Hyperthermia-induced gut microbiome-mediated modulation of host immune system for treatment of gastric / gastric malt / duodenal cancer

Abstract

The present invention provides a hyperthermia-induced gut microbiome- mediated modulation of the host immune system for the treatment of gastric / gastric MALT / Duodenum cancer; the said system uses a wild type / non-pathogenic / attenuated strain coated with iron oxide nanoparticles to specifically target the cancerous tissue by applying an external field around the gastric tissue the said iron oxide generates heat on bacteria surface and causes lysis of bacteria in the tumor microenvironment; the lysis of bacteria triggers various potent immune activator and cytokines to activate antigen-presenting cells including lymphocytes and macrophages; the said antigen presenting cell further, eliminates cancer cell from the gut microenvironment and eventually clean tumor microenvironment. Additionally, the activated lymphocytes will be antigenic memory against bacterial particles and cancer tissues. Furthermore, the said treatment system does not involve the use of any chemotherapeutic drug or radiation treatment and, thus, precludes undesirable side effects of chemotherapy as well as minimizes toxicity.

Description

[0001] Field of the Invention:

[0002] The present invention relates to a hyperthermia-induced gut microbiome- mediated modulation of the host immune system to treat gastric cancer. More particularly, the invention relates to a hyperthermia technology that does not use any chemotherapy and radiation treatment; instead, it uses a wild type / non-pathogenic / attenuated strain of Helicobacter pylori coated with Iron-oxide Nanoparticles to target gastric cancer. Even more particularly, the invention relates to a target drug delivery system that involves magnetic hyperthermia-induced activation of the body's natural immune system by using a non-pathogenic strain of Helicobacter coated with Iron-oxide Nanoparticles to target gastric cancer specifically.

[0003] Background of the Invention:

[0004] Gastric Cancer is the most commonly diagnosed cancer as well as deathleading cancer worldwide. One of the most common risk factors for gastric cancer is Helicobacter pylori. Hyperthermia is the most used and known treatment of cancer and is used with chemotherapy as a combined solution to effectively target cancerous cells. It involves the use of magnetic nanoparticles, and when exposed to the external magnetic field, the nanoparticle generates a high amount of heat to kill the cancerous cell. However, the current hyperthermia solution for cancer treatment faces several challenges and limitations that include lack of specificity towards the malignant cells compared to healthy cells, high rate of adverse effects on the cytoskeleton, organelles, intracellular transport, and R.NA processing, short-term neurological and cognitive dysfunction and causes pain, bleeding, swelling and burning at the treatment site. Apart from Hyperthermia, the other currently used gastric cancer treatments are a combination of chemotherapy with nivolumab to treat gastric cancer, immunotherapy via blockage of HER.2 and VEGF proteins, Surgery, Cytotoxic therapies, targeted drug therapies, and therapies to target DNA repair proteins. These all-existing treatments face limitations and challenges as this solution destroys both malignant and healthy cells, which results in the destruction of metabolically active cells in the body, leading to the suppression of the immune system, system toxicity, and hence increasing the risk of secondary infections in cancer treatments. Therefore, a need arises to develop a novel targeted treatment system to effectively recognize and target cancerous cells without causing any harm to the healthy cells of the body and not cause any adverse reaction during treatment to support patient health. Many researchers and inventors have worked on it and developed some related works, but they lack design skills and are missing some features. Some of such inventions are discussed below.

[0005] Reference has been made to CN112022324A, titled "A CRYOSYSTEM COMPRISING NANOPARTICLES FOR TREATING A BODY PART OF AN INDIVIDUAL BY CRYOTHERAPY", by NANOBACTERIE, dated 2020-06-02, which discloses a cryogenic system comprising nanoparticles for use in the treatment of a body part of an individual by cryotherapy. The system comprises two parts: a) a first part cryoprobe wherein : i) the cryoprobe is adapted for internal cooling since it comprises a penetrating section in communication with a cryogen source, which is at least less than 1 / 10 of the maximum volume of said body part and / or has at least one dimension of less than 1 cm; or ii) a cryoprobe suitable for external cooling since it comprises a non-penetrating segment in communication with a refrigerant source, and b) a second portion, which is: i) an assembly of at least two nanoparticles, characterized in that the assembly comprises at least two nanoparticles bound to each other or associated with each other by a binding or binding material, or ii) at least one nanoparticle comprising iron and at least one further metal other than iron, wherein an assembly of at

[0006] 3 least two nanoparticles or at least one nanoparticle is intended to be cooled by a cryoprobe or by switching on a cryoprobe.

[0007] Another reference has been made to WO2018093864A1, titled NANOPARTICLE CONJUGATES AND USES THEREOF, by MASSACHUSETTS INST TECHNOLOGY, dated 2017-11-15, which discloses the targeted delivery of therapeutic agents to specific cells remains a challenge in drug delivery. Provided herein are nanoparticle-targeting agent conjugates that can be used for the targeted delivery of therapeutic agents to certain cells and target tissues. The conjugates comprise nanoparticles (e.g., metal nanoparticles such as gold nanoparticles) with organic outer shells capable of adsorbing large numbers of therapeutic agents (e.g., small molecule drugs). The nanoparticles are covalently linked to targeting agents (e.g., proteins such as antibodies). The present invention also provides formulations comprising the nanoparticle-targeting agent conjugates, and kits comprising the same. In another aspect, the present invention provides methods of using the conjugates for the delivery of therapeutic agents to cells, and the treatment and / or prevention of diseases (e.g., autoimmune diseases, infectious diseases, proliferative diseases such as cancer). In another aspect, the present invention provides methods of preparing the nanoparticle-targeting agent conjugates described herein.

[0008] Another reference has been made to US2016367608A1, titled "NOVEL LACTOBACILLUS PLANTARUM AND COMPOSITION CONTAINING THE SAME", by CJ CHEILJEDANG CORP, dated 2016-09-01, which discloses, a Lactobacillus plantarum CJLP56 KCTC 11402BP, a composition containing the Lactobacillus for treating intestinal diseases, and a composition containing the Lactobacillus for enhancing immunity.

[0009] Another reference has been made to WO2019072885A1, titled "MAGNETIC NANOPARTICLES FOR THE TREATMENT OF CANCER", by INST

[0010] 4 NAT SANTE RECH MED, INSTITUT NAT DES SCIENCES APPLIQUEES, CENTRE NAT RECH SCIENT, and UNIV TOULOUSE 3 PAUL SABATIER, dated 2018-10-10, which discloses, methods and pharmaceutical compositions for the treatment of cancer in a subject in need thereof. Therapeutic strategies using drugs that cause Lysosomal Cell Death have been proposed for the eradication of resistant cancer cells. Nano therapy based on Magnetic Intra-Lysosomal Hyperthermia (MILH) generated by magnetic nanoparticles (MNPs) that are grafted with ligands of receptors overexpressed in tumors appears to be a very promising therapeutic option. The inventors investigated the mechanisms whereby MILH induces cell death using Gastrin-grafted MNPs specifically delivered to lysosomes of tumor cells from different cancers. The inventors provide evidence that MILH causes cell death through a non-apoptotic signaling pathway. The mechanism of cell death involves temperature elevation at the nanoparticle periphery, which enhances the production of reactive oxygen species through the lysosomal Fenton reaction. Subsequently, MILH induces lipid peroxidation, lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol, including Cathepsin-B which activates Caspase-1 but not apoptotic Caspase-3. Thus, the invention relates to a magnetic nanoparticle grafted with a tumor targeting agent for use in a method for inducing non-apoptotic signaling of cancer cell in a subject afflicted with cancer in need thereof.

