A human immunoglobulin G1 antibody originating from an in vitro-selected Fab phage antibody binds avidly to tumor-associated MUC1 and is efficiently internalized.
We describe the engineering and characterization of a whole human antibody directed toward the tumor-associated protein core of human MUC1. The antibody PH1 originated from the in vitro selection on MUC1 of a nonimmune human Fab phage library. The PH1 variable genes were reformatted for expression as a fully human IgG1. The resulting PH1-IgG1 human antibody displays a 160-fold improved apparent kd (8.7 nmol/L) compared to the kd of the parental Fab (1.4 micromol/L). In cell-binding studies with flow cytometry and immunohistochemistry, PH1-IgG1 exhibits staining patterns typical for antibodies recognizing the tumor-associated tandem repeat region on MUC1, eg, it binds the tumor-associated glycoforms of MUC1 in breast and ovarian cancer cell lines, but not the heavily glycosylated form of MUC1 on colon carcinoma cell lines. In many tumors PH1-IgG1 binds to membranous and cytoplasmic MUC1, with often intense staining of the whole-cell membrane (eg, in adenocarcinoma). In normal tissues staining is either absent or less intense, in which case it is found mostly at the apical side of the cells. Finally, fluorescein isothiocyanate-labeled PH1-IgG1 internalizes quickly after binding to human OVCAR-3 cells, and to a lesser extent to MUC1 gene-transfected 3T3 mouse fibroblasts. The tumor-associated binding characteristics of this antibody, its efficient internalization, and its human nature, make PH1-IgG1 a valuable candidate for further studies as a cancer-targeting immunotherapeutic.
Henderikx
Paula
P
Dyax s.a., Liège, Belgium.
Coolen-van Neer
Nicole
N
Jacobs
Anita
A
van der Linden
Edith
E
Arends
Jan-Willem
JW
Müllberg
Jürgen
J
Hoogenboom
Hennie R
HR
D016475:3T3 Cells; D000818:Animals; D000915:Antibody Affinity; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004705:Endocytosis; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D018613:Microscopy, Confocal; D018396:Mucin-1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009369:Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D014162:Transfection; D014407:Tumor Cells, Cultured
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D018396:Mucin-1