Allogenic mesendritic vector for ovarian cancer

Abstract

A method of preparing an allogenic dendritic stem cell vaccine for treating ovarian cancers is disclosed. The method comprises, (a) collecting blood sample from an allogenic donor in a sterile collection bag, (b) separating viable mesenchymal stem cells inoculums by centrifuging the blood sample, suspending said mesenchymal stem cells inoculum in proliferation medium, plating the cells in a flask and incubated, and collecting cells adherent to the flask and further harvesting and expanding said adherent cells, (c) preparing myeloid dendritic cells by isolating, harvesting and culturing said blood sample source using myeloid dendritic isolation kit, (d) mixing the adherent cells obtained. Finally, (e) the composition of the adherent cells obtained in step (d) to the myeloid dendritic cells obtained in step (c) at a ratio of 90:10 and printing the cellular vectors with patient tumor derived heterogeneous progenitors.

Claims

1. A method of preparation of an allogenic dendritic stem cell vaccine for treating ovarian cancers including allogenic blood source derived mesenchymal stem cells and antigen presenting myeloid dendritic cells, comprising the steps of: a. collecting blood sample from an allogenic donor in a sterile collection bag; b. separating viable, mesenchymal stem cells inoculums by centrifuging 50 ml aliquots of the said blood sample using percoll gradient separation method for buffy coat containing mononuclear cells for separation; suspending 3 ml of the said mesenchymal stem cells inoculums in proliferation medium, plating enumerated round cells in a T25 flask at a density of: 5?10.sup.6 cells/ml and incubating at 37? C. with 5% CO.sub.2 for 24 hours, collecting cells adherent to the flask and further culturing and expanding the said adherent cells with a progenitor selection medium being replenished every alternate day at passage P0; trypsinizing the said adherent cells positive for mesenchymal stem cell markers to subsequent passages of expansion thrice to obtain cells at passages P1, P2 and P3; harvesting and expanding the said adherent cells obtained from the passages P0 and P3 of expansion; c. preparing myeloid dendritic cells by isolating, harvesting and culturing the said blood sample using myeloid dendritic isolation kit; d. mixing the adherent cells obtained at passage P0 and P3 of expansion at a ratio of 50:50; and e. a composition of the adherent cells obtained in step (d) to the myeloid dendritic cells obtained in step (c) at a ratio of 90:10 and priming the cellular vectors (d+e) with patient tumor derived heterogeneous progenitors.

2. The method of claim 1 wherein the said blood sample is collected from a donated umbilical cord from a full-term delivery case with an informed prior consent.

3. A composition of an allogenic dendritic stem cell vaccine for treating ovarian cancers comprising allogenic blood source derived mesenchymal stem cells and antigen presenting myeloid dendritic cells at a ratio of 90:10, together primed with patient tumor derived heterogeneous progenitors, and a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) For a more complete understanding of example embodiments of the present technology, reference is now made to the following descriptions taken in connection with the accompanying drawings in which:

(2) FIG. 1 shows a flowchart illustrating the method of preparation of the vector composition;

(3) FIG. 2 shows the collected cord blood stored in a sterile collection bag;

(4) FIG. 3 shows the cultured mesenchymal stem cells at Passage 1 (P1) and Passage 3 (P3);

(5) FIG. 4 shows a table illustrating the comparative expressions of surface markers on mesenchymal stem cells at Passage 0 (P0) and Passage (P3) harvested and expanded from the cord blood source (Thali.sup.RImage based cytometer data);

(6) FIG. 5 shows cultured mesenchymal stem cells at P3 showing cytoplasmic staining of TIMP-1;

(7) FIG. 6 shows the results of P0 mesenchymal stem cells trans-differentiated in-vitro wherein 6A shows the phase contrast mesenchymal stem cells micrograph (10?); 6B shows Von Kossa stained calcium deposits in mesenchymal stem cells differentiated to osteocytes culture; 6C shows Oil-O-Red stained adipocytes; and 6D shows Alcian blue stained chondrocytic cellular cluster;

(8) FIG. 7 shows the micrograph (10?) of MACS segregated and seeded dendritic cells from cord blood; and

(9) FIG. 8A shows the micrograph (10?) of the mixture of mesenchymal stem cells and dendritic cells at 1:1 ratio; FIG. 8B shows 98% viability of the same mixture stained with Propidium Iodide; and FIG. 8C is a representative photograph of 22% of cells testing positive for cytoplasmic stained green color with FITC conjugated secondary antibody against TIMP-1 antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(10) In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present technology. It will be apparent, however, to the one skilled in the art that the present technology can be practiced without these specific details.

