# Linking insertional mutagenesis and cell function to improve CAR T cell therapy

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2021 · $621,091

## Abstract

Summary
Chimeric antigen receptor-engineered T cells (CAR T cells) provide a breakthrough for personalized cancer
therapy. In this approach, a gene encoding a CAR targeting tumor antigens is delivered into patient T-cells ex
vivo using a lentiviral vector, then cells reinfused into patients. The engineered CAR T cells expand in the patient
and attack and destroy tumor antigen-positive cancer cells. Robust clinical responses are seen with CAR T cell
therapy in some leukemias such as pediatric acute lymphocytic leukemia (ALL), but lower rates of response in
adult ALL, chronic lymphocytic leukemia (CLL) and multiple myeloma. One consequence of integration of a CAR-
encoding lentiviral vector in patient T cells is local disruption of the host genome. We recently published an
example where the resulting insertional mutagenesis bolstered successful therapy--patient T-cell expansion was
associated with an integration event in TET2, which encodes an enzyme involved in CpG demethylation, and
this was mechanistically linked with enhanced T cell function and durable remission. Here we take advantage of
data from insertional mutagenesis of patient CAR T cells to identify genes and pathways of particular importance
for effective anti-tumor activity. TGFBR2 provides a second example of where insertional mutagenesis was
associated with expansion of CAR T cells, and separate studies have also implicated reduced function of this
gene as associated with improved CART function. Intense efforts are now under way to modulate both of these
pathways to enhance therapeutic success. We have completed longitudinal analysis of integration site
distributions in 40 CAR T-treated subjects, targeting both ALL and CLL, and find numerous examples of clonal
expansions in patients successfully responding to therapy, providing a unique window on CAR T cell function.
We have in hand samples from another 266 subjects, some of whom are responders showing long term
persistence of CAR T cells. We have further devised a series of assays in cell culture and mouse models to
modulate activity of targeted genes and characterize CAR T cell proliferation and anti-tumor activity. Thus we
propose to investigate these genes and pathways in detail and develop means for manipulating them clinically.
We propose the following Specific Aims: Aim 1. Elucidate the rules governing superior CAR T cell proliferation
and persistence taking advantage of lentiviral integration as an insertional mutagen. Aim 2. Carry out functional
analyses of genes implicated in vector driving of CAR T cells to identify proteins and pathways important for CAR
T proliferation, persistence and anti-tumor activity. The output of this project will be methods for manipulating
genes and pathways important for effective CAR T proliferation and function, which will then be taken directly
into clinical development.

## Key facts

- **NIH application ID:** 10158019
- **Project number:** 5R01CA241762-03
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Frederic D Bushman
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $621,091
- **Award type:** 5
- **Project period:** 2019-06-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10158019

## Citation

> US National Institutes of Health, RePORTER application 10158019, Linking insertional mutagenesis and cell function to improve CAR T cell therapy (5R01CA241762-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10158019. Licensed CC0.

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