Site-Specific ADC Generation Using SMARTag Technology

Site-Specific ADC Generation Using SMARTag Technology David Rabuka, PhD World ADC Summit San Diego, Oct 2015

SMARTag TM technology: Site-specific protein modification using bioorthogonal chemistry Site-Specific Conjugation Sletten EM and Bertozzi CR Angew. Chem. Int. Ed. 2009, 48, 6974-98. Rabuka D Curr. Opin. Chem. Biol. 2010, 14, 790-6 Patterson DM et. al. ACS Chem. Biol. 2014, 9, 592-605. 1

Benefits of site-specific ADC technology Biologic Advantages Enhanced Potency Improved Biophysical Characteristics Improved PK Increased Serum Half-Life Improved Payload Stability Less TOXICITY Increased TI amount 0 1 2 3 4 5 6 7 8 Lot 1 amount 0 amount 1 2 3 4 5 6 7 8 0 Lot 2 1 2 3 4 5 6 7 8 Lot 3 CMC Advantages Reproducible, simplified Analytics Albers AE, et al. Eur J Med Chem 2014, 88, 3-9 Drake PM, et al. Bioconjugate Chem 2014 25:1331-41 2

SMARTag TM technology: Simple and efficient approach for generating site-specific conjugates CHO Cell SMARTag TM mab SMARTag TM ADC FGE formylglycine generating enzyme (CxPxR specific) protein expression conjugation aldehyde generation in vivo standard purification site-specific payload placement SH H O H O xpxr cysteine (thiol) H O xpxr formylglycine (aldehyde) Rabuka, D., et al., at Protoc. 2012, 7, 1052-67.

The SMARTag TM Platform Enables Optimization Through SAR Exploration Site Selection Cytotoxicity Pharmacokinetics Efficacy Toxicity Linker Composition DAR 4

Hydrazinyl-Iso-Pictet-Spengler (HIPS) Ligation to Formylglycine Gives a Stable Carbon-Carbon Bond H H 2 + R O H H H R H H R Pictet-Spengler reaction H Me H Me + R O H R H Me Me H R Me Me Pictet-Spengler ligation Me H Me Bioconjugation: C-C bond formation Me Me Me Me O H H O Agarwal, P. W. et al. Bioconjugate Chem. 2013, 24, 846 851. Liu, J. et al. Sci Rep. 2015, in press.

A SMARTag HIPS MMAE-conjugate is more stable than a conventional Cys-MMAE conjugate at 37 C in rat plasma 120 HIPS C C conjugation Anti-MMAE signal/anti-fc signal (normalized) 80 60 40 20 0 SMARTag "conjugate"" MMAE- Conven9onal"Cys"Conjugate" TBOS plasma Starting point 7 d 14 d 6

SMARTag TM mab Position Engineering: Tags can be Placed Throughout the Protein SITE SELECTIO SMARTag TM mabs MULTIPLE OPTIMIZED SITES BIOPHYSICAL PROPERTIES Aggregation <5% Antigen binding o Impact Internalization o Impact Melting Temp. o Impact FcRn binding o Impact Immunogenicity* Low Risk *Antitope Episcreen 7

Tag Position Greatly Influences Inhibition of Tumor Growth In Vivo Conjugation Site Matters 1 2 tumor volume (mm 3 ) 1200 900 600 Site 1 Site 2 Site 3 T-DM1 Vehicle 300 3 0 7 14 21 time since dose (d) Site selection dramatically affects tumor growth inhibition Drake PM, et al. Bioconjugate Chem 2014 25:1331-41 8

GPEx Cell Lines Enable Robust SMARTag TM mab Production with High Titers and Tag Conversion PROTEI PRODUCTIO Clone 5 Clone 4 Clone 3 Clone 2 Clone 1 0 1 2 3 4 5 titer (g/l) 75 pg/cell/day O H O H Clone 4 Clone 1 0 10 20 30 40 50 60 70 80 90 aldehyde (%) 9

