# Activating Antigen Carriers Generated With Microfluidic Cell Squeezing Drive Effective Anti-Tumor Responses 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: Activating Antigen Carriers Generated With Microfluidic Cell Squeezing Drive Effective Anti-Tumor Responses - Document type: Poster - Year: 2020 - Outlet: SITC - Cells: RBCs - Cargos: Polymers, Protein, Peptide, DNA - Page URL: https://portal.bio/publications/2020-activating-antigen-carriers-generated-with-microfluidic-cell-squeezing-drive-effectiv--ajkG_REAAC4Ac-A0 - Markdown URL: https://portal.bio/publications/2020-activating-antigen-carriers-generated-with-microfluidic-cell-squeezing-drive-effectiv--ajkG_REAAC4Ac-A0.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65cceeda9be9a5b998b5d250_SITC-2020-SQZ-AAC-Poster-K.-Blagovic-FINAL-1-.pdf ## Summary Katarina Blagovic, Amritha Ramakrishnan, Armon Sharei, Howard Bernstein, Katherine Seidl and Defne Yarar ## Publication text Activating Antigen Carriers Generated With Microfluidic Cell Squeezing Drive Effective Anti-Tumor Responses Katarina Blagovic, Amritha Ramakrishnan, Armon Sharei, Howard Bernstein, Katherine Seidl and Defne Yarar; SQZ Biotechnologies, Watertown, MA, USA SQZ AACs Prime Endogenous CD8+ and CD4+ T Cell Responses to Protein and Peptide Antigens ### Background SQZ-Loaded AACs Have Exposed Phosphatidyl Serine SQZ Technology and AAC Platform SQZ AAC Therapeutic Treatment Slows Tumor Growth Antigen Presentation is Indirect (Through Endogenous Antigen Presenting Cells) Antigen and Adjuvant Loading to Generate Activating Antigen Carriers (AACs) 2 4 1 5 SQZ antigen + adjuvant Engulfment by APCs Prime endogenous T cell response Robust systemic response T cells target tumor cells Tumor cell death RBCs, antigen, and adjuvant suspension High-speed cell deformation 3 Brief membrane disruption 5 Membrane resealing 4 Antigen diffusion into cytosol Tumor Regression Correlates with an Influx of Antigen Specific CD8+ T Cells SQZ AACs are engineered to have increased levels of exposed phosphatidylserine (PS) on the AAC membrane outer leaflet. The surface PS is hypothesized to serve as the ligand recognized by receptors on antigen presenting cells that internalize administered AACs in vivo, similar to that of aged RBCs. (A) Phosphatidyl serine surface levels as assessed by annexin V staining of murine red blood cells squeezed with E7 synthetic long peptide (SLP) and adjuvant (AACs) or left intact (RBCs) is assessed by flow cytometry. (B) Frequency of CD47+ events in intact murine red blood cells (RBCs) or AACs. AACs are efficiently delivered with different payloads by SQZ process. (C) Murine red blood cells squeezed with Cascade Blue-conjugated 3kDa dextran, AF647-conjugated ovalbumin (OVA), AlexaFluor488-conjugated IgG, or empty squeezed. Percent of delivered cells is assessed by flow cytometry. Circulation kinetics of AACs. SQZ process increases the levels of exposed phosphatidylserine (PS) on AACs which resemble aged RBCs, and which are cleared from circulation quickly. (A) The duration of fluorescently (PKH26) labeled AACs or intact red blood cells (RBCs) in the mouse blood stream was determined. AACs are cleared from blood within 60 minutes, while RBCs persist. ### A C SQZ AACs prime endogenous antigen-specific CD8+ and CD4+ T cell responses in vivo. Mice were immunized with AACs squeezed with either protein (ovalbumin) or E7 peptide antigen (tumor antigen). (A) Protein and adjuvant squeezed AACs elicit proliferation of CD8+ and CD4+ cells. OT-II (left) or OT-I (right) cells were labeled with CFSE and adoptively transferred into recipient mice. Proliferation of cells in the spleen was monitored 3 days later. (B, C) Both antigen (protein or peptide) and adjuvant are needed to elicit CD8+ T cell responses in vivo. D E7 peptide PBS AAC Vaccination Ovalbumin ### Cfse Cfse CD4+/CD44Hi CD8+/CD44Hi Ovalbumin Boosting augments antigen-specific CD8+ T cell responses. Boosts were administered to a subset of mice and intracellular cytokine staining was performed 7 days after the final vaccination. (D) Boosting on Day 2 post-prime enhances antigenspecific CD8+ T cell responses (**p<0.01). (E) Repeated dosing of AACs additionally increases E7specific CD8+ T cell responses compared to prime alone (****p<0.0001). Therapeutic immunization delays tumor growth in TC-1 tumor model. Mice were subcutaneously implanted with the TC-1 tumor cell line and therapeutically immunized with (A) a single dose of SQZ AAC- HPV at the indicated doses or (B) one or two doses of 1 x 106 SQZ AAC-HPV. (Left) Tumor growth kinetics for untreated mice and mice immunized 10 days after tumor implant. Day(s) of additional dose(s) indicated. (Right) Survival of untreated (PBS) and SQZ immunized mice. Numbers in brackets indicate the median survival. 10 mice were included per group. Day 0: Tumor implant Day 10: SQZ AAC-HPV administration Measure tumors / body weights 2x per week Day 0: Tumor implant Day 10: SQZ AAC-HPV prime Measure tumors / body weights 2x per week Day 12: SQZ AAC-HPV Boost Day 2 Human SQZ AACs Promote Antigen-Specific Responses Human MoDCs can process and present SQZ AAC-derived antigen on MHC-I to re-stimulate T cell responses in vitro. Medium (neg. control) or AACs loaded with tumor derived antigens (E7) and adjuvant were incubated with monocyte-derived dendritic cells in vitro and then co-cultured with antigen-specific CD8+ T responder cells in the presence of LPS. Antigen-specific responses were determined by IFN-γ ELISA. Human AACs are Efficiently Loaded Ex Vivo Characterization of human AACs. Human AACs were generated by squeezing red blood cells with HPV E7, E6 SLPs and adjuvant. Each color dot represents a different human blood donor. (A) % of cells delivered with FAM-labeled E7 SLP and (B) % of cells positive for annexin V was assessed by flow cytometry. (C) Representative images of AACs generated by SQZ processing of FAM-labeled E7 SLP (top), and unlabeled E7 SLP (bottom). Cells were stained with human erythrocyte surface marker CD235a. Graphs are displaying normalized FAM (green) and PacBlue (blue) fluorescence intensity along a line drawn across length of AAC- HPV. BA Human SQZ AACs are Internalized by DCs In Vitro Internalization of AACs by human dendritic cells results in upregulation of maturation markers. (A) Monocyte-derived dendritic cells were incubated with membrane-labeled AACs loaded with adjuvant and antigen and analyzed by flow cytometry. Cells were incubated at 37ºC or at 4ºC, as shown. (B) Upregulation of maturation markers on the surface of DCs following 48-hour co-culture with AACs. Each dot color represents a different MoDC donor. ### A B SQZ with tumor-specific E7 synthetic long peptide (SLP) and adjuvant Co-culture AAC-fed DCs with E7 antigenspecific CD8+ T cell responders Incubate SQZ-AACs with DCs in vitro Assess IFNγ secretion at 18-24hr with ELISA Isolate human RBCs Activation of T cell responses is essential for effective tumor clearance, however, the generation of targeted, effective antigen presentation to stimulate T cell response remains challenging. Using our proprietary Cell Squeeze® technology, we generate SQZ Activating Antigen Carriers (AACs) from red blood cells (RBCs) squeezed with tumor-specific antigens and adjuvant as a potential approach for tumor-specific cancer vaccines. This method makes the cells appear aged and thus harnesses the natural process of RBC clearance from the body for rapid engulfment in vivo by endogenous professional antigen presenting cells (APCs). AAC uptake, processing, and presentation by APCs drives antigen-specific activation of endogenous T cells against a tumor. Here, we