Triply Enhanced Immunotherapy via Dual Glycan Reforming Integrated with Perforation

Abstract The enhancement of immunotherapy is an emerging direction to develop highly effective and practical cancer therapeutic methods. Here a triply enhanced immunotherapy drug (TEID) is designed for ingeniously integrating in situ dual glycan reforming with perforation on cell membrane. The TEID is composed of galactose and neuraminidase conjugated streptolysin O (SLO‐Gal and SLO‐NEU), which are encapsulated in a hyaluronic acid (HA) shell for targeted recognition to tumor tissue via cell surface CD44. After targeted delivery and HAase‐mediated degradation in the tumor region, the TEID releases SLO‐Gal and SLO‐NEU, which can easily anchor Gal and NEU on the tumor cell membrane via the perforation of SLO to perform dual glycan reforming for the introduction of Gal and the cleavage of sialic acid. The former can activate immune cells to secret cytokines for immune‐killing, and the latter can weaken the immune inhibition to improve the immunotherapeutic efficacy. Meanwhile, the perforation of SLO can promote the delivery of cytokines into the tumor cells to further enhance the efficacy. The designed triply enhanced immunotherapy strategy opens a significant and promising route to promote clinical immunotherapy of cancer.


Table of Contents
CLSM cell imaging and flow cytometric analysis of perforating, SA-cleaving and Gal-introducing functions CCK-8 assay and flow cytometric analysis of the cytotoxicity of SLO-Gal and SLO-NEU . Verification of the triple functions of SLO-Gal and SLO-NEU on MCF-7 cells The flow cytometric analysis of the triple functions of SLO-Gal and SLO-NEU . CLSM verification of the triple functions of TEID and the degraded TEID on MCF-7 cells . Flow cytometric analysis of the triple functions of TEID and the degraded TEID  Annexin V-FITC apoptosis detection kit was from Yeasen Co., Ltd.(China).All solutions were prepared using ultrapure water (≥18 MΩ, Milli-Q, Millipore).
Confocal fluorescence micrographs were acquired on a TCS SP8 confocal laser scanning microscope (CLSM) (Leica, Germany).The UV-vis absorption spectra were recorded using a UV-vis spectrophotometer (Nanodrop-2000C, Nanodrop, USA).Flow cytometric analysis was gained on a CytoFLEX flow cytometer (Beckman-Coulter, USA).Zeta potential analysis was performed on a Zetasizer (Nano-Z, Malvern, UK).Dynamic light scattering (DLS) measurements were performed on a 90 Plus/BI-MAS equipment (Brook haven, USA).CCK8 assays were performed on a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific, USA).
The NEU activity of SLO-NEU was evaluated by incubating 1 mM MuNeuNAc with NEU (1 nM) or SLO-NEU (1 nM) at 37 °C for various times to determine the fluorescence of released methylumbelliferone at 445 nm with 365 nm excitation.
S1] Briefly, 0.16 mL MA was dropwise added in 10 mL 0.02 g mL -1 HA at 4 °C, and the solution was adjusted to pH 8-9 with 5.0 M NaOH.After continuously stirring for 24 h, the resulting polymer was precipitated with acetone, washed with ethanol, re-dissolved in water and lyophilized to obtain methacrylated HA (m-HA).Afterward, 100 U mL -1 SLO-NEU, 100 U mL -1 SLO-Gal (U mL -1 is the concentration of SLO), 0.07 mg glycerol dimethacrylate and Irgacure solution to ultraviolet radiation (20 mV cm -2 ) for 60 s.The resulting TEID were obtained by centrifuging with 3 kDa MWCO membrane to remove the excessive crosslinker and initiator.The Gal@HA, NEU@HA, SLO@HA, SLO-Gal@HA and SLO-NEU@HA were prepared with the same procedure.
CLSM cell imaging and flow cytometric analysis of perforating, SA-cleaving and Gal-introducing functions.After MCF-7 or 4T1 cells were seeded on confocal dishes overnight and washed three times with HBSS, the cells were incubated with PBS, 200 U mL -1 SLO, SLO-NEU, SLO-Gal or 0.1 μM NEU at 37 o C for 30 min, and stained with PI or Cy3-SNA (10 μg mL -1 ) at 4 o C for 60 min to verify the perforating function of SLO or SA-cleaving function of SLO-NEU with CLSM imaging under 535 nm for PI channel or 543 nm for Cy3 channel, respectively.The Galintroducing function of SLO-Gal was verified at 495 nm by incubating seeded MCF-7 or 4T1 cells with 20 U mL -1 GD at 37 o C for 60 min to obtain GD per-treated cells, and then incubating them with PBS, 200 U mL -1 SLO, SLO-Gal or SLO-NEU at 37 o C for 30 min to stain with F-Jac (10 μg mL -1 ) at 4 o C for 60 min.
The function verification of HA degraded TEID was performed by incubating TEID with 0.4 mg mL -1 HAase at pH 6.5 at 37 ºC for 2 h, and then with the seeded MCF-7 or 4T1 cells at 37 o C for 30 min to stain with PI or Cy3-SNA, or with β-galactosidase (GD) treated MCF-7 or 4T1 cells at 37 o C for 30 min stain with F-Jac.The imaging signals were collected from 555 to 625 nm for PI channel, 555 to 640 nm for Cy3 channel and 510 to 600 nm for fluorescein channel.Flow cytometric analysis was performed by collecting these stained cells with centrifugation at 1,000 rpm for 3 min and resuspending them in 500 μL PBS.

