Use of a peptide mimotope to guide the humanization of MRK-16, an anti-P-glycoprotein monoclonal antibody

A mimotope-guided strategy for engineering antibodies directed against orph an targets or antigens that are difficult to purify was developed and used to humanize the murine MRK-16 monoclonal antibody (mAb), MRK-16 recognizes a conformational epitope of a 170-kDa membrane protein, termed P-glycoprote in (P-gp). Elevated expression of P-gp on tumor cells is associated with re sistance to cytotoxic drugs, a major obstacle in chemotherapy. Murine MRK-1 6 was used to enrich and screen a phage-displayed peptide library to identi fy reactive mimotopes, One peptide, termed ALR1, was enriched to a greater extent than others and subsequently was expressed as a fusion protein with glutathione S-transferase. ALR1 fusion protein bound MRK-16 specifically an d inhibited binding of MRK-16 to cells expressing elevated levels of P-gp. To humanize MRK-16, the murine complementarity determining regions were gra fted onto homologous human heavy and light chain variable region frameworks . Framework residues that differed between the murine MRK-16 and the homolo gous human templates were analyzed and subsequently, five framework positio ns potentially important for maintaining the specificity and affinity of MR K-16 were identified. A combinatorial library consisting of 32 variants enc oding all possible combinations of murine and human residues at the five di ffering framework positions was expressed in a phage system. In the absence of purified P-gp, ALR1 fusion protein was used as surrogate antigen to scr een the antibody library to identify the framework combination that most pr eserved the binding activity of the mAb. On the basis of the initial screen ing against the mimotope four antibody variants were selected for further c haracterization. The binding affinity of these variants for the ALR1 fusion protein correlated with their binding to cells expressing elevated levels of P-gp. Thus, peptide mimotopes which can be identified for virtually any antibody including those that recognize conformational or carbohydrate epit opes, can serve as antigen templates for antibody engineering.