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New gene expressed in prostate (NGEP) is a prostate-specific polytopic membrane protein found at high concentrations at cell:cell contact regions. To determine if NGEP is a useful target for antibody-based therapy of prostate cancer, we performed an immunohistochemical analysis of 126 human prostate carcinoma samples using polyclonal anti-NGEP sera and found that 91% of the cancers express NGEP protein. To elucidate the topology of NGEP and guide the development of monoclonal antibodies (mAb) reacting with the extracellular regions of NGEP, a hemagglutinin epitope tag was inserted at several positions within the NGEP sequence. The tagged proteins were expressed in 293T cells and locations of the tags were determined by immunofluorescence in intact or permeabilized cells. The results indicate that NGEP contains eight transmembrane domains with both the NH2 and COOH termini of NGEP located inside the cell. We produced mAb to three regions that are predicted to be intracellular based on the epitope tag data (amino acids 1-352, 441-501, and 868-933), and as predicted, the mAb only detected the protein in permeabilized cells. NGEP is a glycoprotein with predicted glycosylation sites at N809 and N824. When these residues were converted to glutamine, glycosylation was abolished, confirming that the residues are extracellular. Our findings on the expression and the orientation of the NGEP protein serve as an important framework for the development of mAb targeting the extracellular regions of NGEP that could be used for prostate cancer immunotherapy. [Cancer Res 2008;68(15):6306–12]

Supplementary Figure 1, Tables 1-4 from Topology of NGEP, a Prostate-Specific Cell:Cell Junction Protein Widely Expressed in Many Cancers of Different Grade Level

NGEP is a prostate-specific gene identified by analysis of expressed sequence tag databases. RNA analysis revealed two spliced forms of NGEP mRNA: a short form encoding a soluble protein (NGEP-S) and a long form encoding a polytopic membrane protein (NGEP-L). Transient expression of myc epitope–tagged NGEP-L showed that it was localized to the plasma membrane. We have now produced a specific antibody to the COOH terminus of NGEP-L and showed that it detects an ∼100-kDa protein in extracts of normal prostate and prostate cancers that contain high levels of NGEP mRNA. The antibody detects a protein that is highly expressed on the apical and the lateral surfaces of normal prostate and prostate cancer cells by immunohistochemistry. The antibody does not detect a protein in the prostate cancer cell line LNCaP, which has very low NGEP mRNA levels. To study NGEP function, two stable LNCaP cell lines were prepared by transfection with NGEP-L and shown to contain similar amounts of NGEP-L protein as human prostate. Confocal immunofluorescence showed that NGEP-L is present on the plasma membrane of the transfected LNCaP cells and is highly concentrated at cell:cell contact regions. Furthermore, as the cell density increased, the cells formed large aggregates. A specific RNA interference that lowered NGEP-L levels prevented formation of cell aggregates. Our results suggest that NGEP-L has a role in promoting cell contact–dependent interactions of LNCaP prostate cancer cells and also that NGEP is a promising immunotherapy target for prostate cancer. [Cancer Res 2007;67(4):1594–601]

Purpose: To establish monoclonal antibodies (mAb) against megakaryocyte potentiation factor (MPF) and detect MPF in the blood of patients with mesothelioma. Experimental Design: Mice were immunized with a purified recombinant human MPF-rabbit-Fc fusion protein and with MPF. Several hybridomas producing mAbs to MPF were established. A double-determinant (sandwich) ELISA was constructed using mAbs to two different epitopes and used to determine if MPF is present in the serum of patients with mesothelioma. Results: We established seven anti-MPF mAbs whose topographical epitopes were classified into three nonoverlapping groups. All the mAbs reacted with recombinant MPF protein by ELISA. One of the mAbs detected MPF and the mesothelin precursor protein containing MPF in cell lysates on Western blotting. A sandwich ELISA using mAbs to two different epitopes was constructed and used to measure the presence of MPF in the media of various mesothelin-expressing cancer cell lines and in human serum. The ELISA showed that MPF levels were elevated in 91% (51 of 56) of patients with mesothelioma compared with healthy controls. Furthermore, serum MPF fell to normal levels in two patients after surgery for their peritoneal mesothelioma. Conclusions: Using new mAbs to MPF, we showed that MPF is secreted by several mesothelioma cell lines and is frequently elevated in the blood of patients with mesothelioma. Measurement of MPF may be useful in following the response of mesothelioma to treatment.

