Peptide-pulsed Autologous Dendritic Cells Research Articles

The safety and anti-tumor effect of multiple peptides-pulsed dendritic cells combined with induced specific cytotoxic T lymphocytes for patients with solid tumors

ObjectiveThe aim of this study was to explore the safety and efficacy of multiple peptide-pulsed autologous dendritic cells (DCs) combined with cytotoxic T lymphocytes (CTLs) in patients with cancer.MethodsFive patients diagnosed with cancer between November 2020 and June 2021 were enrolled and received DC-CTLs therapy. Peripheral blood was collected and antigenic peptides were analyzed. The phenotype and function of DC-CTLs and the immune status of patients were detected using flow cytometry or IFN-γ ELISPOT analysis.ResultsDCs acquired a mature phenotype and expressed high levels of CD80, CD86, CD83, and HLA-DR after co-culture with peptides, and the DC-CTLs also exhibited high levels of IFN-γ. Peripheral blood mononuclear cells from post-treatment patients showed a stronger immune response to peptides than those prior to treatment. Importantly, four of five patients maintained a favorable immune status, of which one patient’s disease-free survival lasted up to 28.2 months. No severe treatment-related adverse events were observed.ConclusionOur results show that multiple peptide-pulsed DCs combined with CTLs therapy has manageable safety and promising efficacy for cancer patients, which might provide a precise immunotherapeutic strategy for cancer.

A randomised controlled trial of long NY-ESO-1 peptide-pulsed autologous dendritic cells with or without alpha-galactosylceramide in high-risk melanoma

AimWe have previously reported that polyfunctional T cell responses can be induced to the cancer testis antigen NY-ESO-1 in melanoma patients injected with mature autologous monocyte-derived dendritic cells (DCs) loaded with long NY-ESO-1-derived peptides together with α-galactosylceramide (α-GalCer), an agonist for type 1 Natural Killer T (NKT) cells.ObjectiveTo assess whether inclusion of α-GalCer in autologous NY-ESO-1 long peptide-pulsed DC vaccines (DCV + α-GalCer) improves T cell responses when compared to peptide-pulsed DC vaccines without α-GalCer (DCV).Design, setting and participantsSingle-centre blinded randomised controlled trial in patients ≥ 18 years old with histologically confirmed, fully resected stage II–IV malignant cutaneous melanoma, conducted between July 2015 and June 2018 at the Wellington Blood and Cancer Centre of the Capital and Coast District Health Board.InterventionsStage I. Patients were randomised to two cycles of DCV or DCV + α-GalCer (intravenous dose of 10 × 106 cells, interval of 28 days). Stage II. Patients assigned to DCV + α-GalCer were randomised to two further cycles of DCV + α-GalCer or observation, while patients initially assigned to DCV crossed over to two cycles of DCV + α-GalCer.Outcome measuresPrimary: Area under the curve (AUC) of mean NY-ESO-1-specific T cell count detected by ex vivo IFN-γ ELISpot in pre- and post-treatment blood samples, compared between treatment arms at Stage I. Secondary: Proportion of responders in each arm at Stage I; NKT cell count in each arm at Stage I; serum cytokine levels at Stage I; adverse events Stage I; T cell count for DCV + α-GalCer versus observation at Stage II, T cell count before versus after cross-over.ResultsThirty-eight patients gave written informed consent; 5 were excluded before randomisation due to progressive disease or incomplete leukapheresis, 17 were assigned to DCV, and 16 to DCV + α-GalCer. The vaccines were well tolerated and associated with increases in mean total T cell count, predominantly CD4+ T cells, but the difference between the treatment arms was not statistically significant (difference − 6.85, 95% confidence interval, − 21.65 to 7.92; P = 0.36). No significant improvements in T cell response were associated with DCV + α-GalCer with increased dosing, or in the cross-over. However, the NKT cell response to α-GalCer-loaded vaccines was limited compared to previous studies, with mean circulating NKT cell levels not significantly increased in the DCV + α-GalCer arm and no significant differences in cytokine response between the treatment arms.ConclusionsA high population coverage of NY-ESO-1-specific T cell responses was achieved with a good safety profile, but we failed to demonstrate that loading with α-GalCer provided an additional advantage to the T cell response with this cellular vaccine design. Clinical trial registration: ACTRN12612001101875. Funded by the Health Research Council of New Zealand.

Abstract 875: Next generation clonal neoantigen targeting T cells, generated using the PELEUSTM bioinformatics platform and the VELOSTM manufacturing method show superior reactivity and phenotypic characteristics than classical TIL products

