Immunotherapeutic composition and preparation thereof

Abstract

The present disclosure provides an immunotherapeutic composition comprising (i) metallic cations (ii) an organic immunosuppressant (iii) phosphate species. The composition is a) capable of delivering antigens, b) activating immunological mechanisms in the immune cells c) inhibiting immune-suppressive cells in the body. The disclosure also provides a method of preparing said immunotherapeutic composition and a method of treatment of cancer and infectious diseases involving administration of said immunotherapeutic composition loaded with suitable antigens.

Description

[0001] IMMUNOTHERAPEUTIC COMPOSITION AND PREPARATION THEREOF

[0002] FIEED OF THE INVENTION

[0003] The present disclosure relates to the field of immunotherapy. Particularly, the present disclosure provides an immunotherapeutic composition comprising (i) metallic cations including but not limited to calcium, manganese, and zinc; (ii) an organic immunosuppressant (iii) phosphate species, wherein the composition is a) capable of delivering antigens, b) activating immunological mechanisms in the immune cells c) inhibiting immune- suppressive cells in the body. The disclosure also provides a method of preparing said immunotherapeutic composition and application thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] Cancer immunotherapy is a growing field in which the immune system is modulated to destroy tumor cells, resulting in durable antitumor immune responses. One of the major challenges faced in cancer immunotherapy is the lack of immunogenicity for cancer antigens where immune cells are not able to detect the cancer antigens as disease-causing molecules. Moreover, immunosuppression caused by specific types of immune cells such as myeloid-derived suppressor cells (MDSC), M2 polarised macrophages, N2 neutrophils, T regulatory cells, dendritic regulatory cells, and tumor-associated fibroblasts suppresses the native immune response against cancer or chronic infections caused by some types of viruses. Said immunosuppressive cells and proteins in the tumor microenvironment and systemic circulation cause adverse effects on cancer immunotherapy leading to severe suppression of immunity against tumors. Said cells cause significant adverse effects by reducing the efficacy of vaccination and immune checkpoint blockade-mediated T-cell response thereby hampering cancer immunotherapy. Normally, in typical vaccines, ‘adjuvants’ are employed to enhance the immunogenicity of otherwise poorly immunogenic antigens. Adjuvants, through their unique material composition, shape, and biological functionality, cause enhanced activation of key immunologic pathways associated with foreign body response, inflammation, tolllike receptor activation (TLR), Stimulator of interferon genes (STING) activation, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) activation, etc, leading to a stronger immune response against the antigens that are bound to the adjuvant. Some adjuvants used in the vaccines are calcium phosphate, Alum, poly- IC, CpG, Mono -phosphoryl lipid A (MPL), QS21, squalene, saponins, etc. These materials are known to enhance the immunogenicity of antigens. However, these adjuvants may not help overcome the adverse effect played by immunosuppressive cells and proteins in the tumor microenvironment and systemic circulation resulting in poor efficacy of vaccination leading to reduced T cell-mediated response due to uncontrolled immune checkpoint activation.

[0006] Targeting MDSCs represents a promising strategy to enhance immunotherapy efficacy. In the prior art, several nanoparticle-based drug delivery systems were investigated to target MDSCs in cancer. For instance, Liposomes and solid lipid nanoparticles were used to encapsulate immunomodulatory agents such as all-trans retinoic acid (ATRA), which promotes MDSC differentiation into nonimmunosuppressive cells. Similarly, PEGylated lipid nanoparticles were used to deliver siRNA or small-molecule inhibitors to deplete MDSCs or block their signalling pathways in cancer. Biodegradable polymeric nanoparticles such as PLGA were reported for sustained release of agents like doxorubicin or curcumin, which reduce MDSC-mediated immunosuppression. Surface modifications with targeting ligands (e.g., anti-CCR2 or anti-LOXl antibodies) enhance selective MDSC uptake. Among inorganic nanoparticles, Gold and silica nanoparticles were used as carriers for immune checkpoint inhibitors or photothermal therapy agents, for indirect disruption of MDSC function. Iron oxide nanoparticles have been explored for immune cell modulation and tumor imaging. Exosomes or synthetic vesicles were used to deliver miRNA or cytokines that reprogram MDSCs to a pro- inflammatory phenotype, enhancing anti-tumor immunity. However, no adjuvant can simultaneously enhance the immunogenicity of antigens and suppress or eliminate the immune suppressive cellular and non-cellular elements in the tumor microenvironment and systemic circulation to increase the effectiveness and efficacy of cancer immunotherapy.

[0007] Engineered nanoparticles were extensively utilized in cancer imaging and therapy. Traditionally, nanoparticle-based therapeutic approaches have focused on enhancing drug delivery, solubility, stability, and target specificity. However, over the past decade — particularly following the approval of COVID- 19 mRNA vaccines — the use of nanoparticles in vaccines and immunotherapy has emerged as a major area of research. Nanoparticles can serve as immune-modulating adjuvants in vaccines and / or facilitate the delivery of vaccine antigens, such as peptides, mRNA, or DNA, to immune cells, specifically targeting lymph nodes, the spleen, and tissue-resident immune cells. More recently, nanoparticles have also been employed to enhance the immunotherapeutic effects of immune checkpoint blockade antibodies (PD-1, PD-L1, CTLA-4) and CAR-T cell therapy. Furthermore, nanoparticles are now being explored as a promising strategy to overcome immune suppression in cancer, particularly addressing challenges posed by myeloid-derived suppressor cells, M2-polarized macrophages, and regulatory T cells.

[0008] The inventors of the present disclosure aim to address the aforementioned problems, by providing an optimized adjuvant immunotherapeutic composition based on engineered nanoparticles and a method for preparing the same, such that the composition not only enhances the immunogenicity of antigens but also eliminates the immune suppressive cells from the tumor microenvironment as well as systemic circulation, collectively facilitating enhanced vaccine-mediated immune response against cancer and other infectious diseases.

[0009] SUMMARY OF THE INVENTION

[0010] To achieve the objective specified above, the present disclosure provides an immunotherapeutic composition comprising (i) metallic cations of metals (M) (ii) an organic immunosuppressant (OIS), and (iii) a phosphate species.

[0011] In some embodiments, the immunotherapeutic composition comprises (i) metallic cations of metals (M) selected from the group comprising calcium, manganese, zinc, or a combination thereof; (ii) an organic immunosuppressant (OIS), and (iii) a phosphate species having a formula HXPO4 wherein x=0-3.

[0012] In some embodiments, the immunotherapeutic composition comprises, (i) metallic cations of metals (M) selected from the group comprising calcium, manganese, zinc, or a combination thereof; (ii) an organic immunosuppressant (OIS), and (iii) a phosphate species having a formula HXPO4 wherein x=0-3; and wherein the amount of metallic cation ranges from about 0.01 wt% to about 80 wt%; the amount of organic immunosuppressant ranges from about 0.01 wt% to about 60 wt%; and the amount of phosphate species ranges from about 10 wt% to about 80 wt% to the total weight of the composition.

[0013] In some embodiments, the organic immunosuppressant is selected from a group comprising 5-Fluorouracil, gemcitabine, paclitaxel, cyclophosphamide, doxorubicin, capecitabine, cisplatin, doxycycline.

[0014] In some embodiments, the phosphate species comprises phosphate, hydrogen phosphate, dihydrogen phosphate, trihydrogen phosphate, their hydrate, or a combination thereof.

[0015] In some embodiments, the composition optionally comprises one or more polymer capping agent(s) to form a hydrogel, wherein the polymer capping agent is selected from the group comprising polyvinyl alcohol (PVA), PVA modified with chitosan, galactomannan, polyethyleneimine, alginate, pectin, cellulose or a combination thereof.

[0016] In some embodiments, the immunotherapeutic composition is in the form of nanoparticles, microparticles, self-assembled micro-flowers, porous microstructures, needle-like structures, plate-like structures, injectable gels, implantable solid cakes, and wafers.

[0017] In some embodiments, the composition is loaded with one or more antigens, wherein the one or more antigens are cancer antigens selected from the group comprising protein, peptides, DNA, RNA, carbohydrates or antigens for infectious diseases caused by virus, bacteria, fungi.

