Field of the invention
The invention described here relates to the field of immunology and molecular biology. More specifically, the invention pertains to a method for producing a population of mature dendritic cells, wherein the exogenous expression of CTLA-4 protein is downregulated and their use in cancer.

Background of the invention
Dendritic cells (DCs) are antigen-presenting cells (APCs) which play a critical role in the regulation of the adaptive immune response. DCs are capable of capturing antigens, processing them, and presenting them on the cell surface along with appropriate co-stimulation molecules. DCs also play a role in the maintenance of B cell function and recall responses.

Immature DCs originate in the bone marrow and migrate throughout the body. The immature DCs lay dormant there waiting to interact with invading pathogens or other foreign bodies. At this point, the primary function of the immature DC is to capture antigens. DCs are capable of processing both endogenous and exogenous antigens and present peptide in the context of either MHC class I or II. However, most of the methods in the prior art use chemical agents to culture the dendritic cells or use activators in order to stimulate/activate and mature the dendritic cells.

In the case of a wound accompanied by inflammation, DCs are attracted to the area of inflammation and stimulated to capture and internally process antigens. Once captured, the antigen is processed either by an exogenous or endosomal pathway, or by proteosomal pathway. For MHC class I presentation to stimulate CD8+ cytotoxic T cells, the antigen or protein is taken up by phagocytosis or receptor mediated endocytosis into the cytosol. The antigens are further degraded in the cytosol via proteosome and enter the endoplasmic reticulum where peptides bind to newly synthesized MHC class I molecules for presentation on the cell surface. For MHC class II presentation to stimulate CD4+ T helper cells, antigen is taken up by phagocytosis or receptor-mediated endocytosis to endosomes where some proteolysis occurs. The peptides enter a vesicle containing MHC class II where they bind and are transported to the cell surface. A key component on DCs in addition to antigen capture, processing and presentation is the presence of co-stimulatory molecules. DCs maintain on their cell surface co-stimulatory molecules including members of the B7 family, TNF family and intracellular adhesion molecules which are critical to the activation of T cells and for the proper homing of DCs before and after antigen capture. (Eric Wieder, Dendritic Cells: A Basic Review; May 2003).

Two proteins on the surface of T cells: CD28 and cytotoxic T-lymphocyte antigen 4 (CTLA-4) play important roles in the regulation of immune activation and tolerance. CD28 provides positive modulatory signals in the early stages of an immune response, while CTLA-4 signaling inhibits T-cell activation, particularly during strong T-cell responses. (Wolchok et al. The Oncologist 2008;13(suppl 4):2–9). It is also reported that CTLA-4 is highly expressed on freshly isolated monocytes, then down-modulated upon differentiation toward immature DCs (iDCs) and it was markedly upregulated on mature DCs obtained with different stimulations (lipopolysaccharides [LPS], Polyinosinic:polycytidylic acid (poly I:C), cytokines). DCs are shown to secrete functionally active CTLA-4 that interferes with T-cell activation by binding to B7 costimulatory molecules thus inhibiting the immune response (Halpert et al. 2016).

As DCs mature, they acquire the properties necessary to form and transport peptide-loaded MHC class II complexes to the cell surface. Antigen transport to the cell surface coincides with increased expression of co-stimulatory molecules, such as B7-1/CD80 and B7-2/CD86. For instance, CD80 and CD86 bind to both CD28 and cytotoxic T-lymphocyte associated protein 4 (CTLA-4). These ligands are upregulated or overexpressed in response to stimulation of Toll like receptors (TLRs) and are a key to the initiation of effective immune response. These molecules amplify T cell receptor (TCR) signaling and promote T cell activation. In the classical view, immature (non-activated) antigen-loaded DCs present antigens to T cells, which lead to tolerance as opposed to mature DCs, which are geared towards the launching of antigen-specific immunity. Mature DC, present within secondary lymphoid organs, express high levels of costimulatory molecules and are remarkably efficient in antigen presentation. (Palucka et al. J Immunother. 2008; 31(9): 793–805; Nature reviews|Cancer VOLUME 12 | APRIL 2012).

Theoretically there are various other methods by which the down regulation of CTLA-4 expression can be done a) by means of siRNA ablation of CTLA-4 in the differentiated dendritic cells, but this process have lot of difficulties as DC CTLA-4 is very stable, b) CTLA-4 could be knocked down with two different siRNA electroporations but this also seems to be impractical. c) The other method is use of Ipilimumab, an antibody against CTLA-4 which interferes with the co-stimulation required for T-cell activation (Pentcheva-Hoang et al. 2014; Jeanbart and Swartz 2015). But in patients who received Ipilimumab, rare skin reactions, tumor mass liquefication with fatal outcomes, gastritis, aseptic meningitis and CNS inflammation as the immune-related adverse events have been reported (Voskens et al. 2013).

