anti-VEGFR2 Antibody Research Articles

Evaluation of Microwave Ablation in 4T1 Breast Tumor by a Novel VEFGR2 Targeted Ultrasound Contrast Agents

ObjectivesA novel ultrasound contrast agent (UCA) VEGFR2-targeting iron-doped silica (SiO2) hollow nanoparticles (VEGFR2-PEG-HSNs-Fe NPs) was prepared and applied in microwave ablation for breast cancer to investigate its value in the evaluation of effectiveness after tumor ablation.MethodsVEGFR2-PEG-HSNs-Fe NPs were prepared by using nano-SiO2, which was regarded as a substrate and etched by ferrous acetate, and then modified with anti-VEGFR2 antibody. Laser confocal microscope and flow cytometry were used to observe its main physicochemical properties, and biological safety was also investigated. After the xenograft tumor was treated with microwave ablation, the extent of perfusion defect was evaluated by ultrasound by injecting VEGFR2-PEG-HSNs-Fe NPs.ResultsThe average particle size of VEGFR2-PEG-HSNs-Fe was 276.64 ± 30.31 nm, and the surface potential was −13.46 ± 2.83 mV. In vitro, the intensity of ultrasound signal increased with UCA concentration. Good biosafety was performed in in vivo and in vitro experiments. The enhanced ultrasound signal was detected in tumors after injection of VEGFR2-PEG-HSNs-Fe NPs, covering the whole tumor. The lesions, which were incompletely ablated, presented as contrast agent perfusion at the periphery of the tumor, and contrast enhanced ultrasound (CEUS) was performed again after complementary ablation. It was confirmed that all the lesions were completely ablated.ConclusionNano-targeted UCAs VEGFR2-PEG-HSNs-Fe NPs had good biosafety and ability of specific imaging, which might be used as a contrast agent in CEUS to evaluate the efficacy of tumor ablation.

Abstract 2649: An ex vivo 3D tumoroid model of fresh patient tissue (3D-EX) to assess the efficacy of anti-angiogenic compounds in renal cell carcinoma

Abstract Introduction: Renal Cell Carcinoma (RCC) is highly resistant to systemic chemotherapy; however, targeting pro-angiogenic pathways has shown significant clinical benefit. The development of targeted anti-angiogenetic agents has been hampered by the lack of clinically relevant models for screening. We have previously reported that the generation of 3D-tumoroids containing the unaltered tumor stroma could effectively be used to detect tumor response to small molecule inhibitors as well as antibody-based therapeutic agents. In this study we described a novel ex vivo platform to develop anti-angiogenic therapeutic strategies using fresh patient tumoroids of hepatocellular carcinoma and renal cell carcinoma. Materials and Methods: All fresh tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays, 3D tumoroids measuring 150 µm in size were treated with tyrosine kinase inhibitors (TKIs) sunitinib, sorafenib or axitinib. Treatment-mediated changes in endothelial cell viability was assessed by the confocal-based high-content real time imaging platform using fluorescent labeled anti-CD31 and anti-VEGFR2 antibodies, combined with custom image analysis algorithms. Additionally, treatment-mediated changes in tumor immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells, macrophages, cell surface expression of checkpoint proteins as well as T-cell activation were evaluated by multiplex flow cytometry. Results: Our data shows that endothelial cells and capillary structures could clearly be visualized within3D tumoroids by confocal microscopy. Furthermore, we were able to quantify apoptotic cell death in endothelial cells induced by TKI inhibitors. Flow cytometry analysis demonstrated significant changes in T-cell activation upon treatment with TKIs that closely correlated with increased proinflammatory cytokine release. Observations found that the combination of anti-angiogenic treatments and immuno-modulatory agents significantly improved treatment responses. Conclusion: The ex vivo platform described in this study allowed assessment of the effect of anti-angiogenetic TKIs on tumor capillary endothelial cells as well as on tumor resident immune cell populations in intact RCC tumoroids of fresh patient tumor samples. We believe this clinically relevant approach can be used to identify the most effective anti-angiogenic and immunotherapeutic drugs and drug combinations in RCC and may improve personalized therapy for individual patients in clinical studies. Citation Format: Jared Ehrhart, Mibel M. Pabón, Stephen Iwanowycz, Zhisong Tong, Tina Pastoor, Soner Altiok. An ex vivo 3D tumoroid model of fresh patient tissue (3D-EX) to assess the efficacy of anti-angiogenic compounds in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2649.

