A review of lipidation in the development of advanced protein and peptide therapeutics

Abstract

The use of biologics (peptide and protein based drugs) has increased significantly over the past few decades. However, their development has been limited by their short half-life, immunogenicity and low membrane permeability, restricting most therapies to extracellular targets and administration by injection. Lipidation is a clinically-proven post-translational modification that has shown great promise to address these issues: improving half-life, reducing immunogenicity and enabling intracellular uptake and delivery across epithelia. Despite its great potential, lipidation remains an underutilized strategy in the clinical translation of lead biologics. We review how lipidation can overcome common challenges in biologics development as well as highlight gaps in our understanding of the effect of lipidation on therapeutic efficacy, where increased research and development efforts may lead to next-generation drugs.

Fig. 1.

Current challenges and solutions facing biologic therapeutics (a) (i) The majority of biologics are administered through injections while alternative routes including pulmonary, oral, and topical administration remain underused. (ii) Injections can lower therapy effectiveness through increased patient non-compliance (Data from [7]). (b) Half-life of biologic therapeutics are shortened by (i) by proteolysis and (ii & iii) renal clearance from the bloodstream. (c) Biologics can also induce immune reactions. (i) Antigen presenting cells (APC) can present peptide fragments to T-cells inducing an immune response through cytokine release that leads to rapid clearance. (ii) B-cell receptors recognize protein antigens and secrete antigen specific immunoglobulin into the serum which can neutralize the therapeutic activity and mark therapeutics for clearance. (d) Biologics mainly enter cells through endocytosis. Endocytosis requires endosomal escape for the therapeutic to gain access to the cytosol without which the biologic is degraded in the lysosome. (e) Challenges and current solutions for biologic therapeutics. Approaches listed in overlapping regions address multiple challenges. *Denotes controversial data on PEGylation that supports both reductions and increases in immunogenicity of biologic therapeutics.

Fig. 2.

Half-life extension of lipidated therapeutics through albumin binding. (a) non-lipidated molecules have increased rates of (i) proteolysis and (ii) renal clearance. (iii) Lipidation enables reversible albumin binding (iv) reducing renal clearance and degradation by proteases, such as dipeptidyl peptidase 4 (relevant for GLP-1 analogues; PDB ID: 4APD). (b) Seven sites are available on albumin for medium and long-chain fatty acids. Interactions between the lipid carboxylate and polar amino acids at some of these sites (top close-up, dark blue) along with increased nonpolar interactions resulting from longer lipids (bottom close-up) can be leveraged for tighter albumin binding (PDB ID: 1E7H).

Fig. 3.

Enhanced drug delivery due to lipidation. (a) Lipidated therapeutics are routinely injected subcutaneously where they form a drug depot through reversible self-association causing (b) a delay of drug absorption into the body, which accounts, for example, for almost doubling the half-life of liraglutide, a GLP-1 analogue used to treat diabetes and obesity (data taken adapted from [69]). (c) Lipidation also increases cell uptake through (i) a flip-flop mechanism or (ii) endocytosis. (d) Increases in lipid length results in increased cell uptake (data taken adapted from [ to 74]).

Fig. 4.

Oral delivery of peptides enabled by lipidation. (a) Tablet formulations are now possible for lipidated therapeutics, such as semaglutide, a GLP-1 analogue used to treat diabetes. (b) Besides the active ingredient (eg semaglutide), permeation enhancers are needed to increase therapeutic bioavailability such as (c) N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) for semaglutide’s oral formulation. (d) Self-association of lipidated therapeutics may negatively affect permeation of lipidated therapeutics through the gastric endothelium as (e) monomers may be able pass, (f) but not multimers.

Fig. 5.

Changes in drug potency due to lipidation. (a) (i) Variations in lipid length, (ii) site of lipid attachment and (iii) type of linker used to attach the lipid influence resulting drug potency. (b) In some cases, increasing lipid length leads to decreased potency where lipidation at a particular site may result in a series of lipidated analogues with higher potency than at a different site (eg for GLP-1 analogues, lipidation at K36 results in analogues with higher potency than lipidation at K26) (Data adapted from [41]). (c) Longer, hydrophilic linkers allow the attachment of longer lipids at lower potency loss such as the use of a γ-glutamyl linker elongated with amino-3,6-dioxaoctanoic acid (OEG) which results in more potent GLP-1 analogues when compared to the use of only the γ-glutamyl linker or no linker (Data adapted from [33]). (d) In some other cases, because lipidation increases affinity towards the cell membrane, it can lower the energy barrier for the formation of therapeutic-target complex (e) which results in increased drug potency with increasing lipid length (Data adapted from [70,104,105]). (f) In some other cases, increasing the lipid length results in oscillating potencies with maxima and minima (Data adapted from [103]).

Fig. 6.

Reduced immunogenicity due to lipidation. (a) Peptide and protein-based drugs can become immunogenic through the MHC II antigen presentation pathway. (b) Lipidated therapeutics can potentially be presented through both MHC I and MHC II antigen presentation pathways but have been shown to have lower immunogenic potential than non-lipidated counterparts. (c) Comparison of non-lipidated with lipidated therapeutics at different steps in the antigen presentation pathway. (i) Lipidation causes increased antigen uptake by antigen presenting cells (APC) (Data adapted from [124]). (ii) Lipidation have been shown to be resistant to proteolytic degradation, though this has not been shown in APCs specifically (Data adapted from [90,126,127]). Lipidation causes an overall decrease in binding to MHC II molecules (Data adapted from [118]). (iv) Lipidation can greatly decrease the magnitude of T-cell responses (Data adapted from [118]).