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How to Create CRISPR-Edited T Cells More Efficiently for Tomorrow's Cell Therapies
Artisan Bio focuses on developing advanced CRISPR technologies for T cell therapy, utilizing a systems approach that integrates quality by design (QbD) principles. The document outlines their proprietary STAR-CRISPR platform, detailing process development, optimization, and the effectiveness of their editing systems compared to competitors. Additionally, it emphasizes the importance of risk assessment and design of experiments (DOE) for enhancing editing efficiency in cell biology applications.
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How to Create CRISPR-Edited T Cells More Efficiently for Tomorrow's Cell Therapies
- 2. STEMCELL Technologies Global BiotechLeader Supporting Life Science Research
- 3. Immunology Pluripotent Stem Cell Biology Hematopoietic StemCell Biology Intestinal Biology Neural Biology Endothelial Cell Biology Pulmonary Research Mesenchymal Stromal Cell Biology Mammary Research Pancreatic Research CHO Cells & Hybridomas Prostate Research STEMCELL Technologies Major Research Areas
- 4. Source Isolate ● Primarycells STEMCELL Products For Every Step of Your Immune Cell-Based Research Activate, Edit, Expand & Differentiate Analyse & Cryopreserve ● EasySep™ ● RoboSep™ ● SepMate™ ● Lymphoprep™ ● Antibodies ● ELISA kits ● Dyes & stains ● CryoStor™ ● Contract Assay Services ● StemSpan™ ● ImmunoCult™ ● Cytokines ● CRISPR/Cas9
- 5. Confidential | ArtisanBio| info@artisancells.com Creating the Next Generation of CRISPR-edited T cells Steven Loo-Yong-Kee
- 6. Copyright 2022 ArtisanBio. All Rights Reserved. University of Toronto, Chemical Engineering & Applied Chemistry (2014 – 2015) Bioprocess Engineering Lab and BioZone (Centre for Applied Bioscience and Bioengineering) – MEng • Scale-up and automation of protein production in from shake flasks to 80-L STR to be used for the desludging of wastewater. University of Toronto, The Centre for Global Engineering (2015 – 2016) Re-invent the Toilet Challenge – RA • Develop a toilet that burns solid waste streams to generate heat and use the heat to dry new incoming solid waste and pasteurize liquid waste. CCRM/CATCT (2016 – 2020) The Centre for Advanced Therapeutic Cell Technologies – RA, Snr RA, Scientist • Leading collaborative immunotherapy and viral vector development efforts between CCRM and GE Healthcare. Non-viral transfection technologies portfolio manager in cell and gene therapies. Avectas (2020 – 2021) Process Development – Scientist II • Development, optimization, characterization, scale up and tech transfer of SOLUPORE cargo delivery process. Artisan Bio (2021 – Present) Cell Biology and Immunology – Scientist, Scientist II • Development of Artisan’s T cell and iPSC editing platforms, including process development, optimization and tech transfer. Founding member of internal DOE core, offering insights into QbD methodologies. Background and Experience
- 7. Copyright 2022 ArtisanBio. All Rights Reserved. ARTISAN BIO DATA KNOW-HOW Cas9 Cas12 Off Target Data Packs Protocols & Builds Artisan’s team provides: Platform & Licenses Guide Sequences STAR Toronto, ON Louisville, CO A 1-Stop Shop cell therapy powerhouse to drive the cell therapy revolution.
