WO2023148641A1 - Monoclonal antibodies specific to receptor binding domain of sars-cov2 and uses thereof - Google Patents

Abstract

The present invention discloses monoclonal antibodies capable of exalted binding with high affinity to the receptor-binding domain (RBD) of the spike (S) protein of the severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) in order to inhibit the binding of the SARS-CoV-2 to the angiotensin-converting enzyme 2 (ACE2), the functional receptor on the host cells. The present invention also discloses composition comprising mAbs as disclosed herein. The antibodies of the present invention can be used for diagnostic, therapeutic and prophylactic methods.

Description

MONOCLONAL ANTIBODIES SPECIFIC TO RECEPTOR BINDING DOMAIN OF SARS-COV2 AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to monoclonal antibodies against SARS-CoV-2. More specifically, the invention describes antibodies against Receptor Binding Domain (RBD), compositions comprising the antibodies and utilities of said antibodies inimmunoassay methods, kits for detecting COVID-19 infection.

BACKGROUND OF THE INVENTION

Recent human pandemics worldwide, including south-east Asia, have been linked to the emergence of a novel Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2 or CoV-2), at the end of 2019 in Wuhan, China, causing Corona Virus Disease 2019 (COVID-19). SARS-CoV-2 is an enveloped RNA virus and has been classified as a subfamily of Orthocoronavirinae, with similarities of 79.5 % and 96 % of SARS CoV and Bat coronavirus respectively (Current Biology 2020, vol.30, page: 2196-2203).

The global outbreak of SARS-CoV-2 infection is being dealt with by mass vaccination. Thereare a number of vaccine formulations, but concerns remain over the possibility of future recurrences, especially with recent reports of upcoming variants or escape mutants with enhanced infectivity and disease severity. Additionally, till date no effective treatment or prophylaxis is currently available to combat this morbidity inducing virus.

Like other coronaviruses, SARS-CoV-2 is an enveloped virus containing a large, positive- stranded RNA genome that encodes viral replicase proteins and structural proteins including spike (S), membrane (M), envelope (E), nucleocapsid (N), and several uncharacterized proteins. Coronavirus infection is initiated by the attachment of the S protein to the specific host receptor, which triggers a conformational change in the S protein. The S protein of SARS-CoV-2 is a type I transmembrane glycoprotein with a predicted length of 1,255 amino acids that contains a leader (residues 1-14), an ectodomain (residues 15-1190), a transmembrane domain (residues 1191-1227), and a short intracellular tail (residues 1227-1255) (ACS CentralScience. 2020 Vol. 6(10), page: 1722-1734). The SI domain of SARS-CoV-2 S protein mediates virus-binding with angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS- CoV-2 in susceptible cells. The receptor-binding domain (RBD) of SARS-CoV- 2 Sprotein is a major target of neutralizing antibodies induced in patients infected with SARS- CoV-2 and in animals immunized with inactivated viruses or S proteins.

It is envisaged that antibodies targeting the Receptor Binding Domain of the virus, would have several applications. While there are few reports pertaining such antibodies against this domain. None of them are active or have attained clinical significance, so far.

Hence, there is a need for antibodies targeting the Receptor Binding Domain (RBD) of the SARS-CoV-2 virus, with clinical significance.

OBJECT OF THE INVENTION

An object of the present invention is to provide monoclonal antibodies (mAbs) that bind to SARS- CoV-2 virus, particularly to the Receptor Binding Domain of SARS-CoV-2 viruses.

Another object of the present invention is to provide mAbs with increased specificity so that these mAbs form basis for effective prophylactic and therapeutic agents.

Another object of the invention is to provide a composition comprising the mAbs of the presentinvention. Another object of the present invention is to provide diagnostic methods and kits comprising the mAbs of the present invention.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided a monoclonal antibody comprising amino acid sequence selected from Seq. ID No. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19.

According to another aspect of the present invention there is provided a process for producing monoclonal antibodies comprising sequences selected from SEQ. ID. No. 2 to 19, said process comprising the steps of :

(i) introducing a point mutation N501Y in RBD-His polypeptide;

(ii) cloning the mutated polypeptide in a vector and expressing in an expression system to obtain a genetically modified protein;

(iii) producing said monoclonal antibodies using said genetically modifying protein of step (ii) and hybridoma technology.

According to yet another aspect of the present invention there is provided a method of treating a subject infected with SARS-CoV-2 and/or all of the VOCs or reducing the likelihood of infection of a subject at risk of contracting SARS-CoV-2 and/or all of the VOCs, comprising delivering to the subject the monoclonal antibody of the present invention.

According to a further aspect of the present invention there is provided a use of monoclonal antibody of the present invention for treating a subject infected with SARS-CoV-2 and/or all of the VOCs or reducing the likelihood of infection of a subject at risk of contracting SARS-CoV-2 and/or all of the VOCs.

According to yet another aspect of the present invention there is provided a pharmaceutical composition comprising the monoclonal antibody of the present invention, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

The present invention also provides an immunoassay reagent comprising the antibody of the present invention, wherein said reagent is labelled with a detectable label. It is another aspect of the present invention to provide an immunoassay for the detection of SARS-CoV-2 and/or all the VOCs in a test sample, said immunoassay comprising:

(i) contacting a test sample suspected of containing COVID- 19 with a first antibody directed against SARS-CoV-2 core antigen to form a complex between said first antibody and antigen located within said test sample;

(ii) contacting said complex formed in step (i) with the monoclonal antibody of the present invention to form a complex between said antibody and antigen in the complex formed in step (i) wherein said antibody is detectably labelled and

(iii) detecting the label of the complex formed in step (ii).

It is also another aspect of the present invention to provide a vaccine comprising a polypeptide having sequence Seq ID 1, and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF FIGURES

Figure 1 depicts neutralization potential of mAbs.

Figure 2 depicts Pseudo-virus Neutralization potential of mAbs

Figure 3 depicts binding affinity of all mAbs against Wuhan RBD and spike protein.

Figure 4 depicts binding of P4A2 to different RBD proteins.

Figure 5 depicts binding of P4A2 to live virus infected cells as Mean Fluorescenceintensity.

Figure 6 depicts comparative neutralization potential of P4A2 with other FDA approved mAbs neutralization potential of mAbs.

Figure 7 illustrates the Prophylactic and therapeutic potential of P4A2 in Animal models.

Figure 8 illustrates the Binding Affinity of P4A2 to different RBD proteins.

Figure 9 illustrates the Biochemical and structural analysis of P4A2 mAb.

Figure 10 illustrates the ability of P4A2 to protect K18-hACE2 mice against SARS-CoV2 VOCs

Figure 11 illustrates the Neutralization potential of P4A2 mAb against Omicron

BA.l variant

Figure 12 illustrates the binding kinetics of mAb P4A2 to RBDs from various VOC by ELISA and biolayer interferometry (BLI).

Figure 13 illustrates the mutations in RBD associated with different VOC that do not affect binding to P4A2 Fab.

Figure 14 illustrates the Immunofluorescence staining of P4A2 binding to live virus infected VERO E6 cells.

Figure 15 illustrates the Relative change in the body mass and gross morphology of lungs excised from uninfected or infected mice showing pneumonitis and inflammation.

Figure 16 illustrates the Pulmonary pathology of SARS-CoV-2 infected hACE2 mice in prophylactic treatment.

Figure 17 illustrates the Pulmonary pathology of SARS-CoV-2 infected hACE2 mice in therapeutic group.

Figure 18 illustrates the Representative layout and images of FRNT neutralization assay with P4A2 mAb.

Figure 19 illustrates the Effect of predicted mutations on binding of P4A2 mAb.

Figure 20 illustrates the Computational modelling of P4A2 mAb with different Omicron variants.

Figure 21 illustrates the Epitopes of different broadly neutralizing antibodies.

Figure 22 illustrates the Neutralization potential of Humanized P4A2 mAb against different VOCs of SARS-CoV-2

Figure 23 illustrates the Protection potential of Humanized P4A2 mAb against the in vivo infections with Wuhan Virus DETAILED DESCRIPTION OF THE INVENTION

The present specification is accompanied by Sequence listing.

The present invention in general is directed to monoclonal antibodies that are specifically immunoreactive with the Receptor Binding Domain (RBD) of SARS-CoV-2 antigen. More particularly, the SARS-CoV-2 antigen is amino acid residues 470-492 of SARS-CoV-2.

Definitions

Unless otherwise stated, the present invention will be implemented using conventional techniques in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

In order that the present invention may be more readily understood, some scientific and technical terms are defined as follows. Unless otherwise explicitly defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. For definitions and terminology in the art, specific reference can be made to Current Protocols in Molecular Biology (Ausubel) by professionals. The abbreviations of amino acid residues are the standard 3 -letter and/or 1 -letter codes used for any one of the 20 L-amino acids commonly used in the art. The singular forms, “a”, “an” and “the”, used in the present application and the appended claims include plural forms, unless otherwise specified in the context clearly.

The term “about” means a value or an integer within an acceptable error range for the particular value or integer as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” can refer to within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” can refer to a range of up to 5%, 10% or 20% (i.e., ± 5%, ± 10% or ± 20%). When used to connect two or more optional items, the term “and/or” should be understood to mean any one of the optional items or any two or more of the optional items.

As used herein, the term “comprise” or “include” means to include the mentioned elements, integers, or steps, but does not exclude any other elements, integers, or steps. As used herein, the term “comprise” or “include”, unless otherwise indicated, encompasses “consisting of’ the mentioned elements, integers or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to encompass an antibody variable region consisting of the specific sequence.

The term “affinity” as used herein refers to the strength of the sum of all noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, “binding affinity” refers to the intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen) . The affinity of molecule X for its partner Y is generally expressed by the dissociation constant (KD). Methods for determining binding affinity are known in the art, including surface plasmon resonance (e.g., BIACORE) or similar techniques (e.g., ForteBio).

The term “antibody” as used herein refers to any form of antibody having a desirable bioactivity. Therefore, it is used in the broadest sense, including but not limited to a monoclonal antibody (including a full-length monoclonal antibody), a polyclonal antibody, a multispecific antibody (such as a bispecific antibody), a humanized antibody, a fully human antibody, a chimeric antibody, a CrossMab antibody, or a camelized single-domain antibody.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include different antibodies directed against different epitopes (or specific for different epitopes). The modifier “monoclonal” indicates the feature of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be constructed as requiring any particular method to produce the antibody.

The term “pharmaceutically acceptable carrier” refers to a diluent, an adjuvant (e.g., Freund’s adjuvant (complete and incomplete)), a pharmaceutical excipient, a pharmaceutical carrier or a stabilizer, etc., which is administered with an active substance.

The term “pharmaceutical composition” refers to such a composition that exists in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that have unacceptable toxicity to the subject to whom the composition is administrated.

As used herein, “to treat” or “treating” or “treatment” includes 1) therapeutic measures, which cure, alleviate and relieve the symptoms of a diagnosed pathological condition or disease and/or stop the progression of the diagnosed pathological condition or disease, and 2) preventive or prophylactic measures, which prevent and/or slow the development of a pathological condition or disease. Therefore, the subject receiving the treatment includes an individual who has suffered from the disease, an individual who is prone to suffer from the disease, and an individual who wants to prevent the disease. In some embodiments, the present invention relates to the treatment of a disease or condition. In some other embodiments, the present invention relates to the prevention of a disease or condition.

In some embodiments according to the present invention, the “treatment” of a disease or condition refers to the improvement of the disease or condition (i.e., alleviating or preventing or reducing the progression of the disease or at least one of its clinical symptoms). In some other embodiments, “treatment” refers to relieving or improving at least one body parameter, including those physical parameters that may not be discernible by the patient. In some other embodiments, “treatment” refers to the regulation of a disease or condition physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. Methods for evaluating the treatment and/or prevention of a disease are generally known in the art unless explicitly described herein.

In yet other embodiments according to the present invention, “prevention” of a disease or condition includes inhibition of the occurrence or development of the disease or condition or the symptom of a particular disease or condition.

