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Phase 0 trials are designed primarily to evaluate the pharmacodynamic and/or pharmacokinetic properties of selected investigational agents before initiating more traditional phase I testing. One of the major objectives of phase 0 trials is to interrogate and refine a target or biomarker assay for drug effect in human samples implementing procedures developed and validated in preclinical models. Thus, close collaboration between laboratory scientists and clinical investigators is essential to the design and conduct of phase 0 trials. Given the relatively small number of patients and tissue samples, showing a significant drug effect in phase 0 trials requires precise and reproducible assay procedures and innovative statistical methodology. Furthermore, phase 0 trials involving limited exposure of a study agent administered at low doses and/or for a short period allow them to be initiated under the Food and Drug Administration exploratory investigational new drug guidance with less preclinical toxicity data than usually required for traditional first-in-human studies. Because of the very limited drug exposure, phase 0 trials offer no chance of therapeutic benefit, which can impede patient enrollment, particularly if invasive tumor biopsies are required. The challenges to accrual are not insurmountable, however, and well-designed and executed phase 0 trials are feasible and have great potential for improving the efficiency and success of subsequent trials, particularly those evaluating molecularly targeted agents.

Phase 0 clinical trials, developed in response to the United States Food and Drug Administration (FDA)'s recent exploratory Investigational New Drug (IND) guidance, are intended to expedite the clinical evaluation of new molecular entities. The exploratory IND supports the performance of first-in-human testing of new investigational agents at subtherapeutic doses based on reduced manufacturing and toxicologic requirements, allowing the demonstration of drug-target effects and assessment of pharmacokinetic-pharmacodynamic relationships in humans earlier in clinical development. The objectives of a phase 0 cancer clinical trial are to establish at the very earliest opportunity-before large numbers of patients have been accrued and exposed to potential drug-associated toxicity-whether an agent is modulating its target in a tumor, and consequently whether further clinical development is warranted. We review here the fundamental requirements of clinical studies conducted under an exploratory IND and address some common misconceptions regarding oncologic phase 0 trials.

Purpose: Interleukin (IL)-2 therapy is currently used for therapy of renal cell carcinoma (RCC). However, it is only effective in approximately 10% to 15% of patients, showing a need for additional therapies. We have previously described a replication-defective fowlpox vector encoding three costimulatory molecules (B7-1, ICAM-1, and LFA-3), designated rF-TRICOM. Here, we show that intratumoral administration of rF-TRICOM in an orthotopic RCC model effectively enhances tumor immunogenicity and reduces tumor burden in mice and the combination of rF-TRICOM and IL-2 is more effective than either therapy alone., Experimental Design: RCC cells were implanted under the capsule of the kidney, and mice were given rF-TRICOM intratumorally 14 days later. We compared the effect of rF-TRICOM, rF-granulocyte macrophage colony-stimulating factor (GM-CSF), and two doses of IL-2 and combinations of the above on antitumor efficacy and survival. Host CD4(+) and CD8(+) T-cell responses were also evaluated., Results: The results show that (a) systemic IL-2 therapy was moderately effective in the reduction of tumor burden in an orthotopic RCC model; (b) a single intratumoral injection of rF-TRICOM and rF-GM-CSF significantly reduced tumor burden; (c) the addition of systemic IL-2 to intratumoral rF-TRICOM/rF-GM-CSF administration resulted in further reduction of tumor burden, decrease in the incidence of metastasis, and extended survival in tumor-bearing mice above that seen with either treatment alone; and (d) CD8(+) T cells played a critical role in the antitumor effect seen with rF-TRICOM/rF-GM-CSF + IL-2 therapy. Finally, the addition of systemic recombinant IL-15 or intratumoral vector-delivered IL-15 to intratumoral rF-TRICOM/rF-GM-CSF administration resulted in substantially more tumor-free mice than either therapy alone., Conclusions: These studies show that intratumoral administration of rF-TRICOM admixed with rF-GM-CSF is effective at reducing tumor burden in mice and the addition of IL-2 further contributes to this effect. These studies thus form the rationale for combination immunotherapy clinical trials in patients with RCC.

The optimal evaluation of molecularly targeted anticancer agents requires the integration of pharmacodynamic assays into early clinical investigations. Phase '0' trials conducted under the new Exploratory Investigational New Drug Guidance from the US Food and Drug Administration can provide a platform to establish the feasibility of assays for target modulation in human samples, evaluate biomarkers for drug effects and provide pharmacokinetic data. Phase 0 trials could facilitate rational drug selection, identify therapeutic failures early, and might compress timelines for anticancer drug development. We expect that such trials will become a routine part of early-phase oncological drug development in the future.

