The Flipside of the Power of Engineered T Cells: Observed and Potential Toxicities of Genetically Modified T Cells as Therapy
Authors: Felipe Bedoya, Matthew J. Frigault, Marcela V. Maus
Journal: Molecular Therapy (2017)

Autologous T cells modified to recognize novel antigen targets are a novel form of therapy for cancer. We review the various potential forms of observed and hypothetical toxicities associated with genetically modified T cells. Despite the focus on toxicities in this review, re-directed T cells represent a powerful and highly effective form of anti-cancer therapy; we remain optimistic that the common toxicities will become routinely manageable and that some theoretical toxicity will be exceedingly rare, if ever observed. Autologous T cells modified to recognize novel antigen targets are a novel form of therapy for cancer. We review the various potential forms of observed and hypothetical toxicities associated with genetically modified T cells. Despite the focus on toxicities in this review, re-directed T cells represent a powerful and highly effective form of anti-cancer therapy; we remain optimistic that the common toxicities will become routinely manageable and that some theoretical toxicity will be exceedingly rare, if ever observed. Adoptive cellular therapy using chimeric antigen receptor (CAR) T cells has been successful in the treatment of refractory or relapsed (r/r) B cell malignancies. Despite this early success, broader applications of CAR T cell therapy may be limited by the availability of targetable antigens and the mitigation of their various toxicity profiles. Toxicities discussed here included bystander effects leading to systemic inflammatory reactions such as the previously described cytokine release syndrome (CRS), neurologic toxicities, on- and off-target effects of CARs, hypersensitivity reactions to CAR constructs, and theoretical toxicities, including insertional mutagenesis mediated by transgene delivery, autonomous CAR signaling, autoimmunity or graft-versus-host disease (GVHD) caused by T cell products, and generation of a replication-competent virus (Figure 1).1Bonifant C.L. Jackson H.J. Brentjens R.J. Curran K.J. Toxicity and management in CAR T-cell therapy.Mol. Ther. Oncolytics. 2016; 3: 16011Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 2Brudno J.N. Kochenderfer J.N. Toxicities of chimeric antigen receptor T cells: recognition and management.Blood. 2016; 127: 3321-3330Crossref PubMed Scopus (767) Google Scholar Despite the focus on toxicities in this review, we remain optimistic that toxicity mitigation can be overcome through experience in clinical management and optimized T cell engineering strategies, while some of the theoretical toxicities may never materialize in the clinical setting. One well-described CAR toxicity is CRS, which results from activation of bystander innate immune cells, including macrophages. CRS has been observed in trials using CAR constructs targeting various antigens but has become associated with anti-CD19 CARs and the anti-CD19 bispecific T cell engaging antibody, blinatumomab.3Teachey D.T. Rheingold S.R. Maude S.L. Zugmaier G. Barrett D.M. Seif A.E. Nichols K.E. Suppa E.K. Kalos M. Berg R.A. et al.Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine-directed therapy.Blood. 2013; 121: 5154-5157Crossref PubMed Scopus (428) Google Scholar CRS can range from mild flu-like symptoms such as fever, myalgias, fatigue, and mild hypotension to a more serious presentation of severe inflammatory response syndrome (SIRS) involving significant hypotension requiring pressors, vascular leak with associated respiratory failure, coagulopathy, and multi-organ system failure. Cytokine profiling of patients with CRS has repeatedly demonstrated significantly elevated levels of interleukin (IL)-6, IL-10, granulocyte colony-stimulating factor (G-CSF), and interferon (IFN)-γ, with levels often correlating with rapid CAR T cell activation and expansion. The severity of CRS does not appear to correlate with overall disease response, but most responding patients demonstrate some degree of CRS.3Teachey D.T. Rheingold S.R. Maude S.L. Zugmaier G. Barrett D.M. Seif A.E. Nichols K.E. Suppa E.K. Kalos M. Berg R.A. et al.Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine-directed therapy.Blood. 2013; 121: 5154-5157Crossref PubMed Scopus (428) Google Scholar, 4Lee 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 (2054) Google Scholar, 5Kochenderfer J.N. Dudley M.E. Feldman S.A. Wilson W.H. Spaner D.E. Maric I. Stetler-Stevenson M. Phan G.Q. Hughes M.S. Sherry R.M. et al.B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells.Blood. 2012; 119: 2709-2720Crossref PubMed Scopus (1142) Google Scholar, 6Grupp S.A. Kalos M. Barrett D. Aplenc R. Porter D.L. Rheingold S.R. Teachey D.T. Chew A. Hauck B. Wright J.F. 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, 7Maude S.L. Barrett D. Teachey D.T. Grupp S.A. Managing cytokine release syndrome associated with novel T cell-engaging therapies.Cancer J. 2014; 20: 119-122Crossref PubMed Scopus (516) Google Scholar In patients with acute lymphoblastic leukemia (ALL), CRS has been correlated with disease burden at the time of infusion, and experience has demonstrated that more potent conditioning regimens may increase the likelihood of severe CRS and/or CAR-related toxicity.8Herper M. Juno therapeutics stops trial of cancer-killing cells after 3 patient deaths.Forbes. 2016; (Published online July 7, 2016)http://www.forbes.com/sites/matthewherper/2016/07/07/juno-therapeutics-stops-trial-of-cancer-killing-cells-after-3-patient-deaths/#20e164b15d89Google Scholar Patients with severe CRS can also develop a macrophage activation syndrome (MAS) reminiscent of hemophagocytic lymphohistiocytosis, as demonstrated by overlapping cytokine profiles, hyperferritinemia, and evidence of hemophagocytosis on bone marrow biopsy.3Teachey D.T. Rheingold S.R. Maude S.L. Zugmaier G. Barrett D.M. Seif A.E. Nichols K.E. Suppa E.K. Kalos M. Berg R.A. et al.Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine-directed therapy.Blood. 2013; 121: 5154-5157Crossref PubMed Scopus (428) Google Scholar, 7Maude S.L. Barrett D. Teachey D.T. Grupp S.A. Managing cytokine release syndrome associated with novel T cell-engaging therapies.Cancer J. 2014; 20: 119-122Crossref PubMed Scopus (516) Google Scholar In all witnessed cases to date, MAS appears to resolve with the resolution of CRS. Current theories on the mechanism of action include high levels of IFN-γ production in the setting of rapid T cell activation and cytotoxicity, resulting in robust macrophage activation.9Tang Y.M. Xu X.J. Advances in hemophagocytic lymphohistiocytosis: pathogenesis, early diagnosis/differential diagnosis, and treatment.Sci. World J. 2011; 11: 697-708Crossref Scopus (62) Google Scholar Given the importance of IFN-γ in T cell cytotoxicity, and the clinically available anti-cytokine therapies, management of CRS involves the blockade of the pro-inflammatory cytokine IL-6 via blockade of the IL-6 receptor with tocilizumab. Neutralization of IL-6 with siltuximab has also been attempted, but it is not as well established as tocilizumab.7Maude S.L. Barrett D. Teachey D.T. Grupp S.A. Managing cytokine release syndrome associated with novel T cell-engaging therapies.Cancer J. 2014; 20: 119-122Crossref PubMed Scopus (516) Google Scholar High-dose corticosteroids have been used in patients, although there is some evidence that they may have a detrimental effect on T cell proliferation; therefore, their use is reserved for management of continuing severe CRS and/or severe neurologic toxicity.10Davila 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 (1758) Google Scholar Predictive biomarkers of which patients are likely to experience CRS are being explored.11Teachey D.T. Lacey S.F. Shaw P.A. Melenhorst J.J. Maude S.L. Frey N. Pequignot E. Gonzalez V.E. Chen F. Finklestein J. et al.Identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia.Cancer Discov. 