Human Plasma Proteins from Transgenic Animal Bioreactors

Chapter 14

Human Plasma Proteins from Transgenic Animal Bioreactors 1

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R. K. Paleyanda , W. H. Velander , T. Κ. Lee , R. Drews , F. C. Gwazdauskas, J. W. Knight , W. N. Drohan , and H. Lubon 1

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Downloaded by RUTGERS UNIV on December 30, 2017 | http://pubs.acs.org Publication Date: September 1, 1996 | doi: 10.1021/bk-1996-0647.ch014

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Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855 Department of Chemical Engineering, VirginiaPolytechnicInstitute and State University, 153 Randolph Hall, Blacksburg, VA 24061 2

We have evaluated the transgenic animal bioreactor (TAB) system for the production of human Protein C (HPC), an anticoagulant plasma protein. Mouse whey acidic protein (mWAP) gene regulatory sequences targeted expression of mWAP/HPC hybrid genes to the mammary gland of transgenic mice and pigs. The transgenes were stably transmitted through several generations and expression did not adversely affect the health of animals. We purified recombinant HPC from milk which was structurally and enzymatically similar to the human protein, demonstrating the potential of TABs for the production of plasma proteins. The active fraction was found to be only a part of the total protein secreted. At mg/mL levels of secretion, modifications like proteolytic maturation and γ-carboxylation became limiting factors in the production of functional protein. We have proposed and shown that these limitations may be overcome by engineering the posttranslational protein modification capacity of selected organs of the TAB. In the 1980s, genes encoding human plasma proteins traditionally used in transfusion and replacement therapy, such as serum albumin (HSA), Factor VIII (FVIII) and Factor IX (FIX) were cloned. The coding sequences of several other therapeutic proteins, namely human tissue plasminogen activator (tPA), erythropoietin (EPO), a antitrypsin (cÂT), antithrombin III (AT-III), Protein C (HPC), Protein S (HPS), Factor VII (FVII), Factor X (FX), prothrombin, hemoglobin, fibrinogen, lactoferrin and collagen were also cloned. Advances in cell biology, recombinant DNA technology and cell culture reactor technology have resulted in improvements in the expression of complex proteins in vitro. However, the complexity of many therapeutic proteins, coupled with limitations in host cell r

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Corresponding author 0097-6156/%/0647-0205$15.00/0 © 1996 American Chemical Society

Fuller et al.; Agricultural Materials as Renewable Resources ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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intracellular processing and secretory pathways have limited the utility of bacterial or yeast cell production systems, although nonglycosylated proteins like albumin are considered to be good candidates for these systems. Mammalian cell culture systems have been used successfully to produce recombinant FVIII and EPO, which are required in small doses for human administration. Even so, such production methods for FVIII are not as cost-effective as purification by traditional means from pooled human plasma. It is also unlikely that mammalian cell culture will be cost-effective for the production of proteins needed in large quantities namely HSA, a AT, hemoglobin, lactoferrin and collagen. One approach is to direct the expression of human proteins to specific cells or tissues in transgenic animals which are easily accessible and collectible. An example of this is the expression of human hemoglobin in the erythrocytes of mice and pigs (7, 2). Another approach is to produce heterologous proteins in the bodily fluids of animals. This has been demonstrated by the secretion of several human proteins into milk (reviewed in 3), of rFIX (4) and α,ΑΤ (5) into blood, of a Cterminal peptide of FVIII into saliva (6) and of rHPC into urine (Lubon et al., unpublished observations) of transgenic mice and livestock. Some of these attempts have been successful in producing foreign proteins at g/L levels. Leading examples of the potential of thistechnologyare the transgenic sheep secreting 60 g/L of ctjAT into milk (7) and pigs expressing 24% or 32 g/L of human hemoglobin in blood (8). Proteins like tPA (9, 70), cÂT (7), lactoferrin (77) and HSA (72) are good candidates for synthesis in the mammary glands of such "transgenic animal bioreactors" (TAB). All these proteins have a common feature - simple posttranslational modifications. For instance, albumin requires proteolytic processing for maturation, cÂT and lactoferrin require glycosylation, and tPA requires both. As there are other, more complex proteins inVol.ved in hemostasis, we and others undertook to challenge the TAB system to express some of these proteins, including HPC (73-75), FVIII (16, Lubon et al, unpublished observations), FIX (4, 17) and hemoglobin (2).


