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CD39 is involved in adenosine metabolism through conversion of extracellular ATP to adenosine. Because extracellular adenosine plays a critical role in the immune suppression of tumor microenvironment, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is one of the important strategies for tumor therapy. In this study, we developed specific and sensitive mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mCD39 mAbs, which were established by the CBIS method including C39Mab-1 (rat IgG2a, kappa) and C39Mab-2 (rat IgG2a, lambda), reacted with not only mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) but also endogenously mCD39-expressed cell lines, such as L1210 (mouse lymphocytic leukemia) and J774-1 (mouse macrophage-like) cell lines through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constant (KD) of C39Mab-1 and C39Mab-2 for CHO/mCD39 was 7.3 × 10−9 M and 5.5 × 10−9 M, respectively. KD of C39Mab-1 and C39Mab-2 for L1210 was 3.3 × 10−9 M and 3.6 × 10−10 M, respectively. Furthermore, C39Mab-1 could detect the lysate of CHO/mCD39 by western blot analysis. These results indicate that C39Mab-1 and C39Mab-2 are useful for the detection of mCD39 in many functional studies.

The epidermal growth factor receptor (EGFR) is a type I transmembrane protein, which is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. EGFR is a c...

The development of protein-specific antibodies is essential for understanding a wide variety of biological phenomena. Parasitic and viral infections and cancers are known to occur within California sea lion (Zalophus californianus) populations. However, sensitive and specific monoclonal antibodies (mAbs) for the pathophysiological analysis of California sea lion tissues have not yet been developed. A type I transmembrane glycoprotein, podoplanin (PDPN), is a known diagnostic marker of lymphatic endothelial cells. We have previously developed several anti-PDPN mAbs in various mammalian species, with applications in flow cytometry, Western blotting, and immunohistochemistry. In this study, we established a novel mAb against California sea lion PDPN (seaPDPN), clone PMab-269 (mouse IgG1, kappa), using a Cell-Based Immunization and Screening method. PMab-269 is specifically detected in seaPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells using flow cytometry and Western blotting. Moreover, PMab-269 clearly identified pulmonary type I alveolar cells, renal podocytes, and colon lymphatic endothelial cells in California sea lion tissues using immunohistochemistry. These findings demonstrate the usefulness of PMab-269 for the pathophysiological analysis of lung, kidney, and lymphatic tissues of the California sea lion.

CC chemokine receptor 8 (CCR8) belongs to the class A of G protein-coupled receptor. It is highly expressed on Treg and T helper 2 (T

C-C motif chemokine receptor 8 (CCR8) is a G protein-coupled receptor predominantly expressed in regulatory T (Treg) and T helper 2 cells. The evidence that CCR8 expression in Treg is increased in cancers, CCR8 increases migration activity of Treg, and CCR8 induces the anti-apoptotic activity in T cell leukemia and lymphoma suggests that CCR8 is associated with cancer development. Thus, developing a specific monoclonal antibody (mAb) for CCR8 is useful for diagnostic and therapeutic purposes and the anti-CCR8 mAb becomes a remarkable experimental tool for basic research. We previously developed an anti-mouse CCR8 (mCCR8) mAb called C

The epithelial cell adhesion molecule (EpCAM) is a calcium‑independent, homophilic, intercellular adhesion factor classified as a transmembrane glycoprotein. In addition to cell adhesion, EpCAM also contributes to cell signaling, differentiation, proliferation, and migration. EpCAM is an essential factor in the carcinogenesis of numerous human cancers. In the present study, we developed and validated an anti‑EpCAM monoclonal antibody (mAb), EpMab‑16 (IgG2a, kappa), by immunizing mice with EpCAM‑overexpressing CHO‑K1 cells. EpMab‑16 specifically reacted with endogenous EpCAM in oral squamous cell carcinoma (OSCC) cell lines in flow cytometry and Western blot analyses. It exhibited a plasma membrane‑like stain pattern in OSCC tissues upon immunohistochemical analysis. The KD for EpMab‑16 in SAS and HSC‑2 OSCC cells were assessed via flow cytometry at 1.1x10‑8 and 1.9x10‑8 M, respectively, suggesting moderate binding affinity of EpMab‑16 for EpCAM. We then assessed whether the EpMab‑16 induced antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) against OSCC cell lines, and antitumor capacity in a murine xenograft model. In vitro experiments revealed strong ADCC and CDC inducement against OSCC cells treated with EpMab‑16. In vivo experiments on OSCC xenografts revealed that EpMab‑16 treatment significantly reduced tumor growth compared with the control mouse IgG. These data indicated that EpMab‑16 could be a promising treatment option for EpCAM‑expressing OSCCs.

