DESC:

RELATED PATENT APPLICATION(S)
This application claims the priority to and benefit of Indian Patent Application No. 201841020522 filed on November 30, 2018; the disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION
The invention relates to biotechnology, particularly biomedical research or immunotherapy. The present disclosure relates to the field of recombinant proteins for use in treating diseases. More specifically, the invention relates to chimeric vaccine comprising human growth hormone sequences and carrier protein encoding the same and methods for expression and using the same as an immunotherapeutic vaccine either alone or in combination with adjuvants to treat Non Small Cell Lung Cancer and other EGF dependant malignant diseases such as breast, bladder, ovarian, vulvia, colon, pulmonary, brain and oesophagus cancers.

BACKGROUND OF THE INVENTION
Cancer is a leading cause of deaths worldwide. Among all cancers, lung cancer is the most common and deadliest cancer causing approximately 1.6 million deaths accounting 19.4% of the total deaths per year, worldwide (World Health Organization. Lyon, France: International Agency for Research on Cancer; Feb 3, 2014. Press Release No. 224. Available at: http://www.iarc.fr/en/media-centre/pr/2014/pdfs/pr224_E.pdf). In 2017, it is estimated that 1,55,870 deaths occurred due to Lung cancer, accounting for 1 in 4 of all cancer deaths (American Cancer Society. Cancer facts & Figures 2017). In India, lung cancer constitutes 6.9% and 9.3% of cases & deaths respectively from all cancer related deaths in both sexes. Lung cancer classified into two main types, small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), accounting for 15% and 85%, respectively (Sher T et al., Mayo Clin Proc 2008;83:355-67. 10.4065/83.3.3554). NSCLC is further classified into three types: squamous-cell carcinoma, adeno carcinoma, and large-cell carcinoma, in which frequency of EGFR mutations can range from 5-17%.

Over 30 years of intensive research by several investigators, has led to major advancement in the treatment of NSCLC which includes surgery, radiation, chemotherapy, targeted therapy and immunotherapy either alone or in combination. Irrespective of the treatment strategies used, only 5% of the patients have shown maximum upto 5-year survival rate and 35% of the patients with wild type tumors have shown only 1-year survival rate (DeSantis CE et al., 2014. CA Cancer J Clin 2014; 64: 252–71).

Approximately 40% to 80% of NSCLCs, is due to over expressed Epidermal Growth Factor Receptor (EGFR) (Charles N. Prabhakar (2015) Epidermal growth factor receptor in non-small cell lung cancer, Transl Lung Cancer Res. 2015 Apr; 4(2): 110–118). The epidermal growth factor receptor (EGFR) is a transmembrane protein and acts as a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands such as Epidermal Growth Factor (EGF), Transforming Growth Factor (TGF), heparin binding growth factor etc. Though, the exact role of EGF on EGFR is completely unknown, it seems that EGFR together with ligands (EGF), specifically regulates the cellular growth. Due to mutations in EGFR, it triggers inappropriate activation and promotes the uncontrolled cell growth (Avraham R et al., Nat Cancer Mol Cell Biol 2011; 12:104–17.), which in turn results into poor prognosis, lower survival, and resistance to therapy (Hirsch FR et al., J Clin Oncol 2003;21: 3798–807).

One of the approaches to reduce, the uncontrolled growth is to reduce the availability of EGF to EGFR, by producing high titer anti-EGF antibodies against autologous huEGF. In this approach, available EGF in serum binds to anti-EGF antibodies raised against EGF based therapeutic vaccine, thereby it decreases the binding of EGF to the receptor (EGFR), which further prevents the cell proliferation. However, the inherent problem associated with making an anti-EGF based vaccine lies in the fact that it is autologous and present in the human biological system and therefore no anti-EGF antibody (Ab) response is possible. Hence, to prepare an EGF based vaccine formulation, the selection of a carrier protein(s) is essential so that the EGF breaks the self-tolerance and gets recognized as a vaccine antigen by the immune system. Hence, EGF based therapeutic vaccine made, which elicits antibodies against EGF, that blocks EGFR, so as to prevent cell growth and to treat lung cancer. The said therapeutic vaccine is composed of human recombinant EGF conjugated to P64 carrier protein derived from Neisseria meningitidis and emulsified in Montanide ISA51 (US 2013/0039940 A1; US 2010/0196412 A1). Early trials with this EGF-P64 based vaccine (commercially termed as ‘CIMAVAX-EGF’) did show an improved survival rate to some extent in vaccinated subjects, wherein a direct correlation was established between the proportion of antibodies raised in a patient and survival thereof. (Rodríguez, P. C.; Rodríguez, G.; González, G.; Lage, A. (2010). "Clinical development and perspectives of CIMAvax EGF, Cuban vaccine for non-small-cell lung cancer therapy". MEDICC Review. 12 (1): 17–23. PMID 20387330).

However, the problem with the chemical conjugation approach may results in a heterologous conjugation mixture. The yield of final purified homogenous mixture could be less and thereby it may not be feasible to meet the demands of multiple dose requirements to treat cancer patients as well as to produce the vaccine commercially.

Therefore, in the present invention, EGF based chimeric proteins were generated using recombinant DNA technology, using immunopotentiator, flagellin as a carrier protein, which can not only enables human epidermal growth factor to be recognized by the human immune system, but also can acts as an innate immune modulator after binding to TLR5 (Toll Like Receptor 5), present on the Immune cells. Moreover, TLR5 have been demonstrated to possess anti-cancer properties along with the ability to enhance immune response compared to other TLRs or NLRs or CLRs. Specifically, Toll Like Receptor 5 (TLR5) agonists are favourably positioned as potential systemic immunotherapeutic agents because of unusual tissue specificity of expression, uniquely safe profile of induced cytokines, and antitumor efficacy demonstrated in a number of animal models (Li-Fen Lee et al., J Immunol 2000, 164: 2769-2775; Sang Hoon Rhee et al., Gastroenterology 2008, 135:518–528.). In addition to it, newer adjuvants in combination with the vaccine antigen may also be desirable.

Furthermore, as a part of cancer treatment, it is also suggested to replace systemic chemotherapeutic treatments with specific target-based therapies in combination with a therapeutic vaccine against lung cancer. Inventors of the present invention describe the use of recombinant chimeric protein of human epidermal growth factor and its vaccine composition with or without adjuvants as a therapeutic vaccine. The vaccine has been tested in a mice tumor model and proved that vaccine composition slows down the progression of a tumor, while increasing the survival period for at least 1 week. Further, this invention also discloses that the use of vaccine composition, immediately after 1 cycle of TKI (tyrosine kinase inhibitor-based EGFR targeted therapy) treatment, increased survival rate by 30% in tumor induced mice model. Hence, the inventors of this patent application have generated a chimeric protein comprising human recombinant EGF and full length or truncated flagellin protein, that is expressed in E. coli wherein, flagellin tends to impart both adjuvant activity along with anti-tumorigenic activity to the said chimeric protein of recombinant human epidermal growth factor. Flagellin is a bigger molecule by itself and chimeric attachment of flagellin to human epidermal growth factor as a single molecule enables the human immune system to recognize EGF as a foreign vaccine antigen and thereby generates anti-EGF antibodies to treat non-small cell lung cancer in humans.

OBJECTIVES OF THE INVENTION
An important objective of this invention is to generate chimeric protein comprising autologous huEGF, so as to produce anti-EGF antibodies against huEGF.

The object of the present invention is to provide anti-EGF antibodies (antibodies against autologous human growth factor), to make EGF (human growth factor) less available for the EGFR to bind and thereby to prevent cell proliferation.

Another object of the present invention is to provide more effective methods of vaccine preparation through the use of autologous growth factor by using an effective carrier protein and by increasing the number of copies of growth factor or EGF, by which immune system is able to recognize huEGF as an immunogen and stimulate immune cells to produce anti-EGF antibodies.

Yet another objective of the invention is to identify new carrier protein which has immunopotentiating property or adjuvant property for the purpose of generating chimeric protein in fusion with human EGF protein.

One of the objectives of the present invention is to generate chimeric proteins containing EGF and full length or truncated flagellin protein, which function both as a carrier protein & immunopotentiator.

Another objective is also to increase the anti-EGF antibodies, by increasing the number of EGF copies in chimeric fusion.

Another objective of the present invention is to evaluate adjuvant and anti-tumorigenic activity of flagellin or fusion proteins containing full length or truncated flagellin.

Yet another objective of the invention is to provide a vaccine composition with or without adjuvant(s) or any applicable delivery system for prophylaxis and/or therapeutic treatment against non-small cell lung cancer wherein EGF is to be used as a vaccine antigen through chimeric approach with novel immunopotentiators.

Another objective of the invention is to determine the efficacy of the chimeric protein as a therapeutic vaccine, while increasing the survival rate.

Additionally, it is also the objective of the present invention to enable combination of targeted therapies to treat non-small lung cancer along with the therapeutic vaccine of the present invention negating the use of systemic chemotherapeutic drugs.

SUMMARY OF THE INVENTION
The present disclosure is directed towards the construction, expression, purification and the characterization of the recombinant chimeric proteins and their respective methods to assess or evaluate the functional properties. Further, the present invention also discloses therapeutic methods of using the recombinant chimeric proteins to treat chronic diseases, such as, for example, lung, breast, head and neck, bladder, prostate, ovarian, vulva, colonic, colorectal, intestinal, pulmonary, brain, oesophagal and other cancers, in which EGF plays an important role to promote cell growth.

In an illustrative embodiment, the recombinant chimeric protein is an immunogenic protein molecule comprising one or more sequences that fold into one physical structure that exposes desired epitopes. For example, chimeric protein expressing full length or truncated sequence of a carrier protein and expressing one or more copies or sequences of autologous human epidermal growth factor or alternate self antigens and thereof that induces high titer anti-growth factor or anti-EGF antibodies, after being accessible to cells of the immune system. It is contemplated within the scope of the disclosure that growth factors may be selected from human origin and the said sequence can contain functional parts thereof.

In these illustrative embodiments, the sequence of human growth factors or the alternate self antigens are codon optimized for better expression in E. coli and they are genetically synthesized.

In these illustrative embodiments, the expression of one or more copies or sequence of the growth factor(s) can be present at different positions within the sequence of the recombinant chimeric protein, for example, one copy of growth factor present at N-terminus or C- terminus of the carrier protein and another copy of the growth factor have been positioned at the centre of the flagellin, by replacing the hyper variable regions (D2 & D3), or at N- or C- terminus or in place of D3 region itself.

In an illustrative embodiment, the sequence of the growth factor may include a sequence of one or more of the following growth factors, and/or alternative self-antigens such as, but not limited to, EGF, IGF-1, IGF-2. FGFs 1-23, TGF-a, TGF-ß, VEGF-A, VEGF-B, VEGF-C, VEGF-D, PDGF, NGF, EGF, HGF and ILs 1-7.

In other illustrative embodiments, the sequence of carrier protein may include full length or truncated flagellin and it is synthetic in origin, either taken from Gram positive bacteria including without limitation to Bacillus subtilis, Clostridium difficile or ????proteobacteria such as Salmonella typhimurium or Enterica species or ??proteobacteria such as Bordetella.

In the present disclosure, the resultant chimeric protein may be a single polypeptide expressing a one or more copies of growth factor or self antigens thereof within the sequence of the carrier protein. In an illustrative embodiment, the sequence of the recombinant protein expresses both huEGF and full length or truncated flagellin and presents the EGF on a surface of the recombinant protein, as it was recognized by the anti-EGF antibodies via western blot technique.

Further embodiment of the invention describes the method of protein purification comprising cell lysis, denaturation & refolding followed by self-cleavage due to temperature & pH shift, subsequently, further purification techniques such as ion exchange chromatography, size exclusion chromatography or affinity chromatography via chitin binding domain resin or Hydrophobic interaction chromatography or chromatography technique using mixed mode resins etc.

It further illustrates the evaluation of adjuvant property, anti-tumorigenic property of carrier protein i.e full length or truncated flagellin of chimeric protein, by in-vitro assays. Further, it also illustrates the characterization of chimeric protein in terms of molecular weight, EGF site recognition and stability.

