Interference from Fc-Fc Interactions in Bridging Immunogenicity Assays for IgG4 mab Therapeutics

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1 Interference from Fc-Fc Interactions in Bridging Immunogenicity Assays for IgG4 mab Therapeutics November, 2014 Michael Partridge Ph.D.

2 IgG4 Antibody Therapeutics Natalizumab (Tysabri): Marketed Pembrolizumab (Keytruda): Marketed Gemtuzumab ozogamicin (Mylotarg): Withdrawn 85 IgG4 therapeutics under investigation for clinical indications (tabs.craic.com) Regeneron IgG4 therapeutics - Multiple in various stages of non-clinical and clinical development

3 Unusual Features of Human IgG4 Does not activate complement or bind Fc receptors - mab therapeutic where MOA doesn t require CDCC or ADCC Capable of Fab arm exchange to generate a bispecific Interacts via Fc under certain circumstances

4 Human IgG4 Fc Interactions: Background Human IgG4 binds to immobilized IgG via Fc Demonstrated by Rispens et al. with IgG immobilized on Sepharose Rispens et al. (2009) J. Imm.

5 RLU RLU Human IgG4 binds to immobilized IgG Fc in ELISA Low affinity, but specific to IgG4 Underappreciated source of matrix interference IgG4 level in 2% human serum 2000 Specific Fc-Fc REGN IgG4 (ng/ml) Anti-id Capture & Detect Non-Specific CAb, anti-id detect IgG4 mab IgG1 mab (ng/ml) Non-Specific CAb, anti-human IgG detect

6 Interaction with immobilized IgG is an intrinsic property of all human IgG4 IgG4 reported in IgG RF may be problematic

7 Human IgG4 may bind to Antigen-bound IgG4 Sets of IgG1 & IgG4 variants specific for different antigens Only IgG4 binds to Agbound IgG4 Rispens et al. (2009) J. Imm.

8 Denatured IgG Fc Inhibit Human IgG4 Binding to Immobilized Fc RLU IgG4 in human serum binds immobilized Fc Biotin Anti- Human IgG (ug/ml) Human IgG4 0 Untreated IgG Denatured IgG Immobilized IgG

9 Absorbance (215 nm) Non-Covalent Oligomers Inhibit Human IgG4 binding to Immobilized Fc RLU F1 F F3 F Elution Time (min) Un. Den. F1 F2 F3 F4 Mouse IgG Fc Added to Human IgG4 (µg/ml) Biotin Anti- Human IgG4 Human IgG4 66 Native PAGE Immobilized IgG

10 IgG4 Fc-Fc Contacts IgG4 in human serum binds immobilized Fc Denatured IgG Fc inhibits this interaction In Solution Fc Aggregates bind IgG4 Native Fc dimers don t bind IgG4

11 Automation of ADA Assay Regeneron performs fully automated bioanalysis - Allows us to analyze all phase 3 PK and ADA samples in-house - Assays for multiple mab and non-mab biotherapeutics In 2013 we began automating an ADA assay for an IgG4 mab - High background signal observed in negative samples

12 Counts Bridging ADA Assay with IgG4 Labeled Drugs: NQC Signal Increased After Reagent Incubation For automated sample analysis, solutions are prepared many hours in advance S/N S/N NQC LQC (60 ng/ml) 0 0 Hours 6 Hours Labeled Drug Incubation

13 ADA Assay: Labeled IgG4 Drug Fc-Fc Interactions Ruthenium labeled Drug Trace Aggregates could mimic oligomeric Fc ADA Biotinylated Drug Streptavidin coated plates Monomeric IgG4 labeled drugs Fc-Fc interaction? Ru-Drug 1.62 %

14 Background Signal Increases Over Time in Bridging ADA Assay with Labeled IgG4 Drugs 500 Signal 3.0 Fold Signal Change IgG4 IgG Labeled Drug Incubation Time (min) Prior to Sample Addition 5 IgG4 & 5 IgG1 labeled drugs: exogenous & endogenous targets, soluble & membrane bound targets

15 Excess Labeled Drug Co-incubated in Multiple Steps Fc-Fc interactions could form in multiple steps thereby generating signal 1) Reagent Preparation (Automation) 2) Sample Incubation (O/N incubation) 3) Assay Plate Incubation 4) Read Buffer

16 Counts Assay Sensitivity Decreases with Labeled Drug Incubation Time S/N Signal Signal to background CPF NQC PQC 7.5 ng/ml PQC 15 ng/ml Labeled Drug Incubation Time (min) May impact detection of low titer positive samples

17 Counts Labeled IgG4 Drugs: Increasing Signal with Plate Order S/N 175 Signal 1.4 Signal to background 150 X = NQC Plate 1 Plate 2 Plate Plate 1 Plate 2 Plate 3 **p< **p< NS NS Screening CP Exercises: Plate Order No Effect on Screening Cut Point Determination **Student s t-test (NS = not significant)

18 Counts Labeled IgG1 Drugs: No Change in Signal with Plate Order S/N Signal Signal to background 125 X = NQC Plate 1 Plate 2 Plate Plate 1 Plate 2 Plate 3 NS NS NS NS Screening CP Exercises: Plate Order No increase in assay signal with IgG1 drugs NS = not significant