A robust enzyme-linked immunosorbent assay to measure serum ramucirumab concentrations.

Aim: Ramucirumab, an anti-VEGFR2 monoclonal antibody, has been approved for the treatment of metastatic gastric and colorectal cancer. An assay measuring ramucirumab serum concentrations was needed to investigate its pharmacokinetics and concentration-response relationship. Results: An ELISA was developed and validated according to the international guidelines for ligand-binding assays. Ramucirumab calibration standards ranged from 0.125 to 40mg/l. Low, middle and high quality controls were spiked at 0.2, 4 and 8mg/l, respectively. The limits of quantification were established to be 0.125 and 10mg/l for LLOQ and ULOQ, respectively. No cross-reactivity with anti-VEGF or anti-EGFR was detected. Conclusion: This in-house-developed ELISA is sensitive, accurate, reproducible and suitable for pharmacokinetic studies of ramucirumab.

Regorafenib enhances antitumor immunity via inhibition of p38 kinase/Creb1/Klf4 axis in tumor-associated macrophages

BackgroundRegorafenib and other multikinase inhibitors may enhance antitumor efficacy of anti-program cell death-1 (anti-PD1) therapy in hepatocellular carcinoma (HCC). Its immunomodulatory effects, besides anti-angiogenesis, were not clearly defined.MethodsIn vivo antitumor...

VEGFR2-targeted antibody fused with IFNαmut regulates the tumor microenvironment of colorectal cancer and exhibits potent anti-tumor and anti-metastasis activity

Although interferon α (IFNα) and anti-angiogenesis antibodies have shown appropriate clinical benefit in the treatment of malignant cancer, they are deficient in clinical applications. Previously, we described an anti-vascular endothelial growth factor receptor 2 (VEGFR2)-IFNα fusion protein named JZA01, which showed increased in vivo half-life and reduced side effects compared with IFNα, and it was more effective than the anti-VEGFR2 antibody against tumors. However, the affinity of the IFNα component of the fusion protein for its receptor-IFNAR1 was decreased. To address this problem, an IFNα-mutant fused with anti-VEGFR2 was designed to produce anti-VEGFR2-IFNαmut, which was used to target VEGFR2 with enhanced anti-tumor and anti-metastasis efficacy. Anti-VEGFR2-IFNαmut specifically inhibited proliferation of tumor cells and promoted apoptosis. In addition, anti-VEGFR2-IFNαmut inhibited migration of colorectal cancer cells and invasion by regulating the PI3K–AKT–GSK3β–snail signal pathway. Anti-VEGFR2-IFNαmut showed superior anti-tumor efficacy with improved tumor microenvironment (TME) by enhancing dendritic cell maturation, dendritic cell activity, and increasing tumor-infiltrating CD8+ T cells. Thus, this study provides a novel approach for the treatment of metastatic colorectal cancer, and this design may become a new approach to cancer immunotherapy.

Pleiotrophin selectively binds to vascular endothelial growth factor receptor 2 and inhibits or stimulates cell migration depending on ανβ3 integrin expression.

