Methods for isolating, culturing, and genetically engineering immune cell populations for adoptive therapy

Abstract

The present disclosure relates in some aspects to methods, cells, and compositions for preparing cells and compositions for genetic engineering and cell therapy. Provided in some embodiments are streamlined cell preparation methods, e.g., for isolation, processing, incubation, and genetic engineering of cells and populations of cells. Also provided are cells and compositions produced by the methods and methods of their use. The cells can include immune cells, such as T cells, and generally include a plurality of isolated T cell populations or types. In some aspects, the methods arc capable of preparing of a plurality of different cell populations for adoptive therapy using fewer steps and/or resources and/or reduced handling compared with other methods.

1 - 116 . (canceled) 
     
     
         117 . A method for producing genetically engineered T cells, the method comprising:
 (a) performing a first selection in a closed system, said first selection comprising enriching for one of (i) CD4 +  cells and (ii) CD8+ cells from a sample containing primary human T cells, the enrichment thereby generating a first selected population and a non-selected population, wherein the sample is a blood or blood-derived sample from a subject and comprises at least 1×10 8  cells;   (b) performing a second selection in the closed system, said second selection comprising enriching for the other of (i) CD4+ cells and (ii) CD8+ cells from the non-selected population, the enrichment thereby generating a second selected population,   (c) combining the cells of the first selected population and cells of the second selected population to produce a culture-initiating composition;   (d) incubating the culture-initiating composition, comprising cells of the first selected population and cells of the second selected population in a culture vessel under stimulating conditions, thereby generating stimulated cells; and   (e) introducing a genetically engineered antigen receptor into a plurality of the stimulated cells generated in (d),   wherein the method generates an output composition comprising CD4+ T cells and CD8 30   T cells expressing the genetically engineered antigen receptor.   
     
     
         118 . The method of  claim 117 , wherein the sample is a peripheral blood mononuclear cell (PBMC) sample. 
     
     
         119 . The method of  claim 117 , wherein the sample is an apheresis sample or a leukapheresis sample. 
     
     
         120 . The method of  claim 117 , wherein the first selected population and/or the second selected population comprises at least 1×10 7  cells. 
     
     
         121 . The method of  claim 117 , wherein the first selected population and/or the second selected population comprises at least 1×10 6  cells. 
     
     
         122 . The method of  claim 117 , wherein said combining is performed in a closed system. 
     
     
         123 . The method of  claim 117 , wherein the enriching cells in the first selection and the second selection comprises immunoaffinity-based selection. 
     
     
         124 . The method of  claim 117 , wherein the first selection and the second selection are carried out in separate separation vessels, which are operably connected. 
     
     
         125 . The method of  claim 117 , wherein the enriching for the CD4+ cells comprises positive selection based on surface expression of CD4 and the enriching for the CD8 30   cells comprises positive selection based on surface expression of CD8. 
     
     
         126 . The method of  claim 117 , wherein the one of the first selection and the second selection that comprises enriching for the CD8+ cells further comprises enriching for central memory T (T CM ) cells. 
     
     
         127 . The method of  claim 117 , wherein the one of the first selection and the second selection that comprises enriching for the CD8+ cells further comprises enriching for cells expressing a marker selected from the group consisting of CD28, CD62L, CCR7, CD127 and CD27. 
     
     
         128 . The method of  claim 117 , wherein the CD4+ cells to CD8+ cells in the culture-initiating composition are combined at a ratio of 5:1 to 1:5. 
     
     
         129 . The method of  claim 128 , wherein the ratio of CD4+ cells to CD8+ cells is 2:1 to 1:2. 
     
     
         130 . The method of  claim 128 , wherein the ratio of CD4+ cells to CD8+ cells in the culture-initiating composition is about 1:1. 
     
     
         131 . The method of  claim 117 , wherein the stimulating conditions in (d) comprise the presence of an anti-CD3 antibody and an anti-CD28 antibody. 
     
     
         132 . The method of  claim 131 , wherein the stimulating conditions in (d) comprise the presence of a cytokine selected from the group consisting of IL-2, IL-15, IL-7 and IL-21. 
     
     
         133 . The method of  claim 131 , wherein the stimulating conditions in (d) comprise the presence of IL-2, IL-15, and IL-7. 
     
     
         134 . The method of  claim 117 , wherein the genetically engineered antigen receptor comprises a T cell receptor (TCR) or a functional non-TCR antigen receptor. 
     
     
         135 . The method of  claim 117 , wherein the genetically engineered antigen receptor comprises a chimeric antigen receptor (CAR).