Koh Furugaki

Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells

Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.

Bevacizumab improves the delivery and efficacy of paclitaxel

It has been reported that bevacizumab in combination with paclitaxel significantly prolongs progression-free survival compared with paclitaxel alone in the initial treatment for metastatic breast cancer. To understand how bevacizumab enhances the efficacy of paclitaxel, we investigated the mechanism in a MX-1 human breast cancer xenograft model. The antitumor activity of bevacizumab at 5 mg/kg in combination with paclitaxel at 20 or 30 mg/kg was significantly higher than that of either agent alone. First, we measured the paclitaxel concentration in tumor to see whether bevacizumab enhances the activity by increasing the tumor concentration of paclitaxel. When given in combination with bevacizumab, the levels of paclitaxel in the tumor increased. Paclitaxel at 30 mg/kg with bevacizumab showed a similar tumor concentration as paclitaxel alone at either 60 or 100 mg/kg, with a similar degree of tumor growth inhibition. In contrast, no remarkable differences in paclitaxel concentration in the plasma or liver were observed between the paclitaxel monotherapy group and the paclitaxel plus bevacizumab group. An increase in paclitaxel concentration by bevacizumab was also found in another model, A549. In the same MX-1 model, vascular permeability in the tumor was significantly decreased by treatment with bevacizumab. There was no difference in microvessel density between the bevacizumab alone group and the combination group. Results suggest that the synergistic antitumor activity of paclitaxel and bevacizumab in combination may be a result of the increase in paclitaxel concentration in tumor resulting from the downregulation of vascular permeability when co-administered with bevacizumab.

Erlotinib inhibits osteolytic bone invasion of human non-small-cell lung cancer cell line NCI-H292

Previous preclinical and clinical findings have suggested a potential role of epidermal growth factor receptor (EGFR) in osteoclast differentiation and the pathogenesis of bone metastasis in cancer. In this study, we investigated the effect of erlotinib, an orally active EGFR tyrosine kinase inhibitor (TKI), on the bone invasion of human non-small-cell lung cancer (NSCLC) cell line NCI-H292. First, we established a novel osteolytic bone invasion model of NCI-H292 cells which was made by inoculating cancer cells into the tibia of scid mice. In this model, NCI-H292 cells markedly activated osteoclasts in tibia, which resulted in osteolytic bone destruction. Erlotinib treatment suppressed osteoclast activation to the basal level through suppressing receptor activator of NF-κB ligand (RANKL) expression in osteoblast/stromal cell at the bone metastatic sites, which leads to inhibition of osteolytic bone destruction caused by NCI-H292 cells. Erlotinib inhibited the proliferation of NCI-H292 cells in in vitro. Erlotinib suppressed the production of osteolytic factors, such as parathyroid hormone-related protein (PTHrP), IL-8, IL-11 and vascular endothelial growth factor (VEGF) in NCI-H292 cells. Furthermore, erlotinib also inhibited osteoblast/stromal cell proliferation in vitro and the development of osteoclasts induced by RANKL in vitro. In conclusion, erlotinib inhibits tumor-induced osteolytic invasion in bone metastasis by suppressing osteoclast activation through inhibiting tumor growth at the bone metastatic sites, osteolytic factor production in tumor cells, osteoblast/stromal cell proliferation and osteoclast differentiation from mouse bone marrow cells.

Loss of an EGFR-amplified chromosome 7 as a novel mechanism of acquired resistance to EGFR-TKIs in EGFR-mutated NSCLC cells.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) show notable effects against non-small cell lung cancers (NSCLCs) harboring EGFR-activating mutations. However, almost all patients eventually acquire resistance to EGFR-TKIs. In this study, we established novel erlotinib resistant NSCLC cells and examined their resistant mechanisms. Resistant cells were established in 14, 3, and 0 wells exposed to 0.1, 1, and 10 μM erlotinib, respectively. The IC(50) values of these cells were 47- to 1209-fold higher than that of the parent cells. No secondary T790M mutation was detected in any resistant cells. However, in 13/17 resistant cells, EGFR copy number was reduced less than approximately one-eighth of parent cells, and in one resistant cell (B10), >99.99% of the population was EGFR-unamplified cells. Most (97.5%) parent cells showed EGFR amplification, but 2.5% of the population comprised EGFR-unamplified cells. An EGFR-unamplified clone (4D8) isolated from parent cells in erlotinib-free normal medium also showed erlotinib resistance comparable to the resistant B10 cells. Loss of an EGFR-amplified chromosome 7 (EGFR-ampch7) was observed in 4D8 and B10 cells. EGFR-unamplified cells were constantly maintained as a minor population of the parent cells under normal cell culture conditions. In conclusion, loss of an EGFR-ampch7 causes acquired resistance in EGFR-mutated HCC827 cells exposed to a relatively low concentration of erlotinib, but a high concentration prevents the emergence of resistance.

Impact of bevacizumab in combination with erlotinib on EGFR-mutated non-small cell lung cancer xenograft models with T790M mutation or MET amplification.

Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non–small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression‐free survival in patients with EGFR‐mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR‐mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR‐mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug‐cessation period. In the HCC827‐EPR model (DEL+T790M) and HCC827‐vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI‐H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL‐resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation‐ or MET amplification‐positive tumors as long as their growth remained significantly suppressed by ERL.