[0018] In some embodiments, the composition serves as an adjuvant in vaccines, Immunotherapeutic s, immune checkpoint blockade antibodies, surgical procedures, chemotherapy, radiation therapy, nuclear medicine, photodynamic therapy, photothermal therapy, cell therapy using (Dendritic cells) DC, (Chimeric antigen receptor-T cells) CAR-T cells, or for immune regulation.

[0019] In another aspect, the present disclosure provides a formulation comprising the immunotherapeutic composition and biocompatible polymers.

[0020] In some embodiments, the biocompatible polymers are selected from a group comprising polyvinyl alcohol (PVA), polyethylene imine (PEI), polyvinylpyrrolidone (PVP), polylactic glycolic acid (PLGA), chitosan, carboxymethyl cellulose, carboxymethyl chitosan, gelatin, alginate, galactomannan, galactose, hydroxypropyl methylcellulose, cellulose, sucrose, polyacrylate, collagen, fibrin, sodium polyacrylate, acrylate polymers, polyethylene oxide, poly(2-acrylamido-2 methyl- 1 -propanesulfonic acid) (polyAMPS), polyacrylamide, silicone, agarose, methylcellulose, hyaluronan, hydrolyzed polyacrylic nitrile, polyethylene glycol (PEG), polyethylene oxide-co- polypropylene oxide (PPG), poly (amino acids), dextran, proteins, peptide hydrogel, DNA hydrogel or a combination thereof.

[0021] Yet another aspect of the disclosure provides a method for the preparation of immunotherapeutic composition of the present disclosure, wherein the method comprises: i. production of a cationic mixture by mixing of aqueous solutions of metallic salts under constant stirring at room temperature, wherein the metallic salts comprise chloride, nitrate, sulphate salts of metals (M); ii. addition of organic immunosuppressant (OIS) dissolved in a solvent to the cationic mixture under constant stirring at room temperature, resulting in the generation of intermediate complex M1-M2-M3- OIS; iii. addition of phosphate species to the intermediate complex with constant stirring, resulting in the generation of immunotherapeutic composition in the form of a precipitate; iv. isolation of the precipitate by centrifugation, and washing the precipitate.

[0022] In some embodiments, the method comprises the optional addition of a polymer capping agent to the cationic mixture in step (i) or after step (iv).

[0023] Another aspect of the disclosure relates to a method for the preparation of the formulation of the present disclosure comprising loading of the immunotherapeutic composition in a biocompatible polymer, together with adjuvants, followed by physical cross-linking by freeze-thaw cross-linking method or by chemical crosslinking by glutaraldehyde / Boric acid cross-linking chemistry.

[0024] Yet another aspect of the disclosure provides a method of treatment of cancer comprising administration of a therapeutically effective amount of immunotherapeutic composition of the disclosure, with or without coadministration of immunomodulators, therapeutic antibodies, CAR-T cells, oncolytic viruses, adoptive T cell transfer.

[0025] In some embodiments, the cancer comprises both solid and liquid tumors.

[0026] Another aspect of the disclosure provides a method of treatment of cancer comprising the administration of a therapeutically effective amount of the formulation of the present disclosure.

[0027] In some embodiments, the cancer comprises both solid and liquid tumors.

[0028] Yet another aspect of the disclosure provides a method of prevention of infectious diseases by administering a vaccine comprising the immunotherapeutic composition of the present disclosure.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure.1: Flowchart depicting the process steps involved in the making of immunotherapeutic composition Ca-Mn-Zn-5FU-HxPO4. yHiO or M-5F-HPO4, where ‘M’ stands for metal ions.

[0031] Figure.2A: SEM (Scanning Electron Microscopic) images of M-5F-HPO4 particles prepared by the method described in Example 1, showing spherical surface morphology with a size distribution of 100-1000 nm. Figure.2B: Raman spectrum showing the presence of 5FU peak in the prepared M- 5F-HPO4.

[0032] Figure.2C: Hydrodynamic size measurement showing the size distribution of 50- 1000 nm M-5F-HPO4 particles prepared by the method described in Example 1 (i) intensity vs size profile, (ii) number vs size profile (iii) volume vs size profile.

[0033] Figure.2D: X-Ray diffraction spectra (XRD spectra) of M-5F-HPO4 particles prepared by the method described in Example 1.

[0034] Figure.2E: Energy-Dispersive X-ray Spectroscopy (ED AX spectrum) showing the presence of Ca, Mn, Zn, and P peaks.

[0035] Figure.2F: ICP-MS (Inductively coupled plasma mass spectrometry analysis) showing the ratio of Ca, Mn, Zn metal ions and phosphate anions per gram of M- 5F-HPO4 prepared by the method described in Example 1.

[0036] Figure.3A: Bar graph showing the increased expression of CD86 in dendritic cells when treated with M-5F-HPO4 (Ca-Zn-Mn-5FU.HxPO4.yH2O) v / s individual metal ion nanoparticles (Ca-5F-HPO4, Zn-5F-HPO4, Mn-5F-HPO4) by Flow-cytometry demonstrating the synergistic effect conferred by the presence of metal cation complex.

[0037] Figure.3B: Bar graph showing the increased release of IFN-gamma from PBMCs when treated with the M-5F-HPO4 (Ca-Zn-Mn-5FU.HXPO4. yHiO) v / s individual metal ion nanoparticles (Ca-5F.HPO4, Zn-5F.HPO4, Mn-5F.HPO4 ) by ELISPOT analysis demonstrating the synergistic effect conferred by the presence of metal cation complex.

[0038] Figure.4: Nanoparticle conjugate of M-5F-HPO4-OVA and fluorescent microscopy imaging showing co-delivery as mentioned in Example 5.

[0039] Figure.4A: zeta potential of bare M-5F-HPO4.

[0040] Figure.4B: zeta potential of M-5F-HPO4 particle conjugated with OVA T4.

[0041] Figure.4C: zeta potential of M-5F-HPO4 conjugated with OVA N4.

[0042] Figure.4D: Confocal microscopy images of uptake of M-5F-HPO4 conjugated with OVA-AF555 peptide, M-5F-HPO4 (green), OVA-AF555 (red), co-localization (yellow). Figure.5: In vitro characterization of M-5FU-HPO4 in different immune cells activation as shown in Example 6:

[0043] Figure.5A(i): STING activation, IFN-beta release measured by ELISA in JAWS II DC cell line after 24hrs treatment with M-5F-HPO4 compared with positive control C-GAMP (lOug / ml).

[0044] Figure.5A(ii): Increase in CD86 expression by JAWS II DC cell line after 24hrs treatment with M-5F-HPO4, positive control: LPS (Ipg / ml).

[0045] Figure.5A(iii): Increase in mRNA expression of STING in JAWS II DC cell line after 24hrs treatment with M-5F-HPO4, positive control: C-GAMP (lOug / ml).

[0046] Figure.5B(i): Pro-inflammatory cytokine TNFALPHA, IL2, IL6 release by RAW264.7 macrophages after 24hrs treatment with M-5F-HPO4 detected by CBA assay.

[0047] Figure.5B(ii) IFN-gamma release by splenocytes detected by ELISPOT after 24hrs treatment with M-5F-HPO4.

[0048] Figure.6: In-vitro biocompatibility of nanoparticles as shown in example 7

[0049] Figure.6A: Cytotoxicity study showing biocompatibility of M-5F-HPO4in PBMCs.

[0050] Figure.6B: Cytotoxicity study showing biocompatibility of M-5F-HPO4 in Dendritic Cells

[0051] Figure.6C: Cytotoxicity study showing biocompatibility of M-5F-HPO4 in B16F10 melanoma Cells.

[0052] Figure.7a: Photographic image showing injectable gel formulation of M-5F-HPO4 nanoparticles with polymeric capping agents.

[0053] Figure.7b: Photographic image showing injectability of injectable gel formulation of M-5F-HPO4 nanoparticles with polymeric capping agents.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] While the invention has been disclosed with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

[0056] With respect to the use of substantially any plural and / or singular terms herein (such as “a”, “an” and “the”), those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for the sake of clarity. The various singular / plural permutations may be expressly set forth herein for the sake of clarity. The suffix “(s)” at the end of any term in the present disclosure envisages in scope both the singular and plural forms of said term.

