Structural Characterization of the ICOS/ICOS-L Immune Complex Reveals High Molecular Mimicry by Therapeutic Antibodies

ARTICLE
S uc u al cha ac e iza ion o he ICOS/ICOS-L
immune complex e eals high molecula mimic y
by he apeu ic an ibodies
Edu ne Rujas1,2, Hong Cui1, Taylo Sica d1,3, An hony Semesi1& Jean-Philippe Julien 1,3,4✉
The inducible co-s imula o (ICOS) is a membe o he CD28/B7 supe amily, and deli e s a
posi i e co-s imula o y signal o ac i a ed T cells upon binding o i s ligand (ICOS-L). Dys-
egula ion o his pa hway has been implica ed in au oimmune diseases and cance , and is
cu en ly unde clinical in es iga ion as an immune checkpoin blockade. He e, we desc ibe
he molecula in e ac ions o he ICOS/ICOS-L immune complex a 3.3 A esolu ion. A
cen al FDPPPF mo i and esidues wi hin he CC’loop o ICOS a e esponsible o he
specifici y o he in e ac ion wi h ICOS-L, wi h a dis inc ecep o binding o ien a ion in
compa ison o o he amily membe s. Fu he mo e, ou s uc u e and binding da a e eal ha
he ICOS N110 N-linked glycan pa icipa es in ICOS-L binding. In addi ion, we epo c ys al
s uc u es o ICOS and ICOS-L in complex wi h monoclonal an ibodies unde clinical e a-
lua ion in immuno he apy. S ikingly, an ibody pa a opes closely mimic ecep o -ligand
binding co e in e ac ions, in addi ion o con ac ing pe iphe al esidues o con e high binding
a fini ies. Ou esul s unco e key molecula in e ac ions o an immune complex cen al o
human adap i e immuni y and ha e di ec implica ions o he ongoing de elopmen o
he apeu ic in e en ions a ge ing immune checkpoin ecep o s.
h ps://doi.o g/10.1038/s41467-020-18828-4 OPEN
1P og am in Molecula Medicine, The Hospi al o Sick Child en Resea ch Ins i u e, To on o, ON M5G 0A4, Canada. 2Biofisika Ins i u e (CSIC, UPV/EHU)
and Depa men o Biochemis y and Molecula Biology, Uni e si y o he Basque Coun y (UPV/EHU), P.O. Box 644, 48080 Bilbao, Spain. 3Depa men o
Biochemis y, Uni e si y o To on o, To on o, ON M5S 1A8, Canada. 4Depa men o Immunology, Uni e si y o To on o, To on o, ON M5S 1A8, Canada.
✉email: [email p o ec ed]
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Pa hogenic in ec ions igge a se ies o highly egula ed
e en s o ches a ed by he immune sys em o limi hos
damage and p o ide long- e m p o ec ion. Upon encoun e
o o eign an igens, an igen-p esen ing cells (APCs) display spe-
cific pep ide majo his ocompa ibili y complexes (MHC) on hei
su ace ha can be ecognized by he T-cell ecep o s (TCR) o
naï e an igen-specific T cells. Howe e , his fi s signal is no
su ficien o success ul ac i a ion o T cells1, and a second
syne gis ic signal p o ided by in e ac ions o co-s imula o y
ecep o s on T cells is equi ed wi h hei cogna e ligands in
APCs2.
A key molecule ha p o ides his seconda y signal and hence
dic a es T cell a e is he inducible T-cell co-s imula o (ICOS)3
and i s unique ligand (ICOS-L) on APCs4,5. ICOS (CD278) is a
ype I ansmemb ane glycop o ein ha belongs o he CD28
amily o co-s imula o y immuno ecep o s. I is p esen on he T
cell su ace as a disulfide bond-linked homodime 3and i is
apidly up egula ed upon TCR c oss-linking and/o CD28 co-
s imula ion3,5,6. On he o he hand, ICOS-L (CD275) belongs o
he B7 amily and is exp essed on he su ace o APCs5,7,8and
non-hema opoie ic cells unde inflamma o y condi ions9–12.
Binding o ICOS-L o ICOS igge s dis inc in acellula signal-
ing cascades h ough he conse ed mo i s YMFM13, IP ox14, and
KKKY15 in he ICOS cy oplasmic ail. These signaling pa hways
deli e he co-s imula o y signals ha p omo e T cell ac i a ion
and di e en ia ion.
Mul iple s udies suppo a mul i ace ed unc ion o ICOS/
ICOS-L and hence a complex ole in dic a ing he cou se o
adap i e immuni y. Indi iduals wi h null mu a ions in he ICOS
gene16 and ICOS-deficien animal models17–19 exhibi a p o ound
de ec in humo al esponses due o he lack o T ollicula helpe
(T h) cells, a specialized CD4+T cell subse essen ial o ge minal
cen e (GC) o ma ion20. In addi ion o egula ing hymus-
dependen (TD) Ab esponses, ICOS also a ec s Th1, Th2, and
Th17 immuni y21,22 and he homeos asis o egula o y T cells
(T eg)23. The exp ession o ICOS has also been epo ed in inna e
lymphoid cells (ILCs), which expands he ole o his ecep o o
he inna e a m o he immune sys em24. This mul iplici y o oles
unde pins he ele ance o he ICOS/ICOS-L signaling pa hway
and in u n, he emendous po en ial o manipula ing his co-
s imula o y signal in he de elopmen o cance immuno he apies
and in he ea men o au oimmune diseases.
Va ious s udies ha e demons a ed ha an i- umo T cell
esponses in mice can be significan ly boos ed by combining
cy o oxic T lymphocy e-associa ed an igen 4 (CTLA-4) blockade
wi h ICOS engagemen 25,26. In addi ion, eme ging e idence
sugges s ha ICOS blockade holds p omise o he ea men o
inflamma o y diseases such as alle gic as hma24. An i-ICOS
monoclonal an ibody (mAb) he apy ha blocks ICOS signaling
has p o en beneficial in he ansplan field by inducing ole ance
ollowing ca diac allog a in a s27–29. As a consequence, he
numbe o an ibodies a ge ing he ICOS/ICOS-L immune
complex en e ing clinical ials is apidly inc easing. In pa icula ,
he humanized monoclonal an i ICOS-L an ibody p ezalumab
has ecen ly shown e ficacy in he ea men o pa ien s wi h
sys emic lupus e y hema osus (SLE) in a phase Ib clinical ial30.
