Enhancing the half-life of Interleukin 2 by conjugation to the Transthyretin Ligand, TLHE And Enhancing the efficacy of peptides that inhibit COVID 19 viral entry

Author

Arjun D. Patel, University of the PacificFollow

Date of Award

2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

Mamoun M. Alhamadsheh

First Committee Member

William K. Chan

Second Committee Member

Miki S. Park

Third Committee Member

David Thomas

Fourth Committee Member

Qinliang Zhao

Abstract

The central dogma of biology states that genetic information describes the flow ofinformation from DNA to RNA and then finally resting in proteins. The fundamental aspect that underlies all aspects of life is the expression and modification of proteins. One can even argue that there is no life without proteins. As such, many human diseases are either directly or indirectly related to dysfunction of proteins and can potentially be solved through protein therapeutics. Consequently, scientists have begun to harness the diversity of proteins to treat the diseases which plague man in the form of protein therapeutics such as clotting factors, cytokines, and growth factors. Unfortunately, the short circulation half-life of proteins is a major limiting factor which must be overcome before their widespread adoption as a platform for therapeutic development. Contributing factors to this short circulation half-life include renal elimination, proteasomal degradation, and metabolism in the liver. Namely, renal elimination is the main challenge for protein therapeutics and warrants clinicians to resort to higher doses and more frequent administrations to maintain necessary concentrations in the body. Unfortunately, side effects of this approach are dose limiting toxicities and reduced therapeutic outcomes as drug concentrations fluctuate drastically. As a result, addressing the challenge of renal elimination for protein therapeutics would allow for the development of novel treatments which were previously not viable.

The human glomerulus readily filters out any particle smaller than approximately 30 kDa in weight. As a result, strategies adopted all share a common theme of endowing the protein with a greater effective size without compromising their natural activity against the intended target.

The current approaches include conjugation to a polymer (i.e., PEGylation), covalent or non-covalent binding to a larger protein, or conjugating to the neonatal Fc receptor. Major limitations of these approaches include compromised activity caused by steric hinderance rooted inconjugation to moieties of larger size. This issue applies to all of the aforementioned reported approaches wherein activity becomes reduced, thus necessitating higher dosages. Furthermore, other limitations also exist such as humoral immune responses against polymers through anti-PEG antibodies, occurrence of organ damage, and solubility issues.

A novel approach was recently developed by the Alhamadsheh lab which demonstratedthe ability of a small molecule linker termed “Transthyretin Ligand for Half-life Extension” (TLHE) to extend the circulation half-life of Gonadotropin releasing hormone. Most essentially, this was accomplished without compromising the potency or introducing a major sterically bulky group to the original peptide. Furthermore, additional concerns such as solubility issues was demonstrated to not be an issue either. In this work, human Interleukin-2 (IL-2) was chosen as a proof of concept to demonstrate application of the TLHE technology in a protein to address the aforementioned half-life challenge. Previously, a mixture of IL-2 and TLHE-IL-2 was demonstrated to maintain comparable activity to control IL-2 in both in vitro and ex vivo efficacy assays. Furthermore, a pharmacokinetic evaluation in rodents demonstrated significant half-life extension of TLHE-IL-2. The objective of this work was to shift the ratio of the IL-2/TLHE-IL-2 mixture to majority TLHE-IL-2 and or enhance the yield of the mixture for further in vivo efficacy evaluation.

What would happen to a viral infection if suddenly there were a billion fake receptors forevery real target receptor. A version of this question is what led to the development of a novel HIV therapy at Duke University around 1996. Fast forward more than 30 years, man still lacks the proper tools to combat viral infections. One can argue that the Achilles’ heel of viral infections is their need to bind a specific receptor. This protein-protein interaction between viral proteins and human receptors is arguably the fundamental point behind all viral infections. Recently, the COVID-19 pandemic again challenged man to develop new weapons at a revolutionary pace in order to save lives. During this time, the Pentelute lab at Massachusetts Institute of Technology reported a humanized version of the peptide sequence thought to represent the binding face of the human ACE2 receptor41. The 23 amino acid sequence was derived from the α1 helix of ACE2 peptidase domain and referred to as, spike-binding peptide 1 (SBP1). It was this sequence which was postulated to be responsible for binding the receptor binding domain of the notorious COVID-19 spike protein. However, a major limitation of peptide is their short in vivo half-life (through serum proteases and renal filtration). Therefore, the main aim of our proposed research was to employ the TLHE approach to extend the in vivo half-life of the SBP1 peptide. This would allow the creation a COVID-19 entry inhibitor that could help combat the COVID-19 pandemic.

Pages

92

Recommended Citation

Patel, Arjun D.. (2023). Enhancing the half-life of Interleukin 2 by conjugation to the Transthyretin Ligand, TLHE And Enhancing the efficacy of peptides that inhibit COVID 19 viral entry. University of the Pacific, Dissertation. https://scholarlycommons.pacific.edu/uop_etds/4257