Peptide drugs have evolved from replacement therapies to precise targeting of cancer, metabolic and infectious diseases. However, their potential is still limited by pre-systemic degradation, fast renal elimination and poor cellular uptake. Chemical modification has so far been the most flexible tool to address these issues. Modifications can be as small as a backbone single atom substitution or as bulky as large hydrophobic groups. As these molecular changes shield peptides from proteolytic enzymes, adjust amphiphilicity and shield immunogenic epitopes, modified peptides resist the blood and intestinal protease pool, persist in the tissue for long enough to act on low-expressed targets, and even exploit endogenous circulating proteins to remain active for weeks. This modification transforms the conformational ensemble of peptides. In many cases, it increases the peptides' selectivity for the target receptor and decreases off-target binding that might lead to cytokine release or hemolysis. The therapeutic index can thereby be broadened by making a better molecule rather than by increasing the dose, which is particularly valuable for cancer and antiviral indications where the risk/benefit ratio must be carefully assessed. The clinical track record of lipidated GLP-1 analogs, cyclised antibiotics, and PEGylated PDCs has now paved the way to advance next generation ligands to the clinic with less attrition.
Fig. 1 Tumor-homing peptides bind to their receptors on tumor cells and selectively deliver cargoes therein, causing cell damage and death.1,5
Native peptides are, by evolutionary design, superb signalers but abysmal drugs. The exposed termini, flexible backbones and hydrophilic surfaces of peptides are pre-programmed for two key things: Peptides are broken down by aminopeptidases and endopeptidases in combination with renal filtration processes. Unmodified peptides are therefore, in this respect, like biological fireflies: The therapeutic effectiveness of these peptides quickly diminishes after a short pharmacological burst because they fail to maintain the required signaling patterns for disease tissue remodeling. Modification changes that. Strategic modification converts these ephemeral ligands into "long-breath" therapeutics by simultaneously addressing three key, rate-limiting hurdles: such transient ligands can be turned into "long-breath" drugs through strategic modification that overcomes three common, rate-limiting challenges: Strategic modification can be used to combat metabolic instability, poor membrane permeability, and immune recognition. Replacement of L- with D-residues, cyclization of the termini or introduction of N-methylated amides can create steric and electronic obstacles to enzymatic digestion; lipidation, cholesterol conjugation or addition of albumin-binding motifs can increase hydrodynamic size and lipophilicity, which decreases glomerular filtration and enables trans-cellular transport; PEGylation or glycosylation can shield epitopes from dendritic-cell recognition and subsequent anti-drug antibody production. The result is a PK profile that can match patient convenience (once-weekly or even monthly administration) while still maintaining the high potency and receptor selectivity that make peptides attractive.
Linear peptides are hydrolyzed by a well-described mechanism. An endo/exopeptidase relay clips the backbone within minutes of intravenous (IV) dosing, and renal corpuscles filter anything under ~6 kDa in the first pass. Chemists have responded by elaborating a multi-pronged defence. Backbone rigidification is the first line of defence. Head-to-tail or side-chain to side-chain cyclisation locks the peptide into a bioactive conformation, which is no longer recognised by canonical proteases, while simultaneously reducing the entropic penalty of receptor binding. Disulfide, thioether or triazole bridges can be introduced orthogonally, so that they do not interfere with late-stage diversification and late-stage diversification and the macrocyclic architecture remains intact. Stereochemical camouflage is the second line. Strategic substitution of L-amino acids with their D-enantiomers disrupts the stereospecificity of peptidases, with a greater effect when the D-residue is at a canonical cleavage hotspot, such as the P1' position. A complementary tactic is N-methylation of backbone amides, which locks the ψ–φ angles into a cis-like geometry that is sterically inaccessible to protease catalytic triads. The third line of defence is appendage-mediated hitch-hiking. Acylation with C14–C20 fatty acids drives reversible association with serum albumin, creating a circulating depot that releases the peptide slowly and shields it from proteolytic insult. Similarly, cholesterylation or albumin-binding peptides can extend the apparent half-life from minutes to hours or even days without increasing the therapeutic dose. When the above modifications are used in combination -cyclized, D-amino acid containing, and lipidated – the effective half-life can approach that of small-molecule drugs, while still retaining peptide-level selectivity.
