Survivin Peptides Research Articles

IT-09 * PHASE I STUDY OF SAFETY, TOLERABILITY AND IMMUNOLOGIC EFFECTS OF A SURVIVIN PEPTIDE MIMIC VACCINE (SurVaxM) IN PATIENTS WITH RECURRENT MALIGNANT GLIOMA

BACKGROUND: Survivin is an inhibitor of apoptosis protein (IAP) that is highly expressed in malignant gliomas and other cancers. Induction of cytotoxic immunity against survivin is an attractive antitumor strategy. Preclinical studies demonstrated the ability of a KLH-conjugated survivin peptide mimic (SVN53-67/M57-KLH; SurVaxM) to induce an immune response capable of inhibiting the growth of gliomas in mice, and killing human glioma cells ex vivo. The vaccine is predicted to be immunogenic in humans with HLA-A*02, HLA-A*03, HLA-A*24 and other haplotypes and pre-clinical studies demonstrate potent and specific cytokine-supported antitumor CTL responses. METHODS: Nine patients with survivin-positive, recurrent malignant gliomas and either HLA-A*02 or HLA-A*03 haplotypes received a series of 4 subcutaneous injections of SurVaxM at 2 week intervals. MRIs were performed at baseline, week 8, and at subsequent intervals. RESULTS: SurVaxM was well tolerated with no SAE, and mostly grade one AE. 6 of 9 patients experienced grade 1 localized erythema at the injection site, likely related to the vaccine. 3 patients reported grades 1 or 2 fatigue, 2/9 experienced myalgia, possibly related to the study drug. Grade 1 lymphopenia was seen in 3/9 patients and grade 1 or 2 leukopenia was recorded in 3 patients. The only grade 3 AE, a seizure, was not related to the vaccine. The majority of patients developed specific cellular and humoral immune responses to survivin and 3 of 8 patients (all with GBM), who are evaluable for clinical response, have stable disease after more than 12 months of follow-up. All others have had progressive disease. Survival data will be described. CONCLUSION: This study demonstrated the safety and tolerability of SurVaxM in patients with recurrent or progressive malignant glioma following failure of standard therapy. SurVaxM proved to be immunogenic in most patients. A phase II clinical trial of SurVaxM is planned.

Survivin as a Cancer Vaccine Target

Survivin expression is associated with a poor prognosis in many cancers. While survivin is being studied as a potentially important target for cancer therapy, its many biological functions in both normal and cancerous cells remain to be fully elucidated. There are at least six specific survivin splice variants that have been identified to date which appear to be self-regulating and may have distinct functions. Several survivin peptide vaccines are currently under development by different groups. Survivin vaccine strategies for the most part have focused upon particular regional epitopes of the molecule that are bound by MHC class I and can lead to a cytotoxic T cell response. Immunotherapy targeting survivin is still at an early stage of development; however, several agents are progressing through early phase clinical trials. Recent studies using SurVaxM, a multi-epitope cryptic peptide, survivin, mimic show specific CD8+ T cell responses, as well as specific CD4+ T cell stimulation. Currently SurVaxM is in Phase I clinical trials designed to study its safety, tolerability and immunological effects in patients with survivin-positive recurrent malignant gliomas and multiple myeloma.

A “non-fratricidal” αβ- T Cell Receptor That Targets Survivin Expressed By Hematological Malignancies