The further embodiments of the invention also established that the immunogenicity of the chimeric proteins in C57BL/C mice. The protein sequences of SEQ IDs 6 to 10 and 16 to 17 containing nucleic acid sequences, SEQ ID Nos. 1 to 5, 11 and 12 are capable to generate anti-EGF antibodies.

In further embodiment, specific vaccine formulations comprising chimeric proteins with immunomodulator or delivery system have also been made available as one of the embodiments of the present invention optionally in presence of other stabilizers like polyols, sugars such as trehalose or amino acids or combinations.

In one aspect of the invention, there is provided a vaccine composition for immunotherapy against cancer, comprising:
a) vaccine antigen selected from a chimeric protein or construct;
b) adjuvants;
c) stabilizers; and
d) a physiologically acceptable buffer selected from phosphate and citrate.

In some embodiment, the vaccine composition of the present invention comprises the vaccine antigen comprising one or more of the chimeric protein sequences as represented by SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2).

In some other embodiment, the vaccine composition of the present invention comprises chimeric protein as vaccine antigen obtained from a codon optimized gene sequences comprising one or more of as represented by SEQ ID No. 1. (EGFL), SEQ ID No. 2 (E1FS1), SEQ ID No. 3 (E1FS2), SEQ ID No. 4 (E2FS1), SEQ ID No. 5 (E2FS2), SEQ ID No. 11 (BsE2FS1) or SEQ ID No. 12 (EGFL2).

In some embodiment, the vaccine composition of the present invention comprises chimeric protein as vaccine antigen, which comprises of growth factor linked to N-terminal or C-terminal or middle of the full length or truncated carrier protein and is genetically synthesized. In some embodiment of the invention, the chimeric protein is expressed in prokaryotic expression system through a prokaryotic expression plasmid. In some embodiment of the invention, the growth factor is human EGF. In some embodiment of the invention, the human EGF epitopes are exposed on the surface of the chimeric proteins.

In some other embodiment, the composition of the present invention comprises genetically synthesized full length Flagellin protein as carrier protein. In some other embodiment, the carrier protein in the vaccine composition of the present invention is a genetically synthesized truncated Flagellin protein without the hypervariable region which are from amino acid number178 to amino acid number 405. In some embodiment of the invention, the genetically synthesized sequence is selected from Gram positive bacteria including without limitation to Bacillus subtilis, Clostridium difficile or ????proteobacteria such as Salmonella typhimurium or Enterica species or ??proteobacteria such as Bordetella.

In some embodiment, the carrier protein the vaccine composition of the present invention helps the immune system to recognize autologous EGF to induce immune response against EGF and acts as adjuvant to enhance Th2 responses or B cell mediated immune responses.

In some embodiment, the adjuvant present in the composition of the invention is selected from one or more of a group of aluminium salt such as aluminium phosphate or aluminium hydroxide, squalene based adjuvants such as MF59, montanide, RIBI adjuvant, incomplete Freund's, glucans, oil-in-water emulsion, MPL, muramyl dipeptide, muramyl dipeptide derivatives, agonists of TLRs (TLR1 to TLR 13) such as MPL, MDP, Imiquimod, poly (I:C), CpG oligonucleotides, Non-CpG oligonucleotides, saponins such as QS-1, ISCOM, ISCOMATRIX, vitamins or immunomodulants such as cytokines, IL-12, IL-15 etc.

In some embodiment, the stabilizer used in the composition of the present invention is selected from the group comprising one or more of sugar such as 5-40% Trehalose or sugar alcohols such as 5-40% Glycerol or 5-40% Sorbitol.

In some embodiment, the vaccine composition according to invention comprises vaccine antigens of SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2) expressed in E.Coli using the pTWIN1 plasmid and purified proteins by self-cleavage, by temperature from 50-65ºC and pH shift ranges from pH 6.5 to 7.5.

In some embodiment, there is provided a method of isolation and purification of proteins of SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1), SEQ ID No. 17 (EGFL2), SEQ ID No. 18 (FliC), SEQ ID No. 19 (SDM) or SEQ ID 20 (EGF) comprising the following steps:
i. introducing the recombinant expression plasmid into bacterial host cells in high cell density growth media;
ii. isolation of protein either in the form of insoluble protein from inclusion bodies or in the form of soluble protein;
iii. purification of proteins by at least one or two of the following methods: ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction column chromatography.

In some embodiment, the high cell density growth media as used for isolation and purification of chimeric proteins in the claimed invention comprises 1.5% yeast extract, 1.5% Casein, 0.4% glycerol, M9 salt solution, trace elements, 1M MgSO4, 1M CaCl2, Biotin, Thiamine and ampicillin.

In some embodiment of the invention, there are provided vaccine composition comprising the chimeric protein sequence as disclosed in by SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2) which are at least 90% - 96% pure.

In another aspect of the invention there is provided a method of treatment to regulate the tumor growth or to increase the survival rate, comprising therapeutic vaccine composition according to invention with or without Tyrosine Kinase Inhibitors (TKIs). In some embodiment, the Tyrosine Kinase Inhibitor comprises 1st or 2nd or 3rd generation TKIs such as Geftinib, Erlotininb, Afatinib, Dacomitinib, Avitinib, Olmutinib or Nazartinib.

In some embodiment, the vaccine composition according to the present invention is stable for at least 2 years at 2-8°C and up to 1 to 72 weeks at 37°C.

In another aspect of the invention, there are provided vaccine composition with immunogenic molecule, a synthetic construct selected from the recombinant chimeric protein sequences as represented in by SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2); (b) adjuvants, either immunomodulator or delivery system and (c) stabilizers, wherein the said vaccine composition is stable for at least 2 years at 2-8°C and 1 to 2 weeks at 37°C.

In a further illustrative embodiment, a process for treating a tumor induced mouse model is disclosed. In this illustrative embodiment, the process includes administering therapeutic vaccine, which contains growth factor & pharmaceutically acceptable carrier protein that shows an adjuvant property to promote an immune response. Further, it also discloses the use of tyrosine kinase inhibitor, as a first line treatment followed by therapeutic vaccination to treat tumor induced mouse model.

BRIEF DESCRIPTIONS OF FIGURES
Figure 1: Protein expression, purification and characterization of EGFL.
Figure 1A: depicts SDS-PAGE of EGFL chimeric protein expressed in BL21 cells, induced with IPTG.
Figure 1B: represents EGFL (before and after cleavage) and its purified elution fractions by ion-exchange chromatography.
Figure 1C: elucidates characterization of EGFL (before and after cleavage) by western blot using Human Anti-EGF antibody.
Figure 1D: shows purified EGFL by Size Exclusion chromatography.
Figure 1E: shows purified protein EGFL by affinity chromatography using CBD (chitin binding domain) resin.
Figure 1F: depicts the yields of EGFL protein tested in different media.
Figure 2A: SDS-PAGE gel depicts the expression of E1FS1 (refolded, before cleavage) and its self cleaved target protein (after cleavage) and its purified elution fractions both by ion-exchange chromatography followed by SEC.
Figure 2B: elucidates characterization of E1FSI (before and after cleavage) by western blot using Human Anti-EGF antibody.
Figure 2C: SDS-PAGE gel depicts the expression of E2FS1 (refolded, before cleavage) and its self cleaved target protein (after cleavage) and its purified elution fractions both by ion-exchange chromatography followed by SEC.
Figure 2D: elucidates characterization of E2FS1 (before and after cleavage) by western blot using Human Anti-EGF antibody.
Figure 2E: SDS-PAGE gel depicts the expression of FliC (refolded, before cleavage) and its self cleaved target protein (after cleavage) and its purified elution fractions both by ion-exchange chromatography followed by SEC.
Figure 2F: SDS-PAGE gel depicts the expression of SDM (refolded, before cleavage) and its self cleaved target protein (after cleavage) and its purified elution fractions both by ion-exchange chromatography followed by SEC.
Figure 2G: illustrates western blot representing the E. coli expressed recombinant huEGF, recognized by the vaccinated sera raised against EGFL.
Figure 3A: illustrates a bar graph quantification of exposed EGF epitopes in its conformational form of EGFL or E1FS1 or E2FS1 chimeric proteins, anti-EGF antibody using huEGF quantification kit.
Figure 3B: illustrates a bar graph of anti-FliC antibody binding to recombinant full length FliC or truncated flagellin (SDM) or EGFL or E1FS1 or E2FS1 proteins that were captured on to ELISA plates.
Figure 4A: illustrates dose response curve generated by EGFL activity as an immunopotentiator, when stimulated with HEK Blue reporter cells engineered with TLR5. This figure represents dose response curve generated by EGFL activity as an immunopotentiator, when stimulated with HEK Blue reporter cells that were engineered with TLR5. Flagellin (FliC) was taken as a positive control. In graph, X-axis represents concentration of protein in log scale and Y-axis represents % response of protein. Each data set point is represented as a Mean±SD, which was obtained from three independent experiments done in duplicates.
Figure 4B: illustrates dose response curve generated by E1FS1 & E2FS1 activity as an immunopotentiator, when stimulated with HEK Blue reporter cells engineered with TLR5. This figure represents dose response curve generated by E1FS1 & E2FS1 activity as an immunopotentiator, when stimulated with HEK Blue reporter cells that were engineered with TLR5. Truncated Flagellin (SDM) was taken as a control. In graph, X-axis represents concentration of protein in log scale and Y-axis represents % response of protein. Each data set point is represented as a Mean±SD, which was obtained from three independent experiments done in duplicates.
Figure 4C: The graph shows secreted IL-8 cytokine by EGFL, by stimulating genetically engineered HEK Blue reporter cells with TLR5 gene. EGFL secreted approximately 700pg/ml IL-8 cytokine, whereas FliC secreted 200pg/ml. Secretion of IL-8 by TLR5 activated cells indicated anti-tumorigenic properties exhibited by EGFL.
Figure 5: Anti cell prolification growth pattern of NCI-H1975 cells, when treated with (Figure 5A): vaccinated sera against raised against EGFL, E1FS1, E2FS1, FliC and SDM (purified commercial anti-EGF antibody used as positive control); and (Figure 5B): combination of Geftinib and vaccinated sera against EGF, EGFL, E1FS1, E2FS1, FliC and SDM.
Figure 6A: Antibody titer raised against chimeric EGFL in mice.
Figure 6B: ELISA assay to represent EGF specific endpoint titer elicited against immunogenic composition containing EGFL.
Figure 6C: ELISA assay to represent EGF specific endpoint titer elicited against immunogenic composition containing E1FS1.
Figure 6D: ELISA assay to represent EGF specific endpoint titer elicited against immunogenic composition containing E2FS1.
Figure 6E: represents Th1:Th2 index of anti-EGF isotype antibody elicited against chimeric proteins.
Figure 7A: ELISA assay to represent flagellin specific antibody elicited against immunogenic composition containing EGFL
Figure 7B: ELISA assay to represent flagellin specific antibody elicited against immunogenic composition containing E1FS1
Figure 7C: ELISA assay to represent flagellin specific antibody elicited against immunogenic composition containing E2FS1
Figure 8A: ELISA assay to represent EGF specific endpoint titer elicited against EGFL, when mice were vaccinated with 50 µg EGFL/mouse with or without adjuvants. X-axis indicates vaccinated mice sera dilution and Y-axis represents absorbance at 490 nm.
Figure 8B: ELISA assay to represent EGF specific endpoint titer elicited against E1FS1, when mice were vaccinated with 50 µg EGFL/mouse with or without adjuvants. X-axis indicates vaccinated mice sera dilution and Y-axis represents absorbance at 490 nm.
Figure 8C: ELISA assay to represent EGF specific endpoint titer elicited against E2FS1, when mice were vaccinated with 50 µg EGFL/mouse with or without adjuvants. X-axis indicates vaccinated mice sera dilution and Y-axis represents absorbance at 490 nm.
Figure 9: Determination of tumor growth pattern induced by two different cell densities of LLC1 (Lewis Lung Cancer Cell line) in mice at different time points, so as to assess the ideal cell density to generate mice tumor model for therapeutic vaccination. This graph shows that mice started developing tumor from Day 7 onwards. Mice that received 1x106 cells/mouse, shown rapid tumor development, whereas mice that received 0.2x106 cells/mouse have shown slow tumor growth. Hence, to generate proof of concept study of therapeutic vaccination by EGFL, high cell density was selected to generate mice induced tumor model.
Figure 10A: Represents rate of tumor growth before and after treatment. X-axis indicates day on which tumor was measured and Y-axis indicates tumor volume. Tumor size increased drastically in mice after receiving tumor cells upto day 17, thereafter, slow tumor growth was observed immediately after receiving vaccination.
Figure 10B: Anti-EGF antibody response and EGF levels in serum are very well correlated thus proving immunotherapeutic activity of chimeric EGFL.
Figure 11: illustrates the survival graphs of tumor induced mice when treated with EGFL (as depicted in Figure 11A), E1FS1 (as depicted in Figure 11B), and E2FS1 (as depicted in Figure 11C). Further Figure 11D, Figure 11E, and Figure 11F represents tumor induced mice treated with EGF, FliC and SDM respectively used as control.
Figure 12: Depicts survival rate of mice treated with Geftinib alone (Figure 12A) and in combination with EGFL (Figure 12B).

