ABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , Ursidae , Cricetinae , Mice , Animals , Humans , Cricetulus , CHO Cells , Endothelial Cells/metabolism , Membrane Glycoproteins , Antibody Specificity , Transcription FactorsABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , Macrophages , Animals , Cricetinae , Mice , Rats , Antibodies, Monoclonal/pharmacology , CHO Cells , CricetulusABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , Giraffes , Cricetinae , Mice , Animals , Immunohistochemistry , Epitopes , Cricetulus , Membrane Glycoproteins , Antibody Specificity , CHO Cells , Transcription FactorsABSTRACT
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. AntiEpCAM monoclonal antibodies (mAbs) have been developed and have previously demonstrated promising outcomes in several clinical trials. An antiEpCAM mAb, EpMab37 (mouse IgG1, kappa) was previously developed by the authors, using the cellbased immunization and screening method. In the present study, a defucosylated version of antiEpCAM mAb (EpMab37mG2af) was generated to evaluate the antitumor activity against EpCAMpositive cells. EpMab37mG2af recognized EpCAMoverexpressing CHOK1 (CHO/EpCAM) cells with a moderate bindingaffinity [dissociation constant (KD)=2.2x108 M] using flow cytometry. EpMab37mG2af exhibited potent antibodydependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC) for CHO/EpCAM cells by murine splenocytes and complements, respectively. Furthermore, the administration of EpMab37mG2af significantly suppressed CHO/EpCAM xenograft tumor development compared with the control mouse IgG. EpMab37mG2af also exhibited a moderate bindingaffinity (KD=1.5x108 M) and high ADCC and CDC activities for a colorectal cancer cell line (Caco2 cells). The administration of EpMab37mG2af to Caco2 tumorbearing mice significantly suppressed tumor development compared with the control. By contrast, EpMab37mG2af never suppressed the xenograft tumor growth of Caco2 cells in which EpCAM was knocked out. On the whole, these results indicate that EpMab37mG2af may exert antitumor activities against EpCAMpositive cancers and may thus be a promising therapeutic regimen for colorectal cancer.
Subject(s)
Antibodies, Monoclonal , Colorectal Neoplasms , Cricetinae , Humans , Animals , Mice , Antibodies, Monoclonal/therapeutic use , Caco-2 Cells , Heterografts , Epithelial Cell Adhesion Molecule , Cricetulus , Colorectal Neoplasms/drug therapy , Immunoglobulin G , Xenograft Model Antitumor Assays , Cell Line, TumorABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , T-Lymphocytes , Mice , Animals , Cricetinae , CHO Cells , Cricetulus , ImmunizationABSTRACT
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.
Subject(s)
Alanine , Antibodies, Monoclonal , Animals , Rats , Epitope Mapping/methods , Receptors, CXCR6 , Epitopes , Enzyme-Linked Immunosorbent Assay , PeptidesABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , Neoplasms , Animals , CHO Cells , Cricetinae , Cricetulus , Immunohistochemistry , Immunosuppressive Agents , Mice , Rats , Receptors, CCR8 , Receptors, Chemokine/metabolismABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , Osteosarcoma , Animals , Antibody-Dependent Cell Cytotoxicity , Cell Line, Tumor , Cricetinae , Dogs , Heterografts , Humans , Mice , Osteosarcoma/drug therapy , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , ErbB Receptors , Animals , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Cricetinae , Dogs , Heterografts , Mice , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Antibodies, Monoclonal , Antineoplastic Agents , Epitope Mapping , Epitopes/genetics , ErbB Receptors/geneticsABSTRACT
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.
