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1.  Therapeutic T cell engineering 
Nature  2017;545(7655):423-431.
Genetically engineered T cells are powerful new medicines, offering hope for curative responses in patients with cancer. Chimaeric antigen receptors (CARs) are a class of synthetic receptors that reprogram lymphocyte specificity and function. CARs targeting CD19 have demonstrated remarkable potency in B cell malignancies. Engineered T cells are applicable in principle to many cancers, pending further progress to identify suitable target antigens, overcome immunosuppressive tumour microenvironments, reduce toxicities, and prevent antigen escape. Advances in the selection of optimal T cells, genetic engineering, and cell manufacturing are poised to broaden T-cell-based therapies and foster new applications in infectious diseases and autoimmunity.
PMCID: PMC5632949  PMID: 28541315
2.  Fully human CD19-specific chimeric antigen receptors for T-cell therapy 
Leukemia  2017;10.1038/leu.2017.57.
Impressive results have been achieved by adoptively transferring T-cells expressing CD19-specific CARs with binding domains from murine mAbs to treat B-cell malignancies. T-cell mediated immune responses specific for peptides from the murine scFv antigen-binding domain of the CAR can develop in patients and result in premature elimination of CAR-T-cells increasing the risk of tumor relapse. As fully human scFv might reduce immunogenicity, we generated CD19-specific human scFvs with similar binding characteristics as the murine FMC63-derived scFv using human Ab/DNA-libraries. CARs were constructed in various formats from several scFvs and used to transduce primary human T-cells. The resulting CD19-CAR-T-cells were specifically activated by and lysed CD19-positive tumor cell lines and primary CLL cells, and eliminated human lymphoma xenografts in immunodeficient mice. Certain fully human CAR constructs were superior to the FMC63-CAR, which is widely used in clinical trials. Imaging of cell surface distribution of the human CARs revealed no evidence of clustering without target cell engagement, and tonic signaling was not observed. To further reduce potential immunogenicity of the CARs, we also modified the fusion sites between different CAR components. The described fully human CARs for a validated clinical target may reduce immune rejection compared with murine based CARs.
PMCID: PMC5608623  PMID: 28202953
3.  T-cell infiltration and clonality correlate with programmed cell death protein 1 and programmed death-ligand 1 expression in patients with soft tissue sarcomas 
Cancer  2017;123(17):3291-3304.
Metastatic sarcomas have poor outcomes and may be amenable to immunotherapies, however knowledge of the immunologic profiles of soft tissue sarcoma (STS) subtypes is limited.
We identified patients with the common STS subtypes: leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS), well/de-differentiated (WD/DD) liposarcoma and myxoid/round cell liposarcoma (MRCL). Gene expression, IHC for PD-1 and PD-L1, and T cell receptor Vβ gene sequencing was performed on FFPE tumors from 81 patients. Differences in liposarcoma subsets were also evaluated.
UPS and LMS had high expression levels of genes related to antigen presentation and T cell infiltration. UPS had higher levels of PD-L1 (p≤0.001) and PD-1 (p≤0.05) on IHC and had the highest T cell infiltration based on TCR sequencing, significantly more than SS, which had the lowest (p≤0.05). T cell infiltrates in UPS were also more oligoclonal compared with SS and liposarcoma (p≤0.05). A model adjusted for STS histologic subtype found that for all sarcomas, T cell infiltration and clonality were highly correlated with PD-1 and PD-L1 expression levels (p≤0.01).
We provide the most detailed overview of the immune microenvironment in sarcoma subtypes to date. UPS, which is a more highly mutated STS subtype, provokes a substantial immune response, suggesting that it may be well-suited to treatment with immune checkpoint inhibitors. SS and liposarcoma subsets are less mutated but do express immunogenic self-antigens, therefore strategies to improve antigen presentation and T cell infiltration may allow for successful immunotherapy in those diagnoses.
PMCID: PMC5568958  PMID: 28463396
PD-1; PD-L1; sarcoma; T cell receptors; gene expression; pleomorphic; leiomyosarcoma; liposarcoma; synovial; immunotherapy
4.  T‐cell infiltration and clonality correlate with programmed cell death protein 1 and programmed death‐ligand 1 expression in patients with soft tissue sarcomas 
Cancer  2017;123(17):3291-3304.
Patients with metastatic sarcomas have poor outcomes and although the disease may be amenable to immunotherapies, information regarding the immunologic profiles of soft tissue sarcoma (STS) subtypes is limited.
The authors identified patients with the common STS subtypes: leiomyosarcoma, undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS), well‐differentiated/dedifferentiated liposarcoma, and myxoid/round cell liposarcoma. Gene expression, immunohistochemistry for programmed cell death protein (PD‐1) and programmed death‐ligand 1 (PD‐L1), and T‐cell receptor Vβ gene sequencing were performed on formalin‐fixed, paraffin‐embedded tumors from 81 patients. Differences in liposarcoma subsets also were evaluated.
UPS and leiomyosarcoma had high expression levels of genes related to antigen presentation and T‐cell infiltration. UPS were found to have higher levels of PD‐L1 (P≤.001) and PD‐1 (P≤.05) on immunohistochemistry and had the highest T‐cell infiltration based on T‐cell receptor sequencing, significantly more than SS, which had the lowest (P≤.05). T‐cell infiltrates in UPS also were more oligoclonal compared with SS and liposarcoma (P≤.05). A model adjusted for STS histologic subtype found that for all sarcomas, T‐cell infiltration and clonality were highly correlated with PD‐1 and PD‐L1 expression levels (P≤.01).
In the current study, the authors provide the most detailed overview of the immune microenvironment in sarcoma subtypes to date. UPS, which is a more highly mutated STS subtype, provokes a substantial immune response, suggesting that it may be well suited to treatment with immune checkpoint inhibitors. The SS and liposarcoma subsets are less mutated but do express immunogenic self‐antigens, and therefore strategies to improve antigen presentation and T‐cell infiltration may allow for successful immunotherapy in patients with these diagnoses. Cancer 2017;123:3291‐304. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
To the author's knowledge, the current study provides the most comprehensive characterization of the sarcoma tumor immune microenvironment to date through the use of gene expression analysis, immunohistochemistry, and T‐cell receptor sequencing. The results demonstrate that some sarcoma subtypes, such as synovial sarcoma, are immunologically quiet, whereas others, such as undifferentiated pleomorphic sarcoma, are highly inflammatory and could be susceptible to immune checkpoint inhibition.
PMCID: PMC5568958  PMID: 28463396
gene expression; immunotherapy; leiomyosarcoma; liposarcoma; pleomorphic; programmed cell death protein (PD‐1); programmed death‐ligand 1 (PD‐L1); sarcoma; T‐cell receptors
5.  Preserved Activity of CD20-Specific Chimeric Antigen Receptor-Expressing T cells in the Presence of Rituximab 
Cancer immunology research  2016;4(6):509-519.
