Recombinant Proteins: What’s New in the Lab and the Clinic

1. Recombinant Proteins: What’s New in the Lab and the Clinic Transfusion Medicine Residents 2008-09-25 Bill Sheffield

2. PubMed Search 2008-09-19 • Recombinant AND protein AND 2008 • >17,000 articles • Add “AND review” … • Near-ubiquitous use in research, increasing number of approved drugs Zhang AL, Luo JX, Zhang TY, Pan YW, Tan YH, Fu CY, Tu FZ. Recent advances on the GAP promoter derived expression system of Pichia pastoris. Mol Biol Rep. 2008 Sep 10. [Epub ahead of print] PMID: 18781398 [PubMed - as supplied by publisher]

3. What’s a recombinant protein? • A protein made using recombinant DNA • Recombinant DNA: DNA molecules in which sequences are assembled in an order not found in nature, usually for the purposes of facilitating protein production

4. Objectives • To explore general issues common to research and pharmaceutical recombinant protein production • To discuss (a) the recombinant protein drugs currently distributed by CBS and (b) ongoing development of these products • To present published and unpublished research from the Sheffield laboratory relating to recombinant plasma proteins

5. Molecular biology truths • DNA makes RNA makes protein • Protein production from a DNA template = protein expression • Most widely used means to transfer DNA = plasmid • Numerous expression systems available Source: www.artes-biotechnology.com

6. Plasmids • Small, double-stranded, circular DNA elements • Usually propagated in bacteria • Used for recombinant protein production in bacteria or transferred to another host (yeast, cultured mammalian or insect cells) Source: Becker WM, Kleinsmith LJ, Hardin, J. The World of the Cell, 4th edition, 2000. Benjamin/Cummings Pub. Co., Don Mills ON.

7. Why make recombinant proteins? • From a research point of view… • Test ideas about protein structure and function by making single amino acid substitutions (or larger changes) • Has become increasingly “easy” and “inexpensive” • In some cases the natural protein is not easy to obtain in sufficient quantities

8. Why make recombinant proteins? • From a clinical point of view… • Safety (e.g. vs. purifying FVIII from pooled plasma of 1000s of donors) • Natural human material not available due to abundance or source (e.g. G-CSF [filgastrim/Neupogen], erythropoietin [Epotin], interferons, fVIIa [Niastase], tissue plasminogen activator [Activase]) • Cost?

9. Production choices…because expression is “empirical” • Choice of DNA (cDNA, genomic clone, codon usage) • Choice of vector/plasmid (promoter strength, inducibility) • Choice of expression system • Post-translational modifications • Purification strategies (e.g. tags) • Efficacy/immunogenicity considerations

10. Expression systems • Bacteria (usu. E. coli, others possible) • Cheap but can mis-fold, do not glycosylate, inclusion bodies • Yeast (Saccharomyces, Pichia, others) • Fold well but acidify media, glycosylation may be high mannose • Insect Cells (baculovirus system) • Yields can be very high but glycans not well characterized • Cultured Mammalian cells • Chinese Hamster Ovary Cells CHO (FDA happy) • Baby Hamster Kidney BHK (FDA happy) • Human cell lines (HEK 293 gaining acceptance) • Tumour cell lines (research use only) • Weirder stuff • Slime molds • Plants • Transgenic animals (e.g. goats expressing human proteins in their milk)

11. Proteins: linear chains of amino acids Often shown in bar graph form Actually must have appropriate 3D structure to function, avoid clearance, avoid immunogenicity (?) RSA HLAH 6 HV3 RSA His6 A word about protein folding

12. Pluses: Chaperone-mediated folding and “post-translational” modifications (N- and O-linked glycans, S-S bonds, gamma carboxylation (VIIa, IX)) Minuses: Quality control mechanisms lower yields Mammalian cell expression: Pluses and Minuses Annu. Rev. Cell Dev. Biol. 2000. 16:557-589

13. Recombinant CBS products • Factor VIII (Kogenate [FS], Advate, Recombinate) • Factor IX (Benefix) • Factor VIIa (Niastase) • Palivizumab (Synagis) • Humanized monoclonal antibody indicated for the prevention of respiratory syncytial virus RSV LRT infections in at-risk infants • Produced in hybridoma cell line • Some data suggests superiority over RSV-IVIG

14. Recombinant CBS coagulation proteins TF-FVIIa FX FXa FIXa TF-FVIIa FVIIIa PROTHROMBIN IIa IIa FXIa FXa FVa FV FVIII FIX IIa FXIII IIa IIa FIBRINOGEN FIBRIN FXI FXIIIa CROSS-LINKED FIBRIN

