FORM 2
THE PATENTS ACT, 1970
(Act 39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(SECTION 10 & Rule 13)
1. TITLE OF THE INVENTION
FULLY HUMAN MONOCLONAL ANTIBODIES AGAINST CORONAVIRUSES
2. Applicant(s)
NAME NATIONALITY ADDRESS
TECHINVENTION India 1004, The Summit Business Park,
LIFECARE PVT. LTD. Andheri East, Mumbai 400093, India
3. PREAMBLE TO THE DESCRIPTION
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it
is to be performed.

FIELD OF THE INVENTION
[0001] The present invention relates to Bio-Pharmaceuticals. Specifically, the present invention relates to an isolated fully human Monoclonal Antibody or Monoclonal Antibody fragment or an immuno conjugate. Further, the present invention relates to Monoclonal Antibodies or an antigen-binding fragment of the Monoclonal Antibody, its composition and uses thereof against coronaviruses. The present invention further relates to the process of preparation, compositions and use of said Monoclonal Antibodies in the treatment or prevention or diagnosis of coronaviruses infection.
BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] In December 2019, an outbreak occurred in Wuhan located in the Hubei province of China wherein individuals where suffering from pneumonia however the cause was unknown. This situation later on raised intense attention not only within China but internationally. Later, it was confirmed that the causative agent for this pneumonia¬like disease is a coronavirus (COV) belonging to the family Coronaviridae (Jiang, F. et al. J Gen Intern Med. 35, 1545–1549 (2020); Wang Wu et.al JAMA. 2020 May 12;323(18):1843-1844F. Wu, F., Zhao, S., Yu, B. et al. Nature 579, 265–269 (2020). ; Zhou, P., Yang, XL, Wang, XG. et al. Nature 579, 270–273 (2020).). On February 11, 2020, the causative virus was named as SARS COV-2 virus by the World Health Organisation and the syndrome was renamed from 2019- nCoV to COVID-19. COVID-19, a respiratory disease, has led to more than 22,140,472 confirmed cases and over 781,135 deaths globally as of August 19, 2020 (Subodh Kumar et al, Virus Res. 2020 Oct 15; 288: 198141). In India as per the WHO report up until 31st Marh 2021 there have been 2,88,09,339 cases of COVID 19 that are confirmed and about 3,46,759 deaths. India is the second most affected country after the US due to the COVID-19 pandemic (Shil P et.,al Spat Spatiotemporal Epidemiol. 2022 Jun;41:100507).
[0004] 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, F. et al. J Gen Intern Med. 35, 1545–1549

(2020); Siu YL et.al J Virol. 2008 Nov;82(22):11318-30). The most critical role is played by the S protein that allows the attachment of the virus, fusion and entry in to the target cell (Gordon, D.E., Jang, G.M., Bouhaddou, M. et al. Nature 583, 459–468 (2020).; Jiang, F. et al. J Gen Intern Med. 35, 1545– 1549 (2020)). This S protein is densely glycosylated and is a trimeric Class I protein. COV particles are decorated with the club-shaped trimers of the S protein, which is 8–23-nm long (Hoffmann M, et.al Cell. 2020 Apr 16;181(2):271-280.e8.). Among different COV species the size differs from 1100 to 1600 amino acids length approximately, with an estimated molecular mass of up to 220 kDa (Li F. et.al Annu Rev Virol. 2016 Sep 29;3(1):237-261). The S protein undergoes structural rearrangement for the viral membrane to fuse with the membrane of the host cell (Bosch BJ, et.al J Virol. 2003 Aug;77(16):8801-11). 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 (Hoffmann M, et.al Cell. 2020 Apr 16;181(2):271-280.e8 ; Walls AC, et.al Cell. 2020 Apr 16;181(2):281-292.e6). [0005] There have been several serious global health consequences to the spread of the novel coronavirus disease (COVID-19). For the therapeutic or prophylactic action of the said disease several options have been invented such as vaccines, anti-viral, convalescent plasma therapy and antibody/ antibody cocktail & Bispecific antibody. The aim behind all these is to stimulate the patient's immune system to attack the virus. In order to help guide the design of vaccines, neutralizing antibodies (nAbs) targeting the coronavirus, which is the causative agent of the disease could be constructed. Vaccines then prompt the immune system to make antibodies against a virus. There are several reports demonstrating through experimental means wherein broad-neutralizing antibodies are targeting conserved region of RBD within the S1 subunit. The S2 subunit involved in viral membrane fusion could be another target.
[0006] There is increasing need for the discovery of the specific Monoclonal Antibodies which can neutralize the virus (Kumar et.al. Appl. Microbiol. Biotechnol. 2020 Apr; 104 (8):32093228).
[0007] An increase in the effectiveness of the treatment would be observed since Monoclonal Antibodies are highly specific which would in turn make it more potent. It would also prevent the viral escape.
[0008] Experimental animal data show that protection against severe acute respiratory syndrome coronavirus infection with human Monoclonal Antibodies (mAbs) is feasible.

