Polymer-based antibody mimetics (iBodies) target human PD-L1 and function as a potent immune checkpoint blocker.

Immune checkpoint blockade (ICB) using monoclonal antibodies against programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) is the treatment of choice for cancer immunotherapy. However, low tissue permeability, immunogenicity, immune-related adverse effects, and high cost could be possibly improved using alternative approaches. On the other hand, synthetic low-molecular-weight (LMW) PD-1/PD-L1 blockers have failed to progress beyond in vitro studies, mostly due to low binding affinity or poor pharmacological characteristics resulting from their limited solubility and/or stability. Here, we report the development of polymer-based anti-human PD-L1 antibody mimetics (α-hPD-L1 iBodies) by attaching the macrocyclic peptide WL12 to a N-(2-hydroxypropyl)methacrylamide copolymer. We characterized the binding properties of iBodies using surface plasmon resonance, enzyme-linked immunosorbent assay, flow cytometry, confocal microscopy, and a cellular ICB model. We found that the α-hPD-L1 iBodies specifically target human PD-L1 (hPD-L1) and block the PD-1/PD-L1 interaction in vitro, comparable to the atezolizumab, durvalumab, and avelumab licensed monoclonal antibodies targeting PD-L1. Our findings suggest that iBodies can be used as experimental tools to target hPD-L1 and could serve as a platform to potentiate the therapeutic effect of hPD-L1-targeting small molecules by improving their affinity and pharmacokinetic properties.

HPMA copolymer conjugated 5-aminolevulinic acid exhibits superior efficacy for photodynamic therapy with tumor-responsive and targeting properties.

In this study, we developed a nanoformulation of 5-aminolevulinic acid (5-ALA) for tumor-targeted photodynamic therapy, in which 5-ALA was conjugated with a biocompatible polymer N-(2-hydroxypropyl)methacrylamide (HPMA) through the hydrazone bond, i.e., P-ALA. P-ALA behaves as the nano-sized molecule with an average size of 5.5 nm in aqueous solution. P-ALA shows a largely increased release rate in acidic pH than physiological pH, suggesting the rapid release profile in acidic tumor environment. P-ALA did not show apparent cytotoxicity up to 0.1 mg/ml, however, under light irradiation, remarkable cell death was induced with the IC50 of 20-30 µg/ml. More importantly, we found significantly higher tumor accumulation of P-ALA than 5-ALA which benefit from its nano-size by taking advantage of the enhanced permeability and retention (EPR) effect. Consequently, P-ALA exhibited much improved in vivo antitumor efficacy without any apparent side effects. We thus anticipate the application of P-ALA as a nano-designed photosensitizer for anticancer photodynamic therapy.

The development of a high-affinity conformation-sensitive antibody mimetic using a biocompatible copolymer carrier (iBody).

Peptide display methods are a powerful tool for discovering new ligands of pharmacologically relevant targets. However, the selected ligands often suffer from low affinity. Using phage display, we identified a new bicyclic peptide binder of prostate-specific membrane antigen (PSMA), a metalloprotease frequently overexpressed in prostate cancer. We show that linking multiple copies of a selected low-affinity peptide to a biocompatible water-soluble N-(2-hydroxypropyl)methacrylamide copolymer carrier (iBody) improved binding of the conjugate by several orders of magnitude. Furthermore, using ELISA, enzyme kinetics, confocal microscopy, and other approaches, we demonstrate that the resulting iBody can distinguish between different conformations of the target protein. The possibility to develop stable, fully synthetic, conformation-selective antibody mimetics has potential applications for molecular recognition, diagnosis and treatment of many pathologies. This strategy could significantly contribute to more effective drug discovery and design.

Correction: MCC950/CRID3 potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.

[This corrects the article DOI: 10.1371/journal.pbio.3000354.].

Simultaneous Delivery of Doxorubicin and Protease Inhibitor Derivative to Solid Tumors via Star-Shaped Polymer Nanomedicines Overcomes P-gp- and STAT3-Mediated Chemoresistance.