15. FVIII Huge (2332 amino acids, 165 – 280 kDa) B domain is heavily glycosylated and dispensable FVIII circulates as a heterodimer; HC varies in length A1 A2 B C1 C2 A3 Processed during synthesis Variable Heavy Chain A1 A2 B 2 C1 C2 A3 Light Chain

16. Recombinant FVIII products • FVIII circulates complexed to von Willebrand factor • 50: 1 vWF: fVIII • Unstable without vWF • Two recombinant expression strategies • Co-express VIII & vWF in CHO cells, remove vWF before formulation (Genetics Institute/Baxter) • Express fVIII alone (Bayer) • Initial products required stabilization with albumin (HSA) when formulated A1 A2 B 2 C1 C2 A3 VWF

17. FVIII: New developments • Alterations to reduce or eliminate exposure to HSA, FBS or other human plasma proteins in culture or formulation (Kogenate FS, Advate) • Recombinate is still formulated with HSA • Refacto (B-domain deleted rFVIII) • Xyntha (Refacto made without HPP, purified using synthetic affinity ligand, not murine MAb) • In clinical development: PEGLip-FVIII-FS (Bayer), Kogenate FS formulated with pegylated liposomes to increase biological half-life (T&H Sept’08 16 patients) • Pre-clinical: Baxter FVIII modified with “releasable” PEG conjugates • Research: Mutant rFVIII molecules with mutated clearance motifs, stabilized (S-S or fusion protein) A1-A3 linkages

18. FIXa FX FVIIIa 1 415 FIX 1 145 181 415 FIXa S S Factor IX • A vitamin K-dependent clotting factor • 55.4 kDa plasma protein, 5g/mL (90 nM) • Complex - 415 aa, N-, O-glycans, sulpho-tyrosines, -OH aspartate, g-COOH** • Expressed in CHO cells modified to overexpress processing peptidase that removes pro sequence

19. Recombinant factor IX and potential development • BeneFIX • Only recombinant fIX product on market • Product enhancements (2007) allowed more rapid, needleless reconstitution • Baxter (early 2008) announced intention to market rfIX, develop pegylated rfIX with Nektar Therapeutics • Pre-clinical work suggests that activity can be enhanced (3X) by point mutation • Clearance slowed by K5A mutation (Stafford, UNC) or activation peptide deletion (Sheffield, CBS) (?)

20. Blood, July 1, 2002 issue K5A, K5R, WT HufIX made in 293 cells Injected into fIX ko mice to restore physiological [IX] K5R 79% in liver in 2 minutes; K5A 59% K5A initially cleared slower, later faster

21. Recombinant VIIa • Produced by Novo Nordisk in BHK cells • 406 amino acids; rfVII activates easily (autoactivation) by cleavage after Arg152 (no AP) • Activation encouraged by processing procedure to make NovoSeven (Niastase) • FVII also vitamin K dependent, some structural similarities to fIX

22. New developments in FVIIa • NovoSevenRT (Ready to Travel) heat-stable formulation • NovoNordisk has “short-acting” and “long-acting” VIIa in clinical development pipeline • MaxyVII (multiple mutations to increase activity, decrease clearance from circulation) • NN1731 (V158D/E296V/M298Q) in clinical development • Trial of NN1731 vs Niastase in hemophilia A patients with inhibitors being treated for acute joint bleeds commenced recruitment in June 2007 (2 years) • CSL Behring pursuing VIIa-FP, fusion protein to HSA, in preclinical studies (Weimer T et al Thromb Haemostas April 2008; WHC)

23. Production of recombinant proteins by genetically transformed mammalian cells in culture very $$$ Production by transgenic livestock potentially less expensive, higher capacity GTC Biotherapeutics (Framingham, MA) produces recombinant antithrombin (ATryn) in the milk of transgenic goats ATryn approved in Europe, phase III in USA – but indications for this protein are limited GTC continues to pursue rfIX production via this route and may be able to avoid issues that caused AmCross to drop this approach An approach to watch (10 years+)

24. Sheffield lab recombinant proteins • Prelude: Our simplest recombinant protein project • Published: Transforming alpha-1-proteinase inhibitor into a thrombin inhibitor • Submitted: The Trojan rabbit: Transforming albumin into a cross-linked component of a thrombus

25. Prelude: GST fusion proteins • Schistosomal glutathione sulfotransferase (GST) is well expressed in E. coli • Fused mouse fIX cDNA in frame (made one mini-gene with no stop codons between GST and fIX DNA) • Purified on glutathione-Sepharose column • Injected chickens • Harvested antibodies from eggs • Adsorbed vs. GST beads • Obtained specific polyclonal antibody

26. Published: Serpin work • Serpins are a protein family • Are SERine Protease Inhibitors • Many serpins are plasma proteins • Antithrombin, alpha-1-proteinase inhibitor (aka alpha-1-antitrypsin), heparin cofactor II, PAI-1, alpha-2-antiplasmin, C1-esterase inhibitor)… • My lab has attempted protein engineering to alter the specificity of serpins.

