Structure of the LINGO-1-anti-LINGO-1 Li81 antibody complex provides insights into the biology of

52 Pages·2014·8.61 MB·English

JPET Fast Forward. Published on April 22, 2014 as DOI: 10.1124/jpet.113.211771
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JPET #211771 1

Structure of the LINGO-1-anti-LINGO-1 Li81 antibody complex
provides insights into the biology of LINGO-1 and the mechanism of
action of the antibody therapy
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R. Blake Pepinsky*, Joseph W. Arndt, Chao Quan, Yan Gao, Omar Quintero-Monzon, at A
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Xinhua Lee, and Sha Mi E
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Departments of Drug and Molecular Discovery, Biogen Idec, Inc.,
14 Cambridge Center, Cambridge, MA 02142, USA

*Corresponding author

Copyright 2014 by the American Society for Pharmacology and Experimental Therapeutics.
JPET Fast Forward. Published on April 22, 2014 as DOI: 10.1124/jpet.113.211771
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Running Title Page

a) Running title: Structure and mechanism of action of anti-LINGO-1 antibody

b) R. Blake Pepinsky, Biogen Idec, Inc., 14 Cambridge Center, Cambridge, MA 02142
Telephone: [phone], Fax: [phone], email: [email protected]

c) text pages-36
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number of tables-1 nlo
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number of figures-9 (plus 7 supplementary figures) d fro
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number of references-48 jp
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number of words in Abstract-237 p
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number of words in Introduction-664 rn
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number of words in Discussion-1776 (includes citations) rg
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d) LINGO-1, leucine-rich repeat and Ig containing Nogo receptor interacting protein-1; Jo
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CNS, central nervous system; MS, multiple sclerosis; mAb, monoclonal antibody; LRR, ls
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leucine-rich repeat; CHO, chinese hamster ovary; CV, column volumes; PAGE, a
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polyacrylamide gel electrophoresis; SEC, size exclusion chromatography; ELISA, , 2
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enzyme-linked immunosorbent assay; DLS, dynamic light scattering; MR, molecular
replacement; MBP, myelin basic protein; OPC, oligodendrocyte progenitor cell; HC,
heavy chain; LC, light chain; CDR, complementarity determining regions; NgR1, Nogo
receptor interacting protein-1; EM, Electron microscopy.

