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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.

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.

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.

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.

The recent identification of techniques both in the mouse and in man to culture dendritic cells which could be adoptively trasnferred for therapy have opened new avenues of research. We have applied both synthetic and natural or acid eluted peptides from the tumor cell surface in order to elicit effective antitumor responses in vivo and in vitro in murine tumor models as well as in vivo in human preclinical trials. Based on these studies, we will soon initiate a human clinical trial utilizing human dendritic cells pulsed with synthetic melanoma derived peptides presented by HLA-A2. The delivery of genes into human dendritic cells to constitutively express cytokines, costimulatory molecules or tumor antigens represents another potential approach to the development of dendritic cell based therapies.

Dendritic cells (DC), as professional antigen presenting cells (APC), have the capacity to capture antigen and migrate to lymphoid organs, where antigen presentation to and priming of naive T cells takes place (reviewed in 1). It is currently believed that effective induction of antigen-specific T cell responses requires a dynamic dialogue of multiple, sequential interactions between the DC as APC and a given T cell clone in order to prime the cell for expansion and effector function. The first interaction (signal 1) is felt to involve the MHC I/II-peptide on APC with the T cell receptor (TCR) on the effector lymphocyte, with a second signal being the costimulatory interaction between CD80 (B7.1)/CD86(B7.2) on DC with CD28 on the T cell. Signals 1 and 2 combine to activate the T cell, upregulating the expression of CD4OL which can interact with DC-expressed CD40 (reviewed in 2). This DC-T cell interaction results in the generation of an antigen-specific T cell responses. The ability of DC to prime naive T cells has been attributed to several factors, most notably the high levels of expression of MHC and costimulatory molecules such as B7.1 and B7.2. It has also been shown that DC are capable of producing several cytokines, among them IL-12, a cytokine critical for the development of Thl responses (3,4). Recently, ligation of CD40 on both human (5) and murine (6) DC has been reported to result in the upregulation of costimulatory molecules and the induction of IL-12. These findings suggest that the interaction of T cells with DC result in signals to DC which are critical for induction of the cellular immune response. Whereas many of the signals required by T cells for activation and proliferation have been described, the signaling requirements for optimal DC activation remain poorly defined.

Cytotoxic T lymphocytes (CTL) are an important component of the host’s immune response to cancer1,2. A number of genes encoding tumor-associated antigens (TAA) and their peptide products which are recognized by CTL in the context of major histocompatibility complex (MHC) class I molecules have recently been identified3,4. Our group has focused on the translation of these new insights into the development and application of novel immunotherapies.

CD8+ T lymphocytes recognize antigenic peptides presented by major histocompatibility complex (MHC) class I molecules. Individual peptide termini appear to be fixed at the C- and N-terminal ends. In contrast, central peptide side chains residues may point in different directions and exhibit limited flexibility, dependent on the MHC class I structural variation. For instance, position 97 in HLA-A201 has been shown to shift individual peptide species into different coordinations, one oriented towards the peptide N terminus, or more towards the C-terminal end. The conformational shape of such non-anchor peptide residues may affect the affinity of MHC/peptide/TCR interaction, resulting in quantitative, or qualitative different T cell effector functions. To characterize the impact of different amino acid residues occupying position 97 in HLA-A2 on peptide binding and presentation to CTL, we generated a panel of mutated HLA-A2 molecules containing either M, K, T, V, G, Q, W, P or H at position 97. The HLA-A0201 presented melanoma-associated MART-1/Melan-A derived peptide AAGIGILTV was employed to assess the impact of such position-97 mutations on HLA-A2 in peptide binding measured in an HLA-A2 reconstitution assay and presentation to AAGIGILTV-specific polyclonal or clonal T lymphocytes as measured by cytotoxicity, or interferon (IFN)-gamma and granulocyte/ macrophage colony-stimulating factor (GM-CSF) secretion. The high-affinity AAGIGILTV peptide bound to all position-97 mutants, albeit with differential efficiencies, and elicited specific release of IFN-gamma and GM-CSF by CTL. CTL responses were triggered only by the HLA-A2 wild type, by HLA-A2-H97 (histidine position 97 mutant), and HLA-A2-W97. The HLA-A2-M97 presenting molecule elicited enhanced cytokine release and CTL effector functions by polyclonal and by clonal effector T cells. These results indicate that MHC class I-bound peptides can trigger specific cytokine release by effector T cells independently of their ability to induce cytolysis. We conclude that relatively minor changes in the MHC class I peptide binding groove, including substitutions at position 97, can affect recognition by antigen-specific T cells. Mutant MHC class I molecules, such as those described here, may act as partial peptide antagonists and could be useful for inducing T lymphocytes with qualitatively different effector functions.

