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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 CYTOMEGALOVIRUS
2. Applicant(s)
NAME
NATIONALITY
ADDRESS
TECHINVENTION LIFECARE PVT. LTD.
India
1004, The Summit Business Park,
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.
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FIELD OF THE INVENTION
[0001] The present invention related to biopharmaceuticals. Specifically, the present invention relates to an isolated fully human antibody or antibody fragment or an immuno conjugate against Cytomegalovirus (CMV). Further, the present invention relates to antibodies against Cytomegalovirus (CMV) or its antigen-binding fragment of the antibody, its composition and uses thereof. The present invention further relates to the use of said antibodies in the treatment or prevention or diagnosis of Cytomegalovirus infection. The invention also relates to a pharmaceutical composition comprising said antibodies for use in the treatment or prevention or diagnosis of Cytomegalovirus infection.
BACKGROUND OF THE INVENTION
[0002] The background description contains details that could be helpful in comprehending the current invention. It is not a statement of belief that any of the data presented here is previous knowledge or that it relates to the invention currently being claimed, nor it is a statement that any publication directly or implicitly cited in this application is prior knowledge. The contents of these publications are hereby included by reference into this application to more completely explain the state of the art to which this invention relates.
[0003] Cytomegalovirus (CMV) is a prevalent virus that affects individuals across the age group. By the age of 40, CMV infection affects more than half of the population (https://www.cdc.gov/cmv/overview.html). The course of primary CMV infection is typically asymptomatic or subclinical. The most frequent form of CMV infection in people with a healthy immune system is mononucleosis, which is characterized by fever, rash, and leukocytosis. Cytomegalovirus infections come in various forms. Congenital CMV is acquired by a newborn from its mother prior to birth. When someone contracts CMV for the first time, this is referred to be a primary/acquired infection. Although it typically doesn't cause symptoms, some people may exhibit symptoms that resemble mononucleosis. Reactivation of CMV infection can occur when an infection that has been dormant becomes active again because of your immune system being weak. It can happen if you have advanced HIV infection, are in treatment for cancer, or have an organ transplant. Recurring CMV is when the person already has the virus. The virus is dormant and then becomes active due to a weak immune system. Perinatal CMV infection is acquired by exposure to
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infected cervical secretions, breast milk, or blood products. Maternal antibody is thought to be protective, and most exposed term infants are asymptomatic or not infected.(https://www.webmd.com/hiv-aids/aids-hiv-opportunistic-infections-cytomegalovirus: accessed on September 20, 2023)
[0004] According to the literature study, it was also observed that CMV is a significant opportunistic infection in patients with immune systems that have been weakened, such as those caused by HIV, solid organ transplants, and bone marrow transplants. According to this article it may develop with specific organ conditions such hepatitis, pneumonitis, and colitis (Azevedo LS et.al., Clinics (Sao Paulo). 2015 Jul;70(7):515-23). People with compromised immune systems who contract CMV are said to experience more severe symptoms that can affect the eyes, lungs, liver, oesophagus, stomach, and intestines. According to the CDC, CMV can cause lung, liver, spleen, brain, and growth related problems in newborns (https://www.cdc.gov/cmv/overview.html: accessed on September 21, 2023).
