Poster · 2019 · KEYSTONE SYMPOSIA: CANCER VACCINES

SQZ’ing Cells to Engineer a New Generation of Cancer Vaccines

Matthew G. Booty, Defne Yarar, Scott Loughhead, Kelan Hlavaty, Alfonso Vicente-Suarez, Katarina Blagovic, Adam Stockman, Disha Subramanya, Howard Bernstein, and Armon Sharei

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

Publication text

SQZ’d Human Cells Function as APCs SQZing Cells to Engineer a New Generation of Cancer Vaccines Matthew G. Booty1, *, Defne Yarar1, *, Scott Loughhead1, Kelan Hlavaty1, Alfonso Vicente-Suarez1, Katarina Blagovic1, Adam Stockman1, Disha Subramanya1, Howard Bernstein1, and Armon Sharei1 1SQZ Biotechnologies, Watertown, MA, USA, *Co-first authors SQZ’d APCs Prime Endogenous CD8+ T Cell Responses to Protein and Peptide Antigens SQZ’d murine T and B cells prime endogenous CD8+ T cell responses in vivo. Mice were immunized with cells SQZ’d with either protein or peptide antigen, and intracellular cytokine staining was performed on splenocytes 7 days later. (A) Mice were untreated or immunized with T cells (blue) or B cells (orange) SQZ’d with ovalbumin and restimulated with SIINFEKL. (B) Mice were untreated or immunized with T cells or B cells SQZ’d with a synthetic long peptide from the HPV16 E7 antigen and re-stimulated the minimal CD8 epitope for HPV16 E7. SQZ’d human T cells and B cells process and present antigenic epitopes on MHC-I to re-stimulate T cell responses in vitro. T cells and B cells with no antigen exposure (NC), incubated with synthetic long peptides (Endo), and SQZ’d in the presence of synthetic long peptides (SQZ) were co-cultured with relevant antigen-specific CD8+ T cell responders. Antigen-specific responses were determined by IFN-γ ELISA. (A) Cells were SQZ’d with peptides encoding a cytomegalovirus (CMV) antigen and co-cultured with CMV-reactive CD8+ T cells. (B) Cells were SQZ’d with peptides encoding an HPV-16 antigen and cocultured with HPV-reactive CD8+ T cells. All data are representative of more than three independent experiments across multiple human donors.

Background

of sufficient antigen on major histocompatibility complex class I (MHC-I) is one barrier to priming potent effector CD8+ T cell responses. In this work, we use the microfluidics-based Cell SQZ® platform to deliver antigen directly to the cytosol of target antigen presenting cells (APCs), resulting in the enhanced presentation of antigen on MHC-I. In addition to facilitating CD8+ T cell priming by professional APCs, this approach can transform multiple cell types into unorthodox, yet effective APCs capable of priming CD8+ T cell responses in murine and human systems. Therapeutic immunization strategies that elicit robust CD8+ T cell responses remain elusive in the clinic. The presentation Therapeutic BAPC immunization drives expansion of tumor specific CD8+ T cells. (A) Tumor growth kinetics for C57BL/6 mice bearing TC1 tumors that were untreated (black) or immunized with the E7 minimal epitope peptide-pulsed B cells (orange), vaccinated subcutaneously with a synthetic long peptide (SLP) containing a CD8 epitope for HPV16 E7, or vaccinated with B cells SQZ’d with the E7 SLP (blue). (B) TIL analysis from day 27 time point shown as % of CD45 (left) or number of cells per 100 mg tumor mass (right). Tumor Regression is Concomitant with an Influx of Antigen Specific CD8+ T Cells

Using the SQZ platform, we have engineered T cells and B cells to function as potent APCs.
These cells are effective in inhibiting tumor growth in prophylactic and therapeutic settings.
Tumor inhibition activity correlates with an increase in tumor-infiltrating CD8+ T cells.
Human cells function as APCs.

Summary

SQZ’d T Cells (TAPCs) Rapidly Process and Directly Present Antigen Murine T cells SQZ’d with ovalbumin process and present the SIINFEKL epitope within hours. Murine T cells were loaded with ovalbumin. Antigen presentation was assessed at the indicated A

Sqz

Ovalbumin SQZ: No Antigen Epitope Presented on MHC-I (SIINFEKL bound to H-2Kb) 0 hours 2 hours 4 hours 24 hours time points by surface staining with an antibody that binds SIINFEKL bound to MHC-I (25-D1.16). (A) Histograms depicting antigen presentation by fluorescence (B) Quantification of fluorescence from panel (A). No presentation was observed in T cells that were incubated with ovalbumin. Murine T cells SQZ’d with protein antigen directly present to CD8+ T cells in vivo. WT or MHC-I KO mice were immunized with 5x106 T cells either incubated (Endo- TAPC) or SQZ’d (SQZ-TAPC) with ovalbumin. CFSE-labeled OT-I cells were transferred into recipient animals prior to immunization to serve as a traceable responder CD8 population. Spleens were harvested 3 days after immunization and analyzed by flow cytometry. Day 3

