University of Southern California Dissertations and Theses

Trans-epithelial transport of G -CSF -transferrin conjugate

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Trans-epithelial transport of G -CSF -transferrin conjugate

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Content TRANS-EPITHELIAL TRANSPORT OF G-CSF-TRANSFERRIN
CONJUGATE
by
Adam Scott Widera
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
May 2004
Copyright 2004 Adam Scott Widera
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UMI Number: 3140572
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DEDICATION
This work is dedicated to my wife Linda, and my son Ethan, who make life worth
living. I owe it all to them.
I thank my parents. Their undying support made this work possible....
1 1
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ACKNOWLEDGEMENTS
First and foremost I thank my thesis advisor, Wei-Chaing Shen, Ph.D., for his
invaluable support and guidance throughout my doctoral studies
I also acknowledge the support offered by the good folks at the Will Rogers Institute
Pulmonary Research Center o f the USC Keck School o f Medicine. Their faithful
collaboration was instrumental to my success. Special gratitude is offered to
Kwang-Jin Kim, Ph.D., whose close reading o f manuscripts provided me with much
excellent teaching and scientific insight.
I thank the American Foundation for Pharmaceutical Education fo r providing
financial support throughout my graduate studies
I thank David Ann, Ph.D., for providing generous use o f his lab’ s equipment and
supplies
Last, but not least, I thank my labmates including, Karin, Jennica, Maureen, Rana,
Rita, Tin-ten, Wendy, Yun Bai, Li Yun, Jeff, Cindy, Wang Li, Fariba, and Thuy for
making life interesting at USC.
Ill
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TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGMENTS iii
LIST OF FIGURES xi
ABSTRACT xv
1. INTRODUCTION 1
1.2. Transferrin, transferrin-receptor, and associated proteins 1
1.2.1. Structure and function of transferrin 2
1.2.2 Transferrin-receptor 6
1.2.2.1 Structure and function of Transferrin-Receptor 6
1.2.2.2 Regulation of expression and tissue distribution 13
1.2.3 Proteins associated with transferrin and transferrin-receptor 15
1.2.3.1. HFE 15
IV
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1.2.3.2. Cubilin and Megalin 17
1.3. The recycling and sorting of transferrin receptor 21
1.3.1 Rab proteins involved in transferrin trafficking 22
1.3.2 The Rabl 1 interacting proteins 25
1.3.3 Rab5 effectors, EEAl and PI(3)K, involved in early endosome 25
fusion
1.4. Utilization of TfR for drug delivery 28
1.4.1 TfR-based targeting for anti-cancer therapeutics 28
1.4.2 TfR-mediated transepithelial protein-drug delivery 31
1.4.2.1 anti-TfR antibody drug conjugates for transepithelial drug delivery 32
1.4.2.2 Enhancement of TfR-mediated drug delivery 35
1.4.2.2.1 Lysosomotrophic Amines 36
1.4.2.2.2 Lipophilic carboxylic ionophores 36
1.4.2.2.3 Brefeldin-A 37
1.4.3. Modification of Rab-dependent sorting pathways for enhancement 39
of TfR-mediated gastrointestinal transepithelial transport
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1.4.4 Enhancement of TfR-mediated transport in pulmonary epithelium. 42
1. MATERIALS ANB METHODS 48
2.1 Caco-2 Cell Culture 48
2.2 MDCK Cell Culture 48
2.3 Primary Culture of Rat Alveolar Epithelial Cell Monolayers 49
2.4 Measurement of Cell Surface Transferrin Receptor on Caco-2 50
Monolayers
2.5 Measurement of Cell Surface Transferrin Receptor in Rat Alveolar 51
Epithelial Cell Monolayers
2.6 Measurement of Total Cellular Transferrin Receptor in Rat Alveolar 52
Epithelial Cell Monolayers
2.7 TfR-Dependent Uptake of ^^Fe in Caco-2 and Rat Alveolar Epithelial 52
Cell Monolayers
2.8 Immunoflouresence Studies in Rat Alveolar Epithelial Cell 53
Monolayers
2.9 Synthesis of Insulin-Transferrin Conjugate 54
2.10 Cloning and production of G-CSF 56
2.11 Preparation of Tf-G-CSF Conjugate 57
vi
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2.12 Assessment of Apical-to-Basolateral Transcytosis of '^^I-Tf, 60
^^^I-M-Tf, *^^I-G-CSF and ^^^I-G-CSF-Tf across Caco-2 and
MDCK Cell Monolayers
2.13 Assessment of Apical-to-Basolateral Transcytosis of '^^I-Tf, 61
’^^I-GCSF and ‘^^I-Tf-GCSF across Rat Alveolar Epithelial
Cell Monolayers
2.14 Analysis of Transcytosed Proteins 62
2.15 In vivo studies of subcutaneously and orally delivered G-CSF-Tf 63
conjugate in male BDFl mice
2.16 Statistical analyses 64
3. RESULTS 65
3.1 Synthesis and characterization of G-CSF-Tf 65
3.2 The Effect of Wortmannin on TfR-Mediated Transcytosis in 71
Caco-2 Monolayers
3.3 LY294002 Mediated Enhancement of TfR-mediated Transcytosis 74
in Caco-2 Monolayers
3.4 Effect of BFA, AG-10, and LY294002 on TfR-Specific ^^^I-Tf 78
Binding in Caco-2 Monolayers
3.5 Effect of BFA, AG-10, and LY294002 on TfR-specific ^^Fe-Tf 81
uptake in Caco-2 monolayers
V ll
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3.6 Comparison of Transcytosis Enhancers’ Effect on TfR-Mediated 84
Transport in Caco-2 Monolayers
3.7 The effect of LY294002 on TfR-mediated transport of a G-CSF-Tf 87
conjugate across Caco-2 monolayers
3.8 Comparison of the effect of BFA, AG-10, or LY294002 treatment 90
on TfR-mediated Transcytosis in MDCK monolayers
3.9 Effect of BFA, AG-10, and LY294002 on TfR-Specific ‘^^I-Tf . 94
Binding in MDCK Monolayers
3.10 Effect of BFA, AG-10, and LY294002 on TfR-Specific *^^I-Tf 97
Binding in MDCK Monolayers
3.11 Measurement of total surface transferrin receptor in primary cultures 100
of rat alveolar epithelial cell monolayers
3.12 Total cellular TfR in rat alveolar epithelial cell monolayers as a 102
function of culture day
3.13 Rat alveolar epithelial cell monolayers exposed to keratinocyte 103
growth factor during culture retain TfR expression
3.14 Confocal microscopy of keratinocyte growth factor treated rat 103
alveolar epithelial cell monolayers
3.15 Type II cell-like rat alveolar epithelial cell monolayers exhibit 107
polarized distribution of TfR at the cell surface
3.16 Type II cell-like rat alveolar epithelial cell monolayers exhibit 108
polarized TfR-specific ^^Fe-Tf uptake
via
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3.17 Effect of BFA, AG-10, and LY294002 on TEER of type II cell-like 109
rat alveolar epithelial cell monolayers
3.18 Effects of BFA on distribution and endocytosis of TfR in rat 112
alveolar epithelial cell monolayers
3.19 TfR-mediated apical-to-basolateral Tf transport in BFA-treated 117
type n cell-like rat alveolar epithelial cell monolayers
3.20 TfR-mediated apical-to-basolateral transcytosis of In-Tf conjugate 120
across type II cell-like rat alveolar epithelial cell monolayers
3.21 Analysis of ^^^I-In-Tf transcytosed across type II cell-like rat 122
alveolar epithelial cell monolayers
3.22 TfR-Mediated ^^^I-GCSF-Tf transcytosis in type II cell-like rat 122
alveolar epithelial cell monolayers
3.23 Comparison of ^^^I-GCSF-Tf and ^^^I-GCSF apical-to-basolateral 125
transport in AEG monolayers
3.24 Analysis of transcytosed ^^^I-GCSF-Tf 126
3.25 Apical-to-basolateral Transcytosis of G-CSF-Tf and TF across 129
Caco-2 monolayers
3.26 Chromatographic analysis of transcytosed G-CSF-Tf 131
3.27 Analysis of biological activity of transcytosed '^^I-G-CSF-Tf 131
in Caco-2 monolayers
IX
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3.28 Neutrophil proliferation in BDFl mice dosed with subcutaneous 134
and oral G-CSF-Tf
4. DISCUSSION 139
5. CONCLUSIONS 165
6. FUTURE DIRECTIONS 170
7. ALPHABETIZED BIBLIOGRAPHY 174
8. APPENDIX: Numerically Ordered References 216
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LIST OF TABLES A N D FIGURES
Table 1) Total Cellular TfR 102
Figure 1) The classical endocytosis pathway of transferrin-receptor
(TfR).
Figure 2) The x-ray crystal structure of the transferrin receptor
ectodomain.
Figure 3) The sequence and secondary structure of human transferrin 10
receptor.
Figure 4) Synthesis scheme of In-Tf conjugate. 55
Figure 5) Synthesis scheme for G-CSF-Tf. 59
Figure 6) G-CSF-dependent proliferation of NFS-60 cells. 67
Figure 7) SDS-PAGE analysis of G-CSF-Tf. 69
Figure 8) Chromatographic analysis of G-CSF-Tf. 70
Figure 9) Wortmannin induced enhancement of TfR-mediated 72
transcytosis in Caco-2 monolayers.
Figure 10) Wortmannin’s effect on TEER of Caco-2 monolayers. 73
XI
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Figure 11) LY294002 induced enhancement of TfR-mediated 75
transcytosis in Caco-2 monolayers.
Figure 12) LY294002’s effect on TEER of Caco-2 monolayers. 77
Figure 13) The effect of BFA, AG-10, and LY294002 on the surface 79
distribution of TfR in Caco-2 monolayers.
Figure 14) The effect of BFA, AG-10, and LY294002 on the uptake of 82
^^Fe-Tf in Caco-2 monolayers.
Figure 15) Comparison of transcytosis enhancer effect on TfR-mediated 85
transcytosis in Caco-2 monolayers.
Figure 16) LY294002 induced enhancement of TfR-mediated 88
transcytosis of ^^^I-G-CSF-Tf across Caco-2 monolayers.
Figure 17) Comparison of transcytosis enhancer effect on TfR-mediated 91
transcytosis in MDCK monolayers.
Figure 18) Effect of BFA, AG-10, and LY294002 treatment on TEER 93
of MDCK monolayers.
Figure 19) The effect of BFA, AG-10, and LY294002 on the surface 95
distribution of TfR in MDCK monolayers.
Figure 20) The effect of BFA, AG-10, and LY294002 on the uptake of 98
^^Fe-Tf in MDCK monolayers.
Figure 21) Total surface TfR in rat alveolar epithelial cells (AEC) as a 101
function of day in culture.
XII
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Figure 22) Effect of KGF treatment on TfR expression in rat alveolar 104
epithelial cell monolayers.
Figure 23) KGF treatment retains TfR expression in rat alveolar 105
epithelial cell monolayers.
Figure 24) Rat AEC monolayers tested on day 7 for polarized 108
distribution of TfR.
Figure 25) TfR-specific uptake of ^^Fe in rat AEC monolayers on day 7. 110
Figure 26) Effect of BFA, AG-10, and LY294002 treatment on TEER 111
of rat type II cell-like alveolar epithelial cell monolayers.
Figure 27) The effect BFA on surface TfR expression and ^®Fe-Tf 114
uptake in rat alveolar epithelial cells (AEC).
Figure 28) The effects of BFA on ^^^I-Tf transport in rat alveolar 118
epithelial cell monolayers.
Figure 29) *^^I-In-Tf transport in rat alveolar epithelial cell monolayers. 121
Figure 30) Specific TfR-mediated transport of ^^^I-GCSF-transferrin 123
conjugate determined on day 7 and comparison to ’^^I-GCSF.
Figure 31) Gel filtration chromatogram of TfR-mediated transcytosed 127
'^^I-GCSF-Tf.
Figure 32) Evaluation of biological activity of transcytosed 128
GCSF-transferrin.
X lll
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Figure 33) Specific TfR-mediated transport of ^^^I-GCSF-transferrin 130
conjugate determined in Caco-2 monolayers and comparison
to ‘^^I-GCSF.
Figure 34) Gel filtration chromatogram of TfR-mediated transcytosed 132
*^^I-GCSF-Tf.
Figure 35) Evaluation of biological activity of transcytosed G-CSF-Tf. 133
Figure 36) Myelopoietic effect of subcutaneously administered 136
G-CSF-Tf.
Figure 37) Myelopoietic effect of orally administered G-CSF-Tf 137
Figure 38) Dose response effect of orally administered G-CSF-Tf 138
Figure 39) Spacefill model of G-CSF x-ray crystal structure. 140
Figure 40) Possible mechanisms of transcytosis enhancer’s effect on 147
TfR distribution in polarized epithelia.
Figure 41) PI3K Inhibitors as potential TfR-mediated transcytosis 148
enhancers.
Figure 42) Possible G-CSF-Tf fusion constructs. 171
XIV
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ABSTRACT
The purpose of this study was to investigate the transferrin-receptor (TfR)-
mediated transepithelial transport of G-CSF-transferrin (If) conjugate in cultured
enterocyte-like Caco-2 monolayers, rat alveolar epithelial cell monolayers, and the
myelopoietic effect of subcutaneously and orally administered G-CSF-Tf in BDFl
mice.
