initial-evaluation-antibody-conjugates-modified-with-viral-derived

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

In mammalian cells, cytoplasmic protein aggregates generally coalesce to form aggresomal particles. Recent studies indicate that prion-infected cells produce prion protein (PrP) aggresomes, and that such aggregates may be present in the brain of infected mice. The molecular activity of PrP aggresomes has not been fully investigated. We report that PrP aggresomes initiate a cell stress response by activating the RNA-dependent protein kinase (PKR). Activated PKR phosphorylates the translation initiation factor eIF2alpha, resulting in protein synthesis shut-off. However, other components of the stress response, including the assembly of poly(A)+ RNA-containing stress granules and the synthesis of heat shock protein 70, are repressed. In situ hybridization experiments and affinity chromatography on oligo(dT)-cellulose showed that PrP aggresomes bind poly(A)+ RNA, and are therefore poly(A)+ ribonucleoprotein complexes. These findings support a model in which PrP aggresomes send neuronal cells into untimely demise by modifying the cell stress response, and by inducing the aggregation of poly(A)+ RNA.

Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response.

Aggresomes are juxtanuclear inclusion bodies that have been proposed to represent a general cellular response to misfolded proteins in mammalian cells. Yet, why aggresomes are not a pathological characteristic of protein misfolding diseases is unclear. Here, we investigate if a misfolded protein inevitably forms aggresomes in mammalian cells.

Aggresomes do not represent a general cellular response to protein misfolding in mammalian cells.

The observation that PrP is present in the cytosol of some neurons and non-neuronal cells and that the N-terminal signal peptide is slightly inefficient has brought speculations concerning a possible function of the protein in the cytosol. Here, we show that cells expressing a cytosolic form of PrP termed cyPrP display a large juxtanuclear cytoplasmic RNA organelle. Although cyPrP spontaneously forms aggresomes, we used several mutants to demonstrate that the assembly of this RNA organelle is independent from cyPrP aggregation. Components of the organelle fall into three classes: mRNAs; proteins, including the RNAseIII family polymerase Dicer, the decapping enzyme Dcp1a, the DEAD-box RNA helicase DDX6, and the small nuclear ribonucleoprotein-associated proteins SmB/B'/N; and non-coding RNAs, including rRNA 5S, tRNAs, U1 small nuclear RNA, and microRNAs. This composition is similar to RNA granules or chromatoid bodies from germ cells, or planarian stem cells and neurons, which are large ribonucleoprotein complexes predicted to function in RNA processing and posttranscriptional gene regulation. The domain of PrP encompassing residues 30 to 49 is essential for the formation of the RNA particle. Our findings confirm the intriguing relation between PrP and RNA in cells, and underscore an unexpected function for cytosolic PrP: assembling a large RNA processing center which we have termed PrP-RNP for PrP-induced ribonucleoprotein particle.

A large ribonucleoprotein particle induced by cytoplasmic PrP shares striking similarities with the chromatoid body, an RNA granule predicted to function in posttranscriptional gene regulation.

Aggregation of the α-Synuclein (α-Syn) protein, amyloid fibril formation and progressive neurodegeneration are the neuropathological hallmarks of Parkinson's Disease (PD). However, a detailed mechanism of α-Syn aggregation/fibrillogenesis and the exact nature of toxic oligomeric species produced during amyloid formation process are still unknown.

Aggregation and neurotoxicity of recombinant α-synuclein aggregates initiated by dimerization.

The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 ((64)Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high selectivity for IL-3Rα-positive cells. In contrast, 7G3 ACs with the ability to either escape endosome entrapment or traffic to the nucleus was not superior to 7G3-ChAcNLS for increasing intracellular accumulation. Transportation of (64)Cu when complexed to 7G3-ChAcNLS also resulted in increased nuclear and intracellular radioactivity accumulation. Thus, ChAcNLS is a novel mAb functionalizing technology that demonstrates its ability to increase AC intracellular accumulation in target cells through escaping endosome entrapment coupled to nuclear trafficking.

ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation.

There is considerable interest in the development of novel and more efficient delivery systems for improving the efficacy of photodynamic therapy (PDT). The authors in this highlighted issue describe the synthesis and the photobiological characterizations of two photosensitizer (PS) conjugates based on β-carboline derivatives covalently conjugated to folic acid (FA) coupled to bovine serum albumin (BSA) as a carrier system specifically targeting cancer cells overexpressing FA receptor alpha (FRα). Accordingly, only the FA-BSA-β-carboline conjugates are internalized specifically in FRα-positive cells and are proved to be phototoxic. On the other hand, albumin-β-carboline conjugates without FA or β-carboline derivatives alone are not internalized and nontoxic. This conjugate is among the first to produce a conjugate composed of a PS and FA molecules that are directly conjugated to BSA. In addition, the in vitro studies are the first evidence that directly conjugated FA-BSA can be used as carriers to selectively enhance cytotoxicity by PDT relative to unmodified PS or nontargeted BSA-PS. This strategy is a positive step forward for the covalent design and construction of a photodynamic nanomedicine for FR-positive tumors.

