Genetic Modification Of Tumor Cells Research Articles

Non-viral Delivery Systems for Breast Cancer Gene Therapy.

The ever-evolving field of gene therapy promises several innovative treatments for cancer. Advances in genetic modification of tumor cells and micro environment have led to the development of more effective therapeutic strategies with fewer side effects. The development of effective delivery system challenges, remains. Non-viral vectors are interesting due to their bio-safety and their ability to transfer different types of nucleic acids. Examples of these techniques are the use of oligonucleoides, liposomes, nanoparticles, inorganic material, and engineered stem cells. In this review, we focus on recent advances in the intracellular delivery of DNA and siRNA to the cancer cells with emphasis on breast cancer.

Radiotherapy and MVA-MUC1-IL-2 vaccine act synergistically for inducing specific immunity to MUC-1 tumor antigen

BackgroundWe previously demonstrated that tumor irradiation potentiates cancer vaccines using genetic modification of tumor cells in murine tumor models. To investigate whether tumor irradiation augments the immune response to MUC1 tumor antigen, we have tested the efficacy of tumor irradiation combined with an MVA-MUC1-IL2 cancer vaccine (Transgene TG4010) for murine renal adenocarcinoma (Renca) cells transfected with MUC1.MethodsEstablished subcutaneous Renca-MUC1 tumors were treated with 8 Gy radiation on day 11 and peritumoral injections of MVA-MUC1-IL2 vector on day 12 and 17, or using a reverse sequence of vaccine followed by radiation. Growth delays were monitored by tumor measurements and histological responses were evaluated by immunohistochemistry. Specific immunity was assessed by challenge with Renca-MUC1 cells. Generation of tumor-specific T cells was detected by IFN-γ production from splenocytes stimulated in vitro with tumor lysates using ELISPOT assays.ResultsTumor growth delays observed by tumor irradiation combined with MVA-MUC1-IL-2 vaccine were significantly more prolonged than those observed by vaccine, radiation, or radiation with MVA empty vector. The sequence of cancer vaccine followed by radiation two days later resulted in 55–58% complete responders and 60% mouse long-term survival. This sequence was more effective than that of radiation followed by vaccine leading to 24–30% complete responders and 30% mouse survival. Responding mice were immune to challenge with Renca-MUC1 cells, indicating the induction of specific tumor immunity. Histology studies of regressing tumors at 1 week after therapy, revealed extensive tumor destruction and a heavy infiltration of CD45+ leukocytes including F4/80+ macrophages, CD8+ cytotoxic T cells and CD4+ helper T cells. The generation of tumor-specific T cells by combined therapy was confirmed by IFN-γ secretion in tumor-stimulated splenocytes. An abscopal effect was measured by rejection of an untreated tumor on the contralateral flank to the tumor treated with radiation and vaccine.ConclusionsThese findings suggest that cancer vaccine given prior to local tumor irradiation augments an immune response targeted at tumor antigens that results in specific anti-tumor immunity. These findings support further exploration of the combination of radiotherapy with cancer vaccines for the treatment of cancer.

Abstract B185: Induction of specific immunity to MUC1 antigen by tumor irradiation and cancer vaccines in murine tumor models