Anti-bear podoplanin (bPDPN) monoclonal antibodies (mAbs), including PMab-247 and PMab-241, have been previously established. Although PMab-247 has shown positive immunostaining for lymphatic endothelial cells (LECs), type I alveolar cells of the lung, and podocytes of the kidney, PMab-241 stains LECs but does not react with lung type I alveolar cells. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) and the PLAG-like domain (PLD). The binding epitope of PMab-247 was previously determined to include bPDPN residues Asp76, Arg78, Glu80, and Arg82. Among these, Glu80 and Arg82 are included in PLD of bPDPN. The purpose of this study is to determine the binding epitope of PMab-241 and to clarify the difference between these two anti-bPDPN mAbs. Analysis of bPDPN deletion mutants revealed that the N-terminus of the PMab-241 epitope exists between amino acids (aa) 75 and 80 of bPDPN. In addition, analysis of bPDPN point mutants demonstrated that the critical epitope of PMab-241 includes Thr75, Asp76, and Arg78 of bPDPN. The binding epitopes of PMab-241 and PMab-247 seem to overlap, but this slight difference may be sufficient to provide the specificity of PMab-241 to discriminate LECs from type I alveolar cells of the lung.

Antibody–drug conjugates (ADCs), which consist of a monoclonal antibody (mAb), a linker, and a payload, can deliver a drug to cancer tissues. We previously produced an anti-dog podoplanin (dPDPN) mAb, PMab-38, which reacts with dPDPN-expressing canine melanomas and squamous cell carcinomas (SCCs), but not with dPDPN-expressing canine type I alveolar cells or lymphatic endothelial cells, indicating that PMab-38 possesses cancer specificity. In this study, we developed an ADC, P38B-DM1, using the mouse–canine chimeric anti-dPDPN antibody, P38B as the antibody, a peptide linker, and emtansine as the payload using the chemical conjugation by affinity peptide (CCAP) method. We investigated its cytotoxicity against dPDPN-overexpressed Chinese hamster ovary (CHO/dPDPN) cells in vitro and its antitumor activity using a mouse xenograft model of CHO/dPDPN cells. P38B-DM1 showed cytotoxicity to CHO/dPDPN cells in a dose-dependent manner in vitro. Furthermore, P38B-DM1 exhibited higher antitumor activity than P38B in the mouse xenograft model. These results suggest that P38B-DM1, developed using the CCAP method, is useful for antibody therapy against dPDPN-expressing canine SCCs and melanomas.

CD19 is a type I transmembrane glycoprotein belonging to the immunoglobulin superfamily. It is expressed in normal and neoplastic B cells, and it modulates the threshold of B cell activation for amplifying B cell receptor signaling. Blinatumomab (a CD3-CD19-bispecific T cell-engaging antibody) and tisagenlecleucel (genetically modified T cells that express a CD19 chimeric antigen receptor [CART-19]) provide significant benefits for patients with CD19-positive relapsed or refractory B cell malignancies. In this study, we first employed the Cell-Based Immunization and Screening (CBIS) method to produce anti-CD19 monoclonal antibodies using CD19-overexpressing cells for both immunization and screening. One established clone-C19Mab-1-proved to be useful in flow cytometry assays against lymphoma cell lines, such as BALL-1, P30/OHK, and Raji. Second, the extracellular domain of CD19 was immunized into mice, and enzyme-linked immunosorbent assays were performed for the first screening. One established clone-C19Mab-3-was determined to be useful for Western blotting and immunohistochemical analysis. Due to their complementary utility, a combination of C19Mab-1 (established using CBIS) and C19Mab-3 (established using conventional method) could be useful for the pathological analysis of CD19.

