DESC:A FULLY HUMAN THERAPEUTIC MONOCLONAL ANTIBODY AGAINST SARS-CoV-2 VIRUS

This application claims the benefit to Indian Provisional Application No. 202021037301, filed on 29th August, 2020, the contents of which are incorporated by reference herein.

FIELD OF THE INVENTION
Present invention is to provide a method of constructing an anti-SARS-CoV-2 specific scFv recombinant antibody library against SARS-CoV-2 virus. The present invention also relates to the processes of producing a fully human monoclonal antibody to target SARS-COV-2 spike (S) glycoprotein , receptor binding proteins and nucleocap-sid protein (N-protein) expressed in yeast, filamentous fungal cells or Mammalian cells. In particular, the present disclosure provides processes of producing a fully human mon-oclonal antibody expressed in Pichia pastoris. The present invention also relates to a ful-ly human monoclonal antibodies which target S1 , S2 subunits , SARS-COV-2 receptor binding domain (RBD), Non-RBD domain, Envelope (E), Nucleocapsid protein (N-protein) and membrane (M) use as a therapeutic or diagnostic reagents and methods of making the same for the treatment of SARS-COV-2 virus. In particular, the present in-vention is to provide a fully human monoclonal antibodies which targets SARS-COV-2 S1 and S2 subunits, SARS-COV-2 receptor binding domain (RBD), non-RBD domain and nucleoprotein (N) to use as a therapeutic or diagnostic reagents and methods of mak-ing the same for the treatment of SARS-COV-2 virus. The invention provides a rational-ly selected therapeutic cocktail antibodies likely to have greater resistance to SARS-CoV-2 escape.

BACKGROUND OF THE INVENTION
In December 2019, Wuhan in the Hubei province of China became the center of an out-break of pneumonia of unknown cause, which raised intense attention not only within China but internationally. Later, it was confirmed that the causative agent for this pneu-monia-like disease is a coronavirus (CoV) belonging to the family Coronaviridae (Jiang et al., 2020a; C. Wang et al., 2020; F. Wu et al., 2020; Zhou et al., 2020). On February 11, 2020, the World Health Organization named the virus SARS-CoV-2 and renamed the syndrome from 2019-nCoV to COVID-19, short for coronavirus disease 2019 (“WHO. Novel Coronavirus(2019-nCoV) Situation Report – 22. February 11, 2020. https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200211-sitrep-22-ncov.pdf. Accessed May27, 2020.,” n.d.). COVID-19, a respiratory disease, has led to more than 22,140,472 confirmed cases and over 781,135 deaths globally as of Au-gust 19, 2020(Subodh Kumar et al, In Press, Journal Pre-proof, Available online 23 Au-gust 2020)

The viral genome of the coronavirus encodes four structural proteins named spike (S), envelope (E), membrane (M), and nucleocapsid (N), 16 non-structural proteins (nsp1–16), and 8 accessory proteins (Jiang et al., 2020b; Siu et al., 2008). The S protein plays the most critical role in viral attachment, fusion, and entry into the target cell (D. E. Gordon et al., 2020; Jiang et al., 2020b, 2020b)). It is a trimeric class I fusion protein that exists in a metastable prefusion conformation following translation. A substantial struc-tural rearrangement of the S protein is required to fuse the viral membrane with the host cell membrane (Bosch et al., 2003; Hulswit et al., 2016). The size of the densely glyco-sylated S protein ranges between CoV species from approximately 1100 to 1600 amino acids length, with an estimated molecular mass of up to 220 kDa (Hulswit et al., 2016; Li, 2016). CoV particles are decorated with the club-shaped trimers of the S protein, which is 8–23-nm long (Hofmann et al., 2004). The S protein not only determines the host tropism but also is the crucial target for neutralizing antibodies produced by the immune system of the infected host (Hofmann et al., 2004; Walls et al., 2020).