For an effective immune prophylaxis in humans, broad coverage of different strains of coronavirus and control of potential neutralization escape variants will be required. Combinations of virus-neutralizing, noncompeting mAbs may have these properties Bispecific antibody could be produced in large quantities and injected into patients ensuring protection against coronaviruses infection for healthcare workers, elderly immunocompromised people, & others who respond poorly to traditional vaccines or are suspected of a recent exposure to the coronaviruses.
[0009] Although various companies are working on the development of COVID-19 treatment methods, including different antibodies, there is still need to explore the novel ways and compositions, for treating coronaviruses, that could be more economical, easily manufactured and scalable and can overcome deficiencies associated with the known arts. The present invention provides a novel, specific and industrially advantageous Bispecific Antibodies that are capable of neutralizing several variants of coronaviruses infectivity. These Monoclonal Antibodies shall be fulfilling the unmet medical need for coronaviruses prophylaxis and treatment.
OBJECT OF THE INVENTION
[00010] An object of the present invention is to provide fully human Monoclonal
Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno
conjugate.
[00011] Another object of the present invention is to provide fully human Monoclonal
Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno
conjugate having at least 90% identity to one or more sequences selected from SEQ ID
No 1-16.
[00012] An object of the present invention is to provide a composition comprising
isolated fully human Monoclonal Antibody or Monoclonal Antibody fragment,
Bispecific Antibody or an immuno conjugate against coronavirus infection.
[00013] In another object of the present invention provides a process for the production
of a fully human Monoclonal Antibody described herein to target coronavirus
glycoprotein or receptor binding proteins.
[00014] Another object of the present invention provides a composition of fully human
Monoclonal Antibodies which target receptor binding domain (RBD), non-RBD domain,
envelope (E), nucleoprotein (N) and membrane (M) on coronavirus

[00015] Another object of the present invention provides a composition of fully human
Monoclonal Antibodies and its usage as the therapeutic or diagnostic reagents and
methods of making the same for the treatment of coronavirus.
[00016] Another object of the present invention provides a process of neutralization of
fully human Monoclonal Antibody cocktail and its potential to target receptor binding
domain (RBD), non-RBD domain, envelope (E), nucleoprotein (N) and membrane (M)
on coronavirus.
[00017] Another object of the present invention provides composition comprising a
potent combination or cocktail of antibodies as the therapeutic or diagnostic reagents and
methods of making the same for the treatment of coronavirus infection.
SUMMARY OF THE INVENTION
[00018] This summary is provided to introduce a selection of concepts in a simplified
form that are further described below in Detailed Description section. This summary is
not intended to identify key features or essential features of the claimed subject matter,
nor is it intended to be used as an aid in determining the scope of the claimed subject
matter.
[00019] One of the aspect of the present invention is to provide fully human Monoclonal
Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno
conjugate having at least 90% identity to one or more sequences selected from SEQ ID
No 1-16.
[00020] In one of the aspect of the present invention provides a composition comprising
isolated fully human Monoclonal Antibody or Monoclonal Antibody fragment,
Bispecific Antibody or an immuno conjugate described herein against coronavirus
infection.
[00021] In another aspect of the present invention provides a composition of fully
human Monoclonal Antibodies and its usage as the therapeutic or diagnostic reagents
and methods of making the same for the treatment of coronavirus infection.
[00022] In another aspect of the present invention provides a process of neutralization
of fully human Monoclonal Antibody cocktail and its potential to target receptor binding
domain (RBD), non-RBD domain, envelope (E), nucleoprotein (N) and membrane (M)
on coronavirus.

[00023] In another aspect of the present invention provides composition comprising a potent combination or cocktail of antibodies as the therapeutic or diagnostic reagents and methods of making the same for the treatment of coronavirus.
DRAWINGS
[00024] The following drawings form part of the present specification and are included
to further illustrate aspects of the present disclosure. The disclosure may be better
understood by reference to the drawings in combination with the detailed description of
the specific embodiments presented herein.
Figure 1: Gel Electrophoresis results of Total RNA Isolation
Figure 2: Gel Electrophoresis results of Primary PCR amplification
Figure 3: Gel Electrophoresis results of Secondary PCR amplification
Figure 4: SARS COV-2 specific SCFV recombinant antibody library construction
Figure 5(a): Neutralization Assay for C15 and C19 clones on Different SARS CoV-2
spike variant
Figure 5(b): Neutralization Assay for C15 and C19 clones on Different SARS CoV-2 spike variant
Figure 5(c): Comparison of neutralization effect of C15, C19 and ACE2-IgFc (Positive control) on Different SARS CoV-2 spike variants.
Figure 5(d): Comparison of neutralization effect (IC50) of different ScFV including C15 & C19 and ACE2-IgFc (Positive control) on Different SARS CoV-2 spike variants.
Figure 6 (a): Pseudo Virus Neutralization Assay for Omicron Strain
Figure 6 (b): Pseudo Virus Neutralization Assay for Delta Strain
Figure 7(a) : 2D representation of the interaction between the known neutralizing antibody and the spike of SARS-CoV-2.
Figure 7(b) : Interface and epitope region predicted for SARS-CoV-2 bound to M15-VH + M15-VL
Figure 7(c) : Interface and epitope region predicted for SARS-CoV-2 bound to M15-VH + M15-VL and known neutralizing antibody

