First-In-Human, Multicenter Study of SENTI-202, a CD33/FLT3 Selective Off-the-Shelf Logic Gated CAR NK Cell Therapy in Hematologic Malignancies including AML: Correlative Data

Subject 1 1Bx3 Subject 2 1Bx3 Subject 4 1.5Bx3 (Prelim. RP2D) Subject 5 1.5Bx3 (Prelim. RP2D) Subject 7 1Bx5 Subject 6 1.5Bx3 (Prelim. RP2D) Subject 8 1Bx5 Subject 9 1Bx5 Subject 3 1Bx3 Best Response CR MRD- CR MRD- CR MRD- CRh MRD- MLFS SD TBD- continuing Rx after SD / Cycle 1 TBD- continuing Rx after SD / Cycle 1 PD Mutations & cytogenetics at diagnosis ASXL1, ETV6, NRAS FLT3-ITD, TET2 IDH2mut, SRSF2, PSU DIC (2;4)(q31;q21)X2,t(13,21)(q 32;q22)X2/46,XY NRAS, TET2, SRSF2, CEBPA, ASXL1, RUNX1 SF3B1, NRAS, KRAS, GATA2, t(I;16)(q21,q22) RPN1:MECOM, paracentric inversion of chromosome 3, GATA2, NRAS, WT1 RUNX1, NF1, 46,XY,t(4;17)(q31;q11.2) TET2, FLT3-ITD, SRSF2, KRAS, ASXL1, RUNX1, SETBP1 loss of 3q, 5q, 7q; DNMT3A, KRAS, TP53 Mutations & cytogenetics at study entry WT1, ASXL1, EGFR, ETV6, NF1, PFH6, gain of NUP214 (9q34), gain of ABL1 (9q34), gain of CDKN2A (9p21), trisomy 9, TRA/D rearrangement TET2 IDH2mut, SRSF2, add(2)(q33)X2, add(7)(p15)X2, add(13)(q32)X2, +mar/46,XY, tri/tetra/polisomy SH2B3, SRSF2, CEBPA, TET2, ASXL1, RUNX1 GATA2, SF3B1, inv(3)(q21q26.2), der(1;16)(q10;p10), 46,XY,inv(3)(q21q26.2), GATA2, NRAS, WT1 RUNX1, IKZF1, 46,XY,t(4;17)(q31;q11.2) ASXL1, CBL, FLT3, RUNX1, SETBP1, SRSF2, TET2, del(7)(p14p12)/46 CBL, DNMT3A, KRAS, TP53, loss of 5q, 7q. Cell populations: Baseline CD33 (baseline, disease outlined in red) %CD33+ Site: 40% CyTOF: 96.6% Site: 98.8% CyTOF: 99.1% Site: Dim/Partial CyTOF: 67.1% Site: Positive CyTOF: 60.1% Site: Positive CyTOF: 62.3% Site: 88.7% CyTOF: 28.1% Site: 98.5% CyTOF: 72.9% Site: 78.5% CyTOF: 73.1% Site: Positive CyTOF: 97.1% FLT3 (baseline, disease outlined in red) %FLT3+ CyTOF: 23.8% CyTOF: 45.6% CyTOF: 5.0% CyTOF: 0.8% CyTOF: 0.4% CyTOF: 2.7% CyTOF: 2.1% CyTOF: 1.9% CyTOF: 0.1% Responses After SENTI-202 Treatment Cell populations: Best Response (end of Cycle 1 for subjects 8 & 9) ABSTRACT CT143 AACR2025 First-In-Human, Multicenter Study of SENTI-202, a CD33/FLT3 Selective Off-the-Shelf Logic Gated CAR NK Cell Therapy in Hematologic Malignancies including AML: Correlative Data Muharrem Muftuoglu1*, Enping Hong2*, StephenA.Strickland3,AlirezaEghtedar4,Gary Schiller5, NoshaFarhadfar6,AshishR.Bajel7,FarhadRavandi1,Mahesh Basyal1, Li Li1, Lawrence Naitmazi2, RochelleEmery2, BrianS. Garrison2, Timothy Lu2, KanyaRajangam2,Michael Andreeff1 * Denotes co-authorship. 1The University of Texas M.D. Anderson Cancer Center, Houston, TX, 2Senti Biosciences, Inc, South San Francisco, CA, 3SCRI at TriStar Centennial, Nashville, TN, 4Colorado Blood Cancer Institute, Denver, CO, 5David Geffen School of Medicine at UCLA, Los Angeles, CA, 6Sarah Cannon Transplant and Cellular Therapy Program at Methodist Hospital San Antonio, TX, 7Peter MacCallum Cancer Centre, Melbourne, Australia BACKGROUND BASELINE BONE MARROW ANALYSIS SENTIBIOSCIENCES,CONTACT email:[email protected] HSC RETENTION AND IMMUNE REPOPULATION IN cCR SUBJECTS ACKNOWLEDGEMENTS The authors would like to thank all patients, their families, and caregivers for their participation in this