Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody

A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the E GP40 pancarcinoma antigen, was humanized through three-dimensional molecula r modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the V-L and V-H regions were grafted onto the human anti-DNA-ass ociated idiotype immunoglobulin clone, R3.5H5G'CL, Ten amino acids residues within the humanized framework were back-mutated to their corresponding ch NR-LU-13 sequence, because they were predicted to disrupt the canonical cla ssification of the CDRs or were within 5 Angstrom of a CDR. Synthesis of th e V-L and V-H regions was accomplished by recursive PCR, and the dual-chain expression vector p451.C4 was positioned under control of the CMVP+E. We o bserved by competitive ELISA that the recombinant humanized NR-LU-13 (huNR- LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profil e when compared with the murine monoclonal antibody (muNR-LU-10), The huNR- LU-13 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity when assayed against eith er the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected I-131-muNR-LU-1 0 and I-125-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets t o the tumor in athymic BALB/c mice bearing the SW1222 human tumor xenograft . Humanization of the chNR-LU-13 antibody is expected to eliminate an undes ired human anti-mouse antibody response, allowing for repeated i.v. adminis tration into humans.