Tumoricidal activity of human monocytes activated in vitro by free and liposome-encapsulated human lymphokines.

Human peripheral blood mononuclear cells from normal donors obtained by separation on a Percoll gradient were incubated with free or liposome-entrapped lymphokines produced from concanavalin A-stimulated lymphocytes and then were tested for cytotoxic activity against tumor cells. The treated monocytes lysed tumorigenic melanoma and glioblastoma target cells, but had no effect on three types of nontumorigenic target cells. The activation of monocytes to become tumoricidal was caused by macrophage-activating factor (MAF) and not by contamination with endotoxins, concanavalin A, or interferon. The endocytosis of liposomes containing MAF, but not of those containing control supernatants, led to the activation of cytotoxic properties in the monocytes. Activation by liposome-encapsulated MAF was very efficient and required less than 1/800th of the amount of free MAF necessary to achieve the same levels of cytotoxicity. Thus, the encapsulation of mitogen-induced MAF in liposomes could provide an effective approach for the activation of blood monocytes in situ.

E-selectin ligands recognised by HECA452 induce drug resistance in myeloma, which is overcome by the E-selectin antagonist, GMI-1271.

Multiple myeloma (MM) is characterized by the clonal expansion and metastatic spread of malignant plasma cells to multiple sites in the bone marrow (BM). Recently, we implicated the sialyltransferase ST3Gal-6, an enzyme critical to the generation of E-selectin ligands, in MM BM homing and resistance to therapy. Since E-selectin is constitutively expressed in the BM microvasculature, we wished to establish the contribution of E-selectin ligands to MM biology. We report that functional E-selectin ligands are restricted to a minor subpopulation of MM cell lines which, upon expansion, demonstrate specific and robust interaction with recombinant E-selectin in vitro. Moreover, an increase in the mRNA levels of genes involved in the generation of E-selectin ligands was associated with inferior progression-free survival in the CoMMpass study. In vivo, E-selectin ligand-enriched cells induced a more aggressive disease and were completely insensitive to Bortezomib. Importantly, this resistance could be reverted by co-administration of GMI-1271, a specific glycomimetic antagonist of E-selectin. Finally, we report that E-selectin ligand-bearing cells are present in primary MM samples from BM and peripheral blood with a higher proportion seen in relapsed patients. This study provides a rationale for targeting E-selectin receptor/ligand interactions to overcome MM metastasis and chemoresistance.

Isolation of tumoricidal macrophages from lung melanoma metastases of mice treated systemically with liposomes containing a lipophilic derivative of muramyl dipeptide.

The systemic administration of multilamellar liposomes--composed of phosphatidylcholine and phosphatidylserine (7:3 mol ratio), containing the immunomodulator, muramyl tripeptide phosphatidylethanolamine (MTP-PE)--into C57BL/6N mice bearing the syngeneic B16-BL6 melanoma was associated with the eradication of spontaneous lung and lymph node metastases. Immunofluorescence and electron microscopic analyses revealed that 24 hours after the tumor-bearing mice were given iv injections of liposomes, 15% of the alveolar macrophages and 5% of the metastasis-associated macrophages contained phagocytosed liposomes. However, only macrophages isolated from lungs or metastases of mice given injections of liposomes containing MTP-PE (treatment success), but not macrophages from mice treated with empty liposomes (treatment failure), were tumoricidal against the target cells in vitro. These data provide direct evidence that the regression of established metastases, after treatment of tumor-bearing mice with liposomes containing MTP-PE, was associated with tumoricidal macrophages residing within the metastases.

Restoration of interferon alpha potentiation of a recombinant ricin A chain immunotoxin following cytoreduction of xenografts of advanced ovarian tumors.

