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Multi-domain Challenges of Fc Fusion Proteins and Bispecific Antibodies
Hua Tu, Ph.D.
Apr 26, 2016
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LakePharma Is a US-Based Biologics CRO
Belmont, CA
San Carlos, CA
Hayward, CA
Worcester, MA
100 employees at 4 lab sites
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Presentation Outline
Fc fusion engineering
Fab-based bispecific antibodies
FIT-Ig bispecific antibodies
We thank our clients and collaborators for allowing us to share data
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Introduction of Fc Fusion Proteins
Fc fusion proteins as therapeutics
– Etanercept: TNFR-Fc (Amgen)
– Romiplostim: Peptide thrombopoietin (TPO) mimetic fused to Fc (Amgen)
Fc fusion advantages
Fc domain may help stabilize fusion partners
Fc may extend half life in vivo
Dimeric (bivalent) molecule, may increase
potency
Affinity purification
Large body of work on Fc engineering
1.Peyvandi F, Garagiola I, Seregni S. Future of coagulation factor
replacement therapy. J Thromb Haemost.2013;11(suppl 1):84–98.
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Case 1: Making a Functional Active Fc Fusion of a TGF-β
Background on this “TGF-β” molecule
Functional form: disulfide bond linked dimer
Highly active, with therapeutic potential and is novel biomarker
Extremely difficult to express without tags, refolding approach is the industry standard
“TGF-β” Fc fusions are not functionally active
Fc tag often interferes with the biological function when the fusion partner forms dimer on their own.
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Engineering Monomeric Fc (MMFc)
MMFc is a monomerWT Fc is a dimer
1. Non-reducing conditions
2. Reducing conditions
1 2
Binding Curve Graph
Time (sec)
nm
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170
0
1
2
3
MMFc runs as monomer on SEC-HPLC
MMFc binds to ProA as strong as WT Fc
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The TGF-b Monomeric Fc Fusion Is a Dimer with Disulfide Bond
MMFcMMFc
1. Non-reducing conditions
2. Reducing conditions
-2
-1.5
-1
-0.5
0
0.5
1
0 1 2 3 4 5 6 7 8
Delta Body Weight
FC-ctrl Fusion Protein
Monomeric Fc “TGF-β” fusion successfully formed dimer via disulfide bonds on “TGF-β”
Protein is functionally active
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Case 2: A Novel Way to Conjugate to a Recombinant Fc
Belmont Biosciences, Inc
Human Fc contains 20 Lysine residues
Amine conjugation targeting Lysines will create heterogeneity
These Lysine Residues can be replaced to create a Lysine Depleted Variant Fc “LDV-Fc”
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LDV-Fc Behaves Like Regular Fc
Recombinant LDV-Fc purified with protein A ( SDS-PAGE, reducing conditions, coomasie staining).
0
10
20
30
40
50
60
0 20 40 60 80 100m
icro
gram
/mL
Hour post-dosing
Mouse PK Study (i.v. dosing 5 mg/kg)
Group 1
Group 2
Belmont Biosciences, Inc
Normal expression; can be purified by ProA
Blood exposure is similar to a wild-type human Fc after injection in mice
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LDV-Fc enables site-specific amine conjugations at its N-termini
Belmont Biosciences, Inc
= recombinant IgG LDV Fc
= reactive amine group for conjugation
= small molecule conjugate
• LDV has only 2 reactive amine groups available at its N-termini.
• This allows for a highlyhomogeneous conjugatedend product.
Human LDV-FcInter chain disulfide bonds
CH2 CH3
CH2 CH3
CH2 CH3
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Biotinylation of LDV-Fc Results in a Homogeneous End-product
Belmont Biosciences, Inc
The mass difference between the main peaks of LDV with and without biotinylation is 679, suggesting
two biotins are added to each LDV molecule, which is a dimer under non-reducing conditions
LDV Control MW Da Delta MW
Calculated protein mass 51623.4
Observed protein mass 54501.8 +2878.4
Suspected glycosylation (G0F)*2 +2890.6 +12.2
S-S bonds (6) -12.15 0
Biotinylated LDV MW Da Delta MW
Calculated protein mass 51623.4
Observed protein mass 55180.8 +3557.4
Suspected glycosylation (G0F)*2 +2890.6 +666.8
S-S bonds (6) -12.15 +679.0
Biotin (2) +679 0
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Potential Applications:
Several types of molecules can be conjugated to an LDV-Fc
Belmont Biosciences, Inc
Non-natural peptide
siRNA
Optional fused targeting domainor Peptibody at C-terminus
Small molecule
Polymer
LDV-Fc
Available for purchase as a research reagent-or-
Commercial license available
e.g. NHS ester reaction
CH2
CH3
CH3
CH2H2N-
H2N-
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LDV-Fc is developed by Belmont Biosciences, Inc.
