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. 2007 Mar;56(3):303-317.
doi: 10.1007/s00262-006-0180-4.

Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR Nanobodies

Rob C Roovers  1 , Toon Laeremans, Lieven Huang, Severine De Taeye, Arie J Verkleij, Hilde Revets, Hans J de Haard, Paul M P van Bergen en Henegouwen
Affiliations

Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR Nanobodies

Rob C Roovers et al. Cancer Immunol Immunother. 2007 Mar.

Abstract

The development of a number of different solid tumours is associated with over-expression of ErbB1, or the epidermal growth factor receptor (EGFR), and this over-expression is often correlated with poor prognosis of patients. Therefore, this receptor tyrosine kinase is considered to be an attractive target for antibody-based therapy. Indeed, antibodies to the EGFR have already proven their value for the treatment of several solid tumours, especially in combination with chemotherapeutic treatment regimens. Variable domains of camelid heavy chain-only antibodies (called Nanobodies) have superior properties compared with classical antibodies in that they are small, very stable, easy to produce in large quantities and easy to re-format into multi-valent or multi-specific proteins. Furthermore, they can specifically be selected for a desired function by phage antibody display. In this report, we describe the successful selection and the characterisation of antagonistic anti-EGFR Nanobodies. By using a functional selection strategy, Nanobodies that specifically competed for EGF binding to the EGFR were isolated from "immune" phage Nanobody repertoires. The selected antibody fragments were found to efficiently inhibit EGF binding to the EGFR without acting as receptor agonists themselves. In addition, they blocked EGF-mediated signalling and EGF-induced cell proliferation. In an in vivo murine xenograft model, the Nanobodies were effective in delaying the outgrowth of A431-derived solid tumours. This is the first report describing the successful use of untagged Nanobodies for the in vivo treatment of solid tumours. The results show that functional phage antibody selection, coupled to the rational design of Nanobodies, permits the rapid development of novel anti-cancer antibody-based therapeutics.

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Figures

Fig. 1
Fig. 1
Immunisation of Llama glama with EGFR-containing cell preparations induces a strong anti-EGFR humoral immune response. The reactivity of pre-immune (day 0: diamonds) and immune sera (day 28: squares and day 42: triangles) of animals immunised with whole, intact A431 cells (a, b, e), or with A431-derived membrane vesicles (c, d) towards A431 cells (a, c), towards EGFR-negative 3T3 2.2 cells (b, d) and towards purified EGF receptor (e) was determined by whole cell ELISA (a–d) or conventional ELISA (e) as described in Materials and methods
Fig. 2
Fig. 2
Selected Nanobodies specifically recognise the EGFR. a The binding of anti-EGFR Nanobody Ia1 displayed on phage and of control phage (not expressing any antibody fragment) to EGFR-negative cell line 3T3 2.2 and EGFR-expressing cell line Her14 was determined by cell-ELISA. Error bars indicate the standard deviation of three independent results. b FACS staining of cell lines expressing EGFR (A431 and Her14) and of the EGFR-negative cell line 3T3 2.2 with FITC-coupled anti-EGFR Nanobody Ia1 (black line) or control Nanobody (grey fill). c Immuno-precipitation (IP) of EGFR from Hela cell lysates using biotinylated anti-EGFR Nanobody Ia1. Lanes 1–3 total cell lysates used for IP. Lanes 4–6 IP with streptavidin beads only, with an anti-glutathion S transferase (control) Nanobody and with the anti-EGFR Nanobody Ia1, respectively. Blots were stained for EGFR as described in Materials and methods
Fig. 3
Fig. 3
Anti-EGFR Nanobodies compete for EGF binding to the EGFR and trivalent bispecific Nanobodies react simultaneously with EGFR and MSA. a By means of ELISA, the binding of biotinylated EGF to immobilised EGFR was measured in the presence of increasing amounts of a control Nanobody (stripes) or of the anti-EGFR Nanobodies Ia1 (diamonds), IIIa3 (squares) or L2-3.40 (triangles). Background staining was defined as no biotinylated EGF being added (rounds). b Monovalent (rounds), bivalent (diamonds) and trivalent (triangles) variants of anti-EGFR Nanobody Ia1 were tested for their ability to block EGF binding to the EGFR in ELISA. c 3 μg of purified mono-, bi- and trivalent Ia1 Nanobody was size-separated on a 15% poly-acrylamide gel and the gel was stained with coomassie brilliant blue to visualise the proteins. d Trivalent, bispecific Nanobodies (of Ia1, IIIa3 and L2–3.40), or bivalent Ia1 were tested for simultaneous binding to EGFR (coated to the ELISA plate) and biotinylated MSA (used to detect bound Nanobody with alkaline-phosphatase coupled extravidin)
Fig. 4
Fig. 4
Anti-EGFR Nanobodies block EGF-induced EGFR phosphorylation but do not act as receptor agonists. a EGF (50 ng/ml, corresponding to approximately 8 nM) was mixed with increasing amounts of purified, trivalent Nanobodies and added to serum-starved Her14 cells for 15 min. EGFR phosphorylation was then measured in cell lysates by Western blotting. Upper panel staining for phosphorylated tyrosine 1068 of EGFR; middle panel staining for total quantity of EGFR; lower panel staining for total amount of actin. b EGF (8 nM) or trivalent, bispecific Nanobodies (1 μM) were added to serum-starved Her14 cells for 15 min and EGFR phosphorylation was measured in total cell lysates by Western blotting. Blots were stained as described in a
Fig. 5
Fig. 5
Trivalent, bispecific Nanobodies inhibit EGF-dependent growth of Her14 cells in vitro. Increasing quantities (0. 1, 10, 100 and 1,000 nM) of purified trivalent Nanobodies were mixed with EGF (8 nM) and added to serum-starved Her14 cells for 4 days. Total cellular protein was then precipitated with tri-chloro acetic acid (TCA) and stained with sulpho-rhodamine B (SRB) as a measure of total cell number. Background proliferation was determined in the absence of EGF. a trivalent Ia1; b trivalent IIIa3 and c trivalent L2–3.40
Fig. 6
Fig. 6
Trivalent, bispecific Nanobodies inhibit A431 tumour cell growth in vitro and growth of A431 xenografts in athymic mice. a–c A431 epidermoid carcinoma cells were grown in serum-free medium supplemented with insulin, transferrin and selenium. After 24 h, medium was refreshed and dilutions (100, 10, 1 and 0 nM) of trivalent Nanobodies (a Ia1; b IIIa3; c L2–3.40) were added, together with 1 ng/ml (approximately 170 pM) of human EGF. After 2 days of culture, cells were pulsed with [3H]- thymidine and assayed 24 h later for the incorporation of radioactivity in genomic DNA. d Groups of 8 mice were subcutaneously injected with A431 cells (107 cells per mouse). One day later, treatment was started by intra-peritoneal injection of 1 mg of purified trimeric Nanobody. Mice were treated twice weekly with the same quantity of Nanobody for up to 4 weeks (indicated by arrows). Relative tumour volume (RTV) was measured as function of time. Squares solvent control (PBS); triangles IaI; inverse triangles IIIa3 and diamonds L2–3.40. Error bars indicate the standard error of the mean of 8 mice
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