Designing receptor agonists with enhanced pharmacokinetics by grafting macrocyclic peptides into fragment crystallizable regions

Abstract

Short half-lives in circulation and poor transport across the blood–brain barrier limit the utility of cytokines and growth factors acting as receptor agonists. Here we show that surrogate receptor agonists with longer half-lives in circulation and enhanced transport rates across the blood–brain barrier can be generated by genetically inserting macrocyclic peptide pharmacophores into the structural loops of the fragment crystallizable (Fc) region of a human immunoglobulin. We used such ‘lasso-grafting’ approach, which preserves the expression levels of the Fc region and its affinity for the neonatal Fc receptor, to generate Fc-based protein scaffolds with macrocyclic peptides binding to the receptor tyrosine protein kinase Met. The Met agonists dimerized Met, inducing biological responses that were similar to those induced by its natural ligand. Moreover, lasso-grafting of the Fc region of the mouse anti-transferrin-receptor antibody with Met-binding macrocyclic peptides enhanced the accumulation of the resulting Met agonists in brain parenchyma in mice. Lasso-grafting may allow for designer protein therapeutics with enhanced stability and pharmacokinetics.

MET‐Activating Ubiquitin Multimers

“…As an initial trial, we prepared a family of U-body in a straightforward design manner (Figure 1a). [19][20][21]23] The pharmacophore sequence of aMD4 or aMD5 was flanked by "G" or "GG" spacers and it replaced L8-T9 or T9-G10 within the β1-β2 loop (Ub-aMD4_n1-4 and Ub-aMD5_n1-4, referred as naïve U-body, Figure 2a-b). The binding affinity was measured by means of surface plasmon Figure 1.…”

“…The D Tyr located at the N-terminus of the pharmacophore sequence was replaced with L Tyr according to the previous studies. [19][20][21] (b) Construction of the Ub-aMD4 and Ub-aMD5 library. The pharmacophore sequences were flanked by 0 to 3 randomized grafting spacer residues (represented as "X'') and they replaced 0 to 4 consecutive residues within the β1-β2 loop (T7 to G10).…”

“…Recently, it has been shown that such RaPID-derived macrocyclic peptides were graftable to protein scaffolds. [19][20][21]23] The RaPID-derived pharmacophore sequences of the macrocyclic peptides can be genetically implanted into surface-exposed loops of proteins without losing both functionalities of the grafted peptide and the scaffold protein. By this macrocyclic peptide grafting, termed "lassografting (LG)", the RaPID-derived MET-binding peptides have been exploited to develop designer MET agonists using dimeric and self-assembling proteins as scaffolds.…”

“…By this macrocyclic peptide grafting, termed "lassografting (LG)", the RaPID-derived MET-binding peptides have been exploited to develop designer MET agonists using dimeric and self-assembling proteins as scaffolds. [20,21] A recent example showed that the fragment crystallizable (Fc) region of human immunoglobulin (Ig), which is an intrinsically dimeric protein, was turned into MET agonists without a loss of the binding ability to neonatal Fc receptor. [21] In this study, we lasso-grafted the pharmacophore sequence of two distinct MET-binding macrocyclic peptides aMD4 and aMD5 (Figure S1) to ubiquitin (Ub), a robust eukaryotic small protein that is expressed as tandemly conjugated forms, [24,25] to generate Ub-based ligands, referred to as U-body.…”

“…[20,21] A recent example showed that the fragment crystallizable (Fc) region of human immunoglobulin (Ig), which is an intrinsically dimeric protein, was turned into MET agonists without a loss of the binding ability to neonatal Fc receptor. [21] In this study, we lasso-grafted the pharmacophore sequence of two distinct MET-binding macrocyclic peptides aMD4 and aMD5 (Figure S1) to ubiquitin (Ub), a robust eukaryotic small protein that is expressed as tandemly conjugated forms, [24,25] to generate Ub-based ligands, referred to as U-body. Unlike the LG to Fc, the initially designed U-body constructs showed substantially weaker affinity to MET compared with the parental peptides.…”