Surrobodies™ – A Novel Approach to Bispecifics…sealanebio.com/media/Sea Lane IBC DDT deck...

Surrobodies™ – A Novel Approach to Bispecifics…

Ramesh Bhatt, Ph.D.Vice President of ResearchSea Lane Biotechnologies

1Sea Lane – DDD presentation August 8, 2012 Sea Lane Biotechnologies – Proprietary

Presentation Agenda

Introduction to Surrobody structureSurrobody advantages directly address bispecific challengesThree brief bispecific examples

Comparability to antibodiesInhibiting soluble targets in vitro and in vivoTargeting receptors in vitro

SummarySurrobody Drug Conjugate Teaser


SurrobodiesHighly adaptable platform inspired by

endogenous human pre-B Cell Receptor

SurrogateLight Chain

VpreB

λ5

VpreB

λ5

Fused SLCNative SLC


The Sea Lane Advantage

• The invariant surrogate light chain simplifiesand accelerates bispecific development and enables drug conjugation for all existing and future Surrobodies

• Proprietary design of fully human, synthetic libraries mirror natural human diversity enabling rapid, high quality lead generation and simple, comprehensive optimization

• Use of naturally occurring, human germline frameworks provides consistently robust expression characteristics and should reduce immunogenic potential

Surrobody™

SurrogateLight Chain

(blue/yellow)

HeavyChain

(green)


Surrogate Light Chain Only known universal heavy chain partner Expected low immunogenicity Invariant protein that simplifies all steps from discovery through manufacture

Intact FcImparts effector function and favorable half lifeKnown scalable production capable through Protein A

Bispecific/Drug Conjugate ReadyRapid conversion to enhanced format

Intellectual PropertyGranted patents protect basic structure and synthetic human library design

General Surrobody Strengths

Complete Heavy Chain

SLC


Synthetic Human Surrobody LibraryDrives Discovery

~28 billion productive membersPatented human diversity productively matches frameworks and CDRs

Based on functional diversityPowers companion optimization technology

Novel library construction processes maximize library fidelity12 week panning through lead ID cycle timeSurrobodies found against 14 different therapeutic targets


Surrobodies Provide Ideal Path for Bispecific and Drug Conjugate Applications

BispecificsSimplified structure dramatically reduces required resourcesAntibody-like PK and other beneficial CMC qualitiesRobust in vivo efficacyMultiple bispecific formats provide unmatched flexibility

Toxin Drug ConjugationSimplified process utilizing the universal surrogate light chain is applicable to all SurrobodiesBioanalytic assessment supports robust and stable conjugation Resulting toxin conjugated Surrobodies are highly potent


Fc Engineering Improves Bispecific Assembly but Provides an Incomplete Solution

25% Productive

75% Unwanted

A A B B

BA

BABA BA

Fc engineered heteromericHeavy chain assembly

8

Light chain mismatching generates predominant and undesirable products

Sea Lane – DDD presentation August 8, 2012 Sea Lane Biotechnologies – Proprietary

Sea Lane Advantage:The Surrogate Light Chain

9

Invariant surrogate light chain partnering eliminates unproductive combinations caused by mismatching of light chain/heavy chain partners

A A

BA

B B


Bispecific Example #1Comparability to Antibodies


Surrobodies DisplayAntibody-Like Characteristics

BiochemicalLong term RT stability >1yrHigh level protein expression

Many >100mg/L in HEK293

Stable following physical stressfreeze/thaw cyclesLow pH 3 exposureShort term High temp exposure

• at 50°C for 48 hours

High concentration (100mg/ml)

All Surrobody formats maintain thermal stability profiles that are similar to commercial antibodies

Tm >65°C

In vitroHigh affinity binding (subnanomolar)Antagonist and Agonist activities are possibleBroad epitopic range

In VivoNHP half-life ~1-2 wksExcellent efficacy in rodent models of disease


Monovalent Bispecifc Surrobodies Simultaneously Bind Two Distinct Targets

0

1

2

3

4

0.01 0.1 1 10 100

OD

450

nm

[nM]

HGF + PlGF :Fc(bi)

