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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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)
*
*
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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’
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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
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11Case 1: SE-HPLC Profile Showed High Monomeric Content
Molecular weight standardFIT-Ig
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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
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13Case 1: FIT-Ig Has Similar Thermostability as IgG
FIT-Ig
IgG
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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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
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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
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1400
0 2 4 6 8 10 12 14 16 18
Tite
r (
mg/
L)
Time ( day )
FIT-Ig titer
PROPRIETARY INFORMATION OF LAKEPHARMA
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]
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
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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
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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
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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?
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
26Case 2: Extensive Clone Screening Identified Well Behaving Clones
0
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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
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100
120
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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