BACKGROUND Immunotoxins consisting of a toxin from bacteria or plants and a targeting module have been developed as potent anti-cancer therapeutics. The majority of them, especially those in preclinical or clinical testing stages, are fusion proteins of a toxin and antibody fragment. Immunotoxins based on full-length antibodies are less studied, even though the fragment crystallizable (Fc) domain plays an important role in regulating the concentration of immunoglobulin G (IgG) in the serum and in antibody-mediated immune responses against pathogens. RESULTS We devised a method to site-specifically conjugate IgG and another protein using a cysteine residue introduced into the IgG and a bio-orthogonally reactive unnatural amino acid incorporated into the other protein. The human epidermal growth factor receptor 2 (Her2)-targeting IgG, trastuzumab, was engineered to have an unpaired cysteine in the heavy chain, and an unnatural amino acid with the azido group was incorporated into an engineered Pseudomonas exotoxin A (PE24). The two protein molecules were conjugated site-specifically using a bifunctional linker having dibenzocyclooctyne and maleimide groups. Binding to Her2 and interaction with various Fc receptors of trastuzumab were not affected by the conjugation with PE24. The trastuzumab-PE24 conjugate was cytotoxic to Her2-overexpressing cell lines, which involved the inhibition of cellular protein synthesis due to the modification of elongation factor-2. CONCLUSIONS We constructed the site-specifically conjugated immunotoxin based on IgG and PE24, which induced target-specific cytotoxicity. To evaluate the molecule as a cancer therapeutic, animal studies are planned to assess tumor regression, half-life in blood, and in vivo immunogenicity. In addition, we expect that the site-specific conjugation method can be used to develop other antibody-protein conjugates for applications in therapeutics and diagnostics.

中文翻译：

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通过抗 Her2 IgG 和工程化假单胞菌外毒素 A 的位点特异性结合构建免疫毒素。

背景技术由来自细菌或植物的毒素和靶向模块组成的免疫毒素已被开发为有效的抗癌治疗剂。它们中的大多数，尤其是处于临床前或临床测试阶段的那些，是毒素和抗体片段的融合蛋白。基于全长抗体的免疫毒素的研究较少，尽管可结晶片段 (Fc) 结构域在调节血清中免疫球蛋白 G (IgG) 的浓度和抗体介导的针对病原体的免疫反应中起重要作用。结果 我们设计了一种使用引入 IgG 中的半胱氨酸残基和掺入其他蛋白质中的生物正交反应性非天然氨基酸来位点特异性缀合 IgG 和另一种蛋白质的方法。人表皮生长因子受体 2 (Her2) 靶向 IgG、曲妥珠单抗、被工程改造为在重链中具有未配对的半胱氨酸，并且具有叠氮基的非天然氨基酸被掺入工程化的假单胞菌外毒素A（PE24）中。这两个蛋白质分子使用具有二苯并环辛炔和马来酰亚胺基团的双功能接头进行位点特异性结合。与 Her2 的结合以及与曲妥珠单抗的各种 Fc 受体的相互作用不受与 PE24 缀合的影响。曲妥珠单抗-PE24 偶联物对 Her2 过表达细胞系具有细胞毒性，这涉及由于延伸因子 2 的修饰而抑制细胞蛋白质合成。结论 我们构建了基于 IgG 和 PE24 的位点特异性结合免疫毒素，可诱导靶标特异性细胞毒性。为了评估该分子作为癌症治疗剂，计划进行动物研究以评估肿瘤消退、血液中的半衰期和体内免疫原性。此外，我们预计位点特异性偶联方法可用于开发其他抗体-蛋白质偶联物，用于治疗和诊断。