DETAILED DESCRIPTION OF THE INVENTION
Detailed embodiments of the present recombinant chimeric proteins or vaccines are disclosed herein, however, it is to be understood that the disclosed embodiments are merely exemplary, which may be embodied in various forms. Therefore, specific functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the recombinant protein disclosed herein.

The present disclosure provides a construction of recombinant chimeric protein expressing full length or truncated flagellin and a human epidermal growth factor (EGF) is described. Further, in one illustrative embodiment, construction of recombinant chimeric protein comprising one or more copies of growth factor to improve the presentation of the maximum number of growth factor epitopes. It is contemplated within the scope of the disclosure that such recombinant chimeric proteins are highly immunogenic to the human immune system. Preferably, the recombinant proteins confer additional properties to the chimeric protein, for example, high expression yield and ease of manufacture and stability.

According to the disclosure the recombinant proteins, whether one or more copies of growth factor fused with either full length or truncated flagellin elicits broad range of immune responses against EGF (epidermal growth factor). Hence, these EGF based chimeric proteins may be useful in treating chronic diseases, for example, breast, lung, bladder, ovarian, Vulva, colonic, pulmonary, brain, colorectal, intestinal, head and neck, and esophagus cancers, where, EGF is responsible for uncontrolled cell proliferation.

The present invention discloses construction of codon optimized chimeric genes and its expression, purification & characterization. Chimeric gene encodes Epidermal Growth Factor (EGF) and a full length or truncated flagellin (without hypervariable region, from the position 178 to 405 amino acids). The present invention also describes the function of the flagellin both as an adjuvant and anti-tumorigenic property. In another embodiment, the present invention also discloses that the purified chimeric protein was able to elicit high titer antibodies against EGF in C57BL/6 mice and also substantially increased survival rate, by reducing the tumor progression rate.

In one aspect of the invention, there is provided a vaccine composition for immunotherapy against cancer, comprising:
a) vaccine antigen selected from a chimeric protein or construct;
b) adjuvants;
c) stabilizers; and
d) a physiologically acceptable buffer selected from phosphate and citrate.

In some embodiment, the vaccine composition of the present invention comprises the vaccine antigen comprising one or more of the chimeric protein sequences as represented by SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2).

In some other embodiment, the vaccine composition of the present invention comprises chimeric protein as vaccine antigen obtained from a codon optimized gene sequences comprising one or more of as represented by SEQ ID No. 1. (EGFL), SEQ ID No. 2 (E1FS1), SEQ ID No. 3 (E1FS2), SEQ ID No. 4 (E2FS1), SEQ ID No. 5 (E2FS2), SEQ ID No. 11 (BsE2FS1) or SEQ ID No. 12 (EGFL2).

In some embodiment, the vaccine composition of the present invention comprises chimeric protein as vaccine antigen, which comprises of growth factor linked to N-terminal or C-terminal or middle of the full length or truncated carrier protein and is genetically synthesized. In some embodiment of the invention, the chimeric protein is expressed in prokaryotic expression system through a prokaryotic expression plasmid. In some embodiment of the invention, the growth factor is human EGF. In some embodiment of the invention, the human EGF epitopes are exposed on the surface of the chimeric proteins.

In some other embodiment, the composition of the present invention comprises genetically synthesized full length Flagellin protein as carrier protein. In some other embodiment, the carrier protein in the vaccine composition of the present invention is a genetically synthesized truncated Flagellin protein without the hypervariable region which are from amino acid number178 to amino acid number 405. In some embodiment of the invention, the genetically synthesized sequence is selected from Gram positive bacteria including without limitation to Bacillus subtilis, Clostridium difficile or ????proteobacteria such as Salmonella typhimurium or Enterica species or ??proteobacteria such as Bordetella.

In some embodiment, the carrier protein the vaccine composition of the present invention helps the immune system to recognize autologous EGF to induce immune response against EGF and acts as adjuvant to enhance Th2 responses or B cell mediated immune responses.

In some embodiment, the adjuvant present in the composition of the invention is selected from one or more of a group of aluminium salt such as aluminium phosphate or aluminium hydroxide, squalene based adjuvants such as MF59, montanide, RIBI adjuvant, incomplete Freund's, glucans, oil-in-water emulsion, MPL, muramyl dipeptide, muramyl dipeptide derivatives, agonists of TLRs (TLR1 to TLR 13) such as MPL, MDP, Imiquimod, poly (I:C), CpG oligonucleotides, Non-CpG oligonucleotides, saponins such as QS-1, ISCOM, ISCOMATRIX, vitamins or immunomodulants such as cytokines, IL-12, IL-15 etc.

In some embodiment, the stabilizer used in the composition of the present invention is selected from the group comprising one or more of sugar such as 5-40% Trehalose or sugar alcohols such as 5-40% Glycerol or 5-40% Sorbitol.

In some embodiment, the vaccine composition according to invention comprises vaccine antigens of SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2) expressed in E.Coli using the pTWIN1 plasmid and purified proteins by self-cleavage, by temperature from 50-65ºC and pH shift ranges from pH 6.5 to 7.5.

In some embodiment, there is provided a method of isolation and purification of proteins of SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1), SEQ ID No. 17 (EGFL2), SEQ ID No. 18 (FliC), SEQ ID No. 19 (SDM) or SEQ ID 20 (EGF) comprising the following steps:
i. introducing the recombinant expression plasmid into bacterial host cells in high cell density growth media;
ii. isolation of protein either in the form of insoluble protein from inclusion bodies or in the form of soluble protein; and
iii. purification of proteins by at least one or two of the following methods: ion exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction column chromatography.

In some embodiment, the high cell density growth media as used for isolation and purification of chimeric proteins in the claimed invention comprises 1.5% yeast extract, 1.5% Casein, 0.4% glycerol, M9 salt solution, trace elements, 1M MgSO4, 1M CaCl2, Biotin, Thiamine and ampicillin.

In another aspect of the invention, vaccine composition provided with immunogenic molecule, a synthetic construct selected from the recombinant chimeric protein sequences as represented in by SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2); (b) adjuvants, either immunomodulator or delivery system and (c) stabilizers, wherein the said vaccine formulation is stable for at least 2 years at 2-8°C and 1 to 2 weeks at 37°C.

In some embodiment of the invention, the vaccine composition comprises chimeric protein sequence as disclosed in SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2) which are at least 90% - 96% pure.

In another aspect of the invention there is provided a method of treatment to regulate the tumor growth or to increase the survival rate, comprising therapeutic vaccine composition according to invention with or without Tyrosine Kinase Inhibitors (TKIs). In some embodiment, the Tyrosine Kinase Inhibitor comprises 1st or 2nd or 3rd generation TKIs such as Geftinib, Erlotininb, Afatinib, Dacomitinib, Avitinib, Olmutinib or Nazartinib.

In some embodiment, the vaccine composition according to the present invention is stable for at least 2 years at 2-8°C and up to 1 to 2 weeks at 37°C.

EXAMPLES
The invention will now be further illustrated with reference to the following examples.

Recombinant chimeric proteins were created expressing the entire EGF coding region on the full length or truncated flagellin at either at the N or C-terminus or in place of hypervariable D2 & D3 domains or D3 region of flagellin. Chimeric gene constructs containing autologous EGF, either at N- or C-terminal or within the sequence of full length or truncated flagellin. Flagellin in synthetic chimeric protein may contain protein sequence similar to flagellin sequence from Gram positive bacteria including without limitation to Bacillus subtilis, Clostridium difficile or ??proteobacteria such as Salmonella typhimurium or enterica species or ??proteobacteria such as Bordetella. Construction of each chimeric genes of the present invention is as detailed in examples 1.1 to 1.8

In the illustrative embodiments given below, include EGF fused to full length or truncated flagellin either at N- or C- termini of the full length or truncated flagellin with the help of linkers. Additional recombinant EGF is again fused either at N- or C-termini or at the middle of the full length flagellin by restriction enzyme. The addition of the linkers can reduce steric hindrance and may also enable unique restriction sites to be introduced within the linkers to allow subsequent manipulation of the genetic constructs. To generate the following constructs given below, amino acid linkers were used, but not limited to EAAAKP, AS and AAA.

Example 1: Generation of the plasmid encoding chimeric constructs:

All chimeric constructs were first cloned into pUC57 plasmid and then further cloned into pTWIN1 expression plasmid. To generate these various chimeric constructs, synthetic LC-EGFL gene sequence, which was cloned into pUC57 (as pUC57 LC_EGFL or pUC57EGFLWO) was taken as a starting plasmid.

To generate library of EGF based chimeric constructs, pTWIN1 (7375 base pairs, New England Biolabs, Cat#N6951S), a derivative of pBR322 plasmid was used as an E.coli expression vector. Plasmid pTWIN1 express the target gene, under the control of T7 promotor, which is regulated by IPTG or Lactose due to the presence of Lac I gene. The purpose of using the pTWIN1 vector is due to its advantage of controllable self cleavage of modified Sap DnaB (Mathy, S et al., (1999) Gene. 231, 1-13) at the N-terminus target protein or thiol induced cleavage of Mxe GyrA inteins (Evans, T.C et al., (1998) Protein Sci. 7, 2256-2264; Southworth, M.W et al., (1999) BioTechniques. 27, 110-120) at the C-terminus target protein. Further, the presence of the chitin binding domain from Bacillus circulans (Chong, S., et al., (1997) Gene. 192, 271-281; Watanabe, T et al., (1994) J. Bacteriol. 176, 4465-4472) facilitates purification without any use of protease, unlike using tags like His etc.

Example 1.1: Construction of plasmid encoding EGFL Nucleic acid sequence of EGFL includes autologous human EGF at N-terminus of full length flagellin and these two sequences are connected by a linker. The whole gene comprising of 1698bps (Seq ID No. 1) and the corresponding translated protein sequence (SEQ ID No. 6) contains 565 amino acids with a molecular weight of approximately 62 kDa.

EGFL was amplified by PCR from pUC57-LC-EGFL (which was genetically synthesized sequence from Genscript) using appropriate primers to introduce SapI site at the N-terminus of EGFL. This PCR fragment, digested with SapI & PstI restriction enzymes and was ligated into pUC57 & pTWIN1 vectors and named as pUC57 EGFLWO & pTWIN1 EGFL respectively. Further, plasmid pTWIN1 encoding EGFL was transformed into DH5alpha. Single cell colonies were selected from the plate containing 100 µg/mL ampicillin, grown overnight at 37°C. These clones were screened by restriction digestion and confirmed by nucleotide sequencing using the appropriate sequencing primers.

Example 1.2: Construction of plasmid encoding E1FS1 Nucleic acid sequence of E1FS1 consists of autologous human EGF at N-terminus of truncated flagellin with deletion of hypervariable region D2 & D3 region (amino acids from position 178 to 405aas). E1FS1 gene sequence was generated by deleting hypervariable regions D2 & D3 from EGFL by site directed mutagenesis. The whole gene comprising of 1017bps (Seq ID No. 2) and the corresponding translated protein sequence (SEQ ID No. 7) contains 338 amino acids with a molecular weight of approximately 37 kDa.

To generate Plasmid pUC57-E1FS1, hypervariable region (position 178 to 405aas) of flagellin was deleted from pUC EGFL (WO), using appropriate primers and purified the circularized plasmid containing E1FS1, after Dpn digestion. E1FS1 fragment from pUC57 was digested with SapI & PstI, and ligated into pTWIN1 vector. After transformation into DH5alpha, clones were screened by restriction digestion and confirmed by DNA sequence using appropriate sequencing primers.

