Poster · 2022 · FOCIS

SQZ® TAC Cell Therapy Platform Induces Antigen Specific T-regs and Prevents Onset of Type 1 Diabetes in Adoptive Transfer Models

Todd Ashworth, Shinu John, Colin Raposo, Judit Cserny, Gloria Serena, Patricia Cho, Armon Sharei and Howard Bernstein

Cells: RBCs
Cargos: Polymers, Protein, Peptide

Publication text

Abstract

Antigen specific tolerance is a recognized therapeutic mode of promoting durable disease remission in autoimmune diseases with well-defined antigens which could overcome the problems associated with broad immunosuppression. The Cell Squeeze® technology utilizes microfluidic mechanical deformation to engineer cell function. To induce tolerance, red blood cells (RBCs) are engineered to deliver target antigens to generate tolerizing antigen carriers (TACs). The Cell Squeeze technology allows the SQZ®TACs to resemble senescent RBCs, enabling them to be cleared by eryptosis, the physiological mechanism of RBC clearance. Herein, we demonstrate SQZ TACs biodistribute to phagocytic cells in the spleen and liver where antigens are processed and presented to antigen specific T-cells in a tolerogenic manner. SQZ TAC treatment in several accelerated murine models of T1D led to significant delay or prevention of hyperglycemia. In both CD4+ (BDC2.5) and CD8+ (NY8.3) NOD adoptive transfer T1D models, co-delivery of TAC loaded with T1D autoantigens provided protection from early disease onset characterized by increased T-cell apoptosis, tissue specific T-reg induction and decreased effector T-cell pancreatic infiltration. In addition, administration of CD4+ directed TACs with the peptide p31 protected T1D onset in NOD mice co-transferred with both BDC2.5 and NY8.3 T cells, thereby demonstrating TAC induced bystander suppression. Recent preclinical efforts have expanded to include development of a TAC-deamidated gliadin formulation designed to ameliorate gluten sensitivity in patients suffering with Celiac Disease. SQZ® Technology Cytosolic Antigen Delivery (Cell Squeeze® Technology): [TITLE] SQZ® TACs are Efficiently Loaded and Have a Modified Membrane TACs are efficiently delivered. (A) Murine red blood cells squeezed with AlexaFluor-647-conjugated ovalbumin (OVA) or AlexaFluor-647-conjugated IgG. Loading is assessed by flow cytometry. (B) Phosphatidyl serine surface levels assessed by annexin V staining of murine red blood cells squeezed or left intact are assessed by flow cytometry. SQZ® TACs Are Engulfed By Phagocytic Cells in Various Organs Uptake of TACs by antigen presenting cells in different organs. (A-B) PKH26 labeled TACs were injected into B6 mice and uptake in various cell types in spleen (A) and liver (B) was assessed 1h postinjection by flow cytometry. SQZ® TAC-p31 Significantly Delayed Diabetes Onset Induced By BDC2.5 T Cells0 20 40 60 80 100 Blood Glucose Day Post Transfer Blood Glucose (mg/dL) No TAC0 20 40 60 80 100 Disease Incidence Day Post Transfer Percent Disease Free No TAC TAC-p31 delayed onset of hyperglycemia in BDC2.5 transfer model. (A) Schematic of adoptive transfer of activated BDC2.5 chromogranin-specific CD4+ T cells and TAC-p31 (BDC2.5 mimetope, relevant antigen) or TAC-HEL (irrelevant antigen) dosing regimen in NOD.SCID mice. (B-C) Blood glucose (BG) levels (B) and disease incidence (C) in animals that were treated with different TACs or left untreated (No TAC). Dotted line represents BG of 250mg/dL. Mice were considered hyperglycemic when BG levels were >250mg/dL for two consecutive readings. For B and C, n=9 for no TAC and n=10 for TAC-p31 and TAC-HEL. SQZ® TAC-p31 Treatment Decreased Insulitis And Upregulated Antigen-Specific Tregs in PancreasTAC-HEL IL-10 pg/mL ✱✱✱✱Spleen Pancreas %CD90.2 CD4+ (of Live) ✱✱Spleen Pancreas %Foxp3+ (of CD4+ CD90.2+) TAC-p31✱✱ ✱✱ (B) TAC-HEL TAC-p31TAC-HEL 10000 30000 IFNg (pg/ml)TAC-HEL 40000 60000 IFNg (pg/mL) * Treatment with TAC-p31 reduced infiltration of BDC2.5 T cells in pancreas, expanded Tregs and suppressed proinflammatory cytokine production (A) Schematic of activated BDC2.5 T cell adoptive transfer and TAC dosing regimen in NOD.SCID recipients. Mice were euthanized either on day 8 (flow cytometry) or day 15 (histology) post-adoptive transfer. (B) Representative H & E staining of pancreata isolated from TAC-HEL (left) and TAC-p31 (right) treated animals. Arrowhead represents islets of Langerhans. (C) Frequency of BDC2.5 T cells in spleen and pancreas of TAC-HEL and TAC-p31 treated mice were assessed by flow cytometry. (D) Lymphocytes were isolated from the indicated groups and organs and stimulated for 24h with p31 peptide. IFNγ secretion from lymphocyte culture supernatants from spleen (left) and pancreas (right) was measured by cytokine bead array. (E) Frequency of BDC2.5 Tregs in spleen and pancreas of indicated treatment groups as analyzed by flow cytometry. (F) IL-10 production measured by ELISA from culture supernatants of splenocytes stimulated with p31 peptide. (A) (B) % BDC2.5 Tregs BDC2.5 Effector T Cells SQZ® TACs Augmented Antigen-Specific Tregs Capable of Bystander Suppression IFNγ secretion (BDC2.5 T cells) IFNγ secretion (NY8.3 T cells) TAC-p31 alone increased BDC2.5 Tregs and suppressed NY8.3 effectors via bystander suppression . (A) Schematic of BDC2.5 and NY8.3 co-adoptive transfer, TAC dosing regimen and pancreas harvest. (B) Frequency of BDC2.5 Tregs in the indicated TAC treatment group assessed by flow cytometry. (C) The number of BDC2.5 (left) and NY8.3 (right) effector T cells in the indicated TAC treatment groups evaluated by flow cytometry. (D) IFNγ secretion measured by cytokine bead array from culture supernatants of lymphocytes stimulated with p31 (left) and NRPA7 peptide (right) in various TAC treatment groups. NY8.3 Effector T Cells SQZ® TAC Cell Therapy Platform Induces Antigen Specific T-regs and Prevents Onset of Type 1 Diabetes in Adoptive Transfer Models Todd Ashworth, Shinu John, Colin Raposo, Judit Cserny, Gloria Serena, Patricia Cho, Armon Sharei and Howard Bernstein, SQZ Biotechnologies, Watertown, MA, USA Utility of the SQZ® platform to induce durable disease remission in Celiac Disease

