Supplementary Materials for

Extended release of perioperative immunotherapy prevents tumor

recurrence and eliminates metastases

Chun Gwon Park, Christina A. Hartl, Daniela Schmid, Ellese M. Carmona, Hye-Jung Kim, Michael S. Goldberg*

*Corresponding author. Email: michael_goldberg1@dfci.harvard.edu

Published 21 March 2018, Sci. Transl. Med. 10, eaar1916 (2018) DOI: 10.1126/scitranslmed.aar1916

This PDF file includes:

Fig. S1. A hydrogel composed of cross-linked hyaluronic acid is stable but biodegradable in vivo. Fig. S2. The hydrogel scaffold extends the release of biologics and small molecules in vitro. Fig. S3. The hydrogel scaffold extends the local release of biologics and small molecules in vivo. Fig. S4. The activity of a biologic released from the hydrogel is preserved completely. Fig. S5. Multiple systemic administrations of R848 fail to confer survival benefit and are less well tolerated than R848 released from a hydrogel. Fig. S6. STING-RR confers superior efficacy to 2′3′-cGAMP upon extended release from a hydrogel in the perioperative setting. Fig. S7. IVIS imaging confirms that STING-RR must be released from a hydrogel to protect against tumor recurrence and eliminate metastases. Fig. S8. Efficacy of STING-RR loaded in a hydrogel is retained after storage for 1 week at 4°C. Fig. S9. Intraoperative placement of the immunotherapy-loaded hydrogel into the tumor resection site is required for therapeutic benefit. Fig. S10. Extended local release of combination immune checkpoint blockade confers limited survival benefit. Fig. S11. Flow cytometric analysis confirms that NK cells, CD8+ T cells, and CD4+ T cells are depleted after administration of appropriate antibodies. Fig. S12. Extended local release of STING-RR increases the number of activated innate immune cells.

www.sciencetranslationalmedicine.org/cgi/content/full/10/433/eaar1916/DC1

Fig. S13. Extended local release of R848 increases the numbers of several leukocyte subsets in the lung. Fig. S14. Increased concentrations of cytokines are observed in plasma collected at various time points after surgery. Fig. S15. Induction of an adaptive antitumor memory response is confirmed by rejection of 4T1-Luc2 cells inoculated as rechallenge. Fig. S16. Extended local release of agonists of innate immunity does not alter the composition of the blood. Fig. S17. Extended local release of agonists of innate immunity is safe. Fig. S18. Extended local release of agonists of innate immunity is well tolerated. Fig. S19. The response of parental 4T1 cells to R848 released locally from a hydrogel is similar to that of 4T1 cells expressing Luc2. Fig. S20. Localized release of perioperative immunotherapy is efficacious in the B16-BL6 model of spontaneous metastasis. Fig. S21. Extended release of R848 from a scaffold derived from alginate confers survival benefit. Table S1. Quantitation and statistics for a panel of cytokines measured in plasma recovered 3 and 14 days after surgery. Table S2. A table of the antibodies used for flow cytometry experiments.

SUPPLEMENTARY FIGURES

Fig. S1. A hydrogel composed of cross-linked hyaluronic acid is stable but biodegradable in

vivo. (A) Alexa Fluor 750 was covalently conjugated to a hydrogel scaffold, which is shown in ambient light. (B) A hydrogel scaffold to which Alexa Fluor 750 was conjugated (left) and a hydrogel that was not modified (right) are shown by fluorescence IVIS imaging (ex 745 nm, em 780 nm). (C) Quantification of the fluorescence IVIS imaging depicted in Figure 1B reveals the degradation kinetics of the scaffold. (D) Surgical resection of orthotopic 4T1 tumors does not confer meaningful survival benefit in the absence of additional therapy. The graph shows a Kaplan-Meier curve of female BALB/cJ mice inoculated orthotopically with 4T1-Luc2 cells whose tumors were either untreated or surgically resected. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times. (E) To enable evaluation of degradation kinetics in several mice, scaffolds were implanted next to the fourth mammary fat pad of non-tumor-bearing female BALB/cJ mice, and fluorescence IVIS imaging was performed at the indicated time points. (F) Alexa Fluor 750 was administered in solution locally, and fluorescence IVIS imaging was performed at the indicated time points.

