Development of an albumin-binding ligand for prolonging the plasma half-life of peptide therapeutics

Alessandro Zorzi
EPFL, 2017
Thèse EPFL

Résumé

Peptides represent a promising format for the development of therapeutics since they combine the advantages of proteins and small molecules. Several techniques based on rational design or in vitro evolution can be used to develop peptides as therapeutics. A limitation of peptides is a rapid clearance from the blood circulation when applied intravenously, preventing their application in therapies that require prolonged drug exposure. Several methods have been tested to extend the in vivo half-life of peptides, including fusion to long-lived serum proteins, PEGylation and conjugation to non-covalent albumin-binding ligands. A particularly attractive approach is a âpiggy backâ strategy in which peptides bind via a ligand to albumin without increasing significantly their small size. Acylation with fatty acids is the most successful strategy for delaying peptide clearance. The attachment of either myristic or palmitic acid to insulin and GLP-1 led to the approval of three acylated peptide drugs as daily injectable. Nevertheless, acylated conjugates generally present low solubility and weak affinity for albumin. Peptides have been explored as an alternative, as their format offers multiple approaches for affinity maturation as well as an easier synthesis of the conjugates. However, the albumin-binding peptides developed so far do not overcome the disadvantages faced with fatty acids. The first aim of my project was the development of an albumin-binding tag that combines the favorable properties of both fatty acids and peptides, and overcomes at the same time their limitations. This tag was engineered in order to have i) a high affinity for human albumin, ii) a high solubility in physiological solutions, and iii) an efficient synthesis in conjunction with therapeutic peptides. Towards this goal, I created a chimeric format consisting of a fatty acid combined with a short linear peptide. After iterative rounds of affinity screening, an evolved peptide-fatty acid tag able to bind human, rat and rabbit albumin with Kd values of 39, 220 and 320 nM, respectively, was obtained. This makes it the best albumin-binding tag among all of the peptide- or fatty acid-based ligands that have been developed. This tag can be easily synthesized in conjugation with peptides on standard automated synthesizers. Finally, it exhibits a high solubility due to four negatively charged amino acids in the peptide sequence, despite the presence of a 16-carbon palmitic hydrophobic tail. The second aim of my project was the application of this tag to three bioactive bicyclic peptides, previously developed in the laboratory, in order to improve their in vivo half-lives. I demonstrated that the conjugation has, overall, a mild impact on peptidesâ binding affinity towards their targets and human albumin, and increases their proteolytic stability. Pharmacokinetic experiments showed that the tag extends the elimination half-life of bicyclic peptides in rats and rabbits approximately 25-fold to over seven and five hours, respectively. Tag conjugation to a bicyclic peptide targeting coagulation factor XII led to an efficient inhibition of coagulation in rabbits up to eight hours, suggesting a potential application in humans as anti-thrombotic drug. These results indicated that the tag will efficiently prolong the exposure of target proteins to bioactive peptides in humans due to its higher affinity for human albumin than that of rat and rabbit.

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https://infoscience.epfl.ch/record/228335?ln=fr

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Alessandro Zorzi
EPFL, 2017
Thèse EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/228335?ln=fr

À propos de ce résultat

Concepts associés (21)

Concepts associés

Chargement

Publications associées (65)

Publications associées

Chargement

Development of cyclic peptide inhibitors of coagulation factor XII and matrix metalloproteinase 2

