devd br Experimental section br Results
Discussion In our previous work (Rashidi et al., 2018) we isolated a novel glucarpidase whose raised devd did not cross-react with the one in clinical use. In principle, therefore, it would be possible to delay the production of antibodies in a patient by alternating the use of the two versions of CPG2. However, this strategy may not totally solve the problem and in any case does not address the issue of limited protein stability in a patient\'s blood, e.g. due to proteolytic degradation.(Lawrence and Price, 2016) In this study, therefore, we adopted a different approach to produce biobetter glucarpidases, which should both reduce the potential for antibodies production and also protect the protein against endogenous proteases. PEGylation of proteins and other biomolecules is one of the most effective strategies to produce biobetter therapeutics. It improves the pharmacokinetics and pharmacodynamics of the conjugated molecules in relation to the non-conjugated one, increases water solubility and also protects against proteolytic degradation. On the other hand, human serum albumin has a long half-life in the body and proteins fused to this protein also have extended half-lives.(Aggarwal, 2007; Kontermann, 2011) The data presented in Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 and Appendix A indicate that the production of pure and active PEG-CPG2 and HSA-CPG2 was successfully achieved. The mass spectra analysis confirmed the formation of the HSA-CPG2, Appendix B. However, it was important to verify that the conjugated forms of CPG2 retained enzyme activity – it remained possible that attachment of large additional molecules might sterically hinder access to the active site of CPG2. Surprisingly, enzyme activity studies indicate that HSA-CPG2 has slightly increased catalytic activity relative to free CPG2 – the Vmax of the former was 48.72 ± 4.389 μM/min whereas unconjugated CPG2 has a Vmax of 24.35 ± 1.91 μM/min.(Rashidi et al., 2018) In the case of PEGylated CPG2, the catalytic activity found to be slightly lower than that of unconjugated CPG2 (Vmax value of PEG-CPG2: 20.69 ± 1.428 μM/min). Thus, the attached PEG may slightly restrict the access of the substrate to the enzyme active site or slightly alter the enzyme\'s conformation. The results of far UV spectroscopy (CD) suggest that significant structural changes in secondary structure components of each protein from induced by PEGylation and/or HSA conjugation compared with the CD scan and its deduced secondary structure results of free Xen-CPG2 recently published by our group.(Rashidi et al., 2018) To compare the stability of the modified forms of CPG2 with the free enzyme, we incubated them with human blood serum for a total of 14 days. Fig. 7 and Fig. 8 confirm that both HSA-CPG2 and PEG-CPG2 are significantly more stable than free CPG2 and that they also have longer half-lives. In the latter case, PEGylation (incorporation of PEG negative charged molecules) is known to stabilize the conformation of proteins by increasing the intramolecular hydrogen bonding and increasing the hydrophilicity.(Davidson et al., 1998; Robotta et al., 2011). Increasing protein conformation also restricts access of proteases to the conjugated protein. On the other hand, HSA, which is well known to be non-immunogenic and biocompatible, also extends the half-life of the HSA-protein therapeutics.(Cho et al., 2017) A PBMC proliferation assay, where T-cell (in the PBMCs) proliferation and differentiation is induced upon exposure to their cognate antigens, is considered to be a helpful tool during preclinical safety assessment and is used efficiently to evaluate and predict immunological effects of biopharmaceuticals.(Lim et al., 2015) To assess the potential immunotoxicity of the modified CPG2s relative to the free CPG2, we, therefore, carried out an ex-vivo lymphocyte proliferation assay. Our results (Fig. 9) indicated that in case of PEG-CPG2 no significant induction of T-cell proliferation was observed, indicating that the attached PEG molecules are able to mask the immunogenic epitopes in CPG2 that react with immune cells and induce immunogenicity. In contrast, HSA-CPG2 triggered a significant increase in T-cell proliferation compared with the negative control. This unexpected result, which apparently contradicts the protein stability results (Fig. 7, Fig. 8), could be explained by the presence of endotoxin impurities, which might induce immunogenicity. We, however, carried out further purification steps to remove any remaining endotoxin but obtained the same results. Further study is needed to establish the cause of the observed immunogenicity.