In vitro and in vivo characterisation of a recombinant carboxypeptidase G2::anti-CEA scFv fusion protein.

BACKGROUND: There is considerable interest in the specific targeting of therapeutic agents to cancer cells. Of particular promise is a technique known as Antibody-Directed Enzyme Prodrug Therapy (ADEPT). In this approach an enzyme is targeted to the tumour by its conjugation to a tumour specific-antibody tumour. After allowing sufficient time for the conjugate to localise at the tumour and clear from the circulatory system, a relatively non-toxic prodrug is administered. This prodrug is converted to a highly cytotoxic drug by the action of the targeted enzyme localised at the tumour site. OBJECTIVES: To construct gene fusions between the pseudomonad carboxypeptidase G2 (CPG2) gene and DNA encoding MFE-23 (an anti-carcinoembryonic antigen (CEA) single-chain Fv (scFv) molecule), derived from a phage display library. To overexpress the resultant gene fusions in Escherichia coli, and assess the in vitro and in vivo properties of the purified fusion proteins. STUDY DESIGN: To introduce unique cloning restriction sites into the 5'-end of the CPG2 gene by site-directed mutagenesis to facilitate fusion to the 3'-end of the gene encoding MFE-23 (constructs with or without a flexible (Gly4Ser)3 linker-encoding sequence were designed). To overexpress the resultant gene fusions under transcriptional control of the lac promoter and to direct the fusion proteins produced to the periplasmic space of E. coli through translational coupling to the pelB signal peptide. RESULTS: Biologically active recombinant CPG2::MFE-23 scFv fusion proteins were produced in E. coli and shown to possess enzyme and anti-CEA activity. Affinity chromatography followed by size exclusion gel filtration yielded approximately 0.7-1.4 mg/l from shake flask culture. The fusion protein in which the enzyme and antibody moieties were joined by a linker peptide was shown to be effectively localised in nude mice bearing human colon tumour xenografts, giving favourable tumour to blood ratios. CONCLUSION: MFE-23 scFv serves as an ideal candidate for the antibody arm of a bacterially expressed fusion protein with CPG2. The biological properties of this recombinant protein suggest that it may be employed for tumour specific prodrug activation. However, further assessment of its stability and pharmokinetics is required if genetic fusion is to be considered as an alternative to chemical conjugation.

Antibody-enzyme conjugates for cancer therapy.

The use of antibody-enzyme conjugates directed at tumor-associated antigens to achieve site-specific activation of prodrugs to potent cytotoxic species, termed "antibody-directed enzyme prodrug therapy" (ADEPT), has attracted considerable interest since the concept was first described in 1987. Prodrug forms of both clinically used anticancer agents and novel cytotoxic compounds have been developed to take advantage of potential prodrug-generating technology employing a variety of enzymes with widely differing substrate specificities. A particular advantage of the ADEPT approach is that it may allow the use of extremely potent agents such as nitrogen mustards and palytoxin, which are too toxic to be readily used in conventional chemotherapy. Preliminary studies using an antibody-enzyme conjugate constructed with a bacterial enzyme and a murine monoclonal antibody not only have established the value of the ADEPT technique, but also have highlighted the potential problem of immunogenicity of proteins of nonhuman origin. This problem has been tackled in the first instance by the use of immunosuppressive agents, but long-term solutions are being investigated in the development of second-generation ADEPT systems, including the development of human antibody-human enzyme fusion proteins and catalytic antibodies. Such improvements, coupled with further refinement of the prodrug-drug element of the system and the wide variety of antibody-enzyme-drug combinations available, should mean that ADEPT-based approaches will form an important element of the search for the anticancer drugs of the future.

Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase.

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.

Virtual cofactors for an Escherichia coli nitroreductase enzyme: relevance to reductively activated prodrugs in antibody directed enzyme prodrug therapy (ADEPT).

