Antigens For Immunotherapy Research Articles

A recombinant adenovirus vector encoding the light chain of human coagulation factor VII and IgG1 Fc fragment to targeting tissue factor for colorectal cancer immunotherapy in the mouse model

Over expression of tissue factor (TF) occurs in more than 50 % of colorectal cancer (CRC). Therefore, TF represents an attractive target antigen for immunotherapy in CRC. Here, we assessed the safety and efficacy of a recombinant adenovirus vector encoding the light chain of human coagulation factor VII (hfVII-LC) and human IgG1 Fc fragment (hIgG1-Fc), termed "benc vector," by targeting TF in the mouse model with colon cancer. Benc vector was administered intravenously or intratumorally in SCID mice with TF over-expressing HT-29 colon cancer. The safety and efficacy of benc vector were observed during animal experiments. Complete inhibition of tumor growth (5/5) was observed not only in the intravenously injection of benc vector group but also in the intratumorally of benc vector group. We also observed a precautionary effect on lung metastases of HT-29 cells by intratumoral injection of benc vector. In the control group of animals given empty control vector, all animals (5/5) developed lung tumors and exhibited a higher number of nodules after injection with HT-29 cells via the tail vein. In contrast, only three animals (3/5) in the treatment group receiving benc vector had any observable lung metastases and a lower number of nodules. No animals died and no bleeding was observed both in treatment groups and control groups. Moreover, only moderate liver damage was detected in mice receiving benc vector by intravenous injections. Benc vector encoding hfVII-LC and hIgG1-Fc can effectively inhibit tumor growth and metastases in SCID mice with TF over-expressing colon cancer and shows promise as an agent for CRC immunotherapy.

Fine-mapping naturally occurring NY-ESO-1 antibody epitopes in melanoma patients’ sera using short overlapping peptides and full-length recombinant protein

The tumor antigen NY-ESO-1 is one of the most antigenic cancer-testis antigens, first identified by serologic analysis of a recombinant cDNA expression library (SEREX). NY-ESO-1 is expressed in different types of cancers including melanoma. NY-ESO-1-specific spontaneous humoral and cellular immune responses are detected in a large proportion of patients with advanced NY-ESO-1-expressing cancers. Therefore NY-ESO-1 is a good candidate antigen for immunotherapy. Although cellular immune responses to NY-ESO-1 are well characterized, much less is known about the humoral immune responses. In this study, we finely mapped linear antibody epitopes using sera from melanoma patients and shorter overlapping peptide sets. We have shown that melanoma patients’ humoral immune systems responded to NY-ESO-1 differently in each individual with widely differing antibody specificity, intensity and antibody subtypes. This knowledge will help us further understand anti-tumor immunity and may also help us to monitor cancer progress and cancer vaccine efficacy in the future.

Understanding and overcoming the immunosuppressive effects of glioma induced immunosuppression

Meeting abstracts Utilizing tumors as a source of vaccination antigens in immunotherapy has demonstrated promising results with minimal toxicity. Unfortunately, researchers have failed to overcome the overpowering suppressive effects of the tumor microenvironment. Recently we demonstrated that the

Immunotherapy with intralesional Candida albicansantigen in resistant or recurrent warts: A study

Background:Warts are sometimes resistant or they tend to recur after every possible destructive therapy. Immunotherapy with skin-test antigens has been used as a viable therapeutic option in such recalcitrant cases.Aim:The aim of the study was to evaluate the response of resistant or recurrent warts to intralesional Candida albicans antigen immunotherapy.Materials and Methods:A total of 40 patients with resistant or recurrent warts who showed a positive test reaction to C. albicans antigen were given intralesional injections of purified C. albicans antigen solution in a single wart at 3-weekly intervals for a total of three doses. The patients were monitored for resolution of the injected wart as well as other untreated warts. The patients who responded positively were then followed up for any relapses over the next 6 months. Adverse events, if any, were also documented.Results:Of the 40 patients enrolled in the study, 34 completed the total treatment protocol of three injections and 6 months of follow-up. In these 34 patients, 19 (56%) showed a complete resolution of warts at all places on the body. In addition, two patients (6%) showed a partial or complete resolution of the treated wart, but there was no effect on the untreated warts. Thirteenpatients (38%) failed to show any response to the treatment regimen. In all patients showing resolution of all the warts, there were no relapses at any site over the next 6 months of follow-up. The most common adverse effect seen was pain during the intralesional injection.Conclusions:Intralesional Candida immunotherapy seems to be an effective treatment option in more than half of the patients who fail to show a positive response to destructive modes of treatment or in whom there are multiple recurrences.Limitations:The small sample size and lack of control group are the main limitations of the study.