Fig. S2. The hydrogel scaffold extends the release of biologics and small molecules in vitro. Scaffolds were placed in PBS (pH 7.4), and drug release was measured using a fluorescence plate reader (A, B, E) or HPLC (C, D, F). The following payloads were evaluated: (A) anti-PD-1, (B) IL-15sa, (C) lenalidomide, (D) celecoxib, (E) 2’3’-cGAMP (model compound for 2’3’-c-di-AM(PS)2 (Rp,Rp), “STING-RR”), and (F) R848. The experiment was performed with biological replicates (n=4+) three times. Data are presented as mean ± SD.

Fig. S3. The hydrogel scaffold extends the local release of biologics and small molecules in

vivo. (A-C) Biologic and small molecule payloads are homogeneously distributed throughout the hydrogel. Confocal imaging of a hydrogel containing (A) anti-PD-1, (B) IL-15sa, or (C) 2’3’-cGAMP confirms their uniform dissemination. The payloads were conjugated to Alexa Fluor 405, VivoTag 680, and FITC fluorescent dyes, respectively. (D-F) Fluorescently labeled payloads – (D) anti-PD-1, (E) IL-15sa, and (F) 2’3’-cGAMP – were loaded into scaffolds, which were implanted next to the fourth mammary fat pad of non-tumor-bearing female BALB/cJ mice. IVIS imaging was performed at the indicated time points. (G-I) Release kinetics were subsequently quantified for (G) anti-PD-1, (H) IL-15sa, or (I) 2’3’-cGAMP. Payloads administered in solution are depicted in black, and payloads released via hydrogel are depicted in red. Fold difference is indicated for each time point. Statistics were calculated using a two-sided unpaired t-test. Data are presented as mean ± SD (n=5 per group, performed twice as biological replicates). * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001

Fig. S4. The activity of a biologic released from the hydrogel is preserved completely. CD122+ central memory CD8+ T cells were isolated and incubated with IL-15sa that had been either not been entrapped in a hydrogel or entrapped in and released from a hydrogel. The dilution of CellTrace Violet, which was used to label the T cells, is visualized by flow cytometry as a measure of cell proliferation. Pre-incubation of CD8+ T cells with a neutralizing antibody against CD122 prevented stimulation of proliferation by IL-15sa.

Fig. S5. Multiple systemic administrations of R848 fail to confer survival benefit and are

less well tolerated than R848 released from a hydrogel. Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells, and R848 (200 g) was or was not injected intraperitoneally for three consecutive days. (A) A Kaplan-Meier curve illustrates relative survival benefit. (B) The weights of the mice are depicted for the treatment groups. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times.

Fig. S6. STING-RR confers superior efficacy to 2′3′-cGAMP upon extended release from a

hydrogel in the perioperative setting. Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells, and no hydrogel or a hydrogel loaded with 2’3’-cGAMP or STING-RR (100 g) was placed in the resection site. A Kaplan-Meier curve illustrates relative survival benefit. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times. Statistics were calculated relative to no hydrogel using the log-rank (Mantel-Cox) test. ** p≤0.01

Fig. S7. IVIS imaging confirms that STING-RR must be released from a hydrogel to

protect against tumor recurrence and eliminate metastases. Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells. IVIS imaging was performed weekly to assess tumor burden. Data for individual mice are shown for tumors that recurred locally (top) or metastasized to the lung (bottom) after the indicated treatments: no hydrogel, an empty hydrogel plus local administration of STING-RR in solution, or a hydrogel loaded with STING-RR.

Fig. S8. Efficacy of STING-RR loaded in a hydrogel is retained after storage for 1 week at

4°C. Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells, and no hydrogel or a hydrogel loaded with STING-RR that had been stored at 4 C for seven days was placed in the resection site. A Kaplan-Meier curve illustrates relative survival benefit. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times. Statistics were calculated relative to no hydrogel using the log-rank (Mantel-Cox) test. ** p≤0.01

Fig. S9. Intraoperative placement of the immunotherapy-loaded hydrogel into the tumor

resection site is required for therapeutic benefit. Local release of an agonist of innate immunity (as monotherapy) from a hydrogel does not confer therapeutic efficacy in the absence of tumor removal, even if the hydrogel is placed peritumorally. Hydrogels loaded with (A) R848 or (B) STING-RR were placed peritumorally 10 days after orthotopic inoculation of 4T1-Luc2 cells. A Kaplan-Meier curve is shown for the control and treatment groups. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times.

Fig. S10. Extended local release of combination immune checkpoint blockade confers

limited survival benefit. Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells, and no hydrogel or a hydrogel loaded with anti-PD-1 and anti-CTLA-4 (300 g each) was placed in the resection site. A Kaplan-Meier curve illustrates relative survival benefit. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times.

