Sang Jick Kim

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.

Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure.

Recombinant Chinese hamster ovary (rCHO) cells expressing a high level of chimeric antibody were obtained by cotransfection of heavy- and light-chain cDNA expression vectors into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level up to 1.0 microM. To determine the clonal variability within the amplified cell population in regard to antibody production stability, 20 subclones were randomly isolated from the amplified cell population at 1.0 microM MTX (CS13-1.0 cells). Clonal analysis showed that CS13-1.0 cells were heterogeneous with regard to specific growth rate (mu) and specific antibody productivity (qAb), although they were derived from a single clone. The mu and qAb of 20 subclones were in the range of 0.51 to 0.72 day-1 and 10.9 to 19.1 microgram/10(6) cells/day, respectively. During 8 weeks of cultivation in the absence of selective pressure, the mu of most subclones did not change significantly. On the other hand, their qAb decreased significantly. Furthermore, the relative decrease in qAb varied among subclones, ranging from 30% to 80%. Southern and Northern blot analyses showed that this decreased qAb resulted mainly from the loss of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNAs. For the sake of screening convenience, an attempted was made to correlate the initial properties of subclones (such as mu, qAb, and Ig gene copies) with their antibody production stability during long-term culture. Among these initial properties examined, only qAb of subclones could help to predict their stability to some extent. The subclones with high qAb were relatively stable with regard to antibody production during long-term culture in the absence of selective pressure (P < 0. 005, ANOVA). Taken together, the clonal heterogeneity in an amplified CHO cell population necessitates clonal analysis for screening stable clones with high qAb.

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.

Broadly neutralizing anti-hepatitis B virus antibody reveals a complementarity determining region H3 lid-opening mechanism

The humanized monoclonal antibody HzKR127 recognizes the preS1 domain of the human hepatitis B virus surface proteins with a broadly neutralizing activity in vivo. We present the crystal structures of HzKR127 Fab and its complex with a major epitope peptide. In the complex structure, the bound peptide forms a type IV β-turn followed by 310 helical turn, the looped-out conformation of which provides a structural basis for broad neutralization. Upon peptide binding, the antibody undergoes a dramatic complementarity determining region H3 lid opening. To understand the structural implication of the virus neutralization, we carried out comprehensive alanine-scanning mutagenesis of all complementarity determining region residues in HzKR127 Fab. The functional mapping of the antigen-combining site demonstrates the specific roles of major binding determinants in antigen binding, contributing to the rational design for maximal humanization and affinity maturation of the antibody.

Enhanced specific antibody productivity of calcium alginate-entrapped hybridoma is cell line-specific

In order to determine whether the enhanced specific antibody productivity (qMAb) of calcium alginate-entrapped hybridoma is cell line-specific, calcium alginate-entrapped hybridomas (4A2 and DB9G8) were cultivated under the condition where we had previously observed significantly enhancedqMAbof calcium alginate-entrapped S3H5/γ2bA2 hybridoma. Unlike S3H5/γ2bA2 hybridoma, neither 4A2 nor DB9G8 hybridomas showed persistently enhancedqMAbwhen they were entrapped in calcium alginate beads. The enhancedqMAbof entrapped 4A2 and DB9G8 hybridomas, which was 2–3 times higher than theqMAbof free-suspended cells in a control experiment, was observed only during the early stage of the culture. During the early stage of the culture, the viable cell concentration decreased probably due to cell damage during the entrapment process. As cell growth resumed, theqMAbdecreased to the similar level ofqMAbof free-suspended cells within 5–7 days. Thus, we conclude that the enhancedqMAbof calcium alginate-entrapped hybridomas is cell line-specific.

Generation of antibodies recognizing an aberrant glycoform of human tissue inhibitor of metalloproteinase-1 (TIMP-1) using decoy immunization and phage display.

