Nitric oxide sensitizes tumor cells to dendritic cell-mediated apoptosis, uptake, and cross-presentation

Dendritic cells are professional antigen-presenting cells associated with efficient antigen processing and presentation to T cells. However, recent evidence also suggests that dendritic cells may mediate direct tumoricidal functions. In this study, we investigated the mechanism by which murine dendritic cells mediate the apoptotic death of murine lymphoma cell lines, and whether dendritic cell effector function could be enhanced by preconditioning tumor cells with the protein phosphatase inhibitor nitric oxide (NO) by altering the balance of proapoptotic/antiapoptotic proteins in the treated cells. We observed that NO donor compound sensitized lymphomas to dendritic cell-mediated cytotoxicity in vitro. Both immature and spontaneously matured bone marrow-derived dendritic cells (SM-DC) were capable of inducing tumor cell apoptosis, with SM-DCs serving as comparatively better killers. Fas ligand (FasL)-Fas engagement proved important in this activity because elevated expression of membrane-bound FasL was detected on SM-DCs, and dendritic cells derived from FasL-deficient mice were less capable of killing NO-sensitized tumor cells than wild-type dendritic cells. As FasL-deficient dendritic cells were still capable of mediating a residual degree of tumor killing, this suggests that FasL-independent mechanisms of apoptosis are also involved in dendritic cell-mediated tumor killing. Because NO-treated tumor cells displayed a preferential loss of survivin protein expression via a proteasome-dependent pathway, enhanced tumor sensitivity to dendritic cell-mediated killing may be associated with the accelerated turnover of this critical antiapoptotic gene product. Importantly, NO-treated tumor cells were also engulfed more readily than control tumor cells and this resulted in enhanced cross-presentation of tumor-associated antigens to specific T cells in vitro.     

MHC class I and class II presentation of tumor antigen in retrovirally and adenovirally transduced dendritic cells

The unique antigen-presenting capabilities of dendritic cells (DCs) make them an attractive means with which to initiate an antitumor immune response. Using DCs transduced with tumor antigens for immunotherapy has several theoretical advantages over peptide-pulsed DCs including the possibility that transduced DCs are capable of presenting epitopes on both class I and class II MHC molecules. To test this theory, we inserted the human tumor antigen gp100 into mouse DCs transgenic for HLA-DRbeta1*0401 using either adenoviral vector or a VSV-G pseudotyped retroviral vector. DCs transduced with tumor antigen were able to be recognized by both a murine CD8(+) T-cell clone and a murine CD4(+) T-cell line in a cytokine release assay, thereby demonstrating presentation of both MHC class I and class II gp100 epitopes. This study describes the simultaneous presentation of a tumor-associated antigen to both CD4(+) and CD8(+) T cells and lends support to the use of gene-modified DCs as a means to initiate both CD4(+) and CD8(+) antitumor responses.     

Anti-tumor effects of dendritic and tumor cell fusions are not dependent on expression of MHC class I and II by dendritic cells

Vaccination of fusion cells (FCs) made from dendritic and tumor cells elicits anti-tumor effects. We investigated whether major histocompatibility complex (MHC) class I and II play an important role in the induction of anti-tumor immunity by FCs. Immunization with fusion cells composed of syngeneic, allogeneic, or MHC I(-/-)II(-/-) DCs and B16 cells inhibited tumor growth. Elispot assay showed a higher population of interferon-gamma secreting T lymphocytes in mice immunized with fusion cells. These data suggest that anti-tumor effects of DCs and tumor cell fusions are not dependent on the expression of MHC class I and II on DCs.     

Development of monoclonal antibodies to human tumor markers

The development of hybridoma technology has provided the possibility of preparing unlimited amounts of highly specific and homogeneous monoclonal antibodies thus eliminating many of the disadvantages with conventional antisera and rekindling interest in the application of immunodiagnostic and immunotherapeutic approaches to human cancer. Several reports, including our own of monoclonal antibodies to tumor-associated antigens expressed by solid tumors including gastric, colorectal, lung and other various carcinomas, were reviewed from the viewpoint of tumor markers.     