Evaluation of RNA Amplification Methods to Improve DC Immunotherapy Antigen Presentation and Immune Response.

Dendritic cells (DCs) transfected with total amplified tumor cell RNA have the potential to induce broad antitumor immune responses. However, analytical methods required for quantitatively assessing the integrity, fidelity, and functionality of the amplified RNA are lacking. We have developed a series of assays including gel electrophoresis, northern blot, capping efficiency, and microarray analysis to determine integrity and fidelity and a model system to assess functionality after transfection into human DCs. We employed these tools to demonstrate that modifications to our previously reported total cellular RNA amplification process including the use of the Fast Start High Fidelity (FSHF) PCR enzyme, T7 Powerswitch primer, post-transcriptional capping and incorporation of a type 1 cap result in amplification of longer transcripts, greater translational competence, and a higher fidelity representation of the starting total RNA population. To study the properties of amplified RNA after transfection into human DCs, we measured protein expression levels of defined antigens coamplified with the starting total RNA populations and measured antigen-specific T cell expansion in autologous DC-T cell co-cultured in vitro. We conclude from these analyses that the improved RNA amplification process results in superior protein expression levels and a greater capacity of the transfected DCs to induce multifunctional antigen-specific memory T cells.

Genetic Variant as a Selection Marker for Anti–Prostate Stem Cell Antigen Immunotherapy of Bladder Cancer

A monoclonal antibody against prostate stem cell antigen (PSCA) has emerged as a novel cancer therapy currently being tested in clinical trials for prostate and pancreatic cancers, but this treatment is likely to be efficient only in patients with PSCA-expressing tumors. The present study demonstrates that a genetic variant (rs2294008) discovered by bladder cancer genome-wide association studies is a strong predictor of PSCA protein expression in bladder tumors, as measured by two-sided multivariable linear regression (P = 6.46×10−11; n = 278). The association pattern is similar in non-muscle-invasive tumors, stages Ta (P = 3.10×10−5; n = 173) and T1 (P = 2.64×10−5; n = 60), and muscle-invasive tumors, stages T2 (P =.01; n = 23) and T3/4 (P =.03; n = 22). The study suggests that anti-PSCA immunotherapy might be beneficial for bladder cancer patients with high tumor PSCA expression, which is statistically significantly associated with the presence of CT and TT genotypes of a common genetic variant, rs2294008. Future clinical studies will be needed to validate PSCA as a therapeutic target for bladder cancer.

Limited expression of cancer-testis antigens in renal cell carcinoma patients

The aim of this study was to evaluate the frequency of expression of the cancer-testis antigens (CTAs) NY-ESO-1, MAGE-A4 and SAGE, in renal cell carcinoma (RCC) patients compared to that in head and neck cancer (HNC) patients, which represent a positive control with a high incidence of CTA expression, to identify novel target antigens for immunotherapy. We prospectively examined frozen tissue samples collected from surgery or biopsy from 35 RCC and 40 HNC patients. Total RNA was extracted, and real-time reverse transcription-polymerase chain reaction (RT)-PCR was performed to determine the expression of MAGE-A4, NY-ESO-1 and SAGE. MAGE-A4 was not detected in any of the RCC samples, although a low incidence of NY-ESO-1 (5.7%; 2/35) and SAGE (2.9%; 1/35) expression was observed. No samples demonstrated co-expression of the three CTAs. By contrast, a comparatively high incidence of CTA expression was detected in squamous cell carcinoma (SCC) specimens of HNC patients. The actual incidence was 42.5% (17/40) for MAGE-A4, 20% (8/40) for NY-ESO-1 and 15% (6/40) for SAGE. The incidence of co-expression was 7.5% (3/40) for MAGE-A4 and NY-ESO-1, 7.5% (3/40) for MAGE-A4 and SAGE, 7.5% (3/40) for NY-ESO-1 and SAGE, and 2.5% (1/40) for the CTAs. The number of HNC samples positive for MAGE-A4 was significantly higher compared to that of RCC samples. The remaining two antigens, NY-ESO-1 and SAGE, were expressed at high levels in HNC compared to RCC samples. Limited frequency of CTA (NY-ESO-1, MAGE-A4 and SAGE) expression was demonstrated in RCC compared to HNC samples.