In a particular embodiment, the antibody specifically binds at least one epitope formed by amino acid sequence 470-492 (SEQ ID NO: 1). In a more specific embodiment, the antibody is immunoreactive with an epitope formed by amino acid 470-492 of SARS-CoV-2 antigen.

The monoclonal antibodies of the present invention are represented by Seq. ID

Nos 2 to 19.

A summary of the mAbs of the present invention are described herein below Table 1

The present mAbs, were generated by hybridoma technology, against the SARS- CoV-2 RBD which is a synthetic mammalian cell codon optimized nucleic sequence of RBD-His (wt), which does not exist in nature in the present form. A point mutation N501 Y was introduced by genetic engineering. Also, other SARS-

CoV-2 RBD antigens were generated for screening of mAbs.

Thus according to another aspect of the present invention there is provided a process for producing monoclonal antibodies having sequences selected from Seq. ID 2 to 19, said process comprising the steps of : i) introducing a point mutation N501Y in RBD-His polypeptide; ii) cloning the mutated polypeptide in a vector and expressing in an expression system to obtain a genetically modified protein; iii) producing said monoclonal antibodies using said genetically modifying protein of step (ii) and hybridoma technology. The present invention utilizes recombinant RBD-His (N501Y) of SARS-CoV-2, which was cloned in pcDNA3.1 plasmid vector, was expressed and produced in Expi 293 Fmammalian expression system. The genetically modified protein was purified, and their functional viability was checked in different biophysical or biochemical parameters. The genetic engineering liberated the sequences of anti- RBD mu-mAbs against the SARS-CoV-2 depicted the novel sequences which does not exist in the nature. The synthetic sequences of mAbs against genetic engineered RBD polypeptide were further screened for its binding to recombinant RBD N501Y protein corresponds to alpha variant. 40 out of 400 screened synthetic sequences derived from hybridoma clones, showed reactivity in ELISA. The neutralization specificity of the culture supernatants was tested with the live WA 1/2020 SARSCoV-2 and delta variant. Two promising mAbs (P4A2 and P1A2) stood out as lead antibodies showed crossneutralizing activity up to 10,000 dilutions, whereas neutralization potential of the other tested mAbs ranged between 20-1000 dilutions. Purified P4A2 and P1A2 were tested for their breadth and potency against different SARS-CoV2 VOCs i.e. 2019-nCoV, B.l.1.7, B.1.351, P.1, B.1.617, B.1.617.2, B.1.1.529, both the tested mAbs demonstrated the potent neutralizing activity (CPE 100) against all the VOCs tested in the range of (39-156 ng mL'¹) (Figure 1). The purified P4A2 mAb showed strong binding with half-maximal effective concentration [EC50] of 0.012-0.120 mg/ml, where P4A2 binding specificity with B.1.351 and P.l was found to be nanomolar range which is slightly towards the higher concentration as compared to 2019-nCoV, B.1.1.7, B.1.617 and B.1.617.2. Immunofluorescence assay revealed the binding of P4A2 and other mAbs on VOCs infected Vero cells, with B.1.351 variant (Figure 4 and 5). Binding kinetics revealed that P4A2 binds with RBD with nanomolar or subnanomolar affinities [K_D > I O’⁹] from various VOCs (Figure 3 and 8).

Another aspect of the invention provides a monoclonal antibody that specifically immunoreactive with the Receptor Binding Domain (RBD) of SARS-CoV-2, wherein said monoclonal antibody has heavy chain and light chain sequences as: Seq ID No. 2 to 19. These are unique and novel with respect to their gene sequence for both variable light chain (VLC) and variable heavy chain (VHC). The genetically engineered identified sequences frommAbs binds to the specific region of spike (S) protein on the receptor binding domain (RBD) of the SARS- CoV-2 and /or most of the VOCs with high affinity (K_D <1.0 x 10'¹²; Kon 3.709 x 10⁵; K_off <1.0 x IO’⁷) and prevents viral entry into the human host cells Inhibition efficacy of the monoclonal antibodies was demonstrated in neutralization assays in terms of cytopathiceffect (CPE) based assay, using live authentic viruses as well as in in-vitro surrogate assays. Also, the specific binding of monoclonal antibodies (each clone with specific pair of VLC and VHC sequences) has been demonstrated by the ELISA assay and by using other antigen- antibody physicochemical interaction platforms (Figure6,9 and 10).

Furthermore, some of the mAbs (specific pair of VLC and VHC sequences) clone depicted their broadly neutralizing capacity as were cross reactive to the different variants of concern (VOC) including the recent Omicron of SARS-CoV-2. This implies that the generated mAbs sequences may have their utility in the development of alternative therapeutic modalities for passive prophylaxis and targeting.

The anti-RBD mAb with unique and novel sequence, with high affinity, differential binding with both spike and RBD proteins of SARS-CoV-2 (most of the emerging variants and/or VOC) and inhibits the interaction of RBD-ACE2, preventing viral entry in the human host cells. The anti-RBD mAb offer broadly neutralizing capacity as were cross reactive to the different variants of concern (VOC) including the Omicron of SARS-CoV-2. Purified P4A2 and P1A2 tested for their breadth and potency against different SARS-CoV2 VOCs i.e. 2019- nCoV, B.1.1.7, B.1.351, P.l, B.1.617, B.1.617.2, B.1.1.529, both the tested mAbs demonstrated the potent neutralizing activity (CPEwo) against all the VOCs tested in the range of (39-156 ng/ml) (Figure 1, 9 and 12 ).

According to the present invention, the panel of mouse mAbs developed by hybridoma technology binds to the specific region of spike (S) protein on the receptor binding domain (RBD) of the SARS-CoV-2 and/or most of VOCs with high affinity and inhibits the interaction of RBD-ACE2, preventing viral entry in the human host cells (Figure 3). Inhibition efficacy of the monoclonal antibodies were demonstrated in neutralization assays in terms of cytopathic effect-based assay, using live authentic viruses as well as in in vitro surrogate assays. Briefly, IxlO² TCID50 isolate USA-WA1/2020 virus was passaged once in Vero cells and then treated with serum dilutions ranging from 1:20 to 3260 for 90 min before adsorption on Vero cells for 1 hour. The DMEM containing 2% (vol/vol) FBS was added to the cells after washing. After 4-5 days of incubation at 37°C with 5% CO2, the presence of cytopathic effect (CPE) in cells was observed using a microscope. Uninfected Vero E6 cells were considered as control while VERO E6 cells infected with SARS-CoV2 virus wasconsidered as assay negative control(Figure 7, 10,15,16 and 17). The results for neutralization with these novel mAbs has been demonstrated in Figure no. 5.

Also, the specific binding of monoclonal antibodies (each clone) is demonstrated by the ELISA assay and using other antigen- antibody physicochemical interaction platform.

In another aspect, the mAbs of the present invention as described herein may be prepared as immunoassay reagents, more particularly, such reagents preferably are labelled with a detectable label. Apart from the monoclonal antibodies of the present invention, the immunoassay may contain art known components. These may contain enzyme conjugates or gold nanoparticles for uses in ELISA or immune-chromatography i.e. LFA; assays respectively. In still other embodiments, immunoassay reagents of the invention comprise one or more of the antibodies disclosed herein being bound to a solid phase. The immunoassay reagents comprising the antibodies of the present invention may further comprise an additional antibody against an SARS-CoV-2 antigen. For example, such an additional antibody is an additional anti-CoV-2 antibody or a monoclonal targeting different epitopes or a polyclonal antibody used as detection of SARS- CoV-2 antigen in clinical specimen.

The immunoreagents can thus be suitably designed for either detecting an SARS- CoV-2 antigen or antibodies against SARS-CoV-2 that may be present in the test sample.

In yet another embodiment, the present invention envisages compositions comprising the monoclonal antibodies (mAbs) of the present invention, including a polypeptide, antibody, or modulator of the present invention, at a desired degree of purity, and a pharmaceutically acceptable carrier, excipient, or stabilizer. Compositions may also be done to enhance the stability of the polypeptide or antibody during storage, e.g., in the form of lyophilized compositions or aqueous solutions. The preferred form of the pharmaceutical composition of the present invention includes but not limited to injections, saline formulations, aerosol formulations.

The composition may also contain one or more additional therapeutic agents suitable for the treatment of the particular indication, e.g., infection being treated, or to prevent undesired side-effects. Preferably, the additional therapeutic agent has an activity complementary to the polypeptide or antibody of the present invention, and the two do not adversely affect each other.For example, in addition to the polypeptide or antibody of the invention, an additional or secondantibody, anti-viral agent, and/or anti-infective agent may be added to the composition. Such molecules are suitably present in the pharmaceutical composition in amounts that are effectivefor the purpose intended. It has been found that a pharmaceutical composition including a cocktail of mAbs of the present invention such as viz P1A1, P4G12 etc. provides enhanced viral neutralization efficacy.

A further aspect of the invention is directed to an immunoassay for the detection of SARS CoV-2 in a test sample, said immunoassay comprising: i. contacting a test sample suspected of containing COVID- 19 with a first antibody directed against SARS-CoV-2 core antigen to form a complex between said first antibody and antigen located within said test sample; ii. contacting said complex formed in step (i) with the mAbs of the present invention to form a complex between said antibody and antigen in the complex formed in step (i) wherein said antibody may be detectably labelled and iii. detecting the label of the complex formed in step (ii).

In more specific embodiments, the immunoassay may further be characterized in that the firstantibody is directed to the Receptor Binding Domain (RBD) of S ARS CoV-2 antigen. In moreparticular embodiments the antibody employed in step (ii) is labelled with a fluorescent label. In exemplary embodiments, the label can be selected from acridinium, FITC, gold nanoparticles.

In some embodiments, the immunoassay is one in which the antibody of step (i) is coated on solid phase. In specific preferred embodiments, the antibody of step (i) comprises an antibody that is distinct from the antibody of step (ii). Alternatively, the immunoassay is one in which the antibody of step (i) comprises an antibody that is the same as the antibody of step (ii).

Any of the immunoassays of the invention may be used on a test sample obtained from a patientand the method further comprises diagnosing, prognosticating, or assessing the efficacy of a therapeutic/prophylactic treatment of the patient, wherein, if the method further comprises assessing the efficacy of a therapeutic/prophylactic treatment of the patient, the methodoptionally further comprises modifying the therapeutic/prophylactic treatment of the patient as needed to improve efficacy.

Accordingly, to yet another aspect of the present invention there is provided a vaccine composition comprising a polypeptide having sequence Seq ID 1 and a pharmaceutically acceptable carrier. The vaccine according to the present invention may comprise art known excipients such as adjuvants, stabilizers and preservatives. Adjuvants in use include various aluminium salts such as aluminium hydroxide, aluminium phosphate and potassium aluminium sulphate (alum). Stabilizers used can be Monosodium glutamate (MSG), 2-phenoxyethanol, Gelatin, and the like. Several preservatives may be used including thiomersal, phenoxyethanol, and formaldehyde.

Yet another aspect of the present invention is to provide a process of immunization comprising the steps of administering a subject with the monoclonal antibody of the present invention or a vaccine composition comprising the same.

As will be described in further detail herein, it will be understood by those skilled in the art that any of the immunoassays of the invention may be readily adapted for use in an automatedsystem or a semi-automated system.

The unique features of the monoclonal antibodies of the present invention include, but are not limited to: a. High affinity of (> 10"⁹ M) b. Novel epitopes c. Cross -reactivity to other similar antigens (from other VOCs (variants of concern)) d. Broadly neutralizing (<10"¹² gm mL'¹) e. Binds (with equal strength) to all the VOCs (300 Mean Flourescence Intensity) f. Economical in production (~ 25-30 mg per litre of culture soup) g. Easy to manufacture. h. Potent Diagnostic intermediates with high affinity (K_D <1.0 x 10"

12.