Abstract We have previously demonstrated that tumor irradiation potentiates cancer vaccines using genetic modification of tumor cells in murine tumor models. We showed that prior tumor irradiation enhanced the response of mice to intratumoral gene therapy with an IL-2 adenovector for non-immunogenic tumors. To investigate whether tumor irradiation augments the immune response to a specific tumor antigen, we have now tested the efficacy of tumor irradiation to enhance the therapeutic effect of MVA-MUC1-IL2 cancer vaccine (Transgene TG4010) for the treatment of murine renal adenocarcinoma Renca cells transfected with MUC1. Established subcutaneous (s.c.) Renca-MUC1 tumors (18mm3) were irradiated with 8 Gy on day 11 and peritumoral s.c. injections of MVA-MUC1-IL2 vector were administered at 106- 107 PFU on day 12 and 20. Tumor growth delays were monitored by tumor measurements and histological responses were evaluated following treatment with radiation alone, vector alone, radiation + MVA-MUC1-IL2 vector or radiation + MVA empty vector. Histological evaluation of tumors at an early time point, by 2-3 weeks after radiation and cancer vaccine, revealed that tumors treated with radiation and vaccine showed extensive areas of necrosis due to complete tumor destruction. Intense hemorrhages were observed due to disruption of tumor vasculature. Small areas of remaining tumor showed apoptotic tumor cells and degenerating giant tumor cells typical of radiation-induced changes. At the periphery of the tumor and infiltrating the tumor, we observed large bands of inflammatory cells including lymphocytes and macrophages as well as fibroblasts spindled shaped cells. Immuno-histochemical staining with CD45 leukocyte marker and F4/80 macrophage marker confirmed extensive infiltration of leukocytes and macrophages at the periphery and inside of areas of tumor destruction. These alterations were not as pronounced with radiation alone suggesting a drastic effect of the combined radiation + vaccine therapy on the tumor microenvironment. Radiation induced tumor growth delays for about 15 days but longer tumor growth delays of 30-35 days were observed with radiation + MVA-MUC1-IL2. By day 55, over 30% of the mice treated with radiation + MVA-MUC1-IL2 had a complete response with no sign of tumor whereas no responders were observed with either radiation alone or cancer vaccine alone. Complete responders were immune to rechallenge with Renca-MUC1 cells. These findings suggest that tumor irradiation given prior to cancer vaccine augments a specific immune response targeted at a specific tumor antigen that results in specific tumor immunity. The mechanisms of interaction between tumor irradiation and gene-mediated immunotherapy could include radiation-induced alterations in the tumor microenvironment, as those observed histologically in our studies, which could facilitate a more effective anti-tumor immune response. These include radiation-induced apoptosis and necrosis of tumor cells causing tumor-debulking and release of tumor antigens for APC presentation. Inflammatory cells mobilized in the tumor by radiation-induced tissue damage could subsequently be activated by the immune response triggered by the cancer vaccine. These studies support investing further pre-clinical and clinical efforts to combine radiotherapy with cancer vaccines for the treatment of cancer. Citation Format: Gilda Gali Hillman, Matthew D. Fountain, Shoshana E. Rothstein, Lyndsey Reich, Lisa Abernathy, Christopher K. Yunker, Fulvio Lonardo, Philippe Slos. Induction of specific immunity to MUC1 antigen by tumor irradiation and cancer vaccines in murine tumor models. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B185.

A mammalianized synthetic nitroreductase gene for high-level expression

BackgroundThe nitroreductase/5-(azaridin-1-yl)-2,4-dinitrobenzamide (NTR/CB1954) enzyme/prodrug system is considered as a promising candidate for anti-cancer strategies by gene-directed enzyme prodrug therapy (GDEPT) and has recently entered clinical trials. It requires the genetic modification of tumor cells to express the E. coli enzyme nitroreductase that bioactivates the prodrug CB1954 to a powerful cytotoxin. This metabolite causes apoptotic cell death by DNA interstrand crosslinking. Enhancing the enzymatic NTR activity for CB1954 should improve the therapeutical potential of this enzyme-prodrug combination in cancer gene therapy.MethodsWe performed de novo synthesis of the bacterial nitroreductase gene adapting codon usage to mammalian preferences. The synthetic gene was investigated for its expression efficacy and ability to sensitize mammalian cells to CB1954 using western blotting analysis and cytotoxicity assays.ResultsIn our study, we detected cytoplasmic protein aggregates by expressing GFP-tagged NTR in COS-7 cells, suggesting an impaired translation by divergent codon usage between prokaryotes and eukaryotes. Therefore, we generated a synthetic variant of the nitroreductase gene, called ntro, adapted for high-level expression in mammalian cells. A total of 144 silent base substitutions were made within the bacterial ntr gene to change its codon usage to mammalian preferences. The codon-optimized ntro either tagged to gfp or c-myc showed higher expression levels in mammalian cell lines. Furthermore, the ntro rendered several cell lines ten times more sensitive to the prodrug CB1954 and also resulted in an improved bystander effect.ConclusionOur results show that codon optimization overcomes expression limitations of the bacterial ntr gene in mammalian cells, thereby improving the NTR/CB1954 system at translational level for cancer gene therapy in humans.

