This study characterizes the previously reported anti-canine CD20 antibody 4E1-7-B_f and compares this with commercially available anti-human CD20 antibodies, rituximab and an obinutuzumab biosimilar. While the obinutuzumab biosimilar exhibited binding to canine CD20 in a CD20-transduced cell line, canine B-cell lymphoma cell line (CLBL-1/luc), and canine CD21 + B cells from healthy dogs, functional assays revealed the superiority of 4E1-7-B_f in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities over those of the obinutuzumab biosimilar. Epitope analysis suggested an extracellular region on canine CD20 targeted by 4E1-7-B_f. Furthermore, the lipid raft localization of CD20 in CLBL-1/luc cells by treatment with 4E1-7-B_f classified this antibody as a type II anti-CD20 antibody which works with strong ADCC activity, similar to the obinutuzumab biosimilar, unlike rituximab, a type I anti-CD20 antibody, whose main action is CDC activity. These findings underscore the potential clinical utility of 4E1-7-B_f, emphasizing the specificity, potency, and therapeutic promise in canine lymphoma treatment.

Erythropoietin-producing hepatocellular receptor A1 (EphA1) is one of the Eph receptor family members, the largest group of receptor tyrosine kinases. EphA1 is expressed in various tissues and regulates cellular homeostasis by interacting with its membrane-bound ephrin ligands and other receptors. EphA1 critically correlates with the pathogenesis in several disorders, including Alzheimer's disease and cancers. Therefore, establishing sensitive monoclonal antibodies (mAbs) for EphA1 has been desired for basic research, diagnosis, and treatment. In this study, a novel specific and sensitive anti-human EphA1 mAb, clone Ea1Mab-30 (mouse IgG1, kappa), was established by the Cell-Based Immunization and Screening (CBIS) method. Ea1Mab-30 demonstrated reactivity with an EphA1-overexpressed Chinese hamster ovary-K1 cell line (CHO/EphA1), an endogenously EphA1-expressing bladder carcinoma cell line (5637), and a colorectal adenocarcinoma cell line (Caco-2) in flow cytometry. Crossreactivities of Ea1Mab-30 with other Eph receptors were not observed. Furthermore, the values of apparent binding affinity for CHO/EphA1 and 5637 were determined to be 8.9 × 10-9 M and 1.7 × 10-9 M, respectively. Furthermore, Ea1Mab-30 detected EphA1 protein in CHO/EphA1 and 5637 lysates using Western blot analysis. Ea1Mab-30 also clearly stained EphA1 of formalin-fixed paraffin-embedded CHO/EphA1 using immunohistochemistry. Ea1Mab-30, established by CBIS method, could help analyze the EphA1-contributed cellular functions and have potential applications in pathological diagnosis and treatment with specificity and high affinity for EphA1-expressing cells.

Ephrin type A receptor 2 (EphA2) binds to membrane-bound ligands, ephrin A1, A2, and A5, eliciting bidirectional signaling. This signaling regulates many physiological processes, such as tissue development, homeostasis, and regeneration. The dysregulation of the EphA2-ephrins axis contributes to various diseases, including cancers. The high expression of EphA2 is observed in various cancers, which promotes cancer malignancy, whereas its levels are relatively low in most normal adult tissues. Therefore, EphA2 is a promising target for cancer therapy. We developed anti-human EphA2 monoclonal antibodies in this study using the Cell-Based Immunization and Screening method. Among them, a clone Ea2Mab-7 (IgG1, κ) exhibited a high affinity and sensitivity in flow cytometry. The dissociation constant values of Ea2Mab-7 for CHO/EphA2 and MDA-MB-231 cells were determined as 6.2 ± 1.3 × 10-9 M and 1.6 ± 0.4 × 10-9 M, respectively. Furthermore, Ea2Mab-7 can detect endogenous EphA2 in Western blot and immunohistochemistry. Therefore, the Ea2Mab-7 is highly versatile for basic research and is expected to contribute to clinical applications, such as antibody therapy and tumor diagnosis.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL), a B-cell lymphoma in cattle. Previous studies have demonstrated that T cells of BLV-infected cattle show increased expression of immune checkpoint molecules, including programmed death-1 (PD-1), lymphocyte-activation gene-3 (LAG-3), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and T-cell immunoglobulin domain and mucin domain-3 (TIM-3), leading to T-cell exhaustion. However, the key immune checkpoint molecules driving T-cell exhaustion in BLV-induced tumorigenesis remained unclear. In this study, we identified the key immune checkpoint molecules by performing comprehensive flow cytometric analyses of T cells from EBL cattle, and elucidated the phenotype and function of exhausted T cells using a transcriptomic analysis by RNA sequencing and cell culture assays. The comprehensive expression analysis revealed that the proportion of CD4+ and CD8+ T cells co-expressing PD-1 and TIM-3 was significantly increased in the peripheral blood and tumor tissues of EBL cattle compared to healthy cattle. Transcriptomic analysis of PD-1+TIM-3+ T cells revealed the upregulation of genes related to terminal exhaustion and the downregulation of genes related to T-cell differentiation and response in this subset. Additionally, PD-1+TIM-3+ T cells exhibited higher expression of CTLA-4, LAG-3, and Eomes, and lower expression of T-bet, suggesting a terminally exhausted phenotype. Cell culture assays revealed a significant impairment in IFN-γ production in PD-1+TIM-3+ T cells upon stimulation, reflecting severe dysfunction. These findings indicate that PD-1+TIM-3+ T cells play a central role in T-cell exhaustion during BLV-induced tumorigenesis. This study provides valuable insights for future therapeutic strategies against BLV infection.

Polyethylene glycol (PEG) is widely used as a standard stealth polymer, although the induction of anti-PEG antibodies and consequent effects have drawn attention in recent years. To date, several anti-PEG antibodies induced by PEG-modified proteins via the T cell-dependent (TD) pathway, in which affinity maturation occurs, have been reported. In contrast, structures of the naïve anti-PEG antibodies before affinity maturation have not been described in the literature. Here, to understand the details of the naïve anti-PEG antibodies capturing PEG, we studied a naïve anti-PEG antibody induced by a PEG-modified liposome in the absence of affinity maturation via the T cell-independent (TI) pathway. The mutation levels, structures as well as in vitro and in silico binding properties of TI and TD anti-PEG antibodies were compared. The TI anti-PEG antibody showed no mutation and a low binding affinity toward PEG, meanwhile, it allowed PEG chain sliding and weak interaction with the terminal group. Furthermore, the naïve anti-PEG antibodies may obtain high affinities by forming tunnel structures via minimal mutations. This research provides new insights into polymer-antibody interactions, which can facilitate the development of novel stealth polymers that can avoid antibody induction.

Cadherins are key cell adhesion molecules that engage in extracellular homophilic binding. CDH15/M-cadherin is localized to the apical surface of muscle satellite cells (SCs), which play a critical role in tissue regeneration after injury. Although CDH15 is considered a marker of SCs, there is no anti-CDH15 monoclonal antibody (mAb) suitable for flow cytometry. We developed anti-CDH15 mAbs using the Cell-Based Immunization and Screening (CBIS) method containing a flow cytometry-based high-throughput screening. In flow cytometry, a clone Ca15Mab-1 (IgG1, κ) reacted with human CDH15-overexpressed Chinese hamster ovary-K1 (CHO/CDH15) cells. Furthermore, Ca15Mab-1 recognizes endogenous CDH15-expressing human osteosarcoma (Saos-2) and mouse myoblast (C2C12) cell lines. The dissociation constant values of Ca15Mab-1 for CHO/CDH15, Saos-2, and C2C12 were determined as 6.1 × 10-10 M, 7.9 × 10-10 M, and 9.8 × 10-10 M, respectively. Furthermore, Ca15Mab-1 can detect endogenous CDH15 in immunoblotting and immunohistochemistry. Ca15Mab-1, established by the CBIS method, is versatile for basic research and is expected to contribute to clinical studies such as antibody therapy.

Podoplanin (PDPN), also referred to as T1α/Aggrus, is a type I transmembrane sialoglycoprotein that plays a crucial role in invasiveness, stemness, and epithelial-to-mesenchymal transition, all of which contribute to the malignant progression of tumors. Therefore, a monoclonal antibody (mAb) against PDPN has been evaluated in preclinical models as a promising tumor therapy strategy. However, PDPN plays an essential role in normal development, such as in the development of the lungs. On-target toxicity by anti-PDPN mAbs to normal cells should be avoided to minimize adverse effects. A cancer-specific mAb against PDPN, PMab-117 (rat IgM, kappa), was previously established. This study engineered the humanized IgG1 version (humPMab-117) to investigate antitumor activity. Flow cytometry analysis confirmed that humPMab-117 recognized PDPN-overexpressed glioma LN229 (LN229/PDPN) cells as well as PDPN-positive PC-10 (human lung squamous cell carcinoma) and LN319 (human glioblastoma) cells. In contrast, humPMab-117 did not react with normal epithelial cells from the lung bronchus, gingiva, mammary gland, corneal, and normal kidney podocytes, suggesting that humPMab-117 retains cancer-specific reactivity. Furthermore, humPMab-117 effectively induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against LN229/PDPN, PC-10, and LN319 cells. In the xenograft tumor models, humPMab-117 demonstrated strong antitumor efficacy. These results suggest the potential of humPMab-117 as a therapeutic antibody for treating PDPN-positive malignant tumors.

C-C chemokine receptor type 7 (CCR7) is a member of the G protein-coupled receptor family and functions as a lymph node-homing receptor for immune cells. Upon ligand binding, CCR7 promotes the migration of immune cells to secondary lymphoid organs. In cancers, CCR7 has been revealed as a critical molecule in lymph node metastasis. Consequently, anti-CCR7 monoclonal antibodies (mAbs) have been developed as cancer therapeutic agents. In this study, we established an anti-mouse CCR7 (mCCR7) mAb, C7Mab-7 (rat IgG1, kappa) using the Cell-Based Immunization and Screening (CBIS) method. C7Mab-7 demonstrated high sensitivity in flow cytometry. The dissociation constant (K D) value of C7Mab-7 was determined to be 2.5 × 10⁻⁹ M for mCCR7-overexpressed Chinese hamster ovary-K1 (CHO/mCCR7) cells. Furthermore, C7Mab-7 detected mCCR7 with high sensitivity in western blot and immunohistochemistry. C7Mab-7, developed by the CBIS method, accelerates the development of CCR7-targeted antibody therapies and cancer diagnostics.

Erythropoietin-producing hepatocellular receptor B6 (EphB6) is a member of the largest Eph subfamily of receptor tyrosine kinases. EphB6 is widely expressed in various tissues and regulates cellular homeostasis by interacting with its membrane-bound ephrin ligands and other receptors. EphB6 is involved in cancer pathology despite lacking kinase activity. Developing sensitive monoclonal antibodies (mAbs) for EphB6 has been desired for treatment, diagnosis, and further analysis of EphB6. This study established a novel specific and sensitive anti-human EphB6 mAb clone Eb6Mab-3 (mouse IgG1, kappa) by the Cell-Based Immunization and Screening (CBIS) method. In flow cytometry, Eb6Mab-3 demonstrated reactivity with EphB6-overexpressed Chinese hamster ovary-K1 cells (CHO/EphB6) and endogenously EphB6-expressing DLD-1 colorectal cancer cells. Cross-reactivity of Eb6Mab-3 was not observed. Eb6Mab-3 demonstrated a moderate binding affinity (dissociation constant; K D) for CHO/EphB6 (K D: 2.6 ± 1.0 × 10-8 M) and a high binding affinity for DLD-1 (K D: 3.4 ± 1.3 × 10-9 M). Eb6Mab-3 can detect EphB6 protein in CHO/EphB6 lysate in Western blot. Eb6Mab-3, established by the CBIS method, could be valuable for analyzing the EphB6-associated cellular functions and has potential applications in diagnosis and treatment with specificity and high affinity for cancer cells.

Chimeric Antigen Receptor (CAR)-T cell therapy has revolutionized the treatment of CD19-positive B-cell malignancies. However, the field is rapidly evolving to target other antigens, such as podocalyxin (PODXL), a transmembrane protein implicated in tumor progression and poor prognosis in various cancers. This study explores the potential of PODXL-targeted CAR-T cells, utilizing a cancer-specific monoclonal antibody (CasMab) technique to enhance the specificity and safety of CAR-T cell therapy. We developed CAR-T cells based on the single-chain variable fragment (scFv) derived from the cancer-specific monoclonal antibody PcMab-6, which selectively targets glycosylation modifications on PODXL-expressing cancer cells. As a control, CAR-T cells were also generated from PcMab-47, a non-cancer-specific antibody for PODXL. In vitro experiments demonstrated that CAR-T cells based on PcMab-6 exhibited significant antitumor activity with reduced off-target effects on normal cells compared to PcMab-47-derived CAR-T cells. Additionally, to enhance the persistence and therapeutic efficacy of these CAR-T cells, we developed a humanized version of PcMab-6 scFv. The humanized CAR-T cells showed extended antitumor effects in vivo, demonstrating the potential for prolonged therapeutic activity. These findings underscore the utility of CasMab technology in generating highly specific and safer CAR-T cell therapies for solid tumors, highlighting the promise of humanized CAR-T cells for clinical application.

Long-term consequences of SARS-CoV-2 infection affect millions of people and strain public health systems. The underlying pathomechanisms remain unclear, necessitating further research in appropriate animal models. This study aimed to characterize the trajectory of lung regeneration over 112 days in the male hamster model by combining morphological, transcriptomic and functional readouts. We demonstrate that in the acute phase, SARS-CoV-2 Delta-infected, male, aged hamsters show a severe impairment of lung function at rest. In the chronic phase, similar impairments persisted up to 7 weeks post-infection but were only evident after exercise on a rodent treadmill. The male hamster model recapitulates chronic pulmonary fibrotic changes observed in many patients with respiratory long COVID, but lacks extra-pulmonary long-term lesions. We show that sub-pleural and interstitial pulmonary fibrosis as well as alveolar bronchiolization persist until 112 dpi. Interestingly, CK8+ alveolar differentiation intermediate (ADI) cells are becoming less prominent in the alveolar proliferation areas from 28 dpi on. Instead, CK14+ airway basal cells and SCGB1A1+ club cells, expressing cell proliferation markers, mainly populate alveolar bronchiolization areas at later time-points. We postulate that pulmonary fibrosis and SCGB1A1+ club cell-rich areas of alveolar bronchiolization represent potential risk factors for other diseases in long-COVID survivors.

Site-2 proteases (S2Ps), conserved intramembrane metalloproteases that maintain cellular homeostasis, are associated with chronic infection and persistence leading to multidrug resistance in bacterial pathogens. A structural model of how S2Ps discriminate and accommodate substrates could help us develop selective antimicrobial agents. We previously proposed that the Escherichia coli S2P RseP unwinds helical substrate segments before cleavage, but the mechanism for accommodating a full-length membrane-spanning substrate remained unclear. Our present cryo-EM analysis of Aquifex aeolicus RseP (AaRseP) revealed that a substrate-like membrane protein fragment from the expression host occupied the active site while spanning a transmembrane cavity that is inaccessible via lateral diffusion. Furthermore, in vivo photocrosslinking supported that this substrate accommodation mode is recapitulated on the cell membrane. Our results suggest that the substrate accommodation by threading through a conserved membrane-associated region stabilizes the substrate-complex and contributes to substrate discrimination on the membrane.

Glypican 5 (GPC5) is a member of heparan sulfate proteoglycans and is anchored to the plasma membrane via glycosylphosphatidylinositol. GPC5 plays an essential role in kidney, limb, and brain development. Furthermore, GPC5 is expressed in some cancers, but whether it functions as a cancer-promoting or -suppressing factor remains unclear. Therefore, the development of versatile and specific anti-GPC5 monoclonal antibodies (mAbs) is desired to clarify the biological and pathological functions of GPC5. In this study, we successfully established an anti-human GPC5 mAb (clone G5Mab-1) using the Cell-Based Immunization and Screening method. G5Mab-1 is capable of using flow cytometric analysis. G5Mab-1 is specifically bound only to GPC5, not to the other GPC family members. The dissociation constant value of G5Mab-1 for GPC5-overexpressed Chinese hamster ovary K-1 (CHO/GPC5) cells was determined as 9.9 × 10-9 M. Furthermore, G5Mab-1 detected GPC5 in Western blot and immunohistochemistry using CHO/GPC5 cells. Therefore, the G5Mab-1 is highly versatile for basic research and is expected to contribute to clinical applications, such as antibody-based therapy and diagnosis of cancer.

Colorectal carcinoma brain metastases (n = 60) were studied using next-generation sequencing and immunohistochemistry. RAS and BRAF mutations were detected in 58.2% and 7.3% of cases, respectively. Patients with RAS- and BRAF-mutant tumors could potentially benefit from the treatment with inhibitors. TP53 mutations were detected in 69.1% of metastases. Moreover, altered p53 expression was seen in 91.2% of cases. APC mutations were present in 41.8% of tumors. Diffuse nuclear accumulation of β-catenin was seen in 10.2% of metastases, although only 1 CTNNB1 mutant was identified. Nevertheless, targeting p53 and Wnt/β-catenin pathways may have potential therapeutic implications. Casein kinase 1α1 expression indicating susceptibility to protein kinase inhibitors, was seen in 95% metastases including 10 with strong immunoreactivity. The immune checkpoint marker CD276, a promising target for immunotherapy, was present on tumor cells in 50.8% of metastases and on stromal cells in almost all cases. PRAME, another immunotherapy target, was expressed in 21.7% of tumors. HER2 membrane immunostaining detected in 13.3% of cases implicated potential treatment with HER2 inhibitors. Expression of SLFN11, a predictor of response to DNA-damaging chemotherapies, and a biomarker of sensitivity to PARP inhibitors was seen in 8.3% of tumors. In 6.7% of metastases loss or partial loss of MTAP expression suggested sensitivity to PRMT5 inhibitors. CD44v5 expressed in 35% of cases indicated potential therapeutic utility of anti-CD44v5 monoclonal antibody treatment. Identification of predictive biomarkers through genomic profiling and proteomic analyses is a crucial step toward individually tailored therapeutic regimens for patients with colorectal carcinoma brain metastases.

Immune checkpoint inhibitors (ICIs) are widely used for cancer immunotherapy; however, the clinical efficacy of anti-PD-1/PD-L1 monotherapy is generally limited, highlighting the need to develop combination therapies. Dogs develop spontaneous tumors in immunocompetent settings, and anti-PD-1/PD-L1 antibodies exert similar clinical benefits. However, no clinically relevant anti-CTLA-4 antibody has been reported, limiting the value of canine tumors as comparative models for human ICI research. Here, canine CTLA-4 was molecularly characterized, and a caninized anti-CTLA-4 antibody (ca1C5) that blocks CTLA-4/ligand binding was developed. Treatment with ca1C5 increased cytokine production in canine immune cell cultures, and the immunostimulatory effect was enhanced when used in combination with the anti-PD-L1 antibody c4G12. As a proof-of-concept, a veterinary clinical study was conducted to demonstrate the safety and clinical efficacy of anti-CTLA-4 antibody as salvage combination therapy in dogs with advanced tumors refractory to prior c4G12 monotherapy. The combination treatment (c4G12 plus ca1C5) was well-tolerated, and evidence of antitumor activity was observed in one dog with oral malignant melanoma. Further studies are warranted to advance veterinary care for dogs and to better characterize canine ICI models for human onco-immunology research.

Monoclonal antibody (mAb) and cell-based immunotherapies represent cutting-edge strategies for cancer treatment. However, safety concerns persist due to the potential targeting of normal cells that express reactive antigens. Therefore, it is crucial to develop cancer-specific mAbs (CasMabs) that can bind to cancer-specific antigens and exhibit antitumor activity in vivo, thereby reducing the risk of adverse effects. We previously screened mAbs targeting human epidermal growth factor receptor 2 (HER2) and successfully developed a cancer-specific anti-HER2 mAb, H2Mab-250/H2CasMab-2 (mouse IgG1, kappa). In this study, we assessed both the in vitro and in vivo antitumor efficacy of the humanized H2Mab-250 (humH2Mab-250). Although humH2Mab-250 showed lower reactivity to HER2-overexpressed Chinese hamster ovary-K1 (CHO/HER2) and breast cancer cell lines (BT-474 and SK-BR-3) than trastuzumab in flow cytometry, both humH2Mab-250 and trastuzumab showed similar antibody-dependent cellular cytotoxicity (ADCC) against CHO/HER2 and the breast cancer cell lines in the presence of effector splenocytes. In addition, humH2Mab-250 exhibited significant complement-dependent cellular cytotoxicity (CDC) in CHO/HER2 and the breast cancer cell lines compared to trastuzumab. Furthermore, humH2Mab-250 possesses compatible in vivo antitumor effects against CHO/HER2 and breast cancer xenografts with trastuzumab. These findings highlight the distinct roles of ADCC and CDC in the antitumor effects of humH2Mab-250 and trastuzumab and suggest a potential direction for the clinical development of humH2Mab-250 for HER2-positive tumors.

The CXC chemokine receptor 5 (CXCR5) is a member of the G protein-coupled receptor family that is highly expressed in B cells and a subset of T cells, such as T follicular helper cells. Various types of cancers, including non-small cell lung cancer, breast cancer, and prostate cancer, also express CXCR5. Therefore, antibodies that specifically bind to CXCR5 could be useful for clarification of the mechanisms of cancer progression. In this study, we aimed to develop high-affinity monoclonal antibodies targeting mouse CXCR5 (mCXCR5) for flow cytometry. The established anti-mCXCR5 mAb (Cx5Mab-3; rat IgG2b, kappa), demonstrated reactivity with mCXCR5-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/mCXCR5) in flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KD) of Cx5Mab-3 for CHO/mCXCR5 cell is 7.2 × 10−10 M. Furthermore, Cx5Mab-3 did not cross-react with other mouse CC, CXC, CX3C, and XC chemokine receptors. These results indicate that Cx5Mab-3 is useful for detecting mCXCR5 in flow cytometry with high affinity and specificity.

Leukocyte migration is an essential function of innate and adaptive immune responses. Chemokines and their receptors control the migration system. The abundance of chemokines is controlled by atypical chemokine receptors (ACKRs), chemokine receptor-like molecules that do not couple to the G protein signaling pathways. Among them, ACKR4 regulates dendritic cell migration by controlling the ligands and is involved in tumor development in mouse models. Because no anti-mouse ACKR4 (mACKR4) monoclonal antibody (mAb) for flow cytometry has been reported, this study aimed to develop a novel mAb for mACKR4. Among the established anti-mACKR4 mAbs, A4Mab-1 (rat IgG2b, kappa), A4Mab-2 (rat IgG2b, kappa), and A4Mab-3 (rat IgG2b, kappa) recognized mACKR4-overexpressed Chinese hamster ovary-K1 (CHO/mACKR4) by flow cytometry. The dissociation constant (K D) values of A4Mab-1, A4Mab-2, and A4Mab-3 for CHO/mACKR4 were determined as 6.0 × 10-9 M, 1.3 × 10-8 M, and 1.7 × 10-9 M, respectively. Furthermore, A4Mab-1 and A4Mab-2 could detect mACKR4 by western blotting. These results indicated that A4Mab-1, A4Mab-2, and A4Mab-3 help to detect mACKR4 by flow cytometry and western blotting and obtain the proof of concept in preclinical models.

Podoplanin (PDPN) overexpression is associated with poor clinical outcomes in various tumors. PDPN is involved in malignant tumor progression by promoting invasiveness and metastasis. Therefore, PDPN is considered a promising target of monoclonal antibody (mAb)-based therapy. Because PDPN also plays an essential role in normal cells such as kidney podocytes, cancer specificity is required to reduce adverse effects on normal cells. We developed a cancer-specific mAb (CasMab) against PDPN, PMab-117 (rat IgM, kappa), by immunizing rats with PDPN-overexpressed glioblastoma cells. The recombinant mouse IgG2a-type PMab-117 (PMab-117-mG2a) reacted with the PDPN-positive tumor PC-10 and LN319 cells but not with PDPN-knockout LN319 cells in flow cytometry. PMab-117-mG2a did not react with normal kidney podocytes and normal epithelial cells from the lung bronchus, mammary gland, and corneal. In contrast, one of the non-CasMabs against PDPN, NZ-1, showed high reactivity to PDPN in both tumor and normal cells. Moreover, PMab-117-mG2a exerted antibody-dependent cellular cytotoxicity in the presence of effector splenocytes. In the human tumor xenograft models, PMab-117-mG2a exhibited potent antitumor effects. These results indicated that PMab-117-mG2a could be applied to antibody-based therapy against PDPN-expressing human tumors while reducing the adverse effects.

Podoplanin (PDPN) overexpression is associated with poor clinical outcomes in various tumors. PDPN is involved in malignant tumor progression by promoting invasiveness and metastasis. Therefore, PDPN is considered a promising target of monoclonal antibody (mAb)-based therapy. Because PDPN also plays an essential role in normal cells such as kidney podocytes, cancer specificity is required to reduce adverse effects on normal cells. We developed a cancer-specific mAb (CasMab) against PDPN, PMab-117 (rat IgM, kappa), by immunizing rats with PDPN-overexpressed glioblastoma cells. The recombinant mouse IgG2a-type PMab-117 (PMab-117-mG2a) reacted with the PDPN-positive tumor PC-10 and LN319 cells but not with PDPN-knockout LN319 cells in flow cytometry. PMab-117-mG2a did not react with normal kidney podocytes and normal epithelial cells from the lung bronchus, mammary gland, and corneal. In contrast, one of the non-CasMabs against PDPN, NZ-1, showed high reactivity to PDPN in both tumor and normal cells. Moreover, PMab-117-mG2a exerted antibody-dependent cellular cytotoxicity in the presence of effector splenocytes. In the human tumor xenograft models, PMab-117-mG2a exhibited potent antitumor effects. These results indicated that PMab-117-mG2a could be applied to antibody-based therapy against PDPN-expressing human tumors while reducing the adverse effects.

CXC chemokine receptor 1 (CXCR1) is an important regulator for neutrophil granulocyte activation through binding to the ligand interleukin-8 (IL-8). Upon binding to IL-8, CXCR1 activates downstream signaling, critical for innate and adaptive immune responses. The IL-8-CXCR1 axis also plays an important role in tumor progression, especially in the tumor microenvironment. CXCR1 antagonists or anti-IL-8 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for inflammatory diseases and tumors. In this study, we developed novel mAbs for mouse CXCR1 (mCXCR1) using the N-terminal peptide immunization. Among the established anti-mCXCR1 mAbs, Cx1Mab-8 (rat IgG2b, kappa) recognized mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 (LN229/mCXCR1) by flow cytometry. The dissociation constant (K D) values of Cx1Mab-8 for CHO/mCXCR1 and LN229/mCXCR1 were determined as 4.1 × 10-10 M and 1.5 × 10-9 M, respectively. These results indicated that Cx1Mab-8 is useful for detecting mCXCR1 by flow cytometry with high affinity and could contribute to obtaining the proof of concept in preclinical studies.

CD44 is a type I transmembrane glycoprotein associated with poor prognosis in various solid tumors. Since CD44 plays a critical role in tumor development by regulating cell adhesion, survival, proliferation and stemness, it has been considered a target for tumor therapy. Anti‑CD44 monoclonal antibodies (mAbs) have been developed and applied to antibody‑drug conjugates and chimeric antigen receptor‑T cell therapy. Anti-pan‑CD44 mAbs, C44Mab‑5 and C44Mab‑46, which recognize both CD44 standard (CD44s) and variant isoforms were previously developed. The present study generated a mouse IgG2a version of the anti‑pan‑CD44 mAbs (5‑mG2a and C44Mab‑46‑mG2a) to evaluate the antitumor activities against CD44‑positive cells. Both 5‑mG2a and C44Mab‑46‑mG2a recognized CD44s‑overexpressed CHO‑K1 (CHO/CD44s) cells and esophageal tumor cell line (KYSE770) in flow cytometry. Furthermore, both 5‑mG2a and C44Mab‑46‑mG2a could activate effector cells in the presence of CHO/CD44s cells and exhibited complement-dependent cytotoxicity against both CHO/CD44s and KYSE770 cells. Furthermore, the administration of 5‑mG2a and C44Mab‑46‑mG2a significantly suppressed CHO/CD44s and KYSE770 xenograft tumor development compared with the control mouse IgG2a. These results indicate that 5‑mG2a and C44Mab‑46‑mG2a could exert antitumor activities against CD44‑positive cancers and be a promising therapeutic regimen for tumors.

CD44 regulates cell adhesion, proliferation, survival, and stemness and has been considered a tumor therapy target. CD44 possesses the shortest CD44 standard (CD44s) and a variety of CD44 variant (CD44v) isoforms. Since the expression of CD44v is restricted in epithelial cells and carcinomas compared to CD44s, CD44v has been considered a promising target for monoclonal antibody (mAb) therapy. We previously developed an anti-CD44v10 mAb, C44Mab-18 (IgM, kappa), to recognize the variant exon 10-encoded region. In the present study, a mouse IgG2a version of C44Mab-18 (C44Mab-18-mG2a) was generated to evaluate the antitumor activities against CD44-positive cells compared with the previously established anti-pan CD44 mAb, C44Mab-46-mG2a. C44Mab-18-mG2a exhibited higher reactivity compared with C44Mab-46-mG2a to CD44v3–10-overexpressed CHO-K1 (CHO/CD44v3–10) and oral squamous cell carcinoma cell lines (HSC-2 and SAS) in flow cytometry. C44Mab-18-mG2a exerted a superior antibody-dependent cellular cytotoxicity (ADCC) against CHO/CD44v3–10. In contrast, C44Mab-46-mG2a showed a superior complement-dependent cytotoxicity (CDC) against CHO/CD44v3–10. A similar tendency was observed in ADCC and CDC against HSC-2 and SAS. Furthermore, administering C44Mab-18-mG2a or C44Mab-46-mG2a significantly suppressed CHO/CD44v3–10, HSC-2, and SAS xenograft tumor growth compared with the control mouse IgG2a. These results indicate that C44Mab-18-mG2a could be a promising therapeutic regimen for CD44v10-positive tumors.

Cancer-specific monoclonal antibodies (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy are innovative therapeutic strategies for minimizing adverse effects. We previously established a cancer-specific anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody (mAb), H2Mab-250/H2CasMab-2. In flow cytometry and immunohistochemistry, H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, strongly recognizes both breast cancer and normal epithelial cells in flow cytometry. The human IgG1 version of H2Mab-250 (H2Mab-250-hG1) possesses compatible in vivo antitumor effects against breast cancer xenografts to trastuzumab despite the lower affinity and effector activation than trastuzumab in vitro. This study compared the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC) between H2Mab-250-hG1 and trastuzumab. Both H2Mab-250-hG1 and trastuzumab showed ADCC activity against HER2-overexpressed Chinese hamster ovary -K1 and breast cancer cell lines (BT-474 and SK-BR-3) in the presence of human natural killer cells. Some tendency was observed where trastuzumab showed a more significant ADCC effect compared to H2Mab-250-hG1. Importantly, H2Mab-250-hG1 exhibited superior CDC activity in these cells compared to trastuzumab. Similar results were obtained in the mouse IgG2a types of both H2Mab-250 and trastuzumab. These results suggest the different contributions of ADCC and CDC activities to the antitumor effects of H2Mab-250-hG1 and trastuzumab, and indicate a future direction for the clinical development of H2Mab-250-hG1 against HER2-positive tumors.

One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands, CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of proinflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8 for CHO/mCCR5 were determined as 3.5 × 10-8 M and 7.3 × 10-9 M, respectively. Furthermore, both C5Mab-4 and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies.

As aberrant accumulation of RNA-DNA hybrids (R-loops) causes DNA damage and genome instability, cells express regulators of R-loop structures. Here we report that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates R-loop formation. We found that the phosphorylated form of hTERT (p-hTERT) exhibits RdRP activity in nuclear speckles both in telomerase-positive cells and telomerase-negative cells with alternative lengthening of telomeres (ALT) activity. The p-hTERT did not associate with telomerase RNA component in nuclear speckles but, instead, with TERRA RNAs to resolve R-loops. Targeting of the TERT gene in ALT cells ablated RdRP activity and impaired tumour growth. Using a genome-scale CRISPR loss-of-function screen, we identified Fanconi anaemia/BRCA genes as synthetic lethal partners of hTERT RdRP. Inactivation of RdRP and Fanconi anaemia/BRCA genes caused accumulation of R-loop structures and DNA damage. These findings indicate that RdRP activity of p-hTERT guards against genome instability by removing R-loop structures.

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.

A current challenge is the emergence of SARS-CoV-2 variants, such as BQ.1.1 and XBB.1.5, that can evade immune defenses, thereby limiting antibody drug effectiveness. Emergency-use antibody drugs, including the widely effective bebtelovimab, are losing their benefits. One potential approach to address this issue are bispecific antibodies which combine the targeting abilities of two antibodies with distinct epitopes. We engineered neutralizing bispecific antibodies in the IgG-scFv format from two initially non-neutralizing antibodies, CvMab-6 (which binds to the receptor-binding domain [RBD]) and CvMab-62 (targeting a spike protein S2 subunit epitope adjacent to the known anti-S2 antibody epitope). Furthermore, we created a bispecific antibody by incorporating the scFv of bebtelovimab with our anti-S2 antibody, demonstrating significant restoration of effectiveness against bebtelovimab-resistant BQ.1.1 variants. This study highlights the potential of neutralizing bispecific antibodies, which combine existing less effective anti-RBD antibodies with anti-S2 antibodies, to revive the effectiveness of antibody therapeutics compromised by immune-evading variants.