BACKGROUND: We have demonstrated that, in the human ovarian carcinoma cell line (OVCAR-3), recombinant human interferon alpha (rHuIFN-alpha) potentiated in vitro inhibition of protein synthesis by immunotoxins. The antitumor activity of intracavitary immunotoxin administered to nude mice 5 days after tumor cell injection was enhanced by a nontherapeutic dose of rHuIFN-alpha, as evidenced by increased survival time. PURPOSE: Our purpose was to determine the outcome of treatment with immunotoxin and rHuIFN-alpha in xenografts of more advanced tumors. METHODS: At 10 or 15 days after tumor cell injection, nude mice with peritoneal OVCAR-3 xenografts were treated intraperitoneally with immunotoxin or with 454A12 monoclonal antibody (MAb) recombinant ricin A chain (rRA), alone or combined with a nontherapeutic dose of rHuIFN-alpha. The immunotoxin was composed of rRA covalently bound to an anti-CD71 (transferrin receptor) MAb. In other experiments, mice were treated intraperitoneally with cyclophosphamide and cisplatin to reduce tumor size on days 20 and 27 after tumor cell inoculation and then, beginning on day 40, with immunotoxin alone or combined with rHuIFN-alpha. RESULTS: Initiation of treatment 10 days after OVCAR-3 transplantation significantly increased median survival from 41 to 89 days (10% survivors on day 120) with 454A12 MAb rRA alone and to more than 120 days (70% survivors) with 454A12 MAb rRA combined with rHuIFN-alpha (P < .0001). The increase in survival time between tumor-bearing mice treated with immunotoxin combined with rHuIFN-alpha and those treated with immunotoxin alone was statistically significant (P = .017). In contrast, the 15-day transplant tumors were not curable with immunotoxin therapy (survival, 72 days; 0% survivors) and were refractory to rHuIFN-alpha potentiation (survival, 75 days; 0% survivors). After the second course of chemotherapy to reduce the size of the advanced tumors (day 40), during the ascites cell count nadir, initiation of treatment with 454A12 MAb rRA alone or combined with rHuIFN-alpha resulted in significantly different survival times of 129 and 162 days, respectively (P = .0037). Pathologic examination of surviving mice treated with chemotherapy and 454A12 MAb rRA alone or in combination with rHuIFN-alpha revealed that one (17%) of six mice and 11 (65%) of 17 were tumor free, respectively. CONCLUSIONS: The synergy between immunotoxins and IFN-alpha is dependent on tumor burden. These agents are less effective against large tumor burdens (i.e., advanced stage disease), but their beneficial effects re-emerge after cytoreduction by combination chemotherapy. IMPLICATIONS: The ideal setting for testing the efficacy of intracavitary immunotoxin combined with rHuIFN-alpha after front-line chemotherapy is in patients with residual tumor refractory to additional chemotherapy or in those with toxic effects that prevent delivery of effective doses.

Enhancement by recombinant human interferon alfa of the reversal of multidrug resistance by MRK-16 monoclonal antibody.

BACKGROUND: The anti-P-glycoprotein monoclonal antibody MRK-16 mediates the reversal of multidrug resistance. Recombinant human interferon alfa (rHuIFN alpha) enhances the cytotoxic activity of diverse chemotherapeutics and may modulate multidrug resistance. PURPOSE: Our purpose was to determine the outcome of combination treatment with MRK-16, rHuIFN alpha-2a, and cytotoxic agents on tumor cells that express P-glycoprotein (Pgp). METHODS: Three Pgp-expressing, multidrug-resistant human tumor cell lines were used: the MDR1 retrovirus-infected HT-29 colon adenocarcinoma (HT-29mdr1), the doxorubicin (Adriamycin)-resistant MCF-7 (AdrR MCF-7) breast carcinoma, and the de novo Pgp-acquired, HCT-15 colon carcinoma. The parental cell lines HT-29par and MCF-7 were used as controls. The in vitro effects of MRK-16 and rHuIFN alpha-2a were studied on: (a) chemosensitivity of parental and multidrug-resistant cell lines to vincristine, doxorubicin, or paclitaxel (Taxol); (b) intracellular drug concentrations; and (c) Pgp expression. The efficacy of vincristine alone or in combination with MRK-16 and/or rHuIFN alpha-2a was assessed against HT-29mdr1 cells in female, athymic NCr-nu/nu mice. RESULTS: For vincristine, the IC50 (i.e., the concentration that causes 50% inhibition of cell growth) was 7.0 ng/mL in HT-29mdr1 cells. Pretreatment of HT-29mdr1 cells with MRK-16 partially restored vincristine sensitivity (IC50 = 4.8 ng/mL), which was enhanced by noncytotoxic concentrations of rHuIFN alpha-2a (IC50 = 2.9 ng/mL) via a mechanism independent of Pgp modulation or [3H]vincristine efflux. rHuIFN alpha-2a potentiated MRK-16 reversal of multidrug resistance with both doxorubicin and paclitaxel on HT-29mdr1 cells and with vincristine on AdrR MCF-7 and HCT-15 tumor cells. Treatment of mice with 1 mg/kg vincristine weekly for 3 weeks, beginning 10 days after tumor injection, significantly increased the median survival times of the HT-29par tumor-bearing mice (60 days versus 35 days; P < .0001) but was only marginally therapeutic for HT-29mdr1 tumor-bearing mice (52 days versus 46 days). Pretreatment with MRK-16 (500 micrograms) and rHuIFN alpha-2a (5 x 10(4) U), alone or in combination, 24 hours before vincristine therapy did not affect the survival of HT-29par tumor-bearing mice. In contrast, the survival of mice bearing HT-29mdr1 tumors was significantly increased following treatment with MRK-16 before vincristine (80 days; P < .0001). Administration of a nontherapeutic dose of rHuIFN alpha-2a (5 x 10(4) U) with MRK-16 before vincristine treatment further increased the median survival times of HT-29mdr1 tumor-bearing mice (116 days; P < .0001). CONCLUSIONS: MRK-16 used in combination with rHuIFN alpha-2a was significantly more effective than MRK-16 in overcoming multidrug resistance.

