Anti-PEG Antibodies Inhibit the Anticoagulant Activity of PEGylated Aptamers.
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ABSTRACT:
SUBMITTER: Moreno A
PROVIDER: S-EPMC6707742 | biostudies-literature | 2019 May
REPOSITORIES: biostudies-literature
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Project description:UnlabelledAntibodies that specifically bind polyethylene glycol (PEG) can lead to rapid elimination of PEGylated therapeutics from the systemic circulation. We have recently shown that virus-binding IgG can immobilize viruses in mucus via multiple low-affinity crosslinks between IgG and mucins. However, it remains unclear whether anti-PEG antibodies in mucus may also alter the penetration and consequently biodistribution of PEGylated nanoparticles delivered to mucosal surfaces. We found that both anti-PEG IgG and IgM can readily bind nanoparticles that were densely coated with PEG polymer to minimize adhesive interactions with mucus constituents. Addition of anti-PEG IgG and IgM into mouse cervicovaginal mucus resulted in extensive trapping of mucus-penetrating PEGylated nanoparticles, with the fraction of mobile particles reduced from over 95% to only 34% and 7% with anti-PEG IgG and IgM, respectively. Surprisingly, we did not observe significant agglutination induced by either antibody, suggesting that particle immobilization is caused by adhesive crosslinks between mucin fibers and IgG or IgM bound to individual nanoparticles. Importantly, addition of corresponding control antibodies did not slow the PEGylated nanoparticles, confirming anti-PEG antibodies specifically bound to and trapped the PEGylated nanoparticles. Finally, we showed that trapped PEGylated nanoparticles remained largely in the luminal mucus layer of the mouse vagina even when delivered in hypotonic formulations that caused untrapped particles to be drawn by the flow of water (advection) through mucus all the way to the epithelial surface. These results underscore the potential importance of elucidating mucosal anti-PEG immune responses for PEGylated therapeutics and biomaterials applied to mucosal surfaces.Statement of significancePEG, generally considered a 'stealth' polymer, is broadly used to improve the circulation times and therapeutic efficacy of nanomedicines. Nevertheless, there is increasing scientific evidence that demonstrates both animals and humans can generate PEG-specific antibodies. Here, we show that anti-PEG IgG and IgM can specifically immobilize otherwise freely diffusing PEG-coated nanoparticles in fresh vaginal mucus gel ex vivo by crosslinking nanoparticles to the mucin mesh, and consequently prevent PEG-coated nanoparticles from accessing the vaginal epithelium in vivo. Given the increasing use of PEG coatings to enhance nanoparticle penetration of mucosal barriers, our findings demonstrate that anti-PEG immunity may be a potential concern not only for systemic drug delivery but also for mucosal drug delivery.
| S-EPMC5645030 | biostudies-literature
Project description:Despite their unquestionable properties, oligonucleotide aptamers display some drawbacks that continue to hinder their applications. Several strategies have been undertaken to overcome these weaknesses, using thrombin binding aptamers as proof-of-concept. In particular, the functionalization of a thrombin exosite I binding aptamer (TBA) with aromatic moieties, e.g., naphthalene dimides (N) and dialkoxynaphthalenes (D), attached at the 5′ and 3′ ends, respectively, proved to be highly promising. To obtain a molecular view of the effects of these modifications on aptamers, we performed a crystallographic analysis of one of these engineered oligonucleotides (TBA-NNp/DDp) in complex with thrombin. Surprisingly, three of the four examined crystallographic structures are ternary complexes in which thrombin binds a TBA-NNp/DDp molecule at exosite II as well as at exosite I, highlighting the ability of this aptamer, differently from unmodified TBA, to also recognize a localized region of exosite II. This novel ability is strictly related to the solvophobic behavior of the terminal modifications. Studies were also performed in solution to examine the properties of TBA-NNp/DDp in a crystal-free environment. The present results throw new light on the importance of appendages inducing a pseudo-cyclic charge-transfer structure in nucleic acid-based ligands to improve the interactions with proteins, thus considerably widening their potentialities. Graphical abstract End-functionalization with phosphodiester-linked donor-acceptor groups provides novel properties to TBA aptamer. In particular, a comprehensive crystallographic analysis reveals for the first time a secondary low-affinity binding site of a TBA-like aptamer on thrombin exosite II. The presence of the appendages could finely modulate the hydrophobicity of other G-quadruplex-based aptamers.
