Novel anti-Tn single-chain Fv-Fc fusion proteins derived from immunized phage library and antibody Fc domain.

Tn[GalNAc(α1-3)-Ser/Thr] antigen, a tumor-associated carbohydrate antigen, is highly expressed in various tumors and an attractive candidate for cancer immunotherapy. The generation of an anti-Tn antibody is a first step toward the construction of new anticancer molecules. However, because of the simple and small conformation of the Tn molecule, it is difficult to generate an anti-Tn antibody for therapeutic use by conventional hybridoma technology. The purpose of this study was to isolate anti-Tn single-chain antibody fragments (scFv) by phage display technology from a novel immunised library, to attach an antibody constant region (Fc) and to convert them to scFv-Fc fusion proteins. The scFv-Fcs obtained here showed strict specificity against the Tn antigen and also showed antibody-dependent cellular cytotoxicity. These results suggest a potential use of this antibody generating method by phage display and indicate the potential of Fc-fusion proteins as therapeutic candidates.

Mouse-human chimeric anti-Tn IgG1 induced anti-tumor activity against Jurkat cells in vitro and in vivo.

Tn-antigen (alpha-N-acetyl-galactosamine(GalNAc)-Ser/Thr) is a cancer-associated carbohydrate antigen expressed in various epithelial and hematological cancers, and although a number of anti-Tn IgG and IgM antibodies have been generated, they have not been fully validated for cancer immunotherapy. In this study, we generated a novel murine anti-Tn IgG1 monoclonal antibody, KM3413, by immunization of mucins purified from a culture supernatant of LS180: a human colon cancer cell line. The binding of KM3413 was detected against consecutive Tn-antigens (Tn3 and Tn2), but not against monovalent antigens (Tn1). The affinity (K(D)) of KM3413 was determined to be about 10(-7) M with BIAcore. Cross-reactivity against type-A blood antigen, which shares a sugar residue, alpha-linked GalNAc, with Tn-antigen, was not detected. Next, we generated mouse-human chimeric IgG1 of KM3413 (cKM3413) and evaluated its anti-tumor activities against Jurkat: a human T-lymphoid leukemia cell line. In vitro assay revealed that cKM3413 induced antibody-dependent cellular cytotoxicity (ADCC) and direct killing activity with cross-link antibody. Furthermore, treatment of cKM3413 (1 or 10 mg/kg) showed significantly better survival of Jurkat-inoculated C.B-17/lcr-scid Jcl mice compared with controls using PBS treatment (p<0.001). These results suggest that humanized antibody against clustered Tn-antigens is a promising therapeutic antibody against Tn-positive cancers.