hTERT Peptide Research Articles

Phase 1 trial of 4-1BB-based adoptive T-cell therapy targeting human telomerase reverse transcriptase in patients with advanced refractory solid tumors

Background aimsHuman telomerase reverse transcriptase (hTERT) is an attractive target for anti-cancer therapies. We developed an effective method for generating hTERT-specific CD8+ T cells (hTERT-induced natural T cells [TERTiNTs]) using peripheral blood mononuclear cells (PBMCs) from patients with solid cancers and investigated their feasibility and safety. MethodsThis was a single-center phase 1 trial using a 3 + 3 dose escalation design to evaluate six dose levels of TERTiNTs. PBMCs from each patient were screened using an hTERT peptide panel to select those that stimulated CD8+ T cells. The four most stimulatory peptides were used to produce autologous CD8+ T cells from patients refractory or intolerant to standard therapies. Eligible patients received a single intravenous infusion of TERTiNTs at different dose levels (4 × 108 cells/m2, 8 × 108 cells/m2 and 16 × 108 cells/m2). Pre-conditioning chemotherapy, including cyclophosphamide alone or in combination with fludarabine, was administered to induce lymphodepletion. ResultsFrom January 2014 to October 2019, a total of 24 patients with a median of three prior lines of therapy were enrolled. The most common adverse events were lymphopenia (79.2%), nausea (58.3%) and neutropenia (54.2%), mostly caused by pre-conditioning chemotherapy. The TERTiNT infusion was well tolerated, and dose-limiting toxicities were not observed. None of the patients showed objective responses. Seven patients (30.4%) achieved stable disease with a median progression-free survival of 3.9 months (range, 3.2–11.3). At the highest dose level (16 × 108 cells/m2), four of five patients showed disease stabilization. ConclusionsThe generation of TERTiNTs was feasible and safe and provided an interesting disease control rate in heavily pre-treated cancer patients.

Identification of Cancer Cells in the Human Body by Anti-Telomerase Peptide Antibody: Towards the Isolation of Circulating Tumor Cells.

Early detection of tumor cells by identifying universal Tumor Associated Antigens (TAA) can drastically change our diagnostic, theranostic and therapeutic possibilities to cure cancer. Human Telomerase Reverse Transcriptase (hTERT), a hallmark of cancer, could act as an optimal TAA candidate. Here we report about the development of a monoclonal antibody against hTERT peptide (α-hTERT mAb) presented on the surface of cancer cells and its possible applications as a pan-cancer marker. Liquid biopsies, an innovative tool in precision oncology, comprising the noninvasive analysis of circulating tumor-derived material to counteract limitations associated with tissue biopsies. Within the tumor circulome, the US Food and Drug Administration already approved the use of circulating tumor cells (CTCs) as valid liquid biopsies. However, currently CTCs are being trapped using antibodies against specific cancer types, with anti EpCAM as the most common antibody, directed mainly against solid tumors. Moreover, the precision medicine approach is based on specific cancer type directed antibodies. Our novel mAb against the hTERT 16-mer peptide, corresponding to amino acids 611-626, is capable of detecting various types of cancer cells both in vitro and ex vivo from tumors of patients with either hematological or solid tumors. This antibody does not bind to normal lymphocytes cells. Cleavage of our antibody to F(ab')2 fragments increased its binding specificity to the tested cancer cells. Future studies may point to the use of this antibody in the procedure of capturing CTCs.

MAGEA1 and hTERT Peptide Treatment Improves the Potency of The Dendritic Cell- Cytotoxic T Lymphocytes (DC-CTL) Immunotherapy in DAC Treated Acute Myeloid Leukemia.