Survivin is broadly expressed by hematological malignancies as well as by solid tumors and may be a suitable target for T-cell immunotherapy. Previously, the utility of this target has been challenged by the occurrence of “fratricide” when T cells expressing a high avidity survivin-specific T cell receptor (TCR) killed each other because survivin epitopes can be presented by the T cells themselves (Leisegang M et al, J Clin Invest. 2010 Nov;120(11):3869-77). To overcome this obstacle, we used limiting dilution to isolate a new T-cell clone targeting the HLA-A*02-restricted survivin epitope ELT (survivin95-104) and its variant LML (survivin96-10497M) starting from autologous cultures, rather than from the allogeneic cultures previously used for this approach. In 51Chromium (Cr)-release assays, this T-cell clone, with nanomolar avidity, displayed specific killing against the survivin+HLA-A*02+ leukemia cells BV173 (39±16% specific lysis, E:T 40:1) and multiple myeloma cells U266 (20±7%) but not against HLA-A*02– HL-60 cells (2±2%). Furthermore, the colony formation of primary myeloid leukemias was inhibited (>50% reduction) while that of healthy bone marrow (BM) was unaffected. The TCR α- and β-chains were then cloned in an optimized retroviral vector that was used to transduce CD8+ T cells which then efficiently expressed the transgenic αβTCR (89±4%, n=6). As compared to non-transduced (NT) T cells, survivin-αβTCR+ T cells produced significant lysis of BV173 (46±14% vs 8±6%, E:T 20:1, n=12, p<0.001) and U266 (27±12% vs 14±6%, p=0.003) but not of HL-60 (14±7 vs 14±6 %, p=NS). Blocking the target cells with specific anti-MHC class I antibodies confirmed the HLA-restriction of TCR transgenic T cells. Importantly, transgenic cells recapitulated the function of the original clone by inhibiting colony formation (range 32-78% reduction, n=5) of primary myeloid leukemias while preserving normal clonogenic capacity of healthy BM or cord blood (n=5). When tested in vivo in a xenograft model of established systemic acute leukemia (FFLuc+BV173) using bioluminescent imaging, leukemia progression was significantly slower in mice treated with survivin-αβTCR+ versus NT T cells (40x106 ± 71x106 vs. 128 x 106 ± 176 x 106 photons/sec by day 28) (p=0.04) and survival improved (n=12/group, p=0.01). This effect was even more pronounced when T cells were transferred to mice with limited leukemia burden (bioluminescent signal by day 40: 8.1 x 106 ± 9 x 106 vs. 195 x 106 ± 85 x 106 photons/sec) (p=0.003, n=10/group). Overall survival was improved by day 80 (p<0.001) and 3/10 mice treated with TCR+ T cells completely cleared the leukemia. Crucially, the TCR cloned from our autologous culture system produced no fratricidal activity in 51Cr-release assays against HLA-A*02+ activated T cells (1±2%, E:T 20:1, n=7). Activated T cells were only killed by TCR+ T cells when they were also pulsed with survivin peptides (46±12% for LML, 68±14% for ELT, n=7). To elucidate at the molecular level why our “autologous” TCR had selective antitumor activity unlike the fratricidal activity of “allogeneic” TCRs (Leisegang M et al, J Clin Invest. 2010 Nov;120(11):3869-77), we modeled the structure of each TCR-peptide-HLA ternary complex using the Rosetta software. While the overall binding energies of TCR-peptide-HLA interfaces for both TCRs were similar, the “autologous” TCR showed a 48% higher binding energy contribution for the peptide as compared to the fratricidal TCR, whose interaction was primarily with the HLA molecule rather than with the survivin peptide in the HLA-binding groove. In conclusion, we have cloned a novel survivin-TCR with a highly epitope-specific binding mode that can be efficiently expressed in polyclonal T cells and provides antitumor activity in vitro and in vivo without affecting the survival of T cells or normal hematopoietic progenitors. Our results indicate that maximal recognition of the peptide presented in the HLA groove is critical for TCR selectivity. Disclosures: Heslop:Celgene: Patents & Royalties; Cell Medica: Patents & Royalties. Brenner:Celgene: Patents & Royalties, Research Funding. Dotti:Celgene: Patents & Royalties, Research Funding. Savoldo:Celgene: Patents & Royalties, Research Funding.

Clinical and Immunological Response Following hTERT/Survivin mRNA-Loaded Dendritic Cell Vaccination Combined With Ex-Vivo Expanded T Cell Transfer In Melanoma Patients

New immunotherapy strategies have recently been developed combining peptide or dendritic cell (DC) vaccination with infusion of vaccine-primed and ex vivo expanded T cells. The hypothesis is that adoptive transfer of ex vivo expanded tumor specific T cells can improve progression-free and overall survival by restoring anti-tumor immunity. In a phase I/II clinical trial on malignant melanomas stage IV patients received DC vaccination prior to transfer of ex vivo expanded T cells. Our strategy was to target hTERT and survivin since both is highly expressed in most cancers. The vaccine consisted of autologous DCs loaded with hTERT and survivin mRNA. Prior to each DC vaccination the patients received 5 days of Temozolomide treatment to reduce the number of regulatory T cells (Treg). Following 2 monthly DC vaccinations, blood samples were tested for immune response against hTERT and survivin overlapping peptides. Immune responders were offered injection of T cells. The Elutra fraction of T cells was depleted of Treg using Dynabeads CD25 prior to expansion with Dynabeads CD3/CD28 in a WAVE bioreactor. After 10 days the beads were removed and T cells were washed. 3x1010 expanded T cells were injected fresh and DC vaccination was continued. Prior to T cell infusion, the patients received non-myeloablative conditioning with Fludarabine and Cyclophosphamide Here we present the results from three patients receiving expanded T cells. Immune response against hTERT and survivin peptides were detected in blood samples from 7 to 11 weeks of DC vaccination. After 4-7 months of DC vaccination the T cells were expanded for 10 days prior to injection. DC vaccination was continued 1 day after T cell injection. Infused T cells expanded significantly in vivo and in two of the three patients currently tested both patients showed response against hTERT and survivin peptides. Blood samples taken monthly after T cell injection demonstrated immune response against the same peptides. In one of patients the number of Treg was high (&gt; 4%) before and during vaccination and returned to low numbers (&lt;1%) after T cell injection. Since these findings might explain the beneficial effect of the vaccination we are currently investigating if the number of Tregs in blood show the same profile in the two other patients. Progression free survival (PFS) in the three patients was 31,20 and 11 months respectively. Patients with the shortest PFS relapsed very shortly after the T-cell infusion in spite of an objective immunresponse following the last DC vaccine. Metastatic melanoma patients included in this study given DC vaccines without T-cells had a median PFS of 7 months (3-13). We therefore conclude that dendritic cell vaccination combined with ex-vivo expanded T cell transfer can be an efficient immunotherapy strategy in melanoma patients. Disclosures: No relevant conflicts of interest to declare.

