Selective Targeting of TGF- Activation to Treat Fibroinflammatory Airway Disease

Abstract

Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmonary disease (COPD) and currently has no effective treatment. Transforming growth factor-β (TGF-β) has been widely implicated in the pathogenesis of airway remodeling in COPD. TGF-β is expressed in a latent form that requires activation. The integrin αvβ8 (encoded by the itgb8 gene) is a receptor for latent TGF-β and is essential for its activation. Expression of integrin αvβ8 is increased in airway fibroblasts in COPD and thus is an attractive therapeutic target for the treatment of airway remodeling in COPD. We demonstrate that an engineered optimized antibody to human αvβ8 (B5) inhibited TGF-β activation in transgenic mice expressing only human and not mouse ITGB8. The B5 engineered antibody blocked fibroinflammatory responses induced by tobacco smoke, cytokines, and allergens by inhibiting TGF-β activation. To clarify the mechanism of action of B5, we used hydrodynamic, mutational, and electron microscopic methods to demonstrate that αvβ8 predominantly adopts a constitutively active, extended-closed headpiece conformation. Epitope mapping and functional characterization of B5 revealed an allosteric mechanism of action due to locking-in of a low-affinity αvβ8 conformation. Collectively, these data demonstrate a new model for integrin function and present a strategy to selectively target the TGF-β pathway to treat fibroinflammatory airway diseases.

Full text

DOI: 10.1126/scitranslmed.3008074
, 241ra79 (2014);6 Sci Transl Med
et al.Shunsuke Minagawa
Airway Disease
Activation to Treat FibroinflammatoryβSelective Targeting of TGF-
Editor's Summary
smoke and allergens that are involved in the pathogenesis of COPD.
8, reduced airway inflammation and fibrosis in response to a variety of injurious agents including cigaretteβvαmouse
inflammation and fibrosis. This antibody, when administered to mice engineered to express only human and not
, a central mediator of pathologicalβ−a crucial receptor required for activation of transforming growth factor
8. This protein isβvαa monoclonal antibody that locks in a specific inactive conformation of a protein named integrin
engineeredet al.available treatments that ameliorate fibroinflammatory airway narrowing. In a new study, Minagawa
narrowing causes the obstruction responsible for the breathlessness that these patients experience, and there are no
common diseases such as chronic obstructive pulmonary disease (COPD) and severe chronic asthma. Such airway
Narrowing of the airways through accumulation of scar tissue and inflammation results from chronic injury in
Breathing Freely
http://stm.sciencemag.org/content/6/241/241ra79.full.html
can be found at:
and other services, including high-resolution figures,A complete electronic version of this article
http://stm.sciencemag.org/content/suppl/2014/06/17/6.241.241ra79.DC1.html
can be found in the online version of this article at: Supplementary Material
http://www.sciencemag.org/about/permissions.dtl
in whole or in part can be found at: article
permission to reproduce this of this article or about obtaining reprintsInformation about obtaining
is a registered trademark of AAAS. Science Translational Medicinerights reserved. The title
NW, Washington, DC 20005. Copyright 2014 by the American Association for the Advancement of Science; all
last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue
(print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except theScience Translational Medicine
on June 24, 2014stm.sciencemag.orgDownloaded from on June 24, 2014stm.sciencemag.orgDownloaded from

LUNG DISEASE
Selective Targeting of TGF-b Activation to Treat
Fibroinflammatory Airway Disease
Shunsuke Minagawa,
1
* Jianlong Lou,
2
* Robert I. Seed,
1
* Anthony Cormier,
1
Shenping Wu,
3
Yifan Cheng,
3
Lynne Murray,
4,5
Ping Tsui,
4
Jane Connor,
4
Ronald Herbst,
4
Cedric Govaerts,
6
Tyren Barker,
1
Stephanie Cambier,
1
Haruhiko Yanagisawa,
1
Amanda Goodsell,
7
Mitsuo Hashimoto,
1
Oliver J. Brand,
1
Ran Cheng,
1
Royce Ma,
1
Kate J. McKnelly,
1
Weihua Wen,
2
Arthur Hill,
8
David Jablons,
8
Paul Wolters,
7
Hideya Kitamura,
1
Jun Araya,
9
Andrea J. Barczak,
7
David J. Erle,
7
Louis F. Reichardt,
10
James D. Marks,
2
Jody L. Baron,
7
Stephen L. Nishimura
1†
Airway remodeling, caused by inflammation and fibrosis, is a major component of chronic obstructive pulmo-
nary disease (COPD) and currently has no effective treatment. Transforming growth factor–b (TGF-b) has been
widely implicated in the pathogenesis of airway remodeling in COPD. TGF-b is expressed in a latent form that
requires activation. The integrin avb8 (encoded by the itgb8 gene) is a receptor for latent TGF-b and is essential
for its activation. Expression of integrin avb8 is increased in airway fibroblasts in COPD and thus is an attractive
therapeutic target for the treatment of airway remodeling in COPD. We demonstrate that an engineered opti-
mized antibody to human avb8 (B5) inhibited TGF-b activation in transgenic mice expressing only human and
not mouse ITGB8. The B5 enginee red antibody blocked fibroinflammatory responses induced by tobacco
smoke, cytokines, and allergens by inhibiting TGF-b activation. To clarify the mechanism of action of B5, we
used hydrodynamic, mutational, and electron microscopic methods to demonstrate that avb8 predominantly
adopts a constitutively active, extended-closed headpiece conformation. Epitope mapping and functional char-
acterization of B5 revealed an allosteric mechanism of action due to locking-in of a low-affinity avb8 confor-
mation. Collectively, these data demonstrate a new model for integrin function and present a strategy to
selectively target the TGF-b pathway to treat fibroinflammatory airway diseases.
INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is now the third lead-
ing cause of death in the United States (1). A major cause of airflow
obstruction in COPD is airway narrowing caused by peribronchial
chronic inflammation and fibrosis, known as airway remodeling, for
which there is no effective treatments (2).
Airway remodeling severity is linked to cumulative cigarette smoke
exposure and the frequency of sudden declines in lung function known
as exacerbations (3). COPD exacerbations (~50%) are caused by viral
infections (4). Such infections can directly cause increased transforming
growth factor–b1(TGF-b1) expression (5) and experimental airway re-
modeling accompanied by airway hyperresponsiveness (6). Exposing
mice to the viral mimetic polyinosinic:polycytidylic acid [poly(I:C)] accel-
erates airway remodeling in response to cigarette smoke (7). Therefore,
cigarette smoke combined with poly(I:C) models the self-amplifying cycle
of cigarette smoke–induced injury, viral infection, and exacerbations that
culminate in airway wall thickening and obstructive physiology in
humans. TGF-b1andinterleukin-1b (IL-1b) drive this cycle and repre-
sent potential therapeutic targets in the airway remodeling process in
COPD (8, 9). TGF-b is expressed in an inactive (latent) form and must
be activated to have biologic activity. One protein that activates TGF-b in
vivo is the integrin avb8(encodedbytheITGB8 gene), which shows
increased expression in COPD airways (10). Thus, selective inhibition
of avb8-mediated TGF-b activation is a potential therapeutic strategy
(11) that would bypass the toxicities of global TGF-b inhibition (12, 13).
Increased expression of avb8isdrivenbyIL-1b (10, 14). Increased
IL-1b in COPD patient samples is linked to cigarette smoke and
viruses (15, 16). Adenoviral (Ad) delivery of IL-1b leads to increased
avb8-depe ndent TGF-b activation and airway remodeling that is
blocked by conditional deletion of itgb8 in fibroblasts or by neutraliz-
ing pan–TGF-b antibodies (10). Intratracheal delivery of Ad-IL-1b ini-
tiates the avb8-dependent influx of lung dendritic cells (DCs), which
increases adaptive T cell immunity [that is, CD4 T helper 1 (T
H
1) and
T
H
17] and airway inflammation and fibrosis (10).
Lungs of intratracheal Ad-IL-1b–treated mice or IL-1b–stimulated
mouse or human lung fibroblasts demonstrate a vb8- and TGF-b–
dependent increases in the potent DC chemokine CCL20, suggesting
aproximalroleinTGF-b–dependent airway remodeling (10). CCL20
and DCs are increased in COPD lung biospeci mens (17). Thus, CCL20
is a physiologically relevant biomarker of avb8-mediated TGF-b acti-
vation, leading to DC accumulation (17).
We sought to understand the mechan ism by which integrin
avb8 activates TGF-b in fibroinflammatory airway disease to design
a therapeutic strategy for its treatment. TGF-b is maintained in the
inactive (latent) state by the noncovalent association with its propeptide,
1
Department of Pathology, University of California, San Francisco, San Francisco, CA
94110, USA.
2
Department of Anesthesia and Perioperative Care, University of California,
San Francisco, San Francisco, CA 94110, USA.
3
The Keck Advanced Microscopy Laboratory,
Department of Biochemistry and Biophysics, University of California, San Francisco, San
Francisco, CA 94110, USA.
4
Department of Respiratory, Inflammation and Autoimmunity,
MedImmune, Gaithersburg, MD 20878, USA.
5
Department of Respiratory, Inflammation
and Autoimmunity, MedImmune, Cambridge CB21 6GH, UK.
6
Laboratory of Structure and
Function of Biological Membranes, University of Brussels, Brussels 1000, Belgium.
7
Department of Medicine, University of California, San Francisco, San Francisco, CA 94110,
USA.
8
Department of Surgery, University of California, San Francisco, San Francisco, CA
94110, USA.
9
Department of Pulmonary Medicine, Jikei University, Tokyo 105 8461, Japan.
10
Genetics, Development, and Behavioral Sciences, University of California, San Francisco,
San Francisco, CA 94110, USA.
*These authors contributed equally to this work.
†Corresponding author. E-mail: stephen.nishimura@ucsf.edu
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 1
on June 24, 2014stm.sciencemag.orgDownloaded from

