Robbie Majzner

All Publications

• Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1. Nature cancer Mabe, N. W., Huang, M., Dalton, G. N., Alexe, G., Schaefer, D. A., Geraghty, A. C., Robichaud, A. L., Conway, A. S., Khalid, D., Mader, M. M., Belk, J. A., Ross, K. N., Sheffer, M., Linde, M. H., Ly, N., Yao, W., Rotiroti, M. C., Smith, B. A., Wernig, M., Bertozzi, C. R., Monje, M., Mitsiades, C. S., Majeti, R., Satpathy, A. T., Stegmaier, K., Majzner, R. G. 2022

  Abstract

  Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

  View details for DOI 10.1038/s43018-022-00405-x

  View details for PubMedID 35817829

• GD2-CAR T cell therapy for H3K27M-mutated diffuse midline gliomas. Nature Majzner, R. G., Ramakrishna, S., Yeom, K. W., Patel, S., Chinnasamy, H., Schultz, L. M., Richards, R. M., Jiang, L., Barsan, V., Mancusi, R., Geraghty, A. C., Good, Z., Mochizuki, A. Y., Gillespie, S. M., Toland, A. M., Mahdi, J., Reschke, A., Nie, E., Chau, I. J., Rotiroti, M. C., Mount, C. W., Baggott, C., Mavroukakis, S., Egeler, E., Moon, J., Erickson, C., Green, S., Kunicki, M., Fujimoto, M., Ehlinger, Z., Reynolds, W., Kurra, S., Warren, K. E., Prabhu, S., Vogel, H., Rasmussen, L., Cornell, T. T., Partap, S., Fisher, P. G., Campen, C. J., Filbin, M. G., Grant, G., Sahaf, B., Davis, K. L., Feldman, S. A., Mackall, C. L., Monje, M. 2022

  Abstract

  Diffuse intrinsic pontine glioma (DIPG) and other H3K27M-mutated diffuse midline gliomas (DMG) are universally lethal paediatric central nervous system tumours1. We previously discovered that the disialoganglioside GD2 is highly expressed on H3K27M-mutant glioma cells and demonstrated promising preclinical efficacy of GD2-directed chimeric antigen receptor (CAR) T cells2, providing the rationale for a first-in-human Phase 1 clinical trial (NCT04196413). Because CAR T-cell-induced brainstem inflammation can result in obstructive hydrocephalus, increased intracranial pressure, and dangerous tissue shifts, neurocritical care precautions were incorporated. Here we present the clinical experience from the first four patients with H3K27M-mutant DIPG/DMG treated with GD2-CAR T cells (GD2-CART) at dose level 1 (1e6 GD2-CAR T cells/kg administered intravenously). Patients who exhibited clinical benefit were eligible for subsequent GD2-CAR T infusions administered intracerebroventricularly3. Toxicity was largely related to tumor location and reversible with intensive supportive care. On-target, off-tumor toxicity was not observed. Three of four patients exhibited clinical and radiographic improvement. Proinflammatory cytokines were increased in plasma and cerebrospinal fluid (CSF). Transcriptomic analyses of 65,598 single cells from CAR T cell products and CSF elucidate heterogeneity in response between subjects and administration routes. These early results underscore the promise of this approach for H3K27M+ DIPG/DMG therapy.

  View details for DOI 10.1038/s41586-022-04489-4

  View details for PubMedID 35130560

• Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication. Nature medicine Theruvath, J., Menard, M., Smith, B. A., Linde, M. H., Coles, G. L., Dalton, G. N., Wu, W., Kiru, L., Delaidelli, A., Sotillo, E., Silberstein, J. L., Geraghty, A. C., Banuelos, A., Radosevich, M. T., Dhingra, S., Heitzeneder, S., Tousley, A., Lattin, J., Xu, P., Huang, J., Nasholm, N., He, A., Kuo, T. C., Sangalang, E. R., Pons, J., Barkal, A., Brewer, R. E., Marjon, K. D., Vilches-Moure, J. G., Marshall, P. L., Fernandes, R., Monje, M., Cochran, J. R., Sorensen, P. H., Daldrup-Link, H. E., Weissman, I. L., Sage, J., Majeti, R., Bertozzi, C. R., Weiss, W. A., Mackall, C. L., Majzner, R. G. 1800

  Abstract

  The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

  View details for DOI 10.1038/s41591-021-01625-x

  View details for PubMedID 35027753

• Tuning the Antigen Density Requirement for CAR T Cell Activity. Cancer discovery Majzner, R. G., Rietberg, S. P., Sotillo, E. n., Dong, R. n., Vachharajani, V. T., Labanieh, L. n., Myklebust, J. H., Kadapakkam, M. n., Weber, E. W., Tousley, A. M., Richards, R. M., Heitzeneder, S. n., Nguyen, S. M., Wiebking, V. n., Theruvath, J. n., Lynn, R. C., Xu, P. n., Dunn, A. R., Vale, R. D., Mackall, C. L. 2020

  Abstract

  Insufficient reactivity against cells with low antigen density has emerged as an important cause of CAR resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and the CAR construct in axicabtagene-ciloleucel (CD19-CD28z) outperforms that in tisagenlecleucel (CD19-4-1BBz) against antigen low tumors. Enhancing signal strength by including additional ITAMs in the CAR enables recognition of low antigen density cells, while ITAM deletions blunt signal and increase the antigen density threshold. Further, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBz CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunological synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBz-CARs with enhanced capacity to recognize antigen low targets while retaining a superior capacity for persistence.

  View details for DOI 10.1158/2159-8290.CD-19-0945

  View details for PubMedID 32193224

• Clinical lessons learned from the first leg of the CAR T cell journey. Nature medicine Majzner, R. G., Mackall, C. L. 2019; 25 (9): 1341–55

  Abstract

  Chimeric antigen receptor (CAR) T cell therapy for B cell malignancies has surpassed expectations, driving an ever-expanding number of clinical trials and the first US Food and Drug Administration approvals of cell therapies for the treatment of cancer. This experience has illuminated some generalizable requirements for CAR T cell efficacy as well as the interplay between disease biology and clinical outcomes. Major CAR intrinsic variables affecting T cell behavior have been defined, and mechanisms of tumor resistance are increasingly understood. Here, we review the clinical experience with CAR T cells amassed to date, including but not limited to B cell malignancies, emphasizing factors associated with efficacy, resistance and major barriers to success. We also discuss how these insights are driving next-generation clinical trials, including those in solid tumors.