DCs are being studied as adjuvants for vaccines or as a direct therapy to induce immunity against cancer. DCs loaded with tumor lysates, tumor antigen-derived peptides, MHC class I restricted peptides, or whole protein have all been shown to generate anti-cancer immune responses and activity, including in some cases the ability to induce complete regression of existing tumor. DCs, generated ex vivo by culturing hematopoietic progenitor cells or monocytes with cytokine combinations, have been under test as therapeutic vaccines in cancer patients for more than a decade. For instance, IL-12 is a hallmark inflammatory cytokine capable of eliciting potent Th1 immune responses. Binding of IL-12 to its receptor induces a series of intracellular reactions involving the Jak-Stat signaling cascade; these reactions have a direct impact on DC functions, including enhanced IL-12 production, upregulation of the co-stimulatory molecule CD80, increased capacity to stimulate T-cell proliferation, and
production of IFN-?, which may affect the development of adaptive immunity. Human studies depend on the in vitro exposure of monocytes to different cytokine combinations based on granulocyte macrophage colony-stimulating factor (GM-CSF). Together with IL-4, IFNa52, TNF53 or IL-15 GM-CSF can induce the differentiation of inflammatory DCs that can activate T cells. In particular, IL-4 is widely known for its role in Th2 cell polarization and DC maturation. Moreover, IL-4 can also enhance IL-12 production in DCs, in particular, IL-4 induces DC maturation, upregulating expression of MHC class II molecules, co-stimulatory receptors, and IL-12Rß1, which forms the functional high-affinity IL-12 receptor together with IL12Rß2 it has been reported that immature human DCs activated under high-dose IL-4 produce large amounts of IL-12 and small amounts of IL-10, thus preferentially inducing Th1 differentiation (Radice et al., Translational Oncology Vol. 8, No. 4, 2015).

Few prior arts disclose certain methods for producing mature dendritic cells that can be clinically used to inhibit growth of cancer in patients. For instance, US 2017/0037372 Al discloses a composition for preparation of mature dendritic cells from mononuclear cells comprising DC stimulating agents such as TNFa, IL-1ß, IFN?, TLR3 agonist and prostaglandin E2. US’372 further relates to method for in vitro maturation of immature dendritic cells from human mononuclear cells, incubating mononuclear cells with GM-CSF and IL-4 or IL-13 and further incubating with cytokines such as TNFa, IL-1ß, IFN?, TLR3 agonist (poly I:C) and harvesting mature dendritic cells. However, US’372 fails to show the effect of the composition on the expression of CTLA-4.

WO 2016179475 A1 discloses a method for providing an immune response in a subject comprising tumor antigen primed dendritic cell population which is administered in conjunction with Type I interferon (INF), a TLR-7 agonist, a TLR-9 agonist, AIMp1, a TLR-3 agonist, a retinoic acid inducible gene-I (RIG-I)-like receptor ligand or a cytosolic DNA (CDS) receptor ligand. Further, an ex vivo method of culturing antigen specific T-cells in the presence of dendritic cells homogenously loaded with antigen in the presence of poly (I:C) wherein the culturing is done in the presence of CTLA-4 antagonist such as Ipilimumab, Pembrolizumab or Nivolumab. However, WO’475 fails to disclose the combination of poly I:C along with IL-12.

Indian patent Application 2492/MUMNP/2014 discloses a composition comprising cyclic purine dinucleotide (“CDN”) and inactivated tumor cell which expresses and secretes one or more cytokines which stimulate dendritic cell induction, recruitment and/or maturation. Further the inactivated tumor cells are administered with one or more cytokines for DC maturation such as GM-CSF, IL-12, CCL3, CCL20, and CCL21. The composition may further comprise adjuvants, lipids, CTLA-4 pathway antagonists such as Ipilimumab and tremelimumab and TLR agonists such as a TLR-3 agonist e.g. Poly I:C but the prior art fails to suggest the use of poly I:C and IL-12 which downregulates the exogenous expression of CTLA-4.

