A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth in vivo

<p>Abstract</p> <p>Background</p> <p>Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin α5β1 has been shown to play a critical role during angiogenesis. An inhibitor of this integrin, volociximab (M200), inhibits endothelial cell growth and movement <it>in vitro</it>, independent of the growth factor milieu, and inhibits tumor growth <it>in vivo </it>in the rabbit VX2 carcinoma model. Although volociximab has already been tested in open label, pilot phase II clinical trials in melanoma, pancreatic and renal cell cancer, evaluation of the mechanism of action of volociximab has been limited because this antibody does not cross-react with murine α5β1, precluding its use in standard mouse xenograft models.</p> <p>Methods</p> <p>We generated a panel of rat-anti-mouse α5β1 antibodies, with the intent of identifying an antibody that recapitulated the properties of volociximab. Hybridoma clones were screened for analogous function to volociximab, including specificity for α5β1 heterodimer and blocking of integrin binding to fibronectin. A subset of antibodies that met these criteria were further characterized for their capacities to bind to mouse endothelial cells, inhibit cell migration and block angiogenesis <it>in vitro</it>. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models.</p> <p>Results</p> <p>A panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin α5β1 was determined to be 0.59 nM, as measured by BIAcore. This antibody does not significantly cross-react with human integrin, however 339.1 inhibits murine endothelial cell migration and tube formation and elicits cell death in these cells (EC₅₀= 5.3 nM). In multiple xenograft models, 339.1 inhibited the growth of established tumors by 40–60% (<it>p </it>< 0.05) and this inhibition correlates with a concomitant decrease in vessel density.</p> <p>Conclusion</p> <p>The results herein demonstrate that 339.1, like volociximab, exhibits potent anti-α5β1 activity and confirms that inhibition of integrin α5β1 impedes angiogenesis and slows tumor growth <it>in vivo</it>.</p

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth -5

<p><b>Copyright information:</b></p><p>Taken from "A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth "</p><p>http://www.translational-medicine.com/content/5/1/61</p><p>Journal of Translational Medicine 2007;5():61-61.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2235829.</p><p></p>reated with 339.1 (10 mg/kg, intraperitoneally, thrice or twice weekly, respectively) or vehicle control and tumor volume was monitored using vernier calipers. 339.1 inhibits tumor growth relative to control. Results were statistically significant in both settings. A673 tumors were resected from 339.1- and control-treated mice and frozen sections were assessed for vessel density by immunohistochemical staining for CD31, (C). Vessel density was significantly reduced in tumors from animals treated with 339.1, (D)

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth -1

<p><b>Copyright information:</b></p><p>Taken from "A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth "</p><p>http://www.translational-medicine.com/content/5/1/61</p><p>Journal of Translational Medicine 2007;5():61-61.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2235829.</p><p></p> Fc fusion proteins, (A). A subset of competitive antibodies cross-reacted with human integrin. Antibodies were tested by immunohistochemistry for staining of sections from C32 melanoma (α5β1 negative) or MDA-MB-231 breast carcinoma (α5β1 positive) xenografts, (B). The majority of antibodies tested stained murine α5β1 on tumor vasculature, but only antibodies found to cross-react with human α5β1 by ELISA specifically stained MDA-MB-231 xenograft cells as well. IIA1, the mouse parent antibody of volociximab, which recognizes only human integrin, anti-mouse CD31, which stains mouse vessels, and pooled rat IgG are shown as controls

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth -2

<p><b>Copyright information:</b></p><p>Taken from "A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth "</p><p>http://www.translational-medicine.com/content/5/1/61</p><p>Journal of Translational Medicine 2007;5():61-61.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2235829.</p><p></p> the opposite side of transwell membranes in the presence or absence of anti-integrin antibodies. Cells were stained with Calcein AM and visualized by fluorescence microscopy. Migration in quintiplicate wells was quantified using the Discovery-1 system, (B). Antibodies that inhibited binding of α5β1 binding to fibronectin also inhibited migration towards fibronectin

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth -3

<p><b>Copyright information:</b></p><p>Taken from "A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth "</p><p>http://www.translational-medicine.com/content/5/1/61</p><p>Journal of Translational Medicine 2007;5():61-61.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2235829.</p><p></p> assessed for Annexin V-Alexa Fluor 488 binding by flow cytometry. Cells were counterstained with propidium iodide (PI) to follow non-specific death and the percentage of cells that stained positive for Annexin V and negative for PI was plotted. Representative results from three individual experiments are shown, (A). Only antibody 339.1, which does not cross-react with human α5β1, elicited cell death in these cells. A dose response curve for 339.1 in this assay using SVR murine angiosarcoma cells revealed an ECof 5.3 nM, (A)

