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Engineering and Therapeutic Application of Single-chain Bivalent Transforming Growth

Factor-� (TGF-�) Family Traps

John C. Zwaagstra, Traian Sulea, Jason Baardsnes, Anne E.G. Lenferink, Cathy Collins,

Christiane Cantin, Béatrice Paul-Roc, Suzanne Grothe, Sazzad Hossain, Louis-Philippe Richer,

Denis L’Abbé, Roseanne Tom, Brian Cass, Yves Durocher, and Maureen D. O’Connor-

McCourt1

1Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount

Avenue, Montreal, QC, Canada H4P 2R2

Running title: Design and application of single-chain TGF-� family traps

Keywords: TGF-� family receptors: molecular design; traps, therapy, cancer

Financial support: Research funds were provided by the Genome Heath Initiative Canada.

Corresponding author: John C. Zwaagstra, Biotechnology Research Institute, National Research

Council Canada, 6100 Royalmount Avenue, Montreal, QC, Canada H4P 2R2, Tel.: (514) 496-

6384; Fax: (514) 496-5143; E-mail: [email protected]

Disclosure of potential conflicts of interest: No potential conflicts of interest are disclosed herein.

Word count: Main text (minus references): 5,206

Number of Tables: Main text (none), Supplementary (5)

Number of Figures: Main text (6), Supplementary (6)

on December 31, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 4, 2012; DOI: 10.1158/1535-7163.MCT-12-0060

http://mct.aacrjournals.org/

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ABSTRACT

Deregulation of TGF-� superfamily signaling is a causative factor in many diseases.

Here we describe a protein engineering strategy for the generation of single-chain bivalent

receptor traps for TGF-� superfamily ligands. Traps were assembled using the intrinsically

disordered regions (IDRs) flanking the structured binding domain of each receptor as ‘native

linkers’ between two binding domains. This yields traps that are ~ 3-fold smaller than antibodies

and comprised entirely of native receptor sequences. Two TGF-� Type II receptor-based, single-

chain traps were designed, termed (T�RII)2 and (T�RIIb)2, that have native linker lengths of 35

and 60 amino acids, respectively. Both single-chain traps exhibit a 100-1000 fold higher in vitro

ligand binding and neutralization activity compared to the monovalent ectodomain (T�RII-ED),

and a similar or slightly better potency than pan-TGF-� neutralizing antibody 1D11 or an Fc-

fused receptor trap (T�RII-Fc). Despite its short in vivo half-life (< 1hour), which is primarily

due to kidney clearance, daily injections of the (T�RII)2 trap reduced the growth of 4T1 tumors

in BALB/c mice by 50%, an efficacy that is comparable to 1D11 (dosed thrice weekly).

Additionally, (T�RII)2 treatment of mice with established 4T1 tumors (100 mm3) significantly

inhibited further tumor growth, whereas the 1D11 antibody did not. Overall our results indicate

that our rationally designed bivalent, single-chain traps have promising therapeutic potential.




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INTRODUCTION

The TGF-� superfamily includes more than 30 ligands that regulate several physiological

processes, including cell proliferation, migration and differentiation. Perturbation of their levels

and/or signaling gives rise to significant pathological effects. For instance, TGF-� and activin

play critical pathogenic roles in many diseases including cancer and fibrosis (1-6).

Myostatin/GDF8 is a validated therapeutic target for muscle wasting diseases such as muscular

dystrophy (7, 8). Bone morphogenetic proteins (BMPs) have been implicated in cardiovascular

diseases (9) and high levels of BMP2 and BMP4 are expressed in calcified atherosclerotic

plaques and diseased aortic valves (10).

One approach to developing therapeutic agents that inhibit TGF-� family function is to

block ligand access to its receptors, with both anti-ligand antibodies and soluble receptor

ectodomain-based ligand traps being pursued (11-14). Receptor ectodomain-based traps are a

class of therapeutic agents built on structural knowledge of receptor/ligand interactions and can

thus be optimized using protein engineering approaches (15-17). Our prior studies on the kinetics

of TGF-� ligand-receptor interactions indicated that artificial dimerization of the ectodomain of

the TGF-� Type II receptor (T�RII-ED) greatly improved its antagonist potency. This increased

efficacy results from an avidity effect which promotes more effective ligand sequestration due to

decreased ligand dissociation rates (15, 18, 19). Indeed, IgG Fc-dimerized receptor ectodomains

targeting various ligands have emerged as successful therapeutics with some advancing into the

clinic (20-22).

