Four individually druggable MET hotspots mediate HGF-driven tumor progression

Basilico, Cristina; Hultberg, Anna; Blanchetot, Christophe; Jonge, Natalie De; Festjens, Els; Hanssens, Valérie; Osepa, Sjudry Ilona; Boeck, Gitte De; Mira, Alessia; Cazzanti, Manuela; Morello, Virginia; Dreier, Torsten; Saunders, Michael; Haard, Hans de; Michieli, Paolo

doi:10.1172/jci72316

Cited by 44 publications

(72 citation statements)

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“…In line with this, recent literature reports also suggest that plain receptor dimerization without participation of any co-receptor is sufficient for full MET activation. Monoclonal antibodies, for instance, are able to fully trigger MET phosphorylation and cellular responses (42). Even small artificial MET binding macrocycles and DNA aptamers, which are unlikely to bind further co-receptors, are able to elicit full MET activation when dimerized (43,44).…”

Section: Discussionmentioning

confidence: 99%

MET-activating Residues in the B-repeat of the Listeria monocytogenes Invasion Protein InlB

Bleymüller

Lämmermann

Ebbes

et al. 2016

Journal of Biological Chemistry

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Edited by Norma AllewellThe facultative intracellular pathogen Listeria monocytogenes causes listeriosis, a rare but life-threatening disease. Host cell entry begins with activation of the human receptor tyrosine kinase MET through the bacterial invasion protein InlB, which contains an internalin domain, a B-repeat, and three GW domains. The internalin domain is known to bind MET, but no interaction partner is known for the B-repeat. Adding the B-repeat to the internalin domain potentiates MET activation and is required to stimulate Madin-Darby canine kidney (MDCK) cell scatter. Therefore, it has been hypothesized that the B-repeat may bind a co-receptor on host cells. To test this hypothesis, we mutated residues that might be important for binding an interaction partner. We identified two adjacent residues in strand ␤2 of the ␤-grasp fold whose mutation abrogated induction of MDCK cell scatter. Biophysical analysis indicated that these mutations do not alter protein structure. We then tested these mutants in human HT-29 cells that, in contrast to the MDCK cells, were responsive to the internalin domain alone. These assays revealed a dominant negative effect, reducing the activity of a construct of the internalin domain and mutated B-repeat below that of the individual internalin domain. Phosphorylation assays of MET and its downstream targets AKT and ERK confirmed the dominant negative effect. Attempts to identify a host cell receptor for the B-repeat were not successful. We conclude that there is limited support for a co-receptor hypothesis and instead suggest that the B-repeat contributes to MET activation through low affinity homodimerization.

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Section: Discussionmentioning

confidence: 99%

MET-activating Residues in the B-repeat of the Listeria monocytogenes Invasion Protein InlB

Bleymüller

Lämmermann

Ebbes

et al. 2016

Journal of Biological Chemistry

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“…The two antibodies were isolated from antibody libraries made from actively immunized Lama glama with either human CD70 or MET over-expressing cells using phage display. 27,28 Both CD70 (a membrane-bound cytokine) and MET (a tyrosine kinase receptor) play important roles in tumor growth/survival, angiogenesis and metastasis. 29,30 Our acquired candidate llama-derived antibodies were subjected to structure determination by X-ray crystallography.…”

Section: Camelid Iglv Repertoiresmentioning

confidence: 99%

Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform

Klarenbeek

Mazouari²,

Desmyter

et al. 2015

mAbs

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de Haard & Ikbel Achour (2015) Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform, mAbs, 7:4, 693-706, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Camelid immunoglobulin variable (IGV) regions were found homologous to their human counterparts; however, the germline V repertoires of camelid heavy and light chains are still incomplete and their therapeutic potential is only beginning to be appreciated. We therefore leveraged the publicly available HTG and WGS databases of Lama pacos and Camelus ferus to retrieve the germline repertoire of V genes using human IGV genes as reference. In addition, we amplified IGKV and IGLV genes to uncover the V germline repertoire of Lama glama and sequenced BAC clones covering part of the Lama pacos IGK and IGL loci. Our in silico analysis showed that camelid counterparts of all human IGKV and IGLV families and most IGHV families could be identified, based on canonical structure and sequence homology. Interestingly, this sequence homology seemed largely restricted to the Ig V genes and was far less apparent in other genes: 6 therapeutically relevant target genes differed significantly from their human orthologs. This contributed to efficient immunization of llamas with the human proteins CD70, MET, interleukin (IL)-1b and IL-6, resulting in large panels of functional antibodies. The in silico predicted human-homologous canonical folds of camelidderived antibodies were confirmed by X-ray crystallography solving the structure of 2 selected camelid anti-CD70 and anti-MET antibodies. These antibodies showed identical fold combinations as found in the corresponding human germline V families, yielding binding site structures closely similar to those occurring in human antibodies. In conclusion, our results indicate that active immunization of camelids can be a powerful therapeutic antibody platform.