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.

Interleukin-12 (IL-12) promotes specific and long-lasting anti-tumor immunity mediated by T cells in a variety of murine tumor models. IL-12 also synergizes with B7.1 (CD80) co-stimulation to induce proliferation and cytokine production by both human and murine T cells in vitro. We evaluated the combined anti-tumor efficacy of IL-12 and B7.1 gene delivery in two apparently poorly immunogenic tumor models (TS/A and MCA207). In both of these models, expression of B7.1 and production of IL-12 in the inoculum led to improved anti-tumor immunity, with up to 80% long-term tumor-free animals (vs 0-20% of mice remaining tumor free when inoculated with either B7.1- or IL-12-transfected tumors alone). Tumor-free mice were capable of rejecting a subsequent rechallenge with the wild-type tumor in 66% of the cases. Cooperativity was dependent upon the level of IL-12 secreted by engineered cells. IL-12 delivery required B7 expression of therapeutic effects to be observed in these models. Vaccines provided at a site distal to a control, non-transfected tumor slowed (TS/A) or abrogated (MCA207) the progression of wild-type tumors. The synergistic anti-tumor effects associated with combined application of B7.1- and IL-12-transfected tumors were partially negated by systemic administration of the CD28-B7.1/B7.2 antagonist CTLA4-Ig or by inoculation with neutralizing antibodies directed against murine interferon-gamma or tumor necrosis factor-alpha, two cytokines elicited in response to IL-12 stimulation. These data support the potential clinical utility of combined gene therapy using IL-12- and B7.1-engineered autologous cells (tumor or fibroblasts) as a vaccine to elicit specific anti-tumor immunity.

Human melanoma represents the principal cause of death in patients with skin cancer in the United States and Europe. Tumour infiltrating lymphocytes recognizing melanoma have been used to identify the tumour antigens recognized by T-cells in the context of MHC class I or class II molecules. Such antigens include MAGE-1, MAGE-3, MART-1/Melan-A, gp100, tyrosinase, the tyrosinase-related antigen gp75, the antigen gp15 and the mutated CDK4 and beta-catenin gene-products. The identification of these T-cell epitopes provides us with novel reagents for the development of state-of-the-art treatments and for the (immuno-)monitoring of patients with melanoma. In order for treatments, including peptide-based vaccines, to be successful, several conceptual criteria must be met: (1) The patient's tumour must present the relevant epitope(s) integrated into the vaccine, (2) the tumour should express the appropriate restricting major histocompatibility complex (MHC) molecule(s) required for patient cytotoxic T lymphocyte (CTL) reactivity, and (3) the patient's T-cell repertoire should be able to react productively against the melanoma antigens present in the vaccine. Clinical trials implementing peptide-based vaccines or whole protein therapies have been initiated in the United States and Europe. We suggest that such treatments should include the careful monitoring of anti-tumour T-cell responses. This should include examination of melanoma antigen and MHC class I allele expression in the individual patient's tumour, assessment of the status of the peptide transporter molecules TAP1/TAP2 and evaluation of T-cell mediated immune responses reactive against peptides and autologous melanoma. Evaluation of clinical parameters (such as disease-free survival) in conjunction with an examination of immunological parameters may facilitate our understanding of the immune responses against T-cell antigens that are shared among melanoma and normal melanocytes, and may ultimately help to identify the most effective immunotherapy for patients with melanoma.

