ABL001, a Bispecific Antibody Targeting VEGF and DLL4, with Chemotherapy, Synergistically Inhibits Tumor Progression in Xenograft Models

Abstract

Delta-like-ligand 4 (DLL4) is a promising target to augment the effects of VEGF inhibitors. A simultaneous blockade of VEGF/VEGFR and DLL4/Notch signaling pathways leads to more potent anti-cancer effects by synergistic anti-angiogenic mechanisms in xenograft models. A bispecific antibody targeting VEGF and DLL4 (ABL001/NOV1501/TR009) demonstrates more potent in vitro and in vivo biological activity compared to VEGF or DLL4 targeting monoclonal antibodies alone and is currently being evaluated in a phase 1 clinical study of heavy chemotherapy or targeted therapy pre-treated cancer patients (ClinicalTrials.gov Identifier: NCT03292783). However, the effects of a combination of ABL001 and chemotherapy on tumor vessels and tumors are not known. Hence, the effects of ABL001, with or without paclitaxel and irinotecan were evaluated in human gastric or colon cancer xenograft models. The combination treatment synergistically inhibited tumor progression compared to each monotherapy. More tumor vessel regression and apoptotic tumor cell induction were observed in tumors treated with the combination therapy, which might be due to tumor vessel normalization. Overall, these findings suggest that the combination therapy of ABL001 with paclitaxel or irinotecan would be a better clinical strategy for the treatment of cancer patients.

Keywords: VEGF; anti-angiogenesis; delta-like ligand; irinotecan; paclitaxel; therapeutic antibody.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

ABL001 strongly inhibited tumor progression of various human gastric and colon cancer xenograft models. Tumor size was measured twice per week and compared between vehicle (closed circle) and ABL001 (closed triangle) in human gastric cancer (NUGC-3, MKN45, SNU16 for mABL001, and human patient-derived gastric cancer GAPF006 for ABL001) xenograft model (A) and human colon cancer (Colo205, WiDr, SW48, SW620 for mABL001) xenograft model (B). ABL001 treatment significantly delayed tumor progression in different cancer xenograft models compared to control group of vehicle treatment. Error bars: mean ± SEM.

Figure 2

ABL001 in combination with chemotherapy with paclitaxel or irinotecan synergistically inhibited tumor progression in human gastric PDX and colon cancer xenograft models. In GAPF006 human gastric PDX model (A), mice were treated with vehicle (closed circle, black), paclitaxel alone (closed rectangle, green), ABL001 (closed triangle, blue), or a combination of ABL001 and paclitaxel (closed reverse triangle, red). Compared to vehicle, each treatment group inhibited tumor progression (40.33% TGI in paclitaxel, 46.20% TGI in ABL001, and 74.75% TGI in the combination treatment). In the studies using SW48 (B) and SW620 (C) colon cancer xenograft models, mice were treated with vehicle (closed circle, black), irinotecan alone (closed rectangle, green), mABL001 (closed triangle, blue), or a combination of mABL001 and irinotecan (closed reverse triangle, red). In the case of both colon cancer xenograft models, the combination treatment of mABL001 and irinotecan showed the most potent effects on tumor progression (77.7% TGI in SW48 and 94.47% TGI in SW620 xenograft models). Each line represents the average tumor size (mm3) of each treatment group ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by Tukey’s test.

Figure 3

Combination therapy more potently regressed tumor blood vessels in SW620 xenograft model. Representative immunofluorescence images (A) show the tumor vasculature in SW620 tumor tissues stained for CD31, a generally conserved endothelial cell marker (green) and VEGFR-2 (red) with DAPI (blue). Most tumor blood vessels in vehicle group were stained and colocalized with both markers, CD31 and VEGFR-2. The area densities of CD31 (B) and VEGFR-2 (C) positive vessels were measured in each group. After irinotecan treatment, CD31 or VEGFR-2 positive tumor blood vessels were slightly regressed compared to vehicle treatment. However, after mABL001 or the combination treatment of mABL001 and irinotecan, CD31 and VEGFR-2 positive tumor vessels were significantly reduced (B,C). VEGFR-2 expression reduced more rapidly on tumor vessels. Scale bar indicates 200 µm. Error bars: mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001 by Kruskal–Wallis test.

Figure 4

ABL001 significantly reduced DLL4 expression in tumor blood vessels. Representative immunofluorescence images (A) indicate the tumor vasculature in SW620 tumor tissues stained for CD31 (green) and DLL4 (red). The bottom figures (A) are magnified images of the dotted region of the combination treatment of mABL001 and irinotecan. The left image was shown only by red channel, whereas the right one was shown by merged channels (red and green). Similar to VEGFR-2, DLL4 was stained and colocalized on CD31 positive tumor blood vessels. The area density of DLL4 (B) positive vessels was measured in tumors of each group. Compared to vehicle or irinotecan treatment, DLL4 positive tumor vessels were significantly reduced in tumors after mABL001 or the combination treatment. Some tumor vessels were stained only for CD31 but not for DLL4, after mABL001 or the combination treatment group (arrows and dotted box in A). Scale bar indicates 50 µm in the bottom two images and 100 µm in the other images. Error bars: mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001 by Kruskal–Wallis test.

Figure 5

Combination therapy markedly increased apoptotic tumor cells in SW620 xenograft model. Representative immunofluorescence images (A) reveal apoptotic cells stained for activated caspase-3 (green) with DAPI (blue) in SW620 tumor tissues. The area densities of activated caspase-3-positive apoptotic cells were measured in each group (B). Apoptotic cells in tumors were marginally increased after irinotecan or mABL001 treatment, but the increase was not significant compared to vehicle treatment. However, the combination treatment of mABL001 and irinotecan markedly increased the apoptotic cell population in tumors. Scale bar indicates 50 µm. Error bars: mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001 by Kruskal–Wallis test.