Alphavbeta3 Integrin Research Articles

An engineered AAV targeting integrin alpha V beta 6 presents improved myotropism across species

Current adeno-associated virus (AAV) gene therapy using nature-derived AAVs is limited by non-optimal tissue targeting. In the treatment of muscular diseases (MD), high doses are often required but can lead to severe adverse effects. Here, we rationally design an AAV capsid that specifically targets skeletal muscle to lower treatment doses. We computationally integrate binding motifs of human integrin alphaV beta6, a skeletal muscle receptor, into a liver-detargeting capsid. Designed AAVs show higher productivity and superior muscle transduction compared to their parent. One variant, LICA1, demonstrates comparable muscle transduction to other myotropic AAVs with reduced liver targeting. LICA1’s myotropic properties are observed across species, including non-human primate. Consequently, LICA1, but not AAV9, effectively delivers therapeutic transgenes and improved muscle functionality in two mouse MD models (male mice) at a low dose (5E12 vg/kg). These results underline the potential of our design method for AAV engineering and LICA1 variant for MD gene therapy.

Abstract 1091: Distinct mechanisms of orthotopic tumor control by alphavbeta3and HER2 CAR Ts support development of a bispecific CAR T cell therapy for pediatric rhabdomyosarcoma

Abstract Goal: Evaluate αvβ3 and HER2 as antigens for a bispecific chimeric antigen receptor T (CAR T) cell therapy in pediatric rhabdomyosarcoma. Background: Current treatments are ineffective against relapsed/refractory or metastatic rhabdomyosarcoma (RMS). Antigen loss or modulation on tumor cells commonly leads to tumor escape across diverse cancer types after CAR T therapy targeting different antigens. We hypothesize that simultaneously targeting two RMS antigens with a bispecific CAR T will reduce the likelihood of tumor escape. The integrin alphavbeta3 (αvβ3) plays a role in cancer progression, metastasis and neovascularization in several malignancies. HER2 also contributes to cancer progression and metastasis. Methods: Control of intramuscular RMS tumors by αvβ3 or HER2 CAR Ts was assessed in NSG mice, with non-specific CD19 CAR Ts as controls. Tumor burden was assessed weekly by bioluminescence imaging. Persistence and phenotype of CAR Ts were evaluated by flow cytometry. CAR T cell infiltration, tumor antigen expression and residual disease were determined by immunohistochemistry. Results: Six of six pediatric patient-derived RMS cell lines express αvβ3 and/or HER2. αvβ3 and HER2 CAR Ts kill RMS cells in vitro at low effector-to-target ratios. αvβ3 and HER2 CAR Ts independently control—and nearly eliminate—orthotopic RMS tumors. Fewer HER2 CAR Ts (5 × 106) are needed to control tumors than αvβ3 CAR Ts (10 × 106), but this is not due to lack of persistence or exhaustion of αvβ3 CAR Ts. Tumor control by αvβ3 CAR Ts is delayed (>21 days) compared to that by HER2 CAR Ts (7 days), and is robust, despite initial tumor progression following treatment. Importantly, despite strong tumor control by both CAR Ts, at 7 weeks post-treatment residual tumor nests are evident by histology. At this time, CAR Ts are still abundant, are not exhausted and express memory markers, suggesting that the remaining tumor cells are no longer susceptible to CAR T killing. We are currently evaluating the hypothesis that the remaining tumor cells have down-regulated antigen expression. Delayed tumor control by αvβ3 CAR Ts was ameliorated by engineering αvβ3 overexpression in RMS tumors. Conclusions: αvβ3 and HER2 CAR Ts control orthotopic RMS tumors by distinct mechanisms with changes in αvβ3 antigen expression, likely due to tumor progression, one prevalent factor. This is consistent with our prior reports of αvβ3 CAR Ts preventing breast cancer metastases, which naturally upregulate αvβ3 compared to the primary tumor. Despite near elimination of tumors, residual nests of tumor cells are likely to rebound as CAR T resistant tumors, similar to what is seen in patients treated with monospecific CAR Ts. These data support the incorporation of αvβ3 and HER2 CAR Ts into a bispecific CAR T approach to mitigate antigen escape. Our data also support investigation into the role of αvβ3 in RMS. Citation Format: Amanda M. Lulu, Nagyeong Park, Joseph J. Caruso, Dustin A. Cobb, Daniel W. Lee. Distinct mechanisms of orthotopic tumor control by alphavbeta3and HER2 CAR Ts support development of a bispecific CAR T cell therapy for pediatric rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1091.

