Stephen J. Kennel

Targetability of novel immunoliposomes modified with amphipathic poly(ethylene glycol) s conjugated at their distal terminals to monoclonal antibodies

Distearoyl-N-(3-carboxypropionoyl poly(ethylene glycol) succinyl)phosphatidylethanolamine (DSPE-PEG-COOH) was newly synthesized and used to prepare novel immunoliposomes carrying monoclonal antibodies at the distal ends of the PEG chains (Type C). Liposomes were prepared from egg phosphatidylcholine (ePC) and cholesterol (CH) (2;1, m/m) containing 6 mol% of DSPE-PEG-COOH, and a monoclonal IgG antibody, 34A, which is highly specific to pulmonary endothelial cells, was conjugated to the carboxyl groups of DSPE-PEG-COOH to give various amounts of antibody molecules per liposome. Other immunoliposomes with PEG coating (Type B) or without PEG coating (an earlier type of immunoliposome, Type A) were prepared for comparison. The average molecular weight of PEG in Type B or C immunoliposomes was 2000. Type B and Type C liposomes without antibodies showed prolonged circulation time and reduced reticulo-endothelial system (RES) uptake owing to the presence of PEG. These three different types of 34A-immunoliposomes with 30-35 antibody molecules per vesicle were injected into mice to test the immunotargetability to the lung. The efficiency of lung binding of 34A-Type B was one-half of that of 34A-Type A, though a large amount of 34A-Type B remained in the blood circulation for a long time, suggesting that the steric hindrance of PEG chains reduced not only the immunospecific antibody-antigen binding, but also the RES uptake. The degree of lung binding of 34A-Type C was about 1.3-fold higher than that of 34A-Type A, indicating that recognition by the antibodies attached to the PEG terminal was not sterically hindered and that the free PEG (i.e., that not carrying antibody) was effective in increasing the blood concentration of immunoliposomes by enabling them to evade RES uptake. The latter phenomenon was confirmed by using nonspecific antibody-Type C immunoliposomes (14-Type C), which showed a high blood level for a long time. Our approach provides a simple means of conjugating antibodies directly to the distal end of PEG which is already bound to the liposome membrane, and should contribute to the development of superior targetable drug delivery vehicles for use in diagnostics and therapy.

Cationic liposomes enhance targeted delivery and expression of exogenous DNA mediated by N-terminal modified poly(l-lysine)-antibody conjugate in mouse lung endothelial cells

A new and improved system for targeted gene delivery and expression is described. Transfection efficiency of N-terminal modified poly(L-lysine) (NPLL) conjugated with anti-thrombomodulin antibody 34A can be improved by adding to the system a lipophilic component, cationic liposomes. DNA, antibody conjugate and cationic liposomes form a ternary electrostatic complex which preserves the ability to bind specifically to the target cells. At the same time the addition of liposomes enhance the specific transfection efficiency of antibody-polylysine/DNA binary complex by 10 to 20-fold in mouse lung endothelial cells in culture.

Glucose biosensing using an enzyme-coated microcantilever

A microcantilever-based biosensor is described. The enzyme glucose oxidase was immobilized on a micromachined silicon cantilever containing a gold coating, such as those used for atomic force microscopy. Specific, quantifiable deflection of the derivatized cantilevers was observed in the presence of the appropriate analyte. An analysis of the reaction energetics and the expected thermal response of the cantilever indicates that cantilever deflection is not simply a result of reaction-generated heat. This deflection appears to result from surface induced stresses. The combination of a highly specific enzyme and the microcantilever platform provides a unique approach for quantifying enzyme substrates without the complication of sample labeling.

Characterization of antibody covalently coupled to liposomes

Abstract We have explored the covalent coupling of fatty acids to immunoglobulin G(IgG). N -hydroxysuccinimide ester of palmitic acid (NHSP) was used to couple palmitic acid to either a mouse monoclonal antibody to the major histocompatibility antigen, H-2 k , or goat antibody to the major glycoprotein of the Molony Leukemia Virus, gp-70. The reaction was characterized in terms of the time course, input ratio of NHSP to IgG, stoichiometry of the coupling, distribution of palmitic acid in the IgG subunits, and the antigen binding capacity of the coupled antibody. Incorporation of the fatty acid modified IgG into liposomal membranes using a detergent-dialysis method was studied as a function of extent of fatty acid coupling. Finally, the binding of IgG-coated liposomes with cells expressing proper antigens was characterized. The major conclusions were: (1) the optimal molar ratio of NHSP to IgG in the reaction was between 10 and 20, which yields about 4–5 palmitoyl chains per IgG molecule; (2) at this level of coupling, the antigen binding capacity of the IgG antibody decreased about 3–4-fold; (3) incorporation of the coupled antibody into unilamellar liposomes (about 1000 A diameter) can be achieved with a deoxycholate-dialysis method with an optimal lipid-to-protein ratio of 10:1 (w/w); (4) there were about 48 IgG molecules incorporated per liposome under these conditions; (5) the apparent dissociation constant of the liposome-bound antibody under the optimal condition was about 6–7-fold higher than that of the native antibody; (6) binding of antibody to the target cells was accompanied by binding of liposomal lipids; both bindings could be blocked by pretreatment of cell with unmodified antibody.