(11) Reference in this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present technology. The appearance of the phrase in one embodiment in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Moreover, various features are described which may be exhibited by some embodiments and not by others. Similarly, various requirements are described which may be required for some embodiments but not other embodiments.

(12) Moreover, although the following description contains many specifics for the purposes of illustration, anyone skilled in the art will appreciate that many variations and/or alterations to said details are within the scope of the present technology. Similarly, although many of the features of the present technology are described in terms of each other, or in conjunction with each other, one skilled in the art will appreciate that many of these features can be provided independently of other features. Accordingly, this description of the present technology is set forth without any loss of generality to, and without imposing limitations upon, the present technology.

(13) The present invention discloses a new approach of allogenic dendritic-stem cell vaccine platform to treat all kinds of ovarian cancers. The treatment technology proposed is a combination of adult immune privileged mesenchymal stem cells and antigen presenting dendritic cells especially myeloid dendritic cells as the cellular vectors primed to targeted killing of tumour cells. The unique source of cellular derivatives i.e. human umbilical cord blood is one of it's kind and is shown to reside naive and unmanipulated cellular population with therapeutic values attributed.

(14) The abundant allogenic donated cord blood source is used to harvest both dendritic special type and mesenchymal stem cells equilibrated package primed with patient's tumour antigens for a targeted treatment regime. The individual roles of dendritic cells and mesenchymal stem cells in immunomodulation is exploited in developing this new combinatorial platform of cellular vectors for targeted killing of tumour cells in the patient's body. The source proposed here in developing the technology is a biological discard and is available in profusion for proposed clinical application. The main advantage of this composition to treat ovarian cancers will not subject the suffering patients for any longer waiting for dendritic cells to be cultured as in for autologous preparations and also for a culture independent yield used as cellular formulation for clinical applications.

(15) The process of the immune privileged Mesendritic therapeutic vector and the clinical application modality are proprietary in nature of the technology proposed.

(16) The present invention describes a novel composition that can used as vaccines for treating ovarian cancer comprising mesenchymal stem cells and antigen presenting myeloid dendritic cells obtained from an allogenic source of cord blood primed with antigens obtained from autologous tumour cells and a method of preparing vaccines that includes allogenic mesenchymal stem cells and dendritic cells primed with antigens obtained from the autologous tumour cells as referred in FIG. 1.

(17) In the present invention, both the mesenchymal stem cells and antigen presenting dendritic cells are isolated from the allogenic blood source. In one of the embodiment of the invention, the Umbilical Cord Blood samples are collected from full-term delivery cases with previous consent from the mothers according to the Institute's ethical and scientific committee guidelines. FIG. 2 shows the collected cord blood stored in a sterile collection bag.

(18) Preparation of Mesenchymal Stem Cells:

(19) 50 ml aliquots of the collected cord blood from the bag is aspirated out in sterile 50 ml syringes to centrifuge using percoll gradient separation method for buffy coat containing mononuclear cells (MNCs) separation. The 3 ml of buffy coat obtained after centrifugation was suspended in proliferation medium consisting of DMEM-F12 (Gibo, Life Technologies, Carlsbad, Calif., USA) with 20% FBS (GIBCO, USA), 20 ng/ml fibroblast growth factor (FGF)-2 (Chemicon, Millipore, Billerica, Mass., USA), 1% penicillin/streptomycin (Gibco) and was plated in T25 flasks at a density of 5?106 cells/ml.

(20) After 24 hours of incubation at 37? C. with 5% CO.sub.2, the non-adherent cells were washed off and the cells that were attached to the flask were cultured further with medium being replenished every alternate day. The adherent fibroblastic cell type once confluent in the flask (Passage 0, P0) is trypsinized to next passages for expansion of the cells, thrice (Passage 3, P3). FIG. 3 shows the cultured mesenchymal stem cells at Passage 1 (P1) and Passage 3 (P3). The adherent cells obtained at P0 and P3 harvested and expanded from the cord blood source are analyzed for the expressions of surface markers namely CD34, CD90, CD105, HLA-ABC with image cell cytometry (Thali.sup.R, Invitrogen, USA).

