Immunotherapy has demonstrated increasing success in the treatment of cancer, and early signals suggest that immunotherapies will soon provide a fourth axis for the treatment of childhood cancer as well. Several new classes and types of immunotherapeutics are emerging that open up the possibility of applying immunotherapy to an ever-increasing array of targets and cancers. This project leverages the explosion of technologies and insights emerging in the immunotherapy domain to bring new immunotherapy agents to bear on childhood cancer. We seek to answer fundamental questions about the similarities and differences that exist when targeting childhood vs adult cancers with immune based therapies and to extend out understanding of specific factors that limit the effectiveness of immune based therapies for solid tumors compared to liquid tumors. In FY13 our first major accomplishment was the creation of an entirely novel chimeric antigen receptor targeting the CD22 antigen, which is expressed on B cell leukemia, the single most common cancers of childhood. State-of-the-art technology previously employed to generate chimeric antigen receptors targeting other antigens (e.g. CD19 for leukemia) was applied to generate an entire series of new receptors targeting CD22. A variety of scFv antigen binders were tested as were a variety of spacer domains and costimulatory domains. Through iterative testing, we created a CD22 targeted chimeric antigen receptor that shows similar potency to a CD19 targeted receptor, and thus is predicted to have activity in clinical trials. This work yielded the following conclusions, which were critical for optimizing this new therapeutic but also provide more fundamental insights into this emerging class of therapeutics. First, among three candidates tested, the scFv binding the membrane proximal epitope on CD22 was more potent that binders targeting more distal epitopes. Second, higher affinity interactions between the scFv and its target did not translate into improved efficacy. Third, engineering two costimulatory domains into the chimeric antigen receptor resulted in diminished effectiveness compared to one costimulatory domain. This work was published in Blood in 2013 (Haso et al), a patent was filed, and this new receptor has already been licensed by a commercial entity and will be tested in children and young adults in an early phase trial in 2014. A second accomplishment of this project was substantial progress in our laboratory in developing chimeric antigen receptors targeting solid tumors of childhood. We generated chimeric antigen receptors targeting a variety of antigens expressed on pediatric solid tumors, including GD2, B7H3, ALK and FGFR4. These chimeric antigen receptors (also known as CARs) are genetically engineered into a retroviral vector and expressed in human T cells. Through this work, we have conducted extensive comparison studies of a very potent CAR targeting CD19 vs the less potent CAR targeting GD2 to identify those factors limiting the efficacy of the GD2 CAR. Our early hypothesis was that the less effective GD2-CAR CAR was limited by lesser levels of antigen engagement and lesser T cell activation, but we have now made the remarkable observation that GD2-CAR expressing T cells actually appear to be """"""""superactivated"""""""" compared to their CD19-specific counterparts. These GD2-CAR expressing T cells show hallmark features of exhaustion early during their development and postulate that this exhaustion limits their effectiveness in vivo. Ongoing work is underway to specifically delineate the signaling pathways required for exhaustion in this model system, to identify the role that altered metabolism plays in the development of exhaustion and to combine GD2-targeted CAR cells with other agents that may optimize their efficacy in vivo. A third accomplishment was completion of studies that defined the basis for immune escape in murine rhabdomyosarcoma following treatment with theimmune checkpoint inhibitor anti-PD1. Briefly, treatment with anti-PD1 completely prevented development of murine rhabdomyosarcoma in an orthotopic model, but anti-PD1 treatment in the presence of established disease had minimal benefit. We identified that rhabdomyosarcoma potently induced expansion of myeloid derived suppressor cells that are neutrophilic in lineage and traffic to tumors via a CXCL1/CXCR1 axis. This is distinct from MDSCs reported in tumor models that mimic adult tumors, which are primarily monocytic in lineage and traffic to tumor via the CCL/CCR chemokines axes. We next hypothesized that interruption of trafficking of myeloid derived suppressor cells to rhabdomyosarcomas could enhance the efficacy of anti-PD1 therapy. To do this, we created chimeras lacking CXCR2 on hematopoietic cells and we also treated animals with anti-CXCR2 blocking antibodies. In both cases, we observed substantial regression of established rhabdomyosarcoma with anti-PD1 therapy. This work establishes MDSC as important mediators of immune escape in pediatric solid tumors and identified distinctions between the cells induced in adult tumors vs those induced in tumors of childhood. We furthermore provide a basis for using checkpoint inhibitors of the PD1 family in pediatric solid tumors, in conjunction with therapies to diminish myeloid derived suppressors. This work has been submitted for publication and is under revision. A fourth accomplishment in FY13 near completion of a study demonstrating that T cells genetically engineered to recognize a self antigen expressed in the thymus develop T cell leukemia through a process whereby the T cell receptor itself serves as an oncogene. We developed a T cell transgenic mouse whereby the vast majority of T cells have specificity for """"""""survivin"""""""" an anti-apoptotic molecule considered to be a universal tumor antigen. We hypothesized that survivin TCR trasngeic mice would be immune to tumor growth as most tumors express high levels of survivin. Unfortunately and remarkably, these mice did not resist growth of implanted tumors, but rather T cell lymphoblastic leukemia developed in essentially all mice. This was not due to insertional mutagenesis because it occurred in 3 separate founders, and we mapped the insertion in one mouse and noted that it was not near an oncogenic site. Rather, self-reactivity with survivin antigens expressed in the thymus led to expansion of early thymic progenitors via signaling of the transgenic TCR in the thymus in response to thymic expression of the survivin gene. TCR signaling at this early stage of T cell development leads to expansion of progenitors, followed by acquisition of NOTCH mutations in multiple clones. The role for TCR signaling in this process is confirmed by the face that mice that cannot present peptides derived from the survivin antigen within the thymus (e.g. b2m-/- mice) have a substantially diminished incidence of leukemia. Interestingly, we saw similar development of T-cell ALL in small numbers mice with TCR transgenes specific for gp100 and for WT1, implying that gene therapies aimed at expressing TCRs specific for tumor antigens early in thymopoiesis carry a substantial risk for secondary leukemia. These insights provide novel clues to potential dangers of genetic engineering that involves targeting developing thymocytes toward self-antigens. This work is being prepared for publication.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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Haworth, Kellie B; Leddon, Jennifer L; Chen, Chun-Yu et al. (2015) Going back to class I: MHC and immunotherapies for childhood cancer. Pediatr Blood Cancer 62:571-6
Long, Adrienne H; Haso, Waleed M; Shern, Jack F et al. (2015) 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 21:581-90
Cui, Yongzhi; Onozawa, Masahiro; Garber, Haven R et al. (2015) Thymic expression of a T-cell receptor targeting a tumor-associated antigen coexpressed in the thymus induces T-ALL. Blood 125:2958-67
Mackall, Crystal L; Merchant, Melinda S; Fry, Terry J (2014) Immune-based therapies for childhood cancer. Nat Rev Clin Oncol 11:693-703
Orentas, Rimas J; Nordlund, Jessica; He, Jianbin et al. (2014) Bioinformatic description of immunotherapy targets for pediatric T-cell leukemia and the impact of normal gene sets used for comparison. Front Oncol 4:134
Corrigan-Curay, Jacqueline; Kiem, Hans-Peter; Baltimore, David et al. (2014) T-cell immunotherapy: looking forward. Mol Ther 22:1564-74
Highfill, Steven L; Cui, Yongzhi; Giles, Amber J et al. (2014) Disruption of CXCR2-mediated MDSC tumor trafficking enhances anti-PD1 efficacy. Sci Transl Med 6:237ra67
Mackall, Crystal L (2014) Introduction to the series of reviews on ""Antibody derivatives as new therapeutics for hematologic malignancies"". Blood 123:2283-4
Haso, Waleed; Lee, Daniel W; Shah, Nirali N et al. (2013) Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood 121:1165-74
Duncan, Brynn B; Highfill, Steven L; Qin, Haiying et al. (2013) A pan-inhibitor of DASH family enzymes induces immune-mediated regression of murine sarcoma and is a potent adjuvant to dendritic cell vaccination and adoptive T-cell therapy. J Immunother 36:400-11

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