Overcoming a Critical Obstacle Towards Effective and Safe CAR T-Cell Therapeutics
Authors: Mondal Nandini , Silva Mariana , Castano Ana , Maus Marcela V. , Sackstein Robert 
Published: October 2019

Abstract

Chimeric Antigen Receptor (CAR) T-cells have shown great promise in achieving stable remission of B cell acute lymphoblastic leukemia and are being utilized to treat a variety of other hematologic and non-hematologic malignancies. However, this therapy is associated with significant adverse effects (e.g., cytokine release syndrome and neurotoxicity), largely related to the high cell doses requisite for achieving therapeutic response(s). E-selectin, an endothelial molecule expressed on microvessels of bone marrow and tumors, is critical for extravasation of blood-borne cells. E-selectin binds to sialyl Lewis X (sLeX), a sialofucosylated tetrasaccharide motif decorating specialized glycoproteins and glycolipids on circulating cells. Given that better site-specific delivery of administered CAR T-cells would yield more efficacious immunotherapy while limiting off-target effects, we sought to determine whether these cells express E-selectin ligands.
CAR T-cells targeting glioblastoma were created by lentiviral transduction of human T-cells with anti-EGFR-BBz-mCherry CAR construct. Transduced cells (T) and untransduced cells (UT) were subjected to stimulation with anti-CD3/CD28 microbeads followed by culture expansion in presence of interleukin-2 (IL2) for 10 days ("10"). In some cases, cells were expanded for 7 additional days by co-culturing with irradiated U87 glioblastoma cell line ("17"). These cells were compared to resting T-cells (non-expanded; NE). Expression of sLeX on each T-cell population (NE, T10, T17, UT10 or UT17) was measured by flow cytometry.
We observed that NE T-cells display moderate expression of sLeX as previously reported in literature (Silva, Fung et al., J. Immunol., 2017). T10 CAR T-cells display significantly lower sLeX compared to NE T-cells, while T17 CAR T-cells completely lack sLeX. Similar pattern of sLeX expression was observed on UT10 and UT17 populations, indicating that culture expansion progressively depletes sLeX expression on T-cells, regardless of transduction with a CAR construct.
To analyze the capacity of CAR T-cells to bind E-selectin under hemodynamic shear conditions, we utilized a microfluidic flow chamber wherein T-cells are perfused over human umbilical vein endothelial cell (HUVEC) monolayers treated with TNFα to induce E-selectin expression. Consistent with the observed cell surface sLeX levels, NE T-cells display robust rolling interactions on TNFα-stimulated HUVECs, whereas T10 and T17 CAR T-cells exhibit essentially no endothelial interactions. To overcome this shortfall, we assessed whether sLeX expression can be enforced on the CAR T-cell surface via glycoengineering using glycosyltransferase programmed stereosubstitution (GPS), i.e., fucosyltransferase-mediated α(1,3)-fucose installation that converts terminal sialylated type 2 lactosamines to sLeX. GPS engenders markedly increased CAR T-cell sLeX expression, yielding full (100%) sLeX -expression of the CAR T-cell populations, while increasing sLeX expression from a baseline of 20-30% to maximally 60-70% of the NE T-cell populations. The glycoengineered CAR T-cells display markedly enhanced (>20 fold) adhesive interactions on TNFα-stimulated endothelial cells compared to that of unfucosylated CAR T-cells. Collectively, our data indicate that culture expansion induces down-regulation of endogenous α(1,3)-fucosylation, and, importantly, the missing fucose can be restored via GPS. Finally, to assess whether the enforced E-selectin binding has a meaningful biologic effect, we examined homing of GPS-modified cells to an E-selectin bearing bed (marrow) within 24 hours of intravenous administration. We consistently observed >3-fold increase in marrow infiltrates of culture-expanded GPS-modified T-cells compared to their unmodified counterparts.
Overall, these results draw attention to an inherent inability of CAR T-cells to migrate to sites where they are needed. Our data reveal that culture expanded T-cells lack E-selectin binding capacity, however, sLeX display can be enforced by cell surface glycoengineering. Improved E-selectin binding yields superior tissue colonization. As such, glycoengineering CAR T-cells may translate into lowering of cell dosing, thereby improving both the efficacy and safety, and reducing both cell expansion and clinical care costs, of this promising therapeutic approach.

Disclosures
Maus: kite therapeutics: Consultancy, Research Funding; windmil therapeutics: Consultancy; crispr therapeutics: Consultancy, Research Funding; agentus: Consultancy, Research Funding; novartis: Consultancy; adaptimmune: Consultancy. Sackstein:Warrior Therapeutics LLC: Equity Ownership, Patents & Royalties.