NLS-Cholic Acid Conjugation to IL-5RÎ±-Specific Antibody Improves

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NLS-cholic acid conjugation to IL-5R#-specific antibody improves cellular accumulation and in vivo tumor-targeting properties in a bladder cancer model Michel Paquette, Simon Beaudoin, Mylene-Annie Tremblay, Steve Jean, Angel F. Lopez, Roger Lecomte, Brigitte Guérin, M'hamed Bentourkia, Robert Sabbagh, and Jeffrey V. Leyton Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00077 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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Bioconjugate Chemistry

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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NLS-Cholic Acid Conjugation to IL-5Rα-Specific Antibody Improves Cellular Accumulation and In Vivo Tumor-Targeting Properties in a Bladder Cancer Model Michel Paquette†, Simon Beaudoin†, Mylene-Annie Tremblay†, Steve Jean‡, Angel F. Lopez§, Roger Lecomte†,

ıı, ∆

, Brigitte Guérin†,

ıı, ∆

, M’hamed Bentourkia†,

ıı, ∆

, Robert Sabbagh#, and

Jeffrey V. Leyton†,ıı, ∆ †

Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences

(FMHS), Centre Hospitalier Universitaire de Sherbrooke (CHUS), Université de Sherbrooke (UdeS), 3001 12 Avenue Nord, Sherbrooke, Québec, J1H 5N4, Canada ‡

Department of anatomy and cellular biology, FMHS, CHUS, UdeS, 3001 12 Avenue Nord,

Sherbrooke, Québec, J1H 5N4, Canada §

The Centre for Cancer Biology, SA Pathology and the University of South Australia, Frome Rd,

Adelaide, South Australia, 5000, Australia ıı

Sherbrooke Molecular Imaging Centre (CIMS), Centre de recherche du CHUS, 3001 12 Avenue

Nord, Sherbrooke, Québec, J1H 5N4, Canada ∆

Sherbrooke Pharmacology Institute, 3001 12 Avenue Nord, Sherbrooke, Québec, J1H 5N4,

Canada #

Department of Surgery, FMHS, CHUS, UdeS, 3001 12 Avenue Nord, Sherbrooke, Québec, J1H

5N4, Canada Running title: Peptide that enhances antibody endosomal escape and tumor targeting of bladder cancer Corresponding author: Jeffrey V. Leyton PhD, 3001 12e Avenue Nord, Sherbrooke, QC, Canada J1H 5N4. Tel. (819) 346-1110; FAX: (819) 564-5442; [email protected]


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Abstract: Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although, a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs. We developed a composite compound (ChAcNLS) that enables ACs to escape endosome entrapment and route to the nucleus resulting in the increased intracellular accumulation as an interleukin-5 receptor α-subunit (IL-5Rα)-targeted agent for muscle invasive bladder cancer (MIBC). We constructed

64

Cu-A14-ChAcNLS,

64

Cu-

A14-NLS, and 64Cu-A14 and their performance evaluated by employing mechanistic studies for endosome escape coupled to nuclear routing and whether this delivery system results in improved

64

Cu cellular accumulation. ACs consisting of ~20 ChAcNLS or NLS moieties per

64

Cu-A14 were prepared in good yield, high monomer content, and maintained high affinity for

IL-5Rα. Confocal microscopy analysis demonstrated ChAcNLS mediated efficient endosome escape and nuclear localization.

64

Cu-A14-ChAcNLS increased

64

Cu cellular accumulation in

HT-1376 and HT-B9 cells relative to 64Cu-A14 and 64Cu-A14-NLS. In addition, we tested 64CuA14-ChAcNLS in vivo to evaluate its tissue distribution properties and, ultimately tumor uptake and targeting. A model of human IL-5Rα MIBC was developed by implanting NOD/SCID mice with subcutaneous HT-1376 or HT-B9 MIBC tumors, which grow containing high and low IL5Rα-positive tumor cell densities, respectively. ACs were intravenously injected and daily blood sampling, biodistribution at 48 h and 96 h, and positron emission tomography (PET) at 24 h and 48 h performed. Region of interest (ROI) analysis was also performed on reconstructed PET


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images. Pharmacokinetic analysis and biodistribution studies showed that

64

had faster clearance rates from the blood and healthy organs relative to

64

Cu-A14-ChAcNLS

64

Cu-A14. However,

Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to

64

Cu-A14. This

resulted in 64Cu-A14-ChAcNLS having superior tumor/normal tissue ratios at both 48 h and 96 h biodistribution time points. Visualization of AC distribution by PET and ROI analysis confirmed 64

Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to

64

Cu-A14. In addition,

64

Cu-A14 modified with only NLS had poor tumor targeting. This was a result of poor tumor

uptake due to extremely rapid clearance. Thus, the overall findings in this model of human IL5Rα-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting.


