A novel assay for monitoring internalization of nanocarrier coupled antibodies

BACKGROUND: Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA. RESULTS: The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay. CONCLUSION: The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers

Suicide attempts and non-suicidal self-harm: national prevalence study of young adults

Background There are few prevalence studies of suicide attempts and non-suicidal self-harm (NSSH). Aims We aimed to estimate the prevalence of thoughts of NSSH, suicidal thoughts, NSSH and suicide attempts among 18- to 34-year-olds in Scotland. Method We interviewed a representative sample of young adults from across Scotland. Results We interviewed 3508 young people; 11.3 and 16.2% reported a lifetime history of suicide attempts and NSSH, respectively. The first episode of NSSH tended to precede the first suicide attempt by about 2 years. Age at onset of NSSH and suicide attempt was younger in females. Earlier age at onset was associated with more frequent NSSH/suicide attempts. Women are significantly more likely to report NSSH and suicide attempts compared with men. Conclusions One in nine young people has attempted suicide and one in six has engaged in NSSH. Clinicians should be vigilant, as suicide attempts and NSSH are relatively common

Analysis of Single-Cell RNA-Seq Identifies Cell-Cell Communication Associated with Tumor Characteristics

Tumor ecosystems are composed of multiple cell types that communicate by ligand-receptor interactions. Targeting ligand-receptor interactions (for instance, with immune checkpoint inhibitors) can provide significant benefits for patients. However, our knowledge of which interactions occur in a tumor and how these interactions affect outcome is still limited. We present an approach to characterize communication by ligand-receptor interactions across all cell types in a microenvironment using single-cell RNA sequencing. We apply this approach to identify and compare the ligand-receptor interactions present in six syngeneic mouse tumor models. To identify interactions potentially associated with outcome, we regress interactions against phenotypic measurements of tumor growth rate. In addition, we quantify ligand-receptor interactions between T cell subsets and their relation to immune infiltration using a publicly available human melanoma dataset. Overall, this approach provides a tool for studying cell-cell interactions, their variability across tumors, and their relationship to outcome. Tumors are composed of cancer cells and many non-malignant cell types, such as immune and stromal cells. To better understand how all cell types in a tumor cooperate to facilitate malignant growth, Kumar et al. studied communication between cells via ligand and receptor interactions using single-cell data and computational modeling. Keywords: computational analysis; single-cell RNA sequencing; cell-cell communication; ligand-receptor interaction; tumor microenvironment; syngeneic mouse models; cancer patient samplesNational Institute of General Medical Sciences (U.S.) (Grant T32-GM008334)National Cancer Institute (U.S.) (Grant U01-CA215798

Characterization of highly stable liposomal and immunoliposomal formulations of vincristine and vinblastine

Liposome and immunoliposome formulations of two vinca alkaloids, vincristine and vinblastine, were prepared using intraliposomal triethylammonium sucroseoctasulfate and examined for their ability to stabilize the drug for targeted drug delivery in vivo. The pharmacokinetics of both the encapsulated drug (vincristine or vinblastine) and liposomal carrier were examined in Sprague Dawley rats, and the in vivo drug release rates determined. Anti-HER2 immunoliposomal vincristine was prepared from a human anti-HER2/neu scFv and studied for targeted cytotoxic activity in cell culture, and antitumor efficacy in vivo. Nanoliposome formulations of vincristine and vinblastine demonstrated similar pharmacokinetic profiles for the liposomal carrier, but increased clearance for liposome encapsulated vinblastine (t 1/2 = 9.7 h) relative to vincristine (t 1/2 = 18.5 h). Immunoliposome formulations of vincristine targeted to HER2 using an anti-HER2 scFv antibody fragment displayed a marked enhancement in cytotoxicity when compared to non-targeted liposomal vincristine control; 63- or 253-fold for BT474 and SKBR3 breast cancer cells, respectively. Target-specific activity was also demonstrated in HER2-overexpressing human tumor xenografts, where the HER2-targeted formulation was significantly more efficacious than either free vincristine or non-targeted liposomal vincristine. These results demonstrate that active targeting of solid tumors with liposomal formulations of vincristine is possible when the resulting immunoliposomes are sufficiently stabilized

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Modeling chemotherapy-induced stress to identify rational combination therapies in the DNA damage response pathway

Cells respond to DNA damage by activating complex signaling networks that decide cell fate, promoting not only DNA damage repair and survival but also cell death. We have developed a multiscale computational model that quantitatively links chemotherapy-induced DNA damage response signaling to cell fate. The computational model was trained and calibrated on extensive data from U2OS osteosarcoma cells, including the cell cycle distribution of the initial cell population, signaling data measured by Western blotting, and cell fate data in response to chemotherapy treatment measured by time-lapse microscopy. The resulting mechanistic model predicted the cellular responses to chemotherapy alone and in combination with targeted inhibitors of the DNA damage response pathway, which we confirmed experimentally. Computational models such as the one presented here can be used to understand the molecular basis that defines the complex interplay between cell survival and cell death and to rationally identify chemotherapy-potentiating drug combinations