Poster · 2020 · SITC

Microfluidics Cell Squeezing Enables Potent Cellular Vaccines in Murine Models Through Direct Cytosolic Loading and Direct CD8 T Cell Priming

E. Ilker Ozay, Matthew G. Booty, Katarina Blagovic, David Soto, Olivia Pryor, Adam Stockmann, Disha Subramanya, Kelly Volk, Armon Sharei, Howard Bernstein, Defne Yarar, Katherine J. Seidl and Scott M. Loughhead

Cells: T cells, PBMCs, Monocytes, B cells, Dendritic Cells
Cargos: Polymers, Protein, Peptide

Publication text

Microfluidics Cell Squeezing Enables Potent Cellular Vaccines in Murine Models Through Direct Cytosolic Loading and Direct CD8 T Cell Priming E. Ilker Ozay, Matthew G. Booty, Katarina Blagovic, David Soto, Olivia Pryor, Adam Stockmann, Disha Subramanya, Kelly Volk, Armon Sharei, Howard Bernstein, Defne Yarar, Katherine J. Seidl and Scott M. Loughhead All Associated with SQZ Biotechnologies, Watertown, MA, USA SQZ APCs Lead To A Higher Influx of Antigenspecific CD8 T Cells with 15,000X Less Antigen Than The SLP Peptide Vaccine hours, and CD69 expression was examined on the responding OT-I’s to assess their activation. The minimal OVA epitope SIINFEKL was spiked into wells with cultured OT-I cells to elicit maximal CD69 expression as a positive control.

Background

The presentation of sufficient antigen on major histocompatibility complex class I (MHC-I) is essential to prime CD8 T cells. We employ our proprietary technology, Cell Squeeze® to deliver antigens directly to the cytosol of PBMCs to generate antigen presenting cells (APCs) that can robustly present on MHC-I. In addition to facilitating priming by professional APCs, this approach enables subsets within peripheral blood mononuclear cells (PBMCs) to function as conventional and unconventional APCs in both mouse and human preclinical models. Squeezed Mouse Splenocytes Rapidly Process and Present Antigen SQZ Technology and APC Platform Cytosolic Antigen Delivery (Cell Squeeze® Technology): Engineering Cells to Function as Antigen Presenting Cells: Traditionally, cross-presentation is needed for efficient presentation on MHC-I. SQZ delivers materials directly to cytosol, bypassing need for cross-presentation. Viability and cargo delivery in squeezed mouse splenocytes. Splenocytes were left untreated (NC), incubated at room temp. with fluorescent 3kDa dextran (Incub. ctrl) or squeezed in the presence of fluorescently-labeled dextran (Squeeze). Viability and dextran delivery of each cell type were immediately determined by flow cytometry.OT-I Alone Splenocytes (Squeeze + Protein) T Cells B Cells NK Cells Monocytes 2.5 5.0 Splenocytes (Squeeze + mRNA)

% Cd69+

Squeeze + OVA mRNA (g/mL)Squeeze + Protein Antigen Each cell subset within squeezed splenocytes can process and present antigen to CD8 T cells. Splenocytes were squeezed without cargo (Squeeze only) or squeezed in the presence of ovalbumin protein (squeeze+OVA, panel A) or ovalbumin mRNA (squeeze+OVA mRNA, panel B). Following the squeeze, cells were left untouched (Splenocytes) or cell subsets were purified by negative immunomagnetic bead selection (T Cells, B Cells, NK Cells, and Monocytes, panel C). The indicated cells were then co-cultured with transgenic OT-I CD8 T cells for 24 hours, and CD69 expression was examined on the responding OT-Is to assess their activation. The minimal OVA epitope SIINFEKL was spiked into wells with cultured OT-I cells to elicit maximal CD69 expression as a positive control.Untr. SQZ Protein (Ovalbumin) (of CD8+B220-)Untr. SQZ Synthetic Long Peptide

(hpv16 E7)