latency-associated peptide (18). The latency-associated peptides of
TGF-b1andTGF-b3 both contain RGD motifs (18), which bind to
integrin avb8withhighaffinity(19, 20).
The sentinel event in integrin function is ligand binding, widely
thought to be facilitated by integrin “activation” (21). Mechanisms
of integrin activation inferred using biochemical and structural data
from the avb3, a
IIb
b3, and a5b1 integrins support two distinct models
of integrin activation: (i) a “switchblade-like” opening from a compact
(bent) to extended conformation with an “open” headpiece, and (ii) a
subtle headpiece opening occurring in a bent conformation (22–24).
The former model addresses steric constraints imposed by the cell mem-
br a n e because integrin extension increases access of large ligands of
the extracellular matrix to the ligand-binding pocket (24). In either
model, a closed headpiece conformation is thought to be inactive
and of low affinity (22, 25, 26). How these models and assumptions
apply to avb8 is not immediately obvious because of sequence differ-
ences between conformationally important regions of b8 compared
with other b subunits (27, 28).
Integrin headpieces contain the ligand-binding pocket, located at
the interface of the integrin b-subunit head (referred to as bI) and
the a-subunit head domains (21). Interactions between the b-subunit
bIdomaina1anda7 helices regulate integrin activation states and are
influenced by ligand and metal ion occupancy (21, 25). Integrin bI
domains contain three conserved metal binding sites except the bIdo-
main of integrin b8, which only has two because it lacks two critical
aspartate residues of the ADMIDAS cation binding site that allosteri-
cally couples the ligand-binding pocket to the rest of the integrin (24).
As monitored by adhesion or ligand-bin ding assays of non- b8 integrins,
Ca
2+
and Mg
2+
facilitate integrin low-affinity states, and Mn
2+
,high-
affinity states (22). In the presence of Mn
2+
, integrins extend and open
their headpieces (a process enhanced by RGD peptide) by a “swing-
out” of the adjacent hybrid domain (24, 25, 29). A large body of work
suggests that Mn
2+
alters bI a1-a7 helix interactions, causing headpiece
opening (24, 25, 29).
Here, we use hydrodynamic, electron microscopic, and mutational
analyses to demonstrate that integrin avb8 predominantly adopts a
constitutively active conformation with an extended-closed headpiece
and thus does no t fit current models of integrin activation. We affinity-
matured an anti-human b8 monoclonal antibody (37E1) that binds to
the a1 helix of the b8 bI domain to generate B5. B5 causes a b8
headpiece conformational change that efficiently inhibits TGF-b acti-
vation. The relevance of these findings for a therapeutic strategy is dem-
onstrated using bacterial artificial chromosome (BAC) tran sgenic (Tg)
mice expressing only human and not mouse itgb8.Thesemicewere
used to establis h and extens ivel y character ize a physio logic ally rel evant
airway remodeling system induced by a combination of cigarette smoke
and poly(I:C). B5 blocked airway inflammation and fibrosis not only in
cigarette smoke–induced airway disease but also in cytokine- and allergen-
induced airway disease mouse models, suggesting that avb8-mediated
TGF-b activation may be a new therapeutic target for treating airway
remodeling diseases.
RESULTS
An optimized b8 antibody (B5) blocks TGF-b activation
B5 blocks IL-1b–induced airway remodeling. Intratracheal in-
jection of Ad-IL-1b in mice induces airway disease that recapitulates
keyimmunologicandpathologicfeaturesofairwayremodelinginhu-
man COPD by increasing DCs that drive adaptive T
H
1andT
H
1im-
munity (10). We used intratracheal injectio n of Ad-IL-1b in BAC Tg
mice expressing only human and not mouse itgb8 to test the efficacy
of B5 (Fig. 1A). The expression of human ITGB8 in these BAC Tg
mice is at similar levels and expression patterns as in human tissues
(fig. S1), and rescues the developmental lethality of itgb8 knockout
mice (fig. S2) (30–32). This shows that human b8bindstomurine
latency-associated peptide (fig. S3).
B5 demonstrated a significant dose-dependent blockade of phos-
phorylation of the TGF-b signaling effector pSMAD2/3 from lung
homogenates of BAC ITGB8 Tg mice treated by intratracheal injection
of Ad-IL-1b, indicating successful blockade of TGF-b activation (P =
0.03, Fig. 1B). Decreased pSMAD2/3 correlated with dose-dependent
decreases in CCL20 (fig. S4A), decreases in the total number of cells in
bronchoalveolar lavage, and reduced numbers of neutrophils and
macrophages (fig. S4, B and D). B5 effectively inhibited Ad-IL-1b–
dependent inflammation of the airway wall (Fig. 1, C, D, and G), fi-
brosis(Fig.1,E,F,andH),andincreasesinbronchoalveolarlavage
total cells, macrophages, and neutrophils (Fig. 1, I to K). mRNA levels
of ITGB8 and col1a2 were increased by intratracheal injection of Ad-
IL-1b and were blocked by B5 (Fig. 1, L and M). B5 had no effect on
airway histology of control animals (fig. S5). These results demon-
strate that B5 had similar effects on airway remodeling in vivo, as pre-
viously shown with TGF-b neutralizing antibodies or conditional
deletion of itgb8 on fibroblasts (10).
B5 blocks airway disease in a cigarette smoke exacerbation
model of COPD.
We established and characterized a mouse model
of COPD that combined cigarette smoke with poly(I:C) treatment of
BAC ITGB8 Tg mice (7) to test the efficacy of B5. Three weeks of
whole-body cigarette smoke exposure alone caused minimal patholo-
gy, and 2 weeks of intranasal poly(I:C) alone caused vigorous neutro-
philic inflammation but no significant airway remodeling (fig. S6).
Three weeks of cigarette smoke together with intranasally adminis-
tered poly(I:C) (Fig. 2A) caused increased airway remodeli ng; an in-
crease in the number of total cells in bronchoalve olar lavage; an increase
in macrophages, neutrophils, and lymphocytes; and increased expres-
sion of lung IL-1b, IL-17A, and CCL2 (fig. S6). This system therefore
replicated key components of human COPD and COPD-relevant
animal models (17). Over time, total inflammatory cells and macro-
phages increased steadily with a parallel in crease in lung IL-1b and IL-
17 (fig. S7). CCL2 and CCL20 expression peaked at 12 days after smoke
exposure, similar to that observed with the intratr acheal injection of
Ad-IL-1b (fig. S7).
Combined cigarette smoke–poly(I:C) exposure (Fig. 2A) increased
inflammation and fibrosis surrounding small airways of BAC ITGB8 Tg
mice compared with control, untreated BAC ITGB8 Tg mice (P < 0.01,
Fig. 2, B to D and F). These increa ses were inhibited by treatment (in-
traperitoneal) with the B5 antibody (P < 0.01; Fig. 2, B, C, E, and G). Inflam-
mation (total bronchoalveolar lavage cell counts, neutrophils, and
lymphocytes), inflammatory mediators (lung IL-1b, CCL2, CCL20,
and IL-17), TGF-b signaling (pSMAD3), emphysema (airspace enlarge-
ment), and airway obstruction (airway resistance to acetylcholine chal-
lenge) were all increased by cigarette smoke–poly(I:C). B5 inhibited
each of these increases (Fig. 2, H to S). These data demonstrate that B5
blocks airway remodeling, inflammati on, inflammatory cytokine and che-
mokine production, airspace enlargement, and obstructive airway phys-
iology in a model that mimics COPD exacerbation by cigarette smoke.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 2
on June 24, 2014stm.sciencemag.orgDownloaded from

B5 blocks allergic airway inflammation. B5wasusedinanov-
albumin sensitization and challenge model to determine mechanistic
commonalities with IL-1b and cigarette smoke–poly(I:C) mouse
models of airway disease (fig. S8A). Ovalbumin challenge increased in-
flammation surrounding small airways of BAC ITGB8 Tg mice (fig.
S8, B and D), total bronchoalveolar lavage cell counts, eosinophils,
IL-1b, CCL2, and CCL20, all of which were inhibited by B5 (fig. S8,
C to I). These results are consistent with the ability of B5 to block a
central pathway of avb8-dependent airway inflammation, as previ-
ously suggested using conditional deletion of itgb8 (10, 33).
The avb8 integrin has unique structural and functional features
Secreted integrin avb8 is constitutively active. Integrins are
thought to transit between active and inactive states regulated by cat-
ion occupancy, with Mn
2+
being the most strongly activating cation
(34). As a first step to understand the interaction of avb8withTGF-b,
we asked whether Mn
2+
also increases b8 affinity for latency-associated
peptide. We used human 293 cells stably expressing the integrin b8sub-
unit, which binds well to latency-associated peptide in the presence of
Ca
2+
and Mg
2+
(Fig.3A).Mn
2+
did not increas e, but r ather decreased,
cell adhesion (Fig. 3A) and did not increase the already high level of
binding of soluble truncated secreted avb8 to latency-associated pep-
tide (Fig. 3B and fig. S3). In contrast, binding of soluble truncated
avb3 to its ligands was markedly increased by Mn
2+
(Fig. 3B), suggest-
ing that avb8 is constitutively active, in contrast to avb3, which is not.
Integrin activation corresponds to global conformational rearrange-
ments that can be monitored by size exclusion chromatography
because receptor extension leads to axial asymmetry and a larger hy-
drodynamic radius than a compact (bent) form (21). The conforma-
tional heterogeneity of avb8 was investigated by comparing size
exclusion chromatography profiles of secreted avb8withandwithout
a C-terminal clasp (Fig. 3C). The clasp mimics the spatial constraint s
of transmembrane domains that stabilize the bent conformation of
other integrins (21). The size exclusion chromatography profile of
clasped, secreted avb8 was identical to that of the nonclasped form
of this integrin (Fig. 3D and Table 1). Elution peaks did not change
in the presence of RGD peptide with or without Mn
2+
(Fig. 3D and
Table 1), suggesting that avb8, unlike avb3, predominantly adopts a
single cation- and ligand-independent conformation.
Negative staining electron microscopy was used to directly view
conformations of avb8 in size exclusion chromatography fractions.
Computational grouping of well-resolved particles into classes of nearly
Fig. 1. Optimized B5 antibody blocks TGF-b activation in vivo and in-
tratracheal Ad-IL-1b–induced airway inflammation and fibrosis. (A)
Schematic of the creation of a mouse model of airway inflammation using
adenovirally delivered IL-1b (Ad-IL-1b) administered intratracheally. (B)B5
blocks intratracheal Ad-IL-1b–induced pSMAD2/3, demonstrating that neu-
tralization of avb8inhibitsTGF-b activation in vivo. Lung homogenates
from mice treated with B5, compared with IgG2a isotype or wild-type
(WT) non–Ad-IL-1b injected intratracheally, were evaluated by pSMAD2/3
enzyme-linked immunosorbent assay (ELISA). n =4,*P =0.03byanalysis
of variance (ANOVA) and post-test for linear trend. (C to H)B5(DandF)
compared with isotype control (C and E) blocks inflammation of the airway
wall (C, D, and G) and fibrosis (E, F, and H) induced by intratracheally
administered Ad-IL-1b. Results expressed as area of inflammation or fibrosis
per basement membrane (BM) length. Semiquantitative airway mor-
phometry of standard hematoxylin and eosin (H&E)–stained (C and D) or
trichrome-stained (E and F) secti on s . * ** P < 0.0001, by ANOVA and Tukey’s
post-test. Scale bar, 200 mm. (I to M) B5 blocks intratracheal Ad-IL-1b–
induced inflammation in bronchoalveolar lavage. Total cells in bronchoal-
veolar lavage (I), macrophages (J), and neutrophils (K), as well as gene
transcripts of ITGB8 (L) and col1a2 (M), were increased by intratracheal
Ad-IL-1b, and this increase was inhibited by B5. n = 3, Ad-LacZ+isotype– or
Ad-LacZ+B5–treated mice; n =4,Ad-IL-1b+isotype–treated mice; or n =6,Ad-IL-
1b+B5–treated mice. *P < 0.05, ** P < 0.01, ***P < 0.001, by ANOVA and
Tukey’s post-test.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 3
on June 24, 2014stm.sciencemag.orgDownloaded from

