Analytical and Developm ent Challenges w ith Recom binant Antibody - PowerPoint PPT Presentation

Analytical and Developm ent Challenges w ith Recom binant Antibody Mixtures for the Treatm ent of I nfectious Disease and Cancer Torben P. Frandsen, Ph.D. Director of Antibody Chemistry Symphogen A/S WCBP 2011 Meeting January 12-14 Washington D.C. 2 February 2011 Symphogen/1

Unique Approach to Lead Selection Explore m Abs & m Ab m ixtures sim ultaneously � High specificity mAb � Potent � Long half-life � Low immunogenicity � Proven technology � Synergy mAb � Multiple mechanisms of action � Broader inhibitory profile MIXTURE � Drug escape less likely 2 February 2011 Symphogen/2

Antibody m ixtures – Sym phogen’s Approach to Com plex Disease 2 February 2011 Symphogen/3

Broad Pipeline 2 February 2011 Symphogen/4

An I ntegrated Platform Applicable from discovery to com m ercial production of m Abs and m Ab m ixtures 2 February 2011 Symphogen/5

Sym Select™ Outline 2 February 2011 Symphogen/6 Confidential

Exam ple of Sym Select Readout Proliferation Assay Increasing efficacy Mix1 Mix2 150 percentage of untreated control Mix3 Mix4 Mix5 Mix6 Metabolic activity as Mix7 Mix8 100 Mix9 Mix10 Mix11 Mix12 Mix13 Mix14 50 Mix15 Mix16 Mix17 Mix18 Mix19 Mix20 0 Negative Control 0.0001 0.001 0.01 0.1 1 10 100 Antibody Concentration (µg/ml) 2 February 2011 Symphogen/7

Sym press™ Cell Banking & Manufacturing 2 February 2011 Symphogen/8

Manufacturing Approaches for Recom binant Ab Mixtures • Approach 1 − Manufacturing of individual DS − Mix into 1 DP • Approach 2 − Manufacturing of DS from a pWCB − Release & characterization strategy for recombinant Ab mixtures ��� ��� ��� ��� ��� ��� ��� ��� ��� ��� ��� �� �� ��� � �� ��� � �� ����� ������ � �� ��� � �� ����� ������ ��� � ��� � �� � ��� �� �� � ��� �� �� � ��� �� �� � ��� �� � ������ �� � �� ��� � �� ��� � � ������ �� � �� ��� � �� ��� � � ������ �� 2 February 2011 Symphogen/9

Sym press™ Manufacturing Platform Recom binant Plasm ids Host cells polyclonal antibody − FACS-based single- cell cloning Transfec- Manufacturing tion − Automation integrated for higher capacity − Frontloaded quality testing of clones Seed train Cell line expansion − Production capacity measured in scale- down fed batch cultures − Iterative screening of Mixing of pMCB pW CB Cell line banking cell lines mixed compositions 2 February 2011 Symphogen/10

Control of Relative 5:5 mix Antibody Com position 100 80 60 % 40 20 Ab 1 0 W W W W W W F • Two ECHO cell lines producing two Ab 2 e e e e e e e d e e e e e e B k k k k k k a 1 2 3 4 5 6 t different antibodies were mixed in c h different cell ratios and banked as pMCBs 4:6 mix 100 80 60 % 40 • pMCB cells were thawed and cultivated 20 Ab 1 0 W W W W W W F Ab 2 e for 6 weeks followed by 14 days fed-batch e e e e e e d e e e e e e B k k k k k k a 1 2 3 4 5 6 t c h cultivation. 3:7 mix 100 • Antibodies were purified from samples at 80 60 % 40 different time points and antibody 20 Ab 1 0 W W W W W W F e Ab 2 e e e e e e d e e e e e e distribution determined by CIEX B k k k k k k a 1 2 3 4 5 6 t c h 90 y b 2:8 mix 85 d e ‐ 100 R² = 0,997 n i 80 80 60 m % r 40 75 6 e 20 t Ab 1 k 0 e e 70 W W W W W W F d Ab 2 e e e e e e e e d e e e e e e 1 k k k k k k B w 65 b a 1 2 3 4 5 6 t t A c a h f 60 0 X E s I 55 e C 1:9 mix g a 50 t 100 n 80 e 45 c 60 % r 40 e 40 20 P Ab 1 0 W W W W W W F Ab 2 40 50 60 70 80 90 100 e e e e e e e d e e e e e e k k k k k k B a 1 2 3 4 5 6 Expected percentages t c h 2 February 2011 Symphogen/11

Single-Batch Production of 6 -Mix High Batch-to-batch Consistency Relative antibody composition determined by cation exchange chromatography 7 independent fed-batch productions from pWCB 2 February 2011 Symphogen/12

Sym press™ Release & Characterization 2 February 2011 Symphogen/13

Strategy for Release & Characterization 2 February 2011 Symphogen/14

Product Specific Methods. 2 m Ab 1024 992 992 1024 CIEX RP-HPLC 2 February 2011 Symphogen/15

Product Specific Methods. 6 m Ab E F B A C D Separation of 6 Ab using CIEX 2 February 2011 Symphogen/16

