WO2023125964A1

WO2023125964A1 - Neutralizing antibodies against covid-19 and methods of use thereof

Info

Publication number: WO2023125964A1
Application number: PCT/CN2022/144066
Authority: WO
Inventors: Zhiwei Chen; Runhong ZHOU; Biao ZHOU; Mengxiao LUO; Bohao CHEN
Original assignee: Versitech Limited; Centre For Virology, Vaccinology And Therapeutics Limited
Priority date: 2022-01-03
Filing date: 2022-12-30
Publication date: 2023-07-06

Abstract

Disclosed are compositions and methods using antibodies and antibody fragments that bind SARS-CoV-2 spike protein or receptor binding domain (RBD). Generally, the CDRs of the antibodies and antigen binding fragments comprise: (a) the three light chain CDRs selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, and 64; and (b) the three light chain CDRs selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, and 63.In some forms, the antibody is a bispecific antibody.

Description

NEUTRALIZING ANTIBODIES AGAINST COVID-19 AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Application No. 63/296,089, filed on January 3, 2022, U.S. Provisional Application No. 63/301,755, filed on January 21, 2022, and U.S. Provisional Application No. 63/309,492, filed on February 11, 2022, which are incorporated by reference herein in their entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted as an xml file named “UHK_01167_PCT. xml, ” created on December 20, 2022, and having a size of 64, 265 bytes is hereby incorporated by reference pursuant to 37 C. F. R. § 1.834 (c) (1) .

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

After two years of the COVID-19 pandemic, the highly transmissible SARS-CoV-2 and its variant of concerns (VOCs) have resulted in more than 279 million infections with 5.4 million deaths globally by December 26, 2021 (internet site coronavirus. jhu. edu/map. html) . During this period, various types of COVID-19 vaccines have been quickly developed to control the pandemic with over 8.9 billion doses administered in many countries. Although the extensive implementation of vaccination has significantly reduced the rates of hospitalization, severity and death (Baden et al., 2021; Polack et al., 2020; Tanriover et al., 2021; Voysey et al., 2021; Xia et al., 2021) , current vaccines do not confer complete or durable prevention of upper airway transmission of SARS-CoV-2. The numbers of vaccine-breakthrough infections and re-infections, therefore, have been continuously increasing (Abu-Raddad et al., 2021; Birhane et al., 2021; To et al., 2021) . The pandemic situation has been complicated by repeated emergence of new VOCs, including Alpha (B. 1.1.7) , Beta (B. 1.351) , Gamma (P.1) , Delta (B. 1.617.2) and Omicron (B. 1.1.529) (Khan et al., 2021; Tao et al., 2021) , and waning of vaccine-induced immune responses, together with relaxed preventive masking and social distancing (Qiaoli Peng, 2021; Wang et al., 2021d; Zhang et al., 2021) .

After the World Health Organization (WHO) designated the Omicron as a VOC on November 26, 2021, this variant has been quickly found in over 110 countries and is replacing the Delta VOC within a month, becoming the dominant VOC in many places in the South Africa, European countries, and the United States (Frederic Grabowski, 2021; WHO, 2021) . According to the GASAID database, for example, the relative variant genome frequency of the current circulating Delta VOC has declined from 89%to 19.6%while the Omicron VOC has increased from 0%to 67.4%in African countries during the period from October 4, 2021, to December 26, 2021. The rapid global spread of the Omicron VOC has been associated with vaccine-breakthrough infections and re-infections (Espenhain et al., 2021; Pulliam et al., 2021) . Moreover, like previous findings that the Beta VOC compromised vaccine-induced neutralizing antibody (NAb) (Planas et al., 2021b; Zhang et al., 2021; Zhou et al., 2021b) , the Omicron VOC has resulted in even worse NAb evasion due to more than 30 alarming mutations in SARS-CoV-2 spike glycoprotein (Cameroni et al., 2021; Cele et al., 2021; Liu et al., 2021b; Planas et al., 2021a) . Considering that current NAb combination for clinical immunotherapy showed significantly reduced activities (Liu et al., 2021b; Lu et al., 2021) , there is a need for vaccine-induced broadly neutralizing antibody (bNAbs) among elite vaccinees.

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) or S1 subunit (S1) . In particular, disclosed are antibodies and antigen binding fragments thereof comprising six complementarity determining regions (CDRs) .

In some forms, the CDRs can comprise: (a) the three light chain CDRs and the three heavy chain CDRs of antibody ZCB11, (b) the three light chain CDRs and the three heavy chain CDRs of antibody ZCB3, (c) the three light chain CDRs and the three heavy chain CDRs of antibody ZCC10, or (d) the three light chain CDRs and the three heavy chain CDRs of antibody ZCD3, where the antibody or antigen binding fragment thereof binds to SARS-CoV-2 RBD. In some forms, the CDRs can comprise the three light chain CDRs and the three heavy chain CDRs of antibody ZCD4, where the antibody or antigen binding fragment thereof binds to SARS-CoV-2 S1.

In some forms, the CDRs can comprise: (a) the three light chain CDRs of SEQ ID NO: 4 and the three heavy chain CDRs of SEQ ID NO: 3, (b) the three light chain CDRs of SEQ ID NO: 2 and the three heavy chain CDRs of SEQ ID NO: 1, (c) the three light chain CDRs of SEQ ID NO: 6 and the three heavy chain CDRs of SEQ ID NO: 5, or (d) the three light chain CDRs of SEQ ID NO: 8 and the three heavy chain CDRs of SEQ ID NO: 7, where the antibody or antigen binding fragment thereof binds to SARS-CoV-2 RBD. In some forms, the CDRs can comprise the three light chain CDRs of SEQ ID NO: 9 and the three heavy chain CDRs of SEQ ID NO: 10, where the antibody or antigen binding fragment thereof binds to SARS-CoV-2 S1.

In some forms, the antibody or antigen binding fragment thereof comprise the light chain variable region of antibody ZCB11. 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: 4. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody ZCB11. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3. In some forms, the antibody or antigen binding fragment thereof comprise the light chain variable region of antibody ZCB11 and a heavy chain variable region of antibody ZCB11. 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: 4 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3. In some forms, the antibody or antigen binding fragment thereof comprises the antibody ZCB11.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody ZCB3. 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: 2. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody ZCB3. 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 the light chain variable region of antibody ZCB3 and a heavy chain variable region of antibody ZCB3. 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: 2 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 the antibody ZCB3.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody ZCC10. 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 of antibody ZCC10. 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: 5. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody ZCC10 and a heavy chain variable region of antibody ZCC10. 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: 5. In some forms, the antibody or antigen binding fragment thereof comprises the antibody ZCC10.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody ZCD3. 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 of antibody ZCD3. 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: 7. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody ZCD3 and a heavy chain variable region of antibody ZCD3. 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: 7. In some forms, the antibody or antigen binding fragment thereof comprises the antibody ZCD3.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody ZCD4. 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: 10. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody ZCD4. 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: 9. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody ZCD4 and a heavy chain variable region of antibody ZCD4. 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: 10 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9. In some forms, the antibody or antigen binding fragment thereof comprises the antibody ZCD4.

Also disclosed are humanized antibodies or antigen binding fragments thereof comprising one or more human IgG4 constant domains and (a) the light chain variable region and heavy chain variable region of antibody ZCB11, (b) the light chain variable region and heavy chain variable region of antibody ZCB3, (c) the light chain variable region and heavy chain variable region of antibody ZCC10, or (d) the light chain variable region and heavy chain variable region of antibody ZCD3.

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

In some forms, the CDRs can comprise: (a) the three light chain CDRs and the three heavy chain CDRs of antibody P1D9, (b) the three light chain CDRs and the three heavy chain CDRs of antibody P2B4, (c) the three light chain CDRs and the three heavy chain CDRs of antibody P2B11, (d) the three light chain CDRs and the three heavy chain CDRs of antibody P2D9, (e) the three light chain CDRs and the three heavy chain CDRs of antibody P2E7, (f) the three light chain CDRs and the three heavy chain CDRs of antibody P1D6, (g) the three light chain CDRs and the three heavy chain CDRs of antibody P1E7, (h) the three light chain CDRs and the three heavy chain CDRs of antibody P1F3, (i) the three light chain CDRs and the three heavy chain CDRs of antibody P1F8, (j) the three light chain CDRs and the three heavy chain CDRs of antibody P2B10, (k) the three light chain CDRs and the three heavy chain CDRs of antibody P2C2, (l) the three light chain CDRs and the three heavy chain CDRs of antibody P2D4, (m) the three light chain CDRs and the three heavy chain CDRs of antibody P2E2, (n) the three light chain CDRs and the three heavy chain CDRs of antibody P2E6, (o) the three light chain CDRs and the three heavy chain CDRs of antibody P2E10, (p) the three light chain CDRs and the three heavy chain CDRs of antibody P2F2, (q) the three light chain CDRs and the three heavy chain CDRs of antibody P3B2, (r) the three light chain CDRs and the three heavy chain CDRs of antibody P3B7, (s) the three light chain CDRs and the three heavy chain CDRs of antibody P3B11, (t) the three light chain CDRs and the three heavy chain CDRs of antibody P3C7, (u) the three light chain CDRs and the three heavy chain CDRs of antibody P3C11, (v) the three light chain CDRs and the three heavy chain CDRs of antibody P3D2, (w) the three light chain CDRs and the three heavy chain CDRs of antibody P3D10, (x) the three light chain CDRs and the three heavy chain CDRs of antibody P3E2, (y) the three light chain CDRs and the three heavy chain CDRs of antibody P3E4, (z) the three light chain CDRs and the three heavy chain CDRs of antibody P3E6, or (aa) the three light chain CDRs and the three heavy chain CDRs of antibody P3E9, where the antibody or antigen binding fragment thereof binds to SARS-CoV-2 spike protein.

In some forms, the CDRs can comprise: (a) the three light chain CDRs of SEQ ID NO: 12 and the three heavy chain CDRs of SEQ ID NO: 11, (b) the three light chain CDRs of SEQ ID NO: 14 and the three heavy chain CDRs of SEQ ID NO: 13, (c) the three light chain CDRs of SEQ ID NO: 16 and the three heavy chain CDRs of SEQ ID NO: 15, (d) the three light chain CDRs of SEQ ID NO: 18 and the three heavy chain CDRs of SEQ ID NO: 17, the three light chain CDRs of SEQ ID NO: 20 and the three heavy chain CDRs of SEQ ID NO: 19, the three light chain CDRs of SEQ ID NO: 22 and the three heavy chain CDRs of SEQ ID NO: 21, the three light chain CDRs of SEQ ID NO: 24 and the three heavy chain CDRs of SEQ ID NO: 23, the three light chain CDRs of SEQ ID NO: 26 and the three heavy chain CDRs of SEQ ID NO: 25, the three light chain CDRs of SEQ ID NO: 28 and the three heavy chain CDRs of SEQ ID NO: 27, the three light chain CDRs of SEQ ID NO: 30 and the three heavy chain CDRs of SEQ ID NO: 29, the three light chain CDRs of SEQ ID NO: 32 and the three heavy chain CDRs of SEQ ID NO: 31, the three light chain CDRs of SEQ ID NO: 34 and the three heavy chain CDRs of SEQ ID NO: 33, the three light chain CDRs of SEQ ID NO: 36 and the three heavy chain CDRs of SEQ ID NO: 35, the three light chain CDRs of SEQ ID NO: 38 and the three heavy chain CDRs of SEQ ID NO: 37, the three light chain CDRs of SEQ ID NO: 40 and the three heavy chain CDRs of SEQ ID NO: 39, the three light chain CDRs of SEQ ID NO: 42 and the three heavy chain CDRs of SEQ ID NO: 41, the three light chain CDRs of SEQ ID NO: 44 and the three heavy chain CDRs of SEQ ID NO: 43, the three light chain CDRs of SEQ ID NO: 46 and the three heavy chain CDRs of SEQ ID NO: 45, the three light chain CDRs of SEQ ID NO: 48 and the three heavy chain CDRs of SEQ ID NO: 47, the three light chain CDRs of SEQ ID NO: 50 and the three heavy chain CDRs of SEQ ID NO: 49, the three light chain CDRs of SEQ ID NO: 52 and the three heavy chain CDRs of SEQ ID NO: 51, the three light chain CDRs of SEQ ID NO: 54 and the three heavy chain CDRs of SEQ ID NO: 53, the three light chain CDRs of SEQ ID NO: 56 and the three heavy chain CDRs of SEQ ID NO: 55, the three light chain CDRs of SEQ ID NO: 58 and the three heavy chain CDRs of SEQ ID NO: 57, the three light chain CDRs of SEQ ID NO: 60 and the three heavy chain CDRs of SEQ ID NO: 59, the three light chain CDRs of SEQ ID NO: 62 and the three heavy chain CDRs of SEQ ID NO: 61, or the three light chain CDRs of SEQ ID NO: 64 and the three heavy chain CDRs of SEQ ID NO: 63, where the antibody or antigen binding fragment thereof binds to SARS-CoV-2 spike protein.

