Download pdf - BISPECIFIC ANTIBODY CASE STUDIES - LakePharma Case Studies... · CH2 CH3 CH2 CH3 VL A CL VH B CH1 CH2 CH3 VL B CL VH A CH1 ... Antigen 2 Undesired 3.9E-10 6.7E+05 2.6E-04 0.0093 0.9993

BISPECIFIC ANTIBODY CASE STUDIES

Kexin Huang

October 7, 2016

PROPRIETARY INFORMATION OF LAKEPHARMA

2LakePharma Integrated Solutions for Antibody Drug Development

• Antigen design• Custom protein• Custom cell line

• Hybridoma• Phage display• iQue• OCTET• ELISA• Functional• Thermostability

• Mass spec• PTM• Glyco profile• Aggregation• Formulation• Stability• Thermostability

AE AD

ME

PC

BEBF

BP

BA• Transient

expression and purification

• Production yield• Material for testing

• Immunization• Wild type mice• Trianni mice• Hypersensitive mice

• Ab reformatting • Humanization• Affinity maturation• Sequence liability analysis• Bispecific antibody format

• High yield stable cell line• Path to commercialization• Cell culture process • Purification process• Release assays• Tox material generation

• Binding kinetic • FACS• Cell killing, ADCC, CDC• Target neutralization• Cell stimulation assay

AntigenEngineering

Antibody Generation

Antibody Screening

Antibody Optimization

Large scaleantibody Production

Stable Cell LineDevelopment,Process Development

BioanalyticalCharacterization

FunctionalCharacterization


3Antibody Engineering:

Library construction and screening

Epitope binning and mapping

Sequence liability analysis

Antibody humanization

Affinity maturation

Affinity measurement

FcγR panel and FcRn binding assay

Functional assays

Thermostability engineering

Therapeutic developability analysis

Bispecific antibody engineering

Design -- Screen -- Characterization


4Presentation Outline

Brief overview and considerations in bispecific antibody engineering

Case Study on FIT-Ig – Appended IgG Format

Case Study on a Fab Fusion Bispecific antibody – Appended Fab Format


5Bispecific Antibody, What, Why and How

Artificially assembled molecule

combining two specificities

Synergistic effect

– Better than two mAb mixed together

– Simultaneously target two cells

– Simultaneously target two signaling pathways

Application: Cancer therapy and

inflammatory diseases

Spiess, C., et al.,. Mol. Immunol. (2015)

*

*


6Bispecific Antibody = Non-Natural + Multi-Domain

Non-natural

– Non-natural linker between domains

– Non-native neighboring domain

Multi-domain

– Contain multiple Immunoglobulin (Ig) domains

– More than one favorite domain available for pairing

– More than one sub-optimal domain available for pairing

Quality by Design


7Bispecific Antibody Requires Systematic Optimization – Linker

BsAb Name Productivity (mg/L) Purity %Antigen 2

EC50 (ng/mL)

BsAb(A)-6aa-linker 45 >95 2088

BsAb(A)-12aa-linker 120 >95 363

BsAb(B)-6aa-linker 24 >95 34

BsAb(B)-12aa-linker 90 >95 18


8Bispecific Antibody Requires Systematic Optimization – Orientation

BsAb Name Productivity (mg/L) Purity %

scFv (A) @ N 61 >95

scFv (A) @ C 19 >95

scFv (B) @ N 7 >95

scFv (B) @ C 15 >95

scFv (C) @ N 14 >95

scFv (C) @ C 3 >95


9Case 1: FIT-Ig (Fabs-In-Tandem Ig)

Tetravalent bispecific molecule

Symmetric

Correct pairing of VH/VL

Flexibility allowing dual binding

Linker is optionalCH2CH3

CH2CH3

CH2 CH3VHB CH1VLA CL

VLB CL

VHA CH1

Heavy chain

Light chain

Short chain

N’-

N’-

N’-

-C’

-C’

-C’


10Case 1: FIT-Ig Expresses Well Like IgG

FITIG (from HEK293) (NR)FITIG (from HEK293) (R)A generic IgG (NR)A generic IgG (R)

