Preclinical development of SENTI-202, an off-the-shelf Logic Gated CAR-NK cell therapy, for the treatment of CD33/FLT3+ hematologic malignancies including AML

Arrows point to injection days Samples on D7 and D14 are collected prior to re- dosing Preclinical Pharmacology of SENTI-202 Senti Biosciences, Inc. South San Francisco, CA Alba Gonzalez, Enping Hong, Gozde Yucel, Elizabeth Leitner, Pearley Chinta, Han Deng, Ian Li, Alice Lam, Abla Bakir, Brandon Lee, Papia Chakraborty, Carmina Blanco, Chen-Ting Lee, Niran Almudhfar, Mengxi Tian, Wenqi Song, Andrew Banicki, Otto Contreras, Martin Gieldin, Brian Garrison, Timothy K. Lu, Kanya Rajangam AACR Annual Meeting 2023, Orlando, FL Abstract #3195 Preclinical development of SENTI-202, an off-the-shelf logic gated CAR-NK cell therapy, for the treatment of CD33/FLT3+ hematologic malignancies including AML SENTI-202 Logic Gated CAR-NK cells NOT Gated CAR-NK cells for the treatment of CD33/FLT3+ malignancies engineered to reduce on-target/off-tumor toxicities SENTI-202 is a First In Class OR/NOT Logic Gated Off-The-Shelf CAR-NK Cell Therapy SENTI’s novel Logic-gated gene circuits have enhanced tumor targeting. The CD33/FLT3 OR Gate activating CAR successfully enables the targeting of primary AML blasts, LSCs, and MDS cells, while the NOT Gate decreases the killing of HSCs while preserving their function by an inhibitory CAR that detects a protective antigen, EMCN, found in healthy HSCs. SENTI-202 in vivo anti-tumor function and pharmacokinetics Contact: [email protected] (a) SENTI-202 is an off-the-shelf cell therapy that uses healthy donor derived NK cells from peripheral blood mononuclear cells (PBMCs) that are expanded, engineered, and characterized in our optimized GMP-suitable process. All SENTI-202 components are stably expressed within a single expression g-retroviral vector. (b) Expansion and (c) expression of all components of the gene circuit is shown in multiple lots of SENTI-202. Various lots of SENTI-202 were used for in vitro cytotoxicity assays against target cells with or without expression of the protective antigen EMCN. (a) SENTI-202 showed reduced cytotoxicity when co-cultured with EMCN+ target cells, and (b) demonstrated protection of EMCN+ HSCs, (c) while preserving the myeloid colony forming activity of the protected HSCs. SENTI-202 SHOWS REDUCED KILLING OF EMCN+ CELLS In vitro cytotoxicity and cytokine production of SENTI-202 NOT Gate protection of EMCN+ target cells SENTI-202 HAS INCREASED CYTOTOXICITY ON CD33/FLT3+ TARGET CELLS COMPARED TO UNENGINEERED NK CELLS SERIAL KILLING CAPACITY OF SENTI-202 CYTOKINE PRODUCTION OF SENTI-202 Patients with CD33 and/or FLT3 expressing malignancies, including myeloid malignancies such as Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS) or Multiple Myeloma (MM) have very poor prognosis and high clinical unmet need. CD33 and FLT3 are well validated targets for myeloid malignancies, but current therapies targeted against those antigens present considerable limitations. On one hand, the presence of CD33 negative leukemic stem cells (LSCs) can contribute to eventual relapse; on the other hand, expression of CD33 and/or FLT3 by the normal hematopoietic stem cells and progenitor cells (HSCs/HPCs) can lead to bone marrow toxicities, prolonged thrombocytopenia/ neutropenia, and infectious complications. SENTI-202 is undergoing preclinical development to address these challenges and provide a broader therapeutic window, increasing the anti-tumor activity and safety for the treatment of CD33 and/or FLT3 malignancies. SENTI-202 is a first in class Logic-gated CAR-NK product engineered with an OR and a NOT Logic Gate gene circuit approach to enhance therapeutic efficacy and safety, with additional arming via expression of calibrated release IL-15 (crIL-15). A dual targeting activating CAR (aCAR) that