A recombinant PSMA-specific single-chain immunotoxin has potent and selective toxicity against prostate cancer cells

Abstract

Prostate cancer is the most commonly diagnosed form of cancer and the second leading cancer-related death among men in the Western civilization. Since no effective therapy exists for this tumor after progression beyond resectable boundaries, there is an urgent need for new treatment strategies. Prostate specific membrane antigen (PSMA) represents an excellent target on prostate cancer cells, and therefore specific immunotherapy may be a novel therapeutic option for the management of this tumor. We constructed a fully recombinant immunotoxin (A5-PE40) from a single-chain antibody fragment (scFv) against cell-adherent PSMA and a truncated form of Pseudomonas exotoxin A (PE40) lacking its natural binding domain Ia. The scFv A5 was obtained from a mAb elicited with native PSMA by phage display technology and direct selection on cells carrying the antigen. The bacterially expressed and purified immunotoxin A5-PE40 specifically binds to PSMA-positive prostate cancer cells and induces a 50% reduction of viability (IC50) at a concentration of 20 pM, while PSMA-negative cells remain unaffected. Due to its high and specific toxicity this recombinant immunotoxin is a promising candidate for therapeutic applications in patients with prostate cancer.

Fig. 1

Construction scheme for the recombinant immunotoxin A5-PE40. His ₆, hexa-histidine-tag; c-myc, c-myc-tag; V _H and V _L, variable regions of the light and heavy chains of the scFv A5; pel B, leader sequence for periplasmatic protein expression; PE40, truncated ETA fragment consisting of domains II, Ib and III (aa 253–613) but lacking the receptor binding domain Ia

Fig. 2

Binding of A5 and A5-PE40 to PSMA-negative DU 145 cells (a, c) and PSMA-positive LNCaP cells (b, d). Cells were stained with A5 or A5-PE40, mouse-anti-c-myc mAb, and goat anti-mouse Ig-RPE. Histograms represent logarithms of PE-fluorescence on flow cytometer. Negative control with secondary antibody alone

Fig. 3

Binding of A5 (a) and A5-PE40 (b) to PSMA-negative BOSC 23 cells (open circle) and PSMA-transfected BOSC 23 cells (closed circle) at different concentrations. Mean fluorescence intensity values (MFI) were considered after subtraction of the background staining with secondary antibody alone

Fig. 4

Western blot of A5 and A5-PE40 on PSMA: purified PSMA was subjected to SDS-PAGE under reducing conditions and blotted. The blot was incubated with A5 (line 1) or A5-PE40 (line 2), and peroxidase-coupled anti-c-myc mAb, and then developed with 3,3′diamino-benzidine. Line M contains a pre-stained molecular weight marker

Fig. 5

Cytotoxic effects of A5-PE40 after incubation with LNCaP cells for 6, 12, 24 and 48 h. A5-PE40 concentrations were 49 pM (black), 391 pM (grey) and 3,125 pM (white)

Fig. 6

Effects of treatment of PSMA-positive LNCaP cells and PSMA-negative lines DU 145, PC-3, MCF7 and HCT 15. Cells were incubated for 48 h with A5-PE40 at concentrations ranging from 6 to 25,000 pM. Cell growth was measured in WST assays as described. The results are expressed as the percentage of untreated cells. Data represent mean values of three independent determinations each carried out in triplicates. SD are indicated by the error bars

Fig. 7

Inhibition of the cytotoxic effects of A5-PE40 against LNCaP cells by an excess of purified parental 3/A12 antibody. Cells were incubated with A5-PE40 at different concentrations without (black) and with addition of 3A/12 at 300 nM (grey)