Fig. S11. Flow cytometric analysis confirms that NK cells, CD8+ T cells, and CD4+ T cells

are depleted after administration of appropriate antibodies. Plots are shown for leukocytes isolated from blood 10 days after surgery. These data are representative of n=5 mice, and the experiment was performed three times.

Fig. S12. Extended local release of STING-RR increases the number of activated innate

immune cells. Tumors were resected from mice 10 days after orthotopic inoculation of 4T1-Luc2 cells, and scaffolds were placed in the resection site. Spleens were recovered from mice 3 days after surgery for flow cytometry analysis. Increased numbers of leukocytes with activated and effector phenotypes were observed. Quantitation of flow cytometry gating of subsets of (A) NK cells and (B) dendritic cells is shown. KLRG1, killer cell lectin-like receptor G1; MHCII, major histocompatibility complex class II; PDCA1, plasmacytoid dendritic cell antigen-1. The experiment was performed once with n=5 biological replicates. Statistics were calculated using a two-sided unpaired t-test. Data are presented as mean ± SEM. * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001

Fig. S13. Extended local release of R848 increases the numbers of several leukocyte subsets

in the lung. Lungs were recovered on days (A) 3 and (B) 14 post-surgery, and single-cell suspensions were prepared for flow cytometry. Data are presented as mean ± SEM. * p≤0.05, ** p≤0.01

Fig. S14. Increased concentrations of cytokines are observed in plasma collected at various

time points after surgery. Blood was recovered on days (A) 3 and (B) 14 post-surgery, and samples were analyzed by multiplexing laser bead technology. Concentrations of a panel of cytokines are shown. The experiment was performed once with n=5 biological replicates. Statistics were calculated using a two-sided unpaired t-test. Data are presented as mean ± SEM. * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001

Fig. S15. Induction of an adaptive antitumor memory response is confirmed by rejection of

4T1-Luc2 cells inoculated as rechallenge. A Kaplan-Meier curve illustrates that 100% of naïve mice succumb to the lethal challenge, whereas 100% of the mice that had achieved durable survival benefit after treatment with hydrogels loaded with (A) R848 or (B) STING-RR survive the rechallenge. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed once. Statistics were calculated relative to the rechallenged mice using the log-rank (Mantel-Cox) test. *** p≤0.001, **** p≤0.0001

Fig. S16. Extended local release of agonists of innate immunity does not alter the

composition of the blood. Blood was recovered on days 3 and 14 after surgery and treatment with (A) R848 or (B) STING-RR. WBC, white blood cells; RBC, red blood cells; RDW, red blood cell distribution width; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; NE, neutrophils; LY, lymphocytes; MO, monocytes; EO, eosinophils; BA, basophils; MPV, mean platelet volume; PLT, platelets; HCT, hematocrit; MCV, mean corpuscular volume. The experiment was performed once with n=5 biological replicates. Statistics were calculated using a two-sided unpaired t-test. Data are presented as mean ± SEM. Dashed lines indicate established normal ranges.

Fig. S17. Extended local release of agonists of innate immunity is safe. None of the conditions listed impact the concentrations of liver enzymes and BUN measured (A) 3 days post-surgery (R848) or (B) 15 days post-surgery (STING-RR). Data are presented as mean ± SD (n=6+ per group, performed twice as biological replicates). Dashed lines indicate established normal ranges.

Fig. S18. Extended local release of agonists of innate immunity is well tolerated. None of the conditions listed impact the weight of mice longitudinally upon administration of either (A) R848 or (B) STING-RR. The weight loss observed in the first week is related to the stress of the surgery itself, as it is observed in all groups, including the no hydrogel and empty hydrogel negative controls. Data are presented as mean ± SD (sample sizes for each group are provided in Figure 2D, performed at least three times).

Fig. S19. The response of parental 4T1 cells to R848 released locally from a hydrogel is

similar to that of 4T1 cells expressing Luc2. Tumors were resected from mice 10 days after orthotopic inoculation of wild-type 4T1 cells. A Kaplan-Meier curve of female BALB/cJ is shown for the following groups: no hydrogel, an empty hydrogel plus local administration of R848 in solution, or a hydrogel loaded with R848. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times. Statistics were calculated relative to no hydrogel using the log-rank (Mantel-Cox) test. * p≤0.05