CD20 is an attractive immunotherapy target for B-cell non-Hodgkin lymphomas, and adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) targeting CD20 is a promising strategy. A theoretical limitation is that residual serum rituximab might block CAR binding to CD20 and thereby impede T cell–mediated anti-lymphoma responses. The activity of CD20 CAR-modified T cells in the presence of various concentrations of rituximab was tested in vitro and in vivo. CAR binding sites on CD20+ tumor cells were blocked by rituximab in a dose-dependent fashion, although at 37°C blockade was incomplete at concentrations up to 200 μg/ml. T cells with CD20 CARs also exhibited modest dose-dependent reductions in cytokine secretion and cytotoxicity, but not proliferation, against lymphoma cell lines. At rituximab concentrations of 100 μg/ml, CAR T cells retained ≥ 50% of baseline activity against targets with high CD20 expression, but were more strongly inhibited when target cells expressed low CD20. In a murine xenograft model using a rituximab-refractory lymphoma cell line, rituximab did not impair CAR T-cell activity, and tumors were eradicated in > 85% of mice. Clinical residual rituximab serum concentrations were measured in 103 lymphoma patients after rituximab therapy, with the median level found to be only 38 μg/ml (interquartile range 19-72 μg/ml). Thus, despite modest functional impairment in vitro, the in vivo activity of CD20-targeted CAR T cells remains intact at clinically relevant levels of rituximab, making use of these T cells clinically feasible.
PMCID: PMC4891234  PMID: 27197068
Chimeric antigen receptors; Adoptive T cell therapy; Rituximab; Immunotherapy; Lymphoma
6.  Role of memory T cell subsets for adoptive immunotherapy 
Seminars in immunology  2016;28(1):28-34.
Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. Besides the definition of optimal targets and antigen receptors, the differentiation status of transferred T cells is emerging as a crucial parameter for generating cell products with optimal efficacy and safety profiles.
Long-living memory T cells subdivide into phenotypically as well as functionally different subsets (e.g. central memory, effector memory, tissue-resident memory T cells). This diversification process is crucial for effective immune protection, with probably distinct dependencies on the presence of individual subsets dependent on the disease to which the immune response is directed as well as its organ location.
Adoptive T cell therapy intends to therapeutically transfer defined T cell immunity into patients. Efficacy of this approach often requires long-term maintenance of transferred cells, which depends on the presence and persistence of memory T cells. However, engraftment and survival of highly differentiated memory T cell subsets upon adoptive transfer is still difficult to achieve. Therefore, the recent observation that a distinct subset of weakly differentiated memory T cells shows all characteristics of adult tissue stem cells and can reconstitute all types of effector and memory T cell subsets, became highly relevant. We here review our current understanding of memory subset formation and T cell subset purification, and it's implications for adoptive immunotherapy.
PMCID: PMC5027130  PMID: 26976826
7.  Immunotherapy of non-Hodgkin lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor-modified T cells 
Science translational medicine  2016;8(355):355ra116.
CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in B cell malignancies, but factors that impact toxicity and efficacy have been difficult to define because of differences in lymphodepletion regimens and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4+:CD8+ ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin lymphoma after cyclophosphamide (Cy)-based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had markedly increased CAR-T cell expansion and persistence, and higher response rates (50% CR, 72% ORR, n=20) than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR, n=12). The complete response (CR) rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR, n=11). Cy/Flu minimized the effects of an immune response to the murine scFv component of the CAR, which limited CAR-T cell expansion, persistence, and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥ 3 neurotoxicity were observed in 13% and 28% of all patients, respectively. Serum biomarkers one day after CAR-T cell infusion correlated with subsequent development of sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4+:CD8+ ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of a lymphodepletion regimen that improved disease response and overall and progression-free survival.
PMCID: PMC5045301  PMID: 27605551
8.  Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia 
The Journal of Clinical Investigation  null;126(11):4262-4272.
The adoptive transfer of T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) is effective for treating human B cell malignancies. However, the persistence of functional CD19 CAR T cells causes sustained depletion of endogenous CD19+ B cells and hypogammaglobulinemia. Thus, there is a need for a mechanism to ablate transferred T cells after tumor eradication is complete to allow recovery of normal B cells. Previously, we developed a truncated version of the epidermal growth factor receptor (EGFRt) that is coexpressed with the CAR on the T cell surface. Here, we show that targeting EGFRt with the IgG1 monoclonal antibody cetuximab eliminates CD19 CAR T cells both early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functional B cells, without tumor relapse. EGFRt can be incorporated into many clinical applications to regulate the survival of gene-engineered cells. These results support the concept that EGFRt represents a promising approach to improve safety of cell-based therapies.
PMCID: PMC5096899  PMID: 27760047
9.  31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016): part one 
