Immunotherapy using chimeric antigen receptor-modified T cells has demonstrated high response rates in patients with B cell malignancies, and chimeric antigen receptor T cell therapy is now being investigated in several hematologic and solid tumor types. Chimeric antigen receptor T cells are generated by removing T cells from a patient’s blood and engineering the cells to express the chimeric antigen receptor, which reprograms the T cells to target tumor cells. As chimeric antigen receptor T cell therapy moves into later-phase clinical trials and becomes an option for more patients, compliance of the chimeric antigen receptor T cell manufacturing process with global regulatory requirements becomes a topic for extensive discussion. Additionally, the challenges of taking a chimeric antigen receptor T cell manufacturing process from a single institution to a large-scale multi-site manufacturing center must be addressed. We have anticipated such concerns in our experience with the CD19 chimeric antigen receptor T cell therapy CTL019. In this review, we discuss steps involved in the cell processing of the technology, including the use of an optimal vector for consistent cell processing, along with addressing the challenges of expanding chimeric antigen receptor T cell therapy to a global patient population. Immunotherapy using chimeric antigen receptor-modified T cells has demonstrated high response rates in patients with B cell malignancies, and chimeric antigen receptor T cell therapy is now being investigated in several hematologic and solid tumor types. Chimeric antigen receptor T cells are generated by removing T cells from a patient’s blood and engineering the cells to express the chimeric antigen receptor, which reprograms the T cells to target tumor cells. As chimeric antigen receptor T cell therapy moves into later-phase clinical trials and becomes an option for more patients, compliance of the chimeric antigen receptor T cell manufacturing process with global regulatory requirements becomes a topic for extensive discussion. Additionally, the challenges of taking a chimeric antigen receptor T cell manufacturing process from a single institution to a large-scale multi-site manufacturing center must be addressed. We have anticipated such concerns in our experience with the CD19 chimeric antigen receptor T cell therapy CTL019. In this review, we discuss steps involved in the cell processing of the technology, including the use of an optimal vector for consistent cell processing, along with addressing the challenges of expanding chimeric antigen receptor T cell therapy to a global patient population. Chimeric antigen receptor (CAR) T cell therapy is a cellular therapy that redirects a patient’s T cells to specifically target and destroy tumor cells. CARs are genetically engineered fusion proteins composed of (1) an antigen recognition domain derived from a monoclonal antibody and (2) intracellular T cell signaling and costimulatory domains.1Kuwana Y. Asakura Y. Utsunomiya N. Nakanishi M. Arata Y. Itoh S. Nagase F. Kurosawa Y. Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions.Biochem. Biophys. Res. Commun. 1987; 149: 960-968Crossref PubMed Scopus (223) Google Scholar, 2Gross G. Waks T. Eshhar Z. Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity.Proc. Natl. Acad. Sci. USA. 1989; 86: 10024-10028Crossref PubMed Scopus (987) Google Scholar, 3Finney H.M. Lawson A.D. Bebbington C.R. Weir A.N. Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product.J. Immunol. 1998; 161: 2791-2797Crossref PubMed Google Scholar, 4Maher J. Brentjens R.J. Gunset G. Rivière I. Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.Nat. Biotechnol. 2002; 20: 70-75Crossref PubMed Scopus (689) Google Scholar, 5Sadelain M. Brentjens R. Rivière I. The basic principles of chimeric antigen receptor design.Cancer Discov. 2013; 3: 388-398Crossref PubMed Scopus (857) Google Scholar Use of CAR T cells as a treatment for cancer has been most extensively investigated in patients with B cell malignancies, and early results have been encouraging. For example, CAR T cell therapy has demonstrated complete response rates of 69%–90% in pediatric patients with relapsed or refractory acute lymphoblastic leukemia (ALL) in phase 1 trials.6Maude S.L. Frey N. Shaw P.A. Aplenc R. Barrett D.M. Bunin N.J. Chew A. Gonzalez V.E. Zheng Z. Lacey S.F. et al.Chimeric antigen receptor T cells for sustained remissions in leukemia.N. Engl. J. Med. 2014; 371: 1507-1517Crossref PubMed Scopus (3554) Google Scholar, 7Davila M.L. Riviere I. Wang X. Bartido S. Park J. Curran K. Chung S.S. Stefanski J. Borquez-Ojeda O. Olszewska M. et al.Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia.Sci. Transl. Med. 2014; 6: 224ra25Crossref PubMed Scopus (1760) Google Scholar, 8Lee D.W. Kochenderfer J.N. Stetler-Stevenson M. Cui Y.K. Delbrook C. Feldman S.A. Fry T.J. Orentas R. Sabatino M. Shah N.N. et al.T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial.Lancet. 