  View details for DOI 10.1038/s41591-019-0564-6

  View details for PubMedID 31501612

• CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors CLINICAL CANCER RESEARCH Majzner, R. G., Theruvath, J. L., Nellan, A., Heitzeneder, S., Cui, Y., Mount, C. W., Rietberg, S. P., Linde, M. H., Xu, P., Rota, C., Sotillo, E., Labanieh, L., Lee, D. W., Orentas, R. J., Dimitrov, D. S., Zhu, Z., St Croix, B., Delaidelli, A., Sekunova, A., Bonvini, E., Mitra, S. S., Quezado, M. M., Majeti, R., Monje, M., Sorensen, P. B., Maris, J. M., Mackall, C. L. 2019; 25 (8): 2560–74

  View details for DOI 10.1158/1078-0432.CCR-18-0432

  View details for Web of Science ID 000464654200023

• Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline gliomas. Nature medicine Mount, C. W., Majzner, R. G., Sundaresh, S. n., Arnold, E. P., Kadapakkam, M. n., Haile, S. n., Labanieh, L. n., Hulleman, E. n., Woo, P. J., Rietberg, S. P., Vogel, H. n., Monje, M. n., Mackall, C. L. 2018

  Abstract

  Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMGs) with mutated histone H3 K27M (H3-K27M)1-5 are aggressive and universally fatal pediatric brain cancers 6 . Chimeric antigen receptor (CAR)-expressing T cells have mediated impressive clinical activity in B cell malignancies7-10, and recent results suggest benefit in central nervous system malignancies11-13. Here, we report that patient-derived H3-K27M-mutant glioma cell cultures exhibit uniform, high expression of the disialoganglioside GD2. Anti-GD2 CAR T cells incorporating a 4-1BBz costimulatory domain 14 demonstrated robust antigen-dependent cytokine generation and killing of DMG cells in vitro. In five independent patient-derived H3-K27M+ DMG orthotopic xenograft models, systemic administration of GD2-targeted CAR T cells cleared engrafted tumors except for a small number of residual GD2lo glioma cells. To date, GD2-targeted CAR T cells have been well tolerated in clinical trials15-17. Although GD2-targeted CAR T cell administration was tolerated in the majority of mice bearing orthotopic xenografts, peritumoral neuroinflammation during the acute phase of antitumor activity resulted in hydrocephalus that was lethal in a fraction of animals. Given the precarious neuroanatomical location of midline gliomas, careful monitoring and aggressive neurointensive care management will be required for human translation. With a cautious multidisciplinary clinical approach, GD2-targeted CAR T cell therapy for H3-K27M+ diffuse gliomas of pons, thalamus and spinal cord could prove transformative for these lethal childhood cancers.

  View details for PubMedID 29662203

• Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase. Molecular therapy : the journal of the American Society of Gene Therapy Walker, A. J., Majzner, R. G., Zhang, L. n., Wanhainen, K. n., Long, A. H., Nguyen, S. M., Lopomo, P. n., Vigny, M. n., Fry, T. J., Orentas, R. J., Mackall, C. L. 2017

  Abstract

  We explored the utility of targeting anaplastic lymphoma kinase (ALK), a cell surface receptor overexpressed on pediatric solid tumors, using chimeric antigen receptor (CAR)-based immunotherapy. T cells expressing a CAR incorporating the single-chain variable fragment sequence of the ALK48 mAb linked to a 4-1BB-CD3ζ signaling domain lysed ALK-expressing tumor lines and produced interferon-gamma upon antigen stimulation but had limited anti-tumor efficacy in two xenograft models of human neuroblastoma. Further exploration demonstrated that cytokine production was highly dependent upon ALK target density and that target density of ALK on neuroblastoma cell lines was insufficient for maximal activation of CAR T cells. In addition, ALK CAR T cells demonstrated rapid and complete antigen-induced loss of receptor from the T cell surface via internalization. Using a model that simultaneously modulated antigen density and CAR expression, we demonstrated that CAR functionality is regulated by target antigen and CAR density and that low expression of either contributes to limited anti-tumor efficacy of the ALK CAR. These data suggest that stoichiometric relationships between CAR receptors and target antigens may significantly impact the anti-tumor efficacy of CAR T cells and that manipulation of these parameters could allow precise tuning of CAR T cell activity.

  View details for PubMedID 28676342

• CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nature medicine Fry, T. J., Shah, N. N., Orentas, R. J., Stetler-Stevenson, M. n., Yuan, C. M., Ramakrishna, S. n., Wolters, P. n., Martin, S. n., Delbrook, C. n., Yates, B. n., Shalabi, H. n., Fountaine, T. J., Shern, J. F., Majzner, R. G., Stroncek, D. F., Sabatino, M. n., Feng, Y. n., Dimitrov, D. S., Zhang, L. n., Nguyen, S. n., Qin, H. n., Dropulic, B. n., Lee, D. W., Mackall, C. L. 2017

  Abstract

  Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 106 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19dim or CD19- B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22+ cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.

  View details for PubMedID 29155426

• Enhanced safety and efficacy of protease-regulated CAR-T cell receptors. Cell Labanieh, L., Majzner, R. G., Klysz, D., Sotillo, E., Fisher, C. J., Vilches-Moure, J. G., Pacheco, K. Z., Malipatlolla, M., Xu, P., Hui, J. H., Murty, T., Theruvath, J., Mehta, N., Yamada-Hunter, S. A., Weber, E. W., Heitzeneder, S., Parker, K. R., Satpathy, A. T., Chang, H. Y., Lin, M. Z., Cochran, J. R., Mackall, C. L. 2022

  Abstract

  Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.

  View details for DOI 10.1016/j.cell.2022.03.041

  View details for PubMedID 35483375

• Immunotherapy of Neuroblastoma: Facts and Hopes. Clinical cancer research : an official journal of the American Association for Cancer Research Anderson, J., Majzner, R. G., Sondel, P. M. 2022

  Abstract

  While the adoption of multimodal therapy including surgery, radiation, and aggressive combination-chemotherapy has improved outcomes for many children with high-risk neuroblastoma, we appear to have reached a plateau in what can be achieved with cytotoxic therapies alone. Most children with cancer, including high-risk neuroblastoma, do not benefit from treatment with immune-checkpoint-inhibitors (ICI) that have revolutionized the treatment of many highly immunogenic adult solid tumors. This likely reflects the low tumor mutation burden as well as the downregulated MHC-I that characterizes most high-risk neuroblastomas. For these reasons, neuroblastoma represents an immunotherapeutic challenge that may be a model for the creation of effective immunotherapy for other "cold" tumors in children and adults that do not respond to ICI. The identification of strong expression of the disialoganglioside, GD2, on the surface of nearly all neuroblastoma cells provided a target for immune recognition by anti-GD2 mAbs which recruit Fc-receptor-expressing innate immune cells that mediate cytotoxicity or phagocytosis. Adoption of anti-GD2 antibodies into both upfront and relapse treatment protocols has dramatically increased survival rates and altered the landscape for children with high-risk neuroblastoma. This review describes how these approaches have been expanded to additional combinations and forms of immunotherapy that have already demonstrated clear clinical benefit. We also describe the efforts to identify additional immune targets for neuroblastoma. Finally we summarize newer approaches being pursued that may well help both innate and adaptive immune cells, endogenous or genetically engineered, to more effectively destroy neuroblastoma cells, in order to better induce complete remission and prevent recurrence.

