Systemic macrophage activation with liposome-entrapped immunomodulators for therapy of cancer metastasis

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Systemic macrophage activation with liposome-entrapped immunomodulators for therapy of cancer metastasis I.J. Fidler Department of Cell Biology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard (HMB 173), Houston, TX 77030 (USA)

Macrophages

and homeostasis

The primary functions of mononuclear phagocytes are the processes of tissue turnover such as tissue remodelling during embryogenesis, tissue destruction and repair subsequent to injury, and tissue renewal following the removal of damaged or senescent cells (Fidler and Schroit, 1988). Macrophages recognize, phagocytose, and dispose of aged cells, cellular debris and foreign invaders. Macrophages control the metabolism of lipids and iron and the response to injury and inflammation. Macrophages provide the first and second lines of defence against microbial infections and parasitic infestations and also regulate both the afferent and efferent arms of the immune system (Fidler and Schroit, 1988). Finally, the activation of cells of the macrophage-histiocyte series to become bactericidal, viricidal, fungicidal or tumoricidal also enhances host defence against infections and cancer (Fidler, 1985). Because macrophages are able to recognize tumour cells specifically in vivo (Fidler, 1985 ; Fidler and Schroit, 1988), they can be important to host surveillance against autochthonous, transformed neoplastic cells. Studies of skin carcinogenesis induced by ultraviolet radiation in a murine model support this hypothesis. When a low dose of carcinogen was used, treatment of mice with a macrophage stimulant prolonged the latent period to tumour development. Conversely, treatment of mice with a macrophage toxin shortened the period of latency (Norbury and Kripke, 1979). The study of transplantable tumour systems has likewise revealed that the impairment of macrophage function produced an increase in the incidence of spontaneous and experimental metastasis (Mantovani et al., 1980). There are also published reports regarding the efficacy of adoptive transfer of macrophages in inhibiting experimental metastasis (Fidler, 1974).

Activation

of macrophages to the tumoricidal

state

Continuous functions of macrophages, e.g. removal of aged red blood cells from the circulation, are constitutive. In contrast, a rarer function, such as tumour cell destruction, requires that macrophages receive activation signals. There are two major pathways to achieve macrophage activation in vivo. Macrophages can be activated subsequent to contact with microorganisms or their products, such as endotoxin or cell wall skeleton. Although such interactions are common, clinical attempts to activate macrophages systemically by administering microorganisms or their products has resulted in major side effects, such as allergic reactions and granuloma formation (Allison, 1979). Progress was delayed for this reason until the discovery of muramyl dipeptide (MDP), a component of the bacterial cell wall (Lederer, 1980) capable of activating the immune system (Fogler and Fidler, 1984). Both hydrophilic MDP as well as a lipophilic muramyl tripeptide phosphatidyl-ethanolamine (MTP-PE) have potent effects on many host defence cells, including macrophages (Fogler and Fidler, 1984). Although muramyl peptides influence macrophage function in vitro, comparable effects have not been observed in vivo because these peptides are rapidly cleared after parenteral administration. Even when given in high doses, they fail to induce significant macrophage-mediated antitumour activity (Fogler and Fidler, 1984). In vivo macrophage activation can also be produced by lymphokines termed macrophage-activating factors (MAF) such as gamma interferon (IFNy) (Saiki et al., 1985). Activated macrophages recognize and destroy neoplastic cells both in vitro and in vivo without injuring non-tumorigenic cells. The mechanism for this recognition is non-immunological and requires cellto-cell contact @Ebbs, 1974; Bucana et al., 1983).

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This process is independent of major histocompatibility antigens, tumour-specific antigens, cell cycle or transformation phenotypes (Fidler and Schroit, 1988). It is associated with expression of the phospholipid phosphatidylserine (PS) in the external leaflet of a tumour (but not normal) cell’s membrane (Utsugi et al., 1991). This may be one reason that tumour cell resistance to tumoricidal macrophagemediated lysis is rare (Fogler and Fidler, 1985). In fact, tumoricidal human blood monocytes can discriminate between tumorigenic and non-tumorigenic allogeneic target cells even under co-cultivation conditions (Fidler and Kleinerman, 1984). I base this conclusion on the following data. Highly purified preparations of peripheral blood monocytes isolated from normal donors were activated in vitro by incubation with various immunomodulators. The cytotoxic properties of these monocytes were assessed against different combinations of. tumorigenic and non-tumorigenic human target cell populations. In all combinations used, activated monocytes selectively bound to and lysed only neoplastic cells and left non-tumorigenic ceils unharmed (Fidler and Kleinerman, 1984). Since activated macrophages can destroy phenotypically diverse tumour cells, including cells resistant to killing by other host defence mechanisms and anticancer drugs, systemic activation of macrophages is an attractive strategy for destruction of metastatic cells.