Background: Acute myeloid leukemia (AML) is a type of heterogenous malignant hematological disorder. Recently developed immunotherapies such as chimeric antigen receptor T cell (CAR-T) do not demonstrated promising therapeutic results due to the off-target effect. The Dendritic cell-cytotoxic T lymphocyte adoptive immunotherapy (DC-CTL) is one of the recently developed immunotherapies. One of the reasons that DC-CTL does not work well in AML is the lack of antigens with high binding affinity, high antigen presentation potency, and the specificity to AML cells.Methods: DAC was used to treat AML cells to find overexpressed CTAs upon DAC treatment. The overexpression was confirmed at both mRNA and protein level by realtime PCR and western blotting. Peptides was designed by using the NetMHCpan database and EPIP based on the out-screened protein sequences. The peptides were then used to pulse DC-CTL coculture in vitro and tested the cytotoxicity of CTLs in vitro and their cancer inhibition potency in vivo.Results: Two cancer testis antigen (CTA) proteins, MAGEA1 and hTERT, was up-regulated in DAC treated AML cells. DC cells pulsed by the antigen peptides designed based on the sequence of these two proteins demonstrated increased potency to stimulate CTL cells in terms of cytokines secretion. These cytokines included IFN-γ, IL-6, and TNF-α. Moreover, enhanced in vitro cytotoxicity was found in CTL cells treated with peptide pulsed DC cells. AML progress was inhibited by CTA peptides pulsed DC-CTL in a mouse AML model.Conclusions: MAGEA1 and hTERT could possibly serve as specific tumor antigens upon DAC treatment, providing potential targets for the development of immunotherapies for AML in the future.

Targeting Telomerase with an HLA Class II-Restricted TCR for Cancer Immunotherapy

T cell receptor (TCR)-engineered T cell therapy is a promising cancer treatment approach. Human telomerase reverse transcriptase (hTERT) is overexpressed in the majority of tumors and a potential target for adoptive cell therapy. We isolated a novel hTERT-specific TCR sequence, named Radium-4, from a clinically responding pancreatic cancer patient vaccinated with a long hTERT peptide. Radium-4 TCR-redirected primary CD4+ and CD8+ T cells demonstrated in vitro efficacy, producing inflammatory cytokines and killing hTERT+ melanoma cells in both 2D and 3D settings, as well as malignant, patient-derived ascites cells. Importantly, T cells expressing Radium-4 TCR displayed no toxicity against bone marrow stem cells or mature hematopoietic cells. Notably, Radium-4 TCR+ T cells also significantly reduced tumor growth and improved survival in a xenograft mouse model. Since hTERT is a universal cancer antigen, and the very frequently expressed HLA class II molecules presenting the hTERT peptide to this TCR provide a very high (>75%) population coverage, this TCR represents an attractive candidate for immunotherapy of solid tumors.

Predicting long-term results with circulating tumor cells in patients with de novo androgen sensitive prostate cancer treated with hTERT peptides vaccine.

98 Background: Androgen deprivation, radiotherapy and chemotherapy improves survival in advanced prostate cancer (PC), but many patients still progress and experience toxicity from treatment. We investigated the clinical utility of a medical wire, collecting circulating tumor cells (CTCs) from a peripheral vein, to predict oncologic outcomes prior to treatment with a hTERT peptide vaccine. Methods: Twenty-three patients with advanced PC with non-visceral spread and unselected HLA-type were eligible. One patient progressed before initiation of the vaccination (median FU 60 months). The hTERT peptides vaccine schedule started one week after inclusion with a total of 13 vaccinations (N=22). Patients with no signs of deterioration during the vaccine schedule received image-guided radiation to the prostate (2Gy/37f, n=20). Vaccine response was evaluated by T-cell proliferation assay. CTCs were enriched from the peripheral blood using EpCAM as antibody (CellCollector, Gilupi, Potsdam, Germany). Kaplan-Meier outcome curves were constructed for prostate cancer-specific survival (PCSS) and overall survival with stratification by immune response, CTCs (no/yes), PSA and comparison of patient subsets using the log-rank test. Results: Median age of the cohort was 67 years (range 51-78 years).The mean time from CellCollector after ADT to start vaccine was 2,95 months (SD+-1,78 months). The mean yield of CTCs was 3 cells (range 0 - 22). The detection of CTCs showed an inverse trend towards immune response (p=0.07). None of the patients with a positive CellCollector finding and a PSA below ≤0.2 (n=9) had died of prostate cancer during the observation period of 5 years, patients with no CTCS and a PSA >0.2 (n=14) had an estimated median PCSS of 39 months (log-rank test 0=0.007, Fig. 1). The overall survival analysis showed that 6 out of 9 patients with CTCs compared to 5 out of 14 patients in the CTCs negative group were alive at 5 years FU (log-rank-test=0.159). Conclusions: Patients with a de novo diagnozed metastatic prostate cancer and a positive CTC burden do better when subsequently treated with the hTERT vaccine than those with a negative CTC finding. Clinical trial information: NCT01784913.

