Poster · 2022 · SITC

Generation of cell therapies for diverse applications using microfluidic Cell Squeeze® manufacturing technology

Jonathan Gilbert, Maisam Dadgar, Scott Loughhead, Ipsita Roymoulik, Devin Bridgen, Howard Bernstein, Armon Sharei

Publication text

Generation of cell therapies for diverse applications using microfluidic Cell Squeeze® manufacturing technology Jonathan Gilbert PhD, Maisam Dadgar, Scott Loughhead PhD, Ipsita Roymoulik, Devin Bridgen PhD, Howard Bernstein MD PhD, Armon Sharei PhD SQZ Biotechnologies Company, Watertown, MA, USA

Background

Cellular therapies require precise and versatile biological engineering at scale to generate effective and accessible products. However, current cell delivery approaches are often limited in the types of biology and range of cells they can engineer and have significant scale up challenges. Microfluidic deformation using the Cell Squeeze® technology effectively delivers a variety of payloads to diverse cell types at scale while preserving cell health and function. Cell Squeeze® Technology with differentiated capabilities R

Conclusions

We showed highly efficient delivery (>80%) to immune cells, iPSCs, and TILs while also maintaining cell function.
Demonstrated clinical scale engineering (~10B cells/minute) and in use for 3 clinical trials generating distinct

investigational products.

Our point-of-care system demonstrated a fully closed automated unit for manufacturing cell therapies that

integrates cell isolation, cell washing, cell delivery and bag filling with the potential to be operated outside of a clean room. This system also reduced our operator hours by 90% and process time by ~50% to < 6 hours.

These results support the opportunity for continued expansion of the clinical impact of the Cell Squeeze®

technology leveraging the flexibility of the delivery platform.

Interested parties should reach out to SQZ at platform@sqzbiotech.com

or visit https://sqzbiotech.com/manufacturing/ Immune Cells RBCs HSC/iPSCs Gene editing complexes Proteins and peptides Nanomaterials Nucleic Acids Small molecules Robust across material classes Preservation of cell function Applicable to diverse cell types EP Squeeze Electroporation (EP) results in substantial gene misregulation vs. squeezing Cells and target cargo together in suspension Cells are squeezed through SQZ chip at high speeds Cell membranes are temporarily disrupted Target cargo enters the cytosol of the cells Membranes reseal Broaden Therapeutic Possibilities Through Multi-Dimensional Cell Engineering

Transient engineering capabilities could eliminate need for preconditioning and cytokine support
Allows for potential broader applicability of CAR-Ts and TILs, for example, to more tumor types and earlier treatment lines

Potentially Improving Safety and Broadening Applicability of T cell therapies TME Modifying Persistence Homing Checkpoint knockdown Demonstration of enhanced proliferation and killing in the absence of conventional supplemental cytokines

Cell fate reprogramming through multiplexed mRNA delivery
Control of transcription factor timing and ratios
Potential to unlock applications for iPSC-derived therapies and regenerative medicine

without viral methods or complex lengthy small molecule approaches Supporting Creation of New Therapeutic Candidates Single-step delivery of multiple RNAs in iPSCs for cellular reprogramming Co-delivery of 6 transcription factor mRNAs generated neurons expressing mature dopaminergic markers Day1 Day4 10000 15000 20000 25000 30000

