Characterization of the antitumor immune response generated by treatment of murine tumors with recombinant adenoviruses expressing HSVtk, IL-2, IL-6 or B7-1

In a cancer gene therapy model recombinant adenoviruses expressing the herpes simplex virus thymidine kinase (HSVtk) gene were injected into tumors in situ, either alone or in combination with adenoviruses (Avs) engineered to express IL-2, IL-6 or the costimulatory molecule B7-1. HSVtk phosphorylates the prodrug ganciclovir, thus converting it into an antimetabolite which kills not only HSVtk expressing cells, but also by the 'bystander effect', neighboring untransduced tumor cells. The tumors regressed in 80% of mice upon AvTK/ganciclovir treatment: combinations with AvIL-2, AvIL-6, or AvB7-1 did not improve these results. Cured mice were protected from further challenge with wild-type tumor but not from challenges with an unrelated syngeneic tumor cell line. Since cytotoxic T lymphocyte responses in this tumor model were weak, we analyzed cytokine secretion from spleen cells of treated animals. The best correlate of antitumor immunity in this model was enhanced secretion of GM-CSF, while secretion of IL-2, IL-6 and IFN gamma was also frequently increased but not as consistently. The enhanced IFN gamma secretion associated with unchanged IL-4 secretion suggests that AvTK treatment results in a predominantly Th1-mediated antitumor immune response.     

Interleukin-10 gene transfer activates interferon-gamma and the interferon-gamma-inducible genes Gbp-1/Mag-1 and Mig-1 in mammary tumors

Expression of IL-10 as a transgene inhibits murine mammary tumor growth and metastasis. Using differential display methodology, we sought genes whose expression was modulated by IL-10. We compared mRNA isolated from parental murine mammary 66.1 tumors, as well as tumors derived from neo(r)-transfected cells and 6 different IL-10-expressing cell lines. We identified 2 cDNA products that were up-regulated in all 6 IL-10-expressing tumors in comparison to parental and 66-neo tumors. One cDNA corresponds to the murine guanylate-binding protein gene Gbp-1/Mag-1. The other cDNA corresponds to the chemokine Mig-1 (monokine induced by IFN-gamma). Both genes were originally identified in IFN-gamma-activated macrophages or macrophage cell lines. We now report that cultured mammary epithelial tumor cell lines also express both genes in response to treatment with IFN-gamma and LPS. Furthermore, IFN-gamma mRNA is elevated in IL-10-expressing tumors in comparison with parental or neo-transfected tumors. Thus, high-level expression of IL-10 as a transgene results in activation rather than suppression of IFN-gamma as well as 2 IFN-gamma-inducible genes. Up-regulation of host IFN-gamma is critical to anti-tumor activity since IL-10 no longer inhibits tumor growth in hosts with a deletion in the IFN-gamma gene. Additionally, Gbp-1/Mag-1 and Mig-1 gene induction no longer occur in IFN-gamma mutant mice.     

Redirecting effector T cells through their IL-2 receptors

Fusion proteins constructed of a tumor-specific Ab joined to IL-2 (Ab-IL-2) have been used in the past to deliver cytokine directly to the site of tumor cells in vivo. These molecules mimic the activity of IL-2 and assist in activating and expanding antitumor effector cells. To enhance the cytolytic activity of CTL specific for peptide epitopes of the Her-2/neu tumor Ag presented by HLA-A*0201 molecules, a fusion protein was constructed consisting of a single chain Ab specific for Her-2/neu, linked to IL-2 (neu-Ab-IL-2). When added to a mixture of tumor cells and Her-2/neu-specific CTL, the protein was found to augment lysis of tumor cells. In addition, the hybrid molecule also promoted lysis of Her-2/neu expressing tumors by non-tumor-specific cloned T cell lines, including Th1 CD4 cells. Analysis of the mechanism of cytotoxicity revealed that the fusion protein mediates the formation of stable conjugates between T cells expressing IL-2R and tumor cells expressing Her-2/neu, resulting in lysis through the Fas-Fas ligand pathway. Lysis induction was independent of specific engagement by the TCR. When tested for its ability to enhance tumor cell eradication by Her-2/neu-specific CD8+ T cells in an adoptive transfer model in SCID mice, neu-Ab-IL-2 facilitated the elimination of tumor cells in vivo. Surprisingly, the combination of non-tumor-specific CD8+ T cells and fusion protein also induced a significant delay of tumor growth. This represents a novel approach for redirecting non-tumor-specific T cells to eliminate tumors.     

