Cell therapy method for the treatment of tumors

Abstract

T cell responses are often diminished in humans with a compromised immune system. We have developed a method to isolate, stimulate and expand naive cytotoxic T lymphocyte precursors (CTLp) to antigen-specific effectors, capable of lysing tumor cells in vivo. This ex vivo protocol produces fully functional effectors. Artificial antigen presenting cells (AAPCs; Drosophila melanogaster) transfected with human HLA class I and defined accessory molecules, are used to stimulate CD8 + T cells from both normal donors and cancer patients. The class I molecules expressed to a high density on the surface of the Drosophila cells are empty, allowing for efficient loading of multiple peptides that results in the generation of polyclonal responses recognizing tumor cells endogenously expressing the specific peptides. The responses generated are robust, antigen-specific and reproducible if the peptide epitope is a defined immunogen. This artificial antigen expression system can be adapted to treat most cancers in a significant majority of the population.

What is claimed:  
     
         1 . A method for treating a subject with cancer comprising: 
 a. preparing a non-naturally occurring antigen-presenting cell line(nnAPC), wherein said nnAGC is capable of presenting up to about fifteen different peptide molecules associated with said cancer simultaneously wherein said peptide molecules are each about six to twelve amino acids in length,    b. harvesting CD8 +  cells from said subject or a suitable donor;    c. stimulating said CD8 +  cells with said nnAPC cell line;    d. adding said CD8 +  cells to media that contains a cytokine selected from the group consisting of IL-2, IL-7 or conditioned growth medium (CGM), wherein said cytokines can be used individually or in combination;    e. mixing unsuspended peripheral blood monocytes, or CD-8 depleted peripheral blood monocytes collected from said subject or a suitable donor with about 1 to 50 μg/ml of one of said peptides that said nnAPC can simultaneously present;    f. irradiating said peripheral blood monocyte suspension with a sufficient dose of γ-radiation necessary to sterilize all components in the suspension, except the desired peripheral blood monocytes;    g. isolating adherent peripheral blood monocytes;    h. loading said adherent peripheral blood monocytes with about 1 μg/ml to 50 μg/ml of said each peptide;    i. combining said CD8 +  cells with said adherent peripheral blood monocytes at a ratio of about ten CD8 +  cells to one peripheral blood monocyte; and    j. inoculating said subject with CD8 +  suspension.    
     
     
         2 . The method of  claim 1  wherein said nnAPC is capable of presenting up to about ten peptide molecules.  
     
     
         3 . The method of  claim 1  wherein said peptide molecules are about eight to ten amino acids in length  
     
     
         4 . The method of  claim 1  wherein said peptide molecules are in a concentration range of about 10 nM to 100 μM.  
     
     
         5 . The method of  claim 1  wherein said cytokine component is IL-2.  
     
     
         6 . The method of  claim 1  wherein said cytokine component is IL-2 and IL-7 in combination  
     
     
         7 . The method of  claim 1  wherein the dose of γ-radiation is about 3,000 to 7,000 rads.  
     
     
         8 . The method of  claim 1  wherein the dose of γ-radiation is about 5,000 rads.  
     
     
         9 . A method for treating a subject with cancer comprising, 
 a. preparing a non-naturally occurring antigen-presenting cell line(nnAPC), wherein said nnAPC is capable of presenting up to about fifteen different peptide molecules associated with said cancer simultaneously;    b. harvesting CD8 +  cells from said subject;    c. stimulating said CD8 +  cells with said nnAPC cell line for about six to seven days;    d. stimulating said CD8 +  cells with IL-2 and IL-7 in media;    e. mixing peripheral blood monocytes collected from said subject with about 20 μg/ml of each peptide;    f. irradiating said CD8-depleted peripheral blood monocyte suspension with about 5,000 rads of γ-radiation;    g. isolating adherent peripheral blood monocytes;    h. loading said adherent peripheral blood monocytes with about 1 ug/ml to 50 μg/ml of said epitope;    i. combining said CD8 +  cells with said adherent peripheral blood monocytes at a ratio of about ten CD8+ cells to one peripheral blood monocyte;    j. stimulating said combined suspension of CD8 +  cells and peripheral blood monocytes for about six to seven days;    k. stimulating said suspension of CD8 +  cells and peripheral blood monocytes with IL-2 and IL-7 in media;    l. assaying CD8 +  suspension for suitable CTL activity, purity, sterility and endotoxin content; and    m. inoculating said subject with CD8 +  suspension.    
     
     
         10 . The method of  claim 9  wherein each peptide is about eight to ten amino acids in length.  
     
