T-Cell Immunotherapy: Looking Forward
Authors: Jacqueline Corrigan-Curay, Hans-Peter Kiem, David Baltimore, Marina O'Reilly, Renier J Brentjens, Laurence Cooper, Stephen Forman, Stephen Gottschalk, Philip Greenberg, Richard Junghans, Helen Heslop, Michael Jensen, Crystal Mackall, Carl June, Oliver Press, Daniel Powell, Antoni Ribas, Steven Rosenberg, Michel Sadelain, Brian Till, Amy P Patterson, Robert C Jambou, Eugene Rosenthal, Linda Gargiulo, Maureen Montgomery, Donald B Kohn
Journal: Molecular Therapy (2014)

The rapidly expanding field of T-cell immunotherapy has experienced clinical successes along with some serious toxicities. "T Cell Immunotherapy: Optimizing Trial Design," a workshop sponsored by the National Institutes of Health's (NIH's) Office of Biotechnology Activities (OBA), brought together researchers to discuss the scientific advances and share new data on key trial design issues, including the selection of new targets, optimizing the T-cell population, preconditioning regimens, strategies to promote persistence of cells, and analysis and management of acute reactions to T-cell infusions with the goal of identifying best practices and a research agenda that will facilitate further development and maximize the safety of this promising approach. Introduction T-cell immunotherapy for cancer is a rapidly growing field for gene therapy. Broadly, this field can be divided into two approaches—the use of gene-modified T-cell receptors (TCRs) in which recognition of the tumor antigen is in the context of human leukocyte antigens (HLAs) or use of chimeric antigen receptors (CARs) that typically link a single-chain variable region domain of an antibody (scFv) to one or more signaling elements of a TCR complex to allow T-cell activation.1Eshhar Z Waks T Gross G Schindler DG Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors.Proc Natl Acad Sci USA. 1993; 90: 720-724Crossref PubMed Scopus (1062) Google Scholar The decision to use one approach vs. the other may depend on several factors. For example, CARs offer the ability to bind antigens that are not restricted by HLA recognition, and the ability to modify the T-cell signaling moieties may offer "a broader functional effect than transduced" TCRs.2Sadelain M Brentjens R Riviere I The basic principles of chimeric antigen receptor design.Cancer Discov. 2013; 3: 388-398Crossref PubMed Scopus (857) Google Scholar TCRs, however, have the ability to recognize intracellular proteins, in addition to cell surface antigens, providing a broader array of target tumor-associated targets. In 2010, the OBA hosted a meeting to examine the state of the science and key trial design questions for this emerging field.3Ertl HC Zaia J Rosenberg S June C Dotti G Kahn J et al.Considerations for the clinical application of chimeric antigen receptor T cells: observations from a Recombinant DNA Advisory Committee Symposium held June 15, 2010.Cancer Res. 2011; 71: 3175-3181Crossref PubMed Scopus (68) Google Scholar At the time, some clinical benefit and unexpected toxicities highlighted both the therapeutic potential as well as the need to share data and expertise to optimize the safety of trial design. Since 2010, several promising and clinically successful developments have been reported in leading scientific and medical journals4Brentjens R Davila M Riviere I Park J Wang X Cowell L et al.CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.Sci Transl Med. 2013; 5: 177ra38Crossref PubMed Scopus (1502) Google Scholar,5Grupp SA Kalos M Barrett D Aplenc R Porter D Rheingold R et al.Chimeric antigen receptor–modified T cells for acute lymphoid leukemia.N Engl J Med. 2013; 368: 1509-1518Crossref PubMed Scopus (2497) Google Scholar,6Porter D Levine B Kalos M Bagg A June C Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.N Engl J Med. 2011; 365: 725-733Crossref PubMed Scopus (2627) Google Scholar,7Frantz S Engineered T-cell therapy shows efficacy in blood cancer.Nat Biotechnol. 2011; 29: 853-855Crossref PubMed Scopus (7) Google Scholar as well as national media. Given these developments, the OBA and the NIH Recombinant DNA Advisory Committee concluded that it was an opportune time to reconvene the leading experts in the field from the United States to continue to foster sharing of data across protocols and discuss the key issues in trial design, including optimal management of the cytokine release syndrome (CRS) seen in some research participants in response to the expansion of these active T cells. The following summary of the OBA workshop represents the views of the individual authors and not the NIH. The full presentations and slides are available at the OBA's website.8National Institutes of Health Office of Biotechnology Activities. Scientific symposium—T cell