Reversal of drug resistance in a human colon cancer xenograft expressing MDR1 complementary DNA by in vivo administration of MRK-16 monoclonal antibody.

One strategy to overcome multidrug resistance in neoplasia is to inhibit the gp170 glycoprotein (relative molecular mass, 170,000) that functions as a plasma membrane, energy-dependent, drug-efflux pump. The human colon cancer cell line HT-29, which grows as an ascitic tumor in athymic NCr-nu/nu nude mice, was made multidrug resistant by infection with an MDR1 (also known as PGY1) retrovirus. Referred to as HT-29mdr1, it was used to study reversal of drug resistance in vivo by the anti-P-glycoprotein monoclonal antibody MRK-16. Flow cytometry and radioimmunoassay demonstrated a marked increase in MRK-16 reactivity on HT-29mdr1 cells as compared with its reactivity on the parental, uninfected cell line (HT-29par). The 50% inhibitory concentrations (IC50) of vincristine on HT-29par and HT-29mdr1 cells were 2.5 and 15 ng/mL, respectively. The MRK-16 monoclonal antibody did not affect the vincristine sensitivity of the HT-29par cells. Pretreatment of HT-29mdr1 cells with 10 micrograms/mL MRK-16 in tissue culture partially restored the vincristine sensitivity (IC50 = 7 ng/mL). This modulation of vincristine sensitivity by MRK-16 was then tested in vivo. The median survival times of mice given intraperitoneal transplants of 5 x 10(6) HT-29par or HT-29mdr1 were 37 and 39 days, respectively. Treatment of mice with 1 mg/kg vincristine weekly for 3 weeks, beginning 10 days after tumor injection, resulted in a significant increase in the median survival time of the HT-29par tumor-bearing mice (68 days, P less than .0001), but it had no effect on the HT-29mdr1 tumor-bearing mice. However, treatment of mice bearing the HT-29mdr1 tumor with MRK-16 before vincristine therapy reversed the resistance to the drug (median survival time = 64 days, P less than .0001). The MRK-16 monoclonal antibody alone had no effect on the median survival time of mice given an injection of either HT-29par or HT-29mdr1 cells. These results suggest that strategies employing monoclonal antibody against gp170 may be clinically useful to reverse multidrug resistance.

Enhancement of vincristine cytotoxicity in drug-resistant cells by simultaneous treatment with onconase, an antitumor ribonuclease.