K_on

3.709 x 10⁵ and K_off <1.0 x 10"⁷)

In another aspect, the mAbs of the present invention may be utilized as

• passive-immunizing agents for prevention of SARS-CoV-2 infection;

• biological reagents for diagnosis of SARS-CoV2 infection; • immune-therapeutics for early treatment of SARS-CoV-2 infection;

• immuno-probes for studying the immunogenicity, antigenicity, structure, and functionof the SARS-CoV-2 S protein;

• development of alternate therapeutic modalities for passive prophylaxis and targetingescape mutants of SARS-CoV-2;

• development of efficient diagnosis method with the aid of the anti- RBD mAbs in asandwich ELISA or lateral flow assays;

• for alternative immunotherapeutic modalities for passive prophylaxis and targetingSARS-CoV-2 escape mutants;

• as high-quality diagnostic intermediates in a sandwich ELISA or lateral flow assays.

DETAILED DESCRIPTION OF ACCOMPANYING FIGURES

Figure 1 illustrates the Neutralization of authentic SARS-CoV-2 VOCs by mAbs which was determined using virus-induced cytopathic effect (CPE) based assay. The neutralization values are shown in ng/mL and nmol. CPE based neutralization assays were done in triplicates and repeated at least two times.

Figure 2 illustrates the Broad spectrum cross-neutralization potential of mAbs against was accessed in pseudotyped viral neutralization assay, SARS-CoV-2 WA1/2020 strain was used in this assay.

Figure 3 illustrates the Kinetics of isolated mAbs binding to RBD protein was accessed by BLLOctet. For reproducibility of data all the mAb characterization experiments were repeated at least three independent times in triplicates.

Figure 4 illustrates the Binding kinetics of mAbs to RBDs from various VOC by biolayer interferometry (BLI).

Figure 5 illustrates the Immunofluorescence intensity of P4A2 binding to live virus infected VERO E6 cells. The binding of P4A2 to the spike proteins expressed on the surface of Vero E6 cell infected with various VOCs was assessed by immunofluorescence microscopy. Vero E6 cells were infected at a MOI of 0.1 and 0.01. The number of foci at MOI 0.1 were too numerous to be counted. The foci count for the MOI 0.01 are indicated. P4A2 were used as the primary antibody (diluted 1 in 2000) followed by anti-mouse Alexa 488 as the secondary antibody. The foci were counted using AID ELISPOT 8.0 software. Representative images of the experiment performed in triplicates are shown.

Figure 6 illustrates the comparative neutralization potential of P4A2 mAb with the clinical approved or under-development therapies.

Figure 7 illustrates the prophylactic and therapeutic potential of P4A2 in Animal models. In the upper panel body mass of mice from each group was recorded for 6 days post infection and was plotted as percent change with respect to the day 0 body mass (day of infection, dpi). In the lower panel, in therapeutic treatment group, the mAb was administrated 12 h post infection. Percent change in body mass of mice challenged with Wuhan, Kappa, Delta and Beta variants in presence or absence of therapeutic treatment of P4A2 at two different concentrations (5 and 1 mg of mAb per kg body mass) is plotted as a line graph till 6 dpi, IC denotes infection control group, UN represents uninfected animal group, Ab5 and Abl denotes 5 and Img/kg dose of P4A2 mAb, The Y-axis in figure 2C and 2E is correctly represents percentage change in the body mass which was calculated as a percentage change in the body mass of that animal as compared to the body mass recorded on day zero. Figure 8 illustrates the Binding kinetics of mAb P4A2 to RBDs from various VOC by ELISA and biolayer interferometry (BLI). (A) P4A2 was immobilized on antimouse Fc biosensors and was tested using three fold serial dilutions of RBD (starting with 300 nM and going down to 3.3 nM; the five different concentrations were tested are indicated). Data shown is after the reference was subtracted and aligned using Octet Data Analysis software vl l.l (Forte Bio). Curve fitting was done with a 1:1 binding model, and kon, koff, and Kd values were calculated using a global fit.

Figure 9 illustrates the Biochemical and structural analysis which reveals that P4A2 mAb can neutralize all circulating variants of concerns. (A), Neutralization of authentic SARS-CoV-2 VOCs by P4A2 was determined using virus-induced cytopathic effect (CPE) based assay, neutralization values are shown in ng/mL and nmol. (B) Kinetics of P4A2 binding to RBDs from VOC was accessed by Bio-Layer Interferometry (BLI), Octet. (C) Structure of P4A2 Fab in complex with RBD of Alpha variant. The heavy (H) and light (L) chain of P4A2 Fab are coloured in orange and cyan, respectively and the RBD is coloured magenta. (D) The interacting residues from P4A2 paratope and the RBD epitope are displayed in stick representation and coloured according to element. The carbon atoms of H, L chain and RBD are coloured in yellow, cyan and magenta, respectively. (E) Surface representation of the P4A2 paratope with the RBD epitope is shown. 486Phe from RBD is present in a hydrophobic cavity formed on the paratope. (F) Superimposition of the P4A2-Fab:RBD (green) and the ACE2:RBD structures (blue) shows that P4A2 binding to RBD will prevent interaction of the viral protein with the ACE2 receptor. (G) Superimposition of the structure of residues 475-488 and 455-456 of RBD when bound to P4A2 Fab and ACE2 receptor. The carbon atoms of this stretch when bound to Fab and ACE2 are coloured cyan and green, respectively. (H) Computational model of P4A2 Fab bound to spike trimer show that the P4A2 can bind to the RBD of the trimer in both the ACE2 receptor accessible “up” and receptor-inaccessible “down” position. (I) Neutralization of authentic SARSCoV-2 BA.l and Delta by P4A2 was determined using focusreduction neutralization assay, CR3022 mAb was used as experimental negative control, the CR3022 is a potent neutralizing mAb for SARS-CoVl and shows nonneutralizing behaviour against SARS-CoV2. FRNT neutralization assays were done in duplicates and repeated at least three times. (J) Binding avidity (EC50) of P4A2 to different VOCs RBD proteins i. e. WA1/2020, Alpha, Beta, Gamma, Kappa, Delta and BA.l were determined by EEISA, the EC50 values are 0.0093, 0.0099,0.6002, 0.1158, 0.0131, 0.0121 and 0.2616 respectively. (K) Kinetics of P4A2 binding to BA.1 RBD protein was accessed by BLI-Octet. For reproducibility of data, all the P4A2 mAb characterization experiments were repeated at least three independent times in triplicates.

Figure 10 demonstrates the ability of P4A2 to protect K18-hACE2 mice against SARS-CoV2 VOCs. (A), Calu 3 cells were infected with Delta and BA.l variants of SARS CoV-2 at a MOI of 0.5. At 24 h post infection, cells were fixed with chilled methanol, stained with P4A2 antibody and visualized by immunofluorescence microscopy. DAPI was used to stain nuclei. Images were captured at a magnification of lOx. Representative image of uninfected cells (top row), Calu 3 cells infected with delta strain (middle row), and Calu 3 cells infected with BA.l strain of SARS CoV-2 (botom row). Scale bar represents 100 pm. (B), Broad spectrum cross-neutralization potential of P4A2 against Alpha and Beta coronaviruses was accessed in pseudotyped viral neutralization assay, SARS-CoV- 2 WA1/2020 strain was used in this assay. The P4A2 specifically neutralized SARS- CoV2 with an IC50 230 ng/mL. (C-F). P4A2 offers low-dose prophylactic and therapeutic protection in the K18-hACE2 mouse model. P4A2 antibody was infused intravenously into mice as a single dose of 100 pg (5 mg/kg body weight) as prophylactic treatment, 24 h prior to intranasal inoculation with 10⁵ PFU of SARS- CoV-2 Wuhan and Kappa isolate. (C) Body mass of mice from each group was recorded for 6 days post infection. The Y-axis represents the body mass of the animal relative to the body mass of the same animal recorded on day 0 (normalised to 100) (dpi, day of infection). (D) Lung RNA samples were used to evaluate viral load by qPCR for N gene against a known standard. The N gene copy number values obtained were plotted as bar graph mean ± SEM. (E) In therapeutic treatment group, the mAb was administrated 12 h post infection. Percent change in body mass of mice challenged with Wuhan, Kappa, Delta and Beta variants in presence or absence of therapeutic treatment of P4A2 at two concentrations (5 and 1 mg of mAb per kg body mass) is plotted as a line graph till 6 dpi. ‘IC’ denotes infection control group, and Ab5 and Abl denotes 5 and 1 mg/kg dose of P4A2 mAb, respectively. The Y-axis in panel C and E represents percentage change in the body mass of the animal relative to the body mass recorded on day 0. (F) 0 N gene copy number for viral load assessment from the lungs of the mice at 6 dpi. Data is represented as mean ± SEM values for each group. *, P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001 (One-way or Two-way ANOVA).

Figure 11 illustrates the Neutralization potential of P4A2 mAb against Omicron BA.l variant (A), Sotrovimab was immobilized on anti-human Fc biosensor and tested using three-fold serial dilutions of RBD (starting with 300 nM and going down to 3.3 nM; the five different concentrations tested are indicated). Data shown is after the reference was subtracted and aligned using Octet Data Analysis software vl l.l (Forte Bio). Curve fitting was done with a 1:1 binding model, and k_on, koff, and Kd values were calculated using a global fit. (B), Neutralization of authentic SARS CoV-2 BA.l and Delta by P4A2 and Sotrovimab was determined using focus-reduction neutralization assay. CR3022 mAb was used as the negative control. Neutralization assays were done in duplicates and repeated at least three times.

Figure 12 illustrates the Binding kinetics of mAb P4A2 to RBDs from various VOC by ELISA and biolayer interferometry (BLI). (A) P4A2 was immobilized on antimouse Fc biosensor and was tested using three-fold serial dilutions of RBD (starting with 300 nM and going down to 3.3 nM; the five concentrations tested are indicated). Data shown is after the reference was subtracted and aligned using Octet Data Analysis software vl l.l (Forte Bio). Curve fitting was done with a 1 : 1 binding model, and k_on, koff and Kd values were calculated using a global fit. (B) Cross reactive binding potential (with half-maximal effective concentration, EC 50) of P4A2 mAb to different VOCs RBD protein was tested by indirect ELISA. (C) The epitope specificity of P4A2 was evaluated for epitope competition using BLI. RBD- Fc was captured using anti-human Fc biosensor and saturated with P4A2 and the indicated mAbs at a concentration of 40 pg/ml and unbound P4A2 was washed, followed by incubation with 20 pg/ml of ACE2, P4A2 and P5B3 (binds to a topologically distinct, non-competing epitope and served as a control). No binding signal was observed for P4A2 and ACE2.

Figure 13 illustrates the mutations in RBD associated with different VOC which do not affect binding to P4A2 Fab. Using the crystal structure, computational models of P4A2 Fab in complex with the RBD from (A) Beta, (B) Gamma, (C) Delta, (D) Kappa and (E) BA.l VOC were generated. These models show that, for all the VOCs, there are no mutations in the residues that interact with the P4A2 through their side-chain and hence these mutations will not adversely impact P4A2 binding. E484 is mutated to Lys, Gin or Ala in some of the VOCs, but it forms interactions with the P4A2 Fab paratope through the backbone atoms and not through the side chain.

Figure 14 illustrates the Immunofluorescence staining of P4A2 binding to live virus infected VERO E6 cells. Vero E6 cells were infected with Wuhan (A), Delta (B) and BA.l variant of SARS CoV-2 at a MOI of 0.1 (C) and 0.01 (D). Cells were fixed with 7.4% formaldehyde 32 h following infection, stained with mAb P4A2 and observed under an immunofluorescence microscope. DAPI was used to stain nuclei. Images were captured at a magnification of lOx. Scale bar represents 100 pm.