2016; 6: 664-679Crossref PubMed Scopus (632) Google Scholar More extensive reviews of the grading and management of CRS have been published,7Maude S.L. Barrett D. Teachey D.T. Grupp S.A. Managing cytokine release syndrome associated with novel T cell-engaging therapies.Cancer J. 2014; 20: 119-122Crossref PubMed Scopus (516) Google Scholar, 12Lee D.W. Gardner R. Porter D.L. Louis C.U. Ahmed N. Jensen M. Grupp S.A. Mackall C.L. Current concepts in the diagnosis and management of cytokine release syndrome.Blood. 2014; 124: 188-195Crossref PubMed Scopus (1645) Google Scholar and the algorithms for clinical management are still in development by the various sponsors of CAR T cell therapies. One type of unexpected toxicity is the range of transient neurologic complications that have been observed in almost all trials targeting T cells to CD19 with either CAR T cells or bispecific T cell engagers (BiTEs). Manifestations vary and include confusion, obtundation, seizures, hallucinations, aphasia, ataxia, and more recently and rarely, profound cerebral edema. In some instances, these symptoms can correlate with the onset of CRS, but neurologic symptoms can also occur before or following resolution of CRS. These symptoms are usually self-limiting and are managed with high-dose steroids and anti-epileptic drugs as needed; tocilizumab does not appear to have a beneficial effect in ameliorating neurologic toxicity, though it has not been formally tested in this context. Given the toxicity overlap with blinatumomab and the absence of neurotoxicity seen with other CAR constructs, there may be a correlation with single-chain fragment variable (scFv) cross-reactivity with a yet unidentified antigen expressed in the nervous system.13Goebeler M.E. Knop S. Viardot A. Kufer P. Topp M.S. Einsele H. Noppeney R. Hess G. Kallert S. Mackensen A. et al.Bispecific T-cell engager (BiTE) antibody construct blinatumomab for the treatment of patients with relapsed/refractory non-Hodgkin lymphoma: final results from a phase I study.J. Clin. Oncol. 2016; 34: 1104-1111Crossref PubMed Scopus (296) Google Scholar, 14Topp M.S. Gökbuget N. Stein A.S. Zugmaier G. O'Brien S. Bargou R.C. Dombret H. Fielding A.K. Heffner L. Larson R.A. et al.Safety and activity of blinatumomab for adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia: a multicentre, single-arm, phase 2 study.Lancet Oncol. 2015; 16: 57-66Abstract Full Text Full Text PDF PubMed Scopus (879) Google Scholar CAR T cells have been shown to infiltrate the cerebrospinal fluid and central nervous system (CNS); however, their presence does not appear to be directly correlated with neurologic toxicity and may be beneficial in diseases such as ALL, in which the CNS is a sanctuary site for the leukemia cells. Alternatively, CAR T cell neurotoxicity may be associated with cytokines and inflammatory factors released with immune activation. Recent work identified an association of severe CRS with the peak serum levels of 24 cytokines, including IFN-γ, IL-6, sgp130, and soluble IL-6 receptor (sIL-6R).11Teachey D.T. Lacey S.F. Shaw P.A. Melenhorst J.J. Maude S.L. Frey N. Pequignot E. Gonzalez V.E. Chen F. Finklestein J. et al.Identification of predictive biomarkers for cytokine release syndrome after chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia.Cancer Discov. 2016; 6: 664-679Crossref PubMed Scopus (632) Google Scholar It is therefore possible that specific cytokine profiling may help predict which patients are at high risk for neurologic toxicities. For example, it is well established that high levels of IFN-γ in the blood trigger monocyte/macrophage activation and migration to inflamed tissues.15Goldberg M. Belkowski L.S. Bloom B.R. Regulation of macrophage function by interferon-gamma. Somatic cell genetic approaches in murine macrophage cell lines to mechanisms of growth inhibition, the oxidative burst, and expression of the chronic granulomatous disease gene.J. Clin. Invest. 