Human renal cell cancer (RCC) is clearly responsive to immunotherapy. Clinical responses may be mediated by "non-specific" (e.g. natural killer, NK, cells) or "specific" MHC-class-I-restricted tumor-specific CD8+ T lymphocytes. Typically RCC progresses, however, despite significant infiltration of various lymphoid cells. We examined freshly isolated RCC tumor-infiltrating lymphocytes (TIL) derived from seven RCC patients for cytokine expression by the polymerase chain reaction (PCR). Established RCC tumor cell lines derived from these RCC patients were negative for interleukin-2 (IL-2), IL-4, IL-10, and interferon gamma and found to be positive for tumor necrosis factor alpha (TNF alpha), IL-6, IL-1 beta, granulocyte/macrophage-colony-stimulating factor (GM-CSF), and transforming growth factor beta 1 (TGF beta 1) message as detected by PCR. An identical pattern of cytokine mRNA expression was identified in other long-term RCC lines and in normal human kidney cells upon culture, but not in two Wilms tumor cell lines tested. Short-term-, and long-term-established RCC lines, but not Wilms tumor lines, secreted substantial levels of GM-CSF, TNF alpha, IL-1 beta, and IL-6 as detected by enzyme-linked immunosorbent assay. Both RCC lines and Wilms tumor lines secreted TGF beta 1. In comparison, normal kidney cells secreted IL-6 and GM-CSF, but not IL-1 beta, or TFG beta 1 under identical in vitro cell culture conditions. We applied PCR-based methods to characterize the cytokine mRNA expression pattern in immune cells infiltrating into renal cell cancer without the need for expansion of such effector cells in vitro. Examining freshly collected RCC TIL by PCR from patients with primary cell cell cancer, we could demonstrate that such cells, but not lympho-mononuclear cells harvested from normal human kidney tissue, typically exhibit IL-4 and IL-10 mRNA expression.

Immune cell infiltration in various tumors has been described in numerous reports over the past several years. Local as well as systemic anti-tumor directed immunity has been studied in the context of tumor progression and clinical prognosis (1,2). The host immune response elicited by human cancer includes various effector cells: natural killer cells (NK) cells, macrophages, T-lymphocytes, polymorphonuclear neutrophils (PMNs), as well as basophils and eosinophils. The most convincing evidence supporting a beneficial and effective role of the host immune system in cancer has been the demonstration of MHC (major histocompatibility complex) restricted and tumor specific T-cells. This has been found only on occasion in patients with RCC (3–6). Spontaneous tumor regression, response to immunotherapy with interleukin-2, and/or passive immunotherapy by transferring in vitro expanded tumor infiltrating lymphocytes (TIL) may also lead to regression of renal cell cancer (7,8).

Introduction: Limited MHC Class I antigen presentation by tumor cells has consistently been identified as a significant barrier to cancer immunotherapy. Combinational regimens that coordinately enhance tumor-associated antigen (TAA) presentation on tumor MHC Class I molecules and stimulate TAA-specific T cell immunity are expected to yield synergistic clinical benefit. We have previously shown in vitro that the HSP90 inhibitor 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), which is currently being evaluated as an anti-cancer agent in phase I/II clinical trials, is capable of enhancing MHC Class I presentation of EphA2, a TAA that is over expressed in several cancers (Melanoma, lung cancer, sarcomas). To further characterize the therapeutic potential of this approach, we are currently assessing the safety and effectiveness of a rationally-designed combinational therapy incorporating 17-DMAG and EphA2-targeted immunotherapy in murine tumor models. Results: The anti-tumor efficacy of 17-DMAG was evaluated in an MCA205 sarcoma model applied to syngenic mice.17-DMAG facilitated a dose-dependent reduction in EphA2 expression in vivo, which correlated with increased presentation of EphA2 peptides on tumor MHC class I molecules. Increased EphA2 presentation on tumor MHC class I molecules was determined by documenting the increased recognition of treated tumor cells by EphA2-specific T cells.17-DMAG treatment, when combined with 2 different immunotherapy approaches targeting EphA2 (adoptive transfer of EphA2-specific T cells and DC-based vaccine), led to superior anti-tumor impact and in some cases, even complete tumor regression, compared to single-modality approaches. In addition, combination treatment groups had increased infiltration of tumors by CD4+ and CD8+ T cells and decreased Tregs and Myeloid-derived suppressor cells (MDSCs) compared to groups receiving single mode of treatment or untreated progressors. Infiltrating CD8 T cells in the tumor in the combination therapy cohort were specific for EphA2, as assessed by in vitro stimulation. Skewing of the immune response towards a type 1 phenotype was caused by upregulated inflammatory chemokines and their receptors in the TME. 17-DMAG also facilitated vascular remodeling of the tumor with decreased endothelial cell and pericyte coverage coinciding with increased VCAM-1 expression, which may contribute to the enhanced recruitment of antigen-specific T cells. Conclusions: Our studies suggest that conditionally decreased EphA2 protein levels in tumors treated with 17-DMAG is accompanied by increased recognition by therapeutic anti-EphA2 T cells in vivo. Combinational immunotherapies integrating 17-DMAG have superior anti-tumor efficacy compared to single modality approaches and are orchestrated by the vascular and immunologically-preferred alterations in the tumor promoted by 17-DMAG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4399. doi:1538-7445.AM2012-4399