study, as well as clinical and research staff at all participating institutions. Pharmacokinetics assessment was performed by Charles River Laboratories, and pharmacometrics support was provided by Certara. PresentedatTheAACRAnnualMeeting2025,April25-30,Chicago,IL BLAST & LSC REDUCTION IN cCR BONE MARROW HSPC-Driven Immune Repopulation in Peripheral Blood after SENTI-202 Treatment AML blasts Monocytic Erythroid HSC/HSPCs CD4 T cells CD8 T cells NK cells B cells Immune Repopulation by Cell Type Solid lines: Cycle 1 Dashed lines: Cycle 2 Analysis of immune populations in peripheral blood was performed by multicolor flow cytometry. (a) Stacked bar plots showing the concentration of cells measured in blood at the beginning (pre-dose 1) and end (Day 28 / EOT) of each treatment cycle. Subjects that achieved cCR showed robust immune recovery by the end of each cycle, indicating normal hematopoiesis. Recoveries of (b) granulocytes, (c) T cells, (d) NK cells, and (e) B cells are shown over each treatment cycle are shown for all subjects. EOT, end of treatment. a b c d e AML Hematopoietic Monocytes Erythroid CD4 CD8 NK B cells ExpressionLow High Preliminary RP2D cCR TBD PD ORR SD * * * Preliminary RP2D cohort Schedule I Schedule II Schedule I Dose Level 1 Dose Level 2 Legend Subject 1 (CR MRD-) Subject 2 (CR MRD-) Subject 3 (PD) Subject 4 (CR MRD-) Subject 5 (CRh MRD-) Subject 6 (SD) Subject 7 (MLFS) Subject 8 (TBD) Subject 9 (TBD) SENTI-202 is being evaluated after lymphodepletion in patients with R/R AML in an ongoing Phase I clinical trial, SENTI-202- 101 (NCT06325748). SENTI-202 is a Logic Gated CAR NK cell therapy designed to target CD33 and/or FLT3 positive hematologic malignancies including acute myeloid leukemia (AML). To achieve deeper & longer remissions, SENTI-202 was designed to also target leukemic stem cells (LSCs), which are often FLT3+ (+/-CD33+), while enabling long-term multilineage hematopoiesis by maintaining a healthy HSPC population. The SENTI-202 Logic Gated Gene Circuit therefore includes an inhibitory CAR recognizing EMCN (expressed by healthy HSPCs, rarely by AML) to protect HSPCs from potential SENTI-202 mediated off-tumor on-target toxicity even if they express CD33 and/or FLT3 & enable post-treatment hematopoietic recovery. Here, we report preliminary correlative data evaluating hematopoietic cell fractions in peripheral blood (PB) & bone marrow (BM) from 9 treated relapsed/refractory (R/R) AML patients with available data from the ongoing study (April 9th, 2025). Efficacy and clinical results were presented at AACR abstract # CT014 (oral minisymposium # CTMS01). SUBJECT BASELINE AML MARKER EXPRESSION aCAR: activating chimeric antigen receptor; EMCN:endomucin;HSC:hematopoieticstemcell; HSPC: hematopoietic stem and progenitor cell; iCAR: inhibitory chimeric antigen receptor;LSC:leukemicstemcell. DLT:doselimitingtoxicity;MRD:measurableresidualdisease;R/R:relapsedrefractory;RP2D:recommendedphase2dose 1NCT06325748;21-2priorforMDS;3Otherdoselevelsandschedulesmaybeevaluatedbasedonstudydata; 4SubjectsinMRDnegativecompleteremissionmay receiveoneadditionalcycleasconsolidation. SENTI-202 Pharmacokinetics: SENTI-202 is Detected in All