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Shen B.,Peking Union Medical College | Jiang W.,Biopharmagen Corporation | Fan J.,Peking Union Medical College | Dai W.,Peking Union Medical College | And 4 more authors.
PLoS ONE | Year: 2015

Background: Stem cell factor (SCF) can stimulate hematopoietic stem cell (HSC) expansion; however, the specific structural region(s) of SCF protein that are critical for this function are still unknown. A novel monoclonal antibody (named 23C8) against recombinant human SCF (rhSCF) was previously found to inhibit the ability of rhSCF to enhance HSC expansion, making it possible to identify the relevant active region to HSC. Methods: Eleven polypeptides were synthesized, which were designed to cover the full-length ofrhSCF, with overlaps that are at least 3 amino acids long. ELISA was used to identify the polypeptide(s) that specifically react with the anti-SCF. The effects of the synthetic polypeptideson human HSC expansion, or on the ability of the full-length rhSCF to stimulate cell proliferation, were evaluated ex vivo. Total cell number was monitored using hemocytometerwhereas CD34+ cell number was calculated based on the proportion determined via flow cytometry on day 6 of culture. Results: Of all polypeptides analyzed, only one, named P0, corresponding to the SCF proteinsequence at residues 39-56, was recognized by 23C8 mAb during ELISA. P0 stimulated the expansion of CD34+ cells derived from human umbilical cord blood (UCB). Addition ofP0 increased the numbers of total mononucleated cells and CD34+ cells, by ∼2 fold on day 6. P0 also showed partial competition against full-length rhSCF in the ex vivo cell expansionassay. Conclusion: Residues 39-56 of rhSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34+ cells. The peptide P0 is a potential candidate for furtherdevelopment as a synthetic substitute for rhSCF in laboratory and clinical applications. © 2015 Shen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Fan J.,Peking Union Medical College | Ding X.-X.,Peking Union Medical College | Jiang Y.-P.,Peking Union Medical College | Jiang Y.-P.,Biopharmagen Corporation
Fudan University Journal of Medical Sciences | Year: 2012

Objective: To assess the severity of the acute toxicity and sub-acute toxicity of a novel recombinant human granulocyte-colony stimulating factor a (rhG-CSFa) in mice and the dose-effect relationship. Methods: Total 80 mice (equal numbers of male and female) were randomized into eight groups for the acute toxicity study: six groups were subcutaneously or intravenously injected with rhG-CSFa at 34, 345, and 3450 μg/kg, respectively, and two groups were injected with normal saline as the control. The physical status and body weight were monitored for the next 10 days. Another 50 mice (equal numbers of male and female) were randomized into five groups for the sub-acute toxicity study: four groups were subcutaneously injected with rhG-CSFa at 1.1, 11.5, 115, and 1150 μg/kg, respectively, and the other group was treated with normal saline as the control. The mice received injections for 3 weeks. The physical status and body weight were monitored. The mice were sacrificed after the last rhG-CSFa administration. The numeration of leukocyte, bone marrow smear and blood smear were performed for each mouse. Results: Acute toxicity study indicated that no significant acute toxicity was observed in mice received rhG-CSFa administration. There was no significant difference of weight between the control and the treatment groups (P>0.05). Further study of sub-acute toxicity revealed that the absolute number of granular leukocytes in mice from three treatment groups were higher compared with the control group (P<0.01), and the increase of granular leukocytes was rhG-CSFa dose-dependent. No significant abnormalities were observed in this study. Conclusions: No significant toxicity was observed in mice received rhG-CSFa administration in the acute and sub-acute toxicity studies.

Liu X.-X.,Peking Union Medical College | Jiang Y.-P.,Peking Union Medical College | Jiang Y.-P.,Biopharmagen Corporation
Chinese Medical Sciences Journal | Year: 2010