CH2 CH3VHB CH1VLA CL

VLB CL

VHA CH1

Heavy chain

Light chain

Short chain

N’-

N’-

N’-

-C’

-C’

-C’

ProA purification

Transient yield >300 mg/L


11Case 1: SE-HPLC Profile Showed High Monomeric Content

Molecular weight standardFIT-Ig


12Case 1: Antigen Binding Arms Function Independently

Loading Sample ID Sample ID KD (M) kon(1/Ms) kdis(1/s) Full X^2 Full R^2

FIT-Ig Antigen 1 8.7E-11 6.4E+05 5.6E-05 0.008 0.99

FIT-Ig Antigen 2 5.2E-10 2.5E+05 1.3E-04 0.006 0.99

FlowBsAb

FlowAntigen 1

FlowBuffer

FlowAntigen 2

Binding of antigen 2 remains the same whether antigen 1 is present or not

Antigen 2 on rate:

– With Antigen 1: 2.4 E5

– Without Antigen 1: 2.5 E5

FIT-Ig Retains Full Functions of Parental IgGs

Neutralization Potency IC50 (pM) mAb1 mAb2 FIT-Ig

Antigen 1 101 102

Antigen 2 50 54


13Case 1: FIT-Ig Has Similar Thermostability as IgG

FIT-Ig

IgG


14Case 1: FIT-Ig Displays IgG-like PK Profile

0.01

0.1

1

10

100

1000

0 7 14 21 28

Se

rum

co

nce

ntr

atio

n (

ug

/mL

)

Time (day)

FIT1-Ig SC

0.01

0.1

1

10

100

1000

0 7 14 21 28

Seru

m c

oncentr

ation (

ug/m

L)

Time (day)

FIT1-Ig IVParameter Unit

ELISA method

Antigen 1 Capture

Antigen 2 Capture

Cl ml/day/kg 12.2 11.9

Vss ml/kg 131 126

t1/2 day 10.8 10.8

AUClast day*ug/ml 377 385

AUCINF day*ug/ml 411 419

MRTINF day 10.7 10.6

PK parameters Unit ELISA method

Antigen 1 Capture

Antigen 2 Capture

Tmax day 4.00 4.00

Cmax ug/mL 26.9 23.1

Terminal t1/2 day 10.95 10.40

AUClast day*ug/mL 336 289

AUCINF day*ug/mL 406 350

CL/F mL/day/kg 12.4 14.3

F % 103.7 86.4


15Case 1: FIT-Ig CHO-GSN Single Cell Clones can Reach g/L

CHO-GSN stable clones for FIT-Ig can reach 1 g/L before process development

– >90% pure by CE-SDS

– Production titer in shake flask is about 1 g/L

– Cell culture process optimization should increase the titer significantly

0

10

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90

100

0

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2

3

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8

9

0 5 10 15 20

Via

bili

ty (

% )

VC

D (

x 1

0^6

cel

l /m

L)

Time ( day )

Cell growth profile

VCD Viability

0

200

400

600

800

1000

1200

1400

0 2 4 6 8 10 12 14 16 18

Tite

r (

mg/

L)

Time ( day )

FIT-Ig titer


16

FIT-Ig technology is owned by EpimAb Biotherapeutics,

and further developed at LakePharma

This technology is available for licensing

Contact [email protected] or [email protected]