HLA Ligandome Analysis of Acute Myeloid Leukemia (AML) Revealed Novel Tumor-Associated Antigens for Immunotherapy.

AbstractAbstract 3012 Introduction:Eradication of minimal residual disease (MRD) by antigen-specific immunotherapy after conventional chemotherapy and also after allogeneic stem cell transplantation may improve overall survival in acute myeloid leukemia (AML). To achieve this goal there is a need for novel leukemia-associated HLA-presented peptides, which are able to induce a tumor-specific T cell response. Accordingly we here aimed to identify novel leukemia associated antigens employing, for the first time, the approach of direct elution and analysis of naturally processed and presented HLA ligands from the surface of primary AML cells. In particular we focused on peptides derived from AML specific mutated proteins, which are produced only by leukemia cells, only.Methods: HLA class I ligands were isolated from MACS-purified PBMCs of AML patients using immunoprecipitation. Liquid chromatography tandem mass spectrometry (LC-MS/MS) based peptide sequencing was performed to identify HLA presented peptides. The obtained data were mined for leukemia associated peptides by comparison of HLA ligandomes of AML cells with that of PMBCs and granulocytes from healthy donors, investigation of gene expression databases and literature research. All acquired peptide sequences were blasted against the COSMIC database (http://www.sanger.ac.uk/genetics/CGP/cosmic/), to identify sequences from AML specific mutated proteins. Identified mutations were confirmed by PCR. In addition, HLA quantification experiments on the cell surface of AML cells and autologous healthy monocytes and granulocytes were performed using a flow cytometric indirect immunofluorescence assay. Results:Quantitative preliminary results showed similar amounts of HLA class I molecules on AML cells as compared to autologous healthy monocytes and granulocytes lymphocytes (p=0.56, unpaired t-test). For HLA class II we could show a clear upregulation on AML cells compared to autologous healthy leukocytes, not reaching level of significance due to the small cohort examined till now (p= 0.12, unpaired t-test).We were able to identify a total of more than 10.000 peptides from 6 AML patients, including 2 NPM1 mutated AMLs, 1 FLT3-ITD mutated AML, 1 JAK2 mutated AML from osteomyelofibrosis and two AMLs from myelodysplastic syndrome without any cytogenetical aberrations. Using a new approach of spectral counting the obtained sequences could be compared semi-quantitatively to the same amount of peptides obtained from HLA matched healthy volunteers. Thus several new naturally processed and presented leukemia-associated peptides (LEUMAPs) were identified. This comprised, amongst others, ligands of p53, c-myc oncogene, PRAME, RHAMM and NPM1.The sequences of identified LEUMAPs were all verified by LC-MS/MS-based peptide sequencing of their synthetic counterparts. Testing for immunogenicity by ELISPOTs-assay and an in vitro T cell priming approach is presently ongoing. Conclusions:Our analysis revealed several new leukemia associated antigens, which were, for the first time, directly obtained from the HLA ligandomes of AML patients. Quantitative HLA analysis showed no loss or downregulation of HLA molecules on the surface of AML cells, thereby excluding a potential major obstacle for successful immunotherapy. Our results may pave the way for future peptide based immunotherapy approaches to eradicate MRD after obtaining a remission by presently available conventional therapies in AML patients. Disclosures:No relevant conflicts of interest to declare.

Validation of Stereotyped Immunoglobulin Heavy Chain CDR3 Sequences As Candidate Antigens for Immunotherapy of CLL