GPEx Cell Lines Enable Robust SMARTag TM mab Production with Full Conversion of Multiple Tags on a Single Antibody PROTEI PRODUCTIO Clone 3 Clone 2 Clone 1 0.0 0.5 1.0 1.5 2.0 2.5 titer (g/l) Site 2 Site 1 Clone 3 Clone 2 Clone 1 0 10 20 30 40 50 60 70 80 90 aldehyde (%) 10

Exploring the Effects of Linker Composition on SMARTag TM ADCs Mean tumor volume (mm 3 ) 2000 0 Vehicle Linker 1 Linker 2 4AP Various linkers tested for in vivo efficacy, stability, PK against multiple targets 4AP ADC linker had superior in vivo efficacy, biophysical properites 0 0 10 20 30 40 50 60 Days Albers AE, et al. Eur J Med Chem 2014, 88, 3-9. 11

αher2 4AP is highly monomeric and comprises a single light and heavy chain species MW kd SEC-HPLC 238 171 1 2 3 4 HIC-HPLC 117 71 55 41 31 1) αher2 CT (R) 2) αher2 CT-4AP ADC (R) 3) αher2 CT (R) 4) αher2 CT-4AP ADC (R) 1 2 PLRP-HPLC Conjugated HC, expected 51,245 Da LC-MS: DAR 1.8 Light chain, expected 23,443 Da 12

Homogenous ADC Peptide Mapping: Site specific 4AP conjugation (fgly) relative to DM1 conjugation (lysine) HER2 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDVTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQ QHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLFYPREAKVQWKVDALQSGSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFRGEC Kadcyla LC Sequence Coverage = 214 of 214 =.0% HER2 Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGFIKDTYIHWVRQAPGKGLEWVARIYPTGYTRYADSVKGRFTISADTSKTAYLQMSLRAE DTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICVHKPSTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFWYVDGVEVHAKTKPREEQYSTYRVVSVLTVLHQDWLGKEYKCKVSKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKQVSLTCLVKGFYPSDIAVEWESGQPEYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGVFSCSVMHEALHHYTQKSLS LSPGK HC Sequence Coverage = 450 of 450 =.0% HER2 Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDVTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQ QHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLFYPREAKVQWKVDALQSGSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFRGEC αher2 4AP Redwood Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGFIKDTYIHWVRQAPGKGLEWVARIYPTGYTRYADSVKGRFTISADTSKTAYLQMSLRAE DTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICVHKPSTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFWYVDGVEVHAKTKPREEQYSTYRVVSVLTVLHQDWLGKEYKCKVSKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKQVSLTCLVKGFYPSDIAVEWESGQPEYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGVFSCSVMHEALHHYTQKSLS LSPGSLCTPSRGS HC Sequence Coverage = 426 of 456 = 93.4% _ = Conjugation site(s) 13

The αher2 4AP ADC binds antigen equally well as wild-type antibody (trastuzumab) 4 Absorbance (A.U.) 3 2 1 αher2 WT αher2 4AP 0 0.1 1 10 0 ng/ml Binding to HER2-His protein as measured by ELISA 14

ADCs made with 4AP maytansine are highly potent in vitro against multiple cell lines and different target antigens 0.6 0.5 Maytansine αher2 4AP 0.1 Maytansine αcd22 4AP Kadcyla 0.08 IC50 [nm] Drug 0.4 0.3 0.2 IC50 [nm] Drug 0.06 0.04 0.1 0.02 0 CI-87 BT-474 Sk-Br-3 0 Ramos WSU-DLCL2 15

The αher2 4AP ADC does not show bystander killing against antigen-negative cells in vitro HER2- Ramos Cells HER2- WSU-DLCL2 Cells % Viability 50 % Viability 50 αher2 4AP Kadcyla 0 0.001 0.01 0.1 1 10 0 αher2 4AP Kadcyla 0 0.001 0.01 0.1 1 10 0 Concentration Ab [nm] Concentration Ab [nm] 16

The αher2 4AP conjugate shows superior stability relative to Kadcyla in rat plasma at 37 C over 2 weeks 120 Anti-maytansine signal/anti-fc signal (normalized) 80 60 40 20 0 CT 4AP Kadcyla Starting point 7 d 14 d ELISA-based readout comparing maytansine signal to antibody signal. Loss of payload is indicated by a decrease in the maytansine/fc ratio. 17

