FORM 2
THE PATENTS ACT 1970
(39 OF 1970)
COMPLETE SPECIFICATION (SECTION 10)
"COMBINATION CHEMOTHERAPY WITH RECOMBINANT
PROTEIN"
UNICHEM LABORATORIES LIMITED,
15 A COMPANY REGISTERED UNDER THE INDIAN COMPANY ACT,
1956, HAVING ITS REGISTERED OFFICE LOCATED AT
UNICHEM BHAVAN, PRABHAT ESTATE,
S.V. ROAD, JOGESHWARI (WEST),
MUMBAI - 100102, MAHARASHTRA, INDIA
The following specification particularly describes the invention and the manner in
which it is to be performed.

COMBINATION CHEMOTHERAPY WITH RECOMBINANT PROTEIN
Cross Reference to Related Applications
The application claims the benefit of Indian Provisional Application No. 201821022667 filed on Dec 18, 2018 and 201821043455 filed on Nov 19, 2018, the entire contents of which are incorporated herein by reference.
Field of Invention
The present invention relates to biopharmaceutical combinations of recombinant lectin protein with other therapeutic agents and their antiproliferative effects towards controlling uncontrolled proliferation of cells in cancers.
Background of Invention
A cancer starts with a primary tumor, which is swelling or morbid enlargement that results from an overabundance of cell growth and division. Tumors may be benign, pre-malignant or malignant, or can signify a lesion with no cancerous potential. A benign tumor is a non-cancerous tumor and a malignant tumor is a cancerous tumor. Cancer is when abnormal cells divide in an uncontrolled way. Cancer can sometimes spread to other parts of the body - this is called a secondary tumor or a metastasis.
Cancer treatment therapies include surgical removal of the cancerous tissues, chemotherapy, polychemotherapy, radiotherapy, immunotherapy, etc. These therapies are often invasive techniques which also affect the normal healthy cells along with the cancer cells. Also these therapies are used in combination in order to achieve synergistic effect resulting in enhancing the therapeutic potential of constituent drugs, decreasing the dose, reducing the toxicity of the drugs and delaying induction of drug resistance.
Chemotherapies for cancer treatment are decided based on the mode of cancer cell proliferation. Different properties of the chemotherapeutic agents such as anti-metabolic agents, which interfere with the production or use of metabolites required

by cancers; anti-microtubule agents, which interfere with micro-tubule formation, necessary for mitosis; alkylating agents, which are able to interfere with DNA alkylation by blocking DNA replication; platinum based agents which are able to cross-link DNA; antitumor antibiotic agents, and topoisomerase inhibitors, which inhibit the topoisomerase enzyme necessary for DNA replication.
Oncofetal Thomsen-Friedenreich antigen (Galβ-3GalNAc-a-O-Ser/Thr, T or TF), which is expressed in more than 90% of cancers including oral cancer, colon cancer, ovarian cancer as well as bladder cancer and expression of TF is correlated with tumor progression and metastasis. Applicant's previous patent application 350/MUM/2009 discloses a recombinant lectin protein isolated from sclerotial bodies of fungus Sclerotium rolfsii having high binding specificity toward the oncofetal TF carbohydrate antigen.
A combination therapy for cancer treatment is an effective treatment due to undesirable cytotoxicity of healthy cells. The complexity of the disease, its tendency to spread beyond its original site and become resistant to certain drugs and its genetic diversity underscores the need for a variety of approaches to attack it. Combination therapy not only increases the chances of a cure or long-term remission, but also reduces damage to vital organs and tissues more than a single approach. One type of therapy can sometimes make a tumor more vulnerable to a second type. Certain chemotherapy drugs, for example, can increase tumors' vulnerability to radiation therapy. In other cases, drugs may have a synergistic effect when administered together and enhance each other's potency, so the combined effectiveness is greater than the individual impact. Using them in combination has a multiplier effect: their benefits are magnified many times over. US5053386 (1991) refers to the composition and methods of treatment comprising lectins, Abrin and Abrus Agglutinin for the suppression of post-surgical malignant tumor metastasis. It also discloses the use of a protein in combination with either or both radiation treatment and/or chemotherapy.
Molecular and cellular biochemistry (Vol - 394 (1-2); Page - 225-235; Year - 2014) discloses the effects of lectin from Korean mistletoe (Viscum album var. coloratum

agglutinin, VCA) and doxorubicin (DOX) in MCF-7 (estrogen receptor-positive) and MDA-MB231 (estrogen receptor-negative) human breast cancer cells, in combination and alone. Combination of VCA and DOX showed, a strong synergistic effect in cell growth inhibition, compared to VCA or DOX treatment alone.
Oncotarget (Vol: 8 (26), Page: 42466 -42477, Year: 2017) discloses combination treatment with pemetrexed and Sialic acid-binding lectin isolated from Rana catesbeiana oocytes (cSBL) resulting in greater dose-dependent cytotoxicity than combination of pemetrexed and cisplatin, the standard of care in mesothelioma.
However, the difficulty is not all these combination therapies result in beneficial effects. Hence presently research is focused on developing new and useful anti-proliferative combination partners.
To fight a life-threatening disease like cancer, it is the need of an hour to develop best method for its prevention and treatment. Combination therapy seems to be one of such options.
Object of the Invention
The main object of the present invention is to provide an alternative method to treat or prevent growth of cancer cells. More specifically the objective is to develop an affordable and efficient method to treat or prevent growth of cancer cells.
Another object of the present invention is to provide an effective therapy for prevention or treatment of cancer. Precisely the objective of the present invention is to provide a combination therapy for the prevention or treatment of cancer, wherein the combination is a synergistic combination comprising a recombinant lectin protein.
Yet another object of the present invention is to provide a therapeutically effective combination for the prevention and/or treatment of cancer.

Another object of the invention is to provide an effective concentrations of the therapeutically effective synergistic combination for the prevention and/or treatment of cancer.
Yet another object of the invention is to provide a pharmaceutically acceptable formulation of effective concentrations of the therapeutically effective combination for the prevention and/or treatment of cancer.
Yet another object of the invention is to provide combinations of recombinant lectin protein having amino acid sequence of SEQ ID NO: 1 useful in the prevention, treatment or inhibition of proliferation of cancer cells.
Summary of Invention
According to the first aspect of the present invention, there is provided a therapeutically effective combination comprising a recombinant lectin protein and one or more other therapeutic agent.
According to a second aspect of the present invention, there is provided a therapeutically effective combination comprising recombinant lectin protein and one or more other therapeutic agent, wherein the combination is synergistic, and wherein the concentration of recombinant lectin protein is in the range from 0.5 µg/mL to 100 µg/mL. The said therapeutic combination is preferably used in the prevention or treatment of cancer in a subject.
According to the third aspect of the present invention, there is provided the use of recombinant lectin protein for the treatment or prevention of cancer in a subject wherein the recombinant lectin protein is administered in combination with other therapeutic agent, and wherein the other therapeutic agent is administered simultaneously, separately or sequentially.
According to the fourth aspect of the present invention, there is provided a method of treatment or prevention of cancer or inhibition of growth of tumor cells in a subject comprising administering to the subject effective amount of recombinant

lectin protein in combination with other therapeutic agent, wherein the other therapeutic agent is administered simultaneously, separately or sequentially.
According to the fifth aspect of the present invention, there is provided a combination therapy for prevention, proliferation, treatment or to cure cancer or tumor in a subject comprising administration of recombinant lectin protein either in combination with one or more other therapeutic agent or simultaneous, separate or sequential administration of one or more other therapeutic agents.
Yet another aspect of the present invention provides a recombinant lectin protein in combination with other therapeutic agent for the treatment or prevention of cancer in a subject. The other therapeutic agent is administered simultaneously, separately or sequentially. According to the specific aspect of the invention, there is provided a recombinant lectin protein having amino acid amino acid sequence of SEQ ID NO: 1, 2 or 3 in combination with other therapeutic agent for the treatment or prevention of cancer.
According to yet another aspect of the present invention, there is provide a therapeutic agent for the treatment or prevention of cancer, in combination with a recombinant lectin protein.
According to another aspect of the present invention, there is provided a composition comprising recombinant lectin protein in combination with other therapeutic agent and one or more pharmaceutically acceptable excipients.
According to the above aspects of the present invention the other therapeutic agent is an anti-cancer agent selected from antimetabolites, alkylating antineoplastic agents, anti-microtubule agents and topoisomerase I inhibitors, wherein antimetabolite is selected from 5-Flurouracil (5-FU), Gemcitabine, Methotrexate, Pemetrexed or Capacitabine; Alkylating neoplastic agent is a platinum based anti¬neoplastic agent selected from cisplatin or carboplatin; Anti-microtubule agent is selected from Paclitaxel, Docetaxel, Abraxane or Taxotere and Topoisomerase I is selected from Irinotecan or Topotecan.

According to the specific aspect of the present invention, there is provided a synergistic combination comprising recombinant lectin protein and an anticancer agent selected from one or more 5-Flurouracil (5-FU), Gemcitabine, Cisplatin, Paclitaxel, Carboplatin or Irinotecan.
Further according to above aspects of the present invention, the concentration of other therapeutic agent is in the range from 0.001 11M to 1000 µM.
According to yet another specific aspect of the present invention, there is provided a synergistic combination comprising:
a) recombinant lectin protein and Cisplatin for the treatment or prevention of oral cancer, ovary cancer or bladder cancer;
b) recombinant lectin protein and 5-FU for the treatment or prevention of oral, pancreatic or colon cancer;
c) recombinant lectin protein and lrinotecan for the treatment or prevention of colon cancer;
d) recombinant lectin protein and Paclitaxel for the treatment or prevention of ovary or breast cancer;
e) recombinant lectin protein and Gemcitabine for the treatment or prevention of bladder or pancreatic cancer;
f) recombinant lectin protein and carboplatin for the treatment or prevention of breast cancer.
According to another specific aspect of the present invention, there is provided a recombinant lectin protein for the treatment or prevention of:
a) oral cancer, ovary cancer or bladder cancer, wherein recombinant lectin protein is administered in combination with Cisplatin, and wherein Cisplatin is administered simultaneously, separately or sequentially;
b) oral, pancreatic or colon cancer, wherein recombinant lectin protein is administered in combination with 5-FU, and wherein 5-FU is administered simultaneously, separately or sequentially;

