Peptide-loaded Dendritic Cells Research Articles

Identification of CD8+ T-cell epitope from multiple myeloma-specific antigen AKAP4.

Multiple myeloma (MM) is a malignant plasma cell disorder affecting mainly the elderly population. Revolutionary progress in immunotherapy has been made recently, including monoclonal antibodies and chimeric antigen receptor T cell (CAR-T) therapies; however, the high relapse rate remains problematic. Therefore, combination therapies against different targets would be a reasonable strategy. In this study, we present a new X-chromosome encoded testis-cancer antigen (CTA) AKAP4 as a potential target for MM. AKAP4 is expressed in MM cell lines and MM primary malignant plasma cells. HLA-A*0201-restricted cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) transduced with an adenovirus vector encoding the full-length AKAP4 gene were demonstrated to lyse AKAP4+ myeloma cells. Seven of the 12 candidate epitopes predicated by the BIMAS and SYFPEITH algorithms were able to bind HLA-A*0201 in the T2 binding assay, of which only two peptides were able to induce CTL cytotoxicity in the co-culture of peptide-loaded human mature dendritic cells and the autologous peripheral blood mononuclear cells (PBMCs) from the same HLA-A*0201 donor. The AKAP4 630–638 VLMLIQKLL was identified as the strongest CTL epitope by the human IFN-γ ELISPOT assay. Finally, the VLMLIQKLL-specific CTLs can lyse the HLA-A*0201+AKAP4+ myeloma cell line U266 in vitro, and inhibit tumor growth in the mice bearing U266 tumors in vivo. These results suggest that the VLMLIQKLL epitope could be used to develop cancer vaccine or T-cell receptor transgenic T cells (TCR-T) to kill myeloma cells.

Natural high-avidity T-cell receptor efficiently mediates regression of cancer/testis antigen 83 positive common solid cancers

BackgroundT-cell receptor-engineered T cells (TCR-Ts) have achieved encouraging success in anticancer clinical trials. The antigenic targets, however, were primarily focused on human leukocyte antigen (HLA) A*02:01 restricted epitopes from a...

Baseline immunity predicts prognosis of pancreatic cancer patients treated with WT1 and/or MUC1 peptide-loaded dendritic cell vaccination and a standard chemotherapy

ABSTRACT The prognosis of patients with advanced pancreatic cancer is poor despite the recent introduction of immune checkpoint inhibitors. Therefore, the development of new therapeutic approaches is urgently required. In the present phase I/II study, we have evaluated the safety, the efficacy and the prognostic factors of Wilms’ tumor 1 (WT1) and/or mucin 1 (MUC1) peptide-loaded dendritic cell (DC) vaccination in combination with a chemotherapy employing gemcitabine plus nab-paclitaxel or a combination chemotherapy regimen consisting of oxaliplatin, irinotecan, fluorouracil and leucovorin (FOLFIRINOX) in patients with advanced or relapsed pancreatic ductal adenocarcinoma (PDAC). Forty-eight eligible patients were enrolled and received the vaccinations approximately every 2–4 weeks at least seven times. No severe adverse events related to the vaccinations were observed. Median progression free survival and overall survival were 8.1 months and 15.1 months, respectively. DC vaccinations augmented tumor specific immunity which might be related to clinical outcome. The multivariate analyses demonstrated that WT1 or MUC1-specific interferonɤ enzyme-linked immunospot number prior to DC vaccination was an independent prognostic factor related to overall survival. These results indicate that DC-based immunotherapy combined with a conventional chemotherapy is safe and has clinical benefits for patients in advanced stage of PDAC. The precise evaluation of the baseline antitumor specific immunity is critical to predict clinical outcome.

Combining chemotherapy and autologous peptide-pulsed dendritic cells provides survival benefit in stageIV melanoma patients.