The association and clinicopathological significance of Integrin alphavbeta6 and Rac1 expression in gastric carcinoma.

The present study investigate the expression and correlation of ITGB6 and Rac1 proteins in gastric cancer tissues. By exploring the clinical significance and functions of these proteins, we aimed to gain further insights into the mechanisms underlying gastric cancer development. In this study, a total of 198 patients diagnosed with gastric cancer and who underwent gastrectomy between July 2010 to October 2012 were included. The median follow-up time was 52.00 months. To evaluate the factors influencing overall survival, Kaplan-Meier survival curve analysis and Cox regression analysis were conducted. Furthermore, an independent prognostic factor-based nomogram was constructed and validated to predict survival outcomes in gastric cancer patients. In addition, in vitro experiments including CCK8 and Transwell assays were conducted to explore the roles of ITGB6 and Rac1 in gastric cancer. The expression levels of ITGB6 and Rac1 in gastric cancerous and paraneoplastic tissues were detected by immunohistochemistry. The correlation and clinical significance of the two proteins were also investigated. ITGB6 expression showed significant associations with tumor size (P=0.030), pathological grading (P=0.013), location (P=0.031), N stage (P=0.002), and clinical stage (P=0.002). Additionally, we found that tumor size (P=0.013), tumor's anatomical location (P=0.031), N stage (P=0.002), clinical stage (P=0.035), and survival status (P<0.001) were significantly associated with the expression of Rac1. ITGB6 was moderately correlated with Rac1 (r=0.285, P<0.001). Both the Kaplan-Meier survival analysis and Cox regression model analysis demonstrated that the presence of positive expression of ITGB6 and Rac1 proteins served as independent prognostic factors for gastric cancer. These findings highlight the potential of ITGB6 and Rac1 as valuable markers for predicting the prognosis of gastric cancer patients (HR=2.212 P<0.001 and HR=2.073 P=0.001), with a significant poorer trend for 5-year survival (P<0.0001, respectively, the log-rank test). Additionally, subsequent in vitro experiments preliminarily demonstrated that ITGB6 and Rac1 promoted the proliferation, migration and invasion of gastric cancer cells, and ITGB6 may functions via targeting Rac1. ITGB6 and Rac1 are indicators of poor prognosis and tumor progression in gastric cancer patients. The potential signaling pathways associated with both may provide useful targets for the prevention and treatment of gastric cancer.

Abstract B044: Targeting integrin alpha V beta 3 remodels the tumor microenvironment in pancreatic cancer