Differential uptake and cross-presentation of human papillomavirus virus-like particles by dendritic cells and Langerhans cells

A causal link between cervical cancer and high-risk human papillomaviruses has been established. The virus infects basal cells of the mucosa, where Langerhans cells are the resident antigen-presenting cells. Langerhans cells and dendritic cells, which are targeted by vaccination, internalize similar amounts of human papillomavirus virus-like particles (VLPs), albeit through different uptake mechanisms. VLP uptake by dendritic cells results in activation and cross-presentation of MHC class I-restricted peptides with costimulation to T cells. Conversely, VLP uptake by Langerhans cells leads to cross-presentation in the absence of costimulation. Efficient VLP cross-presentation by Langerhans cells with costimulation can be achieved by addition of CD40 ligand. The lack of a protective immune response after viral contact with Langerhans cells may explain why some women fail to mount an immune response against the virus or why the immune responses that do develop may allow the virus to persist. Because VLPs are currently being tested as a vaccine against cervical cancer, our data are very topical and have implications for optimal vaccination strategies against this disease.     

Reactivity of tumor cells in malignant effusions with a panel of monoclonal and polyclonal antibodies

A panel of 13 mouse monoclonal antibodies (mAb) and 1 rabbit polyclonal antibody was tested for reactivity with tumor cells in 26 effusions obtained from patients with carcinoma of ovary, breast, or mesothelioma, using an immunoperoxidase staining reaction. Specific staining of tumor cells but not reactive mesothelial cells was demonstrated with some of the mAb in the panel. In 4 of 26 effusions no evidence of malignancy was obtained after routine cytological staining, but this was reversed on the basis of immunoperoxidase staining of tumor cells with the mAb in the panel. Serial effusions were evaluated in 4 patients during the course of chemotherapy, allowing an assessment of effect of therapy on the antigenic characteristics of the tumor cells. In another 4 patients, the results of immunoperoxidase staining of effusions were compared with those obtained by applying the same antibody panel to solid tumor nodules. There was a tendency to develop changes in the pattern of reactivity during therapy, and the pattern of reactivity was more restricted in tumor nodules than in effusions. One of the mAb in our panel (2G3) was consistently shown to produce strong staining of a high proportion of tumor cells in effusions and tumor nodules from patients with ovarian or breast cancer and may be of value in immunocytochemical diagnosis and therapy of epithelial malignancies.     

Mouse monoclonal antibodies direct phagocytosis of tumor cells by human monocytes

Human peripheral blood monocytes were found to be capable of phagocytizing human B-non Hodgkin's lymphoma (NHL) cells coated with mouse monoclonal antibodies (MoAbs). The MoAbs used recognized idiotypic determinants on the surface immunoglobulin (Ig) of the tumor cells. MoAbs of IgG1 and IgG2a subclass were equally effective in inducing phagocytosis. Preincubation of the monocytes with heat aggregated Ig from human or mouse serum inhibited phagocytosis to the same degree, suggesting involvement of an Fc receptor on the monocytes that binds both human and mouse antibodies.     

Mouse monoclonal antibodies for experimental immunotherapy promotes killing of tumor cells

Monoclonal antibodies (MAbs) produced against a human colon adenocarcinoma cell line, Colo-205, were tested in antibody-dependent macrophage-mediated cytotoxicity (ADMMC) assays. The antibodies C163, C215, C245 (IgG2a isotype); C151, C239, C241, C242 (IgG1); C152 (IgG2b); and C50 (IgM) were evaluated for their ability to promote killing of Colo-205 cells by thioglycollate-elicited peritoneal mouse macrophages. The concentrations of antibodies tested in ADMMC assays ranged from 1.0 ng/ml to 100 micrograms/ml, and the concentration at which 50% of tumor cells were lysed was used to characterize each antibody. Antibodies of the IgG2a isotype promoted the strongest macrophage-mediated tumor cell lysis effects in vitro. MAbs C215, C163, and C245 were also tested in nude mice which had been inoculated with Colo-205 cells. Tumor suppression was observed in mice injected with MAbs, supporting our ADMMC findings in vitro. Animals treated with MAbs had fewer and smaller tumors, and longer periods of latency between the inoculation of tumor cells and development of tumors, compared to mice sham-treated with PBS. However, in a study of established tumors, C215 antibody did not suppress tumor growth. Serum collected from MAb-treated mice promoted lysis of Colo-205 cells in ADMMC assays while serum from sham-treated mice did not.     