BACKGROUND: Onconase, a protein isolated from oocytes and early embryos of the frog Rana pipiens, shares extensive homology with bovine pancreatic ribonuclease (RNase A) and possesses similar enzyme activity. Onconase is cytotoxic toward cancer cells in vitro and exhibits antitumor activity in animal models. In addition, Onconase has been shown to enhance the cytotoxic activity of some chemotherapeutic agents in vitro. PURPOSE: We studied interactions between the cytotoxic effects of Onconase and the chemotherapeutic agent vincristine (VCR) in the treatment of drug-sensitive and multidrug-resistant human colon carcinoma cells in vitro and in mice. METHODS: Transplantable human colon carcinoma cells (HT-29par cells) were infected with a retrovirus containing human mdr1 (also known as MDR1 and PGY1) complementary DNA (encoding P-glycoprotein [P-gp]), and clones that were cross-resistant to colchicine, doxorubicin, and vinblastine were selected (HT-29mdr1 cells). Drug-resistant HT-29mdr1 cells and drug-sensitive HT-29par parental cells were treated with Onconase and/or VCR in vitro at varying concentrations to measure the effects on protein synthesis and cell viability. The impact of Onconase on VCR accumulation in both types of cells was determined in the presence or absence of MRK-16, an anti-P-gp monoclonal antibody capable of reversing the multidrug-resistant phenotype. The antitumor effects of Onconase and/or VCR treatment were assessed in nude mice bearing established HT-29par or HT-29mdr1 intraperitoneal tumors. IC50 values (drug concentrations resulting in 50% inhibition of protein synthesis or cell viability) for Onconase and VCR were determined from semilogarithmic dose-response curves; interactions between the cytotoxic effects of these two agents were evaluated using data from protein synthesis inhibition experiments and a two-way analysis of variance. Survival distributions from in vivo experiments were compared using Cox proportional hazards models. RESULTS: The combination of Onconase and VCR yielded enhanced cytotoxicity in vitro that was independent of P-gp expression. Evaluation of the effects of these two compounds on protein synthesis over a wide range of drug concentrations indicated possible synergistic interactions (i.e., greater than additive effects) in both drug-resistant and drug-sensitive cells. The enhancement of VCR cytotoxicity was dependent on Onconase enzyme activity and was not associated with increased intracellular levels of VCR. Simultaneous treatment of mice bearing HT-29par tumors with Onconase and VCR did not extend their median survival time (MST) significantly (MST with VCR = 66 days; MST with VCR plus Onconase = 69 days; two-tailed P = .57); however, the MST of mice with HT-29mdr1 tumors was extended significantly by this treatment (MST with VCR = 44 days; MST with VCR plus Onconase = 66 days; two-tailed P<.001). CONCLUSION: Combined administration of Onconase and VCR yields enhanced cytotoxicity in vitro and in vivo against human colon carcinoma cells that overexpress the mdr1 gene.

Nonselective destruction of murine neoplastic cells by syngeneic tumoricidal macrophages.

The purpose of these studies was to select in vitro tumor cells that were resistant to macrophage-mediated lysis. Seven different heterogeneous murine neoplasms (four fibrosarcomas, a melanoma, a rhabdomyosarcoma, and an osteogenic sarcoma) and one cloned line of a fibrosarcoma were incubated in vitro with syngeneic tumoricidal macrophages. Surviving tumor cells were recovered and expanded to undergo subsequent interaction with tumoricidal macrophages. After six sequential interactions, all cell lines were examined for their susceptibility to lysis mediated by murine peritoneal exudate macrophages activated with liposomes containing muramyl tripeptide phosphatidylethanolamine. In all eight systems, no significant differences were detected between the parent tumor cells and cells that survived the sequential interactions. Neither macrophage infiltration into s.c. tumors nor the experimental or spontaneous metastatic potentials of the parental tumors differed from the lines established by cells surviving macrophage-mediated lysis. Collectively, the data suggest that tumor cell destruction by activated macrophages is nonselective and does not lead to the development of resistant tumor cells nor to cells with altered metastatic properties.

Activation of tumoricidal properties in mouse macrophages by lymphokines encapsulated in liposomes.

Cell-free culture supernatants rich in macrophage-activating factor (MAF) activity obtained from mitogen-stimulated F344 rat lymphocytes have been encapsulated within liposomes of differing size and lipid composition and their ability to render normal mouse macrophages cytotoxic for tumor cells in vitro has been compared with that of unencapsulated (free) MAF added to the extracellular medium. Normal macrophages from C57BL/6, C3H/Hen, and C57BL/6 X C3H F1 mice treated with liposome-encapsulated MAF exhibited significant in vitro cytotoxicity against syngeneic and allogeneic tumor cells but did not kill nontumorigenic normal cells. Dose-response measurements revealed that liposome-encapsulated MAF was able to render macrophages tumoricidal at concentrations of at least 20,000 times lower than free MAF. Liposomes containing MAF were able to activate macrophages in the presence of p-nitrophenyl-2-O-alpha-L-fucopyranosyl-beta-D-galactopyranoside, a potent inhibitor of free MAF, indicating that encapsulated MAF was protected within liposomes and that liposome-mediated activation was not caused by small amounts of MAF released into the culture medium from "leaky" liposomes. Liposome-encapsulated MAF was also able to activate macrophages which were refractory to activation by free MAF following either removal of presumably surface receptors for MAF by pronase and/or alpha-L-fucosidase or occupation of the MAF receptor on macrophages by fucose-binding plant lectins (Ulex europaeus 1 and Lotus tetragonolobus agglutinins). Also, populations of nontumoricidal inflammatory tissue macrophages, which were inherently unresponsive to free MAF, would be rendered tumoricidal in vitro by incubation with liposome-encapsulated MAF. Collectively, the data suggest that MAF can render macrophages tumoricidal by acting on intracellular sites.