A nitroreductase enzyme has been isolated from Escherichia coli that has the unusual property of being equally capable of using either NADH or NADPH as a cofactor for the reduction of its substrates which include menadione as well as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954). This property is shared with the mammalian enzyme, DT diaphorase. The nitroreductase can, like DT diaphorase, also use simple reduced pyridinium compounds as virtual cofactors. The intact NAD(P)H molecule is not required and the simplest quaternary (and therefore reducible) derivative of nicotinamide, 1-methylnicotinamide (reduced), is as effective as NAD(P)H in its ability to act as an electron donor for the nitroreductase. The structure-activity relationship is not identical to that of DT diaphorase and nicotinic acid riboside (reduced) is selective, being active only for the nitroreductase. Irrespective of the virtual cofactor used, the nitroreductase formed the same reduction products of CB 1954 (the 2- and 4-hydroxylamino derivatives in equal proportions). Nicotinic acid riboside (reduced), unlike NADH, was stable to metabolism by serum enzymes and had a plasma half-life of seven minutes in the mouse after an i.v. bolus administration. NADH had an unmeasurably short half-life. Nicotinic acid riboside (reduced) could also be produced in vivo by administration of nicotinic acid 5'-O-benzoyl riboside (reduced). These results demonstrate that the requirement for a cofactor need not be a limitation in the use of reductive enzymes in antibody directed enzyme prodrug therapy (ADEPT). It is proposed that the E. coli nitroreductase would be a suitable enzyme for ADEPT in combination with CB 1954 and a synthetic, enzyme-selective, virtual cofactor such as nicotinic acid riboside (reduced).

Galactosylated antibodies and antibody-enzyme conjugates in antibody-directed enzyme prodrug therapy.

Antibody directed enzyme prodrug therapy (ADEPT) has been studied as a two- and three-phase system in which an antibody to a tumor-associated antigen has been used to deliver an enzyme to tumor sites where it can convert a relatively nontoxic prodrug to a cytotoxic agent. In such a system, it is necessary to allow the enzyme activity to clear from the blood before prodrug injection to avoid toxicity caused by prodrug activation in plasma. To accelerate plasma clearance of enzyme activity, two approaches have been studied. The studies have been performed with a monoclonal anticarcinoembryonic-antigen antibody fragment A5B7-F(ab')2 conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), in LS174T xenografted mice. In the first approach, a monoclonal antibody (SB43), directed at CPG2, was used, which inactivates CPG2 in vitro and in vivo. SB43 was galactosylated so that it had sufficient time to form a complex with plasma CPG2, resulting in the inactivation and clearance of the complex from plasma via the carbohydrate-specific receptors in the liver. Injection of SB43gal 19 hours after administration of the radiolabeled conjugate reduced the percentage of injected dose per gram in blood without affecting levels in the tumor. The second approach involved galactosylation of the conjugate so that it cleared rapidly from blood via the asialoglycoprotein receptors in the liver. Localization of the radiolabeled conjugate was achieved by blocking this receptor for about 8 hours with a single injection (8 mg/mouse) of an inhibitor that binds competitively to the receptor. This allowed tumor localization of the conjugate followed by a rapid clearance of the galactosylated conjugate from blood as the inhibitor was consumed. A tumor-to-blood ratio of 45:1 was obtained at 24 hours, which increased to 100:1 at 72 hours after the conjugate injection. These accelerated clearance mechanisms have been applied in antitumor studies in ADEPT.

Tumour necrosis factor increases tumour uptake of co-administered antibody-carboxypeptidase G2 conjugate.

Increased tumour uptake of antibodies and antibody-drug conjugates has been demonstrated following pretreatment of animals with recombinant human tumour necrosis factor-alpha (rTNF-alpha) and interleukin 2 immunoconjugates. The experiments reported here were performed to determine whether improved tumour localisation of antibody-carboxypeptidase G2 conjugates could be achieved, with a view to applying this technology to antibody-directed enzyme-prodrug therapy (ADEPT). B6CF1 mice bearing the Ly-2.1+ murine thymoma E3 were simultaneously injected with 2.0 micrograms rTNF-alpha and 3.5 micrograms (74kBq) 125I-labelled murine anti-Ly-2.1-CPG2 conjugate. Mice in control groups received phosphate buffered saline in place of rTNF-alpha. The conjugate corresponded in molecular weight to a mixture of 1:1 and 2:1 (CPG2:IgG) conjugate and retained its antigen binding specificity and enzymic activity in vitro. A significant increase in tumour uptake was observed 24 h after administration when rTNF-alpha-treated animals were compared to controls (28.1 +/- 9.7%/g and 11.6 +/- 2.3%/g, respectively). Other tissues, most notably gut, skin and kidney also showed an increased localisation of conjugate. By 48 h, analysis of tissue:blood ratios demonstrated that although tumour:blood ratios were significantly higher in rTNF-alpha-treated animals (P < 0.05), all the other tissue:blood ratios were not significantly different between the two groups.

