Defne Yarar, Katarina Blagovic, Amritha Ramakrishnan, Katherine Seidl, Howard Bernstein, and Armon Sharei
- Cells
- RBCs
- Cargos
- Polymers, Protein, Peptide, DNA
Publication text
In summary, these results indicate that antigen and adjuvant delivery via AACs to APCs in vivo can effectively prime a potent anti-tumor response in mice and support the further study of SQZ AACs as an immunotherapy for cancer treatment. Activating Antigen Carriers for Cancer Therapy: Preclinical Immune Responses Drive Tumor Regression Defne Yarar, Katarina Blagovic, Amritha Ramakrishnan, Katherine Seidl, Howard Bernstein, and Armon Sharei; SQZ Biotechnologies, Watertown, MA, USA SQZ AACs Prime Endogenous CD8+ T Cell Responses to Protein and Peptide AntigensBackground Murine AACs are Efficiently Loaded and Have a Modified Membrane SQZ 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 5SQZ 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 AAC Treatment Prevents Tumor Formation Prophylactic immunization protects mice from tumor challenge. C57BL/6 mice were immunized with SQZ AACs loaded with adjuvant and tumor antigen. Seven days later, mice were subcutaneously implanted with a tumor cell line expressing the antigen. (Left) Tumor growth kinetics for untreated and SQZ immunized mice; (Right) Survival of untreated and SQZ immunized mice. Numbers in parentheses indicate the number of tumor-free mice at day 60. Day 0: Vaccinate mice w/ SQZ-AAC Day 7: Tumor implant Measure tumors / body weights 2x per week
Pbs (0/10)
250M AACs (10/10) AACs are efficiently delivered. (A) Murine red blood cells SQZ’d with Ax-647-conjugated ovalbumin (OVA) or AlexaFluor-647-conjugated IgG. Loading is assessed by flow cytometry. (B) Phosphatidyl serine surface levels as assessed by annexin V staining of murine red blood cells SQZ’d or left intact is assessed by flow cytometry. Circulation kinetics and site/cell type uptake of AACs. (A) The persistence of fluorescently (PKH26) labeled AACs or intact red blood cells (RBCs) in the mouse blood stream was determined. (B) The organ and (C) cell type (RP Mf-red pulp macrophage; DC-dendritic cell) for AAC uptake was determined following injection into WT mice. Fluorescently-labeled cells were detected by flow cytometry. A AACs are cleared from blood within 60 minutes t1/2=10 min AACs are primarily taken up in liver and spleen
C57bl/6sqz Aac
Pkh26
labeled RBC B C AACs are engulfed by macrophages and DCs
C57bl/6sqz Aac
C SQZ AACs prime endogenous CD8+ T cell responses in vivo. Mice were immunized with cells SQZ’d with either protein (ovalbumin) or peptide antigen (tumor antigen). (A) OT-II (left) or OT-I D Tumor antigen (peptide) PBS AAC Vaccination Ovalbumin
Cfse Cfse
CD4+/CD44Hi CD8+/CD44Hi Ovalbumin (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) Intracellular cytokine staining was performed on splenocytes 7 days later. (D) Boosts were administered to a subset of mice and intracellular cytokine staining was performed 7 days after the final vaccination. Therapeutic immunization delays tumor growth. Mice were subcutaneously implanted with a tumor cell line and therapeutically immunized with (A) a single dose of SQZ AACs at the indicated doses or (B) one to three doses of 1 x 106 SQZ AACs . (Left) Tumor growth kinetics for untreated mice and mice immunized 7 days after tumor implant. Day(s) of additional dose(s) indicated. (Right) Survival of untreated 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 administration Measure tumors / body weights 2x per week Day 0: Tumor implant Day 10: SQZ-AAC prime Measure tumors / body weights 2x per weekDay 12: SQZ-AAC boost Day 14: SQZ-AAC boost Therapeutic SQZ AAC immunization promotes expansion of tumor-specific CD8+ T cells. TIL analysis from day 21 (purple) or day 26 (pink) post SQZ AAC administration shown as (A) total CD8+ T cells per gram tumor mass, (B) % antigenspecific CD8 + T cells, (C) the ratio of tetramer positive CD8+ T cells to T regulatory cells, or (D) the number of tetramer+ cells per gram of tumor tissue versus total tumor weight.
