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// Dadi Zeng 1, * , Qiang Sun 1, * , Ang Chen 1 , Jiangfeng Fan 1 , Xiaopeng Yang 1 , Lei Xu 1 , Peng Du 1 , Weiyi Qiu 1 , Weicai Zhang 1 , Shuang Wang 1 , Zhiwei Sun 1 1 Beijing Institute of Biotechnology, Fengtai District, Beijing 100071, China * These two authors contributed equally to this work Correspondence to: Zhiwei Sun, email: szwyhhh@aliyun.com Shuang Wang, email: 18910810680@163.com Weicai Zhang, email: drzhangweicai@163.com Keywords: CD47, anti-CD47 antibody, phagocytosis, cancer therapy, cell-in-cell Received: March 17, 2016 Accepted: October 17, 2016 Published: November 15, 2016 ABSTRACT CD47/SIRPα interaction serves as an immune checkpoint for macrophage-mediated phagocytosis. Mouse anti-CD47 blocking antibodies had demonstrated potent efficacy in the treatment of both leukemic and solid tumors in preclinical experimentations, and therefore had moved forward rapidly into clinical trials. However, a fully human blocking antibody, which meets clinical purpose better, has not been reported for CD47 up to date. In this study, we reported the isolation of a fully human anti-CD47 blocking antibody, ZF1, from a phage display library. ZF1 displayed high specificity and affinity for CD47 protein, which were comparable to those for humanized anti-CD47 blocking antibody B6H12. Importantly, ZF1 treatment could induce robust, or even stronger than B6H12, phagocytosis of leukemic cancer cells by macrophage in vitro , and protect BALB/c nude mice from cancer killing by engrafted leukemic cells (CCRF and U937) to a similar extent as B6H12 did. Thus, these data provide primary early pre-clinical support for the development of ZF1 as a fully human blocking antibody to treat human leukemia by targeting CD47 molecule.

To clarify the status and distribution of Fas and Fas-Ligand (FasL) in yak's placentomes, immunohistochemistry (IHC) was carried out to analyze the expression and location of Fas and FasL in paraffin embedded sections. The area of positive stained sites was selected and measured using image analyses software (Image Pro-Plus 6.0). So the positive index (PI) was calculated to estimate the intensity of protein expression according to the percentage of positive area in corresponding compartment of the placentomes. In cotyledonary villi, Fas mainly presented on the villous trophoblast cells in early pregnancy. The positive index reached a maximum of 20.7±8.8 at the third month of pregnancy. Then Fas was declined rapidly along with the progress of gestation and the value was 2.8±1.3 after the 7th month of pregnancy. However, in caruncular crypts, Fas was mainly localized to isolated cells or clustered cells of the uterine stroma underlying the caruncular epithelium. The intensity was lower and the positive index was changed between 4.7±0.9 and 8.5±1.6 throughout gestation. For FasL, it gave a distinct immunostained distribution. In cotyledonary villi, FasL was localized dominantly and strongly in the cytoplasm of binuclear, mononuclear and trinuclear trophoblast giant cells (TGC). The positive index of FasL maintained a moderate level all through the gestation. In caruncular crypts, the expression of FasL was weak and the positive index was declined. Only in the first two months, maternal uterine epithelial cells intensely expressed FasL and the index reached to the maximum of 19.8±5.2. The result of subcellular localization of Fas ligand using immunoelectron microscopy technology indicated that FasL was subcellular located in some intracellular vesicles of TGC. This means the vesicles of trophoblast giant cells itself can express FasL. By the TUNEL method, apoptosis was detected in yak placentomes. The amount of apoptotic cells was rare. The fetal chorionic trophoblast cells and caruncular crypt epithelium cells demonstrated higher percentage of apoptosis in middle pregnancy, which suggested that apoptosis plays an important role in placental cellular regeneration. In addition, the apoptosis of maternal caruncular stromal cells provides a local mechanism for maternal immunotolerance to the fetus and this mechanism was mediated by Fas-FasL pathway.

Yak milk is superior to common cow milk in nutrients including protein, fat and calories. However, the milk yield of the yak is very much lower compared with other dairy bovines. To understand the molecular mechanisms of lactogenesis, lactation and mammary gland development, mammary tissue samples were taken from five yaks during a dry period (DP, n = 3) and lactation period (LP, n = 2). Two types of cDNA sequence libraries that reflected the different physiological states of the mammary gland were constructed using RNA sequencing technology. After removing reads containing adapters, reads containing poly-N and low-quality reads from the raw data, 45,423,478 to 53,274,976 clean reads were obtained from these libraries. A total of 74.72% to 80.65% of the high-quality sequence reads were uniquely aligned to the BosGru v2.0 yak reference genome. Using the DESeq R package, 360 differentially expressed genes were detected between the two groups when the adjusted P value (padj < 0.05) was used as the cutoff value; this included 192 upregulated and 168 downregulated genes in the yak mammary gland tissue of the DP compared to the LP. A gene ontology analysis revealed that the most enriched GO terms were protein binding, multi-organism process, immune system and others. KEGG pathway analysis indicated that the differentially expressed genes were mostly enriched in Hippo signaling, insulin signaling, steroid biosynthesis and others. The analysis of the up- and downregulated genes provides important insights into the molecular events involved in lactogenesis, lactation and mammary gland development and will guide further research to enhance milk yield and optimize the constituents of yak milk.