Chinese Hamster Ovary K1 Cells Research Articles

The calcium-binding photoprotein clytin II: Expression of the preferred human codon-optimized clytin II gene in Chinese hamster ovary-K1 cells and its use in the G-protein-coupled receptor assays

Clytin II (CLII) is a Ca2+-binding photoprotein and has been identified as an isotype of clytin I (CLI). CLII consists of apoCLII (an apoprotein) and 2-peroxide of coelenterazine (an adduct of molecular oxygen to coelenterazine), which is identical to the widely used Ca2+-binding photoprotein, aequorin (AQ). However, CLII triggered by Ca2+ exhibits a 4.5-fold higher maximum luminescence intensity (Imax) compared to both AQ and CLI, and it is approximately 5 times less sensitive to Ca2+ than AQ. To confirm the suitability of the preferred human codon-optimized CLII (pCLII) gene for cell-based G-protein-coupled receptor (GPCR) assays, a transformant stably expressing apoprotein of pCLII using the pCLII gene in the mitochondria of CHO–K1 cells was established and in situ regenerated pCLII in the cells were applied to the high-throughput screening system. An ATP-stimulated GPCR assay for endogenous P2Y purinergic receptors was confirmed using the established stable transformant.

PMab-314: An Anti-Giant Panda Podoplanin Monoclonal Antibody.

The giant panda (Ailuropoda melanoleuca) is one of the important species in worldwide animal conservation. Because it is essential to understand the disease of giant panda for conservation, histopathological analyses of tissues are important to understand the pathogenesis. However, monoclonal antibodies (mAbs) against giant panda-derived proteins are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. PDPN is also overexpressed in various human tumors, which are associated with poor prognosis. Here, an anti-giant panda PDPN (gpPDPN) mAb, PMab-314 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening method. PMab-314 recognized N-terminal PA16-tagged gpPDPN-overexpressed Chinese hamster ovary-K1 cells (CHO/PA16-gpPDPN) in flow cytometry. The KD value of PMab-314 for CHO/PA16-gpPDPN was determined as 1.3 × 10-8 M. Furthermore, PMab-314 is useful for detecting gpPDPN in western blot analysis. These findings indicate that PMab-314 is a useful tool for the analyses of gpPDPN-expressed cells.

Cx3Mab-4: A Novel Anti-Mouse CXCR3 Monoclonal Antibody for Flow Cytometry.

C-X-C motif chemokine receptor 3 (CXCR3, CD183) is a G-protein-coupled receptor for CXCL9, CXCL10, and CXCL11. CXCR3 induces chemotaxis of immune cells and promotes inflammation. Various mouse models have been developed to mimic the pathogenesis of diseases and used in the evaluation of therapeutics for these diseases. Although CXCR3 is an attractive target to suppress inflammation, anti-CXCR3 therapeutic agents have not been approved. In this study, we established a novel anti-mouse CXCR3 (mCXCR3) monoclonal antibody, Cx3Mab-4 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Flow cytometric analysis demonstrated that Cx3Mab-4 bound to mCXCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR3) cells, but did not react to parental CHO-K1 cells. The dissociation constant of Cx3Mab-4 was determined as 1.3 × 10-9 M, indicating that Cx3Mab-4 possesses a high affinity to mCXCR3-expressing cells. Cx3Mab-4 could be useful for targeting CXCR3-expressing cells in preclinical mouse models.

Cx4Mab-1: A Novel Anti-Mouse CXCR4 Monoclonal Antibody for Flow Cytometry.

The CXC chemokine receptor 4 (CXCR4, CD184) is a member of the G protein-coupled receptor family that is expressed in most leukocytes. Overexpression of CXCR4 is associated with poor prognosis in not only hematopoietic malignancy but also solid tumors. Because CXCR4 is an attractive target for tumor therapy, reliable preclinical murine models using anti-CXCR4 monoclonal antibodies (mAbs) have been warranted. This study established a novel anti-mouse CXCR4 (mCXCR4) mAb using the Cell-Based Immunization and Screening method. Flow cytometric analysis showed that an anti-mCXCR4 mAb, Cx4Mab-1 (rat IgG2a, kappa), recognized mCXCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR4) cells and endogenously mCXCR4-expressing mouse myeloma P3X63Ag8U.1 (P3U1) cells. Furthermore, Cx4Mab-1 did not recognize mCXCR4-knockout P3U1 cells. The dissociation constants of Cx4Mab-1 for CHO/mCXCR4 and P3U1 were determined as 6.4 × 10-9 M and 2.3 × 10-9 M, respectively, indicating that Cx4Mab-1 possesses a high affinity to both endogenous and exogenous mCXCR4-expressing cells. These results indicate that Cx4Mab-1 could be a useful tool for preclinical mouse models.

