WO2022101839A1

WO2022101839A1 - Anti-sars-cov-2 monoclonal antibodies and cocktail thereof

Info

Publication number: WO2022101839A1
Application number: PCT/IB2021/060495
Authority: WO
Inventors: Sanjeev Kumar Mendiratta; Arun Kumar Singh; Aashini Parikh; Hardik Pandya; Vibhuti SHARMA; Sanjay Bandyopadhyay
Original assignee: Cadila Healthcare Limited
Priority date: 2020-11-12
Filing date: 2021-11-12
Publication date: 2022-05-19

Abstract

The present invention provides monoclonal antibody capable of binding and neutralizing virus of coronavirus family, preferably SARS-CoV-2. It also provides a cocktail of at least two monoclonal antibodies capable of binding and neutralizing virus of coronavirus family, preferably SARS-CoV-2. According to the present invention, the monoclonal antibody or cocktail of at least two monoclonal antibodies can bind and neutralize coronavirus including, but not limited to, SARS-CoV-2, MERS CoV and SARS-CoV. In one aspect, the present invention provides pharmaceutical composition of monoclonal antibody or cocktail of atleast two monoclonal antibody capable of binding and neutralizing virus of coronavirus family, preferably SARS-CoV-2. In another aspect, present invention provides process of preparing monoclonal antibody or cocktail of atleast two monoclonal antibody capable of binding and neutralizing virus of coronavirus family, preferably SARS-CoV-2.

Description

ANTI-SARS-COV-2 MONOCLONAL ANTIBODIES AND COCKTAIL

THEREOF

FIELD OF THE INVENTION

The present invention relates to a monoclonal antibody or cocktail of atleast two monoclonal antibodies capable of binding and neutralizing virus of corona virus family, preferably SARS-CoV-2.

BACKGROUND OF THE INVENTION

Newly identified viruses can be difficult to treat because they are not sufficiently characterized. The emergence of these newly identified viruses highlights the need for the development of novel antiviral strategies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly-emergent coronavirus which causes a severe acute respiratory disease, Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 was first identified from an outbreak in Wuhan, China and as of November 01, 2021, a total of 247,480,119 confirmed cases were reported including 5,015,327 deaths globally, in China, Europe, USA, India and at least 200 other countries and / or territories (1). Clinical features of COVID-19 include fever, dry cough, and fatigue, and the disease can cause respiratory failure resulting in death. Thus far, there has been no vaccine or therapeutic agent to prevent or treat SARS-CoV-2 infection. In view of the continuing threat to human health, there is an urgent need for preventive and therapeutic antiviral therapies for SARS-CoV-2 control. Because this virus uses its spike glycoprotein for interaction with the cellular receptor ACE2 and the serine protease TMPRSS2 for entry into a target cell, this spike protein represents an attractive target for antibody therapeutics (2). Currently, the intermediate host of SARS-CoV-2 is still unknown, and no effective prophylactics or therapeutics are available. It shows an urgent need for the immediate development of vaccines and antiviral drugs for prevention and treatment of CO VID-19 (3). More than 100 pre-clinical or clinical trials are going on, which include repurposing of drugs already approved for different indications such as antimalarial, anti-viral, anti-parasitic drugs, cytokines or complement targeting antibodies, etc. Still, there is a growing need of antivirals against novel coronavirus SARS-CoV-2. While a number of vaccines are in development, therapeutics would still be needed as vaccines even if highly successful, would need a long time to be vaccinate the entire population of 7.5 billion people. Monoclonal antibodies have received less attention even though neutralizing antibodies are a key component of protective immunity for most viral diseases. Neutralizing monoclonal antibodies to SARS-CoV-2 have the potential for both therapeutic and prophylactic applications, and can help to guide vaccine design and development (5). In particular, fully human antibodies that specifically bind to the SARS-CoV-2-Spike protein (SARS-CoV-2-S) with high affinity and that inhibit virus infectivity could be important in the prevention and treatment of COVID-19. At present, two cocktail monoclonal antibodies namely, casirivimab & imdevimab of regeneron pharmaceutical and bamlanivimab & etesevimab of Eli Lilly have been authorized for treatment of SARS-CoV-2. Thus, in view of the continuing threat to human health, there is further need for therapeutic antiviral therapies for SARS-CoV-2 control. Current patent application claims a monoclonal antibody or cocktail of atleast two monoclonal antibodies capable of binding and neutralizing virus of corona virus family, preferably SARS-CoV-2.

SUMMARY OF THE INVENTION

The present invention provides a monoclonal antibody capable of binding and neutralizing virus of coronavirus family, preferably SARS-CoV-2. It also provides a cocktail of at least two monoclonal antibodies capable of binding and neutralizing virus of coronavirus family, preferably SARS-CoV-2. Further, the antibody or cocktail of at least two antibodies of the present invention have reduced binding to FcγRs to minimize its ADCC activity. The present invention may also provide an antibody or cocktail of at least two antibodies to minimize its CDC activity. The present invention may also provide an antibody or cocktail of at least two antibodies to minimize its ADCP activity. The antibody or cocktail of at least two antibodies according to the present invention may have higher affinity towards human neonatal Fc receptor (hFcRn). The antibody or cocktail of at least two antibodies according to the present invention may have long circulating half-life in the body of the patient and it can be given at a reduced dosing frequency. In one of the aspects, the present invention provides pharmaceutical composition of monoclonal antibody or cocktail of atleast two monoclonal antibody capable of binding and neutralizing virus of coronavirus family, preferably SARS-CoV-2.

List of abbreviations used herein the specification

Table 1 : Abbreviations of amino acid as used in the specification

Other abbreviations used herein the specification ADCC: Antibody-dependent cell-mediated cytotoxicity,

ADCP: Antibody-dependent cell-mediated phagocytosis

ADE: Antibody dependent enhancement

AUC_{inf_obs}: Area under the curve from zero till infinite observation

AUC_last: Area under the curve from zero till last observation and BSA: Bovine Serum Albumin CDC: Complement-dependent cytotoxicity

C_max : Maximum Serum Concentration

DMEM: Dulbecco's Modified Eagle Medium

EC₅₀ : Half-maximal Effective concentration

ELISA: Enzyme-linked immunosorbent assay

FBS: Fetal bovine serum

HC: Heavy chain

HR2: heptad-repeat 2 regions

IC₅₀: Half-maximal inhibitory concentration

IgGl: Immunoglobulin 1

LC: Light chain mAb: Monoclonal antibody

MEM: Minimum essential medium

MERS CoV : Middle east respiratory syndrome coronavirus mM: millimolar

MOI: Multiplicity of Infection

N: Number of animals

NHP: Non-human primate nM : nanomolar

RBD: Receptor binding domain rhFcRn: recombinant human neonatal Fc receptor

SARS-CoV: Severe acute respiratory syndrome coronavirus

SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2

SARS-CoV-2-S: Severe acute respiratory syndrome coronavirus 2 spike protein

SD: Standard deviation

SEQ ID No. : Sequence ID number

SOC: Standard of Care

SPR: Surface Plasmon Resonance

TMB: 3, 3', 5, 5'-Tetramethylbenzidin Definitions

The term “antibody” as referred to herein includes whole antibodies and any antigen- binding fragment (i.e.“antigen-binding portion”) or single chains thereof. An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., immune effector cells) and the first component (Clq) of the classical complement system. Herein the present invention, antigen is SARS-CoV-2 or SARS-CoV and antibodies of the present invention are referred as anti SARS-CoV-2 or anti SARS-CoV-2-S.

The term “operatively linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.

The term “Ka” & “Kd” are well known to a skilled person, wherein “Ka” is the association rate of a particular antibody-antigen interaction, whereas the term “Kd” is the dissociation rate of a particular antibody-antigen interaction. The term “K_D” is an affinity rate constant, which is obtained from the ratio of Kd to Ka. It can be measured by using surface plasmon resonance method which is well known in the art. K_D value is a measurement of the binding affinity of the antibody towards its target antigen. The term “K_D” is also defined in WO 2006121168. This patent document is incorporated herein by reference.

The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

The term “bispecific antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a two different antigenic determinants, or epitopes.

The term “recombinant antibody” according to the present invention, includes monoclonal antibodies which are generated recombinantly using synthetic heavy and light chain genes. Recombinant antibodies of this invention are monoclonal antibodies (mAbs) which are not produced using traditional hybridoma- based technologies, and do not need hybridomas and animals in the production process. The term “immune effector function” as used herein is a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include but are not limited to ADCC, ADCP, and CDC. The term also represents a physiological event such as circulating half-life of a drug or targeting of a drug to a particular cell or tissue type.

