Identification Of Novel Tumor-associated Antigens Research Articles

Identification of novel tumor-associated antigens and evaluation of a panel of autoantibodies in detecting oral cancer

BackgroundWe aimed to identify tumor-associated antigen (TAA) biomarkers through bioinformatic analysis and experimental verification, and to evaluate a panel of autoantibodies against tumor-associated antigens (TAAbs) for the detection of oral cancer (OC).MethodsGEO and TCGA databases were used to screen significantly up-regulated genes related to OC, and protein-protein interaction (PPI) analysis and Cystoscope software were used to identify key genes. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of autoantibodies in 173 OC patients and 173 normal controls, and binary logistic regression analysis was used to build a diagnostic model.ResultsUsing bioinformatics, we identified 10 key genes (AURKA, AURKB, CXCL8, CXCL10, COL1A1, FN1, FOXM1, MMP9, SPP1 and UBE2C) that were highly expressed in OC. Three autoantibodies (anti-AURKA, anti-CXCL10, anti-FOXM1) were proven to have diagnostic value for OC in the verification set and the validation set. The combined assessment of these three autoantibodies improved the diagnostic value for OC, with an area under the curve (AUC), sensitivity and specificity of 0.741(95%CI:0.690–0.793),58.4% and 80.4%, respectively. In addition, the combination of these three autoantibodies also had high diagnostic value for oral squamous cell carcinoma (OSCC), with an AUC, sensitivity and specificity of 0.731(95%CI:0.674,0.786), 53.8% and 82.1%, respectively.ConclusionsOur study revealed that AURKA, CXCL10 and FOXM1 may be potential biomarkers and the panel of three autoantibodies (anti-AURKA, anti-CXCL10 and anti-FOXM1) had good diagnostic value for OC.

Identification of Novel Tumor-Associated Antigens in Chronic Lymphocytic Leukemia (CLL) by Serological Proteome Analysis (SERPA)

AbstractAbstract 3878 Introduction:In this study, a serological proteome analysis (SERPA) was applied for the first time to identify novel tumor-associated antigens (Ags) capable of eliciting humoral immune responses in patients with chronic lymphocytic leukemia (CLL). SERPA has been demonstrated to be a valuable method to identify tumor associated Ags in several human solid and hematological malignancies. The identification and characterization of circulating antibodies (Abs) and corresponding Ags in CLL can provide useful information to understand cell transformation, predict clinical outcome, and develop immune-based interventions. Methods:SERPA was performed in 21 untreated patients. Proteins extracted from purified CLL cells were separated by 2-D electrophoresis (2-DE) to obtain proteomic maps which were blotted with corresponding sera by Western Blot to reveal Ab-based reactivity with autologous proteins. To verify the CLL specificity of Abs recognition, 7 out of 21 maps were also probed with sera collected from 7 healthy donors (HD). For identification, Ag spots in WB were aligned with proteins in 2-DE maps. The protein spots corresponding to the assigned Ags were excised from the gel, trypsin digested and analyzed by peptide mass fingerprint by MALDITOF Mass Spectrometry (MS) with the software MASCOT. T cells from 6 CLL patients and 3 HD were stimulated with autologous ENOA-pulsed and control dendritic cells (DC) and evaluated by IFNγ ELISPOT assay. Ags surface expression was analyzed by flow cytometry. Statistical correlations were performed using t-test, Mann-Withney rank sum test and χ2-test. Results:Sixteen out of 21 CLL sera (76%) were immunoreactive and produced a total number of 45 Ag spots, whereas HD sera produced only 3 spots (p<.03). Eleven out of 16 (69%) reactive CLL sera recognized from 2 to 6 different Ags in each individual patients. MS analyses led to the identification of 16 different Ags and many of them were recognized by sera from different patients. Forty-eight percent of CLL sera reacted against α-Enolase (ENOA), whereas none of HD sera was ENOA reactive. The IGHV mutational status was available in 19 CLL patients: 10 were mutated (M), while 9 were unmutated (UM). Interestingly, ENOA was recognized by sera from 7/10 M patients (70%), but only by sera from 3/9 UM patients (33%). Cytofluoroimetric analyses performed in 7 patients showed that ENOA was undetectable on viable CLL cells surface, whereas it was translocated on the membrane of apoptotic CLL cells.Statistical correlation analyses showed that immunoreactive CLL patients are characterized by an early stage of disease. Moreover, ENOA-reactive patients have a better preserved immune system because they have higher numbers of CD3+ (p=.02), CD3+/CD4+ (p=.03) and CD3+/CD8+ (p=.05) cells in the peripheral blood than ENOA-unreactive patients.We also investigated the possibility to induce ENOA-specific T-cell immune responses in 6 CLL patients. ENOA-pulsed DC induced IFNγ production in 4/6 patients (66%). The response was ENOA and CLL specific because: 1) it was not induced by unpulsed DC or DC pulsed with an irrelevant protein; 2) it was not induced when T cells from 3 HD were stimulated with autologous ENOA-pulsed DC. Interestingly, ENOA Abs were detectable by SERPA in 3 out of 4 (75%) patients with ENOA-induced T-cell responses, whereas they were undetectable in patients with unresponsive T cells. Correlations with the IGHV mutational status showed that all patients with ENOA-reactive T cells were M. Conclusions:These results indicate that ENOA is able to elicit specific humoral and cellular immune responses suggesting that this protein can be a promising biomarker and a potential target for immunotherapy in CLL. Disclosures:Massaia:Novartis Farma S.p.A: Honoraria, Research Funding.