Abstract Adoptive transfer of tumor infiltrating lymphocytes (TIL) has generated objective clinical responses in patients with advanced metastatic cancers. Therapeutic exploitation of neoantigens as targets can potentially lead to safer and more effective treatment modalities with reduced toxicities. The Achilles Therapeutics trial NCT03517917 enabled the acquisition of matched tumor specimens and peripheral blood samples from patients undergoing routine surgery and facilitated the development of the proprietary VELOSTM manufacturing process, generating a personalized clonal neoantigen specific T cell product. An in-depth characterization of T cells expanded with the VELOSTM process was performed and compared to a standard TIL product. Samples were obtained from patients with primary NSCLC or metastatic melanoma. TIL were expanded from tumor fragments after dissection in the presence of IL-2. Peptide pools corresponding to the clonal mutations that were identified using the PELEUSTM bioinformatics platform were used to pulse dendritic cells (DC) generated from peripheral blood monocytes from each patient. Clonal neoantigen specific T cells (cNeT) were expanded using the VELOSTM process by co-culture of TIL with the peptide-pulsed autologous DC. As a comparison, TIL were expanded with a rapid expansion protocol (REP-TIL) in the presence of allogeneic feeders, anti-CD3 antibody and high-dose IL-2. Intracellular cytokine staining was performed following rechallenge with individual peptide pools encoding the clonal mutations. Single peptide reactivities were identified using ELISPOT and extended flow cytometric analysis of markers associated with T cell fitness or dysfunction was performed to phenotypically characterize the cNeT, TIL and REP-TIL. Analysis of the immune cell composition showed that cNeT, TIL and REP-TIL have similar CD3+ T cell content (median cNeT 90.2%, TIL 87.3%, REP-TIL 95%, n=6) and are composed of CD4+ and CD8+ T cells (median CD4:CD8 ratio- cNeT 11.1, TIL 2.03 and REP-TIL 4.7, n=6). cNeT showed superior clonal neoantigen specificity compared to TIL or REP-TIL. The proportion of CD3+ T cells responding to clonal neoantigen rechallenge was increased in cNeT (median 24.3%) compared to TIL (median 0.6%) and REP-TIL (median 1.8%) (n=5). The VELOSTM process incorporating the PELEUSTM platform for prediction of clonal neoantigens generates T cell products enriched for clonal neoantigen reactivities and superior phenotypic characteristics compared to conventional TIL. The VELOSTM process is currently being used to manufacture cNeT for two first-in-human studies including NSCLC and melanoma patients (NCT04032847, NCT03997474). Ethical approval: The samples for the study were collected under an ethically approved protocol (NCT03517917). Citation Format: Eleni Kotsiou, Tie Zheng Hou, Joseph Robinson, Sonal Varsani, Theres Oakes, Pablo D. Becker, Shreenal Patel, Jennine Mootien, Andrew Craig, Jane Robertson, Edward Samuel, James Reading, Lyra Del Rosario, Andrew Haynes, Samra Turajlic, Farah Islam, David Lawrence, Mariam Jamal-Hanjani, Martin Foster, Sergio A. Quezada, Katy Newton. Next generation clonal neoantigen targeting T cells, generated using the PELEUSTM bioinformatics platform and the VELOSTM manufacturing method show superior reactivity and phenotypic characteristics than classical TIL products [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 875.

Identification of two HLA-A*0201 immunogenic epitopes of lactate dehydrogenase C (LDHC): potential novel targets for cancer immunotherapy

Lactate dehydrogenase C (LDHC) is an archetypical cancer testis antigen with limited expression in adult tissues and re-expression in tumors. This restricted expression pattern together with the important role of LDHC in cancer metabolism renders LDHC a potential target for immunotherapy. This study is the first to investigate the immunogenicity of LDHC using T cells from healthy individuals. LDHC-specific T cell responses were induced by in vitro stimulation with synthetic peptides, or by priming with autologous peptide-pulsed dendritic cells. We evaluated T cell activation by IFN-γ ELISpot and determined cytolytic activity of HLA-A*0201-restricted T cells in breast cancer cell co-cultures. In vitro T cell stimulation induced IFN-γ secretion in response to numerous LDHC-derived peptides. Analysis of HLA-A*0201 responses revealed a significant T cell activation after stimulation with peptide pools 2 (PP2) and 8 (PP8). The PP2- and PP8-specific T cells displayed cytolytic activity against breast cancer cells with endogenous LDHC expression within a HLA-A*0201 context. We identified peptides LDHC41−55 and LDHC288−303 from PP2 and PP8 to elicit a functional cellular immune response. More specifically, we found an increase in IFN-γ secretion by CD8 + T cells and cancer-cell-killing of HLA-A*0201/LDHC positive breast cancer cells by LDHC41−55- and LDHC288−303-induced T cells, albeit with a possible antigen recognition threshold. The majority of induced T cells displayed an effector memory phenotype. To conclude, our findings support the rationale to assess LDHC as a targetable cancer testis antigen for immunotherapy, and in particular the HLA-A*0201 restricted LDHC41–55 and LDHC288–303 peptides within LDHC.

Vaccination with poly(IC:LC) and peptide-pulsed autologous dendritic cells in patients with pancreatic cancer

BackgroundDendritic cells (DCs) enhance the quality of anti-tumor immune response in patients with cancer. Thus, we posit that DC-based immunotherapy, in conjunction with toll-like receptor (TLR)-3 agonist poly-ICLC, is a promising approach for harnessing immunity against metastatic or locally advanced unresectable pancreatic cancer (PC).MethodsWe generated autologous DCs from the peripheral blood of HLA-A2+ patients with PC. DCs were pulsed with three distinct A2-restricted peptides: 1) human telomerase reverse transcriptase (hTERT, TERT572Y), 2) carcinoembryonic antigen (CEA; Cap1-6D), and 3) survivin (SRV.A2). Patients received four intradermal injections of 1 × 107 peptide-pulsed DC vaccines every 2 weeks (Day 0, 14, 28, and 42). Concurrently, patients received intramuscular administration of Poly-ICLC at 30 μg/Kg on vaccination days (i.e., day 0, 14, 28, and 42), as well as on days 3, 17, 21, 31, 37, and 45. Our key objective was to assess safety and feasibility. The effect of DC vaccination on immune response was measured at each DC injection time point by enumerating the phenotype and function of patient T cells.ResultsTwelve patients underwent apheresis: nine patients with metastatic disease, and three patients with locally advanced unresectable disease. Vaccines were successfully manufactured from all individuals. We found that this treatment was well-tolerated, with the most common symptoms being fatigue and/or self-limiting flu-like symptoms. Among the eight patients who underwent imaging on day 56, four patients experienced stable disease while four patients had disease progression. The median overall survival was 7.7 months. One patient survived for 28 months post leukapheresis. MHC class I –tetramer analysis before and after vaccination revealed effective generation of antigen-specific T cells in three patients with stable disease.ConclusionVaccination with peptide-pulsed DCs in combination with poly-ICLC is safe and induces a measurable tumor specific T cell population in patients with advanced PC.Trial registrationNCT01410968; Name of registry: clinicaltrials.gov; Date of registration: 08/04/2011).