Bevacizumab counteracts VEGF-dependent resistance to erlotinib in an EGFR-mutated NSCLC xenograft model

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), shows superior efficacy in patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations (EGFR Mut+). However, almost all tumors eventually develop resistance to erlotinib. Recently, the Phase II JO25567 study reported significant prolongation of progression-free survival (PFS) by erlotinib plus bevacizumab combination compared with erlotinib in EGFR Mut+ NSCLC. Herein, we established a preclinical model which became refractory to erlotinib after long-term administration and elucidated the mode of action of this combination. In this model, tumor regrowth occurred after remarkable shrinkage by erlotinib; regrowth was successfully inhibited by erlotinib plus bevacizumab. Tumor vascular endothelial growth factor (VEGF) was greatly reduced by erlotinib in the erlotinib-sensitive phase but significantly increased in the erlotinib-refractory phase despite continued treatment with erlotinib. Although EGFR phosphorylation remained suppressed in the erlotinib-refractory phase, phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were markedly higher than in the erlotinib-sensitive phase; among these, pERK was suppressed by erlotinib plus bevacizumab. MVD was decreased significantly more with erlotinib plus bevacizumab than with each drug alone. In conclusion, the erlotinib plus bevacizumab combination demonstrated promising efficacy in the B901L xenograft model of EGFR Mut+ NSCLC. Re-induction of VEGF and subsequent direct or indirect VEGF-dependent tumor growth was suggested as a major mechanism of erlotinib resistance, and erlotinib plus bevacizumab achieved remarkably prolonged antitumor activity in this model.

Increased EGFR Phosphorylation Correlates with Higher Programmed Death Ligand-1 Expression: Analysis of TKI-Resistant Lung Cancer Cell Lines

Despite the recent development of immunotherapies that target programmed death-1 (PD-1) or programmed death ligand-1 (PD-L1) in non-small cell lung cancer (NSCLC) treatment, these therapies are less effective in NSCLC patients with epidermal growth factor receptor (EGFR) mutations. However, the molecular mechanisms underlying this lower efficacy of immunotherapies in EGFR mutant lung cancers are still unclear. In this study, we analyzed PD-L1 protein expression in lung cancer cell lines with EGFR mutations prior to and after acquisition of resistance to EGFR tyrosine kinase inhibitors (TKIs). We found that parental lung cancer cell lines harboring EGFR mutations showed negative (PC9 and H3255 cells) and positive (HCC827 cells) staining for PD-L1 by immunohistochemistry. Comparing PD-L1 expression between EGFR-TKI resistant cell lines and their parental cells, we found that increased phosphorylation of EGFR was related to increased expression of PD-L1. Increased phosphorylation of EGFR was accompanied by the T790M secondary mutation. Acquired resistance cells with MET amplification or EGFR loss both showed decreased phosphorylation of EGFR and decreased PD-L1 expression. Our results indicate that lung cancer cell lines with EGFR mutations (parental cells) do not harbor high PD-L1 protein expression. In addition, EGFR phosphorylation affects PD-L1 expression after acquisition of resistance to EGFR-TKIs.

1207PANTITUMOR ACTIVITY OF BEVACIZUMAB COMBINED WITH ERLOTINIB IN T790M RESISTANCE MUTATION POSITIVE NON-SMALL CELL LUNG CANCER XENOGRAFT MODELS

ABSTRACT Aim: Erlotinib (ERL), a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, benefits patients with non-small cell lung cancer (NSCLC), especially those with EGFR active mutations. However, progressive disease occurs when resistance is acquired by T790M mutation or MET amplification. Bevacizumab (BEV), a humanized anti-vascular endothelial cell growth factor monoclonal antibody, has been demonstrated to be effective in combination with standard chemotherapies for advanced NSCLC patients. We previously reported the promising efficacy of combining ERL and BEV in mouse models of NSCLC harboring EGFR exon 19 deletion. In the present study, we examined the antitumor activity of BEV combined with ERL in NSCLC models harboring T790M resistance mutation. Methods: BALB-nu/nu mice were subcutaneously inoculated with NSCLC cell lines, NCI-H1975 (L858R + T790M mutation) or HCC827-EPR (exon19 deletion + T790M mutation). After randomization on Day 1, BEV (5 mg/kg) was intraperitoneally administered once a week, and ERL (75 mg/kg) was orally given daily. Antitumor activity was evaluated by tumor growth inhibition (TGI) with measuring tumor volume. In tumor tissues, phosphorylations of EGFR signaling molecules were evaluated by western blot analysis, and microvessel density was evaluated by CD31 immunohistochemistry. Results: In the NCI-H1975 model, the TGI on Day 11 of ERL, BEV and combination was 8%, 45% and 35%, respectively. NCI-H1975 tumors were resistant to ERL, and the combination of ERL and BEV did not enhance the antitumor activity of BEV monotherapy. In the HCC827-EPR model, the TGI on Day 50 of ERL, BEV and combination was 96%, 63% and 111%, respectively. HCC827-EPR tumors exhibited significant sensitivity to ERL (p ≤ 0.05) in vivo, and combining the two agents showed significantly higher antitumor activity than each monotherapy (p ≤ 0.05). The phosphorylation levels of EGFR, AKT or ERK in HCC827-EPR tumors were suppressed by ERL. The microvessel density was significantly decreased by BEV in both models. Conclusions: The combination treatment of ERL and BEV has the potential to be effective for ERL-responding NSCLC models with T790M resistance mutation. Disclosure: T. Mitsudomi: Dr.Mitsudomi has received research funds from Astra Zeneca, Chugai Pharma, Boehringer Ingelheim, Roche, Pfizer. All other authors have declared no conflicts of interest.