SARS-CoV-2 challenge in Syrian hamsters was evaluated. To execute these experiments, the inventors employed technology of single B cell antibody gene cloning to generate a panel of SARS-CoV-2 RBD-specific monoclonal HuNAbs from the peripheral blood mononuclear cells (PBMCs) of one acute and three convalescent COVID-19 patients. Since intramuscular or intranasal inoculation of several potent IgG HuNAbs cannot completely prevent SARS-CoV-2 infection in the nasal turbinate (NT) of Syrian hamsters as described in previous studies (Zhou et al. (2021) , Cell Host Microbe, 29 (4) : 551-563; Chan et al. (2020) , Clinical Infectious Diseases 71 (9) : 2428-2446) , the inventors sought to improve the efficacy of HuNAb by converting IgG to IgA. To achieve this goal, the inventors engineered the potent B8-IgG1 into monomeric IgA1 (B8-mIgA1) , monomeric IgA2 (B8-mIgA2) , dimeric (B8-dIgA1) and dimeric IgA2 (B8-dIgA2) and determined their efficacies in the Syrian hamster model against the live intranasal SARS-CoV-2 challenge.

Methods

Human subjects

A total of 4 patients with COVID-19 including 3 convalescent cases and one acute case were recruited between February and May 2020. All patients were confirmed by reverse-transcription polymerase chain reaction (RT-PCR) as described previously (Chan et al., (2020) J Clin Microbiol, 58 (5) : e00310-20) . Clinical and laboratory findings were entered into a predesigned database. Written informed consent was obtained from all patients. This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster, the Hong Kong East Cluster Research Ethics Committee, and the Kowloon West Cluster Research Ethics Committee (UW 13-265, HKECREC-2018-068, KW/EX-20-038 [144-26] ) .

Syrian hamsters

The animal experimental plan was approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR 5359-20) of the University of Hong Kong (HKU) . Male and female golden Syrian hamsters (Mesocricetus auratus) (aged 6–10 weeks) were purchased from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Laboratory Animal Unit (LAU) . The animals were kept in Biosafety Level-2 housing and given access to standard pellet feed and water ad libitum following LAU’s standard operational procedures (SOPs) . The viral challenge experiments were then conducted in the Biosafety Level-3 animal facility following SOPs strictly, with strict adherence to SOPs.

Cell lines

HEK293T cells, HEK293T-hACE2 cells Vero-E6 cells, HK2 cells and Vero-E6-TMPRSS2 cells were maintained in DMEM containing 10%FBS, 2 mM L-glutamine, 100 U/mL/mL penicillin and incubated at 37 ℃ in a 5%CO2 setting (Liu et al., (2019) , JCI Insight 4 (4) : e123158) . Expi293F ^TM cells were cultured in Expi293 ^TM Expression Medium (Thermo Fisher Scientific) at 37 ℃ in an incubator with 80%relative humidity and a 5%CO2 setting on an orbital shaker platform at 125 ±5 rpm/min (New Brunswick innova ^TM 2100) according to the manufacturer’s instructions.

ELISA Analysis of plasma and antibody binding to RBD and trimeric spike

The recombinant RBD and trimeric spike proteins derived from SARS-CoV-2 (Sino Biological) were diluted to final concentrations of 1 μg/mL/mL, then coated onto 96-well plates (Corning 3690) and incubated at 4 ℃ overnight. Plates were washed with PBS-T (PBS containing 0.05%Tween-20) and blocked with blocking buffer (PBS containing 5%skim milk or 1%BSA) at 37 ℃ for 1 hour. Serially diluted plasma samples or isolated monoclonal antibodies were added to the plates and incubated at 37 ℃ for 1 hour. Wells were then incubated with a secondary goat anti-human IgG labelled with horseradish peroxidase (HRP) (Invitrogen) or with a rabbit polyclonal anti-human IgA alpha-chain labelled with HRP (Abcam) and TMB substrate (SIGMA) . Optical density (OD) at 450 nm was measured by a spectrophotometer. Serially diluted plasma from healthy individuals or previously published monoclonal antibodies against HIV-1 (VRC01) were used as negative controls.

Isolation of RBD-specific IgG+ single memory B cells by FACS

RBD-specific single B cells were sorted as described in previous studies (Kong et al., (2016) Immunity 44 (4) : 939-950) . In brief, PBMCs from infected individuals were collected and incubated with an antibody cocktail and a His-tagged RBD protein for identification of RBD-specific B cells. The cocktail consisted of the Zombie viability dye (Biolegend) , CD19-Percp-Cy5.5, CD3-Pacific Blue, CD14-Pacific Blue, CD56-Pacific Blue, IgM-Pacific Blue, IgD-Pacific Blue, IgG-PE, CD27-PE-Cy7 (BD Biosciences) and the recombinant RBD-His described above. Two consecutive staining steps were conducted: the first one used an antibody and RBD cocktail incubation of 30 min at 4 ℃; the second staining involved staining with anti-His-APC and anti-His-FITC antibodies (Abcam) at 4 ℃ for 30 min to detect the His tag of the RBD. The stained cells were washed and resuspended in PBS containing 2%FBS before being strained through a 70-μm cell mesh filter (BD Biosciences) . RBD-specific single B cells were gated as CD19+CD27+CD3-CD14-CD56-IgM-IgD-IgG+RBD+ and sorted into 96-well PCR plates containing 10 μL of RNAase-inhibiting RT-PCR catch buffer (1M Tris-HCl pH 8.0, RNase inhibitor, DEPC-treated water) . Plates were then snap-frozen on dry ice and stored at -80 ℃ until the reverse transcription reaction.

Single B cell RT-PCR and antibody cloning

Single memory B cells were isolated from PBMCs of infected patients were cloned as previously described (Smith et al., (2009) , Nat Protocols, 4: 372-384) . Briefly, one-step RT-PCR was performed on sorted single memory B cell with a gene specific primer mix, followed by nested PCR amplifications and sequencing using the heavy chain and light chain specific primers. Cloning PCR was then performed using heavy chain and light chain specific primers containing specific restriction enzyme cutting sites (heavy chain, 5′-AgeI/3′-SalI; kappa chain, 5′-AgeI/3′-BsiWI) . The PCR products were purified and cloned into the backbone of antibody expression vectors containing the constant regions of human Igγ1 or Igα1 and Igα2. The Igα1 and Igα2 vectors were purchased from InvivoGen (pfusess-hcha1 for IgA1 and pfusess-hcha2m1 for IgA2) . The constructed plasmids containing paired heavy and light chain expression cassettes were co-transfected into 293T cells (ATCC) grown in 6-well plates. Antigen-specific ELISA and pseudovirus-based neutralization assays were used to analyze the binding capacity to SARS-CoV-2 RBD and the neutralization capacity of transfected culture supernatants, respectively.

Genetic analysis of the BCR repertoire

Heavy chain and light chain germline assignment, framework region annotation, determination of somatic hypermutation (SHM) levels (in nucleotides) and CDR loop lengths (in amino acids) were performed with the aid of the IMGT/HighV-QUEST software tool suite (HighV-QUEST) . Sequences were aligned using Clustal W in the BioEdit sequence analysis package (Version 7.2) . Antibody clonotypes were defined as a set of sequences that share genetic V and J regions as well as an identical CDR3.

Antibody production and purification

The paired antibody VH/VL chains were cloned into Igγ, Igα1 or Igα2 and Igk expression vectors using T4 ligase (NEB) . For production of IgG and monomeric IgA, the plasmids with paired heavy chain (IgG, IgA1, IgA2) and light chain genes were co-transfected into Expi293 ^TM expression system (Thermo Fisher Scientific) following the manufacturer’s protocol to produce recombinant monoclonal antibodies. For dIgA antibody production, plasmids of paired heavy chain (IgA1, IgA2) and kappa light chain together with a J chain were co-transfected into Expi293 ^TM expression system (Thermo Fisher Scientific) at the ratio of 1: 1: 1 following the manufacturer’s instructions. Antibodies produced from cell culture supernatants were purified immediately by affinity chromatography using recombinant Protein G-Agarose (Thermo Fisher Scientific) or CaptureSelect ^TM IgA Affinity Matrix (Thermo Fisher Scientific) according to the manufacturer’s instructions, to purify IgG and IgA, respectively. The purified antibodies were concentrated by an Amicon ultracentrifuge filter device (molecular weight cut-off 10 kDa; Millipore) to a volume of 0.2 mL in PBS (Life Technologies) , and then stored at 4 ℃ or -80 ℃ for further characterization.

Size exclusion chromatography

The prepacked HiLoad 26/60 Superdex ^TM 200pg (code No. 17-1071-01, Cytiva) column was installed onto the Amersham Biosciences AKTA FPLC system. After column equilibration with 2 column volumes (CV) of PBS, the concentrated IgA antibodies were applied onto the column using a 500-μl loop at a flow rate of 2 mL/min. Dimers of IgA1 or IgA2 were separated from monomers upon washing with 2 CV of PBS. The milli-absorbance unit at OD 280nm was recorded during the washing process. 2 mL-fractions were collected, pooled, concentrated and evaluated by western blot using mouse anti-IGJ monoclonal antibody [KT109] (Abcam) and rabbit anti-human IgA alpha chain antibody (Abcam) .