Lundqvist, Andreas | van Hoef, Vincent | Zhang, Xiaonan | Wennerberg, Erik | Lorent, Julie | Witt, Kristina | Sanz, Laia Masvidal | Liang, Shuo | Murray, Shannon | Larsson, Ola | Kiessling, Rolf | Mao, Yumeng | Sidhom, John-William | Bessell, Catherine A. | Havel, Jonathan | Schneck, Jonathan | Chan, Timothy A. | Sachsenmeier, Eliot | Woods, David | Berglund, Anders | Ramakrishnan, Rupal | Sodre, Andressa | Weber, Jeffrey | Zappasodi, Roberta | Li, Yanyun | Qi, Jingjing | Wong, Philip | Sirard, Cynthia | Postow, Michael | Newman, Walter | Koon, Henry | Velcheti, Vamsidhar | Callahan, Margaret K. | Wolchok, Jedd D. | Merghoub, Taha | Lum, Lawrence G. | Choi, Minsig | Thakur, Archana | Deol, Abhinav | Dyson, Gregory | Shields, Anthony | Haymaker, Cara | Uemura, Marc | Murthy, Ravi | James, Marihella | Wang, Daqing | Brevard, Julie | Monaghan, Catherine | Swann, Suzanne | Geib, James | Cornfeld, Mark | Chunduru, Srinivas | Agrawal, Sudhir | Yee, Cassian | Wargo, Jennifer | Patel, Sapna P. | Amaria, Rodabe | Tawbi, Hussein | Glitza, Isabella | Woodman, Scott | Hwu, Wen-Jen | Davies, Michael A. | Hwu, Patrick | Overwijk, Willem W. | Bernatchez, Chantale | Diab, Adi | Massarelli, Erminia | Segal, Neil H. | Ribrag, Vincent | Melero, Ignacio | Gangadhar, Tara C. | Urba, Walter | Schadendorf, Dirk | Ferris, Robert L. | Houot, Roch | Morschhauser, Franck | Logan, Theodore | Luke, Jason J. | Sharfman, William | Barlesi, Fabrice | Ott, Patrick A. | Mansi, Laura | Kummar, Shivaani | Salles, Gilles | Carpio, Cecilia | Meier, Roland | Krishnan, Suba | McDonald, Dan | Maurer, Matthew | Gu, Xuemin | Neely, Jaclyn | Suryawanshi, Satyendra | Levy, Ronald | Khushalani, Nikhil | Wu, Jennifer | Zhang, Jinyu | Basher, Fahmin | Rubinstein, Mark | Bucsek, Mark | Qiao, Guanxi | MacDonald, Cameron | Hylander, Bonnie | Repasky, Elizabeth | Chatterjee, Shilpak | Daenthanasanmak, Anusara | Chakraborty, Paramita | Toth, Kyle | Meek, Megan | Garrett-Mayer, Elizabeth | Nishimura, Michael | Paulos, Chrystal | Beeson, Craig | Yu, Xuezhong | Mehrotra, Shikhar | Zhao, Fei | Evans, Kathy | Xiao, Christine | Holtzhausen, Alisha | Hanks, Brent A. | Scharping, Nicole | Menk, Ashley V. | Moreci, Rebecca | Whetstone, Ryan | Dadey, Rebekah | Watkins, Simon | Ferris, Robert | Delgoffe, Greg M. | Peled, Jonathan | Devlin, Sean | Staffas, Anna | Lumish, Melissa | Rodriguez, Kori Porosnicu | Ahr, Katya | Perales, Miguel | Giralt, Sergio | Taur, Ying | Pamer, Eric | van den Brink, Marcel R. M. | Jenq, Robert | Annels, Nicola | Pandha, Hardev | Simpson, Guy | Mostafid, Hugh | Harrington, Kevin | Melcher, Alan | Grose, Mark | Davies, Bronwyn | Au, Gough | Karpathy, Roberta | Shafren, Darren | Ricca, Jacob | Merghoub, Taha | Wolchok, Jedd D. | Zamarin, Dmitriy | Batista, Luciana | Marliot, Florence | Vasaturo, Angela | Carpentier, Sabrina | Poggionovo, Cécile | Frayssinet, Véronique | Fieschi, Jacques | Van den Eynde, Marc | Pagès, Franck | Galon, Jérôme | Hermitte, Fabienne | Smith, Sean G. | Nguyen, Khue | Ravindranathan, Sruthi | Koppolu, Bhanu | Zaharoff, David | Schvartsman, Gustavo | Bassett, Roland | McQuade, Jennifer L. | Haydu, Lauren E. | Davies, Michael A. | Tawbi, Hussein | Glitza, Isabella | Kline, Douglas | Chen, Xiufen | Fosco, Dominick | Kline, Justin | Overacre, Abigail | Chikina, Maria | Brunazzi, Erin | Shayan, Gulidanna | Horne, William | Kolls, Jay | Ferris, Robert L. | Delgoffe, Greg M. | Bruno, Tullia C. | Workman, Creg | Vignali, Dario | Adusumilli, Prasad S. | Ansa-Addo, Ephraim A | Li, Zihai | Gerry, Andrew | Sanderson, Joseph P. | Howe, Karen | Docta, Roslin | Gao, Qian | Bagg, Eleanor A. L. | Tribble, Nicholas | Maroto, Miguel | Betts, Gareth | Bath, Natalie | Melchiori, Luca | Lowther, Daniel E. | Ramachandran, Indu | Kari, Gabor | Basu, Samik | Binder-Scholl, Gwendolyn | Chagin, Karen | Pandite, Lini | Holdich, Tom | Amado, Rafael | Zhang, Hua | Glod, John | Bernstein, Donna | Jakobsen, Bent | Mackall, Crystal | Wong, Ryan | Silk, Jonathan D. | Adams, Katherine | Hamilton, Garth | Bennett, Alan D. | Brett, Sara | Jing, Junping | Quattrini, Adriano | Saini, Manoj | Wiedermann, Guy | Gerry, Andrew | Jakobsen, Bent | Binder-Scholl, Gwendolyn | Brewer, Joanna | Duong, MyLinh | Lu, An | Chang, Peter | Mahendravada, Aruna | Shinners, Nicholas | Slawin, Kevin | Spencer, David M. | Foster, Aaron E. | Bayle, J. Henri | Bergamaschi, Cristina | Ng, Sinnie Sin Man | Nagy, Bethany | Jensen, Shawn | Hu, Xintao | Alicea, Candido | Fox, Bernard | Felber, Barbara | Pavlakis, George | Chacon, Jessica | Yamamoto, Tori | Garrabrant, Thomas | Cortina, Luis | Powell, Daniel J. | Donia, Marco | Kjeldsen, Julie Westerlin | Andersen, Rikke | Westergaard, Marie Christine Wulff | Bianchi, Valentina | Legut, Mateusz | Attaf, Meriem | Dolton, Garry | Szomolay, Barbara | Ott, Sascha | Lyngaa, Rikke | Hadrup, Sine Reker | Sewell, Andrew Kelvin | Svane, Inge Marie | Fan, Aaron | Kumai, Takumi | Celis, Esteban | Frank, Ian | Stramer, Amanda | Blaskovich, Michelle A. | Wardell, Seth | Fardis, Maria | Bender, James | Lotze, Michael T. | Goff, Stephanie L. | Zacharakis, Nikolaos | Assadipour, Yasmine | Prickett, Todd D. | Gartner, Jared J. | Somerville, Robert | Black, Mary | Xu, Hui | Chinnasamy, Harshini | Kriley, Isaac | Lu, Lily | Wunderlich, John | Robbins, Paul F. | Rosenberg, Steven | Feldman, Steven A. | Trebska-McGowan, Kasia | Kriley, Isaac | Malekzadeh, Parisa | Payabyab, Eden | Sherry, Richard | Rosenberg, Steven | Goff, Stephanie L. | Gokuldass, Aishwarya | Blaskovich, Michelle A. | Kopits, Charlene | Rabinovich, Brian | Lotze, Michael T. | Green, Daniel S. | Kamenyeva, Olena | Zoon, Kathryn C. | Annunziata, Christina M. | Hammill, Joanne | Helsen, Christopher | Aarts, Craig | Bramson, Jonathan | Harada, Yui | Yonemitsu, Yoshikazu | Helsen, Christopher | Hammill, Joanne | Mwawasi, Kenneth | Denisova, Galina | Bramson, Jonathan | Giri, Rajanish | Jin, Benjamin | Campbell, Tracy | Draper, Lindsey M. | Stevanovic, Sanja | Yu, Zhiya | Weissbrich, Bianca | Restifo, Nicholas P. | Trimble, Cornelia