Designing a Long Acting Erythropoietin by Fusing Three Carboxyl-Terminal Peptides of Human Chorionic Gonadotropin β Subunit to the N-Terminal and C-Terminal Coding Sequence

A new analog of EPO was designed by fusing one and two CTPs to the N-terminal and C-terminal ends of EPO (EPO-(CTP)3), respectively. This analog was expressed and secreted efficiently in CHO cells. The in vitro test shows that the activity of EPO-(CTP)3 in TFI-1 cell proliferation assay is similar to that of EPO-WT and commercial rHEPO. However, in vivo studies indicated that treatment once a week with EPO-(CTP)3 (15 μg/kg) dramatically increased (~8 folds) haematocrit as it was compared to rHuEPO. Moreover, it was found that EPO-(CTP)3 is more effective than rHuEPO and Aranesp in increasing reticulocyte number in mice blood. The detected circulatory half-lives of rHuEPO, Aranesp, and EPO-(CTP)3 following IV injection of 20 IU were 4.4, 10.8, and 13.1 h, respectively. These data established the rational for using this chimera as a long-acting EPO analog in clinics. The therapeutic efficacy of EPO-CTP analog needs to be established in higher animals and in human clinical trials

Harnessing Soluble NK Cell Killer Receptors for the Generation of Novel Cancer Immune Therapy

The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The NCRs, which include three members; NKp46, NKp44 and NKp30, are critically involved in NK cytotoxicity against different targets, including a wide range of tumor cells derived from various origins. Even though the tumor ligands of the NCRs have not been identified yet, the selective manner by which these receptors target tumor cells may provide an excellent basis for the development of novel anti-tumor therapies. To test the potential use of the NCRs as anti-tumor agents, we generated soluble NCR-Ig fusion proteins in which the constant region of human IgG1 was fused to the extracellular portion of the receptor. We demonstrate, using two different human prostate cancer cell lines, that treatment with NKp30-Ig, dramatically inhibits tumor growth in vivo. Activated macrophages were shown to mediate an ADCC response against the NKp30-Ig coated prostate cell lines. Finally, the Ig fusion proteins were also demonstrated to discriminate between benign prostate hyperplasia and prostate cancer. This may provide a novel diagnostic modality in the difficult task of differentiating between these highly common pathological conditions

Expression of NCRs ligands on human prostate cancer.

<p>(a,b) NKp30-Ig and NKp46D2-Ig specifically bind to human prostate cell lines. PC3/<i>Luc</i> (a) and DU145 (b) cell lines were stained with NKp30-Ig, NKp46D2-Ig or control CD99-Ig, followed by PE-conjugated mouse anti-human IgG1 antibody. Grey histograms represent the background staining by the control CD99-Ig fusion protein and the black empty histograms represent the staining by either NKp30-Ig or NKp46D2-Ig, as indicated in the top of each histogram. This figure represents one experiment out of three performed. (c) Immunohistochemical staining of primary human prostate adenocarcinoma and benign prostate hyperplasia (BPH) by NKp30-Ig and NKp46D2-Ig. Cuts from formalin-fixed and paraffin-embedded human prostate adenocarcinoma (upper panel) and BPH (lower panel) were antigen-retrieved by microwave-citrate treatment. Slides were then stained with NKp30-Ig, negative control CD99-Ig or NKp46D2-Ig, followed by biotinylated-goat-anti-human-Fc and avidin-biotin HRP complex. Substrate for HRP was AEC (red color) and slides were counter-stained with Hematoxylin. Figure shows a representative staining at X400 magnification. Arrow in NKp30-Ig staining of adenocarcinoma (top left panel) points to a representative membrane staining. Staining intensity for top left and top right panels is considered as 2 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002150#s2" target="_blank">Methods</a>). (d) Expression of NKp30 and NKp46 ligands is abundant on malignant prostate tumors. Cuts from different patients suffering from benign (<i>n</i> = 8) or malignant (<i>n</i> = 9) prostate tumors were prepared and stained as above. Staining was performed in triplicates. Analysis of staining intensity (0-3) and percentage of stained tumor cells was performed by two pathologists. Positive staining was defined when staining intensity was above 1 and encompassed at least 50% of the cells, as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002150#s2" target="_blank">material and methods</a>’.</p