Michael F. Maloney, E. Ilker Ozay, Amy M. Merino, Andrea A. Silva, Amber Martin, Sanjana Manja, Madhav Upadhyay, Christine Trumpfheller, Pablo Umana, Armon Sharei, Howard Bernstein, Scott M. Loughhead
- Cells
- T cells, PBMCs, Monocytes, B cells, Dendritic Cells, NK cells
- Cargos
- Polymers, Protein, Peptide, mRNA
Publication text
SQZ™ eAPCs Generated from PBMCs by Delivery of Multiple mRNAs Encoding for Antigens, Costimulatory Proteins, and Engineered Cytokines #211
Background
Antigen-specific CD8+ T cells are critical components of mounting an effective immune response against tumors. Generation of antigen-specific T cells require interactions with multiple signals produced by antigen presenting cells (APCs). These signals are comprised of three components: (signal 1) the peptide-MHC complex binding to the T cell receptor, (signal 2) costimulatory molecules on the surface of APCs, and (signal 3) inflammatory cytokines binding to cognate receptors on T cells. To engineer all major cell subsets of human peripheral blood mononuclear cells (PBMCs) to become enhanced APCs (eAPCs), we used Cell Squeeze® technology to deliver multiple mRNA encoding for non-self antigens (signal 1), CD86 (signal 2), and/or membrane-bound cytokines (signal 3). The signal 3 molecules, membranebound IL-12 (mbIL-12) and membrane-bound IL-2 (mbIL-2), are chimeric proteins designed to increase the localized concentration of the cytokines and limit off-target effects. Flow cytometry and western blots were used to confirm the translation of each of the delivered mRNA. The increased capabilities of these enhanced APCs were assessed in vitro by culturing the APCs with antigen-specific T cells for multiple days before measuring the functionality of antigen-specific T cells via intracellular cytokine staining or ELISA. We demonstrate that Cell Squeeze® processing of PBMCs with mRNA encoding for signals 1, 2, and 3 results in highly effective enhanced APCs in vitro. In a single squeeze process, efficient delivery and translation of up to five mRNA is observed in all major PBMC cell subsets including T cells, B cells, NK cells, and monocytes. Once translated, the chimeric mbIL-2 and mbIL-12 can bind to their cognate receptors. We show that enhanced APCs can present antigenic peptides derived from mRNA encoding for a foreign antigen on MHC complexes in an HLA agnostic manner, which drives antigen-specific T cell responses (i.e. HPV16-specific T cells). The addition of CD86, mbIL-2, and mbIL-12 further enhance the activation of antigen-specific T cells, as measured by an increase in the secretion of the proinflammatory cytokine, IFNγ, upon peptide restimulation. SQZ™ Technology and Enhanced APC Platform Cytosolic Antigen Delivery (Cell Squeeze® Technology): Generation of Enhanced APCs (SQZ™ eAPCs) from PBMCs via Delivery of mRNA: *mb indicates membrane-bound SQZ™ eAPC
Cd86
2 3 Signals CD8 T Cell SQZ™ eAPC mbIL-2 mbIL-12TCR
Mhc-i
Signal 3: mbIL-2 or mbIL-12 Signal 2: CD86 Signal 1: Antigen mRNA CD8 T cell proliferation mRNA-Encoded CD86 and Membrane-bound Cytokines Translated in PBMC Cell Subsets
Cd86
mbIL-2 mbIL-12No mRNA mbIL-2 mRNA Squeeze mbIL-12 mRNA Squeeze 0.1 ng/mL 0.5 ng/mL 1 ng/mL 0.2 0.4 0.6 0.8 HEK-Blue IL-12
Od630 (au)
Background Subtracted Recombinant IL-12 spikeNo mRNA mbIL-2 mRNA Squeeze mbIL-12 mRNA squeeze 0.05 ng/mL 0.1 ng/mL 0.5 ng/mL 0.3 0.6 0.9 1.2 1.5 HEK-Blue IL-2
Od630 (au)