Pleiotrophin (PTN) has a moderate stimulatory effect on endothelial cell migration through ανβ3 integrin, while it decreases the stimulatory effect of vascular endothelial growth factor A (VEGFA) and inhibits cell migration in the absence of ανβ3 through unknown mechanism(s). In the present work, by using a multitude of experimental approaches, we show that PTN binds to VEGF receptor type 2 (VEGFR2) with a KD of 11.6nM. Molecular dynamics approach suggests that PTN binds to the same VEGFR2 region with VEGFA through its N-terminal domain. PTN inhibits phosphorylation of VEGFR2 at Tyr1175 and still stimulates endothelial cell migration in the presence of a selective VEGFR2 tyrosine kinase inhibitor. VEGFR2 downregulation by siRNA or an anti-VEGFR2 antibody that binds to the ligand-binding VEGFR2 domain also induce endothelial cell migration, which is abolished by a function-blocking antibody against ανβ3 or the peptide PTN112-136 that binds ανβ3 and inhibits PTN binding. In cells that do not express ανβ3, PTN decreases both VEGFR2 Tyr1175 phosphorylation and cell migration in a VEGFR2-dependent manner. Collectively, our data identify VEGFR2 as a novel PTN receptor involved in the regulation of cell migration by PTN and contribute to the elucidation of the mechanism of activation of endothelial cell migration through the interplay between VEGFR2 and ανβ3.

Systemic treatment of hepatocellular carcinoma: from sorafenib to combination therapies.

For almost a decade, systemic therapy of advanced hepatocellular carcinoma (HCC) was limited to the tyrosine kinase inhibitor (TKI) sorafenib. Different agents including checkpoint inhibitors, TKIs and anti-VEGFR antibodies demonstrated efficacy in treatment. For the first time, the combination of atezolizumab and bevacizumab, a first-line treatment that is superior to the current standard was identified, potentially changing the way we treat HCC. In this review, we summarize current data on systemic treatment of patients with advanced HCC, focusing on combination therapies comprising immune checkpoint inhibitors, TKIs and locoregional therapies. We elucidate findings from recent trials and discuss such challenges as the lack of predictive biomarkers for identification of subgroups that will benefit from novel treatment strategies.

Hybrid membrane camouflaged copper sulfide nanoparticles for photothermal-chemotherapy of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Biomimetic nanoparticles (NPs) coated with cell membranes show enhanced biocompatibility and specificity for homotypic cells, and have gained considerable attention for targeted anti-tumor therapy. We constructed cancer cell-macrophage hybrid membrane-coated near infrared (NIR)-responsive hollow copper sulfide nanoparticles encapsulating sorafenib and surface modified with anti-VEGFR (CuS-SF@CMV NPs). These CuS-SF@CMV NPs expressed the characteristic membrane proteins of both cancer cells and macrophages, and selectively accumulated in cancer cells in vitro and tumors in vivo, compared to the CuS NPs. In addition, the CuS-SF@CMV NPs achieved synergistic photo-thermal and chemotherapy in cancer cells upon NIR irradiation, with 94.3% inhibition of tumor growth in a murine hepatoma model. While the initial increase in temperature rapidly killed the tumor cells, sorafenib and the anti-VEGFR antibody sustained the tumor killing effect by respectively inhibiting tumor cell proliferation and angiogenesis via the Ras/Raf/MEK/ERK and PI3K/AKT pathways. Taken together, the CuS-SF@CMV NPs have immune evasion, tumor cell targeting and drug loading capacities, along with an inherent photo-thermal conversion ability, making them ideal for synergistic photo-thermal/chemo therapy against HCC. Statement of SignificanceWe created cancer cell-macrophage hybrid membrane-coated hollow CuS NPs encapsulating sorafenib and surface modified with anti-VEGFR antibodies (CuS-SF@CMV). These CuS-SF@CMV NPs enhanced synergistic PTT and chemotherapy against hepatoma cells through homotypic cell targeting, immune escape and inhibition of a tumorigenic signaling pathway. A long-term inhibition of tumor growth and metastasis was achieved owing to the rapid destruction of the cancer cells through photo-thermal conversion by the CuS NPs, and sustained clearance of the tumor cells by sorafenib and anti-VEGFR antibodies.Our findings suggest that CuS-SF@CMV NPs present great treating effects in preclinical models of HCC, providing the framework for further study in clinical trials to improve patient outcome in hepatocellular carcinoma.

Sorafenib-Loaded Long-Circulating Nanoliposomes for Liver Cancer Therapy.

