Structure and function of several anti-dansyl chimeric antibodies formed by domain interchanges between human IgM and mouse IgG2b.
Two pairs of chimeric, domain-switched immunoglobulins with identical murine, anti-dansyl (5-dimethylaminonaphthalene-1-sulfonyl) variable domains have been generated, employing as parent antibodies a human IgM and a mouse IgG2b. The first pair of chimeric antibodies mu mu gamma mu and gamma gamma mu gamma was generated by switching the C mu 3 and C gamma 2 domains between IgM and IgG2b. The second pair of chimeras mu mu gamma gamma and gamma gamma mu mu were formed by switching both C mu 3 and C mu 4 with C gamma 2 and C gamma 3. SDS-polyacrylamide gel electrophoresis and analytical ultracentrifugation showed that over half (57 and 71%) of the two chimeric antibodies possessing the C mu 4 domain and tail piece formed disulfide-linked IgM-like polymers. In contrast, the two chimeric antibodies lacking the C mu 4 domain were almost entirely monomeric. Both monomeric chimeras had reduced ability to activate complement. The chimera gamma gamma mu gamma had no activity under any of the assay conditions, whereas mu mu gamma gamma caused only a small amount of cell lysis but was fully active in consuming complement at 4 degrees C. The polymeric chimera gamma gamma mu mu was much less active than IgM, bound C1 weakly and caused some cell lysis but consumed little complement with soluble antigen. The polymeric chimera mu mu gamma mu bound C1 strongly and was the most active antibody in all assays, even more active than the parental IgG2b and IgM antibodies; it was the only antibody that exhibited antigen-independent activity. The results suggest that C mu 3 alone does not constitute the complement binding site in IgM but requires both C mu 1-2 and C mu 4 for full activity.
Poon
P H
PH
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024, USA.
Morrison
S L
SL
Schumaker
V N
VN
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003167:Complement Activation / Q000276:immunology; D003172:Complement C1 / Q000378:metabolism; D003619:Dansyl Compounds; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology*; D007075:Immunoglobulin M / Q000235:genetics / Q000276:immunology*; D051379:Mice; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D013329:Structure-Activity Relationship; D014461:Ultracentrifugation
D000906:Antibodies; D003172:Complement C1; D003619:Dansyl Compounds; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011993:Recombinant Fusion Proteins