Figure 15 illustrates the relative change in the body mass and gross morphology of lungs excised from uninfected or infected mice showing pneumonitis and inflammation. (A) Body mass of mice from each group (both prophylactic and therapeutic) was recorded for 6 days post infection. The Y-axis represents the body mass of the mouse relative to the body mass of the same mouse recorded on day 0 (normalised to 100) (dpi, day of infection). (B). Schematic representation of the animal challenge experiment performed with prophylactic or therapeutic intervention of P4A2 antibody. Briefly, animals challenged with Wuhan, Kappa, Delta or Beta SARS-CoV-2 strain (10⁵ pfu/ mice) were given prophylactic (1 day prior to challenge) or therapeutic dose (12 h post challenge). (B) Representative images of the excised lung showing inflammation and pneumonitis.

Figure 16 illustrates the Pulmonary pathology of SARS-CoV-2 infected hACE2 mice in prophylactic treatment. Animals challenged with SARS-CoV-2 with or without P4A2 antibody were euthanized on day 6 post infection. The left lower lobe of their lung were fixed in 10% formalin solution and used for H & E staining. (A) Representative transverse section of the lung. Images at showing pneumonitis (magnification = 40x; red arrow), alveolar epithelial injury (blue arrow) and inflammation (black arrow). The stained sections were assessed by blinded-trained histologist on the scale of 0-5 (where 0 represents no feature, while 5 represents the maximum score). (B) The histological scores for each pulmonary pathology was plotted as mean ± SEM. The disease index score was calculated by taking the average score of all pulmonary pathologies (pneumonitis, alveolar epithelial injury and inflammation). Ther, therapeutic; Pro, prophylactic. NS, not statistically significant.

Figure 17 illustrates the Pulmonary pathology of SARS-CoV-2 infected hACE2 mice in therapeutic group. Lung samples from euthanized animals 6 days post challenge were fixed in 10% formalin solution and stained with H & E. (A) Representative transverse section of the H & E stained lung images at 40 x magnification showing pneumonitis (red arrow), alveolar epithelial injury (blue arrow) and inflammation (black arrow). The stained sections were assessed by blinded-trained histologist on the scale of 0-5 (where 0 represents no feature, while 5 represents the maximum score). (B) The histological scores for each pulmonary pathology was plotted as mean ± SEM. The disease index score was calculated by taking the average score of all pulmonary pathologies. Ther, therapeutic; NS, not statistically significant.

Figure 18 illustrates the representative layout and images of FRNT neutralization assay with P4A2 mAb. (A). SARS CoV-2 neutralizing antibodies were used as the positive control [two-fold dilution series starting at 1:20 and ending at 1:640 (highlighted in green)]. Pre-defined virus dilution (virus only control; wells 7 through 11 in rows A and B to get at least 60-200 FFU/well) and only medium (no virus control; added in well 12 in rows A and B) served as the control. P4A2 (rows C and D), Sotrovimab (rows E and F) and CR30322 (rows G and H) as two-fold serial dilutions were used were used in the experiment. The antibody neutralization assays against SARS CoV-2 Delta variant (B.1.617.2) (B) and Omicron variant (B.1.1.529) (C) was done similarly.

Figure 19 illustrates the effect of predicted mutations on binding of P4A2 mAb. (A) Amino acid sequence of the variable regions of heavy and light chain of mAb P4A2 are denoted as P4A2-H and P4A2-L, respectively. Framework regions are shown in black, whereas the complementary determining region 1, 2 and 3 are highlighted in red, blue and green, respectively, (B) The mutations predicted by Maher et al are displayed in stick representation and coloured according to element. The Spike-RBD, heavy and light chains of P4A2 Fab are shown in magenta, orange and cyan, respectively. None of the predicted mutations of the Spike-RBD overlap with the residues that interact with P4A2 Fab and therefore it is possible that these mutations may not reduce the ability of P4A2 to neutralize the corresponding new variants of SARS-CoV-2.

Figure 20 illustrates the Computational modelling of P4A2 mAb with different Omicron variants. Computational models of P4A2 Fab in complex with Spike-RBD from different Omicron lineages BA.l, BA.2, BA.3, BA.4/5, BA.2.75 and BA.2.12.1.

Figure 21 illustrates the epitopes of different broadly neutralizing antibodies. The surface of the Spike-RBD is displayed and the epitope for different mAbs are shown in red colour. The mAbs JMB2002 and S3H3 are not shown here because the epitopes for these two antibodies are outside the RBD. The epitopes of 87G7, 510A5, Cov2-2196, NCV2SG48, NCV2SG53, S2E12, S2K146 and ZWD12 shown some overlap with that of P4A2 but only P4A2 mAb possesses a hydrophobic cleft into which the 486Phe residue is buried. Based on available information, P4A2 forms multiple interactions with its cognate epitope on Spike-RBD and multiple residues present in this epitope are critical for interaction with ACE2.

Figure 22 illustrates the neutralization potential of humanized P4A2 mAb clones against different SARS-CoV2 VOCs tested.

Figure 23 illustrates the protective effect of humanized P4A2 mAb in Animal challenge model. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the exampleswhich follow represent techniques used/followed by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtaina like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Materials and Methods

Viruses and antibodies

SARS-CoV-2 to B.6, and delta lineage viruses were isolated as described previously [Kinetics of viral load, immunological mediators and characterization of a SARS-CoV-2 isolate in mild COVID-19 patients during acute phase of infection | medRxiv. [cited 26 Feb 2022]. Available: https://www.medrxiv.org/content/! 0.1101/2020.11.05.20226621 v2 ; Effectiveness of ChAdOxl nCoV-19 vaccine against SARS-CoV-2 infection during the delta (B.1.617.2) variant surge in India: a test-negative, case-control study and a mechanistic study of post-vaccination immune responses. The Lancet Infectious Diseases. 2021 ;0. doi:10.1016/S1473-3099(21)00680-0 . Leo Poon provided SARS-CoV-2 Omicron isolate (sub-lineage BA.l) [Probable Transmission of SARS-CoV-2 Omicron Variant in Quarantine Hotel, Hong Kong, China, November 2021. Emerg Infect Dis. 2022;28: 460-462. doi:10.3201/eid2802.212422]. Vero E6 or Calu-3 cells were used to propagate SARS-CoV-2 variants. Dr. Raiees Andrabi (Scripps Research Institute, USA) generously provided the full-length spike proteins of SARS-CoV-1, SARS-CoV-2, MERS, HKU1, OC43, NL63, and 229E. CR3022 antibody was purchased commercially (Sino Biologicals). 1162 (isotype IgG), a non-neutralizing antibody was used from the present inventor’s previous study [Identification of an anti-SARS-CoV-2 receptor binding domain directed human monoclonal antibody from a naive semi-synthetic library. J Biol Chem. 2020. doi:10.1074/jbc.AC120.014918]. The pseudotyped viral stocks of all seven alpha and beta coronaviruses were prepared as described in the present inventor’s previous study [Cross -neutralization of SARS-CoV-2 by HIV-1 specific broadly neutralizing antibodies and polyclonal plasma. Immunology; 2020 Dec. doi:10.1101/2020.12.09.418806] . The present inventors have used B .1.1.7 (Alpha), Bl.1.351 (Beta), B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.1.529 (Omicron, BA.l) terminology throughout our manuscript.

Expression and purification of antibody and RBD protein

A synthetic codon optimized (for mammalian cells) nucleotide sequence of RBD- His (wildtype), spike protein (wildtype), and a point mutant of RBD-His (N501 Y) protein (variant B.l.1.7) and RBD corresponding to other variants from SARS- CoV-2, were cloned in pcDNA3.1 plasmid vector. The constructs were expressed and produced in Expi 293 F mammalian expression system [Antibody-mediated broad sarbecovirus neutralization through ACE2 molecular- mimicry. bioRxiv. 2021: 2021.10.13.464254. doi: 10.1101/2021.10.13.464254]. Briefly, the cells were transiently transfected with the plasmids. The supernatant was collected after 5-6 days and the soluble protein was purified using Ni-NTA affinity chromatography (Qiagen, Germany).

Animal ethics statement

All the mice were procured from the Small Animal Facility, THSTI, Faridabad. All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals as promulgated by Committee for the Purpose of Control And Supervision of Experiments on Animals (CPCSEA), Government of India and adopted by the Institution Animal Ethics Committee of THSTI (IAEC Project/Protocol No: THSTI-IAEC-146, IAEC-160). The approval of the Institutional Biosafety Committee (approval # THS-354/2021) and Department of Biotechnology Review Committee on Genetic Manipulation (RCGM approval #: BT/IBKPZ 137/20220) were taken before commencing work. Generation of hybridoma for anti-RBD (N501Y) murine monoclonal antibodies

Six to eight week old, female BALB/c mice were immunized intramuscularly (i.m.) with purified RBD (N501Y) protein (30 pg in 100 pL PBS per animal) along with Quit A adjuvant (InvivoGen, USA). Mice were boosted thrice with the purified protein (30 pg, 15 pg and 7.5 pg in 100 pl PBS per animal), along with Quil A adjuvant. Sera samples from mice were collected three days after the first and second booster. The mouse with the highest titer of serum cross -neutralizing antibodies, was given a final booster injection 4 days before the spleen was aseptically removed. Splenocytes were utilized in the generation of hybridomas using ClonaCell-HY Hybridoma Generation Kit (STEMCELL Technologies, USA), following the manufacturer’s protocol. The well-adapted antibody secreting clones were propagated in tissue culture flasks and vials were stored in liquid nitrogen for future use. Hybridoma clones secreting anti-RBD antibodies were further screened by ELISA.

ELISA

For the screening of hybridoma clone heat inactivated mice sera (dilution starting from 1:100 to 1:218, 700)/ purified mAbs (5 to 0.002 pg mL'¹), ELISA plates were coated with recombinant RBD (N501Y) protein (1 pg mL'¹; 100 pL per well). Plates were blocked with 5% non-fat milk and hybridoma culture supernatant or purified mAbs were added in three-fold serial dilutions. Following incubation for Ih at room temperature, HRP conjugated goat anti-mouse secondary antibody (Jackson Immunoresearch, USA; diluted 1 in 2500) was added to each well. In the case of titration experiments, purified mAbs (100 pL per well) were added as threefold serial dilutions starting with 5 pg mL'¹. Standard protocols of blocking (5% skimmed milk in PBS) and washing of ELISA plates (4 times with PBS-0.05% Tween-20) were followed. All the experiments were repeated at least three times in triplicates.

Purification of anti-RBD (N501Y) murine monoclonal antibodies

Serum-free media designated for monoclonal antibody production was used to propagate the hybridoma cells (0.5 x 10' cells mL^-1) in a T175 tissue culture flask in order to purify the antibodies. For scale up of antibody production, a WHEATON CELLine flask was used for hybridoma culture, following the manufacturer’s instructions. Protein G agarose resin (G-Biosciences) was used to purify the anti- RBD IgG mAbs from the hybridoma culture supernatant. Five column volumes of 1 x PBS were used to wash the beads in the column. Two to three column volumes of 0.1 M glycine (pH 2.5) was added to elute the antibodies, followed by neutralization with IM Tris-HCl (pH 8.0). The purified antibodies were dialyzed against 1 x PBS three times, using dialysis tubing (Thermo Fisher Scientific; MWCO = 10 kDa), and concentrated with a 50 kDa cut-off Amicon Ultra- 15 centrifuge unit (Millipore). A 0.2 pm syringe filter (MDI, India) was used to filter the antibody solutions before they could be used in experiments. NanoDrop spectrophotometer was used to estimate the protein concentration and purified IgG mAbs were analyzed on using 12% Tris-Glycine-SDS-PAGE analysis.

Cytopathic effect-based neutralization assay

Initial screening of heat-inactivated mice serum samples and hybridoma culture supernatants was performed. Briefly, heat-inactivated serum or purified mAbs were serially diluted two or four times and mixed with 100 TCID₅₀ of SARS-CoV-2 isolates. The serum or mAb mixture was transferred to the Vero E6 monolayer seeded in a 96-well plate in triplicate and incubated for Ih. The cell surface was washed with serum-free medium and replenished with fresh complete medium. The plate was further incubated for 72h at 37°C in a humidified CO₂ incubator. The cells were observed for the absence of viral cytopathic effect and was used as an indicative of neutralization. The neutralization titer was defined as the dilution at which no cytopathic effect was seen. All the experiments were repeated at least three times in triplicates.