Background Subtracted Recombinant IL-2 spike B cells T cells NK cells Monocytes Human PBMCs Squeeze mRNAs encoding for CD86, mbIL-2, and mbIL-12 Culture squeezed PBMCs for 4 or 24 hours Harvest cells for flow cytometry at 4 hours Cell Squeeze® technology enables signal 2/3 mRNA delivery to all major cell subsets of human PBMCs resulting in the translation of functional membrane-bound proteins. Isolated human PBMCs were squeezed in the presence of CD86, mbIL-2, and/or mbIL-12 mRNA. A) After squeezing, PBMCs were incubated for 4 or 24 hours to allow for translation of the mRNA. Squeezed PBMCs were stained at 4 hours for major cell subsets (T, B, NK cells, and monocytes) before measuring protein levels by flow cytometry. Mock-squeezed PBMCs were used as a control. B) After squeezing with mbIL-2 or mbIL-12 mRNA, PBMCs were co-culture with HEK-Blue™ IL-2 or IL-12 reporter cells, which express the cognate cytokine receptor. Engagement of cytokine receptors triggers a signaling pathway and induces the production of secreted alkaline phosphatase (SEAP). Activation of the HEK-Blue reporter cells was assessed using a QUANTI-Blue™ assay to measure the levels of SEAP in the co-culture supernatant. Recombinant IL-2 or IL-12 were spiked into cultures as a positive control. Human PBMCs squeezed with mbIL-2 or mbIL-12 mRNA QUANTI-Blue™ Assay + HEK-Blue IL-2 or HEK-Blue IL-12 Co-squeezing mRNAs Encoding HPV16 Antigens + Signal 2/3 mRNA Generates a Robust T Cell Response Human PBMCs from an HLA-A*02 donor Squeezed PBMCs + 0.1% E6 or E7 TCR transduced T cells Re-stimulate CD8+ T cells with E6 or E7 minimal epitopes Assess expansion of HPV16-specific T cells by IFNγ ICS Squeeze mRNAs encoding for E6, E7, CD86, mbIL-2, and mbIL-12 CD8+ T cells transduced with E6 or E7-specific TCR 6 days No mRNA CD86+mbIL-2 E6+E7+CD86+mbIL-2 CD3No mRNA CD86+mbIL-2+mbIL-12 mRNA E6+E7+CD86+mbIL-2+mbIL-12 mRNA 0.4 0.8 1.2 1.6 E629-38 Peptide Restim % IFNγ+ of CD8 T cells % IFNγ+ of CD8+ (No Restim Subtracted) Cultures with E6 TCR transduced T cells No mRNA CD86+mbIL-2 E6+E7+CD86+mbIL-2
Cd3
Cultures with E7 TCR transduced T cellsBNo mRNA CD86+mbIL-2+mbIL-12 mRNA E6+E7+CD86+mbIL-2+mbIL-12 mRNA E711-19 Peptide Restim % IFNγ+ of CD8 T cells % IFNγ+ of CD8+ (No Restim Subtracted)A PBMCs squeezed with mRNAs encoding E6, E7, CD86, mbIL-2 and mbIL-12 are capable of expanding CD8+ T cells transduced with HPV16-specific TCRs. Isolated CD8+ T cells from an HLA-A*02 donor were transduced with HPV16 E6 TCR or E7 TCR lentiviruses. The transduced CD8+ T cells were cultured with autologous PBMCs that were squeezed in the presence of E6+E7 mRNA and/or CD86+mbIL-2+mbIL-12 mRNA. To allow for expansion of antigenspecific CD8+ T cells, squeezed PBMCs were cultured with the E6 TCR or E7 TCR transduced T cells for 6 days. Cells were then restimulated with the E6 or E7 minimal epitopes. Intracellular cytokine staining for IFNγ was done to measure the percentage of E6 TCR T cells (A) or E7 TCR T cells (B). Squeezing all 5 mRNA into PBMCs resulted in a significant increase in the percentage of the cognate HPV16-specific CD8+ T cells compared to E6+E7 mRNA alone or CD86+mbIL-2+mbIL-12 mRNA alone. Translation, Processing, and Presentation of Antigen-Encoded mRNA in SQZ™ eAPCs Human PBMCs squeezed with antigenencoded mRNA activate expanded antigenspecific CD8+ T cells in vitro. Isolated human PBMCs were squeezed in the presence of HPV16 E7 mRNA (A) or CMV pp65 mRNA (B). Squeezed PBMCs were co-cultured with the respective antigen-specific CD8+ T cells that have been expanded with the cognate minimal epitopes. Activation of the antigenspecific CD8+ T cells was assessed by measuring IFNγ levels in the culture supernatants via ELISA. Translation of mRNA was detected by western blot of squeezed PBMC whole cell lysate. Untreated PBMCs were used as a negative control.
