Drug development of peptides started from the discovery of hormones and neurotransmitters and treatment with peptide-based drugs, such as for hormonal replacement, from the 1950s. Peptide-based drug design has been on the rise as the 3D structure of proteins and their functions on the cell surface as well as in cells were described. In particular, for immune response, several proteins are involved on the cell surface, which interact with each other, leading to the formation of the immunological synapse. Peptide-based drugs have the benefits of selectivity and in general, these are nonimmunogenic and can be produced in large quantities. Since most of the peptides lack the tertiary and quaternary structures, they are more stable than antibodies. Peptides have the beneficial attributes of small-molecule drugs and protein therapeutics. However, the disadvantages of peptides are a lack of in vivo enzymatic stability, short half-life, fast renal clearance and formulation issues. A number of strategies have been studied that can be used in the design of peptide-based drugs to overcome the short half-life and low bioavailability. Peptide in vivo stability can be improved by peptide backbone modification. This can be achieved by the introduction of unnatural amino acids or D-amino acids, peptide-bond modification, N- and C-termini modifications and by constraining the peptide backbone by introduction of cyclization, resulting in enzyme stable molecules.
Fig. 1 Mechanism of immunoregulatory peptides.1,2
Linear peptides typically contain between 8 and 20 residues which makes them too small to act as effective B-cell receptor cross-linkers and to bind APCs multivalently. As a result, linear peptides function as haptens rather than true antigens and can only elicit low or short-lived antibody titers without very strong adjuvants. A restricted number of MHC anchor residues exhibited by these peptides means they bind to only a limited range of HLA alleles which results in insufficient population coverage and elevated immune escape risks. Amino acid insertions and deletions (including truncations or shortened linear peptides) have the potential to affect T-cell response, as these changes in amino acid sequence can create new T-cell epitopes, destroy existing epitopes or change HLA binding profiles. Amino acid insertions, deletions and modifications can alter the T-cell-receptor-facing contours of a peptide and thus create a new T-cell epitope. Linear peptides also lack the conformational rigidity necessary to recapitulate native viral or tumor epitopes, resulting in antibodies that recognize the synthetic peptide but do not neutralize the intact pathogen. The requirement for linear peptides to attach themselves to large carrier proteins such as KLH creates a risk that the immune response will shift toward the carrier protein itself and reduce the specificity intended for the peptide.
Typically, peptides face multiple absorption, distribution, metabolism and excretion (ADME) hurdles resulting in their poor drug candidate success rates including low permeability, metabolic instability, short half-life and low residence time in tissues. More than 99% of peptides are poorly permeable to the cell membrane as a result of their high hydrogen-bonding capacity and low lipophilicity. Low oral bioavailability of peptides results from low absorption and high first-pass extraction due to enzymatic- and pH-mediated hydrolysis in the GI tract and liver. Peptide drugs are therefore typically delivered as injectables or other alternative drug delivery routes such as inhaled, buccal, intranasal and transdermal. After injection, linear peptides are rapidly cleared by renal filtration (<5 kDa) and concurrently degraded by ubiquitous serum endo- and exopeptidases, such that the effective half-life is often reduced to minutes. This limits the antigen exposure in lymph nodes and the ensuing window for dendritic cell uptake, processing, and presentation to naïve T cells. Low systemic residence also means higher or repeated doses are required to achieve desired pharmacological effect, thus increasing cost and risk of reactogenicity. Fast degradation also generates truncated fragments that can be presented on MHC molecules as cryptic epitopes, thus skewing the immune repertoire or inducing tolerance rather than activation. Measures such as lipidation, PEGylation, or nanoparticle encapsulation can retard renal clearance, but add complexity to manufacture and regulatory qualification.