The therapeutic options for Covid-19 include vaccines, anti-viral, convalescent plasma therapy and antibody/ antibody cocktail to neutralize the virus. The aim behind all these is to stimulate the patient's immune system to attack the virus.
The novel coronavirus disease (COVID-19) has had devastating global health conse-quences and there is currently no cure and no licensed vaccine. Neutralizing antibodies (nAbs) to the causative agent of the disease, severe acute respiratory syndrome corona-virus-2 (SARS-CoV-2), represent potential prophylactic and therapeutic options and could help guide vaccine design. Vaccines prompt the immune system to make antibod-ies against a virus. Alternatively, monoclonal antibody could be produced in large quan-tities and injected into patients as a treatment. Again, the antibodies gives protection against SARS-CoV-2 infection for healthcare workers, elderly people and others who respond poorly to traditional vaccines or are suspected of a recent exposure to the SARS-CoV-2.

There are reports on broad-neutralizing antibodies to target a conserved region in the RBD of the S1 subunit. The S2 subunit required for viral membrane fusion might be an-other target.
Monoclonal antibody cocktail may also exhibit more potent antivirus activity that could increase the effectiveness of the treatment and prevent the viral escape. Single antibody or monoclonal antibody cocktail, which can either be administered to at-risk people be-fore exposure as a vaccine or as treatment for those already infected can also be an op-portunities for the realistic design of therapeutics specific to novel coronavirus.

Experimental animal data show that protection against severe acute respiratory syn-drome coronavirus (SARS-CoV-2) infection with human monoclonal antibodies (mAbs) is feasible. For an effective immune prophylaxis in humans, broad coverage of different strains of SARS-CoV and control of potential neutralization escape variants will be re-quired. Combinations of virus-neutralizing, noncompeting mAbs may have these proper-ties.
Monoclonal antibodies are highly specific and have been considered against various dis-eases. The recent advancement in the therapeutic protein production platforms could make the monoclonal antibody production at lower production costs and affordable.
Therefore, selection of broad-neutralizing antibodies against SARS-CoV-2 and SARS-CoV is attractive and might be useful for treating not only COVID-19 but also future SARS-related CoV infections.

Prior work has helped to establish the yeast Pichia pastoris as a cost-effective platform for producing functional antibodies that are potentially suitable for research, diagnostic, and therapeutic use. See co-owned U.S. Pat. Nos. 7,935,340; 7,927,863 and 8,268,582, each of which is incorporated by reference herein in its entirety.
Eukaryotes are capable of many of the post-translational modifications such as proteo-lytic processing, folding, di- sulfide bond formation and glycosylation. Thus, many pro-teins that end up as inactive inclusion bodies in bacterial systems are produced as bio-logically active molecules in Pichia pastoris . The methylotrophic yeast Pichia pastoris has become an increasingly popular host for recombinant protein expression in recent times. Glycoengineered Pichia pastoris expression system that could produce glycopro-teins with glycosylation profiles similar to mammalian systems.

Therapeutic glycoproteins produced by the Pichia pastoris platform have shown compa-rable folding, stability, and in vitro and in vivo efficacies in preclinical models to their counterparts produced from the CHO cells. Pichia pastoris offers a cost and time effi-cient alternative platform for therapeutic protein production.
Again, the invention provides competitive screening strategy to isolate human antibodies from a phage display library. Platform Technology for the Development of Novel fully human monoclonal antibodies for therapeutic and diagnostic applications. There are sev-eral advantages of Phage display technology over conventional Hybridoma technology. The antibodies generated through the methods are much more superior than the hybrid-oma technology produced antibodies, a fully human high affinity antibodies isolated from the phage display library which generally has high affinity and lower toxicity
Current challenges in developing neutralizing antibodies against SARS-CoV-2 include mutations in less conserved region of S1 subunit, possibly antigen drift, immunodomi-nant epitope, ADE potentially induced by non-neutralizing antibodies, or increased affin-ity of viral S protein for ACE2 .
Integrating recent technological advances for antibody discovery and helps to define the spike protein as a major site of vulnerability for vaccine design and therapeutic-antibody development. The most potent neutralizing antibodies isolated here also could serve as candidate biologics to prevent or treat SARS-CoV-2 infection.
Therefore, there is an important need to identify and develop neutralizing antibodies which is efficient and cost effective.
The present invention aims to the processes of producing a fully human monoclonal an-tibody to target SARS-COV-2 spike (s) glycoprotein or receptor binding proteins ex-pressed in Pichia pastoris.
The invented, fully human monoclonal antibody expressed in Picha pastoris is highly reproducible and scalable which is a very important factor in manufacturing it at a larger scale during the current pandemic and is cost effective. It shows potential utility as a medical countermeasure.

