Expansion Of Peripheral Blood Lymphocytes (PBLS) From Peripheral Blood

A method for expanding peripheral blood lymphocytes (PBLs) from peripheral blood comprising:

• a. Obtaining a sample of PBMCs from peripheral blood of a patient, wherein said sample is optionally cryopreserved and the patient is optionally pretreated with an ITK inhibitor;
• b. Isolating PBLs from said sample by selecting and removing CD19+ B cells and optionally pretreating said PBLs with an ITK inhibitor at a concentration of between 0.1 nM and 200 nM;
• c. Co-culturing said PBLs with said CD19+ B cells for a period of between 2 to 5 days;
• d. Adding from about 2.5×105 to about 5×105 cells to a gas-permeable container in a first cell culture medium and stimulating said PBLs with 3000 IU/ml IL-2 and anti-CD3/anti-CD28 antibodies immobilized on beads, and and optionally an ITK inhibitor at a concentration of between 0.1 nM and 200 nM, for a period of between 3 to 6 days;
• e. Exchanging the first cell culture medium with a second cell culture medium and additional IL-2 at a concentration of about 3000 IU/ml and optionally an ITK inhibitor at a concentration of between 0.1 nM and 200 nM;
• f. Culturing the PBLs from step (e) for an additional period of between 3 to 6 days with IL-2 and anti-CD3/anti-CD28 antibodies immobilized on beads and optionally an ITK inhibitor at a concentration of between 0.1 nM and 200 nM;
• g. Exchanging the second cell culture medium with a third cell culture medium and adding additional IL-2 at a concentration of 3000 IU/mL, and optionally an ITK inhibitor at a concentration of between 0.1 nM and 200 nM, and culturing the cells for an additional period of between 2 to 5 days;
• h. Isolating the antibody-bound PBLs from the culture in step (g);
• i. Removing the antibodies from the PBLs isolated in step (h);
• and
• j. Harvesting the PBLs,
• wherein the ITK inhibitor is optionally an ITK inhibitor that covalently binds to ITK, and further wherein the PBLs may be optionally transduced with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain variable fragment antibody fused with at least one endodomain of a T-cell signaling molecule.