The term “ADCC” or “antibody dependent cell-mediated cytotoxicity” as used herein is the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

The term “ADCP” or “antibody dependent cell-mediated phagocytosis” as used herein is the cell-mediated reaction wherein nonspecific cytotoxic cells that express FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

Complement-dependent cytotoxicity (CDC) refers to the condition in which the Clq binds the antibody and this binding triggers the complement cascade which leads to the formation of the membrane attack complex (MAC) (C5b to C9) at the surface of the target cell, as a result of the classical pathway complement activation. The term “immune effector cell” as used herein is a cell that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and γδT cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys.

The term “Fc” fragment, whose name reflects its ability to crystallize readily. In human IgG molecules, the Fc region is generated by papain cleavage N-terminal to Cys 226. The Fc region is central to the immune-effector functions of antibodies.

The term “Fc protein” as used herein refers to the portion of a single immunoglobulin heavy chain beginning in the hinge region just upstream of the papain cleavage site and ending at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a portion of a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, and a CH3 domain.

The term “cocktail” as used herein is a mixture of at least two monoclonal antibodies as two independent active drug substances. The cocktail according to the present invention comprises mixture of at least two monoclonal antibodies selected from mAb A, mAb B, mAb C, mAb D, mAb E, mAb F, mAb G, mAb H, mAb I, mAb J, mAb K, mAb L, mAb M, mAb N, mAb O and mAb P. Preferred cocktail comprises at least one monoclonal antibody selected from mAb A, mAb B, mAb C, mAb D, mAb E, mAb F, mAb G and mAb H along with the another antibody selected from mAb I, mAb J, mAb K, mAb L, mAb M, mAb N, mAb O and mAb P. Preferably, the said cocktail is prepared in the formulation buffer. Cocktail is prepared by mixing therapeutic amounts, preferably equipotent amounts of the two active drug substance materials of monoclonal antibodies in the formulation buffer. Finally, it is formulated with other suitable pharmaceutical excipient(s) to prepare a stable pharmaceutical composition which can be used for therapeutic or treatment purpose.

The term “coronavirus” or “CoV” refers to any virus of the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV. SARS-CoV- 2 refers to the newly-emerged coronavirus which was identified as the cause of a serious outbreak starting in Wuhan, China, and which is rapidly spreading to other areas of the globe. SARS-CoV-2 has also been known as 2019-nCoV and Wuhan coronavirus. It binds via the viral spike protein to human host cell receptor angiotensin- converting enzyme 2 (ACE2). The spike protein also binds to and is cleaved by TMPRSS2, which activates the spike protein for membrane fusion of the virus.

The term “treatment” or “therapeutics” as used herein, refers to any treatment of a disease in a mammal, particularly in a human. It includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

The terms “patient” and “subject” are used interchangeably and are used in their conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a composition of the present invention, and includes both humans and non-human animals. Examples of subjects include, but are not limited to, humans, chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, adult, juvenile and new born individuals are of interest.

In the context of the present invention, a “therapeutically effective amount” or “effective amount” of antibody or cocktail of at least two antibodies refers to an amount effective for use in the treatment of SARS-CoV-2 infection and its related clinical manifestations. An “effective amount” of an antibody of the invention, or composition thereof, is an amount that is delivered to a mammalian subject, either in a single dose or as part of a series, which is effective for inducing an immune response against target antigen in said subject. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

A “pharmaceutically effective dose” or “therapeutically effective dose” is that dose required to treat or prevent, or alleviate one or more SARS-CoV-2 related disorder or symptom in a subject, preferably in the present invention, for cancer or infection or autoimmune disease. The pharmaceutically effective dose depends on inter alia the specific compound (herein it is anti-SARS-CoV-2 antibody or its combination or conjugate or bispecific) to administer, the severity of the symptoms, the susceptibility of the subject to side effects, the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration such as health and physical condition, concurrent medication, the degree of protection desired, and other factors that those skilled in the medical arts will recognize.

Table 2: List of amino acid sequence used herein the specification

Table 3: List of nucleotide sequence used herein the specification

DESCRIPTION OF FIGURES

Figure 1 : It depicts vector map of dual expression vector pDual Cov2. It expresses HC and LC together from the single vector.

Figure 2: It illustrates the Clq binding responses of anti-SARS-CoV-2 antibodies mAb A, mAb D and mAb E

Figure 3: It illustrates the Clq binding responses of anti-SARS-CoV-2 antibodies mAb

I, mAb L and mAb M

Figure 4: It illustrates neutralization potency of the anti-SARS-CoV-2 antibodies mAb

A, mAb D, mAb E, mAb I, mAb L and mAb M

Figure 5 : Inhibition of RBD binding to ACE2 in the presence of mAb E and mAb M

Figure 6A: Binding of mAb M on mAb E pre-captured channels

Figure 6B: Binding of mAb E on mAb M pre-captured channels

Figure 7: Neutralization potential of mAb E, mAb M and cocktail of mAb E & mAb M. Data is presented as percentage inhibition of SARS-CoV-2 virus by the antibodies as compared to control (no antibodies).

Figure 8A: Pharmacokinetic (PK) profile of mAb E in hamster serum

Figure 8B: Pharmacokinetic (PK) profile of mAb M in hamster serum

Figure 9A: Pharmacokinetic (PK) profile of mAb E in NHP serum

Figure 9B: Pharmacokinetic (PK) profile of mAb M in NHP serum

EMBODIMENTS OF THE INVENTION

Embodiment 1

The present invention provides monoclonal antibody or cocktail of at least two monoclonal antibodies that binds SARS-CoV-2 spike protein.

Embodiment 2

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:1 and light chain amino acid sequence set forth in SEQ ID NO: 9.

Embodiment 3

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:2 and light chain amino acid sequence set forth in SEQ ID NO: 9.

Embodiment 4

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:3 and light chain amino acid sequence set forth in SEQ ID NO: 9.

Embodiment 5

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:4 and light chain amino acid sequence set forth in SEQ ID NO: 9.

Embodiment 6

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:5 and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 7

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:6 and light chain amino acid sequence set forth in SEQ ID NO:9 Embodiment 8

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:7 and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 9

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 8 and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 10

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 10 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 11

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 11 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 12

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 12 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 13

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 13 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 14

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 14 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 15

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 15 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 16

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 16 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 17

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 17 and light chain amino acid sequence set forth in SEQ ID NO: 18. Embodiment 18

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 1 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 19

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:2 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 20

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:3 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 21

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:4 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 22

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:5 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 23

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:6 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 24

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO:7 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 25

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 8 and light chain amino acid sequence set forth in SEQ ID NO: 18.

Embodiment 26

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NOTO and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 27

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 11 and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 28

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 12 and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 29

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 13 and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 30

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 14 and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 31

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 15 and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 32

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 16 and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 33

The present invention provides monoclonal antibody that binds SARS-CoV-2 spike protein comprising heavy chain amino acid sequence set forth in SEQ ID NO: 17 and light chain amino acid sequence set forth in SEQ ID NO:9.

Embodiment 34

The present invention provides a composition comprising cocktail of at least two monoclonal antibodies that binds SARS-CoV-2 spike protein wherein one antibody comprising heavy chain amino acid sequence set forth in either SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 and light chain amino acid sequence set forth in SEQ ID NO:9 and second antibody comprising heavy chain amino acid sequence set forth in either SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 and light chain amino acid sequence set forth in SEQ ID NO: 18.

In another embodiment, the present invention provides a composition comprising cocktail of at least two monoclonal antibodies that binds SARS-CoV-2 spike protein wherein one antibody comprises heavy chain amino acid sequence set forth in either SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO: 8 and light chain amino acid sequence set forth in SEQ ID NO: 18 and the second antibody comprises heavy chain amino acid sequence set forth in either SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 and light chain amino acid sequence set forth in SEQ ID NO:9.

In another embodiment, the composition according to the present invention comprises one or more monoclonal antibodies and suitable pharmaceutical excipients. The monoclonal antibody according to the present invention is as described in embodiments 1 to 34.

In another embodiment, the composition according to the present invention comprises a cocktail of at least two monoclonal antibodies and suitable pharmaceutical excipients. Cocktail according to the present invention is as described herein elsewhere in the present invention. Embodiment 35

In one of the embodiments, the amino acid sequence of constant region of anti-SARS- CoV-2 antibody of the present invention comprises of the IgG₁, IgG₂, IgG₃, IgG₄, IgG₂/G₄, IgA, IgE, IgM or IgD constant region, preferably the IgG₁ or IgG₄.

Embodiment 36

In another embodiment, one or more anti-SARS-CoV-2 antibodies or cocktail of at least two antibodies of the present invention are provided which has modified or reduced or no ADCC, ADCP and /or CDC activity. In one of the embodiments, the anti-SARS-CoV-2 antibody or cocktail of at least two antibodies has reduced potential to cause any safety issue associated with ADCC, ADCP and / or CDC.

