Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an importan... more 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 tha...
Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an importan... more 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 tha...
Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuz... more Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuzumab and pertuzumab, were clinically approved. We previously developed a highly sensitive and specific anti-HER2 mAb, H2Mab-139 (mouse IgG1, kappa). In this study, we produced a defucosylated IgG2a version of anti‑HER2 mAb (H2Mab-139-mG2a-f) to enhance 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 cells in vitro and exhibited potent antitumor activities in the xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and could be useful for an ant...
Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuz... more Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuzumab and pertuzumab, were clinically approved. We previously developed a highly sensitive and specific anti-HER2 mAb, H2Mab-139 (mouse IgG1, kappa). In this study, we produced a defucosylated IgG2a version of anti‑HER2 mAb (H2Mab-139-mG2a-f) to enhance 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 cells in vitro and exhibited potent antitumor activities in the xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and could be useful for an ant...
The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various huma... more 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 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 and dHER2‐positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3x10-9 M). Furthermo...
The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various huma... more 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 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 and dHER2‐positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3x10-9 M). Furthermo...
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
The CC motif chemokine receptor 3 (CCR3) is a G protein-coupled receptor activated by eotaxin-1-3... more The CC 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 (C 3 Mab-2; rat IgG 2b , kappa) using the Cell-Based Immunization and Screening method and showed that C 3 Mab-2 could detect endogenous and exogenous mCCR3 in flow cytometry. In this study, we showed that C 3 Mab-2 and its recombinant antibody (recC 3 Mab-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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
The CC motif chemokine receptor 3 (CCR3) is a G protein-coupled receptor activated by eotaxin-1-3... more The CC 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 (C 3 Mab-2; rat IgG 2b , kappa) using the Cell-Based Immunization and Screening method and showed that C 3 Mab-2 could detect endogenous and exogenous mCCR3 in flow cytometry. In this study, we showed that C 3 Mab-2 and its recombinant antibody (recC 3 Mab-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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
The CC motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells. CCR8 is also ... more The CC 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 C 8 Mab-3, rat IgG 1 , kappa) using the Cell-Based Immunization and Screening (CBIS) method. We revealed that C 8 Mab-3 and its recombinant antibody (recC 8 Mab-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, C 8 Mab-3 and recC 8 Mab-3 reacted to P388 (a mouse lymphocyte-like cell) and J774-1 (a mouse macrophage-like cell), which express endogenous mCCR8. C 8 Mab-3 also detected exogenous and endogenous mCCR8 using immunocytochemistry. These results suggest that C 8 Mab-3, developed using the CBIS method, is useful for immunocytochemistry against exogenous and endogenous mCCR8.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
The CC motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells. CCR8 is also ... more The CC 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 C 8 Mab-3, rat IgG 1 , kappa) using the Cell-Based Immunization and Screening (CBIS) method. We revealed that C 8 Mab-3 and its recombinant antibody (recC 8 Mab-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, C 8 Mab-3 and recC 8 Mab-3 reacted to P388 (a mouse lymphocyte-like cell) and J774-1 (a mouse macrophage-like cell), which express endogenous mCCR8. C 8 Mab-3 also detected exogenous and endogenous mCCR8 using immunocytochemistry. These results suggest that C 8 Mab-3, developed using the CBIS method, is useful for immunocytochemistry against exogenous and endogenous mCCR8.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, local... more 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 antihuman CCR2 (hCCR2) mAb, C 2 Mab-9 (mouse IgG 1 , kappa), which is applicable for flow cytometry and immunocytochemistry. In this study, we investigated the critical epitope of C 2 Mab-9. We conducted enzymelinked immunosorbent assay (ELISA) using several N-terminal peptides of hCCR2, and demonstrated that C 2 Mab-9 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. C 2 Mab-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 C 2 Mab-9 reaction with U937 cells in flow cytometry. These results indicate that the critical binding epitope of C 2 Mab-9 includes Phe23, Phe24, Asp25, and Tyr26.