(21) FIG. 4 shows a table illustrating the comparative expressions of surface markers on mesenchymal stem cells at P0 and P3 harvested and expanded from the cord blood source (Thali.sup.RImage based cytometer data). With reference to the FIG. 4, mesenchymal stem cells at P0 and P3 are found to show variation in the surface marker expression percentages although no significance is attributed to any factor. Therefore, P0 and P3 mesenchymal stem cells are two different cell populations in terms of the composition expressing varied surface markers and the cell cycle state.

(22) Mesenchymal stem cells at P0 and P3 (70-80% confluent as shown in FIG. 3) are stained with anti-TIMP-1 antibody (Abcam; 1:200); FITC conjugated secondary antibody. Immunofluorescence of the cells as observed, shows more than 15% of the number of cells test positive for TIMP-1 expression at P0 compared to P3 cells as shown in FIG. 5 which shows cultured mesenchymal stem cells at P3 showing cytoplasmic staining of TIMP-1. The positively stained green cells are counted per focus of 100 cells and an average of five foci is compared between P0 and P3 cultures.

(23) Trans-differentiation of the P0 and P3 mesenchymal stem cells in-vitro showed that both the cultures are equally able to become osteocytes, chondrocytes and adipocytes with positive staining for vonkossa, alcain blue and oil-o-red chemicals. FIG. 6 shows the results of P0 mesenchymal stem cells trans-differentiated in-vitro wherein 6A shows the phase contrast mesenchymal stem cellsmicrograph (10?); 6B shows Von Kossa stained calcium deposits in the mesenchymal stem cells differentiated to osteocytes culture; 6C shows Oil-O-Red stained adipocytes; and 6D shows Alcian blue stained chondrocytic cellular cultures.

(24) Preparation of Antigen Presenting Dendritic Cells:

(25) Dendritic Cells suitable for administration to subjects can be isolated or obtained from the allogenic cord blood source by culturing and harvesting using standard techniques known in the prior art. 2?10.sup.9 cells (adjusted with Normal Saline, 10 ml), of Cord blood collected in the bag is prepared for mDCs isolation using myeloid dendritic cell isolation kit (MACS Miltenyi Biotech, Germany). FIG. 7 shows the micrograph (10?) of MACS segregated and seeded dendritic cells from cord blood.

(26) Preparation of Cell Based Vector:

(27) Mesenchymal stem cells at P0 and P3 are mixed in different pre-determined ratios with the myeloid dendritic cells (cell density ratios). Table 1 shows the different compositions of the vector cell base that can be prepared and named accordingly.

(28) TABLE-US-00001 TABLE 1 mDCs Tube Code MSCs ratio ratio given 1 (P0 + P1) (50:50) 1 MD1 2 (P0 + P1) (50:50) 1 MD2 3 (P0 + P1) (50:50) 1 MD3 4 (P0 + P1) (50:50) 1 MD4

Example 1

(29) MD1 mixture is enumerated under the microscope and stained with Propidium Iodide (PI) to assess the viability. More than 98% of the cell mixture is recorded to be viable with only 2% of the cells stained red with PI. The cell mixture (1 ml) is seeded in growth medium containing DMEM-F12 and 10% FBS for overnight in a coated tissue culture plate. After 16 hours of sedimentation of the cells in the plate, the cells are fixed in paraformaldehyde (4%) and stained with anti-human TIMP-1 antibody. The Immunofluorescence of the cells showed 20-22% more cells test positive for cytoplasmic stained green color with FITC conjugated secondary antibody. FIG. 8A shows the photograph of the mesenchymal stem cells and dendritic cells mixture at 1:1 ratio; FIG. 8B shows 98% viability of the same mixture stained with Propidium Iodide; and FIG. 8C shows the photograph of 22% of cells, testing positive for cytoplasmic stained green color with FITC conjugated secondary antibody against TIMP-1 primary antibody.

(30) Preparation of Vector and its Composition:

(31) The biopsy (1-2 inches) of the tumor tissue obtained is macerated in a sterile container and the cell suspension in cell proliferation medium. 50-100 cells of the biopsy counted under the microscope is mixed with the vector cell based ratios as shown in Table 2.