Identification of Novel Tumor-Associated Antigens for Chronic Lymphocytic Leukemia (CLL) Based On HLA Ligandome Analysis – New Targets for Peptide Based Immunotherapy

AbstractAbstract 4119Recurrent disease, selection for chemo resistant clones and inhibition of immune effector functions are limitations of chemotherapeutic treatment of cancer including CLL. This is even more since graft-versus-leukemia effects and remissions after donor lymphocyte infusions have been correlated to long-term CLL free survival. Clonotype analysis of such cases suggested clonally expanded CD8+ T cells that recognize tumor associated antigens (TAAs) presented on HLA (human leukocyte antigen) as mediators of the observed effects, thus making CLL an attractive target for peptide vaccine based immunotherapy. For this goal we established the approach of direct isolation and identification of naturally processed and presented HLA ligands from tissues of interest by affinity chromatography and mass spectrometry. Comparative, semi-quantitative analysis of the HLA ligandomes of malignant and benign samples provided the rationale for selection of potential targets. 42 CLL patients (ages 48–85; median 70 years) of different HLA types and disease stages (Binet A, 24 patients; Binet B, 11 patients; Binet C, 7 patients) were enrolled in this study. Furthermore we collected blood samples from 50 healthy volunteers. HLA class I ligands were isolated from CLL cells as well as benign B cells from healthy donors and unsorted healthy PBMC using a standard affinity chromatography protocol implementing the pan-HLA class I specific antibody W6/32. Liquid chromatography coupled mass spectrometry (LC-MS/MS) based peptide analysis was performed on an LTQ Orbitrap hybrid mass spectrometer followed by annotation of fragment spectra using the MASCOT search algorithm against the human proteome comprised in the SwissProt database. Spectral counting based analysis provided semi-quantitative information regarding the abundance of HLA ligands and their source proteins in the respective ligandomes. In addition, HLA quantification experiments on the cell surface of CLL cells and autologous healthy B cells were performed using a flow cytometric indirect immunofluorescence assay. For this interim analysis we completely analyzed the HLA ligandomes of 7 CLL patients and 10 healthy controls. In total, we identified more than 15.000 different HLA ligands representing more than 7300 different proteins. This comprised more than 6,500 different ligands from CLL cells representing a total of 4,149 source proteins. Comparative analysis of representation in the HLA ligandomes of CLL cells, healthy B cells and healthy PBMC identified 1741 different source proteins as being exclusively expressed in CLL. Semi-quantitative evaluation revealed 138 of these proteins as being highly expressed on CLL (e.g. SET proto-oncogene, Pim-1 Oncogene, Mucin 1). Flow cytomerty based quantification of surface HLA expression revealed similar amounts of HLA class I (p=0.23, unpaired t-test) and II (p=0.33, unpaired t-test) molecules on CLL cells and autologous benign B lymphocytes, with a trend to higher HLA expression on CLL cells.Taken together, the presented strategy enabled the identification of a vast array of both known and novel TAAs and their corresponding naturally processed and presented HLA ligands in CLL. It pinpointed highly overrepresented TAAs for further analysis and immunogenicity testing. Expansion of the dataset after analysis of all enrolled patients will provide an unprecedented in-depth characterization of the HLA ligandome of CLL for future immunotherapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

