Synergy between an antiangiogenic integrin alphav antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases

The suppression and eradication of primary tumors and distant metastases is a major goal of alternative treatment strategies for cancer, such as inhibition of angiogenesis and targeted immunotherapy. We report here a synergy between two novel monotherapies directed against vascular and tumor compartments, respectively, a tumor vasculature-specific antiangiogenic integrin alphav antagonist and tumor-specific antibody-interleukin 2 (IL-2) fusion proteins. Simultaneous and sequential combination of these monotherapies effectively eradicated spontaneous liver metastases in a poorly immunogenic syngeneic model of neuroblastoma. This was in contrast to controls subjected to monotherapies with either an antiangiogenic integrin alphav antagonist or antibody-IL-2 fusion proteins, which were only partially effective at the dose levels applied. Furthermore, simultaneous treatments with the integrin alphav antagonist and tumor-specific antibody-IL-2 fusion proteins induced dramatic primary tumor regressions in three syngeneic murine tumor models, i.e., melanoma, colon carcinoma, and neuroblastoma. However, each agent used as monotherapy induced only a delay in tumor growth. A mechanism for this synergism was suggested because the antitumor response was accompanied by a simultaneous 50% reduction in tumor vessel density and a 5-fold increase in inflammatory cells in the tumor microenvironment. Subsequently, tumor necrosis was demonstrated only in animals receiving the combination therapy, but not when each agent was applied as monotherapy. The results suggest that these synergistic treatment modalities may provide a novel and effective tool for future therapies of metastatic cancer.     

Assessment of oxacillin salt agar for detection of MRSA identified by presence of the mecA gene

Progressive growth of immunogenic murine tumors elicits a tumor-specific but functionally deficient T-cell immune response in the draining lymph nodes. These T cells, referred to as "pre-effector" cells could be induced in vitro to differentiate into mature immune effector cells, capable of mediating the regression of established metastases. Initially, tumor cells were used to stimulate the in vitro maturation of pre-effector cells. Alternatively, we found that pre-effector cells could be activated by sequential stimulation with anti-CD3 and interleukin-2 in the absence of tumor cells. In adoptive immunotherapy, these activated cells mediated therapeutic effects that were exquisitely specific to the tumor that triggered the pre-effector cell response in vivo. Since the anti-CD3 interaction with T cells is polyclonal, the activated lymph node cell population must also contain a significant number of T cells that do not have tumor specificity. In an attempt to selectively activate tumor-sensitized pre-effector cells, we recently utilized superantigenic bacterial toxins as T-cell stimuli for effector cell generation. Superantigens combine with major histocompatibility class II molecules to form the ligands that stimulate T cells bearing distinct T-cell receptor V beta elements. Lymph node cells draining the MCA 205 sarcoma stimulated with staphylococcal enterotoxins A (SEA), B (SEB), or C2 (SEC2) resulted in selective expansions of V beta 3 and 11, V beta 3 and 8, or V beta 8.2 T cells, respectively. Adoptive immunotherapy experiments revealed that SEB- and SEC2-, but not SEA- stimulated cells, mediated tumor-specific eradication of pulmonary metastases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-lived and transferable tumor immunity in mice after targeted interleukin-2 therapy

A major goal of tumor immunotherapy is the induction of tumor-specific T cell responses that are effective in eradicating disseminated tumor, as well as mounting a persistent tumor-protective immunity. We demonstrate here that a genetically engineered fusion protein consisting of human/mouse chimeric anti-ganglioside GD2 antibody and human interleukin-2 is able to induce eradication of established B78-D14 melanoma metastases in immunocompetent syngeneic C57BL/6J mice. This therapeutic effect is mediated by host immune cells, particularly CD8+ T cells and is associated with the induction of a long-lived immunity preventing tumor growth in the majority of animals when challenged up to four months later with B78-D14 cells. This effect was tumor-specific, since no cross-protection against syngeneic, ganglioside GD2+ EL-4 thymoma cells was observed. Furthermore, this tumor-specific protection can be transmitted horizontally to naive, syngeneic SCID mice by passive transfer of CD8+ T lymphocytes derived from immune animals. These results suggest that antibody-targeted delivery of cytokines provides a means to elicit effective immune responses against established tumors in the immunotherapy of neoplastic disease.     

