Antibody-coated Liposomes Research Articles

Endothelial capture using antibodies and nanoparticles in human tissues: Antigen identification and liver segment imaging

Background: The unique phenomenon of endothelial antibody capture (endocapt) leads to site-specific accumulation of antibodies on the endothelium after its locoregional injection. The potential of this phenomenon has already been demonstrated in animal models. In the present study, the translational potential of several human endothelium-specific antibodies for their use in the endocapt-based approach was analysed. Methods: The binding of different endothelium-surface specific monoclonal antibody clones was analysed in human tissue and in endothelial cells using image-based immunofluorescence and the determination of half-maximal effective concentration (EC50). The potential of endocapt-based locoregional application of antibodies or antibody-coated liposomes was analysed ex vivo using isolated mouse liver perfusion and in vivo using superselective injection in tumour models. Results: Eight out of ten antibody clones were assigned to the group of “fast binding antibodies”. Different antibody clones showed various binding kinetics to the same endothelial marker whereas the binding kinetics of single antibody clones was independent from the tissue type. Anti-CD49e, anti-CD31, anti-CD34 and anti-CD102 antibodies showed the lowest EC50 of antibody binding concentration and constant results in EC50 determination of antibody binding to cells and human tissue. Experimental studies using anti-mouse CD49e antibody and coated immunoliposomes confirmed their effective capture by endothelial cells in vitro and in vivo by which fluorescent liver segment labelling was achieved. Conclusions: Our findings identify the high potential of several human antibody clones, especially anti-CD49e, -CD31, -CD34 and -CD102, for endocapt technology. We also propose important translational implications of these antibodies for image-guided liver surgery and for use of nanoparticles/immunoliposomes. Toxicological studies are indispensable for further translational development of new antibodies for endocapt. Statement of SignificanceThe phenomenon of endothelial antibody capture (endocapt) leads to site-specific accumulation of antibodies on the endothelium after its locoregional injection. This phenomenon broadly prevents systemic circulation of the antibody or antibody-drug conjugates. In the present study, our findings identify several human antibody clones promising for endothelial capture technology.This study provided the experimental demonstration of liver segment labelling ex vivo using isolated mouse liver perfusion and in vivo using superselective injection in tumor models. In addition, this study proposed the important translational implications of selected antibodies for image-guided liver surgery and for use of nanoparticles/immunoliposomes.

Inhibition of HIV Virus by Neutralizing Vhh Attached to Dual Functional Liposomes Encapsulating Dapivirine.

Although highly active antiretroviral therapy (HAART) has greatly improved the life expectancy of HIV/AIDS patients, the treatment is not curative. It is a global challenge which fosters an urgent need to develop an effective drug or neutralizing antibody delivery approach for the prevention and treatment of this disease. Due to the low density of envelope spikes with restricted mobility present on the surface of HIV virus, which limit the antibody potency and allow virus mutation and escape from the immune system, it is important for a neutralizing antibody to form bivalent or multivalent bonds with the virus. Liposome constructs could fulfil this need due to the flexible mobility of the membrane with its attached antibodies and the capacity for drug encapsulation. In this study, we evaluated the neutralization activity of a range of liposome formulations in different sizes coated with anti-gp120 llama antibody fragments (Vhhs) conjugated via either non-covalent metal chelation or a covalent linkage. The non-covalent construct demonstrated identical binding affinity to HIV-1 envelope glycoprotein gp120 and neutralizing ability for HIV virus as free Vhh. Although covalently linked Vhh showed significant binding affinity to gp120, it unexpectedly had a lower neutralization potency. This may be due to the comparability in size of the viral and liposome particles restricting the number which can be bound to the liposome surface so involving only a fraction of the antibodies, whereas non-covalently attached antibodies dissociate from the surface after acting with gp120 and free the remainder to bind further viruses. Covalently conjugated Vhh might also trigger the cellular uptake of a liposome-virion complex. To explore the possible ability of the antibody-coated liposomes to have a further function, we encapsulated the hydrophobic antiviral drug dapivirine into both of the non-covalently and covalently conjugated liposome formulations, both of which revealed high efficacy in reducing viral replication in vitro. Thus, dual function liposomes may lead to a novel strategy for the prophylaxis of HIV/AIDS by combining the neutralizing activity of Vhh with antiviral effects of high drug concentrations.