In some forms, the antibody or antigen binding fragment thereof comprise the light chain variable region of antibody P1D9. 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: 12. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P1D9. 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: 11. In some forms, the antibody or antigen binding fragment thereof comprise the light chain variable region of antibody P1D9 and a heavy chain variable region of antibody P1D9. 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: 12 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P1D9.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2B4. 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: 14. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2B4. 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: 13. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2B4 and a heavy chain variable region of antibody P2B4. 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: 14 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2B4.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2B11. 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: 16. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2B11. 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: 15. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2B11 and a heavy chain variable region of antibody P2B11. 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: 16 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2B11.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2D9. 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: 18. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2D9. 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: 17. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2D9 and a heavy chain variable region of antibody P2D9. 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: 18 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2D9.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2E7. 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: 20. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2E7. 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: 19. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2E7 and a heavy chain variable region of antibody P2E7. 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: 20 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 19. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2E7.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P1D6. 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: 22. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P1D6. 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: 21. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P1D6 and a heavy chain variable region of antibody P1D6. 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: 22 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P1D6.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P1E7. 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: 24. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P1E7. 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: 23. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P1E7 and a heavy chain variable region of antibody P1E7. 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: 24 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P1E7.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P1F3. 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: 26. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P1F3. 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: 25. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P1F3 and a heavy chain variable region of antibody P1F3. 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: 26 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P1F3.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P1F8. 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: 28. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P1F8. 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: 27. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P1F8 and a heavy chain variable region of antibody P1F8. 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: 28 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 27. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P1F8.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2B10. 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: 30. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2B10. 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: 29. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2B10 and a heavy chain variable region of antibody P2B10. 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: 30 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2B10.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2C2. 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: 32. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2C2. 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: 31. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2C2 and a heavy chain variable region of antibody P2C2. 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: 32 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2C2.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2D4. 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: 34. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2D4. 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: 33. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2D4 and a heavy chain variable region of antibody P2D4. 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: 34 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2D4.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2E2. 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: 36. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2E2. 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: 35. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2E2 and a heavy chain variable region of antibody P2E2. 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: 36 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 35. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2E2.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2E6. 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: 38. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2E6. 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: 37. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2E6 and a heavy chain variable region of antibody P2E6. 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: 38 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2E6.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2E10. 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: 40. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2E10. 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: 39. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2E10 and a heavy chain variable region of antibody P2E10. 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: 40 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 39. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2E10.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2F2. 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: 42. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P2F2. 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: 41. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P2F2 and a heavy chain variable region of antibody P2F2. 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: 42 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P2F2.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3B2. 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: 44. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3B2. 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: 43. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3B2 and a heavy chain variable region of antibody P3B2. 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: 44 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 43. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3B2.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3B7. 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: 46. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3B7. 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: 45. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3B7 and a heavy chain variable region of antibody P3B7. 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: 46 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3B7.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3B11. 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: 48. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3B11. 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: 47. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3B11 and a heavy chain variable region of antibody P3B11. 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: 48 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3B11.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3C7. 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: 50. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3C7. 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: 49. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3C7 and a heavy chain variable region of antibody P3C7. 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: 50 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3C7.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3C11. 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: 52. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3C11. 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: 51. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3C11 and a heavy chain variable region of antibody P3C11. 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: 52 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3C11.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3D2. 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: 54. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3D2. 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: 53. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3D2 and a heavy chain variable region of antibody P3D2. 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: 54 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 53. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3D2.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3D10. 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: 56. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3D10. 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: 55. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3D10 and a heavy chain variable region of antibody P3D10. 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: 56 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 55. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3D10.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3E2. 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: 58. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3E2. 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: 57. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3E2 and a heavy chain variable region of antibody P3E2. 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: 58 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 57. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3E2.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3E4. 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: 60. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3E4. 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: 59. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3E4 and a heavy chain variable region of antibody P3E4. 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: 60 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 59. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3E4.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3E6. 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: 62. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3E6. 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: 61. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3E6 and a heavy chain variable region of antibody P3E6. 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: 62 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 61. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3E6.

In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3E9. 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: 64. In some forms, the antibody or antigen binding fragment thereof comprises a heavy chain variable region of antibody P3E9. 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: 63. In some forms, the antibody or antigen binding fragment thereof comprises the light chain variable region of antibody P3E9 and a heavy chain variable region of antibody P3E9. 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: 64 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 63. In some forms, the antibody or antigen binding fragment thereof comprises the antibody P3E9.

Also disclosed are humanized antibodies or antigen binding fragments thereof comprising one or more human IgG4 constant domains and (a) the light chain variable region and heavy chain variable region of antibody P1D9, (b) the light chain variable region and heavy chain variable region of antibody P2B4, (c) the light chain variable region and heavy chain variable region of antibody P2B11, (d) the light chain variable region and heavy chain variable region of antibody P2D9, (e) the light chain variable region and heavy chain variable region of antibody P2E7, (f) the light chain variable region and heavy chain variable region of antibody P1D6, (g) the light chain variable region and heavy chain variable region of antibody P1E7, (h) the light chain variable region and heavy chain variable region of antibody P1F3, (i) the light chain variable region and heavy chain variable region of antibody P1F8, (j) the light chain variable region and heavy chain variable region of antibody P2B10, (k) the light chain variable region and heavy chain variable region of antibody P2C2, (l) the light chain variable region and heavy chain variable region of antibody P2D4, (m) the light chain variable region and heavy chain variable region of antibody P2E2, (n) the light chain variable region and heavy chain variable region of antibody P2E6, (o) the light chain variable region and heavy chain variable region of antibody P2E10, (p) the light chain variable region and heavy chain variable region of antibody P2F2, (q) the light chain variable region and heavy chain variable region of antibody P3B2, (r) the light chain variable region and heavy chain variable region of antibody P3B7, (s) the light chain variable region and heavy chain variable region of antibody P3B11, (t) the light chain variable region and heavy chain variable region of antibody P3C7, (u) the light chain variable region and heavy chain variable region of antibody P3C11, (v) the light chain variable region and heavy chain variable region of antibody P3D2, (w) the light chain variable region and heavy chain variable region of antibody P3D10, (x) the light chain variable region and heavy chain variable region of antibody P3E2, (y) the light chain variable region and heavy chain variable region of antibody P3E4, (z) the light chain variable region and heavy chain variable region of antibody P3E6, or (aa) the light chain variable region and heavy chain variable region of antibody P3E9.

In some forms, the antibody or antigen binding fragment thereof attenuates the ability of a ligand of SARS-CoV-2 spike protein or a subunit thereof (e.g., S1 protein) or a component thereof (e.g., 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 of the antibody or antigen binding fragment thereof 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.

In some forms, the bispecific antibody (named ZCB11-P2B11) has the light chain variable region sequence of ZCB11:

Also disclosed are pharmaceutical composition comprising any of the disclosed the antibodies or antigen binding fragments thereof and a physiologically acceptable carrier or excipient. In some forms, the pharmaceutical composition is useful 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 useful in a method of treating COVID-19. In some forms, the pharmaceutical composition is useful in a method of preventing COVID-19.

Also disclosed are methods of detection or diagnosis of SARS-CoV-2 infection, comprising: (a) assaying the presence of SARS-CoV-2 spike protein or a subunit thereof (e.g., S1 protein) or a component thereof (e.g., RBD) in a sample from a subject using the antibody or antigen binding fragment thereof of any one of claims 1-18 and (b) comparing the level of the SARS-CoV-2 spike protein or a subunit thereof (e.g., S1 protein) or a component thereof (e.g., RBD) with a control level, wherein an increase in the assayed level of SARS-CoV-2 spike protein or a subunit thereof (e.g., S1 protein) or a component thereof (e.g., RBD) compared to the control level is indicative of SARS-CoV-2 infection. In some forms, the presence of SARS-CoV-2 spike protein or a subunit thereof (e.g., S1 protein) or a component thereof (e.g., RBD) is assayed by enzyme linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , or fluorescence-activated cell sorting (FACS) .

Also disclosed are methods 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 19 if the subject has a disease characterized by increased expression of SARS-CoV-2 spike protein or a subunit thereof (e.g., S1 protein) or a component thereof (e.g., RBD) . In some forms, the antibody or antigen binding fragment thereof is any one of the disclosed the antibodies or antigen binding fragments thereof.

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-1H are graphs of identification of an elite vaccinee who developed bNAbs. Plasma samples derived from 34 BNT162b2-vaccinees were tested at average 30.7 days (range 7-47 days) after second vaccination (BioNTech-Pfizer) . (FIGs. 1A-1F) Serially diluted plasma samples were subjected to neutralization assay against the pseudotyped SARS-CoV-2 WT (FIG. 1A) and five variants of concern including Alpha (B. 1.1.7) (FIG. 1B) , Beta (B. 1.351) (FIG. 1C) , Gamma (P. 1) (FIG. 1D) , Delta (B. 1.617.2) (FIG. 1E) , and Omicron (B. 1.1.529) (FIG. 1F) . The neutralizing curve of the elite BNT162b2-26 vaccinee (red) was compared with the mean curve of all vaccinees tested (dark black) . (FIG. 1G) Binding activity of spike-specific plasma IgG was determined by ELISA at serial dilutions. The binding curve of the elite BNT162b2-26 vaccinee was presented as red. (FIG. 1H) The neutralization antibody potency index was defined by the ratio of IC50/AUC of anti-spike IgG in BNT162b2-vaccinees. Neutralizing IC50 values represented plasma dilution required to achieve 50%virus neutralization. The area under the curve (AUC) represented the total peak area was calculated from ELISA OD values. Each symbol represented an individual vaccinee with a line indicating the median of each group. The elite BNT162b2-26 vaccinee was presented as red dots.

Figures 2A-2N are graphs of comparison of bNAbs isolated from the elite vaccinee. RBD-specific (FIG. 2A) and spike-specific (FIG. 2B) binding activities of 4 newly cloned NAbs including ZCB3, ZCB11, ZCC10 and ZCD3 were determined by ELISA at serial dilutions. A known NAb ZB8 was included as a control. Neutralizing activities of ZCB3, ZCB11, ZCC10 and ZCD3 were determined against six pseudotyped SARS-CoV-2 variants of concern including D614G (WT) (FIG. 2C) , Alpha (FIG. 2D) , Beta (FIG. 2E) , Gamma (FIG. 2F) , Delta (FIG. 2G) , and Omicron (FIG. 2H) as compared with the control NAb ZB8. Neutralizing activities of ZCB3, ZCB11, ZCC10 and ZCD3 were determined against the same six but authentic SARSCoV-2 variants of concern including D614G (WT) (FIG. 2I) , Alpha (FIG. 2J) , Beta (FIG. 2K) , Gamma (FIG. 2L) , Delta (FIG. 2M) , and Omicron (FIG. 2N) as compared with the control NAb ZB8. The color coding was consistently used in all figures. The dashed line in each graph indicated 50%neutralization.

Figures 3A-3E show naturally occurring mutations or deletions conferring antibody resistance in VOCs. (FIG. 3A) Fold change of IC50 values relative to WT was determined by pseudoviruses carrying individual mutations or deletion against bNAbs ZCB3 and ZCB11 as compared with ZB8. (FIGs. 3B-3C) Antibody competition by SPR between ZCB11 and ZC8 (FIG. 3B) as well as between ZCB11 and ZCB3 (FIG. 3C) . (FIG. 3D) Structural alignment between S2E12 and ZCB11 variable regions. The structure of the ZCB11 variable region predicted by the SWISS-MODEL is superimposed into the structure of S2E12 (PDB: 7K3Q) . Cartoon representation of ZCB11 variable region of heavy chain (VH) is shown in purple and the variable region of light chain (VK) in orange. The S2E12 VH and VK are shown in yellow and green, respectively. The CDRs of VH and VK are labelled. (FIG. 3E) The structure of RBD in complex with the S2E12 variable region (from PDB 7K45) . RBD is shown in cyan with receptor binding motif (RBM) highlighted in light pink and the amino acids whose substitution confers resistance to ZCB11 in (A) are highlighted in red.