- 8. Confidential | ArtisanBio| info@artisancells.com STAR-CRISPR A novel, high performance editing system with enhanced safety and FTO STAR-CRISPR Design 3 Engineered gRNA ensures unique editing system compared to other Cas12a’s STAR-CRISPR 1.0 Available AND Core + Opt + Mods v2 STAR Nuclease 1.0 STAR gRNA 1.0 STAR-CRISPR 2.X → In Optimization Studies
- 9. Copyright 2022 ArtisanBio. All Rights Reserved. Cas9 Cas12a STAR PAM Break site Off-Target (Safety) On-Target (Performance) NGG blunt end * *** TTTN sticky end *** ** TTTN sticky end **** **** STAR-CRISPR OVERVIEW Table adapted from: Huang et al, 2022. Comparison of DNA targeting CRISPR editors in human cells. Mechanism ● STAR-guides architecture results in 40% reduction in off-target vs. wild- type single guide RNA License availability and cost Commercial * */** **** ● Engineered, high performance Cas12a-like architecture not found in nature ● Novel Nuclear Localization tags and orientation increased editing efficiency ● Optimized insertions components (homology arms, template) ● Tuned protocols and experimental designs Artisan Engineering Improvements A high-performance Type 5 nuclease with differentiated IP position and editing performance
- 10. Copyright 2022 ArtisanBio. All Rights Reserved. BOTTOM UP Hypothesis Driven Solution Space Definition Deep literature mining, Discovery analytics, Process flow diagrams Solution Space Reduction Risk Assessment, Scale Down Model, Perturbations, DoE, High-throughput screening, Model fitting Design Space Definition High resolution DoE, Scale Up & Model validation TOP DOWN Data Driven “The key to the systems approach is a ‘big picture’ view – you need to look at how the components of a system interact with each other to achieve an overall result, rather than simply optimizing each component.” Systems Approach Adapted from Dr. P. Chrysanthopoulos (weCANdev.ca)
- 11. Copyright 2022 ArtisanBio. All Rights Reserved. Quality by Design (QbD): Defining the Levers “A systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management.” – ICH Q8(R2) Process Reagents Equipment Materials Product INPUTS OUTPUTS
- 12. Copyright 2022 ArtisanBio. All Rights Reserved. Quality by Design (QbD): Defining the Levers By constructing a correlation map that describes how input variations affect the outputs, the approach of utilizing iterative risk assessments and DoE screening studies allows for a deep process understanding and better control of quality attributes for the intermediate and drug product state. INPUTS OUTPUTS Critical Process Parameters (CPPs) Critical Quality Attributes (CQAs) Process Reagents Equipment Materials Quality Target Product Profile (QTPPs) Critical Material Attributes (CMAs)
- 13. Copyright 2022 ArtisanBio. All Rights Reserved. QbD in Practice: Allogeneic CAR-T Therapy High level view is oversimplified, lacking proper process understanding to ensure consistency and reproducibility of the final product. Adapted from Dr. P. Chrysanthopoulos (weCANdev.ca)
- 14. Copyright 2022 ArtisanBio. All Rights Reserved. Process Development for Improving Editing Efficiency INPUTS OUTPUTS Process Parameters Intermediate Quality Attributes Unit Operation Reagents Equipment Materials Analytics Controlled and Uncontrolled Variables With a similar focus on process understanding, taking a DoE-driven risked-based iterative approach to understanding the correlations between inputs and outputs for Artisan’s STAR-CRISPR editing platform.
- 15. Copyright 2022 ArtisanBio. All Rights Reserved. Process Development and Evolution Knowledge Space Solution Space Design Space Control Space Set Point Process Understanding Process Development Process Characterization Process Validation Input Output