Systemic activation of macrophages by llposomes containing immunomodulators Efforts to activate the tumoricidal properties of macrophages significantly in vivo by the administration of IFNy have not succeeded because lymphokines injected intravenously have an extremely short half-life (Poste et al., 1979). Even under ideal in vitro conditions, a minimum 8-hour interaction time between human monocytes and free lymphokines is required for successful activation (Kleinerman et al., 1983). Similarly, the short halflife of MDP in the blood (< 30 min) does not result in significant monocyte-macrophage activation (Fogler and Fidler, 1984). Advances in liposome technology have provided a means for systemic activation of macrophages. During the last several years, attention has focused on the use of synthetic phospholipid vesicles (liposomes) to deliver drugs to various organ sites in vivo (Daoud et al., 1989). Most attempts to target liposomes to extra circulatory compartments have not succeeded because these vesicles do not extravasate out of the circulation and are rapidly taken up by circulating and fixed phagocytic cells. We have taken advantage of this natural fate of liposomes to deliver biological agents to cells of the reticuloendothelial

system (RES), resulting in the activation of these phagocytic cells to the tumoricidal state (see review in Fidler, 1988). Once phagocytosed, biological compounds are released into the cytoplasm of the phagocyte. Such intracellular delivery avoids the problems of dilution, protein binding, and rapid clearance, and it therefore minimizes undesirable side effects. Preclinical

rodent studies

Several conditions must be satisfied for liposomes to deliver biologically active agents effectively to mononuclear phagocytes in situ. First, the liposomes must readily bind to and be phagocytosed by free and fixed phagocytes ; second, they must prevent degradation of the entrapped drug; third, they must retain the encapsulated agent for delivery to the intracellular compartment of RES cells; and fourth, they must localize to macrophages in organs where metastases occur. The distribution of intravenously administered liposomes is determined by their physical size, composition, charge and size of the inoculum (Poste et al., 1982). Although distribution patterns can be modified by manipulating these parameters, the majority of infused liposomes are removed from the circulation mainly by free and fixed cells of the RES. Certain classes of phosph6lipids are preferentially recognized by macrophages. In particular, the addition of negatively charged phospholipids, such as PS, to liposomes consisting primarily of phosphatidylcholine (PC) greatly increases macrophage binding and phagocytosis (Schroit and Fidler, 1982). Rodent and human macrophages can become tumoricidal following phagocytosis of multilamellar vesicles (MLV) consisting of PC and PS and containing lymphokines (MAF, IFNy), MDP, or lymphokines and/or synthetic immunomodulators (see review in Fidler, 1988). Because the lung is a major site of metastatic disease, we determined which liposomes localized best in the lung after intravenous injection. Large MLV (> 0.1 pm) arrested in the lungs more efficiently than small unilamellar liposomes of identical lipid composition. In addition, liposomes of the same structural class localized in the lungs more efficiently when they contained PS than when they contained only PC. The PC/PS liposomes delivered encapsulated compounds to blood monocytes, which then migrated out of the circulation to differentiate into lung macrophages (Poste et al., 1982; Schroit and Fidler, 1982). The intravenous injection of MDP or MTP-PE in MLV composed of PC/PS produced in situ activation of mouse lung macrophages that persisted for 3 to 4 days. This activation resulted from a direct effect of activator-containing liposomes on macro-

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phages ; it did not depend on an indirect action of the immunomodulator on T cells with a release of lymphokines that activate macrophages. This conclusion is based on data from studies in which lung macrophages from mice with deficient T-cell function were tumoricidal after systemic administration of liposome-encapsulated MTP-PE (but not after control liposomes alone) (Fidler, 1981).

