The dashed arrows show the timing of irinotecan injection, and the solid arrows show the timing of DS\5573a, isotype control, or vehicle injection. study, we aimed to develop new antitumor mAbs by employing cancer cell immunization methods. Many antitumor mAbs were obtained, but we focused specifically on a mouse anti\human B7\H3 mAb (M30), because of the correlation between its expression and tumor progression, and the suitable expression profiles. To generate a therapeutic mAb, M30 was humanized (Hu\M30), and an afucosylated humanized anti\B7\H3 IgG1 antibody, designated DS\5573a, was generated from Hu\M30. In this article, we characterize DS\5573a and show its remarkable antitumor activity against B7\H3\expressing cells and gene BRAF inhibitor (Transgenic, Fukuoka, Japan)19 were s.c. immunized with MCF\7, and mice splenocytes were fused with P3X63Ag8U.1 cells using PEG 4000 (Immuno\Biological Laboratories, Gunma, Japan). One of the hybridoma that produced a mouse IgG2a mAb (M30) was selected because M30 showed antitumor activity against NCI\H322\bearing nude mice. The antigen of M30 was identified as B7\H3 by mass spectrometry.20 Establishment of Hu\M30 and DS\5573a cDNAs of the heavy\ and light\chain variable regions of M30 were obtained by RT\PCR. Hu\M30 was generated using the CDR grafting method. Expression vectors of Hu\M30 were transfected into 293\F cells (Life Technologies, Tokyo, Japan), and Hu\M30 was purified from the supernatant.20 Afucosylated Hu\M30, DS\5573a, was produced by the POTELLIGENT? CHOK1SV expression system (BioWa, La Jolla, CA, USA, and Lonza, Allendale, NJ, USA). Epitope mapping of DS\5573a Full\length human B7\H3 (NCBI Reference Sequence, “type”:”entrez-protein”,”attrs”:”text”:”NP_001019907.1″,”term_id”:”67188443″,”term_text”:”NP_001019907.1″NP_001019907.1: a.a. 27C534), and IgV1 (a.a. 27C139), IgC1 (a.a. 140C244), IgV2 (a.a. 245C357), or IgC2 (a.a. 358C456) domain expression vectors, each with transmembrane/intracellular domain (a.a. 457C534), were transfected into CHO\K1 cells. Each vector had FLAG\tag at the N\terminus. They were then treated with 1 g/mL DS\5573a or human IgG1 isotype control (Enzo Life Sciences, New York, NY, USA), and were stained with FITC\conjugated anti\human IgG (Jackson ImmunoResearch, West Grove, PA, USA). To detect the expressed B7\H3 proteins around the cell surface, cells were treated with 1 g/mL anti\FLAG antibody (Sigma\Aldrich, Tokyo, Japan) or mouse IgG1 isotype control (BD, Tokyo, Japan), and were stained with FITC\conjugated anti\mouse IgG (Cappel, Aurora, OH, USA). Samples were analyzed by Cytomics FC500 MPL (Beckman Coulter, Tokyo, Japan). Biacore assay The BRAF inhibitor binding affinity of DS\5573a or Hu\M30 against recombinant human B7\H3 protein (4IgB7\H3 or 2IgB7\H3) (R&D Systems, Minneapolis, MN, USA) was analyzed by surface plasmon resonance using Biacore 3000 or 4000 (GE Healthcare, Tokyo, Japan). DS\5573a or Hu\M30 was immobilized to sensor chips using a human antibody capture kit (GE Healthcare), and then each recombinant human B7\H3 protein was injected. The M,and represent 51Cr release of each sample, maximum 51Cr BRAF inhibitor release, and background 51Cr release, respectively. Antibody\dependent cellular phagocytosis (ADCP) assay Human macrophages were derived from PBMCs by treatment with 10 ng/mL recombinant human granulocyte M\CSF (PeproTech, Rocky Hill, NJ, USA) and recombinant human M\CSF (PeproTech) for 13 days. The day before the assay, macrophages were treated with 250 U/mL recombinant human interferon\ (PeproTech), and 10 ng/mL M\CSF. Around the assay day, PKH26\labeled target cells (5 104 cells) treated with serial dilutions of antibodies were mixed with macrophages (1 105 cells). After 3 h of incubation at 37C, each sample was stained with APC\labeled anti\human CD11b mAb (BD), and was measured by FACSCanto II (BD) after fixation with 1% paraformaldehyde. All experiments were carried out in triplicate. ADCP activity (%) was calculated using the following formula: evaluation All experimental procedures were carried out according to the in\house guidelines of the Institutional Animal Care and Use Committee of Daiichi Sankyo Co., Ltd. To confirm the efficacy of DS\5573a assays. Table 1 Expression level of B7\H3 in each cancer cell line assays. ALL, acute lymphoblastic leukemia. DS\5573a exerts much more potent human PBMC\mediated ADCC than Hu\M30 Next, to confirm whether Rabbit Polyclonal to CDK8 DS\5573a exerts enhanced ADCC activity as expected, we compared the activity of DS\5573a and Hu\M30 against cancer cell lines with various levels of BRAF inhibitor B7\H3 expression in the presence of human PBMCs. As shown in Figure ?Physique3,3, notably the ADCC activity of DS\5573a and Hu\M30 was found to fit to.