c) colon cancer, wherein recombinant lectin protein is administered in combination with Irinotecan, and wherein Irinotecan is administered simultaneously, separately or sequentially;
d) ovary cancer or breast cancer, wherein recombinant lectin protein is administered in combination with Paclitaxel, and wherein Paclitaxel is administered simultaneously, separately or sequentially;
e) bladder cancer or pancreatic cancer, wherein recombinant lectin protein is administered in combination with Gemcitabine, and wherein Gemcitabine is administered simultaneously, separately or sequentially;
f) breast cancer, wherein recombinant lectin protein is administered in combination with carboplatin, and wherein carboplatin is administered simultaneously, separately or sequentially.
According to yet another specific aspect of the present invention, there is provided a synergistic combination comprising:
a) recombinant lectin protein and Cisplatin for the treatment or prevention of oral cancer, wherein the concentration of recombinant lectin protein is 1 µg/mL to 90 µg/mL and the concentration of Cisplatin is 0.1 µM to 1.5 µM;
b) recombinant lectin protein and Cisplatin for the treatment or prevention of ovary cancer, wherein the concentration of recombinant lectin protein is 1 µg/mL to 20µg/mL and the concentration of Cisplatin is 0.01µM to 5 µM;
c) recombinant lectin protein and Cisplatin for the treatment or prevention of bladder cancer, wherein the concentration of recombinant lectin protein is 5 µg/mL to 80 µg/mL and the concentration of Cisplatin is 1 µM to 500 µM;
d) recombinant lectin protein and 5-FU for the treatment or prevention of oral cancer, wherein the concentration of recombinant lectin protein is 1 µg/mL to 90 µg/mL and the concentration of 5-FU is 0.01 µM to 5 µM;
e) recombinant lectin protein and 5-FU for the treatment or prevention of colon cancer, wherein the concentration of recombinant lectin protein is 1µg/mL to 80 µg/mL and the concentration of 5-FU is 10 µM to 200 µM;

f) recombinant lectin protein and 5-FU for the treatment or prevention of pancreatic cancer, wherein the concentration of recombinant lectin protein is 5 µg/mL to 80 µg/mL and the concentration of 5-FU is 1 µM to 250µM;
g) recombinant lectin protein and Irinotecan for the treatment or prevention of colon cancer, wherein the concentration of recombinant lectin protein is 20 µg/mL to 80 µg/mL and the concentration of Irinotecan is 1 µM to 50 µM;
h) recombinant lectin protein and Paclitaxel for the treatment or prevention of ovary cancer, wherein the concentration of recombinant lectin protein is 1 µg/mL to 20µg/mL and the concentration of Paclitaxel is 0.25 nM to 5 nM;
i) recombinant lectin protein and Paclitaxel for the treatment or prevention of breast cancer, wherein the concentration of recombinant lectin protein is 10 µg/mL to 80 µg/mL and the concentration of Paclitaxel is 0.1 nM to 10 nM;
j) recombinant lectin protein and Gemcitabine for the treatment or prevention of bladder cancer, wherein the concentration of recombinant lectin protein is 2.5 µg/mL to 80 µg/mL and the concentration of Gemcitabine is 1 µM to 300µM;
k) recombinant lectin protein and Gemcitabine for the treatment or prevention of pancreatic cancer, wherein the concentration of recombinant lectin protein is 5 µg/mL to 80 µg/mL and the concentration of Gemcitabine is 1 µM to 25 µM;
1) recombinant lectin protein and carboplatin for the treatment or prevention of breast cancer, wherein the concentration of recombinant lectin protein is 2.5 µg/mL to 80 µg/mL and the concentration of carboplatin is 50 µM to 1000µM.
Description of Invention
Definitions
The terms "Cancer" and "Tumor", are interchangeably used in the present invention, are having same meaning as understood by the person skilled in the art. They form when the normal cells grow out of control and crowd out healthy body cells. Formation of such an enlarged outgrowth affects the normal functioning of the tissue/organ/organism. Cancer can start any place in the body and can also

spread to other parts of the body. When cancer cells spread within the body, it is called metastasis.
The term "recombinant" means a nucleic acid or a polypeptide has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a "recombinant nucleic acid" is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other well-known molecular biological procedures. A "recombinant DNA molecule" is comprised of segments of DNA joined together by means of such molecular biological techniques. The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule.
The term "lectin" as used herein refers to a carbohydrate-binding protein. More specifically the recombinant lectin protein of the present invention is the lectin derived from Sclerotium rolfsii lectin (SRL). Sclerotium rolfsii lectin (SRL) is a lectin that has been isolated from the sclerotial bodies of the soil-borne phytopathogenic fungus S. rolfsii.
The terms "homology" or "homologous" as used herein refer to two or more referenced entities that share at least partial identity over a given region or portion. Areas, regions or domains of homology or identity refer to a portion of two or more referenced entities that are similar or are the same. Thus, where two sequences are identical over one or more sequence regions they share identity in these regions. Substantial homology refers to a molecule that is structurally or functionally conserved such that it has, or is predicted to have, at least a similar structure or function (e.g., a biological function or activity) to a reference molecule. Or it may means that the molecule has a relevant/corresponding region or portion which it shares homology with that of the reference molecule.
In one embodiment, the percentage "homology" between two sequences is determined using the BLASTP algorithm with default parameters (Altschul et al. Nucleic Acids Res. 1997 Sep l;25(17):3389-402). In particular, the BLAST

algorithm can be accessed on the internet using the URL: https://blast.ncbi.nlm.nih.gov/Blast.cgi. In an alternative embodiment, for global sequence alignments, percentage homology between two sequences is determined using the EMBOSS Needle algorithm using default parameters. In particular, the EMBOSS Needle algorithm can be accessed on the internet using the URL: https://www.ebi.ac.uk/Tools/psa/emboss_needle/.
Unless otherwise indicated, the term "homology" is used interchangeably with the term "sequence identity" in the present specification.
The term "recombinant lectin protein" is intended here to cover any pharmaceutically acceptable salt, solvate, hydrate, prodrug, or any other compound which, upon administration to the patient is capable of providing (directly or indirectly) the compound as described herein. The preparation of salts, solvates, hydrates, and prodrugs can be carried out by methods known in the art.
As used herein, the terms "therapies" and "therapy" can refer to any method, composition, and/or active ingredient that can be used in the treatment, prevention and/or management of the disease or one or more symptoms thereof
The term "combination therapy" as used throughout the specification, is meant to encompass the administration to a patient suffering from cancer of the referred therapeutic agents in the same or separate pharmaceutical formulations, and at the same time or at different times. If the therapeutic agents are administered at different times they should be administered sufficiently close in time to provide for the potentiating or synergistic response to occur.
The term "chemotherapy" refers to the use of drugs to treat cancer. As used herein, a "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer.
The therapeutic agent that may be used in combination with recombinant lectin protein is preferably a chemotherapeutic agent. There are no limitations on the chemotherapeutic agent (the 'term' chemotherapeutic agent has the same meaning as that known to a person skilled in the art) that can be used in this invention. Non

limiting examples of such chemotherapeutic agents are alkylating agents such as, e.g., mitomycin C, cyclophosphamide, busulfan, ifosfamide, isosfamide, melphalan, hexamethylmelamine, thiotepa, chlorambucil, mechlorethamine or dacarbazine; antimetabolites such as, e.g., gemcitabine, capecitabine, 5-fluorouracil, cytarabine, 2-fluorodeoxy cytidine, mcthotrcxate, idatrexate, tomudex or trimetrexate; topoisomerase II inhibitors such as, e.g., doxorubicin, epirubicin, etoposide, teniposide or mitoxantrone; topoisomerase I inhibitors such as, e.g., irinotecan (CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38) or topotecan; antimitotic drugs such as, e.g., paclitaxel, docetaxel, vinblastine, vincristine or vinorelbine; and platinum derivatives such as, e.g., cisplatin, oxaliplatin, spiroplatinum or carboplatinum.
The term "radiation therapy" or "radiotherapy" is the treatment of cancer and other diseases with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated by damaging their genetic material, making it impossible for these cells to continue to grow. Although radiation damages both cancer cells and normal cells, the latter are able to repair themselves and function properly. Radiation therapy used according to the present invention may include, but is not limited to, the use of gamma-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is further contemplated that radiotherapy may include the use of radiolabeled antibodies to deliver doses of radiation directly to the cancer site (radioimmunotherapy) and/or include the use of a radiosensitizer.
The term "immunotherapy" rely on the use of immune effector cells and molecules to target and destroy cancer cells. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells

include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.
As used herein, "Pharmaceutically-acceptable salt" refers to salts which retain the biological effectiveness and properties of compounds which are not biologically or otherwise undesirable. Pharmaceutically-acceptable salts refer to pharmaceutically-acceptable salts of the compounds, which salts are derived from a variety of organic and inorganic counter ions well known in the art.
The term "pharmaceutical combination" refers to any combination of a first and a second pharmaceutical ingredient, whether mixed into a single composition or maintained separately. The term "pharmaceutical composition" or "pharmaceutical]y acceptable composition" or "pharmaceutically acceptable formulation" refers to a mixture of a compound disclosed herein with pharmaceutical excipients, such as diluents or carriers (see, for example, Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (Sep. 15, 2012) and handbook of Pharmaceutical Excipients, 6th edition, Raymond Rowe, Pharmaceutical Press (2009)). The pharmaceutical composition facilitates administration of the compound to an organism. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.
The terms "effective" or "therapeutically effective" refer to an effect sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effect of an inventive combination may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the agents being delivered, the disease being treated, the mode of administration, and the patient. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease, disorder or medical condition is reduced or halted.
Combination Index (CI) values were based on Loewe's additivity model were determined to assess the nature of drug-drug interactions that can be additive (CI=1), antagonistic (CI>1), or synergistic (CI<1) for various drug-drug

concentrations and effect leveJs (Fa: fraction affected; inhibition of cancer ceJJ proliferation). CI values were calculated based on linear regression trendlines using the CompuSyn software (ComboSyn Inc., Paramus, N.J.), following the method by Chou et al, whereby the hyperbolic and sigmoidal dose-effect curves are transformed into a linear form (Chou TC (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70: 440-6, instructions also available at ComboSyn, Inc., www.combosyn.com).
A synergistic effect permits the effective treatment of a disease using lower amounts (doses) of individual therapy. The lower doses result in lower toxicity without reduced efficacy. In addition, a synergistic effect can result in improved efficacy. Finally, synergy may result in an improved avoidance or reduction of disease as compared to any single therapy. The term "additive" refers to sum of any two or more therapeutic agents in combination. As used herein, the term "antagonistic" refers to block (e.g., reduces or prevents) a biological activity. The term "inhibit" or "inhibition" means to reduce by a measurable amount.
As used herein, the term "anti-cancer agent" refers to a substance or treatment (e.g., radiation therapy) that inhibits the function of cancer cells, inhibits their formation, and/or causes their destruction in vitro or in vivo.
The term "Cytotoxic" is used in context with the effect of therapeutic agents of the present invention on the cancer cell lines and means 'toxic to cells'. The therapeutic agents that are effective in destroying the cancer cells are called cytotoxic or described as having cytotoxicity.
The term "IC50" or IC50 refers to an inhibitory dose which causes 50% inhibition of a given quantifiably measureable parameter. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical parameter (or component of a parameter, i.e. an enzyme, cell, cell receptor or microorganism) by half.
The term "synergistic" refers to a combination of two or more therapeutic agents, which is more effective than the additive effects of any two or more single agents.