Eradication

of mouse melanoma

metastases

Systemic macrophage activation with liposomes containing lymphokines, MDP, MTP-PE, rIFNy with MTP-PE, and MDP with MAF resulted in eradication of well-established lung and lymph node metastases produced by subcutaneous melanoma (see review in Fidler, 1988). For these studies, the major tumour model was the B 16-BL6 melanoma cell line, which is syngeneic to C,,BL/6 mice. This tumour metastasizes to lymph nodes and the lungs following implantation in the footpad in over 90 % of the mice. Mice were given an intrafootpad injection of melanoma cells, and 4 to 5 weeks later, when the tumours had reached 10 to 12 mm, the leg with tumour was amputated ad mid-femur to include the popliteal lymph node. Three days later, the mice received intravenous injections of MLV containing macrophage activators or saline. Mice were treated twice a week for 4 weeks. All mice treated with either saline, free lymphokines, free MDP or liposomes containing placebo died by day 90 of the experiment (60 days after amputation). Significantly, 70 % of mice given liposome-encapsulated lymphokines and 60 % of mice given liposome-encapsulated MDP survived to 200 days, at which time the experiments were terminated. At the beginning of the treatment, the metastases contained at least 10’ cells. We speculate that the residual tumour burden of the surviving mice must have been reduced to less than 10 viable cells, because the median survival time of mice injected with as few as 10 viable B16 cells (admixed with lo6 dead cells) is 40 to 50 days (Fidler, 1980; Fidler et al., 1982). Studies of mice who had residual metastatic disease treated with liposomal MDP have revealed that the tumour cells in the lesions were still fully susceptible to destruction by activated macrophages (Eppstein et al., 1986). Similar findings demonstrating successful treatment of metastases by the intravenous injection of liposomes containing immunomodulators have also been reported for several murine fibrosarcomas (Eppstein et al., 1986; Lopez-Berestein et al., 1984), melanomas (Phillips et al., 1985), lung carcinoma (Deodhar et al., 1982), liver metastases (Phillips and Tsao, 1989; Brodt et al., 1989), and primary skin cancers (Talmadge et al., 1986). For more information, see other reviews in this issue.

FUNCTIONS

Limitations of liposome-immunomodulator phage-mediated therapy

201 macro-

To determine the limitations of this therapy, the initial liposome injection was administered to groups of mice either 3, 7 or 10 days after resection of the primary melanoma. All mice were treated twice weekly for 4 weeks and then observed daily for up to 250 days. Administration of liposomes containing saline had no therapeutic effect, regardless of the time of initial treatment. Liposomal MTP-PE treatment was effective, but the degree of benefit depended upon the timing of the first treatment. The most improved survival was seen when treatment was begun on day 3 after surgical removal of the primary tumour (65 % survival). When the first treatment began on day 7 after excision of the primary tumour, only 45 % of the mice survived, and when the therapy began on day 10 after excision of the tumour, only 30 % of the mice survived (Fidler, 1986). These data clearly indicated that the tumour burden at initiation of therapy profoundly influenced the outcome of systemic therapy with MLV-containing immunomodulators. Synergistic activation of macrophages by liposomes containing two immunomodulators Many studies have shown that free or liposomeencapsulated lymphokines such as MAF or IFNy and such bacterial products as LPS or MDP can act synergistically to activate macrophages (Saiki et al., 1985). Because liposomes can deliver more than one compound to macrophages in situ, we investigated whether combining IFNy and MDP within the same liposome could produce synergistic activation of lung macrophages in vivo and enhanced eradication of lung melanoma metastases (Fidler et al., 1989). Synergistic activation by free IFNy and MDP has been shown to occur in vitro. However, as noted above, neither the i.v. injection of free IFNy nor of free MDP led to activation of macrophages in situ. The i.v. administration of MLV containing optimal doses of IFNy or MTP-PE rendered lung macrophages tumoricidal. When subthreshhold doses of IFNy and MTP-PE were combined and delivered within the same MLV, significant in situ activation also occurred (Fidler et al., 1989). To test the hypothesis that IFNy and MTP-PE delivered within the same liposome would synergistically activate macrophages in situ, thus destroying a larger metastatic tumour burden, therapy experiments were conducted in mice with relatively large metastatic lesions. Liposomes were injected twice weekly for 4 weeks. Mice receiving saline or MLV containing low concentrations of IFNy or MTP-PE died by day 90 of the study. Treatment with liposomes containing either optimal MTP-PE or IFNy yielded long-term