CLSM imaging of perforating function on NK cells.
After NK cells were incubated with PBS, 200 U mL -1 SLO, SLO-NEU or SLO-Gal at 37 o C for 30 min, the cells were washed three times with HBSS and stained with PI (10 μg mL -1 ) at 4 o C for 60 min.The cells were then seeded on poly-L-lysine treated confocal dishes for 10 min and imaged by CLSM to collect the emission signals from 555 nm to 625 nm under 535 nm excitation.

CCK-8 assay and flow cytometric analysis of the cytotoxicity of SLO-Gal and SLO-NEU. After MCF-7 cells or 4T1 cells seeded on
dishes were incubated with PBS, 0.1 μM Gal, 0.1 μM NEU, 200 U mL -1 SLO, SLO-Gal, SLO-NEU, or the mixture of 100 U mL -1 SLO-Gal and SLO-NEU at 37 o C for 30 min, they were incubated with NK cells with a ratio of 1:1 at 37 °C for 8 h, 12 h, 24 h and 48 h, to analyze the cell viability through CCK8 assay, respectively.The optimal ratio of NK to cancer cells was obtained by incubating 10000 seeded MCF-7 or 4T1 cells with the mixture of 100 U mL -1 SLO-Gal and SLO-NEU at 37 o C for 30 min, and then incubating them with different amounts of NK cells at 37 °C for 24 h to detect the cell viability.
The flow cytometric analysis of the cytotoxicity was performed by incubating the seeded 4T1 cells with PBS, 0.1 μM Gal, 0.1 μM NEU, 200 U mL -1 SLO, SLO-Gal, SLO-NEU, or the mixture of 100 U mL -1 SLO-Gal and SLO-NEU at 37 o C for 30 min, and then with NK cells at 37 °C for 24 h to stain with AnnexinV-FITC/PI.CCK-8 assay of cytotoxicity.Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood obtained from Nanjing Integrated Traditional Chinese and Western Medicine Hospital by density gradient separation with human peripheral blood lymphocyte isolate fluid.
After MCF-7 cells or 4T1 cells seeded on dishes and incubated with the mixture of 100 U mL -1 SLO-Gal and SLO-NEU at 37 o C for 30 min, the cells were respectively incubated with PBS or different effectors (T cells, PBMCs and NK cells) with the ratio of 1:1 at 37 °C for 24 h to detect the cell viability.
Quantification of cytokines secreted from NK cells.After the seeded 4T1 cells were incubated with PBS, 0.1 μM Gal and NEU, 200 U mL -

Figure S3 .
Figure S3.Verification of the triple functions of SLO-Gal and SLO-NEU on MCF-7 cells.a, b) CLSM images of MCF-7 cells after incubated with PBS (Control), SLO, SLO-Gal or SLO-NEU and then stained with PI to verify perforating function (a), and Cy3-SNA to verify SA-cleaving function (b).c) CLSM images of MCF-7 cells and GD per-treated MCF-7 cells after incubated with PBS (Control and GD), SLO, SLO-Gal or SLO-NEU and then stained with F-Jac to verify Gal-introducing function.

Figure S4 .
Figure S4.CLSM images of PI stained NK cells after incubation with SLO, SLO-NEU or SLO-Gal.CLSM images of PI stained NK cells after incubation with 200 U mL -1 SLO, SLO-NEU or SLO-Gal at 37 o C for 30 min.

Figure S5 .
Figure S5.The flow cytometric analysis of the triple functions of SLO-Gal and SLO-NEU.a, b, d, e) Flow cytometric analysis of 4T1 or MCF-7 cells incubated with PBS (Control), SLO, SLO-NEU or SLO-Gal and then stained with PI to verify the perforating function (a, d), and with PBS (Control), SLO, SLO-Gal, NEU or SLO-NEU and then stained with Cy3-SNA to verify the SA-cleaving function (b, e).c, f) Flow cytometric analysis of 4T1 or MCF-7 cells and GD per-treated 4T1 or MCF-7 cells after incubated with PBS (Control and GD), SLO, SLO-Gal or SLO-NEU and then stained with F-Jac to verify Gal-introducing function.

Figure S8 .
Figure S8.Cell viability of tumor cells after immune-killing with NK cells at different ratios of NK to tumor cells.Cell viability of 4T1 cells (a) and MCF-7 cells (b) after performing common immune-killing (black columns), SLO-Gal and SLO-NEU enhanced immune-killing (pink columns) with NK cells.The enhancing efficiency (red columns) at different ratios of NK to cancer cells was calculated with (ViabilityNK -ViabilitySLO-Gal & SLO-NEU + NK) / ViabilityNK.The error bars indicate mean ± SD (n = 3).

Figure S9 .
Figure S9.Flow cytometric scatter plots of the 4T1 cells treated with different components in the absence of NK cells.Flow cytometric scatter plots of 4T1 cells after incubation with Gal, NEU, SLO, SLO-Gal, SLO-NEU or the mixture of SLO-Gal and SLO-NEU and then staining with Annexin V-FITC and PI.

Figure S10 .
Figure S10.Zeta potentials of TEID at different pHs for different times.Zeta potentials of TEID at pH 5.0, 6.5 and 7.4 for different times.

Figure S11 .
Figure S11.CLSM verification of the triple functions of TEID and the degraded TEID on MCF-7 cells.a, b) CLSM images MCF-7 cells after incubated with PBS (Control), TEID, HA degraded TEID and SLO-Gal&SLO-NEU and then stained with PI to verify perforating function (a), Cy3-SNA to verify SA-cleaving function (b).c) CLSM images of GD pre-treated MCF-7 cells after incubated with PBS (Control), TEID, HA degraded TEID and SLO-Gal&SLO-NEU and then stained with F-Jac to verify the Gal-introducing function.