Selective targeting of TGF-β 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.

Fig. 1. Optimized B5 antibody blocks TGF-β activation in vivo and intratracheal Ad-IL-1β–induced airway inflammation and fibrosis

(A) Schematic of the creation of a mouse model of airway inflammation using adenovirally delivered IL-1β (Ad-IL-1β) administered intratracheally. (B) B5 blocks intratracheal Ad-IL-1β–induced pSMAD2/3, demonstrating that neutralization of αvβ8 inhibits TGF-β activation in vivo. Lung homogenates from mice treated with B5, compared with IgG2a isotype or wild-type (WT) non–Ad-IL-1β injected intratracheally, were evaluated by pSMAD2/3 enzyme-linked immunosorbent assay (ELISA). n = 4, *P = 0.03 by analysis of variance (ANOVA) and post-test for linear trend. (C to H) B5 (D and F) 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-1β. Results expressed as area of inflammation or fibrosis per basement membrane (BM) length. Semiquantitative airway morphometry of standard hematoxylin and eosin (H&E)–stained (C and D) or trichrome-stained (E and F) sections. ***P < 0.0001, by ANOVA and Tukey’s post-test. Scale bar, 200 μm. (I to M) B5 blocks intratracheal Ad-IL-1β–induced inflammation in bronchoalveolar lavage. Total cells in bronchoalveolar lavage (I), macrophages (J), and neutrophils (K), as well as gene transcripts of ITGB8 (L) and col1a2 (M), were increased by intratracheal Ad-IL-1β, and this increase was inhibited by B5. n = 3, Ad-LacZ+isotype– or Ad-LacZ+B5–treated mice; n = 4, Ad-IL-1β+isotype–treated mice; or n = 6, Ad-IL-1β+B5–treated mice. *P < 0.05, ** P < 0.01, ***P < 0.001, by ANOVA and Tukey’s post-test.

Fig. 2. B5 blocks airway inflammation and fibrosis induced by cigarette smoke and the viral mimetic poly(I:C)

(A) Schematic showing 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 μm. 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), and lymphocytes (K). (L to O) B5 blocks the expression of cigarette smoke–poly(I:C)–induced IL-1β (L), CCL2 (M), CCL20 (N), and IL-17 (O). ELISAs were performed on whole-lung lysates for expression of IL-1β, IL-17, and CCL2 or on bronchoalveolar lavage for expression of CCL20. (P) Analysis of pSMAD3 immunostaining. (Q to S) Lung phenotyping. (Q) Mean linear intercept; L(m), estimate of airspace enlargement. (R) Airway resistance with increasing acetylcholine (ach) concentrations (log₂). (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.

Fig. 3. Integrin αvβ8 is constitutively active in a high-affinity extended-closed conformation

(A) Adhesion of β8-expressing 293 cells to latency-associated peptide (LAP) or bovine serum albumin (BSA; control), with Ca²⁺/Mg²⁺ or Mn²⁺, and reported as absorbance (A₅₇₀). n = 3 experiments. (B) Binding of soluble αvβ8–alkaline phosphatase (AP) or αvβ3-AP fusion proteins to latency-associated peptide, or to the αvβ3 ligand fibronectin as a control, with Ca²⁺/Mg²⁺ (open bars) or Mn²⁺ (solid bars) reported as A₄₀₅. n = 8 experiments. ***P < 0.001, by ANOVA and Tukey’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 αvβ8 secreted proteins. Clasped protein or unclasped protein in a solution containing Ca²⁺/Mg²⁺, unclasped protein with an RGD peptide in a solution containing Ca²⁺/Mg²⁺ or Mn²⁺. Particles too large to enter the medium are excluded and this volume is denoted as “void volume (V₀)” 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.