Biochemical, morphological, and ultrastructural studies on the uptake of liposomes by murine macrophages.

The interaction of multilamellar liposomes with mouse peritoneal macrophages cultured in vitro has been examined. The principal mechanism of liposome uptake by these cells is by phagocytic engulfment. Studies with radiolabeled liposomes demonstrated that they are incorporated into macrophages as intact structures and that treatment of macrophages with inhibitors of phagocytosis prevents liposome uptake. Incubation of macrophages with liposomes containing encapsulated fluorescein-labeled bovine serum albumin resulted in localization of fluorescence within discrete cytoplasmic vacuoles. Ultrastructural observations confirmed that liposomes were internalized and were enclosed within phagosomes. Electron microscopy also revealed that, by 24 hr following phagocytosis, adjacent phagosomes containing liposomes prepared from bovine brain phosphatidylserine, egg phosphatidylcholine, and lysolecithin (mol ratio, 4.95/4.95/0.1) fused within the cytoplasm. In contrast, phagosomes containing neutral liposomes consisting solely of egg phosphatidylcholine did not fuse and remained as discrete single structures. Negatively charged bovine brain phosphatidylserine/egg phosphatidylcholine/lysolecithin liposomes were phagocytosed at a much faster rate (12 times faster) than were neutral egg phosphatidylcholine liposomes.

Activation of tumoricidal properties in peripheral blood monocytes of patients with colorectal carcinoma.

The purpose of these studies was to determine whether blood monocytes of patients with different stages of colorectal carcinoma could be activated by various immunomodulators to become tumor cytolytic. Monocytes obtained from 12 colorectal carcinoma patients and 8 normal donors were incubated in vitro with free or liposome-encapsulated agents. The cytotoxic properties of the monocytes were determined subsequent to interaction with radioactively labeled allogeneic colon carcinoma cells, melanoma cells, glioblastoma cells, and allogeneic nontumorigenic skin cells. Blood monocytes from normal donors and all colorectal carcinoma patients were activated in vitro to become tumoricidal by immunomodulators in free form or entrapped within liposomes; i.e., the monocytes recognized and lysed tumorigenic cells but not nontumorigenic cells. The tumoricidal activity of monocytes was observed in blood monocytes obtained from patients even after multiple doses of radiotherapy and chemotherapy, and that fact suggests that the in vivo activation of macrophages may be feasible.

Enhanced cytotoxicity against colon carcinoma by combinations of noncompeting monoclonal antibodies to the 17-1A antigen.

The purpose of this report was to investigate the binding specificities and ability to generate antibody dependent cell mediated cytotoxicity (ADCC) or antibody dependent complement mediated cytotoxicity of three novel monoclonal antibodies (mAbs), designated M74, M77, and M79, raised against the colorectal carcinoma antigen 17-1A. As determined by indirect radioimmunoassay, all three mAbs, as well as mAb 17-1A, bound to a similar extent to adherent cultures of the human colon carcinoma cell line, HT-29. Scatchard analysis of direct binding data for mAbs 17-1A, M74, M77, and M79 to HT-29 cells demonstrated equivalent association constants (7.54-9.77 X 10(7) liters/mol) and molecules bound per cell (2.15-2.69 X 10(5)). In contrast to mAbs M77 and M79, mAb M74 inhibited the binding of 125I-labeled mAb 17-1A to HT-29 cells. Similar to mAb 17-1A, incubation of human lymphocytes and blood monocytes with mAbs M74, M77, or M79 generated ADCC activity against HT-29 colon carcinoma cells. Various combinations of noncompeting mAbs to the 17-1A antigen (17-1A and M74; M77 and M79; M74 and either M77 or M79) but not competing mAbs (17-1A and M74; M77 and M79) resulted in a heightened level of ADCC activity. Under optimum conditions (saturation of antigenic sites with mAb), ADCC generated by the combination of noncompeting mAbs to the 17-1A antigen was additive to the activity seen with the respective mAbs alone. Under suboptimum conditions, the combination of noncompeting mAbs to the 17-1A antigen resulted in tumor cell cytotoxicity which was synergistic to the lysis obtained with the respective mAbs alone. No mAb used alone was able to generate antibody dependent complement mediated cytotoxicity against a panel of 17-1A positive colon carcinoma cells. Similarly, no antibody dependent complement mediated cytotoxicity activity was obtained with the combination of competing mAbs to the 17-1A antigen. However, HT-29 cells treated with noncompeting mAbs to the 17-1A antigen antigen were rendered susceptible to lysis by human complement. We conclude that the combination of mAbs recognizing different epitopes on the same tumor antigen could have important implications for the passive immunotherapy of cancer.