Despi e he impo ance o he ICOS/ICOS-L in e ac ion in
modula ing many aspec s o adap i e immuni y and he g owing
e idence o he benefi s o a ge ing his immune complex o
he apeu ic in e en ions, he molecula de ails o how he
ex acellula domains o his ecep o /ligand pai in e ac and
how leading he apeu ic an ibodies ecognize hei a ge s emain
elusi e.
He e, we epo he c ys al s uc u e o he co-complex
be ween human ICOS and i s ligand ICOS-L a 3.3 Å esolu-
ion, which e eals he molecula de ails o immune ecep o
specifici y. Fu he mo e, we desc ibe he s uc u al basis o
he in e ac ions o wo he apeu ic an ibodies wi h ICOS
and ICOS-L, espec i ely. Toge he , ou s uc u al cha ac e iza-
ions unco e he molecula bluep in s o he ICOS/ICOS-L
in e ac ion and a de ailed iew o molecula mimic y achie ed by
he apeu ic an ibodies o a ge he ICOS/ICOS-L signaling axis
in immuno he apy.
Resul s
S uc u e o he ICOS/ICOS-L complex. We de e mined he
molecula basis o he co-s imula o y signal p o ided by ICOS
(Fig. 1a) by sol ing he h ee-dimensional s uc u e o med by he
ex acellula domains o ICOS ( esidues 21–129) and i s ligand
ICOS-L ( esidues 19–248) (Fig. 1b) a 3.3 Å esolu ion by x- ay
c ys allog aphy (Table 1). T unca ion o ICOS a esidue 129 and
he e o e be o e C136, abolished he o ma ion o disulfide-linked
homodime s and was equi ed o ob ain well-di ac ing c ys als.
The c ys al s uc u e e eals ha ICOS adop s he p edic ed
single immunoglobulin (Ig) a iable (V- ype) domain a chi-
ec u e. ICOS-L is o ganized in wo dis inc domains: he mos
apical domain (D1) adop s a V- ype old, whe eas he memb ane-
p oximal domain (D2) adop s a C1- ype old (Fig. 1c).
ICOS/ICOS-L in e ac in a 1:1 ecep o -ligand s oichiome y.
The main binding in e ace is o med by he FDPPPFK mo i
(amino acids 114–120) loca ed in he ICOS FG loop, which
in e ac s wi h esidues om s ands C and C’and loops CC’and
C’D o ICOS-L. These esidues o m a ne wo k o in e ac ions
ha include H-bonds and a oma ic s acking (Fig. 1d, op). Single
alanine subs i u ion o esidues Q50, F114, and F119 d as ically
impac ed he binding o ICOS o ICOS-L o almos unde ec able
le els (Fig. 1e), confi ming he c i ical ole o his in e ace o
ecep o binding as p e iously epo ed31.
ICOS con ains h ee pu a i e N-linked glycosyla ion si es.
Clea elec on densi y was obse ed in he co-complex c ys al
s uc u e o wo N-ace yl glucosamine (GlcNAc) and h ee
mannose esidues o he N-linked glycan a posi ion N110.
Unexpec edly, ICOS-L esidues F122, Q123, and E124 o m H-
bonds wi h his ICOS glycan, which bu ies ~300 Å2o su ace
a ea on ICOS-L (Fig. 1d, bo om). Si e-di ec ed mu agenesis o
knock ou his N-linked glycosyla ion si e (N110Q) in ICOS led
o a as e on- a e and a sligh ly slowe o - a e esul ing in a 4.3-
old imp o emen in binding a fini y o ICOS-L (Fig. 1 ). This
esul sugges s ha he ICOS N110 glycan s e ically ga es ICOS-L
binding and ha he bu ied su ace a ea on ICOS-L ep esen s
me e accommoda ion o he o he wise encumbe ing ICOS glycan
a he binding in e ace.
Compa ison o ICOS and ICOS-L o o he CD28 and B7 amily
membe s. Supe posi ion o he IgV domains o ICOS-L, B7-1,
and p og ammed cell dea h ligand-1 (PDL-1) e ealed ha he
angle o app oach o ICOS o i s ligand di e s conside ably om
ha obse ed in ela ed amily membe s (Fig. 2a). In he CTLA-4/
B7 and PD1/PD-L1 complexes, he ecep o s c oss he IgV
domains o hei cogna e ligands a ~110° and 90°, espec i ely.
Howe e , in he ICOS/ICOS-L complex, he IgV domains c oss a
150° (Fig. 2b).
These di e ences in disposi ion be ween he complexes a e due
o hei dis inc ecep o -ligand binding in e aces. Despi e low
sequence iden i y (~20%), he o e all s uc u e o ICOS and
ICOS-L sha e many simila i ies wi h membe s o hei espec i e
amilies (Fig. 3). Compa ison o ICOS wi h CTLA-4 and CD28
e eal some deg ee o s uc u al homology wi h a oo mean
squa e de ia ion ( .m.s.d) o backbone a oms o 3.2 and 4.5 A,
espec i ely, and a lowe s uc u al simila i y wi h PD1 ( .m.s.d o
5.9 A). No ably, he FDPPPF sequence in ICOS is analogous and
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ema kably s uc u ally conse ed o he in a ian mo i
MYPPPY p esen in he IgV domains o CD2832 and CTLA-
433,34 (backbone .m.s.d o 0.45 and 0.39 A, espec i ely). The
side chains o esidues flanking he h ee cen al p olines a e
simila ly o ien ed in all s uc u es as a esul o he cis- ans-cis
a angemen o p olines. In con as , he analogue mo i
LAPKAQ in PD1 is no conse ed ei he in sequence o in
s uc u e (Supplemen a y Fig. 1). O e all hese mo i s con ibu e
e
WT N110Q
0.0
5.0×10–9
1.0×10–8
1.5×10–8 1×106
8×105
6×105
4×105
2×105
4×10–3
3×10–3
2×10–3
1×10–3
ICOS
KD (M)
WT N110Q
0
ICOS
Kon (M–1s–1)
WT N110Q
0
ICOS
Ko (s–1)
0 100 200 300
0.0
0.1
0.2
0.3
Response (nm)
ICOS WT
0 100 200 300
0.0
0.1
0.2
0.3
Time (s)
Response (nm)
ICOS F114A
0 100 200 300
0.0
0.1
0.2
0.3
ICOS Q50A
0 100 200 300
0.0
0.1
0.2
0.3
Time (s)
ICOS F119A
T-cell
ICOSTCR
MHC
APC
ICOS-L
cd
ab
Fig. 1 Th ee-dimensional s uc u e o he human ICOS/ICOS-L complex. a Schema ic ep esen a ion o he ICOS/ICOS-L co-s imula o y in e ac ion.