The oral route of administration is the ultimate goal in peptide drug delivery and modification has started to breach the multiple physiological barriers represented by the gastrointestinal (GI) tract. Luminal proteases, adhesion to the mucus and the tight-junction fence of the epithelium, limit the uptake of unmodified peptides to<1 % systemically. Mid-chain lipidation or cholesterol conjugation sufficiently increase lipophilicity to allow partitioning of the molecule into the mucus layer and the enterocyte membrane, while backbone cyclization reduces the hydrogen-bond inventory that otherwise precludes passive diffusion through cell membranes. The chemical coupling of larger or hydrophilic sequences to cell-penetrating peptides or ligands for transporters such as PepT1 can provide a Trojan-horse mechanism of entry when the trans-cellular route is not available. On the inside of the enterocyte, the same modifications that confer plasma protease resistance now protect against cytosolic peptidases and the peptide is released intact on the basolateral side of the cell. Parenteral administration routes are also augmented: PEGylation or albumin binding increases the hydrodynamic diameter above the renal filtration threshold, prolonging circulation time and the fraction of peptide that reaches target tissue. Crucially, these improvements in exposure are achieved without the spike in serum concentration that result from the high-dose injection of unmodified peptides and the resulting burst-release, widening the therapeutic window and minimising local reaction at the site of injection.
Peptides may undesirably activate innate and/or adaptive immune responses by multiple mechanisms: for example, amphipathic sequences can self-assemble with serum nucleic acids into nano-complexes recognized by TLRs; hydrophobic patches can mimic pathogen-associated molecular patterns; unnatural linkages and/or D-amino acids can be recognized as damage-associated molecular patterns (DAMPs). In contrast, chemical modification provides means to deactivate those signals. PEGylation sterically masks peptides to prevent opsonisation and dendritic-cell uptake, whereas glycosylation can place "self" markers that bind to inhibitory lectins on macrophages. Insertion of backbone-modified residues such as γ-amino acids or β-amino acids inhibit proteasomal processing and presentation of cryptic T-cell epitopes on MHC-II. If activation of the immune response is the goal, for example for peptide vaccines, the same concepts can be inverted: lipidation can increase depot formation at the injection site and thus improve uptake by antigen-presenting cells, and limiting D-amino acid content can prevent epitope destruction. Modification thus acts as a rheostat and not a binary switch.
Chemical vocabularies for peptide engineers have grown immensely beyond the canonical set of 20 amino acids and in many cases can be pieced together to address almost any pharmacologic liability. Instead of searching for the one "magic bullet" modification, newer designs stack orthogonal chemistries (hydrophilic polymers, macrocyclic locks, lipid anchors, non-natural backbones, stapling hydrocarbons) into a single entity to enable it to escape proteases, cross barriers and silence immunity all at once. The three most commonly called upon platforms are PEGylation, cyclization and lipidation, but even within these three well-travelled classes the implementations are still being refined: branched or dendritic PEGs that cleave at programmed rates, bicyclic architectures that simulate protein quaternary interactions, and lipid tails of varied length that self-insert into endogenous nanodiscs in circulation. Because every modification alters solubility, bulk and receptor interactions, the challenge is to strike a balance between gain-of-function and unanticipated liabilities (loss of potency, changes in signaling bias, off-target tissue sequestration). Iterative design processes now integrate solid-phase synthesis with on-resin "click" diversification to enable screening of hundreds of analogues in parallel for stability, permeability and immunologic footprint before pursuing in animal models. The sections below will delve into the mechanistic rationale behind why PEGylation, cyclization and lipidation have become the workhorses for peptide drug optimization.
