Cleavable PEGylation Enhances the Antitumor Efficacy of Small-Sized Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs) have emerged as a promising class of cancer therapeutics. However, traditional ADCs are often limited by poor tumor penetration due to their large molecular size. While the use of small-sized antibody fragments or analogues can improve tumor permeability, this approach typically results in an extremely shortened blood circulation half-life, which diminishes the therapeutic benefits and brings other metabolic challenges. In addition, the expression of target antigens on normal tissues often leads to unnecessary on-target/off-tumor toxicity. To address these issues, we developed a novel tumor site-specific cleavable PEGylation strategy for small-sized ADC design. The small ADC molecule ZHER2-MMAE was site-specifically PEGylated at its N-terminus with a 20 kDa polyethylene glycol (PEG) chain and a uPA (LSGRSDNH) cleavage sequence was inserted between them (PEG20k-U-ZHER2-MMAE). Our results showed that PEG20k-U-ZHER2-MMAE achieves a similar half-life extension (6.4 and 6.0 h) compared to the conventional PEG20k-ZHER2-MMAE, both representing about a 26-fold improvement compared to ZHER2-MMAE. Importantly, PEG20k-U-ZHER2-MMAE exhibited significantly higher drug accumulation at the tumor site, leading to the complete eradication of NCI-N87 and SK-OV-3 tumors at a dose of 5.5 mg/kg. Additionally, it demonstrated a maximum tolerated dose (MTD) exceeding 35 mg/kg, while the noncleavable PEG20k-ZHER2-MMAE could only slow tumor growth. In addition, compared to ZHER2-MMAE, the in vitro cytotoxic activity of PEG20k-ZHER2-MMAE or PEG20k-U-ZHER2-MMAE was reduced by about 50 times, with the latter expected to reduce the on-target/off-tumor side effects due to the specific activation by uPA at tumor sites. These data fully demonstrate the effectiveness and high safety of our tumor-specific cleavable PEGylation strategy, supporting the potential in the development of next-generation ADCs for cancer therapy.