is primarily by passive diffusion of uncharged
MSA-2, we hypothesized that acidification of
the extracellular environment, such as oc-
curs often in tumors ( 22 ), would facilitate
cellular entry and retention of MSA-2, there-
by increasing its intracellular concentration
and enhancing its apparent cellular potency.
Employing the algorithms of Scottet al.( 23 )
and a binding equation based on Model 3
{fraction saturation = [free MSA-2]^2 /(KD+
[free MSA-2]^2 )},wesimulatedthetheoretical
effects of extracellular pH on the intracellular
[MSA-2] and fractional saturation of STING.
The simulations predicted that when the ratio
of cell membrane permeability of uncharged
to charged MSA-2≥50, varying extracellular
pH within the pathophysiological range (6 to
7.5) will have substantial effects on the intra-
cellular concentrations of monomeric and di-
meric MSA-2 (Fig. 7A) and will thereby shift
the cellular potency of MSA-2 (Fig. 7B). This
observation implies that in vivo, systemic
doses of MSA-2 that are insufficient to induce
detectable STING activation in normal tissues
may elicit substantial STING activation in tis-
sues with an acidified microenvironment.
These predictions were validated qualita-
tively by observations that stepwise reductions
of extracellular pH from 7.5 to 6 increased
MSA-2 potency in both THP-1 cells and mouse
macrophages (Fig. 7, C and D). By contrast,
in THP-1 cells the potency of cGAMP, which
is 100% anionic over the same pH range, was
essentially unchanged (Fig. 7E). Direct pH mea-
surement in vivo confirmed that relevant syn-
geneic mouse tumors were more acidic than
nontumor regions (Fig. 7F). Moreover, higher
MSA-2 concentrations were observed in tumors
than in plasma or other nontumor tissues
(Fig.7G)andwereusedtocalculatetheoret-
ical bioactive MSA-2 dimer levels (dotted line).
The increase in proinflammatory cytokine (IFN-b,
IL-6, and TNF-a) levels also trends higher in
tumors than in various nontumor tissues after
administration of MSA-2 by SC or PO routes
(Fig. 7H and fig. S9, B and C).
MSA-2 enhances in vivo antitumor activity
of anti–PD-1 antibody
We also investigated whether MSA-2 could
enhance antitumor activity of anti–PD-1 in
syngeneic tumor models that are either mod-
erately or poorly responsive to PD-1 blockade.
Combinations of systemically administered
MSA-2 with anti–PD-1 were evaluated in four
syngeneic mouse tumor models: advanced
MC38 (colorectal), CT26 (colorectal), B16F10
(melanoma), and LL-2 (lung) tumors. For each
tumor model, one or more combinations of
anti–PD-1 dosed intraperitoneally and MSA-2
(dosed subcutaneously or orally) were found
to be synergistic in inhibiting tumor growth
and prolonging overall survival compared with
the corresponding monotherapy (Fig. 8, A to
Panet al.,Science 369 , eaba6098 (2020) 21 August 2020 7of10
Fig. 7. Simulated and observed effect of extracellular pH on MSA-2–induced STING activation.
(A) Simulated effect of extracellular pH on intracellular concentration of dimeric and monomeric MSA-2 when
total extracellular [free MSA-2] = 3mM. Assumptions: membrane potential =−40 mV, intracellular pH = 7.2,
permeability ratio of uncharged to charged free MSA-2 = 50. (B) Simulated effect of extracellular pH on
relationship between extracellular [free MSA-2] and STING occupancy (KD=100mM^2 ) in cells. Same assumptions
as in (A); see materials and methods. (CandD) Observed effect of extracellular pH on potency of MSA-2 in
stimulating IFN-bsecretion in THP-1 cells and mouse macrophages, respectively. (E) Observed effect of
extracellular pH on cGAMP potency in THP-1 cells. (F) In vivo pH measurements from tumors (solid circles)
and contralateral skin area (open circles) (n= 9 to 11) in three different syngeneic tumor models. (G)Time
course of MSA-2 concentrations in various tissues after a single SC dose of 50 mg/kg to MC38 tumor-bearing
C57BL6 mice (n= 3). Data were collected from the same experiment depicted in Fig. 3, B and C. Simulated
upper limit for bioactive [dimeric MSA-2] in tumors (dotted line) calculated using Model 3 (Eq. 3 from Fig. 5C).
(H)IFN-blevels in various tissues of MC38 tumor-bearing C57BL6 mice (n= 5, mean ± SD) 4 hours after
the indicated doses of MSA-2 by SC or PO administration. Statistical significance was determined by pairedttest
(F) or one-way ANOVA [(G) and (H)]. *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001.RESEARCH | RESEARCH ARTICLE