Tumor-associated Antigen Epitopes Research Articles

Development of Cell Technologies Based on Dendritic Cells for Immunotherapy of Oncological Diseases.

Immunotherapy using dendritic cell-based vaccination is a natural approach using the capabilities and functions inherent in the patient's immune system to eliminate tumor cells. The development of dendritic cell-based cell technologies evolved as the disorders of dendritic cell differentiation and function in cancer were studied; some of these functions are antigen presentation, priming of cytotoxic T-lymphocytes and induction of antigen-specific immune responses. At the initial stage of technology development, it was necessary to develop protocols for the in vitro generation of functionally mature dendritic cells that were capable of capturing tumor antigens and processing and presenting them in complex with MHC to T-lymphocytes. To achieve this, various forms of tumor-associated antigen delivery systems were tested, including lysates, tumor cell proteins (peptides), and DNA and RNA constructs, and it was shown that the use of DNA and RNA constructs was the most effective method, as it made it possible not only to deliver the most immunogenic epitopes of tumor-associated antigens to dendritic cells, but also to enhance their ability to induce antigen-specific cytotoxic T-lymphocytes. Currently, cell therapy based on dendritic cells is a modern basis for antigen-specific immunotherapy of cancer due to the simplicity of creating DNA and RNA constructs encoding information about both target tumor antigens and regulatory molecules. The potential development of cell technologies based on dendritic cells aims to obtain antigen-specific cytotoxic T-lymphocytes induced by dendritic cells, study their functional activity and develop cell-based therapy.

BIOM-56. THE PRIVATE AND RECURRING ANTIGENIC PROFILE OF MOST AGGRESIVE PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Efficient immunotherapies for pediatric brain tumors face a significant challenge in identifying tumor rejection antigens. To overcome this hurdle, we utilized a comprehensive computer algorithm called the Open Reading Frame Antigen Network (O.R.A.N.). METHODS Our focus was on expressed epitopes, including single nucleotide variations, small indels, gene fusions, novel junctions from alternative splicing, and Tumor Associated Antigens (TAAs), for antigenic epitope predictions. To ensure uniqueness, we compared immunogenic TAA epitopes against a personalized human proteomic library, ensuring they were not shared by other expressed isoforms or genes. The immunological landscape of medulloblastoma (n = 170) and DIPG (n = 30) was explored using immune deconvolution techniques combined with single-cell RNA sequencing integration. RESULTS Our hypothesis was that medulloblastomas and DIPG exhibit diverse immune profiles and express genes that are rarely found in normal tissues, making them potential targets for immunotherapy. In this study, we found that most neoantigens identified in medulloblastoma patients were unique to each individual, emphasizing the need for personalized neoantigen-based therapies. Interestingly, recurring neoantigens were predominantly present in the MB SHH subgroup, while they constituted only a small fraction of putative antigens in WNT, Group 3, and Group 4 patients. On the other hand, recurring Tumor Associated Antigens (TAAs) were more frequently expressed in the latter three molecular subgroups but not in the SHH subgroup. Thus, a combination of personalized neoantigen vaccines and targeting recurring TAAs could potentially maximize the anti-tumor effects. CONCLUSION Medulloblastoma is a disease characterized by its heterogeneity, and current standard treatments do not adequately address this aspect. In the case of DIPG, surgical resection is not a viable option due to its diffuse and infiltrative nature within the brainstem. By leveraging the power of the immune system, cellular immunotherapy holds promise in providing more targeted and personalized treatment options for pediatric brain tumor patients.

