ヘリオス（4593） – Business Briefing for Institutional Investors

April 6, 2022 Company Name: HEALIOS K.K. Representative: Hardy TS Kagimoto, Chairman & CEO (TSE Growth Code: 4593) Business Briefing for Institutional Investors HEALIOS K.K. held a webinar to present an update on its business to institutional investors in Japan. The meeting agenda is provided below, and the presentation materials are available for viewing. HEALIOS K.K. Business Briefing for Institutional Investors Date and Time: Tuesday April 5, 2022, 10:00-12:00 Description: 1. Introduction 2. Overview and Current Status of HLCM051 Clinical Trials 3. iPSC Regenerative Medicine eNK cells & UDC Briefing materials: attached Chairman and CEO Hardy TS Kagimoto, MD Executive Vice President CMO (Chief Medical Officer) Masanori Sawada, MD, PhD, MBA Executive Officer Research field Kouichi Tamura, Ph.D Contact: Department of Corporate Communications, HEALIOS K.K. E-mail: ir@healios.jp 1 Overview and Current Status of HLCM051 Clinical TrialsHEALIOS K.K.CompanyDateApril 5, 2022Executive Vice President CMO (Chief Medical Officer)Masanori Sawada, MD, PhD, MBAHLCM051 ARDS: Target DiseaseAcute Respiratory Distress Syndrome or ARDS is a collective term for respiratory failure that occurs suddenly in critically ill patients (mainly due to severe pneumonia, sepsis, trauma, etc.). Activated inflammatory cells run amok and attack the lungs.It is a disorder with a very high mortality rate (30 ~ 58%) and a poor prognosis, for which there is a need for novel therapies that can improve patient outcomes.At present, there are no therapeutic drugs that can make a direct improvement to a patient’s vital prognosis when ARDS develops.The only symptomatic treatment for respiratory failure includes artificial respiration.Time-dependent change in CT image in ARDS affected lung ECMOArtificial Respiration（出所） Ichikado K BMJ Open. 2012 Mar 1;2(2):e000545© HEALIOS K.K. All rights reserved.2HLCM051 ARDS: Overview of Cell TherapyThe allogeneic bone marrow-derived Multipotent Adult Progenitor Cell product (HLCM051) is expected to restore damaged lung tissue and improve respiratory function by reducing inflammation, regulating immune function, promoting angiogenesis, and protecting and repairing damaged cells and tissues.© HEALIOS K.K. All rights reserved.Partner Company: Athersys, Inc.Head Office Cleveland, Ohio (U.S.A.) NASDAQ：ATHXDeveloped ProductsStem cell product:MultiStem® （proprietary）(Source) Based on materials provided by Athersys3HLCM051 ARDS: Multiple Mechanisms of Action© HEALIOS K.K. All rights reserved.(Source) Athersys4HLCM051 ARDS: Multiple Mechanisms of Action Intravenous HLCM051 first reaches the lungs. It restores lung tissue and improves respiratory function by promoting inflammation reduction, immune regulation, and protection and repair of damaged cells and tissues.© HEALIOS K.K. All rights reserved.5(Source) Ware et al. NEJM 2000; 342: 1334HLCM051 ARDS: ONE-BRIDGE StudyPhase II study investigating the efficacy and safety of HLCM051 in pneumonia induced ARDS patientsARDS trial cohortsCohort 1 : Patients with pneumonia-induced ARDSARDSPatientsCOVID-19TestnegativeRandom:21Efficacy and safety evaluationFrom April 2019 to March 2021Primary endpoint: Ventilator free days (VFD) at Day 28Secondary endpoint: Mortality (at day 28, 60, 90, 180)Cohort 2 : Patients with pneumonia-induced ARDS caused by COVID-19positiveSafety evaluationFrom April 2020 to August 2020Patient enrollment of COVID-19 pneumonia-derived cases (Cohort 2) was performed separately from the conventional clinical trial administration group (Cohort 1). © HEALIOS K.K. All rights reserved.6HLCM05120Standard therapy10HLCM0515HLCM051 ARDS: ONE-BRIDGE Study Results at 180 Days Post AdministrationThe HLCM051 treated group demonstrated a 9-day higher median VFD than the standard therapy group.The treated group saw a 39% reduction in mortality as compared to patients treated with standard therapy. Cohort 1No safety