Example 1.3: Construction of plasmid encoding E1FS2 Nucleic acid sequence of EFS2 consists of autologous human EGF at N-terminus of truncated flagellin with deletion of hypervariable region D3 region (amino acids from position 186 to 285aas). E1FS2 gene sequence was generated by deleting hypervariable regions D3 from EGFL by site directed mutagenesis. The whole gene comprising of 1401bps (Seq ID No. 3) and the corresponding translated protein sequence (SEQ ID No. 8) contains 466 amino acids with a molecular weight of approximately 51 kDa.

To generate Plasmid pUC57-E1FS2, where hypervariable region D3 (position 186 to 285aas) of flagellin was deleted from pUC EGFL (WO), using appropriate primers and purified the circularized plasmid containing E1FS2, after Dpn digestion. E1FS2 fragment from pUC57 was digested with SapI & PstI, and ligated into pTWIN1 vector using the same restriction sites. After transformation into DH5alpha, clones were screened by restriction digestion and confirmed by DNA sequence using appropriate sequencing primers.

Example 1.4: Construction of plasmid encoding E2FS1 Nucleic acid sequence of E2FS1 consists of 2 copies of autologous human EGF and truncated flagellin with deletion of D2 & D3 region. E2FS1, wherein, 1 copy of EGF located at N-terminus and another copy located within the sequence of the flagellin (in place of D2 & D3 region). E2FS1 gene sequence was generated by introducing second copy of EGF into E1FS1 using NheI site as linker. The whole gene comprising of 1185bps (Seq ID No. 4) and the corresponding translated protein sequence (SEQ ID No. 9) contains 394 amino acids with a molecular weight of approximately 43 kDa.

To generate E2FS1, plasmid pUC57-E1FS1 was digested with Nhe1 and used as a vector after gel purification. The second copy of EGF insert, which was amplified from the pUC EGFL (WO or the previous one) with NheI sites at both N- & C-terminus using appropriate primers was ligated. After ligation, E2FS1 was inserted into pTWIN1 plasmid using SapI & PstI from pUC57-E2FS1. Clones were screened by restriction digestion and confirmed by nucleotide sequencing using appropriate primers.

Example 1.5: Construction of plasmid encoding E2FS2 Nucleic acid sequence of E2FS2 consists of 2 copies of autologous human EGF and truncated flagellin with deletion of D3 region. E2FS2, wherein, one copy of EGF located at N-terminus and another copy located within the sequence of flagellin (in place of D3 region). E2FS2 gene sequence was generated by introducing second copy of EGF into E1FS2 with the help of NheI restriction site as a linker. The whole gene comprising of 1569bps (Seq ID No. 5) and the corresponding translated protein sequence (SEQ ID No. 10) contains 522 amino acids with a molecular weight of approximately 57 kDa. The plasmid (pTWIN1-E2FS2) was constructed using sapI & pstI restriction enzymes. and the plasmid was transformed into DH5a. Single cell positive colonies were selected from the plate containing 100 µg/mL ampicillin grown overnight at 37°C. These positive colonies were further confirmed by nucleotide sequencing.

Example 1.6: Construction of plasmid encoding BsE2FS1: Nucleic acid sequence of BsE2FS1 consists of 2 copies of autologous human EGF and flagellin derived from Bacillus subtilis (strain 168), wherein, 1 copy of EGF located at N-terminus and another copy at the C-terminus of flagellin. The whole gene comprising of 1260bps (Seq ID No. 11) and the corresponding translated protein sequence (SEQ ID No. 16) contains 420 amino acids with a molecular weight of approximately 46 kDa.

BsE2FS1 was a synthetic sequence from pUC57 vector was digested with SapI & PstI and ligated into SapI & PstI digested pTWIN1 vector. After ligation, Plasmid pTWIN1 encoding BsE2FS1 was transformed into DH5alpha and positive clones were screened by restriction digestion and confirmed by nucleotide sequencing done using appropriate primers.

Example 1.7 - Construction of plasmid encoding EGFL2: Nucleic acid sequence of EGFL2 includes 2 copies of autologous human EGF both at N- & C- terminus of full length flagellin and these two sequences are connected by a linker. The whole gene comprising of 1863 bps (Seq ID No. 12) and the corresponding translated protein sequence (SEQ ID No. 17) contains 621 amino acids with a molecular weight of approximately 79 kDa.

To generate EGFL2, second copy of EGF was introduced to full length flagellin at the C-terminus using NheI site using FW Nh & Rv Pst in pUC57. Further, EGFL2 sequence from pUC57 was digested with SapI & PstI and ligated into SapI & PstI digested pTWIN1 vector. After ligation, Plasmid pTWIN1 encoding EGFL2 was transformed into DH5alpha and positive clones were screened by restriction digestion and confirmed by nucleotide sequencing done using appropriate primers.

Example 1.8: Construction of plasmid encoding recombinant Full length (FliC) or truncated flagellin: (SDM- deletion of hypervariable region D2 & D3 of flagellin)

Example 1.8a: Nucleic acid sequence of FLiC includes full length flagellin. The whole gene comprising of 1518 bps (Seq ID No. 13) and the corresponding translated protein sequence (SEQ ID No. 18) contains 506 amino acids with a molecular weight of approximately 55 kDa. FliC was amplified by PCR from pUC57-LC-EGFL (genetically synthesized sequence) using appropriate primers. This insert was ligated into SapI & PstI digested pTWIN1 vector. Plasmid pTWIN1 encoding FliC was transformed into DH5alpha and positive clones were screened by digestion (PstI & XbaI) and confirmed by DNA sequencing using the appropriate primers.

Example 1.8b: Construction of truncated Flagellin (SDM) Nucleic acid sequence of SDM EGFL includes truncated flagellin with deletion of hypervariable region D2 & D3 region (amino acids from position 178 to 405aas). The whole gene comprising of 837 bps (Seq ID No. 14) and the corresponding translated protein sequence (SEQ ID No.19) contains 279 amino acids with a molecular weight of approximately 30 kDa.

SDM was amplified from pUC57 E1FS1, clone#5 using appropriate primers. PCR fragment was purified and digested with SapI & PstI and used as insert. This insert was introduced into the SapI & PstI digested pTWIN1 by ligation. After ligation, plasmid pTWIN1 containing SDM was transformed into DH5alpha and positive clones were selected by digestion with NheI & PstI and confirmed by sequencing using appropriate primers.

Example 1.9: Construction of plasmid encoding Epidermal Growth Factor (EGF): Nucleic acid sequence of autologous human EGF comprising of 162bps (Seq ID No. 15) and the corresponding translated protein sequence (SEQ ID No. 20) contains 54 amino acids with a molecular weight of approximately 6.2 kDa.

Plasmid pTWIN1 containing EGF was generated by ligating SapI & PstI digested pTWIN1 vector and also PCR amplified EGF from the plasmid that has genetically synthesized sequence of EGF. After ligation, Plasmid pTWIN1 encoding EGF was transformed into DH5alpha and positive clones were confirmed by digestion (PstI & NheI) and sequence was confirmed by DNA sequencing using the appropriate primers.

The above mentioned chimeric constructs, which were cloned in pTWIN1 expression plasmid were used to express the chimeric protein such as (EGFL, E1FS1, E1FS2, E2FS1, E2FS2, BsE2FS1, EGFL2 etc.,) and to evaluate their efficiency as a therapeutic vaccine candidates. Further, pTWIN1 expression plasmid constructs encoding genes such as [(EGF, full length flagellin (FliC) or truncated Flagellin (SDM)] were also generated to express recombinant proteins such as (EGF, FliC, SDM respectively) and used as controls in both in-vitro and in-vivo studies. A summary is provided as per the below Table 1 for all the Nucleic Acids and recombinant proteins:

Nucleic acid sequence Name of chimeric protein Protein Sequence
Seq ID # Number of base pairs Seq ID # Number of amino acids
Recombinant Chimeric proteins
1 1698 EGFL 6 565
2 1017 E1FS1 7 338
3 1401 E1FS2 8 466
4 1185 E2FS1 9 394
5 1569 E2FS2 10 522
11 1260 BsE2FS1 16 420
12 1863 EGFL2 17 621
Recombinant Proteins
13 1518 FliC 18 506
14 837 SDM 19 279
15 162 EGF 20 54
TABLE 1: Summary of Recombinant chimeric proteins and Recombinant proteins

Example 2: Expression & Characterization of Chimeric Proteins:

Example 2.1: Expression & Characterization of EGFL: The plasmid (pTWIN1-EGFL) was again transformed into E. coli BL21 (DE3) (NEB, Cat#C2527I) or BL21 Rosetta 2 (DE3) pLysS Cat#71401, or RosettaTM 2 (DE3) competent cells (Novagen, Cat#70954). Single colony was isolated from the LB plate with 100 µg/mL amp and further grown in LB medium containing 100 µg/mL amp overnight at 37°C with shaking to get start up culture. The culture was then diluted to 1-10% and allowed to grow until the OD600 reached 0.4 – 0.6. Next, the bacterial culture was induced with 0.2 - 1 mM IPTG for 16-18 h (as shown in Figure 1A). Cells were finally harvested by centrifugation at 12,000 rpm for 20 minutes. To check the expressed protein, SDS-PAGE sample buffer was added to the cell pellet and resolved proteins on SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) under non-reducing conditions. Further, the gel with expressed protein having MW 85kDa (before cleavage) and 62kDa (after cleavage) was transferred on to a nitrocellulose membrane for a western blot. Mouse anti-hEGF antibody (Abcam, Cat#ab10409) and HRP-labeled goat-anti-mouse IgG were used as primary and secondary antibody respectively. 3,3 ´-diaminobenzidine tetrahydrochloride (DAB) was used as a substrate. Western blot analysis determined that the chimeric protein EGFL displays the EGF epitopes in the correct conformation and thus EGFL is able to get recognized by the anti-EGF antibodies, as illustrated in Figure 1C.
In order to increase the EGFL protein yield, BL21 (DE3) cells were grown in different media compositions (PP, LBM9, CYM9 media or enriched media), while testing in different induction protocols, using Lactose as inducer.

PP Media: Single colony was inoculated from a fresh transformed plate into LB Media and incubated for 16-18 hrs at 37?, 250 rpm. Then, 10% inoculum was used for secondary culture in PP media composed of 40% glucose, M9 salt solution, trace elements, 1M MgSO4, Thiamine and ampicillin and incubated at 37?, 250 rpm till the OD reached 6.5. Further it was induced with 0.5% lactose and incubated overnight at 25?. Cells were finally harvested further processed for protein extraction and purification.

LBM9 media: Single colony was inoculated from a fresh transformed plate into 5 ml of LB Media and incubated for 16-18 hrs at 37?, 250 rpm. Then, 10% inoculum was used for secondary culture in LBM9 media composed of 2% LB media, 40% glucose, M9 salt solution, trace elements, 1M MgSO4, 1M CaCl2, Biotin, Thiamine and ampicillin and incubated at 37?, 250 rpm till the OD reached 6.5. Further it was induced with 0.5% lactose and incubated overnight at 25?. Cells were finally harvested by and further processed for protein extraction and purification.

BBIL-CYM9 Media: In this disclosure, bacterial culture was grown in the below mentioned media to obtain high cell density and then induced with lactose to increase the yield. Briefly, single colony was inoculated from a fresh transformed plate into LB Media and incubated for 16-18 hrs at 37?, 250 rpm. Then, 10 % inoculum was used for secondary culture in CYM9 media composed of 1.5% yeast extract, 1.5% Casein, 0.4% glycerol, M9 salt solution, trace elements, 1M MgSO4, 1M CaCl2, Biotin, Thiamine and ampicillin and incubated at 37?, 250 rpm till the OD reached 6.5. Further it was induced with 0.2% lactose and incubated for 3 hrs at 37?. From this, again 10% inoculum was used for sub culture in CYM9 media and induced with 0.2% lactose and incubated for overnight at 25?. Cells were finally harvested and further processed for protein extraction and purification. The present invention also discloses the abovementioned novel media, namely BBIL-CYM9 used with double induction.