Unmet Need in Lung Cancer: Can Vaccines Bridge the Gap?

Despite recent advances in therapy for lung cancer, median survival remains poor. Cancer vaccines might meet the unmet need for new and effective therapies with low toxicity. A better understanding of the immunology of cancer has led to novel strategies for vaccine delivery, including the use of immunogenic adjuvant agents, genetic modification of tumor cells to produce cytokines, viral vectors, and the use of antigen-presenting cells. A number of these strategies have been studied in clinical trials for lung cancer. This review discusses the rationale for vaccine therapy in lung cancer, strategies for vaccine delivery, and clinical trials of vaccines in lung cancer.

Increased induction of antitumor response by exosomes derived from interleukin-2 gene-modified tumor cells

Tumor-derived exosomes (TEX) have been proposed as a new kind of cancer vaccine; however, their in vivo antitumor effects are not satisfactory. In order to further improve the efficacy of vaccination with TEX, we investigated whether interleukin-2 (IL-2) genetic modification of tumor cells can make IL-2 presence in the exosomes, thus increasing antitumor effects of the TEX. E.G7-OVA tumor cells expressing Ovalbumin (OVA) as a tumor model antigen were used to prepare TEX by serial centrifugation and sucrose gradients ultracentrifugation. To demonstrate their antitumor effects, IL-2-containing exosomes (Exo/IL-2) were injected subcutaneously into C57BL/C mice: either bearing tumor or followed by tumor inoculation. We found IL-2 within those exosomes as detected by both ELISA and Western blot. Vaccination with these Exo/IL-2 could induce antigen-specific Th1-polarized immune response and Cytotoxic T lymphocytes (CTL) more efficiently, resulting in more significant inhibition of tumor growth. CD8(+) T cells are the main effector cells, however, CD4(+) T cells, and NK cells are also involved in the induction of antitumor response of this approach. Our results demonstrate that IL-2 genetic modification of tumor cells can make the TEX contain IL-2 with the increased antitumor effects, representing a promising way of exosome-based tumor vaccine.

Enhanced induction of dendritic cell maturation and HLA-A*0201-restricted CEA-specific CD8+ CTL response by exosomes derived from IL-18 gene-modified CEA-positive tumor cells

Dendritic cells (DC)-derived or tumor-derived exosomes are a population of nanometer sized membrane vesicles that can induce specific anti-tumor immunity. However, the immunogenic potential and efficiency of exosomes-based tumor vaccine are not satisfactory enough to achieve a curative effect in clinical trials. In this article we investigated whether IL-18 genetic modification of tumor cells can increase the efficacy of exosomes derived from IL-18 gene-modified tumor cells. We transfected carcinoembryonic antigen (CEA)-expressing tumor cells with a recombinant adenovirus encoding human IL-18 (AdhIL-18) and prepared the exosomes, Exo/IL-18, from IL-18 gene-modified tumor cells. We found that Exo/IL-18 naturally contain CEA and bioactive IL-18. Moreover, Exo-IL-18 are potent in chemoattracting DC and T cells, enhancing the proliferation and Th1 cytokine release of PBMC, and promoting the phenotypic and functional maturation of DC. Furthermore, Exo/IL-18-pulsed DC are quite potent to induce HLA-A*0201-restricted, CEA-specific CD8+ CTL from the PBMC of HLA-A*0201 CEA+ cancer patients in vitro. In almost all of these experiments, Exo/IL-18 show more potent functions than the conventionally prepared exosomes derived from parent tumor cells without IL-18 gene modification. Our findings suggest that Exo/IL-18 has more potent capability to induce specific anti-tumor immunity, and our strategy of IL-18 modification of exosomes is a feasible approach to develop exosomes-based tumor vaccines.

