Kai-Chuan Chen

Antiangiogenesis Targeting Tumor Microenvironment Synergizes Glucuronide Prodrug Antitumor Activity

Purpose: This study is aimed at investigating the in vivo antitumor activity of a novel cell-impermeable glucuronide prodrug, 9-aminocamptothecin glucuronide (9ACG), and elucidating the synergistically antitumor effects of antiangiogenesis therapy by targeting the tumor microenvironment. Experimental Design: We analyzed the antitumor effects of 9ACG alone or combined with antiangiogenic monoclonal antibody DC101 on human tumor xenografts by measuring tumor growth and mouse survival in BALB/c nu/nu nude and NOD/SCID mice. The drug delivery, immune response, and angiogenesis status in treated tumors were assessed by high performance liquid chromatography, immunohistochemistry, and immunofluorescence assays. Results: We developed a nontoxic and cell-impermeable glucuronide prodrug, 9ACG, which can only be activated by extracellular β-glucuronidase to become severely toxic. 9ACG possesses potent antitumor activity against human tumor xenografts in BALB/c nu/nu nude mice but not for tumors implanted in NOD/SCID mice deficient in macrophages and neutrophils, suggesting that these cells play an important role in activating 9ACG in the tumor microenvironment. Most importantly, antiangiogenic monoclonal antibody DC101 potentiated single-dose 9ACG antitumor activity and prolonged survival of mice bearing resistant human colon tumor xenografts by providing strong β-glucuronidase activity and prodrug delivery through enhancing inflammatory cell infiltration and normalizing tumor vessels in the tumor microenvironment. We also show that inflammatory cells (neutrophils) were highly infiltrated in advanced human colon cancer tissues compared with normal counterparts. Conclusions: Our study provides in vivo evidence that 9ACG has potential for prodrug monotherapy or in combination with antiangiognesis treatment for tumors with infiltration of macrophage or neutrophil inflammatory cells.

Directed Evolution of a Lysosomal Enzyme with Enhanced Activity at Neutral pH by Mammalian Cell-Surface Display

Human beta-glucuronidase, due to low intrinsic immunogenicity in humans, is an attractive enzyme for tumor-specific prodrug activation, but its utility is hindered by low activity at physiological pH. Here we describe the development of a high-throughput screening procedure for enzymatic activity based on the stable retention of fluorescent reaction product in mammalian cells expressing properly folded glycoproteins on their surface. We utilized this procedure on error-prone PCR and saturation mutagenesis libraries to isolate beta-glucuronidase tetramers that were up to 60-fold more active (k(cat)/K(m)) at pH 7.0 and were up to an order of magnitude more effective at catalyzing the conversion of two structurally disparate glucuronide prodrugs to anticancer agents. The screening procedure described here can facilitate investigation of eukaryotic enzymes requiring posttranslational modifications for biological activity.

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β -glucuronidase

Development of nonimmunogenic and specific reporter genes to monitor gene expression in vivo is important for the optimization of gene therapy protocols. We developed a membrane-anchored form of mouse β-glucuronidase (mβG) as a reporter gene to hydrolyze a nonfluorescent glucuronide probe (fluorescein di-β-D-glucuronide, (FDGlcU) to a highly fluorescent reporter to assess the location and persistence of gene expression. A functional β-glucuronidase (βG) was stably expressed on the surface of murine CT26 colon adenocarcinoma cells where it selectively hydrolyzed the cell-impermeable FDGlcU probe. FDGlcU was also preferentially converted to fluorescent probe by (βG) on CT26 tumors. The fluorescent intensity in βG-expressing CT26 tumors was 240 times greater than the intensity in control tumors. Selective imaging of gene expression was also observed after intratumoral injection of adenoviral βG vector into carcinoma xenografts. Importantly, mβG did not induce an antibody response after hydrodynamic plasmid immunization of Balb/c mice, indicating that the reporter gene product displayed low immunogenicity. A membrane-anchored form of human βG also allowed in vivo imaging, demonstrating that human βG can be employed for imaging. This imaging system therefore, displays good selectivity with low immunogenicity and may help assess the location, magnitude and duration of gene expression in living animals and humans.

A Humanized Immunoenzyme with Enhanced Activity for Glucuronide Prodrug Activation in the Tumor Microenvironment

Antibody-directed enzyme prodrug therapy (ADEPT) utilizing β-glucuronidase is a promising method to enhance the therapeutic index of cancer chemotherapy. In this approach, an immunoenzyme (antibody-β-glucuronidase fusion protein) is employed to selectively activate anticancer glucuronide prodrugs in the tumor microenvironment. A major roadblock to the clinical translation of this therapeutic strategy, however, is the low enzymatic activity and strong immunogenicity of the current generation of immunoenzymes. To overcome this problem, we fused a humanized single-chain antibody (scFv) of mAb CC49 to S2, a human β-glucuronidase (hβG) variant that displays enhanced catalytic activity for prodrug hydrolysis. Here, we show that hcc49-S2 displayed 100-fold greater binding avidity than hcc49 scFv, possessed greater enzymatic activity than wild-type hβG, and more effectively killed antigen-positive cancer cells exposed to an anticancer glucuronide prodrug as compared to an analogous hβG immunoenzyme. Treatment of tumor-bearing mice with hcc49-S2 followed by prodrug significantly delayed tumor growth as compared to hcc49-hβG. Our study shows that hcc49-S2 is a promising targeted enzyme for cancer treatment and demonstrates that enhancement of human enzyme catalytic activity is a powerful approach to improve immunoenzyme efficacy.

