Cryopreservation of haptenized tumor cells

Abstract

A method of preserving haptenized tumor cells is described. The method employs a freezing medium containing an effective amount of sucrose and human serum albumin in an isotonic buffered saline solution. Cryogenically preserving haptenized cells in such a medium has been found to maintain the integrity of the tumor cells during storage. The haptenized tumor cells also retain cell-associated antigens and haptens, and are as immunogenic, i.e., capable of inducing immunotherapeutic response, as fresh vaccine in a mouse model of metastatic disease. In a specific embodiment, haptenized cells are exposed to a solution of 8% sucrose, 10% human serum albumin in Hank's buffered solution, and then frozen to −80° C. overnight and then stored in a liquid nitrogen freezer. Methods of storing haptenized tumor cells and compositions are also provided.

1 . 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.    
   
   
       2 . The method of  claim 1 , wherein the isotonic buffered saline solution is Hank's buffered solution.  
   
   
       3 . The method of  claim 1 , wherein the freezing medium comprises 8% sucrose, 10% human serum albumin and the isotonic buffered saline solution is Hank's buffered solution.  
   
   
       4 . The method of  claim 1 , wherein the temperature is from about −80° C. to about −196° C.  
   
   
       5 . The method of  claim 1 , wherein at least 70% of the level of at least one tumor cell-associated antigen (TCAA) is preserved after about 3 months storage at a temperature of at least −80° C.  
   
   
       6 . The method of  claim 5 , wherein at least 90% of the TCAA is preserved.  
   
   
       7 . The method of  claim 1 , wherein at least 50% of the haptenized tumor cells are preserved intact after about 3 months storage at a temperature of at least −80° C.  
   
   
       8 . The method of  claim 7 , wherein at least 70% of the haptenized tumor cells are preserved intact.  
   
   
       9 . The method of  claim 1 , wherein the tumor cells are selected from the group consisting melanoma cells, ovarian cancer cells, colorectal cancer cells, small cell lung cancer cells, kidney cancer cells, breast cancer cells, and leukemia cells.  
   
   
       10 . The method of  claim 9 , wherein the tumor cells are melanoma cells.  
   
   
       11 . The method of  claim 1 , wherein the tumor cells are haptenized with at least one hapten selected from the group consisting of DNP, TNP, and sulfanilic acid.  
   
   
       12 . The method of  claim 9 , wherein the tumor cells are haptenized with DNP.  
   
   
       13 . The method of  claim 11 , wherein the tumor cells are haptenized with at least two different haptens.  
   
   
       14 . A method of storage for haptenized tumor cells for use in a vaccine, which method comprises storing a haptenized tumor cells and a freezing medium composition at a temperature below the freezing temperature for at least 3 months.  
   
   
       15 . The method of  claim 14 , wherein the temperature is from about −80° C. to −196° C.  
   
   
       16 . The method of  claim 12 , wherein the tumor cells are haptenized with at least one hapten selected from DNA and sulfanilic acid.  
   
   
       17 . A composition comprising haptenized tumor cells for use in a vaccine and a freezing medium, wherein the freezing medium comprises sucrose, human serum albumin and an isotonic buffered saline solution.  
   
   
       18 . The composition of  claim 15 , wherein the freezing medium comprises 8% sucrose, 10% human serum albumin and the isotonic buffered saline solution is Hank's buffered solution.  
   
   
       19 . The composition of  claim 15 , wherein the tumor cells are selected from the group consisting melanoma cells, ovarian cancer cells, colorectal cancer cells, small cell lung cancer cells, kidney cancer cells, breast cancer cells, and leukemia cells.  
   
   
       20 . The composition of  claim 17 , wherein the tumor cells are melanoma cells.  
   
   
       21 . The composition of  claim 15 , wherein the tumor cells are haptenized with at least one hapten selected from the group consisting of DNP, TNP, and sulfanilic acid.