# Microfluidic cell squeezing enables human PBMCs as drivers of antigen-specific CD8 T cell responses across a broad range of antigens for diverse clinical applications 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: Microfluidic cell squeezing enables human PBMCs as drivers of antigen-specific CD8 T cell responses across a broad range of antigens for diverse clinical applications - Document type: Poster - Year: 2020 - Outlet: SITC - Page URL: https://portal.bio/publications/2020-microfluidic-cell-squeezing-enables-human-pbmcs-as-drivers-of-antigen-specific-cd8-t--ajkG-REAAC0Ac-Am - Markdown URL: https://portal.bio/publications/2020-microfluidic-cell-squeezing-enables-human-pbmcs-as-drivers-of-antigen-specific-cd8-t--ajkG-REAAC0Ac-Am.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65cced7f9be9a5b998b5d244_SITC-2020-APC-Human-Poster-M.-Maloney-FINAL.pdf ## Summary Michael F. Maloney, Scott M. Loughhead, Amritha Ramakrishnan, Carolyne Smith, Anita Venkitaraman, Christian Yee, Miye Jacques, Defne Yarar, Armon Sharei, Howard, Bernstein, Kelan Hlavaty, Melissa Myint, Katherine J. Seidl ## Publication text Microfluidic cell squeezing enables human PBMCs as drivers of antigen-specific CD8 T cell responses across a broad range of antigens for diverse clinical applications Michael F. Maloney, Scott M. Loughhead, Amritha Ramakrishnan, Carolyne Smith, Anita Venkitaraman, Christian Yee, Miye Jacques, Defne Yarar, Armon Sharei, Howard Bernstein, Kelan Hlavaty, Melissa Myint, Katherine J. Seidl All Associated with SQZ Biotechnologies, Watertown, MA, USA ### Background Antigen-specific CD8+ T cell activity is critical for mounting an effective immune response in a wide range of indications, including immune-oncology and infectious diseases. To elicit antigen-specific CD8+ T cell activity, we used microfluidic cell squeezing (Cell Squeeze® technology) to deliver antigens directly to the cytosol of antigen presenting cells (APCs). Direct cytosolic delivery bypasses the need for cross-presentation and efficiently loads antigen into the major histocompatibility complex class I (MHC-I) pathway. The Cell Squeeze® technology enables delivery of various biomaterials into all major cell subsets of human peripheral blood mononuclear cells (PBMCs) in a single SQZ process. Therefore, not only does microfluidic squeezing enable PBMCs to function as unconventional APCs, but it also allows efficient investigation of a wide range of antigens including whole protein, peptides, and mRNA. The ability to utilize various antigen biomaterials in the Cell Squeeze® technology allows for broad application in multiple disease areas. SQZ APCs Are Agnostic to Antigen Biomaterial SQZ Technology and APC Platform Cytosolic Antigen Delivery: Human PBMCs squeezed with SLP or antigen-encoded mRNA re-stimulate T cell responses in vitro. Isolated human PBMCs were either untreated (No Contact) or squeezed in the presence of a CMV pp65 SLP (A), HPV16 E7 SLP (B), Influenza M1 mRNA (C), or HPV16 E7 mRNA (D). Squeezed PBMCs were co-cultured with the respective HLA-restricted antigen-specific CD8+ T cells. Activation of antigen-specific T cells was assessed by IFN-γ ELISA. Squeezed PBMCs Efficiently Translate mRNA Cell Squeeze® technology enables delivery to all major cell subsets of human PBMCs in a single SQZ process. Isolated human PBMCs were squeezed in the presence of 3 kDa dextran fluorescently labeled with Cascade Blue. Squeezed PBMCs were stained for major