In this report we describe a novel design strategy for generating minimized TGF-�

family ligand traps in which bivalency is achieved via covalent linkage of two receptor

ectodomains (C-terminus of one ectodomain linked to N-terminus of the other) in a single




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polypeptide, rather than via dimerization domains. Generally, flexible non-natural polyglycine or

glycine-serine sequences are used as linkers to connect the two folded binding domains in

bivalent fusion proteins (e.g. (23)). In contrast, our strategy exploits the intrinsically disordered

regions (IDRs) that flank the structured, ligand-binding domain of TGF-� family receptor

ectodomains. We speculated that using the cognate human receptor’s IDRs as linkers might bring

advantages relative to artificial Gly/Ser-based linkers in terms of reduced immunogenicity. As

well, improved affinity might be achieved due to the IDRs having structural plasticity ((24) and

references therein) which could promote bridged bivalent binding.

Specifically, we designed traps for the TGF-�s, BMPs, activins, and myostatin using

‘natural sequence’ flexible linkers by fusing the C-terminal disordered region from one receptor

ectodomain to the N-terminal disordered region of the second ectodomain. Using two TGF-�

traps, (T�RII)2 and (T�RIIb)2, as primary examples, we demonstrate that they exhibit improved

ligand binding and neutralization potencies compared to the cognate monovalent receptor

ectodomain. We have also assessed the in vivo efficacy of the (T�RII)2 trap compared to 1D11

antibody, a well studied TGF-� neutralizing antibody with proven efficacy as an anti-cancer

therapeutic (25-29). Using the 4T1 mouse mammary tumor model, we demonstrate that the

single-chain TGF-� trap, but not 1D11 antibody, is able to reduce the growth of established

primary tumors.

MATERIALS AND METHODS

Reagents and cell lines. TGF-� ligands were provided by Dr. Andrew Hinck (U. Texas Health

Science Center, San Antonio). BMP2, T�RII-Fc, and anti-TGF�RII and 1D11 antibodies (R&D

Systems), anti Smad2 and phosph-Smad2 antibodies (New England Biolabs) and human serum




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(Sigma-Aldrich). Mink lung (Mv1Lu) and 4T1 mouse mammary carcinoma cell lines (American

Tissue Culture Collection (ATCC)).

Gene assembly, cloning and production of traps. Gene constructs of T�RII-ED, (T�RII)2 and

(T�RIIb)2 traps are preceded by an N-terminal cassette consisting of the human VEGF signal

peptide/8×His tag/Thrombin or TEV cleavage sites. Gene assemblies were cloned in the pTT

plasmid vector (30) and expressed in modified human embryonic kidney cells (293-EBNA1

clone 6E) expressing EBNA1 (30, 31). Trap protein was purified on Fractogel-Cobalt column

and desalted in PBS (32).

Surface plasmon resonance (SPR) analysis. SPR data was generated using a ProteOn XPR36

instrument (BioRad Inc.) at 25�C using HBST (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA,

0.05% Tween-20) as running buffer. TGF-� (500 resonance units (RUs)) was immobilized onto

a BioRad GLC sensorchip using standard amine coupling methods. TGF-� trap samples were

injected for 120 seconds followed by 600 seconds dissociation at a flow rate of 50 �L/minute.

Specific trap-TGF-� interaction sensorgrams were double-referenced to blank control surface

and buffer blank (BioRad Protein Manager Software v2.1) and normalized to a maximum 100

RU response (BiaEval v3.2, Biacore Inc.).

Competitive SPR analysis. SPR data was generated using a Biacore 3000 instrument. A high

density, amine-coupled 1D11 anti-TGF-� antibody surface (~10,000 RUs) on a Biacore CM-5

sensorchip was used. Solutions containing TGF-� trap variant or 1D11 were pre-incubated with

0.3 nM TGF-� at 5�C and then injected over 1D11-sensorchip under mass-transport limiting




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conditions for 3 minutes with a 30 second dissociation time at a flow rate of 5 �L/minute at

25�C. Sensorgrams were aligned to the injection start point and double-referenced to control

surface and blank injection sensorgrams. The % free TGF-� was calculated based on the

binding-rate value, derived from the double-referenced sensorgram (BiaEval v3.2, Biacore Inc.)

and normalized to the 0.3 nM TGF-� control rate. Binding curves were plotted to determine

binding IC50 values (Supplementary Table S3).

