Liposomal formulations of synthetic MUC1 peptides: effects of encapsulation versus surface display of peptides on immune responses.

Synthetic human MUC1 peptides are important candidates for therapeutic cancer vaccines. To explore whether a human MUC1 peptide BP25 (STAPPAHGVTSAPDTRPAPGSTAPP) can be rendered immunogenic by incorporation in liposomes, the effects of physical association of the peptide with liposomes on immune responses were investigated. Lipid conjugated and nonconjugated MUC1 peptides were incorporated in liposomes with a composition of distearoylphosphatidylcholine/cholesterol/dimyristoylphosphatidylglyc erol (3:1:0.25, molar ratio) containing monophosphoryl lipid A (1% w/w of the total lipids). Liposomes were characterized for peptide retention by HPLC and for surface peptide display of MUC1 epitopes by flow cytometry. C57BL/6 mice were immunized with lipopeptide alone, peptide mixed with peptide-free liposomes, and peptide associated with liposomes in entrapped or surface-exposed forms. T cell proliferative responses, cytokine patterns, and antibody isotypes were studied. Results showed that immune responses were profoundly influenced by the liposome formulations. Physically associated, either encapsulated or surface-exposed, peptide liposomes elicited strong antigen-specific T cell responses, but not lipopeptide alone or peptide mixed with peptide-free liposomes. Analysis of the cytokines secreted by the proliferating T cells showed a high level of IFN-gamma and undetectable levels of IL-4, indicating a T helper type 1 response. Thus, physical association of the peptide with liposomes was required for T cell proliferative responses, but the mode of association was not critical. On the other hand, the nature of the association significantly affected humoral immune responses. Only the surface-exposed peptide liposomes induced MUC1-specific antibodies. A domination of anti-MUC1 IgG2b over IgG1 (94 versus 6%) was observed. Our results support the hypothesis that different immune pathways are stimulated by different liposome formulations. This study demonstrated that a liposome delivery system could be tailored to induce either a preferential cellular or humoral immune response.

Immunogenicity and antitumor activity of a liposomal MUC1 peptide-based vaccine.

A human MUC1-transfected mouse mammary adenocarcinoma cell line (GZHI) was used to develop both subcutaneous and intravenous tumor models. A vaccine formulation comprised of a 24 mer (human MUC1) synthetic peptide encapsulated with monophosphoryl lipid A adjuvant (MPLA) in multilamellar liposomes was tested for immunogenicity and anti-tumor activity. A low dose of the human MUC1 peptide (5 microg) administered in liposomes provided excellent protection of mice in both tumor challenge models. The protective antitumor activity mediated by the liposome formulation correlated with anti-MUC1-specific T-cell proliferation, gamma-interferon (IFN-gamma) production and IgG2a anti-MUC1 antibodies, suggesting a type 1 (T1) T-cell response. In contrast, lack of protection in mice immunized with negative control vaccines correlated with IgG1 anti-MUCI antibody formation, low or no anti-MUC1 IgG2a and low antigen-specific T-cell proliferation, consistent with a type 2 (T2) T-cell response to the tumor.

Cytotoxicity and proliferation of splenocytes and lymph node cells from adjuvant-treated nude mice. Studies of natural and in vitro activation.

The cytotoxicity of NK cells and lymphocytes derived from nonadherent splenocytes (SPL) and regional lymph node cells (LNC) from complete Freund's adjuvant (CFA)-treated athymic nude young (4-6 weeks) and aged (over 1 year) BALB/c nu/nu mice in vitro activated with rIL-2, anti-CD3 mAb or PPD was analyzed and compared to SPL and LNC from age-matched euthymic BALB/c mice. The high natural cytotoxicity to YAC-1 target cells of SPL or LNC could be augmented by 48 h stimulation in vitro with rIL-2, especially when derived from young nude BALB/c mice. The increase in cytotoxic activity was accompanied by increased proliferative activity of both SPL and LNC, which showed statistically significant differences between the rates of stimulation of cells from the young and aged groups. Anti-CD3 mAb strongly activated the cytotoxicity of BALB/c euthymic donor effector cells against P-815 target cells, corresponding to a very high proliferative activity of these cells, but anti-CD3 mAb did not lead to activation of effector cells from nude donors. FACS analyses of antigenic markers similarly showed an increased number of T cells in LNC from aged BALB/c nude donors, which, however, never reached the levels of those of euthymic animals.

