FLT3 OR CD33 NOT EMCN Logic Gated CAR NK Cell Therapy (SENTI 202) for Precise Targeting of AML

FLT3 OR CD33 NOT EMCN Logic Gated CAR-NK Cell Therapy (SENTI-202) for Precise Targeting of AML Brian S. Garrison, PhD1, Han Deng, PhD1, Gozde Yucel, PhD1, Nicholas W. Frankel, PhD1, Marcela Guzman-Ayala, PhD1, Russell Gordley, PhD1, Michelle Hung, PhD1, Derrick Lee1, Marcus Gainer1, Kathryn Loving, PhD1, Jenny Chien1, Tiffany Pan, MS1, Wesley Gorman1, Nelia Leemans, MS1, Alice Lam1, Poornima Ramkumar, PhD1, Travis Wood1, Wilson Wong, PhD2, Philip Lee, PhD1, Tim Lu, MD PhD1,Gary Lee, PhD1 1 Senti Bio, South San Francisco, CA; 2 Department of Biomedical Engineering, Boston University, Boston, MA Abstract: 2799 SENTI-202: Designed to Address Unmet Needs in AML Treatment SENTI-202 Components Treatment Profile Allogeneic CAR-NK cell therapy Initial Indication Relapsed/Refractory AML (r/rAML) Rationale Targeting both FLT3 and CD33 will broadly clear both AML blasts and LSCs, while protecting healthy HSCs via recognition of EMCN Gene Circuit Logic Gating: FLT3 OR CD33 NOT EMCN FLT3-targeting scFv; part of bivalent activating CAR (aCAR) CD33-targeting scFv; part of bivalent activating CAR (aCAR) EMCN inhibitory CAR (iCAR) crIL-15 (novel IL-15 molecule) 1 2 3 4 FLT3 CD33 OR GATE Only (FLT3 OR CD33) FLT3 CD33 EMCN OR/NOT GATE (FLT3 OR CD33) NOT EMCN CD33 No Logic SENTI -202 AML Blasts AML LSCs Healthy HSCs/PCs Respond with OutputsSense Inputs Compute Decision CD33 FLT3 EMCN Antigen: SENTI-202 Schematic SENTI-202: AML Blasts & LSC Clearance While Sparing Healthy HSCs Bivalent FLT3/CD33 aCAR EMCN iCAR Unengineered NK cells Engineered NK cells 0 50 100 150 200 soluable IL-15 (pg/ml/1e6 cells) Secreted IL-15 In SENTI-202, NK cells are engineered with activating and inhibitory chimeric antigen receptors (CARs), imbuing the cells with logic-based sense-and- respond capabilities. By concurrently targeting AML tumor-associated antigens FLT3 and CD33 with a bivalent activating CAR (aCAR), SENTI-202 is designed to target both AML blasts and leukemic stem cells (LSCs), leading to potentially greater efficacy and deeper more sustained remissions. By targeting the safety antigen Endomucin (EMCN) with an inhibitory CAR (iCAR), SENTI-202 is designed to protect hematopoietic stem cells (HSCs) and progenitor cells (PCs) from off-tumor toxicity, potentially enabling a curative cell therapy approach in the absence of transplantation. Single Gene Circuit Expression of all SENTI-202 Components crIL-15 2A Bivalent FLT3/CD33 activating CAR EMCN inhibitory CAR2ASENTI-202 Circuit >70% aCAR + iCAR Co-Expression Robust Cell-Associated and Secreted IL-15 FSC Cell-Associated IL-15 IL-15+ 47.8% aCAR + iCAR 74.9% Soluble IL-15 (pg/ml/1e6 cells) SENTI-202 uses a single gene circuit that encodes for 4 components: (1.) crIL-15, (2.) FLT3 aCAR, (3.) CD33 aCAR, and (4.) EMCN iCAR, which are all expressed in a tricistronic format (see above). FLT3 and CD33 CARs have been combined into a single bivalent FLT3/CD33 aCAR, which is a single aCAR with the ability to bind both FLT3 and CD33. The SENTI-202 circuit enables up to 74.9% of our engineered CAR-NK cells to express both the FLT3/CD33 bivalent aCAR and the EMCN iCAR (left), and express significant levels of IL-15 detected on both the NK cell surface (middle) and in solution (right). Introduction While chimeric antigen receptor (CAR) cell therapies have provided extraordinary clinical responses in some hematological malignancies, developing effective CAR cell therapies for acute myeloid leukemia (AML) has been challenging due to: (a) the lack of a single target antigen robustly expressed across both AML leukemic stem cell (LSC) and leukemic blast cell subpopulations, and (b) the lack of truly AML-specific target antigens, since current proposed targets are also expressed on healthy tissues and may result in off-tumor toxicity. Using Logic Gated gene circuits, Senti Bio is engineering