Optimisation of small-scale coupling of A5B7 monoclonal antibody to carboxypeptidase G2.

Conjugates of F(ab')2 fragment of the monoclonal antibody A5B7 coupled to carboxypeptidase G2 (CPG2) have been produced using the heterobifunctional reagents 2-mercapto-[S-acetyl]acetic acid, N-hydroxysuccinimide ester (SATA) and m-maleimidobenzoyl-N-hydroxysuccinimide ester (SMPB). The effect of various levels of modifying reagent on enzyme activity and antigen binding activity were determined, and it was shown that whilst CPG2 is relatively sensitive to modification, insertion of three maleimide groups per CPG2 resulted in the loss of 30% of enzyme activity; A5B7 F(ab')2 was insensitive to modification, little or no activity being lost. The coupling efficiency of the reaction was shown to be fairly constant over a wide range of substitution levels. There was thus no advantage to be gained in using high substitution levels, which may result in loss of enzyme activity. The formation of undesired high molecular weight aggregates could be controlled by adjustment of the protein concentration during the final coupling step.

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT).

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the "classical nitroreductase" of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated from Walker cells. The reduction of CB1954 by the E. coli enzyme results in the generation of cytotoxic species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active enzyme against CB1954 (kcat = 360 min-1) than Walker DT diaphorase (kcat = 4 min-1) and also has a lower Km for NADH (6 vs 75 microM).

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--II. A comparison of an Escherichia coli nitroreductase and Walker DT diaphorase.

A nitroreductase enzyme that has been isolated from Escherichia coli B is capable of bioactivating CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] to a cytotoxic agent, a property shared with the mammalian enzyme Walker DT diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2] as isolated from Walker cells. In contrast to Walker DT diaphorase, which can only reduce the 4-nitro group of CB1954, the E. coli nitroreductase can reduce either (but not both) nitro groups of CB1954 to the corresponding hydroxylamino species. The two hydroxylamino species are formed in equal proportions and at the same rates. CB1954 is reduced much more rapidly by the E. coli nitroreductase than by Walker DT diaphorase. If the reduction of CB1954 was carried out in the presence of V79 cells (which are insensitive to CB1954) a large cytotoxic effect was evident. This cytotoxicity was only observed under conditions in which the E. coli nitroreductase or Walker DT diaphorase reduced the drug. It is proposed that E. coli B nitroreductase would be a suitable enzyme for antibody-directed enzyme prodrug therapy (ADEPT) in combination with CB1954.

Human immune response to monoclonal antibody-enzyme conjugates in ADEPT pilot clinical trial.

The human immune response to monoclonal antibody-enzyme conjugates has been studied in patients included in the pilot clinical trial of ADEPT. Each patient received murine monoclonal anti-CEA antibody fragments (A5B7-F(ab')2, conjugated to bacterial enzyme, carboxypeptidase G2 (CPG2) followed by a galactosylated monoclonal anti-CPG2 antibody (SB43), 36-48 h after the conjugate. Some patients were also given a dose of 131I-labeled conjugate (4-8 mg, 7-15 mCi) for blood clearance and gamma camera image studies. All patients studied developed human antimouse antibodies (HAMA) and anti-CPG2 antibodies within 10 d after a single course of treatment with the conjugate. In most cases, IgM response was detected at 7 d after the conjugate followed by the IgG response 14 d later. In one patient, HAMA and anti-CPG2 antibodies of the IgG type could still be detected at 10 mo after treatment. Anti-CPG2 antibodies in serum of one patient were found to inhibit CPG2 activity in vitro. Generation of neutralizing antibodies limits the use of repeat cycles of ADEPT in patients. Use of immunosuppressive agents may allow a useful time window for several ADEPT cycle treatments by delaying the appearance of HAMA and anti-CPG2 antibodies. Patients given cyclosporin A before and during ADEPT are currently being studied for HAMA and anti-CPG2 response.