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Introduction

Although armed antibodies (hereinafter antibody-conjugate [AC]) delivering molecules for the imaging or treatment of targeted tissues is now a prominent approach in medicine, enhancing AC cellular retention and tumor uptake is necessary to improve their effectiveness. The increased residence time inside cancer cells and overall tumor uptake can have important implications for improved tumor killing and detection. Since the vast majority of ACs undergo a process known as receptor-mediated endocytosis, it has been perceived that effective drug release (and hence, subsequent cellular accumulation) takes advantage from the catabolic environment within the endosomal-lysosomal intracellular trafficking pathway. 1 However, small molecules and radionuclides transported into cells by ACs are sensitive to a variety of mechanisms that ultimately leads to poor accumulation. Thus, new strategies for intracellular delivery technologies are needed to improve AC tumor cell accumulation and potentially effectiveness for its intended application. The hydrophobic interiors of cellular membranes are barriers for ACs to efficiently access the intracellular environment, which limits controlled placement and accumulation of delivered molecular payloads such as chemotherapeutics and radioisotopes. Upon receptormediated internalization, current ACs are reliant on entrapment inside the endosomal-lysosomal trafficking pathway where cathepsin-mediated degradation is exploited for payload release. Unfortunately, this trafficking pathway often impedes the efficient intracellular accumulation of these payloads multifold. First, ACs may undergo increased recycling, which has been shown to be a limiting factor for tumor imaging and cytotoxic effectiveness. 2, 3 Second, upon degradation these payloads are released near the cell surface where they are actively exported by


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overexpressed membrane associated transport proteins. downregulated.

4

2-4

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Three, cell surface receptors may be

In addition, delivering biological payloads (i.e. toxins) that recognize

intracellular targets can be degraded and rendered inactive.

5-7

Thus, a technology that i) would

enable ACs to efficiently escape the endosomal-lysosomal pathway, and ii) subsequently route to alternative subcellular locations could greatly enhance payload placement and accumulation and, hence, effectiveness. Leading technologies are synthetic peptides or polymers coupled to antibody surface residues enabling ACs with endosome escape or subcellular destination routing controls.

8-12

In

general, these strategies exploit cellular mammalian physiology such as pH-sensitive endosome membrane-destabilizing activities or nuclear and mitochondrial-specific localization sequences. First-generation peptides were developed from a class of agents known as cell-penetrating peptides (CPPs).

13

Although CPP-conjugated antibodies are remarkable for their ability to

‘penetrate’ membranes and accumulate payloads with high cellular accumulation, penetration is indiscriminate.

12, 14-18

In vivo, peptide-ACs suffer from increased accumulation in non-target

tissue resulting in poor tumor targeting. 19, 20 This is most likely caused by a change in the overall AC net charge due to modification with large cationic/anionic peptides. An increase in net charge has been shown to increase AC plasma clearance or increase distribution in normal tissues. 21 Our group recently developed a peptide fusion that does not indiscriminately penetrate cells. This compound is derived from viral mechanisms used for escaping endosomal entrapment and nuclear routing resulting in the ability of ACs to increase cellular accumulation of attached radioisotope and cytotoxic payloads with high target cell selectivity. 22, 23 Cholic acid coupled to a 13 amino acid peptide containing a nuclear localization sequence (Fig. 1; termed ChAcNLS)


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enables ACs to efficiently i) escape endosome entrapment, ii) route to the nucleus, and iii) increase intracellular accumulation of the delivered payload with high target receptor affinity and target cell selectivity in leukemia and breast cancer.

