Development of Highly Effective Anti-Mesothelin hYP218 Chimeric Antigen Receptor T Cells With Increased Tumor Infiltration and Persistence for Treating Solid Tumors.

Mesothelin targeting CAR T cells have limited activity in patients. In this study, we sought to determine if efficacy of anti-mesothelin CAR T cells is dependent on the mesothelin epitopes that are recognized by them. To do so, we developed hYP218 (against membrane-proximal epitope) and SS1 (against membrane-distal epitope) CAR T cells. Their efficacy was assessed in vitro using mesothelin-positive tumor cell lines and in vivo in NSG mice with mesothelin-expressing ovarian cancer (OVCAR-8), pancreatic cancer (KLM-1), and mesothelioma patient-derived (NCI-Meso63) tumor xenografts. Persistence and tumor infiltration of CAR T cells was determined using flow cytometry. hYP218 CAR T cells killed cancer cells more efficiently than SS1 CAR T cells, with a two- to fourfold lower ET50 value (effector-to-target ratio for 50% killing of tumor cells). In mice with established tumors, single intravenous administration of hYP218 CAR T cells lead to improved tumor response and survival compared with SS1 CAR T cells, with complete regression of OVCAR-8 and NCI-Meso63 tumors. Compared with SS1 CAR T cells, there was increased peripheral blood expansion, persistence, and tumor infiltration of hYP218 CAR T cells in the KLM-1 tumor model. Persistence of hYP218 CAR T cells in treated mice led to antitumor immunity when rechallenged with KLM-1 tumor cells. Our results show that hYP218 CAR T cells, targeting mesothelin epitope close to cell membrane, are very effective against mesothelin-positive tumors and are associated with increased persistence and tumor infiltration. These results support its clinical development to treat patients with mesothelin-expressing cancers.

Degradation-Dependent Protein Release from Enzyme Sensitive Injectable Glycol Chitosan Hydrogel.

Glycol chitosan (GC) is a hydrophilic chitosan derivative, known for its aqueous solubility. Previously, we have demonstrated the feasibility of preparing injectable, enzymatically crosslinked hydrogels from HPP [3-(4-Hydroxyphenyl)-propionic acid (98%)]-modified GC. However, HPP-GC gels showed very slow degradation, which presents challenges as an in vivo protein delivery vehicle. This study reports the potential of acetylated HPP-GC hydrogels as a biodegradable hydrogel platform for sustained protein delivery. Enzymatic crosslinking was used to prepare injectable, biodegradable hydrogels from HPP-GC with various degrees of acetylation (DA). The acetylated polymers were characterized using Fourier transform infrared and nuclear magnetic resonance spectroscopy. Rheological methods were used to characterize the mechanical behavior of the hydrogels. In vitro degradation and protein release were performed in the presence and absence of lysozyme. In vivo degradation was studied using a mouse subcutaneous implantation model. Finally, two hydrogel formulations with distinct in vitro/in vivo degradation and in vitro protein release were evaluated in 477-SKH1-Elite mice using live animal imaging to understand in vivo protein release profiles. The lysozyme-mediated degradation of the gels was demonstrated in vitro and the degradation rate was found to be dependent on the DA of the polymers. In vivo degradation study further confirmed that gels formed from polymers with higher DA degraded faster. In vitro protein release demonstrated the feasibility to achieve lysozyme-mediated protein release from the gels and that the rate of protein release can be modulated by varying the DA. In vivo protein release study further confirmed the feasibility to achieve differential protein release by varying the DA. The feasibility to develop degradable enzymatically crosslinked GC hydrogels is demonstrated. Gels with a wide spectrum of degradation time ranging from less than a week and more than 6 weeks can be developed using this approach. The study also showed the feasibility to fine tune in vivo protein release by modulating HPP-GC acetylation. The hydrogel platform therefore holds significant promise as a protein delivery vehicle for various biomedical and regenerative engineering applications. Impact statement The study describes the feasibility to develop a novel enzyme sensitive biodegradable and injectable hydrogel, where in the in vivo degradation rate and protein release profile can be modulated over a wide range. The described hydrogel platform has the potential to develop into a clinically relevant injectable and tunable protein delivery vehicle for a wide range of biomedical applications.

Sensitivity of Mesothelioma Cells to PARP Inhibitors Is Not Dependent on BAP1 but Is Enhanced by Temozolomide in Cells With High-Schlafen 11 and Low-O6-methylguanine-DNA Methyltransferase Expression.

