An Anti‐PSMA Bivalent Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Imaging and Therapy

Abstract

Prostate specific membrane antigen (PSMA) is overexpressed on prostate tumor cells and the neovascular endothelia various solid tumors. A bivalent immunotoxin generated by fusing a fold-back single-chain diabody derived from the Fv fragments of an anti-PSMA monoclonal antibody with a truncated diphtheria toxin (DT) containing the activity and translocation domains [A-dmDT390-scfbDb(PSMA)] might be suitable for targeted therapy of tumors that overexpress PSMA. In this study, a PSMA-positive and a PSMA-negative prostate cancer cell lines were treated with immunotoxin A-dmDT390-scfbDb(PSMA) in order to study the tumor targeting specificity and therapeutic potential of the immunotoxin. The cellular uptake and selective toxicity of the immunotoxin were evident in monolayer cultures of PSMA-positive LNCaP prostate cancer cells but not in cultures of PSMA-negative PC-3 prostate cancer cells. Cellular accumulation of A-dmDT390-scfbDb(PSMA) increased with increasing incubation times and concentrations in LNCaP cells. The proportion of apoptotic LNCaP cells increased upon incubation with increasing doses of the fold-back immunotoxin. Optical imaging and MRI with the Alexa Fluor 680-labeled A-dmDT390-scfbDb(PSMA) confirmed the specific targeting and therapeutic efficacy of this immunotoxin towards PSMA-positive LNCaP solid tumor xenografts in athymic nude mice.

A bivalent recombinant immunotoxin with high potency against tumors with EGFR and EGFRvIII expression

“…[27][28][29][30] The full linear sequence of DT390-BiscFv806 was DT390-V L -(G 4 S) 3 -V H -(G 4 S) 3 -V L -(G 4 S) 3 -V H , which was confirmed by DNA sequencing. The cartoon structure of DT390-BiscFv806 was shown in Figure 1A, presenting a bivalent tandem scFv format.…”

“…Importantly, P. pastoris is capable of folding multi-domain RITs properly by forming disulfide bonds. [27][28][29][30] The expression and purification parameters of this system have been further optimized for production scale up of RITs according to the U.S. FDA GMP guidelines. An example is the anti-CD3 RIT, Resimmune (A-dmDT390-bisFv(UCHT1)), which is under Phase II clinical trials and has been produced at a raw yield of »207 mg/L and a purified yield of »144.2 mg/L after a 163-hour induction period in a single batch of 120 L bioreactor culture.…”

“…Valency, molecular size, and circulation time are 3 key factors affecting the anti-tumor efficacy of a RIT. 29,39,40 Use of biscFv to construct RIT can significantly improve its functional affinity. Under most conditions for bivalent binding, 2 measurable K D Figure 5.…”

“…Our previous studies on different formats of RITs have confirmed the higher binding affinity of bivalent RITs than that of monovalent RITs. 28,29,40 The studies with RITs targeting PSMA (A-dmDT390-scfbDb(PSMA)) or CD3 (A-dmDT390-bisFv(UCHT1)) have shown that the binding affinity and cytoxicity of the bivalent format are >2.5-fold and >10-fold higher, respectively, than that of a monovalent format. 28,29,40 We expected that the bivalent DT390-BiscFv806 would have a high binding affinity and cytotoxicity to the targeted cells.…”

“…In our previous studies, A-dmDT390-scfbDb(PSMA) was confirmed to be highly cytotoxic to PSMA-expressing LNCaP prostate cancer cells (IC 50 , 0.57 nM), but not to PSMA-negative PC-3 cells (IC 50 , >100 nM). 29 High anti-tumor efficacy of DT390-BiscFv806 against established tumor xenografts…”