# Development of antigen multimers for CAR T cell detection and functional profiling

> **NIH NIH R21** · UNIVERSITY OF CHICAGO · 2023 · $239,078

## Abstract

PROJECT SUMMARY
CAR T cell therapy offers new hope to patients with relapsed/refractory B cell malignancies and has become
another weapon in the war on cancer. Despite the success and excitement around CAR therapies, sensitive and
universal CAR-labeling reagents are required for accurate CAR T cell detection and profiling in research,
industry, and in the clinic. To improve CAR T cell therapy efficacy, scientists, clinicians, and the pharmaceutical
industry need accurate and multifunctional CAR-staining reagents to perform downstream assays, conduct
preclinical studies, profile patient biospecimens, and develop new CARs. To overcome this technology gap, we
developed antigen-multimers – high-avidity CAR detection reagents. Akin to conventional MHC multimers, our
preliminary data demonstrate that antigen-multimers sensitively and specifically detect CD19-directed CAR T
cells in commercial infusion products, in peripheral blood, and in tumor biopsies of diffuse large B cell lymphoma
patients. Furthermore, we show that CD19-multimers can be readily employed to isolate CAR T cells from patient
samples at different treatment stages for downstream flow cytometry and single-cell multi-omics analyses. In our
application, based on our exciting preliminary observations, we propose to test the overarching hypothesis that
antigen-multimers will enable CAR T cell detection and isolation and facilitate downstream single-cell analyses
of CAR T cells from patient biospecimens. Our antigen-multimers can be used to generate new knowledge
regarding CAR T cell function and in vivo efficacy.
 In Specific Aim 1, we propose to develop antigen-multimers for high-sensitivity CAR T cell detection and
isolation. In addition to demonstrating the flexibility of our technology by generating 4 different CAR-detecting
antigen-multimers, we will also develop approaches for simultaneously isolating and restimulating CAR T cells
from patient samples in order to enable downstream phenotyping and functional analyses.
 In Specific Aim 2, we propose to develop antigen-multimer-enabled single CAR T cell multicolor flow
cytometry and multi-omics. We will first use CD19-multimers to determine CAR expression, cell number,
differentiation, functional phenotype, and persistence of CAR T cells by multicolor flow cytometry. We will next
use CD19-multimers to isolate CAR T cells from responder and non-responder patients for subsequent single-
cell multi-omics analyses. Leveraging the experience of two investigators with complementary expertise in
immunoengineering (Huang) and translational/clinical CAR T cell therapy research (Kline), as well as the
UChicago Cell Therapy Biobank which contains hundreds of lymphoma and myeloma patient samples treated
with CD19- or BCMA-directed CAR T cell therapy, the proposed experiments will establish antigen-multimers as
a new class of CAR-staining reagents with broad research and clinical applications.

## Key facts

- **NIH application ID:** 10741209
- **Project number:** 1R21AI169159-01A1
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** Jun Huang
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $239,078
- **Award type:** 1
- **Project period:** 2023-06-09 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10741209

## Citation

> US National Institutes of Health, RePORTER application 10741209, Development of antigen multimers for CAR T cell detection and functional profiling (1R21AI169159-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10741209. Licensed CC0.

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