Selective cancer therapy by extracellular activation of a highly potent glycosidic duocarmycin analogue.

Conventional cancer chemotherapy is limited by systemic toxicity and poor selectivity. Tumor-selective activation of glucuronide prodrugs by beta-glucuronidase in the tumor microenvironment in a monotherapeutic approach is one promising way to increase cancer selectivity. Here we examined the cellular requirement for enzymatic activation as well as the in vivo toxicity and antitumor activity of a glucuronide prodrug of a potent duocarmycin analogue that is active at low picomolar concentrations. Prodrug activation by intracellular and extracellular beta-glucuronidase was investigated by measuring prodrug 2 cytotoxicity against human cancer cell lines that displayed different endogenous levels of beta-glucuronidase, as well as against beta-glucuronidase-deficient fibroblasts and newly established beta-glucuronidase knockdown cancer lines. In all cases, glucuronide prodrug 2 was 1000-5000 times less cytotoxic than the parent duocarmycin analogue regardless of intracellular levels of beta-glucuronidase. By contrast, cancer cells that displayed tethered beta-glucuronidase on their plasma membrane were 80-fold more sensitive to glucuronide prodrug 2, demonstrating that prodrug activation depended primarily on extracellular rather than intracellular beta-glucuronidase activity. Glucuronide prodrug 2 (2.5 mg/kg) displayed greater antitumor activity and less systemic toxicity in vivo than the clinically used drug carboplatin (50 mg/kg) to mice bearing human lung cancer xenografts. Intratumoral injection of an adenoviral vector expressing membrane-tethered beta-glucuronidase dramatically enhanced the in vivo antitumor activity of prodrug 2. Our data provide evidence that increasing extracellular beta-glucuronidase activity in the tumor microenvironment can boost the therapeutic index of a highly potent glucuronide prodrug.

A simple method for the production of recombinant proteins from mammalian cells

Expression of recombinant proteins in mammalian cells is useful for obtaining products with normal post‐translational modifications. We describe a simple and economical method for the production of milligram levels of proteins in murine fibroblasts. Retroviral or lipofectAMINE™ (Gibco Laboratories) transduction was employed to generate stable murine‐fibroblast producer cells. Confluent cultures of stable fibroblast clones were maintained for up to 1 month in 0.5% serum. Culture medium was collected every 2–3 days and polyhistidine‐tagged proteins were purified by ammonium sulphate precipitation and Ni2+‐nitrilotriacetic acid affinity chromatography. Highly pure, active, glycosylated recombinant proteins, including human β‐glucuronidase, mouse β‐glucuronidase, aminopeptidase N (CD13) and a single‐chain antibody–enzyme fusion protein, were obtained with yields of 3–6 mg/l of culture medium. Fc‐tagged proteins were also produced and purified in a single step by Protein A affinity chromatography with yields of 6–12 mg/l. The techniques described here allow simple and economical production of recombinant mammalian proteins with post‐translational modifications.

Local enzymatic hydrolysis of an endogenously generated metabolite can enhance CPT-11 anticancer efficacy.

Irinotecan (CPT-11) is a clinically important anticancer prodrug that requires enzymatic hydrolysis by carboxyesterase to generate the active metabolite SN-38. However, SN-38 is further metabolized to inactive SN-38 glucuronide (SN-38G), thus diminishing the levels of active SN-38. Although exogenously administered glucuronide drugs are being investigated for cancer therapy, it is unknown if endogenously generated camptothecin glucuronide metabolites can be used for tumor therapy. Here, we tested the hypothesis that tumor-located hydrolysis of endogenously generated SN-38G can enhance the antitumor efficacy of CPT-11 therapy. EJ human bladder carcinoma cells expressing membrane-tethered β-glucuronidase (EJ/mβG cells) were used to selectively hydrolyze SN-38G to SN-38. Parental EJ and EJ/mβG cells displayed similar in vitro and in vivo growth rates and sensitivities to CPT-11 and SN-38. By contrast, EJ/mβG cells were more than 30 times more sensitive than EJ cells to SN-38G, showing that SN-38 could be generated from SN-38G in vitro. Systemic administration of CPT-11 resulted in tumor-located hydrolysis of SN-38G and accumulation of SN-38 in EJ/mβG subcutaneous tumors. Importantly, systemic administration of CPT-11, which itself is not a substrate for β-glucuronidase, dramatically delayed the growth of EJ/mβG xenografts without increased systemic toxicity. Thus, the anticancer activity of CPT-11 can be significantly enhanced by converting the relatively high levels of endogenously generated SN-38G to SN-38 in tumors. The high concentrations of SN-38G found in the serum of patients treated with CPT-11 suggest that clinical response to CPT-11 may be improved by elevating β-glucuronidase activity in tumors. [Mol Cancer Ther 2009;8(4):940–6]

