Global Manufacturing of CAR T Cell Therapy
2017; Cell Press; Volume: 4; Linguagem: Inglês
10.1016/j.omtm.2016.12.006
ISSN2329-0501
AutoresBruce L. Levine, James Miskin, Keith Wonnacott, Christopher Hunt Keir,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoImmunotherapy 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 density and maintains cell viability.14Powell Jr., D.J. Brennan A.L. Zheng Z. Huynh H. Cotte J. Levine B.L. Efficient clinical-scale enrichment of lymphocytes for use in adoptive immunotherapy using a modified counterflow centrifugal elutriation program.Cytotherapy. 2009; 11: 923-935Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Separation of T cell subsets at the level of CD4/CD8 composition using specific antibody bead conjugates or markers is an additional step that may be performed.15Riddell S.R. Sommermeyer D. Berger C. Liu L.S. Balakrishnan A. Salter A. Hudecek M. Maloney D.G. Turtle C.J. Adoptive therapy with chimeric antigen receptor-modified T cells of defined subset composition.Cancer J. 2014; 20: 141-144Crossref PubMed Scopus (82) Google Scholar Purifying autologous antigen-presenting cells (APCs) from the patient to use for T cell activation would require several additional steps, making it labor intensive and difficult to obtain a potent CAR T cell product.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 For this reason, an approach was developed to activate T cells in a more standardized, efficient manner using, for example, beads coated with anti-CD3/anti-CD28 monoclonal antibodies (Life Technologies). While the use of anti-CD3 antibodies alone or in combination with feeder cells and growth factors, such as IL-2, has been the practice for many years, in comparison to beads coated with anti-CD3/anti-CD28 monoclonal antibodies or cell-based artificial APCs (aAPCs), the activation and ex vivo expansion are suboptimal.16Levine B.L. Bernstein W.B. Connors M. Craighead N. Lindsten T. Thompson C.B. June C.H. Effects of CD28 costimulation on long-term proliferation of CD4+ T cells in the absence of exogenous feeder cells.J. Immunol. 1997; 159: 5921-5930PubMed Google Scholar, 17Suhoski M.M. Golovina T.N. Aqui N.A. Tai V.C. Varela-Rohena A. Milone M.C. Carroll R.G. Riley J.L. June C.H. Engineering artificial antigen-presenting cells to express a diverse array of co-stimulatory molecules.Mol. Ther. 2007; 15: 981-988Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar The beads, or aAPCs, can be easily removed from the culture through magnetic separation.18Levine B.L. Personalized cell-based medicine: activated and expanded T cells for adoptive immunotherapy.BioProcessing J. 2007; 6: 14-19Crossref Google Scholar In the presence of interleukin-2 and aAPCs, T cells can grow logarithmically in a perfusion bioreactor for several weeks.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, 16Levine B.L. Bernstein W.B. Connors M. Craighead N. Lindsten T. Thompson C.B. June C.H. Effects of CD28 costimulation on long-term proliferation of CD4+ T cells in the absence of exogenous feeder cells.J. Immunol. 1997; 159: 5921-5930PubMed Google Scholar, 18Levine B.L. Personalized cell-based medicine: activated and expanded T cells for adoptive immunotherapy.BioProcessing J. 2007; 6: 14-19Crossref Google Scholar, 19Hami L.S. Green C. Leshinsky N. Markham E. Miller K. Craig S. GMP production and testing of Xcellerated T Cells for the treatment of patients with CLL.Cytotherapy. 2004; 6: 554-562Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 20Somerville R.P. Dudley M.E. Bioreactors get personal.OncoImmunology. 2012; 1: 1435-1437Crossref PubMed Scopus (24) Google Scholar The use of aAPCs derived from the chronic myelogenous leukemia cell line K562, which can be engineered to express the required costimulatory ligands, also has been investigated as a method of expanding T cells ex vivo.17Suhoski M.M. Golovina T.N. Aqui N.A. Tai V.C. Varela-Rohena A. Milone M.C. Carroll R.G. Riley J.L. June C.H. Engineering artificial antigen-presenting cells to express a diverse array of co-stimulatory molecules.Mol. Ther. 2007; 15: 981-988Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 21Maus M.V. Thomas A.K. Leonard D.G. Allman D. Addya K. Schlienger K. Riley J.L. June C.H. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB.Nat. Biotechnol. 2002; 20: 143-148Crossref PubMed Scopus (367) Google Scholar Culture conditions may be further refined to polarize T cells to a specific phenotype (i.e., Th2 or Th17) during expansion. Indeed, CAR T cells that were polarized to a Th17 phenotype demonstrated efficacy in a preclinical model, suggesting that T cell polarization is a strategy that may enter the clinic in the future.22Guedan S. Chen X. Madar A. Carpenito C. McGettigan S.E. Frigault M.J. Lee J. Posey Jr., A.D. Scholler J. Scholler N. et al.ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells.Blood. 2014; 124: 1070-1080Crossref PubMed Scopus (223) Google Scholar During the activation process, the T cells are incubated with the viral vector encoding the CAR, and, after several days, the vector is washed out of the culture by dilution and/or medium exchange. The viral vector uses viral machinery to attach to the patient cells, and, upon entry into the cells, the vector introduces genetic material in the form of RNA.23Coffin J. Hughes S. Varmus H. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997Google Scholar In the case of CAR T cell therapy, this genetic material encodes the CAR. The RNA is reverse-transcribed into DNA and permanently integrates into the genome of the patient cells; therefore, CAR expression is maintained as the cells divide and are grown to large numbers in the bioreactor. The CAR is then transcribed and translated by the patient cells, and the CAR is expressed on the cell surface. Lentiviral vectors, which have a safer integration site profile than gammaretroviral vectors,24McGarrity G.J. Hoyah G. Winemiller A. Andre K. Stein D. Blick G. Greenberg R.N. Kinder C. Zolopa A. Binder-Scholl G. et al.Patient monitoring and follow-up in lentiviral clinical trials.J. Gene Med. 2013; 15: 78-82Crossref PubMed Scopus (79) Google Scholar, 25Montini E. Cesana D. Schmidt M. Sanvito F. Bartholomae C.C. Ranzani M. Benedicenti F. Sergi L.S. Ambrosi A. Ponzoni M. et al.The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy.J. Clin. Invest. 2009; 119: 964-975Crossref PubMed Scopus (462) Google Scholar are commonly used in clinical trials of CAR T cell therapies, including CTL019. Other methods of gene transfer, including the Sleeping Beauty transposon system or mRNA transfection, have been investigated as alternative approaches to express a CAR in T cells.26Huls M.H. Figliola M.J. Dawson M.J. Olivares S. Kebriaei P. Shpall E.J. Champlin R.E. Singh H. Cooper L.J. Clinical application of Sleeping Beauty and artificial antigen presenting cells to genetically modify T cells from peripheral and umbilical cord blood.J. Vis. Exp. 2013; : e50070PubMed Google Scholar, 27Singh H. Figliola M.J. Dawson M.J. Olivares S. Zhang L. Yang G. Maiti S. Manuri P. Senyukov V. Jena B. et al.Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells.PLoS ONE. 2013; 8: e64138Crossref PubMed Scopus (132) Google Scholar CAR T cells generated using transient mRNA transfection have been used in the clinic; however, this approach requires several rounds of CAR T cell infusion.28Beatty G.L. Haas A.R. Maus M.V. Torigian D.A. Soulen M.C. Plesa G. Chew A. Zhao Y. Levine B.L. Albelda S.M. et al.Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies.Cancer Immunol Res. 2014; 2: 112-120Crossref PubMed Scopus (602) Google Scholar Furthermore, while the Sleeping Beauty transposon system is considered to be inexpensive and has been tested in early-phase clinical trials, there are still several concerns, including efficiency relative to lentiviral vectors, the unknown potential of insertional mutagenesis, and remobilization of transposons.29Aronovich E.L. McIvor R.S. Hackett P.B. The Sleeping Beauty transposon system: a non-viral vector for gene therapy.Hum. Mol. Genet. 2011; 20: R14-R20Crossref PubMed Scopus (110) Google Scholar Bioreactor culture systems are designed to provide the optimal gas exchange requirements and culture mixing necessary to grow large numbers of cells for clinical use (Figure 2). The WAVE Bioreactor (now known as the Xuri; GE Healthcare Life Sciences), which utilizes a rocking platform, has been used to expand the CD19-targeted CAR T cell therapy CTL019.11Levine 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, 20Somerville R.P. Dudley M.E. Bioreactors get personal.OncoImmunology. 