(b) (c)

(e) (f)

(a) (b)

Preclinical development of a TAC-DAG clinical product to restore peripheral tolerance in CeD (A) Spleen/liver mediated clearance, processing and class II presentation of TAC-derived deamidated gliadin peptides induce antigen dependent T-cell deletion/anergy and expansion of regulatory T-cells post IV transfusion. (B) Significant loading of deamidated alpha gliadin epitopes into TACs can be quantified by ELISA. (C) Schematic overview of an in vitro potency assay used to quantify the relative potency of TAC-DAG in a DQ2.5+ MoDC/Jurkat-luciferase co-culture system. Free or cell squeezed peptide (TAC) is provided for 72 hours following which Jurkat luciferase signals are measured to assess relative potency across various conditions tested. (D) Cell Squeeze® engineering of deamidated alpha gliadin activated antigen specific T-cells in vitro. (B)Free peptide TAC 40000 60000 80000 400000 500000 600000 700000 800000

Rlu

Hel

Deamidated 33-mer1 2 3 4 5 6 Deamidated α-Gliadin (ng/1E9 TAC) Batch number

Summary

Tolerizing Antigen Carriers cell squeezed with either autoantigen or food allergens causal in autoimmune disease expands the therapeutic potential of the SQZ® platform to disease areas beyond Immuno-Oncology. Autoimmune diseases defined by well defined antigens, such as Celiac Disease, have the potential to benefit from TAC therapy given the ability of TACs to activate T-cells in an antigen specific manner. SQZ Biotech plans to leverage the TAC platform for the treatment of Celiac Disease with an IND filing planned for H1 2023.