Live virus focus reduction neutralization assay:

Virus neutralization assay was performed as described previously [ Sub-optimal Neutralisation of Omicron (B.1.1.529) Variant by Antibodies induced by Vaccine alone or SARS-CoV-2 Infection plus Vaccine (Hybrid Immunity) post 6-months | medRxiv. [cited 20 Feb 2022]. Available: https://www.medrxiv.org/content/10.1101/2022.01.04.22268747vl}. Inhibitory concentration was assessed by foci reduction neutralization assay using SARS- CoV-2 delta (Genbank accession no.: MZ356904.1) and Omicron variants, BA.l (GISAID accession no.: EPI_ISL_8764350). The virus neutralization assay was performed in Vero E6 cells. Cells were incubated for 24 hour for Delta and 32 hour for the Omicron variant. The virus stock was propagated in Calu-3 cells (American Type Culture Collection, USA). Control purified IgG was used as negative control. All the experiments were repeated at least three times in triplicates.

Vero E6 cells were maintained in Minimal Essential Medium (MEM) containing 10% heat-inactivated fetal bovine serum (FBS), 1% Penicillin -Streptomycin solution (PS), 1% Non-Essential Amino Acid (NEAA) seeded in a 96-well plate (25,000 cells per well) and incubated overnight at 37°C with 5% CO2. Reagents were two-fold serially diluted (75 pL + 75 pL) in dilution medium (MEM with 1 % FBS) from 5pg mL’¹ to 0.00244 pg mL'¹. Predefined virus dilution (75pL) was added and incubated for 1 h at 37°C with 5% CCE. For the quality control step, predefined SARS-CoV-2 neutralizing antibodies positive serum was used. Virus dilution was determined to get approximately 60-200 foci with cell control. After neutralization incubation virus-reagent mixture was added over cell monolayer for virus adsorption and incubated 1 h at 37°C with 5% CO2. Overlaying medium was added after adsorption incubation and cells were fixed after 24 h. In the case of BA.1 , cells were fixed after 28 h of incubation. Cells were permeabilized with using 100 pL of IMF buffer and incubated at room temperature for 20 min. Cells were stained with the addition of 100 pL of anti-nucleocapsid antibody at 1:2000 dilution for 1 h, followed by 100 pL of 1 :500 dilution of Alexa flour 488 conjugated donkey anti-mouse IgG secondary antibody. After incubation, cells were washed thrice with 1 x PBS and developed foci were quantified by AID iSpot reader (AID GmbH, Germany) using AID EliSpot 8.0 iSpot software. Using AID raw file to find IC50 by using GraphPad Prism (Version 9.3.1) and graphs were plotted with log(inhibitor) versus normalized response - Variable slope parameter. The purified P4A2 antibody was two-fold serially diluted starting from 20 to 0.039 pg mL'¹. The virus neutralization assay was performed in Vero E6 cells. Cells were incubated for 24 h for Delta and 32 h for BA.l variant. The virus stock was propagated in Calu- 3 cells (American Type Culture Collection, USA). Control purified IgG was used as the negative control. All the experiments were repeated at least three times in triplicates.

Pseudovirus assay-based neutralization assay

Full length spike proteins of all alpha and beta coronaviruses were co-transfected in 1.25 x 10⁵ HEK293T cells, with helper plasmid expressing firefly luciferase, an HIV-1 backbone and for SARS-CoV-1 and -2, serine protease TMPRSS2 (CMV- Luc, RA8.2 backbone plasmid, pTMPRSS2). After 68-72hour culture supernatant was collected and stored at -70°C. To access the cross-neutralization potential of P4A2, three-fold serial dilution (starting at 10 μg mL^-1) of P4A2 was performed. The serially diluted P4A2 antibody was mixed with respective pseudotyped viruses for 60 min at 37°C. Pseudovirus/ P4A2 combinations were added to 293T-ACE2 cells pre-seeded (24 h) at 20,000 cells per well. After 48-72 h, relative luminescence unit (RLU) was measured on a luminometer. The percent reduction in neutralization was measured as ratio of relative RLU readout in the presence of P4A2 normalized to RLU readout in the absence of bnAb. Four-parameter logistic regression was used to calculate the half maximum inhibitory concentrations (IC50) (GraphPad Prism version 8.3). 1162 and CR3022 mAb against SARS-CoV-2 were used as assay controls. All the experiments were repeated at least three times in triplicates.

Immunofluorescence microscopy

A 96-well plate was seeded with Vero E6 (25,000 cells per well). Virus suspensions at the indicated MOIs (100 μL per well) were added and the plate was incubated for 1 hour. After a 24 hour incubation, cells were fixed with 7.4% formaldehyde and left overnight. Wells were washed with PBS thrice. The cells were permeabilized with 100 pL buffer (20 mM HEPES, pH 7.5, 0.1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 0.02% sodium azide) at room temperature for 20 min. The cells were incubated with mAb P4A2 (diluted 1 in 2000) at room temperature for 60 min. Secondary antibody (anti-mouse Alexa 488, cat no.: A21206, Invitrogen; 100 pL diluted 1 in 500). Micro-plaques were estimated using AID iSpot Analyzer (Autoimmun Diagnostika GmbH) and foci were counted using AID ELISPOT 8.0 software.

To check the binding of P4A2 with Delta and BA.1 infected Calu 3 cells (90,000 in each well) were seeded in 8-well chamber slides. Cells were incubated for 48 h at 37°C in CO2 incubator. DMEM high glucose with 10% (v/v) FBS was removed 24 h later and cells were washed with lx PBS. Cells were infected at a MOI of 0.5 (DMEM high glucose supplemented with 2% FBS). Delta and BA.l virus (100 μL per well) was added and incubate at 37°C on a rocker. The virus was removed and washed twice with 1 x PBS. 300 pL of 10 % complete DMEM was added and incubated further for 24 h at 37°C.

Biolayer interferometry binding assay

Binding assays were carried out using an Octet Red instrument (ForteBio) using biolayer interferometry (BLI). Briefly, mouse Fc sensors (ForteBio Inc.) were used to capture the P4A2 mAb at 10 pg mL^-1 in 1 x kinetics buffer [1 x PBS, pH 7.4, 0.01% (w/v) BSA and 0.002% (v/v) Tween-20] and incubated at the indicated concentrations of RBD. Associations and dissociations have been reported, depending on the analyte. Data was analyzed using the software ForteBio Data Analysis. The starting concentration of RBD was 300 nM followed by three-fold serial dilution. All the experiments were repeated at least three times.

Purification, crystallization, data collection, refinement and analysis

A total of 30 mg of P4A2 mAb was digested for Fab preparation. The Fab preparation was performed by Pierce Fab Preparation Kit (Cat. no. 44985) as per the manufacturer’s protocol. The purified Fab and RBD proteins were mixed at 1 :1.7 molar ratio and incubated overnight at 4°C. This was then subjected to size exclusion chromatography on 16/600 Superdex 200 column (Cytiva) in a buffer containing 20 mM HEPES pH 7.5 and 150 mM NaCl. The peak corresponding to P4A2 Fab:RBD complex was concentrated to 10 mg mL^-1 and stored at -80°C by flash freezing. The purified complex was subjected to crystallization trials using commercially available screens and trays were set up using Mosquito Crystallization Robot (TTP Labtech). The hits obtained in different screens were further expanded to produce single crystals which were tested for diffraction using a METALJET X-ray home source (Bruker Inc.). The condition that provided crystals with best diffraction quality was composed of 0.2 M magnesium formate dihydrate and 10% PEG 5KMME. These crystals were frozen with 20% glycerol as cryo-protectant. X-ray diffraction data could be collected to a maximal resolution of 3.0 A at the automated ID30A-1 beamline in ESRF, France. The diffraction data was processed using IMOSFLM and AIMLESS programs of the CCP4 suite (Supplementary Table S3).

Table S3. Crystallographic data and refinement statistics for P4A2 Fab:Spike-RBD complex

“Values in parentheses are for the highest resolution shell. where is the integrated intensity of a given reflection.

was calculated using 7% of data excluded from refinement.

The structure was determined by molecular replacement using PHASER and the search model was the STEC90-C11 Fab:RBD complex. This model was subjected to iterative model building and refinement using COOT and PHENIX, respectively and the sequence of the STEC90-C 11 Fab was slowly changed to that of P4A2. The final R_free and R_Work are 28.2 and 23.0%, respectively. The refined structure was deposited with Protein Data Bank with the accession code 7WVL.

The structure was visualized and analysed using PYMOL (Schrodinger Corp.) and the interactions were identified using the CONTACT program of CCP4. Mutations were created in silico in the RBD structure using PYMOL to obtain models of P4A2 Fab bound to RBD corresponding to different SARS-CoV-2 strains and Omicron lineages. These models were subjected to energy minimization using the DESMOND module of the Schrodinger suite (Schrodinger Inc.) and analyzed. For the model of the Spike-RBD from BA.4/5 in complex with P4A2 Fab, the binding energy was calculated using the Molecular Mechanics energies combined with Generalized Bom and Surface Area continuum (MMGBSA method). Two models of P4A2 bound to Spike Trimer with the RBD in the up and down conformation were prepared using 7TM0 and 7T0U and these models were also subjected to energy minimization using the DESMOND module. All the figures were prepared using PYMOL.

Animal protection studies

Eight- to ten- week-old K18-hACE2 transgenic mice were pebbled and randomly allotted to different groups (n = 5) viz., infection control and those receiving P4A2 in different cages. The animal experiments and procedures were performed in accordance with the Institutional Animal Ethics Committee (IAEC), Institutional Biosafety Committee (IBSC) and Review Committee on Genetic Manipulation (RCGM) guidelines. In prophylactic treatment, antibody recipient groups were given intraperitoneal (i.p. ) infusion of P4A2 mAb one day prior to challenge (day ‘-1’), except for the control group where PBS was given (no virus challenge). In therapeutic treatment group, the mAb was administrated 12 hour post infection.

Clinical spectrum of SARS-CoV-2 infection

The mouse experiments were done at the Animal Biosafety Laboratory (ABSL)-3. Change in daily body weight, activity and clinical symptoms of all the animals were monitored post infection. On day 6, all the infected animals were euthanized, the lungs were collected and imaged for gross morphological studies. The right lower lobe of the lung was immersed in a 10% (v/v) neutral formalin solution and subjected to immunohistochemistry analysis. The viral load parameters were analysed using homogenized lung tissues in 2 mL Trizol solution. The homogenates were stored immediately at -80°C till further use. Blood was drawn from the animals via the retro-orbital vein on days ‘-1’ and ‘O’, and via direct heart puncture after euthanizing the animal. Serum samples were stored at -80°C for future experiments.

Quantification of viral load in lung

RNA was isolated from homogenized lung tissues using the Trizol-chloroform technique according to the manufacturer's procedure, and quantified using Nanodrop. The iScript cDNA synthesis kit (Biorad, USA) was used for cDNA synthesis. Briefly, 1 pg total RNA was reverse-transcribed into cDNA. The qPCR was performed on diluted cDNAs (1:5) using the KAPA SYBR FAST qPCR Master Mix (5x) Universal Kit (KK4600) and 7500 Dx real-time PCR equipment (Applied Biosystems, USA). The results were analyzed with SDS2.1 software. For virus load estimation, the CDC-approved SARS-CoV-2 N gene primers 5'- GACCCCAAAATCAGCGAAAT-3' (forward) and 5'- TCTGGTTACTGCCAGTTGAATCTG-3' (reverse) were used as previously described. The logio N copy number of N gene was calculated by using pre-titrated SARS-CoV-2 genomic RNA and expressed as N copy number/' lung mass (mg). To produce the standard curve for absolute quantification, a known copy number of viral RNA was employed as a standard.