CD40, a member of the TNF receptor superfamily, is expressed on B cells, dendritic cells, and some tumor cells, including melanoma and bladder carcinoma. In this study, we report that both mouse and human renal carcinoma cells (RCC) also constitutively express functional CD40. Treatment of mouse RCC with CD40L induced strong expression of genes and proteins for ICAM-1 and Fas, and this expression was further enhanced by combining CD40L with IFN-gamma. Similar effects were demonstrated using an agonist anti-CD40 antibody. The increased levels of Fas expression on RCC after treatment with CD40L plus IFN-gamma resulted in potent killing by either FasL-positive effector cells or agonistic anti-Fas antibody. The combination of CD40L plus IFN-gamma also significantly enhanced killing of RCC by tumor-specific CTL lines. Our results demonstrate that constitutively expressed CD40 is functionally active and may provide a molecular target for the development of new approaches to the treatment of RCC.

Interleukin-2-based regimens of biological therapy have shown some clinical promise for the treatment of kidney cancer in humans, although the mechanisms responsible for tumor regression occurring in these patients remain unclear. Preclinical insight into these mechanisms is limited by a paucity of orthotopic animal models of kidney cancer. We have used streptozotocin, an antibiotic and diabetogenic nitrosamine compound derived from Streptomyces achromogenes to induce new kidney tumors in BALB/c mice. Single or multiple doses of streptozotocin induced kidney tumors in up to 25% of mice by 50-90 weeks of age, with up to 18% characterized as renal cell carcinomas (RCCs). Several transplantable lines were obtained from the RCCs, and one of these lines was subsequently cloned. The initial tumor isolates and sublines were histologically reconfirmed to be RCCs, and all grew progressively but slowly (mean survival times, 57 to >100 days) in vivo after intrarenal implant. None of the primary isolates or sublines revealed mutations in either the VHL or Ras genes, although karyotype analysis and chromosome painting revealed the consistent presence of a submetacentric chromosome resulting from the fusion of chromosomes 16 and 19. Biological characterization of these tumors revealed several features analogous to the growth of human kidney cancers, including a propensity for the formation of lung metastases and high vascularity. This hypervascularity is evident by both gross and microscopic analysis and correlates with the expression of several proangiogenic genes. Overall, the features of orthotopic transplantability, slower in vivo growth (relative to the rapid growth rates of other transplantable mouse kidney tumors), propensity for lung metastases, and hypervascularity may make these tumors valuable models for the study of new therapeutic strategies based on antineovascular agents and antitumor cytokines.

A significant splenomegaly and lymphadenopathy develops during the progressive growth of Lewis Lung (3LL) tumors in mice. Enlarged spleen and lymph nodes occur because of a pronounced increase in granulocytes in these organs. This granulocytosis in spleen and lymph node was not simply due to recruitment of granulocytes from peripheral blood to spleen and lymph nodes, but also a result of development and/or differentiation of granulocytes from the bone marrow. There was a marked increase in development of myeloid lineage cells, whereas lymphoid populations including T cells and B cells, were dramatically decreased in bone marrow and peripheral blood of 3LL tumor-bearing mice. These data demonstrate that host hematopoiesis shifts from lymphoid to granulocytic development in the 3LL tumor-bearing mice. Interestingly, a somatic mutation of N-Ras gene was found in 3LL tumor cells at codon 61, suggesting that mutated N-Ras may contribute to induction of granulocytosis in 3LL tumor-bearing mice.