This technology is available for licensing
Contact Gray Shaw
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Bispecific Antibody = Non-natural + Multi-domain
Spiess, C., et al.,. Mol. Immunol. (2015)
Non-natural
– Non-natural linker between domains
– Non-native neighboring domain
Multi-domain
– Contain multiple Ig domains
– Have more than one favorite domain pair available
– Have more than one sub-optimal domain pair available
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Multi-domain Challenges Surface at Various Stages
• Off rate ranking
• Production yield• Quantification methods• Stability and formulation
• Stable cell line and single cell cloning
• Binding kinetics• Competition assay
Antibody Generation
Antibody Screening
Antibody Optimization
Large scaleantibody Production
Stable Cell LineDevelopment,Process Development
FunctionalCharacterization
• Fc receptor and FcRn binding assay
Case Study 3:
– A Fab fusion bispecific antibody
Case Study 4:
– An Fc fusion bispecific antibody
BioanalyticalCharacterization
• Binding kinetics
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Case 3: Fab-based Bispecific Antibodies
Functional domains can be
– scFv
– Nanobody
– Ligand
– Domain or fragment 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
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Case 3: Fab-based Bispecific Antibodies
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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Each Domain is Functionally Active
Binding to Target cell line
Binding to T cells
BsAb mediated killing assay
Efficacy in xenograft model
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Perfect until CHO Stable Cell Line Development
CHO stable pools showed instability and variability
CHO stable single cell clones displayed variable profiles
Capillary Electrophoresis of single cell clone productions showed profile change
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Titer Quantitation of CM Showed Inconsistent Results
Titer measurements by Octet targeting different domains show uncorrelated results
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
Titer measurement results using different antigens appear uncoupled.
Which measurement is correct?
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
Arm
1
Arm 2
Titer Values
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Different Types of Molecules Found by anti-CH1 Purification
NR
R
NR
R
Target moleculeTarget molecule, heterodimer of Fd and light chain
Undesired moleculeA Fd homodimer purified by anti-CH1
Fd chain should not have formed dimer, but it did
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Undesired Molecule 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
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Undesired Molecule is Fd Dimer
Target molecule: Properly assembled Fab fusion BsAb with two binding specificities
Undesired is a mis-paired Fd dimer
• Has only Fd chain
• Contains one binding specificity through the appended scFv fusion
• Contains CH1 domain, and therefore binds to anti-CH1 resin
Reduced cell productivity
Pool instability
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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
40
60
80
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
Stable clones were found after two rounds of single cell cloning
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Summary
Issue
Native Fd chain and light chain alone are not sufficient to ensure pairing when other pairing domains are present. Undesired dimer may be mediated by appended functional domains such as scFv.
Solutions
Accurate titer measurement
‒ Antigen based measurements are more accurate than constant region based measurements. Ratio of both antigen based measurements is critical.
Clone selection
‒ All binding moieties are vital. Ratio of both antigen based measurements provides important pairing information.
‒ High producing clones can be obtained after extensive screening.
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Case 4: FIT-Ig
Fabs-In-Tandem Ig by EpimAb Biotherapeutics
CH2CH3
CH2CH3
• Tetravalent, bispecific molecule
• Symmetric
• Standard Fc w/o mutations (g1)
• Correct pairing of VH/VL
• Flexibility allowing dual binding
• Linker is optional
CH2 CH3VHB CH1VLA CL
VLB CL
VHA CH1
Heavy chain
Light chain
Short chain
N’-
N’-
N’-
-C’
-C’
-C’
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FITIG (from HEK293) (NR)FITIG (from HEK293) (R)A generic IgG (NR)A generic IgG (R)
FIT-Ig Behaves Like IgG
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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SEC-HPLC
Molecular weight standardFIT-Ig
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Antigen Binding Domains Are Completely Independent
On-rate profiling
Loading Sample ID Sample ID KD (M) kon(1/Ms) kdis(1/s) Full X^2 Full R^2
BsAb Antigen 1 8.7E-11 6.4E+05 5.6E-05 0.0197 0.998
BsAb Antigen 2 5.2E-10 2.5E+05 1.3E-04 0.0343 0.99
Inject BsAb
Inject Antigen 1
Inject Buffer
Inject 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
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FIT-Ig Retains Full Functions of Parental IgGs
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FIT-Ig Displays IgG-like PK Profile
Parameter Unit
ELISA method
IL-17 Capture
IL-20 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 10.60.01
0.1
1
10
100
1000
0 7 14 21 28
Seru
m c
oncentr
ation (
ug/m
L)
Time (day)
FIT1-Ig IV
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 SCPK parameters Unit
ELISA Method
IL-17 Capture
IL-20 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
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FIT-Ig is developed by EpimAb Biotherapeutics
This technology is available for licensing
Contact Stephan Lensky, Ph.D [email protected]
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Summary
Unnatural, multi-domain molecules present new challenges
These challenges can be met successfully through design, engineering and refinement
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LakePharma (CA) Combined with Blue Sky BioServices (MA)
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LakePharma at PEGS
LakePharma booth (#319)
Blue Sky BioServices booth (#241)
Poster #A63
– “Proteomic Sequencing and Resurrection of a Monoclonal Antibody”
– de novo antibody sequencing technology platform
LakePharma Cocktail Party
– Tuesday, April 26th; 4:30 - 6:30pm (right after Tuesday exhibit)
– At Jerry Remy’s, right next to Del Frisco
– Pick up drink tickets at our booths
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Thank You for Your Time!
LakePharma, Inc.Corporate Headquarters
520 Harbor BlvdBelmont, CA 94002
650-288-4891www.lakepharma.com
[email protected]@lakepharma.com