Antitumor efficacy of adenocarcinoma cells engineered to produce interleukin 12 (IL-12) or other cytokines compared with exogenous IL-12

BACKGROUND: Numerous animal model studies have examined the ability of genetically engineered tumor cells to release cytokines and to elicit an immune memory against the parental tumor. Often only a single cytokine is studied, and few comparative studies have been conducted. PURPOSE: We evaluated the antitumor efficacy of adenocarcinoma cells engineered to release interleukin (IL)-12 in a mouse model system. The efficacy of this cytokine was compared with that of other cytokines released by engineered adenocarcinoma cells and that of exogenous IL-12 administered both locally and intraperitoneally. METHODS: BALB/cAnCr mice were inoculated with syngeneic parental mammary adenocarcinoma (TSA) cells in quantities sufficient to lead to tumors in all inoculated mice. TSA cells engineered to release IL-12 (TSA-IL12) were also injected into normal and selectively immunosuppressed BALB/cAnCr mice. Tumor incidence, growth, and rejection patterns were evaluated by the measurement of neoplastic masses and by the study of the histologic and ultrastructural features of the tumor site. The effects of local or intraperitoneal administration of recombinant IL-12 (rIL-12) on tumor-bearing animals were also studied. RESULTS: Most mice rejected TSA-IL12 cells through a CD8-positive, T-lymphocyte-dependent reaction associated with macrophage infiltration, vessel damage, and necrosis. The systemic immunity of mice that had rejected TSA-IL12 cells to a subsequent challenge with parental TSA cells was less efficient than that elicited by TSA cells engineered to release IL-4 or IL-10 but equivalent to that elicited by TSA cells engineered to release IL-2, IL-7, and interferon alfa. Compared with TSA cells engineered to produce other cytokines, TSA-IL12 cells were the most efficient in curing mice with established TSA tumors; injection of 0.1 million proliferating cells contralaterally to the tumor growth area cured five of 15 mice bearing 1-day-old tumors; injection of the same dose of proliferating cells into the tumor growth area cured two of 20 tumor-bearing mice. However, two 5-day courses with a nontoxic dose (0.1 microgram) of rIL-12 given intraperitoneally cured a similar proportion of these animals (six of 20). Only two of 20 mice with 7-day-old TSA tumors were cured by vaccination with proliferating TSA-IL12 cells, whereas 24 of 30 mice with such tumors were cured by intraperitoneal administration of rIL-12. CONCLUSIONS: TSA cells engineered to release IL-12 are rejected by most mice; the ensuing immune memory for TSA parental cells, however, was less efficient than that elicited by proliferating TSA cells engineered to release other cytokines (e.g., IL-4, IL-10, and possibly interferon gamma). The immune reaction elicited by TSA-IL12 cells was the most efficient in curing mice with established TSA tumors; notably though, the same or a better cure rate was obtained with rIL-12 given intraperitoneally.     

A family of bicistronic vectors to enhance both local and systemic antitumor effects of HSVtk or cytokine expression in a murine melanoma model

