AI-enabled discovery and biochemical optimization of minibinders targeting cancer cell-surface proteins
Experimental validation and functional optimization remain bottlenecks in AI-based protein design. We present a scalable workflow for developing AI-designed minibinders against cancer-associated surface proteins. Screening thousands of designs using mammalian cell-surface display identifies several high-affinity PD-L1 minibinders but far fewer for CD276 (B7-H3) and VTCN1 (B7-H4), highlighting substantial target dependence. Interface predicted template modeling (ipTM) scores generated by Chai-1 with ESM embeddings correlate with binding success and capture deleterious effects of interface mutations. Fluorophore-labeled AI-minibinders enable flow-cytometric staining comparable to conventional antibodies. However, when incorporated into chimeric antigen receptors (CAR), some show poor cell-surface trafficking and limited functionality. Redesign through a genetic algorithm-based diversification strategy that preserves the binding interface while changing non-binding surfaces experimentally reveals an isoelectric point (pI) window that improves CAR expression and enhances target-selective tumor cell killing. Our findings identify biochemical optimization beyond the binding interface as a critical requirement for translating AI-minibinders into functional applications.
Authors
- Heiko Rühl (ORCID: https://orcid.org/0000-0003-0672-2453)
- Bianca Broske (ORCID: https://orcid.org/0000-0003-3898-436X)
- Jan M. P. Tödtmann
- Michael Hölzel (ORCID: https://orcid.org/0000-0003-4048-8824)
- Sophie C. Binder (ORCID: https://orcid.org/0009-0004-5573-1131)
- Tobias Bald (ORCID: https://orcid.org/0000-0003-0061-235X)
- Benjamin A. McEnroe (ORCID: https://orcid.org/0009-0009-7574-565X)
- Caroline I. Fandrey (ORCID: https://orcid.org/0000-0001-6513-8877)
- Alexander Semaan (ORCID: https://orcid.org/0000-0001-5424-3217)
- Sebastian Kobold (ORCID: https://orcid.org/0000-0002-5612-4673)
- Katja Blumenstock
- Kristina Markova (ORCID: https://orcid.org/0000-0002-1323-9155)
- Gregor Hagelueken (ORCID: https://orcid.org/0000-0001-8781-5664)
- Matthias Geyer (ORCID: https://orcid.org/0000-0002-7718-5002)
- Alexander Hoch (ORCID: https://orcid.org/0000-0002-4790-5507)
- Johannes Oldenburg (ORCID: https://orcid.org/0000-0002-1585-4100)
- Michelle C.R. Yong
- Dominic Ferber (ORCID: https://orcid.org/0000-0003-2633-2858)
- Julia M. Messmer (ORCID: https://orcid.org/0000-0001-7858-1501)
- Tim Kempchen (ORCID: https://orcid.org/0009-0007-0173-2026)
- Stephan Menzel (ORCID: https://orcid.org/0009-0005-4742-9335)
- Peter Konopka (ORCID: https://orcid.org/0009-0002-2373-2653)
- Prof. Marieta I. Toma
- Elisabeth Tan
- Tim Rollenske (ORCID: https://orcid.org/0000-0002-7921-4778)
- Jonathan L. Schmid-Burgk
- Marie Kleinert
Institutions
- German Cancer Research Center (DE)
- Heidelberg University (DE)
- University Hospital Bonn (DE)
- University Hospital Heidelberg (DE)
- Helmholtz Zentrum München (DE)
- LMU Klinikum (DE)
- German Center for Lung Research (DE)
- Franklin University (US)
- Ludwig-Maximilians-Universität München (DE)
- Saarland University (DE)
Publication Details
- Journal
- Nature Communications
- Published
- 2026-08-20
- DOI
- https://doi.org/10.1038/s41467-026-76760-5
- Primary Topic
- Monoclonal and Polyclonal Antibodies Research
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Melanoma Research Alliance
- Deutsche Forschungsgemeinschaft
- Rheinische Friedrich-Wilhelms-Universität Bonn