- 16. Copyright 2022 ArtisanBio. All Rights Reserved. HLA-E KI NK cell HLA-I KO HLA-II KO CD8+ T Cell CD4+ T Cell GvHD Cytokine Receptor Solid Tumor Site TCR KO Dual CAR Solid Tumor X X X Multiplex Editing Precision Engineering Larger Payloads Patients Need Genome Engineering To Address Medical Needs with Next-Generation Products Medical Need Artisan Solution Relapse caused by “antigen escape” and low solid tumor antigen expression Dual- and Tri-CAR Cell Therapies Cost, long vein-to-vein time, and lack of patient access “Off-The-Shelf” and “FAST” Auto Approaches Overcoming solid tumor microenvironment Tumor homing for intratumoral CAR-T infiltration Low allogeneic persistence Immune evasion edits that knockout HLA-I, HLA- II, and insert HLA-E for NK evasion
- 17. Copyright 2022 ArtisanBio. All Rights Reserved. MULTIPLEX BUILDS: Solid Tumor Homing CAR-T ENGINEERED SOLUTIONS CAR A target highly expressed on solid tumor and limited expression on health tissues. Chemokine Homing Chemokine receptor enables CAR-T to home towards tumors and serves to increase drug concentration and ultimately efficacy. Allo Chassis Allogeneic edits enable “off-the-shelf” therapeutics that decrease manufacturing costs and increase drug availability to patients while preventing GvHD. HLA-II KO HLA-I KO TCR KO Chemokine Homing CD4+ T Cell CD8+ T Cell CAR Graft vs. Host Disease X X X
- 18. Copyright 2022 ArtisanBio. All Rights Reserved. Step 1: Technology transfer of T cell editing process RNP Delivery RNP Preparation STAR Nuclease 1.0 STAR gRNA 1.0 CD3 Isolation EasySep™ Human T Cell Isolation Kit Cell Sourcing Human Peripheral Blood Leukopak, Frozen Design STAR gRNA 1.0 Pre-Editing Cell Culture ImmunoCult™-XF T Cell Expansion Medium ImmunoCult™ Human CD3/CD28 T Cell Activator Human Recombinant IL-2 Cryopreservation CryoStor® CS10 T cell Expansion ImmunoCult™-XF T Cell Expansion Medium Human Recombinant IL-2 Downstream Processing / Analysis Flow Cytometry (% KO, % KI) Image-Based Cell Killing Assay (Cell Potency) Amplicon-Seq (% INDELs) rhAmpSeq (off-target nomination)
- 19. Copyright 2022 ArtisanBio. All Rights Reserved. Step 2: Risk assessment A risk assessment dictates the potential critical process parameters for the intermediate quality attributes. Cell Source Pre-Editing Culture Cargo Preparation Cargo Delivery T cell Expansion Intermediate Quality Attributes Parameter Rank Factor Q 3 Factor R 3 Factor S 3 Factor T 2 Factor U 1 Factor V 2 Factor W 2 Parameter Rank Factor J 1 Factor K 3 Factor L 3 Factor M 2 Factor N OOS Factor O 2 Factor P OOS Parameter Rank Factor B OOS Factor C 3 Factor D OOS Factor E 3 Factor F OOS Factor G OOS Factor H OOS Factor I OOS Parameter Rank Factor X OOS Factor Y OOS Factor Z OOS Factor AA OOS Factor AB OOS Factor AC OOS KI Efficiency KO Efficiency Parameter Rank Factor A OOS Covariates Scale Highly Likely 3 Potentially 2 Unlikely/Unknown 1 Ranking of parameters by likely influence on intermediate quality attributes. Total Rank = KI Efficiency x KO Efficiency Parameters with highest Total Rank promoted to DOE. As a group, decided some factors were out of scope (OOS) for this body of work based on what was being investigated and the focus of the experiment.
- 20. Copyright 2022 ArtisanBio. All Rights Reserved. Step 3: DOE Screening Design Constants Values Cell Source Single Donor, Frozen Leukopak Culturing Reagents Stemcell Technologies Reagents for T Cell Therapy Research Transfection Conditions Buffer Method Levels 3 Factors 6 (5 continuous + 1 discrete) Resolution IV Replicates 3 Center Points 0 Blocks 1 Runs/Block 33 Total Runs 99 Definitive screening allows for main linear and quadratic effects, and two-way interactions characterization Controlled Factors Low Mid High Factor 1 -1 0 1 Factor 2 -1 0 1 Factor 3 -1 0 1 Factor 4 -1 0 1 Factor 5 -1 0 1 Factor 6 -1 - 1 Screening Design Construction Pre-Editing Culture Cargo Preparation Cargo Delivery T cell Expansion Intermediate Quality Attributes VCD/Viability Phenotype Cell Cycle Analysis VCD/Viability Phenotype VCD/Viability Phenotype Cell Cycle Analysis VCD/Viability Phenotype Editing Efficiency KI Efficiency KO Efficiency Analytics