Figure 7(d) : Interface and epitope region predicted for SARS-CoV-2 bound to M19-VH + M15-VL
Figure 7(e) : Interface and epitope region predicted for SARS-CoV-2 bound to M19-VH + M15-VL and known neutralizing antibody.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[00025] The following is a detailed description of embodiments of the disclosure. 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 intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[00026] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[00027] 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 manner in one or more embodiments.
[00028] 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 and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, 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 examples are reported as precisely as practicable.
[00029] As used in the description herein and throughout the claims that follow, the
meaning of "a," "an," and "the" includes plural reference unless the context clearly
dictates otherwise.
[00030] Also, as used in the description herein, the meaning of "in" includes "in" and
"on" unless the context clearly dictates otherwise.
[00031] 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."
[00032] 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 methods 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 examples, or exemplary language (e.g.
"such as") provided with respect to certain embodiments 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 specification should be construed as
indicating any unclaimed element essential to the practice of the invention.
[00033] The description that follows, and the embodiments described therein, is
provided by way of illustration of an example, or examples, of particular embodiments
of the principles and aspects of the present disclosure. These examples are provided for
the purposes of explanation, and not of limitation, of those principles and of the
disclosure.
[00034] The headings and abstract of the invention provided herein are for convenience
only and do not interpret the scope or meaning of the embodiments.
[00035] Various terms as used herein are shown below. To the extent a term used in a
claim is not defined below, it should be given the broadest definition persons in the
pertinent art have given that term as reflected in printed publications and issued patents
at the time of filing.

[00036] The term "Monoclonal Antibody”,” antibody” or “mAb “according to the present invention means, but not limited to, the antibody that the colony of the antibody of basically homology obtains, that is, each antibody forming described 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.
[00037] Monoclonal Antibodies of the invention include, but are not limited to, synthetic antibodies, Monoclonal Antibodies, recombinant produced antibodies, multispecific Monoclonal Antibodies (including Bispecific antibodies), human antibodies, fully human antibodies, chimeric antibodies, intra-bodies, 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 Monoclonal 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 immuno globulin molecule.
[00038] The term “coronavirus” according to the present invention means, but not limited to, a group of virus belonging to the coronoviridae family that are a natural host to animals that causes COVID-19 infection in human body. These include SARS –CoV2, MERS CoV, SADS-CoV, SARS-CoV, HCoV-NL63, HCoV-OC43, HCoV-HKU1 and their respective variants and variants of concern.
[00039] 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 3846 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. [00040] The term “fully human Monoclonal Antibody” or “human Monoclonal Antibody” according to the present invention means, but not limited to, refers to Monoclonal Antibody that comprises a human variable region and, most preferably a

human constant region. In specific embodiments, the terms refer to a Monoclonal Antibody that comprises a variable region and constant region of human origin. [00041] As used herein, the terms "treat," "treatment" and "treating" refer to the treatment of coronavirus. The term "isolated" according to the present invention means, but not limited to, refers to nucleic acid molecule which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, 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 techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, a nucleic acid molecule(s) encoding a Monoclonal Antibody of the invention is isolated or purified. [00042] The term "complementarity determining regions" (CDR) according to the present invention means, but not limited to, refers to sequences within the variable regions of binding proteins, such as immunoglobulins, that usually contribute to a large extent to the antigen binding site which is complementary in shape and charge distribution to the epitope recognized on the antigen. The CDR regions can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, e. g., by solubilisation in SDS. Epitopes may also consist of posttranslational modifications of proteins.
Monoclonal Antibody:
[00043] The present invention provides fully human Monoclonal Antibody or
Monoclonal Antibody fragment, Bispecific Antibody or an immuno conjugate.
[00044] In one embodiment, the present invention provides fully human Monoclonal
Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno
conjugate having at least 90% identity to one or more sequences selected from SEQ ID
No 1-16.
[00045] In one embodiment, the present invention provides a fully human Monoclonal
Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno
conjugate against coronaviruses having at least 90% identity to one or more sequences
selected from SEQ ID No 1-16.