We have recently shown that intra-tumor (i.t.) injection of syngenic dendritic cells (DC) engineered to express the transcription factor Tbet (TBX21) promotes protective Type-1 T cell-mediated immunity via a mechanism that is largely interleukin (IL)-12p70-independent. Since IL-12 is a classical promoter of Type-1 immunity, the current study was undertaken to determine whether gene therapy using combined Tbet and IL-12 cDNA would yield improved anti-tumor efficacy based on the complementary/synergistic action of these biologic modifiers. Mice bearing established s.c. tumors injected with DC concomitantly expressing ectopic Tbet and IL-12 (i.e. DC.Tbet/IL12) displayed superior i.) rates of tumor rejection and extended overall survival, ii.) cross-priming of Tc1 reactive against antigens expressed within the tumor microenvironment, and iii.) infiltration of CD8+ T cells into treated tumors in association with elevated locoregional production of CXCR3 ligand chemokines. In established bilateral tumor models, i.t. delivery of DC.Tbet/IL12 into a single lesion led to slowed growth or regression at both tumor sites. Furthermore, DC.Tbet/IL12 pulsed with tumor antigen-derived peptides and injected as a therapy distal to the tumor site prevented tumor growth and activated robust antigen-specific Tc1 responses. These data support the translational use of combined Tbet and IL-12p70 gene therapy in the cancer setting. In order to target multiple gene pathways which should provide a more beneficial clinical outcome, we have begun combinational immunotherapy with DC.Tbet/IL12 and chemotherapeutic drugs targeting multiple receptor tyrosine kinases (i.e. sunitinib). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1537. doi:1538-7445.AM2012-1537

Interleukin-12 (IL-12) is a heterodimeric cytokine originally defined by its ability to induce the maturation of cytolytic lymphocytes and by its capacity to effectively synergize with IL-2 in the induction of cytolytic activity. Recent studies in mice have demonstrated the ability of IL-12 to cause tumor regression and stimulate long-term antitumor immunity in treated animals. To examine the antitumor effect of direct gene transfer of IL-12 into tumors, we have developed retroviral vectors that coordinately express both subunits of IL-12. An MFG-based retroviral vector was used to generate a recombinant retrovirus in which a long terminal repeat (LTR)-driven polycistronic transcript encodes both subunits of human IL-12: hp35 and hp40 cDNAs are linked and coexpressed using the internal ribosome entry site (IRES) from the encephalomyocarditis virus (DFG-hIL-12). In addition, two IRES sequences were used to express both subunits of IL-12 and a neomycin resistance (neoR) selectable marker gene from the same polycistronic message (TFG-hIL-12). The amphotropic DFG-hIL-12 and TFG-hIL-12 viruses were used to infect both human and murine cell lines as well as primary tumor cultures. The production of human IL-12 by the nonselected, infected cells was measured in both a PHA blast proliferation bioassay and an ELISA and ranged from 15 to 40 ng/10(6) cells per 24 hr. Following G418 selection of TFG-hIL-12-infected cells, the level of expression of IL-12 was significantly higher (up to 120 ng/10(6) cells per 24 hr). The IL-12 protein secreted by the infected cells exhibited all of the biologic activities of recombinant hIL-12: proliferation of activated natural killer (NK) and T cells, stimulation of interferon-gamma (IFN-gamma) induction by NK and T cells, and enhancement of lymphokine-activated killer (LAK) activity. These retroviral vectors expressing human IL-12 should be useful in evaluating the biological properties of IL-12 as well as for use in clinical trials for gene therapy of patients with cancer.

