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Background Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of blood cancers, but its efficacy against solid tumors remains limited. Cytokines are important immune system modulators that can be harnessed to enhance the efficacy of adoptive T cell therapy. 1 Although the combination of adoptively transferred T cells with local cytokine administration or engineered cytokine production has been shown to promote proliferation and effector function of T cells, the systemic toxicity and severe side effects associated with cytokines remain critical challenges to be addressed.2 To address these issues, we developed a cytokine-cell conjugates (CCCs) platform utilizing fucosyltransferase(FT)-mediated cell-surface glycan editing.Methods We first synthesized cytokine-conjugated donor substrate, GDP-Fuc-cytokine via NHS coupling and TCO-Tz ligation ( figure 1).3 Subsequently, FT-mediated direct cell-surface fucosylation transferred Fuc-cytokine to LacNAc and sLacNAc residues in the cell-surface glycocalyx of T cells. With the establishment of the cell-surface conjugation approach, we applied it to construct a small library of CCCs with 17 selected cytokines by conjugating them individually onto day 4 activated P14 cells in 96-well plates. The library was then directly subjected to in vitro screening to identify cytokines that confer enhanced immunity to the modified T cells. Three selection criteria were used for the screening: cell expansion, apoptosis, and target cell killing.Results Notably, Neo4, 4 which was developed to recapitulate the type I signaling pathway of native IL-4, was found to reduce apoptosis, support proliferation, and enhance the cytotoxicity of CD8 T cells upon conjugation. Mechanistically, we discovered that Neo4 can induce a unique spectrum of intracellular signaling cascades, through signal transducer and activator of transcription (STAT) 1, 3, 5, and 6, augmenting the glycolytic metabolism and oxidative phosphorylation. Neo4-conjugated P14 CD8 T cells exhibited increased expression of proliferation and effector function markers, along with decreased markers of exhaustion after co-culturing with tumor cells. These Neo4-conjugated P14 CD8 T cells showed improved efficacy in vivo against solid tumors. When combined with ICB, this approach led to complete tumor regression and sustained antitumor responses (figure 2). Remarkably, Neo4 conjugation not only enhanced the efficacy of ACT but also reduced immune-related adverse events (irAEs) associated with ICB. Furthermore, Neo4-conjugated anti-CD19 human CAR-T cells demonstrated durable antitumor activity after leukemia rechallenge in a mouse model of Nalm6.Conclusions This work establishes a generalizable method for enhancing the efficacy of CAR-T cells, resulting in the eradication of solid tumors, durable immune protection, and reduced toxicity.References Corria-Osorio J, Carmona SJ, Stefanidis E, et al. Orthogonal cytokine engineering enables novel synthetic effector states escaping canonical exhaustion in tumor-rejecting CD8(+) T cells. Nat Immunol. 2023;24:869-883.Propper DJ, Balkwill FR. Harnessing cytokines and chemokines for cancer therapy. Nat Rev Clin Oncol. 2022;19:237–253.Li J, Chen M, Liu Z, Zhang L, Felding BH, Moremen KW, Lauvau G, Abadier M, Ley K, Wu P. A single-step chemoenzymatic reaction for the construction of antibody-cell conjugates. ACS Cent Sci. 2018;4(12):1633-1641.Yang H, Ulge UY, Quijano-Rubio A, et al. Design of cell-type-specific hyperstable IL-4 mimetics via modular de novo scaffolds. Nat Chem Biol. 2023;19:1127–1137.Ethics Approval The Institutional Animal Care and Use Committee (IACUC) is a federally mandated group that oversees the Scripps Animal Care and Use Program. Our goal is to ensure that animals used in research, education, and testing are treated humanely and ethically. All procedures involving animal handling, injection, and monitoring will follow IACUC-approved protocols, as determined by the Scripps Research Institute IBCs and the Institutional Animal Care and Use Committee (IACUC).Abstract 314 Figure 1Construct a cytokine-cell conjugate library to screen for armored T cells with improved immunity in vitroAbstract 314 Figure 2Neo4-conjugation potentiates T cell anti-tumor immunity and enhances synergy with immune checkpoint blockade in vivo