WO2022062803A1

WO2022062803A1 - Neutralizing antibodies against covid-19 and methods of use thereof

Info

Publication number: WO2022062803A1
Application number: PCT/CN2021/114274
Authority: WO
Inventors: Zhiwei Chen; Dongyan Zhou; Runhong ZHOU; FuK Woo CHAN; Li Liu; Kwok Yung Yuen
Original assignee: The University Of Hong Kong
Priority date: 2020-09-24
Filing date: 2021-08-24
Publication date: 2022-03-31

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: 8 and the three heavy chain CDRs of SEQ ID NO: 4, (2) the three light chain CDRs of SEQ ID NO: 5 and the three heavy chain CDRs of SEQ ID NO: 1, (3) the three light chain CDRs of SEQ ID NO:6 and the three heavy chain CDRs of SEQ ID NO: 2, or (4) the three light chain CDRs of SEQ ID NO: 7 and the three heavy chain CDRs of SEQ ID NO: 3, and 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

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) ) . We and others also 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.

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

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

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

(4) the three light chain CDRs of SEQ ID NO: 7 and the three heavy chain CDRs of SEQ ID NO: 3, 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: 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 26-33 of SEQ ID NO: 4, a second heavy chain CDR comprising amino acids 51-57 of SEQ ID NO: 4, and a third heavy chain CDR comprising amino acids 96-114 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 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 97-112 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 27-32 of SEQ ID NO: 6, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 6, and a third light chain CDR comprising amino acids 89-97 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 97-112 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: 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: 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 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 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: 8, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4,

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, or

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.

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.

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.

Figures 1A and 1B show the screening of SARS-CoV-2 neutralizing antibodies from convalescent patients. Humoral immune responses to SARS-CoV2 were analysed in each patient by the endpoint ELISA for binding to viral RBD. The pseudovirus assay was used to measure neutralization activity. Antibody responses from patients were categorized according to disease severity. Figure 1A shows testing of a phage-displayed antibody Fab library (left) and monoclonal phage colonies (right) against RBD by ELISA throughout the panning procedure. Unpanned phage library, 1st round and 2nd round amplified phage polyclones (poly) were tested in 1: 5 dilution by the phage ELISA. An unrelated antigen was used as control. Figure 1B displays ELISA binding of the 384 single colonies (mono) with RBD. The four strongest single phage binders were named according to the clone numbers.

Figures 2A-2F show the efficacy of ZDY20 prophylaxis against live SARS-CoV-2 in Syrian hamsters. Figure 2A shows the experimental schedule. Two groups of hamsters received a single intraperitoneal injection of ZDY20 at doses of 10 mg/kg (n=4) and 5 mg/kg (n=3) at one day before the live virus challenge (-1 dpi) . Another group was given the control antibody VRC01 at 10 mg/kg (n=4) . On Day 0, each hamster was intranasally inoculated with a challenge dose of 100 μL of Dulbecco’s Modified Eagle Medium containing 10 ⁵ PFU of SARS-CoV-2 (HKU-001a strain, GenBank accession no: MT230904.1) . The hamsters were sacrificed on 4 dpi for analysis. Figure 2B shows testing of infectious virions by viral plaque assay in nasal turbinate, trachea and lung tissues. Plaque forming units (PFU) per mg of tissues extractions were compared between different groups in log-transformed units. Statistics were generated using one-way ANOVA comparisons test. *p<0.05; **p<0.01, ***p<0.001. Figure 2C shows the relative viral concentration (normalized by β-actin) as determined in nasal turbinate, trachea and lung tissues by the sensitive RT-PCR assay. Figure 2D shows serum concentration and Figure 2E shows neutralizing activity of the HuNAb ZDY20 as determined by ELISA and pseudovirus assays, respectively. Figure 2F shows comparison of NP ⁺ cells per 50× field in lungs of three experimental groups. Statistics were generated using Kruskal–Wallis test and Dunn’s multiple comparisons test. *p<0.05; **p<0.01, ***p<0.001.

Figure 3 shows robust SARS-CoV-2 infection in nasal turbinate during the HuNAb ZDY20 prophylaxis experiment by IF staining. NP ⁺ cells per 50× field were compared in nasal turbinate of 3 animal groups. Statistical analysis was done by the Kruskal–Wallis test and Dunn’s multiple comparisons test. *p<0.05; **p<0.01, ***p<0.001.

Figures 4A-4F show post-challenge ZDY20 therapy suppresses SARS-CoV-2 replication in lungs and acute lung injury. Figue 4A shows the experimental schedule. After the live intranasal SARS-CoV-2 challenge, three groups of hamsters (n=4 per group) received a single intraperitoneal injection of 10mg/kg ZDY20 at 1dpi, 2dpi and 3dpi, respectively. The hamsters were sacrificed on 4dpi for final analysis. Figure 4B shows viral plaque assay as used to quantify infectious viruses in nasal turbinate, trachea and lung tissues. Log ₁₀-transformed plaque forming units (PFU) per mg of tissue extractions were compared between treated hamsters at different time points and control animals. Statistics were generated using one-way ANOVA comparisons test. *p<0.05; **p<0.01, ***p<0.001. Figure 4C shows use of sensitive RT-PCR to quantify the viral RNA copy numbers (normalized by β-actin) in nasal turbinate, trachea and lung tissues. Figure 4D shows serum concentration and Figure 4E shows neutralizing activity of the HuNAb ZDY20 as determined by ELISA and pseudovirus assays, respectively. Figure 4F shows comparison of NP ⁺ cells per 50× field in lungs of four experimental groups. Statistics were generated using Kruskal–Wallis test and Dunn’s multiple comparisons test. *p<0.05; **p<0.01, ***p<0.001.

Figure 5 shows that post-challenge ZDY20 therapy does not suppress SARS-CoV-2 replication in nasal turbinate by IF staining and correlation analysis. Positive correlation was found between NP ⁺ cells per 50×field in lungs and the amount of peripheral ZDY20.

Figure 6 shows the screening strategy for SARS-CoV-2 neutralizing antibody by phage surface display. A total of 12 convalescent patients were included to collect plasma and PBMC samples. A Fab phage library displaying the variable heavy chain (VH) and light chain (VL) was established from the pooled PBMCs. The recombinant RBD protein of SARS-CoV-2 was used as a bait during the phage display panning procedures. Positive binding clones of single phages were sequenced and the paired VH/VL were reconstructed into antibody for IgG1 expression. Purified IgG1 was validated and functionally tested. Finally, a golden Syrian hamster model was set-up to evaluate both prophylactic and treatment efficacy of the lead HuNAb.