Making bacterial extracts suitable for chromatography.

Despite the exotic appearance and odor of bacterial extracts, it should be remembered that they are predominantly water with only a few percent w/v solids. The twin objectives of preparation prior to chromatography are, therefore, clarification and concentration. It is not the purpose of this chapter to describe cell disruption techniques, but it is worth noting that the method used, whether physical, mechanical, or chemical, can affect the properties of the extract and subsequent clarification, for instance, in relation to viscosity on membrane filtration. The concentration stage can incorporate a primary purification step, for example, in the form of a protein precipitation/resuspension or a batch binding of protein, normally to an ionexchange matrix. The more recent development of fast-flow chromatographic matrices does, however, allow larger process volumes to be applied directly to columns, for example, 200-L bed vol columns can operate at loading flow rates of up to 1000 L/h. The general techniques used for clarification and concentration are summarized in Table 1, and practical examples are described as they relate to pilot and large-scale processing rather than laboratory techniques. Table 1 General Techniques Used for Clarification and Concentration Physical Chemical Clarification Centrifugation Two phase Filtration (P)H Polymers Concentration Filtration Precipitation Batch binding.

Crystallization and preliminary crystallographic analysis of carboxypeptidase G2 from Pseudomonas sp. strain RS-16.

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been crystallized from polyethylene glycol (average Mr 4000) by vapour diffusion. The crystal symmetry is monoclinic C2, with unit cell dimensions a = 206 A, b = 82 A, c = 116 A and beta = 118 degrees. The molecular mass and volume of the unit cell suggest that there are two dimers of the enzyme in the asymmetric unit. The crystals diffract to at least 3.0 A and are suitable for X-ray structure analysis.

Antibody directed enzyme prodrug therapy (ADEPT): a three phase system.

Monoclonal anti-CEA antibody, A5B7, and its fragments conjugated to CPG2 localize to a peak concentration in the LS174T xenografts within 24 h after injection, but enzyme activity persists in plasma such that prodrug injection has to be delayed for 5-6 days in order to avoid toxicity. Injection of prodrug at this time did not result in growth delay of this tumour. A three-phase system has been developed in which residual plasma enzyme was inactivated and cleared by a galactosylated anti-CPG2 antibody, SB43gal, allowing prodrug administration within 24 h after the conjugate. Using this three-phase system, a marked growth delay of this tumour was achieved after a single course of treatment consisting of conjugate injection followed by SB43gal, 19 h later and three doses of the prodrug.

Antibody directed enzyme prodrug therapy (ADEPT): clinical report.

Following an extensive series of studies in nude mice with human xenografts a pilot scale clinical trial of antibody directed enzyme prodrug therapy has been initiated. The principle is to activate a relatively inert prodrug to an active cytotoxin by a tumour located enzyme. In the first stage of the study a prodrug para-N-(mono-2-chloroethyl monomesyl)-aminobenzoyl glutamic acid was administered to six patients with advanced colorectal cancer in a dose escalating protocol. Nausea and vomiting occurred as the only discernible toxic effect at the higher dose levels. Three of these patients and two other patients with advanced disease have proceeded to the second stage of the study in which an antibody-enzyme conjugate was given IV, followed after 36-48 h by a galactosylated anti-enzyme antibody. When plasma enzyme levels had become undetectable the patients received multiple doses of the prodrug. At the lower doses toxicity was minimal as were clinical responses. Two patients received higher doses which resulted in myelosuppression and temporary regression of advanced disease. No complications resulted from administration of the antibody-enzyme complex or enzyme inactivating antibody. The myelosuppression is attributable to the relatively long half-life of the active drug formed from the prodrug used in the present study.

Ablation of human choriocarcinoma xenografts in nude mice by antibody-directed enzyme prodrug therapy (ADEPT) with three novel compounds.

