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 naïve 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 viral infection in a subject 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 viral infection 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 ug/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 the 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 melanoma, comprising the steps of: 
 a. administering to said subject an effective amount of interferon-alfa that is capable of enhancing the expression of tumor antigen on the surface of the tumor; and    b. inoculating said subject with an effective amount of autologous cytotoxic T lymphocytes with specificity for melanoma-associated target antigen.    
     
     
         10 . The method of  claim 9 , further comprising the step of administering to said subject an effective amount of interleukin-2 that is capable of enhancing the in vivo maintenance of the autologous cytotoxic T lymphocytes with specificity for melanoma-associated target antigen.  
     
     
         11 . The method of  claim 10 , wherein the interferon-alfa is selected from interferon-alfa-2a or interferon-alfa-2b.  
     
     
         12 . The method of  claim 10 , wherein the effective amount of interferon-alfa is about 10 MU/m 2 /day and is subcutaneously administered to the subject consecutively from day 5 to day 1 prior to inoculating said subject with the effective amount of autologous cytotoxic T lymphocytes with specificity for melanoma-associated target antigen.  
     
     
         13 . The method of  claim 10 , wherein the effective amount of autologous cytotoxic T lymphocytes with specificity for melanoma-associated target antigen is about 1-10×10 9  cells/infusion.  
     
     
         14 . The method of  claim 10 , wherein the autologous cytotoxic T lymphocytes with specificity for melanoma-associated target antigen are obtained by a method comprising steps of: 
 a) preparing a non-naturally occurring antigen-presenting cell line (nnAPC), wherein said nnAPC is capable of presenting up to about fifteen different epitopes associated with said melanoma simultaneously and where each epitope is a peptide of eight to ten amino acids in length;    b) loading the nnAPC with up to about fifteen different epitopes associated with said melanoma;    c) harvesting CD8 +  cells from said subject;    d) stimulating said CD8 +  cells with the epitope-loaded nnAPC cell line to obtain CD8 +  cells specific for the melanoma;    e) growing the CD8 +  cells specific for the melanoma in media containing IL-2 and IL-7;    f) mixing CD8-depleted peripheral blood monocytes collected from said subject with each epitope used for nnAPC loading;    g) irradiating said CD8-depleted peripheral blood monocytes with γ-radiation;    h) isolating adherent CD8-depleted peripheral blood monocytes;    i) loading said adherent peripheral blood monocytes with each epitope used for nnAPC loading;    j) restimulating said CD8 +  cells specific for the melanoma with the epitope-loaded adherent peripheral blood monocytes;    k) growing the restimulated CD8 +  cells specific for the melanoma in media containing IL-2 and IL-7; and    l) expanding the restimulated CD8 +  cells specific for the melanoma by OKT3 antibody stimulation.    
     
     
         15 . The method of  claim 14 , wherein step 0) can be repeated at least one more time.  
     
     
         16 . The method of  claim 14 , wherein said non-naturally occurring antigen-presenting cell line is loaded with epitopes that are peptides derived from tyrosinase, gp100, and MART-1.  
     
     
         17 . The method of  claim 16 , wherein said non-naturally occurring antigen-presenting cell line is loaded with epitopes comprising amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.  
     
     
         18 . The method of  claim 14 , wherein the effective amount of interleukin-2 is about 3 MIU/day and is subcutaneously administered to the subject consecutively from day 0 to day 28 after inoculating said subject with the effective amount of autologous cytotoxic T lymphocytes with specificity for melanoma-associated target antigen.  
     
     
         19 . The method of  claim 14 , wherein the method is repeated at an interval of about 2 months.  
     
     
         20 . The method of  claim 19 , wherein the method is repeated for at least two cycles and further comprises the step of evaluating a response in said subject after each cycle.  
     
     
         21 . The method of  claim 14 , comprising the steps of: 
 a. subcutaneously administering to the subject 10 MU/m 2 /day of interferon-alpha-2b consecutively from day 5 to day 1 prior to inoculating said subject with autologous cytotoxic T lymphocytes with specificity for melanoma-associated target antigen;    b. introducing about 1-10×10 9  cells/infusion of the autologous cytotoxic T lymphocytes with specificity for melanoma-associated target antigen to the subject; and    c. subcutaneously administering to the subject about 3 MIU/day of interleukin-2 consecutively from day 0 to day 28 following the introducing step.