Hirotake Shiraiwa

A defucosylated anti-CD317 antibody exhibited enhanced antibody-dependent cellular cytotoxicity against primary myeloma cells in the presence of effectors from patients

The humanized monoclonal antibody (mAb) against CD317 antigen (anti‐HM1.24 antibody; AHM), which is highly expressed on multiple myeloma (MM), induces antibody‐dependent cellular cytotoxicity (ADCC). However, the antitumor activity of AHM in the clinical setting has not been clearly demonstrated. In this study, we produced defucosylated AHM and evaluated its potency for clinical application by performing autologous ADCC assays against primary MM cells from patients. Defucosylated AHM that was produced in rat myeloma YB2/0 cells expressing a low level of fucosyltransferase (FUT8) showed significant ADCC activity against three out of six primary MM cells in the presence of autologous PBMC, whereas conventional AHM did not. The results indicate that the potency of AHM to induce ADCC against primary MM cells was insufficient, but was significantly augmented by defucosylation. To generate more homogenous defucosylated monoclonal antibodies (mAb) for fermentation, we disrupted the GFT gene that encodes a GDP‐fucose transporter in a CHO/DXB11 cell line by sequential homologous recombination. Analysis of the N‐linked oligosaccharide in the defucosylated AHM produced by the established GFT(−/−)CHO cell line showed that a majority (93.4%) of the oligosaccharide was fucose free. The GFT(−/−) cells stably produced defucosylated mAb over passages. These results demonstrate that GTF(−/−)CHO‐produced defucosylated AHM (GFTKO‐AHM) will be a promising new therapeutic antibody against MM in the clinical setting. (Cancer Sci 2010)

An anti–glypican 3/CD3 bispecific T cell–redirecting antibody for treatment of solid tumors

An anti–glypican 3/CD3 bispecific T cell–redirecting antibody (ERY974) is a promising therapeutic agent for solid tumors. Double trouble for solid tumors Because the endogenous immune response is not enough to clear a patient’s cancer, therapies are being designed to redirect T cells to tumor cells. This can be done by engineering the cells ex vivo, such as in CAR T cell therapy, or in vivo, such as with bispecific antibodies. Ishiguro et al. describe the development and preclinical testing of a bispecific antibody recognizing CD3 and glypican 3, a common antigen on solid tumors. This bispecific antibody was effective in a variety of mouse cancer models, even when treatment was initiated after the tumor was quite large. Treatment also appeared to be safe when administered to monkeys. These results suggest further development of this antibody for therapeutic use in multiple cancer types. Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell–redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid “on-target off-tumor” toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G–structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein–1) and CTLA-4 (cytotoxic T lymphocyte–associated protein–4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.

Abstract DDT01-05: First-in-class T cell-redirecting bispecific antibody targeting glypican-3: a highly tumor-selective antigen

Immune checkpoint inhibitors such as anti-PD1 antibodies have shown promising clinical responses in several solid tumors, however there remain patients who do not show an adequate response. Recent biomarker studies have revealed that the presence of neoantigens in the tumor can determine the level of response, and thus the next challenge will be how to target tumors with a neoantigen level that is too low to be recognized by endogenous cytotoxic T cells. Hope in this area is offered by a T cell-redirecting antibody (TRAB), which bispecifically engages CD3 and a tumor antigen, even at very low expression levels, to activate the inherent cytolytic potential of T cells against target tumor cells. A TRAB is highly potent because T cells are activated only in the presence of the targeted antigens and are not restricted by the specificity of the T cell receptor. Given this very potent cytotoxicity, the key to successfully achieving strong antitumor efficacy while avoiding on-target off-tumor toxicity is to select a highly tumor-selective antigen. Our fully humanized IgG TRAB recognizes CD3 and a highly tumor-selective antigen, glypican-3 (GPC3), which is a fetal protein expressed in a wide variety of tissues during development but suppressed in most adult tissues. On the other hand, an inct101e in GPC3 expression has been reported in hepatocellular carcinoma, gastric cancer, lung squamous cell carcinoma, and other cancers. In nonclinical in vitro pharmacology studies, the anti-GPC3 TRAB elicited activation and proliferation of T cells and T cell-dependent cellular cytotoxicity against a wide variety of GPC3-expressing tumor cells, and showed long-lasting in vivo efficacy against tumor expressing very low levels of GPC3 at a few thousand molecules per cell. Furthermore, in an immunocompetent mouse model using human CD3 transgenic mice, anti-GPC3 TRAB showed strong antitumor efficacy against poorly immunogenic tumors, whereas both the immune checkpoint inhibitors and a conventional ADCC-inducing antibody recognizing GPC3 did not show significant efficacy. Pharmacokinetics and toxicology studies in nonhuman primates showed a plasma half-life comparable to a standard IgG drug, allowing a QW or Q2W regimen in humans, with toxicity which was manageable and reversible; the main observations of transient cytokine elevation and associated clinical symptoms were markedly reduced by steroid premedication. Our anti-GPC3 TRAB, which is supported by proprietary antibody engineering technology (ART-Ig) that enables large-scale GMP manufacturing, has promise as a new approach in cancer immunotherapy. Citation Format: Takahiro Ishiguro, Yasuko Kinoshita, Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Natsuki Ono, Yoko Kayukawa, Mika Kamata-Sakurai, Hirotake Shiraiwa, Akihisa Kaneko, Werner Frings, Shunichiro Komatsu, Junichi Nezu, Mika Endo. First-in-class T cell-redirecting bispecific antibody targeting glypican-3: a highly tumor-selective antigen. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr DDT01-05.