[0005] According to studies, there is a high seroprevalence of CMV (up to 95%) in India, and in susceptible people such as immunocompromised patients and newborns, CMV can result in serious morbidity (Cherukat J et.al., Asian J Transfus Sci. 2021 Jan-Jun;15(1):113-114). According to reports, women of reproductive age had a prevalence of 80 to 90% of CMV IgG antibodies in various parts of India, according to serological surveys. An average of 2.0–2.5% of pregnancies have a seroconversion risk. The likelihood of intrauterine transmission is unaffected by gestational age, however the clinical outcome for infected infants seems to be worse before 20 weeks. In utero transmission can occur after either primary or recurrent infections because of latency and irregular reactivation. (Chakravarti A et.al., Indian J Med Microbiol. 2009 Jan-Mar;27(1):3-11)
[0006] Compared to recurrent infections (0.15 to 1%), primary infections are transmitted more frequently (15 to 50%) and are more likely to harm the fetus. Ninety percent of newborns with congenital infections are asymptomatic at birth, and 5 to 17% of them go on to experience symptoms including sensorineural hearing loss, chorioretinitis, or neurologic deficits typically during the first two years of life. 10% of neonates with symptoms die, and 90% of those who survive experience severe sequelae. More than 90% of newborns who survive CMV illness suffer late complications, and mortality can reach 30% (Chakravarti A et.al., Indian J Med Microbiol. 2009 Jan-Mar;27(1):3-11).
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[0007] According to a literature review, during primary CMV infection, the host develops antibodies (first IgM and then IgG) that are directed specifically against a number of CMV proteins, including structural tegument proteins, envelope glycoproteins, and nonstructural proteins like the IE1 protein. This humoral immunity is essential for limiting viral spread and possibly helps to reduce the disease's clinical symptoms. IgM antibodies can't pass placental barriers, thus they can't shield the fetus from the infection's clinical effects (Satyen Yash Sanghavi et al., Journal of Clinical and Diagnostic Research. 2019 Nov; 13(11) 10-13). [0008] When HCMV enters the body, a range of immunological reactions are encoded. The HCMV gene targets MHC class I and class II molecules for antigen presentation, uses cytokine mimicry to exert paracrine effects on immune cells, and encodes proteins that counteract a variety of innate immune responses meant to combat the virus. Accordingly, HCMV is necessary for long-term persistence in the host but insufficient to entirely avoid immunosurveillance (Griffiths P et.al., Nat Rev Microbiol. 2021 Dec;19(12):759-773).
[0009] The need for the emergence of monoclonal antibodies is widespread. This is due to reports stating that a pentamer complex made up of gH/gL and the UL128/130/131 locus (UL128L) gene products is necessary for HCMV entrance into epithelial/endothelial cells. Plaque development results from HCMV infection of epithelial cells at low multiplicity of infection (MOI). It was established that, unlike MAbs to gH or gB, all monoclonal antibodies (MAbs) directed to UL128L components of the pentamer were able to suppress plaque formation. The same MAbs produced comparable outcomes when employed to prevent virus transfer from infected endothelium cells to leukocytes (Gerna G et.al., J Virol. 2016 Jun 24;90(14):6216-6223).
[00010] There are no monoclonal antibodies currently available in the market for the treatment for CMV infection. Most of the treatment is done by prescribing Anti-CMV drugs that target the viral DNA polymerase like Ganciclovir. Its antiviral activity requires phosphorylation by the CMV protein kinase, pUL97. Cidofovir is a nucleotide analog, which is already phosphorylated and active. Valganciclovir is extensively used for cytomegalovirus (CMV) infection treatment and prophylaxis after solid organ transplantation (SOT). Foscarnet has a different mode of action as it directly inhibits polymerase function by blocking the pyrophosphate binding site of pUL54 (Huntjens DW et.al., Pharmaceutics. 2023 Jan 3;15(1):163)). The first-line
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options for therapy of CMV disease, as mentioned above, are almost always intravenous administration of Ganciclovir or oral administration of Valganciclovir.
