Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody.
A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular 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-associated idiotype immunoglobulin clone, R3.5H5G'CL. Ten amino acids residues within the humanized framework were back-mutated to their corresponding chNR-LU-13 sequence, because they were predicted to disrupt the canonical classification of the CDRs or were within 5 A of a CDR. Synthesis of the 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 CMV(P+E). We observed by competitive ELISA that the recombinant humanized NR-LU-13 (huNR-LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profile 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 either the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected 131I-muNR-LU-10 and 125I-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets to 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 undesired human antimouse antibody response, allowing for repeated i.v. administration into humans.
Graves
S S
SS
Department of Molecular Biology, NeoRx Corporation, Seattle, Washington 98119, USA.
Goshorn
S C
SC
Stone
D M
DM
Axworthy
D B
DB
Reno
J M
JM
Bottino
B
B
Searle
S
S
Henry
A
A
Pedersen
J
J
Rees
A R
AR
Libby
R T
RT
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics / Q000627:therapeutic use*; D000951:Antigens, Neoplasm / Q000276:immunology; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000276:immunology; D006224:Cricetinae; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004797:Enzyme-Linked Immunosorbent Assay; D000071858:Epithelial Cell Adhesion Molecule; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009374:Neoplasms, Experimental / Q000188:drug therapy*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins