We describe evidence for a novel mechanism of monoclonal antibody (MAb)-directed nanoparticle (immunoliposome) targeting to solid tumors in vivo. Long-circulating immunoliposomes targeted to HER2 (ErbB2, Neu) were prepared by the conjugation of anti-HER2 MAb fragments (Fab ¶ or single chain Fv) to liposome-grafted polyethylene glycol chains. MAb fragment conjugation did not affect the biodistribution or long-circulating properties of i.v.-administered liposomes. However, antibody-directed targeting also did not increase the tumor localization of immunoliposomes, as both targeted and nontargeted liposomes achieved similarly high levels (7-8% injected dose/g tumor tissue) of tumor tissue accumulation in HER2-overexpressing breast cancer xenografts (BT-474). Studies using colloidal gold-labeled liposomes showed the accumulation of anti-HER2 immunoliposomes within cancer cells, whereas matched nontargeted liposomes were located predominantly in extracellular stroma or within macrophages. A similar pattern of stromal accumulation without cancer cell internalization was observed for anti-HER2 immunoliposomes in non-HER2-overexpressing breast cancer xenografts (MCF-7). Flow cytometry of disaggregated tumors posttreatment with either liposomes or immunoliposomes showed up to 6-fold greater intracellular uptake in cancer cells due to targeting. Thus, in contrast to nontargeted liposomes, anti-HER2 immunoliposomes achieved intracellular drug delivery via MAb-mediated endocytosis, and this, rather than increased uptake in tumor tissue, was correlated with superior antitumor activity. Immunoliposomes capable of selective internalization in cancer cells in vivo may provide new opportunities for drug delivery.

Cyclosporin A (CsA) has proven effective as an inhibitor of a variety of T cell responses in vitro, including antigen-specific proliferation, macrophage-mediated antigen presentation, and antigen-and lectin-induced cytokine production, including IL-1, IL-2, IL-3, and IFN-y, but not IFN-a/# (1) . CsA appears to function in the prevention of graft rejection by affecting the early activation of immunocompetent cells, possibly by blocking antigen-specific receptor binding, class I and II expression, inhibition of phospholipase A2 activity, and/or inhibition of synthesis of cytokine mRNA (1-4). The selective suppressive activity of CsA for cell-mediated immunity prompted its use in human organ transplantation (5) .Transforming growth factors (TGFs) are a family of peptides that, under certain conditions, can induce normal cells to express a transformed phenotype (6). At least two types of TGFs have been described : TGF-a, which is homologous to epidermal growth factor (EGF), and TGF-f3. Mature TGF-a is a 25 kD homodimer held together by disulfide bonds that binds to a specific receptor(s) distinct from the TGF-a or EGF receptor (6) . TGF-0 inhibits IL-2-induced T cell proliferation, IL-2-R induction, IL-1-induced thymocyte proliferation, IFNa but not IL-2-induced NK activity, B cell proliferation to growth factors and IFN-y-induced class II antigen expression on Hs294T melanoma cells (7)(8)(9)(10)(11) .In this report, we describe the effects of CsA and TGF-(3 on the production of TNF-a (also referred to as cachectin), TNF-# (lymphotoxin), and IFN-y by human PBMC and murine peritoneal macrophages . Our results demonstrate that CsA and TGF-0 have suppressive effects on cytokine production that are dependent on their selective activity on specific cell types . Materials and MethodsCytokines. Recombinant human IFN-y (rHuIFN-,y) and murine IFN-,y (rMuIFN-y), cloned and expressed in Escherichia coli (12, 13), were purified to >99% purity~Rinder-knecht, E., unpublished results) .

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Epidermoid cervical carcinoma cells (CaSki line) have been established in continuous culture. When leukocytes from cervical cancer patients were incubated with CaSki culture fluid concentrates, inhibition of leukocyte migration was observed in more than 70 percent of the patients tested. By contrast, significantly less inhibition was observed with normal donor leukocytes or leukocytes from patients with other types of cancer. These results were consistent with the expression of tumor-associated antigen by CaSki cells. Analysis of the serum from the donor of the cell line at the time of tumor biopsy, and of CaSki culture fluids, demonstrated the presence of the beta subunit of human chorionic gonadotropin.

Many 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.