Figures 4A-4G illustrate the efficacy of ZCB11 against authentic SARS-CoV-2 Delta and Omicron in golden Syrian hamsters as compared with ZB8. (A) Experimental schedule and color coding for different treatment groups. Three groups of hamsters (n=8) received a single intraperitoneal injection of PBS (grey) , 4.5 mg/kg of ZB8 (purple) or 4.5 mg/kg of ZCB11 (blue) at one day before viral infection (-1 dpi) . 24 hours later (day 0) , each group was divided into two subgroups for intranasal challenge with 105 PFU live SARS-CoV-2 Delta and Omicron variants, respectively. All animals were sacrificed on day 4 for final analysis. (B, E) Daily body weight was measured after viral infection. (C, F) The nucleocapsid protein (NP) subgenomic RNA copy numbers (normalized by β-actin) in lung homogenates were determined by a sensitive RT PCR. (D, G) Live viral plaque assay was used to quantify the number of infectious viruses in lung homogenates. Log10-transformed plaque-forming units (PFU) per mL were shown for each group. LOD: limit of detection. Each symbol represents an individual hamster with a line indicating the mean of each group. The color coding was consistently used in each graph. Statistics were generated using one-way ANOVA followed by Tukey's multiple comparisons test. **p<0.01; ***p<0.001.

Figures 5A-5B are graphs showing the gating strategy for sorting antigen specific memory B cells from the BioNTech-26 vaccinee (FIG. 5A) as compared with a healthy control (FIG. 5B) .

Figures 6A-6G are graphs of binding and neutralizing activities of 14 newly cloned human monoclonal antibodies. (FIGs. 6A-6F) HEK 293T cells were transfected with expression plasmids encoding paired heavy and light chains. Two days after transfection, culture supernatants were subjected to binding test to SARS-CoV-2 Spike (FIG. 6A) , S1 (FIG. 6B) , S2 (FIG. 6C) , RBD (FIG. 6D) and NTD (FIG. 6E) by ELISA, respectively. (FIG. 6F) Neutralization activities of culture supernatants were also determined by the pseudotyped SARS-CoV-2 WT in 293T-ACE2 cells. (FIG. 6G) Antibody binding kinetics by SPR. ZCB11 was captured on protein A covalently immobilized onto a CM5 sensor chip followed by injection of purified soluble SARS-CoV-2 WT RBD at five different concentrations. The black lines indicated the experimentally derived curves while the color lines represented fitted curves based on the experimental data.

Figures 7A-7F are graphs of competition binding assay of newly cloned NAbs with ZB8 including binding between antibodies of ZB8 vs ZCB3 (FIG. 7A) , ZCB3 vs ZB8 (FIG. 7B) , ZB8 vs ZCD3 (FIG. 7C) , ZCD3 vs ZB8 (FIG. 7D) , ZB8 vs ZCB11 (FIG. 7E) , ZCB3 vs ZB11 (FIG. 7F) . The sensorgrams show distinct binding patterns when pairs of testing antibodies were sequentially applied to the purified SARS-CoV-2 RBD covalently immobilized onto a CM5 sensor chip. Color coding curves indicate distinct binding patterns of representative NAbs to RBD with (orange) or without (green) prior incubation with each testing antibody.

Figures 8A-8F is graphs of neutralizing antibody titres in the patients at peak response time against a panel of SARS-CoV-2VOC pseudoviruses, including D614G (WT) (FIG. 8A) , alpha (B. 1.1.7) (FIG. 8B) , beta (B. 1.351) (FIG. 8C) , gamma (P1) (FIG. 8D) , delta (B. 1.617.2) (FIG. 8E) , and omicron (B. 1.1.529) (FIG. 8F) .

Figures 9A and 9B are graphs of longitudinal neutralizing antibody titres of OP1 (FIG. 9A) and OP2 (FIG. 9B) against the full panel of variants of concern.

Figures 10A and 10C are pie charts of the repertoire of heavy chains and light chains, respectively, in the isolated antibodies. Figures 10B and 10D are graphs of the activation of the clonal types of the heavy chains and light chains, respectively, in the isolated antibodies.

Figure 11A is a graph of the mean somatic hypermutation (SHM) of the heavy chains and light chains. Figure 11B is a graph of the mean length of CDR3 of the heavy chains and light chains.

Figures 12A-12D are diagrams of the recombination of the heavy chain VD (FIG. 12A) , VJ (FIG. 12B) , and DJ (FIG. 12C) , and the light chain VJ (FIG. 12D) .

Figures 13A and 13B are graphs of the neutralization of recombinant antibodies against WT and Omicron virus.

Figures 14A and 14B are graphs of the binding affinity of selected recombinant antibodies against WT and Omicron spike proteins.

Figures 15A-15B are graphs of percent neutralization of WT (FIG. 15A) and Omicron (FIG. 15B) pseudovirus over a concentration (μg/mL log ₁₀) of each of the indicated recombinant antibodies.

Figures 16A-16B are graphs of percent neutralization of WT (FIG. 16A) and Omicron (FIG. 16B) live virus over a concentration (μg/mL log ₁₀) of each of the indicated recombinant antibodies.

Figures 17A to 17H are graphs showing a lack of competition for binding between selected recombinant antibodies of ZCB11 vs P1-D9 (FIG. 17A) , P1-D9 vs ZCB11 (FIG. 17B) , ZCB11 vs P2-D9 (FIG. 17C) , P2-D9 vs ZCB11 (FIG. 17D) , P2-E7 vs ZCB11 (FIG. 17E) , ZCB11 vs P2-E7 (FIG. 17F) , P2-D9 vs P2-B4 (FIG. 17G) , and P2-B4 vs P2-D9 (FIG. 17H) , over a period of 140 seconds.

Figures 18A to 18L are graphs showing competition for binding between selected recombinant antibodies of P1-D9 vs P2-B4 (FIG. 18A) , P2-B4 vs P1-D9 (FIG. 18B) , ZCB11 vs P2-B4 (FIG. 18C) , P2-B4 vs ZCB11 (FIG. 18D) , P2-E7 vs P2-B4 (FIG. 18E) , P2-B4 vs P2-E7 (FIG. 18F) , P2-D9 vs P2-E7 (FIG. 18G) , P2-E7 vs P2-D9 (FIG. 18H) , P1-D9 vs P2-D9 (FIG. 18I) , P2-D9 vs P1-D9 (FIG. 18J) , P1-D9 vs P2-E7 (FIG. 18K) , and P2-E7 vs P1-D9 (FIG. 18L) , over a period of 140 seconds.

Figure 19 is a graph of quantified results of measurements of Spike-specific IgG ₊B cells from PBMCs isolated form patients.

Figures 20A-20B are pie charts of the proportion of activated (AM) , tissue-like memory (TLM) , intermediate memory (IM) , and resting memory (RM) B cells in OP1 (FIG. 20A) and OP2 (FIG. 20B) .

Figures 21A-21B are graphs of the percentage of IFN-γ+ cells gated on CD4 (FIG. 21A) and CD8 (FIG. 21B) .

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.

The strikingly high transmissibility and antibody evasion of SARS-CoV-2 Omicron variant have posted great challenges on the efficacy of current vaccines and antibody immunotherapy. Here, we screened 34 BNT162b2-vaccinees and cloned a public broadly neutralizing antibody (bNAb) ZCB11 from an elite vaccinee. ZCB11 neutralized all authentic SARS-CoV-2 variants of concern (VOCs) , including Omicron and OmicronR346K with potent IC ₅₀ concentrations of 36.8 and 11.7 ng/mL, respectively. Functional analysis demonstrated that ZCB11 targeted viral receptor-binding domain (RBD) and competed strongly with ZB8, a known RBD-specific class II NAb. Pseudovirus-based mapping of 57 naturally occurred single mutations or deletions revealed that only S371L resulted in 11-fold neutralization resistance, but this phenotype was not observed in the Omicron variant. Furthermore, prophylactic ZCB11 administration protected lung infection against both the circulating pandemic Delta and Omicron variants in golden Syrian hamsters. These results demonstrated that vaccine-induced ZCB11 is a promising bNAb for immunotherapy against pandemic SARS-CoV-2 VOCs.

SARS-CoV-2 Omicron variants with striking transmissibility and antibody evasion are a public threat for losing COVID-19 pandemic control. Zhou et al. cloned ZCB11, a public human bNAb from a BNT162b2-induced memory B cell of an elite vaccinee. ZCB11 displays ultrabroad and potent neutralization activity overcoming antibody resistant mutations in Omicron and in other variants of concern. ZCB11 protects Golden Syrian hamsters against highly prevalent circulating pandemic Delta and Omicron variants.

ZCB11 is a public human bNAb cloned from a BNT162b2-induced memory B cell. ZCB11 is RBD-specific and neutralizes potently all authentic SARS-CoV-2 VOCs. ZCB11 overcomes naturally occurred single mutations or deletions in SARS-CoV-2 VOCs. ZCB11 protected hamsters against currently circulating pandemic Delta and Omicron variants.

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 (ZCB11, ZCB3, ZCC10, and ZCD3) 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 vaccinees.

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 ZCB11 significantly reduced infection in lungs of hamsters intranasally-challenged with SARS-CoV-2. Moreover, post-challenge ZCB11 therapy suppressed viral loads and lung damage especially when treated within 48-hours. HuNAb ZCB11 prevented entry of pseudovirus and live virus by competing with human cellular receptor ACE2 for RBD binding. These results demonstrated that systemic HuNAb suppresses SARS-CoV-2 replication and lung injury.

Additionally, based on the screening of the 34 BNT162b2-vaccinees and two omicron patients at peak response time, 27 HuNAbs (P1D9, P2B4, P2B11, P2D9, P2E7, P1D6, P1E7, P1F3, P1F8, P2B10, P2C2, P2D4, P2E2, P2E6, P2E10, P2F2, P3B2, P3B7, P3B11, P3C7, P3C11, P3D2, P3D10, P3E2, P3E4, P3E6, and P3E9) were generated that bind to conformational determinants of the spike protein. The disclosed SARS-CoV-2 HuNAbs, each with a distinct sequence, are newly discovered from vaccinees.

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 spike protein or a component thereof such as RBD and are capable of substantially blocking SARS-CoV-2 spike protein or 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 spike protein or RBD’s interaction with ACE2” denotes that the provision of such molecule attenuates SARS-CoV-2 spike protein or 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 viruses.

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 spike protein or a component thereof such as RBD when present in a subject.

Variable domain 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 spike protein or a component thereof such as 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 spike protein or a component thereof such as RBD. The determination of percent identity of two amino acid sequences can be determined by BLAST protein comparison.

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

(1) the light chain CDR1 of anti-SARS-CoV-2 RBD antibody ZCB11, ZCB3, ZCC10, or ZCD3;

(2) a light chain CDR2 of anti-SARS-CoV-2 RBD antibody ZCB11, ZCB3, ZCC10, or ZCD3; and

(3) the light chain CDR3 of anti-human SARS-CoV-2 RBD antibody ZCB11, ZCB3, ZCC10, or ZCD3.

In some preferred forms, 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 ZCB11, ZCB3, ZCC10, or ZCD3;

(2) the heavy chain CDR2 of anti-SARS-CoV-2 RBD antibody ZCB11, ZCB3, ZCC10, or ZCD3; and

(2) the heavy chain CDR3 of anti-SARS-CoV-2 RBD antibody ZCB11, ZCB3, ZCC10, or ZCD3.

In some preferred forms, 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 spike protein antibody P1D9, P2B4, P2B11, P2D9, or P2E7;

(2) a light chain CDR2 of anti-SARS-CoV-2 spike protein antibody P1D9, P2B4, P2B11, P2D9, or P2E7; and

(3) the light chain CDR3 of anti-human SARS-CoV-2 spike protein antibody P1D9, P2B4, P2B11, P2D9, or P2E7.

(1) the heavy chain CDR1 of anti-SARS-CoV-2 spike protein antibody P1D9, P2B4, P2B11, P2D9, or P2E7;

(2) the heavy chain CDR2 of anti-SARS-CoV-2 spike protein antibody P1D9, P2B4, P2B11, P2D9, or P2E7; and

(3) the heavy chain CDR3 of anti-SARS-CoV-2 spike protein antibody P1D9, P2B4, P2B11, P2D9, or P2E7.

