Article pubs.acs.org/bc
High-Affinity VEGF Antagonists by Oligomerization of a Minimal Sequence VEGF-Binding Domain James E. Stefano,*,† Julie Bird,† Josephine Kyazike,† Anthony Wai-Ming Cheng,∥ Ekaterina Boudanova,† Markryan Dwyer,§ Lihui Hou,† Huawei Qiu,† Gloria Matthews,‡ Michael O’Callaghan,⊥ and Clark Q. Pan† †
Protein Engineering, ‡Preclinical Orthopaedics, and §New Biologics, Genzyme Corporation (A Sanofi Company), Framingham, Massachusetts, United States ∥ Department of Pathology, Duke University Medical Center, Durham, North Carolina, United States ⊥ 4s3Bioscience Inc., Medford, Massaschusetts, United States S Supporting Information *
ABSTRACT: Vascular endothelial growth factor (VEGF) neutralizing antagonists including antibodies or receptor extracellular domain Fc fusions have been applied clinically to control angiogenesis in cancer, wet age-related macular degeneration, and edema. We report here the generation of high-affinity VEGF-binding domains by chemical linkage of the second domain of the VEGF receptor Flt-1 (D2) in several configurations. Recombinant D2 was expressed with a 13 a.a. C-terminal tag, including a C-terminal cysteine to enable its dimerization by disulfide bond formation or by attachment to divalent PEGs and oligomerization by coupling to multivalent PEGs. Disulfide-linked dimers produced by Cu2+ oxidation of the free-thiol form of the protein demonstrated picomolar affinity for VEGF in solution, comparable to that of a D2-Fc fusion (sFLT01) and ∼50-fold higher than monomeric D2, suggesting the 26 a.a. tag length between the two D2 domains permits simultaneous interaction of both faces of the VEGF homodimer. Extending the separation between the D2 domains by short PEG spacers from 0.35 kD to 5 kD produced a modest ∼2-fold increase in affinity over the disulfide, thus defining the optimal distance between the two D2 domains for maximum affinity. By surface plasmon resonance (SPR), a larger (∼5-fold) increase in affinity was observed by conjugation of the D2 monomer to the termini of 4-arm PEG, and yielding a product with a larger hydrodynamic radius than sFLT01. The higher affinity displayed by these D2 PEG tetramers than either D2 dimer or sFLT01 was largely a consequence of a slower rate of dissociation, suggesting the simultaneous binding by these tetramers to neighboring surface-bound VEGF. Finally, disulfidelinked D2 dimers showed a greater resistance to autocatalytic fragmentation than sFLT01 under elevated temperature stress, indicating such minimum-sequence constructs may be better suited for sustained-release formulations. Therefore, these constructs represent novel Fc-independent VEGF antagonists with ultrahigh affinity, high stability, and a range of hydrodynamic radii for application to multiple therapeutic targets.
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INTRODUCTION Vascular endothelial growth factor (VEGF) serves the role as a potent angiogenic agent as well as having the capacity for increasing vascular permeability,1,2 thereby increasing the perfusion of serum into tissue. It has been a primary therapeutic target for suppressing angiogenesis in cancer therapy, wet agerelated macular degeneration (AMD), and diabetic retinopathy, conditions whose pathology is directly related to vessel growth. The most common form, VEGF A, is found in multiple splice variants, although the principal form is 165 amino acids in length (VEGF165).3−5 VEGF165 has affinity for the heparin sulfate proteoglycan (HSPG) component of the extracellular matrix due to a heparin binding domain(s) in the C-terminal portion,6,7 which is critical for its mitogenic potential.8 The © 2012 American Chemical Society
angiogenic activity of VEGF is associated with the binding of VEGF receptor 2 (VEGF R2, KDR, or Flk), a tyrosine kinase which induces endothelial cell growth with an EC50 in the subnanomolar range.4,5 However, VEGF shows a higher (pM) affinity for a second receptor, VEGF R1 (or Flt-1), which is required for normal blood vessel formation during development, but whose tyrosine kinase activity is nonessential.9 As a result, Flt-1 is thought to play a role in vivo largely in acting as a decoy to modulate VEGF availability. Two approved biotherapeutics (bevacizumab and ranibizumab) comprising Received: June 7, 2012 Revised: November 6, 2012 Published: November 26, 2012 2354
dx.doi.org/10.1021/bc300301m | Bioconjugate Chem. 2012, 23, 2354−2364
Bioconjugate Chemistry
Article
conjugation to poly(ethylene glycol)s as a strategy for prolonging circulating half-life. Conjugation to multiarm PEGs was performed to assess possible avidity effects and to provide a potential platform for targeted delivery. Finally, the suitability of two compositions for sustained-release formulation was assessed by the resistance to thermal stress.