Luciferase assays. TGF-� and BMP reporter assays were performed using Mv1Lu or

C2C12BRA cells having a PAI-1-luciferase (33) or BMP-luciferase reporter gene(34),

respectively. Cells were treated for 16 hours at 37�C with trap samples plus 20 pM TGF-� or 1

nM BMP2 in DMEM, 1% FBS, 0.1% BSA. Cells were then lysed and luciferase activity was

measured (Promega Corp.). TGF-� neutralization IC50 values are listed in Supplementary Table

S4.

In vivo pharmacokinetics. The (T�RII)2 trap was [125I]-labeled (Pierce IODO-GEN tube kit,

Fisher Sci.). Male Sprague Dawley rats (Charles River, St. Constant, QC) were injected via tail

vein with a single dose of trap (10 mg/Kg unlabeled trap + 15 �Ci [125I]-(T�RII)2). Blood and

urine samples were collected at various time points post injection and counted in a WallacWizard

1470 gamma counter (PerkinElmer Life Sci.). Tissues were removed from animals sacrificed at

selected time points. Trap levels are expressed as microgram equivalents, as calculated from the

cpms.




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In vivo efficacy studies. 4T1 mouse mammary tumor cells (4x104 in 40 �L saline) were

orthotopically implanted in the left #4 inguinal mammary fat pad of 6-8 week old female

BALB/c mice. After 24 hours animals were randomized and treatment was initiated for two

weeks with daily tail vein injections of (T�RII)2 trap (10 mg/Kg) or saline (vehicle control)

whereas 1D11 antibody was administered thrice a week (5 mg/Kg). Animals were euthanized on

day 15 and tumors were weighed and processed for histology or snap-frozen for Q-PCR analysis.

In a second study, mice with established 4T1 tumors (~100 mm3) were randomized and treated

daily (i.p.) with saline or trap (10 mg/Kg) or thrice a week with 1D11 (5 mg/Kg). P-values were

calculated using a two-tailed non-parametric Whitney-Mann test.




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RESULTS

Design of single-chain bivalent traps for TGF-� superfamily ligands

TGF-� superfamily growth factors adopt the cysteine-knot fold and form covalent

disulfide-linked symmetric homodimers (reviewed in (35, 36)). The ectodomains of the TGF-�

superfamily receptors are also structurally conserved and contain a single structured domain

belonging to the snake-toxin family (37). An additional feature is that their ectodomains

typically contain intrinsically disordered regions (IDRs) flanking the structured ligand-binding

domain (Fig. 1A, Supplementary Table S1). Structural elucidation of several TGF-�-superfamily

ligand-receptor complexes confirmed that two receptor ectodomains bind simultaneously to one

homodimeric ligand molecule (38-44).

To generate minimized TGF-� family ligand traps we employed a single-chain approach

in which two ectodomains are fused covalently in a head-to-tail manner. Examination of the

crystal structures of TGF-�-superfamily ligand-receptor complexes shows that the linear distance

between the two receptor ectodomains, when bound to ligand, varies between 45 to 80 Å

(Supplementary Table S2). Thus linkers of different lengths are required for each TGF-� family

member single-chain trap. A comparison of the minimum number of residues required for

linkage versus the number of residues in natural linkers formed by fusing the C-terminal IDR of

the first receptor ectodomain with the N-terminal IDR of the second is shown in Supplementary

Table S2. Using the single-chain TGF-� trap termed (T�RII)2 as an example, this table shows

that the linker assembled by fusion of the IDRs has more than sufficient length (10 plus 25 = 35

a.a. versus the minimal 32 a.a. required length) to span over the TGF-� ligand and enable

bivalent binding. The existence of a splicing variant, termed T�RIIb, which features a 25-residue

insertion in its N-terminal IDR, allowed us to examine the effect of a longer 60 a.a. “natural”




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linker. The resultant traps are shown schematically in Fig. 1B. Molecular mechanics based 3D-

models of the TGF-�-bound (T�RII)2 trap further confirmed that the length of the natural linker

is sufficient to circumvent the ligand (Fig. 1C). A similar approach was used to design traps for

additional TGF-� superfamily ligands, e.g. activin and BMPs (Supplementary Table S2 and Fig.

S1).