A B
C D
Day 0: Tumor implant Day 14: SQZ-AAC vaccination Day 21 Day 26 TIL analysis Human SQZ AACs Promote Antigen-Specific Responses Human MoDCs can process and present SQZ AACderived antigen on MHC-I to re-stimulate T cell responses in vitro. Medium (neg. control) or AACs loaded with cytomegalovirus antigen were incubated with MoDCs and adjuvant in vitro and then co-cultured with CMV antigen-specific CD8+ T responder cells. 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 SQZing red blood cells with fluorescently labeled tumor antigen and adjuvant. (A) % of cells delivered with antigen and (B) % of cells positive for annexin V was assessed by flow cytometry. Data from SQZ’d cells (H-SQZ-AAC, green) and untreated cells (no SQZ, black) are shown. BA Human SQZ AACs are Internalized by DCs In Vitro Human dendritic cells engulf AACs. Monocyte-derived dendritic cells were incubated with SQZ-AACs loaded with fluorescently labeled antigen (Aovalbumin; B-tumor antigen) for 24 hours and imaged by epi-fluorescence microscopy or (B) analyzed by flow cytometry. Cells were incubated at 37ºC (A, B) or on ice (B), as shown.
A B
SQZ with tumor-specific synthetic long peptide (SLP) Co-culture AAC-fed DCs with antigenspecific CD8+ T cell responders Incubate SQZ-AACs with DCs in vitro Assess IFNγ secretion at 18-24hr with ELISA Isolate human RBCs We have developed an approach to generate tumor-specific CD8 T cell responses by directly targeting antigen and adjuvant to antigen presenting cells (APCs) in vivo. This method harnesses the natural process of red blood cell (RBC) clearance from the body to activate the immune response. Using the Cell Squeeze® microfluidics platform, we have generated activating antigen carriers (AACs), engineered from RBCs, that are loaded with antigen and adjuvant. Here, we show that AACs target in vivo APCs, drive endogenous antigen presentation and prime potent anti-tumor T cell responses. These findings support the further study of SQZ AACs as an immunotherapy for human cancer treatment. IFNg (pg/mL)
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.
# Activating Antigen Carriers for Cancer Therapy: Preclinical Immune Responses Drive Tumor Regression
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 for Cancer Therapy: Preclinical Immune Responses Drive Tumor Regression
- Document type: Poster
- Year: 2019
- Outlet: SITC
- Cells: RBCs
- Cargos: Polymers, Protein, Peptide, DNA
- Page URL: https://portal.bio/publications/2019-activating-antigen-carriers-for-cancer-therapy-preclinical-immune-responses-drive-tum--ajkHGxEAACgAc-CL
- Markdown URL: https://portal.bio/publications/2019-activating-antigen-carriers-for-cancer-therapy-preclinical-immune-responses-drive-tum--ajkHGxEAACgAc-CL.md
- Original file: https://portal-bio.cdn.prismic.io/portal-bio/65ccffe69be9a5b998b5d311_SITC-2019-Poster-D.-Yarar.pdf
## Summary
Defne Yarar, Katarina Blagovic, Amritha Ramakrishnan, Katherine Seidl, Howard Bernstein, and Armon Sharei
## Publication text
In summary, these results indicate that antigen and adjuvant delivery via AACs to APCs in vivo can effectively prime a potent anti-tumor response in mice and support the further study of SQZ AACs as an immunotherapy for cancer treatment. Activating Antigen Carriers for Cancer Therapy: Preclinical Immune Responses Drive Tumor Regression Defne Yarar, Katarina Blagovic, Amritha Ramakrishnan, Katherine Seidl, Howard Bernstein, and Armon Sharei; SQZ Biotechnologies, Watertown, MA, USA SQZ AACs Prime Endogenous CD8+ T Cell Responses to Protein and Peptide AntigensBackground Murine AACs are Efficiently Loaded and Have a Modified Membrane SQZ 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 5SQZ 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 AAC Treatment Prevents Tumor Formation Prophylactic immunization protects mice from tumor challenge. C57BL/6 mice were immunized with SQZ AACs loaded with adjuvant and tumor antigen. Seven days later, mice were subcutaneously implanted with a tumor cell line expressing the antigen. (Left) Tumor growth kinetics for untreated and SQZ immunized mice; (Right) Survival of untreated and SQZ immunized mice. Numbers in parentheses indicate the number of tumor-free mice at day 60. Day 0: Vaccinate mice w/ SQZ-AAC Day 7: Tumor implant Measure tumors / body weights 2x per week
