Thomas Tüting

Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope

Enhanced green fluorescent protein (EGFP) is a novel marker gene product, which is readily detectable using techniques of fluorescence microscopy, flow cytometry, or macroscopic imaging. In the present studies, we have examined the immunogenicity of EGFP in murine models. A stable transfectant of the transplantable CMS4 sarcoma of BALB/c origin expressing EGFP, CMS4-EGFP-Zeo, was generated. Splenocytes harvested from mice immunized with a recombinant adenovirus expressing EGFP (Ad-EGFP) were restimulated in vitro with CMS4-EGFP-Zeo. Effector lymphocytes displayed strong cytotoxicity against CMS4-EGFP-Zeo, but not against mock-transfected CMS4-Zeo tumor cells. A number of candidate H2-Kd-binding peptides derived from the EGFP protein were chosen according to an epitope prediction program and synthesized. These peptides were tested for their ability to bind to H2-Kd molecules and stimulate IFNγ-production by splenocytes harvested from Ad-EGFP-immunized mice. Using this methodology, the peptide, HYLSTQSAL (corresponding to EGFP200–208) which strongly binds to H2-Kd molecules, was identified as a naturally occurring epitope of EGFP. These results should facilitate the use of EGFP as a model tumor antigen in BALB/c mice.

Gene-based strategies for the immunotherapy of cancer

Abstract T lymphocytes play a crucial role in the host’s immune response to cancer. Although there is ample evidence for the presence of tumor-associated antigens on a variety of tumors, they are seemingly unable to elicit an adequate antitumor immune response. Modern cancer immunotherapies are therefore designed to induce or enhance T cell reactivity against tumor antigens. Vaccines consisting of tumor cells transduced with cytokine genes in order to enhance their immunogenicity have been intensely investigated in the past decade and are currently being tested in clinical trials. With the development of novel gene transfer technologies it has now become possible to transfer cytokine genes directly into tumors in vivo. The identification of genes encoding tumor-associated antigens and their peptide products which are recognized by cytotoxic T lymphocytes in the context of major histocompatibility complex class I molecules has allowed development of DNA-based vaccines against defined tumor antigens. Recombinant viral vectors expressing model tumor antigens have shown promising results in experimental models. This has led to clinical trials with replication-defective adenoviruses encoding melanoma-associated antigens for the treatment of patients with melanoma. An attractive alternative concept is the use of plasmid DNA, which can elicit both humoral and cellular immune responses following injection into muscle or skin. New insights into the molecular biology of antigen processing and presentation have revealed the importance of dendritic cells for the induction of primary antigen-specific T cell responses. Considerable clinical interest has arisen to employ dendritic cells as a vehicle to induce tumor antigen-specific immunity. Advances in culture techniques have allowed the generation of large numbers of immunostimulatory dendritic cells in vitro from precursor populations derived from blood or bone marrow. Experimental immunotherapies which now transfer genes encoding tumor-associated antigens or cytokines directly into professional antigen-presenting cells such as dendritic cells are under evaluation in preclinical studies at many centers. Gene therapy strategies such as in vivo cytokine gene transfer directly into tumors as well as the introduction of genes encoding tumor-associated antigens into antigen-presenting cells hold considerable promise for the treatment of patients with cancer.

IFN-α-Expressing Tumor Cells Enhance Generation and Promote Survival of Tumor-Specific CTLs

IFN-α gene therapy has been successfully applied in several tumor models. Our studies involving the murine colorectal adenocarcinoma cell line MC38 confirm that IFN-α transduction of a poorly immunogenic tumor cell reduces tumorigenicity and leads to long-lasting tumor immunity. To investigate the effect of IFN-α transduction on the development of antitumor immune responses, we restimulated splenocytes from MC38-immune mice in vitro. Detection of MC38-specific cytotoxicity was markedly enhanced when murine IFN-α2-transduced MC38 (MC38-IFNα) or CD80-transduced MC38 (MC38-CD80) was used for restimulation compared with wild type (MC38-WT) or neomycin resistance gene-transduced MC38 (MC38-Neo) cells. MC38-specific CD8+ CTL line and clone were established from splenocytes of mouse immunized with MC38-IFNα. Stimulation with MC38-IFNα as well as MC38-CD80 enhanced the proliferation of MC38-specific CTLs in vitro much more effectively than stimulation with WT or MC38-Neo (p < 0.05). Coincubation of MC38-specific CTLs with MC38-IFNα or MC38-CD80 resulted in significantly less DNA fragmentation (8.0% and 12.8%, respectively) compared with coincubation of the CTLs with MC38-WT (43.5%; p < 0.001) or MC38-Neo cells (38.1%; p < 0.003). These results suggest that prevention of apoptotic cell death in tumor-specific CTLs may be one mechanism by which IFN-α-expressing tumor cells can promote the generation of antitumor immunity. The effect of IFN-α on CTLs appears to be similar to that of CD80, which also prevents apoptotic cell death after stimulation of T lymphocytes.

