Biologic and Functional Characterization of In Vivo CAR-T cells

Project Summary
Chimeric Antigen Receptor (CAR) T cell therapy has revolutionized treatment for B cell malignancies by
targeting T cytotoxicity to the site of the tumor. Despite the success of CAR-T cells in B cell malignancies, more
than half of patients receiving CAR-T cell treatment fail to achieve long term disease control. Therapeutic
failure can be attributed to many causes including the inverse relationship between CAR-T cell manufacturing
duration and the resulting anti-tumor potency. Additionally, CAR-T cell manufacturing poses barriers to access
such as cost and difficulty meeting supply demand equilibrium. To engineer the next generation of CAR-T cells
with enhanced anti-tumor efficacy and greater patient access, I will generate CD19.28z CAR-T cells in vivo
using modified lentiviral particles engineered to express a T cell targeting antibody fragment, referred to in this
proposal as the Programmable Antibody-mediated Cellular Knock-In of T cells (PACK-IT) system. The PACK-IT
system will be used to explore my central hypothesis: engineering T cells in vivo is feasible and will deliver a
more efficacious CAR-T cells (PACK-IT CAR-T cells) with distinct biologic features, reducing cost and
increasing access. I have demonstrated feasibility of the PACK-IT system to generate functional CD19.28z
CAR T cells in vitro and extended the use of the PACK-IT system to successfully transduce T cells in tumor
bearing mice. Based on the proof-of-concept experiments, I propose to (i) optimize the PACK-IT system in
terms of transduction efficiency, phenotype, and anti-tumor potency of resulting CAR-T cells, (ii) in vivo
comparison of PACK-IT CAR-T cells and those made via conventional manufacturing, and (iii) assess the
impact of armoring PACK-IT CAR-T cells with drug regulatable cytokine receptors on anti-tumor potency in
immunocompetent hosts. Collectively, the proposed work will result in a method to produce CAR-T cells in
vivo, allow rigorous characterization of the impact of eliminating the ex vivo manufacturing process, and
develop PACK-IT CAR-T cells armed with regulatable cytokine receptors to boost T cell function in vivo. The
proposed work will take place at Stanford University School of Medicine, a leading institution in immunology
and immunotherapy and a setting that emphasizes innovation. Dr. Crystal Mackall is the ideal sponsor for this
project due to her extensive track record of mentoring successful physician scientists and her expertise in T
cell biology and translational therapeutics. In addition, I will be supported by a multidisciplinary team including
mentorship from Drs. Howard Chang (genome wide sequencing, engineered lentiviral vectors), Christopher
Barnes (structural virology), and Anusha Kalbasi (engineered cytokine receptors).

Grant Number: 5F30CA291106-02
NIH Institute/Center: NIH
Principal Investigator: Kylie Burdsall