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 spike protein or a component thereof such as 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 forms the Fc region is the native IgG1, IgG2, or IgG4 Fc region. In some forms 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 spike protein or 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 spike protein or RBD antibodies are particularly preferred for in vivo use in humans, however, murine antibodies or antibodies of other species may be advantageously employed for many uses (for example, in vitro or in situ detection assays, acute in vivo use, etc. ) . A humanized antibody may comprise amino acid residue substitutions, deletions, or additions in one or more non-human CDRs. The humanized antibody derivative may have substantially the same binding, stronger binding or weaker binding when compared to a non-derivative humanized antibody. In some forms, 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 some forms, 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 spike protein or 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 spike protein or 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 spike protein or 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 forms, the constant domains of the SARS-CoV-2 RBD antibodies are (or comprise) human IgA, IgD, IgE, IgG or IgM domains. In some forms, human IgG constant domains, especially of the IgG1 and IgG3 isotypes are used, when the humanized SARS-CoV-2 spike protein or RBD antibody 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 some forms, IgG2 and IgG4 isotypes are used when the SARS-CoV-2 spike protein or 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 forms, the SARS-CoV-2 spike protein or RBD antibody contains both the light chain as well as at least the variable domain of a heavy chain. In some forms, the SARS-CoV-2 spike protein or 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 forms, the constant domain is a complement fixing constant domain where it is desired that the antibody exhibits cytotoxic activity, and the class is typically IgG1. In some forms, where such cytotoxic activity is not desirable, the constant domain may be of the IgG2 class. The SARS-CoV-2 spike protein or 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 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 spike protein or RBD, such as other molecules of the immune system. For example, such antibodies may bind to both SARS-CoV-2 spike protein or 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 some forms, 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 spike protein or 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 spike protein or 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 spike protein or 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 spike protein or 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 spike protein or 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 spike protein or 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 spike protein or 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 spike protein or 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 spike protein or 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 some forms, 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 spike protein or 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 forms, 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 some forms 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 forms, 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 some forms, 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 some forms, an antibody derivative will possess a similar or identical function as the parental antibody. In some forms, 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 spike protein or 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 spike protein or 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 spike protein or 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.

Some forms encompass modification of framework residues of the humanized SARS-CoV-2 spike protein or 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) .

Some forms encompass anti-human SARS-CoV-2 spike protein or 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 some forms, 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 some forms, the SARS-CoV-2 spike protein or RBD antibodies or SARS-CoV-2 spike protein or 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 forms, the Fc region is the native IgG1, IgG2, or IgG4 Fc region. In some forms, 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 some forms, 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) .

Some forms include 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 forms the IgG ₁ and/or IgG ₂ domain is deleted for example, Angal, s. et al. describes 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 some forms, 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, a 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 some preferred forms, 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 spike protein or RBD antibody or any of its antigen-binding fragments that immunospecifically binds SARS-CoV-2 spike protein or 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 spike protein or 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 some forms, 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 some forms, 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 forms, 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 forms, 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 spike protein or 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 some preferred forms, 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 forms, 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 spike protein or RBD antibody or an antigen-binding fragment thereof, the nucleic acid can be administered in vivo to promote expression of its encoded antibody, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (See U.S. Patent No. 4,980,286) , or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont) , or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (See e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88: 1864-1868) , etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination.

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

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

In some forms, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete) , excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

Generally, the ingredients of compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compositions can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

A. Inhalation Means

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

1. Dry Powder Inhalers

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

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

(Novartis Ag Corporation Switzerland, Basel, Switzerland) ,

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

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

(Direct-Haler A/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) ) .

2. Pressurized Metered-Dose Inhalers

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

3. Soft Mist Inhalers

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

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

4. Medical Nebulizers

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

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

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

wherein the CDRs comprise:

(a) the three light chain CDRs selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, and 64; and

(b) the three heavy chain CDRs selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, and 63,

wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 spike protein or a component thereof.

2. The antibody or antigen binding fragment thereof of paragraph 1 comprising the light chain variable region of antibody ZCB11.

3. The antibody or antigen binding fragment thereof of paragraph 1 comprising the light chain variable region of antibody ZCB3.

4. The antibody or antigen binding fragment thereof of paragraph 1 comprising the light chain variable region of antibody ZCC10.

5. The antibody or antigen binding fragment thereof of paragraph 1 comprising the light chain variable region of antibody ZCD3.

6. The antibody or antigen binding fragment thereof of paragraph 1 comprising the light chain variable region of antibody ZCD4.

7. The antibody or antigen binding fragment thereof of paragraph 1 comprising the light chain variable region of antibody P1D9.

8. The antibody or antigen binding fragment thereof of paragraph 1 comprising the light chain variable region of antibody P2B4.

9. The antibody or antigen binding fragment thereof of paragraph 1 comprising the light chain variable region of antibody P2B11.

10. The antibody or antigen binding fragment thereof of paragraph 1 comprising the light chain variable region of antibody P2D9.

11. The antibody or antigen binding fragment thereof of paragraph 1 comprising the light chain variable region of antibody P2E7.

12. The antibody or antigen binding fragment thereof of any one of paragraphs 1-11 comprising a heavy chain variable region of antibody ZCB11.

13. The antibody or antigen binding fragment thereof of any one of paragraphs 1-11 comprising a heavy chain variable region of antibody ZCB3.

14. The antibody or antigen binding fragment thereof of any one of paragraphs 1-11 comprising a heavy chain variable region of antibody ZCC10.

15. The antibody or antigen binding fragment thereof of any one of paragraphs 1-11 comprising a heavy chain variable region of antibody ZCD3.

16. The antibody or antigen binding fragment thereof of any one of paragraphs 1-11 comprising a heavy chain variable region of antibody ZCD4.

17. The antibody or antigen binding fragment thereof of any one of paragraphs 1-11 comprising a heavy chain variable region of antibody P1D9.

18. The antibody or antigen binding fragment thereof of any one of paragraphs 1-11 comprising a heavy chain variable region of antibody P2B4.

19. The antibody or antigen binding fragment thereof of any one of paragraphs 1-11 comprising a heavy chain variable region of antibody P2B11.

20. The antibody or antigen binding fragment thereof of any one of paragraphs 1-11 comprising a heavy chain variable region of antibody P2D9.

21. The antibody or antigen binding fragment thereof of any one of paragraphs 1-11 comprising a heavy chain variable region of antibody P2E7.

22. The antibody or antigen binding fragment thereof of paragraph 1,

(1) wherein the CDRs comprise:

(a) the three light chain CDRs of SEQ ID NO: 4 and the three heavy chain CDRs of SEQ ID NO: 3,

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

(c) the three light chain CDRs of SEQ ID NO: 6 and the three heavy chain CDRs of SEQ ID NO: 5, or

(d) the three light chain CDRs of SEQ ID NO: 8 and the three heavy chain CDRs of SEQ ID NO: 7, and

wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 RBD; or

(2) wherein the CDRs comprise the three light chain CDRs of SEQ ID NO: 10 and the three heavy chain CDRs of SEQ ID NO: 9, wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 S1.

23. The antibody or antigen binding fragment thereof of paragraph 1,

(1) wherein the CDRs comprise:

(a) the three light chain CDRs and the three heavy chain CDRs of antibody ZCB11,

(b) the three light chain CDRs and the three heavy chain CDRs of antibody ZCB3,

(c) the three light chain CDRs and the three heavy chain CDRs of antibody ZCC10, or

(d) the three light chain CDRs and the three heavy chain CDRs of antibody ZCD3, and

(2) wherein the CDRs comprise the three light chain CDRs and the three heavy chain CDRs of antibody ZCD4, wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 S1.

24. The antibody or antigen binding fragment thereof of paragraph 1,

wherein the CDRs comprise:

(a) the three light chain CDRs of SEQ ID NO: 12 and the three heavy chain CDRs of SEQ ID NO: 11,

(b) the three light chain CDRs of SEQ ID NO: 14 and the three heavy chain CDRs of SEQ ID NO: 13,

(c) the three light chain CDRs of SEQ ID NO: 16 and the three heavy chain CDRs of SEQ ID NO: 15,

(d) the three light chain CDRs of SEQ ID NO: 18 and the three heavy chain CDRs of SEQ ID NO: 17, or

(e) the three light chain CDRs of SEQ ID NO: 20 and the three heavy chain CDRs of SEQ ID NO: 19, and

wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 spike protein.

25. The antibody or antigen binding fragment thereof of paragraph 1,

wherein the CDRs comprise:

(a) the three light chain CDRs and the three heavy chain CDRs of antibody P1D9,

(b) the three light chain CDRs and the three heavy chain CDRs of antibody P2B4,

(c) the three light chain CDRs and the three heavy chain CDRs of antibody P2B11,

(d) the three light chain CDRs and the three heavy chain CDRs of antibody P2D9, or

(e) the three light chain CDRs and the three heavy chain CDRs of antibody P2E7,

26. The antibody or antigen binding fragment thereof of any one of paragraphs 1-25, wherein the antibody or antigen binding fragment thereof attenuates the ability of a ligand of SARS-CoV-2 spike protein or RBD to bind to ACE2.

27. The antibody or antigen binding fragment thereof of any one of paragraphs 1-26 comprising one or more constant domains from an immunoglobulin constant region (Fc) .

28. The antibody or antigen binding fragment thereof of paragraph 27, wherein the constant domains are human constant domains.

29. The antibody or antigen binding fragment thereof of paragraph 28, wherein the human constant domains are IgA, IgD, IgE, IgG or IgM domains.

30. The antibody or antigen binding fragment thereof of paragraph 29, wherein human IgG constant domains are IgG1, IgG2, IgG3, or IgG4 domains.

31. The antibody or antigen binding fragment thereof of any one of paragraphs 1-30, wherein the antibody or antigen binding fragment thereof is detectably labeled or comprises a conjugated toxin, drug, receptor, enzyme, receptor ligand.

32. The antibody or antigen binding fragment thereof of any one of paragraph 1-31, wherein the antibody is a monoclonal antibody, a human antibody, a chimeric antibody or a humanized antibody.

33. The antibody or antigen binding fragment thereof of any one of paragraphs 1-32, wherein the antibody is a bispecific, trispecific or multispecific antibody.

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

the light chain variable region and heavy chain variable region of antibody ZCB11,

the light chain variable region and heavy chain variable region of antibody ZCB3,

the light chain variable region and heavy chain variable region of antibody ZCC10,

the light chain variable region and heavy chain variable region of antibody ZCD3,

the light chain variable region and heavy chain variable region of antibody ZCD4,

the light chain variable region and heavy chain variable region of antibody P1D9,

the light chain variable region and heavy chain variable region of antibody P2B4,

the light chain variable region and heavy chain variable region of antibody P2B11,

the light chain variable region and heavy chain variable region of antibody P2D9,

the light chain variable region and heavy chain variable region of antibody P2E7,

the light chain variable region of ZCB11 and the heavy chain variable region of antibody P2B11, or

the light chain variable region of SEQ ID NO: 4 and the heavy chain region of SEQ ID NO: 65.

35. A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of paragraphs 1-34 and a physiologically acceptable carrier or excipient.

36. The pharmaceutical composition of paragraph 35 for use in a method of preventing or treating COVID-19 in a subject.

37. The pharmaceutical composition for use of paragraph 36, wherein the subject has COVID-19.

38. The pharmaceutical composition for use of paragraph 37, wherein the subject is at risk of developing COVID-19.

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

40. The pharmaceutical composition of paragraph 35 for use in a method of preventing COVID-19.

41. Use of the antibody or antigen binding fragment thereof of any of paragraphs 1-34 in manufacture of a medicament for preventing or treating COVID-19 in a subject.

42. Use of the antibody or antigen binding fragment thereof of any of paragraphs 1-34 in manufacture of a medicament for treating COVID-19 in a subject.

43. Use of the antibody or antigen binding fragment thereof of any of paragraphs 1-34 in manufacture of a medicament for preventing COVID-19 in a subject.

44. A method of detection or diagnosis of SARS-CoV-2 infection, comprising: (a) assaying the presence of SARS-CoV-2 spike protein or RBD in a sample from a subject using the antibody or antigen binding fragment thereof of any one of paragraphs 1-34 and (b) comparing the level of the SARS-CoV-2 spike protein or RBD with a control level, wherein an increase in the assayed level of SARS-CoV-2 spike protein or RBD compared to the control level is indicative of SARS-CoV-2 infection.

45. The method of paragraph 44, wherein the presence of SARS-CoV-2 spike protein or RBD is assayed by enzyme linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , or fluorescence-activated cell sorting (FACS) .

46. 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 35 if the subject has a disease characterized by increased expression of SARS-CoV-2 spike protein or RBD.

47. The method of paragraph 46, wherein the antibody or antigen binding fragment thereof is the antibody or antigen binding fragment thereof of any one of paragraphs 1-34.