OBJECTS OF THE INVENTION
The object of the present invention is to provide a method of constructing an anti-SARS-CoV-2 specific scFv recombinant antibody library against SARS-CoV-2 virus

Another object of the present invention is to provide a competitive process of screening strategy to isolate human antibodies from a phage display library.

Another object of the present invention is to provide a method of production of a fully human monoclonal antibody to target SARS-COV-2 spike (s) glycoprotein or receptor binding proteins.

Another object of the present invention is to provide a fully human monoclonal antibod-ies which target SARS-COV-2 S1 and S2 subunits, SARS-COV-2 receptor binding do-main (RBD), non-RBD domain and nucleoprotein (N).

Another object of the present invention is to provide the use of Pichia pastoris platform for the production of novel therapeutic fully human monoclonal antibody.

Another object of the present invention is to provide a fully human therapeutic monoclo-nal antibody potently neutralizes SARS-CoV-2 by engaging the S receptor-binding do-main.

Another object of the present invention is to provide a fully human monoclonal antibod-ies targeting Non-RBD regions against SARS-CoV-2

Another object of the present invention is to provide a fully human monoclonal antibod-ies targeting nucleoprotein (N) regions against SARS-CoV-2

Another object of the present invention is to provide a fully human monoclonal antibod-ies which target SARS-COV-2 receptor binding domain (RBD), non-RBD domain, and nucleoprotein (N) use as the therapeutic or diagnostic reagents and methods of making the same for the treatment of SARS-COV-2 virus

Another object of the present invention is to provide a fully human monoclonal antibod-ies which target SARS-COV-2 receptor binding domain (RBD), non-RBD domain, enve-lope (E), nucleoprotein (N) and membrane (M) use as the therapeutic or diagnostic rea-gents and methods of making the same for the treatment of SARS-COV-2 virus

Another object of the present invention is to provide a fully human monoclonal anti-body cocktail having the aforenamed monoclonal antibody in combination with a second monoclonal antibody which binds simultaneously to the spike for neutralizing the SARS-COV-2 virus

Yet another object of the present invention is to provide the potent combination or cock-tail of antibodies as the therapeutic or diagnostic reagents and methods of making the same for the treatment of SARS-COV-2 virus

SUMMARY OF THE INVENTION

In a general aspect the present invention provides a method of constructing an anti-SARS-CoV-2 specific scFv recombinant antibody library against SARS-CoV-2 virus.

In an embodiment, the present invention provides a competitive process of screening strategy to isolate human antibodies from a phage display library.

In an embodiment, the present invention provides a method of production of a fully hu-man monoclonal antibody to target SARS-COV-2 spike (s) glycoprotein or receptor binding proteins.

In another embodiment, the present invention provides a fully human Monoclonal anti-bodies which target SARS-COV-2 S1 and S2 subunits, SARS-COV-2 receptor binding domain (RBD), non-RBD domain and nucleoprotein (N) .

In another embodiment, the present invention provides the use of Pichia pastoris plat-form for the production of novel therapeutic fully human monoclonal antibody.

In another embodiment, the present invention provides a fully human therapeutic mono-clonal antibody potently neutralizes SARS-CoV-2 by engaging the S receptor-binding domain.