Safety, tolerability and immunogenicity of V934/V935 hTERT vaccination in cancer patients with selected solid tumors: a phase I study

BackgroundHuman telomerase reverse transcriptase (hTERT) is an antigen that may represent a target for a novel anti-cancer strategy. A pilot, phase I study tested the safety and feasibility of a prime-boost immunization regimen based on V935, an adenoviral type 6 vector vaccine expressing a modified version of hTERT, administered alone or in combination with V934, a DNA plasmid that also expresses the same antigen and is delivered using the electroporation injection technique.MethodsTreatments: Group #1 received two doses (low-dose: 0.5 × 109 vg, and high-dose: 0.5 × 1011 vg) of V935 followed by a 4-week observation period. Group #2 received three doses of V934-electroporation and two doses of V935 following a 4-week observation period. Doses were low-dose V934 (0.25 mg of plasmid) with low-dose V935 (0.5 × 109 vg); high-dose V934 (2.5 mg of plasmid) with high-dose V935 (0.5 × 1011 vg). Group #3 received five doses of V934-EP and two doses of V935: V934 was administered IM every 2 weeks for five doses. Following a 4-week observation period, V935 was administered IM every 2 weeks for two doses followed by a 4-week observation period. One (1) dose level was tested in treatment group #3: high-dose V934 (2.5 mg of plasmid), in combination with high-dose V935 (0.5 × 1011 vg). Immunogenicity was measured by ELISPOT assay and three pools of peptides encompassing the sequence of hTERT.ResultsIn total, 37 patients affected by solid tumors (prostate cancer in 38%) were enrolled. The safety profile of different regimens was good and comparable across groups, with no severe adverse events, dose-limiting toxicities or treatment discontinuations. As expected, the most common adverse events were local reactions. A significant increase in ELISPOT responses against hTERT peptide pool 2 was observed (p < 0.01), while no evidence of boosting was observed for peptide pools 1 and 3. This was also evident for group #1 and #2 separately. In patients with prostate cancer, there was a significant increase in ELISPOT response against hTERT peptide pool 2 following immunization (p < 0.01), regardless of previous therapy, immunosuppressing agents, or adenoviral type 6 titers at screening.ConclusionOur results suggest the safety and feasibility of V934/V935 hTERT vaccination in cancer patients with solid tumorsTrial Registration Name of the registry: ClinicalTrial.gov Trial registration number: NCT00753415 Date of registration: 16 September 2008 Retrospectively registered URL of trial registry record: https://clinicaltrials.gov/ct2/results?cond=&term=NCT00753415&cntry=&state=&city=&dist=

Improving TCR affinity on 293T cells

This study presents an efficient method to improve TCR affinity, comprising 1) CDR-directed saturation mutation of TCR cDNA, 2) transient TCR display on CD3-expressing HEK293T (CD3-293T) cells by simple plasmid transfection, 3) staining with HLA-tetramers, and 4) multi-round sorting of cells with CD8-independent tetramer binding on a flow cytometer. Using these procedures, we successfully identified mutant TCRs with enhanced binding from an HLA-A*24:02-restricted, human telomerase reverse transcriptase (hTERT)-specific TCR. Two such clones, 2A7A and 2D162, harboring mutations in CDR1 and CDR2 of TCRβ, respectively, were isolated with both showing sequential four amino acid substitutions. When expressed on CD3-293T cells along with wild-type TCRα, the TCR molecules of these mutants as well as their combinatory mutation, bound to HLA-A24/hTERT-tetramers more strongly than the wild-type TCRs, without binding to control tetramers.Besides, in order to facilitate a functional study of TCR, we established an artificial T cell line, designated as CD8I-J2, which expresses a human CD8 and IFN-γ producing cassette by modifying Jurkat-derived J.RT3-T3.5 cells. CD8I-J2 cells expressing wild-type or affinity-enhanced hTERT-specific TCRs were analyzed for their recognition of serially diluted cognate peptide on HLA-A*24:02-transduced T2 cells. CD8I-J2 cells expressing each mutant TCR recognized the hTERT peptide at lower concentrations than wild-type TCR. The hierarchy of peptide recognition is concordant with tetramer binding on CD3-293T cells and none of these mutant TCRs were cross-reactive with irrelevant peptides reported to be present on HLA-A*24:02 molecules as far as tested. These methods might thus be useful for obtaining high affinity mutants from other TCRs of interest.