Endothelial targeting of liposomes encapsulating SOD/catalase mimetic EUK-134 alleviates acute pulmonary inflammation

Production of excessive levels of reactive oxygen species (ROS) in the vascular endothelium is a common pathogenic pathway in many dangerous conditions, including acute lung injury, ischemia-reperfusion, and inflammation. Ineffective delivery of antioxidants to the endothelium limits their utility for management of these conditions. In this study, we devised a novel translational antioxidant intervention targeted to the vascular endothelium using PEG-liposomes loaded with EUK-134 (EUK), a potent superoxide dismutase/catalase mimetic. EUK loaded into antibody-coated liposomes (size 197.8±4.5nm diameter, PDI 0.179±0.066) exerted partial activity in the intact carrier, while full activity was recovered upon liposome disruption. For targeting we used antibodies (Abs) to platelet-endothelial cell adhesion molecule (PECAM-1). Both streptavidin–biotin and SATA/SMCC conjugation chemistries provided binding of 125–150 Ab molecules per liposome. Ab/EUK/liposomes, but not IgG/EUK/liposomes: i) bound to endothelial cells and inhibited cytokine-induced inflammatory activation in vitro; and, ii) accumulated in lungs after intravascular injection, providing >60% protection against pulmonary edema in endotoxin-challenged mice (vs <6% protection afforded by IgG/liposome/EUK counterpart). Since the design elements of this drug delivery system are already in clinical use (PEG-liposomes, antibodies, SATA/SMCC conjugation), it is an attractive candidate for translational interventions using antioxidant molecules such as EUK and other clinically acceptable drugs.

Comparison of the effects of antibody-coated liposomes, IVIG, and anti-RBC immunotherapy in a murine model of passive chronic immune thrombocytopenia

AbstractThe present work evaluated antibody-coated liposomes as a new treatment strategy for immune thrombocytopenic purpura (ITP) through the use of a mouse model of the disease. Effects of antimethotrexate antibody (AMI)–coated liposomes and intravenous immunoglobulin (IVIG)–coated liposomes (15, 30, 60 µmol lipid/kg) were compared with the effects of IVIG (0.4, 1, 2 g/kg) and anti–red blood cell (anti-RBC) monoclonal antibody immunotherapy (TER119, 5, 15, 25, and 50 µg/mouse) on MWReg30-induced thrombocytopenia. Each treatment was found to attenuate thrombocytopenia in a dose-dependent manner and, consistent with previous work, IVIG was found to increase antiplatelet antibody clearance in a dose-dependent manner. TER119 demonstrated greater effects on thrombocytopenia relative to other therapies (peak platelet counts: 224% ± 34% of initial platelet counts for 50 µg TER119/mouse versus 160% ± 34% for 2 g/kg IVIG, 88% ± 36% for 60 µmol lipid/kg AMI-coated liposomes, and 80% ± 25% for 60 µmol lipid/kg IVIG-coated liposomes). However, the effects of TER119 were associated with severe hemolysis, as TER119 decreased RBC counts by approximately 50%. The present work demonstrated that antibody-coated liposomes attenuated thrombocytopenia in this model at a much lower immunoglobulin dose than that required for IVIG effects and, in contrast with TER119, antibody-coated liposomes increased platelet counts without altering RBC counts.

