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

Abstract

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.

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

“…Identification of B-cell epitopes by hybridoma technology has been laborious in the past, but feasibility has vastly improved due to the availability of a generic method for rapid recognition of Bcell epitopes based on a phage display library of scFv antibodies, surface enhanced laser desorption and ionisation (SELDI) affinity mass spectrometry and bioinformatics tools as described previously by our group (Spencer et al, 2002). The CM79 site, a surface-exposed conformational B-cell epitope comprising the Cterminus and an internal sequence of CP has been identified by this method and we have shown previously that the human antibody response to CP is substantially directed towards this CM79 site.…”

“…Construction of pPM331 (encoding MFECP, a recombinant fusion protein of the anti-CEA scFv MFE-23 and CP, an enzyme derived from Variovorax paradoxus, formerly Pseudomonas sp RS 16) has been described previously (Michael et al, 1996). Plasmid pDP161 (encoding MFEdmCP) is identical to MFECP except for a mutation in both regions encoding the discontinuous immunodominant epitope of CP (R162A and G412A) (Spencer et al, 2002). This plasmid was created by isolating a 244 bp SalI/EcoRI fragment from the previously constructed vector carrying G412A (pDP132), and inserting it between the equivalent sites of plasmid pDP130, which carries R162A.…”

“…The anti-CP scFv antibody CM79 was expressed in E. coli TG1 cells with a C-terminal His-tag for purification on IMAC as described previously (Spencer et al, 2002). The cell-free supernatant was concentrated and purified on IMAC (Casey et al, 1995) on an automated Ä KTA purification system with a final yield of purified CM79 of approximately 12 mg l À1 culture supernatant.…”

“…It was postulated that identification and silencing of epitopes recognised by the human immune system could allow repeated treatment with ADEPT if these epitopes could be disrupted without compromise of enzyme activity. Previously, we developed a system for identifying B-cell epitopes and applied it to identify a discontinuous conformational epitope on CP with the CM79 scFv antibody (CM79-identified epitope; Spencer et al, 2002) (Figure 2). In the current paper we modify the CM79-identified epitope to test our hypothesis.…”

Professor Tom Connors and the development of novel cancer therapies by the Phase I/II Clinical Trials Committee of Cancer Research UK

“…Human colon carcinoma xenografts show tumour regression without significant toxicity and this fusion protein is now being used with the bis-iodo-phenol mustard prodrug (ZD2767P, XIV) in a Cancer Research UK Phase I/II trial. Further bioinformatics-led engineering of the enzyme and antibody are in progress to reduce immunogenicity (Boehm et al, 2000;Spencer et al, 2002). Anticancer agents based on an elegant working hypothesis are sometimes successful in the clinic K However, the way they act is often nothing to do with the working hypothesis 3.…”