Effects of Viral Peptide Presentation on CD4+ T Cell Responses to MHC Class II-Restricted Tumor Peptides

Background: Designing a vaccine against a defined tumor is a promising but complicated process that can be used as tumor immunotherapy. The presence of human telomerase reverse transcriptase (hTERT) has been proved in about 85% of tumors; therefore, this enzyme is a promising vaccine target. Most studies in tumor immunotherapy have focused on induction and reinforcement of CD8+ T cell responses against tumor antigens, but some evidence indicates that CD4+ T cell responses are important for induction of CD8+ T cell response and memory. Therefore, in this in vitro study, we combined an hTERT peptide with HIV peptides restricted to HLA-DRB1*11:03/04, the most common MHC class II molecule in Iran, to induce CD4+ T cell responses in healthy individuals and lung cancer patients. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation from nine healthy individuals and six lung cancer patients who were HLA-DRB1*11:03/04 positive. PBMCs were incubated with the tumoral and viral peptides for five days and then exposed to A549 cells to evaluate specific proliferative responses. The proliferation of PBMCs was examined by the MTT assay. Then, levels of secreted interferon-γ in culture media were analyzed by enzyme-linked immunosorbent assay (ELISA). Results: Proliferation of PBMCs was significantly greater in the presence of the tumoral peptide than in controls (P = 0.001). In addition, PBMC proliferation was greater when the viral peptides were added than with the tumoral peptide alone (P = 0.000). The IFN-γ secretion assay results supported the MTT assay (P = 0.000). Conclusions: In this study, we demonstrated that the hTERT-derived peptide induced CD4+ T cell proliferation in healthy individuals and patients with lung cancer. Both PBMC proliferation and IFN-γ secretion were further induced by the addition of tumor and viral peptides in combination.

Abstract IA45: Prospect of a vaccine to prevent cancer in BRCA1-2 mutation carriers

Abstract New, effective immune approaches to treat cancer not only underscore the power of the immune system in the therapeutic setting, but also spark renewed efforts to develop specific immune approaches to prevent cancer. We are focused on developing a novel vaccine that prevents BRCA1/2-related cancer when administered prophylactically to healthy individuals who carry BRCA1/2 mutations a polio vaccine for BRCA1/2-related cancer. An increasing understanding of cancer risk in BRCA1/2 mutation carriers provides an opportunity to test immune prevention in individuals at extremely high risk for developing cancer. It is in this prevention setting for which it is now hypothesized that vaccines will work (as they do in infectious diseases) in contrast to post-exposure vaccines which generally do not, especially in cancer-bearing individuals. In initial efforts, a first-in-human clinical trial is being conducting in which a next-generation DNA vaccine is used to target the telomerase reverse transcriptase hTERT as a potential universal tumor antigen exploitable for cancer prevention. Telomerase is expressed by the vast majority of human cancers including those related to BRCA1/2 mutation, whereas telomerase expression in normal tissue is restricted. Extensive data demonstrate that TERT can be recognized by human cytotoxic T lymphocytes (CTLs) despite being a product of the human genome potentially subject to the forces of immune tolerance. Peptides derived from TERT are naturally processed by tumors, presented on MHC, and can trigger effector functions of specific CTLs. TERT-specific CTLs lyse TERT positive tumor cells in vitro in an antigen-specific, MHC-restricted fashion; except for activated B cells, normal hTERT+ MHC+ cells are not lysed by TERT T cells. Because hTERT is involved in the transformation of cancer cells and is required for oncogenesis, tumor downregulation of hTERT expression in the face of T cell immune responses may be incompatible with ongoing tumor growth, addressing immune escape by antigen-loss. Clinical studies of peptide- and cell-based vaccines demonstrate the immunogenicity of TERT in patients. In multiple studies, we have observed the induction of hTERT-specific T cells both systemically and intratumorally after peptide vaccination. hTERT-specific CD8+ T cells exhibit effector functions in vitro including proliferation, IFNγ production, and tumor lysis. Median overall survival is significantly longer in those patients who achieved an immune response to hTERT peptide compared with patients who did not. Some formulations of TERT vaccination have been immunologically weak. In mice, immunization with optimized TERT DNA that is robustly immunogenic can inhibit growth of preexisting tumors and also prevent formation of cancer in challenge experiments without toxicity. A clinical trial testing a novel DNA formulation is underway and will be discussed. Citation Format: Robert H. Vonderheide. Prospect of a vaccine to prevent cancer in BRCA1-2 mutation carriers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr IA45.

