An anti–glypican 3/CD3 bispecific T cell–redirecting antibody for treatment of solid tumors by Takahiro Ishiguro, Yuji Sano, Shun-ichiro Komatsu, Mika Kamata-Sakurai, Akihisa Kaneko, Yasuko Kinoshita, Hirotake Shiraiwa, Yumiko Azuma, Toshiaki Tsunenari, Yoko Kayukawa, Yukiko Sonobe, Natsuki Ono, Kiyoaki Sakata, Toshihiko Fujii, Yoko Miyazaki, Mizuho Noguchi, Mika Endo, Asako Harada, Werner Frings, Etsuko Fujii, Eitaro Nanba, Atsushi Narita, Akihisa Sakamoto, Tetsuya Wakabayashi, Hiroko Konishi, Hiroaki Segawa, Tomoyuki Igawa, Takashi Tsushima, Hironori Mutoh, Yukari Nishito, Mina Takahashi, Lorraine Stewart, Ehab ElGabry, Yoshiki Kawabe, Masaki Ishigai, Shuichi Chiba, Masahiro Aoki, Kunihiro Hattori, and Junichi Nezu Sci Transl Med Volume 9(410):eaal4291 October 4, 2017 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 1. Generation of ERY974. Generation of ERY974. (A) Schematic illustration of ERY974 structure and the introduced mutations. The two Fab arms share a common light chain, depicted in green, with a variable domain. (B) Flow cytometry results showing specific binding of ERY974 to GPC3 and CD3E in SK-HEP-1 (a parental human liver/ascites adenocarcinoma cell line), SK-HEP-1/hGPC3, (SK-HEP-1 cells that overexpress human GPC3), BAF3 (a parental mouse pro–B cell line), and BAF3/CD3ε (BAF3 cells that overexpress human CD3E). The blue lines indicate staining with a control IgG4 antibody, and the red lines indicate staining with ERY974. FITC, fluorescein isothiocyanate. Takahiro Ishiguro et al., Sci Transl Med 2017;9:eaal4291 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 2. GPC3 expression in normal and tumor tissues. GPC3 expression in normal and tumor tissues. IHC analysis of an anti-GPC3 mouse antibody (mGC33) in a multi-tissue array. Representative photomicrographs of GPC3 staining in tumor and normal tissues are shown. Cytoplasmic H-scores are indicated in parentheses. H-scores were calculated as values between 0 and 300, defined as [1 × (percentage of cells staining at 1+ intensity) + 2 × (percentage of cells staining at 2+ intensity) + 3 × (percentage of cells staining at 3+ intensity) = H-score]. Photos were taken using a 20× objective lens. Takahiro Ishiguro et al., Sci Transl Med 2017;9:eaal4291 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 3. GPC3-dependent TDCC and polyclonal T cell activation induced by ERY974. GPC3-dependent TDCC and polyclonal T cell activation induced by ERY974. (A) Target cells and PBMCs were incubated with various concentrations of ERY974 for 24 hours. A keyhole limpet hemocyanin (KLH)–TRAB antibody was used as a negative control. TDCC was measured as described in Materials and Methods. Data represent means ± SD (n = 3). (B) T cell activation was assessed by measuring CD25 and CD69 levels on CD3+ T cells by flow cytometry. Data represent means ± SD (n = 3). (C) TDCC of ERY974 elicited by CD4+ or CD8+ T cells was measured. Data represent means ± SD (n = 3). (D) Target cells and CFSE-labeled PBMCs were incubated with ERY974 (1 μg/ml) or KLH-TRAB for 3 days. Total numbers of viable CD4+ or CD8+ T cells at day 0 (immediately after reaction started) and day 3 were counted by flow cytometry. Data represent means ± SD (n = 3). (E) Red lines represent the CFSE profiles in the presence of ERY974, and blue lines represent the profile in the presence of KLH-TRAB. The histograms show one representative result of triplicate assays. Takahiro Ishiguro et al., Sci Transl Med 2017;9:eaal4291 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 4. Antitumor efficacy of ERY974 against various cancer types. Antitumor efficacy of ERY974 against various cancer types. (A) GPC3 expression in various cancer cell lines determined by quantitative flow cytometry. (B) IHC results of GPC3 staining in xenograft tissues of indicated cancer cell lines. H-scores in the cytoplasm are indicated in parentheses. Images were taken using a 20× objective. (C) Antitumor efficacy of ERY974 against various xenograft tumors in NOD-SCID