SummaryBackground and ObjectivesWe examined retrospectively whether the combination of standard dacarbazine (DTIC) and/or fotemustine chemotherapy and autologous peptide‐loaded dendritic cell (DC) vaccination may improve survival of stage IV melanoma patients. Furthermore, a small cohort of long‐term survivors was studied in more detail.Patients and methodsBetween 1998 and 2008, 41 patients were vaccinated at least three times with DCs while receiving chemotherapy and compared to all other 168 patients in our database who only received chemotherapy (1993–2008).ResultsMedian life expectancy of patients receiving additional DC‐vaccination was 18 months, compared to eleven months for patients under standard chemotherapy alone. In contrast to patients with other haplotypes, the HLA‐A1/A1 subset of DC‐treated patients showed significantly lower median survival (12 vs. 25 months). Autoantibodies were frequently detected in serum of both vaccinated and non‐vaccinated patients, and there was no correlation between titers, loss or appearance of autoantibodies and survival. Additionally, phenotyping of DCs and PBMCs also did not reveal any conspicuous correlation with survival.ConclusionsCombining standard chemotherapy and DC vaccination appears superior to chemotherapy alone. The impact of HLA haplotypes on survival emphasizes the importance of a careful selection of patients with specific, well‐defined HLA haplotypes for future vaccination trials using peptide‐pulsed DCs, possibly combined with checkpoint inhibitors.

A signal peptide derived from Hsp60 induces protective cytotoxic T lymphocyte immunity against lymphoid malignancies independently of TAP and classical MHC-I

Hsp60sp, a signal peptide derived from the leader sequence of heat shock protein 60kDa (Hsp60), is a Qa-1/HLA-E-binding peptide. We previously showed that Hsp60sp-specific CD8+ T cells are involved in the immunoregulation of autoimmune diseases by controlling the response of self-reactive lymphocytes. Here, we report that Hsp60sp-specific CD8+ T cells killed malignant lymphocytes in vitro independently of transporter associated with antigen processing (TAP) and classical MHC-I expression. Induction of this cytotoxic T lymphocyte (CTL) response in vivo, either by adoptive transfer of in vitro-amplified CTLs or peptide-loaded dendritic cell immunization, resulted in effective control of lymphoid tumors, including TAP- or classical MHC-I-deficient cells. Hsp60sp-specific immune activation combined with programmed cell death protein 1 (PD-1) blocking synergistically restrained mouse lymphoma development. Importantly, Hsp60sp-specific CD8+ T cells did not negatively affect normal tissues and cells. Our data suggest that Hsp60sp-based immunotherapy is an inviting strategy to control lymphoid malignancies.

Intranodal Administration of Neoantigen Peptide-loaded Dendritic Cell Vaccine Elicits Epitope-specific T Cell Responses and Clinical Effects in a Patient with Chemorefractory Ovarian Cancer with Malignant Ascites

ABSTRACT Chemorefractory ovarian cancer has limited therapeutic options. Hence, new types of treatment including neoantigen-specific immunotherapy need to be investigated. Neoantigens represent promising targets for personalized cancer immunotherapy. We here describe the clinical and immunological effects of a neoantigen peptide-loaded DC-based immunotherapy in a patient with recurrent and chemoresistant ovarian cancer. A 71-year-old female patient with chemorefractory ovarian cancer and malignant ascites received intranodal vaccination of DCs loaded with four neoantigen peptides that were predicted by our immunogenomic pipeline. Following four rounds of vaccinations with this therapy, CA-125 levels were remarkably declined and tumor cells in the ascites were also decreased. Concordantly, the tumor-related symptoms such as respiratory discomfort improved without any adverse reactions. The reactivity against one HLA-A2402-restricted neoantigen peptide derived from a mutated PPM1 F protein was detected in lymphocytes from peripheral blood by IFN-γ ELISPOT assay. Furthermore, the neoantigen (PPM1 F mutant)-specific TCRs were detected in the tumor-infiltrating T lymphocytes post-vaccination. Our results showed that vaccination with intranodal injection of neoantigen peptide-loaded DCs may have clinical and immunological impacts on cancer treatment.