[0057] The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0058] Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “containing” or “has” or “having” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0059] Throughout this specification, the term ‘combination thereof or ‘combinations thereof or ‘any combination thereof or ‘any combinations thereof are used interchangeably and are intended to have the same meaning, as regularly known in the field of patent disclosures.

[0060] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / - 10% or less, + / - 5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0061] As used herein, the term “comprising” when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited and that the additional one or more steps may be performed before, between, and / or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc.

[0062] An objective of the present disclosure is to provide an optimized adjuvant immunotherapeutic composition based on engineered nanoparticles such that the composition serves as a stand-alone immunotherapeutic or as a vaccine formulation conjugates with antigens, in that the composition not only enhances the immunogenicity of antigens but also eliminates the immune suppressive cells from the tumor microenvironment as well as systemic circulation, collectively facilitating enhanced immune response against cancer and other infectious diseases. Another objective of the present disclosure is to provide a method for preparing the said immunotherapeutic composition.

[0063] Immunotherapeutic composition:

[0064] To achieve the aforesaid objective, the present disclosure provides an immunotherapeutic composition comprising (i) metallic cations of metals (M) (ii) an organic immunosuppressant (OIS), and (iii) a phosphate species.

[0065] Doped calcium biomineral nanoparticles have been utilized in diverse applications, including multimodal imaging (NIR, CT, MRI), early cancer detection, radio-wave hyperthermia, targeted drug delivery, stem cell tracking, cancer immunotherapy, and MRI-guided tissue regeneration. These versatile nanoparticles function as a host matrix, enabling the incorporation of inorganic elements or organic moieties through a novel doping process within a spontaneous amorphous apatite structure. The inventors of the present disclosure, have made an innovation in doped calcium phosphate chemistry by way of spontaneously organizing the inorganic host Calcium ions together with two other immune activating dopant ions; zinc and manganese using an organic immunosuppressant such as myelosuppressive chemotherapeutic agents comprising 5 -fluorouracil to first precipitate a unique intermediate of (Ca-Zn-Mn)5FU, followed by its nanoprecipitation using hydrogen phosphate hydrate anions to obtain the final novel product of (Ca-Zn-Mn)-5FU- hydrogen phosphate hydrate (Ca-Zn-Mn)5FU.HPO4.xH2O.

[0066] This immunogenic composition is unique and novel with two most critical innovative functional properties of activating both innate and adaptive immune responsive mechanisms in antigen-presenting cells and T cells such as STING activation in dendritic cells (DCs), TLR activation in macrophages, CD86 and Type 1 interferon expression in DCs, Type-II Interferon gamma activation and cytotoxic function in T cells, and at the same time, the 5-FU embedded within the matrix is capable of depleting immunosuppressive myeloid cells in the tumor microenvironment and systemic circulation. The immunotherapeutic composition is biocompatible, non-toxic, and serves not only as an antigen carrier but also as an adjuvant and immunomodulator for cancer and infectious disease immunotherapy. As a carrier and an adjuvant, it not only increases the immunogenicity of antigens but also activates specific immunological pathways such as STING, CD40, CD86, MHC-II, IFN-P and IFN-y in APC and CTC and also reduces or eliminates the immune suppressive cells from the tumor micro -environment, peripheral organs as well as systemic circulation, thereby enhancing the efficacy of cancer immunotherapy .

[0067] As described above, the present disclosure provides an immunotherapeutic composition comprising (i) metallic cations of metals (M) (ii) an organic immunosuppressant (OIS), and (iii) a phosphate species.

[0068] In some embodiments, the immunotherapeutic composition comprises (i) metallic cations of metals (M) selected from the group comprising calcium, manganese, zinc, or a combination thereof; (ii) an organic immunosuppressant (OIS), and (iii) a phosphate species having a formula HXPO4 wherein x=0-3.

[0069] In some embodiments, the immunotherapeutic composition comprises, (i) metallic cations of metals (M) selected from the group comprising calcium, manganese, zinc, or a combination thereof; (ii) an organic immunosuppressant (OIS), and (iii) a phosphate species having a formula HXPO4 wherein x=0-3; and wherein the amount of metallic cations ranges from about 0.01 wt% to about 80 wt%; the amount of organic immunosuppressant ranges from about 0.01 wt% to about 60 wt%; and the amount of phosphate species ranges from about 10 wt% to about 80 wt% to the total weight of the composition.

[0070] In some embodiments, the metallic cations and the organic immunosuppressant form an intermediate complex M1-M2-M3-OIS, that reacts with phosphate species to form a complex of formula Ml-M2-M3-OIS.HxPO4.yH2O wherein x may vary from 0-3 and y may vary from 0-10.

[0071] In some embodiments, x can be 0,1,2 or 3.

[0072] In some embodiments, y can be 0,1,2, 3, 4, 5, 6, 7, 8, 9, 10.

[0073] In some embodiments, Ml, M2, and M3 can be the same metallic cation and it can be any one of calcium, manganese, or zinc.

[0074] In some embodiments, Ml, M2, and M3 can be different such as Ml is either calcium, manganese, or zinc; M2 is either calcium, manganese, or zinc; M3 is either calcium, manganese, or zinc.

[0075] In some embodiments, the amount of metallic cations ranges from about 0.01 wt% to about 80 wt% to the total weight of the composition, including all values therein between.

[0076] In some embodiments, the amount of metallic cations ranges from about 0.1 wt% to about 70 wt% to the total weight of the composition, including all values therein between.

[0077] In some embodiments, the amount of metallic cations ranges from about 1 wt% to about 50 wt% to the total weight of the composition, including all values therein between. In some embodiments, the amount of calcium ion ranges from about 0.1 wt% to about 80 wt% to the total weight of the composition including all values therein between.

[0078] In some embodiments, the amount of calcium ion ranges from about 1 wt% to about 60 wt% to the total weight of the composition including all values therein between. In some embodiments, the amount of zinc ion ranges from about 0.1 wt% to about 50 wt% to the total weight of the composition including all values therein between. In some embodiments, the amount of zinc ion ranges from about 1 wt% to about 30 wt% to the total weight of the composition including all values therein between.

[0079] In some embodiments, the amount of manganese ion ranges from about 0.1 wt% to about 50 wt% to the total weight of the composition including all values therein between.

[0080] In some embodiments, the amount of manganese ion ranges from about 0.1 wt% to about 5 wt% to the total weight of the composition including all values therein between.

[0081] In some embodiments, the amount of organic immunosuppressant ranges from about 0.1 wt% to about 60 wt% to the total weight of the composition including all values therein between.

[0082] In some embodiments, the amount of organic immunosuppressant ranges from about 1 wt% to about 30 wt% to the total weight of the composition, including all values therein between.

[0083] In some embodiments, the amount of phosphate species ranges from about 10 wt% to about 80 wt% to the total weight of the composition, including all values therein between.

[0084] In some embodiments, the amount of phosphate species ranges from about 10 wt% to about 50 wt% to the total weight of the composition, including all values therein between.

[0085] In some embodiments, the composition optionally comprises one or more polymer capping agent(s) to form a hydrogel, wherein the polymer capping agent is selected from the group comprising polyvinyl alcohol (PVA), PVA modified with chitosan, galactomannan, polyethyleneimine, alginate, pectin, cellulose or a combination thereof.

[0086] In some embodiments, the amount of the polymer capping agent ranges from about 1 wt% to about 20 wt% to the total weight of the composition.

[0087] In some embodiments, the amount of the polymer capping agent is about 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt% upto 20 wt% including all values therein between.

[0088] In some embodiments, the organic immunosuppressant is selected from a group comprising 5-Fluorouracil, gemcitabine, paclitaxel, cyclophosphamide, doxorubicin, capecitabine, cisplatin, doxycycline.

[0089] In some embodiments, the organic immunosuppressant is selected from a group comprising 5-Fluorouracil, gemcitabine, paclitaxel, cyclophosphamide.

[0090] In an embodiment, the organic immunosuppressant is 5-Fluorouracil.