  View details for DOI 10.1158/1078-0432.CCR-21-1356

  View details for PubMedID 35435953

• In vivo imaging of nanoparticle-labeled CAR T cells. Proceedings of the National Academy of Sciences of the United States of America Kiru, L., Zlitni, A., Tousley, A. M., Dalton, G. N., Wu, W., Lafortune, F., Liu, A., Cunanan, K. M., Nejadnik, H., Sulchek, T., Moseley, M. E., Majzner, R. G., Daldrup-Link, H. E. 1800; 119 (6)

  Abstract

  Metastatic osteosarcoma has a poor prognosis with a 2-y, event-free survival rate of 15 to 20%, highlighting the need for the advancement of efficacious therapeutics. Chimeric antigen receptor (CAR) T-cell therapy is a potent strategy for eliminating tumors by harnessing the immune system. However, clinical trials with CAR T cells in solid tumors have encountered significant challenges and have not yet demonstrated convincing evidence of efficacy for a large number of patients. A major bottleneck for the success of CAR T-cell therapy is our inability to monitor the accumulation of the CAR T cells in the tumor with clinical-imaging techniques. To address this, we developed a clinically translatable approach for labeling CAR T cells with iron oxide nanoparticles, which enabled the noninvasive detection of the iron-labeled T cells with magnetic resonance imaging (MRI), photoacoustic imaging (PAT), and magnetic particle imaging (MPI). Using a custom-made microfluidics device for T-cell labeling by mechanoporation, we achieved significant nanoparticle uptake in the CAR T cells, while preserving T-cell proliferation, viability, and function. Multimodal MRI, PAT, and MPI demonstrated homing of the T cells to osteosarcomas and off-target sites in animals administered with T cells labeled with the iron oxide nanoparticles, while T cells were not visualized in animals infused with unlabeled cells. This study details the successful labeling of CAR T cells with ferumoxytol, thereby paving the way for monitoring CAR T cells in solid tumors.

  View details for DOI 10.1073/pnas.2102363119

  View details for PubMedID 35101971

• GPC2-CAR T cells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity CANCER CELL Heitzeneder, S., Bosse, K. R., Zhu, Z., Zhelev, D., Majzner, R. G., Radosevich, M. T., Dhingra, S., Sotillo, E., Buongervino, S., Pascual-Pasto, G., Garrigan, E., Xu, P., Huang, J., Salzer, B., Delaidelli, A., Raman, S., Cui, H., Martinez, B., Bornheimer, S. J., Sahaf, B., Alag, A., Fetahu, I. S., Hasselblatt, M., Parker, K. R., Anbunathan, H., Hwang, J., Huang, M., Sakamoto, K., Lacayo, N. J., Klysz, D. D., Theruvath, J., Vilches-Moure, J. G., Satpathy, A. T., Chang, H. Y., Lehner, M., Taschner-Mandl, S., Julien, J., Sorensen, P. H., Dimitrov, D. S., Maris, J. M., Mackall, C. L. 2022; 40 (1): 53-+

  View details for DOI 10.1016/j.ccell.2021.12-005

  View details for Web of Science ID 000744691500012

• GPC2-CAR Tcells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity. Cancer cell Heitzeneder, S., Bosse, K. R., Zhu, Z., Zhelev, D., Majzner, R. G., Radosevich, M. T., Dhingra, S., Sotillo, E., Buongervino, S., Pascual-Pasto, G., Garrigan, E., Xu, P., Huang, J., Salzer, B., Delaidelli, A., Raman, S., Cui, H., Martinez, B., Bornheimer, S. J., Sahaf, B., Alag, A., Fetahu, I. S., Hasselblatt, M., Parker, K. R., Anbunathan, H., Hwang, J., Huang, M., Sakamoto, K., Lacayo, N. J., Klysz, D. D., Theruvath, J., Vilches-Moure, J. G., Satpathy, A. T., Chang, H. Y., Lehner, M., Taschner-Mandl, S., Julien, J., Sorensen, P. H., Dimitrov, D. S., Maris, J. M., Mackall, C. L. 1800

  Abstract

  Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed Tcells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (5,000 molecules/cell, range 1-6* 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR Tcell therapy of solid tumors.

  View details for DOI 10.1016/j.ccell.2021.12.005

  View details for PubMedID 34971569

• CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial. Nature medicine Spiegel, J. Y., Patel, S., Muffly, L., Hossain, N. M., Oak, J., Baird, J. H., Frank, M. J., Shiraz, P., Sahaf, B., Craig, J., Iglesias, M., Younes, S., Natkunam, Y., Ozawa, M. G., Yang, E., Tamaresis, J., Chinnasamy, H., Ehlinger, Z., Reynolds, W., Lynn, R., Rotiroti, M. C., Gkitsas, N., Arai, S., Johnston, L., Lowsky, R., Majzner, R. G., Meyer, E., Negrin, R. S., Rezvani, A. R., Sidana, S., Shizuru, J., Weng, W., Mullins, C., Jacob, A., Kirsch, I., Bazzano, M., Zhou, J., Mackay, S., Bornheimer, S. J., Schultz, L., Ramakrishna, S., Davis, K. L., Kong, K. A., Shah, N. N., Qin, H., Fry, T., Feldman, S., Mackall, C. L., Miklos, D. B. 2021

  Abstract

  Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19- or CD19lo disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial ( NCT03233854 ) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL (n=17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL (n=21), 62% of patients responded with 29% CR. Relapses were CD19-/lo in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22-/lo disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency.

  View details for DOI 10.1038/s41591-021-01436-0

  View details for PubMedID 34312556

• Charting a path for prioritization of novel agents for clinical trials in osteosarcoma: A report from the Children's Oncology Group New Agents for Osteosarcoma Task Force. Pediatric blood & cancer Whittle, S. B., Offer, K., Roberts, R. D., LeBlanc, A., London, C., Majzner, R. G., Huang, A. Y., Houghton, P., Alejandro Sweet Cordero, E., Grohar, P. J., Isakoff, M., Bishop, M. W., Stewart, E., Slotkin, E. K., Greengard, E., Borinstein, S. C., Navid, F., Gorlick, R., Janeway, K. A., Reed, D. R., Hingorani, P. 2021: e29188

  Abstract

  Osteosarcoma is the most common bone tumor in children and young adults. Metastatic and relapsed disease confer poor prognosis, and there have been no improvements in outcomes for several decades. The disease's biological complexity, lack of drugs developed specifically for osteosarcoma, imperfect preclinical models, and limits of existing clinical trial designs have contributed to lack of progress. The Children's Oncology Group Bone Tumor Committee established the New Agents for Osteosarcoma Task Force to identify and prioritize agents for inclusion in clinical trials. The group identified multitargeted tyrosine kinase inhibitors, immunotherapies targeting B7-H3, CD47-SIRPalpha inhibitors, telaglenastat, and epigenetic modifiers as the top agents of interest. Only multitargeted tyrosine kinase inhibitors met all criteria for frontline evaluation and have already been incorporated into an upcoming phase III study concept. The task force will continue to reassess identified agents of interest as new data become available and evaluate novel agents using this method.

