Radiotherapy for genes that cause cancer.

Many approaches to cancer gene radiotherapy are possible. Even existing strategies, devised with other goals in mind, can cause radiosensitization by altering the intrinsic radiosensitivity of tumour cells or by modifying the tumour microenvironment. Small increases in the levels of radiosensitization could, over a fractionated radiation course, have major effects on the probability of tumour cure. The clinical application of gene radiotherapy will require use of in vivo gene delivery systems that still need validation but, because radiation is effective at decreasing tumour burden, genes will not have to be expressed in all cells in order to achieve a cure.

Molecular cloning of CD44R1 and CD44R2, two novel isoforms of the human CD44 lymphocyte "homing" receptor expressed by hemopoietic cells.

In addition to the 85-95 kD CD44 species found on most hemopoietic cell types, the human myelomonocytic cell line KG1a expresses proteins of approximately 115 kD and 130 kD that react with monoclonal antibodies belonging to CD44. The possibility that these higher molecular weight species may represent novel CD44 isoforms containing additional protein sequence was investigated. CD44 cDNA clones were isolated from a plasmid-based expression library prepared from KG1a mRNA. One of the three clones obtained (clone 2.3) was found to encode a CD44 molecule of approximately 130 kD in transfected COS cells. Sequences analysis indicated that the molecule encoded by this cDNA clone, designated CD44R1, was essentially identical to CD44 except for the presence of an additional 132 amino acids inserted into the extracellular domain. This inserted region is rich in serine and threonine residues that may serve as sites of O-linked glycosylation, and contains a potential site of N-linked glycosylation and a potential site of chondroitin sulphate attachment. PCR analysis using primers that flank the inserted region present within CD44R1 identified an additional CD44 isoform, designated CD44R2, that contains only the last 69 amino acids present within the unique region of CD44R1. Peripheral blood mononuclear cells and granulocytes from normal individuals and patients with chronic myelogenous leukemia, polycythemia vera, or acute myelomonocytic leukemia, express both CD44R1 and CD44R2. In contrast, CD44R1 and CD44R2 appear to be differentially expressed in various CD44-positive cell lines. Thus KG1a, and the Epstein-Barr Virus-transformed B cell lines WalkDR4 and Way-1 express both CD44 and the CD44 isoforms CD44R1 and CD44R2, while the myeloid cell lines HL60 and U937 express high levels of CD44, but only very low levels of CD44R1 and CD44R2. The CD44-negative cell lines DHL-4, DHL-10, Jurkat, and K562 are also negative for CD44R1 and CD44R2.

Hyaluronic acid capsule modulates M protein-mediated adherence and acts as a ligand for attachment of group A Streptococcus to CD44 on human keratinocytes.

We used wild-type and isogenic mutant strains of group A Streptococcus (GAS) that expressed M protein, capsule, or both to study the function of M protein and the hyaluronic acid capsular polysaccharide in attachment of GAS to human keratinocytes. Types 6 and 24, but not type 18, M protein were found to mediate attachment of GAS to soft palate or skin keratinocytes, but this interaction was prevented by the hyaluronic acid capsule on highly encapsulated, or mucoid, strains. Monoclonal antibody to CD44, the principal hyaluronic acid-binding receptor on keratinocytes, inhibited attachment of both highly encapsulated and poorly encapsulated wild type strains of GAS, but not the attachment of acapsular mutants. Transfection of K562 cells with cDNA encoding human CD44 conferred the capacity to bind each of six wild-type strains of GAS, but not to bind acapsular mutants. Because, in contrast to other potential adhesins, the group A streptococcal capsule is both highly conserved and surface-exposed, it may serve as a universal adhesin for attachment of diverse strains of GAS to keratinocytes of the pharyngeal mucosa and the skin.

Accessory cell activity of murine tumor-associated macrophages.

The accessory cell activity of macrophages associated with the murine 3-methylcholanthrene-induced fibrosarcoma FSa was investigated. On the basis of Fc receptor expression and phagocytic activity, 20-25% of cells present within enzymatically disaggregated tumor cell suspensions could be classified as macrophages. These cells were approximately 50% I-Ak positive but did not express the Mac-1 antigen. T-cells played an important role in regulating I-Ak expression, and macrophages obtained from tumors grown in nu/nu mice were I-Ak negative. Tumor-associated macrophages were shown to possess potent accessory cell activity and were fully capable of reconstituting the primary anti-calf red blood cell plaque-forming cell (PFC) response of Sephadex G-10-passed spleen cells. This function required the presence of the I-Ak-positive subpopulation, and macrophages treated with anti-Ia serum and complement or obtained from tumors grown in nu/nu hosts lacked accessory cell activity. Tumor-associated macrophages were also able to provide the essential accessory cell function required for cooperation between tumor-specific TH cells and normal B-cells in the generation of an anti-trinitrophenyl (TNP) PFC response in the presence of TNP-coupled FSa antigen. These results suggest that progressive growth of the FSa tumor in vivo cannot be readily attributed to a defect in the accessory cell function of tumor-associated macrophages.

Down-regulation of murine fibrosarcoma transforming growth factor-beta 1 expression by interleukin 7.

BACKGROUND: Cytokine genes encode proteins that modulate immune system responses. Modification of tumor cells by the introduction of cytokine genes has been used as a strategy to augment host immunity. Interleukin 7 (IL-7) gene transfer enhances the immune response to tumor cells and can result in tumor regression. Transforming growth factor-beta 1 (TGF-beta 1) is a potent immunosuppressive cytokine produced by many tumors. We have previously reported that recombinant IL-7 decreases the expression of TGF-beta 1 by murine macrophages. PURPOSE: This study investigates the inhibition of tumor-derived TGF-beta 1 production as a possible mechanism for the enhanced antitumor immunity that accompanies IL-7 gene transfer. METHODS: A fibrosarcoma cell line (FSA-JmIL-7) genetically modified to produce IL-7 was used to evaluate the effects of IL-7 on tumor production of TGF-beta 1. The control cell line (FSA-Jneo) originated from the same parental fibrosarcoma cell line (FSA) and was produced by transduction with the same retroviral vector without the IL-7 gene. FSA-Jneo and FSA-JmIL-7 tumor cells were evaluated for the expression of TGF-beta 1 messenger RNA (mRNA). To determine if the observed change in TGF-beta 1 mRNA was associated with an alteration in protein secretion, we compared supernatants from tumor cell cultures for TGF-beta 1 production. Specific anti-TGF-beta 1 monoclonal antibody (MAb) was used to confirm the role of TGF-beta 1 in these assays. RESULTS: Compared with FSA parental and FSA-Jneo cells, FSA-JmIL-7 cells expressed TGF-beta 1 mRNA at a lower level. Compared with supernatants from FSA-Jneo cells, FSA-JmIL-7 supernatants contained consistently lower levels of TGF-beta 1 activity (P < .05). In addition, FSA-Jneo supernatants suppressed lymphocyte proliferation to a significantly greater degree than supernatants from FSA-JmIL-7 cells (P < .05). Studies with anti-TGF-beta 1 MAb added to the supernatants confirmed the role of TGF-beta 1 in inhibition of lymphocyte proliferation. CONCLUSION: These findings suggest that IL-7 gene transfer inhibits the production of TGF-beta 1 by tumor cells and thus may enhance the efficacy of the host's antitumor immune response. IMPLICATION: The regulation of endogenous tumor-derived cytokines in response to cytokine gene transfer may contribute to altered immune responses in the tumor microenvironment and thus may be an important additional parameter to assess in gene therapy.

Photodynamic therapy-mediated immune response against subcutaneous mouse tumors.

The curative ability of photodynamic therapy (PDT) is severely compromised if treated tumors are growing in immunodeficient hosts. Reconstitution of severe combined immunodeficient (scid) mice with splenocytes from naive immunologically intact BALB/c mice did not improve the response to Photofrin-based PDT of EMT6 tumors growing in these animals. In contrast, adoptive transfer of BALB/c splenocytes containing EMT6 tumor-sensitized immune cells had a dramatic effect on tumor regrowth after PDT. For instance, full restoration of the curative effect of PDT was achieved with scid mice that received splenocytes from BALB/c donors that were cured of EMT6 tumors by PDT 5 weeks before adoptive transfer. Splenocytes obtained from donors cured of EMT6 tumors using X-rays were much less effective. Selective in vitro depletion of specific T-cell populations from engrafting splenocytes indicated that CTLs are the main immune effector cells responsible for conferring the curative outcome to PDT in this experimental model, whereas helper T lymphocytes play a supportive role. The immune specificity of these T-cell populations was demonstrated by the absence of cross-reactivity between the EMT6 and Meth-A tumor models (mismatch between tumors growing in splenocyte donors and recipients). The immunocompetent BALB/c mice that received adoptively transferred splenocytes containing PDT-generated, tumor-sensitized immune cells also benefited from the improved outcome of PDT of tumors they were bearing. This was demonstrated not only with the fairly immunogenic EMT6 tumor model but also with weakly immunogenic Line 1 carcinomas. The results of this study indicate that PDT is a highly effective means of generating tumor-sensitized immune cells that can be recovered from lymphoid sites distant to the treated tumor at protracted time intervals after PDT, which asserts their immune memory character. It is also shown that the treatment of tumors by PDT creates the conditions necessary for converting the inactive adoptively transferred pre-effector, tumor-sensitized immune cells into fully functional antitumor effector cells. An additional finding of this study is the evidence of NK cell activation in PDT-treated Meth-A sarcomas.