1990; 85: 563-569Crossref PubMed Scopus (45) Google Scholar Cells of the myeloid lineage present in the CNS may become activated by these same inflammatory factors, causing a localized cytokine release in the CNS. This may also partially explain the neurologic toxicity observed with blinatumomab.3Teachey D.T. Rheingold S.R. Maude S.L. Zugmaier G. Barrett D.M. Seif A.E. Nichols K.E. Suppa E.K. Kalos M. Berg R.A. et al.Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine-directed therapy.Blood. 2013; 121: 5154-5157Crossref PubMed Scopus (428) Google Scholar Juno Therapeutics reported a series of three cases of lethal neurotoxicity in adult patients with r/r ALL treated with JCAR015, which includes the CD28 and CD3z signaling domains. Although the study went through a temporary clinical hold by the U.S. Food and Drug Administration (FDA), it was rapidly re-instated after the conditioning regimen was modified to eliminate fludarabine; this trial now uses only cyclophosphamide in the conditioning regimen. Because fludarabine has not been independently associated with lethal or severe neurotoxicity as a single agent or with other forms of CAR T cells, it is likely to remain an important drug in the conditioning regimens for other CAR T cells. Since the initial writing of this manuscript, this trial has been placed on clinical hold after more cases of lethal cerebral edema have occurred. One of the most important factors in CAR design is choosing the right antigen target. Ideally, the antigen should be expressed only on the surface of the tumor cells, not on healthy tissue. With few exceptions, such as EGFRvIII in glioblastoma, most antigens that have been selected thus far are also present at some level in normal tissues. Because the main driver initiating CAR T cell activation is the direct engagement of the scFv with its cognate antigen, the coexpression of this antigen on any non-tumor cell leads to simultaneous elimination of both tumor and non-tumor targets; the degree of on-target toxicity is likely related to the affinity of the CAR, the level of antigen expression on the healthy tissue, the potency of the CAR, and the relative functional importance of the healthy tissue target. One notable example is that of a CAR recognizing Her2/neu, an antigen that is frequently overexpressed in epithelial malignancies, including gastric, colon, and breast cancer. In 2010, a single patient with Her2+ metastatic colon cancer was treated with a high dose of Her2-targeted CAR therapy and within 15 min of infusion, developed new respiratory failure requiring intubation and intensive care unit (ICU) management; the patient died from CRS and CAR T cell targeting of Her2 on lung epithelium.16Morgan R.A. Yang J.C. Kitano M. Dudley M.E. Laurencot C.M. Rosenberg S.A. 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 (1746) Google Scholar However, other studies targeting Her2 with lower doses and different CAR constructs, based on either a lower-affinity scFv or less potent signaling domains, show that Her2-targeted CAR T cells may be safe.17Ahmed N. Salsman V.S. Kew Y. Shaffer D. Powell S. Zhang Y.J. Grossman R.G. Heslop H.E. Gottschalk S. HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors.Clin. Cancer Res. 2010; 16: 474-485Crossref PubMed Scopus (276) Google Scholar, 18Ahmed N. Brawley V.S. Hegde M. Robertson C. Ghazi A. Gerken C. Liu E. Dakhova O. Ashoori A. Corder A. et al.Human epidermal growth factor receptor 2 (HER2)-specific chimeric antigen receptor-modified T cells for the immunotherapy of HER2-positive sarcoma.J. Clin. Oncol. 