A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor

Abstract

Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 10 ( 13) Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 μM for the parental clone. Individual, optimized, Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation, and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC 50 values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR, and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy.

Figure 1

Biophysical properties of EI-Tandem. Individual Adnectin subunits derived from the 10th type III domain of human fibronectin were engineered by changing the targeting loops to redirect binding to the human EGFR and IGF-1R. The monospecific Adnectins were linked together by a short glycine-serine linker to create a bispecific molecule and PEGylated to increase serum half-life. Binding affinity was determined using Biacore T100 Evaluation Software. The tandem Adnectin was >98% monomeric as indicated by size exclusion chromatography on a Superdex 200, 5/150 HPLC column and was soluble to greater than 20 mg/ml. The melting profile predicts stability at body temperature and typical yield of purified product is 230 mg per liter of E. coli.

Figure 2

Antiproliferative activity of EI-Tandem. (A) Effect of EI-Tandem on the proliferation of H292 cells. (B) Effects of EI-Tandem on the proliferation of RH41 cells. Cell proliferation was determined by quantification of cellular DNA using CyQUANT NF fluorescent stain. Cells were plated into 96-well microplates and treated with various doses of PEGylated EGFR-Adnectin (○), IGF-IR-Adnectin (□) or EI-Tandem (●), for 72 h prior to quantification of DNA. All constructs were conjugated to a 40 kDa branched PEG at the c-terminal cysteine. Error bars indicate SD. (C) Immunoblot analysis of total EGFR and IGF-IR in DiFi cells demonstrating downregulation of the EGFR after treatment with EI-Tandem. (D) Immunocytochemical staining of EGFR in DiFi cells after treatment with PEGylated EI-Tandem showing disappearance of EGFR from the cells. EGFR is stained red and DNA is stained blue.

Figure 3

Pharmacokinetic profile and in vivo activity of EI-Tandem. (A) Plasma levels of EI-Tandem after dosing i.p. with 100 mg/kg (○) and 10 mg/kg (○) of EI-Tandem. (B) H292 xenografts were either untreated (○); dosed three times a week with 100 mg/kg PEGylated EI-Tandem formulated in PBS (□) or dosed every three days i.p. with panitumumab (○). Dosing schedule of EI-Tandem (I) and panitumumab (x) are indicated on the x-axis. (C) Efficacy of PEGlyated EI-Tandem against RH41 Ewing sarcoma xenografts. Treatments and schedule are as described for the H292 model. The EI-tandem dosed in the RH41 model was the I-GS10-E#4-PEG construct (Table 1). Inhibition of growth was significantly different for panitumumab and EI-Tandem vs. control in H292 and EI-Tandem vs. control in RH41 by a two-tailed paired t-test as described in Experimental Procedures. (D) The effects of EI-Tandem (open bars) and panitumumab (hatched bars) on pEGFR, total EGFR and pErb-B2 in H292 tumors as determined by Meso Scale analysis. “C” signifies control levels (black bars) measured in untreated H292 tumors sampled on day 20 from the experiment shown in (B). Time points indicate hours after the last dose of treatment in the study presented in (B). Error bars indicate SD.

Figure 4

Effect of EI-Tandem on EGFR and IGF-IR crosstalk in vitro and in vivo. (A) BxPC3 cells were serum starved before stimulation with either EGF, IGF-I or a combination of EGF+IGF-I. All treatments were with 1 µM PEGylated Adnectins. Equal amounts of total protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to membrane and probed with various antibodies, followed by GAPDH as an internal loading control. (B) Efficacy of PEGlyated EI-Tandem against BxPC3 xenografts. Animals were untreated (■); dosed three times a week with 100 mg/kg EI-Tandem (□), dosed three times a week with 50 mg/kg EGFR-Adnectin (solid red line), IGFR-Adnectin (solid blue line) or a combination of 50 mg/kg EGFR-Adnectin + 50 mg/kg IGFR-Adnectin (solid green line). For comparison, an EGFR antibody was administered in one group with the optimal dose of 40 mg/kg cetuximab every three days (○). All treatments were by the i.p. route. The data represent the means ± SE of tumor sizes from eight animals in each group. Dosing schedule of EI-Tandem or monospecific Adnectins (I) and cetuximab (x) are indicated on the x-axis. BxPC3 immunoblot and xenograft were carried out with the I-GS10-E#4-PEG construct (Table 1).

Figure 5

The EGFR-Adnectin does not compete for binding of EGFR antibodies to EGFR. (A) Initial injection of the EGFR-Adnectin shows binding to EGFR on the surface of the chip. A second injection of EGFR-Adnectin mixed with an equal amount of cetuximab, panitumumab, or nimotuzumab showed no competition for binding of antibodies to EGFR by the Adnectin. (B) The EI-Tandem Adnectin can bind EGFR and IGF-IR simultaneously. Initial injection of EI-Tandem Adnectin showed binding to EGFR immobilized on the chip surface. A second injection of EI-Tandem plus soluble IGF-IR showed a second binding event for sIGF-IR to other end of the immobilized EI-Tandem Adnectin.