The above cited prior arts disclose certain methods for producing mature dendritic cells using various DC cell activators, such as IL-12, IL-15, IL-18, IL-21, and interferons (IFNs) and inhibitors of DCs however, the use of immune checkpoint inhibitors results in development of autoimmune reactions because of lack of specificity for the tumor cells. Thus, limited clinical success has been achieved thus far, as many patients experienced severe life-threatening toxic side effects experienced severe life-threatening toxic side effects. While dendritic cells are readily matured ex vivo, their phenotype and fate are rarely evaluable; therefore, strategies to ensure that dendritic cells access lymphoid tissues and retain an immunostimulatory phenotype are required.

OBJECT OF THE INVENTION
An object of the invention is to provide a method for producing a population of mature dendritic cells, wherein the exogenous expression of CTLA-4 is downregulated which may be used to inhibit the growth of cancer cells in vitro and in vivo.

SUMMARY OF THE INVENTION
Accordingly, the present invention provides a method for producing a population of mature dendritic cells with downregulated cytotoxic T-lymphocyte associated protein-4 (CTLA-4), said method comprising the steps of:
i) exposing immature dendritic cells (iDC) to tumor antigen to obtain a population of first dendritic cells;
ii) culturing the population of first dendritic cells in presence of Toll-like Receptor-3 (TLR-3) agonist and interleukin to obtain a population of second dendritic cells; and
iii) culturing the population of second dendritic cells in a first culture media to obtain a population of mature dendritic cells with downregulated cytotoxic T-lymphocyte associated protein-4 (CTLA-4).

Additionally, the present invention provides a kit for differentiation of immature dendritic cells (iDC) into mature dendritic cells with downregulated cytotoxic T-lymphocyte associated protein-4 (CTLA-4), said kit comprising: tumor antigen; Toll-like Receptor-3 (TLR-3) agonist; interleukin; and at least one media to support culturing of the immature dendritic cells (iDC) in presence of the tumor antigen, the Toll-like Receptor-3 (TLR-3) agonist and the interleukin.

BRIEF DESCRIPTION OF FIGURES
Fig. 1 (A), (B), (C), (D), (E) depicts maturation status of monocytes differentiated dendritic cells with different maturation stimuli/surface CTLA-4 expression at different stages of differentiation to mature dendritic cells.
Fig. 2 (A), (B), (C), (D), (E) depicts exogenous CTLA-4 expression at different stages of differentiation to mature dendritic cells/exogenous expression of CTLA-4 with different maturation stimuli.
Fig. 3 depicts expression of exogenous CTLA-4 diminished significantly (in poly I: C/IL-12 subgroup).
Fig. 4 depicts release of IL-12p70 (Plots are the representation of various experiments).
Fig. 5 depicts release of TNF alpha (Plots are the representation of various experiments).
Fig. 6 depicts release of IL-4 (Plots are the representation of various experiments).
Fig. 7 depicts T cell proliferation assay (Plots are the representation of various experiments).

DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the present invention provides a method for producing a population of mature dendritic cells with downregulated cytotoxic T-lymphocyte associated protein-4 (CTLA-4), said method comprising the steps of:
i) exposing immature dendritic cells (iDC) to tumor antigen to obtain a population of first dendritic cells;
ii) culturing the population of first dendritic cells in presence of Toll-like Receptor-3 (TLR-3) agonist and interleukin to obtain a population of second dendritic cells; and
iii) culturing the population of second dendritic cells in a first culture media to obtain a population of mature dendritic cells with downregulated cytotoxic T-lymphocyte associated protein-4 (CTLA-4).

In an embodiment of the invention, the step of exposing immature dendritic cells (iDC) to tumor antigen comprises pulsing immature dendritic cells (iDC) with tumor lysate obtained from tissue having tumor, the tumor lysate being enriched in the tumor antigen.

In another embodiment of the invention, wherein pulsing immature dendritic cells (iDC) with tumor lysate is performed at a tumor lysate concentration of 1 µg-5 mg/ml.

In another embodiment of the invention, the tumor antigen is obtained from brain tissue having tumor, prostate tissue having tumor, breast tissue having tumor, pancreatic tissue having tumor or a renal cell having tumor.

In another embodiment of the invention, wherein the Toll-like Receptor -3 (TLR-3) agonist is at least one of Polyinosinic:polycytidylic acid (poly I:C), and RGC100; preferably, Polyinosinic:polycytidylic acid (poly I:C).

In another embodiment of the invention, wherein a concentration of TLR-3 agonist while culturing the population of first dendritic cells is between 1 µg-5 mg/ml, preferably 5 µg/ml.

In another embodiment of the invention, wherein the interleukin is selected from a group comprising interleukin-2, interleukin-12, and interleukin-21; preferably, interleukin -12.

