VEGF-Trap: a VEGF blocker with potent antitumor effects

Abstract

Vascular endothelial growth factor (VEGF) plays a critical role during normal embryonic angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using a humanized monoclonal antibody are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. Previous studies have found that one of the most effective ways to block the VEGF-signaling pathway is to prevent VEGF from binding to its normal receptors by administering decoy-soluble receptors. The highest-affinity VEGF blocker described to date is a soluble decoy receptor created by fusing the first three Ig domains of VEGF receptor 1 to an Ig constant region; however, this fusion protein has very poor in vivo pharmacokinetic properties. By determining the requirements to maintain high affinity while extending in vivo half life, we were able to engineer a very potent high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties. This VEGF-Trap effectively suppresses tumor growth and vascularization in vivo, resulting in stunted and almost completely avascular tumors. VEGF-Trap-mediated blockade may be superior to that achieved by other agents, such as monoclonal antibodies targeted against the VEGF receptor.

Fig 1.

Engineering of VEGF-Traps with improved pharmacokinetics. (A) Schematics of full-length VEGFR1 (red) and VEGFR2 (blue) are provided, indicating their seven Ig domains, transmembrane regions (black bars), and kinase domains (ovals). The parental VEGF-Trap contains the first three Ig domains of VEGFR1 (including the highly basic 10-aa stretch in Ig3, blue box) fused to the Fc portion of human IgG1. VEGF-Trap_ΔB1 is identical to the parental VEGF-Trap, except that the basic stretch in Ig3 has been removed. VEGF-Trap_ΔB2 is the same construct as ΔB1, except that the first Ig domain has been removed. VEGF-Trap_R1R2 possesses the second Ig domain of VEGFR1 and the third Ig domain of VEGFR2 fused to the Fc portion of human IgG1. (B) The four indicated VEGF-Traps were assayed in vitro for their capacity to bind to extracellular matrix, with only the parental VEGF-Trap and VEGF-Trap_ΔB1 demonstrating binding. (C) Pharmacokinetic analysis of the VEGF-Traps reveals that the parental VEGF-Trap has the poorest profile, whereas VEGF-Trap_R1R2 showed the best profile.

Fig 2.

Binding affinity and inhibitory properties of VEGF-Traps. (A) Affinities of indicated VEGF-Traps for VEGF, as determined by using a binding assay that measures unbound VEGF (ordinate) after incubation of 10 pM of human VEGF₁₆₅ with varying concentrations of VEGF-Traps (abscissa). (B) Inhibition of VEGF-induced phosphorylation of VEGFR2 in human umbilical vein endothelial cell phosphorylations using indicated VEGF-Traps at 1.5-fold molar excess, as revealed with immunoblotting assay. (C) Inhibition of VEGF-induced proliferation of fibroblasts containing a chimeric VEGFR2/TrkB receptor, using varying concentrations of VEGF-Traps in the presence of 1.56 nM of VEGF.

Fig 3.

Using blockade of VEGF-induced acute hypotension to pharmacodynamically compare VEGF-Traps. (A) When rats were treated with VEGF-Traps at 25 mg/kg at 1 day before VEGF challenge, VEGF-Trap_R1R2 (n = 8) completely blocked VEGF-induced hypotension, whereas PBS (n = 6) and parental VEGF-Trap (n = 6) were ineffective. ANOVA shows treatment effect, P < 0.007. (B) At a 5-fold lower dose (5 mg/kg), VEGF-Trap_R1R2 was still effective at 1 day (n = 4) or 3 days (n = 3) before the VEGF challenge. ANOVA shows treatment effect, P < 0.03.

Fig 4.

VEGF-Trap_R1R2 dramatically inhibits the s.c. growth and vascularity of implanted tumors from diverse tissues and species. (A) VEGF-Trap _R1R2 substantially blocked the growth of the indicated s.c. implanted tumors, at the indicated doses twice weekly for 2 weeks (C6 and B16F10.9) or 3.0 weeks (A673). Error bars represent standard error of mean, n = five mice/treatment group. The differences between control tumor volumes and VEGF-Trap_R1R2–treated tumor volumes were analyzed by using Student's t tests and found to be significant at the following levels: B16F10 P = 0.01; A673 P = 0.06; C6 P < 0.0001. (B–D) Histological analysis reveals that VEGF-Trap_R1R2 can effectively block blood vessel growth in these implanted tumors. Sections of C6 tumors stained with antibodies to platelet–endothelial cell adhesion molecule reveal that vehicle-treated animals had large tumors that were highly vascularized (B), whereas animals treated with 25 mg/kg VEGF-Trap_R1R2 (C) had tumors that were largely avascular with large areas of necrosis (N). Viable tumor appeared to be vascularized because of cooption of preexisting host vessels (white arrowheads) associated with hypodermal musculature (M) and dermis. Treatment with 2.5 mg/kg VEGF-Trap_R1R2 greatly stunted tumor growth (C) and resulted in large necrotic regions (N), although small pockets of vessels were occasionally apparent (black arrows). (Bar = 100 μm.)

Fig 5.

VEGF-Trap_R1R2 blocks tumor growth (of subcutaneously implanted B16F10.9 cells) at far lower concentrations than DC101, a monoclonal antibody directed to VEGFR2. Mice were treated twice weekly with the indicated dose of VEGF-Trap_R1R2, DC101, or vehicle. After 2.5 weeks, mice were killed, and tumors were excised and measured. Individual tumor volumes are shown (colored bars), as are average tumor volumes for each treatment (black bars) ± SEM, n = six mice/treatment group. Differences between treatment groups were analyzed by using a one-way ANOVA followed by Fisher's protected least significant difference test. Average volume of tumors in all treatment groups is significantly smaller than control tumor volume (P < 0.01). Differences in tumor volume between the high-dose VEGF-Trap, low-dose VEGF-Trap, and high-dose DC101 treatment groups are not significantly different, but they are significantly different from those of the low-dose DC101 treatment group (P < 0.02).