Study On The Immunogenicity Of OsrHSA,its Residual Host Cell Proteins And Humanized Glycosylation In Rice

Rice is a highly self-pollinated crop,which is generally recognized as safe(GRAS)plant by US food and drug administration(FDA)because it does not contain any harmful ingredients.It has been demonstrated that rice endosperm bioreactor for the production of pharmaceutical proteins has the advantages of lower cost,simple extraction and purification process,easy scale,and good safety.It can be one of the best platforms for the production of recombinant protein drugs.Although this expression platform has more advantages over the microbial and animal cell expression platforms,the major concern of using plant bioreactors in the production of pharmaceutical products is the safety of plant-made biopharmaceuticals(PMP).Generally,the safety concern of biopharmaceuticals is mainly due to the host-related,drug-related and processing-related impurities.The protein glycosylation of plant cells has similar sugar skeletons with humans;however,there are the differences in the fucose and xylose residues.Therefore,the immunogenicity of plant glycans becomes some protein structure-related impurities that is one of the key factors for the production of biopharmaceuticals in rice endosperm cells.The previous results of the efficient,stable,and cost-effective expressing recombinant Human Serum Albumin from Oryza Sativa(OsrHSA)in rice endosperm indicated that the protein N-glycosylation pattern in rice seeds is simpler.Due to different glycol forms variation with different protein expression systems,the glycosylation patterns of biopharmaceuticals manufactured in plants adds the plant-specific N-glycans residues,α-1,3-fucose and β-1,2-xylose to the final glycosylated product.The presence of these plant-specific N-glycans could significantly change the stability and activity of biopharmaceuticals.Some studies have demonstrated that the concerns arisen from N-glycosylation can reliably resolve in producing pharmaceuticals by humanization glycosylation type through genetic engineering in plants.Human serum albumin is a major component in plasma,which is a monomer and non-glycosylation protein.It is widely applied with high dosage to treat patients with circulatory dysfunction due to hypovolemia,including patients with cirrhotic ascites and hypoalbuminemia.Recombinant human serum albumin as a substitute could provide more safe and sufficient than plasma-derived albumin.Currently,the large-scale OsrHSA has been produced in transgenic rice endosperm in China,and the Chinese Food and Drug Administration(CFDA)recently approved it for clinical trials in China.However,the safety of the residual Host Cell proteins(HCPs)from OsrHSA is a critical concern.OsrHSA is the first drug entering the clinical trial in China.The data indicated that OsrHSA has similar in physical and biochemical characters to plasma-derived HSA.Therefore,the safety concerns of OsrHSA drug are not from HSA itself,while from the impurities that derived from downstream processing,particularly,residual host cell proteins(HCPs).Whereas,the HCP could not be completely removed from the downstream processing.The potential immunogenicity is the most important risk of the HCPs in OsrHSA.Although the residual HCP concentration in OsrHSA has controlled as low as 1.5 μg/g,the total HCP content is still high because HSA occasionally infuses at more than 50 g/dose in the clinic.HCP content in OsrHSA is still high.In general,the safety of biopharmaceuticals is not only affected by quantity,but also by type of HCPs.Although the HCPs content could be reduced through more complicated processing steps,it could not entirely be removed.The residual HCPs is one of the critical quality attributes(CQA).The potential immunogenicity of HCPs in OsrHSA becomes a risk in a clinical trial.So,it is essential to evaluate the safety of HCP via varies approaches and as one of the criteria of CQA.Up-to-now,it is neither report regarding the immunogenicity of HCP in OsrHSA nor any report on non-clinical or clinical research.Therefore,it is essential to gather direct evidence of the immunogenicity and immunotoxicity of HCPs in OsrHSA using the animal system.In this study,the safety of HCPs,one of the host-related components of the protein drug in rice endosperm cell bioreactors,and protein-related impurity,N-glycan,were investigated.Firstly,we assessed the immunogenicity and immunotoxicity of OsrHSA and its residual HCPs using Sprague-Dawley rat.Several parameters of hematological and biochemical analysis,cytokine,C-reactive protein(CRP),and plasma circulating immune complex(CIC),complement and an anti-drug antibody(ADA),etc.have been analyzed.Then we investigated the humanized glycans in rice endosperm cells using TALEN technology.We knocked out the OsFUT(a-1,3-fucosyltransferase,OsFUT)or