P4.05 - The study of CD8+ T cell response to MHC class I-restricted universal tumoral peptide in accompany with viral peptide

ABSTRACT Background: Breast cancers likes as other human cancers are immunogenic and express tumor-associated proteins that can stimulate and generate immune responses in cancer. One of the important antigen that is express in tumors especially in Brest cancers is human Telomerase reverse transcriptase (hTERT). There are some databases that store the information about T cell epitopes, and we used them to identify immunogenic epitopes in proteins that bind in Class I MHC. in this study we used a peptide derived of hTERT molecule accompany with viral peptide derived of influenza that restricted to HLA-A2 (common MHC class I in Iran) for induction of T CD8+ responses in breast cancer patients. Material and Methods: PBMCs were obtained from blood sample of HLA-A2 positive breast cancer patient incubated with tumor cell line that were transloaded with synthetic peptides from hTERT and viral peptides. Proliferation of T cells analyzed with MTT reaction. After this step, supernatants from previous step evaluated for IFN-γ production by ELISA test. Final step in this study was cytotoxicity assay by measuring LDH on supernatants from first step. Results: As we expected in this study, stimulation and proliferation of T cells occurred. MTT data showed that proliferation of PBMCs incubated with tumoral and viral peptide more than proliferation of PBMCs incubated with tumoral peptide alone. IFN-γ ELISA assessment and LDH cytotoxicity assay confirmed the MTT assay. Conclusion: Our findings showed that the viral peptide intensified CTLs responses to tumoral peptide.

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.

Widespread CD4+ T-cell reactivity to novel hTERT epitopes following vaccination of cancer patients with a single hTERT peptide GV1001

Understanding the basis of a successful clinical response after treatment with therapeutic cancer vaccines is essential for the development of more efficacious therapy. After vaccination with the single telomerase (hTERT) 16-mer peptide, GV1001, some patients experienced clinical responses and long-term survival. This study reports in-depth immunological analysis of the T-cell response against telomerase (hTERT) in clinically responding patients compared with clinical non-responders following vaccination with the single hTERT 16-mer peptide, GV1001. Extensive characterization of CD4+ T-cell clones specific for GV1001 generated from a lung cancer patient in complete remission after vaccination demonstrated a very broad immune response to this single peptide vaccine with differences in fine specificity, HLA restriction, affinity and function. Some CD4+ T-cell clones were cytotoxic against peptide-loaded target cells and also recognized processed recombinant hTERT protein. Furthermore, T-cell responses against several unrelated hTERT epitopes, some of which are novel, were detected, indicating extensive epitope spreading which was confirmed in other clinical responders. In contrast, patients responding immunologically, but not clinically, after vaccination did not display this intramolecular epitope spreading. Multifunctional CD4+ T-cell clones specific for novel hTERT epitopes were generated and shown to recognize a melanoma cell line. Pentamer analysis of T cells in peripheral blood also demonstrated the presence of an important CD8+ T-cell response recognizing an HLA-B7 epitope embedded in GV1001 not previously described. These results indicate that the highly diverse hTERT-specific T-cell response, integrating both T helper and CTL responses, is essential for tumor regression and the generation of long-term T-cell memory.

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.

Dendritic cells transfected with lentiviral vector-encoding hTERT peptide augment antitumor T cell response in vitro

HIV-1 derived lentiviral vectors (LVs) have emerged as a powerful tool for gene delivery. hTERT is an ideal tumor-associated antigen with which to develop a potential dendritic cell (DC) vaccine. The purpose of this study was to construct a recombinant lentivirus vector of the hTERT peptide and to determine the hTERT-specific cytotoxic T lymphocyte response elicited by DCs transfected with hTERT-lentivirus vectors in vitro. LVs encoding the hTERT peptide were constructed, DCs from cord blood were prepared, their morphology was observed and phenotype was analyzed by flow cytometry. Lenti-hTERT was transfected into DCs to construct the DC vaccines. T lymphocytes stimulated with DC vaccines and HepG2 cells (hTERT+) or 293T cells (hTERT-) were co-cultured for 24 h, respectively. The ability to stimulate proliferation of allogeneic T lymphocytes and the killing activity of CTLs activated by these DCs were determined using the MTT method. According to our results, the recombinant vector lenti-hTERT and lenti-hTERT-DC vaccine were successfully constructed. The stimulatory capacity of the lenti-hTERT DCs in the allogeneic T lymphocyte reaction was markedly enhanced compared with the DC control group (P<0.01). Inhibition rates in HepG2 cells of CTLs stimulated with lenti-hTERT-DCs (CTLT) were significantly higher than CTLs stimulated with the control DC group (CTLN) (P<0.01). Inhibition rates in 293T cells of CTLT and CTLN were low and there was no difference between the different DC groups (P>0.05). DCs transfected with the hTERT peptide were capable of eliciting a stronger hTERT-specific CTL response in vitro. Our data indicate that lenti-hTERT-DCs may potentially be used as an effective approach for cancer immunotherapy.