Enriched media: Single colony was inoculated from a fresh transformed plate into LB Media and incubated for 16-18 hrs at 37?, 250 rpm. Then, 10% inoculum was used for secondary culture in enriched media composed of 1.5% yeast extract, 1.5% Casein, 2% glycerol, M9 salt solution, trace elements, 1M MgSO4, Thiamine and ampicillin and incubated at 37? till the OD reaches 0.4-0.6. Culture was then induced with 0.2% lactose and incubated for 3 hrs at 37?. Induced culture was used as inoculum (10%) for inoculating tertiary culture and incubated at 37?, 250 rpm till the OD reaches 3. Further it was induced with 0.2% lactose and incubated overnight at 25?. Cells were finally harvested by centrifugation at highest rpm for 20 minutes and further processed for protein extraction.

The yield of EGFL chimeric protein tested in different media are represented as shown in Table 2 below and the Figure 1F represents the data corresponding to the above Table 2.

S.No Media Inducer Final protein concentration (mg/L)
1 PP 0.5%Lactose 19
2 CYM9 0.5%Lactose 48
3 LB+M9 0.5%Lactose 35
4 Enrich 0.5%Lactose 24
5 LB 0.5 Mm IPTG 15
TABLE 2: Yield of EGFL chimeric protein tested in different media

Example 2.2: Expression & Characterization of E1FS1: The plasmid (pTWIN1-E1FS1) was again transformed into E. coli BL21 (DE3) competent cells. Single colony was isolated and grown in LB medium containing 100 µg/mL amp overnight at 37°C with shaking to get start up culture. The culture was then diluted to 1-10% and allowed to grow until the OD600 reached 0.4 – 0.6. Next, the bacteria were induced with 0.2mM IPTG for 16-18 hours. Cells were finally harvested by centrifugation at 12,000 rpm for 20 minutes. To check the expressed protein, SDS-PAGE sample buffer was added to the cell pellet and ran SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) under non-reducing conditions. The expressed protein was characterized by SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) (as shown in Figure 2A). The gel was transferred to a nitrocellulose membrane for a western blot. Mouse anti-hEGF antibody and HRP-labeled goat-anti-mouse IgG were used as primary and secondary antibody respectively. 3,3 ´-diaminobenzidine tetrahydrochloride (DAB) was used as a substrate. In this disclosure, Western blot analysis determined that the chimeric protein E1FS1 can display the EGF epitopes in the correct conformation and thus E1FS1 is able to get recognized by the anti-EGF antibodies, as illustrated in Figure 2B.

Example 2.3: Expression & Characterization of E2FS1: The plasmid (pTWIN1-E2FS1) was again transformed into E. coli BL21 (DE3) competent cells. Single colony was isolated and grown in LB medium containing 100 µg/mL amp overnight at 37°C with shaking to get start up culture. The culture was then diluted to 1-10% and allowed to grow until the OD600 reached 0.4 – 0.6. Next, the bacteria were induced with 0.2mM IPTG for 16-18 hours. Cells were finally harvested by centrifugation at 12,000 rpm for 20 minutes. To check the expressed protein, SDS-PAGE sample buffer was added to the cell pellet and ran SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) under non-reducing conditions. The expressed protein was characterized by SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) (as shown in Figure 2C). The gel was transferred to a nitrocellulose membrane for a western blot. Mouse anti-hEGF antibody and HRP-labeled goat-anti-mouse IgG were used as primary and secondary antibody respectively. 3,3 ´-diaminobenzidine tetrahydrochloride (DAB) was used as a substrate. In this disclosure, Western blot analysis determined that the chimeric protein E2FS1 can display the EGF epitopes in the correct conformation and thus E2FS1 is able to get recognized by the anti-EGF antibodies, as illustrated in Figure 2D.

Example 2.4: Expression & Characterization of FliC: The plasmid (pTWIN1-FliC) was again transformed into E. coli BL21 (DE3) competent cells. Single colony was isolated and grown in LB medium containing 100 µg/mL amp overnight at 37°C with shaking to get start up culture. The culture was then diluted to 1-10% and allowed to grow until the OD600 reached 0.4 – 0.6. Next, the bacteria were induced with 0.2mM IPTG for 16-18 hours. Cells were finally harvested by centrifugation at 12,000 rpm for 20 minutes. To check the expressed protein, SDS-PAGE sample buffer was added to the cell pellet and ran SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) under non-reducing conditions. The expressed protein was characterized by SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) (as shown in Figure 2E).

Example 2.5: Expression & Characterization of SDM: The plasmid (pTWIN1-SDM) was again transformed into E. coli BL21 (DE3) competent cells. Single colony was isolated and grown in LB medium containing 100 µg/mL amp overnight at 37°C with shaking to get start up culture. The culture was then diluted to 1-10% and allowed to grow until the OD600 reached 0.4 – 0.6. Next, the bacteria were induced with 0.2mM IPTG for 16-18 hours. Cells were finally harvested by centrifugation at 12,000 rpm for 20 minutes. To check the expressed protein, SDS-PAGE sample buffer was added to the cell pellet and ran SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) under non-reducing conditions. The expressed protein was characterized by SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) (as shown in Figure 2F).

Example 2.6: Expression & Characterization of HuEGF: The plasmid (pTWIN1-EGF) was again transformed into E. coli BL21 (DE3) competent cells. Single colony was isolated and grown in LB medium containing 100 µg/mL amp overnight at 37°C with shaking to get start up culture. The culture was then diluted to 1-10% and allowed to grow until the OD600 reached 0.4 – 0.6. Next, the bacteria were induced with 0.2mM IPTG for 16-18 h. Cells were finally harvested by centrifugation at 12,000 rpm for 20 minutes. To check the expressed protein, SDS-PAGE sample buffer was added to the cell pellet and ran SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) under non-reducing conditions. The expressed protein was characterized by SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis). The gel was transferred to a nitrocellulose membrane for a western blot. Mouse vaccinated sera raised against EGFL was used to probe huEGF as a primary antibody and HRP-labeled goat-anti-mouse IgG was used as secondary antibody. 3,3 ´-diaminobenzidine tetrahydrochloride (DAB) was used as a substrate.

In this disclosure, Western blot analysis determined that the anti-EGF antibodies raised against EGFL were able to bind to huEGF, as illustrated in Figure 2G.

Example 3: Isolation, purification & characterization of recombinant proteins:

The given illustrative embodiment discloses the isolation and purification protocol for all recombinant proteins that were disclosed in the present invention.

The E. coli culture pellet was resuspended in lysis buffer (20 mM Tris-Cl, 2 mM EDTA, 150 mM NaCl, pH 8.5) with lysozyme (1 mg/ml). Then, DNase and MgCl2 were added with agitation at 4°C. The cell culture was subjected to sonication with conditions (40 amplitude for 10 seconds ‘ON’ & 10 seconds ‘OFF’, later 70 amplitude for 30 minutes with 10 seconds ‘ON’ and 10 seconds ‘OFF’) and after sonication sample was centrifuged at 12000 rpm for 15-20 minutes. Pellet & Supernatant was separated & checked by SDS-PAGE. Protein was found in supernatant in soluble form and the solution was kept at 4ºC. This protein solution was dialyzed against cleavage buffer (20 mM Tris-Cl, 2 mM EDTA, 150 mM NaCl, pH 6.0 & 5% glycerol) for 48-72 hours at RT. Temperature (from 4ºC to RT) & pH shift (from pH 8.5 to pH range from 6.5 – 7.5) was done to induce Ssp DnaB intein self-cleavage. Finally, the solution was subjected to heat treatment at 50-65°C for 30 minutes and supernatant was collected by centrifugation at 12000 rpm for 10 minutes at 4°C. The supernatant containing desired protein was further subjected to protein purification by ion exchange chromatography (IEC) followed by Size exclusion chromatography (SEC) to achieve higher purity of upto 99% of the targeted protein. Alternatively, higher purity of up to 99% pure protein may also be obtained using either one of the following methods or in combination such as Ion-exchange chromatography or affinity chromatography using CBD (Chitin Binding Domain) resin or hydrophobic interaction chromatography or Size exclusion chromatography or chromatography using mixed mode resins.

Example 3.1: Ion Exchange Chromatography (IEC): The suspension of heated or cleaved mixture was directly injected into a MacroPrep High Q 3HT strong anion exchange resin obtained from Biorad. Resin was equilibrated with 3 volumes of equilibration buffer pH 6.5 (20 mM Tris, 0.2 mM EDTA, 150 mM NaCl, 0.01 % sodium azide, 0.1 % Tween 20 & 0.1 % Triton X-114). The sample was loaded on to the column with a flow rate 0.5 ml/min. The desired chimeric protein that is devoid of intein CBD tag was collected by elution buffer (20 mM Tris-Cl, 0.2 mM EDTA, 400mM NaCl, pH 7.5) with a flow rate 1 mL/min. Purified IEC fractions of chimeric proteins EGFL, E1FS1 and E2FS1 are illustrated in Figure 1B, Figure 2A and Figure 2C respectively and IEC purified recombinant proteins such as FliC, and SDM are illustrated in Figure 2E and 2F respectively.

Example 3.2: Size exclusion chromatography (SEC): Superdex 200 resin packed in XK16/60 column was used to purify the recombinant chimeric protein(s). Initially, column was equilibrated with 10CVs of equilibration buffer (1xPBS, pH 7.4). Sample was loaded at a flow rate of 0.5 ml/min followed by desired protein elution at a flow rate of 0.8 ml/min. Purified SEC fractions of chimeric proteins EGFL, E1FS1 and E2FS1 are illustrated in Figure 1D, Figure 2A and Figure 2C respectively and SEC purified recombinant proteins such as FliC, and SDM are illustrated in Figure 2E and 2F respectively.

Example 3.3: Affinity Chromatography: To remove intein associated chitin binding domain, which was expressed along with chimeric proteins, the affinity column packed with chitin resin was used. The chitin resin was equilibrated with 10 column volumes (CVs) of equilibration buffer (20 mM Tris-Cl, 500 mM NaCl, 1 mM EDTA, 0.1% Tween 20, pH 8.5) followed by sample loading on to the column with a flow rate 0.5 ml/min or less. Column was washed with 10 CVs equilibration buffer. Later 1 bed volume of cleavage buffer was added and flushed through the column quickly and filled the column with 1CV of cleavage buffer (20 mM Tris-Cl, 1M NaCl, 1 mM EDTA, 0.1% Tween 20, pH 6.5 – 7.5) and allowed to incubate at RT for 24-48 hours. Bound fractions were eluted with 3-5 CVs of cleavage buffer. At each step fractions were collected and analysed by SDS-PAGE. Purified fractions of EGFL using affinity chromatography are illustrated in Figure 1E. Protein Yield expressed in LB media with 0.2 mM IPTG induction has been shown in Table 3

S. No Name of the protein % Recovery or % Yield (mg/ml) % Purity
1 EGFL 15 95-99
2 E1FS1 15 90-99
3 E2FS1 16 85-99
4 FliC 12.5 90-99
5 SDM 15 90-99

Table 3: Protein Yield expressed in LB media with 0.2 mM IPTG induction

Example 4: Quantification & Characterization of functional properties of EGF:

Example 4.1: Determination of EGF epitope binding and its quantification by ELISA: To demonstrate, whether the chimeric proteins retain the antigenicity or recognition site, in its folded form, Further, to assess the increased level of EGF recognition, especially, in case of recombinant chimeric protein such as E2FS1, where in 2 copies of EGF were fused was estimated by ELISA using Human EGF ELISA kit (Cat# KHG0061, Invitrogen).

