Present and future for clinical applications of extracellular vesicles

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1 Present and future for clinical applications of extracellular vesicles Natasa Zarovni, Exosomics Siena SpA, Italy xxxxxxxxxxxxxxxxxxx

2 Exosomes have come a long way..as for many misunderstood geniuses, first undervalued and only belatedly exalted, for decades deemed garbage cans of cells, they are now considered modern Mercury, messengers of key information * POC: proof-of-concept; LOC: lab-on-chip; LDT: laboratory developped test

3 Means to frame the size and evolution of the EV field 1. Growth of peer-reviewed publications on exosomes/evs One of the metrics for the evolution and maturation of the fields is the consensus on nomenclature (overlapping clarity) More than 1/3 of all EV pubblications ever date to last 5 years

4 Means to frame the size and evolution of the EV field 2. Geographical dispersion 3. Fields of interest Breakout by the country of source Breakout by areas of focus NIH: Program to Assess the Rigor and Reproducibility of Exosome-Derived Analytes for Cancer Detection, Extracellular RNA CommunicationRole of Exosomes in HIV Pathogenesis ClinicalTrials.gov: biomarkers, gene delivery, regenerative medicine EU: COST, MC-ITN, EuroNANOMED, RIA, IMU Breakout by areas of focus?

5 Diagnostics and Therapy Life Sciences Means to frame the size and evolution of the EV field 1. Growth of peer-reviewed publications on exosomes/evs ReNeuron Biocartis Exosome sciences, Athleon Qiagen Evox Therapeutics Exosome Diagnostics Exosomics Siena ExoVita Biosciences Grail (Illumina) Sysmex Inostics Oncocyte Carisome, Caris LS Foundation Medicine Codiak Sciences Hansabiomed SBI Norgen Biotech Thermo Fisher Exiqon Qiagen 101Bio NEP Hitachi Chemicals JSR

6 Exosomics Siena: Development of EV based cancer diagnostics since 2011 Main R&D areas: Cancer exosome markers: proteins, RNAs, DNA for solid tumor screening and monitoring Assays and devices for quantitative and qualitative exosome based diagnostics Sample type- and purpose-tailored exosomes/evs isolation and preanalytical protocols Products and services: Cancer Dx Immunoassays: Cancer Molecular Dx: Supporting protocols: Services and contracts: - EVScreen TM Exosomics Siena -SeleCTEV TM -SoRT-EV TM -EV isolation protocols and devices -LBP TM -Preanalytical protocols and devices - Contract Research - Dx standards and controls - Purification and quantification - Custom assays

7 Exosomics pipeline: EV based diagnostics for major advancements in cancer care Precision and efficient treatment in cancer requires precision and actionable diagnostics Cancer screening Single entry test for all solid cancers Specific tests to determine tissue origin Non-invasive, allowing best compliance Easy to perform and cheap Cancer diagnostics/monitoring Precision medicine Beyond tissue biopsy Beyond CTCs and ctdna Treatment selection Treatment monitoring Resistance prediction comprehensive patient management and sample-to-decision process Exosomics Siena

8 One Root different directions Transplants Gene Therapy Cosmetics Nutrition & Food industry Vaccines Infective Diseases Immunotherapy Regenerative Medicine CVD Vetenirary Medicine Neurology Metabolic Diseases CID&AID Reproductive Biology (Hu) Oncology Therapy Wellness Medical DX and monitoring Screening and prevention Companion Dx

9 EVs in cell-free regenerative medicine Transplants Gene Therapy EXOSOMES PROs Not immunogenic if extracted from proper source Very stable -long circulating life Vaccines and long shelf life Nutrition& Defined tropism to specific tissue or microenvironment Infective Food Intrinsic Indystry biological activities Diseases Intrinsic ability to cross biologic barriers Vetenirary Specific sorting mechanism of proteins to exosomes that helps CVD Medicine predicting or engineerizing surface moieties Neurology Loadable with nucleic acids and small molecules Less complex then viable cells CID&AID Reproductive Biology (Hu) Cosmetics Immunotherapy Oncology Regenerative Medicine Metabolic Diseases Therapy Wellness Medical DX and monitoring Screening and prevention Companion Dx Exosomics Siena