Product Specific Methods. 2 5 m Ab 25,0 Run 1 Run 2 20,0 Run 3 Run 4 Run 5 Relative area (%) Run 6 15,0 Run7 Run 8 Run 9 10,0 Run 10 5,0 0,0 157 159 160 162 189 191 192 196 197 199 201 202 203 207 240 241 245 293 301 305 306 317 319 321 324 Antibody Light Chain LC MS Persson P, Engström A, Rasmussen LK, Holmberg E, Frandsen TP. 2010. Anal. Chem. 82: 7274-7282. 2 February 2011 Symphogen/17

Multiplex Quantitation of m Abs by MRM Addition of reference peptides m Ab 1 Digestion to Quantitation Signature Peptides m Ab 2 • Identification of unique signature peptides • Addition of fixed conc. of isotopically labeled reference peptides equal in Principle of MRM quantitation sequence to signature peptides • Digestion of sample • Quantitation of each mAb by MRM of the 4 peptides 2 February 2011 Symphogen/18

CMC Regulatory Status for Antibody Mixtures • CMC regulatory path established Based on extensive and constructive FDA dialogue, EMA scientific advice o and interactions with national agencies • FDA endorsem ent of: Two-tiered polyclonal cell bank strategy – pMCB/pWCB concept o Single-batch manufacturing approach o Release and characterization strategy and methods for DS and DP o • Alignm ent betw een FDA and EMA advice • Sym 0 0 1 ( Rozrolim upab) is the first recom binant polyclonal antibody to enter the clinic Phase 1 clinical trial completed in the US o Phase 2 clinical trials ongoing o • Sym 0 0 4 ( anti-EGFR) Phase 1 clinical trial ongoing in the US and EU o 2 February 2011 Symphogen/19

Pharm acology W hy Use Com binations of m Ab? 2 February 2011 Symphogen/20

The Sym 0 0 4 Drug Candidate Sym 0 0 4 is a 1 :1 m ixture of 9 9 2 and 1 0 2 4 bind non- tw o chim eric I gG1 antibodies overlapping epitopes in dom ain 9 9 2 and 1 0 2 4 I I I of the Epiderm al Grow th Factor Receptor ( EGFR) 2.5 2.0 OD 450nm 1.5 Sym004 1.0 992 1024 0.5 0.0 0.0001 0.001 0.01 0.1 1 10 100 Antibody Concentration (µg/ml) Pedersen MW, Jacobsen HJ, Koefoed K, Hey A, Pyke C, Haurum JS and Kragh M. 2010. Cancer Res 70:588-97. 2 February 2011 Symphogen/21

Synergistic I nhibtion of A4 3 1 NS Cells I n Vitro 140 Metabolic activity as percentage of untreated control cells 120 No effect level 100 80 60 Control mAb 992 40 1024 Sym004 20 Cetuximab Panitumumab 0 0.0001 0.01 1 100 Antibody concentration [µg/ml] 2 February 2011 Symphogen/22

Synergy and Superior in vivo Efficacy of Sym 0 0 4 in an A4 3 1 NS Xenograft Model Treatment period 2250 2000 1750 Tumor volume (mm 3 ) 1500 1250 1000 750 500 250 0 0 10 20 30 40 50 60 70 Days post tumor inoculation Control mAb, 50 mg/kg, i.p. twice weekly, N=9 992, 50 mg/kg, i.p. twice weekly, N=9 1024, 50 mg/kg, i.p. twice weekly, N=9 Cetuximab, 50 mg/kg, i.p. twice weekly, N=9 Sym004, 50 mg/kg, i.p. twice weekly, N=9 2 February 2011 Symphogen/23

Sym 0 0 4 I nduces Massive EGFR I nternalization 1 µg/ml of Sym004 2h 1 µg/ml of Cetuximab 2h 3D build of A431NS cells at 600X 2 February 2011 Symphogen/24

Phase 1 / 2 Clinical Study - Ongoing An open-label, multi-center Phase 1/2 dose escalation study to investigate the safety, tolerability and activity (Part B) of multiple doses of Sym004 in patients with advanced solid tumors Patient population Part A (phase 1 component) in a heterogenous population of patients with refractory or recurrent advanced solid tumors and late stage cancer Part B (phase 2 component) in a homogenous patient population with refractory or recurrent advanced mCRC and wild type KRAS Tim eline • IND submitted December 2009 • Cleared FDA January 2010 • Study initiated March 2010 2 February 2011 Symphogen/25

Sym 0 0 4 Principle can be Extended to Other Solid Tum or Targets Like HER2 Xenograft Model of Gastric Cancer Treatment Period 1600 1400 1200 Tumor volume (mm 3 ) 1000 800 600 400 200 0 10 20 30 40 50 60 Days post tumor inoculation 50 mg/kg mAb mixture A i.p. twice weekly 50 mg/kg of Control mAb i.p.twice weekly 50 mg/kg mAb mixture B i.p. twice weekly 50 mg/kg Trastuzumab i.p. twice weekly 50 mg/kg mAb mixture C i.p. twice weekly 2 February 2011 Symphogen/26