[0091] In some embodiments, the composition comprises calcium, manganese, and zinc as metallic cations in a ratio of about 1: 1: 1, 1: 1:0.5, 1: 1:0.25, l:0.5:0.5, 0.5: 1: 1.

[0092] In an embodiment, the composition comprises calcium, manganese, and zinc as metallic cations in a ratio of about 1: 1: 1.

[0093] In an embodiment, the composition comprises calcium, manganese, and zinc as metallic cations in a ratio of about 1: 1:0.5.

[0094] In an embodiment, the composition comprises calcium, manganese, and zinc as metallic cations in a ratio of about 1: 1:0.25.

[0095] In an embodiment, the composition comprises calcium, manganese, and zinc as metallic cations in a ratio of about l:0.5:0.5.

[0096] In an embodiment, the composition comprises calcium, manganese, and zinc as metallic cations in a ratio of about 0.5: 1 : 1.

[0097] In some embodiments, the phosphate species comprises phosphate, hydrogen phosphate, dihydrogen phosphate, trihydrogen phosphate, their hydrate, or a combination thereof.

[0098] In some embodiments, the composition comprises (i) calcium, manganese, and zinc as metallic cations (ii) phosphate or hydrogen phosphate as phosphate species; (iii) 5-Fluorouracil as an organic immunosuppressant. In an embodiment, the immunotherapeutic composition has the formula Ca-Mn-Zn- 5-FU(HxPO4)yH2O where, x=0-3, y=l-10.

[0099] In some embodiments, the immunotherapeutic composition is in an amorphous form comprising nano or micro-sized particles.

[0100] In some embodiments, the immunotherapeutic composition is in a poly crystalline form comprising nano- or micro-sized crystals.

[0101] In some embodiments, the immunotherapeutic composition is in the form of nanoparticles, microparticles, self-assembled micro-flowers, porous microstructures, needle-like structures, plate-like structures, injectable gels, implantable solid cakes, and wafers.

[0102] In some embodiments, the composition is loaded with one or more antigens, wherein the one or more antigens are cancer antigens selected from the group comprising protein, peptides, DNA, RNA, carbohydrates or antigens for infectious diseases caused by viruses, bacteria, fungi.

[0103] In various embodiments, the composition is loaded with one or more antigens related to either cancer comprising but not limited to protein, peptides, DNA, RNA, carbohydrates such as whole tumor lysate, glycolipid antigen, polysaccharide antigen, mucin antigen, glycosylated mucin antigen, gplOO, Trp2, Her2, mutated EGFR, EGFR V-III, mutant RAS, mutant BRAF, mutant MEK, alpha-fetoprotein (AFP), cancer antigen 125 (CA125), cancer antigen 15-3 (CA15- 3), carbohydrate antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), human chorionic gonadotropin (hCG or beta-hCG), pro state- specific antigen (PSA), tumor testis antigen, MARTI, MELAN-A, MAGE- A3, WT1, beta catenin, CDC27, HSP70-2M, EBV-EBNA, MUC-1, mammaglobinA, mesothelin, folate receptor A, etc, or with one or more antigens relating to infectious diseases caused by virus or bacteria, fungi, comprising but not limited to protein, peptides, DNA, RNA, carbohydrates associated with SARS, MARS, Hepatitis-A,B,C, flu virus, human papilloma virus, Cholera, COVID- 19 (corona virus), Dengue, Diphtheria, Hepatitis, Haemophilus influenzae type b (Hib), Human papillomavirus (HPV), Influenza, Japanese encephalitis, Malaria, Measles, Meningococcal meningitis, Mumps, Pertussis, Pneumococcal disease, Poliomyelitis, Rabies, Rotavirus, Rubella, Tetanus, Tick-borne encephalitis, cytomegalovirus, Tuberculosis, Typhoid, Varicella, Yellow Fever, Chikungunya, Enterotoxigenic Escherichia coli, Group A Streptococcus (GAS), Group B Streptococcus (GBS), Herpes Simplex Virus, HIV- 1, Influenza Vaccines, Malaria, Neisseria gonorrhoeae, Nontyphoidal Salmonella Disease, Norovirus, Paratyphoid fever, Respiratory Syncytial Virus (RSV), Schistosomiasis Disease, Shigella, Tuberculosis, etc, in combination with other adjuvants but not limited to CpG, GM-CSF, Saponins, Poly:IC, Aqualene, QS21, Alum, lipid adjuvants, polysaccharides, etc.

[0104] In some embodiments, the immunotherapeutic composition serves as an adjuvant in vaccines, immunotherapeutic s, immune checkpoint blockade antibodies, surgical procedures, chemotherapy, radiation therapy, nuclear medicine, photodynamic therapy, photothermal therapy, cell therapy using DC, Chimeric antigen receptor-T cells CAR-T cells, or for immune regulation.

[0105] In some embodiments, the immunotherapeutic composition serves as an adjuvant performing multiple functions comprising a) carrying and delivering immunogenic antigens to immune cells; b) simultaneously activating co -stimulatory signalling pathways involving components such as CD40, CD80, CD86, MHC-I, MHC-2, STING, IFN-gamma-beta or TLR in antigen-presenting cells such as DC, macrophages or B cells and c) annihilating immune-suppressive cells such as MDSCs, M2 macrophages and N2 neutrophils and T regs in the tumor tissue, bone marrow, blood, spleen and lymph nodes and d) activating cytotoxic T cell-mediated antitumor immunity.

[0106] In another aspect, the present disclosure provides a formulation comprising the immunotherapeutic composition and biocompatible polymers.

[0107] In some embodiments, the formulation comprises biocompatible polymers that are selected from a group comprising polyvinyl alcohol (PVA), polyethylene imine (PEI), polyvinylpyrrolidone (PVP), polylactic glycolic acid (PLGA), chitosan, carboxymethyl cellulose, carboxymethyl chitosan, gelatin, alginate, galactomannan, galactose, hydroxypropyl methylcellulose, cellulose, sucrose, polyacrylate, collagen, fibrin, sodium polyacrylate, acrylate polymers, polyethylene oxide, poly(2-acrylamido-2 methyl- 1 -propanesulfonic acid) (polyAMPS), polyacrylamide, silicone, agarose, methylcellulose, hyaluronan, hydrolyzed polyacrylicnitrile, polyethylene glycol (PEG), polyethylene oxide-co- polypropylene oxide (PPO), poly (amino acids), dextran, proteins, peptide hydrogel, DNA hydrogel or a combination thereof.

[0108] In some embodiments, the formulation is an injectable hydrogel.

[0109] In various embodiments, the injectable hydrogel attracts specific set of immune cells of interest achieved by suitable adjustment of the composition of the hydrogel made of biocompatible polymers comprising but not limited to PVA, PEI, Gelatin, alginate, chitosan. The immune cells attracted can then be activated by the immunotherapeutic composition component of the injectable hydrogel which also eliminates or reduces the presence of undesirable immunosuppressive cells away from the hydrogel in the tumor microenvironment or systemic circulation. The immune cells attracted by the injectable hydrogel comprise but not limited to cytotoxic T cells while the immunosuppressive cells eliminated or reduced by the injectable hydrogel comprise but not limited to MDSCs, M2 macrophages, N2 neutrophils, Tregs.

[0110] In some embodiments, the formulation comprises an adjuvant.

[0111] In some embodiments, the adjuvants are one or more selected from a group comprising CpG, GM-CSF, Saponins, Poly:IC, cytokines such as Interleukins (IL) IL 15, IL2, IL 12, or a combination thereof.

[0112] Yet another aspect of the disclosure provides a method for the preparation of immunotherapeutic composition of the present disclosure, wherein the method comprises: i. production of a cationic mixture by mixing of aqueous solutions of metallic salts under constant stirring at room temperature, wherein the metallic salts comprise chloride, nitrate, sulphate salts of metals (M); ii. addition of organic immunosuppressant (OIS) dissolved in a solvent to the cationic mixture under constant stirring at room temperature, resulting in the generation of intermediate complex M1-M2-M3- OIS; iii. addition of phosphate species to the intermediate complex with constant stirring, resulting in the generation of immunotherapeutic composition in the form of a precipitate; iv. isolation of the precipitate by centrifugation, and washing the precipitate.

