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

LakePharma Is a US-Based Biologics CRO 100 employees at 4 lab sites Belmont, CA San Carlos, CA Hayward, CA Worcester, MA 2

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 3

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 1.Peyvandi F, Garagiola I, Seregni S. Future of coagulation factor replacement therapy. J Thromb Haemost. 2013;11(suppl 1):84 – 98. Affinity purification  Large body of work on Fc engineering  4

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. 5

Engineering Monomeric Fc (MMFc) 1 2 MMFc runs as monomer on SEC-HPLC Binding Curve Graph 3 2 nm 1 WT Fc is a dimer MMFc is a monomer 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 Time (sec) 1. Non-reducing conditions MMFc binds to ProA as strong as WT Fc 2. Reducing conditions 6

The TGF- b Monomeric Fc Fusion Is a Dimer with Disulfide Bond Monomeric Fc “TGF - β” fusion successfully formed dimer via disulfide bonds on “TGF - β”  Protein is functionally active  Delta Body Weight 1 0.5 MMFc MMFc 0 -0.5 -1 -1.5 -2 0 1 2 3 4 5 6 7 8 FC-ctrl Fusion Protein 1. Non-reducing conditions 2. Reducing conditions 7

Case 2: A Novel Way to Conjugate to a Recombinant Fc 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”  Belmont Biosciences, Inc 8

LDV-Fc Behaves Like Regular Fc Normal expression; can be purified by ProA  Blood exposure is similar to a wild-type human Fc after injection in mice  Mouse PK Study (i.v. dosing 5 mg/kg) 60 Group 1 50 Group 2 microgram/mL 40 30 20 10 0 0 20 40 60 80 100 Hour post-dosing Recombinant LDV-Fc purified with protein A ( SDS- PAGE, reducing conditions, coomasie staining). Belmont Biosciences, Inc 9

LDV-Fc enables site-specific amine conjugations at its N-termini • LDV has only 2 reactive amine groups available at its N-termini. • = reactive amine group for conjugation This allows for a highly homogeneous conjugated = recombinant IgG LDV Fc end product. = small molecule conjugate CH2 CH3 Human LDV-Fc Inter chain disulfide bonds CH2 CH3 CH2 CH3 Belmont Biosciences, Inc 10

Biotinylation of LDV-Fc Results in a Homogeneous End-product  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 Belmont Biosciences, Inc 11

Potential Applications: Several types of molecules can be conjugated to an LDV-Fc Small molecule e.g. NHS ester reaction Polymer CH2 CH3 H 2 N- LDV-Fc CH3 CH2 H 2 N- siRNA Optional fused targeting domain Non-natural peptide or Peptibody at C-terminus Available for purchase as a research reagent -or- Commercial license available Belmont Biosciences, Inc 12

LDV-Fc is developed by Belmont Biosciences, Inc. This technology is available for licensing Contact Gray Shaw gshaw@belmontbio.com 13

Bispecific Antibody = Non-natural + Multi-domain 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 Spiess, C., et al.,. Mol. Immunol. (2015) domain pair available 14

Multi-domain Challenges Surface at Various Stages • Production yield • • Stable cell line and Quantification methods • • Off rate ranking Stability and formulation single cell cloning Stable Cell Line Large scale Development, Antibody Antibody Antibody Functional Bioanalytical antibody Generation Screening Optimization Characterization Process Characterization Production Development • Binding kinetics • • Binding kinetics Fc receptor and • Competition assay FcRn binding assay Case Study 3:  – A Fab fusion bispecific antibody Case Study 4:  – An Fc fusion bispecific antibody 15

Case 3: Fab-based Bispecific Antibodies Functional domains can be Function domains Function domains – scFv – Nanobody – Ligand – Domain or fragment promoting dimer formation Fd LC Advantages ‒ Adjustable affinity - Flexibility on the valency ‒ Undesired effector functions removed Function domains Function domains ‒ Better design profile than BiTE format ‒ Two chains in the molecule 16

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  NR R >98% Purity by reducing capillary electrophoresis >98% Purity by SE-HPLC >98% Purity by reducing capillary electrophoresis 17

Each Domain is Functionally Active Binding to Target cell line BsAb mediated killing assay Binding to T cells Efficacy in xenograft model 18

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  19

Titer Quantitation of CM Showed Inconsistent Results Titer measurements by Octet targeting different domains show uncorrelated results Titer Values 80.0 Linear (Y = a * X + b) a 0.0002171 b 0.001482 70.0 LOQ=0.8 ug/mL 60.0 LOD=0.05 ug/mL 50.0 Arm 1 40.0 30.0 Linear (Y = a * X + b) a 0.0009554 20.0 b 0.0020708 10.0 LOQ=0.8 ug/mL LOD=0.2 ug/mL 0.0 0.0 50.0 100.0 150.0 200.0 250.0 Arm 2 Titer measurement results using different antigens appear uncoupled. Which measurement is correct? 20

Different Types of Molecules Found by anti-CH1 Purification NR Target molecule Target molecule, heterodimer of Fd and light chain R NR Undesired molecule A Fd homodimer purified by anti-CH1 R Fd chain should not have formed dimer, but it did 21

Undesired Molecule Loses Binding to One Antigen Target Undesired Antigen 1 Antigen 2 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 NA Antigen 1 Undesired 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 22

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 23

Extensive Clone Screening Identified Well Behaving Clones  Stable clones were found after two rounds of single cell cloning Single cell clone and subclones VCD profile 20 VCD (x10^6 cells/mL) 15 TT5730 - XCX 2A6 10 TT5731 - XCX 3A6 5 TT5688 - XCX 3B6 TT5691 - CL XCX Clone 0 0 5 10 15 20 Time (day) Single cell clone and subclones viability profile 120 100 Viability (%) 80 TT5730 - XCX 2A6 60 TT5731 - XCX 3A6 40 20 TT5688 - XCX 3B6 0 TT5691 - CL XCX 0 5 10 15 20 Time (day) 24

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. 25