CTIM-30. THE PRIVATE AND RECURRING ANTIGENIC PROFILE OF HUMAN MEDULLOBLASTOMA

Abstract BACKGROUND Identifying tumor rejection antigens remains a major barrier to developing effective immunotherapeutics and their application to pediatric brain tumors. We performed a multi-faceted computer algorithm, the Open Reading Frame Antigen Network (O.R.A.N.), on medulloblastoma transcription profiles and predicted antigens across a broad array of antigen classes. METHODS Patient-specific HLA haplotypes were called via customized Optitype and Phlat algorithms. Only expressed mutations such as single nucleotide variations, small indels, gene fusions, and Tumor Associated Antigens (TAAs) were used for antigenic epitope predictions. TAAs were selected only if expressed > 1 transcript per million (TPM) in tumor and the standardized expression across a human tissue database. Immunogenic TAA epitopes were screened against a customized human proteomic library to guarantee that epitopes were not shared by other expressed isoforms or genes. Immune deconvolution with single cell RNASeq integration was leveraged for teasing out medulloblastoma immunologic landscape. RESULTS We hypothesized that medulloblastomas are immunologically heterogeneous and express genes with limited normal tissue expression that may serve as targets for immunotherapy. In this study, most neoantigens from medulloblastoma patients were found private suggesting a personalized neoantigen based therapy was on demand. In addition, recurring neoantigens were common in SHH but composed only a small proportion of putative antigens in WNT, Group 3 and Group 4 patients. However, recurring TAAs were found to be more commonly expressed in the latter three molecular subgroups but not in SHH. Together, combining personalized neoantigen vaccine and taking advantage of recurring TAAs approach may maximize anti medulloblastoma effect. CONCULSION Medulloblastoma is a heterogeneous disease, and the current standard of care treatments don’t take that into account. Newer, and safer, patient-specific treatment approaches are required to treat high-risk patients who are not cured by the standard therapies .Cellular immunotherapy might be key to improving survival and avoiding morbidity.

Dendritic cells transfected with a polyepitope DNA construct stimulate an antitumor cytotoxic response in various tumors.

Dendritic cells (DCs) loaded with tumor-associated antigens (TAAs) are known to be crucial for the antitumor response and are still included in various treatment regimens in cancer immunotherapy research. In the present study, a cell-based protocol was evaluated, involving the use of original DNA constructs encoding the wide range of TAA epitopes expressed on different epithelial cancers. The constructs were transfected into in vitro-generated DCs of patients with various types of cancer, including breast, colorectal and non-small cell lung cancer. The direct cytotoxicity assay of effector cells, activated with the transfected DCs, revealed a significant increase in cytotoxicity against autologous tumor cells. The use of DNA constructs encoding a large number of TAAs for insertion into DCs in vitro, aiming to activate a T-cell response may prove to be a reliable and unified approach for immunotherapy and for the prevention of relapse in patients with epithelial cancers.

Dry Formulation of Virus-Like Particles in Electrospun Nanofibers.

Biologics can be combined with liquid polymer materials and electrospun to produce a dry nanofibrous scaffold. Unlike spray-drying and freeze-drying, electrospinning minimizes the physiological stress on sensitive materials, and nanofiber mat properties such as hydrophobicity, solubility, and melting temperature can be tuned based on the polymer composition. In this study, we explored the dry formulation of a virus-like particle (VLP) vaccine by electrospinning VLP derived from rabbit hemorrhagic disease virus modified to carry the MHC-I gp100 tumor-associated antigen epitope. VLP were added to a polyvinylpyrrolidone (PVP) solution (15% w/v) followed by electrospinning at 24 kV. Formation of a nanofibrous mat was confirmed by scanning electron microscopy, and the presence of VLP was confirmed by transmission electron microscopy and Western blot. VLP from the nanofibers induced T-cell activation and interferon- (IFN-) γ production in vitro. To confirm in vivo cytotoxicity, Pmel mice treated by injection with gp100 VLP from nanofibers induced a gp100 specific immune response, lysing approximately 65% of gp100-pulsed target cells, comparable to mice vaccinated with gp100 VLP in PBS. VLP from nanofibers also induced an antibody response. This work shows that electrospinning can be used to dry-formulate VLP, preserving both humoral and cell-mediated immunity.

Cancer vaccine based on a combination of an infection-enhanced adenoviral vector and pro-inflammatory allogeneic DCs leads to sustained antigen-specific immune responses in three melanoma models