Subject(s)
Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/immunology , Receptors, CCR/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Anti-Idiotypic/pharmacology , Antibodies, Monoclonal/pharmacology , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/therapy , CHO Cells , Colitis/immunology , Colitis/therapy , Cricetinae , Cricetulus , Diabetes Mellitus, Type 2/immunology , Diabetes Mellitus, Type 2/therapy , Enterocytes/immunology , Epitopes/immunology , Flow Cytometry , Humans , Immunoglobulin G/immunology , Mice , Receptors, CCR/antagonists & inhibitors , T-Lymphocytes/drug effectsABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Membrane Glycoproteins/immunology , Sea Lions/immunology , Animals , Antibodies, Monoclonal/biosynthesis , CHO Cells , Cricetinae , Cricetulus , Epitope Mapping , Flow Cytometry , Humans , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/genetics , Mice , Podocytes/immunology , Sea Lions/geneticsABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Immune Checkpoint Inhibitors/immunology , Neoplasms/therapy , Receptors, Immunologic/immunology , Animals , Antibodies, Monoclonal/pharmacology , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , CHO Cells , CTLA-4 Antigen/antagonists & inhibitors , CTLA-4 Antigen/immunology , Cricetinae , Cricetulus , Flow Cytometry , Gene Expression Regulation/immunology , Humans , Immune Checkpoint Inhibitors/pharmacology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Mice , Neoplasms/immunology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Receptors, Immunologic/antagonists & inhibitors , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Receptors, CCR8/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/pharmacology , CHO Cells , Cricetinae , Cricetulus , Flow Cytometry , Gene Expression Regulation/immunology , Humans , Mice , Rats , Receptors, CCR8/antagonists & inhibitorsABSTRACT
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.
Subject(s)
Antibodies, Anti-Idiotypic/pharmacology , Antibodies, Monoclonal/pharmacology , HIV Infections/immunology , Receptors, CCR3/immunology , Animals , Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/immunology , CHO Cells , Cricetulus , Flow Cytometry , HIV Infections/therapy , HIV Infections/virology , Humans , Hypersensitivity/immunology , Hypersensitivity/therapy , Macrophages/immunology , Mice , Receptors, CCR3/antagonists & inhibitorsABSTRACT
The epithelial cell adhesion molecule (EpCAM) is a calciumindependent, 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 antiEpCAM monoclonal antibody (mAb), EpMab16 (IgG2a, kappa), by immunizing mice with EpCAMoverexpressing CHOK1 cells. EpMab16 specifically reacted with endogenous EpCAM in oral squamous cell carcinoma (OSCC) cell lines in flow cytometry and Western blot analyses. It exhibited a plasma membranelike stain pattern in OSCC tissues upon immunohistochemical analysis. The KD for EpMab16 in SAS and HSC2 OSCC cells were assessed via flow cytometry at 1.1x108 and 1.9x108 M, respectively, suggesting moderate binding affinity of EpMab16 for EpCAM. We then assessed whether the EpMab16 induced antibodydependent cellular cytotoxicity (ADCC) and complementdependent 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 EpMab16. In vivo experiments on OSCC xenografts revealed that EpMab16 treatment significantly reduced tumor growth compared with the control mouse IgG. These data indicated that EpMab16 could be a promising treatment option for EpCAMexpressing OSCCs.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents, Immunological/pharmacology , Epithelial Cell Adhesion Molecule/antagonists & inhibitors , Mouth Neoplasms/drug therapy , Squamous Cell Carcinoma of Head and Neck/drug therapy , Animals , Antibodies, Monoclonal/pharmacology , Antibody-Dependent Cell Cytotoxicity/drug effects , Antineoplastic Agents, Immunological/therapeutic use , Apoptosis/drug effects , CHO Cells , Cell Line, Tumor , Cricetulus , Female , Humans , Mice , Mouth Neoplasms/immunology , Mouth Neoplasms/pathology , Squamous Cell Carcinoma of Head and Neck/immunology , Squamous Cell Carcinoma of Head and Neck/pathology , Xenograft Model Antitumor AssaysABSTRACT
