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A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics
Daniel I R Spencer1, Lynda Robson, Des Purdy
1CRC Targeting and Imaging Group, Department of Oncology, Royal Free and University College Medical School, UCL, London, UK.
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.
Insights
Researchers developed a novel fusion protein for cancer therapy, reducing immune reactions. This advancement in antibody-directed enzyme prodrug therapy (ADEPT) aims to improve treatment efficacy by minimizing human anti-CPG2 antibodies (HACA).
Area of Science:
- Biotechnology
- Immunology
- Oncology
Background:
- Antibody-directed enzyme prodrug therapy (ADEPT) utilizes antibodies to deliver drug-activating enzymes to tumor cells for targeted cancer treatment.
- Previous ADEPT trials using carboxypeptidase G2 (CPG2) conjugated to anti-carcinoembryonic antigen (CEA) antibodies showed efficacy but were limited by the development of human antibodies to CPG2 (HACA).
Purpose of the Study:
- To develop a recombinant fusion protein (MFE-CP) combining CPG2 with an anti-CEA single-chain Fv antibody fragment.
- To identify and mitigate the immunogenicity of the MFE-CP therapeutic, specifically addressing HACA formation.
Main Methods:
- Utilized crystal structure of CPG2, filamentous phage technology, and surface-enhanced laser desorption/ionization affinity mass spectrometry to identify HACA-reactive epitopes on MFE-CP.
- Engineered a functional mutant of MFE-CP by modifying identified immunogenic sites.
Main Results:
- Successfully identified a discontinuous epitope on MFE-CP responsible for HACA reactivity.
- The engineered MFE-CP mutant demonstrated a significant reduction in reactivity with HACA-positive patient sera (median reduction of 38.5%).
Conclusions:
- The developed MFE-CP fusion protein and its modified mutant show promise for reducing immunogenicity in ADEPT cancer therapy.
- The methodologies employed are valuable for identifying and engineering protein therapeutics to minimize undesirable immune responses.