An igen-p esen ing cell (APC), T-cell ecep o (TCR), Majo His ocompa ibili y Complex (MHC), Inducible Co-s imula o (ICOS), Inducible Co-s imula o
ligand (ICOS-L). bDomain o ganiza ion o ICOS and ICOS-L; signal pep ide (SP), ex acellula domains (D1-D2), ansmemb ane (TM) domain, and
cy oplasmic ail (CT). Disulfide bonding pa e n is shown wi h ed lines. Blue a ows indica e he leng h o he c ys alized ec odomains. cSeconda y
s uc u e ca oon ep esen a ion o he side iew o he ICOS/ICOS-L complex. ICOS (deep eal, anspa en su ace) adop s a V- ype Ig old while ICOS-L
adop s a V- ype Ig old (whea ) ollowed by a C1- ype Ig domain old (o ange). The helices a e shown in ligh pink. dDe ailed iew o he ecep o -ligand
in e ace. Top: he 114FDPPF119 mo i in he FG loop o ICOS o ms key hyd ophobic in e ac ions wi h a oma ic esidues om ICOS-L shown in s ick
ep esen a ion. Hyd ogen bonds be ween ICOS and ICOS-L a e shown as black dashed lines. Bo om: The blue mesh a ound he N110 glycan depic ed as
s icks ep esen a composi e omi elec on densi y map con ou ed a 1.0 sigma. Colo s a e as in (c). eKine ic binding cu es o ICOS-L o ICOS WT and
ICOS mu an s. Mu a ion o esidues Q50, F114, and F119 abolished binding in BLI. Compa ison o he binding a fini y (K
D
), associa ion (on), and
dissocia ion (k
o
) a es o ICOS-L o WT ICOS and o ICOS N110 glycan knock-ou mu an (N110Q). The mean alues and s anda d de ia ion o h ee
biological eplica es a e shown.
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400 A2(64%), 360 A2(55%), 480 A2(71%), and 230 A2(28%) o
su ace a ea o he binding in e ace o ICOS/ICOS-L, CTLA-4/
B7-1, CTLA-4/B7-2, and PD1/PDL-1, espec i ely.
A unique ea u e o ICOS, howe e , is i s unusually long
s and C’ ha p o udes om he su ace o he ec odomain
leading o he o ma ion o a ligand in e ac ion no ye obse ed
in any o he p e iously sol ed CD28/B7 amily complexes.
Specifically, binding o esidues
66
TKTKGS
71
loca ed in he
ICOS elonga ed s and C’ o ICOS-L bu ies 130 A2o su ace
a ea, accoun ing o ~20% o he o al BSA. Hence, al hough
he FDPPPF mo i cons i u es he co e o he ICOS/ICOS-L
in e ace, esidues TKTKGS in s and C’ oge he wi h esidues
Q50, K52 in s and C con ibu e o he binding specifici y o
ICOS (Fig. 3a).
Alignmen o he B7 amily p o eins wi h ICOS-L show high
s uc u al homology, wi h backbone .m.s.d. anging om 1.1 o
1.6 A. Unsu p isingly, he p ima y s uc u al di e ence obse ed
in ICOS-L wi h amily membe s is ound in he con o ma ion o
loop C’D, which con ac s s and C’o ICOS (Fig. 3b). As a esul ,
he binding in e ace o ICOS-L is shi ed owa d he edge o he
AGFCC’shee and in ol es esidues om s ands F (L114, L116)
and C (Y51, Y53, Q55), as well as loops CC’(K60, V62), C’D (I67,
Q69, N75) and FG (Q118, G121, F122). Consis en ly, mu a ions
in any o hese esidues we e p e iously epo ed o esul in a
subs an ial loss o ecep o binding35.
Oligome ic engagemen o he ICOS/ICOS-L immune com-
plex. Due o he abili y o ICOS o o m disulfide-linked homo-
dime s3and he impo ance o su ace oligome iza ion o igge
i s co-s imula o y signal, we measu ed binding a fini y and
binding a idi y o he ICOS/ICOS-L complex by biolaye in e -
e ome y (BLI) (Fig. 4). Binding o he ICOS-L ec odomain
monome o he ICOS ec odomain immobilized as an a ay on he
biosenso s showed a ela i ely low binding a fini y (K
D
=722 nM)
and e y apid o - a es (K
o
=1.6 × 10−1s−1) (Fig. 4, op panel
and Supplemen a y Table 1). In con as , by flipping he o ien-
a ion o he in e ac ion and measu ing binding a idi y o ICOS
disulfide-linked homodime s o immobilized ICOS-L a ayed on
he biosenso , wo o de s o magni ude di e ence in he appa en
binding a fini y cons an (K
D
=10 nM) was ob ained (Fig. 4,
bo om panel and Supplemen a y Table 1). Such high a idi y
migh eflec clus e ing and oligome iza ion o ligands and
ecep o s a he cell su ace upon T-cell ac i a ion and is in
ag eemen wi h he p opensi y o ICOS and ICOS-L o o m
homodime s a he cell memb ane3,35.
Ou molecula da a p o ide pa ial insigh in o he oligome ic
assembly o ICOS and ICOS-L. Indeed, in he absence o an
in e chain disulfide bond, ICOS exis s as a monome in solu ion
(Supplemen a y Fig. 2a). In ou c ys al s uc u e whe e he ICOS
in e chain disulfide bond has been unca ed, we obse e an
ICOS/ICOS-L in e ace ha bu ies ~420 Å2o BSA (Supplemen-
a y Fig. 2b). This in e ac ion could be indica i e o an ICOS
dime ic in e ace p esen a he cell memb ane; howe e , in his
c ys allog aphic a angemen he ICOS p o ome s a e o ien ed in
a head o ail ashion wi h he C- e minal ails (whe e he cys eine
esidues would media e pu a i e disulfide bonds) poin ing away
om each o he (Supplemen a y Fig. 2c). As such, in he absence
o unc ional da a suppo ing his dime ic in e ace, we p opose
ha he an ipa allel ICOS dime a angemen obse ed in he
c ys al la ice is likely an a e ac o c ys al packing. Al oge he ,
Table 1 Da a collec ion and efinemen s a is ics.