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, local... more 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 antihuman CCR2 (hCCR2) mAb, C 2 Mab-9 (mouse IgG 1 , kappa), which is applicable for flow cytometry and immunocytochemistry. In this study, we investigated the critical epitope of C 2 Mab-9. We conducted enzymelinked immunosorbent assay (ELISA) using several N-terminal peptides of hCCR2, and demonstrated that C 2 Mab-9 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. C 2 Mab-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 C 2 Mab-9 reaction with U937 cells in flow cytometry. These results indicate that the critical binding epitope of C 2 Mab-9 includes Phe23, Phe24, Asp25, and Tyr26.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immuno... more It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immunotherapy. The CC 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 C 8 Mab-1, rat IgG 2a , kappa) using the Cell-Based Immunization and Screening (CBIS) method. We showed that C 8 Mab-1 and its recombinant antibody (recC 8 Mab-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, C 8 Mab-1 and recC 8 Mab-1 specifically reacted to P388 (a mouse lymphocytelike cells) and J774-1 (a mouse macrophage-like cells), which express endogenous mCCR8, in both applications. These results suggest that C 8 Mab-1, developed using the CBIS method, is useful for flow cytometry and immunocytochemistry against exogenous and endogenous mCCR8.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immuno... more It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immunotherapy. The CC 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 C 8 Mab-1, rat IgG 2a , kappa) using the Cell-Based Immunization and Screening (CBIS) method. We showed that C 8 Mab-1 and its recombinant antibody (recC 8 Mab-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, C 8 Mab-1 and recC 8 Mab-1 specifically reacted to P388 (a mouse lymphocytelike cells) and J774-1 (a mouse macrophage-like cells), which express endogenous mCCR8, in both applications. These results suggest that C 8 Mab-1, developed using the CBIS method, is useful for flow cytometry and immunocytochemistry against exogenous and endogenous mCCR8.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. It is expr... more 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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Killer cell lectin-like receptor subfamily G member 1 (KLRG1), a type II transmembrane protein, w... more 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 IgG 1 , 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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 2021
Immune checkpoint inhibitors targeting programmed cell death-ligand 1 (PD-L1), programmed cell de... more 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 antihuman TIGIT (hTIGIT) monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. Mice were immunized with hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hTIGIT), and hybridomas were screened by flow cytometry. One of the mAbs, TgMab-2 (IgG 1 , kappa), specifically and sensitively detects hTIGIT in CHO/hTIGIT and NK cells. The dissociation constants (K D) 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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 2022
The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein. Although EGFR is phy... more 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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 2021
CC chemokine receptor 3 (CCR3), also known as CD193, belongs to class A of G protein-coupled rece... more 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, C 3 Mab-2 (rat IgG 2b , 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, C 3 Mab-2, generated by the CBIS method, can be a valuable tool for detecting mCCR3 on the surface of mouse cells.
HER3 belongs to the epidermal growth factor receptor (EGFR) family and is known to form an active... more 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), H 3 Mab-17 (IgG 2a , kappa), by immunizing mice with HER3-overexpressed CHO-K1 cells (CHO/HER3). H 3 Mab-17 was found to react specifically with endogenous HER3 in colorectal carcinoma cell lines, using flow cytometry. The K D for H 3 Mab-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 H 3 Mab-17 to HER3. Then, we assessed the H 3 Mab-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 H 3 Mab-17 had strongly induced both ADCC and CDC against Caco-2 cells. In vivo experiments on Caco-2 xenografts revealed that H 3 Mab-17 treatment significantly reduced tumor growth compared with the control mouse IgG. These data indicated that H 3 Mab-17 could be a promising treatment option for HER3-expressing colon cancers.
Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an importan... more 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 tha...
Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an importan... more 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 tha...
Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuz... more Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuzumab and pertuzumab, were clinically approved. We previously developed a highly sensitive and specific anti-HER2 mAb, H2Mab-139 (mouse IgG1, kappa). In this study, we produced a defucosylated IgG2a version of anti‑HER2 mAb (H2Mab-139-mG2a-f) to enhance 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 cells in vitro and exhibited potent antitumor activities in the xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and could be useful for an ant...
Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuz... more Two monoclonal antibodies (mAbs) against human epidermal growth factor receptor 2 (HER2), trastuzumab and pertuzumab, were clinically approved. We previously developed a highly sensitive and specific anti-HER2 mAb, H2Mab-139 (mouse IgG1, kappa). In this study, we produced a defucosylated IgG2a version of anti‑HER2 mAb (H2Mab-139-mG2a-f) to enhance 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 cells in vitro and exhibited potent antitumor activities in the xenograft models. These results indicated that H2Mab-139-mG2a-f exerts antitumor effects against HER2-positive human breast cancers and could be useful for an ant...
The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various huma... more 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 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 and dHER2‐positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3x10-9 M). Furthermo...
The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various huma... more 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 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 and dHER2‐positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3x10-9 M). Furthermo...
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
The CC motif chemokine receptor 3 (CCR3) is a G protein-coupled receptor activated by eotaxin-1-3... more The CC 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 (C 3 Mab-2; rat IgG 2b , kappa) using the Cell-Based Immunization and Screening method and showed that C 3 Mab-2 could detect endogenous and exogenous mCCR3 in flow cytometry. In this study, we showed that C 3 Mab-2 and its recombinant antibody (recC 3 Mab-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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
The CC motif chemokine receptor 3 (CCR3) is a G protein-coupled receptor activated by eotaxin-1-3... more The CC 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 (C 3 Mab-2; rat IgG 2b , kappa) using the Cell-Based Immunization and Screening method and showed that C 3 Mab-2 could detect endogenous and exogenous mCCR3 in flow cytometry. In this study, we showed that C 3 Mab-2 and its recombinant antibody (recC 3 Mab-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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
The CC motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells. CCR8 is also ... more The CC 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 C 8 Mab-3, rat IgG 1 , kappa) using the Cell-Based Immunization and Screening (CBIS) method. We revealed that C 8 Mab-3 and its recombinant antibody (recC 8 Mab-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, C 8 Mab-3 and recC 8 Mab-3 reacted to P388 (a mouse lymphocyte-like cell) and J774-1 (a mouse macrophage-like cell), which express endogenous mCCR8. C 8 Mab-3 also detected exogenous and endogenous mCCR8 using immunocytochemistry. These results suggest that C 8 Mab-3, developed using the CBIS method, is useful for immunocytochemistry against exogenous and endogenous mCCR8.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
The CC motif chemokine receptor 8 (CCR8) is highly expressed in regulatory T cells. CCR8 is also ... more The CC 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 C 8 Mab-3, rat IgG 1 , kappa) using the Cell-Based Immunization and Screening (CBIS) method. We revealed that C 8 Mab-3 and its recombinant antibody (recC 8 Mab-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, C 8 Mab-3 and recC 8 Mab-3 reacted to P388 (a mouse lymphocyte-like cell) and J774-1 (a mouse macrophage-like cell), which express endogenous mCCR8. C 8 Mab-3 also detected exogenous and endogenous mCCR8 using immunocytochemistry. These results suggest that C 8 Mab-3, developed using the CBIS method, is useful for immunocytochemistry against exogenous and endogenous mCCR8.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, local... more 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 antihuman CCR2 (hCCR2) mAb, C 2 Mab-9 (mouse IgG 1 , kappa), which is applicable for flow cytometry and immunocytochemistry. In this study, we investigated the critical epitope of C 2 Mab-9. We conducted enzymelinked immunosorbent assay (ELISA) using several N-terminal peptides of hCCR2, and demonstrated that C 2 Mab-9 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. C 2 Mab-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 C 2 Mab-9 reaction with U937 cells in flow cytometry. These results indicate that the critical binding epitope of C 2 Mab-9 includes Phe23, Phe24, Asp25, and Tyr26.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
