Chava Kimchi-Sarfaty, Ph.D.

Division of Hematology Research and Review Office of Blood Research and Review (OBRR)

Center for Biologics Evaluation and Research (CBER) Food and Drug Administration (FDA)

WCBP, January 2015

What is Behind Sequence Variation in Recombinant Therapeutic Proteins?

The findings and conclusions in this presentation have not been formally disseminated by the Food and Drug

Administration and should not be construed to represent any Agency determination or policy.

Disclaimer

Protein therapeutics – benefits and importance

“Silent mutations” – understanding synonymous mutations and polymorphisms ADAMTS13

Codon optimization – recognizing the advantages of

optimized proteins Factor IX

Summary/Conclusions

Today’s Presentation

Protein therapeutics – benefits and importance

“Silent mutations” – understanding synonymous mutations and polymorphisms ADAMTS13

Codon optimization – recognizing the advantages of

optimized proteins Factor IX

Summary/Conclusions

Today’s Presentation

Bioengineered proteins: Why do we need them?

Most proteins are not good drug-products due to:

• Short serum half-life • Poor bioavailability • Lack of post-translational modifications • Inefficient production

Therapeutic-protein development allows for

engineered improvements in product quality, therapeutic outcomes, patient convenience and manufacturing efficiency.

Protein therapeutics are increasingly important in modern medical practice

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2004 2006 2008 2010 2012

YEAR

FDA

Appr

oval

s (B

iolo

gics

/Sm

all M

olec

ules

) More protein therapeutics are entering into clinical practice Protein-based therapeutics have significantly changed the medical landscape for treating complex diseases and managing unmet medical needs.

Cytokines Interleukin-2 Aldesleukin

(Proleukin®) • -Glycosylation; -N-terminal alanine, S125C AA substitution. • Half-life = 85 minutes.

Interferon beta-1b Interferon beta-1b (Betaseron, EXTAVIA®, Ziferon)

• S17C substitution; -Carbohydrate side chains. • Mean half-life = 8 minutes - 4.3 hours.

Interferon beta-1b Interferon beta-1b (Betaferon®)

• S17C substitution; No Met at position 1; -Carbohydrate moieties. • Levels remained above baseline throughout the seven day (168 hour) study period.

Colony stimulating factors (CSF) G-CSF Filgastrim

(Neupogen®) • -Glycosylation; +N-terminal methionine

Erythropoiesis stimulating agent (ESA) Erythropoietin (EPO)

Darbapoetin alfa (Aranesp®)

• +2 N-linked oligosaccharide chains

Coagulation Factors rFVIII-B domain deleted

rFVIII B domain deleted (Refacto®)

• Deletion results in higher secretion

Bioengineered Proteins with Modified Sequences that have been Approved or are under Development

Hormones

Basal Insulins Glargine (Lantus®)

• D21G substitution at A-chain; +2 Arg residues at C-terminus of B-chain. • Relatively constant concentration over 24 hours with no pronounced peak.

Detemir (Levemir®)

• +C14 fatty acid chain to lysine at B29; Thr residue removed at B30; • Relatively constant serum concentration over 24 hours.

Degludec (Tresiba)

• +C16 fatty acid chain to lysine at B29 via Glu spacer; Thr residue removed at B30 • Ultra long half-life of ~ 40 hours.

Short acting insulins

Lispro (Humalog®)

• P28K and K29P substitutions at B-chain; • Absorbed faster and has a shorter half-life due to lack of hexamer formation.

Aspart (Novorapid®, Novolog®)

• P28D substitution at B-chain. • Faster absorption, onset of action, and shorter duration

Glulysine (Apidra®) • N3K and K29E substitutions in B-chain • More rapid onset of action and shorter activity duration

GLP-1 Analogues

Liraglutide (Victoza®) • R34K substitution; C16 fatty acid addition at position 26 • Half-life of 13 hours (vs. half-life of < 2 minutes of human GLP-1)

Parathyroid hormone (PTH)

Teriparatide (Forteo®) • Truncated PTH 1-34aa instead of 1-84aa identical to human PTH.