[00011] Based on an assessment of the literature on earlier unsuccessful clinical trials with different Monoclonal Antibodies. The information about a handful of these MAbs is provided herein. The hMSL-109 MAbs was designed to target the HCMV surface glycoprotein. RG7667 was capable of neutralising HCMV infection in all cell types. CSJ148 was composed of two anti-HCMV human monoclonal antibodies (LJP538 and LJP539). Each antibody was able to bind to and suppress the activity of the pentameric complex (containing of the glycoproteins gH, gL, UL128, and UL131) and glycoprotein B (gB), two important viral glycoproteins. These monoclonal antibodies tailored against CMV infection, however, were ineffective. (Struble EB et.al., Int J Mol Sci. 2021 Aug 13;22(16):8728))
[00012] Although there are different companies working on CMV treatment methods, there is still need to explore the novel ways and compositions of treating CMV that could be easily manufactured and scalable and can overcome deficiencies associated with the known arts. There is an emergence to discover and develop Novel Antibodies domestically. These Human Monoclonal Antibodies is also more sensitive, efficacious and specific to the target site such as cell wall, fragment and sub units. Also the fully human monoclonal antibodies would provide immediate relief by neutralizing the antibodies. The present invention provides a novel, specific and industrially scalable antibody of CMV infection. These antibodies shall be fulfilling the unmet medical need for CMV prophylaxis and treatment.
OBJECTS OF THE INVENTION
[00013] An object of the present invention is to provide an isolated fully human monoclonal antibodies or antibody fragment, bispecific antibody or an immuno conjugate against CMV glycoprotein.
[00014] Another object of invention is to provide an isolated fully human monoclonal antibodies or fully human monoclonal antibody fragments, bispecific antibody or an immuno conjugate against CMV that bind to glycoprotein selected from gH, gL, gB, gO, gM/Gn and pUL128-131.
[00015] Another object of the present invention is to provide Naïve ScFv phage display antibody libraries against CMV glycoprotein.
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[00016] Still another object of the present invention is to provide fully human monoclonal antibody or antibody fragment comprising heavy chain selected from SEQUENCE ID No. 1-16 and light chain selected from SEQUENCE ID No. 17-36.
[00017] Another object of the present invention is to provide a composition comprising fully human monoclonal antibodies or antibody fragment comprising heavy chain selected from SEQUENCE ID No. 1-16 and light chain selected from SEQUENCE ID No. 17-36.
[00018] An object of the present invention is to provide composition comprising fully human monoclonal antibody and antibody fragment against CMV further containing a pharmaceutically acceptable carrier, diluent or adjuvant.
[00019] Another object of the present invention is to provide a method for the preparation of fully human monoclonal antibody and antibody fragment against CMV glycoprotein.
[00020] Another object of the present invention is to provide fully human monoclonal antibodies for the treatment of congenital, perinatal, primary/acquired CMV infection
[00021] Yet another another object of the present invention is an isolated fully human antibody or antibody fragment, an immuno conjugate and/or the pharmaceutical composition of the invention for use in the prevention or treatment or diagnosis of CMV infection.
[00022] Another object of the present invention is to provide a fully human monoclonal antibody composition having the above detailed monoclonal antibody in combination with a second monoclonal antibody which binds simultaneously to glycoprotein for neutralizing the CMV glycoprotein.
SUMMARY OF THE INVENTION
[00023] 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.
[00024] In one aspect of the present invention is to provide an isolated fully human monoclonal antibodies or antibody fragment, bispecific antibody or an immuno conjugate against CMV glycoprotein.
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[00025] Another aspect of the present invention is to provide an isolated fully human monoclonal antibodies or fully human monoclonal antibody fragments, bispecific antibody or an immuno conjugate against CMV that bind to glycoprotein selected from gH, gL, gB, gO, gM/Gn and pUL128-131.
[00026] Another aspect of the present invention is to provide Naïve ScFv phage display antibody libraries against CMV glycoprotein.
[00027] Still another aspect of the present invention is to provide fully human monoclonal antibody or antibody fragment comprising heavy chain selected from SEQUENCE ID No. 1-16 and light chain selected from SEQUENCE ID No. 17-36.
[00028] In one aspect of the present invention is to provide a composition comprising fully human monoclonal Antibodies or antibody fragment comprising heavy chain selected from SEQUENCE ID No. 1-16 and light chain selected from SEQUENCE ID No. 17-36.