Investigation of antibody-coated liposomes as a new treatment for immune thrombocytopenia

Immune thrombocytopenia (ITP) is an autoimmune disease that is mediated by anti-platelet antibodies. It is believed that anti-platelet antibody-opsonized platelets are eliminated through Fcγ receptor-mediated and complement-mediated phagocytosis by macrophages of the reticuloendothelial system (RES). Polyclonal pooled immunoglobulin with high titer for the D-antigen of erythrocytes (i.e., anti-D) has been successfully used to ameliorate ITP. Based on the pathogenesis of ITP and based on the successful application of anti-D for the treatment of ITP, we hypothesized that antibody-coated liposomes may be used to inhibit Fcγ receptor-mediated and complement-mediated phagocytosis, thereby increasing platelet counts in ITP. To test this hypothesis, we have developed a liposome preparation that is coated with a model monoclonal IgG1 antibody. Antibody-coated liposomes were found to inhibit complement deposition and macrophage phagocytosis in vitro. Furthermore, antibody-coated liposomes were also found to attenuate thrombocytopenia in a rat model of ITP, in a dose-dependent manner. The results suggest that antibody-coated liposomes may be used as ‘decoy particles’ to competitively inhibit the destruction of antibody-coated platelets; thus, antibody-coated liposomes may have value in the treatment of ITP.

Endocytosis does not necessarily augment the cytotoxicity of adriamycin encapsulated in immunoliposomes

We studied the relationship between endocytosis and cytotoxicity of adriamycin (ADM) encapsulated in antibody-coated liposomes (immunoliposomes, IL) which are called chemoimmunoliposomes (CIL), by using several human cancer cell lines. IL coated with a monoclonal antibody, HBJ127 (IgG), which recognizes human gp125 antigen, specifically bound to gp125-positive target cancer cell lines, KU-1, T24, MKN-7, SKBr-3 and LS174T. Flow cytometric analysis using IL encapsulating carboxyfluorescein (CF) revealed that efficiencies of endocytosis varied among different cancer cells. The rate of IL internalization was in the order KU-1> T24> MKN-7> SKBr-3> LS174 T . In 1 h incubation at 37°C, all the four cell lines other than LS174T internalized about 60% of IL which were bound on their cell surfaces. KU-1, T24 and MKN-7, but not SKBr-3, significantly processed IL in endosome or lysosome. On the contrary, 80% of CIL bound to LS174T remained on the cell surface even after 2 h incubation. Furthermore, we evaluated the cytotoxic activities of CIL against the same panels of cancer cells. CIL inhibited the growth of all cancer cells tested in antibody-dependent manner, but, contrary to our expectation, KU-1 and T24 cells, which showed significant endocytosis activity, required a 7–14-fold higher amount of ADM binding than LS174T cells with low endocytosis activity for 50% cell growth inhibition. The difference of sensitivity to free ADM was only within 2.3-fold among those cancer cells. These results showing that liposomal ADM endocytosed is less effective than that remaining on the cell surface suggest that endocytosis is not necessarily required for cytotoxicity of CIL.

Assay of cell surface-bound immunoliposomes using monoclonal antibody reactive with a cross-linking reagent.

A monoclonal antibody (mAb) reactive with a crosslinking reagent, N-(m-maleimidobenzoyl)dipalmitoylphosphatidylethanolamine (mMBPE), in liposomes was produced from a hybridoma clone established by a fusion between P3X63Ag8.653 mouse myeloma cells and spleen cells from a BALB/c mouse hyperimmunized with the antibody-coated liposomes containing mMBPE. Using this mAb (termed AL-6), the quantity of immunoliposomes bound on target tumor cells was assessed by flow cytofluorometry. The results obtained using fluorescein isothiocyanate-coupled AL-6 allowed the enumeration not only of the immunoliposomes bound on all tumor cells but also those on individual target tumor cells. The relevance of this assay method was confirmed by a comparison with another assay method of cell-bound liposomes using immunoliposomes containing carboxyfluorescein in the vesicles.