immunotherapy—optimizing trial design.http://osp.od.nih.gov/office-biotechnology-activities/event/2013-09-10-123000-2013-09-11-161500/scientific-symposium-t-cell-immunotherapy-optimizing-trial-designDate: September 2013Google Scholar State of the science The number of CAR and TCR protocols registered with the OBA has continued to increase rapidly (Figure 1); as of the meeting in September 2013 there were 111 protocols, 104 of which targeted cancer, with more than 500 subjects dosed. More than 40 protocols address hematological malignancies, with CD19 being the most common target in these protocols. Among protocols for solid tumors, the melanoma antigens (gp100, MART-1) and cancer-testis antigens predominate for TCRs; for CARs there are multiple targets, with a slight predominance of Her2/neu, GD2, and mesothelin (Figures 2 and 3). Approximately 90% of TCR trials have targeted solid malignancies; approximately 50% of CAR trials have targeted hematological malignancies.Figure 2Chimeric antigen receptor targets for hematological-malignancy protocols registered with the National Institutes of Health's Office of Biotechnology Activities.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Chimeric antigen receptor targets for solid-malignancy protocols registered with the National Institutes of Health's Office of Biotechnology Activities. CEA, carcinoembryonic antigen; EGFR, epidermal growth factor receptor; IL-3, interleukin-3; PSMA, prostate-specific membrane antigen; TAG-72, tumor-associated antigen 72; TNF, tumor necrosis factor; VEGFR, vascular endothelial growth factor receptor.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Steven Rosenberg reviewed the extensive portfolio of National Cancer Institute (NCI) research in this area, beginning with a summary of his research using unmodified tumor-infiltrating lymphocytes (TILs) against melanoma in 1988. He began using lymphodepletion before administration of TILs in 2002 and demonstrated increased efficacy.9Rosenberg S Dudley M Adoptive cell therapy for the treatment of patients with metastatic melanoma.Curr Opin Immunol. 2009; 21: 233-240Crossref PubMed Scopus (487) Google Scholar Dr. Rosenberg has continued to apply this approach to melanoma, including ocular melanoma, as well as metastatic gastrointestinal and human papillomavirus–induced cancers. These studies have demonstrated that in a subset of patients (about 20%), administration of T cells can result in prolonged remissions of five years or longer. The results led to a program of research dedicated to gene-modified T cells that accounts for almost 20% of T-cell immunotherapy protocols registered with the OBA to date. The results of the Rosenberg group's first trials with gene-modified TCRs for melanoma were published in 2006 in Science.10Morgan R Dudley M Wunderlich J Hughes M Yang J Sherry R et al.Cancer regression in patients after transfer of genetically engineered lymphocytes.Science. 2006; 314: 126-129Crossref PubMed Scopus (2107) Google Scholar In a recent TCR study targeting the cancer-testis antigen NY-ESO-1, the overall response rate was 50% in the 19 subjects with melanoma, including 4 with complete remissions, and a 67% overall response for those with synovial sarcoma, including one complete remission, in a population that had multiple prior chemotherapy regimens.11Robbins P Morgan R Feldman S Yang J Sherry R Dudley M et al.Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive in NY-ESO-1.J Clin Oncol. 2011; 29: 917-924Crossref PubMed Scopus (1227) Google Scholar These results contrasted with the MAGE-A3 trial in which an unexpected off-target neurological toxicity was seen.12Morgan R Chinnasamy N Abate-Daga D Gros A Robbins P Zheng Z et al.Cancer regression and neurological toxicity following anti-MAGE-A3 gene therapy.J Immunother. 2013; 26: 133-151Crossref Scopus (240) Google Scholar Rosenberg's group has also developed an extensive portfolio of CAR protocols, focusing primarily on solid tumors, with novel targets such as vascular endothelial growth factor receptor 2 (VEGFR2), epidermal growth factor receptor variant III (EGFRvIII), and mesothelin, as well as new targets in development, such as chondroitin sulfate proteoglycan 4 (CSP4). Antoni Ribas, who uses a vector developed by Rosenberg's lab, described his work on melanoma using a TCR-targeting MART-1 given with lymphodepletion. He has observed a high frequency of tumor responses (9 of 14 subjects with tumor-size reductions), but few responses were durable. He has also recently started enrolling research participants into a trial using a TCR-targeting NY-ESO-1. He noted that one of the aspects being tested is whether fresh cells are potentially more active than cryopreserved cells. Other highlights included clinical results from several investigators targeting CD19 in leukemia and lymphoma. In addition to Dr. Rosenberg's summary of his work in this area,13Kochenderfer J Wilson W Janik J Dudley M Stetler-Stevenson M Feldman S et al.Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19.Blood. 2010; 116: 4099-4102Crossref PubMed Scopus (984) Google Scholar Carl June, Renier Brentjens, Laurence Cooper, Stephen Forman, Michael Jensen, Helen Heslop, and Crystal Mackall summarized their results in ongoing trials using CD19-specific CARs in leukemia and lymphoma.4Brentjens R Davila M Riviere I Park J Wang X Cowell L et al.CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.Sci Transl Med. 2013; 5: 177ra38Crossref PubMed Scopus (1502) Google Scholar,6Porter D Levine B Kalos M Bagg A June C Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.N Engl J Med. 2011; 365: 725-733Crossref PubMed Scopus (2627) Google Scholar,14Brentjens R Riviere I Park J Davila M Wang X Stefanski J et al.Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias.Blood. 2011; 118: 4817-4828Crossref PubMed Scopus (994) Google Scholar Dr. Heslop noted that in a trial comparing first- and second-generation CARs, her group found that the second-generation CAR demonstrated both improved expansion and persistence.15Savoldo B Ramos C Liu E Mims MP Keating MJ Carrum G et al.CD28 costimulation improves expansion and persistence of chimeric antigen receptor–modified T cells in lymphoma patients.J Clin Invest. 2011; 121: 1822-1826Crossref PubMed Scopus (758) Google Scholar In addition, several protocols have established that administration of CAR T cells after stem cell transplant does not interfere with engraftment of the transplant. The investigators presented examples of clinical remissions, but, because the goal is often to establish remission so as to proceed with a curative transplant, the durability of remissions from CAR T cells without subsequent transplant has not yet been determined. However, even in the setting of multiple previous therapies, CD19-specific CARs have shown efficacy. Dr. Brentjens reported that in his protocol with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL), 14 of 16 subjects achieved molecular chronic remissions as assessed by deep-sequencing PCR analysis to search for the malignant clone.16Davila M Riviere I Wang X Bartido S Park J Curran K et al.Efficacy and toxicity management of 19–28z CAR T cell therapy in a B cell acute lymphoblastic leukemia.Sci Transl Med. 2014; 6: 224ra25Crossref PubMed Scopus (1760) Google Scholar Another emerging theme was the responsiveness of ALL to this approach, which was also highlighted in Dr. June's and Dr. Mackall's presentations. Dr. Cooper presented data from ongoing trials infusing CD19-specific CAR+ T cells after autologous and allogeneic hematopoietic stem cell transplantation. The intent was to augment the graft-vs.-tumor effect, recognizing that the current clinical practice for many patients with B-cell malignancies is to infuse tumor-specific T cells as a bridge to transplantation. These trials have advanced a new approach to human gene therapy based on the electrotransfer of DNA plasmids encoding a second-generation CAR stably expressed following transposition from the Sleeping Beauty (SB) system. In parallel to work on CD19-specific CARs, Brian Till highlighted the results of his trials targeting CD20, including a trial that used a third-generation CAR with CD28 and 4–1BB costimulatory domains. Unlike the other trials, which use retroviral vectors or SB transposons, he used an electroporated DNA plasmid. In general, the T cells were well tolerated, with some immediate febrile reactions, and two of the three subjects had prolonged remissions with persistence of the T cells for up to a year.17Till B Jensen M Wang J Qian X Gopal A Maloney D et al.CD20-specific adoptive immunotherapy for lymphomas using a chimeric antigen receptor with both CD28 and 4–1BB domains: pilot clinical trial results.Blood. 2012; 119: 3940-3950Crossref PubMed Scopus (405) Google Scholar However, the DNA plasmid vector was not an efficient vector, and the IL-2 used to promote persistence also led to an increase in T regulatory cells (Tregs). Philip Greenberg highlighted his group's work using a TCR targeting another hematological malignancy antigen, Wilms tumor antigen 1 (WT1), which is highly expressed in leukemia and some solid tumors but is also expressed on some normal tissues. Their trial built on a previous trial using naturally isolated, cloned T cells targeting WT1, which did not show toxicity but had limited efficacy. Using virus-specific T cells, they have recently initiated a trial to test a TCR based on a high-avidity, natural clone. In the solid-tumor area, Dr. Heslop presented a summary of her group's trials for neuroblastoma, targeting GD2 using both virus-specific and non-virus-specific T cells.18Louis C Savoldo B Dotti G Pule M Yvon E Meyers G et al.Antitumor activity and long-term fate of chimeric antigen receptor–positive T cells in patients with neuroblastoma.Blood. 