[0011] Another reference has been made to US2021220480A1, titled "Cancer Treatment and Imaging Methods Using Thermotherapy and Drug Delivery", by PEYMAN GHOLAM A, dated 2021-04-01, which discloses, Cancer treatment and imaging methods using thermotherapy and drug delivery are disclosed herein. In one embodiment, the method comprises the steps of administering a plurality of antibody or aptamer-conjugated nanoparticles, liposomes, and / or micelles containing a medication and / or gene to a patient

[0012] 5 in need thereof so as to target a tumor in the patient, at least some of the antibody or aptamer-conjugated nanoparticles, liposomes, and / or micelles attaching to surface antigens of tumor cells of the tumor so as to form a tumor cell / nanoparticle / liposome / micelle complex; and heating the antibody or aptamer-conjugated nanoparticles, liposomes, and / or micelles using an energy source so as to raise the temperature of the tumor cell / nanoparticle complex, micelle complex, and / or liposome complex, thereby releasing one or more medications from the antibody or aptamer- conjugated nanoparticles, liposomes, and / or micelles, and damaging one or more tumor cell membranes at the tumor site.

[0013] Another reference has been made to JP2023103204A, titled "IRON OXIDE MAGNETIC PARTICLES", by ZTI BIOSCIENCES CO LTD, dated 2023-04-04, which discloses, iron oxide magnetic particles that can be used in various fields. SOLUTION: In iron oxide magnetic particles containing iron oxide and MXn, M includes one or more selected from a group consisting of Cu, Sn, Pb, Mn, Ir, Pt, Rh, Re, Ag, Au, Pd, and Os, the X includes one or more selected from a group consisting of F, Cl, Br, and I, and the n is an integer of 1 to 6. Further, the MXn includes one or more selected from a group consisting of CuF, CuF2, CuF3, CuCI, CuCI2, CuBr, CuBr2, Cui, CuI2, and CuI3.

[0014] Another reference has been made to US20030099725, titled "Herbal compositions useful as chemo preventive and therapeutic agents and methods of manufacturing same", by GLOBAL CANCER STRATEGIES LTD, dated 2001-02-26, which discloses, Compositions derived from traditional Chinese herbal medicines, medicinal plants and extracts thereof, are provided for the prevention and treatment of cancers especially cancer of the lung, esophagus, stomach, oral cavity and prostate as well as for treating Helicobacter pylori infection. The compositions of the invention are obtained through specific techniques and have demonstrated efficacy for

[0015] 6 chemoprevention of cancers of the lung, esophagus, stomach, and the oral cavity as well as exhibiting in vitro activity against prostate cancer and leukemic cell lines. The compositions are useful as adjuncts to conventional surgery or radiotherapy treatments in patients with esophageal cancer. The compositions are effective as a treatment for H. pylori infection. Currently, there is no effective chemo preventive agent for these cancers. The current invention addresses a special need in cancer control. Preferred compositions of the invention contain the herbal ingredients Sophora tonkinensis, Polygonum bistorta, Prunella vulgaris, Sonchus brachyotus, Dictamnus dasycarpus Turcz, and Dioscorea bulbifera.

[0016] Another reference has been made to US20140010844, titled "Tissue Targeted Antigenic Activation of the Immune Response to Treat Cancers", by QU BIOLOG INC, dated 2013-06-20, which discloses, in part methods of treating cancers of a specific organ or tissue by administering a composition that is antigenically specific for one or more microbes that are pathogenic in the specific organ or tissue in which the cancer is situated. The formulations of the invention thereby facilitate activation of an immune response to a cancer in a particular tissue or organ. The compositions may for example include killed or attenuated microbial pathogens, such as whole killed bacterial cells, and may be administered at sites distant from the cancer, for example the skin. In some embodiments, microbial species of endogenous flora that are known to cause infection in the relevant organ or tissue may be used in the formulation of the antigenic compositions. In alternative embodiments, exogenous microbial pathogens that are known to cause infection in the relevant organ or tissue may be used in the formulation of the antigenic compositions. The administration of the immunogenic compositions may be repeated relatively frequently over a relatively long period of time. In embodiments for intradermal or subcutaneous injection, dosages may be adjusted so that injections

[0017] 7 reproduce a consistent, visible, delayed inflammatory immune reaction at the successive site or sites of administration.

[0018] Another reference has been made to WO2007142674, titled "NANO PARTICLE HEATING AND APPLICATIONS THEREOF" by MASSACHUSETTS INST TECHNOLOGY, HAMAD-SCHIFFALI KIMBERLY, and WIJAYA ANDY [US], dated 2006-10-27, which discloses, magnetic nanoparticles, which when placed in a magnetic field are selectively heated at a certain frequency of the magnetic field, as a function of their size, composition, or both. The invention also provides for use of such nanoparticles, in applications including, inter alia, selective nanoparticle heating and applications thereof, hyperthermia induction in cells or tissue, remote alteration of protein structure and / or drug delivery.

[0019] Another reference has been made to WO2017156148A1, titled "FUNCTIONALIZED PRUSSIAN BLUE NANOPARTICES, COMBINATION PRUSSIAN BLUE NANOPARTICLE-BASED NANO-IMMUNOTHERAPHY AND APPLICATIONS THEREOF", by FERNANDES ROHAN, SZE RAYMOND W, CRUZ CONRAD RUSSELL Y, SANDLER ANTHONY D, BOLLARD CATHERINE M, SWEENEY ELIZABETH E, CANO-MEJIA JULIANA, BURGA RACHEL, and DUMONT MATTHIEU F, dated 2017-03-08, which discloses, combination treatment with Prussian blue nanoparticles and at least one immunotherapeutic treatment. Stable, functionalized Prussian blue nanoparticles, including those with enhanced stability under alkaline conditions, and methods of cancer, neoplasm, and tumor treatment using them, including photothermal treatment and combination immunotherapeutic treatments.

[0020] Another reference has been made to KR20170104024A, titled "Nanovesicles derived from Helicobacter pylori and Use thereof", by MD HEALTHCARE INC, and UNIV CHUNG ANG IND ACAD COOP FOUND, dated 2016-03-03, which discloses, nanovesicles isolated from Helicobacter pylori and, more specifically, to nanovesicles isolated from Helicobacter pylori specifically attached to stomach tissue and uses thereof. The nanovesicles isolated from Helicobacter pylori according to the present invention reduces that a material is delivered to an undesirable place by specifically delivering a material for treating or diagnosing stomach diseases to stomach tissue, thereby reducing side effects, and pain and inconvenience of patients. In addition, the nanovesicles can increase treatment efficiency or diagnosis efficiency by delivering the material to specific cells or specific tissue. If it is hard to treat metastatic cancer or the like by an existing anticancer therapy, the nanovesicles can be used for effectively treating metastatic cancer.

[0021] Another reference has been made to JP6469603B2, titled "TREATMENT OF CANCER OR TUMOR INDUCED BY RELEASE OF HEAT GENERATED BY VARIOUS CHAINS OF MAGNETOSOMES EXTRACTED FROM MAGNETOTACTIC BACTERIA AND SUBMITTED TO ALTERNATIVE MAGNETIC FIELD", by NANOBACTERIE, dated 2016-02-22, which discloses, drugs or methods for the treatment of a tumor or tumor cell or cancer of a subject in need by the generation of heat. SOLUTION: The invention relates to a drug or medical device comprising a chain of magnetotactic bacteria for use in the treatment of a tumor by heat treatment, wherein magnetosomes constituting a chain of the magnetosomes possess crystallographic directions orientated in the direction of the chain elongation in a degree that the chain of magnetosomes possess a magnetic anisotropy that is stronger than that of individual magnetosomes.