Characterization of Spontaneous Immune Responses against Long Peptides Derived from Bcl-X(L) in Cancer Patients Using Elispot

In recent years we and others have used the ELISPOT assay successfully to identify novel tumor antigens by the characterization of spontaneous HLA class I restricted immune responses against a number of minimal 9–10 amino acid long peptide epitopes. In the present study, we examined the capability of using longer peptides when scrutinizing Peripheral Blood Mononuclear Cells (PMBC) from melanoma patients for spontaneous immunity by means of ELISPOT IFN-γ secretion assay. To this end, we examined PBMC for the presence of specific T-cell responses against long peptides derived from the tumor associated antigen BCL-X(L). The protein product of the larger BCL-X(L) differs from Bcl-X(S) protein by an inserted region (amino acids 126–188). Thus, we scrutinized eight long peptides covering this inserted region for spontaneous immunity. The peptides were overlapping and consisted of 20–23 amino acids. PBMC were pre-stimulated with peptide-pulsed autologous dendritic cells (DC) and subjected to the IFN-γ ELISPOT assay. Four of the BCL-X(L) derived peptides elicited very frequent responses in several patients. Additionally, in all patients responses against more than one of the peptides could be detected. In conclusion several long BCL-X(L) derived peptide epitopes exist, which may be used in anti-cancer immunity. Furthermore, the ELISPOT assay offers an attractive and sensitive method for the characterization of spontaneous immune reactivity against long peptides.

Identification of Leukemia Associated Antigens From ANKRD17 and CDK4 Using Mass Spectrometry Based Screening,

Abstract 4020 Introduction:The discovery of therapeutically relevant tumor antigens requires the demonstration of endogenous antigen processing and presentation by class I HLA molecules and evidence for the antigen's ability to elicit a specific immune response. Traditional antigen discovery methods have focused on initially generating T cell clones that recognize a target with subsequent testing of sequentially purified peptide pools to determine the chemical structure of the antigen. Generating T cell clones and the subsequent antigen discovery processes are labor-intensive, time-consuming and inefficient because antigen characteristics such as source protein, tissue restriction, HLA-restriction, and binding affinity cannot be determined prior to the generation of the T cell clone. To address these limitations, we developed an antigen discovery method based upon initial identification of HLA-epitopes with subsequent immune characterization that bypasses the need to generate a T cell clone and enabled us to identify and characterize 2 leukemia associated antigens within 1 year. Methods:Wild-type HLA-A0201 was stably transduced into the U937 myeloid leukemia cell line. Approximately 2×109 HLA-A0201+U937 were lysed and the peptide/HLA-A0201 complexes were purified by immunoaffinity column chromatography. The peptides were liberated from the HLA by weak acid elution and analyzed by reverse phase HPLC/tandem mass spectrometry (MS/MS). Collision induced dissociation (CID) MS/MS data were analyzed using the Mascot search engine. From 23 unambiguously sequenced, high-abundance peptides we analyzed 2 peptides, derived from ANKRD17 (UNC-A17 = LLIERGASL) and CDK4 (UNC-CK4 = ALTPVVVTL). These peptides were selected because ANKRD17 is a known source protein for the breast cancer antigen NY-BR-16, and CDK4 initiates entry into S-phase and is currently an investigational therapeutic target for treatment of acute myeloid leukemia (AML). Results:We confirmed CDK4 protein expression and observed variable (1 sample with high expression and 2 samples with low expression) expression of ANKRD17 in 3 of 3 human AML samples using western blots. Using an in-vitro assay, we found that both antigens bound HLA-A0201 with a dissociation t1/2 > 8 hours. Because of this slow off rate, we performed T cell expansion studies from healthy donor peripheral blood mononuclear cells (PBMC). We were able to generate antigen-specific oligoclonal T cell populations for both antigens. These T cell populations demonstrated antigen-specific immune function based upon intracellular interferon-γ flow cytometry measurements using both peptide-pulsed autologous dendritic cells and HLA-A0201+U937 cells as immune targets. We next looked for evidence of in-vivo T cell immunity towards these antigens in 5 patients (2 AML, 1 CML, 1 MDS, 1 MF) who had undergone allogeneic stem cell transplant. Using UNC-A17 and UNC-CK4 peptide loaded, fluorescently conjugated HLA-A0201 tetramers we identified UNC-CK4 specific T cells in 2 samples (0.22% and 0.39% of total CD8+ cells) and UNC-A17 specific T cells in 1 sample (0.64% of total CD8+ cells). Conclusion:Using this novel methodology we were able to identify 2 new leukemia associated antigens by directly examining the target cells for antigen expression and epitope processing. This direct methodology eliminated the need to generate a T cell clone or evaluate an entire peptide pool for evidence of immunogenicity, as the first step in antigen discovery. Refinement of our HPLC-MS techniques will enable us to sequence increasing numbers of peptide epitopes from the source cells. These technical improvements combined with the standard genomics and bioinformatics tools that are currently available will allow us to narrow our focus on the discovery of immunologically relevant tumor associated self antigens and minor histocompatibility antigens that have very narrow tissue expression and favorable HLA binding characteristics. Disclosures:No relevant conflicts of interest to declare.