Primary tumor cells from cancer patients “decorated” with a novel form of CD80 protein effectively induce autologous T-cell responses ex-vivo

2580 Background: The genetic modification of tumor cells is an elaborate process and has several limitations for effective use as cancer immunotherapy in clinical settings. We have recently developed a novel technology, ProtExa, that allows for rapid, efficient, and durable display of exogenous proteins on the cell surface. This technology involves generation of chimeric molecules composed of immunological proteins of interest and core streptavidin, biotinylation of biological surfaces, and decoration with chimeric proteins. In a mouse B lymphoma model, vaccination with tumor cells decorated with a chimeric costimulatory protein (CD80-SA) containing the extracellular portion of human CD80 and core streptavidin was effective in preventing tumor growth in 100% of animals. In this study, we tested whether primary tumor cells resected from cancer patients and decorated with CD80-SA can induce autologous proliferative and cytotoxic T-cell responses ex vivo. Methods: Primary tumor cells and blood samples were c...

Primary tumor cells from cancer patients “decorated” with a novel form of CD80 protein effectively induce autologous T-cell responses ex-vivo

2580 Background: The genetic modification of tumor cells is an elaborate process and has several limitations for effective use as cancer immunotherapy in clinical settings. We have recently developed a novel technology, ProtExä, that allows for rapid, efficient, and durable display of exogenous proteins on the cell surface. This technology involves generation of chimeric molecules composed of immunological proteins of interest and core streptavidin, biotinylation of biological surfaces, and decoration with chimeric proteins. In a mouse B lymphoma model, vaccination with tumor cells decorated with a chimeric costimulatory protein (CD80-SA) containing the extracellular portion of human CD80 and core streptavidin was effective in preventing tumor growth in 100% of animals. In this study, we tested whether primary tumor cells resected from cancer patients and decorated with CD80-SA can induce autologous proliferative and cytotoxic T-cell responses ex vivo. Methods: Primary tumor cells and blood samples were collected from 10 lung and 6 colon cancer patients of various stage (I to IV) who were treatment naïve. T-cell proliferative (CFSE and 3H-thymidine incorporation) and cytolytic (JAM) assays were used.Results: Tumor cells decorated with CD80-SA generated effective proliferative responses [Stimulation Index (SI) >2] in 8 out of 10 lung cancer patients and 3 out of 6 colon cancer patients. These responses varied from patient to patient and ranged from 3 to 50 SI. Cytotoxic T cell responses were informative in 10 lung and 4 colon cancer patients and ranged from 5% to 70% specific killing. Conclusions: Tumor cells decorated with CD80-SA can induce proliferative and cytotoxic responses ex-vivo. The rapid and durable cell-surface display of immunostimulatory proteins using the ProtExä technology will now be utilized in Phase I clinical trials in cancer patients to ascertain tolerability, toxicity and eventual immune mediated anti-tumor activity. This study was supported by Grants from Kentucky Lung Cancer Program and Jewish Hospital Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration ApoImmune, Inc. ApoImmune, Inc. ApoImmune, Inc.

Induction of T-cell antitumor immunity and protection against tumor growth by secretion of soluble human CD70 molecules.