The T Cell Receptor As An Oncogene: Thymic Expression Of Self-Reactive T Cell Receptors Targeting Survivin Induces T-Cell Lymphoblastic Leukmia

Genetic engineering techniques can now efficiently express T cell receptors recognizing tumor antigens on mature T cells and antitumor effects have been observed with this approach, although persistence of such mature T cells is often limiting. To enhance the potential for long-term persistence, some investigators are considering using genetic engineering to express T cell receptors targeting tumor antigen in developing T cells. To test this approach, we generated three founders of TCR transgenic (Tg) mice with specificity for the H-2b restricted immunodominant epitope of murine survivin, a molecule overexpressed by most cancers. The gene was expressed under the human CD2 promoter, which induces expression of the transgene in DN2 thymocytes and has been used successfully in the generation of multiple previous TCR transgenics. In survivin TCR Tg mice, survivin reactive T cells were predominantly CD8+ and mediated specific immune reactivity toward survivin peptides. Some antitumor reactivity was observed, but it was not potent, and the survivin reactive transgenic T cells were unable to mediate objective tumor regression of survivin bearing tumors in vivo. Surprisingly however, spontaneous T cell acute lymphoblastic leukemia (T-ALL) occurred in progeny of all three founders, beginning at 4-6 months of age and by two years of age, all survivin TCR Tg+ mice succumb to T-cell ALL. Both survivin TCR+Rag+/- and survivin TCR+Rag-/- mice develop leukemia at the same rate. The leukemic cells are CD3+, survivin TCR+, and CD8+ or CD4–/CD8–, and are malignant as evidenced by continous growth in vitro without growth factors and the induction of leukemia following inoculation into C57BL/6 immunocompetent recipients. Alpha gene rearrangement analysis in tumor tissues demonstrated oligoclonality, consistent with a model wherein leukemic transformation is not isolated to a rare clone, but occurs with significant frequency in developing thymocytes. Insertional mutagenesis was judged unlikely since leukemia occurred in three separate founders, and was further ruled out by insertion site analysis of one founder, which demonstrated no evidence for integration of the transgene within an oncogenic locus. We thus postulated that the survivin reactive TCR itself was oncogenic, perhaps as a result of signaling via survivin peptides expressed within the thymus. In support of this, we demonstrate that survivin is expressed in thymic tissue, and prior to the development of leukemia, the survivin-specific TCRs signal, as evidenced by NFAT nuclear translocation, and enhanced BrdU incorporation compared to non-transgenic controls. Furthermore, transgenic mice that lack beta2microglobulin, and thus are unable to present survivin peptides, have a significantly diminished rate of leukemia development (see figure). Interestingly, at least one NOTCH1 mutation was also found in all leukemias, with mutations in the PEST domain being most common (8/8), but 5' deletions (19/25) and mutations in the heterodimerization domain also observed. We also observed T cell acute lymphoblastic leukemia with NOTCH mutations in TCRTg+ mice with specificity to other tumor-associated antigens including WT1 and gp100, albeit with lower penetrance. We conclude that genetic engineering aimed at endowing T lymphoid progenitors with the capacity to recognize tumor associated antigens expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Immunomodulatory Monoclonal Antibodies Combined with Peptide Vaccination Provide Potent Immunotherapy in an Aggressive Murine Neuroblastoma Model

Neuroblastoma is one of the commonest extracranial tumors of childhood. The majority of patients present with metastatic disease for which outcome remains poor. Immunotherapy is an attractive therapeutic approach for this disease, and a number of neuroblastoma tumor antigens have been identified. Here, we examine the therapeutic potential of combining immunomodulatory monoclonal antibodies (mAb) with peptide vaccination in murine neuroblastoma models. Neuroblastoma-bearing mice were treated with mAb targeting 4-1BB, CD40, and CTLA-4 alone, or in combination with a peptide derived from the tumor antigen survivin (GWEDPPNDI). Survivin-specific immune response and therapeutic efficacy were assessed. In the Neuro2a model, treatment of established tumor with anti-4-1BB, anti-CD40, or anti-CTLA-4 mAb results in tumor regression and long-term survival in 40% to 60% of mice. This is dependent on natural killer (NK) and CD8(+) T cells and is associated with tumor CD8(+) lymphocyte infiltrate. Successful therapy is achieved only if mAb is given to mice once tumors are established, suggesting dependence on sufficient tumor to provide antigen. In the more aggressive AgN2a and NXS2 models, single-agent mAb therapy provides ineffective therapy. However, if mAb (anti-CTLA-4) is given in conjunction with survivin peptide vaccination, then 60% long-term survival is achieved. This is associated with the generation of survivin-specific T-cell immunity, which again is only shown in the presence of tumor antigen. These data suggest that the combination of antigen and costimulatory mAb may provide effective immunotherapy against neuroblastoma and may be of particular use in the minimal residual disease setting.