[00029] Still another aspect of the present invention is to provide composition comprising fully human monoclonal antibody and antibody fragment against CMV further containing a pharmaceutically acceptable carrier, diluent or adjuvant.
[00030] Further another aspect of the present invention is to provide a method for the preparation of fully human monoclonal antibody and antibody fragment against CMV glycoprotein.
[00031] Another aspect of the present invention is to provide fully human monoclonal antibodies for the treatment of congenital, perinatal, primary/acquired CMV infection.
[00032] Another object of the present invention is to provide a fully human monoclonal antibody composition having the above detailed monoclonal antibody in combination with a second monoclonal antibody which binds simultaneously to glycoprotein for neutralizing the CMV glycoprotein.
[00033] In one aspect of the present invention is to provide an isolated fully human antibody or antibody fragment, an immuno conjugate and/or the pharmaceutical composition of the invention for use in the prevention or treatment or diagnosis of the infection of CMV.
DRAWINGS
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
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understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
Figure 1A-1D : represents the Phage ELISA plate Results performed for approximate 400 clones wherein the yellow color indicates a positive result for binders.
Figure 2A-2B : represents the Repeat Phage ELISA plate results of 132 Clones wherein the yellow color indicates a positive result for binders .
Figure 3A-3B: represents the results of Agarose Gel Electrophoresis after restriction digestion wherein the presence of monoclonal antibody gene within the vector is observed.
DETAILED DESCRIPTION OF THE INVENTION
[00034] 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.
[00035] 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.
[00036] 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.
[00037] 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,
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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. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
[00038] 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.
[00039] Also, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.
[00040] 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."
[00041] 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.
[00042] 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
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for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[00043] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[00044] 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.
[00045] The term "monoclonal antibody”,” antibody” or “MAbs “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. Fully human monoclonal antibodies have high degree of specificity for single antigen. In addition, different from the polyclonal antibody preparations.
[00046] Fully human monoclonal antibodies of the invention include, but are not limited to, synthetic antibodies, fully human monoclonal antibodies, recombinant antibodies, multi-specific fully human monoclonal antibodies (including bi-specific 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 fully human 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.
[00047] 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.
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[00048] The term “fully human monoclonal antibody” or “human monoclonal antibody” according to the present invention means, but not limited to, refers to a fully human monoclonal antibody that comprises a human variable region and, most preferably a human constant region. In specific embodiments, the terms refer to a fully human monoclonal antibody that comprises a variable region and constant region of human origin.
[00049] 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 fully human monoclonal antibodies of the invention are isolated or purified.
[00050] 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.
[00051] The term “Bio-panning” according to the present invention means, but not limited to, refers to an affinity selection technique which selects for peptides that bind to a given target. This technique is often used for the selection of antibodies.
[00052] In one embodiment of the present invention is to provide an isolated fully human monoclonal antibodies or antibody fragment, bispecific antibody or an immuno conjugate against CMV glycoprotein.
[00053] In one embodiment the present invention related to preparation of Single chain fragment variable (ScFv) naïve antibody library wherein the targets of antigen specific phages where CMV glycoproteins such as gH, gL, gB, gO, gM/Gn, pUL128-131.
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[00054] In one embodiment of the present invention is to provide fully human monoclonal antibody or antibody fragment comprising heavy chain selected from SEQUENCE ID No. 1-16 and light chain selected from SEQUENCE ID No. 17-36.
[00055] In another embodiment of the present invention is to provide a composition comprising fully human monoclonal Antibodies or antibody fragment comprising heavy chain selected from SEQUENCE ID No. 1-16 and light chain selected from SEQUENCE ID No. 17-36.
[00056] In one of the embodiment the present invention provides a method of production (Bio-panning method, recombinant phage production & specific ELISA) of antigen specific phages against Cytomegalovirus (CMV), said method comprises use of host cells selected from, cells of mammalian, plant, insect, fungal or bacterial origin. Host cells of the present invention include eukaryotic cells from 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 fully human monoclonal antibodies and other binding proteins.
