VOLUME
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2015
JOURNAL OF CLINICAL ONCOLOGY
UNDERSTANDING THE PATHWAY
Chimeric Antigen Receptor T Cells for Cancer Immunotherapy Kevin J. Curran and Renier J. Brentjens, Memorial Sloan Kettering Cancer Center, New York, NY See accompanying article on page 1688
T cells can be genetically modified to target tumors through the expression of a chimeric antigen receptor (CAR).1 The clinical benefit of CAR T cells has now been reported by several groups targeting the CD19 antigen in patients with hematologic malignancies.2-5 However, in solid tumors, CAR T cells have been less effective at inducing complete tumor responses and have mediated a high rate of on-target/ off-tumor toxicity.6-10 To understand this discrepancy, we must understand the design and implementation of CAR T cells for cancer immunotherapy. The basic design of CAR T cells consists of two fundamental domains: the antigen-binding portion (commonly composed of a single-chain variable fragment [scFv] derived from a monoclonal antibody [mAb]) joined to one or more intracellular T-cell signaling domains. To be effective after infusion, CAR T cells must expand, persist, exhibit enduring antitumor cytotoxicity, withstand and/or counteract an immunosuppressive tumor microenvironment, and overcome targeted tumor antigen escape. In designing CAR T cells for cancer immunotherapy, all of these factors must be harmonized to generate the optimal CAR T cell.
CAR DESIGN: TARGET ANTIGEN
Critical to the successful use of CAR T cells is choosing the proper tumor-associated antigen (TAA) to target. The ideal TAA for CAR T cells has the following key characteristics: expression on all tumor cells including the cancer stem cell, expression on the tumor cell surface, role in tumor cell survival, and lack of expression on normal tissues. Few TAAs are in fact ideal, because most are only variably expressed by the tumor and many are also expressed on one or more normal tissue types. The latter is most apparent when on-target/off-tumor toxicity occurs as in the case of carbonic anhydrase IX–specific CAR T cells or carcinoembryonic antigen-specific CAR T cells after infusion in a patient with renal cell carcinoma or colon cancer, respectively.6,9 In the accompanying article, Ahmed et al11 demonstrate that infusion of human epidermal growth factor receptor 2 (HER2) –specific CAR T cells for patients with relapsed/refractory sarcoma can be safely accomplished. This finding is significant in light of a prior report demonstrating that HER2-specific CAR T cells can lead to pulmonary toxicity and/or multiorgan failure and subsequent death, which has been attributed to unforeseen CAR T-cell recognition of low-level HER2 expression on the lung epithelium.12 This unexpected toxicity highlights the enhanced sensitivity of CAR T cells to low-level antigens Journal of Clinical Oncology, Vol 33, No 15 (May 20), 2015: pp 1703-1706
compared with mAbs when expressed on normal tissues. In contrast, it was found that HER2-specific CAR T cells did not produce cardiotoxicity after infusion, although treatment with the HER2-specific mAb trastuzumab did.13 Taken together, these findings highlight the discrepant safety profiles between mAbs and CAR T-cell therapies, even when they target the same epitope. Although there is a difference between the HER2-specific CARs and HER2 mAbs, the safe use of this target for CAR T cells, as reported by Ahmed et al,11 is an important advancement for the field of cancer immunotherapy. Potential strategies for overcoming the limiting effect of on-target/off-tumor toxicity have included blocking antigenic sites in off-tumor organs by using mAbs or by using CAR T cells, with combinatorial CARs recognizing two separate TAAs.14,15 CAR DESIGN: INTRACELLULAR SIGNALING
Early CAR designs incorporated one intracellular signaling domain, most commonly the immunoreceptor tyrosine-based activation motif of the CD3 chain.1 However, these first-generation CARs suboptimally activated the T cells and failed to demonstrate clinical benefit in early clinical trials.1 Enhancement of T-cell activation, persistence, and antitumor efficacy was mediated in second-generation CARs by including additional signaling domains within the CAR construct, most commonly the CD28 or 4-1BB costimulatory signaling domains. The dramatic complete response rates of 70% to 90% in adult and pediatric patients with relapsed/refractory B-cell acute lymphatic leukemia (B-ALL) following infusion of CD19-specific second-generation CAR T cells has resulted in renewed vigor for expanding CAR T-cell technology to other malignancies.2-4 To this end, second- and even thirdgeneration CAR designs, the latter incorporating two costimulatory signaling domains, have been used in solid tumors (including the CD28-containing HER2-specific CAR used in the accompanying article), albeit with only modest clinical responses. CAR T CELLS: TRANSLATION TO THE CLINIC