10 EVs in cell-free regenerative medicine Transplants Gene Therapy Cosmetics Nutrition& Food Indystry CVD CID&AID Vaccines Vetenirary Medicine CONs Complexity of exosome cargo and exosome activities Possible wide tropism and off-side effects Lack of reliable and efficient methods for obtaining well defined and pure exosome preparations Infective Diseases Reproductive Biology (Hu) Neurology Medical DX and monitoring Immunotherapy Screening and prevention Oncology Regenerative Medicine OPPORTUNITIES: Ex vivo and in vitro modification Fit-to-the purpose purification methods Path to scalable and GMP manufacture Metabolic Diseases Compliance with regulatory reqirements QC testing through manufacturing (DX and potency assays) New sources (plants, platelelts etc) Synergy with drugs/agents Advanced preclinical testing Clinical trial capacity with experienced physicians Companion Dx Exosomics Siena

11 Emerging areas with unmet needs and huge markets Transplants Gene Therapy Cosmetics Nutrition & Food industry Vaccines Infective Diseases Immunotherapy Regenerative Medicine CVD Vetenirary Medicine Neurology Metabolic Diseases Therapy CID&AID Edible exosomes: -Easily available -Well tolerated -Intrinsic activities Wellness Reproductive Biology (Hu) Medical DX and -Assisted reproduction monitoring - Prenatal testing Screening and prevention Oncology CNS exosomes: -Early screening tests - Surrogate markers Companion Dx Exosomics Siena

12 Understudied niches new extraordinary opportunities Transplants Gene Therapy Cosmetics Nutrition & Food industry -Veterinary testing -Animal Vaccines reproduction -Food safety Infective Diseases Immunotherapy Regenerative Medicine CVD Vetenirary Medicine Neurology Metabolic Diseases CID&AID -Differential diagnosis - Treatment monitoring Reproductive -CDx Biology (Hu) Oncology -Activation -Proliferation Therapy Wellness Medical -Metabolism DX and monitoring Screening and prevention Companion Dx Exosomics Siena

13 Two major exploitation paths Transplants Gene Therapy Cosmetics Nutrition & Food industry Vaccines Infective Diseases Immunotherapy Regenerative Medicine CVD Vetenirary Medicine Neurology Metabolic Diseases CID&AID Reproductive Biology (Hu) Oncology Exosomes/EVs as Therapeutic Delivery Vehicles Exosomes/EVs as Circulating Biomarkers Therapy Wellness Medical DX and monitoring Screening and prevention Companion Dx

14 Exosomes have unprecedented advantages in diagnostics Exosomes circulate in periferal blood and in different biological fluid Exosomes as concentrators of biomarkers: Reflect the proteic and genomic content of the parent cell * Enable secretion of otherwise non-soluble molecules Enrich in low-abundant molecular species Can be selectively isolated Enrich in some acknowledged cancer markers

15 Exosomes have unprecedented advantages in diagnostics Exosomes/EVs are complex biomarkers They carry multiple molecular traits that can be detected simultaneously and provide multiple diagnostic readouts Noninvasive testing + High concentration, low matrix effect Improved IVD High specificity and sensitivity

16 What are the most exploited EV biomarkers today? Most work has been done on mirnas EV DNA based assays are the first ones that hit the clinical use: First LDT for Alk in Lung Cancer lunched by Exosome Dx in 2015 First IVD for CDx (EGFR) by Roche Dx FDA approved in 2016

17 First dowel in the puzzle Companion Diagnostics Define the market need: Where are we today? Which alterations are important? Companion Dx pipeline today Targeted therapies are already standard of care Companion Dx pipeline evolution Drugs with required pharmacogenetic companion diagnostic testing according to EMA and FDA From Roche report Most companion Dx assays are performed on tumor tissue Exosomics Siena

18 Beyond oncology what s next in Liquid Biopsy? Transplants CVD CID&AID Reproductive Biology (Hu) Infective Diseases Neurology Immunotherapy... Exosomics Siena