A B
human PBMCs Squeeze mRNA encoding for non-self antigen Co-culture SQZ APCs with antigen-specific CD8+ T cells Assess T cell activation via IFNγ ELISAUntreated pp65 mRNA Squeeze 12000 CMV pp65-reactive T cells IFN (pg/mL)Untreated E7 and E6 mRNA Squeeze 12000 HPV16 E7-reactive T cells IFN (pg/mL)
Summary
Cell Squeezing of human PBMCs with mRNA encoding for signals 1, 2, and 3 has the potential to generate enhanced APCs that drive robust CD8+ T cell response against multiple targets across several disease areas, including HPV16+ tumors. The versatility of the Cell Squeeze® technology potentially enables rapid implementation across multiple diseases by simply changing the mRNA encoded antigen. It also provides a basis for future optimization of T cell activation signals through incorporation of other signaling molecules. PBMCs squeezed with antigen-encoding mRNA restimulate memory CD8+ T cell responses in vitro. Isolated human PBMCs from an HLA-A*02 CMV+ donor were squeezed in the presence of CMV pp65 mRNA. To allow for expansion of antigenspecific CD8+ T cells, squeezed PBMCs were cultured for 5 days. Cells were then restimulated with known HLA-restricted minimal epitopes. Intracellular cytokine staining (ICS) for IFN-γ was done to measure the percentage of CD8+ memory T cells specific for the minimal epitope. Squeezing antigen-encoding mRNA into PBMCs resulted in a significant increase in the percentage of the cognate antigen-specific CD8+ memory T cells compared to the empty squeeze control. Cell Squeeze® mRNA Delivery Stimulates Memory CD8+ T Cells Across Various HLA Haplotypes0
Ifn+cd45ro+
(% of CD3+CD8+) pp65 mRNA + + + + No Peptide pp65265-275 pp65417-426
Hla-a*01
pp65363-373 Empty squeeze CMV pp65 mRNA SQZ pp65265-275 Peptide No Peptide
Cd45ro
Pp65417-426 Peptide
Hla-a*01
pp65363-373 Peptide BA Michael F. Maloney1, E. Ilker Ozay1, Amy M. Merino1, Andrea A. Silva1, Amber Martin1, Sanjana Manja1, Madhav Upadhyay1, Christine Trumpfheller2, Pablo Umana2, Armon Sharei1, Howard Bernstein1, Scott M. Loughhead1 1SQZ Biotechnologies, Watertown, MA USA 2Roche Innovation Center Zurich, Roche Pharma Research and Early Development (pRED), Schlieren, Switzerland
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™ eAPCs Generated from PBMCs by Delivery of Multiple mRNAs Encoding for Antigens, Costimulatory Proteins, and Engineered Cytokines
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™ eAPCs Generated from PBMCs by Delivery of Multiple mRNAs Encoding for Antigens, Costimulatory Proteins, and Engineered Cytokines
- Document type: Poster
- Year: 2021
- Outlet: SITC
- Cells: T cells, PBMCs, Monocytes, B cells, Dendritic Cells, NK cells
- Cargos: Polymers, Protein, Peptide, mRNA
- Page URL: https://portal.bio/publications/2021-sqztm-eapcs-generated-from-pbmcs-by-delivery-of-multiple-mrnas-encoding-for-antigens--ajkGwxEAACcAc99i
- Markdown URL: https://portal.bio/publications/2021-sqztm-eapcs-generated-from-pbmcs-by-delivery-of-multiple-mrnas-encoding-for-antigens--ajkGwxEAACcAc99i.md
- Original file: https://portal-bio.cdn.prismic.io/portal-bio/65cce0de9be9a5b998b5d194_SITC2021eAPCPosterFinal211.pdf
