Applications of HTRF and Tag-lite Assays for HTP Antibody Screening

Applications of HTRF and Tag-lite Assays for HTP Antibody Screening Brigitte Devaux, PhD Bristol Myers Squibb, Redwood City CA HTRF Symposium April 25, 2013 1

Introduction Generate human therapeutic antibodies to large number of targets (soluble or membrane bound) for various therapeutic indications Also produce reagent antibodies for research/development/clinical applications Hybridoma technology Fusion of splenocytes with myeloma partner Need to develop HTP assays amenable to screening hybridoma supernatants from fusion plates Immunization Create potential for screening on multiple antigens or cell types Tagged antigen versus irrelevant Ag with same tag human/cyno/mouse Ag target Conjugated Ag versus un-conjugated Ag (with PEG for example) Homologues from same family Transfectants and cancer cells 2

Hybridoma Workflow Human Ig Tg mice spleens E-Fusion Splenocytes + fusion partner Balb/c spleens HuG/K Screening HTRF Automation-96 ELISA (soluble Ag) Hybridoma-96 HTRF (soluble Ag) AD-384 Antigen Screening FACS (cells) Hybridoma-96 FMAT/Tag-lite (cells) AD-384 Ag specific Hit Picking Automation-24 Rescreen of positive lines Subcloning Expansion Purification

HTP Antigen Screening Assays Requirements: Detect antibody binding to antigen (Soluble or Cell based) Robust & flexible assays No wash steps Work in conditioned media Sensitivity: Adequate for Ab concentration range from 10 ng-10 g /ml +/- response (no need for EC50 determination) Up to 10,000 samples in a run Time sensitive (24 hour turn around) Rapid assay development against a wide range of targets

Presentation Outline HTRF antigen screening assays (3 examples) HTRF with tagged antigens (e.g. His, Fc - Ag1 & 2) HTRF with non-tagged antigen (Ag3) and labeled secondary reagents (non-blocking polyclonal) HTRF with multiple labeled antigens in one well (duplexing, 2 examples) HTRF with Drug Ab IgG4 versus irrelevant IgG4 HTRF with PEG versus non PEG Ag HTRF Blocking assay (1 example) HTRF Tag-Lite (2 examples) Tag-lite with GPCR1 Tag-lite with GPCR2 5

Presentation Outline HTRF antigen screening assays (3 examples) HTRF with tagged antigens (Ag1 & 2) HTRF with non-tagged antigen (Ag3) and labeled secondary reagents (non-blocking polyclonal) HTRF with multiple labeled antigens in one well (duplexing, 2 examples) HTRF with Drug Ab IgG4 versus irrelevant IgG4 HTRF with PEG versus non PEG Ag HTRF Blocking assay (1 example) HTRF Tag-Lite (2 examples) Tag-lite with GPCR1 Tag-lite with GPCR2 6

HTRF 665 Ratio HTRF 665 Ratio HTRF with Tagged Antigens (His, Fc): Ag 1 Tb Streptavidin Tb Biotin anti-his Ab Ag1-His (monomer) Murine Ab in supernatant Anti-mouse Fc AlexaFluor 647 Tb Anti-mouse Fc Tb Murine Ab in Supernatant Ag1-Hu Fc (dimer) Anti-Human Fc D2 12000 10000 8000 6000 4000 2000 0 Ag1-His MAB 1 MAB 2 MAB 3 migg 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 Ag 1 - Hu Fc MAB 1 MAB 2 MAB 3 migg Clone Conc. [ug/ml] Mab1 & 3: Positive Control Mab 2, Mouse IgG : Negative Control Clone Conc. [ug/ml]

AG1-His HTRF 665 Ratio HTRF Ag1-His Fusion Screening at 96 well stage Blue : Positive Control Red : Binding Supernatants Well Identification of Six Ab Supernatants with Binding Activity

Ag1-His HTRF 665 Ratio Ag1-His HTRF 665 Ratio Supernatants tested in Ag1 ELISA and FACS assays at 24 well stage Red : Identified by the HTRF assay at the 96 well stage Ag1-hFc ELISA (OD) CHO-Ag1 FACS MFI Same hits in HTRF and ELISA assays Not all HTRF/ELISA hits bind by FACS

HTRF with Tagged Antigens (His): Ag 2 Antigen has a large number of leucine rich repeats on its extracellular surface Primary screening: HTRF and ELISA in parallel HTRF binding in solution ELISA with Ag coated on plate HTRF Format Tb Anti-mouse Fc Tb mouse anti-his Ag2-His Human Clone in supernatant Anti-Human Fc AlexaFluor 647