In the case of the (T�RII)2 trap, the feasibility of appropriate bivalent ligand binding was

further assessed by molecular dynamics (MD) simulation in explicit solvent. As seen in

Supplementary Fig. S2A, the TGF-�3-bound (T�RII)2 trap attained a stable MD solution

structure which preserves the mode of simultaneous binding observed experimentally for the two

unlinked ligand-binding domains (38, 39). This MD analysis also indicated that the linker of

(T�RII)2 becomes relatively rigid, with only 6 residues experiencing greater mobility than those

of the structured ligand-binding domains of the trap (Supplementary Fig. S2B). In addition, this

analysis indicated that the trap can establish a favorable interaction with TGF-�3, with a highly

favorable solvated interaction energy (SIE) (45) of –25.4 kcal/mol (Supplementary Fig. S2C).

This further substantiates the feasibility of employing natural IDR-based linkers since there were

no significant unfavorable steric and electrostatic contacts predicted between the linker and the

ligand. Based on this MD analysis of the (T�RII)2 trap, we assumed that the 25-residue longer

linker of the (T�RIIb)2 trap will also allow unobstructed binding of TGF-�.

Single-chain bivalent TGF-� traps exhibit improved binding to immobilized TGF-�

compared to monovalent T�RII-ED

To investigate the ability of these ligand traps to bind TGF-�, his-tagged versions of

(T�RII)2 and (T�RIIb)2 traps and T�RII-ED monomer were expressed in modified HEK-293




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cells and purified using cobalt affinity columns. The (T�RII)2 and (T�RIIb)2 proteins migrated

over a 50-60 KDa range due to glycosylation, as assessed by SDS-PAGE (Fig. 2A). The binding

of (T�RII)2, (T�RIIb)2, T�RII-ED and 1D11 (a pan-TGF-�-neutralizing antibody) to

immobilized TGF-� isoforms was compared by surface plasmon resonance (SPR) (Figs. 2B-D).

The sensorgrams representing binding of these proteins to TGF-�1 and TGF-�3 were essentially

identical. The kinetics of T�RII-ED binding to TGF-�1 and -�3 was characteristic of the

monomeric receptor (i.e. fast on- and off-rates, (18)). The binding of T�RII-ED to TGF-�2 was

undetectable, as expected since T�RII has a low affinity for TGF-�2 (15, 46). In contrast to the

monovalent T�RII-ED, both (T�RII)2 and (T�RIIb)2 dissociated from TGF-�1 and -�3 with

slow off-rates, indicating that the linkage of two T�RII ectodomains resulted in improved ligand

binding. This is also supported by the observation that, in contrast to T�RII-ED, (T�RII)2 and

(T�RIIb)2 were both able to bind TGF-�2 (Fig. 2D). Nonetheless, the fast off-rate component in

their TGF-�2 binding sensorgrams suggests that their affinity for TGF-�2 is much weaker than

for TGF-�1/3. The 1D11 antibody exhibited similar binding to TGF-�1 and -�3 as (T�RII)2 and

(T�RIIb)2. However, the slow off-rate with TGF-�2 indicates stronger binding of 1D11 to this

isoform.

Single-chain bivalent TGF-� traps effectively bind/sequester TGF-� in solution

Flowing the traps and 1D11 antibody over immobilized TGF-�1, -�2, and -�3 surfaces

allows for a relative ranking of binding activity, as presented above. However, in this assay

orientation the trap may be interacting not only intramolecularly with a 1:1, trap:ligand

stoichiometry as designed, but also intermolecularly with the two T�RII binding domains within




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the trap contacting neighbouring immobilized TGF-� molecules. In order to determine if the

traps functions in solution as designed, we performed competitive SPR binding experiments in

which the TGF-� trap is bound to ligand in solution thereby removing potential ligand-

immobilization artifacts. In this assay the trap protein is first allowed to bind to a fixed amount of

TGF-� in solution, after which this mixture is flowed over immobilized 1D11 antibody in order

to quantify the amount of ligand left unbound by the trap (T�RII and 1D11 compete for binding

TGF-�, i.e. have overlapping epitopes). The binding IC50s for the various traps, 1D11 and

T�RII-ED monomer to TGF-�2 and TGF-�3 were determined by plotting the percent free TGF-

� over a range of trap concentrations (Figs. 3A and 3B, Supplementary Table S3). Single-chain

(T�RIIb)2 and (T�RII)2 traps were compared with IgG Fc-dimerized T�RII (T�RII-Fc),

monomeric T�RII-ED and 1D11 antibody. As expected, T�RII-ED was least efficient for

binding TGF-�2 and TGF-�3. The two single-chain traps, T�RII-Fc and 1D11 all bound TGF-�3

with similar efficacies, exhibiting average IC50s ranging from 0.1-0.4 nM. In contrast, the

single-chain traps and T�RII-Fc bound weakly to TGF-�2 (IC50s >100 nM), whereas 1D11

bound efficiently to this isoform (IC50 = 1.8 nM). When these results are compared with the

direct binding data in Figs. 2B-D, it is apparent that slow dissociation rates define the

interactions that result in efficient binding/sequestration of ligand in solution, i.e. TGF-�2 with

1D11, and TGF-�1/3 with 1D11, (T�RII)2 and (T�RIIb)2.

