Enhancing adoptive T‐cell therapy with fucoidan‐based IL‐2 delivery microcapsules

Abstract Adoptive cell therapy (ACT) with antigen‐specific T cells is a promising treatment approach for solid cancers. Interleukin‐2 (IL‐2) has been utilized in boosting the efficacy of ACT. However, the clinical applications of IL‐2 in combination with ACT is greatly limited by short exposure and high toxicities. Herein, a complex coacervate was designed to intratumorally deliver IL‐2 in a sustained manner and protect against proteolysis. The complex coacervate consisted of fucoidan, a specific IL‐2 binding glycosaminoglycan, and poly‐l‐lysine, a cationic counterpart (FPC2). IL‐2‐laden FPC2 exhibited a preferential bioactivity in ex vivo expansion of CD8+T cells over Treg cells. Additionally, FPC2 was embedded in pH modulating injectable gel (FPC2‐IG) to endure the acidic tumor microenvironment. A single intratumoral administration of FPC2‐IG‐IL‐2 increased expansion of tumor‐infiltrating cytotoxic lymphocytes and reduced frequencies of myeloid populations. Notably, the activation and persistency of tumor‐reactive T cells were observed only in the tumor site, not in the spleen, confirming a localized effect of FPC2‐IG‐IL‐2. The immune‐favorable tumor microenvironment induced by FPC2‐IG‐IL‐2 enabled adoptively transferred TCR‐engineered T cells to effectively eradicate tumors. FPC2‐IG delivery system is a promising strategy for T‐cell‐based immunotherapies.


Supplemental Figures
Supplementary Fig. 1. Underwater stability of bulk FPC 2 . To assess the water-immiscibility and underwater stability of FPC 2 , the letter 'KIST' was written in a petri-dish containing PBS using bulk FPC 2 through a 22G syringe and monitored for 2 months. Bulk FPC 2 was prepared via the centrifugation of an FPC 2 suspension.
3 Supplementary Fig. 2. Protein protection effects of FPC 2 against proteolysis. Optical microscopic (left) and fluorescent images (right) of the lysates of (a) free BSA-laden collagen gels and (c) FPC 2 -BSA-laden collagen gels after collagenase treatment. Scale bar = 100 μm.
Visualization of (b) the dissolution of BSA-FITC from fully degraded collagen gels and (d) the protective effects of FPC 2 for the encapsulated BSA against proteolysis. /PLL complex coacervates (FPC 2 ) were coated to each well of an ELISA plate, followed by incubation with different concentrations of IL-2 solution (1.25, 2.5, and 5 µg/mL). The amount of bound IL-2 was quantified using a sandwich ELISA method (n = 4). Relative IL-2 binding abilities were normalized to that of the sample with the highest value at each concentration of IL-2. All values shown are means ± SD. Statistical significance was designated as p * < 0.05, p ** < 0.01, ns: not significant.

Supplementary Fig. 9. Precipitation after incubation of heparin and PLL. (a) Incubation
of anionic heparin and cationic PLL caused precipitation rather that coacervation at physiological condition. (b) An excess addition of salt (more than 1 M NaCl) could induce complex coacervation by weakening strong electrostatic interactions. Scale bar = 100 μm.

Underwater stability of bulk FPC 2
To assess the underwater stability of FPC 2 , the letter 'KIST' was written in a petri-dish containing PBS using bulk FPC 2 through a 22G syringe. The written bulk FPC 2 was incubated in PBS at RT and monitored for 2 months. Bulk FPC 2 was prepared by centrifugation of FPC 2 suspension at 12,000 g rpm and 4 °C for 10 min.

Incubation of heparin and PLL
Heparin from porcine intestinal mucosa (Sigma-Aldrich) was incubated with PLL dissolved in PBS or PBS containing 1 M NaCl (Sigma-Aldirch) at various weight ratios of heparin and PLL (0:10-10:0). The mixtures were observed by an optical microscope. Stock solutions of 6.25 mg/mL of each polyelectrolyte were prepared, and the total polyelectrolyte concentration was fixed at 6.25 mg/mL.

IL-2 binding affinity test
200 μL of fucoidan, chondroitin sulfate A from bovine cartilage (Sigma-Aldrich), heparin dissolved in PBS and FPC 2 (the weight ratio of fucoidan and PLL = 7:3) were incubated in a high-binding 96-well plate (Thermo Fisher) at a final concentration of 25 μg/mL overnight at RT. The coated-wells were then washed three times with PBS, and then incubated in 1% BSA (Sigma-Aldrich) in PBS for 1 h at 37 °C to inhibit non-specific binding. After washing with PBS, 200 μL of human interleukin-2 (IL-2, Peprotech) dissolved in PBS was added into each well at 0, 1.25, 2.5, and 5.0 μg/mL and incubated at room temperature for 2 h at 37 °C. Unbound IL-2 was removed by washing three times with PBS, and 100 μL of biotinylated anti IL-2 solution (Peprotech) was added, followed by a further incubation for 90 min at 37°C. After further washing of the wells three times with PBS, 100 μL of avidin-peroxidase (HRP) solution (Peprotech) was added and further incubated for 30 min at 37°C. After again washing three times with PBS, 100 μL of 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]diammonium salt (ABTS) liquid substrate solution (Peprotech) was added followed by a further incubation for 20 min at RT for color development. The IL-2 binding ability of each sample was calculated by measuring the absorbance at 450 nm, with the correction wavelength set at 650 nm. Relative IL-2 binding abilities were normalized to that of the sample with the highest value at each concentration of IL-2. Four independent samples were averaged to obtain each measurement.