| Cryopreservation of haptenized tumor cells -> Monitor Keywords |
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  Cryopreservation of haptenized tumor cellsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Maintaining Blood Or Sperm In A Physiologically Active State Or Compositions Thereof Or Therefor Or Methods Of In Vitro Blood Cell Separation Or TreatmentThe Patent Description & Claims data below is from USPTO Patent Application 20050233301. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The invention relates to compositions and methods for cryo-preservation of haptenized tumor cells. The tumor cell compositions are particularly suitable for an immunotherapeutic vaccine. BACKGROUND OF THE INVENTION [0002] In blood transfusion, bone marrow transplantation, immuno-therapeutic vaccine preparation, or other cell preparations ex vivo, one of the principal problems encountered is that of the preservation of cells. It is critical to be able to preserve cells, under good conditions of viability, for time periods compatible with clinical production and storage, and to make it possible to analyze cell preparations. The most commonly used method of long-term preservation of cells is to freeze and subsequently thaw them. However, during the freezing of cells, lysis of cells and loss of cell integrity may occur. This is often observed by the decrease in intact tumor cells and the concomitant increase in the amount of non-intact tumor cells in a sample of tumor cells. This problem can be even more complex when the cells have been modified or altered prior to preservation, and when the cells are obtained by proteolytic digestion of a tissue or tumor specimen. Preservation of cells under less extreme conditions, for example on ice (about 0.degree. C.), refrigerated (about 4.degree. C.), or at room temperature, prior to use, is also difficult as these storage conditions are effective only for a period of hours. Immunotherapy [0003] The preservation of cells, especially their immunogenicity, is important is in immunotherapy of cancer using tumor cells. The aim of the immunotherapy is to evoke an immune response to the tumor, or to vaccinate against new tumors, by administering tumor cells or tumor cell extracts to the cancer patient. The tumor cells in the composition should contain antigens that are also present in the tumor to be treated, so that the immune response elicited against the antigens in the composition is effective against the tumor. Generally, the cells are recovered from tumors, suspended in a cryopreservation medium and frozen until used for the vaccine preparation. When needed, the cells are thawed, and then stored at temperatures ranging from about 0.degree. C. (on ice) to room temperature until administration. [0004] Immunotherapy regimens using unmodified intact tumor cells prepared from tumors taken from the patient, i.e., autologous tumor cells, have been extensively described in the literature (see, e.g., Berd et al., Cancer Research 1986;46:2572-2577; Hoover et al., Cancer 1985;55:1236-1243; and U.S. Pat. No. 5,484,596 to Hanna et al.). Alternative vaccine compositions based on disrupted cells have also been suggested including, e.g., tumor membranes (see, e.g., Levin et al., In: Human Tumors in Short Term Culture: Techniques and Clinical Applications, P. P. Dendy, Ed., 1976, Academic Press, London, pp. 277-280) or tumor peptides extracted from tumors (see, e.g., U.S. Pat. No. 5,550,214 to Eberlein, and U.S. Pat. No. 5,487,556 to Elliot et al.). The tumor cells can also be modified in some manner to alter or increase the immune response (see, e.g., Hostetler et al., Cancer Research 1989;49:1207-1213; and Muller et al., Anticancer Research 1991; 11: 925-930). Haptenized Tumor Cell Vaccines [0005] One particular form of tumor cell modification that has a pronounced effect on immunotherapy is coupling of a hapten to the tumor cells. An autologous whole-cell vaccine modified with the hapten dinitrophenyl (DNP) has been shown to produce inflammatory responses in metastatic sites of melanoma patients. Adjuvant therapy with DNP-modified vaccine produces markedly higher post-surgical survival rates than those reported after surgery alone. U.S. Pat. No. 5,290,551 to Berd discloses and claims vaccine compositions comprising haptenized melanoma cells. Melanoma patients who were treated with these cells developed a strong immune response. This response can be detected in a delayed-type hypersensitivity (DTH) response to haptenized and non-haptenized tumor cells. More importantly, the immune response resulted in increased survival rates of melanoma patients. [0006] Haptenized tumor cell vaccines have also been described for other types of cancers, including lung cancer, breast cancer, colon cancer, pancreatic cancer, ovarian cancer, and leukemia (see International Patent Publication Nos. WO 96/40173 and WO 00/09140, and U.S. Pat. No. 6,333,028, and the associated techniques and treatment regimens optimized (see International Patent Publication Nos. WO 00/38710, WO 00/31542, WO 99/56773, WO 99/52546, and WO 98/14206). For example, it has been shown that the addition of human serum albumin (HSA) increases the stability of haptenized tumor cell preparations (see WO 00/29554 and U.S. Pat. No. 6,248,585). [0007] It has also been found that haptenization of tumor cell extracts such as plasma membranes and peptides can yield potent immunotherapy vaccines (see International Patent Publication Nos. WO 96/40173 and WO 99/40925, both by Berd et al.). [0008] For haptenized vaccines, the search for storage conditions that preserve the stability of the haptenized cells or extracts also have to take into account that some haptenization reactions may alter or affect the cell viability or integrity. Previous work has suggested that if no measures are taken to increase the