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survival (> 250 days) in only 30 070of these animals. Treatment with liposomes containing both MTP-PE and IFNy produced long-term survival (> 250 days) in 50-60 % of the mice (Fidler et al., 1989). These results, taken together, show that the optimal liposome administration schedule (in mice) is twice weekly for 8 weeks, that tumour burden limits the efficacy of this form of therapy, and that enhanced macrophage activation extends the tumour burden limit that can be effectively treated. Therapy of autochthonous with osteogenic sarcoma

lung metastases in dogs

The natural history of canine osteosarcoma comprises progressive growth of the primary neoplasm and death due to visceral (lung) metastasis (MacEwen et al., 1989), mirroring the same disease process in humans. Surgical removal of the primary tumour yields a median survival of 3 to 6 months, with only 10 070survival after 1 year or longer. MacEwen and co-workers (1989) recently completed a randomized double-blind study of liposome-encapsulated MTPPE for osteosarcoma metastasis in dogs. Twentyseven dogs were entered into the study; all animals had histologically documented osteosarcoma and complete amputation of the primary tumour-bearing limb. Immediately after surgery, dogs were randomized to receive either liposomal MTP-PE or liposomes containing saline (placebo). Intravenous infusions of 2 mg/m2 of MTP-PE (500 mg of phospholipids) were given twice weekly for 8 weeks (total of 16 injections). Fourteen dogs received liposome-MTP-PE ; 13 received liposomes containing saline. The median survival time of dogs receiving the saline placebo was 77 days, and this survival did not differ from that for surgical treatment alone. In contrast, the median survival of dogs treated with liposome-MTP-PE was 222 days. Indeed, 4 dogs in this group were alive and free of disease 1 year after surgery. Also, liposomal MTP-PE did not produce toxic effects and was well tolerated. Since a major limitation for treatment of metastases by macrophage activation is tumour burden, it is probable that the treatment did not benefit some dogs. In other words, the tumour burden (in micro-metastases) exceeded the level that can be eliminated by macrophages. Evidence from this trial suggests that systemic macrophage activation for destruction of metastases should follow other cytoreductive treatments such as‘ chemotherapy (MacEwen et al., 1989). Phase I trial of liposome-encapsulated cancer patients

MTP-PE

The biological effects of liposomes containing MTP-PE in humans were studied in a multicenter

in

phase I trial in cancer patients (Murray et al., 1989; Creaven et al., 1990; Urba et al., 1990). The commercial preparation of liposomal-MTP-PE used in the clinical trial has been used with success to activate human blood monocytes in vitro and murine macrophages both in vitro and in vivo. In addition, this commercial liposome-MTP-PE preparation has been found to activate pulmonary macrophages following intranasal administration (Brownbill et al., 1985), to produce systemic macrophage activation following oral administration (Fidler et al., 1987) and, as described above, to eliminate spontaneous murine melanoma metastases and autochthonous osteogenic sarcoma metastases in dogs (MacEwen et al., 1989). The primary objectives of this phase I study were to determine the toxicity and maximal tolerated dose of MLV-MTP-PE and to examine the biological activity of this preparation. The findings obtained from two parallel phase I trials were similar. Important findings from the trial included the following. (a) Liposome-MTP-PE was safe when given at the dose and schedule described. The major dose-limiting side effects encountered were chills and fever. Toxicity was not cumulative, and the maximal tolerated dose of MTP-PE was 6 mg/m2 (in 1.5 g phospholipids). (b) A consistent increase in absolute white blood count and neutrophils was observed as well as increases in serum ILIP. (c) Liposome-MTP-PE therapy produced tumoricidal activity in the monocytes of 24 of the 28 patients treated. The optimal dose of liposome-MTP-PE for producin monocyte tumoricidal activity was 0.5-2.0 mg/m !3, which was less than the maximum tolerated dose (6 mg/m2/dose). (d) Those patients with increased granulocyte counts showed a significant decrease in serum cholesterol. (e) Significant elevations in Creactive protein, P,-microglobulin, and ceruloplasmin were frequently observed. (f) Biodistribution and pharmacokinetics of liposome-MTP-PE were determined in 4 patients after i.v. infusion of ggmT~labelled MLV. At 6 hours following injection, radioactivity was concentrated in the liver, spleen, nasopharynx, thyroid, and to some extent, the lungs. By 24 hours, this radioactivity had partially cleared. In 2 of the 4 patients studied, lung metastases were imaged, presumably by tumor-associated macrophages that had phagocytosed 99mTc-labelled liposomes (Murray et al., 1989). Conclusions The successful therapy of cancer metastasis must overcome the heterogeneous nature of malignant neoplasms and the continuous emergence of variant cells. In situ macrophage activation with liposomes containing immunomodulators can provide an approach : tumoricidal macrophages destroy malignant