An effective peptide vaccine strategy circumventing heterogeneity of murine myeloid leukemia

Abstract Therapeutic cancer vaccines are an attractive approach for treating malignant tumors, and successful tumor eradication depends primarily on controlling tumor immunosuppression status as well as heterogeneity of tumor cells driven by epigenetic alterations. Here we describe an effective peptide vaccination approach capable of circumventing antigenic heterogeneity by targeting multiple shared tumor antigens simultaneously in murine myeloid leukemia. Peptide-loaded dendritic cell (DC) prime and noninfectious peptide booster heterologous immunizations were assessed for the immunogenicity of polo-like kinase-1 (PLK1)-derived peptides, and targeting multiple shared tumor antigens simultaneously with PD-L1 blockade was evaluated for antitumor effects against murine myeloid leukemia. A synthetic PLK1122 (DSDFVFVVL)-based heterologous vaccination generated large numbers of long-lasting antigen-specific CD8 T-cells eliciting therapeutic effects against various established tumors. The therapeutic efficacy of single antigen-targeting PLK1122-based vaccine with sufficient endurance of PD-L1 blockade toward C1498 leukemia relied on the heterogeneous clonal levels of MHC-I and PD-L1 expression. A novel multi-peptide-based vaccination targeting PLK1 and survivin simultaneously along with PD1 blockade led to complete tumor eradication and long-term survival in mice with clonally heterologous C1498 myeloid leukemia. Our findings suggest that PLK1 could be an attractive immunotherapeutic target antigen for cancer immunotherapy, and that similar strategies would be applicable for the optimization of cancer vaccines for the treatment of numerous viral diseases and malignant tumors.

An altered HLA-A0201-restricted MUC1 epitope that could induce more efficient anti-tumor effects against gastric cancer

MUC1 is a tumor-associated antigen (TAA) overexpressed in many tumor types, which makes it an attractive target for cancer immunotherapy. However, this marker is a non-mutated antigen without high immunogenicity. In this study, we designed several new altered peptides by replacing amino acids in their sequences, which were derived from a low-affinity MUC1 peptide, thus bypassing immune tolerance. Compared to the wild-type (WT) peptide, the altered MUC1 peptides (MUC11081-1089L2, MUC11156-1164L2, MUC11068-1076Y1) showed higher affinity to the HLA-A0201 molecule and stronger immunogenicity. Furthermore, these altered peptides resulted in the generation of more cytotoxic T lymphocytes (CTLs) that could cross-recognize gastric cancer cells expressing WT MUC1 peptides, in an HLA-A0201-restricted manner. In addition, M1.1 (MUC1950-958), a promising antitumor peptide that has been tested in multiple tumors, was not able to induce stronger antitumor responses. Collectively, our results demonstrated that altered peptides from MUC1, as potential HLA-A0201-restricted CTL epitopes, could serve as peptide vaccines or constitute components of peptide-loaded dendritic cell vaccines for gastric cancer treatment.

Preliminary report of a novel formulation of clinical-grade, fully matured, tumor-associated peptide-loaded dendritic cells for cancer immunotherapy

BackgroundWe developed and clinically test a new method to manufacture mature dendritic cells for autologous cell therapy of solid and hematological malignancies.MethodsPeripheral blood monocytes are matured into dendritic cells with GM-CSF/IL-4 mixture, and then activated through IL1b, TNFa, IFNa-2A, and Poly(I:C), then viably frozen until use. DC are injected intra-dermally for a total of 6 injections.ResultsHigh-grade mDC with high pre- and post- freezing yields could be generated. These DC spontaneously produce IL12p70 and they can be further stimulated via CD40. In a patient with advanced pancreatic cancer treated with our mDC formulation, we observed no toxicity but remarkable immune response and objective response in terms of tumor shrinking.ConclusionWe describe a new method to manufacture GMP-grade dendritic cells for autologous therapeutic cancer vaccines. We also show proof-of-principle efficacy in a patient with advanced pancreatic cancer.Trial registrationNCT02705703.