Reverse transcriptase-polymerase chain reaction and nucleotide sequencing The variable region of the immunoglobulin heavy and light chain transcripts expressed in B cell hybridoma P4A2 was RT-PCR amplified and sequenced following the protocol and primers. Briefly, cDNA was synthesized from 10 to 50 snap frozen hybridoma cells, using a commercially available kit (Qiagen, Germany ) with isotype specific antisense primers, each at a concentration of 0.75 pM. The 20 pL reaction was performed at 42°C for 30 min. Reverse transcriptase was inactivated by incubating at 95°C for 3 min. The nested PCR amplification was performed using Q5 DNA polymerase (New England Labs, USA). The cDNA (4 pL) was used as template in a 50 pL first round PCR which comprised of external antisense primer (0.25 p.M) and a cocktail of V_H (or V_L as the case may be) family specific external sense primers, each at a final concentration of 0.1 pM, 1 x Q5 DNA polymerase buffer, dNTPs (200 pM) and Q5 DNA polymerase (0.5 U) as recommended by the manufacturer. Two microliter of the first round PCR product was used as template in a 50 pL second round nested PCR following the protocol described above for the first round. The second round PCR product was column purified following the manufacturer’s instructions (Invitrogen, USA) and sequenced.

Sequence analysis

The nucleotide sequence was analyzed using Sequencher (version 5.4.5; Gene Codes, USA) and MacVector (version 17.5.4; MacVector, USA) software. The V, D and J gene segment assignment was done using IMGT/V-QUEST (https://www.imgt.org/IMGT_vquest/input) and IgBlast

(https://www.ncbi.nlm.nih.gov/igblast/) using default parameters.

Antibody humanization by CDR grafting plus back mutation

The structure of parental antibody was modelled by computer-aided homology modelling program. Humanized antibodies were designed using CDR grafting. Briefly, the CDRs of parental antibody were grafted into the human acceptors to obtain humanized light chains and humanized heavy chains for each parental antibody. 3 heavy chains (VH1, VH2, and VH3) and 3 light chains (VL1, VL2, and VL3) were paired with each other for affinity ranking experiment. PTM risk of all sequences was analyzed, and appropriate PTM removal mutations were designed.

Production of chimeric, humanized antibodies and PTM removal Abs

The DNA sequences encoding the chimeric, humanized antibodies (Hui, 2, and 3- P4A2) and PTM removal Abs heavy and light chains were synthesized and inserted into pcDNA3.4 vector to construct expression plasmids of full-length IgGs. The designed plasmids of heavy and light chain were transfected into appropriate host. The recombinant IgGs secreted to the medium were purified using protein A affinity chromatography. The purified antibody was buffer-exchanged into PBS using PD- 10 desalting column. The concentration and purity of the purified protein were determined by OD₂₈₀ and SDS-PAGE, respectively.

Affinity ranking of chimeric, humanized antibodies and PTM removal Abs The affinity of purified antibody Hui, 2, and 3-P4A2 binding to was individually determined on cognate antigen (Spike/RBD) analyte using a Surface Plasmon Resonance (SPR) biosensor, Biacore 8K (GE Healthcare). Antibodies were immobilized on the sensor chip through Fc capture method. The cognate antigen (Spike/RBD) was used as the analyte. The data of dissociation (kd) and association (k_a) rate constants were obtained using Biacore 8K evaluation software. The equilibrium dissociation constants (KD ) were calculated from the ratio of k_d over k_a. The antibodies were ranked by their dissociation rate constants (off-rates, kd). Based on the ranking result, number of back mutation and antibody yield, the top 3 clones were selected.

Affinity measurement of purified humanized IgGs

The affinity of purified antibody binding to Hu1, 2, and 3-P4A2 was individually determined using a Surface Plasmon Resonance (SPR) biosensor, Biacore T200 (GE Healthcare). Antibodies were captured on the sensor chip through Fc capture method. Hui, 2, and 3-P4A2 was used as the analyte. The data of dissociation (kd) and association (k_a) rate constants were obtained using Biacore T200 evaluation software. The equilibrium dissociation constants (KD) were calculated from the ratio of kd over k_a.

Example 1: Expression and purification of antigen and structural protein

A synthetic Mammalian cell codon optimized nucleic sequence of RBD-His (wt), spike (wt), and a point mutant of RBD-His (N501Y) from SARS-CoV-2, was cloned in pcDNA3.1 plasmid vector. The construct pcDNA3.1 -RBD-His (wt), pcDNA3.1-RBD-His (N501Y) and pcDNA3.1-S-His (wt) was expressed and produced in Expi 293 F mammalian expression system. Briefly, the cells were transiently transfected with the plasmids. The supernatant was collected after 5-6 days, and the soluble protein was purified using Ni affinity chromatographywith Ni++ ions immobilised on a resin by covalent attachment to nitrilotriacetic acid (NTA) (QIAGEN, Germany). The down-stream bioprocess for pure antigen yielded approximately 20mg protein per litre of culture soup.

Example 2: Expression and purification of functional RBD protein

A synthetic, Mammalian cell, codon optimized nucleic sequence of RBD-His (wt), spike (wt),and a point mutant of RBD-His (N501Y) protein (variant B.1.1.7) from SARS-CoV-2, was cloned in pcDNA3.1 plasmid vector. The construct pcDNA3.1-RBD-His (wt), pcDNA3.1- RBD-His (N501Y) and pcDNA3.1-S-His (wt) was expressed and produced in Expi 293 F mammalian expression system. Briefly, the cells were transiently transfected with the plasmids. The supernatant was collected after 5-6 days, and the soluble protein was purified using Ni affinity chromatography with Ni++ ions immobilised on a resin by covalent attachment to nitrilotriacetic acid (NTA) (QIAGEN, Germany),

Example 3: Preparation of hybridoma for anti-RBD (N501Y) murine monoclonal antibodies

A group of female BALB/c mice (age; 6-8 weeks), were immunized subcutaneously (s.c.), withpurified RBD (N501Y) polypeptide (30 pg in 100 pl PBS per animal), along with QuilA adjuvant. Mice were boosted 3 times with antigen (30 pg, 15 pg, 7.5 pg, in 100 pl PBS per animal), along with QuilA adjuvant. Mice were bled 3 days after the last booster and the mouse with the highest titer of serum antibodies to RBD (N501 Y) polypeptide antigen was given final booster injections, intraperitoneally (i.p.), 4 days before aseptically removing the spleen. Splenocytes were utilized in hybridoma generation, for preparation of monoclonal antibody using the Clonacell-HY system, according to manufacturer's instructions. Once established, theclones were expanded in tissue culture flasks and stored in liquid nitrogen for future use. Hybridomas producing anti-flagellin antibodies were rescreened and purified two times by limiting dilution.

Example 4: Purification of anti-RBD (N501Y) murine monoclonal antibodies

In order to purify proposed murine monoclonal antibodies, the hybridoma cells (5 x 10⁵ cells per ml in a T175 tissue culture flask) were cultured in 200 ml SFM medium (Gibco), a serum- free, chemically defined medium that is intended as a monoclonal antibody production medium. The hybridoma culture soup was clarified; filtered and anti-RBD (N501Y) murine mAbs (IgG) were purified by protein G affinity chromatography using Protein G Agarose (G-Biosciences), Purified monoclonal antibodies were dialyzed against phosphate buffered saline (pH 7.4) and concentrated. Protein was quantitated by determining the OD value at 280 nm, using Nanodrop 2000c spectrophotometer and analyzed using 15% Tris-glycine SDS-PAGE.

Example 5: Characterization of anti-RBD (N501Y) murine monoclonal antibodies

Purified monoclonal antibody showed band of heavy and light chains, at their respective molecular weights, when resolved on 15% Tris-glycine, SDS-PAGE. The titers formonoclonalantibodies were determined by ELISA. Antigen-binding property of monoclonal antibodies was detected by ELISA. The anti-RBD (N501Y) murine monoclonal antibody (P4A2, P2A10, P1A2, P4C2 and P1A1) was binding to the RBD (N501Y) polypeptide and also showed cross-reaction with the other VOCsof SARS-CoV-2, Moreover, when recombinant S protein, was used for binding analysis of monoclonal antibodies, showed similar results, as shown by RBD (N501Y).

Example 6: Amplification of antibody variable region genes

Total RNA was extracted from hybridoma cells using TRI reagent. cDNA was generated by reverse transcription SuperScriptTM III Reverse Transcriptase Kit (ThermoFisher Scientific), using the consensus primers for mouse antibody heavy and light variable region genes. The PCR amplified VH and VL were cloned, using RBC T&A cloning kit (Real Biotech Corporation). The variable regions of both the light (VL) and heavy (VH) chains, from all the hybridoma clones, of anti-RBD (N501Y) murine monoclonal antibodies were sequenced. The IMGT/HighV- QUEST analysis tool (available at; http://www.imgt.org/IMGT vquest/vquest), was used for genetic diversity analysis of antibody sequences. Sequence alignment of all the four monoclonal antibodies depicted unique sequence for each.

Example 7: Neutralization potential of monoclonal antibodies

Both the mAbs were purified and tested for its neutralization potential against the wild type SARS-CoV-2. All the tested 4 mAbs showed broad and potent neutralization against SARS- CoV-2 Wuhan and different VOCs in live authentic BSL3 CPE assay. All the mice sera from RBD N501Y mutant protein (B.l.1.7) groups showed high titres of neutralizing antibodies when tested against authentic Wuhan SARS-CoV-2 virus and its Variants of concerns (VOCs)in CPE assay

Table 1: Represents the CPE values at which hundred percent neutralization was observed against authentic SARS-CoV-2 Wuhan and its VOCs viruses. Both mAbs showed a varied degree of broad and potent neutralization potential.

Example 8: Immunofluorescence staining with SARS-CoV2 Wuhan and its

VOCs

Cells were infected with the SARS-CoV-2 Wuhan and its VOC B.l.1.7 and mAb P4A2was used as primary antibody. The Immunofluorescence staining was done using anti-Mouse Flurochrome antibody. The data suggests that the mAb P4A2 binds with both wild typeWuhan and its VOC (Figure 5).

Example 9: Binding Kinetics using Octet Red: Binding assays were carried out using anOctet Red instrument (Forte'Bio) using biolayer interferometry (BLI).

The binding affinity of mAbs was assessed by Octect, Red using anti-mouse Fc sensors. The analytes were used in various concentrations with * fold serial dilution in the PBS buffer background supplemented with 0.1% BSA. Associations and dissociations were recorded for 1500 sec. Data produced was analyzed using the ForteBio Data Analysis software, 10.0 (Forte-Bio Inc). The kinetic parameters were calculated using a global fit 1:1 model (Figure 8).

Example 10: P4A2 and P1A2 binds with the overlapping epitopes in Receptor binding motif

As depicted in BLI based assay, both antibodies also showed inhibition of ACE2 interaction with RBD suggesting both of these antibodies binding epitope overlaps with RBM. In a competition assay, the present inventors have found that P4A2 completely blocked P1A2 from binding the RBD and vice versa. The purified P4A2 mAb showed strong binding with half-maximal effective concentration [EC50] of 0.012-0.120 mg/ml, where P4A2 binding specificity with B.1.351 and P.l was found to be nanomolar range which is slightly towards the higher concentration as compared to 2019-nCoV, B.1.1.7, B.1.617 and B.1.617.2. Immunofluorescence assay showed the binding of P4A2 on VOCs infected Vero cells, similar number of foci throughout all the VOCs tested, with slightly low intense florescence spot signals with B.1.351 variant only.

In line with the ELISA reactivity’s, biolayer interferometry measurements of P4A2 binding kinetics revealed that P4A2 binds with RBD with nanomolar or subnanomolar [KD > 10⁹] affinities from various VOCs.

