Noncovalent scFv multimers of tumor-targeting anti-Lewis(y) hu3S193 humanized antibody.
Single-chain variable fragments (scFvs) of anti-Lewis(y) hu3S193 humanized antibody were constructed by joining the V(H) and V(L) domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V(H) domain were removed (-1 residue, -2 residue) and then joined directly to the V(L) domain. An scFv construct in the reverse orientation with the V(L) joined directly to the V(H) domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.
Power
Barbara E
BE
CSIRO Health Sciences and Nutrition, Parkville, Victoria 3052, Australia. barbara.power@csiro.au
Doughty
Larissa
L
Shapira
Deborah R
DR
Burns
John E
JE
Bayly
Ann M
AM
Caine
Joanne M
JM
Liu
Zhanqi
Z
Scott
Andrew M
AM
Hudson
Peter J
PJ
Kortt
Alexander A
AA
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007983:Lewis Blood Group Antigens / Q000276:immunology*
D007128:Immunoglobulin Fragments; D007983:Lewis Blood Group Antigens; C094162:Lewis Y antigen