its SENTI-202 CAR-NK cell therapy to overcome these long-standing challenges to treating patients with AML. SENTI-202 is a novel off-the-shelf NK cell product candidate engineered to incorporate both OR and NOT Logic Gated CAR gene circuits, wherein the OR GATE is designed to increase AML LSC/blast tumor clearance (to prevent relapse), and the NOT GATE is designed to protect healthy hematopoietic stem cells (HSCs) from off-tumor toxicity, enabling regeneration of a healthy hematopoietic system and mitigating the need for a bone marrow transplant. SENTI-202 is also engineered to express calibrated release IL-15 (crIL-15), a novel engineered IL-15 molecule that enables autocrine and paracrine signaling that imbues NK cells with enhanced anti-tumor activity and persistence. FLT3 OR CD33 Logic Gated CAR-NK Cells Demonstrate Robust Killing Activity 8Day 12 15 19 23 26 NK cells FLT3 “OR” CD33 CAR-NK cells Untreated Groups No Cancer Moderate Tumor Load Significant Tumor Load Sacrificed 0 10 20 30 0 50 100 Survival curve of AML-01043 study * (Pre-Mix Treatment) Days Post Tumor Implantation Probability of Survival Molm13 only Pre-mixed Molm13 + Nv NKsPre-mixed Molm13 + FLT3/CD33 CAR_IL-15 NKs P-Values: *P=0.0143 (Molm13 vs Pre-mix Nv NKs) *P=0.0143 (Molm13 vs Pre-mixed FLT3/CD33 CAR_ IL-15 NKs) *P=0.0253 (Pre-mixed Nv NKs vs Pre-mixed FLT3/CD33 CAR_ IL-15 NKs) * Excluded 1 mouse got only half does of the treatment NK cells CAR-NK cells OR GATED CAR Outperforms Traditional monoCARs 80Percent Killing 60 40 20 0 FLT3 (Only) CAR-NK CD33 (Only) CAR-NK FLT3 OR CD33 CAR-NK FLT3++ CD33++ Leukemia Target Cell Percent Killing 80 60 40 20 0 100 OR GATED CAR-NK Cells Kill AML in Patient Samples AML Patient Sample #770 (M2) #857 (M2) #847 (M3) #837 (M5) #846 (M5) NK cell CAR-NK cell Bivalent FLT3/CD33 CAR-NK Cells Outperform a Bicistronic Approach Target Leukemia Cells: MV4-11 SEM HL60 Bivalent CAR-NK #1 Bivalent CAR-NK #2 Bicistronic CAR-NK NK cell Dual targeting FLT3 OR CD33 Logic Gated CAR-NK cells demonstrate significant in vitro cytotoxicity against primary AML cells in fresh patient samples. 80 60 40 20 0 100Percent Killing CAR-NK Cell Type: : * ** FLT3/CD33 CAR-NK Cell Therapy Significantly Suppresses Tumor and Increases Survival in AML (MOLM-13) Xenotransplantation Model (1.) Untreated (2.) Unengineered NK Cells (3.) FLT3 OR CD33 CAR-NK Cells Treatment Groups FLT3/CD33 CAR-NK Cell Therapy Significantly Suppresses Tumor and Increases Survival in AML (MV4-11) Xenotransplantation Model Days 5 9 13 16 20 23 27 30 (1.) Untreated (2.) Unengineered NK Cells (3.) FLT3 OR CD33 CAR-NK Cells Days Senti’s Novel crIL-15 Enhances NK Cell Persistence and Tumor Killing crIL-15 Enables Both Autocrine and Paracrine Signaling crIL-15 Improves CAR-NK Cell Persistence and Serial Killing ns **** **** More tumor cells added More tumor cells added Un-engineered NKs SENTI CAR-NK + crIL-15 Unengineered NK Cells CAR-NK + wtIL-15 SENTI CAR-NK + crIL-15 5 4 3 2 0 1 0 24 48 72 96 120 144 168 192 Tumor alone + NK cells + CAR-NK cells + wtIL-15 + CAR-NK cells + crIL-15 Relative Tumor Cell Abundance Calibrated release IL-15 (crIL-15) outperforms traditional secreted IL-15 (wtIL-15) within an Incucyte-based serial killing assay, where CAR-NK cells expressing crIL-15 demonstrate enhanced killing and persistence in the secondary and tertiary re-challenges. Microscopy images from the tertiary re-challenge show Senti’s CAR-NK + crIL-15 cells with enhanced killing (red=tumor cells). Senti’s novel crIL-15 product enables both autocrine and paracrine signaling. Purple triangles represent crIL-15 molecules surrounding the CAR-NK cell in the middle, with purple space representing the potential effective area of crIL-15. OR GATED CAR-NK Cells Significantly Kill Leukemia Cell Lines and Secrete Cytokines 80Percent Killing 60 40 20 0 SEM MOLM-13 THP-1Target Cell: Cytokine Expression (pg/ml) 500 1000 1500 0 Gran.B IFN-g TNF-a NK cells GFP-NK cell