Treated Patients Pharmacokinetic (PK) analysis of SENTI-202 in trial subjects. SENTI-202 was detected in all treated subjects, with data shown from (a) Cycle 1 and (b) Cycle 2. SENTI-202 PK profile was consistent with allogeneic NK cell therapy with cells detected in the first 2 weeks in periphery followed by clearance thereafter. (c) PK parameters were analyzed and showed no significant differences in exposure between Schedule I dose levels 1 and 2, or cycles 1 and 2. Estimated LLOQ assumes equivalent DNA loading for each reaction. Note: Subject 1 Cycle 1 samples were processed out of stability window and are excluded from analysis of PK parameters. SENTI-202-101 Dosing and Correlative Sampling Timepoints Bone marrow aspirate collections (CyTOF) Lymphodepletion Whole blood collections (PK and flow cytometry) D0 D28D14 D21D7D3 D10Baseline Whole blood samples are collected at pre-dose and 0.5h/8h/24h after each dose of SENTI-202. For Schedule I, single blood samples are additionally collected at D3 and D10. Dose 1 Dose 2 Dose 3 Dose 4 Dose 5 Dose 1 Dose 3 Dose 3 Schedule I: 3 doses of SENTI-202 (1Bx3, 1.5Bx3) Schedule II: 5 doses of SENTI-202 (1Bx5) SENTI-202 Transgene Copies/µg DNA Cycle 1 Cycle 2 Methods •Whole blood and bone marrow aspirate are collected into sodium heparin tubes, then processed to cryopreserved PBMC/BMMC by Ficoll separation. •Pharmacokinetics was determined by detection of the SENTI-202 transgene in PBMC samples by droplet digital PCR. •BMMC samples were analyzed by a 49-marker mass cytometry (CyTOF) panel. •Additional whole blood samples were collected into Cyto-Chex BCT tubes and analyzed by flow cytometry. PK Parameters Median (range) given for all Dose Level 1 1B/CAR+ NK dose) Dose Level 2 (1.5B CAR+ NK/dose) All Subjects N=9Schedule I (3 doses) N=3 Schedule II (5 doses) N=3 Schedule I (3 doses) N=3 (Preliminary RP2D) Cmax (Copies/µg DNA) 2392 (1567 - 2496) 1358 (380 - 8212) 1739 (676 - 3388) 1878 (380 - 8212) Tmax (Days) 5 (0 - 7) 7 (3 - 14) 3 (0 – 7) 3 (0 - 14) AUCobs (Days*Copies/µg DNA) 6873 (3160 - 11074) 1554 (720 - 29067) 3532 (2133 - 12395) 3593 (720 - 29067) Peripheral clearance (Days) 11 (7 - 15) 14 (14 - 15) 13 (7 – 14) 14 (7 - 15) a b c • CD33, CLL1, and CD123 are expressed highly in all subjects who achieved cCR. • 3 of 4 subjects who achieved cCR expressed FLT3; whereas both the SD and the no-response subject had < 3% FLT3 expression. Note: Subject 3 had a more stem/progenitor-like AML, expressing stem-associated markers (CD34+, CD90+, EMCN+) and mutant TP53, that did not respond to treatment. Subject 3 AML was also 43% EMCN+ (EMCN is rarely expressed in AML). Subject AML heterogeneity The SENTI-202-101 clinical trial enrolled subjects with heterogenous AML profiles. (a) UMAP analysis of AML populations in bone marrow samples at baseline show diverse proteomic features that uniquely identify samples from each subject. (b) Heat map analysis of AML populations in bone marrow samples show the prevalence of AML-associated markers and their heterogeneity of expression across study subjects. cCR, composite complete response; PD, progressive disease / no response; SD, stable disease; TBD, to be determined. a b