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Tumor Evolution

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Tumor Evolution 8/1/2017 Immune Priming with Ultrasound Chandan Guha, MBBS, PhD Conflicts and Grants - NIH (R01 EB009040) - NCI SBIR grant with Professor and Vice Chair, Radiation Oncology Celldex Therapeutics, Inc. Professor, Urology and Pathology - Project Energy with Director, Einstein Institute of Onco-Physics Johnson & Johnson Montefiore Medical Center Albert Einstein College of Medicine, Bronx, NY [email protected] Tumor Evolution Immune Immune Tumor Restoration Surveillance progression Resurgence Living “with” Diagnosis Tumor • Cancer cure restored Living “with” Primary => Mets immune surveillance Mutated Cells • Ablation with Immune Restoration is curative “Killing” tumor Mutated Cells Tumor Ablation Immune Escape (Epigenetic loss – Antigen presentation) Suppression (IL10, TGFß, PD-L1) Cooption (Macrophage, Ectopic Lymphoid structure) Focal Oncology Clinical Adaptive Learning (FOCAL) Cancer Clinic Network 1. Ablative Therapies for local control induces anti-tumoral immunity, which in turn helps local control. 2. Ablative Therapies for systemic immunity: Immune Priming Ablation (IPA) for In Situ Tumor Vaccines a. UPR => ER stress => Antigen Processing / Presentation b. “Eat Me” and DAMP signals c. Reversal of tolerance d. Antigen Presentation (neo-antigens & cryptic antigens) “Focal Therapy for Systemic Cure” 1 8/1/2017 Project ENERGY.01: Proposed Study Design Immuno-Priming Abla on (IPA) Therapies Study 1: 3LL IP only; Biomarker endpoints Immuno-Priming (IP) Best 2 of all LOFU1-2 HT RP IRE1-2 RFP1-2 Digoxin Metformin Metastatic Model Immuno-Priming - Ablation (IPA) 3LL Lewis Lung Study 2: 3LL SBRT (10-20 Gy) RFA IPA; Biomarker, Immune Profile, Tumor control GEM models and survival endpoints LOFU / I RE / Digoxin RapidCap + + Liver (Ch) LOFU Low Energy / Intensity SBRT (10-20 Gy) RFA Focused Ultrasound KPC Best 2 IPA’s HT Histotripsy RP Radiation Priming Energy IPA1-2 Study 3: 3LL; 4T1;GEM + 2 Metastatic (3LL, 4T1) IRE Irreversible Electroportaion GM-CSF IPA + DC Act./Check-Point Inhib.; 1 GEM (CaP / KPC) RFP Radiofrequency Priming +/- Biomarker, Tumor control Anti-PD1 SBRT Stereotactic Body Radiation and survival endpoints Therapy RFA Radio Frequency Ablation Go/No-Go for IRE/RFA Internal R&D (Ethicon) = Go/No-Go for LoFu Licensing External R&D (Electroblate, Varian) NewCo Formation Thorsten Melcher Autologous in situ tumor vaccines Treatment Protocol 1 .8 No Treatment 3 weeks • RT (60 Gy) .6 • Surgery Flt3L (10 µg/day) .4 3 weeks X 10 days C on tro l • RT (60 Gy) • Surgery R T .2 R T + F lt 3L Flt3L (10 µg/day) F lt 3L 3 weeks X 10 days 0 20 4 0 6 0 80 10 0 1 20 140 1 60 1 80 20 0 Sur vi val T ime ( Day s) 2 8/1/2017 RT + Flt3L Improves Survival of Tumor-bearing C57Bl/6 Mice 1 .8 RT + Flt3L .6 C57Bl/6 mice .4 Control (RT+Flt3L - 55% cured) RT .2 RT + Flt3L Flt3L 0 20 40 60 80 100 120 140 160 180 200 Survival Time (Days) Immunodeficient Nude mice (RT+Flt3L - 0% cured) Survival Time (Days) Systemic Effects of Primary Tumor Irradiation Kaplan & Meier Survivorship Function 1 B - RT C - Surgery .8 E - S + Flt3L D1 - RT + Flt3L-10 .6 D2 - RT + Flt3L-30 .4 Surgery + Flt3L .2 0 0 40 80 120 160 Survival Time (Days) Sample size: 29 patients 3 8/1/2017 Nitin Ohri Sample size: 29 patients Ablative SBRT dose fractionation • 34 Gy x 1 Fx • 18 Gy x 3 Fx • 10 Gy x 5 Fx Study Subjects # Demographics Disease Burden Prior Treatment(s) SBRT Clinical Course Right lung squamous cell carcinoma with 50 Gy in 5 Progression at 6 73 year-old 1 multiple lung masses Carboplatin + gemcitabine fractions to weeks, death at 4 Korean male and bilateral RLL mass months mediastinal adenopathy Chemoradiotherapy for Right lung 34 Gy in 1 Partial response 55 year-old localized disease, 2 adenocarcinoma, with fraction to at 2 months, Hispanic female nivolumab for metastatic bilateral lung nodules LUL nodule stable at 4 months disease Chemoradiotherapy for Right lung squamous 50 Gy in 5 80 year-old localized disease, cell carcinoma with fractions to Partial response 3 Caucasian carboplatin + gemcitabine spine and pelvic right lung at 2 months female for metastatic disease, metastases mass nivolumab Right lung 73 year-old Carboplatin/ + 50 Gy in 5 adenocarcinoma with 4 Caucasian pemetrexed, maintenance fractions to PET/CT on 4/27 mediastinal adenopathy female pemetrexed RLL mass and liver metastasis Patient 2 Right lung mass reduced from 5.0 cm (maximum SUV 10.7) to 2.1 cm (maximum SUV 6.5) on first post- treatment PET/CT Target Lesion Total Glycolytic Activity (TGA): 1.0 cc 0.3 cc Other lesions’ TGA: 44.7 cc 4.5 cc 4 8/1/2017 Patient 3 Improvement or resolution of most of the osseous foci Target Lesion Total Glycolytic Activity (TGA): 49.1 cc 6.2 cc Other lesions’ TGA: 130.2 cc 45.4 cc Patient 3 Target Lesion Total Glycolytic Activity (TGA): 49.1 cc 6.2 cc Other lesions’ TGA: 130.2 cc 45.4 cc Energy activated in situ Tumor Vaccines POC Studies Energy Tumor Models End Points Limitation Immunotherapy Single Flt3L • Lewis Lung 1. Primary Tumor • Murine Ectopic Fraction + / - 3LL in C57/Bl6 Growth Transplantation SFRT • BN1LNE Liver 2. Metastases Models CD40L 25-60 Gy (HCC) in 3. Survival 4. Immune assays • RT Dose Balb/c • Fractionation 20 Gy TLR9 • Lewis Lung PTG, Mets, Survival • RT to Draining agonist 3LL in C57/Bl6 Lymph Node 10 Gy Listeria- • TRAMPC1 PTG and Immune PSA TPSA23 Assays • Break Tolerance ADXS31- Prostate to self antigens 142 Cancer • Small Animal 20Gy x 3 PD1-Fc • Lewis Lung PTG, Mets, Survival Treatment 3LL in C57/Bl6 LOFU HIFU • TPSA23 PTG, Immune • Lack of Immune Prostate Assays Surveillance and carcinogenic LOFU SBRT • B16 Melanoma PTG, Mets, Survival environment (10 Gy x 3) Immune Assays 5 8/1/2017 Acoustic priming ULTRASOUND -- ADVANTAGES 1 W/cm2 2000 W/cm2 • US Can do both Imaging and Treatment TM • Quick Tissue Destruction • Bloodless • Precise and Accurate • Non-sterile environment Focal Zone HIFU Beam Peripheral Zone Diagnostic (Imaging) Beam HIFU directed harmlessly across skin and rectum toward the tumor 6 8/1/2017 Therapeutic Ultrasound as an autologous in situ tumor vaccine • HIFU = High Intensity Focused Ultrasound • MRgFUS = MR-guided Focused Ultrasound • TULSA = Transurethral ultrasound ablation • LOFU = Low intensity (energy) focused ultrasound (LOFU coined by Guha