Recent advances in proteolytic stability for peptide, protein, and antibody drug discovery

ABSTRACT

Introduction: To discover and develop a peptide, protein, or antibody into a drug requires overcoming multiple challenges to obtain desired properties. Proteolytic stability is one of the challenges and deserves a focused investigation.

Areas covered: This review concentrates on improving proteolytic stability by engineering the amino acids around the cleavage sites of a liable peptide, protein, or antibody. Peptidases are discussed on three levels including all peptidases in databases, mixtures based on organ and tissue types, and individual peptidases. The technique to identify cleavage sites is spotlighted on mass spectrometry-based approaches such as MALDI-TOF and LC-MS. For sequence engineering, the replacements that have been commonly applied with a higher chance of success are highlighted at the beginning, while the rarely used and more complicated replacements are discussed later. Although a one-size-fits-all approach does not exist to apply to different projects, this review provides a 3-step strategy for effectively and efficiently conducting the proteolytic stability experiments to achieve the eventual goal of improving the stability by engineering the molecule itself.

Expert opinion: Improving the proteolytic stability is a spiraling up process sequenced by testing and engineering. There are many ways to engineer amino acids, but the choice must consider the cost and properties affected by the changes of the amino acids.

KEYWORDS:

• Proteolytic stability
• peptide
• protein
• antibody
• peptidase
• cleavage site
• amino acid replacement

Article highlights

• Despite the significant achievements of peptides, proteins, and antibody drugs in clinical use and marketing, one of the shared challenges among peptides, proteins, and antibody drugs is proteolytic stability that could compromise desired pharmacokinetics, pharmacodynamics, and safety.

• In the human body, cells produce numerous endogenous peptidases with various functions. Peptidases are necessary for living creatures, but peptides, proteins, and antibody drugs with natural amino acids are intrinsically liable to peptidases.

• While peptidase databases provide a full list of peptidases from different species and detailed expression levels at specific intracellular and intercellular locations for in silico prediction, peptidase mixtures, and individual peptidases are the ones used in vitro for experimental analysis.

• Although many sources and software provide in silico cleavage prediction, in vitro experiments are more meaningful than in silico prediction to obtain cleavage sites of peptide, protein, and antibody drugs under biophysical conditions.

• After the identification of cleavage sites, engineering the amino acids around the sites are primarily from P1 and P1ʹ to P2 and P2ʹ, and possibly from P3 and P3ʹ to P4 and P4ʹ. The replacement might change the efficacy, toxicity, and many other properties, while the proteolytic stability is increased. Scientists must balance the proteolytic stability and other desired properties.

• After revealing the key components of proteolytic stability, a 3-step strategy was proposed to conduct the proteolytical stability experiment without overwhelming resources and with balancing under- or over-engineering the sequences.

This box summarizes key points contained in the article.

Acknowledgments

The authors would like to thank Christopher Reutter for editing and proofreading the manuscript.

Declaration of interest

X Lai, J Tang, and M ElSayed are all employees of Eli Lilly and Company. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Reviewer disclosures

Peer reviewers in this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

This manuscript was not funded.