Log10(median) LSCs at baseline are largely non-cycling and not expected to be eliminated by lymphodepleting chemotherapy. a b c d Best Response %CD33+ %FLT3+ %CD33+ FLT3+ Subject 4 cCR 93.2 82.1 76.8 Subject 5 cCR 81.9 6.6 5.4 Subject 6 SD 72.7 40.5 30.7 Subject 1 cCR 98.6 89.5 88.6 Subject 2 cCR 95.4 62.9 62.6 Subject 3 PD 99.5 27.8 27.8 Subject 7 MLFS 61.3 39.0 24.1 Subject 8 TBD 97.6 69.5 68.0 Subject 9 TBD 70.4 23.2 18.4 CD33 & FLT3 expression in LSCs Preliminary RP2D Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Subject 7 Subject 8 Subject 9 LSC (CD34+ CD38low) LSC (GPR56+)AML blast reduction % CD34+ CD38low LSC of AML cells BaselineC1D28 C2D28 100 10 1 0.1 0.01 0 % GPR56+ LSC of AML cells 100 10 1 0.1 0.01 0 BaselineC1D28 C2D28Baseline Best Response* Total Bone Marrow Blasts (%) 100 80 60 40 20 0 e f Analysis of leukemic stem cells (LSCs) in trial subjects. (a) Schematic of clonal repopulation of LSCs after treatment with conventional anti-AML chemotherapy. (b) Cell cycle analysis of CD34+CD38low LSCs, defined by Ki67 and IdU: Ki67- = G0, Ki67+ IdU- = G1, IdU+ = S. Cell cycle analysis determined that most LSCs in treated subjects were quiescent at baseline, and therefore not expected to be eliminated by lymphodepleting chemotherapy such as fludarabine/ cytarabine. (c) LSCs identified in subject bone marrow were shown to express CD33 and FLT3, and can therefore be targeted by SENTI-202. (d) AML blast reduction (measured locally by flow cytometry) was noted in a majority of subjects across all dosing cohorts. LSCs in responders were identified by two different methods: (e) CD34+CD38low and (f) GPR56+, and their proportions in subject bone marrow were shown to decrease at least 10- fold after SENTI-202 treatment. * Subject 5 samples in (e) and (f) had low viability. G0 G1 S phase Time (days) Time (days) SUMMARY cCR TBD PD ORR SD •AML blast reduction was observed in a majority of subjects. 5/7 subjects evaluable for best overall response had blast reduction by CyTOF consistent with clinical response of CR or CRh (composite CR or cCR) or MLFS. 4/4 cCR patients were MRD- as assessed locally, 2/3 in preliminary RP2D cohort. •SENTI-202 was detected in all treated subjects, with comparable PK to other CAR NK cell therapies. •AML in subjects enrolled on the SENTI-202-101 clinical trial was proteomically and mutationally heterogenous, with a unique genetic and proteomic signature for each subject. All subjects expressed CD33 (range 28-99%), and 3/4 cCR subjects expressed FLT3 (range 5-46%). Most LSCs were in G0/G1 at baseline and were not expected to be eliminated by Flu/Ara-C. •In subjects achieving cCR: •A >10-fold decrease in LSCs was observed. •HSPCs were either increased or maintained after SENTI-202 treatment. EMCN+ HSPCs were detectable throughout treatment and differentiated into normal hematopoietic lineages during response. Multiple immune populations were increased in peripheral blood after SENTI-202 treatment. •Correlative data collected during the SENTI-202-101 clinical trial affirms the anti-leukemic effects of SENTI-202 in responders, as well as the repopulation of immune cell subpopulations post-treatment. The observed effects are