Figures 7A-7D show mapping of RBD antigenic determinants of four HuNAbs by yeast surface display. Size and location of the minimal spike fragments that bind to each HuNAb determined by yeast surface display of spike antigenic epitopes. The number (n) indicates the number of reactive clones for each antibody. Figure 7A shows 43 peptides bound by ZDY20, with the peptides overlapping amino acids 337-524 of RBD (corresponding to nucleotides 1021-1586) . Figure 7B shows 56 peptides bound by ZDY28, with the peptides overlapping amino acids 336-523 of RBD (corresponding to nucleotides 1018-1581) . Figure 7C shows 37 peptides bound by ZDY49, with the peptides overlapping amino acids 336-515 of RBD (corresponding to nucleotides 1018-1558) . Figure 7D shows 56 peptides bound by ZDY95, with the peptides overlapping amino acids 336-515 of RBD (corresponding to nucleotides 1018-1558) .

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 (ZDY20, ZDY28 ZDY49 and ZDY95) 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 12 patients.

The disclosed antibody drugs were demonstrated to be effective for SARS-CoV-2 prevention and therapy in the golden Syrian hamster model. Prophylactic intraperitoneal injection of 10 mg/ml ZDY20 significantly reduced infection in lungs of hamsters intranasally-challenged with SARS-CoV-2. Moreover, post-challenge ZDY20 therapy suppressed viral loads and lung damage especially when treated within 48-hours. HuNAb ZDY20 prevented entry of pseudovirus and live virus with IC ₉₀ values of 1.24 μg/ml and 1 μg/ml, respectively, by competing with human cellular receptor ACE2 for RBD binding. These results demonstrated that systemic HuNAb suppresses SARS-CoV-2 replication and lung injury.

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:

ZDY28HV:

ZDY95HV:

ZDY49HV:

ZDY20HV:

ZDY28VK:

ZDY95VK:

ZDY49VK:

ZDY20VK:

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 7. Table 7 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 7 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.

Thus, in addition to antibodies and antigen-binding fragments thereof that possess the CDRs of the anti-SARS-CoV-2 RBD antibodies: ZDY20, ZDY28, ZDY95, and ZDY49, 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 a 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 ZDY20, ZDY28, ZDY95, or ZDY49;

(2) alight chain CDR2 of anti-SARS-CoV-2 RBD antibody ZDY20, ZDY28, ZDY95, or ZDY49; and

(3) the light chain CDR3 of anti-human SARS-CoV-2 RBD antibody ZDY20, ZDY28, ZDY95, or ZDY49.

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 ZDY20, ZDY28, ZDY95, or ZDY49;

(2) the heavy chain CDR2 of anti-SARS-CoV-2 RBD antibody ZDY20, ZDY28, ZDY95, or ZDY49; and

(2) the heavy chain CDR3 of anti-SARS-CoV-2 RBD antibody ZDY20, ZDY28, ZDY95, or ZDY49.

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 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, 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 Fc RIIB 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, and 5,585,089) , 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 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.

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.

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/SCorp 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) ) .

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.

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.

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: 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.

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: 4, a second heavy chain CDR comprising amino acids 51-57 of SEQ ID NO: 4, and a third heavy chain CDR comprising amino acids 96-114 of SEQ ID NO: 4.

4. The antibody or antigen binding fragment thereof of one of paragraphs 1-3 comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

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

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 27-32 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.

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: 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 97-112 of SEQ ID NO: 1.

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: 5.

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: 1.

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: 6, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 6, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 6.

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: 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.

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: 6.

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: 2.

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: 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.

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 26-33 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 97-112 of SEQ ID NO: 3.

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: 7.

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: 1.

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

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

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

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: 7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3.

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 method 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

In this study, we identified a panel of candidate HuNAbs and conducted a thorough investigation of the lead candidate HuNAb ZDY20 at the dose of 10 mg/ml, which is 10,000-fold higher than its IC ₉₀ value of 1 μg/ml against live SARS-CoV-2, in in our established golden Syrian hamster model for COVID-19 (Chan et al., Clinical Infectious Diseases (2020) ) .

Materials and Methods

Patients

A total of 12 patients with COVID-19 were recruited between February and March 2020. All patient cases were confirmed by reverse-transcription polymerase chain reaction (RT-PCR) as described previously (Chan et al., J. Clin. Microbiol., 58: 10.1128/JCM. 00310-20 (2020) ) . 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 University of Hong Kong/Hospital Authority Hong Kong West Cluster, Hong Kong East Cluster Research Ethics Committee, and Kowloon West Cluster Research Ethics Committee (UW 13-265, HKECREC-2018-068, KW/EX-20-038 [144-26] ) .

Construction of a Fab phage display library

Total RNA was extracted from peripheral blood lymphocytes of convalescent SARS-CoV2 patients (RNeasy Mini kit; QIAGEN) , and both total RNA and mRNA was reverse transcribed into cDNA using PrimeScript 1st strand cDNA Synthesis Kit (TaKaRa) . Fd segment (variable and first constant domains) genes and light-chain genes were amplified by using primers specific for the human chain genes (Hust et al., in Antibody Engineering: Methods and Protocols, Second Edition, P. Chames, Ed. (Humana Press Inc., Totowa, NJ, United States, 2012) , chap. 5, pp. 85-107) . Then the amplified chains were assembled into the pComb3X phage display vector using

HiFi DNA Assembly Cloning Kit (NEB) . The assembled products were transformed into Escherichia coli. TG1 (Lucigen) and resulted in a library of 3×10 ⁶ clones. The transformants were expanded into a volume of 2 liters, and the resulting phage was recovered as described previously (Hust et al., in Antibody Engineering: Methods and Protocols, Second Edition, P. Chames, Ed. (Humana Press Inc., Totowa, NJ, United States, 2012) , chap. 5, pp. 85-107) .

Enrichment of antigen-binding clones by in solution panning

This panning procedure was adapted from the previously described ones (Chames, in Antibody Engineering: Methods and Protocols, Second Edition, P. Chames, Ed. (Humana Press Inc., Totowa, NJ, United States, 2012) , chap. 11, pp. 213-224) . For the first round of panning, 100 μl streptavidin magnetic beads (ThermoFisher) were coated with 200 nM biotinylated SARS-CoV-2 Spike protein RBD (ACROBiosystems) which are then blocked with 2%skimmed-milk PBS for an hour. At the same time the 1×10 ¹² phage from Fab phage library were also blocked in 2%skimmed-milk PBS. Then the mixture of phage and biotinylated RBD on beads were incubated for an hour at room temperature (RT) on a rotating wheel. Then the beads are pulled out of solution and washed 5 times with 1 ml of PBS Tween20 (0.1%) and 2 times with 1 ml of PBS. The remaining phage was eluted with 0.1 M trimethylamine (TEA) and 0.2 M Glycine pH 2 sequentially and then immediately neutralized with 1 M Tris base. The eluted phage was amplified by infection of Escherichia coli. TG1 cultures, followed by super-infection with helper phage M13KO7 (NEB) . At

Round

2, 100 nM antigen is used with 50 μl beads. Then 25 times of washes were performed at Round 2 to narrow down the Round 1 phage output to enrich for those that bind specifically to the target. Similarly, at

Round

3, 25 times of washes were performed to narrow down the diversity and enrich for only specific, stronger binders from the phage library. Polyclonal phage ELISA was carried out at Round 2 and if phage were not sufficiently enriched at this stage then a third round was carried out. All the panning procedures were done in solution in low protein binding 1.5 ml Eppendorf tubes.