P

H

HGF

BiotinPlGF

SAHRP

12

HGF + PlGFBispecific


Mammalian Expressed Surrobodies Have Excellent Characteristics

Format

Unique proteins(# preps)

Avg yield

(mg/L)

Intact SgG (%)

High MW“Aggregate”

(%)

Low MW“Protein”

(%)

Thermal Stability

(oC)

RT Stability (months)

Standard Surrobody

194(249)

77 98.2% 1.6% 0.2% >65 24

Bispecific Monovalent

Binding

72(82)

81 88.2% 3.4% 8.4% >65 12

All 16 possible bispecific Vh framework combinations produce comparable yields in HEK293 transient systems


Size Exclusion and Mass Spec Show High Quality Monovalent Bispecific Complexes

mass147000 148000 149000 150000

%

0

100 94.8147981.1

148072.1

SL-183

Bi-specificTarget 1:Target 2Mass Spec analysis:

Virtually all heterodimeric bispecific

SEC analysis: Cotransfection results in single peak at the appropriate size marker

MWStandards

Protein A purifiedBispecific Surrobody


7400 147600 147800 148000 148200 148400 148600 1488

7_sample05 157 (5.321) M1 [Ev-120888,It10] (Gs,0.300,2207:3792,0.30,L30,R30); Cm (154:167)148102.4

148201.7

147400 147600 147800 148000 148200 148400

196 (6.641) M1 [Ev-147837,It10] (Gs,0.300,2015:3843,0.30,L30,R30); Cm (193:205)147800.9

147899.0

147400 147600 147800 148000 148200 148400

184 (6.234) M1 [Ev-142519,It10] (Gs,0.300,2042:3846,0.30,L30,R30); Cm (179:191)147844.1

147942.8

148041.8

147400 147600 147800 148000 148200 148400 148600

_sample08 166 (5.625) M1 [Ev-141161,It10] (Gs,0.300,2158:3753,0.30,L30,R30); Cm (159:182)147884.6

147984.8

147400 147600 147800 148000 148200 148400 1486

87_sample09 206 (6.979) M1 [Ev-140978,It10] (Gs,0.300,2014:3660,0.30,L30,R30); Cm (201:218)147888.8

147987.5

147700 147800 147900 148000 148100 148200 148300

] ( ) ( )148004.5938

148104.2031

7400 147600 147800 148000 148200 148400 148600

118) M1 [Ev-120766,It10] (Gs,0.300,2156:3427,0.30,L30,R30); Cm (145:171)148013.5938

147588.5000

148112.0000

7400 147600 147800 148000 148200 148400 148600

03 190 (6.437) M1 [Ev-158758,It10] (Gs,0.300,2023:3934,0.30,L30,R30); Cm (187:206)148017.5000

148116.7969

147400 147600 147800 148000 148200 148400 148600 148800

175 (5.930) M1 [Ev-146679,It10] (Gs,0.300,2137:4000,0.30,L30,R30); Cm (172:189)148061.0000

148158.7969

H/P H/V H/B5 H/B6

H/D V/B5 V/B6 V/D B5/D

Deglycosylated proteins

(Focusing on region of intact tetramers:

~148,000 Da)

LC-MS Analysis of a Collection of Monovalent Bispecific Surrobodies


Monovalent Bispecific Surrobodies are Stable Following Storage at Room Temp for 12 Months

ELISA binding isotherms show no loss of activity between -80°C and room temperature.

-1 0 1 2 30

1

2

3

4-80oC4oCR.T

log [conc] nM

OD

450n

m

EC50 (nM)

-80°C 1.92

4°C 2.12

Room Temp 1.87

-80°C4°CR.T


The PK Properties of Bispecific Surrobodies are Comparable to Parentals in Cynomolgus

AgentHalf-life(days)

Clearance(mL/hr/kg)

Vss(mL/kg)

Bispecific (HGF/PlGF) 6.04 0.42 77.2

Monospecific PlGF 6.35 0.35 78.5

Monospecific HGF 9.28 0.40 110

HGF IgG 10.88 0.19 75.6

0 10 20 300.1

1

10

100

1000 Bispecific HGF/PlGF

Monospecific PlGFMonospecific HGF

time (days)

ug/m

l

Naïve groups (n=3) of Cynomolgus monkeys were administered single IV dose of Surrobodies (10 mg/kg)Serum was tested over a 28 day periodThe overall PK properties are “Antibody-like”


