Masilamani Anie P, Huber Nathalie, Nagl Constanze, Dettmer-Monaco Viviane, Monaco Gianni, Wolf Isis, Schultze-Seemann Susanne, Taromi Sanaz, Gratzke Christian, Fuchs Hendrik, Wolf Philipp
Department of Urology, Medical Center-University of Freiburg, Freiburg, Germany.
Faculty of Medicine, University of Freiburg, Freiburg, Germany.
Front Pharmacol. 2023 Jul 6;14:1211824. doi: 10.3389/fphar.2023.1211824. eCollection 2023.
Immunotoxins consist of an antibody or antibody fragment that binds to a specific cell surface structure and a cytotoxic domain that kills the cell after cytosolic uptake. Exotoxin A (PE) based immunotoxins directed against a variety of tumor entities have successfully entered the clinic. PE possesses a KDEL-like motif (REDLK) that enables the toxin to travel from sorting endosomes via the KDEL-receptor pathway to the endoplasmic reticulum (ER), from where it is transported into the cytosol. There, it ADP-ribosylates the eukaryotic elongation factor 2, resulting in ribosome inhibition and finally apoptosis. One major problem of immunotoxins is their lysosomal degradation causing the need for much more immunotoxin molecules than finally required for induction of cell death. The resulting dose limitations and substantially increased side effects require new strategies to achieve improved cytosolic uptake. Here we generated an immunotoxin consisting of a humanized single chain variable fragment (scFv) targeting the prostate specific membrane antigen (PSMA) and the de-immunized PE variant PE24mut. This immunotoxin, hD7-1(VL-VH)-PE24mut, showed high and specific cytotoxicity in PSMA-expressing prostate cancer cells. We deleted the REDLK sequence to prevent transport to the ER and achieve endosomal entrapment. The cytotoxicity of this immunotoxin, hD7-1(VL-VH)-PE24mutΔREDLK, was greatly reduced. To restore activity, we added the endosomal escape enhancer SO1861 and observed an up to 190,000-fold enhanced cytotoxicity corresponding to a 57-fold enhancement compared to the initial immunotoxin with the REDLK sequence. A biodistribution study with different routes of administration clearly showed that the subcutaneous injection of hD7-1(VL-VH)-PE24mutΔREDLK in mice resulted in the highest tumor uptake. Treatment of mice bearing prostate tumors with a combination of hD7-1(VL-VH)-PE24mutΔREDLK plus SO1861 resulted in inhibition of tumor growth and enhanced overall survival compared to the monotherapies. The endosomal entrapment of non-toxic anti-PSMA immunotoxins followed by enhanced endosomal escape by SO1861 provides new therapeutic options in the future management of prostate cancer.
免疫毒素由与特定细胞表面结构结合的抗体或抗体片段以及在胞质摄取后杀死细胞的细胞毒性结构域组成。针对多种肿瘤实体的基于外毒素A（PE）的免疫毒素已成功进入临床。PE具有一个类似KDEL的基序（REDLK），该基序使毒素能够从分拣内体通过KDEL受体途径转运到内质网（ER），并从那里转运到胞质溶胶中。在那里，它将真核生物延伸因子2进行ADP核糖基化，导致核糖体抑制并最终引发细胞凋亡。免疫毒素的一个主要问题是它们会被溶酶体降解，这使得诱导细胞死亡最终所需的免疫毒素分子数量要多得多。由此产生的剂量限制和显著增加的副作用需要新的策略来实现改善的胞质摄取。在这里，我们构建了一种免疫毒素，它由靶向前列腺特异性膜抗原（PSMA）的人源化单链可变片段（scFv）和去免疫的PE变体PE24mut组成。这种免疫毒素hD7-1（VL-VH）-PE24mut在表达PSMA的前列腺癌细胞中表现出高特异性细胞毒性。我们删除了REDLK序列以防止转运到内质网并实现内体截留。这种免疫毒素hD7-1（VL-VH）-PE24mutΔREDLK的细胞毒性大大降低。为了恢复活性，我们添加了内体逃逸增强剂SO1861，并观察到细胞毒性增强了高达190,000倍，与带有REDLK序列的初始免疫毒素相比提高了57倍。一项不同给药途径的生物分布研究清楚地表明，在小鼠中皮下注射hD7-1（VL-VH）-PE24mutΔREDLK导致肿瘤摄取最高。与单一疗法相比，用hD7-1（VL-VH）-PE24mutΔREDLK加SO1861联合治疗携带前列腺肿瘤的小鼠导致肿瘤生长受到抑制并提高了总体生存率。无毒抗PSMA免疫毒素的内体截留，随后通过SO1861增强内体逃逸，为前列腺癌的未来治疗提供了新的治疗选择。