Methods included using Caco-2 and rat alveolar epithelial cell monolayers
exhibiting a minimum transepithelial electrical resistance of 500 Qcm^ and 2000
Qcm^, respectively as an in vitro model trans-epithelial transport capability. BDFl
mice were used as an in vivo absorption model. TfR-mediated transcytosis was
measured by using '^^I-G-CSF-Tf and analyzing the downstream compartment by
gamma counter. The efficacy of subcutaneously and orally administered G-CSF-Tf
was determined by performing daily absolute neutrophil counts.
The results obtained from transport experiments in Caco-2 cells and rat
alveolar epithelial cell monolayers revealed that the monolayers that received *^^I-G-
CSF-Tf exhibited significantly higher apical-to-basolateral transport rates compared
to the monolayers that received *^^I-G-CSF. Inclusion of 100-fold excess unlabeled
Tf reduced the extent of ^^^I-G-CSF-Tf transport by 80%. Chromatographic and
bioactivity assays revealed that the protein recovered from the basolateral
compartment was the intact conjugate and it retained full ability to stimulate the
proliferation of the G-CSF dependent cell line, NFS-60, upon reduction.
XV
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Subcutaneous administration of G-CSF-Tf in BDFl mice demonstrated that the
conjugate is able to elicit a statistically significant enhancement in therapeutic effect
relative to filgrastim, which includes a longer duration of action with higher absolute
neutrophil counts. Oral administration of G-CSF-Tf in BDFl mice demonstrated
that G-CSF-Tf is able to elicit a significant, and apparently dose dependent, increase
in absolute neutrophil counts while filgrastim had no effect.
Our data indicate that G-CSF-Tf is transported across Caco-2 monolayers and
ratalveolar epithelial cell monolayers by TfR-specific processes, at a rate that is
significantly higher than the non-specific flux of G-CSF. G-CSF-Tf is also able to
elicit a prolonged myelopoietic effect relative to filgrastim when administered by
subcutaneously or orally in BDFl mice. The development of an orally or pulmonary
bioavailable G-CSF has the potential to provide great benefit to patients under
sustained G-CSF dosing regimes.
XVI
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1. INTRODUCTION
Iron is a fundamental necessity for almost every living organism. The ability
of this metal to readily exist in two common redox states leads to its requirement as a
co-factor in many fundamental biological processes, including O2 absorption, ATP
generation through oxidative phosphorylation, DNA synthesis, etc. However, the
charged and reactive nature of free iron presents a challenge for efficient tissue
distribution and cellular uptake. Transferrin (Tf) and its cognate receptor have been
used by vertebrate species to overcome these barriers for efficient and regulated
intracellular iron delivery. The ubiquitous nature of the TfR and the fact that it is
internalized through endocytic means has made it an essential tool for the general
elucidation of sorting and recycling pathways of endocyticaUy derived vesicles in
epithelial cells. In addition, the high specificity of endocytic uptake of Tf by TfR has
made it a subject of interest for targeted drug delivery via parenteral administration
and for delivery of TfR-targeted drug conjugates across epithelial barriers.
The introduction of this work focuses on the sorting and recycling pathways
of TfR in epithelial cell systems. Special focus is given to the methods by which the
understanding of these pathways can be exploited to achieve higher efficiency in
targeted delivery systems.
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1.2 Transferrin, transferrin-receptor, and associated proteins
1.2.1 Structure and function of transferrin
Transferrins are a structurally related class of metal-binding glycoproteins of
approximately 80 kDa in size whose primary function is the binding and
transportation of non-heme iron (1-5). The transferrins have been classified into
three major sub-classes: serum Tf, lactoferrin, and ovotransferrin. Serum Tf is
responsible for binding and transporting iron through many of the biological fluids
including blood, lymph, cerebrospinal fluid, colostrum, bile, amniotic fluid, and
breast milk (6-10). Lactoferrin is found in breast milk, saliva, and tear secretions; it
is thought to function primarily in these environments as an iron chelator,
sequestering iron to act as a bacteriostatic agent (11-13). Lactoferrin also is
implicated in modulating leukocyte antibacterial activity and may also be involved
with inflammatory responses (14; 15). Unlike serum Tf, lactoferrin does not
contribute iron to erythrocytes, and it is cleared by the reticulo-endothelial system
(16). Ovotransferrin is found in reptile and avian oviduct secretions as well as avian
egg white, and its function is analogous to that of lactoferrin, purported to primarily
serve as a bacteriostat via the sequestration of iron (17-19). In addition to the three
major types of Tf, melanotransferrin (p97) can also be considered to be part of the Tf
family. This fourth member of the Tf family is a structural homologue distinctly
different from the others in that it seems to have little to do with the receptor-specific
cellular uptake of iron (20; 21). Rather, this protein is predominantly localized to the
surface of human melanomas and may assist in rapid cell prohferation via iron
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scavenging and the prevention of lipid peroxidation (22; 23). Considering that serum
Tf has been used extensively in endocytosis research, and since it is the protein that
has been identified as a potential drug carrier candidate, it will be the focus of the
remainder of the review; the other three transferrins will not be discussed further.
The serum transferrins from different species share a high degree of
homology, typically composed of a single chain polypeptide with approximately 700
amino acids. However, there are varying degrees of carbohydrate content between
species (24). Human Tf is glycosylated with identically branched heterosaccharide
chains linked to the polypeptide by aspariginyl-iV-acetylglucosamine bonds at two
separate residues (25) and one O-linked carbohydrate at Ser51 . While the
carbohydrate chains of Tf are seen in all species, they do not affect the ability of Tf
to bind to TfR, nor do they affect they ability of the receptor complex to be
internalized into the cell (26; 27). In addition, in contrast to other proteins, the
desialation of Tf has little effect on the catabolism rate (28).
Transferrin is composed of two homologous globular domains, known as the
N and C lobes, connected by a short Unear peptide region, that function in a similar
fashion. Both lobes of the Tf molecule are capable of binding one iron atom with
similar affinities. However, one of the necessities for stringent binding of iron to Tf
is the presence of synergistic bicarbonate anions (29-31), which allows F e^"^ to
tightly coordinate with the amino acids that are suspected of comprising the iron
binding pocket of the N and C lobes (i.e., two Tyr, one Lys and one His for each
lobe) (32; 33). Lack of the presence of bicarbonate anion also affects the abiUty of
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Tf to properly release Fe^^ once it is bound, with bicarbonate-free Tf demonstrating
poor iron release capabilities (34; 35). In addition, Tf has been suggested as a
suitable carrier for medically therapeutic metal ions, since Tf has the ability to bind
to other metal ions in vitro (36-39). However there is little evidence to suggest that
Tf has significant interactions with metals other than iron under normal in vivo
conditions (7).
One of the unique features of Tf is that the binding of Fe^^ alters the shape of
the molecule, affecting the binding affinity of Tf for its receptor. This
conformational change seems to occur in a stepwise fashion as each iron molecule is
bound (40-42). The iron-loaded Tf, or holotransferrin, binds to the receptor with
greater affinity than mono-transferrin or apo-transferrin (43; 44). Binding of iron
also results in increased stability of the molecule relative to the apo-form, with the
iron-loaded molecule exhibiting increased resistance to thermal or proteolytic
degradation (45; 46).
Transferrin undergoes a conformational change as a result of a trigger-
mechanism that is induced by the decrease in pH during the endocytosis cycle. This
allows for the release of iron into the endosomal compartment of the cell and the
subsequent recycling of the apo-transferrin to the cell surface, where the lowered
affinity of the apo-transferrin for the receptor at extracellular pH causes the release
of Tf from the receptor (47; 48). A schematic of the endocytosis and recycling cycle
for the Tf/TfR complex is shown in Figure 1.
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(5)
©
Clathrln-
coated pit
Figure 1: The classical endocytosis pathway of transferrin-
receptor (TfR). Transferrin (Tf) binds two atoms per molecule.
The iron-loaded Tf is subsequently bound by TfR and the Tf/TfR
complex is internalized via clathrin coated pits. The iron is released
from Tf in the acidic endosomal environment and Tf/TfR is recycled
back to the plasma membrane. Iron-free Tf is then released from TfR
to complete the cycle.
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The liver is the principle site of Tf production and catabolism. This can be most
dramatically demonstrated in the case of human liver transplant patients, who after
receiving a new liver, obtain the electrophoretic profile of the donor’s serum Tf (as
determined by the normal inter-individual variability in post-translational
modification of Tf) (49; 50). In addition to this example, in vitro studies with rat
liver have determined that the rates of incorporation of radiolabeled amino-acids into
transferrin-precursors were nearly identical to the total synthesis values obtained by
in vivo perfusion studies (51-53). While the liver is responsible for the large
majority of Tf catabolism, a significant amount of Tf is also secreted by the liver into
the bile, with estimates for the average normal adult of approximately 100 mg/day
passing into the small intestine (54).
1.2.2 Transferrin-receptor
1.2.2.1 Structure and function
TfR was first recognized through formation of monoclonal antibodies raised from
mice that were immunized against various neoplastic cell lines, with several of the
antibodies recognizing antigenic portions of the protein we now know as TfRl (55-
57). The receptor is a homodimer composed of two identical subunit monomers with
an approximate mass of 90 kDa each (58). The ectodomain of the receptor is
composed of three distinct domains, the protease-like, apical, and helical domains
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(Figure 2). The helical domains of TfR face each other and interact to form the
dimeric receptor (59). The extracellular portion of the receptor is subject to extensive
post-translational modification with three sites of mannose rich iV-linked
glycosylation at residues Asn251, Asn317, and Asn727 and one O-linked
glycosylation on Thrl04 (60). The monomers are covalently linked by two disulfide
bonds at residues Cys 89 and Cys98 (61). Treatment with trypsin cleaves two 70
kDa fragments that retain the ability to bind to Tf. The protein also has a single
transmembrane domain, comprised of residues 68-88 of the polypeptide. Following
this, it has a short cytoplasmic tail, of 5 kDa in size, that is the site of
phosphorylation and acylation of the receptor (61). Unlike Tf, the post-translational
modifications of TfR seem to be crucial for its biological functionality. Specifically,
mutations that eliminate the sites of potential A-linked glycosylation severely limits
the ability of the receptor to bind to Tf.
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Apical
Domain
Domaifi
Figure 2: The x-ray crystal structure of the transferrin receptor
ectodomain. The putative dimeric orientation of two transferrin
receptor molecules is shown as suggested by Lawrence et al. The
receptor is organized into three distinct domains, composed of the
protease-like, apical, and helical domains. The stalk region is also
shown with the suggested orientation relative to the protease-like
domain and the plasma membrane. The image was rendered from
the pdb coordinates of the A subunit of 1CX8 (human TfR,
www.rcsb.org) (Lawrence et al, 1999) using RasMol and Adobe
Photoshop.
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In addition, mutations to the 0-linked glycosylation at Thrl04 suggest that reduction
in this type of carbohydrate linkage may limit the cellular half-life of the receptor
(62; 63). The sequence and secondary structural motifs of TfR is shown in Figure 3.
The receptor has very high affinity for differic-Tf, with binding constants on
the order of 2-7 x 10'® M (64). The exact orientation of Tf binding to TfR is not fully
understood, however the primary recognition site appears to reside on the C lobe of
Tf, with a sequential synergistic effect between the N and C lobes of Tf that
enhances affinity for TfR (65; 66). In addition, site-directed mutagenesis studies
have shown that the central helical domains of TfR, formed by the union of the two
monomers, are key to the ability of the receptor to recognize Tf (67). However,
inspection of the computational union of Tf and the TfR crystal structures
(ectodomain) have also indicated that much favorable interaction occur between the
outward facing apical domains of the receptor and Tf (59). The iron saturation status
of Tf also plays a large role in the ability of TfR to recognize Tf, with apo-Tf having
very little affinity for TfR at physiological pH 7.4 (43). However, at pH 5.0 Tf
forms a very stable complex with TfR, which is the underlying basis for the iron
exchange and transport capability of Tf (68-70).
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Figure 3; The sequence and secondary structure of human transferrin
receptor.
Each residue in the sequence is reported as a single letter code.
Secondary structure is calculated and described according to an
implementation of the method of Kabsch and Sander (1983) Biopolymers
22, 2577-2637. The assignments are: H = helix; B = residue in isolated beta
bridge; E = extended beta strand; G = 310 helix; 1 = pi helix; T = hydrogen
bonded tum; S = bend. From the Protein Data Bank, www.rcsb.org.
