PROJECT SUMMARY
Chimeric receptor antigen (CAR) T cells are transforming cancer treatment by providing tumor-specific,
molecularly targeted therapies. However, even though current clinical applications of CAR T cell-based cancer
immunotherapies such as Kymriah or Yescarta induce remission in most cases, long-term disease control, which
is especially needed in pediatric and young adult cancer patients with high-risk malignancies, remains a major
clinical challenge. In fact, malignant relapse continues to be the leading cause of death post CAR T cell therapy.
Insufficient CAR T cell persistence in vivo is a major obstacle to reducing the risk of relapse and improving
survival. We have developed a novel platform for long-lasting tumor immunosurveillance based on continuous
in vivo generation of naïve CAR T cells. This proposal is driven by the hypothesis, based on our published and
unpublished data, that after the completion of the initial course of intensive chemotherapy long-lasting T cell
immunity to cancer antigens can be established by using hematopoietic stem and progenitor cells (HSPCs)
engineered to express a tumor cell-targeting CAR and delivered into the patient’s thymus. Image-guided
intrathymic injection is a minimally invasive procedure that harnesses the thymus of cancer patients as an in vivo
bioreactor, thus offering an innovative and also relatively simple and low-toxic clinical method for sustainable
production of highly potent naïve designer T cells from genetically manipulated HSPCs. Direct thymic
engraftment of HSPCs (bypassing the bone marrow) eliminates the need for myelo-ablative conditioning while
preserving the desired outcome, i.e., long-term generation of naïve antigen-specific T cells. Thymic engraftment
will be facilitated by thymic irradiation combined with either cell delivery to the thymus by intrathymic injection or
by enhancing the thymic homing capacity of intravenously administered HSPCs by overexpression of thymus-
specific homing molecules. We will focus on CD19 CARs as a model system to establish proof of concept of our
approach because CD19 CARs have become the gold standard for evaluating novel CAR technologies. Our
experimental approaches include strategies designed to allow successful thymic negative selection of CD19
CAR-transduced HSPCs. Over time the project is expected to expand to include a variety of CAR specificities.
CAR T cell development from HSPCs will be analyzed both in vitro and in vivo, including assays assessing
thymic hematopoietic stem cell maintenance and T cell differentiation from HSPCs within the thymic epithelial
microenvironment. We will demonstrate in vivo efficacy (B cell depletion and anti-tumor activity) of the most
promising CAR expression system in syngeneic mouse models. Translational studies in humanized mice,
including a patient-derived pediatric acute lymphoblastic leukemia model, will be performed during the final year
of the project. In sum, this research will test the novel paradigm of CAR T cell development in vivo, promising to
make tumor immunosurveillance by CAR T cells broadly available as post-consolidation therapy of high-risk
malignancies in pediatric and young adult patient populations.