BISPECIFIC ANTIBODY CASE STUDIES - LakePharma Case Studies... · CH2 CH3 CH2 CH3 VL A CL VH B CH1 CH2

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  • 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

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    FIT1-Ig SC

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    Seru

    m c

    oncentr

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    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

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    Tite

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    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

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    Fu

    nctio

    n d

    om

    ain

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    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

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    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.