cell subsets and delivery of 3 kDa dextran was analyzed by flow cytometry. Untreated PBMCs (No Contact) were used as a control. Engineering Cells to Function as Antigen Presenting Cells: No Contact Cascade Blue Dextran T cells (CD3+) B cells (CD19+) Monocytes (CD14+) NK cells (CD56+) Cell Squeeze® Technology Enables Efficient Delivery to All Major Cell Subsets of PBMCs Dextran Delivery in PBMCs Dextran SQZ Delivery and viability for research scale SQZ processes is equivalent to delivery and viability of PBMCs processed at manufacturing scale (>1 billion cells). Utilizing a fully-enclosed cGMP SQZ system, over 1 billion isolated human PBMCs were squeezed in the presence of 3 kDa dextran fluorescently labeled with Cascade Blue. Delivery of dextran was analyzed by flow cytometry. Viability, delivery, and recovery post SQZ are plotted from three manufacturing scale SQZ processes. ### Mhc-i ### Mhc-ii Endocytosis ### Mhc-i Cell Squeeze® Delivery Enhances MHC-I Presentation: Cell Squeeze® delivery of naked, uncomplexed mRNA to PBMCs results in robust protein translation. Isolated human PBMCs were squeezed in the presence of naked, uncomplexed mRNA encoding for EGFP (A), HPV16 E7 (B), or HPV16 E6 (C). (A) EGFP mRNA translation was analyzed 4 hours post SQZ by flow cytometry. (B-C) HPV16 E6 and E7 mRNA translation was detected by Western Blot of squeezed PBMC whole cell lysate. Untreated PBMCs (No Contact) were used as a control. ### Egfp EGFP mRNA Translation in PBMCs No Contact EGFP mRNA ### Sqz ### A B C Cell Squeeze® Technology: Manufacturing Scale SQZ Engineering of Traditional and Nontraditional APCs Manufacturing Scale Performance and RecoveryNo Contact 15000 20000 25000 Squeeze MoDCs Squeeze B Cells 12000 Squeeze T Cells IFN (pg/mL) Squeezed human MoDCs, B cells, or T cells process and present antigenic epitopes on MHC-I to re-stimulate T cell responses in vitro. MoDCs (A), B cells (B), or T cells (C) were isolated from human PBMCs and squeezed in the presence of a CMV pp65 synthetic long peptide (SLP). Squeezed cells were co-cultured CMV pp65-specific CD8+ T cell responders. Activation of CD8+ T cell responders was assessed by IFN-γ ELISA. Untreated PBMCs (No Contact) or PBMCs incubated with the pp65 SLP (Endo) were used as controls. ### A B C ### Summary MoDCs, T cells, or B cells as APCs CD8+ T cells CD8+ T cell activation and IFNγ secretion Cell Squeeze® technology has demonstrated a robust ability to deliver antigen biomaterial to human PBMCs to generate APCs that drive antigenspecific CD8 T cell responses. SQZ APCs have been produced at the manufacturing scale in < 24 hours. The HPV-specific SQZ APC candidate, SQZ- PBMC-HPV, is currently being evaluated in a phase I trial for HPV16+ tumors. 84% 87% 81% 94% 90% 78% Traditionally, cross-presentation is needed for efficient presentation on MHC-I. SQZ delivers materials directly to cytosol, bypassing need for cross-presentation.No Contact Flu M1 mRNA SQZ Flu M1-reactive IFN (pg/mL) A B CNo Contact CMV pp65-reactive ### Hpv16 E7 Slp Sqz HPV16 E7-reactive IFN (pg/mL) D mRNA Encoding AntigenSLP as AntigenNo Contact HPV16 E7 mRNA SQZ 12000 HPV16 E7-reactive IFN (pg/mL)Pre-Squeeze Post-Squeeze % ViabilitySQZ % Recovery Post-SQZNo Contact ### Sqz % Delivery #169 human PBMCs SQZ SLP or mRNA encoding for fulllength antigen Co-culture SQZ APCs with antigen-specific CD8+ T cells Assess T cell activation via IFN-γ ELISA ## 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.