A simple competitive assay to determine peptide affinity for HLA class II molecules: A useful tool for epitope prediction

We have designed a cytometry-based competition assay to evaluate peptide binding to empty recombinant HLA class II molecules. The efficiency of this assay was evaluated using recombinant HLA-DP0401 molecules (HLA-DP) produced in insect cells and 13 peptides from human telomerase reverse transcriptase (hTERT). We demonstrate that our method allowed accurate measurements of peptide Ki values and can thus discriminate strong, moderate and poor HLA-DP binders. In parallel, we showed that among hTERT peptides, the most immunodominant in healthy individuals were those with moderate affinity for HLA-DP while no T cell response could be evidenced against peptides with very strong or very low affinities for HLA-DP. This strongly suggests that the precise determination of peptide affinity with our method can improve HLA class II epitope prediction.

HTERT mRNA dendritic cell vaccination: complete response in a pancreatic cancer patient associated with response against several hTERT epitopes

Immunotherapy targeting the hTERT subunit of telomerase has been shown to induce robust immune responses in cancer patients after vaccination with single hTERT peptides. Vaccination with dendritic cells (DCs) transfected with hTERT mRNA has the potential to induce strong immune responses to multiple hTERT epitopes and is therefore an attractive approach to more potent immunotherapy. Blood samples from such patients provide an opportunity for identification of new, in vivo processed T-cell epitopes that may be clinically relevant. A 62-year-old female patient underwent radical surgery for a pancreatic adenocarcinoma. After relapse, she obtained stable disease on gemcitabine treatment. Due to severe neutropenia, the chemotherapy was terminated. The patient has subsequently been treated with autologous DCs loaded with hTERT mRNA for 3 years. Immunomonitoring was performed at regular intervals following start of vaccination and clinical outcome measured by CT and PET/CT evaluation. The patient developed an immune response against several hTERT-derived Th and CTL epitopes. She presently shows no evidence of active disease based on PET/CT scans. No serious adverse events were experienced and the patient continues to receive regular booster injections. We here provide evidence for the induction of hTERT-specific immune responses following vaccination of a pancreas cancer patient with DCs loaded with hTERT mRNA. These responses are associated with complete remission. A thorough analysis of this patient immune response has provided a unique opportunity to identify novel epitopes, associated with clinical effects. These will be included in future hTERT vaccines.

Immunogenicity of a recombinant lentiviral vector carrying human telomerase tumor antigen in HLA-B*0702 transgenic mice

Over expression of telomerase represents a hallmark of cancer cells and the induction of T cell immunity against this universal tumor antigen have gained promising interest for anticancer immunotherapy. In this study we evaluated a recombinant lentiviral vector expressing the human telomerase reverse transcriptase (lv-hTERT) vaccination in the humanized HLA-B*0702 transgenic (HLA-B7 Tg) mice. A single lv-hTERT vector immunization induces potent and broad HLA-B7-restricted CTL responses against hTERT. Unlike conventional hTERT peptide or DNA immunization, the lv-hTERT vector triggers high and sustained IFN-γ producing CD8 + T cell responses in HLA-B7 Tg mice. The avidity and in vivo cytotoxicity of CD8 + T cells were stronger in lv-hTERT vector-immunized mice than in hTERT peptide or DNA vaccinated groups. The study also showed that the use of prime-boost vaccination drastically improved the magnitude and strength of lentivector-primed CD8 + T cells. Our data indicated that lentiviral delivery of hTERT is suitable for enhancing cellular immunity against hTERT and offers a promising alternative for telomerase-based cancer vaccine.

Schedule Dependent Effects of Adoptively Transferred Autologous T Cells After AutoSCT for Myeloma: Accelerated Immune Recovery Is Associated with Improved Event-Free Survival.

