A CD3 Fusion Receptor (CD3-FR) Uniquely Enables Compatibility of Allogeneic CAR-T and -NK Cells with T Cell Engagers to Enhance Antitumor Function and Limit Antigen Escape

Abstract

Chimeric antigen receptor (CAR) T cells have shown remarkable clinical success in the treatment of many malignancies. However, certain challenges remain, including broad patient access and durability of response. We have shown that a scalable manufacturing platform, where genetically edited induced pluripotent stem cell (iPSC) master cell lines are created to serve as a renewable starting material for the derivation of uniformly engineered CAR T or NK cells, can facilitate broad patient access in an off-the-shelf manner. To improve durability of response, often plagued by antigen heterogeneity found in cancer, we are pursuing several combination strategies, merging cell therapy with therapeutic agents such as checkpoint blockade therapy and monoclonal antibodies to increase anti-tumor activity and multi-antigen targeting of cancer. T cell engagers have also shown remarkable advancements in the treatment of various cancer types, but they are also afflicted by their own challenges and would equally benefit from combination strategies. Unfortunately, T cell engagers are not compatible with allogeneic adoptive cell therapy, as the cells used in the allogenic setting do not express the CD3 molecule used for interaction with the engagers, as the T cell receptor (TCR) that supports CD3 surface expression, is either not expressed in NK cells or has been ablated in T cells to prevent graft-versus-host disease. To investigate the anti-tumor synergy between off-the-shelf cell therapy and T cell engagers, we developed a novel CD3ε fusion receptor (CD3-FR) to uniquely support the expression of a functional CD3 on TCR-less allogeneic T and NK cells and to enable compatibility between allogeneic cell therapy and T cell engagers. Initially, CD3-FR constructs with various co-stimulating endodomains were confirmed to elicit T cell engager-dependent NFAT activity in TCR alpha chain (TRAC) knockout (T-KO) Jurkat cells. Next, iPSCs already containing a CAR inserted into the TRAC locus were engineered to contain CD3-FR. CAR+ CD3-FR+ iPSCs were differentiated into CAR-iT cells, uniformly expressing both modalities (>95% CAR+, >90% CD3ε+, TCR not detected). To demonstrate that CD3-FR+ CAR-iT cells can elicit T cell engager-dependent antitumor activity, CAR antigen (1° Ag) negative but T cell engager (2° Ag) positive target cells were used in various cytotoxicity assays. As expected, control CAR-iT cells failed to kill the 1° Ag negative tumors despite addition of T cell engager targeting 2° Ag. On the other hand, CD3-FR+ CAR-iT cells showed potent activity toward target cells but only in the presence of the T cell engager (>80% cytolysis). Furthermore, when comparing the expression of activation markers in CD3-FR+ CAR-iT cells in co-cultures with or without the T cell engager, we observed an increased proportion of CD69+ (61% vs. 28%) and CD25+ (37% vs. 2%) cells in the CD3-FR+ CAR-iT cell co-cultures containing the T cell engager. Cytokine production was also tested, with increased TNF (5-fold) and IFNγ (7-fold) measured in supernatants from CD3-FR+ co-cultures upon the addition of T cell engager, further demonstrating T cell engager-dependent functionality and specificity of the CD3-FR construct. To test the ability of CD3-FR CAR-iT cells to mitigate 1° Ag escape, we used a target cell population heterogenous for the CAR-specific target (1° Ag, 50% positive) but homogenous for EpCAM (2° Ag, 100% positive). After three days, the standard CAR-iT cells failed to control tumor cell growth due to antigen escape. In contrast, CD3-FR+ CAR-iT cells exhibited robust control (80% cytolysis) of the 1° Ag heterogenous targets in the presence of αEpCAM, indicating mitigation of 1° Ag escape. Lastly, CD3-FR was edited into iPSC-derived NK (iNK) cells to demonstrate compatibility of CD3-FR+ NK cells with T cell engagers. An xCelligence-based killing assay was performed in the presence of a T cell engager, where CD3-FR+ iNK cells showed superior target cell killing (~80% lysis) after 96 hours compared to parental iNK cells (~30% lysis), demonstrating the potential for synergy between T cell engagers and NK cells (Figure 1). Taken together, these studies present a novel opportunity to combine T cell engager technology with off-the-shelf CAR-iT or iNK cell products, uniquely merging two powerful therapeutics whose combination was previously underappreciated in improving cancer therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal