View
219
Download
0
Embed Size (px)
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
FcR 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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
11Case 1: SE-HPLC Profile Showed High Monomeric Content
Molecular weight standardFIT-Ig
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
13Case 1: FIT-Ig Has Similar Thermostability as IgG
FIT-Ig
IgG
PROPRIETARY INFORMATION OF LAKEPHARMA
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
0
10
20
30
40
50
60
70
80
90
100
0
1
2
3
4
5
6
7
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
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 AE@lakepharma.com or stephan.lensky@epimab.com
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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
PROPRIETARY INFORMATION OF LAKEPHARMA
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.