The derivative of protease inhibitor ritonavir (5-methyl-4-oxohexanoic acid ritonavir ester; RD) was recently recognized as a potent P-gp inhibitor and cancerostatic drug inhibiting the proteasome and STAT3 signaling. Therefore, we designed high-molecular-weight HPMA copolymer conjugates with a PAMAM dendrimer core bearing both doxorubicin (Dox) and RD (Star-RD + Dox) to increase the circulation half-life to maximize simultaneous delivery of Dox and RD into the tumor. Star-RD inhibited P-gp activity, potently sensitizing both low- and high-P-gp-expressing cancer cells to the cytostatic and proapoptotic activity of Dox in vitro. Star-RD + Dox possessed higher cytostatic and proapoptotic activities compared to Star-Dox and the equivalent mixture of Star-Dox and Star-RD in vitro. Star-RD + Dox efficiently inhibited STAT3 signaling and induced caspase-3 activation and DNA fragmentation in cancer cells in vivo. Importantly, Star-RD + Dox was found to have superior antitumor activity in terms of tumor growth inhibition and increased survival of mice bearing P-gp-expressing tumors.

Singlet oxygen phosphorescence detection in vivo identifies PDT-induced anoxia in solid tumors.

Real-time surveillance of photodynamic therapy (PDT) has been desired by the research community for a long time. The impact of the treatment is encoded in the phosphorescence kinetics of its main mediator: singlet oxygen. We report successful in vivo measurements of these weak kinetics through the skin of living mice after systemic drug application. Using special high transmission optics centered around 1200, 1270 and 1340 nm, singlet oxygen phosphorescence can be clearly discriminated from other signals. N-(2-Hydroxypropyl)methacrylamide copolymers conjugated with pyropheophorbide-a exhibit highly selective accumulation in tumors. Signals of this drug in tumors were compared to those in normal tissue. In both places, the major part of the signal could be identified as arising from drug still circulating in the bloodstream. Despite high concentrations of extravasated drug in the tumors due to the EPR effect, nearly no signal could be detected from these photosensitizers in vivo, contradicting in vitro experiments. We propose that the reason for this discrepancy is oxygen depletion in tumor tissue in vivo, even at moderate (at PDT scale) illumination intensities, soon after the start of the illumination. These results underline the importance of singlet oxygen surveillance during PDT treatment.

Inhibitor-GCPII Interaction: Selective and Robust System for Targeting Cancer Cells with Structurally Diverse Nanoparticles.

Glutamate carboxypeptidase II (GCPII) is a membrane protease overexpressed by prostate cancer cells and detected in the neovasculature of most solid tumors. Targeting GCPII with inhibitor-bearing nanoparticles can enable recognition, imaging, and delivery of treatments to cancer cells. Compared to methods based on antibodies and other large biomolecules, inhibitor-mediated targeting benefits from the low molecular weight of the inhibitor molecules, which are typically stable, easy-to-handle, and able to bind the enzyme with very high affinity. Although GCPII is established as a molecular target, comparing previously reported results is difficult due to the different methodological approaches used. In this work, we investigate the robustness and limitations of GCPII targeting with a diverse range of inhibitor-bearing nanoparticles (various structures, sizes, bionanointerfaces, conjugation chemistry, and surface densities of attached inhibitors). Polymer-coated nanodiamonds, virus-like particles based on bacteriophage Qß and mouse polyomavirus, and polymeric poly(HPMA) nanoparticles with inhibitors attached by different means were synthesized and characterized. We evaluated their ability to bind GCPII and interact with cancer cells using surface plasmon resonance, inhibition assay, flow cytometry, and confocal microscopy. Regardless of the diversity of the investigated nanosystems, they all strongly interact with GCPII (most with low picomolar Ki values) and effectively target GCPII-expressing cells. The robustness of this approach was limited only by the quality of the nanoparticle bionanointerface, which must be properly designed by adding a sufficient density of hydrophilic protective polymers. We conclude that the targeting of cancer cells overexpressing GCPII is a viable approach transferable to a broad diversity of nanosystems.

N-(2-Hydroxypropyl)methacrylamide-Based Linear, Diblock, and Starlike Polymer Drug Carriers: Advanced Process for Their Simple Production.

We developed a new simplified method for the synthesis of well-defined linear, diblock, or starlike N-(2-hydroxypropyl)methacrylamide (HPMA)-based polymer drug carriers using controlled reversible addition-fragmentation chain transfer polymerization. The prepared monodispersed polymers are after the drug attachment intended for enhanced anticancer therapy. This new approach significantly reduces the number of required synthetic steps and minimizes the consumption of organic solvents during the synthesis. As a result, highly defined linear, diblock, and starlike copolymers designed for pH-triggered drug activation/release in tumor tissue were formed in sufficient amounts for further physicochemical and biological studies. Within the synthesis, we also developed a new procedure for the selective deprotection of tert-butoxycarbonyl hydrazide and amine groups on hydrophilic HPMA copolymers, including the one-pot removal of polymer end groups. We studied and described in detail the kinetics and efficacy of the deprotection reaction. We believe the simplified synthetic approach facilitates the preparation of polymer conjugates bound by the pH-sensitive hydrazone bond and their application in tumor treatment.

