免疫毒素是抗体-毒素结合物，结合到细胞表面抗原后，进入肿瘤细胞内发挥有效的细胞毒作用。免疫毒素展示出有效的细胞毒性，并已获得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，大部分内吞的免疫毒素会在溶酶体内降解，只有约5％的游离毒素逃逸到胞质内发挥细胞毒性。许多研究通过改造毒素片段来减少免疫原性或增加稳定性，但抗体片段如何对免疫毒素的活性作出贡献尚未得到很好地证明。 在本研究中，我们使用32A9和42A1两种具有相似抗原结合能力和内吞率的抗-GPC3抗体构建了scFv-mPE24免疫毒素，并评估了其体外和体内的抗肿瘤活性。随后，对比了32A9 scFv-mPE24和42A1 scFv-mPE24的抗原结合能力、细胞内转运、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评估了32A9 scFv-mPE24和42A1 scFv-mPE24的降解行为。最后，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吞率，但与42A1 scFv-mPE24相比，32A9 scFv-mPE24具有更优越的抗肿瘤活性。我们发现32A9 scFv-mPE24的降解速度更快，有助于高效释放游离毒素，而42A1 scFv-mPE24则相对较慢。这些现象是由于32A9 scFv-mPE24和42A1 scFv-mPE24在溶酶体中降解行为的不同所决定的。此外，32A9对低pH环境敏感，使得32A9结合物在溶酶体中易失去抗原结合并发生降解，从而高效地产生游离毒素发挥细胞毒性，而42A1对酸性环境具有抵抗力，使42A1结合物在溶酶体中相对稳定，延缓了游离毒素的释放。 这些结果表明，基于低pH敏感性的抗体的免疫毒素在溶酶体中降解更快，导致游离毒素有效释放，并改善了细胞毒性，与基于正常抗体的免疫毒素相比。我们的发现表明，在设计免疫毒素和其他依赖溶酶体降解的抗体结合物药物时，低pH敏感性抗体可能具有优势。© 2023. BioMed Central Ltd., part of Springer Nature.

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.© 2023. BioMed Central Ltd., part of Springer Nature.