AbstractAbstract 1775The immunoglobulin gene repertoire in CLL is remarkably restricted with greater than 30% of cases carrying quasi-identical (stereotyped)heavy complementarity-determining region 3 (VH CDR3) sequences. Indeed, cases can be clustered into different subsets based on shared, subset-biased motifs within the clonotypic VH CDR3s, with, notably, only a handful of subsets accounting for almost 10% of all CLL. VH CDR3 stereotypes are more frequent in cases with unmutated IGHV genes (U-CLL) who are associated with adverse prognosis. In principle, VH CDR3 stereotypy might allow to exploit these IG motifs as candidate Tumor Associated Antigens (TAA) for targeted immunotherapy of CLL. The aim of our study was to validate as potential TAA subset-specific IG motifs from major CLL subsets, focusing especially on subsets #1 and #2 that are the largest overall and both associated with aggressive clinical course.We have so far identified, by in silico analysis, 1–3 long peptides (15-mer) encompassing the VH CDR3 protein regions of subsets #1, 2, 4, 6, 8, 10 with (i) high binding score to MHC class II molecules and (ii) also containing minimal HLA class I-specific epitopes (HLA-A2, -A3, -A24, DR1, DR7, DR13 that are most frequent in the Caucasian population).Blood lymphocytes from 18 CLL patients were collected and phenotyped by flow cytometry with appropriate antibodies to assess the expression of stimulatory, co-stimulatory and negative regulatory molecules on both T and B cells. In addition, HLA typing of CLL patients was performed to select patients expressing the aforementioned HLA molecules. Overall, 13/18 patients matched the defined HLA class I and/or class II molecules.Negatively purified T cells from 11 CLL patients expressing HLA-A2 and/or DR13 have been then stimulated in vitro with the synthesized peptides of the specific stereotype (subset #1 and 2) in the presence of culture medium containing 5% of human serum plus IL-2 (20 IU/ml) and IL-15 (10 ng/ml). These T lymphocytes were then weekly stimulated with autologous irradiated antigen presenting cells (APC; monocytes, B cells, etc.) pulsed with the peptides. Starting from the third week of culture, the specific recognition of CDR3-derived TAAs and of tumor cells (autologous CLL cells) by the T cell cultures has been assessed by in vitro functional assays (ELISPOT assay).We were able to isolate CDR3- (subsets #1 and #2) and tumor-specific T cells from 5/11 CLL patients. In addition, in 4 selected patients the Ag- and tumor specific T lymphocytes have been expanded in vitro by Rapid Expansion Protocol (REP), based on the stimulation of T cells with allogeneic irradiated PBMCs from healthy donors plus OKT3 and high doses of IL-2. Using this protocol we were able to obtain large numbers (2–10 ×109) of anti-CDR3 T cells in all 4 cases tested, thereby, in principle, achieving the potential to use this protocol for expanding sufficient cells for clinical applications. Interestingly, post-REP T cell cultures showed enrichment (85–90%) of CD3+CD8+ T cells and down-modulation of negative regulatory molecules, such as CTLA-4, as compared to pre-REP in vitro stimulated T cells. These cells could be expanded in vitro for up to 6 weeks without any decay in proliferation.Taken together, these results indicate that stereotyped VH CDR3 peptide sequences can represent candidate antigens to elicit T cell-mediated anti-CLL responses, especially in poor prognosis cases, where therapeutic innovation is more urgently needed. After validation of this protocol in a larger series, our results may provide the proof of principle for the design of new immunotherapy protocols for CLL, including both active vaccination and adoptive cell therapy. Disclosures:No relevant conflicts of interest to declare.

Optimizing dendritic cell vaccine for immunotherapy in multiple myeloma: tumour lysates are more potent tumour antigens than idiotype protein to promote anti-tumour immunity

Dendritic cells (DCs) are the most potent antigen-presenting cells and are the mediators of T cell immunity. Many investigators have explored the potential of using DCs as a vaccine for tumour-derived antigens in immunotherapy of B cell malignancies, and the results have been disappointing. To search for better tumour antigens to improve the efficacy of DC-based immunotherapy in myeloma, we evaluated and compared the efficacy of the vaccination of DCs pulsed with idiotype (Id) or tumour lysate in the 5TGM1 myeloma mouse model. Our results showed that Id- or tumour lysate-pulsed DC vaccines protected mice efficiently against developing myeloma, retarded tumour growth, induced tumour regression against established tumour and protected surviving mice from tumour rechallenge. The therapeutic responses were associated with an induction of strong humoral immune responses, including anti-Id or anti-lysate antibodies, and cellular immune responses including myeloma-specific CD8(+) cytotoxic T lymphocytes, CD4(+) type 1 T helper cells and memory T cells in mice receiving Id- or tumour lysate-pulsed DC vaccines. In addition, our studies showed that tumour lysate-pulsed DCs were more potent vaccines than the Id-pulsed DC vaccines to promote anti-tumour immunity in the model. This information will be important for improving the strategies of DC-based immunotherapy for patients with myeloma and other B cell tumours.

Is it time to abandon RHAMM/HMMR as a candidate antigen for immunotherapy of acute myeloid leukemia?