The herpes simplex virus-thymidine kinase/ganciclovir (HSVtk/GCV) system produces both direct and immune-mediated tumor cell killing. Here, we compare the efficacy of HSVtk/GCV with cytokines, alone and in combination, on the tumorigenicity and immunogenicity of B16 cells. With respect to single gene modifications, only HSVtk/GCV, or high-level interleukin-2 (IL-2) secretion, completely prevented tumor growth, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) generated the best levels of long-term systemic protection. To augment both local killing and immune activation, we constructed bicistronic constructs that express HSVtk and a cytokine within the same cell. Co-expression of HSVtk with IL-2 or GM-CSF enhanced the local antitumor activity of any gene alone. In a tumor-prevention model, HSVtk killing, in an environment preprimed with GM-CSF, generated the best long-term immune protection. However, in a short-term therapy model, continued IL-2 expression was most effective against 3-day established tumors. This probably reflects differences in the activities of IL-2 and GM-CSF in generating short-term, nonspecific immune stimulation compared to long-term immunological memory, respectively. As a prelude to in vivo delivery experiments, we also demonstrated that these bicistronic cassettes can be packaged normally into retroviral (5 x 10(5) virus/ml from pooled populations) and adenoviral vectors (5 x 10(9) virus/ml) and function as predicted within virally infected cells. This family of bicistronic vectors can be used to stimulate synergy between suicide and cytokine genes, overcomes the problems of delivering two genes on separate vectors, and should allow easier preparation of vectors for the delivery of multiple genes to patients' tumor cells.     

Interleukin-10 gene transfer inhibits murine mammary tumors and elevates nitric oxide

Transfection of cDNA for IL-10 into line 66.1 murine mammary tumor cells results in marked suppression of tumor growth and metastasis. Others have reported that nitric oxide has potent antitumor activity and IL-10 is known to regulate the inducible isoform of nitric oxide synthase (iNOS) expressed in macrophages. We identified nitric oxide production in mammary tumors as indicated by electron paramagnetic resonance detection of nitric oxide-hemoglobin (NO-Hb). IL-10 expression resulted in elevated levels of NO-Hb in mammary tumors. Immunohistochemical examination of mammary tumors for iNOS protein revealed few positively staining cells in parental or control neo-transfected tumors but strong iNOS staining in all IL-10 transfected tumors, consistent with the NO-Hb data. To determine if mammary epithelial tumor cells themselves, express nitric oxide synthase activity, cultured tumor cells were treated with pro-inflammatory cytokines and nitrite accumulation was assessed in the conditioned medium. All IL-10 producing cell lines accumulated uM concentrations of nitrite in response to short term (24 hr) cytokine stimulation. Cells not expressing IL-10 (parental and neo-transfectants) accumulated no nitrite under similar culture conditions. After longer stimulation (48 hr), parental and 66-neo cells accumulated lower amounts of nitrite. IL-10 gene transfer is associated with increased iNOS protein expression and enzymatic activity detected both in vitro and in vivo. Our findings suggest that the antimetastatic and antitumor activity of IL-10 is related to enhanced production of nitric oxide.     

In situ cancer vaccination: an IL-12 defective vector/replication-competent herpes simplex virus combination induces local and systemic antitumor activity

Intratumoral inoculation of replication-competent, attenuated herpes simplex virus (HSV) mutants inhibits tumor growth by direct cytotoxic viral replication and induction of a tumor-specific immune response. To boost the antitumor response, we describe a defective HSV vector encoding IL-12 as an adjuvant to in situ vaccination by the replication-competent HSV helper virus. The defective HSV vector system consists of defective particles containing tandem repeats of the cytokine genes (p40 and p35) in combination with a HSV helper virus. Heterodimeric IL-12 was expressed and secreted after IL-12 defective vector infection of tumor cells. In a syngeneic, bilateral established tumor model with CT26 murine colon carcinoma, unilateral intratumoral inoculation with an IL-12 defective/replication-competent HSV vector combination significantly reduced tumor growth of the inoculated and noninoculated contralateral tumors. This antitumor effect was significantly greater than with a lacZ-defective/replication-competent HSV vector combination, which itself was significantly greater than the mock inoculation. Efficacy is associated with enhancement of tumor-specific CTL activity, including specificity against the CT26 immunodominant MHC class I restricted Ag AH1, and IFN-gamma production. There was no significant tumor growth inhibition after intratumoral inoculation of s.c. CT26 tumors in athymic mice. We conclude that this defective HSV vector system is an effective method for cytokine gene delivery to tumors in situ and IL-12 expression in tumors synergizes the antitumor activity mediated by the replication-competent HSV helper virus.     

