A chimeric anti-CEA antibody with heavy interchain disulfide bonds deleted: molecular characterization and biodistributions in normal and tumor bearing mice.
We have deleted the interchain disulfide bonds in a chimeric anti-CEA antibody (chT84.66) by mutating two cysteines in the heavy chain to glycine residues. The resulting antibody delta SSchT84.66 was expressed in high yield in a bioreactor and purified to homogeneity in a single step on an anti-idiotypic antibody affinity column. The molecular size of the antibody was 150 kDa as judged by gel filtration, SDS gel electrophoresis under non-reducing conditions, and MALDI-TOF/MS. The 150 kDa antibody had nearly identical kinetic (Kon = 1.53 x 10(6) M-1 s-1, .koff = 1.14 x 10(-5) s-1) and affinity constants (Kaff = 1.34 x 10(11) M-1) compared to the parent murine (Kaff = 1.25 x 10(11) M-1) and chimeric (Kaff = 1.16 x 10(11) M-1) antibodies when tested on biosensor chips. When delta SSchT84.66 was conjugated to the isothiocynato derivative of DTPA, radiolabeled with 111In, and injected into either normal or nude mice bearing tumor xenografts, it gave nearly identical biodistributions to chT84.66. delta SSchT84.66 and chT84.66 antibodies gave a maximum tumor uptake of 48 and 74% ID/g, and tumor to blood ratios of 5.3 and 6.2 at 48 h, respectively. We conclude that delta SSchT84.66 irreversibly associates into H2L2 dimers after concentration, that the dimers are stable under both the in vitro and in vivo conditions used in this study, and the properties of the antibody are virtually indistinguishable from the parent chT84.66 antibody.
Neumaier
M
M
Department of Clinical Chemistry, Universitätskrankenhaus Eppendorf, Hamburg, Germany.
Gaida
F J
FJ
Lewis
M R
MR
Hefta
L J
LJ
Shively
L E
LE
Raubitschek
A
A
Shively
J E
JE
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism; D015374:Biosensing Techniques; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009374:Neoplasms, Experimental / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000493:pharmacokinetics; D014018:Tissue Distribution
D000906:Antibodies; D002272:Carcinoembryonic Antigen; D004220:Disulfides; D011993:Recombinant Fusion Proteins