The role of host lymphoid populations in the response of mouse EMT6 tumor to photodynamic therapy.

Photodynamic therapy (PDT) treatment of murine EMT6 mammary sarcoma using Photofrin (10 mg/kg) and light (110 J/cm2) cured all these lesions growing in syngeneic BALB/c mice. In contrast, the same treatment produced initial ablation but no long-term cures of EMT6 tumors growing in either scid or nude mice, the immunodeficient strains sharing the same genetic background with BALB/c mice. No difference was detected in either the level of Photofrin accumulated per g of tumor tissue or the extent of tumor cell killing during the first 24 h after PDT of EMT6 tumors growing in BALB/c or scid mice. The assumption that the difference in tumor cures could be ascribed to the absence of functional lymphoid cells in scid and nude mice was supported by the results of experiments involving the adoptive T-cell transfer into scid mice or bone marrow transfer between BALB/c and scid mice. The adoptive transfer of splenic virgin T lymphocytes from BALB/c mice into scid mice performed 9 days before PDT of EMT6 tumors growing in the recipients was successful in delaying the recurrence of treated tumors. Adoptive transfer done immediately after PDT or 7 days after PDT had no obvious benefit. Even better improvement and a high cure rate of PDT-treated tumors was obtained with scid mice reconstituted with BALB/c bone marrow. In contrast, a marked drop in tumor cure rate was observed with BALB/c mice reconstituted with scid bone marrow. These results suggest that the activity of host lymphoid populations was essential for preventing the recurrence of EMT6 tumors following the PDT treatment used in this study. The contribution of PDT-induced immune reaction may, therefore, be of critical importance for the cure with at least some tumors.

Potentiation of photodynamic therapy-elicited antitumor response by localized treatment with granulocyte-macrophage colony-stimulating factor.

Murine squamous cell carcinoma (SCCVII) cells were genetically engineered to produce marine granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF immunotherapy, based on the peritumoral injection of lethally irradiated GM-CSF-producing SCCVII cells, was examined as adjuvant to photodynamic therapy (PDT) treatment of this tumor. The GM-CSF immunotherapy administered three times in 48-h intervals, starting 2 days before the light treatment, substantially improved the curative effect of Photofrin-mediated PDT. A comparable effect of GM-CSF immunotherapy was observed in the combination with benzoporphyrin derivative-mediated PDT. The tumor-localized GM-CSF immunotherapy alone had no obvious effect on the growth of parental SCCVII tumors. This treatment did not significantly alter the differential peripheral WBC count and appeared not to affect tumor leukocyte infiltration. However, GM-CSF treatment did increase the cytotoxic activity of tumor-associated macrophages against SCCVII tumor cells. It appears, therefore, that tumor-localized immune stimulation by GM-CSF amplifies a PDT-induced antitumor immune reaction, which has a potentiating effect on tumor control.

Effects of IL-3 gene expression on tumor response to irradiation in vitro and in vivo.

Expression of a murine interleukin 3 gene in murine fibrosarcoma cells (FSA-JmIL-3) did not alter their survival after in vitro irradiation. However, FSA-JmIL-3 tumors established in vivo were much more sensitive to irradiation than was the parental tumor. Following 25 Gy of irradiation, parental fibrosarcoma tumors regrew after a growth delay of 10 days, but FSA-JmIL-3 tumors continued to regress. Examination of the cellular composition of tumors following irradiation revealed that, instead of tumor cell repopulation, the FSA-JmIL-3 tumors became heavily infiltrated with lymphocytes, indicating that the effect of irradiation was to allow the IL-3-elicited cellular immune response to infiltrate the tumors and mediate rejection. This study indicates that combining gene immunotherapy approaches with radiotherapy might increase the effectiveness of both, and it seems logical to pursue such treatment options.