Enhancement of interleukin-4-mediated tumor regression in athymic mice by in situ retroviral gene transfer

Intratumoral grafting of genetically engineered cells that produce interleukin-4 (IL-4) has been shown to produce tumor regression as well as prolong survival of mice harboring intracerebral gliomas. We sought to determine whether retroviral-mediated gene delivery into tumor cells in situ resulted in enhanced tumor regression by IL-4. Two mouse fibroblast lines were obtained: they both secreted similar levels of IL-4 but one produced a retrovirus vector bearing the IL-4 gene (CRE-MFG-IL-4 cells), whereas the other did not (NIH3T3-IL-4 cells). In mixed transplantation assays in the subcutaneous flanks of athymic mice, CRE-MFG, IL-4 cells were more effective than NIH3T3-IL-4 cells in inhibiting the growth of rat C6 glioma cells (p < 0.005, ANOVA). Subcutaneous tumors injected with fibroblasts that produced a control retrovirus vector without producing IL-4 (CRE-MFG-LacZ cells) did not inhibit subcutaneous tumor growth. An intracranial assay was used to evaluate survival of athymic mice harboring intracranial gliomas. Three days after implanting rat C6 glioma cells into the right frontal lobes of athymic mice, NIH3T3-IL-4 cells (n = 10) or CRE-MFG-IL-4 cells (n = 10) were stereotactically inoculated into the tumor bed. The average survival of mice treated with CRE-MFG-IL-4 cells was 38 days (+/- 2.4, SE), whereas that of mice treated with NIH3T3-IL-4 cells was 31 days (+/- 0.8, SE) (p < 0.005, ANOVA; p < 0.001, log-rank analysis).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo effects of locally secreted IL-10 on the murine antitumor immune response

BACKGROUND: Interleukin-10 (IL-10) is a cytokine secreted by the TH2 class of murine lymphocytes that suppresses the secretion of interferon-gamma (IFN-gamma) by TH1 lymphocytes and inhibits macrophage-mediated T-cell stimulation and cytotoxicity. The observation that IL-10 is produced by human carcinomas in vitro and in vivo led to the hypothesis that this cytokine plays a role in the suppression of the human anti-tumor immune response. We tested this hypothesis in a murine model. METHODS: To evaluate the effect of IL-10 on the induction of an anti-tumor immune response, mice were immunized with tumor cells transfected with the IL-10 gene and then challenged with parental tumor. The effect of the local secretion of IL-10 on an established immune response was tested by immunizing mice with parental tumor and then challenging with IL-10-secreting tumors. RESULTS: IL-10-secreting tumors were as effective immunogens as control tumors. Immune mice rejected IL-10-secreting tumors as readily as control challenge tumors. In an in vitro assay, IL-10 did not inhibit CD8 lymphocyte secretion of IFN-gamma in response to tumor stimulation. One IL-10-secreting tumor clone regressed when injected into naive mice and induced an antigen-specific immune response capable of protecting mice from subsequent tumor challenge. CONCLUSIONS: The local secretion of IL-10 did not inhibit either the induction of an antitumor immune response or the ability of established effector cells to reject challenge tumors. In contrast to its effect on TH1 lymphocytes, IL-10 does not inhibit IFN-gamma secretion by CD8 lymphocytes.     

A combination of IL-10 and direct contact with plasma cell tumors decreases CD23 expression on splenic B cells

We and others have previously found that splenic B cells from plasma cell tumor-bearing mice exhibit decreased CD23 expression. In the present study we further examined the mechanism of CD23 down-regulation by plasma cell tumors. We show here that although direct contact is required between the tumor cells and B cells, it is not sufficient, since fixed tumor cells do not induce the same reduction in CD23 expression. We have identified IL-10, a cytokine produce by the tumors, as the sole soluble factor that contributes to decreased CD23 expression on B cells induced by plasma cell tumors because 1) Abs to IL-10 prevent the loss of CD23 induced by plasma cell tumors both in vitro and in vivo; 2) engineered IL-10 negative variants of these tumors are reduced in their ability to down-regulate CD23 expression; 3) rIL-10 alone induces partial, but significant, decreases in CD23 expression on normal splenic B cells; and 4) the addition of IL-10 and fixed tumor cells to cultures of normal splenocytes decreases CD23 expression to levels similar to those in cocultures with live tumor cells. Collectively, these results demonstrate that plasma cell tumors down-regulate CD23 expression on B cells by a coordinate mechanism of IL-10 plus contact-mediated events and reveal a novel role for IL-10 in the regulation of CD23 expression on B cells that is suggestive of host B cell activation in the presence of the tumor.     

