Hyo Jeong Hong

Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure

Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 microM. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific antibody productivity (qAb) rapidly increased with increasing MTX level up to 0.08 microM, and thereafter, it gradually increased to 20 microg/10(6) cells/day at 4 microM MTX. Southern blot analysis showed that the enhanced qAb at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 microM MTX in regard to antibody production was investigated during long-term culture in the absence of MTX. The qAb of all subclones significantly decreased during the culture. However, the relative extent of decrease in qAb was variable among the subclones. The HP subclone isolated at 1 microM MTX was most stable and could retain 59% of the initial qAb after 80 days of cultivation. Southern blot analysis showed that this decrease in qAb of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased qAb, the HP subclone isolated at 1 microM MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long-term culture.

TMPRSS4 promotes invasion, migration and metastasis of human tumor cells by facilitating an epithelial–mesenchymal transition

TMPRSS4 is a novel type II transmembrane serine protease found at the cell surface that is highly expressed in pancreatic, colon and gastric cancer tissues. However, the biological functions of TMPRSS4 in cancer are unknown. Here we show, using reverse transcription–PCR, that TMPRSS4 is highly elevated in lung cancer tissues compared with normal tissues and is also broadly expressed in a variety of human cancer cell lines. Knockdown of TMPRSS4 by small interfering RNA treatment in lung and colon cancer cell lines was associated with reduction of cell invasion and cell-matrix adhesion as well as modulation of cell proliferation. Conversely, the invasiveness, motility and adhesiveness of SW480 colon carcinoma cells were significantly enhanced by TMPRSS4 overexpression. Furthermore, overexpression of TMPRSS4 induced loss of E-cadherin-mediated cell–cell adhesion, concomitant with the induction of SIP1/ZEB2, an E-cadherin transcriptional repressor, and led to epithelial–mesenchymal transition events, including morphological changes, actin reorganization and upregulation of mesenchymal markers. TMPRSS4-overexpressing cells also displayed markedly increased metastasis to the liver in nude mice upon intrasplenic injection. Taken together, these studies suggest that TMPRSS4 controls the invasive and metastatic potential of human cancer cells by facilitating an epithelial–mesenchymal transition; TMPRSS4 may be a potential therapeutic target for cancer treatment.

An Anthrax Lethal Factor-Neutralizing Monoclonal Antibody Protects Rats before and after Challenge with Anthrax Toxin

ABSTRACT Lethal factor (LF) is a component of anthrax lethal toxin (LeTx). We generated anti-LF murine monoclonal antibodies (MAbs) that show LeTx-neutralizing activity in vitro and in vivo. Anti-LF MAbs were generated by immunization with recombinant LF, and the MAbs showing LeTx-neutralizing activity in vitro were selected. Two MAbs with the highest affinities, 5B13B1 (dissociation constant [Kd], 2.62 nM) and 3C16C3 (Kd, 8.18 nM), were shown to recognize the same or closely overlapping epitopes on domain III of LF. The 50% inhibitory concentration of 5B13B1 (0.21 μg/ml) was approximately one-third that of 3C16C3 (0.63 μg/ml) in the in vitro LeTx-neutralization assay. The 5B13B1 antibody, which had the highest neutralizing activity, provided perfect protection against LeTx challenge in an in vivo LeTx neutralization assay using Fisher 344 rats. In addition, the antibody showed pre- and postexposure prophylactic effects in the animal experiments. This is the first report that an MAb binding to domain III of LF has neutralizing activity against LeTx. The 5B13B1 antibody may be useful in prophylaxis against anthrax poisoning.