Immunotherapy with dendritic cells and tumor major histocompatibility complex class I-derived peptides requires a high density of antigen on tumor cells

Immunization with dendritic cells and unfractionated MHC class I-binding peptides derived from autologous tumor cells has been shown to induce effective antitumor immunity. However, the importance of the relative abundance of tumor peptides on the surface of tumor cells is not known. We have addressed this question using peptides isolated from three tumor cell lines transfected with a minigene encoding amino acids 33-41 of the lymphocytic choriomeningitis virus glycoprotein (LCMV(33-41)). The three cell lines expressed different levels of MHC class I molecules and had different abilities to stimulate proliferation of LCMV(33-41)-specific T cells in vitro. We found that antitumor immune responses were best elicited by immunizing mice with dendritic cells and synthetic LCMV(33-41) peptide. Peptide preparations from a given tumor cell line conferred protection against challenge with the same tumor cell line. However, protective immunity to a different tumor could be induced only if the cell line used for peptide preparation presented a high relative proportion of LCMV(33-41) in association with MHC class I. Our results suggest that multiple peptide epitopes are required for the induction of an effective antitumor immune response using MHC class I-binding peptides from tumor cells. Also, the ability to induce an antitumor immune response appears to correlate with the proportion, rather than the absolute amount, of tumor-specific peptide in the mixture used for immunization.     

Generation of in vitro B-CLL specific HLA class I restricted CTL responses using autologous dendritic cells pulsed with necrotic tumor lysate

New approaches in the treatment of chronic B lymphocytic leukemia (B-CLL) have led to improved clinical response rates. In this setting there is a need to evaluate novel therapeutic approaches that aim to eradicate minimal residual B-CLL cells following an initial favorable response. The use of tumor lysate-pulsed dendritic cells (DC) represents a potentially important development in the field of cancer vaccination. B-CLL is ideally suited for DC-based vaccination since tumor cells are readily available (peripheral blood) and both known (tumor idiotype) and unknown antigens can be exploited to stimulate immune responses. In the current study we have evaluated the ability to stimulate in vitro autologous immune reactivity against target B-CLL cells using autologous DCs pulsed with B-CLL tumor lysate. Enhanced specific T cell IFN-gamma expression was detected in 9 of 14 patients evaluated. These responses were specific with increased levels of IFN-gamma mRNA measurable in T-cells stimulated with NC-DCs and not unpulsed DCs or DCs pulsed with normal B cell lysate. CTLs demonstrating increased levels of IFN-gamma mRNA also lysed autologous B-CLL targets cells in an MHC class 1-restricted manner by (51)chromium release assay. Priming target leukemic cells with CD40 ligand and IL-4 enhanced CTL killing. The effector CTL displayed negligible toxicity against NK susceptible target cells K-562 and spared CD19(+)CD5(-) normal B cells in cytotoxicity assays. The specificity of the CTL response was confirmed by blocking HLA class I molecules and cold target inhibition assays.     

Enhancement of lung tumor colony formation by treatment of mice with monoclonal antibodies to pulmonary capillary endothelial cells

Two rat monoclonal antibodies, 34A and 201B, have been isolated and shown to bind preferentially to capillary endothelial cells in the lung. Administration of these antibodies to mice increases the number of lung colonies derived from i.v. injection of tumor cells. The antibodies increase lung colonization in C57BL/6 mice following i.v. injection of B16-F10 melanoma cells and in BALB/c mice following injection of line 1 lung carcinoma cells. Neither 34A nor 201B monoclonal antibody binds to B16 melanoma or line 1 carcinoma and so must exert its effect by interaction with endothelial cells. Antibodies injected i.v., s.c., or i.p. are active from 1 h to 1 wk if injected before cell injection. The effect is optimal when 0.1 ml of ascites fluid containing 120 micrograms of antigen binding capacity of both MoAbs 34A and 201B is injected. Significant damage to endothelial cells could not be documented by histopathological examination at the light microscope level or by protein leakage into the air space as measured by lung lavage. However, electron micrographs taken 3 h after monoclonal antibody injection show minor damage to endothelial cell membranes throughout the lung with some areas of mild edema. The increased colonization may be mediated by this subtle damage to endothelial cells, or antibody interactions with endothelial cells may trigger secondary reactions such as altered expression of growth factors.     

