Umbilical Cord Blood derived Natural Killer Cells Provide a Highly Scalable Source For Gene Circuit-Engineered Allogeneic CAR-NK Therapies

AllogeneicCARnaturalkiller(NK)cell-basedadoptivecelltherapyhas shownpromiseinrecentyearsfortreatingcancerpatients.Theunique biologicalpropertiesofUmbilicalCordBlood(UCB)cellsalongwith encouragingresultsfromrecentUCB-derivedNKcell-basedtherapies1make UCBanattractivesourceforobtaininglargenumbersof‘off-the-shelf’NK cells.Topotentiallybroadentargettumortypesandfurtherenhanceanti- tumorefficacy,SentiBiousesitssyntheticbiologyplatformtoprogram ‘genecircuits’towardengineeringoptimalefficacy,precisionandcontrol intomanycellorgene-basedmedicines,includingCAR-NKcells.Here,we studiedthetransductionefficiency,scalabilityandcryopreservationofUCB- derivedNKcellscomparedtoadultperipheralbloodmononuclearcell (PBMC)-derivedNKcellsinthecontextofgenecircuit-engineeredCAR-NK therapy. Senti’sAllogeneic NK Cell Process is Designed to Enable Scalable Manufacturing for Off-The-Shelf NK Cell Therapies METHODS ABSTRACT Cryopreserved NK cells Retain Expandability and Gene Expression Post-Thaw Umbilical Cord Blood derived Natural Killer (UCB-NK) Cells Provide a Highly Scalable Source For Gene Circuit-Engineered Allogeneic CAR-NK Therapies Senti Biosciences, Inc. South San Francisco, CA Dharini Iyer, Wesley Gorman, Carmina Blanco, Travis Wood, Mario Lorente, Charity Vilchez Juang, Denny Nguyen, Brandon Lee, Brett Kiedaisch,Philip Lee ISCT 2021 Abstract# 410 Engineering Smarter Medicines OBJECTIVES •ToevaluateUCB-NKcellsasapotentialcellsourceforgene-circuit engineeredCAR-NKtherapy. •Tocomparetheexpandability,transductionefficiencyandfunctionality ofUCB-NKcellsandPBMC-NKcells. •NKcellswereisolatedfromUCBunitsandperipheralblood leukapheresisfollowingCD3depletionandCD56selection. •IsolatedNKcellswereexpandedonirradiatedgene-modifiedfeedercells andtransducedwithGFP-andCAR-expressingconstructs. •Gene-modifiedNKcellswerecryopreservedpost-transductionand furtherexpandedpost-thaw. Enrichment of NK cells from Adult Peripheral Blood and Umbilical Cord Blood Units We Believe Transduction Enables Robust Expression of Gene Circuits Robust Expansion of NK cells using GMP-compatible G-Rex Vessels Cryopreserved NK cells Retain Expandability and Gene Expression Post-Thaw Process Supports Consistent Expansion, Activation and Stable CAR Expression in NK cells from Multiple Donors SUMMARY REFERENCES Chimericantigenreceptor(CAR)isefficientlytransduced intoPBMC-NKcellsandUCB-NKcellsusingaretroviral vector Transducedcellsretain>90%viabilityandsimilargrowth ratetounmodifiedNKsthroughthe21-dayprocess CARexpressionisstableduringexpansionandfollowing freeze/thaw CARexpressionandfoldexpansionofgene-modified UCB-NKcellsiscomparabletoPBMC-NKcells Multipleenrichmentsfromhealthydonorleukapheresisandcordblood unitsyield>95%NKpurityand<1%Tcells Purifiedcellsfrombothcellsourcesarecryopreservedandthawedwithout lossofviability PBMC-NKcellsandUCB-NKcellsgrowrobustlyonengineeredfeedercells PBMC-NKcellsandUCB-NKcellsdisplayedsimilarfoldexpansionand growthrateoverthe21-daycultureperiod.UCB-NKcellsexpandedbetter (4x)overt+20daysincomparisontoPBMC-NKcells CAR-modifiedPBMC-NKcellsandUCB-NKcellsshowspecificandpotentcytotoxic activityuponco-activationwithMOLM13cancercellsaswellaselevatedcytokine