One of the strategies to promote an antitumor response is the genetic modification of tumor cells to induce expression of costimulatory molecules. We have tested the capacity of a soluble form of CD70 molecule (sCD70). After construction of a vector carrying the sCD70, we obtained stable sCD70-secreting TS/A tumor cells and allogenic MC57 fibroblasts. In all, 45% of wild-type (wt) tumors were rejected in immunocompetent mice when transfected sCD70-secreting cells were injected three times in the periphery of the wt tumors. Furthermore, the sCD70-secreting TS/A cells induced a protective memory against wt TS/A tumor growth: 70% of the wt tumors used for the challenge were rejected by mice, which had rejected tumors 45 days before in the presence of sCD70-secreting TS/A cells. It was also shown that in vitro mock TS/A tumor cell proliferation was inhibited by splenocytes harvested from mice injected with TS/A cells expressing CD70. Growth kinetics of wt TS/A tumors in immunocompetent versus nude mice suggested that T lymphocytes were implicated in the antitumor response, which was confirmed by membrane expression of specific markers. The data suggest that injection of genetically transfected cells secreting sCD70 in the periphery of wt TS/A tumors induces T-cell-mediated inhibition of tumor growth and builds up a protective antitumor memory.

Adenovirus Vector-Mediatedin VivoGene Transfer of OX40 Ligand to Tumor Cells Enhances Antitumor Immunity of Tumor-Bearing Hosts

OX40 ligand (OX40L), the ligand for OX40 on activated CD4+ T cells, has adjuvant properties for establishing effective T-cell immunity, a potent effector arm of the immune system against cancer. The hypothesis of this study is that in vivo genetic engineering of tumor cells to express OX40L will stimulate tumor-specific T cells by the OX40L-OX40 engagement, leading to an induction of systemic antitumor immunity. To investigate this hypothesis, s.c. established tumors of three different mouse cancer cells (B16 melanoma, H-2b; Lewis lung carcinoma, H-2b; and Colon-26 colon adenocarcinoma, H-2d) were treated with intratumoral injection of a recombinant adenovirus vector expressing mouse OX40L (AdOX40L). In all tumor models tested, treatment of tumor-bearing mice with AdOX40L induced a significant suppression of tumor growth along with survival advantages in the treated mice. The in vivo AdOX40L modification of tumors evoked tumor-specific cytotoxic T lymphocytes in the treated host correlated with in vivo priming of T helper 1 immune responses in a tumor-specific manner. Consistent with the finding, the antitumor effect provided by intratumoral injection of AdOX40L was completely abrogated in a CD4+ T cell-deficient or CD8+ T cell-deficient condition. In addition, ex vivo AdOX40L-transduced B16 cells also elicited B16-specific cytotoxic T lymphocyte responses, and significantly suppressed the B16 tumor growth in the immunization-challenge experiment. All of these results support the concept that genetic modification of tumor cells with a recombinant OX40L adenovirus vector may be of benefit in cancer immunotherapy protocols.

Tumor vaccination with cytokine-encapsulated microspheres.

The rapid advances that have taken place in tumor immunology within the past decade have fostered renewed interest in the use of immune-based therapies for the treatment of cancer (,). Numerous strategies, which aim to provoke and augment anti-tumor immunity in cancer patients, have been developed and are being tested in clinical trials (, , ). One strategy that has worked well in preclinical mouse models involves the use of immune stimulatory molecules, i.e., cytokines, to augment the anti-tumor activity of the immune system (, , ). The early studies focused on the systemic administration of selected cytokines to promote a nonspecific augmentation of an already existing anti-tumor immunity (,). While successful in murine models, particularly with interleukin (IL)-2 and -12, the systemic delivery of cytokines has not been efficacious in the clinics due to severe systemic toxicity. Systemic administration of immunotherapeutic agents ignores the paracrine nature of their activity. With the advent of molecular techniques that allowed efficient genetic modification of tumor cells in vitro, cytokine gene-modified tumor cell vaccines became the preferred alternative to systemic therapy. This approach results in the local and sustained release of cytokines by the tumor cells at the vaccine site, which induces the development of a tumor-specific, systemic anti-tumor immunity while circumventing the toxicity associated with systemic delivery (, , , ).