Synergy of pharmacological agents can be determined quantitatively by calculating Combination Index (CI) values.
CI values can be calculated based on the measured IC50 values for two drugs A and B, using the formula as given in below.

Where,
DA(50) = Concentration of drug A in combination with B to produce 50% cytotoxicity
ICA(50) = Concentration of drug A alone to produce 50% cytotoxicity
DB(50) = Concentration of drug B in combination with A to produce 50% cytotoxicity
ICB(50)= Concentration of drug B alone to produce 50% cytotoxicity
Wherein drug A is a lectin of the invention (e.g. a protein of SEQ ID NO: 1) and drug B is the other therapeutic agent that was tested.
Synergism, additive or antagonism is determined based on the following criteria: CI > 1 - antagonism is indicated CI = 1 - additive effected is indicated CI < 1 - synergism is indicated
As such, if the CI value is calculated to be less than one, this indicates that drugs A and B have a synergistic effect.
As used herein, a "subject" refers to an animal that is the object of treatment, observation or experiment. "Animal" includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles and, in particular, mammals. "Mammal" includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such as monkeys, chimpanzees, and apes, and, in particular, humans.

In addition, any drug referred to herein may be in amorphous form or crystalline form either as free compound or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.
Brief Description of the Accompanying Sequences:
SEQ ID NO: 1: represents a variant of the S. rolfsii lectin amino acid sequence (reported as Rec-2 in WO 2010/095143).
SEQ ID NO: 2: represents a variant of the S. rolfsii lectin amino acid sequence (reported as Rec-3 in WO 2010/095143).
SEQ ID NO: 3: represents a variant of the S. rolfsii lectin amino acid sequence (reported in WO 2014/203261).
Recombinant lectin protein having Amino acid sequence of SEQ ID NO: 1
TYKIT VRVYQ TNPDA FFHPV EKTVW KYANG GTWTI TDDQH VLTMG GSGTS GTLRF HADNG ESFTA TFGVH NYKRW CDIVT NLAAD ETGMV INQQY YSQKN REEAR ERQLS NYQVK NAKGR NFQIV YTEAE GNDLH ANLII G
Recombinant lectin protein having Amino acid sequence of SEQ ID NO: 2
VYKIT VRVYQ TNPDA FFHPV EKTVW KYANG GTWSI TDDQH VLTMG GSGTS GTLRF HADNG ESFTA TFGVH NYKRW CDIVT NLAAD ETGMV INQQY YSQKN REEAR ERQLS NYQVK NAKGR NFQIV YTEAE GNDLH ANLII G
Recombinant lectin protein having Amino acid sequence of SEQ ID NO: 3
VYKIT VRVYQ TNPDA FFHPV EKTVW KYADG GTWSI TDDQH VLTMG GSGTS GTLRF HADNG ESFTA TFGVH DYKRW CDIVT DLAAD ETGMV INQEY YSEKD REEAR ERQNS NYEVK DAKGR NFEIV YTEAE GNDLH ADLII G

Detailed description of the invention
The main aspect of the present invention is the therapeutically effective combination comprising recombinant lectin protein and one or more other therapeutic agent.
Yet another aspect of the present invention is the therapeutically effective combination comprising recombinant lectin protein and one or more other therapeutic agent, wherein the combination is synergistic and wherein the concentration of the recombinant lectin protein is in the range from 0.5µg/mL to 100 µg/mL.
Another aspect of the present invention is the use of recombinant lectin protein for the treatment or prevention of cancer in a subject wherein the recombinant lectin protein is administered in combination with other therapeutic agent, and wherein the other therapeutic agent is administered simultaneously, separately or sequentially.
Yet another aspect of the present invention is a method of treatment or prevention of cancer or inhibition of growth of tumor cells in a subject comprising administering to the subject effective amount of recombinant lectin protein in combination with other therapeutic agent, wherein the other therapeutic agent is administered simultaneously, separately or sequentially.
Yet another main aspect of the present invention is a combination therapy for prevention, proliferation, treatment or to cure cancer or tumor in a subject comprising administration of recombinant lectin protein either in combination with one or more other therapeutic agent or simultaneous, separate or sequential administration of one or more other therapeutic agents.
Further aspect of the present invention provides a recombinant lectin protein in combination with other therapeutic agent for the treatment or prevention of cancer in a subject. The other therapeutic agent is administered simultaneously, separately or sequentially.

The therapeutically effective combination, the use, the method of treatment, the combination therapy or the recombinant lectin protein of the present invention has application in the prevention of growth or metastasis of tumor, treatment or to cure cancer. According to some embodiments the synergistic combination may be used for prevention or treatment of adenocarcinoma, Squamous cell carcinoma. Transitional cell carcinoma, Basal cell carcinoma, Sarcomas, Lymphomas, epithelial cell carcinomas or non-epithelial cell carcinomas. According to the particular embodiment the therapeutically effective combination of the present invention may be used for prevention or treatment of pancreatic cancer, abdominal cancer, liver cancer, prostate cancer, oral cancer, colon cancer, ovary cancer, bladder cancer, kidney cancer, stomach cancer, breast cancer, bone marrow cancer, melanoma, leukemia or central nervous system cancer in a subject.
According to the specific embodiment of the present invention the cancer that is prevented or treated is selected from pancreatic cancer, oral cancer, colon cancer, ovary cancer, bladder cancer, breast cancer.
The recombinant lectin protein in the present invention is the protein having amino acid sequence of SEQ ID NO: 1 or may comprise an amino acid sequence having at least 60% homology to SEQ ID NO: 1. In some embodiments, the recombinant lectin protein may comprise an amino acid sequence having at least 60% homology to SEQ ID NO: 2 or 3. In some embodiments the recombinant lectin protein comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%o homology to SEQ ID NO: 1. In some embodiments, the recombinant lectin protein comprises the amino acid sequence of SEQ ID NO: 1, 2 or 3.
Further according to the aspects of present invention the other therapeutic agent is an anti-cancer agent. The anti-cancer agent is used in chemotherapy for the treatment of cancer. In some embodiments the other therapeutic agent may be selected from are alkylating agents such as, e.g., mitomycin C, cyclophosphamide, busulfan, ifosfamide, isosfamide, melphalan, hexamethylmelamine, thiotepa, chlorambucil, mechlorethamine or dacarbazine; antimetabolites such as, e.g., gemcitabine, capecitabine, 5-fluorouracil, cytarabine, 2-fluorodeoxy cytidine,

methotrexate, idatrexate, tomudex or trimetrexate; topoisomerase II inhibitors such as, e.g., doxorubicin, epirubicin, etoposide, teniposide or mitoxantrone; topoisomerase I inhibitors such as, e.g., irinotecan (CPT-11), 7-ethyl-10-hydroxy-camptothecin (SN-38) or topotecan; antimitotic drugs such as, e.g., paclitaxel, docetaxel, vinblastine, vincristine or vinorelbine; and platinum derivatives such as, e.g., cisplatin, oxaliplatin, spiroplatinum or carboplatinum.
As per American cancer society (www.cancer.org) chemotherapeutic agent used for treatment of breast cancers is selected from Paclitaxel Taxol, Docetaxel Taxotere, Doxorubicin, Epirubicin, Cisplatin, Carboplatin, Vinorelbine, Capecitabine, Gemcitabine, Ixabepilone and Eribulin. Likewise for the treatment of Pancreatic cancers chemotherapeutic agent is selected from Gemcitabine, 5-fluorouracil, Oxaliplatin, paclitaxel, Capecitabine, Cisplatin and Irinotecan. Also for the treatment of the oral cavity and oropharynx chemotherapeutic agent is selected from Cisplatin, Carboplatin, 5-Fluorouracil (5-FU), Paclitaxel, Docetaxel and Hydroxyurea. Similarly for treatment of cancers of the colon or rectum chemotherapeutic agent is selected from 5-FU, Capecitabine, Irinotecan, Oxaliplatin and a combination of Trifluridine and Tipiracil. For treatment of cancers of the ovary the drug is selected from Platinum compounds such as Cisplatin or Carboplatin and taxane, such as Paclitaxel or Docetaxel standalone or in combination and for treatment of bladder cancer the chemotherapeutic agent is selected from Gemcitabine, Cisplatin, Methotrexate, Vinblastine, Doxorubicin, Carboplatin or their combinations.
According to the specific embodiments of the present invention, the other therapeutic agent is selected from Paclitaxel, Cisplatin and Carboplatin for breast cancer or ovary cancer, Cisplatin and 5-FU for oral cancer, Cisplatin and Gemcitabine for bladder cancer, 5-FU and Gemcitabine for pancreatic cancer and 5-FU and Irinotecan for colon cancer.
According to some embodiments of the present invention the other therapeutic agent is administered to the subject in same or separate pharmaceutical

formulations, and at the same time or at different times wherein the other therapeutic agent may be administered simultaneously, separately or sequentially.
The term "simultaneous administration," as used herein, means that the composition comprising recombinant lectin protein and the other therapeutic agent are administered with a time separation of no more than about 15 minute(s), more specifically no more than about any of 10, 5, or 1 minutes. When recombinant lectin protein and the therapeutic agent are administered simultaneously, the recombinant lectin protein and the therapeutic agent may be contained in the same composition (e.g., a composition comprising both, recombinant lectin protein and the one or more therapeutic agent) or in separate compositions (e.g., the recombinant lectin protein is contained in one composition and the one or more therapeutic agents are contained in another composition).
In the "sequential administration" the other therapeutic agent is administered either prior to or after the administration of the recombinant lectin protein. In the sequential administration the recombinant lectin protein and the other therapeutic agent/s may administered with a time separation of more than about 15 minutes, more specifically, more than about any of 20, 30, 40, 50 and 60 or more minutes. Either the recombinant lectin protein or the other therapeutic agent/s may be administered first. The recombinant lectin protein and the other therapeutic agent/s are contained in separate compositions, which may be contained in the same or different packages.
In the "separate administration" the recombinant lectin protein and the other therapeutic agent/s may be administered in a sequential manner wherein one is administered first and the other second or vice versa. The time separation between the two administrations is more than 60 minutes.
The recombinant lectin protein when used standalone exhibited effective cytotoxicity. However when combined with other therapeutic agent, the effect on the test cell lines was highly surprising. The combination not only exhibited additive efficacious effect, but high synergism was also observed. The combinations were highly efficacious for inhibition of growth of cancer cells.