Fowlpox-based Survivin vaccination for malignant mesothelioma therapy (P2117)

Abstract Survivin is an attractive candidate for cancer immunotherapy since it is abundantly expressed in most human cancers and absent in normal adult tissues. Malignant mesothelioma (MM) is a deadly cancer associated with asbestos exposure with no successful therapies currently available. We evaluated a Survivin-based vaccine as therapeutic tool for malignant mesothelioma in different MM mouse models. BALB/c mice, injected subcutaneously or intraperitoneally with murine fiber-induced MM tumor cells, were vaccinated with recombinant Fowlpox virus replicons encoding Survivin. Vaccination generated immune responses in both models, which led to delayed tumor growth and improved survival. Flow cytometric and immunofluorescent analyses of tumors from vaccinated mice showed CD8+ T cells infiltration, and real-time PCR demonstrated increased mRNA and protein levels of immunostimulatory cytokines. ELISPOT and intracellular cytokine staining analyses of Survivin peptide pulsed spleen cells from vaccinated mice confirmed antigen-specific, interferon-γ-producing CD8+ T cell responses. In addition pentamer-based flow cytometry showed vaccination generated Survivin specific CD8+ T cells. Importantly, vaccination did not affect fertility or induce autoimmune abnormalities in mice. Our results demonstrate that vaccination with recombinant Fowlpox expressing Survivin improves targeting of aggressive MM cells by the immune system and may form the basis for promising clinical applications.

Abstract 3309: T cell lymphoblastic leukemia in survivin TCR transgenic mice.

Abstract Survivin has been considered a potential tumor antigen due to high expression in most cancers and limited expression in normal tissues. To explore whether survivin reactive TCRs mediate antitumor effects in mice, we generated TCR transgenic (Tg) mice with specificity for the H-2b restricted immunodominant epitope of survivin. Survivin reactive T cells were CD8+ and mediated immune reactivity toward survivin peptide pulsed targets. Some antitumor reactivity was observed, but it was not potent, and the survivin TCR Tg+ cells did not mediate objective tumor regression of survivin bearing tumors. Surprisingly, spontaneous T cell acute lymphoblastic leukemia (T-ALL) was observed beginning at 4-6 months of age in Tg+Rag+/- and Tg+Rag-/- mice. By one year of age, all survivin Tg+ mice succumbed to T-cell ALL. The leukemic cells were CD3+, survivin TCR+, and CD8+ or CD4-/CD8-Occurrence in 3 founders suggests that the transgene itself, rather than insertional mutagenesis, is causative. Given that survivin is expressed in the thymus, we postulated that the TCR-Tg serves as an oncogene via recognition of survivin peptides, which expands early thymic progenitors. In support of this, premalignant survivin TCR Tg+ thymi show expanded CD4-CD8-CD44-CD25- thymocytes and increased BrdU incorporation and leukemia incidence was significantly diminished TCR-Tg+β2m-/- mice. Subsequent to expansion of early thymic progenitors in response to TCR triggering, survivin TCR Tg+ cells acquire NOTCH mutations and upregulate CD25, implicating NOTCH signaling as a 2nd hit in this oncogenic process. At least one NOTCH1 mutation was found in all leukemias, with PEST (8/8), 5’ deletions (19/25) and heterodimerization mutations observed. T-ALL with NOTCH mutations was also observed at reduced frequencies, in TCR Tg mice with specificity to WT1 and gp100. We conclude that genetic engineering aimed at endowing hematopoietic or T lymphoid progenitors with the capacity to recognize tumor antigens expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis. Citation Format: Yongzhi Cui, Masahiro Onozawa, Haiying Qin, Terry Fry, Peter Aplan, Crystal L. Mackall. T cell lymphoblastic leukemia in survivin TCR transgenic mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3309. doi:10.1158/1538-7445.AM2013-3309 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 4746: Modulation of suppressive myeloid populations by tasquinimod.

Abstract Purpose: Suppressive myeloid cells such as myeloid derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) are a major barrier of cancer immunotherapy. These populations suppress immune responses, promote angiogenesis, and facilitate cancer progression and metastasis. Tasquinimod is an experimental anti-angiogenic agent tested in prostate cancer. In the current study, we investigated the novel immunomodulatory effects of tasquinimod. We focused on the modulation of myeloid populations by tasquinimod since tasquinimod binds to the inflammatory protein S100A9, which potentially activates MDSCs. Thus, we studied whether tasquinimod affects the induction/function of MDSCs and TAMs, and whether this agent can facilitate vaccine therapy in prostate and kidney cancer models. Experimental Design: The effects of tasquinimod on myeloid cells were tested in Myc-CaP and TRAMP transplantable prostate cancer models, and the RENCA orthotopic model of renal cell carcinoma. Splenocytes, blood cells, and cells from tumor samples were isolated from tumor bearing mice. The samples were subjected to staining with MDSC markers (Gr1 and CD11b) and macrophage markers (F4/80 and CD206), and to flow cytometry analysis. An immunotherapy combining tasquinimod with a modified survivin peptide vaccine, SurVaxM, was administered in Myc-CaP and TRAMP models. Tumor growth, MDSCs, and TAMs accumulation and their function after different treatments were monitored. Tasquinimod's effect on metastases is currently being tested in a LuCaP 23.1 orthotopic prostate cancer model. Results: Tasquinimod treatment reduced peripheral Gr1−CD11b+ monocytes, but did not affect the number of peripheral Gr1+CD11b+ MDSCs. However, tumor infiltrating Gr1+CD11b+ MDSCs significantly decreased following tasquinimod treatment (60% reduction, p = 0.03). In addition, Gr1−CD11b+ monocytes gained the macrophage marker, F4/80, in tumors, and tasquinimod reduced the M2 macrophages (immunosuppressive macrophages) in both peripheral sites and tumors (56% reduction, p = 0.04). Tasquinimod had a modest effect on the suppressive function of MDSCs. In the vaccine therapy, tasquinimod, combined with SurVaxM, improved antitumor immune responses and reduced tumor growth (48% inhibition, p = 0.03), as compared to vehicle and single treatments (no inhibition by SurVaxM, 15% inhibition by tasquinimod). Conclusions: Our study suggests that tasquinimod does not affect the expansion of Gr1+CD11b+ MDSCs induced by tumor, but interferes with the migration of MDSCs to tumor sites. Moreover, tasquinimod targets M2 macrophages, possibly by depleting the monocytic precursor populations such as Gr1−CD11b+ monocytes. By inhibiting MDSCs and TAMs, tasquinimod has an immunomodulatory potential to target cancer progression and metastasis, and to improve immunotherapies. Citation Format: Li Shen, Michael Ciesielski, Kiersten M. Miles, Robert Fenstermaker, Roberto Pili. Modulation of suppressive myeloid populations by tasquinimod. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4746. doi:10.1158/1538-7445.AM2013-4746

