# Development of a cellular therapy product with single specificity and improved persistence to prevent immunity to biotherapeutics

> **NIH NIH R21** · INDIANA UNIVERSITY INDIANAPOLIS · 2024 · $239,768

## Abstract

Regulatory T cell (Treg) therapy is a highly promising approach for controlling unwanted or pathogenic immune
responses in autoimmune disease, transplantation or in anti-drug antibody (ADA) formation following the
administration of biotherapeutics such as clotting factor VIII (FVIII) replacement therapy in hemophilia A. Lack of
specificity and limited persistence impedes clinical application of polyclonal Treg therapy, which can be surmounted
by expressing antibody-based synthetic receptors such as a chimeric antigen receptor (CAR) or TCR fusion
construct (TRuC) to redirect antigen specificity. TRuCs utilize the internal signaling machinery of a TCR by
reconfiguring endogenous TCR-CD3 signaling to respond to antibody-based recognition. We recently showed in a
preclinical hemophilia A model of FVIII deficiency that engineering antigen specificity by TRuC expressing Tregs
delivers durable suppression of ADA responses by more faithfully mimicking the physiological TCR signaling of
endogenous Tregs. However, dual recognition by both the synthetic receptor and endogenous TCR can increase
the likelihood of off-target suppressive effects and lead to competition for extracellular expression and downstream
signaling mediators.
In this proposal, we will evaluate a novel synthetic hybrid receptor generated by replacing the α and β variable (V)
regions of the TCR with the VH and VL domains of a FVIII specific antibody (FVIII V-swapTCR). A single-step
CRISPR/Cas mediated integration of FVIII V-swapTCR into the TCRα constant (TRAC) locus should abolish
endogenous TCR expression, thus eliminating dual antigen specificity, while allowing for more precise control of
synthetic receptor expression. In Aim 1, we will establish the specificity, and functionality of TCR KO FVIII V-
swapTCR Tregs. We will extensively characterize the phenotype and molecular pathways utilized by TCR KO FVIII
V-swapTCR Tregs. Functional suppression by the single-specific engineered Tregs will be assessed in a model of
ADA formation to FVIII replacement therapy in hemophilia A. In Aim 2, we will explore an innovative single chain
immunocytokine to drive the selective proliferation of TCR KO FVIII V-swapTCR Tregs in vivo, thereby improving
durability of suppression. The results of this study will provide pre-clinical evidence for effective suppression of ADA
responses to biotherapeutics and enable rational therapeutic design and applicability to other immune disorders.
Validating the principles of TCR KO Vswap TCR will provide proof of principle and will be a first step in engineering
single-antigen specific “off the shelf” antigen specific TCR and MHCI KO FVIII V-swap Tregs to overcome
alloreactive barriers, contributing to our long-term goal to generate universally applicable Tregs from healthy donors.

## Key facts

- **NIH application ID:** 10894754
- **Project number:** 5R21HL170146-02
- **Recipient organization:** INDIANA UNIVERSITY INDIANAPOLIS
- **Principal Investigator:** Moanaro Biswas
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $239,768
- **Award type:** 5
- **Project period:** 2023-08-01 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10894754

## Citation

> US National Institutes of Health, RePORTER application 10894754, Development of a cellular therapy product with single specificity and improved persistence to prevent immunity to biotherapeutics (5R21HL170146-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10894754. Licensed CC0.

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