Examples

Example 1: An elite broadly neutralizing antibody protects SARS-CoV-2 Omicron variant challenge

Materials and Methods

Human subjects

A cohort of 34 vaccinees who received two doses of BNT162b2 before June 2021 were recruited for this study. The exclusion criteria include individuals with (1) documented SARS-CoV-2 infection, (2) high-risk infection history within 14 days before vaccination, (3) COVID-19 symptoms such as sore throat, fever, cough and shortness of breath. Clinical and laboratory findings were entered into a predesigned database. Written informed consent was obtained from all study subjects. This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster (Ref No. UW 21-120-452) .

Viruses

Authentic SARS-CoV-2 D614G (MT835143) , Alpha (MW856794) , Beta (GISAID: EPI_ISL_2423556) , Omicron (hCoV-19/Hong Kong/HKU-344/2021; GISAID accession number EPI_ISL_7357684) and Delta (hCoV-19/Hong Kong/HKU-210804-001/2021; GISAID: EPI_ISL_3221329) variants were isolated from respiratory tract specimens of laboratory-confirmed COVID-19 patients in Hong Kong (Lu et al., 2021) . All experiments involving live SARS-CoV-2 followed the approved standard operating procedures of the Biosafety Level 3 facility at The University of Hong Kong (Chan et al., 2021; Chan et al., 2020c) .

Cell lines

HEK293T cells, HEK293T-hACE2 cells and Vero-E6-TMPRSS2 cells were maintained in DMEM containing 10%FBS, 2 mM L-glutamine, 100 U/mL/mL penicillin and incubated at 37 ℃ in a 5%CO2 setting (Liu et al., 2019) . Expi293FTM cells were cultured in Expi293TM Expression Medium (Thermo Fisher Scientific) at 37 ℃ in an incubator with 80%relative humidity and a 5%CO2 setting on an orbital shaker platform at 125 ±5 rpm/min (New Brunswick innovaTM 2100) according to the manufacturer’s instructions.

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

The recombinant RBD and trimeric spike proteins derived from SARS-CoV-2 (Sino Biological) were diluted to final concentrations of 1 μg/mL/mL, then coated onto 96-well plates (Corning 3690) and incubated at 4 ℃ overnight. Plates were washed with PBS-T (PBS containing 0.05%Tween-20) and blocked with blocking buffer (PBS containing 5%skim milk or 1%BSA) at 37 ℃ for 1 h. Serially diluted plasma samples or isolated monoclonal antibodies were added to the plates and incubated at 37 ℃ for 1 h. Wells were then incubated with a secondary goat anti-human IgG labelled with horseradish peroxidase (HRP) (Invitrogen) or with a rabbit polyclonal anti-human IgA alpha-chain labelled with HRP (Abcam) and TMB substrate (SIGMA) . Optical density (OD) at 450 nm was measured by a spectrophotometer. Serially diluted plasma from healthy individuals or previously published monoclonal antibodies against SARS-CoV-2 (B8) were used as negative controls.

Isolation of SARS-CoV-2 spike-specific IgG+ single memory B cells by FACS

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

Single B cell RT-PCR and antibody cloning

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

Genetic analysis of the BCR repertoire

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

Antibody production and purification

The paired antibody VH/VL chains were cloned into Igγ and Igκ expression vectors using T4 ligase (NEB) . Antibodies produced from cell culture supernatants were purified immediately by affinity chromatography using recombinant Protein G-Agarose (Thermo Fisher Scientific) according to the manufacturer’s instructions, to purify IgG. The purified antibodies were concentrated by an Amicon ultracentrifuge filter device (molecular weight cut-off 10 kDa; Millipore) to a volume of 0.2 mL in PBS (Life Technologies) , and then stored at 4 ℃ or -80 ℃ for further characterization.

Pseudovirus-based neutralization assay

The neutralizing activity of NAbs was determined using a pseudotype-based neutralization assay as we previously described (Poeran et al., 2020) . Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively (Poeran et al., 2020) . Viral supernatant was collected at 48 h post-transfection and frozen at -80 ℃ until use. The serially diluted monoclonal antibodies or sera were incubated with 200 TCID50 of pseudovirus at 37 ℃ for 1 hour. The antibody-virus mixtures were subsequently added to pre-seeded HEK 293T-ACE2 cells. 48 hours later, infected cells were lysed to measure luciferase activity using a commercial kit (Promega, Madison, WI) . Half-maximal (IC50) or 90% (IC90) inhibitory concentrations of the evaluated antibody were determined by inhibitor vs. normalized response --4 Variable slope using GraphPad Prism 8 or later (GraphPad Software Inc. ) .

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

The SARS-CoV-2 focus reduction neutralization test (FRNT) was performed in a certified Biosafety level 3 laboratory. Neutralization assays against live SARS-CoV-2 were conducted using a clinical isolate previously obtained from a nasopharyngeal swab from an infected patient (Chu et al., 2020) . The tested antibodies were serially diluted, mixed with 50 μL of SARS-CoV-2 (1×103 focus forming unit/mL, FFU/mL) in 96-well plates, and incubated for 1 hour at 37℃. Mixtures were then transferred to 96-well plates pre-seeded with 1×104/well Vero E6 cells and incubated at 37℃ for 24 hours. The culture medium was then removed, and the plates were air-dried in a biosafety cabinet (BSC) for 20 mins. Cells were then fixed with a 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 sera anti-SARS-CoV-2-N for 1 hour at RT before adding an Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies) . The fluorescence density of SARS-CoV-2 infected cells were scanned using a Sapphire Biomolecular Imager (Azure Biosystems) and the neutralization effects were then quantified using Fiji software (NIH) .

Antibody binding kinetics and competition between antibodies measured by Surface Plasmon Resonance (SPR)

The binding kinetics and affinity of recombinant monoclonal antibodies for the SARS-CoV-2 RBD protein (SinoBiological) were analyzed by SPR (Biacore T200, GE Healthcare) . Specifically, the SARS-CoV-2 RBD 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 250. SPR assays were run at a flow rate of 10 μL/min in HEPES buffer. For conventional kinetic/dose response, serial dilutions of monoclonal antibodies were injected across the spike protein surface for 180s, followed by a 900s dissociation phase using a multi-cycle method. Remaining analytes were removed in the surface regeneration step with the injection of 10 mM glycine-HCl (pH 1.5) for 60s 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. Before evaluating the competition between antibodies, both the saturating binding concentrations of antibodies for the immobilized SARS-CoV-2 RBD protein were determined separately. In the competitive assay, antibodies at the saturating concentration were injected onto the chip with immobilized spike protein for 120s until binding steady-state was reached. The other antibody also used at the saturating concentration was then injected for 120s, followed by another 120s of injection of antibody to ensure a saturation of the binding reaction against the immobilized RBD protein. The differences in response units between antibody injection alone and prior antibody incubation reflect the antibodies’ competitive ability by binding to the RBD protein.

Model building of ZCB11 and structure presentation

A model of ZCB11 variable regions was generated based on the protein sequence by the SWISS-MODEL using the crystal structure of S2E12 Fab fragment (Research Collaboratory for Structural Bioinformatics [RCSB] PDB code 7K3Q) as the template. The structure alignment, cartoon representations, labeling of amino acids in RBD (from PDB 7K45) were generated by PyMOL.

Hamster experiments

In vivo evaluation of monoclonal antibody ZB8 or ZCB11 in the established golden Syrian hamster model of SARS-CoV-2 infection was performed as described previously, with slight modifications (Chan et al., 2020a) . The animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR 5359-20) of the University of Hong Kong (HKU) . Briefly, 6-10-week-old male and female hamsters were obtained from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Centre for Comparative Medicine Research. The hamsters were housed with access to standard pellet feed and water ad libitum until live virus challenge in the BSL-3 animal facility at Department of Microbiology, HKU. The viral challenge experiments were then conducted in our Biosafety Level-3 animal facility following SOPs strictly, with strict adherence to SOPs. The hamsters were randomized from different litters into experimental groups. Experiments were performed in compliance with the relevant ethical regulations (Chan et al., 2020a) . For prophylaxis studies, 24 hours before live virus challenge, three groups of hamsters were intraperitoneally administered with one dose of test antibody in phosphate-buffered saline (PBS) at the indicated dose. At day 0, each hamster was intranasally inoculated with a challenge dose of 100 μL of Dulbecco’s Modified Eagle Medium containing 105 PFU of SARS-CoV-2 Delta variant or Omicron variant under anesthesia with intraperitoneal ketamine (200 mg/kg) and xylazine (10 mg/kg) . The hamsters were monitored daily for clinical signs of disease. Syrian hamsters typically clear virus within one week after SARS-CoV-2 infection. Accordingly, animals were sacrificed for analysis at day 4 after virus challenge with high viral loads (Chan et al., 2020a) . Half the nasal turbinate, trachea, and lung tissues were used for viral load determination by quantitative RT-qPCR assay (Chan et al., 2020b) and infectious virus titration by plaque assay (Chan et al., 2020a) as we described previously.

Quantification and statistical analysis

Statistical analysis was performed using PRISM 8.0 or later. Ordinary one-way ANOVA and multiple comparisons were used to compare group means and differences between multiple groups. Unpaired Student's t tests were used to compare group means between two groups only. A P-value <0.05 was considered significant. The number of independent experiments performed, the number of animals in each group, and the specific details of statistical tests are reported in the figure legends and the Methods section.

RESULTS

Identification of an elite vaccinee who developed bNAbs

To isolate potent bNAbs against currently circulating SASR-CoV-2 VOCs, we searched for elite vaccinees, who had developed potent bNAbs among a Hong Kong cohort of 34 vaccinees, around average 30.7 days (range, 7-47 days) after their second dose of the BNT162b2 vaccination (BioNTech-Pfizer) (Table 1) (Qiaoli Peng, 2021) . 100%subjects developed NAbs against the pseudotyped SARS-CoV-2 wildtype (WT, D614G) (Figure 1A) . To seek for vaccinees with bNAb, we then tested the full panel of pseudotyped SARS-CoV-2 VOCs including Alpha (B. 1.1.7) , Beta (B. 1.351) , Gamma (P. 1) , Delta (B. 1.617.2) and Omicron (B. 1.1.529) (FIGs. 1B-1F) . Only two study subjects (2/34) , BNT162b2-26 and BNT162b2-55, were considered as elite vaccinees who harbored bNAbs with IC90 or IC50 values higher than the mean titers of all VOCs tested in the cohort. BNT162b2-26 displayed significantly high bNAbs titers against the Beta and Delta variants (Figures 1C and 1E, Table 2) , the known most resistant VOC and the dominant VOC, respectively, before the Omicron variant (Baisheng Li, 2021; Wang et al., 2021a) . After measuring binding antibodies to spike protein (Figure 1G) , we calculated the neutralizing potency index as previously described (Garcia-Beltran et al., 2021) . We found that Omicron resulted in the highest reduction of the mean neutralizing potency index as compared with other VOCs (Figure 1H) . BNT162b2-26, however, displayed neutralizing potency index scores consistently higher than the mean ones against all VOCs tested. We, therefore, chose this elite vaccinee for subsequent search of bNAb.

Table 1. Characteristics of BNT162b2 vaccinees

Table 2. Neutralization titers of BNT162b2-26 plasma

Isolation of NAbs against SARS-CoV-2 from the elite vaccinee

With vaccinee informed consent, we obtained another blood sample donated by BNT162b2-26 at day 130 after his second vaccination. Fresh PBMCs from BNT162b2-26 were stained for antigen-specific memory B cells (CD19, CD27, IgG) using the 6xHis-tagged SARS-CoV-2 WT spike as the bait as previously described (Biao Zhou, 2021) . Spike-specific memory B cells were found in BNT162b2-26 but not in the healthy donor (HD) control (Figures 5A-5B) and were sorted into each well with a single B cell for antibody gene amplification. After antibody gene sequencing, we recovered 14-paired heavy chain and light chain for antibody IgG1 engineering. Seven of these 14 paired antibodies including ZCB3, ZCB8, ZCB9, ZCB11, ZCC10, ZCD3, ZCD4 in antibody expression supernatants showed positive responses to WT spike by ELISA 48 hours post transient transfection (Figure 6A) . Five of these seven spike-reactive antibodies including ZCB3, ZCD4, ZCB11, ZCC10 and ZCD3 targeted spike S1 subunit (Figure 6B) , whereas ZCB8 and ZCB9 were S2-specific (Figure 6C) . Moreover, among these five S1-reactive antibodies, only ZCD4 was not specific to RBD (Figures 6D) and none of them interacted with NTD (Figures 6E, Table 3) . Eventually, only four RBD-specific ZCB3, ZCB11, ZCC10 and ZCD3 showed neutralizing activities against WT by the pseudovirus neutralization assay (Figure 6F) . These results demonstrated that RBD-specific NAbs were primarily obtained from memory B cells of BNT162b2-26 at 130 days after his second vaccination.