In another specific embodiment of the present invention, there is provide a synergistic combination comprising recombinant lectin protein and other anti-cancer agent selected from Paclitaxel, 5-FU, Carboplatin, Cisplatin, Irinotecan and Gemcitabine. The combination is used for the treatment or prevention of cancer.
The main aspect of the present invention is the combination comprising recombinant lectin protein having amino acid sequence of SEQ ID NO: 1 and Cisplatin, wherein the combination is used for the treatment or prevention of breast, oral, ovarian or bladder cancer,
Another main aspect of the present invention is the combination comprising recombinant lectin protein and 5-FU, wherein the combination is used for treatment or prevention of pancreatic, oral or colon cancer.
Additional main aspect of present invention is the combination comprising recombinant lectin protein and Irinotecan, wherein the combination is used for treatment or prevention of colon cancer.
Yet another main aspect of the present invention is the combination comprising recombinant lectin protein and Paclitaxel, wherein the combination is used for treatment or prevention of ovarian and breast cancer.
Yet another main aspect of the present invention is the combination comprising recombinant lectin protein and Gemcitabine, wherein the combination is used for treatment or prevention of bladder and pancreatic cancer.
Another main aspect of the invention is the synergistic comprising recombinant lectin protein and carboplatin, wherein the combination is used for treatment or prevention of breast cancer.
According to yet another embodiment the combination therapy may include administration of constituents of the present invention simultaneously with the radiation therapy.
Another aspect of the present invention is the composition comprising recombinant lectin protein in combination with other therapeutic agent and one or more pharmaceutically acceptable excipients.

According to some embodiments of the present invention the composition of recombinant lectin protein and that of other therapeutic agent may be a single composition comprising both lectin protein and therapeutic agent, or may be a separate composition, which is administered simultaneously, separately or sequentially
According to the aspect of the invention the combined preparation is in a suitable pharmaceutically acceptable form/pharmaceutical composition. The preparation can be a suitable oral form or a parenterally administrable form or an implant. The preparation, for example, can be in the form of tablets, capsules, lozenges, suspensions, solutions, emulsions, powders, or syrups for intravenous, intramuscular, intraperitoneal, subcutaneous, intradermal, depot injection, intrathecal, transdermal, sublingual, intrahepatic, oral or by inhalation administration.
According to the aspect of the invention the pharmaceutical composition also comprises one or more pharmaceutically acceptable excipient. Pharmaceutically acceptable excipient refer to non-active pharmaceutical ingredient in the formulation known to the skilled person, such as stabilizer, solubilizer, preservative, disintegrators, binders, fillers, and lubricants or any other known to the person skilled in the art.
The composition used in present invention comprise 50 mM Tris Base (Tromethamine), 150 mM Sodium Chloride with pH 8.0±0.2. However such parenteral composition comprise excipients not limited to Dextrose, Glycerol, Sodium chloride or Mannitol as tonicity modifiers; Ascorbic acid, Acetylcysteine, Sulfurous acid salts (bisulfite, metabisulfite) or Monothioglyercol as antioxidants; Phenol, Metacresol, Benzyl alcohol, Parabens (methyl, propyl, butyl), Chlorobutanol, Thimerosal and Phenylmercuric salts (acetate, borate, nitrate) as antimicrobial agents; Calcium disodium, Ethylenediaminetetra acetic acid (EDTA), Disodium EDTA, Sodium EDTA, Calcium or Diethylenetriaminepenta acetic acid as Chelating agents; Polyoxyethylene sorbitan monooleate (Tween 80), Sorbitan monooleate Polyoxyethylene sorbitan monolaurate (Tween 20), Lecithin,

Polyoxyethylene polyoxypropylene copolymers (Pluronics) or Sorbitan trioleate (span 85) as surfactants; and Propylene glycol, Glycerin, Ethanol, Polyethylene glycol, Sorbitol, Dimethylacetamide or Cremophor EL as co-solvent. Other pharmaceutical compositions are prepared according to the prior knowledge of the person skilled in the art using excipients listed in the standard books and acceptable by the regulatory authorities across countries.
The preferable quantity of recombinant lectin protein and the other therapeutic agent in a pharmaceutical composition varies according to the form of composition, route of administration, type, age and genetic make-up of the subject and stage of cancer and the method of treatment.
According yet another embodiment the effective concentrations of recombinant lectin protein may be in the range from 0.5 µg/mL to 100 µg/mL. In a particular embodiment the effective concentration may be in the range from 1 µg/mL to 90 Hg/mL. According to another embodiment the effective concentration of the other therapeutic may be in the range from 0.001 nM to 1000 µM. A skilled person will be aware of the fact that in case of combinations the effective concentration is the concentration of all the ingredients when combined give synergistic effect. Thus the effective concentration of recombinant lectin protein will differ based on the other therapeutic agent, method of treatment and cancer type. Similarly effective concentration of each therapeutic agent will be different for different cancers, when used along with recombinant lectin protein or individually. For example in the present invention effective synergistic concentration in case of ovary cancer cell lines, of recombinant lectin protein was from 1 µg/mL to 20 µg/mL whereas no synergistic effect was seen for breast cancer cell line at a concentration of 2.5 µg/mL of recombinant lectin protein even if Paclitaxel was used as other therapeutic agent in both the case. Similarly effective synergistic concentrations of Paclitaxel in both these cancer is different. Thus the concentrations of all active agents would vary based on the cancer type and the person skilled in the art is well aware of it.
The present inventors performed several studies on different cancer cell lines to examine the effect of recombinant lectin protein standalone and in combination

with therapeutic agent listed above for the treatment of several cancers. The present inventors selected Gemcitabine and 5-FU for Pancreatic cancer, Paclitaxel and carboplatin for Breast cancer, Cisplatin and 5-FU for oral cancer, 5-FU and Irinotecan for colon cancer, Cisplatin and Paclitaxel for ovarian cancer and Cisplatin and Gemcitabine for bladder cancer.
Examples
The examples in the present invention are for demonstration of the effect of SEQ ID NO: 1 standalone and in combination with representative anti-cancer agents against selected cancer cells. The anti-cancer agents selected here represent each class of chemotherapeutic agent that is antimetabolites, alkylating antineoplastic agents, anti-microtubule agents and topoisomerase I inhibitors. Thus the person skilled in the art will be well aware of the fact that any other anti-cancer agent, not included in the examples below but belonging to the class of chemotherapeutic agent listed above, would demonstrate similar synergistic effect with some degree of concentration variations. Therefore examples listed below are for demonstration purpose only and do not limit the scope of this invention in any manner.
The cell line used in the experiments were procured from American Type Culture Collection (ATCC).
Example 1: Cytotoxic effect of SEQ ID NO: 1, Gemcitabine and 5-Fluorouracil (5-FU) standalone, and synergistic cytotoxic effect of SEQ ID NO: 1 in combination with Gemcitabine and 5-FU on Pancreatic cancer cells (PANC-1 cell line)
A study was conducted to determine the synergistic cytotoxic effects of SEQ ID NO: 1 in combination with chemotherapeutic agents, Gemcitabine and 5-Fluorouracil, in pancreatic cancer cell line (PANC-1). The study was conducted in two phases, described in Example 1-1 and Example 1-2.

Example 1-1 - Measurement of IC50 values of Gemcitabine and 5-FU in the presence of SEQ ID NO: 1 for cytotoxicity in PANC-1 cell lines
A study was performed to determine the effect of the concentration of the recombinant lectin represented by SEQ ID NO: 1 on the IC50 of Gemcitabine for cytotoxicity in PANC-1 cells. Conversely the effect of the concentration of Gemcitabine on the IC50 of the recombinant lectin represented by SEQ ID NO: 1.
Similarly, another study was performed for 5-FU in combination with the recombinant lectin of SEQ ID NO: 1.
The cell were kept in contact with the recombinant lectin of SEQ ID NO: 1 and the chemotherapy agent for 48h and the cytotoxic effect (percentage cytotoxicity) was measured.
The cytotoxic effect was determined by well-known colorimetric MTT assay (Mossman, T. 1983 Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays Journal of Immunological Methods, volume 65, pages 55-63) for assessing cell metabolic activity.
Based on percentage cytotoxicity data, IC50 values were determined using the software GraphPad Prism version 4.01. These determined IC50 values are given in Table 1 and Table 2 below.
Table 1: IC50 values of SEQ ID NO: 1 and Gemcitabine alone and in combination for cytotoxicity in PANC-1 cell lines

Concentration of Gemcitabine (µM) IC50 of SEQ ID NO: 1 (µg/mL)
0 25.060
0.01 55.490
0.1 19.450
1 14.130
5 12.230
25 2.044
50 0.917

Conc of SEQ ID NO: 1 (µg/mL) IC50 of Gemcitabine (µM)
0 7.407
2.5 15.940
5 10.790
10 3.036
20 0.039
40 0.038
80 10.610

Table 2: 1C50 values of SEP ID NO: 1 and 5-Flurouracil (5-FU) alone and in combination against PANC-1 cell lines

Concentration of 5-FU (µM) IC50 of SEQ ID NO: 1 (µg/mL)
0 24.630
0.1 686.900
1 22.490
10 6.842
50 1.690
100 1.070
250 0.098

Conc, of SEQ ID NO: 1 (µg/mL) IC50 of 5-FU (µM)
0 86.99
2.5 296.80
5 1.409
10 1.257
20 0.869
40 8.454
80 0.725

Table 1 and 2 relate to the IC50 of SEQ ID NO: 1 alone or in combination with Gemcitabine or 5-FU. It is observed that IC50 towards pancreatic cancer cells was reduced considerable when recombinant lectin protein having amino acid sequence of SEQ ID NO: 1 was used in combination with Gemcitabine or 5-Flurouracil (5-FU).
Example 1-2 - Calculation of CI values for Gemcitabine and 5-FU with SEP ID NO: 1
In a further study, PANC-1 cells were treated with Gemcitabine (0.01µM- 50µM) in combination with SEQ ID NO: 1 (2.5µg/mL- 80µg/mL) for 48 h. The cytotoxic effect on each combination was determined by MTT assay. Similarly another set of cells were treated with 5-FU (0.1 µM- 250µM) in combination with SEQ ID NO: 1 (2.5µg/mL-80µg/mL).
Using the same method as in Example 1-1, the IC50 value for Gemcitabine at a given concentration of the recombinant lectin of SEQ ID NO: 1 was measured and the IC50 value for the recombinant lectin of SEQ ID NO: 1 at a given concentration of Gemcitabine was determined. The IC50 value for each of Gemcitabine and the

recombinant lectin of SEQ ID NO: 1 alone was also measured. The same was performed for 5-FU in place of Gemcitabine.
Combination Index (CI) values were determined for each drug concentration combination. The CI values were calculated based on the IC50 values using the formula as given in below.