Mutationen und Deletionen des p53 Tumorsuppressorgens sind die haufigsten genetischen Veranderungen bei Tumoren [1], wie z.B. Melanomen, Lungen- und Colontumo-ren. Als Folge der Mutationen konnen Proteine exprimiert werden, die sich vom Wildtyp (wt) durch eine von Minuten auf Stunden verlangerte Halbwertszeit unterscheiden und deshalb immunhistologisch nachgewiesen werden konnen. Uberexprimiertes, mutiertes p53 fuhrt bei 20–30% von Patienten mit p53+ Tumoren (Mamma-, Colo-rektalen Tumoren) zur Bildung von IgG p53-Antikorpern, was eine Th-Zellreaktivitat gegenuber p53 Sequenzen voraussetzt und eine CD8+ T-Zell Reaktivitat nahe legt. Solche CD8+ T-Zellen erkennen 8–10 Aminosauren lange, von einem MHC Klasse I Molekul (HLA Molekul) prasentierten Peptidsequenzen. Die Sequenzen folgen fur die jeweiligen HLA Molekule unterschiedlichen Regeln, dem Peptidbindungsmotiv. Es ist fur das bei Kaukasiern am haufigsten vorhandene HLA-A2 Molekul dadurch bestimmt, das an den ”Ankerpositionen” 2 und 9 der Aminosauresequenz von 1–9, mit denen die Sequenz in den Peptidbindungsspalt des HMC Molekuls hineinragt, spezielle Aminosauren vorhanden sind [2]. Die Bindung eines Peptides an ein MHC Molekul ist eine entscheidende Voraussetzung dafur, das es von T-Zellen (CD8+) erkannt wird.

Mutations of the p53 gene are the most frequently observed genetic changes in human cancers; often leading to an overexpression of the wild-type (wt) p53 protein. Demonstrable T cell reactivity against tumor cells overexpressing wt or mutant p53-derived peptides could support the application of such epitopes in cancer immunotherapies. As the binding of peptide to MHC class I molecules is a prerequisite for antigen-specific T cell recognition, we evaluated the ability of wt and mutant p53 peptides to bind to HLA-A2.1 using two independent flow cytometry-based assay systems, the T2 major histocompatibility complex (MHC) class I peptide stabilization assay (stabilization assay) and the peptide-induced MHC class I reconstitution assay (reconstitution assay). The twenty selected wt sequences each conformed to the previously reported HLA-A2.1 peptide binding motif. Seven of the wt p53 and 2/13 mutant p53 peptides derived from the previously chosen wt peptides bound to HLA-A2.1 in both the stabilization and the reconstitution assays. An additional six wt and six mutant p53 peptides, presumably exhibiting lower affinity for HLA-A2.1, were identified only in the reconstitution assay. Those p53 peptides binding HLA-A2.1 may provide useful immunogens for the generation of HLA-A2.1-restricted cytolytic T lymphocytes in vitro and in vivo.

Tumor vasculature is classified by leaky, chaotic blood vessels consisting of several components including vascular endothelial cells (VEC) and mural cells (smooth muscle cells and pericytes), as well as immune regulatory cells, stem cells and vascular progenitors. Irregular tumor vasculature limits immune effector cell access to the tumor, and may in part be responsible for the modest success observed in many current anti-cancer immunotherapies. Certain drugs targeting tyrosine kinase receptors expressed on tumor stromal cells induce a Type-1 proinflammatory skewing of the tumor microenvironment (TME) with a coordinate reduction in the number of immune regulatory cell populations. A transient normalization of the vasculature may also occur in association with enhanced leukocyte recruitment. We have shown that vaccination against tumor-associated stromal antigens leads to the activation of VCAM-1 expression on tumor vascular endothelial cells and the enhanced recruitment of protective Type-1 T cells into the TME. Currently little is known regarding pericyte biology and their behavior in the TME, with no existing treatments specifically targeting pericytes. We hypothesize that pericytes residing in the TME differentially express antigens that distinguish them from pericytes in healthy tissue, and vaccination specifically targeting tumor pericyte antigens will result in vascular normalization and improved delivery of protective Type-1 T cells into the TME. To address these hypotheses, we examined mRNA expression of pericytes and VEC that have been flow-sorted from human renal cell carcinoma (RCC) tumors and patient-matched normal adjacent kidney (NAK) tissue. We have identified antigens that are differentially expressed by pericytes and/or by VEC in the TME versus NAK (such as RGS5). We are currently investigating the therapeutic efficacy of genetic (lentiviral) vaccines encoding tumor-associated stromal antigens in murine tumor models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3466. doi:10.1158/1538-7445.AM2011-3466