Peptide cyclization is a frequently used peptide modification. Different cyclization strategies include head-to-tail, backbone-to-side chain and side chain-to-side chain cyclization. Peptide cyclization has been used to increase proteolytic stability and cell-permeability as well as mimic and stabilize peptide secondary structure. A peptide sequence by itself and in the absence of attachment to other peptides cannot adopt loop or turn type secondary structures, but cyclization induces the formation of such structures by pre-organizing the intramolecular interactions which otherwise would have required additional residues. Peptide cyclization has also been used to stabilize other types of secondary structure such as α-helix and β-sheet. Cyclic peptides have received significant attention in drug development because of their favorable structural stability, high specificity and better bioavailability in comparison to linear peptides. They are resistant to enzymatic degradation and the cyclic scaffold enables tight binding to diverse biological targets with high affinity, and a number of drugs with cyclic peptide motifs have been developed and approved for clinical use. The number of peptide and protein drugs on the market and under development clearly demonstrates the growing importance of cyclic peptides in drug discovery. Approximately 50 % of all approved peptides have a cyclic structure, the majority being 5–7 membered macrocycles. 39 % of the cyclic peptides are natural, 55 % are analogues and 6 % are heterologous.
Linear peptides frequently fail to recapitulate the exact 3-D conformation adopted by the epitope in its parent protein. The result is that antibodies that recognise the synthetic fragment often do not bind the native surface of the pathogen. Cyclic peptides address this problem by fixing the backbone in a rigid, native-like conformation. This can be achieved by head-to-tail lactamisation, disulfide or hydrocarbon stapling, or thioether bridges. The resulting constraint on the antigen forces it to adopt the critical side-chain geometry required for high-affinity interactions with both B-cell receptors (BCR) and MHC molecules. For instance, cyclic decapeptides mimicking the α-helical CDRH3 loop of the broadly neutralising anti-influenza antibody C05 were shown to bind hemagglutinin (HA) with<100 nM affinity, similar to the full-length antibody, and to retain breadth against H4 and H7 viral subtypes by precluding clashes with hypervariable escape residues. In a separate study, cyclic constructs bearing the group A Streptococcus (GAS) J8 epitope were found to preserve the native α-helix (confirmed by CD spectroscopy), resulting in high-titre IgG responses that opsonised clinical isolates. Computational tools (RosettaRemodel, AlphaFold2) now permit in-silico optimisation of ring size and linker chemistry to ensure that the cyclic scaffold reproduces the exact epitope orientation, while minimising entropic penalties upon receptor binding. The net effect is the generation of antibodies that, in addition to recognising the synthetic peptide, also cross-react strongly with intact pathogens, a necessary requirement for effective vaccine efficacy.
The process of cyclisation provides the antigen with proteolytic, chemical and thermal stability to ensure it arrives intact at professional APCs for germinal centre reaction support. Cyclization of peptides eliminates terminal amino acids which serve as exopeptidase targets while backbone rigidity through this process creates steric hindrance that blocks endopeptidases such as trypsin and chymotrypsin. Serum-stability assays revealed that cyclic lipopeptides maintained >90 % structural integrity after 24 h, while linear analogues were fully degraded after 30 min. The same constructs were resistant to accelerated stress with little oxidation or aggregation, enabling lyophilised, room-temperature storage, a property critical to global vaccine distribution. Lipidated cyclic adjuvants (α-amino-C16 fatty acid conjugates) further improve pharmacokinetics, as they spontaneously self-assemble into nanoparticles readily uptaken by dendritic cells and preferentially transported to the draining lymph nodes for prolonged antigen presentation and heightened T-helper cell activation. The prolonged half-life also translates to a dose-sparing effect (≥10-fold reduction in antigen amount) and the possibility of single-dose immunisation, as has been demonstrated in mice, where cyclic peptide–based vaccines elicited IgG titres comparable to those seen with three boosts of conventional adjuvants.
