Enhancing endosomal escape for an improved cytotoxicity of anti-PSMA immunotoxins against prostate cancer

• Applicant: Professor Dr. Philipp Wolf
• Subject Area: Reproductive Medicine, Urology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 407307550

• Final Report Year: 2023

Final Report Abstract

In recent decades, diagnosis of prostate cancer has improved and treatment of early tumor stages has advanced, e.g. by robot-assisted surgery. Despite new combinatorial therapies, however, this tumor entity remains a deadly disease in advanced, metastatic stages. In the increasing context of personalized cancer therapy, targeted approaches come more and more into focus including the use of immunotoxins. 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. Pseudomonas 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), where it is transported into the cytosol and ADP-ribosylates the eukaryotic elongation factor 2 resulting in ribosome inhibition and apoptosis. One major problem of immunotoxins is their lysosomal degradation causing the need of 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. The aims of the project were to enhance the cytotoxicity of immunotoxins against prostate cancer by endosomal escape. For this, 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, called 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, called hD7-1(VL-VH)- PE24mut REDLK, 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 wild-type immunotoxin with the REDLK sequence. In biodistribution studies with different application forms, subcutaneous injection of hD7-1(VL-VH)-PE24mut REDLK in mice resulted in highest tumor uptake. Treatment of mice bearing prostate tumors with a combination of hD7-1(VL-VH)- PE24mut REDLK and SO1861 resulted in enhanced overall survival and inhibition of tumor growth. With the same strategy, we were able to enhance the cytotoxicity of the targeted toxin EGF-PE24mut REDLK, which binds to the epidermal growth factor receptor (EGFR) on prostate cancer cells. By addition of SO1861 an up to 6,966-fold enhanced cytotoxicity was reached, which was 16- to 300-fold enhanced compared to the targeted toxin with the REDLK motif, called EGF-PE24mut. The endosomal entrapment of non-toxic immunotoxins or targeted toxins followed by enhanced endosomal escape provides new therapeutic options in the management of prostate cancer and could lead to enhanced antitumor activity and reduced off-tumor side effects.

Publications

• Endosomal Entrapment und Enhanced Endosomal Escape von Pseudomonas Exotoxin A basierten Immuntoxinen als neue Strategie bei der Behandlung des fortgeschrittenen Prostatakarzinoms. MD Thesis
  Huber N.
  
• Pseudomonas Exotoxin A Based Toxins Targeting Epidermal Growth Factor Receptor for the Treatment of Prostate Cancer. Toxins, 12(12), 753.
  Fischer, Alexandra; Wolf, Isis; Fuchs, Hendrik; Masilamani, Anie Priscilla & Wolf, Philipp
  
• EGF-basierte Targeted Toxine zur Behandlung des Prostatakarzinoms. Online Presentation, AuF-Symposium, Deutsche Gesellschaft für Urologie, Berlin, 2021.
  Fischer A., Wolf I., Fuchs H., Masilamani A.P. & Wolf P.
  
• Targeted Toxins for the Treatment of Prostate Cancer. Biomedicines, 9(8), 986.
  Wolf, Philipp
  
• Enhanced cytotoxicity of a Pseudomonas Exotoxin A based immunotoxin against prostate cancer by addition of the endosomal escape enhancer SO1861. Frontiers in Pharmacology, 14.
  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
  
• Synergistic Cytotoxicity of a Toxin Targeting the Epidermal Growth Factor Receptor and the Glycosylated Triterpenoid SO1861 in Prostate Cancer. Journal of Cancer, 14(16), 3039-3049.
  Fischer, Alexandra; Masilamani, Anie Priscilla; Schultze-Seemann, Susanne; Wolf, Isis; Gratzke, Christian; Fuchs, Hendrik & Wolf, Philipp