concerns.Cohort 2No deaths, no safety concerns. Primary EndpointVFD (the number of days out of 28 during which a ventilator was not used for the patient)Secondary EndpointMortality(180 days after administration)© HEALIOS K.K. All rights reserved.The ventilator was withdrawn within 28 days for all five patients and in three days or less for three of these patients. Cohort 1HLCM051Standard therapyCohort 2HLCM05120 days11 daysSafetyNo safety issues26.3% 42.9%VFDPrimary EndpointSecondary EndpointMortality(180 days after administration)25 days0%（Source）in-house data7HLCM051 ARDS: Comparison with Historical DataComparison with historical data was a secondary efficacy endpoint of the study protocol• Data source reported in Scientific Reports (Sci Rep. 2021; 11: 20051.) in October 2021• Matching comparison was performed with the data from the paper on which the study design is based.ONE-BRIDGE Study HLCM051(20 subjects)Forward-collected historical data(104 subjects)Matching by Propensity ScoreAbstraction© HEALIOS K.K. All rights reserved.820 subjects were selected from historical data and compared to group HLCM051 of ONE-BRIDGE study (VFD, Mortality)HLCM051 ARDS: Comparison with Historical DataConsistent with the ONE-BRIDGE study, VFD was prolonged and mortality improved.In the matched historical data comparison, the VFD was prolonged by 8.1 days (mean), and the mortality rate was 33.7% lower (reflecting a 56% decline in mortality as compared to the historical data group).Primary EndpointVFD (the number of days out of 28 during which a ventilator was not used for the patient)Secondary EndpointCompared with historical dataHLCM051Matched historical data14.8days6.7日 daysP=0.0110P=0.0536P=0.0110＋＋Mortality(180 days after administration)26.3% 60.0%© HEALIOS K.K. All rights reserved.HLCM051 N=20Matched Historical DataN=20（Source）in-house data9HLCM051 ARDS: Development StatusONE-BRIDGE StudyRegenerative medicine product pre-application consultation with thePMDA was conducted at March end 2022.August 2021Announcement of top-line resultsHealios was advised that when making a future application forapproval for the ARDS indication, it needs to add certain supportingdata to the proposed application data package. Consultations withthe regulator are ongoing.April 2019First patient enrolledMarch 2021Patient enrollment completedPatient enrolmentData analysisPreparation for ApplicationApplicationApproval / Launch*HLCM051 has been designated as an orphan regenerative medicine product for use in the treatment of ARDS by the Ministry of Health, Labor and Welfare.© HEALIOS K.K. All rights reserved.10HLCM051 Ischemic Stroke: OverviewIschemic stroke is caused by a blockage of blood flow in the brain that cuts off the supply of oxygen and nutrients, resulting in tissue loss.The annual number of cases in Japan ranges from 230,000 to 330,000It is estimated that 37.9％ of bedridden patients and 21.7％ of persons who were in need of care were affected by ischemic stroke.Treatment in Accordance with the Period After OnsetPeriod after onset10h20h30h40hThere is a time limitbecause of the risk of cerebral hemorrhage(Source) AthersysClot-dissolving agent*1Mechanical reperfusion *2HLCM051※1 Dissolves blood clots in the brain vessels ※2 Insertion of the catheter into a blood vessel and recovery of the thrombus directly with a wire.Possibility as anew alternative HLCM051 Therapy Could Greatly Extend the Treatment Window for Stroke Patients© HEALIOS K.K. All rights reserved.(Note) This material was prepared to explicitly describe the major therapeutic options for ischemic stroke and their treatment window periods after onset.Appropriate treatments are conducted according to patients’ conditions and classification of their symptoms. Experimental or investigational treatments not included in the above are also performed.11HLCM051 Ischemic Stroke: Mechanism & Neuroprotective Effects in Cerebral Infarction✓ Cerebralinfarction leads to activation oftheperipheralimmune system and release ofproinflammatory cells and cytokines from thespleen (atrophy of the spleen).