1 / 40

Jeffrey S. Isenberg, MD, MPH Chief Science Officer Radiation Control Technologies, Inc.

A CD47-suppressing morpholino kills cancers and protects normal tissues from radiation and chemotherapy. Jeffrey S. Isenberg, MD, MPH Chief Science Officer Radiation Control Technologies, Inc. June 8, 2019. Thrombospondin-1 (TSP1).

blake
Download Presentation

Jeffrey S. Isenberg, MD, MPH Chief Science Officer Radiation Control Technologies, Inc.

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. A CD47-suppressing morpholino kills cancers and protects normal tissues from radiation and chemotherapy Jeffrey S. Isenberg, MD, MPH Chief Science Officer Radiation Control Technologies, Inc. June 8, 2019

  2. Thrombospondin-1 (TSP1) TSP1 is a stress-released protein that binds to the cell receptor CD47 CD47 CD47 is found on all normal and cancer cells Arterioscler Thromb Vasc Biol. 2008 Apr;28(4):615-21

  3. Evolution of the thrombospondin family } Matricellular TSPs amphibians, fish, birds, mammals – all with pressurized circulation Adapted from Adams et al J Mol Biol 2003

  4. The TSP1-CD47 signal appeared along with vascular eNOS TSP Insects Fish Amphibians Reptiles Mammals nNOS Presenilin(TM) Wrapper(IgV) (SIRPβ1) Vertebrates TSP1 nNOS eNOS (SIRPβ1) CD47 (SIRPβ1) βγδ CD47 SIRPα TSP1 eNOS (van Beek 2005; Stork et al. 2009; Watanabe et al. 2010)

  5. Nitric oxide (NO) signaling within arteries targets multiple cells types to promote blood flow Matrix Biol. 2014 Jul;37:92-101

  6. TSP1-CD47 signaling dysregulates vascular homeostasis by blocking eNOS, NO, cGMP, cAMP and increasing reactive oxygen species (ROS) production Matrix Biol. 2014 Jul;37:92-101

  7. Absence of TSP1-CD47 signaling enhances tissue survival to ischemia and anoxia Arterioscler Thromb Vasc Biol. 2008 Apr;28(4):615-21

  8. TSP1-CD47 limits multiple beneficial pathways to promote cell and tissue injury Thrombospondin-1 binding to CD47 limits signaling through the nitric oxide (NO)/cGMP cascade in vascular and some hematopoietic cell lineages. In non-transformed cells TSP1/CD47 signalinglimits stem cell self-renewal. Ligation of CD47 by TSP1 also regulates autophagy signaling and in some cases can induce programmed cell death. CD47 also serves as the counter-receptor for SIRPα. Int J Biochem Cell Biol. 2016 Dec;81(Pt A):184-194

  9. What is a morpholino oligonucleotide • Uncharged backbone • Excellent stability • Specific • Effective • Non-toxic • Versatile • No strong oligo-protein interactions • Reasonable water solubility (to 1 mM) Slide provided by Jon Moulton, PhD, Gene Tools, LLC

  10. The morpholino binds to the native RNA and limits RNA translation. What is morpholino antisense RNA (sense) Morpholino (antisense) Morpholinos are stable and inmmune to enzymatic degradation. Slide provided by Jon Moulton, PhD, Gene Tools, LLC

  11. Blocking translation with a CD47 morpholino oligonucleotide To block translation, a steric-blocking morpholino oligonucleotide: (1) Stops the initiation complex before it reaches the AUG base start sequence of the mRNA. (2) Without reaching the start, the complete ribosome cannot form a complex with the mRNA. (3) Without the complete ribosome-mRNA complex, the protein will not be made. Slide provided by Jon Moulton, PhD, Gene Tools, LLC

  12. Radiation and chemotherapy increase TSP1-CD47 signaling in normal human and cancer cells and tissues CD47 TSP1 Human buttock skin irradiated with 2MED of UV radiation and TSP1 mRNA and protein levels determined. (Adapted from J Dermatol Sci. 2010 Jul;59(1):16-24) CD47 mRNA levels in human cancer cells treated with chemotherapeutic agents. (Adapted from Proc Natl Acad Sci U S A. 2018 Feb 6;115(6))

  13. Absence of TSP1-CD47 signaling protects from high-dose radiation Mice hindlimbs irradiated with 25 Gy. Apoptosis (cell death) demonstrated by intra-nuclear staining of muscle and bone marrow cells. Am J Pathol. 2008 Oct;173(4):1100-12

