backbone phosphate of bound cGAMP (Fig.
2F). This facilitates formation of direct
hydrogen-bond contacts with the guanidi-
nium side chains of R238 from both STING
monomers (Fig. 2, D and F). These residues
are located on opposing ends of thebstrands
from each monomer that make the intersu-
bunit interactions within theb-sheet cap
element (Fig. 2D). By inducing compaction
at the base of the binding site (i.e., through
interactions with both T263 side chains),
mimickingintersubunitstackinginteractions
and orientating both R238 side chains to
induce formation of theb-sheet cap element,
SR-717 is able to serve as a direct cGAMP
mimetic that activates STING by inducing it to
adopt the closed conformation. SR-717 also
forms hydrogen-bond interactions with the
side chain of R232 from only one monomer
(fig. S9B), exactly as is observed for bound
cGAMP (PDB ID 4KSY). Additional stabiliz-
ing interactions are derived from a water-
mediated hydrogen-bond network involving
Val^239 (V239), Ser^241 (S241), Asn^242 (N242),
and Y240 (fig. S9B). The near identical con-
formational change induced by SR-717, as
well as its ability to directly mimic the
binding mode of cGAMP, is highlighted by
an overlay of the SR-717–bound hSTING co-
crystal structure with the cGAMP-bound com-
plex (Fig. 2, B and F). By contrast, the SR-717
complex clearly does not correspond to the
open conformation induced by di-GMP (Fig.
2E; PDB ID 4F9G). Consistent with the ob-
served impact of SR-717 on the thermal sta-
bility of mSTING (Fig. 1D), the 2.5-Å cocrystal
structure of SR-717 bound to mSTING (resi-
dues 154 to 340) (figs. S8B and S10A) is nearly
identical to the induced closed-conformation
hSTING complex (figs. S10B and S11). SR-717
is a stable cGAMP mimetic that activates
STING by inducing the same closed confor-
mation, which thereby provides an avenue to
explore this class of systemic STING agonist
in diverse contexts, including antitumor
immunity.
We used IFN-bprotein concentrations in
circulating plasma as a pharmacodynamic
marker of STING target engagement by SR-
717, which displayed favorable mouse phar-
macokinetic properties (fig. S4A), as well as
robust antitumor activity after intratumoral
delivery in syngeneic B16.F10 melanoma or
MC38 colorectal adenocarcinoma mouse mod-
els (fig. S12, A and B, respectively). Dose-
dependent induction of IFN-bwas observed
after intraperitoneal administration of SR-717
in WT C57BL/6 mice (Fig. 3A). By contrast,
SR-717 did not affect circulating IFN-bor
associated proinflammatory cytokine expres-
sion inStinggt/gtmice (Fig. 3B and fig. S16A),
thereby demonstrating its selective on-target
in vivo activity. Based on the known ability
of cGAS-STING pathway activation in DCs to
stimulate CD8 T cell priming ( 11 , 12 , 23 ), we
elected to use the poorly immunogenic and
highly aggressive syngeneic B16.F10 murine
melanoma model ( 24 ) to evaluate and char-
acterize the antitumor activities of system-
ically delivered closed conformation–inducing
STING agonists. A therapeutic mode of treat-
ment was modeled by initiating treatment
with SR-717 on day 11 when B16.F10 mela-
noma tumors are well established (Fig. 3C).
Notably, efficacious doses of diABZI-2 ( 22 ),
an open conformation–inducing STING ago-
nist that induces equivalent maximal levels
of cell-based activity when compared with
SR-717 (fig. S13, A to C), were found to in-
duce ~20 ng/ml of IFN-b(fig. S13D). By con-
trast, systemic dosing regimens of the closed
conformation–inducing STING agonist SR-717
that resulted in the induction of >0.2 ng/ml
of circulating IFN-b(Fig. 3A and fig. S13D)
were found to be well tolerated and effica-
cious. Specifically, a 30 mg/kg intraperitoneal
once-per-day regimen of SR-717 for 1 week
(Fig. 3C) was found to maximally inhibit
tumor growth (Fig. 3D), as well as lengthen
survival time in tumor-bearing mice (Fig. 3E).
The antitumor efficacy of SR-717 displayed
an obligatory dependence on STING expres-
sion, based on the observed lack of activity
inStinggt/gthost mice (Fig. 3, D and E).