identical conformers (fig. S9) resulted in highly related classes in 20 of
the clasped and 19 of 20 of the unclasped preparations (fig. S9). Bot h
clasped and unclasped versions were almost entirely in an extended-
closed state, similar to the avb3integrininMn
2+
without RGD pep-
tide (21). Unclasped avb8 was more heterogeneous than clasped, and
both contained a second most common form (6% of clasped, 39% of
unclasped) that was extended but likely represented a side view or a
conformational intermediate (Fig. 3E). The bent conformation (0% of
clasped, 5% of unclasped) was a minor population that did not signif-
icantly affect the hydrodynamic profile (Table 1), which would be
expected if the bent conformation was a major population (21). In
addition, RGD peptide would be expected to induce integrin extension
Fi g . 2. B5 blocks airway inflammation and fibrosis induced by
cigarette smoke and the viral mimetic poly(I:C). (A)Schematicshowing
creation of the cigarette smoke intranasal (IN)–poly(I:C)–induced mouse
model of airway remodeling. (B and C) Quantitative airway morphometry
showing cigarette smoke–poly(I:C) (CS+PolyI:C)–induced inflammation of
the airway wall (B) and wall thickening (C), and the effects of B5 compared
to control IgG2a. (D to G) Photomicrographs of mouse lungs treated with
cigarette smoke–poly(I:C) and control (D and F), and cigarette smoke–
poly(I:C) with B5 (E and G); H&E (D and E) and trichrome (F and G). Scale
bar, 75 mm. B5 blocks cigarette smoke–poly(I:C)–induced influx of neutrophils
and lymphocytes. (H to K) Total cells from the bronchoalveolar lavage (BAL)
(H),macrophages(I),neutrophils(J),andlymphocytes(K).(L to O)B5blocks
the expression of cigarette smoke–poly(I:C)–induced IL-1b (L), CCL2 (M),
CCL20 (N), and IL-17 (O). ELISAs were performed on whole-lung lysates for
expression of IL-1b, IL-17, and CCL2 or on bronchoalveolar lavage for expres-
sion of CCL20. (P) Analysis of pSMAD3 immunostaining. (Q to S)Lungphe-
notyping. (Q) Mean linear intercept; L(m), estimate of airspace enlargement.
(R) Airway resistance with increasing acetylcholine (ach) concentrations
(log
2
). (S) Concentration of acetylcholine as provocative challenge doubling
baseline resistance (pc200); room air groups, n = 3; cigarette smoke–poly(I:C)
groups, n = 4 to 5. *P < 0.05, **P < 0.01, ***P < 0.001, by ANOVA and
Bonferroni’s post-test.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 4
on June 24, 2014stm.sciencemag.orgDownloaded from

if a bent species was present (21); avb8 in the presence of RGD peptide
in Ca
2+
/Mg
2+
or Mn
2+
remained extended-closed without headpiece
opening (Fig. 3E). Together, these data demonstrate that the secreted
form of avb8 is constitutively active in a thermodynam ically favored,
stable extended-closed conformation.
avb8 expressed on the cell surface is constitutivel y active. We
next asked whether avb8 was constitutively and fully active on the cell
surface. Other studies have used glycosylation modification of the
head-hybrid domain interface to constitutively activate integrins on
the cell surface (a
IIb
b3, avb3, a5b1) (35, 36). The b8 integrin subunit
contains a natural glycan at an almost identical location as one of
these mutationally introduced glycans in the b1 integrin subunit
(Fig. 4A) (35, 36). To further amplify the effects of activating glycans,
we mutationally introduced a second glycosylation site at the head-
hybrid domain interface (N294). At a homologous residue in the b3
(N303) or an adjacent residue in the b1 (N429 or N333) subunit, an
artificial glycan induces extension, headpiece opening, and constitutive
activation (Fig. 4B) (35, 36). Homology modeling of avb8predictsthat
both the natural and artificial glycan “wedge” expresse d by a vb8would
have the same contact points on the adjacent hybrid domain as avb3
and would thus similarly favor the open headpiece conformation (Fig.
4B). Such swing-out of the hybrid domain increases the affinity of lig-
and binding by the avb3(35), a
IIb
b3(35), and a5b1(36)andthus
would be predicted to also open the avb8headdomain(Fig.4B).
Pools of HT1080 cells transfected with the b8 glycan mutant or
wild-type b8 subunit were surface-biotinylated and immunoprecipi-
tated to reveal glycosylation status (Fig. 4C) and were sorted to equal
levels of surface expression (Fig. 4D). Surface recognition by multiple
domain-specific b8 antibodies (figs. S10 and S11) indicated proper
folding of the glycan mutant. The glycan mutant b8 subunit migrated
on SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels with a
~3-kD larger molecular weight than that of the wild-type b8 subunit.
The deglycosylating amidase, peptide–N-glycosidase F, reduced the
size of the glycan mutant approximately to that of the deglycosylated
wild-type b8 subunit (Fig. 4C). Cell adhesion assays (Fig. 4E) and
TGF-b activation assays (Fig. 4F) revealed no functional differences
between the glycan mutant and wild-type b8 subunits. These data
demonstrate that forced openin g of the hybrid domain does not
Fig. 3. Integrin avb8 is constitutively active in a high-affinity extended-
closed conformation. (A)Adhesionofb8-expressing 293 cells to latency-
associated peptide (LAP) or bovine serum albumin (BSA; control), with
Ca
2+
/Mg
2+
or Mn
2+
, and reported as absorbance (A
570
). n =3experiments.
(B) Binding of soluble avb8–alkaline phosphatase (AP) or avb3-AP fusion
proteins to latency-associated peptide, or to the avb3 ligand fibronectin
as a control, with Ca
2+
/Mg
2+
(open bars) or Mn
2+
(solid bars) reported as
A
405
. n = 8 experiments. ***P <0.001,byANOVAandTukey’s post-test. n.s.,
not significant. (C) Schematic of the domain structure of secreted integrins
with or without a C-terminal clasp with locations of various domains.
Clasped version has a 10–amino acid linker between the acid-base coil.
(D) Size exclusion chromatography of clasped and unclasped avb8 se-
creted proteins. Clasped protein or unclasped protein in a solution
containing Ca
2+
/Mg
2+
,unclaspedproteinwithanRGDpeptideinasolution
containing Ca
2+
/Mg
2+
or Mn
2+
. Particles too larg e to enter the medium are
excluded and this volume is denoted as “void volume (V
0
)” as indicated.
n =3.(E) Negative staining electron microscopy of peak fractions shown
in (D). Representative class averages showing extended-closed or bent
conformations. Cartoon depicts domain structure. Below the micrographs
are shown percentages of each subclass. Scale bar, 10 nm.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 5
on June 24, 2014stm.sciencemag.orgDownloaded from

further increase avb8 function, and are consistent with hydrodynamic
and electron microscopy data, suggesting that the avb8 extended-closed
conformation is constitutively (and maximally) active when on the cell
surface.
B5 selectively targets avb8-mediated TGF-b activation. To de-
velop a b8 antibody suitable for clinical use, we chose to optimize the
neutralizing b8 antibody (clone 37E1) that inhibited TGF-b activation
in coculture assays at high concentrations (20). We reasoned that if
the effects of 37E1 were specific for inhibiting TGF-b activation at
high concentrations, then it would be worth optimizing. Human fetal
tracheal fibroblasts, a model of myofibroblasts [the cell type implicated
in lung fibrosis (19)], were treated with 37E1 or 1D11, a pan–TGF-b
isoform neutralizing antibody, to interrogate the spectrum of genes
influenced by basal autocrine avb8-m ediated TGF-b activation (tables
S1 and S2). Two hundred fifty-two genes were significantly altered
(B statistic >0) by TGF-b antibody treatment compared with control
antibody treatment (table S1). The direction of change [increase ver-
sus decrease in response to b8orTGF-b antibody (100 mg/ml)] was
the same for 251 of these 252 genes (fig. S12), indicating virtually
identical pathways affected by inhibiting avb8orTGF-b.Thus,
Table 1. Size exclusion chromatography of av-integrin constructs.
Condition Elution volume (ml)* Stokes radius (Å)
No Fab/ligand
avb8 (C-terminal clasp) 10.51 ± 0.03 64.25
avb8 (truncated) 10.55 ± 0.02 63.87
Plus ligand (RGD peptide)
avb8 (truncated) Ca
2+
10.53 ± 0.12 63.98
avb8 (truncated) Mn
2+
10.49 ± 0.01 64.28
Plus B5 Fab
avb8 (C-terminal clasp) 10.0 ± 0.02 67.70
†
avb8 (truncated) 10.1 ± 0.03 67.04
†
Plus clone 68 Fab
avb8 (C-terminal clasp) 10.15 ± 0.02 66.78
†
avb8 (truncated) 10.18 ± 0.03 66.53
†
*Mean ± SEM (n ≥ 3). †P < 0.01 by unpaired Student’s t test.
Fig. 4. avb8 is constitutively active on the cell surface. (A)Alignment
of human b8andb1 integrin subunits with positions of N-X-T consensus
(underlined) and N-linked glycosylation sites in green. Native b8glycosyl-
ation site (N414), adjacent to the mutant N429 glycosylation site in the
b1 integrin subunit (49). This is modeled onto a space-filling rendering of
homology-modeled (PyMOL V1.1r1) avb8headpiecebasedontheavb3
crystal structure in a closed-conformation [Protein Data Bank (PDB) 3IJE]
(25) with GlcNAc2Man7 glycan chain (brown) from human CD2 (1GYA)
(49). Green, b-subunit head domain; black, b8-hybrid; blue, av subunit.
(B) Alignment of human b8andb3subunitswithneo-b8glycosylation
site N294 (green) introduced by the N296T (red) mutation compared with
the neo-b3 glycosylation site N303 (green) introduced by the N305T mu-
tation (red) (35). Modeled rendering of the avb8headpiece,asin(A).Gly-
cans in position N414 (brown) and N294 (red) oriented laterally allowing
the closed headpiece conformation. (C) Immunoprecipitation with b8anti-
body of surface-labeled b8-HT1080 cells transfected with WT b8(lanes2
and 4) or N294 mutant (lanes 1 and 3), with or without pepti de– N-
glycosidase F (PNG) (nonreducing SDS-PAGE). *, a size increase of 3 kD in
the neoglycosylated N294 mutant. (D) Histogram overlays of anti-b8–
stained, stably transfected HT1080 pools of WT, N294 glycan mutants, or
mock versus WT cells stained with secondary antibody only (PBS). (E and F)
Cell adhesion (E) or TGF-b activation (F) assays using transfected sorted
pools of HT1080 cells e xpressing equal surface concentrations of WT
b8 or N294 glycan b8 compared with mock-transfected cells. n =3;
*P <0.05,**P < 0.01, ***P <0.001,byANOVAandTukey’sorBonferroni’s
post-test.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 6
on June 24, 2014stm.sciencemag.orgDownloaded from