Polyclonal, monoclonal phage ELISAs and recombinant protein ELISAs

The procedures of phage ELISA were adapted from previously described ones (Chames, in Antibody Engineering: Methods and Protocols, Second Edition, P. Chames, Ed. (Humana Press Inc., Totowa, NJ, United States, 2012) , chap. 11, pp. 213-224) . For polyclonal phage ELISA, the RBD proteins were coated on 96-well enzyme-linked immunosorbent assay plates at 50 ng/well. After blocking with 4%skimmed-milk in PBS, 50 μl phage library or amplified eluted phage were added to the plates at serial dilutions stating from 1: 5 and then incubated for 1 h at RT. Plates were washed and secondary antibody anti-M13 Major Coat Protein Antibody-HRP (Santa Cruz Biotechnology) was added. 3, 3′, 5, 5′-Tetramethylbenzidine Liquid Substrate (Sigma-Aldrich T4444) was used for color development. Optical density at 450 nm was read on a Varioskan LUX Multimode Microplate Reader (ThermoFisher) . For monoclonal phage ELISA, single colony was picked from the plate of eluted phage and cultured in 96-deep-well plates with about 200 μl/well at 37℃, 250 rpm. When the OD600 reached 0.5, each well of the single colony culture was super-infected with helper phage M13KO7 (NEB) and then incubated at 30℃, 250 rpm for 16-18 hours. The supernatant of the single culture was used for ELISA as the polyclonal phage ELISA with 1: 1 dilution with 4%skimmed-milk in PBS. The strong single binders were sent for sequencing. For recombinant protein ELISAs, the RBD or spike proteins were coated on 96-well enzyme-linked immunosorbent assay plates at 50 ng/well. After blocking with 4%skimmed-milk in PBS, 50 μl of NAbs were added to the plates at serial dilutions stating from 10 μg/ml and then incubated for 1 h at 37℃. Plates were washed and secondary antibody anti-human IgG-HRP (Invitrogen) was added. 3, 3′, 5, 5′-Tetramethylbenzidine Liquid Substrate (Sigma-Aldrich T4444) was used for color development. Optical density at 450 nm was read on a Varioskan LUX Multimode Microplate Reader (ThermoFisher) .

Protein expression and purification

The antibody VH/VL and constant region genes were amplified and cloned into expression vector AbvecIgG and AbvecIgKappa using

HiFi DNA Assembly Cloning Kit (NEB) . The plasmids of paired IgH and L genes were co-transfected into Expi293F expression system (Thermo Scientific) following the manufacturer’s protocol to produce recombinant HuNAbs. Antibodies from cell culture supernatants were purified immediately by affinity chromatography using Recombinant Protein G Agarose (Thermo Fisher) according to the manufacturer’s instructions. The purified HuNAbs were concentrated by an Amicon ultracentrifuge filter device (molecular weight cutoff, 50 kDa; Millipore) to a volume of 0.2 ml in PBS (Life Technologies) , and stored at –80℃.

Pseudovirus-based neutralization assay

The neutralizing activity of NAbs was determined using a pseudotype-based neutralization assay as we previously described (Liu et al., Emerging Microbes &Infections 9: 1664-1670 (2020) ) . The pseudovirus was generated through co-transfection of 293T cells with 2 plasmids, 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 (Liu et al., Emerging Microbes &Infections 9: 1664-1670 (2020) ) . Viral supernatant was collected 48 h post-transfection and was frozen at -150℃. The serially diluted NAbs were incubated with 200 TCID ₅₀ of pseudovirus at 37℃ for 1 hour. The HuNAb-virus mixtures were subsequently added into pre-seeded HEK 293T-ACE2 cells. After 48 hours, 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 HuNAbs were determined by log (inhibitor) vs. normalized response --Variable slope using GraphPad Prism 6 (GraphPad Software Inc. ) .

Neutralization activity of HuNAbs against live SARS-CoV-2

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) previously obtained from a nasopharyngeal swab of an infected patient (Chu et al., Lancet Microbe 1: e14-e23 (2020) ) . Serial dilutions of testing antibodies were conducted, mixed with 50 μl of SARS-CoV-2 (1×10 ³ 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 1×10 ⁴/well Vero E6 cells and incubated at 37℃ for 24 hours. After 24 hours, the culture medium of the plates was removed and air-dried in the BSC for 20 mins. Cells were then fixed with 4%paraformaldehyde solution for 30 min and air-dried in the BSC again. Cells were further permeabilized with 0.2%Triton X-100 and incubated with cross-reactive rabbit anti-SARS-CoV-2-N IgG (Sino Biological, Inc) for 1 hour at RT before adding Alexa Fluor 488 goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (Life Technologies) . The numbers of SARS-CoV-2 foci were calculated using the Sapphire Biomolecular Imager (Azure Biosystems) .

Antibody binding kinetics, and competition with receptor ACE2 measured by SPR

The binding kinetics and affinity of HuNAbs to SARS-CoV-2 spike protein (ACROBiosystems) were analyzed by SPR (Biacore 8K, GE Healthcare) . Specifically, 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, a concentration series of HuNAbs 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 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 sensorgram series were fit to a 1: 1 (Langmuir) binding model of interaction. Prior to evaluate competition between antibodies and the human ACE2 peptidase domain, both the saturated binding concentrations of antibodies and ACE2 protein (ACROBiosystems) with immobilized SARS-CoV-2 spike protein were determined separately. In the competitive assay, antibodies at the saturated concentration were injected onto the chip immobilized with spike protein for 120s until binding steady-state was reached. ACE2 protein also at the saturated concentration was then injected for 120s which was followed by another 120s of injection of antibody to make sure the saturated binding reaction against the immobilized spike protein. The differences of response units between ACE2 injection alone and prior antibody incubation reflect the antibodies’ competitive ability against ACE2 in binding with spike protein.

Epitope Mapping Through Yeast Surface Display System

The antigen library displayed on yeast surface was constructed by methods as we and others described previously (Zuo et al., J. Bio. Chem., 286: 33511-33519 (2011) ; Guo et al., J. Acquir. Immune Defic. Syndr., 68: 502-510 (2015) ) . The size of full-length SARS-CoV-2 spike gene library is about 10 ⁶. The conditions of yeast culture and antigen expression induction were described previously (Zuo et al., J. Bio. Chem., 286: 33511-33519 (2011) ; Guo et al., J. Acquir. Immune Defic. Syndr., 68: 502-510 (2015) ; Chao et al., Nature Protocols, 1: 755-768 (2006) ) . Galactose induced yeast library cells (10 ⁶-10 ⁷ cells/test) were blocked by FACS buffer (2%FBS in PBS) for 30 min at 4℃ and then incubated with 2 μg screened anti-RBD (SARS-CoV-2) HuNAbs in 50 μL FACS buffer at 4℃ for 1h. After washing twice, yeasts were incubated with AF546-conjugated goat anti-human IgG secondary antibodies (Invitrogen) for another 1h at 4℃ (0.2 μg/test in 50 μL FACS buffer) . The fluorescence positive (PE channel) cells (50k-100k) were sorted into yeast culture media, re-cultured and re-induced by galactose. The induced yeasts were stained with the same antibodies at the same conditions. The sorted yeast single clones from the second round were verified by FACS. The displayed gene fragments in the plasmids of positive yeasts were amplified by yeast colony PCR and then sequenced. The sequence data was analyzed by the Sequencher 5.4.6. (GeneCodes Corp. ) .

