The peptide targeting motif serves as an address tag to direct immunomodulatory payloads to distinct leukocyte populations, which can in effect turn a global pharmacological intervention into a cell-autonomous manipulation. Peptides allow distinct modules of affinity-matured epitopes, protease-resistant scaffolds, and linker motifs for chemical conjugation to be presented on a single <30-mer scaffold to engage lineage-restricted receptors, with far less risk of immunogenicity or steric hindrance compared to antibodies. This is of particular importance in the context of immune cell targeting, where broader receptor engagement could lead to undesirable outcomes such as cytokine release syndrome or global immune dysregulation. Targeting payloads to individual cell types via peptides therefore allows for an expanded therapeutic window while still maintaining peripheral immune quiescence.
Overview of Cell-Based Drug Delivery Systems1,5
Selectivity in immune cell targeting is critical since the same receptor superfamily (CD28, TNFR etc.) can mediate opposite fates in different lineages (costimulation versus apoptosis in T- and B-cells respectively). Systemically administered agents in the absence of ligand-directed delivery are active in all receptor expressing populations, leading to a fine line between efficacy and toxicity. In this way peptides can serve as molecular zip codes to deliver cytokine antagonists preferentially to activated T-cells in sites of inflammation or tolerogenic siRNA to autoreactive B-cells.
Pan-immune therapies, such as corticosteroids or checkpoint inhibitors, activate T, B, myeloid and innate lymphoid cells indiscriminately. This can lead to side effects such as cytokine storms, opportunistic infections, or end-organ damage. Without cell-type selectivity, immune subsets that are beneficial (e.g. regulatory T cells) are inactivated alongside pathogenic clones. Prolonged high-dose administration required to reach rare cell types further exacerbates an already broad adverse-event profile. As systemic drug levels must exceed activation thresholds for the most refractory cell population, circulating concentrations are often above levels required for endothelial activation, complement recruitment, or neurotoxicity. Attempts to broaden the therapeutic window using high-low alternating dosing schedules still subjects non-target organs to potentially toxic peak concentrations, explaining why spatial rather than temporal control is superior.
Leukocytes include dendritic cells, monocytes, macrophages, neutrophils, eosinophils, mast cells, γδ T cells, mucosal-associated invariant T cells, and numerous B and NK subsets, with different endocytic rates, protease repertoires, and receptor cassettes. A peptide targeting CD11c for instance will be recognized by both tolerogenic DC2s and pro-inflammatory DC1s. High affinity for a target receptor will not ensure functional selectivity. CD14 is expressed on classical monocytes, macrophages and microglia; CD163 on M2 macrophages and certain DC subsets; CD1c on DC2s and activated B cells. Overlaps in surface receptor expression have made design of functionally-selective peptides necessarily rely on Boolean-logic approaches (two-ligand recognition or protease-uncaged second loop) instead of a single "unique" epitope. This in turn means that peptide length and cyclisation strategies must accommodate two binding faces without exceeding the size limit for RMT.
Table 1 Conceptual comparison: systemic vs peptide-targeted immune modulation
| Feature | Systemic therapy | Peptide-targeted approach |
| Cell spectrum | Pan-leukocyte | Subset-restricted |
| Dose requirement | High | Lower (local concentration) |
| Off-target toxicity | Common | Reduced |
| Receptor overlap risk | N/A | Addressed by dual motifs |
| Development time | Long (biologics) | Shorter (solid-phase) |
| Immunogenicity | Variable | Minimal with D-amino acids |
Systemic immunotherapy becomes a cell-type specific treatment when the short peptide sequences binding to cell surface receptors with restricted expression across leukocyte subsets are attached. These targeting peptides act as molecular zip-codes to shuttle siRNA, mRNA or CRISPR RNPs to specific subpopulations such as Tregs, M1 macrophages or cytotoxic T cells while avoiding all other off-target lineages rather than saturating the entire immune compartment. Targeted to its receptor with nanomolar affinity, the same peptide can be additionally engineered to (i) induce internalization via clathrin or caveolin mediated endocytosis and (ii) release its genetic cargo once in the cytosol, thus turning a systemic signal into a lineage-specific instruction.