Cord Blood Lymphocytes After TCR Gene Transduction Are Functional, Retain An Early Differentiation Phenotype and Are Suitable for Adoptive Immunotherapy

Abstract 3117The efficacy of adoptive T cell therapy largely depends on the in vivo persistence of infused cells (Robbins PF et al. J Immunol 2004;173 :7125), that is related to the differentiation stage. The less differentiated central memory (CM) T cells display superior proliferation, persistence and anti-tumor response following infusion when compared to the more differentiated effector memory (EM) T cells. However both T cell receptor (TCR) gene transfer and in vitro expansion of antigen-specific T lymphocytes necessitate lymphocyte activation; with lymphocytes from adult donors this results in accumulation of highly differentiated EM CD45RA+ (EMRA) cells with reduced half life and proliferative capability. Cord blood (CB) lymphocytes are predominantly naïve (N), and we have studied the potential utility of these cells for TCR transduction-based immunotherapy.T cells from CB samples were activated by anti-CD3 antibody plus IL-2, and transduced with a TCR specific for a peptide from the EBV LMP2 molecule using a retroviral vector. Lymphocytes from adult donors were also transduced as controls. We found that after transduction CB and adult lymphocytes showed comparable levels of TCR expression. Moreover, both CB and adult T cells rapidly shifted toward a more mature phenotype, CB cells becoming mainly CM (60%), and adult T cells becoming mainly EM (52%). In keeping with their phenotype, the expression CD57, a marker of replicative senescence was low in CB T lymphocytes (18%) and high in adult T lymphocytes (68%).To asses if the transduced cells were able to respond to specific antigen stimulation, transduced lymphocytes were incubated with peptide-pulsed autologous dendritic cells. CB T cells expanded as effectively as lymphocytes from adult donors with a 50-fold increase in cell count after 3 week culture, with no indication that the expansion was leveling off. At that time point more than 50% of the cells expressed the transduced TCR both in CB and in adult samples.Further, CB cultures were dominated by EM accounting for a mean of 55% of the cells at the end of the culture period with very few EMRA. In contrast, adult blood cell cultures were characterised by a marked increase in EMRA which came to comprise a mean of 66% of the final population. Further phenotypic analysis at the end of the in vitro expansion demonstrated that CD27, a marker of less differentiated T cells, was expressed on 85% of transduced CB T cells but only on 39% of adult T cells, confirming that CB lymphocytes had retained a less differentiated phenotype.To investigate the effector function of the transduced cells we demonstrated that after further stimulations. polyfunctional CB CD8 cells secreting IFNg, IL2 and TNFa increased from 0.8% after the first stimulation to 4.5% after re-stimulation. In contrast, polyfunctional transduced CD8 T cells from adults decreased from 2.0% to 0.9% during the same period. Both transduced CB and adult CD8 cells demonstrated effective HLA-restricted, antigen-specific target cell lysis in a standard chromium release assay.Our results suggest that TCR-transduced CB T cells have greater in vivo expansion potential, making these cells potentially more suitable for adoptive immunotherapy, particularly in those cases where autologous lymphopheresis or donor lymphocyte infusion is not an option. Disclosures:No relevant conflicts of interest to declare.

Identification of a novel conserved HLA-A*0201-restricted epitope from the spike protein of SARS-CoV

BackgroundThe spike (S) protein is a major structural glycoprotein of coronavirus (CoV), the causal agent of severe acute respiratory syndrome (SARS). The S protein is a potent target for SARS-specific cell-mediated immune responses. However, the mechanism CoV pathogenesis in SARS and the role of special CTLs in virus clearance are still largely uncharacterized. Here, we describe a study that leads to the identification of a novel HLA-A*0201-restricted epitope from conserved regions of S protein.ResultsFirst, different SARS-CoV sequences were analyzed to predict eight candidate peptides from conserved regions of the S protein based upon HLA-A*0201 binding and proteosomal cleavage. Four of eight candidate peptides were tested by HLA-A*0201 binding assays. Among the four candidate peptides, Sp8 (S958-966, VLNDILSRL) induced specific CTLs both ex vivo in PBLs of healthy HLA-A2+ donors and in HLA-A2.1/Kb transgenic mice immunized with a plasmid encoding full-length S protein. The immunized mice released IFN-γ and lysed target cells upon stimulation with Sp8 peptide-pulsed autologous dendritic cells in comparison to other candidates.ConclusionThese results suggest that Sp8 is a naturally processed epitope. We propose that Sp8 epitope should help in the characterization of mechanisms of virus control and immunopathology in SARS-CoV infection.

Personalized immunotherapy: a siren myth?

10.2217/PME.09.39 © 2009 Future Medicine Ltd “...the antigenic characteristics of tumor cells can be perceived by the innate and cognate immune systems, which represent the extrinsic barriers against tumor progression, or at least a substantial component of the host–tumor equilibrium.” Antoine Tesniere1,2,3 1U848 INSERM, ‘Cancer, Apoptosis & Immunity, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Universite Paris Sud, Villejuif, France Guido Kroemer1,2,3 1U848 INSERM, ‘Cancer, Apoptosis & Immunity, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Universite Paris Sud, Villejuif, France Thomas Tursz Departement de medecine, Institut Gustave Roussy, Villejuif, France

Cyclin D1 as a Universally-Expressed Mantle Cell Lymphoma-Associated aTumor Antigen for Immunotherapy