Jonas Alfred Karl Wilbs

Peptides represent a promising molecular class for drug development. They combine several strengths of small molecules (e.g. efficient tissue diffusion, low immunogenicity and access to chemical synthesis) and key properties of biologics such as monoclonal antibodies (e.g. high affinity and specificity). Most of the peptide drugs are natural products or derivatives thereof, as for example human peptide hormones or antibacterial peptides. In recent years, a range of methodologies were established to develop peptides binding to therapeutic targets de novo. Our laboratory is specialized on the in vitro evolution of bicyclic peptides by phage display. Bicyclic peptides have two macrocyclic rings that allow for binding protein targets with high affinity and selectivity. The aim of my thesis was to improve the potency, selectivity and stability of phage-selected bicyclic peptide inhibitors of coagulation factor XII (FXII) and matrix metalloproteinase 2 (MMP-2), and to test their therapeutic potential in vivo. Specifically, I planned at improving the bicyclic peptides by substituting natural amino acids with unnatural ones. As described in the following, the substitutions to unnatural amino acids had led to substantial improvements in the bicyclic peptides, and this had enabled me the evaluation of the inhibitors in disease models. In my first and second project, I aimed at improving the potency and stability of a bicyclic peptide FXII inhibitor that was previously developed in our lab by phage display, and to test the therapeutic potential of the resulting peptide in vivo. FXII has recently been identified as a promising target for safe anticoagulation therapy. In the first project, I investigated if the insertion of a single carbon atom into the macrocyclic backbone of a bicyclic peptide FXII inhibitor can improve its binding affinity. Positions within the macrocycle susceptible to atom insertion were first identified using a scanning methodology where glycine mutants were compared to ¿-alanine mutants. Upon insertion of atoms in the backbone using ¿-amino acids or homologated cysteine analogues, two peptides showed 4.7- and 2.5-fold improved Ki values. The better one blocked FXII with a Ki of 1.5 ± 0.1 nM and was more potent than the lead peptide in inhibiting the activation of the intrinsic coagulation pathway. The strategy of ring size variation by one or several atoms should be generally applicable for the affinity maturation of in-vitro-evolved cyclic peptides. In my second project, I aimed at improving further the potency of the bicyclic peptide FXII inhibitor described before, and to additionally improve its proteolytic stability. I achieved both these goals by replacing further natural amino acids to unnatural ones. Sub-nanomolar activity for human and mouse FXII (370 and 450 pM respectively) as well as a high stability (t1/2 > 128 ± 8 h in plasma) permitted preclinical evaluation of the peptide. The inhibitor efficiently blocked intrinsic coagulation in blood plasma from human, mouse and rabbit. I further demonstrated that the peptide reduced experimental thrombosis induced by ferric chloride in mice and suppressed blood coagulation in artificial lungs in rabbits, all without increasing the risk of bleeding. This shows that the optimized bicyclic peptide is a promising candidate for thromboprotection in various medical conditions. In a third project, I aimed at improving the potency and stability of a bicyclic peptide MMP-2 inhibi

EPFL2018

Chargement

New methods for developing (bi)cyclic peptides by phage display

Camille Villequey

Cyclic peptide ligands are a promising molecular format for the development of therapeutics. They combine some of the advantages of large protein therapeutics (high affinity and specificity) and of small molecule drugs (accessibility to chemical synthesis and low immunogenicity). With phage display, large combinatorial libraries of more than one billion mono- and polycyclic peptides can be developed and screened against virtually any target. During phage affinity selections, the physical link between phenotype (encoded peptide) and genotype (phage DNA) allows the identification of enriched peptide binders by sequencing of the phage vector DNA. Based on these concepts, our laboratory has established a robust chemistry in which phage displayed peptides containing two or three cysteines are cyclized or bi-cyclized with thiol-reactive linkers. This procedure has been generally applied for the isolation of potent and selective (bi)cyclic peptide ligands with therapeutic potential. The isolation of binders with optimal properties is highly dependent on the possibility to generate (bi)cyclic peptide libraries with high structural diversity and on the ability to thoroughly decode the phage selection output. In my PhD work, I developed new methods to generate large and structurally diverse libraries and to decode more efficiently phage selected peptides. In my first project, I explore the cyclization of peptides with two chemical bridges as a method to provide rapid access to phage-encoded libraries with high scaffold diversity. To this end, a range of twelve structurally diverse chemicals were tested for thiol-reactivity and phage compatibility. Subsequently, peptide libraries of the format XCXnCXnCX (X = random amino acid, C = cysteine, n = 3 or 4) were cyclized with six of these chemical linkers and panned against the protease plasma kallikrein. The selection yielded inhibitors with remarkable affinity (sub-nM KI), target selectivity (>1000-fold) and proteolytic stability (t1/2 in plasma > 3 days) despite a relatively small molecular mass (~ 1200 Da). In the second project, I developed phage-encoded libraries of small cyclic peptides in order to identify ligands that have the potential to be orally absorbed. A major challenge for the generation of such library is the usually low diversity of compounds due to the limited number of amino acids. In this work, we addressed this limitation by taking advantage of a recently discovered peptide cyclization reaction in which the thiol of a cysteine is ligated with the N-terminal amino group using a chemical reagent. We applied this strategies to two phage-encoded peptide libraries of four and five amino acids (XXXC and XXXXC) that were cyclized with eight chemical linkers yielding a collection of more than 1.3 million compounds. Panning the libraries against the two disease targets plasma kallikrein and coagulation factor XIa yielded ligands with single-digit micromolar affinity. In a third project, I developed a strategy to bypass the bacterial infection step in phage display. Bacterial infection is usually crucial for the propagation and identification of enriched clones by DNA sequencing. However, mutations or chemical modifications of the phage coat proteins, sometimes lead to decreased infection rates and therefore compromise the entire procedure. Consequently, there is an interest for developing methods in which this step could be completely omitted.