Interferon-α gene therapy for cancer : retroviral transduction of fibroblasts and particle-mediated transfection of tumor cells are both effective strategies for gene delivery in murine tumor models

Stable transfection of tumor cells with IFN-α genes has been shown to result in abrogation of tumor establishment and induction of antitumor immunity. However, strategies suitable for the clinical application of IFN-α gene therapy for cancer have not been reported. In this study, we investigated two gene delivery systems capable of mediating the local paracrine production of high levels of biologically active IFN-α in murine tumor models: retroviral transduction of fibroblasts and particle-mediated transfection of tumor cells. In spite of the antiproliferative effects of IFN-α, it was possible to obtain stable retroviral producer cell lines and transduce a variety of murine tumor cells including syngeneic fibroblasts to stably secrete 2000–5000 U (40–100 ng) murine IFN-α/106 cells/24 h. IFN-α transduction of tumor cells abrogated tumorigenicity in establishment models and induced antitumor immunity in several murine tumor model systems. Importantly, IFN-α gene delivery using retrovirally transduced syngeneic fibroblasts was capable of suppressing the establishment of the poorly immunogenic TS/A mouse mammary adenocarcinoma and induced antitumor immunity. Particle-mediated transient transfection of tumor cells using the gene gun led to the production of up to 20000 U IFN-α/106 cells during the first 24 h and proved to be equally effective in suppressing establishment of TS/A adenocarcinoma and inducing antitumor immunity. These results suggest that retroviral transduction of autologous fibroblasts can serve as an effective gene delivery method for IFN-α gene therapy of cancer. Particle-mediated transfection of freshly isolated tumor cells may represent a clinically attractive alternative approach for nonviral gene delivery. Both strategies circumvent the difficulties in routinely establishing primary tumor cell lines from the vast majority of human cancers.

Interferon-alpha gene therapy in combination with CD80 transduction reduces tumorigenicity and growth of established tumor in poorly immunogenic tumor models.

Interferon-alpha (IFN-α) or CD80 transduction of tumor cells individually reduces tumorigenicity and enhances antitumor responses. Here, we report that the combination of IFN-α and CD80 cancer gene therapy in poorly immunogenic murine tumor models, the colorectal adenocarcinoma cell line MC38, and the methylcholanthrene-induced fibrosarcoma cell line MCA205 reduces tumor growth more efficiently without affecting in vitro growth. Wild-type (WT), neomycin-resistance (Neo) gene-, or CD80-transduced tumor cells grew progressively in all immunocompetent mice. In contrast, IFN-α-transduced MC38 or MCA205 cells were rejected in 13 of 15 and seven of 15 mice, respectively. Synergistic effects were observed when IFN-α- and CD80-transduced tumor cells were mixed and inoculated. These admixed cells were rejected by 14 of 15 (MC38) or seven of 15 mice (MCA205), whereas, a mixture of IFN-α and Neo cells or CD80 and Neo cells led to tumors associated with progressive growth. Induction of long-lasting tumor immunity against WT tumor cells was demonstrated by rejection of a subsequent rechallenge in 10 of 13 (MC38) and six of seven (MCA205) tumor-free mice. The therapeutic efficacy with established WT MC38 tumors was shown when mice were treated with a vaccine consisting of repetitive injections of IFN-α- and CD80-transduced MC38 cells into the contralateral flank (P < 0.01). this treatment was associated with accumulation of cd4+, CD8+ cells and dendritic cells within the established tumor, demonstrating induction of antitumor immune responses. Combination gene therapy using IFN-α and CD80 is an effective immune therapy of cancer and could be considered for clinical trials.

Impact of p53-based immunization on primary chemically-induced tumors.