Ag 2 - HTRF & ELISA vs. FACS All HTRF positive are FACS positives Good correlation between HTRF signal and FACS signal FACS positives (also HTRF +) missed by ELISA(OD<0.3)! Blue are FACS positive - subcloned

HTRF 665 Ratio HTRF with Labeled Secondary Reagent Detection: Ag3 Tb or Anti-mouse FC Tb Mouse Clone in Supernatant Ag3 Polyclonal anti-ag3 DL649 Polyclonal Biotinylated anti-ag3 and Streptavidin DL649 6000 HTRF - Ag3 - Labeled vs Biotinylated Secondary Antibody 5000 4000 Labeled (100 ng/ml Ag3) 3000 2000 Labeled (20 ng/ml Ag3) Labeled (5 ng/ml Ag3) Biotinylated (100 ng/ml Ag3) 1000 0 1 to 25 1 to 125 1 to 625 1 to 3125 0 Biotinylated (20 ng/ml Ag3) Biotinylated (5 ng/ml Ag3) Positive Control Clone (Dilution) Direct and indirect labeling of polyclonal generated same results

Ag3 HTRF 650 Ratio Positive Control (1:500) Positive Control (1:10) Potential Hits in Red Negative Control

HTRF with multiple Labeled Antigens in one well Search for anti-id Abs The antigen is an Ab! Do not want Abs binding to Fc domain Duplexing: Drug Ab IgG4 2 assays in 1 Tb Anti-mouse Fc Tb Mouse Clone in Supernatant Drug Ab (IgG4) DL649 AND Irrelevant IgG4 DL488

665 Ratio Drug Ab IgG4 DL 649 665 Ratio Yellow : Selected hits Light Blue : Positive Control Dark Blue or Pink : Not selected 12000 10000 8000 6000 4000 2000 Immunogen : Drug Ab V domain only 0 6000 0 1000 2000 3000 4000 5000 520 Ratio 5000 4000 3000 2000 1000 Immunogen : Drug Ab IgG4 0 0 1000 2000 3000 4000 5000 6000 7000 520 Ratio Irrelevant IgG4 DL 488 More anti-ids obtained when immunizing with V part of the Ab

HTRF with PEG-conjugated Antigen versus non-peg Ag Duplexing: PEG-Ag versus non-peg Ag Positive Control Ab PEG & non-peg Ag selective Sups PEG-Ag Selective Sups Positive Control Ab Non-PEG Ag selective Sups PEG-Ag did not coat to plates by ELISA No problem with HTRF

HTRF Blocking assay Goal: identify anti-ids which bind to Ab when ligand is bound (non-blockers) or anti-ids which interfere with ligand binding (blockers) Tb Anti-mouse FC Tb Mouse Clone in Supernatant Irrelevant Ligand (In Excess) Drug Ab (IgG4) DL649 Or Tb Anti-mouse FC Tb Mouse Clone in Supernatant Ligand (In Excess) Irrelevant Ab (IgG4) DL649

21F3 23C2 11C2 8B5 14H10 16C11 3G4 15F7 27D5 13G6 15F8 22A10 21D2 22G10 38C1 38H2 42C5 42D9 49B3 55D9 4E9 17F9 18H2 25E5 Neg. control HTRF 665 Ratio HTRF Blocking Assay +/- Ligand 16000 HTRF Assay - Drug +/- Ligand - Anti-Id Ab Titration 14000 12000 10000 NB NB 8000 No Ligand - Clone at 5 ug/ml 6000 No Ligand - Clone at 1 ug/ml No Ligand - clone at 0.2 ug/ml 4000 25 ug/ml Ligand - Clone at 5 ug/ml 25 ug/ml Ligand - Clone at 1 ug/ml 2000 25 ug/ml Ligand - Clone at 0.2 ug/ml 0 3169 3190 3201 Preincubation ligand/drug Ab for 45 min Map Clone ID Most clones Most shown clones are are blockers Two Two non-blockers (anti-framework) (anti-framework)

HTRF: Conclusions Advantages Homogenous assay - No wash Sensitive (5ng/ml or less) Rapid (1 hr incubation time) Easily developed and automated Reagents stable Low volume 384 well plates Small reaction volumes Binding in solution Binding antigen to plates for ELISA may block a subset of epitopes Disadvantages Homogenous assay Hook effect Can make distinguishing supernatants based on single point signals difficult Requires antigen to be tagged or labeled May remove access to certain epitopes Matrix (conditioned media) can interfere with HTRF ratio Ideally positive control in media