17Case 2: A Fab Fusion Bispecific Antibody

Functional domains can be

– scFv

– Nanobody

– Ligand

– Domain promoting dimer formation

Advantages

‒ Adjustable affinity - Flexibility on the valency

‒ Undesired effector functions removed

‒ Better design profile than BiTE format

‒ Two chains in the molecule

Fu

nctio

n d

om

ain

s

Fu

nctio

n d

om

ain

s

Fu

nctio

n d

om

ain

s

Fu

nctio

n d

om

ain

s

Fd LC


18Case 2: Good Productivity and Purity in HEK293

Transient production yield in HEK293 is 300 mg/mL

Anti-CH1 affinity purification

Short term stability study did not show increase of aggregate or fragment

>98% Purity by SE-HPLC

>98% Purity by reducing capillary electrophoresis

>98% Purity by reducing capillary electrophoresis

NR R


19Case 2: Each Binding Arm is Independent and Functional Active

Binding to Target cell line

Binding to T cells

BsAb mediated killing assay

Efficacy in xenograft model


20Case 2: Perfect Until CHO Stable Cell Line Development

CHO stable pools showed significant reduced titer

CHO stable pools showed instability after freeze/thaw

CHO stable single cell clones displayed sub-optimal growth profiles


21Case 2: Titer Quantification of CM Showed Inconsistent Results

Titer measurements by Octet using two antigens showed uncorrelated results

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

0.0 50.0 100.0 150.0 200.0 250.0

An

tige

n 1

Antigen 2

Titer Values on Two Random Days

Linear (Y = a * X + b)

a 0.0002171

b 0.001482

LOQ=0.8 ug/mLLOD=0.05 ug/mL

Linear (Y = a * X + b)

a 0.0009554

b 0.0020708

LOQ=0.8 ug/mLLOD=0.2 ug/mL

Which measurement is correct and Why?


22Case 2: Single Cell Clone Productions Showed Profile Change

Capillary Electrophoresis of single cell clone productions showed profile change

Undesired Target

Undesired Target

Undesired Target

Undesired Target

CE-SDS from transient production


23Case 2: Two Types of Molecules Found by anti-CH1 Purification

NR

R

NR

R

Target moleculeA heterodimer of Fd and light chain

Undesired by-productA homodimer of Fd chain purified by anti-CH1

Fd chain should not form dimer but it did


24Case 2: Undesired By-product Loses Binding to One Antigen

Loading Sample ID Sample ID KD (M) kon(1/Ms) kdis(1/s) Full X^2 Full R^2

Antigen 1 Target 5.1E-09 3.1E+05 1.6E-03 0.0392 0.9993

Antigen 1 Undesired NA

Antigen 2 Target 1.8E-10 1.2E+06 2.1E-04 0.0293 0.9982

Antigen 2 Undesired 3.9E-10 6.7E+05 2.6E-04 0.0093 0.9993

Target Undesired

An

tige

n 1

An

tige

n 2


25Case 2: Undesired By-product is a mispaired Fd Dimer

Target molecule: Properly assembled Fab fusion BsAb with two binding arms

Undesired by-product: A mispaired Fd dimer

• Contains one binding arm through the appended functional domains

• Contains CH1 domain, hence binds to anti-CH1 resin with compromise

Reduced cell productivity

Pool instability

Difficulty in CM quantification Target

Undesired

Compromised binding to anti-CH1


26Case 2: Extensive Clone Screening Identified Well Behaving Clones

0

5

10

15

20

0 5 10 15 20

VC

D (

x10

^6 c

ells

/mL)

Time (day)

Single cell clone and subclones VCD profile

TT5730 - XCX 2A6

TT5731 - XCX 3A6

TT5688 - XCX 3B6

TT5691 - CL XCX Clone

0

20

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0 5 10 15 20

Via

bili

ty (

%)

Time (day)

Single cell clone and subclones viability profile

TT5730 - XCX 2A6

TT5731 - XCX 3A6

TT5688 - XCX 3B6

TT5691 - CL XCX


27Summary

Present and Future

Challenges

Product related variants

Solution

Engineering

– Systematic optimization

– Comprehensive analytical profiling early

Development

‒ Antigen based titer measurements vs. constant region based measurements

‒ Pairing indicated by ratio of binding arms

‒ Balance the risk and benefit


28

Antibody Engineering team provides end-to-end services,

from design, screening to analytical characterization

Library construction and screening

Epitope binning and mapping

Sequence liability analysis

Antibody humanization

Affinity maturation

Affinity measurement

FcγR panel and FcRn binding assay

Functional assays

Thermostability engineering

Therapeutic developability analysis

Bispecific antibody engineering

Everything is difficult before it becomes EASY