Table 3. Epitopes of four isolated SARS-CoV-2 specific antibodies

Antibody	Epitope
ZCB3	RBD
ZCB8	S2
ZCB9	S2
ZCB11	RBD
ZCC10	RBD
ZCD3	RBD
ZCD4	S1

Notably, besides the previously published control ZB8 (Biao Zhou, 2021) , ZCB11 had the strongest binding capability to both RBD and Spike with the same EC50 values of 20 ng/ml by ELISA (Figures 2A-2B, Table 4) . Moreover, the binding dynamics of ZCB11 to SARS-CoV-2 RBD was determined using the surface plasmon resonance (SPR) . We found that ZCB11 exhibited the fast-on/slow-off kinetics with an equilibrium dissociation constant (KD) value of 5.75×10-11 M, suggesting an RBD-specific high-binding affinity (Figure 6G and Table 5) . In subsequent quantitative neutralization analysis against WT, we found that two of these four NAbs, ZCB3 and ZCB11, showed high neutralization potency with IC50 values below 100 ng/mL (Figure 2C, Table 6) . Sequence analysis revealed that ZCB3, ZCC10 and ZCD3 utilized IGHV3-53/3-66 heavy chain, whereas their paired light chains had distinct IGKV1-9, IGKV3-20 and IGKV1-27, respectively (Table 3) . In contrast, ZCB11 utilized different IGHV1-58 heavy chain and IGKV3-20 light chain. Our four new NAbs were all considered as public antibodies characterized by a IGHV3-53/3-66 heavy chain with 10-12 residues in the CDR3 region or a IGHV1-58 heavy chain with 15-17 residues in CDR3 region as previous reported by others (Schmitz et al., 2021; Tan et al., 2021; Zhang et al., 2021) . These results demonstrated BNT162b2-26 developed mainly public NAbs after two doses of vaccination.

Table 4. Binding ability of public NAbs to SARS-CoV-2 RBD and spike.

Table 5. Surface plasmon resonance analysis of ZCB11

Table 6. Neutralization IC50 values of public NAbs.

N.A.: Not applicable

Antibody neutralization of SARS-CoV-2 VOCs

To understand the breadth of these four newly cloned public RBD-specific NAbs, we performed SARS-CoV-2 neutralization assays using both pseudoviruses and authentic VOC isolates, including Alpha, Beta, Gamma, Delta and Omicron variants (Figures 2D-2H) . ZB8, a known RBD-specific class II NAb, was included as a positive control. Testing pseudoviruses in 293T-ACE2 cells, we found that ZCB11 was the best bNAb that neutralized all VOCs potently, including the most alarming Omicron variant (Liu et al., 2021b) , with IC50 values of around 30 ng/mL for Gamma and Delta variants 164 and 6 ng/mL for Alpha, Beta and Omicron variants (Figures 2D-2H, Table 6) . ZCB3 was the second best bNAb and neutralized Alpha, Beta, Gamma and Delta variants potently, but not the Omicron variant. ZCC10 and ZCD3 neutralized Alpha, Gamma and Delta variants at relative low potency, but lost neutralization totally against Beta and Omicron variants. Importantly, testing authentic VOC viruses in Vero-E6-TMPRSS2 cells, we consistently found that ZCB11 was the most potent bNAb, followed by ZCB3 (Figures 2I-2N) . The IC50 values of ZCB11 for neutralizing Alpha, Beta, Gamma, Delta, Omicron and OmicronR346K variants were 85.1, 39.9, 56.9, 11.2, 36.8 and 11.7 ng/mL, respectively, which were comparable to the IC50 value of 51 ng/ml for neutralizing the WT (Table 6) . ZCB3 was about 10-fold less potent than ZCB11 for neutralizing Beta and Omicron variants. Notably, the potency of ZCB11 in the pseudovirus assay was higher than that in the authentic virus assay, which was probably related to different target cells used. ZB8 showed unmeasurable and weak neutralization against Delta pseudovirus and Delta authentic virus, respectively. Conversely, ZCC10 and ZCD3 showed weak and unmeasurable neutralization against Gamma pseudovirus and Gamma authentic virus, respectively. These results demonstrated that ZCB11 functioned as an elite bNAb potently neutralized all circulating SARS-CoV-2 VOCs in vitro. Notably, although BNT162b2-26 developed mainly public NAbs, ZCB11 was unlikely dominantly elicited due to the reduced titer against Omicron as compared with WT (Figures 1A and 1F) .

Naturally occurred mutations or deletions conferring antibody resistance

Since Omicron variant escaped from NTD-specific NAbs and majority of known RBD-specific NAbs in the class I, class II, class III and class IV groups (Liu et al., 2021b; Planas et al., 2021a; Yunlong Cao, 2021) , we sought to determine possible mutations or deletions responsible for antibody resistance for ZCB3 and ZCB11 as compared with the control ZB8. We first constructed and tested a large panel of pseudoviruses carrying individual mutations or deletions found in Omicron variant as compared with those previously found in Alpha, Beta, Gamma and Delta variants (Figure 3A) . For ZB8, we consistently found that the E484 is essential for its neutralization activity. E484K in Beta, E484Q in Delta and E484A in Omicron were responsible for the significant ZB8 resistance, followed Q493R for about 10-fold resistance. For ZCB3, none of single mutations or deletions tested conferred resistance for equal to or more than 10-fold. Only and Q493R in Omicron reduced neutralization potency of around 3.5-fold. For ZCB11, only S371L in Omicron showed 11.2-fold resistance (Figure 3A) . Moreover, Q493R, Y505H, T547K and Q954H in Omicron exhibited over 6-fold resistance. Unexpectedly, when all these and other mutations combined in Omicron, they did not confer significant resistance at all. Subsequently, we performed antibody competition by Surface SPR. Although they engaged different clonotype and antibody resistant profiles, ZCB11 exhibited as a strong competitor for WT RBD binding against either ZCB3 or ZB8, respectively (Figures 3B-3C and Figures 7E-7F) , suggesting overlapped antibody binding epitopes in RBD between them. To further predict the binding mode of ZCB11, we searched structural database for RBD specific NAbs with similar B cell clonotype. Interestingly, the patient-derived S2E12 Nab, which used the same IGHV1-58 heavy chain and IGKV3-20 light chain (Tortorici et al., 2020) , shared the high amino acid identity of 82.2%in heavy chain variable regions with ZCB11. A model of ZCB11 variable regions was generated based on the protein sequence by the SWISS MODEL using the crystal structure of S2E12 Fab fragment (Research Collaboratory for Structural Bioinformatics [RCSB] PDB code 7K3Q) as the template. The superimposed ZCB11 and S2E12 variable regions (Figure 3D) showed that the secondary elements and most of loops are relatively conserved, except for the HCDR1 and KCDR3 which contained a single amino acid insertion and deletion, respectively. It is possible that ZCB11 also recognized the convex receptor binding motif (RBM) like S2E12. S477N, Q493R and Y505H mutations that conferred partial ZCB11 resistance in the pseudovirus assay were close to the binding interface between S2E12 and RBM (Figure 3E) .

In vivo efficacy of ZCB11 against SARS-CoV-2 Delta and Omicron variants

To determine the in vivo potency of ZCB11 against the dominant circulating VOCs, we conducted viral challenge experiments using the golden Syrian hamster COVID-19 model as compared with ZB8 (Chan et al., 2020a) . Since ZB8 conferred nearly complete lung protection against SARS-CoV-2 WT intranasal challenge at 4.5 mg/kg as we previously described (Zhou et al., 2021a) , we tested it in parallel with ZCB11 using the same dose according to our standard experimental procedure (Figure 4A) . One day prior vial challenge, three groups of hamsters (n=8) received the intraperitoneal injection of ZCB11, ZB8 and PBS, respectively. Twenty-four hours later, half of the animals (n=4) in each group were separated into subgroups and were challenged intranasally with 105 PFU of SARS-CoV-2 Delta variant and Omicron variant, respectively. Animal body weight changes were measured daily until day 4 when all animals were sacrificed for endpoint analysis. For three subgroups challenged with the SASR-CoV-2 Delta variant, we found that the infection caused around 10%body weight loss overtime in the PBS and ZB8 pre-treatment groups. In contrast, transient and less than 4%body weight decrease was observed for the ZCB11-treated hamsters (Figure 4B) . Moreover, relatively lower sub-genomic viral loads (Figure 4C) and unmeasurable numbers of live infectious viruses (six orders of magnitude drop) (Figure 4D) were achieved by ZCB11 than by ZB8. For hamsters challenged with the SASR-CoV-2 Omicron variant, no significant body weight loss was found in all three subgroups, indicating relatively weaker pathogenicity caused by Omicron than by Delta (Figure 4E) . However, significantly lower sub-genomic viral loads (Figure 4F) and unmeasurable numbers of live infectious viruses (Figure 4G) were achieved only by ZCB11. These results demonstrated that ZCB11 conferred significant protection against both Delta and Omicron variants, whereas ZB8 exhibited only partial protection against Delta but not Omicron. These findings were consistent with in vitro neutralizing activities of ZCB11 and ZB8 against live Delta and Omicron variants, respectively (Figures 2I-2N) . Since the number of infectious viruses in the PBS group of Delta-challenged hamsters was over one order of magnitude higher than that in the PBS group of Omicron-challenged animals (Figure 4D and 4G) , higher amount of ZCB11 might be needed for improved suppression of sub-genomic viral loads against the Delta variant.

DISCUSSION

It remains unclear what kind of human monoclonal NAbs can potently neutralize all current SARS-CoV-2 VOCs including the Omicron Variant. In this study, we showed that the standard two-dose BNT162b2 vaccination was able to induce spike-specific memory B cells, from which we successfully cloned the elite bNAb ZCB11 around 130 days post the second vaccination. We demonstrated that ZCB11 not only neutralized all authentic SARS-CoV-2 VOCs including Omicron and OmicronR346K at comparable high potency in vitro but also protected golden Syrian hamsters against the major circulating Omicron and Delta variants. Till now, few existing NAbs under clinical development have displayed similar neutralization breadth and in vivo potency (Liu et al., 2021b; Planas et al., 2021a) . Since sequence analysis revealed that ZCB11 was a family member of public antibodies with the IGHV1-58 heavy chain and IGKV3-20 light chain, our findings have significant implication to vaccine design for inducing high amounts of ZCB11-like bNAb for broad protection and for clinical development of ZCB11-based immunotherapy against the pandemic SARS-CoV-2 VOCs.

ZCB11 overcomes naturally occurred spike mutations and deletions across current SARS-CoV-2 VOCs. Alpha variant with D614G and N501Y mutations enhanced RBD binding to human ACE2 receptor, transforming it into the most prevalent variant at the early stage of 2021 (Yang et al., 2021) . N501Y alone was found conferring partial resistance to RBD-specific class I 910-30 and NTD-specific 4-18 NAbs (Liu et al., 2021b) . Subsequently, Beta, Gamma and Delta variants displayed the most troublesome mutations including K417N, E484K/Q/A and N501Y, conferring high resistance to RBD-specific class I and class II NAbs (Dejnirattisai et al., 2021b; Hoffmann et al., 2021; Liu et al., 2021b; Wang et al., 2021c) . E484K/Q/Aled to almost complete loss of neutralization by potent RBD-specific class II LY-CoV555 and 2-15 (Liu et al., 2021b) . Attributed probably by antibody evasion, Delta variant, carrying L452R/T478K/D614G/P681R mutations, were found in more than 170 countries and accounted for 99%of newly confirmed cases before the Omicron variant (Li et al., 2021; Liu et al., 2021a; Mlcochova et al., 2021) . After the emergence of the Omicron variant with more than 30 mutations in viral spike protein (Karim and Karim, 2021) , the ongoing wave of COVID-19 pandemic has already been dominated by it over the Delta variant in many countries probably due to further antibody evasion to almost all current vaccines and NAbs including those approved for clinical use or emergency use (Dejnirattisai et al., 2021a; Liu et al., 2021b; Wang et al., 2021d; Yunlong Cao, 2021) . N440K and G446S in Omicron conferred resistance to class III antibodies such as REGN10987 and 2-7 (Liu et al., 2021b) . G142D and del143-145 led to resistance to NTD-specific 4-18 and 5-7, whereas S371L conferred much broader resistance to RBD-specific class I, class III and class IV NAbs including potent Brii-196, REGN10987 and Brii-198 in clinical development (Liu et al., 2021b) . In this study, we consistently found that E484K/Q/Ain Beta, Delta and Omicron variants conferred strong resistance to our RBD-specific class II ZB8 NAb. These resistant mutations, however, did not affect the potency of ZCB11 significantly. Although S371L in Omicron displayed partial resistance (～11-fold) to ZCB11, similar amount of resistance was not observed against Omicron and OmicronR346K that also contained S371L (Figure 2N) . Although ZCB11 shared 86.5%amino acid identity in variable regions with the previously reported ultrapotent S2E12 bNAb (Chen et al., 2021; Tortorici et al., 2020) , it is critical to solve the real structure of RBD ZCB11 Fab complex in future studies to understand if ZCB11 and S2E12 use an identical mode of action, which will be useful for novel vaccine design to elicit ZCB11-like bNAb responses.