2011; 118: 6050-6056Crossref PubMed Scopus (818) Google Scholar,19Pule MA Savoldo B Myers GD Rossig C Russell HV Dotti G et al.Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma.Nat Med. 2008; 14: 1264-1270Crossref PubMed Scopus (921) Google Scholar Their data have demonstrated an association between persistence of T cells and reduced tumor progression. In addition, in research participants with prolonged detection of activated T cells, the presence of central memory T cells was important, raising the question of what the optimal T-cell product is. Other solid-tumor trials discussed included CARs targeting HER2/neu for sarcoma and glioblastoma, including a trial using tri-virus-specific T cells and another trial that combines the CAR with a dominant-negative TGF-β receptor. Data were also presented on first- and second-generation CARs targeting carcinoembryonic antigen (CEA) and prostate-specific membrane antigen. Again, some early indications of clinical efficacy were promising, but an ongoing challenge will be to refine strategies to improve T-cell persistence and efficacy. In some cases, on-target, off-tissue toxicities may ultimately limit the use of certain targets; for example, colitis developed in protocols using CEA-specific TCR and CAR T cells.20Parkhurst M Yang J Langan R Dudley M Nathan D Feldman S et al.T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis.Mol Ther. 2011; 19: 620-626Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar Finally, Dr. Jensen reported his work in glioblastoma using a novel CAR called a zetakine. Instead of an antibody, single-chain target domain, he used a human cytokine, IL-13, with a mutation in the sequence that gave high affinity for IL-13 receptor α2. These cells were infused intracranially, establishing the safety of intracranial administration with some antitumor responses. These talks provided an overview of a field that continues to expand rapidly, in terms of both targets and diseases. Most protocols involve administration of the cells in the setting of lymphodepletion, and some groups, predominantly in protocols for solid tumors, use IL-2 to promote cell persistence. In addition to identifying effective targets that have minimal off-tumor effects, finding the ideal balance between persistence and expansion of T cells without triggering systemic cytokine reactions is a key issue for the field. This may be achieved by such strategies as including the design of the cells, the type of T cells infused, the dose, the immune status of the recipient, and the use of cytokine support. Finally, as with many cancer therapies, some toxicity is likely. Establishing protocols to limit toxicity so that the risk-to-benefit ratio remains favorable is a high priority. Promoting T-cell persistence Persistence of the gene-modified T cells is associated with prolonged remission in subjects,18Louis C Savoldo B Dotti G Pule M Yvon E Meyers G et al.Antitumor activity and long-term fate of chimeric antigen receptor–positive T cells in patients with neuroblastoma.Blood. 2011; 118: 6050-6056Crossref PubMed Scopus (818) Google Scholar and the field has developed strategies to promote persistence. One approach is to create a host environment that is conducive to expansion of the T cells. Expansion should not only promote a rigorous antitumor effect but also lead to the development of a stable population of tumor-specific T cells that can be reactivated in case of recurrence of tumor antigen. Use of selected central memory T cells may be another strategy to promote an enduring T-cell population. The majority of T-cell protocols registered with the OBA to date involve administration of the cells to subjects when they are lymphopenic. For solid-tumor protocols, this involves administration of the T cells after administration of lymphodepleting chemotherapy, such as cyclophosphamide, whereas the protocols for hematological malignancies have most commonly called for administering cells in the posttransplant setting or the use of disease-specific chemotherapy regimens. However, it is important to note that lymphodepletion has not been universally applied, notable exceptions being studies administering virus-specific T cells, or the successful neuroblastoma protocols targeting GD2, which used both virus-specific and non-virus-specific T cells.18Louis C Savoldo B Dotti G Pule M Yvon E Meyers G et al.Antitumor activity and long-term fate of chimeric antigen receptor–positive T cells in patients with neuroblastoma.Blood. 