Embodiment 37

In one of the embodiments, each of the anti-SARS-CoV-2 antibody or cocktail of at least two antibodies of the present invention has a K_D of 10^-8 M or less, more preferably 10^-10 M or less and even more preferably 10^-10 M or less for target antigen of SARS-CoV-2. K_D value is a measurement of the binding affinity of the antibody towards its target antigen. Here, in the current invention target antigen is receptor binding domain (RBD) and S Trimer protein of SARS-CoV-2.

In one of the embodiments, each of the anti-SARS-CoV-2 antibody or cocktail of at least two antibodies of the present invention has a K_D of 10^-7 M or less, more preferably 10’⁹ M or less for recombinant human neonatal Fc receptor (rhFcRn).

In one of the embodiments, each of the anti-SARS-CoV-2 antibody or cocktail of at least two antibodies of the present invention has very less or no binding to recombinant Human FcγRIIIa (Phe), Human FcγRIIa, Human FcγRIIb, Human FcγRI and Clq.

Embodiment 38

In one of the embodiments, each of the anti-SARS-CoV-2 antibody or cocktail of at least two antibodies of the present invention cross-reacts with SARS-CoV-2 spike protein from species other than human.

Embodiment 39

In one of the embodiments, each of the anti-SARS-CoV-2 antibody or cocktail of at least two antibodies of the present invention has higher binding specificity towards SARS-CoV-2 spike protein. Embodiment 40

In one of the embodiments, each of the anti-SARS-CoV-2 antibody or cocktail of at least two antibodies of the present invention has an increased half-life in subject. The term ‘increased’ as referred herein is with respect to the anti-SARS-CoV-2 antibody or cocktail of antibodies known in the art.

In one embodiment, each of the anti-SARS-CoV-2 antibody or cocktail of at least two antibodies according to the present invention may have improved circulating half-life. The term ‘improved’ as referred herein is with reference to the anti-SARS-CoV-2 antibody or cocktail of antibodies known in the art.

Embodiment 41

In one of the embodiments, the anti-SARS-CoV-2 antibody or cocktail of at least two antibodies of the present invention has anti-viral effect or has ability to neutralize SARS-CoV-2 virus. Virus neutralization potency of the anti-SARS-CoV-2 antibodies of the current invention is analysed by measuring IC₅₀ values of said antibodies. Individual monoclonal antibody half maximal inhibitory concentration (IC₅₀) against SARS-CoV-2 S Pseudotyped Luciferase Lenti virus was determined in ACE2 receptor overexpressing 293T cells.

Embodiment 42

In one of the embodiments, the present invention provides a process of making the antibodies as described in any of the embodiments above by cell culture method comprising: i. culturing the host cell expressing monoclonal antibody capable of binding and neutralizing SARS-CoV-2 spike protein; and ii. isolating and recovering monoclonal antibody capable of binding and neutralizing SARS-CoV-2 spike protein expressed in the said host cell.

Embodiment 43

In another embodiment, the present invention provides a process of purifying the antibodies as described in any of the embodiments by suitable chromatography or purification technique.

Embodiment 44

In another embodiment, the present invention provides a kit comprising one or more monoclonal antibodies as per the present invention capable of binding and neutralizing to SARS-CoV-2 spike protein.

In another embodiment, the present invention provides a kit comprising cocktail of at least two monoclonal antibodies as per the present invention capable of binding and neutralizing SARS-CoV-2 spike protein.

Embodiment 45

In one of the embodiments, the present invention provides a method for diagnosing SARS-CoV-2 or other viruses related to coronavirus family using the monoclonal antibodies or cocktail of at least two monoclonal antibodies capable of binding and neutralizing SARS-CoV-2 or other viruses related to coronavirus family.

Embodiment 46

In another embodiment, the present invention provides a method of treating and preventing SARS-CoV-2 or other viruses related to coronavirus family using each of the monoclonal antibody or cocktail of at least two monoclonal antibodies of the present invention, capable of binding SARS-CoV-2 or other viruses related to coronavirus family.

Embodiment 47

In another embodiment, each of the monoclonal antibody or cocktail of at least two monoclonal antibodies of the present invention, capable of binding SARS-CoV-2 or other viruses related to coronavirus family can be used for parenteral administration. Parenteral administration includes intravenous, subcutaneous, intra peritoneal, intramuscular administration or any other route of delivery generally considered to be falling under the scope of parenteral administration and as is well known to a skilled person. Hyaluronidase enzyme can be used to formulate subcutaneous formulation of the present invention. Each of the monoclonal antibody or cocktail of at least two monoclonal antibodies of the present invention, capable of binding SARS-CoV-2 or other viruses related to coronavirus family can be used for intranasally in the form of nasal drops.

Embodiment 48

In one of the embodiment, the present invention is to provide a method of detecting SARS-CoV-2 or other viruses related to coronavirus family using the monoclonal antibodies or cocktail of at least two monoclonal antibodies of the present invention, capable of binding and neutralizing SARS-CoV-2 or other viruses related to coronavirus family.

Embodiment 49

In another embodiment, the present disclosure provides a pharmaceutical composition comprising a monoclonal antibody or cocktail of at least two monoclonal antibodies of the present invention, and a pharmaceutically acceptable carrier or diluent.

DETAILED DESCRIPTION OF THE INVENTION

There is a need for neutralizing therapeutic anti-SARS-CoV-2-Spike protein (SARS- CoV-2-S) antibodies and their use for treating or preventing viral infection. The present invention addresses this need by providing human anti-SARS-CoV-2-S antibodies or cocktail thereof and methods of use for treating viral infections. 2019- nCoV uses the densely glycosylated spike (S) protein to gain entry into host cells. The S protein is a trimeric class I fusion protein that exists in a metastable prefusion conformation that undergoes a substantial structural rearrangement to fuse the viral membrane with the host cell membrane. This process is triggered when the SI subunit binds to a host cell receptor. Receptor binding destabilizes the prefusion trimer, resulting in shedding of the SI subunit and transition of the S2 subunit to a stable postfusion conformation (4). It is well known that 2019-nCoV S and SARS-CoV S share the same functional host cell receptor, ACE2. It is also reported that ACE2 binds to the 2019- nCoV S ectodomain with ~I5 nM affinity, which is ~10- to 20-fold higher than ACE2 binding to SARS-CoV S. The high affinity of 2019-nCoV S for human ACE2 may contribute to the apparent ease with which 2019-nCoV can spread from human to human (4). The anti-SARS-CoV-2-S antibodies or cocktail thereof according to the present invention neutralizes SARS-CoV-2 via binding to spike protein of the virus and inhibits further biological activity of the spike protein. Further, the anti- SARS-CoV-2-S antibodies or cocktail thereof according to the present invention has reduced or no ADCC, ADCP and / or CDC activity. Previous studies focusing on in vitro and in mouse models SARS-CoV infection have indicated the potential risk of antibody dependent enhancement (ADE) hindering the ability of antibody therapeutics to control inflammation in the lung and other organs. Further, ADE may lead to acute respiratory injury, acute respiratory distress syndrome, and other observed inflammation-based sequelae. To mitigate the risk of ADE, the present invention provides anti-SARS-CoV-2-S antibodies or cocktail thereof which has reduced or no binding to FcγRIIIa (Phe), Human FcγRIIa, Human FcγRIIb, Human FcγRI and Clq. Binding of anti-SARS-CoV-2-S antibodies to FcγRIIIa (Phe), Human FcγRIIa, Human FcγRIIb and Human FcγRI is analysed by SPR method as described herein the examples. Binding of anti-SARS-CoV-2-S antibodies to Clq is analysed by ELISA as described herein the examples. In one of the aspects, the anti-SARS-CoV-2-S antibodies or cocktail thereof according to the present invention has a long circulating half-life. The anti-SARS-CoV-2 antibodies of the present invention have higher affinity towards hFcRn and therefore it is expected to have long circulating half-life in vivo. Such antibodies with higher affinity towards hFcRn can provide protection against virus for longer period of time as compared to the antibodies known in the art. Further antibodies or cocktail of at least two antibodies of the current invention, due to its higher FcRn binding affinity is expected to increases transcytosis, thereby providing higher levels of the drug in tissues having high expression of FcRn. Mucosal tissues such as lungs, genital tract and rectum, etc. are known to have high expression of FcRn and are involved in transcytosis of antibodies to prevent infections in these tissues. Due to long circulating half-life, the anti-SARS-CoV-2-S antibodies or cocktail thereof of the present invention can be given to the subject with reduced dose and with a better dose regimen as compared to the current clinically active anti-SARS-CoV antibodies. In one of the aspects, the cocktail of at least two anti-SARS-CoV-2-S antibodies of the present invention comprises equipotent amount of the two anti-SARS-CoV-2-S antibodies. Cocktail preparation comprising equipotent amount of two monoclonal antibodies is illustrated herein examples.