Pseudovirus-based neutralization assay

The neutralizing activity of NAbs was determined using a pseudotype-based neutralization assay as described in inventors’ previous studies (Poeran et al. (2020) , Anesth Analg, 131 (5) , 1337-134177) . Briefly, the pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively (Poeran et al. (2020) , Anesth Analg, 131 (5) , 1337-134177) . Viral supernatant was collected at 48 hour post-transfection and frozen at -80 ℃ until use. The serially diluted monoclonal antibodies or sera were incubated with 200 TCID ₅₀ of pseudovirus at 37 ℃ for 1 hour. The antibody-virus mixtures were subsequently added to pre-seeded HEK 293T-ACE2 cells. 48 hours later, infected cells were lysed to measure luciferase activity using a commercial kit (Promega, Madison, WI) . Half-maximal (IC ₅₀) or 90% (IC ₉₀) inhibitory concentrations of the evaluated antibody were determined by inhibitor vs. normalized response --4 Variable slope using GraphPad Prism 6 or later (GraphPad Software Inc. ) .

Neutralization activity of monoclonal antibodies against authentic SARS-CoV-2

The SARS-CoV-2 focus reduction neutralization test (FRNT) was performed in a certified Biosafety level 3 laboratory. Neutralization assays against live SARS-CoV-2 were conducted using a clinical isolate (HKU-001a strain, GenBank accession no: MT230904.1; SEQ ID NO: 9) previously obtained from a nasopharyngeal swab from an infected patient (Chu et al. (2020) , The Lancet Microbe 1 (1) , e14-e23) . The tested antibodies were serially diluted, mixed with 50 μL of SARS-CoV-2 (1×103 focus forming unit/mL, FFU/mL) in 96-well plates, and incubated for 1 hour at 37 ℃. Mixtures were then transferred to 96-well plates pre-seeded with 1×104/well Vero E6 cells and incubated at 37℃ for 24 hours. The culture media was then removed, and the plates were air-dried in a biosafety cabinet (BSC) for 20 minutes. Cells were then fixed with a 4%paraformaldehyde solution for 30 minutes and air-dried in the BSC again. Cells were further permeabilized with 0.2%Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N for 1 hour at RT before adding an Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies) . The fluorescence density of SARS-CoV-2 infected cells were scanned using a Sapphire Biomolecular Imager (Azure Biosystems) and the neutralization effects were then quantified using Fiji software (NIH) .

Antibody binding kinetics and competition with the ACEW receptor measured by Surface Plasmon Resonance (SPR)

The binding kinetics and affinity of recombinant monoclonal antibodies for the SARS-CoV-2 spike protein (ACROBiosystems) were analyzed by SPR (Biacore 8K, GE Healthcare) . Specifically, the spike protein was covalently immobilized to a CM5 sensor chip via amine groups in 10mM sodium acetate buffer (pH 5.0) for a final RU around 500. SPR assays were run at a flow rate of 30 mL/min in HEPES buffer. For conventional kinetic/dose-response, serial dilutions of monoclonal antibodies were injected across the spike protein surface for 180s, followed by a 600s dissociation phase using a multi-cycle method. Remaining analytes were removed in the surface regeneration step with the injection of 10 mM glycine-HCl (pH 2.0) for 2×30s at a flow rate of 30 μl/min. Kinetic analysis of each reference subtracted injection series was performed using the Biacore Insight Evaluation Software (GE Healthcare) . All sensor gram series were fit to a 1: 1 (Langmuir) binding model of interaction. Before evaluating the competition between antibodies and the human ACE2 peptidase domain, both the saturating binding concentrations of antibodies and of the ACE2 protein (ACROBiosystems) for the immobilized SARS-CoV-2 spike protein were determined separately. In the competitive assay, antibodies at the saturating concentration were injected onto the chip with immobilized spike protein for 120s until binding steady state was reached. ACE2 protein also used at the saturating concentration was then injected for 120 seconds (s) , followed by another 120 seconds of injection of antibody to ensure a saturation of the binding reaction against the immobilized spike protein. The differences in response units between ACE2 injection alone and prior antibody incubation reflect the antibodies’ competitive ability against ACE2 binding to the spike protein.

Hamster experiments

In vivo evaluation of monoclonal antibody B8-IgG1, B8-mIgA1, B8-mIgA2, B8-dIgA1, B8-dIgA2 in the established golden Syrian hamster model of SARS-CoV-2 infection was performed with modifications as described in previous studies (Chan et al. (2020) , Clinical Infectious Diseases 71 (9) : 2428-2446) . Approval was obtained from the University of Hong Kong (HKU) Committee on the Use of Live Animals in Teaching and Research. Briefly, 6-8-week-old male and female hamsters were housed with access to standard pellet feed and water ad libitum until live virus challenge in the BSL-3 animal facility. The hamsters were randomized from different litters into experimental groups. Experiments were performed in compliance with the relevant ethical regulations (Chan et al. (2020) , Clinical Infectious Diseases 71 (9) : 2428-2446) . For prophylaxis studies, 24 hours before live virus challenge, three groups of hamsters were intraperitoneally or intranasally administered with one dose of test antibody in phosphate-buffered saline (PBS) at the indicated dose. At day 0, each hamster was intranasally inoculated with a challenge dose of 100 μL of Dulbecco’s Modified Eagle Medium containing 105 PFU of SARS-CoV-2 (HKU-001a strain, GenBank accession no: MT230904.1; SEQ ID NO: 9) under anesthesia with intraperitoneal ketamine (200 mg/kg) and xylazine (10 mg/kg) . For pre-treatment study, each hamster received one 1.5 mg/kg dose of intraperitoneal B8-IgG1 at 24, 48, and 72 hours (n=4 per group) after virus challenge. The hamsters were monitored twice daily for clinical signs of disease. Syrian hamsters typically clear virus within one week after SARS-CoV-2 infection. Accordingly, animals were sacrificed for analysis at day 4 after virus challenge with high viral loads (Chan et al. (2020) , Clinical Infectious Diseases 71 (9) : 2428-2446) . Half the nasal turbinate, trachea, and lung tissues were used for viral load determination by quantitative SARS-CoV-2-specific RdRp/Hel RT-qPCR assay (Chan et al., (2020) J Clin Microbiol, 58 (5) : e00310-20) and infectious virus titration by plaque assay (Chan et al. (2020) , Clinical Infectious Diseases 71 (9) : 2428-2446) .

Cryo-EM sample preparation and data acquisition

The purified SARS-CoV-2 S-B8 protein complexes were concentrated before being applied to the grids. Aliquots (4 μL) of the protein complex were placed on glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3, 300 mesh) . The grids were blotted and flash-frozen in liquid ethane cooled by liquid nitrogen with a Vitrobot apparatus (Mark IV, ThermoFisher Scientific) . The grids sample quality was verified with an FEI Talos Arctica 200-kV electron microscope (Thermo Fisher Scientific) . The verified grids with optimal ice thickness and particle density were transferred to a Titan Krios operating at 300 kV and equipped with a Cs corrector, a Gatan K3 Summit detector (Gatan Inc. ) and a GIF Quantum energy filter (slit width 20 eV) . Micrographs were recorded in the super-resolution mode with a calibrated pixel size of

Each movie has a total accumulated exposure of 50

fractionated in 32 frames. The final image was binned 2-fold to a pixel size of

AutoEMation was used for the fully automated data collection. The defocus value of each image, which was set from -1.0 to -2.0 μm during data collection, was determined by Gctf. Data collection statistics are summarized in Table 2 below.

Table 2: Statistics of cryo-EM data collection, processing and model refinement

Cryo-EM data processing

The procedure for image processing of SARS-CoV-2 S-B8 complex is presented in FIG. 14. In brief, Motion Correction (MotionCo2) , CTF-estimation (GCTF) and non-templated particle picking (Gautomatch, mrc-lmb. cam. ac. uk/kzhang/) were automatically executed by the TsinghuaTitan. py program (developed by Dr. fang Yang) . Sequential data processing was carried out on RELION 3.0 and RELION 3.1. Initially, 2,436,776 particles were auto picked by Gautomatch or RELION 3.0 from 4213 micrographs. After several 2D classifications, 1,451,176 particles were selected and applied for 3D classification with one class. Two different states were obtained after further 3D classification: 3 RBD in up conformation bound with B8 Fab (3u) , and 2 up RBDs and 1 down RBD with each bound to a B8 Fab (2u1d) . 616, 799 particles for the 2u1d state and 351, 095 particles for the 3u state were subjected to 3D auto-refinement, yielding final resolutions at

and

respectively. Further CTF refinement and Bayesian polishing improved the resolution to

(2u1d, C1 symmetry) and

(3u, C3 symmetry) with better map quality. To improve the RBD-B8 portion map density, focused local search classification was applied for each RBD-B8 portion with an adapted soft mask. The best classes for each RBD-Fab portion were selected and yielded a final resolution at

(RBD-Fab1, up) ,

(RBD-Fab2, up) ,

(RBD-Fab3, down) ,

(RBD-Fab3, up) from 479, 305, 508, 653, 656, 429, and 136, 482 particles, respectively. Further CTF refinement and Bayesian polishing improved the resolution of RBD-Fab2 to

with better map quality. RBD-Fab maps were fitted onto the whole structure map using Chimera, then combined using PHENIX combine_focused_maps. The reported resolutions were estimated with the gold-standard Fourier shell correlation (FSC) cutoff of 0.143 criterion. Data processing statistics are summarized in Table 2.

