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In Vitro Affinity Maturation of a Specificity-Determining Region-Grafted Humanized Anticarcinoma Antibody

Isolation and Characterization of Minimally Immunogenic High-Affinity Variants

¹ Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute,
² Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases,
³ Division of Bioengineering and Physical Sciences, Office of Research Services, Office of the Director, NIH, Bethesda, Maryland, and
⁴ Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska

ABSTRACT

Purpose: HuCC49V10 (V10), a humanized anticarcinoma monoclonal antibody (Ab) CC49, was generated by grafting only the specificity-determining regions (SDRs) of CC49 onto the variable light and variable heavy frameworks of the human Abs LEN and 21/28'CL, respectively. SDRs are those residues of the complementarity-determining regions that are most critical for antigen (Ag) binding. Compared with HuCC49, which was developed by conventional complementarity-determining region grafting, V10 has lower reactivity to the sera from patients who were previously given murine CC49 in clinical trials, although its Ag-binding affinity is 2–3-fold lower than that of HuCC49. To generate variants of V10 with higher Ag-binding affinity and lower sera reactivity, in vitro affinity maturation of V10 was carried out using phage display technique.

Experimental Design: A limited library of Fabs was generated by replacing some of the SDRs with all possible residues located at the corresponding positions in human Abs. The library was enriched, by several rounds of panning, in Fabs that have high affinity for the TAG-72 Ag. The clones encoding the best binders were expressed in insect cells as whole Abs that were purified and characterized.

Results: Competition radioimmunoassay and surface plasmon resonance measurements showed that two of the isolates, V14 and V15, have higher binding affinity than that of V10. In addition, the surface plasmon resonance analysis showed that the variants V14 and V15, compared with the parental V10, have lower reactivity to the anti-V region Abs using sera from patients who received murine CC49.

Conclusions: The two isolates, V14 and V15, which show higher Ag-binding reactivity and lower sera reactivity than the parental V10 Ab, are potentially more useful clinical reagents. These results demonstrate that phage display can be used to isolate variants of an Ab that are potentially less immunogenic in patients than the parental Ab from which they are derived.

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