Immune modulating therapy using UC-MSC-cm to inhibit tumor growth

Abstract

An Umbilical cord derived mesenchymal stem cell (UC-MSC) conditioned media (UC- MSC-CM) composition of the range from 5%-100% possessing immunomodulatory effect inhibiting tumour growth when administered intraperitoneally and intravenously. MSC- CM arrests cell cycle in leukaemia cells (K-562 and HL-60), lung cancer cells (A549 and HOP-62), breast cancer cells (MDA-MB-231 and MCF-7) and cervical cancer cells (HeLa). Elevated levels of IL-6, TIM3 and TIMP2 in MSC-CM confer to its immunomodulatory function by leading to an increase in expression of activation markers, cytotoxic granules, mucosal associated invariant T cells and decreasing the inflammasome pathway activation, consequently decreasing the levels of innate immune inflammasome pathway molecules in treated xenograft tumor tissues. MSC-CM as an adjunct with IL-12 augments cytotoxic potential against cancer cell lines. Tumor inhibition effect of MSC-CM is shown by significant reduction of relative tumor volume which is expressed by fold change of genes involved in various pathways affecting xenograft tumor growth.

Description

Immune modulating therapy using UC-MSC-CM to inhibit tumor growth TECHNICAL FIELD

[0001] The present invention generally relates to the technical field of immunomodulating therapy using UC-MSC-CM. Particularly, the present disclosure provides the composition of MSC-CM which inhibits the tumor growth and causes immunomodulation by decreasing the inflammasome pathway activation, consequently decreasing the levels of innate immune inflammasome pathway molecules and pro- inflammatory cytokines. BACKGROUND

[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] Mesenchymal stromal cells (MSC) are a non-hematopoietic bone marrow cell population and it forms a key component of the osteoblastic niche in hematopoietic microenvironment. MSC have been defined as primitive, multipotent, undifferentiated cells capable of self-renewal, and they have the ability to give rise to different cell lineages (Kode et al., Cytotherapy 11(4): 377-391, 2009. MSCs have been reported to promote the proliferation of melanoma cells (Djouad et al., Blood.2003;102(10):3837–3844. 2003). Mora-Garcia et al, Journal of Translational Medicine, 2016 found that overexpression of immuno- ectoenzymes CD39 and CD73 and immunosuppressor adenosine by MSC in cervical cancer patients led to inhibition of cytotoxic T cells and suppression of anti-tumor mechanisms. Few groups have reported effect of MSC on solid tumors in animal models. deMelo et al (PLoS One , 10(6): e0128922. doi:10.1371 / journal.pone.0128922; 2015) have shown that adipose tissue derived MSCs modified with a lentivector carrying HSV-Tk genes, injected at the tumor site, could successfully reduce tumor size of established intracranial glioma. In another study UC-MSC completely eliminated mammary tumors in rat model without recurrence compared to PBS treated tumors (Tamura et al, The Open Tissue Engineering and Regenerative Medicine Journal, 2011, 4, 39-47 2011). Hepatoma cell growth was significantly inhibited by MSC conditioned media (Lu et al; 2008, Cancer Biol Ther.;7:245–251). It has been reported that upregulated inflammasome activity is correlated to various types of cancers including breast cancer, gastric cancer, brain tumor, and malignant prostate, while inflammasomes also have a protective role in colitis-associated cancer. Pro-carcinogenesis effect of NLRP33 in lung cancer (Thi and Hong, 2017; J Cancer Prevention. 22: 62-73, 2017) and hepatocellular cancer has also been shown by regulating NK cell-mediated immune mechanisms. The NLRP3 inflammasome was shown to promote cell differentiation in gastric cancer through cyclin-D1 (CCND1) activation in addition to IL-1β and IL-18 production. UC- 2MSC show low immunogenicity, tumor tissue tropism and have shown attenuation of tumors in vitro and in vivo models (Tamura et al, 2011). Stem cell-derived exosomes have also shown to be of potential efficacy against breast, prostate and oral cancer (Rosenberger et al, . Scientific Reports 9:663, 2019). These observations have led to increased interest in exploring UC-MSC and their conditioned media MSC-CM as anti-cancer therapy.

[0004] Some studies have demonstrated that in macrophages, Gal-9–TIM-3 interaction downregulates TLR signaling, leading to a decrease in IL-12 and IL-1β production; an increase of IL-23, IL-6, IL-8, and IL-10 production (Sauer, N.,et.al., 2023 Cancer immunology, immunotherapy).

[0005] US 20200230172A1patent application showed ‘STEM CELL CONDITIONED MEDIA FOR CLINICAL AND COSMETIC APPLICATIONS’ through subcutaneous application directed towards treating dermatological conditions.

[0006] US202217977476A patent application disclosed ‘Mesenchymal stem cells for the prevention and targeted treatment of cancer and other disorders’. US2020345775A1 patent application showed ‘MSC- expressed immunomodulators in combination with car-t for cancer therapy’. Patent application CN113881707A explained Product and method for regulating and controlling immunosuppression effect of umbilical cord mesenchymal stem cells and application.

[0007] The present invention utilizes healthy umbilical cord derived MSC as continuous source of MSC (UC-MSC) and its conditioned media (MSC-CM) developed and provided by OCT-Therapy & Research Pvt Ltd. (Mumbai) for evaluating their anti-tumor efficacy against five cancer models viz. leukemia, breast oral, lung, and colon tumor; in vitro assays using cancer cell lines and in vivo assay using cancer xenografts in immunodeficient mouse model. when administered intraperitoneally or intravenously or both which has never been explored before and it’s potential to be used as therapeutic modality.This invention demonstrates NLRP3 inflammasomes as a suitable biomarker in tumor diagnosis and even prognosis. Therefore, present invention is aimed at demonstrating UC- MSC and MSC-CM for anticancer potential through NLRP3 inflammasome and Innate immune pathways. OBJECTS OF THE PRESENT DISCLOSURE

[0008] Some of the objects of the present disclosure, which atleast one embodiment herein satisfies are listed herein below.

[0009] It is an object of the present disclosure to provide for composition of an Umbilical cord derived mesenchymal stem cells (UC-MSC) conditioned media (UC-MSC-CM) that possesses immunomodulatory effect inhibiting tumor growth when administered intraperitoneally or intravenously or both. 3

[0010] It is an object of the present disclosure to provide for composition of MSC-CM that effectively arrests the cell cycle in leukaemia cells (K-562 and HL-60), lung cancer cells (A549 and HOP-62), breast cancer cells (MDA-MB-231 and MCF-7) and cervical cancer cells (HeLa) .

[0011] Yet another objective of present invention is to provide for understanding of immunoregulatory potential of MSC-CM by analysing soluble factor landscape in terms of pro-inflammatory cytokines, TIM3, TIMP-2, activation markers, cytotoxic granules, and immune checkpoint markers.

[0012] Yet another object of present invention is to provide for the composition of MSC-CM that inhibits the growth of tumor by significant reversal of suppression caused in T-cell, B-cell and Myeloid cell mitogenic responses.

[0013] In yet another objective of present invention to provide for the composition of MSC-CM when administered intraperitoneally and intravenously against tumor xenograft showed enhanced the reduction of relative tumor volume.

[0014] Yet another objective of present invention provides that administration of MSC-CM shows decrease in NLRP3, IL-18 and AIM2 inflammasome expression in residual tumor tissues after in vivo treatment by intravenous or intraperitoneal route with MSC-CM and decrease in the levels of IL-6 thereby conferring immunomodulatory function.

[0015] In yet another objective of the present invention it is provided that MSC-CM induces fold change of genes involved in various pathways affecting xenograft tumor growth.

