NK cells revealed significantly reduced IFN-g+
NK cells in TILs (fig. S16C), with a modest but
significant increase of IFN-g+NK cells in
spleen (fig. S16C). Moreover, SR-717 treatment
had no significant effect on the frequency
of polyfunctional CD8 T cell responses (fig.
S16D). We also observed increases in the fre-
quencies of CD8 T cells undergoing apopto-
sis within the dLN (fig. S16E), the magnitude
of which being consistent with those previ-
ously reported for efficacious doses of cGAMP
after intratumoral injection ( 14 ). The anti-
tumor activity derived from STING activation
is dependent on CD11c+CD8aDCs ( 12 )and
involves tumor antigen cross-presentation lead-
ing to the activation of CD8 T cells within the
draining lymph node, as well as the activation
of type I interferon signaling ( 11 , 23 ). Sys-
temic SR-717 administration induces the ac-
tivation of CD11c+CD8aDCs, as determined
by CD80 and CD86 staining intensity (Fig.
4G), and enhances cross-priming of CD8 T cells,
as determined by monitoring the in vivo
proliferation of transferred Thy1.1+OT-I CD8
T cells isolated from mice pretreated with
SR-717 and subsequently injected with oval-
bumin protein (fig. S16, F and G). At this
stage of treatment, OT-I CD8 T cell activation
state was determined to be significantly en-
hancedbytreatmentwithSR-717,asdeter-
mined based on the evaluation of CD44+PD-1+
and granzyme B+CD8 T cell populations, as
well as polyfunctional CD8 T cell responses
(fig. S16, H to J).
Finally, we examined the impact of SR-717
on the expression of critical targets associated
with antitumor immunity. STING pathway
activation can induce mechanisms known to
regulate immune checkpoint protein expres-
sion. STING activation, and the subsequent
induction of type I interferon, can induce
STAT3 phosphorylation ( 29 ), a key regulator
of interferon-dependent PD-L1 expression
( 30 , 31 ). SR-717 was found to induce the ex-
pression of PD-L1 in THP1 cells (Fig. 4H and
fig. S17A) and in primary human PBMCs (fig.
S17B) in a STING-dependent manner (Fig. 4H).
Consistent with previous findings describing
the impact of cGAMP on STING protein lev-
els ( 32 ), and indicative of negative feedback
mechanisms associated with pathway activa-
tion, total STING protein levels were observed
to decrease after treatment with SR-717 (Fig.
4H). In vivo, we observed that intraperitoneal
injection of SR-717 resulted in increased cell-
surface levels of PD-L1 on CD11c+CD8−DCs
but not on CD8+DCs isolated from the inguinal
lymph nodes of B16.F10 tumor bearing mice
(Fig. 4I), even though SR-717 clearly activates
CD8+DCs, which suggests cell type–selective
differences in downstream STING-dependent
signaling. Indoleamine 2,3-dioxygenase 1 (IDO1)
in vivo expression has been demonstrated to
be induced in a STING-dependent manner
( 33 ). SR-717 STING agonist was found to in-
duceIDO1expressioninprimaryhuman
PBMCs (fig. S17C). Taken together, our results
demonstrate that although STING activation
with SR-717 induces the expected stimulatory
events, a corresponding induction of molecules
known to suppress immune responses was also
elicited, albeit in a cell type–selective manner.
These observations have important implications
for the selection of agents and the temporal
design of combination-based clinical trials in-
volving a systemically delivered STING agonist.
To address the limitations of intratumoral
delivery, we have identified the SR-717 chemi-
cal series of functional cGAMP mimetic STING
agonists, which, after systemic administra-
tion, were demonstrated to promote anti-
tumor immunity and activate CD8+T cells
within tumors and the dLN, as well as ac-
tivate NK cells within the dLN. The systemic
administration of SR-717 reduced tumor bur-
den in the B16.F10 melanoma model with
a level of efficacy that was observed to be
superior than what is observed for anti–
PD-1 or anti–PD-L1 therapy in this particular
poorly immunogenic model. Importantly, sys-
temic administration of SR-717 produced
substantial efficacy despite inducing mod-
est levels of IFN-b, suggesting that the
threshold for efficacy in tumor models may
be far lower than previously reported and
can be achieved without considerable tox-
icity. It is also of potential critical impor-
tance that STING activation by SR-717 was
found to induce the expression of PD-L1 in a
STING-dependent fashion. These results have
important implications for the choice of
agent to be combined with a STING agonist,
as well as the relative timing of a dosing reg-
imen, in the context of cancer treatment. Pre-
sumably, it would be unproductive to treat
with an agent that increases the relative
abundance of the target of the second agent.
The ability of SR-717 to induce the cGAMP-
induced closed STING conformation, in con-
trast to open conformation–inducing ligands,
enables exploration of the relative impor-
tance of different potential scaffolding func-
tions in vivo and in the context of systemic
distribution in settings of antitumor immu-
nity and beyond. Differential pathway acti-
vation associated with the recognition of
bacterial-derived CDNs [e.g., di-GMP derived
from commensal bacteria ( 34 )] as compared
with endogenously produced cGAMP, derived
from cytosolic DNA as a result of diverse
pathological events (e.g., genomic instabil-
ity), is readily conceivable and most likely
probable. Each class of agonist may provide
differential therapeutic benefits depending
on the setting.REFERENCES AND NOTES- Q. Chen, L. Sun, Z. J. Chen,Nat. Immunol. 17 ,1142–1149 (2016).
