Safer CARS
Authors: Helen E Heslop
Journal: Molecular Therapy (2010)

Genetically modifying T cells to express synthetic chimeric antigen receptors (CARs)1Sadelain M Riviere I Brentjens R Targeting tumours with genetically enhanced T lymphocytes.Nat Rev Cancer. 2003; 3: 35-45Crossref PubMed Scopus (408) Google Scholar is an attractive strategy for producing antitumor effects. The first-generation CARs usually consisted of the single-chain variable fragment of an antibody specific for tumor antigen linked to the transmembrane and intracellular signaling domains of either CD3ζ or FcRγ. This produces human leukocyte antigen (HLA)-unrestricted activation of the modified cytotoxic T lymphocytes (CTLs) upon encounter with tumor antigen, leading to lysis of tumor cells in vitro and eradication of tumor in mouse models. Because CARs bind to target antigens in an HLA-unrestricted manner, they are resistant to many tumor-immune evasion mechanisms, such as downregulation of HLA class I molecules or failure to process or present proteins. However, efficacy was modest in clinical trials in subjects with lymphoma, neuroblastoma, or ovarian or renal cancer2Till BG Jensen MC Wang J Chen EY Wood BL Greisman HA et al.Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells.Blood. 2008; 112: 2261-2271Crossref PubMed Scopus (566) Google Scholar,3Kershaw MH Westwood JA Parker LL Wang G Eshhar Z Mavroukakis SA et al.A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer.Clin Cancer Res. 2006; 12: 6106-6115Crossref PubMed Scopus (934) Google Scholar,4Lamers CH Sleijfer S Vulto AG Kruit WH Kliffen M Debets R et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.J Clin Oncol. 2006; 24: e20-e22Crossref PubMed Scopus (703) Google Scholar because of incomplete activation of T cells with limited expansion and persistence in vivo. Normal T-cell activation is dependent on receipt of two classes of signal: one through the engagement of the T-cell receptor (TCR) with antigen presented in the context of the major histocompatibility complex and a second through engagement of costimulatory molecules such as CD28, OX40, and CD40L. Tumors often do not express appropriate ligands for costimulatory molecules, and engagement of first-generation CARs in the absence of costimulation leads to anergy and failure of in vivo expansion. To overcome these limitations, second-generation CARs were developed to incorporate the intracellular domains of one or more costimulatory molecules such as CD28, OX40, and 4-1BB within the endodomain, and these improved antigen-specific T-cell activation and expansion.1Sadelain M Riviere I Brentjens R Targeting tumours with genetically enhanced T lymphocytes.Nat Rev Cancer. 2003; 3: 35-45Crossref PubMed Scopus (408) Google Scholar,5Maher J Brentjens RJ 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 (712) Google Scholar Third-generation CARs include a combination of costimulatory endodomains.6Zhao Y Wang QJ Yang S Kochenderfer JN Zheng Z Zhong X et al.A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity.J Immunol. 2009; 183: 5563-5574Crossref PubMed Scopus (240) Google Scholar An alternative approach to providing costimulation is to express CARs in antigen-specific T cells, which will then also be activated and expanded following engagement of their native αβTCR by antigen on professional antigen-presenting cells, with attendant costimulation.7Pule 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 (934) Google Scholar,8Cooper LJ Al Kadhimi Z Serrano LM Pfeiffer T Olivares S Castro A et al.Enhanced antilymphoma efficacy of CD19-redirected influenza MP1-specific CTLs by cotransfer of T cells modified to present influenza MP1.Blood. 2005; 105: 1622-1631Crossref PubMed Scopus (95) Google Scholar In a recently reported clinical trial, subjects receiving Epstein-Barr virus (EBV)-specific CTLs engineered with a CAR (CAR-CTLs) specific for the disialoganglioside antigen GD2a on neuroblastoma cells showed longer in vivo persistence than unselected T cells engineered with a distinguishable GD2-CAR, due to stimulation of the CAR-CTLs by EBV antigen.7Pule 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 (934) Google Scholar More than 10 clinical trials evaluating second- or third-generation CARs are currently listed on ClinicalTrials.gov, and there is some encouraging preliminary evidence of clinical efficacy9Office of Biotechnology Activities. Webcasts of the December 2009 meeting of the Recombinant DNA Advisory Committeehttp://oba.od.nih.gov/rdna/rac_past_meeting_2009_webcasts.html#dec09Google Scholar,10Brentjens R Riviere I Frattini M Wang X Taylor C Olszewska M et al.Marked