Improved tumor localization and radioimaging with chemically modified monoclonal antibodies

A method for the chemical modification of monoclonal antibodies using the heterobifunctional crosslinker succinimidyl 3-(2-pyridyldithio)propionate (SPDP), has been developed which dramatically alters the physiochemical properties of antibody reagents. For these studies, three murine monoclonal antibodies, B72.3, Lym-1, and TNT-1 were used to demonstrate the effects of chemical modification on clearance and biodistribution in tumor-bearing nude mice. In vitro, all three antibodies, modified to the same degree with SPDP, showed equal immunoreactivities and lower non-specific binding. Modified antibodies also were found to have lower isoelectric points compared to unmodified controls. In vivo, modified antibodies unexpectedly were found to have 2-6 times faster clearance in tumor-bearing nude mice similar to rates obtained with their F(ab')2 fragments. Paired-label in vivo biodistribution and external imaging experiments with intact antibodies and F(ab')2 fragments demonstrated that chemically modified antibodies gave 1.5-3 fold higher tumor uptake and retained less activity in normal organs thus markedly increasing the tumor to normal organ ratios. Because of these results, chemically modified antibodies produced clearer images at earlier time points by external scintigraphy. As "stealth" molecules, chemically modified monoclonal antibodies appear to have significantly improved uptake in tumors and faster clearance times compared to native molecules. These results suggest that alteration of the physicochemical properties of monoclonal antibodies may generate improved reagents for in vivo use.     

Diagnostic and therapeutic use of monoclonal antibodies to human tumor antigens

Monoclonal antibodies have been obtained to various differentiation antigens that are more strongly expressed on tumor cells than on normal cells from the adult host, some of these antigens are relatively specific for tumors of a given type, e.g. melanoma. Monoclonal anti-tumor antibodies offer strong promise for clinical use, initially for tumor diagnosis and, ultimately, also for therapy.     

Immunostaining of colorectal cancer with monoclonal anti-CEA antibodies compared to serum and tumor CEA content

Carcinoembryonic antigen was measured in serum and in extracts from 37 colorectal tumors and we found a poor correlation between circulating and tumor CEA. Monoclonal antiCEA antibodies were used in indirect immunoperoxidase staining of the corresponding formalin fixed tissue sections. We found that serum CEA measurement had a sensitivity of only 41.9% as compared to 90.3% for the immunohistochemical staining. The positive and negative predictive values for immunostaining were respectively 96.6% and 72.7%. Immunohistochemical staining of tissue sections with monoclonal anti CEA coupled to other biochemical or immunological assays could be a useful adjunct for the diagnosis of premalignant or slightly modified tissues.     

Identification of tumor vascular antigens by monoclonal antibodies prepared from rat-tumor-derived endothelial cells

We have reported the isolation and specific in vitro properties of tumor-derived endothelial cells (TEC) from rat KMT-17 fibrosarcomas transplanted into rats. To develop antibody-based tumor vascular targeting therapy for solid tumors, we have generated monoclonal antibodies (MAbs) using passive immunization of outside-out membrane vesicles of rat epididymal-fat-pad-derived capillary endothelial cells (FCEC) followed by active immunization of those of rat TEC. The MAbs produced were screened against TEC and FCEC. Of all cultured hybridomas, 75 (3.3%) of the secreted MAbs preferentially recognized TEC. We selected a total of 7 MAbs which detected antigens highly abundant in TEC, although 5 of the 7 MAbs were weakly positive for FCEC in cell-ELISA and FACS analyses. The antigens recognized by these MAbs, with the exception of MAb TES-7, were present on endothelial cells of tumor blood vessels in KMT-17 fibrosarcoma tissues, as shown by immunohistochemical analysis. Antigens of 40- and 80-kDa were recognized by MAbs TES-1, 7, 17, 21 and 26 and by MAbs TES-23 and 27 respectively. Although the function of these antigens, which are preferentially expressed on rat tumor-derived endothelial cells, is still unknown, we believe that future studies of such antigens will help elucidate the role of endothelial cells in tumor vasculature. Our results indicate that MAbs may provide a novel tool for the development of antibody-based therapy targeting tumor vasculature. Int. J. Cancer 77:561–566, 1998. © 1998 Wiley-Liss, Inc.     

CYTOTOXICITY OF INDIRECT IMMUNOTOXIN MEDIATED BY ANTI-GASTRIC CANCER MONOCLONAL ANTIBODIES ON TUMOR CELLS

In the present study, an indirect assay was employed to investigate 5 anti-gastric cancer monoclonal antibodies for their cytotoxic potential as ricin A chain-containing immunotoxins. The tumor cell, were treated with dilutions of tested antibody followed by ricin A chain coupled to goat anti-mouse immunoglobulin. The cytotoxic effect was determined with tetrazolium colorimetric assay. The results showed that among the 5 antibodies chosen, MGb2 and MG7 could be well used for preparation of effective A chain immunotoxins.     

MHC class I genes controlling the metastatic phenotype of tumor cells.