Foxa2

Time after squeeze delivery Normalized to Control fold increase Day1 Day4 TH Time after squeeze delivery 6 TFs Human iPSCs 6 RNA factors Up to 25000x increase in target genes Expression of key dopaminergic markers Maintenance of Normal T cell Proliferation 2 Efficient HSC Engraftment Without Rest 6 Cell Squeeze® Technology vs. Electroporation Automated, Fully Closed, Integrated Manufacturing System for Cell Therapy Products No rest time and faster proliferation Fewer operator-hours Potential for ~ 90% reduction in operator-hours 6 <10hr manufacture time with POC process for SQZ® red blood cell product Potential to operate outside a conventional ISO7 clean room Drivers of Cell Therapy Cost Clean room time GMP Operators Materials/ virus if applicable Spleen chimerism in mouse model measured at wk 8 Ctrl Squeeze EP 10 6 10 7 10 8 10 9 Cell count Cells after 7 days activation 16x Ctrl Squeeze EP Spleen chimerism % human cells ~3x Immune Stem Other Primary Experience with Diverse Cells and Molecules PBMCs + mRNAs Add and upregulate immune stimulatory functions with mRNAs T cells + RNPs Multiplexed gene editing with B2M, TIM-3 and TRAC RNPs DCs + siRNAs siRNA knockdown of cell surface proteins in T cells and DCs 4 T cells + mRNAs Expression of codelivered mRNAs in T cells 3 iPSCs + mRNAs iPSC reprogramming via delivery of 6 transcription factor mRNAs 5 HSCs + mRNAs Upregulation of a cell surface receptor in HSCs via mRNA 6 HSCs + RNPs Multiplexed gene editing with B2M and CCR5 RNPs 6 RBCs + Peptides Introduction of peptide epitopes into RBC antigen carriers 8 RBCs + Peptides + Adjuvant Co-delivery of antigenic peptides and adjuvant 9 Fibroblasts + Proteins Delivery of protein transcription factors 7 Cell Squeeze® system allows for improved product quality and manufacturing cost relative to current processes High-Performance, High-Quality and Flexible Engineering with the Cell Squeeze® GMP System GMP System currently in use to produce 3 investigational cell therapies for Phase 1/ 2 trials in the US, Canada and EU High-Performance High-Quality Flexible Designed for high-throughput, efficient cell engineering to use in a range of cell therapy manufacturing processes

Rapid processing rate: ~10B nucleated cells/min and

capable of up to 1T RBCs per minute

Demonstrated efficient delivery at GMP production scale
Fully closed system maintains aseptic process conditions
Single use, sterile disposable SQZ® kit
Suitable for clean room environments
Built-in integrity test
Potential for seamless system compatibility with

proprietary SQZ® chips

Library of SQZ® chips and disposables may be

leveraged for future applications Advancing the future of integrated, automated cell therapy manufacturing with the SQZ® POC System Potential to drive powerful impacts for cell therapy manufacturing, designed to address primary cost and quality drivers SQZ® Point-of-Care enables more cell engineering possibilities while driving to reduce the time, cost and complexity of manufacturing Potential to eliminate clean room Potential for decentralized or on-site production without a clean room Potentially Faster Manufacturing Potential for improvement in manufacturing speed Removal of Manual Steps Substantial removal of manual steps to improve process reproducibility Fewer Operators Potential for meaningful reduction in operator use and operator-hours New Therapeutic Approaches with Cell Squeeze® Technology: Transient mRNA for Lasting Impact in T cells and iPSCs Presented at ASGCT 2021 Efficient Engineering at GMP Production Scale Cell Squeeze® delivery in PBMCs with the GMP system demonstrated ~90% delivery efficiency, viability and recovery in large-scale, non-clinical production runs (billions of cells) Manufacturing Statistics for SQZ-PBMC-HPV-101 Phase 1/2 Trial Patient Leukapheresis SQZ® Process <24-hours QC and Batch Release Administration to Patient ~1 week Cell Squeeze® delivery step with the GMP system: ~ 1 min

Median viability for final cell therapy product: 91%
Functional delivery of antigenic cargo: HPV16 specific T cell clone assay

verified all patient lots were presenting antigen on MHC-I Manufacturing time and product dose yield:

Median manufacturing time was 17 hours for SQZ-PBMC-HPV and allowed

for a vein-to-vein time of about a week

Median of 5 doses available for each highest dose patient from a single

leukapheresis session Optimized mRNA delivery in iPSCs with >80% efficiency Case Study: Point-of-Care System Per Batch Statistics for SQZ® Engineered Red Blood Cell Product Candidate Per Batch Statistics Current SQZ® Process Process on POC System Average Processing Time 12 hrs 45 mins 5 hrs 45 mins Required operator hours 150 15 Average yield @ highest dose 9 doses 10 doses 0 hour 4 hours 24 hours 48 hours 8 days mbIL-2 Expression Time post squeeze mbIL-2+ (% of CD3+) 0 hour 4 hours 24 hours 48 hours 8 days mbIL-12 Expression Time post squeeze mbIL-12+ (% of CD3+) TIL Phenotype (Blood, Day 5)

