Abstract 2886: A <i>de novo</i> designed miniprotein enables modular and specific CAR-T targeting

Abstract A definitive cure for acute myeloid leukemia (AML) remains elusive. Inter and Intra-patient heterogeneity in antigen expression profiles is a major challenge for AML treatment. Adapter CAR-T cell therapies utilize a chimeric antigen receptor (CAR) which binds to a non-natural small molecule, which in turn can be attached to a soluble affinity domain that binds the tumor antigen. Decoupling cancer cell binding and CAR engagement provides flexibility and control in directing the CAR-T therapy through dosing of the adapter, which can improve safety and decrease T cell exhaustion. A critical challenge for the success of adapter CAR-T is finding an ideal adapter molecule. De novo designed miniproteins are a novel therapeutic modality. They are approximately 45 amino acids in length and adopt a highly stable tertiary structure, are easily produced recombinantly or synthetically, and present many additional advantages over traditional biologics. The structure of the miniprotein is created from scratch using generative artificial intelligence and is therefore controllable, permitting specific design of the conjugation site, as well as precise tuning the affinity. The small size, high solubility, and compact structure confers better biodistribution and lower internalization in the tumor cell surface. Here, we show that miniproteins outperform antibodies as the targeting moiety of an adapter CAR-T therapy, and we demonstrate this using CD123 directed treatment of AML. We engineered AIP26GQ, a 5.6 kDa miniprotein that binds selectively to CD123 with KD = 0.5 pM. AIP26GQ is thermostable, and can be conjugated to a fluorescein molecule (FITC) via an engineered lysine residue. We demonstrated that AIP26GQ can direct anti-FITC CAR-T cells to kill AML model cell lines (MV-4-11) in vitro. We evaluated multiple positions for selective attachment of FITC, and identified the optimal geometric orientation for maximizing CAR-T activation. In vivo efficacy studies using MV-4-11 in NCG mice showed that AIP26GQ-directed CAR-T treatment reduces tumor burden to background levels and the remission is stable at least 15 days after the last dose. In the same study, a talacotuzumab antibody based adapter molecule and a direct anti-CD123 CAR-T were used for comparison. The miniprotein-directed CAR exhibited multiple advantages over both the antibody and the direct CAR-T, including increased efficacy with lower dose required of both the adapter and the CAR-T cells, improved tumor occupation, and less CAR-T exhaustion. Our system combining a FITC-conjugated miniprotein with anti-FITC CAR-T cells yields complete remission in an MV-4-11 xenograft mice model, outperforming both an antibody adapter and direct CAR-T approaches. We demonstrated that the use of controllable CAR-T cells combined with miniprotein adapters exhibits an unprecedented potential for the design of dosable, redirectable and multiplexable cancer treatments. Citation Format: Maria Eugenia Llases, Craig McKay, Sakeena Syed, Vitaliy Andrusyshyn, Edson Carcamo Noriega, James W. Bowman, Tessa Howard, Nathalia Rodriguez, Frank Teets, Ian Truebridge, Xavier Michelet, Zach Caruolo, Lindsay Webb, John Newcomb, Fred Mermelstein, Carl Novina, Christopher Bahl. A de novo designed miniprotein enables modular and specific CAR-T targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2886.