Unconjugated Peptide Research Articles

Conjugation with the Carrier Helped to Reveal acidification-Induced Structural Shift in the Peptide from Phospholipase Domain of Parvovirus B19.

Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144-159; 164; 171-183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).

Peptide-drug conjugate designated for targeted delivery to HER2-expressing cancer cells.

Targeted therapy of the highest globally incident breast cancer shall resolve the issue of off-target toxicity concurring with augmented killing of specific diseased cells. Thus, the goal of this study was to prepare a peptide-drug conjugate targeting elevated expression of HER2 receptors in breast cancer. Towards this, the rL-A9 peptide was conjugated with the chemotherapeutic drug doxorubicin (DOX) through a N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker. The synthesized peptide-drug conjugate, rL-A9-DOX, was characterized by mass spectrometry. Molecular docking studies, based on binding energy data, suggested a stronger interaction of rL-A9-DOX with the HER2 receptor in comparison to the unconjugated peptide, rL-A9. The cytotoxic effect of the rL-A9-DOX conjugate was observed to be higher in HER2-positive SKOV3 cells compared to HER2-negative MDA-MB-231 cells, indicating selective cell killing. Cellular internalization of the rL-A9-DOX conjugate was evident from the flow cytometry analysis, where a noticeable shift in mean fluorescent intensity (MFI) was observed for the conjugate compared to the control group. This data was further validated by confocal microscopy, where the fluorescent signal ascertained nuclear accumulation of rL-A9-DOX. The present studies highlight the promising potential of rL-A9-DOX for targeted delivery of the drug into a defined group of cancer cells.

1169. Unconjugated Multi-epitope Peptides Adjuvanted with ALFQ Induce Durable and Broadly Reactive Antibodies to Human and Avian Influenza Viruses

Abstract Background Traditional influenza vaccine production often leads to mismatch due to antigenic drift, thereby reducing protection. Alternate vaccine strategies that are peptide-based, nanoparticle-based or nucleic acid-based are being developed. Composite multi-epitope peptides provide a cost-effective and easily scalable strategy towards a universal influenza vaccine. In this study, we demonstrate that unconjugated, multi-epitope peptides formulated with the highly potent adjuvant Army Liposome Formulation with QS-21 (ALFQ) generate broadly reactive durable antibodies to human and avian influenza viruses when administered intramuscularly or intradermally. Methods ICR mice were immunized with an unconjugated composite influenza peptide vaccine comprising HA+NA (Flu Pep11) and Matrix (M1/M2/M2e)+T-cell epitope (Flu Pep5906) formulated with ALFQ. A low-dose (1 µg) and high-dose (20 µg) of vaccine was administered intramuscularly or intradermally. Isotype-specific IgG titers to composite peptides, individual epitopes, and multiple strains of influenza A (H1N1, H3N2, H5N1), and B (Yamagata, Victoria) were analyzed by ELISA. Neutralizing titers against Group 1 and Group 2 viruses were determined using microneutralization assay. Results A robust IgG1 and IgG2b antibody response was seen at day 21 post primary immunization and remained steady for all groups up to day 200 (duration of study). IgG2a and IgG3 titers were also generated. Antibodies recognized multiple strains each of human and avian influenza A and influenza B virus subtypes, 200 days post primary immunization, and were comparable between doses and routes. Strong neutralizing titers were shown against Group 1 and 2 influenza viruses. Conclusion An unconjugated peptide vaccine comprising multiple highly conserved epitopes of HA, NA and Matrix formulated with ALFQ adjuvant and administered intramuscularly or intradermally, generated broad and durable antibodies to human and avian influenza viruses. Induced antibodies neutralized seasonal and pandemic influenza strains. These composite peptides adjuvanted with ALFQ generate durable and broad immunity and provide a cost-effective and easily scalable strategy that moves us closer to a universal influenza vaccine. Disclosures All Authors: No reported disclosures

Unconjugated Multi-Epitope Peptides Adjuvanted with ALFQ Induce Durable and Broadly Reactive Antibodies to Human and Avian Influenza Viruses.