In another embodiment, the present invention provides a fully human monoclonal anti-bodies targeting Non-RBD regions against SARS-CoV-2

In another embodiment, the present invention provides a fully human monoclonal anti-bodies targeting nucleoprotein (N) regions against SARS-CoV-2

In another embodiment, the present invention provides a fully human monoclonal anti-bodies which target SARS-COV-2 receptor binding domain (RBD), non-RBD domain, and nucleoprotein (N) use as the therapeutic or diagnostic reagents and methods of mak-ing the same for the treatment of SARS-COV-2 virus

In another embodiment, the present invention provides e a fully human monoclonal anti-bodies which target SARS-COV-2 receptor binding domain (RBD), non-RBD domain, envelope (E), nucleoprotein (N) and membrane (M) use as the therapeutic or diagnostic reagents and methods of making the same for the treatment of SARS-COV-2 virus

In another embodiment, the present invention provides a fully human monoclonal anti-body cocktail having the aforenamed monoclonal antibody in combination with a second monoclonal antibody which binds simultaneously to the spike for neutralizing the SARS-COV-2 virus

In another embodiment, the present invention provides the potent combination or cock-tail of antibodies as the therapeutic or diagnostic reagents and methods of making the same for the treatment of SARS-COV-2 virus

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES:
Fig 1: Isolation of Total RNA.
Fig 2: Primary PCR amplification of antibody genes:
Fig 3: Secondary PCR amplification of antibody genes
Fig 4: Process flow - Method of constructing an anti-SARS-CoV-2 specific scFv recom-binant antibody library

DETAILED DESCRIPTION OF THE INVENTION
The following is a detailed description of some of the embodiments and explanation of the present invention with some examples thereof. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the inten-tion is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable man-ner in one or more embodiments.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the de-sired properties sought to be obtained by a particular embodiment. In some embodi-ments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific exam-ples are reported as precisely as practicable.
The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all ex-amples, or exemplary language (e.g. “such as”) provided with respect to certain embod-iments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specifi-cation should be construed as indicating any non-claimed element essential to the prac-tice of the invention.
The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

Term used herein " monoclonal antibody " or " mAb " represent the antibody that the colony of the antibody of basically homology obtains, that is, each antibody forming de-scribed colony is all identical except the possible naturally occurring sudden change that may exist with small quantity. Monoclonal antibody has high degree of specificity for single antigen. In addition, different from the polyclonal antibody preparations.

Antibodies of the invention include, but are not limited to, synthetic antibodies, mono-clonal antibodies, recombinantly produced antibodies, multispecific antibodies (includ-ing bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibod-ies, intrabodies, single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. The antibodies of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.
The variable heavy chain portion of the Fab is coded for by a combination of 3 genes, called VH (variable heavy), DH (diversity heavy), and JH (joining heavy). The variable light chain portion of the Fab consists of either a kappa chain or a lambda chain coded for by a combination of 2 genes, VL (variable light) and JL (joining light). In the DNA of each B-lymphocyte there are multiple forms of each one of these variable determinant genes. Although the exact number of each gene isn't known and varies from person, there are approximately 38-46 VH genes; 23 DH genes; 6 JH genes; 34-38 kappa VL genes; 5 kappa JL genes; 29-33 lambda VL genes; and 4-5 lambda JL genes.
The terms “fully human antibody” or “human antibody” are used interchangeably herein and refer to an antibody that comprises a human variable region and, most preferably a human constant region. In specific embodiments, the terms refer to an antibody that comprises a variable region and constant region of human origin.
The term “phagemid” vectors used herein is a DNA-based cloning vector, which has both bacteriophage and plasmid properties. It has ability to be packaged into the capsid of a bacteriophage, due to their having a genetic sequence that signals for packaging. Phagemids are used "Phage Display

The term “lymphocyte” as used herein is a type of white blood cell in the immune sys-tem . Lymphocytes include natural killer cells (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity).

The term “Biopanning” used herein is an affinity selection technique which selects for peptides that bind to a given target. This technique is often used for the selection of anti-bodies .
The term “host cell” as used herein refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid mole-cule due to mutations or environmental influences that may occur in succeeding genera-tions or integration of the nucleic acid molecule into the host cell genome.

