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Antibody Discovery and Bispecific Design:
Development Considerations
Aaron K. Sato, Ph.D.
LakePharma, CSO
My First Chemistry Set
The Chemistry Set
In 1914, American chemist John J. Porter produced the first
line of chemistry sets, “Chemcraft.”
Overview
• LakePharma and the Antibody Center
• Antibody Discovery
• Developability
• Bispecific Design and Testing
• Brief Overview of Expression Systems
Company mission is to provide industry-leading protein
engineering and biomanufacturing services
▪ Biologics company focusing on providing integrated solutions
▪ Founded in 2009 and based in San Francisco Bay Area
▪ Named as an Inc. 5000 Fastest Growing Companies in 2015 and 2016
▪ Named on San Francisco Business Times’ list of 100 Fastest Growing
Private Companies in the Bay Area in 2016
▪ Acquired Blue Sky BioServices (Worcester, MA) in March 2016
▪ ~150 employees, 30% have PhDs
▪ LakePharma is the largest US-based biologics CRO, with most complete
capabilities
LakePharma
Who is LakePharma?
Discovery Development ManufacturingEngineering
LakePharma is a biotech company that enables and supports the discovery and development of the biologics of tomorrow
LakePharma’s Integrated Solutions for
Drug Development and Manufacturing
▪ Our technical capabilities cover the entire spectrum of biologics development and analytics
▪ We can take drug program from idea to lead to stable cell line to a formulated protein
LakePharma Antibody Center
Stable Cell Line
Development
Cell-based
Binding Assays
Functional
Assays
Antibody
DiscoveryAntibody
EngineeringCell Biology
Protein &
Analytical Science
(in vitro focus)
Phage Display
Ribosome Display
DNA Cloning
HT Screening
HT Ab fragment
expression/purific
ation (E. coli)
Affinity Maturation
(in vivo focus)
Hybridoma
Campaign
Assay
Development to
Support
Hybridoma
Tech Transfer
Single B-cell
Technologies
▪ Focus on antibody
▪ No downstream development
BioExpression
Assay & Analytics
Bispecific
Antibody
Developability
Assays
Humanization
Immunogenicity
Assessment (in
silico)
High Throughput
Expression
Hybridoma
Sequencing
Recombinant
Antibody
Production
DNA cloning
DNA scale Up
Multiple shots on goal for antibody
discovery and engineering …
Antibody Structure
Human and humanized antibodies
Abgenix, Medarex, PDL and othersSpecialized Antibodies
Fundamental Breakthroughs in
Therapeutic Antibodies
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FDA-approved therapeutic antibodies
Human or humanized mAbs
$37.9B (2012 U.S. sales)
Murine or
chimeric mAbs
New state-of-
the-art for ADCs:
homogeneity,
site-specific
conjugation
ProteinSAR™
bispecifics: optimal
structures for
superior drug-like
properties
Source of Antigen
• Tagged Antigen
– Fc Fusions
– His Tag
– BirA Tag Enzymatic Biotinylation
• Cell-based Antigen
– Stable CHO line over-expressing antigen
– Tumor cell line that expresses antigen
– Primary cells/tissues expressing antigen
Linker-
warhead
Source of Antibodies and
Integration
Engineered
Antibody
Ribosome Display
Phage Display
Cell-Based
Display
B-Cell Based Technologies
Antibody
Engineering
Bispecifics ADCsnnAA
Many
Scaffolds
Phage Display
>109 different antibodies
Library Types
Naïve Immune
Lower affinity to human antigen?
Synthetic
Requires careful design
Higher affinity and broader
epitope space?
Ag Specific Immune
Focused effort
Developability?
Phage Display Selection
Ribosome Display
How & what?
mRNA
Ribosome
Antigen-biotinAntibody
+ Ag No AgCDR H3
Round 1
Round 2
Round 3
Round 4
Streptavidin
capture
PCR amplify Screen output
Affinity Maturation
Naïve Libraries
(Synthetic)
Limited to
recombinant
protein selections
IVTT
Ribosome Display
A Tool for Lead Optimization
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
0 500 1000 1500 2000
KD
(n
M)
Recovered protein (ug/mL)
Parent
New lead
Rd1
Rd2
Rd3
+Ag -Ag
Parent
Improved clones
Cell Binding ELISAKD vs protein recovered
Mouse Hybridoma
(Köhler/Milstein, 1975)
Nobel Prize, 1984
Hybridoma
• High affinity antibodies
specific to Ag
– Cross-reactivity to host
Ag difficult!