Antimetastatic vaccination against Lewis lung carcinoma with autologous tumor cells modified to express murine interleukin 12

Interleukin 12 (IL-12) is a disulfide-linked heterodimer molecule produced predominantly by professional antigen presenting cells. It promotes the induction of sundry biological effects with significant relevance to antitumor immunity, such as enhancing a T(H)1 helper response, an in vivo antiangiogenic effect, induction of adhesion molecules that assist in lymphocyte homing to sites of tumor growth, and a direct stimulatory effect on both T-cells and NK cells. We tested the efficacy of an antimetastatic vaccine composed of autologous murine D122 cells transfected with both subunits of IL-12 cDNA to express biologically-active IL-12 molecule. Expression of IL-12 by D122 cells significantly reduced their tumorigenicity and metastatic potential in immunocompetent syngeneic hosts. Furthermore, vaccination of mice with 2 x 10(6) irradiated IL-12-transfected D122 cells engendered a protective CTL response which rejected a subsequent challenge with parental D122 cells and eradicated lung micrometastasis in animals whose primary tumors have been surgically removed. The antitumor effects of IL-12 were mediated primarily by its ability to induce gammaIFN expression in vivo. CD8+ T-cells as well as NK cells were crucial in the execution of the antitumor effects of IL-12. These results suggest that autologous tumor cells expressing IL-12 by gene transfer are a potent antitumor vaccine able to induce a systemic immune response against poorly immunogenic and spontaneously metastatic tumors.     

Long-term connexin-mediated bystander effect in highly tumorigenic human cells in vivo in herpes simplex virus thymidine kinase/ganciclovir gene therapy

Gene therapy via the herpes simplex virus thymidine kinase (tk) gene and ganciclovir (GCV) treatment eliminates experimental tumors. In this approach, cells expressing the tk gene (tk+) and neighboring tumor cells which do not express the gene are killed. We have demonstrated this bystander effect is enhanced in vitro by gap junctional intercellular communication (GJIC). In order to extend our in vitro results into in vivo situations, we injected into nude mice different ratios of tk+/tk- HeLa cells, either lacking or transfected with connexin43 (Cx43), a gene coding for a gap junction protein. When GCV was administered before tumors were palpable, fewer animals developed tumors, even after a longer period, if the injected cells were mixtures of Cx43(+)-tk+ and Cx43(+)-tk- while tumor growth was not prevented with mixtures of HeLa cells not expressing Cx43, i.e. Cx43(+)-tk+/Cx43(-)-tk-. When GCV was given after the appearance of tumors, the size of the tumors from Cx43- cells was 30% reduced for 3 weeks if 50% of the injected cells were tk+. However, for cells expressing Cx43, the tumor size was 66% reduced if 10% of the cells were tk+. Such a reduction demonstrates a long-term bystander effect which is dependent on Cx43 expression.     

Adenovirus-mediated combined suicide gene and interleukin-2 gene therapy for the treatment of established tumor and induction of antitumor immunity

The antitumor effect of the combined transfer of a suicide gene and a cytokine gene was evaluated in the present study. Adenoviruses expressing Escherichia coli cytosine deaminase (AdCD) and adenoviruses expressing murine interleukin-2 (AdIL-2) were utilized for the treatment of established tumors. The mice were inoculated s.c. with FBL-3 erythroleukemia cells and 3 days later received an intratumoral injection of AdCD in the presence or absence of AdIL-2 followed by intraperitoneal 5-fluorocytosine (5-FC) administration. The results demonstrated that tumor-bearing mice treated with AdCD/5-FC in combination with AdIL-2 showed more potent inhibition of tumor growth and survived much longer than did mice treated with AdCD/5-FC, AdIL-2, adenovirus expressing beta-galactosidase/5-FC or phosphate-buffered saline. The tumor mass showed obvious necrosis and inflammatory cell infiltration, and more CD4+ and CD8+ T cells infiltrating the tumor after combined therapy. The splenic natural killer and cytotoxic T lymphocyte activities increased significantly in the mice after combined therapy with AdCD/5-FC/AdIL-2. Our results demonstrate that therapy combining a suicide gene and IL-2 gene can inhibit the growth of established tumors in mice significantly and induce antitumor immunity of the host efficiently.     

