Kenji Hirotani

Anti-HER3 monoclonal antibody patritumab sensitizes refractory non-small cell lung cancer to the epidermal growth factor receptor inhibitor erlotinib

Human epidermal growth factor receptor (HER) 3 is aberrantly overexpressed and correlates with poor prognosis in non-small cell lung cancer (NSCLC). Patritumab is a monoclonal antibody against HER3 that has shown promising results in early-phase clinical trials, but an optimal target population for the drug has yet to be identified. In the present study, we examined whether heregulin, a HER3 ligand that is also overexpressed in a subset of NSCLC, can be used as a biomarker to predict the antitumorigenic efficacy of patritumab and whether the drug can overcome the epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) resistance induced by heregulin. Patritumab sensitivity was associated with heregulin expression, which, when abolished, resulted in the loss of HER3 and AKT activation and growth arrest. Furthermore, heregulin overexpression induced EGFR TKI resistance in NSCLC cells harbouring an activating EGFR mutation, while HER3 and AKT activation was maintained in the presence of erlotinib in heregulin-overexpressing, EGFR-mutant NSCLC cells. Sustained HER3-AKT activation was blocked by combining erlotinib with either anti-HER2 or anti-HER3 antibody. Notably, heregulin was upregulated in tissue samples from an NSCLC patient who had an activating EGFR mutation but was resistant to the TKI gefitinib. These results indicate that patritumab can overcome heregulin-dependent EGFR inhibitor resistance in NSCLC in vitro and in vivo and suggest that it can be used in combination with EGFR TKIs to treat a subset of heregulin-overexpressing NSCLC patients.

Tumorigenicity depends on angiogenic potential of tumor cells: dominant role of vascular endothelial growth factor and/or fibroblast growth factors produced by tumor cells

The aim of this study was to determine the role of tumor-derived angiogenic factors in solid tumor formation. We compared the angiogenic potential of tumorigenic and non-tumorigenic human tumor cell lines. All tumorigenic cell lines induced angiogenesis in vivo and their angiogenesis-inducing abilities were higher than those of the other non-tumorigenic cell lines. This in vivo angiogenic potential was well correlated with the in vitro endothelial cell growth-stimulating activity contained in the cell extract or conditioned medium of each cell line. The endothelial cell growth-stimulating activities of these cell lines were completely inhibited by neutralizing antibodies to basic fibroblast growth factor (bFGF), acidic FGF (aFGF) or vascular endothelial growth factor (VEGF). Furthermore, the levels of tumor-derived endothelial cell growth-stimulating activities depended on the amounts of angiogenic factors such as VEGF and bFGF produced by tumor cells. Although VEGF transcripts were detected in all of the cell lines by RT-PCR assay, the non-tumorigenic cell lines showed poor productivity of VEGF as well as FGFs and had less or non-potency for endothelial cell growth stimulation. These findings suggest that the increase in production of angiogenic factors by tumor cells is necessary for their in vivo angiogenic and tumorigenic potentials, and that VEGF and FGFs are the major mediators of tumor-induced angiogenesis.

Circulating heregulin level is associated with the efficacy of patritumab combined with erlotinib in patients with non-small cell lung cancer

OBJECTIVES Patritumab is a fully human anti-human epidermal growth factor receptor 3 (HER3) antibody that blocks activation by its ligand, heregulin (HRG). Preclinical studies have demonstrated the efficacy of patritumab in aberrantly high HRG-expressing non-small cell lung cancer (NSCLC). In the phase II randomized, placebo-controlled double-blind study HERALD (n=212 patients with NSCLC), patritumab plus erlotinib did not improve progression-free survival (PFS) compared with placebo plus erlotinib. The current study examined whether soluble HRG (sHRG) level in serum correlated with the efficacy of patritumab plus erlotinib. MATERIALS AND METHODS Serum was obtained from participants prior to treatment (n=202). sHRG level was measured using a validated quantitative immune assay, and correlations with survival were blindly assessed. RESULTS sHRG level was various (-1346-11,772pg/mL). Participants were divided into the sHRG-high or -low subgroups at the concentration defining near the third quartile, 980pg/mL. Patritumab plus erlotinib significantly improved PFS relative to placebo in the sHRG-high subgroup (n=46, hazard ratio 0.42 [0.19-0.96], p=0.0327). In contrast, the HRG-low subgroup (n=148) had no improvement in PFS with patritumab. CONCLUSION sHRG seems to be a predictive biomarker for the efficacy of patritumab plus erlotinib in NSCLC patients.