Chemical attachment of poly(ethylene oxide) chains (PEGylation) is the most common approach to convert a fast clearance peptide to a long-circulating therapeutic. The polymer is often conjugated to the N-terminus, a lysine side-chain or via a thiol-maleimide linkage to cysteine to form an extended hydrophilic brush that increases the hydrodynamic radius above the renal cutoff. This steric effect can improve the residence time from minutes to hours or even days without having to escalate the dose. In addition to the steric repulsion of the PEG corona, the polymer provides a solvation layer that reduces aggregation tendencies and masks epitopes against opsonizing antibodies, thus attenuating both innate and adaptive recognition. Initial fears of completely abolishing receptor affinity have been addressed using cleavable linkers (carbonates, hydrazones, β-eliminative carbamates) that slowly release the polymer once the conjugate has reached its target environment, thus restoring full agonist activity. Branching the topology (Y-shape, comb, dendron) can further enhance the stealth profile while avoiding the increase in viscosity encountered with high-molecular-weight linear PEGs. Critically, this chemistry can be reversed when immune stealth is not desired: Vaccines functionalized with a short PEG spacer show improved lymphatic transport while still retaining the particulate shape needed for dendritic-cell uptake. Process chemists prefer solution phase or resin-based coupling with protecting groups under mildly basic conditions, but enzymatic methods using transglutaminase or sortase A can now be used for site-specific conjugation at internal tags without the need for protecting groups, a method that further reduces batch-to-batch heterogeneity. Overall, PEGylation provides a knob to tune between stealth and visibility, allowing the peptide to evade the systemic filtration while still having the ability to engage its therapeutic target once it encounters the appropriate environmental trigger.
Macrocyclisation transforms the dynamic extended chain of a linear peptide into a conformationally restricted ring, thus frustrating exopeptidases and pre-organising the binding epitope in one fell swoop. Disulfide bridging between two cysteines is the most ancient and still widely used strategy, but the redox lability of the S–S bond under cytosolic reducing conditions has led to efforts to find isosteric replacements (thioethers, triazoles, lactams, olefinic staples) that better resist reduction while preserving the spatial register. Head-to-tail amide cyclisation via coupling of the C-terminal carboxylate to the free N-terminus generates a continuous ring that abolishes both termini, the primary sites of recognition for aminopeptidases and carboxypeptidases. Side-chain-to-side-chain lactam formation between aspartate and lysine (or their higher homologues) narrows the conformational space of the peptide and often improves receptor selectivity by penalising non-productive binding modes. Stapling an α-helical segment with a hydrocarbon bridge at the i, i+4 or i, i+7 positions nucleates the helix even in aqueous solvent, improving proteolytic stability and cell permeability via better membrane partitioning. The cyclisation event is often postponed until after chain assembly, to allow late-stage diversification: the linear precursor is cleaved from resin, cyclised in dilute solution to drive intramolecular condensation over intermolecular, and then purified by reverse-phase chromatography. On-resin variants that use orthogonal protecting groups abbreviate the workflow and reduce the risk of epimerisation, but with a lower overall yield. the size and geometry of the ring has to be fine-tuned to the bioactive conformation of the peptide: if the cycle is too strained it can invert the helical handedness or collapse the binding surface, with concomitant loss of activity. If well optimised, however, cyclic analogues often show orders-of-magnitude improvements in serum half-life and oral bioavailability, establishing macrocyclisation as a keystone of peptide drug design.
Attachment of a saturated or mono-unsaturated fatty acid acyl group (acylation) confers amphipathicity and completely transforms the pharmacokinetic profile of a peptide. The acyl chain non-covalently but strongly associates with serum albumin to effectively hijack its 19-day half-life, while the peptide remains water-soluble and unmasked for receptor binding; its observed clearance rate is now dominated by the slow dissociation rate from the carrier protein. The alkyl chain length and peptide coupling position are optimized to have enough albumin affinity without adversely affecting the potency: myristoylation (C14) provides modest improvement but without compromising aqueous solubility, while stearoylation (C18) leads to a nearly irreversible depot and clearance that may take weeks. Addition of a branch to the acyl group or a single cis-double bond, on the other hand, speeds up the desorption process and thus acts as a safety catch to prevent exaggerated pharmacology. In addition to prolonging the half-life, lipidation can also enhance lymphatic absorption following oral administration, thus circumventing the first-pass metabolism and leading to a higher proportion of the administered dose that eventually reaches the systemic circulation. The hydrophobic patch can also enhance insertion into cell membranes, which is particularly useful for intracellular targets such as cytosolic or organelle-associated proteins; it is important to avoid non-specific membrane disruption, however, which may lead to haemolysis or mitochondrial uncoupling. Chemically, the lipid group can be introduced using an acid-stable amide bond to a lysine side chain, or by using a thioester linkage to a cysteine, the latter being cleaved by endogenous thioesterases. Solid-phase methods that utilize pre-formed lipid building blocks are generally more practical, and also avoid the solubility issues of early solution-phase methods. Overall, lipidation provides a low-cost, scalable method for the production of long-acting peptide therapeutics, which can be more convenient for the end-user due to less frequent dosing.