Interleukin 2 expression by tumor cells alters both the immune response and the tumor microenvironment

Microenvironmental conditions within solid tumors can have marked effects on the growth of the tumors and their response to therapies. The disorganized growth of tumors and their attendant vascular systems tends to result in areas of the tumors that are deficient in oxygen (hypoxic). Cells within these hypoxic areas are more resistant to conventional therapies such as radiation and chemotherapy. Here, we examine the hypoxic state of EMT6 mouse mammary tumors and the location of host cells within the different areas of the tumors to determine whether such microenvironmental conditions might also affect their ability to be recognized by the immune system. Hypoxia within tumors was quantified by flow cytometry and visualized by immunohistochemistry using a monoclonal antibody (ELK3-51) against cellular adducts of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetam ide (EF5), a nitroimidazole compound that binds selectively to hypoxic cells. Thy-1+ cells, quantified using a monoclonal antibody, were found only in the well-oxygenated areas. The location of these Thy-1+ cells was also examined in EMT6 tumors that had been transfected with the gene for interleukin-2 (IL-2) because these tumors contain greatly increased numbers of host cells. Surprisingly, we found that IL-2-transfected tumors had significantly decreased hypoxia compared to parental tumors. Furthermore, using the fluorescent dye Hoechst 33342, an in vivo marker of perfused vessels, combined with immunochemical staining of PECAM-1 (CD31) as a marker of tumor vasculature, we found increased vascularization in the IL-2-transfected tumors. Thus, expression of IL-2 at the site of tumor growth may enhance tumor immunity not only by inducing the generation of tumor-reactive CTLs but also by allowing increased infiltration of activated T cells into the tumors.     

The Friedreich's ataxia mutation confers cellular sensitivity to oxidant stress which is rescued by chelators of iron and calcium and inhibitors of apoptosis

Interleukin 12 (IL-12) exhibits anti-tumor activity in a variety of laboratory models. Although IL-12 itself activates strong anti-tumor activity, the combination of vaccine therapy with IL-2-transduced tumor cells and systemic rIL-12 has been shown to cure tumor-bearing mice more effectively than either rIL-12 or IL-2-transduced tumor vaccines alone. In the present study, regression of brain tumors established in naive mice was obtained by combined administration of an intratumoral injection of a single dose of IL-2-producing glioma cells (SR/IL-2 cells) and recombinant IL-12. Intraperitoneal rIL-12 administration substantially delayed the growth of s.c. inoculated gliomas, but not of gliomas located in the brain. Although vaccination with SR/IL-2 cells alone was not effective against s.c. inoculated gliomas, the combination therapy of vaccination with irradiated SR/IL-2 cells and systemic rIL-12 was more effective than rIL-12 alone. In our brain-tumor model, intratumoral administration of irradiated SR/IL-2 cells and of rIL-12 remarkably prolonged survival as compared with untreated mice. Efficacy was reduced when studies were performed in mice depleted of CD8+ cells or NK cells. Mice cured of their intracerebral tumors by combined administration of SR/IL-2 cells and rIL-12 demonstrated protective immunity upon rechallenge. In summary, the therapeutic potential for control of tumor growth by intratumoral administration of IL-2-producing glioma cells and rIL-12 may be useful in the development of treatment for patients with glioma.     