Cytotoxic cells in immunodeficient athymic mice.

A studies of cytotoxic cells in athymic nude mice demonstrate higher cytotoxic activity of NK cells and macrophages than in their euthymic counterparts. The higher level of endogenous cytotoxic activity can be considered as complementary to the deficiency or lack of thymus dependent T lymphocytes and their functions. However, with increased age of mice some T lymphocytes and their functions can be demonstrated. By stimulation of splenocytes and lymph node cells in vitro with IL-2 or anti CD3 antibody cytotoxic activity towards P-815 (NK resistant, LAK sensitive) target cells can be generated. There exist data, which indicate that the cytotoxic activity is exerted by extrathymic pre-T lymphocytes with TcR gamma delta antigenic phenotype. The differences in transplantability of human tumors in athymic nude mice cannot be explained by defect in antigen recognition and in immune response of athymic nude mice, recipients of the xenografted material. The biological relevance in vivo of high endogenous cytotoxicity of NK cells observed in many strains of athymic nude mice remains obscure. The availability of new immunodeficient mouse models, e.g. scid mice deficient in B and T lymphocytes and with low level of NK cells, in which not only xenografted human tumor grow but human lymphoid cell can be transplanted as well, opens new and broader experimental possibilities, in which new preclinical immunotherapeutical approaches can be applied.

Strain- and age-dependent natural and activated in vitro cytotoxicity in athymic nude mice.

The defect in athymic nude mice with respect to T-lymphocyte number and function is accompanied by increased levels of natural cytotoxicity as well as other immune reactions with potential antitumor effects. With increasing age of immunodeficient mice the take rate of xenotransplanted tumors decreases while the number of cells with T-lymphocyte markers increases and some T-lymphocyte-associated functions become detectable. In the present study the natural cytotoxicity of nonadherent splenocytes from young (4-6 weeks) and aged (about 1 year) BALB/c nu/nu mice was analyzed and compared with that of splenocytes from normal euthymic young and old Balb/c mice on the one hand, and with Bar nu/nu and NCr nu/nu young and old mice on the other. To investigate a possible contribution of T lymphocytes to the cytotoxic effect in athymic mice, LAK cells were generated. For this purpose, the nonadherent splenocytes were exposed in vitro either to IL-2 or to anti-CD3 mAb, specifically to activate T lymphocytes expressing TcR-CD3 in the latter case. Cytotoxic in vitro assays were applied using YAC-1 (NK-sensitive) and P-815 (NK-resistant, LAK-sensitive) target cells in parallel experiments in which splenocytes from young and old donors were used as effector cells. Splenocytes from young immunodeficient mice showed consistently high cytotoxicity with YAC-1 cells. In aged athymic mice, splenocytes stimulated in vitro with anti-CD3 mAb showed cytotoxicity to P-815 (LAK-sensitive) cells. FACS analyses of antigenic markers revealed an increased number of T cells in spleens of aged immunodeficient mice, with differences between mice of the examined strains and a decrease in the number of NK cells in aged mice.

Activity of natural killer (NK) cells in the course of experimental trichinellosis in mice.

B6C3F1 mice were infected with 200 or 500 larvae of Trichinella spiralis per mouse and pulmonary NK cell-mediated clearance of semisyngeneic tumour cells was determined in vivo on days 10, 20, 30, and 60 after the infection. Cytotoxic activity of NK cells in the lungs was substantially elevated on days 20 and 30 after challenge with both "doses" of the parasite. At the same time large granular lymphocytes (LGLs) as well as cells expressing surface asialo-GM1 molecules were isolated in elevated numbers from spleens of the infected as opposed to the normal mice. Expression of other markers of differentiation, such as THy 1, CD4, and CD8 was also enhanced on splenocytes isolated from the infected mice on day 30 but not 20 after administration of the larvae. The present results indicate that NK cell-mediated activity in vivo is stimulated above the baseline level during migration and early muscle phases of the infection with T. spiralis in mice. The possible impact of this effect upon the course of trichinellosis as well as upon the growth of tumours in the infected host is discussed.

Suppression of the activity of natural killer-like cells by peritoneal macrophages obtained from Lewis lung tumor-bearing, Propionibacterium granulosum KP-45-treated, or normal, untreated mice.