(of CD8+B220-) Squeezed Mouse APCs Directly Prime CD8 T Cells in vivo Squeezed murine splenocytes directly present antigen to CD8 T cells in vivo. (A) WT or MHC-I KO mice were immunized with 5x106 splenocytes either incubated (Incubation Control) or squeezed (SQZ) with ovalbumin protein. CFSE-labeled OT-I CD8 T cells were transferred into recipient animals prior to immunization to serve as a traceable responder CD8 population. Lymph nodes were harvested 3 days after immunization and analyzed by flow cytometry. (B) Mice were immunized with splenocytes squeezed with ovalbumin or HPV16 E7 synthetic long peptide (SLP). 7 days after immunization, spleens were harvested and re-stimulated with either the ovalbumin minimal epitope or the minimal epitope for HPV16 E7 to assess the percentage of antigen-specific CD8s. Splenic Response No Immunization → MHC-I-/- Incubation Ctrl. → Wild-type Ovalbumin Squeeze → Wild-type Incubation Ctrl. → MHC-I-/- Ovalbumin Squeeze → MHC-I-/-

Cfse

Lymph Node: Adjuvant Process Enhances Capabilities of SQZ APCs0 Tumor Vol. (mm3) Squeezed Splenocytes (IV) E7 SLP + CpG (Subcutaneous) 16 28 Prime Imm

Til

Analysis Day DayUntr. SLP % CD45+ (of Live) ns *** **Untr. SLP % CD8+ (of CD45+) *** *** *Untr. SLP % E7 Tetramer+ (of CD8+) *** *** * D CB Therapeutic immunization with squeezed splenocytes drives the expansion of tumor specific CD8 T cells. (A) Tumor growth kinetics for mice bearing TC1 tumors that were left untreated (black), vaccinated subcutaneously with an E7 synthetic long peptide plus CpG (orange), or immunized with splenocytes squeezed with the same E7 SLP (teal). (B-D) TIL analysis from the day 28 time point showing the intratumoral influx of CD45+ cells, CD8+ cells, and E7-tetramer+ CD8 T cells. CpG incubation phenotypically and functionally alters squeezed splenocytes resulting in higher antigenspecific CD8 T cells with augmented therapeutic benefit in a tumor model. (A) Squeezed splenocytes were incubated with the adjuvant, CpG. B cell surface expression of APC maturation markers, CD86 and MHC-I, was measured at 24 hours after the incubation. (B) Splenocytes were either squeezed with ovalbumin protein or E7 synthetic long peptide (SLP). Later, they were subjected to CpG incubation prior to injection into mice. Antigen-specific CD8 T cells in spleen were analyzed on day 7 post injection. (C) Tumor growth kinetics for mice bearing TC1 tumors that were left untreated (black), immunized with E7 SLPsqueezed splenocytes incubated with (teal) or without (blue) the CpG.0

Cd86 Mfi

CpG incub. - +0 10000 20000 30000 40000

Mhc-i Mfi

CpG incub. - +

C0.0

Ovalbumin CpG incub.: - - + Peptide Restim. (SIINFEKL) Media Ctrl. Untreated 1x106 squeezed splenocytes0.0 0.2 0.4 0.6 0.8

