Researchers at the University of Bonn have identified the isoelectric point (pI) of AI-designed protein minibinders as a key determinant of whether they function effectively when incorporated into chimeric antigen receptor (CAR) T cells, according to a study published August 20 in Nature Communications. The finding suggests that optimizing binding affinity alone may be insufficient when using generative protein design to build next-generation CARs.
The researchers developed minibinders against three B7-H family proteins expressed on cancer cells — PD-L1 (B7-H1), CD276 (B7-H3), and VTCN1 (B7-H4) — using a computational pipeline combining RFdiffusion for protein backbone generation, ProteinMPNN for sequence design, and AlphaFold2 and Chai-1 for structural evaluation. Candidates were subsequently screened experimentally and shown to recognize endogenous targets on human cancer cells.
The key finding emerged when selected minibinders were incorporated into second-generation CAR constructs. Despite retaining target-binding activity, some produced poor CAR surface expression and reduced tumor-cell killing selectivity. The researchers traced the effect to the pI of the minibinder domain, with highly basic binders showing reduced and heterogeneous presentation on the T-cell surface.
The authors propose that positively charged minibinders undergo nonspecific electrostatic interactions with negatively charged components of the cell surface, interfering with trafficking and membrane presentation. This means that the portions of an AI-designed binder outside its target-binding interface can materially affect the performance of the resulting cell therapy.
To test whether the property could be engineered, the researchers generated approximately 6,000 variants of HM9, a CD276 minibinder with a pI of 9.4, while preserving residues involved in target binding. Computational and experimental screening identified a pI range of approximately 6.5–8.5 as optimal. CAR-T cells incorporating variants within that range showed improved surface expression and greater selectivity for CD276-positive tumor cells across three independent human donors.