Targeted Drug Delivery with Polymers and Magnetic Nanoparticles: Covalent and Noncovalent Approaches, Release Control, and Clinical Studies.

Targeted delivery combined with controlled drug release has a pivotal role in the future of personalized medicine. This review covers the principles, advantages, and drawbacks of passive and active targeting based on various polymer and magnetic iron oxide nanoparticle carriers with drug attached by both covalent and noncovalent pathways. Attention is devoted to the tailored conjugation of targeting ligands (e.g., enzymes, antibodies, peptides) to drug carrier systems. Similarly, the approaches toward controlled drug release are discussed. Various polymer-drug conjugates based, for example, on polyethylene glycol (PEG), N-(2-hydroxypropyl)methacrylamide (HPMA), polymeric micelles, and nanoparticle carriers are explored with respect to absorption, distribution, metabolism, and excretion (ADME scheme) of administrated drug. Design and structure of superparamagnetic iron oxide nanoparticles (SPION) and condensed magnetic clusters are classified according to the mechanism of noncovalent drug loading involving hydrophobic and electrostatic interactions, coordination chemistry, and encapsulation in porous materials. Principles of covalent conjugation of drugs with SPIONs including thermo- and pH-degradable bonds, amide linkage, redox-cleavable bonds, and enzymatically-cleavable bonds are also thoroughly described. Finally, results of clinical trials obtained with polymeric and magnetic carriers are analyzed highlighting the potential advantages and future directions in targeted anticancer therapy.

iBodies: Modular Synthetic Antibody Mimetics Based on Hydrophilic Polymers Decorated with Functional Moieties.

Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named "iBodies", consist of an HPMA copolymer decorated with low-molecular-weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live-cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand.

Highly Effective Synthetic Polymer-Based Blockers of Non-Specific Interactions in Immunochemical Analyses.

In vitro diagnostic methods face non-specific interactions increasing their background level and influencing the efficacy and reproducibility. Currently, the most important and employed blocker of non-specific interactions is bovine serum albumin (BSA), an animal product with some disadvantages like its batch-to-batch variability and contamination with RNases. Herein, we developed amphiphilic water-soluble synthetic copolymers based on the highly biocompatible, non-immunogenic and nontoxic N-2-(hydroxypropyl)methacrylamide (HPMA)-based copolymers or poly(oxazoline)s as highly effective synthetic blockers of non-specific interactions and an effective BSA alternative. The highest blocking capacity was observed for HPMA-based polymers containing two hydrophobic anchors taking advantage of the combination of two structurally different hydrophobic molecules. Polymers prepared by free radical polymerisation with broader dispersity were slightly better in terms of surface covering. The sandwich ELISA evaluating human thyroid-stimulating Hormone in patient samples revealed that the designed polymers can fully replace BSA without compromising the assay results. Importantly, as a fully synthetic material, the developed polymers are fully animal pathogen-free; thus, they are highly important materials for further development.

Polymer-Tethered Quenched Fluorescent Probes for Enhanced Imaging of Tumor-Associated Proteases.

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become the standard of care in cancer surgeries. One of the key parameters to optimize in contrast agents is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker, as well as the positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased overall signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

Polymer-tethered quenched fluorescent probes for enhanced imaging of tumor associated proteases.

Fluorescence-based contrast agents enable real-time detection of solid tumors and their neovasculature, making them ideal for use in image-guided surgery. Several agents have entered late-stage clinical trials or secured FDA approval, suggesting they are likely to become standard of care in cancer surgeries. One of the key parameters to optimize in contrast agent is molecular size, which dictates much of the pharmacokinetic and pharmacodynamic properties of the agent. Here, we describe the development of a class of protease-activated quenched fluorescent probes in which a N-(2-hydroxypropyl)methacrylamide copolymer is used as the primary scaffold. This copolymer core provides a high degree of probe modularity to generate structures that cannot be achieved with small molecules and peptide probes. We used a previously validated cathepsin substrate and evaluated the effects of length and type of linker as well as positioning of the fluorophore/quencher pair on the polymer core. We found that the polymeric probes could be optimized to achieve increased over-all signal and tumor-to-background ratios compared to the reference small molecule probe. Our results also revealed multiple structure-activity relationship trends that can be used to design and optimize future optical imaging probes. Furthermore, they confirm that a hydrophilic polymer is an ideal scaffold for use in optical imaging contrast probes, allowing a highly modular design that enables efficient optimization to maximize probe accumulation and overall biodistribution properties.

Singlet Oxygen In Vivo: It Is All about Intensity-Part 2.

Recently, we reported induced anoxia as a limiting factor for photodynamic tumor therapy (PDT). This effect occurs in vivo if the amount of generated singlet oxygen that undergoes chemical reactions with cellular components exceeds the local oxygen supply. The amount of generated singlet oxygen depends mainly on photosensitizer (PS) accumulation, efficiency, and illumination intensity. With illumination intensities above a certain threshold, singlet oxygen is limited to the blood vessel and the nearest vicinity; lower intensities allow singlet oxygen generation also in tissue which is a few cell layers away from the vessels. While all experiments so far were limited to light intensities above this threshold, we report experimental results for intensities at both sides of the threshold for the first time, giving proof for the described model. Using time-resolved optical detection in NIR, we demonstrate characteristic, illumination intensity-dependent changes in signal kinetics of singlet oxygen and photosensitizer phosphorescence in vivo. The described analysis allows for better optimization and coordination of PDT drugs and treatment, as well as new diagnostic methods based on gated PS phosphorescence, for which we report a first in vivo feasibility test.

Polymer theranostics with multiple stimuli-based activation of photodynamic therapy and tumor imaging.

Background: Efficient theranostic strategies concurrently bring and use both the therapeutic and diagnostic features, serving as a cutting-edge tool to combat advanced cancers. Goals of the Investigation: Here, we develop stimuli-sensitive theranostics consisting of tailored copolymers forming micellar conjugates carrying pyropheophorbide-a (PyF) attached by pH-sensitive hydrazone bonds, thus enabling the tumor microenvironment-sensitive activation of the photodynamic therapy (PDT) effect, fluorescence or phosphorescence. Results: The nanomedicines show superior anti-tumor PDT efficacy and huge tumor-imaging potential, while reducing their accumulation, and potentially side effects, in the liver and spleen. The developed theranostics exhibit clear selective tumor accumulation at high levels in the mouse sarcoma S180 tumor model with almost no PyF found in the healthy tissues after 48 h. Once in the tumor, illumination at λexc = 420 nm reaches the therapeutic effect due to the 1O2 generation. Indeed, an almost complete inhibition of tumor growth is observed up to 18 days after the treatment. Conclusion: The clear benefit of the specific PyF release and activation in the acidic tumor environment for the targeted delivery and tissue distribution dynamics was proved. Conjugates carrying pyropheophorbide-a (PyF) attached by pH-sensitive hydrazone bonds showed their excellent antitumor PDT effect and its applicability as advanced theranostics at very low dose of PyF.

Tumor-specific targeting of polymer drug delivery systems with recombinant proteins bound via tris(nitrilotriacetic acid).

Antibody-mediated targeting is an efficient strategy to enhance the specificity and selectivity of polymer nanomedicines towards the target site, typically a tumor. However, direct covalent coupling of an antibody with a polymer usually results in a partial damage of the antibody binding site accompanied with a compromised biological activity. Here, an original solution based on well-defined non-covalent interactions between tris-nitrilotriacetic acid (trisNTA) and hexahistidine (His-tag) groups, purposefully introduced to the structure of each macromolecule, is described. Specifically, trisNTA groups were attached along the chains of a hydrophilic statistical copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA), and at the end or along the chains of thermo-responsive di-block copolymers based on N-isopropylmethacrylamide (NIPMAM) and HPMA; His-tag was incorporated to the structure of a recombinant single chain fragment of an anti-GD2 monoclonal antibody (scFv-GD2). Static and dynamic light scattering analyses confirmed that mixing of polymer with scFv-GD2 led to the formation of polymer/scFv-GD2 complexes; those prepared from thermo-responsive polymers formed stable micelles at 37 °C. Flow cytometry and fluorescence microscopy clearly demonstrated antigen-specific binding of the prepared complexes to GD2 positive murine T-cell lymphoma cells EL-4 and human neuroblastoma cells UKF-NB3, while no interaction with GD2 negative murine fibroblast cells NIH-3T3 was observed. These non-covalent polymer protein complexes represent a new generation of highly specific actively targeted polymer therapeutics or diagnostics.

