Research And Development Of A New Kind Of Thrombopoietic Factor —Recombinant Human Tyrosyl-tRNA Synthetase

Thrombocytopenia is a common and side effect for cancer patients treated with chemo-or radiotherapy and results in a relative decrease in platelets in the blood. In addition, it is a complication observed in patients with acquired immunodeficiency syndrome, chronic liver disease, idiopathic thrombocytopenic purpura, and myelodysplastic syndrome (MDS). Low platelet counts often cause excess bleeding. Traditionally, platelet transfusion has been used in cases of severe thrombocytopenia, but this approach is limited by availability, cost, potential for infectious side effects and disease transmission, and ineffectiveness due to antibody formation against platelets.A large number of pleiotropic hematopoietic growth factors have been identified to influence various phases of development in megakaryocytic lineage, including recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (c-kit ligand), interleukin 1(IL-1), IL-3, IL-6, IL-11, and thrombopoietin (TPO). Although administration of these cytokines reduces the need for platelet transfusions in patients with severe thrombocytopenia, the pleiotropic effect often results in unwanted or unacceptable toxic effects, including hyperbilirubinemia, rapid induction of anemia, fever, fatigue, hypotension, chills, and headache. For example, data regarding the use the TPO-like synthetic protein, Nplate, has raised safety concerns after almost 10% of the MDS patients (low to intermediate-1 risk) enrolled in the trial developed acute myelogenous leukemia (AML).A lot of research indicate that many aminoacyl tRNA synthetase with cytokine function. As reported that human tyrosyl-tRNA synthetase engineered a single tyrosine to alanine amino acid substitution at position 341 unmasked the ELR motif which delivers thrombopoietic activity. Here, we try to evaluate the protective effect of rhTyrRS (Y341A) against thrombocytopenia induced by chemotherapy. This research would be carried out through three part:1, pharmacodynamics and safety evaluation of rhTyrRS (Y341A); 2, mechanism of signal pathway of rhTyrRS (Y341A); 3, pilot research of rhTyrRS (Y341A);.1. Pharmacodynamics and safety evaluation of rhTyrRS (Y341A)Thrombocytopenia model was induced by cyclophosphamide on day 8, blood samples were collected at regular time point. Mice were sacrificed after treatment and femur bones were dissected. Megakaryocytes were detected by histopathological assay.There was no obvious side effect of rhTyrRS (Y341A) by a large dose injection. Platelet count increased significantly after 7 days injection of rhTyrRS. The injection of rhTyrRS (Y341A) reduced the toxicity of cyclophosphamide on liver and the degree of thrombocytopenia. The pretreatment with rhTyrRS (Y341A) increased the number of megakaryocytes surrounding the bone marrow sinus.The acute toxicity of rhTyrRS (Y341A) (22mg/kg) was detected by a large dose injection. Mice were randomly divided into six groups (n=12/group):PBS only (0μg/kg), 1μg/kg,10μg/kg, 100μg/kg of rhTyrRS (Y341A),5μg/kg TPO, and a sham (saline only) group. All mice received daily intraperitoneal injections based on their group assignment for 7 days.In this chapter we found rhTyrRS (Y341A) might be used in the treatment of chemotherapy induced thrombocytopenia with a dose effect and time effect relationship.3. Mechanism of signal pathway of rhTyrRS (Y341A).Chemoattractant effect of rhTyrRS was detected by migration assay and adhesion assay. Expression of VCAM-1 of HUVEC was detected by RT-PCR, Real-time quantitative PCR and flow cytometry. We analysied rhTyrRS (Y341A) induced nuclearlocalization of NFκB by the method of immunofluorescence.These results indicated that rhTyrRS (Y341A) augments megakaryocyte-like cell motility and adhesion. VCAM-1 induced by rhTyrRS (Y341A) supports adhesion of megakaryocytes. Immunofluorescence assay indicted the nuclearlocalization of NFκB was induced by rhTyrRS (Y341A).In this chapter we found rhTyrRS (Y341A) may promote the migration and adhesion of megakaryocyte through the stimulation of NFκB signal pathway.1. Pilot research of rhTyrRS (Y341A)In laboratory based fermentation, we fermented engineering bacteria in 30-liter fermenter. The optimal parameters of fementation were studied. The culture was centrifuged and sediment was collected. The supernatant was collected after high pressure homogenization. After cation exchange chromatography, gel filtration chromatography and anion exchange chromatography, the purified protein was identified by reductive SDS-PAGE, Western-Blot, N-terminal sequencing, LC/MS, HPLC, endotoxin detection and aminoacylation assay, rhTyrRS (Y341A) was added with mannitol, then freeze-dried and formed a preparation for injection.We got 5.0g purified rhTyrRS (Y341A). Protein recovery rate is greater than 25% and productivity is more than 250mg/L. HPLC showed the purity of rhTyrRS (Y341A) was greater than 95% and the molecular weight of rhTyrRS (Y341A) was 59.16kD. Protein activity was confirmed by aminoacylation assay. ELISA showed IL-8 was induced by rhTyrRS (Y341A)In this chapter we established a procedure for production and verification of rhTyrRS.