10
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1 LYWDDLKRKL SEKLDSTDFT STIKLLNENS YVPREAGSQK DENIALYVEN
HHHHHHHH HHHHHT HH HHHHHTTTGG GSS TTSHH HHHHHHHHHH
51 EFREFKLSKV WRDQHFVKIQ VKDSAQNSVI IVDKNGRLVY LVENPGGYVA
HHHHTT S EEEEEEEEE EE SS EEE EE SSS S B SS BT
101 YSKAATVTGK LVHANFGTKK DFEDLYTPVN GSIVIVRAGK ITFAEKVANA
T EEEEE EEE TTTTTT TTT SSS T TSEEEEES S S HHHHHHHH
151 ESLNAIGVLI YMDQTKFPIV NAELSFFGHA HLGTGDPYTP GFPSFNHTQF
HHTT EEE E TTTS S SS B B SSSS TT S SS SSSS
201 PPSRSSGLPN IPVQTISRAA AEKLFGNMEG DCPSDWKTDS TCRMVTSESK
S SS S S EEEE HHH HHHHHHTB S SSSSS T T SEE TT
251 NVKLTVSNVL KEIKILNIFG VIKGFVEPDH YVWGAQRDA WGPGAAKSGV
EEEEEE EEE EEEEEEEE EE SSBTT EEEEE SS TTTTHH
301 GTALLLKLAQ MFSDMVLKDG FQPSRSIIFA SWSAGDFGSV GATEWLEGYL
HHHHHHHHHH HHHHHHHTT SB EEEE E SGGGTTH HHHHHHGGGT
351 SSLHLKAFTY INLDKAVLGT SNFKVSASPL LYTLIEKTMQ NVKHPVTGQF
TTHHHHB EE EE TT S S S EEEE GG GHHHHHHHHT T B SSSSSB
401 LYQDSNWASK VEKLTLDNAA FPFLAYSGIP AVSFCFCEDT DYPYLGTTMD
SS TT SS B TTTHH HHHHHTS EEEEE SS SSTTBTT
451 TYKELIERIP ELNKVARAAA EVAGQFVIKL THDVELNLDY EEYNSQLLSF
HHHHHTTTT THHHHHHHHH HHHHHHHHHH HHSSS S SHHHHHHHHH
501 VRDLNQYRAD IKEMGLSLQW LYSARGDFFR ATSRLTTDFG NAEKTDRFVM
HHHHHTTHHH HHHSS HH HHHHHHHHHH HHHHHTHHHH HTTTT HHHH
551 KKLNDRVMRV EYHFLSPYVS PKESPFRHVF WGSGSHTLPA LLENLKLRKQ
HHHHHHHHHH TGGGBTTTTT TTT S BTT T STTTHHH HHHHHHHHHT
601 NNGAFNETLF RNQLALATWT IQGAANALSG DVWDIDNEF
SS 3 HHHH HHHHHHHHHH HHHHHHHS S SSS
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Within the past few years a new member of the TfR family was discovered
and denoted as transferrin receptor 2 (TfR2). However, unlike TfRl, TfR2 has a
much lower affinity for Tf (25 fold lower than TfRl) and it seems to be primarily
involved with regulation and maintenance of iron homeostasis in the body, and it
might also play a minor role in the delivery of iron to rapidly dividing tissues. The
TfR2 gene produces two transcripts of 2.5 and 2.9 kb in length. Analysis of the
amino acid sequence suggests that TfR2 is a type II transmembrane glycoprotein,
like TfRl, sharing 45% sequence identity and 66% similarity in the extracellular
domain (71). In contrast to TfRl, TfR2 is primarily expressed in the liver (71; 72).
It lacks iron-regulatory elements in the coding and noncoding regions of its mRNA,
indicating that the expression of TfR2 is not regulated by the iron-responsive protein
feedback mechanism, as seen for TfRl. It would appear from this that TfR2 plays a
minor role in regulating intracellular iron concentrations, or it perhaps responds to a
yet undiscovered intracellular regulatory mechanism (73). In spite of these
differences, TfR2 interacts with Tf in a similar fashion as TfRl. Specifically, TfR2
exhibits changes in binding affinity to Tf as a result of environmental pH and the
iron saturation status of Tf. Specifically, TfR2 does not bind very well to apo-Tf at
physiological pH, demonstrating a preference for holo-Tf, while an acidic milieu
results in preferential binding of the apo-Tf (73). While there has been some
indication that TfR-2 expression levels may be a marker of erythroid precursor cells,
as levels of TfR2 mRNA levels in erythroleukemia marrow samples are significantly
elevated (74; 75), most of the data seem to support a role for TfR2 in modulating
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iron homeostasis. For example, mutations in TfR2 have been associated with a form
of hereditary hemochromatosis that is not associated with forms of hemochromatosis
that arise from mutations in the hemochromatosis protein (HFE) (76-79), suggesting
role for TfR2 in excessive iron uptake. In addition, TfR2 has also been implicated in
regulating expression of hepcidin, a modulator of dietary iron uptake (80).
1.2.2.2 Regulation of expression and tissue distribution
TfRl expression is regulated at the post-transcriptional level by interactions between
iron-regulatory proteins (IRP) and iron-response elements (IRE). The IRE are
portions of the 3’-untranslated portion of TfR mRNA that form secondary structural
features in the shape of five hairpins (81; 82). These hairpins are required for the
iron-dependent regulation of TfR expression. The IRE of TfR mRNA are recognized
and bound by the IRP, thus controlling the extent of TfR mRNA translation and
stability (83). Under iron deprivation conditions, IRP bind to the IRE increasing the
stability of the mRNA and thus up-regulating the expression of TfR (84-86). There
are two distinct IRP that interact with TfR IRE, known as IRPl and IRP2. BRPl and
IRP2 respond to variations in iron concentration via differing mechanisms (83; 87).
IRPl has been considered to be a bifunctional enzyme, functioning in a
similar fashion to aconitase when cellular iron levels are plentiful, with no RNA
binding ability, and subsequently switching to a predominantly RNA binding protein
functionality when cellular iron levels are low (88-92). IRPl also shows dual
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functionality with regard to regulation of intracellular iron concentrations. For
example, not only does it bind the IRE of TfR to up-regulate TfR production, but
evidence suggests that in iron-rich conditions it also binds the 5’-region of ferritin
mRNA to inhibit the complete translation of the ferritin protein(93-96). The
resultant down-regulation of ferritin would conceivably result in the complementary
liberation of free iron from intracellular storage.
In contrast, IRF2 synthesis is directly affected by intracellular iron levels,
with regulation occurring at the ubquitination level (97-99). For example, when iron
stores are plentiful, IRP2 rapidly undergoes ubiquitination and is degraded in the
cellular proteosome machinery (97). In conditions where intracellular iron levels are
low, IRP2 is up-regulated by the renewed production of non-proteosome directed
protein (100; 101). While there are differences in the regulation of IRPl and IRP2 in
response to iron levels, their functional significance in relation to TfR is not
necessarily mutually exclusive (102). While constitutive expression of mutant
inactive IRPl in vitro may adversely affect normal iron-metabolism (103), in vivo
data with mice that are IRPl deficient demonstrate no abnormalities in iron
utilization, implying that when intracellular IRPl is lacking, IRP2 may be sufficient
to successfully regulate TfR expression (104).
The regulation of TfR2 is not well understood at the cellular level. For
example, in contrast to TfRl, chelation of iron does not up-regulate the expression of
TfR2 (105). Indeed, in several instances TfR2 behaves in a manner opposite to
TfRl. For example, in liver development, TfR2 is up-regulated, while TfRl is
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down-regulated; conversely, during erythrocytic differentiation of murine
erythroleukemia cells, expression of TfRl steadily increases, while TfR2 expression
is down-regulated (75).
TfR is expressed in many tissue types in the body, particularly those areas
that feature a normally high turnover of cells. The level of receptors is particularly
high in erythrocyte precursors, placenta, and the liver (106-110). Of particular note
is the presence of TfR in appreciable amounts on in the capillaries of the brain (111),
epithelium of the small intestine (112; 113), type II pneumocytes (114), and
neoplastic carcinomas (115). Knowledge of the TfR present in these tissues had led
to the focus of targeting of TfR for the delivery of therapeutic molecules.
1.2.3 Proteins associated with transferrin and transferrin-receptor
I.2.3.1 HFE
Hereditary hemochromatosis is a genetic disorder that affects approximately 1 in 200
Caucasians. It is a disease characterized by excess dietary iron absorption and iron
deposition in several tissues. Clinical consequences include hepatic failure,
hepatocellular carcinoma, diabetes, cardiac failure, impotence, and arthritis.
Although an increase in plasma iron levels may be evident early in life, in most cases
these clinical symptoms do not present themselves until age 40 to 50, as iron-levels
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are slowly but steadily increased throughout the life of the individual (116; 117).
The symptoms of hereditary hemochromatosis are thought to derive from a defect in
the hemochromatosis gene which results in a decrease in the amount of functional
gene product, known as HFE, a protein closely related to the major
histocompatibility complex class I molecules (118). HFE is preferentially expressed
in duodenal crypt cells and Kupffer cells of the liver, predominantly localized to the
perinuclear region of the cell (119; 120). The HFE protein is a 343-residue type I
transmembrane glycoprotein that contains a membrane bound heavy chain (like
MHC class I proteins) with three extra-cellular domains (121). However, unlike the
MHC class proteins, two of the helices in the extra-cellular domain are spaced much
closer together, eliminating what would normally be the peptide-binding groove for
the MHC class I proteins. This difference reflects the fact that HFE is important for
modulating Tf-mediated iron uptake rather than peptide binding (122; 123). The
majority of hemochromatosis patients have a defect in a single codon of the gene that
results in the mutation of Cys282 to Tyr282, the result of which is the abolishment of
a crucial disulfide bond in HFE (124; 125). Shortly after the discovery of the HFE
gene, it was shown that HFE closely interacts with the Tf receptor (121; 122; 126;
127). In addition, confirmation of HFE’s role in hemochromatosis was provided by
gene knockout studies in mice where the HFE knockout mice demonstrated elevated
Tf saturation and increased iron storage in hepatocytes by 10 weeks of age (128).
HFE binds to TfRl with a high affinity at physiological pH of 7.4, similar in
magnitude to Tf, and it competes with Tf for binding to TfR, reducing the affinity of
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the receptor for Tf in a noncompetitive manner (123; 129). Unlike apo-Tf, HFE has
very low binding affinity for TfR at pH 6.0, which is representative of the
environment that would be seen in the early part of the endosomal pathway (121).
HFE is closely related to the TfR-specific iron uptake pathway (130-132), and it co-
localizes with TfR rich intracellular compartments (127) and with TfR on the
basolateral surface of enterocyte precursor cells (120). It has been shown that HFE
binding to TfR is required for HFE to be transported to TfR positive endosomes and
for the regulation of iron homeostasis in cultured cells, but binding is not required for
HFE to be transported to the basolateral membrane (133). While HFE has been
shown to avidly bind to TfRl, it has very little affinity for TfR2 (134). HFE, through
its interactions with Tf and TfR, is implicated in sensing and regulating iron-levels in
the body, particularly at the level of iron uptake through the duodenal enterocyte. It
is possible that HFE is not the sole mechanism by which the iron-body stores are
regulated, as HFE deficient mice are still capable of regulating uptake of iron from
the diet to some extent (128; 135), but HFE plays a vital role in regulating iron-
stores. The exact mechanism of HFE and Tf/TfR interactions that lead to iron uptake
regulation remains to be discovered.
1.2.3.2 Cubilin and Megalin
Cubilin and megalin are large cell-surface receptor proteins that are the mediators of
endocytic uptake for a wide array of ligands. These two proteins, depending upon
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the tissue, are closely associated but structurally very different. Both receptors are
expressed primarily on the apical surfaces of polarized epithelial cells. Megalin and
cubilin bind distinct ligands with variable affinities. In some cases, the two proteins
act independently of one another, while in other situations the two receptors are co
expressed and act in concert with one another as a dual-receptor complex. The
cubilin/megalin receptor system is of interest to this review because recent evidence
has suggested that this receptor system is a major pathway for the uptake of Tf from
the apical membranes of polarized epithelia.
Cubilin is a 3600 amino acid protein with a non-glycosylated molecular
weight of approximately 400 kDa. Post-translational modification of the protein
results in the mature protein with a molecular weight of 460 kDa (136; 137). The
complete cDNA sequence has been elucidated for human (138), canine (139), and rat
(137); it shows a high degree of homology between the species. Analysis of the
sequences revealed that cubilin is composed of an initial region of roughly 110
amino acids followed by eight epidermal growth factor (EGF)-like domains
followed by 27 CUB (complement subcomponents Clr/Cls, EGF-related sea urchin
protein and bone morphogenic protein-1) domains (140). While cubilin is believed
to be bound to the plasma membrane of the cell at its amino terminus (141), no
transmembrane segments have been identified in the protein, and it does not have a
glycosylphosphatidylinositol (GPI)-anchor. Helical plotting of the amino-terminal
region of cubilin has suggested that an amphipathic helix is formed by the protein
that is similar to the lipid binding regions of apolipoproteins (141). It has been
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suggested that cubilin is anchored to the plasma membrane by the insertion of the
hydrophobic residues of the conserved helices into the lipid bilayer, as is postulated
for other proteins (142-146). In addition, cubilin has a potential palmitoylation site at
the amino terminal region which could further assist in anchoring the protein to the
membrane (147; 148).
Megalin is a 4600 amino acid type I transmembrane receptor protein with a
non-glycosylated molecular weight of roughly 517 kDa. Post-translational
modification results in the mature protein with a molecular weight near 600 kDa.
Megalin belongs to the low-density lipoprotein receptor family (149). The complete
cDNA sequences have been elucidated in rat (150)and human (151); there is 77%
identity between the species. The ectodomain of megalin consists of four lipid
binding regions composed of cysteine-rich complement-type/LDLR class A repeats.
These are separated by 17 EGF-like repeats and eight cysteine-poor spacer regions,
which contain YWTD motifs and are involved in the pH-sensitive release of ligands
in endosomal compartments (152). The short carboxy-terminal cytoplasmic tail of
megalin mediates clustering in clathrin coated pits and it contains certain motifs that
have been implicated in the genesis of signal transduction (153; 154).
Megalin and cubilin are co-expressed at the plasma membrane and
throughout the endosomal system of epithelial cells. In particular, they are expressed
in several absorptive epithelia, including the placental cytotrophoblast (155; 156),
visceral yolk sac (157; 158), renal proximal tubule (157; 159-161), and the small
intestine (162; 163). In the kidney proximal tubule and visceral yolk sac, the two
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receptors have been colocalized at the brush border membrane, coated pits, endocytic
vesicles and dense apical tubules, which comprise the membrane-recycling
machinery in these cells (157; 164; 165). In addition, studies have also suggested
that megalin and cubilin have some limited presence in lysosomal compartments.
