Toon Laeremans

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

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.

Improved tumor targeting of anti–epidermal growth factor receptor Nanobodies through albumin binding: taking advantage of modular Nanobody technology

The ∼15-kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies) can easily be formatted as multivalent or multispecific single-chain proteins. Because of fast excretion, however, they are less suitable for therapy of cancer. In this study, we aimed for improved tumor targeting of a bivalent anti–epidermal growth factor receptor (EGFR) Nanobody (αEGFR-αEGFR) by fusion to a Nanobody unit binding to albumin (αAlb). Biodistributions of αEGFR-αEGFR, αEGFR-αEGFR-αAlb (∼50 kDa), αTNF-αTNF-αAlb (control, binding tumor necrosis factor-α), and the ∼150-kDa anti-EGFR antibody cetuximab were compared in A431 xenograft-bearing mice. The proteins were radiolabeled with 177Lu to facilitate quantification. Tumor uptake of 177Lu-αEGFR-αEGFR decreased from 5.0 ± 1.4 to 1.1 ± 0.1 %ID/g between 6 and 72 h after injection. Due to its rapid blood clearance, tumor-to-blood ratios >80 were obtained within 6 h after injection. Blood clearance became dramatically slower and tumor uptake became significantly higher by introduction of αAlb. Blood levels of αEGFR-αEGFR-αAlb were 21.2 ± 2.5, 11.9 ± 0.6, and 4.0 ± 1.4 and tumor levels were 19.4 ± 5.5, 35.2 ± 7.5, and 28.0 ± 6.8 %ID/g at 6, 24, and 72 h after injection, respectively. Tumor uptake was at least as high as for cetuximab (15.5 ± 3.9, 27.1 ± 7.9, and 25.6 ± 6.1 %ID/g) and significantly higher than for αTNF-αTNF-αAlb. αEGFR-αEGFR-αAlb showed faster and deeper tumor penetration than cetuximab. These data show that simple fusion of αEGFR and αAlb building blocks results in a bifunctional Nanobody format, which seems more favorable for therapy as far as pharmacokinetics and tumor deposition are concerned. [Mol Cancer Ther 2008;7(8):2288–97]

Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation

Single-domain antibodies called nanobodies block P2X7, an inflammatory ion channel, reducing skin and kidney inflammation in mice. Tackling a tough target: An ATP-sensitive channel Injured and dying cells release lots of ATP, which triggers inflammation by binding to the ion channel P2X7. Interfering with this process could treat numerous diseases, but so far small-molecule drugs have not been potent or specific enough. Now, Danquah and colleagues have developed single-domain “mini antibodies” called nanobodies that rise to the challenge. One of their nanobodies blocked the P2X7 channel and inhibited disease in mouse models of kidney inflammation and contact dermatitis. Another nanobody dampened the release of inflammatory messengers from human cells 1000 times more effectively than the small-molecule drugs now under development. Nanobodies can be linked together to prolong their lifetime or confer cell specificity, a useful versatility that increases their appeal. Ion channels are desirable therapeutic targets, yet ion channel–directed drugs with high selectivity and few side effects are still needed. Unlike small-molecule inhibitors, antibodies are highly selective for target antigens but mostly fail to antagonize ion channel functions. Nanobodies—small, single-domain antibody fragments—may overcome these problems. P2X7 is a ligand-gated ion channel that, upon sensing adenosine 5′-triphosphate released by damaged cells, initiates a proinflammatory signaling cascade, including release of cytokines, such as interleukin-1β (IL-1β). To further explore its function, we generated and characterized nanobodies against mouse P2X7 that effectively blocked (13A7) or potentiated (14D5) gating of the channel. Systemic injection of nanobody 13A7 in mice blocked P2X7 on T cells and macrophages in vivo and ameliorated experimental glomerulonephritis and allergic contact dermatitis. We also generated nanobody Dano1, which specifically inhibited human P2X7. In endotoxin-treated human blood, Dano1 was 1000 times more potent in preventing IL-1β release than small-molecule P2X7 antagonists currently in clinical development. Our results show that nanobody technology can generate potent, specific therapeutics against ion channels, confirm P2X7 as a therapeutic target for inflammatory disorders, and characterize a potent new drug candidate that targets P2X7.