Mantle cell lymphoma (MCL) accounts for 5% to 10% of all non-Hodgkin lymphomas and has the worst prognosis among all lymphomas. The hallmark of MCL is a t(11;14) translocation resulting in overexpression of cyclin D1 by tumor cells of virtually all patients. In this study we examined whether cyclin D1 could be an effective tumor-associated antigen for immunotherapy. We identified a cyclin D1 peptide (P101) for HLA-A*0201, created heteroclitic peptide (Py101) for better binding, and generated peptide-specific T-cell lines from HLA-A*0201+ blood donors and MCL patients. After 5 to 7 rounds of in vitro stimulation with peptide-pulsed autologous dendritic cells (DCs), T-cell lines were obtained, which contained about 45% CD4+ and 55% CD8+ T cells. As exemplified by the results of peptide-tetramer staining, the frequencies of peptide-specific CD8+ T cells increased during in vitro stimulation, from 5.7% of specific T cells at fourth stimulation to 19.7% at sixth stimulation. These cell lines proliferated in response to autologous DCs pulsed with cyclin D1 peptide Py101 (but not unpulsed), as measured by 3[H]-thymidine incorporation and CFSE-dilution assays. These results indicate that the T cells are indeed specific for cyclin D1 Py101 peptide. Moreover, the T cells efficiently lysed peptide-pulsed but not unpulsed T2 cell and autologous DCs, and cyclin D1+ and HLA-A*0201+ human MCL lines MINO, SP53, Jeko-1, and Granta 519, and more importantly, HLA-A*0201+ primary lymphoma cells from MCL patients. No killing was observed on HLA-A*0201− primary lymphoma cells or HLA-A*0201+ normal blood cells including B cells. These results indicate that these T cells are potent cytotoxic T cells and recognize cyclin D1 peptides naturally presented by patient lymphoma cells in the context of surface HLA-A*0201 molecules. Flow cytometry analysis was used to examine the expression of granzymes, perforin, and Fas ligand (FasL) by the T cells. Based on these results, T cells may kill their target cells via the perforin/granzyme pathways, because they express perforin and high levels of granzymes but not FasL. Two independent methods were used to examine the cytokine expression profiles of the T cells. Upon restimulation with DCs pulsed with Py101 peptide, but not with unpulsed DCs, 11.2% of the T cells expressed IFN-γ by intracellular cytokine staining. IL-4-expressing T cells were very few (0.3%). To detect cytokine secretion, an ELISPOT assay was used to enumerate IFN-γ-secreting cells. After restimulation with DCs pulsed with cyclin D1 peptides, or with cyclin D1+/HLA-A*0201+ SP53 and cyclin D1+/HLA-A*0201+ primary lymphoma cells from patients, large numbers of IFN-γ-secreting cells were detected. Taken together, our work identifies cyclin D1 as a potentially important antigen for immunotherapy in MCL.

UTY-Coded Minor Histocompatibility Antigens as Targets for a Graft- Versus-Host Reaction against Hematopoietic Cells in the Dog.

Female donors produce a strong graft-versus-leukemia (GvL) effect in male patients. Presumably Y-encoded minor histocompatibility antigens (mHA) are recognized by the alloimmune effector lymphocytes. UTY (ubiquitously transcribed tetratricopeptide repeat, Y-linked) is a potential candidate gene for mHA in dogs and men. In the canine model we could show that the minor histocompatibility antigen UTY might be a potential target to further improve the adoptive immunotherapeutic approach. Five HLA-A2 restricted peptides were used for pulsing canine dendritic cells and stimulating canine T cells. T-cells were separated from the blood of female donors using the MACS technique and effector T-cells were generated by incubation of 2x10*6 CD3+ T-cells/ml with autologous mature, peptide-pulsed dendritic cells (DCs) in X-Vivo 15 medium (+ 160 U/ml human IL-2 and 5 ng/ml human IL-7; ratio T:DCs=10:1). T cells were restimulated weekly with peptide-pulsed DCs. After 3 or 4 weeks in culture T-cells were examined for the CD8/CD4 ratio, cytotoxicity assays were assessed after 3–4 re-stimulations in [51Cr]-release assays. The number of peptide-specific T cells was assessed in IFN-g-ELISPOT-assays. UTY-mRNA expression was determined by RT-PCR in several female and male cell types (BM, PBMCs, monocytes, DCs, B-cells). We were able to expand T-cells from 6 female dogs using peptide-pulsed autologous DCs; 3 out of 5 UTY-derived peptides were able to stimulate a specific autologous T-cell response. The number of CD8+ T-cells could be expanded during four weeks of in vitro culture in 3 dogs. In IFN-g-ELISPOT-assays we could demonstrate that the expanded T-cells secrete IFN-g upon recognition of DCs from DLA-identical male littermates. In addition, these T-cells specifically lysed DLA-identical male BM (25-100%), but not autologous or DLA-identical female BM (0–15%) in [51Cr]-release assays (E:T=80:1). Cell types other than BM and DCs (DLA-identical male and female monocytes, PBMCs, B-cells) were not lysed (0–18%). In summary, we could demonstrate that UTY is a naturally processed and presented antigen in the dog. Our data show that HLA-A2 restricted UTY-peptides immunize specific CTLs from female dogs that efficiently recognize DLA-identical male bone marrow and DCs. These findings suggest that UTY may be employed for studies of immunotherapy in the canine model.

