Poster · 2019 · SITC

Antigen delivery to PBMCs by microfluidic squeezing primes anti-tumor immunity

Matthew G. Booty, Kelan A. Hlavaty, Emrah I. Ozay, Carolyne Smith, Anita Venkitaraman, Christian Yee, Adam Stockman, Disha Subramanya, Olivia Prior, Kelly Volk, Katherine Seidl, Howard Bernstein, Armon Sharei, and Scott Loughhead

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

Publication text

Squeezed Human PBMCs Function as APCs Antigen Delivery to PBMCs by Microfluidic Squeezing Primes Anti-Tumor Immunity Matthew G. Booty1, Kelan A. Hlavaty1, Emrah I. Ozay1, Carolyne Smith1, Anita Venkitaraman1, Christian Yee1, Adam Stockman1, Disha Subramanya1, Olivia Prior1, Kelly Volk1, Katherine Seidl1, Howard Bernstein1, Armon Sharei1, and Scott Loughhead1 1SQZ Biotechnologies, Watertown, MA, USA Squeezed Mouse APCs Directly Prime CD8+ T Cells in vivo Squeezed mouse splenocytes directly present antigen to CD8+ T cells in vivo. (A) WT or MHC-I KO mice were immunized with splenocytes either incubated (Incubation Ctrl.) or squeezed with ovalbumin. 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) WT mice were immunized with splenocytes squeezed with ovalbumin or HPV16 E7 synthetic long peptide. 7 days after immunization, spleens were harvested and re-stimulated with either the ovalbumin minimal epitope (SIINFEKL) or the minimal epitope for HPV16 E7 to assess the percentage of antigen-specific CD8s.

Background

generating potent cancer immunizations. Here, we use microfluidics-based squeezing 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 potent CD8+ T cell priming by professional APCs, this approach can make unfractionated peripheral blood mononuclear cells (PBMCs) effective, unconventional APCs capable of priming CD8+ T cell responses in mouse and human preclinical models. The presentation of sufficient antigen on major histocompatibility complex class I (MHC-I) is a potential barrier to Tumor Regression Is Concomitant with the Influx of Antigen Specific CD8+ T Cells Through the direct cytosolic delivery of antigen, we have engineered unfractionated PBMCs to function as potent antigen presenting cells (APCs). This strategy has demonstrated significant potential to generate CD8+ T cell responses in both mouse and human systems and has been scaled up for clinical implementation (NCT04084951).

Summary

Squeezed Mouse Splenocytes Rapidly Process and Present Antigen of 3 kDa dextran (Squeeze). Viability and dextran delivery of each cell type were immediately determined by flow cytometry. (B) Splenocytes were squeezed without cargo (Squeeze only) or squeezed in the presence of ovalbumin protein (Squeeze + OVA). At the indicated time points, cells were stained with the 25-D1.16 antibody to assess SIINFEKL presentation on MHC-I (H-2Kb) of each cell type. 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 the cytosol, bypassing the need for cross-presentation. Therapeutic Efficacy in Mice Priming of Prophylactic Immunity in Mice Prophylactic immunization protects mice from primary tumor challenge and elicits protective immunological memory. Mice were primed on day -14 and boosted on day -7 with splenocytes squeezed with a synthetic long peptide containing a CD8 epitope for HPV16 E7. On day 0, mice were subcutaneously implanted on the right flank with the HPV16 E6/E7-positive TC-1 tumor cell line. All immunized mice remained tumor free for 60 days and were subsequently rechallenged with TC-1 tumor cells subcutaneously implanted on the left flank. Tumor growth kinetics for untreated and squeeze immunized mice are shown. 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 (blue), or immunized with splenocytes squeezed with the same E7 SLP (green). (B) TIL analysis from the day 28 time point showing the intratumoral influx of CD45+ cells, CD8+ cells, and E7-tetramer+ CD8+ T cells. Therapeutic immunization delays tumor growth and enhances survival when adjuvanted. Mice were subcutaneously implanted with the HPV16 E6/E7-positive TC-1 tumor cell line. On day 10 when tumors were palpable, mice were therapeutically immunized with a single dose of splenocytes squeezed with HPV16 E7 synthetic long peptide. For the standard squeezed splenocyte immunization, adjuvant is incorporated to elicit a productive immune response. As a control, adjuvant was omitted. (A) Tumor growth kinetics for untreated mice and mice immunized with squeezed splenocytes. (B) Survival of untreated and immunized mice. Numbers in brackets indicate the median survival in days. Squeezed mouse splenocytes present antigen on MHC-I. (A) Murine splenocytes were left untreated (NC), incubated at room temperature with fluorescent 3 kDa dextran (Incub. ctrl) or squeezed in the presence CB Squeezed human PBMCs present antigen and billions of human cells can be squeezed in a single manufacturing run. (A) Human PBMCs were squeezed at the research scale with a synthetic long peptide (SLP) containing the HLA-A*02-restricted HPV16 E7 epitope. After squeezing, cells were washed and incubated overnight with HLA-A*02 restricted E7 responder CD8+ T cells. Supernatants were analyzed by ELISA. (B) Human PBMCs were enriched by density centrifugation and cells were squeezed at manufacturing scale with fluorescent dextran. (C) Viability and delivery were determined by flow cytometry immediately following squeeze. Each dot represents a single donor. Delivery was determined based on cells that were not incubated with dextran (not shown). Isolate PBMCs from HLA-A*02+ donor PBMCs Squeeze PBMCs with SLP Co-culture squeezed PBMCs with antigenspecific CD8+ T cell responders Assess IFNγ secretion at 18- 20hr with ELISA

