Metabolic glycan labeling immobilizes dendritic cell membrane and enhances antitumor efficacy of dendritic cell vaccine

Introduction: Immunotherapy has shifted the paradigm for clinical cancer treatment with the success of checkpoint blockades and chimeric antigen receptor (CAR) T therapies. Yet, challenges such as low patient response rates and side effects limit their effectiveness. However, the ability to modulate the function of antigen presenting cells (e.g., dendritic cells (DCs)) and orchestrate the priming of antigen-specific T cells holds promise to improve the overall cytotoxic T lymphocyte (CTL) response and therapeutic efficacy, and has been actively pursued in therapeutic cancer vaccines. Indeed, Sipuleucel-T, one type of DC vaccines for treating prostate cancer, was among the first FDA-approved cancer immunotherapies. Despite some level of success, the use of DC vaccines has been limited by the modest CTL response and therapeutic efficacy. Therefore, we aim to improve DC vaccines by enhancing the activation status of DCs during the ex vivo manufacturing process and further modulate the function of DCs post adoptive transfer. Here we introduce a universal yet facile technology to enable targeted modulation of adoptively transferred DCs in vivo. We utilize metabolic glycoengineering processes of unnatural sugars to label DCs with unique chemical tags (i.e., azido groups). We find that the metabolic glycan labeling process itself is able to reduce the mobility of proteins on DC membrane and upregulate the activation markers of DCs. Moreover, the chemical tags are expressed on the cell membrane in the form of glycoproteins and glycolipids and can mediate targeted conjugation of dibenzocyclooctyne (DBCO)-bearing agents via efficient click chemistry to amplify the overall therapeutic efficacy.

Materials and

Methods:

Methods: Bone marrow derived DCs (BMDCs) were isolated and differentiated from C57BL/6 mice. Then, BMDCs were labeled with Tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz), a commonly used metabolic labeling agent. Activation and antigen presentation of BMDCs after azido labeling were analyzed by FACS assay. For in vivo studies, C57BL/6 mice were subcutaneously injected with EG.7-OVA cells as a tumor model. Tumor efficacy was examined by comparing azido-labeled DCs and cytokines (IL15 and IL2) conjugated DCs verses unmodified DCs.

Results, Conclusions, and Discussions: We report a facile metabolic glycan labeling approach for developing enhanced DC vaccines. Azido-sugars can metabolically label DCs with azido groups, which itself can reduce the membrane mobility of DCs, activate DCs, and improve the antigen presentation and T cell priming ability of DCs. Reduced in membrane mobility can potentially explain why azido-labeled DCs show upregulated expression of activation markers (CD86, MHCII, CD40, and CCR7) as stiffer membranes may contribute to receptor clustering and the activation of downstream signaling pathways. In vivo, azido-labeled antigen-pulsed DCs also result in improved CTL response and antitumor efficacy compared to unlabeled DCs in both prophylactic and therapeutic settings. This facile approach to improving the activation status, antigen presentation ability, and subsequent T cell priming property of DCs, via simple addition of azido-sugars to the culture medium, provides a avenue to developing enhanced DC vaccines.

Moreover, the ability to target immunomodulatory agents (e.g., cytokines) to adoptively transferred DCs in vivo holds promise to amplify CTL response and antitumor efficacy of DC vaccines, but remains challenging. Here we demonstrated that, in addition to the improved activation status, antigen presentation ability, and T cell priming property, azido-labeled DCs also enable conjugation of cytokines such as IL-2 and IL-15 via efficient click chemistry. in vivo targeting of IL-15 to azido-labeled DC vaccines can significantly improve the CTL response and overall antitumor efficacy in both prophylactic and therapeutic settings. This approach can be universally applied to different types of DC vaccines for treating various types of tumors. The benign safety profile also adds to the translation potential of the developed DC vaccines. The DC labeling and targeting technology not only enables the development of enhanced DC vaccines with robust CTL response and therapeutic efficacy, but also provides a platform to manipulate intercellular interactions between DCs and other immune cells.

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