### Pbs (0/10)
250M AACs (10/10) AACs are efficiently delivered. (A) Murine red blood cells SQZ’d with Ax-647-conjugated ovalbumin (OVA) or AlexaFluor-647-conjugated IgG. Loading is assessed by flow cytometry. (B) Phosphatidyl serine surface levels as assessed by annexin V staining of murine red blood cells SQZ’d or left intact is assessed by flow cytometry. Circulation kinetics and site/cell type uptake of AACs. (A) The persistence of fluorescently (PKH26) labeled AACs or intact red blood cells (RBCs) in the mouse blood stream was determined. (B) The organ and (C) cell type (RP Mf-red pulp macrophage; DC-dendritic cell) for AAC uptake was determined following injection into WT mice. Fluorescently-labeled cells were detected by flow cytometry. A AACs are cleared from blood within 60 minutes t1/2=10 min AACs are primarily taken up in liver and spleen
### C57bl/6sqz Aac
### Pkh26
labeled RBC B C AACs are engulfed by macrophages and DCs
### C57bl/6sqz Aac
C SQZ AACs prime endogenous CD8+ T cell responses in vivo. Mice were immunized with cells SQZ’d with either protein (ovalbumin) or peptide antigen (tumor antigen). (A) OT-II (left) or OT-I D Tumor antigen (peptide) PBS AAC Vaccination Ovalbumin
### Cfse Cfse
CD4+/CD44Hi CD8+/CD44Hi Ovalbumin (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) Intracellular cytokine staining was performed on splenocytes 7 days later. (D) Boosts were administered to a subset of mice and intracellular cytokine staining was performed 7 days after the final vaccination. Therapeutic immunization delays tumor growth. Mice were subcutaneously implanted with a tumor cell line and therapeutically immunized with (A) a single dose of SQZ AACs at the indicated doses or (B) one to three doses of 1 x 106 SQZ AACs . (Left) Tumor growth kinetics for untreated mice and mice immunized 7 days after tumor implant. Day(s) of additional dose(s) indicated. (Right) Survival of untreated 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 administration Measure tumors / body weights 2x per week Day 0: Tumor implant Day 10: SQZ-AAC prime Measure tumors / body weights 2x per weekDay 12: SQZ-AAC boost Day 14: SQZ-AAC boost Therapeutic SQZ AAC immunization promotes expansion of tumor-specific CD8+ T cells. TIL analysis from day 21 (purple) or day 26 (pink) post SQZ AAC administration shown as (A) total CD8+ T cells per gram tumor mass, (B) % antigenspecific CD8 + T cells, (C) the ratio of tetramer positive CD8+ T cells to T regulatory cells, or (D) the number of tetramer+ cells per gram of tumor tissue versus total tumor weight.
### A B
### C D
Day 0: Tumor implant Day 14: SQZ-AAC vaccination Day 21 Day 26 TIL analysis Human SQZ AACs Promote Antigen-Specific Responses Human MoDCs can process and present SQZ AACderived antigen on MHC-I to re-stimulate T cell responses in vitro. Medium (neg. control) or AACs loaded with cytomegalovirus antigen were incubated with MoDCs and adjuvant in vitro and then co-cultured with CMV antigen-specific CD8+ T responder cells. 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 SQZing red blood cells with fluorescently labeled tumor antigen and adjuvant. (A) % of cells delivered with antigen and (B) % of cells positive for annexin V was assessed by flow cytometry. Data from SQZ’d cells (H-SQZ-AAC, green) and untreated cells (no SQZ, black) are shown. BA Human SQZ AACs are Internalized by DCs In Vitro Human dendritic cells engulf AACs. Monocyte-derived dendritic cells were incubated with SQZ-AACs loaded with fluorescently labeled antigen (Aovalbumin; B-tumor antigen) for 24 hours and imaged by epi-fluorescence microscopy or (B) analyzed by flow cytometry. Cells were incubated at 37ºC (A, B) or on ice (B), as shown.
### A B
SQZ with tumor-specific synthetic long peptide (SLP) Co-culture AAC-fed DCs with antigenspecific CD8+ T cell responders Incubate SQZ-AACs with DCs in vitro Assess IFNγ secretion at 18-24hr with ELISA Isolate human RBCs We have developed an approach to generate tumor-specific CD8 T cell responses by directly targeting antigen and adjuvant to antigen presenting cells (APCs) in vivo. This method harnesses the natural process of red blood cell (RBC) clearance from the body to activate the immune response. Using the Cell Squeeze® microfluidics platform, we have generated activating antigen carriers (AACs), engineered from RBCs, that are loaded with antigen and adjuvant. Here, we show that AACs target in vivo APCs, drive endogenous antigen presentation and prime potent anti-tumor T cell responses. These findings support the further study of SQZ AACs as an immunotherapy for human cancer treatment. IFNg (pg/mL)
## 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.
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