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

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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

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Symphogen/1 2 February 2011

Analytical and Developm ent Challenges w ith Recom binant Antibody Mixtures for the Treatm ent

  • f 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.

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Symphogen/2 2 February 2011

Unique Approach to Lead Selection

Explore m Abs & m Ab m ixtures sim ultaneously

mAb mAb

MIXTURE

High specificity Potent Long half-life Low immunogenicity Proven technology Synergy Multiple mechanisms of action Broader inhibitory profile Drug escape less likely

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Symphogen/3 2 February 2011

Antibody m ixtures – Sym phogen’s Approach to Com plex Disease

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Broad Pipeline

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An I ntegrated Platform

Applicable from discovery to com m ercial production of m Abs and m Ab m ixtures

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Symphogen/6 2 February 2011

Sym Select™ Outline

Confidential

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Symphogen/7 2 February 2011

Exam ple of Sym Select Readout Proliferation Assay

0.0001 0.001 0.01 0.1 1 10 100 50 100 150

Mix19 Mix1 Mix2 Mix3 Mix5 Mix7 Mix9 Mix11 Mix13 Mix15 Mix16 Mix17 Mix18 Mix4 Mix6 Mix8 Mix10 Mix12 Mix14 Mix20 Negative Control

Antibody Concentration (µg/ml)

Metabolic activity as percentage of untreated control Increasing efficacy

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Symphogen/8 2 February 2011

Sym press™ Cell Banking & Manufacturing

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Symphogen/9 2 February 2011

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

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Symphogen/10 2 February 2011

Sym press™ Manufacturing Platform

− FACS-based single- cell cloning − Automation integrated for higher capacity − Frontloaded quality testing of clones − Production capacity measured in scale- down fed batch cultures − Iterative screening of mixed compositions

Recom binant polyclonal antibody Plasm ids Host cells Transfec- tion Cell line expansion Cell line banking Mixing of cell lines pMCB pW CB Seed train Manufacturing

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Symphogen/11 2 February 2011

Control of Relative Antibody Com position

20 40 60 80 100 W e e k 1 W e e k 2 W e e k 3 W e e k 4 W e e k 5 W e e k 6 F e d B a t c h %

5:5 mix

Ab 1 Ab 2 20 40 60 80 100 W e e k 1 W e e k 2 W e e k 3 W e e k 4 W e e k 5 W e e k 6 F e d B a t c h %

4:6 mix

Ab 1 Ab 2 20 40 60 80 100 W e e k 1 W e e k 2 W e e k 3 W e e k 4 W e e k 5 W e e k 6 F e d B a t c h %

3:7 mix

Ab 1 Ab 2 20 40 60 80 100 W e e k 1 W e e k 2 W e e k 3 W e e k 4 W e e k 5 W e e k 6 F e d B a t c h %

2:8 mix

Ab 1 Ab 2 20 40 60 80 100 W e e k 1 W e e k 2 W e e k 3 W e e k 4 W e e k 5 W e e k 6 F e d B a t c h %

1:9 mix

Ab 1 Ab 2
  • Two ECHO cell lines producing two

different antibodies were mixed in different cell ratios and banked as pMCBs

  • pMCB cells were thawed and cultivated

for 6 weeks followed by 14 days fed-batch cultivation.

  • Antibodies were purified from samples at

different time points and antibody distribution determined by CIEX

‐

R² = 0,997

40 45 50 55 60 65 70 75 80 85 90 40 50 60 70 80 90 100

P e r c e n t a g e s f A b 1 d e t e r m i n e d b y C I E X a t w e e k 6 Expected percentages

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Symphogen/12 2 February 2011

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

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Symphogen/13 2 February 2011

Sym press™ Release & Characterization

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Strategy for Release & Characterization

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Symphogen/15 2 February 2011

Product Specific Methods. 2 m Ab

992 992 1024 1024

CIEX RP-HPLC

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Symphogen/16 2 February 2011

Product Specific Methods. 6 m Ab

Separation of 6 Ab using CIEX

B A C D E F

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Symphogen/17 2 February 2011

Product Specific Methods. 2 5 m Ab

0,0 5,0 10,0 15,0 20,0 25,0 157 159 160 162 189 191 192 196 197 199 201 202 203 207 240 241 245 293 301 305 306 317 319 321 324

Antibody Relative area (%)

Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run7 Run 8 Run 9 Run 10

Light Chain LC MS

Persson P, Engström A, Rasmussen LK, Holmberg E, Frandsen TP. 2010. Anal. Chem. 82: 7274-7282.