In another embodiment of the invention, wherein interleukin-12 is a recombinant interleukin-12 or a native interleukin-12; preferably, a recombinant interleukin-12.

In another embodiment of the invention, wherein a concentration of interleukin while culturing the population of first dendritic cells is between 1 ng-100 ng/ml, preferably, 1ng/ml.

In another embodiment of the invention, wherein the immature dendritic cells (iDC) are obtained by a process comprising the steps of: obtaining plasma blood mononuclear cells (PBMCs) and isolating monocytes from PBMCs; and culturing the monocytes in a growth media with at least one cytokine to obtain immature dendritic cells (iDC).

In another embodiment of the invention, wherein the cytokine is selected from the group comprising granulocyte macrophage-colony stimulating factor, interleukin-3, interleukin-4, interleukin-6, interleukin-15, and combinations thereof, preferably a combination of granulocyte macrophage-colony stimulating factor and interleukin-4.

In another embodiment of the invention, wherein each of granulocyte macrophage-colony stimulating factor and interleukin-4 is a native protein.

In another embodiment of the invention, wherein each of granulocyte macrophage-colony stimulating factor and interleukin-4 is a recombinant protein.

In another embodiment of the invention, wherein the growth media comprises 1 to 100 ng/ml of recombinant granulocyte macrophage-colony stimulating factor and 1 to 100 ng/ml of interleukin-4.

More specifically, the present invention pertains to modifying the process of differentiation of monocytes to dendritic cells which may further down regulate the expression of CTLA-4. The present invention provides a method which will downregulate the exogenous expression of CTLA-4, when DC are TH1 polarized by targeting TLR-3 receptor using TLR-3 agonist such as poly I:C and very high concentration of IL-12 which will reduce the exogenous expression of CTLA-4 to the larger extent.

In an embodiment, the present invention is directed towards a method for producing a population of mature dendritic cells comprising the steps of:
i) preparing an antigen enriched tumor lysate from a tumorous tissue;
ii) obtaining plasma blood mononuclear cells (PBMCs) and then isolating monocytes;
iii) culturing the monocytes in a suitable growth media containing at least one cytokine to obtain immature dendritic cells (iDC);
iv) exposing the immature dendritic cells (iDC) with the antigen enriched tumor lysate;
v) culturing the iDC in the presence of TLR-3 agonist such as poly I:C with at least one interleukin; and
vi) further culturing the iDC population to obtain a population of mature dendritic cells.

The immature DCs may be primed with at least one antigen specific to the diseased cell population, in particular antigen enriched tumor lysate. In particular the dendritic cell population may be loaded ex vivo with tumor lysates, modified tumor antigen, tumor RNA, tumor antigen-derived peptides, MHC class I restricted polypeptides or whole tumor cells, more preferably antigen enriched tumor lysate.

The tumor antigen may be derived from brain tumor (e.g., a glioma), a prostate tumor, a breast tumor (e.g., a triple negative breast cancer), a pancreatic tumor (e.g., a pancreatic ductal adenocarcinoma) or a renal cell tumor. The dendritic cells are primed with substantially all the antigens as may present in the tumor tissue. This approach potentially elicits a polyclonal T-cell response to numerous epitopes in multiple tumor antigens, limiting the chances of immunologic escape.

The mononuclear cells may be obtained commercially or freshly harvested and further monocytes may be isolated. The monocytes may be cultured in a suitable growth media for about 5 days at 5% CO2, 37°C and 95% humidity and may be incubated with suitable nutrient medium containing at least one cytokine selected from, but not limited to, the group comprising GM-CSF, IL-3, IL-4, IL-6 and IL-15, more preferably GM-CSF and IL-4. GM-CSF and IL-4 may be used in its native form or as a recombinant protein, preferably GM-CSF and IL-4 and more preferably rhGM-CSF and rhIL-4.

The concentration of rhGM-CSF might be in the range of 1 ng-100 ng/ml, more preferably 50ng/ml. rhIL-4 might be used at concentrations from 1 ng-100 ng/ml, more preferably 10ng/ml.

The culture may be pulsed with tumor lysate together with adjuvants such has at least one TLR-3 agonist selected from, but not limited to Polyinosinic:polycytidylic acid (poly I:C) and RGC100, more preferably poly I:C along with at least one cytokine selected from, but not limited to IL-2, IL-12 and IL-21, preferably IL-12 and more preferably rhIL-12.

The concentration of poly I:C might be in the range of 1 µg-5 mg/ml, more preferably 5ug/ml. rhIL-12 might be used at concentrations from 1 ng-100 ng/ml, more preferably 1ng/ml.

