Trans-chromosomic (Tc) mice carrying mini-chromosomes with megabase-sized human immunoglobulin (Ig) loci have contributed to the development of fully human therapeutic monoclonal antibodies, but mitotic instability of human mini-chromosomes in mice may limit the efficiency of hybridoma production. Here, we establish human antibody-producing Tc mice (TC-mAb mice) that stably maintain a mouse-derived, engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig-knockout background. Comprehensive, high-throughput DNA sequencing shows that the human Ig repertoire, including variable gene usage, is well recapitulated in TC-mAb mice. Despite slightly altered B cell development and a delayed immune response, TC-mAb mice have more subsets of antigen-specific plasmablast and plasma cells than wild-type mice, leading to efficient hybridoma production. Our results thus suggest that TC-mAb mice offer a valuable platform for obtaining fully human therapeutic antibodies, and a useful model for elucidating the regulation of human Ig repertoire formation.

Ephrin type-B receptor 2 (EphB2) is a member of the Eph family tyrosine kinase receptors. EphB2 binds to ephrin-B1, ephrin-B2, and ephrin-B3, which are critical regulators of vascular and neural development, influencing cell migration and axon guidance. EphB2 is overexpressed in several tumors, including glioma, breast cancer, hepatocellular carcinoma, and malignant mesothelioma, where it functions as a tumor promoter. Therefore, the development of monoclonal antibodies (mAbs) targeting EphB2 is essential for the diagnosis and treatment of EphB2-positive tumors. In this study, we developed novel mAbs for human EphB2 using the Cell-Based Immunization and Screening method. Among the established anti-EphB2 mAbs, Eb2Mab-12 (mouse IgG1, kappa) showed reactivity toward EphB2-overexpressed Chinese hamster ovary-K1 cells (CHO/EphB2) and an endogenously EphB2-expressing cancer cell line (LS174T), as confirmed by flow cytometry. The dissociation constant (KD) values of Eb2Mab-12 for CHO/EphB2 and LS174T were determined to be 1.7 × 10−9 M and 4.4 × 10−10 M, respectively, using flow cytometry. Furthermore, Eb2Mab-12 exhibited no cross-reactivity with other members of the EphA and EphB receptors. These results indicate that Eb2Mab-12 possesses high affinity and specificity in detecting EphB2, suggesting its potential application in tumor therapy.

Erythropoietin-producing hepatocellular (Eph) receptor A3 (EphA3) is a member of the Eph receptor family, which binds to its respective ligands, ephrins. These interactions are essential for normal development and tissue homeostasis. Dysregulation of EphA3 has been reported to be associated with human hematopoietic malignancies, making it a promising target for therapy and diagnosis. Due to the high similarity of the extracellular domain among Eph receptors (more than 33% amino acid identity), generating highly specific monoclonal antibodies (mAbs) is crucial. We developed anti-human EphA3 mAbs in this study using the Cell-Based Immunization and Screening (CBIS) method. Among them, the clone Ea₃Mab-20 (IgG₁, kappa) exhibited high affinity and specificity in flow cytometry. The dissociation constant values of Ea₃Mab-20 for CHO/EphA3 and Jurkat cells were determined to be 9.0 ± 0.3 × 10⁻⁹ M and 1.4 ± 0.1 × 10⁻⁹ M, respectively. Ea₃Mab-20 showed no cross-reactivity with other Eph receptors in flow cytometry. Furthermore, Ea₃Mab-20 demonstrated the suitability for detecting formalin-fixed paraffin-embedded cell samples in immunohistochemistry. Therefore, Ea₃Mab-20 is valuable mAb for basic research and is expected to contribute to the clinical application of mAb for cancer therapy and diagnosis.

Cadherin 26 (CDH26) is a recently identified member of the cadherin superfamily. Although CDH26 gene expression has been reported in association with allergic inflammatory responses, the protein expression levels and the signaling pathways mediated through its interactions with other proteins remain poorly understood. This is primarily due to the lack of monoclonal antibodies (mAbs) that can recognize the intact, cell surface-expressed form of CDH26. In this study, we developed an anti-human CDH26 mAb, Ca₂₆Mab-6 (IgM, kappa), using the Cell-Based Immunization and Screening (CBIS) method. Ca₂₆Mab-6 demonstrated high sensitivity and specificity for CDH26 in flow cytometry, and did not bind to CHO-K1 cells, which overexpress any of the other type I or type II cadherins. Ca₂₆Mab-6 successfully detected endogenous CDH26 protein expression in HepG2, U-87 MG, MCF7, and 293FT cells. The dissociation constant of Ca₂₆Mab-6 was determined to be 9.8 ± 4.8 × 10⁻⁹ M for CDH26-overexpressed CHO-K1 (CHO/CDH26) cells and 3.6 ± 1.0 × 10⁻⁷ M for HepG2 cells. The detection of CDH26 expression in cancer cells may offer new insights into the potential relationship between inflammatory responses and malignant transformation. Therefore, Ca₂₆Mab-6, developed using the CBIS method, is expected to facilitate functional studies of CDH26 and contribute to the development of CDH26-targeted antibody-based therapies.

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

Glypican-1 (GPC1) has emerged as a critical mediator of malignant tumor progression. GPC1 plays essential roles in regulating various signaling pathways involved in tumor cell proliferation, invasiveness, and tumorigenesis. Overexpression of GPC1 in tumors mediates oncogenic transformation, epithelial-to-mesenchymal transition, metastatic dissemination, and therapeutic resistance. Accordingly, GPC1-targeted therapeutic strategies have been investigated in clinical and preclinical studies. However, clinical efficacy has been limited. We previously developed an anti-GPC1 monoclonal antibody (mAb), G₁Mab-28 (mouse IgG₁, κ), which exhibits high affinity and specificity for GPC1. In the present study, we generated recombinant isotype-converted G₁Mab-28, including G₁Mab-28-mG_2a (mouse IgG_2a) and G₁Mab-28-hG₁ (human IgG₁). Both mAbs recognized GPC1-expressing human tumor cell lines, including lung squamous cell carcinoma PC-10 and pancreatic ductal adenocarcinoma PK-45H, by flow cytometry. Moreover, both mAbs exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against those cell lines. In mouse xenograft models, treatment with the mAbs resulted in potent antitumor efficacy against PC-10 and PK-45H tumors. Collectively, these findings support the therapeutic potential of G₁Mab-28 for the treatment of GPC1-positive tumors.