VEGF Antibody Research Articles

68Ga-TRAP-(RGD)3 Hybrid Imaging for the In Vivo Monitoring of αvß3-Integrin Expression as Biomarker of Anti-Angiogenic Therapy Effects in Experimental Breast Cancer.

ObjectivesTo investigate 68Ga-TRAP-(RGD)3 hybrid imaging for the in vivo monitoring of αvß3-integrin expression as biomarker of anti-angiogenic therapy effects in experimental breast cancer.Materials and MethodsHuman breast cancer (MDA-MB-231) xenografts were implanted orthotopically into the mammary fat pads of n = 25 SCID mice. Transmission/emission scans (53 min to 90 min after i.v. injection of 20 MBq 68Ga-TRAP-(RGD)3) were performed on a dedicated small animal PET before (day 0, baseline) and after (day 7, follow-up) a 1-week therapy with the VEGF antibody bevacizumab or placebo (imaging cohort n = 13; therapy n = 7, control n = 6). The target-to-background ratio (TBR, VOImaxtumor/VOImeanmuscle) served as semiquantitative measure of tumor radiotracer uptake. Unenhanced CT data sets were subsequently acquired for anatomic coregistration and morphology-based tumor response assessments (CT volumetry). The imaging results were validated by multiparametric ex vivo immunohistochemistry (αvß3-integrin, microvascular density–CD31, proliferation–Ki-67, apoptosis–TUNEL) conducted in a dedicated immunohistochemistry cohort (n = 12).Results68Ga-TRAP-(RGD)3 binding was significantly reduced under VEGF inhibition and decreased in all bevacizumab-treated animals (ΔTBRfollow-up/baseline: therapy -1.07±0.83, control +0.32±1.01, p = 0.022). No intergroup difference in tumor volume development between day 0 and day 7 was observed (Δvolumetherapy 134±77 μL, Δvolumecontrol 132±56 μL, p = 1.000). Immunohistochemistry revealed a significant reduction of αvß3-integrin expression (308±135 vs. 635±325, p = 0.03), microvascular density (CD31, 168±108 vs. 432±70, p = 0.002), proliferation (Ki-67, 5,195±1,002 vs. 7,574±418, p = 0.004) and significantly higher apoptosis (TUNEL, 14,432±1,974 vs. 3,776±1,378, p = 0.002) in the therapy compared to the control group.Conclusions68Ga-TRAP-(RGD)3 hybrid imaging allows for the in vivo assessment of αvß3-integrin expression as biomarker of anti-angiogenic therapy effects in experimental breast cancer.

Malignant Pleural Effusion and ascites Induce Epithelial-Mesenchymal Transition and Cancer Stem-like Cell Properties via the Vascular Endothelial Growth Factor (VEGF)/Phosphatidylinositol 3-Kinase (PI3K)/Akt/Mechanistic Target of Rapamycin (mTOR) Pathway

Malignant pleural effusion (PE) and ascites, common clinical manifestations in advanced cancer patients, are associated with a poor prognosis. However, the biological characteristics of malignant PE and ascites are not clarified. Here we report that malignant PE and ascites can induce a frequent epithelial-mesenchymal transition program and endow tumor cells with stem cell properties with high efficiency, which promotes tumor growth, chemoresistance, and immune evasion. We determine that this epithelial-mesenchymal transition process is mainly dependent on VEGF, one initiator of the PI3K/Akt/mechanistic target of rapamycin (mTOR) pathway. From the clinical observation, we define a therapeutic option with VEGF antibody for malignant PE and ascites. Taken together, our findings clarify a novel biological characteristic of malignant PE and ascites in cancer progression and provide a promising and available strategy for cancer patients with recurrent/refractory malignant PE and ascites.

Antihypoxic Potentiation of Standard Therapy for Experimental Colorectal Liver Metastasis through Myo-Inositol Trispyrophosphate.

Tumor hypoxia activates hypoxia-inducible factors (Hifs), which induce a range of malignant changes including vascular abnormalities. Here, we determine whether inhibition of the hypoxic tumor response through myo-inositol trispyrophosphate (ITPP), a compound with antihypoxic properties, is able to cause prolonged vascular normalization that can be exploited to improve standard-of-care treatment. We tested ITPP on two syngeneic orthotopic mouse models of lethal colorectal cancer liver metastasis. Tumors were monitored by MRI and analyzed for the hypoxic response and their malignant potential. A Hif activator and in vitro assays were used to define the working mode of ITPP. Hypoxic response and vasculature were re-evaluated 4 weeks after treatment. Finally, we determined survival following ITPP monotherapy, FOLFOX monotherapy, FOLFOX plus Vegf antibody, and FOLFOX plus ITPP, both overlapping and sequential. ITPP reduced tumor load, efficiently inhibited the hypoxic response, and improved survival. These effects were lost when mice were pretreated with a Hif activator. Its immediate effects on the hypoxic response, including an apparent normalization of tumor vasculature, persisted for at least 4 weeks after treatment cessation. Compared with FOLFOX alone, Vegf antibody combined with FOLFOX prolonged survival by <30%, whereas ITPP combined with FOLFOX extended survival by >140%, regardless of whether FOLFOX was given in overlap or after ITPP exposure. Our findings reveal a truly antihypoxic mechanism for ITPP and demonstrate the capacity of this nontoxic compound to potentiate the efficacy of existing anticancer treatment in a way amenable to clinical translation. Clin Cancer Res; 22(23); 5887-97. ©2016 AACR.