CAR-NK cell FLT3 OR CD33 Logic Gated CAR-NK cell therapy shows significant in vivo efficacy within an AML (MOLM-13+luciferase) xenotransplantation model. After 1 month, Untreated (1.) and Unengineered NK cell-treated (2.) groups were lost to disease, while all FLT3 OR CD33 CAR-NK cell- treated mice (3.) survived. FLT3 OR CD33 Logic Gated CAR-NK cell therapy (1.) shows significant in vivo efficacy within an AML (MV4-11+luciferase) xenotransplantation model, with increased survival over Untreated (1.) and Unengineered NK Cell-treated (2.) groups. OR Logic Gating in a single circuit can be accomplished via a bivalent or bicistronic CAR approach. We determined that Bivalent FLT3 OR CD33 CAR-NK cells (blue bars) demonstrate significant in vitro cytotoxicity over bicistronic FLT3 OR CD33 CAR-NK cells (dark gray bar) against multiple leukemia lines. Dual targeting FLT3 OR CD33 Logic Gated CAR-NK cells (green box) show significant killing over FLT3 and CD33 single CAR approaches. Dual targeting FLT3 OR CD33 Logic Gated CAR-NK cells demonstrate significant in vitro cytotoxicity (left) and cytokine secretion (right; THP-1 data shown) against multiple leukemia cell lines (SEM, MOLM-13, THP-1). NOT GATE Significantly Reduces CAR-NK Cell On-Target/Off-Tumor Toxicity NOT GATE Engagement Enables Healthy Cell Protection CAR-NK Cell Healthy Cell Activation CAR (aCAR) Inhibitory CAR (iCAR) Activating (Tumor) Antigen Safety Antigen (Left) OR/NOT GATED CAR-NK cells recognize and kill cancer cells that express aCAR- targeted tumor-associated antigens (e.g. FLT3, CD33; green) and do not express iCAR-targeted safety antigens (e.g. EMCN; purple). (Right) OR/NOT GATED CAR-NK cells do not kill healthy cells expressing the iCAR- targeted safety antigen; iCAR engagement results in inhibition of NK cell activation. Bivalent FLT3 OR CD33 CAR Targets AML LSCs 14 4 6 8 10 12 Healthy HSCs AML LSCs AML Blasts FLT3 Expression (Log2) FLT3 Expression FLT3 CD33 Q1 52.3 Q2 39.3 Q3 0.93 Q4 7.48 AML LSCs 102 103 104 105 101 102 103 104 105 However, NOT GATE Needed to Protect Healthy HSCs Targeting FLT3 enables more comprehensive targeting of AML LSCs, a cell population thought to contribute to relapse in AML patients. Senti has Identified Multiple iCAR Architectures for CAR-NK Cells 50 40 30 20 10 0 NOT GATE Architecture: 1 2 3 4 5 6 7 8 9 10 CAR-Mediated Killing (%) EMCN- EMCN+ Target Cell EMCN Differentiates Healthy HSCs from AML LSCs Healthy HSCs (from healthy donor) AML LSCs (from AML patient) EMCN Side Scatter (SSC) EMCN Side Scatter (SSC) EMCN+ = 0%EMCN+ = 70.2% To design and employ a NOT GATE (iCAR) for protection of healthy HSCs from off-tumor toxicity, we first used a proprietary NOT GATE paired-antigen discovery platform to identify Endomucin (EMCN) as a potential NOT GATE safety antigen robustly expressed on healthy HSCs (left) but not expressed on AML LSCs (right). Using an in vitro NOT GATE cytotoxicity screen, we identified at least 9 different NOT GATE architectures that are functional within the context of CAR-NK cells. All NOT GATED CAR-NK cells consisted of a FLT3 aCAR and an EMCN iCAR with a unique architecture (FLT3 NOT EMCN CAR-NK cells), which were then mixed with FLT3+ SEM target cells (+/- EMCN-expressing versions), and in the presence of a functional iCAR there is reduced killing of the +EMCN target cells (or increased protection of the EMCN+ target cells). NOT GATE Enables Protection of EMCN+ Cells CAR-Mediated Killing (%) FLT3+ EMCN- FLT3+ EMCN+ Target Cell FLT3aCAR: iCAR: FLT3 FLT3 Non-specific EMCN #1 EMCN #2 67% Protection 50% Protection Top iCAR candidate architectures were independently validated using FLT3 NOT EMCN CAR-NK cells against FLT3+ target cells (+/- EMCN-expressing populations). We observed a significant decrease in killing of EMCN- expressing target cells, indicating the NOT GATE enabled up to 67% protection of EMCN+ cells. Importantly, we believe 10-20% protection of healthy