Establishment and characterization of a fucosylated α-fetoprotein-specific monoclonal antibody: a potential application for clinical research

The Lens culinaris agglutinin (LCA)-reactive fraction of α-fetoprotein (AFP-L3) is a well-known cancer biomarker for hepatocellular carcinoma (HCC) with very high specificity. Because LCA recognizes only bi-antennary N-glycans with a core fucose, some of fucosylated AFP in HCC patients may not be detected. Then glycan antibodies, which recognize both specific glycan and protein, are desired for glycobiology. Here, we successfully established a novel glycan antibody for fucosylated AFP and demonstrated its potential clinical application. After immunization with a fucosylated AFP peptide, positive screening was performed for fucosylated AFP peptides using solid-phase enzyme-linked immunosorbent assay (ELISA).
The newly developed antibody was designated: fucosylated AFP-specific mAb (FasMab). Western blot analysis showed that FasMab reacted with AFP produced by HepG2 cells, but not with AFP produced by α-1,6-fucosyltransferase deficient HepG2 cells. The specific binding of FasMab to fucosylated AFP was confirmed with ELISA as well as western blot analysis. A preliminary high sensitivity chemiluminescence enzyme immunoassay kit showed increased levels of fucosylated AFP in the sera of patients with HCC, but not in the sera of normal patients, or patients with chronic liver diseases. Thus, the novel glycan antibody, FasMab, is a promising tool to study fucosylated AFP with clinical and basic research applications.

Regulation of Anti-Tumor Activity Using Monoclonal Antibodies to Alpha-Fetoprotein Receptor and after Immunization with This Protein

Wiskott-Aldrich syndrome (WAS), an inherited blood cell disorder due to mutations of the X-chromosome gene WASP (Wiskott-Aldrich Syndrome Protein), was characterized originally by thrombocytopenia, immunodeficiency and eczema. Whereas platelet dysfunction is severe and consistent, immune defects are clinically variable, ranging from negligible to life-threatening. To understand this heterogeneity, WASP was quantified in peripheral blood mononuclear cells of patients with diverse mutations.
In this study we assessed the relationship between the mutation, protein expression and phenotype in WAS patients. The majority of the patients with missense mutations exhibited mild phenotype, whereas patients with premature stop codon were in most cases severe. We designed a one-step approach intended for use in identifying mutations in samples from newly diagnosed patients.
The approach relies on direct sequencing of amplified exon regions in a staggered schedule that was based on the mutation distribution frequency in previous cases. The method proved to be fast and reliable. Definitive mutation information was generated for each patient studied.

Regulation of Anti-Tumor Activity Using Monoclonal Antibodies to Alpha-Fetoprotein Receptor and after Immunization with This Protein

The object of this work was to study (i) the effect of monoclonal antibodies (mAb) to a receptor (R) of an oncofetal protein of an alpha-fetoprotein (AFP) on the survival rate and sensitivity of tumor target cells to the cytotoxic action of effector cells, (ii) the level of Ab to AFP-R in the blood serum of patients with malignant tumors (iii) the effect of blood serum with a high level of Ab to AFP-R on the survival rate of tumor cells in vitro, and also (iv) the effect of immunization of animals with an AFP-R preparation on subsequent development of a grafted tumor.
It is shown that mAb to AFP-R of clones 2E1, 5C6 and 2B8 effectively bond to both mouse tumor cells and to human tumor cells. Monoclonal Ab to AFP-R of the studied clones do not affect the proliferation of tumor cells of mice and insignificantly inhibit the proliferation of human tumor cells. In patients with malignant tumors, a substantial increase was detected of both the sum Ab to AFP-R, and Ab of the class IgM, and simultaneously an increase of the fraction Ab to AFP-R of the class IgM, which indicates the induction of a primary immune response to AFP-R in such patients.
Separate clones of mAb to AFP-R are capable of activating the immune system in respect to tumor cells, inducing of antibody-dependent cellular cytotoxic activity, but with an increase of the concentration of mAb to AFP-R to 1 &mgr;M, the blocking of the cytotoxic activity of peripheral blood mononuclear cells in respect to human tumor cells is possible. In the case of single immunization of mice with an AFP-R preparation, isolated from tumor tissue of lung cancer of a human, inhibition of the growth of a tumor, grafted four days after the immunization, was observed.