Activation of tumoricidal properties in human blood monocytes by liposomes containing lipophilic muramyl tripeptide.

Peripheral blood monocytes were isolated from normal human donors by separation on a continuous Percoll gradient and adherence to yield preparations of blood monocytes with a high degree of purity (greater than 99%). The monocytes were incubated in vitro with medium alone or with multilamellar liposomes that contained either a lipophilic derivative of muramyl dipeptide, muramyl tripeptide (MTP-PE), or medium. The cytotoxic properties of the monocytes were assessed by an in vitro radioisotope release assay against various allogeneic targets. Monocytes that have phagocytosed liposomes containing MTP-PE were rendered tumoricidal. These monocytes lysed cells of three different tumorigenic lines but not cells of two nontumorigenic lines. The ability of MTP-PE-activated human blood monocytes to recognize and selectively lyse neoplastic cells was also demonstrated under cocultivation conditions. We conclude that human blood monocytes can be rendered tumoricidal after interaction with liposomes containing MTP-PE.

Design of liposomes to improve delivery of macrophage-augmenting agents to alveolar macrophages.

Factors affecting the localization of liposomes injected i.v. in the lung have been studied to identify the optimal type of liposome for delivery of macrophage-activating agents to the lung to augment the tumoricidal activity of alveolar macrophages (AM). Comparison of pulmonary retention of liposomes of differing size, surface charge, and composition following i.v. injection into inbred mice revealed that large multilamellar (MLV) and reversed-phase-evaporation (REV) liposomes are arrested in the lung more efficiently than are small unilamellar liposomes of identical lipid composition. MLV and REV containing negatively charged amphiphiles arrest in the lung more efficiently than do neutral MLV's or REV's or MULV's and REV's containing positively charged amphiphiles. Comparison of the ability of liposomes containing a variety of negatively charged amphiphiles to localize in the lung established that optimal localization was achieved using MLV and REV prepared from phosphatidylserine (PS) and phosphatidylcholine (PC) (3:7 mol ratio) or PS:PC:lysolecithin (4.95:4.95:0.1 mol ratio). The proportion of these liposomes retained in the lung after i.v. injection was constant over a wide dose range (0.02 to 20 mumol phospholipid per mouse), but hemodilution due to i.v. inoculation of liposomes in volumes exceeding 0.2 ml reduced retention in the lung. Uptake of liposomes by AM was demonstrated by showing that i.v. injection of PS:PC MLV liposomes containing fluorescein-labeled bovine serum albumin resulted in localization of fluorescence within AM recovered by pulmonary lavage. Similarly, AM recovered after i.v. injection of PS:PC MLV liposomes containing lymphokine preparations rich in macrophage-activating factor (MAF) activity exhibited tumoricidal activity. In contrast, macrophages recovered from control animals given injections of unencapsulated MAF or liposomes containing lymphocyte supernatants without MAF activity were devoid of cytotoxic activity. Neutral (PC) MLV liposomes containing MAF, which show only very limited retention in the lung, were ineffective in activating AM in situ. We conclude that negatively charged MLV liposomes (PS:PC, 3:7 mol ratio) localize efficiently in the lung and that macrophage-activating agents encapsulated within such liposomes can successfully activate lung macrophages in situ.

Involvement of macrophages in the eradication of established metastases following intravenous injection of liposomes containing macrophage activators.

Liposomes containing encapsulated lymphokines or muramyl dipeptide (MDP), when injected i.v. into C57BL/6 mice, produced significant destruction of established lung and lymph node metastases from a s.c. highly metastatic B16-BL6 melanoma. We present evidence that eradication of the metastases is mediated by the activation of host macrophages to the tumoricidal state. Results from three separate types of experiments support this conclusion. (a) When macrophage-activating agents such as lymphokines of MDP were delivered in liposomes that were not efficiently retained in the lung, little or no activation of lung macrophages was observed, and growth of metastases was unaltered. (b) Eradication of metastases was not observed when tumor-bearing animals were treated with agents that impaired macrophage function (e.g., silica, carrageenan, hyperchlorinated drinking water) prior to systemic therapy with liposome-encapsulated lymphokines or liposome-encapsulated MDP. (c) Macrophages activated in vitro by liposome-encapsulated MDP and then injected i.v. into mice bearing experimental lung metastases also significantly inhibited lung metastases. These results suggest that the augmented host response against pulmonary and lymph node metastases generated by the systemic administration of liposome-encapsulated lymphokines or MDP is mediated via activated cytotoxic macrophages.