Single-chain TGF-� traps are potent neutralizers of TGF-�1 and TGF-�3 in a cell-based

assay




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Neutralization of TGF-�1, -�2, and -�3 by (T�RII)2 and (T�RIIb)2 traps was compared

with T�RII-Fc, T�RII-ED and 1D11 using a cell-based TGF-�-reporter assay (Figs. 3C and 3D,

Supplementary Table S4). The average IC50s of (T�RII)2 for TGF-�3 and -�1 (�0.3 and �1.4

nM, respectively) were similar to those for T�RII-Fc (�0.3 and �0.5 nM for TGF-�3 and -�1,

respectively). The IC50s for (T�RIIb)2 were somewhat lower (�0.05 and �0.1 nM for TGF-�3

and -�1, respectively) and similar to 1D11. In contrast, T�RII-ED was much less potent in

neutralizing TGF-�1/3 (IC50s �100 nM). Finally, TGF-�2 was neutralized by 1D11 but not by

the receptor-based traps (Fig. 3D). Our results demonstrate that linkage of two T�RII

ectodomains with natural linker sequences enhances not only binding to TGF-�1 and -�3 but

also neutralization potency. However, the improvement in TGF-�2 binding (Fig. 2D) was not

sufficient to enable neutralization of this TGF-� isoform.

Our molecular dynamics simulation of the (T�RII)2 trap indicated that the natural linker

becomes rigid and may establish molecular interactions with the ligand (Supplementary Fig.

S2A), which could contribute to an increased affinity and neutralizing potency of this trap. To

test the effect of the linker sequence, we assessed a (T�RII)2 trap in which the 35 a.a. natural

linker was replaced by a 35 a.a. artificial linker (composed of [8×GGGS]GGG). We observed a

~10-fold decrease of the IC50 for TGF-�1 neutralization for the artificial- versus natural-linker

trap (13.3 nM and 1.4 nM, respectively, Supplementary Fig. S3). This suggests that the natural

linker in (T�RII)2 has properties that enhance trap affinity and efficacy.

Serum stability and pharmacokinetic (PK) characteristics of the (T�RII)2 trap




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For our in vivo studies, we focused on the (T�RII)2 trap since its sequence mimics the

predominant T�RII isoform found in tissues, and it showed TGF-� binding characteristics

similar to (T�RIIb)2 (Figs 2B-D, 3A). First, we assessed the susceptibility of (T�RII)2 to

proteolytic degradation in serum. As shown in Fig. 4A, the (T�RII)2 trap showed no evidence of

degradation and retained full neutralization potency after incubation in human serum at 37�C for

up to 7 days. Although stable in serum, the size of the (T�RII)2 trap with glycosylation is

slightly under the ~ 60 KDa molecular cut-off for kidney filtration, hence we anticipated a short

circulation time in vivo. This was confirmed by PK studies which showed that (T�RII)2 has a

half-life of less than one hour, predominantly due to kidney clearance and elimination in the

urine (Figs. 4B-D). This is considerably shorter than antibodies, including 1D11 and the

humanized equivalent GC1008 (fresolimumab), which have half-lives of several days in vivo

(47).

The (T�RII)2 trap reduces growth of 4T1 mammary tumors primarily by reversing TGF-�

mediated immune suppression and angiogenesis

We next tested the in vivo efficacy of the (T�RII)2 trap versus 1D11 antibody using the

syngeneic 4T1 mammary tumor model in BALB/c mice. The tumor-inhibiting action of 1D11

has been well documented using this model (25, 26). 4T1 cells were implanted orthotopically

into the mammary fat pad and treatment was then initiated for a period of two weeks. To

partially compensate for its short half-life we increased the dose (10 mg/Kg) and frequency

(daily) of injection of the (T�RII)2 trap compared to 1D11 (5 mg/Kg, thrice a week). Fig. 5A

shows that, compared to the saline controls, treatment with 1D11 and the trap slowed tumor

growth by �50 %.




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We also compared 1D11 and (T�RII)2 trap efficacies in mice with established 4T1

tumors and found that the trap, but not 1D11, significantly reduced further tumor growth (Fig.

