ALETA-001, a CAR-T Engager protein, or CTE, optimizes anti-CD19 CAR-T celltherapies for lymphoma

ALETA-001, a CAR-T Engager protein, or CTE, optimizes anti-CD19 CAR-T cell therapies for lymphoma Paul D. Rennert, LiheSu, Lan Wu, Roy R. Lobb and Christine Ambrose, Aleta Biotherapeutics, Natick, MA. USA Introduction ChimericAntigenReceptorTcellstargetingmalignanciesexpressingCD19 (CAR19)havebeenwidelysuccessful,withproductsapprovedtotreatBcell lymphomas(NHL)andBcellleukemia.AmajorlimitationofCAR19therapyis thesteeprelapseratewithin6monthsoftreatment,oftenduetothelossor diminutionoftumorcellCD19expression.ALETA-001isaCAR-TEngager protein(CTE)thatcontainstheCD19extracellulardomain(ECD),ananti-CD20 VHHtotargetthissecondantigen,andananti-albuminVHHforhalf-life extension.WhencombinedwithCAR19Tcells,ALETA-001triggerscytotoxicity throughCD19boundtoCD20,thusincreasingtotaltargetantigendensityand preventingrelapseduetolossofCD19expression.ALETA-001willenterCancer ResearchUK-sponsoredPhase1/2clinicaltrialsinCAR19-treatedNHLpatients laterthisyear. ALETA-001 ALETA-001isamultifunctionalbiologicforinjectionthatcontains ananti-CD20llamaVHHlinkedtoanoptimizedCD19proteinand furtherlinkedtoananti-albuminllamaVHH. Figure1.DesignoftheALETA-001CAR-TEngagerprotein. •ALETA-001bindstoCD20onBcelltumorcells,displaysCD19onthetumor cellsurfacetherebyactivatinganti-CD19CARTcells,andbindstoalbumin, providingforalonghalf-lifeuponinjection. •Aleta-001increasesCD19antigendensityand/orreplaceslostCD19 expressionbycoatingcellsurfaceCD20withCD19. •BindstoanyCAR19:Tcell(autologous,allogeneic),NK,γδ,iPSC,orother. •55KDamonomerutilizesstandardbiologicproductionandcanbedosed“off- the-shelf”. •HerewepresentresultsfromALETA-001preclinicalwork thatfurtherelucidatethemechanismsofaction. Figure 6. In vivo CD19-negative lymphoma model anti-albumin VHH CD19-ECDN C anti-CD20 VHH Binds to CD20 -low nMbinding affinity EC50 -sub-nMcytotoxicity IC50 Binds to albumin ~ 12-day T1/2in human ~ 2 days in mouse Binds to anti-CD19 CAR -low nMbinding affinity EC50 Disclosure: all authors are employees and/or stockholders of Aleta Biotherapeutics Inc. no luminescent signal measured -6/8 mice-8/8 mice ALETA-001eliminatesCD19-negativelymphomainCAR-19treated animals:CD19-deficientJeKo-1MantleCellLymphomacells(MCL)were implantedintoNSGmiceandallowedtoestablishfor4daysbeforeCAR-19Tcells andresearch-gradeALETA-001proteinwasinjected.TheCARsweregivenonce, andtheproteindosed3xweeklyuntilday31,atwhichpointdosingstoppedand animalsweremonitoredforcancerrecurrence. Figure5a. TheseresultssupportourclinicalhypothesisthatALETA-001will improvetheeffectivenessofexistinganti-CD19CAR-Tcell therapeutics.ThePhase1/2supportedbyCancerResearchUKwill testthishypothesisinpatientsreceivingCAR-19swhofailtoreacha completeresponseatmonth1orrelapsefromaCRbymonth6.The trialwillbegininthe1H’23. IMAGES DAY Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 untreated CAR19 only CAR19 plus 2.0 mg/kg 606 CAR19 plus 0.4 mg/kg 606 CAR19 plus 0.08 mg/kg 606 CAR19 plus 0.016 mg/kg 606 last ALETA- 001 dose, day 31 no recurrence observed through day 42 Figure 2. Incubation with ALETA-001 binds the CD19 ECD onto CD20, and this increases the amount of detectable CD19 by FACs analysis. Left: JeKo-1, Mantle Cell Lymphoma. Right: Ramos, Hodgkin Lymphoma MOA –1. ALETA-001 increases apparent CD19 density on CD19- positive lymphoma cells. ALETA-001bindingincreasesthedetectableCD19onJeKo-1cellsfrom ~25,000moleculestomorethan200,000molecules,anamountequivalentto thesumofCD19expressed(orangebar)+CD20expressed.Similarly, ALETA-001bindingincreasesthedetectableCD19onRamoscellsfrom ~75,000tonearly250,000,closelyresemblingthesumofCD19(orange)+ CD20(red). Figure 4. Incubation with ALETA-001 increases CAR-19 cytotoxicity against wildtype JeKo-1 lymphoma cells, with more rapid killing at lower E:T ratios measured at 18 hours. Figure 2. Incubation with ALETA-001 replaces CD19 lost from JeKo-1- CD19 Knockout cells at a level higher than native CD19 expression and matching CD20 expression. MOA–2.ALETA-001replacesCD19thatisotherwiselostfrom JeKo-1-CD19knockoutcells,whileexceedingnormalCD19 expression ALETA-001bindingincreasesthedetectableCD19onJeKo-1-CD19 knockoutcellsfrom~0moleculestomorethan250,000molecules,anamount equivalenttoCD20expression(redbar) Figure 3. Incubation with ALETA-001 mediates CAR-19 cytotoxicity against JeKo-CD19-knockout cells, at pg/ml concentrations. Cytotoxicity IC50 mean (ng/ml), sd(n) ALETA- 001 0.13, 0.09 (n=11) ALETA-001 induces CAR-19 T cell cytotoxicity against CD19-negative JeKo-1 cells with an IC50 = 130 pg/ml. UsingE:Tratiosof0.3:1to3:1weobservedmorerapidlymphomacellkilling at18hours,indicatingthatCAR-Tactivationandfunctionalitywereenhanced bythepresenceofALETA-001.Theeffectwasdiscernablewithaslittleas1 ng/mlALETA-001atE:Tratiosof3:1and1:1andwasrobustwith10ng/ml ALETA-001atalltestedE:Tratios.By48hoursallincubationconditionshad “caughtup”andtheeffectwasobscured. MOA–3.ALETA-001increasestheeffectivenessoftheCAR-T cellpoolandsupportsmoreeffectivekillingofwildtypeJeKo-1 cellsthatexpressbothCD19andCD20. MostpatientdiffuselargeBcelllymphomacells(DLBCL) expressmuchhigherlevelsofCD20thanCD19 from N.A.Johnsonet al. 2009. Blood. doi:10.1182/blood-2008-09-177469 Figure6.RescueattemptsforCAR19-relapsedpatientsyieldpooroutcomes CD19CD20 Animalswereuntreated,givenCAR-19alone,orgivenCAR-19+increasing amountsofALETA-001,dosedIVonthehalf-life(thus,every3days).Imaging wasperformedthroughday42posttreatmentinitiation(4daysafterCD19-negative lymphomainfusion).Inthehighestdosegroupsof2mg/kgtherewerenorelapses (8/8cured)andinthenextdosegroup(0.4mg/kg)6of8animalswerecured. #175 ALETA-001 mediates potent cytotoxicity Most patients are CD20-bright, CD19-medium The expression levels differ by up to a log French consortium data US multi-center data

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