(32) TABLE-US-00002 TABLE 2 Tumor cell mDCs number MSCs ratio ratio (autologous) 1 (P0 + P1) (50:50) 1 50-100 2 (P0 + P1) (50:50) 1 50-100 3 (P0 + P1) (50:50) 1 50-100 4 (P0 + P1) (50:50) 1 50-100

Example 2

(33) Tumor cell line ATCC IGROV1 cultured in RPMI 1640 supplemented with 10% Fetal Bovine Serum is sub cultured for 7 days. Cells are trypsinized and washed with the growth medium. Upto 200 cells counted in the growth medium is mixed with MD1 tube cells at room temperature. The stability of the vector prepared is checked for every one hour till 10 hrs of incubation at 37? C. with 5% CO.sub.2 by evaluating the apoptosis. Till 10.sup.th hour of the incubation, there was no abnormal apoptosis observed by Image cytometer using annexin V marker in the preparation establishing the stability of the mixture of cells in the presence of each other presenting as cell based vector candidacy for allogenic infusion targeting the solid tumor tissue.

REFERENCES

(34) 1. Yap T A, Carden C P, Kaye S B. Beyond chemotherapy: targeted therapies in ovarian cancer. Nature Rev Cancer 2009; 9:167-81. [PubMed: 19238149] 2. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin 2008; 58:71-96. [PubMed: 18287387] 3. Santin A D, Hermonat P L, Ravaggi A, et al. Induction of tumour-specific HLA class I-restricted CD8+ cytotoxic T lymphocytes by ovarian tumour antigen-pulsed autologous dendritic cells in patients with advanced ovarian cancer. Am J Obstet Gynecol 2000; 183:601-9. [PubMed: 10992180] 4. Santin A D, Bellone S, Ravaggi A, et al. Induction of ovarian tumour-specific CD8+ cytotoxic T lymphocytes by acid-eluted peptide-pulsed autologous dendritic cells. Obstet Gynecol 2000; 96:422-30. [PubMed: 10960637] 5. Zhao X, Wei Y Q, Peng Z L. Induction of T cell responses against autologous ovarian tumours with whole tumour cell lysate-pulsed dendritic cells. Immunol Invest 2001; 30:33-45. [PubMed: 11419910] 6. Gong J, Nikrui N, Chen D, et al. Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce anti-tumour immunity. J Immunol 2000; 165:1705-11. [PubMed: 10903782] 7. Chiang C L L, Ledermann J A, Rad A N, et al. Hypochlorous acid enhances immunogenicity and uptake of allogeneic ovarian tumour cells by dendritic cells to cross-prime tumour-specific T cells. Cancer Immunol Immunother 2006; 55: 1384-95. [PubMed: 16463039] 8. Hernando J J, Park T W, Kubler K, et al. Vaccination with autologous tumour antigen-pulsed dendritic cells in advanced gynaecological malignancies: clinical and immunological evaluation of a phase I trial. Cancer Immunol Immunother 2002; 51:45-52. [PubMed: 11845259] 9. Cannon M J, Santin A D, O'Brien T J. Immunological treatment of ovarian cancer. Curr Opin Obstet Gynecol 2004; 16:87-92. [PubMed: 15128013] 10. Aggarwal, S. & Pittenger, M. F. (2005). Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, Vol. 105, pp 1815-1822, ISSN 0006-4971, doi: 10.1182/blood-2004-04-1559 11. Barleon, B. et al. (1996). Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood, Vol. 87, pp 3336-3343, ISSN 0006-4971 12. Bian, Z-Y. et al. (2010). Human mesenchymal stem cells promote growth of osteosarcoam: Involvement of interleukin-6 in the interaction between human mesenchymal stem cells and Saos-2. Cancer Sci., Vol. 101, pp 2554-2560, doi: 10.1111/j.1349-7006.2010.01731.x 13. Bisping, G. et al. (2009). Bortezomib, dexamethasone, and fibroblast growth factor receptor 3-specific tyrosine kinase inhibitor in t(4;14) myeloma. Clin. Cancer Res., Vol. 15, pp 520-531, doi: 10.1158/1078-0432.CCR-08-1612. 14. www.clinicaltrials.gov