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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® TAC Cell Therapy Platform Induces Antigen Specific T-regs and Prevents Onset of Type 1 Diabetes in Adoptive Transfer Models 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® TAC Cell Therapy Platform Induces Antigen Specific T-regs and Prevents Onset of Type 1 Diabetes in Adoptive Transfer Models - Document type: Poster - Year: 2022 - Outlet: FOCIS - Cells: RBCs - Cargos: Polymers, Protein, Peptide - Page URL: https://portal.bio/publications/2022-sqz-tac-cell-therapy-platform-induces-antigen-specific-t-regs-and-prevents-onset-of-t--ajkGlxEAACcAc97S - Markdown URL: https://portal.bio/publications/2022-sqz-tac-cell-therapy-platform-induces-antigen-specific-t-regs-and-prevents-onset-of-t--ajkGlxEAACcAc97S.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65ccdb1a9be9a5b998b5d123_FOCISposter_Final.pdf ## Summary Todd Ashworth, Shinu John, Colin Raposo, Judit Cserny, Gloria Serena, Patricia Cho, Armon Sharei and Howard Bernstein ## Publication text ### Abstract Antigen specific tolerance is a recognized therapeutic mode of promoting durable disease remission in autoimmune diseases with well-defined antigens which could overcome the problems associated with broad immunosuppression. The Cell Squeeze® technology utilizes microfluidic mechanical deformation to engineer cell function. To induce tolerance, red blood cells (RBCs) are engineered to deliver target antigens to generate tolerizing antigen carriers (TACs). The Cell Squeeze technology allows the SQZ®TACs to resemble senescent RBCs, enabling them to be cleared by eryptosis, the physiological mechanism of RBC clearance. Herein, we demonstrate SQZ TACs biodistribute to phagocytic cells in the spleen and liver where antigens are processed and presented to antigen specific T-cells in a tolerogenic manner. SQZ TAC treatment in several accelerated murine models of T1D led to significant delay or prevention of hyperglycemia. In both CD4+ (BDC2.5) and CD8+ (NY8.3) NOD adoptive transfer T1D models, co-delivery of TAC loaded with T1D autoantigens provided protection from early disease onset characterized by increased T-cell apoptosis, tissue specific T-reg induction and decreased effector T-cell pancreatic infiltration. In addition, administration of CD4+ directed TACs with the peptide p31 protected T1D onset in NOD mice co-transferred with both BDC2.5 and NY8.3 T cells, thereby demonstrating TAC induced bystander suppression. Recent preclinical efforts have expanded to include development of a TAC-deamidated gliadin formulation designed to ameliorate gluten sensitivity in patients suffering with Celiac Disease. SQZ® Technology Cytosolic Antigen Delivery (Cell Squeeze® Technology): [TITLE] SQZ® TACs are Efficiently Loaded and Have a Modified Membrane TACs are efficiently delivered. (A) Murine red blood cells squeezed with AlexaFluor-647-conjugated ovalbumin (OVA) or AlexaFluor-647-conjugated IgG. Loading is assessed by flow cytometry. (B) Phosphatidyl serine surface levels assessed by annexin V staining of murine red blood cells squeezed or left intact are assessed by flow cytometry. SQZ® TACs Are Engulfed By Phagocytic Cells in Various Organs Uptake of TACs by antigen presenting cells in different organs. (A-B) PKH26 labeled TACs were injected into B6 mice and uptake in various cell types in spleen (A) and liver (B) was assessed 1h postinjection by flow cytometry. SQZ® TAC-p31 Significantly Delayed Diabetes Onset Induced By BDC2.5 T Cells0 20 40 60 80 100 Blood Glucose Day Post Transfer Blood Glucose (mg/dL) No TAC0 20 40 60 80 100 Disease Incidence Day Post Transfer Percent Disease Free No TAC TAC-p31 delayed onset of hyperglycemia in BDC2.5 transfer model. (A) Schematic of adoptive transfer of activated BDC2.5 chromogranin-specific CD4+ T cells and TAC-p31 (BDC2.5 mimetope, relevant antigen) or TAC-HEL (irrelevant antigen) dosing regimen in NOD.SCID mice. (B-C) Blood glucose (BG) levels (B) and disease incidence (C) in animals that were treated with different TACs or left untreated (No TAC). Dotted line represents BG of 250mg/dL. Mice were considered hyperglycemic when BG levels were >250mg/dL for two consecutive readings. For B and C, n=9 