Most public antibodies were RBD-specific class I NAbs (Barnes et al., 2020; Liu et al., 2021b; Schmitz et al., 2021; Tan et al., 2021; Yuan et al., 2021) . Accordingly, public antibody is encoded by B cell clonotypes isolated from different individuals that share similar genetic features (Dong et al., 2021) . In a previous study, 7 of 13 NAbs were found using IGHV3-53/3-66 heavy chain and paired predominantly with IGKV1-9*01 light chain (Liu et al., 2021b) . These NAbs displayed abolished neutralizing activity after K417N in Beta variant was introduced into the pseudovirus neutralization assay. Interestingly, our newly cloned NAbs ZCB3, ZCC10 and ZCD3 utilized the same IGHV3-53/3-66 heavy chain but paired with IGKV1-9, IGKV3-20 and IGKV1-27 light chains, respectively (Table 7) . ZCB3, our second best bNAb, used the identical pair of IGHV3-53/3-66 and IGKV1-9 but did not display neutralization reduction against the K417N pseudovirus. ZCB3, however, showed reduced neutralization potency for over 10-fold against Beta and Omicron variants as compared with ZCB11. More interestingly, our elite bNAb ZCB11 used IGHV1-58 heavy chain and IGKV3-20 light chain, which also belongs to public antibodies reported by other groups (Dong et al., 2021; Schmitz et al., 2021; Tortorici et al., 2020; Wang et al., 2021b) . In these studies, patient-derived S2E12 and vaccine-induced 2C08 NAbs that shared 95%amino acid identity also used the same IGHV1-58 heavy chain and IGKV3-20 light chain. 2C08 was able to prevent challenges against Beta and Delta variants in the hamster model. Like 82.2%amino acid identity between ZCB11 and S2E12, ZCB11 and 2C08 shared 83.8%amino acid identity in their heavy chain variable regions. Their potency difference for neutralizing Omicron remained to be determined. Nevertheless, vaccine design in eliciting high amounts of ZCB11-like bNAb should be considered as a research priority, especially after its clonotype has been found in different ethnic human populations but have not been abundantly induced by current vaccines. Since ZCB11 protected hamsters against both the Delta and Omicron variants, the most dominant circulating SARS-CoV-2 VOCs in the world, our findings warrant the clinical development of ZCB11 and ZCB11-like bNAbs for patient immunotherapy and transmission prevention.

ZCB11 probably represents the broadest breadth among bNAbs reported thus far with comparable potency against all current SARS-CoV-2 VOCs including Omicron and OmicronR346K.

Table 7. Gene family analysis of four neutralizing antibodies

Example 2: Vaccine-breakthrough infection by the SARS-CoV-2 omicron variant elicits broadly cross-reactive immune responses

RESULTS

After the World Health Organization (WHO) designated the omicron variant of concern (VOC) on the 26 November 2021, the extremely rapid spread of this variant is replacing the delta VOC with increased risk of vaccine-breakthrough infection in the South Africa, in European countries and in the United States (Shu and McCauley) . Both vaccine-induced neutralising antibodies (NAbs) and current NAbs in combination therapy have shown significantly reduced activities (Wang et al. (2021e) ; Lu et al. ) . Till now, it remains unclear whether vaccine-induced memory responses can be recalled by the omicron viral infection. We, therefore, investigated the host immune responses in two cases of vaccine-breakthrough omicron infection in Hong Kong.

In mid-November 2021, the first Chinese case of omicron patient (OP1) was diagnosed in a quarantine hotel in Hong Kong (Wong et al. ) . About 9 days after the OP1, omicron patient 2 (OP2) , who was due to a separate transmission event, was also confirmed by sequencing analysis. Based on the vaccination records, OP1 and OP2 were confirmed with omicron infection at 178 and 53 days after the second dose of BNT162b2 and mRNA-1273, respectively (Table 8) . During hospitalisation, both cases presented with mild clinical symptoms not requiring oxygen supplementation or ICU treatment. With patients’ informed consent, we obtained three sequential sera and one peripheral blood mononuclear cell (PBMC) samples from each patient to determine their immune responses recalled by the omicron viral infection.

Table 8. Subject Characterization

IQR: interquartile range

We first measured the neutralising antibody titre (IC ₅₀) in their serum samples against the current panel of SARS-CoV-2VOC pseudoviruses, including alpha (B. 1.1.7) , beta (B. 1.351) , gamma (P1) , delta (B. 1.617.2) and omicron (B. 1.1.529) as compared with D614G (WT) (Figures 8A-8F) . We compared IC ₅₀ values of 34 local vaccinees, whose blood samples were collected around the mean 30 days after the second BNT162b2-vaccination (Pfizer–BioNTech) (Table 8; Peng et al. ) . Consistent with recent preprint publications by others, we found that the omicron variant showed the greatest resistance to BNT162b2 vaccine-induced neutralisation with an average 5.9-fold deficit relative to D614G (Table 9) . Strikingly, however, the breakthrough infection was able to elicit cross-reactive broadly neutralising antibodies (bNAbs) from the unmeasurable level (<1: 20) to the mean IC ₅₀ value of 1: 2929 (range 588.5–5508) at 9 days post symptoms onset (PSO) in OP1 and from the mean IC ₅₀ value of 1: 24.3 to 1: 854.5 at 12 days PSO in OP2, respectively (Figures 9A-9B) . Moreover, the amounts of NAbs in OP1 and OP2 were consistently higher than the mean IC ₅₀ values of BNT162b2 vaccinees across all VOCs tested (Figures 8A-8F) . In particular, there were 121.41-and 74.89-fold higher IC ₅₀ values against beta and omicron in OP1 than those in BNT162b2 vaccinees (Figures 8A-8F) . Besides NAbs against the current panel of VOCs, OP1 also displayed enhanced IC ₅₀ values of NAbs against 15/16 SARS-CoV-2 variants with individual mutations or deletions, including the E484Kmutation, which conferred significant resistance to vaccine-induced NAbs. These results demonstrated that although the omicron VOC evaded BNT162b2 vaccine-induced NAbs, the breakthrough infection could recall cross-reactive bNAbs generally against all current VOCs in both OP1 and OP2.

Table 9. Fold Change of IC ₅₀ Relative to D614G

Variant	Vaccinees
Alpha	1.41
Beta	-3.79
Gamma	1.33
Delta	-3.17
Omicron	-5.90

Multicolour flow cytometry data showed no sign of severe immune suppression in OP1 and OP2 who had normal frequencies of T lymphocyte (no lymphocytopenia) , stable conventional dendritic cell (cDC) : plasmacytoid dendritic cell (pDC) ratio and normal myeloid-derived suppressor cells (MDSCs) similar to mild and healthy subjects, as we described previously (Zhou et al. (2020) ) . We also measured the frequency of Spike-specific IgG ₊ B cells, 13.2%in OP1 and 2.31%in OP2 were relatively higher than the mean 1.12% (range 0.004%–7.92%) found among BNT162b2 vaccinees around their peak responses (Figure 19) . Unlike SARS-CoV-2 infection in unvaccinated patients, who display predominantly tissue-like memory (TLM) B-cell response (Woodruff et al. ) . Spike-specific IgG ₊ B cells from OP1 and OP2 exhibited the dominant phenotype of resting memory (RM) (Figures 20A-20B) , which was commonly found among healthy BNT162b2 vaccinees.

Isolation of SARS-CoV-2 specific monoclonal antibodies (mAbs) . We sorted the antigen-specific immunoglobulin G-positive (IgG+) memory B cells from the PBMCs of OP1 at the single cell level using SARS-CoV-2 spike extracellular domain (S-ECD) as the bait. The S-ECD positive memory B cells accounts for 4.46%of total memory B cells, which is much higher than previous report by us and others (Ju et al., 2020; Liu et al., 2020; Zhou et al., 2022) . From the PBMCs of OP1, we sorted totally 161 S-ECD reactive memory B cells and successfully recovered 122 heavy chain sequences and 129 light chain sequences, respectively (Figures 10A and 10C) . By antibody repertoire analysis, we found that multiple clonotypes were activated in the heavy chain including IGHV1-53, IGHV3-66, IGHV3-11 and IGHV1-69 (Figures 10B and 10D) . However, IGKV1-39 is the most predominant light chain. The mean somatic hypermutation (SHM) for heavy chain ranges from 0.3 %to 13.1 %with the mean value of 4.9 %, which is higher compared to that of light chain with the mean SHM of 3.8 %ranging from 0 %to 12.2 % (Figure 11A) . In addition, the mean length of complementarity determining region 3 (CDR3) ranges from 10 to 34 for heavy chain and 8 to 12 for light chain, with the mean CDR3 length of 14.6 and 9.1 for heavy chain and light chain, respectively (Figure 11B) . The VDJ recombination for heavy chain is also very diverse, with the most common recombination of IGHD3 and IGHV3 for IgH gene family (Figures 12A-12D) . Whereas the recombination of IGKV1 and IGKJ2 is the most common usage for IgK gene family (Figures 12A-12D) . Taken together, these results indicated that multiple antibody clonotypes were induced by vaccine-breakthrough infection of SARS-CoV-2 Omicron variant in this patient.

NAb screening and binding activity of five lead bNAbs to SARS-CoV-2 spikes was performed by ELISA. From the recovered antibody sequences, we successfully expressed 104 recombinant mAbs with natural pairing of VH and VL. 63 mAbs were able to bind to SARS-CoV-2 spike as determined by ELISA. 27 of them showed potent neutralizing capability against pseudotyped SARS-CoV-2 WT, whereas 12 of them were able to neutralize both pseudotyped SARS-CoV-2 WT and Omicron (Figures 13A-13B) . Considering the importance of SARS-CoV-2 Omicron variant, we focused on the most potent broad neutralizing antibodies (P1D9, P2B4, P2B11, P2D9, P2E7) by measuring binding affinity to SARS-CoV-2 both WT and Omicron (Figures 14A-14B) . All five antibodies displayed high binding affinity to WT (D614G) spike with the EC50 ranging from 0.009 to 0.040 μg/ml. Reduced binding affinity to Omicron spike was observed except P2B11, which may be due to the different conformation of soluble Omicron trimer spike from the native spike on viral particles (Table 10) .

Table 10. Binding Affinity of Selected Antibodies

EC50 (μg/ml)	P1D9	P2B4	P2B11	P2D9	P2E7
WT	0.016	0.04	0.011	0.03	0.009
Omicron	0.128	0.158	0.027	1.228	0.045

Potent broad neutralization was seen against against both pseudotyped and live SARS-CoV-2 viruses. For the pseudovirus assay, all five bNAbs can potently neutralize SARS-CoV-2 WT with the IC50 values ranging from 0.004 to 0.008 μg/ml and SARS-CoV-2 Omicron variant with the IC50 values ranging from 0.006 to 0.035 μg/ml (left and right) , respectively (Figures 15A-15B) . Consistently, all five bNAbs show comparable neutralizing capability against authentic SARS-CoV-2 WT, whereas P1D9 and P2D9 had relatively weaker neutralization against the live SARS-CoV-2 Omicron compared to the other antibodies, respectively (Figures 16A-16B; Table 11) .

Table 11. IC50 of Selected Antibodies

No competitive binding was seen between various combinations of antibodies to SARS-CoV-2 RBD, including between ZCB11 and P1D9 (Figures 17A and 17B) , between ZCB11 and P2D9 (Figures 17C and 17D) , between ZCB11 and P2E7 (Figures 17E and 17F) , between P2B4 and P2D9 (Figures 17G and 17H) .