  View details for DOI 10.1002/pbc.29188

  View details for PubMedID 34137164

• Augmenting anti-CD19 and anti-CD22 CAR T-cell function using PD-1-CD28 checkpoint fusion proteins. Blood cancer journal Blaeschke, F., Stenger, D., Apfelbeck, A., Cadilha, B. L., Benmebarek, M., Mahdawi, J., Ortner, E., Lepenies, M., Habjan, N., Rataj, F., Willier, S., Kaeuferle, T., Majzner, R. G., Busch, D. H., Kobold, S., Feuchtinger, T. 2021; 11 (6): 108

  View details for DOI 10.1038/s41408-021-00499-z

  View details for PubMedID 34088894

• Transient rest restores functionality in exhausted CAR-T cells through epigenetic remodeling. Science (New York, N.Y.) Weber, E. W., Parker, K. R., Sotillo, E., Lynn, R. C., Anbunathan, H., Lattin, J., Good, Z., Belk, J. A., Daniel, B., Klysz, D., Malipatlolla, M., Xu, P., Bashti, M., Heitzeneder, S., Labanieh, L., Vandris, P., Majzner, R. G., Qi, Y., Sandor, K., Chen, L., Prabhu, S., Gentles, A. J., Wandless, T. J., Satpathy, A. T., Chang, H. Y., Mackall, C. L. 2021; 372 (6537)

  Abstract

  T cell exhaustion limits immune responses against cancer and is a major cause of resistance to chimeric antigen receptor (CAR)-T cell therapeutics. Using murine xenograft models and an in vitro model wherein tonic CAR signaling induces hallmark features of exhaustion, we tested the effect of transient cessation of receptor signaling, or rest, on the development and maintenance of exhaustion. Induction of rest through enforced down-regulation of the CAR protein using a drug-regulatable system or treatment with the multikinase inhibitor dasatinib resulted in the acquisition of a memory-like phenotype, global transcriptional and epigenetic reprogramming, and restored antitumor functionality in exhausted CAR-T cells. This work demonstrates that rest can enhance CAR-T cell efficacy by preventing or reversing exhaustion, and it challenges the notion that exhaustion is an epigenetically fixed state.

  View details for DOI 10.1126/science.aba1786

  View details for PubMedID 33795428

• How to stop using gadolinium chelates for magnetic resonance imaging: clinical-translational experiences with ferumoxytol. Pediatric radiology Daldrup-Link, H. E., Theruvath, A. J., Rashidi, A., Iv, M., Majzner, R. G., Spunt, S. L., Goodman, S., Moseley, M. 2021

  Abstract

  Gadolinium chelates have been used as standard contrast agents for clinical MRI for several decades. However, several investigators recently reported that rare Earth metals such as gadolinium are deposited in the brain for months or years. This is particularly concerning for children, whose developing brain is more vulnerable to exogenous toxins compared to adults. Therefore, a search is under way for alternative MR imaging biomarkers. The United States Food and Drug Administration (FDA)-approved iron supplement ferumoxytol can solve this unmet clinical need: ferumoxytol consists of iron oxide nanoparticles that can be detected with MRI and provide significant T1- and T2-signal enhancement of vessels and soft tissues. Several investigators including our research group have started to use ferumoxytol off-label as a new contrast agent for MRI. This article reviews the existing literature on the biodistribution of ferumoxytol in children and compares the diagnostic accuracy of ferumoxytol- and gadolinium-chelate-enhanced MRI. Iron oxide nanoparticles represent a promising new class of contrast agents for pediatric MRI that can be metabolized and are not deposited in the brain.

  View details for DOI 10.1007/s00247-021-05098-5

  View details for PubMedID 34046709

• NOT-Gated CD93 CAR T Cells Effectively Target AML with Minimized Endothelial Cross-Reactivity. Blood cancer discovery Richards, R. M., Zhao, F., Freitas, K. A., Parker, K. R., Xu, P., Fan, A., Sotillo, E., Daugaard, M., Oo, H. Z., Liu, J., Hong, W. J., Sorensen, P. H., Chang, H. Y., Satpathy, A. T., Majzner, R. G., Majeti, R., Mackall, C. L. 2021; 2 (6): 648-665

  Abstract

  Chimeric antigen receptor (CAR) T cells hold promise for the treatment of acute myeloid leukemia (AML), but optimal targets remain to be defined. We demonstrate that CD93 CAR T cells engineered from a novel humanized CD93-specific binder potently kill AML in vitro and in vivo but spare hematopoietic stem and progenitor cells (HSPC). No toxicity is seen in murine models, but CD93 is expressed on human endothelial cells, and CD93 CAR T cells recognize and kill endothelial cell lines. We identify other AML CAR T-cell targets with overlapping expression on endothelial cells, especially in the context of proinflammatory cytokines. To address the challenge of endothelial-specific cross-reactivity, we provide proof of concept for NOT-gated CD93 CAR T cells that circumvent endothelial cell toxicity in a relevant model system. We also identify candidates for combinatorial targeting by profiling the transcriptome of AML and endothelial cells at baseline and after exposure to proinflammatory cytokines.CD93 CAR T cells eliminate AML and spare HSPCs but exert on-target, off-tumor toxicity to endothelial cells. We show coexpression of other AML targets on endothelial cells, introduce a novel NOT-gated strategy to mitigate endothelial toxicity, and demonstrate use of high-dimensional transcriptomic profiling for rational design of combinatorial immunotherapies.See related commentary by Velasquez and Gottschalk, p. 559. This article is highlighted in the In This Issue feature, p. 549.

  View details for DOI 10.1158/2643-3230.BCD-20-0208

  View details for PubMedID 34778803

  View details for PubMedCentralID PMC8580619

• Breaking PD-11-mediated Resistance in anti-CD19 and anti-CD22 Car T Cells with PD-1/CD28 Fusion Receptors Blaeschke, F., Apfelbeck, A., Stenger, D., Mahdawi, J., Lepenies, M., Rataj, F., Willier, S., Kaeuferle, T., Gruenewald, T. P., Majzner, R. G., Busch, D. H., Kobold, S., Feuchtinger, T. SPRINGERNATURE. 2020: 258–59

  View details for Web of Science ID 000600556201101

• PET reporter gene imaging and ganciclovir-mediated ablation of chimeric antigen receptor T-cells in solid tumors. Cancer research Murty, S., Labanieh, L., Murty, T., Gowrishankar, G., Haywood, T., Alam, I. S., Beinat, C., Robinson, E., Aalipour, A., Klysz, D. D., Cochran, J. R., Majzner, R. G., Mackall, C. L., Gambhir, S. S. 2020

  Abstract

  Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. Additionally, the potential adverse effects of CAR T-cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a positron emission tomography (PET) reporter gene for imaging of T-cell trafficking in brain tumor patients. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir (GCV). Here we report the molecular engineering, imaging, and GCV-mediated destruction of B7H3 CAR T-cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for GCV. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[18F]-fluoro-3-[hydroxymethyl]butyl)guanine [18F]FHBG of B7H3-sr39tk CAR T-cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T-cells confirmed complete tumor ablation with intraperitoneal GCV administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities.

