Immunohistochemistry Based Companion Diagnostic Strategies For Antibody-Drug Conjugates

1. Immunohistochemistry Based Companion Diagnostic Strategies For Antibody-Drug Conjugates www.flagshipbio.com pathservices@flagshipbio.com

2. Cross disciplinary approach to pathology Pathology In situ hybridization Immunohistochemistry Histology Analysis Biology Special stains Knowledge - Experience - Leadership

3. Cross disciplinary approach to pathology - Internal histology lab - Lab/Assay collaborations - Pathologists (DVM, MD) - Academic collaborations Pathology (Contextual) Analysis (Data) Biology (Interpretive) tIA Solution • Proprietary custom built software • R&D program • Experienced programmers • Image analysis technicians • Biologists (Oncology, Inflammation, Neurobiology, Stem cell) • Academic collaborations Knowledge - Experience - Leadership

4. Typical Workflow in Tissue Studies Histology/IHC Stained slide production Scanning lab Slide Scanning IA algorithms applied to digitized image of tissue Quantitative Data Pathology review IA quantitative data and interpretation delivered to client Flagship biologist data Interpretation Knowledge - Experience - Leadership

5. Diagnostic Tissue Antibodies • Therapeutic antibodies won’t work in formalin tissue • Therapeutic recognizes tertiary and secondary structure • Diagnostic antibodies generally recognize 5-7 linear amino acid unstructured region • Diagnostic antibodies frequently not well tested in tissue samples Knowledge - Experience - Leadership

6. What do I Need for a CDx? • Biomarker test strategy • IHC, FISH, PCR, etc • Choice of test related to nature of target and selection strategy • Analytical validation strategy • Typical of platform used • Requirements highest for PMA • Regulatory strategy • CDER and CDRH are partnered on co-development plan • Correspondence/Notifications/Submissions • Design Control and Commercialization strategy • Device manufacturing • Placement in clinical laboratories Knowledge - Experience - Leadership

7. What do I Need for a CDx? • A disease hypothesis • Rationale for a target disease • Understanding of efficacy mechanism • A biomarker • May be drug target • May be a related factor • An assay to measure a biomarker • Robust • Interpretable • Patients who respond to the drug • Basis for all comparisons • Well defined response criteria needed The efficacy relationship may be clearer for naked antibodies than ADCs The biomarker relationship may differ between a naked antibody and an ADC The assay for an ADC may need to rely on a context dependent measurement The biomarker-efficacy relationship is best determined empirically Knowledge - Experience - Leadership

8. False CDx Assumptions for ADCs 1. The drug target is a selection biomarker -Drug response may be determined by other factors independent of target -The drug target may not be the sole disease driver 2. High biomarker=high response - High levels of the target do not guarantee response -Low levels of the target do not preclude response • The target mechanism is the same across diseases -Different indications require different clinical strategies -Different indications may require different CDx strategies • All IHC based approaches are similar -The antibody reagent used is the key determinant in what is actually assayed -Antibodies assay epitopes, not whole proteins • Positive is positive; negative is negative -How much biomarker is present, may be more critical than mere presence vs absence -The context of the biomarker, not just quantity, is critical to its biological role Knowledge - Experience - Leadership

9. Target/Biomarker-Response Profiles Conceptual Relationship The observed relationship is often more complex Observed Relationships The simplest hypothesis is a direct relationship between the target or biomarker and the observed response Only highly positive patients respond Only truly negative patients do not respond Both high and low expressing patients respond Knowledge - Experience - Leadership

10. Factors Which Affect ADC Response Target Expression Factors • Amount of antigen needed for drug targeting • Tumor microenvironment • Heterogeneity of tumor expression • Accessibility to ADC (vascularization, fibrosis) • Antigen shedding • Inflammatory response Knowledge - Experience - Leadership

11. 3 institute study (6 pathologists) of 800 HER2 biopsies High heterogeneity = high pathologist disconcordance Lab Invest. 2012 Sep;92(9):1342-57 Knowledge - Experience - Leadership

12. Factors Which Affect ADC Response Drug Mechanism of Action • Release of drug: lysosome, other proteases • Binding to tubulin (auristatins) • Behavior of other chemotypes (DNA-crosslinker) • Anti-proliferative effects • Induction of Apoptosis • Synergy with SOC Knowledge - Experience - Leadership

13. Factors Which Affect ADC Response Mechanisms of Resistance • Internalization/cleavage of targeting protein • Premature release of drug • Drug efflux (pumps) • Anti-apoptotic signaling • Low proliferative index • Stromal component • Vascularity • Novel prognostic indicators Knowledge - Experience - Leadership

14. Factors Which Affect ADC Response • Amount/location of antigen needed for drug targeting: Membrane vs Cytoplasm Expression Image Cytoplasm Membrane Scoring Knowledge - Experience - Leadership

15. Factors Which Affect ADC Response • Amount/location of antigen needed for drug targeting: Low levels of membrane expression Image Cytoplasm Membrane Scoring Knowledge - Experience - Leadership

16. Factors Which Affect ADC Response Amount/location of antigen needed for drug targeting: Low prevalence of expression IMAGE ANALYSIS IMAGE

17. Factors Which Affect ADC Response Amount/location of antigen needed for drug targeting: Heterogeneity of expression IMAGE IMAGE ANALYSIS

18. Factors Which Affect ADC Response Amount/location of antigen needed for drug targeting: Expression Compartmentalization IMAGE IMAGE ANALYSIS

19. Factors Which Affect ADC Response Amount/location of antigen needed for drug targeting: Expression Compartmentalization IMAGE IMAGE ANALYSIS

20. Factors Which Affect ADC Response Mechanisms of Resistance: Stromal Factors (Inflammation) IMAGE IMAGE ANALYSIS

21. Factors Which Affect ADC Response Mechanisms of Resistance: Stromal Factors (Fibrosis) IMAGE IMAGE ANALYSIS

22. Factors Which Affect ADC Response Mechanisms of Resistance: Tumor Microenvironment (Hypoxia) IMAGE IMAGE ANALYSIS

23. Factors Which Affect ADC Response Mechanisms of Resistance: Vascularity CD31 Stained Tissue Vessel Proximity Perfusion Map Close Range Medium Range Vessel Far Range Vessel Markup

24. Factors Which Affect ADC Response Mechanisms of Resistance: Novel Biomarkers Biomarker B Biomarker C Biomarker A Biomarker D Biomarker E Biomarker F

25. Understanding the ADC CDx Strategy The context of a biomarker can greatly improve the ability to select patients An H-score can show a linear relationship between biomarker content and the pathological score but does not stratify patients well Incorporation of a weightedvalue which reflects a context-specific attribute of ADC efficacy allows a clear stratification scoring scheme Incorporate Dx classification ruleset Knowledge - Experience - Leadership

26. ADC CDx End Use Concepts 1. All information must be refined into a binary answer -Dx positive or Dx negative, no in between -A line must be drawn in the sand, based on all applicable knowledge 2. All endpoints need to be quantitatively measured - Subjective or qualitative measurements cannot be validated - Better discrimination of a biomarker will discriminate patients better • The assay needs to reflects its intended measurement use -Assay is designed to perform best at the Dx cutoff, not in a wide range • The test is component and application specific -There are no substitutions or reagents, devices, or protocols -The test must be re-validated for any other use (breast vs lung, etc) Knowledge - Experience - Leadership