Intracellular expression and functional properties of an anti-p21Ras scFv derived from a rat hybridoma containing specific lambda and irrelevant kappa light chains.
A rat single-chain Fv (Y238 scFv) was derived from the Y13-238 monoclonal antibody, a non-neutralizing anti-Ras antibody. The Y13-238 hybridoma expresses two functional light chains. N-terminus microsequencing of these chains showed the presence of the Y3 Ag1.2.3 Vkappa chain derived from the rat fusion partner and of a rat Vlambda chain. Primers designed for rat Vlambda amplification allowed the cloning of a functional scFv that could bind p21Ras. The kinetics of interaction of purified Y238 scFv with the p21Ras protein was evaluated by BIAcore with a NTA sensor chip and gave an apparent affinity constant in the nanomolar range (K(D)=4.58+/-0.63 nM). Immunoprecipitation experiments of Y238 scFv expressed in Xenopus laevis oocytes confirmed the specificity of the scFv for the Ras protein. Y238 scFv could be intracellularly expressed in oocytes and in mammaliam cells without adverse effect on the Ras signalling cascade. This scFv was therefore used as control in experiments where another anti-Ras scFv (Y259 scFv, derived from the neutralizing anti-Ras mAb Y13-259) blocked the Ras pathway in vitro and led to tumor regression in a nude mouse model [Cochet, O., Kenigsberg, M., Delumeau, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., Teillaud, J.L., Tocqué, B., 1998. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression. Cancer Res. 58, 1170-1176.]. Finally, BIAcore analyses indicated that the epitopes recognized by Y238 and Y259 scFvs are not overlapping and allowed a more precise definition of the Y13-238 epitope.
Laboratoire de Biotechnologie des Anticorps, Institut Curie, Paris, France.
O
O
Cochet
M
M
Kenigsberg
I
I
Delumeau
M
M
Duchesne
F
F
Schweighoffer
B
B
Tocqué
J L
JL
Teillaud
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D001483:Base Sequence; D015374:Biosensing Techniques; D003001:Cloning, Molecular; D017931:DNA Primers; D018604:Epitope Mapping; D015870:Gene Expression / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007142:Immunoglobulin gamma-Chains / Q000235:genetics / Q000276:immunology*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D016283:Proto-Oncogene Proteins p21(ras) / Q000276:immunology* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology; D017421:Sequence Analysis
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D007142:Immunoglobulin gamma-Chains; D007145:Immunoglobulin kappa-Chains; D011994:Recombinant Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)