As used herein, the terms “treat,” “treatment” and “treating” refer to the treatment of SARS-COV-2
An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreo-ver, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant tech-niques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, a nucleic acid molecule(s) encoding an antibody of the invention is isolated or purified.
A “therapeutic antibody” may refer to any antibody or functional fragment thereof that is used to treat viral infections, cardiovascular disease or other diseases or conditions. Ex-amples of therapeutic antibodies that may be used according to the embodiments de-scribed herein include, but are not limited to fully human monoclonal antibody.
The object of the present invention is to provide a method of constructing an anti-SARS-CoV-2 specific scFv recombinant antibody library against SARS-CoV-2 virus
In an embodiment, the present invention provides a method of production a fully human monoclonal antibody to target SARS-COV-2 spike (s) glycoprotein, receptor binding proteins and nucleocapsid protein (N-protein).

In an embodiment, the present invention provides a method of production a fully human monoclonal Antibody to target S1, S2, receptor binding proteins (RBD) and nucleocapsid protein (N-protein).

In specific embodiments, an antibody of the invention is a fully human antibody, a mon-oclonal antibody, a recombinant antibody, bispecific antibody, or any combination thereof. In particular embodiments, the antibody is a fully human antibody, such as a fully human monoclonal antibody, or antigen binding fragment thereof, that immuno-specifically binds to target SARS-COV-2 spike (s) glycoprotein or receptor binding pro-teins.
Reference will now be made in detail to the exemplary embodiments of the present dis-closure, examples of which are illustrated in the accompanying drawings. Wherever pos-sible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.
The present invention relates to the roles of S protein in receptor binding and membrane fusion indicate that vaccines based on the S protein could induce antibodies to block vi-rus binding and fusion or neutralize virus infection. S protein has therefore been selected as an important target for a fully human antibody development.
Potent combination two antibodies were able to bind simultaneously to the spike, neu-tralizing the virus “synergistically.” In other words, the antibodies were more potent in combination than they were individually.
The S protein molecule contains two subunits S1 and S2. The S1 subunit has an RBD that interacts with its host receptor, whereas the S2 subunit mediates fusion between the virus and host cell membranes for releasing RNA into the cytoplasm for replication.
The sequences of monoclonal antibodies that are effective against SARS-CoV-2 could be cloned and expressed in Pichia pastoris as suitable expression system which could be tested against SARS-CoV-2.

The present invention disclosed a fully Human antibody targeting spike protein of SARS-CoV-2 could potentially inhibit the virus binding to its cellular receptor thereby preventing its entry into the cell.
The present invention provides a fully human monoclonal antibodies which target SARS-COV-2 receptor binding domain (RBD), non-RBD domain, envelope (E), nucleo-protein (N) and membrane (M) protein use as the therapeutic or diagnostic reagents and methods of making the same for the treatment of SARS-COV-2 virus

In a general aspect the present invention provides a method of constructing an anti-SARS-CoV-2 specific scFv recombinant antibody library against SARS-CoV-2 virus.

In an embodiment, the present invention provides a competitive process of screening strategy to isolate human antibodies from a phage display library.
Method of constructing an anti-SARS-CoV-2 specific scFv recombinant antibody library againstSARS-CoV-2 virus

In an embodiment, the present invention provides a method of production of a fully hu-man monoclonal antibody to target SARS-COV-2 spike (s) glycoprotein or Receptor binding proteins.

In another embodiment, the present invention provides a fully human monoclonal anti-bodies which target SARS-COV-2 S1 and S2 subunits, SARS-COV-2 receptor binding domain (RBD), non-RBD domain and nucleoprotein (N) .

