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Published on: October 15, 2016
Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein
Emiliano Pavoni1, Michela Flego, Maria Luisa Dupuis
1Kenton Labs, c/o Sigma-Tau, via Pontina, km 30,400, 00040 Pomezia (RM), Italy. pavoni@kenton.it
Background:
CEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration.
Methods:
The human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized.
Results:
The scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells.
Conclusion:
The binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance.
Insights
Researchers developed a new human antibody fragment (scFv E8) that targets cancer-associated antigen (CEA) with high affinity. This promising immunotherapy candidate demonstrates selective binding to tumor cells and favorable pharmacokinetic properties for potential cancer treatment.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Carcinoembryonic antigen (CEA) is a tumor-associated antigen found on chemotherapy-resistant cancers.
- Developing human anti-CEA single-chain antibody fragments (scFv) is crucial for creating novel monoclonal antibodies with enhanced tumor penetration and blood clearance.
Purpose of the Study:
- To isolate and characterize human anti-CEA scFv antibodies.
- To improve the affinity of anti-CEA scFv antibodies through in vitro mutagenesis.
- To evaluate the potential of these engineered antibodies as anticancer agents.
Main Methods:
- Isolation of the MA39 scFv from a phage library by panning on purified CEA.
- In vitro mutagenesis of the MA39 scFv gene to create a new library.
- Selection and characterization of high-affinity scFv clones, including E8, for CEA epitope recognition.
Main Results:
- The scFv MA39 antibody was affinity-matured, yielding the E8 clone.
- The E8 clone recognizes a shared epitope among CEACAM1, CEACAM3, and CEACAM5.
- E8 demonstrated selective binding to CEA on various human tumor tissues and cells.
Conclusions:
- The scFv E8 antibody exhibits high binding affinity for efficient in vivo antigen capture in CEA-expressing tumor cells.
- E8 is a human, non-immunogenic immunoreagent with selective, high-affinity binding to CEA on metastatic melanoma, colon, and lung carcinomas.
- The small molecular size of E8 facilitates efficient tissue penetration and rapid plasma clearance, making it a potential anticancer compound.
