Melanoma is believed to be a highly immunogenic tumor and recent developments in immunotherapies are promising. IFN-y produced by immune cells has a crucial role in tumor immune surveillance; however, it has also been reported to be pro-tumorigenic. In the current study, we found that IFN-y enhances the expression of CD74, which interacts with its ligand, macrophage migration inhibitory factor (MIF), and thereby activates the PI3K/AKT pathway in melanoma, promoting tumor survival. IFN-y increased phosphorylation of Ala Ser473 and upregulated total cell surface expression of CD74 in human melanoma cell lines tested. CD74 was highly expressed in melanoma tissues. Moreover, the expression of CD74 on tumor cells correlated with plasma IFN-gamma levels in melanoma patient samples. In our analysis of melanoma cell lines, all produced MIF constitutively. Blockade of CD74 MIF interaction reduced Ala phosphorylation and expression of pro-tumorigenic molecules, including IL-6, IL-8, and BCL-2. Inhibition of CD74 MIF interaction significantly suppressed tumor growth in the presence of IFN-gamma in our xenograft mouse model. Thus, we conclude that IFN-y promotes melanoma cell survival by regulating CD74 MIF signaling, suggesting that targeting the CD74 MIF interaction under IFN-y-stimulatory conditions would be an effective therapeutic approach for melanoma.

BACKGROUND:Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is crucial for successful drug development. However, due to immune tolerance, making it difficult to generate antibodies using conventional approaches.METHODOLOGY/FINDINGS:Mixed four human gastric cancer (GC) cell lines were used as the immunogen in A/J mice; sixteen highly positive hybridoma colonies were selected via fluorescence-activated cell sorting-high throughput screening (FACS-HTS) using a total of 20,000 colonies in sixty-seven 96-well plates against live cells (mixed human GC cells versus human PBMC controls). MS17-57 and control commercial Alkaline Phosphatase (ALP) mAbs were used to confirm the target antigens (Ags), which were identified as ALPs expressed on the GC cell surface through a combination of western blot, immunoprecipitation and mass spectrometry (MS). MS identified the Ags recognized by MS17-57 to be two variants of a secreted ALP, PALP and IALP (Placental and intestinal ALP). These proteins belong to a hydrolase enzyme family responsible for removing phosphate groups from many types of molecules. Immunofluorescence staining using MS17-57 demonstrated higher staining of gastrointestinal (GI) cancer tissues compared to normal GI tissues (P<0.03), and confirmed binding of MS17-57 to be restricted to a functional epitope expressed on the cancer cell surface. Proliferation assays using the PALP/IALP-expressing GC cell lines demonstrated that MS17-57 inhibited cell growth by 32 ± 8%. Transwell cell migration assays documented that MS17-57 can inhibit PALP/IALP-expressing GI cancer cell migration by 25 ± 5%. MS17-57 mAb inhibited tumor growth in nude mice.CONCLUSIONS:Our findings indicate that PALP and IALP can be ectopically expressed on extracellular matrix of GI cancers, and that MS17-57 directed against PALP/IALP can inhibit GI cancer cells growth and migration in vitro and in vivo. This investigation provides an example of identification of cancer biomarkers representing promising therapeutic targets using mAb generated through a novel HTS technology.

e22169 Background: There remains considerable interest in generating monoclonal antibody (mAb) directed against specific tumor targets for antigen (Ag) discovery, diagnosis and therapy. In this investigation, mAbs were generated against potentially novel Ags on the cancer cell surface using a flow cytometry-high throughput screening (FACS-HTS) strategy. Methods: Mixed live cells from four human gastric cancer (GC) cell lines were used as the immunogen in A/J mice; Sixteen highly positive hybridoma colonies were selected via FACS-HTS using a total of 20,000 colonies in sixty-seven 96-well plates against live cells (mixed human GC cells versus normal human PBMC). The purified ms17-57 and control commercial ALP mAbs were used to confirm the target Ags, which were identified as alkaline phosphatases (ALPs) expressed on the GC cell surface through a combination of western blot, immunoprecipitation and mass spectrometry (MS). Results: MS identified the Ags recognized by ms17-57 to be two variants of a secreted ALP, PALP and IALP (Placental and Intestinal ALP). These proteins belong to a hydrolase enzyme family responsible for removing phosphate groups from many types of molecules (i.e. dephosphorylation). Immunohistochemical staining using ms17-57 demonstrated significantly higher staining of gastrointestinal (GI; gastric and colorectal) cancer tissues than normal (non-cancer) GI control tissues (P<0.01), and confirmed binding of ms17-57 to be restricted to a functional epitope expressed on the cancer cell surface. Proliferation assay using the PALP/IALP-expressing GC cell lines demonstrated that ms17-57 inhibited cell growth by 32±8%. Transwell cell migration assay documented that ms17-57 can inhibit PALP/IALP-expressing GI cancer cell migration. Conclusions: Our findings indicate that PALP and IALP can be ectopically expressed in the extracellular matrix of GI cancers, and that ms17-57 directed against PALP/IALP identified through HTS can inhibit GI cancer cell growth and migration. This investigation provides an example of identification of cancer biomarkers representing promising therapeutic targets using mAb generated through a novel HTS technology.

8503 Background: Prior reports have suggested a link between specific HLA class II alleles, including HLA-DRB1*1101, and melanoma recurrence. We therefore investigated the relationship between HLA class II alleles and clinical outcome in a large population of patients who presented with localized melanoma. Methods: HLA class II alleles were determined by typing of genomic DNA from patients who presented with localized melanoma (AJCC stage IA-IIC). Patients found to have occult regional disease by sentinel node biopsy were excluded. Log-rank and Cox analysis of outcome versus standard prognostic factors and common HLA class II alleles (=5% of the population) was performed. Results: 1,241 patients underwent HLA typing. The median primary tumor thickness was 1.1 mm, 16% were ulcerated, and the median follow-up was 56 months. Tumor thickness, ulceration, and Clark level were independent predictors of recurrence. The HLA class II allele HLA-DRB1*1101 (11% of the population) independently predicted a higher risk of recurrence [hazard ratio (HR) 2.04, P=0.004] while HLA-DRB1*0401 (20% of the population) predicted a lower risk of recurrence (HR 0.42, P=0.004). HLA- DRB1*1101 was a particularly strong predictor of recurrence in stage I patients (HR 2.73, P<0.0001). Among patients who recurred, HLA- DRB1*1101 was an independent predictor of local-regional vs. distant recurrence (HR 2.7, P=0.007), and was more common in those whose first recurrence was isolated to the regional nodal basin, compared to those whose first recurrence was elsewhere or in multiple locations (24% HLA-DRB1*1101-positive vs. 14%, P<0.009). Conclusions: These results confirm that HLA-DRB1*1101 is a genetic marker of increased risk of melanoma recurrence, while HLA-DRB1*0401 is a marker of decreased risk of recurrence. HLA-DRB1*1101 is a particularly strong marker among the group of patients predicted clinically to be at the lowest overall risk for recurrence, and independently predicts pattern of recurrence (regional nodal failure). This information is of value in the development of surveillance strategies for melanoma patients, as well as the selection, stratification and randomization of early-stage melanoma patients for clinical trials. No significant financial relationships to disclose.