B7-H3 Negatively Modulates CTL-Mediated Cancer Immunity

Purpose: Anti-programmed-death-1 (PD-1) immunotherapy improves survival in non–small cell lung cancer (NSCLC), but some cases are refractory to treatment, thereby requiring alternative strategies. B7-H3, an immune-checkpoint molecule, is expressed in various malignancies. To our knowledge, this study is the first to evaluate B7-H3 expression in NSCLCs treated with anti-PD-1 therapy and the therapeutic potential of a combination of anti-PD-1 therapy and B7-H3 targeting. Experimental Design: B7-H3 expression was evaluated immunohistochemically in patients with NSCLC (n = 82), and its relationship with responsiveness to anti-PD-1 therapy and CD8+ tumor-infiltrating lymphocytes (TILs) was analyzed. The antitumor efficacy of dual anti-B7-H3 and anti-programmed death ligand-1 (PD-L1) antibody therapy was evaluated using a syngeneic murine cancer model. T-cell numbers and functions were analyzed by flow cytometry. Results: B7-H3 expression was evident in 74% of NSCLCs and was correlated critically with nonresponsiveness to anti-PD-1 immunotherapy. A small number of CD8+ TILs was observed as a subpopulation with PD-L1 tumor proportion score less than 50%, whereas CD8+ TILs were still abundant in tumors not expressing B7-H3. Anti-B7-H3 blockade showed antitumor efficacy accompanied with an increased number of CD8+ TILs and recovery of effector function. CD8+ T-cell depletion negated antitumor efficacy induced by B7-H3 blockade, indicating that improved antitumor immunity is mediated by CD8+ T cells. Compared with a single blocking antibody, dual blockade of B7-H3 and PD-L1 enhanced the antitumor reaction. Conclusions: B7-H3 expressed on tumor cells potentially circumvents CD8+-T-cell–mediated immune surveillance. Anti-B7-H3 immunotherapy combined with anti-PD-1/PD-L1 antibody therapy is a promising approach for B7-H3–expressing NSCLCs. Clin Cancer Res; 24(11); 2653–64. ©2018 AACR.

Evaluation of patritumab with or without erlotinib in combination with standard cytotoxic agents against pediatric sarcoma xenograft models

Integrating molecularly targeted agents with cytotoxic drugs used in curative treatment of pediatric cancers is complex. An evaluation was undertaken with the ERBB3/Her3‐specific antibody patritumab (P) either alone or with the ERBB1/epidermal growth factor receptor inhibitor erlotinib (E) in combination with standard cytotoxic agents, cisplatin, vincristine, and cyclophosphamide, in pediatric sarcoma xenograft models that express receptors and ligands targeted by these agents.