Generation of donor-derived Wilms tumor antigen 1–specific cytotoxic T lymphocytes with potent anti-leukemia activity for somatic cell therapy in children given haploidentical stem cell transplantation: a feasibility pre-clinical study

BackgroundThe Wilms tumor antigen 1 (WT1) is over-expressed in a vast majority of adult and childhood acute leukemia and myelodysplastic syndromes, being lowly or transiently expressed in normal tissues and hematopoietic stem cells (HSCs). A number of HLA-restricted WT1 epitopes are immunogenic, allowing the in vitro induction of WT1-specific cytotoxic T lymphocytes (CTLs) from patients and healthy donors. AimThe aim of the study was to investigate the feasibility of producing WT1-specific CTLs suitable for somatic cell therapy to prevent or treat relapse in children with acute myeloid or lymphoblastic leukemia given haploidentical HSC transplantation (haplo-HSCT). MethodsFor WT1-specific CTL production, donor-derived either peripheral blood mononuclear cells (PBMCs) or CD8+ lymphocytes were stimulated with WT1 peptide-loaded donor dendritic cells in the presence of interleukin (IL)-7 and IL-12. Effector cells were re-stimulated once with irradiated donor PBMCs pulsed with WT1-peptides, and then expanded in an antigen-independent way. ResultsWT1-specific CTLs, displaying high-level cytotoxicity against patients’ leukemia blasts and negligible activity against patients’ non-malignant cells, were obtained from both PBMCs and CD8+ lymphocytes. WT1-specific CTLs obtained from PBMCs showed a better expansion capacity and better anti-leukemia activity than those obtained from CD8+ lymphocytes, even though the difference was not statistically significant. In CTLs derived from PBMCs, both CD8+ and CD4+ subpopulations displayed strong anti-leukemia cytotoxic activity. DiscussionResults of this pre-clinical study pave the way to a somatic cell therapy approach aimed at preventing or treating relapse in children given haplo-HSCT for WT1-positive leukemia.

Abstract 3180: Targeting peptide-loaded-DC CIK cells induce a specific antitumor response

Abstract Currently, immunotherapy has become an effective alternative therapeutic approach for cancers. Cytokine-induced killer (CIK) cells have a higher proliferation rate, increased efficacy with few side-effects, and non- MHC-restricted killing after co-culturing with dendritic cells (DCs). However, the specificity and efficacy of DC-CIK treatment still need to be improved. In our study, the antitumor effects of CIK cells co-culturing with DCs pulsed with non-cell derived targeting peptides, which could specifically bind to certain tumor cells, were evaluated. Our results indicated that targeting peptide-loaded DCs could enhance the differentiation and cytotoxicity of CIK cells. CIK cells, which were treated with specific targeting peptide-loaded DCs, could effectively and specifically kill tumor cells in vitro and in vivo, as long as tumor cells were pre-treated with these targeting peptides. Moreover, the CIK cytotoxic effect of this type acquired a certain properties of memory and resistance to the tolerance. In conclusion, targeting peptides could guide DC-CIK more effectively and specifically kill tumor cells and this non-cell derived targeting peptide-loaded-DC CIK may work as a novel means for cancer therapy. Citation Format: Yimin Zhu, Xueyuan Cui, Cuijuan Liu. Targeting peptide-loaded-DC CIK cells induce a specific antitumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3180.