ICOS/ICOSL/VNAR ICOSL/P ezalumab/VNAR ICOS/STIM003/an i-kappa V
H
H
Da a collec ion
Wa eleng h (Å) 1.00000 1.03316 0.97934
Space g oup P4(1)2(1)2 P2(1) C2
Cell dimensions
a,b,c (Å) 104.0, 104.0, 123.1 68.5, 152, 86.7 171.8, 49.0, 91.8
α,β,ɣ(○) 90, 90, 90 90, 104.1, 90 90, 101.9, 90
Resolu ion (Å) 40–3.30 (3.40–3.30) 40–3.15 (3.25–3.15) 40–2.38 (2.48–2.38)
No. molecules in ASU 1 2 1
No. o al eflec ions 128,986 (4688) 209,889 (9398) 201,759 (20,592)
No. unique eflec ions 10,227 (406) 29,746 (1378) 30,150 (3223)
Mul iplici y 11.8 (12.2) 7.0 (7.0) 6.6 (6.0)
Rme ge (%) 26.9 (78.6) 16.8 (67.0) 10.9 (60.8)
Rpim (%) 7.9 (23.0) 6.8 (27.4) 4.5 (25.8)
<I/σI> 10.0 (1.7) 9.4 (1.7) 13.3 (2.1)
CC
1/2
99.3 (62.8) 99.4 (76.2) 99.7 (73.2)
Comple eness (%) 95.7 (97.9) 99.9 (100) 99.2 (94.0)
Refinemen
Non-hyd ogen a oms 3513 11,774 5245
Mac omolecule 3301 11,580 5072
Sol en –– 153
R
wo k
/R
ee
0.224/0.267 0.201/0.256 0.203/0.260
Rms des ia ions
Bond lengh s (Å) 0.01 0.004 0.004
Bond angles (○) 1.14 0.81 0.72
Ramachand an plo
Fa o ed egions (%) 98.1 95.2 97.7
Allowed egions (%) 1.4 4.7 2.3
B- ac o s (Å2)
Wilson B- ac o 86.6 76.7 45.7
A e age B- ac o s 85.8 81.3 49.6
A e age mac omolecule 83.9 80.8 49.6
A e age sol en –– 44.6
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ou da a sugges ha ICOS dime iza ion a he cell su ace
p obably in ol es a limi ed dime ic in e ace o he ec odomain
and ha he ICOS ec odomain is mainly linked h ough he
disulfide bond a he C- e minal ail nea he ansmemb ane
egion (Supplemen a y Fig. 2d).
The apeu ic an ibodies can compe e wi h he ICOS/ICOS-L
in e ac ion. Monoclonal an ibodies STIM003 and p ezalumab
a ge ICOS and ICOS-L, espec i ely and a e cu en ly unde
clinical e alua ion. Using BLI, we confi med ha binding o hese
an ibodies o hei espec i e ligands p e en s he o ma ion o
he ICOS/ICOS-L immune complex (Fig. 5a). To elucida e he
molecula basis o ligand blockade by he an ibodies, we sol ed
he c ys al s uc u es o ICOS/STIM003 Fab and ICOS-L/p e-
zalumab Fab complexes a 2.38 and 3.15 A esolu ion, espec i ely
(Table 1).
Supe posi ion o he ICOS s uc u e, when c ys allized in
complex wi h i s na u al ligand and in complex wi h STIM003,
showed high s uc u al simila i y (backbone .m.s.d o 0.90 A).
The binding in e ace o ICOS in he an ibody complex was
s ikingly simila o he one when bound o i s na u al ligand
(Fig. 5b). In bo h cases, binding is concen a ed in he FG loop o
ICOS. Howe e , he o al bu ied su ace a ea o ICOS is sligh ly
highe o he STIM003 in e ac ion in compa ison o he
in e ac ion wi h ICOS-L (800 o 620 A2, espec i ely), due o
addi ional in e ac ions o he an ibody wi h he C’C”loop and
he C”s and. Despi e he high simila i y o he con ac ed ICOS
su ace, ICOS-L and STIM003 app oach he ecep o wi h
di e en angles o app oach (Fig. 5b). Consequen ly, he ICOS
N110 glycan minimally in e e es wi h binding o STIM003
(Supplemen a y Fig. 3 and Supplemen a y Table 1).
The c ys al s uc u e o he ICOS-L/p ezalumab Fab complex
was ob ained using a sha k Va iable New An igen Recep o
(VNAR) Single Domain36 as a c ys allog aphy chape one
(Supplemen a y Fig. 4a). The c ys al s uc u e e ealed ha ICOS
and p ezalumab bind ICOS-L wi h a simila angle o app oach,
and ha he ICOS IgV domain is in a highly simila con o ma ion
in bo h complexes (backbone .m.s.d o 1.0 A). Bo h molecules
in e ac wi h an o e lapping su ace on ICOS-L; howe e ,
p ezalumab makes addi ional con ac s wi h s and F and loop
C’D o ICOS-L upon binding, leading o a highe BSA compa ed
o he na u al complex (900 A2 s. 580 A2, espec i ely) (Fig. 5c).
As a esul o he ex ended in e ace, p ezalumab binding is
p oximal o N70 (Fig. 6a), a p edic ed N-linked glycosyla ion si e.
Howe e , he c ys al s uc u e e eals almos no in e ac ion wi h
N70 o he N-linked glycan (BSA o 10 and 60 A2, espec i ely)
he e o e sugges ing ha binding o p ezalumab o ICOS-L is
la gely independen o N-glycosyla ion. No ably, he angle
be ween he ICOS-L IgV and IgC domains in he ICOS/ICOS-L
and ICOS-L/p ezalumab Fab c ys al s uc u es di e s by ~14°,
indica ing flexibili y ha can be a ibu ed o a la gely flexible
linke and minimal in e ac ions be ween he wo ICOS-L Ig
domains (Supplemen a y Fig. 4b).