2012; 1: 1435-1437Crossref PubMed Scopus (24) Google Scholar, 30Somerville R.P.T. Devillier L. Parkhurst M.R. Rosenberg S.A. Dudley M.E. Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE® bioreactor.J. Transl. Med. 2012; 10: 69Crossref PubMed Scopus (68) Google Scholar Another culture system that can be used is the G-Rex (Wilson Wolf), which has the ability to expand cells from low seeding densities.31Jin J. Sabatino M. Somerville R. Wilson J.R. Dudley M.E. Stroncek D.F. Rosenberg S.A. Simplified method of the growth of human tumor infiltrating lymphocytes in gas-permeable flasks to numbers needed for patient treatment.J. Immunother. 2012; 35: 283-292Crossref PubMed Scopus (23) Google Scholar, 32Bajgain P. Mucharla R. Wilson J. Welch D. Anurathapan U. Liang B. Lu X. Ripple K. Centanni J.M. Hall C. et al.Optimizing the production of suspension cells using the G-Rex "M" series.Mol. Ther. Methods Clin. Dev. 2014; 1: 14015Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar The G-Rex uses gas-permeable membranes, allowing the flask to be placed directly in a cell culture incubator. One drawback of this system, however, is that the flask must be opened during cell inoculation. The CliniMACS Prodigy (Miltenyi Biotec) is a single device that accomplishes cell preparation, enrichment, activation, transduction, expansion, final formulation, and sampling.33Kaiser A.D. Assenmacher M. Schröder B. Meyer M. Orentas R. Bethke U. Dropulic B. Towards a commercial process for the manufacture of genetically modified T cells for therapy.Cancer Gene Ther. 2015; 22: 72-78Crossref PubMed Scopus (131) Google Scholar This contrasts with other methods, which use separate machines for the cell culture, cell washing, and other steps in the preparation. It was shown recently that the CliniMACs Prodigy is feasible for generating CAR T cells, and this device is expected to be used soon to prepare CAR T cells for clinical trials.34Mock U. Nickolay L. Cheung G. Zhan H. Peggs K. Johnston I. Kaiser A. Pule M. Thrasher A. Qasim W. Automated lentiviral transduction of T cells with CARs using the CliniMACS Prodigy.Blood. 2015; 126: 2043Google Scholar When the cell expansion process is finished, the cell culture, which may reach a volume of up to ≈5 L, must be concentrated to a volume that can be infused into the patient.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, 18Levine B.L. Personalized cell-based medicine: activated and expanded T cells for adoptive immunotherapy.BioProcessing J. 2007; 6: 14-19Crossref Google Scholar The washed and concentrated cells are cryopreserved in infusible medium, and, following product release, the frozen cells are transported to and thawed at the center where the patient will be treated. While aspects of cell washing, isolation, and culture are semi-automated, improving the throughput of currently manual parts of processing will be critical for developing CAR T cell therapies that can be used for a wider range of indications and larger populations. Although protocols for manufacturing clinical-grade CAR T cells have now been established, CAR T cell therapies have been used to treat only a few hundred patients to date. When scaling out this complex manufacturing process to treat more patients in larger trials at an increased number of clinical centers, the process should be carefully evaluated to ensure production efficiency without compromising the integrity and potency of the final product. Because CAR T cells can be used to target several types of cancer, the scale of production for the vector and the CAR T cells also will depend on the incidence of each indication. Additional considerations include generating consistently high-quality vector for predictable genetic modification of cells, understanding the long-term safety of gene therapy, and anticipating global regulatory concerns. In the United States, the viral vector used to transduce the CAR into T cells is considered a key raw material of the CAR T cell manufacturing process, and the modified T cell is considered the investigational final product, also known as the