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8(+) T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8(+) T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-gamma-dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-gamma production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-gamma and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8(+) T cell-mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed by in vitro activated natural killer (NK) cells, but the relevance of this observation to the biological function of NK cells has been unclear. Herein, we have demonstrated the in vivo induction of mouse TRAIL expression on various tissue NK cells and correlated NK cell activation with TRAIL-mediated antimetastatic function in vivo. Expression of TRAIL was only constitutive on a subset of liver NK cells, and innate NK cell control of Renca carcinoma hepatic metastases in the liver was partially TRAIL dependent. Administration of therapeutic doses of interleukin (IL)-12, a powerful inducer of interferon (IFN)-gamma production by NK cells and NKT cells, upregulated TRAIL expression on liver, spleen, and lung NK cells, and IL-12 suppressed metastases in both liver and lung in a TRAIL-dependent fashion. By contrast, alpha-galactosylceramide (alpha-GalCer), a powerful inducer of NKT cell IFN-gamma and IL-4 secretion, suppressed both liver and lung metastases but only stimulated NK cell TRAIL-mediated function in the liver. TRAIL expression was not detected on NK cells from IFN-gamma-deficient mice and TRAIL-mediated antimetastatic effects of IL-12 and alpha-GalCer were strictly IFN-gamma dependent. These results indicated that TRAIL induction on NK cells plays a critical role in IFN-gamma-mediated antimetastatic effects of IL-12 and alpha-GalCer.

The IFN-gamma-inducible proteins monokine induced by IFN-gamma (Mig) and chemokine responsive to gamma-2 (Crg-2) can contribute to IL-12-induced antiangiogenic and leukocyte-recruiting activities, but the extent to which leukocytes vs parenchymal cells in different organs contribute to the production of these molecules remains unclear. The results presented herein show that IFN-gamma-dependent induction of Mig and Crg-2 gene expression can occur in many nonlymphoid organs, and these genes are rapidly induced in purified hepatocytes isolated from mice treated with IL-2 plus IL-12, or from Hepa 1-6 hepatoma cells treated in vitro with IFN-gamma. In addition to depending on IFN-gamma, the ability of IL-12 or IL-2/IL-12 to induce Mig and Crg-2 gene expression in purified hepatocytes also is accompanied by the coordinate up-regulation of the IFN-gamma R alpha and beta-chains, in the absence of IL-12R components. Supernatants of primary hepatocytes obtained from mice treated in vivo with IL-2/IL-12 or from hepatocytes treated in vitro with IFN-gamma contain increased chemotactic activity for enriched human and mouse CD3(+) T cells, as well as mouse DX5(+) NK cells. The hepatocyte-derived chemotactic activity for mouse T cells but not NK cells was ablated by Abs specific for Mig and Crg-2. These results suggest that parenchymal cells in some organs may contribute substantially to initiation and/or amplification of inflammatory or antitumor responses.

IL-7 is a critical cytokine in the development of T and B cells but little is known about its activity on nonhematopoietic cells. An unexpected finding was noted in allogeneic bone marrow transplant studies using IL-7 receptor null (IL-7R alpha(-/-)) mice as recipients. These mice exhibited a significantly greater weight loss after total body irradiation compared with wild type, IL-7R alpha(+/+), mice. Pathological assessment indicated greater intestinal crypt damage in IL-7R alpha(-/-) recipients, suggesting these mice may be predisposed to gut destruction. Therefore, we determined the effect of the conditioning itself on the intestinal tract of these mice. IL-7R alpha(-/-) mice and IL-7R alpha(+/+) mice were irradiated and examined for lesions and apoptosis within the small intestine. In moribund animals, IL-7R alpha(-/-) mice had extensive damage in the small intestine, including marked ablation of the crypts and extreme shortening of villi following 1500 cGy total body irradiation. In contrast, by 8 days after irradiation, the small intestines of IL-7R alpha(+/+) mice had regenerated as distinguished by normal villus length and hyperplastic crypts. Following 750 cGy irradiation, IL-7R alpha(-/-) mice had a higher proportion of apoptotic cells in the crypts and an accompanying increase in the pro-apoptotic protein Bak was expressed in intestinal epithelial cells. These results demonstrate the increased radiosensitivity of intestinal stem cells within the crypts in IL-7R alpha(-/-) mice and a role for IL-7 in the protection of radiation-induced apoptosis in these same cells. This study describes a novel role of IL-7 in nonhematopoietic tissues.