Hamster experiments

In vivo evaluation of NAb ZDY20 in an established golden Syrian hamster model of SARS-CoV-2 infection was performed as described previously with slight modifications (Chan et al., Clinical Infectious Diseases (2020) ) . 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 obtained from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Laboratory Animal Unit and kept in Biosafety Level-2 (BSL-2) housing 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. For prophylaxis studies, 24 hours before live virus challenge, three groups of hamsters were intraperitoneally administered one dose of NAb ZDY20 in phosphate-buffered saline (PBS) at 10 mg/kg, 5 mg/kg, or control antibody VRC01 at 10 mg/kg. On Day 0, each hamster was intranasally inoculated with a challenge dose of 100 μL of Dulbecco’s Modified Eagle Medium containing 10 ⁵ PFU of SARS-CoV-2 (HKU-001a strain, GenBank accession no: MT230904.1) under intraperitoneal ketamine (200 mg/kg) and xylazine (10 mg/kg) anaesthesia. For treatment study, each hamster received one 10 mg/kg dose of intraperitoneal NAb ZDY20 at 24 hours, 48 hours, or 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 (Chan et al., Clinical Infectious Diseases (2020) ) . Accordingly, animals were sacrificed for analysis at day 4 after virus challenge. Half nasal turbinate, trachea, and lung tissues were used for viral load determination by quantitative SARS-CoV-2-specific RdRp/Hel reverse transcription-polymerase chain reaction assay (Chan et al., J. Clin. Microbiol., 58: 10.1128/JCM. 00310-20 (2020) ) and infectious virus titration by plaque assay (Chan et al., Clinical Infectious Diseases (2020) ) .

Histopathology and immunohistochemistry

The lung tissues collected at necropsy were fixed in zinc formalin, then processed into paraffin-embedded tissues blocks. The tissue sections in 4 μm were stained with haematoxylin and eosin (H&E) for light microscopy examination as previously described with modifications. For identification and localization of SARS-CoV-2 nucleocapsid protein (NP) in organ tissues, immunofluorescence staining was performed on deparaffinised and rehydrated tissue sections using rabbit anti-SARS-CoV-2-N protein antibody together with FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, PA, USA) . Briefly, the tissue sections were first treated with Antigen Unmasking Solution in pressure cooker (Vector Laboratories) . 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 antibody rabbit anti-SARS-CoV-2-N antibody (1: 4000 dilution with 1%BSA/PBS) was incubated at 4℃ overnight. This was followed by FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) for 30 min and then mounted with 4’ , 6-diamidino-2-phenylindole (DAPI) . All tissue sections were examined and the images were captured with Olympus BX53 semi-motorized fluorescence microscope using cellSens imaging software.

Statistics and reproducibility

Statistical analysis was performed using PRISM 6.0 or SPSS 26.0. Kruskal–Wallis test and Dunn’s multiple comparisons test were used to compare viral loads in nasal turbinate, trachea and lungs between different groups. Student’s t test was used to determine significant differences between the prophylaxis or treatment groups with the paired-wise control groups. When more than two groups were compared together, the ANOVA analysis was used. P value of <0.05 was considered statistically significant.

Results

Generation of a phage library displaying human antibody Fab of COVID-19 patients

In order to clone SARS-CoV-2-specific HuNAbs, we obtained peripheral blood mononuclear cells (PBMC) from 12 convalescent COVID-19 patients in Hong Kong at a mean duration of 19 (±10.4) days after symptom onset (Table 1) . These included 7 females and 5 males with a mean age of 59 years old (range, 21-75) . Two had severe disease, 7 had mild disease, and 3 were asymptomatic.

Table 1. Demographics of study subjects

The PBMCs of these 12 patients were pooled for the generation of a Fab phage library as only small amounts of PBMCs were obtainable from each patient. ELISA and pseudovirus neutralization assays were performed to measure the antibody titers in each patient’s serum prior to the pooling, which confirmed that each study subject had SARS-CoV-2 RBD-specific binding and neutralizing antibody activities (data not shown) . Consistent with the observations that we and others reported previously, the two severe patients had higher NAb titers than the mild and asymptomatic patients (Riva et al., Nature, 10.1038/s41586-41020-42577-41581 (2020) ; Wang et al., bioRxiv, 2020.2006.2013.150250 (2020) ; Liu et al., Emerging Microbes &Infections 9: 1664-1670 (2020) ) . The mean NAb IC ₅₀ titer was 1: 1753 with a range of 1: 638-1: 5701 (data not shown) . Using the pooled PBMCs, we first set up a Fab phage library consisting of 3×10 ⁶ clones (Fig. 6) . We then developed an in-solution selection method for two rounds of the panning (Fig. 1A) . We were able to pick up 384 single reactive colonies (Fig. 1B) . Next, we tested the binding ability of the phage-displayed-Fab to recombinant SARS-CoV-2 RBD by a monoclonal phage-based ELISA, followed by sequencing 18 single phage colonies that displayed strong RBD-binding ability. Finally, we obtained four pairs of VH/VL from the top four clones (Fig. 1B) , which resulted in four human monoclonal antibodies in the native form of IgG1, named ZDY20, ZDY28, ZDY49, and ZDY95.

Specificity and neutralizing activities of cloned human antibodies against SARS-CoV-2

Sequence analysis revealed that ZDY28, ZDY49 and ZDY95 share high similarity with the same IGHV3-30 and IGKV1-39 (Table 2) .

Table 2. Sequence analysis of four HuNAbs

They share identical CDR3 _H and CDR3 _L but with a few amino acid differences in framework region 1 (FR1) (Table 6) . In contrast, the fourth one namely ZDY20 is distinct from them by employing IGHV3-53 and IGKV1-33, which were recently shown to be associated with higher antiviral activity (Yuan et al., Science, eabd2321 (2020) ; Dalamaga et al., Metabolism 108: 154260 (2020) ) . ZDY20 has a longer CDR3 _H with three additional amino acids compared with other three antibodies (Table 6) .

Table 6. Amino acid sequence alignment of four HuNAbs VH and VK. ZDY28VH (SEQ ID NO: 1) , ZDY95VH (SEQ ID NO: 2) , ZDY49VH (SEQ ID NO: 3) , ZDY20VH (SEQ ID NO: 4) , ZDY28VK (SEQ ID NO: 5) , ZDY95VK (SEQ ID NO: 6) , ZDY49VK (SEQ ID NO: 7) , ZDY20VK (SEQ ID NO: 8) .