Model building and structure refinement

The spike model (PDB code: 6VSB) and the initial model of the B8 Fab generated by SWISS-Model were fitted into the EM density map, and further manually adjusted with Coot. Glocusides were built manually with carbohydrate tool in Coot. The atomic models were refinement using Phenix in real space with secondary structure and geometry restraints. The final structures were validated using Phenix. molprobity. UCSF Chimera, ChimeraX and PyMol were used for map segmentation and figure generation. Model refinement statistics are summarized in Table 2.

SARS-CoV-2 infection of reconstructed human nasal epithelia

MucilAir ^TM, corresponding to reconstructed human nasal epithelium cultures differentiated in vitro for at least 4 weeks, were purchased from Epithelix (Saint-Julien-en-Genevois, France) . The cultures were generated from pooled nasal tissues obtained from 14 human adult donors. Cultures were maintained in air/liquid interface (ALI) conditions in transwells with 700 μL of MucilAir ^TM medium (Epithelix) in the basal compartment, and then kept at 37 ℃ under a 5%CO2 atmosphere. SARS-CoV-2 infection was performed as previously described 50. Briefly, the apical side of ALI cultures was washed 20 minutes (min) at 37 ℃ in Mucilair ^TM medium to remove mucus. Cells were then incubated with 104 plaque-forming units (PFU) of the isolate BetaCoV/France/IDF00372/2020 (EVAg collection, Ref-SKU: 014V-03890) . The viral input was diluted in DMEM medium to a final volume 100 μL, and then left on the apical side for 4 hours at 37 ℃. Control wells were mock treated with DMEM medium (Gibco) for the same duration. Viral inputs were removed by washing twice with 200 μL of PBS (5 min at 37 ℃) and once with 200 μL Mucilair ^TM medium (20 min at 37 ℃) . The basal medium was replaced every 2-3 days. Apical supernatants were harvested every 2-3 days by adding 200 μL of Mucilair ^TM medium on the apical side, with an incubation of 20 min at 37 ℃ prior to collection. For IgA treatment, cultures were washed once and then pretreated with antibodies added to the apical compartment for 1 hour in 50μL. Viral input was then directly added to reach a final volume of 100 μL. The antibodies were added again at day 2 d. p. i. in the apical compartment during an apical wash (20 min at 37 ℃) . To test the effect of dIgA treatment in the presence of mucus, dIgA were added directly to the apical compartment of MucilAir ^TM cultures without an initial wash. After IgA treatment for 1 hour, the virus was added directly to the IgA/mucus mixture and left on the apical side for 4 hours at 37℃. After viral inoculation, a single brief wash was made to remove the viral input while limiting mucus loss. The cultures were then maintained as in the no-mucus condition.

Viral RNA quantification in reconstructed human nasal epithelia

Apical supernatants were collected, stored at -80 ℃ until thawing and were then diluted 4-fold in PBS in a 96-well plate. Diluted supernatants were inactivated for 20 min at 80 ℃. For SARS-CoV-2 RNA quantification, 1 μL of diluted supernatant was added to 4 μL of PCR reaction mix. PCR was carried out in 384-well plates using the Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) with SARS-CoV-2 NP-specific primers (Forward 5’-TAA TCA GAC AAG GAA CTG ATT A-3’ (SEQ ID NO: 10) ; Reverse 5’-CGA AGG TGT GAC TTC CAT G-3’ (SEQ ID NO: 11) on a QuantStudio 6 Flex thermocycler (Applied Biosystems) . A standard curve was established in parallel using purified SARS-CoV-2 viral RNA.

Histopathology and immunofluorescence (IF) staining

The lung and nasal turbinate tissues collected at necropsy were fixed in zinc formalin and then processed into paraffin-embedded tissue blocks. The tissue sections (4 μm) were stained with hematoxylin and eosin (H&E) for light microscopy examination as described in previous studies with modifications (Zhou et al. (2021) , Cell Host Microbe, 29 (4) : 551-563) . For identification and localization of SARS-CoV-2 nucleocapsid protein (NP) in organ tissues, immunofluorescence staining was performed on deparaffinized and rehydrated tissue sections using a rabbit anti-SARS-CoV-2-NP protein antibody together with an AF488-conjugated anti-rabbit IgG (Jackson ImmunoResearch, PA, USA) . Briefly, the tissue sections were first treated with antigen unmasking solution (Vector Laboratories) in a pressure cooker. After blocking with 0.1%Sudan black B for 15 min and 1%bovine serum albumin (BSA) /PBS at RT for 30 min, the primary rabbit anti-SARS-CoV-2-NP antibody (1: 4000 dilution with 1%BSA/PBS) was incubated at 4℃ overnight. This step was followed by incubation with a FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) for 30 min and the sections were then mounted in medium with 4’, 6-diamidino-2-phenylindole (DAPI) . For identification of DC-SIGN expression, we stained the NT slices with rabbit anti-DC-SIGN primary antibody (Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies) according to the manufacturer’s instructions. For identification of ACE2 expression, the goat anti-ACE2 primary antibody (R&D) and Alexa Fluor 568 donkey anti-goat IgG (H+L) secondary antibodies (Invitrogen) according to the manufacturer’s instructions. All tissue sections were examined, and the fluorescence images and whole section scanning were captured using 5×, 10× and 20× objectives with Carl Zeiss LSM 980. NP+ cells per field were quantified based on the mean fluorescence intensity (MFI) using the ZEN BLACK 3.0 and ImageJ (NIH) .

Effects of B8-dIgA on SARS-CoV-2 infection in HK2 cells

HK2 cells were seeded into 24-well plates at the 40-50%confluency and cultured overnight. The B8-dIgA or control dIgA at the concentration of 1, 10, 100, 1000 ng/ml/mL and then mixed with SARS-CoV-2 (1: 10 TCID ₅₀) and incubated for 1 hour at room temperature. The antibody/virus mixture was then added to HK2 cells after the cell culture medium was removed and washed with PBS once and incubated for 1 hour at 37℃. The infectious medium was replaced with fresh medium containing respective concentration of antibody after washing 3 times with PBS. 24 hours later, the infected cells were imaged under fluorescence microscope after staining with AF488-conjugated anti-SARS-CoV-2 NP antibody. Alternatively, the infected cells were lysed and blotted for SARS-CoV-2 NP protein to determine the extent of infection. Tubulin was blotted as the internal control.

B8-dIgA mediated enhancement via CD209

HEK293T cells were seeded into 10-cm dish at 40%confluency and cultured overnight. The HEK293T cells were transfected with human CD209 (Sino Biological) at 70%-90%confluency. The expression of CD209 was measured by flow cytometry. The transfected HEK293T-CD209 cells were seeded into 96-well plates with 2.4×10 ⁴ cells per well and cultured overnight. The HEK293T-CD209 cells were pre-treated with 10 ng/ml/mL of B8-dIgA or control dIgA and incubated for 6 h prior SARS-CoV-2 infection (MOI=0.05) . 24 h later, cells were then fixed with 4%paraformaldehyde solution for 30 min and air-dried in the BSC. Cells were further permeabilized with 0.2%Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N for 1 hour at RT before adding Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies) . The fluorescence density of SARS-CoV-2 infected cells was acquired using a Sapphire Biomolecular Imager (Azure Biosystems) and then the MFI of four randomly selected areas of each sample was quantified using Fiji software (NIH) .

Effects of B8 antibodies on SARS-CoV-2 mediated cell-cell fusion

Vero-E6 TMPRSS2 cells were seeded into 48-well plates and cultured overnight. After treatment with B8 antibodies at the dose of 3000 ng/ml/mL for 1 hour, HEK293T cells transfected with SARS-CoV-2 spike-GFP were added into the treated Vero-E6 TMPRSS2 cells and co-cultured for 48 hours. The cell-cell fusion between Vero-E6 TMPRSS2 and HEK293T-Spike-GFP was then determined under a fluorescence microscope (Nikon ELIPSE) and the images of randomly selected region were captured using 4× and 10× objectives using the Nikon software.

Re-analysis of published nasal brushing single-cell sequencing data

The preprocessed scRNA-seq data from nasal brushing samples of 2 healthy controls and 4 COVID-19 patients were downloaded from Gene Expression Omnibus (GEO) database with accession numbers GSE171488 and GSE164547. Quality control metrics were consistent with the original article (Ahn et al. (2021) , J Clin Invest 131 (13) : e148517) and performed based on the R package Seurat (version 4.0.3) (Hao et al., (2021) , Cell, 184 (13) : 3573-3587) . Harmony (Korsunsky et al., (2019) , Nat Methods, 16 (12) : 1289-1296) was used to integrate the samples based on the top 4000 most variable genes obtained with the FindVariableFeatures () function in Seurat. CD14+ (monocyte) cells were extracted for further analysis. The annotation of the cell type was performed by manually checking the marker genes of each cluster identified from the FindAllMarkers () function in Seurat.