To establish if efficacy was restricted to
subcutaneous tumors, as well as to determine
utility in the context of metastasis, we treated
C57BL/6 mice that had been injected intra-
venously with B16.F10 cells, which are reported
to home to and colonize lung tissue ( 25 ). SR-717
was observed to significantly inhibit the
formation of pulmonary nodules in this model
of metastasis (Fig. 3, F and G), consistent
with the ability of a systemic STING agonist
to control metastasis and B16.F10 tumori-
genesis in a manner that is independent of
tissue type. We investigated the efficacy of
this compound series in the context of oral
delivery, using the active STING agonist ana-
log SR-301 (fig. S14, A and B; EC 50 =0.6mM),
which has appreciable bioavailability (%F =
32.2) and suitable rodent exposure proper-
ties (fig. S4B).
Encouragingly, 15 mg/kg oral once-per-day
dosing with SR-301 for 18 days was found to
maximally reduce tumor burden in the B16.
F10 model (fig. S14, C to E). Based on the more
favorable physicochemical properties, as well
as enhanced uniformity with respect to dos-
age to pharmacodynamic relationships, intra-
peritoneal administration of SR-717 was used
to further characterize antitumor immunity.
Using the B16.F10 model, we compared the
antitumor activity of SR-717 to that observed
for anti–programmed cell death 1 (anti–PD-1)
or anti–programmed cell death 1 ligand 1
(anti–PD-L1) antibody therapy. Under the
constraintsandlimitationsofthismodel,maximal achievable efficacy was observed
with a single-agent therapy consisting of
SR-717 STING agonist. Specifically, SR-717 dis-
played a better level of efficacy to that achieved
by anti–PD-1 or anti–PD-L1 antibody therapy
in this model, with respect to tumor burden
(Fig.3,HandJ,respectively)oroverall
survival (Fig. 3, I and K, respectively). The
observed activity profiles of PD-1–based check-
point blockade in this poorly immunogenic
model is consistent with previous reports
( 24 , 26 ).
We investigated the impact of effective sys-
temic exposure levels of SR-717 on immune
cell activation and immunological mechanisms,
in the context of tumor-bearing mice. Consist-
ent with previous reports associated with in-
tratumoral injection of 2′ 3 ′-cGAMP ( 13 , 27 ),
systemic delivery of SR-717 increased the fre-
quency of activated CD69+CD8 T cells among
isolated tumor infiltrating lymphocytes (TILs)
andwithinisolatedspleensandinguinal
lymph nodes (Fig. 4A). A similar increase was
observed in the frequency of activated CD69+
natural killer (NK) cells within isolated spleens
and inguinal lymph nodes (Fig. 4B), con-
sistent with previous findings describing the
ability of the STING pathway to activate this
cell type ( 28 ). Within the CD45.2+popula-
tion, SR-717 treatment resulted in a signifi-
cant increase in the frequency of CD8 T cells
among TILs and a decrease in the frequen-
cies of NK cells within the draining lymph
node (dLN) and spleen (fig. S16B). Because
of the potential for STING agonism to in-
duce pathological consequences, we next
assessed the infiltration of CD8 T cells into
peripheral tissues. We observed no signif-
icant differences in lymphocyte or CD8 T
cell infiltration into the lung after SR-717
treatment(30mg/kgintraperitoneallyfor
7 days) (fig. S15, A and D), although a small
but statistically significant increase in fre-
quencies of CD44+PD-1+T cells was observed
(fig. S15A). In the liver, a small but statistically
significant increase in CD8 T cell infiltration
was observed (fig. S15, B and D). Measure-
ment of liver enzymes revealed a transient up-
regulation of alanine aminotransferase (ALT)
levels on day 4 after treatment, which returned
to vehicle levels by day 7 after treatment, and
no impact on aspartate transaminase (AST) lev-
els was observed (fig. S15C). Further assess-
ment of T cell function revealed that SR-717
treatment significantly increased the fre-
quency of granzyme B and CD107a+CD8 T
cells in both spleen andtumor(Fig.4,Cand
E). Although we did not observe increases in
granzyme B+NK cells in spleen or tumor (Fig.
4D), we did detect increases in CD107a ex-
pressioninNKcellsinbothtissues(Fig.4F),
suggesting that at the time of analysis, NK
cells had already degranulated in SR-717–
treated animals. Analysis of cytokine-producingChinet al.,Science 369 , 993–999 (2020) 21 August 2020 5of7
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