Peritoneal adherent cells (PAC) obtained from Propionibacterium granulosum KP-45-treated or Lewis lung carcinoma-bearing BDF1 mice suppressed in vitro the NK-like cytotoxic activity of murine splenocytes against YAC-1 tumor target cells. Maximum inhibition occurred when suppressor and effector cells were preincubated together for 18 h, but the effect was demonstrable also when the two groups of cells were mixed only at the onset of the 4-h cytotoxic assay (i.e. without previous contact). Inhibitory cells appeared to be mostly macrophages, as judged by adherence to plastic and morphologic features, and as little as 5 to 20% of PAC, relative to the total number of co-incubated cells, were required for the clear demonstration of the effect. In addition to activated also normal, resident PAC obtained from untreated animals inhibited the NK cell-mediated cytotoxicity, but the effect was significantly pronounced only when 20% of suppressor cells were incubated overnight with effector splenocytes. The results favor the hypothesis that both functionally activated as well as resting macrophages operate as important regulators of the activity of NK cells in vivo.

Augmentation of natural cell activity in tumor-bearing and normal mice by MVE-2.

Mice bearing advanced Lewis lung carcinoma were found to have significantly decreased natural killer (NK) cell activity in spleen and blood. The same pattern of lowered spontaneous NK cell activity was observed in nude mice with advanced human colon carcinoma LS 174 and in C3H mammary tumor virus-positive mice that spontaneously developed mammary adenocarcinomas. Maleic anhydride divinyl ether (MVE-2) usually augments NK cell activity in normal mice. We found that the lower level of spontaneous NK cell activity in tumor-bearing mice could be boosted by a single injection of MVE-2; however, this response was much weaker than that observed in age-matched normal mice. Multiple treatments with MVE-2 which are known to induce hyporesponsiveness to further augmentation of NK cell activity in spleen and blood of normal mice, also produced NK cell hyporesponsiveness in the spleen, bone marrow, and blood of tumor-bearing mice.

Cell regulatory and immunorestorative activity of picibanil (OK432).

Picibanil (OK432), a pharmaceutical preparation of a low virulent Su strain of Streptococcus pyogenes, possesses cell regulatory activity particularly in its ability to augment natural killer (NK) cell activity and to activate macrophages to exert a tumoricidal effect both in vitro and in vivo. It is effective in retarding and/or inhibiting the growth of three different tumors: MBL-2 lymphoma, M109 alveolar adenocarcinoma, and B16 melanoma. The antitumor effect is mediated through regulation of NK cells and macrophages, possibly by its ability to stimulate the production and secretion of interferon and interleukin 1 and 2. It is a very effective adjuvant for tumor cell vaccines that elicit cytotoxic T-cell responses. Following cytoreductive chemotherapy (Cytoxan) Picibanil treatment leads to an earlier reconstitution of both bone marrow cellularity and differentiation to granulocyte-macrophage colonies.

Immune response by biological response modifiers.

Several biological response modifiers (BRMs) were demonstrated to increase myelopoiesis and effector cell responses (M phi and natural killer cell activity) in vivo. The increased myelopoiesis was reflected by an increase in bone marrow cellularity and granulocyte-M phi colony-forming cells (GM-CFU-C). The increase in myelopoiesis appeared to be related to a concomitant increase in colony-stimulated factor (CSF) production and secretion by M phi and bone marrow cells. CSF induction by BRMs increased myelopoiesis and counteracted the myelosuppressive and immunosuppressive effects of cyclophosphamide. CSF induced in vivo by BRMs attained high titers and were maintained over a longer period than exogenously injected CSF, which was rapidly cleared from serum.

Protein deficiency reduces natural antitumor immunity.

Clinical data have shown that neoplastic diseases and/or related therapies frequently result in protein depletion of tumor-bearing patients. Depressions of acquired and specific immunity caused by protein depletion are well known. In an experimental model protein depletion was induced by lack of nutritional protein in otherwise isocaloric conditions in BALB/c and C57BL/6 mice over various time periods (max. 35 days). The results show that natural immune effector cells, natural killer cells, and monocyte/macrophages also during treatment with biological response modifiers (BRM) are depressed in their cytotoxic potentials in vitro and in vivo. Substantial and critical reductions of bone marrow cellularity (bone marrow nucleated cells) were also observed. In contrast, preliminary results show that if, following protein depletion, mice were treated parenterally with amino acids (Neo-aminomel, Boehringer-Ma. Co., FRG) complete restoration of immune parameters takes place. Adequate protein status is shown to be a crucial factor for natural immunity and therapy with BRM.