Metastatic clones of murine tumors that manifest impaired expression of class I MHC antigens do not induce an antitumor CTL activity. Transfection of H-2Kb genes into D122 carcinoma and B16 melanoma clones converted these cells to low metastatic immunogenic clones that can be used to protect in vivo against metastases of parental clones. Amplification of the protective effect can be achieved by combination of syngeneic and allogeneic MHC class I genes. Studying the mechanisms involved in MHC class I suppression in tumor cells, we found that changes in H-2 promoter activity were the cause of low expression. Proteins that might be involved were demonstrated by migration retardation methods. The involvement of the fos-jun complex in regulation of MHC expression is discussed.     

Improving efficacy of interleukin-12-transfected dendritic cells injected into murine colon cancer with anti-CD137 monoclonal antibodies and alloantigens

Intralesional administration of cultured dendritic cells (DCs) engineered to produce IL-12 by in vitro infection with recombinant adenovirus frequently displays eradicating efficacy against established subcutaneous tumors derived from the CT26 murine colon carcinoma cell line. The elicited response is mainly mediated by cytolytic T lymphocytes. In order to search for strategies that would enhance the efficacy of the therapeutic procedure against less immunogenic tumors, we moved onto malignancies derived from the inoculation of MC38 colon cancer cells that are less prone to undergo complete regression upon a single intratumoral injection of IL-12-secreting DCs. In this model, we found that repeated injections of such DCs, as opposed to a single injection, achieved better efficacy against both the injected and a distantly implanted tumor; that the use of semiallogeneic DCs that are mismatched in one MHC haplotype with the tumor host showed slightly better efficacy; and that the combination of this treatment with systemic injections of immunostimulatory anti-CD137 (4-1BB) monoclonal antibody achieved potent combined effects that correlated with the antitumor immune response measured in IFN-gamma ELISPOT assays. The elicited systemic immune response eradicates concomitant untreated lesions in most cases. Curative efficacy was also found against some tumors established for 2 weeks when these strategies were used in combination. These are preclinical pieces of evidence to be considered in order to enhance the therapeutic benefit of a strategy that is currently being tested in clinical trials. Supplementary Material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.     

Retardation of metastatic tumor growth after immunization with metastasis-specific monoclonal antibodies

The influence of 4 murine monoclonal antibodies (MAbs) directed against surface determinants of a metastasizing rat adenocarcinoma (BSp73ASML) on metastatic spread was evaluated and compared to their in vivo binding as well as to the induction of a humoral anti-MAb response, especially with respect to the development of anti-idiotypic (ID) antibodies of the internal image type. In a protocol of explicit immunization, all 4 MAbs transiently inhibited metastatic growth. Survival was prolonged only with one MAb (4.4ASML). With another MAb (1.1ASML), directed against a new variant form of CD44, metastatic growth was accelerated after transient retardation. Retardation of metastatic growth correlated with the humoral anti-MAb response. This accounted for the isotype- as well as for the idiotype-specific response. An exception was noted after immunization with MAb 1.1ASML. Rats with high levels of anti-1.1ASML antibodies, which inhibited binding to the tumor cells (internal image-type antibodies) showed accelerated metastatic spread. Data are interpreted to mean that MAb-induced inhibition of metastatic spread may be based on 2 independent mechanisms: blockade of metastasis-associated epitopes (i.e., with MAb 1.1ASML) and induction of an anti-mouse Ig response. In the latter case it was irrelevant whether the response was isotype- or idiotype-specific.     

Tumor reactive cis-aconitylated monoclonal antibodies coupled to daunorubicin through a peptide spacer are unable to kill tumor cells

Antibody-drug conjugates containing a linkage susceptible to lysosomal hydrolases were constructed by coupling peptide-daunorubicin (DNR) derivatives to MAb. Using a modification in the method of Trouet et al, peptide derivatives of DNR containing the sequences Ala-Leu and Ala-Leu-Ala-Leu linked to drug via their carboxy terminus were prepared. Cleavage of these derivatives by lysosomal enzymes resulting in the release of free DNR was demonstrated. Human antitumor MAb were derivatized with either succinic anhydride or cis-aconitic anhydride to introduce spacer arms for coupling. Binding studies showed that MAb with a decrease of 12-20 amino groups retained greater than 70% of their immunoreactivity, a level deemed acceptable for constructing conjugates. Derivatized and native MAb were conjugated to peptide-DNR via a carbodiimide mediated reaction. None of the conjugates displayed cytotoxicity toward target tumor cell lines in vitro.