[0016] Further objective of present invention provides the composition and manner of use of MSC-CM for treating leukemia, triple negative breast cancer, lung cancer and cervical cancer. SUMMARY

[0017] The present invention generally relates to the technical field of immunomodulating therapy using UC-MSC-CM. Particularly, the present disclosure provides the composition of MSC-CM which inhibits the tumor growth and causes immunomodulation by decreasing the inflammasome pathway activation, consequently decreasing the levels of innate immune inflammasome pathway molecules and pro- inflammatory cytokines.

[0018] An aspect of the present disclosure provides the process to prepare MSC-CM that possesses immunomodulatory effect inhibiting tumor growth when administered intraperitoneally or intravenously or both.

[0019] Another aspect of the present invention discloses that MSC-CM so prepared and used inpresent invention is biocompatible and comprises the optimal concentration of growth factors.In one of the embodiments of the present invention the 5%-100% MSC-CM arrests cell cycle in leukaemia cells (K-562 and HL-60), lung cancer cells (A549 and HOP-62), breast cancer cells (MDA- MB-231 and MCF-7) and cervical cancer cells (HeLa) after treatment with 5%-100% MSC-CM for 48 4hours.

[0020] Yet another aspect of present invention shows that treatment with 5%-100% MSC-CM led to 10- 95% increase of immune -cell mediated kill of 562 and HL-60 leukemia cells.

[0021] Yet another aspect of present invention shows that MSC-CM when administered intraperitoneally against MDA-MB231 breast cancer xenograft, K562 leukemia xenograft, when administered intravenously against HeLa cervical cancer xenograft and against A549 lung cancer xenograft enhanced the reduction of relative tumor volume.

[0022] In another aspect of present invention the MSC-CM showed elevated levels of IL-6, TIM3 and TIMP-2 conferring to its immunomodulatory function.

[0023] In another embodiment of present invention, treatment with 5%-100% MSC-CM led to increase in expression of activation markers, cytotoxic granule (Perforin and Granzyme B) in T cells, and Mucosal-associated invariant T cells (MAIT) in T cell subsets.

[0024] In another embodiment of present invention it is shown that MSC-CM acts as an an adjunct with IL-12 augments cytotoxic potential against cancer cell lines lines like K562, HL-60, A549, MDA-MB- 231and MCF-7.

[0025] Another aspect of present invention shows treatment with 5%-100% MSC-CM led to 10-200% significant reversal of suppression caused in T-cell, B-cell and myeloid cell mitogenic responses in presence of leukemia cell supernatant.

[0026] In yet another embodiment of the present invention the 5%-100% MSC-CM shows decrease in expression of innate immune inflammasome pathway molecules by 88% of NLRP3, 87% of IL-18 and 7% AIM2 in treated xenograft tumor tissues by intravenous or intraperitoneal routes and decrease in the levels of IL-6 thereby conferring immunomodulatory function.

[0027] In yet another aspect of present invention by transcriptomic mapping it is shown that MSC-CM induces fold change of genes involved in various pathways affecting xenograft tumor growth.

[0028] Further aspect of present invention provides the composition and manner of use of MSC-CM for treating leukemia, triple negative breast cancer, lung cancer and cervical cancer. BRIEF DESCRIPTION OF DRAWINGS

[0029] FIG. 1 illustrates the cytocompatibility of MSC-CM on healthy peripheral blood lymphocytes.

[0030] FIG. 2 illustrates effect of MSC CM on the DNA cell cycle of K-562 leukemia cell line FIG.2a shows’Bright Field’ microscopic and flowcytometry analysis and FIG.2b depict the % population of cell cycle phases in cancer cells before and after treatment 5with MSC-CM. .

[0031] FIG. 3 illustrates effect of MSC CM on the DNA cell cycle of HL-60 leukemia cell line FIG.3a shows’Bright Field’ microscopic and flowcytometry analysis and FIG.3b depict the % population of cell cycle phases in cancer cells before and after treatment with MSC-CM..

[0032] FIG. 4 illustrates effect of MSC CM on the DNA cell cycle of A549 lung cancer cell line FIG.4a shows’Bright Field’ microscopic and flowcytometry analysis and FIG.4b depict the % population of cell cycle phases in cancer cells before and after treatment with MSC-CM.

[0033] FIG. 5 illustrates effect of MSC CM on the DNA cell cycle of HOP-62 lung cancer cell line FIG.5a shows’Bright Field’ microscopic and flowcytometry analysis and FIG.5b depict the % population of cell cycle phases in cancer cells before and after treatment with MSC-CM

[0034] FIG. 6 illustrates effect of MSC CM on the DNA cell cycle of MDA-MB-231 breast cancer cell line FIG.6a shows’Bright Field’ microscopic and flowcytometry analysis and FIG.6b depict the % population of cell cycle phases in cancer cells before and after treatment with MSC-CM

[0035] FIG. 7 illustrates effect of MSC CM on the DNA cell cycle of MCF-7 breast cancer cell line FIG.7a shows ‘Bright Field’ microscopic and flowcytometry analysis and FIG.7b depict the % population of cell cycle phases in cancer cells before and after treatment with MSC-CM

[0036] FIG. 8 illustrates evaluation of soluble factors and immune markers in MSC-CM by CBA, Multiplex Assay and ELISA FIG.8a shows levels of cytokines and checkpoint inhibitors and FIG.8b shows the concentration of growth factors in MSC-CM

[0037] FIG.9 to 12 illustrates assessment of immunomodulatory role of MSC-CM on T cell subsets (HI-PBL) 6

[0038] FIG.13 illustrates reversal of immunosuppression after treatment with MSC-CM

[0039] FIG 14 shows the effect of MSC-CM on cytotoxic potential of PBL against K562 and HL- 60 leukemic cells

[0040] FIG 15 shows the effect of MSC-CM on cytotoxic potential of PBL against A549 lung cancer cells

[0041] FIG 16a shows the effect of MSC-CM on cytotoxic potential of PBL against MDA-MB-231 breast cancer cells

[0042] FIG 16b shows the effect of MSC-CM on cytotoxic potential of PBL against MCF-7 breast cancer cells

[0043] FIG 17 shows the effect of MSC-CM and rIL-12 on cytotoxic potential of PBL against A549 lung cancer cells

[0044] FIG 18 shows the effect of MSC-CM and rIL-12 on cytotoxic potential of PBL against MDA-MB-231 breast cancer cells

[0045] FIG 19 shows elucidates the in vivo tumor reduction efficacy of MSC-CM against MDA- MB-231 in NOD-SCID mice.

[0046] FIG 20 shows in vivo efficacy of MSC-CM against MDA-MB-231 xenograft in terms of Relative Tumor Volume (RTV), Average Animal body Weight (grams), % survival, and Tumor Volume (cc).

[0047] FIG 21a shows the in vivo tumor reduction efficacy of MSC-CM against K562 in NOD- SCID mice.

[0048] FIG 21b shows in vivo efficacy of MSC-CM against K562 xenograft in terms of Relative Tumor Volume (RTV), Average Animal body Weight (grams), % survival, and Tumor Volume (cc)

[0049] FIG 22 illustrates the in vivo tumor reduction efficacy of MSC-CM against HeLa in NOD- SCID mice.