2011; 118: 6050-6056Crossref PubMed Scopus (818) Google Scholar Dr. Rosenberg reviewed his group's clinical data, as well as the animal data that support lymphodepletion for promoting antitumor efficacy. As stated earlier, in the TIL melanoma studies, despite administration of 109 to 1010 T cells, the cells did not persist and there were minimal objective responses.21Dudley M Wunderlich J Nishimura M Yu D Yang J Topalian S et al.Adoptive transfer of cloned melanoma-reactive T lymphocytes for the treatment of patients with metastatic melanoma.J Immunother. 2001; 24: 363-373Crossref PubMed Scopus (301) Google Scholar However, when nonmyeloablative (NMA) chemotherapy using cyclophosphamide and fludarabine was added, and the TIL product was generated with a shorter culture time, providing a more diverse TIL population that contained both CD4+ and CD8+ T cells, T-cell persistence was enhanced and 6 of 13 subjects showed objective cancer responses.22Rosenberg S Dudley M Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes.Proc Natl Acad Sci USA. 2004; 101: 14639-14645Crossref PubMed Scopus (315) Google Scholar Dr. Rosenberg's group went on to investigate whether the addition of 2 or 12 Gy of total-body irradiation (TBI) to the NMA chemotherapy would further increase efficacy of TIL transfer in melanoma patients. The response rate for those who received chemotherapy alone was about 49%; the addition of 2 Gy resulted in objective response in 52% of subjects, and 12 Gy of TBI resulted in a 72% objective response rate, with a complete response rate of 40%.23Dudley M Yang J Sherry R Hughes M Royal R Kammula U et al.Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens.J Clin Oncol. 2008; 26: 5233-5239Crossref PubMed Scopus (1084) Google Scholar The addition of TBI to NMA chemotherapy was generally well tolerated, with the exception of one death in a subject with an undetected diverticular abscess in the 12-Gy group. A drawback of escalation to 12 Gy of radiation is the need for autologous peripheral blood stem cell support. An ongoing randomized trial is comparing NMA chemotherapy against NMA and TBI, although preliminary results indicate that the challenges of adding TBI may not be balanced by the improved response. A significant amount of animal work has been done to elucidate the mechanisms that underlie the improved antitumor responses observed with lymphodepletion. These data indicate that lymphodepletion augments the antitumor response by eliminating Tregs, cellular "sinks" for cytokines such as IL-7 and IL-15, and by enhancing antigen-presenting cell activation and availability.24Antony P Piccirillo C Akpinarli A Finkelstein S Speiss P Surman D et al.CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells.J Immunol. 2005; 174: 2591-2601Crossref PubMed Scopus (595) Google Scholar,25Gattinoni L Finkelstein S Klebanoff C Antony P Palmer D Spiess P et al.Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells.J Exp Med. 2005; 202: 907-912Crossref PubMed Scopus (796) Google Scholar,26Paulos CM Wrzesinski C Kaiser A Hinrichs C Chieppa M Cassard L et al.Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling.J Clin Invest. 2007; 117: 492-501Crossref PubMed Scopus (384) Google Scholar This activation of the immune system may be due in part to translocation of bacteria from the gut. It was shown in a mouse model that administration of ciprofloxacin, which is effective against Gram-negative bacteria commonly found in the gut, to an irradiated animal reduced the activated dendritic cells in the spleen and reduced the effectiveness of adoptive cell transfer. Of note, it has been demonstrated that the effect of lymphodepletion is on the host rather than on the tumor. Thus, if one shields the host—in this case, the mouse—and treats the tumor, no effect is seen in these melanoma models. One dilemma is that Tregs are the first T cells to recover after lymphodepletion, and therefore lymphodepletion may foster an environment that works against the antitumor effect. Dr. Rosenberg noted that the NCI group has some data demonstrating an inverse relationship between the recovery of Tregs and objective antitumor response, supporting the importance of eliminating Tregs. However, others questioned whether we clearly understand the role of Tregs, because suppression of a tumor response may depend on whether the Tregs are actually activated and tumor-specific. Therefore, the presence of Tregs may not be absolutely undesirable, as they may also organize the immune response. Gene delivery and design of T cells In addition to host preparation, the design of the T-cell vectors is a critical area of research. Dr. Cooper noted that the ability to stably express transgenes, such as CARs, in T cells has revolutionized adoptive immunotherapy