A comparative study on conbercept and triamcinolone acetoinde intravitreal injection for macular edema secondary to central retinal vein occlusion

Background Macular edema is one of the serious complications of central retinal vein occlusion (CRVO), and the present therapies are laser coagulation and intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs.Conbercept is humanized-monoclonal VEGF antibody and used for the treatment of retinal vascular diseases.However, fewer studies were focused on its application in macular edema secondary to CRVO. Objective The aim of this study was to compare the effectiveness and safety of conbercept with triamcinolone acetonide (TA) by intravitreal injections for macular edema secondary to CRVO. Methods A non-randomized controlled study was carried out under the approval of the informed consent of patients.Sixty eyes of 60 patients with macular edema secondary to CRVO were included in Weifang Yidu Central Hospital from March 2012 to August 2013.The eyes were divided into the conbercept group and TA group with 30 for each group.Conbercept and TA of 0.05 ml were intravitreally injected in different groups, and the best corrected visual acuity (BCVA), central macular thickness (CMT) measured by OCT, intraocular pressure (IOP) and relavant complications were examined before injection and 1 week, 1 month, 3 months and 6 months after injection.The treatment outcomes were compared intergrouply and along with time. Results The BCVA was evidently better in 1 week, 1 month, 3 months and 6 months after injection than that before injection both in conbercept group and TA group (all at P<0.01), and the BCVA of TA group was better than that of conbercept group 1 week after injection (P<0.05). The CMT values of Conbercept were (572.00±100.01), (325.12±91.55), (280.00±92.37), (258.65±88.65), (300.00±87.64)μm, and those of TA group were (570.00±102.21), (345.12±89.31), (290.00±80.27), (309.65±84.13) and (303.00± 90.59)μm, and CMT value after injection was significantly lower in 1 week, 1 month, 3 months and 6 months after injection than that before injection both in the conbercept group and the TA group (all at P<0.05), and CMT value was evidently reduced in the conbercept group compared with the TA group 3 months after injection (P<0.05). The IOP was (15.20±3.52), (21.20±3.80), (26.40±4.00), (23.60±3.73) and (21.50±3.27)mmHg in the TA group before injection and 1 week, 1 month, 3 months and 6 months after injection, showing significnatly elavation after injection (all at P<0.05), and the IOP at different time points was higher in the TA group than that in the conbercept group (all at P<0.05). However, there was no considerable change of IOP before and after injection in conbercept group (all at P<0.05). Conclutions Both conbercept and TA are effective for macular edema secondary to CRVO by intravtreal injection.Compared with TA, conbercept is much safer because of less risk of IOP rising after intravtreal injection. Key words: Antibodies, monoclonal, humanized/therapeutic use; Retinal vein occlusion/complications; Macular edema/drug therapy; Conbercept/therapeutic use; Triamcinolone acetoinde/therapeutic use; Intravitreal injections; Treatment outcome; Comparative studies

Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ) as a therapeutic target to prevent retinal vasopermeability during diabetes

Lipoprotein-associated phospholipase A2 (Lp-PLA2) hydrolyses oxidized low-density lipoproteins into proinflammatory products, which can have detrimental effects on vascular function. As a specific inhibitor of Lp-PLA2, darapladib has been shown to be protective against atherogenesis and vascular leakage in diabetic and hypercholesterolemic animal models. This study has investigated whether Lp-PLA2 and its major enzymatic product, lysophosphatidylcholine (LPC), are involved in blood-retinal barrier (BRB) damage during diabetic retinopathy. We assessed BRB protection in diabetic rats through use of species-specific analogs of darapladib. Systemic Lp-PLA2 inhibition using SB-435495 at 10 mg/kg (i.p.) effectively suppressed BRB breakdown in streptozotocin-diabetic Brown Norway rats. This inhibitory effect was comparable to intravitreal VEGF neutralization, and the protection against BRB dysfunction was additive when both targets were inhibited simultaneously. Mechanistic studies in primary brain and retinal microvascular endothelial cells, as well as occluded rat pial microvessels, showed that luminal but not abluminal LPC potently induced permeability, and that this required signaling by the VEGF receptor 2 (VEGFR2). Taken together, this study demonstrates that Lp-PLA2 inhibition can effectively prevent diabetes-mediated BRB dysfunction and that LPC impacts on the retinal vascular endothelium to induce vasopermeability via VEGFR2. Thus, Lp-PLA2 may be a useful therapeutic target for patients with diabetic macular edema (DME), perhaps in combination with currently administered anti-VEGF agents.

Clinical, Pathological and Immunohistochemical Evaluation of a Primary Hemangiosarcoma in a Pinscher Dog