[0050] FIG 23 shows in vivo efficacy of MSC-CM against HeLa xenograft in terms of Relative Tumor Volume (RTV), Average Animal body Weight (grams), % survival, and Tumor Volume (cc)

[0051] FIG 24 shows effect of MSC-CM on reduction of A549 lung cancer in NOD-SCID mice FIG 25 shows in vivo efficacy of MSC-CM against A549 xenograft in terms of Relative Tumor Volume (RTV), Average Animal body Weight (grams), % survival, and Tumor Volume (cc)

[0052] FIG 26a shows effect of MSC-CM on inflammasome pathway regulation in lung cancer xenograft A549 7

[0053] FIG 26b shows Histochemical scoring (H-score)’ of MSC-CM on inflammasome pathway regulation in lung cancer xenograft A549

[0054] FIG 27a shows efficacy of MSC-CM in tumour reduction of A549 lung cancer xenograft model by showing differential gene expression by RNAseq using NGS of A549 xenograft control versus 5-FU treated mice tumours

[0055] FIG 27b shows efficacy of MSC-CM in tumour reduction of A549 lung cancer xenograft model by showing differential gene expression by RNAseq using NGS of A549 xenograft control versus MSC-CM(i.v.) treated mice tumours

[0056] FIG 27c shows efficacy of MSC-CM in tumour reduction of A549 lung cancer xenograft model by showing differential gene expression by RNAseq using NGS of A549 xenograft control versus MSC-CM(i.p.) treated mice tumours

[0057] FIG 28 shows analysis of soluble factors in mice plasma from K-562 tumor xenograft after MSC-CM treatment

[0058] FIG 29 shows analysis of soluble factors in mice plasma from A549 tumor xenograft after MSC-CM treatment

[0059] FIG 30 shows analysis of soluble factors in mice plasma from HeLa tumor xenograft after MSC-CM treatment DETAILED DESCRIPTION OF THE INVENTION

[0060] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0061] In the following description, the numerous specific details are set forth in order to provide a thorough understanding of embodiments of the present invention. It will be apparent to one skilled in the art that embodiments of present invention may be practiced without some of these specific details. All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0062] In some embodiments, the numbers expressing quantities or dimensions of items, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some 8instances by the term “about”. Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing requirements.

[0063] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all groups used in the appended claims.

[0064] Exemplary embodiments will now be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments are shown. These exemplary embodiments are provided only for illustrative purposes and so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those of ordinary skill in the art. The invention disclosed may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Various modifications will be readily apparent to persons skilled in the art. The general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Moreover, all statements herein reciting embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents as well as equivalents developed in the future (i.e. any elements developed that perform the same function, regardless of structure). Also, the terminology and phraseology used is for the purpose of describing exemplary embodiments and should not be considered limiting. Thus, the present invention is to be accorded the widest scope encompassing numerous alternatives, modifications and equivalents consistent with the principles and features disclosed. For purpose of clarity, details relating to technical 9material that is known in the technical fields related to the invention have not been described in detail so as not to unnecessarily obscure the present invention.

[0065] Thus, for example, it will be appreciated by those of ordinary skill in the art that the diagrams, schematics, illustrations, and the like represent conceptual views or processes illustrating systems and methods embodying the invention.

[0066] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the “invention” may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the “invention” will refer to subject matter recited in one or more, but not necessarily all, of the claims.

[0067] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. If the specification states a component or feature “may”, “can”, “could”, or “might” be included or have characteristic, that particular component or feature is not required to be included or have the characteristic. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0068] The embodiments of present invention ‘Immune modulating therapy using UC-MSC-CM to inhibit tumor growth’ comprise of an Umbilical cord derived mesenchymal stem cells (UC-MSC) conditioned media (UC-MSC-CM) composition possessing immunomodulatory effect inhibiting tumour growth, particularly causes immunomodulation by decreasing the inflammasome pathway activation, consequently decreasing the levels of innate immune inflammasome pathway molecules and pro- inflammatory cytokines.

[0069] Further in one of the embodiments, the clinical grade MSCs are prepared according to Indian Patent No.385309 by the applicants using xeno-free methods, wherein human serum derived from FFP or CDP is used as a growth supplement to culture the umbilical cord derived MSCs. Further as per Indian patent application number 201721005531 and PCT application number PCT / IN2018 / 050078 by the applicants, the process used to prepare MSC-CM used in present invention is described as follows: The conditioned medium is prepared by culturing Mesenchymal Stem cells in a suitable culture medium for around twenty four hours to approximately three days so as to allow cell secretions to leach into the 10culture medium, to form a physiologically balanced composition of beneficial cytokines, growth factors and proteins, also known as conditioned medium. After harvesting the conditioned medium, the supernatant that is free from cellular matter or debris is subjected to filtering to ensure sterility of the conditioned medium. The process includes harvesting the extract such that removing the extract involves removing the cell culture supernatant resulting in a cell-free extract from the cultured cells comprising the harvested conditioned medium. The conditioned media obtained by the present process is tested by in vitro and in vivo methods for safety and efficacy.

[0070] The MSC- CM so prepared and used in the present invention comprises the optimal concentration of growth factors as follows: Human Fibroblast growth factor (hFGF) is in a concentration ranging from 100 to 1500 pg / ml, human Granulocyte Colony Stimulating factor (hGCSF) is in a concentration ranging from 50 to 1500 pg / ml, human Hepatocyte growth factor (hHGF) is in a concentration ranging from 50 to 3000 pg / ml, Interleukin 1 receptor agonist (IL- lRα) is in a concentration ranging from about 100 to 1500 pg / ml, human vascular endothelial growth factor (hVEGF) in a concentration ranging from about 10 to about l000 pg / ml and Interleukin-6 (IL-6) in a concentration ranging from about 100 to 2000 pg / ml. In one of the embodiments of the present invention the5%-100%MSC-CM arrests cell cycle in leukaemia cells (K-562 and HL-60), lung cancer cells (A549 and HOP-62), breast cancer cells (MDA- MB-231 and MCF-7) and cervical cancer cells (HeLa).

[0071] In yet another embodiment of the present invention the 5%-100% MSC-CM shows decrease in NLRP3, IL-18 and AIM2 inflammasome expression in residual tumor tissues after in vivo treatment by intravenous or intraperitoneal route with MSC-CM and decrease in the levels of IL-6 thereby conferring immunomodulatory function.

[0072] In another embodiment of present invention, treatment with 5%-100% MSC-CM led to increase in expression of activation markers, cytotoxic granules, and Mucosal-associated invariant T cells (MAIT) in T cell subsets.

[0073] Further the methodology used in the present invention to show the immunomodulating effect of5%-100%MSC-CM inhibiting tumor growth is exemplified in the following examples.

[0074] Example 1

[0075] Preparation of MSC-CM The Human plasma or cryo-depleted plasma (AB positive) was collected in a closed system by certified blood banks. Blood bags which tested negative for infectious diseases such as HIV 1 and 2, HbsAg, HCV (Hepatitis), syphilis (VDRL) and malarial parasites were further transported cold to the laboratory and processed under sterile conditions using aseptic techniques, to recover serum such that it is suitable to support culture of clinical grade quality human Mesenchymal cells (MSCs) which can be used for 11clinical applications. The serum from pooled AB positive plasma or cryo-depleted plasma (CDP) was recovered and further processed to neutralize viruses, bacteria and mycoplasma as per the method specified by Sprössig, 1976; and Wutzler, 1975. The pooling maybe from between 5 to multiple donor lots as per the processing capacity and is done to reduce lot to lot variability of a biological component. The human serum recovered above is used to supplement cell culture media such as DMEM, αMEM or any suitable media at a percentage between 5-30%. The supplemented culture medium is used to culture adult human stem cells at a cell density ranging from 6000 to 60000 cells / cm2in suitable culture vessels which could be 2 dimensional or 3 dimensional. The cells are incubated between 36-37°C and 4-20% CO2till 70-90% confluent. The stem cell conditioned media harvested from 24 hours to 3 days, is pooled over cumulative passages and across different lots, filtered, aliquoted and stored at -20°C. Total protein content: Total protein content was between 0.5 to 100 mg / ml Cytokine analysis by ELISA or Multiplexing: As an example, the following cytokines were present at the respective concentrations: human Fibroblast growth factor (hFGF) 658 pg / ml, human Granulocyte Colony Stimulating Factor (hGCSF) 960 pg / ml, human Hepatocyte Growth Factor (hHGF) 1175 pg / ml, Interleukin 1 receptor agonist (IL- lRα) 364 pg / ml, Vascular Endothelial Growth Factor (VEGF) 208 pg / ml and Interleukin-6 (IL-6) 962 pg / ml. At least two of these growth factors should be present for the desired efficacy along with an in vitro anti-oxidant activity of between 25-90% using 2,2-diphenyl-1-picryl hydrazyl (DPPH) assay. In ‘Sterility’ testing the composition of MSC-CM is found negative for aerobic and anerobic bacteria, fungus, mycoplasma and endotoxin. The pH ranges between 5.5 to 9.0 and is stable at -20°C for 2 years.