27. 1-Proteinase Inhibitor (1-PI) • 20 M in plasma • 394 amino acids (52 kDa) • Anti-neutrophil elastase k2 = 4 X 109 M-1min-1 • Poor inhibitor of thrombin k2 = 3 X 103 M-1min-1 http://www.expasy.ch/ch2dgifs/PLASMA_HUMAN/PLASMA_HUMAN.gif

28. 1-PI M358R • Good inhibitor of thrombin k2~ 1 X 107 M-1min-1 • Antithrombotic drug candidate? • Good inhibitor of Activated Protein C k2~ 3 X 106 M-1min-1 • How to maximize anti-thrombin activity? Huntington JA et al. Nature 2000

29. Harnessing the HCII “tail” • HCII 1-75 binds thrombin exosite 1 • Needs activation by GAGs • We grafted HCII 1-75 to 1-PIM358R Baglin TP et al. PNAS 2002

30. 394 1 API (M358R) * - - 1 75 Acidic tail HAPI (M358R) * - - HCII 1 480

31. - - 1 75 Acidic tail HAPI (M358R) * Merging extra- and intra-loop approaches • Would making the RCL more HCII-like or more AT-like increase selectivity for thrombin over APC? Filion ML et al. Biochemistry 2004 EGTQATTVTTVGFMPRSTQPE API / HAPI RCL 4 Hopkins PC et al. J Mol Evol 2000 KGTEAAGAMAVVIAPRSLPPE API / HAPI RCL 5

33. Kinetic results

34. TCT • Prothrombin-depleted plasma + thrombin + inhibitor • Measured time to clot • HAPI RCL5 more effective than HAPI RCL4

35. In vivo stability in mice Results were published (Sutherland et al.) in Thromb Haemostas November 2007)

36. Submitted: The Trojan “Bunny” • Albumin, the most abundant plasma protein • Not glycosylated but has many disulphide bonds • Pichia pastoris yeast best system for expression • Pp HSA shown to be biochemically identical to HAS but scale defeated recombinant expression except in Japan (Bipha Corp product approved May 2007) • We use the same system to make recombinant albumin and albumin fusion proteins.

37. Albumin • Most abundant plasma protein protein (35-50 mg/ml) • 584 amino acids, 67 kDa • Slowly cleared (t1/2 = 4-5 days in rabbits, 18 days in humans) • Non-glycosylated

38. RSA HLAH 6 HV3 RSA His6 Genetic fusion to albumin • Concept: Increase molecular volume and/or negative charge • Mechanism: Reduction in glomerular filtration, other potential size-related effects, possible stabilization, masking of labile bonds • Geometry: N-terminal HV3, then GLY6, then RSA, then HIS6

39. Production of albumin fusion proteins in yeast (P. pastoris) • Yeast are simple eukaryotes • Fold complex proteins better than E. coli

40. Interfering with blood clots • Rationale: Speed clot lysis of thrombi • Alpha-2-antiplasmin is cross-linked to fibrin by fXIIIa & inhibits plasmin • Concept: Fool the clot into incorporating a modified albumin that cannot inhibit plasmin (kind of a Trojan horse), make the clot easier to dissolve

41. We fused portions of alpha-2-AP that cannot inhibit plasmin to HSA • Only one, A2AP(13-42)-HSA, was well-expressed • Amino acid sequencing showed that it was a mixture of intact and cleaved termini

42. A2AP(13-42)-HSA, became a substrate for fXIIIa, like native A2AP

43. Plasma clotted with thrombin in the presence of tPA lysed rapidly (3) • Clot lysis was delayed by inclusion of A2AP(13-42) fusion protein (FP) • Submitted to BMC Biotechnology August 19, 2008 • Under review • Not quite a Trojan Horse (yet) but shows promise…more like a Trojan rabbit

44. Objectives achieved? • General issues common to recombinant proteins on an industrial and research scale • CBS’ 4 recombinant drugs (FVIII, FIX, FVIIa, Synagis) and the predicted next new product (PegLip FVIII) • GST-mfIX, HAPI RCL5, and A2AP(13-42)-HSA, 3 examples of recombinant protein expression from my lab • Acknowledgements to my lab • Current members: Varsha Bhakta, Sharon Gataiance, Louise Eltringham-Smith, • Graduates Jason Sutherland (PhD), Marc Filion (MSc)