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Project description:Antibodies that specifically bind polyethylene glycol (PEG), i.e. anti-PEG antibodies (APA), are associated with reduced efficacy and increased risk of serious adverse events for several PEGylated therapeutics. Here, we explored the concept of using free PEG molecules to saturate circulating APA. Surprisingly, we found that 40 kDa free PEG effectively restored the prolonged circulation of PEGylated liposomes in the presence of high titers of pre-existing APA for at least 48 h in mice. In contrast, lower molecular weight free PEG (≤10 kDa) failed to restore circulation beyond a few hours. These in vivo results were consistent with estimates from a minimal physiologically based pharmacokinetic model. Importantly, the infusion of free PEG appeared to be safe in mice previously sensitized by injection of PEGylated liposomes, and free PEG did not elicit excess APA production even in mice with pre-existing adaptive immunity against PEG. Our results support further investigation of high molecular weight free PEG as a potential method to control and overcome high titers of APA, restoring the prolonged circulation of PEGylated liposomes and possibly other PEGylated therapeutics.
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Project description:In the search for optimized thrombin binding aptamers (TBAs), we herein describe the synthesis of a library of TBA analogues obtained by end-functionalization with the electron-rich 1,5-dialkoxy naphthalene (DAN) and the electron-deficient 1,8,4,5-naphthalenetetra-carboxylic diimide (NDI) moieties. Indeed, when these G-rich oligonucleotides were folded into the peculiar TBA G-quadruplex (G4) structure, effective donor-acceptor charge transfer interactions between the DAN and NDI residues attached to the extremities of the sequence were induced, providing pseudo-cyclic structures. Alternatively, insertion of NDI groups at both extremities produced TBA analogues stabilized by π-π stacking interactions. All the doubly-modified TBAs were characterized by different biophysical techniques and compared with the analogues carrying only the DAN or NDI residue and unmodified TBA. These modified TBAs exhibited higher nuclease resistance, and their G4 structures were markedly stabilized, as evidenced by increased Tm values compared to TBA. These favorable properties were also associated with improved anticoagulant activity for one DAN/NDI-modified TBA, and for one NDI/NDI-modified TBA. Our results indicated that TBA pseudo-cyclic structuring by ad hoc designed end-functionalization represents an efficient approach to improve the aptamer features, while pre-organizing and stabilizing the G4 structure but allowing sufficient flexibility to the aptamer folding, which is necessary for optimal thrombin recognition.
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Project description:Pre-existing and induced anti-poly(ethylene glycol) (PEG) antibodies (abs) have been shown to be related with limitation of therapeutic efficacy and reduction in tolerance of several therapeutic agents. However, the current methods to detect anti-PEG abs are tedious and usually lack quantification. A facile, rapid, sensitive, and reliable technique to detect anti-PEG abs is highly desired in both research and clinic settings. In this work, we have presented a surface plasmon resonance (SPR) biosensor technique for the detection of anti-PEG abs and compared three PEG surface chemistries. Methoxy-PEG (mPEG) 5k was found to have the best performance. The detection of anti-PEG abs directly from diluted blood serum was achieved within 40 min. Detection sensitivity is as good as or better than enzyme-linked immunosorbent assay (ELISA). Furthermore, different antibody isotypes can be quantitatively differentiated by adopting secondary antibodies. A pilot study has been performed to analyze clinical blood samples using this technology, demonstrating its potential as a convenient and powerful method to prescreen and monitor anti-PEG abs in the patients before or after they receive treatment with PEG-containing drugs.
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Project description:We and others have recently shown cisplatin resistance-related protein 9 (CRR9)/Cleft Lip and Palate Transmembrane 1-Like (CLPTM1L) to affect survival and proliferation in lung and pancreatic tumor cells. Our research has indicated that CLPTM1L affects multiple survival signaling pathways in tumor cells under oncogenic, genotoxic, and microenvironmental stress. We have confirmed the association of CLPTM1L with pancreatic cancer by demonstrating overexpression of CLPTM1L in pancreatic tumors and poor survival in patients with high tumor expression of CLPTM1L. Predicting a transmembrane structure, we determined that CLPTM1L could be targeted at the plasma membrane. Herein, we describe the development of mAbs targeting CLPTM1L. Lead antibodies inhibited surface accumulation of CLPTM1L, Akt phosphorylation, anchorage-independent growth, and chemotherapeutic resistance in lung and pancreatic tumor cells. Gemcitabine promoted a physical interaction between CLPTM1L and p110α in pancreatic tumor cells, which was inhibited by anti-CLPTM1L. In vivo treatment with anti-CLPTM1L robustly inhibited the growth of both lung and pancreatic adenocarcinoma xenografts. The efficacy of anti-CLPTM1L correlated with specific epitopes representing important targets in human cancers, particularly those driven by KRas, for which effective targeted therapies have been elusive. This study is the first to report cell-surface exposure of the tumor survival protein CLPTM1L and inhibition of the function of surface CLPTM1L with novel, systematically developed inhibitory mAbs establishing proof of concept of clinically practical agents inhibiting this compelling new tumor survival target in cancer. Mol Cancer Ther; 15(5); 985-97. ©2016 AACR.
