Directed evolution of chlorotoxin enhances MMP-2 recognition and improves CAR-T-cell activity in glioblastoma models in vitro

Background: Cysteine-rich miniproteins from venomous animals are promising therapeutic scaffolds due to their compact structure, high stability, and low immunogenicity. Chlorotoxin (CTX), a miniprotein derived from scorpion venom, selectively targets glioblastoma by binding matrix metalloproteinase-2 (MMP-2), an invasion-associated enzyme overexpressed in ∼80% of glioblastomas. CTX has been utilized in tumor imaging, drug delivery, and immunotherapy, including its incorporation into chimeric antigen receptor (CAR) constructs. CTX-based CAR show potent MMP-2-dependent cytotoxicity in glioblastoma models. We hypothesized that affinity maturation of CTX via phage display could enhance MMP-2 binding and improve CAR T-cell performance. Methods: A CTX-based phage display library (∼1.3 million variants) was screened against immobilized MMP-2. A lead variant, CTXA8, was recombinantly expressed and tested for binding specificity against a panel of off-target proteins. Its cellular uptake and localization were evaluated, followed by incorporation into CAR constructs in both single-unit and tandem formats (eCTXA8-CAR). The cytotoxicity of CTX-, CTXA8-, and eCTXA8-CAR T cells was assessed against glioblastoma cell lines and primary patient-derived tumor cells. Results: Screening identified CTXA8, which demonstrated a 4.4-fold increase in MMP-2 affinity and reduced off-target binding relative to CTX. Fluorescent CTXA8 showed 2.4-3.5-fold greater uptake in glioblastoma cells than CTX. Among the CAR constructs tested, eCTXA8 induced the highest IFN-γ release. eCTXA8-CAR T-cells consistently outperformed CTX-CAR and non-transduced T cells in cytotoxicity assays, especially at low effector-to-target ratios. Conclusion: This study demonstrates that directed evolution of CTX can produce high-affinity, selective MMP-2 ligands suitable for next-generation CAR T-cell therapies. CTXA8-based CARs offer enhanced anti-tumor efficacy, supporting their potential in overcoming challenges in solid tumor immunotherapy.