An ibody mimic y o he ICOS/ICOS-L in e ac ion. Rema k-
ably, esidues a he co e o he ICOS/ICOS-L in e ac ion a e also
cen al o he an ibody complexes (ICOS/STIM003 and ICOS-L/
p ezalumab) (Fig. 6a). P ezalumab uses he hea y chain com-
plemen a y de e mining egion (CDR) 3 (HCDR3) o mimic he
cen al ICOS FG loop ha engages wi h ICOS-L. On he o he
hand, HCDR3, LCDR1, and LCDR2 esidues o STIM003
esemble he hyd ophobic esidues o he ICOS-L on shee ha
d i e he in e ac ion wi h ICOS. A key ea u e o he an ibody-
an igen in e ac ions is ha in addi ion o cen al hyd ophobic
con ac s ha esemble he na u al ligands, addi ional hyd ogen
bonds and sal b idges o m in he pe iphe y (Fig. 6b). Indeed,
only fi e hyd ogen bonds a e o med a he binding in e ace
be ween ICOS and ICOS-L. In con as , en hyd ogen bonds and
b
a
Fig. 2 Angle o ligand- ecep o in e ac ions o CTLA-4/B7 amily
membe s. a Top iew ca oon ep esen a ion o ICOS/ICOS-L and
p e iously sol ed CTLA-4/B7-133 and PD1/PDL-169 complexes. The
o ien a ion o he complexes a e based on he s uc u al alignmen o he
ligands (di e en shades o b own). Recep o s a e shown as shades o
g een. S uc u al ea u es a e labeled o help wi h o ien a ion. bAngle o
app oach o he h ee ecep o s o i s ligands calcula ed using Pymol70.
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one sal b idge (ICOS/STIM003), and se en hyd ogen bonds
(ICOS-L/p ezalumab) a e pa o he an ibody-an igen binding
in e aces. These addi ional in e ac ions a e concen a ed in he
enla ged a eas o he an ibody in e aces ha is sol en accessible
in he ICOS/ICOS-L complex (Fig. 5) and a e p esumably
esponsible o he highe binding a fini y o he an ibodies
compa ed o he na u al ligands (Fig. 6c and Supplemen a y
Table 1). No ewo hy, he mimic y o hese wo an ibodies
compa ed o hei cogna e ecep o s is highe han any o he
he apeu ic an ibodies a ge ing ecep o s/ligands in he same
amily o which he h ee-dimensional s uc u e has been
epo ed (Fig. 6d and Supplemen a y Fig. 5).
Discussion
T cell ac i a ion is media ed by he coo dina ed in e play o a
complex ne wo k o ansmemb ane co-s imula o y and co-
inhibi o y ecep o /ligand pai s. When dys egula ed, T cell
esponses can esul in issue damage leading o au oimmuni y
o cance . Thus, modula ion o hese molecula complexes o
ep ess o enhance immune esponses is a powe ul s a egy in
immuno he apy. Impo an examples a e p o ided by he
ema kable imp o emen s in disease ou come ob ained wi h
an ibodies a ge ing CTLA-4 and PD137–40. ICOS, a membe o
his amily, has eme ged as a p omising a ge o immuno he -
apy24–30 because o i s cen al ole in he T/B-cell co-signaling
pa hway3associa ed wi h adap i e and inna e immuni y21,41.
Ou c ys al s uc u e o he ICOS/ICOS-L complex e ealed
ha ICOS adop s a p edic ed35 o e all Ig- old s uc u e simila o
CTLA-4 and CD2842. These h ee su ace ecep o s u ilize a
cen al PPP mo i disposed in a high-ene gy cis- ans-cis con-
o ma ion ha is flanked by a oma ic esidues o engage hei
cogna e ligands32–34. Howe e , we show ha unlike CTLA-4/
CD28, ICOS u ilizes a second se o esidues wi hin i s CC’loop
o con ibu e a conside able ac ion o he con ac s likely
esponsible o i s binding specifici y o ICOS-L. In e es ingly,
iden ifica ion o his second binding in e ace o e s an oppo -
uni y o guide loop g a ing s a egies o he design o soluble
ligands wi h c oss- eac i i y be ween ecep o s. In line wi h his
idea, simul aneous blockade o he co-s imula o y ecep o s ICOS
and CD28 ha e been e ec i e in p olonging ca diac allog a
ICOS
CTLA-4
PD1
ICOSL
B7–1
B7–2
PD1–1
b
a
Fig. 3 Specifici y o he ICOS and ICOS-L in e ac ion. Sequence alignmen o (a), ICOS wi h CTLA-4 and PD1 and b, ICOS-L wi h B7-1, B7-2, and PDL-1.
In a ian esidues (as e isk), and esidues wi h highly (colon) and weakly simila p ope ies (do ) a e indica ed. S ands and αhelixes a e deno ed by
a ows and cylinde s, espec i ely. Seconda y s uc u e ca oon ep esen a ion o (a), he V- ype domains o ICOS in compa ison wi h CTLA-4 (PDB IDs:
1I8L and 1I85) and PD1 (PDB ID: 4ZQK) and (b), ICOS-L in compa ison wi h B7-1 (PDB ID: 1I8L), B7-2 (PDB ID: 1I85) and PDL-1 (PDB ID: 4ZQK). Backbone
.m.s.d. be ween ICOS and ICOS-L and s uc u es o o he amily membe s is shown in pa en heses and we e calcula ed in Pymol70. Residues a e shown as
su ace and colo ed acco ding o hei BSA. A eas wi h highe and lowe BSA in ICOS and ICOS-L wi h espec o he o he amily membe s a e boxed in
ed and blue, espec i ely. The o al BSA o each ec odomain is indica ed.
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su i al in a s28. The e o e, we specula e ha such c oss- eac i e
and bipa a opic molecules could show beneficial he apeu ic
p ope ies o se e al disease indica ions, including allog a
ansplan a ion, au oimmune diseases, and cance .