Fig. 4. αvβ8 is constitutively active on the cell surface

(A) Alignment of human β8 and β1 integrin subunits with positions of N-X-T consensus (underlined) and N-linked glycosylation sites in green. Native β8 glycosylation site (N414), adjacent to the mutant N429 glycosylation site in the β1 integrin subunit (49). This is modeled onto a space-filling rendering of homology-modeled (PyMOL V1.1r1) αvβ8 headpiece based on the αvβ3 crystal structure in a closed-conformation [Protein Data Bank (PDB) 3IJE] (25) with GlcNAc2Man7 glycan chain (brown) from human CD2 (1GYA) (49). Green, β-subunit head domain; black, β8-hybrid; blue, αv subunit. (B) Alignment of human β8 and β3 subunits with neo-β8 glycosylation site N294 (green) introduced by the N296T (red) mutation compared with the neo-β3 glycosylation site N303 (green) introduced by the N305T mutation (red) (35). Modeled rendering of the αvβ8 headpiece, as in (A). Glycans in position N414 (brown) and N294 (red) oriented laterally allowing the closed headpiece conformation. (C) Immunoprecipitation with β8 antibody of surface-labeled β8-HT1080 cells transfected with WT β8 (lanes 2 and 4) or N294 mutant (lanes 1 and 3), with or without peptide–N-glycosidase F (PNG) (nonreducing SDS-PAGE). *, a size increase of 3 kD in the neoglycosylated N294 mutant. (D) Histogram overlays of anti-β8–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-β activation (F) assays using transfected sorted pools of HT1080 cells expressing equal surface concentrations of WT β8 or N294 glycan β8 compared with mock-transfected cells. n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, by ANOVA and Tukey’s or Bonferroni’s post-test.

Fig. 5. Affinity-optimized β8 antibody (B5) inhibits TGF-β–dependent chemokine expression by stimulated human lung fibroblasts

(A) Inhibition of TGF-β activation as measured using B5, an affinity-matured more potent inhibitor of αvβ8-mediated TGF-β activation than 37E1. Cocultures of β8-transfected HT1080 cells with transformed mink lung epithelial TGF-β reporter cells with varying concentrations (μg/ml) of B5 (filled squares) or 37E1 (open squares) reported by relative light units (LU; ×10³). n = 5 experiments. (B) B5 is more potent than 37E1 in blocking TGF-β–dependent CCL20 secretion by IL-1β–stimulated human lung fibroblasts. ELISA for measuring CCL20 in culture supernatant from IL-1β–stimulated normal primary human lung fibroblasts treated with isotype control (open bar), 37E1 (vertical stripes), B5 (filled bar), or 1D11 (horizontal stripes) at 10 μg/ml. Non–IL-1β–treated fibroblast controls do not secrete detectable CCL20. n = 5 different patients. ***P < 0.001, by ANOVA and Tukey’s post-test. n.s., not significant.

Fig. 6. αvβ8 is in a stable extended conformation with a closed headpiece; B5 is associated with inward bending of the β8 head-hybrid domain angle

(A) Ribbon diagram (PyMOL V1.1r1) of the extended, closed structure of the β8 subunit generated by homology modeling (Modeller) (50) to αvβ3 (PDB 3IJE) (25). Modeled β8 (green) with the α1 and α7 helices (red) superimposed on αvβ3 (purple). Red spheres, atoms of the B5 epitope on the α₁ helix (R₁₃₃, F₁₃₇, F₁₃₈). Modeled RGD tripeptide (blue spheres) based on PDB 3ZDX (51) bound to the ligand-binding pocket in complex with the MIDAS Ca²⁺ cation (orange sphere). The distance from the edge of the ligand-binding pocket (A₁₁₅) to R₁₃₃ of the B5 epitope is 28 Å, indicated by dotted arrows. Head, hybrid, and Psi domains are indicated. The αv subunit and leg domains are not included. (B) Image analysis measuring the hybrid-head domain angles of clasped and unclasped αvβ8 with no Fab compared to size exclusion chromatography–purified αvβ8-B5 or αvβ8–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), respectively. **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 (βI) and hybrid domain angles. (C) Rendered αvβ8 space filling model (PyMOL V1.1r1) of the closed headpiece structure of the αvβ8 subunit, as above, with docked prototype Fab (SG/19: PDB 3VI3, translucent gray with black outline) to the B5 epitope (R₁₃₃, F₁₃₇, F₁₃₈) indicated by red spheres, approximating dimensions and orientation of B5 Fab with inward bending of the hybrid domain. Modeled β8 (green) with αv (light blue). RGD tripeptide (orange spheres) bound to ligand-binding pocket and angles of the closed head-hybrid domain (black lines). (D) Space-filling αvβ8 generated homology model, as above, except with a rendering of docked 68 Fab.

Fig. 7. B5 is a noncompetitive allosteric inhibitor that induces a low-affinity state

(A) Latency-associated peptide decreases B5 binding to αvβ8-expressing HT1080 cells. β8-expressing HT1080 cells stained with B5 (2 μg/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 αvβ8-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 mediate cell adhesion, but insufficient to support TGF-β activation. (C) β8-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 < 0.01 by unpaired Student’s t test. (D) B5 induces a low-affinity state maximally inhibiting the binding of soluble αvβ8 to latency-associated peptide 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 concentrations 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-β activation by ~70%, and the addition of RGD peptide completely blocks remaining activation. β8-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 concentrations. 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.