Aiko Nagayama, MD, PhD Ellisen lab Massachusetts General Hospital Cancer Center Chabner Collquium

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1 New biomarkers in a novel antibody-drug conjugate for triple negative breast cancer Aiko Nagayama, MD, PhD Ellisen lab Massachusetts General Hospital Cancer Center Chabner Collquium

2 Financial disclosure A. Nagayama: Own stock of Chugai, Inc.

3 Overview of talk Antibody-drug conjugate Structure and mechanism of action of sacituzumab govitecan Predictive biomarker of sacituzumab govitecan in triple negative breast cancer Synthetic lethal targets of sacituzumab govitecan in triple negative breast cancer Future directions

4 Triple negative breast cancer (TNBC) Absence of ER, PgR and HER2 Constitutes 15% to 20% of all breast cancer Affects younger/african patients Median survival of months from time of metastasis Homologous recombination deficiency and high genomic instability Lehmann, B. D. et al. (2011). J Clin Invest 121(7): Oncotarget May 30;6(15):

5 Latest developments of antibody-drug conjugate Panowski, S. et al. (2014). MAbs 6(1): FDA Approved antibodydrug conjugate Disease Antigen Payload Year of approval Brentuximab vedotin Lymphoma CD30 MMAE 2011 T-DM1 Breast cancer HER2 DM Inotuzumab ozogamicin ALL CD22 Calicheamicin 2017 Gemtuzumab ozogamicin AML CD33 Calicheamicin 2017 Currently in phase 3 for solid tumors Disease Antigen Payload Year of approval Sacituzumab govitecan Breast cancer Trop-2 SN-38 - Rovalpituzumab tesirine Lung cancer DLL3 PBD - Mirvetuximab soravtansine Ovarian cancer FRα DM4 - Depatuxizumab mafodotin Glioblastoma EGFR MMAF - Trastuzumab derutecan Breast cancer HER2 DXd - SYD985 Breast cancer HER2 Duocarmycin - Enfortumab vedotin Urothelial cancer Nectin-4 MMAE -

6 Sacituzumab govitecan (IMMU-132) is a novel antibody-drug conjugate Received Breakthrough Therapy Designation from the U.S. FDA In 2016 for the treatment of patients with TNBC Antibody Anti-Trop2 antibody (hrs7) Payload SN-38 Linker ph-sensitive cleavable linker Average 7.6 molecules of SN-38 are conjugated with antibody Nagayama, A. et al. (2017). Target Oncol 12(6): David M. Goldenberg, T. M. C. et al. (2015). Oncotarget 6(26).

7 Mechanisms of action of antibody-drug conjugate Selective delivery 1.ADC binds to antigen 2.ADC gets internalized by endocytosis 3.Intracellular trafficking from endosome to lysosome 4.Degradation in lysosome and payload release 5.Payload takes action Antibody-dependent cellular cytotoxicity (ADCC) Killing of an antibody-coated target cell by a cytotoxic effector cell by releasing of the cytotoxic granules Bystander killing effect Free drug in the cytosol cross the plasma membrane to access the extracellular milieu and kill neighbor cells Nagayama, A. et al. (2017). Target Oncol 12(6): Chalouni, C. et al. (2018). J Exp Clin Cancer Res 37(1):

8 Trop-2 is a transmembrane glycoprotein overexpressed in epithelial cancers Trop-2 is a 46 kd glycoprotein initially identified in a trophoblast cell Overexpressed in many epithelial cancers including triple negative breast cancer Ca 2+ transducer that depends on a specific protein kinase C phosphorylation site The overexpression of Trop-2 correlates with a poor prognosis in breast cancer David M. Goldenberg, T. M. C. et al. (2015). Oncotarget 6(26). Lin, H. et al. (2013). Exp Mol Pathol 94(1):

9 SN-38 is a potent topoisomerase 1 inhibitor SN-38 is an active metabolite of irinotecan, originally isolated from Camptotheca acuminata SN-38 binds reversibly to the topoisomerase 1 cleavage complex on DNA and slow down DNA relegation by interfacial inhibiting mechanism, which causes S-phase specific cell death MDA-MB-468 Pommier, Y. (2009). Chem Rev 109(7):

10 IMMU-132 induced significant tumor regression in TNBC xenograft models David M. Goldenberg, T. M. C. (2015). Oncotarget 6(26

11 Single-arm, open-label study of sacituzumab govitecan in patients with metastatic triple-negative breast cancer Bardia, A. (2017) San Antonio Breast Cancer Symposium

12 Clinical response and survival outcome of IMMU-132 Typical response rates in this population to standard therapy are 12%, and PFS <3 months Bardia, A. (2017) San Antonio Breast Cancer Symposium

13 Progress in clinical trials for IMMU-132 Cancer type N 3 prior lines of therapy (%) ORR (%) PFS (months) OS (months) Breast (Triple negative) ( ) 12.7 ( ) Breast (Luminal) 54 > ( ) - Urothelial ( ) 16.1 ( ) Lung (Non-small cell) ( ) 9.5 ( ) Lung (Small cell) ( ) 7.5 ( ) Bardia, A, et al. (2017). SABCS. Bardia, A, et al. (2018). ASCO. Tagawa, S. T, et al. (2017). ESMO. Heist, R. S. et al. (2017). J Clin Oncol 35(24): Gray, J. E. et al. (2017). Clin Cancer Res 23(19):

14 Some unanswered questions regarding IMMU Is Trop2 expression predictive of response? 2. What are the tumor-intrinsic pathways/phenotypes predictive of response? 3. What combination therapy strategies might be employed to overcome de novo and acquired resistance?