It may be observed that the addition of such adjuvant such as poly I:C and IL-12 may significantly decrease the effects of inhibitory CTLA-4 by downregulating the exogenous expression of CTLA-4 (as depicted in Fig 3).

Additionally, the present invention provides a kit for differentiation of immature dendritic cells (iDC) into mature dendritic cells with downregulated cytotoxic T-lymphocyte associated protein-4 (CTLA-4), said kit comprising: tumor antigen; Toll-like Receptor-3 (TLR-3) agonist; interleukin; and at least one media to support culturing of the immature dendritic cells (iDC) in presence of the tumor antigen, the Toll-like Receptor-3 (TLR-3) agonist and the interleukin.

The various steps are set out herein below:
i) Preparing an antigen enriched tumor lysate from a tumorous tissue;
Fresh surgical tumor is taken to prepare tumor lysate. The tumor is further homogenized to prepare single cell suspension and it is further mixed with immunogenic reagent and then incubated.

ii) Isolating plasma blood mononuclear cells (PBMC) from blood sample and then isolating monocytes;
The mononuclear cells may be obtained commercially or freshly harvested. Alternatively, the mononuclear cells may also be derived from bone narrow, peripheral blood, umbilical cord and similar sources. The peripheral blood collected may comprise mononuclear cells and other cells. Optionally, an anti-coagulant along with culture media may be added to the blood so obtained so as maintain a healthy population of mononuclear cells. The population of cells so obtained may comprise substantial number of lymphocytes.

The population of Peripheral Blood Mononuclear cells collected as a result of apheresis and Leukopheresis undergoes Density gradient separation to obtain a pure Peripheral Blood Mononuclear cells free of RBC contamination. As a result of Density gradient method, pure peripheral blood mononuclear cells are separated substantially free of RBCs or Plasma. The Peripheral Blood Mononuclear cells are cultured on a substrate coated with an activator such as poly L-lysine and cultured in suitable growth media for 2-4 hours at 370C in CO2 incubator. The culture is washed with the culture media to wash away any other cells like granulocytes, and lymphocytes.

iii) Culturing the monocytes in a suitable growth media containing at least one cytokine to obtain immature dendritic cells (iDC);
The cells may be added with AIMV media along with rhGM-CSF (1ng-100ng/ml) and rhIL-4 (1ng-100ng/ml) and autologous plasma (0.1-10%) and incubated at 370C in CO2 incubator and the culture is replenished every alternate day till the 5th Day.

iv) Feeding immature Dendritic cells (iDC) ex vivo with the antigen enriched and modified tumor lysate;
On the sixth day the culture may be pulsed with enriched modified tissue lysate (1ug-1mg/ml) or tumor lysate derived from malignant tumor.

v) Culturing the iDC in the presence of TLR-3 agonist such as Poly I:C and rhIL-12 recombinant protein;
Further to the mixture, adjuvant such as polyI:C (1ug-5mg/ml) and rhIL-12 (1ng-100ng/ml) may be added. The addition of such adjuvant decreases the effects of inhibitory CTLA-4.

vi) Further culturing the iDC population for 2-days to obtain a population of mature activated dendritic cells.
The immature iDC cells are grown so as to permit sufficient uptake of antigen and its expression on cell surface. After the dendritic cells are “pulsed” with activated tissue lysate it may take two days for the immature dendritic cells to become activated dendritic cells and process the antigen for presentation to naive and memory T-cells and once the DC has engulfed and processed the antigen, mature antigen-primed dendritic cell population obtained.
The population of dendritic cells so obtained after approximately 8 days as mature activated Dendritic cell population is then ready for use.

Assessing immune efficacy of mature dendritic cells and inhibition of cancer
The method of the present invention may be used for treating cancer to achieve a therapeutic effect that is greater than the therapeutic effect achieved by administration of conventional dentritic cell compositions. In this respect, the immune efficacy of given mature dendritic cells is typically measured by IFN-? response that is to assess if immunologically the subject has responded or not. Alternatively, the therapeutic effect or therapeutic index can be determined according to the effect on tumor volume, which can be inferred from tumor area, or staging of cancer. For example, stabilization of the tumor after administration of few doses would represent a significant therapeutic effect as it would be indicative of the fact that the administration of the population of dendritic cells prepared according to the invention has arrested further growth or spread of the tumor and same may be eventually controlled or reduced. Alternatively, the therapeutic effect can be measured by observing the change in the one year survival rate, the five-year survival rate, or ten-year survival rate. An increase in therapeutic effect may also mean decreasing the side-effects and improving quality of life. In a further aspect, the dose of population of dendritic cells used in the present invention far less than what is used in conventional therapies.