C-C motif chemokine receptor 1 (CCR1/CD191) is a member of G-protein-coupled receptors and is expressed on myeloid cells, such as neutrophils and macrophages. Because the CCR1 signaling promotes tumor expansion in the tumor microenvironment (TME), the modification of TME is an effective strategy for cancer therapy. Although CCR1 is an attractive target for solid tumors and hematological malignancies, therapeutic agents for CCR1 have not been approved. Here, we established a novel anti-mouse CCR1 (mCCR1) monoclonal antibody (mAb), C1Mab-6 (rat IgG2b, kappa), using the Cell-Based Immunization and Screening method. Flow cytometry and Western blot analyses showed that C1Mab-6 recognizes mCCR1 specifically. The dissociation constant of C1Mab-6 for mCCR1-overexpressed Chinese hamster ovary-K1 was determined as 3.9 × 10-9 M, indicating that C1Mab-6 possesses a high affinity to mCCR1. These results suggest that C1Mab-6 could be a useful tool for targeting mCCR1 in preclinical mouse models.

CD44 is a type I transmembrane glycoprotein and possesses various isoforms which are largely classified into CD44 standard (CD44s) and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles in tumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully understood. Using peptide immunization, we developed an anti-CD44v4 monoclonal antibody, C44Mab-108, which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined the critical epitope of C44Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine (or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271-290 of human CD44v3-10) and found that C44Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore, these peptides could not inhibit the recognition of C44Mab-108 in flow cytometry and immunohistochemistry. The results indicate that the critical binding epitope of C44Mab-108 includes Asp280 and Trp281 of CD44v3-10.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is associated with a poor prognosis, making it an important therapeutic target. Here, we establish a novel cancer-specific anti-HER2 antibody, H2Mab-214. H2Mab-214 reacts with HER2 on cancer cells, but unlike the therapeutic antibody trastuzumab, it does not react with HER2 on normal cells in flow cytometry measurements. A crystal structure suggests that H2Mab-214 recognizes a structurally disrupted region in the HER2 domain IV, which normally forms a β-sheet. We show that this misfolding is inducible by site-directed mutagenesis mimicking the disulfide bond defects that also may occur in cancer cells, indicating that the local misfolding in the Cys-rich domain IV governs the cancer-specificity of H2Mab-214. Furthermore, we show that H2Mab-214 effectively suppresses tumor growth in xenograft mouse models. Our findings offer a potential strategy for developing cancer-specific therapeutic antibodies that target partially misfolded cell surface receptors.

Monoclonal antibody (mAb)-based and/or cell-based immunotherapies provide innovative approaches to cancer treatments. However, safety concerns over targeting normal cells expressing reactive antigens still exist. Therefore, the development of cancer-specific mAbs (CasMabs) that recognize cancer-specific antigens with in vivo antitumor efficacy is required to minimize the adverse effects. We previously screened anti-human epidermal growth factor receptor 2 (HER2) mAbs and successfully established a cancer-specific anti-HER2 mAb, H2Mab-250/H2CasMab-2 (IgG1, kappa). In this study, we showed that H2Mab-250 reacted with HER2-positive breast cancer cells but did not show reactivity to normal epithelial cells in flow cytometry. In contrast, a clinically approved anti-HER2 mAb, trastuzumab, recognized both breast cancer and normal epithelial cells. We further compared the affinity, effector activation, and antitumor effect of H2Mab-250 with trastuzumab. The results showed that H2Mab-250 exerted a comparable antitumor effect with trastuzumab in the mouse xenograft models of BT-474 and SK-BR-3, although H2Mab-250 possessed a lower affinity and effector activation than trastuzumab in vitro. H2Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

The C-X-C motif chemokine receptor-1 (CXCR1) is a rhodopsin-like G-protein-coupled receptor, expressed on the cell surface of immune cells and tumors. CXCR1 interacts with some C-X-C chemokines, such as CXCL6, CXCL7, and CXCL8/interleukin-8, which are produced by various cells. Since CXCR1 is involved in several diseases including tumors and diabetes mellitus, drugs targeting CXCR1 have been developed. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CXCR1 has been desired for the diagnosis and treatment. This study established a novel anti-mouse CXCR1 (mCXCR1) mAb, Cx1Mab-1 (rat IgG1, kappa), using the Cell-Based Immunization and Screening method. Cx1Mab-1 reacted with mCXCR1-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR1) and mCXCR1-overexpressed LN229 glioblastoma (LN229/mCXCR1) in flow cytometry. Cx1Mab-1 demonstrated a high binding affinity for CHO/mCXCR1 and LN229/mCXCR1 with a dissociation constant of 2.6 × 10-9 M and 2.1 × 10-8 M, respectively. Furthermore, Cx1Mab-1 could detect mCXCR1 by Western blot analysis. These results indicated that Cx1Mab-1 is useful for detecting mCXCR1, and provides a possibility for targeting mCXCR1-expressing cells in vivo experiments.

C-C chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family, is the most common coreceptor for the human immunodeficiency virus type 1. CCR5 is also involved in the pathogenesis of tumors and inflammatory diseases. The CCR5 antagonists including monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCCR5 mAbs, C5Mab-2 (rat IgG2b, kappa), reacted with mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. Using flow cytometry, the dissociation constant (KD) of C5Mab-2 for CHO/mCCR5 was determined as 4.3 × 10-8 M. These results indicated that C5Mab-2 is useful for the detection of mCCR5 in flow cytometry and may be applicable to obtain the proof of concept in preclinical studies.

Immune checkpoint blockade therapy has shown successful clinical outcomes in multiple human cancers. In dogs, several types of tumors resemble human tumors in many respects. Therefore, several groups have developed the anti-dog programmed cell death ligand 1 (dPD-L1) monoclonal antibodies (mAbs) and showed efficacy in several canine tumors. To examine the abundance of dPD-L1 in canine tumors, anti-dPD-L1 diagnostic mAbs for immunohistochemistry are required. In this study, we immunized the peptide in the dPD-L1 intracellular domain, and established anti-dPD-L1 mAbs, L₁Mab-352 (mouse IgG₁, kappa), and L₁Mab-354 (mouse IgG₁, kappa). In enzyme-linked immunosorbent assay, L₁Mab-352 and L₁Mab-354 showed high-binding affinity to the dPD-L1 peptide, and the dissociation constants (K_D) were determined as 6.9 × 10^-10 M and 7.2 × 10^-10 M, respectively. Furthermore, L₁Mab-352 and L₁Mab-354 were applicable for the detection of dPD-L1 in immunohistochemical analysis in paraffin-embedded dPD-L1-overexpressed cells. These results indicated that L₁Mab-352 and L₁Mab-354 are useful for detecting dPD-L1 in immunohistochemical analysis.

Immune checkpoint blockade therapy has shown successful clinical outcomes in multiple human cancers. In dogs, several types of tumors resemble human tumors in many respects. Therefore, several groups have developed the anti-dog programmed cell death ligand 1 (dPD-L1) monoclonal antibodies (mAbs) and showed efficacy in several canine tumors. To examine the abundance of dPD-L1 in canine tumors, anti-dPD-L1 diagnostic mAbs for immunohistochemistry are required. In this study, we immunized the peptide in the dPD-L1 intracellular domain, and established anti-dPD-L1 mAbs, L1Mab-352 (mouse IgG1, kappa), and L1Mab-354 (mouse IgG1, kappa). In enzyme-linked immunosorbent assay, L1Mab-352 and L1Mab-354 showed high-binding affinity to the dPD-L1 peptide, and the dissociation constants (KD) were determined as 6.9 × 10-10 M and 7.2 × 10-10 M, respectively. Furthermore, L1Mab-352 and L1Mab-354 were applicable for the detection of dPD-L1 in immunohistochemical analysis in paraffin-embedded dPD-L1-overexpressed cells. These results indicated that L1Mab-352 and L1Mab-354 are useful for detecting dPD-L1 in immunohistochemical analysis.

CD39 is involved in adenosine metabolism by converting extracellular ATP to adenosine. As extracellular adenosine plays a critical role in the immune suppression of the tumor microenvironment, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is one of the important strategies for tumor therapy. This study developed specific and sensitive mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening method. The established anti-mCD39 mAb, C39Mab-1 (rat IgG2a, kappa), reacted with mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant of C39Mab-1 for CHO/mCD39 was 7.3 × 10-9 M. Furthermore, C39Mab-1 detected the lysate of CHO/mCD39 by western blot analysis. These results indicated that C39Mab-1 is useful for the detection of mCD39 in many functional studies.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is an important target for monoclonal antibody (mAb) therapy. All therapeutic mAbs, including anti-HER2 mAbs, exhibit adverse effects probably due to the recognition of antigens expressed in normal cells. Therefore, tumor-selective or specific mAbs can be beneficial in reducing the adverse effects. In this study, we established a novel cancer-specific anti-HER2 monoclonal antibody, named H2Mab-250/H2CasMab-2 (IgG1, kappa). H2Mab-250 reacted with HER2-positive breast cancer BT-474 and SK-BR-3 cells. Importantly, H2Mab-250 did not react with nontransformed normal epithelial cells (HaCaT and MCF 10A) and immortalized normal epithelial cells in flow cytometry. In contrast, most anti-HER2 mAbs, such as H2Mab-119 and trastuzumab reacted with both cancer and normal epithelial cells. Immunohistochemical analysis demonstrated that H2Mab-250 possesses much higher reactivity to the HER2-positive breast cancer tissues compared with H2Mab-119, and did not react with normal tissues, including heart, breast, stomach, lung, colon, kidney, and esophagus. The epitope mapping demonstrated that the Trp614 of HER2 domain IV mainly contributes to the recognition by H2Mab-250. H2Mab-250 could contribute to the development of chimeric antigen receptor-T or antibody-drug conjugates without adverse effects for breast cancer therapy.

Breast cancer patients with high levels of human epidermal growth factor receptor 2 (HER2) expression have worse clinical outcomes. Anti-HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2-positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti-HER2 mAb, H2 Mab-77 (mouse IgG1 , kappa). This was then altered to produce H2 Mab-77-mG2a -f, an afucosylated mouse IgG2a . In the present work, we examined the reactivity of H2 Mab-77-mG2a -f and antitumor effects against breast cancers in vitro and in vivo. BT-474, an endogenously HER2-expressing breast cancer cell line, was identified by H2 Mab-77-mG2a -f with a strong binding affinity (a dissociation constant [KD ]: 5.0 × 10-9  M). H2 Mab-77-mG2a -f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in Western blot analysis. Furthermore, H2 Mab-77-mG2a -f demonstrated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for BT-474 cells. MDA-MB-468, a HER2-negative breast cancer cell line, was unaffected by H2 Mab-77-mG2a -f. Additionally, in the BT-474-bearing tumor xenograft model, H2 Mab-77-mG2a -f substantially suppressed tumor development when compared with the control mouse IgG2a mAb. In contrast, the HER2-negative MDA-MB-468-bearing tumor xenograft model showed no response to H2 Mab-77-mG2a -f. These findings point to the possibility of H2 Mab-77-mG2a -f as a treatment regimen by showing that it has antitumor effects on HER2-positive breast tumors.

Combination immunotherapy with multiple immune checkpoint inhibitors (ICIs) has been approved for various types of malignancies, including malignant pleural mesothelioma (MPM). Podoplanin (PDPN), a transmembrane sialomucin-like glycoprotein, has been investigated as a diagnostic marker and therapeutic target for MPM. We previously generated and developed a PDPN-targeting Ab reagent with high Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, the effects of anti-PDPN Abs on various tumor-infiltrating immune cells and their synergistic effects with ICIs have remained unclear. In the present study, we established a novel rat-mouse chimeric anti-mouse PDPN IgG2a mAb (PMab-1-mG2a ) and its core-fucose-deficient Ab (PMab-1-mG2a -f) to address these limitations. We identified the ADCC and CDC activity of PMab-1-mG2a -f against the PDPN-expressing mesothelioma cell line AB1-HA. The antitumor effect of monotherapy with PMab-1-mG2a -f was not sufficient to overcome tumor progression in AB1-HA-bearing immunocompetent mice. However, PMab-1-mG2a -f enhanced the antitumor effects of CTLA-4 blockade. Combination therapy with anti-PDPN Ab and anti-CTLA-4 Ab increased tumor-infiltrating natural killer (NK) cells. The depletion of NK cells inhibited the synergistic effects of PMab-1-mG2a -f and CTLA-4 blockade in vivo. These findings indicated the essential role of NK cells in novel combination immunotherapy targeting PDPN and shed light on the therapeutic strategy in advanced MPM.

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.

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.

Receptor proteins, such as epidermal growth factor receptor (EGFR), interact with other proteins in the extracellular region of the cell membrane to drive intracellular signalling. Therefore, analysis of extracellular protein-protein interactions (exPPIs) is important for understanding the biological function of receptor proteins. Here, we present an approach using a proximity biotinylation enzyme (AirID) fusion fragment of antigen binding (FabID) to analyse the proximity exPPIs of EGFR. AirID was C-terminally fused to the Fab fragment against EGFR (EGFR-FabID), which could then biotinylate the extracellular region of EGFR in several cell lines. Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis indicated that many known EGFR interactors were identified as proximity exPPIs, along with many unknown candidate interactors, using EGFR-FabID. Interestingly, these proximity exPPIs were influenced by treatment with EGF ligand and its specific kinase inhibitor, gefitinib. These results indicate that FabID provides accurate proximity exPPI analysis of target receptor proteins on cell membranes with ligand and drug responses.

A cell-surface ectonucleotidase CD39 mediates the conversion of extracellular adenosine triphosphate into immunosuppressive adenosine with another ectonucleotidase CD73. The elevated adenosine in the tumor microenvironment attenuates antitumor immunity, which promotes tumor cell immunologic escape and progression. Anti-CD39 monoclonal antibodies (mAbs), which suppress the enzymatic activity, can be applied to antitumor therapy. Therefore, an understanding of the relationship between the inhibitory activity and epitope of mAbs is important. We previously established an anti-mouse CD39 (anti-mCD39) mAb, C39Mab-1 using the Cell-Based Immunization and Screening method. In this study, we determined the critical epitope of C39Mab-1 using flow cytometry. We performed the PA tag (12 amino acids [aa])-substituted analysis (named PA scanning) and RIEDL tag (5 aa)-substituted analysis (named RIEDL scanning) to determine the critical epitope of C39Mab-1 using flow cytometry. By the combination of PA scanning and RIEDL scanning, we identified the conformational epitope, spanning three segments of 275-279, 282-291, and 306-323 aa of mCD39. These analyses would contribute to the identification of the conformational epitope of membrane proteins.

By converting extracellular adenosine triphosphate to adenosine, CD39 is involved in adenosine metabolism. The extracellular adenosine plays a critical role in the immune suppression of the tumor microenvironment. Therefore, the inhibition of CD39 activity by monoclonal antibodies (mAbs) is thought to be one of the important strategies for tumor therapy. In this study, we developed novel mAbs for mouse CD39 (mCD39) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCD39 mAbs, C39Mab-2 (rat IgG2a, lambda), reacted with mCD39-overexpressed Chinese hamster ovary-K1 (CHO/mCD39) and an endogenously mCD39-expressed cell line (SN36) by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) values of C39Mab-2 for CHO/mCD39 and SN36 were 5.5 × 10-9 M and 4.9 × 10-9 M, respectively. These results indicated that C39Mab-2 is useful for the detection of mCD39 in flow cytometry.

In small animal models of severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infection, ferrets (Mustela putorius furo) have been used to investigate the pathogenesis. Podoplanin (PDPN) is an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) against ferret PDPN (ferPDPN) are useful for the pathological analyses of those tissues. We previously established an anti-ferPDPN mAb, PMab-292 using the Cell-Based Immunization and Screening (CBIS) method. In this study, we determined the critical epitope of PMab-292 using flow cytometry. The ferPDPN deletion mutants analysis revealed that the Val34 is located at the N-terminus of the PMab-292 epitope. Furthermore, the PA tag-substituted analysis (PA scanning) showed that Asp39 is located at the C-terminus of PMab-292 epitope. The epitope sequence (VRPEDD) also exists between Val26 and Asp31 of ferPDPN, indicating that PMab-292 recognizes the tandem repeat of the VRPEDD sequence of ferPDPN.

Abstract Breast cancer patients with high levels of human epidermal growth factor receptor 2 (HER2) expression have worse clinical outcomes. Anti‐HER2 monoclonal antibody (mAb) is the most important therapeutic modality for HER2‐positive breast cancer. We previously immunized mice with the ectodomain of HER2 to create the anti‐HER2 mAb, H₂Mab‐77 (mouse IgG₁, kappa). This was then altered to produce H₂Mab‐77‐mG_2a‐f, an afucosylated mouse IgG_2a. In the present work, we examined the reactivity of H₂Mab‐77‐mG_2a‐f and antitumor effects against breast cancers in vitro and in vivo. BT‐474, an endogenously HER2‐expressing breast cancer cell line, was identified by H₂Mab‐77‐mG_2a‐f with a strong binding affinity (a dissociation constant [K_D]: 5.0 × 10⁻⁹ M). H₂Mab‐77‐mG_2a‐f could stain HER2 of breast cancer tissues in immunohistochemistry and detect HER2 protein in Western blot analysis. Furthermore, H₂Mab‐77‐mG_2a‐f demonstrated strong antibody‐dependent cellular cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC) for BT‐474 cells. MDA‐MB‐468, a HER2‐negative breast cancer cell line, was unaffected by H₂Mab‐77‐mG_2a‐f. Additionally, in the BT‐474‐bearing tumor xenograft model, H₂Mab‐77‐mG_2a‐f substantially suppressed tumor development when compared with the control mouse IgG_2a mAb. In contrast, the HER2‐negative MDA‐MB‐468‐bearing tumor xenograft model showed no response to H₂Mab‐77‐mG_2a‐f. These findings point to the possibility of H₂Mab‐77‐mG_2a‐f as a treatment regimen by showing that it has antitumor effects on HER2‐positive breast tumors.

Podocalyxin (PODXL) overexpression is associated with poor clinical outcomes in various tumors. PODXL is involved in tumor malignant progression through the promotion of invasiveness and metastasis. Therefore, PODXL is considered a promising target of monoclonal antibody (mAb)-based therapy. However, PODXL also plays an essential role in normal cells, such as vascular and lymphatic endothelial cells. Therefore, cancer specificity or selectivity is required to reduce adverse effects on normal cells. Here, we developed an anti-PODXL cancer-specific mAb (CasMab), PcMab-6 (IgG1, kappa), by immunizing mice with a soluble PODXL ectodomain derived from a glioblastoma LN229 cell. PcMab-6 reacted with the PODXL-positive LN229 cells but not with PODXL-knockout LN229 cells in flow cytometry. Importantly, PcMab-6 recognized pancreatic ductal adenocarcinoma (PDAC) cell lines (MIA PaCa-2, Capan-2, and PK-45H) but did not react with normal lymphatic endothelial cells (LECs). In contrast, one of the non-CasMabs, PcMab-47, showed high reactivity to both the PDAC cell lines and LECs. Next, we engineered PcMab-6 into a mouse IgG2a-type (PcMab-6-mG2a) and a humanized IgG1-type (humPcMab-6) mAb and further produced the core fucose-deficient types (PcMab-6-mG2a-f and humPcMab-6-f, respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC). Both PcMab-6-mG2a-f and humPcMab-6-f exerted ADCC and complement-dependent cellular cytotoxicity in the presence of effector cells and complements, respectively. In the PDAC xenograft model, both PcMab-6-mG2a-f and humPcMab-6-f exhibited potent antitumor effects. These results indicated that humPcMab-6-f could apply to antibody-based therapy against PODXL-expressing pancreatic cancers.

The erythropoietin-producing hepatocellular carcinoma (Eph) receptors are the largest receptor tyrosine kinase family. EphB4 is essential for cell adhesion and motility during embryogenesis. Pathologically, EphB4 is overexpressed and contributes to poor prognosis in various tumors. Therefore, specific monoclonal antibodies (mAbs) should be developed to predict the prognosis for multiple tumors with high EphB4 expression, including breast and gastric cancers. This study aimed to develop specific anti-EphB4 mAbs for multiple applications using the Cell-Based Immunization and Screening method. EphB4-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/EphB4) cells were immunized into mice, and we established an anti-EphB4 mAb (clone B4Mab-7), which is applicable for flow cytometry, Western blot, and immunohistochemistry (IHC). B4Mab-7 reacted with endogenous EphB4-positive breast cancer cell line, MCF-7, but did not react with EphB4-knockout MCF-7 (BINDS-52) in flow cytometry. Dissociation constant (KD) values were determined to be 2.9 × 10-9 M and 1.3 × 10-9 M by flow cytometric analysis for CHO/EphB4 and MCF-7 cells, respectively. B4Mab-7 detected the EphB4 protein bands from breast cancer cells in Western blot, and stained breast cancer tissues in IHC. Altogether, B4Mab-7 is very useful for detecting EphB4 in various applications.

In the case of the Japanese encephalitis virus (JEV), the envelope protein (E), a major component of viral particles, contains a highly conserved N-linked glycosylation site (E: N154). Glycosylation of the E protein is thought to play an important role in the ability of the virus to attach to target cells during transmission; however, its role in viral particle formation and release remains poorly understood. In this study, we investigated the role of N-glycosylation of flaviviral structural proteins in viral particle formation and secretion by introducing mutations in viral structural proteins or cellular factors involved in glycoprotein transport and processing. The number of secreted subviral particles (SVPs) was significantly reduced in N154A, a glycosylation-null mutant, but increased in D67N, a mutant containing additional glycosylation sites, indicating that the amount of E glycosylation regulates the release of SVPs. SVP secretion was reduced in cells deficient in galactose, sialic acid, and N-acetylglucosamine modifications in the Golgi apparatus; however, these reductions were not significant, suggesting that glycosylation mainly plays a role in pre-Golgi transport. Fluorescent labeling of SVPs using a split green fluorescent protein (GFP) system and time-lapse imaging by retention using a selective hook (RUSH) system revealed that the glycosylation-deficient mutant was arrested before endoplasmic reticulum (ER)- Golgi transport. However, the absence of ERGIC-53 and ERGIC-L, ER-Golgi transport cargo receptors that recognize sugar chains on cargo proteins, does not impair SVP secretion. In contrast, the solubility of the N154A mutant of E or the N15A/T17A mutant of prM in cells was markedly lower than that of the wild type, and proteasome-mediated rapid degradation of these mutants was observed, indicating the significance of glycosylation of both prM and E in proper protein folding and assembly of viral particles in the ER.

Immunohistochemistry staining is an essential method in pathological diagnoses. Podoplanin (PDPN) is a specific maker of alveolar epithelium, lymphatic vessels, and glomeruli. In this study, we established a novel anti-giraffe PDPN (girPDPN) mAb, PMab-301, using the Cell-Based Immunization and Screening (CBIS) method. PMab-301 (mouse IgG1, kappa) detected girPDPN in various applications, such as flow cytometry, western blot, and immunohistochemistry. PMab-301 specifically stained type-I alveolar cells using formalin-fixed paraffin-embedded giraffe lung tissues. Our findings suggest the potential usefulness of PMab-301 for the pathophysiological analyses of giraffe tissues.

Immune checkpoint inhibitors (ICIs) have been developed for canine tumour treatment, and pilot clinical studies have demonstrated their antitumour efficacy in dogs with oral malignant melanoma (OMM). Although ICIs have been approved for various human malignancies, their clinical benefits in other tumour types remain to be elucidated in dogs. Here, we conducted a clinical study of c4G12, a canine chimeric anti-PD-L1 antibody, to assess its safety and efficacy in dogs with various advanced malignant tumours (n = 12) at the Veterinary Teaching Hospital of Hokkaido University from 2018 to 2023. Dogs with digit or foot pad malignant melanoma (n = 4), osteosarcoma (n = 2), hemangiosarcoma (n = 1), transitional cell carcinoma (n = 1), nasal adenocarcinoma (n = 1), B-cell lymphoma (n = 1), or undifferentiated sarcoma (n = 2) were treated with 2 or 5 mg/kg c4G12 every 2 weeks. Treatment-related adverse events of any grade were observed in eight dogs (66.7%), including elevated aspartate aminotransferase (grade 3) in one dog (8.3%) and thrombocytopenia (grade 4) in another dog (8.3%). Among dogs with target disease at baseline (n = 8), as defined by the response evaluation criteria for solid tumours in dogs (cRECIST), one dog with nasal adenocarcinoma and another with osteosarcoma experienced a partial response (PR), with an objective response rate of 25.0% (2 PR out of 8 dogs; 95% confidence interval: 3.2-65.1%). These results suggest that c4G12 is safe and tolerable and shows antitumor effects in dogs with malignant tumours other than OMM. Further clinical studies are warranted to identify the tumour types that are most likely to benefit from c4G12 treatment.

Immune checkpoint molecules PD-1/PD-L1 cause T-cell exhaustion and contribute to disease progression in chronic infections of cattle. We established monoclonal antibodies (mAbs) that specifically inhibit the binding of bovine PD-1/PD-L1; however, conventional anti-PD-1 mAbs are not suitable as therapeutic agents because of their low binding affinity to antigen. In addition, their sensitivity for the detection of bovine PD-1 is low and their use for immunostaining PD-1 is limited. To address these issues, we established two anti-bovine PD-1 rabbit mAbs (1F10F1 and 4F5F2) and its chimeric form using bovine IgG1 (Boch1D10F1), which exhibit high binding affinity. One of the rabbit mAb 1D10F1 binds more strongly to bovine PD-1 compared with a conventional anti-PD-1 mAb (5D2) and exhibits marked inhibitory activity on the PD-1/PD-L1 interaction. In addition, PD-1 expression in bovine T cells could be detected with higher sensitivity by flow cytometry using 1D10F1. Furthermore, we established higher-producing cells of Boch1D10F1 and succeeded in the mass production of Boch1D10F1. Boch1D10F1 exhibited a similar binding affinity to bovine PD-1 and the inhibitory activity on PD-1/PD-L1 binding compared with 1D10F1. The immune activation by Boch1D10F1 was also confirmed by the enhancement of IFN-γ production. Finally, Boch1D10F1 was administered to bovine leukemia virus-infected cows to determine its antiviral effect. In conclusion, the high-affinity anti-PD-1 antibody developed in this study represents a powerful tool for detecting and inhibiting bovine PD-1 and is a candidate for PD-1-targeted immunotherapy in cattle.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality control process that eliminates misfolded proteins from the ER. DnaJ homolog subfamily C member 10 (ERdj5) is a protein disulfide isomerase (PDI) family member that accelerates ER-associated degradation (ERAD) by reducing disulfide bonds of aberrant proteins with the help of an ER-resident chaperone BiP. However, the detailed mechanisms by which ERdj5 acts in concert with BiP are poorly understood. In this study, we reconstituted an in vitro system that monitors ERdj5-mediated reduction of disulfide-linked J-chain oligomers, known to be physiological ERAD substrates. Biochemical analyses using purified proteins revealed that J-chain oligomers were reduced to monomers by ERdj5 in a stepwise manner via trimeric and dimeric intermediates, and BiP synergistically enhanced this action in an ATP-dependent manner. Single-molecule observations of ERdj5-catalyzed J-chain disaggregation using high-speed atomic force microscopy (HS-AFM) demonstrated the stochastic release of small J-chain oligomers through repeated actions of ERdj5 on peripheral and flexible regions of large J-chain aggregates. Using systematic mutational analyses, ERAD substrate disaggregation mediated by ERdj5 and BiP was dissected at the molecular level.

<jats:p>Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is an important target for monoclonal antibody (mAb) therapy such as trastuzumab. All therapeutic mAbs, including anti-HER2 mAbs, exhibit adverse effects probably due to the recognition of antigens expressed in normal cells. Therefore, tumor-selective or specific mAbs have been desired to reduce adverse effects. In this study, we provide a strategy for the selection of cancer-specific mAb against HER2. We screened more than 200 of anti-HER2 mAbs obtained by our laboratory and established a novel cancer-specific anti-HER2 antibody, H2Mab-250 (IgG1, kappa). H2Mab-250 reacted with HER2-positive breast cancer BT-474 and SK-BR-3 cells. Importantly, H2Mab-250 never showed reactivity to non-transformed normal epithelial cells (HaCaT and MCF 10A) and immortalized normal epithelial cells in flow cytometry. In contrast, most anti-HER2 mAbs including H2Mab-119 (IgG1, kappa) reacted with both cancer and normal epithelial cells. The epitope mapping revealed that H2Mab-250 recognized the domain VI of HER2 and the Trp614 mainly contributes to the recognition by H2Mab-250. In immunohistochemical analysis, H2Mab-250 exhibited a superior reactivity to HER2-positive breast cancer section compared to H2Mab-119. Importantly, H2Mab-250 never showed any reactivity to normal tissues by immunohistochemical analysis. The strategy to select cancer-specific mAbs would contribute to the development of novel antibodies and modalities for cancer therapy.</jats:p>

The clinically approved human epidermal growth factor receptor 2 (HER2)-targeting monoclonal antibodies (mAbs), trastuzumab, and pertuzumab, target domains IV and II, respectively. Trastuzumab is now the standard treatment for HER2-overexpressed breast and gastric cancers, and trastuzumab in combination with pertuzumab showed clinical benefit. However, there still exist patients who do not respond to the therapy. Furthermore, HER2 mutants that cannot be recognized by pertuzumab were found in tumors. Therefore, novel anti-HER2 mAbs and modalities have been desired. In our previous study, we developed a novel anti-HER2 domain I mAb, H2Mab-139 (mouse IgG1, kappa). We herein produced a defucosylated mouse IgG2a type of mAb against HER2 (H2Mab-139-mG2a-f) to enhance antibody-dependent cellular cytotoxicity (ADCC)-mediated antitumor activity. H2Mab-139-mG2a-f exhibits a high binding affinity in flow cytometry with the dissociation constant (KD) determined to be 3.9 × 10-9 M and 7.7 × 10-9 M against HER2-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/HER2) and HER2-positive BT-474 cells, respectively. Moreover, we showed that H2Mab-139-mG2a-f exerted ADCC and complement-dependent cytotoxicity against CHO/HER2 and BT-474 in vitro and exhibited potent antitumor activities in mouse xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and is useful as an antibody treatment for HER2-positive human cancers.

A cancer-specific anti-PDPN mAb, LpMab-23 (mouse IgG1, kappa), was established in our previous study. We herein produced a humanized IgG1 version (humLpMab-23) and defucosylated form (humLpMab-23-f) of an anti-PDPN mAb to increase ADCC activity. humLpMab-23 recognized PDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/PDPN), PDPN-positive PC-10 (human lung squamous cell carcinoma), and LN319 (human glioblastoma) cells via flow cytometry. We then demonstrated that humLpMab-23-f induced ADCC and complement-dependent cytotoxicity against CHO/PDPN, PC-10, and LN319 cells in vitro and exerted high antitumor activity in mouse xenograft models, indicating that humLpMab-23-f could be useful as an antibody therapy against PDPN-positive lung squamous cell carcinomas and glioblastomas.

Carcinoma cells possess high proliferative and invasive potentials and exhibit a resilience against stresses, metabolic disorder, and therapeutic efforts. These properties are mainly acquired by genetic alterations including driver gene mutations. However, the detailed molecular mechanisms have not been fully elucidated. Here, we provide a novel mechanism connecting oncogenic signaling and the tumorigenic properties by a transforming growth factor-β1-stimulated clone 22 (TSC-22) family protein, THG-1 (also called as TSC22D4). THG-1 is localized at the basal layer of normal squamous epithelium and overexpressed in squamous cell carcinomas (SCCs). THG-1 knockdown suppressed SCC cell proliferation, invasiveness, and xenograft tumor formation. In contrast, THG-1 overexpression promoted the EGF-induced proliferation and stratified epithelium formation. Furthermore, THG-1 is phosphorylated by the receptor tyrosine kinase (RTK)-RAS-ERK pathway, which promoted the oncogene-mediated tumorigenesis. Moreover, THG-1 involves in the alternative splicing of CD44 variants, a regulator of invasiveness, stemness, and oxidative stress resistance under the RTK pathway. These findings highlight the pivotal roles of THG-1 as a novel effector of SCC tumorigenesis, and the detection of THG-1 phosphorylation by our established specific antibody could contribute to cancer diagnosis and therapy.