L. | Rosenberg, Steven | Hinrichs, Christian S. | Tsang, Kwong | Fantini, Massimo | Hodge, James W. | Fujii, Rika | Fernando, Ingrid | Jochems, Caroline | Heery, Christopher | Gulley, James | Soon-Shiong, Patrick | Schlom, Jeffrey | Jing, Weiqing | Gershan, Jill | Blitzer, Grace | Weber, James | McOlash, Laura | Johnson, Bryon D. | Kiany, Simin | Gangxiong, Huang | Kleinerman, Eugenie S. | Klichinsky, Michael | Ruella, Marco | Shestova, Olga | Kenderian, Saad | Kim, Miriam | Scholler, John | June, Carl H. | Gill, Saar | Moogk, Duane | Zhong, Shi | Yu, Zhiya | Liadi, Ivan | Rittase, William | Fang, Victoria | Dougherty, Janna | Perez-Garcia, Arianne | Osman, Iman | Zhu, Cheng | Varadarajan, Navin | Restifo, Nicholas P. | Frey, Alan | Krogsgaard, Michelle | Landi, Daniel | Fousek, Kristen | Mukherjee, Malini | Shree, Ankita | Joseph, Sujith | Bielamowicz, Kevin | Byrd, Tiara | Ahmed, Nabil | Hegde, Meenakshi | Lee, Sylvia | Byrd, David | Thompson, John | Bhatia, Shailender | Tykodi, Scott | Delismon, Judy | Chu, Liz | Abdul-Alim, Siddiq | Ohanian, Arpy | DeVito, Anna Marie | Riddell, Stanley | Margolin, Kim | Magalhaes, Isabelle | Mattsson, Jonas | Uhlin, Michael | Nemoto, Satoshi | Villarroel, Patricio Pérez | Nakagawa, Ryosuke | Mule, James J. | Mailloux, Adam W. | Mata, Melinda | Nguyen, Phuong | Gerken, Claudia | DeRenzo, Christopher | Spencer, David M. | Gottschalk, Stephen | Mathieu, Mélissa | Pelletier, Sandy | Stagg, John | Turcotte, Simon | Minutolo, Nicholas | Sharma, Prannda | Tsourkas, Andrew | Powell, Daniel J. | Mockel-Tenbrinck, Nadine | Mauer, Daniela | Drechsel, Katharina | Barth, Carola | Freese, Katharina | Kolrep, Ulrike | Schult, Silke | Assenmacher, Mario | Kaiser, Andrew | Mullinax, John | Hall, MacLean | Le, Julie | Kodumudi, Krithika | Royster, Erica | Richards, Allison | Gonzalez, Ricardo | Sarnaik, Amod | Pilon-Thomas, Shari | Nielsen, Morten | Krarup-Hansen, Anders | Hovgaard, Dorrit | Petersen, Michael Mørk | Loya, Anand Chainsukh | Junker, Niels | Svane, Inge Marie | Rivas, Charlotte | Parihar, Robin | Gottschalk, Stephen | Rooney, Cliona M. | Qin, Haiying | Nguyen, Sang | Su, Paul | Burk, Chad | Duncan, Brynn | Kim, Bong-Hyun | Kohler, M. Eric | Fry, Terry | Rao, Arjun A. | Teyssier, Noam | Pfeil, Jacob | Sgourakis, Nikolaos | Salama, Sofie | Haussler, David | Richman, Sarah A. | Nunez-Cruz, Selene | Gershenson, Zack | Mourelatos, Zissimos | Barrett, David | Grupp, Stephan | Milone, Michael | Rodriguez-Garcia, Alba | Robinson, Matthew K. | Adams, Gregory P. | Powell, Daniel J. | Santos, João | Havunen, Riikka | Siurala, Mikko | Cervera-Carrascón, Víctor | Parviainen, Suvi | Antilla, Marjukka | Hemminki, Akseli | Sethuraman, Jyothi | Santiago, Laurelis | Chen, Jie Qing | Dai, Zhimin | Wardell, Seth | Bender, James | Lotze, Michael T. | Sha, Huizi | Su, Shu | Ding, Naiqing | Liu, Baorui | Stevanovic, Sanja | Pasetto, Anna | Helman, Sarah R. | Gartner, Jared J. | Prickett, Todd D. | Robbins, Paul F. | Rosenberg, Steven A. | Hinrichs, Christian S. | Bhatia, Shailender | Burgess, Melissa | Zhang, Hui | Lee, Tien | Klingemann, Hans | Soon-Shiong, Patrick | Nghiem, Paul | Kirkwood, John M. | Rossi, John M. | Sherman, Marika | Xue, Allen | Shen, Yueh-wei | Navale, Lynn | Rosenberg, Steven A. | Kochenderfer, James N. | Bot, Adrian | Veerapathran, Anandaraman | Gokuldass, Aishwarya | Stramer, Amanda | Sethuraman, Jyothi | Blaskovich, Michelle A. | Wiener, Doris | Frank, Ian | Santiago, Laurelis | Rabinovich, Brian | Fardis, Maria | Bender, James | Lotze, Michael T. | Waller, Edmund K. | Li, Jian-Ming | Petersen, Christopher | Blazar, Bruce R. | Li, Jingxia | Giver, Cynthia R. | Wang, Ziming | Grossenbacher, Steven K. | Sturgill, Ian | Canter, Robert J. | Murphy, William J. | Zhang, Congcong | Burger, Michael C. | Jennewein, Lukas | Waldmann, Anja | Mittelbronn, Michel | Tonn, Torsten | Steinbach, Joachim P. | Wels, Winfried S. | Williams, Jason B. | Zha, Yuanyuan | Gajewski, Thomas F. | Williams, LaTerrica C. | Krenciute, Giedre | Kalra, Mamta | Louis, Chrystal | Gottschalk, Stephen | Xin, Gang | Schauder, David | Jiang, Aimin | Joshi, Nikhil | Cui, Weiguo | Zeng, Xue | Menk, Ashley V. | Scharping, Nicole | Delgoffe, Greg M. | Zhao, Zeguo | Hamieh, Mohamad | Eyquem, Justin | Gunset, Gertrude | Bander, Neil | Sadelain, Michel | Askmyr, David | Abolhalaj, Milad | Lundberg, Kristina | Greiff, Lennart | Lindstedt, Malin | Angell, Helen K. | Kim, Kyoung-Mee | Kim, Seung-Tae | Kim, Sung | Sharpe, Alan D. | Ogden, Julia | Davenport, Anna | Hodgson, Darren R. | Barrett, Carl | Lee, Jeeyun | Kilgour, Elaine | Hanson, Jodi | Caspell, Richard | Karulin, Alexey | Lehmann, Paul | Ansari, Tameem | Schiller, Annemarie | Sundararaman, Srividya | Lehmann, Paul | Hanson, Jodi | Roen, Diana | Karulin, Alexey | Lehmann, Paul | Ayers, Mark | Levitan, Diane | Arreaza, Gladys | Liu, Fang | Mogg, Robin | Bang, Yung-Jue | O’Neil, Bert | Cristescu, Razvan | Friedlander, Philip | Wassman, Karl | Kyi, Chrisann | Oh, William | Bhardwaj, Nina | Bornschlegl, Svetlana | Gustafson, Michael P. | Gastineau, Dennis A. | Parney, Ian F. | Dietz, Allan B. | Carvajal-Hausdorf, Daniel | Mani, Nikita | Velcheti, Vamsidhar | Schalper, Kurt | Rimm, David | Chang, Serena | Levy, Ronald | Kurland, John | Krishnan, Suba | Ahlers, Christoph Matthias | Jure-Kunkel, Maria | Cohen, Lewis | Maecker, Holden | Kohrt, Holbrook | Chen, Shuming | Crabill, George | Pritchard, Theresa | McMiller, Tracee | Pardoll, Drew | Pan, Fan | Topalian, Suzanne | Danaher, Patrick | Warren, Sarah | Dennis, Lucas | White, Andrew M. | D’Amico, Leonard | Geller, Melissa | Disis, Mary L. | Beechem, Joseph | Odunsi, Kunle | Fling, Steven | Derakhshandeh, Roshanak | Webb, Tonya J. | Dubois, Sigrid | Conlon, Kevin | Bryant, Bonita | Hsu, Jennifer | Beltran, Nancy | Müller, Jürgen | Waldmann, Thomas | Duhen, Rebekka | Duhen, Thomas | Thompson, Lucas | Montler, Ryan | Weinberg, Andrew | Kates, Max | Early, Brandon | Yusko, Erik | Schreiber, Taylor H. | Bivalacqua, Trinity J. | Ayers, Mark | Lunceford, Jared | Nebozhyn, Michael | Murphy, Erin | Loboda, Andrey | Kaufman, David R. | Albright, Andrew | Cheng, Jonathan | Kang, S. Peter | Shankaran, Veena | Piha-Paul, Sarina A. | Yearley, Jennifer | Seiwert, Tanguy | Ribas, Antoni | McClanahan, Terrill K. | Cristescu, Razvan | Mogg, Robin | Ayers, Mark | Albright, Andrew | Murphy, Erin | Yearley, Jennifer | Sher, Xinwei | Liu, Xiao Qiao | Nebozhyn, Michael | Lunceford, Jared | Joe, Andrew | Cheng, Jonathan | Plimack, Elizabeth | Ott, Patrick A. | McClanahan, Terrill K. | Loboda, Andrey | Kaufman, David R. | Forrest-Hay, Alex | Guyre, Cheryl A. | Narumiya, Kohei | Delcommenne, Marc | Hirsch, Heather A. | Deshpande, Amit | Reeves, Jason | Shu, Jenny | Zi, Tong | Michaelson, Jennifer | Law, Debbie | Trehu, Elizabeth | Sathyanaryanan, Sriram | Hodkinson, Brendan P. | Hutnick, Natalie A. | Schaffer, Michael E. | Gormley, Michael | Hulett, Tyler | Jensen, Shawn | Ballesteros-Merino, Carmen | Dubay, Christopher | Afentoulis, Michael | Reddy, Ashok | David, Larry | Fox, Bernard | Jayant, Kumar | Agrawal, Swati | Agrawal, Rajendra | Jeyakumar, Ghayathri | Kim, Seongho | Kim, Heejin | Silski, Cynthia | Suisham, Stacey | Heath, Elisabeth | Vaishampayan, Ulka | Vandeven, Natalie | Viller, Natasja Nielsen | O’Connor, Alison | Chen, Hui | Bossen, Bolette | Sievers, Eric | Uger, Robert | Nghiem, Paul | Johnson, Lisa | Kao, Hsiang-Fong | Hsiao, Chin-Fu | Lai, Shu-Chuan | Wang, Chun-Wei | Ko, Jenq-Yuh | Lou, Pei-Jen | Lee, Tsai-Jan | Liu, Tsang-Wu | Hong, Ruey-Long | Kearney, Staci J. | Black, Joshua C. | Landis, Benjamin J. | Koegler, Sally | Hirsch, Brooke | Gianani, Roberto | Kim, Jeffrey | He, Ming-Xiao | Zhang, Bingqing | Su, Nan | Luo, Yuling | Ma, Xiao-Jun | Park, Emily | Kim, Dae Won | Copploa, Domenico | Kothari, Nishi | doo Chang, Young | Kim, Richard | Kim, Namyong | Lye, Melvin | Wan, Ee | Kim, Namyong | Lye, Melvin | Wan, Ee | Kim, Namyong | Lye, Melvin | Wan, Ee | Knaus, Hanna A. | Berglund, Sofia | Hackl, Hubert | Karp, Judith E. | Gojo, Ivana | Luznik, Leo | Hong, Henoch S. | Koch, Sven D. | Scheel, Birgit | Gnad-Vogt, Ulrike | Kallen, Karl-Josef | Wiegand, Volker | Backert, Linus | Kohlbacher, Oliver | Hoerr, Ingmar | Fotin-Mleczek, Mariola | Billingsley, James M. | Koguchi, Yoshinobu | Conrad, Valerie | Miller, William | Gonzalez, Iliana | Poplonski, Tomasz | Meeuwsen, Tanisha | Howells-Ferreira, Ana | Rattray, Rogan | Campbell, Mary | Bifulco, Carlo | Dubay, Christopher | Bahjat, Keith | Curti, Brendan | Urba, Walter | Vetsika, E-K | Kallergi, G. | Aggouraki, Despoina | Lyristi, Z. | Katsarlinos, P. | Koinis, Filippos | Georgoulias, V. | Kotsakis, Athanasios | Martin, Nathan T. | Aeffner, Famke | Kearney, Staci J. | Black, Joshua C. | Cerkovnik, Logan | Pratte, Luke | Kim, Rebecca | Hirsch, Brooke | Krueger, Joseph | Gianani, Roberto | Martínez-Usatorre, Amaia | Jandus, Camilla | Donda, Alena | Carretero-Iglesia, Laura | Speiser, Daniel E. | Zehn, Dietmar | Rufer, Nathalie | Romero, Pedro | Panda, Anshuman | Mehnert, Janice | Hirshfield, Kim M. | Riedlinger, Greg | Damare, Sherri | Saunders, Tracie | Sokol, Levi | Stein, Mark | Poplin, Elizabeth | Rodriguez-Rodriguez, Lorna | Silk, Ann | Chan, Nancy | Frankel, Melissa | Kane, Michael | Malhotra, Jyoti | Aisner, Joseph | Kaufman, Howard L. | Ali, Siraj | Ross, Jeffrey | White, Eileen | Bhanot, Gyan | Ganesan, Shridar | Monette, Anne | Bergeron, Derek | Amor, Amira Ben | Meunier, Liliane | Caron, Christine | Morou, Antigoni | Kaufmann, Daniel | Liberman, Moishe | Jurisica, Igor | Mes-Masson, Anne-Marie | Hamzaoui, Kamel | Lapointe, Rejean | Mongan, Ann | Ku, Yuan-Chieh | Tom, Warren | Sun, Yongming | Pankov, Alex | Looney, Tim | Au-Young, Janice | Hyland, Fiona | Conroy, Jeff | Morrison, Carl | Glenn, Sean | Burgher, Blake | Ji, He | Gardner, Mark | Mongan, Ann | Omilian, Angela R. | Conroy, Jeff | Bshara, Wiam | Angela, Omilian | Burgher, Blake | Ji, He | Glenn, Sean | Morrison, Carl | Mongan, Ann | Obeid, Joseph M. | Erdag, Gulsun | Smolkin, Mark E. | Deacon, Donna H. | Patterson, James W. | Chen, Lieping | Bullock, Timothy N. | Slingluff, Craig L. | Obeid, Joseph M. | Erdag, Gulsun | Deacon, Donna H. | Slingluff, Craig L. | Bullock, Timothy N. | Loffredo, John T. | Vuyyuru, Raja | Beyer, Sophie | Spires, Vanessa M. | Fox, Maxine | Ehrmann, Jon M. | Taylor, Katrina A. | Korman, Alan J. | Graziano, Robert F. | Page, David | Sanchez, Katherine | Ballesteros-Merino, Carmen | Martel, Maritza | Bifulco, Carlo | Urba, Walter | Fox, Bernard | Patel, Sapna P. | De Macedo, Mariana Petaccia | Qin, Yong | Reuben, Alex | Spencer, Christine | Guindani, Michele | Bassett, Roland | Wargo, Jennifer | Racolta, Adriana | Kelly, Brian | Jones, Tobin | Polaske, Nathan | Theiss, Noah | Robida, Mark | Meridew, Jeffrey | Habensus, Iva | Zhang, Liping | Pestic-Dragovich, Lidija | Tang, Lei | Sullivan, Ryan J. | Logan, Theodore | Khushalani, Nikhil | Margolin, Kim | Koon, Henry | Olencki, Thomas | Hutson, Thomas | Curti, Brendan | Roder, Joanna | Blackmon, Shauna | Roder, Heinrich | Stewart, John | Amin, Asim | Ernstoff, Marc S. | Clark, Joseph I. | Atkins, Michael B. | Kaufman, Howard L. | Sosman, Jeffrey | Weber, Jeffrey | McDermott, David F. | Weber, Jeffrey | Kluger, Harriet | Halaban, Ruth | Snzol, Mario | Roder, Heinrich | Roder, Joanna | Asmellash, Senait | Steingrimsson, Arni | Blackmon, Shauna | Sullivan, Ryan J. | Wang, Chichung | Roman, Kristin | Clement, Amanda | Downing, Sean | Hoyt, Clifford | Harder, Nathalie | Schmidt, Guenter | Schoenmeyer, Ralf | Brieu, Nicolas | Yigitsoy, Mehmet | Madonna, Gabriele | Botti, Gerardo | Grimaldi, Antonio | Ascierto, Paolo A. | Huss, Ralf | Athelogou, Maria | Hessel, Harald | Harder, Nathalie | Buchner, Alexander | Schmidt, Guenter | Stief, Christian | Huss, Ralf | Binnig, Gerd | Kirchner, Thomas | Sellappan, Shankar | Thyparambil, Sheeno | Schwartz, Sarit | Cecchi, Fabiola | Nguyen, Andrew | Vaske, Charles | Hembrough, Todd
Journal for Immunotherapy of Cancer  2016;4(Suppl 1):1-106.