Dendritic cells express CD11c and DEC-205, M2 macrophages are over-expressing CD163 and mannose receptor, and neutrophils are expressing CD66b and CXCR2. Peptides that bind to these receptors, but not their natural ligands (e.g. mannose or RGD mimetics), bind to the receptor outside of the natural binding pocket, avoiding competition with serum proteins, but still recruit clathrin. Head-to-tail cyclisation of the epitope prevents degradation by the many aminopeptidases in serum and phagolysosomes, and remains intact until receptor binding. Receptors are then trafficked down different pathways (DEC-205 is recycled to the surface, while mannose receptor is trafficked to late endosomes), which then allows for peptides to contain pH-sensitive histidines or reducible disulfides that release the cargo in early endosomes for DEC-205 targeted cells, or hold the complex until lysosomal proteolysis for mannose-receptor-directed cells. The directed trafficking then allows for the same peptide backbone to carry siRNA to dendritic cells or ROS-generating chemotherapeutics to M2 macrophages, simply by changing the receptor-binding loop.
Passive particles are dependent on size or charge, and are cleared by the first phagocyte that they encounter (commonly Kupffer cells or splenic macrophages), diluting the dose before it reaches lymph-node dendritic cells or tumor-associated neutrophils. The principal advantage of peptide-guided systems is that they circumvent this "first-pass sink" by their requirement for both receptor expression and endocytic competence. As a result the payload is selectively concentrated in the inside of the target subset even when that subset comprises <5 % of total leukocytes. Because peptide-mediated uptake is receptor-driven, the systemic therapeutic window is broadened: lower systemic doses are required, minimizing off-target cytokine storms or complement activation. Charge-driven complexes bind to endothelial glycosaminoglycans which results in entrapment in the lung or kidney. Peptide-targeted carriers are either neutral or only slightly anionic at physiological pH values, thus avoiding electrostatic adsorption. In addition, the peptide itself can be designed to be proteolytically removable after endosomal uptake, thereby uncovering a hidden cationic face that can selectively disrupt the vesicle once inside the target cell. In this "time-bomb" fashion, membrane lysis is confined to the desired leukocyte, even in situations where the target population (say a dendritic cell subset) shares an anatomical space with functionally distinct but regulatory T cells or resting B cells which lack the receptor (thus reducing the danger of global immunosuppression or auto-antibody generation).
Peptides targeted for uptake by specific leukocyte subsets must balance selectivity, kinetics, and safety. The ligand should bind to a cell surface epitope that is lineage-restricted enough to avoid by-stander uptake while also being sufficiently expressed to enable receptor-mediated internalization under shear flow. Because immune cells cycle between resting and activated states, affinity and linker chemistry must be chosen to allow for fast release after endosomal delivery, to avoid receptor saturation and loss of responsiveness with repeated dosing. Finally, the peptide itself should not be detectable by pattern-recognition receptors so as not to turn a delivery vehicle into an unwanted adjuvant.
Lineage-specific vs activation-state markers form the first bifurcation in design: CD45RA differentiates naïve T cells from memory subsets, while PD-1 or 4-1BB indicate exhaustion or activation, respectively. The use of lineage markers alone risks payload delivery to functionally irrelevant cells; activation markers, on the other hand, may be transient or broadly shared across multiple subsets (e.g., CD69 on both NK and T cells), yielding incomplete selectivity. Risks of expression overlap are averted with bivalent or AND-gate architectures: a peptide that simultaneously engages CD11b and the TNF-α-inducible adhesion receptor VCAM-1 will specifically accumulate in inflamed macrophages, but not resting monocytes that only express CD11b alone. Designers must thus map marker density and co-expression by spectral flow cytometry across healthy, inflamed, and tumour tissues, favouring pairs whose overlap coefficient is <0.3 and whose density ratio between target and off-target cells is >5-fold.
