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STEMCELL Technologies
Global Biotech Leader Supporting Life Science Research
Immunology
Pluripotent
Stem Cell Biology
Hematopoietic
Stem Cell 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
Source Isolate
● Primary cells
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
Confidential | ArtisanBio | info@artisancells.com
Creating the Next Generation of
CRISPR-edited T cells
Steven Loo-Yong-Kee
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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.
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
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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)
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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)
Copyright 2022 Artisan Bio. 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)
Copyright 2022 Artisan Bio. 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.
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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)
Copyright 2022 Artisan Bio. 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.
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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
Copyright 2022 Artisan Bio. 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
How to Create CRISPR-Edited T Cells More Efficiently for Tomorrow's Cell Therapies

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  • 6. Copyright 2022 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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 Artisan Bio. 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