Abstract 784▪▪This icon denotes an abstract that is clinically relevant. BACKGROUND:Previously, we showed that adoptive transfer of in vivo vaccine-primed and ex vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at day +12 after transplant increased CD4 & CD8 levels at day +42 and augmented vaccine-specific immune responses in pts with myeloma (MM)[Nat Med 2005; 11: 1230-7]. DESIGN:We conducted a phase I/II 2-arm clinical trial in which 54 pts with advanced MM received autografts after high-dose melphalan followed by infusions of ex-T at day +2 after transplant. Pts also received pretransplant and posttransplant immunizations using a pneumococcal conjugate vaccine (PCV) only (ARM B; n = 26) or the PCV plus an HLA-A2 restricted multipeptide tumor antigen vaccine (3 hTERT peptides + 1 survivin peptide + 1 CMV peptide) for HLA-A2+ patients (ARM A; n = 28). After adoptive transfer of 4.26 × 10e10 costimulated T cells (range 1.59-5.0), the median CD3, CD4 and CD8 counts were 4198, 1545 and 2858 cells per microliter at day +14 post-transplant and 16% of pts developed significant autologous GVHD [Clin Cancer Res 2009;15: 4499 – 507]. Here we first report clinical outcomes from this trial and describe several novel observations about immune reconstitution after day +2 T-cell transfers. CLINICAL RESULTS:For the 54 total transplant pts, the median age was 55 (range 37-68), 28 (52%) were male and 21 (39%) were AA while 32 (59%) were Caucasian and 1 (2%) was Asian. 66% had IgG and 28% had IgA paraproteins while 6% excreted light chains only; 41% had abnormal cytogenetics. With a median followup of 1 year (0.25 - 2.5 years) 50 pts (93%) are alive and 31 (57%) remain event-free (EFS). The projected median EFS is 15 mos with no difference between ARMS A and B. Based on the Cox regression analysis, EFS did not correlate significantly to gender, race nor absolute lymphocyte counts (ALC). However, age correlated inversely to EFS with older pts exhibiting a lower EFS (coefficient = -0.0667, p-value = 0.0398). MM responses at day +60 and day +100 did not correlate to EFS, but the response at day +180 did correlate to EFS (p <0.0001). Notably, higher CD3+ T-cell levels at day +14 and higher CD8+ T-cell levels at day +14 and day +60 were significantly associated with improved EFS (p = 0.0104, 0.00117, 0.0111, respectively). IMMUNOLOGICAL RESULTS:Preliminary results indicated that 6 of 15 pts (40%) in ARM A had positive tetramer responses to hTERT and/or survivin peptides at one or more timepoints after immunization, defined as tetramer staining by flow cytometry > 0.1% (and > 3-fold increase). Based on analysis of the first 36 patients, the mean sum of the antibody responses to 4 of the 7 serotypes of the pneumococcal conjugate vaccine (PCV; serotypes 6B, 14, 19F, 23F) were 83.55 mcg/ml at day +100 and 74.13 mcg/ml at day +180, highly protective levels, compared to 5.17 mcg/ml at enrollment. We also observed several novel features of immune system recovery after early T-cell transfers. Compared to an historical cohort of 103 MM pts who were autografted without ex-T, post-transplant quantitative IgG, IgA and IgM levels at day +180 (and days +60/+100 for IgG and IgA) were significantly higher in the pts who received day +2 T-cell transfers (IgG: 1525 mg/dl vs 1137 mg/dl, p= 0.014; IgA: 264 mg/dl vs 76 mg/dl, p = 0.0029; IgM: 58 mg/dl vs 39 mg/dl, p = 0.029). Furthermore, when IgG recovery was examined for the IgA MM pts only, the IgG level at day +180 was significantly higher in the ex-T trial (mean IgG = 810 mg/dl vs 611 mg/dl, p = 0.0478). Conversely, when IgA recovery was examined for the IgG MM pts only, the IgA level was significantly higher in the ex-T trial at days +60 (mean IgA = 129.82 mg/dl vs 71.70 mg/dl, p = 0.0036), day +100 (mean IgA = 114.43 mg/dl vs 59.44 mg/dl, p = 0.0075) and day +180 (mean IgA = 98.38 mg/dl vs 48.74 mg/dl, p = 0.0037). Remarkably, a pattern of CD8 T-cell “reprogramming” was also observed after day +2 T-cell transfers such that the % of CD8+/CD45RA+ T-cells at day +60 post-transplant was 75% vs 36% in a cohort of MM transplant pts who did not receive ex-T (p< 0.0001). In addition, the log ratio of Teffector/Tregulatory cells was significantly higher among the patients who received early T-cell transfers (2.57 vs. 1.93, p = 0.0066). CONCLUSIONS:Early adoptive transfers of anti-CD3/anti-CD28 costimulated autologous T-cells significantly enhanced post-transplant humoral and cellular immune reconstitution, a pattern which may be associated with better myeloma control and protection from infection. These data may have important implications for efforts to induce clinically significant immune responses to cancer vaccines in the transplant setting. Disclosures:Vonderheide:University of Pennsylvania and Dana Farber Cancer Institute: Patents & Royalties. June:University of Pennsylvania: Patents & Royalties.