An unconjugated composite peptide vaccine targeting multiple conserved influenza epitopes from hemagglutinin, neuraminidase, and matrix protein and formulated with a safe and highly potent adjuvant, Army Liposome formulation (ALFQ), generated broad and durable immune responses in outbred mice. The antibodies recognized specific epitopes in influenza peptides and several human, avian, and swine influenza viruses. Comparable antibody responses to influenza viruses were observed with intramuscular and intradermal routes of vaccine administration. The peptide vaccine induced cross-reactive antibodies that recognized influenza virus subtypes A/H1N1, A/H3N2, A/H5N1, B/Victoria, and B/Yamagata. In addition, immune sera neutralized seasonal and pandemic influenza strains (Group 1 and Group 2). This composite multi-epitope peptide vaccine, formulated with ALFQ and administered via intramuscular and intradermal routes, provides a high-performance supra-seasonal vaccine that would be cost-effective and easily scalable, thus moving us closer to a viable strategy for a universal influenza vaccine and pandemic preparedness.

Improving Stability Enhances In Vivo Efficacy of a PCSK9 Inhibitory Peptide.

Optimization of peptide stability is essential for the development of peptides as bona fide alternatives to approved monoclonal antibodies. This is clearly the case for the many peptides reported to antagonize proprotein convertase subtilisin-like/kexin type 9 (PCSK9), a clinically validated target for lowering cholesterol. However, the effects of optimization of stability on in vivo activity and particularly the effects of binding to albumin, an emerging drug design paradigm, have not been studied for such peptide leads. In this study, we optimized a PCSK9 inhibitory peptide by mutagenesis and then by conjugation to a short lipidated tag to design P9-alb fusion peptides that have strong affinity to human serum albumin. Although attachment of the tag reduced activity against PCSK9, which was more evident in surface plasmon resonance binding and enzyme-linked immunosorbent competition assays than in cellular assays of activity, activity remained in the nanomolar range (∼40 nM). P9-alb peptides were exceptionally stable in human serum and had half-lives exceeding 48 h, correlating with longer half-lives in mice (40.8 min) compared to the unconjugated peptide. Furthermore, the decrease in in vitro binding was not deleterious to in vivo function, showing that engendering albumin binding improved low-density lipoprotein receptor recovery and cholesterol-lowering activity. Indeed, the peptide P9-albN2 achieved similar functional endpoints as the approved anti-PCSK9 antibody evolocumab, albeit at higher doses. Our study illustrates that optimization of stability instead of binding affinity is an effective way to improve in vivo function.

In silico studies of interactions of peptide-conjugated cholesterol metabolites and betulinic acid with EGFR, LDR, and N-terminal fragment of CCKA receptors.

In this work, we designed three new ligands by conjugating cholesterol metabolites 3-hydroxy-5-cholestenoic acid (3-HC) and 3-oxo-4-cholestenoic acid (3-OC) and the natural tri-terpenoid betulinic acid with the tumor-targeting peptide YHWYGYTPQNVI. Molecular interactions with the unconjugated peptide and the conjugates were examined with three receptors that are commonly overexpressed in pancreatic adenocarcinoma cells using ligand docking and molecular dynamics. This study demonstrated the utility of the designed conjugates as a valuable scaffold for potentially targeting EGFR and LDLR receptors. Our results indicate that the conjugates showed strong binding affinities and formation of stable complexes with EGFR, while the unconjugated peptide, BT-peptide conjugate, an 3-HC-peptide conjugate showed the formation of fairly stable complexes with LDLR receptor. For EGFR, two receptor kinase domains were explored. Interactions with the N-terminal domain of CCKA-R were relatively weaker. For LDLR, binding occurred in the beta-propeller region. For the N-terminal fragment of CCKA-R, the conjugates induced significant conformational changes in the receptor. The molecular dynamic simulations for 100ns demonstrate that BT-peptide conjugates and the unconjugated peptide had the highest binding and formed the most stable complexes with EGFR. RMSD and trajectory analyses indicate that these molecules transit to a dynamically stable configuration in most cases within 60ns. NMA analysis indicated that amongst the conjugates that showed relatively higher interactions with the respective receptors, the highest potential for deformability was seen for the N-terminal-47 amino acid region of the CCKA-R receptor with and the lowest for the LDLR-receptor. Thus, the newly designed compounds may be evaluated in the future toward developing drug delivery materials for targeting tumor cells overexpressing LDLR or EGFR.

Conserved Influenza Hemagglutinin, Neuraminidase and Matrix Peptides Adjuvanted with ALFQ Induce Broadly Neutralizing Antibodies.

