Anti-PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR-mutated NSCLC.

Treatment with anti-PD-1 and anti-PD-L1 therapies has shown durable clinical benefit in non-small cell lung cancer (NSCLC). However, patients with NSCLC with epidermal growth factor receptor (EGFR) mutations do not respond as well to treatment as patients without an EGFR mutation. We show that EGFR-mutated NSCLC expressed higher levels of CD73 compared with EGFR WT tumors and that CD73 expression was regulated by EGFR signaling. EGFR-mutated cell lines were significantly more resistant to T cell killing compared with WT cell lines through suppression of T cell proliferation and function. In a xenograft mouse model of EGFR-mutated NSCLC, neither anti-PD-L1 nor anti-CD73 antibody alone inhibited tumor growth compared with the isotype control. In contrast, the combination of both antibodies significantly inhibited tumor growth, increased the number of tumor-infiltrating CD8+ T cells, and enhanced IFN-γ and TNF-α production of these T cells. Consistently, there were increases in gene expression that corresponded to inflammation and T cell function in tumors treated with the combination of anti-PD-L1 and anti-CD73. Together, these results further support the combination of anti-CD73 and anti-PD-L1 therapies in treating EGFR-mutated NSCLC, while suggesting that increased T cell activity may play a role in response to therapy.

Antitumor efficacy of IPI-504, a selective heat shock protein 90 inhibitor against human epidermal growth factor receptor 2-positive human xenograft models as a single agent and in combination with trastuzumab or lapatinib.

IPI-504 is a novel, highly soluble small-molecule inhibitor of heat shock protein 90 (Hsp90), a protein chaperone essential for regulating homeostasis of oncoproteins and cell signaling proteins. Human epidermal growth factor receptor 2 (HER2; ErbB2) oncoprotein, expressed in a subset of metastatic breast cancers, is a Hsp90 client protein. In this study, we investigated the antitumor activity and the mechanism of action of IPI-504 in HER2(+), trastuzumab-sensitive and trastuzumab-refractory cell lines in vitro and in vivo. IPI-504 exhibited potent antiproliferative activities (range of IC(50), 10-40 nmol/L) against several tumor cell lines examined, whereby mechanism of action was mediated through HER2 and Akt degradation. Both intravenous and oral administration of IPI-504 assessed in multiple schedules showed potent tumor growth inhibition in vivo with corresponding degradation of HER2. The tolerability and efficacy of IPI-504 combined with either trastuzumab or lapatinib were also investigated in HER2(+) tumor xenograft models. Combination of IPI-504 with trastuzumab significantly enhanced tumor growth delay and induced greater responses when compared with either agent alone. Although, as expected, trastuzumab alone did not exhibit any significant antitumor activity in the trastuzumab-resistant JIMT-1 model, IPI-504 administered in combination with trastuzumab yielded greater antitumor efficacy than either agent alone. Finally, combination of IPI-504 and lapatinib was well tolerated up to 50 mg/kg IPI-504 and 100 mg/kg lapatinib and resulted in significant delay in tumor growth, including partial and complete tumor responses. These lines of evidence support the development of IPI-504 in HER2-positive breast cancers as a single agent and in combination with either trastuzumab or lapatinib

Antibody-dependent cell-mediated cytotoxicity effector-enhanced EphA2 agonist monoclonal antibody demonstrates potent activity against human tumors.

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated approximately 80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells.

Efficacy and antivascular effects of EphA2 reduction with an agonistic antibody in ovarian cancer.