Bioengineered Proteins with Modified Sequences that have been Approved or are under Development

All Information Publically Available From: Aldesleukin http://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?id=37301

Interferon beta-1b http://dailymed.nlm.nih.gov/dailymed/lookup.cfm?setid=261fde67-efb2-4bd7-947e-4f68a56e76ff

Betaferon http://berlex.bayerhealthcare.com/html/products/pi/Betaseron_PI.pdf

EXTAVIA http://www.rxlist.com/extavia-drug.htm

Filgastrim http://pi.amgen.com/united_states/neupogen/neupogen_pi_hcp_english.pdf

Aranesp http://pi.amgen.com/united_states/aranesp/ckd/aranesp_pi_hcp_english.pdf

Refacto http://www.abopharmaceuticals.com/ProductSheets/Refacto.pdf

Lantus http://products.sanofi.ca/en/lantus.pdf

Levemir http://dailymed.nlm.nih.gov/dailymed/archives/fdaDrugInfo.cfm?archiveid=8918

Tresiba

http://druginfo.nlm.nih.gov/drugportal/ProxyServlet?mergeData=true&objectHandle=DBMaint&APPLICATION_NAME=drugportal&actionHandle=default&nextPage=jsp/drugportal/ResultScreen.jsp&TXTSUPERLISTID=0844439969&QV1=INSULIN+DEGLUDEC

Humalog http://pi.lilly.com/us/humalog-pen-pi.pdf

Novorapid http://www.novonordisk.com/images/diabetes/pdf/Novorapid%20Product%20Monograph.pdf

Novolog http://www.fda.gov/ohrms/dockets/ac/06/briefing/2006-4254b_13_04_KP%20InsulinAspartFDAlabel102005.pdf

Apidra http://products.sanofi.ca/en/apidra.pdf

Victoza http://www.rxlist.com/victoza-drug.htm

Forteo http://www.rxlist.com/forteo-drug.htm

TERTIARY STRUCTURE

HETERO-GENEITY

IMMUNO-GENICITY PK/PD BIOACTIVITY AGGREGA-

TION

MUTATIONS

FUSION PROTEINS

CODON OPTIMIZED PROTEINS

MODERATE

HIGH

MODERATE TO HIGH

LOW

HIGH

HIGH

LOW?

MODERATE TO HIGH

LOW?

LOW TO MODERATE

HIGH

LOW

LOW TO MODERATE

HIGH

LOW

LOW TO MODERATE

MODERATE TO HIGH

MODERATE TO HIGH

Risk Assessment of Three Principal Protein-engineering Strategies

Bioengineered proteins: Platform Technologies for Fusion Proteins

PEGylation

Fc-fusion Albumin-fusion

XTEN-fusion

PolyXen-fusion

Rath T et al (2014) Critical. Rev. Biotech.

Bioengineered proteins: An emerging trend

Platform technology Therapeutic areas Products (number)

PEGylation Cytokines, colony stimulating factors, erythropoiesis stimulating agents, hormones, coagulation factors, oncology 23

Fc-fusion Immunosuppressive, anti-angiogenic, coagulation factors, thrombopoietin stimulating factors, enzyme replacement 12

Albumin-fusion Cytokines, colony stimulating factors, coagulation factors, hormones, oncology 6

XTEN-fusion Coagulation factors, hormones, enzyme replacement proteins 10

PolyXen-fusion Cytokines, colony stimulating factors, erythropoiesis stimulating agents, hormones 4

Protein therapeutics – benefits and importance

“Silent mutations” – understanding synonymous mutations and polymorphisms ADAMTS13

Codon optimization – recognizing the advantages of

optimized proteins Factor IX

Summary/Conclusions

Today’s Presentation

AUG AAG UUU GGC

AUG AAG UUU GGU

Met Phe Gly

Met Lys Phe Gly

Lys A synonymous

change

NO change in primary sequence

AUG AAG UUU GGC

AUG AAG UUU GUC

Met Phe Gly

Met Lys Phe Val

Lys A non-synonymous

change

Primary sequence altered

Even synonymous changes in genetic code can affect protein levels and protein conformation,

producing physiological effects

Breaking the silence. Katsnelson. Nature Medicine 17: 1536-1538. 2011

Synonymous Nucleotide Changes are Not Silent

Understanding “Silent” Mutations – 2013 FDA Report

Synonymous mutations can impact critical cis regulatory sequences: • Splice sites, ESEs and ESSs

altered protein primary structure

• microRNA and exonic TF binding sites

altered gene expression

Methods:

IN SILICO: NNSplice, NetGene2, GeneSplicer, Human Splicing Finder, ESEfinder, RESCUE-ESE, FAS-ESS, ESRsearch, ExonScan IN VITRO: RT-PCR, northern blot, mini-gene splicing assay, FISH, RNA oligonucleotide hybridization & pull down, exon-specific RNAi

Kimchi-Sarfaty C. et al.: Trends Pharmacol Sci.34(10):534-48, 2013

Gene Regulation and mRNA Processing

Synonymous mutations can shape local mRNA structure: i. Unstable mRNA easily degraded -> Low protein

expression ii. Very stable 5’ region -> hinders translation initiation ->

Low protein expression

Methods: • IN SILICO: • IN VITRO: RT-PCR, northern blot, mini-gene splicing assay, FISH, RNA

oligonucleotide hybridization & pull down, exon-specific RNAi

Methods:

IN SILICO: mFold, Kinefold, remuRNA, RNAfold

IN VITRO: endoribonuclease footprinting, transcript labeling & transcription inhibition, quantitative real-time PCR, circular dichroism

Edwards N.C., ... Kimchi-Sarfaty C. : PLoS ONE 7;6 e38864, 2012; Salari R., Kimchi-Sarfaty C. et al.: Nucleic Acids Res, 41:44 - 53 , 2013; Hamasaki-Katagiri N., … and Kimchi-Sarfaty C.: JMB, 425: 4023 - 4033, 2013.

mRNA structure

Major influences of translational speed: i. Synonymous codon choices

ii. Positively charged amino acids interacting with negatively charged 50S exit tunnel

iii. mRNA structure

Methods:

IN SILICO: SeqForm, SeqX

IN VITRO: in vitro translation, polysome profiling, ribosome profiling/footprinting, single molecule translocation, optical tweezers, pulse-chase, cycloheximide treatment, enzymatic digestion, circular dichroism, markers of Unfolded Protein Response

Protein Translation Kinetics

- A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13 - metalloprotease enzyme that cleaves von Willebrand Factor (VWF). VWF assists platelet adhesion to wound sites.

Sadler, Blood (2008)

Synonymous Mutations in ADAMTS13

ADAMTS13 SNPs

ADAMTS13:

Tseng, Kimchi-Sarfaty, Pharmacogenomics, 12(8):1147-60, 2011

Hing et al., BJH 160(6):825-37, 2013; Saini ….Kimchi-Sarfaty C. Proteases in Health and Diseases (2013)

• Located at metalloprotease domain surface • Far from the active site • Naturally occurring variant

Characterizing Pro118Pro mutation (P118P, CCG>CCA) and non-synonymous “control” (P118F, CCG>TTC) using ADAMTS13 protein model and in silico tools

Synonymous mutant: Pro118Pro (P118P, CCG>CCA)

Non-synonymous control: Pro118Phe (CCG>TTC)

• This residue is exposed to the surface, far from the active site

• Phe is similar to Pro in hydrophobic nature, charge

• Phe is more bulky and flexible than Pro

Proline

Phenylalanine

Similar levels of ADAMTS13 mRNA in WT, P118P and P118F

ADAMTS13 mRNA, activity levels, and in vitro translation levels

Different levels of ADAMTS13 activity in WT and P118P

Different in vitro translation levels of WT and P118P ADAMTS13

0

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WT M1 M2

mRN

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T

WT P118P P118F

ADAMTS13 carrying a synonymous mutation has different inactivation rate compared to WT

Our characterization of the synonymous mutation, P118P, in ADAMTS13 demonstrated ~25% increase in expression level and ~25% increase in half-life.

Results and Conclusions

Protein therapeutics – Benefits and Importance

“Silent mutations” – Understanding synonymous mutations and polymorphisms ADAMTS13

Codon optimization – Recognizing the advantages of

optimized proteins Factor IX

Summary/Conclusions

Today’s Presentation

Codon optimization strategies

Expr

essio

n le

vel

Kimchi-Sarfaty et al., Science 315, 525-528, 2007; Gartner J., ... Kimchi-Sarfaty C. et al.: P Natl Acad Sci USA, 110:13481 - 13486, 2013; Sauna E.Z. and Kimchi-Sarfaty C.: Electronic Library of Science (eLS), Elsevier, 2013

Codon Optimization: An Augmentation of Protein Expression through a study of Factor IX

“a sophisticated algorithm that considers all relevant transcription and translation optimization parameters in a single operation and delivers a DNA sequence configured with your specifications, optimized for maximum performance in your system.”