The E484K mutation associated with emerging Omicron in the SARS-CoV2 RBD reduced thebinding of P4A2 mAb but doesn’t affect neutralization potency. Neutralising activity of P4A2was nearly unchanged against B.1.351, P.l variants as compared to other VOCs tested, suggesting that E484K mutation doesn’t have any negative effect on P4A2 neutralization mechanism. Similarly, restoration of E484, K to Q in B.1.351 RBD restored the binding of P4A2 antibody.

Cross-neutralization potential of P4A2 against alpha and beta coronaviruses was assessed by S-mediated entry into cells of either vesicular stomatitis virus (VSV) pseudotyped with MERS-CoV S, OC43 S, SARS-CoV S, SARS-CoV-2 S and HKU4 S in the presence of varying concentrations of P4A2. The P4A2 neutralized the SARS-CoV-2 S with IC50 concentration 200ng/ml, however no cross-neutralization shown by P4A2 with other coronaviruses, showingthat P4A2's epitope is unique to SARS CoV-2.

The potency of P4A2 was evaluated in vivo in a KI 8 hACE-2 mice challenge model of SARS-CoV-2 infection. Eight- to ten-week-old animals were administered intraperitoneal 5.0mg/ kg dose of P4A2, assessed the prophylactic efficacy against SARS-CoV-2 (Wuhan isolate)and Kappa variant. Animals were challenged intranasally with 10⁵ focus-forming units (FFU)of virus, one day after antibody administration. The changes in body weight of all experimentalanimals were monitored on a daily basis till day 6. At days 6-7 post-challenge, additional clinical data were evaluated to estimate the overall disease severity index. On day 6, mice in the infection control group lost more than 10% of their body weight compared to those in the P4A2 treatment group. Furthermore, as compared to the virus challenge group, mice given P4A2 had nearly undetectable viral RNA levels in their lungs (P<0.001).

To further evaluate P4A2's post-exposure therapeutic ability against SARS-CoV- 2 challenge, the same strategy was used as stated above, but P4A2 was given to the mice therapeutically 6- 8 hours after post viral infection. The therapeutic effect of P4A2 was measured against SARS-CoV-2 WA1/2020, B.1.351 (Beta), B.1.617 (Kappa) and B.1.617.2(Delta) VOCs (Figure no.7). Example 11

Characterization of P4A2 Fab

It was found that P4A2 demonstrated exceptional efficient and broad neutralization of both ancestral SARS-CoV-2 WA1/2020 and other VOCs, Alpha, Beta, Kappa and Delta (range = 10-39 ng mL'¹ corresponding to 0.07 to 0.26 nM) (Fig. 9A). The P4A2 binds with RBDs of different VOCs with high affinity and binding assays based on competition suggested that P4A2 ACE2 binding sites overlap (Fig. 9B and Fig. 12).

The crystal structure of P4A2 Fab in complex with the RBD-N501Y (residues 332- 544) of protein was determined to a maximum resolution of 3.0 A (Figure. 9C). The crystal structure shows the electron density for the entire heavy and light chain of the P4A2 Fab. For the RBD, electron density for the first five residues (332-336) was disordered, and the density for residues 526-544 at the C-terminal is missing. The structure showed that the residues 475-489 and 455-456 of the RBD are present close to the P4A2 Fab paratope (Figure. 9D). The residues of the RBD that interact with the paratope of the P4A2 Fab are 455Leu, 456Phe, 483Val, 484Glu, 485Gly, 486Phe, 487Asn and 489Tyr (Fig. 9D). The P4A2 paratope is made up of residues T30, R31, Y32, S33, Y35 and M37 from CDR1 along with N52 from CDR2 and S99 from CDR3 of the heavy chain. The residues involved from CDR1 of the light chain are Y31, T34, L36, Q38, F40 and that from CDR2 are Y53, A54, and N57. Q93 and S95 from CDR3 of the light chain also contribute to the paratope (Fig. 9D). The area of the surface buried due to interaction between RBD and P4A2 o paratope is 1667 A . The key interactions that stabilize the distributed epitopes from the Receptor-binding Motif (RBM) in the P4A2 paratope include (i) the presence of the aromatic ring of 486Phe deep inside a hydrophobic cavity (Fig. 9E) lined by F40L, Y35H and M37H (ii) hydrogen bond formed between backbone carbonyl of R31H and backbone nitrogen of 484Glu, (iii) hydrogen bond formed between backbone carbonyl of 484Glu and backbone nitrogen of S33H, (iv) hydrogen bond formed between backbone nitrogen of 486Phe and side chain of Y35H, (v) hydrogen bond formed between backbone carbonyl of 485Gly and side chain of S99H, (vi) hydrogen bond formed between side chain of 487Asn and backbone carbonyl of S95L, and (vii) hydrophobic interactions formed between 456Phe, 489Tyr, L36L and Y53L.

Some of the residues of RBD that interact with the P4A2 paratope are a part of the RBM that interact with the human ACE2. Based on the crystal structure of the SARS-CoV-2 RBD in complex with human ACE2 (PDB code: 6M0J), the viral protein forms interactions with the human receptor through the following residues: 417Eys, 446Gly, 449Tyr, 453Tyr, 455Leu, 456Phe, 475Ala, 486Phe, 487Asn, 489Tyr, 493Gln, 496Gly, 498Gln, 500Thr, 501Asn, 502Gly and 505Tyr [ J L, J G, J Y, S S, H Z, S F, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020;581. doi: 10.1038/s41586-020- 2180-5]. Among these residues 455Leu, 456Phe, 486Phe, 487Asn and 489Tyr form key hydrophobic and polar interactions with the paratope of P4A2 Fab and therefore, binding of the Fab to the spike-RBD will render these residues inaccessible to the ACE2 receptor. A superimposition of the RBD from the complex with P4A2 Fab onto that from the complex with ACE2 shows that the viral protein bound to P4A2 will be unable to engage with the human receptor due to steric clashes (Fig. 9F). The backbone conformation of the stretch spanning residues 475- 488 and 455-456 from Spike-RBD is similar when bound to P4A2 and to ACE2 (6M0J) with an RMSD of 0.8 A (Fig. 9G). Except for 486Phe which exhibits a different side chain conformation, there is substantial overlap in the side chain orientation for the other residues. The structure of the P4A2:Fab complex was utilized to generate two computational models of the Fab in complex with Spike trimer which showed that P4A2 should be able to bind to the RBD when it is both in the “up” (accessible to ACE2 receptor) or “down” (inaccessible to ACE2 receptor) positions (Fig. 9H). Overall, the composition and conformation of the epitope in RBD and the mode of binding of the Fab ensures that P4A2 interaction will prevent recognition of ACE2 by the spike protein and thus prevent entry of the virus into the host cell.

The computational models of P4A2 Fab in complex with the Spike-RBD from Beta, Gamma, Delta, Kappa and Omicron (BA.l) VOCs were generated. These models show that, for these VOCs, there are no mutations in the residues that interact with the P4A2 through their side-chain. E484 is mutated to Lys, Gin or Ala in some of the VOCs, but this residue forms interactions with the P4A2 Fab paratope through the backbone atoms and not through the side chain (Figure 13).

Overall, the structural analysis provides an explanation regarding the ability of the P4A2 mAb to neutralize the Alpha, Beta, Gamma, Kappa, and Delta variants and also suggests that the mutations in residues of the RBD observed in the BA.1 variant will not abrogate P4A2:spike protein interaction (Figure. 13).

To validate the above inference, the present inventors have tested the neutralization potential of P4A2 with live BA.l virus. The P4A2 neutralized BA.l with an IC50 of 45 ng mL-1 (0.3 nM; Fig. 91), and binds to purified BA.l RBD with high specificity and nanomolar affinity (Fig. 9J and K). The P4A2 shows strong binding specificity in ELISA with half-maximal effective concentration (EC50) of 0.001- 0.600 ng/mL. These values were higher for P4A2 binding with Beta, Gamma and BA.l RBD proteins as compared to RBD from other tested VOCs (Fig. 12B). However, BLI data suggests that P4A2 binds with nanomolar to sub nanomolar affinity to all tested VOCs RBD proteins (Fig 9J and 12A). Further, the neutralizing activity of P4A2 was unchanged against all the VOCs tested, suggesting that slightly higher EC50 binding values for Beta, Gamma and BA.l RBD proteins does not have any negative effect on the P4A2 neutralization mechanism.

It is possible that mutation of the E484 residue to Lys, Lys or Ala may be responsible for the observed reduction in affinity in the case of RBD from Beta, Gamma and BA.l, respectively. From the crystal structure of Spike-RBD: P4A2 Fab complex, it is clear that this residue forms interactions with the Fab paratope through the polypeptide backbone atoms. The side chain is not involved in these interactions, but the change in electrostatic surface of the molecule due to these mutations may increase the Koff leading to a decrease in the equilibrium affinity. However, the affinity is still in the nanomolar range and therefore the E484 mutations have minimal effect on the ability of P4A2 to neutralize the SARS-CoV- 2 virus in cell culture and animal models. This is further corroborated by immunofluorescence data that the number of foci recognized by P4A2 is similar in all the VOC-infected cells and reveals its broad reactivity to cells infected with all VOC studied (Fig. 10A, 5 and 14).

The broad-spectrum antiviral intervention effect of P4A2 against different Alpha and Beta coronaviruses was tested using vesicular stomatitis virus (VSV) pseudotyped viruses; MERS-CoV S, OC43 S, SARS-CoV S, SARS-CoV-2 S and HKU1 S, confirming that the epitope recognised by P4A2 is present only in the SARS-CoV-2 family and no cross-neutralization was seen with other coronaviruses, (Fig. 10B).

Example 13

P4A2 conferring protection in vivo in a K18 hACE-2 mouse challenge model of SARS-CoV-2 infection

Eight to ten- week-old animals were administered a single dose (5 mg/kg) of P4A2 intraperitoneally to assess its prophylactic efficacy (against WA1/2020 SARS- CoV-2 and Kappa variant) and therapeutic effect (against WA 1/2020 SARS-CoV- 2, Kappa, Beta, and Delta). Animals were challenged intranasally with 105 plaqueforming units (PFU) of the virus. In the prophylactic group, antibody was infused one day prior to virus challenge and in therapeutic group antibody was administrated 12 h following infection with the virus. The changes in body weight of experimental animals were monitored on a daily basis for 6 days. Additional clinical data was analysed in order to calculate the overall disease severity index at 6-7 days post-challenge. On day 6, mice in the infection control group lost more than 10% of their body weight compared to those in the P4A2 treatment group (Fig. 10C). Furthermore, as compared to the virus challenge group, mice given P4A2 had significantly reduced viral RNA levels in their lungs (P<0.001; Fig. 10D, 15).

Example 14

Determination of the minimal protective dose for therapeutic protection

To determine the minimal protective dose for therapeutic protection, the present inventors had titrated the passively administered P4A2 at a lower dose (1 mg/kg). A 20 pg dose of the P4A2 was fully protective and sufficient to suppress viral replication in the lungs, confirming the high potency of P4A2 in vivo against the Beta and Delta variant (Fig. 10E, 10F, 15 and 17).

Example 15

Comparison of neutralizing activity of P4A2 and available mABs.

Few mAbs are currently available that effectively neutralize all VOCs [Chen Z, Zhang P, Matsuoka Y, Tsybovsky Y, West K, Santos C, et al. Extremely potent monoclonal antibodies neutralize Omicron and other SARS-CoV-2 variants. medRxiv. 2022; 2022.01.12.22269023. doi: 10.1101/2022.01.12.22269023]. Sotrovimab (S309) is reported to neutralise the BA.l variant, but at a significantly lesser potency than P4A2. Although sotrovimab and P4A2 binds with BA.l RBD with similar affinity (Fig. 11A-C and 18). The observed difference in the neutralization potential of these two mAbs might be because they target different epitopes with different mechanisms of neutralization. The sotrovimab recognizes a glycan epitope, without competing with receptor attachment. However, P4A2 directly binds to the site that competes with virus attachment to host cells. This might be one probable reason that sotrovimab can neutralize the Omicron BA.l variant, but with significantly lesser potency (Table SI).