Identification of Tumor-Associated Antigens from Medullary Breast Carcinoma by a Modified SEREX Approach

Medullary breast carcinoma (MBC) is a relatively rare malignancy with heavy lymphocytic infiltration that despite cytologically anaplastic features and high mitotic index has more favorable prognosis than other types of breast cancer. Lymphocytic infiltration of tumors reflects ongoing immune response against tumor antigens which could represent a great interest as potential targets for cancer immunotherapy. The search for MBC antigens by SEREX methodology has not been successful due to a very high titer of false positive clones, representing immunoglobulin genes. Here, we describe a novel approach for generating cDNA expression libraries from MBC tumor samples which are depleted of IgG cDNA clones and, therefore, are suitable for the identification of novel tumor-associated antigens (TAA) by SEREX approach. Modified methodology allowed us to isolate a panel of known and novel TAA which are currently under further investigation.

Gene Expression Profiling and Real-Time PCR Analyses Make It Possible To Identify Novel Potential Cancer-Testis Antigens in Multiple Myeloma.

The identification of novel tumor-associated antigens is critical for the development of immunotherapeutic strategies. Cancer-testis (CT) antigens represent attractive targets due to their restricted pattern of expression. More than 90 CT genes have been previously classified into four categories according to their expression profiles:i.testis-restricted (expression in testis and tumor samples only),ii.“tissue restricted” (mRNA detected in 2 or fewer non-gametogenic tissues),iii.“differentially expressed” (mRNA detected in three to six non-gametogenic tissues), andiv.“ubiquitously expressed”.Among those, we previously reported that 18 CT genes were expressed by primary myeloma cells (MMC) of more than 10% of patients with multiple myeloma (MM). This study aimed at finding novel putative CT genes expressed in MM using cDNA microarray analysis and real-time RT-PCR validation. Gene expression profiles of 5 testis samples, 64 MMC, 7 normal memory B cell (MB), 7 normal bone marrow plasma cell samples and 23 normal tissue samples available on a public database were obtained using Affymetrix U133AB microarrays. Out of 45000 probe sets of Affymetrix U133 AB chips, we selected 16982 probe sets which had a “Present” Affymetrix Call in MMC of at least 6/64 patients and in 3/5 testis samples. In order to select genes with a similar pattern of expression than the known CT genes, we developed 4 independent filters making it possible to keep a high number of known CT genes while decreasing the total number of probe sets. Firstly, 2514 of 16982 probe sets had a ratio of the mean signal in MMC with a Present call / mean signal in MB > 2.5. Secondly, 541 of these 2514 probe sets had a Present call in less than 7 of the 23 normal tissues. Thirdly, 333 of these 541 probe sets had a ratio of the mean signal in MMC with a Present call / mean signal in MMC with an Absent call > 2.5. Fourthly, we removed genes whose expression profiles were discordant with different probe sets or discordant with data of the literature. The final probe set list contains 88 probe sets which include 13 of 18 known CT genes reported in MM, thus resulting in a 190-fold enrichment. The expression in 13 normal tissues and in MM samples of 21 out of these 75 putative novel CT genes was investigated by real time RT-PCR. Seven genes were ubiquitously expressed or poorly expressed in MMC samples and further deleted. According to the previously defined CT gene categories, we found one novel “testis-restricted” (TEX14), 8 “tissue-restricted” and 5 “differentially expressed” CT genes. Immunogenicity of one gene product - IGSF11 - was already demonstrated in other cancers by identifying a T-cell epitope. Two genes - NLGN4X and FAM133A - are located in X chromosome and 2 genes - CTNNA2 and FAM133A - are expressed only in brain and testis. In conclusion, by analyzing gene expression patterns with Affymetrix microarrays, we found 75 novel putative CT antigen candidates expressed in MMC of 10 to 100% of patients. Real time RT-PCR validation made it possible to confirm the CT status of 14 genes out of the 21 tested. Further studies are warranted to determine their immunogenicity.