A novel approach for modifying tumor cell-derived plasma membrane vesicles to contain encapsulated IL-2 and engrafted costimulatory molecules for use in tumor immunotherapy.

The genetic modification of tumor cells and delivery of cytokines have been proposed as useful strategies in the development of anti-tumor vaccines; however, a number of factors limit their use in clinical settings. To facilitate vaccine development, we explored the possibility of modifying plasma membrane vesicles (PMV) by using a novel chelator lipid, nitrilotriacetic acid ditetradecylamine (NTA-DTDA). Our analyses by flow cytometry show that NTA-DTDA can be incorporated into PMV prepared from murine P815 mastocytoma and that the incorporated NTA-DTDA permits anchoring or "engraftment" onto the vesicle surface of hexahistidine-tagged proteins such as recombinant forms of the costimulatory molecules B7.1 and CD40. The engrafted PMV also can incorporate and deliver the immunostimulatory cytokine Interleukin-2 (IL-2). Our results show that modified PMV derived from P815 cells bind the murine T cell clone D10 in a receptor-ligand dependent manner, inducing cell adhesion and promoting cell survival in vitro. The modified PMV can bind syngeneic T cells, stimulating T cell proliferation and cytotoxic T cell responses. Moreover, when used as vaccines in syngeneic animals, the modified vesicles induce significant protection against challenge with the native P815 tumor. The results indicate that PMV modified by engraftment of recombinant forms of B7.1 and CD40 and incorporation of IL-2 can be used to modulate immune responses, which provides a novel approach for the development of anti-tumor vaccines and cancer immunotherapies.

Antitumor effect of B16 melanoma cells genetically modified with the angiogenesis inhibitor rnasin.

The growth of new blood vessels is an essential condition for the development of tumors with a diameter greater than 1-2 mm and also for their metastatic dissemination. RNasin, the placental ribonuclease inhibitor, is known to have antiangiogenic activity through the inhibition of angiogenin and basic fibroblast growth factor. Nevertheless, the administration of the recombinant form of a protein poses several limitations; as a result, we have studied the antitumor effect of RNasin in a murine gene therapy model. RNasin cDNA was subcloned into the pcDNA3 expression vector, and the resulting recombinant plasmid was used to transfect the B16 murine melanoma cell line. An RNasin inverted construction was used as control. Mice intravenously injected with clones expressing RNasin showed a significant inhibition of tumor metastatic progression with respect to control groups (P<.001) and survived longer (P<.001). Tissue sections from RNasin-expressing cell tumors showed a lower number of blood vessels when compared to tissue sections from mice lungs that had been inoculated with control cell lines. The results of these experiments show that the genetic modification of tumor cells with RNasin cDNA yields a significant antitumor effect, and suggest that this effect is at least partially the result of angiogenesis inhibition.

Anti-tumor immunity induced by in vivo adenovirus vector-mediated expression of CD40 ligand in tumor cells.

CD40 ligand (CD40L), the ligand for CD40 on antigen-presenting cells, is essential for the initiation of antigen-specific T cell responses, an important component of the immune response to tumors. This study is based on the hypothesis that in vivo genetic modification of tumor cells to express CD40L will trigger CD40 on local antigen-presenting cells to present tumor antigen to the cellular immune systems, thus eliciting anti-tumor immunity to suppress growth of the tumor. To examine this concept, subcutaneous tumors of three different murine tumor models in two strains of mice were infected with a recombinant adenovirus (Ad) vector expressing murine CD40L (AdmCD40L). In the B16 (H-2b, melanoma) and CT26 (H-2d, colon cancer) murine models, injection of AdmCD40L into established subcutaneous tumors resulted in sustained tumor regression and tumor-free status in >60% of animals. Intratumoral injection of AdmCD40L also significantly suppressed the growth of established, weakly immunogenic Lewis lung carcinoma (H-2b) tumors, but to a lesser extent. Ex vivo AdmCD40L-transduced tumor cells implanted in syngeneic hosts induced significant antitumor response against preexisting identical tumors at a distant site. Both in vivo and in vitro AdmCD40L modification of tumors to express CD40L elicited tumor-specific cytolytic T lymphocytes responses, and the transfer of spleen cells from treated mice efficiently protected naive mice against a subsequent tumor challenge. These results support the concept that transduction of tumors with a recombinant CD40L adenovirus vector may be a useful strategy for cancer immunotherapy.