15 Trop-2 expression in cancer cell lines and IMMU-132 sensitivity Positivity of Trop-2 expression IC50 of IMMU-132 (nm) 25 R2= IC50 (nm) HCC38 MDAMB Bardia, A. (2017). J Clin Oncol 35(19): MDAMB-468 HCC Trop-2 expression David M. Goldenberg, T. M. C (2015). Oncotarget 6(26).

16 Activity of SN-38 in various breast cancer cell lines IC50 of SN-38 by breast cancer subtype IC50 of SN-38 by TNBC subtypes *p= IC50 (um) IC50 (nm) TNBC Luminal HER2 1 BL1 BL2 M LAR MSL IM Lehmann, B. D.et al. (2011). J Clin Invest 121(7):

17 IC50 distribution of SN-38 in breast cancer cell lines by mutation BRCA 1 BRCA 2 TP53 Breast cancer cell lines Triple negative breast cancer cell lines

18 Response to IMMU-132 in MGH/DFCI TNBC patient cohort ORR, % (n/ N) p value ORR, % (n/ N) p value ORR, % (n/n) p value Overall 31.4 (11/35) - Prior chemotherapy in the metastatic setting gbrca1/2 Age (median) < (9/28) (2/7) Primary tumor subtype Triple negative 25.0 (5/20) Luminal (6/13) ECOG PS (5/12) (6/23) Visceral involvement Yes (11/32) No 0 (0/3) (9/25) (2/10) Prior platinum in any setting Yes (6/22) No (5/13) Prior anthracycline in any setting Yes (10/32) No (1/3) Prior immunotherapy Yes (3/8) No (8/27) Mut 0 (0/1) Wild (6/21) Not tested PIK3CA p (5/12) Mut (1/6) Wild (6/14) Mut 40.0 (4/10) Wild 30.0 (3/10)

19 Biomarker analysis in tumor samples Patients (n=27) Specimens (n=71) Primary breast (n=22) Lymph nodes (n=13) Lung/Pleura (n=17) Others (n=19) Sequencing Total nucleic acid extractions Tissue microarray analysis DNA whole exome sequencing RNA sequencing Immunohistochemistry staining Patients (n=6) Matching pre/post-treatment specimens (n=25)

20 Combination of IMMU-132 and PARP inhibitors in triple negative breast cancer BRCA mutant xenograft model IMMU-132 plus olaparib had a significant antitumor effect in TNBC tumor xenograft models Cardillo, T. M. et al. (2017). Clin Cancer Res 23(13):

21 ATR was the top hit from sirna screening in triple negative breast cancer cell line treated with Camptothecin No repair Cell death Josse, R. et al. (2014). Cancer Res 74(23):

22 CRISPR/Cas9 gene editing for screen CRISPR/Cas9 gene editing technology enabled precise but in the large scale interrogation A Cas9 protein complexed with an ~100 nucleotide guide RNA CRISPR/Cas9 nucleases can be engineered to recognize a target DNA site consisting of a protospacer and a protospacer adjacent motif (PAM) sequence 22 Tsai, S. Q. (2016). Nat Rev Genet 17(5):

23 CRISPR/Cas9 knockout screening to identify synthetic lethal targets of IMMU-132 Negative selection Lentiviral Cas9 Lentiviral genome wide sgrna library DMSO MDA-MB-468 Cas9-expressing MDA-MB-468 Puromycin selection IMMU-132 gdna extraction gdna extraction 12 to 15 cell doublings Comparison of sgrna abundance using deep sequencing

24 Triple negative breast cancer model for CRISPR/Cas9 screen TNBC cell line Subtype Mutations Trop-2 surface expression IC50 of IMMU-132 (nm) IC50 of SN-38 (nm) Doubling time (hr) MDA-MB-468 BL1 PTEN, RB1, SMAD4, TP53 300, ± MDA-MB-468 IC50 (nm) SN-38 + DMSO 0.97 ± 0.26 SN-38 + VE ± 0.04 SN-38 + Olaparib 0.43 ± 0.02

25 Hypothesized distribution of log fold-change in sgrna representation Lopes, R., G, et al. (2016). Nat Rev Mol Cell Biol 17(9):

26 Future directions Validation by custom library Validation in vitro Assessment in genetically manipulated cells Assessment with pharmacological combination Validation in vivo Xenograft mouse model PDX mouse model

27 Summary Sacituzumab govitecan demonstrated the significant clinical activity in triple negative breast cancer Predictive biomarker of IMMU-132 in tumor will be analyzed by WES, RNA-seq, and IHC staining CRISPR/Cas9 knockout screening will be employed to identify synthetic lethal targets of IMMU-132 for potential candidates of combination therapy

28 Acknowledgments Leif Ellisen, MD, PhD Sheng Sun, PhD Mihriban Karaayvaz, PhD Ning Ding, PhD Siang Boon Koh, PhD Thomas Joseph Kania, BS Varunika Vivekanandan, BS Shuxi Qiao, PhD Srinivas Vinod Saladi, PhD Aditya Bardia, MBBS, MD, MPH Dennis Sgroi, MD Gad Getz, PhD