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO–K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

Vibegron shows high selectivity and potent agonist activity for β3-adrenoceptors, irrespective of receptor density.

β3-Adrenoceptor (AR) agonists are used to treat patients with an overactive bladder (OAB). Clinical proof-of-concept data have been obtained for the β3-AR agonists vibegron, mirabegron, solabegron, and ritobegron; however, the selectivities of these agents have not been compared directly under the same experimental conditions. Moreover, the bladders of some patients express lower β3-AR densities than those of healthy individuals, and the β3-AR density might be expected to affect agonist activity. This study assessed the β3-AR selectivities of four β3-AR agonists and examined the effects of β-AR density on their pharmacological profiles. Functional cellular assays were performed using Chinese hamster ovary-K1 cells expressing three human β-AR subtypes transfected with different amounts of plasmid DNA (0.1, 0.05, 0.025 μg/well). The half-maximal effective concentration values, intrinsic activities (IAs), and β3-AR selectivities of vibegron, mirabegron, solabegron, and ritobegron were calculated to assess their pharmacological profiles. The β3-AR selectivities of vibegron, mirabegron, solabegron, and ritobegron were >7937-, 517-, 21.3-, and >124-fold higher than for β1-ARs, and >7937-, 496-, >362- and 28.1-fold higher than for β2-ARs, respectively, under the same experimental conditions. The IAs of mirabegron, solabegron, and ritobegron decreased in line with decreasing receptor density, while the IA of vibegron was maintained at the same level as that of the full agonist isoproterenol at various β3-AR densities. Vibegron has high β3-AR selectivity and exhibits full agonist activity, regardless of the β3-AR density. These results suggest that vibegron is a highly effective and safe drug for treating OAB.

Debottlenecking and reformulating feed media for improved CHO cell growth and titer by data-driven and model-guided analyses.

Designing and selecting cell culture media along with their feeding are a key strategy to maximize culture performance in biopharmaceutical processes. However, the sensitivity of mammalian cells to their culture environment necessitates specific nutritional requirements for their growth and the production of high-quality proteins such as antibodies, depending on the cell lines and operational conditions employed. In this regard, previously we developed a data-driven and in-silico model-guided systematic framework to investigate the effect of growth media on Chinese hamster ovary (CHO) cell culture performance, allowing us to design and reformulate basal media. To expand our exploration for media development research, we evaluated two chemically defined feed media, A and B, using a monoclonal antibody-producing CHO K1 cell line in ambr15 bioreactor runs. We observed a significant impact of the feed media on various aspects of cell culture, including growth, longevity, viability, productivity, and the production of toxic metabolites. Specifically, the concentrated feed A was inadequate in sustaining prolonged cell culture and achieving high titers when compared to feed B. Within our framework, we systematically investigated the major metabolic bottlenecks in the tricarboxylic acid cycle and relevant amino acid transferase reactions. This analysis identified target components that play a crucial role in alleviating bottlenecks and designing highly productive cell cultures, specifically the addition of glutamate to feed A and asparagine to feed B. Based on our findings, we reformulated the feeds by adjusting the amounts of the targeted amino acids and successfully validated the effectiveness of the strategy in promoting cell growth, life span, and/or titer. This article is protected by copyright. All rights reserved.

