Production of Antibodies against Multipass Membrane Proteins Expressed in Human Tumor Cells Using Dendritic Cell Immunization

Takahiko Tamura,Joe Chiba

doi:10.1155/2009/673098

Abstract

Antibody mediated therapeutic strategies against human malignant tumors have been widely authorized and clinically applied to cancer patients. In order to develop methods to generate antibodies reactive to the extracellular domains of multipass plasma membrane proteins specifically expressed in malignant tumors, we examined the use of dendritic cells (DCs) for immunization. DCs were transduced with genes encoding the human six transmembrane epithelial antigen of prostate 1 (STEAP1), STEAP4, and seven transmembrane prostate specific G-protein coupled receptor (PSGR). Mice were immunized with these DCs and followed by repeated booster immunization with plasmids expressing each protein. The immunized mice produced significant amounts of antibodies against these proteins. Our results suggest that DC immunization is an effective method to produce antibodies reactive to extracellular regions of plasma membrane proteins with multiple-transmembrane domains, and may be useful to develop antibody mediated antitumor therapies.

Highlights

In the development of antibody-mediated therapies targeted to human cancer cells, including cancer stem/progenitor cells, the extracellular domains of plasma membrane proteins expressed on cancer cells are promising candidates for antigens against which to be immunized [1], but the high homology of amino acid sequences between human antigens and their homologues in animals to be immunized often hamper efficient antibody production because of immunological tolerance
Flow cytometry analysis showed that LNCaP cells were significantly stained with immunized serum, DU145 cells stained very faintly (Figure 1(c)). These results demonstrate that immunized sera could react with six transmembrane epithelial antigen of prostate 1 (STEAP1) in human prostate cancer cells
We investigated whether dendritic cells (DCs) as immunogens elicited antibody production against multiple-transmembrane proteins expressed in human prostate cancer cells

Summary

Introduction

In the development of antibody-mediated therapies targeted to human cancer cells, including cancer stem/progenitor cells, the extracellular domains of plasma membrane proteins expressed on cancer cells are promising candidates for antigens against which to be immunized [1], but the high homology of amino acid sequences between human antigens and their homologues in animals to be immunized often hamper efficient antibody production because of immunological tolerance. To obtain antibodies reactive to the native extracellular structure of such membrane proteins, immunization by injection of cultured cells expressing the antigen has been used [3]. Large numbers of cells (typically 107-108 cells per animal) are usually needed to prepare for immunization and some modifications of the injected cells are required, for example, genes encoding immunomodulatory cytokines (interleukin-4, and others) or costimulatory molecules are expressed together with the antigen to obtain higher titers. The cells expressing plasma membrane proteins having multiple transmembrane domains such as G-protein coupled receptors (GPCRs) are not always available for immunization. Development of a simple and successful protocol for immunization against human multi-pass membrane proteins is needed in antibody-mediated cancer research

Methods

Results

Conclusion