  View details for DOI 10.1158/0008-5472.CAN-19-3579

  View details for PubMedID 32958548

• Identification of dual positive CD19+/CD3+ T cells in a leukapheresis product undergoing CAR transduction: a case report. Journal for immunotherapy of cancer Schultz, L., Patel, S., Davis, K. L., Ramakrishna, S., Sahaf, B., Bhatia, N., Baggott, C., Erickson, C., Majzner, R. G., Oak, J., Bertaina, A., Mackall, C., Feldman, S. 2020; 8 (2)

  Abstract

  BACKGROUND: Chimeric antigen receptor (CAR) therapy and hematopoietic stem cell transplantation (HSCT) are therapeutics for relapsed acute lymphocytic leukemia (ALL) that are increasingly being used in tandem. We identified a non-physiologic CD19+/CD3+ T-cell population in the leukapheresis product of a patient undergoing CAR T-cell manufacturing who previously received a haploidentical HSCT, followed by infusion of a genetically engineered T-cell addback product. We confirm and report the origin of these CD19+/CD3+ T cells that have not previously been described in context of CAR T-cell manufacturing. We additionally interrogate the fate of these CD19-expressing cells as they undergo transduction to express CD19-specific CARs.MAIN BODY: We describe the case of a preteen male with multiply relapsed B-ALL who was treated with sequential cellular therapies. He received an alphabeta T-cell depleted haploidentical HSCT followed by addback of donor-derived T cells genetically modified with a suicide gene for iCaspase9 and truncated CD19 for cell tracking (RivoCel). He relapsed 6months following HSCT and underwent leukapheresis and CAR T-cell manufacturing. During manufacturing, we identified an aberrant T-cell population dually expressing CD19 and CD3. We hypothesized that these cells were RivoCel cells and confirmed using flow cytometry and PCR that the identified cells were in fact RivoCel cells and were eliminated with iCaspase9 activation. We additionally tracked these cells through CD19-specific CAR transduction and notably did not detect T cells dually positive for CD19 and CD19-directed CARs. The most likely rationale for this is in vitro fratricide of the CD19+ 'artificial' T-cell population by the CD19-specific CAR+ T cells in culture.CONCLUSIONS: We report the identification of CD19+/CD3+ cells in an apheresis product undergoing CAR transduction derived from a patient previously treated with a haploidentical transplant followed by RivoCel addback. We aim to bring attention to this cell phenotype that may be recognized with greater frequency as CAR therapy and engineered alphabetahaplo-HSCT are increasingly coupled. We additionally suggest consideration towards using alternative markers to CD19 as a synthetic identifier for post-transplant addback products, as CD19-expression on effector T cells may complicate subsequent treatment using CD19-directed therapy.

  View details for DOI 10.1136/jitc-2020-001073

  View details for PubMedID 32929049

• Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals. Nature methods Su, Y., Walker, J. R., Park, Y., Smith, T. P., Liu, L. X., Hall, M. P., Labanieh, L., Hurst, R., Wang, D. C., Encell, L. P., Kim, N., Zhang, F., Kay, M. A., Casey, K. M., Majzner, R. G., Cochran, J. R., Mackall, C. L., Kirkland, T. A., Lin, M. Z. 2020

  Abstract

  Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

  View details for DOI 10.1038/s41592-020-0889-6

  View details for PubMedID 32661427

• Immune-Based Approaches for the Treatment of Pediatric Malignancies. Annual review of cancer biology Bosse, K. R., Majzner, R. G., Mackall, C. L., Maris, J. M. 2020; 4: 353-370

  Abstract

  Immune-based therapies have now been credentialed for pediatric cancers with the robust efficacy of chimeric antigen receptor (CAR) T cells for pediatric B cell acute lymphocytic leukemia (ALL), offering a chance of a cure for children with previously lethal disease and a potentially more targeted therapy to limit treatment-related morbidities. The developmental origins of most pediatric cancers make them ideal targets for immune-based therapies that capitalize on the differential expression of lineage-specific cell surface molecules such as antibodies, antibody-drug conjugates, or CAR T cells, while the efficacy of other therapies that depend on tumor immunogenicity such as immune checkpoint inhibitors has been limited to date. Here we review the current status of immune-based therapies for childhood cancers, discuss challenges to developing immunotherapeutics for these diseases, and outline future directions of pediatric immunotherapy discovery and development.

  View details for DOI 10.1146/annurev-cancerbio-030419-033436

  View details for PubMedID 34113750

  View details for PubMedCentralID PMC8189419

• Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors. Nature medicine Theruvath, J. n., Sotillo, E. n., Mount, C. W., Graef, C. M., Delaidelli, A. n., Heitzeneder, S. n., Labanieh, L. n., Dhingra, S. n., Leruste, A. n., Majzner, R. G., Xu, P. n., Mueller, S. n., Yecies, D. W., Finetti, M. A., Williamson, D. n., Johann, P. D., Kool, M. n., Pfister, S. n., Hasselblatt, M. n., Frühwald, M. C., Delattre, O. n., Surdez, D. n., Bourdeaut, F. n., Puget, S. n., Zaidi, S. n., Mitra, S. S., Cheshier, S. n., Sorensen, P. H., Monje, M. n., Mackall, C. L. 2020

  Abstract

  Atypical teratoid/rhabdoid tumors (ATRTs) typically arise in the central nervous system (CNS) of children under 3 years of age. Despite intensive multimodal therapy (surgery, chemotherapy and, if age permits, radiotherapy), median survival is 17 months1,2. We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs. 3,4), which drive oncogenesis in ATRT, but requires residual SWItch/Sucrose Non-Fermentable (SWI/SNF) activity mediated by BRG1/SMARCA4. Consistent with the embryonic origin of ATRT5,6, B7-H3 is highly expressed on the prenatal, but not postnatal, brain. B7-H3.BB.z-chimeric antigen receptor (CAR) T cells administered intracerebroventricularly or intratumorally mediate potent antitumor effects against cerebral ATRT xenografts in mice, with faster kinetics, greater potency and reduced systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. CAR T cells administered ICV also traffic from the CNS into the periphery; following clearance of ATRT xenografts, B7-H3.BB.z-CAR T cells administered intracerebroventricularly or intravenously mediate antigen-specific protection from tumor rechallenge, both in the brain and periphery. These results identify B7-H3 as a compelling therapeutic target for this largely incurable pediatric tumor and demonstrate important advantages of locoregional compared to systemic delivery of CAR T cells for the treatment of CNS malignancies.

  View details for DOI 10.1038/s41591-020-0821-8

  View details for PubMedID 32341579

• Immune receptor inhibition through enforced phosphatase recruitment. Nature Fernandes, R. A., Su, L. n., Nishiga, Y. n., Ren, J. n., Bhuiyan, A. M., Cheng, N. n., Kuo, C. J., Picton, L. K., Ohtsuki, S. n., Majzner, R. G., Rietberg, S. P., Mackall, C. L., Yin, Q. n., Ali, L. R., Yang, X. n., Savvides, C. S., Sage, J. n., Dougan, M. n., Garcia, K. C. 2020

  Abstract

  Antibodies that antagonize extracellular receptor-ligand interactions are used as therapeutic agents for many diseases to inhibit signalling by cell-surface receptors1. However, this approach does not directly prevent intracellular signalling, such as through tonic or sustained signalling after ligand engagement. Here we present an alternative approach for attenuating cell-surface receptor signalling, termed receptor inhibition by phosphatase recruitment (RIPR). This approach compels cis-ligation of cell-surface receptors containing ITAM, ITIM or ITSM tyrosine phosphorylation motifs to the promiscuous cell-surface phosphatase CD452,3, which results in the direct intracellular dephosphorylation of tyrosine residues on the receptor target. As an example, we found that tonic signalling by the programmed cell death-1 receptor (PD-1) results in residual suppression of T cell activation, but is not inhibited by ligand-antagonist antibodies. We engineered a PD-1 molecule, which we denote RIPR-PD1, that induces cross-linking of PD-1 to CD45 and inhibits both tonic and ligand-activated signalling. RIPR-PD1 demonstrated enhanced inhibition of checkpoint blockade compared with ligand blocking by anti-PD1 antibodies, and increased therapeutic efficacy over anti-PD1 in mouse tumour models. We also show that the RIPR strategy extends to other immune-receptor targets that contain activating or inhibitory ITIM, ITSM or ITAM motifs; for example, inhibition of the macrophage SIRPα 'don't eat me' signal with a SIRPα-CD45 RIPR molecule potentiates antibody-dependent cellular phagocytosis beyond that of SIRPα blockade alone. RIPR represents a general strategy for direct attenuation of signalling by kinase-activated cell-surface receptors.