[0076] Example 2

[0077] Evaluation of biocompatibility of MSC-CM by evaluating the effect on healthy peripheral blood mononuclear cells (PBMC) by CCK-8 dye assay: MSC-CM was assessed for safety and biocompatibility by testing it against healthy peripheral blood mononuclear cells (PBMC). PBMC were isolated from peripheral blood of healthy individuals (n=3). PBMC were plated at 5000 cells per well in 96-well flat-bottomed cell culture plate and various concentrations of MSC-CM at 5, 10, 20 and 40% were added to wells. Cells were incubated for 48 hours at 37°C in a humidified atmosphere of 5% CO2 in air. Only PBMC cells served as cell control. CCK-8 dye was added to all wells at 10% final concentration. Cultures were further incubated at 37°C for 3 hours. Later, optical density of wells was read at 450 nm on an ELISA Reader. Assay was conducted as three replicates for each concentration. Data was represented as mean % cell growth and standard error for 3 healthy donors (Figure 1). Figure 1. Cytocompatibility of MSC-CM. Biocompatibility of MSC-CM was assessed by testing on healthy peripheral blood mononuclear cells (PBMC) at concentrations of 5, 10, 20 and 40% and effect on cell viability and metabolic activity were observed. Bar graph shows percent cell growth 12as seen by CCK-8 dye assay. Data indicates cell growth (mean % ± standard error) of PBMC from healthy donors (n=3). Data showed that MSC-CM did not exhibit any significant detrimental effect on viability and metabolic activity of healthy peripheral blood mononuclear cells (Figure 1). This experiment indicated that this compound was non-toxic and safe towards normal immune cells present in healthy peripheral blood. It was interesting to note that at the highest concentration of 40%, MSC-CM was safe towards PBMC (93-128% cell growth).

[0077] Example 3

[0078] Effect of MSC-CM on cell cycle phases and ploidy of cancer cell lines The leukaemia cells (K-562 and HL-60), lung cancer cells (A549 and HOP-62), breast cancer cells (MDA- MB-231 and MCF-7) and cervical cancer cells (HeLa) were treated with and without 20% MSC-CM for 48 hours. The percent populations in different phases of cell cycle were assessed by Propidium Iodide assay and flowcytometry. Analysis was done using ModFit LT™ software. The experiment was conducted in technical replicates (n=2) and figures are representative of one set (Figures 2-7). The Figures 2b to 7b depict the % population of cell cycle phases in cancer cells before and after treatment with MSC-CM. The distinct changes in apoptosis, S-phases and G2 / M phases are highlighted. In one of the embodiments of present invention, the effect of MSC-CM on cell cycle phases and ploidy of K-562 leukemia cells is shown in Figure 2a. Bright field microscopy shows the effect of untreated and treated K562 leukemic cell line with MSC-CM and histograms obtained show cell cycle analysis by flow cytometry. Histograms demonstrate cell cycle phase arrest after 48 hours treatment with MSC-CM. The % populations are mentioned at top right corner of each panel with each phase of cell cycle as shown in Figure 2a. Highlighted rectangles represent cell cycle phase affected with MSC-CM as claimed in present invention. In one of the embodiments of present invention, the effect of MSC-CM on cell cycle phases and ploidy of HL-60 leukemia cells is shown in Figure 3. Bright field microscopy shows the effect of untreated and treated HL-60 leukemic cell line with MSC-CM and histograms obtained show cell cycle analysis by flow cytometry. Histograms demonstrate cell cycle phase arrest after 48 hours treatment with MSC-CM. The % populations are mentioned at top right corner of each panel with each phase of cell cycle as shown in Figure 3. Highlighted rectangles represent cell cycle phase affected with MSC-CM as claimed in present invention.

[0079] In one of the embodiments of present invention, the effect of MSC-CM on cell cycle phases and ploidy of A549 lung cancer cells is shown in Figure 4. Bright field microscopy shows the effect of untreated and treated A549 lung cancer cells with MSC-CM and histograms obtained show cell cycle analysis by flow cytometry. Histograms demonstrate cell cycle phase arrest after 48 hours treatment with MSC-CM. The % populations are mentioned at top right corner of each panel with each phase of cell 13cycle as shown in Figure 4. Highlighted rectangles represent cell cycle phase affected with MSC-CM as claimed in present invention.

[0080] In one of the embodiments of present invention, the effect of MSC-CM on cell cycle phases and ploidy of HOP-62 lung cancer cells is shown in Figure 5. Bright field microscopy shows the effect of untreated and treated HOP-62 lung cancer cells with MSC-CM and histograms obtained show cell cycle analysis by flow cytometry. Histograms demonstrate cell cycle phase arrest after 48 hours treatment with MSC-CM. The % populations are mentioned at top right corner of each panel with each phase of cell cycle as shown in Figure 5. Highlighted rectangles represent cell cycle phase affected with MSC-CM as claimed in present invention.

[0081] In one of the embodiments of present invention, the effect of MSC-CM on cell cycle phases and ploidy of MDA-MB-231 breast cancer cells is shown in Figure 6. Bright field microscopy shows the effect of untreated and treated MDA-MB-231 breast cancer cells with MSC-CM and histograms obtained show cell cycle analysis by flow cytometry. Histograms demonstrate cell cycle phase arrest after 48 hours treatment with MSC-CM. The % populations are mentioned at top right corner of each panel with each phase of cell cycle as shown in Figure 6. Highlighted rectangles represent cell cycle phase affected with MSC-CM as claimed in present invention.

[0082] In one of the embodiments of present invention, the effect of MSC-CM on cell cycle phases and ploidy of MCF-7 breast cancer cells is shown in Figure 7. Bright field microscopy shows the effect of untreated and treated MCF-7 breast cancer cells with MSC-CM and histograms obtained show cell cycle analysis by flow cytometry. Histograms demonstrate cell cycle phase arrest after 48 hours treatment with MSC-CM. The % populations are mentioned at top right corner of each panel with each phase of cell cycle as shown in Figure 7. Highlighted rectangles represent cell cycle phase affected with MSC-CM as claimed in present invention.

[0083] Flow cytometry analysis as depicted in above Figures 2-7 revealed that cell population in either S phase and / or G2 / M phases of cell cycle phases was affected in various cancer cell lines after treatment with 20% MSC-CM for 48 hours.

[0084] Example 4

[0085] Evaluation of soluble factors and immune markers in MSC-CM by CBA, Multiplex Assay and ELISA In yet another embodiment of present invention, the role of T helper (Th) cells in regulating immune responses is identified by the production of various cytokines. The immunoregulatory potential of MSC- CM is analysed by measuring the levels of cytokines for IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL- 17A cytokines using BD™ Cytometric Bead Array (CBA) Human Th1 / Th2 / Th17 Cytokine Kit. 14In yet another embodiment of present invention, the levels of immune check point markers like TIM3, PD- 1 and PDL-1 are measured using MILLIPLEX® Human Immuno-Oncology Checkpoint Protein Panel 1 - Immuno-Oncology Multiplex Assay to check the immunomodulatory ability of MSC-CM (Figure 8). Additionally, MSC-CM was analysed for TIMP-2 and IL-6 by ELISA. In yet a further embodiment of present invention, Figures 8a and 8b clearly show the significant increase in the level of IL-6, TIM-3 and TIMP-2 which contributes to its anti-inflammatory and immunomodulatory effect. IL-6 plays a key role in the triggering of humoral immune response by stimulating the B cell differentiation and secretion of Ig antibodies.