The on-rate for target engagement has to be fast (<1 min) for Circulating T-cells that are only transiently present in capillaries, whereas tissue-resident macrophages can afford a much slower binding. As a result, peptides are usually flanked by Gly-Ser spacers that increase mobility and allow diffusion-limited association rates, and cyclic backbones that sequester the epitope and prevent dissociation. Binding kinetics for slowly recycling targets like PD-1 are improved by bivalent architectures that use avidity to attain sufficiently high levels of uptake, even when the surface density of the receptor is low, such as during down-regulation in chronic inflammation. Intermediate off-rates (10–100 nM) permit multiple rounds of endocytosis per cell, thereby greatly amplifying gene delivery when receptor down-regulation is not limiting. Histidine tails are pH sensitive and protonated in late endosomes, which helps to destabilize the vesicle and release the siRNA directly into the cytosol. As such, this escape mechanism is hard to incorporate into larger antibody conjugates. MMP-cleavable linkers can also be used to activate the compound only in inflamed tissue, thereby guaranteeing gene modulation where protease activity is increased.
Arginine- or lysine-rich sequences can activate Toll-like receptor 7/8 or create amyloid-like fibrils that lead to dendritic cell activation. For this reason, cationic content is limited to a maximum of 40 %, and zwitterionic glutamate residues are added to mask charges while maintaining condensation ability. Furthermore, common T-cell epitopes (e.g., RGD motifs) are avoided to prevent unintended activation of T-cell receptors that could lead to cytokine release. Replacement of L-amino acids with D-amino acids at non-binding locations can frustrate proteasomal processing and thereby decrease the chance of MHC presentation and subsequent anti-peptide antibody production. Backbone protection by cyclization or N-methylation can also be used to resist aminopeptidase degradation while maintaining receptor binding affinity. Such modifications are intended to make the peptide long enough to remain unperceived by the immune system in order to deliver the gene cargo but still be removable in target cells under reducing conditions.
Peptide-based immune delivery is not a platform but a family of architectures, each mediating a tradeoff between molecular precision on one hand and payload volume, manufacturing complexity, and biological risk on the other. Whether the payload is a minimalist peptide–drug conjugate (PDC) with one siRNA cargo or a peptide-dressed liposome packed with CRISPR RNPs, the strategy is the same: use the peptide as the address label and the architecture as the envelope. The envelope has come in different flavors, each with different tradeoffs: covalent conjugates provide stoichiometric precision but limited cargo capacity; nanocarriers provide bulk capacity at the cost of heterogeneity; multitargeting broadens cell coverage but risks receptor saturation. Tailoring the architecture to the immune biology—circulating versus tissue-resident cells, acute versus chronic inflammation, mono- versus multi-lineage targets—rather than to a single engineering metric will therefore be required for success.
Covalent PDCs are typically composed of a 10–30 mer targeting peptide chemically linked to a small molecule immunomodulator (e.g., a JAK inhibitor, STING agonist) or to a short siRNA through a cleavable disulfide or hydrazone bond. The predictable pharmacokinetics, arising from a single molecular species that is measured, cleared and regulated as one analytical entity, is an obvious benefit. The affinity of a peptide can be tuned, from micromolar to picomolar, by cyclization or D-amino acid substitution, to engage even low-density targets such as PD-1 on the surface of exhausted T-cells, without requiring systemic drug levels that would also activate naïve lymphocytes. Conditional linkers (MMP-sensitive, pH-sensitive) have been designed to ensure that the payload is only liberated inside the inflamed lysosome, thereby converting a systemically toxic small molecule to a cell-immanent gene modulator. The peptide is typically cleared from circulation within minutes, meaning that the plasma half-life of the conjugate is effectively governed by the peptide, not the payload, providing an automatic off-switch that avoids the prolonged immune suppression that plagues conventional small-molecule JAK inhibitors. Split-dosing regimens can be used to titrate receptor occupancy in real time, an advantage impossible with long-lived antibodies. The single-molecule format precludes cargoes of >3 kDa; larger biologics (mRNA, CRISPR RNPs) require alternative architectures.