Several likely factors play a role in the clinical efficacy of CAR T cells after adoptive transfer. Paramount is the ability of CAR T cells to expand and/or persist, which has translated into improved response in both hematologic and solid tumors.2-4,10 The addition of costimulatory signaling within the design of the CAR T cell has improved © 2015 by American Society of Clinical Oncology
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Curran and Brentjens
persistence.16 In contrast, the development of an endogenous antiCAR immune response can limit persistence of CAR T cells.3,7,17 To reduce CAR T-cell immunogenicity, the antigen-binding portion of the traditionally murine-derived scFv can be humanized or generated from human scFv phage display libraries.18 The use of prior condi-
A
tioning chemotherapy with respect to CAR T-cell infusion as well as the selection of central memory T-cell phenotype has been positively correlated with T-cell persistence.19,20 Although persistence is important, the optimal duration for CAR T cells circulation is unknown and may serve as a double edged sword by first completely eradicating
C
Identify suitable TAAs
B
Select CAR design
Screen for reactivity to normal cells Select human scFv specific for TAA
Candidates 1 and 2, not uniformly expressed
Select first-, second-, or third-generation CAR signaling domains
Candidate 3, not expressed on normal cells
Candidate 4, expressed on normal cells Candidates 3 and 4, expressed uniformly on tumor cells
E
Optimize efficacy, persistence, and safety using syngeneic murine model Treat murine tumor cell line with murine CAR T cells in immunocompetent murine model Increase antitumor efficacy by employing armored CAR T cells if needed
D
Test in preclinical xenogeneic murine model
Treat human tumor cell line or primary cancer cells with human CAR T cells Costimulatory ligand transgene
Cytokine transgene
G F
Translate to clinical setting to test safety and efficacy
Suicide transgene
Combine with immune checkpoint blockade to enhance efficacy
PD-1/CTLA-4 blocking mAbs
PD-1 CTLA-4 CAR T cell
Fig 1. Algorithm depicting pathway for designing optimal chimeric antigen receptor (CAR) T cells for tumor immunotherapy. (A) First, suitable tumor-associated antigens (TAAs) must be identified, with (B) secondary selection of target antigens based on TAA expression on normal tissues identifying optimal target tumor antigens with limited, if any, expression on nonmalignant cells. (C) Selected CAR constructs will be generated from human single-chain variable fragment (scFv) phage display libraries designed to reduce CAR immunogenicity. (D) CARs targeted to these TAAs need to be studied in xenotransplantation murine tumor models to validate potential efficacy. (E) Whenever possible, murine-derived CAR T cells should be assessed in clinically relevant syngeneic tumor models thereby providing a platform for the exploration of additional genetic modifications (eg, armored CAR T cells) to optimally enhance in vivo antitumor efficacy, persistence, and safety. Having met these criteria, modification of CAR T cells to express either a suicide vector or elimination gene to enhance safety regarding unforeseen on-target/off-tumor toxicity should be included and validated before initial clinical trial application. (F) Clinical trial application with either CAR T cells or armored CAR T cells should be initially assessed with respect to safety and clinical outcomes. (G) If single-agent CAR T-cell therapy is deemed insufficient in this setting, more clinical trials should be conducted that use the rational combination of CAR T-cell therapy and additional agents such as monoclonal antibody (mAb) –mediated immune checkpoint inhibitors (ie, PD-1/PD-L1 and/or CD80/CTLA4 blockade) to protect CAR T cells and further recruit endogenous immune effectors. 1704
© 2015 by American Society of Clinical Oncology
JOURNAL OF CLINICAL ONCOLOGY
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Chimeric Antigen Receptor T Cells for Cancer Immunotherapy
residual tumor cells through greater persistence and second by potentiating on-target/off-tumor toxicities as seen in the correlation between CD19-specific CAR T-cell persistence and long-standing B-cell aplasias.4 Although currently published clinical data from CD19-specific CAR T-cell trials have yet to establish optimal duration for maximal clinical benefit, the future inclusion of suicide or elimination genes such as iCASP9 and huEGFRt, respectively, may circumvent or abort on-target/ off-tumor toxicities once tumor eradication is complete.21,22 CAR T CELLS: ARMORED CARS