Abstract 1482: Anti-GPC3 TRAB, a first-in-class T cell-redirecting bispecific antibody targeting glypican-3 with potent in vitro and in vivo antitumor efficacy against solid tumors

We present efficacy data for the T cell-redirecting antibody (TRAB) with highly potent anti-tumor efficacy. Anti-Glypican-3 (GPC3) TRAB is a humanized IgG4 bispecific antibody that simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface. Anti-GPC3 TRAB utilizes T cells as effectors to induce strong TRAB dependent cellular cytotoxicity (TDCC) in the presence of GPC3-expressing cells. Treatment with anti-GPC3 TRAB first activates T cells by increasing the expression of CD25 and CD69 and also upregulating cytokines IL-2, IL-4, IL-6, IL 10, IFNγ, and TNF, and then it enhances the proliferation of T cells. Anti-GPC3 TRAB showed antitumor activity against xenograft tumors derived from various cancer types — MKN-74 (human gastric adenocarcinoma), PC-10 (human lung squamous cell carcinoma), TOV-21G (human ovarian clear cell carcinoma), and KYSE70 (human esophageal squamous cell carcinoma) — in a NOD-SCID mouse model injected with human T cells. Although recent immunotherapy, as represented by immune check point inhibitors PD-1, PD-L1, and CTLA-4 antibodies, showed promising efficacy in human, not every patient can benefit from this immunotherapy, because the significant efficacy shown in patients by a blockade of immune checkpoints is closely related to the tumor microenvironment. The immune check point inhibitors show high efficacy against inflamed tumors, because these have been sufficiently infiltrated by cytotoxic T cells that recognize cancer-specific antigens. However, they do not have efficacy against non inflamed tumors. In an immunocompetent mouse model using human CD3 transgenic mice, neither the inhibitors that block immune checkpoints (such as PD-1, PD-L1 and CTLA-4) nor a conventional ADCC antibody recognizing GPC3 could show significant efficacy against a poorly immunogenic LLC1/hGPC3 tumor. However, anti-GPC3 TRAB showed efficacy against this poorly immunogenic tumor by utilizing any kind of T cell as effectors irrespective of TCR specificity, including not only CD8-positive but also CD4-positive T cells. The studies we present show that anti-GPC3 TRAB is a promising drug with high efficacy utilizing all kinds of T cells as effectors. The compound is expected to have efficacy even in patients with poorly immunogenic tumors, in which an immune checkpoint blockade fails to show efficacy. Citation Format: Yasuko Kinoshita, Takahiro Ishiguro, Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Natsuki Ono, Yoko Kayukawa, Otoya Ueda, Naoko A. Wada, Hiroshi Hino, Koichi Jishage, Hirotake Shiraiwa, Mika Kamata-Sakurai, Junichi Nezu, Mika Endo. Anti-GPC3 TRAB, a first-in-class T cell-redirecting bispecific antibody targeting glypican-3 with potent in vitro and in vivo antitumor efficacy against solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1482.