A universal influenza candidate vaccine that targets multiple conserved influenza virus epitopes from hemagglutinin (HA), neuraminidase (NA) and matrix (M2e) proteins was combined with the potent Army liposomal adjuvant (ALFQ) to promote induction of broad immunity to seasonal and pandemic influenza strains. The unconjugated and CRM-conjugated composite peptides formulated with ALFQ were highly immunogenic and induced both humoral and cellular immune responses in mice. Broadly reactive serum antibodies were induced across various IgG isotypes. Mice immunized with the unconjugated composite peptide developed antibody responses earlier than mice immunized with conjugated peptides, and the IgG antibodies were broadly reactive and neutralizing across Groups 1 and 2 influenza viruses. Multi-epitope unconjugated influenza composite peptides formulated with ALFQ provide a novel strategy for the development of a universal influenza vaccine. These synthetic peptide vaccines avoid the pitfalls of egg-produced influenza vaccines and production can be scaled up rapidly and economically.

Myristoylation of Novel Protein Kinase C Beta II Peptide Inhibitor is Required for the Attenuation of Phorbol 12‐myristate 13‐acetate‐induced Superoxide Release in Isolated Rat Polymorphonuclear Leukocytes

Polymorphonuclear leukocyte (PMN) superoxide (SO) production by NADPH oxidase (NOX‐2) activation contributes to myocardial ischemia‐reperfusion (I/R) injury. Protein kinase C beta‐II (PKC βII) is a principal mediator of NOX‐2 activation via phosphorylation of NOX‐2 cytosolic protein p47phox. Phorbol 12‐myristate 13‐acetate (PMA) is a known broad‐spectrum PKC agonist that induces PMN SO release. In prior studies, selective PKCβII inhibition with myristoylated PKCβII peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII‐) attenuated PMA‐induced PMN SO release and myocardial I/R injury in a dose‐dependent manner. However, the role of myristoylation mediating the inhibitory effects of myr‐PKCβII‐ on PMN SO release needs to be determined. Therefore, we aim to confirm the role of PKCβII by using myristoylated PKCβII peptide activator (N‐myr‐SVEIWD; myr‐PKCβII+) and myr‐PKCβII‐ that influence PKCβII translocation. Whereas, myr‐scrambled PKCβII‐ (N‐myr‐WNPESLNTE; myr‐PKCβII‐scram) is a control for myristoylation. We hypothesize that myr‐PKCβII+ should augment, myr‐PKCβII‐should attenuate, and myr‐PKCβII‐scram should have no effect on PMA‐induced PMN SO release compared to non‐treated and unconjugated peptide controls. PMNs (5×106) isolated from male Sprague‐Dawley rats (~400g) were incubated for 15 min at 37°C in the presence/absence of SO dismutase (SOD; 10μg/mL, positive control), unconjugated PKCβII+/− (20 μM), myr‐PKCβII+/− (20 μM), or myr‐PKCβII‐scram (20 μM). SO release was evaluated by the absorbance change (at 550 nm) via ferricytochrome c reduction after PMA stimulation (100 nM) for 390 sec. Data were analyzed by ANOVA using Bonferroni‐Dunn post‐hoc analysis. Myr‐PKCβII‐ significantly attenuated PMA‐induced PMN SO release (0.29±0.02; n=36; p<0.05) when compared to myr‐PKCβII+ (0.42±0.03; n=29), myr‐PKCβII‐scram (0.53±0.05; n=10), and non‐treated controls (0.41±0.02; n=55). Unconjugated PKCβII+ (0.41±0.04; n=16) and PKCβII‐(0.40±0.04; n=28) were similar to non‐treated controls. SOD (n=8) significantly reduced SO release by 94±7% compared to all groups (p<0.01). Cell viability determined by 0.2% trypan blue exclusion was similar in all groups, 94±2%. Unexpectedly, myr‐PKCβII‐scram rather than myr‐PKCβII+ PMNs exhibited the highest PMA‐induced PMN SO release but was not significantly different from untreated controls. Additional experiments will determine whether myr‐PKCβII‐scram significantly enhances SO release. Results suggest myr‐ conjugation improved myr‐PKCβII‐ delivery compared to unconjugated PKCβII‐ but does not contribute to the inhibitory effects of PMA‐induced PMN SO release. Therefore, myr‐PKCβII‐ may be an effective therapeutic intervention to limit inflammation‐induced I/R injury.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine. Current license is supported by Young Therapeutics, LLC.