The immunosuppressive tumor microenvironment has a central role in limiting the effectiveness of CAR T cells in vivo.23 This environment is flush with endogenous immunosuppressive cells, including CD4⫹ regulatory T cells, myeloid-derived suppressor cells, plasmacytoid dendritic cells, tumor-associated macrophages, immune checkpoint inhibitors, and immunosuppressive soluble ligands and cytokines such as interleukin-10 (IL-10) or transforming growth factor-.24 The limited clinical benefit of the HER2-specific CAR T cells in the article by Ahmed et al11 could be due, in part, to the inability of CAR T cells to overcome this immunosuppressive tumor microenvironment. As the authors surmise, additional engineering of the CAR T cells or use of immune checkpoint blockade may enhance the effectiveness of the tumor-specific T cells.24-27 To this end, we and others have proposed additional modification of CAR T cells to enhance antitumor efficacy and persistence despite a prevalent hostile tumor microenvironment. As proof of principal, we have addressed the tumor microenvironment through further modification of CAR T cells to secrete the proinflammatory IL-12 cytokine.24 These armored IL-12–secreting CAR T cells are protected from regulatory T cell–mediated inhibition while delivering the IL-12 cytokine within the tumor microenvironment, thus potentially reversing the anergic state of endogenous tumor-infiltrating tumor-targeted T cells. Once reactivated, the endogenous immune system has the potential to target multiple TAAs (epitope spreading) and avert the development of antigen-negative tumor cells as has been seen with the development of CD19– clones after treatment with CD19-specific CAR T cells in patients with relapsed B-ALL.4 DESIGNING CAR T CELLS FOR ADOPTIVE THERAPY OF CANCER IN 2015
After more than 20 years of preclinical evaluation, CAR T-cell immunotherapy is reaching an initial zenith in the setting of relapsed B-ALL, which may well alter the standard of care for this patient population. Expansion of this technology to a broader cohort of malignancies, especially solid tumors, will require a multistep CAR T-cell design REFERENCES 1. Curran KJ, Pegram HJ, Brentjens RJ: Chimeric antigen receptors for T cell immunotherapy: Current understanding and future directions. J Gene Med 14:405-415, 2012 2. Davila ML, Riviere I, Wang X, et al: Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 6:224ra25, 2014 www.jco.org
approach (Fig 1). This approach will first require the identification of suitable TAAs (Fig 1A), with secondary selection of target antigens based on TAA expression on normal tissues identifying optimal target tumor antigens with limited, if any, expression on nonmalignant cells (Fig 1B). Selected CAR constructs will be generated from human scFv phage display libraries designed to reduce CAR immunogenicity (Fig 1C).17 In addition, CARs targeted to these TAAs need to be studied in xenotransplant murine tumor models to validate potential efficacy (Fig 1D). Whenever possible, murine-derived CAR T cells should be assessed in clinically relevant syngeneic tumor models thereby providing a platform for the exploration of additional genetic modifications (eg, armored CAR T cells) to optimally enhance in vivo antitumor efficacy, persistence, and safety (Fig 1E). Having met these criteria, further modification of CAR T cells to express either a suicide vector or elimination gene to enhance safety regarding unforeseen on-target/ off-tumor toxicity should be included and validated before initial clinical trial application (Fig 1E). Investigators in this field must acknowledge that despite preclinical murine tumor models, the ultimate safety and efficacy of this approach using novel CAR T cell–targeted antigens can be accurately assessed only in carefully designed human clinical trials. Clinical trial application with either CAR T cells or armored CAR T cells should be initially assessed with respect to safety and clinical outcomes (Fig 1F). If single-agent CAR T cell therapy is deemed insufficient in this setting, additional clinical trials should be conducted using the rational combination of CAR T cell therapy with additional agents such as mAb-mediated immune checkpoint inhibitors (ie, PD-1/PD-L1 and/or CD80/ CTLA4 blockade) to protect CAR T cells and further recruit endogenous immune effectors (Fig 1G). Collectively, this schematic highlights our current appreciation of CAR T-cell technology with respect to extrapolating this promising adoptive T-cell approach to other hematologic malignancies and, more significantly, to solid tumor malignancies. We anticipate that the outcomes from current and future CAR T-cell clinical trials targeting solid tumors will markedly expand and modify this CAR T-cell design algorithm. AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Disclosures provided by the authors are available with this article at www.jco.org.
