Poster · 2020 · SITC

Generating Enhanced Tumor Infiltrating Lymphocytes through Microfluidic Cell Squeezing

Devin T. Bridgen, Colin Thalhofer, Elaine Ballinger, Rebecca J. Silver, Arindam Bhattacharjee, Ryan Montler, Andrew Weinberg, Armon Sharei, and Jonathan Gilbert

Cells: T cells
Cargos: Polymers, mRNA

Publication text

Overview Generating Enhanced Tumor Infiltrating Lymphocytes Through Microfluidic Cell Squeezing Devin T. Bridgen1*, Colin Thalhofer2*, Elaine Ballinger2, Rebecca J. Silver1, Arindam Bhattacharjee1, Ryan Montler2, Andrew Weinberg2, Armon Sharei1, and Jonathan Gilbert1 1SQZ Biotechnologies, Watertown MA; 2AgonOx, Inc, Portland, OR Background: Tumor Infiltrating Lymphocyte (TIL) therapies have shown significant solid tumor activity in patients, but current TIL compositions require patient lymphodepletion and high dose IL-2 after cell infusion to support clinical activity. Ex vivo engineering of TILs with mRNAs encoding membrane-bound cytokines enables use of potent cytokines, such as IL-2 or IL-12, that have limiting systemic toxicities but critically may become therapeutically feasible in membrane-bound form due to delivering only localized signaling. Engineered TILs have the potential to increase therapeutic functionality while removing the toxicity associated with lymphodepletion and/or IL-2 support, which may expand the patient population and potentially allow for repeat dosing and concomitant treatment with checkpoint therapies. Methods: To transiently overexpress membrane-bound cytokines, we used microfluidic cell squeezing (Cell Squeeze®) to deliver mbIL-2 and mbIL-12 mRNA to the cytosol of expanded tumor-reactive CD8 human TILs (AGX-148) from melanoma patients. After delivery, TILs were cultured in IL-2-free media with or without autologous tumor cells. Exogenous rhIL-2 and rhIL12 were used as a positive control. TILs were characterized over time by flow cytometry and tumor killing was monitored using an IncuCyte. Results: We demonstrated that mbIL-2 and mbIL-12 mRNA constructs delivered simultaneously by microfluidic cell squeezing to human TILs are highly expressed for multiple days while maintaining high viability in vitro. In an autologous tumor cell co-culture, TILs with mbIL-2+12 have increased proliferation over 3 days and increased granzyme B expression. Furthermore, TILs expressing mbIL-2+12 killed more autologous tumor cells over a 24-hour period than unmodified cells. Conclusions: Through microfluidic cell squeeze delivery of mRNAs, we have created enhanced TILs with high levels of membrane-bound cytokines in vitro. These cells can proliferate without exogenous IL-2 and have an improved functionality. Kinetics of mRNA Expression and Viability in Squeezed TILs IncuCyte Tumor Co-Culture Killing Enhanced Tumor Infiltrating Lymphocytes: Summary Engineering TILs with SQZ™ Technology Generation of Tumor-Reactive TILs (AGX-148) Figure 1. AGX-148 TILs from 2 donors were thawed, cultured overnight, and squeezed with no mRNA (Control TIL), or both mbIL-2 mRNA and mbIL-12 mRNA at either 0.25 or 0.5 mg/mL, as indicated. Cells were subsequently cultured without exogenous rhIL2 for 3 days and viability (A), mbIL-2 expression (B), and mbIL-12 expression (C) were quantified by flow. Representative histograms of mbIL-2 (D) or mbIL12 (E) expression are shown 1-day post-squeeze. A) Figure 3. AGX-148 TILs from Figure 2 were analyzed for Granzyme B (GZMB) expression by flow cytometry after 20 hours of co-culture with autologous tumor cells. Figure 4. AGX-148 TILs were cultured in cytokine-free media at a 2:1 ratio of autologous tumor cell lines stained with Cytolight Red and imaged for 24 hours using an IncuCyte fluorescent microscope. Control TIL cells in media with 3000 IU/mL rhIL-2 and 10 ng/mL rhIL-12 were used as a positive control. Representative images at the beginning of the co-culture (0 hours) and after 24 hours are shown from (A) Donor 1 and (B)Donor 2. A red mask was applied to the IncuCyte images and used to calculate % tumor confluence. A) Donor 1 B) Donor 2 Control TIL+ rhIL-2+12Control TIL B) Cell Proliferation in Tumor Co-Culture at 3 Days Granzyme B Expression in Tumor Co-Culture at 20 Hours Figure 2. AGX-148 TILs isolated from three melanoma patients’ tumor tissue were thawed, cultured overnight in rhIL-2 free media, then squeezed with no mRNA (Control TIL), mbIL-2, or both mbIL-2 and mbIL-12 (mbIL-2+12). Cells were cultured for 3 days in the presence of autologous tumor cells. As a positive control, rhIL-2 @ 3000 IU/mL and rhIL-12 @ 10 ng/mL were added to the culture medium where indicated. After 3 days in culture, TILs were analyzed for (A) number of viable CD8+ T cells in each sample and (B) Ki67 expression by flow cytometry. Tumor tissue samples undergo enzymatic digestion, mechanical dissociation, and CD8+ bead enrichment. The AGX-148 TILs (CD39+ CD103+ T cells) are then isolated using a cell sorter. The TILs are expanded using a primary expansion protocol followed by a rapid expansion protocol in G-Rex flasks.0 5×10 4 1×10 5 1.5×10 5 # Viable CD8+ Events0 5×10 3 1×10 4 1.5×10 4 2×10 4 2.5×10 4 # Viable CD8+ Events0 6 12 18 24 -80 -60 -40 -20 Hours % Change in Tumor Confluence0 6 12 18 24 -80 -60 -40 -20 Hours % Change In Tumor Confluence0 % Ki67 Positive0 % Ki67 Positive0 % GZMB Positive0 % GZMB Positive0 % GZMB Positive