ABSTRACTAutologous patient-derived dendritic cells (DCs) modified ex vivo to present tumor-associated antigens (TAAs) are frequently used as cancer vaccines. However, apart from the stringent logistics in producing DCs on a patient basis, accumulating evidence indicate that ex vivo engineered DCs are poor in migration and in fact do not directly present TAA epitopes to naïve T cells in vivo. Instead, it is proposed that bystander host DCs take up material from vaccine-DCs, migrate and subsequently initiate antitumor T-cell responses. We used mouse models to examine the possibility of using pro-inflammatory allogeneic DCs (alloDCs) to activate host DCs and enable them to promote antigen-specific T-cell immunity. We found that alloDCs were able to initiate host DC activation and migration to draining lymph node leading to T-cell activation. The pro-inflammatory milieu created by alloDCs also led to recruitment of NK cells and neutrophils at the site of injection. Vaccination with alloDCs combined with Ad5M(gp100), an infection-enhanced adenovirus encoding the human melanoma-associated antigen gp100 resulted in generation of CD8+ T cells with a T-cell receptor (TCR) specific for the gp10025-33 epitope (gp100-TCR+). Ad5M(gp100)-alloDC vaccination in combination with transfer of gp100-specific pmel-1 T cells resulted in prolonged survival of B16-F10 melanoma-bearing mice and altered the composition of the tumor microenvironment (TME). We hereby propose that alloDCs together with TAA- or neoepitope-encoding Ad5M can become an “off-the-shelf” cancer vaccine, which can reverse the TME-induced immunosuppression and induce host cellular anti-tumor immune responses in patients without the need of a time-consuming preparation step of autologous DCs.

Designing therapeutic cancer vaccines by mimicking viral infections.

The design of efficacious and cost-effective therapeutic vaccines against cancer remains both a research priority and a challenge. For more than a decade, our laboratory has been involved in the development of synthetic peptide-based anti-cancer therapeutic vaccines. We first dedicated our efforts in the identification and validation of peptide epitopes for both CD8 and CD4 T cells from tumor-associated antigens (TAAs). Because of suboptimal immune responses and lack of therapeutic benefit of peptide vaccines containing these epitopes, we have focused our recent efforts in optimizing peptide vaccinations in mouse tumor models using numerous TAA epitopes. In this focused research review, we describe how after taking lessons from the immune system's way of dealing with acute viral infections, we have designed peptide vaccination strategies capable of generating very high numbers of therapeutically effective CD8 T cells. We also discuss some of the remaining challenges to translate these findings into the clinical setting.

Induction of an antitumor response using dendritic cells transfected with DNA constructs encoding the HLA-A*02:01-restricted epitopes of tumor-associated antigens in culture of mononuclear cells of breast cancer patients.

Advances in oncoimmunology related to the definition of the basic mechanisms of the formation of antitumor immune response, as well as the opening of tumor-associated antigens recognized by immune cells, allowed to start developing ways to influence the effector cells of the immune system to generate effective antitumor cytotoxic response. We investigated the possibility to stimulate an antitumor response in a culture of mononuclear cells of breast cancer patients by dendritic cells transfected with HLA-A*02:01-restricted DNA constructs. We isolated dendritic cells from peripheral blood monocytes and delivered our constructs to these cells by magnetic transfection. Additionally, a series of experiments with loading of dendritic cells with autologous tumor cell lysate antigens was conducted. We have shown that dendritic cells transfected with the HLA-A*02:01-restricted DNA constructs are effective in inducing an antitumor response in a culture of mononuclear cells of breast cancer patients. Dendritic cells transfected with DNA constructor dendritic cells loaded with lysate antigens revealed a comparable stimulated cytotoxic response of mononuclear cells to these two ways of antigen delivery. We conclude that using DNA constructs in conjunction with patient stratification by HLA type allows the application of transfected DCs as an effective method to stimulate antitumor immunity in vitro.

Circulating Type-1 Anti-Tumor CD4(+) T Cells are Preferentially Pro-Apoptotic in Cancer Patients.

Cancer patients frequently exhibit a deficiency in Type-1 (but not Type-2 or regulatory) CD4+ T cell responses against tumor-associated antigens (TAA), which may limit protection against disease progression or responsiveness to immunotherapy in these individuals. Since such deficiency was acutely evident in patients with active disease (AD), where chronic stimulation of anti-tumor CD4+ T cells would be expected and activation-induced cell death may be prevalent, we employed MHC Class II-peptide tetramers to characterize the frequency and apoptotic status of TAA- vs. influenza (FluM1) virus-specific CD4+ T cells in the peripheral blood of HLA-DR*0401+ patients with melanoma or renal cell carcinoma. We observed that Flu-specific CD4+ T cells ranged from 0.17 to 3.89%, while up to approximately 1% of CD4+ T cells reacted against individual TAA epitopes derived from the EphA2 or MAGE-6 proteins. The frequencies of EphA2 and MAGE-6-specific CD4+ T cells in patients were significantly correlated with AD and gender of the patient (i.e., females > males), while frequencies of Flu-specific CD4+ T cells were distributed within a normal range in all patients. Notably, patient CD4+ T cells reactive with MHC class II-TAA (but not MHC class II-Flu) tetramers were significantly enriched for a pro-apoptotic (Annexin-V+) phenotype, particularly amongst the Th1 (T-bet+) subset. These results suggest that the preferential sensitivity of TAA (but not viral)-specific CD4+ Th1 cells to apoptosis in melanoma patients with AD will need to be overcome for optimal clinical benefit of immunotherapeutic approaches to be realized.