Abstract B41: Analysis of immune responses and tumor protection in C57/Bl6 mice immunized with altered CD8 survivin peptide ligands.

Abstract Survivin is an intracellular tumor-associated antigen (TAA) that is broadly expressed in a large variety of tumors as well as tumor-associated endothelial cells, but mostly absent in differentiated tissues. The objective of the present work is to analyze the immune response and tumor protection generated in mice following vaccination with the wild-type (WT) survivin-derived peptide SURV20-28 (ATFKNWPFL) and with the two altered peptide ligand (APL) variants SURV20-28-3P (ATPKNWPFL) and SURV20-28-GP (AGPKNWPFL) that were substituted at non-anchor positions and which both displayed higher binding affinity to H-2-Db. C57/Bl6 mice were inoculated subcutaneously with 100 µg of each peptide in combination with the adjuvants CpG and imiquimod. After four doses, blood samples were taken and intracellular cytokine staining (ICS) analysis was performed to detect IFN-γ and TNF-α producing CD8+ T cells following stimulation with the wild-type peptide SURV20-28. Peptide-specific CD8+ T cells were also analyzed by MHC-tetramer staining. Mice were thereafter challenged with a lethal dose of the syngeneic B16 melanoma cells and tumor growth was evaluated twice weekly for a period of 80 days. ICS data showed that, after stimulation with the WT peptide, only low background levels of IFN-γ were detected in mice vaccinated with the WT peptide, the APL SURV-3P or in the control group (treated only with adjuvants), while approximately 1% of all CD8+ T cells specifically produced IFN-γ in mice immunized with SURV20-28-GP. Furthermore, the highest TNF-α responses were also observed in the mice groups treated with the APL SURV20-28-GP. Analysis with H-2Db/SURV20-28 tetramers confirmed that mice immunized with SURV20-28-GP responded with a significantly higher frequency of specific CD8+ T cells. After tumor challenge, mice were sacrificed upon becoming moribund or when the mean tumor diameter reached 10 mm, as permitted by the approved ethical protocol. In this case, 40% of mice vaccinated with either SURV20-28-3P or SURV20-28-GP remained tumor-free during the entire period of the experiment, while all the animals immunized with the wild type SURV20-28 peptide succumbed to tumor development by day 30, at a similar rate than the control group treated only with the two adjuvants. In conclusion, these results suggest that the use of altered survivin peptides, modified at non-anchor positions, can contribute to increased antitumor responses and protect from tumor challenge in a mouse melanoma tumor model. Citation Format: Yago Pico de Coaña, Adil Doganay Duru, Alvaro Lladser, Adnane Achour, Rolf Kiessling. Analysis of immune responses and tumor protection in C57/Bl6 mice immunized with altered CD8 survivin peptide ligands. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B41.

Efficient induction of specific cytotoxic T lymphocytes against gastric adenocarcinoma by a survivin peptide

Survivin has been demonstrated to be an excellent target for immunotherapy in several types of cancer, but little is known of the efficacy of survivin with gastric adenocarcinoma. In this study, a simple method was performed, and relatively high efficacy was shown upon inducing survivin-derived peptide-specific cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells of healthy donors. The induced CTLs exhibited specific lysisagainstHLA-A2 matched tumor cells in vitro, and similar results were demonstrated in primary cell cultures isolated from patients with gastric adenocarcinoma. Up to 30% of randomly selected patients could potentially benefit from immunotherapy targeting survivin. These results suggested that this survivin epitope peptide could be a promising vaccine candidate for immunotherapy for patients with gastric adenocarcinoma.