ADCs made with 4AP maytansine display improved in vivo stability relative to Kadcyla as exemplified by a rat PK study 0000 αcd22 4AP Total Ab αcd22 4AP Total Conjugate αcd22 4AP Total ADC 000 αher2 4AP Total Conjugate αher2 4AP Total Ab αher2 4AP Total ADC 000 Kadcyla Total Ab Kadcyla Total Conjugate Kadcyla Total ADC 000 00 00 ng/ml 00 ng/ml ng/ml 0 0 0 0 5 10 15 20 25 Days post-dose 0 5 10 15 20 25 Days post-dose 0 5 10 15 20 25 Days post-dose Analyte αcd22 4AP T ½ (days) αher2 4AP T ½ (days) Kadcyla T ½ (days) Total antibody 9.5 8.5 8.1 Total conjugate (DAR >0) Total ADC (DAR sensitive) 7.2 7.6 6.5 6.1 6.6 4.9 18

The αher2 4AP conjugate outperformed Kadcyla at equal drug loading, and was similar to Kadcyla at equal antibody loading Kadcyla DAR: 3.5 αher2 4AP DAR: 1.76 120 Body weight was not affected by dosing 1800 1600 Endpoint Tumor volume (mm 3 ) 1400 1200 0 800 600 400 200 0 20 40 60 80 Days Relative body weight (%) 4 stable disease 90 0 20 40 60 80 Days post-dose Vehicle Isotype 4AP, 6 mg/kg αher2 4AP, 3 mg/kg αher2 4AP, 6 mg/kg Kadcyla, 3 mg/kg 19

The αher2 4AP conjugate provided enduring tumor control leading to significant survival benefits compared to Kadcyla Percent survival 50 0 20 40 60 80 Days post-dose Vehicle Isotype 4AP, 6 mg/kg αher2 4AP, 3 mg/kg αher2 4AP, 6 mg/kg Kadcyla, 3 mg/kg 8 mice/group CI-87 xenografts Dosing started when tumors reached ~268 mm 3. Animals dosed 1x/wk for 4 wk. Error bars = S.E.M. 20

The αcd22 4AP conjugate was highly efficacious against WSU-DLCL2 xenografts 2500 Mean tumor volume (mm 3 ) Endpoint 1500 0 500 Vehicle αcd22 4AP 6 complete responses (75%); 3 durable to end of study 0 0 10 20 30 40 50 60 10 mg/kg Days 21

A single dose exploratory rat toxicity study based on the Kadcyla toxicology program was performed Test Article DAR Dose (mg/kg) # of Rats Vehicle n/a 0 5 4AP ADC 1.9 6 5 4AP ADC 1.9 20 5 4AP ADC 1.9 40 5 4AP ADC 1.9 60 5 Kadcyla 3.5 6 5 Kadcyla 3.5 20 5 Kadcyla 3.5 60 5 Single bolus i.v. injection on day 1. Based on Poon et al. 2013, Toxicology and Applied Pharmacology 22

o mortality was observed for rats dosed with a 4AP ADCs, even at 60 mg/kg, which was a lethal dose for Kadcyla Test article Dose (mg/kg) Mortality Vehicle 0 0/5 4AP ADC 6 0/5 4AP ADC 20 0/5 4AP ADC 40 0/5 4AP ADC 60 0/5 Kadcyla 6 0/5 Kadcyla 20 1/5 70 Kadcyla 60 5/5 0 5 10 15 Body weight (% of control) 90 80 Days post-dose Equivalent drug dosing Vehicle 1 Kadcyla, 6 mg/kg Kadcyla, 20 mg/kg Kadcyla, 60 mg/kg αcd22 4AP, 6 mg/kg αcd22 4AP, 20 mg/kg αcd22 4AP, 40 mg/kg αcd22 4AP, 60 mg/kg αher2 4AP, 6 mg/kg αher2 4AP, 20 mg/kg αher2 4AP, 60 mg/kg 4AP ADC treatment caused minimal weight loss, with less impact than Kadcyla at equal drug dosing 23

