An Affibody-Cytotoxin Conjugate-Based Nanoagent for EGFR Targeting and Precise Therapy of Epidermal Squamous Cell Carcinoma

Monomethyl auristatin E (MMAE) is a highly cytotoxic tubulin inhibitor frequently used in antibody-drug conjugates (ADCs) for targeted cancer therapy. However, ADCs face several challenges, including inhomogeneity in the drug-to-antibody ratio (DAR) and potential immunogenicity. Affibodies, a class of small high-affinity proteins (∼6.5 kDa) with nonimmunogenic properties and relatively simple structures, present a promising solution to these issues. Here, we developed an affibody-cytotoxin conjugate-based nanoagent (Z_EGFR:1907-MMAE ADCN) for EGFR targeting and precise therapy of epidermal squamous cell carcinoma (ESCC). In detail, the EGFR-targeting affibody (Z_EGFR:1907-Cys) was conjugated to MMAE via a Cathepsin B-cleavable dipeptide linker to produce an amphiphilic Z_EGFR:1907-MMAE conjugate. It self-assembled into Z_EGFR:1907-MMAE ADCN with a diameter of 113.0 ± 3.4 nm and a PDI of 0.16 ± 0.02 in PBS. Z_EGFR:1907-MMAE ADCN exhibited rapid internalization and significant cytotoxicity (IC₅₀ = 4.08 nM) against EGFR-positive cancer cells in vitro. Furthermore, Z_EGFR:1907-MMAE ADCN also had an excellent tumor-homing ability and demonstrated a high tumor inhibition rate (TIR) of 97.3% in vivo. Such a nanoagent strategy highly improved the therapeutic window and biosafety of MMAE in precise cancer therapy.