In another embodiment, the present invention provides a fully human monoclonal anti-bodies which target SARS-COV-2 receptor binding domain (RBD), non-RBD domain, and nucleoprotein (N) use as the therapeutic or diagnostic reagents and methods of mak-ing the same for the treatment of SARS-COV-2 virus

In another embodiment, the present invention provides e a fully human monoclonal anti-bodies which target SARS-COV-2 receptor binding domain (RBD), non-RBD domain, envelope (E), nucleoprotein (N) and membrane (M) use as the therapeutic or diagnostic reagents and methods of making the same for the treatment of SARS-COV-2 virus

In another embodiment, the present invention provides a fully human monoclonal anti-body cocktail having the aforenamed monoclonal antibody in combination with a second monoclonal antibody which binds simultaneously to the spike for neutralizing the SARS-COV-2 virus

In another embodiment, the present invention provides the potent combination or cock-tail of antibodies as the therapeutic or diagnostic reagents and methods of making the same for the treatment of SARS-COV-2 virus

While the foregoing description discloses various embodiments of the disclosure, other and further embodiments of the invention may be devised without departing from the basic scope of the disclosure. The invention is not limited to the described embodiments, versions or examples, the invention is described hereinafter, with reference to the fol-lowing examples, which are to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art, such examples are illustrative only and should not be construed to the limit of the scope of present invention.

Examples
The present disclosure is further explained in the form of following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifica-tions to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the inven-tion.

Example-1
Phage display libraries to develop fully human monoclonal antibody against SARS-CoV-2 virus.
A fully human monoclonal antibody are developed using transgenic mice or phage dis-play libraries. Herein heavy and light chains of IgG proteins are expressed in structural polymorphic forms. Human IgG allotype is one of the many factors that can contribute immunogenicity.
Phage display libraries to develop fully human monoclonal antibodies. B lymphocytes taken from a SARS-CoV-2 recovered patient. After the RNA isolation, mRNA is extract-ed which is converted to cDNA and eventually into DNA.
From Antibody Library, SARS-CoV-2 neutralizing antibody (nAb) producing clones were screened – sequenced – characterized and expressed into suitable expression vector and host system.

Example -2
Fully human monoclonal antibody expression in Pichia pastoris
Production of recombinant proteins in Pichia pastoris host is both extracellular or intra-cellular manner, but preferably intracellular .The method for production of expressed S-protein comprising the step of preparing pre inoculum of Pichia pastoris which is used for the preparation of inoculum for fermentation and production of desired end product i.e. S-protein, S subunit1 protein, S subunit 2 protein.
The process of the invention is applicable to recombinant Pichia pastoris cells producing S- Protein, or S protein subunit 1 or S protein subunit 2.

Example-3
Fully human monoclonal antibodies targeting Non-RBD regions against SARS-CoV-2
Fully human monoclonal antibodies targeting Non-RBD regions for treating COVID-19 While the RBD is the focus for the development of neutralizing antibodies against SARS-CoV-2, the function of non-RBD regions is poorly understood. However, the Fully human monoclonal antibodies shows the binding on the N-terminal domain (NTD) of the SARS-CoV-2 S protein and exhibits high neutralization potency SARS-CoV-2. The structural analysis has confirmed its binding to the NTD, not the RBD which directly in-teracts with ACE2, demonstrating a new vulnerable epitope in the S1 subunit as a target of neutralizing antibodies (nAbs) for treating SARS-CoV-2.

Example 4
Potent combination or cocktail designed to prevent SARS-CoV-2 infection
Fully human monoclonal antibodies can also work as a potent combination or cocktail to recognize two sections of the receptor-binding domain that don’t overlap.
Combined antibodies were able to bind simultaneously to the spike, neutralizing the vi-rus “synergistically. The antibodies were more potent in combination than they were in-dividually on the SARS-CoV-2 infection.

The two antibodies, together and separately, protected mice from the worst effects of SARS-CoV-2 infection compared with untreated animals, these mice showed less weight loss, produced less of the virus, and had less lung inflammation. Rationally selected therapeutic cocktails likely offer greater resistance to SARS-CoV-2 escape.