Background: Hemangiosarcoma (HSA) is a malignant tumor that arises from the vascular endothelium affecting more often dogs than other species as cats, cows and horses. It comprises approximately 2% of all tumors in dogs. The most common primary site for the HSA in dogs is the spleen, and other locations include the right atrium, pericardium, liver andprostate. Other authors have reported this tumor in lungs, kidney, oral cavity, muscle, bone, urinary bladder, left ventricle, tongue and retroperitoneum. Due to the importance of the HSA in canine species, the aim of this study was to describe the clinical and pathological fndings, besides therapeutic protocol in an unusual case of HSA.Case:A six-year-old male pinscher was referred to the veterinary hospital with a history of cyanosis and choking. The animal was submitted to radiographic examination in lateral cervical view, which identifed the presence of a mass of 1.2 cm in diameter near the pharynx. In order to evaluate the oral cavity, general anesthesia was performed, and it was possible to see a soft, rosy, circumscribed and vascularized lump in pharyngeal region. Due to suspicion of neoplasm, excisional biopsywithout surgical margin was performed. The histopathological exam diagnosed hemangiosarcoma. Immunohistochemistry against vimentin, factor VIII, VEGF and Ki67 was performed and confrmed diagnosis of low grade hemangiosarcoma. Antineoplastic chemotherapy protocol was initiated with doxorubicin and cyclophosphamide every 21 days totaling sixsessions. However, the animal died after the cyclophosphamide intoxication with a three-fold recommended dose (660 mg/m² total in the last session), showing a median survival rate of 220 days.Discussion: The most common primary site for HSA in dogs is the spleen. The pharyngeal location is rare, with only a few reports in literature. In the present case, solitary tumor was observed in pharynx with no involvement of other organs, evidenced by radiographic examination, abdominal ultrasound and echocardiogram, suggesting that pharynx was theprimary location of the tumor. Main features of HSA comprise a solitary nodule or multifocal lesions within the organ or widely disseminated. Histologically, they consist of pleomorphic immature endothelial cells with formation of vascular spaces with variable amount of blood and/or thrombi. In some cases, HSA shows a polymorphic subtype and immunohistochemistry is necessary to provide a defnitive diagnosis. The sample was submitted to histopathological examination which revealed proliferation of endothelial cells with pronounced pleomorphism ranging from polygonals to ovoid, sparse cytoplasm, round to oval nucleus with visible nucleolus, few mitotic fgures, some of them, aberrant, which confrmed diagnosis of HAS. Due to the unusual location, we performed immunohistochemical staining for vimentin, factor VIII,VEGF and Ki67 antibodies to confrm mesenchymal origin of the tumor. In IHC, it was possible to identify positive reac tion for vimentin protein, factor VIII, VEGF and few Ki67 positive cells, confrming histopathological diagnosis. Despite literature describes an aggressive biological behavior of canine HSA, with common occurrence of metastasis, recurrencewas not observed at the site of the removal of the tumor. In histopathological evaluation, it was observed low number ofmitoses, besides the low Ki67 expression on IHC, featuring a low grade tumor with minor ability to metastasize. To the author’s knowledge, this case describes an unusual presentation of HSA, with low metastatic potential, in which chemo therapy protocol achieved survival time of 220 days.Keywords: angiosarcoma, dogs, immunohistochemistry, pharynx.

Effects of strontium ranelate on sutural bone formation: a histological and immunohistochemical study

Abstract Aim Rapid maxillary expansion is performed to correct a skeletal transverse deficiency of the maxilla, which is a frequently- encountered orthodontic anomaly. Strontium ranelate (SrR) is a novel agent that has a dual action, involving anti-resorptive and bone-forming effects. The aim of this study was to evaluate the effects of systemically applied SrR on osteoblastic bone formation after maxillary expansion on the mid-palatal suture of rats using histological and immunohistochemical tests. Materials and methods A total of 24 Wistar rats were randomly divided into two equal groups. In all animals, five-day inter- premaxillary expansion was applied and maintained for a seven-day retention period, during which 625mg/kg/day SrR diluted with saline solution was administered orally to the experimental group. The rats were sacrificed and the tissues prepared for histological and immunohistochemical examinations after the retention period. Results Osteoblastic activity and the width of the blood vessels in the suture area were significantly increased in the SrR group compared with the control group (p &lt; 0.05). Ossification was also observed to be active under light microscopy by staining with hematoxylin and eosin in the experimental group. Immunohistochemical labelling performed using osteonectin, osteocalcin, TGF-β and VEGF antibodies revealed significant immunoreactivity in the experimental group (p &lt; 0.05). Conclusion It may be concluded that SrR contributed to stimulatory osteogenesis in the expansion region. Therefore, a retention period may be shortened and relapse possibly reduced, following the application of SrR after the expansion.

Correlation of Vascular Endothelial Growth Factor Expression and Neovascularization with Colorectal Carcinoma: A Pilot Study

Background: In Egypt, there is an increasing incidence of colorectal cancer (CRC), especially among patients under 40 years of age, affecting a very important age group in our community. The basic pathogenic step in the process of tumor growth, invasion and metastasis is tumor induced angiogenesis. The aim of this study was to evaluate the angiogenesis in colorectal carcinoma by detect the expression of VEGF vascular endothelial growth factor and micro vascular density (MVD) determination with IHC (immunohistochemistry) method and to determine if and how angiogenesis correlates with clinicopathologic parameters. Methods and findings: Sixty five archival, paraffin embedded tissue samples of colorectal carcinoma from Pathology Lab of Suez Canal University Teaching Hospital (Egypt) with complete follow-up files in Oncology Unit from. VEGF and micro vessels were identified immunohistochemically, using monoclonal VEGF antibody and CD34 antibody, respectively. VEGF IHC expression was positive in 94.7% of cases and MVD ranged from 9 to 42 mean 24.55 ± 13.79. A significant positive relation was found between VEGF expression; and histological type, tumor grade, lymph node (LN) involvement, UICC TNM classification, perineural and lympho-vascular invasions (p<0.05). A significant positive relation was found between VEGF expression and MVD (p<0.001). There is a significant correlation has been found between MVD and size of the tumor, histological subtype, tumor grade, LN involvement, UICC TNM classification, vascular and perineural invasion and survival (p<0.05). Conclusions: This study has highlighted the prognostic significance of VEGF and MVD to predict survival in CRC patients, as well as some of traditional prognostic factors as tumor histologic type, tumor size, tumor grade, LN involvement, tumor stage and lympho-vascular and perineural invasion to identify patients at high risk for relapse who may benefit from adjuvant treatment including new therapeutic strategies in the future.

