Giving DNA vaccines a helping hand.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
W J Storkus
Publications and source records attributed to W J Storkus.
DNA immunization targeting the skin: molecular control of adaptive immunity.
DNA-based immunization represents a novel approach for vaccine development. Recombinant DNA techniques are used to clone DNA sequences encoding antigens of choice into eukaryotic expression plasmids, which are readily and economically amplified in bacteria and recovered with a high degree of purity. For immunization, plasmid DNA is either coated onto microscopic gold particles and bombarded into skin using a gene gun or injected into skin or muscle. Expression of administered genes results in the induction of humoral and cellular immune responses against the encoded antigen. DNA immunization is capable of inducing protective immunity in a number of animal models of infectious disease and cancer. Recent studies suggest that antigen-specific cytotoxic T lymphocyte induction occurs through the presentation of appropriate peptides in the context of major histocompatibility complex molecules on bone marrow-derived professional antigen presenting cells. Following DNA inoculation into the skin, Langerhans cells and/or dermal dendritic cells are believed to acquire the newly synthesized antigen, either through direct transfection or via antigen uptake from transfected keratinocytes, and migrate to regional lymph nodes where they stimulate primary T cell responses. The nature of the immune response depends on the route, method, and timing of DNA delivery and can also be influenced by co-delivery of plasmids encoding immunomodulating cytokines like IFN-alpha, IL-2, or IL-12 and costimulatory molecules like B7-1. While many aspects of the biology of cutaneous DNA immunization remain unknown, the skin appears to offer unique potential as a target for DNA-based immunization.
Animals↗
Autologous human dendriphages pulsed with synthetic or natural tumor peptides elicit tumor-specific CTLs in vitro.
The recent identification of tumor-associated antigens and tumor-associated antigen-derived peptide epitopes recognized by cytolytic T lymphocytes (CTLs) in the context of major histocompatibility complex (MHC) class I molecules has prompted the development of peptide-based vaccines for the treatment of human cancers, particularly melanoma. The design of such clinical protocols requires an understanding of the inherent immunogenicity of the peptide(s) and a choice of a facilitating adjuvant promoting cellular immunity against these peptides. We have evaluated the abilities of a series of defined synthetic peptide epitopes derived from MART-1/Melan-A, gp100, tyrosinase, and MAGE-3 or unfractionated peptides naturally presented by melanoma MHC molecules to elicit HLA-A2-restricted and melanoma-reactive CTLs from the peripheral blood of normal donors or patients with metastatic melanoma. Autologous peripheral blood dendritic cells (DCs), which were easily generated from all donors when cultured in the presence of recombinant human interleukin-4 and recombinant human granulocyte-macrophage colony-stimulating factor were pulsed with melanoma peptides and used to "prime" and/or "boost" CTL cultures in vitro. Our results suggest that antimelanoma CTLs may be reproducibly generated in short-term in vitro cultures in this manner using either a subset of the defined synthetic peptides (MART-1/Melan-A27-35, MART-1/Melan-A32-40, gp100(280-288), tyrosinase368-376, and MAGE-3(271-279)) or unfractionated peptides (containing both idiotypic and shared melanoma epitopes) derived from freshly isolated autologous melanoma lesions. These in vitro data support the use of autologous DCs prepulsed with such peptides as an appropriate antigen adjuvant delivery system in melanoma peptide-based vaccines.
Antigens, Neoplasm↗
FLT3 ligand induces the generation of functionally active dendritic cells in mice.
FLT3 ligand (FL) is a recently described hematopoietic growth factor that stimulates the proliferation and differentiation of hematopoietic progenitors. We have investigated the effect of FL on murine hematopoiesis and dendritic cell (DC) generation and accumulation in lymphoid tissues and liver in vivo and in vitro evaluating the morphologic, phenotypic, and functional characteristics of these DC. We have observed extramedullary hematopoiesis in the mouse spleen with all lineages of hematopoietic cells represented after the administration of FL. Injection of FL results in a time-dependent and reversible accumulation of DC in the spleen, bone marrow, lymph nodes, and liver. Both flow cytometry and immunohistochemistry revealed a significant accumulation of DC in these tissues. Results of mixed leukocyte reaction suggested that these cells, isolated from murine bone marrow or spleen, were active as antigen presenting cells. Furthermore, cultivation of splenic and marrow cells with GM-CSF and IL-4 gave rise to large numbers of functionally active mature DC. Thus, the results of this study suggest that FL is a promising growth factor that stimulates the generation of large number of DC and may be a useful cytokine for the immunotherapy of cancer.