FR1 CDR1

ZDY28VH EVQLVQSGGGVVQPGRSLRLSCAAS GFTFSSYG

ZDY95VH......................... ........

ZDY49VH..... E................... ........

ZDY20VH..... ET... LIQ.. G......... . I.V.. NY

FR2 CDR2

ZDY28VH MHWVRQAPGKGLEWVAV ISYDGSNK

ZDY95VH................. ........

ZDY49VH................. ........

ZDY20VH. S............. S. . YSG.. T-

FR3

ZDY28VH YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC

ZDY95VH......................................

ZDY49VH......................................

ZDY20VH......................................

CDR3 Junction region

ZDY28VH ASRSTVVPAAIFAFDI--- WGQGTMVTVSS

ZDY95VH................ --- ...........

ZDY49VH................ --- ...........

ZDY20VH. REGLGDGYNAI. GLGFDL .. R.. L.....

FR1 CDR1

ZDY28VK AIRLTQSPSSLSASVGDRVTITCRAS QSISSY

ZDY95VK.. QM...................... ......

ZDY49VK D. QM...................... ......

ZDY20VK D. VM............. S..... Q.. . D. NN.

FR2 CDR2

ZDY28VK LNWYQQKPGKAPKLLIY AAS

ZDY95VK................. ...

ZDY49VK................. ...

ZDY20VK......... R.. E... N D..

FR3

ZDY28VK SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC

ZDY95VK....................................

ZDY49VK....................................

ZDY20VK N. EA....................... T........

CDR3 Junction region

ZDY28VK QQSYSTPYT FGQGTKLKIT

ZDY95VK......... ..........

ZDY49VK......... ......... K

ZDY20VK.. YG. L... ....... D. R

These four antibodies all had low rates of somatic hypermutation (SHM) in both IGHV (0.35%or 0.69%) and IGKV (0.36%or 1.08%) except that the SHM rate in IGKV of ZDY20 reached 7.89%. We next analysed the binding activity of these four antibodies to recombinant SARS-CoV-2 RBD and spike proteins by ELISA.

Binding profiles of four candidate HuNAbs (ZDY20, 28, 49 and 95) to viral RBD and to viral spike trimer were determined. HIV-1-specific HuNAb VRC01 served as a negative control. In these solid phase ELISA, ZDY28, ZDY49 and ZDY95 showed similar binding ability to RBD (EC ₅₀ 0.012 μg/ml) and to spike (EC ₅₀ 0.1 μg/ml) , which were lower than those of ZDY20 (EC ₅₀ 0.135 μg/ml and 0.52 μg/ml, respectively) (Table 3) . However, the anti-SARS-CoV-2 neutralization activity of ZDY20 (IC ₉₀ 1.24 μg/ml) was lower than those of ZDY28 (IC ₉₀ 7.47 μg/ml) , ZDY49 (IC ₉₀ 17.08 μg/ml) and ZDY95 (IC ₉₀ 13.35 μg/ml) in the pseudovirus assay (Table 3) . ZDY20 and ZDY28 were also tested against the live SARS-CoV-2. ZDY20 exhibited a slightly lower IC ₉₀ (1 μg/ml) than that of ZDY28 (1.28 μg/ml) against live SARS-CoV-2 in Vero-E6 cells (Table 3) . Both neutralization assays were done in triplicated wells.

Table 3. Neutralizing and ELISA binding activities of HuNAbs (μg/ml)

Notably, none of these HuNAbs showed cross-neutralization against the SARS-CoV pseudovirus (data not shown) . Using a random yeast surface display of SARS-CoV-2 spike fragments, we found that all four HuNAbs recognize conformational determinants instead of a small linear epitope because their binding domain on viral RBD required a minimal of 179 amino acid residues (337-524 for ZDY20, 336-523 for ZDY28, and 336-515 for ZDY49 and ZDY95) (Fig. 7) . None of these four HuNAbs recognizes a small linear epitope. We then measured the binding affinity of each antibody to SARS-CoV-2 spike trimer using the surface plasmon resonance (SPR) . Interestingly, unlike its weaker binding activity by ELISA, ZDY20 displayed the highest affinity binding to SARS-CoV-2 spike trimer with a KD value of 8.8 nM, which was lower than those of other three antibodies (ZDY28 10.2 nM, ZDY49 24.4 nM and ZDY95 15 nM) (Table 4) . Furthermore, in the human cellular receptor ACE2 competition assay by SPR, ZDY20 presented the most potent activity followed by ZDY95, ZDY28 and ZDY49. Distinct binding patterns of ACE2 to the spike protein with or without prior incubation were observed with each testing HuNAb. These results suggested that the higher anti-SARS-CoV-2 neutralizing potency of ZDY20 is likely due to its stronger competitive blockade of RBD binding to ACE2.

SARS-CoV-2 infects nasal turbinate robustly despite systemic ZDY20 prophylaxis

To determine the potential role of HuNAb as prophylaxis for SARS-CoV-2 infection, we administered ZDY20 intraperitoneally to golden Syrian hamsters before virus challenge in our Biosafety Level-3 (BSL-3) animal laboratory. Syrian hamsters typically recover from SARS-CoV-2 infection with resolution of clinical signs and clearance of virus shedding within one week after infection as we previously described (Chan et al., Clinical Infectious Diseases (2020) ) . Accordingly, the hamsters were sacrificed for analysis at 4 days post-infection (dpi) when high viral loads and acute lung injury were consistently observed. In this prophylaxis study, each hamster received a single intraperitoneal injection of either 10 mg/kg (n=4) high dose or 5 mg/kg (n=3) low dose of ZDY20 (Fig. 2A) , which were about 10,000-fold or 5,000-fold higher than its IC ₉₀ value of 1 μg/ml tested by the live SARS-CoV-2 in Vero-E6 cells, respectively. Another group of hamsters (n=4) were injected with a control HIV-1-specific HuNAb VRC01 at the high dose of 10 mg/kg (Liu et al., Nature 584: 450-456 (2020) ) . Twenty-four hours after the ZDY20 injection, each animal was challenged intranasally with 10 ⁵ plaque forming units (PFU) of live SARS-CoV-2 (HKU-001a strain) (Liu et al., Nature 584: 450-456 (2020) ; Chan et al., Clinical Infectious Diseases (2020) ) . At 4 dpi, nasal turbinate, trachea and lung tissues were harvested to quantify infectious viruses by plaque assay, viral loads by real-time reverse transcription polymerase chain reaction (RT-PCR) and infected cells by immunoflouresence staining (IF) . We found that infectious plaque-forming units were readily detected in all tissue compartments of VRC01 controlled hamsters tested but not in 3/4 and 0/3 nasal turbinate, 3/4 and 2/3 trachea, and 2/4 and 1/3 lungs of hamsters that were given the high and the low doses of ZDY20, respectively (Fig. 2B) . The more sensitive RT-PCR further revealed that viral RNA copy numbers were reduced in lungs by an average of 3 logs (range, 0.7-4.5) (Fig. 2C) . In contrast, there was no statistically significant viral load reduction in nasal turbinate and trachea in both dose groups, suggesting less preventive efficacy by ZDY20 in upper respiratory portal of viral entry. To determine the role of ZDY20 in protection, we measure antibody concentration and neutralization titer at 0 dpi and 4 dpi. Over 34 μg/ml and 9 μg/ml ZDY20 were found in 3/4 high dose and 2/3 low dose animals at 0 dpi (Fig. 2D, solid symbol and Table 5) , together with mean IC ₅₀ values around 236 and 99, respectively (Fig. 2E, solid symbol) . On 4 dpi, over 26 μg/ml and 5 μg/ml ZDY20 were found in 3/4 high dose and 2/3 low dose animals (Fig. 2D, open symbol and Table 5) , together with mean IC ₅₀ values around 55 and 45, respectively (Fig. 2E, open symbol) . These results demonstrated that most animals maintained peripheral amounts of ZDY20 higher than its IC ₉₀ values during the entire course of experiment.