[0086] Example 5

[0087] Evaluation of immunomodulatory profile of immune cell subsets treated with MSC-CM In yet another embodiment of present invention the immunophenotyping of peripheral blood lymphocytes from healthy individual is done. Immune profile with respect to expression of activation markers, effector function cytotoxic granules (Perforin and Granzyme B) in T cells and Mucosal-associated invariant T cells (MAIT) cell subsets was analyzed. The experiment was conducted in vitro using peripheral blood lymphocytes taken from three healthy individuals (n=3). The analysis was done using FlowJo™ software and data is represented as percent positive cells for different immune subsets marker (Figures 9-12).

[0088] Assessment of immunomodulatory role of MSC-CM on T cell subsets is shown in Figure 9. Peripheral blood lymphocytes from healthy individual or HI-PBL representing immune cells are cultured in presence and absence of 10% MSC-CM for 48 hours. The data is represented as percent positive cells for different T cell subsets CD3, CD4 and CD8 cells alongwith expression of activation markers CD25 and CD69.

[0089] Assessment of immunomodulatory role of MSC-CM on helper T cell subsets is shown in Figure 10. Peripheral blood lymphocytes from healthy individual or HI-PBL representing immune cells were cultured in presence and absence of 10% MSC-CM for 48 hours. The data is represented as percent positive cells for different T cell subsets CD3, CD4 and CD8 cells alongwith expression of cytotoxic effector molecules perforin and granzyme B.

[0090] Assessment of immunomodulatory role of MSC-CM on T cell subsets is shown in Figure 11. Peripheral blood lymphocytes from healthy individual or HI-PBL representing immune cells were cultured in presence and absence of 10% MSC-CM for 48 hours. The data is represented as percent positive cells for Mucosa-Associated Invariant T (MAIT) different cell subsets CD3, CD4 and CD8 cells alongwith expression of activation markers CD25 and CD69.

[0091] Assessment of immunomodulatory role of MSC-CM on T cell subsets is shown in Figure 12. Peripheral blood lymphocytes from healthy individual or HI-PBL representing immune cells were cultured in presence or absence of 10% MSC-CM for 48 hours. The data is represented as percent positive cells for 15Mucosa-Associated Invariant T (MAIT) different cell subsets CD3, CD4 and CD8 cells alongwith expression of cytotoxic effector molecules perforin and granzyme B.

[0092] In yet another embodiment of present invention and as shown in Figures 9-12, it is observed that immunophenotypic profiles of healthy immune cells were maintained or unaltered after MSC-CM treatment. Further, there was marginal increase in expression of activation markers, cytotoxic granules, and Mucosal-associated invariant T cells (MAIT) in T cell subsets after treatment with 10% MSC-CM.

[0093] Example 6

[0094] Reversal of immunosuppression after treatment with MSC-CM as shown in Figure 13. Mononuclear cells isolated from healthy peripheral blood (PBL) were treated with Phytohemagglutinin (PHA), Pokeweed mitogen (PWM) and Granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulation of T-cell, B-cell and myeloid cell mitogens, respectively. Lymphocyte proliferation responses are measured using Tritiated thymidine incorporation assay. Further culture of the same healthy immune cells led to reduction of proliferation in presence of 50% culture supernatant of myeloid cell line OCI- AML2 (Figure 13). It was interesting to note that T-cell and B-cell suppression responses were reversed by 20% MSC-CM (Figure 13). T-cell responses of healthy PBL to PHA were increased in myeloid OCI- AML-2 culture supernatant+ PHA + MSC-CM count per minute (cpm, 11727) compared to myeloid OCI- AML-2 culture supernatant+ PHA (cpm, 10343). Further, B-cell responses of healthy PBL to PWM were increased in myeloid OCI-AML-2 culture supernatant+ PWM + MSC-CM count per minute (cpm, 7794) compared to myeloid OCI-AML-2 culture supernatant+ PWM (cpm, 3281) and only PWM (cpm, 7129). Reversal of B-cell proliferation can be due to presence of significant levels of B-cell stimulatory cytokine IL-6 in MSC-CM, as reported in Figure 8. Further myeloid responses were also increased in myeloid OCI- AML-2 culture supernatant+ GM-CSF + MSC-CM count per minute (cpm, 6478) compared to only GM- CSF (cpm, 715), myeloid OCI-AML-2 culture supernatant+ GM-CSF (cpm, 5108) (Figure 13).

[0095] Example 7

[0096] Effect of MSC-CM on immune cell-mediated cytotoxicity against various tumor targets. Immune cell-mediated cytotoxicity is integral in the immune response to intracellular pathogens and cancer cells in the body. Activated effector cells, including T cells, NK, natural killer T cells (NKT), and granulocytes, can kill targeted cells through unique mechanisms and regulatory pathways. The immunomodulatory function of MSC-CM is shown in Example 1 and confirmed with modulation of immune cell mediated cytotoxicity potential against human erythroleukemia cell line (K-562) which is known to be sensitive to natural killer cell-mediated cytotoxicity. In yet another embodiment of the present invention, the immunomodulatory function of MSC-CM, the cell-mediated cytotoxicity experiments are confirmed by taking freshly isolated peripheral blood lymphocytes (PBL) from healthy individuals (n=2) representing immune cells and are co-cultured with 16various targets such as lung adenocarcinoma cell line (A549), and triple negative breast adenocarcinoma cell line (MDA-MB-231). The immune-cell mediated cytotoxicity is measured by51Chromium release assay in which target cells are labelled with radioactive isotope Chromium-51 or51Cr. PBL are co-cultured in presence or absence of 10% MSC-CM at E:T ratios of 5:1 to 40:1. Each set is conducted in triplicate wells. After incubation for 4 hours, cell free supernatants were collected and analysed on gamma counter. Percent lysis was calculated (Figures 14 -16). % specific lysis = 100 × ((test) – (spontaneous) / (maximal) − (spontaneous)51Cr release

[0097] Figure 14 shows the effect of 10% MSC-CM on cell mediated cytotoxic potential of PBL against K562 and HL-60 leukemic cells. Non-adherent PBLs were co-cultured with either K562 or HL-60 cells for 4 hours at E:T ratios of 5:1 to 40:1 in presence of 10% MSC-CM and cytotoxicity was measured using activity of51Cr in cell free supernatant. Data for K562 target represents mean ± standard error from three experiments independent from different healthy individuals.

[0098] Role of MSC-CM on modulating cytotoxic potential of PBL against lung adenocarcinoma cell line (A549) is shown in Figure 15. Freshly isolated PBL (n=3) are co-cultured with A549 cells for 4 hours at E:T ratios from 5:1 to 40:1 in presence of 10% MSC-CM and cytotoxicity is measured using activity of51Chromium in cell free supernatant. Figure 15 shows representative data from one healthy individual.

[0099] Role of MSC-CM on modulating cytotoxic potential of PBL against breast cancer cell lines MDA-MB-231 and MCF-7 is shown in Figure 16. Freshly isolated PBL (n=2) are co-cultured with TNBC MDA-MB-231 cells (Figure 16 a) or MCF-7 (Figure 16 b) for 4 hours at E:T ratios from 5:1 to 40:1 in presence of 10% MSC-CM and cytotoxicity is measured using activity of51Chromium in cell free supernatant.