Acquisition of chemoresistance in intrahepatic cholangiocarcinoma cells by activation of AKT and extracellular signal-regulated kinase (ERK)1/2.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor and is refractory to conventional chemotherapy. The aim of this study is therefore to elucidate the mechanism of chemoresistance in ICC which is not fully understood. We generated cisplatin resistant ICC cells via long term exposure to cisplatin and found that these cells are also resistant to 5-fluorouracil (5-FU) and gemcitabine. The chemoresistant cells showed enhanced Bcl-2 expression and reduced Bax expression compared to parental ICC cells. In addition, the resistant cells showed enhanced activation of AKT and extracellular signal-regulated kinase (ERK) 1/2. Inhibition of AKT activation by phosphoinocitide 3-kinase (PI3K) inhibitor LY294002 resulted in reduced Bcl-2 expression and enhanced Bax expression and thus induced apoptosis in the resistant cells, whereas inhibition of ERK1/2 activation by mitogen-activated protein kinase (MEK) inhibitor U0126 did not induce apoptosis without affecting the expression of Bcl-2 and Bax but decreased cell growth. Moreover, the inhibition of AKT or ERK1/2 sensitized the resistant cells to cisplatin and therefore resulted in greatly enhanced cisplatin-induced apoptosis and growth inhibition in the cells. The results indicate that AKT and ERK1/2 signaling mediate chemoresistance in the cells and could be important therapeutic targets for overcoming chemoresistance in ICC.

A humanized anti--4-1BB monoclonal antibody suppresses antigen-induced humoral immune response in nonhuman primates.

The interaction of 4-1BB and its ligand plays an important role in the regulation of T-cell–mediated immune responses. In this study, the authors examined the effect of a humanized anti–4-1BB monoclonal antibody (H4B4) on ovalbumin-induced immune responses in baboons. Previously, a mouse monoclonal antibody, 4B4 against the human 4-1BB molecule, was generated and characterized. Based on this antibody, a humanized version of 4B4 monoclonal antibody was constructed and the resultant antibody, H4B4, showed full recovery of the binding activity of the original antibody 4B4: a 1.5-fold increase in affinity for 4-1BB. In addition, H4B4 mediated antibody-dependent cellular cytotoxicity of activated human peripheral blood T cells and CEM cells in a dose-dependent manner. Weekly administration of H4B4 at doses of 1 or 4 mg/kg could suppress immunoglobulin G production against ovalbumin. This was not a result of the overall immune suppression, because the numbers of B and T cells and the total immunoglobulin G production were not altered during treatment with H4B4. These findings suggest that treatment with H4B4 may be a valid therapeutic approach to control unwanted immune responses in persons with autoimmune diseases.

Heat Shock 70‐kDa Protein 8 Isoform 1 Is Expressed on the Surface of Human Embryonic Stem Cells and Downregulated upon Differentiation

The cell‐surface markers used routinely to define the undifferentiated state and pluripotency of human embryonic stem cells (hESCs) are those used in mouse embryonic stem cells (mESCs) because of a lack of markers directly originated from hESC itself. To identify more hESC‐specific cell‐surface markers, we generated a panel of monoclonal antibodies (MAbs) by immunizing the irradiated cell clumps of hESC line Miz‐hES1, and selected 26 MAbs that were able to bind to Miz‐hES1 cells but not to mESCs, mouse embryonic fibroblast cells, and STO cells. Most antibodies did not bind to human neural progenitor cells derived from the Miz‐hES1 cells, either. Of these, MAb 20‐202S (IgG1, κ) immunoprecipitated a cell‐surface protein of 72‐kDa from the lysate of biotin‐labeled Miz‐hES1 cells, which was identified to be heat shock 70‐kDa protein 8 isoform 1 (HSPA8) by quadrupole time‐of‐flight tandem mass spectrometry. Immunocytochemical analyses proved that the HSPA8 protein was also present on the surface of hESC lines Miz‐hES4, Miz‐hES6, and HSF6. Two‐color flow cytometric analysis of Miz‐hES1 and HSF6 showed the coexpression of the HSPA8 protein with other hESC markers such as stage‐specific embryonic antigen 3 (SSEA3), SSEA4, TRA‐1‐60, and TRA‐1‐81. Flow cytometric and Western blot analyses using various cells showed that MAb 20‐202S specifically bound to the HSPA8 protein on the surface of Miz‐hES1, contrary to other anti‐HSP70 antibodies examined. Furthermore, the surface expression of the HSPA8 protein on Miz‐hES1 was markedly downregulated upon differentiation. These data indicate that a novel MAb 20‐202S recognizes the HSPA8 protein on the surface of hESCs and suggest that the HSPA8 protein is a putative cell‐surface marker for undifferentiated hESCs.