Hpv16 E7

CpG incub. - - + Peptide Restim. (E7 minimal) Media Ctrl. Untreated 1x106 squeezed SQZ enables direct cytosolic delivery of antigen which has allowed us to engineer unfractionated PBMCs to function as potent APCs. This strategy has demonstrated significant potential to generate CD8 T cell responses in both mouse and human systems. SQZ APCs are currently being evaluated in a clinical trial for HPV16+ tumors. Summary0 20 40 60 Tumor Volume (mm3) Squeezed splenocytes w/o CpG incub. Squeezed splenocytes w/ CpG incub. Prime C Boosting Further Augments Antigen-specific CD8 T Cell Responses0 20 40 60 Tumor Volume (mm3) P B B0 20 40 60 Percent survival *** **0.0 0.2 0.4 0.6 0.8 Cell Dose (x106): 0 0.1 0.25 1 Prime Only Prime + Boost Untreated Squeezed splenocytes Prime + Boost + Boost Double boosting with E7 SLP-squeezed splenocyte vaccine further enhances antigen-specific CD8 T cells and provides higher antitumor activity and survival. (A) Mice received E7 SLP-squeezed splenocyte vaccine at three different cell doses (0.1, 0.25, 1 M). In addition, they received single or double boosting. E7 Antigenspecific CD8 T cells in spleen were analyzed on day 7 post injection (****p<0.0001). (B) Tumor growth kinetics for mice bearing TC1 tumors that received one dose of splenocyte vaccine (red-orange) or double boosted (teal-blue). (C) Percent survival of the mice cohorts with single vaccination (red-orange) or with additional double boosting (teal-blue). Brackets in red indicate the median day of survival for each vaccination cohort (**p<0.01, ***p<0.001, ****p<0.0001). C #170Untr. SQZ Synthetic Long Peptide

(hpv16 E7)

(of CD8+B220-) Peptide Restim. Media Ctrl.0 20 40 60 Tumor Volume (mm3) Squeezed 1M

0.25m

0.1m

1m P/b/b

0.25m P/b/b

0.1m P/b/b

P B B0 20 40 60

Survival Percent survival Squeezed Untreated [33.5]

1m [42]

0.25m [37.5]

1m P/b/b [60]

0.1m P/b/b [49]

0.1m [38]

0.25m P/b/b [50]

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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.