A Hpv16 E7

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: 15/15 Mice Protected from Primary Tumor Challenge 11/15 Mice Protected from Tumor Rechallenge Each cell subset within squeezed splenocytes presents antigen to CD8+ T cells. Splenocytes were squeezed without cargo (Squeeze only) or squeezed in the presence of ovalbumin protein (Squeeze + OVA). Following the squeeze, the indicated cell subsets were purified by negative immunomagnetic bead selection (T Cells, B Cells, NK Cells, and Monocytes) or were left untouched (Splenocytes). 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-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.

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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.

# Antigen delivery to PBMCs by microfluidic squeezing primes anti-tumor immunity 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: Antigen delivery to PBMCs by microfluidic squeezing primes anti-tumor immunity - Document type: Poster - Year: 2019 - Outlet: SITC - Cells: T cells, PBMCs, Monocytes, B cells, Dendritic Cells - Cargos: Polymers, Protein, Peptide, DNA - Page URL: https://portal.bio/publications/2019-antigen-delivery-to-pbmcs-by-microfluidic-squeezing-primes-anti-tumor-immunity--ajkHFxEAAC4Ac-B- - Markdown URL: https://portal.bio/publications/2019-antigen-delivery-to-pbmcs-by-microfluidic-squeezing-primes-anti-tumor-immunity--ajkHFxEAAC4Ac-B-.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65ccff449be9a5b998b5d30b_SITC-2019-Poster-M.-Booty.pdf ## Summary Matthew G. Booty, Kelan A. Hlavaty, Emrah I. Ozay, Carolyne Smith, Anita Venkitaraman, Christian Yee, Adam Stockman, Disha Subramanya, Olivia Prior, Kelly Volk, Katherine Seidl, Howard Bernstein, Armon Sharei, and Scott Loughhead ## Publication text Squeezed Human PBMCs Function as APCs Antigen Delivery to PBMCs by Microfluidic Squeezing Primes Anti-Tumor Immunity Matthew G. Booty1, Kelan A. Hlavaty1, Emrah I. Ozay1, Carolyne Smith1, Anita Venkitaraman1, Christian Yee1, Adam Stockman1, Disha Subramanya1, Olivia Prior1, Kelly Volk1, Katherine Seidl1, Howard Bernstein1, Armon Sharei1, and Scott Loughhead1 1SQZ Biotechnologies, Watertown, MA, USA Squeezed Mouse APCs Directly Prime CD8+ T Cells in vivo Squeezed mouse splenocytes directly present antigen to CD8+ T cells in vivo. (A) WT or MHC-I KO mice were immunized with splenocytes either incubated (Incubation Ctrl.) or squeezed with ovalbumin. 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) WT mice were immunized with splenocytes squeezed with ovalbumin or HPV16 E7 synthetic long peptide. 7 days after immunization, spleens were harvested and re-stimulated with either the ovalbumin minimal epitope (SIINFEKL) or the minimal epitope for HPV16 E7 to assess the percentage of antigen-specific CD8s. ### Background generating potent cancer immunizations. Here, we use microfluidics-based squeezing 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 potent CD8+ T cell priming by professional APCs, this approach can make unfractionated peripheral blood mononuclear cells (PBMCs) effective, unconventional APCs capable of priming CD8+ T cell responses in mouse and human preclinical models. The presentation of sufficient antigen on major histocompatibility complex class I (MHC-I) is a potential barrier to Tumor Regression Is Concomitant with the Influx of Antigen Specific CD8+ T Cells Through the direct cytosolic delivery of antigen, we have engineered unfractionated PBMCs to function as potent antigen presenting cells (APCs). This strategy has demonstrated significant potential to generate CD8+ T cell responses in both mouse and human systems and has been scaled up for clinical implementation (NCT04084951). ### Summary Squeezed Mouse Splenocytes Rapidly Process and Present Antigen of 3 kDa dextran (Squeeze). Viability and dextran delivery of each cell type were immediately determined by flow cytometry. (B) Splenocytes were squeezed without cargo (Squeeze only) or squeezed in the presence of ovalbumin protein (Squeeze + OVA). At the indicated time points, cells were stained with the 25-D1.16 antibody to assess SIINFEKL presentation on MHC-I (H-2Kb) of each cell type. 