PMCID: PMC5123387
10.  CD19 CAR–T cells of defined CD4+:CD8+ composition in adult B cell ALL patients 
The Journal of Clinical Investigation  null;126(6):2123-2138.
BACKGROUND. T cells that have been modified to express a CD19-specific chimeric antigen receptor (CAR) have antitumor activity in B cell malignancies; however, identification of the factors that determine toxicity and efficacy of these T cells has been challenging in prior studies in which phenotypically heterogeneous CAR–T cell products were prepared from unselected T cells.
METHODS. We conducted a clinical trial to evaluate CD19 CAR–T cells that were manufactured from defined CD4+ and CD8+ T cell subsets and administered in a defined CD4+:CD8+ composition to adults with B cell acute lymphoblastic leukemia after lymphodepletion chemotherapy.
RESULTS. The defined composition product was remarkably potent, as 27 of 29 patients (93%) achieved BM remission, as determined by flow cytometry. We established that high CAR–T cell doses and tumor burden increase the risks of severe cytokine release syndrome and neurotoxicity. Moreover, we identified serum biomarkers that allow testing of early intervention strategies in patients at the highest risk of toxicity. Risk-stratified CAR–T cell dosing based on BM disease burden decreased toxicity. CD8+ T cell–mediated anti-CAR transgene product immune responses developed after CAR–T cell infusion in some patients, limited CAR–T cell persistence, and increased relapse risk. Addition of fludarabine to the lymphodepletion regimen improved CAR–T cell persistence and disease-free survival.
CONCLUSION. Immunotherapy with a CAR–T cell product of defined composition enabled identification of factors that correlated with CAR–T cell expansion, persistence, and toxicity and facilitated design of lymphodepletion and CAR–T cell dosing strategies that mitigated toxicity and improved disease-free survival.
FUNDING. R01-CA136551; Life Science Development Fund; Juno Therapeutics; Bezos Family Foundation.
PMCID: PMC4887159  PMID: 27111235
11.  Inclusion of Strep-Tag II in design of antigen receptors for T cell immunotherapy 
Nature biotechnology  2016;34(4):430-434.
The tactical introduction of Strep-tag II into synthetic antigen receptors provides engineered T cells with a marker for identification and rapid purification, and a functional element for selective antibody coated microbead-driven large-scale expansion. Such receptor designs can be applied to chimeric antigen receptors of different ligand specificities and costimulatory domains, and to T cell receptors to facilitate cGMP manufacturing of adoptive T cell therapies to treat cancer and other diseases.
PMCID: PMC4940167  PMID: 26900664
12.  Engineering CAR-T Cells: Design Concepts 
Trends in immunology  2015;36(8):494-502.
Despite being empirically designed based on a simple understanding of TCR signaling, T cells engineered with chimeric antigen receptors (CARs) have been remarkably successful in treating patients with advanced refractory B cell malignancies. However, many challenges remain in improving the safety and efficacy of this therapy and extending it toward the treatment of epithelial cancers. Other aspects TCR signaling beyond those directly provided by CD3ζ and CD28 phosphorylation strongly influence a T cell’s ability to differentiate and acquire full effector functions. Here, we discuss how the principles of TCR recognition, including spatial constraints, Kon/Koff rates, and synapse formation, along with in-depth analysis of CAR signaling might be applied to develop safer and more effective synthetic tumor targeting receptors.
PMCID: PMC4746114  PMID: 26169254
13.  Human HLA-A*02:01/CHM1+ allo-restricted T cell receptor transgenic CD8+ T Cells specifically inhibit Ewing sarcoma growth in vitro and in vivo 
Oncotarget  2016;7(28):43267-43280.
The endochondral bone protein Chondromodulin-I (CHM1) provides oncogene addiction in Ewing sarcoma (ES). We pre-clinically tested the targetability of CHM1 by TCR transgenic, allo-restricted, peptide specific T cells to treat ES. We previously generated allo-restricted wildtype CD8+ T cells directed against the ES specific antigen CHM1319 causing specific responses against ES. However, utilization of these cells in current therapy protocols is hampered due to high complexity in production, relatively low cell numbers, and rapid T cell exhaustion.
In order to provide off-the-shelf products in the future, we successfully generated HLA-A*02:01-restricted T cell receptor (TCR) transgenic T cells directed against CHM1319 by retroviral transduction.
After short-term expansion a 100% purified CHM1319-TCR-transgenic T cell population expressed a CD62L+/CD45RO and CD62L+/CD45RA+ phenotype. These cells displayed specific in vitro IFNg and granzyme B release in co-culture with HLA-A*02:01+ ES cell lines expressing CHM1. When co-injected with ES cells in Rag2−/−ɣc−/− mice, CHM1-specific TCR-transgenic T cells significantly inhibited the formation of lung and liver metastases in contrast to control mice. Lungs and livers of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host-disease in contrast to all mice, receiving CHM1319-TCR-transgenic T cells.
CHM1319 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic.
PMCID: PMC5190022  PMID: 27281613
Ewing sarcoma; immunotherapy; T cell receptor transgenic T cells; adoptive transfer; allogeneic stem cell transplantation
14.  Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo 
Leukemia  2015;30(2):492-500.