37E1 does not produce off-target effects independent of TGF-b (P <
2×10
−16
, r
2
= 0.88).
Gene array data encouraged engineering of an optimized version of
the 37E1 antibody, B5, which improved the affinity to a range suitable
for in vivo applications (figs. S13 and S14). When 37E1 and B5 were
expressed as single-chain antibody fragments (scFv) on yeast, the ap-
parent binding affinity improved from 117.51 to 15.42 nM, respective-
ly. A second affinity-matured antibody, clone 68, was developed in
parallel on the basis of hybridoma clone 11E8, which cross-competed
with B5 for avb8 binding and therefore was directed against an over-
lapping epitope. Clone 68 had a higher affinity for avb8thandidB5
[as scFv, dissociation constant (K
d
) = 1.42 nM] but did not significant-
ly inhibit avb8-mediated TGF-b activation [inhibition as immuno-
globulin G1 (IgG1) at 10 mg/ml = 0 ± 14%]. In contrast, B5 as an
intact IgG (K
d
of 0.54 nM) displayed a 36-fold increase in ability to
block the binding of avb8-AP to latency-associated peptide relative to
37E1 (fig. S14), which was specific to avb8becauseitdidnotinhibit
the binding of avb6-AP (fig. S15). Optimized B5 markedly increased
(~1000-fold) the ability to block TGF-b activation (P < 0.001, Fig. 5A)
and IL-1b–induced CCL20 production by human lung fibroblasts,
compared with 37E1 (P <0.0001,Fig.5B).Thesedatademonstrate
that the affinity-matured B5 derivative has signific antly impro ved abil-
ities to inhibit key TGF-b–dependent functional endpoints from rele-
vant human cells.
B5 binds to the b8 head-hybrid interface and alters the head
domain conformation .
Hybridoma clone 37E1 recognized only hu-
man and not the mouse b8 subunit, suggesting that the 37E1 binding
epitope depends on species-specific amino acid differences, reflecting
its derivation from mice immunized with human avb8. Chimeric
constructs swapping various regions between mouse and human b8
were expressed on 293 cells and used to identify a critical region (lo-
cated between amino acids 133 and 138) of human b8requiredfor
binding of B5, 37E1, and clone 68 antibodies (fig. S10). The blocking
antibodies B5 and 37E1 and the nonblocking clone 68 recognized var-
ious combinations of the human species-specific residues R
133
,F
137
,
and F
138
,whichwerelocatedontheC-terminala1helixoftheb8
bI domain (fig. S10 and Fig. 6A).
The mechanisms of action of integrin antibodies that function as
allosteric activators and inhibitors have been successfully determined
using size exclusion chromatography and electron microscopy of
integrin–Fab fragment complexes (37). To determine the mechanism
of action of B5, we asked whether B5 induced conformational changes
of avb8 that could be detected by size exclusion chromatography and
electron microscopy that were not seen with Fab from the non–function-
blocking antibody clone 68, which binds to an overlapping epitope.
The avb8–B5 Fab or avb8–clone 68 Fab complex eluted from the size
exclusion chromatography column slightly earlier than avb8 alone,
consistent with the binding of the Fabs, but did not reveal detectable
conformational differences between avb8 bound by B5 or clone 68
(fig. S16 and Table 1).
Electron microscopy of peak size e xclusion c hromatography
fractions of avb8-Fab complexes revealed that both B5 and clone 68
Fabs bound exclusively to the extended conformation at the b8sub-
unit head-hybrid junction oriented away from the ligand-binding
pocket, agreeing well with epitope mapping data (Fig. 6A). Analysis
of class averages of clasped and unclasped preparations revealed that
nearly 100% of the well-resolved classes were extended with a closed
headpiece with or without B5 and clone 68 Fab (Fig. 6B and fig. S17).
Two-dimensional image analysis of classaveragesrevealedthathead-
hybrid domain angles were reduced (~20°) when bound to B5 Fab,
but were not significantly changed by clone 68 Fab (Fig. 6, B to D).
The B5/clone 68 epitope was far (~28 Å) and oriented away from the
ligand-binding pocket, making steric effects of B5 on the RGD ligand-
binding site improbable (Fig. 6, A, C, and D). B5 Fab blocked the
binding of secreted avb8 to latency-associate d peptide nearly as well
as the intact B5 IgG (at 40 mg/ml: B5 Fab 74 ± 10% versus B5 IgG 87 ±
1% inhibition), demonstrating that antibody avidity or the additional
size of the intact IgG was not required for B5 function. Together, these
data suggest that B5 is a noncompetitive allosteric inhibitor; subtle dif-
ferences seen on electron microscopy between the orientations of the
hybrid domain between the blocking B5 and nonblocking clone 68
Fabs reflect conformational changes in the head domain and ligand-
binding pocket.
Homology modeling of avb8 to the closed headpiece conforma-
tions of avb3 (Fig. 4A) reveals a steric clash with a natural glycan
(N414), highly conserved between species, absent from all other in-
tegrin b subunits, located on the hybrid domain close to the vertex
of the head-hybrid domain angle, where the interdomain distances
are predicted to be closest (35). This site faces the a5-b5 loop at the
bottom of t he bI domain, and when a glycan is computationally
docked on this site, several glyc an conformations sterically clash
with the bI head, and thus predicted to open the head-hybrid angle
(Fig. 4B), clearly not seen on any electron microscopy projections
(Fig. 3, D and E). Thus, the natural (N414) (Fig. 4A) and the mutant
(N294) glycans (Fig. 4B) of b8 are almost certainly laterally displaced
in the closed headpiece conformation of avb8, with or without inward
hybrid ben ding by ~20°, as seen with B5. Together, these data suggest
that the closed conformation of avb8 is highly thermodynamically
favored.
B5 is a noncompetitive allosteric inhibitor that induce s a low-
affinity ligand binding state of avb8.
We sought to determine
whether B5 directly competes for latency-associated peptide because
allosteric antibody inhibitors directed to the C-terminal a1helixof
Fig. 5. Affinity-optimized b8 antibody (B5) inhibits TGF-b–dependent
chemokine expression by stimulated human lung fibroblasts. (A) Inhi-
bition of TGF-b activation as measured using B5, an affinity- matured more
potent inhibitor of avb8-mediated TGF-b activation than 37E1. Cocultures
of b8-transfected HT1080 cells with transformed mink lung epithelial TGF-b
reporter cells with varying concentrations (mg/ml) of B5 (filled squares) or
37E1 (open squares) reported by relative light units (LU; ×10
3
). n =5ex-
periments. (B)B5ismorepotentthan37E1inblockingTGF-b–dependent
CCL20 secretion by IL-1b–stimulated human lung fibroblasts. ELISA for
measuring CCL20 in culture supernatant from IL-1b–stimulated normal
primary human lung fibroblasts treated with isotype control (open bar),
37E1 (vertical stripes), B5 (filled bar), or 1D11 (horizontal stripes) at 10 mg/ml.
Non–IL-1b–t reated fibroblast controls do not secrete detectable CCL20. n =
5 different patients. ***P <0.001,byANOVAandTukey’s post-test. n.s., not
significant.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 7
on June 24, 2014stm.sciencemag.orgDownloaded from

other integrins noncompetitively inhibit ligand binding (36, 38).
Latency-associated peptide decreased B5 binding to b8-expressing
HT1080 cells by 29% (Fig. 7A). To determine whether this decreased
binding was due to competitive or noncompetitive inhibition, we used
a linear regression model of a solid-phase ligand-binding assay (Fig. 7B).
Pl o t s rev e a l e d a noncompetitive mode of inhibition by B5 (Fig. 7B),
suggesting that B5 binds to a low-affinity conformation of avb8and
acts as a noncompetitive allosteric inhibitor.
Allosteric inhibitors reduce but do not completely eliminate ligand
binding (36). Inde ed, B5 (or 37E1) maximally blocked receptor b inding,
Fig. 6. avb8 is in a stable extended conformation with a closed
headpiece; B5 is associated with inward bending of the b8 head-
hybrid domain angle. (A) Ribbon diagram (PyMOL V1.1r1) of the
extended, closed structure of the b8 subunit generated by homology
modeling (Modeller) (50)toavb3(PDB3IJE)(25). Modeled b8 (green) with
the a1anda7 helices (red) superimposed on avb3 (purple). Red spheres,
atoms of the B5 epitope on the a1 helix (R
133
,F
137
,F
138
). Modeled RGD
tripeptide (blue spheres) based on PDB 3ZDX (51) bound to the ligand-
bindingpocketincomplexwiththeMIDASCa
2+
cation (orange sphere).
The distance from the edge of the ligand-binding pocket (A
115
)toR
133
of the B5 epitope is 28 Å, indicated by dotted arrows. Head, hybrid, and
Psi domains are indicated. The av subunit and leg domains are not included.
(B) Image analysis measuring the hybrid-head domain angles of clasped
and unclasped avb8 with no Fab compared to size exclusion chromatography–
purified avb8-B5 or avb8–clone 68 Fab complexes. n = 15, 24, 10, 15, 37,
and 30 measurements from class averages of clasped alone (filled squares),
clasped+B5 Fab (open upward triangles), clasped+clone 68 Fab (filled
diamonds), unclasped alone (open downward triangles), unclasped+B5
Fab (closed circles), and unclasped+clone 68 Fab (open squares), respec-
tively. **P < 0.01, ***P < 0.001, by ANOVA and Tukey’s post-test. (Insets)
Representative electron microscopy class averages. Average head-hybrid
domain angles shown below the micrographs. Scale bar, 10 nm. Cartoons
show bound Fab with head (bI) and hybrid domain angles. (C)Rendered
avb8 space filling model (PyMOL V1.1r1) of the closed headpiece structure
of the avb8 subunit, as above, with docked prototype Fab (SG/19: PDB
3VI3, translucent gray with black outline) to the B5 epitope (R
133
,F
137
,
F
138
) indicated by red spheres, approximating dimensions and orientation
of B5 Fab with inward bending of the hybrid domain. Mod eled b8 (green)
with av (light blue). RGD tripeptide (orange spheres) bound to ligand-
binding pocket and angles of the closed head-hybrid domain (black lines).
(D) Space-filling avb8 generated homology model, as above, except with a
rendering of docked 68 Fab.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 8
on June 24, 2014stm.sciencemag.orgDownloaded from