2015; 33: 1688-1696Crossref PubMed Scopus (652) Google Scholar, 19Zhao Y. Moon E. Carpenito C. Paulos C.M. Liu X. Brennan A.L. Chew A. Carroll R.G. Scholler J. Levine B.L. et al.Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor.Cancer Res. 2010; 70: 9053-9061Crossref PubMed Scopus (335) Google Scholar Some antigens that are expressed on normal tissues may still be targetable by CARs without causing dose-limiting toxicity, such as mesothelin and prostate-specific membrane antigen (PSMA) (S.F. Slovin et al., 2013, J Clin Oncol., abstract);20Beatty 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 however, neither of these targets has brought about dramatic clinical efficacy, and it is possible that these two outcomes are inexorably linked. Other examples of on-target, off-tumor toxicities associated with the most commonly targeted antigens used in CAR pre-clinical studies and clinical trials are shown in Table 1.Table 1Examples of On-Target, Off-Tumor Toxicities of CAR T Cell TherapiesTarget AntigenDiseaseOn-Target, Off-Tumor ToxicityReferencesCD19B cell malignanciesB cell aplasiaMaude et al.50Maude S.L. Fitzgerald J.C. Fisher B.T. Li Y. Huang Y.S. Torp K. Seif A.E. Kavcic M. Walker D.M. Leckerman K.H. et al.Outcome of pediatric acute myeloid leukemia patients receiving intensive care in the United States.Pediatr. Crit. Care Med. 2014; 15: 112-120Crossref PubMed Scopus (38) Google ScholarPorter et al.51Porter D.L. Hwang W.T. Frey N.V. Lacey S.F. Shaw P.A. Loren A.W. Bagg A. Marcucci K.T. Shen A. Gonzalez V. et al.Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.Sci. Transl. Med. 2015; 7: 303ra139Crossref PubMed Scopus (1160) Google ScholarHER2/ERBB2colon cancerlethal pulmonary failureMorgan et al.16Morgan R.A. Yang J.C. Kitano M. Dudley M.E. Laurencot C.M. Rosenberg S.A. 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 (1746) Google ScholarCarbonic anhydrase-IX (CA-IX)renal cancerbile duct epithelium infiltration (cholestasis)Lamers et al.52Lamers C.H.J. Sleijfer S. Vulto A.G. Kruit W.H. Kliffen M. Debets R. Gratama J.W. Stoter G. Oosterwijk E. 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 (694) Google ScholarLamers et al.53Lamers C.H. Sleijfer S. van Steenbergen S. van Elzakker P. van Krimpen B. Groot C. Vulto A. den Bakker M. Oosterwijk E. Debets R. Gratama J.W. Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity.Mol. Ther. 2013; 21: 904-912Abstract Full Text Full Text PDF PubMed Scopus (488) Google ScholarCD30Hodgkin's lymphomalymph node, tonsils, thymus infiltrationWang et al.54Wang C. Wu Z. Wang Y. Guo Y. Dai H. Wang X.H. Li X. Zhang Y.J. Zhang W.Y. Chen M.X. et al.Autologous T cells expressing CD30 chimeric antigen receptors for relapsed or refractory Hodgkin's lymphoma: an open-label phase I trial.Clin. Cancer Res. 2016; (Published online August 31, 2016)https://doi.org/10.1158/1078-0432.CCR-16-1365Crossref Scopus (213) Google ScholarCD123acute myeloid leukemiaelimination of hematopoietic stem cell and myeloid compartmentsMardiros et al.55Mardiros A. Dos Santos C. McDonald T. Brown C.E. Wang X. Budde L.E. Hoffman L. Aguilar B. Chang W.C. Bretzlaff W. et al.T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.Blood. 2013; 122: 3138-3148Crossref PubMed Scopus (261) Google ScholarGill et al.56Gill S. Tasian S.K. Ruella M. Shestova O. Li Y. Porter D.L. Carroll M. Danet-Desnoyers G. Scholler J. Grupp S.A. et al.Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells.Blood. 2014; 123: 2343-2354Crossref PubMed Scopus (326) Google ScholarKappa light chainnon-Hodgkin's lymphoma/multiple myelomaelimination of kappa-expressing B and plasma cellsRamos et al.57Ramos C.A. Savoldo B. Torrano V. Ballard B. Zhang H. Dakhova O. Liu E. Carrum G. Kamble R.T. Gee A.P. et al.Clinical responses with T lymphocytes targeting malignancy-associated κ light chains.J. Clin. Invest. 2016; 126: 2588-2596Crossref PubMed Scopus (207) Google Scholar Open table in a new tab In the case of CD19 antigen (expressed on the B cell lineage), some on-target, off-tumor toxicities, such as B cell aplasia, are clinically manageable and may not be unacceptably detrimental following CAR T cell therapy.21Maude 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 (3552) Google Scholar, 22Porter D.L. Levine B.L. Kalos M. Bagg A. June C.H. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.N. Engl. J. Med. 2011; 365: 725-733Crossref PubMed Scopus (2624) Google Scholar, 23Barrett D.M. Singh N. Porter D.L. Grupp S.A. June C.H. Chimeric antigen receptor therapy for cancer.Annu. Rev. Med. 2014; 65: 333-347Crossref PubMed Scopus (272) Google Scholar Patients with B cell aplasia often receive intravenous immunoglobulin (IVIG), but this may not be necessary in all patients (S.J. Schuster et al., 2016, J Clin Oncol., abstract). The prolonged absence of CD19-expressing cells reflects the exceptional capacity of CAR T cells to persist and function in vivo. Given the marked toxicities described thus far and the apparent power of T cell-based therapy, the importance of pre-clinical assays to determine the potential for on-target, off-tumor expression of antigens is apparent. Various approaches are under way to screen for antigens expressed exclusively in tumors (Figure 2). Bulk- and single-cell deep sequencing technologies such as DNA sequencing (DNA-seq), RNA sequencing (RNA-seq), and tumor exome analysis have yielded a better understanding of unique tumor antigens.24Srivastava P.K. Neoepitopes of cancers: looking back, looking ahead.Cancer Immunol. Res. 2015; 3: 969-977Crossref PubMed Scopus (47) Google Scholar, 25Cohen C.J. Gartner J.J. Horovitz-Fried M. Shamalov K. Trebska-McGowan K. Bliskovsky V.V. Parkhurst M.R. Ankri C. Prickett T.D. Crystal J.S. et al.Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes.J. Clin. Invest. 2015; 125: 3981-3991Crossref PubMed Scopus (265) Google Scholar An established database of proteins expressed by healthy tissues, on a single-cell level, would complement this approach. Although most CAR targets have been limited to natural cell-surface antigens in the form of proteins, it is possible to target post-translational modifications of proteins26Posey Jr., A.D. Schwab R.D. Boesteanu A.C. Steentoft C. Mandel U. Engels B. Stone J.D. Madsen T.D. Schreiber K. Haines K.M. et al.Engineered CAR T cells targeting the cancer-associated Tn-glycoform of the membrane mucin MUC1 control adenocarcinoma.Immunity. 2016; 44: 1444-1454Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 27Kumaresan P.R. Manuri P.R. Albert N.D. Maiti S. Singh H. Mi T. Roszik J. Rabinovich B. Olivares S. Krishnamurthy J. et al.Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection.Proc. Natl. Acad. Sci. USA. 2014; 111: 10660-10665Crossref PubMed Scopus (140) Google Scholar and glycosphingolipids with CARs.28Prapa M. Caldrer S. Spano C. Bestagno M. Golinelli G. Grisendi G. Petrachi T. Conte P. Horwitz E.M. Campana D. et al.A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing.Oncotarget. 2015; 6: 24884-24894Crossref PubMed Scopus (44) Google Scholar T cells can also be genetically modified with natural or engineered T cell receptors (TCRs), which recognize peptides presented in the context of major histocompatibility complex (MHC). By combining genetic analysis of mutations in cancer cells with immune-peptidome analysis by mass spectrometry, a wave of new peptide antigen targets that can be recognized by endogenous TCRs29Gfeller D. Bassani-Sternberg M. Schmidt J. Luescher I.F. Current tools for predicting cancer-specific T cell immunity.OncoImmunology. 