mice inoculated with human T cells. Mean tumor volume values are shown + SD (n = 5). *P < 0.05 between vehicle and ERY974 group by Wilcoxon test. Arrows indicate timing of ERY974 administration. (D) Antitumor efficacy of ERY974 against large tumors. When the mean volume of implanted KYSE70 tumors reached more than 600 mm3, vehicle or ERY974 (1 mg/kg) was administered 3 to 5 hours after the T cell injection. Mean tumor volumes in each group are shown + SD (n = 4). Images represent tumor burden over time in one representative animal dosed with ERY974 on day 29. (E) Antitumor efficacy of ERY974 against GPC3-negative cell xenograft tumors. Mean values for tumor volume in each group are shown + SD (n = 5). Takahiro Ishiguro et al., Sci Transl Med 2017;9:eaal4291 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 5. Antitumor efficacy of ERY974 in immunocompetent human CD3 transgenic mice. Antitumor efficacy of ERY974 in immunocompetent human CD3 transgenic mice. (A) Histopathological analysis of Hepa1-6/hGPC3 and LLC1/hGPC3 tumors. Tumor tissue samples taken 3 days after administering vehicle or ERY974 (5 mg/kg) were stained as indicated. H&E, hematoxylin and eosin. (B) Gene expression analysis in Hepa1-6/hGPC3 and LLC1/hGPC3 tumors. RNA from tumors treated with vehicle or ERY974 was used for RNA-seq. Each group was tested in triplicate (n = 3). Z scores were calculated using log2-transformed fragments per kilobase of exon per million mapped fragments values for all target genes. (C) Antitumor efficacy of ERY974 and immune checkpoint inhibitors. Values represent means + SD (n = 5). *P < 0.05 between vehicle group and the antibody treatment group at day 25 determined by Dunn’s multiple comparisons test. Arrows indicate the timing of antibody administration. Takahiro Ishiguro et al., Sci Transl Med 2017;9:eaal4291 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 6. Safety assessment in cynomolgus monkey. Safety assessment in cynomolgus monkey. (A) Binding affinity of ERY974 to human or cynomolgus monkey epitope peptides derived from GPC3 or CD3 was measured by surface plasmon resonance analysis. KD, dissociation constant. (B) Change in serum interleukin (IL)–6 concentration over time in a single-dose study. Five animals per sex per group received a single administration of ERY974. Necropsies were performed on day 22 after the dose. (C) Serum IL-6 concentration (red and black lines) over time in a repeated-dose study. Three animals per sex received doses (blue bars) that increased daily by about threefold to 1.5 mg/kg. Necropsies were performed on day 10. Takahiro Ishiguro et al., Sci Transl Med 2017;9:eaal4291 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

Fig. 7. Effect of premedication on antitumor efficacy and cytokine release. Effect of premedication on antitumor efficacy and cytokine release. (A) Antitumor efficacy of ERY974 in huNOG mice. Mean tumor volume values are shown + SD (n = 4 or 5). *P <0.05 between vehicle and ERY974 groups by Dunnett’s multiple comparisons test. Arrows indicate ERY974 administration. (B) IL-6 and IL-2 induction. Blood was collected 17 hours before and 2, 6, and 24 hours after ERY974 administration. Plasma concentrations of IL-6 and IL-2 are shown. Values represent means + SD (n = 5). (C) Influence of DEX premedication on antitumor efficacy. huNOG mice bearing tumors were administered vehicle, DEX premedication alone, or ERY974 (1 mg/kg) with or without DEX premedication. Mean tumor volume values are shown + SD (n = 4 or 5). *P < 0.05 between the vehicle and ERY974 groups by Dunnett’s multiple comparisons test. (D) Effect of DEX on induction of IL-6 and IL-2. Blood was collected 19 hours before and 6 and 24 hours after the first dose of ERY974, and 19 hours before and 6 hours after the second dose (149 and 174 hours after the first dose, respectively) of ERY974. Values represent means + SD (n = 5). Takahiro Ishiguro et al., Sci Transl Med 2017;9:eaal4291 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works