A) B)0

% Ki67 Positive C)

D)0 1 2 3

% Viability0 1 2 3 % mbIL-2 Expression0 1 2 3 % mbIL-12 Expression Squeeze mbIL-2+12 (0.25mg/mL) Squeeze mbIL-2+12 (0.5mg/mL) mbIL-2 mbIL-12 E) + rhIL-2+12 24 Hours 0 Hours24 Hours 0 Hours Control TIL Squeeze mbIL-2 Squeeze mbIL-2+12 Control TIL + rhIL-2+12 IL-2 Available to TILs in Patient Proposed mbIL-2-TILs: Cells supported with consistent and localized IL-2 for >1 day Current Systemic IL-2 Support: Proleukin dosing every 8 to 12 hours for up to 6 doses unless toxicity limit reached. Systemic exposure with elimination half life of <1.5hrs. 8-12 hrs TIL isolation and expansion Patient preconditioning Systemic IL-2 (Proleukin) TIL Treatment Overview Conceptual Kinetics: mbIL-2 vs Systemic IL-2 Tumor Processing Tumor-Reactive TIL Isolation TIL Expansion Cells and target mRNA together in suspension Cells are squeezed through chip at high speeds Cell membranes are temporarily disrupted Target mRNA enters the cytosol of the cells Membranes reseal mbIL-2 mbIL-12 Expression of target mRNA

Cell Squeeze® Technology enables efficient primary immune cell engineering while preserving cell function
Can efficiently deliver a variety and mixtures of cargo types, including proteins, peptides, nucleic acids, and RNPs
cGMP technology in use in multiple clinical trials and can process >10B cells/min
Membrane bound cytokines localize to the surface of the target TIL, minimizing toxicity and enabling use of multiple

potent cytokines like IL-2 and IL-12

Enhanced TILs express mRNA for membrane-bound cytokines IL-2 and IL-12

for multiple days, exposing TILs to cytokine support for similar lengths of time to current systemic IL-2 treatment (typically 6 doses over 2-3 days)

When co-cultured with autologous tumor in cytokine-free media, enhanced

TILs proliferate, as well as upregulate Ki67 and Granzyme B. This reflects an enhanced ability to proliferate and kill tumor cells.

Enhanced TILs kill autologous tumor cells in cytokine-free media at similar

rates to TILs supported with exogenous IL-2+IL-12 Together, enhanced TILs made from CD8+ human AGX-148 TILs have the potential to generate a differentiated TIL product with increased potency and free from the toxicity profile of systemic IL-2. Building upon the clinically implemented Cell Squeeze® Technology, enhanced TILs could expand the patient population eligible for adoptive cell transfer (ACT) by removing the requirements for high-dose IL-2, which is associated with severe side effects and currently limits the potential of TIL adoptive cell therapies. mbIL-2 mbIL-12 Control TIL+ rhIL-2+12Control TIL Squeeze mbIL-2 mbIL-2 TILs Current TILs + rhIL-2+120 2×10 4 4×10 4 6×10 4 # Viable CD8+ Events

Cd103

Cd39

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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.