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 the cytosol, bypassing the need for cross-presentation. Therapeutic Efficacy in Mice Priming of Prophylactic Immunity in Mice Prophylactic immunization protects mice from primary tumor challenge and elicits protective immunological memory. Mice were primed on day -14 and boosted on day -7 with splenocytes squeezed with a synthetic long peptide containing a CD8 epitope for HPV16 E7. On day 0, mice were subcutaneously implanted on the right flank with the HPV16 E6/E7-positive TC-1 tumor cell line. All immunized mice remained tumor free for 60 days and were subsequently rechallenged with TC-1 tumor cells subcutaneously implanted on the left flank. Tumor growth kinetics for untreated and squeeze immunized mice are shown. 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 (blue), or immunized with splenocytes squeezed with the same E7 SLP (green). (B) TIL analysis from the day 28 time point showing the intratumoral influx of CD45+ cells, CD8+ cells, and E7-tetramer+ CD8+ T cells. Therapeutic immunization delays tumor growth and enhances survival when adjuvanted. Mice were subcutaneously implanted with the HPV16 E6/E7-positive TC-1 tumor cell line. On day 10 when tumors were palpable, mice were therapeutically immunized with a single dose of splenocytes squeezed with HPV16 E7 synthetic long peptide. For the standard squeezed splenocyte immunization, adjuvant is incorporated to elicit a productive immune response. As a control, adjuvant was omitted. (A) Tumor growth kinetics for untreated mice and mice immunized with squeezed splenocytes. (B) Survival of untreated and immunized mice. Numbers in brackets indicate the median survival in days. Squeezed mouse splenocytes present antigen on MHC-I. (A) Murine splenocytes were left untreated (NC), incubated at room temperature with fluorescent 3 kDa dextran (Incub. ctrl) or squeezed in the presence CB Squeezed human PBMCs present antigen and billions of human cells can be squeezed in a single manufacturing run. (A) Human PBMCs were squeezed at the research scale with a synthetic long peptide (SLP) containing the HLA-A*02-restricted HPV16 E7 epitope. After squeezing, cells were washed and incubated overnight with HLA-A*02 restricted E7 responder CD8+ T cells. Supernatants were analyzed by ELISA. (B) Human PBMCs were enriched by density centrifugation and cells were squeezed at manufacturing scale with fluorescent dextran. (C) Viability and delivery were determined by flow cytometry immediately following squeeze. Each dot represents a single donor. Delivery was determined based on cells that were not incubated with dextran (not shown). Isolate PBMCs from HLA-A*02+ donor PBMCs Squeeze PBMCs with SLP Co-culture squeezed PBMCs with antigenspecific CD8+ T cell responders Assess IFNγ secretion at 18- 20hr with ELISA ### A Hpv16 E7 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: 15/15 Mice Protected from Primary Tumor Challenge 11/15 Mice Protected from Tumor Rechallenge Each cell subset within squeezed splenocytes presents antigen to CD8+ T cells. Splenocytes were squeezed without cargo (Squeeze only) or squeezed in the presence of ovalbumin protein (Squeeze + OVA). Following the squeeze, the indicated cell subsets were purified by negative immunomagnetic bead selection (T Cells, B Cells, NK Cells, and Monocytes) or were left untouched (Splenocytes). 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-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. ## 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.