TGF-b activation, or TGF-b–dependent gene expression by 60 to 90%
(Figs. 1B, 4F, and 7, C to E, and figs. S12, S14, and S15). We hypothe-
sized that inducti on of a lo w-affinity state that changes the shape of the
ligand-binding pocket of avb8 would increase the ability of a linear
RGD peptide to compete for latency-associated peptide binding. An
RGD peptide based on the latency-associated peptide of the TGF-b
1 RGD binding loop normally blocks avb8–latency-associated peptide
interactions at high concentrations, because RGD peptides must b e con-
strained into conformations that mimic the natural ligands from which
they were derived to achieve high affinity for integrins (39). Satu rat i ng
concentrations of B5 had no significant effect on cell adhesion, but
allowed RGD peptide to block adhesion completely at concentrations
where RGD peptide alone had minimal effect, suggesting that B5 in-
duced a low-affinity state where binding of a linear RGD peptide could
more easily compete with latency-associated peptide (Fig. 7C). B5
maximally blocked soluble avb8 receptor binding or TGF-b activation
by 92 or 68%, respectively, and the remaining binding or activation
could be blocked completely by high concentrations of RGD peptide
(Fig. 7, D and E). Together, these data demonstrate that B5 allosteri-
cally induces a low-affinity state that has minimal impact on cell ad-
hesion, but markedly inhibits soluble receptor binding and TGF-b
activation.
DISCUSSION
This study addresses the hypothesis that selective targeting of TGF-b
activation with an b8 antibody can treat airway remodeling. Airway
remodeling in severe asthma and COPD is refractory to current thera-
pies. Here, we present development of an antibody, B5, that selec tively
Fig. 7. B5 is a noncompetitive allosteric inhibitor that induces a low-
affinity state. (A) Latency-associated peptide decreases B5 binding to
avb8- expressing HT1080 cells. b8-expressing HT1080 cells stained with B5
(2 mg/ml) with increasing concentrations of latency -associated peptide (LAP)
reported as mean fluorescence intensity (MFI) (n =6).(B) Lineweaver-Burk
plots of solid-phase binding assays of avb8-AP binding to latency-associated
peptide with two different concentrations of B5 (squares), or RGD peptide
(circles) as a competitive inhibitor control, or no inhibitor (triangles-dotted
line). B5 plots show similar x intercepts as uninhibited receptor but different
slopes and y intercepts consistent with noncompetitive inhibition. RGD
plots intersect above the x axis with the uninhibited receptor consistent
with a competitive mode of inhibition. Representative of two experiments
with similar results. (C to E) B5 induces low-affinity binding sufficient to me-
diate cell adhesion, but insufficient to support TGF-b activation. (C) b8-
expressing 293 cells adhered to latency-associated peptide with saturating
concentrations of RGD peptides and B5 (filled squares, solid line) or isotype
control (open squares, hashed line). Assays performed as in Fig. 3A. n =3.
**P <0.01byunpairedStudent’s t test. (D) B5 induces a low-affinity state
maximally inhibiting the binding of soluble avb8 to latency-associated pep-
tide by 92% in the presence of RGE peptide (filled inverted triangles, hashed
lines); remaining binding blocked completely by RGD peptide (filled
squares, solid line). Boxed magnified area of the highest B5 and RGD/E con-
centrations shows small amount of residual binding remaining with B5 and
RGE peptide completely blocked by B5+RGD. Isotype with RGD peptide
(open squares, solid line) or RGE peptide (open inverted triangles, hashed
line). *P = 0.039 by nonlinear regression and F test of the bottom of each
data set. (E) B5 blocks TGF-b activation by ~70%, and the addition of RGD
peptide completely blocks remaining activation. b8-transfected 293 cells
with RGD peptide and saturating concentrations of B5 (filled squares, solid
line) or isotype control (open squares, hashed lines) at the indicated con-
centrations. Mock-transfected 293 cells with RGD peptide and saturating
concentrations of B5 (filled diamonds, solid line) or isotype control (open
triangles, hashed lines). n =4.***P < 0.001, by ANOVA and Tukey’s post-test
of the highest concentration of RGD peptide and antibodies.
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 9
on June 24, 2014stm.sciencemag.orgDownloaded from

blocks avb8-mediated TGF-b activation, airway inflammation, and fi-
brosis in vivo. We use mouse models that phenocopy many of the
relevant features of human airway disease to illuminate the mechanistic
connections between TGF-b and the pathologic, cellular, and bio-
chemical endpoints that are altered in human COPD specimens.
Therapeutic effects of B5 in disease models
We have recently characteri zed Ad-IL-1b and chronic ovalbumin air-
way remodeling mouse models and found that they mimic key path-
ologic, chemokine, and cytokine features of human airway remodeling
in both COPD and asthma (10). Here, extensive characterization dem-
onstrates the validity of the combined cigarette smoke–poly(I:C) air-
way remodeling system because proinflammatory and profibrogenic
cytokines that are increased in human disease biospecimens [that is,
IL-1b (16), CCL2 (40), CCL20 (17), and IL-17 (41)] were also in-
creased in mice exposed to cigarette smoke–poly(I:C). These increases
were not observed in mice treated only with cigarette smoke, and were
greatly amplified by the concomitant exposure to the viral mimetic
poly(I:C). These data add to the evidence that viral infection plays
an important role as a cofactor in airway remodeling. B5 treatment
decreased airway inflammation and inflammatory mediator produc-
tion in mice exposed to Ad-IL-1b,ovalbumin,andcigarettesmoke–
poly(I:C), as well as chemokine production by stimulated human lung
fibroblasts, suggesting that B5 inhibits a central and common fibro-
inflammatory mechanism involving lung fibroblasts.
These data support a general model of the pathologic function of
avb8inTGF-b activation in the airway, whereby active IL-1b and
TGF-b togethe r increase fibro blast chemokine ex pression , DC recruit-
ment, and priming of pathologic adaptive immunity, which culminates
in obstructive airway physiology. This mechanistic cascade assumes that
DC behavior is indirectly influenced by fibroblasts. However, avb8-
mediated TGF-b activation directly by DCs has also been shown to increase
priming of a/b CD4
+
IL-17–producing cells in asthma models (33), sug-
gesting that B5 also has the capacity to directly influence DC function
(10, 33). Whatever the case, the function of avb8 by the relatively
restricted cell types that support avb8-mediated TGF-b activation shares
in the common alterations of proinflammatory cytokines. In particular,
IL-17A provides a common mec hanistic basis for the therapeutic effects
of B5 in a variety of injury and fibroinflammation models in the lung
and other organs where avb8 is expressed (that is, kidney and liver).
However, current mouse models do not replicate the full spectrum
of human COPD (that is, progressive fibrotic airway wall thickening
and physiologic fixed airway obstruction). These differences reflect
fundamental biologic and anatomic differences between humans
and mice. For instance, mice lack subsegmental and respiratory bron-
chioles, which are the major sites of fixed airway obstruction in hu-
mans (42). Despite these shortcomings, the inhibiting effects of B5 on
cigarette smoke–poly(I:C)–indu ced airspace enlargement and airway
hyperresponsiveness in mice suggest that B5 may delay disease pro-
gression or prevent the exacerbation of COPD symptoms. The preven-
tion of airspace enl argemen t by B5 is notable because loss of function
of the TGF-b–activating integrin avb6 causes spontaneous airspace
enlargement (43), demonstrating a fundamental difference in the
biology of avb8comparedtoavb6.
The role of integrin conformation in avb8 function
Mechanism-of -action studies performed on the process of therapeutic
development of B5 reveal that avb8 is in a constitutive ly active
extended-closed conformation. This conclusion is reinforced by (i)
lack of evidence that avb8 assumes either a bent or an extended-open
conformation by size exclusion chromatography and negative electron
microscopy staining; (ii) avb8 has a naturally occurring glycan wedge
(N414) in a nearly identical location to a mutationally introduced gly-
can that constitutively activates and opens the a5b1 integrin headpiece
but does not cause headpiece opening of avb8; (iii) introduction of a
second glycan (N294) that constitutively activates the b3integrinsbut
does not further increase the functio n of avb8. These data reinforce
predictions of an extended constitutively active avb8conformation
based on domain-swapping of the “knee” region of the b8 subunit (44)
and add to the debate about the relative importance of “swi tchblade” and
bent integrin activation models (22, 25, 26) by demonstrating that the
extended-closed conformation can be stable and functional.
The constitutive activity of avb8 raises questions about how avb8-
mediated TGF-b activation is regulated. Current evidence suggests
that metalloproteolytic cleavage of latent TGF-b bound to the cell sur-
face is involved in the activation mechanism and thus could indirectly
modulate avb8function(20). Hence, avb8 could position latency-as-
sociated peptide for metallopr oteases to cleave the latency-associated
peptide and release mature TGF-b from the cell surface (20). Evidence
would argue against mechanical forces induced from the integrin to
latent TGF-b as causing avb8-med iated TGF-b activation. Such
mechanical forces require dynamic integrin conformational changes
or tension transduced from the cytoskeleton to integrin cytoplasmic
domains to cell- or matrix-tethered latent TGF-b. These dynamic con-
formational changes are not seen with a vb8 because it adopts mainly
a single conformation, and the b8 cytoplasmic domain is not required
for cell adhesion and TGF-b activation (20).
Conformational changes induced by B5
The B5 epitope located at the head-hybrid junction would be pre-
dicted to be altered by hybrid domain swing-out, as illustrated by
the a5b1 glycan (N429) wedge mutant (36). B5 retains its ability to
bind to and inhibit the function of the b8 glycan mutant, suggesting
that the b8 hybrid domain does not swing-out even with two glycans
occupying the vert ex of the head-h ybrid do main angle. N egativ e elec-
tron microscopy staining is consistent with a stabilizing effect of B5 on
an exaggerated closed-head conformation with a ~20° inward swi ng of
the hybrid domain. This conformation was not well represented in neg-
ative electron microscopy stains when bound to the non–function-
blocking clone 68 Fab, suggesting that it is not thermodynamically
favored and thus represents a rare low-affinity conformer.
Regulation of avb8 affinity for binding to latent TGF-b
Integrin hybrid domain motions are coupled to changes in the ligand-
binding pocket. Exactly how the inward hybrid domain motion seen
with the B5 Fab is coupled to changes in the ligand-binding pocket
remains to be determined, but some predictions can be made from
the extensive structural literature involving other integrins. In partic-
ular, two of the homologous residues comprising the B5 epitope (R
133
and F
137
)onthea1helixofbI domains of other integrins are involved
in allosteric effects on ligand binding and affect hybrid domain mo-
tions, but none cause inward bending of the hybrid domain (36, 37).
Positional changes of the a1 helix affect the neighboring a7helix,
which is directly coupled to the hybrid domain. Such coordinated
changes would be required for inw ard bending of the hybrid domain.
The lack of conservation of the b8 ADMIDAS domain indicates that
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 10
on June 24, 2014stm.sciencemag.orgDownloaded from