Purpose: Mesothelin is a cell surface protein that is highly expressed in some malignant tumors, and is a promising target for immunotherapy. Recent data suggests that mesothelin is an adhesive protein and may have a role in the metastases of ovarian cancer. Although a few monoclonal antibodies (MAb) to mesothelin have been produced, they have limitations for the study of expression of native mesothelin because of their low affinity or reactivity only with denatured mesothelin protein. We have produced novel MAbs to mesothelin to help study mesothelin function and to develop improved diagnosis and immunotherapy of mesothelin-expressing tumors. Experimental Design: Mesothelin-deficient mice were immunized with plasmid cDNA encoding mesothelin, and boosted with a mesothelin-rabbit IgG Fc fusion protein prior to cell fusion. Hybridomas were screened by an ELISA using plates coated with mesothelin-Fc protein. Results: Seventeen hybridomas producing anti-mesothelin antibodies were established and shown to react with two epitopes on mesothelin. One group reacts with the same epitope as the low affinity antibody K1 that was originally used to identify mesothelin. The other is a new group that reacts with a new epitope. One antibody from each group was chosen for further study and shown to react strongly on ELISA, on immunohistochemistry, and by fluorescence-activated cell sorting on living cells. Conclusion: Our two newly established MAbs, MN and MB, have different and useful properties compared with current antibodies used for the detection of mesothelin by immunohistochemistry, fluorescence-activated cell sorting, ELISA, and Western blotting.

Purpose: The immunoglobulin superfamily receptor translocation associated 2 (IRTA2) gene encodes a cell surface receptor homologous to the family of Fc receptors. Because of the restricted expression of mRNA in B cell–lineage cells, IRTA2 is a new potential target for the immunotherapy of B cell malignancies. To study the expression of the IRTA2 gene product, we produced monoclonal antibodies (MAbs) specific to IRTA2. Experimental Design: A mouse used for cell fusion was DNA-immunized with an expression plasmid encoding the IRTA2 cDNA. The reactivity of MAbs secreted from the hybridomas were characterized with recombinant proteins of IRTA family members in an enzyme immunoassay and a fluorescence-activated cell sorter (FACS). Nineteen human lymphoma cell lines and blood cells from five patients with hairy cell leukemia (HCL) were analyzed with IRTA2 expression using FACS. Results: Three MAbs (F25, F56, and F119) were selected based on their specific reactivity with recombinant IRTA2 and lack of cross-reactivity with other IRTA family members. In a FACS analysis, MAbs F56 and F119 detected IRTA2 expression in six of seven B cell non–Hodgkin's lymphoma and one of six Burkitt's lymphoma cell lines. Reverse transcriptase-PCR experiments and Western blotting using MAb F25 confirmed the expression profile. We also found that HCL cells from five patients expressed IRTA2. Conclusions: Our results provide the first evidence that IRTA2 is expressed on the surface of human lymphoma cell lines and HCL cells. IRTA2 could be useful as a new target for immunotherapy.