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Project description:Vaccine-induced sterilizing protection from infection by Plasmodium parasites, the pathogens that cause malaria, will be essential in the fight against malaria as it would prevent both malaria-related disease and transmission. Stopping the relatively small number of parasites injected by the mosquito before they can migrate from the skin to the liver is an attractive means to this goal. Antibody-eliciting vaccines have been used to pursue this objective by targeting the major parasite surface protein present during this stage, the circumsporozoite protein (CSP). While CSP-based vaccines have recently had encouraging success in disease reduction, this was only achieved with extremely high antibody titers and appeared less effective for a complete block of infection (i.e., sterile protection). While such disease reduction is important, these and other results indicate that strategies focusing on CSP alone may not achieve the high levels of sterile protection needed for malaria eradication. Here, we show that monoclonal antibodies (mAbs) recognizing another sporozoite protein, TRAP/SSP2, exhibit a range of inhibitory activity and that these mAbs may augment CSP-based protection despite conferring no sterile protection on their own. Therefore, pursuing a multivalent subunit vaccine immunization is a promising strategy for improving infection-blocking malaria vaccines.
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Project description:PTK7, a catalytically defective receptor protein tyrosine kinase, promotes angiogenesis by activating KDR through direct interaction and induction of KDR oligomerization. This study developed anti-PTK7 monoclonal antibodies (mAbs) to regulate angiogenesis by inhibiting PTK7 function. The effect of anti-PTK7 mAbs on vascular endothelial growth factor (VEGF)-induced angiogenic phenotypes in human umbilical vascular endothelial cells (HUVECs) was examined. Analysis of mAb binding with PTK7 deletion mutants revealed that mAb-43 and mAb-52 recognize immunoglobulin (Ig) domain 2 of PTK7, whereas mAb-32 and mAb-50 recognize Ig domains 6-7. Anti-PTK7 mAbs inhibited VEGF-induced adhesion and wound healing in HUVECs. mAb-32, mAb-43, and mAb-52 dose-dependently mitigated VEGF-induced migration and invasion in HUVECs without exerting cytotoxic effects. Additionally, mAb-32, mAb-43, and mAb-52 inhibited capillary-like tube formation in HUVECs, and mAb-32 and mAb-43 suppressed angiogenesis ex vivo (aortic ring assay) and in vivo (Matrigel plug assay). Furthermore, mAb-32 and mAb-43 downregulated VEGF-induced KDR activation and downstream signaling and inhibited PTK7-KDR interaction in PTK7-overexpressing and KDR-overexpressing HEK293 cells. Thus, anti-PTK7 mAbs inhibit angiogenic phenotypes by blocking PTK7-KDR interaction. These findings indicate that anti-PTK7 mAbs that neutralize PTK7 function can alleviate impaired angiogenesis-associated pathological conditions, such as cancer metastasis.
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Project description:Humans commonly have low level antibodies to poly(ethylene) glycol (PEG) due to environmental exposure. Lipid nanoparticle (LNP) mRNA vaccines for SARS-CoV-2 contain small amounts of PEG, but it is not known whether PEG antibodies are enhanced by vaccination and what their impact is on particle-immune cell interactions in human blood. We studied plasma from 130 adults receiving either the BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines or no SARS-CoV-2 vaccine for PEG-specific antibodies. Anti-PEG IgG was commonly detected prior to vaccination and was significantly boosted a mean of 13.1-fold (range 1.0-70.9) following mRNA-1273 vaccination and a mean of 1.78-fold (range 0.68-16.6) following BNT162b2 vaccination. Anti-PEG IgM increased 68.5-fold (range 0.9-377.1) and 2.64-fold (0.76-12.84) following mRNA-1273 and BNT162b2 vaccination, respectively. The rise in PEG-specific antibodies following mRNA-1273 vaccination was associated with a significant increase in the association of clinically relevant PEGylated LNPs with blood phagocytes ex vivo. PEG antibodies did not impact the SARS-CoV-2 specific neutralizing antibody response to vaccination. However, the elevated levels of vaccine-induced anti-PEG antibodies correlated with increased systemic reactogenicity following two doses of vaccination. We conclude that PEG-specific antibodies can be boosted by LNP mRNA vaccination and that the rise in PEG-specific antibodies is associated with systemic reactogenicity and an increase of PEG particle-leukocyte association in human blood. The longer-term clinical impact of the increase in PEG-specific antibodies induced by lipid nanoparticle mRNA vaccines should be monitored. It may be useful to identify suitable alternatives to PEG for developing next-generation LNP vaccines to overcome PEG immunogenicity in the future.
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