regression of adenopathy following infusion of autologous T cells genetically targeted to the CD19 antigen in a patient with bulky CLL. Abstract submitted to the 13th annual meeting of the American Society of Gene and Cell Therapy, Washington, DC, 17–22 May 2010,, 2010Google Scholar However, in this issue of Molecular Therapy, two recent serious adverse events (SAEs) are reported in subjects enrolled in these trials, raising concerns about these receptors. The first event, described by Morgan and colleagues at the National Institutes of Health (NIH),11Morgan RA Yang JC Kitano M Dudley ME Laurencot CM Rosenberg SA Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2.Mol Ther. 2010; 18: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1796) Google Scholar occurred in a patient with widely metastatic colon cancer who received more than 1010 T cells modified with a CAR targeting HER2 containing two costimulatory moieties (CD28 and 4-1BB) after intensive lymphodepletion. The subject developed pulmonary toxicity within 15 minutes in association with very high cytokine levels, followed by cardiac arrest, and died 4 days later. After extensive investigation and analysis of autopsy specimens, the investigators concluded that the toxicity may have been due to targeting of low levels of HER2 on pulmonary endothelium by transgenic T cells. In the second report, Brentjens and colleagues from Memorial Sloan–Kettering Cancer Center describe a patient with bulky chronic lymphocytic leukemia and extensive previous treatment who received autologous T cells transduced with a CD19-28ζ CAR at a dose of 3 × 107 cells/kg after lymphodepletion with cyclophosphamide.12Brentjens R Yeh R Bernal Y Riviere I Sadelain M Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial.Mol Ther. 2010; 18: 666-668Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar This patient developed fever, hypotension, and dyspnea 20 hours after infusion, which rapidly progressed. Elevated cytokine levels were seen before the T-cell infusion, but an autopsy failed to reveal an obvious cause of death. The investigators concluded that low-grade sepsis was the most likely trigger in this heavily pretreated immunosuppressed patient but also considered the possibility that a cyclophosphamide-induced "cytokine storm" may have enhanced the in vivo activation of modified T cells.12Brentjens R Yeh R Bernal Y Riviere I Sadelain M Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial.Mol Ther. 2010; 18: 666-668Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar Although the adaptive immune response is considered a highly targeted system, it should be noted that "on-target but off-organ" effects have been observed in other studies with both native and genetically modified T cells. For example, when T cells specific for minor histocompatibility antigens were given to treat leukemic relapse, severe toxicity resulted from the unexpected presence of the target antigens on lung tissue.13Warren EH Fuji N Akatsuka Y Cahney CN Mito JL Loeb KR et al.Therapy of relapsed leukemia after allogeneic hematopoietic cell transplant with T cells specific for minor histocompatibility antigens.Blood. 2010; (e-pub ahead of print 13 January 2010)Google Scholar Similarly, when T cells were genetically modified with MART-1-specific TCRs, impressive regression of melanoma was observed, but it was associated in some cases with toxicities to melanin-expressing cells in the inner ear and the retina.14Johnson LA Morgan RA Dudley ME Cassard L Yang JC Hughes MS et al.Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen.Blood. 2009; 114: 535-546Crossref PubMed Scopus (1108) Google Scholar Finally, a CAR targeting carbonic anhydrase IX expressed by renal carcinoma cells produced liver toxicity because the target antigen was also expressed on bile duct epithelium.4Lamers CH Sleijfer S Vulto AG Kruit WH Kliffen M Debets R et al.Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.J Clin Oncol. 2006; 24: e20-e22Crossref PubMed Scopus (703) Google Scholar Morbidity due to bystander inflammation has also been reported during therapeutic responses to EBV-specific T lymphocytes.15Heslop HE Slobod KS Pule MA Hale GA Rousseau A Smith CA et al.Long term outcome of EBV specific T-cell infusions to prevent or treat EBV-related lymphoproliferative disease in transplant recipients.Blood. 2010; 115: 925-935Crossref PubMed Scopus (649) Google Scholar Notably, however, none of these examples was associated with the rapid and fulminant onset observed with CAR–T-cell infusion described by Morgan et al., and all were either self-limited or responsive to steroids. Indeed, the rapid onset of the CAR-associated SAE at the NIH is most reminiscent of the SAEs reported with a superagonist CD28 antibody.16Suntharalingam G Perry MR Ward S Brett SJ Castello-Cortes A Brunner MD et al.Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412.N Engl J Med. 2006; 355: 1018-1028Crossref PubMed Scopus (1694) Google Scholar In addition to these explanations, there is a concern that second- and third-generation receptors may be too easily triggered by low avidity "off-target" binding to produce a potent activation signal that leads to a lethal cytokine storm. It is still unclear whether the combination of lymphodepletion and the presence of costimulatory moieties in the second- and third-generation CARs used might have contributed to the cytokine storm. Because the increased potency of second- and third-generation CARs has considerable potential benefits for patients with malignant disease, how could the safety profile be enhanced? For many T-cell therapies, such as donor lymphocyte infusion after hematopoietic stem cell transplant, the side effects correlate with the cell numbers infused,17Kolb HJ Graft-versus-leukemia effects of transplantation and donor lymphocytes.Blood. 2008; 112: 4371-4383Crossref PubMed Scopus (485) Google Scholar so one possibility is to evaluate lower doses of T cells to prevent severe immediate toxicity. Should the subsequent expansion of infused CAR-specific T cells produce delayed but more indolent toxicities, several therapeutic maneuvers are available. These include corticosteroids in doses used to treat graft-versus-host disease (GvHD) or antibodies to T cells such as alemtuzumab (CAMPATH-1H), each of which will deplete the majority of circulating transduced cells. After the fatality in the CD19-28ζ CAR study, the regimen was modified so that cells are infused over 2 days, with one-third given on the first day and two-thirds on the second day.12Brentjens R Yeh R Bernal Y Riviere I Sadelain M Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial.Mol Ther. 2010; 18: 666-668Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar The NIH investigators also concluded that for future first-in-human studies with CARs, a more restricted dose-escalation scheme should be evaluated.12Brentjens R Yeh R Bernal Y Riviere I Sadelain M Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial.Mol Ther. 2010; 18: 666-668Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar A second approach is to include a safety switch mediated by a second transgene in the construct to allow the cells to be destroyed on exposure to a specific signal. The herpes simplex viral thymidine kinase (TK) gene—the product of which phosphorylates nucleoside prodrugs such as ganciclovir and acyclovir to the active moiety, which interferes with DNA polymerase—has been introduced into allogeneic T lymphocytes used as donor lymphocyte infusions following stem cell transplantation. If the infused T cells induce GvHD, administration of the prodrug causes T-cell death and abrogates GvHD. This approach has now reached a phase III clinical trial.18Ciceri F Bonini C Stanghellini MT Bondanza A Traversari C Salomoni M et al.Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I-II study.Lancet Oncol. 2009; 10: 489-500Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar Because the TK suicide gene is potentially immunogenic and requires a prodrug that would otherwise have its own therapeutic applications, investigators are also developing alternatives such as inducible caspase 9 (icasp9). Because caspase 9 is a naturally occurring component of the caspase pathway, it should be nonimmunogenic. Moreover, icasp9 is highly effective in producing rapid (less than 120 minutes) apoptosis even in nondividing cells.19Tey SK Dotti G Rooney CM Heslop HE Brenner MK Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation.Biol Blood Marrow Transplant. 2007; 13: 913-924Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar The molecule can be triggered by administration of a small-molecule dimerizer that brings together two nonfunctional icasp9 molecules to form the active enzyme, and the approach is currently being evaluated in the clinic. A follow-up Recombinant DNA Advisory Committee meeting will be held in June 2010 to focus on how best to develop a safe approach to implementing these promising new therapeutics, and it is a great credit to all investigators involved that they have been so forthcoming in providing detailed reports of SAEs. With their help, we should speedily resolve these remaining concerns so that CARS will become both safe and effective components of cancer therapy.