A Novel Anti-CD44 Variant 9 Monoclonal Antibody C44Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers

Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has been shown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3-10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay and characterized their applications as flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C44Mab-1 (IgG1, kappa), reacted with a peptide of the variant 9-encoded region, indicating that C44Mab-1 recognizes CD44v9. C44Mab-1 could recognize CHO/CD44v3-10 cells or colorectal cancer cell lines (COLO201 and COLO205) in flow cytometric analysis. The apparent dissociation constant (KD) of C44Mab-1 for CHO/CD44v3-10, COLO201, and COLO205 was 2.5 × 10-8 M, 3.3 × 10-8 M, and 6.5 × 10-8 M, respectively. Furthermore, C44Mab-1 was able to detect the CD44v3-10 in western blotting and the endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C44Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers.

Development of a Novel Anti-CD44 Variant 7/8 Monoclonal Antibody, C44Mab-34, for Multiple Applications against Oral Carcinomas

Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker as it plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mouse with a CD44 variant (CD44v3-10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide that covers both variant 7- and variant 8-encoded regions, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO) cells or the oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3-10 and HSC-3 was 1.4 × 10-9 and 3.2 × 10-9 M, respectively. C44Mab-34 could detect CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications and is expected to be useful in the application of OSCC diagnosis and therapy.

Enhanced cell growth, production, and mAb quality produced in Chinese hamster ovary-K1 cells by supplementing polyamine in the media.

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. KEY POINTS: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing.

Assessment of the Macrophage Scavenger Receptor CD163 in Mediating Glaesserella parasuis Infection of Host Cells.

The macrophage CD163 surface glycoprotein is a member of the SRCR family class B, which has been identified as the key trigger in host-pathogen interactions, but its specific roles in sensing Glaesserella parasuis (G. parasuis) infection are largely unknown. Here, we investigated porcine CD163 in mediating the adhesion and immune response of G. parasuis using in vitro host-bacteria interaction models. CD163-overexpressing Chinese hamster ovary K1 cells (CHO-K1) showed obvious subcellular localization in the cytoplasm, especially in the cytomembrane. Although detection using scanning electron microscopy (SEM) confirmed the bacterial adhesion, there was no significant difference in the adhesion of G. parasuis to CHO-K1 cells between the presence and absence of CD163. In addition, similar results were observed in 3D4/21 cells. Meanwhile, bindings of G. parasuis to nine synthetic peptides, the bacterial binding motifs within SRCR domains of CD163, were weak based on a solid-phase adhesion assay and agglutination assay. Moreover, CD163 had no effect on the expression of G. parasuis-induced inflammatory cytokines (IL-6, INF-γ, IL-10, IL-4 and TGF-β) in CHO-K1 cells. In conclusion, these findings indicate that porcine CD163 plays a minor role in sensing G. parasuis infection.

Development of a Novel Anti-CD44 Variant 4 Monoclonal Antibody C44Mab-108 for Immunohistochemistry.

CD44 has been known as a marker of tumor-initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in the malignant progression of cancers by promoting stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and the establishment of the therapy. However, the function of the variant 4-encoded region has not been elucidated. Therefore, specific monoclonal antibodies (mAbs) against variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) mAbs by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterize them. One of the established clones (C44Mab-108; IgG1, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The KD of C44Mab-108 for CHO/CD44 v3-10 was 3.4 × 10-7 M. In western blot analysis, C44Mab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, C44Mab-108 stained formalin-fixed paraffin-embedded (FFPE) oral squamous carcinoma tissues in immunohistochemistry. These results indicated that C44Mab-108 is useful to detect CD44v4 in immunohistochemistry using FFPE tissues.

Antigenicity and immunogenicity of SARS-CoV-2 surface glycoprotein fragment in CHO cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein that projects from the virus surface is highly immunogenic. It is considered to be the target of many neutralizing antibodies as well as a target in vaccine design efforts. Evaluation the immunogenicity of a recombinant fragment of the spike protein (rfsp) that is comprised of Receptor Binding Domain (RBD), S1/S2 cleavage site, and fusion peptide (FP) as immunogenic proteins of SARS-COV-2, in BALB/c mice and evaluation of the efficacy of epitopes rfsp as a multi-subunit chimeric vaccine. The present study made use of CHO-K1 (Chinese hamster ovary K1) cells to create a cell line for constant expression rfsp. The rfsp was purified with Ni-NTA chromatography and confirmed by Western blotting. The immunogenicity and neutralizing antibody efficacy of rfsp were evaluated in BALB/c mice. ELISA was employed to test rfsp via sera of COVID-19 convalescent patients infected with SARS-CoV-2 alpha and delta variants. Our results showed significant differences in antibody titers in immunized mice compared to the control groups and neutralizing antibodies were positive, sera from mice immunized are capable of bound SARS-CoV-2 virus, chimer peptide is capable bound antibodies patients infected with SARS-CoV-2 and patients infected with delta variant SARS-CoV-2. Overall, these results indicate that rfsp protein would be a novel potential antigen candidate for the development of a subunit SARS CoV-2 vaccine and rfsp has the potential to be a useful option for the development of the assays for serodiagnosis of SARS-CoV-2 infection.