The epidermal growth factor receptor (EGFR) is a type I transmembrane protein, which is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. EGFR is a crucial mediator of cell growth and differentiation and forms homodimers or heterodimers with other HER family members to activate downstream signaling cascades. We previously established an anti-human EGFR (hEGFR) monoclonal antibody (mAb), clone EMab-134 (mouse IgG

The classic method for identifying the epitope that monoclonal antibodies (mAbs) bind uses deletion mutants and point mutants of the target protein. However, determining the epitope of mAbs-reactive membrane proteins is often challenging. We recently developed the RIEDL insertion for epitope mapping (REMAP) method to identify mAb-binding epitopes. Herein, we first checked the reactivity of an anti-epidermal growth factor receptor (EGFR) mAb (EMab-51) to several EGFR deletion mutants such as EGFR/dN152, EGFR/dN313, EGFR/dN370, EGFR/dN375, EGFR/dN380, and EGFR/dN482. We found the N-terminus of the EMab-51-binding epitope between residues 375 and 380 of EGFR. We next produced EGFR/dN313 mutants with the RIEDL peptide tag inserted at each possible position of 375-AFRGDSFTHTPPLDP-389. EMab-51 lost its reactivity with the mutants having a RIEDL tag inserted at each position of 377-RGDSFTHTPP-386, whereas LpMab-7 (an anti-RIEDL mAb) detected every mutant. Thus, using the REMAP method, we identified the EMab-51-binding epitope of EGFR as 377-RGDSFTHTPP-386.

CC chemokine receptor 9 (CCR9) belongs to the beta chemokine receptor family and is mainly distributed on the surface of immature T lymphocytes and enterocytes. This receptor is highly expressed in rheumatoid arthritis, colitis, type 2 diabetes, and various tumors. Therefore, more sensitive monoclonal antibodies (mAbs) need to be developed to predict the prognosis of many high CCR9 expression diseases. Because CCR9 is a structurally unstable G protein-coupled receptor, it has been difficult to develop anti-CCR9 mAbs using the traditional method. This study developed anti-human CCR9 (hCCR9) mAbs for flow cytometry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with hCCR9-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hCCR9), and hybridomas showing strong signals from CHO/hCCR9 and no signals from CHO-K1 cells were selected by flow cytometry. We established an anti-hCCR9 mAb, C

Podoplanin (PDPN)/T1alpha is expressed on lymphatic endothelial cells, type I alveolar cells of the lungs, and podocytes of the kidney. PDPN possesses three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) of the N-terminus and the PLAG-like domains (PLDs). We previously reported an anti-goat PDPN (gPDPN) monoclonal antibody (mAb), PMab-235, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-235 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-235 remains to be elucidated. In this study, we investigated the epitopes of PMab-235 using enzyme-linked immunosorbent assay and immunohistochemistry. The results revealed that the critical epitope of PMab-235 produced by CBIS method is Arg75, Leu78, and Pro79 of gPDPN, which is included in PLD. The findings of our study can be applied to the production of more functional anti-gPDPN mAbs.

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed in normal tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, and renal podocytes. The overexpression of PDPN in cancers is associated with hematogenous metastasis by interactions with the C-type lectin-like receptor 2 (CLEC-2). We have previously reported the development of a mouse monoclonal antibody (mAb) clone, PMab-38 (IgG1, kappa), against dog PDPN (dPDPN). PMab-38 reacted strongly with canine squamous cell carcinomas and melanomas, but not with lymphatic endothelial cells, indicating its cancer specificity. In this study, we developed and produced several mouse-canine chimeric antibodies originating from PMab-38. A mouse-canine chimeric antibody of subclass A (P38A) and a mouse-canine chimeric antibody of subclass B (P38B) were transiently produced using ExpiCHO-S cells. Core-fucose-deficient P38B (P38Bf) was developed using FUT8 knockout ExpiCHO-S cells. We compared the binding affinities, antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) of P38A, P38B, and P38Bf against Chinese hamster ovary (CHO)/dPDPN cells. Flow cytometry analysis showed that the KD of P38A, P38B, and P38Bf were 1.9 × 10−7, 5.2 × 10−9, and 6.5 × 10−9, respectively. Both P38B and P38Bf revealed high ADCC activities against CHO/dPDPN cells; P38Bf demonstrated significantly higher ADCC compared with P38B, especially at low concentrations. P38B and P38Bf exhibited higher CDC activities against CHO/dPDPN cells. Conversely, P38A did not exhibit any ADCC or CDC activity. In summary, P38Bf is a good candidate for antibody therapy against dPDPN-expressing canine cancers.

