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

Transfusion Medicine Residents

2008-09-25

Bill Sheffield

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]

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

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To present published and unpublished research from the Sheffield laboratory relating to recombinant plasma proteins

Source: www.artes-biotechnology.com

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

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.

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

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?

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

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)

A word about protein folding• Proteins: linear

chains of amino acids

• Often shown in bar graph form

• Actually must have appropriate 3D structure to function, avoid clearance, avoid immunogenicity (?)

HV3 RSA HisHis66

RSA

HLAH6

Mammalian cell expression: Pluses and Minuses

• 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 Annu. Rev. Cell Dev. Biol. 2000. 16:557-589

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

FIBRINOGEN FIBRIN

TF-FVIIa

FX

FXa FVFVa

FIX

FIXa

IIa

PROTHROMBINIIa

FXa

TF-FVIIa

FXIa

FXI

IIa

FVIIIa

FVIII

IIa

CROSS-LINKED FIBRIN

FXIIIa

FXIIIIIa

Recombinant CBS coagulation proteins

A1 A2 B C1 C2

Huge (2332 amino acids, 165 – 280 kDa)B domain is heavily glycosylated and dispensableFVIII circulates as a heterodimer; HC varies in length

A3

A1 A2 B

C1 C2

Variable Heavy Chain

Light Chain

2

A3

FVIII

Processed during synthesis

A1 A2 B

C1 C2

2

A3

VWF

• 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

Recombinant FVIII products

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

Factor IX

• A vitamin K-dependent clotting factor• 55.4 kDa plasma protein, 5g/mL (90 nM)• Complex - 415 aa, N-, O-glycans, sulpho-

tyrosines, -OH aspartate, COOH**• Expressed in CHO cells modified to overexpress

processing peptidase that removes pro sequence

FIX

FIXaS S

11 415415

11 181181 415415145145

FIXaFIXa FXFX

FVIIIaFVIIIa

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) (?)

• 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

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

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)

An approach to watch (10 years+)• 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

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

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

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.

1-Proteinase Inhibitor (1-PI)

http://www.expasy.ch/ch2dgifs/PLASMA_HUMAN/PLASMA_HUMAN.gif

• 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

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

Harnessing the HCII “tail”

• HCII 1-75 binds thrombin exosite 1• Needs activation by GAGs

• We grafted HCII 1-75 to 1-PI M358R

Baglin TP et al. PNAS 2002

* APIAPI (M358R) (M358R) 1 394

HHCII CII

* HAPIHAPI (M358R) (M358R)

1 75

1 480

Acidic Acidic tail tail

- -

- -

k2 (M-1min-1)  

Thrombin APC Selectivity Ratio

1-PI M358R 1.0 0.5 107 1.4 0.1 106 7.9

HAPI M358R 2.30 22 108 1.8 0.1 106 128

Merging extra- and intra-loop approaches

* HAPI (M358R) HAPI (M358R)

1 75

Acidic Acidic tail tail

- -

EGTQATTVTTVGFMPRSTQPE

KGTEAAGAMAVVIAPRSLPPEHopkins PC et al. J Mol Evol 2000

Filion ML et al.Biochemistry 2004 API / HAPI RCL 4

API / HAPI RCL 5

• Would making the RCL more HCII-like or more AT-like increase selectivity for thrombin over APC?

-thrombin APC-thrombin /APC

k2

( 106

M-1min-1)SI

k2

( 106

M-1min-1)

1-PI

M358R 11 0.5 3.2 0.3 1.4 0.1 7.9

HAPI M358R

230 22b 2.9 0.1 1.8 0.1 128

HAPI RCL4

22 4 26 1.6 0.01 0.002 2,200

HAPI RCL5

140 5 2.1 0.2 0.013 0.002 10,769

Kinetic results

TCT• Prothrombin-

depleted plasma + thrombin + inhibitor

• Measured time to clot

• HAPI RCL5 more effective than HAPI RCL4

In vivo stability in mice

Results were published (Sutherland et al.) in Thromb Haemostas November 2007)

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.

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

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

HV3 RSA HisHis66

RSA

HLAH6

• Geometry: N-terminal HV3, then GLY6, then RSA, then HIS6

Production of albumin fusion proteins in yeast (P. pastoris)

• Yeast are simple eukaryotes

• Fold complex proteins better than E. coli

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

• 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

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

• 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

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)