A type of sorafenib- (SOR-) loaded long-circulating nanoliposome was constructed, and the targeting performance and antitumor effects of the prepared liposome were evaluated in the present study. Polyethylene glycol- (PEG-) modified long-circulating nanoliposomes (LC-NPs) were designed and prepared using reverse evaporation, and the LC-NPs were used for delivering sorafenib (LC-PEG-SOR-NPs). Then, the anti-VEGFR antibody as a targeting moiety was chemically coupled with LC-PEG-SOR-NPs to form liver cancer-targeted nanoliposomes (anti-VEGFR-LC-PEG-SOR-NPs). The drug entrapment and loading efficiency were measured. And the cancer-targeting performance and therapeutic efficiency were evaluated both in vitro and in vivo. The anti-VEGFR-LC-PEG-SOR-NPs with an average of 119.8 ± 4.2 nm showed a uniform spherical structure. The drug entrapment and loading efficiency were 92.5% and 18.5%, respectively. The killing efficiency of anti-VEGFR-LC-PEG-SOR-NPs was up to 18% after incubating with liver cancer cells for 72 h. Furthermore, the anti-VEGFR-LC-PEG-SOR-NPs could actively target at the tumor region and could efficiently inhibit tumor growth with negligible side effects. This newly designed nanoliposomes had desirable dispersibility, high drug entrapment efficiency, tumor targeting and therapeutic efficiency, and good safety. As a biocompatible nanocomposite, it was promising to become a novel and useful tumor-targeting nanodrug for liver cancer therapy.

Anti-VEGF/VEGFR2 Monoclonal Antibodies and their Combinations with PD-1/PD-L1 Inhibitors in Clinic.

The vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway is one of the most important pathways responsible for tumor angiogenesis. Currently, two monoclonal antibodies, anti-VEGF-A antibody Bevacizumab and anti-VEGFR2 antibody Ramucizumab, have been approved for the treatment of solid tumors. At the same time, VEGF/VEGFR2 signaling is involved in the regulation of immune responses. It is reported that the inhibition of this pathway has the capability to promote vascular normalization, increase the intra-tumor infiltration of lymphocytes, and decrease the number and function of inhibitory immune cell phenotypes, including Myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and M2 macrophages. On this basis, a number of clinical studies have been performed to investigate the therapeutic potential of VEGF/VEGFR2-targeting antibodies plus programmed cell death protein 1 (PD-1)/ programmed cell death ligand 1 (PD-L1) inhibitors in various solid tumor types. In this context, VEGF/VEGFR2- targeting antibodies, Bevacizumab and Ramucizumab are briefly introduced, with a description of the differences between them, and the clinical studies involved in the combination of Bevacizumab/ Ramucizumab and PD-1/PD-L1 inhibitors are summarized. We hope this review article will provide some valuable clues for further clinical studies and usages.

Endothelium capture-based liver segment imaging using vascular endothelial growth factor receptor 2 in preclinical ex vivo models.

BackgroundNear‐infrared (NIR) imaging of liver segments provides substantial information for surgeons performing liver resection. It was hypothesized that ramucirumab, an endothelium‐specific antibody approved by the Food and Drug Administration, could be used for liver segment imaging using the endothelium capture principle.MethodsThe capture efficacy of anti‐vascular endothelial growth factor receptor (VEGFR) 2 monoclonal antibodies (mAbs) and segment imaging were studied in a mouse model. Binding of ramucirumab in human and porcine tissues was studied using immunofluorescence staining. Isolated porcine liver perfusion was used to analyse the labelling and NIR imaging of selected liver segments.ResultsVEGFR2 is well expressed on the endothelium of the smallest microvascular blood vessels in mouse, porcine and human liver tissues, as well as in human liver tumours. Perfusion of selected segments in the isolated liver model showed high capture of the anti‐VEGFR2 (clone 522302) mAb and ramucirumab in mice and pigs respectively. NIR imaging of selected segments was achieved using isolated porcine liver perfusion with IRDye® 800CW‐conjugated ramucirumab.ConclusionVEGFR2 is well expressed on the smallest microvascular blood vessels and can capture antibodies during single intravascular passages with high efficacy. The ex vivo imaging of a selected segment using endothelial capture of ramucirumab demonstrates the potential of this antibody for intraoperative navigation in liver surgery. Surgical relevanceImaging of liver segments provides substantial information for surgeons when performing liver resection.The antivascular endothelial growth factor receptor (VEGFR) 2 antibody ramucirumab conjugated with near‐infrared dye could visualize selected liver segments using an endothelial capture‐based approach in an isolated perfusion liver model.The ex vivo imaging of a selected segment using endothelial capture of ramucirumab demonstrates the potential of this anti‐VEGFR2 antibody for intraoperative navigation in liver surgery.