Catalytically Active Peptide–Gold Nanoparticle Conjugates: Prospecting for Artificial Enzymes

AbstractThe self‐assembly of peptides onto the surface of gold nanoparticles has emerged as a promising strategy towards the creation of artificial enzymes. The resulting high local peptide density surrounding the nanoparticle leads to cooperative and synergistic effects, which result in rate accelerations and distinct catalytic properties compared to the unconjugated peptide. This Minireview summarizes contributions to and progress made in the field of catalytically active peptide–gold nanoparticle conjugates. The origin of distinct properties, as well as potential applications, are also discussed.

Peptide–Gold Nanoparticle Conjugates as Artificial Carbonic Anhydrase Mimics

We herein describe the design and synthesis of a catalytically active peptide–gold nanoparticle conjugate (Pep-Au-NP) that binds Zn(II) within its peptide monolayer and develops carbonic anhydrase activity. Specifically, a modified variant of the β-sheet forming IHIHIQI-peptide (IHQ), which forms an interstrand 3-His Zn(II)-binding site, was used as a ligand for spherical gold nanoparticles (Au-NPs). The resulting immobilized peptide maintains its ability to form β-sheets, as determined by circular dichroism (CD)-spectroscopy and, thus, maintains its ability to form Zn(II)-binding sites. The addition of Zn(II)-ions to the peptide–gold nanoparticle conjugates (Au@IHQ-NP) resulted in significant improvements in rates of ester hydrolysis of 4-nitrophenyl acetate (4-NPA) and the hydration of CO2 compared to the unconjugated peptide variants. Recycling of the catalyst revealed that Au@IHQ-NP remains intact with at least 94% of its initial activity after five rounds of CO2 hydration. The herein reported results reveal that Pep-Au-NPs are able to perform reactions catalyzed by natural metalloenzymes and open up new possibilities for the implementation of these conjugates.

Synthesis and Characterization of Peptide-Chitosan Conjugates (PepChis) with Lipid Bilayer Affinity and Antibacterial Activity.

Antimicrobial peptides appear among innovative biopolymers with potential therapeutic interest. Nevertheless, issues concerning efficiency, production costs, and toxicity persist. Herein, we show that conjugation of peptides with chitosans can represent an alternative in the search for these needs. To increase solubility, deacetylated and degraded chitosans were prepared. Then, they were functionalized via N-succinimidyl- S-acetylthiopropionate or via glutathione (GSH), an endogenous peptide linker. To the best of our knowledge, it is the first time that GSH is used as a thiolating agent for the conjugation of peptides. Next, thiolated chitosans were conjugated through a disulfide bond with designed short-chain peptides, one of them derived from the antimicrobial peptide Jelleine-I. Conjugates and respective reaction intermediates were characterized by absorciometry, attenuated total reflectance-Fourier transform infrared, and 1H NMR. Zeta potential measurements showed the cationic nature of these biomacromolecules and their preferential partitioning to Gram-positive bacterial-like model membranes. In vitro investigation using representative Gram-positive and -negative bacteria ( Staphylococcus aureus and Escherichia coli, respectively) showed that the conjugation strategies lead to enhanced activity in relation to the unconjugated peptide and to the unconjugated chitosan. The obtained products showed selectivity toward S. aureus at low cytotoxicity as determined in NIH/3T3 cells. Overall, our study demonstrates that an appropriate choice of antimicrobial peptide and chitosan characteristics leads to increased antimicrobial activity of the conjugated product and represents a strategy to modulate the activity and selectivity of antimicrobials resorting to low-cost chemicals. The present proposal starts from less expensive raw materials (chitosan and short-chain peptide), is based on aqueous solvents, and minimizes the use of reactants with a higher environmental impact. The final biopolymer contains the backbone of chitosan, just 3-6% peptide derived from royal jelly and GSH, all of them considered safe for human use or as a physiological molecule.

A method for confirming CJC-1295 abuse in equine plasma samples by LC-MS/MS.

CJC-1295 is a peptide-based drug that stimulates the production of growth hormone (GH) from the pituitary gland. It incorporates a functional maleimido group at the C-terminus that allows it to covalently bind plasma proteins such as serum albumin. These CJC-1295-protein conjugates have a much greater half-life compared to the unconjugated peptide and are capable of stimulating GH production for more than six days in humans after a single administration. Conjugated CJC-1295 is difficult to detect in blood by mass spectrometry due to its low abundance, high molecular weight, and conjugation to a range of different protein substrates. Previously we described a screening procedure for the detection of CJC-1295 in equine plasma using an immuno-PCR assay. Here we demonstrate the confirmation of CJC-1295 in equine plasma by LC-MS/MS after immuno-affinity capture and tryptic digestion. Using this method, CJC-1295 was identified down to concentrations as low as 180pg/mL in 1mL of equine plasma.