# Generating Enhanced Tumor Infiltrating Lymphocytes through Microfluidic Cell Squeezing 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: Generating Enhanced Tumor Infiltrating Lymphocytes through Microfluidic Cell Squeezing - Document type: Poster - Year: 2020 - Outlet: SITC - Cells: T cells - Cargos: Polymers, mRNA - Page URL: https://portal.bio/publications/2020-generating-enhanced-tumor-infiltrating-lymphocytes-through-microfluidic-cell-squeezin--ajkGxxEAACkAc99v - Markdown URL: https://portal.bio/publications/2020-generating-enhanced-tumor-infiltrating-lymphocytes-through-microfluidic-cell-squeezin--ajkGxxEAACkAc99v.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65cce1649be9a5b998b5d199_SITC2021TILPosterFinal165.pdf ## Summary Devin T. Bridgen, Colin Thalhofer, Elaine Ballinger, Rebecca J. Silver, Arindam Bhattacharjee, Ryan Montler, Andrew Weinberg, Armon Sharei, and Jonathan Gilbert ## Publication text Overview Generating Enhanced Tumor Infiltrating Lymphocytes Through Microfluidic Cell Squeezing Devin T. Bridgen1*, Colin Thalhofer2*, Elaine Ballinger2, Rebecca J. Silver1, Arindam Bhattacharjee1, Ryan Montler2, Andrew Weinberg2, Armon Sharei1, and Jonathan Gilbert1 1SQZ Biotechnologies, Watertown MA; 2AgonOx, Inc, Portland, OR Background: Tumor Infiltrating Lymphocyte (TIL) therapies have shown significant solid tumor activity in patients, but current TIL compositions require patient lymphodepletion and high dose IL-2 after cell infusion to support clinical activity. Ex vivo engineering of TILs with mRNAs encoding membrane-bound cytokines enables use of potent cytokines, such as IL-2 or IL-12, that have limiting systemic toxicities but critically may become therapeutically feasible in membrane-bound form due to delivering only localized signaling. Engineered TILs have the potential to increase therapeutic functionality while removing the toxicity associated with lymphodepletion and/or IL-2 support, which may expand the patient population and potentially allow for repeat dosing and concomitant treatment with checkpoint therapies. Methods: To transiently overexpress membrane-bound cytokines, we used microfluidic cell squeezing (Cell Squeeze®) to deliver mbIL-2 and mbIL-12 mRNA to the cytosol of expanded tumor-reactive CD8 human TILs (AGX-148) from melanoma patients. After delivery, TILs were cultured in IL-2-free media with or without autologous tumor cells. Exogenous rhIL-2 and rhIL12 were used as a positive control. TILs were characterized over time by flow cytometry and tumor killing was monitored using an IncuCyte. Results: We demonstrated that mbIL-2 and mbIL-12 mRNA constructs delivered simultaneously by microfluidic cell squeezing to human TILs are highly expressed for multiple days while maintaining high viability in vitro. In an autologous tumor cell co-culture, TILs with mbIL-2+12 have increased proliferation over 3 days and increased granzyme B expression. Furthermore, TILs expressing mbIL-2+12 killed more autologous tumor cells over a 24-hour period than unmodified cells. Conclusions: Through microfluidic cell squeeze delivery of mRNAs, we have created enhanced TILs with high levels of membrane-bound cytokines in vitro. These cells can proliferate without exogenous IL-2 and have an improved functionality. Kinetics of mRNA Expression and Viability in Squeezed TILs IncuCyte Tumor Co-Culture Killing Enhanced Tumor Infiltrating Lymphocytes: Summary Engineering TILs with SQZ™ Technology Generation of Tumor-Reactive TILs (AGX-148) Figure 1. AGX-148 TILs from 2 donors were thawed, cultured overnight, and squeezed with no mRNA (Control TIL), or both mbIL-2 mRNA and mbIL-12 mRNA at either 0.25 or 0.5 mg/mL, as indicated. Cells were subsequently cultured without exogenous rhIL2 for 3 days and viability (A), mbIL-2 expression (B), and mbIL-12 expression (C) were quantified by flow. Representative histograms of mbIL-2 (D) or mbIL12 (E) expression are shown 1-day post-squeeze. A) Figure 3. AGX-148 TILs from Figure 2 were analyzed for Granzyme B (GZMB) expression by flow cytometry after 20 hours of co-culture with autologous tumor cells. Figure 4. AGX-148 TILs were cultured in cytokine-free media at a 2:1 ratio of autologous tumor cell lines stained with Cytolight Red and imaged for 24 hours using an IncuCyte fluorescent microscope. Control TIL cells in media with 3000 IU/mL rhIL-2 and 10 