Competitive binding was seen between various combinations of antibodies to SARS-CoV-2 RBD, including between P1D9 and P2B4 (Figures 18A and 18B) , between ZCB11 and P2B4 (Figures 18C and 18D) , between P2E7 and P2B4 (Figures 18E and 18F) , between P2D9 and P2E7 (Figures 18G and 18H) , between P1D9 and P2D9 (Figures 18I and 18J) , and between P1D9 and P2E7 (Figures 18K and 18L) .

We further measured their cross-reactive T-cell responses to the Spike and nucleocapsid (NP) peptide pools derived from the SARS-CoV-2 wildtype as compared with BNT162b2 vaccinees by intracellular cytokine staining (ICS) . The cytomegalovirus (CMV) pp65 peptide pool was used as a positive control. We found that Spike and NP-specific CD4 IFN-γ responses were 0.61%and 0.12%in OP1 and 0.15%and 0.10%in OP2, respectively (Figure 21A) . Spike-and NP-specific CD8 IFN-γ responses were 0.56%and 0.11%in OP1 and 0.10%and 0.08%in OP2 (Figure 21B) , respectively. Moreover, the Spike-specific CD4 and CD8 T-cell responses were relatively higher in OP1 or comparable in OP2 as compared with mean values in BNT162b2 vaccinees (CD4 T: mean 0.19%and CD8 T: mean 0.10%) . As much weaker or unmeasurable T-cell responses were found in severe COVID-19 patients around the same period PSO (Zhou et al. (2020) ; Rydyznski Moderbacher et al. ) , T-cell responses in OP1 and OP2 probably also contributed to disease progression control. As the omicron variant caused a higher rate of vaccine breakthrough infection and reinfection than the delta variant (Espenhain et al. ) , it is worrisome if such infections would lead to more severe sickness or death due to immune escape. In this study, we demonstrated that the omicron breakthrough infection rapidly recalled vaccine-induced memory bNAbs and T-cell immune responses, which very likely contributed to protection in OP1 and OP2. Our finding provides a probable immune mechanism underlying a recent report that most omicron patients had no signs of severe COVID-19 as compared with the delta variant (Espenhain et al. ) . Our findings, therefore, re-emphasize the importance of complete vaccination coverage among human populations, especially in developing countries. Notably, the ongoing adaptive evolution of SARS-CoV-2 created an unprecedented demand of vaccines against VOCs (Rochman et al. ) . As similarly high amounts of bNAbs against both omicron and other VOCs were detected in OP1 and OP2, the rapid development of omicron-based vaccine is a reasonable strategy as a booster vaccine to elicit and sustain long-term cross-protective immunity against COVID-19.

Cross-reactive immune responses elicited by vaccine-breakthrough infection of the SARS-CoV-2 omicron variant. Characteristics of two omicron patients and 34 BNT162b2 vaccinees (Table 8) . Neutralising antibody titres among the BNT162b2 vaccinees (grey) (n = 34) and two omicron patients (OP1: red and OP2: blue) at the peak response time. Neutralising antibody titres represent serum dilution required to achieve 50%virus neutralisation (IC ₅₀) . The numbers indicate the fold of enhancement of IC ₅₀ values relative to mean titre measured among BNT162b2 vaccinees (Figures 8A-8F) . Fold-change of mean IC ₅₀ values relative to the SARS-CoV-2 D614 G strain among the BNT162b2 vaccinees (Table 9) . Longitudinal neutralising antibody titres (IC ₅₀) of OP1 and OP2 against the full panel of variants of concern (VOCs) (Figures 9A-9B) . Each symbol with colour coding represents an individual VOC. Dash line indicates the limit of detection (LOD) (1: 20) .

PBMCs were isolated from the blood collected at 11 and 12 days PSO of OP1 and OP2, respectively. PBMCs were further subjected to the measurement of antigen-specific B-and T-cell responses (Figures 19, 20A-20B, and 21A-21B) . The gating strategy for SARS-CoV-2 Spike-specific B cells by flow cytometry. AF488 and AF647 double-positive cells were defined as Spike-specific cells. Quantified results are shown (Figure 19) . Phenotypes of Spike-specific B cells were defined by using CD21 and CD27 markers. Pie chart shows the proportion of activated (AM) , tissue-like memory (TLM) , intermediate memory (IM) and resting-memory (RM) B cells (Figures 20A-20B) . PBMCs were subjected to the ICS assay against Spike or NP or CMV peptide pools. IFN-γ ₊ cells were gated on CD4 and CD8 T cells, respectively. Quantified results depict the percentage of IFN-γ ₊ cells (Figure 21) .

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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:

(a) the three light chain CDRs selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, and 64; and

(b) the three heavy chain CDRs selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, and 63,

wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 spike protein or a component thereof.
The antibody or antigen binding fragment thereof of claim 1 comprising the light chain variable region of antibody ZCB11.
The antibody or antigen binding fragment thereof of claim 1 comprising the light chain variable region of antibody ZCB3.
The antibody or antigen binding fragment thereof of claim 1 comprising the light chain variable region of antibody ZCC10.
The antibody or antigen binding fragment thereof of claim 1 comprising the light chain variable region of antibody ZCD3.
The antibody or antigen binding fragment thereof of claim 1 comprising the light chain variable region of antibody ZCD4.
The antibody or antigen binding fragment thereof of claim 1 comprising the light chain variable region of antibody P1D9.
The antibody or antigen binding fragment thereof of claim 1 comprising the light chain variable region of antibody P2B4.
The antibody or antigen binding fragment thereof of claim 1 comprising the light chain variable region of antibody P2B11.
The antibody or antigen binding fragment thereof of claim 1 comprising the light chain variable region of antibody P2D9.
The antibody or antigen binding fragment thereof of claim 1 comprising the light chain variable region of antibody P2E7.
The antibody or antigen binding fragment thereof of any one of claims 1-11 comprising a heavy chain variable region of antibody ZCB11.
The antibody or antigen binding fragment thereof of any one of claims 1-11 comprising a heavy chain variable region of antibody ZCB3.
The antibody or antigen binding fragment thereof of any one of claims 1-11 comprising a heavy chain variable region of antibody ZCC10.
The antibody or antigen binding fragment thereof of any one of claims 1-11 comprising a heavy chain variable region of antibody ZCD3.
The antibody or antigen binding fragment thereof of any one of claims 1-11 comprising a heavy chain variable region of antibody ZCD4.
The antibody or antigen binding fragment thereof of any one of claims 1-11 comprising a heavy chain variable region of antibody P1D9.
The antibody or antigen binding fragment thereof of any one of claims 1-11 comprising a heavy chain variable region of antibody P2B4.
The antibody or antigen binding fragment thereof of any one of claims 1-11 comprising a heavy chain variable region of antibody P2B11.
The antibody or antigen binding fragment thereof of any one of claims 1-11 comprising a heavy chain variable region of antibody P2D9.
The antibody or antigen binding fragment thereof of any one of claims 1-11 comprising a heavy chain variable region of antibody P2E7.
The antibody or antigen binding fragment thereof of claim 1,

(1) wherein the CDRs comprise:

(a) the three light chain CDRs of SEQ ID NO: 4 and the three heavy chain CDRs of SEQ ID NO: 3,

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

(c) the three light chain CDRs of SEQ ID NO: 6 and the three heavy chain CDRs of SEQ ID NO: 5, or

(d) the three light chain CDRs of SEQ ID NO: 8 and the three heavy chain CDRs of SEQ ID NO: 7, and

wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 RBD; or

(2) wherein the CDRs comprise the three light chain CDRs of SEQ ID NO: 10 and the three heavy chain CDRs of SEQ ID NO: 9, wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 S1.
The antibody or antigen binding fragment thereof of claim 1,

(1) wherein the CDRs comprise:

(a) the three light chain CDRs and the three heavy chain CDRs of antibody ZCB11,

(b) the three light chain CDRs and the three heavy chain CDRs of antibody ZCB3,

(c) the three light chain CDRs and the three heavy chain CDRs of antibody ZCC10, or

(d) the three light chain CDRs and the three heavy chain CDRs of antibody ZCD3, and

wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 RBD; or

(2) wherein the CDRs comprise the three light chain CDRs and the three heavy chain CDRs of antibody ZCD4, wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 S1.
The antibody or antigen binding fragment thereof of claim 1,

wherein the CDRs comprise:

(a) the three light chain CDRs of SEQ ID NO: 12 and the three heavy chain CDRs of SEQ ID NO: 11,

(b) the three light chain CDRs of SEQ ID NO: 14 and the three heavy chain CDRs of SEQ ID NO: 13,

(c) the three light chain CDRs of SEQ ID NO: 16 and the three heavy chain CDRs of SEQ ID NO: 15,

(d) the three light chain CDRs of SEQ ID NO: 18 and the three heavy chain CDRs of SEQ ID NO: 17, or

(e) the three light chain CDRs of SEQ ID NO: 20 and the three heavy chain CDRs of SEQ ID NO: 19, and

wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 spike protein.
The antibody or antigen binding fragment thereof of claim 1,

wherein the CDRs comprise:

(a) the three light chain CDRs and the three heavy chain CDRs of antibody P1D9,

(b) the three light chain CDRs and the three heavy chain CDRs of antibody P2B4,

(c) the three light chain CDRs and the three heavy chain CDRs of antibody P2B11,

(d) the three light chain CDRs and the three heavy chain CDRs of antibody P2D9, or

(e) the three light chain CDRs and the three heavy chain CDRs of antibody P2E7,

wherein the antibody or antigen binding fragment thereof binds to SARS-CoV-2 spike protein.
The antibody or antigen binding fragment thereof of any one of claims 1-25, wherein the antibody or antigen binding fragment thereof attenuates the ability of a ligand of SARS-CoV-2 spike protein or RBD to bind to ACE2.
The antibody or antigen binding fragment thereof of any one of claims 1-26 comprising one or more constant domains from an immunoglobulin constant region (Fc) .
The antibody or antigen binding fragment thereof of claim 27, wherein the constant domains are human constant domains.
The antibody or antigen binding fragment thereof of claim 28, wherein the human constant domains are IgA, IgD, IgE, IgG or IgM domains.
The antibody or antigen binding fragment thereof of claim 29, wherein human IgG constant domains are IgG1, IgG2, IgG3, or IgG4 domains.
The antibody or antigen binding fragment thereof of any one of claims 1-30, 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 1-31, 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 1-32, 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

the light chain variable region and heavy chain variable region of antibody ZCB11,

the light chain variable region and heavy chain variable region of antibody ZCB3,

the light chain variable region and heavy chain variable region of antibody ZCC10,

the light chain variable region and heavy chain variable region of antibody ZCD3,

the light chain variable region and heavy chain variable region of antibody ZCD4,

the light chain variable region and heavy chain variable region of antibody P1D9,

the light chain variable region and heavy chain variable region of antibody P2B4,

the light chain variable region and heavy chain variable region of antibody P2B11,

the light chain variable region and heavy chain variable region of antibody P2D9,

the light chain variable region and heavy chain variable region of antibody P2E7,

the light chain variable region of ZCB11 and the heavy chain variable region of antibody P2B11, or

the light chain variable region of SEQ ID NO: 4 and the heavy chain region of SEQ ID NO: 65.
A pharmaceutical composition comprising the antibody or antigen binding fragment thereof of any one of claims 1-34 and a physiologically acceptable carrier or excipient.
The pharmaceutical composition of claim 35 for use in a method of preventing or treating COVID-19 in a subject.
The pharmaceutical composition for use of claim 36, wherein the subject has COVID-19.
The pharmaceutical composition for use of claim 37, wherein the subject is at risk of developing COVID-19.
The pharmaceutical composition of claim 35 for use in a method of treating COVID-19.
The pharmaceutical composition of claim 35 for use in a method of preventing COVID-19.
Use of the antibody or antigen binding fragment thereof of any of claims 1-34 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 1-34 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 1-34 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 spike protein or RBD in a sample from a subject using the antibody or antigen binding fragment thereof of any one of claims 1-34 and (b) comparing the level of the SARS-CoV-2 spike protein or RBD with a control level, wherein an increase in the assayed level of SARS-CoV-2 spike protein or RBD compared to the control level is indicative of SARS-CoV-2 infection.
The method of claim 44, wherein the presence of SARS-CoV-2 spike protein or RBD is assayed by enzyme linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , or fluorescence-activated cell sorting (FACS) .
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 35 if the subject has a disease characterized by increased expression of SARS-CoV-2 spike protein or RBD.
The method of claim 46, wherein the antibody or antigen binding fragment thereof is the antibody or antigen binding fragment thereof of any one of claims 1-34.