High resolution computed tomography and MRI for monitoring lung tumor growth in mice undergoing radioimmunotherapy: Correlation with histology

A model lung tumor system has been developed in mice for the evaluation of vascular targeted radioimmunotherapy. In this model, EMT-6 mammary carcinoma tumors growing in the lung are treated with 213Bi, an alpha particle emitter, which is targeted to lung blood vessels using a monoclonal antibody. Smaller tumors (< 100 microm in diameter) are cured, but larger tumors undergo a period of regression and then regrow and ultimately prove lethal. The goal of this work was to determine if external imaging with MRI or CT could be used routinely to monitor the growth/ regression of lung tumors in live mice. To attempt to evaluate individual tumors in vivo, animals were initially imaged with magnetic resonance imaging (MRI). High resolution MRI images could be obtained only after sacrifice when lungs were not moving. In contrast, high resolution computed tomography (CT) produced evaluable images from anesthetized animals. Serial CT images (up to 5/animal) were collected over a 17 day period of tumor growth and treatment. When tumored animals became moribund, animals were sacrificed and lungs were inflated with fixative, embedded in paraffin, and then sectioned serially to compare the detection of tumors by high resolution CT with detection by histology. CT proved most useful in detecting lung tumors located in the hilar area and least useful in detecting serosal surface and anterior lobe tumor foci. Overall, CT images of live animals revealed tumors in approximately 2/3 of cases detected in histologic serial sections when relatively few tumors were present per lung. Detection of lesions and their resolution post therapy were complicated due to residual hemorrhagic, regressing tumor nodules and the development of lung edema both of which appeared as high density areas in the CT scans. We conclude that the microCT method used could identify some lung tumors as small as 100 microm in diameter; however, no concrete evaluation of therapy induced regression of the tumors could be made with CT analyses alone.

Highly efficient immunoliposomes prepared with a method which is compatible with various lipid compositions

Monoclonal antibody was conjugated to N-glutaryl-phosphatidylethanolamine in the presence of octylglucoside by using N-hydroxysulfosuccinimide as a carboxyl-activation reagent. The conjugated antibody was then incorporated into liposomes by a simple dialysis method. The method is mild and is compatible with various lipid compositions of the liposomes. We have prepared immunoliposomes containing a lung endothelium-specific monoclonal antibody and showed excellent target binding (approximately 75% injected dose) of the immunoliposomes in mouse. Immunoliposomes can be prepared to contain other acidic lipids such as phosphatidylserine and various amounts of cholesterol. The presence of 20% or more cholesterol in liposomes resulted in high level of target binding. We have used in these experiments a new radioactive lipid-phase marker, 111In-DTPA-SA, which was very stable in vivo. The halflife of clearance in mouse exceeded 3 weeks.

Vascular targeted radioimmunotherapy with 213Bi-An α-particle emitter

Abstract To destroy both tumor blood vessels and adjacent tumor cells, an α-particle emitter, 213 Bi, has been targeted with a monoclonal antibody (MAb) to vessels that feed lung tumors in mice. Animals, bearing approximately 100 EMT-6 carcinomas each of 50–400 cells in size in the lung, that were treated with 120 μCi of 213 Bi-MAb 201B were all cured of their disease. Animals treated when tumors were larger (10 3 –10 4 cells) had extended life spans, but a small number of residual tumors eventually killed the animals. Significant extension of life span was also induced with another tumor model—rat tracheal carcinoma growing in the lungs of SCID mice that were then treated with 136 μCi 213 Bi-MAb 201B. These studies indicate that attack of both blood vessels and tumor cells simultaneously is an effective mode of cancer treatment.

Integrin Regulation by Vascular Endothelial Growth Factor in Human Brain Microvascular Endothelial Cells ROLE OF α6β1 INTEGRIN IN ANGIOGENESIS

The precise role of vascular endothelial growth factor (VEGF) in regulating integrins in brain microvascular endothelial cells is unknown. Here, we analyzed VEGF effects on integrin expression and activation in human brain microvascular endothelial cells (HBMECs). Using human cDNA arrays and ribonuclease (RNase) protection assays, we observed that VEGF up-regulated the mRNA expression of α6 integrin in HBMECs. VEGF significantly increased α6β1 integrin expression, but not α6β4 integrin expression in these cells. Specific down-regulation of α6 integrin expression by small interfering RNA (siRNA) oligonucleotides inhibited both the capillary morphogenesis of HBMECs and their adhesion and migration. Additionally, VEGF treatment resulted in activation of α6β1 integrins in HBMECs. Functional blocking of α6 integrin with its specific antibody inhibited the VEGF-induced adhesion and migration as well as in vivo angiogenesis, and markedly suppressed tumor angiogenesis and breast carcinoma growth in vivo. Thus, VEGF can modulate angiogenesis via increased expression and activation of α6β1 integrins, which may promote VEGF-driven tumor angiogenesis in vivo.

