Anticancer vaccine and diagnostic methods and reagents

Abstract

The invention provides a method of priming T cells against tumor antigens comprising by obtaining naïve CD4 + or CD8 + T cells from at least one healthy individual, obtaining at least one protein or peptide from at least one cancerous cell; obtaining antigen presenting cells (APCs), culturing the APCs with the at least one protein or peptide, and adding the T cells to the culture of the APCs. The primed T cells can then be employed to identify the antigens or therapeutically as prophylaxis or treatment for cancers. The invention also provides cyclin molecules, and fragments derived from cyclin molecules, as tumor antigens. The invention provides a method for diagnosing a malignant or pre-malignant condition within a patient. The invention also provides a method for vaccinating a patient against malignancies comprising introducing a protein or peptide consisting essentially of all or an immunogenic fragment of a cyclin protein into the patient.

What is claimed is:  
     
         1 . A method for diagnosing a malignant or pre-malignant condition within a patient comprising assaying for the expression of a cyclin molecule within the patient, whereby deregulated cyclin expression supports a diagnosis of a malignant or pre-malignant condition.  
     
     
         2 . The method of  claim 1 , wherein the condition is diagnosed within a biopsy comprising tissue excised from the patient.  
     
     
         3 . The method of  claim 1 , wherein the condition is lung cancer or precancer, breast cancer or precancer, pancreatic cancer or precancer, or colon cancer or precancer.  
     
     
         4 . The method of  claim 1 , wherein the condition is a malignancy or premalignancy associated with altered or absent p53 production.  
     
     
         5 . The method of  claim 1 , wherein the cyclin molecule is cyclin B1.  
     
     
         6 . A method for diagnosing a malignant or pre-malignant condition within a patient comprising assaying for immunoreactivity to a cyclin molecule within the patient, whereby the presence of immunoreactivity supports a diagnosis of a malignant or pre-malignant condition.  
     
     
         7 . The method of  claim 6 , where the immunoreactivity is humoral immunoreactivity.  
     
     
         8 . The method of  claim 6 , where the immunoreactivity is cellular immunoreactivity.  
     
     
         9 . The method of  claim 6 , comprising assaying for the serum titer of antibodies specific for a cyclin molecule within the patient, whereby elevated antibody titer expression supports a diagnosis of a malignant or pre-malignant condition.  
     
     
         10 . The method of  claim 9 , wherein the assay is accomplished by ELISA.  
     
     
         11 . The method of  claim 6 , wherein the condition is lung cancer or precancer, breast cancer or precancer, pancreatic cancer or precancer, or colon cancer or precancer.  
     
     
         12 . The method of  claim 6 , wherein the condition is a malignancy or premalignancy associated with altered or absent p53 production.  
     
     
         13 . The method of  claim 6 , wherein the cyclin molecule is cyclin B1.  
     
     
         14 . A method for vaccinating a patient against malignancies comprising introducing a peptide consisting essentially of all or an immunogenic fragment of a cyclin protein into a patient under conditions sufficient for the patient to develop an immune response to the cyclin protein.  
     
     
         15 . The method of  claim 14 , wherein the peptide consists essentially of all or an immunogenic fragment of a cyclin A, D1, B1, or E protein.  
     
     
         16 . The method of  claim 14 , wherein the peptide consists essentially of a mature cyclin B1 protein.  
     
     
         17 . The method of  claim 14 , wherein the peptide consists essentially of a fragment of from about 5 to about 15 contiguous amino acids of a wild-type human cyclin B1 peptide and which has from 0 to about 5 single amino acid substitutions relative to the wild-type sequence.  
     
     
         18 . The method of  claim 14 , wherein the peptide comprises an amino acid sequence as set forth in SEQ ID NOs: 1-8.  
     
     
         19 . A method of priming T cells against tumor antigens comprising, obtaining naïve CD4 +  or CD8 +  T cells from at least one healthy individual, obtaining at least one protein or peptide from at least one cancerous cell; obtaining antigen presenting cells (APCs), culturing the APCs with the at least one protein or peptide, and adding the T cells to the culture of the APCs, whereby the T cells are primed against the at least one protein or peptide.  
     
