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.

Trans-chromosomic (Tc) mice carrying mini-chromosomes with human immunoglobulin (Ig) loci have contributed to the development of fully human therapeutic monoclonal antibodies (Abs); however, we previously observed that mitotic instability of human mini-chromosomes in mice has limited the efficiency of hybridoma production. Here, we established a new generation of human Ab producing Tc mice (TC-mAb mice), which 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 revealed that the human Ig repertoire, including variable gene use, was well recapitulated in TC-mAb mice. Despite slightly altered B cell development and a delayed immune response, immunized TC-mAb mice exhibited more subsets of antigen-specific plasmablast and plasma cells compared with wild-type mice, leading to high efficiency hybridoma production. Thus, TC-mAb mice offer a valuable platform to obtain fully human therapeutic Abs and to elucidate the regulation of human Ig repertoire formation.

We previously generated fully human antibody-producing TC-mAb mice for obtaining potential therapeutic monoclonal antibodies (mAbs). In this study, we investigated 377 clones of fully human mAbs against a tumor antigen, epithelial cell adhesion molecule (EpCAM), to determine their antigen binding properties. We revealed that a wide variety of mAbs against EpCAM can be obtained from TC-mAb mice by the combination of epitope mapping analysis of mAbs to EpCAM and native conformational recognition analysis. Analysis of 72 mAbs reacting with the native form of EpCAM indicated that the EpCL region (amino acids 24–80) is more antigenic than the EpRE region (81–265), consistent with numerous previous studies. To evaluate the potential of mAbs against antibody–drug conjugates, mAbs were directly labeled with DM1, a maytansine derivative, using an affinity peptide-based chemical conjugation (CCAP) method. The cytotoxicity of the conjugates against a human colon cancer cell line could be clearly detected with high-affinity as well as low-affinity mAbs by the CCAP method, suggesting the advantage of this method. Thus, this study demonstrated that TC-mAb mice can provide a wide variety of antibodies and revealed an effective way of identifying candidates for fully human ADC therapeutics.

The properties of the variable domain of heavy-chain (VHH) antibodies are particularly relevant in cancer therapy. To isolate tumor cell-specific VHH antibodies, VHH phage libraries were constructed from multiple tumor cells. After enriching the libraries against particular tumor cell lines, a next-generation sequencer was used to screen the pooled phages of each library for potential antibody candidates. Based on high amplification folds, 50 sequences from each library were used to construct phylogenetic trees. Several clusters with identical CDR3 were observed. Groups X, Y, and Z were assigned as common sequences among the different trees. These identical groups over the trees were considered to be cross-reactive antibodies. To obtain monoclonal antibodies, we assembled 200 sequences (top 50 sequences from each library) and rebuilt a combined molecular phylogenetic tree. Groups were categorized as A–G. For each group, we constructed a phagemid and determined its binding specificity with tumor cells. The phage-binding results were consistent with the phylogenetic tree-generated groups, which indicated particular tumor-specific clusters; identical groups showed cross-reactivity. The strategy used in the current study is effective for screening and isolating monoclonal antibodies. Specific antibodies can be identified, even when the target markers of cancer cells are unknown.

Objective: We aimed to establish reversibly immortalized cell lines from human uterine and ovary cells using the Sendai virus (SeV) vector. The immortalized cells derive from normal and benign ovarian epithelial cells and endometrial epithelial cells. Furthermore, we sought to elucidate the mechanisms of carcinogenesis using the immortalized cell lines. Methods: Cells were collected at the time of surgery after obtaining patient consent. The cells used in this study were as follows: ovarian epithelial cells (normal epithelium, Ov n; normal epithelium with germline BRCA1 or BRCA2 mutation, Ov BRCA1 2; ovarian endometrioma, Ov endo; mucinous cystadenoma; Ov m), normal fallopian tube (FT) cells, and endometrial epithelium (normal epithelium, Em n). These cells were infected with temperature-sensitive SeV vectors carrying three immortalization genes, Bmi-1, hTERT, and SV40T.The presence of infection was confirmed through Green Fluorescent Protein (GFP) and Orange Fluorescent Protein (OFP). Immunoreactivity to the anti-human EpCAM antibody (a marker derived from epithelial carcinoma) in each SeV-infected cell was confirmed through flow cytometry. QH and multicolor FISH staining were performed for karyotyping of metaphase chromosomes in each cell line to determine chromosome number and structural abnormalities. Human transcriptome sequencing analysis was performed with NovaSeq 6000 (Illumina) using total RNA from each cell line. Some genes that showed significant expression in each cell line were subjected to real-time PCR (RT-PCR). Results: We established the immortalized cell lines from human uterine and ovarian tissues. SeV-infected cells exhibited GFP and OFP fluorescence, while non-infected cells did not. SeV infection allowed all primary cell lines to grow for 25 or more passages, while non-infected SeV cells lacked the proliferative capacity and showed senescence-like morphology. SeV-infected cells senesced in a temperature-dependent manner. Ov n SeV- infected cells ion causedshowed a small increase in chromosome structural abnormalities. But, Ov BRCA1 and 2 SeV-infected cells showed larger than thatand . Llong-term passaged cells did not show immune response to anti-human EpCAM antibodies in normal cells. Eleven Three genes were predominantly expressed in Ov BRCA1 and Ov BRCA2 cells and were not expressed in Ov n cells. SomeTwo out of ththe three genesem were found to be predominantly expressed in Ov endo cells compared to the expression in the Ovn cell line. Furthermore, RT-PCR results also indicated substantially higher expression of two genes in Ov BRCA1/2 cells and in Ov endo cells compared to the expression in Ov n cells. Conclusion: We succeeded in the reversible immortalization of endometrial and ovarian epithelial cells by using SeV infection. We identified several candidate genes that may be involved in the oncogenic mechanism of ovarian cancer associated with endometriosis or germline BRCA1 and BRCA2. Citation Format: Masayo Okawa, Hiroaki Komatsu, Kohei Hikino, Yuki Iida, Masayo Hosokawa, Mayumi Sawada, Akiko Kudoh, Jun Chikumi, Shinya Sato, Genki Hichiwa, Yasuhiro Kazuki, Kanako Kazuki, Fuminori Taniguchi, Mitsuo Oshimura, Tasuku Harada. Establishment and characterization of reversibly immortalized endometrial and ovarian epithelial cell lines using Sendai virus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1214.