Schematic illustration of PDCs designed to covalently target SARS-CoV-2 PLpro, featuring the inhibitor GRL0617 conjugated to sulfonium-linked peptides based on the PLpro-specific substrate sequence LRGG2,5
10–100 copies of a peptide are tethered to liposomes, polymeric micelles, or inorganic nanoparticles using maleimide-PEG linkers. Multivalency converts low-affinity monovalent interactions in the µM range to high-avidity interactions in the nM range without increasing the systemic dose of the targeting agent, which allows targeting of low-density receptors such as CD1c on dendritic cells. The surface density of the targeting peptide is also tunable: dense (50–100 peptides) creates 80–120 nm clusters that remain in the lymph nodes, while sparse (10–20 peptides) creates 30–50 nm particles that penetrate the inflamed joint synovium; the former is good for vaccine adjuvants, the latter for rheumatoid arthritis siRNA. Cleavable (hydrazone or disulfide) linkers are often used to shed the peptide after transcytosis, which restores the native particle surface charge and reduces off-target adhesion in healthy tissue. Multivalent particles are avidly phagocytosed by macrophages. This is a useful property for particles designed to deliver IL-10 siRNA to M1 macrophages, but it is a liability when T-cell engagement is desired. Developers have accordingly engineered additional "stealth" layers of short zwitterionic peptides or PEG brushes that are selectively repellant to phagocytes while leaving T-cell receptor engagement intact. Dual-density surfaces may also be used, where high-avidity clusters serve to capture target cells while low-density linker peptides enable deep tissue penetration (at the cost of some specificity).
Chronic inflammation is mediated by multiple lineages (Th1, Th17, Treg, M1, M2) with partially overlapping surface markers. Targeting a single receptor risks incomplete coverage. Bivalent peptides with two different motifs (e.g. CD3 for T-cells + CD11b for monocytes) implement an OR-logic gate that broadens coverage but keeps it cell-type specific. AI-aided design ensures both arms maintain affinity even in close proximity, while cleavable linkers can be used to release them sequentially: after the first arm is shed following engagement on T-cells, re-binding to healthy tissue is prevented while the second arm remains bound and active on monocytes. The architecture also lowers the chance of escape through antigen loss, a common mode of resistance against single-receptor approaches. Too broad a target (CD11b) would lower the effective dose on target lineages, while too narrow a target (CD1c) would risk excluding pathogenic subsets. Developers therefore combine activation-state AND-logic: a peptide might bind CD11b on all monocytes but only release its payload where MMP-9 activity is high, thus focusing the gene silencing to inflamed macrophages and sparing resting monocytes. Alternatively, density-based gating takes advantage of the 10-fold higher PD-1 expression on exhausted T-cells compared to naïve T-cells to achieve functional selectivity without molecular engineering. These approaches trade off some coverage for predictable efficacy across patient heterogeneity.
Immune cell targeting is a zero sum game: a peptide must be able to find its target in the presence of look-alikes, bind tightly enough to be internalized, yet release quickly enough not to get dragged into lysosomes or back to the circulation. Each improvement in affinity or stability comes at the cost of increased off-target uptake, reduced circulation time or side immune activation. Leukocytes live in a world of hours: they circulate, become activated and die in this time frame, so the opportunity for productive engagement is extremely short. A peptide optimized for its target in splenic T-cells can thus completely miss its target in the inflamed joint synovium, where the same receptor may be buried beneath glycocalyx. Successful design therefore requires treating the immune system as a moving target – designing for dynamics, not for static binding.
CD11b marks not only inflammatory monocytes, but also neuro-protective microglia; PD-L1 is up-regulated on both tolerogenic dendritic cells and exhausted T cells. Selecting for a single epitope thus enriches a mixed population in which some members promote disease and others limit it. Designers use Boolean-logic approaches—concatenating two moderate-affinity peptides separated by a protease-cleavable bridge—so that only cells that express both epitopes internalize the full payload, sharpening therapeutic index without enlarging the peptide beyond renal filtration limits. Even dual-motif constructs can still bind "bystander" subsets that transiently express one marker during inflammation. Incorporating a shear-activated lipophilic anchor that inserts only under arterial flow further restricts uptake to circulating pathogenic cells while sparing tissue-resident sentinels, so that the pharmacokinetics match the biological context.