[0022] Another reference has been made to US11000596B2, titled "Compositions and methods for the prevention and treatment of cancer", by UTI LP, UTI LTD PARTTIERSHIP, dated 2018-11-26, which discloses, Conventional cancer immunotherapy falls short at efficiently expanding T

[0023] 9 cells that specifically target cancerous cells in numbers sufficient to significantly reduce the tumor size or cancerous cell number in vivo. To overcome this limitation, provided herein are nanoparticles coated with MHC class I and / or class II molecules presenting tumor-specific antigens and co-stimulatory molecules and their use to expand antigen-specific anti- tumorigenic T cells to levels not achieved in current immunotherapeutic techniques. These antigen-specific anti-tumorigenic T cells include cytotoxic T cells, effector T cells, memory T cells, and helper T cells that are necessary to initiate and maintain a substantial immune response against metastatic or non-metastatic cancerous, pre-cancerous, or neoplastic cells in vivo. The present invention describes a systemic approach to targeting cancerous or pre-cancerous cells that are circulating cells, as in lymphomas, migratory metastatic cells, and solid tumors.

[0024] Another reference has been made to CN 109331179A, titled "Application of composite magnetic nanoparticles", by CHINESE PLA GENERAL HOSPITAL, dated 2018-10-25, which discloses, an application of composite magnetic nanoparticles in killing Helicobacter pylori (H. pylori). Zn0.5Fe2.5O4@SiO2 composite magnetic nanoparticles are of a uniform core-shell structure under an electron microscope and have good super paramagnetism. The Zn0.5Fe2.5O4@SiO2 composite magnetic nanoparticles can effectively inhibit the growth of in vitro H. pylori in an alternating magnetic field, and affect the drug sensitivity of H. pylori to amoxicillin. Under suitable dosage and heating condition, the Zn0.5Fe2.5O4@SiO2 composite magnetic nanoparticles can achieve intra gastric heating without causing gastric mucosal damage.

[0025] Another reference has been made to WO2024039151A1, titled "SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES FOR IMMUNOTHERAPY", by SEOUL NATIONAL UNIV R & DB FOUNDATION [KR]; BRAIN & BEYONDS CO LTD [KR], dated 2023-08-11, which discloses, a

[0026] 10 composition for cancer immunotherapy, comprising superparamagnetic nanoparticles as an active ingredient, a composition for treating immune diseases, a composition for enhancing immunity, a composition for suppressing a decrease in immune function or enhancing immune function, the composition being administered in combination with a chemotherapeutic anticancer drug, a composition for suppressing a decrease in immune function, caused by anticancer treatment or enhancing immune function, and a composition for ameliorating, treating or preventing decreased immune responses in individuals with cancer. The superparamagnetic nanoparticles according to the present invention have magnetically-induced heating characteristics and high biocompatibility, thus being applicable through in vivo injection, and can induce an increase in immune cells, thus being effectively used in the treatment of cancer and immune diseases.

[0027] Another reference has been made to CA3132653A1, titled "CANCER. TREATMENT", by STIMIT CORP, dated 2020-05-01, which discloses, technologies for treatment and / or prevention of cancer and metastatic tumors. For example, in some embodiments, a biomaterial (e.g., polymeric biomaterial) or scaffold that can stimulate innate immunity is administered in the absence of an immunomodulatory payload to a target site in a subject following tumor removal (e.g., by surgical resection). Such technologies can reduce or inhibit incidence of tumor regrowth and / or metastasis.

[0028] Another reference has been made to US10682421B2, titled "Nanoparticle composition for use in targeting cancer stem cells and method for treatment of cancer", by UNIV CITY HONG KONG, dated 2018-03-08, which discloses, a composition in the form of a nanoparticle. The nanoparticle composition has a diameter from 5 to 500 nanometers. The nanoparticle composition has i) a central core portion including magnetic Fe3O4 nanoparticles adapted to act as a heat source when subjected to a magnetic field and a

[0029] 11 chemotherapeutic agent configured to treat cancer tissues, ii)— a shell portion including a shell member encapsulating said core portion, and iii)— antibodies configured to target cancer stem cells and adhered to surface of said shell member. The chemotherapeutic agent is a heat shock protein inhibitor and is releasable on activation of the heat source due to the magnetic field, and the shell member is made of silica or a silica-based material. Surface of the nanoparticle is modified with the antibodies capable of binding with a cluster of differentiation molecules on the cell surface of the target cancer stem cells, whereby by way of combination of specificity of the nanoparticle composition due to the antibody, thermo-therapeutic effect of the Fe3O4 nanoparticles, and release of the heat shock protein inhibitor on site at the target cancer stem cells, inhibition of the target cancer stem cells is synergistically and additionally enhanced is increased.

[0030] Another reference has been made to CN104667277A, titled "Application of using protein-coated iron-based magnetic nano-particle as magnetic hyperthermia agent", by UNIV NORTHWESTERN, dated 2015-02-11, which discloses, an application of using a protein -coated iron-based magnetic nano-particle as a magnetic hyperthermia medium. By taking the ironbased magnetic nano-particle as a core, the surface of the magnetic nanoparticle core is coated with protein to form stable magnetic hydrosol. The magnetic hyperthermia medium has good biocompatibility, stable magnetic soil and high magnetic hyperthermia performance, can be used for effectively killing cancer cells, and has a wide application prospect in magnetic hyperthermia and hyperthermia chemotherapy as well as integrated anti-cancer diagnosis and treatment.

[0031] Another reference has been made to JP2007031393A, titled "MAGNETIC NANOPARTICLE FOR THERMOTHERAPY OF TUMOR", by FUJIFILM CORP, dated 2005-07-29, which discloses, the magnetic nanoparticles for use in a thermotherapy of tumor is such that the surface of magnetic

[0032] 12 nanoparticles 1-100 nm in average size is coated with a compound having aggregation-inhibitory effect, and an antibody bindable selectively to cancer cells is bound to the compound.

[0033] Another reference has been made to US11939595B2, titled "Nanoscale artificial antigen presenting cells", by UNIV JOHNS HOPKINS, dated 2019- 08-23, which discloses, nano-scale Artificial Antigen Presenting Cells (aAPC), which deliver stimulatory signals to lymphocytes, including T- helper lymphocytes, for use as a powerful tool for immunotherapy.

[0034] Another reference has been made to CN101247836A, titled "Nanoparticle active ingredients conjugates", by MAGFOR.CE NANOTECHNOLOGIES AG, dated 2006-04-12, which discloses, to nanoparticles, whereby at least one therapeutically active substance is bonded to the nanoparticles and the release of the therapeutically active ingredient is brought about or initiated by an alternating magnetic field. The invention further relates to pharmaceutical compositions, in particular, injection solutions comprising said nanoparticle and the use thereof for the treatment of cancer.

