Influence of different conjugation methods for activating antibodies on polymeric nanoparticles: Effects for polyclonal expansion of human CD8+ T cells.

Particle-based systems have become a state-of-the-art method for in vitro expanding cytotoxic T cells by tailoring their surface with activating molecules. However, commonly used methods utilize facile carbodiimide chemistry leading to uncontrolled orientation of the immobilized antibodies on the particle surface that can lead to poor binding to target cells. To address this, selective coupling strategies utilizing regioselective chemical groups such as disulfide bridges offer a simple approach. In this work we present a set of methods to investigate the effect of polymeric nanoparticles, conjugated with either regioselective- or randomly-immobilized antiCD3 and antiCD28 antibodies, on the activation potential, expansion and expression of activation markers in T cells. We show that nanoparticles with well-oriented monovalent antibodies conjugated via maleimide require fewer ligands on the surface to efficiently expand T cells compared to bivalent antibodies randomly-immobilized via carbodiimide conjugation. Analysis of the T cell expression markers reveal that the T cell phenotype can be fine-tuned by adjusting the surface density of well-oriented antibodies, while randomly immobilized antibodies showed no differences despite their ligand density. Both conjugation techniques induced cytotoxic T cells, evidenced by analyzing their Granzyme B secretion. Furthermore, antibody orientation affects the immunological synapse and T cell activation by changing the calcium influx profile upon activation. Nanoparticles with well-oriented antibodies showed lower calcium influx compared to their bivalent randomly-immobilized counterparts. These results highlight the importance of controlling the antibody density and orientation on the nanoparticle surface via controlled coupling chemistries, helping to develop improved particle-based expansion protocols to enhance T cell therapies.

A versatile method for conjugating lipid nanoparticles on T cells through combination of click chemistry and metabolic glycoengineering.

Cell-mediated drug delivery by conjugating nanomedicine to the surface of living cells is a promising strategy for enhancing the efficacy of both drug delivery and cell therapy. It exploits the tissue homing properties of the specific cell types to overcome in vivo barriers and forms a drug depot by directly putting the therapeutic payload in target cells. An important concern of developing this system is the method to conjugate nanoparticles on cells. Herein, we developed a bioorthogonal T cell conjugation strategy using SPAAC click chemistry, which allows controllable and highly efficient conjugation without affecting the viability and functions of the cytotoxic T lymphocytes. Azide groups were incorporated on the surface of T cells through metabolic glycoengineering, followed by reacting with dibenzylcyclooctyne (DBCO) modified lipid nanoparticles (LNPs). LNPs can be conjugated to T cells, allowing for the loading of different drug molecules on the cells. The metabolic engineering and click reaction approach provides a simple and versatile strategy to conjugate NPs to living cells and enable the development of sophisticated therapeutic cell products.

Enhancing Adoptive Cell Therapy by T Cell Loading of SHP2 Inhibitor Nanocrystals before Infusion.

Whereas adoptive T cell therapy has been extensively studied for cancer treatment, the response is still limited primarily due to immune dysfunction related to poor cell engraftment, tumor infiltration and engagement, and lack of a target. In addition, the modification of therapeutic T cells often suffers from being complex and expensive. Here, we present a strategy to load T cells with SHP099, an allosteric SHP2 inhibitor, to enhance the therapeutic efficacy of the T cells. Remote-loading of SHP099 into lipid nanoparticles decorated with triarginine motifs resulted in nanocrystal formation of SHP099 inside the lipid vesicles and allowed high loading efficiency and prolonged retention of SHP099 nanocrystals within T cells. Cell-loaded SHP099 enabled sustained inhibition of the PD-1/PD-L1 signaling and increased cytolytic activity of the T cells. We show in a mouse model that tumor-homing T cells can circulate with the cargos, improving their tumor accumulation compared to systemically administered lipid nanoparticles. On an established solid tumor model, adoptively transferred SHP099 loaded T cells induced complete tumor eradication and durable immune memory against tumor rechallenging on all treated mice by effectively inhibiting the PD-1/PD-L1 checkpoint signal. We demonstrate that the combination of T cell therapy with SHP2 inhibition is a promising therapeutic strategy, and the lipid nanocrystal platform could be generalized as a promising approach for T cell loading of immunomodulatory drugs.

A quantitative ex vivo study of the interactions between reconstituted high-density lipoproteins and human leukocytes.

Knowledge of the interactions between nanoparticles and immune cells is required for optimal design of nanoparticle-based drug delivery systems, either when aiming to avoid phagocytic clearance of the nanoparticles or promote an immune response by delivering therapeutic agents to specific immune cells. Several studies have suggested that reconstituted high-density lipoproteins (rHDL) are attractive drug delivery vehicles. However, detailed studies of rHDL interactions with circulating leukocytes are limited. Here, we evaluated the association of discoidal rHDL with leukocytes in human whole blood (HWB) using quantitative approaches. We found that while the rHDL of various lipid compositions associated preferentially with monocytes, the degree of association depended on the lipid composition. However, consistent with the long circulation half-life of rHDL, we show that only a minor fraction of the rHDL associated with the leukocytes. Furthermore, we used three-dimensional fluorescence microscopy and imaging flow cytometry to evaluate the possible internalization of rHDL cargo into the cells, and we show increased internalization of rHDL cargo in monocytes relative to granulocytes. The preferential rHDL association with monocytes and the internalization of rHDL cargo could possibly be mediated by the scavenger receptor class B type 1 (SR-BI), which we show is expressed to a higher extent on monocytes than on the other major leukocyte populations. Our work implies that drug-loaded rHDL can deliver its cargo to monocytes in circulation, which could lead to some off-target effects when using rHDL for systemic drug delivery, or it could pave the way for novel immunotherapeutic treatments aiming to target the monocytes.