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ChAcNLS, a novel modification to antibody-conjugates permitting target cell-specific endosomal escape, localization to the nucleus and enhanced total intracellular accumulation Simon Beaudoin, Andreanne Rondeau, Olivier Martel, MarcAndre Bonin, Johan E van Lier, and Jeffrey V. Leyton Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00075 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 26, 2016
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Molecular Pharmaceutics
ChAcNLS, a novel modification to antibody-conjugates permitting target cell-specific endosomal escape, localization to the nucleus and enhanced total intracellular accumulation Simon Beaudoina, Andreanne Rondeaua, Olivier Martela, Marc-Andre Boninb, Johan E. van Liera,c, and Jeffrey V. Leytona,c* a
Departément de médecine nucléaire et radiobiologie, bPlateforme de synthèse de peptides et de sondes d'imageries, Faculté de médecine et sciences de la santé, Université de Sherbrooke and c Centre de’Imagerie Moléculaire de Sherbrooke (CIMS), 3001 12e Avenue Nord, Sherbrooke, Québec, Canada J1H5N4 *Address correspondence to: Jeffrey V. Leyton PhD Département de médecine nucléaire et radiobiologie Université de Sherbrooke 3001, 12e Avenue Nord, Sherbrooke, QC, Canada J1H5N4 Tel. (819) 820-6868; FAX: (819) 564-5442 E-mail:
[email protected] Keywords: nuclear localization, endosome escape, intracellular accumulation, antibody-conjugates
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Molecular Pharmaceutics
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Abstract
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The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in
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humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and
4
reduced systemic toxicity that occurs with standard non-targeted therapies. One area that can further
5
clinical success for ACs will be to increase their intracellular accumulation. However, entrapment
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and degradation in the endosomal-lysosomal pathway, which ACs are reliant for the depositing of
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their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative
8
approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We
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hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected
10
trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in
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target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the
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nuclear localization sequence (NLS) from SV-40 large T-antigen, termed ChAcNLS. ChAcNLS was
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conjugated to the mAb 7G3 (7G3-ChAcNLS) that has nanomolar affinity for the cell-surface
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leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-
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ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-
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positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell
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fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to
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determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition,
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the radioisotope copper-64 (64Cu) was also utilized as a surrogate molecular cargo to evaluate
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nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped
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endosome entrapment and degradation resulting in a unique intracellular distribution pattern. MAb
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modification with ChAcNLS maintained 7G3 nanomolar affinity and produced high selectivity for
23
IL-3Rα-positive cells. In contrast, 7G3 ACs with the ability to either escape endosome entrapment or
24
traffic to the nucleus was not superior to 7G3-ChAcNLS for increasing intracellular accumulation.
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Transportation of
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intracellular radioactivity accumulation. Thus, ChAcNLS is a novel mAb functionalizing technology
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that demonstrates its ability to increase AC intracellular accumulation in target cells through
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escaping endosome entrapment coupled to nuclear trafficking.
64
Cu when complexed to 7G3-ChAcNLS also resulted in increased nuclear and
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Molecular Pharmaceutics
1. Introduction
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Antibody-conjugates (ACs) have impacted the healthcare industry for their ability to deliver
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attached molecular payloads, such as chemotherapeutics and radioisotopes, selectively against cancer
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cells but new directions to advance AC efficacy must be sought to improve responses in tumor
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regression and duration. The enhancement of AC intracellular retention in target cells is one area that
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could increase their effectiveness.1 Upon receptor-mediated internalization, ACs are typically
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trapped inside endosomes and trafficked through the endosomal-lysosomal pathway.2 Lysosomes are
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membrane-enclosed organelles that contain an array of digestive enzymes and receive proteins
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transported by endosomes through vesicle membrane fusion and results in the release of active drug
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catabolites. Despite this method of targeted intracellular drug accumulation, most patients eventually
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progress.3-6 Important factors that cause cancer cell resistance have been shown to be the
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overexpression of multidrug resistant (MDRs) transporters and the down regulation of target receptor
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cell surface levels for chemotherapeutics7, 8 and intracellular dehalogenation for radioiodinated ACs.9
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Receptor recycling pathways and their increased use by cancer cells has also been implicated to
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reduce the intracellular accumulation of the internalized AC.10 Therefore, avoiding entrapment in
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these intracellular pathways is an important area to improve the cellular accumulation of transported
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payloads and for maximizing AC activity.