medicinal product in the European Union. In contrast to the final CAR T cell product, which must be individually generated for each patient, the viral vector encoding the CAR can be made in large quantities and stored at −80°C for 4 years in our experience. Other reports suggest that frozen viral vector stocks are stable for up to 9 years at this temperature.35Lamers C.H. van Elzakker P. Luider B.A. van Steenbergen S.C. Sleijfer S. Debets R. Gratama J.W. Retroviral vectors for clinical immunogene therapy are stable for up to 9 years.Cancer Gene Ther. 2008; 15: 268-274Crossref PubMed Scopus (15) Google Scholar, 36Przybylowski M. Hakakha A. Stefanski J. Hodges J. Sadelain M. Rivière I. Production scale-up and validation of packaging cell clearance of clinical-grade retroviral vector stocks produced in cell factories.Gene Ther. 2006; 13: 95-100Crossref PubMed Scopus (27) Google Scholar As with the CAR T cell manufacturing process, generation of the vector stocks must take place in Good Manufacturing Practice (GMP) facilities. The sterility of the vector is crucial because the final CAR T cell product cannot be sterilized by filtration; manufacture of the vector under controlled, clean room conditions with minimal open processing and sterile filtration during the final aseptic stages of production, all supported and verified by an array of safety testing, ensures sterility and the absence of packaging cells from the final vector product. In addition, use of a third-generation minimal lentiviral vector, incorporating key safety features, enhances safety.37Kim V.N. Mitrophanous K. Kingsman S.M. Kingsman A.J. Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1.J. Virol. 1998; 72: 811-816Crossref PubMed Google Scholar, 38Dull T. Zufferey R. Kelly M. Mandel R.J. Nguyen M. Trono D. Naldini L. A third-generation lentivirus vector with a conditional packaging system.J. Virol. 1998; 72: 8463-8471Crossref PubMed Google Scholar In our experience, the batch manufacture of viral vector for cellular therapies takes a minimum of 2 weeks. Most of this time is spent growing adequate numbers of cells, such as HEK293T cells, to produce large quantities of replication-defective viral vector.39Wang X. Olszewska M. Qu J. Wasielewska T. Bartido S. Hermetet G. Sadelain M. Rivière I. Large-scale clinical-grade retroviral vector production in a fixed-bed bioreactor.J. Immunother. 2015; 38: 127-135Crossref PubMed Scopus (45) Google Scholar Starting from a cryopreserved aliquot of an appropriate working cell bank, the cells are expanded in culture for several days to the appropriate number for production, allowing considerable expansion from the original number of cells seeded. The cells are then transfected with plasmids that collectively result in the production of the minimal lentiviral vector. These plasmids are typically (1) a Gag/Pol packaging construct that encodes the viral structural proteins (Gag) and enzymes (Pol); (2) a construct encoding a suitable envelope glycoprotein from a heterologous source, resulting in vector particle pseudotyping (e.g., VSV-G); (3) a construct for the expression of viral accessory protein Rev; along with (4) a vector plasmid encoding the CAR construct as well as other sequences required for efficient reverse transcription, RNA packaging, and integration.40Schambach A. Zychlinski D. Ehrnstroem B. Baum C. Biosafety features of lentiviral vectors.Hum. Gene Ther. 2013; 24: 132-142Crossref PubMed Scopus (109) Google Scholar The vector systems should employ a number of key safety features that collectively prevent the reacquisition of replication competence (e.g., codon-optimized Gag/Pol that minimizes homology between vector components to prevent recombination, self-inactivating long terminal repeat sequences, and removal of all unnecessary sequences and accessory genes).41Yu S.F. von Rüden T. Kantoff P.W. Garber C. Seiberg M. Rüther U. Anderson W.F. Wagner E.F. Gilboa E. Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells.Proc. Natl. Acad. Sci. USA. 1986; 83: 3194-3198Crossref PubMed Scopus (402) Google Scholar, 42Kotsopoulou E. Kim V.N. Kingsman A.J. Kingsman S.M. Mitrophanous K.A. A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene.J. Virol. 