Quantification of Human EGF by ELISA: The assay was done as per the instructions given in the manual. Briefly, 96 well plate coated with anti-EGF antibody (capture antibody) was used. Later, Standards, control (EGF) or pre diluted Recombinant protein samples such as EGFL or E1FS1 or E2FS1 or BsE2FS1 or EGFL2 (100µl of each sample/per well) were added to each well and incubated it for 2 hours. The plate was washed four times with PBS containing 0.5% Tween (PBST). Further, Biotin conjugated anti-Hu EGF antibody (100 µl/well) was added and incubated the plate at room temperature for 1hr. After washing the plate, 100 µl of 1 x streptavidin-HRP conjugate was added and incubated it for 30 minutes. Finally, chromogen (TMB) was used as a substrate. After 30 minutes of incubation, reaction was stopped with stop solution and read the absorbance at 450 nm. These results show that the EGF epitopes of chimeric protein was been able to recognized by anti-EGF antibodies. Further, there was increase in the EGF concentration, where 2 copies of EGF introduced in the chimeric construct sequence (E2FS1). Table 4, as given below, shows the theoretical equimolar ratios of EGF & FliC or SDM in each chimeric construct and the estimated EGF concentration by ELISA. Figure 3A illustrates the estimated EGF concentration reflects antigenicity or epitopes exposed, but not the protein concentration.

S. No Fusion Proteins Theoritical EGF: FliC emperical ratio (mg/ml) Calculated /predicted EGF in 50µg chimeric proteins Estimated EGF in 50µg chimeric proteins *
EGF FliC/SDM EGF EGF
1 EGF 1 NA 50µg 35µg
2 FliC NA 1 --- ---
3 SDM NA 1 --- ---
4 EGFL 0.1 (10%) 0.88 (88%) 5µg 0.225µg
5 E1FS1 0.174 (17.4%) 0.81 (81%) 8.5µg 0.925
6 E2FS1 0.29 (29%) 0.69 (69%) 14.4µg 1.325
Table 4: Theoretical equimolar ratios of EGF & FliC or SDM in each chimeric construct and the estimated EGF concentration by ELISA.

Example 4.2: Relative estimation of Full Length or truncated Flagellin by ELISA: To assess, the relative amount of flagellin epitopes exposed in chimeric proteins, 96 well plate was coated with 100 µl of each protein at equal concentration (1 µg/ml), diluted in PBS, pH 7.4. Plate was incubated for overnight at 4C. Subsequently 3 washes with PBST were carried out and then the plates were blocked with a solution of 1 x PBS containing 0.5% skim milk, incubated them during a period of 1 hour at RT. Next step, the plates were incubated with 50 ul/well of anti-mouse-FliC antibody HRP conjugate (1:5000 dilution) for 1 hour at RT. Reaction color was developed with TMB 50 ul/well, incubated for 30 minutes at RT. Optical density was measured at 450 nm in an ELISA plate reader.

The results were demonstrated the activity and the efficiency of the chimeric proteins, as these proteins maintain EGF recognition sites for the capture anti-EGF antibody. It also demonstrated that there is an increase in the concentration of EGF, as the number of EGF copies increase, further, FliC or SDM used as carrier protein in these chimeric proteins were also recognized by the anti-FliC antibodies, as demonstrated by antibody capture assay (ELISA).Furthermore, these results showed that EGFL and E2FS1 elicited less anti-FliC compared to E1FS1, as illustrated in Figure 3A.

Example 4.3: Adjuvant activity of FliC or SDM in chimeric constructs: Flagellin has the ability to bind to TLR5 receptors that are present on the surface of the immune cells, so as to stimulate immune response. TLR5 (Toll Like Receptor 5) receptor is an (extracellular) pattern recognition receptor (PRRs) recognizes D1 domain of flagellin and stimulates cascade of signalling pathways to induce immune response. To demonstrate whether the generated chimeric fusion proteins in the present invention stimulate TLR5 receptor or not, we stimulated TLR5 reporter cell lines (i.e HEK-Blue Human TLR5 reporter cell lines) with chimeric fusion proteins of the present invention described in the following section

Example-4.3a: Cell lines and treatments: HEK – Blue Human TLR5 reporter cell lines purchased from Invivogen, California, USA. These cells were prepared by co-transfection of human TLR5 gene and codon-optimized SEAP (secreted embryonic alkaline phosphatase) as a reporter gene into HEK 293 cells. Cell lines were cultured in DMEM, 4.5 g/l glucose, 2-4 mM L-glutamine, 10% (v/v) fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, 100 µg/ml Normocin™. Upon stimulation of cells with chimeric fusion proteins containing full length or truncated flagellin, NFkB gets activated, which in-turn secrets SEAP and it was measured in cell supernatant using spectrophotometer at wavelength 630-650 nm.

Example-4.3b: TLR5 Specific Reporter Assay: HEK – Blue Human TLR5 reporter cells (5x104 / well) were plated and cultured overnight in a humidified CO2 incubator at 37ºC. Next day, cells were treated with various concentrations of chimeric fusion protein/s, (0.0001 to 1000 ng/ml) and cultured for 16 hours. Cells were also treated with Flagellin (FliC) or truncated flagellin (SDM) as a positive control. EGF was also tested for TLR5 activity, but EGF didn’t show any activity (hence results not shown). Supernatant was collected and treated with Quanti blue detection reagent at 37ºC for 15-30 minutes. Absorbance was read at 630 nm. Dose response curve was generated by plotting concentration of protein on X-axis and % response on the Y-axis is as shown in Figure 4A and Figure 4B. To generate dose response curve, highest absorbance shown at tested concentration was taken as 100% response and the least absorbance was taken as 0% response. Effective concentration at 50% (EC50) response was determined from the dose response curve.

In the present invention, proteins showed maximum 100% response with a high absorbance at 1000 ng/ml concentration (reached plateau at this concentration in sigmoidal curve), whereas, 0% response with a low absorbance at 0.0001 ng/ml concentration. These results indicated EC50 as 3.5, 0.006, 0.01, 0.99 & 0.08 ng/ml of EGFL, E1FS1, E2FS1, FliC & SDM respectively (concentration at which protein(s) showed half maximal response).

In conclusion, the results obtained indicated that the chimeric fusion proteins EGFL / E1FS1/ E2FS1 of the present invention were able to stimulate immune cells that express TLR5 receptors, which in-turn determines its ability to enhance immune response and thereby has the capacity to act as an adjuvant as a whole for elevated immune responses, as illustrated in Figure 4A and Figure 4B.

Example 4.3c: Anti-tumorigenic Property Assay (In-Vitro): Usually, activated macrophages or immune cells that express TLR5, secretes IL-8, which in-turn induces physiological responses that lead to the recruitment of granulocytes, neutrophils at the site of infection or it leads to phagocytosis or chemotaxis. Secretion of IL-8 is also a measure of anti-tumorigenic property. In the present invention, to confirm IL-8 secreted by activated immune cells with TLR5 was measured by ELISA, which in-turn signifies the anti-tumorigenic property of the chimeric fusion proteins of the present invention as disclosed in Example 4C above. To estimate IL-8, supernatant collected from stimulated HEK Blue reporter cells with chimeric protein(s) or flagellin was used and performed ELISA as disclosed in the below sections.

Example 4.3d: Determination of IL-8 by ELISA: To quantify human Interleukin 8 (hu IL-8) in cell culture supernatant, Enzyme Linked Immunosorbent Assay (ELISA) kit purchased from Thermo scientific (Cat#EH2IL8) was used. All reagents, standards, samples were prepared as per the instruction manual. A series of 2.5fold dilutions of standard ranging from (0, 25.6, 64, 160, 400 & 1000 pg/ml) and similarly, 4-fold dilutions (1:4, 1:16 & 1:64) of cell supernatant were prepared to quantitate IL-8 concentration secreted by cells after TLR5 stimulation. Briefly, anti-huIL-8 pre-coated 96 well strip plate was used and to this, 50 µL standard or samples were added to each well. The plate was incubated at room temperature for 1 hour and washed the plate thrice. Later, 50 µL/well of biotinylated antibody diluted as per manual instructions was added to each well and incubated at room temperature for 1 hour. Later, 100 µL of streptavidin-HRP was added to each well and incubated at room temperature for 30 minutes. Finally, after washes, 100 µL of TMB solution was added to each well and incubated at RT for 15 minutes. Reaction was stopped by the addition of 100 µL of stop solution to each well and the plate was read at 450 nm. Figure 3 represents, bar graph of IL-8 cytokine levels secreted by chimeric protein and FliC (as a positive control).

Example 4.4: Anti-Cell Proliferation Assay: Several antitumor therapies target the EGFR pathway as a way of treatment. However, the use of tyrosine kinase inhibitors (TKIs) namely Geftininb or Erlotinib etc., are one among are the standard treatment in advanced NSCLC with EGFR mutations (Rosell R, et al., 2012; Maemondo M, et al., 2010). To know the effect on mechanism of action of mouse anti-EGF antibodies raised against recombinant chimeric proteins (EGFL or E1FS1 or E2FS1 or BsE2FS1 or E1FS2 or E2FS2 or EGFL2), EGFR mutant NSCLC cell lines (NCI-H1975, ATCC, 10×103 cells or PC9 Cells, ATCC, 3×103 cells) were treated and analyzed for anti-cell proliferation. Cell growth and viability is assessed by MTT assay. As the EGF is considered to boost the cell proliferation in cancer cells, this experiment is conducted to analyse the anti-proliferative effect of sera raised against chimeric proteins in the presence of EGF on human lung cancer cell line.

Anti-proliferative activity of Most known TKIs:
In-Vitro Assay: Lung Cancer Cells (NCI-H1975, ATCC, 10x103 cells or PC9 Cells, ATCC, 3×103 cells) were seeded in 96 well plate using RPMI 1640 complete media (10% FBS and 1% PenStrep), allowed to attach for 24 hours at 37? in 5% CO2 incubator. Media was discarded and treated with EGF or mouse anti-sera raised against recombinant chimeric proteins (EGFL or E1FS1 or E2FS1 or BsE2FS1 or E1FS2 or E2FS2) or EGFR TKIs (Gefitinib at IC50 – Inhibitory concentration that causes 50% deaths) either individually or in combinations for 72 hours in RPMI 1640 media without FBS. Mouse unvaccinated sera and anti-sera raised against recombinant proteins (EGF or FliC or SDM) were used as controls. After 72 hours of treatment, supernatant was discarded and cells were incubated with 100 µls of MTT (Thiazolyl Blue Tetrazolium Bromide) Reagent at a concentration of 10 mg/ml for 2-3 hours incubation at 37?. Plate was observed for formazan crystals and MTT reagent was removed. Crystals were dissolved in 50 µls of DMSO and read at 540 nm. This assay revealed that the vaccinated sera raised against recombinant chimeric proteins were able to suppress the stimulating effects of human EGF (hEGF) on cell proliferation, whereas unvaccinated sera had no effect on cell proliferation. Further, combination treatment of Geftinib along with vaccinated sera raised against chimeric proteins shown more anti-cell proliferation effect compared to cells that are treated with either vaccinated sera alone or with Geftinib treatment alone at IC50 (Figure 5A and Figure 5B). The results of Anti-Proliferation Assay have been shown in Tables 5 and 6.

S.No Description % Proliferation

Controls (Unstimulated Cells, No EGF)
1 Cells Alone 100
2 Cells + Unvaccinated Sera 99.5 ± 2.082
EGF (10ng) stimulated Cells with vaccinated mice sera
3 Cells 115.4 ± 5.092
4 Cells + Unvaccinated Sera 110 ± 2.517
5 Cells + Vaccinated Sera against EGF 104.8 ± 7.017
6 Cells + Vaccinated Sera against EGFL 90.33 ± 6.429
7 Cells + Vaccinated Sera against E1FS1 97.65 ± 14.66
8 Cells + Vaccinated Sera against E2FS1 92.39 ± 10.05
9 Cells + Vaccinated Sera against FliC 102.5 ± 10.61
10 Cells + Vaccinated Sera against SDM 97 ± 5.657
TABLE 5: Anti-Proliferation Assay

S.No Description % Growth Inhibition

Controls (Unstimulated Cells, No EGF)
1 Cells + Unvaccinated Sera + Geftinib 25.11 ± 2.113
EGF (10 ng) Stimulated Cells, treated with Geftinib at IC50 alone
2 Cells + Unvaccinated Sera + Geftinib 49.85 ± 0.2192
EGF (10 ng) Stimulated Cells, treated with combination of Geftinib at IC50 and vaccinated mice sera
3 Cells + Vaccinated Sera against EGF 41.06 ± 1.615
4 Cells + Vaccinated Sera against EGFL 32.59 ± 1.294
5 Cells + Vaccinated Sera against E1FS1 35.15 ± 1.396
6 Cells + Vaccinated Sera against E2FS1 26.91 ± 1.067
7 Cells + Vaccinated Sera against FliC 38 ± 1.51
8 Cells + Vaccinated Sera against SDM 39.44 ± 1.564
TABLE 6: Anti-Proliferation Assay along with Geftinib

Example 5: Evaluation of mice immune response by the Immunotherapeutic vaccine candidates comprising human epidermal growth factor

In order to demonstrate whether recombinant chimeric proteins (EGFL or E1FS1 or E2FS1) are able to elicit antibodies against huEGF, vaccination study was performed in C57BL/6 mice.

