A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy

Abstract

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC₅₀ value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Keywords: PE; PSMA (prostate-specific membrane antigen); immunotoxins; prostate carcinoma; sdAbs (single-domain antibodies).

Figure 1

Characterization of the immunotoxin JVM-PE24X7. (A) An illustrated structure of JVM-PE24X7 constructed by fusing the sdAb (dark blue) with the toxin PE24X7 (mint green). (B) SDS-PAGE and (C) Western blot analysis of purified JVM-PE24X7 (lane 1), PE24X7 (lane 2) and JVM (lane 3). The proteins were loaded onto a 12.5% polyacrylamide gel with protein weight standards and detected by Coomassie brilliant blue staining or Western blot analysis using a mouse anti-His monoclonal antibody.

Figure 2

Expression levels of PSMA in a variety of PCa cells. (A) LNCaP, C4-2B, 22Rv1 and PC-3 cells were fixed on slides and sequentially incubated with an anti-PSMA antibody and a FITC-labeled secondary antibody (green) DiI was used for staining of cell membrane (red). DAPI is used for nuclear staining (blue) (magnification 2400×). Scale bar: 21.5 μm. (B) Whole-cell lysates from LNCaP, C4-2B, 22Rv1 and PC-3 cells were analyzed by Western blotting.

Figure 3

JVM-PE24X7 binding assays towards the PSMA receptor by ELISA. (A) Protein-based ELISA in which the human PSMA protein was coated on plates to determine the binding affinity of JVM-PE24X7 for the PSMA protein. (B&C) Cell-based ELISA in which PSMA-positive LNCaP cells or PSMA-negative PC3 cells were seeded onto the plate to determine the binding of JVM-PE24X7 to the PSMA receptor in the natural environment. (D) The K_D data were calculated using GraphPad Prism.

Figure 4

Fluorescence microscopy and flow cytometry analyses of JVM-PE24X7 internalization and accumulation in LNCaP and PC-3 cells. (A) LNCaP cells were treated with 0.5 μM rhodamine-labeled JVM-PE24X7 for 10 min, 30 min, 60 min, or 180 min. (B) LNCaP cells were treated with 0, 0.01, 0.1 or 0.5 μM rhodamine-labeled JVM-PE24X7 for 180 min. (C) PC-3 cells were treated with 0.5 μM rhodamine-labeled JVM-PE24X7 for 180 min. After washing with phosphate-buffered saline (PBS), cells were fixed and incubated with DAPI. The cells were observed under a fluorescence microscope (magnification 400×). Scale bar: 10 μm. (D) LNCaP cells were treated with 0.01, 0.1, or 0.5 μM FITC-labeled JVM-PE24X7 for 180 min. (E) LNCaP cells were treated with 0.5 μM FITC-labeled JVM-PE24X7 for 10, 30, 60 or 180 min. (F) PC-3 cells were treated with 0.5 μM FITC-labeled JVM-PE24X7 for 180 or 300 min. After washing with PBS, the cells were collected and analyzed by flow cytometry. (G–I) The fluorescence data in D–F were all quantified. Error bars, means ± SEM (n = 3). Statistical significance was calculated by Student’s t-test: * p < 0.05; **** p < 0.0001. ns = no significance.

Figure 5

PSMA-positive LNCaP, C4-2B, and 22Rv1 cells and PSMA-negative PC-3 cells were incubated with (A) JVM-PE24X7 or (B) PE24X7 for 72 h or (C) preincubated with 1.0 μM JVM for 1 h before JVM-PE24X7 was added. Then, the cell viabilities were determined by MTT assay. (D) EC₅₀ data were calculated with GraphPad Prism. Error bars, means ± SEM (n = 5).

Figure 6

Representative flow cytometry plots of PSMA-positive C4-2B cells and PSMA-negative PC-3 cells treated with JVM-PE24X7, JVM or PE24X7. (A) C4-2B cells were treated with 100 pM JVM-PE24X7, JVM or PE24X7 for 24, 48 or 72 h. (B) PC-3 cells were treated with 100 nM JVM-PE24X7, JVM or PE24X7 for 72 h, stained with annexin V-APC/PI and analyzed for apoptosis by flow cytometry.

Figure 7

Cytotoxicity of JVM-PE24X7 visualized by using live/dead staining. PSMA-positive LNCaP, C4-2B, and 22Rv1 cells and PSMA-negative PC-3 cells were incubated with various concentrations of JVM-PE24X7 (0 M, 0.1 pM, 10 pM, 1 nM or 100 nM) and then visualized by microscopic imaging. The live and dead cells were stained with calcein-AM (green) and EthD-1 (red), respectively. Scale bar: 50 μm.

Figure 8

In vivo antitumor activity of JVM-PE24X7. Mice bearing C4-2B tumors were treated with the immunotoxin JVM-PE24X7 or PBS on days 1, 4, 7, 10 and 13. (A) Changes in tumor volume and (B) body weight in the mice were measured every other day. Arrows indicate the time points of JVM-PE24X7 injection. (C) Histological analysis of the tumor and major tissues after treatment with PBS and JVM-PE24X7 (400×). Scale bar: 100 μm. Error bars, means ± SEM (n = 5). Statistical significance was calculated by Student’s t-test: * p < 0.05; **** p < 0.0001.