In T-cell lines dually sensitized with antigens from EBV and CMV or from EBV and WT1 the T-cells specific for CMV or WT1 are often restricted by hla alleles that differ from those restricting T-cells specific for EBV

Background & Aim Virus-specific T-cells generated from transplant or 3rd party donors can induce durable remissions of post-transplant severe infections or EBV lymphomas. T-cells simultaneously sensitized with antigens from multiple viruses have also shown promise. We hypothesized that in any individual donor, immunogenic peptides from different viruses might elicit T-cell responses restricted by different HLA alleles. In HLA non-identical patients, the T-cells, specific for one virus or tumor antigen will be effective only if they are restricted by an HLA allele shared by the patient. Methods, Results & Conclusion We analyzed HLA restrictions (HLA-R) of 42 CTL lines generated by dual sensitization with either autologous EBV-transformed B-cells (BLCL) loaded with a pool of overlapping 15-mer peptides spanning the sequence of CMVpp65 (BLCL/CMV CTL) (n=20) or autologous BLCL loaded with a pool of 15-mers spanning the oncofetal protein WT-1 (BLCL/WT1 CTL) (n=22). EBV CTL lines were generated by sensitization with BLCL alone. The HLA-R of the CMVpp65-specific T-cells(CMV-T), WT1 specific T cells (WT1-T) and EBV specific T cells (EBV-T) in each T cell product were assessed by their cytotoxic activity against a panel of Cr51 labeled peptide-loaded dendritic cells and BLCL sharing a single HLA allele with the T cell donor. In 13/20 BLCL/CMV CTL(65%) and 17/22 BLCL/WT1 CTL(77%) the CMV-T or WT1-T were restricted by single HLA alleles. In 10/20(50%) BLCL/CMV CTL, CMV-T and EBV-T in the same line were restricted by the same HLA allele. However, in the other 10(50%) the CMV-T were restricted by an HLA allele different from that of the EBV-T. Similarly, in 13/22(59%) BLCL/WT1 CTL, WT1-T and EBV-T in the same line were restricted by different HLA alleles. In 2/4 BLCL/CMV CTL and 2/5 BLCL/WT1 CTL, in which the HLA-R of CMV-T or WT1-T differed from that of EBV-T in the same culture, the HLA allele differentially presenting CMV or WT1 antigen but not an EBV antigen was a prominent restricting allele of EBV-T in the EBV CTL of the same origin. This suggests that while immunodominant EBV and CMV epitopes presented by certain prevalent HLA alleles such as HLA B0702 or HLA A0201 often induce responses to both viruses, in certain dual-sensitized cultures, immunogenic epitopes from different antigens may compete for presentation by a specific HLA allele or differ in their potential to induce a dominant T-cell response.

Isolation and enrichment of mouse insulin-specific CD4+ T regulatory cells

Polyclonal T regulatory cells (Treg - CD4+CD25+CD127lowFoxp3+) are used in several protocols for the treatment of type 1 diabetes (T1D), multiple sclerosis and graft-versus host disease in clinical trials. However, general opinion is that autoantigen-specific Treg could be more efficient in autoimmunity suppression due to their direct effect on pathogenic autoantigen-specific effector T cells. This study describes isolation and expansion of insulin-specific Treg in vitro. Insulin-specific Treg are uniformly distributed in lymphoid tissues however their number is extremely low. To enrich the proportion of insulin-specific Treg, pure CD4+ cells were co-cultured with insulin B chain peptide-loaded dendritic cells, isolated from mice that develop T1D spontaneously – NOD mice. Insulin-specific CD4+ cell expansion peaked after 48 h of incubation and was in favour of Treg. These cells were then sorted using insulin peptide-loaded MHC class II tetramers and cultured in vitro for 48 h in the presence of TCR stimulators, TGF-β and IL-2. The proportion of gained insulin-specific cells with T regulatory phenotype (CD4+CD25highCD127lowGITR+FoxP3+) was in average between 93% and 97%. These cells have shown potent in vitro suppressive effect on T effector cells, produced IL-10 and TGF-β and expressed PD-1 and CD39. Further proliferation of these insulin-specific Treg required the presence of dendritic cells, anti-CD3 antibody and IL-2. This study provides new, reproducible experimental design for the enrichment and expansion of insulin-specific Treg that can be used for the cell-based therapy of autoimmunity.