Introduction: EphA2 and HSP90 play important roles in tumor growth, survival and metastasis. An effective therapeutic approach would promote decreased expression of EphA2 on tumor cells, while increasing presentation of EphA2-derived peptides on tumor MHC class I complexes, thereby enhancing the recognition of tumors by anti-EphA2 CD8+ T effector cells. Aim: We are assessing the safety and effectiveness of the HSP90 inhibitor 17-DMAG in enhancing anti-tumor immunotherapy with EphA2-specific T cells in murine tumor models. Methods: We tested the effect of 17-DMAG on tumor cell expression of EphA2 and the MHC I antigen-processing machinery in vitro. Efficacy of 17-DMAG in an MCA205 sarcoma model was also evaluated in vivo. Results: 17-DMAG facilitated a dose-dependent reduction in EphA2 expression, but had no adverse effects on the antigen-processing machinery of the tumors. Similar effects were seen in an MCA205 sarcoma model in vivo, where reduction in EphA2 levels in tumors corresponded with increased recognition by EphA2-specific CD8+ T cells in vitro. 17-DMAG treatment also facilitated increased infiltration of CD4 and CD8 T cells in the tumor, compared to untreated cohorts. Conclusion: 17-DMAG treatment promotes immunologically-preferred alterations tumor cells in vitro as well as In vivo. Our preliminary in vivo studies suggest that decreased EphA2 levels in tumors is accompanied by increased presentation of EphA2 peptides in tumor MHC class I complexes. Further in vivo studies are warranted to assess the net anti-tumor impact of combinational immunotherapies integrating 17-DMAG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1805. doi:10.1158/1538-7445.AM2011-1805

Interleukin-12 (IL-12) is a B-cell and monocyte-derived 75 kDa heterodimeric cytokine released early after immune stimulation. It promotes the cytolytic maturation and proliferation of T and natural killer (NK) cells and release of interferon-gamma from these effectors. Furthermore, IL-12 appears to stimulate the production of a Th1 immune response. We have examined the effects of IL-12 on the proliferation and lytic activity of fresh peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs). IL-12 stimulates proliferation of PBMCs by as much as 10-fold after T-cell receptor (TCR) ligation induced by anti-CD3 or phytohemagglutinin. In contrast, IL-12 promotes only marginal proliferation of resting PBMCs. IL-12 modulates IL-2-induced lymphokine-activated killer (LAK) cell activity: it inhibits IL-2 LAK when added early to culture but augments LAK activity in PBMCs preactivated by IL-12. IL-12 induces the proliferation (three- to fourfold above background) and enhances the cytolytic activity (two- to fourfold) of TIL lines and melanoma-derived, peptide-specific T-cell clones that have been recently restimulated with autologous tumor. These results suggest that IL-12 may serve in vivo to amplify and focus the cellular immune response by selectively inducing the outgrowth and enhancing the lytic potential of T and NK cells that have received the proper coactivation signals.