     
         11 . A method for treating a subject with melanoma comprising, 
 a. preparing a non-naturally occurring antigen-presenting cell line(nnAPC), wherein said nnAPC is capable of presenting up to about fifteen different peptide molecules associated with said melanoma simultaneously where each peptide is eight to ten amino acids in length;    b. harvesting CD8 +  cells from said subject;    c. stimulating said CD8 +  cells with said nnAPC cell line for about six to seven days;    d. stimulating said CD8 +  cells with IL-2 and IL-7 in media;    e. mixing peripheral blood monocytes collected from said subject with about 20 μg/ml of each peptide said nnAPC can present;    f. irradiating said CD8-depleted peripheral blood monocyte suspension with about 5,000 rads of γ-radiation;    g. isolating adherent peripheral blood monocytes;    h. loading said adherent peripheral blood monocytes with about 1 ug/ml to 50 μg/ml of said epitope;    i. combining said CD8 +  cells with said adherent peripheral blood monocytes at a ratio of about ten CD8+ cells to one peripheral blood monocyte;    j. stimulating said combined suspension of CD8 +  cells and peripheral blood monocytes for about six to seven days;    k. stimulating said suspension of CD8 +  cells and peripheral blood monocytes with IL-2 and IL-7 in media;    l. assaying CD8 +  suspension for suitable CTL activity, purity, sterility and endotoxin content; and    m. inoculating said subject with CD8 +  suspension.    
     
     
         12 . The method of  claim 11  wherein said nnAPC present ten peptides.  
     
     
         13 . The method of  claim 12  wherein said peptides are Tyrosinase 369-377 , Tyrosinase 207-216 , gp100 209-217,  gp100 154-162 , MART-1 27-35 , HER-2/neu 789-797 , HER-2/neu 369-377 , C-lectin 8-16 , Pec60 20-29 , and Pec60 25-35 .  
     
     
         14 . A non-naturally occurring antigen-presenting cell (nnAPC) derived from  Drosophila melanogaster  cells transfected with nucleic acid that expresses human class I HLA molecules, binding molecules, and co-stimulatory molecules, wherein said nnAPC is capable of presenting up to fifteen different peptide molecules that are encoded by said nucleic acid simultaneously.  
     
     
         15 . The non-naturally occurring antigen-presenting cell nnAPC of  claim 14  wherein said nnAPC is capable of presenting up to ten different peptide molecules simultaneously.  
     
     
         16 . The non-naturally occurring antigen-presenting cell nnAPC of  claim 15  wherein said nnAPC is presents the following ten peptide molecules simultaneously, Tyrosinase 369-377 , Tyrosinase 207-216 , gp100 209-217 , gp100 154-162 , MART-1 27-35 , HER-2/neu 789-797 , HER-2/neu 369-377 , C-lectin 8-16 , Pec60 20-29 , and Pec60 25-33.    
     
     
         17 . A method for manufacturing non-naturally occurring antigen-presenting cell (nnAPC) capable of presenting up fifteen peptide molecules simultaneously, said method comprising of the step, 
 a. preparing a insect cell line from  Drosophila melanogaster  eggs;    b. growing said insect cells a media that is suitable for growing insect cells, preferably Schneider™'s Drosophila Medium;    c. making a pRmHa-3 plasmid from a pRmHa-1 expression vector, where said pRmHa-3 plasmid includes a metallothionein promoter, metal response consensus sequences and an alcohol dehydrogenase gene bearing a polyadenylation signal isolated from  Drosophila melanogaster;      d. inserting into said pRmHa-3 plasmid complementary DNA for human class I HLA A2.1, B7.1, B7.2, ICAM-1, β-2 microglobulin and LFA-3, wherein A2.1 can be substituted with any human class I DNA sequence;    e. transfecting said insect cells with a phshneo plasmid and said pRmHa-3 plasmid containing complementary DNA;    f. creating nnAPC by contacting said insect cells with CuSO 4  to induce expression of the transfected genes in said insect cells.    
     
     
         18 . The method of  claim 17  wherein said nnAPC is capable of presenting up to ten peptides.  
     
     
         19 . The method of  claim 17  wherein said insect cell line is prepared by growing cells for twelve days, selecting desired cells with peptides that are capable of identifying said desired cells, and expanding said desired cells with OKT3 and IL-2.  
     
     
         20 . The method of  claim 19  wherein said peptides capable of identifying said desired cells are tetramers.  
     
     
         21 . The method of  claim 1  wherein said peptide molecules of step (a) are selected from the peptides having an amino acid sequence set forth in SEQ ID NO: 1 through 42.  
     
     
         22 . The method of  claim 9  wherein said peptide molecules of step (a) are selected from the peptides having an amino acid sequence set forth in SEQ ID NO: 1 through 42.  
     
     
         23 . The method of  claim 17  wherein said peptide molecules of step (a) are selected from the peptides having an amino acid sequence set forth in SEQ ID NO: 1 through 42.