Example 5
Anti-SARS-CoV-2 specific scFv antibody library Construction
A. Collection of blood samples and Lymphocyte Isolation:
Volunteers who were recovered from the SARS-CoV-2 infection were selected as donors for blood collection. Thirty (30) ml of whole blood was collected from ten (10) SARS-CoV-2 survivors in vacutainers and were processed immediately for isolation of lym-phocytes by Ficoll Paque separation method. The isolated lymphocytes were counted (Table: 1) and stored at -80oC, if not used immediately, for further use.

Table: 1: The cell count of the lymphocytes isolated from the blood samples of ten (10) SARS-CoV-2 survivors.
Sr. No Patient ID Lymphocyte count Storage
1 IK ~ 25 Million Pellet stored with Trizol @-80C
2 SS ~27 Million Pellet stored with Trizol @-80C
3 SHS ~24 Million Pellet stored with Trizol @-80C
4 TS ~26 Million Pellet stored with Trizol @-80C
5 TP ~27 Million Pellet stored with Trizol @-80C
6 SA ~23 Million Pellet stored with Trizol @-80C
7 MR ~28 Million Pellet stored with Trizol @-80C
8 AA ~ 32 Million Pellet stored with Trizol @-80C
9 RN ~31 Million Pellet stored with Trizol @-80C
10 NS ~26 Million Pellet stored with Trizol @-80C

B. Total RNA Isolation from Lymphocytes:
Total RNA from lymphocytes were isolated by Trizol method. After RNA isolation, samples were checked by electrophoresis on 1 % agarose gel (Fig.1). All the RNA sam-ples were stored at -80oC, RNAse inhibitor were added in each RNA sample before stor-ing at -80oC.
Sr. No Patient ID QC Storage
1 IK Ran on agarose gel Stored @ -80C with RNAse inhib-itor
2 SS Ran on agarose gel Stored @ -80C with RNAse inhib-itor
3 SHS Ran on agarose gel Stored @ -80C with RNAse inhib-itor
4 TS Ran on agarose gel Stored @ -80C with RNAse inhib-itor
5 TP Ran on agarose gel Stored @ -80C with RNAse inhib-itor
6 SA Ran on agarose gel Stored @ -80C with RNAse inhib-itor
7 MR Ran on agarose gel Stored @ -80C with RNAse inhib-itor
8 AA Ran on agarose gel Stored @ -80C with RNAse inhib-itor
9 RN Ran on agarose gel Stored @ -80C with RNAse inhib-itor
10 NS Ran on agarose gel Stored @ -80C with RNAse inhib-itor

Fig 1: Isolation of Total RNA. The lymphocytes from donor samples were used for iso-lation of total RNA using Trizol method.

C. Amplification of the antibody genes:
The total RNA which was isolated from the lymphocytes was used for cDNA preparation using One Step RT PCR Kit, (Qiagen, Germany) followed by primary PCR amplifica-tion of antibody genes viz: Variable heavy (VHs) and Variable kappa and Variable lambda (VKs /VLs) by using specific primer sets. The primary PCR products of about 750 bp (Fig -2) was used as template for secondary PCR using the primer sets and with specific restriction sites.(As Fig 3) The amplified product (750 bp) was used for further cloning steps
Fig 2: Primary amplification of antibody genes: The antibody specific genes were PCR amplified using primers specific for VH and VK/VL.

Fig 3: Secondary amplification of antibody genes: The antibody specific genes were PCR amplified (Fig -3) using primers specific for VH and VK/VL with specific restriction sites suitable for cloning in appropriate phagemid vector.

D. Cloning of antibody genes and ScFv library banking:
The PCR amplified genes were cloned in phagemid vector (pSEX, Progen) which were then transformed into E. coli XL1B strain. The transformants were selected by growing on 2YT media with antibiotic resistance. The bacteriophage M13K07 (Invitrogen, Ger-many) was used as helper phage for the expression of different antibody clones. The pool of these transformants was used to prepare pool of recombinant phages particles which can be used for bio-panning.