Preparation of antibodies

Antibodies of the present invention can be prepared using recombinant technology using expression vector. Any expression vector known to the skilled person can be used in the present invention, and the choice of the expression vector is dependent on the nature of the host cell of choice. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present) known to persons skilled in the art. For example, to express the antibodies, DNAs encoding full-length light and heavy chains, can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The heavy chain of the antibodies described herein can be used to create full- length antibody genes of any antibody isotype by inserting them into expression vectors already encoding full-length light chain of the desired isotype. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques such as lipid mediated transfection, electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Introduction of the vector in host cells can be effected by, but not limited to, calcium phosphate transfection, virus infection, DEAE dextran mediated transfection, lipofectamin transfection or electroporation, and any skilled person can select and use an introduction method suitable for the expression vector and host cell used. Preferably, the vector contains one or more selectable markers, but is not limited thereto, and a vector containing no selectable marker may also be used.

The present invention provides a host cell that comprises the expression vector transformed into a host cell to produce the monoclonal antibody of the present invention. In the present invention, the host cell may comprise cells of mammalian, plant, insect, fungal or bacterial origin, but is not limited thereto. A mammalian cell can be selected from, but is not limited thereto, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK 293 cells and HEK293T cells. Suitable mammalian host cell known to skilled person can be used for the development of antibodies and their cocktail of the present invention. Antibodies produced according to the present invention can be further produced by known cell culture techniques for large scale antibody production. Antibodies can be recovered from the culture medium using standard protein purification methods. When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Antibodies of the invention can be tested for binding to SARS-CoV-2 by, for example, standard ELISA.

In some instances, one or more framework region amino acid residues of the human immunoglobulin are also replaced by corresponding amino acid residues of the non- human antibody (so called “back mutations”). In addition, phage display libraries can be used to vary amino acids at chosen positions within the antibody sequence. The properties of a humanized antibody are also affected by the choice of the human framework. Furthermore, humanized and chimerized antibodies can be modified to comprise residues that are not found in the recipient antibody or in the donor antibody in order to further improve antibody properties, such as, for example, affinity or effector function.

In one of the embodiments, the anti-SARS-CoV-2 antibody according to the present invention is monoclonal antibody or bispecific antibody or polyclonal antibody, preferably monoclonal antibody.

Immunoconjugates and Bispecific antibodies

An immunoconjugate comprising an antibody of the present invention, or antigen- binding portion thereof, linked to another therapeutic agent, such as a cytotoxin or a radioactive isotope can also be developed. A bispecific molecule comprising an antibody, or antigen-binding portion thereof, of the present invention, linked to a second functional moiety having a different binding specificity than said antibody, or antigen-binding portion thereof can be developed.

Nucleic acid molecules encoding anti-SARS-CoV-2 antibodies, vectors and host cells

In one embodiment, the present invention provides nucleic acid molecules encoding the antibodies, or antigen-binding portions thereof as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors. In the present application, pZRCIII vector is used for the cloning and expression of anti-SARS-CoV-2 antibodies of the present invention. pZRCIII vector is described in patent document WO 2012/046255 A2. The host cell according to the present invention is prokaryotic or eukaryotic cell, preferably the host cell is an E. coli cell or a mammalian cell, such as a CHO cell or its variants.

Combination of anti-SARS-CoV-2 antibodies of the present invention with other drugs In one of the aspects, the anti-SARS-CoV-2 antibodies of the present invention can be administered to the patient in combination with standard of care. Standard of care (SOC) can be either hydroxychlorquine or any suitable antiviral agents, e.g., remdesivir, and/ or ritonavir, and/ or liponavir and/ or anti-viral monoclonal antibodies, and/ or any anti-inflammatory therapy such as anti-IL-6 or anti-TNF alpha or such, and/ or Inteferons selected from IFN alpha, IFN alpha 2b, IFN alpha 2a, PEGylated IFN alpha 2a, PEGylated IFN alpha 2b or any other therapy being given to the COVID-19 patient as a part of SOC. According to the present invention, SOC is to be administered as per local regulatory guideline / institute SOP.

Preparation of cocktail of anti-SARS-CoV-2 antibodies of the present invention

The cocktail of anti-SARS-CoV-2 antibodies of the present invention_manufacturing was performed by mixing two independent anti-SARS-CoV-2 monoclonal antibodies in equal amount to prepare the formulated bulk solution. The final cocktail preparation can be prepared either in liquid or lyophilized form by filling the 0.2 pm sterile filtered formulated bulk solution (cocktail preparation) in the glass vials. The cocktail anti- SARS-CoV-2 antibodies was further exemplified in example 8 of the present application.

Here, the present invention is illustrated with the following non-limiting examples which should not be interpreted as limiting the scope of the invention in any way.

EXAMPLES

Example 1: Generation of monoclonal antibodies

Example 1.1 Generation of dual expression vector constructs containing light and heavy chain genes of anti-SARS-CoV-2 antibodies

Chemically synthesized light and heavy chain genes were used for dual vector construction of the mAb candidates. Light chain genes and heavy chain genes (IgGl wild type and variants with or without HR2 peptide of nCov2 Spike protein fusion), cloned in pMA/pMK vectors were obtained from Geneart, Germany. The brief description of various vector constructs is given in Table 4.

Light chain genes, LC-1 (SEQ ID No. 19) and LC-2 (SEQ ID no. 20) were isolated from these constructs by restriction digestion with HindIII and XmaI. These digested light chain genes were individually ligated, with HindIII and XmaI digested pDual cloning vector.

The ligation product was transformed in E. coli Topi OF’ and the transformants were scored on the basis of antibiotic resistance. The clones were analysed by restriction digestion and DNA sequencing by Sanger’s method. These intermediate vectors containing light chain genes in transcriptional assembly no.l were named as pDual Cov2A-LC and pDual Cov2B-LC.

To prepare final constructs having both the light chain and heavy chain genes, plasmid DNA of intermediate pDualCov2A-LC vector was digested with MluI and EcoRI to allow the cloning of heavy chain genes mAb A (SEQ ID No. 21), mAb D (SEQ ID No. 22), mAb E (SEQ ID No. 23), mAb F (SEQ ID No. 24), mAb G (SEQ ID No. 25) and mAb H (SEQ ID No. 26). Similarly, Plasmid DNA of intermediate pDual Cov2 B-LC vector was also digested with MluI and EcoRI to allow the cloning of heavy chain genes mAb I (SEQ ID No. 27), mAb L (SEQ ID No. 28), mAb M (SEQ ID No. 29), mAb N (SEQ ID No. 30), mAb O (SEQ ID No. 31) and mAb P (SEQ ID No. 32).

To prepare heavy chain genes for cloning in pDual vector, Clal restriction enzyme site of these genes was replaced with MluI restriction enzyme site by PCR followed by digestion of the PCR products with MluI and HindIII.

The digested heavy chain genes (SEQ ID No.21, 22, 23, 24, 25 and 26) were ligated individually, with the MluI and EcoRI digested pDual Cov2A-LC vector and in the same way, digested heavy chain genes (SEQ ID No. 27, 28, 29, 30, 31 and 32) were ligated individually, with the MluI and EcoRI digested pDual Cov2B-LC vector. The ligation products were transformed in E.coli Topi OF’ and transformants were scored based on the ampicillin resistance. The clones were confirmed on the basis of restriction digestion and Sanger sequencing. The dual plasmids encoding Cov2A-LC with different variants [HC of mAb A, HC of mAb D, HC of mAb E, HC of mAb F, HC of mAb G and HC of mAb H] of Cov2 heavy chain gene were named as pDual Cov2 A3 [Corresponding to amino acid sequence of LC-1 and HC mAb A; sequence ID NO. 9 and 1], pDual Cov2 A5 [Corresponding to amino acid sequence of LC-1 and HC mAb D; sequence ID NO. 9 and 4], pDual Cov2 A7 [Corresponding to amino acid sequence of LC-1 and HC mAb E; sequence ID NO. 9 and 5], pDual Cov2 A12 [Corresponding to amino acid sequence of LC-1 and HC mAb F; sequence ID NO. 9 and 6] , pDual Cov2 A4 [Corresponding to amino acid sequence of LC- 1 and HC mAb G; sequence ID NO. 9 and 7] and pDual Cov2 A6 [Corresponding to amino acid sequence of LC-1 and HC mAb H; sequence ID NO. 9 and 8]. The dual plasmids encoding Cov2B-LC with different variants [HC of mAb I, HC of mAb L, HC of mAb M, HC of mAb N, HC of mAb O and HC of mAb P] of Cov2 heavy chain gene were named as pDual Cov2 B14 [Corresponding to amino acid sequence of LC-2 and HC mAb I; sequence ID NO. 18 and 10], pDual Cov2 B9 [Corresponding to amino acid sequence of LC-2 and HC mAb L; sequence ID NO. 18 and 13], pDual Cov2 B10 [Corresponding to amino acid sequence of LC-2 and HC mAb M; sequence ID NO. 18 and 14], pDual Cov2 B15 [Corresponding to amino acid sequence of LC-2 and HC mAb N; sequence ID NO. 18 and 15], pDual Cov2 B8 [Corresponding to amino acid sequence of LC-2 and HC mAb O; sequence ID NO. 18 and 16], and pDual Cov2 B13[Corresponding to amino acid sequence of LC-2 and HC mAb P; sequence ID NO. 18 and 17]. The brief description of various vector constructs is given in Table 4 with corresponding amino acid sequences of HC and LC encoded by the vectors. The representative vector map of the prepared constructs is given as Figure 1.