[0035] Another reference has been made to CN101247836A, titled "Compositions and Methods for Delivering Biopolymers", by University of Michigan, dated 2020-01-10, which discloses, nanoparticles complexed with biomacromolecule agents configured for treating, preventing or ameliorating various types of disorders, and methods of synthesizing the same. SOLUTION: A composition comprises a nanoparticle, where an antigen is complexed with the nanoparticle, where an adjuvant is complexed or admixed with the nanoparticle, where the nanoparticle is selected from the group consisting of sHDL nanoparticles, fullerenes, and the like. The antigen is selected from the group consisting of antigens against PCSK9 and antigens against gplOO melanoma. The adjuvant is selected from among CPG, polylC, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, and the like.

[0036] Another reference has been made to US20190091350A1, titled "Cancer Treatment Methods Using Thermotherapy And / or Enhanced Immunotherapy", by PEYMAN GHOLAM A, dated 2018-11-26, which discloses, the method comprising the steps of administering a plurality of nanoparticles to target a tumor in a patient, the nanoparticles being coated with an antitumor antibody, cell penetrating peptides (CPPs), and a polymer, and the nanoparticles containing medication and / or gene, and a dye or indicator in the polymer coating, at least some of the nanoparticles attaching to surface antigens of tumor cells so as to form a tumor cell / nanoparticle complex; exciting the nanoparticles using an ultrasound source generating an ultrasonic wave so as to peel off the polymer coating of the nanoparticles, thereby releasing the dye or indicator into the circulation of the patient and the medication and / or gene at the tumor site; and imaging a body region of the patient so as to detect the dye or indicator released into the circulation of the patient.

[0037] Another reference has been made to US11820822B2, titled "Methods for sensitizing cancer cells to T cell-mediated killing by modulating molecular pathways", by DANA FARBER. CANCER INST INC, dated 2018-06-05, which discloses, identification of biomarkers, and methods of modulate thereof, for sensitizing cancer cells to T cell-mediated killing. For example, the present invention, in part, comprises methods of sensitizing cancer cells in a subject afflicted with a cancer to cytotoxic T cell-mediated killing comprising administering to a subject a therapeutically effective amount of an agent that modulates the biomarker.

[0038] 14 Another reference has been made to US2023067643A1, titled "IRON OXIDE NANOPARTICLE FOR TARGETED CHEMO-IMMUNOTHERAPY", by UNIV WASHINGTON, dated 2022-08-23, which discloses, Multifunctional nanoparticle for targeted therapeutic drug delivery, comprising (a) an iron oxide core having silica-polyethylene glycol coupled thereto to provide an iron oxide coated core, (b) a cytotoxic agent reversibly associated with the coated core; (c) an immunomodulating agent reversibly associate with the coated core; and (d) a tumor targeting agent associated with the coated core. Methods for using the nanoparticle to treat cancers and methods for making the nanoparticle.

[0039] However, none of the above discussed invention relates to a Hyperthermia induced gut microbiome-mediated system to treat gastric cancer bv activating natural immunity and cell mediated, said system facilitates hyperthermia induced activation to activate cell mediated immunity for gastric cancer treatment and does not uses any chemotherapeutic drug and radiation treatment: said system uses a wild type / non-pathoaenic / attenuated strain of Helicobacter pylori coated with Iron-oxide Nanoparticles to specifically targeting gastric cancerous tissue bv magnetic hyperthermia induced activation of the natural immune system. Additionally, the said Iron-oxide Nanoparticles are MRI sensitizers and hyperthermia responsive moiety, so, on applying a high- freouencv external magnetic field, the Iron-oxide nanoparticles generate heat and the Ivsis bacterial cell, and the Ivsed bacteria are exposed to the tumor microenvironment and triggers various immune cell gene expression changes such as, upregulation of IFN- v, TNF-B, IL-6 etc. The activated Antigen Presenting Cell including microphages start cleaning tumor microenvironment bv flushing out the foreign cell including cancerous cell. Furthermore, the

[0040] 15 present invention uses hyperthermia as standalone treatment via activating human body's natural and cell mediated immunity, while the above discussed invention uses chemotherapy drugs for the target drug therapy for cancer treatment, said invention does not use any chemotherapeutic drugs and precludes undesirable side effects of chemotherapy.

[0041] Objective of the Invention:

[0042] The main objective of the invention is to provide a hyperthermia induced gut microbiome mediated system to treat Gastric / Gastric MALT / Duodenal cancer.

[0043] Another objective of the invention is to provide a treatment method that uses wild type / non-pathogenic / attenuated strain of Helicobacter pylori coated with Iron-Oxide Nanoparticles to specifically target gastric cancer.

[0044] Another objective of the invention is to provide a tissue targeted drug delivery treatment system to specifically target gastric tissue for the efficient treatment of Gastric / Gastric MALT / Duodenal cancer and minimizing the toxicity.

[0045] Another objective of the invention is to provide a magnetic hyperthermia induced activation of body's natural and cell mediated immune system for specifically targeting gastric cancerous cell.

[0046] Another objective of the invention is to provide Hyperthermia as standalone treatment without involving chemotherapy for the effective treatment of gastric cancer.

[0047] Another objective of the invention is to provide chemotherapy and radiation free treatment for gastric cancer.

[0048] 16 Another objective of the invention is to provide system for activating innate immunity for the target killing of malignant cancerous cell without affecting the normal healthy cells.

[0049] Another objective of the invention is to provide iron oxide nanoparticles, that on applying an external magnetic field generate heat.

[0050] Another objective of the invention is to provide magnetic induced activation of immune system on applying a high frequency external magnetic field.

[0051] Another objective of the invention is to activate B and T-cells against cancer tissue using hyperthermia.

[0052] Another objective of the invention is to activate B and T-cells against cancer tissue using tissue microbiome.

[0053] Another objective of the invention provides generation of memory (immunization) in situ against the tumor tissue and the tissue microbiome.

[0054] Summary of the Invention:

[0055] The present invention provides a hyperthermia induced gut microbiome- mediated system to treat gastric cancer. The said system activates human body's innate immunity to treat gastric cancer and does not use any chemotherapeutic drug and radiation treatment. The said system uses a wild type / non-pathogenic / attenuated strain of Helicobacter pylori strain coated with Iron-Oxide Nanoparticles to specifically target gastric cancerous tissue. The said Iron-Oxide nanoparticles are MR.I sensitizers as well as hyperthermia responsive moiety, and on applying an external magnetic field the Iron-oxide nanoparticles generate heat on the surface of bacteria, causing lysis of bacterial cell, the lysed bacteria is spillage around the tumor microenvironment and trigger various immune cell gene expression changes, such as upregulates IFN- y, TNF-p, IL-6 etc., which further activates various antigen presenting cells including macrophages;

[0056] 17 the activated microphages start cleaning of tumor microenvironment by flushing out the foreign particulates including malignant cancer cells from the gut microenvironment.

[0057] Statement of the Invention:

[0058] Accordingly, the present invention provides a hyperthermia induced gut microbiome-mediated system to treat gastric cancer, the said system activates human body's innate immunity to treat gastric cancer without using any chemotherapy drug and radiation treatment, the said system establishes hyperthermia as standalone treatment of the cancer and it uses a non-pathogenic strain of Helicobacter pylori coated with Iron Oxide Nanoparticles to specifically target gastric cancer, by the magnetic hyperthermia induced activation of the human body's natural immune system; the said IONPS are MR.I sensitizers and hyperthermia responsive, and on applying a high frequency external magnetic field, the IONPs generate heat on the bacteria surface, causing lysis of the bacteria; the lysed bacteria spillage around the tumor microenvironment and activates various antigen presenting cell including microphages, which then start cleaning the tumor microenvironment by flushing out the foreign particulates including cancer cells; moreover, the developed system does not use any chemotherapy drugs and radiation treatment, and hence it precludes the undesirable adverse effects of the chemotherapy.