Amajor dilemma in the treatment of acute myeloid leukemia (AML) is the fact that current chemotherapies lead to complete remission in the majority of patients but overall survival at five years is less than 25%.[1][1] Treatment failure is ascribed to the presence of small numbers of tumor cells (

TCL1: a shared tumor-associated antigen for immunotherapy against B-cell lymphomas

AbstractImmunotherapy with therapeutic idiotype vaccines offers promise for treatment of B-cell malignancies. However, identification of novel immunogenic lymphoma-associated antigens that are universally expressed is necessary to overcome the barriers of patient-specific idiotype vaccines. Here, we determined whether T-cell leukemia/lymphoma 1 (TCL1) oncoprotein encoded by the TCL1 gene could be a target for immunotherapy of B-cell malignancies. We show that TCL1 mRNA and protein are selectively expressed in normal B cells but markedly hyperexpressed in multiple human B-cell lymphomas, including follicular lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, diffuse large B-cell lymphoma, and splenic marginal zone B-cell lymphoma. We demonstrated that TCL1-specific CD8+ T cells can be generated from HLA-A*0201 (HLA-A2)+ normal donors and identified TCL171-78 (LLPIMWQL) as the minimal epitope recognized by these T cells. More importantly, TCL171-78 peptide-specific T cells were present in the peripheral blood and tumor-infiltrating lymphocytes of lymphoma patients, could be expanded in vitro, and lysed autologous tumor cells but not normal B cells in an HLA-A2–restricted manner. Our results suggest that TCL1 is naturally processed and presented on the surface of lymphoma cells for recognition by cytotoxic T cells and can serve as a novel target for development of immunotherapeutic strategies against common B-cell lymphomas.

Targeting the MAGE A3 antigen in pancreatic cancer

Pancreatic cancer is the fourth-leading cause of death in the United States and one of the most aggressive known malignancies. New and innovative advances in treatment are desperately needed. One promising area of investigational treatment for pancreatic cancer involves the use of immunotherapy. The development of immunotherapy for pancreatic cancer has been hampered by difficulty in generating tumor-reactive lymphocytes from resected specimens and by a lack of appropriate target antigens expressed on tumor cells. Innovative strategies have been developed with the use of peripheral blood lymphocytes that are genetically engineered to express T-cell receptors targeting common tumor antigens, including cancer-testis antigens, such as the MAGE-A3 antigen. Cancer-testis antigens pose excellent targets for immunotherapy because they are expressed in cancer and in the testis, an immune-privileged site, but have limited expression in normal tissue. An additional advantage in targeting cancer-testis antigens for immunotherapy is that their expression can be selectively up-regulated in tumor cells via epigenetic regulation with chromatin remodeling agents. Current interest in targeting cancer-testis antigens in pancreatic cancer is well-founded because cancer-testis antigens have been shown to be expressed in pancreatic cancer as potential targets for therapy. In our studies, we validated the expression pattern of cancer-testis antigens in resected specimens of pancreatic cancer and tested the hypothesis that treatment of pancreatic cancer cells with chromatin remodeling agents would render them more sensitive to antigen-specific T lymphocytes. We focused predominately on the MAGE-A3 antigen because it is highly expressed in pancreatic cancer, and several immunotherapeutic strategies are in clinical trials targeting this specific antigen. The results of these studies have important translational implications and provide the rationale for combined treatment with chromatin remodeling agents and immunotherapeutic approaches for pancreatic cancer.

Successful therapeutic vaccination with integrase defective lentiviral vector expressing nononcogenic human papillomavirus E7 protein

Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, one of most common cancer among woman worldwide, and represents an important risk factor associated with other anogenital and oropharyngeal cancers in men and women. Here, we designed a therapeutic vaccine based on integrase defective lentiviral vector (IDLV) to deliver a mutated nononcogenic form of HPV16 E7 protein, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer, fused to calreticulin (CRT), a protein able to enhance major histocompatibility complex class I antigen presentation (IDLV-CRT/E7). Vaccination with IDLV-CRT/E7 induced a potent and persistent E7-specific T cell response up to 1 year after a single immunization. Importantly, a single immunization with IDLV-CRT/E7 was able to prevent growth of E7-expressing TC-1 tumor cells and to eradicate established tumors in mice. The strong therapeutic effect induced by the IDLV-based vaccine in this preclinical model suggests that this strategy may be further exploited as a safe and attractive anticancer immunotherapeutic vaccine in humans.

