Antibody conjugates directed against intercellular adhesion molecule(ICAM-1) or platelet-endothelial cell adhesion molecule (PECAM-1) have formed the basis for drug delivery vehicles that are specifically recognized and internalized by endothelial cells. There is increasing evidence that ICAM-1 and PECAM-1 may also play a role in cell scavenger functions and pathogen entry. To define the mechanisms that regulate ICAM-1 and PECAM-1 internalization, we examined the uptake of anti-PECAM-1 and anti-ICAM-1 conjugates by endothelial cells. We found that the conjugates must be multimeric, because monomeric anti-ICAM-1 and anti-PECAM-1 are not internalized. Newly internalized anti-ICAM-1 and anti-PECAM-1 conjugates did not colocalize with either clathrin or caveolin, and immunoconjugate internalization was not reduced by inhibitors of clathrin-mediated or caveolar endocytosis, suggesting that this is a novel endocytic pathway. Amiloride and protein kinase C (PKC) inhibitors, agents known to inhibit macropinocytosis,reduced the internalization of clustered ICAM-1 and PECAM-1. However,expression of dominant-negative dynamin-2 constructs inhibited uptake of clustered ICAM-1. Binding of anti-ICAM-1 conjugates stimulated the formation of actin stress fibers by human umbilical vein endothelial cells (HUVEC). Latrunculin, radicicol and Y27632 also inhibited internalization of clustered ICAM-1, suggesting that actin rearrangements requiring Src kinase and Rho kinase (ROCK) were required for internalization. Interestingly, these kinases are part of the signal transduction pathways that are activated when circulating leukocytes engage endothelial cell adhesion molecules, suggesting the possibility that CAM-mediated endocytosis is regulated using comparable signaling pathways.

Background Tocilizumab, a monoclonal antibody directed against the interleukin-6 receptor, has been proposed to mitigate the cytokine storm syndrome associated with severe COVID-19. We aimed to investigate the association between tocilizumab exposure and hospital-related mortality among patients requiring intensive care unit (ICU) support for COVID-19. Methods We did a retrospective observational cohort study at 13 hospitals within the Hackensack Meridian Health network (NJ, USA). We included patients (aged ≥18 years) with laboratory-confirmed COVID-19 who needed support in the ICU. We obtained data from a prospective observational database and compared outcomes in patients who received tocilizumab with those who did not. We applied a multivariable Cox model with propensity score matching to reduce confounding effects. The primary endpoint was hospital-related mortality. The prospective observational database is registered on ClinicalTrials.gov , NCT04347993 . Findings Between March 1 and April 22, 2020, 764 patients with COVID-19 required support in the ICU, of whom 210 (27%) received tocilizumab. Factors associated with receiving tocilizumab were patients' age, gender, renal function, and treatment location. 630 patients were included in the propensity score-matched population, of whom 210 received tocilizumab and 420 did not receive tocilizumab. 358 (57%) of 630 patients died, 102 (49%) who received tocilizumab and 256 (61%) who did not receive tocilizumab. Overall median survival from time of admission was not reached (95% CI 23 days–not reached) among patients receiving tocilizumab and was 19 days (16–26) for those who did not receive tocilizumab (hazard ratio [HR] 0·71, 95% CI 0·56–0·89; p=0·0027). In the primary multivariable Cox regression analysis with propensity matching, an association was noted between receiving tocilizumab and decreased hospital-related mortality (HR 0·64, 95% CI 0·47–0·87; p=0·0040). Similar associations with tocilizumab were noted among subgroups requiring mechanical ventilatory support and with baseline C-reactive protein of 15 mg/dL or higher. Interpretation In this observational study, patients with COVID-19 requiring ICU support who received tocilizumab had reduced mortality. Results of ongoing randomised controlled trials are awaited. Funding None.

Drug targeting to a highly expressed, noninternalizable determinant up-regulated on the perturbed endothelium may help to manage inflammation and thrombosis. We tested whether inter-cellular adhesion molecule-1 (ICAM-1) targeting is suitable to deliver antithrombotic drugs to the pulmonary vascular lumen. ICAM-1 antibodies bind to the surface of endothelial cells in culture, in perfused lungs, and in vivo. Proinflammatory cytokines enhance anti-ICAM binding to the endothelium without inducing internalization. (125)I-labeled anti-ICAM and a reporter enzyme (beta-Gal) conjugated to anti-ICAM bind to endothelium and accumulate in the lungs after intravenous administration in rats and mice. Anti-ICAM is seen to localize predominantly on the luminal surface of the pulmonary endothelium by electron microscopy. We studied the pharmacological effect of ICAM-directed targeting of tissue-type plasminogen activator (tPA). Anti-ICAM/tPA, but not control IgG/tPA, conjugate accumulates in the rat lungs, where it exerts plasminogen activator activity and dissolves fibrin microemboli. Therefore, ICAM may serve as a target for drug delivery to endothelium, for example, for pulmonary thromboprophylaxis. Enhanced drug delivery to sites of inflammation and the potential anti-inflammatory effect of blocking ICAM-1 may enhance the benefit of this targeting strategy.

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