Gene-mediated enzyme prodrug therapy (GDEPT) seeks to increase the therapeutic index of anti-neoplastic agents by promoting selective activation of relatively nontoxic drug derivatives at sites of specific enzyme expression. Glucuronide prodrugs are attractive for GDEPT due to their low toxicity, bystander effect in the interstitial tumor space and the large range of possible glucuronide drug targets. In this study, we expressed human, murine and Esherichia coli beta-glucuronidase on tumor cells and examined their in vitro and in vivo efficacy for the activation of glucuronide prodrugs of 9-aminocamptothecin and p-hydroxy aniline mustard. We show that (1) fusion of beta-glucuronidase to the Ig-like C(2)-type and Ig-hinge-like domains of the B7-1 antigen followed by the B7-1 transmembrane domain anchored high levels of active murine and human beta-glucuronidase on cells, (2) strong bystander killing of tumor cells was achieved in vitro by murine beta-glucuronidase activation of prodrug, (3) potent in vivo anti-tumor activity was achieved by prodrug treatment of tumors that expressed murine beta-glucuronidase and (4) the p-hydroxy aniline prodrug was more effective in vivo than the 9-aminocamptothecin prodrug. Our results demonstrate that surface expression of murine beta-glucuronidase for activation of a glucuronide prodrug of p-hydroxy aniline mustard may be useful for more selective therapy of cancer.

β-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of β-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of β-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol-glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. β-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the fluorochrome to nucleophilic moieties located near β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified β-glucuronidase or β-glucuronidase-expressing CT26 cells (CT26/mβG) but not on bovine serum albumin or non-β-glucuronidase-expressing CT26 cells used as controls. β-glucuronidase-activated FITC-TrapG did not interfere with β-glucuronidase activity and could label bystander proteins near β-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mβG tumors, but only NIR-TrapG could image CT26/mβG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing β-glucuronidase activity in vivo.

Systemic injection of therapeutic antibodies may cause serious adverse effects due to on-target toxicity to the antigens expressed in normal tissues. To improve the targeting selectivity to the region of disease sites, we developed protease-activated pro-antibodies by masking the binding sites of antibodies with inhibitory domains that can be removed by proteases that are highly expressed at the disease sites. The latency-associated peptide (LAP), C2b or CBa of complement factor 2/B were linked, through a substrate peptide of matrix metalloproteinase-2 (MMP-2), to an anti-epidermal growth factor receptor (EGFR) antibody and an anti-tumor necrosis factor-α (TNF-α) antibody. Results showed that all the inhibitory domains could be removed by MMP-2 to restore the binding activities of the antibodies. LAP substantially reduced (53.8%) the binding activity of the anti-EGFR antibody on EGFR-expressing cells, whereas C2b and CBa were ineffective (21% and 9.3% reduction, respectively). Similarly, LAP also blocked 53.9% of the binding activity of the anti-TNF-α antibody. Finally, molecular dynamic simulation showed that the masking efficiency of LAP, C2b and CBa was 33.7%, 10.3% and −5.4%, respectively, over the binding sites of the antibodies. This strategy may aid in designing new protease-activated pro-antibodies that attain high therapeutic potency yet reduced systemic on-target toxicity.

The survival of prostate cancer (PrCa) patients is associated with the transition to hormone-independent tumor growth and metastasis. Clinically, the dysregulation of androgen action has been associated with the formation of PrCa and the outcome of androgen deprivation therapy in PrCa. CCAAT/enhancer binding protein delta (CEBPD) is a transcription factor that has been reported to act as an oncogene or tumor suppressor, depending on the extra- and intracellular environments following tumorigenesis. We found that androgen can activate CEBPD transcription by direct binding of the androgen receptor (AR) to the CEBPD promoter region. Increases of suppressor of zeste 12 (SUZ12) and enhancer of zeste homolog 2 (EZH2) attenuated the androgen-induced transcription of CEBPD. Importantly, the increases in E2F1, SUZ12 and EZH2 as well as the inactivation of CEBPD were associated with the clinicopathological variables and survival of PrCa patients. We revealed that caspase 8 (CASP8), an apoptotic initiator, is responsive to CEBPD induction. Reporter and in vivo DNA-binding assays revealed that CEBPD directly binds to and activates CASP8 reporter activity. A prodrug system was developed for therapeutic application in AR-independent or androgen-insensitive PrCa to avoid the epigenetic effects on the suppression of CEBPD expression. Our results showed that the combination of a perforin (PF)-CEBPD prodrug (which increases the level of procaspase-8) and a PF-granzyme B prodrug (which activates CASP8 and caspase 3 (CASP3)) showed an additive effect in triggering the apoptotic pathway and enhancing apoptosis in PrCa cells.

During rheumatoid arthritis (RA) treatment, long-term injection of antitumor necrosis factor α antibodies (anti-TNFα Abs) may induce on-target toxicities, including severe infections (tuberculosis [TB] or septic arthritis) and malignancy. Here, we used an immunoglobulin G1 (IgG1) hinge as an Ab lock to cover the TNFα-binding site of Infliximab by linking it with matrix metalloproteinase (MMP) -2/9 substrate to generate pro-Infliximab that can be specifically activated in the RA region to enhance the selectivity and safety of treatment. The Ab lock significantly inhibits the TNFα binding and reduces the anti-idiotypic (anti-Id) Ab binding to pro-Infliximab by 395-fold, 108-fold compared with Infliximab, respectively, and MMP-2/9 can completely restore the TNFα neutralizing ability of pro-Infliximab to block TNFα downstream signaling. Pro-Infliximab was only selectively activated in the disease site (mouse paws) and presented similar pharmacokinetics (PKs) and bio-distribution to Infliximab. Furthermore, pro-Infliximab not only provided equivalent therapeutic efficacy to Infliximab but also maintained mouse immunity against Listeria infection in the RA mouse model, leading to a significantly higher survival rate (71%) than that of the Infliximab treatment group (0%). The high-selectivity pro-Infliximab maintains host immunity and keeps the original therapeutic efficiency, providing a novel strategy for RA therapy.