Quantification and statistical analysis

Statistical analysis was performed using PRISM 6.0 or later. Ordinary one-way ANOVA and multiple comparisons were used to compare group means and differences between multiple groups. Unpaired Student's t tests were used to compare group means between two groups only. A p-value <0.05 was considered significant. The number of independent experiments performed, the number of animals in each group, and the specific details of statistical tests are reported in the brief description of the figures and the Methods section.

B8VH:

C5VH:

B7VH:

B4VH:

B8VK:

C5VK:

B7VK:

B4VK:

Results

Characterization of human monoclonal antibodies from COVID-19 patients

To isolate human monoclonal antibodies (MAbs) , peripheral blood mononuclear cells (PBMCs) were obtained from one acute (P4) and three convalescent (P1-P3) COVID-19 patients in Hong Kong at a mean 73.5 (+25) days after symptoms onset (Table 3) . Enzyme-linked immunosorbent assay (ELISA) and pseudovirus neutralization assays revealed that all patient sera showed SARS-CoV-2 RBD-and spike-specific binding (FIG. 1A and 1B) and neutralizing antibody (NAb) activities (FIG. 1C) . The mean NAb IC ₅₀ titer was 1: 1753 with a range of 1: 638-1: 5701. Flow cytometry was then used to sort SARS-CoV-2-specific immunoglobulin positive (IgG+) memory B cells from individual PBMC samples using two fluorescent-conjugated RBD probes. The percentage of RBD-binding IgG+ memory B cells ranged from 0.19%to 0.52% (FIG. 2A and FIG 2B) . A total of 34 MAbs were successfully cloned from these patients, including 3 from P1, 8 from P2, 17 from P3 and 6 from P4. It was confirmed that 18 of these MAbs exhibited RBD-specific binding activities detected by ELISA (FIG. 2C) . No clear dominance of heavy (H) chain gene family was found among these 4 subjects by sequence analysis (FIG. 2D, top panels) . VLK1, however, was the most used variable gene family for the light (L) chain (FIG. 2D, bottom panels) . The average somatic hypermutation (SHM) rate ranged from 0%to 12.2%for the H chain and from 0.7%to 7.9%for the L chain (FIGs. 2E-2H) . The average complementarity-determining region 3 (CDR3) lengths ranged from 12.3 to 17.4 for the H chain and 8.4 to 9.4 for the L chain, respectively (FIGs. 2I-2L) . These results suggested overall comparable degrees of affinity maturation in these RBD-specific human MAbs obtained from individual memory B cells of four Hong Kong patients.

Table 3. Demographics of study subjects

Specificity and potency of SARS-CoV-2-specific human neutralizing antibodies

To determine the antiviral activities of these 18 RBD-specific human MAbs, binding and neutralization assays were performed. Five of them, namely A6-IgG1, B4-IgG1, B7-IgG1, B8-IgG1 and C5-IgG1 displayed RBD-and spike-specific binding by ELISA (FIGs. 3A and 3B) and neutralizing activities against both pseudotyped and authentic viruses (FIGs. 3C and 3D and FIG. 3E) . Interestingly, the four most potent HuNAbs, B4-IgG1, B7-IgG1, B8-IgG1 and C5-IgG1, all came from patient P3 (FIG. 3E) . Sequence analysis revealed strong similarities between B7-, B8-, and C5-IgG1, which all contained an IGHV1-69 heavy chain gene and an IGKV3 kappa light chain gene. B4-IgG1 contained distinct IGHV3-66 and IGKV1-33 genes with CDR3 lengths of 12 amino acids (aa) and 9 aa, and somatic hypermutation (SHM) rate of 3.8%and 4.6%, respectively (Table 4) . B7 and B8 were the most similar, as both contained IGHV1-69 and IGKV3-11 though B8 had a shorter CDR3 (14 aa vs 18 aa) and higher SHM (4.8%vs 0.0%) than those of B7 in the heavy chain. In the L chain, B7 and B8 shared a 9 aa CDR3, but B8 had a higher SHM rate than that of B7 (1.7%vs 0.7%) C5-IgG1 had a similar IGHV1-69 light chain gene with a 16 aa CDR3 and 2.4%SHM, but a different IGKV3-20 with a 9 aa CDR3 and 2.8%SHM. By ELISA, these four P3-derived HuNAbs bound to the SARS-CoV-2 RBD with half-maximal effective concentration (EC50) values ranging from 0.02 to 0.06 μg/ml, indicating stronger binding than that of the P4-derived A6-IgG1 (0.3 μg/ml) (FIG. 3A and Table 5) . P3-derived MAbs also exhibited stronger binding activities to the spike, with the EC50 values ranging from 0.018 to 0.06 μg/ml, compared to A6-IgG1 (17.94 μg/ml) (FIG. 3B and Table 5) . Neutralizing assays using pseudoviruses revealed that these four potent HuNAbs had IC ₅₀ values ranging from 0.0095 to 0.038 μg/ml, and IC90 values ranging from 0.046 to 0.136 μg/ml, respectively (FIG. 3C, and Table 6) . Moreover, B8 proved to be the most potent HuNAb, capable of inhibiting authentic SARS-CoV-2 with an IC50 value of 0.013 μg/ml and an IC ₉₀ value of 0.032 μg/ml, respectively (FIG. 3D and Table 6) . Next, it was determined if these HuNAbs could compete with ACE2 for RBD binding by surface plasmon resonance (SPR) . It was found that all of them strongly competed with ACE2 (FIGs. 4A-4D) . In line with the ELISA results, B8-IgG1 displayed the best KD value for RBD binding (169 pM) (FIGs. 5A-5D, Table 7) and the strongest competition with ACE2 (FIGs. 4A-4D) , which explained its potent neutralizing activity. As B4-IgG1 displayed only partial competition for RBD binding with the other antibodies (FIGs. 6A-6P) , antibody synergy experiments were performed using the pseudotype neuralization assay. No significant synergistic effects were found between any pairs of these four HuNAbs (FIGs. 7A-7F) . These results demonstrated that B4-, B7-, B8-and C5-IgG1 were all RBD-specific and competed with ACE2 for similar sites on the RBD.

Table 4. Sequence analysis of four HuNAbs

Table 5. Binding abilities of mAbs to different SARS-Cov-2 epitopes

Table 6. Neutralizing and ELISA binding activities of HuNAbs (μg/ml)

Table 7. Surface plasmon resonance analysis of RBD-specific NAbs

Table 8. Amino acid sequence alignment of four HuNAbs VH and VK. B8VH (SEQ ID NO: 1) , C5VH (SEQ ID NO: 2) , B7VH (SEQ ID NO: 3) , B4VH (SEQ ID NO: 4) , B8VK (SEQ ID NO: 5) , C5VK (SEQ ID NO: 6) , B7VK (SEQ ID NO: 7) , B4VK (SEQ ID NO: 8) .

B8-IgG1 pre-exposure prophylaxis and post-exposure treatment in the golden Syrian hamster model

To determine the efficiency of B8-IgG1 in pre-exposure prophylaxis and post-exposure treatment against live intranasal SARS-CoV-2 infection, B8-IgG1 was administered intraperitoneally in golden Syrian hamsters, before or after viral challenge in our Biosafety Level-3 (BSL-3) animal laboratory (FIG. 8A) . In the pre-exposure prophylaxis group (G1, n = 4) , each hamster received a single intraperitoneal injection of 1.5 mg/kg B8-IgG1. In the post-exposure treatment groups, each hamster received a single intraperitoneal injection of 1.5 mg/kg B8-IgG1 at day 1 (G2, n = 4) , day 2 (G3, n = 4) or day 3 (G4, n = 4) post-infection (dpi) , respectively (FIG. 8A) . The challenge dose was 10 ⁵ plaque-forming units (PFU) of live SARS-CoV-2 (HKU-001a strain) (Zhou et al. (2021) , Cell Host Microbe, 29 (4) : 551-563; Chan et al. (2020) , Clinical Infectious Diseases 71 (9) : 2428-2446) . Another group of hamsters (G0, n = 4) received PBS injection as a no-treatment control. One G4 animal died accidentally during the procedure. Since Syrian hamsters recover quickly from SARS-CoV-2 infection, with resolution of clinical signs and clearance of virus shedding within one week after infection (Plante et al. (2020) , Nature 592: 116-121; Chan et al. (2020) , Clinical Infectious Diseases 71 (9) : 2428-2446) , the animals were sacrificed at 4 dpi for HuNAb efficacy analysis, at a time when high viral loads and acute lung injury were consistently observed. At 4 dpi, NT and lung tissues were harvested to quantify infectious viruses by measuring PFUs, viral RNA loads by real-time reverse-transcription polymerase chain reaction (RT-PCR) and infected cells by immunofluorescence (IF) staining of viral nucleocapsid protein (NP) -positive cells as described previously (Zhou et al. (2021) , Cell Host Microbe, 29 (4) : 551-563; Chan et al. (2020) , Clinical Infectious Diseases 71 (9) : 2428-2446) .