     
         20 . The method of  claim 19 , wherein the T cells are CD4 +  T cells  
     
     
         21 . The method of  claim 19 , wherein the at least one protein or peptide is obtained by lysing the cancerous cell to obtain a lysate and extracting the protein or peptide from the lysate.  
     
     
         22 . The method of  claim 19 , wherein the T cells are CD8 +  T cells.  
     
     
         23 . The method of  claim 19 , wherein the at least one protein or peptide is obtained by extracting HLA class I molecules from the cancerous cell and eluting the protein or peptide from the extracted HLA class I molecules.  
     
     
         24 . The method of  claim 19 , wherein the at least one protein or peptide is fractionated.  
     
     
         25 . The method of  claim 19 , wherein the at least one protein or peptide is obtained from a tumor comprising the at least one cancerous cell.  
     
     
         26 . The method of  claim 19 , wherein the APCs are dendritic cells.  
     
     
         27 . The method of  claim 26 , wherein the dendritic cells are generated in vitro.  
     
     
         28 . The method of  claim 19 , wherein the APCs are cultured with the at least one protein or peptide in the presence of tumor necrosis factor α.  
     
     
         29 . The method of  claim 19 , wherein the T cells are added to the culture of the APCs in the presence of one or more cytokines.  
     
     
         30 . The method of  claim 29 , wherein the cytokines are selected from the group of cytokines consisting of IL-1β, IL-2, and IL-4, and IL-7.  
     
     
         31 . The method of  claim 19 , wherein the T cell/APC culture is restimulated by introducing autologous macrophages, loaded with the at least one protein or peptide, or irradiated cancerous cells.  
     
     
         32 . The method of  claim 19 , wherein the T cell/APC culture is restimulated more than one time at intervals of from about 7 to about 10 days.  
     
     
         33 . A method of identifying tumor antigens comprising, obtaining naïve CD4 +  or CD8 +  T cells from at least one healthy individual, obtaining at least one protein or peptide from at least one cancerous cell; obtaining antigen presenting cells (APCs), culturing the APCs with the at least one protein or peptide, and adding the T cells to the culture of the APCs, whereby the T cells are primed against the at least one protein or peptide, and assessing the peptide sequence of the stimulatory molecules.  
     
     
         34 . The method of  claim 33 , wherein the T cells are CD4 +  T cells  
     
     
         35 . The method of  claim 33 , wherein the at least one protein or peptide is obtained by lysing the cancerous cell to obtain a lysate and extracting the protein or peptide from the lysate.  
     
     
         36 . The method of  claim 33 , wherein the T cells are CD8 +  T cells.  
     
     
         37 . The method of  claim 33 , wherein the at least one protein or peptide is obtained by extracting HLA class I molecules from the cancerous cell and eluting the protein or peptide from the extracted HLA class I molecules.  
     
     
         38 . The method of  claim 33 , wherein the at least one protein or peptide is fractionated.  
     
     
         39 . The method of  claim 33 , wherein the at least one protein or peptide is obtained from a tumor comprising the at least one cancerous cell.  
     
     
         40 . The method of  claim 33 , wherein the APCs are dendritic cells.  
     
     
         41 . The method of  claim 41 , wherein the dendritic cells are generated in vitro.  
     
     
         42 . The method of  claim 33 , wherein the APCs are cultured with the at least one protein or peptide in the presence of tumor necrosis factor α.  
     
     
         43 . The method of  claim 33 , wherein the T cells are added to the culture of the APCs in the presence of one or more cytokines.  
     
     
         44 . The method of  claim 43 , wherein the cytokines are selected from the group of cytokines consisting of IL-1β, IL-2, and IL-4, and IL-7.  
     
     
         45 . The method of  claim 33 , wherein the T cell/APC culture is restimulated by introducing autologous macrophages, loaded with the at least one protein or peptide, or irradiated cancerous cells.  
     
     
         46 . The method of  claim 33 , wherein the T cell/APC culture is restimulated more than one time at intervals of from about 7 to about 10 days.