CC chemokine receptor type-2 (CCR2) belongs to the G protein-coupled receptors superfamily, local... more 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 antihuman CCR2 (hCCR2) mAb, C 2 Mab-9 (mouse IgG 1 , kappa), which is applicable for flow cytometry and immunocytochemistry. In this study, we investigated the critical epitope of C 2 Mab-9. We conducted enzymelinked immunosorbent assay (ELISA) using several N-terminal peptides of hCCR2, and demonstrated that C 2 Mab-9 recognizes 11-29 and 21-39 amino acids of hCCR2. We further performed ELISA using 20 peptides, which include alanine substitution of hCCR2. C 2 Mab-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 C 2 Mab-9 reaction with U937 cells in flow cytometry. These results indicate that the critical binding epitope of C 2 Mab-9 includes Phe23, Phe24, Asp25, and Tyr26.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immuno... more It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immunotherapy. The CC 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 C 8 Mab-1, rat IgG 2a , kappa) using the Cell-Based Immunization and Screening (CBIS) method. We showed that C 8 Mab-1 and its recombinant antibody (recC 8 Mab-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, C 8 Mab-1 and recC 8 Mab-1 specifically reacted to P388 (a mouse lymphocytelike cells) and J774-1 (a mouse macrophage-like cells), which express endogenous mCCR8, in both applications. These results suggest that C 8 Mab-1, developed using the CBIS method, is useful for flow cytometry and immunocytochemistry against exogenous and endogenous mCCR8.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immuno... more It has been widely accepted that monoclonal antibody (mAb) is an effective tool for cancer immunotherapy. The CC 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 C 8 Mab-1, rat IgG 2a , kappa) using the Cell-Based Immunization and Screening (CBIS) method. We showed that C 8 Mab-1 and its recombinant antibody (recC 8 Mab-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, C 8 Mab-1 and recC 8 Mab-1 specifically reacted to P388 (a mouse lymphocytelike cells) and J774-1 (a mouse macrophage-like cells), which express endogenous mCCR8, in both applications. These results suggest that C 8 Mab-1, developed using the CBIS method, is useful for flow cytometry and immunocytochemistry against exogenous and endogenous mCCR8.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
CD10 is a glycosylated transmembrane protein and is known as a membrane endopeptidase. It is expr... more 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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy
Killer cell lectin-like receptor subfamily G member 1 (KLRG1), a type II transmembrane protein, w... more 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 IgG 1 , 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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 2021
Immune checkpoint inhibitors targeting programmed cell death-ligand 1 (PD-L1), programmed cell de... more 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 antihuman TIGIT (hTIGIT) monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. Mice were immunized with hTIGIT-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hTIGIT), and hybridomas were screened by flow cytometry. One of the mAbs, TgMab-2 (IgG 1 , kappa), specifically and sensitively detects hTIGIT in CHO/hTIGIT and NK cells. The dissociation constants (K D) 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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 2022
The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein. Although EGFR is phy... more 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.
Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 2021
CC chemokine receptor 3 (CCR3), also known as CD193, belongs to class A of G protein-coupled rece... more 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, C 3 Mab-2 (rat IgG 2b , 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, C 3 Mab-2, generated by the CBIS method, can be a valuable tool for detecting mCCR3 on the surface of mouse cells.
HER3 belongs to the epidermal growth factor receptor (EGFR) family and is known to form an active... more 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), H 3 Mab-17 (IgG 2a , kappa), by immunizing mice with HER3-overexpressed CHO-K1 cells (CHO/HER3). H 3 Mab-17 was found to react specifically with endogenous HER3 in colorectal carcinoma cell lines, using flow cytometry. The K D for H 3 Mab-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 H 3 Mab-17 to HER3. Then, we assessed the H 3 Mab-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 H 3 Mab-17 had strongly induced both ADCC and CDC against Caco-2 cells. In vivo experiments on Caco-2 xenografts revealed that H 3 Mab-17 treatment significantly reduced tumor growth compared with the control mouse IgG. These data indicated that H 3 Mab-17 could be a promising treatment option for HER3-expressing colon cancers.
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Papers by Yukinari Kato