[0113] In some embodiments of the method, the metals comprise calcium, manganese, zinc, or a combination thereof.

[0114] In some embodiments of the method, the organic immunosuppressant is selected from a group comprising 5 -Fluorouracil, gemcitabine, paclitaxel, cyclophosphamide, doxorubicin, capecitabine, cisplatin, doxycycline, and wherein the solvent comprises water, phosphate buffer saline (PBS), dimethylsulfoxide (DMSO), or a combination thereof.

[0115] In some embodiments of the method, the phosphate species comprise phosphate, hydrogen phosphate, dihydrogen phosphate, trihydrogen phosphate, their hydrate, or a combination thereof.

[0116] In some embodiments of the method described above, optionally a polymer capping agent is added to the cationic mixture in step (i) or after step (iv).

[0117] In some embodiments of the method described above, conjugation of the precipitated immunotherapeutic composition to an antigen by adsorption is done at a temperature ranging from about -5°C to about 10°C for a period ranging from about 12 hours to about 24 hours.

[0118] Yet another aspect of the disclosure, relates a method for the preparation of the formulation of the present disclosure comprising loading of the immunotherapeutic composition of the present disclosure in biocompatible polymer, together with adjuvants, followed by physical cross-linking by freeze-thaw cross-linking method or by chemical cross-linking by glutaraldehyde / Boric acid cross-linking chemistry. In some embodiments of the method for the preparation of the formulation, the immunotherapeutic composition comprises (i) metallic cations of metals (M) selected from the group comprising calcium, manganese, zinc or a combination thereof; (ii) an organic immunosuppressant (OIS), and (iii) a phosphate species having a formula HXPC)4 wherein x=0-3; and wherein the amount of metallic cation ranges from about 0.01 wt% to about 80 wt%; the amount of organic immunosuppressant ranges from about 0.01 wt% to about 60 wt%; and the amount of phosphate species ranges from about 10 wt% to about 80 wt% to the total weight of the composition.

[0119] In some embodiments of the method, the biocompatible polymer is selected from a group comprising polyvinyl alcohol (PVA), polyethylene imine (PEI), polyvinylpyrrolidone (PVP), polylactic glycolic acid (PLGA), chitosan, carboxymethyl cellulose, carboxymethyl chitosan, gelatin, alginate, galactomannan, galactose, hydroxypropyl methylcellulose, cellulose, sucrose, polyacrylate, collagen, fibrin, sodium polyacrylate, acrylate polymers, polyethylene oxide, poly(2- acrylamido-2 methyl- 1 -propanesulfonic acid) (polyAMPS), polyacrylamide, silicone, agarose, methylcellulose, hyaluronan, hydrolyzed polyacrylic nitrile, polyethylene glycol (PEG), polyethylene oxide-co- polypropylene oxide (PPG), poly (amino acids), dextran, proteins, peptide hydrogel, DNA hydrogel or a combination thereof; and wherein the adjuvant is selected from a group comprising CpG, GM-CSF, Saponins, Poly:IC, cytokines such as Interleukins (IL) IL15, IL2, IL12, or a combination thereof.

[0120] Yet another aspect of the disclosure provides a method of treatment of cancer comprising the administration of a therapeutically effective amount of immunotherapeutic composition of the disclosure.

[0121] In some embodiment of said method of treatment of cancer, the method comprises administration of a therapeutically effective amount of immunotherapeutic composition of the disclosure with or without co-administration of immunomodulators .

[0122] In some embodiment of said method of treatment of cancer, immunomodulators comprise checkpoint inhibitors, therapeutic antibodies, Toll-like Receptor (TLR) Agonists, adoptive T cell transfer comprising CAR-T cell therapy, Tumor- Infiltrating Lymphocyte (TIL) Therapy; oncolytic viruses.

[0123] In an embodiment of the method of treatment of cancer, the method comprises coadministration of the vaccine comprising the immunotherapeutic composition carrying cancer antigen along with immune checkpoint blockade therapy such as anti-PDl antibodies.

[0124] In some embodiments, the cancer comprises both solid and liquid tumors.

[0125] In some embodiments, the solid tumors comprise solid breast cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, osteosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, glioblastoma, medulloblastoma, and meningioma, melanoma.

[0126] In some embodiments, the liquid tumours comprise acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute promyelocytic leukemia (APL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B- ALL), juvenile myelomonocytic leukemia (JMML), hairy cell leukemia (HCL), multiple myeloma, Waldenstrom macroglobulinemia, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma.

[0127] Yet another aspect of the disclosure relates to a method of treatment of cancer by immunotherapy comprising administration of a vaccine comprising the immunotherapeutic composition or injectable hydrogel of the present disclosure carrying cancer antigen.

[0128] Another aspect of the disclosure provides a method of treatment of cancer comprising administration of a therapeutically effective amount of the formulation of the present disclosure.

[0129] In some embodiments, the cancer comprises both solid and liquid tumors.

[0130] In some embodiments, the solid tumors comprise solid breast cancer, lung cancer, colorectal cancer, prostate cancer, pancreatic cancer, osteosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, glioblastoma, medulloblastoma, and meningioma, melanoma.

[0131] In some embodiments, the liquid tumours comprise acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute promyelocytic leukemia (APL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell acute lymphoblastic leukemia (B- ALL), juvenile myelomonocytic leukemia (JMML), hairy cell leukemia (HCL), multiple myeloma, Waldenstrom macroglobulinemia, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma.

[0132] Yet another aspect of the disclosure provides a method of prevention of infectious diseases by administering a vaccine comprising the immunotherapeutic composition of the present disclosure.

[0133] In various embodiments, the infectious diseases comprise but are not limited to viral diseases such as bacterial diseases such as SARS, MARS, Hepatitis-A,B,C, flu virus, cytomegalovirus, human papillomavirus, Cholera, COVID- 19 (corona virus), Dengue, Diphtheria, Hepatitis, Haemophilus influenzae type b (Hib), Human papillomavirus (HPV), Influenza, Japanese encephalitis, Malaria, Measles, Meningococcal meningitis,, Mumps, Pertussis, Pneumococcal disease, Poliomyelitis, Rabies, Rotavirus, Rubella, Tetanus, Tick-borne encephalitis, Tuberculosis, Typhoid, Varicella, Yellow Fever, Chikungunya, Enterotoxigenic Escherichia coli, Group A Streptococcus (GAS), Group B Streptococcus (GBS), Herpes Simplex Virus, HIV-1, Influenza Vaccines, Malaria, Neisseria gonorrhoeae, Nontyphoidal Salmonella Disease, Norovirus, Paratyphoid fever, Respiratory Syncytial Virus (RSV), Schistosomiasis Disease, Shigella, Tuberculosis.

[0134] In various embodiments of the method of treatment, the immunotherapeutic composition is administered via sub-cutaneous route, intracranial route, oral, intranasal route, intradermal route, sublingual route, buccal routes, rectal route, intravenous route, intramuscular route, etc

[0135] In some embodiments, the immunotherapeutic composition of the present disclosure is capable of a) delivering antigens, b) activating immunological mechanisms in the immune cells c) inhibiting immune-suppressive cells from the tumor microenvironment as well as systemic circulation in the body, thereby enhancing vaccine-mediated immune response against cancer and other infectious diseases.

[0136] The immunotherapeutic composition is suitable for use as an antigen carrier and adjuvant for vaccines and immune-modulator for cancer and infectious disease immunotherapy. Specifically, the immunotherapeutic composition with antigen when administered in vivo, not only delivers the antigen to antigen-presenting cells (APC) for processing and further presentation to attract immune cells such as cytotoxic T cells (CTC) but also simultaneously activates specific immunological pathways such as STING, CD40, CD80, CD83, CD86, MHC-II, IFN-a -0, -y, pro- inflammatory cytokines in APC and CTC and also kills the immune-suppressive cells such as MDSCs, M2 macrophages, N2 neutrophils, regulatory T cells, regulatory dendritic cells in both cancer microenvironment, peripheral organs and systemic circulation.