Animals↗
Genetically modified bone marrow-derived dendritic cells expressing tumor-associated viral or "self" antigens induce antitumor immunity in vivo.
The clinical application of synthetic tumor peptide-based vaccines is currently limited to patients with specified major histocompatibility complex (MHC) class I alleles. Such logistic limitations may be overcome using tumor gene-based approaches. Here we describe the effective generation of dendritic cells (DC) expressing tumor peptide-MHC complexes as a result of particle-mediated transfer of genes encoding tumor-associated antigens (TAA). Bone marrow-derived DC were transfected with plasmid DNA encoding the tumor-associated viral antigen E7 derived from human papilloma virus (HPV) 16. When applied as a vaccine, these genetically modified DC induced antigen-specific CD8+ cytotoxic T lymphocytes (CTL) in vivo and promoted the rejection of a subsequent, normally lethal challenge with an HPV 16-transformed tumor cell line. Of greatest interest, immunization of mice with syngeneic DC genetically modified to enhance their presentation of a constitutive "self" epitope derived from the tumor-suppressor gene product p53 caused a significant reduction in the in vivo growth of a chemically induced p53-positive sarcoma. These results suggest that cancer vaccines consisting of DC genetically modified to express TAA of viral or "self" origin effectively induce antitumor immunity in vivo.
Animals↗
Bone marrow-derived dendritic cells serve as potent adjuvants for peptide-based antitumor vaccines.
Dendritic cells (DCs) are considered the most effective antigen-presenting cells (APCs) for primary immune responses. Since presentation of antigens to the immune system by appropriate professional APCs is critical to elicit a strong immune reaction and DCs seem to be quantitatively and functionally defective in the tumor host, DCs hold great promise to improve cancer vaccines. Even though they are found in lymphoid organs, skin and mucosa, the difficulty of generating large numbers of DCs has been a major limitation for their use in vaccine studies. A simple method for obtaining DCs from mouse bone marrow cells cultured in the presence of GM-CSF + interleukin 4 is now available. In four different tumor models, mice injected with DCs grown in GM-CSF plus interleukin 4 and prepulsed with a cytotoxic T lymphocyte-recognized tumor peptide epitope developed a specific cytotoxic T lymphocyte response and were protected against a subsequent tumor challenge with tumor cells expressing the relevant tumor antigen. Moreover, treatment of day 5-14 tumors with peptide-pulsed DCs resulted in sustained tumor regression in five different tumor models. These results suggest that presentation of tumor antigens to the immune system by professional APCs is a promising method to circumvent tumor-mediated immunosuppression and is the basis for ongoing clinical trials of cancer immunotherapy with tumor peptide-pulsed DCs.
Animals↗
Development of dendritic cell-based genetic vaccines for cancer.
Explore the source record for details and available documents.
Animals↗
Dendritic cell based therapy of cancer.
Explore the source record for details and available documents.
Animals↗
Gene-based strategies for the immunotherapy of cancer.
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 pre-clinical 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.
Animals↗
Interferon-alpha 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-alpha 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-alpha 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-alpha in murine tumor models: retroviral transduction of fibroblasts and particle-mediated transfection of tumor cells. In spite of the antiproliferative effects of IFN-alpha, 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-alpha/10(6) cells/24 h. IFN-alpha transduction of tumor cells abrogated tumorigenicity in establishment models and induced antitumor immunity in several murine tumor model systems. Importantly, IFN-alpha 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 20,000 U IFN-alpha/10(6) 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-alpha 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.
Animals↗
Murine models of cancer cytokine gene therapy using interleukin-12.
