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

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

Background: Podocalyxin (PODXL) has been identified as a promising therapeutic target and a potential diagnostic biomarker in various tumors. Despite the therapeutic potential of anti-PODXL monoclonal antibodies (mAbs), their further development has been limited by concerns regarding potential on-target off-tumor toxicities. To minimize adverse effects on normal tissues, developing a cancer-specific mAb (CasMab) against PODXL is essential. Methods: Our group established a cancer-specific anti-PODXL mAb, PcMab-60 (IgM, κ), through the screening of over one hundred hybridoma clones. In this study, PcMab-60 was engineered into a humanized IgG₁-type mAb (humPcMab-60), and its antitumor activity was examined using mouse xenograft models of pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer. Results: HumPcMab-60 retains cancer-specific reactivity; humPcMab-60 reacted to PDAC cell lines (PK-45H and MIA PaCa-2) and the colorectal cancer cell line (Caco-2), but not to a normal lymphatic endothelial cell line in flow cytometry. Furthermore, humPcMab-60 exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against PODXL-expressing cell lines and showed antitumor effects against the tumor xenografts. Conclusions: A humanized anti-PODXL CasMab, humPcMab-60, could be a promising mAb-based tumor therapy.

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

Background: Podocalyxin (PODXL) has been identified as a promising therapeutic target and a potential diagnostic biomarker in various tumors. Despite the therapeutic potential of anti-PODXL monoclonal antibodies (mAbs), their further development has been limited by concerns regarding potential on-target off-tumor toxicities. To minimize ad-verse effects on normal tissues, the development of a cancer-specific mAb (CasMab) against PODXL is essential. Methods: We established a cancer-specific anti-PODXL mAb, PcMab-60 (IgM, κ), through the screening of over one hundred hybridoma clones. In this study, we engineered PcMab-60 into a humanized IgG1-type mAb (humPcMab-60) and examined its antitumor activity against mouse xenograft models of pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer. Results: HumPcMab-60 retains the can-cer-specific reactivity; humPcMab-60 reacted to PDAC cell lines (MIA PaCa-2 and PK-45H) and colorectal cancer cell line (Caco-2), but not to a normal lymphatic endothelial cell line in flow cytometry. Furthermore, humPcMab-60 exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against PDOXL-expressing cell lines, and showed the antitumor effect against the tumor xenografts. Conclusions: A humanized anti-PODXL CasMab, humPcMab-60, could be a promising mAb-based tumor therapy.

Erythropoietin-producing hepatocellular receptor A2 (EphA2) has emerged as a key mediator that promotes tumor malignant progression. EphA2 overexpression and its non-canonical signaling lead to oncogenic transformation, metabolic reprogramming, resistance to treatments, and metastasis. Therefore, strategies targeting EphA2 have been evaluated in clinical trials. However, the clinical effects were not sufficient. An anti-EphA2 monoclonal antibody (mAb), Ea₂Mab-7 (mouse IgG₁, κ), demonstrated high affinity and specificity among Eph receptors. In this study, we produced recombinant class-switched Ea₂Mab-7 variants, including Ea₂Mab-7-mG_2a (mouse IgG_2a) and Ea₂Mab-7-hG₁ (human IgG₁). Both Ea₂Mab-7-mG_2a and Ea₂Mab-7-hG₁ recognized human triple-negative breast cancer MDA-MB-231, pancreatic cancer MIA PaCa-2, and colorectal cancer HCT-15 in flow cytometry. Furthermore, both Ea₂Mab-7-mG_2a and Ea₂Mab-7-hG₁ exerted significant antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against these tumors. In mouse xenograft models of breast, pancreatic, and colorectal cancers, both mAbs demonstrated antitumor activity. These results indicate the potential of Ea₂Mab-7 variants for the treatment of EphA2-positive cancers.