Podoplanin (PDPN) is expressed in type I alveolar cells of lung but not in type II alveolar cells. PDPN is also known as a specific lymphatic endothelial cell marker because PDPN is not expressed in vascular endothelial cells. PDPNs of several animals have been characterized using specific anti-PDPN monoclonal antibodies (mAbs): PMab-1, PMab-2, PMab-32, PMab-38, PMab-44, and PMab-52 for mouse, rat, rabbit, dog, bovine, and cat PDPNs, respectively. In this study, we investigated the possible crossreaction between these anti-PDPN mAbs and tiger PDPN. Flow cytometry and western blot analyses revealed that the anti-cat PDPN mAb PMab-52 (IgM, kappa) reacted with tiger PDPN, which is overexpressed in Chinese hamster ovary-K1 cells. Using immunohistochemical analysis, type I alveolar cells of the tiger lung were strongly detected by PMab-52. These results indicate that PMab-52 may be useful for the detection of tiger PDPN.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Since both DG and PA serve as lipidic second messengers, DGK plays a pivotal role in regulating the balance of two signaling pathways mediated by DG and PA in cellular functions. Reportedly, DGKγ, one of the 10 mammalian DGK isozymes, is involved in leukemic cell differentiation, mast cell function, and membrane traffic. Transfection studies using tagged expression vectors and immunohistochemistry on rat tissues revealed that DGKγ localizes to the cytoplasm, plasma membrane, and Golgi apparatus. However, a limited number of studies reported the detailed localization of native protein of DGKγ in human tissues and cells. In this study, we developed a novel anti-DGKγ monoclonal antibody, DgMab-6, which is very useful in immunocytochemistry of human cultured cells.

Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in several body tissues, including podocytes of renal glomerulus, type I alveolar cells of lung, and lymphatic endothelial cells. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) presented on platelets. Monoclonal antibodies (mAbs) against human-, mouse-, rat-, rabbit-, dog-, bovine-, and cat-PDPN have already been established. However, anti-horse PDPN mAbs have not yet been developed. In this study, we immunized mice with synthetic horse PDPN peptides and developed anti-horse PDPN mAbs. One of the established mAbs, PMab-202 (IgG1, kappa), was specifically able to detect horse PDPN in Chinese hamster ovary/horse PDPN (CHO/horPDPN) cells in flow cytometry experiments. PMab-202 was also able to detect endogenous horse PDPN expressed in and a horse kidney cell line, FHK-Tcl3.1, in flow cytometry and Western blot analyses. PMab-202 is expected to prove useful in investigating the function of horse PDPN.

Immune checkpoint inhibitors targeting programmed cell death-ligand 1 (PD-L1), programmed cell death-1 (PD-1), and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) recently made a significant survival rate improvement in cancer treatment. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is expressed in T and NK cells related to their activities. It has a single extracellular immunoglobulin domain, a type 1 transmembrane domain, and a single intracellular ITIM. TIGIT binds with poliovirus receptor (PVR) or PVR2, resulting in suppressing T and NK cell activities. Some studies showed that the combined use of a TIGIT inhibitor with another immune checkpoint inhibitor enhanced antitumor activities more strongly than their single use. Therefore, TIGIT should be a new target for immunotherapy. In this study, we developed new anti-human TIGIT (hTIGIT) monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. Mice were immunized with hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hTIGIT), and hybridomas were screened by flow cytometry. One of the mAbs, TgMab-2 (IgG

