1. by Adrienne Sallets, Sophie Robinson, Adel Kardosh, and Ronald Levy
Enhancing immunotherapy of STING agonist for lymphoma in preclinical models
by Adrienne Sallets, Sophie Robinson, Adel Kardosh, and Ronald Levy
BloodAdv
Volume 2(17):
September 11, 2018
© 2018 by The American Society of Hematology

2. Adrienne Sallets et al. Blood Adv 2018;2:2230-2241
© 2018 by The American Society of Hematology

3. STING toxicity on lymphoma vs immunotherapeutic effect.
STING toxicity on lymphoma vs immunotherapeutic effect. (A) The Sting gene was knocked out of BL370 lymphoma cells using the CRISPR/cas9 system. Depicted is a western blot showing STING expression in BL3750 WT and STING KO clones, as well as splenocytes isolated from WT or STING KO mice. (B) Viability of WT and KO BL3750 cells after incubation with 20 µg/mL STINGa for 24 hours (n = 3). Statistical significance was calculated by a Student t test. Error bars are standard error of the mean (SEM). (C) STING knockout (KO) or wild-type (WT) BL3750 cells were implanted SC on STING KO or WT mice. Each mouse was implanted with 2 tumors, 1 on each side of the abdomen. At day 6, 8, and 10 after tumor implantation, 20 µg STINGa was injected into 1 tumor. Tumor growth of both the injected and distant tumors was monitored (5-10 mice per group). Statistical significance was calculated using 2 way analysis of variance (ANOVA). Error bars are SEM. ***P < ns., not significant.
Adrienne Sallets et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

4. STING in situ vaccination is improved when combined with CpG, anti-OX40, or anti-GITR.
STING in situ vaccination is improved when combined with CpG, anti-OX40, or anti-GITR. (A) Six- to 8-week-old BALB/C mice were implanted with 5 × 106 A20 cells on both flanks. One tumor was used as injection site and the other was monitored for systemic effect. Mice were treated on days 6, 8, and 10 after tumor implantation with IT injection of 5 μg STINGa and/or IT injection of 5 μg CpG (B); SC injection of 8 μg anti-OX40 antibodies (C); SC injection of 50 μg anti-GITR antibodies (D); IT injection of 5 μg Resiquimod (E). These experiments were reproduced at least twice. Error bars are SEM. Shown data are 1 representative experiment with 10 mice per group. Statistical significance of tumor growth was calculated by 2-way ANOVA. ***P < .001.
Adrienne Sallets et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

5. The systemic antitumor response is T-cell mediated.
The systemic antitumor response is T-cell mediated. BALB/C mice were implanted with 5 × 106 A20 cells on both flanks. (A) Local (treated site) and systemic effect of the treatment in the context of CD4 and/or CD8 T-cell depletion. Mice were treated with IT injection of STINGa and SC injection of anti-GITR. Treatment was given on day 6, 8, and 10 after tumor implantation. Depleting antibodies for CD4 and/or CD8 T-cell depletion were given on day 4, 5, 6, 11, and 14. One experiment with 10 mice per group. Statistical significance of tumor growth was calculated using 2-way ANOVA. (B-D) Tumor-specific CD4 and CD8 T cells in the spleen. On day 6, 8, and 10, mice were treated with IT injection of STINGa and/or SC injection of anti-GITR. On day 17, spleens were harvested. INF-γ–producing cells were monitored after restimulation with tumor cells (A20) or unrelated tumor cells (4T1). (B) Raw data of representative samples. (C) Percentage of CD44+INF-γ+ cell among the CD8 T cells. Statistical significance was calculated using 1-way ANOVA. (D) Percentage of CD44+INF-γ+ cell among the CD4 T cells. Statistical significance was calculated using 1-way ANOVA. (E) Treated tumor were harvested at 2 and 4 hours after IT injection of STINGa or vehicle control. Graphs illustrate percentage of CD8 T cell, CD4 T cell, Tregs (CD4+FoxP3+), and CD4eff (CD4+FoxP3−). Statistical significance was calculated using 1-way ANOVA. Error bars are SEM. *P < .05; **P < .01; ***P < .001.
Adrienne Sallets et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