INTRODUCTION: BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase thought to be involved in DNA double-strand break repair, is frequently mutated in mesothelioma. Because poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPIs) induce synthetic lethality in BRCA1/2 mutant cancers, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesothelioma and if combination therapy with temozolomide (TMZ) would be beneficial. METHODS: A total of 10 patient-derived mesothelioma cell lines were generated and characterized for BAP1 mutation status, protein expression, nuclear localization, and sensitivity to the PARPIs, olaparib, and talazoparib, alone or in combination with TMZ. BAP1 deubiquitinase (DUB) activity was evaluated by ubiquitin with 7-amido-4-methylcoumarin assay. BAP1 knockout mesothelioma cell lines were generated by CRISPR-Cas9. Because Schlafen 11 (SLFN11) and O6-methylguanine-DNA methyltransferase also drive response to TMZ and PARPIs, we tested their expression and relationship with drug response. RESULTS: BAP1 mutations or copy-number alterations, or both were present in all 10 cell lines. Nonetheless, four cell lines exhibited intact DUB activity and two had nuclear BAP1 localization. Half maximal-inhibitory concentrations of olaparib and talazoparib ranged from 4.8 µM to greater than 50 µM and 0.039 µM to greater than 5 µM, respectively, classifying them into sensitive (two) or resistant (seven) cells, independent of their BAP1 status. Cell lines with BAP1 knockout resulted in the loss of BAP1 DUB activity but did not increase sensitivity to talazoparib. Response to PARPI tended to be associated with high SLFN11 expression, and combination with temozolomide increased sensitivity of cells with low or no MGMT expression. CONCLUSIONS: BAP1 status does not determine sensitivity to PARPIs in patient-derived mesothelioma cell lines. Combination of PARPI with TMZ may be beneficial for patients whose tumors have high SLFN11 and low or no MGMT expression.

Injectable nanocomposite analgesic delivery system for musculoskeletal pain management.

Musculoskeletal pain is a major health issue which results from surgical procedures (i.e. total knee and/or hip replacements and rotator cuff repairs), as well as from non-surgical conditions (i.e. sympathetically-mediated pain syndrome and occipital neuralgia). Local anesthetics, opioids or corticosteroids are currently used for the pain management of musculoskeletal conditions. Even though local anesthetics are highly preferred, the need for multiple administration presents significant disadvantages. Development of unique delivery systems that can deliver local anesthetics at the injection site for prolonged time could significantly enhance the therapeutic efficacy and patient comfort. The goal of the present study is to evaluate the efficacy of an injectable local anesthetic nanocomposite carrier to provide sustained analgesic effect. The nanocomposite carrier was developed by encapsulating ropivacaine, a local anesthetic, in lipid nanocapsules (LNC-Rop), and incorporating the nanocapsules in enzymatically crosslinked glycol chitosan (0.3GC) hydrogels. Cryo Scanning Electron Microscopic (Cryo SEM) images showed the ability to distribute the LNCs within the hydrogel without adversely affecting their morphology. The study demonstrated the feasibility to achieve sustained release of lipophilic molecules from the nanocomposite carrier in vitro and in vivo. A rat chronic constriction injury (CCI) pain model was used to evaluate the efficacy of the nanocomposite carrier using thermal paw withdrawal latency (TWL). The nanocomposite carriers loaded with ropivacaine and dexamethasone showed significant improvement in pain response compared to the control groups for at least 7 days. The study demonstrated the clinical potential of these nanocomposite carriers for post-operative and neuropathic pain. STATEMENT OF SIGNIFICANCE: Acute or chronic pain associated with musculoskeletal conditions is considered a major health issue, with healthcare costs totaling several billion dollars. The opioid crisis presents a pressing clinical need to develop alternative and effective approaches to treat musculoskeletal pain. The goal of this study was to develop a long-acting injectable anesthetic formulation which can sustain a local anesthetic effect for a prolonged time. This in turn could increase the quality of life and rehabilitation outcome of patients, and decrease opioid consumption. The developed injectable nanocomposite demonstrated the feasibility to achieve prolonged pain relief in a rat chronic constriction injury (CCI) model.

Nanostructures for the Inhibition of Viral Infections.

Multivalent interactions are omnipresent in biology and confer biological systems with dramatically enhanced affinities towards different receptors. Such multivalent binding interactions have lately been considered for the development of new therapeutic strategies against bacterial and viral infections. Multivalent polymers, dendrimers, and liposomes have successfully targeted pathogenic interactions. While a high synthetic effort was often needed for the development of such therapeutics, the integration of multiple ligands onto nanostructures turned to be a viable alternative. Particles modified with multiple ligands have the additional advantage of creating a high local concentration of binding molecules. This review article will summarize the different nanoparticle-based approaches currently available for the treatment of viral infections.

Toward multifunctional "clickable" diamond nanoparticles.