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The functionalization of monoclonal antibodies (mAbs) with cell-penetrating peptides has
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resulted in remarkable increases in intracellular accumulation when cells are treated with these types
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of ACs.11-16 However, this advancement in AC cellular accumulation has been mostly for allowing
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mAbs to access and target specific molecules inside cells that would otherwise be unavailable for
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antibodies to target. Of the few reports that attempt to utilize ACs equipped with cell-penetrating
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peptides as therapeutic agents against cell surface cancer-specific receptors,17-20 all suffered from
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high accumulation in non-target cells or tissues and thus limited in their application for targeted
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delivery.
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shown impressive abilities to escape endosomes and enter the cytoplasm while maintaining target
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cell selectivity.18, 21 However, it is yet to be determined whether increased escape by these ACs
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corresponds to an increase in intracellular accumulation.
Recent advancements whereby ACs functionalized with pH-sensitive polymers have
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Another recent advancement has been to empower ACs to achieve multi-selective targeting
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by attaching peptides that harbor compartment-localizing amino acids.22, 23 In particular to this study,
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the nuclear localization signal (NLS) sequence from SV-40 Large T-antigen has previously been
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incorporated into synthetic peptides and conjugated to proteins and demonstrated the ability to direct
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the transport of proteins into the nucleus.24 Although, the optimized NLS sequence is 25 amino acids
3
long,25 we have previously utilized the mAb, 7G3, conjugated to a 13-mer peptide
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(CGYGPKKKRKVGG) harboring a segment of the NLS (underlined) sufficient for nuclear
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translocation.26-30 7G3 is specific for the leukemic blasts and leukemic stem cells marker IL-3Rα.31,
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32
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cell selectivity.33 7G3-NLS was used to deliver the radioisotope cargo indium-111 (111In) inside the
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nucleus. Molecular damage by
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they travel only nanometer-micrometer distances they are more effective if delivered inside the
An advantage of this short sequence is that it does not penetrate cells and allows mAbs to maintain 111
In is due to its emissions of energetic Auger electrons.34 Because
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nucleus.34 Unfortunately, cytotoxicity was not overwhelming relative to standard
111
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evidence suggested it was due to ineffective nuclear localization caused by entrapment in the
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endosomal-lysosomal and/or recycling pathways.27, 28, 30 This prompted our current efforts to develop
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an AC that gains the ability to effectively escape endosome entrapment. Furthermore, we wanted to
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determine if the combination of both endosome escape and nuclear trafficking enhances intracellular
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accumulation in order to establish a unique innovation in AC design and support future studies
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whereby ACs are equipped with cytotoxic molecular payloads and tested for improved therapeutic
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activity.
In-7G3 and the
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In this report, we present the initial characterization of the bile acid, cholic acid (ChAc)-NLS
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fusion compound (ChAcNLS) conjugated to 7G3. In general, bile acids are known for their role in
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dietary lipid breakdown.35 However, bile acids have been shown to be essential for viral escape from
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endosomes into the cytoplasm through a non-detergent mechanism.36, 37 Non-enveloped viruses that
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cannot rely on membrane fusion, recruit ChAc to trigger ceramide formation in endosome
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membranes. Increased ceramide in the endosome forms channels or makes membrane flip-flop
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sufficient for protein traversal.38-41 42 Although this mechanism is not completely resolved, this led us
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to hypothesize that ChAc functionalization of 7G3 could allow for the selective disruption of
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endosomal membranes and not plasma membranes. Coupled to NLS, ChAcNLS conjugation to 7G3
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could create an AC with the additional ability to efficiently localize to the nucleus of cells with intact
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receptor selectivity. As a proof-of-principle model system, we used TF-1a leukemic cells that
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express IL-3Rα. We examined the selectivity of 7G3-ChAcNLS by comparing its treatment on IL-
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3Rα-negative cells or by functionalizing isotype-matched non-specific mAbs with ChAcNLS. We
31
examined the efficiency of 7G3-ChAcNLS by comparing it to unmodified 7G3 and different 7G3 4 ACS Paragon Plus Environment
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Molecular Pharmaceutics
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ACs with either limited endosome escape or nuclear translocation capabilities. Importantly, we
2
evaluated what were the effects of endosome escape coupled to nuclear translocation on intracellular
3
accumulation. For extending this technology to molecular payloads, we tested the TF-1a nuclear and
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intracellular accumulation of transported radioisotope copper-64 (64Cu).