  14. CD47 morpholino treatment protects tissues and blood vessels from high-dose radiation Mice exposed to 25 Gy to the hindlimb develop tissue necrosis, fibrosis and loss of blood flow. Mice treated with a CD47 morpholino locally prior to high-dose regional radiation are protected from injury and show enhanced blood flow. Laser Doppler images showing radiation-driven decreases in hindlimb blood flow after radiation. Treatment with a CD47 morpholino delivered regionally preserved limb blood flow after high-dose radiation. Sci Transl Med. 2009 Oct 21;1(3):3ra7

  15. CD47 morpholino treatment increases survival to lethal whole body radiation @ 24 h 7.6 Gy TBI Mice treated with a CD47 morpholino had greater survival to lethal whole body radiation, along with increased circulating white cells and less proximal GI tract cell death/apoptosis (white dots) on TUNEL assay. Esophagus - TUNEL staining @ 24 h WT WT + IR WT + IR+ CD47M Sci Rep. 2013;3:1038

  16. CD47 morpholino treatment protects the gastro-intestinal tract from lethal whole body radiation • Mice treated with lethal whole body radiation experienced loss of GI tract villi • Mice treated with radiation and a CD47 morpholino maintained GI track villi • Images of H&E staining of intestinal tissue 14 days post-irradiation. Autophagy. 2012 Nov;8(11):1628-42

  17. CD47 morpholino treatment provides protection to the lungs following lethal whole body radiation Cell death/apoptosis is seen by white dots on TUNEL assay in lungs following lethal whole body radiation. Treatment with a CD47 morpholino (10 uM) decreases cell death/apoptosis in the lung. TUNEL staining of lung tissue 24-48 h post lethal whole body radiation treatment (upper images). Quantification of same in bar graph. Autophagy. 2012 Nov;8(11):1628-42

  18. CD47 morpholino treatment given after lethal whole body radiation increases survival Mice given the CD47 morpholino after lethal radiation survived better than untreated thus showing how the morpholino could be employed in military and nuclear industry settings. Sci Rep. 2013;3:1038

  19. CD47 morpholino treatment blocks radiation injury by increasing protective autophagy TUNEL apoptosis SQSTM1 expression • WT mice were treated with saline or 10 µM CD47 morpholino and 48 h later challenged with lethal total body irradiation (7.6 Gy) • SQSTM1 targets other proteins that bind to it for selective autophagy Autophagy. 2012 Nov;8(11):1628-42

  20. CD47 morpholino treatment protects from doxorubicin-mediated cardiac cell death Cardiac tissue Cardiac myocytes Cardiac myocytes Breast Cancer Res Treat. 2018 Nov;172(1):69-82

  21. CD47 morpholino treatment protects from doxorubicin-mediated cardiac fibrosis Doxorubicin-mediated cardiac fibrosis in tumor-bearing animals • Doxorubicin- and chemotherapy-mediated heart injury causes heart failure/death • The CD47 morpholino prevents these devastating cardiac complications Breast Cancer Res Treat. 2018 Nov;172(1):69-82

  22. CD47 morpholino treatment enhances protective autophagy in normal cells and tissues TSP1 Radiation CD47 morpholino Expert Opin Ther Targets. 2013 Jan;17(1):89-103; Breast Cancer Res Treat. 2018 Nov;172(1):69-82

  23. Absence of TSP1-CD47 signaling increases radiation-mediated tumor killing WT and TSP1 -/- mice received 10 Gy of radiation to melanoma-bearing hindlimbs and tumor volumes measured. Similar results were obtained when this experiment was performed tumor-bearing in WT and CD47 -/- mice (Cancer Res. 2014 Dec 1;74(23):6771-83). Am J Pathol. 2008 Oct;173(4):1100-12

  24. Treatment with a CD47 morpholino increases radiation-mediated tumor killing B16 Melanoma Head and Neck Squamous Cell Carcinoma • Solid tumor regrowth after radiation was essentially stopped in mice that received a CD47 morpholino prior to tumor radiation (red arrows). • Treatment with a CD47 morpholino alone (single 10 µM dose) decreased tumor growth modestly (green arrow). Sci Transl Med. 2009 Oct 21;1(3):3ra7

  25. CD47 morpholino treatment protects normal CD68+ macrophages from cancer-targeting radiation tumor + radiation + CD47 morpholino tumor + radiation tumor Sci Transl Med. 2009 Oct 21;1(3):3ra7

