Small-molecule-regulated Gene Circuit for Controlling Cytokine Expression in Cell Therapies

DO NOT DISTRIBUTE | SENTI BIOSCIENCES Small-molecule-regulated gene circuit for controlling cytokine expression in cell therapies Michelle Hung1, Divya Israni2, Huishan Li2, Yin Yin Chong1, Poornima Ramkumar1, Allison Drain2, Allison Quach1, Mengxi Tian1, Rishi Savur1, Niran Almudhfar1, Chen-Ting Lee1, Abla Bakir1, Edwin Cruz1, Carmina Blanco1, Travis Wood1, Mario Lorente1, Brandon Lee1, Brett Kiedaisch1, Russell M. Gordley1, Marcela Guzman Ayala1, Ahmad Khalil1, Gary Lee1, Tim Lu1 1 Senti Biosciences, Inc. 2 Boston University 1 Presented by: Michelle Hung May 14, 2021 DO NOT DISTRIBUTE | SENTI BIOSCIENCES 2 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Disclosures o Michelle Hung is a paid employee of Senti Biosciences, Inc o This presentation included verbal remarks by the presenter that are not included here DO NOT DISTRIBUTE | SENTI BIOSCIENCES 3 Challenge SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Executive Summary Our Solution Design and Build Regulator Dial – a gene circuit to regulate production of potent immune effectors using FDA- approved small molecule drugs Test Regulator Dial in primary immune cells and Learn how different properties of the gene circuit can be optimized to achieve desired results Optimized Regulator Dial gene circuit enabled Grazoprevir dose-dependent control of IL-12 production in vivo Safely improve efficacy of cell-based immunotherapies for solid tumors DO NOT DISTRIBUTE | SENTI BIOSCIENCES 4 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Persistence Immune Cell Recruitment and Tumor Killing Direct Killing Engineered Cell Engineered Cells Cancer Cell Engineered Cell Cancer Cell Attack Why arm cell-based immunotherapies with cytokines? DO NOT DISTRIBUTE | SENTI BIOSCIENCES 5 Controlling IL-12 arming in cell-based immunotherapies SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES The benefit of controlling the arming of cell-based immunotherapies Dose Controlled Armed CAR-immune cells (IV) OFF Dose Small Molecule Drug (Oral) Little/no immune effectors secreted in absence of small molecule drug Presence of small molecule triggers controlled secretion of immune effectors in the tumor ON o IL-12 is a highly potent immune activator with the potential to stimulate the tumor immunity cycle o Overexpressing IL-12 from adoptive T cell therapies using a poorly regulated promoter has resulted in significant clinical toxicities (Zhang et al., Clin. Can. Res. 2015) o Narrow therapeutic window associated with IL-12 has limited success to date DO NOT DISTRIBUTE | SENTI BIOSCIENCES 6 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial Transcription Factor Parts Controlled Payload Regulator Dial is intended to enable control of gene expression via FDA approved small molecules o Regulator Dial Transcription Factor uses a highly specific ZF DNA binding domain linked to an activator that drives target gene expression without impacting the rest of the transcriptome o Controlled payload is a cytokine driven by a promoter that contains a binding site for the Regulator Dial DNA binding domain cytokine payload Clinically-driven design of synthetic gene regulatory programs in human cells. Divya V. Israni, Hui-Shan Li, Keith A. Gagnon, Jeffry D. Sander, Kole T. Roybal, J. Keith Joung, Wilson W. Wong, Ahmad S. Khalil. bioRxiv 2021.02.22.432371 DO NOT DISTRIBUTE | SENTI BIOSCIENCES 7 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial Transcription Factor Parts Controlled Payload Regulator Dial is intended to enable control of gene expression via FDA approved small molecules o Regulator DNA binding domain is linked to the transcriptional activation domain by a protease cleavable linker o When the protease is active, the DNA binding domain is not linked to the transcriptional activation domain, and is unable to activate transcription o Cytokine payload is OFF cytokine payload Clinically-driven design of synthetic gene regulatory programs in human cells. Divya V. Israni, Hui-Shan Li, Keith A. Gagnon, Jeffry D. Sander, Kole T. Roybal, J. Keith Joung, Wilson W. Wong, Ahmad S. Khalil. bioRxiv 2021.02.22.432371 Absence of Small Molecule Drug DO NOT DISTRIBUTE | SENTI BIOSCIENCES 8 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial Transcription Factor Parts Controlled Payload Regulator Dial is intended to enable control of gene expression via FDA approved small molecules o NS3i