[0137] EXAMPLES

[0138] While the present disclosure is susceptible to various modifications and alternative forms, specific aspects thereof have been shown by way of examples (and drawings) described in detail below. However, it should be understood that it is not intended to limit the invention to the forms disclosed, on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention as defined by the appended claims. The present disclosure is therefore further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.

[0139] Materials: All the chemicals used for nanoparticle preparations were procured from Sigma Aldrich / Merck (India). 5-Flourouracil was procured from TCI Pvt. Ltd. (India). Cell lines such as B16F10, RAW 264.7, JAWS-II DC cell line were purchased from the American Type Culture Collection (ATCC), and Peripheral blood mononuclear cells and bone marrow-derived Dendritic cells were cultured from human volunteers or animals at Amrita Institute of Medical Sciences, with approval from Institute Ethics committee. Methods: The average particle size of the nanoparticles was analyzed using dynamic light scattering (DLS, Nano ZS, Zetasizer-Nanoseries, Malvern, UK). Zeta potential was determined using Nano ZS, Zetasizer-Nanoseries (Malvern, UK). The scanning electron microscopy (SEM) images were obtained using a SEM-JSM- 6490LA (JEOL, Japan. The X-ray diffraction (XRD) pattern, analyzed using PAN analytical X Pert-pro system fitted with Cu-Ka source indicated an amorphous structure. The material composition was analyzed by energy dispersive X-ray (EDX) spectral analysis (OXFORD) and inductively coupled plasma spectroscopy, ICP-AES (Thermo Electron IRIS INTERPID II XSP DUO). The Raman spectrum was obtained using a Cora-Compact Raman Analyzer (Anton Paar). Invitro activation studies of immune cell marker expression (CD86) were analyzed using flow cytometry (BD FACS Lyric). Thl / Th2 Cytokine analysis was done by ELISA. STING expression was done using RT PCR analysis. IFN gamma expression studies were done using the ELISPOT technique. The uptake study was done using Confocal microscopy.

[0140] Example: 1. Preparation of (Ca-Zn-Mn)-5FU.H PO4, yH2O nanoparticles with x =1, y = 2 (M-5F-HPO4) containing Ca, Mn, and Zn in the ratio of 1: 1:0.5.

[0141] This example describes the formation of immunotherapeutic composition. 10 mL each of 0.1 M calcium nitrate, zinc nitrate, and manganese nitrate were mixed under magnetic stirring at 500 rpm for 15 minutes to achieve a homogeneous solution of cations. To this, a clear solution of 5 -fluorouracil, dissolved in 30 mL of PBS or DMSO-PBS mixture (5FU stock:50 mg / ml), was added dropwise to obtain a final drug concentration of 10 mg / mL, and the mixture was stirred well at 500 rpm (Process steps as in Figure 1). A light-white and translucent colloidal precipitate formed immediately after the addition of 5 -fluorouracil solution to the solution of cations, indicating 5-fluorouracil reaction with metal cations, thereby forming the intermediate Ca-Zn-Mn-5-fluorouracil. After 30 minutes of stirring to homogenize this intermediate, an additional dose of phosphate anion in the form of 0.05 M, 15 mL diammonium monohydrogen phosphate was added. This resulted in a highly colloidal white precipitate of (Ca-Zn-Mn)-5-FU.HPO4. 2H2O, referred to as M-5F- HPO4. The precipitate was centrifuged and washed several times with double- distilled water, and the elemental composition was estimated by ICP or ED AX to confirm the cation ratio of Ca, Mn, and Zn as 1: 1:0.5. Preferentially, a polymer capping agent such as PVA (1-5%) can be added to the cationic mixture to avoid uncontrolled precipitation.

[0142] Example: 2. Preparation of (Ca-Zn-Mn)-5FU.H PO4, yH2O nanoparticles (M-5F- HPO4), containing Ca, Mn, and Zn in the ratio of 1:1:0.25.

[0143] In a typical synthesis of nanoparticles containing weight percentages of three cations, Ca, Mn, and Zn, in a 1: 1:0.25 ratio, first, 10 mL of 0.1 M calcium nitrate, 10 mL of 0.1 M manganese nitrate, and 2.5 mL of zinc nitrate were mixed under magnetic stirring at 500 rpm for 15 minutes to obtain a homogeneous solution of cations. To this mixture, a clear solution of 5 -Fluorouracil dissolved in 30 mL of phosphate buffer or DMSO with phosphate (5FU stock:50 mg / ml), was added drop wise to achieve a final drug concentration of 10 mg / mL and stirred thoroughly at 500 rpm. A light white and translucent colloidal precipitate formed immediately after the addition of 5-FU to the metal ion solution, indicating the reaction of 5-FU with metal ions to form the intermediate Ca-Zn-Mn-5-FU. After 30 minutes of stirring, this intermediate homogenized, and an additional dose of phosphate anions in the form of 0.05 M, 10 mL diammonium hydrogen phosphate was added to achieve a final pH of 10. This resulted in a colloidal white precipitate of M-5F- HPO4 containing Ca, Mn, and Zn in the ratio of 1:1:0.25, referred to as M-5F- HPO4. The precipitate was then centrifuged and washed several times with double distilled water, and the elemental composition was estimated using ICP or ED AX to confirm the cation ratio of Ca, Mn, and Zn as 1: 1:0.25.

[0144] Example: 3. Preparation of Ca-Mn-Zn-5-FU(HPO4).xH2O nanoparticle vaccine with tumor antigens.

[0145] A representative cancer vaccine for melanoma was prepared using Ca-Zn-Mn- 5FU.HPO4.2H2O (M-5F-HPO4) as adjuvant material and TRP2 as melanoma antigen. In a typical preparation, lOOpg of TRP2 was mixed with 500 pg of M-5F- HPO4 and incubated at 4°C under stirring for 12 hrs in a rotating spinner at 25 RPM. Additionally, in some preferred reactions, additional TLR agonists — CpG, Poly- EC-LC, and GM-CSF — have been added as secondary adjuvant molecules. The final product has been homogenized and is being used directly for vaccination. For the animal study, three doses of the vaccine, equivalent to 20 mg / kg NP (M-5F- HPO4)adjuvant and 5 mg / kg of TRP2 peptide, have been injected subcutaneously into mice models of melanoma in therapeutic settings. Tumor volumes, T cell infiltration, and MDSC depletion have been monitored periodically in vaccinated animals.

[0146] Example: 4. Morphology, chemical composition, and physicochemical characteristics.

[0147] Scanning electron microscopic (SEM) images of prepared M-5F-HPO4 particles showed a spherical surface morphology with a size distribution of 100-1000 nm (Figure 2a). The presence of 5FU in the prepared particles was confirmed by Raman spectroscopy (Figure 2b). The particle formed showed a narrow size distribution in dynamic light scattering (DLS) data similar to SEM analysis (Figure 2c). X-ray diffraction (XRD) pattern (Fig 2d), Energy Dispersive X-ray analysis (EDAX) (Figure 2e), and Inductively coupled plasma mass spectrometry (ICP-MS) (Figure 2f) analysis revealed the phase and chemical structure along with the elemental composition of the nanoparticles prepared in Examples 1 and 2.

[0148] Example: 5. Synergistic immune response by a combination of Ca-Zn-Mn-5FU HPO4.

[0149] A key inventive aspect of the said nanoparticle was the use of three specific metal ions — Ca, Zn, and Mn — to achieve a synergistic effect in immune activation within antigen-presenting cells (DC) and mononuclear cells, including lymphocytes. To demonstrate this, the expression of CD86 in DC and IFN-gamma release from PBMC were monitored using flow cytometry and ELISpot analysis, respectively, following treatment with nanoparticles comprising calcium, 5FU, and phosphate species (Ca-5F-HPO4); zinc, 5FU, and phosphate species (Zn-5F-HPO4); manganese, 5FU, and phosphate species (Mn-5F-HPO4) and the exemplary immunotherapeutic composition of the present disclosure Ca-Zn-Mn-5FU-HPO4 (M-5F-HPO4). Compared to individual Ca-5F-HPO4, Zn-5F-HPO4, or Mn-5F- HPO4, M-5F-HPO4, exhibited a synergistically higher expression of CD86, a co- stimulatory marker in dendritic cells (DC), as well as enhanced IFN-gamma release in peripheral blood mononuclear cells (PBMC), as shown in Figure 3.