[00046] In another embodiment, the present invention provides a fully human monoclonal antibodies against coronaviruses having at least 90% identity to one or more sequences selected from SEQ ID No 1-16.
[00047] In one embodiment, the present invention provides a fully human Monoclonal Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno conjugate against coronaviruses infection having at least 90% identity to one or more heavy chain sequences selected from SEQ ID No 1-12.
[00048] In another embodiment, the present invention provides a fully human Monoclonal Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno conjugate against coronaviruses infection having at least 90% identity to one or more light chain sequences selected from SEQ ID No 13-16.
[00049] In one embodiment, the present invention provides a fully human Monoclonal Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno conjugate against coronaviruses infection having heavy chain sequences at least 90% identity to one or more sequence selected from SEQ ID No 1-12 and light chain sequences at least 90% identity to one or more light chain sequences selected from SEQ ID No 13-16.
[00050] In one embodiment, the present invention provides a composition comprising an isolated fully human Monoclonal Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno conjugate against coronaviruses infection. [00051] In one embodiment, the present invention provides a composition comprising an isolated fully human Monoclonal Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno conjugate for the manufacture of pharmaceutical composition against coronaviruses.
[00052] In one embodiment, the present invention provides a composition comprising an isolated fully human Monoclonal Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno conjugate for the manufacture of pharmaceutical composition useful for the treatment, prevention or diagnosis of coronaviruses. [00053] In one embodiment, the present invention provides a fully human Monoclonal Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno conjugate against coronaviruses infection having heavy chain sequences at least 90% identity to one or more sequence selected from SEQ ID No 1-12 and light chain

sequences at least 90% identity to one or more light chain sequences selected from SEQ
ID No 13-16 for the manufacture of pharmaceutical composition against coronaviruses.
[00054] In one embodiment, the present invention provides a fully human Monoclonal
Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno
conjugate against coronaviruses infection having heavy chain sequences at least 90%
identity to one or more sequence selected from SEQ ID No 1-12 and light chain
sequences at least 90% identity to one or more light chain sequences selected from SEQ
ID No 13-16 useful for the treatment, prevention or diagnosis of coronaviruses.
[00055] In one embodiment, the present invention provides a production method for
fully human Monoclonal Antibody or Monoclonal Antibody fragment, Bispecific
Antibody or an immuno conjugate.
[00056] In an embodiment, the present invention provides a production process for fully
human Monoclonal Antibody described herein targeting sites including but not limited
to coronaviruses spike (s) glycoprotein (SI and S2 subunit), receptor binding proteins
(RBD) and nucleocapsid protein (N-protein).
[00057] In an embodiment the present invention provides a production process for fully
human monoclonal antibody wherein its sequences have a 90% identity or more
sequences selected from Sequence ID No 1-16.
[00058] In one embodiment the present invention provides a composition comprising
fully human Monoclonal Antibodies demonstrating neutralizing properties to be used for
the treatment or prevention or diagnosis of coronaviruses infection.
[00059] In further embodiment, the present invention provides a composition
comprising isolated fully human Monoclonal Antibody or Monoclonal Antibody
fragment or an immuno conjugate of the invention as a medicament for use in the
prevention or treatment or diagnosis of the infection of the coronaviruses.
[00060] In one embodiment the coronavirus is selected from group comprising of S ARS
-Cov2, MERS CoV, SADS-CoV, SARS-CoV, HCoV-NL63, HCoV-OC43, HCoV-
HKU1 and their respective variants, variants of concern or combination thereof.
[00061] In one of the embodiment the coronavirus is preferably SARS-Cov-2.
Process:
[00062] In one of the embodiment of the present invention provides a process for the
preparation of fully human monoclonal antibody by screening against coronaviruses

spike (s) glycoprotein (SI and S2 subunit), receptor binding proteins (RBD) and
nucleocapsid protein (N-protein).
[00063] In another embodiment the present invention provides a process for the
production of fully human monoclonal antibody that has 90% sequence identity to Seq
ID No 1- 16.
[00064] In one embodiment, the present invention provides a process of production of
a fully human Monoclonal Antibody to inhibit virus binding to cellular receptor on
coronaviruses.
[00065] In one embodiment, the present invention provides a process of production of
a fully human Monoclonal Antibody to inhibit virus interaction with cellular receptor
targeting coronaviruses spike (s) glycoprotein (SI and S2 subunit), receptor binding
proteins (RBD) and nucleocapsid protein (N-protein) of coronaviruses.
[00066] In one of the embodiments the present invention provides a process of
production of a fully human Monoclonal Antibody against coronaviruses, said method
comprising at least one of the steps of;
a. Blood Collection
b. RNA Isolation
c. cDNA Conversion and Monoclonal Antibody Genes Amplification by PCR
d. Monoclonal Antibody Genes Cloning and scFv library banking
e. Monoclonal Antibody Selection by Bio-panning
f Screening by ELISA
g. DNA sequencing
h. Expression & Purification in IgG Format [00067] In another embodiment, the present invention provides a process for the detection of neutralization activity by interaction of fully human Monoclonal Antibody cocktail with a secondary Monoclonal Antibody produced in an animal model. [00068] In another embodiment, the present invention provides a composition comprising fully human Monoclonal Antibodies which target coronaviruses 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 coronaviruses virus.