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Symphogen/18 2 February 2011

Multiplex Quantitation of m Abs by MRM

  • Identification of unique signature

peptides

  • Addition of fixed conc. of isotopically

labeled reference peptides equal in sequence to signature peptides

  • Digestion of sample
  • Quantitation of each mAb by MRM of

the 4 peptides

Digestion to Signature Peptides m Ab 2 m Ab 1 Quantitation Principle of MRM quantitation Addition of reference peptides

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Symphogen/19 2 February 2011

CMC Regulatory Status for Antibody Mixtures

  • CMC regulatory path established
  • Based on extensive and constructive FDA dialogue, EMA scientific advice

and interactions with national agencies

  • FDA endorsem ent of:
  • Two-tiered polyclonal cell bank strategy – pMCB/pWCB concept
  • Single-batch manufacturing approach
  • Release and characterization strategy and methods for DS and DP
  • 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
  • Phase 2 clinical trials ongoing
  • Sym 0 0 4 ( anti-EGFR)
  • Phase 1 clinical trial ongoing in the US and EU
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Symphogen/20 2 February 2011

Pharm acology W hy Use Com binations of m Ab?

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Symphogen/21 2 February 2011

The Sym 0 0 4 Drug Candidate

Sym 0 0 4 is a 1 :1 m ixture of tw o chim eric I gG1 antibodies 9 9 2 and 1 0 2 4 9 9 2 and 1 0 2 4 bind non-

  • verlapping epitopes in dom ain

I I I of the Epiderm al Grow th Factor Receptor ( EGFR)

0.0001 0.001 0.01 0.1 1 10 100 0.0 0.5 1.0 1.5 2.0 2.5

Sym004 992 1024

Antibody Concentration (µg/ml) OD450nm

Pedersen MW, Jacobsen HJ, Koefoed K, Hey A, Pyke C, Haurum JS and Kragh M. 2010. Cancer Res 70:588-97.

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Symphogen/22 2 February 2011

0.0001 0.01 1 100 20 40 60 80 100 120 140

992 1024 Sym004 Cetuximab Panitumumab Control mAb

Antibody concentration [µg/ml]

Metabolic activity as percentage

  • f untreated control cells

Synergistic I nhibtion of A4 3 1 NS Cells I n Vitro

No effect level

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Symphogen/23 2 February 2011

10 20 30 40 50 60 70 250 500 750 1000 1250 1500 1750 2000 2250

Sym004, 50 mg/kg, i.p. twice weekly, N=9 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

Treatment period

Cetuximab, 50 mg/kg, i.p. twice weekly, N=9

Days post tumor inoculation Tumor volume (mm3)

Synergy and Superior in vivo Efficacy of Sym 0 0 4 in an A4 3 1 NS Xenograft Model

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Symphogen/24 2 February 2011

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

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Symphogen/25 2 February 2011

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
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Symphogen/26 2 February 2011

Sym 0 0 4 Principle can be Extended to Other Solid Tum or Targets Like HER2

10 20 30 40 50 60 200 400 600 800 1000 1200 1400 1600

50 mg/kg of Control mAb i.p.twice weekly 50 mg/kg mAb mixture A i.p. twice weekly 50 mg/kg mAb mixture B i.p. twice weekly 50 mg/kg mAb mixture C i.p. twice weekly 50 mg/kg Trastuzumab i.p. twice weekly

Treatment Period

Xenograft Model of Gastric Cancer

Days post tumor inoculation

Tumor volume (mm3)

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Symphogen/27 2 February 2011

Conclusions

  • The necessary technologies allowing generation of

recombinant antibody mixtures have been developed

− Symplex™, High through-put isolation and generation of lead Ab − SymSelect™, Rational functional testing and selection − Sympress™, Cost–effective, commercial scale manufacturing

  • Manufacturing of recombinant antibody mixtures with high

batch-to-batch consistency have been demonstrated

  • A release and characterization strategy for recombinant

antibody mixtures have been implemented

  • Improved efficacy and synergy using mixtures of antibodies

have been successfully demonstrated in several cancer models

  • Recombinant antibody mixtures under evaluation in

different clinical trials

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Symphogen/28 2 February 2011

A Sym phony of Natural Antibodies

Contact: tpf@symphogen.com

Thank You

Our website: www.symphogen.com