T Cell Responses in Patients Vaccinated with Telomerase (hTERT)-mRNA Transfected Dendritic Cells.

Abstract 373▪▪This icon denotes an abstract that is clinically relevant.Two cancer patients were vaccinated with dendritic cells (DC) loaded with telomerase (hTERT) mRNA to investigate the safety, tolerability and immunological response to vaccination prior to the start of a new phase I/II clinical trial. Following written informed consent one primary lung adenocarcinoma with metastasis and one patient with a relapsed pancreatic ductal type of adenocarcinoma, were treated with autologus monocyte-derived DC transfected with mRNA encoding hTERT. The patients first received four weekly injections administered intradermally followed by monthly booster injections. Peripheral blood mononuclear cells (PBMC) at each vaccination time point were tested in vitro with transfected DC and a panel of 24 overlapping hTERT peptides. In addition, hTERT-specific CD8+ T cells were monitored by pentamer staining. The treatment was well tolerated with minor side effects. Immune responses against telomerase-transfected DC and some of the overlapping hTERT peptides were detected in both patients. We also detected hTERT-specific CD8+ T cells in both patients by pentamer staining in post-vaccination samples. The lung cancer patients obtained a stable disease that lasted 18 months while the patient with pancreas cancer who started the DC vaccination in July 2007 following palliative chemotherapy, still is in stable disease by continuously boost vaccination. T-cell responses against telomerase epitopes have also been identified in both non-vaccinated cancer patients and cancer patients previously vaccinated with telomerase peptide. Since patients with these findings often show extraordinary clinical courses of their disease we hypothesize that it exists a high degree of immunogenicity and HLA promiscuity for some telomerase epitopes. In this study we have shown that vaccination with hTERT-mRNA transfected DC is safe and able to induce robust immune responses to several telomerase T-cell epitopes both in CD4+ and CD8+ T cells. This opens up the possibility for a broad clinical application of mRNA hTERT DC vaccines. Furthermore, responding T cells identified in these patients are strong candidates for T-cell receptor cloning and the receptors identified can thereafter be transferred into T cells creating the next generation of immuno-gene therapy with retargeted T cells. Disclosures:No relevant conflicts of interest to declare.

Coadministration of Telomerase Genetic Vaccine and a Novel TLR9 Agonist in Nonhuman Primates

The human telomerase reverse transcriptase (hTERT) is an attractive target for human cancer vaccination because its expression is reactivated in most human tumors. We have evaluated the ability of DNA electroporation (DNA-EP) and adenovirus serotype 6 (Ad6) to induce immune responses against hTERT in nonhuman primates (NHPs) (Macaca mulatta). Vaccination was effective in all treated animals, and the adaptive immune response remained detectable and long lasting without side effects. To further enhance the efficacy of the hTERT vaccine, we evaluated the combination of hTERT vaccine and a novel TLR9 agonist, referred to as immunomodulatory oligonucleotide (IMO). Monkeys were dosed weekly with IMO concurrently with the vaccine regimen and showed increases in cytokine secretion and activation of natural killer (NK) cells compared with the group that received vaccine alone. Using a peptide array, a specific profile of B-cell reactive epitopes was identified when hTERT vaccine was combined with IMO. The combination of IMO with hTERT genetic vaccine did not impact vaccine-induced TERT-specific cell-mediated immunity. Our results show that appropriate combination of a DNA-EP/Ad6-based cancer vaccine against hTERT with IMO induces multiple effects on innate and adaptive immune responses in NHPs.