CD44 is widely expressed on the surface of most tissues and all hematopoietic cells, and regulates many genes associated with cell adhesion, migration, proliferation, differentiation, and survival. CD44 has also been studied as a therapeutic target in several cancers. Previously, an anti‑CD44 monoclonal antibody (mAb), C44Mab‑5 (IgG1, kappa) was established by immunizing mice with CD44‑overexpressing Chinese hamster ovary (CHO)-K1 cells. C44Mab‑5 recognized all CD44 isoforms, and showed high sensitivity for flow cytometry and immunohistochemical analysis in oral cancers. However, as the IgG1 subclass of C44Mab‑5 lacks antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC), the antitumor activity of C44Mab‑5 could not be determined. In the present study, we converted the mouse IgG1 subclass antibody C44Mab‑5 into an IgG2a subclass antibody, 5‑mG2a, and further produced a defucosylated version, 5‑mG2a‑f, using FUT8‑deficient ExpiCHO‑S (BINDS‑09) cells. Defucosylation of 5‑mG2a‑f was confirmed using fucose‑binding lectins, such as AAL and PhoSL. The dissociation constants (KD) for 5‑mG2a‑f against SAS and HSC‑2 oral cancer cells were determined through flow cytometry to be 2.8x10‑10 M and 2.6x10‑9 M, respectively, indicating that 5‑mG2a‑f possesses extremely high binding affinity. Furthermore, immunohistochemical staining using 5‑mG2a‑f specifically stained the membranes of oral cancer cells. In vitro analysis demonstrated that 5‑mG2a‑f showed moderate ADCC and CDC activities against SAS and HSC‑2 oral cancer cells. In vivo analysis revealed that 5‑mG2a‑f significantly reduced tumor development in SAS and HSC‑2 xenografts in comparison to control mouse IgG, even after injection seven days post‑tumor inoculation. Collectively, these results suggest that treatment with 5‑mG2a‑f may represent a useful therapy for patients with CD44‑expressing oral cancers.

The epidermal growth factor receptor (EGFR), a transmembrane receptor and member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases, is a critical mediator of cell growth and differentiation. EGFR forms homo- or heterodimers with other HER family members to activate downstream signaling cascades in a number of cancer cells. In a previous study, the authors established an anti-EGFR monoclonal antibody (mAb), EMab-134, by immunizing mice with the ectodomain of human EGFR. EMab-134 binds specifically to endogenous EGFR and can be used to detect receptor on oral cancer cell lines by flow cytometry and western blot analysis; this antibody is also effective for the immunohistochemical evaluation of oral cancer tissues. In the present study, the subclass of EMab-134 was converted from IgG1 to IgG2a (134-mG2a) to facilitate antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The dissociation constants (KDs) of EMab-134 and 134-mG2a against EGFR-expressing CHO-K1 (CHO/EGFR) cells were deter-mined by flow cytometry to be 3.2×10−9 M and 2.1×10−9 M, respectively; these results indicate that 134-mG2a has a higher binding affinity than EMab-134. The 134-mG2a antibody was more sensitive than EMab-134 with respect to antigen detection in oral cancer cells in both western blot analysis and immunohistochemistry applications. Analysis in vitro revealed that 134-mG2a contributed to high levels of ADCC and CDC in experiments targeting CHO/EGFR, HSC-2, and SAS cells. Moreover, the in vivo administration of 134-mG2a significantly inhibited the development of CHO/EGFR, HSC-2, and SAS mouse xenografts in comparison to the results observed in response to EMab-134. Taken together, the findings of the present study demonstrate that the newly-formulated 134-mG2a is useful for detecting EGFR by flow cytometry, western blot analysis and immunohistochemistry. Furthermore, the in vivo results suggested that it may also be useful as part of a therapeutic regimen for patients with EGFR-expressing oral cancer.

Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses as a marker for lymphatic endothelial cells. We recently have developed anti-PDPN mAbs against many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, and bear. However, anti-sheep PDPN (sPDPN) has not yet been established. In this study, we used the Cell-Based Immunization and Screening method for the development of anti-sPDPN mAbs. RAP14 tag was added to N-terminus of sPDPN, and anti-RAP14 tag mAb (PMab-2) was used to detect the expression level of sPDPN in flow cytometry and western blot. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. One of the mAbs, PMab-256 (IgG1, kappa), specifically detected CHO/sPDPN cells by flow cytometry and western blot. Furthermore, PMab-256 stained type I alveolar cells of lung, renal glomerulus and Bowman's capsule, and lymphatic endothelial cells of lung and colon. Our findings suggest the potential usefulness of PMab-256 for the functional analyses of sPDPN.

Oral cancers constitute approximately 2% of all cancers, with the most common histological type being oral squamous cell carcinoma (OSCC), representing 90% of oral cancers. Although diagnostic technologies and therapeutic techniques have progressed, the survival rate of patients with OSCC is still 60%, whereas the incidence rate has increased. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is detected in normal tissues such as heart, breast, and pancreas as well as in many cancers, including lung, renal, breast, colorectal, and oral cancers. This glycoprotein is associated with the progression, metastasis, and poor outcomes of oral cancers. PODXL overexpression was strongly detected using our previously established anti-PODXL monoclonal antibody (mAb), PcMab-47, and its mouse IgG2a-type, 47-mG2a. In previous studies, we also generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines, in order to investigate the function of PODXL in the proliferation of oral cancer cells. The growth of SAS/PODXL-KO cell lines was observed to be lower than that of parental SAS cells. For this study, PODXL-KO OSCC cell lines were generated using HSC-2 cells, and the role of PODXL in the growth of OSCC cell lines in vitro was assessed. Decreased growth was observed for HSC-2/PODXL-KO cells compared with HSC-2 parental cells. The influence of PODXL on tumor growth of OSCC was also investigated in vivo, and both the tumor volume and the tumor weight were observed to be significantly lower for HSC-2/PODXL-KO than that for HSC-2 parental cells. These results, taken together, indicate that PODXL plays an important role in tumor growth, both in vitro and in vivo., Highlights • PODXL-knock out cell lines were established using HSC-2. • The growth of HSC-2/PODXL-KO cells were lower than that of parental HSC-2. • Tumor volumes of HSC-2/PODXL-KO cells were lower than that of HSC-2. • PODXL has an important role in tumor growth in vitro and in vivo.

The alpha-thalassemia/mental-retardation-syndrome-X-linked (ATRX) gene is located on the q arm of the X chromosome. ATRX gene mutations were first discovered in pancreatic neuroendocrine tumors, and subsequently in other cancer subtypes, including gliomas. Molecular subgrouping of gliomas has been more important than conventional histological classifications. Mutations in the isocitrate dehydrogenase (IDH), telomerase reverse transcriptase (TERT) promoter, and ATRX and the codeletion of chromosomes 1p/19q are used as biomarkers for diagnosing the subtypes of diffuse gliomas. We recently developed a sensitive monoclonal antibody (mAb) AMab-6 against ATRX by immunizing mice with recombinant human ATRX. AMab-6 can help to detect ATRX mutations via Western blotting and immunohistochemical analyses. In this study, we characterized the binding epitope of AMab-6 using enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemical analysis, and found that Gln2368 of ATRX is critical for AMab-6 binding to ATRX. Our findings could be applied to the production of more functional anti-ATRX mAbs., Highlights • ATRX gene is located on the q arm of the X chromosome. • AMab-6 detected ATRX in Western blot and immunohistochemical analyses sensitively. • Gln2368 of ATRX is critical for AMab-6-binding to ATRX. • A blocking peptide completely neutralized AMab-6 reaction by IHC.

CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s), which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C44Mab-5 (IgG1, kappa), recognized both CD44s and CD44v3-10. C44Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C44Mab-5 detected 166/182 (91.2%) of oral cancers. These results suggest that the C44Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.