stability of haptenized melanoma vaccine preparations, they might have a cell integrity duration of less than four hours after hapten modification. Also, some haptens or haptenization procedures render the cells more fragile than others. For example, while preparations of DNP-modified cells can be stable for at least 18 hours when stored at 4.degree. C., some procedures for sulfanilic acid (SA) conjugation render the cells more fragile, and the SA-modified cells may in some cases only be stable for less than 2 hours at 4.degree. C. [0009] However, whether utilizing modified or unmodified tumor cells, in order to elicit a successful immune response against the tumors of the patient after administration, the amount and immunogenicity of the antigens in the tumor cell composition should be retained as much as possible during preparation and storage of the composition. The tumor antigens should also remain associated with the cells. [0010] Thus, there is a need in the art for an effective treatment for cells to be stored and preserved prior to delivery as an immunotherapy vaccine. There is also a need for a treatment that preserves the integrity, antigen-content and immunogenicity of such cells for vaccines prior to administration, and methods for designing tumor cell preparations and formulations to obtain optimal immune response. The present invention advantageously addresses these and other needs in the art. SUMMARY OF THE INVENTION [0011] The present invention is based, in part, on a cryopreservation method found to preserve a haptenized tumor cell vaccine in terms of the number of intact tumor cells; the density of various tumor-cell associated antigens, including haptens; and the in vivo immunogenicity and immunotherapeutic potential of the tumor cell vaccine. The present invention therefore advantageously provides a method of treating tumor cells or tumor cell extracts for their preservation and/or storage prior to use in anti-tumor vaccines. [0012] Accordingly, the invention provides a method of preserving haptenized tumor cells, which method comprises: (i) contacting the haptenized tumor cells with a freezing medium, wherein the freezing medium comprises sucrose, human serum albumin and an isotonic buffered solution; and (ii) freezing the tumor cells, whereby the immunogenicity of the tumor cells is preserved. In one embodiment, the isotonic buffered saline solution is Hank's buffered solution. For example, the freezing medium may comprise an 8% sucrose, 10% human serum albumin-supplemented Hank's buffered solution. The storage temperature can be from about -20.degree. C. to about -196.degree. C., preferably -80.degree. C. to about -196.degree. C. In one embodiment, at least 70%, preferably at least 90%, of the level of at least one tumor cell-associated antigen (TCAA) is preserved after about 3 months storage at a temperature, e.g., of -80.degree. C. or less. In another embodiment, at least 50%, preferably at least 70%, of the haptenized tumor cells are preserved intact after about 3 months storage, e.g., at a temperature -80.degree. C. or less. The tumor cells may, for example, be melanoma cells, ovarian cancer cells, colorectal cancer cells, small cell lung cancer cells, kidney cancer cells, breast cancer cells, or leukemia cells. In a particular embodiment, the tumor cells are melanoma cells. The tumor cells are haptenized with at least one hapten, which can be selected from, e.g., DNP, TNP, and sulfanilic acid. In a particular embodiment, the hapten is DNP. In another particular embodiment, the tumor cells are haptenized with at least two different haptens. [0013] The invention also provides for a method of storage for haptenized tumor cells for use in a vaccine, which method comprises storing a haptenized tumor cells and freezing medium composition at a temperature below the freezing temperature for at least 3 months. In one embodiment, the temperature is from about -80.degree. C. to -196.degree. C. The tumor cells may be haptenized with, for example, at least one hapten selected from DNA and sulfanilic acid. [0014] The invention also provides for a composition comprising haptenized tumor cells for use in a vaccine and freezing medium, wherein the freezing medium comprises sucrose, human serum albumin and an isotonic buffered saline solution. Preferably, the freezing medium comprises 8% sucrose, 10% human serum albumin and the isotonic buffered saline solution is Hank's buffered solution. The tumor cells can be, for example, melanoma cells, ovarian cancer cells, colorectal cancer cells, small cell lung cancer cells, kidney cancer cells, breast cancer cells, or leukemia cells. In a particular embodiment, the tumor cells are melanoma cells. The tumor cells are haptenized with at least one hapten, which can be selected from, e.g., DNP, TNP, and sulfanilic acid. In a particular embodiment, the hapten is DNP. In another particular embodiment, the tumor cells are haptenized with at least two different haptens. [0015] The Drawings, Detailed Description, and Examples will further explain the present invention. BRIEF DESCRIPTION OF THE DRAWINGS [0016] FIG. 1 shows the relapse-free survival rate of mice after surgical removal of primary 410.4 mammary adenocarcinoma tumors and treatment with cryogenically preserved irradiated DNP-modified tumor cells as compared to control mice treated with saline. [0017] FIG. 2 shows the relapse-free survival rate of mice treated with cryogenically preserved haptenized tumor cell vaccine as compared to mice treated with fresh haptenized tumor cell vaccine. [0018] FIG. 3 shows the relapse-free survival rate of mice treated with cryogenically preserved haptenized tumor cell vaccine as compared to mice treated with cryogenically preserved non-haptenized tumor cell vaccine. Continue reading... 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