# Microfluidics Cell Squeezing Enables Potent Cellular Vaccines in Murine Models Through Direct Cytosolic Loading and Direct CD8 T Cell Priming 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: Microfluidics Cell Squeezing Enables Potent Cellular Vaccines in Murine Models Through Direct Cytosolic Loading and Direct CD8 T Cell Priming - Document type: Poster - Year: 2020 - Outlet: SITC - Cells: T cells, PBMCs, Monocytes, B cells, Dendritic Cells - Cargos: Polymers, Protein, Peptide - Page URL: https://portal.bio/publications/2020-microfluidics-cell-squeezing-enables-potent-cellular-vaccines-in-murine-models-throug--ajkHBREAACsAc-BV - Markdown URL: https://portal.bio/publications/2020-microfluidics-cell-squeezing-enables-potent-cellular-vaccines-in-murine-models-throug--ajkHBREAACsAc-BV.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65ccf0199be9a5b998b5d25c_SITC-2020-APC-Murine-Poster-E.-I.-Ozay-FINAL.pdf ## Summary E. Ilker Ozay, Matthew G. Booty, Katarina Blagovic, David Soto, Olivia Pryor, Adam Stockmann, Disha Subramanya, Kelly Volk, Armon Sharei, Howard Bernstein, Defne Yarar, Katherine J. Seidl and Scott M. Loughhead ## Publication text Microfluidics Cell Squeezing Enables Potent Cellular Vaccines in Murine Models Through Direct Cytosolic Loading and Direct CD8 T Cell Priming E. Ilker Ozay, Matthew G. Booty, Katarina Blagovic, David Soto, Olivia Pryor, Adam Stockmann, Disha Subramanya, Kelly Volk, Armon Sharei, Howard Bernstein, Defne Yarar, Katherine J. Seidl and Scott M. Loughhead All Associated with SQZ Biotechnologies, Watertown, MA, USA SQZ APCs Lead To A Higher Influx of Antigenspecific CD8 T Cells with 15,000X Less Antigen Than The SLP Peptide Vaccine hours, and CD69 expression was examined on the responding OT-I’s to assess their activation. The minimal OVA epitope SIINFEKL was spiked into wells with cultured OT-I cells to elicit maximal CD69 expression as a positive control. ### Background The presentation of sufficient antigen on major histocompatibility complex class I (MHC-I) is essential to prime CD8 T cells. We employ our proprietary technology, Cell Squeeze® to deliver antigens directly to the cytosol of PBMCs to generate antigen presenting cells (APCs) that can robustly present on MHC-I. In addition to facilitating priming by professional APCs, this approach enables subsets within peripheral blood mononuclear cells (PBMCs) to function as conventional and unconventional APCs in both mouse and human preclinical models. Squeezed Mouse Splenocytes Rapidly Process and Present Antigen SQZ Technology and APC Platform Cytosolic Antigen Delivery (Cell Squeeze® Technology): Engineering Cells to Function as Antigen Presenting Cells: Traditionally, cross-presentation is needed for efficient presentation on MHC-I. SQZ delivers materials directly to cytosol, bypassing need for cross-presentation. Viability and cargo delivery in squeezed mouse splenocytes. Splenocytes were left untreated (NC), incubated at room temp. with fluorescent 3kDa dextran (Incub. ctrl) or squeezed in the presence of fluorescently-labeled dextran (Squeeze). Viability and dextran delivery of each cell type were immediately determined by flow cytometry.OT-I Alone Splenocytes (Squeeze + Protein) T Cells B Cells NK Cells Monocytes 2.5 5.0 Splenocytes (Squeeze + mRNA) ### % Cd69+ Squeeze + OVA mRNA (g/mL)Squeeze + Protein Antigen Each cell subset within squeezed splenocytes can process and present antigen to CD8 T cells. Splenocytes were squeezed without cargo (Squeeze only) or squeezed in the presence of ovalbumin protein (squeeze+OVA, panel A) or ovalbumin mRNA (squeeze+OVA mRNA, panel B). Following the squeeze, cells were left untouched (Splenocytes) or cell subsets were purified by negative immunomagnetic bead selection (T Cells, B Cells, NK Cells, and Monocytes, panel C). The indicated cells were then co-cultured with transgenic OT-I CD8 T cells for 24 hours, and CD69 expression was examined on the responding OT-Is to assess their activation. The minimal OVA epitope SIINFEKL was spiked into wells with cultured OT-I cells to elicit maximal CD69 expression as a positive control.Untr. SQZ Protein (Ovalbumin) (of CD8+B220-)Untr. SQZ Synthetic Long Peptide ### (hpv16 E7) (of CD8+B220-) Squeezed Mouse APCs Directly Prime CD8 T Cells in vivo Squeezed murine splenocytes directly present antigen to CD8 T cells in vivo. (A) WT or MHC-I KO mice were immunized with 5x106 splenocytes either incubated (Incubation Control) or squeezed (SQZ) with ovalbumin protein. CFSE-labeled OT-I CD8 T cells were transferred into recipient animals prior to immunization to serve as a traceable responder CD8 population. Lymph nodes were harvested 3 days after immunization and analyzed by flow cytometry. (B) Mice were immunized with splenocytes squeezed with ovalbumin or HPV16 E7 synthetic long peptide (SLP). 