Influence of the Assembly State on the Functionality of a Supramolecular Jagged1-Mimicking Peptide Additive.

Expanding the bioactivation toolbox of supramolecular materials is of utmost relevance for their broad applicability in regenerative medicines. This study explores the functionality of a peptide mimic of the Notch ligand Jagged1 in a supramolecular system that is based on hydrogen bonding ureido-pyrimidinone (UPy) units. The functionality of the peptide is studied when formulated as an additive in a supramolecular solid material and as a self-assembled system in solution. UPy conjugation of the DSLJAG1 peptide sequence allows for the supramolecular functionalization of UPy-modified polycaprolactone, an elastomeric material, with UPy-DSLJAG1. Surface presentation of the UPy-DSLJAG1 peptide was confirmed by atomic force microscopy and X-ray photoelectron spectroscopy analyses, but no enhancement of Notch activity was detected in cells presenting Notch1 and Notch3 receptors. Nevertheless, a significant increase in Notch-signaling activity was observed when DSLJAG1 peptides were administered in the soluble form, indicating that the activity of DSLJAG1 is preserved after UPy functionalization but not after immobilization on a supramolecular solid material. Interestingly, an enhanced activity in solution of the UPy conjugate was detected compared with the unconjugated DSLJAG1 peptide, suggesting that the self-assembly of supramolecular aggregates in solution ameliorates the functionality of the molecules in a biological context.

Glycolipid-peptide conjugate vaccines enhance CD8+ T cell responses against human viral proteins

An important goal of vaccination against viruses and virus-driven cancers is to elicit cytotoxic CD8+ T cells specific for virus-derived peptides. CD8+ T cell responses can be enhanced by engaging help from natural killer T (NKT) cells. We have produced synthetic vaccines that induce strong peptide-specific CD8+ T cell responses in vivo by incorporating an NKT cell-activating glycolipid. Here we examine the effect of a glycolipid-peptide conjugate vaccine incorporating an NKT cell-activating glycolipid linked to an MHC class I-restricted peptide from a viral antigen in human peripheral blood mononuclear cells. The vaccine induces CD1d-dependent activation of human NKT cells following enzymatic cleavage, activates human dendritic cells in an NKT-cell dependent manner, and generates a pool of activated antigen-specific CD8+ T cells with cytotoxic potential. Compared to unconjugated peptide, the vaccine upregulates expression of genes encoding interferon-γ, CD137 and granzyme B. A similar vaccine incorporating a peptide from the clinically-relevant human papilloma virus (HPV) 16 E7 oncoprotein induces cytotoxicity against peptide-expressing targets in vivo, and elicits a better antitumor response in a model of E7-expressing lung cancer than its unconjugated components. Glycolipid-peptide conjugate vaccines may prove useful for the prevention or treatment of viral infections and tumors that express viral antigens.

Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex.

The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12 nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.

Study of (Cyclic Peptide)-Polymer Conjugate Assemblies by Small-Angle Neutron Scattering.

We present a fundamental study into the self-assembly of (cyclic peptide)-polymer conjugates as a versatile supramolecular motif to engineer nanotubes with defined structure and dimensions, as characterised in solution using small-angle neutron scattering (SANS). This work demonstrates the ability of the grafted polymer to stabilise and/or promote the formation of unaggregated nanotubes by the direct comparison to the unconjugated cyclic peptide precursor. This ideal case permitted a further study into the growth mechanism of self-assembling cyclic peptides, allowing an estimation of the cooperativity. Furthermore, we show the dependency of the nanostructure on the polymer and peptide chemical functionality in solvent mixtures that vary in the ability to compete with the intermolecular associations between cyclic peptides and ability to solvate the polymer shell.

The molecular basis for the prolonged blood circulation of lipidated incretin peptides: Peptide oligomerization or binding to serum albumin?