markerspostactivation. PBMC-NKcellsandUCB-NKcellstransducedwithCARconstructsexhibitedstableCAR expression,consistentgrowthkineticsandviabilityoverfreeze/thaw.NKsfrombothcell sourcesshowedsimilarexpandabilityandgrowthprofilepre-freezeandpost-thaw. PBMC-NKcellsandUCB-NKcellsfrommultipledonors(n=3)exhibitstable andconsistentCARexpression,expansion(TCplate)andgrowthrateacross multiplepassagesduringthe21-daycultureperiod. G-Rexvesselshaveagas permeablesurfaceandalarge liquidvolumethatsupports expansionofsuspensioncells withminimalmediaexchange G-Rexvesselssupportrobust expansionofPBMC-NKcellsand UCB-NKcells BetterexpansionofUCB-NK (p<0.05)cellsseeninG-Rex vesselscomparedtoPBMC-NK cells Achieved highly efficient retroviral vector mediated transduction of allogeneic UCB-derived NK cells through transduction optimization studies Process yielded consistent growth rate of gene modified CAR-NK cells and stable transgene expression between freeze/thaw Product attributes were comparable between PBMC-NK cells and UCB-NK cells and across multiple donors Gene-modified CAR-NK cells show potent anti-tumor activity against target cancer cells in an in vitro cell-based cytotoxicity assay Large scale expansion of UCB-NK cells and PBMC-NK cells provide a scalable source for gene circuit-engineered allogeneic CAR-NK therapies Process Flow CD56 CD3 PBMC-NKs UCB-NKs 1.LiuE,MarinD,BanerjeeP,etal.UseofCAR-transducednaturalkillercellsinCD19-positivelymphoid tumors.NEnglJMed.2020;382(6);545-553.doi:10.1056/NEJMoa1910607 Contact: [email protected] NK Cell Purity NK Cell Viability CAR-NK cell killing of MOLM13 cancer cells NK Cell Expansion CAR Expression Population Doubling Pre- Transduction Post- Transduction Post-Thaw Fold Expansion * Isolated NK cells Transduction Expansion Cryopreservation Isolated NK cells Transduction Expansion Cryopreservation Isolated NK cells Transduction Expansion Cryopreservation Isolated NK cells Transduction Expansion Cryopreservation Isolated NK cells Transduction Expansion NK function (Pre-freeze) 97.8% 99.9% NK Cell Viability 1 10 10 0 10 00 10 000 0 7 10 17 21 Unmodified NK Cells Transduced CAR-NK Cells <1% <1% Expansion of CAR-NK Cells Days in Culture 107 106 105 104 103 102 101 100 PeripheralBlood 107 106 105 104 103 102 101 100 Umbilical Cord 3.0M2.0M1.0M0 39% 45% 107 106 105 104 103 102 101 100 107 106 105 104 103 102 101 100 3.0M2.0M1.0M0 2.0M1.5M1.0M500K02.0M1.5M1.0M500K0 MFI MFI Fold Expansion 1 10 10 0 10 00 10 000 0 7 10 17 21 Transduction Transduction Fold Expansion Unengineered Transduced CAR-NK Cells Forward Scatter (FSC) CAR Lentivi rus + Purified NK cells Viral Vector CAR-NK cells High purity and functional NK cells isolated from peripheral blood Initial cell pack Purified NK cells Vectors optimized for NK cell transduction Proprietary expansion and cryopreservation process for >100 doses per batch and high post-thaw potency “Off-the-shelf” infusion at clinical site Healthy donor 1. Starting Cells 4. Cryopreserved Drug Product 2. Gene Modification 3. Expansion 4. Cryopreservation Patient Dosing Patient PBMC-NKUCB-NK 0 20 40 60 80 100NK Cell Viability Pre-Freeze Post-Thaw Process Designed to Enable Scalable Manufacturing for Off-The-Shelf CAR-NK Cell Therapies

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