show that AACs target in vivo APCs, drive endogenous antigen presentation, and prime potent anti-tumor T cell responses in mice. In a human in vitro model, AACs cocultured with APCs drive T cell activation. These findings support the further study of SQZ AACs as an immunotherapy for human cancer treatment. CRBCs % Annexin V+RBCs ### % Cd47+ Uptake of AACs by endogenous APCs. (A) The organ and cell type (RPM-red pulp macrophage; DC-dendritic cell) for AAC uptake was determined following injection into WT mice. AACs are primarily taken up in liver and spleen (left). AACs are engulfed by macrophages and DCs (right). In vivo maturation of APCs. (B) Upregulation of maturation markers on endogenous splenic APCs following uptake of AACs loaded with adjuvant and antigen (E7 SLP). Empty squeezed RBCs were used as a control. ### C57bl/6sqz Aac AACs are Rapidly Cleared from CirculationCD8+ DC CD11b+ DC ### Rpm 12000 16000 CD86 geometric MFI ### Cd86cd8+dc CD11b+DC ### Rpm 10000 20000 40000 80000 MHC-II geometric MFI Empty SQZ ### Sqz Aac-hpv* ### Mhc-ii AAC Uptake Results in Upregulation of Maturation Markers of Endogenous APCs ### Bpbs P ### P/b/b/b % IFN+ / CD44hi (of CD8+) ****0 10 20 30 40 Tumor Volume (mm3)0 20 40 60 Percent survival PBS [36] ### 100m Sqz Aac-hpv Prime [38] ### 100m Sqz Aac-hpv Prime + Boost Day 2 [51] PacBlue- CD235a ### Fam-e7 ### Slp CRBCs % DeliveryRBCs % Annexin V+0.1 1 10 100 1000 10000 20000 30000 40000 50000 60000 AAC (x106) ### Mfi (a.u.) (Gated on live CD11c+) PKH26 labeled AACs (37C) AACs (4C) PKH26 labeled AACs (4C)Empty SQZ 0.5 1.0 1.5 2.0 2.5 ### Cd86 Fold Change from Control Media **Empty SQZ 1.8 ### Mhc-ii Fold Change from Control Media *Neg control IFN (pg/mL) Summary: These results indicate that AACs loaded with antigen and adjuvant can effectively drive antigen presentation and prime a potent antigen-specific anti-tumor response in mice. In vitro, antigen and adjuvant loaded AACs can induce antigen-specific CD8+ T cell responses following co-culture with human APCs. Together this supports the further study of SQZ AACs as an immunotherapy for cancer treatment.0 10 20 30 40 Tumor Volume (mm3)0 20 40 60 Percent survival PBS [32.5] ### 1b Sqz Aac-hpv [57] ### 250m Sqz Aac-hpv [47] ### 100m Sqz Aac-hpv [39.5] 1B – 1x109 250M – 250x106 100M – 100x106 t1/2=10 min ### C57bl/6sqz Aac ### Pkh26 labeled RBCLiver Spleen Bone marrow Lung Lymph node 2×106 4×106 6×106 8×106 1×107 CD45+PKH26+ cells/organRPM DC B cells ### %pkh26+ (Gated on Live CD45+ cells) Liver (AAC) Liver (Control) Spleen (AAC) Spleen (control)Dextran ### Ova IgG % Delivery Empty SQZ 0 50 100 150 200 Normalized Intensity (AU) Distance (Pixels) 0 50 100 150 200 Normalized Intensity (AU) Distance (Pixels)PBS Adjuvant E7 peptide E7 peptide + adjuvant 0.8Adjuvant Ovalbumin Ovalbumin + adjuvant 1.0 % IFN+ / CD44HI (of CD8+) E P – Prime B – BoostPBS P ### P/b % IFN+ / CD44hi (of CD8+) ** Therapeutic SQZ AAC immunization promotes the expansion of tumor-specific CD8+ T cells. Tumor infiltrating lymphocyte (TIL) analysis from day 21 (teal) or day 26 (blue) post SQZ-AAC-HPV administration shows a significant increase in: (A) Total number of CD8+ T cells per gram of tumor tissue. (B) % of antigen-specific CD8+ T cells. (C) Ratio of tetramer positive CD8+ T cells to T regulatory cells. At least 100-fold increase in E7-specific CD8+ T cells/Treg ratio is observed compared to PBS controls. ### A B C Day 0: Tumor implant Day 14: SQZ-AAC vaccination Day 21 Day 26 TIL analysis ### Aac0 Recruitment of Ag-specific CD8 TILs % Tetramer+ (Gated on Live CD8+ T cells) - *1 2 3 2×10 6 4×10 6 6×10 6 8×10 6 Recruitment of CD8+ T Cells No. of CD8+ T cells/g tissue - * ### Aac0.01 0.1 Ratio of Tet+CD8 Tcells/Tregs Tetramer+ T cells/Tregs * *** ### Aac ## 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.