recognizes both CD33 and FLT3 tumor antigens improves the anti-tumor activity, ensuring the targeting of AML blasts and LSCs. Additionally, an inhibitory CAR (iCAR) that recognizes endomucin (EMCN), a protective antigen expressed on the surface of healthy cells prevents the CAR- mediated cytotoxicity of healthy target cells including HSCs/HSPCs, improving the safety potential and reducing on-target/off-tumor toxicities. crIL-15 provides NK cell activation and persistence. Lots of SENTI-202 and control unengineered NK cells (different healthy donors) CYTOTOXICITY ON PRIMARY AML SAMPLES (a) SENTI-202 demonstrated increased serial killing capacity, retaining the ability to kill AML target cells over multiple rounds of rechallenge. (b) SENTI-202 also demonstrated increased cytokine and cytotoxic molecule production during cytotoxicity assays compared to unengineered NK cells. SENTI-202 CAR-NK CELLS HAVE INCREASED PERSISTENCE IN VITRO In vitro persistence and pSTAT5 signaling Preclinical development and in-house manufacturing capabilities SENTI’s state of the art manufacturing facility and optimized GMP-compatible process enables large scale production of SENTI- 202 product from various healthy NK donors. Selected NK donors have been evaluated pre-clinically with demonstrated activity and tolerability. IND-enabling studies are underway and on track for IND-submission in 2023. SENTI-202 clinical evaluation Phase 1 dose-escalation evaluation of SENTI-202 safety and efficacy is being planned in patients with CD33 and/or FLT3 expressing malignancies. SENTI-202 CAR-NK CELLS HAVE INCREASED PSTAT5 11.7% 4.26% 58.6% 20.9% 50.2% 14.1% Round 1 Round 2 Round 3 Unengineered NK SENTI-202 SENTI-202 SHOWS PROTECTION OF EMCN+ HSCs COMPARED TO EMCN- LEUKEMIA TARGETS EMCN-APC SEM- CD33/EMCN SEM-CD33 SEM WT pSTAT5 CD56 SENTI-202 Uengineered NK SENTI-202 REDUCES TUMOR AML TUMOR GROWTH AND PROLONGS MICE SURVIVAL SENTI-202 IN VIVO KINETICS AND PERSISTENCE In vivo treatment of SENTI-202 (a) decreases MV4-11 tumor burden and prolongs survival, with donor-to-donor differences in in vivo performance. (b) SENTI-202 cellular PK is evaluated in vivo via flow cytometry, showing persistence over multiple doses and eventual clearance. Summary and next steps Lentivir us Outpatient use potential Patient Isolate from selected donors Thaw and infuse Scalable ~21 Day Process NK Cells Selected Donor Gene Circuit Engineered CAR- NK cells Engineer CryopreserveExpand Off-The-Shelf Gene Circuits 1 2 3 4 5 Easy to thaw vials Final product harvested and cryopreserved >100 doses per batch NK cells isolated from peripheral blood of selected donors NK cells efficiently engineered with Gene Circuits High post-thaw potency SENTI-202 is engineered with crIL-15, which releases wild type IL-15 by the action of endogenous proteases to provide autocrine and paracrine support to SENTI-202 and other immune cells. Consistent with this feature, (a) SENTI-202 has increased persistence compared to unengineered NK cells when cultured in cytokine-free conditions, and (b) multiple lots of SENTI-202 demonstrated endogenous phospho-STAT5 signaling by flow cytometry. Bivalent CD33 and/or FLT3 CAR NK SENTI-202 0 20 40 60% Cytotoxicity (Leukemia) ns Bivalent CD33 and/or FLT3 CAR NK SENTI-202 0 10 20 30 40 50% Cytotoxicity (EMCN+ HSC) ✱✱ a b c Various lots of SENTI-202 manufactured using different NK donors were used for in vitro cytotoxicity assays. SENTI-202 demonstrated increased cytotoxicity against the AML target cell lines (a) Molm-13, (b) MV4-11, and (c) the SEM cell line engineered to express CD33 (SEM-CD33+) compared to donor-matched unengineered NK cells. b ca ba ba Multiple lots of SENTI-202 were used for in vitro cytotoxicity assays against 7 primary myeloid malignancy tumor-derived samples (6 AML and 1 MDS). SENTI-202 consistently demonstrated increased cytotoxicity