BACKGROUND: EphA2 is an oncoprotein and tyrosine kinase receptor that is overexpressed in ovarian and many other cancers. We investigated the effects of reduced EphA2 levels on tumor growth and the tumor microenvironment in an orthotopic ovarian cancer model. METHODS: The effect of the EphA2-agonistic monoclonal antibody EA5, alone or in combination with paclitaxel, on the growth of ovarian cancer cells (SKOV3ip1, HeyA8, and HeyA8MDR [taxane-platinum resistant]) was determined in vitro and in vivo by immunoblotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and immunohistochemical analysis. Expression of EphA2 and markers of angiogenesis (CD31, vascular endothelial growth factor [VEGF], and basic fibroblast growth factor), proliferation (proliferating cell nuclear antigen), and endothelial cell apoptosis (CD31-terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate nick-end labeling colocalization) and phosphorylation of Src were analyzed by immunoblotting, immunohistochemistry, immunofluorescence, and in situ hybridization in tumors from treated mice. Statistical tests were two-sided. RESULTS: EA5 antibody treatment led to a more than 90% reduction in EphA2 expression in HeyA8 tumors in vivo. In mice bearing orthotopic SKOV3ip1 or HeyA8 tumors, 4 weeks of EA5 treatment resulted in tumors that weighed 31% and 45% less, respectively, than those in control (IgG-treated) mice (95% confidence interval [CI] = -0.09% to 71% and 20% to 70%, P = .27 and .01, respectively). Combination therapy with EA5 and paclitaxel reduced tumor weight by 77% and 80% (95% CI = 63% to 91% and 68% to 91%), respectively, compared with paclitaxel alone and by 92% and 88% (95% CI = 87% to 97% and 80% to 94%), respectively, compared with IgG alone. Combination therapy also reduced the weight of HeyA8MDR tumors by 47% (95% CI = 24% to 72%) compared with paclitaxel. Mice bearing SKOV3ip1 or HeyA8 tumors that were treated with combination therapy survived longer than those treated with paclitaxel alone (median survival = 144 versus 69 days and 46 versus 37 days, respectively). EA5-treated tumors had reduced microvascular density, proliferation, and VEGF protein and mRNA levels, with increased endothelial cell apoptosis. EphA2 was associated with Src, which was rapidly dephosphorylated after EA5 treatment. CONCLUSIONS: EA5 in combination with paclitaxel decreased tumor growth in an orthotopic ovarian cancer mouse model through antiangiogenic mechanisms associated with reduced levels of VEGF and phosphorylated Src. Humanized antibody constructs against EphA2 are worthy of future study.

Differential EphA2 epitope display on normal versus malignant cells.

The EphA2 receptor tyrosine kinase is overexpressed in many different types of human cancers where it functions as a powerful oncoprotein. Dramatic changes in the subcellular localization and function of EphA2 have also been linked with cancer, and in particular, unstable cancer cell-cell contacts prevent EphA2 from stably binding its ligand on the surface of adjoining cells. This change is important in light of evidence that ligand binding causes EphA2 to transmit signals that negatively regulate tumor cell growth and invasiveness and also induce EphA2 degradation. On the basis of these properties, we have begun to target EphA2 on tumor cells using agonistic antibodies, which mimic the consequences of ligand binding. In our present study, we show that a subset of agonistic EphA2 antibodies selectively bind epitopes on malignant cells, which are not available on nontransformed epithelial cells. We also show that such epitopes arise from differential cell-cell adhesions and that the stable intercellular junctions of nontransformed epithelial cells occlude the binding site for ligand, as well as this subset of EphA2 antibodies. Finally, we demonstrate that antibody targeting of EphA2 decreases tumor cell growth as measured using xenograft tumor models and found that the mechanism of antibody action relates to EphA2 protein degradation in vivo. Taken together, these results suggest new opportunities for therapeutic targeting of the large number of different cancers that express EphA2 in a manner that could minimize potential toxicities to normal cells.

Effects of the cyclooxygenase inhibitor, piroxicam, in combination with chemotherapy on tumor response, apoptosis, and angiogenesis in a canine model of human invasive urinary bladder cancer.

The objectives of this study were: (a) to determine the antitumor activity and toxicity of a cyclooxygenase inhibitor (piroxicam) combined with cisplatin chemotherapy in dogs with naturally-occurring, invasive transitional cell carcinoma (TCC) of the urinary bladder; and (b) to determine the effects of this treatment on prostaglandin E(2) concentration, tumor cell proliferation and apoptosis, and angiogenesis. Pet dogs with naturally-occurring invasive TCC underwent complete tumor staging before and after 10 weeks of piroxicam/cisplatin treatment. Prostaglandin E(2) concentrations were determined by immunoassay in snap-frozen tumor tissues. Apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling assay), proliferation (proliferating cell nuclear antigen), and microvessel density were determined in formalin-fixed tissues. Urine basic fibroblast growth factor and vascular endothelial cell growth factor concentrations were determined by immunoassay. Partial remission (> or =50% reduction in tumor volume) was noted in 6 of 12 dogs treated with piroxicam/cisplatin. Renal toxicity was dose-limiting. Apoptotic index doubled with treatment in 11 of 12 dogs but was not associated with tumor response. Proliferative index decreased in five dogs, and tumor decreased in size in three of the five dogs. Change in urine basic fibroblast growth factor and vascular endothelial cell growth factor was associated with tumor response. microvessel density was not associated with tumor response. In conclusion, piroxicam/cisplatin had antitumor activity against canine TCC, a disease that closely mimics human invasive urinary bladder cancer. Strategies to prevent renal toxicity of this protocol are needed. Induction of tumor apoptosis and reduction in angiogenic factor concentrations were observed, but additional studies are needed to further define the mechanisms of the antitumor activity of piroxicam/cisplatin.