Immunotherapy has demonstrated increasing success in the treatment of cancer, and emerging clinical studies illustrate the potential for immunotherapy to provide a fourth pillar for treatment of childhood cancer (surgery, radiation therapy, chemotherapy and immunotherapy). 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 this explosion of technologies and insights in the immunotherapy domain to bear on the problem childhood cancer. We seek to answer fundamental questions about similarities and differences between childhood vs adult cancers as it relates to immune based therapies and to extend our understanding of specific factors that limit the effectiveness of immune based therapies for solid tumors compared to liquid tumors. A major accomplishment was publication of a report that defined myeloid derived suppressor cells as the basis for immune escape in murine rhabdomyosarcoma following treatment with the immune checkpoint inhibitor anti-PD1. Anti-PD1 is part of a new class of immunotherapeutics that circumvent immunosuppressive mechanisms used by cancer and allow natural, endogenous antitumor immune responses to emerge. Response rate of approximately 10-30% have been reported in malignant melanoma, lung cancer and renal cell carcinoma. This work used anti-PD1 in a model of a pediatric solid tumor, namely rhabdomyosarcoma. Briefly, treatment of mice with anti-PD1 prior to tumor inoculation completely prevented development of cancer, but delaying anti-PD1 treatment for even 7 days after tumor inoculation resulted in minimal benefit. We identified that rhabdomyosarcoma-induced expansion of myeloid derived suppressor cells that traffic to tumors via a CXCL1/CXCR1 axis. We therefore 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 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 also sought to determine whether the CXCL/CXCR axis was likely to play a role in human pediatric sarcomas. We first demonstrated that human sarcoma cell lines produce several CXCR ligands and furthermore, discovered that serum from patients treated on trials of immunotherapy had elevated levels of CXCL1 and CXCL8. Moreover, among patients with metastatic sarcoma, elevated CXCL8 levels were associated with poor prognosis. We therefore believe that the CXCL/CXCR chemokine axis represents a potential basis for developing therapies that could be used to augment the efficacy of immunotherapy for childhood cancer. This work was published as the cover article in Science Translational Medicine in 2014 and was also highlighted in Nature Reviews Cancer. A second accomplishment is 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 in which 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 transgenic 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. Rather, self-reactivity withsurvivin 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 fact that mice which cannot present peptides derived from the survivin antigen within the thymus (e.g. b2m-/- mice) have a substantially diminished incidence of 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. A third accomplishment identification of a fundamental structure problem associated with chimeric antigen receptors that limits their functionality and is present in the vast majority of CARs generated. We believe that this is a previously unappreciated problem that likely has limited success so far with all chimeric antigen receptors with the exception of the CD19-CAR. This work demonstrated that the ineffective GD2-CAR is actually superactivated compared to the highly effective CD19-CAR even during in vitro culture prior to exposure to antigen. This was confirmed using numerous approaches including expression of activation markers (CD25, CD69, loss of IL7R and downregulation CD27), as well as by direct demonstration of phosphorylation of the synthetic zeta chain incorporated into the CAR construct. We further went on to demonstrate that this tonic, antigen-independent activation results from signaling via the CAR itself in the absent of antigen and leads to T cell exhaustion, thus limiting the functionality of the CAR expressing cell. We demonstrated that the tonic signaling results from aggregation of the GD2-CAR within the cell membrane, which does not occur with the CD19-CAR. Furthermore, the aggregation is due to factors contained within the framework region of the scFv, as substitution of the framework regions from the GD2-CAR into the CD19-CAR replicates the phenomena. Unfortunately, we could not address the questions of whether the phenomena could be reversed with the use of framework regions from CD19-CAR, as this construct was not expressed on the cell surface. Thus, this work identified a novel cause of CAR failure, notably T cell exhaustion due to aggregation of the scFv within the cell membrane. The work went on to demonstrate that signaling moieties contained within the receptor could either augment or diminish the exhaustion the occurred in these cells. CD28 costimulation augmented the exhaustion whereas 4-1BB costimulation diminished exhaustion. This effect was sufficient to convert the GD2-CAR from a wholly ineffective agent against osteosarcoma xenografts to one with substantial antitumor activity. We went on to compare gene expression profiles in previously activated non-exhausted cells versus exhausted cells versus 4-1BB rescued cells. We identified a series of novel genes that were previously not associated with exhaustion as differentially expressed in exhausted versus rescued T cells. Work is underway to explore the biology of T cell exhaustion using this model system as a basis for discovery of fundamental principles.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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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
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
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
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
Mackall, Crystal L (2014) Introduction to the series of reviews on ""Antibody derivatives as new therapeutics for hematologic malignancies"". Blood 123:2283-4
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; Merchant, Melinda S; Fry, Terry J (2014) Immune-based therapies for childhood cancer. Nat Rev Clin Oncol 11:693-703
Corrigan-Curay, Jacqueline; Kiem, Hans-Peter; Baltimore, David et al. (2014) T-cell immunotherapy: looking forward. Mol Ther 22:1564-74
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
Mackall, Crystal; Maris, John (2013) Q&A: Crystal Mackall, John Maris on pediatrics. Cancer Discov 3:961

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