Immune response of mice against recombinant chimeric proteins:
Example 5.1: Immunization: C57BL/6 mice (4-8week old, female) mice were purchased and maintained in the animal care facility under standard approved protocols. All procedures involving mice were carried out with the approval of Institutional Animal Ethics Committee. For this, C57BL/C mice (6-8 week old, female, n=6) were injected intra-muscularly with chimeric proteins (EGFL or E1FS1 or E2FS1, 50 µg/mouse/100 µl) on day 0, 14 and 28. Mice were bled one day before each immunization and 14 days after last immunization. Sera was separated from blood and used to evaluate the total anti-EGF/anti-FliC antibody titer & its antibody isotypes (IgG1, IgG2a or IgG3) by ELISA. In this experiment, control group of mice were also injected with EGF or FliC or SDM. Group of mice, received EGF were tested at three different concentrations (5, 10 & 16 µg/mouse/100 µl), which is equivalent to 50 µg of EGFL, E1FS1 & E2FS1 respectively. These equivalent EGF concentrations generated based on the equimolar ratio of EGF & FliC or SDM from chimeric proteins. It was established that chimeric proteins elicited high titer anti-EGF antibodies. Anti-EGF antibody titer elicited by EGFL (25 µg/mouse) in mice is represented as in Figure 6A. End point titer representing anti-EGF specific antibody raised against chimeric proteins EGFL, E1FS1, and E2FS1 and are depicted in Figure 6B, Figure 6C and Figure 6D respectively.

Vaccine formulation: Chimeric protein (50µg/mouse of EGFL or E1FS1 or E2FS1) in Phosphate buffered saline, pH 7.4 with 5-40% trehalose mixed with aluminium hydroxide in 1:1 ratio with a recommended aluminium content.

Anti-EGF End Point Titer: Pooled serum samples were used to determine anti-EGF antibody titer. To perform ELISA, 96 well microtiter plates were coated with huEGF at a concentration [1 or 10 µg/ml, 100 µl/well either in PBS or carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6)] and kept at 4ºC for overnight. Next day, plates were washed with washing buffer (PBST) and blocked with blocking buffer (PBS with 1% BSA) at RT for 1-2 hour ELISA plates were washed again with wash buffer (PBS, 0.1% Tween™20) and added serially diluted (dilutions from 1;50 to 819200 in PBS, 0.1% BSA, 0.05% Tween™20, 0.02% sodium azide) sera from hyper immunized mice and incubated at RT for 1 hour. After one hour incubation, wells were washed and added Goat Anti- mouse IgG HRP conjugate antibody (dilution 1:5000) and kept for 1 hour incubation at RT. After incubation, wells were washed, and developed with TMB as a substrate. Absorbance was read at 450 nm. Threshold (Mean + 3SD) was established by taking the absorbance of negative control (PBS) group. Anti-EGF end point titers were determined after considering the threshold value.

End point titer representing anti-EGF antibody elicited against chimeric proteins EGFL, E1FS1, and E2FS1 and are depicted in Figure 7A, Figure 7B and Figure 7C respectively. These results demonstrated that EGFL and E2FS1 elicits lesser anti-FliC antibodies as compared to E1FS1.

Anti-FliC End Point Titer: Pooled serum samples were used to determine background elicited against full length or truncated flagellin. To perform this, 96 well microtiter plates were coated with FliC at a concentration [1 or 10 µg/ml, 100 µl/well either in PBS or carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6)] and kept at 4ºC for overnight. Next day, plates were washed with washing buffer (PBST) and blocked with blocking buffer (PBS with 1% BSA) at RT for 1-2 hour. ELISA plates were washed again with wash buffer (PBS, 0.1% Tween™20) and added 4-fold serial dilutions (in PBS, 0.1% BSA, 0.05% Tween™20, 0.02% sodium azide) of hyper immunized sera (1;50 to 819200) and incubated at RT for 1 hour. After one hour incubation, wells were washed and added Goat Anti-mouse IgG HRP conjugate (dilution 1:5000) antibody and kept for 1 hour incubation at RT. After incubation, wells were washed, and developed with TMB as a substrate. Absorbance was read at 450 nm. Threshold (Mean + 3SD) was established by taking the absorbance of negative control (PBS) group. Anti-FliC end point titers were determined after considering the threshold value.

IgG sub class (Isotype) determination: In order to demonstrate, the type of IgG subclass, pooled mice hyper immunized serum samples were used to perform ELISA. Microtiter (96 well) plates were coated with huEGF at a concentration [1 or 10µg/ml, 100µl/well either in PBS or carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6)] and kept at 4ºC for overnight. Next day, plates were washed with washing buffer (PBST) and blocked with blocking buffer (PBS with 1% BSA) at RT for 1-2 hour. ELISA plates were washed again with wash buffer (PBS, 0.1% Tween™20) and added serially diluted (in PBS, 0.1% BSA, 0.05% Tween™20, 0.02% sodium azide) sera from hyper immunized mice and incubated at RT for 1 hour. After one hour incubation, wells were washed and appropriate dilutions of anti mouse IgG isotype subclass antibodies (IgG1 or IgG2a or IgG3) were added for incubation at RT. Wells were washed and incubated 1 hour with Goat Anti- mouse IgG HRP conjugate secondary antibody. After incubation, wells were washed, and developed with TMB as a substrate. Absorbance was read at 450 nm. Threshold (Mean + 3SD) value was established by taking the absorbance of negative control (PBS) group. Th1/Th2 index was calculated using the formula as [IgG2a+IgG3)/2]/[(IgG1). This assay demonstrated that all chimeric proteins biased towards Th2 responses, as indicated by Th1/Th2 index, which was ?1, which is illustrated in Figure 6E.

Example 6: Immunogenicity of chimeric proteins in combination with adjuvant/s:

The term "adjuvant" as used herein refers to a specific stimulator of the immune cells which when combined with the chimeric vaccine antigens of the present invention provides an even more enhanced antigen specific immune response. In this illustrative embodiment,, to enhance the immune response of chimeric protein(s), apart from the presence of flagellin in the chimeric vaccine antigen, various adjuvants (aluminum hydroxide and MDP, GMDP and their derivatives, MPL, poly (I:C), CpG etc.,) were co-administered with the chimeric vaccine antigens either alone or in combination with delivery system (emulsions Oil in Water or Water in Oil etc.,) or combination of one or more of the adjuvants.

Vaccination: To demonstrate enhanced immune response in the presence of adjuvants, C57BL/C mice (6-8week old, female, n=6) were injected intra-muscularly either with chimeric proteins (EGFL or E1FS1 or E2FS1, 50µg/mouse) alone or in combination with aluminium hydroxide (1:1) on day 0 and 14. Blood was collected on day 28 and separated serum for further antibody titer analysis by ELISA as mentioned in example 5.1. These results indicated that anti-EGF antibody titers were further enhanced in the presence of adjuvants as an additional component of the vaccine composition comprising the chimeric proteins of the present invention. EGF specific antibody titers obtained when chimeric protein EGFL or E1FS1 and E2FS1 tested in combination with alum are represented in Figure 8A, Figure 8B and Figure 8C respectively.

Example 7: Therapeutic Vaccination in Tumor Induced Mice Model:

The main objective of this experimental procedure was to demonstrate that the chimeric proteins comprising autologous huEGF are able to show antitumor effect against EGF dependent tumors. To conduct therapeutic vaccination against chimeric proteins, tumor induced mice model (syngeneic model) was used and the protocol is as mentioned below.

Example 7.1 Cell lines: LLC1 (Lewis Lung Carcinoma 1) cell lines were obtained from the American Type Culture Collection (ATCC Cat# CRL-1642). LLC1 cells were cultured in DMEM medium (Invitrogen). Medium was supplemented with 10% fetal bovine serum (Invitrogen), 100 IU/mL of penicillin, and 100 µg/mL of streptomycin (Invitrogen) and cells grown at 37°C in a humidified incubator containing 5% CO2. C57BL/6 mice (5-6 week old, female, n=15) were injected with 0.2 × 106 or 1 × 106 LLC1 cells/0.2 ml PBS, on day 0 via subcutaneous route (s.c). Simultaneously, control group mice (n=10) were also injected with PBS and maintained. Body weight and tumor size were measured twice a week using weighing balance and vernier callipers, respectively. Tumor volume was calculated using the formula w2×l2 where w = width and l = length. Mice developed tumor from day 7 onwards. Mice that received 1×106 cells/mouse, shown rapid tumor development, whereas mice that received 0.2×106 cells/mouse have shown slow tumor growth (Figure 9). Hence, high cell density (1×106 cells/ mouse) was selected to generate mice induced tumor model, for therapeutic vaccination study by chimeric proteins comprised of EGF.

Example 7.2 Therapeutic Vaccination against EGFL: C57BL/6 mice were injected with LLC1 (1×106/0.2ml) cells resuspended in PBS, on day 0 via subcutaneous route (s.c). Tumor volume was measured and mice bearing tumor size ranging less than 2 cm were selected for therapeutic vaccination study. Further, these tumor induced C57BL/6 mice were randomized into four groups (n=15), based on the tumor size. First group (i.e tumor induced, untreated, n=15) of mice were used as controls, to compare the survival rate. Other three groups of mice were treated with 3 doses of EGFL or EGF or FliC, at an interval of 14 days, starting on day 10. Second group (i.e tumor induced, treated, n=15) of mice were treated with immunotherapeutic vaccine candidate 50 µg/mouse of EGFL. Third group of mice received EGF at 5 µg/mouse, which is of equivalent to EGF concentration in EGFL, interms of equimolar ratio. Fourth group of mice received FliC at 50 µg/mouse. EGF & FliC were tested as controls in third & fourth group respectively, as these two proteins are part of chimeric protein, a therapeutic vaccine candidate. Mice were also observed daily for any abnormal behavioural patterns, clinical signs and mortality. Survival rate was determined among groups.

Mice treated with EGFL showed approximately 20% survival benefit compared to untreated mice (mice received cells alone) as shown in Figure 11A, which is statistically significant (p value = 0.02), according to Log rank (Mantel Cox), with Hazard Ratio 2.84. Mice received EGF & FliC didn’t show significant survival benefit showing Hazard Ratio (mentioned in Figures as HR) of 0.93 and 1.0 respectively, as shown in Figures 11D and 11E respectively.

Example 7.3 Therapeutic Vaccination against E1FS1: C57BL/6 mice were injected with LLC1 (1x106/0.2ml) cells resuspended in PBS, on day 0 via subcutaneous route (s.c). Tumor volume was measured and mice bearing tumor size ranging less than 2 cm were selected for therapeutic vaccination study. Further, these tumor induced C57BL/6 mice were randomized into four groups (n=15), based on the tumor size. First group (i.e tumor induced, untreated, n=15) of mice were used as controls, to compare the survival rate. Other three groups of mice were treated with 3 doses of E1FS1 or EGF or SDM, at an interval of 14 days, starting on day 10. Second group (i.e tumor induced, treated, n=15) of mice were treated with immunotherapeutic vaccine candidate 50 µg/mouse of E1FS1. Third group of mice received EGF at 10 µg/mouse, which is of equivalent to EGF concentration in E1FS1, interms of equimolar ratio. Fourth group of mice received SDM at 50 µg/mouse. EGF & SDM were tested as controls in third & fourth group respectively, as these two proteins are part of chimeric protein, a therapeutic vaccine candidate. Mice were also observed daily for any abnormal behavioural patterns, clinical signs and mortality. Survival rate was determined among groups.

Mice treated with E1FS1 showed 20 % survival benefit, compared to untreated mice (mice received cells alone), which is statistically significant, according to Log rank (Mantel Cox) test with a p value 0.01) and showed, Hazard ratio 2.3 as shown in Figure 11B. Mice received EGF & SDM didn’t show significant survival benefit showing Hazard Ratio of 0.93 and 0.63 respectively as shown in Figure 11D and Figure 11 F respectively.