Generation of CD8+ Cytotoxic T Cell Clones Recognizing BMI1-Derived Peptides

Leukemic stem cells (LSCs) resistant to conventional chemotherapeutic drugs can cause relapse of disease even after intensive treatment. Therefore, novel therapeutic strategies targeting LSCs are needed. The polycomb group gene BMI1 was initially identified as a cooperating oncogene in Eμ/myc transgenic mice. The protein product of BMI1 has been shown to repress the products of the CDKN2 tumor suppressor gene, p16 (INK4A) and p14 (ARF), and is required for self-renewal of both normal hematopoietic stem cells as well as LSCs. BMI1 expression level has been correlated with prognosis in myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). Furthermore, higher BMI1 expression in the tumorigenic cancer stem cell population has been demonstrated in brain tumor, breast cancer, and head and neck cancer. Thus, BMI1 represents a potential attractive target for cancer stem cell-specific immunotherapy. To further evaluate BMI1 potential, we generated CD8+BMI1-specific cytotoxic T cell clones. Two BMI1-encoded peptides (CLPSPSTPV, BMI1271-279, and TLQDIVYKL, BMI174-82) with predicted high affinity binding to the class I MHC molecule HLA-A*0201 were identified using publicly available prediction algorithms, and high-affinity binding to HLA-A*0201 was confirmed using an MHC stabilization assay on T2 cells. CD8+ T cells were negatively enriched from peripheral blood mononuclear cells (PBMC) from three normal human HLA-A 0201+ donors and stimulated at 10-day intervals with BMI1 peptide-pulsed autologous dendritic cells or PBMC for two or three cycles. Reactivity of the resulting T cell lines for the corresponding BMI1 peptides was subsequently assessed by ELISPOT assay, and was detected in lines from two of the three donors for CLPSPSTPV and in a line from one of the three donors for TLQDIVYKL. A CTL clone specific for CLPSPSTPV was isolated from one of the donors and evaluated in further studies. A peptide titration assay demonstrated that the clone had high affinity (peptide concentration required for half maximal lysis at E/T=10:1, 0.1 nM). In standard cytotoxicity assays, this CTL clone efficiently killed CLPSPSTPV peptide-pulsed T2 cells and 6 of 9 HLA-A*0201-positive or -transduced BMI1+ myeloid leukemia cell lines, and did not kill nonmalignant HLA-A2+ cells, including fibroblasts, PHA-blasts, and EBV-transformed B cells. Quantitative RT-PCR analysis of BMI1 expression was performed on a commercial multiple tissue cDNA panel, 39 primary leukemia/lymphoma samples (4 ALL, 22 AML, 4 CLL, 4 DLBCL, 4 FL, 1 MCL), and 31 solid tumor lines (21 Renal cell ca., 1 Ovarian ca., 3 Breast ca., 4 Melanoma, 1 Colon ca., 1 Pancreas ca.). All samples had detectable levels of BMI1 expression. Among normal tissues, testis had the highest level of BMI1 expression. Of the primary leukemia/lymphoma samples, four of 22 AML cells and one of four CLL cells expressed high levels of BMI1, and all solid tumor lines had low levels of BMI1 expression. When tested for cytotoxicity against five HLA-A*0201+ primary leukemia cells (3 AML, 1 ALL, 1 CML-BC), the BMI1-specific CTL clone killed 3 of 5 efficiently. The clone was also tested for recognition of 14 HLA-A*0201+ solid tumor lines (6 Renal cell ca., 2 Breast ca. 2 Melanoma, 1 Ovarian ca., 1 Colon ca., 2 Medulloblastoma), but only killed 2 of the 14 lines. These results suggest that CTL therapy targeting BMI1 may have utility as a novel strategy for eliminating LSCs. Future studies will determine whether this clone can specifically recognize leukemic stem cells and inhibit the engraftment of human AML in NOD/SCID mice.

Aurora-a Kinase: A Novel Target of Cellular Immunotherapy for Human Leukemia

[Purpose] Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family, and the Aur-A gene is located at chromosome 20q13. Aur-A is mainly expressed in the G2/M phase of the cell cycle and regulates mitotic cell division in normal cells. Aur-A is expressed exclusively in testis in normal tissues, but is abundantly expressed in various kinds of cancer including hematological malignancies. Its overexpression usually accompanies chromosomal abnormality related with the poor prognosis. Furthermore, the silencing of Aur-A gene has disrupted the proliferation of cancer cells and augmented their susceptibility to anti-cancer agents. These data strongly suggest that Aur-A is one of the crucial oncognes and a rational target for cancer treatment. Thus, we postulated that Aur-A might be an ideal target for cancer immunotherapy, and attempted to verify the feasibility of cellular immunotherapy for leukemia targeting Aur-A.[Methods] Nine-mer peptides derived from Aur-A which were algorithmically predicted to bind to HLA-A*0201 molecule were synthesized and assessed their binding affinities by the stabilization assay with T2 (HLA-A*0201) cells. Aur-A-specific cytotoxic T lymphocytes (CTLs) were generated by stimulation of CD8+ T cells with Aur-A peptide-pulsed autologous dendritic cells. Cytotoxicity of Aur-A-specific CTLs was defined by standard 51Cr-release assay. HLA class I restriction was examined with anti-HLA class I antibody and target-specificity of Aur-A-specific CTLs was examined by cold target inhibition assay with 51Cr-labeled HLA-A*0201-positive leukemia cell lines in combination with Aur-A peptide-loaded 51Cr-unlabeled HLA-A*0201-positive lymphoblastoid cell lines. The expressions of Aur-A mRNA and Aur-A protein in leukemia cells and normal cells were assessed by semi-quantitative real-time PCR and Western blotting. The frequencies of Aur-A-specific CTL precursors in the peripheral blood of healthy individuals and patients with leukemia were measured by tetramer staining.[Results] We established an HLAA* 0201-restricted and Aur-A207-215 peptide (YLILEYAPL)-specific CTL line (designated as AUR-1) by repeated stimulations of CD8+ T cells with Aur-A207-215 peptide-pulsed peripheral blood mononuclear cells from an HLA-A*0201-positive healthy individual. AUR-1 exerted cytotoxicity to various kinds of leukemia cell lines in an HLA-A*0201- restricted fashion. AUR-1 could discriminate freshly isolated leukemia cells from normal cells and normal mitotic cells, according to the expression levels of Aur-A mRNA. By using freshly isolated leukemia cells from CML patients' bone marrow mononuclear cells (BMMCs), we further investigated whether AUR-1 could lyse the leukemic progenitor cells. The CD34+CD38low fraction of CML BMMCs which include leukemic stem cells but not the normal counter part cells overexpressed Aur-A mRNA. Additionally AUR-1 could lyse CD34+ CML BMMCs, but not normal CD34+ cells. Finally, by using tetramer assay, Aur-A207-215-specific CTL precursors were detected more frequently in the peripheral blood of HLA-A*0201-positive patients with leukemia than that of healthy individuals.[Conclusion] This is the first report that Aur-A-specific CTLs can exert the cytotoxicity against leukemic stem cells but not normal hematopoietic stem cells. Results in this study suggests that the cellular immunotherapy targeting Aur-A is a promising strategy for the treatment of human leukemia.