6. Biopanning and rapid analysis of selective interactive ligands (BRASIL)
The antigens from SARS-CoV-2 virus viz: spike protein (S1, S2 & RBD) and N protein (Sino Biological Inc) were selected for bio-panning of recombinant phages from above ScFv library (pool of VHs, VKs & VLs). The target proteins were coated on immune tubes and recombinant phages prepared from above library were allowed to bind to coat-ed target antigens. After removal of unbound phage with various washing steps, the bound phages were eluted and used for next round of bio-panning. The enrichment of bound phages was done by three rounds of bio-panning with reduced concentrations of target antigens (20 to 1 micrograms/ml).
A. Primary Screening by Phage Enzyme-linked immunosorbent assay (ELISA)
The recombinant phages obtained after three rounds of bio-panning were screened for their antigen specificity by phage ELISA (enzyme-linked immunosorbent assay). In this screening procedure the target antigens (S1, S2, RBD & N protein) were coated on ELI-SA plate against which the recombinant phages were allowed to bound. The un-bound phages removed by multiple washing steps. The bound phages were detected using anti M13K07-HRP antibody (Invitrogen, Germany). About 3% clones were found to be posi-tive as a result of phage ELISA.
B. Secondary Screening
The ELISA positive clones were screened further by sequencing antibody genes using Sanger’s method these clones were then checked for virus neutralization assay. Estab-lishment of the novel lead SARS-CoV-2 binders as in fig 4.

Fig 4: Process flow - Method of constructing an anti-SARS-CoV-2 specific scFv re-combinant antibody library

Although the preferred embodiments of the present invention and their respective varia-tions have been described, people having ordinary skills in the art would envision vari-ous modifications of those embodiments. Accordingly, the present invention should not be limited to precise forms and manners in the above disclosure and description but should simply be taken by way of examples. Thus, the present invention can be varied and modified without departing the true scope and spirit thereof as defined in the ap-pended claims.
,CLAIMS:WE CLAIM:
1. A method of constructing an anti-SARS-CoV-2 specific scFv recombinant antibody library comprising the steps of:
i) collection of blood samples and Lymphocyte Isolation, wherein the cell count of the lymphocytes isolated from the blood samples of SARS-CoV-2 survivors;
ii) isolation of Total RNA from lymphocytes of SARS-CoV-2 survivors;
iii) primary and secondary amplification of antibody genes by using specific primer sets; and
iv) amplified genes are cloned in pSEX phagemid vector followed by transformation into E. coli XL1B strain for the preparation of recombinant phage library.

2. The anti-SARS-CoV-2 specific scFv recombinant antibody library as claimed in claim 1,wherein antibody genes are specific to the variable region of heavy chain and light chain of SARS-CoV-2 antibodies.

3. The method for constructing an anti-SARS-CoV-2 specific scFv recombinant antibody library as claimed in claim 1 is used further for biopanning and rapid analysis of selec-tive interactive ligands (BRASIL), wherein the steps comprising of,
i) S1, S2, RBD & N protein as a target proteins for phage binding ,wherein the concen-trations of target proteins is about 20 to is about 1 micrograms/ml;
ii) target proteins were coated on immune tubes and recombinant antibodies were al-lowed to bind to coated target antigens;
iii) removal of unbound phage with various washing steps and the bound phages were
eluted ;
iv) biopanning enriched recombinant phages expressing antibodies obtained after Phage
ELISA screening; and
v) validation of SARS-CoV-2 neutralizing Antibodies (nAbs) targeting against spike protein (S) and nucleoprotein (N) protein of SARS-CoV-2.

4. The method for constructing an anti-SARS-CoV-2 specific ScFv recombinant anti-body library as claimed in claim 1 and 3, wherein the recombinant antibody is a fully Human antibody targeting spike protein and N protein of SARS-CoV-2.

5. The method for constructing an anti-SARS-CoV-2 specific ScFv recombinant anti-body library as claimed in claim 4, wherein the fully human antibodies targeting against spike protein and N protein of SARS-CoV-2 can be used for therapeutic and diagnostic purposes.