Engineering a Programmed Death-Ligand 1-Targeting Monobody via Directed Evolution for SynNotch-Gated Cell Therapy

Introduction: Chimeric Antigen Receptor T (CAR T) cell therapy is a revolutionary treatment option for cancer therapy. It has shown remarkable success in treating hematological cancers, but solid tumors remain challenging due to factors such as the lack of tumor-specific antigens and the immunosuppressive tumor microenvironment. PD-L1, an immune checkpoint inhibitor, provides negative regulatory feedback that suppresses T cell activation, offering cancer cells a survival advantage. Blocking the PD-1/PD-L1 axis with immune checkpoint blockade (ICB) has proven effective in treating several cancers. This success underscores the significance of the PD-1/PD-L1 axis in cancer therapy. Targeting PD-L1 as a CAR-T cell antigen is an intriguing strategy. By focusing on cancer cells overexpressing PD-L1, CAR T cells can attack cancer cells and neutralize the immunosuppressive PD-1/PD-L1 axis, mitigating T cell exhaustion. However, this approach is risky due to potential on-target off-tumor effects, as PD-L1 is expressed in various non-cancerous cells. Thus, no FDA-approved CAR T cell therapy targets PD-L1, highlighting the need for design strategies to avoid off-target toxicity.

Materials and

Methods: To minimize non-specific toxicity, our study employed two strategies: (1) designing a CAR based on a PD-L1-recognizing monobody with stronger affinity at the lower pH typical in the tumor microenvironment, and (2) integrating SynNotch, which recognizes a clinically validated tumor-associated antigen (TAA) CD19 introduced into target cancer cells to form an IF THEN gate with PD-L1 for high precision control.

Results, Conclusions, and Discussions: Combining rational design and directed evolution of the human fibronectin-derived monobody scaffold, “PDbody” was engineered to bind to PD-L1 with a preference for slightly lower pH, which is typical in the tumor microenvironment (Figure 1A and 1B). PDbody was further utilized as a CAR to target the PD-L1-expressing triple negative MDA-MB-231 breast cancer cell line. To mitigate on-target off-tumor toxicity associated with targeting PD-L1, a Cluster of Differentiation 19 (CD19)-recognizing SynNotch IF THEN gate was integrated into the system (Figure 1C). This CD19-SynNotch PDbody-CAR system was then expressed in primary human T cells to target CD19-expressing MDA-MB-231 cancer cells. These CD19-SynNotch PDbody-CAR T cells demonstrated both specificity and efficacy in vitro, accurately eradicating cancer targets in cytotoxicity assays (Figure 1D). Moreover, in an in vivo bilateral murine tumor model, they exhibited the capability to effectively restrain tumor growth. Overall, CD19-SynNotch PDbody-CAR T cells represent a distinct development over previously published designs due to their increased efficacy, proliferative capability, and mitigation of off-tumor toxicity for solid tumor treatment.

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