AUTHOR CONTRIBUTIONS Manuscript writing: All authors Final approval of manuscript: All authors
3. Lee DW, Kochenderfer JN, Stetler-Stevenson M, 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 385:517-528, 2015 4. Maude SL, Frey N, Shaw PA, et al: Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371:1507-1517, 2014 5. Kochenderfer JN, Dudley ME, Kassim SH, et al: Chemotherapy-refractory diffuse large B-cell lym-
phoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol 33:540-549, 2015 6. Lamers CH, Sleijfer S, Vulto AG, et al: Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: First clinical experience. J Clin Oncol 24:e20-e22, 2006 7. Kershaw MH, Westwood JA, Parker LL, et al: A phase I study on adoptive immunotherapy using
© 2015 by American Society of Clinical Oncology
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gene-modified T cells for ovarian cancer. Clin Cancer Res 12:6106-6115, 2006 8. Park JR, Digiusto DL, Slovak M, et al: Adoptive transfer of chimeric antigen receptor re-directed cytolytic T lymphocyte clones in patients with neuroblastoma. Mol Ther 15:825-833, 2007 9. Parkhurst MR, Yang JC, Langan RC, et al: T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 19:620-626, 2011 10. Louis CU, Savoldo B, Dotti G, et al: Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood 118:6050-6056, 2011 11. Ahmed N, Brawley V, Hedge M, et al: Human epidermal growth factor receptor 2 (HER2)–specific chimeric antigen receptor-modified T cells for the immunotherapy of osteosarcoma. J Clin Oncol 33: 1688-1696, 2015 12. Morgan RA, Yang JC, Kitano M, et al: Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 18:843-851, 2010 13. Seidman A, Hudis C, Pierri MK, et al: Cardiac dysfunction in the trastuzumab clinical trials experience. J Clin Oncol 20:1215-1221, 2002 14. Lamers CH, Sleijfer S, van Steenbergen S, et al: Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: Clinical evaluation and
management of on-target toxicity. Mol Ther 21:904912, 2013 15. Kloss CC, Condomines M, Cartellieri M, et al: Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol 31: 71-75, 2013 16. Savoldo B, Ramos CA, Liu E, et al: CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J Clin Invest 121:1822-1826, 2011 17. Lamers CH, Willemsen R, van Elzakker P, et al: Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells. Blood 117:72-82, 2011 18. Dao T, Yan S, Veomett N, et al: Targeting the intracellular WT1 oncogene product with a therapeutic human antibody. Sci Transl Med 5:176ra33, 2013 19. Brentjens RJ, Rivière I, Park JH, et al: Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 118:4817-4828, 2011 20. Berger C, Jensen MC, Lansdorp PM, et al: Adoptive transfer of effector CD8⫹ T cells derived from central memory cells establishes persistent T cell memory in primates. J Clin Invest 118:294-305, 2008
21. Di Stasi A, Tey SK, Dotti G, et al: Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 365:1673-1683, 2011 22. Wang X, Chang WC, Wong CW, et al: A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood 118:1255-1263, 2011 23. Vesely MD, Kershaw MH, Schreiber RD, et al: Natural innate and adaptive immunity to cancer. Annu Rev Immunol 29:235-271, 2011 24. Pegram HJ, Lee JC, Hayman EG, et al: Tumortargeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood 119:4133-4141, 2012 25. Pegram HJ, Park JH, Brentjens RJ: CD28z CARs and armored CARs. Cancer J 20:127-133, 2014 26. Hoyos V, Savoldo B, Quintarelli C, et al: Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia 24:1160-1170, 2010 27. Dotti G, Gottschalk S, Savoldo B, et al: Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev 257: 107-126, 2014
DOI: 10.1200/JCO.2014.60.3449; published online ahead of print at www.jco.org on April 20, 2015
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JOURNAL OF CLINICAL ONCOLOGY
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Chimeric Antigen Receptor T Cells for Cancer Immunotherapy
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Chimeric Antigen Receptor T Cells for Cancer Immunotherapy The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I ⫽ Immediate Family Member, Inst ⫽ My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or jco.ascopubs.org/site/ifc. Kevin J. Curran No relationship to disclose Renier J. Brentjens Stock or Other Ownership: Juno Therapeutics Honoraria: Pfizer Consulting or Advisory Role: Juno Therapeutics Research Funding: Juno Therapeutics Patents, Royalties, Other Intellectual Property: IP on CAR T cells; licensed patent (Juno)
www.jco.org
© 2015 by American Society of Clinical Oncology
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Curran and Brentjens
Acknowledgment We thank Hollie Pegram for designing the figure and editing the manuscript.
