NOT gate gene circuits expand the range of tumor-associated antigens addressable by CAR-NK and -T cell therapies

Nicholas W. Frankel1, Assen Roguev2,1, Marcus Gainer1, Han Deng1, Priscilla Wong1, Gozde Yucel1, Alice Lam1, Yongshuai Li1, Michelle Hung1, Derrick Lee1, Chen-Ting Lee1, Andrew Banicki1, Mengxi Tian1, Niran Almudhfar1, Wenqi Song1, Lawrence Naitmazi1, Brian Garrison1, Kanya Rajangam1, Timothy K. Lu1 SITC 2023 San Diego Abstract #257 1Senti Biosciences, Inc. South San Francisco, CA Logic-gated gene circuits expand the range of tumor-associated antigens addressable by CAR-NK and -T cell therapies On-target / off-tumor killing leading to poor therapeutic window Heterogeneous antigen expression leading to tumor escape Inhibitory CAR: NOT GATE Bivalent activating CAR: OR GATE Only ~5% of potential surface proteins have been utilized in CAR cell therapies due to 2 key limitations Logic gating can expand the range of potential therapeutics into the “empty” space Activating CAR (aCAR) recognizes a tumor associated antigen (TAA) Logic gating broadens tumors addressable by CAR cell therapies NOT gate design leverages a two-receptor system with armoring Senti products deploy NOT gates for selective tumor killing Many tumor-associated antigens are expressed on normal tissues In vitro protection of healthy primary human HSC fraction expressing EMCN 0 5 10 15 20 25 30 35 40 45 50 CD33 and/or FLT3 CAR-NK Cells SENTI-202 CAR-NK Cells % Killing (EMCN+ HSCs) 0 20 40 60 80 100 CEA CAR-NK Cells CEA NOT VSIG2 CAR-NK Cells VSIG2- CEA+ VSIG2+ CEA+ Selective killing of TAA+ cancer cells with decreased killing of TAA+PA+ model healthy cells with addition of inhibitory CAR construct 98% protection of healthy cells 0 10 20 30 40 50 60 70 80 90 CEA CAR-T Cells only CEA NOT VSIG2 CAR-T Cells VSIG2– CEA+ VSIG2+ CEA+ 76% protection of healthy cells NOT gated CAR-T construct achieves robust protection of model healthy cells while selectively and effectively killing cancer cells Take aways Many tumor-associated antigens are also expressed on healthy cells, risking on-target/off-tumor toxicity. VSIG2+ : VSIG2- at Takedown 98% Enrichment of VSIG2+ cells Logic gating will help design cell therapies that address more tumor types by increasing the therapeutic window. Senti’s NOT gate (used in SENTI-202 and -401) protects healthy cells in vitro and in vivo without interfering with cancer killing and works in T or NK cells. NK cells T cells No treatment Senti-401 treatment Avoidance of on-target, off- tumor toxicity may be challenging in solid tumors due to broad expression of putative TAAs Solid tumor TAAs targeted in CAR-T clinical trials Source: Barros et al., Cancers (Basel). 2022. doi:10.3390/ cancers14112667 Normal tissues with potential off-target toxicity Source: Kwon et al., Nat Biotechnol. 2023. doi: 10.1038/ s41587-023-01686-y 81% of solid tumor TAAs are “dirty” (at least 10% expression in normal tissue) % of cells in tissue expressing TAA 100% 50% 0% No. trials Logic-gated CARs that use NOT gates to shut down killing in normal tissues is a potential strategy to mitigate this risk 2GeneFab Alameda, CA Robust NOT gated CAR circuits are validated in both NK and T cells inhibitory CAR (iCAR) recognizes a protective antigen (PA) selectively expressed on healthy cells TAA+ TAA+ PA+ CAR-NK cells NOT gated CAR-NK cells aCAR PA TAA iCAR NOT gate architecture Inhibitory CAR recruits phosphatases to the immune synapse via ITIMs (immunoreceptor tyrosine-based inhibitory motifs), which inactivate kinases required for the response of the activating CAR on a target cell by target cell basis. High-throughput functional screening with a mixed-target NOT gate assay TAA+ TAA+ PA+ “cancer” target cell “healthy” target cell Mix target cells 1:1 Identification of successful NOT gates that enrich model healthy target cells Target cells only Custom liquid handling robotics Enrichment of healthy model cells (% pts.) NOT gate constructs Simultaneous screening of large arrays of NOT gate candidate in a single run with a single operator (240 per run is possible) 1 2 3 Target cells only Optimization of NOT gates using an automated HTS system Binder - target discovery - scFv design - affinity Structure - hinge - transmembrane Signaling - number of ITIMs - ITIM sequences - linkers iCAR points of optimization Multi payload gene circuit in a single DNA construct SENTI-202 for AML, MDS, and other blood cancers SENTI-401 for CRC and other solid tumors aCAR synergy Selective killing of CEA+ cancer cells along with protection of CEA+VSIG2+ model healthy cells in a mixed solid tumor in vivo model Effective killing of patient-derived AML cells, including both leukemic stem cells (LSCS) and blasts, and MDS, including a rare sample that expresses EMCN (Patient A) Patient A* B C D E F G AML: Blast LSC MDS AML Blasts LSCs Healthy HSCs/PCs FLT3, CD33 OR GATE ONLY (FLT3 OR CD33) FLT3, CD33, EMCN OR/NOT GATE (FLT3 OR CD33) NOT EMCN CD33 NO LOGIC SENTI-202 FLT3 Is Expressed on LSCs but It Is Also Highly Expressed on Healthy Hematopoietic Stem Cells FLT3 and/or CD33 targeting bivalent aCAR EMCN targeting iCAR Calibrated release IL-15 Colorectal cancer CEA VSIG2 CEACAM5: 85-90% of CRC and 40-60% of other solid tumors including lung cancer1 VSIG2 was identified by bioinformatics using single cell RNA sequencing and validated as protective antigen with immunohistochemistry Healthy colon epithelium CEA targeting aCAR VSIG2 targeting iCAR Calibrated release IL-15 IL-21 TA A Model healthy cells (TAA+PA+) Cancer cells (TAA+ PA-) Model healthy cells (TAA+PA+) Cancer cells (TAA+PA-) Cytokine multi-arming (as needed) Blast LSC Blast LSC Blast LSC Blast LSC Blast LSC Killing of EMCN+ AML cells confirmed that activating the NOT gate does not interfere with endogenous, NK cell-mediated cancer cell killing capabilities ß Increasing potential for off-target toxicity TAA+ TAA+ PA+ CAR-T cells NOT gated CAR-T cells *This rare patient sample expressed over 97% EMCN+ Target cell killing (%) Target cell killing (%)

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