Migration of Acute Lymphoblastic Leukemia Cells into the Central Nervous System Is Regulated By VEGF

In acute lymphoblastic leukemia (ALL), central nervous system (CNS) directed therapy is required to achieve long-term remission and survival even in patients without detectable CNS disease, indicating subclinical CNS manifestation in many patients. Hence, prophylactic CNS therapy is indispensable but bears the risk of secondary neoplasms and neurocognitive deficits in ALL survivors. Therefore, a better understanding of mechanisms mediating CNS involvement in ALL are needed in order to identify potential targets for prophylactic and therapeutic intervention.In this study, we transplanted primary patient B cell precursor (BCP) ALL cells onto NOD/SCID mice and investigated engraftment of human leukemia cells in the recipient's peripheral blood (PB), bone marrow (BM), spleen (S), and meninges by flowcytometry staining for huCD19. Upon disease onset, we identified meningeal infiltration of human ALL cells together with leukemia engraftment in BM, S and PB in a subset of samples (CNSpos) in contrast to absent CNS involvement despite high leukemia cell infiltration of BM, S and PB in other recipients (CNSneg). CNSpos and CNSneg phenotypes were consistently observed in subsequent xenograft passages. In CNSpos animals meningeal leukemia infiltration was also detected by immunohistochemistry on brain sections and meningeal enhancement was detected by small animal magnetic resonance imaging in CNSpos recipients.We further characterized ALL cells isolated from meningeal and BM infiltrates by gene expression profiling and identified the gene coding for vascular endothelial growth factor A (VEGF) to be highly expressed in ALL cells isolated from the CNS as compared to BM derived cells. Differential expression of VEGF was validated by qPCR and confirmed in independent sample cohorts. Most interestingly, reported functions of VEGF include regulation of cellular growth, vascular permeability, and trans-endothelial cell migration, and elevated VEGF protein levels have previously been described in cerebrospinal fluid specimens collected from ALL and AML patients with CNS leukemia.VEGF signals through its receptors 1 or 2 (VEGFR1/2). On all primary ALL samples, only expression of VEGFR1 but not VEGFR2 was detected. We used the BCP-ALL cell line Nalm-6, which also expresses VEGF and VEGFR1 and mediates a clear CNSpos phenotype upon engraftment. However, cellular survival, proliferation, apoptosis, and metabolic activity were not affected, neither upon exposure to VEGF or the antagonizing VEGF antibody bavacizumab, nor by stable VEGF overexpression or knock-down, thus indicating absence of autocrine VEGF/VEGFR1 signaling.We further analyzed whether paracrine VEGF signaling through VEGFR2, which upon activation mediates endothelial cell permeability, is involved in trans-endothelial migration of ALL cells leading to leukemia infiltration of the CNS. Brain endothelial cells (bEND.3) incubated with VEGF showed increased phosphorylation of VEGFR2 downstream signaling molecules (Src, AKT), which indicates activated signaling mediating cellular permeability.Moreover, we modeled migration of ALL cells through brain endothelial cells in a transwell assay and observed significantly increased migration of Nalm-6 ALL cells through bEND.3 monolayers upon exposure to VEGF or upon overexpression of VEGF as compared to controls. Vice versa, significantly lower numbers of migrated leukemia cells were detected after incubation with bevacizumab or upon VEGF knock down indicating VEGF dependent trans-endothelial migration.Thus, we identified high expression of VEGF in CNS derived leukemia cells, absent autocrine signaling of VEGF on ALL cells and, most importantly, VEGF dependent trans-endothelial migration of ALL cells indicating VEGF as a possible mediator of CNS leukemia.Finally, we investigated the impact of VEGF on CNS leukemia manifestation in vivo. Recipient animals transplanted with 3 CNSpos primograft samples (4 experiments, one in repetition) were treated with bevacizumab or control. In all experiments, anti-VEGF treatment significantly reduced the leukemia burden exclusively in the CNS but not in BM, S, and PB compartments, indicating that transmigration of leukemia cells and CNS manifestation in ALL is mediated by VEGF. Thus, targeting of VEGF signaling may serve as a novel strategy to control CNS leukemia in patients. DisclosuresNo relevant conflicts of interest to declare.

Hypoxia inhibits nephrogenesis through paracrine Vegfa despite the ability to enhance tubulogenesis

Reduced nephron number predisposes to hypertension and kidney disease. Interaction of the branching ureteric bud and surrounding mesenchymal cells determines nephron number. Since oxygen supply may be critical for intrauterine development, we tested whether hypoxia and hypoxia-inducible factor-1α (HIF-1α) influence nephrogenesis. We found that HIF-1α is required for branching of MDCK cells. In addition, culture of metanephric mouse kidneys with ureteric bud cell-specific stabilization or knockout of HIF-1α revealed a positive impact of HIF-1α on nephrogenesis. In contrast, widespread stabilization of HIF-1α in metanephric kidneys through hypoxia or HIF stabilizers impaired nephrogenesis, and pharmacological HIF inhibition enhanced nephrogenesis. Several lines of evidence suggest an inhibitory effect through the hypoxia response of mesenchymal cells. HIF-1α was expressed in mesenchymal cells during nephrogenesis. Expression of the anti-branching factors Bmp4 and Vegfa, secreted by mesenchymal cells, was increased upon HIF stabilization. The conditioned medium from hypoxic metanephric kidneys inhibited MDCK branching, which was partially rescued by Vegfa antibodies. Thus, the effect of HIF-1α on nephrogenesis appears context dependent. While HIF-1α in the ureteric bud is of importance for proper branching morphogenesis, the net effect of hypoxia-induced HIF activation in the embryonic kidney appears to be mesenchymal cell-dependent inhibition of ureter branching.