2000; 74: 4839-4852Crossref PubMed Scopus (200) Google Scholar, 43Zufferey R. Dull T. Mandel R.J. Bukovsky A. Quiroz D. Naldini L. Trono D. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.J. Virol. 1998; 72: 9873-9880Crossref PubMed Google Scholar Within 48 hr of transfection, the production cells begin to release CAR-expressing lentiviral vector, which can be collected from the culture medium.39Wang X. Olszewska M. Qu J. Wasielewska T. Bartido S. Hermetet G. Sadelain M. Rivière I. Large-scale clinical-grade retroviral vector production in a fixed-bed bioreactor.J. Immunother. 2015; 38: 127-135Crossref PubMed Scopus (45) Google Scholar Over the course of several days, medium exchange allows for the harvest of multiple batches of vector-containing medium, typically two harvests. After filtration to remove production cells and debris, the viral vector is purified through downstream processing to enrich for the viral vector while removing impurities and to formulate the vector into an appropriate storage buffer. In our experience, the vector may be frozen at this point to allow for the production of multiple sub-batches to make larger quantities of the final vector product for increased economic efficiency. Once the target quantity of vector is available, a further GMP process, involving sterilizing filtration and vialing under aseptic conditions, is performed. Once production is complete, the vector is cryopreserved until later use. It is important to establish quality control testing for safety, sterility, purity, potency, identity, and titer so that manufacturing centers can be assured that each batch of vector meets defined standards before it is used to transduce T cells.44Cross P.J. Levine B.L. Assays for the release of cellular gene therapy products.in: Dropulic B. Carter B.J. Concepts in Genetic Medicine. John Wiley & Sons, 2006: 307-318Google Scholar These quality control tests are described in guidance documents written by the U.S. Food and Drug Administration (FDA) and are briefly summarized here. Safety testing, which can involve preclinical experimentation or toxicity studies in animals, aims to determine that the product is safe for humans when administered appropriately. The sterility of the product is tested to ensure that it is free of contaminating microorganisms; in the case of gammaretroviral vectors and lentiviral vectors, assays for replication-competent retroviruses/lentiviruses (RCRs/RCLs) and mycoplasma testing of the cells and media used in the process help determine that the final CAR T cell product is free from adventitious agents and safe for infusion into patients. Impurities in the vector product are broadly tested to (1) verify consistent purification afforded by the vector manufacturing process, and (2) consequently ensure that the quality of the vector is consistent prior to its use in the T cell manufacturing process. Impurity testing includes testing for process-related impurities, such as Benzonase (Merck KGaA; used to degrade and facilitate the removal of DNA) and bovine serum albumin (originating from fetal bovine serum), and it also includes characterization of both residual host cell DNA and residual plasmid DNA. In addition, the T cells are tested for bacterial and fungal contamination from the cell culture process. The potency of the vector product is tested to assess whether it functions as anticipated, and its identity is proven through relevant physical, chemical, or biological tests. For example, the titer of viral vector can be measured by analyzing the percentage of healthy donor cells transduced by predefined MOIs, and this can verify whether a particular batch of vector is expected to optimally transduce patient cells.39Wang X. Olszewska M. Qu J. Wasielewska T. Bartido S. Hermetet G. Sadelain M. Rivière I. Large-scale clinical-grade retroviral vector production in a fixed-bed bioreactor.J. Immunother. 2015; 38: 127-135Crossref PubMed Scopus (45) Google Scholar In early clinical trials performed at the University of Pennsylvania, multiple vector suppliers were used during the generation of the CD19-targeting therapy CTL019. Using vectors from different suppliers as starting material raises additional q
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