Anoikis is a form of apoptosis induced in normal cells as a result of loss of their adhesion to substrate. In the present study, we have tested whether tumor cells are also sensitive to anoikis and whether selection of tumor cells for resistance to anoikis could increase their metastatic ability. In vitro cultured Cloudman S91 melanoma cells are strongly adherent to the plastic. Prevention of their adherence by rocking or by covering culture plates with polyhydroxyethylmethacrylate resulted in induction of anoikis and death of almost all cells. Their death was prevented in the presence of caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone. To select anoikis-resistant cells, S91 cells floating in the culture medium were sequentially isolated and transferred for seven generations. As a result, a new subline of S91 cells capable of growing in free cell suspension was selected. These S91 nonadherent (S91Nadh) cells were completely resistant to anoikis and manifested higher metastatic ability than S91Adh cells. Anoikis resistance of S91Nadh cells was not attributable to their resistance to other apoptotic signals in vitro, and they showed no increase in their survival in vivo in the lungs after i.v. inoculation. Increased metastatic potential of the anoikis-resistant S91Nadh cells was associated with various phenotypic changes, including increased proliferation and loss of VLA-4 integrin expression because of down-regulation of the VLA-49alpha (CLD49d) gene. In parallel, they showed a reduction in homotypic aggregation and binding to endothelial cells, increased Matrigel invasiveness, and decreased matrix metalloproteinase-2 and matrix metalloproteinase-9 activity that paralleled up-regulation of the TIMP-1 gene. S91Nadh cells also manifested changes in cell surface carbohydrates, such as appearance of alpha-galactosyl epitopes as a result of up-regulation of the alpha1,3-galactosyltransferase gene and concomitant reduction in cell membrane sialylation. Thus, selection of S91 melanoma cells for anoikis resistance resulted in an increase in their metastatic potential in parallel with multiple alterations in their phenotypic properties.

Using a novel transgenic mouse model of spontaneous mammary carcinoma, we show here that the IL-12/pulse IL-2 combination can induce rapid and complete regression of well-established autochthonous tumor in a setting where the host immune system has been conditioned by the full dynamic process of neoplastic progression and tumorigenesis. Further, this regimen inhibits neovascularization of established mammary tumors, and does so in conjunction with potent local induction of genes encoding the IFN-gamma- and TNF-alpha-inducible antiangiogenic chemokines IFN-inducible protein 10 and monokine induced by IFN-gamma. In contrast to untreated juvenile C3(1)TAg mice in which histologically normal mammary epithelium predictably undergoes progressive hyperplasia, atypical changes, and ultimately transition to overt carcinoma, the current studies also demonstrate a unique preventative therapeutic role for IL-12/pulse IL-2. In juvenile mice, early administration of IL-12/pulse IL-2 markedly limits the expected genetically programmed neoplastic transition within the mammary epithelium and does so in conjunction with enhancement of constitutive Fas and pronounced induction of local Fas ligand gene expression, T cell infiltration, and induction of apoptosis within the mammary epithelium. These events occur in the absence of a durable Ag-specific memory response. Thus, this novel model system demonstrates that the potent therapeutic activity of the IL-12/pulse IL-2 combination rapidly engages potent apoptotic and antiangiogenic mechanisms that remain active during the delivery of IL-12/pulse IL-2. The results also demonstrate that these mechanisms are active against established tumor as well as developing preneoplastic lesions.

CD40 stimulation, by either antibody or ligand, has been shown to inhibit the growth of a variety of neoplastic cells, both in vivo and in vitro. In this study, we assessed the effects of CD40 stimulation using a murine agonistic CD40 monoclonal antibody (MoAb) (FGK115) or a soluble recombinant murine CD40 ligand (srmCD40L) in both lethally irradiated and nonirradiated BALB/c mice. Toxicity after CD40 stimulation was not observed in nonirradiated animals receiving up to 100 microg of the agonist anti-CD40 MoAb. However, as little as 10 microg of the agonistic anti-CD40 MoAb induced acute toxicity resulting in 100% morbidity of lethally irradiated animals by 4 days after irradiation. Histological evaluation of animals receiving anti-CD40 MoAb revealed severe intestinal lesions with disruption of the villi, goblet cell depletion, and crypt hyperplasia of the small intestine, colon, and cecum. Delaying the administration of anti-CD40 MoAb or reducing the amount of irradiation given resulted in increased survival and less severe lesions. Analysis of serum cytokine levels in lethally irradiated mice receiving agonistic anti-CD40 showed a marked increase of interferon (IFN)-gamma. Lethally irradiated IFN-gamma knockout mice given the agonistic anti-CD40 MoAb demonstrated significant increases in survival and minimal gut lesions compared with wild-type mice receiving the same regimen, suggesting that IFN-gamma plays a major role in this toxic reaction. These results indicate that CD40 stimulation using agonistic antibodies following lethal irradiation leads to a fatal, cytokine-induced disease affecting the intestine.

