Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids - PowerPoint PPT Presentation

Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids Developing a Functional Precision Medicine Platform for Immuno-Oncology Russell W. Jenkins, MD PhD Clinical Fellow/Instructor Massachusetts General Hospital Cancer Center Dana-Farber/Harvard Cancer Center Laboratory of David A. Barbie, MD (DFCI/Broad Institute) 2017 Chabner Colloquium Boston, MA 10-30-2017

Disclosures Russell W. Jenkins, MD has disclosed no relevant financial relationships.

Background – PD-1/PD-L1 axis in cancer • Despite the unprecedented success of PD-1/PD-L1 blockade in melanoma (and other cancers), innate resistance occurs in the majority of patients – Robert et al. NEJM 2015 (KEYNOTE-006) – Reck et al. NEJM 2016 (KEYNOTE- 024) • Robust biomarkers to guide treatment and drug development are lacking – Nishino et al. Nat Rev Clin Onc 2017 • There is a great unmet need to develop functional precision medicine approaches to guide and accelerate translational efforts (novel IO agents, combos) Confidential

Background – Novel cancer models • Existing methods to assess anti-tumor immune responses in patients rely on remote assessment in plasma/whole blood or static measurements from biopsies (DNA, RNA, IHC/IF) • Novel ex vivo systems (e.g. CTCs, PDXs, organoids) have the potential to improve personalized cancer therapy, but lack features of the native tumor immune microenvironment, and often take several weeks to generate sufficient material for drug testing – Friedman et al . Nat Rev Cancer 2015 Confidential

Background - Precision Medicine • “The essential job of precision medicine is to match the right drugs to the right patients.” • Technologies that enable the ex vivo study of response to anti-PD-1 and combination therapies would provide a major advance Confidential

Modeling the Immune Tumor Microenvironment • Requirements for modeling immune checkpoint blockade include: – (1) Viable (fresh) tumor tissue – (2) Culture system that recapitulates the tumor microenvironment • 3D growth • model extracellular matrix – (3) Immune cells : autologous, antigen- experienced immune cells to mimic the native tumor microenvironment (ideally from the tumor itself) Confidential

Isolation of organotypic tumor spheroids PDOTS MDOTS Jenkins et al., Cancer Discovery, accepted DFCI US Patent WO2016112172A1

MDOTS/PDOTS Confidential

Immune Cell Profiling of MDOTS Flow cytometry S1 Patient- Murine- derived derived Tumor Specimen >100 μm S2 MDOTS/PDOTS Physical + Sequential 40-100 μm enzymatic Microfluidic filtration Serial Microscopy dissociation 3D Culture S3 (Live/Dead Analysis) Bioplex Cytokine Profiling of Conditioned Media <40 μm Confidential

MDOTS Retain Autologous Immune Cells B16F10 MDOTS MC38 MDOTS 1) MDOTS from 2 different syngeneic implantable tumor models retain autologous lymphoid and myeloid cells 2) No significant difference in different spheroid fractions Confidential Pat Lizotte, William Walker, Jiehui Deng

Ex vivo 3D culture and cytokine/chemokine profiling Conditioned Medium 0.5 mm CD45 EpCAM 25 μm

Ex Vivo Culture of MDOTS/PDOTS

MDOTS respond to ex vivo PD-1 blockade Control Control Control Control Anti-PD-1 Anti-PD-1 Anti-PD-1 Anti-PD-1 Amir Aref, Pat Lizotte, Elena Ivanova, Cloud Paweletz

Intrinsic resistance to PD-1 blockade αPD -1 IgG Live Dead MC38 50 μm Control Control Anti-PD-1 Anti-PD-1 B16F10 Response/resistance confirmed in additional models: GL621 (sensitive) CT26 (partially resistant) LLC (resistant)

Tank-binding kinase 1 structure/function TBK1 • Key signaling kinase in innate immunity • Activates IRF3, NF- κ B, autophagy • Co-opted by oncogenic KRAS Barbie et al, Nature 2009 Eck and Barbie Lab collaboration Tu, Zhu et al., Cell Rep 2013 Yang et al., Cancer Immunol Res 2016