Cd62l+cd45ro+

TCM-like

Cd62l-cd45ro+

TEM-like

Cd62l-cd45ro-

TEFF-like % total (gated on hCD45+ cells) ns ✱✱✱✱ ✱✱✱✱ Proportion Human Cells (Peripheral Blood) 0 1 2 3 4 5 6 Day Proportion of human CD45+ cells (gated on total CD45+ cells) Control mbIL-2 mbIL-2+12 Control+rhIL-2 IFN-γ ELISA IFN-γ (pg/mL) IFN-γ ELISA Tumor + Control TILs Tumor + mbIL-2/12 TILs (no exogenous cytokines) Tumor cells Caspase-3/7 mbIL-2/12 TILs exhibit improved persistence and memory-like phenotype in a mouse model Matched Patient Tumor Tumor and TIL co-culture in cytokine-free media Activation Tumor Killing SQZ® mRNA Engineered TIL mbIL-12 Oct4

Nanog

Sox2

Myc

Tert

0.0 0.5 1.0 1.5 fold change Minimal fold change in gene expression: squeeze compared to control Presented at ESMO 2022 Presented at ISSCR 2022 Presented at ASGCT 2022

References

[1]: Presented at AACR 2022 [2]: Presented at ASGCT 2021 [3]: Presented at SITC 2021 [4]: Sharei et al. 2015. PLOS One. https://doi.org/10.1371/journal.pone.0118803 [5]: Presented at ISSCR 2022 [6]: Presented at ASGCT 2022 [7]: Sharei et al. 2013. PNAS. https://doi.org/10.1073/pnas.1218705110 [8]: Raposo et al. 2022. Front. Immunol. https://doi.org/10.3389/fimmu.2022.869669 [9]: Presented at AACR 2022 Potential near-term clinical implementation Planned to be clinically implemented in 1H 2023 with SQZ TAC Program

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Some publications reference SQZ Biotechnologies, a company Dr. Armon Sharei and colleagues previously founded. Portal is the next generation company leveraging Dr. Sharei's platform technology.