✓ Spleen-induced proinflammatory mediators contribute to exacerbation of blood-brain barrier (BBB) disruption and central nervous system (CNS) inflammation mediated by injurious microglia (M1).✓ Intravenously administered HLCM051 reduces the destruction of the BBB by promoting the release of anti-inflammatory mediators from the spleen while suppressing atrophy of the spleen, and reduces inflammation of the CNS by promoting the environment for nerve cell regeneration.M1 Microglia： injurious propertiesM2 Microglia： Protection© HEALIOS K.K. All rights reserved.(Source) Stroke. 2018 May;49(5):1058-106512HLCM051 Ischemic Stroke: Mechanism Involving the SpleenOffner et al. focused on the peripheral immune system (especially the spleen) and involvement of the peripheral immune system after acute central nervous system injury and stroke.Offner et al. (2009) found that peripheral immune organs, including the spleen, shrank by about 20 ~ 40% within 72 ~ 96 h of the onset of stroke in rodents.© HEALIOS K.K. All rights reserved.13(Source) Offner et al.（2009）(Source) Walker P., et al., Exp Neurology, 2010HLCM051 Ischemic Stroke: The Importance of Protecting the Penumbra Region in the Treatment of Cerebral InfarctionPerfusionDWIIschemic coreIschemicpenumbraThe penumbra is an area of the brain in the early stage after the onset of cerebral infarction in which blood flow is reduced and the brain is in a state of ischemia, but cells are not necrotized.(Source) Neurology Handbook Differential Diagnosis and Treatment (5th ed.)14© HEALIOS K.K. All rights reserved.HLCM051 Ischemic Stroke: Results of Double-blind Study Conducted by AthersysThe proportion of patients who achieved Excellent Outcome was statistically significant in the group of patients who received MultiStem within 36 hours of the onset of cerebral infarctionAnalysis of the Double-blind study conducted by AthersysOverview of the AnalysisMultiStem(n=31)16.1%p=0.020%Day 90Placebo(n=19)29.0%p=0.010%Day 365TrialThe placebo-controlled double-blind Phase 2 study conducted by Athersys in the US and the UK（MASTERS study)SubjectsAdministered MultiStem or Placebo within 36 hours of the onset of strokeEndpointProportion of subjects with an Excellent Outcome on Day 90 and Day 365* is defined as mRS score of ≤1 (scale, 0 to 6), NIHSS score of ≤1 (scale, 0 to 42), and BI score of ≥95 (scale, 0 to 100).(Source) This material was based on Lancet Neurol. 2017 May;16(5):360-368; 16 360–68 Supplementary appendix Table 5© HEALIOS K.K. All rights reserved.15HLCM051 Ischemic Stroke: TREASURE Study OverviewPlacebo-Controlled, Double-Blind, Phase 2/3 Efficacy and Safety Trial of HLCM051 (MultiStem®) in Patients With Ischemic StrokeTREASURE: TReatment Evaluation of Acute Stroke Using REgenerative cellsAcute ischemic stroke (within 36 hours)Estimated Enrollment: 220 subjectsMain Inclusion Criteria:✓ Clinical diagnosis ofcerebral cortical ischemic stroke✓ 20 years of age or older✓ A modified Rankin Scale (mRS) of 0 or 1 prior to the onset of ischemic allowedstroke© HEALIOS K.K. All rights reserved.✓ NIHSS 8 – 20 at baselineRandomized(１：１）✓ tPA or mechanical thrombectomy HLCM051(N=110)Placebo(N＝110)Primary Endpoint・EfficacyProportion of subjects with an excellent outcomedefined by functional assessment [Day 90 ]・SafetyComparison between the HLCM051 and placebo groups in key adverse eventsSecondary Endpoints (examples)・Proportion of subjects with an excellent outcomedefined by functional assessments [Day 365]・Proportion of subjects exhibiting functional outcome throughout the range of mRS scores by shift analysis[Days 90 and 365]16HLCM051 Ischemic Stroke: TREASURE Study Implementation System✓ 48 medical institutions across Japan participated in the TREASURE study✓ Subject enrollment period: November 2017 to March 202112312４６6４１© HEALIOS K.K. All rights reserved.17HLCM051 Stroke: Topline Results in