IL-18 is considered to be a strong cofactor for CD4+ T helper 1 (Th1) cell induction. We have recently reported that IL-18 can induce IL-13 production in both NK cells and T cells in synergy with IL-2 but not IL-12, suggesting IL-18 can induce Th1 and Th2 cytokines when accompanied by the appropriate first signals for T cells. We have now found that IL-18 can act as a cofactor to induce IL-4, IL-10 and IL-13 as well as IFN-gamma production in T cells in the presence of anti-CD3 monoclonal antibodies (mAb). IL-18 can rapidly induce CD40 ligand (CD154) mRNA and surface expression on CD4+ but not CD8+ T cells. The administration of IL-18 alone in vivo significantly increased serum IgE levels in C57BL/6 (B6) and B6 IL-4 knockout mice. Furthermore, the administration of IL-18 plus IL-2 induced approximately 70-fold and 10-fold higher serum levels of IgE and IgG1 than seen in control B6 mice, respectively. IgE and IgG1 induction in B6 mice by administration of IL-18 plus IL-2 was eliminated by the pretreatment of mice with anti-CD4 or anti-CD154, but not anti-CD8 or anti-NK1.1 mAb. These results suggest that IL-18 can induce Th2 cytokines and CD154 expression, and can contribute to CD4+ T cell-dependent, IL-4-independent IgE production.

The liver is a complex organ composed of hepatic parenchymal cells and a variety of non-parenchymal cells that consist of endothelial cells, Kupffer cells, and several subsets of resident lymphocytes, including natural killer (NK), T, and NK1.1+/CD3+ (NK/T) cells. The regulation of these various lymphoid subpopulations and their relative contributions to antiviral, antitumor and pathogenic inflammatory responses in the liver remain topics of much interest. Studies from our laboratory have shown that various immune stimulants and cytokines can augment liver-associated NK activity at least partially through the mobilization of NK cells from the bone marrow to the liver. The mobilization process can be dependent on the induction of interferon (IFN)-gamma and/or tumor necrosis factor-alpha and on very late activation antigen-4/vascular cell adhesion molecule-1 interaction. The induction of IFN-gamma by cytokines such as interleukin (IL)-12 also rapidly triggers the induction of chemokine genes in parenchymal cells that may contribute to the localization of NK and T cells. Both IL-2 and IL-12 trigger changes in the number and functions of liver-associated leukocyte subsets, and induce antimetastatic effects that are likely mediated through several direct and indirect mechanisms. The overall goal of these studies is to understand the interactions and functions of liver-associated NK1.1+ cells in the context of innate and adaptive immune responses to neoplasia.

The 5' flanking region of the C3(1) component of the rat prostate steroid binding protein (PSBP) has been used to successfully target the expression of the SV40 large T-antigen (Tag) to the epithelium of both the mammary and prostate glands resulting in models of mammary and prostate cancers which histologically resemble the human diseases. Atypia of the mammary ductal epithelium develops at about 8 weeks of age, progressing to mammary intraepithelial neoplasia (resembling human ductal carcinoma in situ [DCIS]) at about 12 weeks of age with the development of invasive carcinomas at about 16 weeks of age in 100% of female mice. The carcinomas share features to what has been classified in human breast cancer as infiltrating ductal carcinomas. All FVB/N female mice carrying the transgene develop mammary cancer with about a 15% incidence of lung metastases. Approximately 10% of older male mice develop anaplastic mammary carcinomas. Unlike many other transgenic models in which hormones and pregnancy are used to induce a mammary phenotype, C3(1)/Tag mice develop mammary tumors in the mammary epithelium of virgin animals without hormone supplementation or pregnancy. Although mammary tumor development appears hormone-responsive at early stages, invasive carcinomas are hormone-independent, which corresponds to the loss of estrogen receptor-alpha expression during tumor progression. Molecular and biologic factors related to mammary tumor progression can be studied in this model since lesions evolve over a predictable time course. Genomic alterations have been identified during tumor progression, including an amplification of the distal portion of chromosome 6 containing ki-ras and loss of heterozygosity (LOH) in other chromosomal regions. We have demonstrated that stage specific alterations in the expression of genes which are critical regulators of the cell cycle and apoptosis are functionally important in vivo. C3(1)/Tag mice appear useful for testing particular therapies since growth of the mammary tumors can be reduced using chemopreventive agents, cytokines, and an anti-angiogenesis agent.