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to the development of trastuzumab–deruxtecan, an antibody-drug conjugate, the targetable HER2-positive breast cancer patients have been expanded. To evaluate the developing modalities using anti-HER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs against mouse HER2 (mHER2) should be established. This study developed anti-mHER2 mAbs using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs, H2Mab-300 (rat IgG2b, kappa) and H2Mab-304 (rat IgG1, kappa), reacted with mHER2-overexpressed Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial cell) via flow cytometry. Furthermore, these mAbs never recognized mHER2-knockout NMuMG cells. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) values of H2Mab-300 and H2Mab-304 for CHO/mHER2 were 1.2 × 10−9 M and 1.7 × 10−9 M, respectively. The KD values of H2Mab-300 and H2Mab-304 for NMuMG were 4.9 × 10−10 M and 9.0 × 10−10 M, respectively. These results indicated that H2Mab-300 and H2Mab-304 could apply to the detection of mHER2 using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

Zinc ions (Zn2+) are vital to most cells, with the intracellular concentrations of Zn2+ being tightly regulated by multiple zinc transporters located at the plasma and organelle membranes. We herein present the 2.2-3.1 Å-resolution cryo-EM structures of a Golgi-localized human Zn2+/H+ antiporter ZnT7 (hZnT7) in Zn2+-bound and unbound forms. Cryo-EM analyses show that hZnT7 exists as a dimer via tight interactions in both the cytosolic and transmembrane (TM) domains of two protomers, each of which contains a single Zn2+-binding site in its TM domain. hZnT7 undergoes a TM-helix rearrangement to create a negatively charged cytosolic cavity for Zn2+ entry in the inward-facing conformation and widens the luminal cavity for Zn2+ release in the outward-facing conformation. An exceptionally long cytosolic histidine-rich loop characteristic of hZnT7 binds two Zn2+ ions, seemingly facilitating Zn2+ recruitment to the TM metal transport pathway. These structures permit mechanisms of hZnT7-mediated Zn2+ uptake into the Golgi to be proposed.

Podoplanin (PDPN) is a prognostic factor and is involved in several mechanisms of tumor progression in human squamous cell carcinoma (SCC). Canine non-tonsillar SCC (NTSCC) is a common oral tumor in dogs and has a highly invasive characteristic. In this study, we investigated the function of PDPN in canine NTSCC. In canine NTSCC clinical samples, PDPN overexpression was observed in 80% of dogs with NTSCC, and PDPN expression was related to ki67 expression. In PDPN knocked-out canine NTSCC cells, cell proliferation, cancer stemness, and migration were suppressed. As the mechanism of PDPN-mediated cell proliferation, PDPN knocked-out induced apoptosis and G2/M cell cycle arrest in canine NTSCC cells. These findings suggest that PDPN promotes tumor malignancies and may be a novel biomarker and therapeutic target for canine NTSCC.

Mucosal melanoma metastasizes at an early stage of the disease in human and dog. We revealed that overexpression of podoplanin (PDPN) in tumor invasion fronts (IFs) was related to poor prognosis of dogs with mucosal melanoma. Moreover, PDPN expressed in canine mucosal melanoma cells promotes proliferation and aggressive amoeboid invasion by activating Rho-associated kinase (ROCK)-myosin light chain 2 (MLC2) signaling. PDPN-ROCK-MLC2 signaling plays a role in cell cycle arrest and cellular senescence escape as a mechanism for regulating proliferation. PDPN induces amoeboid invasion in the IFs of mouse xenografted tumor tissues, similar to canine mucosal melanoma clinical samples. We further identified that PDPN expression was related to poor prognosis of human patients with mucosal melanoma, and human mucosal melanoma with PDPN high expression enriched gene signatures related to amoeboid invasion, similar to canine mucosal melanoma. Overall, we propose that PDPN promotes canine and human mucosal melanoma metastasis by inducing aggressive amoeboid invasion and naturally occurring canine mucosal melanoma can be a novel research model for PDPN expressing human mucosal melanoma. Implications: PDPN could be a new therapeutic target to restrict the metastatic dissemination of canine and human mucosal melanoma.

CC chemokine receptor 6 (CCR6) is a member of the G-protein-coupled receptor family that is highly expressed in B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. CCR6 has been revealed to have important functions in many pathological conditions, such as cancer, intestinal bowel disease, psoriasis, and autoimmune diseases. The only CCR6 chemokine ligand, CC motif chemokine ligand 20 (CCL20), is also involved in pathogenesis by interacting with CCR6. The CCL20/CCR6 axis is drawing attention as an attractive therapeutic target for various diseases. In this study, we developed novel monoclonal antibodies (mAbs) against human CCR6 (hCCR6) using the peptide immunization method, which are applicable to flow cytometry and immunohistochemistry. The established anti-hCCR6 mAb, clone C6Mab-19 (mouse IgG1, kappa), reacted with hCCR6-overexpressed Chinese hamster ovary-K1 (CHO/hCCR6), human liver carcinoma (HepG2), and human differentiated hepatoma (HuH-7) cells in flow cytometry. The dissociation constant (KD) of C6Mab-19 was determined as 3.0 × 10-10 M for CHO/hCCR6, 6.9 × 10-10 M for HepG2, and 1.8 × 10-10 M for HuH-7. Thus, C6Mab-19 could bind to exogenously and endogenously expressed hCCR6 with extremely high affinity. Furthermore, C6Mab-19 could stain formalin-fixed paraffin-embedded lymph node tissues from a patient with non-Hodgkin lymphoma by immunohistochemistry. Therefore, C6Mab-19 is suitable for detecting hCCR6-expressing cells and tissues and could be useful for pathological analysis and diagnosis.

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC with peritoneal metastasis exhibits a poor prognosis due to the lack of effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed in tumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeted monoclonal antibodies (mAbs) is important for GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of the clones (C44Mab-94; IgG1, kappa) recognized the variant-8-encoded region and peptide, indicating that C44Mab-94 is a specific mAb for CD44v8. Furthermore, C44Mab-94 could recognize CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and NUGC-4) in flow cytometric analyses. C44Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastric cancer cells. These results indicate that C44Mab-94 is useful for detecting CD44v8 in a variety of experimental methods and is expected to become usefully applied to GC diagnosis and therapy.

Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer, and has been revealed as the second-highest expression of CD44 in cancers. CD44 has been investigated as a cancer stem cell marker of HNSCC and plays a critical role in tumor malignant progression. Especially, splicing variant isoforms of CD44 (CD44v) are overexpressed in cancers and considered a promising target for cancer diagnosis and therapy. We developed monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3–10-overexpressed PANC-1 cells. Among the established clones, C44Mab-18 (IgM, kappa) reacted with CHO/CD44v3–10, but not with CHO/CD44s and parental CHO-K1 using flow cytometry. The epitope mapping using peptides that cover variant exon-encoded regions revealed that C44Mab-18 recognized the border sequence between variant 10 and the constant exon 16-encoded sequence. These results suggest that C44Mab-18 recognizes variant 10-containing CD44v, but not CD44s. Furthermore, C44Mab-18 could recognize the human oral squamous cell carcinoma (OSCC) cell line, HSC-3, in flow cytometry. The apparent dissociation constant (KD) of C44Mab-18 for CHO/CD44v3–10 and HSC-3 was 1.6 × 10−7 M and 1.7 × 10−7 M, respectively. Furthermore, C44Mab-18 detected CD44v3–10 but not CHO/CD44s in Western blotting, and endogenous CD44v10 in immunohistochemistry using OSCC tissues. These results indicate that C44Mab-18 is useful for detecting CD44v10 in flow cytometry and immunohistochemistry.

Although immune checkpoint inhibitors (ICIs), such as the anti-programmed death-ligand 1 (PD-L1) antibody, have been developed for the treatment of canine malignant melanoma, desirable clinical efficacies have not been achieved. Recent studies in humans have suggested that radiation therapy (RT) combined with ICIs induces robust systemic antitumour immunity in patients with cancer. This study retrospectively examined the therapeutic efficacy of combination therapy (hypofractionated RT and anti-PD-L1 antibody [c4G12]) in dogs with pulmonary metastatic oral malignant melanoma. The intrathoracic clinical benefit rate (CBR)/median overall survival (OS) in the no RT (n = 20, free from the effect of RT), previous RT (n = 9, received RT ≤8 weeks prior to the first c4G12 dose), and concurrent RT (n = 10, c4G12 therapy within ±1 week of the first RT fraction) groups were 10%/185 days, 55.6%/283.5 days (p < 0.05 vs. no RT group), and 20%/129 days (p > 0.05 vs. no RT group), respectively. The adverse events were considered to be tolerable in the combination therapy. Thus, hypofractionated RT before the initiation of c4G12 therapy can be an effective approach for enhancing the therapeutic efficacy of immunotherapy, with acceptable safety profiles. Further prospective clinical studies are required to confirm the findings of this study.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

Sheep have been used as a large animal experimental model for studying infectious diseases. However, due to a lack of staining antibodies and reagents, immunological studies on sheep have not progressed. The immunoinhibitory receptor programmed death-1 (PD-1) is expressed on T lymphocytes. The interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. We previously reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections using anti-bovine PD-L1 monoclonal antibodies (mAbs). Furthermore, we found that blocking antibodies against PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy of cattle. However, the immunological role of the PD-1/PD-L1 pathway in chronic diseases of sheep remains unknown. In this study, we identified cDNA sequences of ovine PD-1 and PD-L1 and examined the cross-activity of anti-bovine PD-L1 mAbs against ovine PD-L1 as well as the expression of PD-L1 in ovine listeriosis. The amino acid sequences of ovine PD-1 and PD-L1 share a high degree of identity and similarity with homologs from ruminants and other mammalian species. Anti-bovine PD-L1 mAb recognized ovine PD-L1 on lymphocytes in the flow cytometric assay. Furthermore, an immunohistochemical staining confirmed the PD-L1 expression on macrophages in the brain lesions of ovine listeriosis. These findings indicated that our anti-PD-L1 mAb would be useful for analyzing the ovine PD-1/PD-L1 pathway. Further research is needed to determine the immunological role of PD-1/PD-L1 in chronic diseases such as BLV infection through experimental infection of sheep.

<jats:p>The Epidermal Growth Factor Receptor (EGFR) overexpression or its mutation mediates the sustaining proliferative signaling in cancers. Human EGFR-targeting monoclonal antibody (mAb) therapy such as cetuximab has been approved for clinical use in patients with colorectal cancers and head and neck squamous cell carcinomas. A reliable preclinical mouse model is essential to further develop the mAb therapy against EGFR. However, a mAb against mouse EGFR (mEGFR) for flow cytometry has not been established. In this study, we developed a specific and sensitive mAb for mEGFR using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mEGFR mAb, EMab-300 (rat IgG1, kappa) reacted with mEGFR-overexpressed Chinese hamster ovary-K1 (CHO/mEGFR) and endogenously mEGFR-expressed cell lines, including NMuMG (a mouse mammary gland epithelial cell) and Lewis lung carcinoma cells by flow cytometry. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) of EMab-300 for CHO/mEGFR and NMuMG was 4.3 &amp;times; 10&amp;minus;8 M and 1.9 &amp;times; 10&amp;minus;8 M, respectively. These results indicated that EMab-300 applies to the detection of mEGFR by flow cytometry, and is expected in the use of preclinical study.</jats:p>

Antigen-binding fragments (Fabs) of antibodies are both key biopharmaceuticals and valuable tools for basic life science. To streamline the production of diverse Fabs by capitalizing on standard and highly optimized protein production protocols, we here explore a method to prepare recombinant Fabs as secreted fusion proteins with an N-terminal human growth hormone domain and an octa-histidine tag. These tagged Fabs can be purified with standard immobilized metal chelate affinity chromatography. We first demonstrated Fab overproduction using the rat monoclonal antibody NZ-1. Optimization of linker residues enabled the complete removal of the tags by TEV protease, leaving only two additional residues at the N-terminus of the heavy chain. We purified NZ-1 Fab at ∼4 μg/mL of culture supernatant and further confirmed that the NZ-1 Fab from the fusion protein maintained its native fold and binding affinity for target cell-surface antigens. We also showed that several other Fabs of mouse IgG1s, the major subclass in mice, could be produced with the same procedure. Our preparation method can provide greater flexibility in functional and structural modifications of target Fabs because specialized purification techniques are not necessary.

Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, elucidating the function of each CD44 isoform in a tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through the immunization of CD44v3-10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3), as determined via an enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3-10) or some cancer cell lines (COLO205 and HSC-3) via flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3-10, COLO205, and HSC-3 was 1.5 × 10-9 M, 6.3 × 10-9 M, and 1.9 × 10-9 M, respectively. C44Mab-6 could detect the CD44v3-10 in Western blotting and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments and is expected for the application of tumor diagnosis and therapy.

Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker and plays tumor promotion and drug resistance roles in various cancers. In particular, the splicing variants are overexpressed in many carcinomas and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant's (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-3; IgG1, kappa) recognized peptides of the variant-5-encoded region, indicating that C44Mab-3 is a specific mAb for CD44v5. Moreover, C44Mab-3 reacted with CHO/CD44v3-10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent KD of C44Mab-3 for CHO/CD44v3-10 and PK-1 was 1.3 × 10-9 M and 2.6 × 10-9 M, respectively. C44Mab-3 could detect the exogenous CD44v3-10 and endogenous CD44v5 in Western blotting and stained the formalin-fixed paraffin-embedded pancreatic cancer cells but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that C44Mab-3 is useful for detecting CD44v5 in various applications and is expected to be useful for the application of pancreatic cancer diagnosis and therapy.

CC chemokine receptor 6 (CCR6) is one of the members of the G-protein-coupled receptor (GPCR) family that is upregulated in many immune-related cells, such as B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. The coordination between CCR6 and its ligand CC motif chemokine ligand 20 (CCL20) is deeply involved in the pathogenesis of various diseases, such as cancer, psoriasis, and autoimmune diseases. Thus, CCR6 is an attractive target for therapy and is being investigated as a diagnostic marker for various diseases. In a previous study, we developed an anti-mouse CCR6 (mCCR6) monoclonal antibody (mAb), C6Mab-13 (rat IgG1, kappa), that was applicable for flow cytometry by immunizing a rat with the N-terminal peptide of mCCR6. In this study, we investigated the binding epitope of C6Mab-13 using an enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) method, which were conducted with respect to the synthesized point-mutated-peptides within the 1-20 amino acid region of mCCR6. In the ELISA results, C6Mab-13 lost its ability to react to the alanine-substituted peptide of mCCR6 at Asp11, thereby identifying Asp11 as the epitope of C6Mab-13. In our SPR analysis, the dissociation constants (KD) could not be calculated for the G9A and D11A mutants due to the lack of binding. The SPR analysis demonstrated that the C6Mab-13 epitope comprises Gly9 and Asp11. Taken together, the key binding epitope of C6Mab-13 was determined to be located around Asp11 on mCCR6. Based on the epitope information, C6Mab-13 could be useful for further functional analysis of mCCR6 in future studies.

Antibodies against immune checkpoint molecules restore T-cell function by inhibiting the binding of PD-1 and PD-L1 and have been shown to exert therapeutic effects in various human cancers. However, to date, no monoclonal antibody that recognizes feline PD-1 or PD-L1 has been reported, and there are many unknowns regarding the expression of immune checkpoint molecules and their potential as therapeutic targets in cats. Here we developed anti-feline PD-1 monoclonal antibody (1A1-2), and found that the monoclonal antibody against anti-canine PD-L1 (G11-6), which was previously developed in our laboratory, cross-reacted with feline PD-L1. Both antibodies inhibited the interaction of feline PD-1 and feline PD-L1 in vitro. These inhibitory monoclonal antibodies augmented the interferon-gamma (IFN-γ) production in activated feline peripheral blood lymphocytes (PBLs). Furthermore, for clinical application in cats, we generated a mouse-feline chimeric mAb by fusing the variable region of clone 1A1-2 with the constant region of feline IgG1 (ch-1A1-2). Ch-1A1-2 also augmented the IFN-γ production in activated feline PBLs. From this study, 1A1-2 is first anti-feline PD-1 monoclonal antibody with the ability to inhibit the interaction of feline PD-1 and PD-L1, and the chimeric antibody, ch-1A1-2 will be a beneficial therapeutic antibody for feline tumors.

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.

One of G protein-coupled receptors, CC chemokine receptor 3 (CCR3), is expressed in eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 levels in the serum of colorectal cancer patients are significantly higher than in control groups. Moreover, CCR3 is essential for recruiting eosinophils into the lung. Therefore, CCR3 is considered both a therapeutic target for colorectal cancer and allergic diseases. Previously, we established anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), by immunizing a rat with an N-terminal peptide of mCCR3. These mAbs can be used in flow cytometry and enzyme-linked immunosorbent assays. In this study, we performed the epitope mapping of C3Mab-6 and C3Mab-7 using alanine scanning. The reactivity between these mAbs and point mutants of mCCR3 were analyzed using flow cytometry. The results indicated that Phe3, Asn4, Thr5, Asp6, Glu7, Lys9, Thr10, and Glu13 of mCCR3 are essential for C3Mab-6 binding, whereas Phe15 and Glu16 are essential for C3Mab-7 binding.

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.

<jats:p>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 &amp;times; 10&amp;minus;9 M and 5.5 &amp;times; 10&amp;minus;9 M, respectively. KD of C39Mab-1 and C39Mab-2 for L1210 was 3.3 &amp;times; 10&amp;minus;9 M and 3.6 &amp;times; 10&amp;minus;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.</jats:p>

We recently developed a novel anti-human C-C chemokine receptor 9 (hCCR9) monoclonal antibody (mAb), C9Mab-11, which is applicable to flow cytometry, western blotting, and enzyme-linked immunosorbent assay (ELISA). This study aims to identify the binding epitope of C9Mab-11 by using 1 × and 2 × alanine (or glycine) substituted-hCCR9 peptides (1 × and 2 × Ala-scan) by ELISA. According to the 1 × Ala-scan analysis, the response of C9Mab-11 was diminished against M13A of the hCCR9 peptide, but was not eliminated. In the 2 × Ala-scan analysis, the reactions were abolished in the substitution of P11A-N12A, N12A-M13A, and M13A-A14G of hCCR9 N-terminal peptides. The results indicate that the binding epitope of C9Mab-11 includes Pro11, Asn12, Met13, and Ala14 of hCCR9, with the region around Met13 being particularly important. The successful identification of the C9Mab-11 epitope might be useful for the future pathophysiological analysis of hCCR9.

Interactions between programmed death 1 (PD-1) and PD-ligand 1 (PD-L1) cause functional exhaustion of T cells by inducing inhibitory signals, thereby attenuating effector functions of T cells. We have developed an anti-bovine PD-L1 blocking antibody (Ab) and have demonstrated that blockade of the interaction between PD-1 and PD-L1 reactivates T-cell responses in cattle. In the present study, we examined the potential utility of PD-1/PD-L1-targeted immunotherapy in enhancing T-cell responses to vaccination. Calves were inoculated with a hexavalent live-attenuated viral vaccine against bovine respiratory infections in combination with treatment with an anti-PD-L1 Ab. The expression kinetics of PD-1 in T cells and T-cell responses to viral antigens were measured before and after vaccination to evaluate the adjuvant effect of anti-PD-L1 Ab. PD-1 expression was upregulated in vaccinated calves after the administration of a booster vaccination. The activation status of CD4+, CD8+, and γδTCR+ T cells was enhanced by the combination of vaccination and PD-L1 blockade. In addition, IFN-γ responses to viral antigens were increased following combinatorial vaccination with PD-L1 blockade. In conclusion, the blockade of the PD-1/PD-L1 interaction enhances T-cell responses induced by vaccination in cattle, indicating the potential utility of anti-PD-L1 Ab in improving the efficacy of current vaccination programs.

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.

CD44 is a cell surface glycoprotein, and its isoforms are produced by the alternative splicing with the standard and variant exons. The CD44 variant exon-containing isoforms (CD44v) are overexpressed in carcinomas. CD44v6 is one of the CD44v, and its overexpression predicts poor prognosis in colorectal cancer (CRC) patients. CD44v6 plays critical roles in CRC adhesion, proliferation, stemness, invasiveness, and chemoresistance. Therefore, CD44v6 is a promising target for cancer diagnosis and therapy for CRC. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary (CHO)-K1 cells. We then characterized them using enzyme-linked immunosorbent assay, flow cytometry, western blotting, and immunohistochemistry. One of the established clones (C44Mab-9; IgG1, kappa) reacted with a peptide of the variant 6-encoded region, indicating that C44Mab-9 recognizes CD44v6. Furthermore, C44Mab-9 reacted with CHO/CD44v3-10 cells or CRC cell lines (COLO201 and COLO205) by flow cytometry. The apparent dissociation constant (KD) of C44Mab-9 for CHO/CD44v3-10, COLO201, and COLO205 was 8.1 × 10-9 M, 1.7 × 10-8 M, and 2.3 × 10-8 M, respectively. C44Mab-9 detected the CD44v3-10 in western blotting, and partially stained the formalin-fixed paraffin-embedded CRC tissues in immunohistochemistry. Collectively, C44Mab-9 is useful for detecting CD44v6 in various applications.

Human epidermal growth factor receptor 2 (HER2) is a cell surface type I transmembrane glycoprotein that is overexpressed on a variety of solid tumors and transduces the oncogenic signaling upon homo- and heterodimerization with HER families. Anti-HER2 monoclonal antibodies (mAbs) including trastuzumab and its antibody-drug conjugate have been shown to improve patients' survival in HER2-positive breast, gastric, and lung cancers. Canine tumors have advantages as naturally occurring tumor models, and share biological and histological characteristics with human tumors. In this study, we generated a defucosylated version of mouse-dog chimeric anti-HER2 mAb (H77Bf) derived from H2Mab-77 (mouse IgG1, kappa). H77Bf possesses the high binding affinity (a dissociation constant: 8.7 × 10-10 M) for a dog HER2 (dHER2)-expressing canine fibroblastic tumor cell line (A-72). H77Bf exhibited antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity for A-72 cells. Moreover, intraperitoneal administration of H77Bf significantly suppressed the development of A-72 tumor compared with the control dog IgG in a mouse xenograft model. These results indicate that H77Bf exerts antitumor activities against dHER2-expressing canine cancers, which could provide a valuable information for canine cancer treatment.

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.

Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, which is highly expressed on tumor cells. As EpCAM plays a crucial role in cell adhesion, survival, proliferation, stemness, and tumorigenesis, it has been considered as a promising target for tumor diagnosis and therapy. Anti‑EpCAM monoclonal antibodies (mAbs) have been developed and have previously demonstrated promising outcomes in several clinical trials. An anti‑EpCAM mAb, EpMab‑37 (mouse IgG1, kappa) was previously developed by the authors, using the cell‑based immunization and screening method. In the present study, a defucosylated version of anti‑EpCAM mAb (EpMab‑37‑mG2a‑f) was generated to evaluate the antitumor activity against EpCAM‑positive cells. EpMab‑37‑mG2a‑f recognized EpCAM‑overexpressing CHO‑K1 (CHO/EpCAM) cells with a moderate binding‑affinity [dissociation constant (KD)=2.2x10‑8 M] using flow cytometry. EpMab‑37‑mG2a‑f exhibited potent antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) for CHO/EpCAM cells by murine splenocytes and complements, respectively. Furthermore, the administration of EpMab‑37‑mG2a‑f significantly suppressed CHO/EpCAM xenograft tumor development compared with the control mouse IgG. EpMab‑37‑mG2a‑f also exhibited a moderate binding‑affinity (KD=1.5x10‑8 M) and high ADCC and CDC activities for a colorectal cancer cell line (Caco‑2 cells). The administration of EpMab‑37‑mG2a‑f to Caco‑2 tumor‑bearing mice significantly suppressed tumor development compared with the control. By contrast, EpMab‑37‑mG2a‑f never suppressed the xenograft tumor growth of Caco‑2 cells in which EpCAM was knocked out. On the whole, these results indicate that EpMab‑37‑mG2a‑f may exert antitumor activities against EpCAM‑positive cancers and may thus be a promising therapeutic regimen for colorectal cancer.

C-C chemokine receptor 9 (CCR9) is a receptor for C-C-chemokine ligand 25 (CCL25). CCR9 is crucial in the chemotaxis of immune cells and inflammatory responses. Moreover, CCR9 is highly expressed in tumors, including several solid tumors and T-cell acute lymphoblastic leukemia. Several preclinical studies have shown that anti-CCR9 monoclonal antibodies (mAbs) exert antitumor activity. Therefore, CCR9 is an attractive target for tumor therapy. In this study, we conducted the epitope mapping of an anti-mouse CCR9 (mCCR9) mAb, C9Mab-24 (rat IgG2a, kappa), using the 1× alanine (1× Ala)- and 2× alanine (2× Ala)-substitution methods via enzyme-linked immunosorbent assay. We first performed the 1× Ala-substitution method using one alanine-substituted peptides of the mCCR9 N-terminus (amino acids 1-19). C9Mab-24 did not recognize two peptides (F14A and F17A), indicating that Phe14 and Phe17 are critical for C9Mab-24-binding to mCCR9. Furthermore, we conducted the 2× Ala-substitution method using two consecutive alanine-substituted peptides of the mCCR9 N-terminus, and showed that C9Mab-24 did not react with four peptides (M13A-F14A, F14A-D15A, D16A-F17A, and F17A-S18A), indicating that 13-MFDDFS-18 is involved in C9Mab-24-binding to mCCR9. Overall, combining, the 1× Ala- or 2× Ala-scanning methods could be useful for understanding for target-antibody interaction.

Colorectal cancer (CRC) is the third most common type of cancer and the second leading cause of cancer deaths worldwide. Surgery or surgery plus radiotherapy and/or chemotherapy for patients with metastatic CRC (mCRC) were accepted as the main therapeutic strategies until the early 2000s, when targeted drugs, like cetuximab and bevacizumab, were developed. The use of targeted drugs in clinical practice has significantly increased patients' overall survival. To date, the emergence of several types of targeted drugs has opened new possibilities and revealed new prospects for mCRC treatment. Therapeutic strategies are continually being updated to select the most suitable targeted drugs based on the results of clinical trials that are currently underway. This review discusses the up-to date molecular evidence of targeted therapy for mCRC and summarizes the Food and Drug Administration-approved targeted drugs including the results of clinical trials. We also explain their mechanisms of action and how these affect the choice of a suitable targeted therapy.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) in cattle and is widespread in many countries, including Japan. Recent studies have revealed that the expression of immunoinhibitory molecules, such as programmed death-1 (PD-1) and PD-ligand 1, plays a critical role in immunosuppression and disease progression during BLV infection. In addition, a preliminary study has suggested that another immunoinhibitory molecule, T-cell immunoglobulin domain and mucin domain-3 (TIM-3), is involved in immunosuppression during BLV infection. Therefore, this study was designed to further elucidate the immunoinhibitory role of immune checkpoint molecules in BLV infection. TIM-3 expression was upregulated on peripheral CD4+ and CD8+ T cells in BLV-infected cattle. Interestingly, in EBL cattle, CD4+ and CD8+ T cells infiltrating lymphomas expressed TIM-3. TIM-3 and PD-1 were upregulated and coexpressed in peripheral CD4+ and CD8+ T cells from BLV-infected cattle. Blockade by anti-bovine TIM-3 monoclonal antibody increased CD69 expression on T cells and gamma interferon (IFN-γ) production from peripheral blood mononuclear cells from BLV-infected cattle. A syncytium formation assay also demonstrated the antiviral effects of TIM-3 blockade against BLV infection. The combined inhibition of TIM-3 and PD-1 pathways significantly enhanced IFN-γ production and antiviral efficacy compared to inhibition alone. In conclusion, the combined blockade of TIM-3 and PD-1 pathways shows strong immune activation and antiviral effects and has potential as a novel therapeutic method for BLV infection. IMPORTANCE Enzootic bovine leukosis caused by bovine leukemia virus (BLV) is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BLV-host interactions are complex. Previously, it was found that immune checkpoint molecules, such as PD-1, suppress BLV-specific Th1 responses as the disease progresses. To date, most studies have focused only on how PD-1 facilitates escape from host immunity in BLV-infected cattle and the antiviral effects of the PD-1 blockade. In contrast, how T-cell immunoglobulin domain and mucin domain-3 (TIM-3), another immune checkpoint molecule, regulates anti-BLV immune responses is rarely reported. It is also unclear why PD-1 inhibition alone was insufficient to exert anti-BLV effects in previous clinical studies. In this study, the expression profile of TIM-3 in T cells derived from BLV-infected cattle suggested that TIM-3 upregulation is a cause of immunosuppression in infected cattle. Based on these results, anti-TIM-3 antibody was used to experimentally evaluate its function in influencing immunity against BLV. Results indicated that TIM-3 upregulation induced by BLV infection suppressed T-cell activation and antiviral cytokine production. Some T cells coexpressed PD-1 and TIM-3, indicating that simultaneous inhibition of PD-1 and TIM-3 with their respective antibodies synergistically restored antiviral immunity. This study could open new avenues for treating bovine chronic infections.

Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has progressed dramatically in the past decade, partly due to the success of immunotherapies using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1 therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1 and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 share high sequence identities (66-83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor-ligand binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established an anti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PD-L1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in a feline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm, FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-γ stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1 expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5, 80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor-angiotensin-converting enzyme-2 (ACE2) as the first step in viral cell entry. SARS-CoV-2 spike protein expression in the ACE2-expressing cell surface induces cell-cell membrane fusion, thus forming syncytia. To exert its fusogenic activity, the spike protein is typically processed at a specific site (the S1/S2 site) by cellular proteases such as furin. The C488 residue, located at the spike-ACE2 interacting surface, is critical for the fusogenic and infectious roles of the SARS-CoV-2 spike protein. We have demonstrated that the C488 residue of the spike protein is involved in subcellular targeting and S1/S2 processing. C488 mutant spike localization to the Golgi apparatus and cell surface were impaired. Consequently, the S1/S2 processing of the spike protein, probed by anti-Ser-686-cleaved spike antibody, markedly decreased in C488 mutant spike proteins. Moreover, brefeldin-A-mediated endoplasmic-reticulum-to-Golgi traffic suppression also suppressed spike protein S1/S2 processing. As brefeldin A treatment and C488 mutation inhibited S1/S2 processing and syncytia formation, the C488 residue of spike protein is required for functional spike protein processing.

The CC chemokine receptor 9 (CCR9), also known as CD199, is one of chemokine receptors. The CC chemokine ligand 25 (CCL25) is known to be the only ligand for CCR9. The CCR9-CCL25 interaction plays important roles in chemotaxis of lymphocytes and tumor cell migration. Therefore, CCR9-CCL25 axis is a promising target for tumor therapy and diagnosis. In this study, we established a sensitive and specific monoclonal antibody (mAb) against mouse CCR9 (mCCR9) using N-terminal peptide immunization method. The established anti-mCCR9 mAb, C9Mab-24 (rat immunoglobulin [IgG]2a, kappa), reacted with mCCR9-overexpressed Chinese hamster ovary-K1 (CHO/mCCR9) and mCCR9-endogenously expressed cell line, RL2, through flow cytometry. Kinetic analyses using flow cytometry showed that the dissociation constants (KD) of C9Mab-24 for CHO/mCCR9 and RL2 cell lines were 6.0 × 10-9 M and 4.7 × 10-10 M, respectively. Results indicated that C9Mab-24 is useful for detecting mCCR9 through flow cytometry, thereby providing a possibility for targeting mCCR9-expressing cells in vivo experiments.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a global pandemic of coronavirus disease 19. Coronaviruses, including SARS-CoV-2, use RNA-dependent RNA polymerase (RdRP) for viral replication and transcription. Since RdRP is a promising therapeutic target for infection of SARS-CoV-2, it would be beneficial to develop new experimental tools for analysis of the RdRP reaction of SARS-CoV-2. Here, we succeeded to develop novel mouse monoclonal antibodies (mAbs) that recognize SARS-CoV-2 nsp12, catalytic subunit of the RdRP. These anti-nsp12 mAbs, RdMab-2, -13, and -20, specifically recognize SARS-CoV-2 nsp12 by western blotting analysis, while they exhibit less or no cross-reactivity to SARS-CoV nsp12. In addition, SARS-CoV-2 nsp12 was successfully immunoprecipitated using RdMab-2 from lysates of cells overexpressing SARS-CoV-2 nsp12. RdMab-2 was able to detect SARS-CoV-2 nsp12 transiently expressed in established culture cells such as HEK293T cells by indirect immunofluorescence technique. These novel mAbs against SARS-CoV-2 nsp12 are useful to elucidate the RdRP reaction of SARS-CoV-2 and biological cell response against it.

The CC chemokine receptor 3 (CCR3) is a receptor for CC chemokines, including CCL5/RANTES, CCL7/MCP-3, and CCL11/eotaxin. CCR3 is expressed on the surface of eosinophils, basophils, a subset of Th2 lymphocytes, mast cells, and airway epithelial cells. CCR3 and its ligands are involved in airway hyperresponsiveness in allergic asthma, ocular allergies, and cancers. Therefore, CCR3 is an attractive target for those therapies. Previously, anti-mouse CCR3 (mCCR3) monoclonal antibodies (mAbs), C3Mab-3 (rat IgG2a, kappa), and C3Mab-4 (rat IgG2a, kappa) were developed using the Cell-Based Immunization and Screening (CBIS) method. In this study, the binding epitope of these mAbs was investigated using flow cytometry. A CCR3 extracellular domain-substituted mutant analysis showed that C3Mab-3, C3Mab-4, and a commercially available mAb (J073E5) recognized the N-terminal region (amino acids 1–38) of mCCR3. Next, alanine scanning was conducted in the N-terminal region. The results revealed that the Ala2, Phe3, Asn4, and Thr5 of mCCR3 are involved in C3Mab-3 binding, whereas Ala2, Phe3, and Thr5 are essential to C3Mab-4 binding, and Ala2 and Phe3 are crucial to J073E5 binding. These results reveal the involvement of the N-terminus of mCCR3 in the recognition of C3Mab-3, C3Mab-4, and J073E5.