[0059] Brief Description of the Drawings:

[0060] Figure 1: represents work flow of magnetic hyperthermia induced activation of the body's natural immune system by helicobacter pylori coated Iron-Oxide Nanoparticle facilitating immune-clearance of cancer cells malignant cancerous cell.

[0061] 18 Figure 2: represents experimental results of iron oxide nanoparticles wherein figure 2(a) is a dynamic light scattering graph that exhibits hydrodynamic diameter of 74.68 nm, and the figure 2(b) is high resolution transmission electron microscopy (HR-TEM) study data showing the size of obtained IONPS (Iron oxide nanoparticles) i.e. 5 nm. Further, the figure 2(c) represents energy dispersive spectroscopy (EDS) spectra that indicates presence of iron in the iron oxide nanoparticles (IONPs).

[0062] Figure 3: represents analytical studies data of the iron oxide nanoparticles; wherein figure 3(a) shows FTIR analysis data that exhibits specific functional groups peaks present in the IONPs; and figure 3 (b) represents Raman spectroscopy analysis data of iron oxide and the figure 3(c) represents XRD analysis data showing crystallite structure of IONPs.

[0063] Figure 4: represents hyperthermia and magnetic studies of iron oxide nanoparticles; wherein figure 4(a) shows hyperthermia studies data of iron oxide and figure 4(b) represents magnetic studies data of iron oxide respectively.

[0064] Figure 5: represents cytotoxic studies both in presence and absence of hyperthermia.

[0065] Figure 6: represents electron microscopic images of samples including uncoated bacteria (a), IONPs coated bacteria (b), magnetic hyperthermia treated IONPs coated bacteria (c), and magnetic hyperthermia treated IONPs coated bacteria (d).

[0066] Figure 7: represents growth curve and growth pattern of bacteria; wherein the figure 7(a) shows growth curve of control uncoated helicobacter pylori bacteria and IONPs coated helicobacter pylori. Further, the figure 7(b) illustrates growth pattern of bacteria after undergoing hyperthermia treatment.

[0067] 19 The figures are merely for illustration purpose and shall not be construed to limit the scope of the invention.

[0068] Detailed Description of the Invention:

[0069] It should be noted that the particular description and embodiments set forth in the specification below are merely exemplary of the wide variety and arrangement of instructions which can be employed with the present invention. The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. All the features disclosed in this specification may be replaced by similar other or alternative features performing similar or same or equivalent purposes. Thus, unless expressly stated otherwise, they all are within the scope of present invention. Various modifications or substitutions are also possible without departing from the scope or spirit of the present invention. Therefore, it is to be understood that this specification has been described by way of the most preferred embodiments and for the purposes of illustration and not limitation.

[0070] The present invention provides a hyperthermia induced gut microbiome- mediated system to treat gastric cancer, the said system uses a non- pathogenic strain of Helicobacter pylori coated with Iron-Oxide Nanoparticles to specifically target gastric cancerous cell by magnetic hyperthermia-induced activation of the human body's natural immune system in presence of a high frequency external magnetic field; the said iron-oxide nanoparticles (IONPS) are MR.I sensitizers and hyperthermia responsive moiety therefore they are used to visualize the infected tissues.

[0071] The helicobacter pylori bacteria are firstly coated with the iron oxide nanoparticles and then is directly delivered to the gastric tissue for treatment; wherein the coating method of iron oxide nanoparticles or IONPs on the bacterial surface comprises steps of:

[0072] 20 I. harvesting helicobacter pylori bacteria;

[0073] II. the harvested bacteria are washed thrice with phosphate buffered saline (PBS) or distilled water;

[0074] III. sonication of iron oxide nanoparticles;

[0075] IV. STPP is added dropwise to the bacterial solution while stirring continuously;

[0076] V. adding the sonicated iron oxide nanoparticles to the solution;

[0077] VI. after addition of iron oxide nanoparticles to the solution, the prepared solution is stirred for 2 hours;

[0078] VII. after stirring, the entire mixture is incubated at 37°C overnight;

[0079] VIII. after the incubation, the solution is washed again with PBS to remove any unbound IONPS or other unwanted components from the reaction;

[0080] IX. finally, the purified solution containing the H. pylori cells coated with IONPs is stored.

[0081] The present invention provides immune activating effect of hyperthermia- treated iron oxide nanoparticles (IONPs) coated with H. pylori bacteria. T and B cells were initially exposed to hyperthermia-treated IONPs coated H. pylori for six hours, a period anticipated to stimulate immune cell activation and cytokine production. Subsequently, these activated immune cells were co-cultured with both conditioned (containing hyperthermia-lysed bacterial particles) and unconditioned AGS gastric adenocarcinoma cells. To quantify the immune response, IL-6 and TGF-p cytokines within the conditioned media were labeled with fluorescent antibodies and analyzed using flow cytometry. Elevated levels of these cytokines in the flow cytometry data indicated a very strong immune response triggered by the hyperthermia- treated bacteria. Additionally, these bacteria effectively stimulated T and B cell activation, demonstrating their potential to modulate the immune system and effectively target gastric cancer cells.

[0082] 21 The materials used for immune activation are T cells (CD4+ cells) and B cells as immune cell sources, and an AGS cell line representing human gastric adenocarcinoma cells as a target cell line. Iron oxide nanoparticles coated with H. pylori bacteria were employed as the immunogenic stimulus. Conditioned media, containing hyperthermia-lysed bacterial particles, utilized for cytokine analysis. Fluorescent antibodies specific for IL-6 and TGF-p cytokines employed for cytokine detection, while a flow cytometry instrument used for cytokine quantification.

[0083] The present invention provides the method of immune activation that comprises steps of (a) Immune cell interaction involves initial exposure of T and B cells to hyperthermia-treated IONPS coated H. pylori for six hours. It is anticipated that this incubation would activate immune cells, stimulating cytokine production. Subsequently, activated B and T cells exposed to both conditioned and unconditioned AGS cells, and immune activation levels for IL-6 and TGF-p checked in both cell types.

[0084] (b) After the immune cell interaction, cytokine analysis is conducted using flow cytometry. Specific cytokines, IL-6 and TGF-p, within the conditioned media labeled with fluorescent antibodies. The labeled cytokines then quantified in terms of amount and type using flow cytometry.

[0085] (c) Now, flow cytometry data is analyzed to detect and quantify IL-6 and TGF-p production by T and B cells. Elevated levels of these cytokines were indicative of a strong immune response initiated by the immune cells.

[0086] (d) Increased cytokine levels suggested that hyperthermia-treated bacteria effectively activated both T and B cells. These findings indicated the potential of these bacteria to modulate the immune system and induce a response against gastric tumor cells, as represented by the AGS cell line.

[0087] 22 Flow cytometry analysis of cytokine levels offers valuable insights into the immune response triggered by hyperthermia-treated IONPS coated with H. pylori. The said method of immune activation allows for the assessment of the efficacy of these bacteria in activating immune cells and potentially targeting gastric cancer cells.