  View details for DOI 10.1038/s41586-020-2851-2

  View details for PubMedID 33087934

• Immune-Based Approaches for the Treatment of Pediatric Malignancies ANNUAL REVIEW OF CANCER BIOLOGY, VOL 4 Bosse, K. R., Majzner, R. G., Mackall, C. L., Maris, J. M., Jacks, T., Sawyers, C. L. 2020; 4: 353–70

  View details for DOI 10.1146/annurev-cancerbio-030419-033436

  View details for Web of Science ID 000518890900019

• c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature Lynn, R. C., Weber, E. W., Sotillo, E. n., Gennert, D. n., Xu, P. n., Good, Z. n., Anbunathan, H. n., Lattin, J. n., Jones, R. n., Tieu, V. n., Nagaraja, S. n., Granja, J. n., de Bourcy, C. F., Majzner, R. n., Satpathy, A. T., Quake, S. R., Monje, M. n., Chang, H. Y., Mackall, C. L. 2019

  Abstract

  Chimeric antigen receptor (CAR) T cells mediate anti-tumour effects in a small subset of patients with cancer1-3, but dysfunction due to T cell exhaustion is an important barrier to progress4-6. To investigate the biology of exhaustion in human T cells expressing CAR receptors, we used a model system with a tonically signaling CAR, which induces hallmark features of exhaustion6. Exhaustion was associated with a profound defect in the production of IL-2, along with increased chromatin accessibility of AP-1 transcription factor motifs and overexpression of the bZIP and IRF transcription factors that have been implicated in mediating dysfunction in exhausted T cells7-10. Here we show that CAR T cells engineered to overexpress the canonical AP-1 factor c-Jun have enhanced expansion potential, increased functional capacity, diminished terminal differentiation and improved anti-tumour potency in five different mouse tumour models in vivo. We conclude that a functional deficiency in c-Jun mediates dysfunction in exhausted human T cells, and that engineering CAR T cells to overexpress c-Jun renders them resistant to exhaustion, thereby addressing a major barrier to progress for this emerging class of therapeutic agents.

  View details for DOI 10.1038/s41586-019-1805-z

  View details for PubMedID 31802004

• Low CD19 Antigen Density Diminishes Efficacy of CD19 CAR T Cells and Can be Overcome By Rational Redesign of CAR Signaling Domains Majzner, R. G., Rietberg, S. P., Labanieh, L., Sotillo, E., Weber, E. W., Lynn, R. C., Theruvath, J. L., Yuan, C. M., Xu, P., Nguyen, S. M., Shah, N. N., Stetler-Stevenson, M., Fry, T. J., Lee, D. W., Mackall, C. L. AMER SOC HEMATOLOGY. 2018

  View details for DOI 10.1182/blood-2018-99-115558

  View details for Web of Science ID 000454837602339

• CAR T Cell Therapy for Neuroblastoma FRONTIERS IN IMMUNOLOGY Richards, R. M., Sotillo, E., Majzner, R. G. 2018; 9

  View details for DOI 10.3389/fimmu.2018.02380

  View details for Web of Science ID 000447371900002

• CAR T Cell Therapy for Neuroblastoma. Frontiers in immunology Richards, R. M., Sotillo, E., Majzner, R. G. 2018; 9: 2380

  Abstract

  Patients with high risk neuroblastoma have a poor prognosis and survivors are often left with debilitating long term sequelae from treatment. Even after integration of anti-GD2 monoclonal antibody therapy into standard, upftont protocols, 5-year overall survival rates are only about 50%. The success of anti-GD2 therapy has proven that immunotherapy can be effective in neuroblastoma. Adoptive transfer of chimeric antigen receptor (CAR) T cells has the potential to build on this success. In early phase clinical trials, CAR T cell therapy for neuroblastoma has proven safe and feasible, but significant barriers to efficacy remain. These include lack of T cell persistence and potency, difficulty in target identification, and an immunosuppressive tumor microenvironment. With recent advances in CAR T cell engineering, many of these issues are being addressed in the laboratory. In this review, we summarize the clinical trials that have been completed or are underway for CAR T cell therapy in neuroblastoma, discuss the conclusions and open questions derived from these trials, and consider potential strategies to improve CAR T cell therapy for patients with neuroblastoma.

  View details for DOI 10.3389/fimmu.2018.02380

  View details for PubMedID 30459759

  View details for PubMedCentralID PMC6232778

• Tumor Antigen Escape from CAR T-cell Therapy. Cancer discovery Majzner, R. G., Mackall, C. L. 2018

  Abstract

  Emerging data from chimeric antigen receptor (CAR) T-cell trials in B-cell malignancies demonstrate that a common mechanism of resistance to this novel class of therapeutics is the emergence of tumors with loss or downregulation of the target antigen. Antigen loss or antigen-low escape is likely to emerge as an even greater barrier to success in solid tumors, which manifest greater heterogeneity in target antigen expression. Potential approaches to overcome this challenge include engineering CAR T cells to achieve multispecificity and to respond to lower levels of target antigen and more efficient induction of natural antitumor immune responses as a result of CAR-induced inflammation. In this article, we review the evidence to date for antigen escape and downregulation and discuss approaches currently under study to overcome these obstacles.Significance: Antigen escape and downregulation have emerged as major issues impacting the durability of CAR T-cell therapy. Here, we explore their incidence and ways to overcome these obstacles in order to improve clinical outcomes. Cancer Discov; 8(10); 1-8. ©2018 AACR.