Mono alen binding o ICOS o ICOS-L showed low a fini y
and as binding kine ics, while bi alen binding esul ed in a wo-
o de s o magni ude s onge binding kine ics. Rapid o -
a es migh be impo an o allow T h-cell mo ili y in o he B cell
zone in he GC h ough a dynamic o ma ion and dis up ion o T
cell-APC con ac s acili a ing as an igen scanning. This p ocess
is go e ned by ICOS/ICOS-L in e ac ion in a TCR-independen
manne 43. Howe e , e ficien co-s imula o y signaling elies on
a idi y o he o ma ion o oligome ic s uc u es wi h high s a-
bili y a he immunological synapse. In e es ingly, his depen-
dence on binding a idi y obse ed o ICOS has been p e iously
epo ed o CTLA-4 homodime s, bu no o CD28 homo-
dime s44. Indeed, CTLA-4 and B7-1 can o m a zippe -like oli-
gome iza ion o disulfide-linked CTLA-4 homodime s and B7-1
homodime s33. On he con a y, CD28 homodime s a e incom-
pa ible wi h he simul aneous binding o wo B7-1 molecules due
o clashes o he memb ane-p oximal domain o he ligand42. Ou
c ys al s uc u e o he ICOS/ICOS-L monome ic complex did
no allow o define a biologically ele an dime in e ace o he
ICOS ec odomain; indeed, a mu a ion emo ing he in e chain
disulfide bond in ICOS was equi ed o ob ain well-di ac ing
c ys als, esul ing in he ICOS ec odomain being monome ic in
solu ion and limi ed in o ma ion could be de i ed om he
c ys al packing in e aces. Simila ly, monome ic o ms o CTLA-
4 in solu ion ha e been epo ed in he absence o he disulfide
linkage32,45. This obse a ion, oge he wi h he capaci y o ICOS
and CTLA-4 o simul aneously bind wo ligand molecules, sug-
ges ha ICOS and ICOS-L may le e age a pe iodic a angemen
o ecep o -ligand complexes a he T-cell-APC in e ace simila
o he one epo ed o he CTLA-4/B7-1 complex. Howe e ,
u u e s udies will be equi ed o p ecisely define he oligome ic
assembly o hese molecules a he immune synapse.
A no able ea u e unco e ed by ou ICOS/ICOS-L co-complex
s uc u e is he p esence o ICOS N-linked glycan N110 a he
binding in e ace. The N110Q mu a ion emo ing his N-linked
glycan esul ed in a 4.3- old imp o emen in binding a fini y o
ICOS o ICOS-L. Such a ole o N-linked glycosyla ion mod-
ula ing ecep o binding was also epo ed in p e ious s udies
whe e deglycosyla ion o CD28 was ound o enhance binding o
CD80 on APC46, and a hypoglycosyla ed o m o B7-2 showed
educed binding o CD28 and CTLA-447. Impo an ly, abno mal
glycosyla ion o cell su ace p o eins can influence signaling
pa hways implica ed in cell su i al and g ow h-p omo ing se -
e al diso de s, including cance 48. Fo example, a ecen s udy
epo ed ha N-glycan modifica ion o PD-L1 on iple-nega i e
b eas cance cells was essen ial o PD-1 in e ac ion and he e-
o e T cell exhaus ion49. I emains o be de e mined how ICOS
glycosyla ion is impac ed in dys egula ed cells, and how such
modifica ions in he p esence and composi ion o he N110 gly-
can may impac disease p og ession. None heless, ou findings
con ibu e u he e idence o pos - ansla ion modifica ions, and
pa icula ly glycobiology in modula ing binding h esholds, in
his case ele an o T cell ac i a ion.
The apeu ic an ibodies wi h an an agonis ic mode o ac ion
o en compe e wi h na u al ligands50, and hei e ficacy is linked
o he ex en o epi ope o e lap wi h he na u al ligand oo p in .
Ye , s e ic o e lap is a ely achie ed by exac mimic y o he
na i e molecula in e ac ions. S ikingly, ou c ys allog aphic
s udies e ealed ha wo he apeu ic an ibodies, STIM003 and
p ezalumab, con ac almos all esidues in ol ed in he ICOS/
ICOS-L immune complex in e ac ion. Compa ison o o he
he apeu ic an ibody-ligand s uc u es in he checkpoin blockade
amily (PD1, PDL-1, and CTLA-4) e ealed ha he le el o
mimic y obse ed o STIM003 and p ezalumab is ema kable.
Acco dingly, ou s uc u al da a sugges ha he epo ed
an agonis ic e ec o p ezalumab51 is likely a ibu ed o e ficien
ou compe ing ICOS o binding o ICOS-L, in addi ion o pos-
sible s e ic hind ance ha block ecep o -ligand clus e ing a he
memb ane su ace. The le el o mimic y achie ed by he ICOS
and ICOS-L he apeu ic an ibodies desc ibed he e app oaches
some o he p e ious epo s in he field o in ec ious diseases
whe e an ibodies agains i al en elope p o eins p ecisely a ge
specific conse ed ecep o -in e ac ing esidues o a oid i al
escape52–55. The molecula p inciples de i ed om ou s uc u es
will con inue o in o m de no o s uc u e-based s a egies o he
design o biologics ha showcase na u al mimic y, as also
ecen ly exemplified by he design o selec i e mimics o IL-2 and
IL-1556.
A de ailed analysis o he binding in e ace o ICOS/STIM003
and ICOS-L/p ezalumab also e ealed a sligh ly la ge oo p in
compa ed o he binding in e ace o he ecep o -ligand pai , and
he p esence o addi ional hyd ogen bonds and sal b idges in he
pe iphe y o he co e in e ac ion. In he BioMu a sequence
da abase o cance pa ien s57, eigh single nucleo ide a ia ions
(SNVs) compiled in he ICOS sequence (S76P, G70R, N73Y,
K78N, F114V, P116S, P116H, and P117A) and wo in he ICOS-L
sequence (Y51C and Y65H) we e iden ified as an ibody-con ac
esidues (Supplemen a y Fig. 6a). How hese SNVs will impac
he apeu ic esponses is an a ea o u u e in es iga ion now
possible om he p ecise delinea ion o hese an ibody epi opes.
Toge he , ou da a p o ide c i ical knowledge o be e
unde s and he molecula basis o he APC/T cell in e ac ion, and
he a omic bluep in s o he design o nex -gene a ion biologics
o modula e he ICOS/ICOS-L he apeu ic axis.