- 21. Copyright 2022 ArtisanBio. All Rights Reserved. Step 4: Model Fit and Critical Factors Identification CAR KI RMSE 0.08 R-Squared 0.89 P-value < 0.001 KI Efficiency Model Fit Directionality of critical factors identified, and initial predictive model created Identified significant main factor for KI efficiency within the solution space of this DOE. Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Solution Space Definition Solution Space Reduction
- 22. Copyright 2022 ArtisanBio. All Rights Reserved. Step 4: Model Fit and Critical Factors Identification Triple KO RMSE 0.0922 R-Squared 0.91 P-value < 0.0001 KO Efficiency Model Fit Directionality of critical factors identified, and initial predictive model created Preliminary operating range identified with significant decrease in KO efficiency at low levels of respective factors. Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Solution Space Definition Solution Space Reduction
- 23. Copyright 2022 ArtisanBio. All Rights Reserved. Initial data displaying high editing efficiency potential Non-viral Insertion (3.2 kb) + Triple Knock-Out (3 gRNA) % Knock-Out (flow) Compan y Product CTX-110 CTX-120 CTX-130 Max 90.8% Allo Chassis % Knock-In (flow) Company Product UCAR19 CTX-110 Allo Chassis +miniplasmid ɑβT cells ɑβT cells Editing that partners have indicated they have obtained internally with Cas9 after optimization DOE design identifies significant factors and high-impact interactions, laying the groundwork for efficient on-target editing in T cells using Artisan’s STAR-CRISPR Platform. Competitor data references publicly available disclosures from other CRISPR editing technologies and the companies using their platforms
- 24. Copyright 2022 ArtisanBio. All Rights Reserved. Next Steps: Process optimization, characterization and validation Knowledge Space Solution Space Design Space Control Space Set Point Process Understanding Input Output Process Development Process Characterization Process Validation
- 25. Copyright 2022 ArtisanBio. All Rights Reserved. Systems approach is applicable across cell types: HSC Fold Expansion and CAR KI Factor 1 Factor 2 Factor 3 Early data showing improved HSC cell health and insert expression Response 1 Response 2 Response 3 Response 4 Multiple factors identified that are critical for the chosen responses Single and Triple Knock Out Flow cytometry Insertion (3.5 kb) Flow cytometry CD34 expression Flow cytometry 78.5% 19.5% Max 27.2% 95.4% 97.5% No loss of CD34 expression post-editing HSCs edited with CD19- CAR template into B2M Singleplex KO: B2M Multiplex KO: B2M, CIITA, TRAC
- 26. Copyright 2022 ArtisanBio. All Rights Reserved. Systems approach is applicable across cell types: iPSC Preliminary data of iPSC editing showing improved performance compared to alternatives Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Factor 6 Factor 3 Viability Yield KI DoE identified multiple factors that positively impact KI efficiency data to further improve editing efficiency 3.4% 13.1% 29.3% PoC DoE 1 DoE 2 % Knock-In (flow) % Single HLA-I Knock-Out (flow) Cas9 Company Nuclease STAR- CRISPR MAD7 Company Nuclease HiFi Cas9 Cas9 MAD7 Cas-Clover Insert 1 Insert 2 STAR- CRISPR % Knock-In (flow) Competitor data references publicly available disclosures from other CRISPR editing technologies and the companies using their platforms
- 27. Copyright 2022 ArtisanBio. All Rights Reserved. Systems Approach • QbD principles provide the framework for DOE deployment for process development • Systems approach is iterative • DOE is the fastest, cheapest (leanest) way to results • Deployed with great success within Artisan enabling accelerated process understanding to improve editing efficiency Know-How Summary Protocols & Builds Platform & Licenses
- 28. Copyright 2022 ArtisanBio. All Rights Reserved. Process and Analytical Development Cell Biology and Immunology Synthetic Biology Acknowledgements Panos Chrysanthopoulos Kristina Collavino Stephie Leung Hala Abdouni Tolga Barker Connor Yanchus Luca Orlando Calley Hirsch Jessica Schwaber Josh Miller Javier Hernandes Alica Luca Roland Baumgartner Business Development Laurén Kinner