Abstract In pancreatic ductal adenocarcinoma (PDAC) the tumor microenvironment (TME) plays a crucial role in therapy resistance, metastasis, and recurrence. ProAgio is a novel therapeutic cytotoxin rationally designed to bind outside the RGD ligand pocket of the alphaV-beta3 integrin pair and then kill cells expressing this target. Pre-clinical studies demonstrated the safety and anti-tumor efficacy of ProAgio in animal models, and ProAgio is currently being tested in a Phase I clinical trial. The effect of ProAgio on TME cellular components has not been closely examined. We hypothesized that ProAgio remodels the PDAC TME to decrease stromal density and local immunosuppression by killing stromal cells thought to express the integrin pair, such as cancer associated fibroblasts (CAFs), proliferating endothelial cells, and tumor associated macrophages (TAMs). To identify potential cellular targets of ProAgio in the PDAC TME, we re-analyzed publicly available single cell RNA-seq datasets of human and mouse PDAC samples. We assessed integrin co-expression using flow cytometry, performed immunophenotyping and histopathological analysis to identify TME cellular and structural alterations induced by ProAgio. Integrins αV and β3 were both expressed at the RNA level in TAMs, myeloid cells, lymphocytes (T, B and NK), endothelial cells and CAFs. In the autochthonous KPC mouse model, we confirmed that granulocytes, macrophages, monocytes, CD4+ and CD8+T cells co-expressed integrin αVβ3 at the protein level. Amongst CAFs, inflammatory CAFs (iCAFs) had the highest percentage of cells co-expressing αV and β3 integrins. No anti-tumor effect was observed after 14 days of ProAgio treatment in a syngeneic KPC-derived PDAC orthotopic model, however, in the KPC autochthonous model the same treatment retarded tumor growth within 1 week. ProAgio treated autochthonous tumors displayed no changes in immune components or CAF subsets, but increased collagen area was observed. In addition, ProAgio treatment resulted in higher blood vessel area while integrin β3 protein levels remained stable. In conclusion, short-course ProAgio remodels the TME and delays tumor growth in an autochthonous PDAC model with mature stromal components, but does not deplete CAFs, TAMs or collagen area. Future studies will seek to refine our understanding of the cell subtypes affected by more prolonged ProAgio treatment and to determine the cellular and inflammatory mediators responsible for these changes. Citation Format: Mayrel Palestino Dominguez, Philip Homan, Xianyu Zhang, Sandra Navas Reyes, Theresa Guerin, Laura Bassel, Liu Zhi-ren, Serguei Kozlov, Christine Alewine. Targeting integrin alpha V beta 3 remodels the tumor microenvironment in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B044.

Colorectal polyps: Targets for fluorescence-guided endoscopy to detect high-grade dysplasia and T1 colorectal cancer.

Differentiating high-grade dysplasia (HGD) and T1 colorectal cancer (T1CRC) from low-grade dysplasia (LGD) in colorectal polyps can be challenging. Incorrect recognition of HGD or T1CRC foci can lead to a need for additional treatment after local resection, which might not have been necessary if it was recognized correctly. Tumor-targeted fluorescence-guided endoscopy might help to improve recognition. Selecting the most suitable HGD and T1CRC-specific imaging target from a panel of well-established biomarkers: carcinoembryonic antigen (CEA), c-mesenchymal-epithelial transition factor (c-MET), epithelial cell adhesion molecule (EpCAM), folate receptor alpha (FRα), and integrin alpha-v beta-6 (αvβ6). En bloc resection specimens of colorectal polyps harboring HGD or T1CRC were selected. Immunohistochemistry on paraffin sections was used to determine the biomarker expression in normal epithelium, LGD, HGD, and T1CRC (scores of 0-12). The differential expression in HGD-T1CRC components compared to surrounding LGD and normal components was assessed, just as the sensitivity and specificity of each marker. 60 specimens were included (21 HGD, 39 T1CRC). Positive expression (score >1) of HGD-T1CRC components was found in 73.3%, 78.3%, and 100% of cases for CEA, c-MET, and EpCAM, respectively, and in <40% for FRα and αvβ6. Negative expression (score 0-1) of the LGD component occurred more frequently for CEA (66.1%) than c-MET (31.6%) and EpCAM (0%). The differential expression in the HGD-T1CRC component compared to the surrounding LGD component was found for CEA in 66.7%, for c-MET in 43.1%, for EpCAM in 17.2%, for FRα in 22.4%, and for αvβ6 in 15.5% of the cases. Moreover, CEA showed the highest combined sensitivity (65.0%) and specificity (75.0%) for the detection of an HGD-T1CRC component in colorectal polyps. Of the tested targets, CEA appears the most suitable to specifically detect HGD and T1 cancer foci in colorectal polyps. An in vivo study using tumor-targeted fluorescence-guided endoscopy should confirm these findings.