© 2015 by American Society of Clinical Oncology
JOURNAL OF CLINICAL ONCOLOGY
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33
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NUMBER
15
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MAY
20
2015
JOURNAL OF CLINICAL ONCOLOGY
UNDERSTANDING THE PATHWAY
Chimeric Antigen Receptor T Cells for Cancer Immunotherapy Kevin J. Curran and Renier J. Brentjens, Memorial Sloan Kettering Cancer Center, New York, NY See accompanying article on page 1688
T cells can be genetically modified to target tumors through the expression of a chimeric antigen receptor (CAR).1 The clinical benefit of CAR T cells has now been reported by several groups targeting the CD19 antigen in patients with hematologic malignancies.2-5 However, in solid tumors, CAR T cells have been less effective at inducing complete tumor responses and have mediated a high rate of on-target/ off-tumor toxicity.6-10 To understand this discrepancy, we must understand the design and implementation of CAR T cells for cancer immunotherapy. The basic design of CAR T cells consists of two fundamental domains: the antigen-binding portion (commonly composed of a single-chain variable fragment [scFv] derived from a monoclonal antibody [mAb]) joined to one or more intracellular T-cell signaling domains. To be effective after infusion, CAR T cells must expand, persist, exhibit enduring antitumor cytotoxicity, withstand and/or counteract an immunosuppressive tumor microenvironment, and overcome targeted tumor antigen escape. In designing CAR T cells for cancer immunotherapy, all of these factors must be harmonized to generate the optimal CAR T cell.
CAR DESIGN: TARGET ANTIGEN
Critical to the successful use of CAR T cells is choosing the proper tumor-associated antigen (TAA) to target. The ideal TAA for CAR T cells has the following key characteristics: expression on all tumor cells including the cancer stem cell, expression on the tumor cell surface, role in tumor cell survival, and lack of expression on normal tissues. Few TAAs are in fact ideal, because most are only variably expressed by the tumor and many are also expressed on one or more normal tissue types. The latter is most apparent when on-target/off-tumor toxicity occurs as in the case of carbonic anhydrase IX–specific CAR T cells or carcinoembryonic antigen-specific CAR T cells after infusion in a patient with renal cell carcinoma or colon cancer, respectively.6,9 In the accompanying article, Ahmed et al11 demonstrate that infusion of human epidermal growth factor receptor 2 (HER2) –specific CAR T cells for patients with relapsed/refractory sarcoma can be safely accomplished. This finding is significant in light of a prior report demonstrating that HER2-specific CAR T cells can lead to pulmonary toxicity and/or multiorgan failure and subsequent death, which has been attributed to unforeseen CAR T-cell recognition of low-level HER2 expression on the lung epithelium.12 This unexpected toxicity highlights the enhanced sensitivity of CAR T cells to low-level antigens Journal of Clinical Oncology, Vol 33, No 15 (May 20), 2015: pp 1703-1706
compared with mAbs when expressed on normal tissues. In contrast, it was found that HER2-specific CAR T cells did not produce cardiotoxicity after infusion, although treatment with the HER2-specific mAb trastuzumab did.13 Taken together, these findings highlight the discrepant safety profiles between mAbs and CAR T-cell therapies, even when they target the same epitope. Although there is a difference between the HER2-specific CARs and HER2 mAbs, the safe use of this target for CAR T cells, as reported by Ahmed et al,11 is an important advancement for the field of cancer immunotherapy. Potential strategies for overcoming the limiting effect of on-target/off-tumor toxicity have included blocking antigenic sites in off-tumor organs by using mAbs or by using CAR T cells, with combinatorial CARs recognizing two separate TAAs.14,15 CAR DESIGN: INTRACELLULAR SIGNALING
Early CAR designs incorporated one intracellular signaling domain, most commonly the immunoreceptor tyrosine-based activation motif of the CD3 chain.1 However, these first-generation CARs suboptimally activated the T cells and failed to demonstrate clinical benefit in early clinical trials.1 Enhancement of T-cell activation, persistence, and antitumor efficacy was mediated in second-generation CARs by including additional signaling domains within the CAR construct, most commonly the CD28 or 4-1BB costimulatory signaling domains. The dramatic complete response rates of 70% to 90% in adult and pediatric patients with relapsed/refractory B-cell acute lymphatic leukemia (B-ALL) following infusion of CD19-specific second-generation CAR T cells has resulted in renewed vigor for expanding CAR T-cell technology to other malignancies.2-4 To this end, second- and even thirdgeneration CAR designs, the latter incorporating two costimulatory signaling domains, have been used in solid tumors (including the CD28-containing HER2-specific CAR used in the accompanying article), albeit with only modest clinical responses. CAR T CELLS: TRANSLATION TO THE CLINIC