The epithelial cell adhesion molecule (EpCAM) is a stem cell and carcinoma antigen, which mediates cellular adhesion and proliferative signaling by the proteolytic cleavage. In contrast to low expression in normal epithelium, EpCAM is frequently overexpressed in various carcinomas, which correlates with poor prognosis. Therefore, EpCAM has been considered as a promising target for tumor diagnosis and therapy. Using the Cell-Based Immunization and Screening (CBIS) method, we previously established an anti-EpCAM monoclonal antibody (EpMab-37; mouse IgG1, kappa). In this study, we investigated the antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and an antitumor activity by a defucosylated mouse IgG2a-type of EpMab-37 (EpMab-37-mG2a-f) against a breast cancer cell line (BT-474) and a pancreatic cancer cell line (Capan-2), both of which express EpCAM. EpMab-37-mG2a-f recognized BT-474 and Capan-2 cells with a moderate binding-affinity [apparent dissociation constant (KD): 2.9 × 10−8 M and 1.8 × 10−8 M, respectively] by flow cytometry. EpMab-37-mG2a-f exhibited ADCC and CDC for both cells by murine splenocytes and complements, respectively. Furthermore, administration of EpMab-37-mG2a-f significantly suppressed the xenograft tumor development compared with the control mouse IgG. These results indicated that EpMab-37-mG2a-f exerts antitumor activities and could provide valuable therapeutic regimen for breast and pancreatic cancers.

Human epidermal growth factor receptor 2 (HER2) has been studied in many human cancer types, and its overexpression and/or gene mutation contribute to the poor prognosis. Therefore, HER2 is an important therapeutic target in various cancer types, including breast and gastric cancers. We previously developed an anti-HER2 monoclonal antibody (mAb), H2Mab-77 (mouse IgG1, kappa), which detects HER2 and dog HER2 (dHER2) with high sensitivity and specificity. In this study, we produced a defucosylated mouse-dog chimeric anti-HER2 mAb (H77Bf), and investigated the reactivity against canine osteosarcoma D-17 cells by flow cytometry. Furthermore, we showed that H77Bf exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in vitro and exhibited the potent antitumor activity in vivo. These results suggest that H77Bf exerts antitumor effects against dHER2-expressing canine tumors and could be valuable as part of an antibody treatment regimen for them.

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.

The C-C chemokine receptor 9 (CCR9) belongs to the G-protein-coupled receptor superfamily, and is highly expressed on the T cells and intestinal cells. CCR9 regulates various immune responses by binding to the C-C chemokine ligand, CCL25, and is involved in inflammatory diseases and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR9 is necessary for treatment and diagnosis. In this study, we established a specific anti-human CCR9 (hCCR9) mAb; C9Mab-11 (mouse IgG2a, kappa), using the synthetic peptide immunization method. C9Mab-11 reacted with hCCR9-overexpressed Chinese hamster ovary-K1 (CHO/hCCR9) and hCCR9-endogenously expressed MOLT-4 (human T-lymphoblastic leukemia) cells in flow cytometry. The dissociation constant (KD) of C9Mab-11 for CHO/hCCR9 and MOLT-4 cells were determined to be 1.2 × 10-9 M and 4.9 × 10-10 M, respectively, indicating that C9Mab-11 possesses a high affinity for both exogenously and endogenously hCCR9-expressing cells. Furthermore, C9Mab-11 clearly detected hCCR9 protein in CHO/hCCR9 cells using western blot analysis. In summary, C9Mab-11 can be a useful tool for analyzing hCCR9-related biological responses.

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor family member that is highly expressed in B lymphocytes, certain subsets of effector and memory T cells, and immature dendritic cells. CCR6 has only one chemokine ligand, CCL20. The CCL20-CCR6 axis has been recognized as a therapeutic target for autoimmune diseases and tumor. This study developed specific monoclonal antibodies (mAbs) against mouse CCR6 (mCCR6) using the peptide immunization method. The established anti-mCCR6 mAb, C6Mab-13 (rat IgG1, kappa), reacted with mCCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCCR6), and mCCR6-endogenously expressed P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells in flow cytometry. The dissociation constant (KD) of C6Mab-13 for CHO/mCCR6 cells was determined to be 2.8 × 10-9 M, indicating that C6Mab-13 binds to mCCR6 with high affinity. In summary, C6Mab-13 is useful for detecting mCCR6-expressing cells through flow cytometry.

An anti-mouse CXC chemokine receptor 6 (mCXCR6) monoclonal antibody (mAb), Cx6Mab-1, was developed recently. Cx6Mab-1 is applicable for flow cytometry, Western blotting, and enzyme-linked immunosorbent assay. The purpose of this study is to determine the binding epitope of Cx6Mab-1 using 2 × alanine mutated mCXCR6. Analysis of flow cytometry revealed that Cx6Mab-1 did not recognize S8A-A9G, L10A-Y11A, D12A-G13A, and H14A-Y15A mutants of mCXCR6. The results clearly indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Asp12, Gly13, His14, and Tyr15 of mCXCR6. The successful determination of the Cx6Mab-1 epitope might contribute to the pathophysiological investigation of mCXCR6.

CC chemokine receptor type-2 (CCR2) is a member of the G protein-coupled receptors, and is mainly expressed on cell surface of immune cells. CCR2 binds to its ligand, C-C motif chemokine 2 (also named as monocyte chemoattractant protein-1), which involves in the tumor progression by modulating the tumor microenvironment. Therefore, the monoclonal antibody (mAb) targeting CCR2 could be one of the strategies for cancer treatment. In this study, we investigated the critical epitope of C2Mab-6, an anti-mouse CCR2 (mCCR2) mAb developed by N-terminal peptides immunization. We first performed enzyme-linked immunosorbent assay (ELISA) using N-terminal peptides of mCCR2 and demonstrated that C2Mab-6 recognizes 1-19 amino acids of mCCR2. We further performed ELISA using 20 alanine-substituted peptides of mCCR2. C2Mab-6 lost the reaction to the alanine-substituted peptides of D3A, N4A, M6A, P8A, Q9A, and F10A. These results indicate that the binding epitope of C2Mab-6 includes Asp3, Asn4, Met6, Pro8, Gln9, and Phe10 of mCCR2.

Tall cell carcinoma with reversed polarity (TCCRP) is a rare histologic type of low-grade breast cancer, consisting of tall columnar cells with reversed nuclear polarity and characterized by frequent IDH2 mutations. We herein report 3 cases of TCCRP with sequencing analyses of the IDH2 gene and immunohistochemical examination using monoclonal antibodies (11C8B1) against IDH2 R172. IDH2 R172 mutations were detected in all 3 resected tumors (R172S in 2 tumors and R172T in 1 tumor), and the presence of these mutations was confirmed by IDH2 R172 immunohistochemistry. Tumor cells of TCCRP showed strong and diffuse staining for the antibody against IDH2 R172. In 1 case, tumor tissue from 2 core needle biopsy samples collected on different days were also immunohistochemically positive for IDH2 R172. These results indicate that IDH2 R172 immunohistochemistry is suitable for the detection of TCCRP in both resection and biopsy samples. In addition, a literature review revealed that R172S and R172T account for 76% of IDH2 mutations in TCCRP, suggesting that 11C8B1, which reacts with R172S and R172T, was likely most sensitive for IDH2-mutated TCCRP among many available antibodies for IDH2 R172. Furthermore, the combination of 2 or more antibodies against IDH2 R172 could be more effective for detecting TCCRP mutation. However, it is important to note that IDH2 R172 immunohistochemistry is not absolute, because IDH2 wild type is found in a small proportion (10%) of cases, and a few cases of IDH2-mutated TCCRP may harbor rare subtypes of R172 that are not covered by available antibodies.

CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, and is localized on cell surface of tumor cells and some immune cells, including monocytes and macrophages. CCR2 is a receptor for monocyte chemoattractant protein-1/C-C motif chemokine 2, and is involved in the progression of various diseases such as cancers. Therefore, the development of CCR2-targeted monoclonal antibody (mAb) is desired. Its characterization, including epitope of mAb, is very important for antibody applications. In this study, we investigated the critical epitope of K036C2, which is a commercially available anti-human CCR2 (hCCR2) mAb. We conducted enzyme-linked immunosorbent assay (ELISA) using three N-terminal peptides of hCCR2 and demonstrated that K036C2 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. K036C2 lost the reaction to the alanine-substituted peptides of D25A, Y26A, D27A, G29A, and A30G. These results indicate that the critical binding epitope of K036C2 includes Asp25, Tyr26, Asp27, Gly29, and Ala30 of hCCR2.

The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, cytotoxic T lymphocytes (CTLs), and natural killer cells. CXCR6 plays critical roles in local expansion of effector-like CTLs in tumor microenvironment to potentiate the antitumor response. Therefore, the development of anti-CXCR6 monoclonal antibodies (mAbs) is essential to evaluate the immune microenvironment of tumors. Using N-terminal peptide immunization, we previously developed an anti-mouse CXCR6 (mCXCR6) mAb, Cx6Mab-1 (rat IgG1, kappa) , which is useful for flow cytometry and western blotting. In this study, we determined the critical epitope of Cx6Mab-1 by enzyme-linked immunosorbent assay (ELISA) using the 1 × alanine scanning (1 × Ala-scan) method or the 2 × alanine scanning (2 × Ala-scan) method. Although we first performed ELISA by 1 × Ala-scan using one alanine-substituted peptides of mCXCR6 N-terminal domain (amino acids 1-20), we could not identify the Cx6Mab-1 epitope. We next performed ELISA by 2 × Ala-scan using two alanine (or glycine) residues-substituted peptides of mCXCR6 N-terminal domain, and found that Cx6Mab-1 did not recognize S8A-A9G, A9G-L10A, L10A-Y11A, and G13A-H14A of the mCXCR6 N-terminal peptide. The results indicate that the binding epitope of Cx6Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Gly13, and His14 of mCXCR6. Therefore, we could demonstrate that the 2 × Ala scan method is useful for determining the critical epitope of mAbs.

Immune checkpoint molecules have received attention as targets of cancer immunotherapy. Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is one of the immune checkpoint molecules expressed in CD4+ T, CD8+ T, and natural killer (NK) cells. KLRG1 exhibits antiviral and antitumor immunity, and its expression in T and NK cells is upregulated by viral infectious diseases and some tumors. Thus, monoclonal antibodies (mAbs) for KLRG1 would be useful tools for the diagnosis and immunotherapy against viral infectious diseases and cancers. We have developed anti-human KLRG1 (hKLRG1) mAb (clone KLMab-1, mouse IgG1, kappa) by the Cell-Based Immunization and Screening method. We have also demonstrated that KLMab-1 recognizes both exogenous and endogenous hKLRG1 in flow cytometry. In this study, we first showed that KLMab-1 and its recombinant mAb (recKLMab-1) bound to exogenous hKLRG1 overexpressed in Chinese hamster ovary (CHO)-K1 cells, but not in parental CHO-K1 cells, in immunocytochemistry. We next showed that both mAbs detected endogenous hKLRG1 expressed in human NK cells. These results demonstrate that KLMab-1 and recKLMab-1 are available for immunocytochemistry.

Glioblastoma is a devastating malignant brain tumor with a poor prognosis despite standard therapy. Podoplanin (PDPN), a type I transmembrane mucin-like glycoprotein that is overexpressed in various cancers, is a potential therapeutic target for the treatment of glioblastoma. We previously reported the efficacy of chimeric antigen receptor (CAR)-T cells using an anti-pan-PDPN monoclonal antibody (mAb; NZ-1)-based third-generation CAR in a xenograft mouse model. However, NZ-1 also reacted with PDPN-expressing normal cells, such as lymphatic endothelial cells, pulmonary alveolar type I cells, and podocytes. To overcome possible on-target-off-tumor effects, we produced a cancer-specific mAb (CasMab, LpMab-2)-based CAR. LpMab-2 (Lp2) reacted with PDPN-expressing cancer cells but not with normal cells. In this study, Lp2-CAR-transduced T cells (Lp2-CAR-T) specifically targeted PDPN-expressing glioma cells while sparing the PDPN-expressing normal cells. Lp2-CAR-T also killed patient-derived glioma stem cells, demonstrating its clinical potential against glioblastoma. Systemic injection of Lp2-CAR-T cells inhibited the growth of a subcutaneous glioma xenograft model in immunodeficient mice. Combination therapy with Lp2-CAR-T and oncolytic virus G47Δ, a third-generation recombinant herpes simplex virus (HSV)-1, further inhibited the tumor growth and improved survival. These findings indicate that the combination therapy of Lp2-CAR-T cells and G47Δ may be a promising approach to treat glioblastoma.

Paratuberculosis is a chronic enteritis of ruminants caused by the facultative intracellular pathogen Mycobacterium avium subsp. paratuberculosis. The Th1 response inhibits the proliferation of M. avium subsp. paratuberculosis during the early subclinical stage. However, we have previously shown that immune inhibitory molecules, such as prostaglandin E2 (PGE2), suppress M. avium subsp. paratuberculosis-specific Th1 responses as the disease progresses. To date, the mechanism underlying immunosuppression during M. avium subsp. paratuberculosis infection has not been elucidated. Therefore, in the present study, we investigated the function of cytotoxic T-lymphocyte antigen 4 (CTLA-4) expressed by peripheral blood mononuclear cells (PBMCs) from cattle with paratuberculosis because CTLA-4 expression is known to be elevated in T cells under an M. avium subsp. paratuberculosis experimental infection. M. avium subsp. paratuberculosis antigen induced CTLA-4 expression in T cells from cattle experimentally infected with M. avium subsp. paratuberculosis. Interestingly, both PGE2 and an E prostanoid 4 agonist also induced CTLA-4 expression in T cells. In addition, a functional assay with a bovine CTLA-4-immunogobulin fusion protein (CTLA-4-Ig) indicated that CTLA-4 inhibited gamma interferon (IFN-γ) production in M. avium subsp. paratuberculosis-stimulated PBMCs, while blockade by anti-bovine CTLA-4 monoclonal antibody increased the secretion of IFN-γ and tumor necrosis factor alpha production in these PBMCs. These preliminary findings show that PGE2 has immunosuppressive effects via CTLA-4 to M. avium subsp. paratuberculosis. Therefore, it is necessary to clarify in the future whether CTLA-4-mediated immunosuppression facilitates disease progression of paratuberculosis in cattle.

Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various types of cancer, including breast, gastric, lung, colorectal and pancreatic cancer. A humanized anti‑HER2 monoclonal antibody (mAb), trastuzumab, has been shown to improve survival of patients in HER2‑positive breast and gastric cancer. An anti‑HER2 mAb, H2Mab‑77 (mouse IgG1, kappa) was previously developed. In the present study, a defucosylated version of mouse‑dog chimeric anti‑HER2 mAb (H77Bf) was generated. H77Bf possesses a high binding‑affinity [a dissociation constant (KD): 7.5x10‑10 M, as determined by flow cytometric analysis] for dog HER2‑overexpressed CHO‑K1 (CHO/dHER2) cells. H77Bf highly exerted antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) for CHO/dHER2 cells by canine mononuclear cells and complement, respectively. Moreover, administration of H77Bf significantly suppressed the development of CHO/dHER2 xenograft tumor in mice compared with the control dog IgG. H77Bf also possesses a high binding‑affinity (KD: 7.2x10‑10 M) for a canine mammary gland tumor cell line (SNP), and showed high ADCC and CDC activities for SNP cells. Intraperitoneal administration of H77Bf in mouse xenograft models of SNP significantly suppressed the development of SNP xenograft tumors compared with the control dog IgG. These results indicated that H77Bf exerts antitumor activities against dHER2‑positive canine cancers, and could be valuable treatment regimen for canine cancers.

Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The overexpression of PDPN contributes to the malignant progression of tumors. Therefore, the development of anti-PDPN monoclonal antibodies (mAbs) to animals is essential to evaluate the pathogenesis and cellular functions. Using peptide immunization, we previously developed an anti-elephant PDPN (elePDPN) mAb, PMab-295, which is useful for flow cytometry, Western blotting, and immunohistochemistry. In this study, we determined the critical epitope of PMab-295 by enzyme-linked immunosorbent assay (ELISA). We performed ELISA with the alanine-substituted peptides of elePDPN extracellular domain (amino acids 38-51), and found that PMab-295 did not recognize the alanine-substituted peptides of M41A, P44A, and E47A. Furthermore, these peptides could not inhibit the recognition of PMab-295 to elePDPN-expressing cells by flow cytometry and immunohistochemistry. The results indicate that the binding epitope of PMab-295 includes Met41, Pro44, and Glu47 of elePDPN.

Podoplanin (PDPN) is an essential marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) that can specifically recognize PDPN in immunohistochemistry are important to analyze the development of tissues and the pathogenesis of diseases, including cancers. We have developed anti-PDPN mAbs against many animal species; however, mAbs that can recognize elephant-derived membrane proteins and distinguish the specific cell types in immunohistochemistry are limited. In this study, a novel anti-elephant PDPN (elePDPN) mAb, PMab-295 (IgG1, kappa), was established using the peptide immunization method. PMab-295 recognized both elePDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous elePDPN-expressed LACF-NaNaI cells by flow cytometry and western blotting. Kinetic analyses using flow cytometry showed that the KD of PMab-295 for CHO/elePDPN was 1.5 × 10-8 M. Furthermore, PMab-295 detected elePDPN-expressing cells using immunohistochemistry. These results showed the usefulness of PMab-295 to investigate the molecular function of elePDPN and the pathogenesis of diseases.

The CC chemokine receptor type-2 (CCR2) is one of the members of the G protein-coupled receptor superfamily, which are expressed on the cell surface of immune and tumor cells. CCR2 binds to the C-C motif chemokine ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP-1), which is produced by various cells, including tumor and immune-related cells. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR2 has been desired for treatment and diagnosis. In this study, we established a specific antihuman CCR2 (hCCR2) mAb, C2Mab-9 (mouse IgG1, kappa), using the synthetic peptide immunization method. Flow cytometric and immunocytochemical results showed that C2Mab-9 reacted with hCCR2-expressing U937 (human histiocytic lymphoma) and natural killer cells. Furthermore, C2Mab-9 showed the moderate binding affinity for both cells. Conclusively, C2Mab-9 can be a useful tool for analyzing hCCR2-related biological responses.

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.

Recently, research has been conducted with chimeric antigen receptor (CAR)-T cells to improve efficacy against solid tumors. Humanized CAR improved the long-term survival of CAR-T cells in patients' peripheral blood, resulting in increased therapeutic efficacy. Therefore, the humanization of the CAR-gene sequence is considered an effective method. Podoplanin (PDPN) is a glycosylated transmembrane protein that is highly expressed in solid tumors and is associated with poor prognosis in patients with cancer. Therefore, PDPN is considered a biomarker and good target for cancer treatment with CAR-T cells. Previously, an anti-PDPN CAR was generated from a conventional non-humanized antibody-NZ-1, the only anti-PDPN antibody for which a CAR was produced. In this study, we investigated other anti-PDPN CARs from the antibody NZ-27, or humanized NZ-1, to enhance the therapeutic potential of CAR-T cells. The CAR signal intensity was enhanced by the efficient expression of CAR proteins on the T-cell surface of NZ-27 CAR-T cells, which show tumor-specific cytotoxicity, proinflammatory cytokine production, and anti-tumor activity against PDPN-expressing tumor xenografts in mice that were significantly better than those in non-humanized NZ-1 CAR-T cells. This article is protected by copyright. All rights reserved.

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.

CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, localized on cell surface of some immune-related cells, including monocytes and macrophages. CCR2 and its ligand CCL2 are involved in the progression of various diseases such as cancers. Therefore, CCR2-targeted monoclonal antibodies (mAbs) are needed for treatment and diagnosis. Previously, we successfully developed an anti-human CCR2 (hCCR2) mAb, C2Mab-9 (mouse IgG1, kappa), which is applicable for flow cytometry and immunocytochemistry. In this study, we investigated the critical epitope of C2Mab-9. We conducted enzyme-linked immunosorbent assay (ELISA) using several N-terminal peptides of hCCR2, and demonstrated that C2Mab-9 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. C2Mab-9 lost the reaction to the alanine-substituted peptides of F23A, F24A, D25A, Y26A, and D27A. Among them, F23A, F24A, D25A, and Y26A did not block the C2Mab-9 reaction with U937 cells in flow cytometry. These results indicate that the critical binding epitope of C2Mab-9 includes Phe23, Phe24, Asp25, and Tyr26.

Chinese hamster (Cricetulus griseus) and golden hamster (Mesocricetus auratus) are important animal models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, which affect several organs, including respiratory tract, lung, and kidney. Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The development of anti-PDPN monoclonal antibodies (mAbs) for these animals is essential to evaluate the pathogenesis by SARS-CoV-2 infections. Using the Cell-Based Immunization and Screening method, we previously developed an anti-Chinese hamster PDPN (ChamPDPN) mAb, PMab-281 (mouse IgG3, kappa), and further changed its subclass into IgG2a (281-mG2a-f), both of which can recognize not only ChamPDPN but also golden hamster PDPN (GhamPDPN) by flow cytometry and immunohistochemistry. In this study, we examined the critical epitope of 281-mG2a-f, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with peptides derived from ChamPDPN and GhamPDPN extracellular domain, and found that 281-mG2a-f reacted with the peptides, which commonly possess the KIPFEELxT sequence. Next, we analyzed the reaction with the alanine-substituted mutants, and revealed that 281-mG2a-f did not recognize the alanine-substituted peptides of I75A, F77A, and E79A of ChamPDPN. Furthermore, these peptides could not inhibit the recognition of 281-mG2a-f to ChamPDPN-expressing cells by flow cytometry. The results indicate that the binding epitope of 281-mG2a-f includes Ile75, Phe77, and Glu79 of ChamPDPN, which are shared with GhamPDPN.

The CXC chemokine receptor 6 (CXCR6) is a member of the G protein-coupled receptor family that is highly expressed in helper T type 1 cells, natural killer cells, cytotoxic T lymphocytes, and various type of cells in tumor microenvironment (TME). CXCR6 has been proposed as a therapeutic target against tumors through regulation of the tumor TME. In this study, we developed specific and sensitive monoclonal antibodies (mAbs) for mouse CXCR6 (mCXCR6), which are useful for flow cytometry and Western blotting by N-terminal peptide immunization into rat. The established anti-mCXCR6 mAb, Cx6Mab-1 (rat IgG1, kappa), reacted with not only mCXCR6-overexpressed Chinese hamster ovary-K1 (CHO/mCXCR6) but also mCXCR6-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasm) and J774-1 (mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KD) of Cx6Mab-1 for CHO/mCXCR6, P388, and J774-1 cells were 1.7 × 10-9 M, 3.4 × 10-7 M, and 3.8 × 10-7 M, respectively. Furthermore, Cx6Mab-1 could detect endogenous mCXCR6 in P388 and J774-1 cells by Western blotting. These results indicated that Cx6Mab-1 is useful for detecting mCXCR6 by flow cytometry and Western blotting, and provides a possibility for targeting CXCR6-expressing cells in vivo experiments.

T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) is one of the immune checkpoint molecules. TIGIT is expressed in T or natural killer (NK) cells and is upregulated in several cancers. Because TIGIT suppresses the antitumor activity of the T or NK cells by binding to its ligand, such as CD155, CD112, and CD113, TIGIT can be a molecular marker or a therapeutic target for cancer immunotherapy. We previously developed an anti-human TIGIT (hTIGIT) monoclonal antibody (mAb; clone TgMab-2; mouse IgG1, kappa) by the Cell-Based Immunization and Screening method. TgMab-2 binds to hTIGIT with high binding affinity in flow cytometry. In this study, we investigated the availability of TgMab-2 and its recombinant mAb (recTgMab-2) in immunocytochemistry. We found that TgMab-2 and recTgMab-2 bind to hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells, but not parental CHO-K1 cells, indicating that both mAbs specifically recognize hTIGIT. Furthermore, both mAbs recognized endogenous hTIGIT expressed in human NK cells in immunocytochemistry. These results demonstrate that TgMab-2 and recTgMab-2 are applicable for immunocytochemistry against hTIGIT.

CD44 is a cell surface glycoprotein, which is expressed on normal cells, and overexpressed on cancer cells. CD44 is involved in cell adhesion, migration, proliferation, survival, stemness, and chemo-resistance. Therefore, CD44 is thought to be a promising target for cancer diagnosis and therapy. In this study, we established anti-CD44 monoclonal antibodies (mAbs) by immunizing mice with a CD44 variant (CD44v3-10) ectodomain and screening using enzyme-linked immunosorbent assay. We then characterized them using flow cytometry, Western blotting, and immunohistochemistry. One of the established clones (C44Mab-46; IgG1, kappa) reacted with CD44 standard isoform (CD44s)-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44s) or esophageal squamous cell carcinoma (ESCC) cell lines (KYSE70 and KYSE770). The apparent KD of C44Mab-46 for CHO/CD44s, KYSE70, and KYSE770 was 1.1 × 10-8 M, 4.9 × 10-8 M, and 4.1 × 10-8 M, respectively. C44Mab-46 detected CD44s of CHO/CD44s and KYSE70, and CD44 variants of KYSE770 in Western blot analysis. Furthermore, C44Mab-46 strongly stained the formalin-fixed paraffin-embedded ESCC tissues in immunohistochemistry. Collectively, C44Mab-46 is very useful for detecting CD44 in various applications.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.

Golden (Syrian) hamster (Mesocricetus auratus) is a small animal model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Pathological analyses of the tissues are required to understand the pathogenesis of SARS-CoV-2 and the evaluation of therapeutic modalities, including neutralizing monoclonal antibodies (mAbs). However, mAbs that recognize the golden hamster-derived antigens and distinguish specific cell types, such as the pneumocytes, are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-Chinese hamster (Cricetulus griseus) PDPN mAb PMab-281 (IgG3, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. A defucosylated mouse IgG2a version of PMab-281 (281-mG2a-f) was also developed. The 281-mG2a-f strongly recognized both the Chinese hamster and the golden hamster PDPN using flow cytometry and could detect lung type I alveolar epithelial cells, lymphatic endothelial cells, and Bowman's capsules in the kidney from the golden hamster using immunohistochemistry. These results suggest the usefulness of 281-mG2a-f for analyzing the golden hamster-derived tissues and cells for SARS-CoV-2 research.

CD10 is a cell surface metalloendopeptidase that cleaves and degrades many secreted physiologically active peptides by its enzymatic activity. Although CD10 expression has been found in various types of cells, its expression is increased in several cancers, including renal cancer. In this study, the antitumor activity of a novel anti-human CD10 monoclonal antibody (mAb) was investigated. A defucosylated mouse IgG2a version of C10Mab-31 (31-mG2a-f) was created from an anti-CD10 mAb, C10Mab-31 (IgG1, kappa). Both C10Mab-31 and 31-mG2a-f specifically reacted with endogenous CD10 in renal cancer cells, VMRC-RCW, with the dissociation constant (KD) values of 6.3 × 10-9 M and 1.1 × 10-9 M, respectively, indicating high binding affinity. To further examine the anti-CD10 mAb-mediated effector functions, the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) were examined. The 31-mG2a-f significantly exhibited ADCC and CDC against VMRC-RCW cells in vitro. Furthermore, 31-mG2a-f exhibited antitumor activities in mouse xenografts of VMRC-RCW cells. These results suggest that 31-mG2a-f exerts antitumor activities against CD10-expressing renal cancers and could be a valuable therapeutic candidate for treating them.

It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immunotherapy. The C-C motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells and many cancers and is associated with the progression of the cancers. However, its role in cancer progression remains unclear. Thus, the development of mAbs for CCR8 leads to cancer immunotherapy and elucidation of unknown mechanisms of CCR8-dependent cancer progression. In this study, we have developed an anti-mouse CCR8 (mCCR8) mAb (clone C8Mab-1, rat IgG2a, kappa) using the Cell-Based Immunization and Screening (CBIS) method. We showed that C8Mab-1 and its recombinant antibody (recC8Mab-1) bind to mCCR8-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/mCCR8), but not to the parental CHO-K1 cells, in flow cytometry and immunofluorescence. Moreover, C8Mab-1 and recC8Mab-1 specifically reacted to P388 (a mouse lymphocyte-like cells) and J774-1 (a mouse macrophage-like cells), which express endogenous mCCR8, in both applications. These results suggest that C8Mab-1, developed using the CBIS method, is useful for flow cytometry and immunocytochemistry against exogenous and endogenous mCCR8.

The C-C motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells. CCR8 is also expressed in many cancers and is associated with those progression. The development of monoclonal antibodies (mAbs) for CCR8 leads to cancer immunotherapy and elucidation of unknown mechanisms of CCR8-dependent cancer progression. In this study, we have developed an anti-mouse CCR8 (mCCR8) mAb (clone C8Mab-3, rat IgG1, kappa) using the Cell-Based Immunization and Screening (CBIS) method. We revealed that C8Mab-3 and its recombinant antibody (recC8Mab-3) bind to mCCR8-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/mCCR8), but not to the parental CHO-K1 cells, in flow cytometry. In addition, C8Mab-3 and recC8Mab-3 reacted to P388 (a mouse lymphocyte-like cell) and J774-1 (a mouse macrophage-like cell), which express endogenous mCCR8. C8Mab-3 also detected exogenous and endogenous mCCR8 using immunocytochemistry. These results suggest that C8Mab-3, developed using the CBIS method, is useful for immunocytochemistry against exogenous and endogenous mCCR8.

The epidermal growth factor receptor (EGFR) is involved in tumor malignancy through gene amplification and/or protein overexpression. An anti-human EGFR (hEGFR) monoclonal antibody (clone EMab-134), which explicitly detects hEGFR and dog EGFR (dEGFR), was previously developed. 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 CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, it was shown that 134-mG2a-f reacts with a canine fibroblastic tumor cell line (A-72) using flow cytometry and immunocytochemistry. Furthermore, 134-mG2a-f exerted ADCC and CDC on A-72 cell line. The administration of 134-mG2a-f significantly inhibited the A-72 xenograft growth. These results suggest that 134-mG2a-f exerts antitumor effects on dEGFR-expressing canine fibroblastic tumors.

The CC chemokine receptor type-2 (CCR2) belongs to the G-protein-coupled receptor superfamily, expressed on the cell surface of immune cells and tumors. CCR2 binds to the CC motif chemokine 2/monocyte chemoattractant protein-1, a CC chemokine, which is produced by various cells, including immune-related cells and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR2 has been desired for treatment and diagnosis. This study established a novel, specific, and sensitive anti-mouse CCR2 (mCCR2) mAb; C2Mab-6 (rat IgG1, kappa), using the mCCR2 synthetic peptide immunization method. C2Mab-6 reacted with mCCR2-overexpressed Chinese hamster ovary-K1 cells and L1210 (murine leukemia) cells, which express endogenous mCCR2 in flow cytometry. Furthermore, C2Mab-6 showed a high binding affinity for both cells. Hence, C2Mab-6 can be a useful tool for analyzing mCCR2-related biological responses, using flow cytometry.

The CC chemokine receptor type-4 (CCR4) belongs to the G-protein-coupled receptor superfamily, expressed on the cell surface of T cells and its malignancy. Two CCR4 ligands (CCL17 and CCL22) bind to CCR4 that mediate physiological and pathological functions of T cell immune responses. Anti-CCR4 monoclonal antibody (mAb) mogamulizumab is approved for adult T cell leukemia/lymphoma and cutaneous T cell lymphomas. In addition, mogamulizumab can deplete regulatory T cells, implying the application to solid tumors as an immunomodulator. Therefore, the development of sensitive mAbs for CCR4 has been desired for basic research, diagnosis, and therapy. In this study, a specific, and sensitive anti-mouse CCR4 (mCCR4) mAb, C4Mab-1 (rat IgG1, kappa), was established using N-terminal peptide immunization. C4Mab-1 reacted with mCCR4-overexpressed Chinese hamster ovary (CHO)-K1 cells, P388 (mouse lymphoid neoplasm), and J774-1 (mouse macrophage-like) cells in flow cytometry. Kinetic analyses using flow cytometry showed that KDs of C4Mab-1 for CHO/mCCR4, P388, and J774-1 cells were 4.2 × 10-9 M, 5.4 × 10-7 M, and 1.1 × 10-6 M, respectively. C4Mab-1 could be a valuable tool for elucidating mCCR4-related biological responses.

C-C motif chemokine receptor 9 (CCR9) is a G protein-coupled receptor, which is highly expressed in T-lymphocytes and different cancer cells. CCR9 aggravates immune diseases and cancer progression and is considered a biomarker and a therapeutic target of diseases. The development of specific monoclonal antibody (mAbs) for human CCR9 (hCCR9) is required to diagnose and treat immune diseases and cancers. Previously, we established the cell-based immunization and screening (CBIS) method, which does not need purified target proteins. Anti-hCCR9 mAb (clone C9Mab-1; mouse IgG1, kappa) was also developed using the CBIS method. C9Mab-1 is usable for flow cytometry against exogenously and endogenously expressing hCCR9. This study showed that C9Mab-1 and its recombinant antibody (recC9Mab-1) specifically detected exogenous hCCR9 stably overexpressed in Chinese hamster ovary (CHO)-K1 cells and endogenous hCCR9 expressed in a human T-lymphoblastic leukemia cell line MOLT-4 cells through immunocytochemistry. This study provides a new application of C9Mab-1 and recC9Mab-1 in immunocytochemistry.