Role of pericytes in the development of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is caused by loss-of-function mutations in CCM1, CCM2, or CCM3 genes of endothelial cells. It is characterized by pericyte deficiency. However, the role of pericytes in CCMs is not yet clarified. We found pericytes in Cdh5Cre ERT2 ;Ccm1 fl/fl (Ccm1 ECKO ) mice had a high expression of PDGFRβ. The inhibition of pericyte function by CP-673451 aggravated the CCM lesion development. RNA-sequencing analysis revealed the molecular traits of pericytes, such as highly expressed ECM-related genes, especially Fn1. Furthermore, KLF4 coupled with phosphorylated SMAD3 (pSMAD3) promoted the transcription of fibronectin in the pericytes of CCM lesions. RGDS peptide, an inhibitor of fibronectin, decreased the lesion area in the cerebella and retinas of Ccm1 ECKO mice. Also, human CCM lesions had abundant fibronectin deposition, and pSMAD3- and KLF4-positive pericytes. These findings indicate that pericytes are essential for CCM lesion development, and fibronectin intervention may provide a novel target for therapeutic intervention in such patients.

Uterine administration of C-X-C motif chemokine ligand 12 increases the pregnancy rates in mice with induced endometriosis

To study the effect of intrauterine injection of C-X-C motif chemokine ligand 12 (CXCL12), also known as a stem cell chemoattractant (stromal cell-derived factor 1), on fertility and endometrial receptivity in mice with endometriosis. Laboratory study. Academic Medical Center. Fifty-six mice underwent chemotherapy and bone marrow transplantation. Thirty-six of these mice underwent either surgery to induce endometriosis (n = 20) or sham surgery (n = 16). Injection of CXCL12 as a potential therapeutic agent to improve fertility in endometriosis. Pregnancy rate, bone marrow-derived cell (BMDC) recruitment and endometrial receptivity markers. The mice with or without endometriosis received a single uterine injection of either CXCL12 or placebo. Uterine injection of CXCL12 increased the pregnancy rates in a mouse model of endometriosis. Mice were euthanized after delivery, and implantation markers homeobox A11, alpha-v beta-3 integrin, and progesterone receptor were analyzed by immunohistochemistry, whereas green fluorescent protein positive BMDC recruitment was quantified by immunohistochemistry and immunofluorescence. The sham surgery groups without endometriosis had the highest cumulative pregnancy rate (100%) regardless of CXCL12 treatment. The endometriosis group treated with placebo had the lowest pregnancy rate. An increased pregnancy rate was noted in the endometriosis group after treatment with CXCL12. There was also an increase in BMDC recruitment and endometrial expression of progesterone receptor and alpha-v beta-3 integrin in the endometriosis group that received CXCL12 compared with that in the endometriosis group that received placebo. Uterine injection of CXCL12 increased the pregnancy rates in a mouse model of endometriosis. These results suggest that CXCL12 has a potential role as a therapeutic agent in women with infertility related to endometriosis and potentially other endometrial receptivity defects.

Molecular imaging of alphaVbeta3 integrin for evaluation of myocardial injury after acute myocardial infarction