Antisense RNA to treat HIV infections

Although it may be possible to target externally synthesized antisense RNA molecules to the right cell by using antibody-coated liposomes [6], the inhibitory effect achieved in this experiment was only transient, probably because of the short half-life of these molecules. It would therefore seem better to produce the antisense RNA inside the virus-infected cell in order to achieve a constant inhibitory concentration. To achieve this, experiments in vitro use vectors, often of retroviral origin, to stably transduce and express the antisense sequence of interest

Antibody-coated liposomes as a particulate solid phase for immunoassays: Measurement of urinary ‘micro-albumin’

A novel use of liposomes as a solid phase material achieving separation in immunoassays is described. Antibody-coated liposomes were prepared and used as a particulate solid phase in a radioimmunoassay procedure for urinary albumin. The assay was compared to a liquid phase albumin radioimmunoassay. The potential benefits of liposomes over other particulate solid phases are discussed. The use of liposomes in this manner need not be restricted to radioimmunoassay but shoulb also be applicable to other immunoassays using alternative non-isotopic labels.

PH-sensitive immunoliposomes mediate target-cell-specific delivery and controlled expression of a foreign gene in mouse.

A plasmid containing the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under the control of a mammalian cAMP-regulated promoter was entrapped in H-2Kk antibody-coated liposomes composed of dioleoyl phosphatidylethanolamine, cholesterol, and oleic acid (pH-sensitive immunoliposomes). The entrapped or free DNA was injected intraperitoneally into immunodeficient (nude) BALB/c mice bearing ascites tumor generated by H-2Kk-positive RDM-4 lymphoma cells. About 20% of the injected immunoliposomes were taken up by the target RDM-4 cells. Uptake was much less when liposomes without antibody were used. The presence of the targeting antibody on liposomes also significantly decreased the nonspecific uptake of liposomes by the spleen. Significant CAT enzyme activity was detected in RDM-4 cells from mice treated with DNA entrapped in the pH-sensitive immunoliposomes. Furthermore, CAT expression in RDM-4 cells was under the control of cAMP, as only the cells from mice injected with 8-bromo-cAMP and 3-isobutyl-1-methylxanthine showed CAT activity. CAT activity in liver and spleen was much lower (by factors of 12 and 5, respectively) than in the RDM-4 cells, and the activities in these reticuloendothelial organs were not regulated by cAMP. CAT activity in RDM-4 cells from mice injected with DNA entrapped in pH-insensitive immunoliposomes (containing phosphatidylcholine in place of phosphatidylethanolamine) was approximately one-fourth that in RDM-4 cells from mice injected with pH-sensitive immunoliposomes, indicating the superior delivery efficiency of the pH-sensitive liposomes. These results are discussed in terms of the DNA-carrier potential of immunoliposomes in therapy of cancer and genetic diseases.

Target-sensitive immunoliposomes: preparation and characterization.

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody-coated liposomes: The role of non-specific antibody adsorption

Incubation of small unilamellar liposomes composed ofequimolar phospholipid and cholesterol with mouse IgG or mouse monoclonal IgG 1 for up to 24 h resulted in considerable (34-89%) adsorption of the protein onto the liposomal surface. Immunoglobulin remained adsorbed after exposure to mouse plasma and, in the case of monoclonal (anti-HBSAg) IgG 1, was able to mediate association of the liposomes with the antigen. Extensive immunoglobulin adsorption suggests caution in interpreting co valent linkage values obtained upon prolonged incubation of chemically modified antibodies with liposomes. It may also be a preferred alternative in preparring targeted liposomes for intravenous use when chemically modified antibodies promote premature clearance of the antibody-liposome complex from the circulation.

Lymph node localization of non-specific antibody-coated liposomes

Subcutaneously injected small unilamellar liposomes are drained into the lymphatics and localized in the regional lymph nodes, and thus they can be used for the detection of metastatic spread in breast cancer patients and for delivery of drugs to diseased lymph nodes (1–8). An aqueous phase marker, [ 125I]-polyvinylpyrrolidone, and a lipid phase marker, [ 3H]-cholesterol, were used to study the lymph node localization of IgG-coated liposomes injected subcutaneously into mouse and rat footpads. The results show that human immunoglobulin G (IgG) coated liposomes are rapidly removed from the site of injection and are localized in the regional lymph nodes to a greater extent than control liposomes (i.e. liposomes without IgG). Free IgG was found to inhibit the uptake of IgG-coated liposomes by the lymph nodes. The localization of IgG-coated liposomes in the regional lymph nodes is influenced by charge of the liposomes. The results presented here suggest that antibody-coated liposomes may provide a more efficient way of delivering therapeutic agents to the lymph nodes in the treatment of diseases such as breast cancer with lymph node involvement. Similarly, monoclonal antibody-coated liposomes containing lymphoscintigraphic material may improve the detection of lymph node metastases.