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 (mAb), clone EMab-134 (mouse IgG1, kappa), which specifically detects both hEGFR and dog EGFR (dEGFR). The defucosylated mouse IgG2a version of EMab-134 exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf), and the reactivity of E134Bf against a canine mammary gland tumor cell line (SNP) was examined by flow cytometry. Furthermore, E134Bf highly exerted ADCC and CDC for SNP cells. The administration of E134Bf with canine mononuclear cells significantly suppressed the SNP xenograft growth. These results suggest that E134Bf exerts antitumor effects against dEGFR-expressing canine mammary gland tumors and could be valuable as part of an antibody treatment regimen for them.

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 C8Mab-2 (rat IgG2b, kappa) that was applicable to flow cytometric analysis for both endogenous and exogenous mCCR8. This study showed that C8Mab-2 and recombinant C8Mab-2 (recC8Mab-2) were specifically bound to exogenously expressed mCCR8 in mCCR8-overexpressed Chinese hamster ovary-K1 cells. In addition, we found that C8Mab-2 and recC8Mab-2 recognized endogenous mCCR8 in P388 (a mouse lymphocyte-like cell line) and J774-1 cells (a mouse macrophage-like cell line). These data demonstrate that C8Mab-2 and recC8Mab-2 are useful for immunocytochemical analysis.

The CC chemokine receptor 3 (CCR3) is a member of the G protein-coupled receptor family that is highly expressed in eosinophils and basophils. CCR3 has been proposed as a therapeutic target for human immunodeficiency virus and allergy diagnosis. Therefore, in this study, we developed specific and sensitive monoclonal antibodies (mAbs) for mouse CCR3 (mCCR3), which are useful for flow cytometry by peptide immunization. The established anti-mCCR3 mAbs, C3Mab-6 (rat IgG1, kappa) and C3Mab-7 (rat IgG1, kappa), reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3), in addition to mCCR3-endogenously expressed cell lines, such as P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) through flow cytometry. Kinetic analyses using flow cytometry indicated that the dissociation constants (KDs) of C3Mab-6 for CHO/mCCR3, P388, and J774-1 cells were 8.7 × 10-9 M, 1.4 × 10-7 M, and 1.7 × 10-7 M, respectively, whereas the KDs of C3Mab-7 for these cell lines were 3.7 × 10-9 M, 5.1 × 10-7 M, and 3.1 × 10-7 M, respectively. Results also indicated that C3Mab-6 and C3Mab-7 are useful for detecting cells expressing CCR3 through flow cytometry, thereby making them potentially beneficial for treating CCR3-expressing cells.

CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. It is expressed on predifferentiated lymphocyte progenitor, epithelial, stromal, and tumor cells. Therefore, antibodies against CD10 are used for diagnosing follicular lymphoma and solid tumors, including renal carcinomas. In this study, we developed an anti-human CD10 monoclonal antibody, clone C10Mab-31 (IgG1, kappa), which detects CD10 by flow cytometry and shows high affinity for CD10-overexpressed CHO-K1 (CHO/CD10) cells. Furthermore, the defucosylated mouse IgG2a version of C10Mab-31 (31-mG2a-f) exhibits antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antitumor activities in mouse xenografts of CHO/CD10 cells. These results indicate that 31-mG2a-f exerts antitumor effects against CD10-expressing tumors and could be valuable as part of an antibody treatment regimen for them.

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.

CC chemokine receptor 3 (CCR3) belongs to the G protein-coupled receptor family and is highly expressed in eosinophils and basophils. CCR3 is essential for recruiting eosinophils into the lung. Moreover, CCR3 was found in the serum of colorectal cancer patients higher than in the control group. Therefore, CCR3 will be a useful target for asthma and colorectal cancer diagnosis. This study developed a specific and sensitive monoclonal antibody (mAb) for mouse CCR3 (mCCR3), which is useful for flow cytometry using the Cell-Based Immunization and Screening method. The established anti-mCCR3 mAb, C3Mab-3 (rat IgG2a, kappa), reacted with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells through flow cytometry. C3Mab-3 also reacted with P388 (mouse lymphoid neoplasma) and J774-1 (mouse macrophage-like) cells, which express mCCR3 endogenously. Kinetic analyses using flow cytometry indicated that KDs of C3Mab-3 for CHO/mCCR3, P388, and J774-1 cells were 4.3 × 10-8 M, 2.6 × 10-7 M, and 2.4 × 10-7 M, respectively. C3Mab-3 could be a valuable tool for elucidating mCCR3-related biological response using flow cytometry.

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.

Various proteins are highly expressed in cancer (e.g., epidermal growth factor receptor); however, the majority are also expressed in normal cells, although they may differ in expression intensity. Recently, we reported that CD271 (nerve growth factor receptor), a glycosylated protein, increases malignant behavior of cancer, particularly stem-like phenotypes in squamous cell carcinoma. CD271 is expressed in squamous cell carcinoma and in normal epithelial basal cells. Glycosylation alterations generally occur in cancer cells, therefore, we attempted to establish a cancer-specific anti-glycosylated CD271 antibody. We purified recombinant glycosylated CD271 protein, immunized mice with the protein, and screened hybridomas using an ELISA assay with cancer cell lines. We established a clone G4B1 against CD271 which is glycosylated with O-glycan and sialic acid. The G4B1 antibody reacted with the CD271 protein expressed in esophageal cancer, however, not in normal esophageal basal cells. This specificity was confirmed in hypopharyngeal and cervical cancers. G4B1 antibody recognized the fetal esophageal epithelium and Barrett's esophagus, which possess stem cell -like characteristics. In conclusion, G4B1 antibody could be useful for precise identification of dysplasia and cancer cells in SCC.

CD20, which is expressed on B lymphocytes, has been studied as a therapeutic target for B cell lymphomas and autoimmune disorders. Identifying the binding epitopes of monoclonal antibodies (mAbs) can contribute to our understanding of their functions. We have previously developed an anti-CD20 mAb (clone C20Mab-11) using a Cell-Based Immunization and Screening (CBIS) method. In this study, we aimed to determine the binding epitopes of anti-CD20 mAbs, such as C20Mab-11 and 2H7, using the His-tag insertion for epitope mapping (HisMAP). The results showed that 171-NPSE-174 and 168-EPANPSE-174 in the second loop of CD20 were essential for C20Mab-11 binding and 2H7 binding, respectively. Although we developed many mAbs that recognize conformational epitopes using the CBIS method, there are many difficulties in epitope mapping for these mAbs. HisMAP could be useful for determining the conformational epitopes of other mAbs against membrane proteins.

The C-C motif chemokine receptor 3 (CCR3) is a G protein-coupled receptor activated by eotaxin-1-3, MCP-2-4, and RANTES. CCR3 is associated with allergic diseases and cancer development and is highly expressed in eosinophils, basophils, and cancer cells. Besides, research on the physiological roles of CCR3 is ongoing. Thus, specific monoclonal antibodies (mAbs) for CCR3 would be useful for diagnostic and therapeutic purposes and for unraveling the function of CCR3. We previously developed an anti-mouse CCR3 (mCCR3) mAb (C3Mab-2; rat IgG2b, kappa) using the Cell-Based Immunization and Screening method and showed that C3Mab-2 could detect endogenous and exogenous mCCR3 in flow cytometry. In this study, we showed that C3Mab-2 and its recombinant antibody (recC3Mab-2f) specifically recognized endogenous mCCR3 in P388 (a mouse lymphocyte-like cell line) and J774-1 (a mouse macrophage-like cell line) cells and are usable in immunocytochemistry.

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein. Although EGFR is physiologically essential in normal cells, it contributes to tumor malignancy through gene amplification and/or protein overexpression, which augment signaling cascades in tumor cells. We previously developed an anti-human EGFR (hEGFR) monoclonal antibody (mAb), EMab-134 (mouse IgG1, kappa), which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity. The mouse IgG2a version of EMab-134 (134-mG2a) has antitumor effects toward mouse xenografts of hEGFR-expressing oral squamous cell carcinomas. Furthermore, 134-mG2a-f, the defucosylated version of 134-mG2a, exhibits antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells and antitumor activities in mouse xenografts of CHO/dEGFR cells. Herein, the reactivity of 134-mG2a-f against canine cancer cells with endogenous dEGFR was first examined by flow cytometry and immunocytochemistry. In vitro analysis demonstrated that 134-mG2a-f highly exerted ADCC and CDC for a canine osteosarcoma cell line, D-17, which expresses endogenous dEGFR. Moreover, in vivo administration of 134-mG2a-f significantly suppressed the development of D-17 compared with the results in response to control mouse IgG. These results suggest that 134-mG2a-f exerts antitumor effects against dEGFR-expressing canine cancers, and could be valuable as part of an antibody treatment regimen for them.

CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. CD10 is expressed on predifferentiated lymphocyte progenitor, epithelial, stromal, and tumor cells. Antibodies against CD10 are used for the diagnosis of follicular lymphoma. Anti-human CD10 monoclonal antibody (clone MME/1870) can be used for Western blotting and immunohistochemical analyses. This study examined the critical epitope of MME/1870 using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with deletion mutants, and MME/1870 reacted to the 501-520 amino acid sequence of CD10. Next, we analyzed the reaction to 20 point mutants, and MME/1870 did not recognize the alanine-substituted peptides of Y507A, I511A, I512A, and L515A. These results indicate that the binding epitope of MME/1870 includes Tyr507, Ile511, Ile512, and Leu515 of CD10.

Trophoblast cell surface antigen 2 (TROP2) has been reported to be overexpressed in many cancers, and is involved in cancer cell proliferation, invasion, and metastasis. We previously developed a highly sensitive anti-TROP2 monoclonal antibody (mAb) (clone TrMab-6; mouse IgG2b, kappa) using a Cell-Based Immunization and Screening method. TrMab-6 is useful for investigations using flow cytometry, Western blotting, and immunohistochemistry and possesses antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against TROP2-expressing triple-negative breast cancer (TNBC) cell lines, such as MDA-MB-231 and MDA-MB-468. This study investigated whether TrMab-6 possesses in vivo antitumor activities via ADCC/CDC activities using mouse xenograft models of TNBC cell lines. In vivo experiments on MDA-MB-231 and MDA-MB-468 xenografts revealed that TrMab-6 significantly reduced tumor growth compared with normal mouse IgG treatment. The findings of this study suggest that TrMab-6 is a promising treatment option for TROP2-expressing TNBC.

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are required for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, sheep, and California sea lion PDPNs. However, no anti-Steller sea lion PDPN (stePDPN) antibody has been developed. Immunohistochemical analysis showed that an anti-California sea lion PDPN mAb (PMab-269) reacted with type I alveolar cells from the Steller sea lion lung, renal glomeruli and Bowman's capsules from kidney, and lymphatic endothelial cells from the colon, indicating that PMab-269 is useful for detecting stePDPN.

CD20 is expressed in the B lymphocyte, and an effective target for the detection and treatment of B cell lymphomas. Therefore, CD20 has been studied as a therapeutic target of B cell lymphomas and autoimmune disorders. Specific anti-CD20 monoclonal antibodies (mAbs), such as rituximab, ofatumumab, veltuzumab, and ocaratuzumab, have been developed. Revealing the recognition mechanism of antigen by mAbs could contribute to understanding the function of mAbs and could be useful for the development of vaccine. Rituximab is a mouse-human chimeric anti-CD20 mAb, which was developed and approved for the treatment of the B cell malignancies. Hence, the binding epitope of rituximab for CD20 has been studied. Some reports show that 170-ANPS-173, especially Ala170 and Pro172 of CD20 are important for rituximab binding. However, only phage display results showed that 182-YCYSI-186 of CD20 is also important for rituximab binding to CD20. In this study, we tried to determine the binding epitope of rituximab for CD20 using histidine-tag insertion for epitope mapping (HisMAP) method. The results showed that two regions of CD20 (169-PANPSE-174 and 183-CYSIQ-187) are important for rituximab-binding for CD20.

Podoplanin (PDPN) is a cell-surface mucin-like glycoprotein that plays a critical role in tumor development and normal development of the lung, kidney, and lymphatic vascular systems. PDPN is overexpressed in several tumors and is involved in their malignancy. PDPN induces platelet aggregation through binding to platelet receptor C-type lectin-like receptor 2. Furthermore, PDPN modulates signal transductions that regulate cell proliferation, differentiation, migration, invasion, epithelial-to-mesenchymal transition, and stemness, all of which are crucial for the malignant progression of tumor. In the tumor microenvironment (TME), PDPN expression is upregulated in the tumor stroma, including cancer-associated fibroblasts (CAFs) and immune cells. CAFs play significant roles in the extracellular matrix remodeling and the development of immunosuppressive TME. Additionally, PDPN functions as a co-inhibitory molecule on T cells, indicating its involvement with immune evasion. In this review, we describe the mechanistic basis and diverse roles of PDPN in the malignant progression of tumors and discuss the possibility of the clinical application of PDPN-targeted cancer therapy, including cancer-specific monoclonal antibodies, and chimeric antigen receptor T technologies.

Abstract Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.

Recent evidence indicates that RNA-dependent RNA polymerase (RdRP) activity of human telomerase reverse transcriptase (hTERT) regulates expression of target genes and is directly involved in tumor formation by telomere independent manner. Non-canonical function of hTERT has been considered as a therapeutic target for cancer therapy. We have previously shown that hTERT phosphorylation at threonine 249 (p-hTERT), which promotes RdRP activity, is an indicator of an aggressive phenotype and poor prognosis in liver and pancreatic cancers, using two cohorts with small sample sizes with polyclonal p-hTERT antibody. To clarify the clinical relevance of p-hTERT, we developed a specific monoclonal antibody and determined the diagnostic and prognostic value of p-hTERT in cancer specimens using a large cohort. A monoclonal antibody for phosphorylated hTERT (p-hTERT) at threonine 249 was developed and validated. The antibody was used for the immunohistochemical staining of formalin-fixed paraffin-embedded specimens from 1,523 cases of lung, colon, stomach, pancreatic, liver, breast, and kidney cancers. We detected elevated p-hTERT expression levels in cases with a high mitotic activity, high pathological grade, and high nuclear pleomorphism. Elevated p-hTERT expression was an independent prognostic factor for lung, pancreatic, and liver cancers. Furthermore, p-hTERT expression was associated with immature and aggressive features, such as adenosquamous carcinoma (lung and pancreas), invasive type of cancer (lung), high serum alfa-fetoprotein level (liver), and triple-negative status (breast). In conclusion, RdRP activity indicated by p-hTERT expression predicts aggressive cancer phenotypes in various types of cancer. Thus, p-hTERT is a novel biomarker for the diagnosis of aggressive cancers with a poor prognosis. This article is protected by copyright. All rights reserved.

Background: Human epidermal growth factor receptor 3 (HER3) has been found to be overexpressed in many cancers such as lung, breast, and colon cancers. High expression of HER3 is thought to be a negative prognostic factor in several solid tumors including colorectal cancer. Moreover, HER3 is associated with drug resistance to EGFR- and HER2-targeted therapy in some cancers. Therefore, HER3 is thought to be an important therapeutic target. Purpose: We aimed to develop a novel anti-HER3 monoclonal antibody (mAb), and investigated its antitumor activities against colorectal cancer. Methods: We employed the Cell-Based Immunization and Screening (CBIS) method using HER3-overexpressed CHO-K1 cells for producing anti-HER3 mAbs. Anti-HER3 mAbs were screened using flow cytometry (FCM). Then, we examined the antibody‑dependent cellular cytotoxicity (ADCC) and the complement‑dependent cytotoxicity (CDC) activities of anti-HER3 mAbs for Caco‑2 (a human colorectal adenocarcinoma cell line). Moreover, a mouse xenograft model of Caco-2 was used for examining the antitumor activity. Results: We developed an anti-HER3 mAbs, H₃Mab-17 (IgG_2a, kappa) using CBIS method. H₃Mab-17 reacted with HER3-expressing cells in FCM. In vitro analysis demonstrated that H₃Mab-17 showed the ADCC and CDC activities against Caco-2. H₃Mab-17 significantly reduced tumor development in Caco-2 xenograft compared with control mouse IgG. Conclusion: We have successfully established a novel anti-HER3 mAb (H₃Mab-17), which could be a useful antibody-based therapy for patients with HER3-expressing colorectal cancers.

CC motif chemokine receptor 8 (CCR8), a G protein-coupled receptor (GPCR), is highly expressed in regulatory T cells, T helper 2 cells, and cancer cells. It plays important role in allergic inflammation and cancer development. Therefore, specific monoclonal antibodies (mAbs) for CCR8 would be useful for diagnostic and therapeutic purposes of the diseases. However, the production of mAbs for GPCRs has remained very difficult. We have developed a novel method for the development of mAbs, named the Cell-Based Immunization and Screening (CBIS) method. In the present study, an SD rat was immunized with mouse CCR8-overexpressed CHO-K1 cells (CHO/mCCR8). The hybridomas expressing anti-mCCR8 mAbs were screened by using CHO/mCCR8 and CHO-K1 cells. We obtained 73 strongly anti-mCCR8 mAb-expressing hybridomas from 1,916 hybridomas, and we finally established C₈Mab-2 (IgG_2b, kappa). C₈Mab-2 selectively reacted to CHO/mCCR8 cells in a dose-dependent manner, but not to CHO-K1 cells, in flow cytometry. C₈Mab-2 also recognized endogenous mCCR8 in a mouse lymphocyte-like cell line (P388) and a mouse macrophage-like cell line (J774-1). Furthermore, C₈Mab-2 visualized mCCR8 in CHO/mCCR8, P388, and J774-1 cells in immunocytochemistry. In conclusion, we developed the anti-mCCR8 mAb, C₈Mab-2, which is available for detecting endogenous and exogenous mCCR8 in flow cytometry and immunocytochemistry. C₈Mab-2 would be usable for diagnosis and medical treatment for allergic inflammation and cancer in mouse models.

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR) monoclonal antibody, EMab-134, which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity by flow cytometry, western blotting and immunohistochemistry. In this study, we produced a defucosylated mouse–dog chimeric anti-EGFR monoclonal antibody, E134Bf. Kinetic analysis of the interactions of E134Bf with the canine osteosarcoma cell line (D-17) and canine fibroblastic cell line (A-72) cells was conducted by flow cytometry. The K_D for the interaction of E134Bf with the D-17 and A-72 cells was 5.5 × 10⁻¹⁰ M and 6.0 × 10⁻¹⁰ M, respectively, indicating that E134Bf exhibits high affinity for D-17 and A-72 cells. Furthermore, E134Bf highly exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 and A-72 cells. In vivo administration of E134Bf significantly suppressed the development of D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable as part of an antibody treatment regimen for dogs.

Trophoblast cell surface antigen 2 (TROP2) has become one of the effective therapeutic targets by antibody-drug conjugates (ADCs) such as sacituzumab govitecan which US FDA approved in 2020. TROP2 is reported to be overexpressed, and involved in tumor cell proliferation, invasion, metastasis, and poor prognosis in several types of solid tumor. Several clinical trials of anti-TROP2 ADCs are ongoing worldwide in patients with breast and lung cancer. Here, using a Cell-Based Immunization and Screening (CBIS) method, we developed a novel anti-TROP2 monoclonal antibody (clone TrMab-6; mouse IgG_2b, kappa). TrMab-6 was found to be applicable for many experiments, including flow cytometry, Western blotting, and immunohistochemistry. Furthermore, we investigated the potential of TrMab-6 for in vitro antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) activities, and in vivo antitumor activities. TrMab-6 strongly induced anti-tumor effects against breast cancer cell lines, including MCF7, MDA-MB-231, and MDA-MB-468 in vitro. In vivo experiments revealed that TrMab-6 significantly reduced tumor growth on breast cancer xenograft models. These results indicated that TrMab-6 could be a promising treatment option for TROP2-expressing breast cancers.

<jats:title>Abstract</jats:title><jats:p>Immune checkpoint inhibitors (ICIs) such as anti-PD-L1 antibodies are widely used to treat human cancers, and growing evidence suggests that ICIs are promising treatments for canine malignancies. However, only some canine oral malignant melanoma (OMM) cases respond to ICIs. To explore biomarkers predictive of survival in dogs with pulmonary metastatic OMM receiving the anti-PD-L1 antibody c4G12 (<jats:italic>n</jats:italic> = 27), serum concentrations of prostaglandin E2 (PGE<jats:sub>2</jats:sub>), cytokines, chemokines, and growth factors were measured prior to treatment initiation. Among 12 factors tested, PGE<jats:sub>2</jats:sub>, interleukin (IL)-12p40, IL-8, monocyte chemotactic protein-1 (MCP-1), and stem cell factor (SCF) were higher in OMM dogs compared to healthy dogs (<jats:italic>n</jats:italic> = 8). Further, lower baseline serum PGE<jats:sub>2</jats:sub>, MCP-1, and vascular endothelial growth factor (VEGF)-A concentrations as well as higher IL-2, IL-12, and SCF concentrations predicted prolonged overall survival. These observations suggest that PGE<jats:sub>2</jats:sub> confers resistance against anti-PD-L1 therapy through immunosuppression and thus is a candidate target for combination therapy. Indeed, PGE<jats:sub>2</jats:sub> suppressed IL-2 and interferon (IFN)-γ production by stimulated canine peripheral blood mononuclear cells (PBMCs), while inhibition of PGE<jats:sub>2</jats:sub> biosynthesis using the COX-2 inhibitor meloxicam in combination with c4G12 enhanced Th1 cytokine production by PBMCs. Thus, serum PGE<jats:sub>2</jats:sub> may be predictive of c4G12 treatment response, and concomitant use of COX-2 inhibitors may enhance ICI antitumor efficacy.</jats:p>

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR) monoclonal antibody, namely EMab-134, which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity. In this study, we produced a defucosylated mouse-dog chimeric anti-EGFR monoclonal antibody, namely E134Bf. In vitro analysis revealed that E134Bf highly exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against a canine osteosarcoma cell line (D-17) and a canine fibroblastic cell line (A-72), both of which express endogenous dEGFR. Moreover, in vivo administration of E134Bf significantly suppressed the development of D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable as part of an antibody treatment regimen for dogs.

CD20 is a glycosylated transmembrane protein and is expressed on normal B cells and B cell malignancies. Therapeutic antibodies against CD20 are developed and used in clinic. The understanding of antibody-antigen binding by revealing the epitope is essential for future application to antibody technology. Previously, we developed an anti-human CD20 monoclonal antibody, C20Mab-60 (IgG2a, kappa), using the Cell-Based Immunization and Screening (CBIS). C20Mab-60 can be used for flow cytometry, Western blot, and immunohistochemical analyses. In this study, we examined the critical epitope of C20Mab-60 using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. We performed ELISA with deletion mutants, and C20Mab-60 reacted to the 160-179 amino acids sequence of CD20. Next, we analyzed the reaction to 20 point mutants, and C20Mab-60 did not recognize the alanine-substituted peptides of N171A, P172A, S173A, and E174A. The results indicate that the binding epitope of C20Mab-60, developed by CBIS, includes Asn171, Pro172, Ser173, and Glu174 of CD20.

Human epidermal growth factor receptor 2 (HER2) is a type I transmembrane 185 kDa protein expressed in various types of normal or cancer cells. Overexpression of HER2 is found in many cancers and is related to cell proliferation, differentiation, and migration. We recently developed a novel anti-HER2 monoclonal antibody, H2Mab-181, by immunizing mice with the purified recombinant extracellular domain of HER2. H2Mab-181 can specifically and sensitively detect HER2 not only in flow cytometry and Western blotting for gastric cancer cell lines, but also in immunohistochemical analyses for gastric cancer tissues. In this study, we analyzed the binding epitope of H2Mab-181 to HER2 using enzyme-linked immunosorbent assay (ELISA). Results showed that the H2Mab-181 epitope was determined to be Gly383, Asp384, Ala386, Asn388, and Pro391 by ELISA.

One of G protein-coupled receptors, CCR9, is mainly expressed in the thymocytes and the small bowel. The ligand of CCR9 is CCL25 (TECK), and the CCR9-CCL25 axis controls T cell maturation and intestinal immune response. CCR9 is related to graft-versus-host disease and autoimmune diseases. Recent studies have been reported that CCR9 is also associated with tumor proliferation, apoptosis, migration, and drug resistance. Therefore, CCR9-targeting therapy is receiving a lot of attention. Previously, we developed an anti-human CCR9 (hCCR9) monoclonal antibody, C9Mab-1 (IgG1, kappa), which can be used for flow cytometry, by immunizing mice with hCCR9-overexpressed Chinese hamster ovary-K1 cells. In this study, we examined the critical epitope of C9Mab-1, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with deletion mutants, and C9Mab-1 reacted to the 1-20 amino acids sequence of hCCR9. Next, we analyzed the reaction to 20 point mutants, and C9Mab-1 did not recognize the alanine-substituted peptides of I10A, P11A, N12A, M13A, A14G, D16A, and Y17A. The results indicate that the binding epitope of C9Mab-1 includes Ile10, Pro11, Asn12, Met13, Ala14, Asp16, and Tyr17 of hCCR9.

CD20 is one of the B-lymphocyte antigens and an effective target for the detection and treatment of B cell lymphomas; specific and sensitive monoclonal antibodies (mAbs) are required thus for their diagnosis. Recently, we developed a novel anti-CD20 mAb (clone C20Mab-60), which is not only useful for flow cytometry but also for Western blot and immunohistochemical analyses. However, the epitope of C20Mab-60 has not been determined. To clarify the binding region of mAbs against their target molecules, it is essential to understand the pharmacological function of each mAb. In this study, we aimed to identify the epitope of C20Mab-60 for CD20 using the novel histidine tag (His-tag) insertion for epitope mapping (HisMAP) method. We first established an anti-His-tag mAb, HisMab-1 (mouse IgG2b, kappa), by immunizing mice with recombinant proteins containing an N-terminal His-tag. Although HisMab-1 detected the 4x, 5x, and 6xHis tag-inserted CD20 proteins using flow cytometry, 5xHis tag was selected. While HisMab-1 recognized all the 5xHis tag-inserted CD20 from the 142nd to the 183rd amino acid (aa), C20Mab-60 did not react with the 5xHis tag-inserted CD20 from the 171st to the 174th aa. These results indicate that the main epitope of C20Mab-60 for CD20 is a peptide from 171st to 174th aa of CD20. HisMAP method could be advantageous in the determination of the critical epitope of functional mAbs against many target molecules.

Diacylglycerol kinase (DGK) is a lipid kinase that converts diacylglycerol (DG) to phosphatidic acid (PA). Since both DG and PA serve as intracellular second messenger molecules, DGK plays a pivotal role in balancing these two signaling pathways. Of the DGK family, DGKη is classified as a type II DGK. Reportedly, DGKη is expressed ubiquitously through mammalian tissues and cells. Previous studies using cDNA transfection methods reported cytoplasmic localization of DGKη in cultured human cells. However, subcellular localization of native protein is still unknown. Recently, we established a human DGKη-specific monoclonal antibody, DhMab-4. In this study, we examined subcellular localization of native protein of DGKη using DhMab-4 by immunocytochemistry in human cultured cells.

Since the World Health Organization 2016 classification (2016 WHO), genetic status has been incorporated into the diagnosis of Grade 2/3 gliomas (lower-grade gliomas). Therefore, immunohistochemistry (IHC) of IDH1-R132H, ATRX, and p53 have been used in place of genetic status. We report the associations between histological findings, IHC, and genetic status. We performed IHC of IDH1-R132H, ATRX, and p53 in 76 lower-grade gliomas and discussed its validity based on the 2016 WHO and the upcoming 2021 WHO classification. The sensitivity and specificity of anti-ATRX, p53, and IDH1-R132H IHC were 40.9%/98.1%, 78.6%/85.4%, and 90.5%/84.6%, respectively. Among 21 IDH1-mutant gliomas without 1p/19q codeletion, two gliomas (9.5%) mimicked the so-called classic for oligodendroglioma (CFO) in their morphology. Of the 42 gliomas with 1p/19q codeletion, four cases were difficult to diagnose as oligodendroglioma through morphological examination. Moreover, there were three confusing cases with ATRX mutations but with retained ATRX-IHC positivity. The lessons learned from this study are as follows: (1) ATRX-IHC and p53-IHC should be supplementary to morphological diagnosis, (2) rare IDH mutations other than IDH1 R132H should be considered, and (3) there is no complete alternative test to detect molecular features of glioblastoma under the 2021 WHO classification.

Our previous studies demonstrate the therapeutic efficacy against bovine diseases of an anti-bovine programmed death-ligand 1 (PD-L1) chimeric antibody. In humans, PD-1 and PD-L1 antibodies are more effective when combined with an antibody targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and these combination therapies are therefore clinically used. Here we generated an anti-bovine CTLA-4 chimeric antibody (chAb) to enhance the therapeutic efficacy of the PD-L1 antibody. We further analyzed the effects of dual blockade of CTLA-4 and PD-1 pathways on T-cell responses. The established anti-bovine CTLA-4 chAb showed comparable blocking activity on the binding of bovine CTLA-4 to CD80 and CD86 as the anti-bovine CTLA-4 mouse monoclonal antibody. Anti-bovine CTLA-4 chAb also significantly increased IL-2 production from bovine peripheral blood mononuclear cells (PBMCs). Further, the combination of anti-CTLA-4 chAb with anti-PD-L1 chAb significantly upregulated IL-2 production by PBMCs. These results suggest that the combination of antibodies have higher potential to enhance immune responses against pathogens compared with single administration.

Podoplanin is expressed in various human tumors where it promotes tumor progression, epithelial-mesenchymal transition, and distant metastasis. Podoplanin is also expressed in cancer-associated fibroblasts and induces tumor malignancy. The objective of this study was to evaluate podoplanin expression in various types of feline tumor tissues. Immunohistochemical analysis revealed that podoplanin was expressed in cells of 13/15 (87%) squamous cell carcinomas and 5/19 (26%) fibrosarcomas. Moreover, cancer-associated fibroblasts expressed podoplanin in most tumor types, including 18/21 (86%) mammary adenocarcinoma tissues. Our findings demonstrate that various types of feline tumor tissues expressed podoplanin, indicating the importance of the comparative aspects of podoplanin expression, which may be used as a novel research model for podoplanin biology.

Podocalyxin (PODXL) is a type I transmembrane sialoglycoprotein that is overexpressed in human cancers, including breast, oral, and lung. PODXL promotes tumor progression, and its expression is associated with poor prognosis. Since PODXL is expressed in normal cells, including kidney podocytes and vascular endothelial cells (VECs), cancer-specific monoclonal antibodies (mAbs) are necessary to reduce the adverse effects of antibody therapy on PODXL-expressing cancers. Previously, we established a cancer-specific mAb against PODXL, PcMab-60 (mouse IgM, kappa), by immunizing mice with soluble PODXL produced by LN229 glioblastoma cells. PcMab-60 reacted with PODXL-expressing cancer cells, but did not react with VECs. In this study, we investigated an epitope of PcMab-60 using flow cytometry, surface plasmon resonance (SPR), and enzyme-linked immunosorbent assay (ELISA). The results of SPR revealed that the PcMab-60 epitope consisted of Thr105, Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, Pro116, and Thr117. In contrast, the results of ELISA revealed that the PcMab-60 epitope consisted of Arg109, Gly110, Gly111, Gly112, Ser113, Gly114, Asn115, and Pro116. These results demonstrate the cancer-specific epitope, which was recognized by PcMab-60.

CD44 is a type I transmembrane protein expressed in various kinds of normal cancer cells, including pancreatic, breast, and oral cancers. CD44 is associated with cancer progression, metastases, and treatment resistance. CD44 consists of 20 exons, and various isoforms exist due to alternative splicing of the central 10 exons. Some splicing variants show cancer-specific expression patterns and are related to prognosis of patients with cancer. Therefore, CD44 targeting therapy has been attracting attention. In a previous study, we established an anti-CD44 monoclonal antibody, C44Mab-46 (IgG1, kappa), useful for flow cytometry, Western blotting, and immunohistochemistry by immunizing mice with CD44v3-10 ectodomain. This study investigated the binding epitope of C44Mab-46 using enzyme-linked immunosorbent assay (ELISA) and the surface plasmon resonance (SPR) with the synthesized peptide. ELISA results using deletion mutants showed that C44Mab-46 reacted with the amino acids (aa) of 161-180 aa of CD44. Further examination of the C44Mab-46 epitope using ELISA with point mutants in 161-180 aa of CD44 demonstrates that the C44Mab-46 epitope comprised Thr174, Asp177, and Val178. The SPR with point mutants in 161-180 aa of CD44 demonstrated that the C44Mab-46 epitope comprises Thr174, Asp175, Asp176, Asp177, and Val178. Together, the C44Mab-46 epitope was determined to be located in exon 5 of CD44.