Abstract Background 68Ga-NODAGA-RGD is a positron emission tomography (PET) tracer targeting αvβ3 integrin that is upregulated during angiogenesis. αvβ3 integrin expression increases early after acute myocardial infarction (AMI), and has been proposed as a marker of myocardial repair. Purpose We prospectively evaluated the uptake of 68Ga-NODAGA-RGD and its association to left ventricular function after human AMI. Methods Thirty patients underwent PET at 7.7±3.8 days after primary percutaneous coronary intervention for ST-elevation AMI. Resting myocardial perfusion was evaluated using 15O-water PET followed by evaluation of 68Ga-NODAGA-RGD uptake 60–75 minutes after injection of 179 MBq of tracer. Left ventricular function was evaluated by transthoracic echocardiography on the day of PET, and at 6-month follow-up. The definition of the ischemic area at risk and remote myocardial segments was based on the culprit coronary arterial segments in invasive angiography. 68Ga-NODAGA-RGD images were co-registered with perfusion images and uptake was measured as the standardized uptake value in the segment with the highest uptake (SUVmax) in ischemic area at risk, and the mean standardized uptake value (SUVmean) in remote segments. In addition, we calculated 68Ga-NODAGA-RGD uptake corrected to the mean myocardial blood flow (MBF) in the area at risk (SUVmax/MBFmean) to account for reduced distribution of tracer in non-viable tissue. Results Mean age of patients was 64±9 years, and 90% were males. Uptake of 68Ga-NODAGA-RGD was low in the remote myocardium, but focally increased in the ischemic area at risk (Figure 1). SUVmax in the ischemic area at risk was higher than SUVmean of the remote myocardium (0.73±0.16 vs. 0.51±0.11, p&amp;lt;0.001). 68Ga-NODAGA-RGD SUVmax did not correlate with MBF in the area at risk. Univariable predictors of 68Ga-NODAGA-RGD SUVmax in the area at risk included peak Troponin T (p&amp;lt;0.001), peak pro-BNP (p&amp;lt;0.001), low global longitudinal strain (p=0.01), and low regional longitudinal strain in the area at risk (p=0.02). In multivariable analysis, peak pro-BNP independently predicted SUVmax in the area at risk (p&amp;lt;0.001). At follow-up, left ventricular ejection fraction increased by 1.6±6.9% and global longitudinal strain by 0.5±3.2%. In univariable analysis, SUVmax and SUVmax/MBFmean in the area at risk predicted improvement of global longitudinal strain at 6 months after AMI (p=0.04 and p&amp;lt;0.001, respectively). Conclusion 68Ga-NODAGA-RGD uptake shows increased αvβ3 integrin expression in the ischemic area at risk early after reperfused AMI that is associated with the extent of myocardial injury, both regional and global systolic dysfunction, and increased left ventricular filling pressure. Increased 68Ga-NODAGA-RGD uptake in ischemic myocardium at risk predicts left ventricular function improvement at 6 months after AMI. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Academy of Finland, Finnish Foundation for Cardiovascular Research

Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXβ2.

SUMMARYAllosteric activation and silencing of leukocyte β2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXβ2 ligand-binding domain—the αX I-domain—corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH− and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXβ2. This work highlights how intrinsically flexible pH− and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.

Targeting of the alphav beta3 integrin complex by CAR-T cells leads to rapid regression of diffuse intrinsic pontine glioma and glioblastoma

BackgroundDiffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are two highly aggressive and generally incurable gliomas with little therapeutic advancements made in the past several decades. Despite immense initial success...

Pharmacological characterisation of GSK3335103, an oral αvβ6 integrin small molecule RGD-mimetic inhibitor for the treatment of fibrotic disease

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality. Activation of the pro-fibrotic cytokine transforming growth factor-β (TGFβ) via the alpha-V beta-6 (αvβ6) integrin has been identified as playing a key role in the development of fibrosis. Therefore, a drug discovery programme to identify an orally bioavailable small molecule αvβ6 arginyl-glycinyl-aspartic acid (RGD)-mimetic was initiated. As part of a medicinal chemistry programme GSK3335103 was identified and profiled in a range of pre-clinical in vitro and in vivo systems. GSK3335103 was shown to bind to the αvβ6 with high affinity and demonstrated fast binding kinetics. In primary human lung epithelial cells, GSK3335103-induced concentration- and time-dependent internalisation of αvβ6 with a rapid return of integrin to the cell surface observed after washout. Following sustained engagement of the αvβ6 integrin in vitro, lysosomal degradation was induced by GSK3335103. GSK3335103 was shown to engage with the αvβ6 integrin and inhibit the activation of TGFβ in both ex vivo IPF tissue and in a murine model of bleomycin-induced lung fibrosis, as measured by αvβ6 engagement, TGFβ signalling and collagen deposition, with a prolonged duration of action observed in vivo. In summary, GSK3335103 is a potent αvβ6 inhibitor that attenuates TGFβ signalling in vitro and in vivo with a well-defined pharmacokinetic/pharmacodynamic relationship. This translates to a significant reduction of collagen deposition in vivo and therefore GSK3335103 represents a potential novel oral therapy for fibrotic disorders.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

