Multi-domain Challenges of Fc Fusion Proteins and Bispecific Antibodies

1. Multi-domain Challenges of Fc Fusion
Proteins and Bispecific Antibodies
Hua Tu, Ph.D.
Apr 26, 2016

2. 2
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 100microgram/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 highly
homogeneous conjugated
end 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 domain
or 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
gshaw@belmontbio.com

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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 scale
antibody
Production
Stable Cell Line
Development,
Process
Development
Functional
Characterization
• Fc receptor and
FcRn binding assay
 Case Study 3:
– A Fab fusion bispecific antibody
 Case Study 4:
– An Fc fusion bispecific antibody
Bioanalytical
Characterization
• 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
Functiondomains
Functiondomains
Functiondomains
Functiondomains
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/mL
LOD=0.05 ug/mL
Linear (Y = a * X + b)
a 0.0009554
b 0.0020708
LOQ=0.8 ug/mL
LOD=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
Arm1
Arm 2
Titer Values

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Different Types of Molecules Found by anti-CH1 Purification
NR
R
NR
R
Target molecule
Target molecule, heterodimer of Fd and light chain
Undesired molecule
A 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
Antigen1Antigen2

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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
VCD(x10^6cells/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
Viability(%)
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 standard
FIT-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 10.6
0.01
0.1
1
10
100
1000
0 7 14 21 28
Serumconcentration(ug/mL)
Time (day)
FIT1-Ig IV
0.01
0.1
1
10
100
1000
0 7 14 21 28
Serumconcentration(ug/mL)
Time (day)
FIT1-Ig SC
PK 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
stephan.lensky@epimab.com

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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!
hua.tu@lakepharma.com
LakePharma, Inc.
Corporate Headquarters
520 Harbor Blvd
Belmont, CA 94002
650-288-4891
www.lakepharma.com
inquiries@lakepharma.com
tech@lakepharma.com