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

Using stable isotopes to assess surface water source dynamics and hydrological connectivity in a high-latitude wetland and permafrost influenced landscape

The research has been supported by the NERC/JPI SIWA project (NE/M019896/1); grant issued in accordance with Resolution of the Government of the Russian Federation No. 220 dated 9 April 2010, under Agreement No. 14.B25.31.0001 with Ministry of Education and Science of the Russian Federation dated 24 June 2013 (BIO-GEO-CLIM); grant RFBR No 17-05-00-348a; grant FCP “Kolmogorov” 14.587.21.0036, grant RNF No 15-17-1009, and grant RFBR No 17-55-16008. Stable water isotope data are available in the Natural Environment Research Council (NERC) Environmental Information Data Centre (EIDC) data repository (title: “Stable water isotopes in Western Siberian inland waters”, permanent identifier: https://doi.org/10.5285/ca17e364-638d-4949-befb-b18b3770aec6). We would like to acknowledge the Arctic-GRO and IAEA for their publicly available databases providing supporting data for our analyses. Stream flow data at Nikolskoe was provided by Sergey Vorobiev. Liliya Kovaleva is acknowledged for the artwork in Figure 9. We would like to thank the two anonymous reviewers and the handling editors for their constructive comments that improved the manuscript.Peer reviewedPublisher PD

Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis

AbstractMany targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell. To generate single chain Fv (scFv) antibodies capable of triggering receptor-mediated endocytosis, we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell. Using this methodology, we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library. For this work, an immunotherapeutic was generated from one of these scFv (F5), which bound to ErbB2 (HER2/neu). The F5 scFv was reengineered with a C-terminal cysteine, expressed at high levels in Escherichia coli, and coupled to sterically stabilized liposomes. F5 anti-ErbB2 immunoliposomes were immunoreactive as determined by surface plasmon resonance (SPR) and were avidly internalized by ErbB2-expressing tumor cell lines in proportion to the levels of ErbB2 expression. F5-scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin. This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles

High riverine CO2 emissions at the permafrost boundary of Western Siberia

Acknowledgements: The study was part of the JPI Climate initiative, financially supported by VR (the Swedish Research Council) grant no. 325-2014-6898 to J.K. Additional funding from RNF (RSCF) grant no. 18-17-00237, RFBR grant no. 17-55-16008 and RF Federal Target Program RFMEFI58717X0036 ‘Kolmogorov’ to O.S.P. and S.N.K. as well as NERC grant no. NE/M019896/1 to C.S. is acknowledged. The authors thank A. Sorochinskiy and A. Lim for assistance in the field, as well as M. Myrstener, M. Klaus and S. Monteux for advice on data analysis. L. Kovaleva is acknowledged for artwork.Peer reviewedPostprin

Study of the process e+e−→ppˉe^+e^-\to p\bar{p} in the c.m. energy range from threshold to 2 GeV with the CMD-3 detector

Using a data sample of 6.8 pb$^{-1}$ collected with the CMD-3 detector at the VEPP-2000 $e^+e^-$ collider we select about 2700 events of the $e^+e^- \to p\bar{p}$ process and measure its cross section at 12 energy ponts with about 6\% systematic uncertainty. From the angular distribution of produced nucleons we obtain the ratio $|G_{E}/G_{M}| = 1.49 \pm 0.23 \pm 0.30$

Liposome-based drug delivery in breast cancer treatment

Drug delivery systems can in principle provide enhanced efficacy and/or reduced toxicity for anticancer agents. Long circulating macromolecular carriers such as liposomes can exploit the 'enhanced permeability and retention' effect for preferential extravasation from tumor vessels. Liposomal anthracyclines have achieved highly efficient drug encapsulation, resulting in significant anticancer activity with reduced cardiotoxicity, and include versions with greatly prolonged circulation such as liposomal daunorubicin and pegylated liposomal doxorubicin. Pegylated liposomal doxorubucin has shown substantial efficacy in breast cancer treatment both as monotherapy and in combination with other chemotherapeutics. Additional liposome constructs are being developed for the delivery of other drugs. The next generation of delivery systems will include true molecular targeting; immunoliposomes and other ligand-directed constructs represent an integration of biological components capable of tumor recognition with delivery technologies

Tumour-targeted nanomedicines: principles and practice

Drug targeting systems are nanometre-sized carrier materials designed for improving the biodistribution of systemically applied (chemo)therapeutics. Various different tumour-targeted nanomedicines have been evaluated over the years, and clear evidence is currently available for substantial improvement of the therapeutic index of anticancer agents. Here, we briefly summarise the most important targeting systems and strategies, and discuss recent advances and future directions in the development of tumour-targeted nanomedicines