Recognition and management of vascular lesions in von Willebrand disease

Recognition and management of vascular lesions in von Willebrand disease

Cisplatin decreases HOXA13 and alphaVBeta3 integrin levels in the uterus

ObjectiveTo examine the effects of cisplatin on uterine histology and implantation molecules and the possible protective role of recombinant Klotho administration on uterine histology and uterine receptivity in mice exposed to cisplatin. Materials and methodsThis study was conducted using thirty-two adult female mice assigned to four groups with 8 mice in each group. Saline was given to the 1st group, cisplatin to the 2nd group, recombinant mouse Klotho to the 3rd group and recombinant mouse Klotho plus cisplatin to the 4th group. Uterine tissues were examined for damage histologically and immunobiologically for the uterine receptivity markers HOXA13 and alphaVBeta3 integrin. ResultsApoptosis, degeneration, decrease in uterine thickness and uterine absence of gland scores were higher in the cisplatin group (3rd group) compared to the saline group (1st group) (cisplatin vs. saline p < 0.0001 for all parameters). In the recombinant Klotho plus cisplatin group (4th group), scores of apoptosis, degeneration, reduction in uterine thickness and uterine absence of gland were lower than the group receiving only cisplatin (cisplatin plus recombinant Klotho vs cisplatin, p = 0.006 for apoptosis; p = 0.017 for degeneration; p = 0.011 for the reduction in uterine thickness; p = 0.002 for the absence of gland). However, HOXA13 and alphaVBeta3 integrin staining levels were not different between the cisplatin group (group 3) and the cisplatin plus recombinant Klotho group (group 4) (p = 0.980 and p = 0.762, respectively.) ConclusionCisplatin has adverse effects on the uterus. Administration of recombinant Klotho was found to attenuate the cisplatin-induced damage but failed to preserve levels of the implantation molecules HOXA13 and alphaVbeta3. Further studies examining the effect of cisplatin toxicity using other implantation markers along with functional studies are needed.

Fuzzy binding model of molecular interactions between tropoelastin and integrin alphaVbeta3

Tropoelastin is the highly flexible monomer subunit of elastin, required for the resilience of the extracellular matrix in elastic tissues. To elicit biological signaling, multiple sites on tropoelastin bind to cell surface integrins in a poorly understood multifactorial process. We constructed a full atomistic molecular model of the interactions between tropoelastin and integrin αvβ3 using ensemble-based computational methodologies. Conformational changes of integrin αvβ3 associated with outside-in signaling were more frequently facilitated in an ensemble in which tropoelastin bound the integrin’s α1 helix rather than the upstream canonical binding site. Our findings support a model of fuzzy binding, whereby many tropoelastin conformations and defined sites cooperatively interact with multiple αvβ3 regions. This model explains prior experimental binding to distinct tropoelastin regions, domains 17 and 36, and points to the cooperative participation of domain 20. Our study highlights the utility of ensemble-based approaches in helping to understand the interactive mechanisms of functionally significant flexible proteins.

Downregulation of the αvβ6 Integrin via RGD Engagement Is Affinity and Time Dependent.