6. Dissection of the immune response in the nontreated tumor.
Dissection of the immune response in the nontreated tumor. Mice bearing 2 A20 tumors were treated as in Figure 1C with IT STINGa and/or SC anti-GITR. One day posttreatment and 1 week posttreatment, mice were sacrificed and the draining lymph node of the treated site and the nontreated tumor were excised for cell population analysis. (A) Schedule of injection and organs harvest. (B-I) Cell populations in the nontreated tumor. (B) Infiltrating T cells. T-cell subsets among the T cells 1 day posttreatment (C) and 1 week posttreatment (D). Percentage of ICOS+ (E), CD69+ (F), Ki67+ (G) among CD8 T cells. Percentage of PD-1hi and PD1low among CD8 T cells 1 day posttreatment (H) and 1 week posttreatment (I). Represented are data pulled from 2 experiments with 3 mice per group. Error bars are SEM. Statistical significance was calculated using 1-way ANOVA. *P < .05; **P < .01; ***P < .001.
Adrienne Sallets et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

7. Dissection of the immune response in the draining lymph node of the treated tumor.
Dissection of the immune response in the draining lymph node of the treated tumor. Cell population in the draining lymph node of treated tumor harvested as described in Figure 4A. (A) Percentage of CD11b+ cells. (B) CD86+ cells among CD11b+ cells. T-cell subset 1 day posttreatment (C) and 1 week posttreatment (D). (E) CD8/Treg ratio. ICOS+ (F) CD60+ (G), Ki67+ (H), PD-1+ (I) positive cells among CD8 T cells. Represented are data pulled from 2 experiments with 3 mice per group. Error bars are SEM. Statistical significance was calculated using 1-way ANOVA. *P < .05; **P < .01; ***P < .001.
Adrienne Sallets et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

8. Expending in situ vaccination in a second treated site improve the treatment.
Expending in situ vaccination in a second treated site improve the treatment. (A-B) Six- to 8-week-old BALB/C mice were implanted with 5 × 106 A20 cells on both flanks. One tumor was used as injection site and the other was monitored for systemic effect. Mice were treated with IT injection of 5 μg STINGa and SC injection of 50 μg anti-GITR. Mice were treated every other day 3 or 9 times. (A) Schedule of injections. (B) Growth curve of the treated and nontreated tumor. Data represent 1 experiment with 10 mice per group. (C-D) Six- to 8-week-old BALB/C mice were implanted with 5 × 106 A20 cells at 3 sites on the abdomen. Two tumors were used as injection site; the tired 1 was monitored for systemic effect. Mice were treated with IT injection of 1 μg STINGa and SC injection of 10 μg anti-GITR antibodies. One group of mice was treated every other day 3 times in the tumor 1. A second group of mice was treated 3 times every other day in tumor 1 and then received another 3 injections every other day in the second tumor. (C) Schedule of the injection. (D) Growth curve of the 3 tumors and survival of the mice. Data represent 1 experiment with 10 mice per group. Error bars are SEM. Statistical significance of tumor growth was calculated using 2-way ANOVA. Survival significance was calculated using Mantel-Cox. *P < .05; ***P < .001.
Adrienne Sallets et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology

9. PD-1 blockade improve STING + anti-GITR therapy.
PD-1 blockade improve STING + anti-GITR therapy. Mice bearing 2 A20 tumors were treated as described in Figure 1C. Mice were treated with IT STINGa and IP anti-PD-1, IT STING and IP anti-GITR, IP anti-GITR and IP anti-PD-1, or, the triple combination, IT STINGa, IP anti-GITR and IP anti-PD-1. Treatment efficacy was assessed measuring tumor growth at the treated site (A), tumor growth at the distant nontreated site (B), and overall survival (C). (A-B) One experiment with 10 mice per group. Experiment was repeated twice. Error bars are SEM. Statistical significance of tumor growth was calculated using 2-way ANOVA. (C) Data pulled from 2 experiment with 10 mice per group. Survival significance was calculated using Mantel-Cox. *P < .05; **P < .01; ***P < .001.
Adrienne Sallets et al. Blood Adv 2018;2:
© 2018 by The American Society of Hematology