The role of plasma membrane receptors and the kinetics of macrophage activation by lymphokines encapsulated in liposomes.

The kinetics of activation of tumoricidal functions in mouse macrophages incubated with macrophage-activating factors (MAF) released by mitogen-stimulated lymphocytes (free MAF) and MAF encapsulated with liposomes (liposome-MAF) have been compared. Development of tumoricidal activity requires incubation of macrophages with free or liposome-encapsulated MAF for a minimum of 4 hr. Macrophages incubated with MAF for 4 hr were not cytotoxic when tumor target cells were added immediately after removal of MAF, but they were highly cytotoxic when allowed to complete a "lag" phase before being exposed to tumor cells. The duration of the lag phase varied with different activation protocols. The levels of cytotoxic activity induced by liposome-encapsulated MAF was consistently higher than that obtained with free MAF. Studies using inhibitors of endocytosis demonstrated that internalization of the liposome carrier is required for activation by liposome-MAF and that activation does not result from MAF leaking from liposomes and binding to MAF receptors on either the plasma membrane or the membrane of endocytic vesicles. Comparison of the efficiency of macrophage activation by MAF encapsulated in liposomes of differing internal volume revealed that large multilamellar and large unioligolamellar liposomes were more efficient in activating peritoneal exudate macrophages than were small unilamellar liposomes. Measurement of the volume of liposome contents internalized by macrophages from these three types of liposomes revealed that maximum cytotoxicity required internalization of a given volume of MAF-containing lymphocyte supernatants, after which no further increase in cytotoxicity occurred.

Synergism between human recombinant gamma-interferon and muramyl dipeptide encapsulated in liposomes for activation of antitumor properties in human blood monocytes.

Highly purified human blood monocytes, isolated by centrifugal elutriation under endotoxin-free conditions, were activated in vitro by combining subthreshold amounts of human recombinant gamma-interferon (r-IFN-gamma) and muramyl dipeptide (MDP) to become tumor cytotoxic against allogeneic A375 melanoma cells. Only intact r-IFN-gamma and MDP produced synergism for human monocyte activation. Neither pH 2-treated r-IFN-gamma and intact MDP nor heat-treated IFN-gamma and intact MDP, nor intact IFN-gamma and the biologically inactive stereoisomer of MDP, N-acetylmuramyl-D-alanyl-D-isoglutamine, produced activation of blood monocytes. The encapsulation of intact r-IFN-gamma and MDP within the same preparation of multilamellar liposomes was synergistic for monocyte activation. These data show that synergism for monocyte activation can be produced by human r-IFN-gamma and MDP produced synthetically can be simultaneously delivered to monocytes. Because both r-IFN-gamma and MDP can now be produced in large standardized quantities their synergism for activation of tumoricidal properties in human monocytes could be of clinical significance.

Enhanced therapeutic efficacy against an ovarian tumor xenograft of immunotoxins used in conjunction with recombinant alpha-interferon.