OsXylT(β-1,2-xylosyltransferase,OsXylT)genes and simultaneously knock-in humanα-1,6-fucosyltransferase(FUT8)or galactosyltransferase(GalT)genes into rice genome.The main results are as followings:During our experimental period,no unexpected animal death or abnormal reactions were observed in the HCP group or the negative controls.Body weights of the animals of all groups were increased during the treatment period except at Day 42.We found that the increase of body weight in male animals were obviously more than that of the female animals.However,no obvious increase of body weights was found between HCP and Control groups.No significant changes of any hematological and coagulation-related parameters were found between HCP and negative control groups.The results indicated that residual HCP could not lead to hematological changes.To assess the immunotoxicity of OsrHSA and its HCP,CD4+ and CD8+ T cells were monitored.The results indicated that no significant difference of CD8+ T cells in the HCP was found compared with the negative control at Day 15.However,CD4+T cells and the ratio of CD4+/CD8+ T cells showed significant differences between the HCP and control groups at Day 15.Despite the significant difference in the ratio of CD4+/CD8+ at Day 15,the percentage of lymphocyte T cells was in the normal clinical range,while these changes did not present the correlations between drug dose and administration time.Therefore,these changes were not recognized to be related to the immunotoxicity and the drug.Further studies on a group of highly linked to inflammation cytokines,including Interleukin-4(IL-4),IL-5,IL-6,IL-10,IL-13,Tumor Necrosis Factor(TNF-α),Interferon(IFN-γ),and IL-1β,we found that none of these cytokines were detected in any of the treatment groups,proposing that the HCPs from OsrHSA and OsrHSA had no immunotoxicity and exclude the potential risk of "cytokine storm".To determine the immunogenicity of residual HCP,we monitored the titer and the incidence of anti-HCP,anti-OsrHSA and anti-pHSA antibodies at Day 14,Day 28 and Day 41 after administration.In OsrHSA and Plasma-derived HSA(pHSA)groups,only one animal developed anti-HCP antibody.However,the incidence is 2/10 and the titer is less than 1.0.Although HCP-specific antisera were identified,both the incidence and the titer were very low,indicating that the residual HCPs have low immunogenicity.The C reaction protein(CRP)level is corresponding for the response to inflammation development.Notably,CRP did not increase;instead,it was significantly decreased in female animals in HCP group at Day 15.At D42,the CRP level were significantly decreased in OsrHSA and pHSA groups.Although CRP significantly reduced in the HCP and pHSA groups of female animals at D15,no correlation between drug and administration course were found.Increase of CIC level could develop anti-drug antibody(ADA).To monitor the ADAs of the HCPs and OsrHSA,we determined the level of CIC at Day 15 and Day 42.No obvious drug-related CIC change was observed in the HCP and OsrHSA groups compared with the negative control.To further evaluate whether an immune reaction was activated to response to an antigen(such as HCPs),the levels of the complement 3 and complement 4(C3 and C4)were monitored.The results indicated that the C3 level in the pHSA group was significantly decreased at Day 15 and Day 42 but decreased only in female OsrHSA animals at Day 42.The C4 was not detected in any of the treatment groups.To explore the pathological changes when high dose of OsrHSA and pHSA administration,we observed the potential target organs,including liver,kidney and spleen.No significant drug-related pathological differences observed between the HCP group and the negative control.Besides,we did not find significant changes in organ weights and coefficient ratios in the HCP group.Drug-related pathological changes,including the increase of the organ-to-body weight ratios of the liver,spleen and kidneys in the pHSA and OsrHSA groups,were observed.OsrHSA-related pathology findings were noted in liver,kidney,and spleen with similar incidence and severity between pHSA and OsrHSA animals.These included hepatocytes hypertrophy,renal tubule degeneration/regeneration,glomerular mesangial cell and matrix increase,extramedullary hematopoiesis in hepatic sinusoidal or portal area and spleen in the OsrHSA and pHSA groups.The incidence and severity of pathological changes were dose related and were consistent with biology of albumin after daily dosing.Those pathological changes could highly related to high dose of HSA to increase of osmotic pressure.These changes could not be happened because HSA is used for treatment of patients with circulatory dysfunction due to hypovolemia.Furthermore,except renal tubular protein cast,all the above changes were not noted at the end of recovery