A Novel TBK1/IKK ε inhibitor to Modulate the TME Tumor Cell T cell TBK1/ IKKε TBK1/ IKKε Compound 1 IL-2, IFNγ CCL5, IL-6 Tumor Growth T cell activation IC 50 EC 50 TBK1 1.0 nM 183 nM Zhu et al. CD Yu et al. Nat (251 nM*) IKKε 5.6 nM 2014 Comm 2014 Confidential Susanna Stinson, David Dornan (Gilead Sciences)

Ex vivo identification of effective combination therapy Tumor Cell T cell Control α PD-1 + Cmpd1 TBK1/ IKKε TBK1/ IKKε IL-2, IFNγ CCL5, IL-6 Tumor Growth T cell activation Live 50 μm Dead

Dual TBK1-PD1 inhibition is partially dependent on CD8 T cells Live/Dead Imaging (in device) Terminal Media Ex Vivo Culture 23-plex murine Collection (5 days) MDOT BioPlex analysis Tumor S +/- Cmpd1, anti-PD-1, anti-CD8 Confidential

Patient-derived organotypic tumor spheroids

Patient-derived tumor samples (PDOTS) Serial ex vivo cytokine/chemokine profiling predicts in vivo MDSC infiltration and resistance Granulocytic MDSCs Monocytic MDSCs Malignant pleural effusion (thyroid cancer) Co-induction of immune-suppressive cytokines/chemokines ex vivo more generally associated with short PFS

Summary • Ex vivo assessment of tumor immune response to PD- 1 blockade is possible • Further prospective validation is necessary to determine the utility of this system as a predictive tool • Important potential of this system to identify and personalize effective combination therapies • Co-targeting TBK1 with immune checkpoint blockade may have clinical impact

Acknowledgements DFCI/Belfer Center for Applied Science s* Barbie Lab Massachusetts General Hospital Amir R. Aref, PhD* David A. Barbie, MD Genevieve Boland, MD PhD Cloud P. Paweletz, PhD Israel Canadas, PhD Keith Flaherty, MD Pat Lizotte, PhD Tran Thai Hans Vitzthum Elena Ivanova, PhD Shunsuke Kitajima, PhD Michal Barzily-Rokni, PhD Wei Huang, MD Rohit Thummalapalli Marc Hammond William Walker, Lauren Keogh, Bob Jones Mai Hoang, MD Mark Bittinger, PhD MIT Viswanath Gunda, PhD Sangeetha Palakurthi, PhD Roger Kamm, PhD Sareh Parangi, MD Vivek Sivathanu Ryan Sullivan, MD Dana-Farber Cancer Institute Don Lawrence, MD Kwok Wong, MD PhD* (now at NYU) Broad Institute David Fisher, MD PhD Pasi Janne, MD PhD* William Kim, PhD BWH F. Stephen Hodi, MD Adriano Piris, MD Gordon Freeman, PhD DFCI/Broad Institute Wistar Institute Michaela Bowden, PhD Eli Van Allen, MD Gao Zhang, PhD Willa Zhou Diana Miao NJ Institute of Technology Lisa Cameron, PhD (now at Duke) Meng Xiao He Tian Tian Hongye Liu, PhD Alicia Smart Patrick Ott, MD James Cleary, MD PhD Gilead Sciences Bruce Chabner, MD Charles Yoon, MD David Dornan The organizers of the Jochen Lorch, MD Susanna Stinson Glenn Hanna, MD 2017 Chabner Colloquium DF/HCC Medical Oncology Manisha Thakuria, MD Fellowship Nicole LeBoeuf, MD Ann LaCasce, MD Guilherme Rabinowits, MD M. Dror Michaelson, MD PhD Our patients, their families, and the amazing Brian C. Miller, MD PhD Robert J. Mayer, MD clinical and clinical research support staff at Thanh U. Barbie, MD T32 Training Grant William G. Richards, PhD DF/HCC (MGH, BWH, and DFCI) Svenson Family Fellowship Raphael Bueno, MD