Several likely factors play a role in the clinical efficacy of CAR T cells after adoptive transfer. Paramount is the ability of CAR T cells to expand and/or persist, which has translated into improved response in both hematologic and solid tumors.2-4,10 The addition of costimulatory signaling within the design of the CAR T cell has improved © 2015 by American Society of Clinical Oncology
Information downloaded from jco.ascopubs.org and provided by at NEW YORK UNIVERSITY MED CTR on May 18, 2015 Copyright © 2015 Americanfrom Society of Clinical Oncology. All rights reserved. 128.122.253.212
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Curran and Brentjens
persistence.16 In contrast, the development of an endogenous antiCAR immune response can limit persistence of CAR T cells.3,7,17 To reduce CAR T-cell immunogenicity, the antigen-binding portion of the traditionally murine-derived scFv can be humanized or generated from human scFv phage display libraries.18 The use of prior condi-
A
tioning chemotherapy with respect to CAR T-cell infusion as well as the selection of central memory T-cell phenotype has been positively correlated with T-cell persistence.19,20 Although persistence is important, the optimal duration for CAR T cells circulation is unknown and may serve as a double edged sword by first completely eradicating
C
Identify suitable TAAs
B
Select CAR design
Screen for reactivity to normal cells Select human scFv specific for TAA
Candidates 1 and 2, not uniformly expressed
Select first-, second-, or third-generation CAR signaling domains
Candidate 3, not expressed on normal cells
Candidate 4, expressed on normal cells Candidates 3 and 4, expressed uniformly on tumor cells
E
Optimize efficacy, persistence, and safety using syngeneic murine model Treat murine tumor cell line with murine CAR T cells in immunocompetent murine model Increase antitumor efficacy by employing armored CAR T cells if needed
D
Test in preclinical xenogeneic murine model
Treat human tumor cell line or primary cancer cells with human CAR T cells Costimulatory ligand transgene
Cytokine transgene
G F
Translate to clinical setting to test safety and efficacy
Suicide transgene
Combine with immune checkpoint blockade to enhance efficacy
PD-1/CTLA-4 blocking mAbs
PD-1 CTLA-4 CAR T cell
Fig 1. Algorithm depicting pathway for designing optimal chimeric antigen receptor (CAR) T cells for tumor immunotherapy. (A) First, suitable tumor-associated antigens (TAAs) must be identified, with (B) secondary selection of target antigens based on TAA expression on normal tissues identifying optimal target tumor antigens with limited, if any, expression on nonmalignant cells. (C) Selected CAR constructs will be generated from human single-chain variable fragment (scFv) phage display libraries designed to reduce CAR immunogenicity. (D) CARs targeted to these TAAs need to be studied in xenotransplantation murine tumor models to validate potential efficacy. (E) Whenever possible, murine-derived CAR T cells should be assessed in clinically relevant syngeneic tumor models thereby providing a platform for the exploration of additional genetic modifications (eg, armored CAR T cells) to optimally enhance in vivo antitumor efficacy, persistence, and safety. Having met these criteria, modification of CAR T cells to express either a suicide vector or elimination gene to enhance safety regarding unforeseen on-target/off-tumor toxicity should be included and validated before initial clinical trial application. (F) Clinical trial application with either CAR T cells or armored CAR T cells should be initially assessed with respect to safety and clinical outcomes. (G) If single-agent CAR T-cell therapy is deemed insufficient in this setting, more clinical trials should be conducted that use the rational combination of CAR T-cell therapy and additional agents such as monoclonal antibody (mAb) –mediated immune checkpoint inhibitors (ie, PD-1/PD-L1 and/or CD80/CTLA4 blockade) to protect CAR T cells and further recruit endogenous immune effectors. 1704
© 2015 by American Society of Clinical Oncology
JOURNAL OF CLINICAL ONCOLOGY
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Chimeric Antigen Receptor T Cells for Cancer Immunotherapy
residual tumor cells through greater persistence and second by potentiating on-target/off-tumor toxicities as seen in the correlation between CD19-specific CAR T-cell persistence and long-standing B-cell aplasias.4 Although currently published clinical data from CD19-specific CAR T-cell trials have yet to establish optimal duration for maximal clinical benefit, the future inclusion of suicide or elimination genes such as iCASP9 and huEGFRt, respectively, may circumvent or abort on-target/ off-tumor toxicities once tumor eradication is complete.21,22 CAR T CELLS: ARMORED CARS
