Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering

Lüdel F, Bufe S, Bleymüller WM, et al. Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering. Antibodies. 2019;8(4): 48.Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter kD , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion. spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter k D , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion.</jats:p

Identification of a high affinity binding site for abscisic acid on human lanthionine synthetase component C-like protein 2

Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian receptor mediating the functional effects of the universal stress hormone abscisic acid (ABA) in mammals. ABA stimulates insulin independent glucose uptake in myocytes and adipocytes via LANCL2 binding in vitro, improves glucose tolerance in vivo and induces brown fat activity in vitro and in vivo. The emerging role of the ABA/LANCL2 system in glucose and lipid metabolism makes it an attractive target for pharmacological interventions in diabetes mellitus and the metabolic syndrome. The aim of this study was to investigate the presence of ABA binding site(s) on LANCL2 and identify the amino acid residues involved in ABA binding. Equilibrium binding assays ([3H]-ABA saturation binding and surface plasmon resonance analysis) suggested multiple ABA-binding sites, prompting us to perform a computational study that indicated one putative high-affinity and two low-affinity binding sites. Site-directed mutagenesis (single mutant R118I, triple mutants R118I/R22I/K362I and R118I/S41A/E46I) and equilibrium binding experiments on the mutated LANCL2 proteins identified a high-affinity ABA-binding site involving R118, with a KD of 2.6 nM ± 1.2 nM, as determined by surface plasmon resonance. Scatchard plot analysis of binding curves from both types of equilibrium binding assays revealed a Hill coefficient >1, suggesting cooperativity of ABA binding to LANCL2. Identification of the high-affinity ABA-binding site is expected to allow the design of ABA agonists/antagonists, which will help to understand the role of the ABA/LANCL2 system in human physiology and disease

Non-Agonistic Bivalent Antibodies That Promote c-MET Degradation and Inhibit Tumor Growth and Others Specific for Tumor Related c-MET

The c-MET receptor has a function in many human cancers and is a proven therapeutic target. Generating antagonistic or therapeutic monoclonal antibodies (mAbs) targeting c-MET has been difficult because bivalent, intact anti-Met antibodies frequently display agonistic activity, necessitating the use of monovalent antibody fragments for therapy. By using a novel strategy that included immunizing with cells expressing c-MET, we obtained a range of mAbs. These c-MET mAbs were tested for binding specificity and anti-tumor activity using a range of cell-based techniques and in silico modeling. The LMH 80 antibody bound an epitope, contained in the small cysteine-rich domain of c-MET (amino acids 519–561), that was preferentially exposed on the c-MET precursor. Since the c-MET precursor is only expressed on the surface of cancer cells and not normal cells, this antibody is potentially tumor specific. An interesting subset of our antibodies displayed profound activities on c-MET internalization and degradation. LMH 87, an antibody binding the loop connecting strands 3d and 4a of the 7-bladed β-propeller domain of c-MET, displayed no intrinsic agonistic activity but promoted receptor internalization and degradation. LMH 87 inhibited HGF/SF-induced migration of SK-OV-3 ovarian carcinoma cells, the proliferation of A549 lung cancer cells and the growth of human U87MG glioma cells in a mouse xenograft model. These results indicate that c-MET antibodies targeting epitopes controlling receptor internalization and degradation provide new ways of controlling c-MET expression and activity and may enable the therapeutic targeting of c-MET by intact, bivalent antibodies

Anti-Cancer Auto-Antibodies: Roles, Applications and Open Issues

Auto-antibodies are classically associated with autoimmune diseases, where they are an integral part of diagnostic panels. However, recent evidence is accumulating on the presence of auto-antibodies against single or selected panels of auto-antigens in many types of cancer. Auto-antibodies might initially represent an epiphenomenon derived from the inflammatory environment induced by the tumor. However, their effect on tumor evolution can be crucial, as is discussed in this paper. It has been demonstrated that some of these auto-antibodies can be used for early detection and cancer staging, as well as for monitoring of cancer regression during treatment and follow up. Interestingly, certain auto-antibodies were found to promote cancer progression and metastasis, while others contribute to the body’s defense against it. Moreover, auto-antibodies are of a polyclonal nature, which means that often several antibodies are involved in the response to a single tumor antigen. Dissection of these antibody specificities is now possible, allowing their identification at the genetic, structural, and epitope levels. In this review, we report the evidence available on the presence of auto-antibodies in the main cancer types and discuss some of the open issues that still need to be addressed by the research community