May 2022 90 and 365-day top-line results of the TREASURE study planned for May 2022.Development PlanMarch 2022Last patient final hospital follow-up visit was completed.March 2021Last patient treatedNovember 2017First patient enrolledAugust 2021Patient enrollmentcompletedMay 2022 Top-line dataPatient enrolmentObservationData analysisPreparation for ApplicationApplicationApproval / LaunchNon-clinical / CMC package submittedConsulting with regulatory authoritiesThe approval period may be shortened from 12 months to 6 months by the SAKIGAKE Designation System© HEALIOS K.K. All rights reserved.18iPSC Regenerative MedicineeNK Cells & UDCCompanyHealios K. K.DateApril 5, 2022Executive Officer Research fieldKouichi Tamura, Ph.DProgram OverviewSomatic Stem CelliPSC PlatformInflammatory ConditionsMultistem®•Ischemic stroke • ARDSImmuno-OncologyReplacement TherapiesiPSC eNKUniversal Donor Cell (UDC)iPSC-derived, gene-engineered NK cells for: • Lung cancer• Liver cancer• UDC-pancreatic islets for diabetes• UDC-photoreceptors and RPE1 for retinal disease• Other non-disclosed• Liver buds1 for liver diseaseNear term revenue &Commercial capabilitiesInnovative best in class programs1Future migration to UDC platform© HEALIOS K.K. All rights reserved.20iPSC eNKImmuno-Oncology© HEALIOS K.K. All rights reserved.21The Promise of NK Cells as a Treatment for Solid TumorsKey Facts about Cancer and the Unmet NeedThe Potential for Natural Killer (NK) Cells • Solid tumors are the number one cause of death in • Offer tremendous promise as a new therapeutic Japan (~90% of cancer deaths)approach to treating solid tumors.• Cancer is the leading cause of death worldwide, accounting for nearly 10 million deaths in 20201• The economic impact of cancer is significant and increasing: The total annual economic cost of cancer in 2010 was estimated at US$ 1.16 trillion1•Innate, central role in a cell mediated defense system in humans, and attack cancer cells and virus-infected cells.• Reported advantages over T cell-based therapies:• Broad mechanism to recognize tumor cells• Fewer adverse effects (e.g. CRS & GVHD)• Less exhaustion1https://www.who.int/news-room/fact-sheets/detail/cancer© HEALIOS K.K. All rights reserved.22Healios’ Three-Pronged NK Cell Therapy ApproachA gene-engineered iPSC-NK cell platform designed not only with enhanced cytotoxicity but also with recruitment and trafficking properties Tumor cellDendritic cellsCytotoxic T-cellsRecruit and ActivateNK cell Recognize and AttackMigrate and Infiltratevessel© HEALIOS K.K. All rights reserved.23Key Characteristics of the Healios iPSC eNK Cell PlatformCD16; for enhanced ADCCReceptor for enhanced recognition and cytotoxicityMultiple Product CandidatesReceptor for improved migration into solid tumorsStand-alone eNK productCAR-eNK products against specific targets✓ CAR eNK cell therapy✓ Dual CAR eNK✓ Bispecific CAR eNKIn combination with antibody therapiesIL-15; for improved persistence and survivalChemokine release to recruit host immune cells © HEALIOS K.K. All rights reserved.24Core eNK Platform Technologies & CompetenciesEngineered iPSC LinesDifferentiation of NK Cells With Enhanced FunctionalityProcess Optimization, Scale Up & ManufacturingGCTP/GMP Manufacturing @ HEALIOS Facilityin Kobe, JapanNK differentiationProprietary, engineered iPSC lines✓ Enhanced cytotoxic activity✓ Enhanced proliferative potential and prolonged survival✓ Recruitment of patient immune cells✓ Migration and invasion of solid tumorsMaster cell banks established for NK cell productioneNK• Optimization of differentiation induction conditions• Confirmation of killing function, maintenance of proliferation and survival, migration and invasion• Ability to attract immune cells• Efficacy and safety in animal models• Quality standards strategyValidated CARs for Multiple Products…Product 1Product 2Product n© HEALIOS K.K. All rights reserved.25Cytotoxicity and Cytokine Production of eNK Against A549 (Lung Cancer)Cytotoxicity (LDH assay)Cytokine Production (co-cultured with A549))%(yticxoitotyC5040302010010,000/)Lmgp( γ-NFI8,0006,0004,0002,0000iNKeNKiNKeNKeNK: gene-edited and functionally enhanced NK cells derived from iPSCiNK: gene un-edited iPSC derived NK cellsEnhanced cytotoxicity and IFN-γ production were observed with eNK© HEALIOS K.K. All rights reserved.