Spontaneous and vaccine induced AFP-specific T cell phenotypes in subjects with AFP-positive hepatocellular cancer.

We are investigating the use of Alpha Fetoprotein (AFP) as a tumor rejection antigen for hepatocellular carcinoma (HCC). We recently completed vaccination of 10 AFP+/HLA-A2.1+ HCC subjects with AFP peptide-pulsed autologous dendritic cells (DC). There were increased frequencies of circulating AFP-specific T cells and of IFNgamma-producing AFP-specific T cells after vaccination. In order to better understand the lack of association between immune response and clinical response, we have examined additional aspects of the AFP immune response in patients. Here, we have characterized the cell surface phenotype of circulating AFP tetramer-positive CD8 T cells and assessed AFP-specific CD4 function. Before vaccination, HCC subjects had increased frequencies of circulating AFP-specific CD8 T cells with a range of naïve, effector, central and effector memory phenotypes. Several patients had up-regulated activation markers. A subset of patients was assessed for phenotypic changes pre- and post-vaccination, and evidence for complete differentiation to effector or memory phenotype was lacking. CD8 phenotypic and cytokine responses did not correlate with level of patient serum AFP antigen (between 74 and 463,040 ng/ml). Assessment of CD4+ T cell responses by ELISPOT and multi-cytokine assay did not identify any spontaneous CD4 T cell responses to this secreted protein. These data indicate that there is an expanded pool of partially differentiated AFP-specific CD8 T cells in many of these HCC subjects, but that these cells are largely non-functional, and that a detectable CD4 T cell response to this secreted oncofetal antigen is lacking.

Dacarbazine (DTIC) versus vaccination with autologous peptide-pulsed dendritic cells (DC) in first-line treatment of patients with metastatic melanoma: a randomized phase III trial of the DC study group of the DeCOG

Background: This randomized phase III trial was designed to demonstrate the superiority of autologous peptide-loaded dendritic cell (DC) vaccination over standard dacarbazine (DTIC) chemotherapy in stage IV melanoma patients.Patients and methods: DTIC 850 mg/m2 intravenously was applied in 4-week intervals. DC vaccines loaded with MHC class I and II-restricted peptides were applied subcutaneously at 2-week intervals for the first five vaccinations and every 4 weeks thereafter. The primary study end point was objective response (OR); secondary end points were toxicity, overall (OS) and progression-free survival (PFS).Results: At the time of the first interim analysis 55 patients had been enrolled into the DTIC and 53 into the DC-arm (ITT). OR was low (DTIC: 5.5%, DC: 3.8%), but not significantly different in the two arms. The Data Safety & Monitoring Board recommended closure of the study. Unscheduled subset analyses revealed that patients with normal serum LDH and/or stage M1a/b survived longer in both arms than those with elevated serum LDH and/or stage M1c. Only in the DC-arm did those patients with (i) an initial unimpaired general health status (Karnofsky = 100) or (ii) an HLA-A2+/HLA-B44− haplotype survive significantly longer than patients with a Karnofsky index <100 (P = 0.007 versus P = 0.057 in the DTIC-arm) or other HLA haplotypes (P = 0.04 versus P = 0.57 in DTIC-treated patients).Conclusions: DC vaccination could not be demonstrated to be more effective than DTIC chemotherapy in stage IV melanoma patients. The observed association of overall performance status and HLA haplotype with overall survival for patients treated by DC vaccination should be tested in future trials employing DC vaccines.

Identification of a major histocompatibility complex class I‐restricted T‐cell epitope in the tumour‐associated antigen, 5T4