HSCs may be clinically relevant and allow full reconstitution of a healthy hematopoietic system in patients. NK Cells Cancer Cells Healthy Cells + + Healthy Cells Spared Cancer Cells Killed Blood Collection I.V. Injection NOT GATE NK cells Control CAR-NK cells 0 20 40 60 80 100 NK cell treatment Target cell %Her2+ Peripheral blood, day 21 post implantation NOT GATE NK cells Control CAR-NK cells NOT GATE NK cells Control CAR-NK cells 0 10 20 30 40 50 60 70 NK cell treatment Target cells (% of CD45+ cells) Day 21 target cells (% of CD45+) Healthy cells (Her2+) Cancer cells (Her2–) ns NALM6 Target Cell %HER2+ 20 40 60 80 100 Target Cells (%CD45+)10 20 30 40 50 60 70 Healthy Cell (HER2+) Cancer Cell (HER2-) Using HER2 as a model iCAR target antigen, we demonstrate in vivo NOT GATE protection of HER2+ target cells against CAR-NK cell toxicity. NOT GATED CAR-NK cells and NALM6 target cells (50% HER2+ and 50% HER2-) are injected (left), and at day 21 peripheral blood analysis 94% of the remaining NALM6 cells are HER2+ (instead of 50%), indicating HER2+ target cells are protected from killing. Analysis of HER2+/HER2- target cell frequencies as part of total CD45+ shows a similar result (right). While a FLT3 CAR enables targeting of AML LSCs, this may lead to off-tumor toxicity against healthy HSCs, which also express FLT3. P-Values: (3) Vs (1): p<0.0143 (3) Vs (2): p<0.0253 (2) Vs (1): p<0.0143 (1.) (2.) (3.) 0 20 40 60 80 0 25 50 75 100 Time (days) Percent Survival Untreated CAR-NK cell-treated NK cell-treated Survival Probability (%) Survival Probability (%) Kaplan-Meier Survival Curve Kaplan-Meier Survival Curve P-Values: (3) Vs (1): p<0.01 (3) Vs (2): p<0.01 (2) Vs (1): p<0.01 Day Post Tumor Implantation (1.) (2.) (3.) FLT3 OR CD33 NOT EMCN CAR-NK cells demonstrated 47% protection of healthy HSCs/MPPs compared to equivalent CAR-NK cells without the EMCN NOT GATE within an in vitro cytotoxicity assay. CAR-NK groups show similar killing of MV4- 11 AML cells (not shown). Note: MPP = Multipotent Progenitor EMCN NOT GATE Protects Healthy HSCs/MPPs Summary o SENTI-202 is an allogeneic Logic Gated FLT3 OR CD33 NOT EMCN CAR-NK cell therapy product candidate designed to treat r/rAML patients in need o SENTI-202 uses a single gene circuit expressed from a tricistronic design encoding: o 1. crIL-15: enhances CAR-NK cell activity and persistence o 2. Bivalent FLT3/CD33 activating CAR (aCAR): enables targeting of AML LSCs and blast cells o 3. EMCN inhibitory CAR (iCAR): enables protection of healthy HSCs from off-tumor toxicity o FLT3 OR CD33 Logic Gated CAR-NK cells demonstrate: o Enhanced cytotoxicity over more traditional FLT3 or CD33 single CAR approaches across multiple cell lines o Significant in vitro cytotoxicity against AML patient tumor samples and cell lines o Significant in vivo efficacy within AML (MOLM-13 and MV4-11) xenotransplantation models o Bivalent FLT3/CD33 CARs possess significant killing over a bicistronic FLT3/CD33 CAR approach o crIL-15 provides CAR-NK cells with enhanced killing activity and persistence o NOT GATE Logic Gated CAR-NK cells demonstrate: o Significant in vivo protection of safety antigen-expressing target cells (model antigen = HER2) o Multiple iCAR architectures are functionally active in FLT3 NOT EMCN CAR-NK cells o Significant in vitro protection of healthy HSC/MPP cells from aCAR-mediated toxicity Allogeneic CAR-NK cell 1 2 3 4 EMCN iCAR FLT3 OR CD33 aCAR crIL-15 NOT GATE Engagement Enables In Vivo Healthy Cell Protection NK Cells HSC/MPP CAR-Mediated Killing (%) 50 40 30 20 10 0 47% Protection FLT3 OR CD33 CAR-NK FLT3 OR CD33 NOT EMCN CAR-NK Engineered NK cell Healthy HSC/MPP Healthy HSC/MPP CAR-NK NOT GATED CAR-NK This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N91021C00026 Activating CAR Engagement Triggers Cancer Cell Killing Cytotoxic granulesCytokines CAR-NK Cell AML Cancer Cells Activation

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