Me hods
ICOS and ICOS-L exp ession and pu ifica ion. The ec odomains o human ICOS
(Unip o KB Q9Y6W8) ( esidues 21–138) and ICOS-L (Unip o KB O75144)
( esidue 19–248) ollowed by a obacco e ch i us (TEV) clea age si e we e used o
a monome ic a ian o Venus58 o p omo e exp ession o he glycop o eins. Genes
we e syn hesized a GeneA (Li e Technologies) and cloned in o he pHLsec
exp ession ec o con aining a C- e minal His
6x
ag o downs eam pu ifica ion.
P o eins we e exp essed in HEK 293F cells (The moFishe Scien ific) ollowing
s anda d p o ocols59: abou 200 mL o cells we e seeded a a densi y o 0.8 × 106
cells/mL and incuba ed wi h 125 pm oscilla ion a 37 °C, 8% CO
2
, and 70%
0 100 200 300
0.0
0.1
0.2
Time (s)
Response (nm)
0 100 200 300
0.0
0.1
0.2
0.3
Time (s)
Response (nm)
Biosenso
Biosenso
ICOS
ICOS-L
A ini y
A idi y/appa en a ini y
Fig. 4 Binding o ecombinan ICOS and ICOS-L ec odomains. Schema ic
expe imen al se -up (le ) and kine ic binding cu es ( igh ) o immobilized
ICOS and ICOS-L as analy e ( op) and in e ed sys em (bo om) measu ed
by BLI.
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humidi y in a Mul i on P o shake (In o s HT). Twen y- ou hou s a e seeding,
cells we e ansien ly ans ec ed using 50 μgo fil e ed DNA p eincuba ed o
10 min a oom empe a u e (RT) wi h he ans ec ion eagen Fec oPRO (Poly-
plus T ans ec ions) in a 1:1 a io. Cell suspensions we e ha es ed by cen i uga ion
a 5000 × g o 15 min a e 6–7 days and he supe na an s we e passed h ough a
HisT ap Ni-NTA column (GE Heal hca e) a 4 ml min−1. A e washing he
column wi h 20 mM T is pH 9.0, 150 mM NaCl, 5 mM imidazole, ICOS-Venus,
and ICOS-L-Venus we e elu ed wi h an inc easing g adien o imidazole (up
o 500 mM). F ac ions con aining p o ein we e pooled and diges ed o 1 h a RT
wi h he TEV p o ease. Diges ed p o ein was eco e ed in he flow h ough o a
second HisT ap Ni-NTA column, concen a ed and loaded on o a Supe dex
200 Inc ease size exclusion column (GE Hea hca e) in 20 mM T is pH 9.0, 150 mM
NaCl bu e .
Exp ession and pu ifica ion o ICOS and ICOS-L complexes. Fo s uc u e
de e mina ion o he ICOS/ICOS-L complex, a sha k Va iable New An igen
Recep o (VNAR) Single Domain a ge ing he cons an domain o ICOS-L was
used36. The e na y complex was p oduced by co- ans ec ing he DNA encoding
ICOS ( esidues 21–129): ICOS-L ( esidues 19–248):VNAR in a 3:1:4 a io.
Simila ly, he ICOS-L/p ezalumab complex was p oduced in he p esence o VNAR
by co- ans ec ing ICOS-L:Fab
HC
: Fab
LC
:VNAR in a 2:2:1:4 a io. The ICOS/
STIM003 complex was ob ained by co- ans ec ion o ICOS C136AC137A N23Q
( esidues 21–138):Fab
HC
:Fab
LC
in a 2:2:1 a io. Cys mu a ions we e equi ed o
inc ease sample homogenei y and ob ain well-di ac ing c ys als. The DNA a ios
used in each ans ec ion was selec ed based on he exp ession yields o he indi-
idual p o eins wi h he aim o achie e simila exp ession le els be ween hem
when co- ans ec ing. In o de o ob ain samples o homogeneous glycan com-
posi ion ha would allow downs eam p ocessing and e ficien c ys al packing, he
h ee complexes we e exp essed in HEK 293S cells (Gn I−/−). A e ha es ing he
cells, he supe na an was loaded on o a HisT ap Ni-NTA column and he complex
elu ed wi h a g adien o imidazole. Upon bu e exchange o emo e he imidazole,
Venus and glycans we e clea ed by incuba ing wi h TEV and EndoH, espec i ely
o 1 h a 37 °C. The diges ed complex was collec ed om he flow h ough o a
second HisT ap Ni-NTA column, concen a ed and loaded on o a Supe dex 200
Inc ease size exclusion column (GE Hea hca e) in 20 mM T is pH 9.0, 150 mM
NaCl bu e . In addi ion, o he ICOS/ICOS-L complex, he ac ions con aining
p o ein we e bu e exchanged o 20 mM T is pH 9.0, loaded on a MonoQ ion
exchange column and elu ed wi h a 0–50% linea g adien o 1 M po assium
chlo ide in 20 mM T is pH 9.0 bu e .
ICOS-L ICOS-L
P ezalumab-LC
P ezalumab-HC
c
ICOS
90°
a
0 50 100 150 200
0.0
0.1
0.2
0.3
Response (nm)
0 50 100 150 200
0.00
0.05
0.10
0.15
Response (nm)
ICOS
ICOS +
STIM003
ICOS-L
ICOS-L +
P ezalumab
ICOS-L ICOS
ICOS
STIM003-HC
STIM003-LC
ICOS
ICOS-L
90°
b
Fig. 5 Recogni ion o ICOS and ICOS-L by he apeu ic an ibodies. a An ibody binding compe i ion o ICOS-L (immobilized) o ICOS o ICOS +STIM003
Fab (le ); and ICOS (immobilized) o ICOS-L o ICOS-L +P ezalumab Fab ( igh ). Compa ison be ween he s uc u es o he ICOS/ICOS-L complex and
b he ICOS/STIM003 complex and c he ICOS-L/p ezalumab complex. The ligh and he hea y chains o STIM003 a e colo ed in ligh and da k pink,
espec i ely while he p ezalumab ligh and he hea y chains a e colo ed in g ay and blue, espec i ely. ICOS (deep eal) and ICOS-L (whea ) a e o a ed
90° abou a ho izon al axis o e eal he binding su ace o he an ibodies. Epi ope aces o STIM003 (pink) and ICOS-L (whea ) a e depic ed on he
su ace o ICOS, and p ezalumab (blue) and ICOS (deep eal) on he su ace o ICOS-L.