Aberrant glycosylation of human tissue inhibitor of metalloproteinase-1 (TIMP-1) by N-acetylglucosaminyltransferase-V (GnT-V) was previously reported to be related to cancer progression. Here, we report on the antibodies recognizing the structural features initiated by an addition of N-linked β(1,6)-N-acetylglucosamine (GlcNAc) branch by GnT-V on TIMP-1. Two glycoforms of TIMP-1, TIMP1-L produced in Lec4 cells without GnT-V activity and TIMP1-B in GnT-V overexpressing transfectant cells, were purified from culture supernatant and used for generation of antibodies. TIMP1-L was injected in the left hind footpad of mice as decoy and TIMP1-B in the right hind footpad as immunogen. Phage-displayed scFv library was constructed from the B cells retrieved from the right popliteal lymph nodes and subjected to panning and screening. Phage ELISA of individual clones revealed the scFv clones with preferential binding activity to TIMP1-B, and they were converted into an scFv-Fc format for further characterization of binding specificity. Glycan binding assay of an antibody, 1-9F, revealed its differential specificity toward an extension of glycan structure initiated with β(1,6)-GlcNAc linkage and terminally decorated with a sialic acid. This study demonstrates feasibility of a new strategy combining decoy immunization with phage display for the efficient generation of antibodies tracking down structural features of different glycoforms.

Inhibition of 4‐1BBL‐regulated TLR response in macrophages ameliorates endotoxin‐induced sepsis in mice

Activation of Toll‐like receptor (TLR) signaling rapidly induces the expression of inflammatory genes, which is persistent for a defined period of time. However, uncontrolled and excessive inflammation may lead to the development of diseases. 4‐1BB ligand (4‐1BBL) plays an essential role in sustaining the expression of inflammatory cytokines by interacting with TLRs during macrophage activation. Here, we show that inhibition of 4‐1BBL signaling reduced the inflammatory responses in macrophages and ameliorated endotoxin‐induced sepsis in mice. A 4‐1BB‐Fc fusion protein significantly reduced TNF production in macrophages by blocking the oligomerization of TLR4 and 4‐1BBL. Administration of 4‐1BB‐Fc suppressed LPS‐induced sepsis by reducing TNF production, and the coadministration of anti‐TNF and 4‐1BB‐Fc provided better protection against LPS‐induced sepsis. Taken together, these observations suggest that inhibition of the TLR/4‐1BBL complex formation may be highly efficient in protecting against continued inflammation, and that 4‐1BB‐Fc could be a potential therapeutic target for the treatment of inflammatory diseases.

Humanization by Guided Selections

Guided selection provides a powerful tool for humanization of the preexisting nonhuman antibodies as exemplified by HUMIRA, the worlds first human antibody approved. This chapter describes the sequential guided selection procedure in which mouse VL and VH domains are replaced sequentially with human VL and VH, respectively to derive completely human antibody. The detailed protocols for construction of phage-displayed antibody library, panning, screening, and characterization, are included to achieve successful selection of human antibody with similar characteristics to original mouse antibody.

Heterogeneity within DHFR-Mediated Gene Amplified Population of Cho Cells Producting Chimeric Antibodies

Recombinant Chinese hamster ovary (CHO) cells expressing a high level of chimeric antibody were obtained by cotransfection 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, and 1.0 μM. In order to determine the clonal variability within amplified cell population in regard to antibody production stability, 20 subclones were randomly isolated from amplified cell population at 1 μM MTX and their antibody production stability was characterized. During 8 weeks of cultivation in the absence of selective pressure, the specific antibody productivity (qAb) decreased significantly. However, the relative extent of decrease in qAb was varied among subclones, ranging from 30 % to 80 %, showing that clonal analysis allows one to screen the clone with better stability. Southern and Northern blot analyes showed that this decreased qAb resulted mainly from the loss of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNAs. Furthermore, since the stability of subclones regarding qAbis not related with either their qAb or Ig gene copies at the beginning of long-term culture, their stability during long-term culture cannot be predicted based on theirinitial qAb or Ig gene copies.

Effect of Cloned Gene Dosage on the Expression Level and Stability of Chimeric Antibody Producing CHO Cells

Recombinant Chinese hamster ovary (CHO) cells expressing high level of chimeric antibody against S surface antigen of hepatitis B virus (HBV) were obtained using stepwise methotrexate (MTX)-dihydrofolate reductase (dhfr) mediated gene amplification procedure and subsequent cell cloning by limiting dilution method. Specific growth rate of amplified clone was inversely proportional to the MTX level used in gene amplification. Specific antibody productivity (q Ab) of amplified clones rapidly increased with MTX level up to 0.32 μM and thereafter, q Ab became saturated at 20 μg/106cells/day. The heavy chain gene copy number of amplified clones directly correlated with q Ab. The q Ab of all tested clones gradually decreased during the long-term culture in the absence of MTX, which resulted from the loss of antibody cDNA. Despite the decreased q Ab, the 1 μM amplified clone could maintain high volumetric antibody productivity over three months because cell growth improved during long-term culture in the absence of MTX.