  26. CD47 morpholino treatment increases calreticulin signaling and CD68+ macrophage phagocytosis and killing of cancer Macrophage killing of cancer Calreticulin signaling • The CD47 morpholino attacks cancer through at least 3 distinct pathways: (1) macrophage phagocytosis, (2) T-cell cancer killing and (3) calreticulin signaling • CD47 antibodies only work through macrophages and do not protect non-cancer tissues/organs from radiation and chemotherapy Breast Cancer Res Treat. 2018 Nov;172(1):69-82

  27. CD47 morpholino treatment increases CD8+ T-cell killing of cancer cells RTCA assay Target cell viability Cell-derived LDH levels at 24 h as a measure of cell cytotoxicity. Simply treating killer CD8+ T cell with a CD47 morpholino enhances elimination of 15-12RM fibrosarcoma cells, and this process is further enhanced with radiation treatment. Cancer Res. 2014 Dec 1;74(23):6771-83

  28. CD47 morpholino treatment increases CD8+ T-cell invasion of irradiated cancers Fibrosarcomas Cancer Res 2014;74:6771-6783

  29. 2.2 1.76 Tumor Volume (cm3) 1.32 Tumor Only 10 Gy + vehicle 10 Gy + CD47 Morpholino .88 .44 0 0 3 6 9 12 15 Days Post-Radiation CD47 morpholino treatment plus radiation activates T-cell killing of cancers SCC VII in athymic C3H mice (that lack functional T cells) SCC VII in C3H mice (that have T cells) Cancer Res. 2014 Dec 1;74(23):6771-83

  30. CD47 morpholino treatment alone eliminates hepatic carcinoma Control Sustained CD47 morpholino treatment: - 5 µM i.p. q 4 d for 40 days CD47 morpholino PBS Mismatch control CD47 morpholino Similar effects were obtained in nude mice transplanted with human hepatic carcinomas! This study also indicates that a CD47 morpholino can be given chronically without complications! Hepatology. 2014 Jul;60(1):179-91

  31. CD47 signaling controls radiation sensitivity through regulation of metabolic pathways Antioxid Redox Signal. 2017 Oct 20;27(12):874-911

  32. CD47 signaling controls in vivo radiation sensitivity through regulation of metabolic pathways WT and CD47 -/- mice received lethal 7.6 Gy total body irradiation and metabolic analysis of tissue samples done 24 h later. Numerous key metabolites were preserved in CD47 -/- mice post-lethal total body irradiation. J Biol Chem. 2015 Oct 9;290(41):24858-74.

  33. CD47 morpholino preliminary toxicology • Study performed in cooperation with experts from the Radiation and Nuclear Countermeasures Program (RNCP, Bethesda, MD) and the NIH • Purpose: To test the hypothesis that systemic administration to mice of a supra-therapeutic dose of a CD47 morpholino oligonucleotide was safe Study Design: Age and gender matched mice (n=8/group) Treatment: Single dose, 40 µM CD47 morpholino in normal saline or same volume normal saline vehicle ( 4X the normal therapeutic dose of 10 µM) Administration route: Intraperitoneal injection Study length: 4 weeks post-administration Clinical end points: Change in weight, food consumption, interaction with peers, vocalizations, grooming behavior, posture, nasal discharge, response to handling Anatomic end points: Prosection-observed changes in gross morphology of vital organs (heart, lungs, liver, intestine, kidneys, spleen, brain, skeletal muscle, bone)

  34. CD47 morpholino toxicology • Weekly treatment with a CD47 morpholino for 24 weeks to Sickle Cell Disease (SCD) mice • and control mice mitigated SCD-related complications and had no untoward effects (J.S. Isenberg, personal communication)

  35. Advantages of the CD47 morpholino • A unique sequence and the only one that effectively targets the human RNA sequence; cannot be duplicated • NIH patented in the United States and Internationally • Licensed by Radiation Control Technologies, Inc (RCTI) • Robust basic science and proof-of-principal under multiple applications in animal and cell models • Multiple therapeutic applications: (1) cancer treatment with radiation, (2) cancer treatment with chemotherapy, (3) as a solo agent or with other biologics • Only CD47-targeting agent that harnesses multiple innate immune cell types (macrophages, T-cells, and NK cells to kill cancer • Only agent that protects normal tissues/organs from radiation and chemotherapy injury

