Production of "biobetter" variants of glucarpidase with enhanced enzyme activity.

Glucarpidase, also known as carboxypeptidase G2, is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels "biobetter" variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. Km, Vmax and Kcat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase. Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase.

Modifying an immunogenic epitope on a therapeutic protein: a step towards an improved system for antibody-directed enzyme prodrug therapy (ADEPT).

Carboxypeptidase G2 (CP) is a bacterial enzyme, which is targeted to tumours by an antitumour antibody for local prodrug activation in antibody-directed enzyme prodrug therapy (ADEPT). Repeated cycles of ADEPT are desirable but are hampered by human antibody response to CP (HACA). To address this, we aimed to identify and modify clinically important immunogenic sites on MFECP, a recombinant fusion protein of CP with MFE-23, a single chain Fv (scFv) antibody. A discontinuous conformational epitope at the C-terminus of the CP previously identified by the CM79 scFv antibody (CM79-identified epitope) was chosen for study. Modification of MFECP was achieved by mutations of the CM79-identified epitope or by addition of a hexahistidine tag (His-tag) to the C-terminus of MFECP, which forms part of the epitope. Murine immunisation experiments with modified MFECP showed no significant antibody response to the CM79-identified epitope compared to A5CP, an unmodified version of CP chemically conjugated to an F(ab)(2) antibody. Success of modification was also demonstrated in humans because patients treated with His-tagged MFECP had a significantly reduced antibody response to the CM79-identified epitope, compared to patients given A5CP. Moreover, the polyclonal antibody response to CP was delayed in both mice and patients given modified MFECP. This increases the prospect of repeated treatment with ADEPT for effective cancer treatment.

A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics.

Antibodies are highly specific recognition molecules which are increasingly being applied to target therapy in patients. One type of developmental antibody-based therapy is antibody directed enzyme prodrug therapy (ADEPT) for the treatment of cancer. In ADEPT, an antibody specific to a tumor marker protein delivers a drug-activating enzyme to the cancer. Subsequent intravenous administration of an inactive prodrug results in drug activation and cytotoxicity only within the locale of the tumor. Pilot clinical trials with chemical conjugates of the prodrug activating enzyme carboxypeptidase G2 (CPG2) chemically conjugated with an antibody to and carcinoembryonic antigen (CEA), have shown that CPG2-mediated ADEPT is effective but limited by formation of human antibodies to CPG2 (HACA). We have developed a recombinant fusion protein (termed MFE-CP) of CPG2 with an anti-CEA single chain Fv antibody fragment and we have developed methods to address the immunogenicity of this therapeutic. A HACA-reactive discontinuous epitope on MFE-CP was identified using the crystal structure of CPG2, filamentous phage technology and surface enhanced laser desorption/ionization affinity mass spectrometry. This information was used to create a functional mutant of MFE-CP with a significant reduction (range 19.2 to 62.5%, median 38.5%) in reactivity with the sera of 11 patients with post-therapy HACA. The techniques described here are valuable tools for identifying and adapting undesirable immunogenic sites on protein therapeutics.

Novel prodrugs of alkylating agents derived from 2-fluoro- and 3-fluorobenzoic acids for antibody-directed enzyme prodrug therapy.

The synthesis of six novel fluorinated potential prodrugs for antibody-directed enzyme prodrug therapy is described. The [2- and 3-fluoro-4-[bis(2-chloroethyl)amino]benzoyl]-L-glutamic acid (9 and 21), their bis(mesyloxy)ethyl derivatives (7 and 19), and their chloroethyl (mesyloxy)-ethyl derivatives (8 and 20) are bifunctional alkylating agents in which the activating effect of the ionized carboxyl function is masked through an amide bond to the glutamic acid residue. These compounds were designed to be activated to their corresponding benzoic acid alkylating agents at a tumor site by prior administration of a monoclonal antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2). The synthesis of the analogous novel parent drugs 2- and 3-fluoro-4-[bis(2-chloroethyl)amino]benzoic acid (12 and 24), their bis(mesyloxy)ethyl derivatives (10 and 22), and their chloroethyl (mesyloxy)ethyl derivatives (11 and 23) is also described. The viability of a colorectal cell line was monitored with the six potential prodrugs in the presence of CPG2 and with the parent drugs alone. Compounds 19-21 demonstrated substantial prodrug activity, with activation by CPG2 leading to cytotoxicities comparable to those of 22-24, respectively. The Km and kcat values for 7-9 and 19-21 were determined for CPG2. All potential prodrugs except 7 proved to be excellent substrates. A comparison of the relative chemical reactivity of the compounds as determined by their half-life measurements showed that the 2-fluoro substituent deactivated while the 3-fluoro substituent activated the alkylating moieties.

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.

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.

Methotrexate pharmacokinetics following administration of recombinant carboxypeptidase-G2 in rhesus monkeys.

PURPOSE: Carboxypeptidase-G2 (CPDG2) is a bacterial enzyme that rapidly hydrolyzes methotrexate (MTX) into inactive metabolites. As an alternative form of rescue after high-dose MTX (HDMTX), CPDG2 has more potential advantages than standard leucovorin (LV) rescue. In this study, the plasma pharmacokinetics of MTX with and without CPDG2 were evaluated in adult rhesus monkeys. MATERIALS AND METHODS: The plasma pharmacokinetics of MTX were determined in groups of animals that had received a 300-mg/m2 loading dose of MTX followed by a 60-mg/m2/h infusion during an 18-hour period. One group received CPDG2 at the end of the infusion, and the other group served as a control. Two additional animals with high titers of anti-CPDG2 antibody also were studied. RESULTS: During infusion, the steady-state MTX plasma concentration was 11.3 +/- 4.8 mumol/L. Without CPDG2, the postinfusion plasma MTX concentration remained above 0.1 mumol/L for more than 6 hours. After the administration of 50 U/kg of CPDG2, plasma MTX concentrations decreased to nontoxic levels (less than 0.05 mumol/L) within 30 minutes. The initial half-life (t1/2 alpha) of MTX decreased from 5.8 +/- 2.1 minutes to 0.7 +/- 0.02 minutes after enzyme administration. The postinfusion area under the plasma concentration time curve of MTX was 301 +/- 171 mumol/L/min without CPDG2 compared with 19.6 +/- 6.1 mumol/L/min with CPDG2. The immunogenicity studies performed indicated that although animals developed anti-CPDG2 antibodies, none of them manifested allergic symptoms. The effectiveness of CPDG2 was diminished but not eliminated in animals with high titers of anti-CPDG2 antibody. CONCLUSIONS: CPDG2 is capable of rapidly decreasing plasma MTX concentrations to nontoxic levels. The administration of CPDG2 seems safe, well tolerated, and it may be useful as an alternative to LV rescue.