The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375–394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377-RGDSFTHTPP−386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments., Highlights • We previously produced EMab-134, a novel sensitive and specific anti-EGFR mAb. • EMab-134 is useful in Western blot, flow cytometry, and IHC analyses. • A blocking peptide neutralized EMab-134 reaction against oral cancer cells. • The minimum epitope of EMab-134 was found to be 375-RGDSFTHTPP−384 sequence.

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on normal renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells. Increased expression of PDPN in cancers is associated with poor prognosis and hematogenous metastasis through interactions with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously reported a novel PMab-48 antibody, which is an anti-dog PDPN (dPDPN) monoclonal antibody (mAb) recognizing PDPN expressed in lymphatic endothelial cells. However, the binding epitope of PMab-48 is yet to be clarified. In this study, an enzyme-linked immunosorbent assay and flow cytometry were used to investigate epitopes of PMab-48. The results revealed that the critical epitope of PMab-48 comprises Asp29, Asp30, Ile31, Ile32, and Pro33 of dPDPN.

Podocalyxin (PODXL) overexpression is associated with progression, metastasis, and poor outcomes in cancers. We recently produced the novel anti-PODXL monoclonal antibody (mAb) PcMab-47 (IgG1, kappa). Herein, we engineered PcMab-47 into 47-mG2a, a mouse IgG2a-type mAb, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed 47-mG2a-f, a core fucose-deficient type of 47-mG2a to augment its ADCC. Immunohistochemical analysis of oral cancer tissues using PcMab-47 and 47-mG2a revealed that the latter stained oral squamous cell carcinoma (OSCC) cells in a cytoplasmic pattern at a much lower concentration. PcMab-47 and 47-mG2a detected PODXL in 163/201 (81.1%) and in 197/201 (98.0%) OSCC samples, respectively. 47-mG2a-f also detected PODXL in OSCCs at a similar frequency as 47-mG2a. In vitro analysis revealed that both 47-mG2a and 47-mG2a-f exhibited strong complement-dependent cytotoxicity (CDC) against CHO/hPODXL cells. In contrast, 47-mG2a-f exhibited much stronger ADCC than 47-mG2a against OSCC cells, indicating that ADCC and CDC of those anti-PODXL mAbs depend on target cells. In vivo analysis revealed that both 47-mG2a and 47-mG2a-f exerted antitumor activity in CHO/hPODXL xenograft models at a dose of 100 μg or 500 μg/mouse/week administered twice. 47-mG2a-f, but not 47-mG2a, exerted antitumor activity in SAS and HSC-2 xenograft models at a dose of 100 μg/mouse/week administered three times. Although both 47-mG2a and 47-mG2a-f exerted antitumor activity in HSC-2 xenograft models at a dose of 500 μg/mouse/week administered twice, 47-mG2a-f also showed higher antitumor activity than 47-mG2a. These results suggested that a core fucose-deficient anti-PODXL mAb could be useful for antibody-based therapy against PODXL-expressing OSCCs.

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L1Mab-13 (IgG1, kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L1Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L1Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L1Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is extensively expressed by normal lymphatic endothelial cells, renal podocytes, and pulmonary type I alveolar cells. Nevertheless, increased expression of PDPN in malignant tumors not only associates with poor prognosis but also facilitates hematogenous metastasis through interaction with C-type lectin-like receptor-2 presented on platelets, followed by PDPN-mediated platelet activation. We previously reported a novel PMab-38 antibody, an anti-dog PDPN (dPDPN) monoclonal antibody, which specifically recognizes PDPN in squamous cell carcinomas melanomas and cancer-associated fibroblasts in canine cancer tissues. However, the specific binding with the epitope of PMab-38 remains undefined. In this study, flow cytometry was utilized to investigate the epitope of PMab-38, which was determined using a series of deletion or point mutants of dPDPN. The results revealed that the critical epitope of PMab-38 is Tyr67 and Glu68 of dPDPN.