19 The impact of liquid biopsy for precision medicine in cancer: alternative and complementary to conventional tissue biopsy Tissue Biopsy vs. Liquid Biopsy Invasive, painful Expensive & time consuming Re-biopsy often not possible or accepted Not suitable for cancer monitoring Does not address tumor heterogeneity Jamal-Hanjani et al Clinical Cancer Res 2015 Minimally invasive, no risk for patients Cheap and quick Re-biopsy is not a problem Trully suitable for cancer monitoring Addresses tumor heterogeneity Exosomics Siena

20 Lot of dowels are still missing across the patient management cycle and all can be prompted by liquid biopsy Relapse detection (minimal residual disease) Personalized treatment optimisation Exosomics Siena

21 Major sources of cancer biomarkers for liquid biopsy Circulating cell-free nucleic acids DNA, also RNA? (mrnas, microrna and long non coding RNA) Most studied source of genetic alterations today but has serious limitations (next slides) Circulating tumor cells Cancer cells released by the primary tumor in circulation. Great investments but expectations not fullfilled Diaz & Bardelli Journal of Clinical Oncology Exosomics Siena Extracellular vesicles Lipid vesicles containing proteins and nucleic acids (RNAs and DNA) and other markers categories. Offer unprecedented advantages (see next slides) 21

22 ctdna is not a true tumor biopsy (circulating tumor) ctdna (cell free) cfdna! cfdna might increase during inflammation, infections etc...creating huge background Rapid clearance (unprotected): 2 hours cfdna(non tumor originated) is the vast majority (95% to 99%) of the total DNA extracted from blood ctdna cannot be selectively isolated Exosomics Siena 22

23 Mutation detection is a technical challenge Detecting a disease related mutation in a genome is like finding a needle in a haystack. Tumor tissue biopsy is challenging by itself detecting one needle in hundreds of haystacks Exosomics Siena

24 that is further exacerbated on ctdna Biofluid biopsy multiplies the challenge - detecting one needle in thousands, millions of haystacks Tumor derived DNA is hugely diluted in the blood - if you use total circulating DNA (cfdna) the amount of tumor derived DNA (ctdna) and the sensitivity of one-in-amillion assays may not be sufficient to enable detection of some alterations Exosomics Siena

25 Alternative sources of tumoral DNA in blood Circulating tumor cells Extracellular vesicles - Extremely rare (5-20 cells/ml of plasma) - Low sample recovery, particular harvesting protocols - Expensive and cumbersome analysis(up to 50 ml of plasma required) / low consensus - Total EVs are /ml, tumor-evs estimated /ml - Easy to recover, concentrate and analyze from blood -Heterogeneous population dependent on approach can be an obstacle but we turn it into a huge advantage 25

26 Different timelines, performance and readiness level

27 Exosomes as superior Liquid Biopsy substrate?? 27

28 EVs can be selectively recovered from biofluid samples Gyorgy, 2011 Mathivanan, 2010 We need smart approaches that take into consideration EV heterogeneity Exosomics Siena

29 Challenges for optimal exploitation for biomarker discovery and transfer to a clinical diagnostis Small size vast repertoire of cargo molecules Bulk analysis Vesicles heterogeneity Low fractiion of vesicles carrying dsease relevant marker

30 True exosome diagnostics enabling technical requirement: streamlined and fit to the purpose exosome isolation and rapid ans sensitive quantification of associated molecules

31 True exosome diagnostics enabling technical requirement: streamlined and fit to the purpose exosome isolation and rapid and sensitive quantification of associated molecules

32 PCR remains a Key Tool with outreach vs. NGS

33 Do we need advanced tecnhnologies for detection of EV markers? Shao et al. Nat Med 2012 Im et al. Nat Biotech 2014 Zhao et al. Lab Chip 2016 Cretichet al. Trend Biotech 2015

34 Novel nanovaccines and cell free therapies

35 Clinical trials are a reality Kooijmans et al. 2016

36 While regulatory status still needs to be defined, at least in Europe ISEV position paper 2016

37 EVs have gone commercial already... EXOFACE Exosome moisturizer MISSION PERFECTION SERUM Clarins...or are about to

38 There is still a long way to run The Hype Cycle (representing the maturity, adoption and application) The expectations of EV technologies The expectations are high, some technological components are not ready on the market great opportunity for who comes first to fulfill the niches... 38

39 ...but we need to be carefull