[0150] Example: 6. Delivering tumor antigens to DC for the process of antigen presentation and activation of immune cells.

[0151] Antigen delivery to immune cells such as dendritic cells and macrophages, B cells for the process of antigen presentation by the cells is the first step in proper vaccination. The M-5F- HPO4 nanoparticles prepared by the aqueous wet-chemical method were conjugated to OVA (N4, T4 OVA) peptides by simple adsorption in 4 °C for a minimum of 12 hours by continuous rotation of 25 RPM. The conjugation was evaluated by the increased negative zeta potential of the material (Figure 4a, 4b, 4c). The nanoparticles were first fluorescently conjugated with FITC and a fluorescently tagged OVA peptide (OVA AF555) was then conjugated with the nanoparticles by the above simple adsorption method. The conjugated nanoparticles were then treated with RAW 264.7 macrophage cell line under in vitro conditions for a duration of 24 hrs in D-MEM complete media (DMEM basal media with 10% FBS and 0.1% pen strep). These cells were then imaged by confocal microscopy to visualize the co-delivery of nanoparticle (green) and OVA peptide (red) (Figure 4d). These nanoparticles activated the intracellular immunological signalling in immune cells (DC / PBMC / Macrophages / B cells, T cells) as evaluated by CD86, STING, IFN-Beta, Thl / Th2 cytokine, IFN gamma expression, shown in Figure 5. Example: 7. In vitro biocompatibility and toxicity of tri-metallic-fluorouracil- hydrogen phosphate hydrate in immune cells and cancer cells.

[0152] The M-5F-HPO4 nanoparticles are biocompatible with immune cells such as DC (JAWS II DC) and PBMCs (Figure 6. a, b) at lower concentrations when treated for a duration of 24hrs. Whereas the said nanoparticles showed cytotoxicity in cancer cell lines (B 16F10) when treated for 24 hours. (Figure 6. c).

[0153] Example: 8. Formulation of M-5F-HPO4 nanoparticles with polymeric capping agent forming injectable hydrogels.

[0154] This example depicts the preparation of a polymer formulation of inventive nanoparticles, capped with a suitable biocompatible polymer such as PVA, after the formation of such nanoparticles and washing step, by adding the nanoparticles in polymer solution containing 0.1 to 20 w / w% polymer, preferentially 5% in the example here (Figure.7). This polymeric formulation can be either a free-flowing formulation or made into thick injectable or implantable gel consistency by freezethaw or lyophilization process for subcutaneous injection or surgical implantation. Example: 9. Effect of nanoparticles in Myeloid cells and lymphoid cells in the Tumor.

[0155] The efficacy of the nanoparticle, M-5F-HPO4, in depleting MDSC cells while activating T cells has been studied in a therapeutic setting using a melanoma tumor model. Animals are divided into five groups: (a) Untreated control, (b) Standard vaccine, (c) M-HPO4, (d) 5FU Control, and (e) M-5FU-HPO4.

[0156] After tumor induction, treatment has been administered subcutaneously when the tumor size has reached 30 mm3. Standard vaccine animals have received a vaccine formulation with TRP2 and GP100 as antigens and CpG as the adjuvant. 5FU Control animals have been given free 5FU (100 mg / kg). M-HPO4 group animals have received a vaccine formulation with TRP2 and GP100 as antigens and (Ca- Zn-Mn).HxPO4,yH2O (nanoparticle formulation without 5FU), while M-5FU- HPO4 animals have been treated with a vaccine formulation containing TRP2, GP100 as antigens, and (Ca-Zn-Mn)-5FU.HxPO4, yHiO as the adjuvant. The concentration of nanoparticles has been maintained at 20 mg / kg, and antigens at 5 mg / kg. The untreated control group has not received any treatment.

[0157] All animals have been euthanized when the tumor size in the control group has reached the humane endpoint. Tumor tissue and spleen have been harvested and processed into a single-cell suspension for FACS analysis.

[0158] The lymphoid and myeloid populations of the tumor have been analyzed using flow cytometry. The total immune cell population in the tumor, identified by the CD45 marker, has been found to be higher in the M-5F-HPO4 group. M-MDSCs and PMN-MDSCs, identified using the markers CDl lb+ Ey6C+ and CDl lb+ Ey6G+, respectively, have shown significant reductions in the M-5F-HPO4 group compared to other groups. M2 macrophages, identified as CD45+ CDl lb+ F480+ CD206+ CD86-, have also been significantly reduced in the M-5F-HPO4 group. Conversely, an increase in the Ml macrophage phenotype, identified as CD45+ CD1 lb+ F480+ CD206- CD86+, has been observed in the M-5F-HPO4 group compared to control groups. Tumor-infiltrated CD3+ cells have been found to be higher in the M-5F- HPO4 group than in the control groups, along with a similar increase in CD4+ cells, CD8+ cells, CD4+ CD69+ cells, and CD8+ CD69+ cells in the tumor. These enhanced immune cell expressions and the reduction in suppressor cell populations have significantly contributed to the overall reduction in total tumor volume.

[0159] Example 10: In vivo systemic immune modulation

[0160] The lymphoid and myeloid populations of the spleen in vaccinated animals have been analyzed by flow cytometry. A reduction in overall circulating MDSCs as well as splenic MDSCs has been observed in the M-5F-HPO4 group. M2 macrophages have also been reduced, while Ml macrophages have increased in the spleen. Splenic CD3+ cells are higher in the M-5F-HPO4 group compared to control groups. A similar increase in CD4+ cells, CD8+ cells, CD4+ CD69+ cells, and CD8+ CD69+ cells in the spleen has also been observed for the M-5F-HPO4 group. Example 11: Antitumor immune response of M-5F-HPO4

[0161] M-5F-HPO4 admixed with tumor antigen has demonstrated controlled tumor growth up to 24 days post-treatment by depleting immune-suppressive cells such as MDSCs and M2 macrophages, while concurrently activating CD4 and CD8 T cells against cancer compared to control groups.

[0162] Reference throughout this specification to “some embodiments”, “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0163] Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.

[0164] As used herein, the terms “include” (any form of “include”, such as “include”), “have” (and “have”), “comprise” etc. any form of “having”, “including” (and any form of “including” such as “including”), “containing”, “comprising” or “comprises” are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0165] Any discussion or reference of documents, acts, materials, devices, articles, and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application. Particularly, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. ADVANTAGES OF THE INVENTION

[0166] The immunotherapeutic composition is biocompatible, non-toxic, and serves not only as an antigen carrier but also as an adjuvant and immunomodulator for cancer and infectious disease immunotherapy. As a carrier and an adjuvant, it not only increases the immunogenicity of antigens but also activates specific immunological pathways such as STING, CD40, CD86, MHC-II, IFN-P and IFN-y in APC and CTC and also reduces or eliminates the immune suppressive cells from the tumor micro-environment, peripheral organs as well as systemic circulation, thereby enhancing the efficacy of cancer immunotherapy.

Claims

The claim:

1. An immunotherapeutic composition comprising (i) metallic cations of metals (M) selected from the group comprising calcium, manganese, zinc or a combination thereof; (ii) an organic immunosuppressant (OIS), and (iii) a phosphate species having a formula HXPO4 wherein x=0-3; and wherein the amount of metallic cations range from about 0.01 wt% to about 80 wt%; the amount of organic immunosuppressant ranges from about 0.01 wt% to about 60 wt%; and the amount of phosphate species ranges from about 10 wt% to about 80 wt% to the total weight of the composition.

2. The immunotherapeutic composition as claimed in claim 1, wherein the metallic cations and the organic immunosuppressant form an intermediate complex M1-M2-M3-OIS, that reacts with phosphate species to form a complex of formula Ml-M2-M3-OIS.HxPO4.yH2O wherein x may vary from 0-3 and y may vary from 0-10.

3. The immunotherapeutic composition as claimed in claims 1 to 2, wherein the composition optionally comprises one or more polymer capping agent(s) to form a hydrogel, wherein the polymer capping agent is selected from the group comprising polyvinyl alcohol (PVA), PVA modified with chitosan, galactomannan, polyethyleneimine, alginate, pectin, cellulose or a combination thereof.