Coencapsulation of tumor lysate and CpG-ODN in PLGA-microspheres enables successful immunotherapy of prostate carcinoma in TRAMP mice

Biodegradable poly(lactide-co-glycolide) (PLGA) microspheres (MS) deliver antigens and toll like receptor (TLR) ligands to antigen presenting cells (APC) in vitro and in vivo. PLGA-MS-microencapsulated model antigens are efficiently presented on MHC class I and II molecules of dendritic cells and stimulate strong cytotoxic and T helper cell responses enabling the eradication of pre-existing model tumors. The application of tumor lysates as a source of antigen for immunotherapy has so far not been very successful also due to a lack of suitable delivery systems. In this study we used PLGA-MS with co-encapsulated tumor lysates and CpG oligodeoxynucleotides (CpG-ODN) as well as microencapsulated polyI:C in order to elicit anti-tumor responses. Immunization of mice with such mixtures of MS yielded substantial cytotoxic T cell (CTL) responses and interfered with tumor growth in TRAMP mice, a pre-clinical transgenic mouse model of prostate carcinoma, which has previously resisted dendritic cell‐based therapy. As an important step towards clinical application of PLGA-MS, we could show that γ-irradiation of PLGA-MS sterilized the MS, without reducing their efficacy in eliciting CTL and anti-tumor responses in subcutaneous tumor grafts. Since PLGA is approved for clinical application, sterilized PLGA-MS containing tumor lysates and TLR ligands hold promise as anti-tumor vaccines against prostate carcinoma in humans.

RHAMM/HMMR (CD168) is not an ideal target antigen for immunotherapy of acute myeloid leukemia

Criteria for good candidate antigens for immunotherapy of acute myeloid leukemia are high expression on leukemic stem cells in the majority of patients with acute myeloid leukemia and low or no expression in vital tissues. It was shown in vaccination trials that Receptor for Hyaluronic Acid Mediated Motility (RHAMM/HMMR) generates cellular immune responses in patients with acute myeloid leukemia and that these responses correlate with clinical benefit. It is not clear however whether this response actually targets the leukemic stem cell, especially since it was reported that RHAMM is expressed maximally during the G2/M phase of the cell cycle. In addition, tumor specificity of RHAMM expression remains relatively unexplored. Blood, leukapheresis and bone marrow samples were collected from both acute myeloid leukemia patients and healthy controls. RHAMM expression was assessed at protein and mRNA levels on various sorted populations, either fresh or after manipulation. High levels of RHAMM were expressed by CD34(+)CD38(+) and CD34(-) acute myeloid leukemia blasts. However, only baseline expression of RHAMM was measured in CD34(+)CD38(-) leukemic stem cells, and was not different from that in CD34(+)CD38(-) hematopoietic stem cells from healthy controls. RHAMM was significantly up-regulated in CD34(+) cells from healthy donors during in vitro expansion and during in vivo engraftment. Finally, we demonstrated an explicit increase in the expression level of RHAMM after in vitro activation of T cells. RHAMM does not fulfill the criteria of an ideal target antigen for immunotherapy of acute myeloid leukemia. RHAMM expression in leukemic stem cells does not differ significantly from the expression in hematopoietic stem cells from healthy controls. RHAMM expression in proliferating CD34+ cells of healthy donors and activated T cells further compromises RHAMM-specific T-cell-mediated immunotherapy.

Abstract 1585: Primary and metastatic uveal melanoma cell lines express MelanA that can be used as a target antigen for transfection of mature dendritic cells