consistent with the pharmacodynamic action of the Logic Gate in SENTI-202 in sparing EMCN+ HSPCs while selectively killing CD33+ and/or FLT3+ AML tumor cells. SENTI-202 is a First-in-Class Off-the-Shelf Logic-Gated Selective CD33 OR FLT3 NOT EMCN CAR NK Cell Therapy for Blood Cancers SENTI-202-101 Study Design Clinical data presented at Clinical Trials Minisymposium (CTMS01, abstract CT014); preliminary RP2D identified as Schedule I, Dose Level 2 SENTI-202 Transgene Copies/µg DNA LLOQ LLOQ Schedule I Schedule II Schedule I Dose Level 1 Dose Level 2 Legend Preliminary RP2D Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Subject 7 Subject 8 Subject 9 AML blasts Monocytic Erythroid HSC/HSPCs CD4 T cells CD8 T cells NK cells B cells •AML blast reduction observed in a majority of subjects. •LSCs were decreased by ~10 fold or greater in subjects achieving cCR. Subjects achieving cCR showed robust recovery of immune populations in the periphery, driven by retention and differentiation of HSPCs. Disappearance of AML population Baseline C1D28 a b Baseline C1D28 C2D28 % HSPC of total cells 10 1 0.1 0.01 0.001 100 HSPC (CD34+ CD38low) Analysis of HSPCs and hematopoietic differentiation in the bone marrow of trial subjects. (a) HSPCs were identified as CD34+CD38-/low hematopoietic cells, and the proportion of HSPCs in responder bone marrow was either increased or maintained during SENTI-202 treatment. (b) CyTOF analysis identified cell populations in the classical hematopoietic differentiation hierarchy in healthy and diseased samples. Representative analysis was performed on responder subjects 1 and 2, showing that (c) EMCN+ hematopoietic populations (blue contours) were retained after SENTI-202 treatment. (d) UMAP analysis of subjects 1 and 2 showed the preservation of hematopoietic trajectories in responders, with progenitor cell types enriched at baseline and more differentiated populations appearing after treatment. Prelim. RP2D Low sample viability Subject 1Subject 2 Baseline C1D28 C2D28 Subject 1Subject 2 Subject 1Subject 2 c d Combined AML and healthy bone marrow samples • Subjects achieving cCR retained HSPCs in their bone marrow. • Observed preservation of hematopoietic trajectories in responders. • Progenitor cells are enriched at baseline and preserved during SENTI-202 treatment. C2D28 Low sample viability C1D28 sample (PR) used due to low baseline sample viability. Monocytes CMP/GMP Pro-erythroblasts MEPs Polychromatic erythroblasts pDCs / DCs Early myeloid progenitors MPPs Pre-B-cells HSCs/HSPCs (EMCN+) Pro-B cells Orthochromatic erythroblasts CLPs Mature B-cells* * Subject 5 samples had low viability. * * Subject 5 samples had low viability. * Subject 1 (CR MRD-) Subject 2 (CR MRD-) Subject 3 (PD) Subject 4 (CR MRD-) Subject 5 (CRh MRD-) Subject 6 (SD) Subject 7 (MLFS) Subject 8 (TBD) Subject 9 (TBD) RP2D Subject 1 (CR MRD-) Subject 2 (CR MRD-) Subject 3 (PD) Subject 4 (CR MRD-) Subject 5 (CRh MRD-) Subject 6 (SD) Subject 7 (MLFS) Subject 8 (TBD) Subject 9 (TBD) RP2D * End of cycle 1 for patients 8 & 9 Schedule I Schedule II Schedule I Dose Level 1 Dose Level 2 Legend Preliminary RP2D *

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