Surrobody Thermostability is Comparable to Marketed Antibodies

SL-0

02

SL-0

12

SL-1

76

SL-1

86

SL-1

88

Erbi

tux

Her

cept

in50

55

60

65

70

Tm° C

Target Format

SL-002 PlGF

MonospecificSL-012 HGF

SL-176 ErbB3

SL-186PlGFHGF

Bispecific v1

SL-188 Bispecific v2

Tm was calculated in a fluorescent-based assay of agents in PBS


Bispecific Example #2Targeting Two Soluble Factors

in vitro and in vivo


GF x Ligand Bispecific Properties are Similar to Parental Surrobodies

All values [nM]Ligand-1 Ligand-2 GF

Molecule(specificity)

CellIC50

ELISA Binding

ELISA Inhibition

ELISA Binding

CellIC50

SL-429(Ligand) 0.281 0.020 0.156

SL-516(GF) 0.086 0.139

SL-634(Ligand x GF)MonovalentBispecific

Not Tested 0.048 0.955 0.055 2.602


Anti-GF/Ligand Bispecific Captures the Synergistic Benefits of Dual Inhibition In Vivo

Mice were treated 2x weekly by iv injection. Groups received 5mg/kg each, except bispecific which was 10mg/kg

21Sea Lane – DDD presentation August 8, 2012

Colo205 Day 54

0

500

1000

1500

2000

100%TGI

73%

α-ligand

58%

α-GF

94%

α-GF + α-Ligandcocktail

96%

α-Ligand/α-GFBispecific

Median tumorgrowth inhibition

0%

PBS

Tum

or v

olum

e (m

m3 )

+

Sea Lane Biotechnologies – Proprietary

Anti-GF/Ligand Bispecific Captures the Synergistic Benefits of Dual Inhibition In Vivo

Colo205 Xenograft

1 8 15 22 29 36 43 50 570

500

1000

1500

2000

PBS

Anti-LigandAnti-GF (n = 9)

Anti-GF/Ligand BispecificAnti-GF + Anti-Ligand

Day of study

Mea

n tu

mor

vol

ume

(mm

3 ) ± S

EM

Mice were treated 2x weekly by iv injection. Groups received 5mg/kg each, except bispecific which was 10mg/kg


Bispecific Example #3In vitro Targeting

ErbB3 x Growth Factor Receptor


ErbB3 Summary

ErbB3 represents a pivotal node in regulation of cell proliferation

Implicated in the generation of resistance to a number of key oncology agents

Sea Lane’s anti-ErbB3 is a potent inhibitor with a novel mechanism of action in ErbB2 overexpressing cells (Molecular Cancer Therapeutics; July, 2012)

Sea Lane’s anti-ErbB3 Surrobodies synergistically enhance the activity of ErbB2 and EGFR antibodies

Bispecific ErbB3 x GFR surrobodies capably capture a similarly synergistic cocktail


Anti-ErbB3 Surrobodies Enhance the Activity of Trastuzumab

-4 -2 0 2

0

20

40

60

80

log [SgG/IgG] nM

Perc

ent I

nhib

ition

-4 -2 0 2

0

20

40

60

80

log [SgG/IgG] nM

Perc

ent I

nhib

ition SL-175

Trastuzumab

SL-175*+Trastuzumab

Pertuzumab+Trastuzumab

Pertuzumab

Unstimulated NRG-stimulated

* Nearly identical results were obtained using SL-176Foreman, et. al., Molecular Cancer Therapeutics 2012

SKBR3 Cells


Anti-ErbB3 Surrobody enhancement of anti-ErbB2 provides the rational basis for an ErbB3 x ErbB2 bispecific


Anti-ErbB3 Surrobodies Enhance the Activity of Cetuximab

-2 -1 0 1 2 3

0

50

100

SL-175+CetuximabSL-176+CetuximabCetuximab

SL-175SL-176

log [SgG/IgG] nM

Perc

ent I

nhib

ition

Anti-ErbB3 Surrobody enhancement of anti-EGFR provides the rational basis for an ErbB3 x EGFR bispecific