Cytokine-induced killer cells co-cultured with non-cell derived targeting peptide-loaded dendritic cells induce a specific antitumor response

ABSTRACTCancer is a severe lethal disease. Currently, immunotherapy has become an effective alternative therapeutic approach for cancers. Cytokine-induced killer (CIK) cells have a higher proliferation rate, increased efficacy with few side-effects, and non-MHC-restricted killing after co-culturing with dendritic cells (DCs). Therefore, it has been widely studied and applied in the treatment of cancers. In our study, we explored the antitumor effects of CIK cells co-culturing with DCs pulsed with non-cell derived targeting peptides, which could specifically bind to certain tumor cells. Our results indicated that targeting peptide-loaded DCs could enhance the differentiation and cytotoxicity of CIK cells. Moreover, CIK cells, which were treated with specific targeting peptide-loaded DCs, could effectively and specifically kill tumor cells in vitro and in vivo, as long as tumor cells were pre-coated with the specific binding peptides. In conclusion, targeting peptides could guide DC-CIK to effectively and specifically kill tumor cells which were pre-coated with these targeting peptides and non-cell derived targeting peptide-loaded-DC-CIK may work as a novel means for cancer therapy.

Towards a dendritic cell-based vaccine for the treatment of multiple sclerosis (MS): interim safety data of the first dose cohort of the MS-tolDC phase I clinical trial

Towards a dendritic cell-based vaccine for the treatment of multiple sclerosis (MS): interim safety data of the first dose cohort of the MS-tolDC phase I clinical trial

Detection of ABCB5 tumour antigen-specific CD8+ T cells in melanoma patients and implications for immunotherapy.

ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour-initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti-ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5-reactive T cells are present in human melanoma patients and tested the applicability of ABCB5-derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)-γ and tumour necrosis factor (TNF)-α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co-cultured with ABCB5 antigen-loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5-reactive CD8+ T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART-1)-reactive CD8+ T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART-1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide-loaded autologous DC. As ABCB5-reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5-specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma.

Early-Life Gut Dysbiosis: A Driver of Later-Life Fibrosis?

Using a novel mouse model of scleroderma induced by immunization with topoisomerase-I peptide-loaded dendritic cells, Mehta etal. found that early-life antibiotic exposure resulted in increased later-life fibrosis in the skin and lungs. These observations advance the novel concept that gut microbiome alterations caused by early-life exposures may contribute to scleroderma pathogenesis, and warrant in-depth characterization and validation in complementary disease models.

Early-Life Antibiotic Exposure Causes Intestinal Dysbiosis and Exacerbates Skin and Lung Pathology in Experimental Systemic Sclerosis

Patients with systemic sclerosis (SSc) display altered intestinal microbiota. However, the influence of intestinal dysbiosis on the development of experimental SSc remains unknown. Topoisomerase I peptide-loaded dendritic cell immunization induces SSc-like disease, with progressive skin and lung fibrosis. Breeders were given streptomycin and pups continued to receive antibiotic (ATB) until endpoint (lifelongATB). Alternately, ATB was withdrawn (earlyATB) or initiated (adultATB) during adulthood. Topoisomerase I peptide-loaded dendritic cell (no ATB) immunization induced pronounced skin fibrosis, with increased matrix (Col1a1), profibrotic (Il13, Tweakr), and vascular function (Serpine1) gene expression. Remarkably, earlyATB exposure was sufficient to augment skin Col5a1 and Il13 expression, and inflammatory cell infiltration, which included IL-13+ cells, mononuclear phagocytes, and mast cells. Moreover, skin pathology exacerbation was also observed in lifelongATB and adultATB groups. Oral streptomycin administration induced intestinal dysbiosis, with exposure limited to early life (earlyATB) being sufficient to cause long-term modification of the microbiota and a shift toward increased Bacteroidetes/Firmicutes ratio. Finally, aggravated lung fibrosis and dysregulated pulmonary T-cell responses were observed in earlyATB and lifelongATB but not adultATB-exposed mice. Collectively, intestinal microbiota manipulation with streptomycin exacerbated pathology in two distinct sites, skin and lungs, with early life being a critical window to affect the course of SSc-like disease.