Abstract IA22: Preclinical modeling of adjuvant and metastatic antiangiogenic therapy: Relevance for better predicting clinical outcomes

Abstract Over the last decade my lab has developed a number of models involving treatment of mice with either early stage microscopic metastatic disease for adjuvant therapy or late stage overt metastatic disease for metastatic therapy studies1-3. More recently, models of neoadjuvant therapy have been developed as well with the lab of Dr. John Ebos4. The rationale for utilizing the first two models is that they may be superior in predicting future activity in patients enrolled in randomized phase III adjuvant or metastatic therapy clinical trials, in comparison to conventional treatment models involving mice with unresected established primary tumors, and evaluating the effect on the primary tumor growth only1. As an example, we reported that sunitinib (or pazopanib) or DC101, the VEGFR-2 antibody were all devoid of anti-tumor activity when treating mice with advanced metastatic breast cancer after primary tumor resection, whereas in contrast, all showed efficacy when treating orthotopic primary tumors in control experiments5. Combining chemotherapy with sunitinib also did not improve outcomes in the metastatic setting. In contrast, combining chemotherapy with DC101 caused a small but statistically significant benefit in survival. These results retrospectively correlated with outcomes of four metastatic breast cancer phase III trials evaluating sunitinib alone or in combination with chemotherapy in the metastatic setting (all were negative) or multiple phase III trials evaluating the VEGF antibody, bevacizumab, with chemotherapy, which showed variable benefits in improving PFS in metastatic breast cancer5. With respect to adjuvant therapy modelling, we reported in 2009 that adjuvant sunitinib therapy of mice with microscopic metastases after resection of orthotopic primary human breast cancer xenografts resulted in a worsened survival outcome, with accelerated progression of metastatic disease2. On the basis of the results we raised a cautionary “flag” about the rationale of antiangiogenic drugs in the clinic for adjuvant setting6. As is now well known, there have been multiple adjuvant trials evaluating bevacizumab plus chemotherapy in postsurgical early stage colorectal or breast cancer, as well as sorafenib in hepatocellular carcinoma, and all of these trials have failed to meet their primary endpoint of a benefit in disease free survival7. This has provoked considerable discussion and debate about the basis for such failures in contrast to the same drugs/therapies showing efficacy in the more advanced metastatic settings of the same malignancies. One basis for the modest positive effects noted of such therapies in the metastatic setting, and for the failure in the adjuvant setting, concerns the impact that “vessel co-option”, especially in distant metastases, may have on therapeutic outcomes. Evidence is growing that a variety of tumors and especially overt metastases in certain sites such as the lungs, liver, and brain are minimally or non-angiogenic and instead “hijack” the existing vasculature in such organ sites8. The same may be the case for microscopic metastases. Consequently, there will be growing interest in evaluating whether vessel co-option can be therapeutically targeted (and also what the implications may be for drug-induced vascular normalization). In this regard there are a number of strategies being evaluated such as the impact of metronomic chemotherapy and targeting other pro-angiogenic factors/pathways beyond VEGF such as ang2/tie29, which may be effective as an adjuvant therapy strategy9.

High glucose-induced changes in hyaloid-retinal vessels during early ocular development of zebrafish: a short-term animal model of diabetic retinopathy.

Although a variety of animal models have been used to test drug candidates and examine the pathogenesis of diabetic retinopathy, time-saving and inexpensive models are still needed to evaluate the increasing number of therapeutic approaches. We developed a model for diabetic retinopathy using the early stage of transgenic zebrafish (flk:EGFP) by treating embryos with 130 mM glucose, from 3-6 days post fertilisation (high-glucose model). On day 6, lenses from zebrafish larvae were isolated and treated with 3% trypsin, and changes in hyaloid-retinal vessels were analysed using fluorescent stereomicroscopy. In addition, expression of tight junction proteins (such as zonula occludens-1), effects of hyperosmolar solutions and of hypoxia, and Vegf expression were assessed by RT -PCR. NO production was assessed with a fluorescent substrate. Effects of inhibitors of the VEGF receptor, NO synthesis and a VEGF antibody (ranibizumab) were also measured. In this high-glucose model, dilation of hyaloid-retinal vessels, on day 6, was accompanied by morphological lesions with disruption of tight junction proteins, overproduction of Vegf mRNA and increased NO production. Treatment of this high-glucose model with an inhibitor of VEGF receptor tyrosine kinase or an inhibitor of NO synthase or ranibizumab decreased dilation of hyaloid-retinal vessels. These findings suggest that short-term exposure of zebrafish larvae to high-glucose conditions could be used for screening and drug discovery for diabetic retinopathy and particularly for disorders of retinal vessels related to disruption of tight junction proteins and excessive VEGF and NO production.

Nucleoside Reverse Transcriptase Inhibitors Suppress Laser-Induced Choroidal Neovascularization in Mice.