Ultrasound-Targeted Microbubble Destruction Enhances the Antitumor Efficacy of Doxorubicin in a Mouse Hepatocellular Carcinoma Model

The aim of the study described here was to investigate whether ultrasound-mediated microbubble destruction (UTMD) of targeted microbubbles conjugated with an anti-vascular endothelial growth factor receptor 2 (anti-VEGFR2) antibody can enhance the therapeutic effect of doxorubicin (DOX) on a mouse hepatocellular carcinoma (HCC) model bearing HEP-G2-RFP tumors. The growth of liver tumors in mice was inhibited by using Visistar VEGFR2 plus ultrasound irradiation and by DOX alone. DOX plus UTMD had an inhibitory effect on tumor growth beginning on the seventh day of treatment, while Visistar VEGFR2 alone and DOX alone had inhibitory effects beginning on the 11th day. DOX + UTMD significantly decreased tumor volume and tumor weight compared with DOX alone (p < 0.05) and Visistar VEGFR2 alone (p < 0.05). Compared with DOX alone and Visistar VEGFR2 alone, DOX + UTMD had the highest inhibitory effect on tumor angiogenesis and the highest apoptosis index. UTMD-targeted microbubbles can significantly enhance the antitumor effect of DOX on a mouse HCC model, inhibit angiogenesis and induce apoptosis in tumor cells.

Loss of Endothelial Endoglin Promotes High-Output Heart Failure Through Peripheral Arteriovenous Shunting Driven by VEGF Signaling.

ENG (endoglin) is a coreceptor for BMP (bone morphogenetic protein) 9/10 and is strongly expressed in endothelial cells. Mutations in ENG lead to the inherited vascular disorder hereditary hemorrhagic telangiectasia characterized by local telangiectases and larger arteriovenous malformations (AVMs); but how ENG functions to regulate the adult vasculature is not understood. The goal of the work was to determine how ENG maintains vessel caliber in adult life to prevent AVM formation and thereby protect heart function. Genetic depletion of endothelial Eng in adult mice led to a significant reduction in mean aortic blood pressure. There was no evidence of hemorrhage, anemia, or AVMs in major organs to explain the reduced aortic pressure. However, large AVMs developed in the peripheral vasculature intimately associated with the pelvic cartilaginous symphysis-a noncapsulated cartilage with a naturally high endogenous expression of VEGF (vascular endothelial growth factor). The increased blood flow through these peripheral AVMs explained the drop in aortic blood pressure and led to increased cardiac preload, and high stroke volumes, ultimately resulting in high-output heart failure. Development of pelvic AVMs in this region of high VEGF expression occurred because loss of ENG in endothelial cells leads to increased sensitivity to VEGF and a hyperproliferative response. Development of AVMs and associated progression to high-output heart failure in the absence of endothelial ENG was attenuated by targeting VEGF signaling with an anti-VEGFR2 (VEGF receptor 2) antibody. ENG promotes the normal balance of VEGF signaling in quiescent endothelial cells to maintain vessel caliber-an essential function in conditions of increased VEGF expression such as local hypoxia or inflammation. In the absence of endothelial ENG, increased sensitivity to VEGF drives abnormal endothelial proliferation in local regions of high VEGF expression, leading to AVM formation and a rapid injurious impact on heart function.