Although alloreactive T cells are required for the induction of graft-versus-host disease (GVHD), other factors can influence outcome in murine models of the disease. Lethal total body irradiation (TBI) conditioning regimens followed by reconstitution with allogeneic lymphohematopoietic cells results in the generation of donor anti-host cytotoxic T lymphocyte (CTL)-mediated solid organ (gut, liver, skin) destruction. In contrast, donor anti-host CTL-mediated hematopoietic failure is the primary cause of morbidity following sublethal TBI. To determine the role of interferon (IFN)-gamma in graft-versus-host reactions against hematopoietic and solid organ targets, we used IFN-gamma knockout mice as donors in both lethal TBI and bone marrow transplantation (BMT) rescue and sublethal TBI models. In this report, we show that CD4+ T cells from IFN-gamma knockout (KO) mice resulted in accelerated GVHD after lethal TBI/BMT using a single major histocompatibility class II mismatch model. In marked contrast, the use of these same IFN-gamma KO CD4+ donor cells in combination with sublethal TBI significantly ameliorated GVHD-associated mortality. In these recipients, severe anemia, bone marrow aplasia, and intestinal lesions were observed in the presence but not the absence of donor-derived IFN-gamma. Administration of anti-IFN-gamma antibodies to sublethally irradiated recipients of wild-type donor cells confirmed the role of IFN-gamma depletion in CD4+ T cell-mediated GVHD. In conclusion, the extent of conditioning markedly affects the role of IFN-gamma in GVHD lesions mediated by CD4+ T cells. In models using sublethal TBI, the absence of IFN-gamma is protective from GVHD, whereas in lethal TBI situations, the loss is deleterious.

The role of Fas in the regulation of solid tumor growth was investigated. Murine renal carcinoma (Renca) cells were constitutively resistant to Fas-mediated killing in vitro, but exhibited increased expression of Fas and sensitivity to Fas-mediated killing after exposure to IFN-gamma and TNF. Transfected Renca cells overexpressing Fas were efficiently killed in vitro upon exposure to anti-Fas Ab (Jo2). When Fas-overexpressing Renca cells were injected into syngenic BALB/c mice, there was a consistent and significant delay in tumor progression, reduced metastasis, and prolonged survival that was not observed for Renca cells that overexpressed a truncated nonfunctional Fas receptor. The delay of in vivo tumor growth induced by Fas overexpression was not observed in IFN-gamma-/- mice, indicating that IFN-gamma is required for the delay of in vivo tumor growth. However, there was a significant increase of infiltrated T cells and in vivo apoptosis in Fas-overexpressing Renca tumors, and Fas-overexpressing Renca cells were also efficiently killed in vitro by T cells. In addition, a strong therapeutic effect was observed on Fas-overexpressing tumor cells by in vivo administration of anti-Fas Ab, confirming that overexpressed Fas provides a functional target in vivo for Fas-specific ligands. Therefore, our findings demonstrate that Fas overexpression on solid tumor cells can delay tumor growth and provides a rationale for therapeutic manipulation of Fas expression as a means of inducing tumor regression in vivo.

Mice deficient in lymphotoxin (LT)-alpha lack peripheral lymph nodes and Peyer's patches and have profound defects in development of follicular dendritic cell networks, germinal center formation, and T/B cell segregation in the spleen. Although LTalpha is known to be expressed by NK cells as well as T and B lymphocytes, the requirement of LTalpha for NK cell functions is largely unknown. To address this issue, we have assessed NK cell functions in LTalpha-deficient mice by evaluating tumor models with known requirements for NK cells to control their growth and metastasis. Syngeneic B16F10 melanoma cells inoculated s.c. grew more rapidly in LTalpha-/- mice than in the wild-type littermates, and the formation of experimental pulmonary metastases was significantly enhanced in LTalpha-/- mice. Although LTalpha-/- mice exhibited almost a normal total number of NK cells in spleen, they showed an impaired recruitment of NK cells to lung and liver. Additionally, lytic NK cells were not efficiently produced from LTalpha-/- bone marrow cells in vitro in the presence of IL-2 and IL-15. These data suggest that LTalpha signaling may be involved in the maturation and recruitment of NK cells and may play an important role in antitumor surveillance.