Three novel prodrugs have been designed for use as anticancer agents. Each is a bifunctional alkylating agent which has been protected to form a relatively inactive prodrug. They are designed to be activated to their corresponding alkylating agents at a tumour site by prior administration of an antitumour antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in a two-phase system called antibody-directed enzyme prodrug therapy (ADEPT). The Km and Vmax values for three different antibody-CPG2 conjugates were determined in relation to each prodrug. The Km values ranged from 4.5-12 mumol/l and the Vmax from 0.5-1.6 mumol/U/min. Athymic Nu/Nu mice with palpable transplanted human choriocarcinoma xenografts, which are resistant to conventional chemotherapy, were treated with anti-human chorionic gonadotropin antibodies conjugated to CPG2. This was followed by each of the three novel prodrugs. Significant increase in survival was obtained in three of the regimens tested using only one course of treatment. This demonstrates the potential of a tumour-localised bacterial enzyme to activate protected alkylating agents in order to eradicate an established human xenograft.

Disposition of the prodrug 4-(bis (2-chloroethyl) amino) benzoyl-L-glutamic acid and its active parent drug in mice.

A novel therapy for improving selectivity in cancer chemotherapy aims to modify distribution of a cytotoxic drug by generating it selectively at tumour sites. In this approach an antibody-enzyme conjugate is allowed to localise at the tumour sites before injecting a prodrug which is converted to an active drug specifically by the targeted enzyme in the conjugate. We present here pharmacokinetic studies on the prodrug 4-(bis (2-chloroethyl) amino) benzoyl-L-glutamic acid and its activated derivative, benzoic acid mustard. The glutamic acid is cleaved from the prodrug to form the active drug by carboxypeptidase G2 (CPG2), an enzyme from Pseudomonas sp., which is not found in mammalian cells. The prodrug and its parent active drug were rapidly distributed in plasma and tissues after administration of prodrug or active drug (41 mumol kg-1 intraperitoneally) to mice bearing human choriocarcinoma xenografts. Prodrug and active drug both followed a two-compartment kinetic model. Prodrug was eliminated more rapidly (t1/2 alpha = 0.12 h, t1/2 beta = 0.70 h) than active drug (t1/2 alpha = 0.37 h, t1/2 beta = 1.61 h). Conversion of the prodrug to the activated parent drug was detected within 5 min of administration to mice which had previously received a F(ab')2-anti-human chorionic gonadotrophin antibody (W14A) conjugated to the enzyme, CPG2 (1,000 U kg-1). Tumour was the only tissue that activated all the prodrug reaching the site. It contained the highest concentration of targeted enzyme conjugate capable of catalysing the reaction of prodrug to drug. Plasma and other tissues were also capable of activating the prodrug but active drug production was limited by the amount of enzyme present. The active drug measured in plasma and tissues other than tumour was attributable to residual antibody-enzyme conjugate at non-tumour sites. Low levels of conjugate in tissues and plasma militate against the advantage of tumour localised enzyme therefore necessitating removal of non-localised enzyme.

Carboxypeptidase G2 and trimetrexate cause growth delay of human colonic cancer cells in vitro.

MAWI colonic cancer cells respond to sequential treatment in vitro with carboxypeptidase G2 and trimetrexate by a delay in cell growth as measured by cell numbers, but an increase in incorporation of 75-Se-selenomethionine per cell. The cells are not methionine auxotrophs.

The potential of carboxypeptidase G2: antibody conjugates as anti-tumour agents. II. In vivo localising and clearance properties in a choriocarcinoma model.

The in vivo localising and clearance properties of conjugates of the folate-degrading enzyme carboxypeptidase G2 (CPG2) with anti-human chorionic gonadotrophin (W14A) were measured in nude mice bearing CC3 choriocarcinoma xenografts. Conjugates of W14A-F (ab')2 fragment coupled to CPG2 localised in tumour as effectively as native antibody alone but showed lower uptake in other major tissues. The clearance rates of conjugates prepared with intact antibody or F (ab')2 fragment were shown to be up to five-fold faster than for native antibody and two-fold compared to F (ab')2 fragment. Molecular weight analysis of residual conjugate in the blood showed that no degradation of conjugate to its component molecules occurred during circulation. It was concluded that F (ab')2: CPG2 conjugates offered the greatest potential for targeting applications.