ArticleFrontiers in bioengineering and biotechnology2026
Directed evolution of chlorotoxin enhances MMP-2 recognition and improves CAR-T-cell activity in glioblastoma models
Article in Frontiers in bioengineering and biotechnology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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