22, 23

Importantly, these actions increased

cytotoxicity up to 100-fold when Trastuzumab-emtanisne was modified with ChAcNLS.

22

Cholic acid and the peptide are linked via an N-terminal cysteine in a juxtapose configuration. 23 The cysteine reacts with a maleimide on the chosen crosslinker sulfosuccinimidyl-4-(Nmalemidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) that is initially conjugated to antibody surface lysines. ChAcNLS has a molecular weight of 1.8 kDa and its ability to increase cellular accumulation and cytotoxicity is proportional to the quantity of peptide conjugated on to the AC. 23 Figure 1.

Figure 1. Schematic representation of ChAcNLS conjugated to surface lysines via the crosslinker sulfo-SMCC.

One model system utilized by our group is the transportation of copper-64 (64Cu) by the monoclonal antibody (mAb) A14 that is specific against the interleukin-5 receptor α-subunit (IL5Rα). IL-5Rα is preferentially expressed in muscle invasive bladder cancer (MIBC) relative to non-invasive tumors and healthy urothelial tissues and is a vital component for driving MIBC progression.

24, 25

Addition of IL-5 to MIBC cells amplified components associated with tumor

invasion such as enhanced cellular migration, expression of matrix metalloproteinases, and the arrest of cellular proliferation.

26

We addressed the potential of IL-5Rα as a target for ACs by

evaluating its protein expression, internalization, and expression turnover characteristics. In


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patient MIBC specimens IL-5Rα expression was elevated but predominantly localized in the paranucleus or in the cytoplasm with much less amounts present on the cell surface.

25

The

MIBC cell lines HT-1376 and HT-B9 also contained similar IL-5Rα expression localization patterns. Binding studies with

64

Cu-A14 revealed that HT-1376 and HT-B9 cells contained

modest cell surface expression levels of 616 and 157 receptors per cell, respectively. Interestingly, IL-5Rα underwent rapid internalization and re-expression when incubated with 64

Cu-A14. Nonetheless, 64Cu accumulated only in HT-B9 cells whereas HT-1376 cells were able

to effectively export 64Cu. HT-1376 and HT-B9 cells were also used to develop a pathologically similar model of human IL-5Rα-positive MIBC. For example, HT-1376 xenografts were comprised of >66% of IL-5Rα-positive tumor cells that were disseminated throughout. In contrast, HT-B9 tumors were comprised of 11% IL-5Rα-positive tumor cells and present as small ‘island’ populations. This difference in tumor cell density was similar to the heterogeneity seen in MIBC patients.

25 64

Cu-A14 was intravenously injected into these tumor-bearing mice

and positron emission tomography (PET) performed. PET images showed 64Cu-A14 could only visualize HT-1376 tumors and not HT-B9 tumors. This was due to slow clearance from the blood pool. In this study we characterized a ChAcNLS-modified AC for the first time in vivo. We used the model system of

64

Cu-A14 and IL-5Rα-positive MIBC with the goal of determining

whether ChAcNLS has attractive tumor targeting properties. In this report, fluorescence and genetic methods were used to evaluate the ability of A14-ChAcNLS to escape endosome entrapment and localize to the nucleus, and radioimmunoassays to determine

64

Cu cellular

accumulation. PET imaging, biodistribution, and pharmacokinetic (PK) analysis were used to determine the impact on tumor and normal organ uptake, and hence, tumor targeting. Lastly,


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since modification with peptides containing NLS sequences is one of the original strategies to develop technologies to improve tumor targeting of ACs,

27, 28 64

Cu-A14-NLS was also

constructed and characterized.

Results and Discussion

Preparation of 64Cu-A14 conjugates The purity of 64Cu-A14, 64Cu-A14-NLS, and 64Cu-A14-ChAcNLS was ≥95% and ≥99% by ITLC and SDS-PAGE, respectively (Fig. 2A). The calculated retention factor (Rf) values revealed A14-NLS and A14-ChAcNLS both contained 20 ± 2 moles of NLS and ChAcNLS per mole of antibody, respectively (Fig. 2A). Specific activities ranged from 220 - 310 MBq/mg and remained stable in PBS as

NLS-Cholic Acid Conjugation to IL-5RÎ±-Specific Antibody Improves

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