e) Neuropharmacology
JPET Fast Forward. Published on April 22, 2014 as DOI: 10.1124/jpet.113.211771
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ABSTRACT
Multiple sclerosis (MS) is an autoimmune-inflammatory induced disease of the central
nervous system (CNS) with prominent demyelination and axonal injury. While most
MS therapies target the immunological response, there is a large unmet need for
treatments that can promote CNS repair. LINGO-1 (leucine-rich repeat and Ig
containing Nogo receptor interacting protein-1) is a membrane protein selectively
expressed in the CNS that suppresses myelination, preventing the repair of damaged
axons. We are investigating LINGO-1 antagonist antibodies that lead to remyelination as
a new paradigm for treatment of individuals with MS. The anti-LINGO-1 Li81 antibody D
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(BIIB033) is currently in clinical trials and is the first MS treatment targeting CNS lo
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repair. Here, to elucidate the mechanism of action of the antibody, we solved the crystal fro
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structure of the LINGO-1/Li81 Fab complex and used biochemical and functional jp
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studies to investigate structure-function relationships. Li81 binds to the convex surface sp
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of the leucine-rich repeat domain of LINGO-1 within repeats 4-8. Fab binding blocks urn
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contact points used in the oligomerization of LINGO-1 and produces a stable complex .o
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containing 2 copies each of LINGO-1 and Fab that results from a rearrangement of t A
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contacts stabilizing the quaternary structure of LINGO-1. The formation of the T
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LINGO-1/Li81 Fab complex masks functional epitopes within the Ig domain of LINGO- rn
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1 that are important for its biological activity in oligodendrocyte differentiation. These on
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studies provide new insights into the structure and biology of LINGO-1 and how Li81 rc
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mAb can block its function. , 2
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JPET Fast Forward. Published on April 22, 2014 as DOI: 10.1124/jpet.113.211771
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Introduction
LINGO-1 (LERN1, LRRN6A) is a 581 amino acid membrane associated-
glycoprotein selectively expressed on neurons and oligodendrocytes in the CNS (Mi et al.
2005). It contains a large extracellular domain with 12 leucine rich repeat (LRR) motifs
flanked by N- and C-terminal capping modules, one immunoglobulin (Ig) domain of the
I1 subtype, and a stalk region that is attached to a transmembrane region and a short
cytoplasmic tail (Mi et al., 2004; Mosyak et al., 2006). LINGO-1 suppresses
oligodendrocyte differentiation thereby preventing axonal myelination (Mi et al. 2007,
2009). Blocking its function leads to robust myelination in vitro and in animal models of D
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demyelination (Mi et al. 2005, 2007, 2009). The biological effects of LINGO-1 lo
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antagonists have been validated using siRNA, a dominant negative construct lacking the fro
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cytoplasmic tail, a soluble LINGO-1-Fc fusion protein in which the LINGO-1 jp
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extracellular domain is genetically linked to the hinge region and Fc of human IgG1, and sp
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anti-LINGO-1 mAbs, Fabs, Fab2s, and PEG-Fabs (Mi et al., 2005, 2007, 2009; Ji et al., urn
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2006; Fu et al., 2008; Pepinsky et al., 2011; Cen et al., 2013). Of these, antibodies were .o
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preferred because of their potency, selectivity, and duration of treatment effects due to t A
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long serum half-life (Aires da Silva et al., 2008; Pepinsky et al., 2011). The anti-LINGO- T
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1 antibody Li81 was isolated using Fab phage display technology and engineered into a rn
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human IgG1 aglycosyl framework (Pepinsky et al., 2011; Hoet et al., 2005). We are on
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currently investigating Li81 mAb (BIIB033) in clinical trials as a potential treatment to rc
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repair neuronal damage that occurs in the CNS of individuals with MS (Mi et al., 2013). , 2
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The clinical trial.gov number for the Phase 2 MS study is SYNERGY: NCT01864148.
The use of antibodies as drugs that target antigens expressed in the CNS is
challenging because of poor exposure to the CNS following systemic administration due
to the blood-brain barrier (Shen et al., 2004; Levites et al., 2006; Garg and Balthasar,
2009; Pepinsky et al., 2011). Current estimates are that only 0.1% of antibody drug
levels in blood reach the CNS. Consistent with low levels reported by other labs, we
