Efficacy of Cotargeting Angiopoietin-2 and the VEGF Pathway in the Adjuvant Postsurgical Setting for Early Breast, Colorectal, and Renal Cancers - PubMed (original) (raw)

Efficacy of Cotargeting Angiopoietin-2 and the VEGF Pathway in the Adjuvant Postsurgical Setting for Early Breast, Colorectal, and Renal Cancers

Florence T H Wu et al. Cancer Res. 2016.

Erratum in

Abstract

Antiangiogenic tyrosine kinase inhibitors (TKI) that target VEGF receptor-2 (VEGFR2) have not been effective as adjuvant treatments for micrometastatic disease in phase III clinical trials. Angiopoietin-2 (Ang2) is a proangiogenic and proinflammatory vascular destabilizer that cooperates with VEGF. The purpose of this study was to test whether CVX-060 (an Ang2-specific CovX-body) can be combined with VEGFR2-targeting TKIs (sunitinib or regorafenib) to successfully treat postsurgical metastatic disease in multiple orthotopically implanted human tumor xenograft and syngeneic murine tumor models. In the MDA-MB-231.LM2-4 human breast cancer model, adjuvant sunitinib was ineffective, whereas adjuvant CVX-060 delayed the progression of pulmonary or distant lymphatic metastases; however, overall survival was only improved with the adjuvant use of a VEGF-A/Ang2-bispecific CovX-body (CVX-241) but not when CVX-060 is combined with sunitinib. Adjuvant CVX-241 also showed promise in the EMT-6/CDDP murine breast cancer model, with or without an immune checkpoint inhibitor (anti-PD-L1). In the RENCA model of mouse renal cancer, however, combining CVX-060 with sunitinib in the adjuvant setting was superior to CVX-241 as treatment for postsurgical lung metastases. In the HCT116 and HT29 xenograft models of colorectal cancer, both CVX-060 and regorafenib inhibited liver metastases. Overall, our preclinical findings suggest differential strategies by which Ang2 blockers can be successfully combined with VEGF pathway targeting in the adjuvant setting to treat micrometastatic disease-particularly, in combination with VEGF-A blockers (but not VEGFR2 TKIs) in resected breast cancer; in combination with VEGFR2 TKIs in resected kidney cancer; and as single agents or with VEGFR2 TKIs in resected colorectal cancer. Cancer Res; 76(23); 6988-7000. ©2016 AACR.

©2016 American Association for Cancer Research.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

S.R. Pirie-Shepherd is an employee of Pfizer. U. Emmenegger reports receiving commercial research grants from Johnson and Johnson, Bayer, and Astellas and is a consultant/advisory board member for Bayer, Johnson and Johnson, Amgen, Sanofi, Astellas, Ferring, and Novartis. R.S. Kerbel has received speakers bureau honoraria from Boehringer Ingelheim, Regeneron, and Eli Lilly and is a consultant/advisory board member for Triphase Accelerator and Angiocrine Biosciences. No potential conflicts of interest were disclosed by the other authors.

Figures

Figure 1

Figure 1

Sunitinib versus Ang2-targeting CVX-060 in an orthotopic primary breast tumor model. Two experiments involving 10-day (A and B) or 17-day (C and D) treatment of mice bearing LM2-4luc16 breast tumors with vehicle (negative control), CVX-2000 (scaffold antibody for CVX-060), CVX-060 (Ang2-specific CovX-body), sunitinib (broad-spectrum TKI that potently inhibits VEGFR2), or CVX-060 + sunitinib. A and C, Tumor growth over time. Final tumor volumes were compared by t tests (*, P< 0.05; **, P < 0.01; n = 5; therapy vs. vehicle). Means ± SEM are depicted. Arrows, dosing schedules.B and D, Tumor bioluminescence, measured at 26 and 28 DPI, respectively, were log-transformed prior to t tests (*,P < 0.05; **, P < 0.01; ***,P < 0.001; ‡, P < 0.10; n = 5; vs. vehicle unless otherwise indicated).E, ELISA measurements of murine Ang2 in lung homogenates derived from healthy (tumor-free) control mice versus mice bearing LM2-4luc16 breast tumors after 10-day treatments with vehicle alone, CVX-060 (30 mg/kg/wk), sunitinib (60 mg/kg/d), or CVX-060 + sunitinib. Ang2 protein levels normalized to total protein levels (pg/mg) were compared by_t_ tests. Posttreatment tumor volumes are shown as mean ± SEM.

Figure 2

Figure 2

Effects of adjuvant Ang2-targeting CVX-060 versus sunitinib therapy on distant dissemination of breast cancer and postsurgical OS. Two experiments where orthotopic primary LM2-4luc16 breast tumors averaging 400 mm3 were resected at around 21 DPI to model the adjuvant therapy setting. Sunitinib (SU30, 30 mg/kg/d; SU60, 60 mg/kg/d), CVX-060 (30 mg/kg/wk), and their combinations were given as 1-week (A and B;n = 5–7) or 3-week (C andD; n = 8) adjuvant treatments. A, Bioluminescent quantification of thoracic metastatic burden. Log-transformed fluxes were analyzed by the Mann–Whitney test. Geometric means are depicted. B and D, Kaplan–Meier analysis of OS.C, Kaplan–Meier analysis of TTP of distant thoracic metastases. TTP and OS curves were subjected to the Gehan–Breslow–Wilcoxon test.