In this study four murine IL-12 naked DNA expression plasmids (pIL-12), containing both the p35 and p40 subunits, were shown to induce systemic biological effects in vivo after intradermal injection. Three of the four IL-12 expression vectors augmented NK activity and induced expression of the IFN-gamma and IFN-gamma-inducible Mig genes. Both IL-12 p70 heterodimer and IFN-gamma proteins were documented in the serum within 24 h after intradermal injection of the pIL-12o- plasmid, which also induced the highest level of NK activity in the spleen and liver among the IL-12 constructs. Interestingly, both p40 mRNA expression at the injection site and serum protein levels followed a biphasic pattern of expression, with peaks on days 1 and 5. Subsequent studies revealed that the ability of intradermally injected pIL-12o- to augment NK lytic activity was prevented by administration of a neutralizing anti-IL-12 mAb. Finally, injection of the pIL-12o- into BALB/c IL-12 p40-/- mice also resulted in a biphasic pattern of IL-12 p70 appearance in the serum, and induced IFN-gamma protein and activated NK lytic activity in liver and spleen. These results demonstrate that injection of delivered naked DNA encoding the IL-12 gene mediates the biphasic systemic production of IL-12-inducible genes and augments the cytotoxic function of NK cells in lymphoid and parenchymal organs as a direct result of transgene expression. The results also suggest that these naked DNA plasmids may be useful adjuvants for vaccines against infectious and neoplastic diseases.

IL-13 and IL-4 have similar biological activities and are characteristic of cytokines expressed by Th2 cells. In contrast, IL-12 and IL-18 have been shown to be strong cofactors for Th1 cell development. In this study, we found strong induction of IL-13 mRNA and protein by IL-2 + IL-18 in NK and T cells. In contrast, IL-12 did not enhance the IL-13 production induced by IL-2 alone. Moreover, IL-13 mRNA and protein expression induced by IL-2 + IL-18 in purified NK and T cells obtained from IFN-gamma knockout (-/-) mice were greater than seen in purified cells from normal controls. In contrast, IL-10 production induced by IL-2 and/or IL-12 was not significantly different in IFN-gamma (-/-) mice and normal controls. These results suggest IL-13 expression induced by IL-2 + IL-18 may be regulated by IFN-gamma in vivo, while IL-10 expression may be IFN-gamma-independent. Thus, depending upon the cell type, IL-18 may act as a strong coinducer of Th1 or Th2 cytokines. Our findings suggest that IL-12 and IL-18 have different roles in the regulation of gene expression in NK and T cells.

NK cells have been shown to be important antitumor or antiviral effector cells in the liver. In the present study we have examined the factors that regulate the initial recruitment and subsequent fate of hepatic NK and T cells in mice treated with IL-12 or IL-2. Daily administration of IL-12 caused a rapid initial increase in NK cells followed by a subsequent decrease that coincided with an accumulation of T cells. The recruitment of hepatic NK cells by IL-12, but not the subsequent T cell infiltrate, was abrogated in IFN-gamma(-/-) mice. In contrast, daily administration of IL-2 caused a sustained increase in liver-associated NK cells that was not diminished in IFN-gamma(-/-) mice. The IL-12-induced recruitment in both hepatic NK and T cells was abrogated by in vivo treatment with anti-VCAM-1 mAbs, while treatment with anti-ICAM-1 Abs decreased only the recruitment of T cells in the IL-12-treated mice. The rapid loss of newly recruited hepatic NK cells in IL-12-treated mice did not occur in SCID mice or in B.MRL-Fas(lpr) (Fas-) and B6Smn.C3H-Fasl(gld) (FasL-) mutant mice, suggesting that T cells can actively eliminate hepatic NK cells through a Fas-dependent mechanism. These findings also imply that during the endogenous innate immune response to infectious agents or tumors or in the host response induced by cytokine therapies, the biologic effects of NK cells may be limited by T cell-mediated effects.

Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-gamma or TNF-alpha, and the combination of IFN-gamma + TNF-alpha synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-gamma and TNF-alpha are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-gamma and TNF-alpha.