To evaluate the efficacy of nucleoside reverse transcriptase inhibitors (NRTIs) in the laser-induced mouse model of choroidal neovascularization (CNV). We evaluated the NRTIs lamivudine (3TC), zidovudine (AZT), and abacavir (ABC) and the P2X7 antagonist A438079. Choroidal neovascularization was induced by laser injury in C57BL/6J wild-type, Nlrp3-/-, and P2rx7-/- mice, and CNV volume was measured after 7 days by confocal microscopy. Drugs were administered by intravitreous injection immediately after the laser injury. Vascular endothelial growth factor-A in RPE-choroid lysates was measured 3 days after laser injury by ELISA. HEK293 cells expressing human and mouse P2X7 were exposed to the selective P2X7 receptor agonist 2', 3'-(benzoyl-4-benzoyl)-ATP (Bz-ATP) with or without 3TC, and VEGF-A levels in media were measured by ELISA. Intravitreous injection of 3TC, AZT, and ABC significantly suppressed laser-induced CNV in C57BL/6J wild-type and Nlrp3-/- mice (P < 0.05) but not in P2rx7-/- mice. Intravitreous injection of A438079 also suppressed the laser-induced CNV (P < 0.05). The NRTIs 3TC, AZT, and ABC blocked VEGF-A levels in the RPE/choroid after laser injury in wild-type (P < 0.05) but not P2rx7-/- mice. Moreover, there was no additive effect of 3TC on CNV inhibition when coadministered with a neutralizing VEGF-A antibody. Stimulation of human and mouse P2X7-expressing HEK293 cells with Bz-ATP increased VEGF secretion (P < 0.001), which was abrogated by 3TC (P < 0.001). Stimulation of primary human RPE cells with Bz-ATP increased VEGFA and IL6 mRNA levels, which were abrogated by 3TC. Multiple clinically relevant NRTIs suppressed laser-induced CNV and downregulated VEGF-A, via P2X7.

Sequential Therapy with Saratin, Bevacizumab and Ilomastat to Prolong Bleb Function following Glaucoma Filtration Surgery in a Rabbit Model.

To determine if sequential treatment with Bevacizumab (Avastin), a monoclonal, VEGF antibody that blocks angiogenesis; Saratin, a 12 kD polypeptide with anti-inflammatory and anti-thrombotic properties; and Ilomastat, a matrix metalloproteinase (MMP) inhibitor, prolongs bleb life following glaucoma filtration surgery (GFS) in a rabbit model. Thirty-two New Zealand White rabbits (eight rabbits per group) underwent GFS in the left eye. Group 1 received a perioperative injection of both Saratin and Bevacizumab, and later, subconjuctival injections of Ilomastat on days 8 and 15. Group 2 received only Saratin perioperatively, and also received Ilomastat injections on days 8 and 15. Group 3, the negative control, received a single perioperative injection of Balanced Saline Solution (BSS) along with post-operative BSS injections on days 8 and 15. Group 4, the positive control, received topical treatment with Mitomycin-C (MMC) at the time of surgery with no further treatment. Blebs were evaluated by an observer masked to treatment every third day. Histology was obtained on two eyes in each group on post-op day twelve as well as all eyes following bleb failure. Eyes in group 1 had a mean bleb survival time of 29 ± 2.7 days, whereas those in group 2 that received the experimental treatment without Bevacizumab had a mean survival time of 25.5 ± 2.7 days. An ANOVA test showed that the Saratin/Ilomastat/Bevacizumab group demonstrated a significant prolongation of bleb survival compared to the BSS control—mean survival time of 19.7 ±2.7 days—(p = 0.0252) and was not significantly different from the MMC positive control group (p = 0.4238)—mean survival time of 32.5 ± 3.3. From tissue histology at day 12, the four different groups showed marked differences in the cellularity and capsule fibrosis. The MMC eyes showed minimal cellularity, were avascular and had minimal fibrous tissue. BSS group showed high cellularity, moderate to high fibrosis, and thicker and more defined capsules than either of the treatment groups and the positive control. Both the Saratin/Ilomastat/Bevacizumab and Saratin/Ilomastat only eyes showed moderate cellularity with minimal fibrosis, with less cellularity and fibrosis present in the triple treatment group. Sequential therapy with multiple agents, including Bevacizumab, prolonged bleb function following GFS in the rabbit model and were significantly better than the negative BSS control. The experimental group did not show the same surface tissue histological thinning and side effects associated with MMC treatment.

Abstract 295: Delivery of checkpoint inhibitor antibodies and other therapeutics directly to tumors by encoding them within the oncolytic adenovirus enadenotucirev

