Microfluidic Precision Engineered Artificial Antigen Presenting Cells for Cellular Immunotherapy

Introduction: Cellular immunotherapy is commonly applied for cancer treatment and autoimmune disease therapy. One method, called adoptive cell transfer (ACT), involves extracting and activating immune cells and then reinfusing them into the patient. Natural antigen-presenting cells (APCs) such as dendritic cells (DCs) are typically used to expand T cells in this process, but they have limited availability and time-consuming culture processes. To overcome these problems, we developed the microfluidic process to generate cell-sized single-compartment multisomes (SCMs) and decorated them with antigen-presenting ligands for artificial APCs (aAPCs). Our results demonstrate that aAPCs can bind and interact with T cells and cause their expansion. Additionally, using the same platform, we conjugated glycoprotein A repetitions predominant (GARP) to the CUVs to form artificial GARP-presenting cells (aGPCs) and aimed to induce regulatory T cells (Tregs) expansion to develop an autoimmune disease therapy. The results show that aGPCs can expand the Treg population both in vitro and in vivo. The future directions of this project are to further optimize the preparation of the system by modularizing the artificial cells with different protein conjugation methods and adding a hydrogel component inside the artificial cells to tune their mechanical rigidity. We believe that this platform can be used to produce the aAPC or aGPC preparation that mimics cells, is stable, easy to produce in large quantities, and capable of expanding tumor-specific CD8 T cells or Treg cells for cellular immunotherapy.

Materials and

Methods: To create the artificial cells, we first generate double-emulsion droplets (DEDs) by a flow-focusing device with three different phases (Fig. 1a): the inner aqueous phase (sucrose), the oil phase (containing lipids and cholesterol), and the viscous external phase (glycerol). The DEDs were then functionalized with target protein ligands (including anti-CD28, anti-LFA-1, MHCII-NYESO complex, anti-CD3, or GARP, Figure 1b) and converted into single compartment multisomes (SCMs), which were incubated with human peripheral blood mononuclear cells (PBMCs) from healthy donors for 5 days. The NYESO-specific T cells were analyzed by flow cytometry, with several control groups added to verify the effect of membrane fluidity and size including antibody-coated polystyrene beads, no antigen (NA), and free molecule groups (suspended in the solution only). The Treg cells were analyzed by flow cytometry as well and verified the expression level of transcription factor FoxP3.

Results, Conclusions, and Discussions: Immunotherapy of cancer using cells is an expensive and cumbersome process. The present project aims at developing artificial cells that can be produced inexpensively via microfluidic engineering-based methods. We have created uniform lipid bilayer aAPCs that are highly effective in activating specific T cells in PBMC. The results show a significant increase in antigen-specific CD8 T cell expansion (233-fold, Fig. 2a) and CD4 T cell expansion (16-fold, Fig. 2b). Additionally, the activated T cells by our aAPCs are not only antigen-specific but also cytotoxic, as evidenced by the upregulated secretion of granzyme B (Fig. 2c). By employing the same strategy, we generate the aGPCs which can be used for Treg expansion. In Fig. 3, the aGPC groups have a higher percentage of the Treg population and higher expression of FoxP3. To further improve the preparation of aAPCs or aGPCs, two more strategies were planned. Firstly, using different protein conjugation methods to modularize the artificial cell system and make the system more convenient to use. Secondly, forming a hydrogel core within the artificial cells to adjust the stiffness. With these findings, it is believed that this technology can greatly improve the quality of both cancer cellular immunotherapy and autoimmune disease treatment.

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