Several issues are to be considered in designing a cyclic peptide vaccine with respect to the particular vaccine in development. The first one of course is the identification of immuno-dominant domains of epitopes that are able to elicit protective immune response in terms of humoral immunity and/or cell mediated immunity against the antigen of interest. Immunodominant epitopes can be selected with respect to B cells, cytotoxic or helper T cells. For instance, one of the best approaches is to prepare a protective monoclonal antibody against the conserved regions, that may be useful in the design of protective as well as therapeutic vaccine against cancers. In this case, the selection of immunodominant B cell epitopes will be the key factor. However, vaccines against intracellular pathogens like viruses or against cancers may aim to identify epitopes that elicit cytotoxic T cell responses. As will be described in detail in the succeeding section, for efficient induction of either B-cell or cytotoxic T cell responses, a robust helper T cell responses are needed. Thus, epitope selection will have to be aimed not only at the required effector response (B cells or cytotoxic T cells) but also for helper T cell responses. Once a set of epitopes that will specifically induce the desired subsets of immune responses is identified, the next step would be to identify the right epitope(s) or peptide(s) that will activate T cells to the extent that protective immunity can be conferred.
Fig. 2 Schematic illustration of the cyclic peptide-based nanovaccine and its components.3,4
Recent study shows that cyclic peptides can be used as antigenic scaffolds and built-in adjuvants, and can elicit potent humoral and cellular immunity against previously so-called non-peptide-vaccinable pathogens. A prominent example is the Group A Streptococcus (GAS)-targeting cyclic deca-peptide-lipid conjugate (VC-13). This conjugate self-assembles into 200–400 nm nanoparticles that co-deliver B-cell epitopes (J8 and NS1) and a universal T-helper epitope (PADRE), and activate TLR-2/-4 through its C16-lipid tail. Mice required only a single subcutaneous immunisation to generate IgG titres comparable to that of three boosts with Freund's complete adjuvant, and sera strongly potently opsonically killed clinical GAS isolates. Notably, the cyclic scaffold retained the α-helical conformation of the native epitope (as shown by CD spectroscopy), proving that conformational fidelity can be maintained after macrocyclisation. In a second example, a GnRH-cyclic lipopeptide vaccine targeted against hormone-dependent cancers elicited high-titre anti-GnRH antibodies that suppressed testosterone production in male mice after a single prime-boost schedule, showing the platform's applicability to disease areas beyond infectious disease. Finally, the same cyclic adjuvant core was successfully complexed with the SARS-CoV-2 RBD and a cocaine hapten. The resulting nanoparticles induced neutralising antibodies against SARS-CoV-2 and anti-cocaine IgG antibodies capable of sequestering the drug from systemic circulation. Taken together, these studies establish cyclic peptides as a universal delivery vehicle that combines antigen display, adjuvanticity, and lymph-node targeting in a single chemically defined entity.
Epitope selection guided by immunoinformatics C-terminal truncation to minimal B- and T-cell epitopes MHC promiscuity prediction for >120 HLA alleles to cover maximum population Secondary structure prediction of the native epitope by RosettaRemodel or AlphaFold2 Cyclic peptide stapling of the core epitope to recapitulate predicted secondary structure while removing protease-sensitive flanks from both sides Site-specific insertion of D-amino acids, N-methyl amino acids, or fluorinated aromatic side chains at solvent-accessible epitope surface to increase peptide stability while maintaining antigenicity Staple chemistry depends on the peptide: head-to-tail cyclization via lactams for short alpha helices, disulfide or thioether bridges for cysteine-rich motifs, or hydrocarbon staples for long peptides Inclusion of a lipidation or PEGylation moiety at the C-terminus by click chemistry to generate particles of 200–500 nm for optimal lymph node retention UPLC-MS/NMR analysis to ensure >95 % purity, correct disulfide connectivity, and structural integrity In vitro dendritic-cell uptake and murine immunogenicity screening to fine-tune the lead candidate Our process pipeline takes 4 weeks from lead selection to preclinical immunogenicity testing. Our synthetic design principles create peptide cyclic epitopes that are structurally constrained and mimic the natural conformation, leading to a superior antigen presentation to the immune system.
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