Methods of making chimeric antigen receptor-expressing cells

A method of making a population of cells that express a chimeric antigen receptor (CAR), the method comprising:

• (i) contacting a population of cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the cells;
• (ii) contacting the population of cells with a nucleic acid molecule encoding the CAR, thereby providing a population of cells comprising the nucleic acid molecule;
• and
• (iii) harvesting the population of cells for storage or administration;
• wherein step (ii) is performed together with step (i) or less than 18 hours after the beginning of step (i), and step (iii) is performed no later than 48 hours after the beginning of step (i).