Molecular Chaperone GRP78/BiP Interacts with the Large Surface Protein of Hepatitis B Virus In Vitro and In Vivo

ABSTRACT The proper folding and assembly of viral envelope proteins are mediated by host chaperones. In this study, we demonstrated that an endoplasmic reticulum luminal chaperone GRP78/BiP bound specifically to the pre-S1 domain of the L protein in vitro and in vivo where complete viral particles were secreted, suggesting that GRP78/BiP plays an essential role in the proper folding of the L protein and/or assembly of viral envelope proteins.

Generation and characterization of a novel tetravalent bispecific antibody that binds to hepatitis B virus surface antigens

Hepatitis B virus (HBV) infection is a worldwide public health problem affecting about 350 million people. HBV envelope contains three surface antigens, called pre-S1, pre-S2 and S. For the prophylaxis of HBV infection, only an anti-S monoclonal antibody was tested for the protective efficacy against HBV infection, but it was shown to be incomplete. In addition, some immune escape mutants carrying mutations on the S antigen were reported. Therefore, a multivalent bispecific antibody rather than a single monoclonal antibody would be more beneficial for the prophylaxis of HBV infection. We have generated a novel tetravalent bispecific antibody with two binding sites for each of the S and pre-S2 antigens. Each of the antigen-binding sites was composed of a single-chain Fv (ScFv). The tetravalent antibody was generated by constructing a single gene encoding a single-chain protein. This protein consisted of an anti-S ScFv whose carboxyl end was tethered, through a 45 amino acid linker, to the amino terminus of anti-preS2 ScFv that in turn was joined to the hinge region of human gamma1 constant region. The single-chain protein was expressed in Chinese hamster ovary cells and secreted in culture supernatant as a homodimeric molecule. The tetravalent bispecific antibody showed both anti-S and anti-pre-S2 binding activities. In addition, the binding affinity of the bispecific antiboy for HBV particles was greater than that of either parental antibody. The tetravalent bispecific antibody is a potentially useful reagent for the prevention and treatment of HBV infection.

Limitations to the development of humanized antibody producing Chinese hamster ovary cells using glutamine synthetase-mediated gene amplification.

Recombinant Chinese hamster ovary (CHO) cells expressing a humanized antibody were obtained by transfection of an antibody expression vector (pKC‐GS‐HC‐huS) into CHO‐K1 cells and subsequent glutamine synthetase (GS)‐mediated gene amplification in media containing different concentrations of methionine sulfoximine (MSX). Concentrations consisted of 25, 200, 500, and 1000 μM of MSX. The highest producer (HP) subclones were isolated from each MSX level by the limiting dilution method and were characterized with respect to antibody production. No positive relationship was observed between specific antibody productivity (qAb) and MSX concentration. Furthermore, it was found that the antibody production stability of these subclones was very poor even in the presence of selection pressure. During long‐term cultures in the presence of the corresponding concentrations of MSX, qAb of all HP subclones significantly decreased for the first six passages and thereafter stabilized. Southern and slot blot analyses showed that the loss of antibody gene copies was only partially responsible for the decreased qAb. Fluorescence in situ hybridization (FISH) analysis revealed some cytogenetic features indicative of antibody production instability. Unstable chromosomal structures including dicentrics, rings, and extremely long chromosomes were observed. Amplified sequences enclosed in nuclear projections were often observed. The telomeric repeat sequence, which may be involved in the stabilization of amplified arrays, was found to be absent at the ends of most marker chromosomes. Furthermore, FISH analysis revealed that the overall chromosome content was duplicated in some HP subclones. When metaphase of 12 high producing parental clones was examined, the frequency of occurrence of the polyploidy was 25%. Taken together, the data obtained here suggests that instability could be a concern in the development of CHO cells with GS‐mediated gene amplification.

Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody

The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody (AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn97 by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with 125I or 131I showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.