Adoptive T-cell therapy with gene-modified T-cells expressing a tumor-reactive T-cell receptor (TCR) or chimeric antigen receptor (CAR) is a rapidly growing field of translational medicine and has shown success in the treatment of B-cell malignancies and solid tumors. In all reported trials, patients have received T-cell products comprised of random compositions of CD4+ and CD8+ naïve and memory T-cells, meaning that each patient received a different therapeutic agent. This variation might have influenced the efficacy of T-cell therapy, and complicates comparison of outcomes between different patients and across trials. We analyzed CD19 CAR-expressing effector T-cells derived from different subsets (CD4+/CD8+ naïve, central memory, effector memory). T-cells derived from each of the subsets were efficiently transduced and expanded, but showed clear differences in effector function and proliferation in vitro and in vivo. Combining the most potent CD4+ and CD8+ CAR-expressing subsets resulted in synergistic antitumor effects in vivo. We show that CAR-T-cell products generated from defined T-cell subsets can provide uniform potency compared with products derived from unselected T-cells that vary in phenotypic composition. These findings have important implications for the formulation of T-cell products for adoptive therapies.
PMCID: PMC4746098  PMID: 26369987
15.  Designing Chimeric Antigen Receptors to Effectively and Safely Target Tumors 
The adoptive transfer of T cells engineered to express artificial chimeric antigen receptors (CARs) that target a tumor cell surface molecule has emerged as an exciting new approach for cancer immunotherapy. Clinical trials in patients with advanced B cell malignancies treated with CD19-specific CAR-modified T cells (CAR-T) have shown impressive antitumor efficacy, leading to optimism that this approach will be useful for treating common solid tumors. Because CAR-T cells recognize tumor cells independent of their expression of human leukocyte antigen (HLA) molecules, tumors that escape conventional T cells by downregulating HLA and/or mutating components of the antigen processing machinery can be eliminated. The ability to introduce or delete additional genes in T cells has the potential to provide therapeutic cell products with novel attributes that overcome impediments to immune mediated tumor elimination in immunosuppressive tumor microenvironments. This review will discuss recent concepts in the development of effective and safe synthetic CARs for adoptive T cell therapy (ACT).
PMCID: PMC4397136  PMID: 25621840
16.  Safety of targeting ROR1 in primates with chimeric antigen receptor-modified T cells 
Cancer immunology research  2014;3(2):206-216.
Genetic engineering of T cells for adoptive transfer by introducing a tumor-targeting chimeric antigen receptor (CAR) is a new approach to cancer immunotherapy. A challenge for the field is to define cell surface molecules that are both preferentially expressed on tumor cells and can be safely targeted with T cells. The orphan tyrosine kinase receptor ROR1 is a candidate target for T-cell therapy with CAR-modified T cells (CAR-T cells) since it is expressed on the surface of many lymphatic and epithelial malignancies and has a putative role in tumor cell survival. The cell surface isoform of ROR1 is expressed in embryogenesis but absent in adult tissues except for B-cell precursors, and low levels of transcripts in adipocytes, pancreas, and lung. ROR1 is highly conserved between humans and macaques and has a similar pattern of tissue expression. To determine if low-level ROR1-expression on normal cells would result in toxicity or adversely affect CAR-T cell survival and/or function, we adoptively transferred autologous ROR1 CAR-T cells into nonhuman primates. ROR1 CAR-T cells did not cause overt toxicity to normal organs and accumulated in bone marrow and lymph node sites where ROR1-positive B cells were present. The findings support the clinical evaluation of ROR1 CAR-T cells for ROR1+ malignancies and demonstrate the utility of nonhuman primates for evaluating the safety of immunotherapy with engineered T cells specific for tumor-associated molecules that are homologous between humans and nonhuman primates.
PMCID: PMC4324006  PMID: 25355068
Chimeric antigen receptor; ROR1; Animal model; Cellular immunotherapy; Leukemias and lymphomas
17.  The non-signaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity 
Cancer immunology research  2014;3(2):125-135.
The use of synthetic chimeric antigen receptors (CAR) to redirect T cells to recognize tumor provides a powerful new approach to cancer immunotherapy; however the attributes of CARs that ensure optimal in vivo tumor recognition remain to be defined. Here, we analyze the influence of length and composition of IgG-derived extracellular spacer domains on the function of CARs. Our studies demonstrate that CD19-CARs with a long spacer from IgG4 hinge-CH2-CH3 are functional in vitro but lack antitumor activity in vivo due to interaction between the Fc domain within the spacer and the Fc receptor-bearing myeloid cells, leading to activation-induced T-cell death. We demonstrate that in vivo persistence and antitumor effects of CAR-T-cells with a long spacer can be restored by modifying distinct regions in the CH2 domain that are essential for Fc receptor binding. Our studies demonstrate that modifications that abrogate binding to Fc receptors are crucial for CARs in which a long spacer is obligatory for tumor recognition as shown here for a ROR1-specific CAR. These results demonstrate that the length and composition of the extracellular spacer domain that lacks intrinsic signaling function can be decisive in the design of CARs for optimal in vivo activity.
PMCID: PMC4692801  PMID: 25212991
Cancer immunotherapy; chimeric antigen receptors (CAR); adoptive T-cell therapy; tumor immunology
18.  Outcomes of acute leukemia patients transplanted with naive T cell–depleted stem cell grafts 
The Journal of Clinical Investigation  2015;125(7):2677-2689.
BACKGROUND. Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HCT). In mice, naive T cells (TN) cause more severe GVHD than memory T cells (TM). We hypothesized that selective depletion of TN from human allogeneic peripheral blood stem cell (PBSC) grafts would reduce GVHD and provide sufficient numbers of hematopoietic stem cells and TM to permit hematopoietic engraftment and the transfer of pathogen-specific T cells from donor to recipient, respectively.
METHODS. In a single-arm clinical trial, we transplanted 35 patients with high-risk leukemia with TN-depleted PBSC grafts following conditioning with total body irradiation, thiotepa, and fludarabine. GVHD prophylactic management was with tacrolimus immunosuppression alone. Subjects received CD34-selected PBSCs and a defined dose of TM purged of CD45RA+ TN. Primary and secondary objectives included engraftment, acute and chronic GVHD, and immune reconstitution.
RESULTS. All recipients of TN-depleted PBSCs engrafted. The incidence of acute GVHD was not reduced; however, GVHD in these patients was universally corticosteroid responsive. Chronic GVHD was remarkably infrequent (9%; median follow-up 932 days) compared with historical rates of approximately 50% with T cell–replete grafts. TM in the graft resulted in rapid T cell recovery and transfer of protective virus-specific immunity. Excessive rates of infection or relapse did not occur and overall survival was 78% at 2 years.
CONCLUSION. Depletion of TN from stem cell allografts reduces the incidence of chronic GVHD, while preserving the transfer of functional T cell memory.
FUNDING. NIH, Burroughs Wellcome Fund, Leukemia and Lymphoma Society, Damon Runyon Cancer Research Foundation, and Richard Lumsden Foundation.
PMCID: PMC4563691  PMID: 26053664
19.  Analyzing Cellular Immunity to AAV in a Canine Model Using ELISpot Assay 
Adeno-associated viral vector (AAV) mediated gene transfer represents a promising gene replacement strategy for treating various genetic diseases. One obstacle in using viral-derived vectors for in vivo gene delivery is the development of host immune responses to the vector. Recent studies have demonstrated cellular immune responses specific to capsid proteins of various AAV serotypes in animal models and in human trials for different diseases. We developed a canine specific ELISpot assay to detect such immunity in dogs received AAV treatment. Here, we describe in detail the use of a constructed panel of overlapping peptides spanning the entire VP1 sequence of AAV capsid protein to detect specific T cell responses in peripheral blood in dogs following intra-muscular injection of AAV. This high-throughput method allows the identification of T cell epitopes without the need for large cell numbers and the need for MHC matched cell lines.