on Day 42,indicating reversibility of the effects.However,no obvious differences in the incidence and degree of precancerous lesions were observed between the pHSA and OsrHSA groups.Our results indicated that the safety of OsrHSA is highly comparable to that of pHSA.To obtain humanized glycan in recombinant protein,we used Transcription Activator-Like Effector Nuclease(TALEN)technology to engineer human glycan in a plant cell.Nine TALEN vectors targeting OsFUT and 11 TALEN vectors targeting OsXylT genes were constructed and the transformation was carried out.The transformants with the hpt positive and the GalT or FUT8 expression cassettes were identified under hygromycin selection.The total of 10 independent transformants containing FUT8 gene were validated from 67 hpt resistance transformants.46 transgenic plants were confirmed carrying GalT gene from 207 hpt resistance transformants.The site-specific integration events from both GalT and FUT8 gene positive transformants were performed using TALENs site-specific primers.Unfortunately,we did not find any insertion of the human GalT or FUT8 was integrated into proposed TALEN sites through any DSB.The results indicated that transformants carrying either GalT or FUT8 gene were randomly integrated into rice genome.To further check whether both genes are expressed,we detected the GalT and FUT8 gene expression of T1 seeds by Western blotting.The results showed that three out of 10 and 23 out of 46 lines were confirmed to overexpress FUT8 and GalT,respectively.The further study determines the N-glycosylation patterns using MALDI-TOF mass spectrometry.According to the results,all overexpression FUT8 lines showed different plant type glycans.In overexpression of FUT8 line FF1-141-6,the plant type glycan residues were decreased 5%comparison with TP309.Notably,the total of plant-type glycans,such as GnMX,MMX,and GnGnX,were significantly reduced,which could be caused by the overexpressed mammalian FUT8 gene.Notably,obvious reduction of plant type glycans observed in all the transgenic lines expressing GalT,which is attributed to the expression of GalT in the transgenic endosperm.It has previously reported that the human β-1,4-galactose acts on the central domain of GlcNAcMan5GlcNAc2 to inhibit the formation of plant-type N-glycans residues.Therefore,the significant reductions of the plant-specific N-glycans in all transgenic lines expressing GalT can be well explained by the expression of the GalT in rice seeds.We found that at least one or two types of plant type glycan were reduced in transgenic lines,XG4-133-1 and XG2-118-9.In addition to the significant reduced plant-specific N-glycans,the galactosylated and sialylated N-glycan structures were presented in the transgenic lines overexpressed GalT gene.In XG2-118-9 transgenic line,26.44%out of 81%of the total mammalian N-glycans were sialylated.It is the first report to detect the sialic acid in transgenic endosperm in where expression of GalT.It has been reported that the galactosylated structures serve as substrates for the following sialylation.However,previous researches showed achieving the sialylated N-glycans in the cereal crops is difficult since those lack of the genes corresponding to sialyation pathway.On the other hand,it has been reported that the O.sativa has the genes that encode the sialyltransferase-like genes.We obtained sialylated N-glycans in the transgenic rice with the overexpression of GaIT.Our results indicated that rice has more advantages over other cereal crops.The absence of sialic acid on plant-made biopharmaceuticals is a most critical difference of plant-made pharmaceuticals as an animal-based bioreactor.The absence of the sialic acid on glycosylated plant-derived pharmaceuticals affects their circulating half-life and biological activity.Our finding provides the evidence that humanized glycan could be achieved in rice endosperm cell.Our results again demonstrate that sialyltransferase-like genes in rice genome can act on the sialylation in the presence of galactose as substrate via engineered GalT gene in the rice genome.Taken together,our results demonstrated that the residual HCPs from OsrHSA have low immunogenicity,indicating that the rice endosperm is one of the best host options for plant molecular pharming.In addition,our results suggested that the safety of OsrHSA is highly equivalent to that of pHSA.We validated the hypothesis that the residual HCPs of OsrHSA from rice could be safe for human beings.We concluded that we obtained the sialic acid glycan structure using overexpression of GlaT in rice endosperm cell and significant reduction of plant type glycans,β-1,2-xylose.Those results demonstrated that rice HCP is lower immunogenicity and immunotoxicity in the animal system and humanization of glycosylation in rice endosperm cell could be achieved.