Epitope-based peptide immunotherapy is no longer the naive, labile mimicry of protein antigen epitopes it once was. Through informed design, short stretches of amino acids can be made to arbitrate and direct specific immunological outcomes in a drug-like manner. The aim might be to reactivate inert cytotoxic T cells against a tumor antigen, to dampen the reactivity of autoreactive lymphocytes in an organ-specific autoimmune disease, or to deliver cytotoxic agents to the draining lymph node. In all cases, a rational peptide alteration is combined with chemical drug-like qualities: biological recognition in the form of T-cell receptor and/or MHC engagement, alongside sufficient metabolic stability to remain intact in the enzyme- and reactive-oxygen species-rich milieu of inflamed tissue. Incorporation of non-natural amino acids, backbone cyclisation, or lipid tails can all be deployed to this end. Such designs act as situational flags: danger signals in the context of cancer, expanding the antigenic target beyond classical tumor driver mutations, or "self" decoys in the context of autoimmunity to recalibrate regulatory pathways. Further control can be obtained through peptide formulation into immunomodulatory delivery vehicles, which exploit self-assembling nanofibrils, for example, or albumin carrier systems, to target and localize specific anatomical compartments in which immune fate is determined, thus conferring enhanced efficacy whilst leaving peripheral immunity intact. The sections below elaborate on the application of these principles in three clinical paradigms: vaccination to prevent established tumors, tolerogenic therapy to treat autoimmune disease, and targeted cell killing through peptide-drug conjugates.
Modified peptides are engineered to circumvent this problem by acting as altered-peptide ligands that both increase MHC anchor affinity and retain T-cell contact residues, thereby expanding the diversity of reactive T cells to include a broader repertoire than the limited clone population initially activated by the endogenous epitope. When a single substitution is combined with N-terminal lipidation or C-terminal amidation, the serum stability and dendritic-cell uptake are also improved, allowing the epitope to reach the draining lymph nodes in an immunogenic form instead of being lost to the injection-site interstitium. Longer peptide formats from twenty to forty residues require intracellular processing by antigen-presenting cells, synchronizing CD4 helper and CD8 cytotoxic priming and preventing the tolerance often caused by direct loading of short, pre-processed 8–10-mers onto peripheral MHC-I molecules. Backbone cyclisation in these extended formats "locks" the peptide into a bioactive conformation resistant to proteolysis during transit, but not so stable that it will not unfold inside the proteasome to yield the correct cleavage products. Toll-like receptor agonists co-assembled into nanoparticles with the same peptides confer a particulate size geometry that is additionally reminiscent of viruses, creating a type-I interferon environment critical for effector memory differentiation. Because peptide chemistry is modular, patient-specific neoepitopes identified by sequencing can be produced in weeks, opening the door to genuinely personalized vaccination schedules that are continually re-formulated as tumors evolve under immune pressure. Clinical results to date suggest that modified peptide vaccines act synergistically with checkpoint blockade, transforming "cold" lesions with only exhausted T cells into "hot" microenvironments rich in cytolytic and pro-inflammatory mediators, extending progression-free survival without the off-tissue toxicity of systemic cytokine therapy.