# Generation of cell therapies for diverse applications using microfluidic Cell Squeeze® manufacturing technology This page is a text version of a Portal Biotechnologies publication. The bibliographic record is labeled in the text so it can be read without the surrounding website. ## Bibliographic record - Title: Generation of cell therapies for diverse applications using microfluidic Cell Squeeze® manufacturing technology - Document type: Poster - Year: 2022 - Outlet: SITC - Page URL: https://portal.bio/publications/2022-generation-of-cell-therapies-for-diverse-applications-using-microfluidic-cell-squeeze--ajkGgBEAACsAc96W - Markdown URL: https://portal.bio/publications/2022-generation-of-cell-therapies-for-diverse-applications-using-microfluidic-cell-squeeze--ajkGgBEAACsAc96W.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65ccd6169be9a5b998b5d09c_SITC_2022_Platform_Poster_FINAL.pdf ## Summary Jonathan Gilbert, Maisam Dadgar, Scott Loughhead, Ipsita Roymoulik, Devin Bridgen, Howard Bernstein, Armon Sharei ## Publication text Generation of cell therapies for diverse applications using microfluidic Cell Squeeze® manufacturing technology Jonathan Gilbert PhD, Maisam Dadgar, Scott Loughhead PhD, Ipsita Roymoulik, Devin Bridgen PhD, Howard Bernstein MD PhD, Armon Sharei PhD SQZ Biotechnologies Company, Watertown, MA, USA ### Background Cellular therapies require precise and versatile biological engineering at scale to generate effective and accessible products. However, current cell delivery approaches are often limited in the types of biology and range of cells they can engineer and have significant scale up challenges. Microfluidic deformation using the Cell Squeeze® technology effectively delivers a variety of payloads to diverse cell types at scale while preserving cell health and function. Cell Squeeze® Technology with differentiated capabilities R ### Conclusions - We showed highly efficient delivery (>80%) to immune cells, iPSCs, and TILs while also maintaining cell function. - Demonstrated clinical scale engineering (~10B cells/minute) and in use for 3 clinical trials generating distinct investigational products. - Our point-of-care system demonstrated a fully closed automated unit for manufacturing cell therapies that integrates cell isolation, cell washing, cell delivery and bag filling with the potential to be operated outside of a clean room. This system also reduced our operator hours by 90% and process time by ~50% to < 6 hours. - These results support the opportunity for continued expansion of the clinical impact of the Cell Squeeze® technology leveraging the flexibility of the delivery platform. - Interested parties should reach out to SQZ at platform@sqzbiotech.com or visit https://sqzbiotech.com/manufacturing/ Immune Cells RBCs HSC/iPSCs Gene editing complexes Proteins and peptides Nanomaterials Nucleic Acids Small molecules Robust across material classes Preservation of cell function Applicable to diverse cell types EP Squeeze Electroporation (EP) results in substantial gene misregulation vs. squeezing Cells and target cargo together in suspension Cells are squeezed through SQZ chip at high speeds Cell membranes are temporarily disrupted Target cargo enters the cytosol of the cells Membranes reseal Broaden Therapeutic Possibilities Through Multi-Dimensional Cell Engineering - Transient engineering capabilities could eliminate need for preconditioning and cytokine support - Allows for potential broader applicability of CAR-Ts and TILs, for example, to more tumor types and earlier treatment lines Potentially Improving Safety and Broadening Applicability of T cell therapies TME Modifying Persistence Homing Checkpoint knockdown Demonstration of enhanced proliferation and killing in the absence of conventional supplemental cytokines - Cell fate reprogramming through multiplexed mRNA delivery - Control of transcription factor timing and ratios - Potential to unlock applications for iPSC-derived therapies and regenerative medicine without viral methods or complex lengthy small molecule approaches Supporting Creation of New Therapeutic Candidates Single-step delivery of multiple RNAs in iPSCs for cellular reprogramming Co-delivery of 6 transcription factor mRNAs generated neurons expressing mature dopaminergic markers Day1 Day4 10000 15000 20000 25000 30000 ### Foxa2 Time after squeeze delivery Normalized to Control fold increase Day1 Day4 TH Time after squeeze delivery 6 TFs Human iPSCs 6 RNA factors Up to 25000x increase in target genes Expression of key dopaminergic markers Maintenance of Normal T cell Proliferation 2 Efficient HSC Engraftment Without Rest 6 Cell Squeeze® Technology vs. Electroporation Automated, Fully Closed, Integrated Manufacturing System for Cell Therapy Products No rest time and faster proliferation Fewer operator-hours Potential for ~ 90% reduction in operator-hours 6 <10hr manufacture time with POC process for SQZ® red blood cell product Potential to operate outside a conventional ISO7 clean room Drivers of Cell Therapy Cost Clean room time GMP Operators Materials/ virus if applicable Spleen chimerism in mouse model measured at wk 8 Ctrl Squeeze EP 10 6 10 7 10 8 10 9 Cell count Cells after 7 days activation 16x Ctrl Squeeze EP Spleen chimerism % human cells ~3x Immune Stem Other Primary Experience with Diverse Cells and Molecules PBMCs + mRNAs Add and upregulate immune stimulatory functions with mRNAs T cells + RNPs