NIH grant: Synthetic costimulation and signaling calibration for chimeric T cell receptors (1K22CA303684-01A1)

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NIH grant 1K22CA303684-01A1: Synthetic costimulation and signaling calibration for chimeric T cell receptors
Institute: {'code': 'CA', 'abbreviation': 'NCI', 'name': 'National Cancer Institute', 'admin_org_id': '12500008', 'admin_funding_url': ''}. PI: Sylvain  Simon. Award: $192024.
Start: 2026-07-16T00:00:00.
Project Summary
The adoptive transfer of T cells engineered with synthetic chimeric antigen receptors (CARs) has been effective
in treating B cell malignancies. However, relapses are frequent and often due to CAR T cells failing to recognize
tumor cells with low or negative CD19 expression. This highlights the need for immune receptors with enhanced
sensitivity and multispecificity. To address this issue, we developed a bispecific chimeric T cell receptor (Bi-
ChTCR), consisting of the α and β constant chains of the TCR fused to two tumor-specific binding domains. Bi-
ChTCRs engaged with CD3 subunits to mimic canonical TCR structures, induced potent proximal signaling and
formed an organized immune synapse. Bi-ChTCR T cells (Bi-ChTCR-T) showed superior antigen sensitivity
compared to CAR designs and maintained similar sensitivity to monospecific ChTCR formats, likely due to their
bivalent architecture, where bispecific CARs are often compromised. Although Bi-ChTCR-T lack intrinsic
costimulation, they demonstrated enhanced anti-tumor efficacy against heterogeneous tumors compared to
mono- and bispecific CAR T cells with costimulatory domains. This may be due to more efficient recruitment of
costimulatory molecules or to the superior engagement of CD3 chains, which contain a total of 10
immunoreceptor tyrosine-based activation motifs (ITAMs). However, most tumor cells lack costimulatory ligands,
and it is likely that providing additional costimulation to Bi-ChTCR-T would be beneficial in particular for targeting
antigen low tumor cells. Therefore, our studies propose to use synthetic biology and gene editing to design CD3
subunits that will assemble within the Bi-ChTCR complex to modulate T cell signaling and augment Bi-ChTCR-
T anti-tumor activity. In Aim 1, we will fuse signaling domains from costimulation molecules to synthetic
CD3 subunits to provide cell intrinsic costimulation to Bi-ChTCR-T. We will use a pooled screen approach to
identify CD3-fusion candidates that converge in augmenting Bi-ChTCR T cells functions in vitro, using a repetitive
stimulation assay, and in vivo, using a stress test mouse model. In Aim 2, we will systematically inactivate ITAM
sequences from CD3 subunits to provide a rheostat for Bi-ChTCR signaling and will investigate Bi-ChTCR T
cells antigen sensitivity, persistence and resistance to exhaustion. Finally, Aim 3 will use CyTOF to elucidate
signaling events in Bi-ChTCR T cells expressing CD3-fusion proteins and ITAM-inactivated subunits and
integrate these events with T cell phenotype and functions providing mechanistic insights into their superior anti-
tumor activity. Findings from these studies will facilitate clinical translation of the Bi-ChTCR platform.
I am committed to securing an independent academic position and establishing a research program that
integrates fundamental T cell biology with synthetic biology to enable the pre-clinical development of next-
generation cell therapies for cancer. To support this goal, I have developed a comprehensive training plan that
includes both didactic coursework and hands-on experience to acquire expertise in 1) genomic screening and
computational methods, 2) expand my knowledge in protein design, and 3) cultivate management skills.

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