Gene therapy with a single chain interleukin 12 fusion protein induces T cell-dependent protective immunity in a syngeneic model of murine neuroblastoma

A major goal of tumor immunotherapy is the effective eradication of established metastases associated with the induction of a T cell-mediated protective immunity. We achieved this in a poorly immunogenic murine neuroblastoma model by gene therapy with a single chain interleukin 12 (scIL-12) fusion protein that assures equal expression of its p35 and p40 subunits. Thus, NXS2 hybrid neuroblastoma cells (C1300 x dorsal root ganglion cells), which form experimental bone marrow and liver metastases in syngeneic A/J mice, were transduced with a gene encoding murine interleukin 12, monomerized by introduction of a protein linker between the p35 and p40 protein chains of this heterodimeric cytokine. We demonstrate for the first time that subcutaneous vaccination with these transduced cells induces a protective immunity, as indicated by the complete absence of liver and bone marrow metastasis after challenge with NXS2 wild-type tumor cells. Furthermore, vaccination of animals with established liver and bone marrow metastases completely eradicated liver metastases and suppressed bone marrow metastases. The local and systemic immune response against scIL-12-transduced NXS2 cells is largely dependent on CD8(+) T cells. This was demonstrated in vivo by depletion of immunocompetent A/J mice with monoclonal anti-CD4 and anti-CD8 antibodies and in vitro by specific major histocompatibility complex, class I-restricted CD8(+) T cell-mediated killing of NXS2 and their parental C1300 neuroblastoma cells. In conclusion, we demonstrate successful anti-tumor immunotherapy with an scIL-12 fusion protein that could facilitate clinical application of interleukin 12 gene therapy.     

Combination therapy with suicide and cytokine genes for hepatic metastases of lung cancer

Metastases of lung cancer are a major cause of treatment failure. To evaluate the therapeutic efficacy of gene therapy in metastatic lung cancer, we used adenoviral (ADV) mediated transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene and the cytokine gene interleukin-2 (IL-2) to treat a murine model of metastatic lung cancer in the liver. Hepatic metastases were established by intrahepatic implantation of LL2 cells in syngeneic recipient mice. One week after tumor implantation, various replication defective ADV vectors were injected intratumorally. Treatment with a vector expressing the HSV-tk followed by ganciclovir administration with ADV.tk resulted in significant regression of tumor (p<0.01) as well as prolongation of survival (p<0.001). While a vector expressing mouse IL-2 ADV.IL-2 alone was ineffective, combination therapy with HSV-tk resulted in further tumor regression and improvement of animal survival (p<0.05). These results demonstrate that suicide and cytokine genes can be utilized in combination to treat metastatic lung cancer in vivo.     

Natural killer cell-mediated eradication of neuroblastoma metastases to bone marrow by targeted interleukin-2 therapy

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody-IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell-dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell-deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell-stimulating agents, such as poly I:C or recombinant mouse interferon-gamma. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.     

Systemic targeting of liposome-encapsulated immunomodulators to macrophages for treatment of cancer metastasis

The therapy of cancer is, in reality, the design of therapeutic strategies for therapy of metastatic disease. Metastases consist of unique subpopulations of tumor cells that are derived from the primary tumor, colonize distant target organs, and are able to subvert host immune responses, establish necessary angiogenesis, and obtain a sufficient nutrient supply while evolving to become autonomous from homeostatic mechanisms that function within normal, differentiated tissues. Attempts at eradication of metastases by conventional therapies have generally been unsuccessful due to genetic instability and heterogeneity of metastatic tumors; these properties lead to the emergence of tumor cells that are resistant to most conventional treatments. It may be possible to circumvent this heterogeneity by the activation of tissue macrophages to the tumoricidal state. Activated macrophages are able to kill tumor cells while sparing normal tissues, and efficient activation can be achieved by encapsulation of synthetic muramyl tripeptide analogues into multilamellar vesicles composed of phospholipids. Systemic administration of these liposome-encapsulated compounds leads to tumoricidal activation of alveolar and peritoneal macrophages and eradication of established tumor metastasis in numerous animal tumor models, and this form of therapy is enhanced by combination with parenteral administration of cytokines. Phase III clinical trials of recurrent osteosarcoma are currently in progress. Modulation of the tumor microenvironment by activated macrophages may prove to be an additional modality in treatment strategies that combine the use of biological response modifiers with conventional therapies.     