The prognosis of advanced mesothelioma is poor. Podoplanin (PDPN) is highly expressed in most malignant mesothelioma. This study aimed to evaluate the potential alpha-radioimmunotherapy (RIT) with a newly developed anti-PDPN antibody, NZ-16, compared with a previous antibody, NZ-12. METHODS: The in vitro properties of radiolabeled antibodies were evaluated by cell binding and competitive inhibition assays using PDPN-expressing H226 mesothelioma cells. The biodistribution of 111In-labeled antibodies was studied in tumor-bearing mice. The absorbed doses were estimated based on biodistribution data. Tumor volumes and body weights of mice treated with 90Y- and 225Ac-labeled NZ-16 were measured for 56 days. Histologic analysis was conducted. RESULTS: The radiolabeled NZ-16 specifically bound to H226 cells with higher affinity than NZ-12. The biodistribution studies showed higher tumor uptake of radiolabeled NZ-16 compared with NZ-12, providing higher absorbed doses to tumors. RIT with 225Ac- and 90Y-labeled NZ-16 had a significantly higher antitumor effect than RIT with 90Y-labeled NZ-12. 225Ac-labeled NZ-16 induced a larger amount of necrotic change and showed a tendency to suppress tumor volumes and prolonged survival than 90Y-labeled NZ-16. There is no obvious adverse effect. CONCLUSIONS: Alpha-RIT with the newly developed NZ-16 is a promising therapeutic option for malignant mesothelioma.

INTRODUCTION: Chronic infections lead to the functional exhaustion of T cells. Exhausted T cells are phenotypically differentiated by the surface expression of the immunoinhibitory receptor, such as programmed death-1 (PD-1). The inhibitory signal is produced by the interaction between PD-1 and its PD-ligand 1 (PD-L1) and impairs the effector functions of T cells. However, the expression dynamics of PD-L1 and the immunological functions of the PD-1/PD-L1 pathway in chronic diseases of pigs are still poorly understood. In this study, we first analyzed the expression of PD-L1 in various chronic infections in pigs, and then evaluated the immune activation by the blocking assay targeting the swine PD-1/PD-L1 pathway. METHODS: In the initial experiments, anti-bovine PD-L1 monoclonal antibodies (mAbs) were tested for cross-reactivity with swine PD-L1. Subsequently, immunohistochemical analysis was conducted using the anti-PD-L1 mAb. Finally, we assessed the immune activation of swine peripheral blood mononuclear cells (PBMCs) by the blockade with anti-PD-L1 mAb. RESULTS: Several anti-PD-L1 mAbs tested recognized swine PD-L1-expressing cells. The binding of swine PD-L1 protein to swine PD-1 was inhibited by some of these cross-reactive mAbs. In addition, immunohistochemical analysis revealed that PD-L1 was expressed at the site of infection in chronic infections of pigs. The PD-L1 blockade increased the production of interleukin-2 from swine PBMCs. CONCLUSIONS: These findings suggest that the PD-1/PD-L1 pathway could be also involved in immunosuppression in chronic infections in pigs. This study provides a new perspective on therapeutic strategies for chronic diseases in pigs by targeting immunosuppressive pathways.

To clarify the binding region of monoclonal antibodies (mAbs) to target molecules, it is very essential to understand the pharmacological function of each mAb. Although deletion mutants and point mutants are usefully utilized for epitope mapping, we often experience the difficulty of determining the mAb epitope against membrane proteins. We aimed to develop a novel method to determine the binding region of mAbs using epitope tag system. We first checked the reactivity of an anti-CD44 mAb (C44Mab-5) to several deletion mutants of CD44. We then employed the RIEDL tag system ("RIEDL" peptide and LpMab-7 mAb). We inserted the "RIEDL" peptide into the CD44 protein from the 21st to 41st amino acid (AA). The transfectants produced were stained by LpMab-7 and C44Mab-5 in flow cytometry. C44Mab-5 did not react with 30th-361st AA of the deletion mutant of CD44. Furthermore, the reaction of C44Mab-5 to RIEDL tag-inserted CD44 from 25th to 36th AA was lost, although LpMab-7 detected most of the RIEDL tag-inserted CD44 from 21st to 41st AA. The epitope of C44Mab-5 for CD44 was determined to be the peptide from 25th to 36th AA of CD44 using RIEDL insertion for epitope mapping (REMAP) method. The REMAP method might be useful for determining the critical epitope of functional mAbs against many target molecules.

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.

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 IgG1), by immunizing mice with the ectodomain of hEGFR. In this study, the subclass of EMab-134 was converted from IgG1 to IgG2a (134-mG2a) and further defucosylated (134-mG2a-f) to facilitate antibody-dependent cellular cytotoxicity (ADCC). Although 134-mG2a-f was developed against hEGFR, it was shown to cross-react with dog EGFR (dEGFR) using flow cytometry. The dissociation constant (KD) of 134-mG2a-f against dEGFR-overexpressed CHO-K1 (CHO/dEGFR) cells was determined by flow cytometry to be 3.3 × 10-9 M, indicating that 134-mG2a-f possesses a high binding affinity to dEGFR. Analysis in vitro revealed that 134-mG2a-f contributed to high levels of ADCC and complement-dependent cytotoxicity (CDC) in experiments targeting CHO/dEGFR cells. Furthermore, the in vivo administration of 134-mG2a-f significantly inhibited the development of CHO/dEGFR in comparison with the results observed in response to control mouse IgG. Taken together, the findings of this study demonstrate that 134-mG2a-f could be useful as part of a therapeutic regimen for dEGFR-expressing canine cancers.

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in a variety of cancer types, including breast, lung, gastric, pancreatic, and colorectal cancers. Trastuzumab, a humanized anti-HER2 monoclonal antibody (mAb), has been shown to provide significant survival benefits in HER2-overexpressing breast cancer and gastric cancer patients. Previously, an anti-HER2 mAb, H2Mab-41 (IgG2b, kappa), was developed in our laboratory and its antitumor activity was demonstrated in mouse xenograft models of human colon cancer. The present study aimed to investigate the ability of H2Mab-41 to induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in dog HER2 (dHER2)-overexpressed cell lines, and thus exert its antitumor activity against dHER2-overexpressed tumors in vivo. Flow cytometry results demonstrated the cross-reactivity of H2Mab-41 with dHER2. Further evaluation of interaction between H2Mab-41 and dHER2-overexpressed CHO-K1 (CHO/dHER2) cells indicated moderate binding affinity of H2Mab-41 toward dHER2, with a dissociation constant (KD) of 2.6 × 10-8 M. In vitro analysis revealed that the administration of H2Mab-41 induced high levels of ADCC and CDC in CHO/dHER2 cells. Furthermore, intraperitoneal administration of H2Mab-41 in mouse xenograft models of CHO/dHER2 resulted in significant inhibition of tumor development compared to the control mouse IgG. Thus, the findings of the present study demonstrated the in vivo safety and efficacy of H2Mab-41, highlighting its suitability to be included as a part of a therapeutic regimen for dHER2-expressing canine cancers.

Human epidermal growth factor receptor 2 (HER2) is a type I transmembrane 185 kDa protein. HER2 is expressed in a variety of normal tissue types and cancer cells. HER2 is associated with cell proliferation, differentiation, and migration. The overexpression of HER2 has been observed in a number of cancers, including breast and gastric cancers. Gastric cancer is one of the most common cancers worldwide, with an annual case rate of ∼1 million people diagnosed with the disease. Trastuzumab is a humanized anti-HER2 monoclonal antibody (mAb) that has been utilized in gastric cancer therapy. In this study, we have developed a novel anti-HER2 mAb, H2Mab-181 (IgG1, kappa), through the immunization of mice with a purified recombinant extracellular domain of HER2. H2Mab-181 can specifically and sensitively detect HER2 in both flow cytometry and Western blot applications in gastric cancer cell lines and can also be utilized in immunohistochemical analyses of gastric cancer tissues. Together, H2Mab-181 could be useful for the diagnosis and therapy in gastric cancers.

HER3 belongs to the epidermal growth factor receptor (EGFR) family and is known to form an active heterodimer with other three family members EGFR, HER2, and HER4. HER3 is overexpressed in lung, breast, colon, prostate, and gastric cancers. In the present study, we developed and validated an anti‑HER3 monoclonal antibody (mAb), H3Mab‑17 (IgG2a, kappa), by immunizing mice with HER3‑overexpressed CHO‑K1 cells (CHO/HER3). H3Mab‑17 was found to react specifically with endogenous HER3 in colorectal carcinoma cell lines, using flow cytometry. The KD for H3Mab‑17 in CHO/HER3 and Caco‑2 (a colon cancer cell line) were determined to be 3.0x10‑9 M and 1.5x10‑9 M via flow cytometry, respectively, suggesting high binding affinity of H3Mab‑17 to HER3. Then, we assessed the H3Mab‑17 antibody‑dependent cellular cytotoxicity (ADCC) and complement‑dependent cytotoxicity (CDC) against Caco‑2, and evaluated its antitumor capacity in a Caco‑2 xenograft model. In vitro experiments revealed H3Mab‑17 had strongly induced both ADCC and CDC against Caco‑2 cells. In vivo experiments on Caco‑2 xenografts revealed that H3Mab‑17 treatment significantly reduced tumor growth compared with the control mouse IgG. These data indicated that H3Mab‑17 could be a promising treatment option for HER3‑expressing colon cancers.

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that plays an important role in normal epidermal cell physiology. EGFR is overexpressed in cancer cells and has a number of mutations that implicate tumor malignancy, development, and poor patient prognosis; thus, EGFR is an attractive target for cancer therapy. At present, anti-EGFR monoclonal antibodies (mAbs) have been approved and are used for treating patients with a variety of EGFR-expressing cancers. Epitope mapping is important in identifying the therapeutic mechanism of anti-EGFR mAbs; however, the development of epitope mapping techniques lags behind the development of antimolecular target mAbs, including anti-EGFR mAbs. Hence, in this study, a novel epitope mapping method, RIEDL insertion for epitope mapping (REMAP) method, was developed. The results of this study demonstrated that the critical epitope of anti-EGFR mAb EMab-134 is Gly378, Asp379, Ser380, Phe381, Thr382, His383, Thr384, Pro385, and Pro386 of EGFR. The REMAP method could be useful for determining the critical epitope of functional mAbs against many target molecules.

CD44 functions as a major hyaluronan receptor on most cell types, with roles in cell adhesion, migration, proliferation, differentiation, and survival. The CD44 gene comprises 20 exons, with alternative splicing producing many different isoforms. CD44 variant isoforms exhibit tissue-specific expression patterns and have been studied as therapeutic targets for several cancers; therefore, anti-CD44 monoclonal antibodies (mAbs) are useful for investigating CD44 expression in various cancers. Previously, we established an anti-CD44 mAb, C44Mab-46 (IgG1, κ), by immunizing mice with the CD44v3-10 ectodomain. Although C44Mab-46 recognized all CD44 isoforms, the binding epitope of C44Mab-46 has not been determined. In this study, we first checked the reactivity of C44Mab-46 to several CD44v3-10 deletion mutants such as dN79, dN124, dN147, and dN224. We found the N-terminus of the C44Mab-46-binding epitope between residues 147 and 224 of CD44v3-10. We next investigated this epitope using a novel mapping system: RIEDL insertion for epitope mapping (REMAP) method. We constructed 31 CD44 standard (CD44s) mutants where the RIEDL tag was inserted into the expected epitope region in CD44s. We observed that the C44Mab-46 epitope constituted five amino acids: 174-TDDDV-178 of CD44s. Thus, the REMAP method could be used to determine mAb binding epitopes for membrane proteins.

Podoplanin (PDPN) plays a pivotal role in platelet aggregation, embryo development, and tumor progression. PDPN is universally expressed in many mammalian species, and is considered a typical lymphatic endothelial cell marker. We have previously developed the mouse anti-California sea lion (Zalophus californianus) PDPN (seaPDPN) monoclonal antibody (mAb), clone PMab-269, which is suitable for different experimental applications, including flow cytometry, Western blotting, and immunohistochemistry. In this study, we identified the PMab-269 epitope of the seaPDPN by enzyme-linked immunosorbent assay using deletion mutants and point mutants generated for seaPDPN. Our results demonstrated that PMab-269 recognized the peptide, corresponding to the amino acids 63-82 of seaPDPN. Furthermore, the reactions of PMab-269 to seven alanine-substituted peptides, such as P68A, D76A, F77A, H78A, L79A, E80A, and D81A, were abolished among 20 alanine-substituted peptides. We identified the seven amino acids (Pro68, Asp76, Phe77, His78, Leu79, Glu80, and Asp81) as the critical epitope targeted by PMab-269. The successful identification of the PMab-269 epitope might contribute to the pathophysiological investigations of seaPDPN.

Trophoblast cell surface antigen 2 (TROP2), reported to be overexpressed in several types of cancer, is involved in cell proliferation, invasion, metastasis, and poor prognosis of many types of cancer. Previously, a highly sensitive anti‑TROP2 monoclonal antibody (clone TrMab‑6; mouse IgG2b, κ) was developed using a Cell‑Based Immunization and Screening (CBIS) method. TrMab‑6 was useful for investigations using flow cytometry, western blot, and immunohistochemistry. The aim of the present study was to investigate whether TrMab‑6 possesses in vitro antibody‑dependent cellular cytotoxicity (ADCC) or complement‑dependent cytotoxicity (CDC) activities or in vivo antitumor activities using mouse xenograft models of TROP2‑overexpressed CHO‑K1 (CHO/TROP2) and breast cancer cell lines, including MCF7, MDA‑MB‑231, and MDA‑MB‑468. In vitro experiments revealed that TrMab‑6 strongly induced ADCC and CDC activities against CHO/TROP2 and the three breast cancer cell lines, whereas it did not show those activities against parental CHO‑K1 and MCF7/TROP2‑knockout cells. Furthermore, in vivo experiments on CHO/TROP2 and MCF7 xenografts revealed that TrMab‑6 significantly reduced tumor growth, whereas it did not show antitumor activities against parental CHO‑K1 and MCF7/TROP2‑knockout xenografts. The findings suggest that TrMab‑6 is a promising treatment option for TROP2‑expressing breast cancers.

In our previous study, a microfluidic system was developed based on podoplanin detection for capturing circulating tumor cells (CTCs), derived from malignant pleural mesothelioma (MPM). However, non-epithelioid MPM shows low podoplanin protein expression compared with that in epithelioid MPM; thus, some CTC populations may be missed. To overcome this limitation, a new CTC-detection chip was developed by combining the conventional podoplanin antibody (clone: NZ-1.2) with an epidermal growth factor receptor (EGFR)-targeted antibody (cetuximab). The cell-capture efficiency of the Cocktail-chip reached 100% in all the histological MPM cell lines. The median CTC-counts from 19 patients with MPM (epithelioid/non-epithelioid: 10/9) with the NZ-1.2- and Cocktail-chips were 1 and 3 (P=0.311) in 1 ml peripheral blood, 1.5 and 2 (P=0.332) in epithelioid MPM, and 1 and 3 (P=0.106) in non-epithelioid MPM, respectively. Overall, the Cocktail-chip showed an improved ability to detect more CTCs in patients with non-epithelioid MPM compared with that in the conventional NZ-1.2-chip, showing non-significant, but higher CTC detection. Furthermore, CTC-counts, determined using the Cocktail-chip were significantly correlated with the clinical stage of non-epithelioid MPM. In epithelioid MPM, the Cocktail-chip achieved a CTC-detection efficiency equivalent to that in the conventional NZ-1.2-chip. The Cocktail-chip enabled sensitive CTC detection of all histological MPM, including the non-epithelioid subtype, which may provide a foundation for the diagnosis, treatment, and prognosis of MPM progression.

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, C9Mab-1 (IgG1, kappa), which detected hCCR9 in MOLT-4 leukemia T lymphoblast cells and CHO/hCCR9 cells by flow cytometry. Our study showed that an anti-hCCR9 mAb was developed more rapidly by the CBIS method than the previous method.

Podoplanin (PDPN) plays an important role in the development of many normal tissues and is expressed in various cancers. We have previously developed multiple monoclonal antibodies (mAbs) against PDPNs from a variety of animal species and characterized each of these PDPNs using the anti-PDPN mAbs. In this study, we evaluated whether these anti-PDPN mAbs possess cross-reactivity with ferret PDPN (ferPDPN) using flow cytometry. Comprehensive analysis using 17 differing anti-PDPN mAbs available for immunohistochemistry use, demonstrated that the anti-bear PDPN mAb (clone PMab-241) strongly cross-reacts with ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells. Immunohistochemistry analysis demonstrated intense PMab-241 staining within Bowman's capsules and glomeruli of the ferret kidney, and lymphatic endothelial cells of the ferret lung. These results demonstrate that PMab-241 is suitable for the detection of PDPN in ferret tissues.

The development of specific antibodies is essential to understand a wide variety of biological phenomena and pathophysiological analyses. Podoplanin (PDPN), a type I transmembrane glycoprotein, is known as a diagnostic marker. Anti-PDPN monoclonal antibodies (mAbs) against many species, such as human, mouse, rat, rabbit, dog, bovine, cat, tiger, horse, pig, goat, alpaca, Tasmanian devil, bear, whale, and sheep, have been established in recent studies. However, sensitive and specific mAbs against elephant PDPN (elePDPN) have not been established. Thus, this study established a novel mAb against African savanna elephant (Loxodonta africana) PDPN using the Cell-Based Immunization and Screening method. elePDPN-overexpressed Chinese hamster ovary-K1 (CHO/elePDPN) cells were immunized, and mAbs were screened against elePDPN using flow cytometry. One of the mAbs, PMab-265 (IgM, κ), specifically detected CHO/elePDPN cells by flow cytometry. These findings suggested the potential usefulness of PMab-265 for the functional analyses of elePDPN.

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 3 (CCR3), also known as CD193, belongs to class A of G protein-coupled receptors and is present in high levels in eosinophils, basophils, and airway epithelial cells. CCR3 is considered the therapeutic target for human immunodeficiency virus (HIV) infections and allergic diseases; therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR3 has been desired. This study aimed to establish a specific and sensitive mAb against mouse CCR3 (mCCR3) useful for flow cytometry analysis by employing the Cell-Based Immunization and Screening (CBIS) method. The generated anti-mCCR3 mAb, C3Mab-2 (rat IgG2b, kappa), was found to react with mCCR3-overexpressed Chinese hamster ovary-K1 (CHO/mCCR3) cells, according to flow cytometric analysis. Also, it reacted with P388 (mouse lymphoid neoplasm) or J774-1 (mouse macrophage-like) cells, which express endogenous mCCR3. Taken together, C3Mab-2, generated by the CBIS method, can be a valuable tool for detecting mCCR3 on the surface of mouse cells.

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

Killer cell lectin-like receptor subfamily G member 1 (KLRG1), a type II transmembrane protein, was identified as an inhibitory receptor expressed on natural killer (NK) cells and certain T cells. The protein regulates effector functions and developmental processes in these cells. In this study, we established a specific and sensitive monoclonal antibody (mAb) for human KLRG1 (hKLRG1), which is useful for flow cytometry, using a Cell-Based Immunization and Screening (CBIS) method. The established anti-hKLRG1 mAb, KLMab-1 (mouse IgG1, kappa), reacted with overexpressed hKLRG1 in Chinese hamster ovary-K1 (CHO/hKLRG1) and human NK cells, which also expressed endogenous hKLRG1 as confirmed by flow cytometry. KLMab-1, which was established by the CBIS method, could be useful for elucidating the hKLRG1-related biological response by flow cytometry.

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 (IgG1, kappa), specifically and sensitively detects hTIGIT in CHO/hTIGIT and NK cells. The dissociation constants (KD) of TgMab-2 for CHO/hTIGIT cells were determined to be 3.5 × 10-9 M. These results suggest that TgMab-2, which was developed by CBIS method, is useful for analyzing the function of hTIGIT by flow cytometry.

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 (TH2) cells recruited to the inflammation site and is implicated in allergy and asthma. Recently, CCR8+Treg cells have been suggested to be a master regulator in the immunosuppressive tumor microenvironment; therefore, developing sensitive monoclonal antibodies (mAbs) for CCR8 has been desired. This study established a specific and sensitive mAb for mouse CCR8 (mCCR8), which is useful for flow cytometry by using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mCCR8 mAb, C8Mab-2 (rat IgG2b, kappa), reacted with mCCR8-overexpressed Chinese hamster ovary-K1 (CHO/mCCR8) cells and P388 (mouse lymphoid neoplasma) or J774-1 (mouse macrophage-like) cells, which express endogenous mCCR8 by flow cytometry. C8Mab-2, which was established by the CBIS method, could be useful for elucidating the mCCR8-related biological response by flow cytometry.

TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overexpressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, and immunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2-overexpressed CHO-K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO-K1 cells were selected using flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse IgG1 kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore, TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may be a valuable tool for the detection of TROP2 in cancer.

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteritis of ruminants. Previous studies have shown that programmed death-ligand 1 (PD-L1) is associated with the disease progression, and PD-L1 blockade activates MAP-specific Th1 responses in vitro. Here, we performed anti-PD-L1 antibody administration using 2 MAP-infected cattle at the late subclinical stage of infection. After administration, bacterial shedding was reduced or maintained at a low level. Additionally, MAP-specific Th1 cytokine production was upregulated, and CD69 expression was increased in T cells. Collectively, the treatment has a potential as a novel control method against Johne's disease.

Immunotherapy targeting programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) represents promising treatments for human cancers. Our previous studies demonstrated PD-L1 overexpression in some canine cancers, and suggested the therapeutic potential of a canine chimeric anti-PD-L1 monoclonal antibody (c4G12). However, such evidence is scarce, limiting the clinical application in dogs. In the present report, canine PD-L1 expression was assessed in various cancer types, using a new anti-PD-L1 mAb, 6C11-3A11, and the safety and efficacy of c4G12 were explored in 29 dogs with pulmonary metastatic oral malignant melanoma (OMM). PD-L1 expression was detected in most canine malignant cancers including OMM, and survival was significantly longer in the c4G12 treatment group (median 143 days) when compared to a historical control group (n = 15, median 54 days). In dogs with measurable disease (n = 13), one dog (7.7%) experienced a complete response. Treatment-related adverse events of any grade were observed in 15 dogs (51.7%). Here we show that PD-L1 is a promising target for cancer immunotherapy in dogs, and dogs could be a useful large animal model for human cancer research.

Trophoblast cell‑surface antigen 2 (TROP2) is a type I transmembrane glycoprotein that is overexpressed in a number of cancer types, including triple‑negative breast cancer. The current study aimed to develop a highly sensitive and specific monoclonal antibody (mAb) targeting TROP2, which could be used to evaluate TROP2 expression using flow cytometry, western blot analysis and immunohistochemistry by employing the Cell‑Based Immunization and Screening (CBIS) method. The established anti‑TROP2 mAb, TrMab‑6 (mouse IgG2b, κ), detected TROP2 on PA‑tagged TROP2‑overexpressing Chinese hamster ovary‑K1 (CHO/TROP2‑PA) and breast cancer cell lines, including MCF7 and BT‑474 using flow cytometry. Western blot analysis indicated a 40 kDa band in lysates prepared from CHO/TROP2‑PA, MCF7 and BT‑474 cells. Furthermore, TROP2 in 57/61 (93.4%) of the breast cancer specimens was strongly detected using immunohistochemical analysis with TrMab‑6. In conclusion, the current study demonstrated that TrMab‑6 may be a valuable tool for the detection of TROP2 in a wide variety of breast cancer types.

Purpose: CD44 is widely expressed on the surface of most tissues and all hematopoietic cells. CD44 plays important roles in cell proliferation, adhesion, and migration. Previously, we developed an anti-CD44 monoclonal antibody, C₄₄Mab-5 (IgG₁, kappa) by immunizing mice with CD44‑overexpressing Chinese hamster ovary (CHO)-K1 cells. In this study, we converted the mouse IgG₁ subclass antibody C₄₄Mab‑5 into an IgG_2a subclass antibody, 5‑mG_2a, and further produced a defucosylated version, 5‑mG_2a‑f and investigate its antitumor activities against oral cancers. Methods: To generate 5‑mG_2a‑f, appropriate V_H cDNA of mouse C₄₄Mab‑5 and C_H of mouse IgG_2a were subcloned into pCAG‑Neo vector, and light chain of C₄₄Mab‑5 was subcloned into pCAG‑Ble vector. Vectors were transfected into BINDS‑09 (FUT8‑deficient ExpiCHO‑S cells) using the ExpiCHO Expression System. Mouse xenograft models of HSC-2 and SAS (human oral cancer cell lines) were used for examining the antitumor activity. Results: 5-mG_2a-f demonstrated a sensitive and specific reaction against oral cancer cells in flow cytometry and immunohistochemical analyses. The sensitivity of 5‑mG_2a‑f was similar with that of C₄₄Mab‑5. In vitro analysis demonstrated that 5‑mG_2a‑f showed moderate ADCC and CDC activities against HSC-2 and SAS. 5-mG_2a-f significantly reduced tumor development in HSC-2 and SAS xenografts in comparison to control mouse IgG. Conclusion: 5‑mG_2a‑f may be a useful antibody-based therapy for patients with CD44-expressing oral cancers.

Bovine leukemia virus (BLV) infection is a bovine chronic infection caused by BLV, a member of the genus Deltaretrovirus. In this study, we examined the immunomodulatory effects of GS-9620, a toll-like receptor (TLR) 7 agonist, in cattle (Bos taurus) and its therapeutic potential for treating BLV infection. GS-9620 induced cytokine production in peripheral blood mononuclear cells (PBMCs) as well as CD80 expression in CD11c+ cells and increased CD69 and interferon (IFN)-γ expressions in T cells. Removing CD11c+ cells from PBMCs decreased CD69 expression in T cells in the presence of GS-9620. These results suggest that TLR7 agonism promotes T-cell activation via CD11c+ cells. Analyses using PBMCs from BLV-infected cattle revealed that TLR7 expression in CD11c+ cells was upregulated during late-stage BLV infection. Furthermore, GS-9620 increased IFN-γ and TNF-α production and inhibited syncytium formation in vitro, suggesting that GS-9620 may be used to treat BLV infection.

Combination treatment approaches are increasingly considered to overcome resistance to immunotherapy targeting immunoinhibitory molecules such as programmed death (PD)-1 and PD-ligand 1 (PD-L1). Previous studies have demonstrated that the therapeutic efficacy of anti-PD-L1 Abs is enhanced by combination treatment with cyclooxygenase-2 inhibitors, through downregulation of the immunosuppressive eicosanoid PGE2, although the underlying mechanism remains unclear. In this study, we show that serum PGE2 levels are upregulated after anti-PD-L1 Ab administration in a bovine model of immunotherapy and that PGE2 directly inhibits T cell activation via its receptor E prostanoid (EP) 4. Additionally, anti-PD-L1 Ab induces TNF-α production and TNF-α blockade reduces PGE2 production in the presence of anti-PD-L1 Ab, suggesting that anti-PD-L1 Ab-induced TNF-α impairs T cell activation by PGE2 upregulation. Our studies examining the therapeutic potential of the dual blockade of PD-L1 and EP4 in bovine and murine immune cells reveal that the dual blockade of PD-L1 and EP4 significantly enhances Th1 cytokine production in vitro. Finally, we show that the dual blockade decreases tumor volume and prolongs survival in mice inoculated with the murine lymphoma cell line EG7. Altogether, these results suggest that TNF-α induced by anti-PD-L1 Ab treatment is associated with T cell dysfunction via PGE2/EP4 pathway and that the dual blockade of PD-L1 and EP4 should be considered as a novel immunotherapy for cancer.

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells and several tumor cells, including melanoma and lung cancer cells. A strong correlation has been reported between PD-L1 expression in tumor cells and negative prognosis in cancer patients. Previously, we established an anti-PD-L1 monoclonal antibody (mAb), L1Mab-13 (IgG1, kappa), by immunizing mice with PD-L1-overexpressing CHO-K1 cells. L1Mab-13 specifically reacts with endogenous PD-L1 in lung cancer cell lines in flow cytometry and Western blot applications, and stains a plasma membrane-like pattern in lung cancer tissues via immunohistochemical analysis. In this study, we investigated whether L1Mab-13 reacts with oral cancer cell lines and exerts antitumor activities. Because L1Mab-13 lacks antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), we first converted the subclass of L1Mab-13 from IgG1 into IgG2a (13-mG2a), and further produced a defucosylated version (13-mG2a-f) using FUT8-deficient ExpiCHO-S (BINDS-09) cells. Defucosylation of 13-mG2a-f was confirmed using fucose-binding lectins, such as Aleuria aurantia and Pholiota squarrosa lectins. The dissociation constants (KD) for 13-mG2a-f in SAS and HSC-2 oral cancer cells were determined via flow cytometry to be 2.8 × 10-9 M and 4.8 × 10-9 M, respectively, indicating that 13-mG2a-f possesses extremely high binding affinity. In vitro analysis demonstrated that 13-mG2a-f showed moderate ADCC and CDC activities against SAS and HSC-2 oral cancer cells. In vivo analysis revealed that 13-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. Taken together, these data demonstrate that treatment with 13-mG2a-f may represent a useful therapy for patients with PD-L1-expressing oral cancers.

Diacylglycerol kinase δ (DGKδ) is a type II DGK, which catalyzes diacylglycerol phosphorylation to produce phosphatidic acid. DGKδ is expressed in several types of tissues and organs including the stomach, testis, bone marrow, and lymph node. Here, we established an anti-human DGKδ (hDGKδ) mAb, DdMab-1 (mouse IgG2a, kappa), which is useful for Western blot analysis. We also introduced deletion or point mutations to hDGKδ, and performed western blotting to determine the binding epitope of DdMab-1. DdMab-1 reacted with the dN670 mutant, but not with the dN680 mutant, indicating that the N-terminus of the DdMab-1 epitope is mainly located between amino acids 670 and 680 of the protein. Further analysis using point mutants demonstrated that R675A, R678A, K679A, and K682A mutants were not detected, and V680A was only weakly detected by DdMab-1, indicating that Arg675, Arg678, Lys679, Val680 and Lys682 are important for binding of DdMab-1 to hDGKδ.

Overexpression of podocalyxin (PODXL) is associated with progression, metastasis, and poor outcomes in several cancers. PODXL also plays an important role in the development of normal tissues. For antibody-based therapy to target PODXL-expressing cancers using monoclonal antibodies (mAbs), cancer-specificity is necessary to reduce the risk of adverse effects to normal tissues. In this study, we developed an anti-PODXL cancer-specific mAb (CasMab), named as PcMab-60 (IgM, kappa) by immunizing mice with soluble PODXL, which is overexpressed in LN229 glioblastoma cells. The PcMab-60 reacted with the PODXL-overexpressing LN229 (LN229/PODXL) cells and MIA PaCa-2 pancreatic cancer cells in flow cytometry but did not react with normal vascular endothelial cells (VECs), whereas one of non-CasMabs, PcMab-47 showed high reactivity for not only LN229/PODXL and MIA PaCa-2 cells but also VECs, indicating that PcMab-60 is a CasMab. Next, we engineered PcMab-60 into a mouse IgG2a-type mAb, named as 60-mG2a, to add antibody-dependent cellular cytotoxicity (ADCC). We further developed a core fucose-deficient type of 60-mG2a, named as 60-mG2a-f, to augment its ADCC activity. In vivo analysis revealed that 60-mG2a-f exerted antitumor activity in MIA PaCa-2 xenograft models at a dose of 100 μg/mouse/week administered three times. These results suggested that 60-mG2a-f could be useful for antibody-based therapy against PODXL-expressing pancreatic cancers.

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.

The epithelial cell adhesion molecule (EpCAM), which is a calcium-independent homophilic intercellular adhesion factor, contributes to cell signaling, differentiation, proliferation and migration. EpCAM is essential for carcinogenesis in numerous types of human cancer. The purpose of the present study was to establish an anti-EpCAM monoclonal antibody (mAb) for targeting colorectal adenocarcinomas. Thus, an anti-EpCAM mAb, EpMab-16 (IgG2a, κ), was established by immunizing mice with EpCAM-overexpressing CHO-K1 cells, and validated using flow cytometry, western blot, and immunohistochemical analyses. EpMab-16 reacted with endogenous EpCAM specifically in a colorectal adenocarcinoma cell line as determined by flow cytometry and western blot analyses. Immunohistochemical analysis demonstrated that EpMab-16 stained a plasma membrane-like pattern in clinical colorectal adenocarcinoma tissues. The dissociation constant (KD) for EpMab-16 in a Caco-2 colorectal adenocarcinoma cell line determined by flow cytometry was 1.8×10-8 M, suggesting moderate binding affinity of EpMab-16 for EpCAM. Whether the EpMab-16 induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against Caco-2 or antitumor activity was then assessed in a murine xenograft model. In vitro experiments revealed strong ADCC and CDC induction in Caco-2 cells by EpMab-16 treatment. In vivo experiments in a Caco-2 ×enograft model demonstrated that EpMab-16 treatment significantly reduced tumor growth compared with that in mice treated with the control mouse IgG. These results suggested that EpMab-16 may be a promising treatment option for EpCAM-expressing colorectal adenocarcinomas.