2015; 385: 517-528Abstract Full Text Full Text PDF PubMed Scopus (2055) Google Scholar, 9Maude S.L. Pulsipher M.A. Boyer M.W. Grupp S.A. Davies S.M. Phillips C.L. Verneris M.R. August K.J. Schlis K. Driscoll T.A. et al.Efficacy and safety of CTL019 in the first US phase II multicenter trial in pediatric relapsed/refractory acute lymphoblastic leukemia: results of an interim analysis.Blood. 2016; 128: 2801Crossref Google Scholar, 10Grupp S.A. Laetsch T.W. Buechner J. Bittencourt H. Maude S.L. Verneris M.R. Myers G.D. Boyer M.W. Rives S. De Moerloose B. et al.Analysis of a global registration trial of the efficacy and safety of CTL019 in pediatric and young adults with relapsed/refractory acute lymphoblastic leukemia (ALL).Blood. 2016; 128: 221Crossref Google Scholar The development of CAR T cell therapy has now expanded beyond phase 1 trials and moved into phase 2 multi-site trials (NCT02435849 and NCT02228096), and a major consideration for academic institutions and industry is how to scale out the production of CAR T cells in an efficient, effective manner. Here we describe the process of manufacturing CAR T cells, and we discuss regulatory concerns that must be addressed to successfully produce CAR T cells for larger numbers of patients. The production of CAR T cells requires several carefully performed steps, and quality control testing is performed throughout the entire protocol.11Levine B.L. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.Cancer Gene Ther. 2015; 22: 79-84Crossref PubMed Scopus (89) Google Scholar First, the process involves using leukapheresis to remove blood from the patient’s body, separate the leukocytes, and return the remainder of the blood to the circulation.12Smith J.W. Apheresis techniques and cellular immunomodulation.Ther. Apher. 1997; 1: 203-206Crossref PubMed Scopus (11) Google Scholar After a sufficient number of leukocytes have been harvested, the leukapheresis product is enriched for T cells (Figure 1). This process involves washing the cells out of the leukapheresis buffer, which contains anticoagulants.13Lee G. Arepally G.M. Anticoagulation techniques in apheresis: from heparin to citrate and beyond.J. Clin. Apher. 2012; 27: 117-125Crossref PubMed Scopus (118) Google Scholar Enrichment of lymphocytes can be accomplished subsequently through counterflow centrifugal elutriation, which separates cells by size and and cell Zheng Z. H. J. B.L. of lymphocytes for use in using a counterflow centrifugal Full Text Full Text PDF PubMed Scopus Google Scholar of T cell the of using antibody or is an that be C. A. A. M. therapy with chimeric antigen receptor-modified T cells of J. 2014; 20: PubMed Scopus Google Scholar cells from the patient to use for T cell several steps, and to a CAR T cell B.L. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.Cancer Gene Ther. 2015; 22: 79-84Crossref PubMed Scopus (89) Google Scholar For this an to T cells in a more for example, with monoclonal the use of or in with cells and such as has been the for in to with monoclonal or the and are B.L. M. N. T. of proliferation of T cells in the of Immunol. 1997; Google Scholar, A. cells to express a of Ther. Full Text Full Text PDF PubMed Scopus Google Scholar The or can be from the through B.L. and expanded T cells for J. 6: Google Scholar In the of and T cells can in a for several B.L. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.Cancer Gene Ther. 2015; 22: 79-84Crossref PubMed Scopus (89) Google Scholar, B.L. M. N. T. of proliferation of T cells in the of Immunol. 1997; Google Scholar, B.L. and expanded T cells for J. 6: Google Scholar, C. N. K. S. production and testing of T for the treatment of patients with 6: Full Text Full Text PDF PubMed Scopus Google Scholar, 2012; 1: PubMed Scopus Google Scholar The use of derived from the leukemia cell which can be engineered to express the costimulatory has been investigated as a of expanding T cells A. cells to express a of Ther. Full Text Full Text PDF PubMed Scopus Google Scholar, K. K. of and T lymphocytes by expressing for the T-cell receptor, and Biotechnol. 