Where,
DA(50) = Concentration of drug A in combination with B to produce 50% cytotoxicity
ICA(50) = Concentration of drug A alone to produce 50% cytotoxicity
DB(50) = Concentration of drug B in combination with A to produce 50% cytotoxicity
ICB(50) = Concentration of drug B alone to produce 50% cytotoxicity
Wherein drug A is SEQ ID NO: 1 and drug B is other therapeutic agent that was tested.
Synergism, additive or antagonism were determined based on the following criteria:
CI > 1 - antagonism is indicated
CI = 1 - additive effected is indicated
CI < 1 - synergism is indicated
The results of combination index calculations for study on combination of SEQ ID NO: 1 with Gemcitabine and 5-FU against PANC-1 cell lines is tabulated below.

Table 3: CI values of combination of SEQ ID NO: 1 and Gemcitabine after 48 hours inPANC-1 cell line

2.5
Conc, of 5
SEQ ID 10
NO: 1 20
(µg/mL) 40
80

Concentration of Gemcitabine 0.01 0.1 1 (µM)
5 25 50
9.131 6.897 3.532 2.231 2.231 6.819
4.598 3.363 1.504 0.786 0.785 3.320
3.929 2.842 1.205 0.572 0.572 2.804
3.690 2.656 1.098 0.496 0.496 2.620
2.409 1.657 0.525 0.087 0.087 1.631
2.267 1.547 0.461 0.042 0.042 1.521

According to the aspect of the invention synergism was observed for both the combinations against pancreatic cell line as tabulated above. Pairs of concentrations which showed synergistic effect (i.e. CI < 1) are underlined. For the combination of SEQ ID NO: 1 and Gemcitabine, effective concentration towards synergistic cytotoxic effect was observed for SEQ ID NO: 1 concentration:
For 5µg/mL, 10µg/mL and 20µg/mL of SEQ ID NO: 1 with 5µM and 25µM of Gemcitabine
For 40µg/mL and 80µg/mL of SEQ ID NO: 1 with lµM, 5µM and 25µM of Gemcitabine
Antagonism was observed at all concentrations of SEQ ID NO: 1 with 0.01 µM, 0.1 µM and 50µM of Gemcitabine. At 2.5µg/mL of SEQ ID NO: 1, antagonism was observed for all concentrations of Gemcitabine. Antagonism as also observed for 5 µg/mL and 10µg/mL of SEQ ID NO: 1 with 1µM of Gemcitabine.
Additive effect was observed at 20 µg/mL of SEQ ID NO: 1 with 1 µM of Gemcitabine.

Table 4: CI values of combination of SEQ ID NO: 1 and 5-FU against PANC-1 cell lines

2.5
Conc, of 5
SEQ ID 10
NO: 1 20
(µg/mL) 40
80

Concentration of 5-FU (µM) 0.1 1 10 50 100 250
126.454
7.440
4.637 28.357 | 28.306 28.177 30.696 28.129

0.944 0.941 0.932 1.099 0.402 0.929 0.288

0.298 0.296 0.291

3.715 0.086 0.084 0.079 0.172 0.078
3.604 0.060 0.059 0.054 0.145 0.052
3.429 0.020 0.018 0.014 0.102 0.012

According to the aspect of the invention synergism was observed for both the combinations against pancreatic cell line as tabulated above. Pairs of concentrations which showed synergistic effect are underlined. For the combination of SEQ ID NO: 1 and 5-FU, effective concentration towards synergistic cytotoxic effect was observed for SEQ ID NO: 1 concentration:
For 5µg/mL of SEQ ID NO: 1 with 1 µM to 50 µM, and 250 µM of 5-FU.
For 10 µg/mL to 80 µg/mL of SEQ ID NO: 1 with 1 µM to 250 µM of 5-FU.
Antagonism was observed at lower concentrations of both SEQ ID NO: 1 and 5-
FU.
Additive effect was observed at 5µg/mL of SEQ ID NO: 1 with 100 µM of 5-FU.
The results of Tables 1 to 4 demonstrate that SEQ ID NO: 1 shows synergistic effects in combination with Gemcitabine and 5-Fluorouracil. The combinations were highly efficacious towards inhibiting the growth of cancer cells. The concentration of SEQ ID NO: 1 used was from about 2.5 µg/mL to about 80 µg/mL. The concentration of other therapeutic agent/s i.e. other than SEQ ID NO: 1, according to the present invention is from 1µM to about 250µM.

Example 2: Cytotoxic effect of SEQ ID NO: 1, paclitaxel and carboplatin standalone, synergistic cytotoxic effect of SEQ ID NO: 1 in combination with paclitaxel and carboplatin on Breast cancer cells (MDA-MB-231 cell line)
A study was conducted to determine the synergistic cytotoxic effects of SEQ ID NO: 1 in combination with chemotherapeutic agents, paclitaxel and carboplatin, in a breast cancer cell line (MDA-MB-231). The study was conducted in two phases, described in Example 2-1 and Example 2-2.
Example 2-1 - Measurement of IC50 values for MDA-MB-231 cells of Paclitaxel and Carboplatin in the presence of SEQ ID NO: 1
A study was performed to determine the effect of the concentration of the recombinant lectin represented by SEQ ID NO: 1 on the IC50 of Paclitaxel for cytotoxicity in MDA-MB-231 cells. Conversely the effect of the concentration of Paclitaxel on the IC50 of the recombinant lectin represented by SEQ ID NO: 1.
Similarly another study was performed for Carboplatin in combination with the recombinant lectin of SEQ ID NO: 1.
This was performed using the same techniques as in Example 1-1. The determined IC50 values are given in Table 5 and Table 6 below.
Table 5: IC50 values of SEQ ID NO: 1 and Paclitaxel alone and in combination against MDA-MB-231 cell lines.

Concentration of Paclitaxel (nM) IC50 of SEQ ID NO: 1 (µg/mL)
0 19.800
0.001 25.370
0.01 21.220
0.1 13.700
1 8.720
5 5.775
10 2.248

Conc, of SEQ ID NO: 1 (µg/mL) IC50 of Paclitaxel (nM)
0 1.638
2.5 3.980
5 9.307
10 0.389
20 0.123
40 3.56 xl0-5
80 5.75 xl0-10

Table 6: IC50 values of SEQ ID NO: 1 and Carboplatin alone and in combination against MPA-MB-231 cell lines.

Concentration of Carboplatin (µM) IC50 of SEQ ID NO: 1 (µg/mL)
0 14.310
10 7.188
50 3.584
100 4.290
200 2.073
500 0.523
1000 0.044

Conc, of SEQ ID NO: 1 (µg/mL) IC50 of Carboplatin (µM)
0 477.100
2.5 226.100
5 3.985
10 4.925
20 8.444
40 1.421
80 5.874

Table 5 and 6 relate to IC50 of SEQ IP NO: 1 alone and in combination with Paclitaxel or Carboplatin. It is observed that IC50 towards breast cancer cells was reduced considerably when SEQ IP NO: 1 was used in combination with Carboplatin or Paclitaxel.
Example 2-2 - Calculation of CI values for Paclitaxel and Carboplatin with SEQ IP NO: 1
In a further study, MP A-MB-231 cells were treated with Paclitaxel (0.001nM-1 0nM) in combination with SEQ IP NO: 1 (2.5µg/mL- 80µg/mL) for 48h. Similarly another set of cells were treated with Carboplatin (10µM- 1000µM) in combination with SEQ IP NO: 1 (2.5µg/mL- 80µg/mL). Combination Index values were determined for each drug combination and resultant antagonistic, additive or synergistic effect was determined, as described in Example 1-2. The results are tabulated below.

Table 7: CI values of combination of SEQ ID NO: 1 and Paclitaxel against MDA-MB-231 cell line.

2.5
Conc, of 5
SEQ ID 10
NO: 1 20
(µg/mL) 40
80

Concentration of Paclitaxel (nM) 0.001 0.01 0.1 1 5 10
6 824 14.241 1.823 1.453 1.281 1.281
6.106 12.843 1.564 1.227 1.072 1.072
4.803 10.305 1.094 0.819 0.692 0.692
3.940 8.625 0.782 0.549 0.440 0.440
3.430 7.631 0.598 0.389 0.292 0.292
2.819 6.441 0.378 0.197 0.114 0.114

According to the aspect of the invention, synergism was observed for both the combinations against the breast cancer cell line as tabulated above. Pairs of concentrations which showed synergistic effect (i.e. CI < 1) are underlined. For the combination of SEQ ID NO: 1 and Paclitaxel, effective concentration towards synergistic cytotoxic effect was observed for SEQ ID NO: 1 concentration:
For 10µg/mL to 80µg/mL of SEQ ID NO: 1 with 1 nM to 10 nM of Paclitaxel.
For 20 µg/mL to 80µg/mL of SEQ ID NO: 1 with 0.1 nM of Paclitaxel.
Antagonism was observed at lower concentrations of both SEQ ID NO: 1 (2.5 µg/mL and 5 µg/mL) and Paclitaxel (0.001 nM and 0.01 nM).
Additive effect was observed at 5µg/mL of SEQ ID NO: 1 with 5 nM and 10 nM of Paclitaxel and 10 µg/mL of SEQ ID NO: 1 with 0.1 nM of Paclitaxel.

Table 8: CI values of combination of SEQ ID NO: 1 and Carboplatin against MDA-MB-231 cell line.

2.5
Conc, of 5
SEQ ID 10
NO: 1 20
(µg/mL) 40
80

Concentration of Carboplatin (µM) 10 50 100 200 500 1000
2.005 1.214 0.515 0.518 0.529 0.507
1.501 0.843 0.261 0.263 0.273 0.254
1.600 0.916 0.311 0.313 0.323 0.304
1.290 0.687 0.154 0.157 0.165 0.148
1.073 0.528 0.045 0.047 0.055 0.040
1.006 0.478 0.011 0.013 0.021 0.006

The synergism was observed for both the combinations against breast cancer cell line as tabulated above. Pairs of concentrations which showed synergistic effect (i.e. CI < 1) are underlined. For the combination of SEQ ID NO: 1 and Carboplatin, effective concentration towards synergistic cytotoxic effect was observed for SEQ ID NO: 1 concentration:
For 2.5µg/mL to 80 µg/mL of SEQ ID NO: 1 with 100 µM to 1000 µM of Carboplatin.
For 5µg/mL to 80 µg/mL of SEQ ID NO: 1 with 50 µM of Carboplatin.
Antagonism was observed at 2.5µg/mL to 20 µg/mL of SEQ ID NO: 1 with 10 µM of Carboplatin. Antagonism was also observed at 2.5^ig/mL of SEQ ID NO: 1 with 50 µM of Carboplatin.
Additive effect was observed at 40µg/mL to 80 µg/mL of SEQ ID NO: 1 with 10 µM of Carboplatin
Results demonstrated that SEQ ID NO: 1 shows synergistic effects in combination with Paclitaxel or Carboplatin.