Podoplanin (PDPN) is a highly O-glycosylated glycoprotein that is utilized as a specific lymphatic endothelial marker under pathophysiological conditions. We previously developed an anti-human PDPN (hPDPN) monoclonal antibody (mAb), clone LpMab-3, which recognizes the epitope, including both the peptides and the attached disialy-core-l (NeuAcα2-3Galβl-3 [NeuAcα2-6]GalNAcαl-O-Thr) structure at the Thr76 residue in hPDPN. However, it is unclear if the mAb binds directly to both the peptides and glycans. In this study, we synthesized the binding epitope region of LpMab-3 that includes the peptide (-67LVATSVNSV-T-GIRIEDLP84-) possessing a disialyl-core-1 O-glycan at Thr76, and we determined the crystal structure of the LpMab-3 Fab fragment that was bound to the synthesized glycopeptide at a 2.8 Å resolution. The six amino acid residues and two sialic acid residues are directly associated with four complementarity-determining regions (CDRs; H1, H2, H3, and L3) and four CDRs (H2, H3, L1, and L3), respectively. These results suggest that IgG is advantageous for generating binders against spacious epitopes such as glycoconjugates.

Podoplanin (PDPN), a small transmembrane mucin-like glycoprotein, is ectopically expressed on tumor cells. PDPN is known to be linked with several aspects of tumor malignancies in certain types of human and canine tumors. Therefore, it is considered to be a novel therapeutic target. Monoclonal antibodies targeting PDPN expressed in human tumor cells showed obvious anti-tumor effects in preclinical studies using mouse models. Previously, we generated a cancer-specific mouse-dog chimeric anti-PDPN antibody, P38Bf, which specifically recognizes PDPN expressed in canine tumor cells. In this study, we investigated the safety and anti-tumor effects of P38Bf in preclinical and clinical trials. P38Bf showed dose-dependent antibody-dependent cellular cytotoxicity against canine malignant melanoma cells. In a preclinical trial with one healthy dog, P38Bf administration did not induce adverse effects over approximately 2 months. In phase I/II clinical trials of three dogs with malignant melanoma, one dog vomited, and all dogs had increased serum levels of C-reactive protein, although all adverse effects were grade 1 or 2. Severe adverse effects leading to withdrawal of the clinical trial were not observed. Furthermore, one dog had stable disease with P38Bf injections. This is the first reported clinical trial of anti-PDPN antibody therapy using spontaneously occurring canine tumor models.

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 determined by flow cytometry to be 3.2x10‑9 M and 2.1x10‑9 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.

Podoplanin (PDPN), a 36-kDa type I transmembrane O-glycoprotein, is expressed in normal cells, including renal epithelial cells (podocytes), lymphatic endothelial cells, and pulmonary type I alveolar cells, and in cancer cells, including brain tumors and squamous cell lung carcinomas. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets, and PDPN/CLEC-2 interaction facilitates blood/lymphatic vessel separation. We previously produced an anti-human PDPN monoclonal antibody (mAb), clone NZ-1 (rat IgG2a, lambda) and its rat-human chimeric mAbs (NZ-8/NZ-12), which neutralize PDPN/CLEC-2 interactions and inhibit platelet aggregation and cancer metastasis. In this study, we first developed a humanized anti-human PDPN mAb, named as NZ-27. We further produced a core-fucose-deficient version of NZ-27, named as P1027 and a core-fucose-deficient version of NZ-12, named as NZ-12f. We investigated the binding affinity, antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antitumor activity of P1027 and NZ-12f. We demonstrated that the binding affinities of P1027 and NZ-12f against LN319 (a human glioblastoma cell line) are 1.1 × 10-8 and 3.9 × 10-9 M, respectively. ADCC reporter assays demonstrated that NZ-12f shows 1.5 times higher luminescence than P1027. Furthermore, NZ-12f showed 2.2 times higher ADCC than P1027, whereas both P1027 and NZ-12f showed high CDC activities against LN319 cells. Using LN319 xenograft models, P1027 and NZ-12f significantly reduced tumor development in an LN319 xenograft model compared with control human IgG. Treatment with P1027 and NZ-12f may be a useful therapy for patients with PDPN-expressing cancers.

The MAP tag system comprises a 14-residue peptide derived from mouse podoplanin and its high-affinity monoclonal antibody PMab-1. We determined the crystal structure of PMab-1 complexed with the MAP tag peptide and found that the recognition required only the N-terminal 8 residues of MAP tag sequence, enabling the shortening of the tag length without losing the affinity for PMab-1. Furthermore, the structure illustrated that the MAP tag adopts a U-shaped conformation when bound by PMab-1, suggesting that loop-inserted MAP tag would assume conformation compatible with the PMab-1 binding. We inserted the 8-residue MAP tag into multiple loop regions in various proteins including fibronectin type III domain and G-protein-coupled receptors and tested if they maintain PMab-1 reactivity. Despite the conformational restraints forced by the insertion position, all MAP-inserted mutants were expressed well in mammalian cells at levels comparable to the non-tagged proteins. Furthermore, the binding by PMab-1 was fully maintained even for the mutant where MAP tag was inserted at a structurally restricted β-hairpin, indicating that the MAP tag system has unique feature that allows placement in the middle of protein domain at desired locations. Our results indicate the versatile utility of the MAP tag system in 'site-specific epitope insertion' application.

Affinity tag systems are an essential tool in biochemistry, biophysics, and molecular biology. Although several different tag systems have been developed, the epitope tag system, composed of a polypeptide "tag" and an anti-tag antibody, is especially useful for protein purification. However, almost all tag sequences, such as the FLAG tag, are added to the N- or C-termini of target proteins, as tags inserted in loops tend to disrupt the functional structure of multi-pass transmembrane proteins. In this study, we developed a novel "RIEDL tag system," which is composed of a peptide with only five amino acids (RIEDL) and an anti-RIEDL monoclonal antibody (mAb), LpMab-7. To investigate whether the RIEDL tag system is applicable for protein purification, we conducted the purification of two kinds of RIEDL-tagged proteins using affinity column chromatography: whale podoplanin (wPDPN) with an N-terminal RIEDL tag (RIEDL-wPDPN) and human CD20 with an internal RIEDL tag insertion (CD20-169RIEDL170). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-169RIEDL170 were efficiently purified in one-step purification procedures, and were strongly detected by LpMab-7 using Western blot and flow cytometry. These results show that the RIEDL tag system can be useful for the detection and one-step purification of membrane proteins when inserted at either the N-terminus or inserted in an internal loop structure of multi-pass transmembrane proteins.

The now clinically-used anti-epidermal growth factor receptor (EGFR) monoclonal antibodies have demonstrated significant efficacy only in patients with metastatic colorectal cancer (mCRC), with wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS). However, no effective treatments for patients with mCRC with KRAS mutated tumors have been approved yet. Therefore, a new strategy for targeting mCRC with KRAS mutated tumors is desired. In the present study, we examined the anti-tumor activities of a novel anti-EGFR monoclonal antibody, EMab-17 (mouse IgG2a, kappa), in colorectal cancer (CRC) cells with the KRAS p.G13D mutation. This antibody recognized endogenous EGRF in CRC cells with or without KRAS mutations, and showed a high sensitivity for CRC cells in flow cytometry, indicating that EMab-17 possesses a high binding affinity to the endogenous EGFR. In vitro experiments showed that EMab-17 exhibited antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities against CRC cells. In vivo analysis revealed that EMab-17 inhibited the metastases of HCT-15 and HCT-116 cells in the livers of nude mouse metastatic models, unlike the anti-EGFR monoclonal antibody EMab-51 of subtype mouse IgG1. In conclusion, EMab-17 may be useful in an antibody-based therapy against mCRC with the KRAS p.G13D mutation.

Diacylglycerol kinase (DGK) η is classified as a type II DGK and catalyzes diacylglycerol phosphorylation to produce phosphatidic acid. DGKη has been reported to be highly expressed in the hippocampus and cerebellum. Although a DGKη-specific monoclonal antibody (mAb) is necessary to reveal the association between the expression of DGKη and diseases, an anti-DGKη mAb for immunohistochemistry has not been developed. Recently, we established a specific antihuman DGKη (hDGKη) mAb, DhMab-1 (mouse IgG2a, kappa). For epitope mapping of DhMab-1, here we produced deletion or point mutants of hDGKη and performed Western blotting to determine the binding epitope of DhMab-1. DhMab-1 reacted with the dN755 mutant, but not with the dN760 mutant, indicating that the N-terminus of the DhMab-1 epitope is mainly located between amino acids 755 and 760 of the protein. A more detailed analysis using point mutants demonstrated that seven mutants, that is, A751G, I755A, D756A, P757A, D758A, L759A, and D760A, were not detected by DhMab-1. These results indicate that Ala751, Ile755, Asp756, Pro757, Asp758, Leu759, and Asp760 are important for DhMab-1 binding to hDGKη.

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in glioblastoma as well as breast, gastric, lung, colorectal, and pancreatic cancers. Its expression is associated with poor clinical outcomes. Anti-HER2 antibodies have provided significant survival benefits to patients with HER2-overexpressing breast and gastric cancers. We recently developed an anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, kappa), by immunizing mice with the extracellular domain of HER2, which is expressed in LN229 glioblastoma cells. In this study, we investigated the antitumor activity of H2Mab-19 in an LN229 glioblastoma xenograft model. H2Mab-19 showed high binding affinity (KD: 1.1 × 10-8 M) against LN229 cells. Furthermore, H2Mab-19 significantly reduced tumor development in an LN229 xenograft. These results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing glioblastomas.

Sensitive and specific monoclonal antibodies (mAbs) are needed for detecting CD20. This antigen, one of several B lymphocyte antigens, is a target for every application used in the diagnosis of B cell lymphoma. Many anti-CD20 mAbs have been established, although applications of these antibodies are limited. This study aims to establish sensitive and specific anti-CD20 mAbs suitable for broad application, such as flow cytometry, Western blotting, and immunohistochemical analyses. Using the Cell-Based Immunization and Screening (CBIS) method, all procedures were performed by utilizing CD20-stable transfectants, and a clone, C20Mab-60 (IgG2a, kappa), was developed. In flow cytometry, C20Mab-60 detected overexpression of CD20 in LN229 cell and endogenous CD20 in BALL-1 (a human B cell leukemia cell line) but did not react with CD20-knockout BALL-1 (BINDS-24), indicating specificity for CD20. In Western blotting, C20Mab-60 detected CD20-overexpressing Chinese hamster ovary-K1, BALL-1, and Raji (a human Burkitt's lymphoma cell line) displaying both sensitivity and specificity. Furthermore, B cell but not T cell lymphomas were strongly stained with C20Mab-60 in immunohistochemical analyses. C20Mab-60, which was developed by CBIS method, is shown to be useful for the detection of cells expressing CD20 in lymphoma tissues by flow cytometry, Western blotting, and immunohistochemical analyses.

Human epidermal growth factor receptor 2 (HER2) is reported to be overexpressed in breast cancers and is associated with poor clinical outcome. Trastuzumab is a humanized anti-HER2 antibody that offers significant survival benefits to patients with HER2-overexpressing breast cancer. In this study, a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, kappa) was developed. Antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antitumor activity of H2Mab-19 were investigated using both breast cancer and oral cancer cell lines. H2Mab-19 demonstrated cytotoxicity in BT-474 (a human breast cancer cell line) and HSC-2 or SAS (human oral cancer cell lines). H2Mab-19 also possessed both ADCC and CDC activity against BT-474, HSC-2, and SAS cell lines. In comparison to control mouse IgG, H2Mab-19 significantly reduced tumor development in BT-474, HSC-2, and SAS xenografts. Collectively, these results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing breast and oral cancers.

Diacylglycerol kinase (DGK) plays a pivotal role in intracellular signaling pathways in mammals. Activated G protein-coupled receptor activates phospholipase C (PLC) through heterotrimeric G protein, following which PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DGK catalyzes DG phosphorylation to produce phosphatidic acid. DG and phosphatidic acid function as second messengers and their intracellular concentrations are regulated by DGK; therefore, DGK plays an important role in regulating many biological processes. There are ten DGK isozymes, of which DGKη is classified as a type II DGK. Reports have shown that DGKη is associated with several diseases; for example, it is highly expressed in the hippocampus and cerebellum and is a key element in bipolar disorder. Although a DGKη-specific monoclonal antibody (mAb) is necessary to reveal the association between the expression of DGKη and diseases, an anti-DGKη mAb for immunohistochemistry has not yet been established. In this study, we established a specific anti-human DGKη (hDGKη) mAb, DhMab-4 (mouse IgG2b, kappa). DhMab-4 strongly stained Purkinje cells of human cerebellum in immunohistochemistry analysis. For epitope mapping of DhMab-4, we produced deletion or point mutants of hDGKη and performed western blotting to determine the binding epitope of DhMab-4. DhMab-4 reacted with dN745 mutant but not with dN750 mutant, indicating that the N-terminus of the DhMab-4 epitope is located between amino acids 745 and 750. More detailed analysis using point mutants demonstrated that five mutants, that is, D747A, P748A, F749A, G750A, and T752A, were not detected by DhMab-4. These results indicate that Asp747, Pro748, Phe749, Gly750, and Thr752 are important for DhMab-4 binding to hDGKη.

Anti-podoplanin (PDPN) monoclonal antibodies (mAbs) are needed as markers for lymphatic endothelial cells or type I alveolar cells in immunohistochemical analyses. We have developed anti-PDPN mAbs for many species, including humans, mice, rats, rabbits, dogs, cats, bovines, pigs, Tasmanian devils, alpacas, tigers, whales, goats, horses, and bears. This study develops and characterizes anti-sheep PDPN (sPDPN) mAbs using Cell-Based Immunization and Screening (CBIS) method. A RAP14 tag was added to the N-terminus of sPDPN, and an anti-RAP14 tag mAb (PMab-2) was used to measure the expression level of sPDPN in flow cytometry and Western blots. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells and screened mAbs against sPDPN using flow cytometry. Two of the mAbs, PMab-253 (immunoglobulin M [IgM], kappa) and PMab-260 (IgM, kappa), detected CHO/sPDPN cells specifically using flow cytometry and Western blots. Both PMab-253 and PMab-260 stained the renal glomerulus and Bowman's capsule, lymphatic endothelial cells of the lung and colon, and type I alveolar cells of the lung, suggesting PMab-253 and PMab-260, which were developed by CBIS method, can be applied to functional analyses of sPDPN. We also determined the binding epitope of PMab-253 and PMab-260 using flow cytometry. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-253 and PMab-260 epitope exists between amino acids 110 and 115 of sPDPN. Analysis of sPDPN point mutations revealed that the critical epitope of PMab-253 and PMab-260 includes Thr112 and Ser113 of sPDPN, indicating that the PMab-253 and PMab-260 epitope are independent of the platelet aggregation-stimulating (PLAG) domain or the PLAG-like domain of sPDPN.

Trastuzumab is a humanized antibody against human epidermal growth factor receptor 2 (HER2) that offers significant survival benefits to patients with HER2-overexpressing breast or gastric cancer. HER2 is also known to be overexpressed in colon cancers. In this study, a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, κ) was characterized for its anticancer activity in colon cancers. H2Mab-19 showed both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activities against Caco-2, a colon cancer cell line. Furthermore, H2Mab-19 significantly reduced tumor development in a Caco-2 xenograft model. These results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing colon cancers.

CD20 is one of several B-lymphocyte antigens that has been shown to be an effective target for the detection and treatment of B-cell lymphomas. Sensitive and specific monoclonal antibodies (mAbs) are required for every application used for the diagnosis of B-cell lymphoma. Although many anti-CD20 mAbs have been established, the types of applications, those anti-CD20 can be used in, are limited. In this study, we aimed to establish novel anti-CD20 mAbs to be used for broad applications, such as flow cytometry, western blot, and immunohistochemical analyses, using the Cell-Based Immunization and Screening (CBIS) method. One of the established mAbs, C20Mab-11 (IgM, kappa), detected overexpression of CD20 in CHO-K1 or LN229 cell lines, indicating that C20Mab-11 is specific for CD20. In western blot analyses, C20Mab-11 detected not only overexpression of CD20 in CHO-K1 or LN229 cell lines, but also CD20 of BALL-1 and Raji cells with both sensitivity and specificity. Furthermore, C20Mab-11 strongly stained B cells of the lymph follicle and B cell lymphomas in immunohistochemical analyses. These results indicate that C20Mab-11 develped by CBIS method, is useful for the detection of CD20 in lymphoma tissues by flow cytometry, western blot, and immunohistochemical analyses and potentially could be beneficial for the treatment of B cell lymphomas.

Lymphoma is the most common hematological cancer in dogs. Canine diffuse large B cell lymphoma shows a relatively good response to treatment with multi-agent cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy; however, the 2-year survival rate is as low as 20%. For human B cell type lymphoma, the anti-CD20 chimeric antibody, rituximab, was developed two decades ago. The combination of rituximab and CHOP chemotherapy was highly successful in improving patient prognosis. However, no anti-canine CD20 antibody is available for the treatment of canine lymphoma. During this study, a rat anti-canine CD20 monoclonal antibody was established. We also generated a rat-canine chimeric antibody against canine CD20 designed for clinical application. This chimeric antibody (4E1-7-B) showed in vitro antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against the canine B cell lymphoma cell line CLBL-1. Moreover, to obtain stronger ADCC activity, a defucosylated 4E1-7-B antibody (4E1-7-B_f) was also generated, and it showed tenfold stronger ADCC activity compared with 4E1-7-B. 4E1-7-B_f as well as 4E1-7-B suppressed the growth of CLBL-1 tumors in an immunodeficient xenotransplant mouse model. Finally, a single administration of 4E1-7-B_f induced considerable peripheral B cell depletion in healthy beagles. Thus, 4E1-7-B_f is a good antibody drug candidate for canine B cell type lymphoma.

The purpose of this study is to evaluate outflow pathways from subconjunctival blebs and to identify their identity. Post-mortem porcine (n = 20), human (n = 1), and bovine (n = 1) eyes were acquired, and tracers (fluorescein, indocyanine green, or fixable/fluorescent dextrans) were injected into the subconjunctival space to create raised blebs where outflow pathways were visualized qualitatively and quantitatively. Rodents with fluorescent reporter transgenes were imaged for structural comparison. Concurrent optical coherence tomography (OCT) was obtained to study the structural nature of these pathways. Using fixable/fluorescent dextrans, tracers were trapped to the bleb outflow pathway lumen walls for histological visualization and molecular identification using immunofluorescence against lymphatic and blood vessel markers. Bleb outflow pathways could be observed using all tracers in all species. Quantitative analysis showed that the nasal quadrant had more bleb-related outflow pathways compared to the temporal quadrant (nasal: 1.9±0.3 pathways vs. temporal: 0.7±0.2 pathways; p = 0.003). However, not all blebs resulted in an outflow pathway (0-pathways = 18.2%; 1-pathway = 36.4%; 2-pathways = 38.6%; and 3-pathways = 6.8%). Outflow signal was validated as true luminal pathways using optical coherence tomography and histology. Bicuspid valves were identified in the direction of flow in porcine eyes. Immunofluorescence of labeled pathways demonstrated a lymphatic (Prox-1 and podoplanin) but not a blood vessel (CD31) identity. Therefore, subconjunctival bleb outflow occurs in discrete luminal pathways. They are lymphatic as assessed by structural identification of valves and molecular identification of lymphatic markers. Better understanding of lymphatic outflow may lead to improved eye care for glaucoma surgery and ocular drug delivery.

BACKGROUND: Canine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression. RESULTS: ChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR < 0.005). Interestingly, the majority of genes overexpressed in HSA tumor tissue were associated with extracellular matrix (ECM) remodeling. This observation correlated well with our histological analysis, which found that HSA tumors contain a rich and complex collagen network. Additionally, we characterized the protein expression patterns of two highly overexpressed molecules identified in ChRO-seq analysis, podoplanin (PDPN) and laminin alpha 4 (LAMA4). We found that the expression of these two ECM-associated factors appeared to be largely limited to transformed endothelial cells within the HSA lesions. CONCLUSION: Outcomes from this study suggest that ECM remodeling plays an important role in HSA progression. Additionally, our study identified two potential novel biomarkers of HSA, PDPN and LAMA4. Interestingly, given that function-blocking anti-PDPN antibodies have shown anti-tumor effects in mouse models of canine melanoma, our studies raise the possibility that these types of therapeutic strategies could potentially be developed for treating canine HSA.

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.

An antisheep podoplanin (sPDPN) monoclonal antibody (mAb), PMab-256, has recently been established. PMab-256 shows positive immunostaining for lymphatic endothelial cells, lung type I alveolar cells, and kidney podocytes. PDPN possesses three platelet-aggregation-stimulating (PLAG) domains, PLAG1, PLAG2, and PLAG3, and a PLAG-like domain (PLD). The binding epitope of many anti-PDPN mAbs is located in PLAG domains or PLD. The purpose of this study is to determine the binding epitope of PMab-256. Analysis of sPDPN deletion mutants revealed that the N-terminus of the PMab-256 epitope exists between amino acids 75 and 80 of sPDPN. Furthermore, analysis of sPDPN point mutations demonstrated that the critical epitope includes Thr80 of sPDPN, indicating that the PMab-256 epitope is in the PLD of sPDPN.

Overexpression of human epidermal growth factor receptor 2 (HER2) has been reported in breast cancer, gastric, lung, colorectal, oral, and pancreatic cancers. HER2 expression is associated with poor clinical outcomes. An anti-HER2 humanized antibody, trastuzumab, has improved survival rates in patients with HER2-overexpressing breast and gastric cancers. Previously, we established a novel anti-HER2 monoclonal antibody (mAb), H2Mab-19 (IgG2b, kappa). It has also been characterized for breast, oral, and colon cancers. In this study, we investigated the antitumor activities of H2Mab-19 in pancreatic cancer xenograft models. We selected MIA PaCa-2, a pancreatic cancer cell line which expresses HER2. H2Mab-19 showed high binding affinity (KD: 1.2 × 10-8 M) against MIA PaCa-2 cells. Furthermore, H2Mab-19 significantly reduced tumor development in a MIA PaCa-2 xenograft model. These results suggest that treatment with H2Mab-19 may be a useful therapy for patients with HER2-expressing pancreatic cancers.

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are needed for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, and sheep PDPNs. However, no antilion PDPN (lioPDPN) antibody has been developed. In this study, possible cross-reaction between available anti-PDPN mAbs and lioPDPN was examined. Immunohistochemical analysis showed that antitiger PDPN mAb PMab-231 (IgG2a, kappa) reacted with type I alveolar cells from lion lung, indicating that PMab-231 is useful for the detection of lioPDPN.

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.

The expression of human epidermal growth factor receptor 2 (HER2) has been reported to be overexpressed in several cancers, such as breast, lung, gastric, pancreatic, and colorectal cancers, and be associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H2Mab-41 (IgG2b, kappa), and the antitumor activity of H2Mab-41 was investigated using mouse xenograft models. Caco-2 cells (human colon cancer cell line), which expresses HER2, were subcutaneously implanted into the flanks of nude mice. H2Mab-41 and control mouse IgG were injected three times into the peritoneal cavity of mice. H2Mab-41 significantly reduced tumor development of Caco-2 xenograft in comparison with the control mouse IgG on days 5, 8, 11, 15, and 19. Taken together, these results suggest that H2Mab-41 is useful for antibody therapy against HER2-expressing colon cancers.

Podoplanin (PDPN), a small transmembrane mucin-like glycoprotein, is ectopically expressed. It is also known to be linked with several aspects of tumor malignancy in some types of human tumors, including invasion, metastasis, and cancer stemness. However, there are few reports on the expression of dog PDPN (dPDPN) in canine tumors, and the association between dPDPN and tumor malignancy has not been elucidated. We identified that 11 out of 18 types of canine tumors expressed dPDPN. Furthermore, 80% of canine malignant melanoma (MM), squamous cell carcinoma, and meningioma expressed dPDPN. Moreover, the expression density of dPDPN was positively associated with the expression of the Ki67 proliferation marker. The silencing of dPDPN by siRNAs resulted in the suppression of cell migration, invasion, stem cell-like characteristics, and cell viability in canine MM cell lines. The suppression of cell viability was caused by the induction of apoptosis and G2/M phase cell cycle arrest. Overall, this study demonstrates that dPDPN is expressed in various types of canine tumors and that dPDPN silencing suppresses cell viability through apoptosis and cell cycle arrest, thus providing a novel biological role for PDPN in tumor progression.

Malignant pleural mesothelioma (MPM) has extremely limited treatment despite a poor prognosis. Moreover, molecular targeted therapy for MPM has not yet been implemented; thus, a new targeted therapy is highly desirable. Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer therapy that combines the specificity of antibodies for targeting tumors with toxicity induced by the photoabsorber after exposure to NIR-light. In this study, we developed a new phototherapy targeting podoplanin (PDPN) for MPM with the use of both NIR-PIT and an anti-PDPN antibody, NZ-1. An antibody-photosensitizer conjugate consisting of NZ-1 and phthalocyanine dye was synthesized. In vitro NIR-PIT-induced cytotoxicity was measured with both dead cell staining and luciferase activity on various MPM cell lines. In vivo NIR-PIT was examined in both the flank tumor and orthotopic mouse model with in vivo real-time imaging. In vitro NIR-PIT-induced cytotoxicity was NIR-light dose dependent. In vivo NIR-PIT led to significant reduction in both tumor volume and luciferase activity in a flank model (p < 0.05, NIR-PIT group versus NZ-1-IR700 group). The PDPN-targeted NIR-PIT resulted in a significant antitumor effect in an MPM orthotopic mouse model (p < 0.05, NIR-PIT group versus NZ-1-IR700 group). This study suggests that PDPN-targeted NIR-PIT could be a new promising treatment for MPM.

Circulating tumor cell (CTC) is a potentially useful surrogate of micro-metastasis, but detection of rare tumor cells contaminated in a vast majority of normal hematologic cells remains technical challenges. To achieve effective detection of a variety of CTCs, we have developed a novel microfluidic system (CTC-chip) in which any antibody to capture CTCs is easily conjugated. In previous studies, we employed an antibody (clone E-1) against podoplanin that was strongly expressed on mesothelioma cells. The CTC-chip coated by the E-1 antibody (E1-chip) provided a modest sensitivity in detection of CTCs in malignant pleural mesothelioma (MPM). Here, to achieve a higher sensitivity, we employed a novel anti-podoplanin antibody (clone NZ-1.2). In an experimental model, MPM cells with high podoplanin expression were effectively captured with the CTC-chip coated by the NZ-1.2 antibody (NZ1.2-chip). Next, we evaluated CTCs in the peripheral blood sampled from 22 MPM patients using the NZ1.2-chip and the E1-chip. One or more CTCs were detected in 15 patients (68.2%) with the NZ1.2-chip, whereas only in 10 patients (45.5%) with the E1-chip. Of noted, in most (92.3%, 12/13) patients with epithelioid MPM subtype, CTCs were positive with the NZ1.2-chip. The CTC-count detected with the NZ1.2-chip was significantly higher than that with the E1-chip (p = 0.034). The clinical implications of CTCs detected with the NZ1.2-chip will be examined in a future study.

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 member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases; it is a transmembrane receptor involved in cell growth and differentiation. EGFR homodimers or heterodimers in combination with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many types of cancer, including oral squamous cell carcinoma (OSCC). The present study produced novel anti-EGFR monoclonal antibodies (mAbs) possessing antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and investigated antitumor activity. Mice were immunized with an EGFR-overexpressed glioblastoma cell line, LN229 (LN229/EGFR), after which ELISA was performed using recombinant EGFR. mAbs were subsequently selected according to their efficacy for LN229/EGFR, as determined via flow cytometry. After determining the subclass of mAbs, the EMab-17 (IgG2a, kappa) clone exhibited ADCC and CDC activities against two OSCC cell lines, HSC-2 and SAS. Furthermore, EMab-17 exerted antitumor activities against mouse xenograft models using HSC-2 and SAS, indicating that EMab-17 may be used in an antibody-based therapy for EGFR-expressing OSCC.

The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses reveal that phosphorylation of hTERT at threonine 249 occurs more frequently in aggressive cancers. Using CRISPR/Cas9 genome editing, we introduce substitution mutations at threonine 249 in the endogenous hTERT locus and find that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase and terminal transferase activities. Cap Analysis of Gene Expression (CAGE) demonstrates that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomere independent manner.

Pancreatic ductal adenocarcinoma (PDAC) is among the cancers with the poorest prognoses due to its highly malignant features. BTB and CNC homology 1 (BACH1) has been implicated in RAS-driven tumor formation. We focused on the role of BACH1 in PDAC, more than 90% of which have KRAS mutation. Knockdown of BACH1 in PDAC cell lines reduced cell migration and invasion, in part, by increasing E-cadherin expression, whereas its overexpression showed opposite effects. BACH1 directly repressed the expression of FOXA1 that is known to activate the expression of CDH1 encoding E-cadherin and to inhibit epithelial-to-mesenchymal transition. BACH1 also directly repressed the expression of genes important for epithelial cell adhesion including CLDN3 and CLDN4. In a mouse orthotopic implantation model, BACH1 was required for the high metastatic ability of AsPC-1 cells. IHC analysis of clinical specimens with a newly developed anti-BACH1 mAb revealed that high expression of BACH1 is a poor prognostic factor. These results suggest that the gene regulatory network of BACH1 and downstream genes including CDH1 contribute to the malignant features of PDAC by regulating epithelial-to-mesenchymal transition. SIGNIFICANCE: Greater understanding of the gene regulatory network involved in epithelial-to-mesenchymal transition of pancreatic cancer cells will provide novel therapeutic targets and diagnostic markers.

Podoplanin (PDPN) is a small mucin-type transmembrane glycoprotein, which was first discovered in podocytes of the kidney. PDPN is a specific lymphatic endothelial marker and is also known as T1alpha, a marker of lung type I alveolar cells, or Aggrus, a platelet aggregation-inducing factor. PDPN possesses three platelet aggregation-stimulating (PLAG) domains and PLAG-like domains (PLDs), which bind to C-type lectin-like receptor-2. Previously, we developed a novel anti-whale PDPN (wPDPN) monoclonal antibody (mAb) PMab-237 using the Cell-Based Immunization and Screening (CBIS) method and the RIEDL tag of Arg-Ile-Glu-Asp-Leu sequence. PMab-237 detected wPDPN by flow cytometry, western blot, and immunohistochemical analyses. However, the specific binding epitope of PMab-237 for wPDPN remains unknown. In this study, deletion mutants and point mutants of wPDPN with N-terminal RIEDL tag were produced to analyze the PMab-237 epitope using flow cytometry. The analysis of deletion mutants showed that the N-terminus of the PMab-237 epitope exists between the 80th amino acid (AA) and the 85th AA of wPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-237 includes Leu82 and Thr84 of wPDPN, indicating that the PMab-237 epitope is located in the PLD of wPDPN.

Purpose: Podoplanin is expressed in normal tissues including renal podocytes and lymphatic endothelial cells. To investigate the expression and function of horse podoplanin (horPDPN), sensitive and specific mAbs against horPDPN are necessary. In this study, we aimed to develop useful anti-horPDPN mAbs for many applications such as flow cytometry (FCM), western blot (WB), and immunohistochemistry (IHC). Methods: We employed a conventional immunization method using synthetic peptides or Cell-Based Immunization and Screening (CBIS) method using horPDPN-expressed mammalian cells for producing anti-horPDPN mAbs. Anti-horPDPN mAbs were screened using enzyme-linked immunosorbent assay or FCM. Established anti-horPDPN mAbs were characterized using FCM, WB, and IHC. Results: We developed two anti-horPDPN mAbs, PMab-202 using the peptide immunization and PMab-219 using CBIS method. PMab-202 reacted with horPDPN in FCM and WB, but did not stain horPDPN in IHC. In contrast, PMab-219 detected horPDPN in not only FCM and WB, but also IHC. Conclusion: We have successfully established mouse anti-horPDPN mAbs, PMab-202 and PMab-219. PMab-219 is applicable for FCM, WB, and IHC analyses. CBIS method could be more advantageous to establish immunohistochemistry-applicable mAbs for elucidating the pathophysiological function of horPDPN.