PMCID: PMC4516170  PMID: 21956501
adeno-associated virus; AAV; peptide library; dog; ELISpot assay
20.  Engineering Human Peripheral Blood Stem Cell Grafts That Are Depleted of Naïve T Cells and Retain Functional Pathogen-Specific Memory T cells 
Graft-versus-host disease (GVHD) is a frequent major complication of allogeneic hematopoietic cell transplantation (HCT). The development of approaches that selectively deplete T cells that cause GVHD from allogeneic stem cell grafts and preserve T cells specific for pathogens may improve HCT outcomes. It has been hypothesized that the majority of T cells that can cause GVHD reside within the naïve T cell (TN) subset, and previous studies performed in mouse models and with human cells in vitro support this hypothesis. As a prelude to translating these findings to the clinic, we developed and evaluated a novel, two-step, clinically compliant procedure for manipulating peripheral blood stem cells (PBSC) to remove TN, preserve CD34+ hematopoietic stem cells, and provide for a fixed dose of memory T cells (TM) that includes T cells with specificity for common opportunistic pathogens encountered after HCT. Our studies demonstrate effective and reproducible performance of the immunomagnetic cell selection procedure for depleting TN. Moreover, after cell processing the CD45RA-depleted PBSC products are enriched for CD4+ and CD8+ TM with a central memory phenotype and contain TM cells that are capable of proliferating and producing effector cytokines in response to opportunistic pathogens.
PMCID: PMC3985542  PMID: 24525279
21.  Adoptive Therapy with Chimeric Antigen Receptor Modified T Cells of Defined Subset Composition 
Cancer journal (Sudbury, Mass.)  2014;20(2):141-144.
The ability to engineer T cells to recognize tumor cells through genetic modification with a synthetic chimeric antigen receptor has ushered in a new era in cancer immunotherapy. The most advanced clinical applications are in in targeting CD19 on B cell malignancies. The clinical trials of CD19 CAR therapy have thus far not attempted to select defined subsets prior to transduction or imposed uniformity of the CD4 and CD8 cell composition of the cell products. This review will discuss the rationale for and challenges to utilizing adoptive therapy with genetically modified T cells of defined subset and phenotypic composition.
PMCID: PMC4149222  PMID: 24667960
22.  Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells 
Immunological reviews  2014;257(1):127-144.
A major advance in adoptive T-cell therapy (ACT) is the ability to efficiently endow patient’s T cells with reactivity for tumor antigens through the stable or regulated introduction of genes that encode high affinity tumor-targeting T-cell receptors (TCRs) or synthetic chimeric antigen receptors (CARs). Case reports and small series of patients treated with TCR- or CAR-modified T cells have shown durable responses in a subset of patients, particularly with B-cell malignancies treated with T cells modified to express a CAR that targets the CD19 molecule (1-8). However, many patients do not respond to therapy and serious on and off-target toxicities have been observed with TCR- and CAR-modified T cells (9-13). Thus, challenges remain to make ACT with gene-modified T cells a reproducibly effective and safe therapy and to expand the breadth of patients that can be treated to include those with common epithelial malignancies. This review discusses research topics in our laboratories that focus on the design and implementation of ACT with CAR-modified T cells. These include cell intrinsic properties of distinct T-cell subsets that may facilitate preparing therapeutic T-cell products of defined composition for reproducible efficacy and safety, the design of tumor targeting receptors that optimize signaling of T-cell effector functions and facilitate tracking of migration of CAR-modified T cells in vivo, and novel CAR designs that have alternative ligand binding domains or confer regulated function and/or survival of transduced T cells.
PMCID: PMC3991306  PMID: 24329794
gene therapy; immunotherapies; T cells; cancer; T-cell receptors; chimeric antigen receptors
24.  Tetramer guided, cell sorter assisted production of clinical grade autologous NY-ESO-1 specific CD8+ T cells 
Adoptive T cell therapy represents an attractive modality for the treatment of patients with cancer. Peripheral blood mononuclear cells have been used as a source of antigen specific T cells but the very low frequency of T cells recognizing commonly expressed antigens such as NY-ESO-1 limit the applicability of this approach to other solid tumors. To overcome this, we tested a strategy combining IL-21 modulation during in vitro stimulation with first-in-class use of tetramer-guided cell sorting to generate NY-ESO-1 specific cytotoxic T lymphocytes (CTL).
CTL generation was evaluated in 6 patients with NY-ESO-1 positive sarcomas, under clinical manufacturing conditions and characterized for phenotypic and functional properties.
Following in vitro stimulation, T cells stained with NY-ESO-1 tetramer were enriched from frequencies as low as 0.4% to >90% after single pass through a clinical grade sorter. NY-ESO-1 specific T cells were generated from all 6 patients. The final products expanded on average 1200-fold to a total of 36 billion cells, were oligoclonal and contained 67-97% CD8+, tetramer+ T cells with a memory phenotype that recognized endogenous NY-ESO-1.
This study represents the first series using tetramer-guided cell sorting to generate T cells for adoptive therapy. This approach, when used to target more broadly expressed tumor antigens such as WT-1 and additional Cancer-Testis antigens will enhance the scope and feasibility of adoptive T cell therapy.
Electronic supplementary material
The online version of this article (doi:10.1186/s40425-014-0036-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4196009  PMID: 25317334
Adoptive T cell therapy; NY-ESO-1; Synovial sarcoma; Myxoid; Liposarcoma; Immunotherapy; Antigen specific T cells; Tetramer; Influx cell sorting
25.  Pancreatic Ductal Adenocarcinoma Contains an Effector and Regulatory Immune Cell Infiltrate that Is Altered by Multimodal Neoadjuvant Treatment 
PLoS ONE  2014;9(5):e96565.
The immune response to pancreatic ductal adenocarcinoma (PDA) may play a role in defining its uniquely aggressive biology; therefore, we sought to clearly define the adaptive immune infiltrate in PDA.
We used immunohistochemistry and flow cytometry to characterize the immune infiltrate in human PDA and compared our findings to the patients’ peripheral blood.
In contrast to the myeloid cell predominant infiltrate seen in murine models, T cells comprised the majority of the hematopoietic cell component of the tumor stroma in human PDA. Most intratumoral CD8+ T cells exhibited an antigen-experienced effector memory cell phenotype and were capable of producing IFN-γ. CD4+ regulatory T cells (Treg) and IL-17 producing T helper cells were significantly more prevalent in tumor than in blood. Consistent with the association with reduced survival in previous studies, we observed higher frequencies of both myeloid cells and Treg in poorly differentiated tumors. The majority of intratumoral T cells expressed the co-inhibitory receptor programmed death-1 (PD-1), suggesting one potential mechanism through which PDA may evade antitumor immunity. Successful multimodal neoadjuvant therapy altered the immunoregulatory balance and was associated with reduced infiltration of both myeloid cells and Treg.
Our data show that human PDA contains a complex mixture of inflammatory and regulatory immune cells, and that neoadjuvant therapy attenuates the infiltration of intratumoral cells associated with immunosuppression and worsened survival.
PMCID: PMC4008589  PMID: 24794217

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