HLA-A2-restricted point-mutated Survivin peptide elicited specific cytotoxic T lymphocyte reaction against hepatocarcinoma

Objective To investigate the anti-hepatocarcinoma(anti-HCC) function of HLA-A2-restricted point-mutated Survivin peptide induced CTLs.Methods The HLA-A2-restricted Survivin nonapeptides were evaluated using bioinformatics software.The binding affinity of Survivin peptide to HLA-A2 molecular was determined with flow cytometry analysis.After peptide-induced CTLs were generated in vitro,flow cytometry and ELISA were performed to detect the levels of IFN-γ,which were secreted by reactive CTLs.Peptide-induced CTLs were co-cultured with hepatoma cell lines HepG2 and BEL-7402.The rates of tumor cells lysis were assayed using CytoTox 96(R) and the morphological changes of tumor cells were observed with inverted phase contrast microscope.Results Point-mutated Survivin nonapeptide Sur79M2 (KMSSGCAFL) was filtered out,which was shown higher scores compared with the wild-type peptide Sur79.Consistent with the results of software analysis,Sur79M2 showed higher binding ability in T2 binding assays.At the same time,Sur79M2-induced CTLs could release a large number of IFN-γ after incubated with target cells rather than Sur79.When co-cultured with HCC cell lines HepG2 and BEL-7402,Sur79M2-induced CTLs effectively lysis HepG2 on HLA-A2-restricted manner without killing effect on BEL-7402 that do not express HLA-A2 molecules.Conclusion Sur79M2 could elicited specific cytotoxic T lymphocytes in vitro,which were able to specifically kill HCC cell lines on HLA-A2-restricted manner. Key words: Survivin; Anti-hepatocarcinoma; Point-mutated peptide; HLA-A2-restricted

Metastatic melanoma patients treated with dendritic cell vaccination, Interleukin-2 and metronomic cyclophosphamide: results from a phase II trial

Dendritic cells (DC) are the most potent antigen presenting cells and have proven effective in stimulation of specific immune responses in vivo. Competing immune inhibition could limit the clinical efficacy of DC vaccination. In this phase II trial, metronomic Cyclophosphamide and a Cox-2 inhibitor have been added to a DC vaccine with the intend to dampen immunosuppressive mechanisms. Twenty-eight patients with progressive metastatic melanoma were treated with autologous DCs pulsed with survivin, hTERT, and p53-derived peptides (HLA-A2(+)) or tumor lysate (HLA-A2(-)). Concomitantly the patients were treated with IL-2, Cyclophosphamide, and Celecoxib. The treatment was safe and tolerable. Sixteen patients (57%) achieved stable disease (SD) at 1st evaluation and 8 patients had prolonged SD (7-13.7months). The median OS was 9.4months. Patients with SD had an OS of 10.5months while patients with progressive disease (PD) had an OS of 6.0months (p=0.048) even though there were no differences in prognostic factors between the two groups. Despite the use of metronomic Cyclophosphamide, regulatory T cells did not decrease during treatment. Indirect IFN-γ ELISPOT assays showed a general increase in immune responses from baseline to the time of 4th vaccination. Induction of antigen-specific immune responses was seen in 9 out of 15 screened HLA-A2(+) patients. In conclusion, the number of patients obtaining SD more than doubled and 6-month survival significantly increased compared to a previous trial without Cyclophosphamide and Celecoxib. A general increase in immune responses against the tested peptides was observed.

T Cell Lymphoblastic Lymphoma Resulting From Expression of Self-Reactive TCRs During Early Thymopoiesis

Abstract 2057Survivin has been considered a potential tumor antigen due to high expression in most cancers and limited expression in normal tissues. To explore the potential for survivin reactive TCRs to mediate antitumor effects in mice, we generated several founders of TCR transgenic (Tg) mice with specificity for the H-2b restricted immunodominant epitope of survivin. In survivin TCR Tg mice, survivin reactive T cells were predominantly CD8+ and mediated specific immune reactivity toward survivin peptide pulsed targets. Some antitumor reactivity was observed, but it was not potent, and the survivin reactive transgenic T cells were unable to mediate objective tumor regression of survivin bearing tumors in vivo. Surprisingly, spontaneous T cell acute lymphoblastic leukemia (T-ALL) was observed beginning at 4–6 months of age in both survivin TCR+Rag+/− and survivin TCR+Rag−/− mice. By one year of age, all mice had succumbed to T-cell ALL. The leukemic cells were CD3+, survivin TCR+, and CD8+ or CD4−/CD8−. Analysis of alpha gene rearrangements in tumor tissues revealed oligoclonality but the cells were malignant since they grew continuously in vitro without growth factors and induced tumors in C57BL/6 immunocompetent recipients. The occurrence of T-ALL in 3 founders suggests that the transgene itself, rather than insertional mutagenesis, is causative. We postulate that the survivin reactive TCR serves as an oncogene via recognition of survivin peptides within the thymus, leading to expansion of early thymic progenitors. In support of this, survivin itself is expressed in thymic tissue and premalignant survivin TCR Tg+ thymi show expanded frequencies and absolute numbers of CD4−CD8−CD44−CD25− thymocytes and increased BrdU incorporation within this subset compared to controls. Subsequent to the premalignant phase characterized by expansion of early thymic progenitors, surviving TCR Tg+ cells acquired NOTCH mutations and upregulated CD25, consistent with NOTCH signaling as a 2nd hit in this oncogenic process. At least one NOTCH1 mutation was found in all leukemias, with mutations in the PEST domain being most common (8/8), but 5’ deletions (19/25) and mutations in the heterodimerization domain were also observed. Interestingly, T cell acute lymphoblastic leukemia with NOTCH mutations were also observed, albeit at reduced frequencies, in TCR Tg mice with specificity to WT1 and gp100. We propose a 2-hit model of oncogenesis for self-reactive TCR expression in the thymus. Early thymic expression of TCRs recognizing self antigens expressed in the thymus induces proliferation of early thymocytes, followed by acquisition of NOTCH mutations and ultimately lymphoblastic leukemia. We conclude that genetic engineering aimed at endowing hematopoietic or T lymphoid progenitors with the capacity to recognize tumor antigens expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis. Disclosures:No relevant conflicts of interest to declare.