Table 4: Vector Construct IDs with their corresponding mAh ID and SEQ ID No. of HC and LC

Example 1.2 Generation of cell lines expression anti-SARS-CoV-2 antibodies

This example describes the generation of stable transfected cell lines expressing full- length humanized anti-SARS-CoV-2 antibodies. All vector constructs as described in example 1.1 were used for transfections. Plasmids were linearized with Pvul restriction enzyme prior to transfection. Chinese hamster ovary (CHO), which is one of the suitable hosts for expression of monoclonal antibodies, was used for cell line generation. CHO cells were seeded ~24 hours prior to transfection at a density of 0.5 million / ml to have cells in the exponential phase. Transfections were performed using Neon Transfection system (Invitrogen) by electroporation technique following manufacturer’s instructions. Post transfection, cells were plated in 24 well cell culture plates containing 1 ml of pre-warmed ProCHO5 Serum Free media (Lonza, Switzerland) containing selection pressure and incubated in a humidified incubator at 37 °C in presence of 5 % CO₂. The cell numbers of all the transfected pools were regularly monitored and regular media exchanges were given. Once the cells recovered from transfection, cells were further expanded to 6 well culture plates, T-flasks and culti-tubes (TPP).

Fed-batch cultures were performed for transfected pools of all anti-SARS-CoV-2 antibody candidates in culti tube (TPP) for recombinant protein production. Cells were seeded at a density of 0.3x 10^-6 cIells / ml in ActiPro production medium from Hyclone, GE. Culti tubes were incubated in a humidified Kuhner shaker at 37 °C temperature, 5 % CO₂ level with shaking speed of 230 RPM. A fixed daily feeding regimen was followed during the culture for all the pools using chemically defined feeds from Hyclone, GE. After 72 hours of culture, feeding was initiated and continued till the batch was harvested.

Upon harvest, the culture supernatants were collected for antibody purification by Protein A affinity chromatography. These purified antibody candidates were further tested for various in-vitro assays as described in below examples.

Example 2: Determination of kinetic rate constants of anti-SARS-CoV-2 antibodies to RBD and S Trimer protein of SARS-CoV-2 using the surface plasmon resonance method

Binding kinetics and affinities for anti-SARS-CoV-2 antibodies were assessed using surface plasmon resonance technology on a ProteOn XPR 36 instrument (Bio-Rad) using a GLC sensor chip in filtered and degassed PBS T running buffer (0.005 % (v/v) Tween 20, pH 7.4).

To study RBD (receptor binding domain) binding of anti-SARS-CoV-2 antibodies, the sensor surfaces were prepared by immobilizing with receptor binding domain (RBD) protein of SARS-CoV-2(Cat No: SPD-C82E9; Make: Aero Biosystem) on to the chip surface using the standard amine coupling chemistry. Following surface activation, the remaining active carboxyl groups on the GLC chip surface were later blocked by injecting 1M ethanolamine, pH 8.0 for 5 minutes. The binding of different concentrations of anti-SARS-CoV-2 antibodies to the immobilized RBD was analyzed at a flow rate of 50 μL / min with an association time of 300s and dissociation time of 600s at 25 °C. At the end of each cycle, the chip surface was regenerated using 18 seconds injection of 4 M MgCl₂. The kinetic parameters were obtained by fitting the double-reference subtracted data to a 1:1 binding model using ProteOn Manager software v 3.1.0.6. The kinetic constants of the antibody preparations binding to SARS-CoV-2 spike protein RBD is shown in Table 5. Table 5: Kinetic rate constants of anti-SARS-CoV-2 antibodies for RBD protein of SARS-CoV-2

To study the binding of anti-SARS-CoV-2 antibodies to S Trimer protein, the sensor surfaces were prepared by immobilizing with S Trimer protein (Cat No: SPN-C52H8;

Make: Aero Biosystem) on to the chip surface using the standard amine coupling chemistry. Following surface activation, the remaining active carboxyl groups on the GLC chip surface were later blocked by injecting 1 M ethanolamine, pH 8.0 for 5 minutes. The binding of different concentration of anti-spike mAbs to immobilized S Trimer protein was analyzed at a flow rate of 50 μL / min with an association time of 300s and dissociation time of 600s at 25 °C. At the end of each cycle, the chip surface was regenerated using 18 seconds injection of 4 M MgCl₂ The kinetic parameters were obtained by fitting the double-reference subtracted data to a 1: 1 binding model using ProteOn Manager software v 3.1.0.6. The kinetic constants of the antibody preparations binding to S Trimer is shown in Table 6.

Table 6: Kinetic rate constants of anti-SARS-CoV-2 antibodies for S Trimer protein of SARS-CoV-2

Example 3: Determination of kinetic rate constants of anti-SARS-CoV-2 antibodies to recombinant human neonatal Fc receptor (rhFcRn)

The kinetic constants for the binding of antibodies to recombinant human neonatal Fc receptor (hFcRn) were determined by Surface Plasmon Resonance-based measurement using the ProteOn XPR36 (Bio-Rad). Recombinant rhFcRn receptor (Sino Biologies) was immobilized on a GLC chip following manufacturer’s instruction. To measure the association rate constant (k_assoc) and dissociation rate constant (k_dissoc), five dilutions of the antibodiy preparations were injected at a flow rate of 100 μL / min with an association time of 180s and dissociation time of 600s. Binding were analyzed using PBS (pH 6.0, 0.005 % Surfactant P20) at 25 °C. After each sample run, the chip surface was regenerated using PBS, pH 7.4. The data, in the form of sensograms, was analysed using the data-fitting programs in the ProteOn system. The kinetic constants of rhFcRn binding to different antibodies is shown in Table 7. Table 7: Kinetic rate constants of anti-SARS-CoV-2 antibodies for hFcRn

Example 4: Determination of kinetic rate constants of anti-SARS-CoV-2 antibodies to recombinant Human FcγRIIIa (Phe), Human FcγRIIa and Human FcγRIIb

The kinetic constants for the binding of antibodies to recombinant Human FcγRIIIa - Phenylalanine (rhFcγRIIIa-Phe), Human FcγRIIa and Human FcγRIIb were determined by Surface Plasmon Resonance -based measurement using the ProteOn XPR36 (Bio-Rad). Recombinant hFcγRIIIa-Phe, hFcγRIIa and hFcγRIIb receptors (Sino Biologies) were immobilized on a GLC chip in different channels following manufacturer’s instruction. To measure the association rate constant (k_assoc) and dissociation rate constant (k_dissoc), five dilutions of affinity purified antibodies were prepared and injected at a flow rate of 50 μL / min with an association time of 240s and dissociation time of 600s. Binding assessments were carried out using PBS (pH 7.4, 0.005 % Surfactant P20) at 25 °C. After each sample run, the chip surface was regenerated using 1 M NaCl and Acetate buffer pH 5.0. The data, in the form of sensograms, was analysed using the data-fitting programs in the ProteOn system. The kinetic constants of Recombinant hFcγRIIIa-Phe, hFcγRIIa and hFcγRIIb binding to different antibodies is shown in Tables 8, 9 and 10. mAb E and mAb M did not show any binding to hFcγRIIIa-Phe, hFcγRIIa and hFcγRIIb even when analyzed at higher concentrations.

Table 8: Kinetic rate constants of anti-SARS-CoV-2 antibodies for Recombinant hFcγRIIIa-Phe

Table 9: Kinetic rate constants of anti-SARS-CoV-2 antibodies for Recombinant hFcγRIIa

Table 10: Kinetic rate constants of anti-SARS-CoV-2 antibodies for Recombinant hFcγRIIb

Example 5: Determination of kinetic rate constants of anti-SARS-CoV-2 antibodies to recombinant Human FcγRI First, Anti-SARS-CoV-2 monoclonal antibodies were captured (≈200 RU), vertically, on a RBD protein of SARS-CoV-2 (Cat No: SPD-C82E9; Make: Aero Biosystem) immobilized chip to study the interaction between anti-SARS-CoV-2 antibodies and the analyte, rhFcγRI, following manufacturer’s instructions. Solution of rhFcγRI at different concentrations was injected horizontally over the chip at a flow rate of 50 μL / min with an association time of 240s and dissociation time of 600s. Reactions were conducted at pH 7.4. Following each sample run, the chip surface was regenerated with 4 M MgCl₂. The data, in the form of sensograms, was analyzed using the data-fitting programs in the ProteOn system. The kinetic constants of different antibody preparations binding to hFcγRIa is shown in Table 11. mAb E and mAb M showed poorer binding affinities as compared to their wild type variants.