Infectious virus, measured by PFU, was readily detected in all tissue compartments of G0 hamsters but not in the lungs of 75%G1, 100%G2, 75%G3 and 0%G4 animals, nor in the NT of 50%G1, 75%G2, 50%G3 and 25%G4 animals (FIGs. 8B and 8D. The decrease in PFU was of 2-3 orders of magnitude, suggesting efficient viral suppression in the lungs for the G1, G2, and G3 groups. A sensitive RT-PCR assay further demonstrated that viral RNA copy numbers were decreased in the lungs by 3 orders of magnitude in 50%of G1 hamsters (FIG. 8C) . In contrast, there was no significant viral RNA reduction in the NT of G1 animals (FIG. 8F) , suggesting lower efficacy of B8-IgG1 to prevent viral entry in the URT than in the lungs. There were slight but not significant viral RNA load decreases in both lungs and NT of G2 and some G3 animals. The number of infected cells or foci in these two tissue compartments were then evaluated by anti-NP antibody staining. A clear decrease of NP-positive cells or foci was observed in the lungs of G1 and G2 hamsters (figure not shown) . Abundant NP-positive cells with a diffuse distribution, however, were readily detected in the NT of all the challenged hamsters (figure not shown) . These results demonstrated that systemic B8-IgG1 injection was effective at reducing productive SARS-CoV-2 infection in the lungs when used for pre-exposure prophylaxis and early treatment especially within 48 hours post infection, but was insufficient to prevent viral infection in the NT.

To determine the correlates of B8-IgG1-mediated protection, the antibody concentration in serum, lung homogenate and NT homogenates were also measured at 0 and 4 dpi for all experimental animals. On average, 4, 257 ng/ml and 2, 101 ng/ml B8-IgG1 were found in animal sera at 0 and 4 dpi (Table 9) . On 4 dpi, lung and NT homogenates contained 128 ng/ml and 20 ng/ml in G1, 238 ng/ml and 86 ng/ml in G2, 229 ng/ml and 93 ng/ml in G3, and 192 ng/ml and 46 ng/ml in G4 animals, respectively. These results demonstrated that most animals maintained higher peripheral B8-IgG1 antibody concentration, while a decreasing concentration gradient was observed in the lungs and NT during infection. The concentrations of B8-IgG1 measured in NT homogenates were close to the neutralization IC ₉₀ measured in vitro (32 ng/ml) , explaining why infectious virus was undetectable in the PFU assay. These findings are in line with results obtained for other potent IgG HuNAbs administered systemically. The B8-IgG1 concentrations measured in the NT appeared insufficient to completely block infection in vivo, as indicated by the presence of NP-positive cells scattered throughout the NT.

Table 9: B8-IgG1 concentrations in different compartments of hamsters.

N.A: not applicable

Pre-exposure prophylaxis by monomeric B8-mIgA1 and B8-mIgA2 in Syrian hamsters

Since systemic administration of the RBD-specific neutralizing B8-IgG1 did not suppress SARS-CoV-2 nasal infection effectively, we sought to construct various types of IgA for mucosal intervention. For this purpose, we engineered B8-IgG1 into monomeric B8-mIgA1 and B8-mIgA2, and then into dimeric B8-dIgA1 and B8-dIgA2 by introducing the J chain. By in vitro characterization, purified B8-mIgA1, B8-mIgA2, B8-dIgA1 and B8-dIgA2 retained similar binding to RBD and spike by ELISA as compared to B8-IgG1 (FIGs. 9A and 9B, Table 10) , and comparable antiviral activities based on neutralization IC ₅₀ and IC ₉₀ values (FIGs. 9C and 9D and Table 11) . That said, B8-mIgA2 and B8-dIgA2 showed slightly more potent IC ₉₀ activities than B8-IgG1 in the pseudovirus neutralization assay using 293T-ACE2 cells as targets (Table 11) . After introducing J chain, the proper dimer formation of B8-dIgA1 and B8-dIgA2 was confirmed by size exclusion chromatography analysis (FIGs 9E and 9F) . Furthermore, B8-mIgA1, B8-mIgA2, B8-dIgA1 and B8-dIgA2 also retained comparable competition with ACE2 for binding to spike by SPR analysis (FIGs 9G-9J) . Therefore, the engineered IgA had the expected structural properties, and showed antiviral activities as potent as those of B8-IgG1 in vitro.

Table 10: Binding of B8 HuNAbs to SARS-CoV-2 RBD and spike.

Table 11: Neutralization potency of B8 HuNAbs

[Rectified under Rule 91, 21.03.2022]
The monomeric B8-mIgA1 and B8-mIgA2 in the hamster model was then evaluated, using a higher 4.5 mg/kg dose via either intranasal or intraperitoneal injection, and used the same amount of intranasal B8-IgG1 inoculation as a control (FIGs. 10A-10G) . Interestingly, while changes in total RdRp and subgenomic sgNP viral RNA loads were not obvious (FIG. 10B-10C) , B8-IgG1 and B8-mIgA1 (both i.n. and i.p. ) , but not B8-mIgA2, were able to significantly suppress infectious virus production (PFU) in the lungs of 75%infected hamsters by 2 orders of magnitude (FIG. 10D) . Sporadic infected cell foci were still detected in the lung sections by anti-NP staining (figures not shown) , suggesting that protection conferred by B8-IgG1 and B8-mIgA1 was not complete. The figures not shown are confocal images showing SARS-CoV-2 infection at 4 dpi in both lung and NT of infected Syrian hamsters pre-treated with B8-mIgA1 or B8-mIgA2 as determined by anti-NP immunofluorescence (IF) staining. The SARS-CoV-2 NP and cell nuclei were stained with rabbit anti-SARS-CoV-2 NP (green) and DAPI (blue) , respectively. B8-mIgA2 was not able to suppress viral RNA load nor PFU in the challenged hamsters, regardless of the route of antibody injection. On the other hand, like B8-IgG1, both B8-mIgA1 and B8-mIgA2 did not achieve significant viral suppression in the NT. After intranasal administration of either B8-mIgA1 or B8-mIgA2, some hamsters even showed a trend of slightly increased infectious virus production in NT, though this did not reach statistical significance (FIGs. 10E-10G) . Among these animals, the NP-positive cells were detected readily in the NT, as demonstrated by the whole section scanning, indicating the comparable distribution compared with the B8-IgG1 and no-treatment groups (figure not shown) . These results were consistent with many NP-positive cells observed in diffusely infected areas of NT by classic IF (figure not shown) . These results demonstrated that B8-mIgA1 was more potent than B8-mIgA2 at limiting SARS-CoV-2 infection in the lungs, but that both mIgA did not prevent nor significantly limit SARS-CoV-2 nasal infection.

B8-dIgA1 and B8-dIgA2 mediate enhancement of SARS-CoV-2 nasal infection and injury in Syrian hamsters

Next, the effects of dimeric B8-dIgA1 and B8-dIgA2 in Syrian hamsters were tested. To improve protective efficacy of intranasal dIgA treatment, a 3-fold higher dosage group of 13.5 mg/kg was included besides the 4.5 mg/kg group (FIGs. 11A-11H) , and the interval between dIgA and virus inoculation was shortened to 12 hours (FIG. 11A) . Both RdRp and sgNP viral RNA loads dropped significantly in the lungs of hamsters that received the high dose of B8-dIgA2 compared to the no-treatment group (FIGs. 11B and 11C) . Both B8-dIgA1 and B8-dIgA2 at the high dose also suppressed infectious viruses (PFU) in the lungs of 75%and 100%treated hamsters, respectively (FIG. 11D) . High dose B8-dIgA1 and B8-dIgA2 also decreased the number of NP-positive cells or foci in the lungs, with a more marked change for B8-dIgA2 (figures not shown) . The figures not shown are confocal and HE images showing SARS-CoV-2 infection at 4 dpi in both lung and NT of infected Syrian hamsters pre-treated with B8-dIgA1 or B8-dIgA2 by confocal and HE microscope. Representative images (100×) of infected foci in lungs (A) and NT (B) from each group as determined by anti-NP immunofluorescence (IF) staining were shown. The figures not shown also show that SARS-CoV-2 infection results in more extensive and severe damage of the NT epithelium in B8-dIgA-administrated animals (10× HE images) . Unexpectedly, however, significantly enhanced SARS-CoV-2 nasal infection and tissue damage were observed in most infected hamsters included the low and high dose groups of both B8-dIgA1 and B8-dIgA2, in all the four assays used (FIGs. 11E-11G) . High dose administration of B8-dIgA1 or B8-dIgA2 resulted in increased PFU production in the NT by 37-fold and 81-fold, respectively, compared to the no-treatment group (FIG. 11G) . Since the model showed comparable NT PFU on day 2 and day 4, this level of enhanced infection was unusual. It was also not observed with B8-IgG1 or monomeric B8-mIgA1 and B8-mIgA2 treatment, as described above. Moreover, the distribution of NP-positive cells in hamsters treated with dimeric B8-IgA was broader and reached deeper into NT tissue compared to the no-treatment group, as shown by whole section scanning (figures not shown) , which was associated with more severe and extensive epithelium desquamation and luminal cell debris (figure not shown) . The density of nasal NP+ cells was also significantly higher in B8-dIgA2-treated hamsters than in PBS-treated animals (FIG. 11H) . It is therefore conceivable that treatment with dimeric B8-IgA expanded the type and distribution of target cells in the nasal epithelium. It was confirmed that control dimeric dIgA1 and dIgA2 did not enhance SARS-CoV-2 infection under the same experimental conditions (FIGs. 12A-12G) . These results demonstrated that, instead of inducing viral suppression, pre-exposure dimeric B8-dIgA1 and B8-dIgA2 enhanced SARS-CoV-2 nasal infection and injury significantly in Syrian hamsters, which was consistently found in three independent experiments.