Ingegnerizzazione di mAb per scopi teraupeutici

La quinta edizione di Farmacologia generale e molecolare mantiene l'impostazione originale che ne ha fatto un volume unico nel panorama editoriale, ponendo al centro dell'attenzione il bersaglio molecolare del farmaco anziché il composto chimico. Al tempo stesso si presenta ai lettori profondamente rinnovata: oltre ai necessari aggiornamenti di una materia in rapido sviluppo, che hanno portato a un'ampia revisione di tutti i capitoli, nell'ultimo decennio è mutato il contesto biomedico, spostando l'interesse sul malato che è, e deve sempre più essere, protagonista attivo della scelta terapeutica. Questa lenta ma progressiva rivoluzione culturale ha portato cambiamenti anche nel modo di concepire e sviluppare i farmaci, nella legislazione che governa la loro immissione in commercio e nell'assetto della filiera produttiva del farmaco. Inserendo nuovi capitoli e nuovi Autori, il libro affronta questi temi e le nuove tecnologie che offrono una conoscenza sempre più approfondita dei determinanti molecolari delle patologie e della loro modulazione farmacologica, una maggiore efficacia e specificità nell'azione complessiva dei farmaci e una minore tossicità. Tra le principali novità, vi sono l'ampia rivisitazione degli aspetti riguardanti il drug discovery, temi innovativi portati dalla nanotecnologia e dalle biotecnologie e la discussione degli sviluppi della farmacologia verso la terapia personalizzata. Capitoli inediti trattano la farmacologia di genere e gli aspetti tossicologici della farmacologia e della farmacoterapia. Il libro si conclude con l'analisi critica del futuro della farmacologia mondiale, di particolare utilità per chi vuole intraprendere la carriera di farmacologo. Farmacologia generale e molecolare dispone online, su studenti33.it, di preziosi contenuti aggiuntivi: l'iconografia completa del volume e una ricca selezione di approfondimenti per la preparazione avanzata in ambito farmacologico, quasi un secondo volume che completa il testo cartaceo

Inhibition of the MET Kinase Activity and Cell Growth in MET-Addicted Cancer Cells by Bi-Paratopic Linking.

Andres F, Iamele L, Meyer T, et al. Inhibition of the MET Kinase Activity and Cell Growth in MET-Addicted Cancer Cells by Bi-Paratopic Linking. Journal of molecular biology. 2019;431(10):2020-2039.MET, the product of the c-MET proto-oncogene, and its ligand hepatocyte growth factor/scatter factor (HGF/SF) control survival, proliferation and migration during development and tissue regeneration. HGF/SF-MET signaling is equally crucial for growth and metastasis of a variety of human tumors but resistance to small-molecule inhibitors of MET kinase develops rapidly and therapeutic antibody targeting remains challenging. We made use of the designed ankyrin repeat protein (DARPin) technology to develop an alternative approach for inhibiting MET. We generated a collection of MET-binding DARPins covering epitopes in the extracellular MET domains and created comprehensive sets of bi-paratopic fusion proteins. This new class of molecules efficiently inhibited MET kinase activity and downstream signaling, caused receptor downregulation and strongly inhibited the proliferation of MET-dependent gastric carcinoma cells carrying MET locus amplifications. MET-specific bi-paratopic DARPins may represent a novel and potent strategy for therapeutic targeting of MET and other receptors, and this study has elucidated their mode of action. Copyright © 2019. Published by Elsevier Ltd