Purpose: In this study, the optimization of antitumor therapy with tumor necrosis factor-α (TNF-α) was attempted. Experimental Design: Using the phage display technique, we created a lysine-deficient mutant TNF-α (mTNF-K90R). This mutant had higher affinities to both TNF receptors, despite reports that certain lysine residues play important roles in trimer formation and receptor binding. Results: The mTNF-K90R showed an in vivo therapeutic window that was 13-fold higher than that of the wild-type TNF-α (wTNF-α). This was due to the synergistic effect of its 6-fold stronger in vitro bioactivity and its 2-fold longer plasma half-life derived from its surface negative potential. The reason why the mTNF-K90R showed a higher bioactivity was understood by a molecular modeling analysis of the complex between the wTNF-α and TNF receptor-I. The mTNF-K90R, which was site-specifically mono-PEGylated at the NH2 terminus (sp-PEG-mTNF-K90R), had a higher in vitro bioactivity and considerably longer plasma half-life than the wTNF-α, whereas the randomly mono-PEGylated wTNF-α had 6% of the bioactivity of the wTNF-α. With regard to effectiveness and safety, the in vivo antitumor therapeutic window of the sp-PEG-mTNF-K90R was 60-fold wider than that of the wTNF-α. Conclusions: These results indicated that this functionalized TNF-α may be useful not only as an antitumor agent but also as a selective enhancer of vascular permeability in tumors for improving antitumor chemotherapy.

Background: Pancreatic Ductal Adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States and has a very poor survival rate due to late diagnosis. MUC13 is a recently identified high molecular weight glycoprotein that is upregulated in PDAC and its progression is allowed via alterations of multiple signaling pathways. MUC13 is aberrantly expressed in PDAC and generally correlates with increased expression of HER2, however, the underlying mechanism remains poorly understood. MUC13 consists of three EGF-like domains that may serve as a ligand for EGF receptors, such as HER2, and modulate EGFR signaling pathways. We sought to better characterize the interaction of MUC13 with HER2 in PDAC. Methods: MUC13 and HER2 interaction was studied using reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, Western blotting, co-capping assays in human PDAC cell lines and immunohistofluorescence techniques in human tissues. Tissue microarrays prepared from formalin-fixed, paraffin-embedded specimens of PDAC were assessed for expression of MUC13 and HER2 using our own laboratory generated anti-MUC13 mouse monoclonal antibody (MAb) through confocal immunofluorescence. The association of MUC13 and HER2 co-localization with nuclear chromatin organization was analyzed to study the stage or degree of aggressiveness of the pancreatic cancer using Dapi stained confocal images of tissues. Results: MUC13 co-localizes and interacts with HER2 in PDAC cell lines. The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at Tyrp1248 in PDAC cells, leading to stimulation of HER2 signaling cascade including, ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling, motility and invasion of PDAC cells - all collectively contributing to PDAC progression. The interaction between MUC13-HER2 binding resulting in their tumorigenic characteristics likely occurs at the 1st and 2nd but not the 3rd domains of MUC13 as the EGF 1 and 2 deletion mutant constructs of MUC13 failed to promote proliferation and invasion of cells. These phenotypic effects of MUC13-HER2 co-localization could be effectively compromised by depleting MUC13. MUC13-HER2 co-localization also held true in PDAC human tissues with a strong functional correlation that contributed to an increased degree of disorder and cancer aggressiveness. Conclusions: 1) MUC13 can be detected in formalin-fixed paraffin-embedded tissues using our anti-MUC13 MAb. 2) MUC13 co-localizes and activates HER2 and its downstream signaling cascade promoting PDAC progression in both cell lines and human tissue. 3) This process is reversed by depletion of MUC13. 4) This MUC13-HER2 interaction may potentially be manipulated for targeted therapeutics in patients harboring PDAC. Citation Format: Sheema Khan, Mara C. Ebeling, Mohammad Sikander, Murali M. Yallapu, Tomoko Ise, Satoshi Nagata, Stephen W. Behrman, Nadeem Zafar, Jim Y. Wan, Hemendra M. Ghimire, Peeyush Sahay, Prabhakar Pradhan, Meena Jaggi, Subhash C. Chauhan. MUC13 interaction with receptor tyrosine kinase HER2 drives pancreatic ductal adenocarcinoma progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4592.