To validate the role of B8-dIgA1 and B8-dIgA2, the B8-IgA concentrations in the serum at

day

0 and 4 dpi were measured, and in the lung and NT homogenates at 4 dpi. B8-dIgA1 and B8-dIgA2 were primarily detected in lung homogenates at 4 dpi and were apparently undetectable in the serum and NT homogenates (Table 12) . The enhanced viral replication in NT probably exhausted B8-dIgA1 and B8-dIgA2 locally, through antibody-virus complex formation and clearance 30. To address this possibility, five groups of

Syrian hamsters (n=4 per group) were treated separately with each antibody at the 4.5 mg/kg dose. Twelve hours after the inoculation, antibody concentrations were readily detected in each tissues compartment (Table 13) . The highest concentrations of dIgA antibodies were found in lung homogenates, followed by nasal washes, NT homogenates, and serum. These results suggest that B8-dIgA1 and B8-dIgA2 concentrations in the NT and lungs were still above their neutralization IC ₉₀ values at the time of viral challenge, indicating that the results obtained in our experiments could not be explained by the insufficient amounts of antibodies.

B8-dIgA1-and B8-dIgA2-mediated enhancement of SARS-CoV-2 infection via CD209. Since B8 antibodies share the same binding site to the RBD domain, the possible mechanisms of B8-dIgA1-and B8-dIgA2-enhanced infection were investigated. First, it was consistently found that 10 ng/ml B8-dIgA1 or B8-dIgA2 completely neutralized SARS-CoV-2 infection in human renal proximal tubule cells (HK-2) , as measured in the immunofluorescence assay (figure not shown) . The figure not shown are confocal images showing potent neutralization of live SARS-CoV-2 infection by B8-dIgA1 and B8-IgA2 in human kidney cell line HK-2. The IF staining of SARS-CoV-2 NP in infected HK-2 cells pre-treated with different dose of antibody were indicated. The representative image of each group was shown. The B8-mIgA2 and B8-dIgA2 neutralizing capacity in the MucilAir ^TM model was also tested, consisting of a reconstructed human nasal epithelium, which contained goblet, ciliated, and basal cells (FIG. 13A) . Both B8-mIgA2 and B8-dIgA2 neutralized SARS-CoV-2 in a dose-dependent fashion, when compared to a dIgA2 control antibody. Similar experiments were carried out in the presence of the mucus naturally secreted by goblet cells, to determine whether dIgA interaction with the mucus may alter their neutralization capacity. However, B8-dIgA2 showed the same neutralization capacity in the presence and absence of mucus. These results demonstrated that B8-dIgA1 and B8-dIgA2 did not enhance SARS-CoV-2 infection in either human HK-2 or primary airway epithelial cells, which primarily expressed human ACE2 as a viral receptor. Next, attention was placed on the ACE2-independent mechanisms that might be associated with dimeric IgA-mediated enhancement of SARS-CoV-2 infection. Considering that mucosal monocyte-derived dendritic cells (DC) could mediate SARS-CoV-1 infection and dissemination in rhesus monkeys as early as 2 dpi, the role of DC-expressed surface receptors was investigated. CD209 (DC-SIGN) was focused on because this lectin is known to act as a cellular receptor for secretory IgA (Baumann et al. (2010) , Immunol Lett, 131: 59-66) . By IF staining, intranasal administration of B8-dIgA2 alone did not increase CD209 expression in the NT of treated hamsters (figure not shown) . Upon SARS-CoV-2 infection, however, a noted increase was observed in CD209-positive cells in olfactory epithelium devoid of ACE2 expression (figure not shown) . Importantly, most CD209+cells were positive for NP (figure not shown) , indicating that these CD209+ cells were likely permissive to SARS-CoV-2 infection. It was determined whether B8-dIgA1 and B8-dIgA2 could enhance SARS-CoV-2 infection in 293T cells expressing human CD209 or CD299 but not ACE2. Using a low MOI of 0.05, it was found that pre-incubation of B8-dIgA1 and B8-dIgA2 enhanced live SARS-CoV-2 infection significantly in 293T cells expressing human CD209, as determined by increased viral NP production (FIG. 13B) . Interestingly, human CD299, a type II integral membrane protein that is 77%identical to CD209, did not show similar activities in the same experiment (FIG. 13B) . Control dIgA1 and dIgA2 did not show any enhancement in NP+ cell detection compared with virus only. Considering that CD209+ DCs promote HIV-1 transmission to CD4+ T cells via cell-cell contacts, it was speculated that B8-dIgA1 and B8-dIgA2 might not be able to block the similar process for SARS-CoV-2. Indeed, by testing the B8 antibodies at concentrations 100-times higher than IC ₉₀ neutralization values (around 3000 ng/ml) , none of B8-IgG1, B8-mIgA1, B8-mIgA2, B8-dIgA1 and B8-dIgA2 could block cell-cell fusion (figure not shown) . Taken together, these results demonstrated that B8-dIgA1-and B8-dIgA2-enhanced SARS-CoV-2 nasal infection likely involved viral capture and infection of mucosal CD209+ cells, followed by more robust infection of ACE2+ epithelial cells through trans-infection via cell-cell spread in NT.

Cryo-EM analysis of the spike-B8 complex

To understand the potential mechanism of action of B8 HuNAb, a cryo-EM single-particle analysis of B8 Fab bound to the SARS-CoV-2 spike ectodomain trimer was conducted (FIG. 14) . Two B8-spike complex structures were determined based on 351, 095 and 616, 799 particles collected, respectively (Table 2) . One structure at

resolution contained the spike with all three RBDs adopting the “up” conformation (3u) , where each “up” RBD was bound by one B8 Fab (figure not shown) . The other structure at

resolution contained one spike trimer with 2 RBDs in the “up” conformation and 1 RBD in the “down” conformation (2u1d) , where each RBD was also bound by one B8 Fab despite the presence of two distinct RBD conformations (figure not shown) . After superimposing the “3u” and “2u1d” spikes, a ～53-degree rotation was observed between the “up” RBD (red color) in the 3u spike trimer and the “down” RBD (gray) in the 2u1d spike trimer (FIG. 15A) . The B8 Fab appeared to bind to the receptor-binding motif (RBM) of the RBD through its heavy chain for most of the interactions (FIGs. 15B and 15C) . Therefore, the cellular receptor ACE2 would clash with the B8 Fab due to the overlap of their respective epitopes on the RBM (FIGs. 15B-15D and Table 12) . The elucidation of the epitope revealed that B8 could be grouped into the SARS-CoV-2 neutralizing antibody class II (Wang et al. (2021) , Immunity, 54: 1611-1621) . These structural findings were further supported by neutralization assays using a panel of pseudoviruses containing naturally occurring mutations. Indeed, the E484K mutation from the South African SAΔ9 strain, which is located within the B8-binding interface, caused a major loss of neutralizing potency for all the B8 isotypes tested: IgG1, mIgA1, mIgA2, dIgA1 and dIgA2 (Table 13) . The comparable neutralization profiles of these NAbs against the full panel of viral variants also indicated that the conformation of key RBD-binding residues remained unchanged after engineering of the constant regions of these B8 isotypes.

Table 12: Contacts between SARS-CoV-2 RBD and B8 Fab (distance cutoff

) .

RBD	Heavy chain	RBD	Light chain
V445	E1	E484	W94
G446*	K98	G485	W94
Y449*	N31, Y32, K98	F486*	N93, W94
N450	N31	N487*	N93
L452	N31, F55, L101	Y489*	S92, N93
F456*	L104	T500*	T56
T470	F55
I472	T57
V483	N59
E484	R50, L52, T57, N59, F103
C488	F103
Y489*	F103, L104
F490	F55, L101
L492	L101, A102
Q493*	A102, L104
S494	N31, L101

Table 13: Neutralization of SARS-CoV-2 variants by B8-derived HuNAbs.

Less than -3.0 = resistant and Greater than 0 = sensitive

It remains to be determined whether CD209+ DCs are abundantly recruited to the nasal mucosa in SARS-CoV-2-infected humans. It is, however, known that myeloid DCs are increased in the nasal epithelium upon infection (Liu, et al. (2016) , Mucosal Immunol 9: 1089-1101; Hartmann, et al. (2021) , Clin Vaccine Immunol 13: 1278-12863) . Finally, preliminary analysis of the human nasal cytology data (under accession code EGAS00001004082) revealed the presence of increased CD209+ DCs in addition to abundant ACE2, TMPRSS2, and furin expression in the apical side of multiciliated cells of SARS-CoV-2-infected human subjects (FIGs. 16A-16C) .