2002; 20: PubMed Scopus Google Scholar be to T cells to a or CAR T cells that to a demonstrated efficacy in a that T cell is a that the in the S. X. A. C. J. A.D. J. N. et chimeric antigen receptors 2014; PubMed Scopus (223) Google Scholar the the T cells are with the vector the several the vector is out of the by The vector to to the patient cells, into the cells, the vector in the of J. S. H. Scholar In the of CAR T cell this the The is into and into the of the patient CAR is as the cells and are to numbers in the The CAR is and by the patient cells, and the CAR is the cell which have a G. A. K. G. C. A. G. et and in clinical Gene Med. 2013; PubMed Scopus Google Scholar, M. F. M. F. A. M. et of is by vector design and in a of gene Clin. PubMed Scopus Google Scholar are in clinical trials of CAR T cell including CTL019. of gene including the or have been investigated as to express a CAR in T S. H. of and antigen cells to genetically T cells from and 2013; Google Scholar, H. S. G. S. B. et of T cells expressing chimeric antigen receptor using and antigen 2013; PubMed Scopus Google Scholar CAR T cells generated using have been in the this requires several of CAR T cell G. Chew A. Y. B.L. S.M. et chimeric antigen receptor T cells in solid Res. 2014; PubMed Scopus Google Scholar the is to be and has been in clinical are several including to the of and of The a vector for gene 20: PubMed Scopus Google Scholar are to the optimal requirements and to numbers of cells for clinical use (Figure The as the which a has been to the CAR T cell therapy B.L. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.Cancer Gene Ther. 2015; 22: 79-84Crossref PubMed Scopus (89) Google Scholar, 2012; 1: PubMed Scopus Google Scholar, M.R. S.A. scale of lymphocytes for cell therapy in the Transl. Med. 2012; PubMed Scopus Google Scholar that can be is the which has the to cells from J. Sabatino M. R. S.A. of the of tumor lymphocytes in to numbers for patient 2012; PubMed Scopus Google Scholar, R. J. B. X. K. C. et the production of cells using the Ther. Clin. 2014; 1: Full Text Full Text PDF PubMed Scopus Google Scholar The the to be in a cell of this is that the must be cell The is a single that cell and A.D. M. B. M. Orentas R. B. a process for the of genetically T cells for Gene Ther. 2015; 22: PubMed Scopus Google Scholar This with which use separate for the cell cell and steps in the that the is for CAR T cells, and this is to be to CAR T cells for clinical G. H. K. I. A. M. A. of T cells with CARs using the 2015; Scholar the cell process is the cell which a of to must be to a that can be into the B.L. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.Cancer Gene Ther. 2015; 22: 79-84Crossref PubMed Scopus (89) Google Scholar, B.L. and expanded T cells for J. 6: Google Scholar The and cells are in product the cells are to and the center the patient be of cell and are the of of processing be for CAR T cell that can be for a of and larger for manufacturing CAR T cells have now been CAR T cell have been to a patients to out this manufacturing process to more patients in larger trials an number of clinical the process be carefully to production the and of the CAR T cells can be to target several of the scale of production for the vector and the CAR T cells the of vector for of cells, the safety of gene and global regulatory In the the vector to the CAR into T cells is a of the CAR T cell manufacturing and the T cell is the as the product in the In to the CAR T cell which must be generated for the vector the CAR can be in and for in our that vector are for to this S. R. J.W. for clinical therapy are for to Gene Ther. PubMed Scopus Google Scholar, M. A. Stefanski J. J. Sadelain M. Rivière I. and of cell of vector in cell Ther. PubMed Scopus Google Scholar As with the CAR T cell manufacturing of the vector must in The of the vector is the CAR T cell product be by of the vector with processing and the of and by an of safety and the of cells from the vector In use of a safety K. S.M. for a vector 1998; PubMed Google Scholar, T. R. M. R.J. M. vector with a 1998; PubMed Google Scholar In our the of vector for cellular a of 2 of this is numbers of cells, such as cells, to produce of X. Olszewska M. J. T. Bartido S. G. Sadelain M. Rivière I. vector production in a 2015; PubMed Scopus Google Scholar from a of an cell the cells are expanded in for several to the number for from the number of cells The cells are with that in the production of the are (1) a that the proteins and (2) a a from a in vector a for the of along with a vector the CAR as as for and A. B. C. of Gene Ther. 2013; PubMed Scopus Google Scholar The vector a number of safety that the of that vector to and of and S.F. T. C. M. for of into Natl. Acad. Sci. USA. PubMed Scopus Google Scholar, S.M. 1 vector that a PubMed Scopus Google Scholar, R. T. R.J. A. vector for and in gene 1998; PubMed Google Scholar of the production cells to which can be from the X. Olszewska M. J. T. Bartido S. G. Sadelain M. Rivière I. vector production in a 2015; PubMed Scopus Google Scholar the of several for the of of After to remove production cells and the vector is through processing to for the vector removing and to the vector into an In our the vector be this to for the production of to larger of the vector product for the target of vector is a and is production is the vector is is to quality control testing for and that manufacturing can be that of vector is to T B.L. for the of cellular gene therapy