Circulating leukocytes, on the other hand, transit <0.1 s in cerebral or splenic capillaries. This requires on-rates to approach antibody affinities, which is no small feat for a 12-mer peptide. Cyclisation or head-to-tail stapling of peptides pre-organizes the binding loop for insertion into the target, which speeds association without the burden of extra molecular weight. By contrast, tissue-resident macrophages sit in low-flow environments where engagement times are longer and lower-affinity interactions are feasible. The same peptide can be synthetically modified to include a cleavable PEG shield, which is shed only after extravasation and thus tunes affinity for anatomical location. Internalization is expected, but escape from endosomes must then occur before the cell drifts out of the drug-perfused region. Peptides modified with histidine clusters that protonate at pH 6.0 or disulfide bonds that are rapidly reduced by cytosolic glutathione are thus released into the cytosol within minutes. This avoids the "hit-and-run" problem, in which a leukocyte is tagged but not modulated. Because migration velocities can be >10 µm/min, kinetics of release are as important as binding specificity.
Activation of a receptor does not assure an immune response: an siRNA targeting IRF4 delivered into inflammatory monocytes, for example, might show uptake but if the target gene is not rate-limiting in that cytokine environment then phenotype modulation will not be achieved. Designers circumvent this by conjugating an uptake reporter (fluorophore-quencher peptides) to a functional readout (cytokine secretion, surface marker changes) to assure that peptide delivery does indeed lead to modulation, even though uptake may not be a rate-limiting step in functional efficacy. The same immune cell targeting peptide may elicit opposite immune modulations if used to carry a STING agonist or a JAK inhibitor, for example. The intended payload will have defined downstream signalling and thus peptide design must consider cargo chemistry: stretches of cationic residues to condense siRNA cargo, for example, can have activating TLR7/8 activity if not masked, for example, thus negating the anti-inflammatory purpose. Trade-off between cationic content using norleucine substitutions or use of self-immolative spacers to mask cationic faces until cytosolic release are approaches to consider peptide architecture with regard to desired payload immunogenicity.
Peptide guided immunotherapy is best applied to a disease lesion which features a sharply demarcated, receptor-defined cell population whose surface antigen is either lineage-restricted (CD19 on B-cells) or activation-induced (PD-L1 on exhausted T-cells) and whose density is above a threshold for receptor-mediated endocytosis. In such conditions, a 15–30 mer peptide can be used as a "homing-then-release" vector, binding to its target receptor while passing the capillary in transit, then releasing its payload from inside the acidic endosome so that siRNA, cytokine antagonists, or tolerogenic mRNA are released only inside the pathogenic cell. This architecture has particular appeal for chronic autoimmune settings (rheumatoid arthritis, lupus nephritis) where repeat dosing is necessary and where off-target immune suppression would be clinically unacceptable.
An ideal target is either constitutively lineage-restricted (CD1c on DCs, CD19 on B-cells) or selectively up-regulated on pathogenic activation (CD69 on NK cells, PD-1 on exhausted T-cells). Peptides identified by phage display against recombinant ectodomains are negative-selected on off-target leukocytes to exclude cross-reactive sequences. Cyclization or D-amino acid substitution then locks the epitope to ensure that the peptide does not lose its nanomolar affinity when conjugated to bulky CRISPR RNPs. Epitope mapping also avoids sequences overlapping native ligand binding, so specificity is not lost due to competition from physiological concentrations of cytokines. In rheumatoid arthritis, CD11b-high inflammatory monocytes are a driver of joint destruction; in multiple sclerosis, PD-1-high exhausted T-cells lose their tumor suppressive function. Targeting delivery of TNF-α siRNA to the former or PD-1 mRNA to the latter therefore results in a mechanistic cure rather than symptomatic treatment. Receptor density is validated by quantitative flow cytometry on patient synovial fluid or CSF, to ensure that the chosen marker is present at >10 000 copies per cell, enough to support peptide-mediated uptake without saturating the limited transporters.
