Lymphoid tissue targeting of anti-HIV drugs using liposomes

Abstract

Considering that HIV-1 accumulates and replicates actively within lymphoid tissues, any strategy that will decrease viral stores in these tissues might be beneficial to the infected host. Follicular dendritic cells (FDC), B lymphocytes, antigen-presenting cells like macrophages, and activated CD4(+) T cells are abundant in lymphoid tissues, and all express substantial levels of the HLA-DR determinant of the major histocompatibility complex class II (MHC-II). Monocyte-derived macrophages, which are also CD4(+) and express HLA-DR, are considered to be the most frequent hosts of HIV-1 in tissues of infected individuals. This chapter describes a method for the generation of sterically stabilized immunoliposomes grafted with anti-HLA-DR antibodies that allows efficient delivery of drugs to lymphoid tissues. The method first involves the production of murine HLA-DR (clone Y-17, IgG(2b)) and human HLA-DR (clone 2.06, IgG(1)) antibodies from hybridomas in mice and their purification from ascites fluids. This step is followed by the production of Fab' fragments of antibodies 2.06 and Y-17 that are grafted at the surface of sterically stabilized immunoliposomes instead of the complete IgG to reduce their immunogenicity. The preparation of sterically stabilized liposomes, the composition of which allows an efficient entrapment and retention of several drugs, by the method of thin lipid film hydratation followed by extrusion through polycarbonate membranes is then described. This step is followed by the removal of unencapsulated drug, when present, by low-speed centrifugation of the liposomal preparation through a Sephadex G-50 column. These liposomes contain a fixed amount of poly(ethylene glycol) chain terminated by a maleimide reactive group for the coupling of Fab' fragments. The procedure for the coupling of Fab' fragments at the surface of sterically stabilized liposomes and the removal of uncoupled fragments of antibodies is described. In vitro binding studies of sterically stabilized immunoliposomes to cell lines expressing different surface levels of the mouse or human HLA-DR determinant of MHC-II demonstrate that these liposomes are very specific. When compared with conventional liposomes, the subcutaneous administration in the upper back, below the neck, of mice of anti-HLA-DR immunoliposomes resulted in a 2.9 and 1.6 times greater accumulation in the cervical and brachial lymph nodes, respectively. The use of sterically stabilized immunoliposomes increases 2 to 4.6 times the concentration of liposomes in all tissues, with a peak accumulation at 240 h in brachial, inguinal, and popliteal lymph nodes and at 360 h or greater in cervical lymph nodes. A single bolus injection of indinavir given subcutaneously to mice results in no significant drug levels in lymphoid organs. Most of the injected drug accumulates in the liver and is totally cleared within 24 h postadministration. In contrast, sterically stabilized immunoliposomes are very efficient in delivering high concentrations of indinavir to lymphoid tissues for at least 15 days postinjection. The drug accumulation in all tissues leads to a 21- to 126-fold increased accumulation when compared with the free agent. Anti-HLA-DR immunoliposomes containing indinavir are as efficient as the free agent in inhibiting HIV-1 replication in PM1 cells that express high levels of cell surface HLA-DR. Sterically stabilized anti-HLA-DR immunoliposomes mostly accumulate in the cortex in which follicles (B cells and FDCs) are located, and in parafollicular areas in which T cells, interdigitating dendritic cells, and other accessory cells are abundant. The delivery of drugs in this area of the lymph nodes could represent a convenient strategy to inhibit more efficiently HIV-1 replication. Although the method described in this chapter is specific to the coupling of anti-HLA-DR antibodies, any antibody fragment or peptide specific for an antigen present in relatively large quantities at the surface of lymphoid cells, that is anchored to the surface of sterically stabilized liposomes with an appropriate coupling method, can be used to concentrate drugs within target tissues and improve the therapeutic effect of drugs.