2016; 5: e1177691Crossref PubMed Scopus (29) Google Scholar, 30Leone P. Shin E.C. Perosa F. Vacca A. Dammacco F. Racanelli V. MHC class I antigen processing and presenting machinery: organization, function, and defects in tumor cells.J. Natl. Cancer Inst. 2013; 105: 1172-1187Crossref PubMed Scopus (327) Google Scholar has potential for therapeutic development. Furthermore, new molecular methods that allow for the analysis of post-translationally modified (PTM) proteins has further increased the repertoire of new candidates amenable for T cell recognition. Cobbold et al.31Cobbold M. De La Peña H. Norris A. Polefrone J.M. Qian J. English A.M. Cummings K.L. Penny S. Turner J.E. Cottine J. et al.MHC class I-associated phosphopeptides are the targets of memory-like immunity in leukemia.Sci. Transl. Med. 2013; 5: 203ra125Crossref PubMed Scopus (145) Google Scholar emphasized the increasing importance of these post-translational modifications in human malignancies. They demonstrated that phosphopeptide antigens derived from cancer-related phosphoproteins could serve as immunologic signatures of the "transformed self" and were immunogenic in patients with and without cancer. These neo-antigen phosphopeptides were then identified on primary human malignant tissue and were often derived from oncogenes linked to leukemogenesis, making them exciting targets for future T cell-targeted therapy independent of overall mutational load.31Cobbold M. De La Peña H. Norris A. Polefrone J.M. Qian J. English A.M. Cummings K.L. Penny S. Turner J.E. Cottine J. et al.MHC class I-associated phosphopeptides are the targets of memory-like immunity in leukemia.Sci. Transl. Med. 2013; 5: 203ra125Crossref PubMed Scopus (145) Google Scholar Most of the neoantigens studied are derived from modified intracellular proteins and have been proposed and tested as candidates for TCR-engineered T cells. Nevertheless, the discovery of neoantigens on the surface of cancer cells will likely provide a valuable source of targets for CAR T cells. Recent work has demonstrated the possibility of scFv fragments capable of identifying intracellular neoantigens loaded on endogenous MHC.32Ikeda H. Akahori Y. Yoneyama Orito Y. Miyahara Y. Amaishi Y. Okamoto S. Mineno J. Takesako K. Fujiwara H. et al.Immunotherapy with chimeric antigen receptor targeting intracellular WT1 gene product complexed with HLA-a*24:02 molecule.Blood. 2015; 126: 4292Google Scholar The possibility of CAR T cells being able to target both extra-cellular and intra-cellular proteins is promising, though screening for on-target toxicities may be more challenging to carry out with available, practical assays in the pre-clinical setting. One benefit of CAR-based approaches is that their specificity is dictated by antibody-like binding, independent of MHC expression. Whereas CAR T cell therapy has yet to demonstrate clear off-target effects mediate by inappropriate scFv recognition of a non-target antigen, TCR-modified T cell therapies have revealed the possibility of TCR promiscuity and the risk of off-target effects. One example of such off-target toxicities was observed in patients treated with human leukocyte antigen (HLA)-A1-restricted, MAGE-A3-specific, enhanced-affinity TCR-modified T cells. Despite extensive pre-clinical safety studies, 5 days following T cell infusion, both infused patients died of acute cardiac failure and robust T cell infiltration. No MAGE-A3 expression could be identified on cardiac myocytes. Further investigation revealed that the affinity enhancement of the TCR resulted in recognition of a similar peptide expressed in cardiac muscle, leading to acute cardiac rejection.33Cameron B.J. Gerry A.B. Dukes J. Harper J.V. Kannan V. Bianchi F.C. Grand F. Brewer J.E. Gupta M. Plesa G. et al.Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells.Sci. Transl. Med. 2013; 5: 197ra103Crossref PubMed Scopus (447) Google Scholar Following these unexpected deaths, an extensive investigation was undertaken, with initial experiments demonstrating no cross-reactivity with standard myocyte cell culture models. Only the use of highly purified human cardiomyocytes derived from induced pluripotent stem cells revealed the r