Poster · 2021 · AACR

Peripheral Blood Mononuclear Cells Engineered via Microfluidic Cell Squeeze® Technology to Generate APCs that Drive Mutant-KRas-specific CD8+ T Cells

Carolyne Smith, Defne Yarar, Amritha Ramakrishnan, Kelan Hlavaty, Scott Loughhead, Armon Sharei, Katherine Seidl, Howard Bernstein

Publication text

Peripheral Blood Mononuclear Cells Engineered via Microfluidic Cell Squeeze® Technology to Generate APCs that Drive Mutant-KRas-specific CD8+ T Cells Carolyne Smith, Defne Yarar, Amritha Ramakrishnan, Kelan Hlavaty, Scott Loughhead, Armon Sharei, Katherine Seidl, Howard Bernstein; SQZ Biotechnologies, Watertown, MA, USA SQZ™ PBMC-KRas G12D Activates KRAS Mutations are Shared Across Multiple Cancer Types Squeezed Mouse APCs Inhibit Tumor Growth and Drive an Influx of Antigen-specific CD8 T Cells Therapeutic immunization with squeezed splenocytes inhibits tumor growth and drives the expansion of tumor specific CD8 T cells. (A) Tumor growth kinetics for mice bearing TC-1 tumors that were left untreated (black), vaccinated subcutaneously with an E7 synthetic long peptide plus CpG (orange), or immunized with splenocytes squeezed with the same E7 SLP (teal). (B-D) TIL analysis from the day 28 time point showing the intratumoral influx of CD45+ cells, CD8a+ cells, and E7-tetramer+ CD8 T cells. KRAS is frequently mutated in lung, pancreatic, and colon cancers. The frequencies of KRAS G12D, G12V, G12C, G13D, and G13R mutations in NSCLC, PDA, and colon cancer were extracted from the COSMIC database (https://cancer.sanger.ac.uk/ cosmic). These frequencies were multiplied by the cancer incidences (AACR.org) to determine the estimated incidence per year.0 10,000 20,000 30,000 40,000 50,000 Colon cancer Pancreatic cancer

Nsclc

G12d G12v G12c

G13d G12r

Estimated Incidence per Year (in USA)Media Irrelevant

Wt1-16

G12d1-16

G12d2-19

G12d2-29

SFU / 1 x 106 cells Human PBMCs present KRas G12D-derived antigen to KRas-specific CD8 T cells. HLA- A*11 PBMCs squeezed with media containing KRas G12D peptides of various lengths were co-cultured with KRas G12D-specific CD8 T cells generated in A*11 transgenic mice. Activation of T cells was assessed by ELISpot analysis (IFN). PBMCs incubated with media supplemented with free peptide containing the KRas G12D minimal epitope (Positive Ctrl) and PBMCs squeezed with media, E7-derived peptide (irrelevant), or WT KRas peptide (WT 1-16) were used as controls. Each dot shows the response derived from a different PBMC donor. G12D Functional Response KRasG12D-Specific CD8+ T cells KRasG12D peptide squeezed PBMC

Summary

Here we demonstrate that SQZ™ APCs targeting KRas can directly present antigen to and activate KRas G12D and KRas G12V mutant-specific CD8 T cells. This work lays a strong foundation for developing a potentially potent cell therapy to treat KRAS mutant associated cancers. SQZ™ PBMC-KRas G12V Activates Human PBMCs present KRas G12V-derived antigen to KRas-specific CD8 T cells. HLA-A*11 PBMCs squeezed with media containing KRas G12V peptides of various lengths were co-cultured with KRas G12V-specific CD8 T cells generated in A*11 transgenic mice. Activation of T cells was assessed by ELISpot analysis (IFN). PBMCs incubated with media supplemented with free peptide containing the KRas G12V minimal epitope (Positive Ctrl) and PBMCs squeezed with media, E7-derived peptide (irrelevant), or WT KRas peptide (WT 1-16) were used as controls. Each dot shows the response derived from a different PBMC donor.Media