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2. Materials and methods
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2.1 Human Cells and mAbs
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TF-1a leukemia and Raji Burkitt’s lymphoma cells were obtained from the American Type
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Tissue Collection (Manassas, VA). TF-1a cells are positive for IL-3Rα by flow cytometry
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(corresponding to 7.8 x 103 receptors/cell for 111In-labeled chimeric version of 7G3,43 and Raji cells
12
are IL-3Rα-negative. Cells were cultured in RPMI 1640 medium supplemented with 1%
13
penicillin/streptomycin, 1% non-essential amino acids, 1% sodium pyruvate, 10% heat inactivated
14
FBS, and 0.2% amphotericin B (Wisent, Quebec, Canada). 7G3 and isotype control mIgG2a were
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purchased from BD Biosciences (Ontario, Canada).
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2.2 7G3-ChAcNLS construction
18 19
ChAcNLS was designed with the nuclear localization sequence from SV-40 large T antigen
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with GYG and GG residues at the N- and C-terminus as spacers, respectively (Fig. 1A). The N-
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terminus was capped with a cysteine for conjugation to ChAc and 7G3. Controls included 7G3
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conjugated with ChAc or NLS only and with compounds ChAc-CGYGPLKLRKVGG (ChAcLeu)
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and ChAc-CGYGPDapKDapRKVGG (Dap=2,3-diaminopropionic acid; ChAcDap). Control
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ChAcLeu was to conjugate 7G3 with a compound that enabled endosome escape but did not possess
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a functional NLS but was of similar size. Control ChAcDap was used to conjugate 7G3 with a
26
compound that enabled endosome escape without NLS capabilities but possessed the same net
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charge (+5) as NLS. 7G3 or IgG2a was first maleimide activated for conjugation to the N-terminal
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cysteine of the peptide. Maleimide groups were introduced into 7G3 or IgG2a by reaction of 10
29
mg/mL 7G3 in PBS, pH 7.6, with 10-, 25-, or 50-fold molar excess of sulfosuccinimidyl-4-(N-
30
maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC; VWR, Quebec, Canada) at room
31
temperature for 1 h. Maleimide-derivatized mAbs were purified on a Sephadex G-50 (Sigma5 ACS Paragon Plus Environment
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Aldrich) column eluted with PBS, pH 7.0. Fractions containing maleimide-7G3 or –IgG2a were
2
transferred to a Centricon YM-100 ultrafiltration device (EMD Millipore, Ontario, Canada) and
3
concentrated to 10 mg/mL, which was then reacted with 100-fold molar excess of ChAcNLS or
4
control compounds for 18 h at 4°C. ACs were purified from excess peptide and concentrated in PBS,
5
pH 7.4, by ultrafiltration through a Centricon-YM-100. For peptide synthesis and 7G3-ChAcNLS
6
characterizations please refer to Supporting Information.