<p>Background: Cancer stem cells contribute to tumorigenesis, metastasis, and chemoresistance. A pentaspan membrane glycoprotein CD133 has been used for the isolation of stem-like cells from several cancers.</p><p>Purpose: In this study, we aimed to develop sensitive and specific anti-CD133 mAbs, which exerts anti-tumor and anti-metastasis activities.</p><p>Methods: Cell-Based Immunization and Screening (CBIS) method was employed for the development of anti-CD133 mAbs. LN229/CD133 glioblastoma cells were immunized into mice, and FCM was used for the first screening. WB and IHC screenings were further performed. Human colon cancer cell lines were used for examining the anti-tumor and the anti-metastasis activities of anti-CD133 mAbs.</p><p>Results: We established a novel anti-CD133 mAb, CMab-43 (IgG_2a, kappa), which demonstrated a sensitive and specific reaction against colon cancer cells in FCM, WB, and IHC analyses. CMab-43 showed cancer-specific staining patterns in colon cancer tissues. Furthermore, CMab-43 significantly reduced tumor development of colon cancer cell xenografts, and inhibited experimental metastasis of colon cancer cells.</p><p>Conclusion: CMab-43 is useful for many applications and exerts anti-tumor or anti-metastasis activities. CMab-43 could be advantageous for antibody therapy against CD133-expressing colon cancers.</p>

An affinity tag system requires both high affinity and specificity. The RAP tag epitope DMVNPGLEDRIE, derived from rat podoplanin (PDPN), is specifically recognized by PMab-2 monoclonal antibodies in rats. Here, we demonstrated that high levels of PMab-2 can be produced in Nicotiana benthamiana and plant-derived PMab-2 possesses similar activity to CHO-derived PMab-2, and the RAP tag presents a useful tagging system for detecting and purifying proteins from plant cells. The heavy chain of PMab-2 fused with KDEL, an endoplasmic reticulum retention sequence, and the light chain of the antibody were introduced into N. benthamiana by agroinfiltration. The expression of PMab-2 peaked 4 days after agroinfiltration, and approximately 0.3 mg/g fresh weight of the antibody was accumulated. After purification, the plant-derived PMab-2 successfully recognized rat PDPN expressed in CHO-K1 cells and exhibited almost the same binding activity as CHO-derived PMab-2. The RAP-tagged proteins expressed in plant cells were specifically recognized by PMab-2. These results indicate that PMab-2 can accumulate at high levels in N. benthamiana and is easily purified and that the RAP tagging system presents a useful tool for detecting and purifying proteins of interest in plant cells.

Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on lymphocytes. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. In our previous studies, we have developed anti-bovine PD-L1 monoclonal antibodies (mAbs) and reported that the PD-1/PD-L1 pathway was closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Furthermore, we found that blocking antibodies that target PD-1 and PD-L1 restore T-cell functions and could be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway for chronic equine diseases, including tumors, remains unclear. In this study, we identified cDNA sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 mAbs against EqPD-L1 using in vitro assays. In addition, we evaluated the expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). The amino acid sequences of EqPD-1 and EqPD-L1 share a considerable identity and similarity with homologs from non-primate species. Two clones of the anti-bovine PD-L1 mAbs recognized EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine PD-1/PD-L1. Of note, immunohistochemistry confirmed the PD-L1 expression in EMM tumor tissues. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These findings showed that our anti-PD-L1 mAbs would be useful for analyzing the equine PD-1/PD-L1 pathway. Further research is warranted to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horses.

Many strategies have been tried to produce monoclonal antibodies (mAbs); however, there have been several problems about focusing on molecular targets and screening methods. For instance, the high tumor/normal ratio of antigen expression using DNA microarray has been thought to be important when we determine the molecular targets for antibody-drug. Although many antigens are expressed highly in tumors, those antigens have been removed from the candidates of antibody-drug targets because they were also expressed in normal tissues. We recently established a novel technology to produce a cancer-specific monoclonal antibody (CasMab). The post-translational difference such as glycans can be utilized to produce the CasMab, although the protein possesses the same amino acid sequence in both cancer and normal cells. We have already produced CasMabs against several glycoproteins such as podoplanin, which is expressed in both cancer and normal cells. Those CasMabs possess antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) in vitro and anti-tumor effect in xenograft models in vivo. In conclusion, the CasMab technology is the platform to develop cancer-specific mAbs, which could attack only cancer cells without side effects.

Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E2 (PGE2) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE2 in immune dysfunction and the relationship between PGE2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE2 and the PD-1/PD-L1 pathway.

Podoplanin (PDPN) is a mucin-type membrane glycoprotein, and possesses three platelet aggregation-stimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 at the N-terminus of PDPN, and one or two PLAG-like domains (PLDs) in the middle of PDPN. PDPN is expressed on normal tissues, such as podocytes of the kidney and type I alveolar cells of the lung, and is also overexpressed in numerous malignant cancers. Previously, we reported a novel anti-alpaca podoplanin (aPDPN) monoclonal antibody (mAb), PMab-225, using Cell-Based Immunization and Screening (CBIS) method. PMab-225 specifically detected aPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/aPDPN) cells using flow cytometry and western blotting, and strongly stained alpaca tissues such as lung type I alveolar cells by immunohistochemistry. However, the specific binding epitope of aPDPN for PMab-225 remains unclear. Thus, in this study, a series of deletion or point mutations of aPDPN were utilized for investigating the binding epitope of PMab-225 using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-225 epitope might exist between 80 amino acid (aa) and 85 aa of aPDPN. Furthermore, the analysis of point mutants demonstrated that Thr84 of aPDPN, which exists in PLD, could be included in the critical epitope of PMab-225.

The highly O-glycosylated membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in several malignant cancers, such as oral cancer, lung cancer, germinal neoplasia, mesothelioma, and brain tumor. The expression of PDPN is strongly associated with cancer progression and poor prognosis. PDPN possesses three tandem repeats of platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) and PLAG-like domain (PLD), and binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation. We have previously established a novel anti-Tasmanian devil PDPN (tasPDPN) monoclonal antibody (mAb), PMab-233, which specifically detects tasPDPN using flow cytometry, Western blot, and immunohistochemical analyses. However, the specific binding epitope of tasPDPN for PMab-233 remains to be clarified. Herein, a series of deletion or point mutants of tasPDPN were utilized for investigating the binding epitopes of PMab-233 using flow cytometry. The findings of this study demonstrated that Asp30, Thr33, and Thr34 of tasPDPN, which are located in PLAG1, are responsible for the binding of PMab-233 to tasPDPN.

Podoplanin (PDPN), which is a mucin-type membrane glycoprotein, is expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also overexpressed in several malignant cancers, and its expression is associated with cancer progression and poor prognosis. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain (PLD), which binds to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antihorse PDPN (horPDPN) monoclonal antibody (mAb), PMab-219, using Cell-Based Immunization and Screening (CBIS) method. PMab-219 specifically detected horPDPN-overexpressed Chinese hamster ovary (CHO)-K1 (CHO/horPDPN) cells and FHK-TcL3.1, a horse kidney cell line, using flow cytometry. In addition, PMab-219 strongly stained horse tissues such as renal podocytes or lymphatic endothelial cells by immunohistochemistry. However, the specific binding epitope of PMab-219 for horPDPN remains to be clarified. In this study, a series of deletion mutants or point mutants of horPDPN were produced for analyzing the PMab-219 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-219 epitope exists between 55th amino acid (aa) and 60th aa of horPDPN. Furthermore, the analysis of point mutants demonstrated that the critical epitope of PMab-219, which was developed by CBIS method, could include Val59, Arg61, Ser62, and Thr63 of horPDPN, indicating that PMab-219 epitope is independent of PLAG domain or PLD of horPDPN.

Podoplanin (PDPN)/T1alpha/Aggrus, a small mucin-type transmembrane glycoprotein, has been shown to be expressed on lymphatic endothelial cells and epithelial cells of many organs. PDPN is also upregulated in many cancers, and is involved in cancer metastasis and malignant progression. Human PDPN possesses three platelet aggregation-stimulating (PLAG) domains and the PLAG-like domain, which bind to C-type lectin-like receptor-2 (CLEC-2). Previously, we reported a novel antipig PDPN (pPDPN) monoclonal antibody (PMab-210) using Cell-Based Immunization and Screening (CBIS) method. PMab-210 specifically detected pPDPN-overexpressed Chinese hamster ovary (CHO)-K1 cells by flow cytometry and Western blot analysis. Immunohistochemical analyses demonstrated that PMab-210 stained pulmonary type I alveolar cells strongly and renal corpuscles weakly in pig or microminipig. However, the specific binding epitope of PMab-210 for pPDPN could not be determined by enzyme-linked immunosorbent assay using a series of pPDPN peptides. In this study, deletion mutants or point mutants of pPDPN were produced for analyzing the PMab-210 epitope using flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-210 epitope exists between 45th amino acid (aa) and 50th aa of pPDPN. In addition, the analysis of point mutants demonstrated that the critical epitope of PMab-210 could include Glu47, Asp48, Tyr49, Thr50, and Val51 of pPDPN, indicating that PMab-210 epitope is located in PLAG3 domain of pPDPN.

Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.

Coxsackievirus and adenovirus receptor (CAR) is a single-pass transmembrane protein that is associated with adenoviral infection. CAR is involved in the formation of epithelial tight junctions and promotes tumor growth in some cancers. Previously, we developed mouse monoclonal antibodies against human CAR and found that one, mu6G10A, significantly inhibited tumor growth in xenografts of human cancer cells. Herein, we generated and characterized a mouse-human chimeric anti-CAR antibody (ch6G10A) from mu6G10A. ch6G10A had binding activity, inducing antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, and in vivo anti-tumor activity against CAR-expressing prostate cancer DU-145 cells. In addition, cancer tissue array analysis confirmed that CAR is highly expressed in neuroendocrine lung cancers including small cell lung cancer, and treatment with ch6G10A effectively inhibited in vivo subcutaneous tumor growth of NCI-H69 small cell lung cancer cells in nude mice. Moreover, treatment with mu6G10A effectively inhibited both in vivo orthotopic tumor growth and distant metastatic formation in mouse xenograft models of a highly metastatic subline of human small cell lung cancer DMS273 cells. These results suggest that targeting therapy to CAR with a therapeutic antibody might be effective against several cancer types including small cell lung cancer.

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 on lymphatic endothelial cells, podocytes of the kidneys, and type I alveolar cells of the lungs. PDPN, a platelet aggregation-inducing factor, comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported a mouse antipig PDPN (pPDPN) monoclonal antibody (mAb) clone, PMab-213, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-213 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-213, which was developed by CBIS method, remains to be elucidated. Therefore, this study aimed to investigate the epitope of PMab-213 using enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemical analyses. The results revealed that the critical epitopes of PMab-213 are Pro53, Arg54, Arg56, and Tyr60 of pPDPN.

Podoplanin (PDPN)/T1alpha is a type I transmembrane sialoglycoprotein, which is expressed on podocytes of the kidneys and type I alveolar cells of the lungs. PDPN is also known as Aggrus, a platelet aggregation-inducing factor, which comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains (PLDs) in the middle of the PDPN protein. We have previously established a mouse anti-bear PDPN (bPDPN) monoclonal antibody (mAb) clone, PMab-247 using the Cell-Based Immunization and Screening (CBIS) method. PMab-247 is very useful in flow cytometry, Western blotting, and immunohistochemical (IHC) analyses; however, the binding epitope of PMab-247 has not been elucidated. In this study, we aimed to investigate the epitope of PMab-247 using enzyme-linked immunosorbent assay and IHC analyses. The results revealed that the critical epitopes of PMab-247 are Asp76, Arg78, Glu80, and Arg82 of bPDPN. The Glu80 and Arg82 are included in PLD of bPDPN. The findings of our study can be applied to the production of more functional anti-bPDPN mAbs.

Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.

Podoplanin (PDPN) is utilized as a specific marker of type I alveolar cells of lung and lymphatic endothelial cells of every tissue. Therefore, sensitive and specific monoclonal antibodies detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for Western blot, flow cytometry, and immunohistochemical analyses. In this study, immunohistochemical analyses showed that PMab-247 strongly detected bPDPN, which is expressed in type I alveolar cells and lymphatic endothelial cells of bear lung and podocytes of bear kidney. These findings suggest that PMab-247 could be useful for pathophysiological analyses using immunohistochemistry.

The diacylglycerol kinases (DGKs) catalyze the phosphorylation of the cell membrane lipid diacylglycerol (DG), which is important in lipid biochemistry and signal transduction into phosphatidic acid. DG-mediated signal transduction downstream of the T cell receptor has been reported to be terminated by DGKζ, 1 of 10 DGK isoforms in most cases. We previously established an anti-DGKζ monoclonal antibody (mAb) DzMab-1 (rat IgG1, kappa), which reacts with both mouse DGKζ and human DGKζ (hDGKζ). In this study, we characterized the binding epitope of DzMab-1 using Western blotting, and found that Met1 and Pro3 residues of hDGKζ are important for facilitating DzMab-1 binding to hDGKζ. Furthermore, DzMab-1 was shown to be useful for immunohistochemical analyses for formalin-fixed paraffin-embedded HeLa cells. These findings could be applied for the production of more functional anti-hDGKζ mAbs.

Diacylglycerol kinase (DGK) is an enzyme that converts diacylglycerol (DG) to phosphatidic acid (PA). As both DG and PA serve as lipidic second messengers, DGK plays a pivotal role in controlling the balance of two signaling pathways mediated by DG and PA in cellular functions. DGKζ, one member of the mammalian DGK family, is reported to contain a nuclear localization signal, which suggests its functional role in the nucleus. Previously, morphological studies using tagged expression vectors and immunostaining of rat tissues or cells have revealed that DGKζ localizes mainly to the nucleus. However, a limited number of studies reported the detailed localization of native protein of DGKζ in human tissues and cells. In this study, we developed a novel anti-human DGKζ monoclonal antibody, DzMab-1, which is very advantageous in immunocytochemistry of human cultured cells.

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.

Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN), which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing mAbs against tasPDPN were screened using flow cytometry. One of the mAbs, PMab-233 (IgG1, kappa), specifically detected CHO/tasPDPN cells by flow cytometry and recognized tasPDPN protein by western blotting. Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.

Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies. However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing anti-aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG2b, kappa), specifically detected CHO/aPDPN cells via flow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques.

Sensitive and specific monoclonal antibodies (mAbs) against not only human but also mouse, rat, rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. However, anti-goat PDPN (gPDPN) has not been established yet. PDPN has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses; therefore, mAbs for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues are needed. Although we recently demonstrated that an anti-bovine PDPN mAb, PMab-44 cross-reacted with gPDPN, PMab-44 did not detect lymphatic endothelial cells in immunohistochemistry. In this study, we immunized mice with gPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/gPDPN) cells, and screened mAbs against gPDPN using flow cytometry. One of the mAbs, PMab-235 (IgG1, kappa), specifically detected CHO/gPDPN cells by flow cytometry. Furthermore, PMab-235 strongly detected lung type I alveolar cells, renal podocytes, and lymphatic endothelial cells of colon by immunohistochemistry. These findings suggest that PMab-235 may be useful as a lymphatic endothelial cell marker for goat tissues.

Hepatocyte growth factor (HGF) is secreted as an inactive single-chain HGF (scHGF); however, only proteolytically processed two-chain HGF (tcHGF) can activate the MET receptor. We investigated the localization of tcHGF and activated/phosphorylated MET (pMET) using a tcHGF-specific antibody. In day 16.5 mouse embryos, total HGF (scHGF + tcHGF) was mainly localized in smooth muscle cells close to, but separate from, MET-positive epithelial cells in endodermal organs, including the stomach. In the adult stomach, total HGF was localized in smooth muscle cells, and tcHGF was mainly localized in the glandular base region. Immunostaining for pMET and Lgr5-driven green fluorescent protein (GFP) indicated that pMET localization overlapped with Lgr5+ gastric stem cells. HGF promoted organoid formation similar to EGF, indicating the potential for HGF to promote the survival and growth of gastric stem cells. pMET and tcHGF localizations changed during regeneration following gastric injury. These results indicate that MET is constantly activated in gastric stem cells and that the localization of pMET differs from the primary localization of precursor HGF but has a close relationship to tcHGF. Our results suggest the importance of the microenvironmental generation of tcHGF in the regulation of development, regeneration, and stem cell behavior.

Activation of hepatocyte growth factor (HGF) by proteolytic processing is triggered in cancer microenvironments, and subsequent signaling through the MET receptor is involved in cancer progression. However, the structure of HGF remains elusive, and few small/medium-sized molecules can modulate HGF. Here, we identified HiP-8, a macrocyclic peptide consisting of 12 amino acids, which selectively recognizes active HGF. Biochemical analysis and real-time single-molecule imaging by high-speed atomic force microscopy demonstrated that HiP-8 restricted the dynamic domains of HGF into static closed conformations, resulting in allosteric inhibition. Positron emission tomography using HiP-8 as a radiotracer enabled noninvasive visualization and simultaneous inhibition of HGF-MET activation status in tumors in a mouse model. Our results illustrate the conformational change in proteolytic activation of HGF and its detection and inhibition by a macrocyclic peptide, which may be useful for diagnosis and treatment of cancers.

Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolar cells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for immunohistochemical analyses for cetacean tissues.

Podoplanin (PDPN) has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses. Therefore, sensitive and specific monoclonal antibodies (mAbs) targeting PDPN are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues. Recently, anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse were established in our studies. However, anticetacean (whale) PDPN (wPDPN) has not been established yet. In this study, we immunized mice with wPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/wPDPN) cells, and screened mAbs against wPDPN using flow cytometry. One of the mAbs, PMab-237 (IgG1, kappa), specifically detected CHO/wPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-237 for the functional analyses of wPDPN.

Podoplanin is a type I transmembrane sialomucin-like glycoprotein that is highly expressed in malignant mesothelioma. The rat-human chimeric antibody NZ-12 has high affinity for human podoplanin and antibody-dependent cellular cytotoxicity and is applicable for radioimmunotherapy (RIT) to enhance the antitumor effect. In the present study, we evaluated the in vivo and in vitro properties of radiolabeled NZ-12 and the antitumor effect of RIT with 90 Y-labeled NZ-12 in an NCI-H226 (H226) malignant mesothelioma xenograft mouse model. 111 In-labeled NZ-12 bound specifically to H226 cells with high affinity, and accumulation was high in H226 tumors but low in major organs. RIT with 90 Y-labeled NZ-12 significantly suppressed tumor growth and prolonged survival without body weight loss and obvious adverse effects. Higher podoplanin expression levels were observed in human mesothelioma specimens, suggesting higher tumor accumulation of 90 Y-labeled NZ-12 in patients compared with the H226 tumor xenografts. Our findings suggest that 90 Y-labeled NZ-12 is a promising RIT agent as a new therapeutic option for malignant mesothelioma that warrants further clinical studies to evaluate the dosimetry and efficacy in patients.

Diacylglycerol kinase (DGK) converts diacylglycerol (DG) into phosphatidic acid (PA). DGKα, 1 of the 10 DGK isozymes, is involved in T cell function. In the present study, we describe a specific monoclonal antibody DaMab-8 (mouse IgG1, kappa) against DGKα, which is extremely useful for performing immunohistochemical analysis for T cells in oropharyngeal squamous cell carcinomas. Furthermore, we characterized the binding epitope of DaMab-8 using Western blotting and found that the sites Asn610, Leu611, Trp612, Gly613, Asp614, His619, Tyr623, and Gly624 of DGKα are important for facilitating the DaMab-8 binding to the DGKα protein. Thus, DaMab-8 could be advantageous for immunohistochemical analyses toward clarifying the distribution of DGKα-expressing T cells in every pathophysiological tissue.

Podoplanin (PDPN) is expressed on podocytes of the kidneys, type I alveolar cells of the lungs, and lymphatic endothelial cells. PDPN comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously reported on an anti-tiger PDPN (tigPDPN) monoclonal antibody (mAb), PMab-231, which was developed using the Cell-Based Immunization and Screening (CBIS) method. PMab-231 is very useful in flow cytometry, Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-231 remains to be elucidated. This study aimed to investigate the epitopes of PMab-231, which was developed by CBIS method, using enzyme-linked immunosorbent assay. The results revealed that the critical epitopes of PMab-231 are Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35, Gly36, and Glu38 of tigPDPN, which is corresponding to PLAG1/2. The findings of our study can be applied to the production of more functional anti-tigPDPN mAbs.

Diacylglycerol kinase (DGK) converts diacylglycerol (DG) into phosphatidic acid (PA). Both DG and PA serve as lipidic second messengers; therefore, DGK plays a critical role in regulating the balance of two signaling pathways mediated by DG and PA in cellular functions. DGK gamma (DGKγ), 1 of the 10 mammalian DGK isozymes, has been reported to be involved in membrane traffic, mast cell function, and leukemic cell differentiation. We previously developed a novel anti-DGKγ monoclonal antibody (mAb), DgMab-6, which is very useful in immunocytochemistry of human cultured cells. In this study, we characterized the binding epitope of DgMab-6 using Western blotting, and Glu12 is important for facilitating the DgMab-6 binding to the DGKγ protein. These results might lead to further development for sensitive and specific mAbs against DGKγ.

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.

An antibody fragment that recognizes the tertiary structure of a target protein with high affinity can be utilized as a crystallization chaperone. Difficulties in establishing conformation-specific antibodies, however, limit the applicability of antibody fragment-assisted crystallization. Here, we attempted to establish an alternative method to promote the crystallization of target proteins using an already established anti-tag antibody. The monoclonal antibody NZ-1 recognizes the PA tag with an extremely high affinity. It was also established that the PA tag is accommodated in the antigen-binding pocket in a bent conformation, compatible with an insertion into loop regions on the target. We, therefore, explored the application of NZ-1 Fab as a crystallization chaperone that complexes with a target protein displaying a PA tag. Specifically, we inserted the PA tag into the β-hairpins of the PDZ tandem fragment of a bacterial Site-2 protease. We crystallized the PA-inserted PDZ tandem mutants with the NZ-1 Fab and solved the co-crystal structure to analyze their interaction modes. Although the initial insertion designs produced only moderate-resolution structures, eliminating the solvent-accessible space between the NZ-1 Fab and target PDZ tandem improved the diffraction qualities remarkably. Our results demonstrate that the NZ-1-PA system efficiently promotes crystallization of the target protein. The present work also suggests that β-hairpins are suitable sites for the PA insertion because the PA tag contains a Pro-Gly sequence with a propensity for a β-turn conformation.

Horse podoplanin (horPDPN), a type I transmembrane sialoglycoprotein, is expressed on the podocytes of the kidneys, alveolar type I cells of the lungs, and lymphatic endothelial cells. PDPN is a platelet aggregation-inducing factor, and it primarily possesses three platelet aggregation-stimulating (PLAG) domains, that is, PLAG1, PLAG2, and PLAG3, at the N-terminus and several PLAG-like domains. In a previous study, we reported on a mouse anti-horPDPN monoclonal antibody (mAb) clone, PMab-202. Although the effectiveness of PMab-202 in flow cytometry and Western blotting is known, its exact binding epitope remains unknown to date. In this study, enzyme-linked immunosorbent assay and flow cytometry were used to identify the epitope of PMab-202. We found that the critical epitopes of PMab-202 include Lys64, Thr66, and Phe70 of horPDPN. We believe that our findings can be applied in the production of more functional anti-horPDPN mAbs.

Cancer stem cells contribute to tumorigenesis, metastasis, recurrence, and chemoresistance. CD133/prominin-1-a pentaspan membrane glycoprotein-has been used as a stem cell biomarker for the isolation of stem-like cells from a variety of normal and pathological tissues. In our previous studies, we developed several anti-CD133 monoclonal antibodies using Cell-Based Immunization and Screening (CBIS) methods, followed by characterization of their efficacy by flow cytometry, western blotting, and immunohistochemical analyses. One of the 100 clones, CMab-43 (IgG2a, kappa), demonstrated a sensitive and specific reaction against colon cancer cells. This study aimed to investigate the antitumor activity of CMab-43. Caco-2 cells (human colon cancer cell line) were subcutaneously implanted into the flanks of nude mice. CMab-43 and control mouse IgG were injected three times into the peritoneal cavity of mice. Tumor formation was observed in the control and CMab-43-treated mice of Caco-2 xenograft models. CMab-43 significantly reduced tumor development of Caco-2 xenograft in comparison with the control mouse IgG on days 12, 14, and 17. Our results cumulatively suggest that CMab-43 is useful for antibody therapy against CD133-expressing colon cancers.

Podoplanin (PDPN) is a type I transmembrane heavily glycosylated sialoglycoprotein that is expressed in normal tissues such as pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells. PDPN overexpression in cancerous tissue is associated with hematogenous metastasis through interactions with the C-type lectin-like receptor 2 (CLEC-2). Previously, we have reported the development of a mouse monoclonal antibody (mAb), PMab-38 (IgG1, kappa) against dog PDPN (dPDPN). PMab-38 was found to strongly react with canine squamous cell carcinomas (SCCs) and melanomas; however, it showed no reaction with lymphatic endothelial cells. Recently, we have developed and produced the mouse-canine mAb of subclass B, P38B that showed antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against Chinese hamster ovary (CHO)/dPDPN cells. In the present study, we investigated the antitumor activity using mouse xenograft model. To induce ADCC activity by P38B, canine mononuclear cells were injected surrounding the tumors in a xenograft model. It was demonstrated that P38B exerted antitumor activity against the mouse xenograft model using CHO/dPDPN. These results suggest that P38B is useful for antibody therapy against dPDPN-expressing canine SCCs and melanomas.

Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol (DG) to phosphatidic acid (PA). Both DG and PA serve as signaling molecules; therefore, DGK functions as a key enzyme between DG- and PA-mediated signaling. DGKα, one of the 10 DGK isozymes, is involved in T cell function and has been shown to localize in the cytoplasm and nucleus. Furthermore, DGKα translocates to the plasma membrane in response to T cell receptor stimulation. Recently, we developed a specific monoclonal antibody (mAb), DaMab-2 (mouse IgG1, kappa), against DGKα. DaMab-2 is very useful in immunocytochemical analysis using HeLa cells. In this study, we characterized the binding epitope of DaMab-2 using Western blot and revealed that Cys246, Lys249, Pro252, and Cys253 of DGKα are important for DaMab-2 binding to the DGKα protein. Our findings can be applied for the production of more functional anti-DGKα mAbs.

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 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 (IgG2b, kappa), could specifically detect CHO/pPDPN cells through flow cytometry and detect pPDPN through western blot analysis. KD of PMab-213 for CHO/pPDPN was determined to be 2.1 × 10-9 M, indicating a high affinity for CHO/pPDPN. Furthermore, PMab-213 strongly stained lymphatic endothelial cells, renal podocytes, and type I lung alveolar cells through immunohistochemistry. PMab-213 is expected to be useful in investigating the function of pPDPN.

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.

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.

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.

Bovine leukemia virus (BLV) is a retrovirus that infects B cells in cattle and causes bovine leukosis after a long latent period. Progressive exhaustion of T cell functions is considered to facilitate disease progression of BLV infection. Programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) are immunoinhibitory receptors that contribute to T-cell exhaustion caused by BLV infection in cattle. However, it is unclear whether the cooperation of PD-1 and LAG-3 accelerates disease progression of BLV infection. In this study, multi-color flow cytometric analyses of PD-1- and LAG-3-expressing T cells were performed in BLV-infected cattle at different stages of the disease. The frequencies of PD-1+LAG-3+ heavily exhausted T cells among CD4+ and CD8+ T cells was higher in the blood of cattle with B-cell lymphoma over that of BLV-uninfected and BLV-infected cattle without lymphoma. In addition, blockade assays of peripheral blood mononuclear cells were performed to examine whether inhibition of the interactions between PD-1 and LAG-3 and their ligands by blocking antibodies could restore T-cell function during BLV infection. Single or dual blockade of the PD-1 and LAG-3 pathways reactivated the production of Th1 cytokines, interferon-γ and tumor necrosis factor-α, from BLV-specific T cells of the infected cattle. Taken together, these results indicate that PD-1 and LAG-3 cooperatively mediate the functional exhaustion of CD4+ and CD8+ T cells and are associated with the development of B-cell lymphoma in BLV-infected cattle.

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, is a bovine chronic infection that is endemic in Japan and many other countries. The expression of immunoinhibitory molecules is upregulated in cattle with Johne's disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E2 (PGE2) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE2 were performed to investigate the immunosuppressive effect of PGE2 during Johne's disease. In vitro PGE2 treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 and programmed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) from healthy cattle. PGE2 was upregulated in sera and intestinal lesions of cattle with Johne's disease. In vitro stimulation with Johnin purified protein derivative (J-PPD) induced cyclooxygenase-2 (COX-2) transcription, PGE2 production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with M. avium subsp. paratuberculosis Therefore, Johnin-specific Th1 responses could be limited by the PGE2 pathway in cattle. In contrast, downregulation of PGE2 with a COX-2 inhibitor promoted J-PPD-stimulated CD8+ T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade induced J-PPD-stimulated CD8+ T-cell proliferation and interferon gamma production in vitro Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies enhanced J-PPD-stimulated CD8+ T-cell proliferation in vitro, suggesting that the blockade of both pathways is a potential therapeutic strategy to control Johne's disease. The effects of COX-2 inhibition warrant further study as a novel treatment of Johne's disease.

Podoplanin is distinctively overexpressed in oral squamous cell carcinoma than oral benign neoplasms and plays a crucial role in the pathogenesis and metastasis of oral squamous cell carcinoma but its diagnostic application is quite limited. Here, we report a new near-infrared fluorescence imaging method using an indocyanine green (ICG)-labeled anti-podoplanin antibody and a desktop/a handheld ICG detection device for the visualization of oral squamous cell carcinoma-xenografted tumors in nude mice. Both near-infrared imaging methods using a desktop (in vivo imaging system: IVIS) and a handheld device (photodynamic eye: PDE) successfully detected oral squamous cell carcinoma tumors in nude mice in a podoplanin expression-dependent manner with comparable sensitivity. Of these 2 devices, only near-infrared imaging methods using a handheld device visualized oral squamous cell carcinoma xenografts in mice in real time. Furthermore, near-infrared imaging methods using the handheld device (PDE) could detect smaller podoplanin-positive oral squamous cell carcinoma tumors than a non-near-infrared, autofluorescence-based imaging method. Based on these results, a near-infrared imaging method using an ICG-labeled anti-podoplanin antibody and a handheld detection device (PDE) allows the sensitive, semiquantitative, and real-time imaging of oral squamous cell carcinoma tumors and therefore represents a useful tool for the detection and subsequent monitoring of malignant oral neoplasms in both preclinical and some clinical settings.

Telomerase reverse transcriptase (TERT) is important for the biology of diffuse gliomas. TERT promoter mutations are selectively observed among 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene- (IDH-) wildtype glioblastoma (GBM). However, TERT transcripts range widely in various cancers including gliomas, and TERT protein expression has been rarely investigated thus far. It would be thus critical to examine the expression level of TERT in tumors in addition to its mutational status, and sensitive and specific methods are urgently needed to examine TERT protein expression for the assessment of TERT biology in gliomas. Using our newly developed TERT-specific monoclonal antibody (TMab-6) applicable to human tissue, we found an unexpected increase in TERT expression in TERT-wildtype as well as TERT-mutated gliomas and in tumor vasculature. This is the first extensive analysis on the expression of TERT immunoreactivity in human glioma tissue, suggesting that TERT protein expression may be regulated by several mechanisms in addition to its promoter mutation.

INTRODUCTION: Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway in immunosuppression in bovine mycoplasmosis. METHODS: In the initial experiments, we used enzyme-linked immunosorbent assay to measure interferon-γ (IFN-γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis. RESULTS: Expectedly, IFN-γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD-1+ CD4+ and PD-L1+ CD14+ cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD-1+ CD4+ and PD-1+ CD8+ T cells were negatively correlated with IFN-γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD-1/PD-L1 pathway in vitro by anti-bovine PD-1- and anti-bovine PD-L1 antibodies significantly upregulated the production of IFN-γ from anti-mycoplasma-specific cells. CONCLUSIONS: These results suggest that the PD-1/PD-L1 pathway could be involved in immune exhaustion of bovine mycoplasma-specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways.

Ectodomain shedding (shedding) is a post-translational modification, which liberates the extracellular domain of membrane proteins through juxtamembrane processing executed mainly by the ADAM (a disintegrin and metalloprotease) family of metalloproteases. Because shedding alters characteristics of cells in a rapid and irreversible manner, it should be strictly regulated. However, the molecular mechanisms determining membrane protein susceptibility to shedding (shedding susceptibility) are largely unknown. Here we report that alternative splicing can give rise to both shedding-susceptible and shedding-resistant CADM1 (cell adhesion molecule 1) variant proteins. We further show that O-glycans adjacent to the shedding cleavage site interfere with CADM1 shedding, and the only 33-bp alternative exon confers shedding susceptibility to CADM1 by inserting five non-glycosylatable amino acids between interfering O-glycans and the shedding cleavage site. These results demonstrate that shedding susceptibility of membrane protein can be determined at two different levels of its biosynthesis pathway, alternative splicing and O-glycosylation.

HGF-Met signaling contributes to various biological events by controlling cell migration. Since the abnormal activation of Met receptor causes cancer progression, inhibitors such as neutralizing antibodies are regarded as promising therapeutics. HGF is secreted as a single-chain (sc) precursor and is processed by extracellular proteases to generate disulfide-bonded two-chain (tc) HGF. Although this proteolytic processing of HGF is necessary for its biological activity, exactly how the proteolysis leads to the conversion of HGF to the active form is still unclar due to the lack of structural information. In order to gain insights about this point, we generated 6 antibodies against HGF. All antibodies recognized different epitopes on the native HGF protein and showed distinct effects when tested in a cell-based HGF-Met signaling assay. They included one antibody (t1E4) that strongly blocks Met activation by tcHGF, as well as one antibody (t8E4) exclusively recognizing the active tcHGF but not inactive scHGF. Thus, a panel of anti-HGF antibodies suitable for probing the structural mechanism of HGF activation were obtained.

Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanin-induced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 and NZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73% (11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against podoplanin-positive MPM cells mediated by rat NK (CD161a(+)) cells, but not murine splenocytes or human mononuclear cells. Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanin-transfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8 preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated by human NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin with therapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM.