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Bellato, Federica (2019) Targeting dendritic cells with mannosylated cationic glycopolymers for nucleic acid-mediated cancer immunotherapy. [Ph.D. thesis]

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Abstract (italian or english)

Tumour immunology has been changing the landscape of modern medicine and anticancer therapy, by developing a variety of tumor antigenic peptide-based and nucleic acid-based treatments. Among the several nucleic acid-based therapeutics developed so far, short single-stranded synthetic DNA (ssDNA) molecules, known as CpG oligodeoxynucleotides (ODNs), was found to stimulate B cells and plasmacytoid dendritic cells (pDCs) and activate both innate and adaptative immune responses, thus raising interest for infectious diseases, cancers, and allergy treatment.1 Furthermore, delivery of pDNA or mRNA encoding Tumor Associated Antigens (TAAs) to Dendritic Cells (DCs) is an emerging and promising alternative to the delivery of the parent antigenic peptide for antitumor vaccination, because they have shown to be safer and to provide a higher protein expression upon internalization by Antigen Presenting Cells (APCs).2 Nucleic acids uptake by APCs results in the translation in antigens that are presented through the Major Histocompatibility Complex (MHC) to the immune system cells. This strategy allows to trigger both a direct immune response and a long-term memory against cancer cells. To date, lipid-based systems and viral vectors have dominated the nucleic acid delivery scenario. However, recently synthetic positively charged polymeric carriers have drawn increasing attention, since they provide opportunities for improved safety, greater flexibility and more facile manufacturing while preventing the enzymatic degradation of genetic material and mediating its cellular uptake. The specific term “polyplex” has been coined to identify the supramolecular structures derived from the electrostatic interactions between those polymers and nucleic acids.
In this PhD project, Reversible Addition Fragmentation chain Transfer (RAFT) polymerization3 has been exploited for the synthesis of two generations of diblock and triblock copolymers designed to deliver pDNA encoding TAAs to antigen presenting cells and to trigger the direct immune response and a long-term memory against cancer. The 1st generation of these novel materials was designed with a poly-cationic agmatine acrylamide block to condense oligonucleotides, and with a mannosylated block to actively and selectively target Mannose Receptor (MR)4 expressed on DCs. Furthermore, a 2nd generation of polymers was further engineered by adding a butyl acrylate-based hydrophobic block with the aim of enhancing the endosomal escape properties of this family of carriers. A small library of three cationic di-block copolymers (Man15-b-Agm12, Man29-b-Agm25 and Man58-b-Agm45) and two tri-block copolymers (Man29-b-Agm29-b-But9 and Man62-b-Agm52-b-But32) was obtained via fast RAFT polymerization using D-Mannose acrylamide (Man), Agmatine acrylamide (Agm), and butyl acrylate (But) as monomers. Polymers were generated using identical monomer feed ratios but increasing total monomers units (corresponding to different final polymer molecular weights) to elucidate the effect of the polymer length on nucleic acids complexation and delivery to cells, and glycopolyplexes (GPPs) stability.
Initially, a short single strand DNA (ssDNA, 19 bases) as model for CpG ODN, was used to generate the GPPs. The optimum nitrogen to phosphate (N/P) ratio to achieve complete ssDNA complexation was evaluated by gel electrophoresis. Furthermore, DLS and TEM characterization confirmed the assembly and the narrow size distribution in the range of 25-55 nm, which was maintained over time also in the presence of serum proteins. The strong electrostatic interactions between polymers and ssDNA were confirmed by the Zeta potential reduction of 4-11 mV for 1st generation and of 11-18 mV for the 2nd generation polymers after ssDNA complexation. The association strength and the stability of polymers/ssDNA glycopolyplexes was investigated by the heparin displacement assay. All tested systems required a concentration of heparin at least 6-fold higher than its physiological concentration in the blood (0.15 IU/mL) to induce total ssDNA displacement, with di-block co-polymers retaining ssDNA more strongly as compared to the tri-blocks. Furthermore, polymers ability to cause red blood cells lysis ex vivo was correlated with their potential to induce endosomal