small molecule suppresses protease activity, resulting in an intact transcription factor o Regulator Dial transcription factor can activate transcription o Cytokine payload is ON Regulator Dial Transcription Factor Parts Controlled Payload cytokine payload + NS3 inhibitor Clinically-driven design of synthetic gene regulatory programs in human cells. Divya V. Israni, Hui-Shan Li, Keith A. Gagnon, Jeffry D. Sander, Kole T. Roybal, J. Keith Joung, Wilson W. Wong, Ahmad S. Khalil. bioRxiv 2021.02.22.432371 Presence of Small Molecule Drug DO NOT DISTRIBUTE | SENTI BIOSCIENCES 9 Ideal properties of Regulator Dial SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial is intended to enable control of gene expression via FDA approved small molecules o Versatile: transcriptional regulation has the potential to control any payload of interest; can regulate multiple payloads o Safe: small molecule is FDA approved (hepatitis drug); NS3 protease incorporates mutations to avoid immunogenicity o Dose Dependent: payload level depends on small molecule dose o Convenient: orally-dosed favorable PK profile Clinically-driven design of synthetic gene regulatory programs in human cells. Divya V. Israni, Hui-Shan Li, Keith A. Gagnon, Jeffry D. Sander, Kole T. Roybal, J. Keith Joung, Wilson W. Wong, Ahmad S. Khalil. bioRxiv 2021.02.22.432371 DO NOT DISTRIBUTE | SENTI BIOSCIENCES 10 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Can Regulator Dial control gene expression in primary immune cells? Fluorescent reporter Cells transduced with lentivirus encoding Regulator Dial - ASV + ASV o NS3 inhibitors are a family of well tolerated, orally available, FDA-approved small molecules o Here, Asunaprevir (ASV) was the NS3 inhibitor used DO NOT DISTRIBUTE | SENTI BIOSCIENCES 11 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Can Regulator Dial control gene expression in primary immune cells? Fluorescent reporter Regulator Dial induced a 3.3-fold increase in fluorescent reporter upon small molecule treatment - ASV + ASV initial Regulator Dial 0 10000 20000 30000 40000 50000 Background subtracted fluorescence (AU) untreated + small molecule Cells transduced with lentivirus encoding Regulator Dial DO NOT DISTRIBUTE | SENTI BIOSCIENCES 12 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Can Regulator Dial control gene expression in primary immune cells? Fluorescent reporter - ASV + ASV High basal activity is potentially toxic for potent immune effectors such as IL-12 initial Regulator Dial 0 10000 20000 30000 40000 50000 Background subtracted fluorescence (AU) untreated + small molecule Cells transduced with lentivirus encoding Regulator Dial DO NOT DISTRIBUTE | SENTI BIOSCIENCES 13 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES o Initial regulator dial promoter had high basal activity, which could be potentially toxic if a potent immune effector such as IL-12 is being controlled o We identified alternative minimal promoters that could have lower basal activity Can promoter optimization reduce basal activity of the Regulator Dial? optimize DO NOT DISTRIBUTE | SENTI BIOSCIENCES 14 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Can promoter optimization reduce basal activity of the Regulator Dial? optimized Basal expression was reduced by 20-fold upon promoter optimization - ASV leaky Regulator Dial optimized Regulator Dial 0 5000 10000 15000 20000 Background subtracted fluorescence (AU) Cells transduced with lentivirus encoding Regulator Dial DO NOT DISTRIBUTE | SENTI BIOSCIENCES 15 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES optimized Optimized Regulator Dial induced an 11-fold increase in fluorescent reporter upon small molecule treatment - ASV + ASV How well does the optimized Regulator Dial induce gene expression? tuned Regulator Dial 0 1000 2000 3000 4000 5000 Background subtracted fluorescence (AU) untreated + small molecule Cells transduced with lentivirus encoding Regulator Dial DO NOT DISTRIBUTE | SENTI BIOSCIENCES 16 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial Small molecule sensitivity Regulator Dial small molecule sensitivity 0.01 0.1 10 0 2000 4000 6000 8000 10000 Asunaprevir NS3i Concentration (uM)Fluorescent reporter (AU) Regulator Dial small molecule sensitivity Fluorescent reporter DO NOT DISTRIBUTE | SENTI BIOSCIENCES 17 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial Small molecule sensitivity Regulator Dial small molecule sensitivity 0.01 0.1 10 0 2000 4000 6000 8000 