Intra-tumor Catabolites (fate of ADC) can Predict ADC Efficacy. Donglu Zhang, Ph.D. Genentech Feb 21, 2017 World ADC Berlin-2017

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1 Intra-tumor Catabolites (fate of ADC) can Predict ADC Efficacy Donglu Zhang, Ph.D. Genentech Feb 21, 2017 World ADC Berlin

2 Outline ADC structure and mechanism of action (MOA) Can we use PK or PK-PD (modeling) to predict ADC efficacy? Examples showing [tumor catabolite] efficacy correlation Implications in preclinical ADC discovery Discussion points 2

3 Basic ADC Structures Antibody (anti-her2, CD22 ) Antibody Linker Drug payload, Linker Drug (MMAE) catabolite, drug Val-Cit-PAB-MMAE A microtubule inhibitor Antibody Linker Disulfide-PBD A DNA alkylator Drug (pyrrolobenzodiazepine, PBD) LC HC THIOMAB MW 150,000 Da MMAE, DM1 are regular small molecules PBD are covalent binders They have different cell retention kinetics Different MOA Zhang_Genentech_World ADC 3

4 ADC Mechanism of Action Christina Peters and Stuart Brown. "Antibody drug conjugates as novel anticancer chemotherapeutics." Bioscience reports 35.4 (2015)(modified) 4

5 Which [ADC analyte] in Circulation Correlates with Efficacy or Toxicity? Multiple bioanalytical assays (ELISA, LC/MS) Kaur et al, Bioanalysis, 2013; Kamath & Iyer, Pharm Res, 2015; Xu et al, Anal Biochem 2011 ADC review:.additional work is still needed to better understand the best analytes to use for exposure-response correlations.. -Amrita Kamath Do ADC species in plasma reflect what are in the tumor or other tissues? AUC, Cmax, Cmin-dependent Prodrug PK does not drive efficacy It is important to note that till date there is no consensus on which analyte is the best one to correlate with ADC pharmacology. The process involved in plasma-to-tumor exchange of ADCs and released drug are complex and mostly nonlinear. Consequently, the plasma concentrations of these analytes do not accurately represent their concentrations inside the tumor. Khot A, Sharma S. Shah DK (2015) Bioanalysis Zhang_Genentech_World ADC 5

6 Is C min of Tab Really Correlated with ADC Efficacy? It is difficult to determine C min for a drug with long t 1/2 Trough concentrations (C min ) can not be predicted C min depends on payload class 6

7 What ADC Species Contribute to Efficacy? ADC species in circulation Unconjugated antibody no impact Unconjugated payload limited impact (often concentration too low, may contribute to toxicity) Conjugated antibody and conjugated drugs can carry payload to sites of action The correlation of exposure of any circulating ADC species with efficacy is not known The payload at the site of action is responsible for efficacy and at peripheral sites for toxicity Zhang_Genentech_World ADC 7

8 Example: Cyclopropyl- and Cyclobutyl-containing ADCs Show Distinct Efficacy in Xenograft Mouse Models General procedures: After a single IV dose, (1) plasma and tissues were taken, (2) DAR or total mab, and payload drugs were determined. Zhang et al. (2016) Drug Metab Dispos 8

9 Normalized Average DAR (%) Similar Systemic Exposures but Different Intra-tumor Catabolites From Cyclopropyl- and Cyclobutyl-ADCs in Mouse ADC Time (h) mab (nm) 5 mg/kg Plasma Liver Tumor Plasma Cyclobutyl- Cyclopropy l Similar exposure would predict similar efficacy Methyl Cyclopropyl Cyclobutyl Days after dose ADC Cyclobutyl- Cyclopropyl- Time (h) PBD-dimer (nm) Cyclopropyl-thiol (nm) Plasma Liver Tumor Plasma Liver Tumor <LLOQ <LLOQ <LLOQ But only intra-tumor catabolism analysis rationalizes efficacy difference 9

10 Absorbance, mau Did Cyclopropyl Thiol Bind to DNA Oligo Model? 4.0e4 3.6e4 3.2e4 2.8e4 2.4e4 2.0e4 1.6e4 1.2e4 Oligo 2 Oligo1 DNA Oligo 1: 5 TATAGAATCTATA 3 DNA Oligo 2: 3 ATATCTTAGATAT e4 3.8e4 3.0e4 2.2e4 1.4e4 Insert: charge Z = e4 2.6e4 2.2e4 1.8e4 1.4e4 1.0e4 Oligo 2 Oligo1 Minimal adducts HPLC Time (min) Prediction: ADCs prepared from cyclopropyl-containing PBD should be inactive. Zhang et al., 2016, ACS Med Chem Lett 10

11 Differentiation of Tumor from Organs and Between Organs A WSU-DLCL2 Xenograft Model Controls Vehicle B 12,000 PBD/cell or 140 ng PBD/cm 3 to sustain this efficacy! Tissue free [PBD] does not reflect cellular [PBD] (<1%). PBD was accumulated in tumor but not in normal tissues. DNA covalent-bound [PBD] differentiate tumor from normal tissues and between normal tissues. This analysis has potential utility in tox studies, what is the DNA binding threshold for organ toxicity? C DNA-bound PBD 41 PBDs/10 6 bp Free PBD from organic extraction 1 PBD/10 6 bp or ~300 PBD/cell in liver and lung Ma et al., 2016, Drug Metab Dipos 22

12 Intra-tumor [PBD] Correlates with efficacy of PBD-ADCs Zhang_Genentech_World ADC 12

13 Tumor Catabolites Have Different Kinetic Profiles From Circulating ADC Conjugates T-[3H]DM1 or T-SPP-[3H]DM1 concentrations in plasma/tumors of Xenograft mice Anti-CD70-[14C]MMAF concentrations in plasma/tumors of Xenograft mice Circulating ADCs DAR analysis Erickson et al., 2012, Mol Cancer Ther Alley et al., 2009, JPET DM1 and MMAF are different from DNA alkylators that have prolonged cellular retention times. 13

14 Points for Discussion for ADC Discovery and Development? The correlation of exposure of any circulating ADC species with efficacy (toxicity) is not known The payload at the site of action is responsible for efficacy and toxicity Questions: How does PK analysis help in understanding efficacy in ADC discovery and development? Why are we trying so hard to look for efficacy driver: systemic Cmax or AUC? Should we consider how systemic exposures affect payload delivery to the site of action? Is the method of calculation for therapeutic index valid? Systemic AUC or Cmax in tox/auc or Cmax in human Zhang_Genentech_World ADC 14

15 Summary For PBD, (1) [intratumor catabolite] correlated with tumor growth inhibition/reduction (2) Stability and concentrations of circulating ADC can not support the observed efficacy How can tumor catabolite analysis be routinely performed? In pre-clinic In clinic? Systemic PK can be used to ensure plasma stability of ADC 15

16 Acknowledgement Cyrus Khojasteh Yong Ma Buyun Chen Hoa Le Chenghong Zhang Sudheer Bobba Pingping Lu Liling Liu Peter Fan Emile Plise Jonathan Cheong Jonathan Wang Dewakar Sangaraju Xiao Ding Marcel Hop Pete Dragovich Tom Pillow Shang-Fan Yu Carter Fields Gail Phillips Hans Erickson Jack Sadowsky Dick Vandlen Keyang Xu Dian Su Surider Kaur Geoff Del Rosario Jeff Lau Zhonghua Pei Andy Polson 16

17 Thank You! 17