CFSE labeled OT-Is
A.T. of OVA SQZ’d TAPCs

or control (Endo) T cells Day 0 Assess OT-I proliferation in spleen WT or MHC-I KO Recipients

Cfse

OT-I Proliferation in Spleen

Sqz-tapc

↓ WT Endo-TAPC → WT

Sqz-tapc

↓

Mhc-i-/-

Endo-TAPC → MHC-I-/- SQZ Platform Engineering Cells (All Types) to Function as Antigen Presenting Cells: Cytosolic Antigen Delivery: Traditionally, cross-presentation is needed for efficient presentation on MHC-I. SQZ delivers materials directly to cytosol, bypassing need for cross-presentation. SQZ’d APCs Are Therapeutic Therapeutic immunization delays tumor growth and drives the expansion of tumor specific CD8+ T cells. Mice were subcutaneously implanted with the HPV16 E6/E7-positive TC-1 tumor cell line and therapeutically immunized with a single dose of 1x106 of the indicated cells SQZ’d with an HPV16 E7 synthetic long peptide. (Left) Tumor growth kinetics for untreated mice and mice immunized 10 days after tumor implant. (Right) Survival of untreated and SQZ immunized mice. Numbers in brackets indicate the median survival.

A B

SQZ’d APCs Prime Protective Immunity Prophylactic immunization protects mice from tumor challenge. C57BL/6 mice were immunized with either T cells or B cells SQZ’d with a synthetic long peptide containing a CD8 epitope for HPV16 E7. Seven days later, mice were subcutaneously implanted with the HPV16 E6/E7-positive TC-1 tumor cell line. (Left) Tumor growth kinetics for untreated and SQZ immunized mice; number of tumor free (TF) at day 50 shown. (Right) Survival of untreated and SQZ immunized mice. Numbers in brackets indicate the median survival. Isolate T cells from HLA-A02+ donor PBMCs SQZ T or B cells with synthetic long peptide (SLP) Co-culture T-APCs with antigenspecific CD8 T cell responders Assess IFNg secretion at 18-20hr with ELISA T cellsA BCMV B cells

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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.