Cubilin has recently been shown to be a receptor for Tf. In an elegant study
by Kozyraki et a l, the endocytosis of Tf by cubilin was shown to be accomplished in
a megalin-dependant fashion (166). This hypothesis is supported by the finding that
lysosomes of human, dog, and mouse renal proximal tubules strongly accumulate Tf,
whereas no Tf is detectable in the endosomal/lysosomal system of the renal tubule
epithelium of dogs with deficient surface expression of cubilin. Elevated levels of Tf
are seen in the urine of these dogs. Mice with deficient expression of megalin also
excrete excessive levels of Tf in the urine. In addition, it was demonstrated that in
megalin-deficient murine kidney-proximal-tubule epithelial cells, Tf is localized to
the lumenal plasma membrane, but not in endocytic vesicles, indicating defective
internalization of the cubilin/megalin complex. Cubilin may be responsible for a
major portion of the catabolism of Tf in the body. After the liver, the kidney is the
site of the majority of Tf catabolism. In addition, the cubilin-mediated endocytosis
and lysosomal-routing of Tf in the kidney may have an indirect down-regulating
effect on the intestinal uptake of iron via the maintenance of plasma iron levels.
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1 3 The Recycling of Transferrin Receptors
Transferrin receptors have been studied extensively in filter-grown Madin-Darby
Canine Kidney (MDCK) cells, a well characterized model system to study vesicular
transport in polarized epithelial cells (167). In MDCK cells, endocytosis occurs
from both apical and basolateral plasma membrane (168; 169) and the basolateral
and apical endosomes appear to be interconnected (170-172). For example, Odorizzi
et al. reported the colocalization of the apically and basolaterally internalized Tf
receptors in the endosomal tubules in the cytoplasm of MDCK cells (172). The
internalized Tf receptors were transported to the basolateral membrane in a signal
dependent manner (172). However, Tf recycling in MDCK cells has been shown to
occur from both the basolateral early endosomes and the endosomes located close to
the centrioles in the cells (173; 174). Sheff et al. described two Tf recycling
pathways with different kinetics in MDCK cells (174). In their analysis, they used a
model that was defined by a series of first order rate constants for the transfer of the
ligands, such as Tf, between different compartments. The majority of Tf (65%) was
found to be recycled from the early endosomes within 10 minutes after
internalization (174). The slow phase of Tf recycling occurs from the recycling
compartment (174). A recent investigation of Tf recycling in CHO cells in which
perinuclear RE was removed microsurgically, showed the existence of distinct
recycling endosome populations in the cytoplasm (175). The endosomes appeared to
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correspond to the early endosomes (EEAl-positive), and the recycling endosomes
(EEAl-negative), which recruited Rabl 1 with time (175).
1.3.1 Rab Proteins Involved in Transferrin Trafficking
Rab proteins belong to a family of monomeric GTPases with molecular masses of
20-40 kDa that are involved in different intracellular processes including regulation
of vesicular trafficking pathways (176). The small GTPases Rab4, Rab5, and Rabl 1
have been studied extensively, and Tf in the endocytotic route has been found in
structures containing Rab5, Rab4 and R abll. There are three endosomal
populations. These are characterized by structures which contain only RabS, both
Rab5 and Rab4, and both Rab4 and R abll, respectively (177). RabS, which is
localized primarily to the early endosomes, can be detected in low abundance in
Rab 11-positive recycling endosomes (178; 179). Tf colocalizes with RabS in
clathrin coated vesicles and within RabS-positive endosomes (177; 179). It was
reported that overexpression of the dominant positive mutant of RabS, RabS(Q79L)
inhibits the exit of Tf from the RabS-positive endosomes to the Rabl 1-positive
recycling endosomes (180). On the other hand, the rates of TfR-mediated
transcytosis and recycling were not affected by the overexpression of RabS(Q79L)
in Hela cells. This suggests that the GTP hydrolysis by RabS is not rate-limiting for
the endocytosis of TfR (181), and Tf receptors that are accumulated in the early
endosomes may be recycled back to the plasma membrane by the fast recycling
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pathway. After the discovery of RabS isoforms, RabSb and RabSc, the RabS protein
was renamed RabSa (182; 183). These three isoforms share all the structural features
required for the regulation of the endocytosis. Both RabSb and RabSc increased the
rate of Tf endocytosis in vivo (182). They were also shown to stimulate the early
endosome fusion in vitro (182). However, the RabS isoforms are phosphorylated by
different kinases in vitro (184).
Rab4 is associated with early endosomes (18S) and recycling endosomes, but
not with plasma membrane (186). It appears to be less abundant on recycling
endosomes compared to early endosomes (174). In addition, it is phosphorylated by
p34cdc2 kinase (187). In MDCK cells cotransfected with human TfR and either
Rab4 or the dominant positive mutant of Rab4(Q67L), Tf receptors have been found
to redistribute from the basolateral surface to the apical surface (188). An
enhancement of the apically-directed transcytosis of Tf has also been observed in the
transfected MDCK cells and Rab4 (188). Expression of the dominant negative
mutant of Rab4(S22N) or of the chimeric NHRab4cbvn, in which the carboxyl-
terminal Cys-Gly-Cys motif of Rab4 was replaced with the transmembrane domain
of cellubrevin, did not show any effect on Tf transcytosis in MDCK cells (188; 189).
These results suggest that not only binding to GTP, but also the routing between
membrane and cytosol phases, are important for the function of Rab4. However, the
role of Rab4 in the TfR recycling is not completely understood. Chavrier et al. has
suggested that Rab4 is involved in Tf transport between early and recycling
endosomes (190).
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The presence of Rabl 1-positive membranes which are enriched in actin,
annexin II and t-SNARE syntaxin 13 has been reported (179). This is consistent
with the role of actin filaments in TfR recycling (191). The polarized recycling of Tf
in MDCK cells was inhibited by latmnculin B, which depolymerizes the actin
filaments (192). In Caco-2 cells latrunculin A, a latmnculin B analog, caused 20%
missorting of pre-intemalized Tf to the apical side (193).
R abll has been detected in several different cell types, including epithelial
cells (194). The expression of Rabl 1 in the apical vesicular population in epithelial
cells was reported by Goldenring et al (194). Other studies also reported the
presence of R abll in the pericentriolar recycling endosome (180; 195; 196) and
trans-Golgi network (197; 198). Rabl 1 is also involved in the recycling of
intemalized Tf (180). Expression of a dominant negative mutant of R abll inhibited
the slow phase of Tf recychng (180). The constitutively inactive mutant of Rabl 1
(Rabl 1S25N) was shown to block the Tf recycling at both 16°C and 37°C, while the
wild type Rabl 1 and the dominant positive mutant form of Rabl 1 (Rabl 1Q70L)
inhibited the Tf recycling only at 37°C (196). Therefore, the activation of Rabl 1 by
GTP appears to be required for the exit of Tf from early endosomes towards either
the recycling endosome or the plasma membrane (196).
The Rabl 1 subfamily consists of two isoforms, Rabl la and Rabl lb, with a
homology of about 89%. Rabl lb has been shown to colocalize with intemalized Tf
in the pericentriolar recycling endosome and is essential for recycling of Tf to the
plasma membrane (199). Rab25, a small GTPase closely related to R abll,
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colocalizes with R abll in MDCK cells (167). The rate of IgA transcytosis and of
apical recycling of intemalized ligands was decreased upon the overexpression of
Rab25 in MDCK cells (167). The basolateral recycling of intemalized Tf was not
affected by overexpression of Rab25, suggesting that Rab25 may play a role in
apically directed trafficking (167).
1.3.2 The R ab ll Interacting Proteins
A number of rabl 1 interacting proteins including R ipll (200), Rab Coupling Protein
(RC?) (201), Rabl 1-Family Interacting Proteins, Rabl 1-FIPl, Rabl 1-FIP2, Rabl 1-
FIP3 (202), and Rabl 1-FIP4 (203) have been identified. The interactions between
Rabl 1 and Rabl 1-FEPs primarily involve the C-terminal domains of the FBPs, which
includes the Rab binding domain (RBD) (201-204).
RCP interacts with both Rab4 and R abll. It is likely to be involved in Tf
recycling (201). Rabl 1-FIP2 and Rabl 1-FIP3 have been localized to the rabl 1-
positive compartment in MDCK and HeLa cells (202). Rabl 1-F1P2 appeared to be
involved in recycling of Tf (204). Rabl 1-FIP4 colocalizes with both Rabl 1 and Tf
in HeLa cells (203). It interacts with Rabl 1 in a GTP-dependent manner (203). The
expression of a mutant form of Rabl 1-F1P4 (rabl l-FIP4(C-ter)) did not show any
effect on Tf recycling in HeLa cells but dispersed the Rabl 1-positive compartment
toward the peripheral region of the cell (203).
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1.3.3 RabS Effectors, EEAl and PI(3)K, Involved in Early Endosome Fusion
Early endosomal antigen 1 (EEAl), a hydrophilic 162kD protein localized to the
early endosomal membrane, plays a critical role in the fusion of early endosomes
(181; 205; 206). It has also been found as a homodimer in the cytosolic pool (206;
207). EEAl functions as a tethering protein for two RabS positive membranes (208)
which may facilitate the pairing of SNARE proteins (209) and subsequently
membrane fusion (207; 210). It contains two binding sites for RabS at the N and C
termini (208). Association of EEAl with the endosomal membrane appears not to be
dependent on its interaction with RabS (211). However, the interaction between
RabS and EEAl may regulate early endosome fusion (211). According to Stenmark
et al., the endosomal localization of EEAl requires the presence of a FYVE domain
(212), a cysteine-rich domain at the C-terminal of EEAl protein which binds two
zinc ions (20S; 212) and has a binding site for PI(3)P (213-216). The interaction
between the FYVE domain and PI(3)P is not only specific (217), but also necessary
and sufficient for the binding of EEAl to the endosomal membranes (211). PI(3)P
on the early endosomes or endocytic vesicles could be generated by the recruitment
of PI(3)kinase hVPS34 by RabS-GTP (218), which limits the recruitment of EEAl to
a specific region of the membrane (20S; 208). In addition, the interaction between
hVPS34 and RabS may be mediated by hVPS34-associated protein plSO (218).
So far, two distinct PI(3)Ks, hVPS34 and p8Sa-pl lOp, have been found to bound to
RabS (218). Class-HI PI(3)K hVPS34 is the only PI(3)K required for early
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endosome fusion, and PI(3)P is the most important 3'-phosphatidylinositide in the
fusion process (218). The class-I PI(3)K, p85a-pl lOp, which produces
phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate,
may be involved in the formation of CCVs at the plasma membrane (218). The
involvement of p85a-pll0P in Tf recycling has also been demonstrated. Activation
of class-I PI(3)K by insulin receptor enhanced the rate of Tf recycling while
antibodies against the pi 10a subunit of PI(3)K inhibited Tf recycling (219; 220).
The involvement of PI(3)Ks in Tf transcytosis has also been studied by using
PI(3)K inhibitors, such as wortmannin and LY294002. Wortmannin is a
noncompetitive, irreversible inhibitor of mammalian PI(3)Ks at low concentration
(100 nM), while the inhibitory effect of LY294002 is more reversible and specific
for PI(3)Ks (221-223). The rate of the Tf recycling was reduced by Wortmannin and
LY294002 (224-227).
According to van Dam et al. Tf receptors may be recycled back to the plasma
membrane via two different independent recycling pathways that require the
activities of PI(3)K and dynamin (228). Tf receptor recycling from perinuclear
recycling endosomes was inhibited upon the overexpression of a dominant negative
dynamin-1 mutant (dynts), which accumulates clathrin-coated buds on endosomes
(229). An accumulation of endocytosed Tf in the early endosomes was observed in
the presence of LY294002 indicating that PI(3)K activity is required for the Tf exit
from early endosomes (228). No interference between LY294002 treatment and Tf
transport between either early endosome and recycling endosome or recycling
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endosome and the plasma membrane was reported, suggesting that LY294002 may
inhibit the Tf recycling from the early endosomes to the plasma membrane (228).
The combination of LY294002 and BFA, which appeared to inhibit the assembly of
clathrin coats on recycling endosomes, had a synergistic inhibitory effect on Tf
recycling (228).
1.4 Utilization of TfR for drug delivery
TfR targeted therapy has emerged as an interesting drug-delivery tool with dual
functionality. For example, targeting of TfR can lead to delivery of therapeutic
agents into tissues of choice or across epithelial barriers of choice. The seemingly
contradictory effects can be achieved by focusing the targeting strategies toward
different aspects of TfR-related biology. This section focuses on recent advances in
TfR-facilitated drug delivery.
1.4.1 TfR-based targeting for anti-cancer therapeutics
One of the most well explored avenues for TfR-based drug targeting strategies is the
use of anti-cancer based therapeutics conjugated to Tf (or TfR recognizing antibody)
to preferentially direct the drug to TfR-rich cancer cells. High levels of TfR
expression have been demonstrated in many tumors (108),(230-234) and importantly,
studies have also shown that the TfR is expressed more abundantly in malignant
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tissues than their normal counterparts (108; 115; 233; 235). TfR is also more
abundantly expressed in rapidly dividing cells than quiescent cells (236; 237)
because of its pivotal role in iron uptake and the absolute requirement for iron in
rapid cell proliferation. Therefore, the TfR expressed on cancer cells has been seen as
a suitable target for the delivery of therapeutics by receptor-mediated endocytosis.