753P - Prognostic and predictive value of baseline alpha-fetoprotein (AFP) in patients with advanced hepatocellular carcinoma (HCC) treated with ramucirumab from two phase III studies (REACH, REACH-2)

753P - Prognostic and predictive value of baseline alpha-fetoprotein (AFP) in patients with advanced hepatocellular carcinoma (HCC) treated with ramucirumab from two phase III studies (REACH, REACH-2)

Anti-VEGFR2 Antibody-modified Micelle for Triggered Drug Delivery and Effective Therapy of Choroidal Neovascularization.

This study aimed to examine whether DC101 (anti-VEGFR2 antibody)- modified micelles have applications as novel drug delivery devices, which allow small molecule antiangiogenic agents to deliver to angiogenic sites on a murine laser-induced choroidal neovascularization (CNV) model. CNV was induced by photocoagulation on the unilateral eye of each mouse under anesthesia. Immediately after laser coagulation, E7974-loaded DC101-modified micelles and motesanib-loaded DC101-modified micelles were intravitreally administrated. Two weeks after photocoagulation, CNV was visualized using fluorescein-conjugated dextran (MW=2,000 kDa), and the CNV area was measured in retinal pigment epithelium (RPE)-choroidal flat mounts. Intravitreal administration of both DC101-modified micelles loaded with E7974 at 2 µM and motesanib at 2 µM significantly reduced CNV area in the murine laser-induced CNV model at a clearly lower concentration than the effective dose of each agent. These results suggest that DC101-modified micelle might be effective drug carrier system for treating CNV and other ocular angiogenic diseases.

Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells.

Background and Aims Vascular endothelial growth factor (VEGF) receptors (VEGFR1 and VEGFR2) bind VEGF-A with high affinity. This study sought to determine the relative contributions of these two receptors to receptor-mediated endocytosis of VEGF-A and to clarify their endocytic itineraries in rat liver sinusoidal endothelial cells (LSECs). Methods Isolated LSECs and radiolabeled VEGF-A were used to examine surface binding and receptor-mediated endocytosis. Quantitative real time RT-PCR (Q-RT-PCR) and Western blotting were applied to demonstrate receptor expression. Results Q-RT-PCR analysis showed that VEGFR1 and VEGFR2 mRNA were expressed in LSECs. Ligand saturation analysis at 4°C indicated two different classes of [125I]-VEGFA binding sites on LSECs with apparent dissociation constants of 8 and 210 pM. At 37°C, LSECs efficiently took up and degraded [125I]-VEGF-A for at least 2 hours. Uptake of [125I]-VEGF-A by LSECs was blocked by dynasore that inhibits dynamin-dependent internalization, whereas inhibition of cysteine proteases by leupeptin inhibited degradation without affecting the uptake of [125I]-VEGF-A, suggesting that it is degraded following transport to lysosomes. Incubation of LSECs in the continued presence of a saturating concentration of unlabeled VEGF-A at 37°C was associated with a loss of as much as 75% of the total VEGFR2 within 30 min as shown by Western blot analysis, whereas there was no appreciable decrease in protein levels for VEGFR1 after 120 min incubation, suggesting that VEGF-A stimulation downregulates VEGFR2, but not VEGFR1, in LSECs. This possibility was supported by the observation that a hexapeptide that specifically blocks VEGF-A binding to VEGFR1 caused a marked reduction in the uptake of [125I]-VEGF-A, whereas a control peptide had no effect. Finally, live cell imaging studies using a fluorescently labeled anti-VEGFR2 antibody showed that VEGFR2 was transported via early and late endosomes to reach endolysosomes where degradation of the VEGFR2 takes place. Conclusion Our studies suggest that, subsequent to VEGF-A binding and internalization, the unoccupied VEGFR1 may recycle to the cell surface allowing its reutilization, whereas the majority of the internalized VEGFR2 is targeted for degradation.

A VEGFR2-MICA bispecific antibody activates tumor-infiltrating lymphocytes and exhibits potent anti-tumor efficacy in mice.

MHC class I-related chain A (MICA) is one of the major ligands for natural killer group 2 member D (NKG2D), which is an activating NK receptor. MICA is expressed on the surface of human epithelial tumor cells, and its shedding from tumor cells leads to immunosuppression. To activate immune response in the tumor microenvironment, we designed an anti-VEGFR2-MICA bispecific antibody (JZC01), consisting of MICA and an anti-VEGFR2 single chain antibody fragment (JZC00) and explored its potential anti-tumor activity. JZC01 targeted vascular endothelial growth factor receptor 2 (VEGFR2) and inhibited tumorigenesis by blocking the VEGFR2 signaling pathway. Additionally, JZC01 promoted NK and CD8+ T cells to release IFN-γ and engaged activated lymphocytes to lysis of VEGFR2-expressing tumor cells. The in vivo anti-tumor activity of JZC01 was investigated by establishing a Lewis lung cancer cell-transplanted mouse model. It effectively reduced the tumor vascular density and increased the infiltration and activation of NK and CD8+ T cells in the tumor microenvironment. Thus, JZC01 functions in anti-tumor angiogenesis and anti-tumor immune activation, and showed improved anti-tumor efficacy combined with docetaxel, which provides a new insight into anti-tumor therapy.

Abstract 2131: Significant combination benefit of anti-VEGFR antibody and oncogene-targeted agents in EGFR or ALK mutant NSCLC cells

Abstract Background: Non-small cell lung cancers (NSCLCs) harboring driver oncogene such as epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) rearrangement are sensitive to the corresponding molecular targeted tyrosine kinase inhibitors (TKIs). However, acquired resistance inevitably develops, and more effective treatment strategies are needed. Anti-vascular endothelial growth factor (VEGF) antibody bevacizumab significantly prolongs progression free survival when added to first-generation EGFR TKIs in EGFR mutant NSCLC. However, it remains unknown whether anti-VEGF receptor (VEGFR) antibody enhances the efficacy of molecular targeted agents as well. In this study, we investigated combinations with an anti-VEGFR antibody and EGFR or ALK TKIs in preclinical models Materials and Methods: We treated mice bearing subcutaneous tumor cell lines harboring EGFR mutation (PC-9, H3255) or ALK rearrangement (ABC-11, H3122) using combinations of anti-mouse VEGFR antibody DC101 and erlotinib (EGFR TKI) /alectinib (ALK TKI). Neovascularization of the tumors were determined by CD31 immunostaining. To determine the effects of VEGFR antibody on cancer cell itself, we also examined the efficacy of the combinations in vitro. Results: Combination therapy demonstrated significantly better tumor inhibition compared to erlotinib or alectinib alone in all the examined xenograft tumors. CD31 positive blood vessel staining was significantly reduced in the tumors treated with combination therapy. Mutant EGFR transfection in HEK293T cells increased VEGF expression in the cell culture supernatant. Furthermore, MTT assay showed significantly enhanced cell growth inhibition when anti-human VEGFR antibody ramucirumab combined with erlotinib (against PC-9 or H3255 cells) or alectinib (against ABC-11 or H3122 cells) suggesting that anti-VEGFR antibody affects not only on tumor vasculature but cancer cell itself. Consistent with this finding, combination with siVEGFR and erlotinib also inhibited the cell proliferation significantly better than erlotinib alone in PC-9 cells. Conclusion: A significant combination benefit was observed with anti-VEGFR2 antibody and EGFR or ALK TKI both in vivo and in vitro, suggesting anti-VEGFR antibody inhibits not only the blood vessels but may also exert direct anticancer effects. Combination therapy of erlotinib or alectinib and anti-VEGFR2 antibody could be a promising treatment strategy for oncogene addicted NSCLC. Citation Format: Hiromi Watanabe, Eiki Ichihara, Hiroe Kayatani, Hisao Higo, Go Makimoto, Hirohisa Kano, Kazuya Nishii, Naofumi Hara, Kiichiro Ninomiya, Toshio Kubo, Kadoaki Ohashi, Kammei Rai, Katsuyuki Hotta, Masahiro Tabata, Yoshinobu Maeda, Katsuyuki Kiura. Significant combination benefit of anti-VEGFR antibody and oncogene-targeted agents in EGFR or ALK mutant NSCLC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2131.

