In vitro-engineered non-antibody protein therapeutics

Abstract

Antibodies have proved to be a valuable mode of therapy for numerous diseases, mainly owing to their high target binding affinity and specificity. Unfortunately, antibodies are also limited in several respects, chief amongst those being the extremely high cost of manufacture. Therefore, non-antibody binding proteins have long been sought after as alternative therapies. New binding protein scaffolds are constantly being designed or discovered with some already approved for human use by the FDA. This review focuses on protein scaffolds that are either already being used in humans or are currently being evaluated in clinical trials. Although not all are expected to be approved, the significant benefits ensure that these molecules will continue to be investigated and developed as therapeutic alternatives to antibodies. Based on the location of the amino acids that mediate ligand binding, we place all the protein scaffolds under clinical development into two general categories: scaffolds with ligand-binding residues located in exposed flexible loops, and those with the binding residues located in protein secondary structures, such as α-helices. Scaffolds that fall under the first category include adnectins, anticalins, avimers, Fynomers, Kunitz domains, and knottins, while those belonging to the second category include affibodies, β-hairpin mimetics, and designed ankyrin repeat proteins (DARPins). Most of these scaffolds are thermostable and can be easily produced in microorganisms or completely synthesized chemically. In addition, many of these scaffolds derive from human proteins and thus possess very low immunogenic potential. Additional advantages and limitations of these protein scaffolds as therapeutics compared to antibodies will be discussed.

Same-Day Imaging Using Small Proteins: Clinical Experience and Translational Prospects in Oncology

“…From this, targetspecific binders are selected via phage display, yeast surface display, or ribosome/messenger RNA display. Most sdAbs and engineered scaffold proteins are easily produced in microbial systems, are stable and soluble, and show good binding affinity and specificity (3). So far, several sdAbs and scaffold proteins, selected against different targets, have been used as probes for SPECT and PET imaging (4,5).…”

“…To mimic the clinical situation, binders with approximately equal affinity to human and murine receptors were generated. An anti-IGF-1R Affibody molecule, ZIGF1R:4551, was labeled using 99m Tc(CO) 3 and demonstrated receptor-specific uptake in both IGF-1R-expressing tumors and IGF-1R-expressing tissues (Fig. 4A) (28).…”

“…Attractive alternative targeting proteins, with a further reduction in size, are Camelid single-domain antibody fragments (sdAbs) and several nonimmunoglobulin protein scaffolds such as Affibody molecules (Affibody AB), anticalins, designed ankyrin repeat proteins (DARPins; Molecular Partners AG), and fibronectin type III (FN3, Adnectin; Adnexus) ( Fig. 2; Table 1) (3). Their low molecular weight enables fast penetration in tumor tissues, and their lack of a fragment crystallizable region further improves clearance.…”

Engineering repeat proteins of the immune system

“…In the search for better recognition agents, many different scaffolds have emerged; some notable examples are peptide and DNA aptamers, affimers, and anticalins, among others. The motivation and results of this research have been extensively reviewed in recent years . among these scaffolds, repeat proteins such as designed ankyrin repeat proteins (DARPins) tetratricopeptide repeat proteins (TPRs), and armadillo repeats have emerged as a promising class of scaffolds.…”

Beyond function: Engineering improved peptides for therapeutic applications

“…Expressing the variants in a library on the surface of individual cells allows the library to be easily interrogated for desired characteristics, and the DNA encoding for a desired variant can be recovered after growing the cell or performing PCR. Several different cell‐surface display systems have been used for directed evolution of peptides, including systems involving display on phage, yeast, and bacteria . One of the main limitations of cell‐surface display systems is that the library size is limited by the transformation efficiency of the cells involved in the display.…”