Tumor cells cotransduced with B7.1 and gamma-IFN induce effective rejection of established parental tumor.

Genetic modification of tumor cells with the gene for the B7.1 or with the genes for cytokines results in increased tumor cell immunogenicity. In the work reported here, immunization of naive animals with either B7.1 or gamma-IFN gene-modified MCA106 tumor cells effectively protects the host from subsequent challenge with parental tumor. The same treatment fails to induce regression of established tumors, although tumor-specific CTL are generated in the tumor-bearing animals. In contrast, a large tumor burden of the MCA106 fibrosarcoma can be successfully eliminated by treatment with MCA106 tumor cells cotransduced with the B7.1 and gamma-IFN genes. Antitumor immunity induced by the cotransductants is primarily dependent on CD8+ T cells and partly on CD4+ T cells and NK cells, and the enhanced therapeutic effect may be attributed to the in vivo increase of CTL precursors following treatment. The gamma-IFN and B7.1 genes must be expressed on the same tumor cell for optimal therapeutic effect. Our results suggest that tumor vaccines with a potent immunoprotective effect do not necessarily have therapeutic potential and that weakly immunogenic tumors may be rendered highly immunogenic by cotransfection with the genes for B7.1 and gamma-IFN.

Induction of antitumor immune response in the mouse model after vaccination with B7.1 expressing tumor cells

The aim of the study was the induction of an antitumor immune response by genetic modification of tumor cells. This was done by transfecting the costimulatory molecule B7.1 into a murine tumor cell line SCCVII/SF in order to increase T cell recognition and to install an immunologic memory. One cohort of immunocompetent mice C3 H/HeN were injected with B7.1 expressing tumor cells, while the control group received parental B7.1 negative tumor cells. In a second step those immunized mice were rechallenged by parental tumor cells and tumor growth was compared to a new control group. Transfection with B7.1 prohibits outgrowth of the SCCVII cell line. Animals that have been vaccinated in this way are partially immune towards a secondary exposition to B7.1 negative tumor cells. Outgrowth of this recurrent tumor is slowed down. Such an immunization builds up an immunologic memory. Vaccination with B7.1 expressing tumor cells lead to a partial protective tumor immunity in the SCCVII-C3 H/HeN mouse model.

Genetic modification of a carcinoma with the IL-4 gene increases the influx of dendritic cells relative to other cytokines.