Multiplexed gene editing with B2M, TIM-3 and TRAC RNPs DCs + siRNAs siRNA knockdown of cell surface proteins in T cells and DCs 4 T cells + mRNAs Expression of codelivered mRNAs in T cells 3 iPSCs + mRNAs iPSC reprogramming via delivery of 6 transcription factor mRNAs 5 HSCs + mRNAs Upregulation of a cell surface receptor in HSCs via mRNA 6 HSCs + RNPs Multiplexed gene editing with B2M and CCR5 RNPs 6 RBCs + Peptides Introduction of peptide epitopes into RBC antigen carriers 8 RBCs + Peptides + Adjuvant Co-delivery of antigenic peptides and adjuvant 9 Fibroblasts + Proteins Delivery of protein transcription factors 7 Cell Squeeze® system allows for improved product quality and manufacturing cost relative to current processes High-Performance, High-Quality and Flexible Engineering with the Cell Squeeze® GMP System GMP System currently in use to produce 3 investigational cell therapies for Phase 1/ 2 trials in the US, Canada and EU High-Performance High-Quality Flexible Designed for high-throughput, efficient cell engineering to use in a range of cell therapy manufacturing processes - Rapid processing rate: ~10B nucleated cells/min and capable of up to 1T RBCs per minute - Demonstrated efficient delivery at GMP production scale - Fully closed system maintains aseptic process conditions - Single use, sterile disposable SQZ® kit - Suitable for clean room environments - Built-in integrity test - Potential for seamless system compatibility with proprietary SQZ® chips - Library of SQZ® chips and disposables may be leveraged for future applications Advancing the future of integrated, automated cell therapy manufacturing with the SQZ® POC System Potential to drive powerful impacts for cell therapy manufacturing, designed to address primary cost and quality drivers SQZ® Point-of-Care enables more cell engineering possibilities while driving to reduce the time, cost and complexity of manufacturing Potential to eliminate clean room Potential for decentralized or on-site production without a clean room Potentially Faster Manufacturing Potential for improvement in manufacturing speed Removal of Manual Steps Substantial removal of manual steps to improve process reproducibility Fewer Operators Potential for meaningful reduction in operator use and operator-hours New Therapeutic Approaches with Cell Squeeze® Technology: Transient mRNA for Lasting Impact in T cells and iPSCs Presented at ASGCT 2021 Efficient Engineering at GMP Production Scale Cell Squeeze® delivery in PBMCs with the GMP system demonstrated ~90% delivery efficiency, viability and recovery in large-scale, non-clinical production runs (billions of cells) Manufacturing Statistics for SQZ-PBMC-HPV-101 Phase 1/2 Trial Patient Leukapheresis SQZ® Process <24-hours QC and Batch Release Administration to Patient ~1 week Cell Squeeze® delivery step with the GMP system: ~ 1 min - Median viability for final cell therapy product: 91% - Functional delivery of antigenic cargo: HPV16 specific T cell clone assay verified all patient lots were presenting antigen on MHC-I Manufacturing time and product dose yield: - Median manufacturing time was 17 hours for SQZ-PBMC-HPV and allowed for a vein-to-vein time of about a week - Median of 5 doses available for each highest dose patient from a single leukapheresis session Optimized mRNA delivery in iPSCs with >80% efficiency Case Study: Point-of-Care System Per Batch Statistics for SQZ® Engineered Red Blood Cell Product Candidate Per Batch Statistics Current SQZ® Process Process on POC System Average Processing Time 12 hrs 45 mins 5 hrs 45 mins Required operator hours 150 15 Average yield @ highest dose 9 doses 10 doses 0 hour 4 hours 24 hours 48 hours 8 days mbIL-2 Expression Time post squeeze mbIL-2+ (% of CD3+) 0 hour 4 hours 24 hours 48 hours 8 days mbIL-12 Expression Time post squeeze mbIL-12+ (% of CD3+) TIL Phenotype (Blood, Day 5) ### Cd62l+cd45ro+ TCM-like ### Cd62l-cd45ro+ TEM-like ### Cd62l-cd45ro- TEFF-like % total (gated on hCD45+ cells) ns ✱✱✱✱ ✱✱✱✱ Proportion Human Cells (Peripheral Blood) 0 1 2 3 4 5 6 Day Proportion of human CD45+ cells (gated on total CD45+ cells) Control mbIL-2 mbIL-2+12 Control+rhIL-2 IFN-γ ELISA IFN-γ (pg/mL) IFN-γ ELISA Tumor + Control TILs Tumor + mbIL-2/12 TILs (no exogenous cytokines) Tumor cells Caspase-3/7 mbIL-2/12 TILs exhibit improved persistence and memory-like phenotype in a mouse model Matched Patient Tumor Tumor and TIL co-culture in cytokine-free media Activation Tumor Killing SQZ® mRNA Engineered TIL mbIL-12 Oct4 ### Nanog ### Sox2 ### Myc ### Tert 0.0 0.5 1.0 1.5 fold change Minimal fold change in gene expression: squeeze compared to control Presented at ESMO 2022 Presented at ISSCR 2022 Presented at ASGCT 2022 ### References [1]: Presented at AACR 2022 [2]: Presented at ASGCT 2021 [3]: Presented at SITC 2021 [4]: Sharei et al. 2015. PLOS One. https://doi.org/10.1371/journal.pone.0118803 [5]: Presented at ISSCR 2022 [6]: Presented at ASGCT 2022 [7]: Sharei et al. 2013. PNAS. https://doi.org/10.1073/pnas.1218705110 [8]: Raposo et al. 2022. Front. Immunol. https://doi.org/10.3389/fimmu.2022.869669 [9]: Presented at AACR 2022 Potential near-term clinical implementation Planned to be clinically implemented in 1H 2023 with SQZ TAC Program ## Provenance Some publications reference SQZ Biotechnologies, a company Dr. Armon Sharei and colleagues previously founded. Portal is the next generation company leveraging Dr. Sharei's platform technology.