Table 5. Average concentration of ZDY20 and neutralizing titer in hamster plasma

Next, to understand if ZDY20 protects infection-induced lung injury in hamsters, we performed pathological analysis on lung specimens. Images of hamster lung tissues were visualized by H&E and IF (data not shown) . In control animals, H&E staining showed large patchy alveolar wall and alveolar space infiltration and exudation. Some blood vessels in the lung section showed vasculitis and endotheliitis. In high dose ZDY20-treated hamsters, the lung tissues showed no apparent peribronchiolar infiltration, and the bronchiolar epithelium appeared normal. Alveoli showed mild septal infiltration and congestion. In low dose ZDY20-treated hamster, some bronchiolar epithelial cells were swollen and detached with mild peribronchiolar infiltration and mild alveolar septal infiltration. For viral NP antigen in the sections (50×) stained by IF, the lung of control hamster showed diffuse NP expression in large areas of alveoli and in bronchiolar epithelium. No NP expression was seen in lung sections of hamster treated by high dose antibody (10 mg/ml) . In low dose antibody treated hamster, NP expression was occasionally observed in small area of alveoli and in the epithelium of a few bronchiolar sections.

As compared with uninfected healthy animals, hamsters treated with the high dose ZDY20 showed mild interstitial alveoli inflammation with minor septal infiltration and congestion. There was no apparent peribronchiolar infiltration and the bronchiolar epithelia appeared normal. Hamsters treated with the low dose showed limited areas of bronchiolar epithelial cell swelling and detachment, together with mild peribronchiolar infiltration and mild alveolar septal infiltration. In contrast, control hamsters showed large patchy areas of alveolar wall and alveolar space involvement by inflammatory infiltrates and exudation. IF staining of viral NP antigen in both lung and nasal turbinate tissues as compared with uninfected healthy animals. In the high dose group, few viral NP expression was seen in lung sections. In the low dose group, small amount of viral NP was observed in localized areas of alveoli and in the epithelia of a few bronchiolar sections. In contrast, control hamsters showed diffuse NP expression in extensive areas of alveoli and in the bronchiolar epithelia. The mean number of NP ⁺ cells per 50x field in lungs was significantly lower in the high-dose group than that in control animals (Fig. 2F) . Unexpectedly, nasal turbinate showed robust and diffuse viral NP expression in extensive areas lining the stratified squamous epithelia in both high dose and low dose groups of ZDY20-treated animals similar to untreated control hamsters (data not shown) . Images of IF staining showed the abundancy and distribution of NP ⁺ cells in nasal turbinate of each control animal. SARS-CoV-2 N protein (NP) was stained green and cell nuclei were counter-stained by DAPI. Images of IF staining showed the abundancy and distribution of NP ⁺ cells in nasal turbinate of each animal that received the high dose of 10 mg/kg ZDY20. Images of IF staining showed the abundancy and distribution of NP ⁺ cells in nasal turbinate of each animal that received the low dose of 5mg/kg ZDY20. Moreover, no significant difference in NP ⁺ cells per 50x field was found between treated and control animals when nasal turbinate tissues were analyzed (Fig. 3) . Collectively, these results demonstrated that prior administration of even 10 mg/ml ZDY20 did not prevent robust SARS-CoV-2 infection in nasal turbinate but suppressed productive infection in lungs in the hamster model.

Post-challenge ZDY20 therapy reduces SARS-CoV-2 infection in lungs but much less in nasal turbinate

To explore the therapeutic potential of ZDY20, we infected four groups of hamsters (n=4 per group) as described above. Three groups of hamsters were then treated intraperitoneally with a single high dose of 10 mg/kg at 1, 2 and 3 dpi, respectively (Fig. 4A) . The fourth group was VRC01 control using the same dose at 1 dpi. All animals were consistently sacrificed on 4 dpi for measurements of infectious virus titres and viral RNA copy numbers in lung, trachea and nasal turbinate tissues (Figs. 4B and 4C) . At 1 dpi, ZDY20-treated hamsters showed no measurable infectious viruses in 4/4 nasal turbinate, 4/4 trachea and 3/4 lungs by the plaque assay (Fig. 4B, circles) . The mean viral load reduction was only 0.7 logs in both nasal turbinate (range, 0.1-1.0 logs) (p<0.05) and trachea (range, 0.3-1.5 logs) , but was 1.2 logs (range, 0.8-1.8 logs) in lungs (p<0.01) as compared with the control group (Fig. 4C, circles) . At 2 dpi, ZDY20-treated hamsters presented no measurable infectious viruses in 3/4 nasal turbinate, 3/4 trachea and 2/4 lungs (Fig. 4B, squares) . The mean viral load reduction was 1 log (range, 0.3-1.5logs) in trachea (p<0.05) , but not significantly in nasal turbinate and in lungs as compared with the control group (Fig. 4C, squares) . At 3 dpi, ZDY20-treated hamsters had no measurable infectious viruses in 1/4 nasal turbinate, 3/4 trachea and 1/4 lungs (Fig. 4B, triangles) . The mean viral load reduction was not significant in nasal turbinate, trachea and in lungs as compared with the control group (Fig. 4C, triangles) . These kinetics results demonstrated that earlier commencement of treatment within 2 dpi resulted in more significant reduction of infectious virus. For the significant viral load reduction, commencement of treatment within 1 dpi is likely necessary. To determine the role of ZDY20 in protection, we measure antibody concentration and neutralization titer at 4 dpi. Over 40 μg/ml ZDY20 were found in 4/4 treated animals at 1 dpi, 3/4 treated animals at 2 dpi and 2/4 treated animals at 3 dpi (Fig. 4D and Table 5) , together with mean IC ₅₀ values of 169, 62 and 69 in each corresponding group (Fig. 4E) , respectively.