Tumor cells genetically modified with certain cytokine genes gain immunogenic properties that allow the development of systemic anti-tumor immunity. Whether different cytokines may influence infiltration of transduced tumors by dendritic cells (DC) has not been investigated. Therefore, we analyzed the C26 murine colon carcinoma genetically modified to release interleukin (IL)-2, IL-4, IL-12, granulocyte colony-stimulating-factor (CSF) or granulocyte-macrophage (GM)-CSF for immunostaining with the monoclonal antibody NDLC145 recognizing the DEC205 determinant which, on tumor sections, is virtually restricted to DC. Infiltrating leukocytes were also characterized for expression of co-stimulatory molecules like CD54, CD86 and major histocompatibility complex class II. The intratumoral DC content was dependent on the type of transduced cytokines with C26/IL-4 being the most abundant in DEC205+ cells. The effect of IL-4 in recruiting DC did not depend on the type of tumor since it was confirmed in the TSA mammary carcinoma. In comparison with C26/GM-CSF, C26/IL-4 had more B7.2+ cells but less Ia+ cells. Furthermore, the hypertrophic skin overlaying tumors producing GM-CSF showed numerous Langerhans cells stained by NDLC145 and the draining lymph nodes showed abundance and paucity of DC in C26/GM-CSF and C26/IL-4, respectively. When injected into the ear pinna, C26/GM-CSF stimulated, whereas C26/IL-4 inhibited DC-mediated priming of delayed-type hypersensitivity reaction by 2,4-dinitro-1-fluorobenzene. These findings prove that transduced cytokines differently influence DC recruitment at the tumor site and DC function in nearby tissues. Along with the other leukocytes and their secondary produced cytokines, DC create an environment in which T cells can be differently modulated. Such a phenomenon may have implications on genetic modification of tumor cells to be used as cancer vaccine.

Secretion of Biologically Active Superantigens by Mammalian Cells

The genetic modification of tumor cells to secrete immune regulatory molecules can elicit a potent antitumor immune response. Bacterial superantigens are among the most potent T cell mitogens. Activation of tumor-sensitized T cells by bacterial superantigens can lead to immune effector cells with potent and specific in vivo antitumor activity. Retrovirus vectors encoding for the bacterial superantigens SEA and SEC2 were constructed, and recombinant retrovirus stocks were generated. SEA and SEC2 could be detected in the culture supernatant of tumor cells after a single exposure to retrovirus. Molecular analysis of the genetically modified cells revealed intact proviral DNA and abundant vector-derived superantigen RNA. Biologic activity was apparent for both superantigens. Secretion of biologically active superantigen by mammalian cells has not been reported previously, and this will enable investigating the potential for superantigen gene therapy.

Immune reactions induced by interleukin-2 transfected colorectal cancer cells in vitro: predominant induction of lymphokine-activated killer cells.

One aim of the genetic modification of tumor cells is the generation of immunogenic variants that can be used for the induction of immune responses against tumors. We engineered the human colorectal carcinoma cell line SW480 by means of plasmid transfection to secrete interleukin (IL)-2. Transfection of SW480 cells resulted in stable IL-2 secretion at 5-30 ng/ml per 10(5) cells in 24 h and, unexpectedly, in CD54 expression on the cell surface. SW480 variants expressing IL-2 and CD54 were tested for their capacity to induce T lymphocyte activation in vitro in comparison to untransfected and CD54 transfected cells. The cytolytic effector function of a class I MHC restricted CD8+, peptide antigen specific T cell clone was augmented following expression of CD54. IL-2 secreting SW480 variants did not further increase antigen-dependent cytolysis. Primary activation of resting T lymphocytes was assessed following allogeneic stimulation. When compared with unmodified SW480 cells, CD54 expressing variants did not initiate T cell proliferation. In contrast, IL-2 secreting SW480 cells strongly promoted primary T cell proliferation. Similarly, exogenous IL-2 and SW480 cells induced T cell proliferation which was not only due to IL-2 but was dependent on tumor cells. However, following the initial wave of cell growth in response to IL-2 secreting SW480 cells T lymphocytes could not be restimulated with SW480 or IL-2 secreting variants and could not be further expanded. T cells initially activated by IL-2 secreting SW480 cells exhibited cytolytic activity towards SW480 cells. This reactivity, however, was transient and completely blocked by K562 cells, suggesting MHC-unrestricted, nonspecific cytotoxicity. We conclude that endogenous IL-2 secretion by the colorectal carcinoma cell line SW480 does not result in the activation of MHC restricted specific T lymphocytes but predominantly induces lymphokine-activated killer cells. Considering that tumor cell vaccines are aimed at inducing tumor-specific immune responses, our in vitro observation would rather argue against the in vivo application of such a tumor cell modification in colorectal cancer.