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 agent, 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 Immu31 (hImmu31) was constructed by grafting the complementarity determining regions (CDRs) of murine variable domains for the heavy (VH) and kappa (Vkappa) chain to the respective human VH and Vkappa framework regions (FRs). The cDNA encoding the VH and Vkappa of Immu31 was cloned by reverse transcription-PCR from hybridoma cells, and a chimeric Immu31 (cImmu31) composed of murine V and human C domains was constructed. Competitive ELISA assays showed identical AFP binding activity between the chimeric and murine Abs, confirming the authenticity of the cloned V genes. Based on sequence homology, the EU FR1, FR2, and FR3 and the NEWM FR4 were selected as the scaffold for grafting VH CDRs and REI FRs for Vkappa 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 constructed and expressed, showed comparable immunoreactivity in a competitive binding 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.
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Z
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Immunomedics, Inc., Morris Plains, New Jersey 07950, USA.
Losman
M J
MJ
Eliassen
K C
KC
Hansen
H J
HJ
Goldenberg
D M
DM
Leung
S O
SO
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D000509:alpha-Fetoproteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D000509:alpha-Fetoproteins