determined that Li81 mAb levels in brain and spinal cord following systemic
administration of the antibody in rats were 0.15% of blood levels, but despite the low
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CNS exposure, Li81 treatment led to the repair of damaged CNS neurons with a direct
dependence of dose on the extent of CNS repair (Pepinsky et al., 2011). These findings
provided clear evidence that remyelination that results from blocking of LINGO-1
function is directly linked to the binding of Li81 to LINGO-1. Cellular responses that
are mediated by LINGO-1 result from direct binding of LINGO-1 to growth factor
receptors to block their function. To date four LINGO-1 signaling pathways have been
elucidated (Mi et al., 2013). Structure activity relationship studies suggest that the Ig
domain of LINGO-1 is sufficient for its activity (Mi et al. 2010, Bourikas et al., 2010,
this study). The crystal structure of the LINGO-1 ectodomain revealed that the protein D
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self- associates to form a ring-shaped tetramer (Mosyak et al., 2006). In the structure, lo
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potential function binding sites on the Ig domain are solvent exposed, providing a model fro
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for how LINGO-1 functions. How the Li81 antibody blocks LINGO-1 function is jp
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unknown. sp
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Here, to investigate the mechanism of action of the Li81 antibody we solved the urn
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crystal structure of the LINGO-1 ectodomain/Li81 Fab complex and discovered that the .o
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binding of Li81 to LINGO-1 blocked contacts that allowed LINGO-1 to form the t A
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tetrameric state seen in the published LINGO-1 crystal structure. Li81 Fab treatment T
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prevented the formation of LINGO-1 homo-oligomers in solution and on transfected rn
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cells expressing full length LINGO-1, and led to a rearrangement in the quaternary on
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structure of LINGO-1, which provides a model for how the antibody inhibits LINGO-1 rc
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function in oligodendrocytes. These studies provide a detailed assessment of domains , 2
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within the LINGO-1 structure that are important for function and expand our
understanding of how they contribute to the complex biology of LINGO-1.
JPET Fast Forward. Published on April 22, 2014 as DOI: 10.1124/jpet.113.211771
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Materials and Methods
LINGO-1 production. LINGO-1-Fc produced by fusing the extracellular portion of
human LINGO-1 (residues 1-488) to the hinge and Fc region of human IgGl was
expressed in Chinese hamster ovary (CHO) cells and purified from clarified and filtered
cell culture medium on recombinant Protein A Sepharose Fast Flow (GE Heathcare) at a
loading density of 6 mg of fusion protein/ml of resin. The LINGO-1-Fc was eluted from
the column with 25 mM NaH PO pH 2.8, 100 mM NaCl and neutralized by adding 0.05
2 4
volume of 0.5 M NaH PO pH 8.6. Peak protein-containing fractions were identified by
2 4
absorbance at 280 nm, pooled, and dialyzed against PBS. The preparation was 0.2 µm D
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filtered, aliquoted, frozen on dry ice, and stored at -70°C. The protein concentration was lo
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calculated using an extinction coefficient of ε2800.1% = 1.04 using computer Beckman d fro
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Coulter DU-800 Spectrophotometer NetA software that compensates for baseline shifting jp
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by subtracting absorbance due to turbidity from the value at 280 nm. LINGO-1 sp
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ectodomain was generated from LINGO-1-Fc by digestion with chymotrypsin or urn
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endoproteinase Lys-C as indicated. For limited proteolysis with chymotrypsin, LINGO- .o
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1-Fc (2.5 mg/ml) in PBS was diluted with 2 volumes of 100 mM Tris HCl pH 7.5, 10 t A
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mM CaCl . Chymotrypsin (Roche) was added to a 1:500 enzyme:protein ratio (w/w) and T
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incubated for 3 h at room temperature. PMSF was added to a final concentration of 0.5 rn
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mM and an additional 100 mM NaCl was added. The sample was loaded onto a Protein A on
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Sepharose column (8 mg protein/ml resin) equilibrated in PBS. The flow through rc
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fraction was pooled, concentrated using an Amicon ultra-15 centrifugal filter device (10 , 2
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kDa membrane) and loaded onto a Superdex 200 size exclusion chromatography column.
Peak fractions were pooled, aliquoted, and stored at -70°C. The protein concentration
was calculated using an extinction coefficient of ε 0.1% = 0.9. For limited proteolysis
