Maximizing Assembly and Yield of Unmodified Bispecific Antibodies

Transcription

1 Maximizing Assembly and Yield of Unmodified Bispecific Antibodies Pauline Malinge Head of Molecular Interaction Facility 1

2 Outline Introduction The κλ body format The κλ body platform Bispecific Antibody Discovery Light chain driven antigen specificity: arm identification Bispecific assembly and high yield expression in CHO cells Conclusions 2

3 body format Bispecific antibodies undistinguishable from a human mab NovImmune body : One common heavy chain Two different light chains, one kappa and one lambda A different specificity driven by each arm in combination VH+VL Fully native IgG: no mutation, no linker Common HC Nature Communications, Fischer et al

4 body platform mab discovery and arm combination for bispecific generation A A VH A V A B B B B VH V scfv Phage Display Library (size >10 11 ) Fixed VH mabs Same HC Ab Mixture BiAb 4

5 body platform mab discovery and arm combination for bispecific generation A A VH A V A B B B B VH V scfv Phage Display Library (size >10 11 ) Fixed VH mabs Same HC 1. Target specific mab identification Ab Mixture BiAb 5

6 Light chain driven antigen specificity Light chains fulfill important functions Drive the specificity of each arm in combination with the fixed VH Allow generic and scalable purification scfv Phage Libraries Diversity introduced only in the VL 6

7 Counts % in h ib itio n Light chain driven antigen specificity Target binding and neutralization (functional assay) hmsln IgG binding at the cell surface (FACS) Blocking MUC16-MSLN interaction MUC Benchmark Neg Ctrl MSLN A n tib o d y L o g [M ] Blocking mabs identified without LO and with affinities similar to benchmark 7

8 M F I Light chain driven antigen specificity Affinities obtained directly from libraries in the nm range (from 0.5 to 900nM) Sub-nM obtained affinities via lead optimization High diversity of sequences and germlines identified Over 120 validated mabs against 14 targets, all sharing the same heavy chain CD19 CD47 EpCAM EGFR MSLN A n tib o d y L o g [M ] < 1nM M 8

9 Light chain driven antigen specificity Affinity determination: two different platforms Biacore Surface Plasmon Resonance Interaction under flow Comparative study Octet BioLayer Interferometry Dip and Read Assay Higher throughput Flow channel Octet validation for mab affinity characterization 96/384 well plate Association Dissociation 9

10 Light chain driven antigen specificity Biacore Octet K D = 0.68 ± 0.01 nm (n=2) kon = /Ms koff = /s K D = 0.64 ± 0.04 nm (n=2) kon = /Ms koff = /s Similar data generated Octet platform selected for high throughput mab affinity screening 10

11 Light chain driven antigen specificity Affinity determination (hmsln) K D = M Kon = /Ms koff = /s Weak K D = M kon = /Ms koff = /s AFFINITY MATURATION K D = M kon = /Ms koff = /s Strong Two rounds of lead optimization with diversity introduced only in the light chain 11

12 Light chain driven antigen specificity Characterization of IgG-FcR interaction for selected binders (FcR and FcRn) Human FcRn: ph dependent binding hfcrn biot mab K D = M kon = /Ms koff = /s K D = M kon = /Ms koff = /s 12 Expected kinetic data for higg1 on hfcrn J Immunol Methods, Malinge et al. 2012

13 body platform mab discovery and arm combination for bispecific generation A A VH A V A B B B B VH V scfv Phage Display Library (size >10 11 ) Fixed VH mabs Same HC Ab Mixture BiAb 2. Bispecific assembly and expression 13

14 body platform Culture Supernatant Manufacturing purification process (GE Healthcare resins) Protein A affinity Flow through: contaminants, free light chains Sequential affinity chromatography Protein A KappaSelect KappaSelect affinity Flow through: mab LambdaFabSelect Same process for all -bodies PA BiAb PA BiAb PA BiAb PA BiAb LambdaFabSelect affinity Flow through: mab pi 99% purity 14

15 Bispecific assembly and high yield expression Bispecific body generation 3 steps: Step 1. Cloning of the fixed heavy chain and selected kappa and lambda light chains Novimmune proprietary tri-cistronic vector Step 2. Transient or stable expression in mammalian cells Generation of a mixture of mabs and BiAb in a single cell 25% 25% Step 3. Scalable and generic downstream process Process ensuring high purity and integrity Affinity purification applicable to all bodies 50% BiAb 15

16 Bispecific assembly and high yield expression Step 2 CHO Cell Transfection CHO Pool Generation by selection Clonal CHO Cell Line Generation Productivity Screening Productivity screening light chain ratio Harvest IgG Quantitation PA Purification IgG Quantitation LC Ratio 16

17 m A b T ite r (g /L ) O c te t Ig G T ite r (g /L ) CHO pool screening based on the light chain ratio Octet IgG quantitation (Protein A biosensors) Correlation Octet/PA-HPLC Pool IgG Productivity (FOG) 4 3 Y = *X y = 0.74x R 2 = g/l of total IgG m A b T ite r (g /L ) P A H P L C 0 Ig G T it e r a t H a r v e s t Similar pool ranking between Octet and PA-HPLC IgG quantitations 16 best producer pools selected for light chain ratio determination 17

18 CHO pool screening based on the light chain ratio Expression of a mixture of mabs, BiAb, dimers and free light chains in the supernatant 25% 25% 50% In theory, best clone = 25% mab + 25% mab + 50% BiAb Goal: Selection of high producer CHO pools that express the best LC ratio early in the cell line development process 18

19 CHO pool screening based on the light chain ratio 1 st step: Protein A purification 2 nd step: Determination of the light chain ratio High throughput screening assay development on the Octet 2 assays to determine the and the LC concentrations Biot. Ligand Biot. Ligand LC ratio determined 50% Ab Mixture 19

20 AU CHO pool screening based on the light chain ratio 1. Correlation Agilent/HIC/Octet Agilent Profile HIC Profile Correlation HC y = 1.10x 1.36 R 2 = LC HC LC LC 83% 17% Time Good correlation between HIC and Octet 20

21 CHO pool screening based on the light chain ratio 2. Correlation Agilent/Octet Agilent Profile Octet Correlation LC HC HC LC 52% LC 48% 50% y = 1.02x 2.18 R 2 = Similar LC ratio obtained with the Agilent and the Octet Validation of the Octet method for light chain ratio determination 21

22 Ig G T ite r (g /L ) D is tr ib u tio n % Ig G T ite r (g /L ) Pool screening and CHO cell line generation 3 CHO Pool Generation 2 50% 1 0 Ig G T it e r a t H a r v e s t Clonal CHO Cell Line Generation CHO cell line generation: 2-4 g/l of total IgG g/l of body 0 Ig G T it e r - b o d y T it e r 0 m Ab - b o d y m Ab 22

23 Conclusions The body platform scfv Phage Display Octet implementation for high throughput Target specific mab identification Target binding Affinity determination FcR interaction body expression and purification CHO Pool screening: - Productivity - LC ratio 23

24 Conclusions Set up of a platform for body generation Light chain driven antigen binding with: High affinity and specificity Neutralization activity Lead diversity Simple bispecific antibody generation: Tri-cistronic vector expression system High throughput screening using the Octet Generic and robust purification process 24

25 Acknowledgements Marie Kosco-Vilbois Nicolas Fischer Giovanni Magistrelli Yves Poitevin Soheila Josserand Coralie Borrossi Guillemette Pontini Pierre-Alexis Cayatte Gérard Didelot Thank you for your attention 25