（Source）in-house data26Enforced Expression of Chemokine Receptor A for MigrationRecombinant chemokine AMeasure the relative migration as the ratio of the number of cells that migrated to the lower chamber with and without chemokine A.)i Aenkomehc on sv( sllec detiargmevit laerPeripheral blood NKiNKeNK© HEALIOS K.K. All rights reserved.（Source）in-house data27eNK efficiently migrates to chemokine ARecruitment of Immune Cells by Forced Expression of Chemokine B Recruitment AssessmentiPSC-NKChemokine B-expressed iPSC-NKMediumChamberHumanperipheral bloodMonocyte(CFSE )Semi-permeable membraneRecruitmentiPSC-NK orChemokine B-expressed iPSC-NK✓ Chemokine B is an attractant for T cells and dendritic cells expressing their receptors.✓ Chemokine B-expressing iPSC-NKs recruit human immune cells.eNK shows the potential to recruit T-cells and Dendritic cells.© HEALIOS K.K. All rights reserved.（Source）in-house data28Time Lapse Imaging of Cancer Cell Attack by Healios iPSC-NK cellsiNK killing lung cancer cells (A549)© HEALIOS K.K. All rights reserved.（Source）in-house data29Time Lapse Imaging of Cancer Spheroid Co-cultured with iPSC-NK Cells: Cytotoxicity and Destruction of the SpheroidFrom 24 hours until 85 hours after co-culture Green: apoptotic cellsiNKeNK© HEALIOS K.K. All rights reserved.eNK more rapidly killed cancer cells and destroyed the spheroid（Source）in-house data3030In Vitro Evidence of Anti-tumor Effect as Mono- and Combination Therapy (Lung, A549)0h86h (3.5 days) Green : apoptotic cellseNK onlyeNK with anti-EGFR antibodyAnti-EGFR antibody only© HEALIOS K.K. All rights reserved.eNK cells have killed the cancer cellsThe lung cancer cells were efficiently killed and the lung cancer cell spheroid was destroyed.The cancer cells survived and the cancer cell spheroid expanded for 86 hours.（Source）in-house data31Establishment of Orthotopic Lung Cancer Mouse ModelLung cancer cellsivNormal lungA549 transplanted lungTissue sectionNOG mouseTransplantedcell number1x104 cells1x105 cells1x106 cellsThe cancer nodules were observed in lung tissue（Source）in-house data32The cancer nodules were diffusely observed throughout the lung© HEALIOS K.K. All rights reserved.Anti-tumor Effect of eNK Cells in Tumor Bearing Mice (Lung)Luc-A549（wtEGFR）H1975-luc（EGFR-L858R）HCC827-luc（EGFR-Del19）Luc-A549（wtEGFR）H1975-luc（EGFR-L858R）HCC827-luc（EGFR-Del19）eNK can suppress the tumor growth (A549) or eliminate the tumor (H1975, HCC827)© HEALIOS K.K. All rights reserved.（Source）in-house data33Anti-tumor Effect of eNK Cells in Tumor Bearing Mice (Hepatocellular Carcinoma; HCC, sc)[Day]-40２ 4711142128▲: HepG2 Transplantation (sc)▬: IL-2/IL-15 (ip)△: Vehicle (it)▲: eNK cells (it)■: Blood collection,Tumor size measurementVehicle: HepG2 (sc)eNK (High dose): eNK cells (it)eNK (Middle dose)eNK (Low dose)Intra-tumor injection of eNK cells suppressedtumor growthControl (n=5)eNK細胞 (n=5)16001200800400]3mm[lemuov romuT0-70-4Days post injection of eNK cells [Days]2114728© HEALIOS K.K. All rights reserved.（Source）in-house data34eNK Cell Production Method: Upstream Process① Upstream Process: Preclinical scale 3D Perfusion Bioreactor System Mass production using 3L BioreactoriPSC Sphere FormationHPC DifferentiationInductionNK cellDifferentiationInductionNK cell ExpansionProduction of 100 billion NK cellsusing two 3L-bioreactorsPerfusion System BasedAutomatic Medium exchange500mL Single UseBioreactorWaste out Feed in 3L Single Use Bioreactor＊Illustration of Perfusion System (adopted from homepage of SATAKE MultiMix)iPSC SphereHPCNK cell© HEALIOS K.K. All rights reserved.rebmun lll eCebaVKNi]roitcaeroBLm0003 /sllec[1.E+111.E+101.E+095×1010cells0204060NK