The immunosuppressive tumor microenvironment has a central role in limiting the effectiveness of CAR T cells in vivo.23 This environment is flush with endogenous immunosuppressive cells, including CD4⫹ regulatory T cells, myeloid-derived suppressor cells, plasmacytoid dendritic cells, tumor-associated macrophages, immune checkpoint inhibitors, and immunosuppressive soluble ligands and cytokines such as interleukin-10 (IL-10) or transforming growth factor-.24 The limited clinical benefit of the HER2-specific CAR T cells in the article by Ahmed et al11 could be due, in part, to the inability of CAR T cells to overcome this immunosuppressive tumor microenvironment. As the authors surmise, additional engineering of the CAR T cells or use of immune checkpoint blockade may enhance the effectiveness of the tumor-specific T cells.24-27 To this end, we and others have proposed additional modification of CAR T cells to enhance antitumor efficacy and persistence despite a prevalent hostile tumor microenvironment. As proof of principal, we have addressed the tumor microenvironment through further modification of CAR T cells to secrete the proinflammatory IL-12 cytokine.24 These armored IL-12–secreting CAR T cells are protected from regulatory T cell–mediated inhibition while delivering the IL-12 cytokine within the tumor microenvironment, thus potentially reversing the anergic state of endogenous tumor-infiltrating tumor-targeted T cells. Once reactivated, the endogenous immune system has the potential to target multiple TAAs (epitope spreading) and avert the development of antigen-negative tumor cells as has been seen with the development of CD19– clones after treatment with CD19-specific CAR T cells in patients with relapsed B-ALL.4 DESIGNING CAR T CELLS FOR ADOPTIVE THERAPY OF CANCER IN 2015
After more than 20 years of preclinical evaluation, CAR T-cell immunotherapy is reaching an initial zenith in the setting of relapsed B-ALL, which may well alter the standard of care for this patient population. Expansion of this technology to a broader cohort of malignancies, especially solid tumors, will require a multistep CAR T-cell design REFERENCES 1. Curran KJ, Pegram HJ, Brentjens RJ: Chimeric antigen receptors for T cell immunotherapy: Current understanding and future directions. J Gene Med 14:405-415, 2012 2. Davila ML, Riviere I, Wang X, et al: Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 6:224ra25, 2014 www.jco.org
approach (Fig 1). This approach will first require the identification of suitable TAAs (Fig 1A), with secondary selection of target antigens based on TAA expression on normal tissues identifying optimal target tumor antigens with limited, if any, expression on nonmalignant cells (Fig 1B). Selected CAR constructs will be generated from human scFv phage display libraries designed to reduce CAR immunogenicity (Fig 1C).17 In addition, CARs targeted to these TAAs need to be studied in xenotransplant murine tumor models to validate potential efficacy (Fig 1D). Whenever possible, murine-derived CAR T cells should be assessed in clinically relevant syngeneic tumor models thereby providing a platform for the exploration of additional genetic modifications (eg, armored CAR T cells) to optimally enhance in vivo antitumor efficacy, persistence, and safety (Fig 1E). Having met these criteria, further modification of CAR T cells to express either a suicide vector or elimination gene to enhance safety regarding unforeseen on-target/ off-tumor toxicity should be included and validated before initial clinical trial application (Fig 1E). Investigators in this field must acknowledge that despite preclinical murine tumor models, the ultimate safety and efficacy of this approach using novel CAR T cell–targeted antigens can be accurately assessed only in carefully designed human clinical trials. Clinical trial application with either CAR T cells or armored CAR T cells should be initially assessed with respect to safety and clinical outcomes (Fig 1F). If single-agent CAR T cell therapy is deemed insufficient in this setting, additional clinical trials should be conducted using the rational combination of CAR T cell therapy with additional agents such as mAb-mediated immune checkpoint inhibitors (ie, PD-1/PD-L1 and/or CD80/ CTLA4 blockade) to protect CAR T cells and further recruit endogenous immune effectors (Fig 1G). Collectively, this schematic highlights our current appreciation of CAR T-cell technology with respect to extrapolating this promising adoptive T-cell approach to other hematologic malignancies and, more significantly, to solid tumor malignancies. We anticipate that the outcomes from current and future CAR T-cell clinical trials targeting solid tumors will markedly expand and modify this CAR T-cell design algorithm. AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Disclosures provided by the authors are available with this article at www.jco.org.
