School of Basic Medical Sciences and Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Personalized Cancer Medicine, Nanjing Medical University, 101 Longmian Road, Xuehai Building, Nanjing, 211166, Jiangsu, People's Republic of China.
Department of Gynecology Oncology, Changzhou Maternal and Child Health Care Hospital, Changzhou Medical Center, Nanjing Medical University, Changzhou, China.
J Transl Med. 2023 Aug 25;21(1):572. doi: 10.1186/s12967-023-04210-7.
Immunotoxins are antibody-toxin conjugates that bind to surface antigens and exert effective cytotoxic activity after internalization into tumor cells. Immunotoxins exhibit effective cytotoxicity and have been approved by the FDA to treat multiple hematological malignancies, such as hairy cell leukemia and cutaneous T-cell lymphoma. However, most of the internalized immunotoxin is degraded in lysosomes, and only approximately 5% of free toxin escapes into the cytosol to exert cytotoxicity. Many studies have improved immunotoxins by engineering the toxin fragment to reduce immunogenicity or increase stability, but how the antibody fragment contributes to the activity of immunotoxins has not been well demonstrated.
In the current study, we used 32A9 and 42A1, two anti-GPC3 antibodies with similar antigen-binding capabilities and internalization rates, to construct scFv-mPE24 immunotoxins and evaluated their in vitro and in vivo antitumor activities. Next, the antigen-binding capacity, trafficking, intracellular protein stability and release of free toxin of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 were compared to elucidate their different antitumor activities. Furthermore, we used a lysosome inhibitor to evaluate the degradation behavior of 32A9 scFv-mPE24 and 42A1 scFv-mPE24. Finally, the antigen-binding patterns of 32A9 and 42A1 were compared under neutral and acidic pH conditions.
Although 32A9 and 42A1 had similar antigen binding capacities and internalization rates, 32A9 scFv-mPE24 had superior antitumor activity compared to 42A1 scFv-mPE24. We found that 32A9 scFv-mPE24 exhibited faster degradation and drove efficient free toxin release compared to 42A1 scFv-mPE24. These phenomena were determined by the different degradation behaviors of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 in lysosomes. Moreover, 32A9 was sensitive to the low-pH environment, which made the 32A9 conjugate easily lose antigen binding and undergo degradation in lysosomes, and the free toxin was then efficiently produced to exert cytotoxicity, whereas 42A1 was resistant to the acidic environment, which kept the 42A1 conjugate relatively stable in lysosomes and delayed the release of free toxin.
These results showed that a low pH-sensitive antibody-based immunotoxin degraded faster in lysosomes, caused effective free toxin release, and led to improved cytotoxicity compared to an immunotoxin based on a normal antibody. Our findings suggested that a low pH-sensitive antibody might have an advantage in the design of immunotoxins and other lysosomal degradation-dependent antibody conjugate drugs.
免疫毒素是一种抗体-毒素缀合物，它与表面抗原结合，并在进入肿瘤细胞后发挥有效的细胞毒性作用。免疫毒素具有有效的细胞毒性，并已被 FDA 批准用于治疗多种血液恶性肿瘤，如毛细胞白血病和皮肤 T 细胞淋巴瘤。然而，大多数内化的免疫毒素在溶酶体中降解，只有约 5%的游离毒素逃逸到细胞质中发挥细胞毒性。许多研究通过工程化毒素片段来降低免疫原性或增加稳定性来改进免疫毒素，但抗体片段如何对免疫毒素的活性做出贡献尚未得到很好的证明。
在本研究中，我们使用了两种具有相似抗原结合能力和内化率的抗 GPC3 抗体 32A9 和 42A1，构建了 scFv-mPE24 免疫毒素，并评估了它们的体外和体内抗肿瘤活性。接下来，我们比较了 32A9 scFv-mPE24 和 42A1 scFv-mPE24 的抗原结合能力、运输、细胞内蛋白稳定性和游离毒素的释放，以阐明它们不同的抗肿瘤活性。此外，我们使用溶酶体抑制剂来评估 32A9 scFv-mPE24 和 42A1 scFv-mPE24 的降解行为。最后，我们在中性和酸性 pH 条件下比较了 32A9 和 42A1 的抗原结合模式。
尽管 32A9 和 42A1 具有相似的抗原结合能力和内化率，但 32A9 scFv-mPE24 的抗肿瘤活性优于 42A1 scFv-mPE24。我们发现 32A9 scFv-mPE24 的降解速度更快，能够有效地释放游离毒素，而 42A1 scFv-mPE24 则不然。这些现象是由 32A9 scFv-mPE24 和 42A1 scFv-mPE24 在溶酶体中的不同降解行为决定的。此外，32A9 对低 pH 环境敏感，这使得 32A9 缀合物在溶酶体中容易失去抗原结合并发生降解，从而有效地产生游离毒素发挥细胞毒性作用，而 42A1 对酸性环境有抵抗力，这使得 42A1 缀合物在溶酶体中相对稳定，延迟了游离毒素的释放。
这些结果表明，一种低 pH 敏感的基于抗体的免疫毒素在溶酶体中降解更快，导致有效的游离毒素释放，从而比基于正常抗体的免疫毒素具有更高的细胞毒性。我们的研究结果表明，一种低 pH 敏感的抗体在免疫毒素的设计和其他依赖溶酶体降解的抗体偶联药物的设计中可能具有优势。