  View details for PubMedID 30135176

• Anti-GD2 chimeric antigen receptor T cells as a potent immunotherapy regimen in xenograft models of histone 3 K27M mutant diffuse midline glioma Mount, C. W., Majzner, R., Sundaresh, S., Arnold, E. P., Kadapakkam, M., Haile, S., Labanieh, L., Woo, P., Rietberg, S. P., Vogel, H., Monje, M., Mackall, C. L. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-958

  View details for Web of Science ID 000468818902457

• B7-H3 CAR T CELLS MEDIATE IN VITRO AND IN VIVO ACTIVITY AGAINST NEUROBLASTOMA XENOGRAFTS Kadapakkam, M., Majzner, R., Xu, P., Mackall, C. WILEY. 2018

  View details for Web of Science ID 000428851200460

• Programming CAR-T cells to kill cancer. Nature biomedical engineering Labanieh, L., Majzner, R. G., Mackall, C. L. 2018; 2 (6): 377-391

  Abstract

  T cells engineered to express chimeric antigen receptors (CARs) that are specific for tumour antigens have led to high complete response rates in patients with haematologic malignancies. Despite this early success, major challenges to the broad application of CAR-T cells as cancer therapies remain, including treatment-associated toxicities and cancer relapse with antigen-negative tumours. Targeting solid tumours with CAR-T cells poses additional obstacles because of the paucity of tumour-specific antigens and the immunosuppressive effects of the tumour microenvironment. To overcome these challenges, T cells can be programmed with genetic modules that increase their therapeutic potency and specificity. In this Review Article, we survey major advances in the engineering of next-generation CAR-T therapies for haematologic cancers and solid cancers, with particular emphasis on strategies for the control of CAR specificity and activity and on approaches for improving CAR-T-cell persistence and overcoming immunosuppression. We also lay out a roadmap for the development of off-the-shelf CAR-T cells.

  View details for DOI 10.1038/s41551-018-0235-9

  View details for PubMedID 31011197

• Programming CAR-T cells to kill cancer NATURE BIOMEDICAL ENGINEERING Labanieh, L., Majzner, R. G., Mackall, C. L. 2018; 2 (6): 377–91

  View details for DOI 10.1038/s41551-018-0235-9

  View details for Web of Science ID 000435474500008

• CAR T CELLS TARGETING B7-H3, A PAN-CANCER ANTIGEN, DEMONSTRATE POTENT PRECLINICAL ACTIVITY AGAINST PEDIATRIC SOLID TUMORS AND BRAIN TUMORS Majzner, R., Nellan, A., Heitzeneder, S., Theruvath, J., Mackall, C. WILEY. 2018

  View details for Web of Science ID 000428851200005

• ANTI-GD2 CHIMERIC ANTIGEN RECEPTOR T CELLS AS A POTENT IMMUNOTHERAPY REGIMEN IN XENOGRAFT MODELS OF HISTONE 3 K27M MUTANT DIFFUSE MIDLINE GLIOMA Mount, C., Majzner, R., Sundaresh, S., Arnold, E., Kadapakkam, M., Haile, S., Labanieh, L., Woo, P., Rietberg, S., Vogel, H., Monje, M., Mackall, C. OXFORD UNIV PRESS INC. 2018: 56

  View details for Web of Science ID 000438339000131

• Durable regression of Medulloblastoma after regional and intravenous delivery of anti-HER2 chimeric antigen receptor T cells JOURNAL FOR IMMUNOTHERAPY OF CANCER Nellan, A., Rota, C., Majzner, R., Lester-McCully, C. M., Griesinger, A. M., Levy, J., Foreman, N. K., Warren, K. E., Lee, D. W. 2018; 6: 30

  Abstract

  Standard-of-care therapies for treating pediatric medulloblastoma have long-term side effects, even in children who are cured. One emerging modality of cancer therapy that could be equally effective without such side effects would be chimeric antigen receptor (CAR) T cells. Knowing that human epidermal growth factor receptor 2 (HER2) is overexpressed in many medulloblastomas and has been used as a CAR T target before, we sought to evaluate the efficacy of more sophisticated anti-HER2 CAR T cells, as well as the feasibility and efficacy of different routes of delivering these cells, for the treatment of pediatric medulloblastoma.Daoy, D283 and D425 medulloblastoma cell lines were characterized by flow cytometry to evaluate HER2 expression. Anti-tumor efficacy of HER2-BBz-CAR T cells in vitro was performed using cytokine release and immune cytotoxicity assays compared to control CD19 CAR T cells. In vivo, Daoy and D283 tumor cells were orthotopically implanted in the posterior fossa of NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice and treated with regional or intravenous HER2-BBz-CAR T cells or control CD19 CAR T cells. Non-human primates (NHPs) bearing ventricular and lumbar reservoirs were treated with target autologous cells bearing extracellular HER2 followed by autologous HER2-CAR T cells intraventricularly. Cerebrospinal fluid and blood were collected serially to measure the persistence of delivered cells and cytokines.HER2-BBz-CAR T cells effectively clear medulloblastoma orthotopically implanted in the posterior fossa of NSG mice via both regional and intravenous delivery in xenograft models. Intravenous delivery requires a log higher dose compared to regional delivery. NHPs tolerated intraventricular delivery of autologous cells bearing extracellular HER2 followed by HER2-BBz-CAR T cells without experiencing any systemic toxicity.HER2-BBz-CAR T cells show excellent pre-clinical efficacy in vitro and in mouse medulloblastoma models, and their intraventricular delivery is feasible and safe in NHPs. A clinical trial of HER2-BBz-CAR T cells directly delivered into cerebrospinal fluid should be designed for patients with relapsed medulloblastoma.

  View details for PubMedID 29712574

• Neurotoxicity Associated with a High-Affinity GD2 CAR-Letter. Cancer immunology research Majzner, R. G., Weber, E. W., Lynn, R. C., Xu, P. n., Mackall, C. L. 2018; 6 (4): 494–95

  View details for PubMedID 29610423

• Harnessing the Immunotherapy Revolution for the Treatment of Childhood Cancers CANCER CELL Majzner, R. G., Heitzeneder, S., Mackall, C. L. 2017; 31 (4): 476-485

  Abstract

  Cancer immunotherapies can be classified into agents that amplify natural immune responses (e.g., checkpoint inhibitors) versus synthetic immunotherapies designed to initiate new responses (e.g., monoclonal antibodies [mAbs], chimeric antigen receptors [CARs]). Checkpoint inhibitors mediate unprecedented benefit in some adult cancers, but have not demonstrated significant activity in pediatric cancers, likely due their paucity of neoantigens. In contrast, synthetic immunotherapies such as mAbs and CAR T cells demonstrate impressive effects against childhood cancers. Intense efforts are underway to enhance the effectiveness of pediatric cancer immunotherapies through improved engineering of synthetic immunotherapies and by combining these with agents designed to amplify immune responses.

  View details for DOI 10.1016/j.ccell.2017.03.002

  View details for Web of Science ID 000398670600005

  View details for PubMedID 28366678

• Post-Transplantation Cyclophosphamide after Bone Marrow Transplantation Is Not Associated with an Increased Risk of Donor-Derived Malignancy BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Majzner, R. G., Mogri, H., Varadhan, R., Brown, P., Cooke, K. R., Bolanos-Meade, J., Swinnen, L., Kanakry, J., Luznik, L., Jones, R. J., Fuchs, E., Ambinder, R., Kasamon, Y., Symons, H. J. 2017; 23 (4): 612-617

  Abstract

  Post-transplantation cyclophosphamide (PTCy) can be used for graft-versus-host disease (GVHD) prophylaxis alone or in combination with other agents and is associated with excellent rates of engraftment and acute and chronic GVHD, as well as absence of post-transplantation lymphoproliferative disease. No study has previously evaluated the risk for developing donor-derived malignancy (DDM) in patients who receive PTCy. Giving chemotherapy in the immediate post-transplantation period carries with it a theoretic risk of disturbing the graft at a time of increased hematopoietic stress and causing or accelerating the development of malignancy. From 2000 to 2011, 789 patients underwent allogeneic transplantation and received PTCy at the Johns Hopkins Hospital. There were 4 cases of DDM identified among this large population, which is similar to or below the rate of DDM published in the literature. We found that the estimated cumulative incidence by competing risk analysis of DDM is 1.4% (SE, 1.02%). The use of PTCy does not appear to increase the risk of DDM.