[00069] In another embodiment, the present invention provides the potent combination
or cocktail of antibodies as the therapeutic or diagnostic reagents and methods of making
the same for the treatment of coronaviruses.
[00070] In another embodiment of the present invention provide a detection process
described herein for the interaction of spike protein including coronaviruses SI and S2
subunits, coronaviruses receptor binding domain (RBD), non-RBD domain and
nucleoprotein (N) on coronaviruses and fully human monoclonal antibody.
[00071] In another embodiment, the present invention provide a process for the
determination of reduction activity of the fully human monoclonal antibody to the spike
proteins on coronaviruses.
[00072] In another embodiment of the present invention the reduction activity of fully
human monoclonal antibody was demonstrated by transformation, transfection, post
transfection collection, filtration and detection.
[00073] In another embodiment of the present invention the reduction activity for the
fully human monoclonal antibody was transfected into appropriate mammalian vector.
Host:
[00074] In one of the embodiment the present invention provides a method of production of a fully human Monoclonal Antibody that has 90% sequence identity to Seq ID No 1-16., said method comprises use of host cells selected from, but not limited to, cells of mammalian, plant, insect, fungal or bacterial origin. Host cells of the present invention include but are not limited to eukaryotic cells such as mammalian cells, e.g. hamster, rabbit, rat, pig, mouse, etc.; avian cells, e.g. duck, chicken, quail, etc.; insect cells or other animal cells; plant cells and fungal cells, e.g. corn, tobacco, Saccharomyces cerevisiae, Pichia pastoris; prokaryotic cells such as E. coli; and other cells used in the art for the production of Monoclonal Antibodies and other binding proteins.
Composition/Formulation:
[00075] In one embodiments, the present invention provides composition comprising at least one of an isolated fully human Monoclonal Antibody or Monoclonal Antibody fragment or an immune conjugate of the invention.

[00076] In another embodiment the present invention provides a pharmaceutical
composition of fully human monoclonal antibody that has 90% sequence identity to
SeqIDNo 1-16.
[00077] In one embodiments, the present invention provides composition comprising at
least one of an isolated fully human Monoclonal Antibody or Monoclonal Antibody
fragment or an immuno conjugate of the invention useful for the treatment or prevention
or diagnosis of coronaviruses.
[00078] In yet another embodiment, the present invention provides composition
comprising at least one of an isolated fully human Monoclonal Antibody or Monoclonal
Antibody fragment or an immuno conjugate of the invention further containing a
pharmaceutically acceptable carrier, diluent or adjuvant.
[00079] In yet another embodiment, the present invention provides composition
comprising at least one of an isolated fully human Monoclonal Antibody or Monoclonal
Antibody fragment or an immuno conjugate of the invention as active ingredient and
further comprising pharmaceutically acceptable carrier selected from but not limited to,
buffer, antioxidant, preservative, isotonic agent and chelating agent.
[00080] In yet another embodiment, the present invention provides a pharmaceutical
composition in the form of fully liquid, lyophilized and other suitable form.
[00081] In another one the present invention, the present invention provides a
composition for the determination of the potential of fully human Monoclonal Antibody
to neutralize target sites such as coronaviruses SI and S2 subunits, receptor binding
domain (RBD), non-RBD domain and nucleoprotein (N) on coronaviruses and fully
human monoclonal antibody.
[00082] Another object of the present invention is to provide a composition comprising
a fully human monoclonal antibody cocktail in combination with other antibodies
targeted towards coronaviruses.
Diagnostics:
[00083] In another one the present invention, the present invention provides a diagnostic kit containing at least one of an isolated fully human Monoclonal Antibody or Monoclonal Antibody fragment or an immuno conjugate of the invention wherein the aforementioned fully human Monoclonal Antibody sequences have 90% identity to SEQ ID No 1-16.

[00084] In another one the present invention, the present invention provides a diagnostic
kit containing at least one of an isolated fully human Monoclonal Antibody or
Monoclonal Antibody fragment or an immuno conjugate of the invention described
herein are packed in suitable containers and labelled for diagnosis, prevention and/or
treatment of coronaviruses
[00085] In further embodiment, the present invention provides a detection process of
the fully human monoclonal antibody described herein for the presence of a
coronaviruses in a sample.
[00086] Yet another object of the present invention is to provide the potent combination
or cocktail of Monoclonal Antibodies comprising Monoclonal Antibodies as the
therapeutic or diagnostic reagents and methods of making the same for the treatment of
coronaviruses.
[00087] In another embodiment of the present invention provides a diagnostic kit
comprising one or more of the fully human monoclonal antibody sequence that targets
coronaviruses receptor binding domain (RBD), non-RBD domain, and nucleoprotein (N)
of coronavirus in sample and shall be used for the treatment of COVID-19.
[00088] In another embodiment of the present invention, provides a diagnositic kit
comprising fully human monoclonal antibody that shall be used for the diagnosis and
prevention of coronaviruses infection.
[00089] 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 following 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
[00090] Step 1: 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 COV2 survivors in vacutainers and were processed immediately for isolation of

