Methods of obtaining antigen-specific t cell populations

Abstract

The invention provides a method of obtaining a population of antigen-specific T cells from peripheral blood of a host. An embodiment of the method of the invention comprises (i) dividing PBMCs from peripheral blood of a host into more than one sub-population; (ii) contacting the PBMCs with an antigen and IL-2; (iii) obtaining a sample of PBMCs from each sub-population; (iv) identifying an antigen-reactive sub-population by determining by high throughput quantitative PCR the expression of a factor produced by the PBMCs of each sample; (v) dividing the antigen-reactive sub-population into microcultures; (vi) identifying the antigen-reactive microculture; and (vii) expanding the microculture, thereby obtaining a population of T cells specific for the antigen. The invention also provides a population of T cells obtained by the inventive method, a pharmaceutical composition comprising the same, and a method of treating a disease in a host using the pharmaceutical composition. Related isolating and screening methods are further provided.

1 . A clinical grade population of antigen-specific T cells obtained by a method comprising:
 (i) dividing peripheral blood mononuclear cells (PBMCs) from peripheral blood of a host into more than one sub-population;   (ii) contacting the PBMCs of each sub-population with an antigen and Interleukin-2 (IL-2);   (iii) obtaining a sample of the contacted PBMCs from each sub-population;   (iv) identifying an antigen-reactive sub-population by determining by high throughput quantitative PCR (HT-qPCR) the expression of a factor produced by the PBMCs of each sample;   (v) dividing the antigen-reactive sub-population into microcultures;   (vi) identifying an antigen-reactive microculture; and   (vii) expanding the microculture, thereby obtaining a clinical grade population of T cells specific for the antigen.   
     
     
         2 . The population of  claim 1 , wherein the method is carried out in less than about 7 weeks. 
     
     
         3 . The population of  claim 2 , wherein the method is carried out in about 5 to about 6 weeks. 
     
     
         4 . The population of any of  claim 1 , wherein (i) to (iv) is carried out within about 1 week. 
     
     
         5 . The population of any of  claim 1 , wherein (i) to (vi) is carried out in about 30 days or less. 
     
     
         6 . The population of  claim 1 , wherein the number of PBMCs of the antigen-reactive sub-population identified in (iv) is less than about 10% of the number of PBMCs of (i). 
     
     
         7 . The population of  claim 6 , wherein the number of PBMCs of the antigen-reactive sub-population identified in (iv) is less than about 1% of the number of PBMCs of (i). 
     
     
         8 . The population of  claim 1 , wherein the PBMCs are divided into about 96 sub-populations. 
     
     
         9 . The population of  claim 1 , wherein about 3×10 5  PBMCs are contacted in (ii). 
     
     
         10 . The population of  claim 9 , wherein each sample of (iii) comprises about 1×10 5  PBMCs. 
     
     
         11 . The population of  claim 1 , comprising contacting each sample of (iii) with an antigenic peptide presented by a carrier cell prior to (iv). 
     
     
         12 . The method population of  claim 1 , wherein the factor is Interferon-γ (IFN-γ). 
     
     
         13 . The population of  claim 1 , wherein the PMBCs are contacted in (ii) with a viral antigen or a cancer antigen. 
     
     
         14 . The population of  claim 13 , wherein the cancer antigen is selected from the group consisting of gp100, NY-ESO-1, MART-1, MAGE-A1, and mesothelin. 
     
     
         15 . The population of  claim 14 , wherein the epitope is gp100 154-162  (SEQ ID NO: 2), NY-ESO-1 157-165  (SEQ ID NO: 6), MAGE-A1 278-286  (SEQ ID NO: 10), mesothelin 18-26  (SEQ ID NO: 11), or mesothelin 21-29  (SEQ ID NO: 12). 
     
     
         16 . The population of  claim 13 , wherein the antigen is an influenza viral antigen. 
     
     
         17 . (canceled) 
     
     
         18 . The population of  claim 1 , wherein the population of antigen-specific T cells is greater than about 90% clonal. 
     
     
         19 . The population of  claim 18 , wherein the population of antigen-specific T cells is about 99% clonal. 
     
     
         20 . The population of  claim 1 , wherein the antigen-specific T cells have high functional avidity for the antigen, recognize tumor cells expressing the antigen, and/or are CD27 + . 
     
     
         21 . The population of  claim 20 , wherein the antigen-specific T cells recognize target cells pulsed with about 10 −10  to about 10 −11  M antigen. 
     
     
         22 . The population of  claim 20 , wherein at least 80% of the antigen-specific T cells are CD27 +  T cells. 
     
     
         23 . The population of  claim 1 , wherein the antigen-specific T cells are CD8 +  T cells or CD4 +  T cells. 
     
     
         24 . A pharmaceutical composition comprising the population of  claim 1  and a pharmaceutically acceptably carrier. 
     
     
         25 . A method of treating a disease in a host, comprising administering to the host the pharmaceutical composition of  claim 24  in an amount effective to treat the disease in the host. 
     
     
         26 . The method of  claim 25 , wherein the antigen-specific T cells of the population are autologous to the host. 
     
     
         27 . The method of  claim 25 , wherein the disease is a viral disease or a cancer. 
     
     
         28 . The method of  claim 27 , wherein the cancer is selected from a group consisting of melanoma, breast cancer, colorectal cancer, esophageal cancer, gastric cancer, non-small cell lung cancer, a sarcoma, pancreatic cancer, mesothelioma, and ovarian cancer. 
     
     
         29 . A method of isolating antigen-specific T cells from peripheral blood of a host, comprising:
 (i) dividing peripheral blood mononuclear cells (PBMCs) from peripheral blood of a host into more than one sub-population;   (ii) contacting the PBMCs with an antigen and Interleukin-2 (IL-2);   (iii) obtaining a sample of the contacted PBMCs from each sub-population;   (iv) identifying an antigen-reactive sub-population by determining by high throughput quantitative PCR (HT-qPCR) the expression of a factor produced by the PBMCs of each sample;   (v) dividing the antigen-reactive sub-population into microcultures; and   (vi) identifying an antigen-reactive microculture;   
       whereupon T cells specific for the antigen are isolated from the peripheral blood. 
     
     
         30 . A method of screening candidate cancer antigen epitopes, comprising:
 (i) dividing PBMCs from peripheral blood of a host into more than one sub-population;   (ii) contacting the PBMCs with one or more candidate cancer antigen epitopes and IL-2;   (iii) obtaining a sample of the contacted PBMCs from each sub-population; and   (iv) identifying an antigen-reactive sub-population by determining by high throughput quantitative PCR (HT-qPCR) the expression of a factor produced by the PBMCs of each sample.   
     
     
         31 . The method of  claim 30 , wherein, when a cancer antigen epitope is identified, the method further comprises:
 (v) dividing the antigen-reactive subpopulation into microcultures;   (vi) identifying the antigen-reactive microculture; and   (vii) expanding the microculture; thereby obtaining a population of T cells specific for the cancer antigen epitope.   
     
     
         32 . The method of  claim 31 , further comprising assaying the population for tumor reactivity against a tumor cell line which expresses the cancer antigen epitope. 
     
     
         33 . The method of  claim 31 , further comprising determining the cancer antigen of which the cancer antigen epitope is a part, thereby identifying a cancer antigen.