Many anti-cancer agents have been considered for conjugation to Tf by
varying methods, including direct chemical linkage (238; 239), liposomal packaging
of toxin and linkage to Tf (240; 241), conjugation of DNA/polylysine complexes to
Tf (242-245), and conjugation of liposome/DNA complexes to Tf (246-252).
A great variety of cytotoxic agents have been conjugated to Tf and
investigated as potential anti-cancer therapeutics. Some of the more notable
examples have been methotrexate (238; 240), doxorubicin (238; 239), cisplatin
(253), ricin A (254-257), daunorubicin (241; 258), and toxin CRM107 (259; 260).
Conjugation of these toxins to Tf has the dual benefits of reducing toxicity in
undesired tissues and increasing the targeting efficiency to the cancerous cells. Of
special note is that conjugation to Tf significantly enhances the effectiveness of these
agents in many multi-drug resistant cell lines. For example, Tf-doxorubicin was 5 to
10 times more effective than doxorubicin-control in killing doxorubicin-resistant cell
lines, whereas in doxorubicin-sensitive cell lines, the conjugate was only 4 to 5 times
more effective than control (261). The Tf-based enhancement effect becomes even
more dramatic as one considers the very highly resistant KB cell line. In this case,
the Tf-doxorubicin conjugate exhibited an IC50 value as low as 0.025 pM, while the
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un-conjugated doxorubicin demonstrated no cytotoxic effects with concentrations as
high as 1 |iM (262). The exact mechanism by which Tf conjugation allows
doxorubicin to effectively bypass the drug-resistance machinery of the resistant cell
lines is not fully understood; however, it is conceivable that internalization of the Tf-
doxorubicin conjugate allows for sequestration into the endocytic pathway, away
from drug efflux proteins (such as PGP and/or MDR), which normally reside at the
plasma membrane.
A variation on the theme of using toxins conjugated to Tf has been
investigated to increase efficiency of uptake at the cellular surface. This approach
relies on the formulation of the toxin in liposomal delivery vehicles to further
enhance the efficiency of cellular uptake. For example, methotrexate that has been
formulated in a liposome complex and conjugated to Tf demonstrated a higher
efficiency of cellular uptake and cytotoxicity compared to free methotrexate and
methotrexate-liposome control (240). In a similar fashion, when daunorubicin-
containing liposomes are conjugated to 0X26 ant-rat TfR antibody, they show an
increased delivery and uptake in the CNS relative to the non-OX26 containing
liposomes (241).
An alternate approach toward treating cancerous tissues relies upon gene
therapy to correct errors in genes, such as the tumor repressor gene p53, rather than
killing the cell. Tf-conjugation has been investigated as means to overcome some of
the limitations of cationic liposome-based p53 gene delivery vehicles, such as poor
stability in serum, binding to serum proteins, and lack of specific targeting (263-
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267). Tissue culture experiments have revealed that the Tf conjugate significantly
enhanced the transfection efficiency of the p53 gene in cancerous cell lines
compared to the control DNA-liposome (265). This enhancement was seen even in
the presence of high concentrations of serum. In addition, experiments with nude
mice containing subcutaneous DU-145 tumors revealed that administration of Tf-
liposome conjugate was sufficient for high levels of p53 gene expression in the
tumor. This contrasts with the non-Tf bearing p53-liposome which did not
demonstrate any p53 gene expression in the tumor (267). This strategy has even
developed to the point where beneficial effects are seen in human patients. For
example, when a cationic liposome (carrying p53 DNA) was investigated as a Tf
conjugate, it demonstrated high efficiency in p53 gene delivery to human head and
neck, prostate cancers, as well as long-term therapeutic benefits. For example, when
Tf-liposome therapy was combined with conventional radiation therapy, complete
tumor regression was observed in human prostate cancer and the treatment group
showed no signs of relapse up to six months later (266). Using similar plasma
membrane permeability-enhancing logic as the DNA/liposome complexes, reports
have also focused on DNA/polycation complexes conjugated to Tf to increase
uptake. Comparable benefits in enhanced uptake have been observed for the
polylysine and polyethylenimine containing complexes (242; 245; 268-273). Further
developments in Tf-based targeting of anti-cancer drugs will most likely play
important roles in anti-cancer therapy in the future.
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1.4.2 TfR-mediated transepithelial protein-drug delivery
Modem biotechnology has produced many potent and beneficial drags, yet most of
the products suffer from limited choices regarding potential routes of administration.
The large sizes and charges of protein drags prevents their passage across epithelial
barriers. TfR has emerged as a potential mediator to enable the transport of these
large molecules across epithelial barriers.
1.4.2.1 anti-TfR antibody drug conjugates for transepithelial drug delivery
The capillaries of the brain have been well known to have relatively high levels of
TfR, and since the blood brain barrier (BBB) effectively excludes many therapeutic
drags, including almost all peptide and protein-based therapeutics, targeting of the
BBB’s TfR for drag delivery has been seen as an attractive strategy. However,
utilizing a Tf-based drag conjugation approach is seen as undesirable for trans-BBB
delivery because under physiologic conditions the TfR of the BBB are postulated be
nearly saturated with Tf, due to the relatively high amounts of Tf in serum (274). A
viable altemative to target BBB TfR has been shown to be antibodies that
specifically recognize TfR, with or without the presence of bound Tf (275-278). In
particular, 0X26, a monoclonal antibody for rat TfR that has been the subject of
most of the research in this area, demonstrated an ability to preferentially target the
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TfR of the BBB (111). OX-26 is postulated to not interfere with the normal
functioning of iron uptake via TfR, since the antibody binds an extra-cellular region
that is distinct from the binding pocket for Tf (111).
Upon binding to TfR at the BBB, 0X26 exhibits superior transport abilities
into the CNS. Studies performed with isolated bovine brain capillaries demonstrated
that nearly 50% of bound radiolabeled 0X26 was taken up via endocytosis during a
two hour incubation period (279). In addition, carotid artery perfusion studies
indicate that more than 65% of the administered 0X26 passed through the BBB and
into the extracellular space. This value was significantly higher than for the control
proteins, bovine serum albumin and IgG, thus implying that transcytosis via TfR
significantly enhanced the transport of 0X26 across the BBB. Intravenous bolus
administration of 0X26 also resulted in a steady accumulation of radiolabeled 0X26
in the brain during a five-hour time period, while radiolabeled 0X26 was rapidly
cleared from serum after injection. This suggests that 0X26 rapidly binds TfR at the
BBB and is subsequently endocytosed across the BBB in a more gradual fashion
(279).
The next logical course of action was to determine if 0X26 could enable the
efficient CNS uptake of therapeutics that would normally exhibit poor CNS-
bioavailability. The strategy most commonly reported in the literature involved the
creation of OX26-therapeutic conjugates based on strepavidin/biotin linker
technology. The avidin/biotin linkage between 0X26 and the therapeutic entity has
been created using chemical based and genetic-cloning based strategies. Several
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promising 0X26 conjugates have been created, using a vasoactive intestinal peptide
analog (280), brain-derived neurotrophic factor (BDNF) (281-283), and basic
fibroblast growth factor (bFGF) as the therapeutic cargo. (284; 285). Depending
upon the pharmacokinetic properties desired, the conjugates have been prepared with
avidin-modified 0X26 biotin conjugated to the drug directly or via a PEB 2000
spacer. When BDNF-PEG2000-SA-OX26 was administered to rats for one week
after an induced episode of transient forebrain eschemia, a neuroprotective effect
was observed. Specifically, the neuronal density in the hippocampus of the BDNF-
PEG2000-SA-OX26 treatment group was maintained, while the group receiving pure
BDNF exhibited a 68% decrease compared to control (286). In addition, BDNF-
0X26 conjugate demonstrated effectiveness in ameliorating the effects of acute
stroke. For example, when adult rats were subjected to 24 hours of middle cerebral
artery occlusion, the infarct volume was found to decrease in a BDNF-OX26 dose
dependent fashion. Reduction in stroke volume was significant even if
administration of BDNF-OX26 was delayed for one to two hours after the occlusion
event (281; 282; 287; 288).
bFGF is an attractive drug candidate for 0X26 conjugation because it retains
receptor-binding affinity and has increased brain uptake following conjugation to
OX26-SA. Studies also have demonstrated that bFGF-OX26 conjugate protects
cortical cell cultures against hypoxia/reoxygenation insult in a dose-dependent
manner in vitro (285). In addition, in vivo studies showed that a single intravenous
injection of bFGF-OX26, equivalent to a dose of 25 pg/kg bFGF, produces an 80%
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reduction in infarct volume in the brains of rats subjected to permanent occlusion of
the middle cerebral artery. This result paralleled with a significant improvement of
neurologic deficit. The neuroprotective action was also time-dependent, with a 67%
reduction in stroke volume if bFGF-OX26 was administered 60 min after arterial
occlusion, whereas no significant reduction was observed if treatment was delayed
by 2 h (284; 285).
In addition to the delivery of large therapeutic molecules across the BBS,
TfR based transcytosis has also been explored for targeted gene delivery to the CNS.
For example, high levels of reporter gene expression have been observed when
plasmids encoding for luciferase or p-galactosidase are enclosed in liposomes that
are conjugated to 0X26 via a PEG2000-thio-ether linkage were administered
intravenously to rats (289-291).
1.4.2.2 Enhancement of TfR-mediated drug delivery
In addition to the use of Tf-drug conjugates to target therapeutics to select tissues
from the blood stream, Tf based conjugates have also been considered for the
systemic non-invasive delivery of therapeutics. Several absorptive epithelia have
been shown to express TfR, such as the small intestine (112) and type II
pneumocytes (114), providing a potential target to enable transepithelial delivery of
large bio-active molecules that would normally have negligible bio-availability when
administered orally. One of the problems associated with this approach is the fact
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that most of the surface TfR in differentiated epithelial cells lies at the basolateral, or
serosal, membrane (292). This would tend to preclude the ability to achieve apical-
to-basolateral transcytosis of Tf-drug conjugates. While there is indication that
transepithelial transport may still be feasible (especially in areas where large
numbers of semi-differentiated cells may occur, such as the crypt of Lieberkuhnin of
the small intestine), enhancers of Tf-conjugate transepithelial transport have been
examined as a means to overcome the problem of basolateral-localized TfR polarity.
1.4.2.2.1 Lysosomotrophic Amines
Lysosomotrophic amines (such as ammonium chloride and chloroquine) have been
considered as a means to enhance Tf-drug conjugate transepithelial transport. Most
of these agents increase intraendosomal and intralysosomal pH, with a concomitantly
enhanced accumulation of radiolabeled Tf inside the cell. However, these agents also
seem to cause a non-specific enhancement of ^^^Tf transcytosis. For example when
MDCK monolayers were treated with NH4CI (20 mM), a 7-fold increase in
basolateral-to-apical transcytosis was observed. The increase in transcytosis was
thought to be derived from the overloading of labeled ^^Tf inside the cell (293). The
serious drawback to these agents is the nonspecific alteration of membrane
permeability and integrity.
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1.4.2.2.2 Lipophilic carboxylic ionophores
Carboxylic ionophores, such as monensin and nigericin, exhibit effects similar to
lysosomotrophic amines in altering the pH of endosomal and lysosomal
compartments (294). Monensin, is a Na'^-ionophore that can elicit a variety of
effects in mammalian cells, including alterations of vacuolization and vesicle sorting
in the Golgi and trans-Golgi networks. Treatment of MDCK monolayers with 6 pM
monensin resulted in a 24-fold increase in TfR-mediated transcytosis. The large
increase in TfR-mediated transcytosis appeared to originate from a different mode of
action than lysosomotrophic amines. This was presumed to result from the effects of
monensin on trans-Golgi sorting, since TfR is routed to the TGN as a part of the
normal course of its intracellular sorting (295; 296). The development of monensin
as a useful therapeutic agent has been hampered by the highly lipophilic nature of the
drug, which is reflected in its poor stability and potentially short serum half-life.
1.4.2.2.3 Brefeldin-A
Another transcytosis enhancing reagent that has been shown to have an effect in
modulating the trans-Golgi network is the fungal metabolite, brefeldin-A (BFA).
BFA has been extensively used in the study of intracellular trafficking events (297).
It has dramatic effects on the structure and function of the Golgi complex and the
endosomal sorting pathways (298-300). For example, BFA causes the rapid
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rearrangement of the Golgi, through microtubule dependent processes, causing
Golgi-specific markers to redistribute in a retrograde fashion back to the
endoplasmic reticulum (301; 302). In addition, the trans-Golgi network tubulates in
the presence of BFA, forming a fused trans-Golgi/endosomal network that appears to
exclude vesicles that are lysosomal in nature (298; 303). While the lysosomes do not
appear to fuse with the trans-Golgi/endosomal tubulates, they do appear to form an
extensive microtubule dependent network in the presence of BFA (298). In addition
to these effects, BFA also blocks the anterograde transport of sorted proteins from
the endoplasmic reticulum (301; 302). Unlike the reagents discussed above, BFA
does not cause a change in intra-lysosomal or intra-endosomal pH (298).
In polarized epithelial cells, BFA treatment significantly up-regulates the
basolateral-to-apical transcytosis of internalized TfR and bulk plasma membrane
(304). In spite of these observations, the Golgi of some cell lines seem to be
resistant to the effects of BFA. For example, the Golgi of MDCK cells largely
resists the effects of BFA (305). Nonetheless, BFA causes significant alteration of
trans-Golgi and endosomal function in MDCK cells (305). The fact that BFA causes
significant enhancement of TfR-specific transcytosis in this cell line would indicate
that this enhancement depends upon sorting events at the TGN/endosome level.