Fig. S20. Localized release of perioperative immunotherapy is efficacious in the B16-BL6

model of spontaneous metastasis. Tumors were resected from mice when tumor volumes reached ~650 mm3 after subcutaneous inoculation of B16-BL6 melanoma cells. A Kaplan-Meier curve is shown for no hydrogel, an empty hydrogel plus local administration of R848 in solution, or a hydrogel loaded with R848. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times. Statistics were calculated relative to the group treated with hydrogel containing the indicated agonist of innate immunity using the log-rank (Mantel-Cox) test. * p≤0.05

Fig. S21. Extended release of R848 from a scaffold derived from alginate confers survival

benefit. (A) A hydrogel scaffold derived from alginate extends the release of R848 in vitro. Scaffolds were placed in PBS (pH 7.4), and drug release was measured using HPLC. The experiment was performed with biological replicates (n=4+) three times. Data are presented as mean ± SD. (B) A Kaplan-Meier curve is shown for mice after resection of orthotopic 4T1-Luc2 tumors and treatment with no hydrogel, an alginate hydrogel loaded with DMSO vehicle (“empty alginate hydrogel”), or an alginate hydrogel loaded with R848. The number of mice per group (n) and median survival (ms) are listed. The experiment was performed at least three times. Statistics were calculated relative to the group treated with hydrogel containing R848 using the log-rank (Mantel-Cox) test. * p≤0.05

Table S1. Quantitation and statistics for a panel of cytokines measured in plasma recovered

3 and 14 days after surgery. These data are presented graphically in Fig. 5G.