Abstract 3203: Intratumoral STING activation normalizes tumor vasculatures and synergizes with anti-angiogenic therapy to enhance cancer immunity

Abstract Purpose: The stimulator of interferon genes (STING) signaling pathway is a critical link between innate and adaptive immunity, and induces anti-tumor immune responses. STING is expressed in vasculatures, but its role in tumor angiogenesis has not been elucidated. Here we investigated STING-induced tumor vascular remodeling and the potential of STING-based combination immunotherapy. Experimental Design: We examined the endothelial STING expression pattern and clinical implications in human malignancies. STING-induced vascular remodeling was studied using STING-deficient or wild-type mice. Implanted LLC tumors and transgenic MMTV-PyMT breast tumors were intratumorally injected with STING agonists (cGAMP or RR-CDA), with or without anti-VEGFR2 antibody and/or immune checkpoint inhibitors. The tumor microenvironment was evaluated by histologic and immune profiling analyses. Results: Endothelial STING expression was correlated with enhanced T-cell infiltration, reduced lymphovascular invasion, and prolonged survival in human colon and breast cancer. Intratumoral STING activation normalized tumor vasculatures, as shown by increased pericyte coverage and intact basement membrane, which was mediated by upregulation of vascular stabilizing genes (e.g., Angpt1, Pdgfrb, and Col4a) and endothelial-lymphocyte interaction genes. These effects were dependent on type I interferon signaling and CD8+ T cells. STING activation also induced M1-like macrophage polarization within tumors. Notably, STING-based immunotherapy was maximally effective when combined with VEGFR2 blockade and/or immune checkpoint blockade (αPD-1 or αCTLA-4), leading to complete regression of immunotherapy-resistant tumors. Conclusions: Our data show that intratumoral STING activation can normalize tumor vasculature and the tumor microenvironment, providing a rationale for combining STING-based immunotherapy and anti-angiogenic therapy. Citation Format: Hannah Yang, Hyojoong Kim, Joo Hoon Kim, Hong Jae Chon, Chan Kim. Intratumoral STING activation normalizes tumor vasculatures and synergizes with anti-angiogenic therapy to enhance cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3203.

MMP14-Containing Exosomes Cleave VEGFR1 and Promote VEGFA-Induced Migration and Proliferation of Vascular Endothelial Cells.

PurposeInvestigate the impact matrix metalloproteinase 14 (MMP14) delivered via exosomes produced by corneal fibroblasts on vascular endothelial growth factor receptor 1 (VEGFR1) cleavage on endothelial cells, and other key processes of angiogenesis.MethodsProteolysis of VEGFR1 and R2 by the catalytic domain of MMP14 was investigated via immunocytochemistry with anti-VEGFR1, anti-VEGFR2, and anti-MMP14 antibodies. Exosomes were isolated via precipitation and serial ultracentrifugation from wild-type (WT) and MMP14 exon4-deficient corneal fibroblasts. Transmission electron microscopy and nanotracking analysis were used to characterize the isolated exosomes. The presence of MMP14 in exosomes from WT fibroblasts was confirmed by Western blotting. VEGFR1 cleavage upon treatment with WT-derived exosomes, Δexon4-derived exosomes, or the pan-MMP inhibitor GM60001 was examined via in vitro proteolysis analysis using recombinant mouse (rm) VEGFR1/R2. Endothelial cell migration and proliferation were investigated using a Boyden chamber assay and BrdU incorporation, respectively.ResultsWT-derived exosomes specifically cleaved rmVEGFR1 in vitro, whereas Δexon4-derived exosomes did not. Treatment with the pan-MMP inhibitor GM6001 effectively inhibited VEGFR1 cleavage by WT-derived exosomes, confirming the role of MMP14 in this cleavage. WT-derived exosomes induced greater endothelial cell migration (P < 0.01) and proliferation (P < 0.5) compared to Δexon4-derived exosomes.ConclusionsMMP14-containing exosomes may be involved in the regulation of corneal neovascularization through degradation of VEGFR1 and VEGFA-induced endothelial cell proliferation and migration.