10000 Asunaprevir NS3i Concentration (uM)Fluorescent reporter (AU) Regulator Dial small molecule sensitivity ASV Cmax Fluorescent reporter DO NOT DISTRIBUTE | SENTI BIOSCIENCES 18 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial Small molecule sensitivity Senti’s Design, Build, Test, Learn cycle was applied to improve Regulator Dial small molecule sensitivity Regulator Dial Small molecule sensitivity 0.01 0.1 10 0 2000 4000 6000 8000 10000 Asunaprevir NS3i Concentration (uM)Fluorescent reporter (AU) Regulator Dial small molecule sensitivity ASV Cmax DO NOT DISTRIBUTE | SENTI BIOSCIENCES 19 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial Small molecule sensitivity Senti’s Design, Build, Test, Learn cycle was applied to improve Regulator Dial small molecule sensitivity Regulator Dial Small molecule sensitivity ASV Cmax 0.01 0.1 10 0 2000 4000 6000 8000 10000 Asunaprevir NS3i Concentration (uM)Fluorescent reporter (AU) Regulator Dial small molecule sensitivity DO NOT DISTRIBUTE | SENTI BIOSCIENCES 20 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial Small molecule sensitivity Senti’s Design, Build, Test, Learn cycle was applied to improve Regulator Dial small molecule sensitivity Regulator Dial Small molecule sensitivity ASV Cmax 0.01 0.1 10 0 2000 4000 6000 8000 10000 Asunaprevir NS3i Concentration (uM)Fluorescent reporter (AU) Regulator Dial small molecule sensitivity DO NOT DISTRIBUTE | SENTI BIOSCIENCES 21 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES 0.01 0.1 10 0 20000 40000 60000 80000 uM Asunaprevir Fluorescent reporter (AU) Asunaprevir sensitivity Regulator Dial sensitivity to alternative NS3 inhibitors Multiple NS3i small molecules were chosen based on IC50s against NS3 and human PK data ASV Cmax 0.01 0.1 10 0 20000 40000 60000 80000 uM Danoprevir Fluorescent reporter (AU) Danoprevir sensitivity DPV Cmax 0.01 0.1 10 0 20000 40000 60000 80000 uM Grazoprevir Fluorescent reporter (AU) Grazoprevir sensitivity GRZ Cmax Regulator Dial showed high sensitivity to Grazoprevir at clinically relevant levels Asunaprevir sensitivity Danoprevir sensitivity Grazoprevir sensitivity DO NOT DISTRIBUTE | SENTI BIOSCIENCES 22 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial enables control of IL-12 production in primary immune cells Regulator Dial dose-dependently controlled IL-12 production in primary immune cells at clinically relevant concentrations of Grazoprevir IL-12IL-12 IL-12 0.01 0.1 10 0 5000 10000 15000 20000 25000 uM Grazoprevir pg/mL IL-12 Cmin Cmax Regulator Dial control of IL-12 Cmin to Cmax are the average minimal and maximal serum concentrations of GRZ in patients treated daily with the clinical dose (ZEPATIER ®) DO NOT DISTRIBUTE | SENTI BIOSCIENCES 23 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES In Vivo Regulator Dial control of hIL-12 production 0 1 2 3 4 8 collect plasma cardiac puncture Day Regulator Dial cells dosing Grazoprevir dosing Vehicle 50mg/kg 75mg/kg 10 100 1000 10000 IL12 pg/mL Day 4 Vehicle 50mg/kg 75mg/kg 10 100 1000 10000 IL12 pg/mL Day 8 IL12 Expression Level (pg/mL) IL12 Expression Level (pg/mL) Regulator Dial enabled dose dependent and reversible IL-12 production in vivo In vivo Study Design hIL-12 in mouse plasma DO NOT DISTRIBUTE | SENTI BIOSCIENCES 24 Armed, Controlled Therapy enabled by Regulator Dial SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Regulator Dial could enable us to control expression of potent immune effectors for immune cell therapies Regulator Dial has the potential to enable the following benefits: o Optimized for safe, low expression of potent cytokines in the absence of small molecule drug and strong, dose dependent induction of cytokine production in the presence of small molecule drug o Versatile to regulate potentially any payload of interest o Convenient regulation by orally dosed, FDA-approved small molecule DO NOT DISTRIBUTE | SENTI BIOSCIENCES 25 SMALL-MOLECULE-REGULATED GENE CIRCUIT FOR CONTROLLING CYTOKINE EXPRESSION IN CELL THERAPIES Acknowledgements Thank you to the fantastic team at Senti Biosciences and our collaborators in the Khalil lab at Boston University! See our other Senti Abstracts: Title: Precise Targeting of AML with First-in-Class OR / NOT Logic- Gated Gene Circuits in CAR-NK Cells Garrison et al. (abstract 77) Title: Precise Tumor Targeting with NOT Logic-Gated Chimeric Antigen Receptor Gene Circuits Frankel et al. (abstract 960)

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