Cytokine gene therapy, in particular IL-12 gene therapy, is one of the more novel and promising approaches in cancer therapy based on significant preclinical data derived mainly from murine tumor models. IL-12 is a heterodimeric cytokine that requires the simultaneous expression of both the p35 and p40 chains from the same cell for production of biologically active IL-12. A variety of biological functions have been attributed to IL-12 including the induction of IFN-gamma production and the promotion of predominantly Th1-type immune responses to antigens. Our previous studies using systemic administration of recombinant murine IL-12 have demonstrated profound antitumor efficacy against all tumors tested with the concomitant long-lived specific antitumor immunity in some cases. To determine whether the local secretion of IL-12 achieved by gene transduction has significant antitumor effects, fibroblast cell lines or murine tumor cell lines were transduced with expression plasmids or the retroviral vector TFG-mIL-12-Neo and inoculated intradermally (i.d.). Our first study using IL-12-transfected NIH3T3 cells admixed with the murine melanoma, BL-6, showed that local IL-12 expression suppresses tumor growth and promotes the acquisition of specific antitumor immunity. Subsequent studies showed that IL-12 gene therapy is also effective in treating established day 3 tumors. CD4+ and CD8+ T cells, as well as NK cells, appear to play important roles in the observed antitumor effects resulting from IL-12 paracrine secretion. Administration of neutralizing antibody specific for IFN-gamma also abrogated some of the IL-12-associated antitumor effects. Finally, this IL-12 gene therapy strategy to elicit an antitumor immune response was more effective when used in combination with the transduction of tumor cells with B7.1. Based on these promising results, a clinical protocol for the treating patients with cancer using genetically engineered fibroblasts to express IL-12 has been initiated at our institution.
Animals↗
IL-12-engineered dendritic cells serve as effective tumor vaccine adjuvants in vivo.
The recent identification of tumor-associated antigens (TAA) and TAA-derived peptides presented by MHC molecules to T cells has provided the tools to design and test clinical vaccines for treating human malignancies, such as melanoma. While the most effective adjuvant for use in vaccine formulation remains unclear, autologous dendritic cells (DC) appear to be good candidate adjuvants. We have previously shown that syngeneic bone marrow-derived DC when pulsed ex vivo with relevant TAA-derived peptides can effectively vaccinate mice against a subsequent challenge with tumor or can effectively treat animals bearing established tumors. In this report, we have engineered murine interleukin-12 (mIL-12), a potent stimulator of cell-mediated immunity, into murine DC using retroviral-mediated or plasmid-based transfection procedures. Transfectants produced up to 25 ng rIL-12/10(6) cells/48 hours. These engineered cells are capable of promoting enhanced anti-tumor, antigen-specific CTL responses compared to nontransduced DC.
Adjuvants, Immunologic↗
Cytokine gene therapy of cancer using interleukin-12: murine and clinical trials.
Explore the source record for details and available documents.
Animals↗
Tumor escape from immune recognition: lethal recurrent melanoma in a patient associated with downregulation of the peptide transporter protein TAP-1 and loss of expression of the immunodominant MART-1/Melan-A antigen.
In the last few years, mutiple protein target antigens for immunorecognition by T cells have been identified on human melanoma. How melanoma lesions escape from functional antigen-specific immune recognition remains poorly understood. We have identified the concomitant loss of the immunodominant T cell-defined MART-1/Melan-A antigen and downregulation of the TAP-1 gene in a recurrent metastatic melanoma that was resected in 1993. This phenotype was not observed for an earlier autologous melanoma lesion resected in 1987. The "antigen loss" could be restored in the variant tumor cell line by simultaneously providing both the MART-1/Melan-A gene (by retroviral transfer) and the TAP-1 gene (by a bioballistic approach) resulting in tumor cell sensitivity to MART-1/Melan-A-specific cytotoxic T lymphocytes. This suggests that tumor escape from immune surveillance may have occurred in vivo as a sequential result of (a) antigen loss, and (b) downregulation of the peptide-transporter protein TAP-1 expression by this patient's tumor over a 6-yr period from 1987 to 1993. These results suggest that the characterization of the T cell response to melanoma in individual patients and definition of the immunologically relevant genetic defects in tumors may be required to select the most effective therapeutic strategies for a given patient.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Multiple melanoma-associated epitopes recognized by HLA-A3-restricted CTLs and shared by melanomas but not melanocytes.