[00057] In one embodiment the present invention related to preparation of Single chain fragment variable (ScFv) naïve antibody library wherein the targets for these antigen specific phages are CMV glycoproteins.
[00058] In one embodiment of the present invention is the preparation of ScFv naïve antibody library by isolation of lymphocytes and RNA using genome extraction kit
[00059] Another aspect of the present invention states using highly specific primers that amplify the heavy and light chains of CMV glycoproteins on the CMV monoclonal antibodies.
[00060] In another embodiment of the present invention provides the ScFv antibody library prepared by performing gene amplification through molecular biology techniques such as Primary and Secondary PCR and sequencing.
[00061] In another embodiment of the present invention provides antigen specific phages from the ScFv Antibody Library by performing bio panning method multiple times for screening of fully human monoclonal antibodies against CMV glycoprotein.
[00062] In another embodiment of the invention the antigen specific phages containing the fully human monoclonal antibodies specific to glycoprotein where then maintained in glycerol stock.
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[00063] In another embodiment antigen specific phages were confirmation by plating the colonies and then inoculating into ELISA pates. The positive binders from primary phage ELISA were reconfirmed by repeat phage ELISA.
[00064] In another embodiment of the invention, further analysis was done by determining the OD of the ELISA positive binders, upon restriction digestion, which were then selected for antibody gene sequencing.
[00065] Further embodiment of the invention is the isolation of the plasmid of interest from the phages (Phagemids or plasmids) containing the fully human monoclonal antibody genes and then subjecting it to restriction digestion using restriction enzymes specific to the desired fully human monoclonal antibody genes.
[00066] In another embodiment of the invention the screened and digested fully human monoclonal antibody genes were loaded into gel and checked under UV light for the presence of bands of desired antibody gene fragment.
[00067] In another embodiment of the present invention is to provide composition comprising fully human monoclonal antibody and antibody fragment against CMV further containing a pharmaceutically acceptable carrier, diluent or adjuvant.
[00068] In one embodiment of the present invention is to provide isolated fully human antibody or antibody fragment, an immuno conjugate and/or the pharmaceutical composition for the treatment of congenital, perinatal, primary/acquired CMV infection.
[00069] Another embodiment of the present invention is to provide a fully human monoclonal antibody composition having the above detailed monoclonal antibody in combination with a second monoclonal antibody which binds simultaneously to glycoprotein for neutralizing the CMV glycoprotein.
[00070] In another embodiment of the present invention, provide use of fully human monoclonal antibodies for the diagnosis of the CMV based infection in patients that show the symptoms of the presence of CMV.
[00071] Furthermore, another embodiment of the present invention is detection via the fully human monoclonal antibodies as an additional method to the laboratory SOPs that is normally used to detect the presence of CMV.
[00072] Additional embodiment of the present invention is the use of the fully human monoclonal antibodies in the prevention of appearance of the symptoms related to CMV infection by create an immunity against the CMV virus reinfection.
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EXAMPLES
[00073] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
[00074] Step 1: Bio panning for primary screening to produce ScFv Antibody Library
1. The first step in making antibody libraries is isolation of lymphocytes from human blood, followed by total RNA isolation, primary & secondary PCR for antibody gene amplification.
2. The amplified antibody gene sequences are than restriction digested using NcoI/HindIII and Mlu/NotI, and cloned into appropriate vector such as pSEX vector.
3. The cloned vector is transformed into XL-I blue bacterial host to generate the phage library. Glycerol stocks of the phage library are prepared for further use and long-term storage.
4. The immunotube used for the bio panning was coated with gB antigen and the phages prepared from the library were added on to it.
5. The unbound phages that are not specific to the antigen gB are removed and the bound antigen specific phages were eluted
6. The stringency of the Bio-panning method was done by reducing the concentration of the coated antigen on the immunotube. The concentration of the gB antigen where 10 μg/ml in round 1, 7.5 μg /ml in round 2 and 5 μg/ml in round 3.
