Abstract 5240: Novel CD3-Fusion Receptor enables combination of T-cell engagers and allogeneic CAR T cells to promote enhanced antitumor activity and overcome antigen escape
Authors: Lu Dan , Peralta Eigen , Chu Hui-Yi , Park Soo , Tsuda Masanao , Avramis Earl , Denholtz Matthew , Witty Alec , Lee Tom , Valamehr Bahram 
Published: March 2024

Chimeric antigen receptor (CAR) T cells have shown remarkable clinical success in the treatment of hematologic malignancies. However, the durability of response of CAR T therapy can be impaired by heterogenous expression of the CAR-specific target antigen (1° Ag) on tumors. In combination with a CAR, T-cell engagers (TCEs) that target additional tumor antigen (2° Ag) can be a unique approach to address tumor heterogeneity and overcome antigen escape.

Engineered induced pluripotent stem cell (iPSC) master cell lines are a renewable source material from which iPSC-derived CAR T (CAR-iT) cells can be manufactured and administered in an off-the-shelf manner. In developing allogeneic CAR-iT cells, the T-cell receptor (TCR) must be eliminated to prevent graft-versus-host disease. However, the absence of TCR expression leads to loss of surface CD3 and a corresponding loss of TCE compatibility. Here we discuss a novel CD3ε fusion receptor (CD3-FR) with a modified transmembrane and endodomain, which allows for CD3 surface expression on TCR-less T cells and effective combination of off-the-shelf allogeneic CAR-T cells and TCEs. CAR+ CD3-FR+ iPSCs were differentiated into CAR-iT cells, uniformly expressing both modalities (&gt;95% CAR+, &gt;90% CD3ε+) and absent of TCR. To demonstrate that CD3-FR+ CAR-iT cells can elicit TCE-dependent activity, 1° Ag- (CD19-)/2° Ag+ (EpCAM+) target cells were used in cytotoxicity assays. As shown, CD3-FR- CAR-iT cells failed to kill 1° Ag- target cells. In contrast, CD3-FR+ CAR-iT cells demonstrated potent activity toward the target cells (&gt;80% cytolysis) but only in the presence of the TCE targeting 2° Ag.

To test the ability of CD3-FR+ CAR-iT cells to mitigate antigen escape, we used a tumor cell population heterogenous for the CAR-specific target (50% 1° Ag-) but homogenous for TCE-specific target (100% 2° Ag+). In co-culture assays, CD3-FR- CAR-iT cells failed to prevent tumor cell growth due to 1° Ag- tumor escape. However, CD3-FR+ CAR-iT cells in the presence of TCE exhibited robust control (80% cytolysis) of the heterogenous tumor, mitigating 1° Ag- tumor escape via targeting of 2° Ag. Next, we tested the ability of CD3-FR+ CAR-iT cells to control heterogeneous tumor in vivo in a disseminated leukemia model. As expected, CD3-FR+ CAR-iT cells exhibited significantly better tumor growth inhibition in the presence of TCE (p-value &lt;0.005) compared to controls lacking the CD3-FR modality. Ex vivo analysis of tumor in the bone marrow confirmed 1° Ag- tumor escape in the control treated mice, but not in mice treated with CD3-FR+ CAR-iT and TCE targeting 2° Ag.

Taken together, these studies present a novel opportunity to leverage a novel CD3 fusion receptor and enable combination of TCEs with engineered off-the-shelf CAR T-cell products, uniquely harnessing two potent therapeutic modalities for improving cancer therapy.

Citation Format: Dan Lu, Eigen Peralta, Hui-Yi Chu, Soo Park, Masanao Tsuda, Earl Avramis, Matthew Denholtz, Alec Witty, Tom Lee, Bahram Valamehr. Novel CD3-Fusion Receptor enables combination of T-cell engagers and allogeneic CAR T cells to promote enhanced antitumor activity and overcome antigen escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5240.