Epitope Mapping of an Anti-EpCAM Monoclonal Antibody (EpMab-37) Using the Alanine Scanning Method.

The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, and plays critical roles in cell adhesion, proliferation, and tumorigenesis. EpCAM has been considered as a promising target for tumor diagnosis and therapy. Anti-EpCAM monoclonal antibodies (mAbs) have been developed for EpCAM-overexpressed tumors, and several clinical trials have demonstrated promising outcomes. We previously established an anti-EpCAM mAb, EpMab-37 (mouse IgG1, kappa), using the Cell-Based Immunization and Screening method. EpMab-37 was revealed to recognize the conformational epitope of EpCAM. In this study, we determined the critical epitope of EpMab-37 by flow cytometry using the 1 × alanine scanning (1 × Ala-scan) and the 2 × alanine scanning (2 × Ala-scan) method. We first performed flow cytometry by 1 × Ala-scan using one alanine (or glycine)-substituted EpCAM mutants, which were expressed on Chinese hamster ovary-K1 cells, and found that the EpMab-37 did not recognize the R163A mutant of EpCAM. We next performed flow cytometry by 2 × Ala-scan using two alanine (or glycine) residues-substituted EpCAM mutants, and confirmed that EpMab-37 did not recognize R163A-including mutants of EpCAM. The results indicated that the critical binding epitope of EpMab-37 includes Arg163 of EpCAM.

Screening for microbial potentiators of neutral lipid degradation in CHO-K1 cells.

A cell-based assay was conducted to screen microbial culture broths for potentiators of neutral lipid degradation in Chinese Hamster Ovary K1 cells. A total of 5,363 microbial cultures from fungi and actinomycetes were screened in this assay. Brefeldin A (1) from fungal cultures was found to promote the degradation of triacylglycerol (TG) with an EC50 of 2.6 µM. Beauveriolides I (2), III (3), beauverolides A (4), B (5), and K (6) from fungal cultures showed potentiating effect on cholesteryl ester (CE) degradation with EC50s ranging from 0.02 to 0.13 µM. Among these compounds, 2 and 6 exhibited the strongest activities (EC50, 0.02 µM). From actinomycete cultures, oxohygrolidin (7) (EC50 for TG and CE, > 1.7 and 0.8 µM, respectively) and hygrolidin (8) (EC50 for TG and CE, 0.08 and 0.004 µM, respectively) promoted degradation of CE more preferably than TG.

Antitumor Activity of an Anti-EGFR/HER2 Bispecific Antibody in a Mouse Xenograft Model of Canine Osteosarcoma.

The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanized anti-EGFR and anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs) have been shown to improve patients' survival. Canine tumors resemble human tumors in the initiation and progression. We previously established a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf) and a mouse-dog chimeric anti-HER2 mAb (H77Bf), which exerted antitumor activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognized dog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow cytometry. E134Bf-H77scFv also reacted with dEGFR/dHER2-positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3 × 10-9 M). Furthermore, E134Bf-H77scFv exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in the presence of canine mononuclear cells and complement, respectively. Moreover, administration of E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early compared with the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable treatment regimen for canine tumors.

Identification of the Binding Epitope of an Anti-mouse CCR4 Monoclonal Antibody, C4Mab-1.