Artificial human antigen-presenting cells are superior to dendritic cells at inducing cytotoxic T-cell responses.

Peptide recognition through the MHC class I molecule by cytotoxic T lymphocytes (CTLs) leads to the killing of cancer cells. A potential challenge for T-cell immunotherapy is that dendritic cells (DCs) are exposed to the MHC class I-peptide complex for an insufficient amount of time. To improve tumour antigen presentation to T cells and thereby initiate a more effective T-cell response, we generated artificial antigen-presenting cells (aAPCs) by incubating human immature DCs (imDCs) with poly(lactic-co-glycolic) acid nanoparticles (PLGA-NPs) encapsulating tumour antigenic peptides, followed by maturation with lipopolysaccharide. Tumour antigen-specific CTLs were then induced using either peptide-loaded mature DCs (mDCs) or aAPCs, and their activities were analysed using both ELISpot and cytotoxicity assays. We found that the aAPCs induced significantly stronger tumour antigen-specific CTL responses than the controls, which included both mDCs and aAPCs loaded with empty nanoparticles. Moreover, frozen CTLs that were generated by exposure to aAPCs retained the capability to eradicate HLA-A2-positive tumour antigen-bearing cancer cells. These results indicated that aAPCs are superior to DCs when inducing the CTL response because the former are capable of continuously presenting tumour antigens to T cells in a sustained manner. The development of aAPCs with PLGA-NPs encapsulating tumour antigenic peptides is a promising approach for the generation of effective CTL responses in vitro and warrants further assessments in clinical trials.

Abstract 692: Pappalysin-1 is a suitable target for T cell receptor transgenic T cells to kill Ewing sarcoma in vivo and in vitro

Abstract Pregnancy-associated plasma protein-A (PAPPA), also known as pappalysin, is a member of the insulin like growth factor (IGF) family. PAPPA acts as a protease, cleaving IGF inhibitors, i.e. IGF binding proteins (IGFBPs), thereby releasing IGFs from IGFBPs. The insulin/IGF-axis is involved in cancer in general and in Ewing sarcoma (ES) in particular. ES is a highly malignant bone tumor characterized by early metastatic spread. PAPPA stimulates normal bone growth, and is also associated with various cancers. In particular, PAPPA is overexpressed and required for proliferation in ES. We isolated HLA-A*02:01+/peptide restricted T cells from A*02:01- healthy donors directed against PAPPA, generated by priming with A*02:01+ PAPPA peptide-loaded dendritic cells. After T cell receptor (TCR) identification, retrovirally TCR transduced CD8+ T cells were assessed for their in vitro specificity and in vivo efficacy in human ES bearing Rag2-/-γc-/- mice. Engraftment in mice and tumor infiltration of TCR transgenic T cells in the mice was evaluated. The TCR transgenic T cell clone PAPPA-2G6 demonstrated specific reactivity towards HLA-A*02:01+/PAPPA+ ES cell lines. We furthermore detected circulating TCR transgenic T cells in the blood in Rag2-/-γc-/- mice and in vivo engraftment of the in bone marrow. Tumor growth in mice with xenografted ES was significantly reduced after treatment with PAPPA-2G6 TCR transgenic T cells compared to controls, and tumors from treated mice revealed tumor infiltrating PAPPA-2G6 TCR transgenic T cells. In summary, we demonstrate that PAPPA is a promising target for TCR based immunotherapy of ES. We demonstrate that TCR transgenic T cells recognize this target, home to the tumor, and causes tumor regression in a preclinical mouse model. Citation Format: Uwe Thiel, Andreas Kirschner, Melanie Thiede, Thomas GP Grünewald, Rebeca Alba Rubio, Günther Richter, Thomas Kirchner, Dirk Busch, Poul Sorensen, Stefan Burdach. Pappalysin-1 is a suitable target for T cell receptor transgenic T cells to kill Ewing sarcoma in vivo and in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 692. doi:10.1158/1538-7445.AM2017-692