[0100] In yet another embodiment of the present invention, the data as depicted in Figures 13-16 shows that 10% MSC-CM augments cytotoxicity potential against cancer cell lines. This observation is also confirmed in the presence of recombinant IL-12. (Figures 17 and 18). IL-12 being T-cell stimulator, MSC- CM acts in synergy or as an adjunct to provide enhanced T-cell mediated cytotoxicity of tumor target cells. Role of MSC-CM on modulating cytotoxic potential of PBL against Lung cancer cell line (A549) is shown in Figure 17. Freshly isolated PBL (n=2) are co-cultured with A549 cells for 4 hours at E:T ratios from 5:1 to 40:1 in presence of 10% MSC-CM and in presence or absence of IL-12. Cytotoxicity is measured using activity of51Chromium in cell free supernatant. Role of MSC-CM in modulating cytotoxic potential of PBL against TNBC cell line (MDA-MB-231) is shown in Figure 18. Freshly isolated PBL (n=2) are co-cultured with TNBC MDA-MB-231 cells for 4 hours at E:T ratios from 5:1 to 40:1 in presence of 10% MSC-CM and or IL-12. Cytotoxicity is measured using activity of51Chromium in cell free supernatant. 17

[0101] Example 8

[0102] Elucidation of anti-cancer efficacy of MSC-CM against immunodeficient mouse model of various cancers In yet another embodiment of the present invention, for in vivo efficacy studies, immunodeficient mice (NOD / SCID) were grafted with human cancer cell lines to develop cell line-derived xenograft or CDX model for testing anti-cancer efficacy of MSC-CM. Later, tumor bearing mice at 8 to 10 weeks of age (n=6 per group) were randomized and MSC-CM treatment was started on day1 of the experiment. To evaluate the tumor reduction efficacy of MSC-CM against human breast cancer model, MDA-MB-231 xenograft was administered with MSC-CM by intratumoral (i.t.) and intraperitoneal (i.p.) routes. The positive control group was treated with Adriamycin (ADR) (Table 1). Tumor measurements were carried out to determine tumor growth and tumor volume using digital Vernier calliper. Mice were then observed at regular intervals over a period of 32 days for body weight, tumor volume and mortality. a) MDA-MB-231 model was administered with MSC-CM by both intraperitoneal and intratumoral routes and the positive control group was treated with Adriamycin. Table 1 MSC-CM MSC-CM Days ADR (i.t.) (i.p.) 1 1.00 1.00 1.00 50.41 0.80 0.619 0.43 0.86 0.61 12 0.30 0.75 0.48 15 0.26 0.97 0.46 18 0.20 0.86 0.43 21 0.15 0.86 0.4324 0.14 0.89 0.45 27 0.11 0.83 0.37 30 0.11 0.91 0.43 Note: NOD-SCID Mice per group, n=6 Positive Control Adriamycin (ADR): 2.5 mg / kg Intravenous (i.v.) weekly for 3 weeks MSC-CM: 200 μl Intraperitoneal (i.p.) twice a week X 4 weeks MSC-CM: 200 μl Intratumoral (i.t.) twice a week X 4 weeks As per NCI guidelines, T / C ≤ 0.2 (highlighted yellow) is considered to demonstrate highly significant activity. T / C ≤ 0.42 (highlighted blue) is considered to demonstrate significant activity. In yet another embodiment of present invention Figure 19 elucidates the in vivo tumor reduction efficacy of MSC-CM against MDA-MB-231 xenograft. MDA-MB-231 breast cancer xenograft model are administered with MSC-CM (200 μl) by i.t. and i.p. route and the positive control group was treated 18with ADR. For every group, the mice used were n=6. These mice were monitored till day 30 post-dosing. Difference in Mean Relative Tumor Volume (RTV) between the groups was calculated for day 30 for all the groups by Kruskal-Wallis H Test using IBM SPSS Statistics.21 software for data analysis (*p=0.002). Figure 20 shows in vivo efficacy of MSC-CM against MDA-MB-231 xenograft in terms of Relative Tumor Volume (RTV), Average Animal body Weight (grams), % survival, and Tumor Volume (cc). It was interesting to note that administration of MSC-CM to breast xenograft tumor bearing mice by intraperitoneal route (p=0.002) enhanced the reduction of relative tumor volume (RTV) in comparison to tumor bearing control. Body weight and survival was not affected by MSC-CM i.p. treatment. (Figures 19, 20 and Table 1). In yet another embodiment of present invention, for in vivo efficacy studies, to evaluate the tumor reduction efficacy of MSC-CM against human leukemia model, K562 xenograft was administered with MSC-CM by intraperitoneal (i.p.) route. The positive control group was treated with 5-Flurouracil (5-FU) (Table 2). Tumor measurements were carried out to determine tumor growth and tumor volume using digital Vernier calliper. Mice were observed at regular intervals over a period of 30 days for body weight, tumor volume and mortality. b) K562 Leukemia xenograft model was administered with MSC-CM by intraperitoneal route and the positive control group was treated with 5-FU. Table 2: Days 5-FU MSC-CM 1 1.00 1.00 50.60 0.749 0.37 0.47 12 0.26 0.34 15 0.22 0.28 18 0.14 0.17 21 0.14 0.15 24 0.16 0.13 27 0.13 0.09 30 0.12 0.08 Note: NOD-SCID Mice per group, n=6 Positive Control 5-Flurouracil (5-FU): 5 mg / kg Intraperitoneal (i.p.) for 9 days MSC-CM: 200 μl Intraperitoneal (i.p.) thrice a week X 4 weeks As per NCI guidelines, T / C ≤ 0.2 (highlighted yellow) is considered to demonstrate highly significant activity. T / C ≤ 0.42 (highlighted blue) is considered to demonstrate significant activity. Figure 21 shows In vivo efficacy of MSC-CM against K562 leukemia xenograft. It is interesting to note that administration of MSC-CM to leukemia xenograft tumor bearing mice by intraperitoneal 19route (p=0.007) enhanced the reduction of relative tumor volume (RTV) in comparison to tumor bearing control (Figures 21a, 21b and Table 2). c. MSC-CM against HeLa xenograft In yet another embodiment of the present invention, for in vivo efficacy studies, immunodeficient mice (NOD / SCID) were grafted with human cancer cell lines to develop cell line-derived xenograft or CDX model for testing anti-cancer efficacy of MSC-CM. Later, tumor bearing mice at 8 to 10 weeks of age (n=6 per group) were randomized and MSC-CM treatment was started after 12 days when the tumor was of advanced size. This was to evaluate whether MSC-CM will be effective on tumors of advanced sizes. To evaluate the tumor reduction efficacy of MSC-CM against human cervical cancer model, HeLa xenograft was administered with MSC-CM by intravenous (i.v.) route and the positive control group was treated with Adriamycin (ADR) (Table 3). Tumor measurements were carried out to determine tumor growth and tumor volume using digital Vernier calliper. Mice were then observed at regular intervals over a period of 32 days for body weight, tumor volume and mortality. Table 3 Days ADR MSC-CM 11.00 1.0050.78 0.9090.48 0.80120.25 0.6615 0.19 0.64 18 0.12 0.50 21 0.14 0.55 25 0.15 0.55 29 0.14 0.42 32 0.15 0.40 Note: NOD-SCID Mice per group, n=6 Positive Control Adriamycin (ADR): 2.5 mg / kg Intravenous (i.v.) weekly for 3 weeks MSC-CM: 200 μl Intravenous (i.v.) twice a week X 4 weeks As per NCI guidelines, T / C ≤ 0.2 (highlighted yellow) is considered to demonstrate highly significant activity. T / C ≤ 0.42 (highlighted blue) is considered to demonstrate significant activity. In yet another embodiment of the present invention, Figure 22 elucidates the in vivo tumor reduction efficacy of MSC-CM against HeLa xenograft. HeLa cervical xenograft mice are administered with MSC-CM (200 μl) by intravenous (i.v.) route and the positive control group was treated with ADR. Difference in Mean RTV between the groups was calculated for day 28 for all the groups by Kruskal- Wallis H Test using IBM SPSS Statistics.21 software for data analysis (*p=0.002). In yet another embodiment of the present invention, Figure 23 elucidates the in vivo tumor reduction 20efficacy of MSC-CM against HeLa xenograft. HeLa cervical xenograft model was administered with MSC-CM (200 μl) by intravenous (i.v.) route and the positive control group was treated with ADR. For every group, the mice used were n=6. These mice were monitored till day 32 post-dosing. The data is represented as Relative Tumor Volume (RTV), Average Animal Weight (grams), % survival, and Tumor Volume (cc). Difference in Mean RTV between the groups was calculated for day 28 for all the groups by Kruskal-Wallis H Test using IBM SPSS Statistics.21 software for data analysis (*p=0.002). As per the results shown in above Figures 22 & 23, MSC-CM demonstrated 65% reduction in aggressive HeLa tumor growth. In 4 out of 6 mice population the Xenograft tumor reductions were 51.1, 61.4, 88.4, and 60.2% which is at par with the standard chemotherapeutic drug. As per NCI guidelines for activity criteria, T / C ≤ 0.42 is considered to demonstrate significant activity. Post-termination of the tumor efficacy studies, retro‐orbital blood from each mouse from each group was collected and xenograft tumors were preserved for the preparation of histopathology tissue sections and NGS. d. A549 Lung xenograft model was administered with MSC-CM by intravenous and intraperitoneal route and the positive control group were treated with 5-FU. To evaluate the tumor reduction efficacy of MSC-CM against human lung cancer model, A549 xenograft is administered with MSC-CM by intravenous (i.v.) and intraperiptoneal (i.p.) routes. The positive control group are treated with 5-Fluorouracil (5-FU), (Table 4) Tumor measurements are carried out to determine tumor growth and tumor volume using digital Vernier calliper. Mice are then observed at regular interval for period of 23 days for body weight, tumor volume and mortality. Based on Humane time point, since post 18 days tumor in all groups started increasing while by 23 days it grew beyond permissible limits, experiment was terminated. On day 18, MSC-CM by i.v. route caused better tumor reduction (79%) than MSC-CM by i.p. route (62%) or standard drug 5-FU (64%), (Table 4). Table 4 MSC-CM MSC-CM Days 5-FU (i.v.) (i.p.) 1 1.00 1.00 1.00 5 1.28 0.76 1.02 91.08 0.62 0.9712 0.94 0.54 0.99 15 0.25 0.16 0.34 18 0.36 0.21 0.38 21 0.83 0.50 0.98 23 0.78 0.51 0.98Note: NOD-SCID Mice per group, n=6 21Positive Control 5-Flurouracil (5-FU): 20 mg / kg Intraperitoneal (i.p.) thrice a week X 4 weeks MSC-CM: 200 μl Intravenous (i.v.) twice a week X 4 weeks MSC-CM: 200 μl Intraperitoneal (i.p.) thrice a week X 4 weeks As per NCI guidelines, T / C ≤ 0.2 (highlighted yellow) is considered to demonstrate highly significant activity. T / C ≤ 0.42 (highlighted blue) is considered to demonstrate significant activity. In yet another embodiment of present invention, Figures 24 and 25 elucidate the in vivo tumor reduction efficacy of MSC-CM against A549 Lung xenograft. A549 lung xenograft model was administered with MSC-CM (200 μl) by intravenous (i.v.) route, intraperitoneal (i.p.) route and the positive control group were treated with 5-FU. For tumor control and MSC-CM group, the mice used were n=6. For 5-FU group mice used were n=5. These mice were monitored till day 23 post-dosing. Since tumors in tumor bearing control grew beyond Humane end point, mice were sacrified at day 23. The data is represented as Relative Tumor Volume (RTV). Statistical analysis showed that by student’s t test difference between RTV of tumor bearing control group and RTV of MSC-CM (i.v.) group was p=0.0095 (Figure 24).

