Enhancing CAR T Cell Homing Through Glycoengineering

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The importance of T cell homing to the tumor site is a critical aspect of the anti-tumor immune cellular response. Most of the recent advances in cancer immunotherapy have focused on generating potent tumor specific T cells and overcoming tumor-induced T cell inhibition and tolerance. However, a rate-limiting step to establishing efficacious cellular therapy for the treatment of malignancies is the lack of T cell homing to the tumor site. To circumvent this obstacle, we investigated fucosylation as a mechanism to enhance cytotoxic T lymphocyte (CTL) homing. Fucosylation, a process mediated by fucosyltransferases, adds fucose sugar groups to cell surface glycoproteins, increasing their affinity to selectins on tumor cells, endothelial cells, and within inflamed tissues. Our preclinical work using ex vivo fucosylation of tumor-specific polyclonally-expanded CTL and regulatory T cells (Treg) showed enhanced CTL homing to bone marrow and tumor sites, and Treg homing to graft-versus-host disease involved tissues, respectively. Furthermore, fucosylation of cord blood (CB) products enhanced the homing and engraftment of CB stem cells to marrow in patients with hematologic malignancies undergoing CB transplants. What remains unknown is whether fucosylation of CAR T cells will enhance their homing into tumor tissue. Without this knowledge, the effectiveness of CAR T cells as an immunotherapeutic strategy for the treatment of cancer will likely remain limited. Our long-term objective is to develop effective cellular immunotherapy for cancer. In this proposal, we will investigate the role of fucosylation in the setting of acute myeloid leukemia (AML), using a fucosylated CAR T cell approach (i.e. fucosylated 8F4-CAR T cells) which targets an antigen that our group discovered, PR1. We hypothesize that immunotherapy using fucosylated 8F4-CAR T cells will inhibit AML in vivo, with minimal off-target toxicity. In our first aim, we will study the effects of ex vivo fucosylation on 8F4-CAR T cell efficacy and safety, and in the second aim, we will determine whether engineering 8F4-CAR T cells to endogenously express fucosyltransferase VII will enhance their anti-tumor activities. We will employ an AML xenograft mouse model in both aims, and will elucidate the mechanisms by which fucosylation augments T cell activity. Our proposed research is significant, in that fucosylation is expected to enhance not only T cell homing and entry into tumor sites, but also their cytolytic machinery, thereby improving their efficacy. Furthermore, since the engineering of an adequate dose of tumor-specific effector cells is at times a limiting step to adoptive cellular therapy, fucosylation will allow for the engineering and infusion of fewer tumor-targeting T cells, as more fucosylated T cells are expected to home favorably to the malignant milieu. Underscored by the fact that stem cell fucosylation has already demonstrated safety and efficacy clinically, we believe our innovative approach may greatly facilitate the effectiveness of T cell therapy in AML as well as other malignant diseases, and could transform our current approach to cellular immunotherapy.