Table 11: Kinetic rate constants of anti-SARS-CoV-2 antibodies for Recombinant hFcγRI

Example 6: Clq binding by ELISA

An ELISA based method was used to analyze binding of anti-SARS-CoV-2 mAbs to Clq.

Briefly, ELISA plates were coated using different concentrations of Covid mAbs ranging from 60 to 0.363 μg / ml in PBS (pH 7.4) for mAb A, mAb D, mAb L and mAb I and from 350 to 2.11 μg / ml in PBS (pH 7.4) for mAb E and mAb M. The plates were incubated for 2 hours at 37 °C in an incubator in static condition. This was followed by blocking the plates with 2 % of skimmed milk in phosphate buffered saline containing 0.05 % Tween (IX PBST). This was followed by plate washing using 1X PBST as washing solution. After washing, 8 μg / ml Clq (Sigma Aldrich) was added to the plate, which would bind to the Fc portion of the antibodies. The plates were incubated in plate shaker at room temperature for 1 hour. After incubation, the plates were washed to remove unbound Clq from the wells. The detection was accomplished by addition of Anti Clq HRP conjugated antibody (Abeam). Post incubation at room temperature for 30 minutes, plates were washed. Subsequently Femto substrate was added to each well and incubated for 5 minutes. The luminescence was read in a microplate reader.

Highly reduced Clq binding was observed for mAb E and mAb M candidates as shown in Figures 2 and 3. Table 12 shows the EC₅₀ values obtained for various anti- SARS-CoV-2 antibodies using SoftMax Pro software.

Table 12: EC₅₀ values of anti-SARS-CoV-2 mAbs for Clq binding

Example 7: Determination of anti-viral effect of anti-SARS-CoV-2 antibodies by virus neutralization assay

An in vitro neutralization assay utilizing SARS-CoV-2-S pseudotyped luciferase lentivirus was used to investigate the ability of a panel of anti-SARS-CoV-2-S monoclonal antibodies to neutralize SARS-CoV-2. The pseudotyped virus, Cat. No. CoV-002, was procured from Creative Biogene 45-1 Ramsey Road Shirley, NY 11967, USA. For the infection a 293T cell line stably overexpressing human ACE-2 cell surface receptor protein: 293T-hACE2. MF was used. 293T cells stably expressing huACE2 were seeded in a T75 cm² flask. To seed the cells, media was removed from the cells and trypsin was added and incubated for 2-3 minutes at 37 °C until the cells dislodged. 5 mL of complete DMEM was added to inactivate the trypsin, and pipetted up and down to distribute the cells. The cells were counted and plated so as to have 10,000 cells / well in a 96-well black polystyrene microplate. Simultaneously in a V- bottom 96 well plate, a dilution of each monoclonal antibody was prepared in DMEM media supplemented with 10 % FBS. The antibody preparations were diluted to a starting concentration of 1 mg / mL and diluted serially by 10-fold dilutions to form a concentration range of 1x10⁶ ng / mL to 0.001 ng / mL. The SARS-CoV-2-S pseudotyped luciferase lentivirus was thawed on ice and diluted to obtain a 50 MOI. The pseudovirus were incubated with the antibody dilutions for 60 minutes at 37 °C. After the incubation 100 p L of antibody / pseudovirus mixtures were transferred to the cells and the plate was incubated at 37 °C, 5 % CO₂ for 24 hours. After the 24 hour incubation, supernatant was removed from the cell wells and replaced with 100 μL of DMEM media supplemented with 10 % FBS. The cell wells were further incubated for a period of 48 hours at 37 °C, 5 % CO₂. Post the incubation, 100 μL of firefly luciferase substrate was added to all the wells and kept on a shaker for a further 30 minutes to check the transduction efficacy. The plate was read in a SpectraMax i3x plate reader. The obtained luminescence readings were plotted against the concentrations to determine the IC₅₀ values for each sample. The neutralization potency in the form of IC₅₀ values of the SARS-CoV-2 antibodies is shown in Figure 4.

Example 8: Manufacturing of antibody cocktail comprising mAb E & mAb M

The manufacturing of cocktail of mAb E & mAb M was performed by mixing two independent monoclonal antibodies mAb E and mAb M in equal amount to prepare the formulated bulk solution. Formulated bulk solution of mAb E, was mixed with an equal volume of the formulated bulk solution of mAb M through gentle stirring at around 250 rpm for about 20 minutes. At the end of mixing, sampling was performed to analyze pH, osmolality and protein concentration, as in-process tests for monitoring purpose. The formulated bulk solution was filtered through 0.2 pm sterilizing grade filter and the filtered bulk was collected in a pre-sterilized container placed under laminar air flow. Filling in vials was performed under laminar air flow using peristaltic pump through in-line 0.2 pm sterilizing grade filter at the desired fill volume for preparation of different strengths. After filling, vials were stoppered under laminar air flow. Vials were then sealed with flip-off seals. Sealed vials were checked manually for proper sealing. Visual inspection of vials was performed to check the presence of any visible particles and lower or higher volume, if any. The drug product (cocktail of mAb E & mAb M) in vials were stored between +2 °C and +8 °C. In the same manner, cocktail of other anti-SARS-CoV-2 antibodies of the present invention can be prepared following the process as described herein the current example. Preferably, cocktail of the current invention includes one antibody selected from mAb A to mAb H and second antibody selected from mAb I to mAb P of the current invention.

Example 9: RBD-ACE2 binding inhibition by ELISA

A competitive ELISA was used to assess the inhibition potential of the mAbs towards RBD binding to ACE receptor. In brief, ACE-2 protein (Aero Biosystems, USA) was coated onto ELISA plates (Greiner, Germany) at a concentration of 1 pg/mL in phosphate buffered saline solution followed by an incubation (for 12-72 hours) at 2- 8°C in a humidified chamber. The plate was then blocked using 2.0 % solution of BSA (SD Fine Chem, India). Post blocking, 100 ng/mL of biotinylated RBD (Aero Biosystems, USA) was incubated with different concentrations of mAb E (1500 ng/mL to 2.059 ng/mL, 3 fold serial dilutions) and mAb M (800 ng/mL to 3.277 ng/mL, 2.5 fold serial dilutions) on a plate shaker for 60 mins at room temperature to allow binding of the mAbs to RBD. This mixture was then loaded onto ACE-2 coated plate to allow the unbound RBD to bind to the coated ACE2. Peroxidase conjugated streptavidin at 1/50,000 dilution was used for detection. 3,3',5,5'-Tetramethylbenzidine (TMB) was used as a substrate (Sigma Aldrich, USA). 1 N sulfuric acid was used to stop the reaction. Absorbance was read at 450 nm in a multi-mode plate reader (Molecular devices, USA). Inhibition of RBD binding to ACE-2 in presence of cocktail of mAb E & mAb M was measured in terms of IC₅₀ (i.e., concentration of mAb inhibiting RBD-ACE2 interaction by 50%) values by plotting four parameter fit curves of antibody concentration vs. % inhibition as shown in figure 5. Both the mAbs were able to bind to the spike protein RBD and inhibit its binding to ACE2 receptor at sub- nanomolar IC₅₀ concentrations.

Example 10: Epitope binding using the surface plasmon resonance method

Epitope binding was performed for the two mAbs (mAb E and mAb M) using ProteOn XPR36 instrument to analyze and confirm that the two antibodies bind to unique epitopes on RBD. The RBD protein (Cat No: SPD-C82E9; Make: Aero Biosystems, USA) was immobilized on a GLC sensor chip surface using standard amine coupling chemistry. Running buffer used was lOmM Phosphate Buffered Saline (PBS) (10mM phosphate buffer, pH 7.4, 150mM NaCl, 0.005% Tween 20). Initially, mAh E and mAh M were run at saturating concentrations (at 50 nM) on different channels which were immobilized with RBD. In the subsequent run, mAb E and mAb M were individually run at a concentration of 50 nM over both mAb E and mAb M captured channels. Binding patterns were then analysed. Binding of mAb M was seen on mAb E pre-captured channels. In a similar fashion, binding of mAb E was observed on mAb M pre-captured channels indicating that both the antibodies bind to distinct epitopes of RBD on the spike protein as shown in sensograms (Figures 6A and 6B).