A RANTES-antibody fusion protein retains antigen specificity and chemokine function

The successful eradication of cancer cells in the setting of minimal residual disease may require targeting of metastatic tumor deposits that evade the immune system. We combined the targeting flexibility and specificity of mAbs with the immune effector function of the chemokine RANTES to target established tumor deposits. We describe the construction of an Ab fusion molecule with variable domains directed against the tumor-associated Ag HER2/neu, linked to sequences encoding the chemokine RANTES (RANTES.her2.IgG3). RANTES is a potent chemoattractant of T cells, NK cells, monocytes, and dendritic cells, and expression of RANTES has been shown to enhance immune responses against tumors in murine models. RANTES.her2.IgG3 fusion protein bound specifically to HER2/neu Ag expressed on EL4 cells and on SKBR3 breast cancer cells as assayed by flow cytometry. RANTES.her2.IgG3 could elicit actin polymerization of THP-1 cells and transendothelial migration of primary T lymphocytes. RANTES.her2.IgG3 prebound to SKBR3 cells also facilitated migration of T cells. RANTES.her2.IgG3 bound specifically to the CCR5 chemokine receptor, as demonstrated by flow cytometry, and inhibited HIV-1 infection via the CCR5 coreceptor. RANTES.her2.IgG3, alone or in combination with other chemokine or cytokine fusion Abs, may be a suitable reagent for recruitment and activation of an expanded repertoire of effector cells to tumor deposits.     

Multicomponent gene therapy vaccines for lung cancer: effective eradication of established murine tumors in vivo with interleukin-7/herpes simplex thymidine kinase-transduced autologous tumor and ex vivo activated dendritic cells

Multiple antitumor modalities may be necessary to overcome lung tumor-mediated immunosuppression and effectively treat non-small cell lung cancer (NSCLC). To evaluate a multimodality gene therapy approach for control of local tumor growth, a weakly immunogenic murine alveolar cell carcinoma, L1C2, was transduced with either the interleukin-7/hygromycin-herpes simplex thymidine kinase (IL-7/HyHSVtk) internal ribosome entry site (IRES) retroviral vector or a vector containing the HyHSVtk, but not the IL-7 gene. Of the many cytokines available for gene transfer, IL-7 was chosen for these studies because it both stimulates CTL responses and down-regulates tumor production of the immunosuppressive peptide TGF-beta. Following selection in hygromycin, IL-7 transduction was confirmed by ELISA. Clones produced 1.25 to 10 ng of IL-7/ml/10(6) cells per 24 h. In vitro, genetically modified tumor cells were significantly more sensitive to ganciclovir (GCV) than unmodified parental tumor cells. The in vivo growth of ex vivo modified L1C2 cells was evaluated. There was a dose-response relationship between the amount of IL-7 secreted in vitro and the growth of genetically modified murine tumor in vivo. Transduced tumor cells regressed in mice following GCV therapy. Although ex vivo gene modification of tumor cells led to complete resolution of the tumor following implantation in vivo, IL-7 and HSVtk gene modified tumor cells were not effective in treating established parental tumors. However when 5 x 10(5) bone marrow-derived, in vitro activated dendritic cells (DC) were administered in combination with transduced tumor and GCV, 5 day old established tumors were eradicated in 80% of mice. These studies suggest that multicomponent vaccines may facilitate improved host responses by replacing host immune deficits and thus could have a role in adjuvant therapy and local control of NSCLC.     

Suppression of human prostate carcinoma metastases in severe combined immunodeficient mice by interleukin 2 immunocytokine therapy

Immunocytokines are antibody-cytokine fusion proteins that combine the unique targeting ability of antibodies with the multifunctional activities of cytokines to activate effector cells in the tumor microenvironment. Here, we demonstrate the therapeutic efficacy of a tumor-specific immunocytokine, huKS1/4-IL2, which effectively inhibited growth and dissemination of lung and bone marrow metastases of human prostate carcinoma in severe combined immunodeficient mice. This antitumor effect was specific and highly effective, irrespective of reconstitution of these mice with human lymphokine-activated killer cells. Survival times of mice treated with huKS1/4-IL2 were increased 4-fold as compared with animals treated with a mixture of the corresponding antibody and recombinant human interleukin-2 (rhIL2). A persistent antitumor response after treatment with the huKS1/4-IL2 immunocytokine in B, T, and natural killer cell-deficient severe combined immuodeficient-BEIGE mice, depleted of granulocytes, implies a major role for macrophages in this treatment effect. Our data demonstrate that immunocytokine-directed interleukin-2 therapy to tumor sites is an immunotherapeutic approach with potent effects against disseminated metastases of human prostate carcinoma and suggest that this treatment could be effective in an adjuvant setting for patients with minimal residual disease.     

Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells

A variety of tumors are potentially immunogenic but do not stimulate an effective anti-tumor immune response in vivo. Tumors may be capable of delivering antigen-specific signals to T cells, but may not deliver the costimulatory signals necessary for full activation of T cells. Expression of the costimulatory ligand B7 on melanoma cells was found to induce the rejection of a murine melanoma in vivo. This rejection was mediated by CD8+ T cells; CD4+ T cells were not required. These results suggest that B7 expression renders tumor cells capable of effective antigen presentation, leading to their eradication in vivo.     

Reovirus therapy of tumors with activated Ras pathway

Human reovirus requires an activated Ras signaling pathway for infection of cultured cells. To investigate whether this property can be exploited for cancer therapy, severe combined immune deficient mice bearing tumors established from v-erbB-transformed murine NIH 3T3 cells or human U87 glioblastoma cells were treated with the virus. A single intratumoral injection of virus resulted in regression of tumors in 65 to 80 percent of the mice. Treatment of immune-competent C3H mice bearing tumors established from ras-transformed C3H-10T1/2 cells also resulted in tumor regression, although a series of injections were required. These results suggest that, with further work, reovirus may have applicability in the treatment of cancer.     

Induction of tumor immunity by photodynamic therapy

The mechanism of tumor destruction by photodynamic therapy (PDT) incorporates a variety of events leading to inactivation of tumor cells. The unique feature of PDT is the mobilization of the host to participate in the eradication of treated cancer. A critical element is the induced inflammation at the treated site associated with massive invasion of activated myeloid cells. In addition to further destruction of cancer cells, conditions are created for the presentation of tumor antigens with subsequent activation of lymphoid cells, leading to tumor-specific immunity. This inflammation-primed immune development process results in generation of tumor-specific immune memory cells that appear to be elicited against both strongly and poorly immunogenic PDT-treated cancers. Once generated by PDT, it is conceivable that these immune cells (especially if further expanded and activated by adjuvant immunotherapy) can be engaged in additional eradication of disseminated and/or metastatic lesions of the same cancer. A number of immunotherapy regimens have already been proven effective in enhancing the curative effect of PDT with various animal tumor models. Inflamed cancerous tissue at the PDT-treated site appears to exert powerful attracting signals for immune cells activated by different immunotherapy regimens.     

Dendritic cells augment granulocyte-macrophage colony-stimulating factor (GM-CSF)/herpes simplex virus thymidine kinase-mediated gene therapy of lung cancer

Lung cancer, the leading cause of cancer death in the United States, is resistant to most currently available therapies. To evaluate a multicomponent gene therapy approach that replaces tumor-bearing host immune deficits, we genetically modified Line 1 (L1C2), a weakly immunogenic alveolar cell carcinoma cell line. L1C2 was transduced ex vivo with a retroviral construct that contained two components: a cytokine gene (granulocyte-macrophage colony-stimulating factor) and a drug sensitivity gene (herpes simplex virus thymidine kinase). The third component of this therapy, in vitro-activated syngeneic bone marrow-derived dendritic cells, was included to augment antigen presentation. The addition of ganciclovir (GCV) caused the lysis of transduced tumor cells, resulting in the release of potential tumor antigens. Ex vivo-transduced tumor cells regressed in vivo following GCV therapy but were not effective in the treatment of established parental tumors. To treat established tumors, dendritic cells were administered in combination with transduced tumor cells and GCV. A total of 50% of these mice rejected the 5-day-old established tumors and were immune to rechallenge with parental L1C2 cells. Thus, this multicomponent gene therapy system leads to both the regression of established tumors and enhanced immunogenicity in this weakly immunogenic murine lung cancer model.     