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“…In addition, 6 new VH families were identified from 73 FabVλ (VH families [11][12][13][14][15][16], also binding to ECD-GCGR. ScFv recognizing ECD-GCGR was measured using SPR and revealed off rates…”

Section: Dna Immunization Raises Target-specific Immune Responses Agamentioning

confidence: 99%

“…PBLs were isolated from 400 ml blood collected 4 d after completion of all immunizations; RNA was isolated, cDNA amplified and 4 Fab libraries (llamas 14 and 73) were constructed as previously described. 12 Two scFv libraries were constructed from llama 73 by amplification of the heavy chain (VH) and the light chain (Vλ and Vk) from the primary Fab libraries, digestion with restriction enzymes SfiI/NotI for VH and ApaLI/AscI for Vλ and Vk, extracted and ligated into the pSc vector. Vector pSc was derived from the pCB3 phagemid vector and has the LacZ promoter, RBS, gene3 leader, SfiI/NotI restriction sites, (Gly 4 Ser) 3 linker, ApaLI/AscI restriction sites, His 6 , c-myc-tag, amber stop and gene3.…”

Section: Immunization and Library Constructionmentioning

confidence: 99%

“…3,10,11 Active immunization of outbred llamas and phage display for the generation of potent therapeutic mAbs against membrane proteins has proven powerful. 12,13 Here, we describe the successful combination of DNA immunization of outbred llamas with scFv-phage display, and selections using viruslike particles (VLP) for the identification of glucagon receptor-specific antagonistic mAbs.…”

Section: Introductionmentioning

confidence: 99%

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DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops

Woning

Boeck

Blanchetot

et al. 2016

mAbs

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Haard (2016) DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops, mAbs, 8:6, 1126-1135, DOI: 10.1080/19420862.2016 ABSTRACTThe identification of functional monoclonal antibodies directed against G-protein coupled receptors (GPCRs) is challenging because of the membrane-embedded topology of these molecules. Here, we report the successful combination of llama DNA immunization with scFv-phage display and selections using virus-like particles (VLP) and the recombinant extracellular domain of the GPCR glucagon receptor (GCGR), resulting in glucagon receptor-specific antagonistic antibodies. By immunizing outbred llamas with plasmid DNA containing the human GCGR gene, we sought to provoke their immune system, which generated a high IgG1 response. Phage selections on VLPs allowed the identification of mAbs against the extracellular loop regions (ECL) of GCGR, in addition to multiple VH families interacting with the extracellular domain (ECD) of GCGR. Identifying mAbs binding to the ECL regions of GCGR is challenging because the large ECD covers the small ECLs in the energetically most favorable 'closed conformation' of GCGR. Comparison of Fab with scFv-phage display demonstrated that the multivalent nature of scFv display is essential for the identification of GCGR specific clones by selections on VLPs because of avid interaction. Ten different VH families that bound 5 different epitopes on the ECD of GCGR were derived from only 2 DNA-immunized llamas. Seven VH families demonstrated interference with glucagon-mediated cAMP increase. This combination of technologies proved applicable in identifying multiple functional binders in the class B GPCR context, suggesting it is a robust approach for tackling difficult membrane proteins.

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Four individually druggable MET hotspots mediate HGF-driven tumor progression

Cited by 44 publications

References 50 publications

MET-activating Residues in the B-repeat of the Listeria monocytogenes Invasion Protein InlB

MET-activating Residues in the B-repeat of the Listeria monocytogenes Invasion Protein InlB

Camelid Ig V genes reveal significant human homology not seen in therapeutic target genes, providing for a powerful therapeutic antibody platform

DNA immunization combined with scFv phage display identifies antagonistic GCGR specific antibodies and reveals new epitopes on the small extracellular loops

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