Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activation in vivo and inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1beta (IL-1beta). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1beta-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2-4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approach in vivo may lead to novel therapeutic strategies in the treatment of cardiovascular disease.

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Recombinant full-length CD4 expressed in Spodopterafrugiperda 9 cells with the baculovirus system was electroinserted in erythrocyte (RBC) membranes. Of the inserted CD4, 70% was "correctly" oriented as shown by fluorescence quenching experiments with fluorescein-labeled CD4. The inserted CD4 displayed the same epitopes as the naturally occurring CD4 in human T4 cells. Double-labeling experiments (12'I-CD4 and 51Cr-RBC) showed that the half-life of CD4 electroinserted in RBC membrane in rabbits was approximately 7 days. Using the fluorescence dequenching technique with octadecylrhodamine B-labeled human immunodeficiency virus (HIV)-1, we showed fusion of the HIV envelope with the plasma membrane of RBC-CD4, whereas no such fusion could be detected with RBC. The dequenching efficiency of RBC-CD4 is the same as that of CEM cells. Exposure to anti-CD4 monoclonal antibody OKT4A, which binds to the CD4 region that attaches to envelope glycoprotein gp120, caused a significant decrease in the dequenching of fluorescence. In vitro infectivity studies showed that preincubation of HIV-1 with RBC-CD4 reduced by 80-90% the appearance ofHIV antigens in target cells, the amount of viral reverse transcriptase, and the amount of p24 core antigen produced by the target cels. RBC-CD4, but not RBCs, aggregated with chronically HIV-i-infected T cells and caused formation of giant cells. These data show that the RBC-CD4 reagent is relatively long lived in circulation and efficient in attaching to HIV-1 and HIV-infected cells, and thus it may have value as a therapeutic agent against AIDS.

CD4 is an integral membrane glycoprotein that acts as the cellular receptor for human immunodeficiency virus (HIV). A cDNA encoding full-length CD4 was inserted into the genome ofAutographa californica nuclear polyhedrosis virus under transcriptional regulation of the viral polyhedrin gene promoter. The recombinant virus was used to infect insect cells, which resulted in the abundant expression of CD4 as evaluated by flow cytometry and immunoblot analysis. Recombinant CD4 expressed on the surface of infected insect cells was immunologically indistinguishable from human CD4 when using 11 different anti-CD4 monoclonal antibodies. The extraction of infected cells by phase-transition separation with Triton X-114 followed by immunoaffinity chromatography yielded a single protein detected by NaDodSO4/PAGE using silver staining. N-terminal sequence analysis of the purified recombinant protein showed that CD4 produced in Sf9 cells is efficiently cleaved from the precursor protein. Immunoblot analysis under nondenaturing conditions showed that the purified protein reacted with the anti-CD4 monoclonal antibody Leu-3a. The potential use of the recombinant membraneassociated CD4 in anti-HIV therapy is discussed.

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