Nanobody adaptor-directed CAR T cells enable precise and safe targeting of solid tumors

Background: Chimeric antigen receptor (CAR) T cell therapies have produced durable remissions in hematologic malignancies but have demonstrated only limited efficacy in solid tumors, largely due to antigen heterogeneity, restricted tumor penetration, and on-target, off-tumor toxicities. To address these limitations, we developed an adaptable CAR T cell platform that employs small sized tumor-targeting nanobody adaptors bearing a C-terminal cMyc tag in combination with T cells expressing a cMyc-specific CAR. By switching the connecting nanobody, this design enables tunable target engagement and versatility across antigens. This approach establishes a framework for extending CAR T therapy beyond hematologic malignancies while maintaining safety and specificity. Furthermore, it provides a robust platform to investigate and optimize the interplay between tumor penetration, cytotoxicity, and therapeutic efficacy in preclinical models. Methods: Mesothelin (MSLN) was selected as a model tumor-associated antigen, and a high-affinity MSLN-targeting nanobody (JZQ-B4) was used as the adaptor molecule. The cMyc CAR was constructed to incorporate a single-chain variable fragment (scFv) derived from the 9E10 antibody, a CD28 co-stimulatory domain, and a CD3ζ signaling domain. In vitro studies assessed adaptor-mediated tumor decoration and cMyc CAR T cell cytotoxicity. In vivo biodistribution and targeting were assessed using 18 F -labeled JZQ-B4 PET imaging. Antitumor efficacy of the adaptable CAR T platform was assessed in NSG mice bearing subcutaneous NCI-H226 xenografts. JZQ-B4 adaptors were continuously delivered via subcutaneous osmotic pumps (5 µg/day for 4 weeks), followed by intravenous injection of 1 × 10 7 cMyc CAR T cells starting one day after pump implantation. A conventional MSLN CAR incorporating the same JZQ-B4 nanobody directly into the CAR construct served as a comparative control. Results: PET/CT imaging with 18 F-labeled JZQ-B4 nanobody demonstrated highly selective accumulation in MSLN-positive lung cancer xenografts, with minimal uptake in MSLN-knockout tumors. Unlabeled JZQ-B4 adaptors bound MSLN-positive tumor cells with picomolar affinity and sustained surface retention, thereby enabling efficient tumor decoration and adaptor-dependent recognition by cMyc CAR T cells. Adaptor engagement elicited potent, antigen-specific CAR T cell activation and cytotoxicity in vitro, with negligible activity against antigen-negative targets. In vivo, adaptor-redirected cMyc CAR T cells achieved robust tumor infiltration and durable tumor control while sparing normal tissues, leading to significantly prolonged overall survival compared to groups receiving either adaptors or CAR T cells alone. In contrast, conventional high-affinity MSLN CAR T cells failed to control tumor growth and induced severe systemic toxicity due to on-target, off-tumor activity, characterized by widespread CAR T cell expansion across multiple normal organs. Conclusions: Adaptor-directed targeting represents a clinically adaptable and broadly applicable strategy to enhance the precision and safety of CAR T cell therapy for solid tumors. This modular platform preserves antitumor efficacy while mitigating on-target, off-tumor toxicities and provides a tunable framework to optimize the therapeutic window across diverse solid malignancies.