for certain malignancies. Recombinant fusion genes constructed to recognize tumor-associated antigens (e.g., TCR and CAR) have been constitutively expressed in T cells using Moloney murine leukemia virus (MMLV)-based retroviruses, HIV-based lentiviruses, and DNA plasmids, including the SB transposon/transposase system. Until recently, retroviral transduction by recombinant MMLV-derived vectors has been the most common method for delivery of transgenes intended to be integrated into the T-cell genome. Lentiviral vectors have also been successfully used in the clinic. Both approaches are appealing, and at this time there appears to be equipoise regarding the therapeutic potential of these two viral systems for genetic modification of T cells to express CARs. Transduction using retroviral and lentiviral vectors can be highly efficient, and it is possible to integrate multiple copies of a transgene in a given T cell, which provides for a high level of expression of the transduced gene product. The manufacture of clinical-grade retroviral and lentiviral vector virions is quite similar, although retroviral vectors may be produced from stable packaging cell lines, whereas to date most lentiviral vectors have been produced by transient transfection. Overall, transduction of T cells with recombinant retrovirus and lentivirus involve similar packaging protocols, utilize similar integration mechanisms, and lead to similar transduction efficiencies. Thus, both viral-based approaches to gene transfer are appealing for the human application of CAR+ T cells, although some individual investigators have strong preferences. DNA transposons now offer an alternative to viral-based gene transfer. Supercoiled plasmids can be directly electroporated into T cells using commercial devices, thus eliminating much of the labor and safety concerns associated with generating recombinant viral particles. DNA transposons, such as those derived from the SB system, insert into the genome via a copy-and-paste mechanism when a transposase is (transiently) available to catalyze the reaction. Dr. Cooper's group has successfully used SB to integrate a CD19-specific CAR into human T cells in four human trials under investigational new drug applications. Unlike retroviral/lentiviral integration into transcriptionally active sites, the SB transposon appears to randomly integrate at TA dinucleotide repeats and is typically present at one or two copies per T-cell genome. As with viral-based gene transfer, there is the possibility that a transposon may cause genotoxicity resulting in oncogenesis. However, because the SB system does not readily target transcriptional or promoter elements, it appears suitable for human application. Furthermore, the relatively low cost of generating DNA plasmids for use in compliance with current good manufacturing practice (GMP), in contrast to the cost and complexity of producing clinical-grade virus, renders the SB system an attractive and nimble approach to generate and modify vectors for delivery of therapeutic genes. In summary, the investigator has available multiple approaches to genetically modify T cells. The use of a particular approach will depend on resident expertise and the desired T-cell product. Design of CARs. CARs are recombinant receptors for antigens that retarget and eventually reprogram T-cell function. Unlike the physiological TCR for antigens, which signals T-cell activation through the associated CD3 complex, CARs possess in a single molecule the ability to trigger multiple antigen-specific T-cell functions. The CARs that have recently shown impressive clinical outcomes in research participants with B-cell malignancies are "second-generation CARs," to distinguish them from earlier forms of activating fusion receptors, which only initiate T-cell activation and are now referred to as "first-generation CARs."27Sadelain M Brentjens R Riviere I The promise and potential pitfalls of chimeric antigen receptors.Curr Opin Immunol. 2009; 21: 215-223Crossref PubMed Scopus (364) Google Scholar Michel Sadelain described how the incorporation of co-stimulatory receptor signaling domains into the cytoplasmic tails of CAR ("embedded costimulation") greatly increased the potency of CAR-modified T cells in preclinical models.4Brentjens R Davila M Riviere I Park J Wang X Cowell L et al.CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.Sci Transl Med. 2013; 5: 177ra38Crossref PubMed Scopus (1502) Google Scholar,28Maher J Brentjens R Gunset G Riviere 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,29Brentjens R Santos E Nikhamin Y Yeh R Matsushita M La Perle K et al.Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts.Clin Cancer Res. 2007; 13: 5426-5435Crossref PubMed Scopus (340) Google Scholar Sev