[0088] In the present invention the helicobacter coated Iron-oxide nanoparticles are directly delivered to the gastric tissue, and on applying an high frequency external magnetic field the said IONPs generates heat and raises temperature of the tumor tissue for the treatment; further, the IONPs, generates heat on the bacterial surface and causing lysis of the bacteria, this lysed bacteria spillage around the tumor microenvironment and these spilled bacterial metabolites cytosolic material in the tumor microenvironments trigger various immune cell gene expression changes such as, upregulation of IFN- y, TNF-p, IL-6 etc., these activation draw antigen presenting cell including microphages into the tumor microenvironment, and subsequently, the activated microphages start cleaning the tumor microenvironment by flushing out foreign particulates including malignant cancerous cell.

[0089] The iron-oxide nanoparticles comprise size between 1 nm to 100 nm preferably size of about 5 nm, said iron oxide nanoparticles are MR.I sensitizers and are used to visualize the infected gastric tissue. Further, the IONPs also exhibits hyperthermia response during hyperthermia and therefore, in presence of a strong high frequency external magnetic field a heat dissipation is processed around the Iron oxide Nanoparticles, which results into the heat generation on the surface of Helicobacter pylori and causing lysis of bacterial membrane. This lysed bacterium is exposed to tumor microenvironment.

[0090] 23 On exposing to the tumor microenvironment, various immune potent activator and cytokines including IFN- y, TNF-p, IL-6 etc. are upregulated and the activation of these potent activator and cytokines triggers activation of various Antigen Presenting cell including macrophages; the Antigen Presenting cell now start recognizing the foreign particulate including cancer cell and thus, eliminates these cancerous cells from the gut microenvironment, and eventually clean the tumor microenvironment.

[0091] Experimental Result:

[0092] The experimental result of the present invention involves, a graphical representation of Dynamic light scattering (DLS) showing the hydrodynamic diameter of 74.68 nm 2(a), high resolution transmission electron microscopic (HR-TEM) image showing the size of IONPS i.e. 5nm 2(b), Energy-dispersive X-ray spectroscopy (EDS) shows the presence of iron in IONPs 2(c).

[0093] The Fourier transform infrared (FTIR) showing the specific functional group peaks present in IONPs and the graph plotted between wavenumber (cnrx) at X-axis and transmittance (%) in the Y-axis 3(a).

[0094] The Raman shifts and relative intensity, provides information about the iron-oxide nanoparticles composition, structure, and properties of a sample. The fig. 3(b) shows the graphical representation between Raman shift and relative intensity. The x-axis represents the Raman shift, measured in wavenumbers (cm-1) and y-axis represents the relative intensity of the scattered light. It indicates the energy difference between the incident laser light and the scattered light. Each peak in the spectrum (518.36, 578.95, 686.06, 777.87, 888.04, 953.53, 985.35, 1226.58, 1454.80 and 1559.46) corresponds to a specific vibrational mode of the nanoparticles.

[0095] 24 The graph between '2 theta and intensity' 3(c) representing X-ray Diffraction (XR.D) analysis of the crystallite structure of IONPS. '2 theta' on X-axis represents angle between diffracted x-ray with incident x-ray and intensity on Y-axis represents strength of the diffracted X-ray beam detected at a specific 20 angle.

[0096] The fig. 4(a) and fig. 4(b) shows the hyperthermia and magnetic studies of IONPs respectively. The fig. 4(a) represents the hyperthermia studies showing the increase in temperature (on Y-axis) at various frequencies (on X-axis). The fig. 4(a) represents magnetic studies of the IONPs, the X-axis represents the magnetic field and Y-axis represents the magnetization. The VSM (Vibrating sample magnetometer) measures the magnetic properties and magnetization of the IONPs 4(b) comes out to be 50.56 emu / g.

[0097] The cytotoxicity studies carried out both in presence and absence of Hyperthermia. A graph, shown in fig. 5 between cell viability on Y-axis and concentration on X-axis, with and without hyperthermia, provides insights into how IONPs affects cells under different conditions. The Control Group (as shown in fig. 5) represents cell viability in the absence of the IONPs. Control Group serves as a baseline for comparison. As the concentration of the substance increases, cell viability decreases. This indicates that higher concentrations are more toxic to the cells. The hyperthermia enhances cytotoxicity, the cell viability graph for the hyperthermia group will be lower than the control group at all concentrations, as shown in fig. 5. This suggests that hyperthermia increases the toxic effects of the IONPs.

[0098] The fig. 7(a) represents the growth curve of control uncoated H. pylori and IONPs coated H. pylori and fig. 7(b) illustrates the growth pattern of bacteria after hyperthermia treatment. The uncoated H. pylori line 7(a) represents the typical growth pattern of H. pylori under standard conditions

[0099] 25 and IONPS coated H. pylori demonstrates the growth curve of H. pylori bacteria coated with iron oxide nanoparticles.

[0100] The table below shows the percentage of IL-6 and TGF-p positive populations, indicating immune activation under different conditions.

[0101] Table

[0102] Additionally, on generating heat on the bacterial surface, the bacterial membrane damages and the spilled bacterial metabolites cytosolic material distributed in the tumor microenvironment and trigger various potent immune activator and cytokines such as upregulation of IFN- y, TNF-p, IL- 6 etc., which further draw Antigen Presenting Cells including macrophages into the tumor microenvironment. Subsequently, the activated macrophages start cleaning the tumor microenvironment by flushing out the foreign particulates including cancer cells.

[0103] Referring to figure 1, that represents work flow of magnetic hyperthermia induced activation of the body's natural immune system by helicobacter pylori coated Iron-Oxide Nanoparticle facilitating immune-clearance of cancer cells malignant cancerous cell, wherein the said Iron-Oxide Nanoparticles are loaded on the bacterial surface and are directly delivered to the targeted gastric tissue. Thereafter, a high frequency external magnetic field is applied around the targeted gastric tissue for magnetic hyperthermia induced activation the immune system; In presence of external magnetic field, the heat dissipates is processed around the said Iron oxide nanoparticles and which results into heat generation on the bacterial surface.

[0104] Referring to figure 2, that represents analytical studies data of the iron oxide nanoparticles; the figure 2(a) shows a dynamic light scattering graph which exhibits a hydrodynamic diameter of approximately 74.68 nm. The figure 2(b) represents high resolution transmission electron microscopic (HR-TEM) image of IONPS, where the HR-TEM image shows IONPS particle comprising a size of about 5 nm. Further, the figure 2(c) is the EDS spectra image that indicates presence of iron in the IONPs samples.

[0105] Referring to figure 3, that represents analytical studies data of iron oxide nanoparticles (IONPs); the figure 3 (a) represents FTIR spectra indicating presence of specific functional group peaks in the IONPs; and the figure 3 (b) represents Raman spectra of the lOPs. Furthermore, the figure 3(c) shows the XRD analysis spectra indicating presence of crystallite structure of IONPs.

[0106] 27 Referring to figure 4, that represents hyperthermia and magnetic studies of the iron oxide nanoparticles, the fig. 4(a) represents the hyperthermia studies showing the increase in temperature (on Y-axis) at various frequencies (on X-axis). The fig. 4(a) represents magnetic studies of the IONPS, the X-axis represents the magnetic field and Y-axis represents the magnetization. The VSM (Vibrating sample magnetometer) measures the magnetic properties and magnetization of the IONPs 4(b) comes out to be 50.56 emu / g.

[0107] Referring to figure 5, that represents cytotoxic studies data of the samples involving immune cell activation both in presence of hyperthermia and in absence of hyperthermia; the figure-5 shows MTT assay data that represents metabolic activity of the samples including iron oxide coated nanoparticles helicobacter pylori bacteria; MTT assay represents the cytotoxicity studies both in presence and absence of hyperthermia; the x axis represents the concentration of MTT reagent in pg / ml used, and on the other side y axis representing percentage of cell viability of samples including both with hyperthermia and without hyperthermia. The figure 5 further illustrate that the hyperthermia treated samples has relatively high cytotoxicity rate as compared to the samples that are not treated with hyperthermia.