The arginyl-glycinyl-aspartic acid (RGD) integrin alpha-v beta-6 (αvβ6) has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesized to be pivotal in the development of fibrosis and other diseases. In this study, αvβ6 small molecule inhibitors were characterized in a range of in vitro systems to determine affinity, kinetics, and duration of TGFβ inhibition. High αvβ6 binding affinity was shown to be correlated with slow dissociation kinetics. Compound 1 (high αvβ6 affinity, slow dissociation) and SC-68448 (low αvβ6 affinity, fast dissociation) induced concentration- and time-dependent internalization of αvβ6 in normal human bronchial epithelial (NHBE) cells. After washout, the αvβ6 cell surface repopulation was faster for SC-68448 compared with compound 1 In addition, αvβ6-dependent release of active TGFβ from NHBE cells was inhibited by compound 1 and SC-68448. After washout of SC-68448, release of active TGFβ was restored, whereas after washout of compound 1 the inhibition of TGFβ activation was maintained and only reversible in the presence of a lysosomal inhibitor (chloroquine). However, SC-68448 was able to reduce total levels of αvβ6 in NHBE cells if present continuously. These observations suggest αvβ6 can be degraded after high affinity RGD binding that sorts the integrin for lysosomal degradation after internalization, likely due to sustained engagement as a result of slow dissociation kinetics. In addition, the αvβ6 integrin can also be downregulated after sustained engagement of the RGD binding site with low affinity ligands that do not sort the integrin for immediate lysosomal degradation. SIGNIFICANCE STATEMENT: The fate of RGD integrin after ligand binding has not been widely investigated. Using the αvβ6 integrin as a case study, we have demonstrated that RGD-induced downregulation of αvβ6 is both affinity and time dependent. High affinity ligands induced downregulation via lysosomal degradation, likely due to slow dissociation, whereas sustained low affinity ligand engagement was only able to decrease αvβ6 expression over longer periods of time. Our study provides a potential unique mechanism for obtaining duration of action for drugs targeting integrins.

Abstract 6206: The xl313 cryptic collagen epitope regulates ovarian tumor growth by a yap dependent mechanism

Abstract Structural changes in the extra cellular matrix (ECM) can lead to the exposure of cryptic regulatory sites that play important roles in angiogenesis, inflammation, tumor growth, and tumor metastasis. Previously, we discovered that a unique cryptic regulatory site (XL313 cryptic epitope) containing the amino acid sequence RGDKGE, is generated during tumor development, and we found that this collagen epitope promotes angiogenesis and inflammation in vivo. Here, we provide the first evidence that ovarian carcinoma cells can bind the XL313 collagen epitope through integrin alpha-V beta-3 and that selectively blocking cellular interactions with the XL313 epitope significantly inhibits ovarian tumor growth and the formation of ascites fluid in vivo. To understand the cellular mechanism(s) by which antagonists of the XL313 epitope inhibit these pathological processes, we examined down-stream signaling events that were altered following blocking ovarian tumor cell interaction with the XL313 collagen epitope. Importantly, selectively targeting the XL313 epitope significantly reduced nuclear accumulation of the transcriptional co-activator YAP. In addition, blocking cellular interaction with the XL313 epitope resulted in enhanced YAP phosphorylation at serine-127. Interestingly, specific targeting of the XL313 epitope also enhanced LATS1 phosphorylation, which is a key effector molecule in the hippo- signaling pathway. Taken together, our data provide new cellular and molecular insight for understanding the role of the XL313 cryptic collagen epitope in controlling ovarian tumor growth, and suggest that selectively targeting the XL313 epitope-integrin-signaling cascade may represent a novel strategy to inhibit ovarian cancer progression. Citation Format: Xianghua Han, Jennifer M. Caron, Peter C. Brooks. The xl313 cryptic collagen epitope regulates ovarian tumor growth by a yap dependent mechanism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6206.

A positron emission tomography imaging study to confirm target engagement in the lungs of patients with idiopathic pulmonary fibrosis following a single dose of a novel inhaled \u03b1v\u03b26 integrin inhibitor