Gene Expression Profiling IdentifiesBAX-δas a Novel Tumor Antigen in Acute Lymphoblastic Leukemia

The identification of new tumor-associated antigens (TAA) is critical for the development of effective immunotherapeutic strategies, particularly in diseases like B-cell acute lymphoblastic leukemia (B-ALL), where few target epitopes are known. To accelerate the identification of novel TAA in B-ALL, we used a combination of expression profiling and reverse immunology. We compared gene expression profiles of primary B-ALL cells with their normal counterparts, B-cell precursors. Genes differentially expressed by B-ALL cells included many previously identified as TAA in other malignancies. Within this set of overexpressed genes, we focused on those that may be functionally important to the cancer cell. The apoptosis-related molecule, BAX, was highly correlated with the ALL class distinction. Therefore, we evaluated BAX and its isoforms as potential TAA. Peptides from the isoform BAX-delta bound with high affinity to HLA-A*0201 and HLA-DR1. CD8+ CTLs specific for BAX-delta epitopes or their heteroclitic peptides could be expanded from normal donors. BAX-delta-specific T cells lysed peptide-pulsed targets and BAX-delta-expressing leukemia cells in a MHC-restricted fashion. Moreover, primary B-ALL cells were recognized by BAX-delta-specific CTL, indicating that this antigen is naturally processed and presented by tumor cells. This study suggests that (a) BAX-delta may serve as a widely expressed TAA in B-ALL and (b) gene expression profiling can be a generalizable tool to identify immunologic targets for cancer immunotherapy.

HLA‐restricted specific tumor cytolysis by autologous T‐lymphocytes infiltrating metastatic bone malignant fibrous histiocytoma of lymph node

Malignant fibrous histiocytoma (MFH) of the bone is a high-grade sarcoma characterized histologically by the composition of fibroblasts and pleomorphic cells with a prominent storiform pattern. Despite institution of multi-modality treatments, the prognosis for patients with this tumor remains unsatisfactory. In the present study, towards the goal of developing active immunotherapy for those affected, we established four cell lines from a 53-year-old woman who suffered from MFH of the humerus with lymph node metastases. MFH2003 and MFH2003-B7.1 were the primary lesion-derived cell line and its B7.1-transfectant, respectively. B2003-EBV and TIL2003 were a B cell line established from peripheral blood lymphocytes and a T cell line established from tumor-invaded lymph nodes, respectively. MFH2003 cells could be maintained over a period of 1 year in vitro, and could be xenotransplanted into nude mice. The phenotype of cells analyzed by immunostaining was similar to the original tumor. TIL2003 cells were all CD8+ and specifically recognized MFH2003 cells and MFH2003-B7.1 cells, but not B2003-EBV cells. An anti-HLA-class I monoclonal antibody completely blocked the anti-MFH2003 response of TILs2003. These findings indicate the existence of an anti-MFH specific immune response in the microenvironment of metastatic lymph nodes. The present autologous cell lines provide the basis for identification of novel tumor-associated antigens and may be helpful in the establishment of immunotherapy for patients with MFH of the bone.

Insertion of interleukin-2 ( IL-2) and interleukin-12 ( IL-12) genes into vaccinia virus results in effective anti-tumor responses without toxicity

Identification of novel tumor-associated antigens (TAA) capable of eliciting T-cell responses has renewed interest in the development of anti-tumor vaccines. The insertion of genes encoding specific TAA into a vaccinia virus (rVV) is one approach to vaccination since large amounts of foreign DNA can be stably integrated into the poxvirus genome. Recent reports have documented an increased therapeutic effectiveness of poxvirus-based vaccines when additional treatment with cytokines, such as interleukin-2 (IL-2) or interleukin-12 (IL-12) were used, but the combination of these cytokines as adjuvants for a rVV encoding TAA have not been previously reported. The combination of IL-2 and IL-12 at single regimen systemic doses was toxic and sometimes fatal, manifesting largely as segmental epithelial apoptosis of the large bowel. To explore the local delivery of both cytokines to the site of vaccination, the genes encoding IL-2 and IL-12 were inserted into vaccinia virus along with a model tumor antigen gene. This construct contained five heterologous genes: LacZ (the model antigen), gpt (reporter gene), IL-2, and the two IL-12 subunit genes ( p35 and p40). Treatment with this recombinant virus resulted in a reduced number of pulmonary metastases, improved survival, and minimal toxicity in a murine tumor model. The use of vaccinia virus for the insertion of other heterologous gene combinations may provide a powerful and less toxic approach for novel vaccination strategies in the treatment and prevention of cancer.