[0108] Referring to figure 6, that represents electron microscopic images of the bacteria without coating with IONPs, electron microscopic image of bacteria coated with IONPs, and magnetic hyperthermia treated IONPs coated bacteria. The figure (a) shows the electron microscopic image of helicobacter pylori bacteria without coating with the IONPs, figure (b) is the electro microscopic image of IONPs coated helicobacter pylori bacteria and figures (c) and (d) represents magnetic hyperthermia treated IONPs coated bacteria.

[0109] 28 Referring to figure 7, that represent growth curve of control uncoated helicobacter pylori bacteria as well as Iron oxide nanoparticles coated helicobacter pylori bacteria. The fig. 7(a) represents the growth curve of control uncoated H. pylori and IONPS coated H. pylori and fig. 7(b) illustrates the growth pattern of bacteria after hyperthermia treatment. The uncoated H. pylori line 7(a) represents the typical growth pattern of H. pylori under standard conditions and IONPs coated H. pylori demonstrates the growth curve of H. pylori bacteria coated with iron oxide nanoparticles.

[0110] So accordingly, the invention provides a hyperthermia induced gut microbiome-mediated system to treat gastric cancer, the said system uses a non-pathogenic Helicobacter pylori strain coated with Iron-Oxide Nanoparticles for targeting gastric cancerous cell; said Iron-Oxide nanoparticles are MRI-sensitizers and hyperthermia responsive moiety, and on applying a high frequency external magnetic field the said Iron-Oxide nanoparticles generate heat on bacterial surface leading to lysis of bacterial cell, and the lysed bacteria spillage around the tumor microenvironment and further trigger various potent immune activator and cytokines including upregulation of IFN- y, TNF-p, IL-6 etc.; the activation of potent immune activator further activates various antigen presenting cells including macrophages in the tumor microenvironment, and these Antigen presenting cell start flushing out the foreign particulates including malignant cancer cells. Additionally, the said system does not use any chemotherapy drug and radiation treatment to kill cancerous cell, rather the invention activates natural immunity for the killing of cancerous cell including benign and malignant tumor cells.

[0111] In an exemplary embodiment of the invention, said A Hyperthermia- induced gut microbiome-mediated modulation of host immune system for treatment of gastric cancer comprising:

[0112] 29 -coating Helicobacter pylori bacteria with iron oxide nanoparticles having a size of about 5 nm;

[0113] -delivering the lONP-coated Helicobacter pylori bacteria to gastric tissue;

[0114] -applying a high-frequency external magnetic field to the gastric tissue to generate heat on the surface of the lONP-coated Helicobacter pylori bacteria,

[0115] -thereby causing lysis of the bacteria and releasing bacterial components; -the thermal annihilation of the bacteria will result in spillage of the foreign particles to surrounding tissue environment;

[0116] -inducing an immune response in the gastric tissue through the activation of antigen-presenting cells by the released bacterial components;

[0117] -stimulate immune response through chemotaxis and cytokine secretion; -the activated macrophages will cleanse the tumor microenvironment by flushing out the foreign particulates including cancer cells.

[0118] In another embodiment, said microbiome-mediated immune activation comprising: coating wild type / non-pathogenic / attenuated strain Helicobacter pylori bacteria with iron oxide nanoparticles (IONPS) on the surface of bacteria; delivering the coated bacteria to a gastric tumor site; generate heat on the surface of bacteria via an external magnetic field; causing bacterial lysis and release of bacterial components; lysed bacteria spillage around the tumor microenvironment activates the natural immunity of the body to target and eliminate cancer cells.

[0119] In another embodiment, said spilled bacterial metabolites and cytosolic materials within the tumor microenvironment trigger immune cell gene expression changes, including the upregulation of IFN-y, TNF-p, and IL-6, leading to enhanced anti-tumor immunity.

[0120] In another embodiment, said coating of IONPs on the bacterial surface comprises steps of:

[0121] 30 a) harvesting helicobacter pylori bacteria; b) the harvested bacteria are washed thrice with phosphate-buffered saline (PBS) / distilled water / any other suitable buffer; c) sonication of iron oxide nanoparticles; d) sodium tripolyphosphate (STPP) is added dropwise to the bacterial solution while stirring continuously; e) adding the sonicated iron oxide nanoparticles to the bacterial solution containing the H. pylori; f) the prepared solution is stirred for 2 hours, it allows sufficient interaction for the IONPS to attach to the bacterial surface; g) after stirring, the entire mixture is incubated at 37°C overnight; h) after the incubation, the solution is washed again with PBS to remove any unbound IONPs or other unwanted components from the reaction; i) finally, the purified solution containing the H. pylori cells coated with IONPs is stored.

[0122] In another embodiment, said immune activation comprising : T cells (CD4+ cells) and B cells as immune cell sources, and an AGS cell line representing human gastric adenocarcinoma cells as a target cell line; iron oxide nanoparticles coated with H. pylori bacteria were employed as the immunogenic stimulus; conditioned media, containing hyperthermia-lysed bacterial particles, utilized for cytokine analysis; fluorescent antibodies specific for IL-6 and TGF-p cytokines employed for cytokine detection, and a flow cytometry instrument for cytokine quantification.

[0123] In another embodiment, said immune activation method comprises the steps of: a) T and B cells are first exposed to hyperthermia-treated lONP-coated H. pylori for 6 hours.

[0124] 31 b) during this incubation, the immune cells become activated and produce cytokines. c) after the incubation, activated B and T cells are exposed to AGS cells (conditioned and unconditioned). d) immune activation for IL-6 and TGF-p is checked for B and T cells. e) specific cytokines (IL-6 and TGF-P) within the conditioned media are labeled with fluorescent antibodies. f) flow cytometry is then used to quantify the amount and types of cytokines present. g) the flow cytometry data is analyzed to detect and quantify IL-6 and TGF-p produced by T and B cells. h) elevated levels of these cytokines indicate a strong immune response by the immune cells. i) increased cytokine levels suggest that the hyperthermia-treated bacteria effectively activate T and B cells. j) this indicates the potential of these bacteria to modulate the immune system and induce a response against gastric tumor cells (AGS cell line).

[0125] In another embodiment, said treatment of gastric cancer as claimed in claim 1, comprising steps of: a) iron oxide nanoparticles are coated onto the surface of a wild type / non-pathogenic / attenuated strain of Helicobacter pylori bacteria, resulting in iron oxide nanoparticle-coated Helicobacter pylori. b) the iron oxide nanoparticle-coated Helicobacter pylori are delivered to the targeted gastric tissue. c) a high-frequency external magnetic field is applied to the targeted gastric tissue.

[0126] 32 d) the iron oxide nanoparticles on the Helicobacter pylori generate heat in the presence of the external magnetic field, causing bacterial membrane lysis. e) the lysed bacteria are released into the tumor microenvironment. f) exposure to lysed Helicobacter pylori upregulates various potent immune activators and cytokines, including IFN-y, TNF-p, and IL-6. g) the upregulation of these immune activators and cytokines triggers the activation of various antigen-presenting cells, including macrophages. h) macrophages recognize and eliminate cancer cells from the gut microenvironment, resulting in tumor clearance.