Establishment of HLA-DR4 Transgenic Mice for the Identification of CD4+ T Cell Epitopes of Tumor-Associated Antigens

Reports have shown that activation of tumor-specific CD4+ helper T (Th) cells is crucial for effective anti-tumor immunity and identification of Th-cell epitopes is critical for peptide vaccine-based cancer immunotherapy. Although computer algorithms are available to predict peptides with high binding affinity to a specific HLA class II molecule, the ability of those peptides to induce Th-cell responses must be evaluated. We have established HLA-DR4 (HLA-DRA*01:01/HLA-DRB1*04:05) transgenic mice (Tgm), since this HLA-DR allele is most frequent (13.6%) in Japanese population, to evaluate HLA-DR4-restricted Th-cell responses to tumor-associated antigen (TAA)-derived peptides predicted to bind to HLA-DR4. To avoid weak binding between mouse CD4 and HLA-DR4, Tgm were designed to express chimeric HLA-DR4/I-Ed, where I-Ed α1 and β1 domains were replaced with those from HLA-DR4. Th cells isolated from Tgm immunized with adjuvant and HLA-DR4-binding cytomegalovirus-derived peptide proliferated when stimulated with peptide-pulsed HLA-DR4-transduced mouse L cells, indicating chimeric HLA-DR4/I-Ed has equivalent antigen presenting capacity to HLA-DR4. Immunization with CDCA155-78 peptide, a computer algorithm-predicted HLA-DR4-binding peptide derived from TAA CDCA1, successfully induced Th-cell responses in Tgm, while immunization of HLA-DR4-binding Wilms' tumor 1 antigen-derived peptide with identical amino acid sequence to mouse ortholog failed. This was overcome by using peptide-pulsed syngeneic bone marrow-derived dendritic cells (BM-DC) followed by immunization with peptide/CFA booster. BM-DC-based immunization of KIF20A494-517 peptide from another TAA KIF20A, with an almost identical HLA-binding core amino acid sequence to mouse ortholog, successfully induced Th-cell responses in Tgm. Notably, both CDCA155-78 and KIF20A494-517 peptides induced human Th-cell responses in PBMCs from HLA-DR4-positive donors. Finally, an HLA-DR4 binding DEPDC1191-213 peptide from a new TAA DEPDC1 overexpressed in bladder cancer induced strong Th-cell responses both in Tgm and in PBMCs from an HLA-DR4-positive donor. Thus, the HLA-DR4 Tgm combined with computer algorithm was useful for preliminary screening of candidate peptides for vaccination.

Generation of CTL responses against pancreatic cancer in vitro using dendritic cells co-transfected with MUC4 and survivin RNA

Pancreatic cancer (PC) is one of the most devastating human malignancies without effective therapies. Tumor vaccine based on RNA-transfected dendritic cells (DCs) has emerged as an alternative therapeutic approach for a variety of human cancers including advanced PC. In the present study we compared the cytotoxic T lymphocyte (CTL) responses against PC cells in vitro, which were induced by DCs co-transfected with two mRNAs of tumor associated-antigens (TAA) MUC4 and survivin, versus DCs transfected with a single mRNA encoding either MUC4 or survivin. DCs co-transfected with two TAA mRNAs were found to induce stronger CTL responses against PC target cells in vitro, compared with the DCs transfected with a single mRNA. Moreover, the antigen-specific CTL responses were MHC class I-restricted. These results provide an experimental foundation for further clinical investigations of DC vaccines encoding multiple TAA epitopes for metastatic PC.