G12v

G12v+g12d

SFU / 1 X 106 cells G12V Functional ResponseMedia

G12d

G12v+g12d

12000 14000 SFU / 1x 106 cells G12D Functional Response Human PBMCs present multiple KRas mutant antigens to KRas-specific CD8 T cells. HLA-A*11 PBMCs squeezed with media containing KRas G12D and G12V peptides were co-cultured with KRas G12D-specific (left panel) or KRas G12V-specific (right panel) CD8 T cells generated in A*11 transgenic mice. Activation of T cells was assessed by ELISpot analysis (IFN). PBMCs incubated with media supplemented with free peptide containing the minimal epitope (G12D or G12V variants) and PBMCs squeezed with media were used as controls. Each dot shows the response derived from a different PBMC donor. KRasG12D+G12V peptide squeezed

Pbmc

KRasG12D-Specific CD8+ T cells or KRasG12V-Specific CD8+ T cells G12V Functional ResponseMedia Irrelevant

Wt1-16

G12v1-16

G12v2-19

SFU / 1 x 106 cells KRasG12V-Specific CD8+ T cells KRasG12V peptide squeezed PBMC SQZ™ PBMC-KRas G12V+G12D Activates #15240 Tumor Vol. (mm3) Untreated Squeezed Splenocytes (IV) E7 SLP + CpG (Subcutaneous) 16 28 Prime Imm

Til

Analysis Day DayUntreated % CD45+ (of Live) ns *** **Untreated % CD8a+ (of CD45+) *** *** *Untreated % E7 Tetramer+ (of CD8a+) *** *** *

A Dcb

Cell Squeeze® Technology Enables Efficient Delivery to All Major Cell Subsets of PBMCs

Abstract

KRAS mutations are shared across multiple cancer types and are most commonly found in cancers of the lung, pancreas, and colon. While recently developed drugs blocking signaling driven by KRAS G12C mutations have shown promise in the clinic, there is a great need for therapies targeting the more common G12V and G12D mutations that are not easily targeted with small molecules. We utilize our microfluidic Cell Squeeze® technology to deliver antigens directly into the cytosol of peripheral blood mononuclear cells (PBMCs) for the generation of cancer antigen-targeted cellular vaccines. This method bypasses the need for cross-presentation and directly enables efficient antigen presentation on MHC-I in multiple cell types. Murine and human in vitro studies have demonstrated that this allows SQZ PBMCs to drive CD8+ T cell activation. Here, we demonstrate the delivery of various antigens, including KRas-derived peptides containing the most common mutations (G12D and G12V) to PBMC cell subsets. In addition, we find that human PBMCs squeezed with either KRasG12V or KRasG12D peptides can specifically stimulate KRasG12V- and KRasG12D- reactive CD8+ T cell responders. When KRasG12V and KRasG12D peptides are co-delivered via microfluidic squeezing, the human PBMCs enable activation of CD8+ T cells of both specificities. SQZ™ Technology and APC Platform Cytosolic Antigen Delivery: Engineering Cells to Function as Antigen Presenting Cells:

Mhc-i

Mhc-ii

Endocytosis

Mhc-i

Cell Squeeze® Delivery Enhances MHC-I Presentation: Traditionally, cross-presentation is needed for efficient presentation on MHC-I. Cell Squeeze® technology delivers materials directly to cytosol, bypassing need for cross-presentation. No Contact Cascade Blue Dextran T cells (CD3+) B cells (CD19+) Monocytes (CD14+) NK cells (CD56+) Dextran Delivery in PBMCs Dextran 84% 87% 81% 94% 90% Cell Squeeze® technology enables delivery to all major cell subsets of human PBMCs in a single squeeze process. Isolated human PBMCs were squeezed in the presence of 3 kDa Cascade Blue-labeled dextran, stained for major cell subsets and analyzed by flow cytometry. Untreated PBMCs (No Contact) were used as a control. Squeeze for cytosolic antigen delivery

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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.