280
with endoproteinase Lys-C (Wako), LINGO-1-Fc at 7.5 mg/ml in PBS was incubated for
2 h at room temperature with a 1:1000 enzyme:protein ratio (w/w). Leupeptin was added
to a final concentration of 0.2 mM and an additional 100 mM NaCl was added. The
sample was loaded onto a Protein A Sepharose column (8 mg protein/ml resin)
equilibrated in 20 mM Na HPO pH 7.5, 250 mM NaCl. The flow through fraction was
2 4
JPET Fast Forward. Published on April 22, 2014 as DOI: 10.1124/jpet.113.211771
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pooled, and loaded onto a Ni-NTA Superflow column (Qiagen) at 3 mg protein/ml resin.
The column was washed with 3 column volumes of 20 mM Na HPO pH 7.5, 250 mM
2 4
NaCl and the LINGO-1 was step eluted with the same buffer containing 20 mM
imidazole. The elution pool was concentrated using Amicon ultra-15 centrifugal filter
devices (10 kDa membrane) and loaded onto a Superdex 200 size exclusion
chromatography column. Peak fractions were pooled, aliquoted, and stored at -70°C. The
sequence of LINGO-1-Fc is shown below (ectodomain in black, Fc in green). Observed
cleavage sites for chymotrypsin and endoproteinase Lys-C are shown with red and blue
arrowheads, respectively. Predicted N-linked glycosylation sites are noted in red. N466 D
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is not occupied with a glycan (experimentally determined). lo
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For structural work, the LINGO-1-Fc producing CHO cells were cultured in the presence
of kifunensine to reduce the complexity of the carbohydrates (Yu et al., 2011). The same
methods were used for purification of LINGO-1-Fc and generation of the ectodomain
fragments for LINGO-1-Fc produced in the presence and absence of kifunensine.
Truncated versions of LINGO-1 ectodomain containing the LRR domain alone (residues
1-382), Ig domain alone (residues 383-460), and Ig domain (residues 383-460) with the
RKH sequence (residues 423-425) mutated to EKV were fused to the hinge and Fc region
JPET Fast Forward. Published on April 22, 2014 as DOI: 10.1124/jpet.113.211771
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of human IgGl, expressed in CHO cells, and purified from culture medium on Protein A
Sepharose as described above.
Li81 Fab production. The Li81 mAb was purified from clarified and filtered Chinese
hamster ovary (CHO) cell culture medium on recombinant Protein A Sepharose Fast
Flow. The Li81 mAb was dialyzed into 10 mM sodium acetate pH 3.6. Li81 Fab2
fragment was generated from the mAb by digestion with pepsin at an enzyme:protein
ratio of 1:500 for 4 h at 37°C. Tris-HCl was added to 300 mM, to quench the reaction and
the sample dialyzed against 50 mM Tris-HCl pH 8.9, 50 mM NaCl. The sample was
subjected to anion-exchange chromatography on a Fractogel EMD TMAE (M) (40-90 D
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µm, Merck) column equilibrated in 50 mM Tris-HCl pH 8.9, 50 mM NaCl. The column lo
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was washed with 50 mM Tris-HCl pH 8.9, 50 mM NaCl and the Fab2 eluted with 20 mM fro
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Na2HPO4 pH 7.5, 150 mM NaCl (PBS). Li81 Fab fragment was generated from the Fab2 jpe
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by digestion with papain at an enzyme:Fab2 ratio of 1:300. Fab2 in 10 mM Na2HPO4 pH spe
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7.5, 5 mM EDTA pH 7.5, 20 mM cysteine-HCl, 20 mM NaOH was incubated with urn
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papain at 37°C for 2.5 h and quenched with the thiol protease inhibitor E64. The sample .o
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was dialyzed against PBS. t A
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SDS-Polyacrylamide Gel Electrophoresis. Samples were subjected to SDS-PAGE on T
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4-20% Tris-glycine gradient gels (Invitrogen) and stained with Coomassie brilliant blue. rn
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Non-reduced samples were diluted with Laemmli non-reducing sample buffer, and heated on
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at 75°C for 5 min prior to analysis. Reduced samples were treated with sample buffer rc
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containing 2% 2-mercaptoethanol and heated at 95°C for 2 min. Electronic images of , 2
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stained gels were acquired with the true color setting on a HP Scanjet G4050 scanner
(Hewlett Packard). For densitometry, the gel images were acquired and band intensities
quantified using a GS-800 Calibrated Densitometer (Bio-Rad) and the Quantity One 1-D
Analysis software (Bio-Rad). LINGO-1 and Li81 concentrations in the LINGO-1/Li81
Fab complex were calculated by interpolation of measured densities from standard curves
of densities of LINGO-1 and Li81 Fab standards, which were run on the same gel.
Analytical Size Exclusion Chromatography and Light Scattering. Size Exclusion
Chromatography (SEC) was carried out on a BioSep-SEC-S3000 column, 300 x 7.8 mm
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(Phenomenex) in 20 mM sodium phosphate, 150 mM NaCl, pH 7.2, using a flow rate of
0.6 ml/min on a Waters Alliance instrument (Waters 2790, MA). The column effluent
was monitored by UV detection at 280 nm. Static light scattering was synchronized with
SEC and measured on-line using a Precision PD2100 Detector (Precision Detectors,