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2.3 Exposure of cells to mAb conjugates (Supplemental Fig. 1 in the Supporting Information)
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Unless indicated, 5 x 106 TF-1a cells were exposed to 200 nmol/L of ACs in RPMI/10%FBS
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for 1 h at 37°C. Cells were then washed 3x in ice-cold PBS and suspended in RPMI/10%FBS for an
12
additional 1 h at 37°C as a post-incubation. This allowed for the evaluation of internalization and
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intracellular accumulation of the ACs to be monitored by the methods utilized in this study. Cells
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were then centrifuged and washed in ice-cold PBS for further processing. Assays were performed in
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triplicate and repeated a minimum of three times.
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2.4 7G3-ChAcNLS target cell selectivity by confocal microscopy
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To determine 7G3-ChAcNLS selectivity for IL-3Rα-positive TF-1a cells, cells were
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suspended in 0.1 mL of PBS containing 0.25% Trypsin (Wisent Bio Products, Quebec, Canada) and
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0.25% EDTA and incubated at 37°C for 3 min, which was used to remove surface bound ACs.
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Trypsin was neutralized with the addition of 0.4 mL of RPMI/10% FBS. Very similar trypsin
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procedures have previously been reported.44-46 Trypsinized cells were fixed in 1% paraformaldehyde/
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1% sucrose on ice for 30 min. Cells were then washed and permeabilized with 0.15% Triton X for 5
25
min. Cells were then washed and suspended in 0.5 mL PBS containing 2 µg/mL of anti-murine Fc
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secondary polyclonal antibody conjugated to Alexa Fluor 647 (AF647; Life technologies, Ontario,
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Canada) for 1 h at room temperature in the dark. In addition, to control for potential non-specific
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trafficking activity by the positive charged ChAcNLS on fixed cells, non-treated TF-1a cells were
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also fixed and permeabilized. The fixed and permeabilized TF-1a cells were post-treated with
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ChAcNLS-7G3 for 1 h at 37°C followed by washing and staining with AF647 antibodies. Cells were
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stained with propidium iodide (PI) 5 min prior to analysis. For confocal microscopy, cells were 6 ACS Paragon Plus Environment
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Molecular Pharmaceutics
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mounted onto glass slides with SlowFade mounting media (Life technologies) and covered with a
2
glass coverslip. All images were acquired on a FV1000 scanning confocal microscope (Olympus,
3
Tokyo, Japan) coupled to an inverted microscope using a 63x oil immersion objective. PI
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fluorescence was detected with a 488 nm argon laser and a spectral scanning prism fixed for 600-650
5
nm. AF647 fluorescence was detected using a 633 nm helium-neon laser and a spectral scanning
6
prism fixed for 650-700 nm. Fluorescence emissions from PI and AF647 were collected sequentially.
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Serial horizontal optical sections of 1024 x 1024 pixels with 2-times line averaging were taken at 0.5
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µm intervals through the entire cell thickness. Images were acquired in the same day with identical
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instrument settings. Images are presented as the stacked z-projections (maximum intensity) from 4
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consecutive slices. Each slice was focused for maximum intensity without photobleaching. The
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consequence is that low fluorescence intensity is not captured causing some cells to appear negative.
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Images were pseudocolored and merged (FluoView; Olympus). The ability of the 7G3 conjugates to
13
accumulate inside cells was determined by counting the proportion of AF647 fluorescence positive
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cells and visually analyzing fluorescence intensity.
15 16
2.5 Intracellular accumulation by flow cytometry and Western blot
17 18
Treated cells were fixed and permeabilized and incubated with AF647 as described in section
19
2.4. AC accumulation was calculated as the geometric mean fluorescence intensity (MFI) normalized
20
to background MFI levels within the intact fixed and permeabilized cell population (as determined
21
by the forward scatter/side scatter distribution47 and positive PI staining) incubated with AF647-only
22
as determined by CellQuest Pro software (BD Biosciences).
