Intracellular expression and functional properties of an anti-p21(Ras) 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 a ntibody, a non-neutralizing anti-Pas antibody. The Y13-238 hybridoma expres ses two functional light chains. N-terminus microsequencing of these chains showed the presence of the Y3 Ag1.2.3 V kappa: chain derived from the rat fusion partner and of a rat Vh chain. Primers designed for rat Vh amplifica tion allowed the cloning of a functional scFv that could bind p21(Ras). The kinetics of interaction of purified Y238 scFv with the p21(Ras) protein wa s evaluated by BIAcore(R) with a NTA sensor chip and gave an apparent affin ity constant in the nanomolar range (K-D = 4.58 +/- 0.63 nM). Immunoprecipi tation experiments of Y238 scFv expressed in Xenopus laevis oocytes confirm ed the specificity of the scFv for the Ras protein. Y238 scFv could be intr acellularly expressed in oocytes and in mammaliam cells without adverse eff ect on the Ras signalling cascade. This scFv was therefore used as control in experiments where another anti-Ras scFv (Y259 scFv, derived from the neu tralizing 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., Delume au, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., T eillaud, J.L., Tocque, B., 1998. Intracellular expression of an antibody fr agment-neutralizing p21 ras promotes tumor regression. Cancer Res. 58, 1170 -1176.]. Finally, BIAcore(R) analyses indicated that the epitopes recognize d by Y238 and Y259 scFvs are not overlapping and allowed a more precise def inition of the Y13-238 epitope. O 1999 Elsevier Science Ltd. All rights res erved.