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cells in vitro and in vivo while leaving non-neoplastic cells uninjured. Moreover, macrophage-mediated lysis of tumour cells is not associated with the development of significant tumour cell resistance. Intravenously administered liposomes are removed from the circulation by phagocytic cells of the RES. The endocytosis of liposomes with immunomodulators produces tumoricidal macrophages in situ; thus, as has been shown in several studies, multiple administrations of such liposomes can bring about eradication of cancer metastases in multiple murine tumour systems and in dogs with spontaneous osteosarcoma. Infusion of liposomes containing MTP-PE have also been shown to activate cytotoxic properties in blood monocytes of cancer patients. Findings from phase I clinical trials indicate that at the minimum dose sufficient to produce desirable biological responses, these liposomes are well tolerated and non-toxic. Although the initial findings reported from our laboratory suggest a promising role for the use of macrophages to eradicate metastases, this mode of therapy must be combined with other treatment modalities, such as surgery, radiotherapy and chemotherapy, to reduce the tumour burden first; the activated macrophages can then lyse tumour cells that are resistant to other agents. Supported by grant R35-CA 42107from the National Cancer Institute, National Institutes of Health, and by funds from the June and Richard Anderson Endowment for MelanomaResearch.

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Phillips, N.C., Mora, M.L., Chedid, L., Lefrancier, P. & Bernard, J.M. (1985), Activation of tumoricidal activity and eradication of experimental metastases by freeze-dried liposomes containing a new lipophilic muramyl dipeptide derivative. Cancer Res., 45, 128-134. Poste, G., Kirsh, R. & Fidler, I.J. (1979), Rapid decay of tumoricidal activity and loss of responsiveness to lymphokines in inflammatory macrophages. Cancer Res., 39, 2582-2587. Poste, G., Bucana, C.D., Raz, A., Bugelski, P., Kirsh, R. & Fidler, I.J. (1982), Analysis of the fate of systemically administered liposomes and implications for their use in drug delivery. Cancer Res., 42, 1412-1422. Saiki, I., Sone, S., Fogler, W.E., Kleinerman, E.S., LopezBerestein, G. & Fidler, I.J. (1985), Synergism between human recombinant gamma-interferon and muramyl dipeptide encapsulated in liposomes for activation of antitumor properties in human blood monocytes. Cancer Res., 45, 6188-6193. Schroit, A.J. & Fidler, I.J. (1982), Effects of liposome structure and lipid composition on the activation of the tumoricidal properties of macrophages by liposomes containing muramyl dipeptide. Cancer Res., 42, 161-167. Talmadge, J.E., Lenz, B.F., Klabansky, R., Simon, R., Riggs, C., Guo, S., Oldham, R.K. & Fidler, I.J. (1986), Therapy of autochthonous skin cancers in mice with intravenously injected liposomes containing muramyltripeptide. Cancer Res., 46, 1160-I 163. Urba, W.J., Hatmann, L.C., Longo, D.L., Steis, R.G., Stnith, II, l.W., Kedar, I., Creekmore, S., Sznol, M., Conlon, K., Kopp, W.C., Huber, C., Herold, M., Alvord, W.G., Snow, S. & Clark, J.W. (1990), Phase I and immunomodulatory study of a muramyl peptide, muramyl tripeptide phosphatidylethanolamine. Cancer Res., 50, 2979-2986. Utsugi, T., Schroit, A,J., Connor, J., Bucana, C.D. & Fidler, I.J. (1991), Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res., 51, 3062-3066.