group) • SST = Sonic Stress Therapy • APT – Acoustic Priming Therapy LOFU parameters Condition Condition #2 Condition #3 Condition #4 Condition #5 #1 Duty Cycle (%) 1 25 50 75 100 Power (W) 32 16 8 4 2 Time (ms) 1000 625 1250 2500 5000 Thermal 0.32 2.5 5 7.5 10 Energy (J) Peak Negative Pressure 8.14 6.08 4.58 3.34 2.46 (MPa) Thermal Energy Mechanical Energy The “sonic stress” of LOFU Gene function Genes affected by LOFU treatment 1. Protein Folding DNAJB1, HSPH1, HSPE1, HSPB1, HSPD1, HSPA4L, CRYAB, HSPA6, HSPA7, HSP90AA1, HSP90AA4P, DNAJA4, FKBP4, LGSN, PTGES3 2. Cell cycle regulation IER5, JUN,CACYBP, GPRC5A, RRAD, WEE2 3. Cytokines IL8 4. Receptors CSF2RB, IL7R, NPR1, RXFP2, FLT4, ITGA2 5. Cytoskeleton integrity FAM101B, TCP1 6. Transcriptions ATF3, ANKRD1, EYA4, KAT2A 7. Transporters SLC22A2, SLC22A16, RHAG 8. Apoptosis regulation NLRC4, ANGPTL4, BAG3 9. Peptidase NAALADL1, MEP1A, PLOD2 7 8/1/2017 Gene Ontology Figure 2 Gene ontology GoTerm network. After LOFU treatment, the gene response showed extensive upregulation of genes that are related to unfolded protein. Gene Expression with qRT-PCR 10.000 9.000 8.000 7.000 6.000 5.000 4.000 Control 3.000 Treated 2.000 1.000 0.000 IL8 JUN ATF3 HSPE1 HSPB1 HSPA6 HSPA7 HSPD1 HSPH1 CSF2RB HSPA4L DNAJB1 DNAJA4 ANKRD1 HSP90AA1 HSP90AA4P Figure 4 qRT-PCR of select genes from the RNA sequencing. Genes were selected from pathways highlighted in the KEGG pathway analysis and validated using qRT-PCR. The data correlated well with the RNA sequencing results. HSPA6 and HSPA7 were highly regulated to 200+ and 25+ fold respectively. Increase in Hsp70 mRNA expression after LOFU treatment 140.00 120.00 Hspa1a e g 132.37 n 100.00 a Hspa1b h 80.00 C 80.77 d l 60.00 o F 40.00 A N R 20.00 m 0.00 Hsp expression -20.00 80 Hspa1b 70 e Hspb1-1 s a 60 Hspb1-2 e r c 50 Hspd1 n i Hsph1 d l 40 o f Hsp90aa1 30 A Dnajb5 N R 20 m 10 0 0 5 10 15 20 25 30 Time post-LOFU (h) 8 8/1/2017 Immunomodulation of tumor cells Surface Calreticulin (CRT) Intracellular HSP70 In vitro Effect of LOFU on Cell Surface Markers Summary of in vitro data under JJI-ENERGY ➢ LoFu is non-ablative ➢ LoFu induces “sonic stress” on cancer cells ➢ Sonic stress signature is consistent across cell types and indicative of immune stimulation 3 Watt LoFu 5 Watt LoFu 3LL 4T1 TPSA23 3LL 4T1 TPSA23 6h 24h 6h 24h 6h 24h 6h 24h 6h 24h 6h 24h HSP70 ER Stress CRT BiP MHC I Co-Stimulatory CD86 Death Fas Receptors CD40 Inhibitory PD-L1 0 5 10 20 30 No change TBD SRS Ch-RT LOFU Induce Cell death Calreticulin membrane Translocation HSP activation “danger” signal HMGB1 release TL4 binding “eat me” signal 9 8/1/2017 Treat the tumor draining lymph node (TDLN – immune privilege site) – Reprogram tolerogenic DCs (IDO inhibitors) – Inhibit regulatory T cells (Treg) – Reverse T-cell anergy in TDLN – LOFU primary tumor T cell activation vs. T cell anergy Full stimulation Re-stimulation CD3 CD28 T cell Activation + CD4 T cell Cytokine production Cytokine production Cell Proliferation Cell Proliferation Re-stimulation Anergic stimulus T cell Anergy CD4+ T cell Hyporesponsive Hyporesponsive phenotype phenotype T cell Activation vs.
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