6A). This difference may be due to better penetration into tumors of the smaller trap compared to

1D11 antibody. We also assessed lung metastases in these mice and found that both the trap and

1D11 appeared to reduce the number of lung nodules relative to controls (Fig. 6B). This may be

due to the trap and 1D11 having access to disseminating 4T1 tumor cells, either within the blood

or at the secondary site. Taken together, these studies show primarily that, despite its rapid

elimination in vivo, the (T�RII)2 trap reduces the growth of newly implanted or established

primary tumors, indicating a remarkable potency for this trap.

To probe the mechanisms underlying the reduction of 4T1 tumor growth we analyzed the

effect of the (T�RII)2 trap on 4T1 cells in vitro and on tumors from the animals in which

treatment was initiated the day after tumor cell implantation. In 4T1 cells, the (T�RII)2 trap

effectively antagonized TGF-�1 and -�3 signaling, and partially antagonized TGF-�2 signaling,

as measured by expression of the TGF-� response marker PAI-1 (Supplementary Fig. S4B).

Similarly, the (T�RII)2 trap inhibited TGF-�1 and -�3 mediated Smad2 phosphorylation in 4T1

cells (Supplementary Fig. S4C). In contrast, we were unable to detect a significant decrease in

PAI-1 levels in 4T1 tumors from mice treated with trap or 1D11 compared to the saline controls

(Fig. 5B). Furthermore, Ki67 staining of the tumors did not reveal any differences in cell

proliferation (Supplementary Fig. S5). This was expected since 4T1 cells are not growth

inhibited in vitro by any of the TGF-� isoforms (Supplementary Fig. S4A). These results

therefore suggest that the observed tumor growth reduction is not due to inhibition of TGF-�

signaling within tumor cells or direct effects on tumor cell proliferation, but may be due to TGF-

� neutralization effects on other cell types in the tumor microenvironment. In support of this, we




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observed a significant increase in infiltrating T cells (as detected by CD3 staining) in tumors of

both trap and 1D11 treated mice (Fig. 5C). This correlated with increased granzyme B (a

cytotoxic T cell effector molecule) and Rae-1� (an immune cell activator) mRNA levels,

particularly in tumors from trap-treated mice (Fig. 5B). Additionally, trap and 1D11 treated

tumors exhibited a marked reduction in blood vessel density (Fig. 5D). Together these results

indicate that, similar to 1D11, the (T�RII)2 trap inhibits tumor growth by reversing TGF-�

mediated immune suppression and angiogenesis. Overall, these results demonstrate the

promising potential of the (T�RII)2 trap to act as a therapeutic addressing TGF-�-driven

diseases.

DISCUSSION

Here we present a novel design strategy for the generation of single-chain, bivalent

receptor ectodomain-based traps that neutralize members of the TGF-� superfamily of ligands.

Our approach exploits the presence of intrinsically disordered regions (IDRs) which flank either

side of the structured ligand binding domain, a feature shared by the receptor ectodomains from

this family. Fusion of the C-terminal IDR of one ectodomain with the N-terminal IDR of the

second ectodomain thus forms a bivalent trap that has two structured binding domains connected

by a natural flexible linker.

This design strategy was supported by empirical estimates based on known atomic

distances between two receptor domains when simultaneously bound to ligand, and which

indicated that natural flexible linkers formed by joining receptor IDRs should have sufficient

length to span over the ligand dimer (Supplementary Table S2). In the simplest case, affinity

enhancement can be achieved if the linkers act purely as flexible polypeptides that constrain the




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distance between two receptor binding domains, thereby increasing the effective concentration

(Ceff) of the second binding domain (48, 49). However, our results demonstrate that, when

linkers are composed of natural IDR sequences, they may contribute to enhancement of the

trap’s affinity and neutralization efficacy through additional effects beyond simply acting as a

leash. The first evidence alluding to this is provided by the MD simulation of the (T�RII)2 trap

which indicated that, upon binding TGF-�, the natural linker does not remain free in space but

rather becomes rigid, establishing potential molecular interactions with the ligand

(Supplementary Fig. S2A). This notion is supported by our data showing that the replacement of

the natural linker of the (T�RII)2 trap by an artificial linker caused a 10-fold decrease in TGF-�1

neutralization efficacy (Supplementary Fig. S3). These results suggest that IDR-based linkers

may take on a folded conformation that is adapted to interact with its target, a well-recognized

capability of intrinsically disordered proteins (50). These findings highlight a select advantage of

utilizing a linker composed of natural IDR sequences.