B. in Scholar quality control are in by the and and are which can or toxicity in to that the product is for The of the product is to that is of in the of and for and testing of the cells and in the process that the CAR T cell product is from and for into patients. in the vector product are to (1) consistent by the vector manufacturing and (2) that the quality of the vector is consistent to use in the T cell manufacturing testing testing for such as to and the of and from and of both cell and In the T cells are for and from the cell The of the vector product is to as and is through or For example, the of vector can be by the of cells by and this can a of vector is to patient X. Olszewska M. J. T. Bartido S. G. Sadelain M. Rivière I. vector production in a 2015; PubMed Scopus Google Scholar In early clinical trials performed the of vector the of the therapy CTL019. from as the of vector and is that a single vector is for the of CTL019 cells in and clinical Additionally, the vector be to such as as as the production our to a vector manufacturing process that can global requirements and that has the process (Figure In our experience with vector by we that consistent vector quality in the CAR T cell manufacturing of sufficient size to clinical and the process has been and to vector in a of a manufacturing several This a and to and a have been demonstrated to a of for that manufacturing are for and processing is in a single The processing processing and the have been to that in is and that the product target and and in the T cell manufacturing In the is by a testing which that vector is in for an The of the CTL019 vector in a and both This an of the in which a to which we can patient The demonstrated that the vector can be with and manufacturing are both and a vector that can be and and the in process the vector for CAR T cell large-scale manufacturing and The scale of vector production to into the size of the patient an this our is to clinical as as and production in the development of production that an quality of functional vector a larger The most in CAR T cell manufacturing are vector of and and that is into in the target this which the CAR into the a process by the vector and several the vector J. S. H. Scholar of is that the of to cells, to a of cells, including and M. F. M. a and gene 2013; Google Scholar as T cell is for and the vector is cell B.L. Performance-enhancing drugs: design and production of redirected chimeric antigen receptor (CAR) T cells.Cancer Gene Ther. 2015; 22: 79-84Crossref PubMed Scopus (89) Google Scholar by the of vector into cells an is a with N. S. as and of a of a Full Text PDF PubMed Scopus Google Scholar, M. S. of the by in PubMed Scopus Google Scholar from cellular more that a of M. F. M. a and gene 2013; Google Scholar for to be the vector such as the that is the and the cell or that is CAR T cell has been in patients with CAR T cells in our experience in a J. G. G. A.N. M. et safety and of chimeric antigen receptor T Transl. Med. 2012; PubMed Scopus Google Scholar be are in the vector vector the of K. G. Feldman S.A. S.A. S.A. et in T cells in clinical is to the testing Ther. 2012; 20: Full Text Full Text PDF PubMed Scopus Google Scholar The and to be in the for CAR T cell therapy are carefully for In to testing for throughout the vector manufacturing process and the cell the that patients be for for to to to for in and vector gene therapy Scholar the safety of using for cell and gene extensive As of using requirements the For example, a trial has been to patients the CD19 CAR T cell therapy CTL019 for treatment This patients of have been with CTL019 for B cell The primary of this is to describe to be to CD19 CAR T cell such as the development of malignancies, or of a or or of a hematologic The of this are the of the CTL019 cells in blood and the efficacy of the The of CTL019 cells be by using to the CD19 CAR Additionally, the of patients or experience be as as the of patient from the of CAR T cells are and be in patients with CAR T cells. the design and of for CAR T cell therapy to along with quality and safety testing and patient that patient safety is the major with out the production of CAR T cell is the from a process a single academic institution to a process that can be and treatment (Figure effective the and treatment involved is to that the is and patients are throughout the in a global manufacturing process of CAR T cells be by a of both the product and the process in to the target product and quality For the target product T cells that are of and in this product quality cellular and functional are quality of CTL019 that are and such as T cell or or and quality be with process to a consistent manufacturing process and control that a the of T cells in the product and of is to be that for CAR and from product to As more experience is and to