for no TAC and n=10 for TAC-p31 and TAC-HEL. SQZ® TAC-p31 Treatment Decreased Insulitis And Upregulated Antigen-Specific Tregs in PancreasTAC-HEL IL-10 pg/mL ✱✱✱✱Spleen Pancreas %CD90.2 CD4+ (of Live) ✱✱Spleen Pancreas %Foxp3+ (of CD4+ CD90.2+) TAC-p31✱✱ ✱✱ (B) TAC-HEL TAC-p31TAC-HEL 10000 30000 IFNg (pg/ml)TAC-HEL 40000 60000 IFNg (pg/mL) * Treatment with TAC-p31 reduced infiltration of BDC2.5 T cells in pancreas, expanded Tregs and suppressed proinflammatory cytokine production (A) Schematic of activated BDC2.5 T cell adoptive transfer and TAC dosing regimen in NOD.SCID recipients. Mice were euthanized either on day 8 (flow cytometry) or day 15 (histology) post-adoptive transfer. (B) Representative H & E staining of pancreata isolated from TAC-HEL (left) and TAC-p31 (right) treated animals. Arrowhead represents islets of Langerhans. (C) Frequency of BDC2.5 T cells in spleen and pancreas of TAC-HEL and TAC-p31 treated mice were assessed by flow cytometry. (D) Lymphocytes were isolated from the indicated groups and organs and stimulated for 24h with p31 peptide. IFNγ secretion from lymphocyte culture supernatants from spleen (left) and pancreas (right) was measured by cytokine bead array. (E) Frequency of BDC2.5 Tregs in spleen and pancreas of indicated treatment groups as analyzed by flow cytometry. (F) IL-10 production measured by ELISA from culture supernatants of splenocytes stimulated with p31 peptide. (A) (B) % BDC2.5 Tregs BDC2.5 Effector T Cells SQZ® TACs Augmented Antigen-Specific Tregs Capable of Bystander Suppression IFNγ secretion (BDC2.5 T cells) IFNγ secretion (NY8.3 T cells) TAC-p31 alone increased BDC2.5 Tregs and suppressed NY8.3 effectors via bystander suppression . (A) Schematic of BDC2.5 and NY8.3 co-adoptive transfer, TAC dosing regimen and pancreas harvest. (B) Frequency of BDC2.5 Tregs in the indicated TAC treatment group assessed by flow cytometry. (C) The number of BDC2.5 (left) and NY8.3 (right) effector T cells in the indicated TAC treatment groups evaluated by flow cytometry. (D) IFNγ secretion measured by cytokine bead array from culture supernatants of lymphocytes stimulated with p31 (left) and NRPA7 peptide (right) in various TAC treatment groups. NY8.3 Effector T Cells SQZ® TAC Cell Therapy Platform Induces Antigen Specific T-regs and Prevents Onset of Type 1 Diabetes in Adoptive Transfer Models Todd Ashworth, Shinu John, Colin Raposo, Judit Cserny, Gloria Serena, Patricia Cho, Armon Sharei and Howard Bernstein, SQZ Biotechnologies, Watertown, MA, USA Utility of the SQZ® platform to induce durable disease remission in Celiac Disease ### (b) (c) ### (e) (f) ### (a) (b) ### (a) (b) Preclinical development of a TAC-DAG clinical product to restore peripheral tolerance in CeD (A) Spleen/liver mediated clearance, processing and class II presentation of TAC-derived deamidated gliadin peptides induce antigen dependent T-cell deletion/anergy and expansion of regulatory T-cells post IV transfusion. (B) Significant loading of deamidated alpha gliadin epitopes into TACs can be quantified by ELISA. (C) Schematic overview of an in vitro potency assay used to quantify the relative potency of TAC-DAG in a DQ2.5+ MoDC/Jurkat-luciferase co-culture system. Free or cell squeezed peptide (TAC) is provided for 72 hours following which Jurkat luciferase signals are measured to assess relative potency across various conditions tested. (D) Cell Squeeze® engineering of deamidated alpha gliadin activated antigen specific T-cells in vitro. (B)Free peptide TAC 40000 60000 80000 400000 500000 600000 700000 800000 ### Rlu ### Hel Deamidated 33-mer1 2 3 4 5 6 Deamidated α-Gliadin (ng/1E9 TAC) Batch number ### Summary Tolerizing Antigen Carriers cell squeezed with either autoantigen or food allergens causal in autoimmune disease expands the therapeutic potential of the SQZ® platform to disease areas beyond Immuno-Oncology. Autoimmune diseases defined by well defined antigens, such as Celiac Disease, have the potential to benefit from TAC therapy given the ability of TACs to activate T-cells in an antigen specific manner. SQZ Biotech plans to leverage the TAC platform for the treatment of Celiac Disease with an IND filing planned for H1 2023. ## 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.