# Imaging-guided fine tuning of CAR T cell affinity to limit T cell cytotoxicity to tumor antigen

> **NIH NIH R01** · WEILL MEDICAL COLL OF CORNELL UNIV · 2022 · $517,929

## Abstract

The proposed work is to take quantitative approach to study the effect of interplay between chimeric antigen
receptor (CAR) affinity and antigen density on CAR T cell efficacy and toxicity. Aside from a limited number of
surface-expressed tumor neoantigens, there is a widely acknowledged paucity of tumor-specific targets.
Selectively tuning a CAR’s affinity for its target molecule can maintain its ability to lyse tumor cells exhibiting
high density target expression while preventing this occurrence in normal cells with lower, basal expression.
However, our current understanding of how the threshold for T cell activation and cytotoxicity is defined by the
interplay between CAR affinity and antigen density is still in its infancy. Relevant studies to date have
examined this relationship using CARs with crude affinity variations and target cells devoid of quantitative,
antigen density measurements. Furthermore, the potential for systemic off-tumor toxicity mediated by affinity-
tuned CAR T cells has yet to be evaluated in a pre-clinical environment where the CAR in question can cross-
react with natural levels of its cognate host antigen, thereby rigorously performing risk assessment and
therapeutic index studies of off-tumor CAR reactivity. To systematically examine the affinity tuned CAR
paradigm, we have designed a CAR that specifically targets intercellular adhesion molecule (ICAM)-1, a
molecule that is over-expressed in multiple tumors but which retains weak expression in healthy tissue. This
CAR’s antigen recognition domain is derived from the inserted (I) domain of an integrin called lymphocyte
function-associated antigen (LFA)-1, which we previously adapted by introducing point mutations such that
step-wise variations of ICAM-1 affinity could be derived ranging from 1 mM to 1 nM Kd or 106-fold. Human
LFA-1 I domain binds murine and human ICAM-1 with comparable affinity, allowing simultaneous in vivo
evaluation of CAR reactivity against human ICAM-1 expressing, transplanted tumor tissue alongside any
potential on-target, off-tumor reactivity/toxicity against murine ICAM-1. We have also recently developed and
evaluated a genetic reporter, human somatostatin receptor 2 (SSTR2), that enables quantitative, non-invasive
real-time monitoring and tracking of T cells using positron emission tomography-computed tomography
(PET/CT) and clinically approved imaging reagents. In this proposal, we will 1) determine threshold CAR
affinity that restricts CAR T cell cytotoxicity to tumors with over-expressed antigens, and 2) examine the
influence of CAR affinity and antigen density on spatiotemporal kinetics of T cell expansion and contraction,
and on the rate of tumor elimination, relapse, and systemic toxicity. The outcome of this study has the potential
to significantly impact the study of CAR T cells by providing a quantitative framework for CAR T cell design and
evaluation to maximize their therapeutic index.

## Key facts

- **NIH application ID:** 10321237
- **Project number:** 5R01CA217059-05
- **Recipient organization:** WEILL MEDICAL COLL OF CORNELL UNIV
- **Principal Investigator:** Moonsoo M. Jin
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $517,929
- **Award type:** 5
- **Project period:** 2018-01-01 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10321237

## Citation

> US National Institutes of Health, RePORTER application 10321237, Imaging-guided fine tuning of CAR T cell affinity to limit T cell cytotoxicity to tumor antigen (5R01CA217059-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10321237. Licensed CC0.

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