When the target antigen is common to multiple lineages (CD11b on monocytes, macrophages, and neutrophils), peptide binding dilutes the dose and creates the risk of neutropenia. In such cases, antibody–drug conjugates with Fc-mediated recycling or bispecific antibodies that bind two markers at once will often provide better selectivity than a single-peptide ligand. Analogously, when the receptor is covered by a dense glycocalyx (CD3 on naïve T-cells in lymph nodes), the peptide may be unable to access its epitope. In this scenario, larger and more penetrating carriers may be preferred. When the therapeutic intent is acute, whole-body immune modulation (cytokine storm in sepsis), receptor-limited transcytosis is too slow. Systemic JAK inhibitors or anti-TNF antibodies have faster pharmacokinetics than peptide-guided siRNA, and off-target toxicity is considered an acceptable trade-off. On the other end of the spectrum, when a more localized, chronic inflammation is the target (psoriatic skin, synovial joint), the modular, repeat-dose nature of peptide conjugates provides a safety and manufacturing advantage that outweighs its lower payload capacity.
Table 2 Decision matrix for peptide suitability in immune cell-specific delivery.
| Decision axis | Favors peptide use | Suggests alternative |
| Target specificity | Lineage-restricted | Shared across subsets |
| Receptor density | Moderate & recycling | Very low or saturated |
| Payload size | siRNA, antisense, CRISPR RNP | Large mAb or DNA |
| Systemic urgency | Local modulation needed | Rapid global suppression |
| Manufacturing timeline | Rapid iteration required | Long antibody development |
Immune cell-specific drug delivery requires a level of targeting precision that is difficult to achieve with conventional delivery approaches. Immune cells are highly heterogeneous, dynamically regulated, and often share overlapping surface markers, making non-specific uptake a persistent challenge. Many targeting strategies fail because peptide designs do not adequately account for these biological complexities. Our targeting peptide services support immune-targeted delivery programs by integrating immune biology, peptide engineering, and feasibility assessment into a unified, selectivity-first development approach.
Target-Driven Sequence Engineering: Effective immune targeting begins with a deep understanding of immune cell surface markers and their functional roles. Our target-driven sequence engineering approach designs peptides around markers that are both sufficiently selective and functionally relevant for drug internalization. Peptide sequences are engineered to engage immune cell receptors in a manner that supports productive uptake without triggering unintended activation or signaling. This biology-first design strategy helps ensure that targeting interactions translate into meaningful delivery rather than non-specific binding.
Selectivity-Focused Optimization: In immune-targeted delivery, maximizing selectivity is often more important than maximizing uptake. Our selectivity-focused optimization strategy prioritizes reducing off-target interactions across closely related immune cell subsets. Affinity, binding kinetics, and peptide physicochemical properties are carefully tuned to align with the expression density and internalization behavior of the intended immune cell population. This minimizes cross-reactivity and helps preserve functional specificity in complex immune environments.
Marker Evaluation: Not all immune cell markers are equally suitable for peptide-mediated targeting. Our feasibility assessment includes a comprehensive marker evaluation that examines expression patterns across immune cell subsets, activation states, and tissues. Markers with high overlap across cell types or significant expression in non-target tissues are flagged early, helping teams avoid pursuing targets that are unlikely to deliver meaningful selectivity regardless of peptide optimization.
Early Off-Target Risk Assessment: Off-target immune engagement can lead to unintended modulation or toxicity. We therefore emphasize early off-target risk assessment to identify peptide designs or targeting strategies that may interact with unintended immune populations. By identifying these risks early, we help refine design strategies or redirect development efforts before extensive optimization or scale-up is undertaken.
If your immune-targeted delivery program shows limited selectivity, unexpected immune engagement, or inconsistent targeting across immune cell populations, an early technical discussion can help clarify whether target choice or peptide design is the limiting factor. Discuss your immune cell targeting strategy with our team to evaluate feasibility, assess off-target risks, and define a peptide design approach aligned with your immunology program’s objectives.
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