BFA significantly alters the intracellular sorting of internalized proteins in
polarized epithelial cells. For example, BFA treatment results in the missoiting of
basolateral LDL receptors (306), reduces the efficiency of polymeric immunoglobin
receptors to the cell surface (307). In addition, the effects of BFA on protein sorting
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appear to be much more sensitive at the apical surface, as the concentration of BFA
required to see effects at the apical surface is much lower than for the basolateral
surface (308; 309). This is significant when considering BFA as an enhancer of TfR-
mediated drug delivery. The relative preferential alteration of events at the apical
surface would imply the ability to achieve net enhancement of TfR-mediated
transcytosis in the apical-to-basolateral direction. This effect has been demonstrated
in two highly polarized cell systems, MDCK cells (292) and primary cultured rat
type n pneumocytes (310). In both of these systems, BFA treatment results in the
specific enhancement of apical-to-basolateral transport of conjugates of Tf-insulin
and Tf-GCSF (filgrastim) (292; 310). The rates of apical-to-basolateral transport of
the conjugates are significantly higher in the presence of BFA than for comparable
molar concentrations of the native protein. The enhancement of transport appears to
result from the transient missoiting of basolateral TfR to the apical surfaces of the
cells, from whence they are rapidly intemalized and redirected back to the
basolateral membrane, releasing the Tf-protein-drug conjugate into the basolateral
fluid. This is evidenced by results which demonstrate that BFA-treatment does not
increase the static level of TfR at the apical surface, as measured by *^^I-Tf binding
at 4° C, but rather increases the rate of TfR internalization, as measured by ^^Fe-Tf
uptake. While BFA does affect the trans-epithelial electrical resistance (TEER) of
the monolayers, the effect is transient and TEER retums to normal levels once the
BFA is removed (292; 310).
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1.4.3 Modification of Rab-dependent sorting pathways for enhancement of
TfR-mediated gastrointestinal transepithelial transport
While investigations into BFA-induced enhancement of TfR-mediated
transport have demonstrated a significant elevation in Tf-protein-drug conjugate
transport in polarized epithelia, the fact that BFA is a fungal metabolite makes it
undesirable in some respects. For example, BFA affects many properties of
intracellular organelles, even those that might be unrelated to TfR-based sorting or
transcytosis events. The non-specific side effects of BFA prompted efforts to
discover agents that would have the potential to be more specific in their mode of
enhancement of TfR-mediated transcytosis. As discussed in section 2, several small
GTPases of the Rab family have been shown to be involved in the endocytosis and
transcytosis of TfR. Therefore one can pose the hypothesis that chemical entities
that inhibit or alter the actions of key Rabs, or their downstream effectors, could
possibly control the sorting and recycling events of TfR to elicit a specific
enhancement of TfR-transcytosis.
Recent reports have focused on the ability of AG-10 (aka tyrphostin- 8 [T-8 ],
or 4-hydroxybenzylidene-malononitrile), a known GTPase inhibitor, to increase the
transport of an insulin-Tf conjugate across Caco-2 monolayers (in the apical-to-
basolateral direction) and enhance the oral absorption of the conjugate in
streptozotocin-induced diabetic rats across the GI epithelium (311; 312). In Caco-2
monolayers, treatment with AG-10 induced a 20-fold enhancement of TfR-mediated
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transport of insulin (via the insulin-Tf conjugate) compared to control, while similar
treatment with BFA only resulted in a 3-fold enhancement of insulin transport (312).
This enhancement of transport was determined to be dependent upon TfR-mediated
processes, since an insulin-albumin conjugate demonstrated no transport across the
monolayers, and treatment with AG-10 had no effect on its transport properties
(312). The results from the tissue-culture experiments have been further validated by
in vivo uptake studies. In diabetic rats, the orally administered insulin-Tf conjugate
demonstrated an extended duration of action, with hypoglycemic effects observed 11
hours after administration (113). In addition, AG-10 exhibited the ability to enhance
the hypoglycemic effect of orally administered insulin-Tf conjugate, relative to BFA-
treated and enhancer-free insulin-Tf treatment groups. The effects of AG-10 were
also dose dependent, indicating the observed results resulted from of the effects of
AG-10 (312).
In an effort to further increase the specificity of enhancement of TfR-
mediated transcytosis, downstream effectors of Rab proteins have been considered as
potential targets for inhibition. One of the downstream effectors of Rab proteins are
the phosphatidylinositol 3-kinases (PDK). As noted earlier, PDK’s are required for
cellular membrane trafficking and they have been implicated in the recycling of TfR.
For example, the PDK inhibitors, LY294002 and wortmannin, have been shown to
reduce the rate of Tf recycling (224-227; 313; 314), whereas activation of PDK
activity is known to accelerate the rate of TfR recycling (315; 316). One can
hypothesize that inhibition of recycling of basolaterally derived TfR may result in an
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increase of TfR txaffic to the apical membrane, thus increasing the potential for
enhancement of TfR-mediated transcytosis. Recent results have suggested that
LY294002 is capable of enhancing TfR-mediated transcytosis in a manner that is
similar to AG-10 and different from the mode of action of BFA. For example,
LY294002 specifically enhanced TfR-mediated transcytosis of ^^^I-Tf and ^^^I-Tf-
GCSF conjugate in Caco-2 monolayers, yet demonstrated less of an effect in MDCK
monolayers. This contrasts with BFA, which has much greater enhancement ability
in MDCK monolayers compared to Caco-2 (292; 312). Caco-2 and MDCK are
known to have distinct TfR-sorting pathways, i.e. predominately direct TfR sorting
in MDCK cells, and both indirect and direct sorting in Caco-2 (312). This suggests
that AG-10 and LY294002 affect one type of sorting mechanism, while BFA affects
another. This hypothesis has been supported by a recent report, which demonstrated
that endocytosed TfR are recycled via distinct dynamin and PDK dependent
pathways, where BFA and LY294002 exhibited distinct yet synergistic inhibitory
effects (228).
1.4.4 Enhancement of TfR-mediated transport in pulmonary epithelium.
The large surface area of the lower respiratory tract presents a challenge to
pulmonary host defense. In contrast to upper airways, the epithelium of alveoli is
devoid of the protective mucus barrier and the ciliated cells that enable the constant
clearance of foreign material in the upper airways. While alveolar macrophages may
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provide a formidable first line of defense against pathogenic organisms, of particular
concern is the presence of excessive levels of free iron in the surface fluid lining the
alveolar epithelium in the distal lung (317). Increased levels of free iron in the distal
lung (as a consequence of pathological conditions such as smoking or chronic
inhalation of metallic dusts) have been associated with tissue injury and fibrosis,
primarily due to iron's ability to catalyze the formation of highly reactive hydroxyl
radicals (317-319). Excessive levels of free iron in the lower lung have also been
associated with facilitating the growth of intracellular mycobacteria (318; 320-322).
The task of sequestering free iron lies with the iron-binding proteins, of
which Tf is the primary constituent in the surface fluid lining the airspaces of the
lower lung (323). Tf is an 80 kDa glycoprotein that is responsible for transporting
iron, via its two ferric binding sites, throughout the vasculature (324). In addition to
this primary role, Tf has been implicated as the major source of antioxidant capacity
in the lung (325). Indeed, the levels of Tf, as a percentage of total protein, detected
in bronchoalveolar lavage fluid have been found to be very high (4.0-5.6%) as
compared to values for plasma (3.0%) (326; 327). The presence of such high levels
of apo-Tf in surface fluid lining the alveolar epithelium suggests that there may be a
specialized transport mechanism for Tf from the systemic circulation to the alveolar
fluid. The very tight epithelial barrier of the alveoli excludes a passive paracellular
transport route for the large Tf molecule (328), consistent with a hypothesis that the
transport of Tf into, and possibly out of, alveolar lining fluid might be regulated by
an active transcellular transport process. Endocytosis of Tf by the TfR and
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subsequent transcytosis into alveolar lining fluid would be a likely mechanism for
this process. However, the extent and nature of TfR distribution among the cells of
the rat alveolar epithelium remain unknown.
Primary cultures of rat type II alveolar epithelial cells (AEC) have been
shown to exhibit many of the properties of alveolar epithelium in vivo, including
apparent transdifferentiation from the type II cell-like to the type I cell-like
phenotype within 3-4 days in culture (329-332). Type I pneumocytes comprise
nearly 97% of the surface area of the pulmonary alveoli, yet they are slightly
outnumbered by the more numerous type II cells. The thin expansive type I cells are
thought to primarily serve to enable efficient gas exchange, while the cuboidal type
n cells exhibit essential secretory functions, such as the secretion of pulmonary
surfactant and surfactant proteins.
As a part of this thesis, we investigated the synthesis and distribution of TfR
in polarized alveolar epithelial cell monolayers. Primary cultures of rat alveolar type
n cells were used as a model system to study TfR production in type II cell-like and
type I cell-like monolayers. Under normal culture conditions, the type II
pneumocytes progress toward the type I cell-like phenotype as the cells develop tight
junctions and the monolayers develop high transepithelial electrical resistance
(TEER) (329). This transition to the type I cell-like phenotype early in the culture
period had retarded development of a model system for studying the transport
properties of monolayers with the type II cell-like phenotype. However,
keratinocyte growth factor (KGF) has recently been shown to cause retention of the
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type n cell-like phenotype in culture and prevent progression to the type I cell-like
phenotype while retaining high TEER levels (333-336). KGF exposure of AEC
monolayers was used in our study as a means to investigate the degree of TfR
synthesis in cells exhibiting the type II cell-like phenotype.
The pulmonary epithelium has become an attractive target for systemic
delivery of protein based therapeutics. The large surface area (~ 100 m^) and
extensive vascularization in the lung provide an ideal site for drug absorption.
Several protein drugs have been reported to have relatively high bioavailabilities
when delivered via the pulmonary route. However, conclusive knowledge regarding
the mechanisms of transport through the pulmonary epithelial barrier remains
elusive. In light of this, we have sought to develop a pulmonary delivery strategy
that is targeted to a known transcytosis pathway, namely, transcytosis of the TfR, in
order to obtain more predictable delivery qualities suitable for protein drugs of
varying characteristics.
TfR-mediated drug delivery has advantages over other protein drug delivery
strategies. For example, in contrast to penetration enhancers, TfR transcytosis
neither alters the cellular plasma membrane nor dismpts the cellular tight junctions.
As TfR endocytosis is an endogenous transport process, it is probable that a protein
drug delivery scheme targeting TfR would have fewer side effects compared to other
methods. In addition, Tf is an advantageous protein carrier molecule because it is
inherently resistant to enzymatic degradation processes (337-339). Primary cultures
of rat type II alveolar epithelial cells (AEC) have been shown to exhibit many of the
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properties of alveolar epithelium in vivo, including apparent transdifferentiation from
the type II cell-like to the type I cell-like phenotype within 3-4 days in culture (329-
332). Type I pneumocytes comprise nearly 97% of the surface area of pulmonary
alveoli, yet they are slightly outnumbered by type II cells. In addition to active N a"^
absorption afforded by both types of pneumocytes (340-342), the thin expansive type
I cells are thought to primarily serve to enable efficient gas exchange, while the
cuboidal type II cells exhibit essential secretory functions, such as secretion of
pulmonary surfactant and surfactant proteins.
Primary cultured type II pneumocytes usually acquire the type I cell-like
phenotype and morphology as the cells develop tight junctions leading to monolayers
with high transepithelial electrical resistance (TEER) (329). This transition to the
type I cell-like phenotype had retarded development of a model system for studying
transport properties of monolayers with the type II cell (-like) phenotype. In this
regard, keratinocyte growth factor (KGF) has recently been shown to cause retention
of the type H cell-like phenotype in culture, while retaining high TEER levels (333-
336). Thus, KGF-treated AEC monolayers were used in this study as a means to
investigate the degree of TfR transcytosis in cells exhibiting the type II cell-like
phenotype.
Our first report (114) demonstrated that TfR expression is primarily
limited to AEC monolayers of the type II cell-like phenotype, and that TfR
expression is predominantly localized to the basolateral membrane. KGF treatment
(10 ng/mL) of AEC monolayers was found to restore TfR expression and TfR-
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mediated Tf endocytosis. Later studies utilized these previous findings to develop a
TfR-mediated protein drug delivery strategy that targets TfR in type II cell-like AEC
monolayers. (343)
TfR has developed as a potential ligand to enable drug targeting and delivery
of therapeutic agents that would normally suffer from poor pharmacokinetic
characteristics. TfR-directed targeting has enabled the efficient delivery of
therapeutic agents to sites of interest, including the central nervous system and
malignant tissues. In addition, by utilizing knowledge of the intracellular sorting and
recycling pathways of TfR, including Rab and PI(3)K mediated processes, one can
maximize the transepithelial delivery of peptide-based therapeutics. Depending
upon the desired result, apparently paradoxical effects can be achieved. For
example, TfR-based strategies can achieve accumulation of the carried-drug within
targeted tissues or delivery of the therapeutic entity across tissues of interest. Further
understanding of the intra-cellular events that govern the destiny of intemalized TfR
will result in ever increasing interest in TfR as a pharmaceutically relevant molecule.
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2. MATERIALS AND METHODS
2.1 Caco-2 Cell Culture
Caco-2 cells (American Type Culture Collection, Rockville, MD; passage
number 20) were grown on 0.4 pm pore-size polycarbonate Trans well (Costar,
Cambridge, MA) filters to confluence in Dulbecco’s modified eagle media
supplemented with 10% fetal bovine serum (FBS), glutamine (2 mm), non-essential
amino acids (0.1 mM), penicillin (50 U/mL) and streptomycin (50 pg/mL). All cell
culture reagents were obtained from Invitrogen/Life Technologies (Carlsbad, CA).