The antitumor effects of two immunotoxins were evaluated in vitro and in vivo against the human ovarian carcinoma cell line, OVCAR-3. The immunotoxins used were composed of recombinant ricin A chain (rRTA) covalently attached to a monoclonal antibody directed toward the human transferrin receptor (45412/rRTA, also called 454A12 MAB-rRTA by Cetus Corporation) or Pseudomonas exotoxin coupled to an anticarcinoma monoclonal antibody (NR-LU-10/PE). Preliminary characterization of the NR-LU-10 antigen by immunoprecipitation and cellular fluorescence demonstrated two dominant cell surface polypeptide moieties with molecular weights of 40,000 and 45,000 and a minor component with a molecular weight of 33,000. The immunotoxins were used alone or in combination with recombinant human alpha-interferon (rhIFN-alpha). Protein synthesis was inhibited in a dose-dependent manner in OVCA-3 cells incubated in vitro with either NR-LU-10/PE or 454A12/rRTA (50% inhibitory concentrations, 1 and 75 ng/ml, respectively). Unconjugated NR-LU-10 or 454A12 abrogated the activity of the relevant immunotoxins. Concomitant incubation in vitro of OVCAR-3 cells with NR-LU-10/PE or 454A12/rRTA and a noncytotoxic concentration of rhIFN-alpha potentiated the inhibitory activity of the immunotoxins via a mechanism independent of antigenic upregulation. This potentially synergistic combination was then tested in vivo. The median survival time (MST) of mice given injections i.p. of 4 x 10(6) OVCAR-3 cells was 46 days. Cohorts of mice that received intracavitary treatment beginning 5 days posttumor cell inoculation with either 0.25 or 0.5 microgram of NR-LU-10/PE every other day for a total of 10 treatments exhibited a significantly increased MST of 63 and 104 days, respectively (P less than 0.0001). Likewise, the i.p. injection of either 2.5 or 10 micrograms of 454A12/rRTA given in an identical schedule resulted in a MST of 89 and greater than 120 days, respectively (P less than 0.0001). When rhIFN-alpha was administered i.p. in conjunction with those doses of either immunotoxin, a significant increase in the MST was observed in comparison with mice given immunotoxin alone. The combination of 5 x 10(4) units of rhIFN-alpha and 0.25 microgram of NR-LU-10/PE resulted in 67% long-term survivors (greater than 120 days) compared with only 13% survival of mice given the immunotoxin alone. Similarly, 2.5 micrograms of 454A12/rRTA plus rhIFN-alpha resulted in an enhanced therapeutic response (89% long-term survivors) when compared with 454A12/rRTA alone (29%).(ABSTRACT TRUNCATED AT 400 WORDS)

Continuous administration of endostatin by intraperitoneally implanted osmotic pump improves the efficacy and potency of therapy in a mouse xenograft tumor model.

In the first Phase I clinical trials of endostatin as an antiangiogenic therapy for cancer, the protein was administered as an i.v. bolus for approximately 20-30 min each day. This protocol was based on experimental studies in which animals were treated by s.c. bolus once a day. However, it was not clear in the previous studies whether this schedule could be maximized further. Therefore, we developed experimental models involving continuous administration of endostatin to determine the potency and efficacy of this approach. Endostatin was administered to tumor-bearing mice either s.c. or i.p. in single bolus doses. The efficacy of these regimens was compared with endostatin administered continuously via an i.p. implanted mini-osmotic pump. Our results show that endostatin remains stable and active in mini-osmotic pumps for at least 7 days. We show that endostatin injected i.p. is rapidly cleared within 2 h, whereas endostatin administered continuously via mini-osmotic pump maintains systemic concentrations of 200-300 ng/ml for the duration of administration. Furthermore, continuous i.p. administration of endostatin results in more effective tumor suppression at significantly reduced doses (5-fold), compared with bolus administration. Additional experiments using a human pancreatic cancer model in severe combined immunodeficient mice showed that there was a significant decrease in the microvessel density between the treatment groups and the control group. These data show that continuous administration of human endostatin results in sustained systemic concentrations of the protein leading to: (a) increased efficacy manifested as increased tumor regression; and (b) an 8-10-fold decrease in the dose required to achieve the same antitumor effect as the single daily bolus administration of endostatin. On the basis of this approach, an additional clinical trial has been designed and initiated and is under way in two countries.

Antibodies to phospholipids and liposomes: binding of antibodies to cells.

Binding of two monoclonal anti-liposome antibodies to the surface of cultured murine peritoneal macrophages was investigated by indirect immunofluorescence and enzyme-linked immunosorbent assay. Neither antibody bound to cultures of freshly explanted, nonadherent macrophages, but immunoreactivity was observed following cell adherence to tissue culture plastic. Fluorescent microscopic evaluation revealed heterogeneity in staining patterns of the antibodies on adherent cells. Binding both to viable and fixed adherent macrophages was observed even after a 10,000-fold dilution of antibody. Treatment of adherent macrophage cultures with trypsin increased antibody binding. Further treatment of trypsinized-macrophages with alkaline phosphatase or neuraminidase did not affect antibody binding, but phospholipase D and, to a greater extent, phospholipase C resulted in a marked decrease in cellular binding. The data indicate that antibodies produced against liposomes appear to bind to surface phospholipids of macrophages, but binding can be influenced by the physiological state of the macrophage and overlying cell surface proteins.