HSCI Harvard Stem Cell Institute

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1 HSCI Harvard Stem Cell Institute Receptor targeted and encapsulated MSC for tumors in the brain Khalid Shah Massachusetts General Hospital Harvard Medical School Harvard Stem Cell Institute

2 Acknowledgements Yanni Zhu Dan Stuckey Sung Hugh Choi Wanlu Du Clemens Reinshagen Nusrat Jehan Jordi Martinez Deepak Bhere Nabeel Nissar Cody Cameron Raj Vatsa Jae Woo Priyanka Ravikumar Hiroaki Wakimoto, MGH Jacob Hooker, MGH Umar Mehmood, MGH Ramon Alamay, Barcelona, Spain Peter Sorger, HMS, Boston Jack Lawler, BIDMC, Boston Glenn Prestwich, Utah Biotime

3 Human MSC migrate to tumors in the brain MSC Tumor Week 2 Sasportas and Kasmieh et al, PNAS 29

4 Engineering Stem cells for tumor therapy Secretion of therapeutic proteins NATURE REVIEWS CANCER PERSPECTIVES OPINION Stem cell-based therapies for cancer treatment: separating hope from hype Daniel Stuckey and Khalid Shah

5 Engineering Stem cells for tumor therapy Stem Cell mediated suicide therapy Stem Cell mediated nanoparticle delivery Stuckey and Shah, Nature Reviews Cancer 214

6 Receptor Targeted Therapies for tumors Tumor cell Endothelial cell Tumor cell EGFR CD36 R Nectin 1 DR4/5 IL13 R IL2R TRAIL Bimodal TRAIL/HSV-TK ohsv-trail EGFR Nb-TRAIL Interleukin (IL)-13 PE IL24/MDA7 Bi-specific ENb-TRAIL IL13-PE IL24/MDA7 aatsp1 S- TRAIL TSP-1 Engineered Stem cell Oncolytic herpes virus (ohsv)-trail Engineered Stem cell

7 Glioma cells (Fluc-mCherry) Neural stem cells (TRAIL-IRES-GFP) 9 intervals for 54h

8 Stem Cell released S-TRAIL induces apoptosis in glioma cells % Fluc signal intensity (Relative to control day 2) NSC-GFP NSC-S-TRAIL NSC-S-TRAIL 18 NSC-GFP Day 1 Day 2 Day 3 Bagci et al, Oncogene 212

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11 Mouse model of glioma resection: Mimicking a clinical scenario of tumor debulking Cranial window GBM in a cranial windows Resection cavity Excised GBM

12 % Fluc signal intensity (normalized to before res.) Residual Tumor Volume (Fold D2) un-resected resected Mouse model of glioma resection: Mimicking a clinical scenario of tumor debulking Bioluminescence MRI Preresection L Postresection Preresection Post resection Days post glioma cell implantation Pre-resection Post-resection

13 % glioma cell viability; Fluc intensity (normalized to control at 8 hr) Fold changes in caspase-3/7 activity (normailized to control at 8 hr) secm encapsulated SC migrate to tumors in the brain and induce apoptosis in tumor cells Glioma cells day 4 Encapsulated SC Glioma cell viability Caspase 3/7 activation hr 16 hr 24 hr * * SC-GFP SC-S-TRAIL * * 8 hr 16 hr 24 hr * Control S-TRAIL PARP Cleaved Caspase 8 (full length) Tubulin

14 % Fluc signal intensity (normalized to day 1) secm encapsulated SC- S-TRAIL significantly increase survival of mice with resected gliomas Percent survival Resection unresected resected+sc-gfp-rluc in secm resected+sc-s-trail in suspension resected+sc-s-trail in secm Days post glioma cell implantation SC-GFP-Fluc 1 in gel SC-GFP-Fluc 8 in suspension day 1 day 7 day 14 day 21 day 28

15 Percent survival GCV group PBS group Standard uptake Value Standardized Uptake Value Bi-modal Stem cells co-expressing HSV-TK and S-TRAIL have anti-tumor effects and can be eradicated post treatment HSV-TK expession selectively sensitizes cells to ganciclovir (GCV) and can be utilized as a PET marker using [18F]FHBG as a substrate. Stem Cell HSV-TK Tumor Cell MSC-TR-TK MSC-TR-TK+GCV Baseline L Day TK+ cells R Head L Day 11.8 R Post-GCV Gall bladder and bowel TRAIL A L R L R B.1 1 Control 8 GCV mmsc-tr-tk Days after tumor implantation C D

16 TRAIL sensitive and resistant GBM lines

17 GBM4 GBM8 GBM-18 Patient derived primary GBM cells have varying invading capabilities in vitro and in vivo GBM4 BT74 GBM8 LN229 U87 U87 LN229 Primary lines Day 1 Day 3 Day 5 Day 8 Established lines Day 1 Day 3 Day 5 Day 8 2X 2X 1X 2X 2X 1X

18 secm encapsulated SC in the resection cavity track invading GBM cells in the brain *

19 Targeting Cell Proliferation and Death Pathways in cancer cells

20 P e r c e n t s u r v i v a l % distribution in cell cycle Both HDAc inhibitor and Cisplatin sensitize TRAIL resistant GBMs to TRAIL mediated apoptosis Control MS-275 DR4 a-tubulin DR5 a-tubulin 57kD 44kD 5kD 48kD 4kD 5kD 1% 9% 8% 7% 6% 5% 4% 3% 2% 1% % 6% 25% 69% Control G2 28% S 66% G1 4% 18% 32% 17% Cisplatin 48h Cisplatin 72h S-TRAIL MS275 + S-TRAIL 1 MSC-GFP+Cisplatin MSC-S-TRAIL MSC-S-TRAIL+Cisplatin 5 Bagci-Onder et al Oncogene Days (post-resection) Redjal et al Stem Cells. 214