Genetic modification of a murine fibrosarcoma to produce interleukin 7 stimulates host cell infiltration and tumor immunity.

Retroviral-mediated gene transfer was used to introduce and express the gene for murine interleukin 7 (IL-7) in a fibrosarcoma tumor (FSA). The tumorigenicity of these genetically modified FSA cells was greatly decreased in immunologically intact syngeneic mice but was unaltered in T-cell-deprived mice. IL-7-infected tumors that did grow in intact animals from large size inocula did so slowly and had a high incidence of spontaneous regression. Furthermore, mice that had rejected tumors became specifically immune to challenge with uninfected parental tumor cells. IL-7-infected FSA growing in intact mice were heavily infiltrated with host T-cells that were presumably responsible for slow growth and tumor regression, and tumor cells were in the minority. Fluorescence-activated cell sorter analysis showed that there was a 530% increase in T-cells in IL-7-infected FSA compared with control tumors. CD8+ T-cells were particularly elevated, but CD4+ lymphocytes were also increased in number, as were eosinophils and basophils. The CD4+:CD8+ ratio in IL-7-infected FSA was 1:1.7 in comparison to 1:0.6 in control tumors. Lymphocytes isolated from IL-7-producing tumors had greatly enhanced cytotoxicity towards uninfected, parental FSA cells. Killing of non-cross-reacting fibrosarcoma line was also increased but to a much lesser extent. Injection of recombinant human IL-7 directly into established FSA tumors slowed their growth and, in a significant number of instances, caused complete regression. Mice that had rejected tumor became specifically immune. The dose that was needed for this effect was, however, somewhat large: 20 micrograms twice daily for 10 days. This result contrasts with the efficacy of IL-7 gene infection in stimulating responses to the same tumor. These considerations make IL-7 a good candidate for tumor-directed cytokine gene therapy.

Altered patterns of CD44 epitope expression in human chronic and acute myeloid leukemia.

Abnormal expression of different isoforms of CD44 has been found to characterize many types of malignant cells although data for human acute and chronic myeloid leukemia is limited. In this study, we have identified significant, albeit variable, increases in these diseases of the frequency of both light density and CD34+ cells expressing two particular CD44 epitopes, neither of which is commonly found on normal human marrow cells. One of these epitopes is unique to exon v10-containing isoforms of CD44. The other is located in the common region of CD44 and was previously revealed on T cells only after their activation. Interestingly, another T cell activation-associated epitope was found to be expressed on a high proportion of normal marrow cells including the CD34+ subset and this remained the case for most of the primary leukemic samples evaluated. As expected, >90% of cells in all primary normal and leukemic samples expressed high levels of CD44, as shown by their reactivity with an antibody specific for the CD44 hyaluronan-binding site. To begin investigating how expression of the CD44 epitopes seen more commonly on leukemic than on normal CD34+ cells may be modulated, and to identify potentially associated effects on the hyaluronan-binding ability of the CD44 expressed, the effect of phorbol ester treatment on these properties of CD44 were examined. For these studies, a panel of five different human leukemic cell lines that were found to exhibit different patterns of CD44 expression and function in the absence of phorbol ester were used. Both the level and the hyaluronan-binding properties of CD44 could be stimulated in some, but not all, of these leukemic cell lines. Taken together, our findings indicate that CD44 expression is perturbed in a variety of leukemic populations suggesting a possible relationship to some of the pathogenetic features they share.

Mechanisms mediating the effects of IL-3 gene expression on tumor growth.

IL-3 gene expression within tumors leads to host-cell infiltration, particularly by macrophages, slower tumor growth, and enhanced immunogenicity. Surprisingly, tumor-associated macrophages (TAMs) from within FSAN-JmIL3 tumors had decreased expression of TNF-alpha and iNOS. On short-term culture, TAMs from FSAN-JmIL3 tumors regained their capacity to produce TNF-alpha and NO, indicating that they were primed in vivo. In vitro experiments were unable to demonstrate differences between FSAN-JmIL3 and FSAN tumor cells in their ability to stimulate TNF-alpha production by TAMs. In the absence of evidence that TAM activation was responsible for the slower growth of FSAN-JmIL3 tumors, the response of tumor cells to these effector molecules was studied. TNF-alpha and NO were cytotoxic for FSAN-JmIL3 cells but growth stimulatory for FSAN. These tumor-related phenotypic changes may contribute as much if not more than functional changes in host infiltrating cells to the slower growth of FSAN-JmIL3 tumors in vivo.