Singlet Oxygen In Vivo: It Is All about Intensity.

The presented work addresses the influence of illumination intensity on the amount and locations of singlet oxygen generation in tumor tissue. We used time-resolved optical detection at the typical emission wavelength around 1270 nm and at 1200 nm where there is no singlet oxygen phosphorescence to determine the phosphorescence kinetics. The discussed data comprise in vivo measurements in tumor-laden HET-CAM and mice. The results show that illumination that is too intense is a major issue, affecting many PDT treatments and all singlet oxygen measurements in vivo so far. In such cases, photosensitization and oxygen consumption exceed oxygen supply, limiting singlet oxygen generation to the blood vessels and walls, while photosensitizers in the surrounding tissue will likely not participate. Being a limitation for the treatment, on one hand, on the other, this finding offers a new method for tumor diagnosis when using photosensitizers exploiting the EPR effect. In contrast to high-intensity PDT, some papers reported successful treatment with nanoparticular drugs using much lower illumination intensity. The question of whether, with such illumination, singlet oxygen is indeed generated in areas apart from vessels and walls, is addressed by numerical analysis. In addition, we discuss how to perform measurements at such low intensities.

Tumor Stimulus-Responsive Biodegradable Diblock Copolymer Conjugates as Efficient Anti-Cancer Nanomedicines.

Biodegradable nanomedicines are widely studied as candidates for the effective treatment of various cancerous diseases. Here, we present the design, synthesis and evaluation of biodegradable polymer-based nanomedicines tailored for tumor-associated stimuli-sensitive drug release and polymer system degradation. Diblock polymer systems were developed, which enabled the release of the carrier drug, pirarubicin, via a pH-sensitive spacer allowing for the restoration of the drug cytotoxicity solely in the tumor tissue. Moreover, the tailored design enables the matrix-metalloproteinases- or reduction-driven degradation of the polymer system into the polymer chains excretable from the body by glomerular filtration. Diblock nanomedicines take advantage of an enhanced EPR effect during the initial phase of nanomedicine pharmacokinetics and should be easily removed from the body after tumor microenvironment-associated biodegradation after fulfilling their role as a drug carrier. In parallel with the similar release profiles of diblock nanomedicine to linear polymer conjugates, these diblock polymer conjugates showed a comparable in vitro cytotoxicity, intracellular uptake, and intratumor penetration properties. More importantly, the diblock nanomedicines showed a remarkable in vivo anti-tumor efficacy, which was far more superior than conventional linear polymer conjugates. These findings suggested the advanced potential of diblock polymer conjugates for anticancer polymer therapeutics.

Removable nanocoatings for siRNA polyplexes.

To assist in overcoming the inherent instability of nucleic acid-containing polyplexes in physiological solutions, we have here set out to develop removable nanocoatings for modifying the surface of siRNA-based nanoparticles. Here, N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymers containing carbonylthiazolidine-2-thione (TT) reactive groups in their side chains bound via disulfide spacers to the polymeric backbone were synthesized, and these copolymers were used to coat the surface of polyplexes formed by the self-assembly of anti-Luciferase siRNA with the polycations polyethylene imine (PEI) and poly(HPMA)-grafted poly(l-lysine) (GPL). The coating process was monitored by analyzing changes in the weight-average molecular weight (M(w)), the hydrodynamic radius (R(h)), and the zeta-potential (ζ) of the polyplexes, using both static (SLS) and dynamic (DLS) light scattering methods. The outlined methods resulted in the attachment of, on average, 28 polymer molecules to the surface of the polyplexes, forming a ∼5-nm-thick hydrophilic stealth coating. Initial efforts to develop RGD-targeted coated polyplexes are also described. Atomic force microscopy (AFM) showed an angular polyplex structure and confirmed the narrow size distribution of the coated nanoparticles. The stability of the polymer-coated and uncoated polyplexes was evaluated by gel electrophoresis and by turbidity measurements, and it was found that modifying the surface of the siRNA-containing polyplexes substantially improved their stability in physiological solutions. Using polymer-coated GPL-based polyplexes containing anti-Luciferase siRNA, we finally also obtained some initial proof-of-principle for time- and concentration-dependent target-specific gene silencing, suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.