Autoimmune diseases present the opposite challenge from cancers, as the aim is to dampen an existing antigen-specific response. Modified peptides can have partial agonist/antagonist activity, which inactivates the immune synapse, inducing T cell anergy or deletion or converting T cells into FoxP3+ regulatory T cells (Tregs). Tactics include modifying charged residues in anchor positions to weaken but not completely disrupt MHC binding (biasing the signal toward inhibitory phosphatases rather than the canonical calcium influx). N-methylating the peptide backbone or incorporating D-amino acids can also reduce the half-life of peptide in APCs to decrease the duration of signaling, reducing the risk of epitope spreading. Lipidation of the construct can help cross the blood–thymus barrier, as the attached acyl chain results in transient binding of the peptide to HDL particles, which are transported into the thymic and splenic niches where central and peripheral tolerance are induced. Displaying the modified peptide on the surface of nanoparticles at high valency can create an avidity-dependent effect: high-valency binding to low-affinity autoreactive TCRs preferentially engages inhibitory co-receptors, such as PD-1 and LAG-3, biasing the cell toward producing IL-10 rather than IFN-γ. To avoid tolerance induction of unwanted clones, the peptide is usually modified to contain both the disease relevant epitope and a promiscuous MHC-II helper epitope from a highly-expressed self protein to recruit bystander suppressor CD4+ T cells. Trials in type-I diabetes, multiple sclerosis and uveitis have shown these modified peptide tolerogens can prevent new lesion formation without causing widespread immunodeficiency.
In the conjugate paradigm, the peptide is repurposed as an addressable warhead that brings immunomodulatory payloads to the point of immune synapse. The payload is covalently attached via a linker whose chemistry is designed to be stable in circulation but labile in the tumor, causing minimal off target effects. Targeting sequences (frequently discovered using phage display) target antigens that are over-expressed on malignant or stromal cells, and when internalized bring the drug to the tumor microenvironment (TME) to trigger immunogenic cell death (ICD) or to silence immunosuppressive myeloid subsets. In a similar manner, autoimmunity peptides may target to inflamed vasculature or gut-associated lymphoid tissue (GALT), and when internalized release a drug that is an inhibitor of inflammatory cytokines (eg. IL-1, IL-6) thus suppressing the autoimmunity without the opportunistic infections seen with global biologics. Linker chemistries may also be designed to cleave in response to pathological features: hydrazones, which are acid labile, release the drug in the acidic TME, and valine-citrulline motifs are protease labile and can be used as a proxy for cathepsins which are overexpressed in activated macrophages. Since peptides can be immunogenic, the design may incorporate D- or β-amino acids to disrupt helper T-cell epitopes to prevent a loss of efficacy due to neutralizing antibodies that preclude repeat dosing. The feasibility of the approach has been established clinically: patients can tolerate doses of cytotoxic drugs that would be otherwise unacceptable if free in the circulation since the peptide limits exposure to the antigen-positive lesion and toxicity is transient cytopenia. The modular synthesis of conjugates also portends integration with other immunotherapy modalities including checkpoint antibodies, bispecific T-cell engagers or mRNA vaccines to make peptide-drug conjugates a potentially useful immunotherapeutic tool.
Fig. 2 Schematic representation of a peptide–drug conjugate (PDC), comprising three key components: a homing peptide for targeted delivery, a cleavable or stable linker, and a therapeutic payload.2,5
Drug development often faces barriers like instability and poor bioavailability, which peptides alone cannot overcome. Modified peptides provide solutions by improving circulation time, delivery, and therapeutic potency. Our modification services include PEGylation, cyclization, and lipidation, tailored to research or clinical applications. These modifications make peptides more effective in cancer immunotherapy, autoimmune research, and vaccine development. By ensuring durability and enhanced delivery, modified peptides play a critical role in advancing innovative drug pipelines worldwide.
Products & Services:
Advantages:
Modified peptides improve drug design and therapeutic success. Collaborate with us to optimize modifications that ensure reliable results for immunotherapy and beyond.
1. Why modify peptides?
To improve stability, delivery, and potency.
2. What modifications are available?
PEGylation, lipidation, cyclization, phosphorylation.
3. Are modifications customizable?
Yes, tailored to your sequence.
4. Do you validate modified peptides?
Yes, via HPLC/MS testing.
References