lymphocytes by Ficoll Paque separation method. The isolated lymphocytes were counted and stored in trizol at -8O0C, if not used immediately, for further use. [00091] Step 2: 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.l). All the RNA samples were stored at -80°C, RNAse inhibitor were added in each RNA sample before storing at -80°C.
[00092] Step 3: 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
amplification 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
[00093] Step 4: Cloning of antibody genes and ScFv library banking Eight phage displayed antibody gene libraries were constructed in pSEX81 phagemid vector and transformed in E. coli XL IB to get diversity. The transformants were selected by growing on 2YT media with antibiotic resistance. The bacteriophage M13K07 (Invitrogen, Germany) was used as helper phage for the expression of different antibody clones. The pool of these transformants was used to prepare pool of recombinant phage particles which can be used for bio-panning
[00094] Step 5: Biopanning and screening of binders against SARS COV- 2 antigens.
The pools of phage preparations from these libraries were used for bio-panning against SI, S2 & RBD target antigens 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 coated 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).

[00095] Step 6: 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 (SI, S2, RBD & N protein) were coated on ELISA plate against which the recombinant phages were allowed to bound. The un-bound phages were removed by multiple washing steps. The bound phages were detected using anti M13K07HRP antibody (Invitrogen, Germany). . Over 1500 clones were screened by phage ELISA which resulted in forty-five (45) high reactive clones. [00096] Step 7: Secondary Screening by Sanger's sequencing The CDR diversity of these binders was confirmed by antibody gene sequencing from which four clones carried novel CDR (Table 1). These clones with novel CDR were selected to check their potential virus neutralizing abilities. These clones were then checked for virus neutralization assay. Establishment of the novel lead S ARS COV-2 binders as in fig 4.
[00097] Step 8: Construction and Screening of COVID Specific scFv Libraries:
Eight phage displayed antibody gene libraries were constructed in pSEX81 phagemid vector and transformed in E. coli XL1B to get diversity. Over 1500 clones were screened by phage ELISA which resulted in forty-five (45) high reactive clones. The CDR diversity of these binders was confirmed by antibody gene sequencing from which four clones carried novel CDR (Table 1). These clones with novel CDR were selected to check their potential virus neutralizing abilities.
[00098] Step 9: SARS-COV-2 Neutralization Assay
The neutralization potential of candidate scFvs (Figure 5a, 5b and 5c) was studied. Pseudo viruses using S-protein of SARS-CoV-2 (Wuhan strain) & its variants (Alpha, Delta and Omicron), VSV-G pseudotyped viruses were used as control, followed by incubation with serial dilutions of candidate scFvs (C15, C16, C17 & C19). The reaction mixture was then overlayed on HEK293T expressing ACE2.The GFP positive cell count and luciferase activity as a measure of infectivity was found to be decreasing with increase in ScFv concentration, indicating neutralization potential of these ScFvs. The neutralization potential of all 4 tested ScFv checked against Omicron and Delta variant is expressed in the form of IC50 (Figure 5d). Out of the four scFv tested only CI 5 & CI 9 have shown potential neutralization against all the four variants of S ARS CoV 2 (Wuhan,

Alpha, Delta & Omicron) (Figure 5). ScFv CI5 exhibited the strongest neutralization potential of all candidate scFvs, followed by CI9, against all the tested variants (Figure 5a, 5b). The neutralization potential of these two scFv was more towards Omicron and Delta in comparison to Alpha and Wuhan variants (Figure 5d). A representative image at 4x magnification showing the neutralization potential of CI5 and C19 against Delta and Omicron Variant is shown here (Figure 6a and 6b). These differences in the neutralization potential of the scFvs against SARS CoV2 variants can be attributed to the accumulation of different mutations in these variants.
[00099] Step 10: In Silico Analysis To Determine Antigen Antibody Interaction Four variable heavy chains (VH) and light chains (VL) are created in this study, ABodyBuilder-ML modelled all heavy and light chain structures. There were 12 antibodies structures. The RBD of both SARS-CoV-2 and MERS viruses is comparable, indicating that common antibodies can target both spike proteins. Spike (SI) protein was selected from each PDB structures as a docking target. In this study, the antibody's VH (Heavy chain) and VL (Light chain) variable domain sequences were utilised to predict the CDR regions (CDR1, CDR2, and CDR3).
The binding free energy of the complexes were calculated, M19-VH and M15-VL had the most favourable average binding free energy, when it is bound with the spike (SI) proteins of SARS-CoV-2 and MERS, respectively. M15-VH and M15-VL had the lowest average binding energies of-19.72 kcal/mole and -22.48 kcal/mole, making them the most potent antibody against both viruses. Fig 7 (b-d) Later, the SASA of each residue of both the spike proteins were determined in order to identify the interfacial residues. Ranks were assigned on %SASA calculated based on CDR involved at the interface of the antigen and antibody.
Complexes of antigen of SARS-CoV-2 and MERS with M15-VH + M15-VL and M19-VH + M15-VL were selected for the Molecular dynamic simulation for 100 ns. The antigen bound to M19-VH + M15-VL was steady, whereas the one bound to M15-VH + M15-VL fluctuated and changed conformationally, suggesting M15-VH binding to the SARS-CoV-2 protein was more stable. The RMSD of these antibodies with the MERS increased over time, but M19-VH + M15 - VL was more stable in the last 10 ns, indicating significant MERS protein binding,
Clustering was performed on the antigen-antibody complexes, the middle structure of most populated clusters was selected from each antigen-antibody complex. The binding