Example 11: SARS-CoV-2 live virus plaque reduction neutralization test (PRNT)

Vero E6 cells (ATCC) were maintained in MEM (Sigma) containing 1% penicillin- streptomycin antibiotic (Hi-media laboratories) and supplemented with 10% Fetal bovine serum (Hyclone), in a 5% CO₂ environment at 37 °C and passaged every 2-3 days. The assay medium was Minimum essential medium MEM (Sigma), containing 1% penicillin-streptomycin (Hi-media laboratories) and supplemented with 2% FBS (Hyclone). The SARS-CoV-2 isolate (Strain: NIV-2020-770) was sourced from ICMR- NIV Pune, India. Vero E6 cells (1.0x10⁶ per well) were seeded in 24- well plates in maintenance medium for 24 hrs at 37 °C in a 5% CO₂ incubator. Next day, monoclonal antibodies were serially diluted in assay medium (range of 1x10⁶ ng/mL to 0.001 ng/mL). SARS-CoV-2 virus was added to each dilution at 0.01 MOI, except the cell controls (wells containing cells only). The mixtures of virus and antibody were incubated for 60 min at 37 °C in a 5 % CO₂ incubator. Following completion of incubation the virus and antibody mixtures were added to the pre-seeded Vero E6 cells by first discarding the supernatant followed by replacement with medium containing 2 % carboxy methylcellulose. The plates were incubated for a further 72 hours in a CO₂ humidified incubator at 37 °C. Post incubation the cells were fixed with 4 % formaldehyde and the plaques were enumerated by staining with crystal violet stain. The number of plaques were counted and the percentage inhibition was calculated in comparison to the number of plaques obtained in the wells that contained only the virus but no antibody (positive control). The data was plotted using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA) and the IC₅₀ was calculated. mAb E, mAb M and cocktail of mAb E & mAb M exhibited potent neutralization activity against live SARS-CoV-2 virus in sub-picomolar range (Figure 7). The IC₅₀ of individual antibodies and the cocktail ranged from 0.13 to 0.25 ng/mL as shown in Table 13.

Table 13: IC₅₀ value of Anti-SARS-CoV-2 mAbs

Example 12: Pharmacokinetic study in hamsters

A pharmacokinetic study was carried out in hamsters to understand the pharmacokinetic profiles of monoclonal antibodies mAb E and mAb M. In this study, mAb E and mAb M were administered as cocktail via intraperitoneal (I.P.) route in Golden Syrian Hamsters. 18 female hamsters, aged 11-12 weeks were divided into four groups - 50 mg/kg (25 mg/kg of mAb E + 25 mg/kg of mAb M), 5 mg/kg (2.5 mg/kg of mAb E + 2.5 mg/kg of mAb M), 1.0 mg/kg (0.5 mg/kg of mAb E + 0.5 mg/kg of mAb M), and 0 mg/kg, (placebo), each group consisting of five animals, except for the placebo which consisted of only three animals. The total duration of the study was seven days. Blood was withdrawn from the animals at Day 0 (before administration), Day 1 (24 hrs), Day 3 (72 hrs), Day 5 (120 hrs) and Day 7 (168 hrs) and serum was isolated. The concentration of each mAb in the serum was estimated by ELISA. For the placebo group, samples for pharmacokinetic study were collected only before administration and on Day 7 (168 hrs). Two separate ELISA methods were used to detect mAb E and mAb M antibodies. In both the assays, SARS-CoV-2 (COVID- 19) SI protein (Aero Biosystems, USA) was used as a coating reagent. Post 13-72 hours of incubation in humidified chamber at 2-8 °C, ELISA plates (Greiner, Germany) were blocked using 2 % BSA in PBST (PBS containing 0.05 % Tween 20) (SD Fine Chem). Subsequently, the calibration curves were prepared separately for mAb E and mAb M (ranging from 50 to 0.049 ng/mL) in pooled, naive, hamster serum diluted 500 times (in 0.1% BSA in PBST (PBS containing 0.05 % Tween 20) and the serum samples containing cocktail of mAb E & mAb M were added to the plates at an appropriate dilutions. Detection was accomplished using 1/100,000 diluted peroxidase conjugated goat anti-human lambda light chain secondary antibody for mAb E (Novus Biologies, USA); and 1/35,000 diluted goat anti-human kappa light chain secondary antibody for mAb M (Novus Biologies, USA) to specifically detect the two antibodies. 3,3',5,5'-Tetramethylbenzidine (TMB) was used as a substrate. The reaction was stopped using IN sulfuric acid. The ELISA plates were read in multi-mode reader (Molecular devices, USA) at 450 nm. The pharmacokinetic profiles of mAb E and mAb M are shown in Figures 8 A and 8B. The statistical analysis of pharmacokinetic parameters was performed using Phoenix WinNonlin Software (Table 14 and 15). Dose dependent increase in C_max (Maximum Serum Concentration), AUC_last (Area under the curve from zero till last observation) and AUC_{inf_obs} (Area under the curve from zero till infinite observation) was observed across all doses. The half-life (ti/2) of the antibodies could not be calculated as the clearance phase had not been reached at day 7.

Table 14: Pharmacokinetic parameters of mAb E

Table 15: Pharmacokinetic parameters of mAb M

Example 13: Non-human primate pharmacokinetic (PK) study

A study to assess the PK profile of mAb E and mAb M in rhesus monkeys was conducted at two doses administered intravenously. A total of four female animals were divided in two groups based on dose. A single dose of I.V infusion of cocktail of mAb E & mAb M was administered at 400 mg/kg (200 mg/kg of each antibody) and 100 mg/kg (50 mg/kg of each antibody) doses in the two groups respectively. Blood was drawn and serum was collected from the animals prior to dosing (0 hrs), after dosing at 5 mins, 6 hrs (Day 1), 24 hrs (Day 1), Day 3, 5, 7, 14, 21, 28, 35, 42 and Day 113 for estimation of mAb E and mAb M in serum using ELISA. To estimate the concentration of mAb E and mAb M in monkey serum, two different assays were developed specific for mAb E antibody and mAb M antibody respectively. 96-well ELISA plates (Greiner) were coated with 1.0 pg/mL SARS-CoV-2 SI protein, His Tag (Aero Biosystems) in phosphate buffer saline and incubated for 13-72 hours at 2-8 °C in a humid chamber. The plates were blocked with 2.0 % solution of BSA (SD Fine Chem) in PBS with 0.05% Tween 20 (Sigma.). Calibration curve ranging from (50 ng/mL to 0.195 ng/mL for mAb E and 50 ng/mL to 0.098 ng/mL for mAb M) were prepared in naive pooled monkey serum diluted 500 times in assay diluent (0.1% BSA in PBST). The study samples were appropriately diluted as per requirement and added to the plates. Goat anti-human lambda light chain secondary antibody (Novus Biologicals) at 1/75,000 dilution was used for detection of mAb E, while goat anti- human kappa light chain secondary antibody (Novus Biologicals) was used for detection of mAb M antibody at 1/37500 dilution. TMB (Sigma Aldrich) was used as a substrate. The reaction was stopped by addition of stop solution (containing 1 N H2SO4) and the absorbance of the plate(s) was read at 450 nm in plate reader. The pharmacokinetic profiles of mAb E and mAb M are shown in Figures 9A and 9B respectively. The statistical analysis of pharmacokinetic parameters was performed using Phoenix WinNonlin Software (Table 16A and 16B). Dose dependent increase in C_max, AUCiast and AUC_{inf_obs} was observed across all doses. The half-life (ti/2) of the antibodies was found to be >30 days across all the doses with both the antibodies indicating long half-life of the cocktail drug.

Table 16A: Pharmacokinetic parameters of mAb E Antibody in non-human primate (NHP)

Table 16B: Pharmacokinetic parameters of mAb M Antibody in non-human primate (NHP)

References incorporated in current patent application:

1. https://www.worldometers.info/coronavirus/ 2. Hoffman et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Cellpress, 271-280, 16 April 2020

3. Tai et al., Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine, Cellular and Molecular Immunology (2020).

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

5. Marston HD, Paules CI, Fauci AS. Monoclonal antibodies for emerging infectious diseases — borrowing from history. N Engl J Med. 2018;378(16): 1469- 1472.