# SQZ’ing Cells to Engineer a New Generation of Cancer Vaccines 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: SQZ’ing Cells to Engineer a New Generation of Cancer Vaccines - Document type: Poster - Year: 2019 - Outlet: KEYSTONE SYMPOSIA: CANCER VACCINES - Cells: T cells, PBMCs, Monocytes, B cells, Dendritic Cells - Cargos: Polymers, Protein, Peptide - Page URL: https://portal.bio/publications/2019-sqz-ing-cells-to-engineer-a-new-generation-of-cancer-vaccines--ajkHJBEAACcAc-Co - Markdown URL: https://portal.bio/publications/2019-sqz-ing-cells-to-engineer-a-new-generation-of-cancer-vaccines--ajkHJBEAACcAc-Co.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65cd01669be9a5b998b5d31e_Keystone-Cancer-Vaccines-2019-Poster-M.-Booty-and-D.-Yarar.pdf ## Summary Matthew G. Booty, Defne Yarar, Scott Loughhead, Kelan Hlavaty, Alfonso Vicente-Suarez, Katarina Blagovic, Adam Stockman, Disha Subramanya, Howard Bernstein, and Armon Sharei ## Publication text SQZ’d Human Cells Function as APCs SQZing Cells to Engineer a New Generation of Cancer Vaccines Matthew G. Booty1, *, Defne Yarar1, *, Scott Loughhead1, Kelan Hlavaty1, Alfonso Vicente-Suarez1, Katarina Blagovic1, Adam Stockman1, Disha Subramanya1, Howard Bernstein1, and Armon Sharei1 1SQZ Biotechnologies, Watertown, MA, USA, *Co-first authors SQZ’d APCs Prime Endogenous CD8+ T Cell Responses to Protein and Peptide Antigens SQZ’d murine T and B cells prime endogenous CD8+ T cell responses in vivo. Mice were immunized with cells SQZ’d with either protein or peptide antigen, and intracellular cytokine staining was performed on splenocytes 7 days later. (A) Mice were untreated or immunized with T cells (blue) or B cells (orange) SQZ’d with ovalbumin and restimulated with SIINFEKL. (B) Mice were untreated or immunized with T cells or B cells SQZ’d with a synthetic long peptide from the HPV16 E7 antigen and re-stimulated the minimal CD8 epitope for HPV16 E7. SQZ’d human T cells and B cells process and present antigenic epitopes on MHC-I to re-stimulate T cell responses in vitro. T cells and B cells with no antigen exposure (NC), incubated with synthetic long peptides (Endo), and SQZ’d in the presence of synthetic long peptides (SQZ) were co-cultured with relevant antigen-specific CD8+ T cell responders. Antigen-specific responses were determined by IFN-γ ELISA. (A) Cells were SQZ’d with peptides encoding a cytomegalovirus (CMV) antigen and co-cultured with CMV-reactive CD8+ T cells. (B) Cells were SQZ’d with peptides encoding an HPV-16 antigen and cocultured with HPV-reactive CD8+ T cells. All data are representative of more than three independent experiments across multiple human donors. ### Background of sufficient antigen on major histocompatibility complex class I (MHC-I) is one barrier to priming potent effector CD8+ T cell responses. In this work, we use the microfluidics-based Cell SQZ® platform to deliver antigen directly to the cytosol of target antigen presenting cells (APCs), resulting in the enhanced presentation of antigen on MHC-I. In addition to facilitating CD8+ T cell priming by professional APCs, this approach can transform multiple cell types into unorthodox, yet effective APCs capable of priming CD8+ T cell responses in murine and human systems. Therapeutic immunization strategies that elicit robust CD8+ T cell responses remain elusive in the clinic. The presentation Therapeutic BAPC immunization drives expansion of tumor specific CD8+ T cells. (A) Tumor growth kinetics for C57BL/6 mice bearing TC1 tumors that were untreated (black) or immunized with the E7 minimal epitope peptide-pulsed B cells (orange), vaccinated subcutaneously with a synthetic long peptide (SLP) containing a CD8 epitope for HPV16 E7, or vaccinated with B cells SQZ’d with the E7 SLP (blue). (B) TIL analysis from day 27 time point shown as % of CD45 (left) or number of cells per 100 mg tumor mass (right). Tumor Regression is Concomitant with an Influx of Antigen Specific CD8+ T Cells - Using the SQZ platform, we have engineered T cells and B cells to function as potent APCs. - These cells are effective in inhibiting tumor growth in prophylactic and therapeutic settings. - Tumor inhibition activity correlates with an increase in tumor-infiltrating CD8+ T cells. - Human cells function as APCs. ### Summary SQZ’d T Cells (TAPCs) Rapidly Process and Directly Present Antigen Murine T cells SQZ’d with ovalbumin process and present the SIINFEKL epitope within hours. Murine T cells were loaded with ovalbumin. Antigen presentation was assessed at the indicated A ### Sqz Ovalbumin SQZ: No Antigen Epitope Presented on MHC-I (SIINFEKL bound to H-2Kb) 0 hours 2 hours 4 hours 24 hours time points by surface staining with an antibody that binds SIINFEKL bound to MHC-I (25-D1.16). (A) Histograms depicting antigen presentation by fluorescence (B) Quantification of fluorescence from panel (A). No presentation was observed in T cells that were incubated with ovalbumin. Murine T cells SQZ’d with protein antigen directly present to CD8+ T cells in vivo. WT or MHC-I KO mice were immunized with 5x106 T cells either incubated (Endo- TAPC) or SQZ’d (SQZ-TAPC) with ovalbumin. CFSE-labeled OT-I cells were transferred into recipient animals prior to immunization to serve as a traceable responder CD8 population. Spleens were harvested 3 days after immunization and analyzed by flow cytometry. Day 3 1. CFSE labeled OT-Is 2. A.T. of OVA SQZ’d TAPCs or control (Endo) T cells Day 0 Assess OT-I proliferation in spleen WT or MHC-I KO Recipients ### Cfse OT-I Proliferation in Spleen ### Sqz-tapc ↓ WT Endo-TAPC → WT ### Sqz-tapc ↓ ### Mhc-i-/- Endo-TAPC → MHC-I-/- SQZ Platform Engineering Cells (All Types) to Function as Antigen Presenting Cells: Cytosolic Antigen Delivery: Traditionally, cross-presentation is needed for efficient presentation on MHC-I. SQZ delivers materials directly to cytosol, bypassing need for cross-presentation. SQZ’d APCs Are Therapeutic Therapeutic immunization delays tumor growth and drives the expansion of tumor specific CD8+ T cells. Mice were subcutaneously implanted with the HPV16 E6/E7-positive TC-1 tumor cell line and therapeutically immunized with a single dose of 1x106 of the indicated cells SQZ’d with an HPV16 E7 synthetic long peptide. (Left) Tumor growth kinetics for untreated mice and mice immunized 10 days after tumor implant. (Right) Survival of untreated and SQZ immunized mice. Numbers in brackets indicate the median survival. ### A B SQZ’d APCs Prime Protective Immunity Prophylactic immunization protects mice from tumor challenge. C57BL/6 mice were immunized with either T cells or B cells SQZ’d with a synthetic long peptide containing a CD8 epitope for HPV16 E7. Seven days later, mice were subcutaneously implanted with the HPV16 E6/E7-positive TC-1 tumor cell line. (Left) Tumor growth kinetics for untreated and SQZ immunized mice; number of tumor free (TF) at day 50 shown. (Right) Survival of untreated and SQZ immunized mice. Numbers in brackets indicate the median survival. Isolate T cells from HLA-A02+ donor PBMCs SQZ T or B cells with synthetic long peptide (SLP) Co-culture T-APCs with antigenspecific CD8 T cell responders Assess IFNg secretion at 18-20hr with ELISA T cellsA BCMV B cells ## 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.