# Peripheral Blood Mononuclear Cells Engineered via Microfluidic Cell Squeeze® Technology to Generate APCs that Drive Mutant-KRas-specific CD8+ T Cells 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: Peripheral Blood Mononuclear Cells Engineered via Microfluidic Cell Squeeze® Technology to Generate APCs that Drive Mutant-KRas-specific CD8+ T Cells - Document type: Poster - Year: 2021 - Outlet: AACR - Page URL: https://portal.bio/publications/2021-peripheral-blood-mononuclear-cells-engineered-via-microfluidic-cell-squeeze-technolog--ajkG5xEAACwAc9_t - Markdown URL: https://portal.bio/publications/2021-peripheral-blood-mononuclear-cells-engineered-via-microfluidic-cell-squeeze-technolog--ajkG5xEAACwAc9_t.md - Original file: https://portal-bio.cdn.prismic.io/portal-bio/65cce8779be9a5b998b5d210_AACR-2021-PBMC-KRas-Poster-C.-Smith-FINAL.pdf ## Summary Carolyne Smith, Defne Yarar, Amritha Ramakrishnan, Kelan Hlavaty, Scott Loughhead, Armon Sharei, Katherine Seidl, Howard Bernstein ## Publication text Peripheral Blood Mononuclear Cells Engineered via Microfluidic Cell Squeeze® Technology to Generate APCs that Drive Mutant-KRas-specific CD8+ T Cells Carolyne Smith, Defne Yarar, Amritha Ramakrishnan, Kelan Hlavaty, Scott Loughhead, Armon Sharei, Katherine Seidl, Howard Bernstein; SQZ Biotechnologies, Watertown, MA, USA SQZ™ PBMC-KRas G12D Activates KRAS Mutations are Shared Across Multiple Cancer Types Squeezed Mouse APCs Inhibit Tumor Growth and Drive an Influx of Antigen-specific CD8 T Cells Therapeutic immunization with squeezed splenocytes inhibits tumor growth and drives the expansion of tumor specific CD8 T cells. (A) Tumor growth kinetics for mice bearing TC-1 tumors that were left untreated (black), vaccinated subcutaneously with an E7 synthetic long peptide plus CpG (orange), or immunized with splenocytes squeezed with the same E7 SLP (teal). (B-D) TIL analysis from the day 28 time point showing the intratumoral influx of CD45+ cells, CD8a+ cells, and E7-tetramer+ CD8 T cells. KRAS is frequently mutated in lung, pancreatic, and colon cancers. The frequencies of KRAS G12D, G12V, G12C, G13D, and G13R mutations in NSCLC, PDA, and colon cancer were extracted from the COSMIC database (https://cancer.sanger.ac.uk/ cosmic). These frequencies were multiplied by the cancer incidences (AACR.org) to determine the estimated incidence per year.0 10,000 20,000 30,000 40,000 50,000 Colon cancer Pancreatic cancer ### Nsclc ### G12d G12v G12c ### G13d G12r Estimated Incidence per Year (in USA)Media Irrelevant ### Wt1-16 ### G12d1-16 ### G12d2-19 ### G12d2-29 SFU / 1 x 106 cells Human PBMCs present KRas G12D-derived antigen to KRas-specific CD8 T cells. HLA- A*11 PBMCs squeezed with media containing KRas G12D peptides of various lengths were co-cultured with KRas G12D-specific CD8 T cells generated in A*11 transgenic mice. Activation of T cells was assessed by ELISpot analysis (IFN). PBMCs incubated with media supplemented with free peptide containing the KRas G12D minimal epitope (Positive Ctrl) and PBMCs squeezed with media, E7-derived peptide (irrelevant), or WT KRas peptide (WT 1-16) were used as controls. Each dot shows the response derived from a different PBMC donor. G12D Functional Response KRasG12D-Specific CD8+ T cells KRasG12D peptide squeezed PBMC ### Summary Here we demonstrate that SQZ™ APCs targeting KRas can directly present antigen to and activate KRas G12D and KRas G12V mutant-specific CD8 T cells. This work lays a strong foundation for developing a potentially potent cell therapy to treat KRAS mutant associated cancers. SQZ™ PBMC-KRas G12V Activates Human PBMCs present KRas G12V-derived antigen to KRas-specific CD8 T cells. HLA-A*11 PBMCs squeezed with media containing KRas G12V peptides of various lengths were co-cultured with KRas G12V-specific CD8 T cells generated in A*11 transgenic mice. Activation of T cells was assessed by ELISpot analysis (IFN). PBMCs incubated with media