ng/mL rhIL-12 were used as a positive control. Representative images at the beginning of the co-culture (0 hours) and after 24 hours are shown from (A) Donor 1 and (B)Donor 2. A red mask was applied to the IncuCyte images and used to calculate % tumor confluence. A) Donor 1 B) Donor 2 Control TIL+ rhIL-2+12Control TIL B) Cell Proliferation in Tumor Co-Culture at 3 Days Granzyme B Expression in Tumor Co-Culture at 20 Hours Figure 2. AGX-148 TILs isolated from three melanoma patients’ tumor tissue were thawed, cultured overnight in rhIL-2 free media, then squeezed with no mRNA (Control TIL), mbIL-2, or both mbIL-2 and mbIL-12 (mbIL-2+12). Cells were cultured for 3 days in the presence of autologous tumor cells. As a positive control, rhIL-2 @ 3000 IU/mL and rhIL-12 @ 10 ng/mL were added to the culture medium where indicated. After 3 days in culture, TILs were analyzed for (A) number of viable CD8+ T cells in each sample and (B) Ki67 expression by flow cytometry. Tumor tissue samples undergo enzymatic digestion, mechanical dissociation, and CD8+ bead enrichment. The AGX-148 TILs (CD39+ CD103+ T cells) are then isolated using a cell sorter. The TILs are expanded using a primary expansion protocol followed by a rapid expansion protocol in G-Rex flasks.0 5×10 4 1×10 5 1.5×10 5 # Viable CD8+ Events0 5×10 3 1×10 4 1.5×10 4 2×10 4 2.5×10 4 # Viable CD8+ Events0 6 12 18 24 -80 -60 -40 -20 Hours % Change in Tumor Confluence0 6 12 18 24 -80 -60 -40 -20 Hours % Change In Tumor Confluence0 % Ki67 Positive0 % Ki67 Positive0 % GZMB Positive0 % GZMB Positive0 % GZMB Positive ### A) B)0 % Ki67 Positive C) ### D)0 1 2 3 % Viability0 1 2 3 % mbIL-2 Expression0 1 2 3 % mbIL-12 Expression Squeeze mbIL-2+12 (0.25mg/mL) Squeeze mbIL-2+12 (0.5mg/mL) mbIL-2 mbIL-12 E) + rhIL-2+12 24 Hours 0 Hours24 Hours 0 Hours Control TIL Squeeze mbIL-2 Squeeze mbIL-2+12 Control TIL + rhIL-2+12 IL-2 Available to TILs in Patient Proposed mbIL-2-TILs: Cells supported with consistent and localized IL-2 for >1 day Current Systemic IL-2 Support: Proleukin dosing every 8 to 12 hours for up to 6 doses unless toxicity limit reached. Systemic exposure with elimination half life of <1.5hrs. 8-12 hrs TIL isolation and expansion Patient preconditioning Systemic IL-2 (Proleukin) TIL Treatment Overview Conceptual Kinetics: mbIL-2 vs Systemic IL-2 Tumor Processing Tumor-Reactive TIL Isolation TIL Expansion Cells and target mRNA together in suspension Cells are squeezed through chip at high speeds Cell membranes are temporarily disrupted Target mRNA enters the cytosol of the cells Membranes reseal mbIL-2 mbIL-12 Expression of target mRNA - Cell Squeeze® Technology enables efficient primary immune cell engineering while preserving cell function - Can efficiently deliver a variety and mixtures of cargo types, including proteins, peptides, nucleic acids, and RNPs - cGMP technology in use in multiple clinical trials and can process >10B cells/min - Membrane bound cytokines localize to the surface of the target TIL, minimizing toxicity and enabling use of multiple potent cytokines like IL-2 and IL-12 1. Enhanced TILs express mRNA for membrane-bound cytokines IL-2 and IL-12 for multiple days, exposing TILs to cytokine support for similar lengths of time to current systemic IL-2 treatment (typically 6 doses over 2-3 days) 1. When co-cultured with autologous tumor in cytokine-free media, enhanced TILs proliferate, as well as upregulate Ki67 and Granzyme B. This reflects an enhanced ability to proliferate and kill tumor cells. 1. Enhanced TILs kill autologous tumor cells in cytokine-free media at similar rates to TILs supported with exogenous IL-2+IL-12 Together, enhanced TILs made from CD8+ human AGX-148 TILs have the potential to generate a differentiated TIL product with increased potency and free from the toxicity profile of systemic IL-2. Building upon the clinically implemented Cell Squeeze® Technology, enhanced TILs could expand the patient population eligible for adoptive cell transfer (ACT) by removing the requirements for high-dose IL-2, which is associated with severe side effects and currently limits the potential of TIL adoptive cell therapies. mbIL-2 mbIL-12 Control TIL+ rhIL-2+12Control TIL Squeeze mbIL-2 mbIL-2 TILs Current TILs + rhIL-2+120 2×10 4 4×10 4 6×10 4 # Viable CD8+ Events ### Cd103 ### Cd39 ## 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.