2. M. H. Christensen, S. R. Paludan,Cell. Mol. Immunol. 14 ,4– 13
(2017).
3. K. J. Mackenzieet al.,Nature 548 , 461–465 (2017).
4. S. M. Hardinget al.,Nature 548 , 466–470 (2017).
5. M. De Ceccoet al.,Nature 566 ,73–78 (2019).
6. D. A. Sliteret al.,Nature 561 , 258–262 (2018).
7. M. C. C. Canessoet al.,Mucosal Immunol. 11 ,820– 834
(2018).
8. L. Sun, J. Wu, F. Du, X. Chen, Z. J. Chen,Science 339 , 786– 791
(2013).
9. H. Ishikawa, Z. Ma, G. N. Barber,Nature 461 ,788– 792
(2009).
10. H. Ishikawa, G. N. Barber,Nature 455 , 674–678 (2008).
11. M. B. Fuerteset al.,J. Exp. Med. 208 , 2005– 2016
(2011).
12. S. R. Wooet al.,Immunity 41 , 830–842 (2014).
13. L. Corraleset al.,Cell Rep. 11 , 1018–1030 (2015).
14. K. E. Sivicket al.,Cell Rep. 29 , 785 – 789 (2019).
15. C. Vanpouille-Boxet al.,Nat. Commun. 8 , 15618 (2017).
16. C. Pantelidouet al.,Cancer Discov. 9 , 722–737 (2019).
17. J. Conlonet al.,J. Immunol. 190 , 5216–5225 (2013).
18. P. Gaoet al.,Cell 154 , 748–762 (2013).
19. Q. Yinet al.,Mol. Cell 46 , 735–745 (2012).
20. S. Ouyanget al.,Immunity 36 , 1073–1086 (2012).
21. X. Zhanget al.,Mol. Cell 51 , 226–235 (2013).
22. J. M. Ramanjuluet al.,Nature 564 , 439–443 (2018).
23. M. S. Diamondet al.,J. Exp. Med. 208 , 1989– 2003
(2011).
24. M. A. Curran, W. Montalvo, H. Yagita, J. P. Allison,Proc. Natl.
Acad. Sci. U.S.A. 107 , 4275–4280 (2010).
25. A. Razet al.,Cancer Res. 40 , 1645–1651 (1980).
26. S. Kleffelet al.,Cell 162 , 1242–1256 (2015).
27. O. Demariaet al.,Proc. Natl. Acad. Sci. U.S.A. 112 ,
15408 – 15413 (2015).
28. A. Marcuset al.,Immunity 49 , 754–763.e4 (2018).
29. J. Ahn, S. Son, S. C. Oliveira, G. N. Barber,Cell Rep. 21 ,
3873 – 3884 (2017).
30. A. Garcia-Diazet al.,Cell Rep. 19 , 1189–1201 (2017).
31. T. L. Songet al.,Blood 132 ,1146–1158 (2018).
32. H. Konno, K. Konno, G. N. Barber,Cell 155 , 688– 698
(2013).
33. H. Lemoset al.,J. Immunol. 192 , 5571–5578 (2014).
34. O. Danilchanka, J. J. Mekalanos,Cell 154 , 962– 970
(2013).
ACKNOWLEDGMENTS
We thank A. Theofilopoulos for invaluable discussion and
insight.Author contributions:L.L.L. and E.N.C. conceived of,
initiated, and coordinated the project. L.L.L., E.N.C., J.R.T.,
H.M.P., P.G.S., and A.K.C. contributed to conceptualization.
E.N.C., C.Y., V.F.V., Y.J., M.K., A.M.G.A., W.V., S.A., D.L., N.N.,
L.P., B.B., P.S., F.M.-P., and E.H. conducted research. L.L.L.,
J.R.T., H.M.P., P.G.S., A.C., T.Y., K.J., S.J., and A.K.W. were
involved with project administration. L.L.L., E.N.C., J.R.T., V.F.V.,
H.M.P., P.G.S., C.Y., D.W., and M.Ki. were involved with the
interpretation of data. L.L.L. and E.N.C. wrote the original draft.
L.L.L., E.N.C., J.R.T., V.F.V., and H.M.P. were involved with
review and editing.Competing interests:L.L.L., E.N.C., A.K.C.,
M.Ku., A.M.G.A., H.M.P., P.G.S., C.Y., and W.V. are inventors on
patent application PCT/US2019/018899 submitted by The
Scripps Research Institute that covers small-molecule agonists
of STING.Data and materials availability:All data are
available in the manuscript or in the supplementary materials.
Noncommercial reagents described in this manuscript are
available from L.L.L., H.M.P., or J.R.T. under a material transfer
agreement with The Scripps Research Institute. Coordinates
for the following crystal structure complexes have been
deposited in the RCSB Protein Data Bank: hSTING:SR-717
(PDB ID 6XNP) and mSTING:SR-717 (PDB ID 6XNN).
SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/369/6506/993/suppl/DC1
Materials and Methods
Figs. S1 to S19
Table S1
References
View/request a protocol for this paper fromBio-protocol.24 February 2020; accepted 9 July 2020
10.1126/science.abb4255Chinet al.,Science 369 , 993–999 (2020) 21 August 2020 7of7
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