Table SI

Table SI: Comparative neutralization potential of P4A2 mAb with the clinical approved or under development therapies

P4A2 exhibits strong interactions with residues of RBD that are critical for binding to the ACE2 receptor. Hence, the mutations in the RBD that reduce recognition by P4A2 may also plausibly adversely impact binding to the ACE2 receptor. Therefore, it is possible that this mAb may be able to neutralize new variants that may arise in the future. Maher et al; predict that the following mutations will perturb the ability of therapeutic mAbs to recognize Spike-RBD: A344S, R346K, K417T/N, K444N, G446V, L452R/Q, L455F, G476S, S477I, T478K, V483F, E484K/Q, F490S and S494L/P. These mutations are predicted to adversely affect the activity of sotrovimab, letesevimab, imdevimab, bamlanivimab, casirivimab and CT-P59. However, the present inventors observed that none of these mutations will lead to the loss of stabilizing interactions or will give rise to steric clashes at the P4A2 Fab:RBD interface. As a result, these mutations in the RBD will probably not reduce the ability of P4A2 to neutralize the SARS-CoV-2. (Fig. 19) [Predicting the mutational drivers of future SARS-CoV-2 variants of concern. Sci Transl Med. 2022;14: eabk3445. doi: 10. 1126/scilranslmed.abk3445\. Recently new lineages of Omicron have emerged namely BA.2, BA.2.75, BA.2.12.1BA.3, BA.4, and BA.5 [The recently emerged BA.4 and BA.5 lineages of Omicron and their global health concerns amid the ongoing wave of COVID-19 pandemic - Correspondence. Int J Surg. 2022; 103: 106698] (Fig 20). The 486Phe residue in BA.4 and BA.5 is substituted with Vai. Since the side chain of Vai is also hydrophobic P4A2 may retain substantial affinity for Spike-RBD. Also as expected, 486PheVal results in reduction of affinity of Spike-RBD for ACE2 receptor, but the R493Q reversion mutation reinstates the affinity [Antibody evasion by SARS-CoV-2 Omicron subvariantsBA.2.12.1,BAA andBA.5.Nature.2022;608:603-608].R493Q may also attenuate thepossiblereduction in affinity ofSpike-RBD forP4A2 due to F486V sincetheshorterGin sidechainwillbecloserto theparatopesurfaceand can form new hydrogen bondswith residuesofCDR LI ofP4A2.P4A2 may therefore,retain significantcapacity to bind to Spike protein from BA.4/5 and furtherstudiesare required to ascertain the ability ofP4A2 to neutralize these strains.

ThecomparisonofP4A2withotherknownbroadlyneutralizingantibodiesshows thatepitopes of 87G7,510A5,Cov2-2196,NCV2SG48,NCV2SG53,S2E12, S2K146andZWD12 show varyingdegreesofoverlapwiththatofP4A2.Among these mAbs,the P4A2 isthe only one thatformsmultiple interactionswith its cognateepitopeonSpike-RBD which iscomposedofresiduesthatarecriticalfor interactionwithACE2(Fig.21andTableS2

Table S2. Comparative epitope analysis of P4A2 mAb with other known ACE2 inhibiting antibodies

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2 Omicron sublineages. bioRxiv. 2022; 2022.05.08.491108. doi: 10.1101/2022.05.08.491108

2. WestendorfK, Zentelis S, WangL, FosterD, Vaillancourt P, WigginM, etal. LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants. bioRxiv. 2022; 2021.04.30.442182. doi:10.1101/2021.04.30.442182

3. Wang L, Fu W, Bao L, Jia Z, Zhang Y, Zhou Y, et al. Selection and structural bases of potent broadly neutralizing antibodies from 3-dose vaccinees that are highly effective against diverse SARS-CoV-2 variants, including Omicron sublineages. Cell Res. 2022;32: 691-694. doi:10.1038/s41422-022-00677-z

4. Park Y-J, De Marco A, Starr TN, Liu Z, Pinto D, Walls AC, et al. Antibody-mediated broad sarbecovirus neutralization through ACE2 molecular mimicry. bioRxiv. 2021; 2021.10.13.464254. doi:10.1101/2021.10.13.464254

5. Chi X, Yan R, Zhang J, Zhang G, Zhang Y, Hao M, et al. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science. 2020;369: 650-655. doi: 10.1126/science.abc6952 6. Guo H, Gao Y, Li T, Li T, Lu Y, Zheng L, et al. Structures of Omicron spike complexes and implications for neutralizing antibody development. Cell Reports. 2022;39: 110770. doi:10.1016/j.celrep.2022.110770

7. Du W, Hurdiss DL, Drabek D, Mykytyn AZ, Kaiser FK, Gonzalez-Hernandez M, et al. An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern. Sci Immunol. 2022;7: eabp9312. doi:10.1126/sciimmunol.abp9312

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Overall,the presentinventorshave found thatP4A2 may representan viable therapeutic option which is required to reduce the impact of the COVID19 pandemiconhumanhealth acrosstheglobe.Overall,thepresentinvention shows thatP4A2 aloneisefficaciousin providingprotection from thetested VOCsand thisabilitymayprobablyberetainedformajorityofthefutureVOCs.Humanized P4A2 mAb may be used alone or in conjunction with other non-competing antibodies as an effective prophylactic or therapeutic strategy against current and future variants of SARS-CoV-2. The present studies with the P4A2 mAb also reinforce the idea that therapeutic molecules that bind to regions on the target protein that are critical for natural function will generally be less vulnerable to loss of sensitivity due to mutations in the target protein.

Example 16:

Production and characterizations of three variants of humanized P4A2 mAbs The chimeric and humanized antibodies were expressed and purified. The purified

IgG migrated as -150 kDa band in SDS-PAGE under non-reducing condition, -25, -50 kDa band under reducing condition. Evaluating by the SDS-PAGE result, the purity of IgG is > 90%. The affinity of cognate antigen (Spike/RBD) to humanized Abs (Hui, 2, and 3-P4A2) were determined after recombinant expression. Hu3- P4A2 showed promising neutralization and protection in both in vitro and in vivo assays from SARS-CoV-2 virus and its VOCs (Figure 22 and 23).

Claims

Claims:

1. A monoclonal antibody comprising sequences selected from SEQ. ID No. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19.

2. The monoclonal antibody as claimed in claim 1, having a heavy chain sequence of SEQ. ID No. 3 and light chain sequence of SEQ. ID No. 2.

3. The monoclonal antibody as claimed in claim 1, having a heavy chain sequence of SEQ. ID No. 4 and light chain sequence of SEQ. ID No. 5.

4. The monoclonal antibody as claimed in claim 1, having a heavy chain sequence of SEQ. ID No. 6 and light chain sequence of SEQ. ID No. 7.

5. The monoclonal antibody as claimed in claim 1, having a heavy chain sequence of SEQ. ID No. 8 and light chain sequence of SEQ. ID No. 9.

6. The monoclonal antibody as claimed in claim 1, having a heavy chain sequence of SEQ. ID No. 11 and light chain sequence of SEQ. ID No. 10.

7. The monoclonal antibody as claimed in claim 1, having a heavy chain sequence of SEQ. ID No. 13 and light chain sequence of SEQ. ID No. 12.

8. The monoclonal antibody as claimed in claim 1, having a heavy chain sequence of SEQ. ID No. 15 and light chain sequence of SEQ. ID No. 14.

9. The monoclonal antibody as claimed in claim 1, having a heavy chain sequence of SEQ. ID No. 17 and light chain sequence of SEQ. ID No. 16.

10. The monoclonal antibody as claimed in claim 1, having a heavy chain sequence of SEQ. ID No. 19 and light chain sequence of SEQ. ID No. 18.

11. The monoclonal antibody as claimed in any of the preceding claims, wherein said antibody binds to at least one epitope formed by amino acid sequence having sequence SEQ ID NO: 1.

12. The monoclonal antibody as claimed in any of the preceding claims, wherein said antibody is immunoreactive with an epitope formed by amino acid sequence having sequence SEQ ID NO: 1.

13. The monoclonal antibody as claimed in any of the preceding claims, wherein said monoclonal antibody binds to the specific region of spike (S) protein on the receptor binding domain (RBD) of the SARS-CoV-2 and the VOCs with high affinity and prevents viral entry into the human host cells.

14. The monoclonal antibody as claimed in any of the preceding claims, wherein said monoclonal antibody possesses neutralizing activity against SARS-CoV-2 and VOCs.

15. The monoclonal antibody as claimed in any of the preceding claims 13 and 14, wherein said VOCs are selected from B.1.1.7, B.1.351, P.1, B.1.617, B.1.617.2, B.1.1.529.

16. A process for producing monoclonal antibodies comprising sequences selected from SEQ. ID. No.2 to 19, said process comprising the steps of :

(i) introducing a point mutation N501Y in RBD-His polypeptide;

(ii) cloning the mutated polypeptide in a vector and expressing in an expression system to obtain a genetically modified protein;

(iii) producing said monoclonal antibodies using said genetically modifying protein of step (ii) and hybridoma technology.

17. A method of treating a subject infected with SAR.S-CoV-2 and/or VOCs or reducing the likelihood of infection of a subject at risk of contracting SAR.S-CoV-2 and/or VOCs, comprising delivering to the subject the monoclonal antibody of any one of claims 1-15.

18. Use of monoclonal antibody of any one of claims 1-15 for treating a subject infected with SAR.S-CoV-2 and/or all of the VOCs or reducing the likelihood of infection of a subject at risk of contracting SAR.S-CoV-2 and/or all of the VOCs.

19. A pharmaceutical composition comprising the monoclonal antibody as claimed in claims 1 to 15, and a pharmaceutically acceptable carrier, excipient, or stabilizer.

20. The pharmaceutical composition as claimed in claim 19, wherein said composition is a lyophilized composition or an aqueous solution.

21. The pharmaceutical composition as claimed in claim 19, wherein said composition is in the form of injections, saline formulations or aerosol formulations.

22. The pharmaceutical composition as claimed in claim 19, 20 or 21, wherein said composition further comprises additional therapeutic agents selected from second antibody, anti-viral agent, and/or anti-infective agent.

23. An immunoassay reagent comprising the antibody as claimed in claims 1 to 15, wherein said antibody is labelled with a detectable label.

24. The immunoassay reagent as claimed in claim 23, wherein said antibody is bound to a solid phase.

25. An immunoassay for the detection of SARS CoV-2 and/or its VOCs in a test sample, said immunoassay comprising:

(i) contacting a test sample suspected of containing COVID- 19 with a first antibody directed against SARS -CoV-2 core antigen to form a complex between said first antibody and antigen located within said test sample;

(ii) contacting said complex formed in step (i) with the monoclonal antibody as claimed in claims 1 to 15 to form a complex between said antibody and antigen in the complex formed in step (i) wherein said antibody is detectably labelled and

(iii) detecting the label of the complex formed in step (ii).

26. The immunoassay as claimed in claim 25, wherein said first antibody is directed to the Receptor Binding Domain (RBD) of SARS CoV-2 antigen.

27. The immunoassay as claimed in claim 25 or 26, wherein said antibody employed in step (ii) is labelled with a fluorescent label.

28. The immunoassay as claimed in claim 27, wherein said label is selected from acridinium, FITC and gold nanoparticles.

29. The immunoassay as claimed in any claim 25 to 28, wherein said antibody of step (i) is coated on solid phase.

30. The immunoassay as claimed in any claim 25 to 29, wherein said antibody of step (i) comprises an antibody that is distinct from the antibody of step (ii).

31. The immunoassay as claimed in any claim 25 to 30, wherein said antibody of step (i) comprises an antibody that is the same as the antibody of step (ii).

32. The immunoassay as claimed in any claim 25 to 31 further comprises diagnosing, prognosticating, or assessing the efficacy of a therapeutic/prophylactic treatment of the patient.

33. A vaccine comprising a polypeptide having sequence Seq ID 1, and a pharmaceutically acceptable carrier.