title: Comparative biological properties of a recombinant chimeric anti-carcinoma mAb and a recombinant aglycosylated variant.
abstract: It has been demonstrated previously that the degree of glycosylation of a molecule may alter its pharmacokinetic properties and, in the case of an antibody, its metabolism and other biological properties. Transfectomas producing aglycosylated chimeric B72.3(gamma 1) pancarcinoma monoclonal antibody (mAb) were developed by introduction of the eukaryotic expression construct pECMgpB72.3 HuG1-agly, into SP2/0 murine myeloma cells producing the chimeric kappa chain of mAb B72.3. After cell cloning, one subclone with the highest binding to the TAG-72-positive human colon carcinoma was designated mAb aGcB72.3, and its biological and biochemical properties were compared with those of the chimeric B72.3(gamma 1), designated mAb cB72.3. Polyacrylamide gel electrophoresis showed that under non-reducing conditions, the molecular masses of the aGcB72.3 and cB72.3 mAbs were 162 kDa and 166 kDa respectively. The heavy chain of mAb aGcB72.3 had a slightly faster mobility than that of cB72.3, while the mobility of the light chains of the two chimeric mAbs was similar. No difference was observed in the isoelectric points of either chimeric mAb. Liquid competition radioimmunoassays demonstrated that the aGcB72.3 and cB72.3 mAbs have comparable binding properties to TAG-72. These studies demonstrate that aglycosylation of the chimeric IgG1 mAb B72.3 at the CH2 domain, as has been shown for other mAbs [Dorai H., Mueller B., Reisfeld R. A., Gillies S. D. (1991) Hybridoma 10:211; Morrison S. L., Oi V. T. (1989) Adv Immunol 44:65], eliminates antibody-dependent cell-mediated cytotoxicity activity, but does not substantially alter affinity or plasma clearance in mice. These studies also demonstrate for the first time (a) no difference in plasma clearance of an aglycosylated and a chimeric mAb in a primate after i.v. inoculation; (b) a difference (P less than or equal to 0.05) in mice in the more rapid peritoneal clearance of a chimeric mAb versus an aglycosylated chimeric mAb; (c) higher (0.05 less than or equal to P less than or equal to 0.1) tumor: liver ratios at 24, 72 and 168 h using 111In-labeled aglycosylated chimeric mAb versus chimeric mAb. Since the liver is the major site of metastatic spread for most carcinomas, slight differences in tumor to normal liver ratios may be important in diagnostic applications. These studies thus indicate that comparative analyses of a novel recombinant construct (i.e., aglycosylated) and its standard chimeric counterpart require documentation in more than one system and are necessary if one is ultimately to define optimal recombinant/chimeric constructs for diagnosis and therapy in humans.
authors.affiliation: Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
authors.forename: P H
authors.initials: PH
authors.lastname: Hand
authors.forename: B
authors.initials: B
authors.lastname: Calvo
authors.forename: D
authors.initials: D
authors.lastname: Milenic
authors.forename: T
authors.initials: T
authors.lastname: Yokota
authors.forename: M
authors.initials: M
authors.lastname: Finch
authors.forename: P
authors.initials: P
authors.lastname: Snoy
authors.forename: K
authors.initials: K
authors.lastname: Garmestani
authors.forename: O
authors.initials: O
authors.lastname: Gansow
authors.forename: J
authors.initials: J
authors.lastname: Schlom
authors.forename: S V
authors.initials: SV
authors.lastname: Kashmiri
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000920:Antibody-Dependent Cell Cytotoxicity; D001483:Base Sequence; D002277:Carcinoma / Q000276:immunology*; D003110:Colonic Neoplasms / Q000276:immunology*; D005260:Female; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D008253:Macaca mulatta; D008657:Metabolic Clearance Rate; D051379:Mice; D008969:Molecular Sequence Data; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000276:immunology / Q000493:pharmacokinetics; D014018:Tissue Distribution
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins