Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation

Danquah, Welbeck; Meyer-Schwesinger, Catherine; Rissiek, Björn; Pinto, Carolina Ferreira; Serracant-Prat, Arnau; Amadi, Miriam; Iacenda, Domenica; Knop, Jan-Hendrik; Hammel, Anna; Bergmann, Philine; Schwarz, Nicole; Assunção, Joana; Rotthier, Wendy; Haag, Friedrich; Tolosa, Eva; Bannas, Peter; Boué‐Grabot, Éric; Magnus, Tim; Laeremans, Toon; Stortelers, Catelijne; Koch-Nolte, Friedrich

doi:10.1126/scitranslmed.aaf8463

Cited by 141 publications

(158 citation statements)

References 50 publications

Supporting

Mentioning

153

Contrasting

Order By: Relevance

“…Furthermore, the high selectivity often associated with such biologics alongside their ability to access functional crevices may provide further opportunities to modulate ion channel function. Indeed, the targeting of both ligand and voltage-gated ion channels by Nanobodies and scFv’s, respectively, has already demonstrated this potential (Danquah et al, 2016; Harley et al, 2016). …”

Section: Resultsmentioning

confidence: 99%

Affimer proteins are versatile and renewable affinity reagents

Tiede

Bedford

Heseltine

et al. 2017

eLife

172

162

View full text Add to dashboard Cite

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.DOI: http://dx.doi.org/10.7554/eLife.24903.001

show abstract

Section: Resultsmentioning

confidence: 99%

Affimer proteins are versatile and renewable affinity reagents

Tiede

Bedford

Heseltine

et al. 2017

eLife

172

162

View full text Add to dashboard Cite

show abstract

“…This limited sample represents an interesting mix of ligand-dependent and -independent modes-of-action for the blocking of RON signaling. Many examples document the ability of the outbred camelid’s immune response to generate nanobodies against challenging targets including ion channels (29–31), GPCRs (32, 33), small molecules and toxins (34, 35), viruses (34, 36) and enzymes (2). To our knowledge, this is the first example to illustrate the potential extent of an outbred camelid’s functional immune response in terms of sequence diversity.…”

Section: Discussionmentioning

confidence: 99%

Large Diversity of Functional Nanobodies from a Camelid Immune Library Revealed by an Alternative Analysis of Next-Generation Sequencing Data

et al. 2017

Self Cite

View full text Add to dashboard Cite

Next-generation sequencing (NGS) has been applied successfully to the field of therapeutic antibody discovery, often outperforming conventional screening campaigns which tend to identify only the more abundant selective antibody sequences. We used NGS to mine the functional nanobody repertoire from a phage-displayed camelid immune library directed to the recepteur d’origine nantais (RON) receptor kinase. Challenges to this application of NGS include accurate removal of read errors, correct identification of related sequences, and establishing meaningful inclusion criteria for sequences-of-interest. To this end, a sequence identity threshold was defined to separate unrelated full-length sequence clusters by exploring a large diverse set of publicly available nanobody sequences. When combined with majority-rule consensus building, applying this elegant clustering approach to the NGS data set revealed a wealth of >5,000-enriched candidate RON binders. The huge binding potential predicted by the NGS approach was explored through a set of randomly selected candidates: 90% were confirmed as RON binders, 50% of which functionally blocked RON in an ERK phosphorylation assay. Additional validation came from the correct prediction of all 35 RON binding nanobodies which were identified by a conventional screening campaign of the same immune library. More detailed characterization of a subset of RON binders revealed excellent functional potencies and a promising epitope diversity. In summary, our approach exposes the functional diversity and quality of the outbred camelid heavy chain-only immune response and confirms the power of NGS to identify large numbers of promising nanobodies.

show abstract

“…Next, sdAbs were phage displayed and biopannings (27) were performed on hCD20 pos CHO-K1 cells. After each panning round, different colonies were randomly selected and their sdAbs-containing periplasmatic extracts were analyzed by cell ELISA for specific binding to hCD20 pos CHO-K1 cells (27).…”

Section: Generation Of Anti-hcd20 Sdabsmentioning

confidence: 99%

Theranostic Radiolabeled Anti-CD20 sdAb for Targeted Radionuclide Therapy of Non-Hodgkin Lymphoma

Krasniqi

D’Huyvetter

Xavier

et al. 2017

Molecular Cancer Therapeutics

View full text Add to dashboard Cite

Anti-CD20 radioimmunotherapy is an effective approach for therapy of relapsed or refractory CD20 pos lymphomas, but faces limitations due to poor tumor penetration and undesirable pharmacokinetics of full antibodies. Camelid single-domain Ab fragments (sdAb) might circumvent some of the limitations of radiolabeled full antibodies. In this study, a set of hCD20-targeting sdAbs was generated, and their capacity to bind hCD20 was evaluated in vitro and in vivo. A lead sdAb, sdAb 9079, was selected on the basis of its specific tumor targeting and significant lower kidney accumulation compared with other sdAbs. SdAb 9079 was then radiolabeled with 68 Ga and 177 Lu for PET imaging and targeted therapy. The therapeutic potential of 177 Lu-DTPA-sdAb was compared with that of 177 Lu-DTPA-rituximab and unlabeled rituximab in mice bearing hCD20 pos tumors. Radiolabeled with 68 Ga, sdAb 9079 showed specific tumor uptake, with very low accumulation in nontarget organs, except kidneys. The tumor uptake of 177 Lu-DTPA-sdAb 9079 after 1.5 h was 3.4 AE 1.3% ID/g, with T/B and T/M ratios of 13.3 AE 4.6 and 32.9 AE 15.6. Peak tumor accumulation of 177 Lu-DTPA-rituximab was about 9 times higher, but concomitantly with high accumulation in nontarget organs and very low T/B and T/M ratios (0.8 AE 0.1 and 7.1 AE 2.4). Treatment of mice with 177 Lu-DTPA-sdAb 9079 significantly prolonged median survival compared with control groups and was as effective as treatment with rituximab or its 177 Lu-labeled variant. Taken together, sdAb 9079 displays promising features as a theranostic drug to treat CD20 pos lymphomas.

show abstract

Nanobodies that block gating of the P2X7 ion channel ameliorate inflammation

Cited by 141 publications

References 50 publications

Affimer proteins are versatile and renewable affinity reagents

Affimer proteins are versatile and renewable affinity reagents

Large Diversity of Functional Nanobodies from a Camelid Immune Library Revealed by an Alternative Analysis of Next-Generation Sequencing Data

Theranostic Radiolabeled Anti-CD20 sdAb for Targeted Radionuclide Therapy of Non-Hodgkin Lymphoma

Contact Info

Product

Resources

About