AUTHOR CONTRIBUTIONS Manuscript writing: All authors Final approval of manuscript: All authors
3. Lee DW, Kochenderfer JN, Stetler-Stevenson M, 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 385:517-528, 2015 4. Maude SL, Frey N, Shaw PA, et al: Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med 371:1507-1517, 2014 5. Kochenderfer JN, Dudley ME, Kassim SH, et al: Chemotherapy-refractory diffuse large B-cell lym-
phoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol 33:540-549, 2015 6. Lamers CH, Sleijfer S, Vulto AG, et al: Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: First clinical experience. J Clin Oncol 24:e20-e22, 2006 7. Kershaw MH, Westwood JA, Parker LL, et al: A phase I study on adoptive immunotherapy using
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gene-modified T cells for ovarian cancer. Clin Cancer Res 12:6106-6115, 2006 8. Park JR, Digiusto DL, Slovak M, et al: Adoptive transfer of chimeric antigen receptor re-directed cytolytic T lymphocyte clones in patients with neuroblastoma. Mol Ther 15:825-833, 2007 9. Parkhurst MR, Yang JC, Langan RC, et al: T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 19:620-626, 2011 10. Louis CU, Savoldo B, Dotti G, et al: Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood 118:6050-6056, 2011 11. Ahmed N, Brawley V, Hedge M, et al: Human epidermal growth factor receptor 2 (HER2)–specific chimeric antigen receptor-modified T cells for the immunotherapy of osteosarcoma. J Clin Oncol 33: 1688-1696, 2015 12. Morgan RA, Yang JC, Kitano M, et al: Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 18:843-851, 2010 13. Seidman A, Hudis C, Pierri MK, et al: Cardiac dysfunction in the trastuzumab clinical trials experience. J Clin Oncol 20:1215-1221, 2002 14. Lamers CH, Sleijfer S, van Steenbergen S, et al: Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: Clinical evaluation and
management of on-target toxicity. Mol Ther 21:904912, 2013 15. Kloss CC, Condomines M, Cartellieri M, et al: Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol 31: 71-75, 2013 16. Savoldo B, Ramos CA, Liu E, et al: CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J Clin Invest 121:1822-1826, 2011 17. Lamers CH, Willemsen R, van Elzakker P, et al: Immune responses to transgene and retroviral vector in patients treated with ex vivo-engineered T cells. Blood 117:72-82, 2011 18. Dao T, Yan S, Veomett N, et al: Targeting the intracellular WT1 oncogene product with a therapeutic human antibody. Sci Transl Med 5:176ra33, 2013 19. Brentjens RJ, Rivière I, Park JH, et al: Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 118:4817-4828, 2011 20. Berger C, Jensen MC, Lansdorp PM, et al: Adoptive transfer of effector CD8⫹ T cells derived from central memory cells establishes persistent T cell memory in primates. J Clin Invest 118:294-305, 2008
21. Di Stasi A, Tey SK, Dotti G, et al: Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 365:1673-1683, 2011 22. Wang X, Chang WC, Wong CW, et al: A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood 118:1255-1263, 2011 23. Vesely MD, Kershaw MH, Schreiber RD, et al: Natural innate and adaptive immunity to cancer. Annu Rev Immunol 29:235-271, 2011 24. Pegram HJ, Lee JC, Hayman EG, et al: Tumortargeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood 119:4133-4141, 2012 25. Pegram HJ, Park JH, Brentjens RJ: CD28z CARs and armored CARs. Cancer J 20:127-133, 2014 26. Hoyos V, Savoldo B, Quintarelli C, et al: Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia 24:1160-1170, 2010 27. Dotti G, Gottschalk S, Savoldo B, et al: Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev 257: 107-126, 2014
DOI: 10.1200/JCO.2014.60.3449; published online ahead of print at www.jco.org on April 20, 2015
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Chimeric Antigen Receptor T Cells for Cancer Immunotherapy
AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Chimeric Antigen Receptor T Cells for Cancer Immunotherapy The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I ⫽ Immediate Family Member, Inst ⫽ My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or jco.ascopubs.org/site/ifc. Kevin J. Curran No relationship to disclose Renier J. Brentjens Stock or Other Ownership: Juno Therapeutics Honoraria: Pfizer Consulting or Advisory Role: Juno Therapeutics Research Funding: Juno Therapeutics Patents, Royalties, Other Intellectual Property: IP on CAR T cells; licensed patent (Juno)
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Curran and Brentjens
Acknowledgment We thank Hollie Pegram for designing the figure and editing the manuscript.
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