Figure 3

Figure 3

Adjuvant bispecific targeting of VEGF-A and Ang2 via CVX-241 after resection of primary breast tumors. A and B, Kaplan–Meier analysis of OS. Orthotopic primary breast tumors averaging 400 mm3were resected at 21 or 10 DPI in the LM2-4 (A; n = 8) or EMT-6/CDDP (B; n = 7–8) models, respectively. Adjuvant therapies were initiated 1 or 2 days later, involving sunitinib (VEGFR2 TKI; 60 mg/kg/d), CVX-060 (anti-Ang2; 30 mg/kg/wk), CVX-241 (anti-VEGF/Ang2; 30 mg/kg/wk), 10F.9G2 (anti-PD-L1; 5 mg/kg, 2×/wk), or combinations thereof. P values were derived using the log-rank test. Of the mice that had survived until the termination date of each experiment (62 and 45 days post-resection in A and B, respectively), none had visible lung metastases by necropsy. C andD, ELISA measurements of circulating murine Ang2 and VEGF protein in endpoint plasma samples from the EMT-6/CDDP experiment above. Medians with interquartile ranges are shown. P values were derived from_t_ tests on log-transformed data.

Figure 4

Figure 4

Regorafenib versus Ang2-targeting CVX-060 in orthotopic primary colorectal tumor models. Mice bearing established orthotopic colorectal HCT116luctumors (A and C) or HT29luc tumors (D) were treated for 3 weeks with vehicle, regorafenib (15 mg/kg/d), CVX-060 (30 mg/kg/wk), or regorafenib +CVX-060. A,B, and D, Primary cecal tumors were visualized and quantified by bioluminescent imaging. Log-transformed fluxes were compared by one-way ANOVA with Bonferroni correction (**, P ≤ 0.01; ***, P ≤ 0.001). Geometric means ± SEM are shown.C, Spontaneous liver micrometastases as detected by IHC staining of HLA+ tumor cells. Trends of treatment-related reductions were not statistically significant by the Kruskal–Wallis test (P > 0.05). Geometric means are depicted.

Figure 5

Figure 5

Effects of adjuvant-like regorafenib and Ang2-targeting CVX-060 therapies on colorectal cancer liver metastases. Mice were treated for 31 days with vehicle (n = 10), DC101 (0.8 mg, 2×/wk; n = 10), regorafenib (15 mg/kg/d; n = 10), CVX-060 (30 m/kg/wk;n = 10), or regorafenib + CVX-060 (n = 9) after intrasplenic HCT116luc implantation. Experimental liver metastases were detected by IHC staining for HLA+ tumor cells.A, Overall metastatic burden was subjected to one-way ANOVA with Dunnett posttest (*, P < 0.05); means ± SEM are depicted. **B**, Size distribution analysis of metastatic nodules was subjected to one-way ANOVA with Dunnett posttest. *, _P_< 0.05 versus vehicle for large (>5 mm2) nodules;‡, P < 0.05 versus vehicle for small (<0.5 mm2) nodules; means ± SEM are depicted.**C**, Representative HLA-stained liver sections. Black, gray, and white arrows, respectively, typical large (>5 mm2), medium (0.5–5 mm2), and small (<0.5 mm2)-sized metastatic nodules, for which ×100 magnifications are shown directly below (with corresponding black, gray, or white borders). "P" and "R" indicate a "pushing growth pattern" and "replacement growth pattern," respectively, as defined (38).

Figure 6

Figure 6

Combining Ang2 targeting and VEGF/VEGFR2 targeting in the metastatic (unresected) and adjuvant (postsurgical) settings of a renal cancer model. A,Mice with unresected kidney tumors were treated with vehicle, DC101 (anti-VEGFR2, 0.8 mg, 2×/wk), sunitinib (VEGFR2 TKI, 60 mg/kg/d), CVX-060 (anti-Ang2, 30 mg/kg/wk), or sunitinib plus CVX-060 beginning 9 days after orthotopic implantation of RENCAluc cells. Arrows, treatment durations. Differences in OS were subjected to log-rank (_P_log-rank) and Gehan–Breslow–Wilcoxon (_P_GBW) tests. B andC, Adjuvant therapies began 3 days after resection of orthotopic kidney tumors. Mice received vehicle, sunitinib (60 mg/kg/d), sunitinib + CVX-060 (30 mg/kg/wk), or CVX-241 (anti-Ang2/VEGF-A, 30 mg/kg/wk). Bioluminescent images of postsurgical lung metastases are shown (B). Kaplan–Meier survival analysis is shown (C), with arrows indicating dosing intervals (solid segments indicate continuous therapy; dotted orange segment indicates "2d-off/5d-on" weekly cycles).

Figure 7

Figure 7

Tissue concentrations of murine VEGF, Ang2, EPO, and PDGF-BB after adjuvant VEGF pathway targeting with or without Ang2 neutralization in a renal cancer model. Left kidneys bearing RENCAluc primary tumors were resected prior to adjuvant administration of vehicle, sunitinib, sunitinib + CVX-060, or CVX-241. Most mice, with the exception of two cases of suspected combination drug toxicity (Supplementary Fig. S8), reached endpoint with comparable metastatic burden in the lungs. From these mice, nontumor–bearing right kidneys were collected at endpoint and homogenized. Murine VEGF (A), PDGF-BB (B), Ang2 (C), and EPO (D) were measured from kidney homogenates by ELISA and normalized to total protein (pg/mg). Mean ± SEM and P values from t tests are depicted.

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