Discussion

In this study, the preventive potential of a potent RBD-specific NAb B8 primarily in the forms of monomeric and dimeric IgA against live intranasal SARS-CoV-2 infection in the golden Syrian hamster model as compared with B8-IgG1 was tested (Zhou et al. (2021) , Cell Host Microbe, 29 (4) : 551-563; Chan et al. (2020) , Clinical Infectious Diseases 71 (9) : 2428-2446) . While these B8-IgA antibodies maintained neutralizing activities against SARS-CoV-2 in vitro similar to those of B8-IgG1, they displayed distinct in vivo effects, with clear differences in their capacity to modulate viral infection in the NT. Pretreatment by intranasal administration of 4.5 mg/kg of monomeric B8-mIgA1 or B8-mIgA2 did not significantly reduce infectious virus production in the NT homogenates. On the contrary, the antibody isotype had a marked effect, as intranasal administration of 4.5 mg/kg and 13.5 mg/kg dimeric B8-dIgA1 or B8-dIgA2 paradoxically increased the amount of infectious virus (PFU) in NT homogenates. This enhancing effect was not observed with several intranasal IgG HuNAbs previously tested by the inventors and other groups (Zhou et al. (2021) , Cell Host Microbe, 29 (4) : 551-563; Yang et al., (2020) , Antib Ther, 3: 205-212) . Mechanistically, instead of neutralization, virus-bound B8-dIgA1 and B8-dIgA2 used CD209 as an alternative receptor to infect non-ACE2 cells. CD209+ cells were increased and permissive to viral infection in the olfactory epithelium of Syrian hamsters upon SARS-CoV-2 infection, suggesting that this cell population could contribute to viral mucosal seeding. Indeed, it was found that CD209 expressing cells could be infected in vitro by live SARS-CoV-2 at 0.05 MOI in the presence of B8-dIgA1 and B8-dIgA2. Since none of the B8-based MAbs could prevent SARS-CoV-2 cell-to-cell transmission, even at high concentration in vitro, virus-laden mucosal CD209+ cells might trans-infect ACE2+ cells through cell-to-cell contacts in NT, resulting in enhanced infection and injury. Cryo-EM analysis further indicated that B8 is a typical class II HuNAb that binds to the SRAS-CoV-2 spike RBD in either a 3u or a 2u1d mode. These findings, therefore, reveal a previously unrecognized pathway for RBD-specific dimeric IgA-mediated enhancement of SARS-CoV-2 nasal infection and injury in Syrian hamsters.

The role of dimeric IgA has been explored primarily for mucosal transmitted viruses. At the mucosal surface, the major IgA type is the secretory form, which is generated from dIgA by the acquisition of a secretory component upon endocytosis and secretion by epithelial cells. In the simian AIDS macaque model, neutralizing dIgA given directly into the rectal lumen can prevent viral acquisition in rhesus monkeys challenged via the mucosal route (Watkins et al. (2013) , AIDS, 27: F13-20) . Although the administered dIgA did not contain the secretory component (SC) , they might have associated with free SC, which is present in mucosal secretions such as human lung lavages (Merrill et al., (1980) , Am Rev Respir Dis, 122: 156-161) . Neutralizing dIgA1 and dIgA2 could be protective through several mechanisms, including direct virus neutralization, virion capture, or the inhibition of virion transcytosis across the epithelium (Corthesy (2013) , Front Immunol, 4: 185) . In this macaque study, however, Watkins et al. demonstrated that the dimeric HGN194 dIgA2 protected only 1/6 animals in a rectal challenge model (Watkins et al. (2013) , AIDS, 27: F13-20) . Recently, Taylor et al. found an increase in virion number and penetration depth in the transverse colon and mesenteric lymph nodes, after mucosal treatment with the HGN194 dIgA2 compared to a PBS control (Taylor et al., (2021) , PLoS Pathog, 17: e1009632) . The authors suggested that virus-specific dIgA somehow mediated the delivery of virus immune complexes to the mesenteric lymph nodes for systemic infection. In the current study, the inventors report that SARS-CoV-2 may subvert the action of potent neutralizing antibodies, as pretreatment with neutralizing B8-dIgA1 and B8-dIgA2 induced a more robust nasal infection via a previously unrecognized mode of viral enhancement.

SARS-CoV-2 engages CD209+ cells to evade ACE2-dependent neutralizing B8-dIgA1 and B8-dIgA2 for enhanced NT infection and injury. Previous studies have indicated various scenarios for ADE occurrence in viral infections. The well-known dengue ADE has been associated with poorly neutralizing cross-reactive antibodies against a heterologous viral serotype, leading to increased infection of FcγR-expressing cells (Beltramello et al. (2010) , Cell Host &Microbe 8: 271-283) . Recent findings suggested that an increase in afucosylated antibodies contribute to dengue ADE (Bournazos et al. (2021) , Science 372, 1102-1105) . In contrast, vaccine-associated enhanced respiratory disease induced by respiratory syncytial virus has not been found to be antibody-dependent (van Erp et al., (2019) , Front Immunol 10: 548) . For SARS and MERS, ADE observed in vitro depended on binding of the antibody Fab to the virus and the binding of the Fc component to FcγR on target cells (Wan et al. (2020) J Virol 94: e02015-02019. One study found that spike IgG antibody abrogated wound-healing responses in SARS-CoV-1-infected Chinese macaques (Liu et al. (2019) JCI Insight 4: e123158) . In the case of COVID-19, vaccination and passive immunization studies have not revealed ADE of disease severity (Haynes et al., (2020) Sci Transl Med 12) . Comprehensive studies, however, are necessary to define the clinical correlates of protective immunity against SARS-CoV-2, especially in the context of vaccine breakthrough infections. During natural infection, one study indicated that the increase in afucosylated antibodies might contribute to COVID-19 severity (Larsen et al. (2021) , Science 371) . To date, four classes of potent HuNAbs have been isolated from convalescent COVID-19 patients (Cao et al., (2020) , Cell, 182: 73-84; Wang et al. (2021) , Immunity, 54: 1611-1621) . The molecular mechanism of neutralization for most potent HuNAbs was primarily through blocking the interaction between ACE2 and the spike RBD. Currently, systemic RBD-specific HuNAb treatment remains to be improved for therapeutic suppression of SARS-CoV-2 replication in the NT or URT, both in animal models and human trials (Zhou et al. (2021) , Cell Host Microbe, 29 (4) : 551-563; Chan et al. (2020) , Clinical Infectious Diseases 71 (9) : 2428-2446; Baum et al., (2020) , Science, 370: 1110-1115) . One limitation is the insufficient amounts of HuNAbs distributed on the nasal mucosal surface for protection (Zhou et al. (2021) , Cell Host Microbe, 29 (4) : 551-563) . Other reasons might include alternative entry pathways engaged by SARS-CoV-2 to evade HuNAbs. To this end, Liu et al. reported recently that antibodies against the spike N-terminal domain (NTD) induced an open conformation of the RBD and thus enhanced the binding capacity of the spike to the ACE2 receptor, leading to increased viral infectivity (Liu et al. (2021) , Cell 184: 3452-3466) . Yeung et al. demonstrated that SARS-CoV-2 could engage soluble ACE2 (sACE2) and then bind alternate receptors for viral entry, through interaction between a spike/sACE2 complex with the angiotensin II AT1 receptor, or interaction between a spike/sACE2/vasopressin complex with the AVPR1B vasopressin receptor, respectively (Yeung et al., (2021) , Cell, 184: 2212-2228 e2212) . In this study, the inventors found that, in the presence of potent neutralizing B8-dIgA1 or B8-dIgA2 antibodies, SARS-CoV-2 used the cellular receptor CD209 for capture or infection, which likely expanded the use of CD209+ cells as target cells, leading to enhanced NT infection and trans-infection. Interestingly, a preprint report suggests that cells expressing CD209 can be infected directly by SARS-CoV-2 through an interaction of the spike with the NTD instead of the RBD (Soh et al., (2020) , bioRxiv, 2020.2011.2005.369264) . This mode of action, however, was unlikely to explain the current findings, because no enhancement of SARS-CoV-2 nasal infection was found in presence of control dIgA1 and dIgA2. The current results rather suggest that the direct binding of virus-bound B8-dIgA1 or virus-bound B8-dIgA2 to CD209 is a likely pathway, resulting in the more severe SARS-CoV-2 nasal infection and damage. In line with these results, a previous study demonstrated that dIgA itself can use CD209 as a cellular receptor IgA (Baumann et al. (2010) , Immunol Lett, 131: 59-66) . During the entry process, since neither B8-dIgA1 nor B8-dIgA2 could prevent virus cell-to-cell transmission, infected mucosal CD209+ cells might enable a more robust viral transmission to ciliated nasal epithelial cells in NT, which show the highest expression of ACE2 and TMPRSS2 receptors (Baum et al., (2020) , Science, 370: 1110-1115) . In support of this notion, previous studies indicated that mucosal DCs can capture HIV-1 through binding of its envelope glycoproteins to CD209 and efficiently transfer the bound virions to CD4+ T cells, in a process called trans-enhancement or trans-infection (Geijtenbeek et al., (2000) Cell 100: 587-597) . The trans-infection markedly decreased the neutralization efficiency of potent NAbs directed at HIV (Bracq, et al., (2018) , Front Immunol, 9: 260) . Moreover, although monocyte-derived DCs (MDDCs) cannot support productive SARS-CoV-2 replication (Yang et al. (2020) , J Infect Dis, 222: 734-745) , a recent study demonstrated that MDDCs could mediate efficient viral trans-infection of the Calu-3 human respiratory cell line (Thepaut, et al. (2021) , PLoS Pathog 17, e1009576) . The findings of the current study showing increased number of infectious viruses in NT, therefore, have significant implications for SARS-CoV-2 transmission, COVID-19 pathogenesis, and immune interventions.