As a demonstration of the general applicability of this IDR-linker design strategy, in

addition to TGF-�, we designed single-chain traps for other TGF-� superfamily members, e.g.

BMP and activin (Supplementary Fig. S1). To confirm affinity enhancement, we produced a

BMPR1a receptor-based trap, termed (BMPR1a)2, and determined that it neutralized BMP-2

more effectively than monovalent BMPR1a-ED (Supplementary Fig. S6). Taken together, our

data indicate that a receptor trap design strategy that employs linkers composed of natural IDRs

provides a generally applicable engineering strategy for effective traps for the TGF-�

superfamily of ligands.

With respect to their ability to neutralize TGF-�1 and -�3 in cell-based assays, the

(T�RII)2 and (T�RIIb)2 traps behaved similarly to 1D11 antibody (Fig. 3C and Supplementary




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Table S4). However, in contrast to 1D11, these traps were unable to neutralize TGF-�2 (Fig.

3D). This distinction is consistent with the fact that these traps display faster TGF-�2

dissociation rates (Fig. 2D) and lower potencies in a competitive binding assay (Fig. 3B), as

compared to 1D11.

In vivo we observed that the (T�RII)2 trap and 1D11 reduced the growth of 4T1 tumors

to a similar extent when treatment was initiated the day following tumor cell implantation (Fig.

5A). This implies that the TGF-�1 and/or -�3 isoforms play a greater role in tumor growth than

TGF-�2 in this tumor model. Importantly, these results demonstrate that the (T�RII)2 trap can

reduce tumor growth as effectively as 1D11, despite its short circulating half-life (< 1 hour).

Augmentation of the half-life of the trap, e.g. by PEGylation, may improve its in vivo potency

further. On the other hand, rapid clearance of the (T�RII)2 trap from the circulation may prove

advantageous for disease indications where short term TGF-� neutralization is preferred.

Our analysis of the mechanisms underlying in vivo efficacy indicate that the (T�RII)2

trap enhanced a T cell-mediated immune response in the tumor microenvironment and reduced

blood vessel formation, which concurs with the study of Nam et al. (25) using 1D11. Notably,

although the effect of the (T�RII)2 trap and 1D11 on the growth of newly implanted 4T1 tumors

was not detectably different, we observed that the T cell activity markers, granzyme B and Rae-

1�, were augmented more significantly in tumors from trap-treated compared to 1D11-treated

mice (Fig. 5B). As further evidence of the higher efficacy of the trap, in animals with established

4T1 tumors, we demonstrated that the (T�RII)2 trap, but not 1D11 antibody, reduced primary

tumor growth (Fig. 6A). One explanation is that the smaller (T�RII)2 trap may better penetrate

established tumors compared to 1D11 antibody, providing a potential therapeutic advantage.




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In summary, we present a novel approach for the design of single-chain bivalent receptor

traps for TGF-� family ligands. In particular, we demonstrate the neutralization potency, serum

stability and in vivo efficacy of selected TGF-� traps. Several characteristics make these traps

attractive candidates for therapeutic or diagnostic applications. These advantages include: 1)

neutralization potencies in the nM to sub-nM range, 2) a single chain nature for ease of

production and further engineering (i.e. fusion, PEGylation), and 3) natural sequence

compositions reducing potential immunogenicity.

Acknowledgments

We thank Alina Burlacu for help in trap production and Mario Mercier and Cynthia Hélie

for their assistance in animal studies. We gratefully acknowledge Genzyme Inc. (Boston, MA,

USA) for supplying the 1D11 antibody used in animal studies.

Grant Support

Research funds were provided by the Genome Heath Initiative Canada. This is NRCC

publication no. 53131.




19

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22

FIGURE LEGENDS

Figure 1. Design of single-chain bivalent traps for TGF-�-superfamily ligands. (A) Structured

and unstructured, intrinsically disordered regions (IDRs) in the TGF-�-family receptor

ectodomains. Residue numbers indicate the boundaries of the disordered segments (line) that

precede the transmembrane (TM) region (see Supplementary Table S1 for sequence details). (B)

In-line fused receptor T�RII/b ectodomains as single-chain traps against TGF-�. The point of

fusion is indicated (slash). The native, 25 a.a. insertion in the linker of (T�RIIb)2 is shown in

grey. (C) Molecular models for the (T�RII)2 single-chain trap in complex with TGF-�3 (point of

fusion, red dot; polypeptide chain direction, grey arrowhead; structured domains, magenta;

intervening linker, black). The ligand dimer is rendered with its monomers in yellow and orange.