Interactions of immunoliposomes with target cells.

We have covalently attached a monoclonal antibody (11-4.1) against the murine major histocompatibility antigen, H-2Kk, on the surface of liposomes. The interaction of these antibody-coated liposomes (immunoliposomes) with target cells, RDM-4 lymphoma (H-2Kk), was investigated. About 90% of the immunoliposomes taken up by target cells at 4 degrees C could be removed by a mild protease treatment of the cells, whereas only 30% of the uptake at 37 degrees C was labile to the same treatment. Furthermore, the uptake of immunoliposomes at 37 degrees C was inhibitable by cytochalasin B or by a combination of 2-deoxyglucose and NaN3. These results suggest that immunoliposome binding to the target cell surface is the primary uptake event at 4 degrees C and that the surface-bound liposomes are rapidly internalized by the cells at 37 degrees C, probably via an endocytic pathway. Studies with fluorescence microscopy of target cells treated with immunoliposomes containing carboxyfluorescein also supported this conclusion. If endocytosis is the mechanism by which immunoliposomes gain entry into target cells, the efficacy of a cytotoxic drug encapsulated would depend on the resistance of the drug to lysosomal inactivation and its ability to escape from the lysosomal system. Consistent with this notion, we observed that methotrexate encapsulated in liposomes bearing 11-4.1 antibody specifically inhibited deoxy[6-3H]uridine incorporation into DNA in target RDM-4 cells but not in P3-X63-Ag8 myeloma cells (H-2Kd) at the same doses. The observed cytotoxic effect of encapsulated methotrexate could be reversed by the treatment of cells with a lysosomotropic amine, chloroquine, which has been shown to increase the intralysosomal pH of mammalian cells. On the other hand, cytosine-beta-D-arabinofuranoside encapsulated in immunoliposomes showed no target-specific killing, probably because the drug is readily inactivated in the lysosomal system. These results are discussed in terms of the drug carrier potential of immunoliposomes.

Targeting of monoclonal antibody-coated liposomes to sheep red blood cells

A monoclonal mouse anti-sheep red blood cell specific antibody IgG 2b was esterified with palmitic acid which served as a hydrophobic anchor for successfull incorporation into the liposomal membrane. The formation of coated liposomes by dialyzing the mixed antibody/lipid/detergent micelles against phosphate buffer was simplified b by using the same detergent as for the antibody derivatization. No purification step of any intermediate product was necessary. Targeting of the resulting vesicles to sheep red blood cells occured with high efficiency compared with control liposomes. The uptake was fast and specific as demonstrated with sheep and horse red blood cells by the use of radioactively labelled liposomes and by scanning electron microscopy.

Damage of liposomes in antibody-dependent cell-mediated cytotoxicity.

Lymphoid cells bearing Fc receptors are able to lyse antibody-coated animal target cells in an antibody-dependent cell-mediated cytotoxicity reaction. The results presented here show that liposomes consisting of sphingomyelin, cholesterol, and cardiolipid and coated by cardiolipidic antibodies could be destroyed by spleen or thymus cells. No alteration of liposomes was observed when normal rabbit serum was used or when the effector cell population was depleted of cells bearing Fc receptors. The lysis of antibody-coated liposomes by effector cells could be carried out in two steps. In the first step, the fixation of antibodies on the cardiolipidic antigens could lead to a reorganization of the liposomal membrane. In the second step, the effector Fc-receptor-bearing cells might amplify this alteration of the liposomes.