It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if an antibody is disclosed and discussed and a number of modifications that can be made to a number of molecules including the antibody are discussed, each and every combination and permutation of antibody and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials. These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

It must be noted that as used herein and in the appended claims, the singular forms “a, ” “an, ” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” includes a plurality of such antibodies, reference to “the antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises, ” means “including but not limited to, ” and is not intended to exclude, for example, other additives, components, integers or steps.

“Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

Unless the context clearly indicates otherwise, use of the word “can” indicates an option or capability of the object or condition referred to. Generally, use of “can” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of the word “may” indicates an option or capability of the object or condition referred to. Generally, use of “may” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of “may” herein does not refer to an unknown or doubtful feature of an object or condition.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about, ” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. It should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. Finally, it should be understood that all ranges refer both to the recited range as a range and as a collection of individual numbers from and including the first endpoint to and including the second endpoint. In the latter case, it should be understood that any of the individual numbers can be selected as one form of the quantity, value, or feature to which the range refers. In this way, a range describes a set of numbers or values from and including the first endpoint to and including the second endpoint from which a single member of the set (i.e. a single number) can be selected as the quantity, value, or feature to which the range refers. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Although the description of materials, compositions, components, steps, techniques, etc. can include numerous options and alternatives, this should not be construed as, and is not an admission that, such options and alternatives are equivalent to each other or, in particular, are obvious alternatives. Thus, for example, a list of different antibodies does not indicate that the listed antibodies are obvious one to the other, nor is it an admission of equivalence or obviousness.

Every antibody disclosed herein is intended to be and should be considered to be specifically disclosed herein. Further, every subset of antibodies that can be identified within this disclosure is intended to be and should be considered to be specifically disclosed herein. As a result, it is specifically contemplated that any antibody, or subset of antibodies can be either specifically included for or excluded from use or included in or excluded from a list of antibodies.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

An antibody or antigen binding fragment thereof comprising six complementarity determining regions (CDRs) ,

wherein the CDRs comprise:

(1) the three light chain CDRs of SEQ ID NO: 5 and the three heavy chain CDRs of SEQ ID NO: 1,

(2) the three light chain CDRs of SEQ ID NO: 6 and the three heavy chain CDRs of SEQ ID NO: 2,

(3) the three light chain CDRs of SEQ ID NO: 7 and the three heavy chain CDRs of SEQ ID NO: 3, or

(4) the three light chain CDRs of SEQ ID NO: 8 and the three heavy chain CDRs of SEQ ID NO: 4, and

wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 RBD.
The antibody or antigen binding fragment thereof of claim 1, wherein the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-32 of SEQ ID NO: 5, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 5, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 5.
The antibody or antigen binding fragment thereof of one of claims 1 or 2, wherein the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-33 of SEQ ID NO: 1, a second heavy chain CDR comprising amino acids 51-58 of SEQ ID NO: 1, and a third heavy chain CDR comprising amino acids 97-110 of SEQ ID NO: 1.
The antibody or antigen binding fragment thereof of one of claims 1, 2, or 3 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5.
The antibody or antigen binding fragment thereof of one of claims 1 or 2-4 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1.
The antibody or antigen binding fragment thereof of claim 1, wherein the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-33 of SEQ ID NO: 6, a second light chain CDR comprising amino acids 51-53 of SEQ ID NO: 6, and a third light chain CDR comprising amino acids 90-98 of SEQ ID NO: 6.
The antibody or antigen binding fragment thereof of one of claims 1 or 6, wherein the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-33 of SEQ ID NO: 2, a second heavy chain CDR comprising amino acids 51-58 of SEQ ID NO: 2, and a third heavy chain CDR comprising amino acids 97-112 of SEQ ID NO: 2.
The antibody or antigen binding fragment thereof of one of claims 1, 6, or 7 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6.
The antibody or antigen binding fragment thereof of one of claims 1 or 6-8 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.
The antibody or antigen binding fragment thereof of claim 1, wherein the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-32 of SEQ ID NO: 7, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 7, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 7.
The antibody or antigen binding fragment thereof of one of claims 1 or 10, wherein the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-33 of SEQ ID NO: 3, a second heavy chain CDR comprising amino acids 51-58 of SEQ ID NO: 3, and a third heavy chain CDR comprising amino acids 97-113 of SEQ ID NO: 3.
The antibody or antigen binding fragment thereof of one of claims 1, 10, or 11 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7.
The antibody or antigen binding fragment thereof of one of claims 1 or 10-12 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3.
The antibody or antigen binding fragment thereof of claim 1, wherein the three light chain CDRs comprise a first light chain CDR comprising amino acids 27-32 of SEQ ID NO: 8, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 8, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 8.
The antibody or antigen binding fragment thereof of one of claims 1 or 14, wherein the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-32 of SEQ ID NO: 4, a second heavy chain CDR comprising amino acids 50-56 of SEQ ID NO: 4, and a third heavy chain CDR comprising amino acids 95-106 of SEQ ID NO: 4.
The antibody or antigen binding fragment thereof of one of claims 1, 14, or 15 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.
The antibody or antigen binding fragment thereof of one of claims 1 or 14-16 comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4.
The antibody or antigen binding fragment thereof of claim 1 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1.
The antibody or antigen binding fragment thereof of claim 1 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.
The antibody or antigen binding fragment thereof of claim 1 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3.
The antibody or antigen binding fragment thereof of claim 1 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4.
The antibody or antigen binding fragment thereof of any one of claims 1-21, wherein the antibody or antigen binding fragment thereof attenuates the ability of a ligand of SARS-CoV-2 RBD to bind to ACE2.
The antibody or antigen binding fragment thereof of any one of claims 1-22 comprising one or more constant domains from an immunoglobulin constant region (Fc) .
The antibody or antigen binding fragment thereof of claim 23, wherein the constant domains are human constant domains.
The antibody or antigen binding fragment thereof of claim 24, wherein the human constant domains are IgA, IgD, IgE, IgG or IgM domains.
The antibody or antigen binding fragment thereof of claim 25, wherein human IgG constant domains are IgG1, IgG2, IgG3, or IgG4 domains.
The antibody or antigen binding fragment thereof of any one of claims 1-26, wherein the antibody or antigen binding fragment thereof is detectably labeled or comprises a conjugated toxin, drug, receptor, enzyme, receptor ligand.
The antibody or antigen binding fragment thereof of any one of claim 1-27, wherein the antibody is a monoclonal antibody, a human antibody, a chimeric antibody or a humanized antibody.
The antibody or antigen binding fragment thereof of any one of claims 1-28, wherein the antibody is a bispecific, trispecific or multispecific antibody.
A humanized antibody or antigen binding fragment thereof comprising one or more human IgG4 constant domains and

a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1,

a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2,

a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3, or

a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4.
A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of claims 1-30 and a physiologically acceptable carrier or excipient.
The pharmaceutical composition of claim 31 for use in a method of preventing or treating COVID-19 in a subject.
The pharmaceutical composition for use of claim 32 wherein the subject has COVID-19.
The pharmaceutical composition for use of claim 32 wherein the subject is at risk of developing COVID-19.
The pharmaceutical composition of claim 31 for use in a method of treating COVID-19.
The pharmaceutical composition of claim 31 for use in a method of preventing COVID-19.
Use of the antibody or antigen binding fragment thereof of any of claims 1-30 in manufacture of a medicament for preventing or treating COVID-19 in a subject.
Use of the antibody or antigen binding fragment thereof of any of claims 1-30 in manufacture of a medicament for treating COVID-19 in a subject.
Use of the antibody or antigen binding fragment thereof of any of claims 1-30 in manufacture of a medicament for preventing COVID-19 in a subject.
A method of detection or diagnosis of SARS-CoV-2 infection, comprising: (a) assaying the presence of SARS-CoV-2 RBD in a sample from a subject using the antibody or antigen binding fragment thereof of any one of claims 1-30 and (b) comparing the level of the SARS-CoV-2 RBD with a control level, wherein an increase in the assayed level of SARS-CoV-2 RBD compared to the control level is indicative of SARS-CoV-2 infection.
The use of claim 38, wherein the presence of SARS-CoV-2 RBD is assayed by enzyme linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , or fluorescence-activated cell sorting (FACS) .
A pharmaceutical composition for use in a method of treating a subject infected by or at risk for infection by SARS-CoV-2, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 31 if the subject has a disease characterized by increased expression of SARS-CoV-2 RBD.
The method of claim 42 wherein the antibody or antigen binding fragment thereof is the antibody or antigen binding fragment thereof of any one of claims 1-30.