ECSTASY, an adjustable membrane-tethered/soluble protein expression system for the directed evolution of mammalian proteins

We describe an adjustable membrane-tethered/soluble protein screening methodology termed ECSTASY (enzyme cleavable surface tethered all-purpose screening system) which combines the power of high-throughput fluorescence-activated cell sorting of membrane-tethered proteins with the flexibility of soluble assays for isolation of improved mammalian recombinant proteins. In this approach, retroviral transduction is employed to stably tether a library of protein variants on the surface of mammalian cells via a glycosyl phosphatidylinositol anchor. High-throughput fluorescence-activated cell sorting is used to array cells expressing properly folded and/or active protein variants on their surface into microtiter culture plates. After culture to expand individual clones, treatment of cells with phosphatidylinositol-phospholipase C releases soluble protein variants for multiplex measurement of protein concentration, activity and/or function. We utilized ECSTASY to rapidly generate human β-glucuronidase variants for cancer therapy by antibody-directed enzyme prodrug therapy with up to 30-fold greater potency to catalyze the hydrolysis of the clinically relevant camptothecin anti-cancer prodrug as compared with wild-type human β-glucuronidase. A variety of recombinant proteins could be adjustably displayed on fibroblasts, suggesting that ECSTASY represents a general, simple and versatile methodology for high-throughput screening to accelerate sequence activity-based evolution of mammalian proteins.

Synthesis and Antitumor Properties of BQC-Glucuronide, a Camptothecin Prodrug for Selective Tumor Activation

Major limitations of camptothecin anticancer drugs (toxicity, nonselectivity, water insolubility, inactivation by human serum albumin) may be improved by creating glucuronide prodrugs that rely on beta-glucuronidase for their activation. We found that the camptothecin derivative 5,6-dihydro-4H-benzo[de]quinoline-camptothecin (BQC) displays greater cytotoxicity against cancer cells than the clinically used camptothecin derivatives SN-38 and topotecan even in the presence of human serum albumin. We synthesized the prodrug BQC-glucuronide (BQC-G), which was 4000 times more water soluble and 20-40 times less cytotoxic than BQC. Importantly, even in the presence of human serum albumin, BQC-G was efficiently hydrolyzed by beta-glucuronidase and produced greater cytotoxicity (IC50 = 13 nM) than camptothecin, 9-aminocamptothecin, SN-38, or topotecan (IC50 > 3000, 1370, 48, and 28 nM, respectively). BQC-G treatment of mice bearing human colon cancer xenografts with naturally or artificially elevated beta-glucuronidase activity produced significant antitumor activity, showing that BQC-G is a potent prodrug suitable for selective intratumoral drug activation.

PET Imaging of b-Glucuronidase Activity by an Activity-Based 124 I-Trapping Probe for the Personalized Glucuronide Prodrug Targeted Therapy

Beta-glucuronidase (βG) is a potential biomarker for cancer diagnosis and prodrug therapy. The ability to image βG activity in patients would assist in personalized glucuronide prodrug cancer therapy. However, whole-body imaging of βG activity for medical usage is not yet available. Here, we developed a radioactive βG activity–based trapping probe for positron emission tomography (PET). We generated a 124I-tyramine–conjugated difluoromethylphenol beta-glucuronide probe (TrapG) to form 124I-TrapG that could be selectively activated by βG for subsequent attachment of 124I-tyramine to nucleophilic moieties near βG-expressing sites. We estimated the specificity of a fluorescent FITC-TrapG, the cytotoxicity of tyramine-TrapG, and the serum half-life of 124I-TrapG. βG targeting of 124I-TrapG in vivo was examined by micro-PET. The biodistribution of 131I-TrapG was investigated in different organs. Finally, we imaged the endogenous βG activity and assessed its correlation with therapeutic efficacy of 9-aminocamptothecin glucuronide (9ACG) prodrug in native tumors. FITC-TrapG showed specific trapping at βG-expressing CT26 (CT26/mβG) cells but not in CT26 cells. The native TrapG probe possessed low cytotoxicity. 124I-TrapG preferentially accumulated in CT26/mβG but not CT26 cells. Meanwhile, micro-PET and whole-body autoradiography results demonstrated that 124I-TrapG signals in CT26/mβG tumors were 141.4-fold greater than in CT26 tumors. Importantly, Colo205 xenografts in nude mice that express elevated endogenous βG can be monitored by using infrared glucuronide trapping probes (NIR-TrapG) and suppressed by 9ACG prodrug treatment. 124I-TrapG exhibited low cytotoxicity allowing long-term monitoring of βG activity in vivo to aid in the optimization of prodrug targeted therapy. Mol Cancer Ther; 13(12); 2852–63. ©2014 AACR.