D, and fig. S10). Combination regimens with
lower MSA-2 levels achievedthesameefficacy
end point with improved tolerability (body
weight) compared with the higher MSA-2
monotherapies required for regression in these
anti–PD-1 refractory models (Fig. 8E). Fur-
ther, combinations of MSA-2 and anti–PD-1
increased tumor CD8+T cell infiltration in
the LL-2 tumor model (Fig. 8F). Moreover, T
cell and immunodeficient mice (NCr nude
and NSG) bearing MC38 tumors exhibited
only partial tumor regression in response to
MSA-2 doses that are typically sufficient to
induce complete regression in C57BL6 mice
(Fig. 8G), despite evident target engagement
(changes in both cytokine levels and body
weight; fig. S11). These observations, com-
bined with the MC38 rechallenge results (Fig.
3K), suggest that both innate and adaptive
immune function contribute to STING agonist–
driven tumor regression. However, further in
vitro and in vivo studies are required to
more fully understand the immunopharma-
cology and toxicology of MSA-2 and related
compounds.
Outlook
We report the identification of an orally avail-
able, non-nucleotide–based STING agonist,
MSA-2, in a high-throughput cell-based pheno-
typic screen. When used as a single agent in
mice, systemically administered MSA-2 in-
duced tumor regression with durable anti-
tumor immunity and was well tolerated. In
mouse tumor models that are poorly responsive
to PD-1 blockade, combinations of MSA-2 with
an anti–PD-1 immune checkpoint inhibitor were
superior to monotherapy in inhibiting tumor
growth and prolonging survival. Experiments
with STING-deficient and immunodeficient
mice showed that the immunogenic and anti-
tumor activities of MSA-2 are mediated by
STING and involve both the innate and adaptive
immune systems. These observations strongly
supporttheconceptthatinductionofhost
adaptive antitumor immunity via pharmaco-
logical activation of STING can overcome tu-
mor resistance to checkpoint inhibitors.
First-generation CDN-based STING ago-
nists currently undergoing clinical trials are
dosed by intratumor injection. A recent study
by Sivicket al.( 9 ) highlighted the challenges
associated with attaining optimum drug levels
by direct intratumor dosing, a critical require-
ment to balance the immunogenic and cell-
ablative effect of STING activation. The ease of
MSA-2 PO administration and the correspond-
ing pharmacokinetic profile allow fine-tuning
of the magnitude of STING agonism in various
tissues in vivo.
Notably, MSA-2 preferentially targets tumor
tissue because of its distinctive mechanism
of action. In-depth molecular mechanism of
action studies revealed that MSA-2 is not an
active ligand of STING but rather a prodrug
of sorts. In solution, monomeric MSA-2 exists
in equilibrium with a noncovalent dimer of
MSA-2, which is a potent STING agonist. In
fact, covalently tethered dimers of MSA-2 ana-logs, another previously unidentified class of
STING agonists, exhibited nanomolar affinity
for STING. Simulations and experimental analy-
ses predicted that MSA-2, a weak acid, would
exhibit substantially higher cellular potencyPanet al.,Science 369 , eaba6098 (2020) 21 August 2020 8of10
Fig. 8. Therapeutic activity of systemic MSA-2 regimens combined with PD-1 blockade in mouse tumor
models.Survival curves of B16F10 (A), LL-2 (B), advanced MC38 (C), and CT-26 (D) tumor-bearing mice treated
with vehicle + Iso. control (anti-mouse IgG1 monoclonal antibody), muDX400 (anti-mouse PD-1 monoclonal
antibody), or MSA-2 at the indicated doses (SC or PO; colored as in Fig. 1) alone or in combination with muDX400
(n= 10 per group). Vehicle was dosed SC or PO and antibodies were dosed intraperitoneally (IP). (E) Tolerability,
illustrated by percentage of body weight change of mice in (C), on day 2 for the indicated doses. (F) Quantification of
CD8+T cell infiltration into LL-2 tumors treated as in (B) (n=4).(G) Tumor volume in T cell–deficient NCr nude
and NSG or immune-competent C57BL6 mice after vehicle or MSA-2 administration (n=10,mean±SEM).Statistical
significance was determined by log-rank Mantel-Cox test for (A) to (D), one-way ANOVA for (E), and unpaired
Student’sttest for (F) and (G). ns, not significant; *P<0.05;**P< 0.01; ***P< 0.001; ****P<0.0001.RESEARCH | RESEARCH ARTICLE