Example 7.4 Therapeutic Vaccination against E2FS1: C57BL/6 mice were injected with LLC1 (1×106/0.2ml) cells resuspended in PBS, on day 0 via subcutaneous route (s.c). Tumor volume was measured and mice bearing tumor size ranging less than 2cm were selected for therapeutic vaccination study. Further, these tumor induced C57BL/6 mice were randomized into four groups (n=15), based on the tumor size. First group (i.e tumor induced, untreated, n=15) of mice were used as controls, to compare the survival rate. Other three groups of mice were treated with 3 doses of E2FS1 or EGF or SDM, at an interval of 14 days, starting on day 10. Second group (i.e tumor induced, treated, n=15) of mice were treated with immunotherapeutic vaccine candidate 50 µg/mouse of E2FS1. Third group of mice received EGF at 16 µg/mouse, which is of equivalent to EGF concentration in E2FS1, interms of equimolar ratio. Fourth group of mice received SDM at 50 µg/mouse. EGF & SDM were tested as controls in third & fourth group respectively, as these two proteins are part of chimeric protein, a therapeutic vaccine candidate. Mice were also observed daily for any abnormal behavioural patterns, clinical signs and mortality. Survival rate was determined among groups.

Mice treated with E2FS1 showed approximately 20% survival benefit, compared to untreated mice (mice received cells alone), showed Hazard ratio of 2.2 as shown in Figure 11C.

Example 7.5 Therapeutic Vaccination via Combination therapy: To demonstrate, the efficacy these chimeric proteins in tumor induced mice, after targeted therapy treatment or while undergoing targeted therapy treatment, tumor induced mice were generated and randomized into three groups (n=15 each) as in Example 7.2. One group (i.e untreated, n=15) of tumor induced mice were used as controls, to compare the survival rate. Remaining groups were treated either with targeted therapy (Gefitinib treatment) or therapeutic vaccination (with chimeric proteins, EGFL or E1FS1 or E2FS1) or in combination (Gefitinib treatment followed by therapeutic vaccination with EGFL or E1FS1 or E2FS1). In the present invention, gefitinib (Gef, 2.5 mg/mouse/dose) treatment was administered on day 5 for up to 3-4 weeks (5 days/week). Therapeutic vaccination was given with EGFL or E1FS1 or E2FS1 (50 µg/mouse/dose) on day 7 or 9, followed by 3 vaccinations at an interval of 14 days (i.e at day 7, 21, 35 & 49). Mice treated with combination therapy received both targeted therapy for 3-4 weeks along with 3 doses of EGFL or E1FS1 or E2FS1 (50 µg/mouse/dose) vaccination. Body weight and tumor size were measured twice a week using weighing balance and vernier callipers, respectively. Though the tumor size started to grow drastically across all mice that received LLC1 cells from Day 7 to day 17, thereafter there was a slow growth in treated mice that received either targeted therapy or immunotherapy or combinational therapy. Further, significant slow growth was observed in mice which received combination therapy compared to tumor induced mice (untreated mice) (Figure 10A).

Mice were also observed daily for any abnormal behavioural patterns, clinical signs and mortality & mice survival rate was determined among groups. Combination of Targeted therapy & immunotherapy showed better survival rate. EGFL protein survived for longer period showing 20% more survival benefit compared to mice received LLC1 cells alone as shown in Figure 11A. Further, mice received combination therapy, i.e. Geftinib treatment followed by EGFL treatment showed 50% survival benefit as compared to Geftinib alone with a hazard raio 2.3, as shown in Figure 12A and Figure 12B. The order of survival rate EGFL+Gef (50%) > Gef (19%) > EGFL > LLC1. Similar results were found for the E1FS1 and E2FS1 chimeric proteins. In conclusion, these results demonstrated that EGF based Chimeric fusions proteins are in synergy with targeted therapy.

Example 7.6 Anti-EGF titer determination after therapeutic Vaccination: Mice were bled at one day before each vaccination from all groups (both treated, untreated and other control groups), as mentioned in examples 7.2 to 7.5. Serum was collected from blood and used to estimate mouse EGF levels and mouse anti-EGF antibodies by ELISA as given in Example 5.1 and 7.7. Vaccinated groups either individually or combination with Gefitinib induced high anti-EGF antibodies. Mice received combination therapy clearly showed an inverse relation between anti-EGF antibody titer and EGF levels (Figure 10B) estimated in the treated mice (either drug alone or fusion protein or combination therapy), which in turn benefited with increased survival rate (Figure 12A and Figure 12B).

Example 7.7 Murine EGF Quantitation: To evaluate the correlation between the EGF levels and anti-EGF immune response in blood, serum was separated from blood, EGF levels were estimated in both treated & untreated groups using individual mouse sera. To quantify mouse EGF levels, Mouse EGF quantikine ELISA kit (Cat#MEG00) or DY2028 was purchased from R&D systems. All reagents, standard dilutions, Controls and samples were prepared as per the instruction manual. Pre-coated microtiter strips were used to perform ELISA. Serial dilutions of standard or samples prepared as per instructions were added to each well (100µl/well) and incubated plate for 2hrs at RT. After five washes with washing buffer, 100 µL of Mouse EGF Conjugate was added to each well and incubated plate for 2hrs at RT. After washing the plate, Substrate Solution 100 µL was added to each well and incubated for 30 minutes at room temperature. Finally, reaction was stopped by the addition of stop solution. Optical density was measured at wavelength 450nm and subtracted these readings from 540 or 570nm. Standard graph was plotted by taking EGF standard concentrations on X-axis and optical density on the Y-axis. EGF levels in samples were also calculated by using the regression equation. Figure 12A and Figure 12B showed that combination of targeted therapy & immunotherapy showed better survival rate.

References:

1. Rosell R, Carcereny E, Gervais R, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation positive non–small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 2012; 13: 239–246.

2. Maemondo M, Inoue A, Kobayashi K, et al. Gefitinib or chemotherapy for non–small-cell lung cancer with mutated EGFR. N Engl J Med. 2010; 362: 2380–2388.

3. Sang Hoon Rhee, Eunok IM, and Charalabos Pothoulakis; Toll-Like Receptor 5 Engagement Modulates Tumor Development and Growth in a Mouse Xenograft Model of Human Colon Cancer. Gastroenterology 2008, 135:518–528.

4. Li Fen Lee, Ronald P Hellendall, Ying Wang, J. Stephen Haskill, Naofumi Mukaida, Kauji Matsushima, Jenny Y-P Ting (2000). IL-8 reduced tumorigenicity of human Ovarian cancer In Vivo due to neutrophil infiltration. J Immunol 2000; 164:2769-2775.

,CLAIMS:We claim:
1. A vaccine composition for immunotherapy against cancer, comprising:
a. vaccine antigen, the said vaccine antigen is a chimeric protein or construct;
b. adjuvants;
c. stabilizers; and
d. a physiologically acceptable buffer selected from phosphate and citrate.

2. The vaccine composition as claimed in claim 1, wherein the vaccine antigen comprising one or more of the chimeric protein sequences as represented by SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2).

3. The vaccine composition as claimed in claim 2, wherein the chimeric protein is obtained from a codon optimized gene sequences comprising one or more of as represented by SEQ ID No. 1. (EGFL), SEQ ID No. 2 (E1FS1), SEQ ID No. 3 (E1FS2), SEQ ID No. 4 (E2FS1), SEQ ID No. 5 (E2FS2), SEQ ID No. 11 (BsE2FS1) or SEQ ID No. 12 (EGFL2).

4. The vaccine composition as claimed in claim 2, wherein the chimeric protein comprises of growth factor linked to N-terminal or C-terminal or middle of the full length or truncated carrier protein and is genetically synthesized.

5. The vaccine composition as claimed in claim 2, wherein the chimeric protein is expressed in prokaryotic expression system through a prokaryotic expression plasmid.

6. The vaccine composition as claimed in claim 4, wherein the growth factor is human EGF.

7. The vaccine composition as claimed in claim 6, wherein the human EGF epitopes are exposed on the surface of the chimeric proteins.
8. The vaccine composition as claimed in claim 4, wherein the carrier protein is a genetically synthesized full length Flagellin protein.

9. The vaccine composition as claimed in claim 4, wherein the carrier protein is a genetically synthesized truncated Flagellin protein without the hypervariable region that are from amino acid number 178 to amino acid number 405 or from amino acid number 186 to amino acid number 285.

10. The vaccine composition as claimed in claim 8 or 9, wherein the genetically synthesized sequence is selected from Gram positive bacteria including without limitation to Bacillus subtilis, Clostridium difficile, ????proteobacteria such as Salmonella typhimurium, Enterica species or ??proteobacteria such as Bordetella.

11. The vaccine composition as claimed in claim 8 or 9, wherein the carrier protein helps the immune system to recognize autologous EGF to induces immune response against EGF, and acts as adjuvant to enhance Th2 responses or B cell mediated immune responses.

12. The vaccine composition as claimed in claim 1, wherein the adjuvant is selected for a group comprising one or more of a group of aluminium salts such as aluminium phosphate or aluminium hydroxide, squalene based adjuvants such as MF59, montanide, RIBI adjuvant, incomplete Freund's, glucans, oil-in-water emulsion, MPL, muramyl dipeptide, muramyl dipeptide derivatives, agonists of TLRs (TLR1 to TLR 13) such as MPL, MDP, Imiquimod, poly (I:C), CpG oligonucleotides, Non-CpG oligonucleotides, saponins such as QS-1, ISCOM, ISCOMATRIX, vitamins or immunomodulants such as cytokines, IL-12, IL-15 etc.

13. The vaccine composition as claimed in claim 1, wherein the stabilizers comprises one or more of sugars such as 5-40% Trehalose or sugar alcohols such as 5-40% Glycerol or 5-40% Sorbitol.

14. The vaccine composition as claimed in claim 2, wherein the said vaccine antigens of SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2) are expressed in E.Coli using the pTWIN1 plasmid and purified proteins by self-cleavage, by temperature from 50-65ºC and pH shift ranges from pH 6.5 to 7.5.

15. A method of isolation and purification of proteins of SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1), SEQ ID No. 17 (EGFL2), SEQ ID No. 18 (FliC), SEQ ID No. 19 (SDM) or SEQ ID 20 (EGF) comprising the following steps:
i. introducing the recombinant expression plasmid into bacterial host cells in high cell density growth media;
ii. isolation of protein either in the form of insoluble protein from inclusion bodies or in the form of soluble protein;
iii. purification of chimeric proteins by at least one or two of the following methods: ion exchange chromatography, size exclusion chromatography, affinity chromatography or hydrophobic interaction column chromatography.

16. The vaccine composition as claimed in claim 2, wherein each of the chimeric protein sequence as disclosed in SEQ ID No. 6. (EGFL), SEQ ID No. 7 (E1FS1), SEQ ID No. 8 (E1FS2), SEQ ID No. 9 (E2FS1), SEQ ID No. 10 (E2FS2), SEQ ID No. 16 (BsE2FS1) or SEQ ID No. 17 (EGFL2) are at least 90% - 99% pure.

17. A method of treatment to regulate the tumor growth or to increase the survival rate, comprising therapeutic vaccine composition with or without Tyrosine Kinase Inhibitors (TKIs).

18. The method as claimed in claim 17, wherein the Tyrosine Kinase Inhibitors is comprising 1st or 2nd or 3rd generation TKIs such as Geftinib, Erlotininb, Afatinib, Dacomitinib, Avitinib, Olmutinib, Nazartinib.

19. The method as claimed in claim 15, wherein the high cell density growth media comprises 1.5% yeast extract, 1.5% Casein, 0.4% glycerol, M9 salt solution, trace elements, 1M MgSO4, 1M CaCl2, Biotin, Thiamine and ampicillin.

20. The vaccine composition as claimed in claim 1, wherein the said composition is stable for at least 2 years at 2-8°C and up to 2 weeks at 37°C.

Dated this 30th day of November 2018.

Afzal Hasan
IN/PA-1328
of HASAN AND SINGH
for BHARAT BIOTECH INTERNATIONAL LIMITED