Day 3 No hydrogel R848-loaded hydrogel Fold increase Statistics

Average Std. Error Average Std. Error

Eotaxin 661.63 82.44 643.6 82.1 0.97 0.6972 ns

G-CSF 451.86 257.44 584.47 202.75 1.29 0.338 ns

IP-10 211.36 66.06 388.75 150.95 1.84 0.0151 *

IL-1a 219.46 141.46 161.58 152.67 0.74 0.4929 ns

MIG 105.8 31.77 222.49 96.73 2.1 0.0112 *

IL-15 47 32.86 198.73 108.56 4.23 0.0056 **

MIP-2 169.5 77.69 85.29 56.56 0.5 0.0506 ns

KC 103.62 37.54 118.12 50.57 1.14 0.606 ns

MIP-1B 106.7 24.57 99.91 46.55 0.94 0.7447 ns

IL-6 24.64 21.84 73.98 38.72 3 0.0187 *

IL-2 8.33 2.69 51.05 46.23 6.13 0.0463 *

MCP-1 21.09 13.64 44.49 38.07 2.11 0.1906 ns

IL-7 9.68 5.44 38.56 24.46 3.98 0.0124 *

IL-9 5.22 5.1 34.79 26.68 6.67 0.0229 *

IL-12 (p70) 8.27 2.7 23.69 16.09 2.86 0.0392 *

IL-13 31.57 4.95 22.72 10.06 0.72 0.0615 ns

IL-1B 25.37 12.54 21.38 17.73 0.84 0.6606 ns

IL-5 16.38 8.02 10.21 4.36 0.62 0.0872 ns

IL-10 10.81 1.76 15.17 10.01 1.4 0.3052 ns

M-CSF 14.37 10.47 4.06 2.35 0.28 0.0161 *

TNF-α 13.15 2.84 9.76 2.53 0.74 0.0281 *

IL-12(p40) 9.37 4.54 4.37 2.21 0.47 0.032 *

RANTES 8.4 5.51 8.6 9.97 1.02 0.97 ns

LIF 2.84 1.69 7.58 3.08 2.66 0.005 **

IFN-γ 4.97 2.05 4.66 6.54 0.94 0.9126 ns

IL-4 0.61 0.37 1.25 0.86 2.04 0.1224 ns

IL-17 1.85 0.72 1.13 0.83 0.61 0.1407 ns

VEGF 1.22 0.33 1 0.25 0.82 0.1558 ns

ns = not significant * p≤0.05, ** p≤0.01

Day 14 No hydrogel R848-loaded hydrogel Fold

increase Statistics

Average Std. Error Average Std. Error

IL-1a 533.12 431.99 1072.61 459.88 2.01 0.1035 ns

Eotaxin 675.67 124.36 806.71 94.2 1.19 0.106 ns

G-CSF 627.79 285.67 606.01 204.61 0.97 0.913 ns

IP-10 181.67 16.25 290.02 33.29 1.6 0.0006 ***

MIG 114.38 26.87 167.19 12.53 1.46 0.0026 **

KC 70.23 67.43 159.87 49.92 2.28 0.0529 ns

IL-15 30.52 22.92 124.85 23.3 4.09 0.0005 ***

MIP-2 88.42 44.76 112.01 62.96 1.27 0.5944 ns

MIP-1B 66.92 32.77 83.05 24.67 1.24 0.3999 ns

IL-13 24.7 10.74 41.19 6.15 1.67 0.0223 *

IL-1B 12.15 6.52 37.37 14 3.08 0.0062 **

IL-12(p40) 5.41 1.88 35.81 23.83 6.62 0.0238 *

MCP-1 18.62 7.2 35.66 8.9 1.91 0.0124 *

IL-9 24.51 18.66 9.14 4.9 0.37 0.163 ns

IL-5 21.37 6.45 21.47 4.55 1 0.9789 ns

IL-7 1.93 1.29 11.78 4.99 6.12 0.0033 **

TNF-α 6.63 2.36 11.65 1.83 1.76 0.0038 **

M-CSF 6.23 2.99 10.65 6.52 1.71 0.2321 ns

IL-6 7.06 3.8 9.25 4.61 1.31 0.5482 ns

IL-2 5.68 4.2 8.09 2.98 1.42 0.4482 ns

IL-12 (p70) 6.91 2.44 8 0 1.16 0.4468 ns

IL-10 2.24 0.14 7.13 2.77 3.18 0.0226 *

RANTES 3.5 2.43 3.67 1.62 1.05 0.9204 ns

LIF 1.83 0.89 2.93 1.29 1.6 0.1932 ns

IL-17 0.78 0.48 1.38 0.72 1.77 0.1938 ns

VEGF 1.13 0.37 1.34 0.52 1.19 0.4925 ns

IFN-γ 0.65 0.23 0.72 0.48 1.1 0.8239 ns

IL-4 0.2 0.09 0.41 0.23 2.11 0.0926 ns

ns = not significant

* p≤0.05, ** p≤0.01, *** p≤0.001

Table S2. A table of the antibodies used for flow cytometry experiments.

Protein Color Clone Dilution Vendor

1 CD45 BV785 30-F11 1:200 BioLegend 2 TCR β BV510 H57-597 1:200 BioLegend 3 TCR β PE/Cy7 H57-597 1:200 BioLegend 4 CD4 BV605 GK1.5 1:200 BioLegend 5 CD4 FITC GK1.5 1:400 BioLegend 6 CD8 (alpha) BV650 53-6.7 1:200 BioLegend 7 CD8 (alpha) AF700 53-6.7 1:200 BioLegend 8 NK cell (NKp46) PE 29A1.4 1:100 BioLegend 9 CD11b BV421 M1/70 1:300 BioLegend 10 CD11b BV785 M1/70 1:300 BioLegend 11 CD11c PerCP/Cy5.5 N418 1:100 BioLegend 12 CD19 APC/Cy7 6D5 1:100 BioLegend 13 F4/80 PE/Cy7 BM8 1:100 BioLegend 14 Gr1 FITC RB6-8C5 1:1000 BioLegend 15 CD25 BV785 PC61 1:100 BioLegend 16 CD69 PerCP/Cy5.5 H1.2F3 1:200 BioLegend 17 CD62L APC/Cy7 MEL-14 1:70 BioLegend 18 CD62L PE MEL-14 1:200 BioLegend 19 CD44 APC IM7 1:400 BioLegend 20 CD122 PerCP/Cy5.5 TM-β1 1:50 BioLegend 21 GITR PerCP/Cy5.5 DTA-1 1:200 BioLegend 22 Ly6C FITC HK1.4 1:100 BioLegend 23 CD27 BV650 LG.3A10 1:100 BD Biosciences 24 KLRG1 BV605 2F1/KLRG1 1:200 BioLegend 25 CD40 PE/Cy7 43182 1:200 BioLegend 26 CD86 BV510 GL-1 1:200 BioLegend 27 MHC2 FITC 39-10-8 1:500 BioLegend 28 PDCA1 PE 129C1 1:200 BioLegend 29 CD103 BV605 2E7 1:200 BioLegend 30 B220 APC/Cy7 RA3-6B2 1:200 BioLegend 31 FoxP3 APC FJK-16s 1:70 eBioscience 32 FoxP3 PE MF-14 1:70 BioLegend 33 IFN-γ BV421 XMG1.2 1:70 BioLegend 34 IL2 PE/Cy7 JES6-5H4 1:70 BioLegend 35 Granzyme B FITC NGZB 1:70 eBioscience 36 GM-CSF PerCP/Cy5.5 MP1-22E9 1:70 BioLegend