supplemented with free peptide containing the KRas G12V minimal epitope (Positive Ctrl) and PBMCs squeezed with media, E7-derived peptide (irrelevant), or WT KRas peptide (WT 1-16) were used as controls. Each dot shows the response derived from a different PBMC donor.Media ### G12v ### G12v+g12d SFU / 1 X 106 cells G12V Functional ResponseMedia ### G12d ### G12v+g12d 12000 14000 SFU / 1x 106 cells G12D Functional Response Human PBMCs present multiple KRas mutant antigens to KRas-specific CD8 T cells. HLA-A*11 PBMCs squeezed with media containing KRas G12D and G12V peptides were co-cultured with KRas G12D-specific (left panel) or KRas G12V-specific (right panel) CD8 T cells generated in A*11 transgenic mice. Activation of T cells was assessed by ELISpot analysis (IFN). PBMCs incubated with media supplemented with free peptide containing the minimal epitope (G12D or G12V variants) and PBMCs squeezed with media were used as controls. Each dot shows the response derived from a different PBMC donor. KRasG12D+G12V peptide squeezed ### Pbmc KRasG12D-Specific CD8+ T cells or KRasG12V-Specific CD8+ T cells G12V Functional ResponseMedia Irrelevant ### Wt1-16 ### G12v1-16 ### G12v2-19 SFU / 1 x 106 cells KRasG12V-Specific CD8+ T cells KRasG12V peptide squeezed PBMC SQZ™ PBMC-KRas G12V+G12D Activates #15240 Tumor Vol. (mm3) Untreated Squeezed Splenocytes (IV) E7 SLP + CpG (Subcutaneous) 16 28 Prime Imm ### Til Analysis Day DayUntreated % CD45+ (of Live) ns *** **Untreated % CD8a+ (of CD45+) *** *** *Untreated % E7 Tetramer+ (of CD8a+) *** *** * ### A Dcb Cell Squeeze® Technology Enables Efficient Delivery to All Major Cell Subsets of PBMCs ### Abstract KRAS mutations are shared across multiple cancer types and are most commonly found in cancers of the lung, pancreas, and colon. While recently developed drugs blocking signaling driven by KRAS G12C mutations have shown promise in the clinic, there is a great need for therapies targeting the more common G12V and G12D mutations that are not easily targeted with small molecules. We utilize our microfluidic Cell Squeeze® technology to deliver antigens directly into the cytosol of peripheral blood mononuclear cells (PBMCs) for the generation of cancer antigen-targeted cellular vaccines. This method bypasses the need for cross-presentation and directly enables efficient antigen presentation on MHC-I in multiple cell types. Murine and human in vitro studies have demonstrated that this allows SQZ PBMCs to drive CD8+ T cell activation. Here, we demonstrate the delivery of various antigens, including KRas-derived peptides containing the most common mutations (G12D and G12V) to PBMC cell subsets. In addition, we find that human PBMCs squeezed with either KRasG12V or KRasG12D peptides can specifically stimulate KRasG12V- and KRasG12D- reactive CD8+ T cell responders. When KRasG12V and KRasG12D peptides are co-delivered via microfluidic squeezing, the human PBMCs enable activation of CD8+ T cells of both specificities. SQZ™ Technology and APC Platform Cytosolic Antigen Delivery: Engineering Cells to Function as Antigen Presenting Cells: ### Mhc-i ### Mhc-ii Endocytosis ### Mhc-i Cell Squeeze® Delivery Enhances MHC-I Presentation: Traditionally, cross-presentation is needed for efficient presentation on MHC-I. Cell Squeeze® technology delivers materials directly to cytosol, bypassing need for cross-presentation. No Contact Cascade Blue Dextran T cells (CD3+) B cells (CD19+) Monocytes (CD14+) NK cells (CD56+) Dextran Delivery in PBMCs Dextran 84% 87% 81% 94% 90% Cell Squeeze® technology enables delivery to all major cell subsets of human PBMCs in a single squeeze process. Isolated human PBMCs were squeezed in the presence of 3 kDa Cascade Blue-labeled dextran, stained for major cell subsets and analyzed by flow cytometry. Untreated PBMCs (No Contact) were used as a control. Squeeze for cytosolic antigen delivery ## 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.