Development of a Novel Covalent Folate-Albumin-Photosensitizer Conjugate.

In vivo imaging of estrogen receptor (ER) densities in human breast cancer is a potential tool to stage disease, guide treatment protocols and follow-up on treatment outcome. Both positron emission tomography (PET) and fluorescence imaging have received ample attention to detect ligand-ER interaction. In this study we prepared BODIPY-estradiol conjugates using 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as fluorescent probe and estradiol derivatives as ligand and established their relative binding affinity (RBA) for the ERα. The synthesis of the conjugates involves attachment of a BODIPY moiety to the C17α-position of estradiol using Sonogashira or click reactions of iodo-BODIPY or aza-BODIPY with various 17α-ethynylestradiol (EE2) derivatives. The highest RBA for the ERα was observed with the EE2-BODIPY conjugate (7) featuring a linear eight carbon spacer chain. Cell uptake studies and in vivo imaging experiments in an ER-positive mouse tumor model are in progress.

BODIPY-17α-ethynylestradiol conjugates: Synthesis, fluorescence properties and receptor binding affinities.

Despite the high interest and concern due to an increasing incidence and death rate, patients who develop muscle invasive bladder cancer (MIBC) have few options available. However, the past decade has produced many candidate bladder tumor-specific markers but further development of these markers is still needed for creating effective targeted medications to solve this urgent need. Interleukin-5 receptor α-subunit (IL-5Rα) has recently been reported to be involved in MIBC progression. Thus, we aimed to validate IL-5Rα as a target for antibody-conjugates to better manage patients with MIBC. Patients were recruited and their tumors were processed for IL-5Rα immunohistochemical analysis. NOD/SCID mice were also heterotopically implanted with the human MIBC HT-1376 and HT-B9 cell lines and established xenografts immunohistochemically evaluated for IL-5Rα and compared against patient tumors. Using the mAb A14, an antibody-drug conjugate (ADC) and a radiolabeled immunoconjugate (RIC) were developed by conjugating to vinblastine and to the positron emitter copper-64 (Cu), respectively. As a proof-of-concept for ADC and RIC efficacy,cytotoxicity andpositron emission tomography (PET) imaging in tumor-bearing mice were performed, respectively. In addition, as rapid internalization and accumulation are important components for effective antibody-conjugates, we evaluated these aspects in response to IL-5 andCu-A14 treatments. Our findings suggest that although IL-5Rα protein expression is preferentially increased in MIBC, it is rapid IL-5Rα-mediated internalization allowing vinblastine-A14 to have cytotoxic activity andCu-A14 to detect MIBC tumors. This is the first report to elucidate the potential of IL-5Rα as an attractive MIBC target for antibody-conjugate applications.

Article	Year
Prion protein aggresomes are poly(A)+ ribonucleoprotein complexes that induce a PKR-mediated deficient cell stress response. Biochimica et biophysica acta\| PubMed ID: 18023289	2008
Aggresomes do not represent a general cellular response to protein misfolding in mammalian cells. BMC cell biology\| PubMed ID: 18937858	2008
A large ribonucleoprotein particle induced by cytoplasmic PrP shares striking similarities with the chromatoid body, an RNA granule predicted to function in posttranscriptional gene regulati... Biochimica et biophysica acta\| PubMed ID: 19014979	2009
Aggregation and neurotoxicity of recombinant α-synuclein aggregates initiated by dimerization. Molecular neurodegeneration\| PubMed ID: 23339399	2013
ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation. Molecular pharmaceutics\| PubMed ID: 27112376	2016
Development of a Novel Covalent Folate-Albumin-Photosensitizer Conjugate. Photochemistry and photobiology\| PubMed ID: 27221219	2016
BODIPY-17α-ethynylestradiol conjugates: Synthesis, fluorescence properties and receptor binding affinities. Bioorganic & medicinal chemistry letters\| PubMed ID: 28049591	2017
Targeting IL-5Rα with antibody-conjugates reveals a strategy for imaging and therapy for invasive bladder cancer. Oncoimmunology\| PubMed ID: 29123949	2017

Simon Beaudoin

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