Technical F ield

This invention is concerned with development of a vaccine from bacteriocin belong to ¾ Lactobacillus acidophilus LA-S 2 strain known as lactic acid bacteria for use in the treatment of cancer, especially colon cancer.

The invention particularly focuses on the use with a view to immunotherapy as vaccine in treatment of colon cancer by obtaining the monoclonal antibodies against bacteriocin belong to Lactobacillus acidophilus LA-S 2.

TE C H NICAL BAC KG R OU ND

C olon cancer (large bowel cancer) or colorectal cancer is cancerous growths seen in colon, rectum and appendix. Colon cancer is the third most frequent cancers in the western world and ranks second among cancers leading cause of death. This cancer mostly arises from

¾ adenoma polyps occurring in the large intestine. P olyps are tissue swelling seen in the cavities of digestive tract. Polyps are known benign tumours among the people but the adenoma polyps are tumours that cause cancer known as malignant tumours among the population. C olon cancer is treated with surgical methods: tumour forming cancer and an amount of healthy tissue around it is taken. According to the state of stage, chemotherapy

1&\ and adjuvant chemotherapy is applied. In patients seen metastasis (spread to other organs), chemotherapy and radiotherapy besides the surgery are also applied to extend the patient's life expectancy. Although surgical method and chemotherapy, due to the reappearance of the disease, 50% of patients die within the next 5 years after diagnosis. Although chemotherapy means drug therapy, it is the term used for the treatment performed by using cancer drugs affecting cancer cells. Drugs used for its are also called "anti-cancer drugs ". It may not always be possible to provide the desired benefits of chemotherapy drugs. If cancer tumours can not be destroyed by treatment or it is delayed for treatment, cancer cells can spread throughout the body via blood vessels or lymph nodes. In this case the situation called metastasis (spread) would have occurred. Because chemotherapy drugs affect the patient's immune system, resistance to infectious diseases of patients is reduced during chemotherapy. T his effect is mostly seen between the 5 ^th and 1 5 ^th days after chemotherapy. Therefore, especially during this period, patients treated with chemotherapy should stay away from people with infectious diseases and should pay more attention to hygiene rules. In addition, some chemotherapy drugs in some patients may also lead to fatigue, nausea, vomiting and hair loss.

In recent years, the method called immunotherapy in cancer treatment is used as a treatment method which destroy cancer cells by strengthening the immune system of patient ¾ when used in the early stages of cancer. Also, it is used as an adjuvant therapy to prevent recurrence of the disease after chemotherapy and surgical intervention.

For this purpose, several vaccines used for immunotherapy are produced. T he production of these vaccines is relatively complicated and expensive due to the need to multicellular organisms cells. Therefore, there is a need for new and effective vaccines to be obtained

3ft, easily. Immunotherapy is applied three different ways. The antibodies by the vaccine developed through molonoclonal antibody method is given to the patient and it is tried to treat by allowing the antibodies to fight with cancerous structures. The non-specific immunotherapy, on the other hands, is a treatment applied after chemotherapy or simultaneous with chemotherapy. It is seen some side effects. By means of cancer vaccines

3¾ as finally methods, immune system is tried to stimulate with an antigen that is injected into the body to trigger the study mechanisms of immune system.

In state of art; patent of No. US 7887794 mentions to prevent and treat colon cancer by creating the apoptosis effect with Lactobacillus acidophilus, which is one of lactic acid bacteria. Apoptosis is one of the main types of programmed cell death. This type is the 1&\ normal way to get rid of had abnormal and not needed cells in the body. Patent of No.

US 20140205572 mentions from that mixture obtained by using Lactobacillus acidophilus and other bacteria is potentiated the effect of chemotherapy and thus the chance of treating the colon cancer is increased. On the other hand, patent of No. US 20140348792 mentions from that reducing the amount of LTA (lipoteichoic acid) on surface of bacterium by modifying the genetics of Lactobacillus acidophilus NC K2025 strain can be used in removing of the tumours. As a result, studies on the use of these bacteria for the purpose of immunotherapy are ongoing on cancer treatment especially on treatment of colon cancer. Therefore, It is needed to new structurings to be eliminate the disadvantages mentioned above and to be provide solutions to existing systems. m O BJ E CT O F T H E INV E NTIO N

The present invention is concern with development of vaccine that meet the above mentioned requirements, eliminate all the disadvantages and providesome additional advantages to use on colon cancer treatment from anti-idiotypic monoclonal antibodies obtained against bacteriocin LA-S 2 belong to Lactobacillus acidophilus LA-S 2 strain. Object of the invention develops a new vaccine which can be obtained more easily and economically in terms of culturing of the bacteria, bacteriocin production and preparation for vaccine when the other vaccine materials is kept in mind for use on cancer treatment with immunotherapy method.