7 days after immunization, spleens were harvested and re-stimulated with either the ovalbumin minimal epitope or the minimal epitope for HPV16 E7 to assess the percentage of antigen-specific CD8s. Splenic Response No Immunization → MHC-I-/- Incubation Ctrl. → Wild-type Ovalbumin Squeeze → Wild-type Incubation Ctrl. → MHC-I-/- Ovalbumin Squeeze → MHC-I-/- ### Cfse Lymph Node: Adjuvant Process Enhances Capabilities of SQZ APCs0 Tumor Vol. (mm3) Squeezed Splenocytes (IV) E7 SLP + CpG (Subcutaneous) 16 28 Prime Imm ### Til Analysis Day DayUntr. SLP % CD45+ (of Live) ns *** **Untr. SLP % CD8+ (of CD45+) *** *** *Untr. SLP % E7 Tetramer+ (of CD8+) *** *** * D CB Therapeutic immunization with squeezed splenocytes drives the expansion of tumor specific CD8 T cells. (A) Tumor growth kinetics for mice bearing TC1 tumors that were left untreated (black), vaccinated subcutaneously with an E7 synthetic long peptide plus CpG (orange), or immunized with splenocytes squeezed with the same E7 SLP (teal). (B-D) TIL analysis from the day 28 time point showing the intratumoral influx of CD45+ cells, CD8+ cells, and E7-tetramer+ CD8 T cells. CpG incubation phenotypically and functionally alters squeezed splenocytes resulting in higher antigenspecific CD8 T cells with augmented therapeutic benefit in a tumor model. (A) Squeezed splenocytes were incubated with the adjuvant, CpG. B cell surface expression of APC maturation markers, CD86 and MHC-I, was measured at 24 hours after the incubation. (B) Splenocytes were either squeezed with ovalbumin protein or E7 synthetic long peptide (SLP). Later, they were subjected to CpG incubation prior to injection into mice. Antigen-specific CD8 T cells in spleen were analyzed on day 7 post injection. (C) Tumor growth kinetics for mice bearing TC1 tumors that were left untreated (black), immunized with E7 SLPsqueezed splenocytes incubated with (teal) or without (blue) the CpG.0 ### Cd86 Mfi CpG incub. - +0 10000 20000 30000 40000 ### Mhc-i Mfi CpG incub. - + ### C0.0 Ovalbumin CpG incub.: - - + Peptide Restim. (SIINFEKL) Media Ctrl. Untreated 1x106 squeezed splenocytes0.0 0.2 0.4 0.6 0.8 ### Hpv16 E7 CpG incub. - - + Peptide Restim. (E7 minimal) Media Ctrl. Untreated 1x106 squeezed SQZ enables direct cytosolic delivery of antigen which has allowed us to engineer unfractionated PBMCs to function as potent APCs. This strategy has demonstrated significant potential to generate CD8 T cell responses in both mouse and human systems. SQZ APCs are currently being evaluated in a clinical trial for HPV16+ tumors. Summary0 20 40 60 Tumor Volume (mm3) Squeezed splenocytes w/o CpG incub. Squeezed splenocytes w/ CpG incub. Prime C Boosting Further Augments Antigen-specific CD8 T Cell Responses0 20 40 60 Tumor Volume (mm3) P B B0 20 40 60 Percent survival *** **0.0 0.2 0.4 0.6 0.8 Cell Dose (x106): 0 0.1 0.25 1 Prime Only Prime + Boost Untreated Squeezed splenocytes Prime + Boost + Boost Double boosting with E7 SLP-squeezed splenocyte vaccine further enhances antigen-specific CD8 T cells and provides higher antitumor activity and survival. (A) Mice received E7 SLP-squeezed splenocyte vaccine at three different cell doses (0.1, 0.25, 1 M). In addition, they received single or double boosting. E7 Antigenspecific CD8 T cells in spleen were analyzed on day 7 post injection (****p<0.0001). (B) Tumor growth kinetics for mice bearing TC1 tumors that received one dose of splenocyte vaccine (red-orange) or double boosted (teal-blue). (C) Percent survival of the mice cohorts with single vaccination (red-orange) or with additional double boosting (teal-blue). Brackets in red indicate the median day of survival for each vaccination cohort (**p<0.01, ***p<0.001, ****p<0.0001). C #170Untr. SQZ Synthetic Long Peptide ### (hpv16 E7) (of CD8+B220-) Peptide Restim. Media Ctrl.0 20 40 60 Tumor Volume (mm3) Squeezed 1M ### 0.25m ### 0.1m ### 1m P/b/b ### 0.25m P/b/b ### 0.1m P/b/b ### P B B0 20 40 60 Survival Percent survival Squeezed Untreated [33.5] ### 1m [42] ### 0.25m [37.5] ### 1m P/b/b [60] ### 0.1m P/b/b [49] ### 0.1m [38] ### 0.25m P/b/b [50] ## 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.