Expansion [days]Flow Cytometric Analysis of NK Cell94.4%CD5699.5%99.4%GZBPerforin（Source）in-house data35eNK Cell Production Method: Downstream Process② Downstream Process: Cell Harvest, Aseptic Filling and Finish, CryopreservationUpstream ProcessCell Harvest: Concentrate・WashCryoprotectant MixingAseptic Filling and FinishCryopreservationStorageCold chainCryoprotectant Design (Healios KK Original)Automatic Filling and Finish Machine (Healios KK Original)3L Single Use BioreactorClosed system: Automatic Cell Washing Concentration SystemComposition Optimization: Design of Experiments by AI AnalysisCRF Program OptimizationControlled Rate FreezerStorage: Liquid Nitrogen Tank LDH assay(A549)Cetuximab(-)Cetuximab(+)31.6925.3223.10iyticxootyCti)ssyl f o%(12.1750.040.030.020.010.00.0（Source）in-house dataPrimary NKHealios eNKIn this process, cryopreserved samples show high cytotoxicity36© HEALIOS K.K. All rights reserved.iPSC-Derived Product Manufacturing: A New and Dedicated GMP Facility To control the schedule and quality of clinical trial product manufacturing,Healios established a new facility for cell processing and manufacturing (CPC) in Kobe, Japan.Proprietary, automated 3D perfusion bioreactor system for eNK production3D manufactured eNKfinished product© HEALIOS K.K. All rights reserved.37Market Leading Range of Functional Enhancements HealiosA社Enhanced FunctionHLCN061(eNK)HLS CAReNKiPS cell①iPS cell②iPS cell③B社iPS cellAC社ーMigration into solid tumorRecruitment of host immune cellEnhancement of NK cell function and survivalCARAntibody Dependent Cell CytotoxicityClinical Stage✔✔✔✔–✔✔✔✔✔-✔✔✔-✔✔✔-✔✔?-✔✔?-✔P1HLCN061 (eNK) is expected to have an advantageous effect by enhancing tumor infiltration and immune cell recruitment through the introduction of chemokine receptor A and chemokine B© HEALIOS K.K. All rights reserved.（Source） Adapted by Healios from public information 38Summary: iPSC eNK Immuno-Oncology• Unique Approach: A gene engineered iPSC-NK cell platform designed not only with enhanced cytotoxicity but also with recruitment and trafficking properties•Initial Target Indications: Lung cancer, liver cancer, other non-disclosed• Promising in vitro and in vivo evidence• Robust and advanced manufacturing processes and infrastructure in place• Multiple strong collaborations• Near-term regulatory milestones: Pre-IND: 2022, IND: 2024© HEALIOS K.K. All rights reserved.39Universal Donor Cell (UDC)Replacement Therapies© HEALIOS K.K. All rights reserved.40Hypo-immune Universal Donor Cell (UDC) PlatformWorld-leading engineered “universal” iPSC platform: “UDC”Allogeneic iPS Cell LinePatientImmune responseAllogeneic hiPS cellsRequires additional immunosuppressive drug••Heavy patient burdenShort efficacy durationPatientReduce immune responseAllogeneic hiPS cellsGene-edited iPS cell lineHealios Universal Donor Cell LineReduce or eliminate immunosuppressive drug requirementReduce patient burdenIncrease efficacy duration••Targeted cell programming through gene-editing・In October 2020, Healios established a clinical grade universal donor IPS cell line that can be clinically applied to humans in each of Japan, the United States and Europe.・ Master Cell Bank established in 2021・Healios has led the development of high-quality, universal donor iPS cells in accordance with global standards.・Consultations with the FDA and PMDA led to no concerns inrelation to clinical use of UDC derived therapeutics.・The UDC line differentiates readily into various in-house madecells (e.g. NK cells, liver progenitor cells, vascular endothelial cells, etc.).・Active discussions with several companies and academic institutions in relation to use with various therapeutic candidates.© HEALIOS K.K. All rights reserved.41Hypo-immune UDC: Engineered Genetic ProfileGene Editing Procedure for Healios UDCHLA-G• Off-the-shelf, scalable and cost-efficient• Address broadest population with single product• Enhanced level and duration of efficacyClinical grade line and Master Cell Bank established in 2020/2021© HEALIOS K.K. All rights reserved.