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.

Versatile online SPE-HPLC method for the analysis of Irinotecan and its clinically relevant metabolites in biomaterials.

Monitoring levels of Irinotecan and its metabolites during cancer therapy could help link broad interpatient variations in antitumor activity and toxicity to the patient's metabolic status. We have developed and validated a versatile and highly sensitive method for the simultaneous determination of Irinotecan and its clinically relevant metabolites 7-ethyl-10-hydroxy-camptothecin (SN-38) and SN-38 glucuronide. Sample clean-up involves precipitation by acetone/methanol/0.5 M trichloroacetic acid at 4:4:2 v/v followed by extraction of the metabolites on an SPE column by 20% methanol in 25 mM KH2 PO4 pH 2.9. Online transfer to an analytical µBondapak C18 column, elution with 24% acetonitrile (ACN) in 0.1 M KH2 PO4 pH 2.9 and fluorescence detection with excitation at 375 nm and emission at 430 nm for SN-38 glucuronide and Irinotecan or 540 nm for SN-38 results in high sensitivity (1-2 pg) and short (∼10 min) run times. The method was used to determine the degree of SN-38 glucuronidation in mice after Irinotecan administration and in cultured cancer cells exposed to SN-38. The method may be used to better understand Irinotecan metabolism, personalize therapy, and develop Irinotecan-based tumor targeting therapies.

A humanized immunoenzyme with enhanced activity for glucuronide prodrug activation in the tumor microenvironment.

Antibody-directed enzyme prodrug therapy (ADEPT) utilizing ß-glucuronidase is a promising method to enhance the therapeutic index of cancer chemotherapy. In this approach, an immunoenzyme (antibody-ß-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 ß-glucuronidase (hßG) 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 hßG, and more effectively killed antigen-positive cancer cells exposed to an anticancer glucuronide prodrug as compared to an analogous hßG immunoenzyme. Treatment of tumor-bearing mice with hcc49-S2 followed by prodrug significantly delayed tumor growth as compared to hcc49-hßG. 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.

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 beta-glucuronidase (EJ/mbetaG cells) were used to selectively hydrolyze SN-38G to SN-38. Parental EJ and EJ/mbetaG cells displayed similar in vitro and in vivo growth rates and sensitivities to CPT-11 and SN-38. By contrast, EJ/mbetaG 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/mbetaG subcutaneous tumors. Importantly, systemic administration of CPT-11, which itself is not a substrate for beta-glucuronidase, dramatically delayed the growth of EJ/mbetaG 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 beta-glucuronidase activity in tumors.

Effect of pH and human serum albumin on the cytotoxicity of a glucuronide prodrug of 9-aminocamptothecin.

PURPOSE: 9-aminocamptothecin glucuronide (9ACG) is a prodrug of 9-aminocamptothecin (9AC) that displays potent antitumor activity against human tumor xenografts in nude mice. Camptothecins exist in a pH dependent equilibrium between active lactone and inactive carboxy forms that can be altered by binding to human serum albumin (HSA). Here we investigated the influence of pH and HSA on the lactone-carboxy equilibrium, HSA binding, and cytotoxicity of 9ACG. METHODS: Microfiltration and HPLC were used to measure the influence of pH on lactone to carboxy conversion and HSA binding of 9ACG as compared to other camptothecins. In vitro cytotoxicity of drugs was determined against EJ human bladder carcinoma cells and CL1-5 human lung cancer cells. RESULTS: The rate of lactone to carboxy conversion was similar for 9ACG and 9AC. Decreasing the pH from 7.6 to 6.0 increased the equilibrium levels of the lactone forms of the drugs from 20 to almost 95% of total drug. HSA moderately diminished the amount of free 9ACG lactone but did not change the ratio of 9ACG lactone to 9ACG carboxy. Consistent with the effect of pH on lactone levels, lowering the pH of EJ human bladder carcinoma cells from 7.6 to 6.8 decreased the IC(50) of 9ACG from 480 to 98 nM and 9AC from 33 to 12 nM. Activation of 9ACG by human beta-glucuronidase anchored on the surface of EJ cells further decreased its IC(50) value to 26 nM. Although HSA significantly decreased the cytotoxicity of 9AC and 9ACG, activation of 9ACG at cancer cells with an antibody-beta-glucuronidase immunoconjugate produced greater cytotoxicity than 9AC. CONCLUSIONS: Acidification and targeted delivery of beta-glucuronidase can enhance 9ACG cytotoxicity even in the presence of HSA.

Mast cells mediate malignant pleural effusion formation.

Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1ß, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable.

Stability of the new prodrug 9-aminocamptothecin glucuronide (9ACG) in the presence of human serum albumin.

9-aminocamptothecin glucuronide (9ACG) is a new water-soluble prodrug of 9-aminocamptothecin (9AC) that is a substrate for beta-glucuronidase and displays potent antitumor activity against human tumor xenografts. The lactone ring of camptothecins (CPTs) is required for antitumor activity but spontaneously opens under physiological conditions to an inactive carboxy form. The carboxy form of many CPTs, including 9AC, preferentially binds to human serum albumin (HSA), which further reduces the equilibrium amount of active lactone and greatly decreases antitumor efficacy. In this study, we examined the hypothesis that the unique structure of 9ACG might alter prodrug interaction with HSA and increase 9ACG lactone stability as compared with 9AC. HPLC analysis revealed that HSA did not affect the equilibrium level of 9ACG lactone whereas both CPT lactone and 9AC lactone were greatly reduced in the presence of HSA as compared to their equilibrium levels in PBS. Similar results were found in human serum and whole blood. The lactone ring of 9ACG also opened more slowly (t(1/2)=50 min) as compared with 9AC (t(1/2)=20 min) in the presence of HSA. Both 9ACG lactone and 9ACG carboxy bound HSA with similar affinities (K(D) approximately 4.5 x 10(-5)M(-1)). Binding of 9ACG to HSA reduced prodrug toxicity to cancer cells by about 10-fold in vitro. Injection of HSA into nude mice prolonged the half-life of 9ACG by about 3-fold, indicating that albumin-bound 9ACG lactone may act as a depot of active prodrug in vivo. Our results suggests that in contrast to CPT and 9AC, HSA does not appear to adversely affect 9ACG and may enhance the selective antitumor activity of 9ACG in tumors that contain beta-glucuronidase.

The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

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 ß-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 ß-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.

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 beta-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 beta-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.

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 Ni(2+)-nitrilotriacetic acid affinity chromatography. Highly pure, active, glycosylated recombinant proteins, including human beta-glucuronidase, mouse beta-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.