Immune modulating reagents that trigger the T cell costimulatory molecules GITR and OX40 are being evaluated as anti-cancer agents in early-stage clinical trials. However, little pre-clinical information is available regarding the efficacy and mechanism(s) of action for these agents in the setting of advanced, well-established disease. To further characterize the molecular, cellular, and treatment-associated consequences of GITR and OX40 engagement, novel agonistic reagents directed against murine GITR and OX40 (ligand-Fc fusion proteins) were recently constructed and characterized in vitro. We now show that the growth of well-established, day 17 sarcomas is significantly inhibited or ablated by a short course of either treatment, with OX40L-Fc demonstrating superior anti-tumor efficacy over GITRL-Fc at comparable doses. Despite diminished frequencies of CD4+Foxp3+ regulatory T cells (Treg) in the tumor microenvironment (TME) 3 days post-treatment, the presence of myeloid-derived suppressor cells (MDSC) and inhibitory cytokines such as IL-10 and TGFβ appears unaltered and effector T cell production of the Type-1 cytokine IFNγ remains low. Treatment with either GITR or OX40 agonists, however, induces a coordinate increase in CCR7+ dendritic cells in the tumor-draining lymph nodes (TDLN), followed approximately 7 days after treatment by profound expansion of effector T cells expressing the Type-1 chemokine receptor CXCR3. By day 10 post-treatment, particularly with the OX40 agonist, T cells displaying a recently-activated effector phenotype accumulate in the tumor microenvironment and secrete high levels of IFNγ. Collectively, these data suggests a model in which the first level effect of costimulatory treatment is to direct CCR7+ dendritic cells from the TME to the draining lymph nodes. A secondary, systemic response reflects the expansion of highly-reactive CXCR3+ Type-1 T cells in the TDLN and their subsequent trafficking into the TME. The therapeutic efficacy demonstrated in this model by OX40L-Fc, and to a lesser extent GITRL-Fc, strongly supports the translation of such modalities into human clinical trials, while the mechanistic insight gained from these studies suggests that successful immunotherapies will license both local, and perhaps more importantly, systemic Type-1 T cell immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2938.

T-bet is a T-box transcription factor (Tbx21) previously identified as a master regulator of Type-1 polarization in T cells. We have previously demonstrated that ectopic expression of hT-bet converts dendritic cells (DC) into Type 1-biasing APCs. In the current study, we investigated whether mouse DC overexpressing mT-bet promote superior Type 1 anti-tumor T cell responses when applied as an intratumoral therapy. CD11c+ DCs were then purified and transduced with recombinant adenovirus to express high levels of mT-bet. DC. mT-bet stimulated stronger Tc1 and Th1 T cell responses when compared with control DC and retained the capacity to kill and then uptake tumor cells/debris, allowing for the cross-priming of specific T cell responses in vitro. Surprisingly, in contrast to human DC. T-bet, CD40L-activated DC. mT-bet produced elevated levels (vs. control DC) of IL-12p70/TNF-α. DC. mT-bet exerted potent anti-tumor activity in the (day 7 established) s.c. CMS4 sarcoma after intratumoral administration. These anti-tumor effects were mediated primarily by Tc1 cells, however, both CD4+ T cells and NK cells appeared to also be involved in therapeutic response. In situ imaging suggested that DC. mT-bet treatment inhibited the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells and tumor-associated vascular structures. Collectively, these findings suggest that DC. mT-bet-based therapy has the potential to promote superior Type-1 anti-tumor immunity in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3824.

Structural characteristics of major histocompatibility complex class I antigens associated with natural killer (NK)-resistance phenomena were examined. Previous research has shown that transfection of class I genomic DNA clones into class I-deficient, NK-sensitive target cell lines results in transfectants exhibiting class I+, NK-resistant phenotypes. In contrast to the HLA-A3, -B7, -B27, and -Bw58 class I molecules, the HLA-A2 class I molecules were shown not to protect target cells from NK activity. Here we show that this nonprotective phenotype maps to the alpha 1 domain of the HLA-A2 molecule by examining the NK-protective capacity of the natural interdomain recombinant HLA-Aw69 molecule. HLA-Aw69, which consists of an alpha 1 domain exhibiting homology with HLA-Aw68, and alpha 2/alpha 3/transmembrane-cytoplasmic domains, exhibiting homologies with HLA-A2, mimics HLA-Aw68 and provides HLA-A,B null target cell (C1R) transfectants with increased resistance to NK. Further, the inability of transfected HLA-A2 to confer protection against NK activity can be completely attributed to the expression of a "nonpermissive" residue at position 74 in the alpha 1 domain. Site-directed mutation of the His-74 residue in HLA-A2 to the Asp-74 (HLA-A3, -Aw68, -Aw69, -B7) residue generates a mutant that provides C1R cell line transfectants an NK-resistant phenotype. As His-74 blocks access to a side pocket in the HLA-A2 antigen-binding cleft, these results support the critical involvement of residues within the peptide-binding groove of class I molecules in determining the NK susceptibility phenotype of class I+ target cells.