Tag-lite Technology: Applied to two GPCR Targets Mix and Read Alexafluor labeled Secondary Ab Ligand or Ab binding to GPCR target Tb-Labeled Receptor F1 F2 HTRF signal when binding occurs Clip, Snap or Halo enzyme (20 kd)

500.0 250.0 125.0 62.5 31.3 15.6 7.8 3.9 2.0 1.0 0.5 0.0 HTRF 665 Ratio 200.0 100.0 50.0 25.0 12.5 6.3 3.1 1.6 0.8 0.4 0.2 0.0 HTRF 665 Ratio HTRF 665 Ratio GPCR 1 HTRF Ligand & Ab Binding to Cells Tag-lite - Cell Comparison 16000 14000 12000 10000 Tag-lite - Primary Ab Titration 8000 6000 4000 2000 293-GPCR1-Tb 293-GPCR1 (Unlabeled) 8000 7000 6000 0 5000 14000 12000 10000 Labeled Ligand [nm] Tag-lite - Unlabeled Ligand Titration 4000 3000 2000 1000 0 10 3.3 1.1 0.37 0.12 0.04 0 Primary Ab (ug/ml) Positive Control A Positive Control B 8000 6000 4000 2000 293-GPCR1-Tb 0 Unlabeled Ligand [nm] (10 nm Labeled Ligand)

Tag-lite GCR1 HTRF 665 ratio CHO/GPCR1 Signal Taglite CHO/GPCR1 Binding of Ab supernatants CHO/GPCR1 - Tag-lite GPCR1-Tb CHO-GPCR1 - FMAT Blue Squares : Positive Controls Red Squares : Taglite Positives GMAP Well CHO-S Signal Many non-specific hits identified when FMAT was used Results much cleaner with Tag-lite 25

Ab Binding on Various Cell Lines - FMAT Clone CHO-Ag CHO 293-Ag 293 293Ag Snap 293Ag SnapTb 293-irrAg SnapTb Pos.Control + - + - + + - MuIgG (-cont) - - - - - - - Ab Clone 1 + + - - - - - Ab Clone 2 + - + - - - + Ab Clone 3 - - + - + + - Ab Clone 4 + + + + + + + + - + - + - + Original screening run in // by FMAT and Tag-lite 26

GPCR 2 - HTRF Ligand Binding No ligand binding cell assay available due to low affinity binding of ligand to GPCR receptor No possibility to assess blocking activity of antibodies or small molecule inhibitors Labeled Ligand Titration Unlabeled Ligand Competition (50 nm labeled Ligand) (Negative Control) 50 nm ligand chosen for further inhibition experiments (balancing the needs for maximizing the signal and minimizing ligand concentration) Cold ligand IC50 = ~ 3nM High concentrations of ligand are needed

HTRF 665 Ratio Inhibition by Blocking Antibodies 3000 Taglite - GPCR2 - Blocking Antibodies 2500 2000 1500 1000 Anti-Ligand Anti-GPCR2 Mouse IgG 500 0 10 5 2.5 1.3 0.6 0.3 0.16 0.08 0.04 0.02 0.01 0 Antibody (ug/ml) Anti-ligand Ab preincubated with ligand for 15 minutes Anti-GPCR2 and migg preincubated with cells for 15 minutes Both anti-ligand Ab and anti-gpcr2 Ab demonstrate significant inhibition of ligand binding, while irrelevant mouse IgG does not Anti-GPCR2 does not appear to reach 100% inhibition, even when saturating (background is ~500)

HTRF GPCR2 (665 ratio) Blocking with Anti-GPCR2 Ligand Supernatants ELISA (binding) versus Tag-lite (blocking) Most blocking supernatants (HTRF Ratio < 2000) have higher Elisa OD signals ELISA GPCR2 Ligand (OD)

Taglite Conclusions Strengths: Easy, rapid, robust assay development Faster to execute than FMAT Less influenced by non-specific cell binding Can examine ligand/receptor interactions, even in cases where ligand binding assay could not be established using other means due to low affinity binding of ligand Can use same cells to look at binding and signaling Weaknesses Presence of Snap tag on the receptor can affect antibody binding Labeling ligand can alter its properties

Thanks to Matthew Tomlinson Scott Kurose Edwin Haghnazari & Na Tang From Cisbio Paula So Andrea Tatum Anan Chuntharapai Helen Cai Erika Meaddough Peter Brams Keith Joho Nils Lonberg 31