MA). Molecular weights were calculated using Discovery 32 Light Scattering Analysis
Software.
Biacore measurements. Solution-phase affinity measurements were performed on a
Biacore 3000 instrument (Biacore AB, Uppsala, Sweden). Human LINGO-1 ectodomain
was immobilized on CM5 chips using amine-coupling chemistry in Biacore buffer (10 D
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mM HEPES, pH 7.2, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20). Binding lo
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experiments were run in Biacore buffer containing 0.05% bovine serum albumin. For fro
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measuring LINGO-1-LINGO-1 interactions, serial 2-fold dilutions of the analyte human jp
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LINGO-1 ectodomain from 0.2 to 2 µM was used. For affinity of Li81 Fab, serial 2-fold sp
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dilutions of the Li81 Fab from 0.1 to 1 nM was tested. Data were analyzed with urn
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BIAevaluation 3.0 Software. Competition analysis of Li81 Fab with 1A7 were performed .o
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at a fixed concentration of 10 nM LINGO-1-ectodomain with 1 µM 1A7 Fab, or 25 nM t A
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and 1 µM 1A7 mAb on CM5 chips coated with Li81 and 1A7 mAbs using the amine- T
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coupling method. rn
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Cross-linking studies. LINGO-1 ectodomain (14 µM) alone in 20 mM sodium on
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phosphate, 150 mM NaCl, 35 mM HEPES, pH 7.5, in the presence of 28 µM Li81 Fab, rc
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or within the SEC purified complex was incubated for 70 min at room temperature with 1 , 2
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mM bissulfosuccinimidyl suberate (BS3) (Thermo Scientific). The reaction was stopped
with 50 mM ethanolamine pH 8.0. LINGO-1 ectodomain was preincubated with Fab for
30 min at room temperature prior to treatment. For cross-linking on cells, stable CHO-
cells expressing HA tag full length LINGO-1 (residues 1-581 with HA tag engineered at
the N-terminus) were maintained at 37°C in Alpha plus Eagle Minimum Essential
Medium containing 10% fetal bovine serum and 400 µg/ml Geneticin (Invitrogen).
Cross-linking was performed on 6-well plates. Cells were seeded at 4 x 105 cells/well
and incubated in medium without Geneticin for 2 days. Cells were washed twice with 2
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mM Hepes, pH 7.5, 150 mM NaCl, 1 mM CaCl , 1 mM MgCl , and 25 µg/ml human
2 2
serum albumin, then treated with 10 µg/ml Li81 Fab for 15 min at room temperature and
cross-linked at 4°C for 1 h with 1 mM BS3. The reaction was stopped with 50 mM
ethanolamine pH 8.0. Cells were lysed in 1 ml of RIPA lysis buffer (50 mM Tris HCl,
pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM
MgCl , 5% Glycerol) and samples were clarified by centrifugation in an eppendorf
2
centrifuge. Cell supernatants were immunoprecipitated with 15 µl of anti-HA affinity
Matrix (Roche, Cat. No. 1815016), washed 3 times with 0.3x lysis buffer, and analyzed
by western blotting using horseradish peroxidase conjugated-rat-anti-HA antibody for D
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detection. Similar results were generated when cells were treated with Li81 Fab for 3 h lo
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at 37°C prior to cross-linking (data not shown). fro
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Crystallization and Data Collection. 5.6 mg of SEC-purified LINGO-1 ectodomain jp
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from LINGO-1-Fc cultured in the presence of kifunensine and digested with sp
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chymotrypsin was added to 4.2 mg of Li81 Fab. The sample incubated for 1 h at room urn
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temperature, then concentrated to 2 ml in an Amicon Ultra-15 centrifugal filter device .o
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and applied to a 1.6 cm x 60 cm Superdex 200 column. Fractions containing the t A
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LINGO-1/ Li81 Fab complex were pooled, dialyzed against 10 mM HEPES pH 7.5, 150 T
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mM NaCl, and 0.2 µm filtered. The complex was concentrated to 4.5 mg/ml. The rn
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LINGO-1/Li81 Fab complex was crystallized by the nanodroplet vapor diffusion method on
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at a temperature of 297 K by mixing 200 nl of 4.3 mg/ml LINGO-1/Li81 Fab solution (10 rc
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mM Tris pH 8.0, 150 mM NaCl) with 200 nl of the reservoir solution containing 36% , 2
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pentaerythritol propoxylate (5/4 PO/OH), 0.2 M sodium thiocyanate, 0.1 M HEPES pH
7.0. LINGO-1/ Li81 Fab crystals were transferred to an oil-based cryoprotectant (66.5%
paratone, 28.5 paraffin oil, 5% glycerol) prior to harvesting and flash freezing in liquid
nitrogen.
Diffraction data were collected at a wavelength of 1.000 Å at the Advanced
Photon Source (APS) on beam line LRL-CAT. The data set was collected at 100 K using
a MARmosaic 225 detector. Data were integrated, reduced, and scaled using HKL2000