Proteins and Bispecific Antibodies Hua Tu, Ph.D. Apr 26, 2016 - - PowerPoint PPT Presentation

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 monomer WT Fc is a dimer

• 1. Non-reducing conditions
• 2. Reducing conditions

1 2

Binding Curve Graph Time (sec) nm 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 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

MMFc MMFc

• 1. Non-reducing conditions
• 2. Reducing conditions
• 2
• 1.5
• 1
• 0.5

0.5 1 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).

10 20 30 40 50 60 20 40 60 80 100 microgram/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-Fc

Inter 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

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

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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

• r 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 CH2

H2N- 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

Function domains Function domains Function domains Function domains

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

Arm 1 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 Antigen 1 Antigen 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

5 10 15 20 5 10 15 20

VCD (x10^6 cells/mL) Time (day)

Single cell clone and subclones VCD profile

TT5730 - XCX 2A6 TT5731 - XCX 3A6 TT5688 - XCX 3B6 TT5691 - CL XCX Clone 20 40 60 80 100 120 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

CH2 CH3 CH2 CH3

• Tetravalent, bispecific molecule
• Symmetric
• Standard Fc w/o mutations (g1)
• Correct pairing of VH/VL
• Flexibility allowing dual binding
• Linker is optional

CH2 CH3 VHB CH1 VLA CL VLB CL VHA CH1

Heavy chain Light chain Short chain

N’- N’- N’-

• C’
• C’
• C’

27 FITIG (from HEK293) (NR) FITIG (from HEK293) (R) A generic IgG (NR) A generic IgG (R)

FIT-Ig Behaves Like IgG

CH2 CH3 VHB CH1 VLA 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 7 14 21 28

Serum concentration (ug/mL) Time (day)

FIT1-Ig IV 0.01 0.1 1 10 100 1000 7 14 21 28

Serum concentration (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