  36. CD47 PMO Benefits • Advantages of the CD47 Morpholino: • •       No influence on red blood cells (RBCs) as they have no nucleus.  The morpholino works by stopping CD47 protein production vis inhibiting RNA translation in the nucleus. • •       CD47 Morpholino has a unique sequence and the only one that effectively targets the human RNA sequence; No other sequence works and it cannot be duplicated.  Anyone can manufacture an antibody against CD47 – there is only one Morpholino sequence targeting CD47 that works. • •       NIH patented in the United States and Internationally • •       Licensed by Radiation Control Technologies, Inc (RCTI)  • •       Robust basic science and proof-of-principal under multiple applications in animal and cell models • •       Multiple therapeutic applications: (1) cancer treatment with radiation, (2) cancer treatment with chemotherapy, (3) as a solo agent or (4) with other biologics • •       The morpholino is the only CD47-targeting agent that harnesses multiple innate immune cell types (macrophages, T-cells, and NK cells) to kill cancer • •       Only known agent that protects normal tissues/organs from both radiation and chemotherapy injury • •       CD47 Antibodies in clinic are toxic: since the antibody binds to RBCs, there is a marked side-effect rapid RBC clearance/destruction and anemia. This leads to increased transfusion requirements. Also, the CD47 antibody greatly complicated standard ABO blood typing and compatibility testing for transfusions. These toxicities significantly limit the possible maximum dosage of the CD47 antibody.

  37. Additional advantages of the CD47 morpholino • Stable long-term and deliverable via multiple routes including topically, subcutaneously and intravenously • Wide tissue penetration allowing for robust treatment beyond the vasculature of parenchymal cells, tissues and organs • Safe in animal at 4x therapeutic dosage • Safe in animals when given weekly for over half a year • Currently 3 companies have GMP production capabilities for morpholinos providing increased bandwidth: Sarepta (and their CRO partners), Ajinomoto (Tokyo), and Marlin Biotech

  38. Additional advantages of morpholinos as a class of drugs • Morpholinos, as a drug class, although new, have been approved for clinical use and shown safe in adults and children • A morpholino oligonucleotide made by Sarepta Therapeutics has been approved by the US FDA for treatment of Duchenne muscular dystrophy • The oligo, eteplirsen (Exondys51), is a splice-modifying morpholino correcting frameshift mutations by causing exon 51 of dystrophin to be skipped

  39. Current CD47-SIRP-α Abs have clinical morbidity Recombinant SIRPα extracellular domains • TTI-621 (Trillium Therapeutics, Phase I trials NCT02663518 and NCT02890368). TTI-621 induced anemia in non-human primates (Clin Cancer Res 2017). Dose limiting thrombocytopenia at 0.3 mg/kg (2016 ASH abstract). Grade 1,2 fatigue and loss of appetite reported in phase I with intratumoral injection (2017 ASH abstract) • ALX148 (Alexo Therapeutics, phase 1 NCT03013218) • Hu5F9-G4A (Forty Seven Inc., phase 1 NCT02216409, NCT02678338, NCT02953509,NCT03248479, phase Ib/II NCT02953782). Induced anemia in nonhuman primates (PLoS One 2015). For 16 patients in Hu5F9-G4 trial, there were 11 incidences of G1 and 5 incidences of G2 anemia (JCO 2016) • CC-90002, Celgene Corporation, Phase I trials NCT02367196, NCT02488811, NCT02641002). • Humanized monoclonal antibody targeting SIRP-α: OSE-172 (Effi-DEM, preclinical) • Bispecific CD47 antibody (NI-1701, Novimmune SA, preclinical) • Other preclinical antibodies: Arch Oncology, Janssen Research & Development, Tarnhelm Therapeutics, Surface Oncology, Synthon, Zai Lab Humanized CD47 or SIRP-α antibodies Given role TSP1-CD47 play in blocking nitric oxide and VEGF, CD47 Abs could cause complications such as hypertension, thrombosis, stroke, or decreased wound healing. The CD47 morpholino always improves healing and blocks cardiovascular disease.

  40. Recent and future developmental milestones of CD47 morpholino • Satisfied past-due NIH licensing fees ($233K) and licensing fees ($30K0 and revised existing milestone timeline with NIH • NIH License now in good standing • Engaged morpholino experts Drs. Jon and Hong Moulten to provided key developmental support • Engaged Dr. David Roberts (NIH), co-inventor of the technology along with Dr. Isenberg, to provided scientific input • IND-enabling tox/pharmacokinetic studies of CD47 morpholino • Manage CMC (GMP manufacturing), pharmacological and toxicological studies to comply with FDA's IND guidelines • Building a managerial team with biotechnology/pharmaceutical experience • Identify investors for ongoing development

More Related