C-C chemokine receptor 4 (CCR4) is one of G protein-coupled receptors, and interacts with chemokines, CCL17 and CCL22. CCR4 is expressed on T cells such as helper T type 2 cells, regulatory T cells, and interleukin 17-producing T helper cells. CCR4 is associated with T cells trafficking into the tumor microenvironment, and is associated with tumor progression or metastasis. Therefore, CCR4 may be a potential therapeutic option for T cell malignancies. C4Mab-1 is a novel anti-mouse CCR4 (mCCR4) monoclonal antibody produced by mCCR4 N-terminal peptide immunization. C4Mab-1 is useful for flow cytometric analysis. In this study, we conducted the epitope mapping of C4Mab-1 using enzyme-linked immunosorbent assay (ELISA) and peptide blocking assay. The result of ELISA indicated that Thr7, Asp8, and Gln11 of mCCR4 are the critical amino acids for the C4Mab-1 binding. Furthermore, peptide blocking assay by flow cytometry showed that Thr7, Asp8, and Gln11 of mCCR4 are essential for C4Mab-1 binding to mCCR4-overexpressed Chinese hamster ovary-K1 (CHO/mCCR4) cells, and Val6, Thr9, and Thr10 are involved in the C4Mab-1 binding to CHO/mCCR4 cells. These results indicate that the critical binding epitope of C4Mab-1 includes Thr7, Asp8, and Gln11 of mCCR4.

Development of a Novel Anti-EpCAM Monoclonal Antibody for Various Applications.

The epithelial cell adhesion molecule (EpCAM) is a cell surface glycoprotein, which is widely expressed on normal and cancer cells. EpCAM is involved in cell adhesion, proliferation, survival, stemness, and tumorigenesis. Therefore, EpCAM is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-EpCAM monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established recombinant anti-EpCAM mAbs, recEpMab-37 (mouse IgG1, kappa), reacted with EpCAM-overexpressed Chinese hamster ovary-K1 cells (CHO/EpCAM) or a colorectal carcinoma cell line (Caco-2). In contrast, recEpMab-37 did not react with EpCAM-knocked out Caco-2 cells. The KD of recEpMab-37 for CHO/EpCAM and Caco-2 was 2.0 × 10−8 M and 3.2 × 10−8 M, respectively. We observed that EpCAM amino acids between 144 to 164 are involved in recEpMab-37 binding. In Western blot analysis, recEpMab-37 detected the EpCAM of CHO/EpCAM and Caco-2 cells. Furthermore, recEpMab-37 could stain formalin-fixed paraffin-embedded colorectal carcinoma tissues by immunohistochemistry. Taken together, recEpMab-37, established by the CBIS method, is useful for detecting EpCAM in various applications.

Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody Exerted Antitumor Activities in Mouse Xenograft Models of Canine Mammary Gland Tumor.

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy through gene amplification and/or protein overexpression. In our previous study, we developed an anti-human EGFR (hEGFR) monoclonal antibody, clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 (134-mG2a-f) exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed Chinese hamster ovary-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, the reactivity of 134-mG2a-f against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f highly exerted ADCC and CDC for SNP. The administration of 134-mG2a-f significantly suppressed the SNP xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine mammary gland tumors, and could be valuable as part of an antibody treatment regimen for them.

Development of a Monoclonal Antibody PMab-292 Against Ferret Podoplanin.

Ferrets (Mustela putorius furo) have been used as small animal models to investigate severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infections. Pathological analyses of these tissue samples, including those of the lung, are, therefore, essential to understand the pathogenesis of SARS-CoVs and evaluate the action of therapeutic monoclonal antibodies (mAbs) against this disease. However, mAbs that recognize ferret-derived proteins and distinguish between specific cell types, such as lung epithelial cells, are limited. Podoplanin (PDPN) has been identified as an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-ferret PDPN (ferPDPN) mAb PMab-292 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. PMab-292 recognized ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells by flow cytometry and Western blotting. The kinetic analysis using flow cytometry showed that the KD of PMab-292 for CHO/ferPDPN was 3.4 × 10-8 M. Furthermore, PMab-292 detected lung type I alveolar epithelial cells, lymphatic endothelial cells, and glomerular/Bowman's capsule in the kidney using immunohistochemistry. Hence, these results propose the usefulness of PMab-292 in analyzing ferret-derived tissues for SARS-CoV-2 research.