Methodologies for the isolation of alternative binders with improved clinical potentiality over conventional antibodies

Abstract

The availability of binders to different functional domains of the same protein or to physiologically co-operating proteins allows for the simultaneous inhibition of independent downstream signaling pathways. This multi-target approach represents a promising therapeutic strategy, as demonstrated in the case of the synergistic effect of anti-Her2 treatment based on the combined use of the trastuzumab and pertuzumab monoclonal antibodies that induce cellular cytotoxicity and impair the receptor dimerization, respectively. Therefore, a reliable selection method for the recovery of epitope-specific antibodies is highly needed. Animal immunization with short peptides resembling the epitope sequence for raising conventional antibodies represents an alternative. Panning phage displayed libraries of recombinant antibodies such as scFvs and nanobodies or of other peptide collections is another option. Although recombinant antibodies can provide the same specificity as conventional antibodies, they offer at least two further advantages: i) the protocols for the selection of epitope-specific antibodies can be rationally designed, and ii) their expression as multivalent, bispecific and biparatopic molecules is feasible. This review will analyze the recent literature concerning technical aspects related to the isolation, the expression as multivalent molecules, and the therapeutic applications of binders able to interfere with antigen functional domains. The term binder will be preferred when possible to include those molecules, such as peptides or affibodies, with at least some proven practical uses.

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

“…The A10 construct has been reported as an example.a) Fc-tagged antibodies were first expressed and purified in small-scale to assess the protein accumulation in the lysates (L) and bound to the beads after washing (B). Numbering indicates either contemporary expression induction of Fc-VHH and sulfhydryl oxidase (1) or to anticipated sulfhydryl oxidase expression (2). For large-scale production, the constructs were affinity-purified using Protein A and their polydispersity was successively evaluated by analytical SEC.…”

“…Although IgG antibodies recovered from hybridoma cells still dominate the field, alternative formats know a constantly increasing success and recombinant Fab, single-chain (scFv) and single-domain (VHH) antibodies have demonstrated their reliability in basic research as well as in clinical applications [1,2]. In the case of VHHs, their reduced mass is a decisive advantage to obtain faster clearance for in vivo imaging, a better penetration in solid tumors and even the permeation across the blood brain barrier [3][4][5].…”

Isolation and Epitope Mapping of Staphylococcal Enterotoxin B Single-Domain Antibodies

“…Use of a sandwich-format panning protocol as described in this work was successful in identifying binders to a new epitope which would have otherwise been lost due to their relatively lower affinity. Binders to different epitopes are critical for many applications [23]. …”

Molecular imaging with endogenous and exogenous ligands: The instance of antibodies, peptides, iodide and cupric ions

“…A popular example is offered by radiolabeled antibodies and peptides [22][23][24][25][26][27][28][29][30], which are commonly considered as endogenous molecules. The receptor-like mechanism responsible for the accumulation of these molecules onto the target is particularly simple and, when there exists a sufficient number of cellular receptors, this would make these endogenous molecules a perfect choice as imaging probes.…”