such coordinated positional changes are different for b8compared
withotherintegrins.Inallotherintegrinb-subunits, the ADMIDAS
cation links the top of the a1 helix to the cation of the MIDAS do-
main, which forms the edge of the ligand-binding pocket that coor-
dinates the aspartate of RGD (29). We speculate that binding of B5 to
the C terminus of the a1 helix affects the positioning of the top of
the b8 a1 helix, which is relatively nonconstrained by the lack of the
ADMIDAS, changing the shape of the ligand-binding pocket into a
low-affinity state. Such allosteric effects are consistent with the ob-
served attenuated latency-associated peptide binding to the avb8-B5
complex and the improved ability of linear RGD peptides to inhibit
the residual binding to this complex.
Separation of TGF-b activation and cell adhesion
functions of avb8
Therapeutics targeting avb8-mediated TGF-b activation but not cell
adhesion likely have improved safety profiles compared to therapies
that inhibit both of these functions. For example, genetic deficiency
of avb8 is associated with a paradoxical increase in TGF-b activation
in the kidney, presumably due to unmasking of avb8-independent
pathways activated by TGF-b (45). Our results suggest that allosteric
inhibition of avb8 by B5 produces highly specific targeting of the
TGF-b pathwayonlyinthecellsexpressingavb8 with minimal non–
TGF-b–spec ific effects , providing a safer therapy than ligand-mim etic
avb8 inhibitors. This expectation is borne out by the observ ation that
B5 does not appear to cause toxicities in treated mice.
Mechanistically, avb8-mediated TGF-b activation differs from avb8-
mediated cell adhesion to latency-asso ciated peptide in that adhesion
was not affected by affinity changes induced by B5. Cell adhesion re-
quires integrin clustering, which effectively increases receptor avidity
(46). However, clustering is apparently not required for avb8-mediated
TGF-b activation, because B5 blocks TGF-b activation under conditions
where it does not block adhesion. This suggests that the TGF-b–activating
function of avb8 requires a high-affinity state, whereas cell adhesion
requires only a low-affinity state. This property of B5 could explain why
the transcriptomes of fibroblasts treated with B5 or anti–TGF-b are
almost identical, with no integrin adhesion–specific effects.
In summary, the constitutively active integrin avb8 can be targeted
by an allosteric inhibitor to prevent experimental airway disease. B5
has now been fully humani zed and is currently in preclinic al development.
MATERIALS AND METHODS
Study design
We sought to develop a therapeutic strategy to selectively target the
TGF-b pathway to treat airway remodeling in COPD. To accomplish
this, studies were designed in five parts: (i) to determine the active
conformation of the TGF-b activating integrin avb8; (ii) to develop
a therapeutic neutralizing antibody designed to the active confor-
mation; (iii) to test the mechanism of action of the therapeutic anti-
body; (iv) to develop airway disease models that are relevant to human
pathophysiology; (v) to use the therapeutic antibody for efficacy and
mechanism-of-action studies in mice designed to regulate human
avb8 under the contro l of the human promoter. Sample sizes were pre-
determined for cell culture, biochemistry, and murine experiments
based on large expected effect sizes (based on pilot studies d >0.8)re-
quiring an n =3to6(a =0.5,b =0.8).Fortheovalbuminexperiment,
effect sizes were only moderate, requirin g n = 12. Data collection was
terminated when these sample sizes were met, with the exception of
secreted receptor data for which unanticipated interassay variation
necessitated larger sample sizes (n = 8). For studies using primary hu-
man cells, a minimum of three (gene array) and a maximum of five
normal donors (CCL20 ELISA) were predetermined on the basis of
practical considerations of maintaining simultaneously multiple primary
cultures. No formal outlier analysis was performed.
Production of BAC ITGB8 Tg mice
Tg mice expressing human ITGB8 were generated to test the efficacy
of B5 in disease models. The human BAC successfully rescued the le-
thal phenotype associated with deficiency of mouse itgb8. Genetic
germline deletion of itgb8 is lethal in mice by postnatal day 0 in inbred
strains and lethal within 3 months in mixed or outbred strains (30, 32).
Mice die of complications of developmental defects in vasculogenesis
or neonatal hemorrhage/neurogenesis (30, 32). DC-specific deletion
(CD11c-cre) results in mice that develop autoimmune colitis and die
by 6 months (31). A human chromosome BAC (RP11-431K20) con-
taining the entire 80-kb ITGB8 gene and 70 and 30 kb of 5′ and 3′
flanking regions, respectively, was purified (Nucleobond, Clontech),
determined to be intact by pulse-field electrophoresis, microinjected
into pronuclei of FVB/N mice zygotes in microinjection buffer [10 mM
tris, 0.1 mM EDTA (pH 7.4)], and surgically transferred to oviducts
of pseudopregnant females. Twenty-eight pups survived and tail tip
DNA was screened using BAC end primers, 5′ BAC end pair (forward
5′-CCTGTGTAACTACCACCAC-3′; reverse 5′-CTCACTTGACAA-
TCTAGTCCTC-3′), and 3′ BAC end pair (forward 5′-CAACCAA TC-
AGTAGCACCC-3′; reverse 5′-CTCAGTGAGATCTGTTATGAAC-3′).
Four founders (lines A to D) had completely integrated copies of
the full-length transgene and were crossed to C57B/6 itgb8
+/−
mice.
Lines B to D successfully transmitted the transgene to progeny to gen-
erate BAC; itgb8
+/−
mice on a mixed FVB/C57B/6 background. Sibling
matings generated mice heterozygous or homozygous for the BAC
and homozygous for the itgb8 knockout allele.
Antibody dosing
Initial dosing for B5 was based on dose-response curves generated
with the B-line of mice where 7 mg/kg outperformed lower doses after
IT-Ad-Il-1b provocation (Fig. 1B and fig. S4). To replicate these find-
ings in an independent line of BAC Tg mice, C-line mice were dosed
with B5 or isotype control (7 mg/kg), and the experiment was re-
peated (Fig. 1, I to K). The timing of the first antibody administration
was based on the observation that the maximal inflammation in the
Ad-IL-1b model occurs at 7 days after intratracheal injection (10).
Thus, administration of B5 was initiated at 7 days after intratracheal
injection. In the cigarette smoke–poly(I:C) model, B5 or isotype anti-
body administration was begun 1 week after cigarette smoke exposure,
at a point when significant lung neutrophilia was observed. For the
ovalbumin experiment, antibody dosing was initiated 1 day before an-
tigen challenge.
Cigarette smoke and poly(I:C) exposure
B-line mice were exposed using a whole-body cigarette smoke expo-
sure system (Teague Enterprises) within a barrier facility. Mice are
acclimated using increasing smoke exposuresfor5daysstartingata
total suspended particulates (TSP) of 40 mg/m
3
for 1 hour, and in-
creasing incrementally to final smoke exposures of 100 TSP using
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 11
on June 24, 2014stm.sciencemag.orgDownloaded from