Example 3: Cytotoxic effect of SEQ ID NO: 1, 5-FU and Cisplatin standalone and synergistic cytotoxic effect of SEQ ID NO: 1 in combination with 5-FU and Cisplatin on Oral cancer cells (KB cell line).
A study was conducted to determine the synergistic cytotoxic effects of SEQ ID NO: 1 in combination with chemotherapeutic agents, 5-FU and Cisplatin, in an oral cancer cell line (KB cell line). The study was conducted in two phases, described in Example 3-1 and Example 3-2.
Example 3-1 - Measurement of IC50 values for KB cells of 5-FU and Cisplatin in the presence of SEQ ID NO: 1
A study was performed to determine the effect of the concentration of the recombinant lectin represented by SEQ ID NO: 1 on the 1C50 of 5-FU for cytotoxicity in KB cells. Conversely the effect of the concentration of 5-FIJ on the IC50 of the recombinant lectin represented by SEQ ID NO: 1.
Similarly another study was performed for Cisplatin in combination with the recombinant lectin of SEQ ID NO: 1.
This was performed using the same techniques as in Example 1-1. The determined IC50 values are given in Table 9 and Table 10 below.
Table 9: IC50 values of SEQ ID NO: 1 and 5-FU alone and in combination against KB cell lines

Concentration of 5-FU (µM) IC50 of SEQ ID NO: 1 (µg/mL)
0 17.3800
0.01 0.5800
0.1 0.9080
0.25 0.5010
0.5 0.1141
1 0.0555
5 0.0871

Conc, of SEQ ID NO: 1 (µg/mL) ICso of 5-FU (µM)
0 0.01498
5 0.00010
10 5.31 xlO"6
20 1.34 x 10'5
30 1.19xl0"6
40 5.22 x lO"11
90 1.40 x lO"8

Table 10: IC50 values of SEQ ID NO: 1 and Cisplatin alone and in combination against KB cell lines

Concentration of Cisplatin (µM) IC50 of SEQ ID NO: 1 (µg/mL)
0 38.110
0.1 21.190
0.25 11.240
0.5 2.694
0.75 0.597
1.0 0.316
1.5 0.303

Conc, of SEQ ID NO: 1 (µg/mL) IC50 of Cisplatin (µM)
0 0.72200
5 0.37490
10 0.26880
20 0.13390
30 0.06616
40 0.00712
90 0.00993

Table 9 and 10 relate to IC50 of SEQ ID NO: 1 alone and in combination with 5-FU or Cisplatin. It is observed that IC50 towards oral cancer cells was reduced considerably when SEQ ID NO: 1 was used in combination with 5-FU and Cisplatin.
Example 3-2 - Calculation of CI values for 5-FU and Cisplatin with SEQ ID NO: 1
In a further study, KB cells which were treated were treated with 5-FU (0.01 µM to 5.0 µM) in combination with SEQ ID NO: 1 (5.0 µg/mL - 90 µg/mL) for 48 h. Similarly another set of cells were treated with Cisplatin (0.1 µM to 1.5 µM) in combination with SEQ ID NO: 1 (5.0 µg/mL- 90 µg/mL). Combination Index values were determined for each drug combination and result antagonistic, additive or synergistic effect was determined, as described in Example 1-2.
The results are tabulated below.

Table 11: CI values of combination of SEQ ID NO: 1 and 5-FU against KB cell line

5
Conc, of 10
SEQ ID 20
NO: 1 30
(µg/mL) 40
90

Concentration of 5-FU (µM) 0.01 0.1 0.25 0.5 1 5
0.0406 0.0338 0.0343 0.0335 0.0334 0.0334
0.0596 0.0526 0.0532 0.0523 0.0522 0.0522 0.0288
0.0360 0.0292 0.0297 0.0289 0.0288

0.0136 0.0069 0.0075 0.0066 0.0066 0.0066
0.0102 0.0035 0.0041 0.0033 0.0032 0.0032
0.0120 0.0054 0.0059 0.0051 0.0050 0.0050

Table 12: CI values of combination of SEQ ID NO: 1 and Cisplatin against KB cell lines

5
Conc, of 10
SEQ ID 20
NO: 1 30
(µg/mL) 40
90

Concentration of 0.1 0.25 Cisplatin (µM) 0.5 0.75 1 1.5
1.364 0.967 1.135 0.777 0.845 0.699 0.571 0.577

0.535 0.414 0.308 0.313
0.627 0.469 0.269 0.169 0.081 0.085
0.543 0.394 0.204 0.195 0.109 0.101 0.100 0.026 0.018 0018 0.030 0.022 0.022
0.532 0.384

0.531 0.383 0.195

According to the aspect of the invention synergism was observed for both the combinations against KB cell lines as tabulated above. Pairs of concentrations which showed synergistic effect (i.e. CI < 1) are underlined. The combination of SEQ ID NO: 1 and 5-FU exhibited synergism at all the concentrations (SEQ ID NO: 1 - from 5.0 µg/mL to 90 µg/mL and 5-FU - from 0.01 µM to 5 µM) that were studied. Whereas in case of combination of SEQ ID NO: 1 and Cisplatin synergism was observed at concentrations:
10 µg/mL to 90 µg/mL of SEQ ID NO: 1 and 0.1 µM to 1.5 µM of Cisplatin, and

5 µg/mL of SEQ ID NO: 1 and 0.5µM to 1.5 µM of Cisplatin.
Antagonism was observed at lower concentration of both SEQ ID NO: 1 and Cisplatin.
Example 4: Cytotoxic effect of SEQ ID NO: 1, 5-FU and Irinotecan standalone and synergistic cytotoxic effect of SEQ ID NO: 1 in combination with 5-FU and Irinotecan on Colon cancer cells (HT-29 Cell Line):
A study was conducted to determine the synergistic cytotoxic effects of SEQ ID NO: 1 in combination with chemotherapeutic agents, 5-FU and Irinotecan, in a colon cancer cell line (HT-29). The study was conducted in two phases, described in Example 4-1 and Example 4-2.
Example 4-1 - Measurement of IC50 values for HT-29 cells of 5-FU and Irinotecan in presence of SEQ ID NO: 1
A study was performed to determine the effect of the concentration of the recombinant lectin represented by SEQ ID NO: 1 on the IC50 of 5-FU for cytotoxicity in HT-29 cells. Conversely the effect of the concentration of 5-FU on the IC50 of the recombinant lectin represented by SEQ ID NO: 1.
Similarly another study was performed for Irinotecan in combination with the recombinant lectin of SEQ ID NO: 1.
This was performed using the same techniques as in Example 1-1. The determined IC50 values are given in Table 13 and Table 14 below.

Table 13: IC50 of SEQ ID NO: 1 and 5-FU alone and in combination against HT-29 cell lines

Concentration of 5-FU (µM) IC50 of SEQ ID
NO: 1 (µg/mL)
0 25.470
5 22.990
10 6.374
50 1.667
100 1.387
150 1.216
200 1.334

Conc, of SEQ ID NO: 1 (µg/mL) IC50 of 5-FU (µM)
0 137.300
1 172.600
5 25.860
10 4.513
20 0.282
40 1.241
80 4.884

Concentration of Irinotecan (µM) IC50 of SEQ ID NO: 1 (µg/mL)
0 18.320
0.1 73.480
1 36.280
5 23.520
10 9.995
25 8.258
50 1.395

Conc, of SEQ ID NO: 1 (µg/mL) ICso of Irinotecan (µM)
0 16.200
1 49.240
5 2.779
10 5.449
20 1.338
40 0.278
80 0.167

Table 13 and 14 relate to IC50 of SEQ ID NO: 1 alone and in combination with 5-FU or Irinotecan. It was observed that the IC50 towards colon cancer cells was reduced considerably when SEQ ID NO: 1 was used in combination with 5-FU and Irinotecan.

Example 4-2 - Calculation of CI values for 5-FU and Irinotecan with SEQ ID NO: 1
In a further study, HT-29 cells were treated with 5-FU (5µM - 200µM) in combination with SEQ ID NO: 1 (lµg/mL - 80µg/mL) for 48h. Similarly another set of cells were treated with Irinotecan (0.1 µM - 50µM) in combination with SEQ ID NO: 1 (lµg/mL - 80µg/mL). Combination Index values were determined for each drug combination and resultant antagonistic, additive, or synergistic effect was determined, as described in Example 1-2. The results are tabulated below.
Table 15: CI values of combination of SEQ ID NO: 1 and 5-FU against HT-29 cell lines

1
Conc, of 5
SEQ ID 10
NO: 1 20
(µg/mL) 40
80

Concentration of 5-FU (µM) 5 10 50 100 150 200
3.294 1.261 0.965 0.907 0.920 0.970
1.822 0.486 0.291 0.253 0.262 0.295
1.405 0.266 0.100 0.068 0.075 0.103
1.380 0.253 0.089 0.057 0.064 0.092
1.365 0.245 0.082 0.050 0.057 0.085
1.375 0.251 0.087 0.055 0.0619 0.0898

According to the aspect of the invention, synergism was observed for both the combination against the colon cancer cell line as tabulated above. Pairs of concentrations which showed synergistic effect (i.e. CI < 1 ) are underlined. For the combination of SEQ ID NO: 1 and 5-FU, effective concentration towards synergistic cytotoxic effect was observed for SEQ ID NO: 1 concentration:
For 5 ng/mL to 80 [ig/mL of SEQ ID NO: 1 with 10 nM to 200 \xM of 5-FU concentration.
For 1 |ag/mL of SEQ ID NO: 1 with 50 nM to 200 \xM of 5-FU concentration.

Antagonism was observed at all concentrations of SEQ ID NO: 1 with 5 µM 5-FU concentration and at combination comprising 1 µg/mL of SEQ ID NO: 1 and 10 µM 5-FU concentration.
Table 16: CI values of combination of SEQ ID NO: 1 and Irinotecan against HT-29 cell lines

1
Conc, of 5
SEQ ID 10
NO: 1 20
(µg/mL) 40
80

Concentration of Irinotecan (µM) 0.1 1 5 10 25 50
19.321 4.894 5.723 4.446 4.117 4.082
11.039 2.492 2.983 2.227 2.031 2.011
8.226 1.676 2.052 1.472 1.323 1.307
5.243 0.811 1.065 0.673 0.572 0.562
4.860 0.700 0.939 0.571 0.476 0.466
3.347 0.261 0.438 0.165 0.095 0.087

The synergistic effect was observed for both the combinations against the colon cancer line as tabulated above. Pairs of concentrations which showed synergistic effect (i.e. CI < 1) are underlined. For the combination of SEQ ID NO: 1 and Irinotecan, effective concentration towards synergistic cytotoxic effect was overserved for SEQ ID NO: 1 concentration:
For 20 µg/mL to 80 µg/mL of SEQ ID NO: 1 with 1µM - 50 µM (except 5 µM) of Irinotecan concentration.
Antagonism was observed with SEQ ID NO: 1 concentration at 1 µg/mL to 80 µg/mL with 0.1 µM or Irinotecan and at 1 µg/mL - 10 µg/mL of SEQ ID NO: 1 with 0.01 to 50 µM of Irinotecan
Additive effect was observed at 20 µg/mL of SEQ ID NO: 1 with 5 µM of Irinotecan.