The Caco-2 cells were typically seeded at 4.2 x 1 0 "^ cells/cm^. The monolayers were
also allowed to differentiate for seven days after reaching confluence, which was
typically 7-8 days post-seeding. The cells were grown in a 5% CO2 in air tissue-
culture incubator at 37° C. Media containing 10% FBS was changed every other
day. The trans-epithelial electrical resistance was measured with an epithelial
voltohmmeter (EVOM, World Precision Instruments, West Haven, CT). The
monolayers typically established maximum resistances of 500 Q®cm^.
2.2 MDCK Cell Culture
MDCK cells were cultured in polycarbonate Trans well filters (0.4 pm pore
size) in a 5% CO2 in air tissue-culture incubator at 37° C. The cells were seeded at a
density of 2.2 x 10“ * cells/cm^ in minimal essential medium (MEM) containing 10%
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FBS, glutamine (2 mm), penicillin (50 U/mL) and streptomycin (50 p,g/mL). In
order for the cells to properly develop tight junctions, a feeding protocol was used
that sequentially reduced the percentage of FBS within the culture media. For
example, after the initial seeding, the cells were allowed to grow for three days in
10% FBS containing MEM and then the media was changed to MEM with 2.5%
FBS for two days with daily feeding, and then changed to 1% FBS containing MEM
prior to using the monolayers in transport experiments. The monolayers were used
at day 7 to 8 post seeding, on average. The TEER was typically 2.5 to 3.0 kO®cm^
when the monolayers were used in transepithelial transport studies or TfR
distribution studies.
2.3 Primary Culture of Rat Alveolar Epithelial Cell Monolayers
Lungs of specific pathogen-free Sprague-Dawley male rats were perfused via the
pulmonary artery and lavaged with Ca^'^/Mg^'^ -free Ringer’s solution. The lungs
were then instilled with porcine pancreatic elastase (2.5 U/mL, Worthington
Biochemical, Lakewood NJ) for 20 minutes at 37°C. Minced lung tissue blocks were
sequentially filtered through 100, 40, and 10 |xm Nitex membranes. Alveolar
macrophages were removed from the crude cell mixture by IgG panning (344).
Partially purified type U pneumocytes were plated onto tissue culture-treated 12 mm
polycarbonate filter (Transwells, 0.4 fxm pore size. Costar Coming, Cambridge, MA)
at a density of 1.5 million cells/cm^ in culture medium composed of a 1; 1 mixture of
49
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DMEM and Ham’s F-12 (Sigma Chemical, St. Louis, MO) supplemented with 10%
newbom bovine serum, 1.25 mg/mL bovine serum albumin, 100 U/mL penicillin,
and 100 ng/mL streptomycin. Monolayers were fed on days three and five (or six)
with fresh culture medium. When KGF (hKGF, Calbiochem, San Diego, CA) was
added to the media bathing the AEC monolayers, it was included from day zero
onward at a concentration of 10 ng/mL and replenished with each feeding. The
TEER was measured with an epithelial voltage-ohm meter (EVOM, World Precision
Instruments, Sarasota, FL). Rat AEC monolayers typically achieved TEER > 2,500
Ocm^ and PD values of 10.2 ± 0.5mV. Animal experiments were compliant with the
Principles of Laboratory Animal Care' (NIH Publication #85-23) and approved by
the lACUC at USC.
2.4 Measurement of Cell Surface Transferrin Receptor on Caco-2 Monolayers
Human iron-loaded Tf (Sigma) was radiolabeled with (ICN, Irvine, CA) using
chloramine-T catalyzed modification (345), followed by purification by Sephadex G-
50 column chromatography and subsequent dialysis in phosphate buffered saline
(PBS, pH 7.8). Caco-2 monolayers were washed once with serum-free culture media
(DMEM) supplemented with 0.1% BSA and incubated at 37°C in the same media for
one hour to ensure removal of endogenous Tf. Monolayers were subsequently
treated with BFA (1.0 pg/mL), AG-10 (500 pM), or LY294002 (100 pM) and
incubated at 37° C for two hours to allow for uptake of the drugs. Transcytosis
50
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enhancer-containing media were then removed and replaced with fresh media
containing 1.5 |ig/mL in either the apical or basolateral compartment and
incubated at 4°C for two hours. Non-specific binding was measured in parallel by the
simultaneous addition of 100-fold excess unlabeled Tf in the respective bathing
fluid. TfR-specific ^^^I-Tf binding was determined as the difference between total
and nonspecifically bound ^^^I-Tf. After incubation, monolayers were washed three
times with ice-cold PBS, pH 7.4. ^^^I-Tf bound to the monolayers was measured
with a Packard Gamma Counter (Packard Instruments, Meriden, CT).
2.5 Measurement of Cell Surface Transferrin Receptor in Rat Alveolar
Epithelial Cell Monolayers
Human iron-loaded Tf (Sigma) was radiolabeled and purified as mentioned above.
AEC monolayers were washed once with serum-free culture media (MDSF)
supplemented with 0.1% BSA and incubated at 37°C in the same media for one hour
to ensure removal of endogenous Tf. Monolayers were subsequently dosed with
media containing 1.5 (ig/mL '^^I-Tf and incubated at 4°C for two hours to determine
total surface receptor binding. Non-specific binding was measured as above. TfR-
specific '■^^I-Tf binding was determined as the difference between total and
nonspecifically bound ^^^I-Tf. In experiments where apical or basolateral binding
was measured, dosing of *^^I-Tf was restricted to that respective bathing fluid, as
mentioned previously. In experiments that determined total surface binding, both
51
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apical and basolateral compartments were dosed with After incubation,
monolayers were washed as above and radioactivity associated with the monolayers
was measured with a Gamma Counter.
2.6 Measurement of Total Cellular Transferrin Receptor in Rat Alveolar
Epithelial Cell Monolayers
Total cellular TfR was determined using the method described by Lamb et al. (346).
Briefly, solubilized total cellular TfR was prepared from AEC monolayers by
detaching the cells using 0.1% Triton X-100 in 10 mM Tris-HCl, 150 mM NaCl, pH
8.0 and homogenizing the mixture by twenty passages through a Balch-press
homogenizer. Solubilized TfR was incubated for two hours on ice with 3.2 pg/mL
i25i_Tf, with or without 100-fold molar excess unlabeled Tf. ^^^I-Tf /TfR complexes
were precipitated by addition of equal volumes of 2.0 mg/mL IgG and 45%
(NH4)2S0 4 . Radioactivity in the precipitate was measured using the Packard Gamma
Counter. In addition, specific TfR binding was determined as in the surface receptor
studies. Total protein content was measured with a Pierce BCA protein assay kit.
52
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2.7 TfR-Dependent Uptake of ^® Fe in Caco-2 and Rat Alveolar Epithelial Cell
Monolayers
Human Apo-Tf (Sigma) was loaded with (Perkin Elmer, Wellesley, MA) as
previously described (347). AEC monolayers were washed as in the binding
studies described above. Monolayers were treated with BFA (l|ig/mL) in the apical
compartment and subsequently dosed with 2 jig/mL ^^Fe-Tf on either the apical or
basolateral surface. Non-TfR-mediated ^^Fe uptake was determined by parallel
experiments that included 1 0 0 -fold molar excess of unlabeled holo-transferrin in the
apical fluid. Caco-2 monolayers were incubated for six hours with ^^Fe-Tf and then
washed three times with ice-cold PBS. Cell monolayers were excised and associated
^® Fe was counted with a Packard Gamma Counter to determine uptake.
2.8 Inununoflouresence Studies in Rat Alveolar Epithelial Cell Monolayers
AEC monolayers were fixed with 3.7% formaldehyde at 18 hours post seeding
(dayl) or day 7 of culture and quenched with 50 mM NH4CI. The monolayers were
stained with mouse monoclonal antibody OX-26 (courtesy of W. Pardridge, UCLA),
which is specific for rat TfR, in conjunction with the secondary antibody, FTTC-
labeled goat anti-mouse immunoglobulin (Sigma). The monolayers were
subsequently excised and mounted, apical side up, onto glass slides with Prolong
Antifade Mounting medium (Molecular Probes, Eugene, OR). Samples were imaged
53
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with a Nikon PCM Quantitative Measuring High-Performance Confocal System
equipped with a filter for FTTC attached to a Nikon TE300 Quantum upright
microscope. Confocal images were compiled using Adobe Photoshop 5.0.
2.9 Synthesis of Insulin-Transferrin Conjugate
Recomhinant human insulin (Sigma) was covalently linked to iron-loaded Tf using
the disulfide bifunctional linker, N-succinimidyl 3 -(2-pyridyldithio) propionate
(SPDP) according to the reaction scheme shown in Figure 4. Insulin has three amine
groups that are susceptible to modification by SPDP. In order to obtain a defined
product, the reaction scheme utilizes the difference in pKa between the two a-amino
groups of the N-terminal residues of the A and B chain of insulin and the 8 -amino
group of lysine B29 (see Figure 4). For example, the two a-amino groups were
blocked by adding 5 mg of dimethylmaleic anhydride (DMMA) three times over the
course of 30 minutes (once every 10 minutes) to a mixture of 10 mg of insulin in one
mL of 0.1 M NaHCOs. The reaction was held at pH 6 .8-7.0 to preferentially target
the a-amino groups of insulin. Subsequently, the pH was adjusted to 9.0 and SPDP
(2.5 mg in dimethyl formamide) was added and allowed to react with the e-amino
group of insulin for 1 h at 37° C. Excess SPDP was then removed from the reaction
mixture by overnight dialysis against PBS (pH. 8.0) at 4° C. The ratio of insulin to
PDP was spectrophotometrically determined to be 1 : 1.
54
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(H V M i) . SPHP
* ¥ « 3y V A ' ^ OVf^BNGIyAA^ IM V H rovA s » ^
IiKiiliii Irs-HIP
1 3 - ^ - 2 " ^ ^.MTOIiCIISay m . N t K P C H < H S H
TT-FBP IF-SH
IM V m N G IyA jx^^ ,
3. T T - S H + ll"FEiP —— - ►
b h V
Figure 4. Synthesis scheme of In-Tf conjugate. 1. Synthesis of PDP-
insulin 2. Synthesis of Tf-SH 3. Synthesis of In-Tf.
55
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The SPDP modified insulin was next coupled to free-sulfhydryl containing Tf (Tf-
SH). Tf-SH was synthesized by modifying Tf with SPDP. Human serum apo-Tf (20
mg/mL in PBS, pH 7.0) was loaded with iron by incubating the protein with ferric
ammonium citrate (10 mg/mL in PBS; added to apo-Tf in a 1:10 ratio of ferric
ammonium citrate : apo-Tf) at 37° C for 2 h. The reaction mixture was then dialyzed
over night to remove excess ferric ammonium citrate. The iron-loaded Tf (holo-Tf)
was then modified with SPDP (1.6 mg in di-methyl formamide) at 4° C for 30 min.
The reaction mixture was then dialyzed overnight against PBS (pH 8.0).
Tf-SH was generated subsequently by reducing PDP-Tf with dithiothreitol (25 mM)
for 15 minutes. Tf-SH was then purified from the excess dithiothreitol by size
exclusion chromatography (Sephadex G-50). The purified Tf-SH was immediately
added to two-fold molar excess PDP-insulin and the reaction mixture was well-
stirred for 2 h at 4° C. The reaction was quenched by adding N-ethyl maleimide to a
final concentration of 1 mg/mL. The conjugate was purified by Sephadex G-50
chromatography in PBS (pH 8.0) mobile phase.
2.10 Cloning and production of G-CSF
Human G-CSF was cloned by RT-PCR from human bladder carcinoma cell line
5637 (ATCC), as described by Souza et al (348) and subsequently sub-cloned into
56
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the pGEX-4T-l expression vector (Amersham Pharmacia) as a GST fusion protein in
BL21 E. coli. The expression of the fusion protein was accomplished hy growing
the transformed BL21 bacteria in LB broth at 37° C until A600nm = 0.5 followed by
induction of GST-G-CSF synthesis with 0.1 mM isopropyl P-D-thiogalactoside
(IPTG) for four hours. The bacteria were then harvested, resuspended in PBS, and
lysed via sonication. Triton X-100 was added to the sonicate for a final
concentration of 1% and gently mixed on ice for 30 min. GST-G-CSF fusion protein
was purified from the crude sonicate with Glutathione-Sepharose 4B, washed
extensively with PBS, and incubated overnight with 5U/mg of thrombin (Amersham)
to liberate G-CSF from the matrix. The minute amount of thrombin was removed
from the eluent via Benzamidine Sepharose 6B (Amersham) treatment. The purity
of G-CSF was verified via SDS-PAGE and the yield was estimated by measuring the
absorbance at 280nm, with an extinction coefficient of 15820 M'^ The yield of G-
CSF from this procedure was typically 0.5 mg/L.