Tumors with reduced expression of a cytotoxic T lymphocyte recognized antigen lack immunogenicity but retain sensitivity to lysis by cytotoxic T lymphocytes

A murine solid tumor was transfected to express various levels of an allogeneic major histocompatibility complex class I gene (K216), in order to test the effect of the level of antigen expression on immunogenicity and sensitivity to lysis by cytotoxic T lymphocytes (CTL). The growth rates of clones of tumor cells expressing different levels of the transfected gene were similar in vitro and in nude mice. Although all tumor cells, including cells freshly isolated from growing tumors, were equally sensitive to lysis by specific CTL, only tumor cells expressing the highest level of the K216 antigen stimulated CTL and were rejected by normal mice. In contrast, tumor cells expressing lower levels of antigen failed to immunize for CTL and grew progressively in normal mice, despite retaining expression of the transfected gene and remaining fully sensitive to CTL-mediated lysis; thus, the threshold of antigen needed to stimulate CTL responses was considerably higher than that needed to lyse tumor cells. Reduction of K216 antigen expression from 100-fold to 40-fold above background, impaired significantly the ability of the tumor cells to induce a K216-specific immune response, while tumor cells expressing K216 at levels 2-fold above background were as susceptible to CTL-mediated lysis as tumor cells expressing 50-fold more antigen. The important implication of these findings is that some tumors occurring in nature may not be immunogenic but nevertheless express antigens which are potential targets for immune therapy.     

Spatial and temporal expression of angiogenic molecules during tumor growth and progression

The growth and metastasis of cancer directly correlates with tumor angiogenesis. A better understanding of the expression of regulatory factors controlling angiogenesis is important in exploiting this process therapeutically. Our present study demonstrates that small tumors (3-4 mm in diameter) express more basic fibroblast growth factor (bFGF) and interleukin 8 (IL-8) than large tumors (> 10 mm in diameter), whereas more vascular endothelial growth factor (VEGF) is expressed in large tumors. Immunostaining showed a heterogeneous distribution of angiogenic factors within the tumor; expression of bFGF and IL-8 was highest on the periphery of a large tumor, where cell division is maximum. VEGF expression was higher in the center of the tumor. In vitro studies demonstrated that sparse cultures of tumor cells expressed higher levels of bFGF and IL-8 than confluent cultures. In contrast, the expression of bFGF and IL-8 was not diminished in tumor cells growing on confluent monolayers of normal cells. VEGF expression was upregulated by cell density irrespective of contact with tumor cells or normal cells. These results demonstrate that the expression of different angiogenic factors in tumor cells can be regulated by their proximity to other tumor cells or host cells.     

A double recombinant adenovirus expressing the costimulatory molecule B7-1 (murine) and human IL-2 induces complete tumor regression in a murine breast adenocarcinoma model

Tumors that express tumor-specific antigens can maintain growth in an immunocompetent organism. Current hypotheses tend toward T cell anergy as a key component for the inhibition of immunoreactivity against such tumors. Anergy is thought to occur from hyperactive stimulation of the TCR in the absence of costimulation (costimulation leads to proliferation via IL-2 production). Subcutaneous injection of transgenic polyoma middle T transformed breast adenocarcinoma tumor cells (PyMT) in the hind flank of FVB/n mice results in the formation of tumor nodules at this site. We determined the MHC class I and class II, B7-1, and B7-2 expression in the tumor cells by flow cytometry and showed positive staining for only MHC class I. We show that a single E1-deleted adenovirus constructed to express both the costimulatory molecule B7-1 (murine) and human IL-2 genes (Ad5E1 mB7-1/human IL-2) elicits a very potent antitumor response when administered intratumorally. Ad5E1 mB7-1/human IL-2 induced rapid and complete regression (100%) of all tumors compared with Ad5 E1 mB7-1 (38%), Ad CAIL-2 (42%), and Ad5E1 dl70-3 (control vector) (0%). All mice that exhibited complete tumor regression were fully protected in tumor cell challenge experiments. The systemic immunity generated by intratumoral administration of the Ad vectors was associated with a strong anti-PyMT CTL response. These observations indicate that augmenting the immunogenicity of the tumor with coincident expression of B7-1 in combination with IL-2 may prove beneficial in direct tumor immunotherapy.     