free energy for antibodies (M15-VH + M15-VL and M19-VH + M15-VL, respectively) bound to the SARS-CoV-2 and MERS protein was calculated by MM/GBSA method. Fig 7(c and e) The antibody M19-VH + M15-VL bound to the MERS antigen had comparable total binding free energy to the antibody M15-VH + M15-VL bound to the protein of SARS. The binding free energy was significantly lower in the antibody Ml 5-VH + M15-VL complexed with MERS. This suggested that M19-VH + M15-VL antibody had the strongest binding with the spike protein of SARS while it also showed comparable binding with MERS.
[000100] Table 1: IN SILICO ANALYSIS BASED PREDICTION OF CDR
REGIONS

Name Type CDR1 CDR2 CDR3
M15-VH VH GDTFRTY IPTFVT FGHQMVRYYHYYGMDV
M16-VH VH AGSISSGGY CDDGN DSSGGVIIN
M19-VH VH GFTFSNNGNF VGGSGN GPAY
M20-VH VH GYTFTSY NPSGGS VGGQEQWLDYGMDV
M15-VL VL RASQGISNYLA AASTLQS QQLNSYPLT
M16-VL VL RASQSIDRYLA DTSHRAT QQRYNWPVT
M19-VL VL QASQDISNYLD DASNLEA QQYDNNPLT

[000101] SEQUENCE LISTING

Chain Number Sequence Sequence code
HEAVY CHAIN SEQUENCES
1. Heavy Chain 1 EVQLVQSGAEVKKPGSSVKVSCKTSGDTFRTYGI TWVRQAPGQGLEWMGGIIPTFVTANYAQKFQGR VTITADESTRTAYMELSSLRSEDTAVYYCATFGH QMVRYYHYYGMD VWGQGTTVTVS S 1
2. Heavy Chain 2 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFRTYGI TWVRQAPGQGLEWMGGIIPTFVTANYAQKFQGR VTITADESTRTAYMELSSLRSEDTAVYYCATFGH QMVRYYHYYGMD VWGQGTTVTVS S 2
3. Heavy Chain 3 QMQLVQSGAEVKKPGSSVKVSCKTSGDTFRTYG ITWVRQAPGQGLEWMGGIIPTFVTANYAQKFQG RVTITADESTRTAYMLSSLRSEDTAVYYCATFGH QMVRYYHYYGMD VWGQGTTVTVS S 3
4. Heavy Chain 4 QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYY MHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELS SLRSEDTAVYYC ARV GGQEQWLD YGMD VWGQGTTVTVS S 4
5. Heavy Chain 5 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYY MHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELS SLRSEDTAVYYC ARV GGQEQWLD YGMD VWGQGTTVTVS S 5
6. Heavy Chain 6 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYY MHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELS SLRSEDTAVYYC ARV GGQEQWLD YGMD VGQGTTVTVS S 6
7. Heavy Chain 7 QVQLRQQSGPGLVKPAQTLSLTCTVSAGSISSGG YCWSWIRQLPGKGLEWIGYICDDGNTYYNPSLK 7