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Fab exp ession and pu ifica ion. The Fab hea y and ligh chains o STIM003 and
p ezalumab we e cloned in o cus om pcDNA3.4 exp ession ec o s a GeneA
(Li e Technologies). Fabs we e ansien ly exp essed in 200 mL HEK 293F cells
(The moFishe Scien ific) by co- ans ec ing 90 μg o he LC and he HC in a 1:2
a io wi h Fec oPRO (Polyplus T ans ec ions). Pu ifica ion o he Fabs was pe -
o med using a KappaSelec a fini y column (GE Heal hca e) and 100 mM glycine
pH 2.2 as he elu ion bu e . Elu ed ac ions we e immedia ely neu alized wi h 1
M T is-HCl pH 9.0 and u he pu ified using a MonoS ion exchange column and a
Supe dex 200 Inc ease size exclusion column (GE Heal hca e).
C ys alliza ion and X- ay da a collec ion. Pu ified complexes we e concen a ed,
mixed 1:1 wi h mo he liquo and se up in si ing d op apo di usion c ys al-
liza ion expe imen s. The ICOS/ICOS-L/VNAR complex was concen a ed o 10
mg/mL and c ys als g ew in 0.1 M HEPES, pH 7.0, and 30% ( / ) Je amine ED-
2001 and we e c yop o ec ed wi h 15% ( / ) glyce ol. X- ay di ac ion da a we e
collec ed a he 17-ID-B synch o on beamline a he Ad anced Pho on Sou ce
(APS) a he A gonne Na ional Labo a o y. The ICOS-L/p ezalumab/VNAR
complex was concen a ed o 12 mg/mL and g own in 0.2 M di-ammonium a a e
and 20% (w/ ) PEG 3350 and c yop o ec ed wi h 10% ( / ) glyce ol. X- ay di -
ac ion da a we e collec ed a he 23-ID-B synch o on beamline a APS. To
de e mine he s uc u e o he ICOS/STIM003 complex, he pu ified complex was
incuba ed wi h a a iable hea y-chain (V
H
H) domain specific o he human kappa
ligh chain as a c ys alliza ion chape one60 in a 1:5 a io o 30 min a RT ollowed
by gel fil a ion ch oma og aphy (Supe dex 200 Inc ease size exclusion column, GE
Heal hca e) in 20 mM T is pH 9.0, 150 mM NaCl bu e . The pu ified sample was
concen a ed o 4 mg/mL and c ys als we e ob ained in 0.2 M di-sodium hyd ogen
phospha e and 20% (w/ ) polye hylene glycol 3350 and c yop o ec ed wi h 10% ( /
) glyce ol. X- ay di ac ion da a we e collec ed a he FMX synch o on beamline
a he Na ional Synch o on Ligh Sou ce II (NSLS-II) a B ookha en Na ional
Labo a o y (BNL). Da a om he h ee complexes we e p ocessed using XDS61 and
he s uc u es we e sol ed by molecula eplacemen using Phase 62. CTLA-433,
B7-H363, B7-1 D233, Fabs om ou in e nal da abase, and VNAR ype 164 we e
used as sea ch models o ICOS, ICOS-L D1, ICOS-L D2, he Fabs, and VNAR,
espec i ely. The efinemen o he s uc u es was ca ied ou by i e a i e ounds o
phenix. efine65 and manual building in Coo 66. Rep esen a i e elec on densi y o
he h ee s uc u es is shown in Supplemen a y Fig. 6b–d. EMBL-EBI-PDBePISA67
was used o calcula e he epo ed bu ied su ace a ea. Access o all so wa e was
suppo ed h ough SBG id68.
Biolaye in e e ome y. Binding a fini y measu emen s we e conduc ed by BLI
using an Oc e RED96 BLI sys em (Pall Fo eBio) in PBS pH 7.4, 0.01% BSA, and
0.002% ( / ) Tween. Ni-NTA biosenso s we e used o bind His- agged ICOS and
ICOS-L p o eins. A signal esponse o 0.8 nm was eached be o e ans e ing he
loaded biosenso s o wells con aining a 1:2 se ial dilu ions o he Fabs o o he
0.0 0.2 0.4 0.6 0.8 1.0
Ipilimumab
T emelimumab
A ezolizumab
A elumab
BMS-936559
Du alumab
Pemb olizumab
Ni olumab
P ezalumab
STIM003
Mimic y sco e
c
Time (s)
Response (nm)
ICOS-L s P ezalumab
0 100 200 300
0.0
0.1
0.2
0.3
0.4
0.5
0 100 200 300
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Response (nm)
Time (s)
ICOS s STIM003
62 nM
31 nM
15 nM
7.5 nM
3.7 nM
1.9 nM
31 nM
15 nM
7.5 nM
3.7 nM
1.9 nM
d
ab
Fig. 6 Rema kable ecep o mimic y by an ibodies a ge ing ICOS/ICOS-L. a An ibody mimic y o he main hyd ophobic/a oma ic in e ace o he ICOS/
ICOS-L complex by STIM003 and p ezalumab. HCDR3 o p ezalumab mimics he FG loop o ICOS o media e in e ac ion wi h ICOS-L (le panel).
STIM003 in e ac ions wi h he FG loop o ICOS a e media ed by esidues om HCDR3, LCDR1 and LCDR2 ( igh panels). C i ical esidues o binding a e
shown in s icks and he BSA o hese and he es o esidues in ol ed in binding a e plo ed (bo om). The BSA o he ICOS/ICOS-L in e ac ion is included
o di ec compa ison. bAddi ional H-bonds (black dashes) be ween he an ibodies and ICOS and ICOS-L in a eas ou side he co e in e ace o he
ecep o /ligand complex. cKine ics o p ezalumab and STIM003 binding o ICOS-L and ICOS, espec i ely. dCompa ison o binding mimic y by
he apeu ic an ibodies a ge ing he CD28/B7 supe amily calcula ed as he ac ion o esidues wi h >1 Å2o BSA o he ecep o /ligand pai ha a e
con ac ed by an ibody binding as de ailed in Supplemen a y Fig. 5. Colo coding is as in Fig. 4.
NATURE COMMUNICATIONS | h ps://doi.o g/10.1038/s41467-020-18828-4 ARTICLE
NATURE COMMUNICATIONS | (2020) 11:5066 | h ps://doi.o g/10.1038/s41467-020-18828-4| www.na u e.com/na u ecommunica ions 9