Yuji Sano

Combining Onartuzumab with Erlotinib Inhibits Growth of Non-Small Cell Lung Cancer with Activating EGFR Mutations and HGF Overexpression

Erlotinib, a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR-TKI), benefits survival of patients with non–small cell lung cancer (NSCLC) who harbor activating EGFR mutations. However, elevated expression of hepatocyte growth factor (HGF), a ligand of the MET receptor tyrosine kinase, causes erlotinib resistance. Because onartuzumab, a monovalent antibody to MET, blocks HGF-induced MET activation, the addition of onartuzumab to erlotinib may improve therapeutic efficacy. We engineered the human NSCLC cell line PC-9 (MET-positive cells harboring an exon 19 deletion of EGFR) to overexpress hHGF and evaluated the effects of an onartuzumab and erlotinib combination in vitro and in vivo in xenograft models. A stable clone of PC-9/hHGF was less sensitive to erlotinib than the parental PC-9, and the addition of onartuzumab to erlotinib suppressed the proliferation of these cells in vitro. In PC-9/hHGF xenograft tumors, onartuzumab or erlotinib alone minimally inhibited tumor growth; however, combining onartuzumab and erlotinib markedly suppressed tumor growth. The total MET protein level was decreased in PC-9/hHGF cells, because MET is constitutively phosphorylated by autocrine HGF, leading to its ubiquitination and degradation. Onartuzumab reduced phospho-MET levels, inhibited MET ubiquitination, and consequently restored MET protein levels. Moreover, in NSCLC clinical specimens harboring activating EGFR mutations, more than 30% of patients expressed high levels of HGF. Our findings raised the possibility that patients with NSCLC with EGFR mutations who express high levels of HGF may benefit from onartuzumab and erlotinib combination therapy, and that HGF can be a novel biomarker for selecting such patients. Mol Cancer Ther; 14(2); 533–41. ©2014 AACR.

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 2728: Onartuzumab (MetMAb) restores sensitivity to erlotinib in EGFR mutant NSCLC cells expressing HGF

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Onartuzumab (MetMAb) is a humanized monovalent monoclonal antibody that blocks binding of HGF to Met. The combination of onartuzumab + erlotinib is currently being evaluated in a Phase III clinical trial for 2nd/3rd line NSCLC (1). EGFR-activating mutations, including deletions of exon 19 and L858R point mutations, are associated with better response to erlotinib. However, EGFR mutant NSCLC tumors with high HGF expression are less responsive to EGFR small molecule inhibitors (2) (3). Since onartuzumab blocks HGF-induced Met activation, the addition of onartuzumab to erlotinib may be more beneficial than erlotinib alone in such patients. To test this hypothesis preclinically, we engineered the human NSCLC cell line PC9 (exon 19 deletion of EGFR and Met positive) to express hHGF endogenously, and evaluated combination effects of onartuzumab + erlotinib in vitro and in vivo. Methods: PC-9 cells were transfected with hHGF expression plasmids, and a stable clone was selected. For in vitro analyses, parental PC-9 cells and PC-9/hHGF cells were treated with various concentrations of erlotinib combined with 30μg/mL of onartuzumab, and cytotoxicity was examined. Additionally, the phosphorylation status of Met, EGFR, ERK and AKT was examined by western blot analysis of whole cell lysates. For in vivo analyses, PC-9/hHGF cells were inoculated subcutaneously into nude mice. After tumors reached approximately 200 mm3 in volume, onartuzumab (30 mg/kg, IP, Q3W) was administered alone or in combination with erlotinib (50 mg/kg, PO, daily) and tumor volume was measured over time. Results: One stable clone with high expression of hHGF was selected by hHGF ELISA and western blotting. This PC-9/hHGF clone was less sensitive to erlotinib compared to the parental PC-9 in vitro. Adding onartuzumab to erlotinib suppressed proliferation of these PC9/hHGF cells. pERK and pAKT levels were significantly reduced when PC-9/hHGF cells were treated with onartuzumab + erlotinib. In PC-9/hHGF xenograft tumors, onartuzumab or erlotinib alone had modest effects on tumor growth. However, combining onartuzumab and erlotinib dramatically suppressed tumor growth. Conclusions: Overexpression of hHGF decreases response of PC9 cells to erlotinib. Onartuzumab + erlotinib was more efficacious than erlotinib alone on PC9/hHGF cells in vitro and in vivo. These data support the hypothesis that addition of onartuzumab to erlotinib may enhance efficacy of erlotinib in EGFR mutant NSCLC tumors. References: (1) [NCT01456325][1] at www.clinicaltrials.gov (2) S. Yano et al. Cancer Res 68 (2008) (3) A. B. Turke et al. Cancer Cell 17 (2010) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2728. doi:1538-7445.AM2012-2728 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01456325&atom=%2Fcanres%2F72%2F8_Supplement%2F2728.atom

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 3653: Combining ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, with chemotherapy profoundly improved antitumor efficacy over its monotherapy in xenograft model

Background: ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody (TRAB) currently in Phase 1 clinical trial (NCT02748837). ERY974 consists of a common light chain and two different heavy chains that respectively recognize glypican-3 (GPC3) and CD3. The Fc portion of ERY974 is modified to lose FcγR binding to prevent GPC3-independent Fc-mediated effector function. However, binding activity to FcRn, an important factor in the PK profile of IgG, is maintained. ERY974 simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface, and induces TRAB-dependent cellular cytotoxicity mediated by the potent effector function of T cells. ERY974 shows strong antitumor activity against gastric, lung, ovarian, head & neck, and esophageal cancer-derived xenograft tumors in a non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse model injected with human T cells. Cancer immunotherapy, as represented by immune checkpoint inhibitors such as PD-1, PD-L1, and CTLA-4 antibodies, has recently been demonstrating remarkable clinical benefit in various tumor types. However, the number of patients who have survival benefit is limited, and combining cancer immunotherapy with other agents is required to improve the efficacy. Although ERY974 monotherapy is expected to show clinical activity based on the preclinical data, we examined whether further improvement of ERY974-induced efficacy is attained by combination with chemotherapy. Method & Results: We evaluated the combination effect of ERY974 with chemotherapy against xenograft tumors of MKN45 (gastric cancer) or NCI-H446 (lung cancer) either in a NOD-SCID mouse model injected with human T cells or in a humanized non-obese diabetic/shi-scid/IL-2Rγnull model in which differentiated human T cells are constitutively supplied. Although ERY974 monotherapy shows only minor antitumor effect against MKN45 and NCI-H446, combination therapy remarkably enhanced efficacy. In particular, ERY974 in combination with paclitaxel or cisplatin in NCI-H446 tumors caused a tumor disappearance without regrowth for a long period. Conclusion: These preclinical data suggest the possibility that the strategy of combining ERY974 with chemotherapy may succeed in increasing the clinical benefit. Now the combination effect is being further investigated to clarify the mechanism. Citation Format: Yuji Sano, Yumiko Azuma, Toshiaki Tsunenari, Yasuko Kinoshita, Yoko Kayukawa, Hironori Mutoh, Yoko Miyazaki, Takahiro Ishiguro, Shohei Kishishita, Yoshiki Kawabe, Mika Endo. Combining ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, with chemotherapy profoundly improved antitumor efficacy over its monotherapy in xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3653. doi:10.1158/1538-7445.AM2017-3653

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.