Codon Optimization Commercial Method: Gene Optimizer®’s considerations

Total number of publications in PubMed

Publications that involve codon optimized genes

Increased Interest in Codon Optimization

Gene (exon )

Protein

Activated protein (FIXa): serine protease, 461 amino acids, 55 kDa

GLA (Ca2+ binding) Pre-pro

leader EGF-like domain

Activation Peptide

Catalytic domain

Cleavage GLA (Ca2+ binding) Pre-pro

leader seq

EGF-like domain

Catalytic domain

S S

HB

Gene F9

Incidence 1 : 30,000 males

Treatment pd or rFIX,

gene therapy (clinical trials)

Examples of Regulated Biologics

BeneFIX (Wyeth) AlphaNine (Grifols)

Mononine (Behring)

Inhibitor Development 3-5%

Factor IX and Hemophilia B patients

Hamasaki-Katagiri….Kimchi-Sarfaty: Haemophilia, 2012

The four codon sliding average relative synonymous codon usage (RSCU) was calculated across optimized and non-optimized transcripts. The translation rate and consequently, folding of FIX, may be affected by RSCU patterns.

00.20.40.60.8

11.21.41.61.8

2

1 101 201 301 401

Amino Acid No.

+/-4

Cod

on R

SCU

ave

rage

WT

Opt

Optimized F9 utilizes more common codons

60.9% of the codons are altered, equating to 22.5% of the nucleotides

Codon Substitutions between WT and Optimized FIX

Aptamer binding

Codon Optimization Assumes Synonymous Mutations have No Consequences

WT

WT

Opt

Opt

Opt

Extra Intra

FIX-V5

0

20

40

60

80

100

120

WT Opt#2

% F

IX s

peci

fic a

ctiv

ity

Opt WT

FIX WT 58.1 13.9 3.1 9.4 3.9 11.6 5.55 17.7FIX Codon Optimized 43.1 12.7 3.5 10.9 3.3 26.5 5.16 16.1

% peak areaK1K2

(6Gla)R-K1K2 (6Gla)

K1K2 (5Gla)

K1K2' (5Gla)

R-K1K2 (5Gla)

K1 K2 (3,4Gla)

Total Gla

Pro-peptide %

FIX WT 70 21.4 8.6 5.61FIX Codon Optimized 32.8 34 33.2 5

% peak areaK3

(6Gla)K3

(5Gla)K3

(4Gla)Total Gla

γ-carboxylation level of K1K2 peptide and propeptide determined by Lys-C peptide map

γ-carboxylation level of K3 peptide determined by Lys-C peptide map

GLA status of WT vs Codon Optimized Factor IX

Wild type (WT) and optimized (Opt) factor IX activity in the presence of Hemophilia B patient plasma containing inhibitory antibodies

Structural/ Conformational Differences: Inactivation Curves

Protein therapeutics – Benefits and Importance

“Silent mutations” – Understanding synonymous mutations and polymorphisms ADAMTS13

Codon optimization – Recognizing the advantages of

optimized proteins Factor IX

Summary/Conclusions

Today’s Presentation

Designing proteins with synonymous mutations that have a minimal

impact on protein characteristics/safety while improving production could

be a wise approach for better recombinant drug development; note that

some other characteristics may change

We have evidence indicating that one version of gene-optimized F9 yields

a protein product with unique conformation and functionality relative to

WT F9

Katsnelson: Nature Medicine 17 (12) , 2011; Sauna and Kimchi-Sarfaty: Nature Review Genetics, 12:683, 2011

Results and Conclusions

Robert Peters

My lab Zuben Sauna Daron Freedberg Kazuyo Takeda Mansoor Khan Basil Golding

Anton Komar

The work is supported by: FDA CBER, ModScience AHA 13GRNT17070025 NIH 1R15HL121779-01A1

Mark Welch and Claes Gustafsson

Acknowledgements

Teresa Przytycka