Subsequently, we performed pathological analysis on lung specimens to understand if ZDY20 protects against tissue injury in hamsters. Images of hamster lung tissues were visualized by H&E and IF (data not shown) . In control animals, H&E staining results showed peribronchiolar infiltration and bronchiolar epithelial cell death, diffuse alveolar wall thickening, patchy areas of alveolar space infiltration and exudation. Ocasional blood vessels showed vasculitis. In ZDY20-treated hamsters, all the lung tissues showed much milder inflammation. There was no apparent peribronchiolar infiltration. The bronchiolar epithelium appeared normal with occasionally observed cell death. Alveolar structure showed moderate degree of alveolar wall thickening and capillary congestion. No alveolar space infiltration or exudation and limited vasculitis were observed. For IF stained viral NP antigen in the sections (50×) , the lungs of control hamsters showed NP expression in bronchiolar epithelium and diffuse NP expression in large areas of alveoli. In ZDY20-treated hamsters, NP expression was much confined to small area of alveoli or in the epithelia of bronchioles.

The H&E staining sections of control hamsters showed acute lung injury with peribronchiolar infiltration, bronchiolar epithelial cell death, diffuse alveolar wall thickening, patchy areas of alveolar space infiltration, exudation and vasculitis. In contrast, less severe histopathological changes were observed in all 3 groups of ZDY20-treated hamsters. There was no apparent peribronchiolar infiltration in hamsters treated at 1 dpi. The bronchiolar epithelia appeared normal with only occasionally observed cell death in hamsters treated at 2 dpi. Moderate alveolar wall thickening and capillary congestion without alveolar space infiltration or exudation and limited vasculitis were observed in hamsters treated at 3 dpi. Corroboratively, abundant SARS-CoV-2 NP expression in the bronchiolar epithelium and alveoli was observed in lungs of control hamsters. In ZDY20-treated hamsters, NP expression was confined to small areas of alveoli at 1 dpi and was more frequently found in the bronchiolar epithelia at 2 dpi and 3 dpi. The mean number of NP ⁺ cells per 50×field of lung tissues at 1 dpi was significantly lower than that of the control group (Fig. 4F) . Furthermore, when we performed IF staining of viral NP antigen in nasal turbinate tissues, robust and diffuse viral NP expression in extensive areas lining the stratified squamous epithelia were found in infected hamsters treated with 10 mg/ml ZDY20 either at 1 dpi or 3 dpi similar to untreated control hamsters. Abundant NP ⁺ cells were observed in nasal turbinate of presentative hamsters treated at 1dpi and 3dpi as compared with the control animal. SARS-CoV-2 N protein (NP) was stained green and cell nuclei were counter-stained by DAPI. Collectively, these results demonstrated that post-challenge treatment with 10 mg/ml ZDY20 suppressed robust SARS-CoV-2 infection in lungs but not in nasal turbinate in the hamster model. Lastly, when we included all preventive and treated animals for analysis, the amount of peripheral ZDY20 was correlated negatively with NP ⁺ cells in lungs (Fig. 5) but not in nasal turbinate (data not shown) .

Discussion

SARS-CoV-2 is characterized by a burst in upper-respiratory portal for high transmissibility. To obtain human neutralizing antibodies (HuNAbs) for entry protection, HuNAbs that bind to conformational determinants of viral receptor binding domain (RBD) were generated. HuNAb ZDY20 prevented pseudovirus and live virus entry with IC ₉₀ values of 1.24 μg/ml and 1 μg/ml, respectively, by competing with human cellular receptor ACE2 for RBD binding. Prophylactic intraperitoneal injection of 10mg/ml ZDY20 significantly reduced infection in lungs but not nasal turbinate of hamsters intranasally-challenged with SARS-CoV-2. Although post-challenge ZDY20 therapy suppressed viral loads and lung damage, robust infection was found in nasal turbinate treated within 1-3 days. These results demonstrated that systemic HuNAb suppresses SARS-CoV-2 replication and lung injury.

ZDY20 represents the most promising class of HuNAbs that bind to the conformational determinants of the SARS-CoV-2 RBD overlapping with the cellular entry receptor ACE2 binding site (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) ) . Both SARS-CoV-2 and SARS-CoV use ACE2 to initiate infection, despite that they share only 40%amino acid identity in the RBD external subdomain (Zhou et al., Immunity 53: 1-14 (2020) ; Wan et al., J. Virol., e00127-00120 (2020) ; Chan et al., Emerg. Microbes Infect. 9: 221-236 (2020) ; Lan et al., Nature 581: 215-220 (2020) ) . Structural analysis indicated that residues in the SARS-CoV-2 RBD that are essential for ACE2 binding are highly conserved or share similar side chain properties with those in the SARS-CoV RBD (Lan et al., Nature 581: 215-220 (2020) ) . During the natural course of infection, there was cross-reactivity between antibodies to SARS-CoV-2 and SARS-CoV, but only antibodies from COVID-19 patients neutralized SARS-CoV-2 (Casadevall et al., J. Clin. Invest. (2020) ) . Critically, most SARS-CoV-2 HuNAbs primarily target distinct RBD epitopes overlapping with the ACE2 binding site (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) ) . This feature is critical because the ability of HuNAbs to compete with ACE2 for binding with the viral RBD often reflects their neutralizing potency. In particular, the potency of RBD-specific HuNAbs is higher than those targeting the N terminal domain (NTD) (Liu et al., Nature 584: 450-456 (2020) ; Chi et al., Science 369: 650-655 (2020) ; Rogers et al., Science 369: 956-963 (2020) ) . These results corroborate with our previous findings on SARS-CoV in that the RBD contains the major antigenic determinants for inducing potent NAbs (Chen et al., J. Virol. 79: 2678-2688 (2005) ; Yi et al., J. Virol. 79: 11638-11646 (2005) ) . In this study, we consistently found that the RBD-specific ZDY20 displayed stronger neutralizing activity through better competition for the ACE2 binding site. Our in vivo analysis of ZDY20, therefore, should have important implications for other HuNAbs that have similar functional properties and are now under clinical development (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) ) .