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For Western Blot analysis, after trypsin neutralization, cells were washed with PBS and lysed
24
in RIPA buffer. Total protein concentration was determined using the BCA Protein Assay Reagent
25
(VWR). 10 µg of protein were loaded into wells in a 12% SDS gel and electrophoresed at 180 V for
26
1 h. The gel was transferred on to a PVDF membrane at 100 V for 1 h. PVDF membranes were then
27
washed in TBS/0.1% Tween 20 and placed into a 5% milk/TBS/0.1%Tween 20 blocking solution for
28
1 h followed by washing 3x in TBS/0.1% Tween 20. Membranes were then incubated in 2.5%
29
milk/PBS containing 1 µg/mL of polyclonal rabbit antibodies against murine Fc conjugated to
30
horseradish peroxidase (HRP; Life technologies). HRP signal was then developed by enhanced
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chemiluminescence (BioRad). Probing for anti-Actin (Sigma-Aldrich) as the loading control was
2
performed under similar procedures.
3 4
2.6 Nuclear fractionation and enrichment for analyzing nuclear localization and accumulation
5
(Suppl. Fig. 1)
6 7
To isolate the cell nuclei, cells were washed in ice-cold PBS and then lysed by suspending in
8
0.05% NP-40, 10 mmol/L Tris pH 7.5, 10 mmol/L NaCl, 3 mmol/L MgCl2 buffer on ice for 10 min.
9
Lysed cells were then centrifuged at 80g for 5 min. The nuclei pellet was then washed without NP-40
10
and centrifuged at 80g for an additional 5 min. Nuclei were washed 3x in ice cold PBS. The degree
11
of isolated nuclei enrichment was determined by Western Blot and flow cytometry. Western Blots
12
were performed as previously described with the exception that mAbs used were specific for the
13
well-established nuclear marker lamin A/C (Santa Cruz Biotechnology Inc., Dallas, TX) or plasma
14
membrane/late-endosome/lysosomal marker Lamp 1 (Sigma-Aldrich). These two markers ensure
15
nuclei are free of contaminants from the plasma membrane, late-endosomes or lysosomes and for
16
loading control and analysis by densitometry. Membranes were washed and developed as previously
17
described. For flow cytometric analysis, changes in FSC vs SSC from intact cells and in isolated
18
nuclei were compared (Suppl. Fig. 2). The smaller nuclei population is distinct from intact cells.
19
Nuclei were stained with PI, which binds to DNA to confirm DNA is contained in the nuclei
20
population. Nuclei were then processed and analyzed by confocal microscopy and flow cytometry to
21
determine nuclear localization and accumulation, respectively, as previously described for intact
22
cells.
23 24
2.6 Radioactivity cargo studies
25 26
Approximately 1.5 mg of 7G3 (5 µg/µL in 50 mM NaHCO3 buffer, pH 7.5) was reacted with
27
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA; for complexing copper-64 (64Cu)).
28
DOTA-conjugates were purified on an Amicon Ultra centrifugal filter (Millipore). Approximately
29
500 µg of DOTA-conjugates were reacted with sulfo-SMCC and conjugated to ChAcNLS or NLS as
30
previously described and then purified by centrifugation. 50 µg of DOTA-ChAcNLS-conjugates
31
were placed in 50 µL of 0.1 M ammonium acetate, pH 5.5 for 1 h at room temperature with 8 MBq
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Molecular Pharmaceutics
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of 64CuCl2. The radioimmunoconjugates were purified by dilution in PBC followed by centrifugation
2
in Amicon centrifugal filters. Radiochemical purity was determined by instant thin layer
3
chromatography developed in 100 mM sodium citrate, pH 5.5. The final radiochemical purity of all
4
radioimmunoconjugates was >98%. 100 nmol/L of radioimmunoconjugates were added to 4 x 106
5
TF-1a cells and incubated as described in section 2.3. Cells were fractionated as described in section
6
2.4 and nuclear radioactivity in each fraction measured in a gamma counter and reported as counts
7
per min (CPM) per fraction. We considered intracellular accumulation as the product of the
8
radioactivity in nuclear and cytoplasmic fractions.
9 10
2.7 Statistical analysis
11 12
Data are presented as the mean ± SD. Significant differences were tested using an unpaired,
13
two-sided Student’s t-test (p