In mice as well as humans, cytotoxic T lymphocytes (CTL) specific for wild‐type‐sequence (wt) p53 peptides have been shown to react against a wide range of tumors, but not normal cells. As such, they are attractive candidates for developing broadly applicable cancer vaccines. Of particular interest is the potential of using p53‐based vaccines in high‐risk individuals to prevent cancer. Methylcholanthrene, an immunosuppressive polycyclic hydrocarbon carcinogen implicated as a causative agent in human cancers, has long been used to induce murine tumors with a high incidence of genetic alterations and sensitivity to wt p53‐specific CTL. To analyze the potential of p53‐based vaccines on primary tumors, we evaluated the efficacy of DNA and dendritic cell vaccines targeting wt p53 peptides given to methylcholanthrene‐treated mice in the protection or therapy settings. The results indicate that the efficacy of these vaccines relative to reducing tumor incidence were severely compromised by vaccine‐induced tumor escape. As compared to tumors induced in non‐immunized mice, a higher incidence of epitope‐loss tumors was detected in tumors from the immunized mice. The increase in tumor escape arose as a consequence of either increased frequencies of mutations within/flanking p53 epitope‐coding regions or downregulation of expression of the major histocompatibility complex Class I molecules that present these epitopes for T cell recognition These findings are consistent with current views of immunoselection occurring in patients receiving tumor peptide‐based immunotherapy, and impact on the design and implementation of p53‐based vaccines, in particular, those aimed at treating individuals at high risk for developing cancer.

DNA Vaccines Targeting Dendritic Cells for the Immunotherapy of Cancer

Cytotoxic T lymphocytes (CTL) play a crucial role in the host’s immune response to cancer. The adoptive transfer of tumor-specific CTL can mediate the regression of established tumors in experimental animal models1 as well as in some patients with melanoma2. Recently, a number of genes encoding tumor-associated antigens (TAA) and their peptide products, which are recognized by cytotoxic T lymphocytes in the context of major histocompatibility complex (MHC) class I molecules, have been identified for both murine and human tumors3,4. These insights now need to be translated into the development and application of novel immunotherapies designed to elicit tumor antigen-specific T cells. Dendritic cells (DC) are believed to be critical for the induction of primary, cell-mediated immune responses5,6. Using freshly isolated, as well as, cultured DC pulsed with peptides constituting relevant CTL-defined epitopes, we and others have been able to induce protective and therapeutic antitumor immune responses in mouse tumor models7–13. Furthermore, autologous cultured human DC pulsed with synthetic melanoma peptides were able to stimulate antigen-specific CTL capable of lysing HLA-matched allogeneic melanoma cells that naturally express these epitopes in vitro 14–16. As an alternative to synthetic peptides, that may restrict the immune response to defined tumor-associated epitopes with known MHC restriction, the use of plasmid DNA or recombinant viruses encoding tumor-associated antigens has recently been investigated for the immunotherapy of cancer17–22. Direct inoculation of naked DNA into the skin or muscle of animals results in both humoral and cellular immune responses.

Identification of a 17β-hydroxysteroid dehydrogenase type 12 pseudogene as the source of a highly restricted BALB/c Meth A tumor rejection peptide

Mass spectrometric analysis identified the peptide recognized by a cytotoxic T lymphocyte (CTL) specific for the chemically induced BALB/c Meth A sarcoma as derived from a 17β-hydroxysteroid dehydrogenase type 12 (Hsd17b12) pseudogene present in the BALB/c genome, but only expressed in Meth A sarcoma. The sequence of the peptide is TYDKIKTGL and corresponds to Hsd17b12114–122 with threonine instead of isoleucine at codon 114 and is designated Hsd17b12114T. Immunization of mice with an Hsd17b12114T peptide-pulsed dendritic cell-based vaccine or a non-viral plasmid construct expressing the Hsd17b12114T peptide protected the mice from lethal Meth A tumor challenge in tumor rejection assays. A Hsd17b12114–122 peptide-pulsed vaccine was ineffective in inducing resistance in mice to Meth A sarcoma. These results confirm the immunogenicity of the identified tumor peptide, as well as demonstrate the efficacies of these vaccine vehicles. These findings suggest that the role of the human homolog of Hsd17b12, HSD17B12, as a potential human tumor antigen be explored.