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Human Protein C We have generated transgenic animals for HPC (13-15), FVIII, FIX and fibrinogen (Velander and Lubon, unpublished observations). This report will focus on the expression of HPC in TABs. HPC is an anticoagulant vitamin K-dependent protein that is synthesized in the liver and undergoes extensive co- and/or post-translational modification (18). This processing includes the proteolytic cleavage of a signal peptide and a propeptide, propeptide-directed vitamin K-dependent γ-carboxylation of glutamic acid residues (GLA), glycosylation at four N-linked sites, βhydroxylation of Asp and disulfide bond formation. Endoproteolytic removal of the Lys -Arg dipeptide produces a disulfide-linked heterodimer composed of a 21 KDa light chain, and a 41 kDa heavy chain containing the N-terminal activation peptide and serine protease domain. After activation of the zymogen by a thrombinthrombomodulin complex at the endothelial cell surface, activated HPC (APC) regulates the coagulation cascade by inactivating coagulation factors VIII and V , which are necessary for the efficient generation of factor X and thrombin, respectively. As described earlier, HPC has been used in replacement therapy for homozygous and heterozygous HPC deficiency and in the treatment of coumarin71

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Ind.uced skin necrosis (75). APC has also been shown to prevent the extension of venous thrombi in dogs and rhesus monkeys, to protect baboons from septic shock due to lethal E. coli infusions and to delay thrombotic occlusion in a baboon arterial shunt model (75). Inhibition by APC of disseminated intravascular coagulation and microarterial thrombosis in rabbits has also been reported (79).


Animals Transgenic for HPC Most mammalian cell lines engineered to express rHPC secrete only partially active molecules or low levels of fully active molecules (20-22). We therefore decided to target the expression of HPC to the mammary gland of transgenic animals, as proteins secreted within this organ are largely separated from the bloodstream and considerable amounts of protein, up to 100 mg/mL are found in the milk of certain species. In our initial experiments we used the promoter and gene of a mouse whey acidic protein (mWAP) to direct mammary-specific and lactogenic hormoneInd.ucible expression. The DNA construct contained a 2.5 kb mWAP promoter, the 1.5 kb HPC cDNA inserted at the Kpnl site in the first exon of the 3.0 kb mWAP gene, and 1.6 kb mWAP 3' flanking sequences (13). Mice transgenic for the cDNA construct secreted 3-10 μg/mL rHPC into the milk. To increase the concentration of rHPC in the milk of transgenic mice, we cloned additional 5' flanking sequences of the mWAP promoter (23) and used a construct containing 4.1 kb mWAP promoter sequences, the 9.0 kb HPC gene and 0.4 kb of HPC 3' flanking sequences (75). mWAP-promoter directed expression of the HPC gene was mainly restricted to the lactating mammary gland as detected by northern blot analysis, with "leakage" of expression in the salivary gland and kidney, at less than 0.1% of mammary expression. In the mammary gland, we detected rHPC in the epithelial cells lining the alveoli and in the milk-filled lumina, Figure 1. The developmental pattern of transgene expression differed from that of the endogenous mWAP gene, in that rHPC transcripts appeared earlier in pregnancy than mWAP, with no major Ind.uction during lactation. This suggested that the 4.1 kb promoter fragment also did not contain all the regulatory elements responsible for developmental regulation of the transgene similar to the mWAP gene. The precocious expression of the transgene did not compromise the health or nursing abilities of the transgenic females, but this may not hold true when proteins with potentBiol.ogicalactivity, like EPO, are expressed. Thus, the lack of strict tissue-specific and developmental regulation of transgene expression could be a limitation of the TAB system. In fact, the health of the animals will be a major guideline in the stable production of therapeutic proteins from TABs. Characterization of rHPC Structure. Using immunoaffinity chromatography, we purified rHPC from pooled milk of HPC cDNA mice which had about 70% of the anticoagulant activity of HPC, as assayed by activated partial thromboplastin time (APTT) assays (75). Low concentrations of protein in the milk of cDNA mice did not allow further characterization. rHPC was secreted at higher concentrations of 0.1 to 0.7 mg/mL,