  View details for DOI 10.1016/j.bbmt.2016.12.640

  View details for Web of Science ID 000397364300011

  View details for PubMedID 28062216

• Assessment of programmed death-ligand 1 expression and tumor-associated immune cells in pediatric cancer tissues. Cancer Majzner, R. G., Simon, J. S., Grosso, J. F., Martinez, D. n., Pawel, B. R., Santi, M. n., Merchant, M. S., Geoerger, B. n., Hezam, I. n., Marty, V. n., Vielh, P. n., Daugaard, M. n., Sorensen, P. H., Mackall, C. L., Maris, J. M. 2017

  Abstract

  Programmed death 1 (PD-1) signaling in the tumor microenvironment dampens immune responses to cancer, and blocking this axis induces antitumor effects in several malignancies. Clinical studies of PD-1 blockade are only now being initiated in pediatric patients, and little is known regarding programmed death-ligand 1 (PD-L1) expression in common childhood cancers. The authors characterized PD-L1 expression and tumor-associated immune cells (TAICs) (lymphocytes and macrophages) in common pediatric cancers.Whole slide sections and tissue microarrays were evaluated by immunohistochemistry for PD-L1 expression and for the presence of TAICs. TAICs were also screened for PD-L1 expression.Thirty-nine of 451 evaluable tumors (9%) expressed PD-L1 in at least 1% of tumor cells. The highest frequency histotypes comprised Burkitt lymphoma (80%; 8 of 10 tumors), glioblastoma multiforme (36%; 5 of 14 tumors), and neuroblastoma (14%; 17 of 118 tumors). PD-L1 staining was associated with inferior survival among patients with neuroblastoma (P = .004). Seventy-four percent of tumors contained lymphocytes and/or macrophages. Macrophages were significantly more likely to be identified in PD-L1-positive versus PD-L1-negative tumors (P < .001).A subset of diagnostic pediatric cancers exhibit PD-L1 expression, whereas a much larger fraction demonstrates infiltration with tumor-associated lymphocytes. PD-L1 expression may be a biomarker for poor outcome in neuroblastoma. Further preclinical and clinical investigation will define the predictive nature of PD-L1 expression in childhood cancers both at diagnosis and after exposure to chemoradiotherapy. Cancer 2017. © 2017 American Cancer Society.

  View details for PubMedID 28608950

• New developments in immunotherapy for pediatric solid tumors. Current opinion in pediatrics Schultz, L. M., Majzner, R. n., Davis, K. L., Mackall, C. n. 2017

  Abstract

  Building upon preclinical advances, we are uncovering immunotherapy strategies that are translating into improved outcomes in tumor subsets. Advanced pediatric solid tumors carry poor prognoses and resultant robust efforts to apply immunotherapy advances to pediatric solid tumors are in progress. Here, we discuss recent developments in the field using mAb and mAb-based therapies including checkpoint blockade and chimeric antigen receptors (CARs).The pediatric solid tumor mAb experience targeting the diganglioside, GD2, for patients with neuroblastoma has been the most compelling to date. GD2 and alternative antigen-specific mAbs are now being incorporated into antibody-drug conjugates, bispecific antibodies and CARs for treatment of solid tumors. CARs in pediatric solid tumors have not yet achieved comparative responses to the hematologic CAR experience; however, novel strategies such as bispecific targeting, intratumoral administration and improved understanding of T-cell biology may yield enhanced CAR-efficacy. Therapeutic effect using single-agent checkpoint blocking antibodies in pediatric solid tumors also remains limited to date. Combinatorial strategies continue to hold promise and the clinical effect in tumor subsets with high antigenic burden is being explored.Pediatric immunotherapy remains at early stages of translation, yet we anticipate that with advanced technology, we will achieve widespread, efficacious use of immunotherapy for pediatric solid tumors.

  View details for DOI 10.1097/MOP.0000000000000564

  View details for PubMedID 29189429

• Pulmonary function after hematopoietic stem cell transplantation is significantly better in pediatric recipients following reduced toxicity compared with myeloablative conditioning BONE MARROW TRANSPLANTATION Majzner, R., Sandoval, C., Dozor, A. J., Jin, Z., Van de Ven, C., Dalal, R., Morris, E., Harrison, L., Wolownik, K., Fabricatore, S., Baxter-Lowe, L. A., Cairo, M. S. 2016; 51 (11): 1530-1532

  View details for DOI 10.1038/bmt.2016.172

  View details for Web of Science ID 000387243700025

  View details for PubMedID 27322849

• T cell depletion utilizing CD34(+) stem cell selection and CD3(+) addback from unrelated adult donors in paediatric allogeneic stem cell transplantation recipients BRITISH JOURNAL OF HAEMATOLOGY Geyer, M. B., Ricci, A. M., Jacobson, J. S., Majzner, R., Duffy, D., van de Ven, C., Ayello, J., Bhatia, M., Garvin, J. H., George, D., Satwani, P., Harrison, L., Morris, E., Semidei-Pomales, M., Schwartz, J., Alobeid, B., Baxter-Lowe, L. A., Cairo, M. S. 2012; 157 (2): 205-219

  Abstract

  CD34-selected haploidentical and unrelated donor allogeneic stem cell transplantation (AlloSCT) in paediatric recipients is associated with sustained engraftment and low risk of acute graft-versus-host disease (aGVHD), but limited by delayed immune reconstitution and increased risk of viral and fungal infection. The optimal dose of donor T cells to prevent graft failure and minimize risk of early opportunistic infection and post-transplant lymphoproliferative disorder (PTLD), while avoiding severe aGVHD, remains unknown. We prospectively studied CD34-selected 8-10/10 human leucocyte antigen (HLA)-matched unrelated donor (MUD) peripheral blood stem cell transplantation (PBSCT) in a cohort of 19 paediatric AlloSCT recipients with malignant (n = 13) or non-malignant (n = 6) diseases. T cells were added back to achieve total dose 1·0-2·5 × 10(5)  CD3(+) /kg. GVHD pharmacoprophylaxis consisted only of tacrolimus. All patients engrafted neutrophils. Probabilities of grade II-IV aGVHD, limited chronic GVHD (cGVHD), and extensive cGVHD were 15·8%, 23·3%, and 0%, respectively. One patient developed PTLD. One-year infection-related mortality was 5·6%. T cell immune reconstitution was delayed. One-year overall survival was 82·3%. Five patients with malignant disease ultimately died from progressive disease. CD34-selected MUD PBSCT using a defined dose of T cell add-back resulted in high rates of engraftment and low risk of grade II-IV aGVHD, early transplantation-related mortality, and extensive cGVHD.

  View details for DOI 10.1111/j.1365-2141.2012.09048.x

  View details for Web of Science ID 000302062800009

  View details for PubMedID 22313507