Abstract 3092: U3-1402a, a novel HER3-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent antitumor efficacy

Background HER3 (human epidermal growth factor receptor 3) is a member of HER family, and overexpressed in breast cancer, NSCLC, melanoma, gastric cancer and pancreatic cancer patients` tissues. U3-1402a is an antibody-drug conjugate (ADC) comprised of a fully human anti-HER3 monoclonal immunoglobulin G1 (IgG1) antibody (U3-1287) covalently conjugated via a cleavable peptide linker to exatecan derivative (DXd). The DXd is released after internalization of U3-1402a and leads to apoptosis of the target tumor cells by the inhibition of topoisomerase I. This ADC achieves a high drug-to-antibody-ratio (DAR 7 to 8) with homogeneous conjugation with the topoisomerase I inhibitor. The aim of this study was to preclinically evaluate the efficacy of U3-1402a in breast cancer models. Materials and methods In order to evaluate the pharmacological potential of U3-1402a, in vitro and in vivo studies were performed. In vitro growth inhibition assay evaluated the sensitivity of U3-1402a in HER3-positive human breast cancer cell line (HCC1569) and HER3-negative human cervical carcinoma cell line (C33A). Cells were treated with U3-1402a or MAAA-1181 (payload of U3-1402a) depending on its concentration (U3-1402a: 0.153 to 10 000 ng/mL, MAAA-1181: 2.44 to 160,000 pg/mL). In vivo growth inhibition study evaluated the dose-dependent sensitivity of U3-1402a in HER3-positive breast cancer xenograft model, MDA-MB-453. In addition, several xenograft models with different HER3 expression were tested with its sensitivity to U3-1402a. These models were HCC1569 (human breast cancer cell line, HER3 IHC score 3+), MDA-MB-453 (human breast cancer cell line, HER3 IHC score 2+), NIBIO-G016 (human gastric cancer patient-derived xenograft, HER3 IHC score 1+) and MDA-MB-231 (human breast cancer cell line, HER3 IHC score 0). R esults In vitro study, U3-1402a exhibited anti-tumor killing activity in HER3-positive human breast cancer cell line, HCC1569. C-33A human cervical carcinoma cell line was not sensitive to U3-1402a even MAAA-1181 itself exhibited anti-tumor killing activity to this cell line. In vivo study, U3-1402a showed dose-dependent anti-tumor killing activity in a HER3-positive breast cancer MDA-MB-453 xenograft model. Finally, in vivo tumor regression was only observed in HER3 2+ and 3+ models. Conclusions U3-1402a preclinically exhibited its efficacy in breast cancer model in vitro and in vivo. In vivo efficacy was correlated with HER3 expression. These studies suggest that U3-1402a, a novel HER3-targeting ADC, would be efficacious in a broader patient population with HER3 expression like breast cancer, melanoma, NSCLC, gastric cancer and pancreatic cancer. Citation Format: Suguru Ueno, Kenji Hirotani, Reimar Abraham, Sabine Blum, Birgit Frankenberger, Mauricio Redondo-Muller, Johannes Bange, Yusuke Ogitani, Akiko Zembutsu, Koji Morita, Takashi Nakada, Shuji Majima, Yuki Abe, Toshinori Agatsuma. U3-1402a, a novel HER3-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent antitumor efficacy [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 3092. doi:10.1158/1538-7445.AM2017-3092

Development of DS-5573a: A novel afucosylated mAb directed at B7-H3 with potent antitumor activity

B7‐H3 is highly overexpressed in a variety of human clinical tumors, and its expression is significantly associated with poor outcomes. In our study, we aimed to develop new antitumor mAbs by employing cancer cell immunization, and succeeded in generating a mouse anti‐human B7‐H3 antibody (M30) that shows antitumor activity. M30 was humanized (Hu‐M30), and an afucosylated Hu‐M30 (DS‐5573a) was also generated. To assess the potency of DS‐5573a as a therapeutic mAb, we characterized this mAb and evaluated its antitumor activity in vitro and in vivo. Flow cytometry analysis showed that B7‐H3 proteins were expressed on various types of cancer cell lines broadly, and DS‐5573a binds to IgC1 and IgC2 domains of human B7‐H3. Antibody‐dependent cellular cytotoxicity activity of DS‐5573a was drastically enhanced against medium to high B7‐H3‐expressing cancer cell lines MDA‐MB‐231 and NCI‐H322. DS‐5573a also induced high antibody‐dependent cellular cytotoxicity activity against low B7‐H3‐expressing cancer cell line COLO205, whereas Hu‐M30 induced little activity against it. In addition, DS‐5573a was found to be a novel anti‐B7‐H3 antibody which showed antibody‐dependent cellular phagocytosis activity. Furthermore, DS‐5573a showed dose‐dependent and significant antitumor efficacy (0.03–3 mg/kg) in MDA‐MB‐231‐bearing SCID mice (which have functional natural killer cells and macrophages), but little antitumor efficacy in NOG mice (which lack natural killer cells and have reduced macrophage function). These results suggest that antitumor activity of DS‐5573a is mediated by effector cells, and this mAb could be a promising antitumor therapy for patients with a wide range of B7‐H3‐expressing tumors.

