KnotBodiesTM: creating ion channel blocking antibodies by fusing Knottins into

peripheral CDR loops

Aneesh Karatt Vellatt

Confidential

Precision Medicine and Ion Channel Retreat 2017

Vancouver

Antibody discovery at IONTAS

Target Selection Protein

production Lead

Isolation Primary Screen

Lead Optimisation

Secondary Screening

Lead/back up

Production

Mammalian display

KnotBodiesTM

Research

Phage library

4 x 1010 clones

High proportion of

insert

IgG

Fab scFv

Bi-specific

Antibody drug discovery company founded in Oct 2012

Powerful track record – 24 antibody discovery projects with 14 European and US organisations to Q1 2017

100% record of success!

Targeting ion channels with antibodies

Challenging target class for antibody generation Difficult to express and purify, low stability Limited epitope availability Dynamic molecules with multiple conformations

Plasma

membrane

VGSC

Conus snail

Casewell et al Trends in Ecology and Evolution (2013)

Mambalgin1

ASIC

blocker

Velvet Tarantula

Sea anemone

Black Mamba

Ziconotide

Cav2.2

blocker

ShK

Kv1.3

blocker

ProTx-II

Nav1.7

blocker

Knottins: nature’s ion channel inhibitor scaffold

30-40 amino acids, 3-4 disulfide bonds Forms a conserved (Inhibitory Cystine Knot) structural motif high sequence and functional diversity despite the structural

conservation Also found in non venomous species and modulate wide variety

of biological functions

PcTx1

ASIC1 blocker

Huwentoxin-IV

Nav blocker

ω-conotoxin-MVIIA

: Cav blocker

Knottins: therapeutic development

Ziconotide (PRIALT®; Primary Alternative to Morphine) ω-conotoxin-MVIIA, blocks N-type calcium channels Approved for the treatment of neuropathic pain

Dalazatide (ShK-186)

Engineered ShK toxin, blocks Kv1.3 In phase II trials for the treatment of psoriasis

Most naturally occurring knottins lack exquisite specificity Huwentoxin-IV block potently both Nav1.7 and Nav1.2 ShK (non-engineered) toxin equally blocks Kv1.3 and Kv1.1

Evolved to paralyse prey hence not specific and requires further engineering for therapeutic use

Challenges in knottin engineering and therapeutic development

Limited compatibility with robust library selection technologies that can sample large mutant libraries

Rational design strategies are laborious and exert less control over the specificity of new binders

Chemical synthesis can be complex and expensive

Half life of minutes to hours: too short for a drug that is expensive to synthesise

Scaffolds within scaffolds: Combining the benefits of knottins and antibodies

KnotBodiesTM concept: Insert knottins into peripheral antibody CDR loops

Engineer other CDR loops for improved potency

and selectivity using phage display technology

Knottins Natural blockers of ion channels

Lack specificity, short half life

Difficult to engineer

Antibodies Large binding surface providing specificity

Amenable to engineering using in vitro

selection technologies

Long half life

Ecballium elaterium (Jumping cucumber)

EETI-II: Trypsin

inhibitor (as model

knottin)

Specific ion channel modulators with long half life!

Inserting EETI-II knottin into VL CDRs

CDR1 CDR3 FR1 FR2 FR3 FR4

Knottin Randomised linkers joining

EETI-II knottin to VL framework

Variation in recipient VL

scaffold

VH VL

Fv

VL

VH

H chain

L chain

IgG

Selecting antibodies by phage display

McCafferty et al (1990) Nature 348 p552-4

Immobilised antigen

(Trypsin)

Genotype Genotype + Phenotype Select for phenotype

i.e. antigen binding

AntibodydisplayedonphageaspIIIfusion

Geneencodingthedisplayedantibody

SigP

Promoter

GeneIII

PhagedisplayVector

VH VL

Antibody(inscFvformat)

0

50000

100000

150000

200000

250000

300000

A B C

Tryp

sin

bin

din

g (F

U)

EETI-II Direct phage display

EETI-II CDR1 Fusion

EETI-II CDR2 Fusion

Insertion into antibody CDRs make knottins amenable to phage display!