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 antiCD44 monoclonal antibody (mAb), C44Mab5 (IgG1, kappa) was established by immunizing mice with CD44overexpressing Chinese hamster ovary (CHO)-K1 cells. C44Mab5 recognized all CD44 isoforms, and showed high sensitivity for flow cytometry and immunohistochemical analysis in oral cancers. However, as the IgG1 subclass of C44Mab5 lacks antibodydependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC), the antitumor activity of C44Mab5 could not be determined. In the present study, we converted the mouse IgG1 subclass antibody C44Mab5 into an IgG2a subclass antibody, 5mG2a, and further produced a defucosylated version, 5mG2af, using FUT8deficient ExpiCHOS (BINDS09) cells. Defucosylation of 5mG2af was confirmed using fucosebinding lectins, such as AAL and PhoSL. The dissociation constants (KD) for 5mG2af against SAS and HSC2 oral cancer cells were determined through flow cytometry to be 2.8x1010 M and 2.6x109 M, respectively, indicating that 5mG2af possesses extremely high binding affinity. Furthermore, immunohistochemical staining using 5mG2af specifically stained the membranes of oral cancer cells. In vitro analysis demonstrated that 5mG2af showed moderate ADCC and CDC activities against SAS and HSC2 oral cancer cells. In vivo analysis revealed that 5mG2af significantly reduced tumor development in SAS and HSC2 xenografts in comparison to control mouse IgG, even after injection seven days posttumor inoculation. Collectively, these results suggest that treatment with 5mG2af may represent a useful therapy for patients with CD44expressing oral cancers.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents, Immunological/therapeutic use , Hyaluronan Receptors/antagonists & inhibitors , Mouth Neoplasms/drug therapy , Squamous Cell Carcinoma of Head and Neck/drug therapy , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/isolation & purification , Antineoplastic Agents, Immunological/pharmacology , CHO Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Cricetulus , Female , Humans , Hyaluronan Receptors/metabolism , Mice , Mouth Neoplasms/immunology , Mouth Neoplasms/pathology , Squamous Cell Carcinoma of Head and Neck/immunology , Squamous Cell Carcinoma of Head and Neck/pathology , Xenograft Model Antitumor AssaysABSTRACT
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 antiEGFR monoclonal antibody (mAb), EMab134, by immunizing mice with the ectodomain of human EGFR. EMab134 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 EMab134 was converted from IgG1 to IgG2a (134mG2a) to facilitate antibodydependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity (CDC). The dissociation constants (KDs) of EMab134 and 134mG2a against EGFRexpressing CHOK1 (CHO/EGFR) cells were determined by flow cytometry to be 3.2x109 M and 2.1x109 M, respectively; these results indicate that 134mG2a has a higher binding affinity than EMab134. The 134mG2a antibody was more sensitive than EMab134 with respect to antigen detection in oral cancer cells in both western blot analysis and immunohistochemistry applications. Analysis in vitro revealed that 134mG2a contributed to high levels of ADCC and CDC in experiments targeting CHO/EGFR, HSC2, and SAS cells. Moreover, the in vivo administration of 134mG2a significantly inhibited the development of CHO/EGFR, HSC2, and SAS mouse xenografts in comparison to the results observed in response to EMab134. Taken together, the findings of the present study demonstrate that the newlyformulated 134mG2a 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 EGFRexpressing oral cancer.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Squamous Cell/drug therapy , ErbB Receptors/metabolism , Mouth Neoplasms/drug therapy , Animals , Apoptosis/drug effects , CHO Cells , Carcinoma, Squamous Cell/metabolism , Cell Line , Cell Proliferation/drug effects , Cricetulus , Female , Immunoglobulin G/metabolism , Immunohistochemistry/methods , Mice , Mice, Inbred BALB C , Mice, Nude , Mouth Neoplasms/metabolism , Xenograft Model Antitumor Assays/methodsABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/immunology , Endothelial Cells/immunology , Epitopes/immunology , Membrane Glycoproteins/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibody Specificity/immunology , Autoantibodies/biosynthesis , Autoantibodies/immunology , CHO Cells , Cats , Cattle , Cricetinae , Cricetulus , Dogs , Epitope Mapping , Flow Cytometry , Goats , Horses/immunology , Humans , Mice , Platelet Aggregation/immunology , Podocytes/immunology , Rabbits , Rats , Sheep/immunology , Swine/immunology , TigersABSTRACT
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.