It is now clear that antitumor effects can be induced in a wide array of cancers using immunotherapeutic approaches. Immunotherapy has had documented efficacy in malignant melanoma, renal cell carcinoma, squamous cell carcinoma, prostate cancer, leukemia, neuroblastoma, and breast cancer, to name a few. Furthermore, 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 seeks to leverage the explosion of technologies and insights emerging in the immunotherapy domain to bring new immunotherapy agents to bear on childhood cancer. In addition, we seek to answer fundamental questions about the similarities and differences that exist when targeting childhood vs adult cancers with immune based therapies.In FY12 we extended our studies using murine models of pediatric cancer to identify biologies associated with immune escape in these diseases. Specifically, using two murine models of rhabdomyosarcoma, we demonstrated that the immune checkpoint inhibitor anti-PD1 diminished rhabdomyosarcoma growth when administered in the context of established disease and prevented the development of new tumors. We further 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. If trafficking of myeloid derived suppressor cells to rhabdomyosarcomas is inhibited by blocking the CXCL axis, complete regression of established tumors is induced with anti-PD1 therapy. Thus, we have established evidence the MDSC are 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, perhaps in conjunction with therapies to diminish myeloid derived suppressors. This work is being prepared for publication.A second accomplishment in this project in FY12 has been the development of a genetically engineered mouse that accurately models pediatric tumors in mice. These findings were a discovery in the true sense because we did not expect these mice to develop cancer, yet they reproducibly develop cancers that closely resemble T cell acute lymphoblastic leukemia, thus providing potential new insights into the oncogenesis of this disease. Specifically 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 these mice would be immune to tumor growth as most tumors express high levels of survivin. Unfortunately and remarkably, expression of this T cell receptor did not prevent tumor growth but induced T cell lymphoblastic leukemia in essentially all mice. This was not due to insertional mutagenesis because it occurred in 3 separate founders, but rather appears to be due to self-reactivity with survivin antigens expressed in the thymus that led to expansion of early thymic progenitors followed by acquisition of NOTCH mutations. We saw similar development of T-cell ALL in small numbers mice with TCR transgenes specific for gp100 and for WT1, thus implying that gene therapies aimed at expressing TCRs specific for tumor antigens early in thymopoiesis carry a substantial risk for secondary leukemia. Furthermore, we demonstrated that when such mice cannot present peptides derived from the survivin antigen within the thymus (e.g. b2m-/- mice) that the incidence of leukemia is substantially diminished. We also demonstrate evidence for signaling of the transgenic TCR in the thymus aswell that thymic expression of the survivin gene. 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 of this project is embodied by the substantial progress our laboratory has made during FY12 in developing chimeric antigen receptors targeting childhood tumors. We have developed chimeric antigen receptors targeting CD19, CD22 on pediatric leukemia, GD2 on pediatric solid tumors, B7H3, ALK and FGFR4 on pediatric solid tumors. These chimeric antigen receptors (also known as CARs) are genetically engineered into a retroviral vector and expressed in human T cells. The most potent of these agents thus far is CD19-CAR, which also encodes for CD28. This agent induced potent antitumor effects in mice bearing CD19+ acute lymphoblastic leukemia. On this basis, we have initiated a clinical trial of CD19-CAR therapy in childhood ALL and the first patient has been enrolled. We have also identified in the murine models a potent and unexpected role for CD4+ cells bearing chimeric antigen receptors in eradicating leukemia. On this basis, we have worked to optimize CD4 transduction in our clinical trial. We also created a CD22 chimeric antigen receptor and demonstrated substantial antitumor activity against pediatric acute lymphoblastic leukemia in vitro and in xenograft models. We observe that CD22 is consistent expressed in pediatric ALL, albeit at lower levels that CD19 levels. Nonetheless, there is potent killing of pediatric ALL blasts by CD22-chimeric antigen receptors, with equal activity regardless of affinity, regarding of the presence or absence of a spacer domain, and thus far regardless of whether the costimulatory domain is 41BB or CD28. These results have been submitted for publication and we plan to initiate a clinical trial of CD22-CAR therapy in pediatric ALL in FY13.Through have also had important negative results that have steered us in new directions. This is based upon the observation that chimeric antigen receptors targeting leukemia are typically more potent in eradicating established tumors than chimeric antigen receptors targeting solid tumors. In particular, although GD2 directed chimeric antigen receptors have potent killing activity in vitro, there are unable to eradicate established solid tumors in vivo in xenograft models. We believe that this is an important observation that must be understood if we are to exploit the power of chimeric antigen receptor therapy for pediatric solid tumors. Specifically, we are seeking to determine to what extent the differences observed related to less potent antigens vs a preferential capacity for the solid tumor to escape immune destruction via CAR therapy. Current studies are generating solid tumors that express high levels of CD19 in an effort to determine what factors limit rejection of solid tumors in these models.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011073-05
Application #
8552967
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2012
Total Cost
$1,459,080
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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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