## Summary
Michael F. Maloney, E. Ilker Ozay, Amy M. Merino, Andrea A. Silva, Amber Martin, Sanjana Manja, Madhav Upadhyay, Christine Trumpfheller, Pablo Umana, Armon Sharei, Howard Bernstein, Scott M. Loughhead
## Publication text
SQZ™ eAPCs Generated from PBMCs by Delivery of Multiple mRNAs Encoding for Antigens, Costimulatory Proteins, and Engineered Cytokines #211
### Background
Antigen-specific CD8+ T cells are critical components of mounting an effective immune response against tumors. Generation of antigen-specific T cells require interactions with multiple signals produced by antigen presenting cells (APCs). These signals are comprised of three components: (signal 1) the peptide-MHC complex binding to the T cell receptor, (signal 2) costimulatory molecules on the surface of APCs, and (signal 3) inflammatory cytokines binding to cognate receptors on T cells. To engineer all major cell subsets of human peripheral blood mononuclear cells (PBMCs) to become enhanced APCs (eAPCs), we used Cell Squeeze® technology to deliver multiple mRNA encoding for non-self antigens (signal 1), CD86 (signal 2), and/or membrane-bound cytokines (signal 3). The signal 3 molecules, membranebound IL-12 (mbIL-12) and membrane-bound IL-2 (mbIL-2), are chimeric proteins designed to increase the localized concentration of the cytokines and limit off-target effects. Flow cytometry and western blots were used to confirm the translation of each of the delivered mRNA. The increased capabilities of these enhanced APCs were assessed in vitro by culturing the APCs with antigen-specific T cells for multiple days before measuring the functionality of antigen-specific T cells via intracellular cytokine staining or ELISA. We demonstrate that Cell Squeeze® processing of PBMCs with mRNA encoding for signals 1, 2, and 3 results in highly effective enhanced APCs in vitro. In a single squeeze process, efficient delivery and translation of up to five mRNA is observed in all major PBMC cell subsets including T cells, B cells, NK cells, and monocytes. Once translated, the chimeric mbIL-2 and mbIL-12 can bind to their cognate receptors. We show that enhanced APCs can present antigenic peptides derived from mRNA encoding for a foreign antigen on MHC complexes in an HLA agnostic manner, which drives antigen-specific T cell responses (i.e. HPV16-specific T cells). The addition of CD86, mbIL-2, and mbIL-12 further enhance the activation of antigen-specific T cells, as measured by an increase in the secretion of the proinflammatory cytokine, IFNγ, upon peptide restimulation. SQZ™ Technology and Enhanced APC Platform Cytosolic Antigen Delivery (Cell Squeeze® Technology): Generation of Enhanced APCs (SQZ™ eAPCs) from PBMCs via Delivery of mRNA: *mb indicates membrane-bound SQZ™ eAPC
### Cd86
2 3 Signals CD8 T Cell SQZ™ eAPC mbIL-2 mbIL-12TCR
### Mhc-i
Signal 3: mbIL-2 or mbIL-12 Signal 2: CD86 Signal 1: Antigen mRNA CD8 T cell proliferation mRNA-Encoded CD86 and Membrane-bound Cytokines Translated in PBMC Cell Subsets
### Cd86
mbIL-2 mbIL-12No mRNA mbIL-2 mRNA Squeeze mbIL-12 mRNA Squeeze 0.1 ng/mL 0.5 ng/mL 1 ng/mL 0.2 0.4 0.6 0.8 HEK-Blue IL-12
