169 Microfluidics cell squeezing enables human PBMCs as drivers of antigen-specific CD8 T responses across broad range of antigens for diverse clinical applications

Abstract

BackgroundAntigen-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.MethodsTo elicit antigen-specific CD8+ T cell activity, we used microfluidics cell squeezing (Cell Squeeze®) 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® platform is generally agnostic to cell type and material. Therefore, not only does microfluidic squeezing enable cell subsets within human peripheral blood mononuclear cells (PBMCs) to function as unconventional APCs, but it also enables us to efficiently investigate a wide range of antigens including whole protein, peptides, and mRNA. This ‘plug and play’ nature of the platform allows for broad application in multiple disease areas.ResultsIn human cells, we demonstrated that microfluidic squeezing of PBMCs enables effective delivery to the major cell subsets including T cells, B cells, NK cells and monocytes. Delivery of CMV and HPV16 synthetic long peptides (SLPs) resulted in robust in vitro responses of both CD8+ T cell clones and patient-derived memory populations. To broaden the impact of our PBMC-based cell therapy approach, we investigated several other antigens relevant to other disease areas. Additional materials we delivered via squeezing and demonstrated antigen presentation include neoantigens, M1 Influenza mRNA, and pp65 SLP. Cell Squeeze® platform is simple to use and amenable to scale up. We demonstrated that delivery and viability for research scale process (~2 × 106 cells) is equivalent to delivery and viability of PBMCs processed at manufacturing scale (~1 × 109 cells).ConclusionsMicrofluidic cell squeezing of human PBMCs with antigenic material can be tailored to produce APCs that drive robust CD8+ T cell response against targets across multiple disease areas and has been scaled up for clinical use. SQZ-PBMC-HPV are currently under clinical evaluation for treatment of HPV16+ tumors.

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“…During this time, payload molecules, in suspension with the cells, are free to migrate through the disrupted cell membranes, remaining inside as these gaps seal quickly after squeezing (Figure 2C). Recently, Squeeze ® technology was used ex vivo to demonstrated that different antigens (synthetic long peptides for Cytomegalovirus (CMV) and HPV16 + tumours, neoantigens and M1 influenza mRNA) delivered to human PBMCS resulted in robust antigen-specific CD8 + T-cell responses highlighting the potential to leverage their platform across a wide variety of diseases [210]. In a separate study, the Cell Squeeze ® platform was used to demonstrate that murine or human red blood cells could be converted into activating antigen carriers (AACs).…”