Antitumor Activities in Mouse Xenograft Models of Canine Mammary Gland Tumor by Defucosylated Mouse-Dog Chimeric Anti-Epidermal Growth Factor Receptor Antibody (E134Bf).

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.

Antitumor Activities in Mouse Xenograft Models of Canine Fibroblastic Tumor by Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody.

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.

Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody (134-mG2a-f) Exerts Antitumor Activities in Mouse Xenograft Models of Canine Osteosarcoma.

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.

Safety verification of carbon-ion radiotherapy for patients with cardiac implantable electronic devices (CIEDs).

According to guidelines, carbon-ion beam therapy is considered to carry a high safety risk for patients with cardiac implantable electronic devices (CIEDs), although the actual impacts remain unclear. In this study, we investigated the safety of carbon-ion beam therapy in patients with CIEDs. Patients with CIEDs who underwent carbon-ion therapy at Gunma University Heavy Ion Medical Center between June 2010 and December 2019 were identified and investigated for abnormalities in the operation of their CIEDs, such as oversensing and resetting during irradiation, and abnormalities in operation after treatment. In addition, the risk of irradiation from carbon-ion beam therapy was evaluated by model simulations. Twenty patients (22 sites) with CIEDs were identified, 19 with pacemakers and one with an implantable cardioverter-defibrillator (ICD). Treatments were completed without any problems, except for one case in which the treatment was discontinued because of worsening of the primary disease. Monte Carlo simulation indicated that the carbon beam irradiation produced neutrons at a constant and high level in the irradiation field. Nevertheless, with the distances between the CIEDs and the irradiation fields in the analyzed cases, the quantity of neutrons at the CIEDs was lower than that within the irradiation. Although carbon-ion beam therapy can be safely administered to patients with CIEDs, it is advisable to perform the therapy with sufficient preparation and backup devices because of the risks involved.

An Anti-HER2 Monoclonal Antibody H2Mab-41 Exerts Antitumor Activities in Mouse Xenograft Model Using Dog HER2-Overexpressed Cells.

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.

Defucosylated Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 134-mG2a-f Exerts Antitumor Activities in Mouse Xenograft Models of Dog Epidermal Growth Factor Receptor-Overexpressed Cells.

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.

Epitope Mapping of an Anti-Human Epidermal Growth Factor Receptor Monoclonal Antibody (EMab-51) Using the RIEDL Insertion for Epitope Mapping Method.

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.

Development of a Novel Anti-HER2 Monoclonal Antibody H2Mab-181 for Gastric Cancer.

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.

Epitope Mapping of an Antihuman EGFR Monoclonal Antibody (EMab-134) Using the REMAP Method.

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.

Epitope Mapping of the Anti-California Sea Lion Podoplanin Monoclonal Antibody PMab-269 Using Alanine-Scanning Mutagenesis and ELISA.

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.

Development of Anti-Human CC Chemokine Receptor 9 Monoclonal Antibodies for Flow Cytometry.

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.

Ferret Podoplanin Is Detected by PMab-241 in Immunohistochemistry.

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.

Anti­HER3 monoclonal antibody exerts antitumor activity in a mouse model of colorectal adenocarcinoma.

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 anti­HER3 monoclonal antibody (mAb), H3Mab­17 (IgG2a, kappa), by immunizing mice with HER3­overexpressed CHO­K1 cells (CHO/HER3). H3Mab­17 was found to react specifically with endogenous HER3 in colorectal carcinoma cell lines, using flow cytometry. The KD for H3Mab­17 in CHO/HER3 and Caco­2 (a colon cancer cell line) were determined to be 3.0x10­9 M and 1.5x10­9 M via flow cytometry, respectively, suggesting high binding affinity of H3Mab­17 to HER3. Then, we assessed the H3Mab­17 antibody­dependent cellular cytotoxicity (ADCC) and complement­dependent cytotoxicity (CDC) against Caco­2, and evaluated its antitumor capacity in a Caco­2 xenograft model. In vitro experiments revealed H3Mab­17 had strongly induced both ADCC and CDC against Caco­2 cells. In vivo experiments on Caco­2 xenografts revealed that H3Mab­17 treatment significantly reduced tumor growth compared with the control mouse IgG. These data indicated that H3Mab­17 could be a promising treatment option for HER3­expressing colon cancers.

Development of an Anti-Elephant Podoplanin Monoclonal Antibody PMab-265 for Flow Cytometry.

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.

Development of Monoclonal Antibody PMab-269 Against California Sea Lion Podoplanin.

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.

An anti­TROP2 monoclonal antibody TrMab­6 exerts antitumor activity in breast cancer mouse xenograft models.

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 anti­TROP2 monoclonal antibody (clone TrMab­6; mouse IgG2b, κ) was developed using a Cell­Based Immunization and Screening (CBIS) method. TrMab­6 was useful for investigations using flow cytometry, western blot, and immunohistochemistry. The aim of the present study was to investigate whether TrMab­6 possesses in vitro antibody­dependent cellular cytotoxicity (ADCC) or complement­dependent cytotoxicity (CDC) activities or in vivo antitumor activities using mouse xenograft models of TROP2­overexpressed CHO­K1 (CHO/TROP2) and breast cancer cell lines, including MCF7, MDA­MB­231, and MDA­MB­468. In vitro experiments revealed that TrMab­6 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 CHO­K1 and MCF7/TROP2­knockout cells. Furthermore, in vivo experiments on CHO/TROP2 and MCF7 xenografts revealed that TrMab­6 significantly reduced tumor growth, whereas it did not show antitumor activities against parental CHO­K1 and MCF7/TROP2­knockout xenografts. The findings suggest that TrMab­6 is a promising treatment option for TROP2­expressing breast cancers.

Development of Anti-human T Cell Immunoreceptor with Ig and ITIM Domains (TIGIT) Monoclonal Antibodies for 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.

Development of Anti-Mouse CC Chemokine Receptor 8 Monoclonal Antibodies for 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.

Development of Anti-Mouse CC Chemokine Receptor 3 Monoclonal Antibodies for Flow Cytometry.

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.