Abstract We are developing “armed” versions of the oncolytic adenovirus, enadenotucirev (EnAd), that will selectively infect and deliver immunotherapeutics to tumours following systemic dosing. EnAd is a potent, chimeric Ad11p/Ad3 adenovirus active against a range of epithelial cancer cells. In normal cells, EnAd is attenuated and shows little or no activity by either cytotoxicity assay or qPCR. In vivo, EnAd shows efficacy in a range of xenograft human tumor models following intra-tumoural, intravenous and intra-peritoneal injection and is currently being evaluated clinically for treatment of several different epithelial cancers. Data from ongoing clinical studies have shown that i.v. dosed EnAd infects and selectively replicates in tumor cells, producing significant amounts of viral protein (hexon). This is associated with CD8 cell accumulation and also indicates that transgene encoded proteins will be made in significant amounts by tumors following i.v. delivery of an armed EnAd virus. To develop armed EnAd variants we have developed a novel, efficient cloning system for rapid generation of viruses that can produce antibodies and other payloads under the control of the virus replication cycle or exogenous promoters. As an initial exemplification of the platform we have successfully produced EnAd variants encoding full-length (NG-135) and ScFv (NG-76) forms of anti-human VEGF antibodies. These have similar virus activity profiles to EnAd in cancer cell lines in vitro (virus replication, gene expression and oncolytic action), but also express and release the respective anti-VEGF antibody forms into the culture supernatant of tumor cells but not non-transformed cells. Using HCT-116 or DLD human colon carcinoma xenograft models we have shown that the virus infection profile following intra-tumoral injection is also similar to the parental EnAd virus (virus replication and hexon gene expression). Anti-VEGF antibody expression could be detected in the tumor tissue as both mRNA and functional antibody. Antibodies were detectable early (within 3 days of infection) and sustained over several weeks. Using an orthotopic A549 lung tumor model, NG-135 virus dosed i.v. following the development of tumors was able to decrease tumor burden by &amp;gt;90%. Viruses similarly expressing IgG1 or ScFv versions of anti-PDL1 and anti-CTLA4 checkpoint inhibitor antibodies have also been made and shown to produce functional antibodies that inhibit the respective receptor-ligand interactions in a range of in vitro assays, including upregulation of T-cell responses. In conclusion, our data show that EnAd can be modified to produce different antibody “payloads” following infection of human tumor cells in vitro and in vivo. Evaluation of the in vivo impact of these armed oncolytic viruses on the growth and microenvironment of tumors is now in progress. Citation Format: Brian R. Champion, Prithvi Kodialbail, Sam Illingworth, Nalini Rasiah, Daniel Cochrane, John Beadle, Kerry Fisher, Alice Brown. Delivery of checkpoint inhibitor antibodies and other therapeutics directly to tumors by encoding them within the oncolytic adenovirus enadenotucirev. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 295. doi:10.1158/1538-7445.AM2015-295

Secalonic Acid-D Represses HIF1α/VEGF-Mediated Angiogenesis by Regulating the Akt/mTOR/p70S6K Signaling Cascade.

Tumor angiogenesis is a validated target for therapeutic intervention, but agents that are more disease selective are needed. Here, we report the isolation of secalonic acid-D (SAD), a mycotoxin from a novel source that exhibits potent antiangiogenic antitumor activity. SAD inhibited multiple HIF1α/VEGF-arbitrated angiogenesis dynamics as scored in human umbilical vascular endothelial cells and human MCF-7 breast tumor xenografts. Similarly, SAD suppressed VEGF-induced microvessel sprouting from rat aortic ring and blood vessel formation in the Matrigel plug assay in C57/BL6J mice. Under normoxic or hypoxic conditions, SAD inhibited cell survival through the Akt/mTOR/p70S6K pathway, with attendant effects on key proangiogenesis factors, including HIF1α, VEGFR, and MMP-2/MMP-9. These effects were reversed by cotreatment with the Akt inhibitors perifosine and GSK69069 or by the addition of neutralizing VEGF antibodies. The apoptotic properties of SAD were determined to be both extrinsic and intrinsic in nature, whereas the cell-cycle inhibitory effects were mediated by altering the level of key G1-S transition-phase proteins. In experimental mouse models of breast cancer, SAD dosing produced no apparent toxicities (either orally or intraperitoneal) at levels that yielded antitumor effects. Taken together, our findings offered a preclinical validation and mechanistic definition of the antiangiogenic activity of a novel mycotoxin, with potential application as a cancer-selective therapeutic agent.

Complementary effects of bevacizumab and MMC in the improvement of surgical outcome after glaucoma filtration surgery.

To determine the optimum administration route of bevacizumab after glaucoma filtering surgery (GFS) and to investigate whether a reduced dose of mitomycin-C (MMC) in combination with bevacizumab could improve surgical outcome with a reduced incidence of side-effects. Plasma levels of bevacizumab were determined via ELISA after intracameral (IC), subconjunctival (SC) and intravitreal (IV) injections in mice, subjected to a mouse model of GFS. Application of MMC was compared to bevacizumab (SC, 25 μg) and to the combined use of both adjuvants. Surgical sponges soaked in MMC 0.02% or 0.01% were exposed to the sclera for 1 or 2 min. Treatment outcome was studied by bleb investigation. The three administration routes of bevacizumab equally improved surgical outcome. The VEGF antibody was detected at relatively high levels in plasma shortly after IV injection, whereas it was minimally absorbed after IC and SC injections. Both bevacizumab (SC) and MMC 0.02% (2 min) similarly increased bleb area. As compared to MMC, the combined injection with bevacizumab induced an additional effect on surgical outcome. Exposure of MMC 0.02% for 1 or 2 min together with bevacizumab equally improved surgical outcome, but 2 min application induced corneal toxicity. The combined use of bevacizumab and 1-min MMC 0.01% also improved surgical outcome compared to monotherapy, although to a lesser extent than the combination with 1-min MMC 0.02%. Adjunctive bevacizumab not only enhances the beneficial effect of MMC on surgical outcome, but also allows reducing the administration time of MMC 0.02%, thereby eliminating its toxic effects on the cornea.