An HER3-targeting antibody–drug conjugate incorporating a DNA topoisomerase I inhibitor U3-1402 conquers EGFR tyrosine kinase inhibitor-resistant NSCLC

EGFR tyrosine kinase inhibitors (TKIs) are standard therapy for EGFR-mutant non-small cell lung cancer (NSCLC); however, these tumours eventually acquire chemoresistance. U3-1402 is an anti-HER3 antibody–drug conjugate with a novel topoisomerase I inhibitor, DXd. In the current study, we evaluated the anticancer efficacy of U3-1402 in EGFR-mutant NSCLC cells with acquired resistance to EGFR-TKIs. HCC827GR5 and PC9AZDR7 are EGFR-TKI-resistant clones for gefitinib and osimertinib, respectively. U3-1402 alone or in combination with the EGFR-TKI erlotinib demonstrated potent anticancer efficacy in HCC827GR5 cells using an in vitro growth inhibition assay and in vivo xenograft mouse model. U3-1402 induced apoptosis in HCC827GR5 cells accompanying phosphorylation of histone H2A.X, a marker of DNA damage, but did not block HER3/PI3K/AKT signalling. Further, we found using flow cytometry that the cell surface HER3 expression level in HCC827GR5 cells was twice that found in HCC827 cells, indicating internalization of U3-1402 was increased in resistant cells. In addition, administration of U3-1402 notably repressed growth of EGFR-TKI osimertinib-resistant PC9AZDR7 xenograft tumours, and that PC9AZDR7 cells expressed five times greater cell surface HER3 than PC9 cells. Furthermore, using immunofluorescent microscopy, HER3 was observed predominantly in the nucleus of PC9 cells, but was localized in the cytoplasm of PC9AZDR7 cells. This finding indicates that altered trafficking of the HER3-U3-1402 complex may accelerate linker payload cleavage by cytoplasmic lysosomal enzymes, resulting in DNA damage. Our results indicate that administration of U3-1402 alone or in combination with an EGFR-TKI may have potential as a novel therapy for EGFR-TKI-resistant EGFR-mutant NSCLC.

Abstract 44: U3-1402, a novel HER3-targeting ADC, and a novel DNA topoisomerase I inhibitor inhibit the growth of non-small cell lung cancer with EGFR mutation