Objective: To study the pharmacokinetics of a novel recombinant human granulocyte colony-stimulating factor (rhG-CSFa) in rats and to determine the proteolytic rates of rhG-CSFa in the whole blood and serum of rats in vitro. Methods: The pharmacokinetics of rhG-CSFa and conventional (wild type, WT) granulocyte colony-stimulating factor (G-CSF) were investigated in Sprague-Dawley rats which received either intravenous or subcutaneous injection of rhG-CSFa or WT G-CSF at three different doses (20, 50, or 100 μg/kg). The blood samples of rats were collected at multiple time points (from 0.08 to 12 h) and the concentrations of rhG-CSFa and WT G-CSF in serum were determined with a sandwich enzyme-linked immunosorbent assay (ELISA). For the study of proteolytic rates in vitro, the concentrations of rhG-CSFa or WT G-CSF were determined at 3-minute intervals after addition of the respective drug to rat's whole blood or serum. Results: Pharmacokinetic analysis of serum rhG-CSFa or WT G-CSF levels indicated that, at each dose tested, for either route of drug administration, the area under concentration-time curve values and the maximum serum concentration of rhG-CSFa were higher than those of WT G-CSF, and the serum half life of rhG-CSFa was longer than that of WT G-CSF. Subsequent in vitro whole blood and serum stability study showed that the rates of drug degradation in WT G-CSF were 1.8 folds and 1.5 folds higher than those in rhG-CSFa, respectively. Conclusion: rhG-CSFa has better serum and whole blood stability in vitro and higher bioavailability in vivo as compared to WT G-CSF.

Fan J.,Peking Union Medical College | Ding X.,Peking Union Medical College | Jiang Y.,Peking Union Medical College | Jiang Y.,Biopharmagen Corporation
Journal of Hematology and Oncology | Year: 2012

Stem cell factor (SCF) activates hematopoietic stem cell (HSC) self-renewal and is being used to stimulate the ex vivo expansion of HSCs. The mechanism by which SCF supports expansion of HSCs remains poorly understood. In cord blood ex vivo expansion assays, a newly produced anti-SCF monoclonal antibody (clone 23C8) was found to significantly inhibit the expansion of CD34+ cells. This antibody appears to bind directly to a part of SCF that is critical for biological activity toward expansion of CD34+ cells, which is located in the first 104 amino acids from the NH2-terminus. © 2012 Fan et al.; licensee BioMed Central Ltd.

Ren Z.,Biopharmagen Corporation | Ren Z.,Peking Union Medical College | Wang Y.,Nanjing Medical University | Jiang W.,Biopharmagen Corporation | And 4 more authors.
PLoS ONE | Year: 2014

Background: Angiogenesis has become an attractive target in cancer treatment. Endostatin is one of the potent antiangiogenesis agents. Its recombinant form expressed in the yeast system is currently under clinical trials. Endostatin suppresses tumor formation through the inhibition of blood vessel growth. It is anticipated that combined therapy using endostatin and cytotoxic compounds may exert an additive effect. In the present study, we expressed and purified recombinant human endostatin (rhEndostatin) that contained 3 additional amino acid residues (arginine, glycine, and serine) at the amino-terminus and 6 histidine residues in its carboxyl terminus. The recombinant protein was expressed in E. Coli and refolded into a soluble form in a large scale purification process. The protein exhibited a potent anti-tumor activity in bioassays. Furthermore, rhEndostatin showed an additive effect with chemotherapy agents including cyclophosphamide (CTX) and cisplatin (DDP).Methods: rhEndostatin cDNA was cloned into PQE vector and expressed in E. Coli. The protein was refolded through dialysis with an optimized protocol. To establish tumor models, nude mice were subcutaneously injected with human cancer cells (lung carcinoma A549, hepatocellular carcinoma QGY-7703, or breast cancer Bcap37). rhEndostatin and/or DDP was administered peritumorally to evaluate the rate of growth inhibition of A549 tumors. For the tumor metastasis model, mice were injected intravenously with mouse melanoma B16 cells. One day after tumor cell injection, a single dose of rhEndostatin, or in combination with CTX, was administered intravenously or at a site close to the tumor.Results: rhEndostatin reduced the growth of A549, QGY-7703, and Bcap37 xenograft tumors in a dose dependent manner. When it was administered peritumorally, rhEndostatin exhibited a more potent inhibitory activity. Furthermore, rhEndostatin displayed an additive effect with CTX or DDP on the inhibition of metastasis of B16 tumors or growth of A549 tumors.Conclusion: Soluble rhEndostatin exhibits a potent anti-tumor activity in mouse xenograft models and it also has an additive effect with CTX and DDP, implying possible applications in clinical settings. Copyright: © 2014 Ren et al.

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