Injectable formulations for an intravitreal sustained-release application of a novel single-chain VEGF antibody fragment

Sustained-release formulations of a single-chain anti-VEGF-A antibody fragment were investigated in vitro toward their potential use for intravitreal applications. The hydrophobic polyester hexylsubstituted poly(lactic acid) (hexPLA) was selected as the sustained-release excipient for its biodegradability and semi-solid aggregate state, allowing an easy and mild formulation procedure. The lyophilized antibody fragment ESBA903 was micronized and incorporated into the liquid polymer matrix by cryo-milling, forming homogeneous and injectable suspensions. The protein showed excellent compatibility with the hexPLA polymer and storage stability at 4°C for 10weeks. Additionally, hexPLA shielded the incorporated active substance from the surrounding medium, resulting in a better stability of ESBA903 inside the polymer than after its release in the buffer solution. Formulations of ESBA903 with hexPLA having drug loadings between 1.25% and 5.0% and polymer molecular weights of 1500g/mol, 2500g/mol, 3500g/mol and 5000g/mol were investigated regarding their in vitro release. All formulations except with the highest molecular weight formed spherical depots in aqueous buffer solutions and released the antibody fragment for at least 6–14weeks. The polymer viscosity derived from the molecular weight strongly influenced the release rate, while the drug loading had minor influence, allowing customization of the release profile and the daily drug release. Size exclusion chromatography and SDS–PAGE revealed that the antibody fragment structure was kept intact during incorporation and release from the liquid matrix. Furthermore, the released protein monomer maintained its high affinity to human VEGF-A, as measured by surface plasmon resonance analysis. Formulations of ESBA903 in hexPLA meet the basic needs to be used for intravitreal sustained-release applications in age-related macular degeneration treatment.

"Arming" the chimeric oncolytic adenovirus enadenotucirev to deliver checkpoint inhibitors and other therapeutics directly to tumours

Enadenotucirev (EnAd; formerly called ColoAd1) is a potent, chimeric Ad11p/Ad3 adenovirus active against a range of epithelial cancer cells, with a shorter time-to-lysis than either wild type Ad11p, Ad3 or Ad5. In normal cells, EnAd is attenuated and shows little or no activity by either cytotoxicity or by qPCR. In vivo, EnAd shows efficacy in a range of xenograft human tumour models following intra-tumoural, intravenous and intra-peritoneal injection [1] and is currently being evaluated clinically for treatment of several different epithelial cancers. Data from an ongoing clinical mechanism of action study have shown that i.v. dosed EnAd infects and replicates in tumour cells, producing significant amounts of viral protein (hexon), indicating that transgene encoded proteins will also be made in significant amounts by tumours following i.v. delivery of an armed EnAd virus. To develop 'armed' variants for delivery of therapeutic agents that enhance EnAd's anti-tumour activity, we have developed a novel, efficient cloning system for rapid generation of modified viruses that can be dosed systemically to deliver immunomodulatory antibodies into tumours. We chose to first encode anti-VEGF antibodies since, unlike immunomodulators, they could be readily evaluated in vivo in immunodeficient mouse human tumour xenograft models. We have successfully produced EnAd variants encoding full-length (NG-135) and ScFv (NG-76) forms of anti-human VEGF antibodies which have similar virus activity profiles to EnAd in cancer cell lines in vitro (virus replication, gene expression and oncolytic action), but also express and release the respective anti-VEGF antibody forms into the culture supernatant. Using either HCT-116 or DLD human colon carcinoma xenograft models we have shown that the virus infection profile following intra-tumoural injection is similar to the parental EnAd virus (virus replication and hexon gene expression). Anti-VEGF antibody expression by these tumours could be detected in the tissue as both mRNA and functional antibody. Antibodies were detectable early (within 3 days of infection) and expression was sustained over several weeks. Furthermore, low levels of anti-VEGF antibody were detectable in the blood. Production and evaluation of viruses similarly expressing checkpoint inhibitor antibodies is now in progress, together with evaluation of anti-VEGF armed oncolytic viruses for their impact on the growth and microenvironment of tumour xenografts.

CN-20 * BEVACIZUMAB CAUSING RECURRENT CEREBRAL INFARCTION IN A PATIENT WITH GLIOBLASTOMA: A PREVIOUSLY UNDOCUMENTED PHENOMENON

BACKGROUND: Bevacizumab, an anti-angiogenic VEGF antibody, is utilized for various malignancies, including glioblastoma multiforme (GBM). There has long been a correlation between bevacizumab therapy and CNS complications, including ischemic stroke and intracranial hemorrhage. Data on the pathophysiology of this correlation are scarce, and it has proven difficult to ascribe causality. To our knowledge there has never been a documented case of recurrent ischemia temporally associated with recurrent bevacizumab therapy. RESULTS: SS is a 70 year-old man with GBM. He received concomitant radiation and temozolamide (TMZ), followed by adjuvant TMZ and bevacizumab. After 5 cycles of TMZ/bevacizumab, he developed acute-onset left hemiparesis, upon which an MRI demonstrated an acute ischemic infarction. Angiography demonstrated mild attenuation of distal branches within the tumor bed. Chemotherapy was temporarily suspended, but after nearly two months, he had clinically recovered somewhat from the infarction. His TMZ/bevacizumab was restarted as his MRIs had been favorable to that point, with little disease progression. However, shortly thereafter, he had acute recurrence of his hemiparesis, and MRI again demonstrated acutely restricted diffusion. He was therefore switched to an alternative regimen that did not include bevacizumab. CONCLUSIONS: Here we describe a case of an ischemic stroke with radiographic correlate while on bevacizumab, with clinical improvement arrested by the development of a recurrent stroke when bevacizumab was restarted. Interestingly, the distal arteries within the stroke bed appeared somewhat attentuated, suggestive of radiation vasculopathy. This case, describing a previously undocumented phenomenon, raises the possibility of a synergistic effect of bevacizumab and radiation vasculopathy. It may be that radiation vasculoapthy alone is typically insufficient to cause ischemia, but the addition of bevacizumab may precipitate a sufficiently anti-vascular milieu as to cause frank and sudden-onset ischemia. In implicating bevacizumab, future directions may include large-scale studies to explore the temporal association of bevacizumab and ischemic stroke.