4. The immunotherapeutic composition as claimed in claim 3, wherein the amount of the polymer capping agent ranges from about 1 wt% to about 20 wt% to the total weight of the composition.

5. The immunotherapeutic composition as claimed in claims 1 to 4, wherein the organic immunosuppressant is selected from a group comprising 5- Fluorouracil, gemcitabine, paclitaxel, cyclophosphamide, doxorubicin, capecitabine, cisplatin, doxycycline.

6. The immunotherapeutic composition as claimed in claims 1 to 5, wherein the composition comprises calcium, manganese, and zinc as metallic cations in a ratio of about 1: 1: 1, 1: 1:0.5, 1: 1:0.25, l:0.5:0.5, 0.5: 1: 1.

7. The immunotherapeutic composition as claimed in claims 1 to 6, wherein the composition comprises (i) calcium, manganese, and zinc as metallic cations (ii) phosphate or hydrogen phosphate as phosphate species; (iii) 5- Fluorouracil as an organic immunosuppressant.

8. The immunotherapeutic composition as claimed in claim 7, wherein the immunotherapeutic composition has the formula Ca-Mn-Zn-5- FU(HxPO4)yH2O where, x=0-3, y=l-10.

9. The immunotherapeutic composition as claimed in claims 1 to 8, wherein the immunotherapeutic composition is in an amorphous form comprising nano or micro-sized particles.

10. The immunotherapeutic composition as claimed in claims 1 to 9, wherein the immunotherapeutic composition is in a polycrystalline form comprising nano- or micro-sized crystals.

11. The immunotherapeutic composition as claimed in claims 1 to 10, wherein the immunotherapeutic composition is in the form of nanoparticles, microparticles, self-assembled micro-flowers, porous microstructures, needle-like structures, plate-like structures, injectable gels, implantable solid cakes, and wafers.

12. The immunotherapeutic composition as claimed in claims 1-11, wherein the composition is loaded with one or more antigens, wherein the one or more antigens are cancer antigens selected from the group comprising protein, peptides, DNA, RNA, carbohydrates or antigens for infectious diseases caused by virus, bacteria, fungi.

13. The immunotherapeutic composition as claimed in claims 1-12, wherein the immunotherapeutic composition serves as an adjuvant in vaccines, immunotherapeutic s, immune checkpoint blockade antibodies, surgical procedures, chemotherapy, radiation therapy, nuclear medicine, photodynamic therapy, photothermal therapy, cell therapy using DC, CAR- T cells, or for immune regulation.

14. A formulation comprising the immunotherapeutic composition as claimed in claims 1-13 and biocompatible polymers, wherein the biocompatiblepolymers are selected from a group comprising polyvinyl alcohol (PVA), polyethylene imine (PEI), polyvinylpyrrolidone (PVP), poly lactic glycolic acid (PLGA), chitosan, carboxymethyl cellulose, carboxymethyl chitosan, gelatin, alginate, galactomannan, galactose, hydroxypropyl methylcellulose, cellulose, sucrose, polyacrylate, collagen, fibrin, sodium polyacrylate, acrylate polymers, polyethylene oxide, poly(2-acrylamido-2 methyl- 1 -propanesulfonic acid) (polyAMPS), polyacrylamide, silicone, agarose, methylcellulose, hyaluronan, hydrolyzed polyacrylic nitrile, polyethylene glycol (PEG), polyethylene oxide-co-polypropylene oxide (PPG), poly (amino acids), dextran, proteins, peptide hydrogel, DNA hydrogel or a combination thereof.

15. The formulation as claimed in claim 14, wherein the formulation is an injectable hydrogel.

16. The formulation as claimed in claims 14 to 15, wherein the formulation comprises an adjuvant selected from a group comprising CpG, GM-CSF, Saponins, Poly:IC, cytokines such as Interleukins (IL) IL15, IL2, IL12, or a combination thereof.

17. A method for the preparation of immunotherapeutic composition as claimed in claims 1-13, wherein the method comprises:(i) production of a cationic mixture by mixing of aqueous solutions of metallic salts under constant stirring at room temperature, wherein the metallic salts comprise chloride, nitrate, sulphate salts of metals (M);(ii) addition of organic immunosuppressant (OIS) dissolved in a solvent to the cationic mixture under constant stirring at room temperature, resulting in the generation of intermediate complex M1-M2-M3- OIS;(iii) addition of phosphate species to the intermediate complex with constant stirring, resulting in the generation of immunotherapeutic composition in the form of a precipitate;(iv) isolation of the precipitate by centrifugation, and washing the precipitate.

18. The method as claimed in claim 17, wherein the metals comprise calcium, manganese, zinc, or a combination thereof.

19. The method as claimed in claim 17, wherein the organic immunosuppressant is selected from a group comprising 5 -Fluorouracil, gemcitabine, paclitaxel, cyclophosphamide, doxorubicin, capecitabine, cisplatin, doxycycline, and wherein the solvent comprises water, phosphate buffer saline (PBS), dimethylsulfoxide (DMSO), or a combination thereof.

20. The method as claimed in claim 17, wherein the phosphate species comprise phosphate, hydrogen phosphate, dihydrogen phosphate, trihydrogen phosphate, their hydrate, or a combination thereof.

21. The method as claimed in claim 17, wherein optionally a polymer capping agent is added to the cationic mixture in step (i) or after step (iv).

22. The method as claimed in claim 17, comprising conjugation of the precipitated immunotherapeutic composition to an antigen by adsorption at a temperature ranging from about -5°C to about 10°C for a period ranging from about 12 hours to about 24 hours.

23. A method for the preparation of the formulation as claimed in claims 14-16 comprising loading of the immunotherapeutic composition in biocompatible polymer, together with adjuvants, followed by physical cross-linking by freeze-thaw cross-linking method or by chemical crosslinking by glutaraldehyde / Boric acid cross-linking chemistry.

24. The method as claimed in claim 23, wherein the immunotherapeutic composition is an immunotherapeutic composition defined in claims 1 to 13, wherein the biocompatible polymer is selected from a group comprising polyvinyl alcohol (PVA), polyethylene imine (PEI), polyvinylpyrrolidone (PVP), polylactic glycolic acid (PLGA), chitosan, carboxymethyl cellulose, carboxymethyl chitosan, gelatin, alginate, galactomannan, galactose, hydroxypropyl methylcellulose, cellulose, sucrose, polyacrylate, collagen, fibrin, sodium polyacrylate, acrylate polymers, polyethylene oxide, poly(2-acrylamido-2 methyl- 1 -propanesulfonic acid) (polyAMPS), polyacrylamide, silicone, agarose, methylcellulose, hyaluronan, hydrolyzed polyacrylic nitrile, polyethylene glycol (PEG), polyethylene oxide-co- polypropylene oxide (PPO), poly (amino acids), dextran, proteins, peptide hydrogel, DNA hydrogel or a combination thereof; and wherein the adjuvant is selected from a group comprising CpG, GM-CSF, Saponins, Poly:IC, cytokines such as Interleukins (IL) IL15, IL2, IL12, or a combination thereof.

25. A method of treatment of cancer comprising administration of a therapeutically effective amount of immunotherapeutic composition as claimed in claims 1-11, or immunotherapeutic composition as claimed in claims 12-13, with or without co-administration of immunomodulators comprising, therapeutic antibodies, CAR-T cells, oncolytic viruses, adoptive T cell transfer.

26. The method as claimed in claim 25, wherein the cancer comprises both solid and liquid tumors.

27. A method of treatment of cancer comprising administration of a therapeutically effective amount of the formulation as claimed in claims 14- 16.

28. The method as claimed in claim 27, wherein the cancer comprises both solid and liquid tumors.

29. Use of the immunotherapeutic composition as claimed in claims 1-13 for the treatment of cancer comprising solid or liquid tumors.

30. Use of the formulation as claimed in claims 16-18 for the treatment of cancer comprising solid or liquid tumors.

31. Use of the immunotherapeutic composition as claimed in claim 12, for the prevention of infectious diseases.

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