be the and be M. B.L. S. Grupp S.A. A. T cells with chimeric antigen receptors have and can in patients with leukemia.Sci. Transl. Med. 3: PubMed Scopus Google Scholar, B.L. M. A. Chimeric antigen receptor-modified T cells in leukemia.N. Engl. J. Med. PubMed Scopus Google Scholar such as our with are for a manufacturing In the of CAR T cell product has the that patient is to to the manufacturing we have our for CTL019 manufacturing and vector production that we to the manufacturing results of CAR T cell therapy patient this therapy has the for clinical which to global regulatory and The patient by CAR T cell a to a regulatory and regulatory compliance is a for the development of this and this be an in a global The has been cell and gene for and has a number of are with or In major has a as clinical trial For example, compliance as by a the compliance through a the more that are involved in clinical the more the manufacturing process to be to of the and regulatory in the of cell and gene of the global regulatory including of of and and of and as and and the and to an with a to discuss in the of and CAR T cell have a of this regulatory are and is experience we can a of in product development and a by the this are the requirements with manufacturing that For example, and and patient and requirements can This is the and product are is that the of the the or and the requirements for quality control of requirements with quality requirements can be is for the and of must be as has and to in cell and gene therapy the requirements for the or for cell global to of regulatory we are for CAR T cell manufacturing using that with the quality requirements for major global regions using the global are several of to CAR T cell and to a larger patient population. is the of T cells for CAR T cell The cell for CAR T cell of and T cells the in the blood of the the use of a of CAR T cells in patients with and has been C. T. K. B. N. C. S. et with chimeric antigen receptor T cells of Clin. 2015; Scholar the of T cell becomes an in CAR T cell manufacturing to be to the steps to CAR T cells in a of consideration for the of manufacturing a CAR T cell therapy both and For example, to CAR T cell therapy is which in a of patients. is by the of from the CAR T cells and tumor cells. most of are with treatment D.W. R. N. M. Grupp S.A. C.L. in the and management of 2014; PubMed Scopus Google Scholar has been that to CAR T cells in patients be to the safety of CAR T cell a into the CAR is a that is being investigated as a to specifically CAR T cells in a and as CAR T cell therapy is investigated in an clinical B. C. A. M. J. G. T lymphocytes with and a gene to and PubMed Scopus Google Scholar, C. Y. Wang J. X. S.A. D.M. et a chimeric antigen receptor and an to the efficacy and safety of T cell for 2013; PubMed Scopus Google Scholar For example, the safety has been to in patients cell A. G. Y. A. C. K. B. et as a safety for cell Engl. J. Med. PubMed Scopus Google Scholar of CAR T cells the of CAR T tumor of the safety of CAR T cell therapy involves the of the T cells. This be for CAR T cells solid which have the that CAR T cells be to target expressing and the CAR T cells tumor cells expressing of M. M. M. Sadelain M. antigen recognition with signaling tumor by engineered T Biotechnol. 2013; PubMed Scopus Google Scholar antigen recognition be to CARs for the treatment of solid In our has been in clinical trials using CAR T cells to target solid tumor cells are to the solid tumor is the use of CAR T cells. This to the which the of the cells to A. B. C. R. S. G. S. C. M. J. In of of and safety of an T-cell 2014; Scholar, B. C. I. S. C. R. et T-cell manufacturing for T-cell Res. 2015; PubMed Scopus Google Scholar CAR T cells have efficacy in is a of use in the results of clinical trial use have been A. B. C. R. S. G. S. C. M. J. In of of and safety of an T-cell 2014; Scholar, S. S. H. S. K. G. K. S. et clinical of engineered T cells in 2015; Google Scholar the quality of the patient cells for CAR the efficacy of the CAR T cell product be more has been that the of an T cell is with CAR T cell M. M. et T cell by signaling of chimeric antigen Med. 2015; PubMed Scopus Google Scholar the of of cell and of cell to the of cells that the and be to the CAR T cell the of CAR T cells in patients in the with B cell malignancies, out CAR T cell manufacturing of the safety and efficacy of CAR T cell in larger of patients the a number of manufacturing and regulatory challenges to be to a cellular therapy with a manufacturing process to a patient population. We are manufacturing the CD19 CAR T cell therapy CTL019 in a that the process of using the therapy we have with academic institutions and to both the product and process to that our are to high regulatory and manufacturing and addressing concerns the process of this to more patients.