Viral interleukin 10 (IL-10), the human herpes virus 4 cellular IL-10 homologue, induces local anergy to allogeneic and syngeneic tumors

After the cloning of murine cytokine synthesis inhibitory factor, it was recognized that a homologous open reading frame was encoded within the Epstein-Barr virus (human herpes virus 4). This viral protein has now been termed viral interleukin 10 (vIL-10) to reflect its protein sequence homology to "cellular" IL-10 (cIL-10, either murine or human IL-10). It is now widely accepted that vIL-10 shares many functions with cIL-10, principally, the ability to enhance survival of newly infected B cells and to diminish the production of IFN-gamma and IL-2 during ongoing immune reactions. The immunomodulatory effect of locally secreted vIL-10 and murine IL-10 (mIL-10) was examined in tumor models using CL8-1 (a BL6 melanoma cell line transfected with the H-2Kb class I gene) in syngeneic animals. Although parental BL6 tumor cells grow in immunocompetent syngeneic hosts, CL8-1 are rejected. To achieve local secretion of vIL-10, we generated vIL-10 retroviral vectors. While nontransduced CL8-1 cells (1 x 10(4)) failed to grow when injected intradermally in C57BL/6 mice, CL8-1 cells (1 x 10(4)) transduced with vIL-10 formed palpable tumors and eventually killed 80% of injected animals. Suppression of tumor rejection was also noted when CL8-1 tumors with or without vIL-10 transfection were admixed with syngeneic vIL-10-transfected fibroblasts and inoculated. Since the in vitro proliferation of the tumor was not altered after transduction with the vIL-10 gene and injection of vIL-10-transduced CL8-1 does not affect the rejection of nontransduced CL8-1 inoculated at a distant site, local vIL-10 secretion appears to suppress the process of immune rejection of the target cells in a dose-dependent manner. Similar results were observed for the H-2b MCA105 sarcoma tumor model in allogeneic BALB/c mice (H-2d). Although all animals that received nontransfected MCA105 rapidly rejected these tumors, MCA105 sarcomas transfected with vIL-10 remained palpable for up to 37 d. The local immunosuppressive effect of gene-delivered vIL-10 could be neutralized by anti-human IL-10 monoclonal antibody or could be reversed by the systemic administration of IL-2 or IL-12. In marked contrast, mIL-10 transfection of CL8-1 significantly suppressed tumor growth and frequently led to the rejection of tumor. Similar results were obtained for the murine tumor cell lines MCA102.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intratumoral coinjection of adenoviral vectors expressing IL-2 and IL-12 results in enhanced frequency of regression of injected and untreated distal tumors

We have studied the ability of adenoviral (Ad) vectors expressing the cytokines IL-2 or IL-12 to mediate regression of established tumors in a mouse model of mammary adenocarcinoma. Previous results indicated that intratumoral injection of vectors expressing IL-2 (AdCAIL-2), or IL-12 (AdmIL-12.1) induced complete tumor regression in approximately 30-40% of treated animals. In the current studies, we investigated the mechanism of tumor killing in responding animals and the efficacy of AdIL-2 and AdIL-12 vector administration in combination compared with the use of either vector alone. Animals bearing subcutaneous mammary tumors were injected intratumorally with Ad vectors expressing IL-2 or IL-12 or were coinjected with both vectors. Animals receiving the combination treatment responded substantially better than animals which had received either vector alone, with 65% of animals treated with both vectors undergoing complete tumor regression. In all three treatment regimens, tumor regression was associated with the presence of specific antitumor antigen cytotoxic T-lymphocytes (CTLs), which secreted elevated levels of IFN-gamma. Consistent with circulating CTLs being involved in regression, when animals bearing bilateral tumors were inoculated in a single tumor with IL-2 or IL-12 expressing vectors, both tumors regressed in many cases. Again, treatment with both AdCAIL-2 and AdmIL-12.1 was most effective, with 63% of animals undergoing complete regression of both treated and untreated tumors, compared to 18 or 22% of animals injected with either AdCAIL-2 or AdmIL-12.1 alone. These data indicate that the combination of IL-2 and IL-12 is a more effective inducer of antitumor immune responses than either one alone, and that the resulting antitumor responses are effective in mediating the regression of distal untreated tumors, a property which may aid in the treatment of metastatic disease.