Podoplanin (PDPN) is expressed in several normal tissues, such as lymphatic endothelial cells, podocytes of renal glomerulus, and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. Although monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, dog PDPN, and bovine PDPN have been established, anticat PDPN (cPDPN) mAbs have not been developed. In this study, we immunized mice with Chinese hamster ovary (CHO)-K1 cell lines expressing cPDPN, and developed anti-cPDPN mAbs. One of the clones, PMab-52 (IgM, kappa), detected cPDPN specifically in flow cytometry and Western blot analysis. PMab-52 is also useful for detecting feline squamous cell carcinoma cells in immunohistochemical analysis. PMab-52 is expected to be useful for investigating the function of cPDPN in feline carcinomas.

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG2a, kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG1, kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H2Mab-77 (IgG1, kappa) was selected. Finally, immunohistochemical analyses with H2Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H2Mab-77 to detect HER2 in pathological analyses of breast cancers.

A type I transmembrane sialoglycoprotein podoplanin (PDPN) is expressed in several normal cells, including podocytes of the kidney, type I alveolar cells of the lung, and lymphatic endothelial cells. We recently produced an anti-bovine PDPN (bovPDPN) monoclonal antibody (mAb), PMab-44, by immunizing mice with recombinant proteins of bovPDPN. In this study, we determined the critical epitope of PMab-44 for the recognition of bovPDPN using many deletion mutants and point mutants of bovPDPN. Flow cytometric analyses revealed that the epitope of PMab-44 was Glu46-Thr50, which corresponds to platelet aggregation-stimulating (PLAG) domain-3. The important amino acids in the PMab-44 epitope were determined to be Glu46, Tyr48, and Thr50. Western blot analysis also confirmed these results, indicating that the PLAG domain of bovPDPN is also important in immunogenicity for producing useful anti-PDPN mAbs.

The interaction between podoplanin (PDPN) and C-type lectin-like receptor 2 (CLEC-2) is involved in tumor malignancy. We have established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-21, one of the mouse antipodoplanin mAbs, is of the IgG2a subclass, and its minimum epitope was determined to be Thr76-Arg79 of the human podoplanin. Importantly, sialic acid is linked to Thr76; therefore, LpMab-21 is an antiglycopeptide mAb (GpMab). In this study, we investigated whether LpMab-21 shows antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cell lines in vitro and also studied its antitumor activities using a xenograft model. LpMab-21 showed high ADCC and CDC activities against not only podoplanin-expressing Chinese hamster ovary cells but also LN319 glioblastoma cells and PC-10 lung cancer cells, both of which endogenously express podoplanin. Furthermore, LpMab-21 decreased tumor growth in vivo, indicating that LpMab-21 could be useful for antibody therapy against human podoplanin-expressing cancers.

Podoplanin (PDPN) is known to be expressed in normal tissues, including lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPN have already been established; however, mAbs against pig PDPN (pPDPN) are lacking. In the present study, mice were immunized with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and hybridomas producing mAbs against pPDPN were identified by flow cytometric screening. One of the mAbs, PMab-213 (IgG

Podoplanin (PDPN) is a type I transmembrane glycoprotein that is expressed in normal tissues, including renal corpuscles and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine PDPNs have already been established; however, antipig PDPN (pPDPN) mAbs have not. We therefore immunized mice with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN), and screened hybridomas, which are producing anti-pPDPN mAbs. One of mAbs, PMab-210 (an IgG1, kappa), was able to specifically detect CHO/pPDPN cells by flow cytometry and detect pPDPN by Western blot analysis. Furthermore, PMab-210 strongly stained type I lung alveolar cells and weakly stained renal corpuscles by immunohistochemistry. PMab-210 is expected to be useful in investigating the function of pPDPN.

Podoplanin (PDPN) is expressed in type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. We have characterized the PDPNs of various animal species using specific anti-PDPN monoclonal antibodies (mAbs). In this study, we investigated whether these anti-PDPN mAbs cross-react with goat PDPN (gPDPN). Flow cytometry demonstrated that the anti-bovine PDPN mAb PMab-44 (IgG1, kappa) reacts with gPDPN, which is overexpressed in CHO-K1 cells. Using immunohistochemical analysis, type I alveolar cells of goat lung were strongly detected by PMab-44. These results indicate that PMab-44 is useful for investigating gPDPN.

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L