Promiscuous survivin peptide induces robust CD4+ T‐cell responses in the majority of vaccinated cancer patients

CD4(+) T cells have been shown to be crucial for the induction and maintenance of cytotoxic T cell responses and to be also capable of mediating direct tumor rejection. Therefore, the anticancer therapeutic efficacy of peptide-based vaccines may be improved by addition of HLA class II epitopes to stimulate T helper cells. Survivin is an apoptosis inhibiting protein frequently overexpressed in tumors. Here we describe the first immunological evaluation of a survivin-derived CD4(+) T cell epitope in a multipeptide immunotherapy trial for prostate carcinoma patients. The survivin peptide is promiscuously presented by several human HLA-DRB1 molecules and, most importantly, is naturally processed by dendritic cells. In vaccinated patients, it was able to induce frequent, robust and multifunctional CD4(+) T cell responses, as monitored by IFN-γ ELISPOT and intracellular cytokine staining. Thus, this HLA-DR restricted epitope is broadly immunogenic and should be valuable for stimulating T helper cells in patients suffering from a wide range of tumors.

First-in-human trial focusing on the immunologic effects of the survivin-derived multiepitope vaccine EMD640744.

2515 Background: Survivin is a promising antigen for cancer immunotherapy based on two major criteria: tumor cells depend on its actions, and it has demonstrated immunogenicity in patients with different cancers. Here we report immunomonitoring results of a first-in-man Phase I study (EMR 200032-001) with EMD640744, a cocktail of survivin-derived partially modified HLA class I-restricted peptides in Montanide ISA 51 VG. This multicenter, open-label, parallel group, randomized study compared the immunologic efficacy, safety, tolerability and clinical activity of 3 dosages of sc EMD640744 (30, 100 or 300 µg) in patients with different types of metastatic or locally advanced solid tumors positive for at least one relevant HLA antigen (A1, A2, A3, A24, B7). Methods: Routine immunomonitoring to analyze peptide-specific T cell responses at baseline and at weeks 4, 8, 12, 16, and 17 was performed by IFN-γ ELISpot and peptide/HLA-multimer staining (primary endpoint). Additional analyses were performed by intracellular cytokine staining (ICS) to IL-2, IFNγ, TNFα and degranulation marker CD107a after a single in vitro presensitization for 2 weeks with EMD640744. Results: Routine monitoring displayed T cell responses against the vaccine in 14 of 38 patients (37%) assessed by IFNγ-ELISpot and in 31 of 42 patients (74%) by HLA-multimer staining; 16 responses (38%) were clearly de novo induced. ICS revealed 3 relevant findings: 1) 15 of 22 (68%) analyzed patients showed vaccine-specific CD4+ T cells that could be blocked by anti-HLA-Class II antibodies; 2) survivin-specific CD8+ and CD4+ specific T cells often were polyfunctional, responding with degranulation and production of one or more cytokines; and 3) CD8+ responses against native homologues of modified vaccination peptides were demonstrated in 12 of 13 analyzed patients, and CD4+ responses in 5 of 9 patients. Dose dependency was not observed. Conclusions: Vaccination with EMD640744 elicited de novo T cell responses against survivin peptides in many patients, demonstrating immunological efficacy of EMD640744. The capability of EMD640744 to promote CD4-specific T cell help might explain the remarkable number of successfully induced immune responses.

Abstract 5516: Therapeutic vaccination with the survivin-derived multi-epitope vaccine EMD640744 in patients with advanced solid tumors

Abstract Background: Survivin is a promising tumor-associated antigen for cancer immunotherapy that fulfills two major criteria for this purpose: (1) tumor cells depend on the actions of survivin (inhibition of apoptosis, unrestricted proliferation and angiogenesis); and (2) the protein has demonstrated immunogenicity in patients with different cancers. Here we report results of a first-in-man Phase I study with EMD640744, a cocktail of survivin-derived partially modified HLA class I-restricted peptides in Montanide® ISA 51 VG. Methods: This multicenter, open-label, parallel group, randomized study compared the immunologic efficacy, safety, tolerability and clinical activity of three dosages of EMD640744 (30, 100, or 300 µg) in patients with different types of metastatic or locally advanced solid tumors who were positive for at least one relevant HLA antigen (A1, A2, A3, A24, B7). Patients received weekly s.c. injections of EMD640744 for 8 weeks followed by injections every 4 weeks until tumor progression. For the assessment of the primary endpoint, PBMC samples were prepared at baseline and after 4, 8, 12, 16, and 17 weeks. Peptide-specific T cell responses were analyzed by IFN-γ ELISpot. As a secondary analysis, peptide/HLA-multimer staining was performed. Frequencies of CD8+ T cells specific for patient-relevant single HLA-restricted peptides and EMD640744 were determined ex vivo and after short-term in vitro presensitization with EMD640744. Native homologues of modified vaccination peptides were also included in the analysis. Results: Of 66 patients screened, 53 were treated, and a majority was eligible for response analysis: 38 patients were analyzed by ELISpot and 42 by multimers, with positive ex vivo responses observed in 7 (18%) and 14 (33%) patients, respectively. Combining ex vivo data with results after in vitro stimulation, T cell responses were detected in 14 (37%) and 31 (74%) patients by ELISpot and multimer analysis, respectively. Multimer staining revealed a de novo induction of T cells against survivin peptides in at least 16 patients (38%) whereas pre-existent responses were seen in only 4 patients (10%). T cell responses were detected in all dose groups with similar frequencies. Five out of 10 HLA-A2+ patients reacting against the modified A2-binding peptide in ELISpot assays showed responses against its native homologue. The best tumor response according to RECIST was stable disease in 28% of patients. The most frequent treatment-related adverse events (AEs) were Grade 1-2 local injection site reactions and 2 patients experienced treatment-related Grade 3 AEs (granuloma at injection site and thrombosis). Conclusion: Vaccination with EMD640744 was safe and well tolerated and elicited de novo T cell responses against survivin peptides, demonstrating immunological efficacy of EMD640744 in cancer patients. 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 5516. doi:10.1158/1538-7445.AM2011-5516