Vascular Immunotargeting of Glucose Oxidase to the Endothelial Antigens Induces Distinct Forms of Oxidant Acute Lung Injury: Targeting to Thrombomodulin, But Not to PECAM-1, Causes Pulmonary Thrombosis and Neutrophil Transmigration

Oxidative endothelial stress, leukocyte transmigration, and pulmonary thrombosis are important pathological factors in acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Vascular immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) to the pulmonary endothelium causes an acute oxidative lung injury in mice.(1) In the present study we compared the pulmonary thrombosis and leukocyte transmigration caused by GOX targeting to the endothelial antigens platelet-endothelial cell adhesion molecule (PECAM) and thrombomodulin (TM). Both anti-PECAM and anti-TM delivered similar amounts of (125)I-GOX to the lungs and caused a dose-dependent, tissue-selective lung injury manifested within 2 to 4 hours by high lethality, vascular congestion, polymorphonuclear neutrophil (PMN) sequestration in the pulmonary vasculature, severe pulmonary edema, and tissue oxidation, yet at an equal dose, anti-TM/GOX inflicted more severe lung injury than anti-PECAM/GOX. Moreover, anti-TM/GOX-induced injury was accompanied by PMN transmigration in the alveolar space, whereas anti-PECAM/GOX-induced injury was accompanied by PMN degranulation within vascular lumen without PMN transmigration, likely because of PECAM blockage. Anti-TM/GOX caused markedly more severe pulmonary thrombosis than anti-PECAM/GOX, likely because of TM inhibition. These results indicate that blocking of specific endothelial antigens by GOX immunotargeting modulates important pathological features of the lung injury initiated by local generation of H(2)O(2) and that this approach provides specific and robust models of diverse variants of human ALI/ARDS in mice. In particular, anti-TM/GOX causes lung injury combining oxidative, prothrombotic, and inflammatory components characteristic of the complex pathological picture seen in human ALI/ARDS.

LaPO4 nanoparticles doped with actinium-225 that partially sequester daughter radionuclides.

Nanoscale materials have been envisioned as carriers for various therapeutic drugs, including radioisotopes. Inorganic nanoparticles (NPs) are particularly appealing vehicles for targeted radiotherapy because they can package several radioactive atoms into a single carrier and can potentially retain daughter radioisotopes produced by in vivo generators such as actinium-225 ((225)Ac, t(1/2) = 10 d). Decay of this radioisotope to stable bismuth-209 proceeds through a chain of short-lived daughters accompanied by the emission of four α-particles that release >27 MeV of energy. The challenge in realizing the enhanced cytotoxic potential of in vivo generators lies in retaining the daughter nuclei at the therapy site. When (225)Ac is attached to targeting agents via standard chelate conjugation methods, all of the daughter radionuclides are released after the initial α-decay occurs. In this work, (225)Ac was incorporated into lanthanum phosphate NPs to determine whether the radioisotope and its daughters would be retained within the dense mineral lattice. Further, the (225)Ac-doped NPs were conjugated to the monoclonal antibody mAb 201B, which targets mouse lung endothelium through the vasculature, to ascertain the targeting efficacy and in vivo retention of radioisotopes. Standard biodistribution techniques and microSPECT/CT imaging of (225)Ac as well as the daughter radioisotopes showed that the NPs accumulated rapidly in mouse lung after intravenous injection. By showing that excess, competing, uncoupled antibodies or NPs coupled to control mAbs are deposited primarily in the liver and spleen, specific targeting of NP-mAb 201B conjugates was demonstrated. Biodistribution analysis showed that ∼30% of the total injected dose of La((225)Ac)PO(4) NPs accumulated in mouse lungs 1 h postinjection, yielding a value of % ID/g >200. Furthermore, after 24 h, 80% of the (213)Bi daughter produced from (225)Ac decay was retained within the target organ and (213)Bi retention increased to ∼87% at 120 h. In vitro analyses, conducted over a 1 month interval, demonstrated that ∼50% of the daughters were retained within the La((225)Ac)PO(4) NPs at any point over that time frame. Although most of the γ-rays from radionuclides in the (225)Ac decay chain are too energetic to be captured efficiently by SPECT detectors, appropriate energy windows were found that provided dramatic microSPECT images of the NP distribution in vivo. We conclude that La((225)Ac)PO(4)-mAb 201B conjugates can be targeted efficiently to mouse lung while partially retaining daughter products and that targeting can be monitored by biodistribution techniques and microSPECT imaging.