A protein actuator platform for Boolean logic-gated, cell-specific targeting and therapeutic payload delivery

Background

Cell surface proteins, collectively known as the “surfaceome,” are critical targets in the development of therapeutics and diagnostics, particularly for diseases such as cancer. Many modern therapies, including monoclonal antibodies and engineered immune cells like CAR-T cells, rely on recognizing specific antigens present on the surface of diseased cells to achieve targeted action. However, a significant challenge in this field is that most surface antigens are not exclusive to diseased cells; they are often shared with healthy tissues. This overlap leads to “on-target/off-tumor” toxicity, where therapies inadvertently attack normal cells, resulting in severe side effects and limiting the therapeutic window. As a result, there is a pressing need for technologies that can more precisely discriminate between healthy and diseased cells, ideally by integrating multiple markers to enhance specificity and safety.

Current approaches to improving cell targeting specificity often involve the use of single-antigen recognition systems, which are inherently limited by the lack of truly disease-specific markers. Even advanced strategies, such as dual-targeting CAR-T cells or bispecific antibodies, typically rely on co-expression of two antigens but may not strictly require both for activation, leading to residual off-target effects. Moreover, these systems can be hampered by the complexity of engineering and controlling protein interactions in the oxidizing extracellular environment, where many protein-based switches are less efficient or prone to unintended activation. The lack of robust, conditional systems that can function as precise Boolean logic gates—activating only when multiple, specific conditions are met—remains a major bottleneck in the field. This gap underscores the need for more sophisticated molecular tools that can tightly control therapeutic activity, minimizing collateral damage to healthy tissues while maintaining efficacy against target cells.

Technology

This technology enables highly specific targeting of cell populations through a system that uses conditional split inteins—such as the NrdJ-1 intein—fused to distinct targeting elements. Each fragment of the split intein is engineered to bind a separate cell-surface antigen, and only when both antigens are present on a cell do the fragments come into close proximity, triggering a protein trans-splicing reaction. This reaction assembles a user-defined protein actuator—such as a toxin, antibody epitope, or receptor ligand—directly at the cell surface. The system operates as a Boolean logic gate, most notably an AND gate, ensuring that the active protein is only produced in cells expressing both target antigens. The targeting elements can be proteins, small molecules, or nucleic acids, and the system is compatible with various actuator payloads. It can be produced recombinantly or semisynthetically and is suitable for both ex vivo and localized in vivo applications.

What differentiates this technology is its unprecedented specificity and programmability for cell targeting, achieved by integrating Boolean logic into protein actuator assembly. Unlike conventional approaches that rely on single-antigen recognition—which often leads to off-target effects due to antigen overlap between healthy and diseased cells—this system requires the simultaneous presence of two distinct antigens, dramatically reducing unintended activation. The engineered split inteins are optimized for tight conditionality and robust function in the oxidized extracellular environment, making the system practical for therapeutic use. Experimental validation demonstrates that only double-positive cells (e.g., those expressing both HER2 and EGFR) activate the actuator, enabling selective delivery of cytotoxic agents or diagnostic probes. The platform’s modularity allows adaptation to different antigens and payloads, and its compatibility with systems like SpyTag/SpyCatcher further expands its utility for covalent labeling and targeted delivery. This makes it a powerful tool for precision medicine, particularly in cancer immunotherapy and synthetic biology, where minimizing off-target effects is critical.

Benefits

• Enables highly specific targeting of cells by requiring the presence of two distinct surface antigens (Boolean AND logic gating), reducing off-target effects.
• Generates functional protein actuators only at desired cell subpopulations, improving therapeutic precision and safety.
• Uses engineered conditional split inteins optimized for efficient protein trans-splicing in the oxidized extracellular environment.
• Supports versatile payloads including toxins, antibody epitopes, and receptor ligands for diverse therapeutic and diagnostic applications.
• Compatible with various targeting elements such as proteins, small molecules, and nucleic acids for flexible cell targeting strategies.
• Validated experimentally to selectively activate and deliver cargo or induce cytotoxicity in double-positive cells, minimizing impact on non-target cells.
• Applicable to ex vivo and localized in vivo therapies, including cancer immunotherapy and synthetic biology platforms.
• Can be combined with covalent labeling systems (e.g., SpyTag/SpyCatcher) for enhanced payload delivery and modular design.

Applications

• Targeted cancer cell cytotoxicity
• Precision cell surface labeling
• Selective therapeutic protein delivery
• Cell-specific immune activation
• Logic-gated cellular diagnostics

Additional info

This system utilizes conditional split NrdJ-1 inteins to generate functional protein actuators through Boolean logic-gated trans-splicing. Fragments fused to targeting elements bind specific cell-surface antigens. When complementary antigens are present, proximity triggers splicing, assembling actuators like toxins or ligands. Operating as an AND gate, this mechanism ensures cell-specific activation in oxidized extracellular environments, enhancing therapeutic precision and minimizing off-target effects in synthetic biology and immunotherapy.