Humanization of Immu31, an alpha-fetoprotein-specific antibody

Immu31 is a murine monoclonal antibody (Ab) specific for alpha-fetoprotein (AFP), a tumor-associated marker. The excellent tumor targeting ability of Immu31 has led to the development of a Immu31-based radioimmunodiagnostic a gent, AFP-Scan, for hepatocellular carcinoma and other AFP-producing tumors . To enhance the capability of Immu31-based immunoconjugates being used in diagnostic and therapeutic procedures in humans, a humanized version of Imm u31 (hImmu31) was constructed by grafting the complementarity determining r egions (CDRs) of murine variable domains for the heavy (VH) and kappa (V ka ppa) chain to the respective human VH and V kappa framework regions (FRs). The cDNA encoding the VH and V kappa of Immu31 was cloned by reverse transc ription-PCR from hybridoma cells, and a chimeric Immu31 (cImmu31) composed of murine V and human C domains was constructed. Competitive ELISA assays s howed identical AFP binding activity between the chimeric and murine Abs, c onfirming the authenticity of the cloned V genes. Based on sequence homolog y, the EU FR1, FR2, and FR3 and the NEWM FR4 were selected as the scaffold for grafting VH CDRs and REI FRs for V kappa CDRs of Immu31. The amino acid residues in murine FRs that are considered to be in contact with the CDRs of the Ab were maintained in the humanized version. hImmu31, thus construct ed and expressed, showed comparable immunoreactivity in a competitive bindi ng ELISA assay to that of murine Immu31 and cImmu31. High-level production was achieved by expressing hImmu31 in a dhfr-based amplifiable system, and the productivity has exceeded 100 mg/liter in terminal cultures.