Monoclonal antibodies against autocrine motility factor suppress gastric cancer.

Autocrine motility factor (AMF), which is a secreted form of phosphoglucose isomerase, is mainly secreted by various tumors and has cytokine-like activity. AMF is known to stimulate proliferation, survival and metastasis of cancer cells, and angiogenesis within a tumor. The present study investigated whether inhibition of AMF using targeted-antibodies was able to suppress the growth of cancer. A migration assay using a Boyden chamber was utilized to measure the activity of AMF on the motility of cancer cells. A recombinant human AMF (rhAMF) prepared from E. coli transformed with the pET22b-AMF vector increased the motility of MDA-MB-231 and A549 cells, but it did not affect that of NCI-N87 or HepG2 cells, which exhibited the ability to secrete high amounts of their own endogenous AMF into the culture medium. The extent to which the AMF receptor was expressed on cancer cells did not correlate clearly with the cell motility stimulated by rhAMF. In A549-xenografted nude mice treated with sunitinib or cetuximab, a decrease in the plasma AMF concentration was accompanied by a reduction in tumor weight, suggesting an association between the plasma AMF concentration and anticancer activity. A monoclonal antibody (9A-4H), which revealed a high binding affinity for E. coli-derived rhAMF, significantly suppressed the growth of tumors in Balb/c nude mice transplanted with the human gastric cancer cell line NCI-N87, to the similar extent as trastuzumab, an anticancer antibody. The present study suggests, for the first time, that an antibody specific to AMF may be a therapeutic agent for gastric cancer.

Production and Purification of Recombinant Glucocerebrosidase in Transgenic Rice Cell Suspension Cultures.

Gaucher disease, which is caused by deficiency of glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy. Plant-based systems produce glycoproteins and can be combined with targeting strategies to generate proteins with terminal mannose structures for macrophage uptake. However, the gliding step for the purification is essential since the produced protein still exists inside cells. In the case of rice-amylase 1A (RAmy1A) secretion signal peptide, GCD protein is secreted outside of cells and simplifies the purification step. Here, an established cell line was confirmed as having fundamental characteristics of growth and production. GCD from transgenic calli was examined by Western blot analysis and compared with that from Chinese hamster ovary (CHO) cells. Calli expressing high levels of GCD were used to establish suspension cell lines. Growth and production characteristics were investigated in suspension cell cultures. Production of GCD in suspension cultures was confirmed upon induction for 12-24 h. The amount of GCD in medium increased until 60-84 h and decreased thereafter. Purification of GCD was performed in three steps (ion exchange, hydrophobic interaction, and size exclusion chromatography) and verified. Purified GCD was able to hydrolyze the synthetic substrate. Thus, a rice expression system could be a suitable alternative to GCD expression in mammalian cells.

Glycan structure and serum half-life of recombinant CTLA4Ig, an immunosuppressive agent, expressed in suspension-cultured rice cells with coexpression of human ß1,4-galactosyltransferase and human CTLA4Ig.

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4Ig(P)) reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4Ig(M)). However, limitations of hCTLA4Ig(P) include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing (ß1-2)-xylose and α1,3-fucose, which cause immunogenic reactions in humans. In the present study, human ß1,4-galactose-extended hCTLA4Ig(P) (hCTLA4Ig(P)-Gal) was expressed through the coexpression of human ß1,4-galactosyltransferase (hGalT) and hCTLA4Ig in an attempt to overcome these unfavorable effects. The results indicated that both encoding hGalT and hCTLA4Ig were successfully coexpressed, and the analysis of N-glycan and its relative abundance in purified hCTLA4Ig(P)-Gal indicated that not only were the two glycans containing (ß1-4)-galactose newly extended, but also glycans containing both ß1,2-xylose and α1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4Ig(P), respectively. Unlike hCTLA4Ig(P), hCTLA4Ig(P)-Gal was effective as an acceptor via (ß1-4)-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4Ig(P)-Gal in Sprague-Dawley rats was 1.9 times longer than that of hCTLA4Ig(P), and the clearance pattern of hCTLA4Ig(P)-Gal was close to that for hCTLA4Ig(M). These results indicate that the coexpression with hGalT and hCTLA4Ig(P) is useful for both reducing glycan immunogens and increasing in vivo stability. This is the first report of hCTLA4Ig as an effective therapeutics candidate in glycoengineered rice cells.

Immunogenicity and safety of Vi capsular polysaccharide typhoid vaccine in healthy persons in Korea.

The purpose of this study was to evaluate the immunogenicity and safety of Salmonella Typhi Vi capsular polysaccharide vaccine (Vi vaccine) in Korea. The immunogenicity of a single dose of Vi vaccine was evaluated in 157 subjects (75 children and 82 adults) before and at 1, 6, and 12 months after vaccination. Immunogenicity was measured with a passive hemagglutination assay (PHA), quantified as geometric mean titers (GMTs) and seroconversion rates. The safety of the vaccine was investigated by determining adverse reactions occurring within 4 h, 3 days, and 1 month after injection. The seroconversion rate for children and adults 1 month after vaccination was 96.92% and 89.02%, respectively. In the case of children, the GMTs of Vi antibodies before vaccination were 5.87 +/- 1.34 and 142.59 +/- 2.39 at one month after vaccination. For adults, the GMTs before and one month after vaccination were 5.58 +/- 1.28 and 58.56 +/- 3.67, respectively. Vi antibodies persisted for as long as 6 and 12 months after vaccination. All adverse reactions in adults and children were minor and did not require treatment. The Vi CPS vaccine was safe and immunogenic in adults and children older than 5 years.

Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures.

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4I g) fusion protein, a novel immunosuppressive agent, was expressed in transgenic rice cell suspension culture and its characteristics and in vitro activities were investigated. The expression vector pMYN409 was constructed to express hCTLA4I g under the control of rice alpha-amylase 3D (RAmy3D) promoter. Transgenic calli were prepared by particle bombardment mediated transformation and were screened for hCTLA4I g expression using ELISA. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4I g into the media up to 31.4 mg/L in flask culture. The rice-derived hCTLA4Ig (hCTLA4IgP) was purified from the culture media with affinity chromatography using protein A and compared with CHO-derived hCTLA4Ig (hCTLA4IgM). Recombinant hCTLA4IgP has molecular weight of approximately 50 kDa on SDS-PAGE under reducing condition, which is a little different from that of hCTLA4IgM probably due to the difference of carbohydrate chain structures. Purified hCTLA4IgP was biologically active and was confirmed to suppress T-cell proliferation.

Characterization of human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) expressed in transgenic rice cell suspension cultures.

The avidity for CD80Ig/CD86Ig and the in vitro immunosuppressive effect of recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin, produced by transgenic rice cell suspension cultures (hCTLA4Ig(P)) with CHO-derived recombinant hCTLA4Ig (hCTLA4Ig(M)), were measured. Surface plasmon resonance (SPR) was used for kinetic binding analysis: hCTLA4Ig(P) and hCTLA4Ig(M) had higher avidity for CD80Ig/CD86Ig than for CD28Ig, and the avidity for CD80Ig/CD86Ig was similar. hCTLA4Ig(P) and hCTLA4Ig(M) had similar in vitro immunosuppressive activity against the expression of T cell-derived cytokines, such as IL-2, IL-4, and IFN-gamma, but did not suppress the expression of macrophage-derived cytokines, including TNF-alpha and IL-1beta, as well as NO. Thus the immunosuppressive mechanism of hCTLA4Ig(P) is also T cell-specific and it could therefore be used as an immunosuppressive agent with an equivalent potency to that of hCTLA4Ig(M).