The dendritic cell composition used in the claimed method may be administered in a predetermined quantity/dose to a subject in need thereof. Thus, the therapeutic method allows an increase in the therapeutic index by reducing the number and/or severity of side-effects while still enjoying the activation of immune system and anticancer benefit of administering a dendritic cell-based composition/population. Preferably 6 dosages may be taken with a gap of two weeks between two dosages. Each dosage may comprise of millions of mature primed dendritic cells obtained from the above mentioned method.

ADVANTAGES
i) The present invention is an easiest practical method which down regulates the exogenous expression of CTLA-4.
ii) The method comprises a plurality of tumor antigens and represents substantially all the antigens present in a cancerous tissue. Such dendritic cells are found to provide better therapeutic effect as compared to conventional compositions which employ a specific antigen and the dendritic cells are primed accordingly.
iii) The mature dendritic cells may be used for inhibiting cancer growth.

EXAMPLES
The following examples are given by the way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

1. CULTURE OF MATURE DENDRITIC CELLS FROM BLOOD MONOCYTES
a) Isolation of PBMCs
i) Aseptically spike one of the access ports in the leukapheresis bag and transfer the leukapheresis product pre-diluted with nutrient medium into a sterile 50 ml tube (Take the sterile tube according to the volume of mobilized leukapheresis product).
ii) Gently mix the bottle of ficoll (Density Gradient Medium). Add 12 ml of histopaque into sterile 50 ml conical tube.
iii) Gently layer 30 ml of the diluted leukapheresis product to each sterile 50 ml conical tube containing Ficoll. Be careful not to disturb the interface between the dendity gradient medium and cell suspension.
iv) Centrifuge the 50 ml conical tubes at 2200 rpm for 20 min at room temperature with no brake.

b) Heat inactivation of Plasma
i) Carefully remove the upper layer (plasma) into a sterile 50 ml tube.
ii) Keep the Plasma for heat inactivation at 56 degree for 30 minutes in Running water Bath.
iii) After Inactivation centrifuge it at 5000 rpm for 30 minutes at 4 degree.
iv) After centrifugation take out the supernatant and filter it with 0.45 micron syringe filter (Low Protein Binding Filter), keep it in another sterile tube with proper Barcode labelling.
v) Carefully harvest the cloudy layer of PBMCs from each tube and transfer to new sterile 50 ml tubes.
vi) Add Nutrient media to each tube to a final volume of 50 ml. Mix gently by inversion.
vii) Centrifuge the cells at 1200 rpm for 10 min at room temperature with brakes.
viii) Remove the supernatants from each tube. Resuspend the cell pellets from each tube and add Nutrient media to a final volume of 50 ml. Mix gently by inversion.
ix) Centrifuge the cells at 1200 rpm for 5 min with brakes.
x) Remove the supernatants from each tube. Resuspend and pool together the cell suspensions into one 50 ml conical tube.
xi) Bring the volume of the pooled PBMCs up to 50 ml with more nutrient media.
xii) Resuspend the cell pellet in 50 ml RPMI.
xiii) Mix gently to ensure uniform cell suspension.

2. Day 1: Differentiation of Dendritic Cells from Monocytes
i) Mix it with pipette so that single cell suspension is maintained, mix thoroughly.
ii) Plate 1 x 10 6 precursor cells/ cm2 in nutrient media in six well culture dish. For six doses of vaccine we use six culture plates (Six well plates). These were not total PBMC, but monocytes. The precursors are plated at 1 x 10 6 precursor cells/ cm2.
iii) Incubate the culture dish for 2 hr at 37 °C in the tissue culture incubator containing 5% CO2 and 95% humidity to allow the monocytes to adhere to the plates.
iv) When the incubation is complete, remove the culture dish from the incubator and wash thrice to remove the non-adherent cells using pre-warmed nutrient media or PBS.
v) After the third wash, add 3ml of nutrient medium with 10% autologous plasma supplemented by GM-CSF (1-100ng/ml) with other constituents and IL4 (1-100ng/ml) with other constituents are growth factor for differentiation.
vi) Incubate the culture plate for 2 days at 37 °C in the tissue culture incubator containing 5% CO2 and 95% humidity.

Day 3: Feeding of Dendritic Cells with complete Medium
We need to replace the complete medium with same volume of nutrient medium and 10% of autologous plasma and growth factors.