IL-12 is a potent immunoregulatory cytokine that has been shown to mediate tumor regression in a variety of tumor models. We describe the construction of AdCMV-IL-12, a recombinant adenovirus that encodes both subunits of IL-12 under transcriptional control of the CMV promoter. This recombinant virus efficiently infects a wide variety of cell types leading to the production of high levels of biologically active IL-12. Because the liver is a primary site of infection after i.v.-administered adenovirus, we tested the therapeutic efficacy of this virus in a murine hepatic metastasis tumor model. Systemic administration of AdCMV-IL-12 dramatically inhibited the formation of 3-day Renca hepatic metastases (mean of 16 metastases per liver) compared with the control virus AdCMV-betagal (mean of 209) or vehicle alone (mean of 272). Histologic analysis indicated that metastatic growth inhibition was accompanied by a dramatic perivascular infiltrate consisting of T cells, macrophages, and neutrophils. Therapeutic efficacy was not diminished in animals depleted of CD4+ or CD8+ T cells, or in SCID mice, even after NK cell ablation. In the latter case, a hepatic perivascular infiltrate composed of macrophages and neutrophils was observed after AdCMV-IL-12-treatment, while numerous activated Kupffer cells were noted in the hepatic parenchyma. Analysis of therapy-induced changes in hepatic gene expression demonstrated increased levels of IP-10 and Mig RNAs, but no increase in iNOS, Fas, or FasL RNA levels was observed. Our data suggest a model of metastatic growth inhibition mediated by nonlymphocyte effector cells including macrophages and neutrophils and that may involve anti-angiogenic chemokines.

The regulation of tumor growth by cytokine-induced alterations in host effector cell recruitment and activation is intimately associated with leukocyte adhesion and angiogenic modulation. In the present study, we have developed a novel tumor model to investigate this complex series of events in response to cytokine administration. Gelatin sponges containing recombinant human basic fibroblast growth factor (rhFGFb) and B16F10 melanoma cells were implanted onto the serosal surface of the left lateral hepatic lobe in syngeneic C57BL/6 mice. The tumor model was characterized by progressive tumor growth initially localized within the sponge and the subsequent development of peritoneal carcinomatosis. Microscopic examination of the sponge matrix revealed well developed tumor-associated vascular structures and areas of endothelial cell activation as evidenced by leukocyte margination. Treatment of mice 3 days after sponge implantation with a therapeutic regimen consisting of pulse recombinant human interleukin-2 (rhIL-2) combined with recombinant murine interleukin-12 (rmIL-12) resulted in a marked hepatic mononuclear infiltrate and inhibition of tumor growth. In contrast to the control group, sponges from mice treated with rhIL-2/rmIL-12 demonstrated an overall lack of cellularity and vascular structure. The regimen of rhIL-2 in combination with rmIL-12 was equally effective against gelatin sponge implants of rhFGFb/B16F10 melanoma in SCID mice treated with anti-asialo-GM1 in the absence of a mononuclear infiltration, suggesting that T, B, and/or NK cells were not the principal mediators of the anti-tumor response in this tumor model. The absence of vascularity within the sponge after treatment suggests that a potential mechanism of rhIL-2/rmIL-12 anti-tumor activity is the inhibition of neovascular growth associated with the establishment of tumor lesions. This potential mechanism could be dissociated from the known activities of these two cytokines to induce the recruitment and activation of host effector cells. Moreover, this model provides a unique opportunity to study the cellular and molecular mechanism(s) underlying both tumor angiogenesis and leukocyte recruitment to metastatic lesions.

Administration of recombinant human interleukin-7 (rhIL-7) to mice increases the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakaryocyte macrophage [CFU-GEMM]) from the bone marrow (BM) to peripheral organs, including blood, and also increases the number of primitive progenitor and stem cells in the peripheral blood (PB). We now report that combined treatment of mice with rhIL-7 and recombinant human granulocyte-colony stimulating factor (rhG-CSF) stimulates a twofold to 10-fold increase in the total number of PB CFU-c, and a twofold to fivefold increase in the total number of PB CFU-spleen at day 8 (CFU-S8) over the increase stimulated by rhIL-7 or rhG-CSF alone. In addition, the quality of mobilized cells with trilineage, long-term marrow-repopulating activity is maintained or increased in mice treated with rhIL-7 and rhG-CSF compared with rhIL-7 or rhG-CSF alone. These differences in mobilizing efficiency suggest qualitative differences in the mechanisms by which rhIL-7 and rhG-CSF mobilize progenitor cells, in fact, the functional status of progenitor cells mobilized by rhIL-7 differs from that of cells mobilized by rhG-CSF in that the incidence of actively cycling (S-phase) progenitors obtained from the PB is about 20-fold higher for rhIL-7-treated mice than for mice treated with rhG-CSF. These results suggest the use of rhIL-7-mobilized progenitor/stem cells for gene-modification and tracking studies, and highlight different functions and rates of repopulation after reconstitution with PB leukocytes obtained from mice treated with rhIL-7 versus rhG-CSF.