The role of VH CDR3 in determining Tn‐antigen specificities of MLS128 and 83D4 monoclonal antibodies (1007.1)

Abstract

Tn‐antigen (GalNAcα‐Ser/Thr) is associated with carcinomas. MLS128 and 83D4 are Tn‐antigen specific monoclonal antibodies. They bind carbohydrate epitopes consisting of three or two consecutive Tn‐antigens (Tn3 or Tn2) with different affinities. Since VH sequences of both mAbs are nearly identical except a few amino acids in the CDR3, the aim of this study was to evaluate the role of the VH CDR3 in determining their Tn‐antigen specificities.The VH domain of 83D4 was cloned into pCI‐MLS128 VL‐Fc to construct pCI/MLS128/83D4 VH CDR3 scFv‐Fc vector. CHO cells transfected with the vector were cultured in a selective medium to screen clones expressing MLS128/83D4 VH CDR3 scFv‐Fc protein. Fc‐levels in media of stable clones were determined by ELISA. Of several stable clones producing the MLS128/83D4 VH CDR3 scFv‐Fc protein at 2 µg/ml levels established, one clone was chosen for purification and characterization of the MLS128/83D4 VH CDR3 scFv‐Fc protein. From 330 ml of culture medium, 70 μg of the scFv‐Fc protein were purified to near homogeneity by Protein A‐Sepharose chromatography. Binding activity of three scFv‐Fc proteins (MLS128/83D4 VH CDR3, MLS128, 83D4) was compared by ELISA using Tn2‐ and Tn3 peptides.