The molecular characterization of melanoma-associated Ags allowed the definition of several HLA class I-presented peptides recognized by T cells. However, no HLA-A3.1-restricted melanoma epitopes have been identified to date. To gain insight into the HLA-A3.1-restricted T cell epitope repertoire of human melanoma, we analyzed the immunologic reactivity of CTLs isolated from tumor-involved or tumor-free lymph nodes in two HLA-A3.1+ melanoma patients. Three CTL lines, clonal or highly oligoclonal in their TCR composition, and two CTL clones were selected for HLA class I-restricted lysis of the autologous tumor and then tested for the recognition of HLA-A3+ and HLA-A3- normal or neoplastic cells of the melanocyte lineage. One CTL recognized a unique HLA-A3.1-restricted Ag expressed only by the autologous tumors, while all the other CTLs defined three HLA-A3.1 epitopes shared by melanomas, but not by melanocytes. Moreover, the epitopes of two CTL lines with different specificity were reconstituted by nonoverlapping fractions of HLA-A3+ melanoma-derived peptides resolved by reverse phase-HPLC, indicating that distinct naturally processed peptides were specifically recognized on melanoma cells in association with HLA-A3.1 molecules. These novel lineage-unrelated HLA-A3.1-restricted melanoma epitopes do not derive from MAGE, BAGE, or GAGE gene families, as evaluated by the COS-7 transfection assay. Our data show that CTLs may recognize HLA-A3.1-class 1 complexes presenting melanoma (but not melanocyte)-associated epitopes that are either unique to a given patient's tumor or that are shared between multiple melanomas.
Animals↗
Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines.
The BALB/c Meth A sarcoma carries a p53 missense mutation at codon 234, which occurs in a peptide, termed 234CM, capable of being presented to cytotoxic T lymphocytes (CTL) by H-2Kd molecules (Noguchi, Y., E.C. Richards, Y.-T. Chen, and L.J. Old. 1994. Proc. Natl. Acad. Sci. USA. 91:3171-3175). Immunization of BALB/c mice with bone marrow-derived dendritic cells (DC), generated in the presence of granulocyte macrophage colony-stimulating factor and interleukin 4, and prepulsed with the Meth A p53 mutant peptide, induced CTL that specifically recognized peptide-pulsed P815 cells, as well as Meth A cells naturally expressing this epitope. Immunization with this vaccine also protected naive mice from a subsequent tumor challenge, and it inhibited tumor growth in mice bearing day 7 subcutaneous Meth A tumors. We additionally determined that immunization of BALB/c mice with DC pulsed with the p53 peptide containing the wild-type residue at position 234, 234CW, induced peptide-specific CTL that reacted against several methylcholanthrene-induced BALB/c sarcomas, including CMS4 sarcoma, and rejection of CMS4 sarcoma in vaccination and therapy (day 7) protocols. These results support the efficacy of DC-based, p53-derived peptide vaccines for the immunotherapy of cancer. The translational potential of this strategy is enhanced by previous reports showing that DC can readily be generated from human peripheral blood lymphocytes.
Amino Acid Sequence↗
Class I-like CD1A-C do not protect target cells from NK-mediated cytolysis.
Previous studies have validated the capacity of classical (HLA-A,B,C) and nonclassical (HLA-G) MHC class I antigens expressed by target cells to influence natural killer (NK) cell-mediated cytolysis. Generally, elevated expression of these HLA class I molecules is correlated with enhanced resistance to lysis by NK cells. In this study, we have evaluated the effect of transfected class I-like CD1A, CD1B, and CD1C molecules on C1R (HLA-A,B null) target cell sensitivity to natural killing. We report that while each of these molecules was expressed at physiologically relevant levels on the cell surface of clonal transfectants, no change was observed in target cell susceptibility to fresh or lymphokine-activated NK-mediated lysis.
Antigens, CD1↗
Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity.
Cytotoxic T lymphocytes (CTLs) are a critical component of the immune response to tumors. Tumor-derived peptide antigens targeted by CTLs are being defined for several human tumors and are potential immunogens for the induction of specific antitumor immunity. Dendritic cells (DC) are potent antigen-presenting cells (APCs) capable of priming CTL responses in vivo. Here we show that major histocompatibility complex class I-presented peptide antigen pulsed onto dendritic APCs induces protective immunity to lethal challenge by a tumor transfected with the antigen gene. The immunity is antigen specific, requiring expression of the antigen gene by the tumor target, and is eliminated by in vivo depletion of CD8+ T cells. Furthermore, mice that have rejected the transfected tumor are protected from subsequent challenge with the untransfected parent tumor. These results suggest that immunization strategies using antigen-pulsed DC may be useful for inducing tumor-specific immune responses.
Amino Acid Sequence↗