5T4 is a surface glycoprotein expressed on placental trophoblasts and also on a wide range of human carcinomas. Its highly restricted expression on normal tissues and broad distribution on many carcinomas make 5T4 a promising target for cancer immunotherapy. In the current study, we set out to investigate whether a 5T4-specific cytotoxic T lymphocyte (CTL) repertoire exists in healthy individuals. CD4-depleted peripheral blood mononuclear cells (PBMCs) from blood donors were screened using an ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay. A panel of overlapping peptides, spanning the full length of the 5T4 protein, was used as a source of antigen. In the process of screening, one out of 30 blood donors demonstrated a positive ex vivo IFN-gamma ELISPOT response to a single 5T4 peptide. A polyclonal T-cell line was derived from this donor by culturing PBMCs with autologous peptide-pulsed dendritic cells (DCs). The resulting polyclonal T-cell line and clones were tested in a 51Cr-release assay and by ELISPOT and were shown to be peptide specific. Furthermore, antigen-presenting cells (APCs), infected with a viral vector expressing 5T4, were able to stimulate IFN-gamma production by the peptide-specific T-cell clones. A minimal CD8 epitope, PLADLSPFA, has been identified and found to be restricted through human leucocyte antigen (HLA) Cw7. Subsequently, we have demonstrated that HLA-Cw7-positive colorectal cancer patients vaccinated with a recombinant vaccinia viral vector encoding 5T4 (TroVax) are capable of mounting a strong IFN-gamma ELISPOT response to this novel CTL epitope. These findings have potential application in cancer immunotherapy in terms of subunit vaccine design and the monitoring of immune responses induced in patients by 5T4-based therapies.

Sensitive detection of human papillomavirus type 16 E7-specific T cells by ELISPOT after multiple in vitro stimulations of CD8+ T cells with peptide-pulsed autologous dendritic cells

BackgroundCervical cancer is the second most common gynecological cancer amongst women world-wide. Despite optimized protocols, standard treatments still face several disadvantages. Therefore, research aims at the development of immune-based strategies using tumor antigen-loaded dendritic cells for the induction of cellular anti-tumor immunity.ResultsIn this study, we used dendritic cells loaded with the HLA-A2-restricted HPV type 16 E711–20 peptide in order to induce an in vitro CD8+ T cell response. For this purpose, peptide-pulsed dendritic cells were co-cultured with autologous CD8+ T cells. After 5 weekly stimulations with peptide-pulsed mature dendritic cells, cultured T cells were analyzed for antigen specificity by an IFN-γ ELISPOT assay. Using this ELISPOT assay, we were able to detect E7-specific IFN-γ-secreting CD8+ T cells in 5/5 healthy donors.ConclusionWe show that peptide-pulsed mature dendritic cells are able to stimulate a HPV type 16 E7 peptide-specific immune response in vitro. These experiments describe an efficient culture protocol for antigen-specific T cells for use in pre-clinical vaccination research and confirm the need for sensitive T cell assays for detection of tumor-specific immune responses in vitro.

Technological advances in adoptive immunotherapy

Adoptive immunotherapy is an attractive and elegant strategy for treating a variety of life-threatening diseases. Several approaches have been developed to generate antigen-specific CD4+ and CD8+ T cells for adoptive T-cell therapy in cancer and infectious diseases. Currently, many approaches are based on either the use of autologous peptide pulsed dendritic cells as antigen-presenting cells or nonspecific expansion of T cells. Unfortunately, current approaches lack the ability to serve as reproducible and economically viable methods. Several groups are developing new artificial approaches to overcome problems associated with dendritic cells and the nonspecific expansion of T-cell clones in order to make adoptive immunotherapy more feasible and effective. Thus, by increasing the availability of adoptive immunotherapy, we will be able to better determine the efficacy of the approaches in the treatment of a variety of diseases. In this review, we focus on technological advances that will facilitate adoptive immunotherapy. Specifically, we summarize current strategies which are either based on artificial antigen-presenting cells or on T-cell receptor gene transfer.

Dacarbacine (DTIC) versus vaccination with autologous peptide-pulsed dendritic cells (DC) as first-line treatment of patients with metastatic melanoma: Results of a prospective-randomized phase III study

7508 Background: Progress in the treatment of patients with metastatic melanoma has been very limited over the last two decades. Vaccination with autologous DC was reported as being able to induce some clinical regression in early clinical phase I/II studies. Methods: Patients with advanced metastatic melanoma, HLA-A1, -A2, -A3, -A24 or -B44 positivity, no brain or bone metastases and any previous systemic chemotherapy were randomised to receive DTIC (850 mg/m2 d1, every 28 days with 4-week intervals (arm A) or intradermal vaccination with autologous peptide-pulsed dendritic cells in 2-week intervals for the first 5 vaccinations and every 4 weeks thereafter (arm B). DC were generated from peripheral blood after leukapheresis and culturing monocytes in-vitro in GM-CSF, IL-4 (d1-d5) and a maturing cytokine cocktail (IL-1β, IL-6, TNFα and PGE2) on day 6. Matured DC were pulsed subsequently with peptides derived from MAGE-1, MAGE-3, Melan-A, gp100, tyrosinase and influenza proteins as a control depending on the individual HLA-type (d6). Primary study endpoint was objective response to treatment (OR), secondary endpoints included safety, overall and progression-free survival. The planned accrual per arm was set at 119 patients under the basic assumption that the response rate would double from 15 % for treatment with DTIC to 30 % for DC-vaccination to reach significance at 0.05 with 80% power. Results: In the time period between 1/2001 and 6/2003 a total of 108 (intention-to-treat population, ITT; 55 DTIC, 53 DC) patients were randomized from 1 Swiss and 5 German centers with experience in DC generation and therapy. Accrual was halted prematurely by the data monitoring board because data indicated that the study goals could not be achieved. OR (CR+PR) were 5.5% in arm A and 3.8% in arm B, respectively (ITT). There was no statistically significant difference for response, toxicity, overall and progression-free survival between both study arms. Subset analysis indicated a trend for benefical effect of HLA-A2 and a disadvantage for HLA-B44. Conclusion: DC-based vaccination is as poorly active as standard DTIC chemotherapy in metastatic melanoma. Whether further selection of patients and improved DC generation protocols will lead to a statistical benefit remains topic of further studies. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Merix