Abstract The most common intra-ocular malignancy is uveal melanoma (UM). Metastatic disease occurs in approximately 50% of UM patients and presents most frequently in the liver. Novel therapies that prevent the development of metastases are needed and immunotherapy is a potential option. Because of its origin in the immune-privileged eye, UM may be particularly responsive to T cell-based immunotherapy. MelanA is frequently over-expressed by human cutaneous melanomas. MelanA expression has also been detected in UM primary and metastatic tumor-tissue sections and could serve as a target antigen for immunotherapy of UM. Here we report that primary and metastatic UM cell lines express MelanA and that this protein can be used as a target antigen for transfection of mature dendritic cells (mDC). MelanA expression was confirmed in primary and metastatic UM cell lines using intracellular flow cytometry and quantitative PCR (qPCR). In addition, sequencing of UM-derived MelanA demonstrated that MelanA in primary and metastatic UM cells has an identical sequence as compared to the published MelanA consensus sequence. As an initial step towards activating UM-specific T cells by UM-derived mRNA transfected DC we are generating MelanA mRNA transfected mDC. To facilitate both MHC class I and class II presentation of MelanA epitopes after mRNA electroporation into mDC, we included the endosomal targeting sequence DCLamp linked to the MelanA protein. Monocytes were isolated from leukapheresis products of healthy donors by density centrifugation and plastic adherence. Monocytes were differentiated to monocyte-derived DC by culturing with IL-4 and GM-CSF for 6 days to obtain immature DC (iDC). iDCs were matured with a cytokine mixture containing IL-4, GM-CSF, IL-1α, IL-6, TNFα, and PGE2 for 48 hours. The resulting mDC expressed cell surface maturation markers CD25, CD83, and CD86 as confirmed by flow cytometry. mDC were transfected with either MelanA-mRNA of MelanA-DCLamp-mRNA, using an established electroporation protocol. To assess transfection efficiency and monitor MelanA expression in transfected mDC, MelanA-mRNA and MelanA-DCLamp-mRNA expression was demonstrated by qPCR and MelanA-protein expression was shown by intracellular flow cytometry at different time points post-transfection using variable mRNA concentrations. The detected amount of MelanA-mRNA and protein was dependent on the amount of transfected mRNA and presented transiently. Subsequently, total RNA was isolated from MelanA+ primary and metastatic UM cell lines, then reversely transcribed into cDNA, amplified by PCR and in vitro transcribed to mRNA. As confirmed by qPCR, MelanA-mRNA was present in the pool of UM-derived mRNA and can be used for transfection of mDC. Collectively, these data demonstrate that mDC can be transfected with MelanA-mRNA and suggest MelanA could serve as a target for immunotherapy of UM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1585. doi:1538-7445.AM2012-1585

Exploiting the glioblastoma peptidome to discover novel tumour-associated antigens for immunotherapy

Peptides presented at the cell surface reflect the protein content of the cell; those on HLA class I molecules comprise the critical peptidome elements interacting with CD8 T lymphocytes. We hypothesize that peptidomes from ex vivo tumour samples encompass immunogenic tumour antigens. Here, we uncover >6000 HLA-bound peptides from HLA-A*02(+) glioblastoma, of which over 3000 were restricted by HLA-A*02. We prioritized in-depth investigation of 10 glioblastoma-associated antigens based on high expression in tumours, very low or absent expression in healthy tissues, implication in gliomagenesis and immunogenicity. Patients with glioblastoma showed no T cell tolerance to these peptides. Moreover, we demonstrated specific lysis of tumour cells by patients' CD8(+) T cells in vitro. In vivo, glioblastoma-specific CD8(+) T cells were present at the tumour site. Overall, our data show the physiological relevance of the peptidome approach and provide a critical advance for designing a rational glioblastoma immunotherapy. The peptides identified in our study are currently being tested as a multipeptide vaccine (IMA950) in patients with glioblastoma.

Fluctuation of symptomatic VAS score during immunotherapy on allergic rhinitis

To understand the fluctuation of symptomatic visual analogue scale(VAS)score and its reasons during the allergic rhinitis patients treated with single-mite antigen immunotherapy. Three hundred and seventy six patients with allergic rhinitis were treated with single-mite antigen immunotherapy and their symptoms were evaluated by VAS score and chart, the relative factors which could affect the symptom were followed up. During the allergic rhinitis patients treated with single-mite antigen immunotherapy, the symptomatic VAS score appeared fluctuation from the 8th to 34th time of therapy. The VAS score increased at the 8 th, 18 th, 22 th, 25 th, 29 th, and 34 th time. Afterwards, it generally decreased to a lowest level of the whole treatment process in the last. The comprehensive analysis implied that the factors resulting in VAS score fluctuation were patients expectation, emotion, socio-economic conditions, living environment, re-contact of allergen and children's unclear complain. Patients' expectation, emotion, socio-economic conditions, living environment, re-contact of allergen and children's unclear complain can result in symptomatic VAS score fluctuation. It prompts that health education be necessary before the treatment of allergic rhinitis with single-mite antigen immunotherapy.

Targeting SSX cancer–testis antigens for immunotherapy

SSX family proteins are differentially expressed in prostate tumor tissues, and could provide potential targets for cancer treatment using immune therapies, according to a study published in Cancer Research.