0

50000

100000

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1 8 15 22 29 36 43 50 57 64 71 78 85 92

Tryp

sin

bin

din

g (F

U)

Clone number

VL-CDR1 2 rounds phage (18/94 binders)

0

50000

100000

150000

200000

250000

1 8 15 22 29 36 43 50 57 64 71 78 85 92

Tryp

sin

bin

din

g (F

U)

Clone number

VL-CDR2 2 rounds phage (42/94 binders)

Monoclonal ELISA of trypsin binding KnotBodiesTM

Confirm specificity with EETI-II

loop 1 (trypsin binding loop)

mutation

CPRILMRC

CGAILMRC

0

10

20

30

40

50

60

70

80

90

100

"Selected linker"

Gly4Ser linker (Gly4Ser)2 linker

"Selected linker"

Gly4Ser linker (Gly4Ser)2 linker

KB_A07 KB_A12

No

rmal

ise

d t

rysp

in b

ind

ing

(%)

Correct linkers are important for function

KB_A12( 2.5Å) KB_A05 (1.95Å)

Crystal structure of KnotBodiesTM

Crystal structure of KnotBodiesTM

Demonstrating the capabilities of the KnotBodyTM format

(i) Improving the existing knottin binding by introducing additional VH contacts

(ii) Create a bispecific molecule by introducing a VH that binds to different target

(iii) Alter the specificity of the original knottin scaffold by loop diversification

(iv) Generate ion channel blocking KnotBodies

0

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350000

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B0

6

A1

0

B0

7

C0

1

E06

D0

8

F11

D0

9

C0

6

E08

F02

H0

7

A0

7

D0

1

H0

5

F07

C0

2

H0

1

E07

C1

0

G0

3

B0

8

D1

0

E10

B0

3

B1

1

F05

E12

D1

2

G1

2

B1

2

A0

3

Tryp

sin

bin

din

g (F

U)

Clone ID

KB_A07

Affinity ranking using ELISA

KB_A12

Improved affinity of KnotBodyTM through partner VH selection

Ch

an

ge

in

SP

R Affinity improved clone

KB_A07 (Parent clone) KB_A12 (parent clone)

Trypsin

binding

Off-rate analysis using SPR

Time (S)

Trypsin dissociation

cMET

Gas

6

FGFR

4

Tryp

sin

0

10000

20000

30000Parent KnotBody

Bin

din

g (

FU

)

cMET

Gas

6

FGFR

4

Tryp

sin

0

120000

240000

360000

Bin

din

g (

FU

)

cMET bi-specfic KnotBody

cMET

Gas

6

FGFR

4

Tryp

sin

0

120000

240000

360000

Bin

din

g (

FU

)

Gas6 bi-specific KnotBody

cMET

Gas

6

FGFR

4

Tryp

sin

0

120000

240000

360000

Bin

din

g (

FU

)

FGFR4 bi-specific KnotBody

VL VH VL

VL VL VH VH

Bispecific binding through partner VH selection

Altering specificity of knottin “donor”

KB_A12 KnotBody

Randomise L1 loop of EETI-II donor-> 4 x 109 library

(loop lengths= 6, 8, 9, 10 using VNS codons)

Altering specificity of knottin “donor”

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Clone number

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600000

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47

Clone number

c-Met b-galactosidase

23/47 unique 11/47 unique

-CPRILMRC-

An

tig

en

bin

din

g (

FU

)

L1 loop

Role of potassium channels in T effector cell signaling

Activated CM

Naïve

effector Naïve

Central

memory

Effector

memory

Activated EM

Kv1.3 Model from: Murray et al. (2015) J. Med Chem.

pore

ShK

mutant

Autoreactive TEM cells depend heavily on Kv1.3, while naïve and central memory T cells depends on KCa3.1

Autoreactive TEM cells can be eliminated via selective inhibition of Kv1.3

Measuring Kv1.3 ion channel currents using automated patch clamp

control, t = 0 min

0.5 nM, t = 4 min

5 nM, t = 8 min

50 nM, t = 12 min

500 nM, t = 16 min

100 ms

0.5

nA

-80 mV

+30 mV, every 10 s

Sophion QPatch

-10 -9 -8 -7 -6 -5

0

25

50

75

100

Log [M]

% c

urr

en

t re

ma

inin

g

KB_HsTX1IC50 = 3.9 nM

-10 -9 -8 -7 -6 -5

0

25

50

75

100

Log [M]

% c

urr

en

t re

ma

inin

g

KB_ShKIC50 = 8.6 nM

-10 -9 -8 -7 -6 -5

0

25

50

75

100

Log [M]