（Source）in-house data42Removal of Polymorphic HLAs and Addition of Immunosuppressive GenesResults of gene editing in clinical grade UDCHLA protein knock-outImmunosuppression-related molecules knock-inHLA-AHLA-BHLA-CHLA-DR,DP,DQHLA-GPD-L1PD-L2Parent-iPSCClinical grade UDClessExpressionHigh© HEALIOS K.K. All rights reserved.Post-gene editing disappearance of HLA proteins and enhanced expression of immunosuppression-related genes■Control■Target protein antibody（Source）in-house data43UDC Production Process Checklist①Confirmation of gene editing②Absence of malignant mutations③ Retention of iPS cell propertiesself-renewalQuality check itemConfirmation of gene editingExpression level of HLA proteinsTransgene expressionGene mutationAttributionpluripotencyContentsConfirmation of target region sequenceLoss of HLA Class I expressionLoss of HLA Class Ⅱ expressionExpression of immune suppression associated moleculesExpression of suicide genesNo off target issuesNormal karyotypeNo cancer associated genesSterilityEndotoxin freeMycoplasma freeGene expression analyses (Comparison with the parent cell line)Expression of undifferentiated markersPluripotency (three germ layer differentiation）Absence of immunogenicityFunction of suicide genes© HEALIOS K.K. All rights reserved.（Source）in-house data44Confirmation of Hypo-immunity CD4+ T cell activationE/T= 10:1CD8+ T cell activationE/T= 10:1)%( noitlaupop llec Tde tavitcA1614121086420)%( noitlaupop llec Tde tavitcA)%( yticxoitotyc98765432102520151050T cell only+THP-1+parental+class I/II DKO +clinical gradeT cell only+THP-1+parental+class I/II DKO +clinical gradeUDCUDCNK cell cytotoxicityE/T= 5:1Clinical grade UDC did not show any activation of T or NK cells parentalclass I/II DKOclinical grade cloneclinical grade UDC© HEALIOS K.K. All rights reserved.（Source）in-house data45Evaluation of UDC Inducible Suicide Gene (In Vitro)Mechanism of ActionAdministration of a drug induces dimerizationSuicide geneDimerizationInduction of Cell DeathCulture of UDCsCell viability (ATP assay)Administration of DrugReduction in Viability1.210.80.60.40.20)latot/evil( oitarDeath of UDCs© HEALIOS K.K. All rights reserved.00.0030.010.03Inducer of dimerization (nM)0.10.31（Source）in-house data46Suicide Gene Function In VivoUDCTransplantation at Day 0Administration of drugbetween Day 18-42Death of transplanted cellsTermination at Day 71✓ Gross examinations✓ Mass weight✓ Histopathological examinations of massImmunodeficient mouse)%( amotaret fi onossergeR© HEALIOS K.K. All rights reserved.DrugDrugBefore Gene edit iPSCUDC（Source）in-house data47Hypo-immune UDC: Examples Of Cell TherapiesUniversal Donor Cells (UDC)Pancreatic β cells Photoreceptor cellsRPE cellsLiver budsSuccessfully differentiated from UDCsFuture migration to UDC platform© HEALIOS K.K. All rights reserved.（Source）in-house data and Joint research data48Conclusion• Continued pioneering in cell therapy• Uniquely positioned to leverage strong Japanese proficiencies• Substantial infrastructure to support multiple programs across development stages• Global strategy• Building a commercial organization to launch MultiStem® for ARDS & stroke in Japan • Accelerating innovative iPSC platform development for immuno-oncology & cell replacement therapies• Focus on clinical development of engineered-NK (eNK) cells for solid tumors in Japan and US• Advancement of therapies derived from proprietary hypo-immune Universal Donor Cell (UDC) line • Continued investment in precision manufacturing capabilities and strengths in Japan to support future global supplyCommitted to transforming the lives of patients by creating, developing and commercializing cutting edge cell therapy technologies© HEALIOS K.K. All rights reserved.49＜Contact information＞Corporate CommunicationsHEALIOS K.K.Press contact: pr@healios.jpInvestor contact: ir@healios.jphttps://www.healios.co.jp/contact/