Hybrid incretin peptides are a new generation of drugs for the treatment of diabetes and obesity. Despite their biological potency, the effectiveness of these peptides as drugs is limited by their short circulation time in blood (typically within minutes). In this work, we show that lipid conjugated forms of a GLP-1/GIP/glucagon hybrid peptides stay in circulation for hours. We studied the oligomerization and albumin-binding of the unconjugated hybrid peptide as well as its lipidated variants. These lipidated peptides differ in the N-terminal mutation, the position of lipidation and the linkage to lipid. We found that these lipidated peptides form stable oligomers at concentrations above 1mg/mL. This concentration range is relevant to formulation and storage of the peptides. We observed no binding between the peptide oligomers and human serum albumin. However, at the expected therapeutic concentration range (~10–100ng/mL), the oligomers dissociate into monomers. The monomers of lipidated peptides bind to albumin. We have determined the dissociation constants of binding between the lipidated peptides and serum albumin. The dissociation constants of albumin-binding of our lipidated peptides are all very close and similar to that of the fatty acid binding of albumin. Our findings suggest that the monomeric lipidated peptides bind to HSA mainly by the fatty acid chain. Therefore, albumin binding is likely to be a universal mechanism of the prolonged circulating duration of lipidated pharmaceutical peptides.

Targeting inhibition of Foxp3 by a CD28 2′-Fluro oligonucleotide aptamer conjugated to P60-peptide enhances active cancer immunotherapy

The specific inhibition of Treg function has long been a major technical challenge in cancer immunotherapy. So far no single cell-surface marker has been identified that could be used to distinguish Treg cells from other lymphocytes. The only available specific marker mostly expressed in Treg is Foxp3, which is an intracellular transcription factor. A targeting molecule able to penetrate the membrane and inhibit Foxp3 within the cell is needed. P60-peptide is able to do that, but due to lack of target specificity, the doses are extremely high. In this study we have shown as a proof of concept that P60 Foxp3 inhibitor peptide can be conjugated with a CD28 targeting aptamer to deliver the peptide to CD28-expressing cells. The AptCD28-P60 construct is a clinically feasible reagent that improves the efficacy of the unconjugated P60 peptide very significantly. This approach was used to inhibit Treg function in a vaccination context, and it has shown a significant improvement in the induced immune response, entailing a lower tumor load in an antigen-specific cancer vaccine protocol.

Multivalent targeting of AT1 receptors with angiotensin II-functionalized nanoparticles

The angiotensin II receptor type 1 (AT1R) is a G protein-coupled receptor of paramount significance since it is overexpressed in a number of diseased tissues that are highly attractive for nanoparticle targeting. However, it is also expressed at physiological levels in healthy tissue. Multivalent interactions mediated by multiple AT1R-binding moieties per nanoparticle could promote a high binding avidity to AT1R overexpressing cells and concomitantly spare off-target tissue. To investigate the feasibility of this approach, angiotensin II was thiolated and conjugated to PEGylated quantum dots. Nanoparticle binding, uptake and affinity to several cell lines was investigated in detail. The colloids were rapidly taken up by clathrin-mediated endocytosis into AT1R-expressing cells and showed no interaction with receptor negative cells. The EC50 of the thiolated angiotensin II was determined to be 261 nM, whereas the ligand-conjugated Qdots activated the receptor with an EC50 of 8.9 nM. This 30-fold higher affinity of the nanoparticles compared to the unconjugated peptide clearly demonstrated the presence of multivalent effects when using agonist-targeted nanoparticles. Our study provides compelling evidence that, despite being immediately endocytosed, Ang II-coupled nanoparticles exert potent multivalent ligand–receptor interactions that can be used to establish high affinities to an AT1R overexpressing cell and tissue.

Tolerogenic modulation of the immune response by oligoglycerol- and polyglycerol-peptide conjugates.

Peptide-based therapy is a promising strategy for antigen-specific immunosuppression to treat or even heal autoimmune diseases with significantly reduced adverse effects compared to conventional therapies. However, there has been no major success due to the drawbacks of native peptides, i.e., limited bioavailability. Considering the importance and limitations of peptide-based therapies for treatment of autoimmune diseases, we designed and constructed oligoglycerol (OG)- and polyglycerol (PG)-based peptide conjugates. They were evaluated for their biological activity (in vitro and in vivo), bioavailability, and tolerogenic potential. Among the OG- and PG-peptide constructs, PG-peptide constructs exhibited an extended bioavailability compared to OG-peptide constructs and unconjugated peptide. Interestingly, size, structure, and linker chemistry played a critical role for the tolerogenic capacity of the constructs. The PG-peptide construct bound via an ester linkage was the most tolerogenic conjugate, while the PG-peptide construct bound via an amide induced stronger proliferation, but also higher TNF production and lower frequencies of Foxp3(+) regulatory T-cells. Therefore, we conclude that PG-peptide conjugates bound via an ester linkage are not only promising candidates for tolerogenic vaccination, but also open a new avenue toward the application of peptides for the treatment of autoimmune diseases.