Tumor-specific cytokine release by donor T cells induces an effective host anti-tumor response through recruitment of host naive antigen presenting cells

We recently reported that tumor eradication induced by immunotherapy (IT) in a congenic mouse model using tumor infiltrating lymphocytes (TIL) + recombinant interleukin-2 (rIL-2) is dependent on recruitment of naive host immune cells at the tumor sites. The recruitment of host immune cells was induced mainly through a local secretion of interferon-gamma (IFN-gamma) produced by donor T cells. We now further investigated how a non-specific inflammatory response progresses to a host T-cell-mediated tumor-specific response. In cross-over experiments using MCA-105 and MCA-205 sarcoma tumors, pulmonary metastatic disease was eradicated only in mice treated with tumor-matched TIL + rIL-2. In vitro, TIL stimulated with the tumor of origin secreted relatively high levels of IFN-gamma and granulocyte-macrophage colony stimulating factor (GM-CSF) compared to TIL stimulated with mismatched tumor cells. In lungs of tumor-bearing mice treated with matched TIL + rIL-2, significant increases in the percentages of IFN-gamma, GM-CSF and tumor necrosis factor-alpha (TNF-alpha) positive cells were detected, as well as of macrophages, natural killer (NK) cells and dendritic cells. Depletion of macrophages or NK cells did not inhibit the efficacy. In contrast, depletion of dendritic cells partially inhibited the efficacy of the treatment. Combined depletion of dendritic cells and macrophages abrogated more than 80% of the efficacy. Our data suggest that successful IT may require 3 steps: (1) release of inflammatory cytokines by donor TIL after restimulation by tumor cells; (2) infiltration of host immune cells in response to local cytokine production; and (3) activation of tumor-specific host immune cells by dendritic cells and to a lesser extent by macrophages.     

Modulation of tumor cell response to chemotherapy by the organ environment

The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors. The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential. While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes. The organ microenvironment can also influence the response of metastases to chemotherapy. It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy. We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma. The tumor cells were implanted subcutaneously or into different visceral organs. Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not. In contrast, sensitivity to 5-FU did not differ between these sites of growth. The differences in response to DXR between s.c. tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution. The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression of mdr1 mRNA. However, the organ-specific mechanism for upregulating mdr1 and P-glycoprotein has yet to be elucidated.     

Construction and characterization of a triple-recombinant vaccinia virus encoding B7-1, interleukin 12, and a model tumor antigen

BACKGROUND: Construction of recombinant viruses that can serve as vaccines for the treatment of experimental murine tumors has recently been achieved. The cooperative effects of immune system modulators, including cytokines such as interleukin 12 (IL-12) and costimulatory molecules such as B7-1, may be necessary for activation of cytotoxic T lymphocytes. Thus, we have explored the feasibility and the efficacy of inclusion of these immunomodulatory molecules in recombinant virus vaccines in an experimental antitumor model in mice that uses Escherichia coli beta-galactosidase as a target antigen. METHODS: We developed a "cassette" system in which three loci of the vaccinia virus genome were used for homologous recombination. A variety of recombinant vaccinia viruses were constructed, including one virus, vB7/beta/IL-12, that contains the following five transgenes: murine B7-1, murine IL-12 subunit p35, murine IL-12 subunit p40, E. coli lacZ (encodes beta-galactosidase, the model antigen), and E. coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene). The effects of the recombinant viruses on lung metastases and survival were tested in animals that had been given an intravenous injection of beta-galactosidase-expressing murine colon carcinoma cells 3 days before they received the recombinant virus by intravenous inoculation. RESULTS: Expression of functional B7-1 and IL-12 by virally infected cells was demonstrated in vitro. Lung tumor nodules (i.e., metastases) were reduced in mice by more than 95% after treatment with the virus vB7/beta/IL-12; a further reduction in lung tumor nodules was observed when exogenous IL-12 was also given. Greatest survival of tumor-bearing mice was observed in those treated with viruses encoding beta-galactosidase and B7-1 plus exogenous IL-12. CONCLUSION: This study shows the feasibility of constructing vaccinia viruses that express tumor antigens and multiple immune cofactors to create unique immunologic microenvironments that can modulate immune responses to cancer.     