### Od630 (au)
Background Subtracted Recombinant IL-12 spikeNo mRNA mbIL-2 mRNA Squeeze mbIL-12 mRNA squeeze 0.05 ng/mL 0.1 ng/mL 0.5 ng/mL 0.3 0.6 0.9 1.2 1.5 HEK-Blue IL-2
### Od630 (au)
Background Subtracted Recombinant IL-2 spike B cells T cells NK cells Monocytes Human PBMCs Squeeze mRNAs encoding for CD86, mbIL-2, and mbIL-12 Culture squeezed PBMCs for 4 or 24 hours Harvest cells for flow cytometry at 4 hours Cell Squeeze® technology enables signal 2/3 mRNA delivery to all major cell subsets of human PBMCs resulting in the translation of functional membrane-bound proteins. Isolated human PBMCs were squeezed in the presence of CD86, mbIL-2, and/or mbIL-12 mRNA. A) After squeezing, PBMCs were incubated for 4 or 24 hours to allow for translation of the mRNA. Squeezed PBMCs were stained at 4 hours for major cell subsets (T, B, NK cells, and monocytes) before measuring protein levels by flow cytometry. Mock-squeezed PBMCs were used as a control. B) After squeezing with mbIL-2 or mbIL-12 mRNA, PBMCs were co-culture with HEK-Blue™ IL-2 or IL-12 reporter cells, which express the cognate cytokine receptor. Engagement of cytokine receptors triggers a signaling pathway and induces the production of secreted alkaline phosphatase (SEAP). Activation of the HEK-Blue reporter cells was assessed using a QUANTI-Blue™ assay to measure the levels of SEAP in the co-culture supernatant. Recombinant IL-2 or IL-12 were spiked into cultures as a positive control. Human PBMCs squeezed with mbIL-2 or mbIL-12 mRNA QUANTI-Blue™ Assay + HEK-Blue IL-2 or HEK-Blue IL-12 Co-squeezing mRNAs Encoding HPV16 Antigens + Signal 2/3 mRNA Generates a Robust T Cell Response Human PBMCs from an HLA-A*02 donor Squeezed PBMCs + 0.1% E6 or E7 TCR transduced T cells Re-stimulate CD8+ T cells with E6 or E7 minimal epitopes Assess expansion of HPV16-specific T cells by IFNγ ICS Squeeze mRNAs encoding for E6, E7, CD86, mbIL-2, and mbIL-12 CD8+ T cells transduced with E6 or E7-specific TCR 6 days No mRNA CD86+mbIL-2 E6+E7+CD86+mbIL-2 CD3No mRNA CD86+mbIL-2+mbIL-12 mRNA E6+E7+CD86+mbIL-2+mbIL-12 mRNA 0.4 0.8 1.2 1.6 E629-38 Peptide Restim % IFNγ+ of CD8 T cells % IFNγ+ of CD8+ (No Restim Subtracted) Cultures with E6 TCR transduced T cells No mRNA CD86+mbIL-2 E6+E7+CD86+mbIL-2
### Cd3
Cultures with E7 TCR transduced T cellsBNo mRNA CD86+mbIL-2+mbIL-12 mRNA E6+E7+CD86+mbIL-2+mbIL-12 mRNA E711-19 Peptide Restim % IFNγ+ of CD8 T cells % IFNγ+ of CD8+ (No Restim Subtracted)A PBMCs squeezed with mRNAs encoding E6, E7, CD86, mbIL-2 and mbIL-12 are capable of expanding CD8+ T cells transduced with HPV16-specific TCRs. Isolated CD8+ T cells from an HLA-A*02 donor were transduced with HPV16 E6 TCR or E7 TCR lentiviruses. The transduced CD8+ T cells were cultured with autologous PBMCs that were squeezed in the presence of E6+E7 mRNA and/or CD86+mbIL-2+mbIL-12 mRNA. To allow for expansion of antigenspecific CD8+ T cells, squeezed PBMCs were cultured with the E6 TCR or E7 TCR transduced T cells for 6 days. Cells were then restimulated with the E6 or E7 minimal epitopes. Intracellular cytokine staining for IFNγ was done to measure the percentage of E6 TCR T cells (A) or E7 TCR T cells (B). Squeezing all 5 mRNA