Survivin TCR transgenic mice develop T cell lymphoblastic lymphoma (48.25)

Abstract Survivin has been considered a potential tumor antigen due to high expression in most cancers and limited expression in normal tissues. To explore the potential for survivin reactive TCRs to mediate antitumor effects in mice, we generated several founders of TCR transgenic mice with specificity for the H-2b restricted immunodominant epitope of survivin. In transgenic mice, survivin reactive T cells were predominantly CD8+ and mediated specific immune reactivity toward survivin peptide pulsed targets. Some antitumor reactivity was observed, but it was not potent, and the survivin reactive transgenic T cells were unable to mediate objective tumor regression of survivin bearing tumors in vivo. Spontaneous T cell acute lymphoblastic leukemia (T-ALL) was observed at 4-6 months of age in offspring of all 3 founders and was found in both survivin TCR+Rag+/- and survivin TCR+Rag-/- mice. Flow cytometry demonstrated that these lymphoblastic cells were CD3+ and survivin TCR+, and were variably CD8+ or CD4-/CD8-. Analysis of alpha gene rearrangements revealed oligoclonality but the cell were malignant, grew continuously in vitro and induced tumors in C57BL/6 recipients. The occurrence of T-ALL in 3 founders suggests that the transgene itself, rather than insertional mutagenesis, is causative. We postulate that self-reactive TCR serves as an oncogene if expressed early during thymopoiesis. TCR signaling may cooperate with NOTCH activation and/or other events to generate the malignancy.

Altered decamer and nonamer from an HLA-A0201-restricted epitope of Survivin differentially stimulate T-cell responses in different individuals

Survivin is a universal tumor antigen that is being currently targeted in vaccine approaches against cancer. Our study here examined the immunogenicity of a novel variant of an HLA-A0201-binding decamer peptide from region 95 to 104 of Survivin (ELMLGEFLKL) with a T → M modification at position 3 in the peptide. We found that this new modified 10-mer peptide had enhanced HLA-A0201 binding and induced a stronger T-cell response over its wild type counterpart peptide (ELTLGEFLKL) in select HLA-A0201 + normal donors. In addition, when compared to the previously characterized altered 96–104 peptide (LMLGEFLKL) from the same region of Survivin currently used in vaccine trials, we found that both peptides had similar immunogenicity, but donor T cells preferentially reacted strongly to either one or the other, but not strongly to both. These results suggest that these two closely related Survivin peptides yield distinct T-cell responses and that most individuals dominantly respond to one or the other altered peptide. We also found a novel association between positive reactivity to the new altered decamer Survivin peptide in some individuals and their expression of the HLA-C0701 allele along with HLA-A0201. Thus, vaccinating with both the 10-mer and 9-mer peptides would be required to immunize a maximum number of individuals in the HLA-A0201 + population and could lead to more consistent T-cell responses against this region of Survivin.

Inhibitor of apoptosis protein family as diagnostic markers and therapeutic targets of colorectal cancer

The apoptosis and antiapoptotic signaling pathways are important for regulating carcinogenesis and cancer progression, and for determining prognosis. Molecules involved in apoptosis represent potential cancer diagnostic markers and therapeutic targets. The inhibitor of apoptosis protein (IAP) family includes several important molecules involved in apoptosis that might represent such targets. Increasing evidence has demonstrated that the IAP family of proteins is integral for antiapoptotic and nuclear factor-κB signal transduction, and enhanced expression of IAPs contributes to colon carcinogenesis and its poor prognosis, as well as to drug resistance of tumors. X-linked IAP, cIAP1, cIAP2, and survivin are prognostic markers of colorectal cancer, and survivin and cIAP2 are also utilized to predict the effect of anticancer treatment in colorectal cancer patients. Novel therapies such as YM155 and LY2181308 targeting survivin, AEG35156 and phenoxodiol targeting X-linked IAP, AT-406 as a Smac mimetic, and survivin peptides are currently being evaluated in clinical trials. This report reviews the involvement of the IAP family in colorectal adenocarcinoma in order to summarize the role of the IAP family members as diagnostic and therapeutic targets, and to provide an overview of the future course of research in this area.