The receptor tyrosine kinase EphA2 is over-expressed in numerous carcinomas in association with poor clinical prognosis. While pharmacologic agents that antagonize EphA2-mediated signaling could be contemplated as a therapy, a more preferred result would be to decrease tumor cell EphA2 expression while coordinately enhancing specific immune reactivity to tumor cells. We show that specific mAb triggers the internalization of EphA2, resulting in increased processing of EphA2 protein and presentation of EphA2-derived epitopes in tumor cell HLA class I complexes to CD8+ T cells (detected by IFN-γ production and CD107b translocation). Ab-enhanced CTL recognition is blocked by tumor cell co-treatment with Pseudomonas Exotoxin-A and proteasome inhibitors, but not by chloroquine, suggesting that internalized EphA2 complexes are rapidly translocated into the cytoplasm for delivery of peptides into the class I biosynthetic route. When combined with vaccines designed to augment the frequency of circulating anti-EphA2 CD8+ T cells, this agonist strategy may define an effective therapy for patients harboring EphA2+ cancers.

Many receptor tyrosine kinases (RTK) are overexpressed/dysregulated in advanced cancers, serving as negative prognostic indicators. We evaluated the immunogenicity of dendritic cell (DC)/RTK(EphA2, EGFR, Her2/neu)-based vaccines in HLA-A2 (HHD) Tg mice. To enhance the stimulatory capacity of BM-derived DCs, these cells were infected with a recombinant adenovirus encoding mIL-12p70 (Ad.IL12), prior to being pulsed with RTK-derived peptides. We also evaluated a cohort of DC co-infected with both Ad.IL12 and Ad.EphA2. Each of these DC preparations (vs. control DCs) were injected three times s.c. into HHD mice on a weekly schedule. DTH reactions were monitored 48–72h after each vaccination and splenocytes were harvested on d28 and d60 to assess specific CD8+ T cell reactivity (IFN-γ ELISA). Autoimmunity pathology in RTK+ normal tissues (i.e. kidney, liver, lung) were also evaluated for immune infiltration and tissue destruction by (immuno)histochemistry. Vaccines induced specific DTH responses and Tc1 responses in HHD mice, in association with an increased accumulation of CD8+ T cells around proximal endothelial cells (PEC) in the kidney and in the interstitial lung at d28, in the absence of tissue pathology. DC/RTK-based vaccines may promote specific Type-1 immunity that has the potential to eradicate RTK+ tumors, while sparing normal RTK+ tissues. This work was supported by NIH R01 grant CA114071 (W.J.S.)

Pathogen-derived 'helper' sequences promote anti-tumor immune responses induced by a naked DNA vaccine ( ).

BACKGROUND: A novel tyrosine kinase receptor EphA2 is expressed at high levels in advanced and metastatic cancers. We examined whether vaccinations with synthetic mouse EphA2 (mEphA2)-derived peptides that serve as T cell epitopes could induce protective and therapeutic anti-tumor immunity. METHODS: C57BL/6 mice received subcutaneous (s.c.) vaccinations with bone marrow-derived dendritic cells (DCs) pulsed with synthetic peptides recognized by CD8+ (mEphA2671-679, mEphA2682-689) and CD4+ (mEphA230-44) T cells. Splenocytes (SPCs) were harvested from primed mice to assess the induction of cytotoxic T lymphocyte (CTL) responses against syngeneic glioma, sarcoma and melanoma cell lines. The ability of these vaccines to prevent or treat tumor (s.c. injected MCA205 sarcoma or B16 melanoma; i.v. injected B16-BL6) establishment/progression was then assessed. RESULTS: Immunization of C57BL/6 mice with mEphA2-derived peptides induced specific CTL responses in SPCs. Vaccination with mEPhA2 peptides, but not control ovalbumin (OVA) peptides, prevented the establishment or prevented the growth of EphA2+ or EphA2-negative syngeneic tumors in both s.c. and lung metastasis models. CONCLUSIONS: These data indicate that mEphA2 can serve as an attractive target against which to direct anti-tumor immunity. The ability of mEphA2 vaccines to impact EphA2-negative tumors such as the B16 melanoma may suggest that such beneficial immunity may be directed against alternative EphA2+ target cells, such as the tumor-associated vascular endothelial cells.

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- I/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-A 27-35 , MART-1/Melan-A 32-40 , gp100 280- 288 , tyrosinase 368-376 , 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.