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth -6

<p><b>Copyright information:</b></p><p>Taken from "A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth "</p><p>http://www.translational-medicine.com/content/5/1/61</p><p>Journal of Translational Medicine 2007;5():61-61.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2235829.</p><p></p>-Fc homodimer or an irrelevant Fc fusion protein, (A). Hybridomas were found to produce antibodies with specific preferences for each protein. Supernatants were further tested for binding to murine EC's by flow cytometry, (B). The majority of antibodies tested recognized cell surface integrin. Antibodies purified from supernatants that recognized murine EC's by flow cytometry and showed a preference for α5β1 heterodimer were compared for their capacity to inhibit the binding of α5β1-Fc to mouse fibronectin by competitive ELISA, (C). Many antibodies inhibited integrin-fibronectin binding; one antibody, 321.1, was found to promote binding. Antibodies were assessed for binding to cellular integrin α5β1 on SVR cells by flow cytometry, (D). Most of the antibodies that bound integrin α5β1 by ELISA also bind cell surface integrin

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth -0

<p><b>Copyright information:</b></p><p>Taken from "A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth "</p><p>http://www.translational-medicine.com/content/5/1/61</p><p>Journal of Translational Medicine 2007;5():61-61.</p><p>Published online 27 Nov 2007</p><p>PMCID:PMC2235829.</p><p></p>-Fc homodimer or an irrelevant Fc fusion protein, (A). Hybridomas were found to produce antibodies with specific preferences for each protein. Supernatants were further tested for binding to murine EC's by flow cytometry, (B). The majority of antibodies tested recognized cell surface integrin. Antibodies purified from supernatants that recognized murine EC's by flow cytometry and showed a preference for α5β1 heterodimer were compared for their capacity to inhibit the binding of α5β1-Fc to mouse fibronectin by competitive ELISA, (C). Many antibodies inhibited integrin-fibronectin binding; one antibody, 321.1, was found to promote binding. Antibodies were assessed for binding to cellular integrin α5β1 on SVR cells by flow cytometry, (D). Most of the antibodies that bound integrin α5β1 by ELISA also bind cell surface integrin

Second asymptomatic carotid surgery trial (ACST-2) : a randomised comparison of carotid artery stenting versus carotid endarterectomy

Background: Among asymptomatic patients with severe carotid artery stenosis but no recent stroke or transient cerebral ischaemia, either carotid artery stenting (CAS) or carotid endarterectomy (CEA) can restore patency and reduce long-term stroke risks. However, from recent national registry data, each option causes about 1% procedural risk of disabling stroke or death. Comparison of their long-term protective effects requires large-scale randomised evidence. Methods: ACST-2 is an international multicentre randomised trial of CAS versus CEA among asymptomatic patients with severe stenosis thought to require intervention, interpreted with all other relevant trials. Patients were eligible if they had severe unilateral or bilateral carotid artery stenosis and both doctor and patient agreed that a carotid procedure should be undertaken, but they were substantially uncertain which one to choose. Patients were randomly allocated to CAS or CEA and followed up at 1 month and then annually, for a mean 5 years. Procedural events were those within 30 days of the intervention. Intention-to-treat analyses are provided. Analyses including procedural hazards use tabular methods. Analyses and meta-analyses of non-procedural strokes use Kaplan-Meier and log-rank methods. The trial is registered with the ISRCTN registry, ISRCTN21144362. Findings: Between Jan 15, 2008, and Dec 31, 2020, 3625 patients in 130 centres were randomly allocated, 1811 to CAS and 1814 to CEA, with good compliance, good medical therapy and a mean 5 years of follow-up. Overall, 1% had disabling stroke or death procedurally (15 allocated to CAS and 18 to CEA) and 2% had non-disabling procedural stroke (48 allocated to CAS and 29 to CEA). Kaplan-Meier estimates of 5-year non-procedural stroke were 2·5% in each group for fatal or disabling stroke, and 5·3% with CAS versus 4·5% with CEA for any stroke (rate ratio [RR] 1·16, 95% CI 0·86-1·57; p=0·33). Combining RRs for any non-procedural stroke in all CAS versus CEA trials, the RR was similar in symptomatic and asymptomatic patients (overall RR 1·11, 95% CI 0·91-1·32; p=0·21). Interpretation: Serious complications are similarly uncommon after competent CAS and CEA, and the long-term effects of these two carotid artery procedures on fatal or disabling stroke are comparable