Sustained tumor regression of human colorectal cancer xenografts using a multifunctional mannosylated fusion protein in antibody-directed enzyme prodrug therapy.

PURPOSE: Antibody-directed enzyme prodrug therapy (ADEPT) requires highly selective antibody-mediated delivery of enzyme to tumor. MFE-CP, a multifunctional genetic fusion protein of antibody and enzyme, was designed to achieve this by two mechanisms. First by using a high affinity and high specificity single chain Fv antibody directed to carcinoembryonic antigen. Second by rapid removal of antibody-enzyme from normal tissues by virtue of post-translational mannosylation. The purpose of this paper is to investigate these dual functions in an animal model of pharmacokinetics, pharmacodynamics, toxicity, and efficacy. EXPERIMENTAL DESIGN: MFE-CP was expressed in the yeast Pichia pastoris and purified via an engineered hexahistidine tag. Biodistribution and therapeutic effect of a single ADEPT cycle (1,000 units/kg MFE-CP followed by 70 mg/kg ZD2767P prodrug at 6, 7, and 8 hours) and multiple ADEPT cycles (9-10 cycles within 21-24 days) was studied in established human colon carcinoma xenografts, LS174T, and SW1222. RESULTS: Selective localization of functional enzyme in tumors and rapid clearance from plasma was observed within 6 hours, resulting in tumor to plasma ratios of 1,400:1 and 339:1, respectively for the LS174T and SW1222 models. A single ADEPT cycle produced reproducible tumor growth delay in both models. Multiple ADEPT cycles significantly enhanced the therapeutic effect of a single cycle in the LS174T xenografts (P = 0.001) and produced regressions in the SW1222 xenografts (P = 0.0001), with minimal toxicity. CONCLUSIONS: MFE-CP fusion protein, in combination with ZD2767P, provides a new and successful ADEPT system, which offers the potential for multiple cycles and antitumor efficacy. These results provide a basis for the next stage in clinical development of ADEPT.

Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein.

MFE-CP is a recombinant antibody-enzyme fusion protein used for antibody-mediated delivery of an enzyme to cancer deposits. After clearance from normal tissues, the tumor-targeted enzyme is used to activate a subsequently administered prodrug to give a potent cytotoxic in the tumor. MFE-CP localizes to cancer deposits in vivo, but we propose that its therapeutic potential could be improved by N-glycosylation, obtained by expression in Pichia pastoris. Glycosylation could enhance clearance from healthy tissue and result in better tumor:normal tissue ratios. To test this, glycosylated MFE-CP was expressed and purified from P. pastoris. The resultant MFE-CP fusion protein was enzymatically active and showed enhanced clearance from normal tissues in vivo. Furthermore, it showed effective tumor localization. This favorable glycosylation pattern was analyzed by tandem mass spectrometry. High-resolution, high-detection sensitivity collision-induced dissociation experiments proved essential for this task. Results showed that of the three potential N-glycosylation sites only two were consistently occupied with oligomannose structures. Asn-442 appeared the most heterogeneously populated with oligomannose carbohydrates extending from 5 to 13 units in length. Asn-484 was found only in its nonglycosylated form. There was less heterogeneity at Asn-492, which was glycosylated with oligosaccharide structures ranging from 8 to 10 mannose units. Nonglycosylated forms of Asn-442 and Asn-492 were not observed.

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.