Interstitial brachytherapy procedures for brain tumors

This report characterizes the immunological host response to a syngeneic murine mammary carcinoma along with variants genetically modified to express B7-1 or secrete GM-CSF and interleukin-12 (IL-12). MT-901 is a subline of a mammary adenocarcinoma that was chemically induced in the Balb/c host. It was found to be weakly immunogenic by immunization/ challenge experiments, and it induced tumor-specific T-cell responses in lymph nodes (LN) draining progressive subcutaneous tumors. Tumor clones expressing B7-1 or secreting GM-CSF exhibited reduced tumorigenicity without completely abrogating tumor growth, whereas IL-12 elaboration lead to complete tumor growth inhibition. In vivo subcutaneous inoculation of a transgenic cell clone secreting GM-CSF (240 ng/10(6) cells/24 hours) resulted in significantly enhanced T-cell reactivity of tumor-draining lymph node (TDLN) cells as compared to wild-type TDLN cells. This finding was obtained from observations assessed by several different methods, including: 1) in vitro cytotoxicity, 2) in vitro interferon-gamma release, and 3) adoptive transfer in mice with established tumor. Moreover, the transfer of activated LN cells derived from mice inoculated with GM-CSF-secreting tumor cells resulted in the prolonged survival of animals with macroscopic metastatic disease, which was not evident utilizing LN cells from mice inoculated with wild-type tumor. By contrast, clones that expressed B7-1 or IL-12 (4 ng/10(6) cells/24 hours) did not elicit enhanced tumor-reactive TDLN cells compared with wild-type tumor when assessed in the adoptive transfer model. The autocrine secretion of GM-CSF by transduced tumor cells was found to serve as an effective immune adjuvant in the host response to this weakly immunogenic tumor.     

Administration of recombinant interleukin 12 prevents outgrowth of tumor cells metastasizing spontaneously to lung and lymph nodes

The present study investigates the ability of recombinant interleukin 12 (rIL-12) to modulate the growth of a primary tumor as well as the outgrowth of metastatic tumor cells in an ovarian carcinoma (OV-HM) model. This aggressive tumor displayed rapid growth of the primary tumor mass, high incidence of metastases to lung and lymph nodes, and invasion from the primary s.c. site to the peritoneal cavity. Starting 12 days after s.c. tumor cell implantation, several i.p. injections of rIL-12 at 2-3 day intervals resulted in regression of growing tumors. These treated mice did not show signs of metastases or tumor recurrence at the original site. One month after tumor implantation, untreated mice did not have visible lung metastasis, but some did have palpable lymph nodes. At this stage, the primary tumors of animals without palpable lymph nodes were surgically resected. When examined 2 months later, most animals had developed lymph node and lung metastases. In contrast, rIL-12 injections after tumor resection inhibited the development of metastases in both lung and lymph nodes. This contrasted with the failure of IL-2 to prevent metastases. Even for mice already showing signs of lymph node metastases or invasion of the abdominal wall, rIL-12 administration after tumor resection prevented further invasion to the peritoneal cavity and growth of metastatic tumor cells in lung. It was somewhat surprising that the IL-12 treatment of animals after 1 month of tumor growth without resection also resulted in complete tumor regression, as well as eradication of micrometastasis that would have occurred before the treatment. Moreover, they exhibited resistance to a rechallenge with the same tumor but not with a second tumor. Thus, this tumor system provides a relevant model to clinical situations in terms of treatment of advanced tumors and metastases. These results also indicate that IL-12 can induce a curative immune response, even in the face of an aggressive micrometastasizing tumor.     

Divergent effects of TNF alpha in the adoptive immunotherapy of a murine sarcoma

We have previously described an in vitro sensitization (IVS) procedure which enabled the generation of therapeutic T cells from tumor-bearing mice for adoptive immunotherapy. The procedure involved culture of tumor-draining lymph node (TDLN) cells with irradiated tumor in the presence of interleukin-2 (IL-2). The availability of many recombinant cytokines affords an opportunity to examine their effects on the immune response to tumor. In this study, we investigated the effect of tumor necrosis factor-alpha (TNF alpha) on the generation and function of IVS cells utilized in adoptive immunotherapy of the murine MCA 106 sarcoma. TNF alpha administered iv at nontherapeutic doses was found to enhance the antitumor efficacy mediated by IVS cells plus IL-2 in the treatment of pulmonary metastases. In contrast, TNF alpha administration to mice bearing progressive footpad tumors had inhibitory effects on the sensitization of tumor-reactive cells in TDLN since IVS cells generated from these animals displayed a diminished antitumor effect. This effect appeared to be due to a reduced number of tumor-reactive lymphoid cells in the TDLN since TNF alpha added to IVS cultures did not alter the antitumor efficacy of the resultant IVS effector cells. These findings indicate the divergent effects of TNF alpha on the immune response to tumor and adoptive immunotherapy with IVS cells.