4AP ADC effects on the liver and platelets 00 ight ontrol) of (% control) of control) ALT (% of control) ht of control) t weight (% control) of control) (% of control) % of eight dy control) of of weight (% control) (% of of control) (% of control) ody y weight (% (% of of control) of (% f weight control) of Body control) (% weight Body weight of control) (% of control) (% of control) y ody weight weight Body (% of (% weight control) of control) (% of control) 90 90 0 1500 6 20 60 6 20 60 6 20 40 60 90 90 90 Dose 80 (mg/kg) 90 90 90 90 90 80 90 90 90 90 9080 90 90 80 90 90 8090 Body Body weight weight Body (% weight (% of Body of control) (% weight of control) (% of control) Body Body weight weight (% Body of (% control) weight of control) (% of control) 90 90 90 80 80 80 control) of control) (% of control) Body weight (% of control) Platelets (thousand/µl) 0 500 90 90 0 Body weight (% of control) 0 6 20 60 6 20 60 0 6 20 40 60 90 80 Dose (mg/kg) Vehicle 1 Kadcyla, Vehicle 6 mg/kg 1 Kadcyla, Vehicle Kadcyla, 120 mg/kg 6 mg/kg Kadcyla, Vehicle Kadcyla, 160 6 mg/kg 20 mg/kg Vehicle Vehicle 1 Kadcyla, Vehicle 1 1 αcd22 Kadcyla, 1 Vehicle Vehicle 4AP, 20 6 Kadcyla, 16 60 6 mg/kg Kadcyla, mg/kg Vehicle 1 αcd22 Kadcyla, 6 4AP, 1 Vehicle 6 Kadcyla, 1 6 20 6 20 Vehicle Kadcyla, Vehicle 160 20 6 Vehicle Kadcyla, 60 Kadcyla, 1 20 6 mg/kg αcd22 4AP, mg/kg 20 mg/kg Kadcyla, Kadcyla, αcd22 20 20 1 4AP, 6 6 20 1 Vehicle 20 20 m 60 Kadcyla, 6 mg/kg 1 20 Kadcyla, Vehicle Kadcyla, αcd22 αcd22 160 660 20 mg/kg 6 20 mg/kg Vehicle 604AP, 4AP, Vehicle 6 40 m Vehicle Kadcyla, Vehicle 1 1 6 mg/kg mg/kg Kadcyla, αcd22 4AP, mg/kg 40 Kadcyla, Kadcyla, 6020 mg/kg αcd22 Kadcyla, 4AP, 120 1 1 60 6 mg/kg 60 Vehicle 4AP, 20 6 14AP, 20 606 m Kadcyla, 1 αcd22 Kadcyla, 6 40 6 60 20 6 m αher2 20 6 mg/kg 60 20 660 6 4AP, mg/kg mg/k αcd22 Kadcyla, 60 mg/kg 40 20 6 αcd22 αher2 4AP, 4AP, 6 20 6 mg/kg 20 60 6 40 αcd22 Kadcyla, 60 mg/kg 4AP, 20 6 Kadcyla, Kadcyla, Kadcyla, αcd22 20 60 4AP, 20 m Kadcyla, αher2 60 20 mg/kg mg/kg αher2 αcd22 660 20 Kadcyla, 60 20 Kadcyla, 4AP, 20 6 mg/kg 4AP, 4AP, 4AP, 20 20 40 αcd22 Kadcyla, αcd22 4AP, 60 4AP, 60 mg/kg 40 4AP, 20 40 6 606 40m Kadcyla, 6 20 6 mg/kg Kadcyla, Kadcyla, αcd22 Kadcyla, 60 40 αcd22 60 20 αher2 60 4AP, 20 αher2 4AP, 20 4AP, mg/kg 6 mg/kg αcd22 αher2 αcd22 40 m αcd22 4AP, 4AP, Kadcyla, 4AP, 6 60 60 660 40 mg/kg 20 60 60 40 mg/kg 20 mg/kg αcd22 αher2 6 αcd22 60 6 20 4AP, 4AP, 40 20 40 60 m 4AP, 6 mg/kg mg/kg 6 mg/kg 20 20 40 40 60 αcd22 40 20 6 60 4AP, αher2 4AP, 6 mg/kg 20 6 m αcd22 αher2 αcd22 αcd22 60 4AP, mg/kg 20 6 m 4AP, 20 αcd22 60 60 60 40 4AP, 20 20 40 40 4AP, 40 mg/kg mg/k αher2 4AP, 4AP, 20 20 6 αcd22 mg/kg 60 60 60 20 6 αcd22 αcd22 4AP, 6 4AP, 20 mg/kg 40 40m αcd22 4AP, αcd22 4AP, 60 60 60 60 mg/kg 40 mg/kg mg/kg 6 αher2 αcd22 4AP, 20 60 40 6 60 mg/kg mg/kg αher2 4AP, 4AP, 60 660 20 mg/kg mg/kg 6 20 60 40 αher2 4AP, 4AP, 4AP, 4AP, 20 20 6 60 60 m αcd22 αher2 4AP, αcd22 αcd22 αher2 60 6 mg/kg 6 60 αher2 4AP, 6 mg/kg αher2 4AP, mg/kg 20 20 4AP, 6 60 αher2 αher2 4AP, 60 4AP, 4AP, 4AP, 60 20 mg/k 20 20 6 mg/kg 6 m m αher2 αher2 4AP, 4AP, 4AP, 20 4AP, 660 mg/kg 60 20 60 20 6 m m αher2 4AP, 4AP, 60 60 20 mg/kg αher2 αher2 4AP, 4AP, 4AP, 60 4AP, 20 mg/kg 60 60 20 αher2 4AP, 60 mg m αher2 αher2 4AP, 4AP, 60 mg 60 Vehicle 1 Kadcyla, 6 mg/kg Kadcyla, 20 mg/kg Vehicle 1 Kadcyla, 60 mg/kg Kadcyla, 6 mg/kg αcd22 4AP, 6 mg/kg Vehicle Kadcyla, 1 20 mg/kg αcd22 4AP, 20 mg/kg Vehicle Kadcyla, 1660 mg/kg mg/kg αcd22 Kadcyla, 4AP, 40 620 mg/kg 6 mg/kg αcd22 Kadcyla, 4AP, 20 60 60 mg/kg 20 mg/kg Kadcyla, αher2 αcd22 4AP, 4AP, 60 mg/kg 6 40 mg/kg mg/kg mg/kg αher2 αcd22 4AP, 4AP, 620 20 60 mg/kg mg/kg αcd22 αher2 αcd22 αher2 4AP, 4AP, 20 60 40 6 mg/kg mg/kg αcd22 αher2 4AP, 40 60 20 mg/kg αcd22 αher2 4AP, 60 660 mg/kg mg/kg αher2 4AP, 620 mg/kg αher2 4AP, 20 60 mg/kg αher2 4AP, 60 mg/kg 24

