Nanogel-Based Immunologically Stealth Vaccine Targets Macrophages in the Medulla of Lymph Node and Induces Potent Antitumor Immunity

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1 Nanogel-Based Immunologically Stealth Vaccine Targets Macrophages in the Medulla of Lymph Node and Induces Potent Antitumor Immunity Daisuke Muraoka, Naozumi Harada,, Tae Hayashi, Yoshiro Tahara,, Fumiyasu Momose, Shin-ichi Sawada,, Sada-atsu Mukai,, Kazunari Akiyoshi,, and Hiroshi Shiku Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie , Japan, Basic and Preclinical Research, ImmunoFrontier, Inc., Tokyo , Japan, Department of Polymer Chemistry, Kyoto University Graduate School of Engineering, Kyoto , Japan, ERATO, Japan Science and Technology Agency (JST), Tokyo , Japan Supporting Information S1

2 Supporting Information Figure S1. The CHP nanogel does not stimulate macrophages (CD11b + ) and DCs (CD11c + ). PBS as vehicle, CHP (200 g), CpG ODN (50 g), or poly-iclc RNA (50 g) was subcutaneously injected into the back of BALB/c mice. After 20 h, whole lymph node cells were isolated from the DLN, stained with anti-cd11b, CD11c, CD80, and CD86 antibodies, and analyzed using flow cytometry. Upregulation of CD80 and CD86 by CpG ODN and poly-iclc RNA indicates the activation of macrophages and DCs. S2

3 Supporting Information Figure S2. High performance size exclusion chromatography (HPSEC) of the complex between CHP nanogel and LPA. HPSEC data of the FAM-labeled LPA, CHP nanogel, and CHP:FAM-LPA complex. Absorbance in the samples was measured at 495 nm, where FAM shows absorbance, but the CHP nanogel does not. The peak of the CHP:FAM-LPA complex was detected at an elution time of approximately 16 min. When measured at an appropriate wavelength, the CHP nanogel is usually detected at this elution time. Therefore, this result indicates that the FAM-LPA was coeluted with the CHP nanogel which is indicative of the formation of a stable complex between the LPA and CHP. The labeled MAGE-A4 LPA alone and labeled merk2 LPA alone were both undetectable because of their very low solubility. S3

4 Supporting Information Figure S3. HPSEC of the CHP:LPA complex incubated with mouse serum. Mouse serum was centrifuged at 1,200 x g for 15 min, filtrated through a m pore filter, and diluted in PBS at a concentration of 20%. A complex between fluorescent dye FAM-labeled LPA and CHP nanogel was mixed with the diluted serum and incubated at 37 C for 1, 24, and 48 h. The stability of CHP:LPA complex was then evaluated by HPSEC (Shimadzu Co., Kyoto, Japan) on a TSK-gel G3000SWXL column (TOSOH Co., Tokyo, Japan). Fluorescence of FAM-LPA was detected using an in-line fluorescent detector at the excitation/emission wavelength of 495/525 nm. The peak areas of the LPA complexed with CHP were calculated to be 29, 24, and 23% after the incubation in 20% serum for 1, 24, and 48 h, S4

5 respectively, as compared to the peak area of the complex in PBS. The LPA/CHP complex still remained over 20 % after 40 h in the presence of serum. S5

6 Supporting Information Figure S4. Histochemical analysis of cells incorporating the CHP: FAM-labeled LPA in the DLN. For histochemistry, a inguinal DLN was collected from a BALB/c mouse 16 h after subcutaneous injection of the CHP:FAM-LPA, and was subjected to preparation of cryosections. Resulting O.C.T. compound-embedded cryosections were stained with 4',6 diamidino 2 phenylindole (DAPI) and observed under a fluorescent microscope. Left, DAPI staining. Center, FAM-LPA. Right, merged. Boxes indicate the area magnified. Scale bar = 400 (top) and 50 (bottom) mm. S6

7 Supporting Information Figure S5. Isolation of MSMs and MCMs. CD11b + cells was isolated using anti-cd11b microbeads from the total cell suspension prepared from the DLN. Subsequently, the cells were stained with FAM-labeled anti-cd11b, PE-labeled anti-cd169, PerCP-Cy5.5-labeled anti-f4/80 mabs, and sorted on a FACSAria cell sorting system. S7

8 Supporting Information Figure S6. Depletion of lymph node macrophages by clodronate liposomes. Clodronate liposomes or control liposomes were subcutaneously injected into the footpad of BALB/c mice. After 6 days, total cells were prepared from the DLN, and the frequency of macrophages (a) and DCs (b) was determined using flow cytometry. p-values were determined by Student s t test. *p < n.s., not significant. Similar result was also obtained 24 h after the injection of clodronate liposomes (not shown). S8

9 Supporting Information Figure S7. The IFA:LPA vaccine is cross-presented by DCs. (a c) The IFA:LPA vaccine with CpG ODN was subcutaneously injected into the footpad of BALB/c mice. 18 h later, the fractions containing macrophages and DCs were isolated from the DLN. These cells were cocultured as APCs with merk2-specific DUC18 CD8 + T cells for 72 h in vitro. (a, b) Proliferation of DUC18 CD8 + T cells was measured using a CFSE dilution assay. The numbers shown in histograms indicate the percentages of proliferating cells. (c) The concentration of IFN- in the culture supernatant was determined using ELISA. The data shown in b and c are mean SD of triplicates. The results are representative of one of at least two experiments. p-values were determined by Dunnett's multiple comparison test. *p < S9

10 Supporting Information Figure S8. Specific CD8 + T cell response induced by the CHP nanogel-based vaccine is greater than that induced by a saline-based vaccine. Vaccineinduced CD8 + T cell response. BALB/c mice were injected with either the CHP:MAGE-A4 LPA vaccine or the LPA dissolved into saline followed by the injection with CpG ODN (50 g). Seven days after vaccination, splenocytes were isolated and restimulated with MAGE-A4 LPA in vitro. Frequency of activated specific CD8 + T cells was quantified by intracellular IFN- staining followed by flow cytometry (two mice per group). S10

11 Supporting Information Figure S9. Human macrophages possess substantial crosspresenting activity in the presence of a TLR agonist. (a, b) Stimulation of human macrophages by poly-iclc RNA. Human monocyte-derived macrophages were stimulated with poly-iclc RNA (10 g/ml) in vitro. Upregulation of CD80 and CD86 was analyzed using flow cytometry to determine the activation status of the cells. (a) Histograms showing the level of CD80 and CD86 expression in the treated (blue) and untreated cells (grey). (b) Mean fluorescence intensity (MFI). (c, d) Stimulation of antigen-specific CD8 + T cells by human macrophages treated the CHP:human NMW LPA complex and poly-iclc RNA in vitro. Macrophages were cocultured with NY-ESO-1-specific (c) or MAGE-A4-specific (d) human S11

12 CD8 + T cells, and activation of the CD8 + T cells was determined by IFN- enzyme-linked immunospot (ELISPOT) assay. The data are mean SD of triplicates and are representative of at least two independent experiments. p-values were determined by Student s t test. *p < S12