Requirement of Tumor-Associated Antigen-Specific CD4 + T Cells for an Efficient Dendritic Cell Vaccine in Antitumor Immunotherapy

Evaluation of: Aarntzen EH, De Vries IJ, Lesterhuis WJ et al. Targeting CD4(+) T-helper cells improves the induction of antitumor responses in dendritic cell-based vaccination. Cancer Res. 73(1), 19-29 (2012). The induction of antitumor immune responses in patients is one of the most sought-after goals in cancer treatment. The proposed clinical approach is to use autologous mature dendritic cells (DCs) loaded with class I peptides from tumor-associated antigens (TAAs), as DCs are the most efficient immune cells to generate strong and specific cytotoxic CD8(+) T-lymphocyte (CTL) responses. To optimize DC-based cancer vaccines, Aarntzen et al. evaluated the necessity to stimulate TAA-specific CD4(+) T-helper cells to reinforce antitumor CTL responses. They demonstrated that intranodal injection of DCs pulsed with class I- and II-restricted TAA epitopes increases TAA-specific CTL responses and induces better clinical responses in stage III/IV melanoma patients than the use of DCs pulsed with class I-restricted TAA epitopes alone. This study highlights the interest of concomitant stimulation of TAA-specific CD4(+) and CD8(+) T cells for DC-based antitumor immunotherapy.

Identification of a novel HLA-A2-restricted mutated Survivin epitope and induction of specific anti-HCC CTLs that could effectively cross-recognize wild-type Survivin antigen.

Peptide vaccine based on tumor-associated antigen (TAA), which usually belongs to self-antigen with poor immunogenicity, has been considered as an attractive option for treatment of malignant tumors. The ideal TAA epitopes should have stable affinity to major histocompatibility complex (MHC) molecules and elicit strong anti-tumor immune response. Although point-mutation technology of TAA peptide may increase the binding capability to MHC molecules, some previous studies have revealed that part of the variant peptides results in lymphocyte not to effectively cross-recognize and kill the target tumor expressed wild-type TAA. Here, we designed a novel HLA-A2-restricted mutated TAA Survivin epitope nonapeptide Sur79L2 (KLSSGCAFL) that showed higher binding ability compared to wild-type peptide Sur79 (KHSSGCAFL) in T2-binding assays. To investigate whether Sur79L2 can induce Survivin-specific anti-hepatocellular carcinoma (HCC) response, we stimulated tumor-associated lymphocytes from a HCC patient with Sur79L2 in vitro. IFN-γ release and cytotoxicity assays showed Sur79L2 could effectively cross-recognize and lysis T2 cell plus peptide Sur79 and HCC cell lines (expression of wild-type Survivin antigen) in an HLA-A2-restricted manner. In contrast, peptide Sur95 (ELTLGEFLKL) that has been reported as a very promising anti-tumor epitope in a variety of tumors except HCC were not able to generate detectable cytotoxic immune responses against HCC in this study. Our results suggest that point-mutated peptide Sur79L2 is a new HLA-A2-restricted CTL epitope and may be useful for the immunotherapy for patients with HCC.

Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma

BackgroundThis study evaluated the safety and immune responses to an autologous dendritic cell vaccine pulsed with class I peptides from tumor-associated antigens (TAA) expressed on gliomas and overexpressed in their cancer stem cell population (ICT-107).MethodsTAA epitopes included HER2, TRP-2, gp100, MAGE-1, IL13Rα2, and AIM-2. HLA-A1- and/or HLA-A2-positive patients with glioblastoma (GBM) were eligible. Mononuclear cells from leukapheresis were differentiated into dendritic cells, pulsed with TAA peptides, and administered intradermally three times at two-week intervals.ResultsTwenty-one patients were enrolled with 17 newly diagnosed (ND-GBM) and three recurrent GBM patients and one brainstem glioma. Immune response data on 15 newly diagnosed patients showed 33 % responders. TAA expression by qRT-PCR from fresh-frozen tumor samples showed all patient tumors expressed at least three TAA, with 75 % expressing all six. Correlations of increased PFS and OS with quantitative expression of MAGE1 and AIM-2 were observed, and a trend for longer survival was observed with gp100 and HER2 antigens. Target antigens gp100, HER1, and IL13Rα2 were downregulated in recurrent tumors from 4 HLA-A2+ patients. A decrease in or absence of CD133 expression was seen in five patients who underwent a second resection. At a median follow-up of 40.1 months, six of 16 ND-GBM patients showed no evidence of tumor recurrence. Median PFS in newly diagnosed patients was 16.9 months, and median OS was 38.4 months.ConclusionsExpression of four ICT-107 targeted antigens in the pre-vaccine tumors correlated with prolonged overall survival and PFS in ND-GBM patients. The goal of targeting tumor antigens highly expressed on glioblastoma cancer stem cells is supported by the observation of decreased or absent CD133 expression in the recurrent areas of gadolinium-enhanced tumors.