¾ Another object of this invention is to investigate whether a treatment of relatively less side- effects is successful only with immunotherapy without needing to chemotherapy and surgical intervention.

The present invention is concerned with because of the natural antimicrobial agent of bacteriocin LA-S 2 belong to Lactobacillus acidophilus LA-S 2 strain, use of this bacteriocin as ft, antigen, which means that its use as vaccine material, and then its use in immunotherapy treatment for colon cancer. With reference to realize all the advantages to be understood above mentioned and from the following detailed description comprises the following steps:

1 . Determination of antimicrobial activity on various indicator microorganisms of Lactobacillus acidophilus strains,

¾ 2. Determination of the growth kinetics of Lactobacillus acidophilus strains and determination of the effective factors in these changes by examining of changes occurring in their specifications such as optical density, viability, pH, antimicrobial activity, bacteriocin titer during growth,

3. P urifying and characterization of bacteriocins belong to Lactobacillus acidophilus strains &\ having antimicrobial activity and determination of their antimicrobial activity spectrum by determining of their molecular weights,

4. Determination of biological barriers resistance of Lactobacillus acidophilus LA-S 2 strains,

5. Determination of viability of Lactobacillus acidophilus LA-S 2 strain as the flow cytometric under certain conditions,

6. Examining of cell surface structures of Lactobacillus acidophilus LA-S 2 strain and purification of cell surface molecules and components,

7. Determination of mode of action of bacteriocin belong to Lactobacillus acidophilus LA-S 2 strain,

8. Examination of the ability to adhere to cancer cells of Lactobacillus acidophilus LA-S 2 strain and determination of factors that influence the adhesion,

9. Determination of inhibition by Lactobacillus acidophilus LA-S 2 strain of cell attachment and cell invasion of enterovirulent and enteropathogenic bacteria which is effective on colon cancer, 10. In vitro examination of anti-carcinogenic effects of Lactobacillus acidophilus LA-S 2 strain on particularly colon cancer cells and some other cancer cells,

1 1 . In vitro and In vivo determination of interactions of Lactobacillus acidophilus LA-S 2 strain with cytokines and immune system which is effective in cancer proliferation

¾ 12. In vivo determination of immune response against bacteriocins purified from

Lactobacillus acidophilus LA-S 2 strain,

13. Obtaining of monoclonal antibodies against bacteriocins purified from Lactobacillus acidophilus LA-S 2 strain and in vivo determination of anti-carcinogenic effect,

14. In vivo determination of a nti- inflammatory and anti-carcinogenic effects of Lactobacillus ft, acidophilus LA-S 2 strain on animal models.

Structural and characteristic features and all the advantages of the invention will be more clearly understood through the detailed description written below and therefore evaluation should be made taking into account this detailed description.

DETAIL E D DE S C RIPTIO N OF T H E INVE NTIO N ¾ In this detailed description, the issue of invention, structurings preferred of vaccine development for use in cancer treatment from the LA-S 2 bacteriocins belonging to Lactobacillus acidophilus LA-S 2 strains, are described only for the better understanding of the subject and in way to be created no limiting effect

In this study; determination of the use possibilities for immunotherapy practices on colon &\ cancer as vaccine material (immunizer/antigen) of Lactobacillus acidophilus strains with probiotic character which is the focus of attention because of dietetic, prophylactic and therapeutic characteristics in terms of human health and bacteriocins obtained from these strains and examination of their application possibilities for the purpose of development of immunotherapeutic cancer vaccine (in particular the cancer vaccine for immunotherapy practices on colon cancer) were aimed.

C urrently, there are 5 different model immunotherapeutic cancer vaccines:

I Tumor cell vaccines

I Dendritic cell vaccines

I Antigen vaccines

i Anti-idiotypic vaccines

I DNA vaccines In this case study, antigen vaccine resulting in ine production of monoclonal antibody will be obtained at the first stage and then when second time antibody production with these antibodies was provided, anti-idiotypic vaccine will be obtained.