Molecular modeling was performed as described in Supplementary Methods.

Figure 2. Evaluation of HEK-293 cell produced traps. (A) (T�RII)2 and (T�RIIb)2 trap proteins

after purification on a cobalt affinity column, as detected by Coomassie-staining of samples run

in a SDS-polyacrylamide gel under non-reducing and reducing conditions. Molecular size

markers (KDa) are shown on the left. SPR sensorgrams showing direct binding of single-chain

TGF-� traps (T�RII)2 and (T�RIIb)2, T�RII-ED monomer, and 1D11 antibody to immobilized

TGF-�1 (B), TGF-�2 (D) and TGF-�3 (C).

Figure 3. Competitive SPR analysis of binding of various TGF-� traps to TGF-� isoforms in

solution. The graphs show the percent free TGF-� after prebinding TGF-�3 (A) or TGF-�2 (B)

to increasing amounts of trap or 1D11 antibody. Binding is normalized relative to TGF-�




23

incubated without trap or 1D11 (100% free TGF-�). Supplementary Table S5 lists the average

binding IC50 values derived from the competition graphs. Neutralization of TGF-�3 (C) and

TGF-�2 (D) by various traps or 1D11 antibody, as determined by a luciferase reporter assay

using Mv1Lu cells. Shown are representative graphs of the TGF-�-signaling response (relative to

the maximum response) for cells treated with TGF-�3 or TGF-�2 and increasing amounts of trap

or 1D11. The average IC50 values, determined from neutralization curves for TGF-�1, -�2 and -

�3, are given in Supplementary Table S4.

Figure 4. The (T�RII)2 trap is stable and retains full activity in human serum. (A) Top panel:

western blot analysis of trap protein (detected by anti-T�RII antibody) after incubation in 90%

human serum at 37�C for the indicated number of days. Lower panel: TGF-�1 neutralization

curve for the 7-day trap sample and non-treated control. (B-D) Pharmacokinetic (PK) assessment

of the (T�RII)2 trap in vivo following a single injection in rats. (B) Time course for trap levels in

blood plasma over a 24 hour period. The insert graph shows trap level kinetics during the first 4

hours. Trap levels are expressed as microgram equivalents, as calculated from the cpms. (C)

Concentrations of trap in various tissues at 20 minutes and 24 hour time points. (D) Trap

amounts in urine collected over the indicated time intervals

Figure 5. Anti-tumor efficacies and modes of action comparing (T�RII)2 trap and 1D11

antibody treatments in a mice developing 4T1 mammary tumors. (A, left panel) Mice were

treated with (T�RII)2 trap, 1D11 antibody or saline control (10 mice/cohort). Shown is the

average tumor volumes +/- SEM. (A, right panel) Scatter plot of final tumor volumes of each




24

mouse in the cohorts after 14 days of treatment (Bar, median). P-values were calculated using

two-tailed non-parametric Whitney-Mann test. (B) Relative quantities (RQ) of PAI-1, granzyme

B (GZMB) and Rae-1� transcripts in harvested tumors, as determined by Q-PCR. (C) Evaluation

of infiltrating T-lymphocytes in 4T1 tumors by CD3 staining (red). The lower graph shows the

number of CD3+ cells in tumor sections (5 high power fields (HPF) per tumor section). (D)

Comparison of blood vessel density (red stain) in representative tumor sections stained with

CD31 antibody. Immunohistochemistry staining and Q-PCR experiments were performed as

described in Supplementary Methods.

Figure 6. Anti-tumor efficacies comparing (T�RII)2 trap and 1D11 antibody treatments in a

mice with established 4T1 mammary tumors. (A) Left panel: Growth of 4T1 tumors in mice

treated with (T�RII)2 trap, 1D11 antibody or saline over a 19-day period (10 mice/cohort,

average tumor volumes normalized to initial tumor volume prior to treatment ± SD). Right

panel: scatter plot of final tumor volumes (B) Quantitation of lung metastasis in mice with

established 4T1 tumors after 19 days of treatment (Bar, median).




Published OnlineFirst May 4, 2012.Mol Cancer Ther John C Zwaagstra, Traian Sulea, Jason Baardsnes, et al.

) Family Trapsβ (TGF-βBivalent Transforming Growth Factor-Engineering and Therapeutic Application of Single-chain

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