Correlation of survival with tumor antigen expression in patients with newly diagnosed glioblastoma receiving a multi-epitope pulsed dendritic cell vaccine.

2087 Background: This study evaluated the safety and immune responses to an autologous dendritic cell vaccine pulsed with class I peptides from tumor associated antigens (TAA) expressed on gliomas and overexpressed in their cancer stem cell population (ICT-107). These TAA epitodes AIM-2. TRP-2, HER2 and AIM-2, are also overexpressed on glioblastoma multiforme (GBM) cancer stem cells. Methods: TAA epitopes included HER2, TRP-2, gp100, MAGE-1, IL13Rα2, and AIM-2. HLA-A1 and/or HLA-A2 positive glioblastoma (GBM) patients with gross total resection were eligible. Mononuclear cells from leukapheresis were differentiated into dendritic cells, pulsed with TAA peptides, and administered intradermally three times at two-week intervals in the axilla region after a standard treatment with concurrent temozolomide (TMZ) and radiation therapy for newly diagnosed (ND-GBM). Results: Twenty-one patients were enrolled with 17 ND-GBM and three recurrent GBM patients and one brainstem glioma. Immune response data on 15 newly diagnosed patients showed 33% responders. TAA expression by qRT-PCR showed all patient tumors expressed at least three TAA with 75% expressing all six. Correlations of increased PFS and quantitative expression of MAGE1, AIM-2, gp100 and HER2 were observed. A decrease or absence of CD133 expression was seen in five patients who underwent a second resection. As of February 1, 2012, six of 16 ND-GBM patients showed no evidence of tumor recurrence (44-63 months). Median progressive free survival (PFS) in newly diagnosed patients was 16.9 months with a two-year PFS rate was 43.8% (95%CI,19.8-66.0 ). The median overall survival rate (OS) was 38.4 months and a two-year OS was 80.3% (95%CI,58.6-96.7). Conclusions: Expression of four ICT-107 targeted antigens in the pre-vaccine tumors correlated with prolonged overall survival and PFS in ND-GBM patients. The goal of targeting tumor antigens highly expressed on glioblastoma cancer stem cells is supported by the observation of decreased or absent CD133 expression in the recurrent areas of gadolinium-enhanced tumor.

Glioma-associated antigens associated with prolonged survival in a phase I study of ICT-107 for patients with newly diagnosed glioblastoma.

2042 Background: Active immunotherapy offers the potential for high tumor-specific toxicity and sustained tumoricidal activity. We developed a dendritic cell (DC) immunotherapy to target specific glioma-associated antigens and induce a potent T cell response. Methods: In this study HLA-A2 or A1 positive patients were vaccinated with DC pulsed with MHC class I peptides from tumor associated antigens (TAA) expressed on gliomas. TAA epitopes included HER2, TRP-2, gp100, MAGE-1, IL13Rα2, and AIM-2. TRP-2, HER2 and AIM-2, are also overexpressed on glioblastoma multiforme (GBM) cancer stem cells. In this phase I trial, mononuclear cells isolated by leukapheresis from newly diagnosed GBM patients and differentiated into DC, were pulsed with TAA peptides, and administered intradermally three times at two week intervals. Results: 16 newly diagnosed GBM patients were enrolled since May 2007 with last follow up as of 1/28/2011. Vaccination was followed by adjuvant chemotherapy. TAA expression by qRT-PCR showed all patients expressed at least three TAA with 75% expressing all six, 87% expressing five and 94% expressing four. Correlations of increased PFS and quantitative expression of MAGE1 (p=0.03), AIM2 (p=0.003), gp100 (p=0.05) and HER2 (p=0.04) was observed. Variable changes in antigen expression were observed in the recurrent tumors. 7 of 15 tested (47%) had increased IFN gamma immune responses to a pool of the peptides. Immune responders showed a trend toward better survival. Six patients continue to show no tumor recurrence, three of whom have gone almost four years (43.5-49.8 m) and three more than 2.5 years (31.1-34.2 m). Median progression-free survival time was 16.9 months with median OS survival time not reached. At two years, the PFS rate was 43.75%; the two-year OS rate was 80.2%. Conclusions: Expression of four ICT-107 targeted antigens in the pre-vaccine tumors correlate with prolonged survival as measured by PFS and OS in newly diagnosed GBM patients. A randomized placebo controlled phase II trial is currently underway.