Incorporation by reference

The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

Equivalents

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

435 440 445

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Claims

WE CLAIM:

1. A monoclonal antibody or cocktail of at least two monoclonal antibodies that binds SARS-CoV-2 spike protein which is selected from the group comprising of: a) a heavy chain amino acid sequence set forth in SEQ ID NO:1 and a light chain amino acid sequence set forth in SEQ ID NO:9; b) a heavy chain amino acid sequence set forth in SEQ ID NO:2 and a light chain amino acid sequence set forth in SEQ ID NO:9; c) a heavy chain amino acid sequence set forth in SEQ ID NO:3 and a light chain amino acid sequence set forth in SEQ ID NO:9; d) a heavy chain amino acid sequence set forth in SEQ ID NO:4 and a light chain amino acid sequence set forth in SEQ ID NO:9; e) a heavy chain amino acid sequence set forth in SEQ ID NO:5 and a light chain amino acid sequence set forth in SEQ ID NO:9; f) a heavy chain amino acid sequence set forth in SEQ ID NO:6 and a light chain amino acid sequence set forth in SEQ ID NO:9; g) a heavy chain amino acid sequence set forth in SEQ ID NO:7 and a light chain amino acid sequence set forth in SEQ ID NO:9; h) a heavy chain amino acid sequence set forth in SEQ ID NO:8 and a light chain amino acid sequence set forth in SEQ ID NO:9; i) a heavy chain amino acid sequence set forth in SEQ ID NO:10 and a light chain amino acid sequence set forth in SEQ ID NO: 18; j) a heavy chain amino acid sequence set forth in SEQ ID NO: 11 and a light chain amino acid sequence set forth in SEQ ID NO: 18; k) a heavy chain amino acid sequence set forth in SEQ ID NO:12 and a light chain amino acid sequence set forth in SEQ ID NO: 18; l) a heavy chain amino acid sequence set forth in SEQ ID NO:13 and a light chain amino acid sequence set forth in SEQ ID NO: 18; m) a heavy chain amino acid sequence set forth in SEQ ID NO:14 and a light chain amino acid sequence set forth in SEQ ID NO: 18; n) a heavy chain amino acid sequence set forth in SEQ ID N0:15 and a light chain amino acid sequence set forth in SEQ ID NO: 18; o) a heavy chain amino acid sequence set forth in SEQ ID NO: 16 and a light chain amino acid sequence set forth in SEQ ID NO: 18; p) a heavy chain amino acid sequence set forth in SEQ ID NO: 17 and a light chain amino acid sequence set forth in SEQ ID NO: 18; q) a heavy chain amino acid sequence set forth in SEQ ID NO: 1 and a light chain amino acid sequence set forth in SEQ ID NO: 18; r) a heavy chain amino acid sequence set forth in SEQ ID NO:2 and a light chain amino acid sequence set forth in SEQ ID NO: 18; s) a heavy chain amino acid sequence set forth in SEQ ID NO:3 and a light chain amino acid sequence set forth in SEQ ID NO: 18; t) a heavy chain amino acid sequence set forth in SEQ ID NO:4 and a light chain amino acid sequence set forth in SEQ ID NO: 18; u) a heavy chain amino acid sequence set forth in SEQ ID NO:5 and a light chain amino acid sequence set forth in SEQ ID NO: 18; v) a heavy chain amino acid sequence set forth in SEQ ID NO:6 and a light chain amino acid sequence set forth in SEQ ID NO: 18; w) a heavy chain amino acid sequence set forth in SEQ ID NO:7 and a light chain amino acid sequence set forth in SEQ ID NO: 18; x) a heavy chain amino acid sequence set forth in SEQ ID NO:8 and a light chain amino acid sequence set forth in SEQ ID NO: 18; y) a heavy chain amino acid sequence set forth in SEQ ID NO: 10 and a light chain amino acid sequence set forth in SEQ ID NO:9; z) a heavy chain amino acid sequence set forth in SEQ ID NO: 11 and a light chain amino acid sequence set forth in SEQ ID NO:9; aa) a heavy chain amino acid sequence set forth in SEQ ID NO: 12 and a light chain amino acid sequence set forth in SEQ ID NO:9; bb) a heavy chain amino acid sequence set forth in SEQ ID NO: 13 and a light chain amino acid sequence set forth in SEQ ID NO:9; cc) a heavy chain amino acid sequence set forth in SEQ ID NO: 14 and a light chain amino acid sequence set forth in SEQ ID NO:9; 109 dd) a heavy chain amino acid sequence set forth in SEQ ID NO: 15 and a light chain amino acid sequence set forth in SEQ ID NO:9; ee) a heavy chain amino acid sequence set forth in SEQ ID NO: 16 and a light chain amino acid sequence set forth in SEQ ID NO:9; ff) a heavy chain amino acid sequence set forth in SEQ ID NO: 17 and a light chain amino acid sequence set forth in SEQ ID NO:9; wherein the said monoclonal antibody or cocktail of at least two monoclonal antibodies a) to ff) has a K_D of 10^-7 M or less, more preferably 10^-10 M or less and more preferably 10^-12 M or less for target antigen of SARS-CoV-2. A composition comprising cocktail of at least two monoclonal antibodies that binds SARS-CoV-2 spike protein wherein one antibody comprises a heavy chain amino acid sequence set forth in either SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO: 8 and light chain amino acid sequence set forth in SEQ ID NO:9 and the second antibody comprises a heavy chain amino acid sequence set forth in either SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 and light chain amino acid sequence set forth in SEQ ID NO: 18. A composition comprising cocktail of at least two monoclonal antibodies that binds SARS-CoV-2 spike protein wherein one antibody comprises a heavy chain amino acid sequence set forth in either SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 and a light chain amino acid sequence set forth in SEQ ID NO: 18 and the second antibody comprises a heavy chain amino acid sequence set forth in either SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 and a light chain amino acid sequence set forth in SEQ ID NO: 9. The composition as claimed in claim 2 or 3, wherein the composition comprises one or more monoclonal antibodies as claimed in claims 1 to 3 and suitable pharmaceutical excipients. The composition as claimed in any preceding claims which comprises a cocktail of 110 at least two monoclonal antibodies and suitable pharmaceutical excipients.

6. The antibody as claimed in any preceding claims comprising amino acid sequence of constant region of anti-SARS-CoV-2 antibody selected from IgG₁, IgG₂, IgG₃, IgG₄, IgG₂/G₄, IgA, IgE, IgM or IgD constant region, preferably the IgG₁ or IgG₄ constant region. 7. The anti-SARS-CoV-2 antibodies or cocktail of at least two antibodies as claimed in any preceding claims, having modified or reduced or no ADCC, ADCP and /or CDC activity.

8. The anti-SARS-CoV-2 antibody or cocktail of at least two antibodies as claimed in any preceding claims, having a K_D of 10^-7 M or less, more preferably 10^-9M or less for recombinant human neonatal Fc receptor (rhFcRn). 9. The anti-SARS-CoV-2 antibody or cocktail of at least two antibodies as claimed in any preceding claims which has reduced or no binding to recombinant Human FcγRIIIa (Phe), Human FcγRIIa, Human FcγRIIb, Human FcγRI and Clq.

10. The anti-SARS-CoV-2 antibody or cocktail of at least two antibodies as claimed in any preceding claims which cross-reacts with SARS-CoV-2 spike protein from species other than human.

11. The anti-SARS-CoV-2 antibody or cocktail of at least two antibodies as claimed in any preceding claims which has higher binding specificity towards SARS-CoV-2 spike protein.

12. The anti-SARS-CoV-2 antibody or cocktail of at least two antibodies as claimed in any preceding claims having an increased half-life in subject.

13. The anti-SARS-CoV-2 antibody or cocktail of at least two antibodies as claimed in any preceding claims which has an anti-viral effect or has an ability to neutralize SARS-CoV-2 virus.

14. A process of making the antibodies as described in any preceding claims by cell culture method comprising: a) culturing the host cell expressing monoclonal antibody capable of binding and neutralizing SARS-CoV-2 spike protein; and b) isolating and recovering monoclonal antibody capable of binding and neutralizing SARS-CoV-2 spike protein expressed in the said host cell. 111

15. The process of purifying the antibodies as described in any preceding claims by suitable chromatography or purification technique.

16. A kit comprising one or more monoclonal antibodies or cocktail of at least two monoclonal antibodies as claimed in any preceding claims capable of binding and neutralizing to SARS-CoV-2 spike protein.

17. A method for diagnosing SARS-CoV-2 or other viruses related to coronavirus family using the monoclonal antibodies or cocktail of at least two monoclonal antibodies as claimed in any preceding claims, capable of binding and neutralizing SARS-CoV-2 or other viruses related to coronavirus family.

18. A method of treating and preventing SARS-CoV-2 or other viruses related to coronavirus family using each of the monoclonal antibody or cocktail of at least two monoclonal antibodies as claimed in any preceding claims, capable of binding SARS-CoV-2 or other viruses related to coronavirus family.

19. The monoclonal antibody or cocktail of at least two monoclonal antibodies as claimed in any preceding claims, capable of binding SARS-CoV-2 or other viruses related to coronavirus family can be used for parenteral administration that includes intravenous, subcutaneous, intra peritoneal, intramuscular administration or any other route of delivery. 0. The pharmaceutical composition comprising a monoclonal antibody or cocktail of atleast two monoclonal antibodies as claimed in any preceding claims, and a pharmaceutically acceptable carrier or diluent.