In our SARS-CoV-2-infected hamsters, we showed that systemic ZDY20 prophylaxis did not significantly prevent viral infection in the nasal turbinate tissues. Several potent RBD-specific HuNAbs have been evaluated for SARS-CoV-2 prevention in various animal models (Shi et al., Nature 584: 120-124 (2020) ; Zost et al., Nature 584: 443-449 (2020) ; Liu et al., Nature 584: 450-456 (2020) ; Rogers et al., Science 369: 956-963 (2020) ) . Most of these studies, however, have focused on viral load reduction in the lungs without detailed examination of the viral replication in upper respiratory tract tissues. Using a macaque model for SARS-CoV, we previously demonstrated that epithelial cells lining salivary gland ducts are early target cells of SARS-CoV infection in the upper respiratory tract (Liu et al., J. Virol. 85: 4025-4030 (2011) ) , and such infection may serve as a critical founder portal for systemic viral dissemination in vivo (Liu et al., Mucosal. Immunol. 9: 1089-1101 (2016) ) . During SARS-CoV-2 infection, viral replication in the upper respiratory tract and shedding into salivary droplets have implications not only for the rapid dissemination of the pandemic, but also for the justified use of deep throat saliva as a non-invasive and rapid way of diagnosis (To et al., Lancet Infect Dis 20: 565-574 (2020) ) . Since COVID-19 patients have the highest viral load and viral shedding in saliva at or soon after presentation, effective inhibition of SARS-CoV-2 replication in the upper respiratory tract would be critical for optimized infection control. Based on this consideration, we tested the effects of ZDY20 as a prophylaxis at a high dose of 10 mg/ml, which is 10,000-fold higher than its in vitro IC ₉₀ value of 1 μg/ml. The mean viral loads were significantly reduced in the lungs but not in the nasal turbinate and trachea, although serum neutralization IC ₅₀ values maintained over 1: 200 at the time of virus challenge and over 1: 50 at the time of animal sacrifice, respectively. At the latter time point of 4 dpi, a large number of infected NP ⁺ cells were detected in the nasal turbinate tissues. These results indicated that systemic administration of 10 mg/ml ZDY20 remained insufficient to achieve complete protection against SARS-CoV-2 replication in upper respiratory tract. Similarly, a recent preprint article indicated that SARS-CoV-2 viral loads were detectable in nasopharyngeal swabs in monkeys that received at -3 dpi a high dose of 50 mg/kg of the REGN-COV2 antibody cocktail (Baum et al., bioRxiv, doi. org/10.1101/2020.1108.1102.233320 (2020) ) . Moreover, an intraperitoneal dose of 50 mg/kg treatment with the RBD-specific CB6 antibody that also has an IC ₉₀ value of 1 μg/ml reduced but did not completely prevent histopathological changes in the lungs of SARS-CoV-2-infected monkeys (Shi et al., Nature 584: 120-124 (2020) ) . Collectively, these data highlighted the urgent need to explore the use of mucosal immunity (e.g. sIgA) in the prevention of SARS-CoV-2 infection as sterile protection could not be achieved despite such high doses of systemic HuNAb. Mucosal protection is a serious challenge because one-third of convalescent COVID-19 patients had serum IC ₅₀ values less than 1: 50 (Robbiani et al., Nature 584: 437-442 (2020) ) , and therefore, they may be susceptible to re-infection. Indeed, we have recently documented the world’s first reported case of SARS-CoV-2 re-infection in a Hong Kong patient, whose serum NAb titer decreased from 1: 40 to <1: 10 within 140 days after an acute asymptomatic infection, had virus shedding in the upper respiratory tract due to reinfection by another strain of SARS-CoV-2 (To et al., Clin. Infect. Dis., ciaa1275 (2020) ) .

In our post-challenge treatment kinetics study, we showed that early use of ZDY20, preferably within 2 dpi, suppressed infectious virus, viral loads and acute lung injury in the hamsters. Several studies have evaluated the potency of RBD-specific HuNAbs for treating pre-infected mice, hamsters and monkeys (Shi et al., Nature 584: 120-124 (2020) ; Cao et al., Cell 182: 73-84 e16 (2020) ; Wu et al., Science 368: 1274-1278 (2020) ; Baum et al., bioRxiv, 10. 1101/2020. 1108. 1102. 233320 (2020) ) , yet few performed a treatment kinetics study. In hACE2-transgenic mice, a 20 mg/kg injection of BD-368-2 at 2 hours post infection (hpi) reduced the lung viral loads by about 3 logs (Cao et al., Cell 182: 73-84 e16 (2020) ) . In another study, after a 25 mg/kg dose of a noncompeting pair of HuNAbs given at 12 hpi, viral loads were reduced for over 2 logs yet there were still 10 ⁶ copies/g of lung tissues at 3 days after virus challenge in hACE2-transgenic mice (Wu et al., Science 368: 1274-1278 (2020) ) . In monkey models, a preprint article indicated that SARS-CoV-2 viral loads were detected in nasopharyngeal swabs at 4 dpi in animals that received a very high dose of 150 mg/kg of the REGN-COV2 antibody cocktail at 1 dpi (Baum et al., bioRxiv, 10. 1101/2020. 1108. 1102.233320 (2020) ) . Besides these HuNAb studies, for convalescent plasma therapy, we previously reported that convalescent sera of infected hamsters reduced SARS-CoV-2 replication but not acute lung injury (Chan et al., Clinical Infectious Diseases (2020) ) . Since the mortality rate of critically ill COVID-19 patients might be related to the IgG antibody levels in the transfused convalescent plasma (Joyner et al., medRxiv, doi. org/10. 1101/2020. 08. 12. 20169359 (2020) ) , it might be necessary to develop high-titer HuNAbs as specific antivirals for COVID-19 early treatment. In this study, by initiating ZDY20 treatment at the different time points of 1 dpi, 2 dpi and 3 dpi, we demonstrated the benefits of commencing HuNAb treatment as early as possible in terms of better viral load reduction and alleviation of acute lung injury. Nevertheless, treatment started as late as 3 dpi still resulted in better histopathological changes in the lungs, and therefore should still be considered in COVID-19 patients who presented later in the course of disease as long as the HuNAb does not promote acute lung injury (Liu et al., JCI Insight 4: e123158 (2019) ) .

In summary, we report robust SARS-CoV-2 infection in nasal turbinate in the presence of potent systemic neutralizing antibodies in both prophylaxis and therapeutic studies in golden Syrian hamsters. Apparently, RBD-specific HuNAb ZDY20 at the concentration of 10,000-fold higher than its IC ₉₀ value is insufficient for sterile protection of the upper respiratory tract. Despite the presence of systemic serum ZDY20, high viral loads and infected cells as well as some infectious viruses may persist at least for four days in nasal turbinate. Nevertheless, systemic ZDY20 is able to suppress SARS-CoV-2 replication and acute injury in lungs of experimental hamsters. These findings have important implications to viral transmission, patient management, vaccine development and control of re-infection.

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

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

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

(4) the three light chain CDRs of SEQ ID NO: 7 and the three heavy chain CDRs of SEQ ID NO: 3, 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: 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 2, wherein the three heavy chain CDRs comprise a first heavy chain CDR comprising amino acids 26-33 of SEQ ID NO: 4, a second heavy chain CDR comprising amino acids 51-57 of SEQ ID NO: 4, and a third heavy chain CDR comprising amino acids 96-114 of SEQ ID NO: 4.
The antibody or antigen binding fragment thereof of one of claims 1-3 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 1-4 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, 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 6, 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 97-112 of SEQ ID NO: 1.
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: 5.
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: 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 27-32 of SEQ ID NO: 6, a second light chain CDR comprising amino acids 50-52 of SEQ ID NO: 6, and a third light chain CDR comprising amino acids 89-97 of SEQ ID NO: 6.
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: 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, 10, or 11 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 10-12 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 14, 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 97-112 of SEQ ID NO: 3.
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: 7.
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: 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: 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 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 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: 8, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4,

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, or

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.
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 method 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.