• Multiple immunization
approaches
– Recombinant protein
– Cells
– DNA
• Source of diversity
– Mouse, Rat, Rabbit,
Chicken, …
– Humanized equivalents
• Requires humanization
• Good precedence for
developability
– The “in vivo filter”
The Process of Humanization
Example: An Approach to Humanization
MuHC HC-1 HC-2 HC-3 HC-4
MuLC 1 2 3 4 5
LC-1 6 7 8 9 10
LC-2 11 12 13 14 15
LC-3 16 17 18 19 20
LC4 21 22 23 24 25
• DNA Synthesis
– 4 HC and 4 LC grafts
with murine HC/LC
controls
• Express Antibodies
• 5x5 matrix = 25 IgGs
• Test for: (vs. muParent)
– Assembly/Titer
– Affinity: SPR
– Thermal Stability
– FACS Binding
– Functional Activity
Immortalized Human B-cell Technologies
• After immortalized
through various methods,
screening workflow
similar to hybridoma
• Limited exposure to this
technology
• Pro: Human Antibody
• Con: Immunization?
Cell-based Display Technologies
Bacterial
Advantages over phage display?
Yeast
FACS driven selections
Affinity maturation
Mammalian
More difficult, but worth it
Eukaryotic Processing Machinery
Like the “in vivo filter”?
Developability Assessment
In silico DNA Sequence check and Protein Sequence
Hot Spot Analysis, followed by Mass Spec verification
✓ DNA codon preference
✓ Protein sequence hot spot analysis
Productivity Readiness Check
✓ Transient production in HEK293 or CHO system
Integrity and Stability Check (to demonstrate whether
the antibody is stable biochemically)
✓ Intact Mass/Peptide Mapping/ PTM by Mass
Spec
✓ DSF/DLS – Thermostability assessment
✓ Aggregation/Fragmentation/PTM and Glycan
profiling over 2 weeks incubation under stressed
conditions
✓ SE-HPLC/LabChip CE-SDS
PK Readiness Check
✓ Poly-specificity ELISA, Surface hydrophobicity
assay – Specificity requirement
P o ly -S p e c if ic ity E L IS A
OD
45
0
BsA
b
Ref
1
Ref
2
Ref
3
Ref
4P
C
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
1 5 0 u g /m L
5 0 u g /m L
1 7 u g /m L
2 n d A b o n ly
HPLC
DSF/SLS
DLS
CE-SDS
mass23140 23160 23180 23200 23220 23240 23260 23280 23300 23320 23340 23360
%
0
100
PP9650_DG_R_20170426_SEC 399 (3.577) M1 [Ev-623677,It19] (Gs,0.800,1034:3697,0.20,L33,R33); Sb (15,2.00 ); Cm (395:399) 1: TOF MS ES+ 9.13e623206.4
23185.0
23264.6
23228.2 23245.8
mass48400 48450 48500 48550 48600 48650 48700 48750 48800 48850 48900 48950 49000 49050 49100 49150
%
0
100
PP9650_DG_R_20170426_SEC 345 (3.114) M1 [Ev-383616,It27] (Gs,0.800,1132:2662,0.50,L33,R33); Sb (15,2.00 ); Cm (342:347) 1: TOF MS ES+ 6.15e648600.0
48570.5
48783.5
48658.5
48630.0
48722.048691.0 48754.5
48843.0
48814.5
Heavy ChainLight ChainIntact Mass
Case Study: mAb PPXXXX Ready
to Move Forward
Sample ID %LMWC %(HC+LC) %MMWC %HMWC
mAb reduced N/A 99.3 0.7 N/A
mAb non-reduced 6.5 93.5 N/A N/A
✓– Purity
requirement
rCE>95%
✓– Purity
requirement
nrCE>90%
Case Study: mAb PPXXXX Ready
to Move Forward
3
min0 2.5 5 7.5 10 12.5 15 17.5 20 22.5
mAU
0
100
200
300
400
500
21
Peak # Peak Size
(kDa)
Peak Area %
Peak ID Notes
1 ~1500 1.4 Aggregate
2 ~250 2.3 Aggregate
3 ~150 96.3 Monomer
✓SE-HPLC (Mab-Pac1) – Monomeric state
Case Study: mAb PPXXXX Ready
to Move Forward
✓ Intact mass measurement – Integrity of the molecule
23639.1
36180.4
48508.9
WangS_i5615_PP6410red_Ldp800_34_01_15896.d: +MS, 12.3-14.2min, Smoothed (0.19,1,GA), Baseline subtracted(0.00), Deconvoluted (MaxEnt)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
4x10
Intens.
10000 20000 30000 40000 50000 60000 70000 80000 90000 m/z
23639.1
23794.4
WangS_i5615_PP6410red_Ldp800_34_01_15896.d: +MS, 12.3-14.2min, Smoothed (0.19,1,GA), Baseline subtracted(0.00), Deconvoluted (MaxEnt)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
4x10
Intens.
23400 23600 23800 24000 24200 m/z
48508.9
48636.9
WangS_i5615_PP6410red_Ldp800_34_01_15896.d: +MS, 12.3-14.2min, Smoothed (0.19,1,GA), Baseline subtracted(0.00), Deconvoluted (MaxEnt)
0.0
0.5
1.0
1.5
2.0
4x10
Intens.