Toxicokinetics of the αher2 4AP in non-human primates reveals excellent stability of the conjugate 0000 10 mg/kg total antibody 10 mg/kg total conjugate 000 ng/ml 00 0 0 200 300 400 Hours post-dose 25

The αher2 4AP was very well tolerated in cynomolgus safety studies up to 60 mg/kg Alanine amino transferase (IU/L) 160 140 120 80 60 40 20 0 Day 1 Day 3 Day 5 Platelets (% of predose levels) 120 80 60 40 20 0 26

The SMARTag TM Platform Summary: Optimizing ADC Components PROTEI PRODUCTIO GPEx CELL LIE EGIEERIG SITE SELECTIO MULTIPLE OPTIMIZED PLACEMETS SITE-SPECIFIC COJUGATIO STABLE, PROPRIETARY CHEMISTRIES MODULAR LIKERS SOLUBILITY, RELEASE TRIGGER CYTOTOXI MULTIPLE PAYLOADS 27

Acknowledgements Catalent Biologics West Dr. Penelope Drake Dr. Robyn Barfield Dr. Yun Kim Dr. Al Garofalo Dr. Jesse McFarland Dr. Romas Kudirka Dr. Leon Xu Stefanie Banas Adam Carlson Beatty Huang Lesley Jones Wes Zmolek Catalent Biologics Madison Dr. Gregory Bleck Dr. Dona York Additional Funding 28

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