Repeat proteins have recently been of great interest as potential alternatives to immunoglobulin antibodies due to their unique structural and biophysical features. We here present the development of a binding scaffold based on variable lymphocyte receptors, which are nonimmunoglobulin antibodies composed of Leucine-rich repeat modules in jawless vertebrates, by module engineering. A template scaffold was first constructed by joining consensus repeat modules between the N- and C-capping motifs of variable lymphocyte receptors. The N-terminal domain of the template scaffold was redesigned based on the internalin-B cap by analyzing the modular similarity between the respective repeat units using a computational approach. The newly designed scaffold, termed “Repebody,” showed a high level of soluble expression in bacteria, displaying high thermodynamic and pH stabilities. Ease of molecular engineering was shown by designing repebodies specific for myeloid differentiation protein-2 and hen egg lysozyme, respectively, by a rational approach. The crystal structures of designed repebodies were determined to elucidate the structural features and interaction interfaces. We demonstrate general applicability of the scaffold by selecting repebodies with different binding affinities for interleukin-6 using phage display.

Design of a binding scaffold based on variable lymphocyte receptors of jawless vertibrates by module engineering

Antibodies for therapeutic use are being continuously approved and their demand has been steadily growing. As known, the golden standard for monoclonal antibody (mAb) purification is Protein A affinity chromatography, a technology that has gained high interest because of its great performance and capabilities. The main concerns are the elevated resins costs and their limited lifetime compared to other resins (e.g. ion exchange chromatography). Great efforts have been carried out to improve purification conditions, such as resin characterization and designing alkali/acid stable resins with a longer lifetime. Modification of Protein A ligands and alternative formats such as monoliths membranes and microshperes have been tested to increase the purification performance. New technology has been proposed to improve the large-scale separation; in addition, alternative ligands have been suggested to capture mAbs instead of Protein A ligand; however, most of the information is locked by pharmaceutical companies. This mini review summarizes and describes the advances, results, and impact on the Protein A chromatography purification processing.

Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies

The need for atom-precise biomolecule modification, and particularly the irreversible formation of covalent bonds to specific amino acids in proteins, has become an essential issue in the fields of pharmaceuticals and chemical biology. For example, antibody-drug conjugates (ADCs) are increasingly common entries into the clinical oncology pipeline. Herein, we report a new method of affinity peptide mediated regiodivergent functionalization (AJICAP™) that enables the synthesis of ADCs from native IgG antibodies. We succeeded in introducing thiol functional groups onto three lysine residues in IgGs using Fc affinity peptide reagents without antibody engineering. A cytotoxic molecule was then connected to the newly introduced thiol group, and both a surface plasmon resonance binding assay and in vivo xenograft mouse model results showed that the resulting ADC could selectively target and kill HER2-positive cells. Our strategy provides a new approach for constructing complex antibody-derived biomolecules.

AJICAP: Affinity Peptide Mediated Regiodivergent Functionalization of Native Antibodies.

Peptides are widely used in pharmaceutical industry as active pharmaceutical ingredients, versatile tools in drug discovery, and for drug delivery. They find themselves at the crossroads of small molecules and proteins, possessing favorable tissue penetration and the capability to engage into specific and high-affinity interactions with endogenous receptors. One of the commonly employed approaches in peptide discovery and design is to screen combinatorial libraries, comprising a myriad of peptide variants of either chemical or biological origin. In this review, we focus mainly on recombinant peptide libraries, discussing different platforms for their display or expression, and various diversification strategies for library design. We take a look at well-established technologies as well as new developments and future directions.

https://www.mdpi.com/1422-0067/21/1/215/pdf

Evolving a Peptide: Library Platforms and Diversification Strategies

Review on biomimetic affinity chromatography with short peptide ligands and its application to protein purification.

Alternative Affinity Ligands for Immunoglobulins

Development and Characterization of Peptide Ligands of Immunoglobulin G Fc Region

Bee venom immunotherapy is the main treatment option for bee sting allergy. Its major limitations are the high percentage of allergic side effects and long duration, which are driving the development of novel therapeutic modalities. Three general approaches have been evaluated including the use of hypoallergenic allergen derivatives, adjunctive therapy, and alternative delivery routes. This article reviews preclinical and clinical evidence on the therapeutic potential of these new therapies. Among hypoallergenic derivatives, hybrid allergens showed a markedly reduced IgE reactivity in mouse models. Whether they will offer therapeutic benefit over extract, it is still not known since clinical trials have not been carried out yet. T cell epitope peptides have proven effective in small clinical trials. Major histocompatibility complex class II restriction was circumvented by using long overlapping or promiscuous T cell epitope peptides. However, the T cell–mediated late-phase adverse events have been reported with both short and longer peptides. Application of mimotopes could potentially overcome both T cell– and IgE-mediated adverse events. During this evolution of vaccine, there has been a gain in safety. The efficacy was further improved with the use of Toll-like receptor-activating adjuvants and delivery systems. In murine models, the association of allergen Api m 1 with cytosine-guanosine rich oligonucleotides stimulated strong T-helper type-1 response, whereas its encapsulation into microbubbles protected mice against allergen challenge. An intralymphatic administration of low-dose vaccine has shown the potential to decrease treatment from 5 years to only 12 weeks. Bigger clinical trials are needed to follow up on these results.

Bee Venom Immunotherapy: Current Status and Future Directions

In general, relatively few panning attempts using combinatorial peptide phage display libraries yield specific binders. Major factors affecting the outcome of screening are diversity (complexity) of the library, type of randomization and conformation of peptides, quality and purity/homogeneity of the target material, as well as affinity selection strategy and conditions enforced during individual panning steps. In our experience, certain groups of structurally and/or functionally related target molecules have worked out better for the selection of peptide ligands than others. Therefore, we suggest that the type of target molecule itself may also be an important factor limiting isolation of peptide ligands and can even assist in predicting the selection outcome. Here, we summarize data from 87 selections on diverse set of 23 protein targets performed in our laboratory. It appears that especially the antibodies and the enzymes whose natural substrates are peptides or proteins tend to give better yields in affinity selections in contrast to proteins involved in protein-protein interactions or lipid metabolizing enzymes.

/pdf/type-of-target-molecule-influences-yield-in-peptide-phage-4cclhman1h.pdf

Type of target molecule influences yield in peptide phage display affinity selections

The present invention relates to ligand that may be a peptide compound as well as peptoid or retro-inverso analogues thereof with binding affinity for the Fc region of immunoglobulins. The invention further relates to the application of such peptides and variants thereof for purification of immunoglobulins on the basis of affinity chromatography, non-covalent antibody labelling, antibody detection or immobilization of antibodies to solid support.

Nika Kruljec

Papers

Alternative Affinity Ligands for Immunoglobulins

Development and Characterization of Peptide Ligands of Immunoglobulin G Fc Region

Bee Venom Immunotherapy: Current Status and Future Directions

Type of target molecule influences yield in peptide phage display affinity selections

Affinity ligands for antibody fc region