[0103] Example 9

[0104] Elucidation of the mechanistic role of MSC-CM on NLRP3 innate inflammasome pathway in A549 lung cancer xenograft model in mice Tumors of A549 lung cancer xenograft in immunodeficient NOD-SCID mice treated with MSC-CM (administered by intraperitoneal and intravenous routes) along with tumor bearing control and positive control 5-Fluorouracil (5-FU) were preserved as paraffin blocks and its tissue sections were stained by immunohistochemistry to analyse the expression of NLRP3, IL-18, and AIM2 markers along with H & E and secondary control. Data shows that NLRP3 and its downstream molecule IL-18 is found to be reduced after treatment with MSC-CM in comparison to the standard chemotherapeutic drug. In yet another embodiment of present invention, the AIM2 inflammasome molecule exhibited marginal decrease. Image acquisition was done by ZEISS Axio Imager.Z1 upright microscope at different magnifications and representative images are shown for each group at 40X magnification (Figure 26). Expression of NLRP3 pathway molecules in tissue sections of MSC-CM treated A549 lung cancer xenograft in NOD-SCID Mice is shown in Figure 26a. A549 lung cancer xenograft model is administered with MSC-CM (200 ul) by intraperitoneal (i.p.) and intravenous (i.v.) routes and the positive control group are treated with 5-FU. Expression of NLRP3, IL-18, and AIM2 is assessed along with H & E and secondary control. Representative images are shown for each group at 40X magnification in Figure 26a. Semi-quantitative histology scoring was done to confirm the expression of markers in the tissue sections of different groups. The histology scoring was based on the percentage of cells staining at intensity level. Further interpretation of immunoreactivity was calculated by ‘Histochemical scoring (H-score)’ 22which incorporates both the staining intensity and percentage of stained cells at each intensity level. The score ranges from 0 to 300 (Figure 26b). Histology scoring of tissue sections of MSC-CM treated A549 lung cancer xenograft in NOD-SCID Mice is shown in Figure 26b. A549 lung cancer xenograft model was administered with MSC-CM (200 ul) by intraperitoneal (i.p.) and intravenous (i.v.) routes and the positive control group was treated with 5-FU. Semi-quantitative scoring of immunohistochemical staining was done to assess the expression of NLRP3, IL-18, and AIM2 based on percentage of cells staining at intensity level in different groups. Similarly, bar graphs represent H-Score assessment for NLRP3, IL-18 and AIM2 markers (Figure 26b). MSC-CM showed 88% decrease in NLRP3 from 180 to 20, IL-18 showed 87% decrease from 175 to 10 and AIM2 inflammasome expression showed 7% decrease from 150 to 140 in residual tumor tissues after treatment of mice with MSC-CM by intravenous route.

[0105] Example 10

[0106] Transcriptomic mapping of residual A549 xenograft tumors after treatment with MSC-CM using RNAseq study. The paired-end data of A549 xenograft tumors (two mice each) procured from tumor control group, positive control 5-FU treated group, MSC-CM treated by intravenous (i.v.) as well as intraperitoneal (i.p.) groups from Example 8d, were sequenced using NovaSeq 6000 (Illumina) platform platform. High quality reads were obtained after removal of adapter sequences, ambiguous reads (reads with unknown nucleotides “N” larger than 5%), and low-quality sequences (reads with more than 10% quality threshold (QV) < 25 phred score) using Trimmomatic (v0.39). The filtered HQ reads were aligned to the GRCh38 reference genome108 / fasta / homo_sapiens / dna / Homo_sapiens.GRCh38.dna.toplevel.fa.gz), using STAR (v 2.7.10a) at default parameters. Mapped read counts for individual genes were obtained using featureCounts (v 2.0.3). Differentially expressed genes (DEG) were identified using DESeq2 R package (v 1.40.2). The expression profile of treated tumor samples was compared against tumor control. The read counts were normalized for all the samples and differentially expressed Ensembl gene IDs were identified by using the DESeq2 package. Log2 Fold Change (log2FC) values greater than zero were considered up-regulated whereas less than zero were down-regulated along with P-value threshold of 0.05 for statistically significant results. The Ensembl gene IDs with differential expression were annotated with Ensembl BiomartHeatmap plot was constructed using pHeatmap R package for the top 50 genes involved in Signaling pathways considering the log-transformed and normalized value of genes based on Pearson uncentered distance and average linkage method. 23Figure 27 demonstrates heat maps of differential genes from comparisons of A549 xenograft tumors (two mice each) procured from tumor control group, positive control 5-FU treated group , MSC-CM treated by intravenous (i.v.) as well as intraperitoneal (i.p.) groups. Heatmaps intensity depicts fold change of genes involved in various pathways affecting xenograft tumor growth. It is evident that combination of tumor control versus MSC-CM by i.v. route had most differential expression of genes compared to tumor control versus 5-FU treated mice tumors or combination of tumor control versus MSC-CM by i.p. route.