Example 5: Cytotoxic effect of SEQ ID NO: 1, Cisplatin and Paclitaxel standalone and synergistic cytotoxic effect of SEQ ID NO: 1 in combination with Cisplatin and Paclitaxel on Ovarian cancer cells (PA-1 Cell Line):
A study was conducted to determine the synergistic cytotoxic effects of SEQ ID NO: 1 in combination with chemotherapeutic agents, Cisplatin and Paclitaxel, in ovarian cancer cell line (PA-1 cell line). The study was conducted in two phases, described in Example 5-1 and Example 5-2.
Example 5-1 - Measurement of IC50 values of Cisplatin and Paclitaxel in the presence of SEQ ID NO: 1 for cytotoxicity in PA-1 cell lines
A study was performed to determine the effect of the concentration of the recombinant lectin represented by SEQ ID NO: 1 on the IC50 of Cisplatin for cytotoxicity in MDA-MB-231 cells. Conversely the effect of the concentration of Cisplatin on the IC50 of the recombinant lectin represented by SEQ ID NO: 1.
Similarly another study was performed for Paclitaxel in combination with the recombinant lectin of SEQ ID NO: 1.
This was performed using the same techniques as in Example 1-1. The determined IC50 values are given in Table 17 and Table 18 below.
Table 17: IC50 of SEQ ID NO: 1 and Cisplatin alone and in combination against PA-1 cell lines

Concentration of Cisplatin (µM) IC50 of SEQ ID NO: 1 (µg/mL)
0 12.070
0.01 8.520
0.1 6.033
0.25 4.455
0.5 0.912
1 0.321
5 0.0036

Conc, of SEQ ID NO: 1 (µg/mL) IC50 of Cisplatin (µM)
0 0.08566
1 0.716
2.5 0.314
5 0.093
10 0.0042
15 0.0005
20 0.0001

Table 18: IC50 of SEQ ID NO: 1 and Paclitaxel alone and in combination against PA-1 cell lines.

Concentration of Paclitaxel (µM) IC50 of SEQ ID NO: 1 (µg/mL)
0 8.822
0.01 5.794
0.1 6.545
0.25 4.893
0.5 3.917
1 3.031
5 0.709

Conc, of SEQ ID NO: 1 (µg/mL) IC50 of Paclitaxel
0 4.044
1 3.622
2.5 3.478
5 0.222
10 5.37 xl0-4
15 4.13 x 10-6
20 1.02 x 10-6

Table 17 and 18 relate to IC50 values of SEQ ID NO: 1 alone and in combination with Cisplatin or Paclitaxel. It is observed that IC50 against ovarian cancer cells was reduced considerably when SEQ ID NO: 1 was used in combination with Cisplatin or Paclitaxel.
Example 2-2 - Calculation of CI values for Cisplatin and Paclitaxel with SEQ ID NO: 1
In a further study, PA-1 cells were treated with Cisplatin (0.01µM - 5 µM) in combination with SEQ ID NO: 1 (1 µg/mL- 20µg/mL) for 48h. Similarly another set of cells were treated with Paclitaxel (0.01 nM- 5 nM) in combination with SEQ ID NO: 1 (1 µg/mL- 20µg/mL). Combination Index values were determined for each drug combination and resultant antagonistic, additive or synergistic effect was determined, as described in Example 1-2. The results are tabulated below.

Table 19: CI values of combination of SEQ ID NO: 1 and Cisplatin against PA-1 cell lines

1
Conc, of 2.5
SEQ ID 5
NO: 1 10
(µg/mL) 15
20

Concentration of Cisplatin (µM) 0.01 0.1 0.25 0.5 1 5
2.131 1.330 0.891 0.714 0.707 0.706
1.753 1.049 0.663 0.507 0.501 0.500
1.513 0.870 0.518 0.376 0.370 0.369
0.974 0.469 0.192 0.081 0.076 0.076
0.884 0.402 0.138 0.032 0.027 0.027
0.836 0.367 0.109 0.005 0.0009 0.0004

According to the aspect of the invention, synergism was observed for both the combinations against the ovarian cancer cell line as tabulated above. Pairs of concentrations which showed synergistic effect (i.e. CI < 1) are underlined. For the combination of SEQ ID NO: 1 and Cisplatin, effective concentration towards synergistic cytotoxic effect was observed for SEQ ID NO: 1 concentration:
For all concentrations tested (1 µg/mL to 20 µg/mL) of SEQ ID NO: 1 with 0.25 µM to 5µM of Cisplatin.
For 5 µg/mL to 20 µg/mL of SEQ ID NO: 1 with 0.1 µM of Cisplatin.
For 10 µg/mL to 20 µg/mL of SEQ ID NO: 1 with 0.01 µM of Cisplatin.
Antagonism was observed at 1 µg/mL to 5 µg/mL of SEQ ID NO: 1 with 0.01 µM of Cisplatin. Antagonism was observed at 1 µg/mL of SEQ ID NO: 1 with 0.1 µM of Cisplatin.
Additive effect was observed at 2.5 µg/mL of SEQ ID NO: 1 with 0.1 µM of Cisplatin.

Table 20: CI values of combination of SEQ ID NO: 1 and Paclitaxel against PA-1 cell lines

1
Conc, of 2.5
SEQ ID 5
NO: 1 10
(µg/mL) 15
20

Concentration of Paclitaxel (nM) 0.01 0.1 0.25 0.5 1 5
2.141 2.082 0.748 0.657
2.302 2.240 0.838 0.742 0.657 0.742 0.657 0.742
1.947 1.892 0.640 0.555 0.555 0.555
1.737 1.686 0.523 0.444 0.444 0.444
1.547 1.499 0.417 0.344 0.344 0.344
1.048 1.009 0.140 0.080 0.0803 0.0803

According to the aspect of the invention, synergism was observed for both the combinations against the ovarian cancer cell line as tabulated above. Pairs of concentrations which showed synergistic effect (i.e. CI < 1) are underlined. For the combination of SEQ ID NO: 1 and Paclitaxel, effective concentration towards synergistic cytotoxic effect was observed for SEQ ID NO: 1 concentration:
For all concentrations tested (1 µg/mL to 20 µg/mL) of SEQ ID NO: 1 with 0.25 nM to 5 nM of Paclitaxel.
Antagonism was observed at 1 µg/mL to 15 µg/mL of SEQ ID NO: 1 with 0.01 nM to 0.1 nM of Paclitaxel. Additive effect was observed at 20 \iM of SEQ ID NO: 1 with 0.01 nM to 0.1 nM of Paclitaxel.
Results demonstrated that SEQ ID NO: 1 shows synergistic effects in combination with Cisplatin or Paclitaxel.
Example 6: Cytotoxic effect of SEQ ID NO: 1, Cisplatin and Gemcitabine standalone and synergistic cytotoxic effect of SEQ ID NO: 1 in combination with Cisplatin and Gemcitabine on Bladder cancer cells (T24 Cell Line):
A study was conducted to determine the synergistic cytotoxic effects of SEQ ID NO: 1 in combination with chemotherapeutic agents, Cisplatin and Gemcitabine, in

a bladder cancer cell line (T24 cell line). The study was conducted in two phases, described in Example 6-1 and Example 6-2.
Example 6-1 - Measurement of IC50 values for T24 cells of Cisplatin and Gemcitabine in the presence of SEQ ID NO: 1
A study was performed to determine the effect of the concentration of the recombinant lectin represented by SEQ ID NO: 1 on the IC50 of Cisplatin for cytotoxicity in T24 cells. Conversely the effect of the concentration of Cisplatin on the IC50 of the recombinant lectin represented by SEQ ID NO: 1.
Similarly another study was performed for Gemcitabine in combination with the recombinant lectin of SEQ ID NO: 1.
This was performed using the same techniques as in Example 1-1. The determined IC50 values are given in Table 21 and Table 22 below.
Table 21: IC50 of SEQ ID NO: 1 and Cisplatin alone and in combination against T24 cell lines

Concentration Cisplatin (µM) of ICso of SEQ ID NO: 1 (µg/mL)
0 11.470
1 11.460
10 2.375
25 1.062
50 1.081
100 0.944
500 0.716

Cone, of SEQ ID NO: 1 (µg/mL) ICso of Cisplatin (µM)
0 39.020
2.5 22.870
5 3.79500
10 0.09708
20 0.03436
40 0.08363
80 0.01342

Table 22: IC50 of SEQ ID NO: 1 & Gemcitabine alone & in combination against T24 cell lines

Concentration of Gemcitabine (µM) IC50 of SEQ ID NO: 1 (µg/mL)
0 10.350
1 2.242
10 0.892
50 0.626
100 0.155
200 0.149
300 0.029

Conc, of SEQ ID NO: 1 (µg/mL) IC50 of Gemcitabine (µM)
0 15.420
2.5 0.802
5 0.14090
10 0.00032
20 0.00002
40 6.05 x 10-19
80 7.18 xl0-10

Table 21 and 22 relate to IC50 of SEQ ID NO: 1 alone and in combination with Cisplatin and Gemcitabine. The IC50 against bladder cancer cells was reduced considerably when SEQ ID NO: 1 was used in combination with Cisplatin and Gemcitabine.
Example 6-2 - Calculation of CI values for Cisplatin and Gemcitabine with SEQ ID NO: 1
In a further study, T24 cells were treated with Cisplatin (1 µM - 500 µM) in combination with SEQ ID NO: 1 (2.5µg/mL- 80µg/mL) for 48h. Similarly another set of cells were treated with Gemcitabine (1 µM- 300µM) in combination with SEQ ID NO: 1 (2.5µg/mL- 80µg/mL). Combination Index values were determined for each drug combination and resultant antagonistic, additive or synergistic effect was determined, as described in Example 1-2. The results are tabulated below.