[00075] Step 2: Secondary screening of the antigen specific phages/ binders/ clones by ELISA
1. The antigen specific phage preparation that was isolated by bio panning method are checked for specificity by ELISA. The concentration of gB antigen was 300 ng /100μl in 1xPBS per well coated on ELISA plate along with the positive control of M13KO7 as 109 and 1010 particles/ml and negative control as 1xPBS.
2. Approximate 400 clones were screened by Phage ELISA and got 132 clones as a positive binder against gB antigen of CMV (Fig. 1A-1D). These clones were labelled as gb-NL-C1 to gb-NL-C132
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3. The stock of the antigen specific phages as well as E. Coli clone stocks was frozen stored in the form of glycerol stock. A small quantity of the frozen stock was diluted and plated to form isolated colonies.
4. The isolated colonies where than screened by performing repeat ELISA (Fig. 2A-2B). In repeat phage ELISA, appx 103 clones were successfully got repeated.
5. Out of these 103 repeat phage ELISA clones of gB, 60 clones were selected for plasmid isolation on the basis of highest O.D. and restriction digestion was done for 20 clones to check the presence of plasmid and gene of interest before sending for sequencing.
[00076] Step 3: Plasmid isolation and Restriction digestion of clones obtained by secondary screening
1. After analyzing sequencing data of 60 clones through bioinformatics tools, we got 16 Heavy chains and 20 Light chains (one light chain sequence is repeating) that were novel.
2. The plasmid containing the sequence of interest were isolated and then subjected to restriction digestion using NcoI/NotI restriction endonucleases.
3. The cleaved plasmid was then loaded into the gel wells and checked for the presence of the cloned gene on gel doc (Fig. 3A-3B).
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SEQUENCE LISTING
SEQ NO
AMINO ACID SEQUENCE
SEQ ID NO
Heavy Chain 1
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIHEVSNRFSGVSHRFSGSGSGTDFTLKISRVEAEDVGLYHCMQGLQFPRTFGQGTKLEIK
1
Heavy Chain 2
DIQMTQSPSSLSASIGDRVTITCRASQSISTYLNWYQQKPGKAPQLLIYGASSLQSGVPSRFAGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK
2
Heavy Chain 3
QSVLTQPPSLSVSPGQTVSITCSGDKLGAKYVSWYQQKSGQPPTLVIYQNTERPSGLPERFSGSNSGTTATLTISGTLSVDEADYYCQAWDSTTYVFGTGTKVTVL
3
Heavy Chain 4
SELTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEAEYSCSSFTTNTQWVFGGGTQVTVL
4
Heavy Chain 5
DFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSIWVFGGGTKLTVL
5
Heavy Chain 6
QSALTQPASVSESPGKTVTISCTRSSGRIASYYVQWYQQRPGSAPTTVIFEDNQRPSGVPDRFSGSIDSSPNSASLTISGLKTEDEAVYYCQSYDSSNHNWVFGGGTKLTVL
6
Heavy Chain 7
DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQLRPGQSPRRLIYQVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCIQGTHWPGIFGQGTKLEIK
7
Heavy Chain 8
SELTQPASVSVALGQTAKIPCGGNNIGSRNVHWYQKKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWNSNSDHHVVFGGGTKLTVL
8
Heavy Chain 9