Identification and characterization of 114/A10, an antigen highly expressed on the surface of murine myeloid and erythroid progenitor cells and IL-3-dependent cell lines.

Monoclonal antibody (mAb) 114/A10, raised against the murine bone marrow-derived multipotential hemopoietic progenitor cell line B6SUtA, identifies an antigen highly expressed by various interleukin-3 (IL-3)-dependent cell lines, the myelomonocytic cell line WEHI-3, and a large proportion of primary myeloid and erythroid colony-forming cells. Spleen- and bone marrow-derived 114/A10-positive cells were shown to selectively proliferate in vitro in response to pokeweed mitogen-stimulated spleen cell-conditioned medium or recombinant IL-3. Western blot analysis indicated that the antigen recognized by mAb 114/A10 has a mean relative molecular mass of approximately 150,000, although it is extremely heterogeneous in nature, and differs greatly in size range among different cell lines.

Identification and characterization of CD44RC, a novel alternatively spliced soluble CD44 isoform that can potentiate the hyaluronan binding activity of cell surface CD44.

Soluble CD44 proteins generated by proteolytic cleavage or aberrant intron retention have been shown to antagonize the ligand binding activity of the corresponding cell surface receptor, inducing apoptosis and inhibiting tumor growth. Interestingly, such findings appear to contradict recent studies demonstrating a correlation between the presence of high levels of soluble CD44 in the serum of cancer patients and poor prognosis. In the present study, we report the cloning of a novel, naturally occurring, differentially expressed, soluble CD44 isoform, designated CD44RC, which, in contrast to previously described soluble CD44 proteins, can dramatically enhance the hyaluronan binding activity of cell surface CD44. Sequence analysis suggests that CD44RC is generated by an alternative splicing event in which the 3' end of CD44 exon 2 is spliced into an internal splice acceptor site present within exon 18, altering reading frame and giving rise to a soluble protein with a unique COOH terminus. Functional studies suggest that CD44RC enhances hyaluronan binding by adhering to chondroitin sulfate side-chains attached to cell surface CD44, generating a multivalent complex with increased avidity for hyaluronan.

Combining radiation therapy with interleukin-3 gene immunotherapy.

The goal of this study was to explore immunological strategies to increase local and systemic tumor control in patients receiving radiation therapy. In previous studies, interleukin-3 (IL-3) gene expression within murine tumors was shown to increase their response to irradiation through immune mechanisms. In this study, the efficacy of systemically administered IL-3 gene-transduced irradiated tumor cell vaccines was tested for their ability to augment radiation responses against established immunogenic (FSAR) and nonimmunogenic (FSAN) tumors. Vaccines of irradiated FSAR/FSAN or FSAN-JmIL-3/FSAR-JmIL-3 cells were given intraperitoneally just before and after local irradiation of parental tumors with diameters of 8 mm, as well as in two booster doses. The IL-3 gene-transduced tumor cell vaccines were more effective than the parental vaccines at delaying tumor growth after irradiation, although no complete cures resulted. Responses were largely specific to the tumor type, indicating that tumor-specific immunity was enhanced by IL-3 vaccine administration. When the experiment was repeated in the C3H/HeJ mice, which are deficient in tumor necrosis factor-alpha production, the vaccines were still effective, but less so than in C3H/HeN mice. Systemic IL-3 vaccine treatment increased intratumoral levels of intercellular adhesion molecule-1, Mac-1, EB22/5.3, tumor necrosis factor-alpha, and IL-1 mRNA in irradiated tumors, indicating that cellular infiltration was part of the response. The study demonstrates that local radiation therapy can enhance the efficacy of genetically altered vaccine-based immunotherapy for cancer by decreasing tumor burden. At the same time, tumor cell vaccines may improve the cure rate of local radiation therapy by eliminating residual cancer cells. Although less effective than intratumoral gene expression, administration of IL-3 gene-transduced tumor cell vaccines is clinically a more feasible strategy that may be useful in situations in which the tumor load is small.

Molecular mechanisms regulating TNF-alpha production by tumor-associated macrophages.

The molecular mechanisms that regulate the production and/or functional activity of intratumoral tumor necrosis factor-alpha (TNF-alpha) remain poorly defined. To begin to address this issue we have examined the level of TNF-alpha mRNA and protein produced by macrophages present within immunogenic Fsa-R and non-immunogenic Fsa-N tumors grown in syngeneic Lps(d) C3H/HeJ and Lps(n) C3H/HeN mice. The results obtained indicate that macrophages isolated from tumors grown in Lps(d) C3H/HeJ mice express 5-10-fold less TNF-alpha than equivalent cells present in tumors grown in Lps(n) C3H/HeN mice. These data suggest that the mechanisms that operate within the tumor microenvironment to induce the production of TNF-alpha act, at least in part, via the same signal transduction pathway that is defective in Lps(d) C3H/HeJ mice. Interestingly, despite such differences in TNF-alpha production, tumors inoculated into C3H/HeJ and C3H/HeN mice grew at a similar rate and contained an almost identical proportion of macrophages. Moreover, tumor cells purified from tumors grown in C3H/HeJ and C3H/HeN mice produced similar quantities of the TNF-alpha-inducible cytokine GM-CSF. Thus, although differences in the level of TNF-alpha produced within tumors grown in C3H/HeN and C3H/HeJ mice are readily demonstrable, such differences appear to have little direct impact on the outcome of tumor growth.

Targeting gene therapy to cancer: a review.

In recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root. This is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide. This report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment. In order to specifically target malignant cells while at the same time sparing normal tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation. Although the efficient delivery of DNA to tumor sites remains a formidable task, progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods. In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be combined in the future for effective therapy. To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements. Another novel possibility that will be discussed is the regulation of therapeutic gene products by tumor-specific gene splicing. Gene expression could also be targeted at conditions specific to the tumor microenvironment, such as glucose deprivation and hypoxia. We have concentrated on hypoxia-targeted gene expression and this report will discuss our progress in detail. Chronic hypoxia occurs in tissue that is more than 100-200 microns away from a functional blood supply. In solid tumors hypoxia is widespread both because cancer cells are more prolific than the invading endothelial cells that make up the blood vessels and because the newly formed blood supply is disorganized. Measurements of oxygen partial pressure in patients' tumors showed a high percentage of severe hypoxia readings (less than 2.5 mmHg), readings not seen in normal tissue. This is a major problem in the treatment of cancer, because hypoxic cells are resistant to radiotherapy and often to chemotherapy. However, severe hypoxia is also a physiological condition specific to tumors, which makes it a potentially exploitable target. We have utilized hypoxia response elements (HRE) derived from the oxygen-regulated phosphoglycerate kinase gene to control gene expression in human tumor cells in vitro and in experimental tumors. The list of genes that have been considered for use in the treatment of cancer is extensive. It includes cytokines and costimulatory cell surface molecules intended to induce an effective systemic immune response against tumor antigens that would not otherwise develop. Other inventive strategies include the use of internally expressed antibodies to target oncogenic proteins (intrabodies) and the use of antisense technology (antisense oligonucleotides, antigenes, and ribozymes). This report will concentrate more on novel genes encoding prodrug activating enzymes, so-called suicide genes (Herpes simplex virus thymidine kinase, Escherichia coli nitroreductase, E. (ABSTRACT TRUNCATED)

The effects of cytokine gene transfer into tumors on host cell infiltration and regression.

New strategies are becoming available that promise to revolutionize cancer immunotherapy. Although the task of generating what is in essence a pathogenic autoimmune anti-tumor response in the face of local and systemic immune suppression is likely to remain a formidable one, advances in molecular strategies for enhancing tumor immunity have been made that show considerable promise, in particular those based on gene transfer technology. For example, introduction of certain cytokine genes into murine tumor cells have been shown to enhance tumor immunogenicity and induce regression. Caution is needed in properly interpreting the relevance of observations derived from murine models for human cancer, but clinical trials are underway that will test the utility of cytokine gene therapy for cancer and that will generate data that will be useful for the design of future strategies. Because of the magnitude of the problem of inducing tumor regression, it is argued that, even if genetically engineering can be used to successfully enhance anti-tumor immunity, combination of such strategies with other existing conventional anti-cancer therapies, that increase the effectiveness of both, may be necessary to reliably achieve cure.