into PBMCs resulted in a significant increase in the percentage of the cognate HPV16-specific CD8+ T cells compared to E6+E7 mRNA alone or CD86+mbIL-2+mbIL-12 mRNA alone. Translation, Processing, and Presentation of Antigen-Encoded mRNA in SQZ™ eAPCs Human PBMCs squeezed with antigenencoded mRNA activate expanded antigenspecific CD8+ T cells in vitro. Isolated human PBMCs were squeezed in the presence of HPV16 E7 mRNA (A) or CMV pp65 mRNA (B). Squeezed PBMCs were co-cultured with the respective antigen-specific CD8+ T cells that have been expanded with the cognate minimal epitopes. Activation of the antigenspecific CD8+ T cells was assessed by measuring IFNγ levels in the culture supernatants via ELISA. Translation of mRNA was detected by western blot of squeezed PBMC whole cell lysate. Untreated PBMCs were used as a negative control.
### A B
human PBMCs Squeeze mRNA encoding for non-self antigen Co-culture SQZ APCs with antigen-specific CD8+ T cells Assess T cell activation via IFNγ ELISAUntreated pp65 mRNA Squeeze 12000 CMV pp65-reactive T cells IFN (pg/mL)Untreated E7 and E6 mRNA Squeeze 12000 HPV16 E7-reactive T cells IFN (pg/mL)
### Summary
Cell Squeezing of human PBMCs with mRNA encoding for signals 1, 2, and 3 has the potential to generate enhanced APCs that drive robust CD8+ T cell response against multiple targets across several disease areas, including HPV16+ tumors. The versatility of the Cell Squeeze® technology potentially enables rapid implementation across multiple diseases by simply changing the mRNA encoded antigen. It also provides a basis for future optimization of T cell activation signals through incorporation of other signaling molecules. PBMCs squeezed with antigen-encoding mRNA restimulate memory CD8+ T cell responses in vitro. Isolated human PBMCs from an HLA-A*02 CMV+ donor were squeezed in the presence of CMV pp65 mRNA. To allow for expansion of antigenspecific CD8+ T cells, squeezed PBMCs were cultured for 5 days. Cells were then restimulated with known HLA-restricted minimal epitopes. Intracellular cytokine staining (ICS) for IFN-γ was done to measure the percentage of CD8+ memory T cells specific for the minimal epitope. Squeezing antigen-encoding mRNA into PBMCs resulted in a significant increase in the percentage of the cognate antigen-specific CD8+ memory T cells compared to the empty squeeze control. Cell Squeeze® mRNA Delivery Stimulates Memory CD8+ T Cells Across Various HLA Haplotypes0
### Ifn+cd45ro+
(% of CD3+CD8+) pp65 mRNA + + + + No Peptide pp65265-275 pp65417-426
### Hla-a*01
pp65363-373 Empty squeeze CMV pp65 mRNA SQZ pp65265-275 Peptide No Peptide
### Cd45ro
Pp65417-426 Peptide
### Hla-a*01
pp65363-373 Peptide BA Michael F. Maloney1, E. Ilker Ozay1, Amy M. Merino1, Andrea A. Silva1, Amber Martin1, Sanjana Manja1, Madhav Upadhyay1, Christine Trumpfheller2, Pablo Umana2, Armon Sharei1, Howard Bernstein1, Scott M. Loughhead1 1SQZ Biotechnologies, Watertown, MA USA 2Roche Innovation Center Zurich, Roche Pharma Research and Early Development (pRED), Schlieren, Switzerland
## 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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