SELTQPASVSVAPGQTASISCEGNNIGTKGVHWYQQNPGQAPVLVVFDGGDRPSGIPERFSGSSSGNMATLIISRVEAGDEADYYCQVWDSNSNHVFGTGTKVTVL
9
17
Heavy Chain 10
SYELTQPPSASGSPGQSVTISCTGTSSDVGAYNYVSWYQQHPGKAPKLIIYEVTKRPSGVLDRFSGSKSGTSASLAISGLQSEGDAHYYCATYDDSLQSYVFGTGTKVAVL
10
Heavy Chain 11
DIVMTQSPLSLPVTLGQPASISCRSNQSLLHSSGYTYLNWFHQRPGQSPRRLIYKVSNRDSGVPDRFRGSGSGTAFTLEFSRVEAEDLGVYYCMQGTHWPRTFGQGTKLEIK
11
Heavy Chain 12
NFMLTQPHSVSESPGKTVIISCTRSSGNIASNYVQWYQLRPGNAPTTVIYEDNQRPSGVPDRFSGSIDTSSNSAFLTISGLKTEDEADYYCQSYDSSNWVLGGGTKLTVL
12
Heavy Chain 13
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIHEVSNRFSGVSHRFSGSGSGTDFTLKISRVEAEDVGLYHCMQGLQFPRTFGQGTKLEIK
13
Heavy Chain 14
DIQMTQSPSSLSASIGDRVTITCRASQSISTYLNWYQQKPGKAPQLLIYGASSLQSGVPSRFAGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK
14
Heavy Chain 15
QSVLTQPPSLSVSPGQTVSITCSGDKLGAKYVSWYQQKSGQPPTLVIYQNTERPSGLPERFSGSNSGTTATLTISGTLSVDEADYYCQAWDSTTYVFGTGTKVTVL
15
Heavy Chain 16
SELTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEAEYSCSSFTTNTQWVFGGGTQVTVL
16
Light Chain 3
DFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSIWVFGGGTKLTVL
17
Light Chain 4
QSALTQPASVSESPGKTVTISCTRSSGRIASYYVQWYQQRPGSAPTTVIFEDNQRPSGVPDRFSGSIDSSPNSASLTISGLKTEDEAVYYCQSYDSSNHNWVFGGGTKLTVL
18
Light Chain 5
DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQLRPGQSPRRLIYQVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCIQGTHWPGIFGQGTKLEIK
19
18
Light Chain 6
SELTQPASVSVALGQTAKIPCGGNNIGSRNVHWYQKKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWNSNSDHHVVFGGGTKLTVL
20
Light Chain 7
SELTQPASVSVAPGQTASISCEGNNIGTKGVHWYQQNPGQAPVLVVFDGGDRPSGIPERFSGSSSGNMATLIISRVEAGDEADYYCQVWDSNSNHVFGTGTKVTVL
21
Light Chain 8
SYELTQPPSASGSPGQSVTISCTGTSSDVGAYNYVSWYQQHPGKAPKLIIYEVTKRPSGVLDRFSGSKSGTSASLAISGLQSEGDAHYYCATYDDSLQSYVFGTGTKVAVL
22
Light Chain 9
DIVMTQSPLSLPVTLGQPASISCRSNQSLLHSSGYTYLNWFHQRPGQSPRRLIYKVSNRDSGVPDRFRGSGSGTAFTLEFSRVEAEDLGVYYCMQGTHWPRTFGQGTKLEIK
23
Light Chain 10
NFMLTQPHSVSESPGKTVIISCTRSSGNIASNYVQWYQLRPGNAPTTVIYEDNQRPSGVPDRFSGSIDTSSNSAFLTISGLKTEDEADYYCQSYDSSNWVLGGGTKLTVL
24
Light Chain 11
DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIHEVSNRFSGVSHRFSGSGSGTDFTLKISRVEAEDVGLYHCMQGLQFPRTFGQGTKLEIK
25
Light Chain 12
DIQMTQSPSSLSASIGDRVTITCRASQSISTYLNWYQQKPGKAPQLLIYGASSLQSGVPSRFAGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK
26
Light Chain 13
QSVLTQPPSLSVSPGQTVSITCSGDKLGAKYVSWYQQKSGQPPTLVIYQNTERPSGLPERFSGSNSGTTATLTISGTLSVDEADYYCQAWDSTTYVFGTGTKVTVL
27
Light Chain 14
SELTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEAEYSCSSFTTNTQWVFGGGTQVTVL
28
Light Chain 15
DFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSIWVFGGGTKLTVL
29
Light Chain 16
QSALTQPASVSESPGKTVTISCTRSSGRIASYYVQWYQQRPGSAPTTVIFEDNQRPSGVPDRFSGSIDSSPNSAS
30
19
LTISGLKTEDEAVYYCQSYDSSNHNWVFGGGTKLTVL
Light Chain 17
DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQLRPGQSPRRLIYQVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCIQGTHWPGIFGQGTKLEIK
31
Light Chain 18
SELTQPASVSVALGQTAKIPCGGNNIGSRNVHWYQKKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWNSNSDHHVVFGGGTKLTVL
32
Light Chain 19
SELTQPASVSVAPGQTASISCEGNNIGTKGVHWYQQNPGQAPVLVVFDGGDRPSGIPERFSGSSSGNMATLIISRVEAGDEADYYCQVWDSNSNHVFGTGTKVTVL
33
Light Chain 20
SYELTQPPSASGSPGQSVTISCTGTSSDVGAYNYVSWYQQHPGKAPKLIIYEVTKRPSGVLDRFSGSKSGTSASLAISGLQSEGDAHYYCATYDDSLQSYVFGTGTKVAVL
34
Light Chain 21
DIVMTQSPLSLPVTLGQPASISCRSNQSLLHSSGYTYLNWFHQRPGQSPRRLIYKVSNRDSGVPDRFRGSGSGTAFTLEFSRVEAEDLGVYYCMQGTHWPRTFGQGTKLEIK
35
Light Chain 22
NFMLTQPHSVSESPGKTVIISCTRSSGNIASNYVQWYQLRPGNAPTTVIYEDNQRPSGVPDRFSGSIDTSSNSAFLTISGLKTEDEADYYCQSYDSSNWVLGGGTKLTVL
36
[00077] 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.
[00078] 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.
20
ADVANTAGES OF THE INVENTION
[00079] The present invention focuses on providing a composition of fully human monoclonal antibodies that is reproducible and scalable.
[00080] The present invention also has a composition of fully human monoclonal antibodies that is sensitive and specific when interacting with target sites on CMV glycoprotein.
[00081] The present invention also provides a method for the production of fully human monoclonal antibodies that is consistent when maintained in simultaneous production batches.
[00082] The present invention also provides for a novel and industrially advantageous fully human monoclonal antibodies that has the ability of neutralization of cytomegalovirus (CMV).
[00083] The present invention also provides the use of fully human monoclonal antibodies that shall also fulfill the unmet need for CMV treatment, diagnosis and prevention for various types of CMV infection.
Dated: October 28, 2024
For TECHINVENTION LIFECARE PVT LTD
Aasiya Choudhary
21
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 from SEQUENCE ID No. 1-36.
2. The antibody as claimed in claim 1, wherein the heavy chain sequences are selected from SEQ ID No 1-16.
3. The antibody as claimed in claim 1, wherein the light chain sequences are selected from SEQ ID No 17-36.
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 1-16 and light chain sequences have at least 90% identity to one or more light chain sequences selected from SEQ ID No 17-36.
5. The antibody as claimed in any of the preceding claim 1-4 for the manufacture of pharmaceutical composition against Cytomegalovirus.
6. The antibody as claimed in any of the preceding claim 1-4 useful for the treatment, prevention or diagnosis of Cytomegalovirus.
7. The antibody as claimed in any of the preceding claim 1-5, wherein the cytomegalovirus is selected from the Herpesviridae family.
8. A pharmaceutical composition comprising the antibody as claimed in any of the claims 1-6 and a pharmaceutically acceptable carrier.
9. 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.
10. 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. Monoclonal Antibody Genes Amplification by PCR
d. Monoclonal Antibody Genes Cloning
e. Monoclonal Antibody Gene Selection by Bio-panning
f. Screening by ELISA