48400 48600 48800 m/z
Heavy
Chain
Heavy Chain Light Chain
Measured Mass 48636.90 Da 23794.40 Da
Predicted Mass 48634.90 Da 23794.53 Da
Delta Mass -2.00 Da 0.13 Da
Light Chain
Case Study: mAb PPXXXX Ready
to Move Forward
Peak # pI Peak Area % Peak ID Notes
1-2 --- 15.38 Acidic Peak Group3 8.28 65.28 Main Peak
4-5 --- 19.34 Basic Peak Group
✓ cIEF – Charge variants profiling
Case Study: mAb PPXXXX Ready
to Move Forward
✓DSC –
Thermostability
ANALYSIS
Peak # Tm (°C) ΔHcal (kJ/mol)
1 71.3 438
2 80.7 1487
3 82.1 808
✓DSF – Thermostability and
Aggregation onset
Tagg266: 70.7 °C
MethodLoading
Sample IDSample ID KD (M) kon(1/Ms) kdis(1/s) Full X^2 Full R^2
Octet Antigen PPXXXX 1.30E-11 1.60E+05 2.00E-06 0.0217 0.9998
✓Affinity measurement against Antigen in BLI or SPR – Affinity requirement
Case Study: mAb PPXXXX Ready
to Move Forward
Peak # Peak Size
(kDa)
Peak
Area %Peak ID
1 ~400 3.2 Aggregate
2 ~150 96.8 Monomer
3
Analysis
✓Poly-specificity ELISA, Surface hydrophobicity assay – Specificity requirement
5x uncoated
background
5x
uncoated
background
HEK293 WCL and BVP ELISA showed
consistent results, which serve as good
PK indicators.
Case Study: mAb PPXXXX Ready
to Move Forward
Countless Bispecifics Scaffolds
are Continually Being Developed
Speiss et al 2015 review
• Biology may
dictate the best
format to use
• Is there a one
size fits all
format?
• Ideally, it would
be good to have
a variety of
formats
available
An Example of Specialization
Bispecific T-cell Engager
Many Different Bispecifics are in
Clinical Trials
Kontermann et al 2015 review
Bispecifics by Activity
Dual Targeting
Act directly on multiple target
structures
Coupled with Delivery of X
Bi-Epitopics/Paratopics
Dual binding to non-overlapping target epitopes
Coupled with Delivery of X
X
X
Focus on Dual Targeting
Dual Targeting
Same Cell
Soluble Ligand & Cell Target
Different Cells
Bispecific T-cell Engagers (BiTEs)
Bispecific NK-cell Engagers
All others that do not recruit immune cells
My Bispecific Risk Scorecard
Evaluation Criteria
Criteria Grade
Cost of Goods (0-3, Low to High)
Ease of use (0-3, Easy to Hard)
Time to discovery (0-3, Short to Long)
“Does it look like an antibody?” (0, Yes; 2, No)
Potential for immunogenicity (0-3, Low to High)
Biophysical properties/stability (0-3, Stable to Unstable)
Advantage over cocktail of antibodies (0, Yes; 1, No)
Half-life extension (0, Yes; 3, No)
Total
Case Study:
Lower is
better!
My Bispecific Risk Scorecard
Evaluation Criteria
Criteria Grade
Cost of Goods (0-3, Low to High) 1
Ease of use (0-3, Easy to Hard) 1
Time to discovery (0-3, Short to Long) 1
“Does it look like an antibody?” (0, Yes; 2, No) 2
Potential for immunogenicity (0-3, Low to High) 2
Biophysical properties/stability (0-3, Stable to Unstable) 0
Advantage over cocktail of antibodies (0, Yes; 1, No) 0
Half-life extension (0, Yes; 3, No) 0
Total 7
Case Study: IgG-scFv: EGFR x IGFR, IGFR (Dual Epitopes)Morrison-type Bispecific
My Bispecific Risk Scorecard
Evaluation Criteria
Criteria Grade
Cost of Goods (0-3, Low to High) 1
Ease of use (0-3, Easy to Hard) 1
Time to discovery (0-3, Short to Long) 1
“Does it look like an antibody?” (0, Yes; 2, No) 2
Potential for immunogenicity (0-3, Low to High) 2
Biophysical properties/stability (0-3, Stable to Unstable) 0
Advantage over cocktail of antibodies (0, Yes; 1, No) 0
Half-life extension (0, Yes; 3, No) 0
Total 7
Case Study: FIT-Ig
Case 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’
Case 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
Case 1: SE-HPLC Profile Showed
High Monomeric Content
Molecular weight standardFIT-Ig
Case 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
Flow
BsAb
Flow
Antigen 1
Flow
Buffer
Flow
Antigen
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
Case 1: FIT-Ig Has Similar
Thermostability as IgG
FIT-Ig
IgG
Case 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 28Seru
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
Expression Systems
Contact Information
Aaron K. Sato, Ph.D.
LakePharma
CSO
P: 650-489-9125 Ext. 250
F: 888-635-3618
520 Harbor Blvd, Belmont, CA 94002
http://www.linkedin.com/in/aaronsato