3R4F cigarettes. Full-dose exposures were begun in we ek 1 with 5 hours
of continuous exposure, with rest on weekends. In week 2, intranasal
doses of poly(I:C) (Invivogen, 50 mg per dose) were given on days
9 and 12, and again in week 3 on days 15 and 18.
Electron microscopy
Samples were applied to g low-dischar ged carbon -coated coppe r grids,
washed, stained with 0.75% (w/v) uranylformate, and aspirated to dry-
ness (47). Images were taken on a Tecnai T12 electron microscope
(FEI Company) equipped with a LaB6 filament and operating at a
120-kV acceleration voltage. Micrographs were recorded using a 4K
charge-coupled device camera (UltraScan 4000, Gatan Inc.), where
one pixel equals 2.21 Å on the specimen. Particles were selected man-
ually. Individual particles were windowed out from two binned images
and wer e subject ed to six cycles of mul tirefer ence align ment and K-means
classification using SPIDER to generate well-resolved class averages
(48). The number of class-averag es to capture the sample heterogene ity
was adjusted between 20 and 50. Manual selection was used to assign
obviously related extended conformations into subclasses: extended-
closed, extended-intermediate, and bent. Bent conformations were
assigned when the headpiece-tailpiece angle was less than 90°. The head-
hybrid angles were determined from class averages where the head
and hybrid axis was oriented to allow for definitive angle assignment
[ImageJ 1.42q, National Institutes of Health (NIH)]. Images where the
assignment was ambiguous were not included in the analysis. Individ-
ual angles were measured from class averages where the head and
hybrid domains were well-resolved (ImageJ).
Statistical analysis
For microarray analysis, the data set was normalized using Lowess
normalization. No backgroun d subtraction was performed, and
median feature pixel intensity was used as the raw signal before nor-
malization. A one-way ANOVA line ar model was fit to each compar-
ison of interest to estimate log
2
fold change and calculated B statistic.
All procedures were carried out using functions in the R package limma
inBioconductor.Toregressthelogratiosof(anti-b8/control) against
the log ratios of (anti–TGF-b/control ) among differentiall y expressed
genes, we used log
2
(anti-b8/control) = a + b *log
2
(anti–TGF-b/control)
to obtain intercept and slope estimates. To identify differentially ex-
pressed genes, a B statistic of B ≥ 0 was used. All other data are re-
ported as means ± SEM. Comparisons between two different groups
were determined using Student’s t test. One-way ANOVA was used
for multiple comparisons, and Tukey’sorBonferroni’s post hoc test
was used to test for statistical significance, as indicated. Significance
was defined as P < 0.05. All statistical analyses were performed using
the software package Prism 4.0b (GraphPad Software).
SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/6/241/241ra79/DC1
Materials and Methods
Fig. S1. ITGB8 BAC Tg mice express avb8 at similar expression levels and tissue distribution to
humans.
Fig. S2. ITGB8 BAC transgene rescues early lethality of mouse itgb8 deficiency.
Fig. S3. Secreted human avb8 integrin–placental AP fusion proteins bind to murine latency-
associated peptide.
Fig. S4. Dose response of B5 antibody treatment of intratracheal Ad-IL-1b–injected B-line BAC
ITGB8 Tg mice.
Fig. S5. Antibody treatment with B5 does not have any effect on lung morphology.
Fig. S6. Cigarette smoke and poly(I:C) synergistically produce airway disease that resembles
COPD in humans.
Fig. S7. Effects of combined exposure of cigarette smoke and poly(I:C) on inflammation and
inflammatory mediators.
Fig. S8. B5 antibody treats allergic airway inflammation.
Fig. S9. Electron microscopy of integrin avb8.
Fig. S10. b8 antibody epitope mapping.
Fig. S11. Non–function-blocking antibodies binding to the Psi, hybrid, or epidermal growth
factor (EGF) 1–2 domains.
Fig. S12. Genome-wide comparison of the effects of b8 and TGF-b neutralizing antibodies on
human fibroblast gene expression.
Fig. S13. V
H
and V
L
sequences of 37E1 and B5.
Fig. S14. B5 improves the ability of 37E1 to inhibit the binding of soluble avb8 to latency-
associated peptide.
Fig. S15. B5 specifically blocks binding of avb8, and not avb6, to latency-associated peptide.
Fig. S16. Gel filtration of clasped or unclasped avb8 in complex with B5 Fab.
Fig. S17. Electron microscopy of integrin avb8.
Table S1. Fibroblast differentially expressed gene array data.
Table S2. Autocrine TGF-b activation mediated by avb8 in human fetal tracheal fibroblasts.
References (52–62)
REFERENCES AND NOTES
1. Centers for Disease Control and Prevention (CDC), Chronic obstructive pulmonary disease
among adults—United States, 2011. MMWR Morb. Mortal. Wkly. Rep. 61, 938–943 (2012).
2. D. S. Postma, W. Timens, Remodeling in asthma and chronic obstructive pulmonary disease.
Proc. Am. Thorac. Soc. 3, 434–439 (2006).
3. M.K.Han,E.A.Kazerooni,D.A.Lynch,L.X.Liu,S.Murray,J.L.Curtis,G.J.Criner,V.Kim,
R.P.Bowler,N.A.Hanania,A.R.Anzueto,B.J.Make,J.E.Hokanson,J.D.Crapo,E.K.Silverman,
F. J. Martinez, G. R. Washko; COPDGene Investigators, Chronic obstructive pulmonary disease
exacerbations in the COPDGene study: Associated radiologic phenotypes. Radiology 261,
274–282 (2011).
4. T. M. Wilkinson, G. C. Donaldson, S. L. Johnston, P. J. Openshaw, J. A. Wedzicha, Respiratory
syncytial virus, airway inflammation, and FEV1 decline in patients with chronic obstructive
pulmonary disease. Am. J. Respir. Crit. Care Med. 173, 871–876 (2006).
5. E. W. Uhl, W. L. Castleman, R. L. Sorkness, W. W. Busse, R. F. Lemanske Jr., P. K. McAllister,
Parainfluenza virus-induced persistence of airway inflammation, fibrosis, and dysfunction
associated with TGF-beta 1 expression in brown Norway rats. Am. J. Respir. Crit. Care Med.
154, 1834–1842 (1996).
6. P. J. Sterk, Virus-induced airway hyperresponsiveness in man. Eur. Respir. J. 6, 894–902 (1993).
7. M. J. Kang, C. G. Lee, J. Y. Lee, C. S. Dela Cruz, Z. J. Chen, R. Enelow, J. A. Elias, Cigarette
smoke selectively enha nces viral PAMP– and virus-induced pulmonary innate immune
and remodeling responses in mice. J. Clin. Invest. 118 , 2771–2784 (2008).
8. A. Churg, S. Zhou, X. Wang, R. Wang, J. L. Wright, The role of interleukin-1b in murine
cigarette smoke-induced e mphysema and s mall airway remodeling. Am. J. Respir. Cell
Mol. Biol. 40, 482–490 (2009).
9. M. Podowski, C. Calvi, S. Metzger, K. Misono, H. Poonyagariyagorn, A. Lopez-Mercado, T. Ku,
T. Lauer, S. McGrath-Morrow, A. Berger, C. Cheadle, R. Tuder, H. C. Dietz, W. Mitzner, R. Wise,
E. Neptune, Angiotensin receptor blockade attenuates cigarette smoke–induced lung injury
and rescues lung architecture in mice. J. Clin. Invest. 122,229–240 (2012).
10. H.Kitamura,S.Cambier,S.Somanath,T.Barker,S.Minagawa,J.Markovics,A.Goodsell,
J. Publicover, L. Reichardt, D. Jablons, P. Wolters, A. Hill, J. D. Marks, J. Lou, J. F. Pittet, J. Gauldie,
J. L. Baron, S. L. Nishimura, Mouse and human lung fibroblasts regulate dendritic cell
trafficking, airway inflammation, and fibrosis through integrin avb8–mediated activation of
TGF-b. J. Clin. Invest. 121, 2863–2875 (2011).
11. S. L. Nishimura, Integrin-mediated transforming growth factor-b activa tion, a potential
therapeutic target in fibrogenic disorders. Am. J. Pathol. 175, 1362 –1370 (2009).
12. A.Vitsky,J.Waire,R.Pawliuk,A.Bond,D.Matthews,E.Lacasse,M.L.Hawes,C.Nelson,
S. Richards, P. A. Piepenhagen, R. D. Garman, L. Andrews, B. L. Thurberg, S. Lonning, S. Ledbetter,
M. C. Ruzek, Homeostatic role of transforming growth factor- b in the oral cavity and esoph-
agus of mice and its expression by mast cells in these tissues. Am. J. Pathol. 174, 2137–2149
(2009).
13. M. J. Anderton, H. R. Mellor, A. Bell, C. Sadler, M. Pass, S. Powell, S. J. Steele, R. R. Roberts,
A. Heier, Induction of heart valve lesions by small-molecule ALK5 inhibitors. Toxicol. Pathol. 39,
916–924 (2011).
14. J. A. Markov ics, J. Araya, S. Cambier, S. Soma nath, S. Gli ne, D. Jablons, A. Hill, P. J. Wolters,
S. L. Nishimura, Interleukin-1b induces increased transcription al activat ion of the
transforming growth factor-b-activa ting int egrin subunit b8 through altering chromatin
architecture. J. Biol. Chem. 286,36864–36874 (2011).
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 12
on June 24, 2014stm.sciencemag.orgDownloaded from