19 Confirmation Assay Without Drug With Drug Unlabeled Drug Fc-Fc contacts generate signal Unlabeled drug competes for Fc-Fc contacts, reducing signal

20 Counts Confirmation Assay with IgG4 Labeled Drug # ** Without Drug With Drug ** Without Drug 10 minutes 60 minutes %Inhibition 10 mins = 12% 60 mins = 34% With Drug %Inhibition dependent on reagent prep. time **p<0.001, Student s t-test

21 Counts Confirmation Assay with IgG4 Labeled Drug # %Inhibition 6 mins = 10% 37 mins = 27% Without Drug With Drug Without Drug With Drug 6 minutes 37 minutes %Inhibition increases with reagent prep. time

22 Counts Confirmation Assay with IgG1 Labeled Drug %Inhibition 10 mins = 5% 45 mins = 0% Without Drug With Drug Without Drug With Drug 10 minutes 45 minutes %Inhibition constant with reagent prep. time

23 Confirmation Assay: Reagent Incubation Signal increase greater without excess unlabeled drug Longer reagent incubation time = larger %Inhibition Reagent incubation time needs to be controlled -Confirmation cut point determination -Confirmation assay

24 % Inhibition Impact on Confirmation Assay Also Observed in Plate Order IgG1 Drug IgG4 Drug ** ** Plate 1 Plate 2 Plate 3-10 Plate 1 Plate 2 Plate 3 NS NS NS Confirmation assay plate order **p<0.001 Even plate order can impact %Inhibition in confirmation cut point determination **Student s t-test

25 For IgG4 Drugs, Plate Run Order Impacts %Inhibition in Confirmation Cut Point Determination %Inhibition Plate Order: IgG4 Drugs Drug 1 Drug 2 Drug 3 Drug 4 *p= &2<3 p=0.052 NS p=0.066 NS p=0.876 NS %Inhibition Plate Order: IgG1 Drugs Drug 5 Drug 6 Drug 7 p=0.29 NS p=0.61 NS p=0.326 NS Drug 1: plate 3 has a statistically higher %Inhibition. Drugs 2 and 3: plate order effect is not significant, but close. *ANOVA (NS = not significant)

26 How Does This Affect Confirmation Cut Point? 2 analysts, 2 days, 54 individuals +/- drug 6-10 mins vs 20 minutes reagent incubation % INH Confirmation Cut Point* Reagent Incubation Estimated Observed 6-10 Mins Mins % difference is within precision limits of the assay *FP Rate = 0.1 %

27 Labeled Drug Solution Preparation Scheme IgG4 Drugs Solutions: 2X Tris (acid Rx) 4X Bio-Drug 4X Ru-Drug Screen: Combine three solutions 2:1:1 before adding to acidified sample Confirmation: Divide 2X Tris, add unlabeled drug to one. Combine three solutions 2:1:1 before adding to acidified sample

28 Counts Separating Bio-Drug & Ru-Drug Solutions Reduces Negative Control Signal Hours 6 Hours Combined Bio-Drug & Ru-Drug Separate Bio-Drug & Ru-Drug Containing Tris Separate Tris, Bio-Drug, Ru-Drug &

29 Excess Labeled Drug Co-incubated in Sample: Long Incubations 1) Reagent Preparation (Automation) 2) Sample Incubation (O/N incubation) 3) Assay Plate Incubation 4) Read Buffer

30 Counts Overnight Sample Incubation in Serum Does Not Generate High Signal Without Serum With Serum (3.3%) hr 2 hr O/N 0 1 hr 2 hr O/N Sample Plate Incubation Time Bio & Ru-IgG4 Bio & Ru-IgG1

31 Serum Reduces IgG4 Fc Interactions Signal Drug 1 (IgG1) Drug 2 (IgG1) Drug 3 (IgG1) Drug 4 (IgG4) Drug 5 (IgG4) Drug 6 (IgG4) Signal (Counts) Hu SRM Mky SRM Buffer Zero incubation time 500 IgG IgG4 Human Serum (3%) Monkey Serum (3%) Buffer Human IgG µg/ml in serum 3-15 µg/ml in 3.3% serum Monkey (& rat) serum also block signal

32 Counts IgG4 Fc-Fc Interacts with Other IgGs IgG1:IgG4 Pairing S/N 400 Signal 2.5 Signal to background Labeled Drug Incubation Time (mins) Bio & Ru-IgG4 Bio & Ru-IgG1 Bio-IgG4 & Ru-IgG1 Bio-IgG1& Ru-IgG4

33 Conclusions Fc interactions between human IgG4 labeled drugs can cause increased signal over time in bridging assays Decreases assay sensitivity Detection of low titer antibodies Major impact - Confirmation assay Prepare labeled drug solutions separately Cons of bridging ADA assays Fc Interference in specific disease indications Affects IgG4 formulations?

34 Acknowledgements Elif Karayusuf Olena Dziadiv Onson Luong John Garlits Thanoja Sirimanne Jihua Chen Bob Dreyer Ganga Dhulipala Erica Pyles Enoch Shum Uma Vijayam Giane Sumner Manoj Rajadhyaksha Al Torri