% c

urr

en

t re

ma

inin

g

KB_KalioTxIC50 = 430 nM

Functional inhibition of Kv1.3 by KnotBodiesTM

ShK KnotBody Kaliotoxin KnotBody

-10 -9 -8 -7 -6 -5

0

25

50

75

100

Log [M]

% c

urr

en

t re

ma

inin

g

KB_HsTX1IC50 = 3.9 nM

-10 -9 -8 -7 -6 -5

0

25

50

75

100

Log [M]

% c

urr

en

t re

ma

inin

g

KB_ShKIC50 = 8.6 nM

-10 -9 -8 -7 -6 -5

0

25

50

75

100

Log [M]

% c

urr

en

t re

ma

inin

g

KB_KalioTxIC50 = 430 nM

Functional inhibition of Kv1.3 by KnotBodiesTM

ShK KnotBody inhibited cytokine and Granzyme B secretion by activated PBMCs

Functional inhibition of ASIC1a by KnotBodiesTM

KnotBody A07_PcTX1 KnotBody A12_PcTX1

Therapeutic areas: neuropathic pain, neurological disorders

PcTX1 (Psalmotoxin) - ASIC1a toxin blocker

-9 -8 -7 -6 -5

0

25

50

75

100

Log [M]

% c

urr

en

t re

ma

inin

g

IC50 = 98 nM

-9 -8 -7 -6 -5

0

25

50

75

100

Log [M]

% c

urr

en

t re

ma

inin

g

IC50 = 68 nM

Rapid generation of cell lines for specificity screening

CHO-S cells

Suspension cells

Easy to culture

No dissociation required

Direct use in APC

pINT_ IC Vectors

pIONTAS IC Vectors

Transient expression

Polyclonal stable expression using transposase system

Expression from a specific locus using nuclease mediated integration

MaxCyte STX Electroporation

High transfection efficiency and scalability

High cell viability

Efficient multi plasmid co-transfection

Test Parameters

Kv1.3-CHO Stable Cell Line

(CRL)

Kv1.3 Transient MaxCyte

(24 h after EP)

Kv1.3 + Transposase MaxCyte

(2 weeks after EP)

Cell Viability (%) ≈ 99 ≈ 99 ≈ 98

Mean IK+ (nA) 1.5 ± 1.3 4.7 ± 3.1 2.8 ± 3.4

% IK+ >0.5nA 91 88 87

% seal >1 G 89 83 88

I-V V1/2 (mV) -1.2 2.5 1.1

I-V slope (mV) 26.7 21.7 20.4

Comparison with monoclonal stable cell line

Data obtained using Sophion QPatch, Single hole mode

-12 -11 -10 -9 -8 -7

0

25

50

75

100

Log [M]

% c

urr

en

t re

main

ing

Kaliotoxin

Kv1.1 Transient

Kv1.3 Stable

Kaliotoxin

Kv1.3-CHO stable

Kv1.3 Transient

Kv1.3 +

Transposase

Kv1.1 Transient

IC50 (nM) 0.78 0.68 0.56 2.5

Evaluating the pharmacology of Kaliotoxin

-12 -11 -10 -9 -8 -7

0

25

50

75

100

Log [M]

% c

urr

en

t re

main

ing

Kaliotoxin

Kv1.3 Transient

Kv1.3 +Transp

Kv1.3 Stable

Tapping into nature’s ion channel scaffold

Antibody acquires the ion channel modulating functionality of the knottin

Knottin gains half-life improvement

Increased affinity and specificity from other CDRs

Potential to engineer knottin

Novel bi-specific format

KnotBodiesTM: Summary

Acknowledgement

• John McCafferty

• Damian Bell • Sachin Surade • Edward Masters • Alice Luther • Rachael Leah • Tim Leutkens

• Peter Slavny

• Naja Møller Sørensen • Daniel Sauter

• Payal Roychoudhuri • Caoimhe Nic An tSaoir • Jason Marks

Thank You

Expression and purification of Fab formatted KnotBodiesTM

KnotBody Fabs express well

100 % monomer on SEC

Tm1=76.0°C, Tm2=81.3°C

N.R

R

N.R

R

N.R: Non reducing SDS PAGE

R: Reducing SDS PAGE

Size exclusion chromatography (SEC) profile SDS PAGE

Comparison of KnotBody with bovine antibody with ultralong CDR3

Reshaping antibody diversity

(2013) Wang et al Cell, 153 p1379-1393