A431 cells

Sea Lane Biotechnologies – Proprietary 26Sea Lane – DDD presentation August 8, 2012

Bispecific ErbB3 x GFR Surrobody Captures Complimentary Efficacy of Parental Cocktail

Anti-GFR ( ) Surrobody combined with anti-ErbB3 ( ) Surrobody

SL-433 (Monovalent bispecific)

0.01 0.1 1 10 100 1000

0

20

40

60

80

SL-176 (Anti-ErbB3)

SL-396 (Anti-GFR)

SL-396 + SL-176

[SgG] nM

Per

cent

Inhi

bitio

n

SL-433


Summary

The Surrogate Light Chain is a natural universal Heavy Chain partner

Sea Lane’s fully human synthetic library drives Surrobody discovery

High level, high quality transient expression accelerates Surrobodies through in vitro and in vivo research

Bispecific Surrobodies are highly efficacious with characteristics that are comparable to antibodies

The Surrobody structure is amenable to additional formats that enable optimal therapeutic approaches including:

Multivalent bispecifics

Drug conjugation


Surrobody Drug Conjugates

Boosting the anti-tumor abilities of Surrobodies


Surrobody Drug Conjugates

Invariant surrogate light chain partner is the ideal site for drug conjugation

ALL Surrobodies use the exact same light chain

One solution will work across the entire platform from monospecific to bispecific agents

Light chain has been shown to be more desirable for conjugation than heavy chain


Surrobody Drug ConjugationResearch Milestones

Engineered Surrobody for drug conjugationModified surrogate light chain allows for 2 toxins per SurrobodyPossible to further modify to add more toxinToxin addition efficiency appears excellent

Demonstrated cellular potencies in vitroPOC target: GFR driven cell proliferation

Bispecific drug conjugation comparable to monospecific Surrobodies


Toxins Conjugate Robustly to Surrobodies

Modified Surrobody w/o toxinModified Surrobody plus toxin

0 1 2drugs per molecule

• Analysis of unoptimized Drug Conjugation shows predominance of Surrobodies containing two or one toxins per molecule

• Toxin conjugate: MonomethylAuristatin E (MMAE)


Possible to modify to add more toxinsPerforms as well as, or better than toxin conjugated antibody (not shown)

Surrobody Drug Conjugates More Potently Inhibit In Vitro Proliferation


0.0001 0.001 0.01 0.1 1 10 100 1000

0

20

40

60

80

100

Parent SurrobodyEngineered SLC controlEngineered SLC #1 + ConjugateEngineered SLC #2 + Conjugate

[IgG/SgG] in nM

per

cen

t in

hib

itio

n


Potency[pM] Efficacy

Parent 0.615 20%

Engineered SLC control 0.382 19%

Engineered SLC #1 + Conjugate

0.058 96%

Engineered SLC #2 + Conjugate

0.156 99%

+1 +20Drug permolecule

“A” x “B”


“A” x “C”


“B” x “C”

Blue tracing is Unmodified SgG/ Red tracing is toxin (MMAE) conjugated

Bispecific Surrobody Toxin Conjugation is Comparable to Monospecific Conjugation

Heteromeric Fc Bispecific Surrobodies


“A” x “N”


“N” x “A”


“B” x “N”

In unoptimized conjugation reactions almost all the surrobodies are conjugated with most carrying two toxin molecules


Acknowledgements

Technology DevelopmentLi XuSandra WangGordon Leung

Protein SciencesChuck HannumHieu Tran

DiscoveryAaron KurtzmanHelena YeeDavid Duffin

Program TeamsDanying CaiMedini GorePamela ForemanPhil Kobel

ManagementLawrence HorowitzMichael Horowitz


Contact Information

Sea Lane Biotechnologies2450 Bayshore Parkway, Suite 200Mountain View, CA 94043USAMichael Horowitz Ramesh BhattGC/COO VP of [email protected] [email protected](650) 336-1532 (650) 325-7402


Surrobodies™ – A Novel Approach to Bispecifics…sealanebio.com/media/Sea Lane IBC DDT deck...

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