[0127] In another embodiment, said method provides memory generation / immunization against tumor.

[0128] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from this invention in its broader aspects and, therefore, the aim in the present invention is to cover all such changes and modifications as fall within the true spirit and scope of this invention.

[0129] Advantages of the Invention:

[0130] 1. Tissue targeted Delivery: The direct delivery of helicobacter coated Iron-oxide nanoparticles to the gastric tissue provides efficient treatment and minimizes toxicity.

[0131] 2. Microbiome-mediated therapy: The Helicobacter pylori is natural habitat and causative agent of gastric cancer, the application of natural microbiome of the tumor tissue activates innate immunity as well as eliminates tumor tissue.

[0132] 33 Magnetic Hyperthermia: On applying external magnetic field, the targeted IONPS generate heat in the tumor microenvironment and triggers body's immune response. Hyperthermia as standalone treatment: The generated heat by IONPs raises temperature of the local tumor tissue and causes lysis of bacterial tissue, which eventually leads to activation of antigen presenting cell to effectively clean tumor microenvironment. Chemotherapy free-treatment: The use of magnetic hyperthermia as standalone treatment without using any chemotherapy, facilitates adverse reaction free treatment. Radiation free treatment. Immunization of body against tumor - Coating IONPs with on H. pylori creates a targeted delivery system that can stimulate the immune system. These can be used to present antigens to the immune cells, effectively mimicking an infection and triggering a cell- mediated immune response. When the IONPs coated with H. pylori antigens are introduced into the body, they can be taken up by antigen-presenting cells (APCs) such as dendritic cells. These APCs process the antigens and present them on their surface, which is recognized by T cells, particularly cytotoxic T cells (CD8+ T cells). The presentation of these antigens leads to the activation of T cells, which then proliferate and target cells expressing the same antigens, such as cells infected with H. pylori or even cancer cells that may share similar antigens.

[0133] 34

Claims

We Claim:

1. A Hyperthermia-induced gut microbiome-mediated modulation of host immune system for treatment of gastric / gastric MALT / duodenal cancer comprising:- coating Helicobacter pylori bacteria with iron oxide nanoparticles having a size of about 5 nm;- delivering the lONP-coated Helicobacter pylori bacteria to gastric tissue;- applying a high-frequency external magnetic field to the gastric tissue to generate heat on the surface of the lONP-coated Helicobacter pylori bacteria,- thereby causing lysis of the bacteria and releasing bacterial components;- the thermal annihilation of the bacteria will result in spillage of the foreign particles to surrounding tissue environment;- inducing an immune response in the gastric tissue through the activation of antigen-presenting cells by the released bacterial components;- stimulate immune response through chemotaxis and cytokine secretion;- the activated macrophages will cleanse the tumor microenvironment by flushing out the foreign particulates including cancer cells.

2. The Hyperthermia-induced gut microbiome-mediated modulation for treatment of gastric cancer as claimed in claim 1, wherein said microbiome-mediated immune activation comprising: coating wild type / non-pathogenic / attenuated strain Helicobacter pylori bacteria with iron oxide nanoparticles (IONPS) on the surface of bacteria; delivering the coated bacteria to a gastric tumor site; generate heat on the surface of bacteria via an external magnetic field; causingbacterial lysis and release of bacterial components; lysed bacteria spillage around the tumor microenvironment activates the natural immunity of the body to target and eliminate cancer cells.

3. The Hyperthermia-induced gut microbiome-mediated modulation for treatment of gastric cancer as claimed in claim 1, wherein said, spilled bacterial metabolites and cytosolic materials within the tumor microenvironment triggers immune cell gene expression changes, including the upregulation of IFN-y, TNF-p, and IL-6, leading to enhanced anti-tumor immunity.

4. The Hyperthermia-induced gut microbiome-mediated modulation for treatment of gastric cancer as claimed in claim 1, wherein said coating of IONPS on the bacterial surface comprises steps of: a) harvesting helicobacter pylori bacteria; b) the harvested bacteria are washed thrice with phosphate buffered saline (PBS) / distilled water / suitable buffer; c) sonication of iron oxide nanoparticles; d) sodium tripolyphosphate (STPP) is added dropwise to the bacterial solution while stirring continuously; e) adding the sonicated iron oxide nanoparticles to the bacterial solution containing the H. pylori; f) the prepared solution is stirred for 2 hours, it allows sufficient interaction for the IONPs to attach to the bacterial surface; g) after stirring, the entire mixture is incubated at 37°C overnight; h) after the incubation, the solution is washed again with PBS to remove any unbound IONPs or other unwanted components from the reaction; i) finally, the purified solution containing the H. pylori cells coated with IONPs is stored.

5. The Hyperthermia-induced gut microbiome-mediated modulation for treatment of gastric cancer as claimed in claim 1, wherein said immune activation comprising : T cells (CD4+ cells) and B cells as immune cell sources, and an AGS cell line representing human gastric adenocarcinoma cells as a target cell line; iron oxide nanoparticles coated with H. pylori bacteria were employed as the immunogenic stimulus; conditioned media, containing hyperthermia-lysed bacterial particles, utilized for cytokine analysis; fluorescent antibodies specific for IL-6 and TGF-p cytokines employed for cytokine detection, and a flow cytometry instrument for cytokine quantification.

6. The Hyperthermia-induced gut microbiome-mediated modulation for treatment of gastric cancer as claimed in claim 1, wherein said immune activation method comprises the steps of: a) T and B cells are first exposed to hyperthermia-treated IONP- coated H. pylori for 6 hours. b) during this incubation, the immune cells become activated and produce cytokines. c) after the incubation, activated B and T cells are exposed to AGS cells (conditioned and unconditioned). d) immune activation for IL-6 and TGF-p is checked for B and T cells. e) specific cytokines (IL-6 and TGF-P) within the conditioned media are labeled with fluorescent antibodies. f) flow cytometry is then used to quantify the amount and types of cytokines present. g) the flow cytometry data is analyzed to detect and quantify IL-6 and TGF-p produced by T and B cells. h) elevated levels of these cytokines indicate a strong immune response by the immune cells.i) increased cytokine levels suggest that the hyperthermia-treated bacteria effectively activate T and B cells. j) this indicates the potential of these bacteria to modulate the immune system and induce a response against gastric tumor cells (AGS cell line).

7. The Hyperthermia-induced gut microbiome-mediated modulation for treatment of gastric cancer as claimed in claim 1, comprising steps of: a) iron oxide nanoparticles are coated onto the surface of wild type / non-pathogenic / attenuated strain of helicobacter pylori bacteria, resulting in iron oxide nanoparticle-coated helicobacter pylori. b) the iron oxide nanoparticle-coated helicobacter pylori are delivered to the targeted gastric tissue. c) a high-frequency external magnetic field is applied to the targeted gastric tissue. d) the iron oxide nanoparticles on the helicobacter pylori generate heat in the presence of the external magnetic field, causing bacterial membrane lysis. e) the lysed bacteria are released into the tumor microenvironment. f) exposure to lysed helicobacter pylori upregulates various potent immune activators and cytokines, including IFN-y, TNF-p, and IL- 6. g) the upregulation of these immune activators and cytokines triggers the activation of various antigen-presenting cells, including macrophages. h) macrophages recognize and eliminate cancer cells from the gut microenvironment, resulting in tumor clearance.The Hyperthermia-induced gut microbiome-mediated modulation for treatment of gastric cancer as claimed in claim 1, wherein said method provides memory generation / immunization against tumor.

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