Day 5/6: Harvesting of Immature Dendritic Cells
The culture plates needs to be replenished with complete medium (complete Medium: 10ml of Nutrient medium+2 ml of autologous plasma+ growth factors)

Day 6: Maturation and Antigen Loading of Dendritic Cells
i) Add tumor lysate with a concentration (1ug-5mg/ml) per well and incubate it for 4 hours at 37 c and 5% CO2.
ii) After incubation of 4 hours, all the plates are taken out and supplemented with TLR-3 agonist that is Poly I:C (1ug-100ug/ml) and rhIL-12 (1ng-100ng/ml).
iii) Keep the plates for incubation for 24-36 hours in 5% CO2 at 37 degree°C.

Day 8: Harvesting and Cryopreservation of Dendritic Cells
i) Prepare DC Freezing Solution using 70% autologous plasma and 10% DMSO and 20% Nutrient Medium. Centrifuge the DC Freezing Solution at 2,000 × g for 20 min at 4 °C to remove any particulate material. Transfer supernatant to a new labeled 15 ml conical tube. Store at 4 °C until use. When the incubation is finished, harvest and pool all the wells containing DCs pulsed with the tumor antigens into 50 ml conical tubes.
ii) Centrifuge the tubes at 1500 rpm for 12 min with brakes. Remove the supernatants. Resuspend the cells pellet in with nutrient medium and mix thoroughly. Count the number of cells and determine cell viability. Calculate total number of viable cells.
iii) Resuspend the cell pellets in DC Freezing Solution at 4 - 20 x 106 cells/ml and aliquot 1 ml to each 1.8 ml CryoTube vial labelled.
iv) Use controlled rate freezer, to freeze down the aliquots of DC vaccines and finally cryopreserved in vapor phase of Liquid Nitrogen.

c) Protocol for Preparation of Tumor Lysate
i) Fresh surgical tumor needs to be collected in a Transport medium.
ii) Collected tumor in medium needs to be transported in dry ice/ freezing temperature to the central lab.
iii) Surgical tumor needs to be taken out in a Biosafety cabinet and washed properly with PBS/ Normal Saline and weighed on the weighing balance.
iv) Washed tissue is minced into very small pieces aseptically.
v) Minced tissue is homogenized with homogenizer to make a single cell suspension.
vi) Single cell suspension needs to be mixed with the Immunogenic reagent and to be kept for 1 hour at 37 degree centigrade and 5% CO2 in the incubator. After 1 hour of incubation the mixed suspension to be washed thrice with PBS and pellet down the cell suspension.
vii) Cells pellet needs to be mixed with sterile PBS (5-10mlVolume adjusted accordingly) and after that it be freezed /thawed (2 minutes in Liquid Nitrogen and 4 minutes 37 Degree Water Bath) six times in a continuous fashion.
viii) After the freeze thaw suspension of killed tumor cells to be centrifuged at 5000 RPM for 10 minutes.
ix) Gently take out the supernatant and pass it through 0.22 Micron syringe filter (Low Protein Binding Filter).
i) The tumor lysate needs to be checked for endotoxin and quantified by Bradford Methods and then to be stored at -20 degree centigrade till use.

d) Amplification of the Immunogenicity by oxidizing the Tumor Lysate by oxidizing agents.
1. The harvested mature dendritic cells were tested for Immunogenicity based on their ability to proliferate T cell. (Mixed Lymphocyte Reaction Assay).
2. Three types of Dendritic cells were prepared:
a) Immature Dendritic Cells were loaded with tumor lysate (Control).
b) Immature Dendritic Cells were loaded with tumor lysate treated with 3 concentrations of NaOCl (25µMol, 50 µMol, and 100 µMol).
c) Immature Dendritic Cells were loaded with HClO4 (25µMol, 50 µMol, and 100 µMol).
3. The Dendritic Cells so prepared were mature Dendritic Cells and they were incubated with T cell culture to check their ability to proliferate T cells.
4. Tests were performed in 96 well flat bottom plates, as same medium were used that served for T cell culture: RPMI 1640 medium was supplemented with 2 mM L-glutamine, Primocin and 5% heat-inactivated human serum. T cell enriched fractions were obtained as 1 h non adherent fraction of PBMC. T-cells were brought to a concentration of 2x106 cells/ml, DC of all three types were used at 2x105 cells/ml. A ratio of 20:1 of T-cells to DC, whereas triplicates were carried out. T cells with DC not treated with NaOCl and HClO4 served as control. Cells were pulsed for 4 days of incubation at 370C. The assay is performed by the addition of a premixed optimized Dye Solution. Culture wells of a 96-well plate usually containing various concentration test substance. During 4-hour incubation, living cells convert the tetrazolium component of the Dye Solution into a formazan product. The solubilization solution/stop mix is then added to the culture wells to solubilize the formazan product, and the absorbance at 595nm is recorded using a 96-well plate reader. The 595nm absorbance reading is directly proportional to the number of cells normally used in proliferation assays. Although the absorbance maximum for the formazan product is 595nm and pure solutions appear blue, the color at the end of the assay may not be blue and depends on the quantity of formazan present relative to other components (including serum, acidified phenol red and unreduced MTT) in the culture medium.