15. S. Eltom, C. S. Stevenson, J. Rastrick, N. Dale, K. Raemdonck, S. Wong, M. C. Catley, M. G. Belvisi,
M. A. Birrell, P2X7 receptor and caspase 1 activation are central to airway inflammation ob-
served after exposure to tobacco smoke. PLOS One 6, e24097 (2011).
16. E. Sapey, A. Ahmad, D. Bayley, P. Newbold, N. Snell, P. Rugman, R. A. Stockley, Imbalances
between interleukin-1 and tumor necrosis factor agonists and antagonists in stable COPD.
J. Clin. Immunol. 29, 508–516 (2009).
17. I. K. Demedts, K. R. Bracke, G. Van Pottelberge, D. Testelmans, G. M. Verleden, F. E. Vermassen,
G. F. Joos, G. G. Brusselle, Accumulation of dendritic cells and increased CCL20 levels in the
airways of patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med.
175,998–1005 (2007).
18. M. Shi, J. Zhu, R. Wang, X. Chen, L. Mi, T. Walz, T. A. Springer, Latent TGF-b structure and
activation. Nature 474, 343–349 (2011).
19. J. Araya, S. Cambier, A. Morris, W. Finkbeiner, S. L. Nishimura, Integrin-mediated transforming
growth factor-b activation regulates homeostasis of the pulmonary epithelial-mesenchymal
trophic unit. Am. J. Pathol. 169, 405–415 (2006).
20. D.Mu,S.Cambier,L.Fjellbirkeland,J.L.Baron,J.S.Munger,H.Kawakatsu,D.Sheppard,
V. C. Broaddus, S. L. Nishimura, The integrin avb8 mediates epithelial homeostasis through
MT1-MMP–dependent activation of TGF-b1. J. Cell Biol. 157,493–507 (2002).
21. J. Takagi, B. M. Petre, T. Walz, T. A. Springer, Global conformational rearrang ements in
integrin extracellular domains in outside-in and inside-out signaling. Cell 110, 599–511
(2002).
22. X. Dong, L. Z. Mi, J. Zhu, W. Wang, P. Hu, B. H. Luo, T. A. Springer, aVb3 integrin crystal
structures and their functional implications. Biochemistry 51, 8814–8828 (2012).
23. F. Ye, G. Hu, D. Taylor, B. Ratnikov, A. A. Bobkov, M. A. McLean, S. G. Sligar, K. A. Taylor, M. H. Ginsberg,
Recreation of the terminal events in physiological integrin activation. J. Cell Biol. 188, 157–173
(2010).
24. J. A. Askari, P. A. Buckley, A. P. Mould, M. J. Humphries, Linking integrin conformation to
function. J. Cell Sci. 122, 165–170 (2009).
25. J. P. Xiong, B. Mahalingham, J. L. Alonso, L. A. Borrelli, X. Rui, S. Anand, B. T. Hyman, T. Rysiok,
D. Müller-Pompalla, S. L. Goodman, M. A. Arnaout, Crystal structure of the complete integrin
aVb3 ectodomain plus an a/b transmembrane fragment. J. Cell Biol. 186,589–600 (2009).
26. T. Schürpf, T. A. Springer, Regulation of integrin affinity on cell surfaces. EMBO J. 30, 4712–4727
(2011).
27. M. Moyle, M. A. Napier, J. W. McLean, Cloning and expression of a divergent integrin subunit
b
8
. J. Biol. Chem. 266, 19650–19658 (1991).
28. S. L. Nishimura, D. Sheppard, R. Pytela, Integrin avb8. Interaction with vitronectin and func-
tional divergence of the b8 cytoplasmic domain. J. Biol. Chem. 269, 28708–28715 (1994).
29. J. P. Xiong, T. Stehle, R. Zhan g, A. Joachimiak, M. Frech, S. L. Goodman, M. A. Arnaout,
Crystal structure of the extracellular segment of integrin aVb3 in complex with an Arg-Gly-
Asp ligand. Science 296,151–155 (2002).
30. P. Aluwihare, Z. Mu, Z. Zhao, D. Yu, P. H. Weinreb, G. S. Horan, S. M. Violette, J. S. Munger,
Mice that lack activity of avb6- and avb8-integrins reproduce the abnormalities of Tgfb1-and
Tgfb3-null mice. J. Cell Sci. 122,227–232 (2009).
31. M. A. Travis, B. Reizis, A. C. Melton, E. Masteller, Q. Tang, J. M. Proctor, Y. Wang, X. Bernstein,
X. Huang, L. F. Reichardt, J. A. Bluestone, D. Sheppard, Loss of integrin a
v
b
8
on dendritic
cells causes autoimmunity and colitis in mice. Nature 449, 361–365 (2007).
32. J.Zhu,K.Motejlek,D.Wang,K.Zang,A.Schmidt,L.F.Reichardt,b8 integrins are re-
quired for vascu lar morphogenesis in mouse embryos. Development 129, 2891–2903
(2002).
33. M. Kudo, A. C. Melton, C. Chen, M. B. Engler, K. E. Huang, X. Ren, Y. Wang, X. Bernstein, J. T. Li,
K. Atabai, X. Huang, D. Sheppard, IL-17A produced by ab T cells drives airway hyperrespon-
siveness in mice and enhances mouse and human airway smooth muscle contraction.
Nat. Med. 18,547–554 (2012).
34. I. D. Campbell, M. J. Humphries, Integrin structure, activation, and interactions. Cold Spring
Harb. Perspect. Biol. 3, a004994 (2011).
35. B. H. Luo, T. A. Springer, J. Takagi, Stabilizing the open conformation of the integrin
headpiece with a glycan wedge increases affinity for ligand. Proc. Natl. Acad. Sci. U.S.A.
100, 2403–2408 (2003).
36. M. Nagae, S. Re, E. Mihara, T. Nogi, Y. Sugita, J. Takagi, Crystal structure of a5b1 integrin
ectodomain: Atomic details of the fibronectin receptor. J. Cell Biol. 197, 131–140 (2012).
37. X. Chen, C. Xie, N. Nishida, Z. Li, T. Walz, T. A. Springer, Requirement of open headpiece
conformation for activation of leukocyte integrin a
X
b
2
. Proc. Natl. Acad. Sci. U.S.A. 107,
14727–14732 (2010).
38. A. P. Mould, S. K. Akiya ma, M. J. Humphries, The inhibitory anti-b1 integrin monoclonal
antibody 13 recognizes an epitope that is attenuated by ligand occupancy. Evidence
for allosteric inhibition of integrin function. J. Biol. Chem. 271 , 20365–20374 (1996).
39. J. S. Tung, J. A. Jakubowski, W. F. Heath, B. G. Utterback, D. K. Herron, Correlation of mo-
lecular shape with GPIIb-IIIa receptor antagonist activity in RGD peptides. Receptor 3, 343–356
(1993).
40. S. F. Liu, C. H. Chin, C. C. Wang, M. C. Lin, Correlation between serum biomarkers and BODE
index in patients with stable COPD. Respirology 14, 999–1004 (2009).
41. C.Doe,M.Bafadhel,S.Siddiqui,D.Desai,V.Mistry,P.Rugman,M.McCormick,J.Woods,R.May,
M. A. Sleeman, I. K. Anderson, C. E. Brightling, Expression of the T helper 17-associated cytokines
IL-17A and IL-17F in asthma and COPD. Chest 138, 1140–1147 (2010).
42. J. A. Vanoirbeek, M. Rinaldi, V. De Vooght, S. Haenen, S. Bobic, G. Gayan-Ramirez, P. H. Hoet,
E. Verbeken, M. Decramer, B. Nemery, W. Janssens, Noninvasive and invasive pulmonary
function in mouse models of obstructive and restrictive respiratory diseases. Am. J. Respir.
Cell Mol. Biol. 42,96–104 (2010).
43. D.G.Morris,X.Huang,N.Kaminski,Y.Wang,S.D.Shapiro,G.Dolganov,A.Glick,D.Sheppard,
Loss of integrin avb6-mediated TGF-b activation causes Mmp12-dependent emphysema.
Nature 422,169–173 (2003).
44. B. J. Smagghe, P. S. Huang, Y. E. Ban, D. Baker, T. A. Springer, Modulation of integrin ac-
tivation by an entropic spring in the b-knee. J. Biol. Chem. 285, 32954–32966 (2010).
45. S. Khan, S. Lakhe-Reddy, J. H. McCarty, C. M. Sorenson, N. Sheibani, L. F. Reichardt, J. H. Kim,
B. Wang, J. R. Sedor, J. R. Schelling, Mesangial cell integrin avb8 provides glomerular endo-
thelial cell cytoprotection by sequestering TGF-b and regulating PECAM-1. Am. J. Pathol. 178,
609–620 (2011).
46. T. A. Bunch, Integrin aIIbb3 activation in Chinese hamster ovary cells and platelets in-
creases clustering rather than affinity. J. Biol. Chem. 285, 1841–1849 (2010).
47. M. Ohi, Y. Li, Y. Cheng, T. Walz, Negative staining and image classification—Powerful tools
in modern electron microscopy. Biol. Proced. Online 6,23–34 (2004).
48. J. Frank, M. Radermacher, P. Penczek, J. Zhu, Y. Li, M. Ladjadj, A. Leith, SPIDER and WEB:
Processing and visualization of images in 3D electron microscopy and related fields.
J. Struct. Biol. 116,190–199 (1996).
49. B. H. Luo, K. Strokovich, T. Walz, T. A. Springer, J. Takagi, Allosteric b
1
integrin antibodies
that stabilize the low affinity state by preventing the swing-out of the hybrid domain.
J. Biol. Chem. 279, 27466–27471 (2004).
50. A. Sali, T. L. Blundell, Comparative protein modelling by satisfaction of spatial restraints.
J. Mol. Biol. 234,779–815 (1993).
51. J. Zhu, J. Zhu, T. A. Springer, Complete integrin headpiece opening in eight steps. J. Cell
Biol. 201, 1053–1068 (2013).
52. W. E. Finkbeiner, in The Lung: Scientific Foundations, R. G. Crystal, Ed. (Lippincott-Raven
Publishers, Philadelphia, PA, 1997), pp. 415–433.
53. T. Oida, H. L. Weiner, TGF-b induces surface latency associated peptide expression on murine
CD4 T cells independent of Foxp3 induction. PLOS One 5, e15523 (2010).
54. A. Razai, C. Garcia-Rodriguez, J. Lou, I. N. Geren, C. M. Forsyth, Y. Robles, R. Tsai, T. J. Smith,
L. A. Smith, R. W. Siegel, M. Feldhaus, J. D. Marks, Molecular evolution of antibody affinity
for sensitive detection of botulinum neurotoxin type A. J. Mol. Biol. 351, 158–169 (2005).
55. J. Araya, S. Cambier, J. A. Markovics, P. Wolters, D. Jablons, A. Hill, W. F inkbeiner, K. Jones,
V. C. Broaddus, D. Sheppard , A. Barzcak, Y. Xiao, D. J. Erle, S. L. Nishimura, Squamous
metaplasia amplifies pathol ogic epithelial-mesenchymal interactions in COPD patien ts.
J. Clin. I nvest. 117, 3551–3562 (2007).
56. J. S. Munger, J. G. Harpel, F. G. Giancotti, D. B. Rifkin, Interactions between growth factors
and integrins: Latent forms of transforming growth factor-b are ligands for the integrin
avb1. Mol. Biol. Cell 9, 2627–2638 (1998).
57. J. W. Kehoe, N. Velappan, M. Walbolt, J. Rasmussen, D. King, J. Lou, K. Knopp, P. Pavlik, J. D. Marks,
C. R. Bertozzi, A. R. Bradbury, Using phage display to select antibodies recognizing post-
translational modifications independently of sequence context. Mol. Cell. Proteomics 5,
2350–2363 (2006).
58. M. T. Ganter, J. Roux, B. Miyazawa, M. Howard, J. A. Frank, G. Su, D. Sheppard, S. M. Violette,
P. H. Weinreb, G. S. Horan, M. A. Matthay, J. F. Pittet, Interleukin-1b causes acute lung injury
via avb5 and avb6 integrin–dependent mechanisms. Circ. Res. 102, 804–812 (2008).
59. M. Kolb, P. J. Margetts, D. C. Anthony, F. Pitossi, J. Gauldie, Transient expression of IL-1b in-
duces acute lung injury and chronic repair leading to pulmonary fibrosis. J. Clin. Invest.
107, 1529–1536 (2001).
60. K. H. Shalaby, L. G. Gold, T. F. Schuessler, J. G. Martin, A. Robichaud, Combined forced
oscillation and forced expiration measurements in mice for the assessment of airway hyper-
responsiveness. Respir. Res. 11, 82 (2010).
61. J. C. Hogg, F. Chu, S. Utokaparch, R. Woods, W. M. Elliott, L. Buzatu, R. M. Cherniack, R. M. Rogers,
F. C. Sciurba, H. O. Coxson, P. D. Paré, The nature of small-airway obstruction in chronic ob-
structive pulmonary disease. N. Engl. J. Med. 350, 2645–2653 (2004).
62. S. E. Gline, S. Cambier, C. Govaerts, S. L. Nishimura, A 50-Å separation of the integrin a
v
b
3
extracellular domain C termini reveals an intermediate activation state. J. Biol. Chem. 279,
54567–54572 (2004).
Acknowledgments: WethankJ.Munger(NewYorkUniversity)forTMLCreportercells,H.Weiner
(Harvard Medical School) for the murine TGF-b1–expressing P3U1 cells, X. Huang [University of
California, San Francisco (UCSF) Sandler Airway Physiology Core], and S. Farr-Jones for editorial
assistance. Funding: This work was supported by NIH HL113032, HL063993, HL090662,
NS044155, UCTRDRP, UCSF Academic Senate, UCOP POC award, Sponsored Research agreement
from MedImmune, LLC (S.L.N.), UCSF Liver Center (P30DK026743, to S.L.N. and J.L.B.), UCSF PBBR
(Y.C.), and Nina Ireland Lung Disease Program (P.W.). Author contributions: S.M. performed all in
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 13
on June 24, 2014stm.sciencemag.orgDownloaded from

vivo experiments and helped to establish the BAC Tg mouse line. J.L., W.W., and J.D.M. helped to
conceive and perform affinity maturation and antibody engineering and to create and prepare
recombinant proteins including integrins and IgGs. R.I.S. performed size exclusion chromatography
experiments, developed ELISAs, and conceived electron microscopy experiments. S.W. and Y.C.
performed and int erpreted electron microscopy experim ents. L.M., P.T., J.C., and R.H. performed
optimization of IgG, affinity measurements, and oversight of IgG production. C.G. and A.C.
provided structural and computational expertise. T.B., S.C., R.M., and K.J.M. performed secreted
binding assays and TGF-b activation assays, and established and characterized the BAC Tg line.
R.C., H.Y., A.G., M.H., H.K., and O.J.B. performed extensive characterization of BAC Tg mice. L.F.R.
provided itgb8
+/−
mice. A.H., D.J., and P.W. provided fresh human lung tissue. J.A., A.J.B., and
D.J.E. performed microarray experiments. J.L.B. helped to produce original hybridomas, and
S.L.N. conceived and oversaw the entire project, and wrote and prepared the manuscript.
Competing interests: Some authors are listed on the following U.S. patents: “Integrin avb8
neutralizing antibody” and “Antibodies that bind integrin avb8” (Nos. 61/305,749 and 61/428,814,
respectively, to J.L., J.D.M., J.L.B., and S.L.N.), and IgG vectors (U.S. Patent Application No. 61/305,749
to J.D.M.). Some of this work was funded by a sponsored research agreement from MedImmune,
LLC. J.L., J.D.M., J.L.B., and S.L.N. have received royalty payments from the Regents of the University
of California for b8 neutralizing antibodies used in this publication. The other authors declare no
competing interests. Data and materials availability: All reagents are property of the UC Regents
and can be obtained under a material transfer agreement.
Submitted 19 November 2013
Accepted 30 May 2014
Published 18 June 2014
10.1126/scitranslmed.3008074
Citation: S.Minagawa,J.Lou,R.I.Seed,A.Cormier,S.Wu,Y.Cheng,L.Murray,P.Tsui,
J. Connor, R. Herbst, C. Govaerts, T. Barker, S. Cambier, H. Yanagisawa, A. Goodsell,
M.Hashimoto,O.J.Brand,R.Cheng,R.Ma,K.J.McKnelly,W.Wen,A.Hill,D.Jablons,
P. Wolters, H. Kitamura, J. Araya, A. J. Barczak, D. J. E rle, L. F. R eichardt, J. D . Marks,
J. L. Baron, S. L. Nishimura, Selective targeting of TGF-b activation to treat fibroinflammatory
airway disease. Sci. Transl. Med. 6, 241ra79 (2014).
RESEARCH ARTICLE
www.ScienceTranslationalMedicine.org 18 June 2014 Vol 6 Issue 241 241ra79 14
on June 24, 2014stm.sciencemag.orgDownloaded from