The Construction Of Recombinant Long-Acting Hyaluronidase And Their Application In Subcutaneous Administration And Antitumor Therapy

By degrading hyaluronan in extracellular matrix, hyaluronidase can facilitate therapeutic agents diffusion, and has been applied in subcutaneous administration and antitumor therapy. However, the bioavailabilities of SC-administered macro molecules would be lower than the IV route in general. On the other hand, the application in antitumor therapy was limited as the short in vivo circulation time. Focusing on the primary restrictions in these two areas, we constructed novel recombinant hyaluronidase and explored whether these reconstruction techniques could improve stability in the dermis and enhance antitumor efficacy in vivo.This thesis mainly included the following issues:1. The fused gene fragments of IgK-rhPH20, IgK-rhPH20-HSA and IgK-rhPH20-Fc were successfully constructed and ligated to the cloning vector pMD18-T and expression vector pMH3, respectively. The sizes of the gene fragments resulting from EcoR I and Not I double digestion were 1438 bp,2012 bp and 2077 bp, and PCR verifications were as expected. After gene transfection, two-cycle clone selection and adaption to suspension culture, three stable and high-level expression clones for each protein were chosen from thousands of clones and were used for subsequent expression. Average expression levels of CHO KSD (18 U/24h·mL) derived clones were higher than CHO S (12 U/24h·mL) derived ones. The addition of IRES-eGFP to the expression vection facilitate clones screening by flow cytometer, which was high-efficient and convenient. The influence of expression vector skeleton was also explored, which indicated the higher GC content, the high expression level of protein, as the DNA was open for transcription factor.2. The basic expression levels of batch culture in 40-mL flasks for each of the selected clones were 402,596, and 737 U/mL for rhPH20, rhPH20-HSA and rhPH20-Fc, respectively. In this study, expression of the new protein was undertaken in CHO cells cultured in a 5-L disposable bioreactor. Purification was carried out by a series of chromatographic methods to obtain high-purity products of 61 kDa (rhPH20),79 kDa (rhPH20-HSA) and 190 kDa (rhPH20-Fc). The specific activity of rhPH20-Fc was 35,600 U/mg, higher than that of rhPH20-HSA (10,000 U/mg). Lyophilized formulation was optimized to control appearance and maintain the activity.3. The chimeric proteins rhPH20-HSA and rhPH20-Fc performed fairly well as spreading factors in short-term trypan blue intradermal (ID) infusion in comparison with recombinant hyaluronidase (rhPH20). They extended the channel opening from 24 hours (rhPH20) to 85-120 hours in vivo. Co-administration of rhPH20-Fc with two biomacromolecular pharmaceuticals, Stelara (150 KDa) and TNFRII-Fc-ILlra (TFI,250 kDa), through an ID route increased the bioavailability from 86% to 93% and from 64% and 97%, respectively, compared with rhPH20. The pharmacokinetic profile of ID administrated larger TFI was significantly improved through cooperation with the long-acting hyaluronidase.4. Natural hyaluronidase PH20 and long-acting PH20-Fc were constructed and evaluated as diffusion promoter for typical therapeutics agents in different nanoscales: doxorubicin (～1.5×1.0×0.7 nm) in chemotherapy, trastuzumab (10～15 nm) in biotherapy, and gold nanorods (～100× 35 nm) in thermotherapy. In traditional 2D culture, PH20 and PH20-Fc showed little influence on the cytotoxicity due to the lack of tumor microenvironment. On the contrary, the cytotoxicity of these three therapeutic agents on 3D MCTS could be enhanced after treatment with PH20 and PH20-Fc, since the HA degradation could facilitate the therapeutics penetration and accumulation. Further in vivo evaluation revealed that the significantly prolonged circulation time of PH20-Fc opened the time window to accumulate in tumor tissues, which paved the way for subsequent HA degradation. As a result, co-administration with PH20-Fc further inhibited tumor growth on the tumor xenografts, demonstrating the success of utilizing PH20-Fc to improve antitumor efficiency. Meanwhile, we also noticed the PH20-Fc performances varied among these three therapeutic agents due to their different nanoscales. Trastuzumab with a best promotion on the penetration and accumulation benefited most from PH20-Fc.