Tumor Antigen Epitopes Interpreted by the Immune System as Self or Abnormal-Self Differentially Affect Cancer Vaccine Responses

Epitope selection is an important consideration in the design of cancer vaccines, but factors affecting selection are not fully understood. We compared the immune responses to peptides and glycopeptides from the common human tumor antigen MUC1, a mucin that is coated with O-linked carbohydrates in its variable number of tandem repeats (VNTR) region. MUC1 expressed on tumor cells is characteristically underglycosylated, creating peptide and glycopeptide neoepitopes that are recognized by the immune system. The response to VNTR peptides is weaker in MUC1-transgenic mice (MUC1-Tg mice) than in wild-type (WT) mice, whereas the response to VNTR glycopeptides is equally strong in the two strains. Thus, glycopeptides seem to be recognized as foreign, whereas peptides, although immunogenic, are perceived as self. To explore this further, we generated MUC1 peptide- and glycopeptide-specific T-cell receptor transgenic mice and studied the function of their CD4 T cells when adoptively transferred into MUC1-Tg or WT mice. Peptide-specific T-cell precursors were not centrally deleted in MUC1-Tg mice and did not acquire a T regulatory phenotype. However, their response to the cognate peptide was reduced in MUC1-Tg mice compared with WT mice. In contrast, glycopeptide-specific CD4 T cells responded equally well in the two hosts and, when simultaneously activated, also enhanced the peptide-specific T-cell responses. Our data show that the immune system differentially recognizes various epitopes of tumor-associated antigens either as self or as foreign, and this controls the strength of antitumor immunity. This represents an important consideration for designing safe and effective cancer vaccines.

Immune response correlation with progression-free survival in glioblastoma following dendritic cell immunotherapy (ICT-107).

2097 Background: Active immunotherapy offers the potential for high tumor-specific toxicity and sustained tumoricidal activity. We therefore have developed this protocol to exploit the most current knowledge of specific glioma-associated antigens and used these antigens to induce a potent T cell response. Methods: The goal of this study is to administer dendritic cells pulsed with tumor associated antigens (TAA) expressed on gliomas to therapeutically vaccinate patients. TAA epitopes included HER2, TRP-2, gp100, MAGE-1, IL13R alpha, and AIM-3. In this phase I trial, sponsored by ImmunoCellular, HLA-A1 and/or HLA-A2 positive patients with newly diagnosed or recurrent brainstem glioma or glioblastoma were eligible. Mononuclear cells were isolated by leukapheresis, differentiated into dendritic cells in culture, pulsed with TAA peptides, and then administered intradermally three times at two week intervals. Results: Twenty-one patients with one screen failure, 16 males and 5 females, were enrolled between November 2006 and January 2010. There were 17 newly diagnosed supratentorial glioblastoma multiforme (GBM), 3 patients with recurrent GBM, and 1 brainstem glioma. Vaccination was followed by adjuvant chemotherapy. Immune response data on 17 glioblastoma patients showed 35% responders (> 1.5 fold increase IFN g production after vaccination = responsder by tetramer/FACS) with prolong survival in 6 responder and 11 nonresponder (114 and 87 wks: p = 0.046). Irrespective of immune responsiveness, 6 of 16 newly diagnosed GBM patients continue to show no signs of tumor recurrence, three of whom have gone more than two years with no disease progression (135-169 weeks). The median progression-free survival time in this group was 13.7 months and RT/TMZ regimen 6.9 months (EORTC/NCIC). Data on 20 patients and 57 courses of dendritic cell vaccines demonstrated zero grade 3/4 toxicities that were attributable to the vaccination. Conclusions: Survival and progression-free survival compare favorably to previously performed immunotherapy trials for glioblastoma. In addition, positive antitumor cytokine response correlated with longer progression-free survival. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration ImmunoCellular Therapeutics ImmunoCellular Therapeutics ImmunoCellular Therapeutics ImmunoCellular Therapeutics

Development of a transgenic mouse with high affinity TCR reactive to human tyrosinase epitope (41.39)