Table 23: CI values of combination of SEQ ID NO: 1 and Cisplatin against T24 cells lines

2.5
Conc, of 5
SEQ ID 10
NO: 1 20
(µg/mL) 40
80

Concentration of Cisplatin (µM) 1 10 25 50 100 500
2.171 1.194 1.004 0.210 1.001 1.003 1.000
0.915 0.733 0.324
0.208 0.210 0.207

0.199 0.095 0.094 0.095 0.093
0.736 0.201 0.097 0.095 0.097 0.095
0.717 0.188 0.085 0.083 0.085 0.083
0.685 0.166 0.065 0.063 0.065 0.063

According to the aspect of the invention, synergism was observed for both the combinations against the bladder cancer cell line as tabulated above. Pairs of concentrations which showed synergistic effect (i.e. CI < 1) are underlined. For the combination of SEQ ID NO: 1 and Cisplatin, effective concentration towards synergistic cytotoxic effect was observed for SEQ ID NO: 1 concentration:
For 5 µg/mL to 80 µg/mL of SEQ ID NO: 1 with all tested concentrations (1 µM -500 µM) of Cisplatin.
Antagonism was observed at 2.5 µM of SEQ ID NO: 1 with 1µM and 10 µM of Cisplatin.
Additive effect was observed at 2.5 µg/mL of SEQ ID NO: 1 with 25 µM - 500 µM of Cisplatin.

Table 24: CI values of combination SEQ ID NO: 1 and Gemcilabine against T24 cell lines

2.5
Conc, of 5
SEQ ID 10
NO: 1 20
(µg/mL) 40
80

Concentration of Gemcitabine (µM) 1 10 50 100 200 300
0.280 0.228 0.217 0.217 0.217 0.086 0.217 0.086
0.143 0.096 0.086 0.086

0.116 0.070 0.061 0.060 0.060 0.060
0.068 0.024 0.015 0.015 0.015 0.015
0.067 0.024 0.014 0.014 0.014 0.014
0.055 0.012 0.003 0.003 0.003 0.003

The synergism was observed for both the combinations against bladder cancer cell line as tabulated above. Pairs of concentrations which showed synergistic effect (i.e. CI < 1) are underlined. For the combination of SEQ ID NO: 1 and Gemcitabine, synergistic cytotoxic effect was observed at all concentrations (2.5µM - 80 µM of SEQ ID NO: 1 with 1 µM - 300 µM of Gemcitabine.

Summary of Examples
As an overview the concentrations at which synergism was observed in the cell lines tested for present invention is summarized in the table below:
Table 25. Concentrations of SEQ ID NO: 1 in combination with different therapeutic agents showing synergistic effect on oral, colon, ovary and bladder cancer cell lines.

Cancer Cell Lines Therapeutic agents Cone, of SEQ ID NO: 1 Conc. of other therapeutic agents
Pancreas PANC-1 Gemcitabine 5 to 20 µg/mL 5 µM to 25 µM

40 to 80 µg/mL 1 µM to 25 µM

5-FU 10to80µg/mL lµMto250µM

5 µg/mL 1 to 50 µM & 250 µM
Breast MDA-
MB-231 Paclitaxel 20 to 80 µg/mL 0.1 µMto 10 µM

10 µg/mL 1 nMto 10 µM

Carboplatin 5 to 80 µg/mL 50µMto 1000 µM

2.5 µg/mL l00µMtol00µM
Oral KB 5-FU 5 to 90µg/mL 0.01 to 5 µM

Cisplatin 10to90µg/mL 0.1 to 1.5 µM

5 µg/mL 0.5 to 1.5 µM
Colon HT-29 5-FU 5 to 80 µg/mL 10 to 200 µM

1 µg/mL 50 to 200 µM

Irinotecan 40 to 80 µg/mL 1 to 50 µM

20 µg/mL 1 and 10 to 50 µM
Ovary PA-1 Cisplatin 1 to 2.5 µg/mL 0.25 to 5 µM

5 µg/mL 0.1 to5 µM

10 to 20 µg/mL 0.01 to 5 µM

Paclitaxel 1 to 20 µg/mL 0.25 to 5 nM
Bladder T-24 Cisplatin 5 to 80 µg/mL 1 to 500 µM

Gemcitabine 2.5 to 80 µg/mL 1 to 300 µM

Claims
We claim,
1. A therapeutically effective combination comprising a recombinant lectin protein and one or more other therapeutic agent.
2. A therapeutically effective combination comprising recombinant lectin protein and one or more other therapeutic agent, wherein the combination is synergistic and wherein the concentration of recombinant lectin protein is in the range from 0.5 µg/mL to 100 µg/mL.
3. The therapeutically effective combination as claimed in claim 1 or 2, where the combination is used for prevention or treatment of cancer in a subject.
4. Use of recombinant lectin protein for the treatment or prevention of cancer comprising administration of therapeutically effective amount of recombinant lectin protein in a subject wherein the recombinant lectin protein is administered in combination with other therapeutic agent, and wherein the other therapeutic agent is administered simultaneously, separately or sequentially.
5. A method for treatment or prevention of cancer in a subject comprising administering to the subject effective amount of recombinant lectin protein in combination with other therapeutic agent, wherein the other therapeutic agent is administered simultaneously, separately or sequentially.
6. A combination therapy for prevention, proliferation, treatment or to cure cancer or tumor in a subject using recombinant lectin protein, wherein the combination therapy comprises administration of recombinant lectin protein either in combination with one or more other therapeutic agent or simultaneous, separate or sequential administration of one or more other therapeutic agents in a subject.
7. The therapeutically effective combination as claimed in claim 1 to 3, the use as claimed in claim 4, the method of treatment as claimed in claim 5 and the combination therapy as claimed in claim 6, wherein the other therapeutic agent

is an anti-cancer agent selected form an antimetabolite, an alkylating antineoplastic agent, an anti-microtubule agent and/or a topoisomerase 1 inhibitors.
8. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 7, wherein the antimetabolite is selected from 5-Flurouracil (5-FU), Gemcitabine, Methotrexate, Pemetrexed or Capacitabine.
9. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 7, wherein the alkylating neoplastic agent is a platinum based anti-neoplastic agent selected from cisplatin or carboplatin.
10. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 7, wherein one or more Anti-microtubule agent is selected from Paclitaxel, Docetaxel, Abraxane or Taxotere.
11. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 7, wherein one or more Topoisomerase I is selected from Irinotecan or Topotecan.
12. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim any one of the previous claims, wherein the concentration of the other therapeutic agent is in the range from 0.001 nM to 1000 |iM.
13. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 9, wherein the anticancer agent is cisplatin, and wherein the combination is used for the treatment or prevention of oral, ovarian or bladder cancer in a subject.
14. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 13, wherein the concentration of Cisplatin is in the range of 0.01 µM to 500 µM.

15. A recombinant lectin protein for the treatment or prevention of oral, ovarian or bladder cancer in a subject, wherein the recombinant lectin protein is administered simultaneously, separately or sequentially with cisplatin.
16. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 8, wherein the anticancer agent is 5-FU, and wherein the combination is used for treatment or prevention of oral, pancreatic or colon cancer in a subject.
17. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 16, wherein the concentration of 5-FU is in the range of 0.01 \xM to 250 \iM.
18. A recombinant lectin protein for treatment or prevention of oral, pancreatic or colon cancer in a subject, wherein the recombinant lectin protein is administered simultaneously, separately or sequentially with 5-FU.
19. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 11, wherein the anticancer agent is Irinotecan, and wherein the combination is used for treatment or prevention of colon cancer in a subject.
20. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 19, wherein the concentration of Irinotecan is in the range of 0.1 µM to 50 µM.
21. A recombinant lectin protein for the treatment or prevention of colon cancer in a subject, wherein the recombinant lectin protein is administered simultaneously, separately or sequentially with Irinotecan.
22. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 10, wherein the anticancer agent is Paclitaxel, and wherein the combination is used for treatment or prevention of ovarian or breast cancer in a subject.

23. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 22, wherein the concentration of Paclitaxel is in the range of 0.001 nM to 10 nM.
24. A recombinant lectin protein for the treatment or prevention of ovary or breast cancer in a subject, wherein the recombinant lectin protein is administered simultaneously, separately or sequentially with Paclitaxel.
25. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 8, wherein the anticancer agent is Gemcitabine, wherein the combination is used for treatment or prevention of bladder or pancreatic cancer in a subject.
26. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 25, wherein the concentration of Gemcitabine is in the range of 0.01 \xM to 300 \xM.
27. A recombinant lectin protein for the treatment or prevention of bladder or pancreatic cancer in a subject, wherein the recombinant lectin protein is administered simultaneously, separately or sequentially with Gemcitabine.
28. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 9, wherein the anticancer agent is carboplatin, and wherein the combination is used for treatment or prevention of breast cancer in a subject.
29. The therapeutically effective combination, the use, the method of treatment and the combination therapy as claimed in claim 28, wherein the concentration of Carboplatin is in the range of 10 |iM to 1000 |iM.
30. A recombinant lectin protein for the treatment or prevention of breast cancer in a subject, wherein the recombinant lectin protein is administered simultaneously, separately or sequentially with Carboplatin,
31. The therapeutically effective combination, the use, the method of treatment, or the combination therapy, wherein the recombinant lectin protein is a protein having amino acid sequence of SEQ ID NO: 1, 2 or 3 or having at least 60%, 70%, 80%, 90%, 95%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 1,2 or 3.

32. A composition comprising recombinant lectin protein in combination with other therapeutic agent.
33. The composition as claimed in claim 32, wherein the composition further comprises one or more pharmaceutically acceptable excipients.
34. The composition as claimed in claim 32, wherein the other therapeutic agent is wherein the other therapeutic agent is selected from one or more 5-Flurouracil (5-FU), Gemcitabine, cisplatin, Paclitaxel, carboplatin or Irinotecan..
35. The composition as claimed in claim 32, wherein the composition is used for the treatment or prevention of cancer in a subject.
36. A Recombinant lectin protein having amino acid sequence of SEQ ID NO: 1, 2 or 3 in combination with other therapeutic agent for the treatment or prevention of cancer.
37. The recombinant lectin protein as claimed in claim 36, wherein the other therapeutic agent is selected from one or more 5-Flurouracil (5-FU), Gemcitabine, cisplatin, Paclitaxel, carboplatin or Irinotecan.
38. A Recombinant lectin protein in combination with other therapeutic agent for the treatment or prevention of cancer.
39. The recombinant lectin protein as claimed in claim 38, wherein the other therapeutic agent is selected from one or more 5-Flurouracil (5-FU), Gemcitabine, cisplatin, Paclitaxel, carboplatin or Irinotecan.
40. A therapeutic agent for the treatment or prevention of cancer, in combination with a recombinant lectin protein, wherein the therapeutic agent is selected from one or more of 5-Flurouracil (5-FU), Gemcitabine, cisplatin, Paclitaxel, Carboplatin or Irinotecan.