Generation, characterization, and in vivo studies of humanized anticarcinoma antibody CC49.
Monoclonal antibody (MAb) CC49 reacts with tumor-associated glycoprotein (TAG)-72, a human pancarcinoma antigen. In clinical trials, radiolabeled CC49 has shown excellent tumor localization; however, many of the patients receiving MAb CC49 develop a human antimouse antibody response. In an attempt to prevent this antiimmunoglobulin response, we have developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto the variable light (VL) and variable heavy (VH) frameworks of the human MAbs LEN and 21/28' CL, respectively, while retaining those murine framework residues that may be required for the integrity of the antigen combining-site structure. The HuCC49 MAb was compared with native murine CC49 (nCC49) and chimeric CC49 (cCC49), using a variety of assays. SDS-PAGE analysis under nonreducing conditions showed that the HuCC49 MAb has virtually identical mobility to that of cCC49. Under reducing conditions, the HuCC49 yielded two bands of approximately 25-28 and approximately 50-55 kDa, characteristic of heavy and light immunoglobulin chains. In competition radioimmunoassays, HuCC49 completely inhibited the binding of 125I-labeled nCC49 to TAG-72, although 23- to 30-fold more HuCC49 was required to achieve a level of competition similar to those of cCC49 and nCC49. The relative affinity of HuCC49 was 2- to 3-fold less than those of the cCC49 and nCC49 MAbs, respectively. The plasma clearance in mice of HuCC49 was virtually identical to that of cCC49. Biodistribution studies demonstrated equivalent tumor-targeting of HuCC49 and cCC49 to human colon carcinoma xenografts. These studies thus suggest that HuCC49 and genetically modified molecules, such as sFv and domain-deleted immunoglobulins developed by using the HuCC49 variable region as a cassette, may be potentially useful in both diagnostic and therapeutic clinical trials in patients with TAG-72-positive tumors.
Kashmiri
S V
SV
Laboratory of Tumor Immunology and Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Shu
L
L
Padlan
E A
EA
Milenic
D E
DE
Schlom
J
J
Hand
P H
PH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry* / Q000493:pharmacokinetics; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D000951:Antigens, Neoplasm / Q000276:immunology; D001483:Base Sequence; D001667:Binding, Competitive / Q000276:immunology; D005260:Female; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D006023:Glycoproteins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C047052:tumor-associated antigen 72