Nanodiamonds (NDs) are among the most promising new carbon based materials for biomedical applications, and the simultaneous integration of various functions onto NDs is an urgent necessity. A multifunctional nanodiamond based formulation is proposed here. Our strategy relies on orthogonal surface modification using different dopamine anchors. NDs simultaneously functionalized with triethylene glycol (EG) and azide (-N3) functions were fabricated through a stoichiometrically controlled integration of the dopamine ligands onto the surface of hydroxylated NDs. The presence of EG functionalities rendered NDs soluble in water and biological media, while the -N3 group allowed postsynthetic modification of the NDs using "click" chemistry. As a proof of principle, alkynyl terminated di(amido amine) ligands were linked to these ND particles.

Inhibition of type 1 fimbriae-mediated Escherichia coli adhesion and biofilm formation by trimeric cluster thiomannosides conjugated to diamond nanoparticles.

Recent advances in nanotechnology have seen the development of a number of microbiocidal and/or anti-adhesive nanoparticles displaying activity against biofilms. In this work, trimeric thiomannoside clusters conjugated to nanodiamond particles (ND) were targeted for investigation. NDs have attracted attention as a biocompatible nanomaterial and we were curious to see whether the high mannose glycotope density obtained upon grouping monosaccharide units in triads might lead to the corresponding ND-conjugates behaving as effective inhibitors of E. coli type 1 fimbriae-mediated adhesion as well as of biofilm formation. The required trimeric thiosugar clusters were obtained through a convenient thiol-ene "click" strategy and were subsequently conjugated to alkynyl-functionalized NDs using a Cu(I)-catalysed "click" reaction. We demonstrated that the tri-thiomannoside cluster-conjugated NDs (ND-Man3) show potent inhibition of type 1 fimbriae-mediated E. coli adhesion to yeast and T24 bladder cells as well as of biofilm formation. The biofilm disrupting effects demonstrated here have only rarely been reported in the past for analogues featuring such simple glycosidic motifs. Moreover, the finding that the tri-thiomannoside cluster (Man3N3) is itself a relatively efficient inhibitor, even when not conjugated to any ND edifice, suggests that alternative mono- or multivalent sugar-derived analogues might also be usefully explored for E. coli-mediated biofilm disrupting properties.

Boronic acid-modified lipid nanocapsules: a novel platform for the highly efficient inhibition of hepatitis C viral entry.

The search for viral entry inhibitors that selectively target viral envelope glycoproteins has attracted increasing interest in recent years. Amongst the handful of molecules reported to show activity as hepatitis C virus (HCV) entry inhibitors are a variety of glycan-binding proteins including the lectins, cyanovirin-N (CV-N) and griffithsin. We recently demonstrated that boronic acid-modified nanoparticles are able to reduce HCV entry through a similar mechanism to that of lectins. A major obstacle to any further development of these nanostructures as viral entry inhibitors is their only moderate maximal inhibition potential. In the present study, we report that lipid nanocapsules (LNCs), surface-functionalized with amphiphilic boronic acid (BA) through their post-insertion into the semi-rigid shell of the LNCs, are indeed far superior as HCV entry inhibitors when compared with previously reported nanostructures. These 2(nd) generation particles (BA-LNCs) are shown to prevent HCV infection in the micromolar range (IC50 = 5.4 µM of BA moieties), whereas the corresponding BA monomers show no significant effects even at the highest analyzed concentration (20 µM). The new BA-LNCs are the most promising boronolectin-based HCV entry inhibitors reported to date and are thus observed to show great promise in the development of a pseudolectin-based therapeutic agent.

Phenylboronic-acid-modified nanoparticles: potential antiviral therapeutics.

Phenylboronic-acid-modified nanoparticles (NPs) are attracting considerable attention for biological and biomedical applications. We describe here a convenient and general protocol for attaching multiple copies of para-substituted phenylboronic acid moieties onto either iron-oxide-, silica- or diamond-derived NPs. The boronic acid functionalized NPs are all fabricated by first modifying the surface of each particle type with 4-azidobenzoic ester functions. These azide-terminated nanostructures were then reacted with 4-[1-oxo-4-pentyn-1-yl) amino]phenylboronic acid units via a Cu(I) catalyzed Huisgen cycloaddition to furnish, conveniently, the corresponding boronic-acid modified NPs (or "borono-lectins") targeted in this work. The potential of these novel "borono-lectins" as antiviral inhibitors was investigated against the Hepatitis C virus (HCV) exploiting a bioassay that measures the potential of drugs to interfere with the ability of cell-culture-derived JFH1 virus particles to infect healthy hepatocytes. As far as we are aware, this is the first report that describes NP-derived viral entry inhibitors and thus serves as a "proof-of-concept" study. The novel viral entry activity demonstrated, and the fact that the described boronic-acid-functionalized NPs all display much reduced cellular toxicities compared with alternate NPs, sets the stage for their further investigation. The data supports that NP-derived borono-lectins should be pursued as a potential therapeutic strategy for blocking viral entry of HCV.