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease with poor prognosis and a significant unmet medical need. This study evaluated the safety, pharmacokinetics (PK) and target engagement in the lungs, of GSK3008348, a novel inhaled alpha-v beta-6 (αvβ6) integrin inhibitor, in participants with IPF.MethodsThis was a phase 1b, randomised, double-blind (sponsor unblind) study, conducted in the UK (two clinical sites, one imaging unit) between June 2017 and July 2018 (NCT03069989). Participants with a definite or probable diagnosis of IPF received a single nebulised dose of 1000 mcg GSK3008348 or placebo (ratio 5:2) in two dosing periods. In period 1, safety and PK assessments were performed up to 24 h post-dose; in period 2, after a 7-day to 28-day washout, participants underwent a total of three positron emission tomography (PET) scans: baseline, Day 1 (~ 30 min post-dosing) and Day 2 (~ 24 h post-dosing), using a radiolabelled αvβ6-specific ligand, [18F]FB-A20FMDV2. The primary endpoint was whole lung volume of distribution (VT), not corrected for air volume, at ~ 30 min post-dose compared with pre-dose. The study success criterion, determined using Bayesian analysis, was a posterior probability (true % reduction in VT > 0%) of ≥80%.ResultsEight participants with IPF were enrolled and seven completed the study. Adjusted posterior median reduction in uncorrected VT at ~ 30 min after GSK3008348 inhalation was 20% (95% CrI: − 9 to 42%). The posterior probability that the true % reduction in VT > 0% was 93%. GSK3008348 was well tolerated with no reports of serious adverse events or clinically significant abnormalities that were attributable to study treatment. PK was successfully characterised showing rapid absorption followed by a multiphasic elimination.ConclusionsThis study demonstrated engagement of the αvβ6 integrin target in the lung following nebulised dosing with GSK3008348 to participants with IPF. To the best of our knowledge this is the first time a target-specific PET radioligand has been used to assess target engagement in the lung, not least for an inhaled drug.Trial registrationclinicaltrials.gov: NCT03069989; date of registration: 3 March 2017.

Accelerated blood clearance of targeted ultrasound contrast reduced molecular imaging signal intensity: Secreted Frizzled Related Protein-2 signal remained significantly higher than signal from either Vascular Endothelial Growth Factor Receptor-2 or alphaVbeta3 integrin.

Multiple doses of polyethylene glycol (PEG) decorated pharmaceuticals cause accelerated blood clearance (ABC) due to the generation of antibodies reactive to the PEG moiety. Using molecular imaging to monitor response to therapy could be complicated by the ABC effect due to PEG chains in microbubble lipid shells. Our objective was to measure the half-life of targeted contrast flowing through non-tumor tissue during longitudinal imaging studies, and to determine which targeted agent returned the highest signal intensity within tumors. The molecular imaging signals from contrast agents targeted to three distinct molecular targets, Secreted Frizzled-Related Protein-2 (SFRP2), Vascular Endothelial Growth Factor Receptor-2 (VEGFR2), AlphaVBeta3 Integrin (avb3) were all significantly correlated to contrast half-life. The molecular imaging signal from SFRP2 remained significantly higher than the signal returned by ultrasound contrast targeted to either VEGFR2 or avb3 before and after restricting analyses to imaging exams with similar half-lives. We hypothesize that increasing immune clearance rates during our longitudinal studies limited the amount of targeted contrast able to perfuse tumor vasculature, and that this resulted in a global dose-dependent decrease in molecular imaging signals. Molecular imaging may underestimate biomarker levels as longitudinal studies progress and as contrast half-lives decrease, unless contrast dosing is normalized by the amount of contrast able to reach the tumor and surrounding tissue rather than by the injected dosage.

Angiopoietin-Tie Signaling Pathway in Endothelial Cells: A Computational Model.

SummaryThe angiopoietin-Tie signaling pathway is an important vascular signaling pathway involved in angiogenesis, vascular stability, and quiescence. Dysregulation in the pathway is linked to the impairments in vascular function associated with many diseases, including cancer, ocular diseases, systemic inflammation, and cardiovascular diseases. The present study uses a computational signaling pathway model validated against experimental data to quantitatively study various mechanistic aspects of the angiopoietin-Tie signaling pathway, including receptor activation, trafficking, turnover, and molecular mechanisms of its regulation. The model provides mechanistic insights into the controversial role of Ang2 and its regulators vascular endothelial protein tyrosine phosphatase (VE-PTP) and Tie1 and predicts synergistic effects of inhibition of VE-PTP, Tie1, and Tie2 cleavage on enhancing the vascular protective actions of Tie2.