membrane disruption. Particularly, the capacity of the butyl acrylate-based hydrophobic portion to induce endosomolysis under early endosomes mimicking conditions (pH 6) was confirmed by the higher percentage of induced hemolysis for Man29-b-Agm29-b-But9 and Man62-b-Agm52-b-But32 respect to 1st generation polymers. However, a good compromise between biocompatibility and endosomolytic activity of our systems was confirmed by in vitro viability assay performed with the polyplexes in the polymer concentrations range 0.005-0.2 mg/mL. In vitro uptake studies were performed either on model cell lines CHO (Chinese hamster ovary cells, wild type) and CHO-MR+ (Mannose Receptor expressing CHO cells) or on immortalized dendritic cells DC2.4. The internalization of glycopolyplexes loaded with Cy3-ssDNA was evaluated by flow cytometry after 30 minutes or 1 hour of incubation with cells. As expected, a certain degree of selectivity in the recognition of mannose receptor-expressing cells by the GPPs was observed after 30 minutes of incubation, with the 1st generation low molecular weights polymers outperforming (around 4.5-fold increase in the uptake with respect to free ssDNA) the high molecular weight (2.5-fold increase in the uptake with respect to free ssDNA) and the butyl block-bearing polymers (1.5-fold increase in the uptake with respect to free ssDNA).
The successful complexation and delivery of high molecular weight nucleic acid by the glycopolymers was a key for transfection capabilities. 1st and 2nd generation glycopolymers were used to complex a model plasmid DNA encoding Enhanced Green Fluorescent Protein (pEGFP, 4759 bp). Agarose gel electrophoresis showed that all glycopolymers form stable complexes with pEGFP at relatively low N/P ratios of 5 for Man15-b-Agm12/pEGFP and Man29-b-Agm25/pEGFP, and 2.5 for Man58-b-Agm45/pEGFP, Man29-b-Agm29-b-But9/pEGFP and Man62-b-Agm52-b-But32/pEGFP. The stability of the systems in presence of poly-anionic competitive agents was also investigated, showing the release of pDNA at 1 IU/mL heparin concentration, which is 6-fold above its physiological concentration. The DLS and TEM analysis of the resulting GPPs showed the presence of mixed rod- and toroidal- shaped complexes with a size in the range 100-1000 nm. Moreover, in vitro transfection efficiency was evaluated on both CHO/CHO-MR+ and immortalized dendritic cell lines at a selected nitrogen to phosphorous ratio for each polymer. Flow cytometry and confocal microscopy pointed out that Man29-b-Agm29-b-But9/pEGFP, Man62-b-Agm52-b-But32/pEGFP and Man58-b-Agm45/pEGFP complexes yielded the best transfection performance. In light of these evidences, these materials were selected for further investigation on dendritic cells, using plasmid encoding full-length ovalbumin (pOVA), a well-known immunogenic protein, to trigger an in vitro immune response against it. The immunological studies highlighted that the expression on DCs of the costimulatory clusters of differentiation CD86, necessary for T cell activation, was increased by cell incubation with Man58-b-Agm45/pOVA and Man62-b-Agm52-b-But32/pOVA GPPs. Furthermore, these two GPPs were found to induce a slightly higher presentation of the SIINFEKL ovalbumin antigenic epitope by MHC I complex with respect to free pOVA and Man29-b-Agm29-b-But9/pOVA. Finally, these two polymeric platforms were tested in vivo on C57BL/6 mice bearing B16-OVA melanoma tumor to investigate the applicability of these novel systems in the anticancer immunotherapy field. Both prophylactic and therapeutic approaches showed a higher efficacy of Man58-b-Agm45/pOVA in comparison to Man62-b-Agm52-b-But32/pOVA GPPs in controlling the tumor growth or reducing the tumor volume. In addition, immunological analysis on tumor and spleen samples showed a high level of specific activation of T cells against SIINFEKL antigenic peptide only in samples derived from mice treated with Man58-b-Agm45/pOVA, confirming it as a promising candidate for cancer vaccination.

EPrint type:Ph.D. thesis
Tutor:Salmaso, Stefano
Supervisor:Mastrotto, Francesca
Data di deposito della tesi:02 December 2019
Anno di Pubblicazione:30 September 2019
Key Words:immunotherapy, gene delivery, anticancer vaccination, block co-polymers
Settori scientifico-disciplinari MIUR:Area 03 - Scienze chimiche > CHIM/09 Farmaceutico tecnologico applicativo
Area 03 - Scienze chimiche > CHIM/08 Chimica farmaceutica
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze del Farmaco
Codice ID:12307
Depositato il:02 Feb 2021 10:45
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