This study was performed by experiments conducted under two main experimental groups including in vitro and in vivo studies, microbiological, bioprocess and cell culture studies have taken part. In the scope of in vivo studies; on the other hand, immunization, hybridoma and monoclonal antibody and experimental animal model studies have taken part.

At microbiological studies which are the first stage of in vitro studies; antimicrobial activities ft, of Lactobacillus acidophilus strains had been determined against indicator microorganisms with agar-disc diffusion method. As test microorganisms, in particularly, pathogenic flora bacteria which situated intestinal flora and is effective on colon cancer, pathogens generating adverse effects on the gastrointestinal tract and pathogens which lead to food - borne poisoning had been selected. Following studies conducted to determine the ¾ antimicrobial activity, the growth kinetics of Lactobacillus acidophilus strains had been determined in order to determination the logarithmic phase that especially bacteriocins were produced in the most intense way. During the growth, changes occurring in optical density, pH, viable count, antimicrobial activity spectrums and antimicrobial activity (the formation of inhibition zones and bacteriocin titres) of each strains had been detected with samples taken &\ beginning of every hour. In the second stage, bacterial bioprocess studies have been passed in order to obtain the bacteriocins of strains shown antimicrobial activity. Bacteriocins had been extracted and purified partially from strains with wide range spectrum of antimicrobial activity by applying downstream processing such as centrifugation, dialysis and lyophilisation. Identification of activity bands, molecular weights and activity spectrums belong to bacteriocins purified from producer bacteria cells had been determined with Tris- tricine S DS -PAG E method, which is a different form of sodium dodecyl sulfate polyacrylamide gel electrophoresis (S DS -PAG E ) and is applied to determine especially the proteins having the molecular weight between 1 -10 kDa, and also with Mass S pectrometry Analysis. Obtained bacteriocins had been characterized in terms of thermal resistance at different pH, pH sensitivity, stability and susceptibility to proteolythic, lipolythic and amylolithic enzymes. At the cell culture studies which are last stage of the in vitro studies, the viability of bacteriocin producer strains in different periods of time under certain circumstances had been determined with flow cytometric method and then, acid and bile salt tolerance, hydrophobicity, <f -galactosidase activities of these strains had been examined to . determine the biological barrier resistance. Also, the cell surface structures, adhesion properties and factors affecting adhesion and mode of action of bacteriocin-producing strains had been determined. At the same time, the effectiveness of lactic bacteria strains used in this study had been examined in terms of inhibition of the roles of enterovirulent and enteropathogenic on cell attachment and cell invasion. At the cell culture studies, anti- ¾ carcinogenic effects of Lactobacillus acidophilus strains had been examined on mainly colon cancer cells and various other cancer cells as in vitro. After data obtained from in vitro experiments were evaluated, in vivo experiments had been carried out. Lactobacillus acidophilus LA-S 2 strain and its bacteriocin given best results in terms of the properties such as viability, biological barrier resistance, adhesive effect, antimicrobial activity spectrum, etc ft, after the cell culture and bioprocess studies, had been selected to use in in vivo experiments. At immunization studies which are the first stage of in vivo studies, after the selected strain and its bacteriocin are combined with various adjuvants, they had been used as immunizer (antigen) according to an immunization procedure established for in vivo animal experiments and thus immunization of experimental animals has been provided. ¾ During the immunization, acquired immune responses against bacteriocins presented as antigen had been measured. At the second stage of in vivo studies following the immunization, the antibody titres had been tested by obtaining hybridoma and monoclonal antibodies with monoclonal antibody techniques. T he obtained antibodies had been evaluated in terms of anti-carcinogenic effect. In addition, the interactions of Lactobacillus &\ acidophilus LA-S 2 strain with immune system and effective cytokines in cancer proliferation hade been determined and evaluated as in vivo at this stage. T he adhesion features and cell surface structures had been examined by means of monoclonal antibodies against Lactobacillus acidophilus LA-S 2 and its bacteriocin in the scope of in vivo experiments. E specially on colon cancer, anti-inflammatory and anti-cancerogenic effects of the strain and its bacteriocin had been tried to determine on two different animal models in the scope of experimental animal model studies which are the last stage of in vivo studies and also this study. As a result of these investigations, the immunity had been provided in experimental animals immunized by bacteriocin LA-S 2 used as antigen. Also, the significant reductions had been determined in terms of both the formation of colitis and the progress on colon cancer in experimental animals with colon cancer, which had been weakened the immune system, treated with vaccine that the monoclonal antibodies generated against this antigen were used.