Background HER3, a member of the HER family, is overexpressed in non-small cell lung cancer (NSCLC), especially in those with EGFR mutation. Anti-HER3 antibody therapies, including patritumab, are effective, but limited in their efficacy, for patients with NSCLC. U3-1402 is a novel ADC composed of an anti-HER3 antibody (patritumab) and a novel potent topoisomerase I inhibitor DX-8951. U3-1402 achieved a high drug-to-antibody ratio (DAR: 7-8), because it is homogeneously conjugated with the payload. Here, we aimed to preclinically evaluate U3-1402’s efficacy in NSCLC, especially in those with EGFR mutation. Materials and methods An in vitro growth inhibition assay was used to evaluate the sensitivity of 14 NSCLC cell lines to U3-1402. Cells were treated with U3-1402 at different concentrations (0-10 μg/ml) over 7 days; 50% growth inhibitory concentration relative to control (IC50) was calculated. PC9, HCC827, HCC827GR5, Ma70, Ma70GR, 11-18, H1650, HCC4006, and H1975 cells had the EGFR mutation. HCC827GR and Ma70GR were EGFR-TKI resistant clones, with MET genomic amplification and an unknown resistance mechanism, established from HCC827 and Ma70 cells, respectively. HER3 mRNA expression levels were measured by quantitative reverse transcription-PCR (qRT-PCR) and the ratio was calculated against house-keeping genes in each cell line. Results The in vitro growth inhibition assay indicated that HCC827GR5, Ma70GR, and 11-18 cells were sensitive to U3-1402 (IC50 values 1.0, 5.2, 3.2 μg/ml, respectively). However, other cells were relatively resistant to U3-1402, having IC50 values greater than 10 μg/ml. Notably, HCC827GR5 cells were more sensitive to U3-1402 than parental HCC827 cells were. Specifically, 1.0 μg/ml U3-1402 reduced the viable cell proportion to 50% of control in HCC827GR5 cells, but the effect was limited to 95% of control in HCC827 parental cells. Furthermore, EGFR-TKI erlotinib increased sensitivity to U3-1402 in HCC827GR5 cells. Specifically, 1.0 μM erlotinib reduced the viable cell proportion to 69% of control, and 1.0 μg/ml U3-1402 reduced it to 50% of control in HCC827GR5 cells, but both agents combined reduced it to 3% of control. In an effort to clarify the underlying mechanism by which EGFR-TKI resistant HCC827GR5 cells were more sensitive to U3-1402 than parental HCC827 cells were, we evaluated the HER3 mRNA expression in both cell lines. HCC827GR5 cells had significantly higher levels of HER3 mRNA than parental HCC827 cells did (1.85 vs 0.65, t-test; p = 0.003). Conclusions U3-1402 preclinically exhibited its efficacy in NSCLC with EGFR mutation. Its efficacy was enhanced by EGFR-TKI combination. Sensitivity to U3-1402 might depend on HER3 expression levels. These results provide a rationale for U3-1402 alone or in combination with EGFR-TKI to be investigated in patients with NSCLC with EGFR mutation and aberrant HER3 expression. Citation Format: Kimio Yonesaka, Koji Haratani, Kenji Hirotani, Kazuhiko Nakagawa. U3-1402, a novel HER3-targeting ADC, and a novel DNA topoisomerase I inhibitor inhibit the growth of non-small cell lung cancer with EGFR mutation [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 44. doi:10.1158/1538-7445.AM2017-44

In vivo malignant phenotype of hst-1-transfected cells regulated by paracrine endothelial cell growth stimulation by HST-1.

The hst-1 gene product, one of the fibroblast growth factor family proteins, has transforming and angiogenic activities. The BALB/c 3T3 cell line was transfected with an expression vector harboring human hst-1 cDNA and the malignant properties of two stably transfected clones, TC-1 and TC-2, were examined. The stimulating activity of TC-1-conditioned medium for endothelial cell DNA synthesis was approximately four times stronger than that of TC-2-conditioned medium and correlated with hst-1 mRNA expression levels. Other than endothelial cell growth stimulation, these two clones had similar typical in vitro transformed properties, with identical doubling times and morphological changes. When nude mice were injected s.c. with these clones, TC-1 cells revealed faster tumor formation and growth, compared to TC-2 cells which had less potential to promote endothelial cell growth. Furthermore, the life span of mice injected i.v. with TC-1 cells was shorter than those with TC-2 cells, resulting from progressive tumor growth in the lungs. This advanced malignant in vivo behavior of TC-1 cells may be mediated by the high angiogenic potential of TC-1 cells secreting larger amounts of HST-1 compared to TC-2 cells, suggesting that angiogenesis contributes to malignant progression of tumors.