Abstract Most tumor-associated antigen epitopes recognized on human melanomas by autologous T lymphocytes are derived from nonmutated self-proteins. Given the belief that T cells bearing high affinity T cell receptor (TCR) would be more effective at targeting self antigens expressed by tumors, understanding tolerance with reference to T cells expressing high affinity TCRs against self-tumor antigens is important for optimizing the anti-tumor T cell response. Thus, using a high affinity TCR isolated from a class-I restricted CD4+ T cells from tumor infiltrating lymphocytes of a patient with metastatic melanoma and reactive to human tyrosinase-derived peptide YMDGTMSQV we developed a novel TCR transgenic mice. Our results show that HLA-A2 restricted TCR passed thymic selection in C57BL/6J (H2-Kb) wild type mice and could be tracked in the periphery. Around 25% of the peripheral CD4+ T cells and more than 75% of the peripheral CD8+ T cells from these TCR transgenic mice expressed functional human tyrosinase TCR. Since tyrosinase is a self-antigen and C57BL/6J mice lack HLA-A2/tyrosinase complexes on cells in their thymus -we developed HLA-A2+TCR+ double transgenic mice by breeding our TCR transgenic mouse and HLA-A2 transgenic mouse strains. Surprisingly, more than 90% of TCR transgenic T cells in the HLA-A2+TCR+ double transgenic mice lacked expression of CD4 or CD8 co-receptors and had CD3+CD4-CD8- phenotype. These double transgenic strains also developed spontaneous autoimmune vitiligo. Thus, our novel TCR transgenic mouse model would allow us to undertake various developmental and translational studies.

Human Langerhans-Type Dendritic Cells Break Tolerance against the Tumor Antigen, WT1, by a Largely IL-15-Dependent Mechanism.

The Wilms' tumor 1 (WT1) gene encodes a transcription factor essential for cell growth and development, but overexpression in many leukemias, myelodysplastic syndromes, and solid tumors correlates with poor prognosis. The WT1 protein could therefore serve as a therapeutic target, if tolerance could be broken against this self-differentiation, tumor-associated antigen (TAA). Vaccination protocols using peptide-pulsed dendritic cells (DCs) to induce anti-tumor responses present only defined epitopes restricted to certain human leukocyte antigens (HLA). In contrast, vaccination with mRNA-electroporated DCs supports processing and presentation of multiple TAA epitopes tailored to patient-specific HLA. Human Langerhans-type dendritic cells (LCs) derived from CD34+ hematopoietic progenitors are superior to other conventional DC subtypes, like monocyte-derived DCs (moDCs), in producing IL-15 to generate MHC-restricted, Ag-specific cytolytic T lymphocytes (CTL). Mature LCs transcribe 3 to 5-fold higher amounts of IL-15R-alpha mRNA and express the highest levels of IL-15R-alpha protein compared with other conventional DCs as measured by real-time RT-PCR, Western blot, and immunofluorescence microscopy (P<0.001 to 0.05, n=3–4 independent expts). Anti-IL-15R-alpha blockade also significantly reduces but does not completely eliminate the capacity of LCs to stimulate CTL. This indicates that LCs have a greater capacity than moDCs for presenting IL-15 in trans to stimulate lytic T cell responses. We therefore compared the ability of LCs and moDCs electroporated with WT1 mRNA to break tolerance against this self-differentiation TAA by generating CTLs against WT1+ tumor cells. After a single 7-day round of stimulation at T cell:LC ratio of only 10:1 to 30:1 and in the absence of exogenous IL-15, T cells stimulated by WT-1-electroporated LCs lyse 85–95% of a WT-1+ tumor cell line. MoDCs are completely incapable of generating WT-1-specific CTL under comparable conditions and require exogenous IL-15 10ng/ml to generate 70–80% specific lysis of WT-1+ targets. Lysis of a WT-1- HLA-A*0201+ target cell line or NK cell-sensitive LCL721.221 targets is at background levels (<5%) in all cases. Most importantly, WT-1-electoporated LCs also induce 63% lysis (+/− 11 SEM) of primary WT1+ HLA-A*0201+ blasts from a patient with AML after 7 days' coculture at a T cell:LC ratio of 10:1 without exogenous IL-15. These results demonstrate a major role for the IL-15R-alpha-IL-15 complex on LCs in expanding anti-tumor cytolytic T cells and breaking tolerance against self-differentiation Ags like WT1. These data also support the use of IL-15-producing LCs in active immunization or expansion of T cells ex vivo for adoptive immunotherapy.