CLAIMS:
1. A method for producing a population of mature dendritic cells with downregulated cytotoxic T-lymphocyte associated protein-4 (CTLA-4), said method comprising the steps of:
i) exposing immature dendritic cells (iDC) to tumor antigen to obtain a population of first dendritic cells;
ii) culturing the population of first dendritic cells in presence of Toll-like Receptor-3 (TLR-3) agonist and interleukin to obtain a population of second dendritic cells; and
iii) culturing the population of second dendritic cells in a first culture media to obtain a population of mature dendritic cells with downregulated cytotoxic T-lymphocyte associated protein-4 (CTLA-4).

2. The method as claimed in claim 1, wherein the step of exposing immature dendritic cells (iDC) to tumor antigen comprises pulsing immature dendritic cells (iDC) with tumor lysate obtained from tissue having tumor, the tumor lysate being enriched in the tumor antigen.

3. The method as claimed in claim 2, wherein pulsing immature dendritic cells (iDC) with tumor lysate is performed at a tumor lysate concentration of 1 µg-5 mg/ml.

4. The method as claimed in claim 1, wherein the tumor antigen is obtained from brain tissue having tumor, prostate tissue having tumor, breast tissue having tumor, pancreatic tissue having tumor or a renal cell having tumor.

5. The method as claimed in claim 1, wherein the Toll-like Receptor -3 (TLR-3) agonist is at least one of Polyinosinic:polycytidylic acid (poly I:C), and RGC100; preferably, Polyinosinic:polycytidylic acid (poly I:C).

6. The method as claimed in claim 1, wherein a concentration of TLR-3 agonist while culturing the population of first dendritic cells is between 1 µg-5 mg/ml, preferably 5 µg/ml.

7. The method as claimed in claim 1, wherein the interleukin is selected from a group comprising interleukin-2, interleukin-12, and interleukin-21; preferably, interleukin -12.

8. The method as claimed in claim 7, wherein interleukin-12 is a recombinant interleukin-12 or a native interleukin-12; preferably, a recombinant interleukin-12.

9. The method as claimed in claim 1, wherein a concentration of interleukin while culturing the population of first dendritic cells is between 1 ng-100 ng/ml, preferably, 1ng/ml.

10. The method as claimed in claim 1, wherein the immature dendritic cells (iDC) are obtained by a process comprising the steps of:
obtaining plasma blood mononuclear cells (PBMCs) and isolating monocytes from PBMCs; and
culturing the monocytes in a growth media with at least one cytokine to obtain immature dendritic cells (iDC).

11. The method as claimed in claim 10, wherein the cytokine is selected from the group comprising granulocyte macrophage-colony stimulating factor, interleukin-3, interleukin-4, interleukin-6, interleukin-15, and combinations thereof, preferably a combination of granulocyte macrophage-colony stimulating factor and interleukin-4.

12. The method as claimed in claim 11, wherein each of granulocyte macrophage-colony stimulating factor and interleukin-4 is a native protein.

13. The method as claimed in claim 11, wherein each of granulocyte macrophage-colony stimulating factor and interleukin-4 is a recombinant protein.

14. The method as claimed in claim 11, wherein the growth media comprises 1 to 100 ng/ml of recombinant granulocyte macrophage-colony stimulating factor and 1 to 100 ng/ml of interleukin-4.

15. A kit for differentiation of immature dendritic cells (iDC) into mature dendritic cells with downregulated cytotoxic T-lymphocyte associated protein-4 (CTLA-4), said kit comprising:
i) tumor antigen;
ii) Toll-like Receptor-3 (TLR-3) agonist;
iii) interleukin; and
iv) at least one media to support culturing of the immature dendritic cells (iDC) in presence of the tumor antigen, the Toll-like Receptor-3 (TLR-3) agonist and the interleukin.