TRVTISADTSTNQFSLKLSSVTAADTAVYYCGRD SSGGVIINWGQGTLVTVSS
8. Heavy Chain 8 QMQLRQQSGPGLVKPAQTLSLTCTVSAGSISSGG YCWSWIRQLPGKGLEWIGYICDDGNTYYNPSLK TRVTISADTSTNQFSLKLSSVTAADTAVYYCGRD SSGGVIINWGQGTLVTVSS 8
9. Heavy Chain 9 EVQLRQQSGPGLVKPAQTLSLTCTVSAGSISSGG YCWSWIRQLPGKGLEWIGYICDDGNTYYNPSLK TRVTISADTSTNQFSLKLSSVTAADTAVYYCGRD SSGGVIINWGQGTLVTVSS 9
10. Heavy Chain 10 QVQLVQSGAEVKKPGASVTLSCKASGFTFSNNG NFAMHWLRQAPGQRPEWMGWIVGSGNINYSPT LQGRIAITSDTSANTIYMELSSLKSEDTAVYYCTK GP AYWGQGTLVTVS S 10
11. Heavy Chain 11 EVQLVQSGAEVKKPGASVTLSCKASGFTFSNNG NFAMHWLRQAPGQRPEWMGWIVGSGNINYSPT LQGRIAITSDTSANTIYMELSSLKSEDTAVYYCTK GP AYWGQGTLVTVS S 11
12. Heavy Chain 12 QMQLVQSGAEVKKPGASVTLSCKASGFTFSNNG NFAMHWLRQAPGQRPEWMGWIVGSGNINYSPT LQGRIAITSDTSANTIYMELSSLKSEDTAVYYCTK GP AYWGQGTLVTVS S 12
LIGHT CHAIN SEQUENCES
13. Light Chain 1 IRMTQSPSSLSASVGDRVTITCQASQDISNYLDWH QQKAGEAPKLLIYDASNLEAGVPSRFSGSGSGTD FTFTISSLQPEDIATYY CQQYDNNPLTFGPGTKLEIK 13
14. Light Chain 2 EIVMTQSPATLSLSPGERATLSCRASQSIDRYLAW YQQRPGQAPRLLIYDTSHRATGAPARFSGSGSGT DFTLTISNLETEDFAV YYCQQRYNWPVTFGGGTKVEIR 14

15. Light Chain 3 EIVMTQSPATLSLSPGERATLSCRASQSVSSSYLS WYQQKPGQAPRLLIYGASTRATGIPARFSGSGSG TDFTLTIS SLQPEDFAV YYCQQDYNLPPLSAEGPKWISN 15
16. Light Chain 4 DIQLTQSPSFLSASVGDRVTITCRASQGISNYLAW YQQKPGKAPKFLIYAASTLQSGVPRFSGSGSGTEF TLTISSLQPEDFASYY CQQLNSYPLTFGGGTKVEIK 16
[000102] Although the preferred embodiments of the present invention and their respective variations have been described, people having ordinary skills in the art would envision various modifications of those embodiments.
[000103] 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 appended claims.

ADVANTAGES OF THE PRESENT INVENTION
[000104] The present invention provides an isolated fully human Monoclonal Antibody
or Monoclonal Antibody fragment, Bispecific Antibody or an immuno conjugate against
coronavirus that satisfies the need of COVID-19 treatment.
[000105] The present invention provides composition comprising at least one of an
isolated fully human Monoclonal Antibody or Monoclonal Antibody fragment or an
immuno conjugate of the current invention that satisfies existing need of COVID-19
treatment.
[000106] The present invention provides a novel, specific and industrially advantageous
Monoclonal Antibody that shall be fulfilling the unmet medical need for COVID-19
prophylaxis and treatment.

Claims We claim
1. A fully human Monoclonal Antibody or Monoclonal Antibody fragment, Bispecific Antibody or an immuno conjugate having at least 90% identity to one or more sequences selected from SEQ ID No 1-16.
2. The antibody as claimed in claim 1, wherein the heavy chain sequences are selected from SEQ ID No 1-12.
3. The antibody as claimed in claim 1, wherein the light chain sequences are selected from SEQ ID No 13-16.
4. The antibody as claimed in claim 1, wherein the heavy chain sequences have at least 90% identity to one or more sequence selected from SEQ ID No 1-12 and light chain sequences have at least 90% identity to one or more light chain sequences selected from SEQ ID No 13-16.
5. The antibody as claimed in any of the preceding claim 1-4 for the manufacture of pharmaceutical composition against coronaviruses.
6. The antibody as claimed in any of the preceding claim 1-4 useful for the treatment, prevention or diagnosis of coronaviruses.
7. The antibody as claimed in any of the preceding claim 5-6, wherein the coronaviruses selected from group comprising of SARS-CoV, MERS-Cov, SARS-Cov-2, or combination thereof.
8. The antibody as claimed in any of the preceding claim 1-5, wherein the coronavirusis is preferably SARS-Cov-2.
9. A pharmaceutical composition comprising the antibody as claimed in any of the claims 1-6 and a pharmaceutically acceptable carrier.
10. The composition as claimed in claim 8, wherein the pharmaceutically acceptable carrier is selected from but not limited to, buffer, antioxidant, preservative, isotonic agent and chelating agent.
11. The antibody as claimed in any of the preceding claim 1-4, wherein the process comprising of at least one of the following steps:
a. Blood Collection
b. RNA Isolation
c. cDNA Conversion and Monoclonal Antibody Genes Amplification by PCR

d. Monoclonal Antibody Genes Cloning and scFv library banking
e. Monoclonal Antibody Selection by Bio-panning
f. Screening by ELISA
g. DNA sequencing
h. Expression & Purification in IgG Format