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Figure 1. Immunohistochemical localization of rHPC in mammary gland Paraffin-embedded sections of mammary gland from mid-lactation (day 10) mice were probed with a sheep anti-HPC polyclonal antibody and detected by the Ind.irect immunoperoxidaseTech.nique (Vector Labs.). Sections were counterstained with hematoxylin. The arrows denote the darkly staining rHPC detected predominantly in the alveolar lumina and localized in secretion vacuoles of alveolar epithelial cells. (A, B) Mammary gland of control mouse (C, D) Mammary gland of mWAP/HPC transgenic mouse. Initial magnification = 100 X (A, C), 400 X (B, D).



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when the genomic sequences were expressed (75). This is the first report of the secretion of mg/mL amounts of a human vitamin K-dependent plasma protein into mouse milk. Western blot analysis of milk proteins separated by SDS-PAGE revealed rHPC single, heavy and light chain forms similar to plasma-derived HPC, although they migrated with slightly increased electrophoretic mobility, Figure 2, lanes 3, 7. rHPC consisted of 30 to 40% single chain form, which is two-to-three times more than that observed in plasma HPC. The two-dimensional gel electrophoresis of mouse whey proteins and western blot detection revealed that rHPC was more heterogenous than HPC (24). The predominant single and heavy chain polypeptides of mouse rHPC were more basic than that of HPC, Figure 3. The HPC heavy chain polypeptides resolved at an isoelectric point of 4.7-5.5, while mouse rHPC resolved at pH 5.3-6.2. The observed heterogeneity of rHPC was not attributable to alternate splicing of mRNAs, as confirmed by sequencing of the exon-exon junctions. However, this possibility cannot be ruled out entirely for all precursor RNA molecules. These differences could be due to species-specific posttranslational modifications of rHPC like proteolytic processing, glycosylation or γ-carboxylation in the mouse mammary gland. For example, a decrease in sialic acid or GLA content could affect the electrophoretic mobility of mouse rHPC. Enzymatic deglycosylation with N-glycosidase F, which cleaves between the internal N-acetylglucosamine and asparagine residues of most glycoproteins, showed that these molecular weight and charge disparities were in part due to glycosylation (75). The carbohydrate composition of rHPC produced in cell culture systems was also responsible for the altered electrophoretic mobility as compared to HPC (27, 22). Thus, recombinant proteins produced in transgenic animals have different patterns of glycosylation from their human counterparts. Amino-terminal sequence analysis of rHPC purified by immunoaffinity chromatography showed that in 60-70% of the purified protein, the cleavage of the signal peptide and propeptide, and removal of the connecting KR dipeptide had occurred at the appropriate sites. The increased amount of single chain protein and the presence of propeptide on 30-40% rHPC suggested that at mg/mL levels of expression, mammary epithelial cells were incapable of the efficient proteolytic processing of the rHPC precursor. This result was not unexpected as studies with several different mammalian cell lines, including CHO, CI27 and BHK-21 cells have shown inefficient proteolytic processing of rHPC. Hepatocytes of the human liver also do not completely process the KR dipeptide, as 5-15% of the single chain form has been detected in plasma (20-22). Activity. Despite the observed differences between mouse rHPC and HPC, the enzymatic domain was functional, as the K of rAPC for the synthetic tripeptide substrate

Human Plasma Proteins from Transgenic Animal Bioreactors

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