[0107] Example 11

[0108] Cytokine analysis in mice sera: Mouse cytokines were studied in plasma of tumor-bearing mice treated with MSC-CM. Plasma samples were tested using MILLIPLEX® Multiplex for Luminex® Immunoassays (MCYTOMAG- 70K, Merck). Labelled analytes were run on reader calibrated prior with standard beads (Luminex 200, Millipore, USA). Data was analyzed using Belysa® Immunoassay Curve Fitting Software, Version 1.2, Millipore, USA. Figure 28 demonstrates that expression of sIL-β, sIL-6 and sMCP-1 were unaffected in plasma derived from K-562 xenograft in immunodeficient mice treated with MSC-CM. Figure 29 demonstrates that expression of sIFN-γ and sIL-β was significantly reduced in plasma derived from A549 xenograft in immunodeficient mice treated with MSC-CM. Figure 30 demonstrates that expression of pro-inflammatory cytokine IL-6 was significantly reduced while sIFN-γ and monocyte chemoattractant protein-1 (MCP-1) levels were unaffected in plasma derived from HeLa xenograft in immunodeficient mice treated with MSC-CM.

[0109] ADVANTAGES Various embodiments of present invention elucidate the salient features and advantages which are listed as follows: 1. MSC-CM causes cell growth inhibition by mainly arrest in synthesis phase or G2 / M phases of cell cycle in leukemia, lung and breast cancer cell lines. 2. MSC-CM showed elevated levels of IL-6, TIMP-2 and TIM-3, conferring to its immunomodulatory function. 3. MSC-CM is non-toxic to the healthy immune cells and maintained the overall immune repertoire. 4. MSC-CM demonstrates enhancement in anti-cancer immunity potential in healthy PBL against leukemia, lung and breast cancer cell lines. 5. MSC-CM exhibits significant anti-cancer activity in in-vivo xenograft models of leukemia (K-562), triple negative breast cancer (MDA-MB-231), lung cancer (A549) and cervical cancer (HeLa). 6. MSC-CM exhibits decrease in expression of innate immune inflammasome pathway molecules NLRP3, AIM2 and IL-18 indicating its role as a potential candidate for NLRP3 inhibitor. 24

[0110] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art. Moreover, in interpreting both the specification and the claims, all the terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components or steps may be present, or utilised or combined with other elements, components or steps that are not expressly referenced. Where the specification claims refers to atleast one of something selected from the group consisting of A, B, C….and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

[0111] While some embodiments of the present disclosure have been illustrated and described, those are completely exemplary in nature. The disclosure is not limited to the embodiments as elaborated herein only and it would be apparent to those skilled in the art that numerous modifications besides those already described are possible without departing from the inventive concepts herein. All such modifications, changes, variations, substitutions, and equivalents are completely within the scope of the present disclosure. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. For the Applicants Meera Sharma Regd. Patent Agent[IN / PA-3053] Dated: 16thSeptember, 2024 25

Claims

We claim:

1. An Umbilical cord derived mesenchymal stem cells (UC-MSC) conditioned media (UC-MSC-CM) composition of the range from 5%-100% possessing immunomodulatory effect inhibiting tumour growth wherein, MSC-CM arrests cell cycle in leukaemia cells (K-562 and HL-60), lung cancer cells (A549 and HOP-62), breast cancer cells (MDA-MB-231 and MCF-7) and cervical cancer cells (HeLa); MSC-CM showed elevated levels of IL-6, TIM3 and TIMP-2 conferring to its immunomodulatory function; Presence of 5%-100% MSC-CM led to an increase in expression of activation markers(8 to 223%), cytotoxic granules (2 to 161%), and Mucosal-associated invariant T cells (MAIT) in T cell subsets(6 to 80%); MSC-CM led to significant reversal of suppression caused in T-cell, B-cell and Myeloid cell mitogenic responses in presence of leukemia cell supernatant; MSC-CM as an adjunct with IL-12 augments cytotoxicity potential against cancer cell lines; MSC-CM when administered intraperitoneally and intravenously against tumor xenograft showed led to significant reversal of suppression caused in T-cell, B-cell and Myeloid cell mitogenic responses in presence of leukemia cell supernatant; MSC-CM show decrease in expression of innate immune inflammasome pathway molecules by 88% of NLRP3, 87% of IL-18 and 7% AIM2 in treated xenograft tumor tissues; and MSC-CM induced fold change of genes involved in various pathways affecting xenograft tumor growth.

2. The MSC-CM as claimed in claim1, wherein, arrests cell cycle and augments cytotoxic potential with recombinant IL-12 in cell population of leukaemia cells (K-562 and HL-60), lung cancer cells (A549 and HOP-62), breast cancer cells (MDA-MB-231 and MCF-7) and cervical cancer cells(HeLa) cell lines after treatment with 5%-100% MSC-CM for 48 hours.

3. The MSC-CM as claimed in claim1, wherein, treatment with 5%-100% MSC-CM led to 10-95% increase of immune -cell mediated kill of 562 and HL-60 leukemia cells.

4. The MSC-CM as claimed in claim1, wherein, the pro-inflammatory cytokine IL-6 in a range from 1500-15000 pg / ml that plays a key role in the triggering of humoral immune response by stimulating the B cell differentiation and secretion of antibodies.

5. The MSC-CM as claimed in claim 1, wherein, elevated levels of TIM-3 in a range from 50-3000 pg / ml was found in MSC-CM that confers immunomodulatory function in leukemia, lung, breast and cervical cancer.

266. The MSC-CM as claimed in claim 1, wherein, elevated levels of TIMP-2 in a range from 2000- 25000 pg / ml was found in MSC-CM that confers immunomodulatory function in leukemia, lung, breast and cervical cancer.

7. The MSC-CM as claimed in claim1, wherein, treatment with 5%-100% MSC-CM led to increase in expression of activation markers (CD25 and CD69) by 8 to 223%, cytotoxic granules (perforin and granzyme B) by 2 to 161%, and Mucosal-associated invariant T cells (MAIT) in T cell subsets by 6 to 80% and that the immunophenotypic profile of healthy immune cells was maintained after MSC- CM treatment.

8. The MSC-CM as claimed in claim1, wherein, treatment with 5%-100% MSC-CM led to 10-200% significant reversal of suppression caused in T-cell, B-cell and myeloid cell mitogenic responses in presence of leukemia cell supernatant.

9. The MSC-CM as claimed in claim1, wherein, MSC-CM as an adjunct with IL-12 augments cytotoxicity potential against cancer cell lines like K562, HL-60, A549, MDA-MB-231and MCF-7.

10. The MSC-CM as claimed in claim1, wherein, MSC-CM when administered intraperitoneally against MDA-MB231 breast cancer xenograft, K562 leukemia xenograft, when administered intravenously against HeLa cervical cancer xenograft and A549 lung cancer xenograft enhanced the reduction of relative tumor volume.

11. The MSC-CM as claimed in claim1, wherein, treatment with 5%-100% MSC-CM shows decrease in expression of innate immune inflammasome pathway molecules by 88% of NLRP3, 87% of IL- 18 and 7% AIM2 in treated xenograft tumor tissues by intravenous or intraperitoneal routes.

12. The MSC-CM as claimed in claim1, wherein, treatment with 5%-100% MSC-CM induced fold change of genes involved in various pathways affecting xenograft tumor growth when administered by intravenous or intraperitoneal routes.

13. The composition as claimed in any preceding claim, as and when used in treating leukemia, triple negative breast cancer, lung cancer and cervical cancer. For the Applicants Meera Sharma Regd. Patent Agent[IN / PA-3053] Dated: 16thSeptember, 2024 27

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