an IgG and its affinity-matured Fab fragment against VEGF have been approved for cancer and wet age-related macular degeneration (AMD), respectively. Strategies to exploit the natural function of Flt-1 in regulating VEGF by the construction of soluble Flt-1 receptor decoys have focused on defining the minimal sequence required for highaffinity VEGF binding. Davis-Smyth demonstrated, through a series of deletions and swaps with other PDGF receptor family domains, that deletion of domain 2 (D2) obliterated binding to VEGF, but inclusion of Flt flanking domains 1 and 3 were required to replicate the high (pM) affinity of the full-length ectodomain.10 While such constructs demonstrated efficacy in preclinical models, high doses were required,11and these constructs demonstrated poor pharmacokinetic profiles.12 Moreover, continuous low-level expression of Flt(D1-D3) by adenoviral gene therapy was associated with morbidity and ascites formation.13 The crystal structure of a VEGF fragment with a monomeric Flt-1 D2 fragment showed two D2 domains interacting with each of the outside faces of the VEGF homodimer, but monomeric D2 was reported to have ∼100fold lower binding than the complete receptor ectodomain. A low picomolar affinity was observed only with a monomeric D1-D3 fragment or a dimeric (D2-D3) Fc fusion.14 An Fc fusion protein including the second domain of VEGF R1 and the homologous third domain of VEGF R2 (VEGF Trap, aflibercept) was subsequently found to circumvent the toxicity and PK issues with Flt(D1-D3)12 and was approved for AMD and is currently in clinical trials for macular edema. More recently, the reported requirement for both D2 and D3 for full VEGF binding activity was challenged by the finding that a forced homodimer, containing only the D2 domain of Flt-1 fused to human IgG Fc through a 9Gly linker (sFLT01), demonstrated high-affinity binding comparable to two- and three-domain constructs.15 A spacer sequence in addition to a CH3 domain which provided for formation of a noncovalent dimer were found to be required for high-affinity (pM) binding, suggesting that simultaneous binding to both chains of the VEGF homodimer is required for high affinity. A clinical trial of this molecule in gene therapy for AMD is in progress. All VEGF antagonists currently in the clinic utilize an IgG Fc domain to provide for a long circulating half-life intended to ensure reasonable target exposure by systemic administration. However, systemic exposure to all antiVEGF agents is associated with a number of potentially mechanism-related side effects including hypertension, complications in wound healing, intestinal perforations, and nephrotic syndrome,16,17 a subset of which may also have a component related to the Fc effector function. These side effects may restrict the use of these molecules from otherwise potentially treatable indications linked to VEGF activity, but which would require long-term administration. This class includes osteoarthritis, where subchondral bone damage may induce the expression of VEGF, which would elicit serum effusion into the joint, a possible direct source of pain. In addition, angiogenesis and growth of demyelinated nerve in the subchondral bone in the presence of VEGF also suggests VEGF may be a useful therapeutic target in this context.18 The means to restrict potential side effects for this and other indications may benefit from the targeted delivery of the antiVEGF reagents to critical sites of action such as the tumor periphery or the joint. Here, we investigate the quaternary structure requirements for highaffinity VEGF binding for Flt-1 domain 2 (D2) through dimerization of the minimal sequence structure as well as
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EXPERIMENTAL PROCEDURES Expression and Purification of D2. Oligonucleotides corresponding to amino acids 133−226 of VEGF R1 (Flt-1) isoform 1 (SwissProt P17948) with a Kozak sequence (GCCACCATGG) and the Flt-1 signal peptide sequence were synthesized and Gateway-cloned into a modified commercially available vector containing a CMV immediate early enhancer-promoter (pCEP4, Invitrogen). HEK293-EBNA monolayers were transfected using Lipofectamine-2000 and conditioned medium harvested after 3 and 6 days, pooled, and stored at −80 °C. The medium was concentrated ∼10-fold by centrifugal ultrafiltration (10 kDa MWCO) and applied to a 1 mL antiProtein C (HPC4) mAb affinity column (Roche 11815024001) equilibrated with 150 mM NaCl, 5 mM CaCl2, 50 mM Tris pH 7.2 (EB) at 0.5 mL/min. The column was washed with 20CV EB and protein eluted with 10CV EB + 5 mM EDTA without CaCl2. The eluate was concentrated by centrifugal ultrafiltration and stored at −80 °C. Preparation of Conjugates. Homobifunctional and 4-arm maleimide PEGs were obtained commercially (Thermo Scientific, Rockfield, IL; Jenchem, Allen TX; NOF Corp., White Plains, NY). Purified D2 in 25 mM NaCl, 2 mM EDTA, and 25 mM Na phosphate pH7 was reduced for 1 h with 2 mM TCEP at 25 °C, desalted by a single dilution and centrifugal ultrafiltration on Vivaspin 4 5 kDa MWCO filters at 4 °C (Corning) prewashed with water. The product was reacted for 4 h in molar excess over PEG maleimide groups as described in the text and purified by SEC over a Superdex 200 column (G.E. Healthcare) using PBS as the mobile phase at 0.4 mL/min. Peaks were collected using A214, concentrated by centrifugal ultrafiltration, and flash frozen at −80 °C. Concentrations were determined by an ultra micro BCA method using BSA as a standard, with the final A562 measured using a 720 nm reference wavelength on a Nanodrop 1000 spectrophotometer (Thermo Scientific). Assays were performed in triplicate. All values were within the standard curve and showed a c.v. of