EPFL2017

Chargement

Conformationally constrained peptide ligands offer an attractive format for the development of therapeutics. They can bind with high affinity and selectivity to protein targets, they are small enough to diffuse into tissues, they can be synthesized chemically, and their degradation products are not toxic. In recent years, a number of novel techniques were innovated to develop peptide-based ligands. One such technique is the generation of bicyclic peptides by phage display. This technique is well established in our laboratory and has led to the isolation of high-affinity antagonist for a range of important therapeutic targets. In a first project I aimed at developing bicyclic peptide ligands of the epidermal growth factor receptor Her2. Aberrant expression of Her2 has been implicated in various malignancies including breast cancer. In the treatment of this cancer type several Her2 specific antibodies were proved to be efficient. Due to their small size, bicyclic peptides could potentially offer advantages over antibodies such as ease of synthesis and conjugation, higher molecule-permass ratios, and better tumor penetration. I panned a large bicyclic peptide phage library against the extra-cellular domain of Her2 and obtained a range of peptide sequences binding to Her2 in a specific manner. After affinity maturation, a bicyclic peptide that bound Her2 with a Kd of 304 nM could be obtained. This peptide ligand offers a valuable starting point for further improvement and the development of high-affinity Her2 binders with potential application for tumor imaging and therapy. A second project was triggered by my observation that it is relatively difficult to generate high-affinity bicyclic peptide ligands to proteins with flat and featureless surfaces such as Her2. The major reason for the limited binding affinity of bicyclic peptides was supposed to lie in the lack of a defined secondary structure in solution and the resulting entropic penalty upon binding. To overcome this problem, I proposed to evolve ligands based on a-helices. Chemically stabilized a-helices, also named stapled peptides, were previously developed by rational design. I proposed to evolve a-helical peptides by phage display wherein the helix, is stabilized in a chemical reaction prior to phage panning. I tested this strategy by affinity maturing an alpha-helical peptide binding to beta-catenin. The project resulted with a 250-fold improved ligand of beta-catenin. In a third project, I developed a novel constrained peptide format that I dubbed âhelix-loopâ motif. A stabilized alpha-helix was expanded with a peptide loop in order to increase the number of amino acids that can formcontacts with a target. The loop was linked to either the N- or C-terminal end of the helix and via a cysteine residue to the chemical linker stabilizing the alpha-helix. Libraries were created adding randomized loops with different length to either side of the peptide. Ligands with improved affinity were isolated against the cancer target beta-catenin. In summary, I have exploited the existing bicyclic peptide format to evolve bicyclic peptide ligands of Her2. In addition, I have developed a new approach to affinity mature stabilized helical peptides by phage display as well as a new constrained peptide format. This new approach should be applicable for affinity maturation of any stabilized alpha-helix. The new peptide format promises the generation of high affinity ligands to any protein target, including Her2.

EPFL2014

Chargement

Development of cyclic peptide inhibitors of coagulation factor XII and matrix metalloproteinase 2

Jonas Alfred Karl Wilbs

EPFL2018

New methods for developing (bi)cyclic peptides by phage display

Camille Villequey

EPFL2017

EPFL2014