Comparative studies on biological activity of /+/R and /-/S enantiomers of cyclophosphamide and ifosfamide. I. Antitumour effect of cyclophosphamide and ifosfamide enantiomers.

The differences in antitumour effect of /+/R and /-/S enantiomers of both cyclophosphamide and ifosfamide were detected. In the case of ifosfamide in all five tested tumour models (Leukemia L1210, P388, Lewis lung carcinoma, 16/C mammary adenocarcinoma and B16 melanoma) the /-/S form exerted not only higher antitumour effects than /+/R form, but revealed higher therapeutic indices as well. The same appeared to be true for /-/S enantiomer of cyclophosphamide in three models of solid tumours. In L1210 and P-388 ascitic leukemia models /+/R and /-/S cyclophosphamide exerted the same antitumour effect.

Antitumor activity of optical isomers of cyclophosphamide, ifosfamide and trofosfamide as compared to clinically used racemates.

The relationship between enantiomeric homogeneity of three oxazaphosphorine drugs: cyclophosphamide, ifosfamide and trofosfamide and their antitumor activity was evaluated by standard screening tests against four in vivo transplantable tumor models: L 1210 and P 388 lymphoid leukemias, Lewis lung carcinoma and 16/C line of mouse mammary adenocarcinoma. It was shown that the stereodifferentiation of anti-tumor effect of enantiomers was not outstanding although quite consistently in favour of levorotatory forms. The only exception was seen for cyclophosphamide enantiomers tested against leukemias where R/+/form was more effective than S/-/or racemate.

Pharmacokinetic and therapeutic activity of polyinosinic-polycytidylic acid stabilized with poly-L-lysine in carboxymethylcellulose [poly(I,C)-LC].

Polyinosinic-polycytidylic acid stabilized with poly-L-lysine in carboxymethylcellulose [poly(I,C)-LC] significantly augmented natural killer (NK) cell activity in several tissues. Macrophage (M phi) tumoricidal activity was also markedly increased. Both effector cells were active for 9 days. Poly(I,C)-LC also increased effector cell response in vitro. Injections of poly(I,C)-LC resulted in elevated effector cell responses in four of five routes tested. Treatment with poly(I,C)-LC led to an earlier reconstitution of bone marrow cells, NK cell activity, and M phi effector cell activity in mice pretreated with cyclophosphamide (Cytoxan). Combined treatment of MBL-2 tumor cells with cytoreductive chemotherapy and poly(I,C)-LC resulted in an enhanced therapeutic response.

LL2 cell line derived from transplantable murine Lewis lung carcinoma--maintenance in vitro and growth characteristics.

LL2 is a new in vitro cell line derived from Lewis lung carcinoma passaged routinely in C57BL mice. It has been in continuous culture for more than 1 year and has survived 72 subcultivations. The cells grow in semi-suspension culture being loosely connected with a culture vessel's surface. The cell line is hypotetraploid, with modal chromosome number 72. Tumorigenicity and metastasibility of the cells are retained but lowered as compared with original in vivo tumor line. The LL2 cell line is being used in studies on phenotypic characteristics (markers) related to its pattern of growth.

Stimulatory effect of Propionibacterium granulosum KP-45, glucan and pyran copolymer on the activity of natural killer (NK) cells in murine lungs.

Propionibacterium granulosum KP-45, glucan and pyran copolymer stimulated the elimination of 75Selenomethionine-labelled 3LL tumor cells from murine lungs, as measured 4 hr after intravenous injection of these cells into 16- to 25-week-old B6DF1 mice. This effect was most pronounced 4 to 6 days following intravenous administration of the above biological response modifiers and disappeared 6 to 8 days later. Intraperitoneal injection of all three agents produced only insignificant stimulation results. Spontaneous clearance of 3LL cells from lungs of 8-week-old B6DF1 mice was significantly more effective than in animals over 16 weeks old. Cyclophosphamide suppressed the elimination of tumor cells from lungs in both young and older mice and neutralized the stimulatory effect of P. granulosum KP-45 and glucan. The results suggest that the effector cells responsible for the clearance of radiolabelled 3LL cells from lungs of B6DF1 mice are at least similar to natural killer (NK) lymphocytes.