21 EGFR specific nanobodies for targeted EGFR tumor therapy - Conventional monoclonal antibodies have limited success due their high molecular weight, preventing their delivery across the intact or partially disrupted blood-brain barrier and their physical diffusion into the tissues - - Camels and llamas possess fully functional antibodies that lack light chains. These heavychain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). - Nanobodies have - high target specificity - low inherent toxicity - readily access receptor clefts and are extremely stable - have a low immunogenic potential

22 Nanobody expression (fold Day 7) D 7 D 14 D 21 D 7 D 14 D 21 Characterizing EGFR specific Nanobodies CMV SS 7D12/9G8 IRES egfp LV-ENb1 CMV SS 7D12/38G7 IRES egfp LV-Enb2 Nanobody concentration (nm): EGF (5 ng/ml): ENb2 ENb EGFR py 168 EGFR Akt pser NSC-ENb1 NSC-ENb2 Akt pmapk p44/42 MAPK p44/42

23 TRAIL variant of EGFR nanobodies simultaneously targets A EGFR and death receptors on tumor cells Input IP (EGFR) EGFR nanobody S-TRAIL EGFR C T NT EGFR C T NT DR5 c-parp DR5 46kD 42kD α-tubulin IP (DR5) DR5 C T NT EGFR EGFR DR5 IgG 46kD 42kD

24 Blocking EGFR with Cetuximab significantly reduces ENb-TRAIL mediated apoptosis Caspase 3/7 Activity (Fold Control) Cetuximab ENb-TRAIL EGFR DR5 ENb-TRAIL : Cetuximab (µg/ml) : Casp8 Cleaved Casp8 α-tubulin ENb-TRAIL : Cetuximab (µg/ml) :

25 Percent survival TRAIL variant of EGFR specific nanobodies has A anti-tumor effect Tumor volumes (Fluc intensity x1) No. of cleaved Caspase-3 +ve cells * p= * p=.2 NSC-GFP NSC-ENb NSC-ENb2-TRAIL NSC-GFP NSC-ENb2 NSC-ENb2-TRAIL NSC-GFP NSC-ENb2 NSC-ENb2-TRAIL * 1 * 5 Control ENb2 ENb2-TRAIL Days Elapsed

26 Brain metastasis Parenchymal -brain tissue Leptomeningeal -linings of the brain Brain metastases are most common in patients with lung cancers, breast cancers and melanoma. 2-4% of patients with metastatic breast cancer develop brain metastasis. Majority of patients exhibit multiple tumors at the time of metastasis diagnosis.

27 Developing and Characterizing an imageable E metastatic brain tumor model Fluc bioluminescence in vivo [photons/5min (x 1 6 )] Inject MDA-Br2ma cells via intracarotid artery Fluc imaging/histology mcherry CD31 Merged Parenchymal metastatic foci d d2 d12 d23 d45 Leptomeningeal metastasis Vascular co-option Days after implantation e mcherry CD31

28 Therapeutic SC-TRAIL eradicate breast metastatic tumors in the brain Percent survival NSC-GFP NSC-TRAIL Expression of serpins, protects metastatic cancer cells from death signals and allow for efficient vascular co-option and colonization of tumor cells in the brain (Valiente et al., 214). mcherry GFP Cleaved caspase3 Merged S-TRAIL DR mcherry- metastatic tumor foci GFP- engineered stem cells 5 NSC-S-TRAIL NSC Control Days after tumor injection

29 Oncolytic Virus Therapy

30 Screening for ohsv and TRAIL resistant GBM cells

31 control ohsv ohsv- TRAIL % Rluc activity (fold control 12h) control ohsv ohsv- TRAIL ohsv-trail downregulates ERK and upregulates JNK and P38 and primes ohsv and TRAIL resistant GBM to Caspase mediated apoptosis Phospho- ERK1/2 ERK 1/2 p44 p42 p44 p control ohsv ohsv-trail Phospho-p38 Procaspase 8 Cleaved caspase Phospho-JNK JNK P38 p54 p46 p54 p46 Procaspase 9 Cleaved caspase 9 Cleaved PARP Bcl2 4 2 * * hours after infection * * Tamura et al Oncogene. 212

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33 Viral yield from MSC (x1^6 PFU/ml) MSC loaded with ohsv allow replication and release of viral particles in vitro and in vivo Fluc signal intensity (fold to uninfected GBM cells) h 48 h 12 h Gli36vIII-GFL 1.2 U87-GFL Time (hours)

34 Cell counts (fold to total cell number) Fluorescence imaging reveals the dynamics of MSC-oHSV mediated infection of GBM cells in vivo Fig h 48 h 72 h b.8.6 GFP+ mcherry+ GFP+ and mcherry Time (hours)

35 Fluc signal intensity Percent survival Fig. 3 secm encapsulated SC-oHSV have anti-tumor efficacy in mouse models of GBM resection Time course of ohsv spread in the resction cavity D1 D2 D3 D5 D7 D9 D12 PBS ohsv-fluc MSC-oHSV- Fluc ohsv-mch MSC-oHSV-mCh 1.6E+6 MSC-oHSV-Fluc 4 1.4E+6 1.2E+6 1.E+6 ohsv-fluc E+5 Days post-implantation 6.E+5 4.E+5 2.E+5.E Time post-resection (days)

36 Summary Developing tumor models that mimic the clinical scenarios are critical to developing efficacious treatments for GBMs The incorporation of mechanism based biological therapies is important for the advancement of the field Stem cells have the potential to be used as a therapeutic protein factories that can molecularly target proliferating GBM cells Non-invasive real time imaging provides key information on fate of GBM and therapeutic efficacy of stem cells