against AML blasts, LSCs, and MDS cells when compared to donor- matched unengineered NK cells. ba ba Blast, AML #102, M1LSC, AML #102, M1Blast, AML #757, M4LSC, AML #757, M4Blast, AML #406, M0LSC, AML #406, M0 MDS #325 0 20 40 60 80 100% Cytotoxicity Donor 21 SENTI-202 in vitro cytotoxicity assay Non-Engineered NK SENTI-202 ✱✱ ✱✱✱ ✱✱✱✱ ✱✱✱ ✱✱ ✱ ✱ Blast, AML #400, M5LSC, AML #400, M5Blast, AML #406, M0LSC, AML #406, M0Blast, AML #797, M4LSC, AML #797, M4Blast, AML #865, M1LSC, AML #865, M1 0 20 40 60 80 100% Cytotoxicity Donor 27 SENTI-202 in vitro cytotoxicity assay ✱✱ ✱✱ ✱✱ ✱✱✱ ✱✱ ✱ ✱✱✱✱ ✱✱✱ Non-engineered NK SENTI-202 % Cytotoxicity HSPC only HSPC + SENTI-202 Lot 1HSPC + SENTI-202 Lot 2 0 50 100 150 200 Human Colony-Forming Unit CFC assay Myeloid Colonies HSPC only HSPC + SENTI-202 Lot 1 HSPC + SENTI-202 Lot 2 ns ns c 0 20 40 60 80 100 120 0 10 20 30 40 50 60 70 80 90 100 Days after tumor implantation Probability of Survival Survival Proportions MV4-11 AML model Vehicle Non-engineered NK3 SENTI-202 NK3 Non-engineered NK4 SENTI-202 NK4 10 20 30 40 50 60 70 80 90100110120 2nd treatment1st treatment3rd treatment 105 106 107 108 109 1010 1011 1012 1013 Days Post Tumor Implantation Tumor burden (BLI flux photons/sec) Vehicle Control 10 20 30 40 50 60 70 80 90100110120 2nd treatment1st treatment3rd treatment 105 106 107 108 109 1010 1011 1012 1013 Day Post Tumor Implantation Non-Engineered NK (NK3) 10 20 30 40 50 60 70 80 90100110120 2nd treatment1st treatment3rd treatment 105 106 107 108 109 1010 1011 1012 1013 Day Post Tumor Implantation SENTI-202 (NK3) 10 20 30 40 50 60 70 80 90100110120 2nd treatment1st treatment3rd treatment 105 106 107 108 109 1010 1011 1012 1013 Day Post Tumor Implantation Non-Engineered NK (NK4) 10 20 30 40 50 60 70 80 90100110120 2nd treatment1st treatment3rd treatment 105 106 107 108 109 1010 1011 1012 1013 Day Post Tumor Implantation SENTI-202 (NK4) Pharmacokinetics and Pharmacodynamics of SENTI-202 No NK NK1 NK2 NK3 NK4 NK27 0 20 40 60 80TNFα (pg/mL) TNFα, MOLM-13 targets Non-engineered NK SENTI-202 No NK NK1 NK2 NK3 NK4 NK27 0 100 200 300 400IFNγ (pg/mL) IFNγ, MOLM-13 targets Non-engineered NK SENTI-202 No NK NK1 NK2 NK3 NK4 NK27 0 500 1000 1500GM-CSF (pg/mL) GM-CSF, MOLM-13 targets Non-engineered NK SENTI-202 No NK NK1 NK2 NK3 NK4 NK27 0 20 40 60 80 100IL-10 (pg/mL) IL-10, MOLM-13 targets Non-engineered NK SENTI-202 105 106 107 108 109 1010 Time after NK cell thaw Total Cell Yield NK Growth Curve (N = 5 donors/runs) SENTI-202 NK1 SENTI-202 NK2 SENTI-202 NK3 SENTI-202 NK4 SENTI-202 NK27 Unengineered NK1 Unengineered NK2 Unengineered NK3 Unengineered NK4 Unengineered NK27 Thaw Activation Transduction 0 10 20 30 40 % IL-15 expression (various lots of SENTI-202) % IL-15 (surface) 0 5 10 15 20 Secreted IL-15 (pg/mL/1e6 NK cells 72h) IL-15 (secretion) Viability FSC NK1 NK2 NK3 NK4 NK20 NK22 NK23 NK24 NK27 NK28 0 20 40 60 80 NOT Gate protection of EMCN+ targets Lots of SENTI-202 (different NK healthy donors) % CAR-mediated cytotoxicity SEM-CD33+ SEM-CD33+/EMCN+ ✱✱ ✱✱✱✱ ✱✱✱✱ ✱ ✱✱✱ ✱✱✱✱✱ ✱✱ ✱✱✱✱ ✱✱✱✱ NK1 NK2 NK3 NK4 NK20 NK22 NK23 NK24 NK27 NK28 0 20 40 60 80 100 Cytotoxicity (MOLM-13) Lots of SENTI-202 (different NK healthy donors) % Cytotoxicity Non-engineered NK SENTI-202 ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱ NK1 NK2 NK3 NK4 NK20 NK22 NK23 NK24 NK27 NK28 0 20 40 60 80 100% Cytotoxicity Cytotoxicity (MV4-11) Non-engineered NK SENTI-202 ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱ ✱✱✱✱ ✱ NK1 NK2 NK3 NK4 NK20 NK22 NK23 NK24 NK27 NK28 0 20 40 60 80 100% Cytotoxicity Cytotoxicity (SEM-CD33+) Non-engineered NK SENTI-202 ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ ✱✱✱✱ NK1 NK2 NK3 NK4 NK27 IFNg GM-CSF IL-10 TNFa Perforin IL-6 IL-15 IL-1b Granzyme B IL-2 MOLM-13 NK1 NK2 NK3 NK4 NK27 MV4-11 0 2 4

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