2.11 Preparation of Tf-G-CSF Conjugate
GCSF (Amgen, Thousand Oaks, CA or alternatively the cloned product that
was synthesized) was covalently linked to iron-loaded human Tf through disulfide
linker chemistry (Figure 5). Briefly, a 20 mg/mL solution of iron-loaded Tf in PBS
(pH 7.4) was mixed with a ten-fold molar excess of the hetero-bifunctional cross-
linking agent A-Succinimidyl 3-(2-pyriyldithio)propionate (SPDP) (Pierce,
57
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Rockford, IL) at 4° C for 30 minutes. The reaction mixture was then dialyzed
overnight against PBS (pH 8.0) to remove the excess SPDP. The final ratio of SPDP
to Tf was determined to be 2:1. GCSF was next modified with the homo-
bifunctional linker agent 1,4-Di- [3 ’ -(2 ’ -pyridyldithio)propionamido]butane
(DPDPB) (Pierce) by targeting the free sulfhydryl group of Cysl7 in GCSF. A ten
fold molar excess of DPDPB was added to a 10 mg/mL solution of GCSF in PBS
(pH 7.4) and allowed to react at room temperature until a maximum absorbance at
343nm was obtained. The DPDPB-modified GCSF was subsequently dialyzed
overnight against PBS (pH 8.0, 4° C) to remove excess linker agent. The ratio of
DPDPB to GCSF was determined to be 1 : 1. S PDP-Tf was reduced with 25 mM
dithiothreitol (Sigma) to generate the reactive sulfhydryl species and reacted with 10-
fold molar excess DPDPB-GCSF to form the GCSF-Tf conjugate with a 22.8
angstrom aliphatic spacer. Excess GCSF was needed to limit the extent of unwanted
Tf-Tf crosslinking. The reaction was quenched by addition of 1 mg/mL of N-
ethylmaleimide, followed by overnight dialysis against PBS (pH 8.0,4° C). The Tf-
GCSF conjugate was purified by gel filtration on a Sephacryl S-200 column.
58
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A)
Cysl7-SH
+ D PD PB
iCysl7-SS(CH2)2CONH(CH2)2NHCO(CH2)2SSPyr
. ( i. M L
i ^ Cysl7-SS(CH2)2CONH(CH2)2NHCO(CH2)2SSPyr
i
GCSi
N H C 0 (CH2)2SH
Cysl7-SS(CH2)2CONH(CH2)2NHCO(CH2)2SS(CH2)2CO
G-CSF-Tf
Figure 5: Synthesis scheme for G-CSF-Tf. A) G-CSF is modified with
DPDPB by targeting the free sulfhydiyl group of Cys 17. B) DPDPB-modified
G-CSF is combined with reduced SPDP-modified Tf to form G-CSF-Tf.
59
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2.12 Assessment of Apical-to-Basolateral Transcytosis of ^^^I-Tf, ^^®I-In-Tf,
G-CSF and ^^®I-G-CSF-Tf across Caco-2 and MDCK Cell Monolayers
lodinated G-CSF was obtained for the in vitro transport studies by purifying r-
metHu-G-CSF from commercial filgrastim preparations (Amgen). Specifically,
filgrastim solution was dialyzed against lOmM acetate buffer, pH 4.0, to remove
excipients. Following this, the recovered solution was concentrated in Amicon 3,000
MWCO concentrators, and the G-CSF was iodinated by the chloramine-T catalyzed
method. G-CSF and Tf-G-CSF were iodinated using the chloramine-T method as
described above for the preparation of ^^^I-Tf. Transport studies were conducted on
two week-old Caco-2 monolayers, 6 or 7 days after they had exhibited signs of tight
junction development. Monolayers were washed once with DMEM and incubated at
37° C for 45 min to deplete endogenous Tf. Media were subsequently replaced and
the monolayers were treated with ^^^I-Tf, ^^^I-G-CSF or ^^^I-Tf-G-CSF in the apical
compartment (1.5 jig/mL). Non-specific transport was measured in parallel by the
inclusion of 100-fold molar excess of unlabeled Tf. A transcytosis enhancer, i.e,
BFA (1.6 pg/mL), AG-10 (500 pM), wortmannin (1 or 10 pM), or LY294002 (100
mM) was included in the apical media at the beginning of the transport study. At 2,
4, and 6 h post-dosing, 500 pL samples were collected from the basolateral
compartment and replenished with an equal volume of fresh DMEM. Samples were
subjected to 15% trichloroacetic acid (TCA) precipitation and radioactivity of the
pellet was measured with a Packard gamma counter. The extent of TfR-mediated
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transcytosis was determined by subtracting non-specific transport (inclusive of
excess unlabeled Tf) from total transport.
2.13 Assessment of Apical-to-Basolateral Transcytosis of ^^® I-T f, ^^®I-GCSF and
i25i-Tf.GCSF across Rat Alveolar Epithelial Cell Monolayers
GCSF and Tf-GCSF were iodinated using the chloramine-T method as described
above for the preparation of ^^^I-Tf. Transport studies were conducted on day 6 or 7
rat AEG monolayers, with or without KGF treatment from day 0. Monolayers were
washed once with MDSF and incubated at 37° C for 45 minutes to deplete
endogenous Tf. Media were subsequently replaced and the monolayers were treated
with ^^^I-Tf, ^^^I-GCSF or *^^I-Tf-GCSF in the apical compartment (1.5 jxg/mL).
Non-specific transport was measured in parallel by the inclusion of KXTfold molar
excess of unlabeled Tf. A transcytosis enhancer, i.e., BFA (1.0 pg/mL), AG-10 (500
pM), wortmarmin (1 or 10 pM), or LY294002 (100 mM) was included in the apical
media at the beginning of the transport study. At 2,4, and 6 hours post-dosing, 500
pL samples were collected from the basolateral compartment and replenished with
an equal volume of fresh MDSF. Samples were subjected to 15% trichloroacetic
acid (TCA) precipitation and radioactivity of the pellet was measured with a Packard
gamma counter. The extent of TfR-mediated transcytosis was determined by
subtracting non-specific transport (inclusive of excess unlabeled Tf) from total
transport.
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2.14 Analysis of Transcytosed Proteins
Transcytosed proteins were analyzed in separate transport studies via size exclusion
chromatography and GCSF-dependent cell proliferation assays. Basolateral fluids
were collected after a six hour transport study as described above and subjected to
Sephacryl S-200 column chromatographic analysis, where stock ^^^I-labeled proteins
(i.e.^^^I-Tf, ^^^I-GCSF and ^^^I-GCSF-Tf) were used for determination of appropriate
elution volumes. Biological activity assays were conducted for transcytosed
GCSF and *^^I-GCSF-Tf by measuring proliferation of the murine myeloblastic cell
line NFS-60 (349) (courtesy of Dr. J. Dile, St. Jude’s Children’s Research Center,
Memphis, TN). NFS-60 cells that had been cultured in RPMI-1640 medium,
supplemented with both 10% FBS and 10% WEHI-3 (ATCC) conditioned medium
(CM), were washed three times with serum- and WEHI-3 CM-free RPMI-1640 and
aliquoted to 96 well microtiter plates at a density of 1 x 10^ cells/mL. These cells
were spiked with 20 )iL of medium that had been previously recovered from the
basolateral compartments in the conjugate transport studies and concentrated 10-fold
with a Centricon centrifugal concentrator apparatus (Amicon, Bedford, MA). The
samples were incubated at 37°C in a 5% CO2 incubator for 48 hours. A MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was
subsequently performed essentially as previously described by Mosmann (350).
Briefly, cells were incubated with phenol-red free RPMI media (Invitrogen,
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Carlsbad, CA) containing 1 mg/mL MTT for two hours. The formazan crystals that
formed were then dissolved in isopropanol (Sigma) and absorbance was measured at
570nm on a Dynatech M70 microplate reader.
2.15 In vivo studies of subcutaneously and orally delivered G-CSF-Tf conjugate
in male BDFl mice
Specific pathogen-free BDFl male mice (Charles River Laboratories, Wilmington,
MA) were used at 6-8 weeks of age. BDFl mice were chosen due to their relatively
robust stimulatory response to human G-CSF. The mice were allowed to acclimate
for several days prior to beginning experiments. Animal experiments were
compliant with the 'Principles of Laboratory Animal Care' (NDH Publication #85-23)
and approved by the lACUC at USC. The mice were fasted for 12 h prior to dosing.
The mice were individually weighed and the dosages were adjusted accordingly.
The mice weight was typically between 18-20 g. The treatment groups (n = 5)
received a single subcutaneous (SC) injection or oral administration (via gavage
needle) on day 0 with G-CSF-Tf, filgrastim, or vehicle control (PBS). Since the
molecular weight of G-CSF-Tf is about 5-fold higher than that of filgrastim, G-CSF-
Tf was administered at 5 mg/kg SC and filgrastim was given at 1 mg/kg SC.
Similarly, G-SCF-Tf was given at a dose of 50 mg/kg orally and filgrastim was given
at 10 mg/kg orally. The concentrations of the dosing solutions were adjusted so the
dosage volume in all cases was 100 pL. G-CSF-Tf and filgrastim were formulated
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with sodium bicarbonate (30 mg/mL) in PBS prior to oral administration. Sodium
bicarbonate was included to neutralize stomach acid in order to prevent hydrolysis of
the protein-drug.
Blood samples were collected daily from the tails of the mice into micro
tubes that had been pretreated with heparin. Total white blood cell counts (WBC)
were performed manually with a hemacytometer. The samples were diluted 20-fold
and lysed in an acidic crystal-violet solution (0.1% crystal violet, 1% acetic acid, in
water) prior to being loading in the hemacytometer. The percentage of poly
morphonuclear neutrophils (FMN) amongst the leucocytes was determined manually
with Wright-stained blood-smear glass slides that were examined with a lOOx oil
immersion lens on an Olympus BH-2 microscope. The absolute number of
neutrophils was determined by applying the observed FMN percentage against the
total WBC count for each sample (351).
2.16 Statistical analyses
Data are presented as mean (±SEM). One-way analysis of variance (ANOVA) was
used to determine statistical significance among group (n > 3) means using Tukey’s
post-hoc tests. p<0.05 was considered significant.
64
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RESULTS
3.1 Synthesis and characterization of G-CSF-Tf
The G-CSF that was cloned, expressed, and purified from E. coli was analyzed to
verify that the biological activity was maintained through the purification process.
As seen in Figure 6 A, the purified G-CSF closely matched the Neupogen standard in
the ability to stimulate proliferation of NFS-60 cells, a murine cell line that is
specifically stimulated by murine and human G-CSF. In addition when the purified
G-CSF is analyzed in a dose dependant MTT assay, the ED50 appears to closely
match the values that have been reported for Neupogen, with an ED50 of
approximately 0.06 ng/mL (Figure 6 B).
G-CSF-Tf conjugate was synthesized using disulfide DPDPB linker chemistry that
targeted the free sulfhydryl moiety of Cys 17 on G-CSF, while using the
heterobifunctional linker SPDP to create a Tf that contained reactive sulfhydryl
species. The free sulfhydryl containing Tf was formed by a short reducing reaction
under mild DTT solution (25 mM).
SDS-PAGE demonstrated that the GCSF-Tf conjugate was dominated by
cross-hnked species, with the majority of the non-reduced conjugate showing
coomassie blue staining near or above the 205 kDa molecular weight standard band
65
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(Figure 7, lane 1), indicating the presence of oligomers of Tf in the conjugate. While
the band that appears for the conjugate is broad, it does not appear to have significant
amounts of free Tf or G-CSF. However, when the conjugate is reduced by a short
incubation with DTT, the conjugate is cleaved to liberate its two constituent proteins.
As seen in lane two of Figure 7, two bands appear near 80 kDa and 20 kDa, which
coincide with the expected molecular weights of Tf and G-CSF.
The conjugate also demonstrates a distinct elution profile when analyzed by
size exclusion chromatography. For example, when the conjugate was applied to a
40 mL Sephacryl 200 column, the conjugate eluted at fraction 17 while standard
solutions of Tf and G-CSF eluted at fractions 19 and 23 respectively, as shown by
absorbance of the fractions at 280 nm (Figure 8). In addition, when the eluted
fractions are analyzed for ability to stimulate proliferation of NFS-60 cells, the
absorbance at 570 nm displayed a maximum at fraction 17 (Abs570n m = 0.2) and
appeared to coincide with the maxima observed for the conjugate concentration
curve (Abs. at 280 run). The conjugate also appeared to be well separated from un
reacted free G-CSF, with only a minimal amount of cell-proliferation ability
demonstrated at fraction 23 (Abs570n m = 0.04).
66
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Figure 6. G-CSF-dependent proliferation of NFS-60 cells.
(A) NFS-60 cells were seeded at 20,000 cells/mL and treated with
commercially available G-CSF (filgrastim) or G-CSF derived
purified from E.coli through a GST-G-CSF fusion protein construct
at a dose of 1 ng/mL. Cell concentration was determined daily with a
Coulter counter. (B) G-CSF dose dependent assay of NFS-60 cell
prohferation. NFS-60 cells were seeded in 96-well plates at a density
of 10,O C X ) cells/mL. The wells were spiked (in triplicate) with
varying concentrations of G-CSF and incubated at 37 °C in 5% CO2
for 48 hours. The growth media was then replaced with phenol-red
free media containing 1 mg/ml MTT and allowed to incubate for 2
hours. After incubation, the MTT-containing media was replaced
with 2-propanol to dissolve the formazan crystals. Absorbance was
measured at 570 nm.
67
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45 -
40 -
35 -
-X- Filgrastim
Recombinant GCSF
Control
30 -
« 20 -
15 - I
10
0 24 48 72 96
Culture Time (hrs)
NFS-60 MTT Assay
1.2
h- 0.8
O 0.6
is 0.4
< 0.2
0.000 0.005 0.01 0.02 0.04 0.08 0.16 0.31 0.63 1.25 2.5
Lyophilized G-CSF (ng/mL)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
205 kDa
• i M 121 kDa
70 kDa
52 kDa
35 kDa
29 kDa
21 kDa
1
Figure?. SDS-PAGE analysis of G-CSF-Tf. G-CSF-
Tf was analyzed under non reducing (lane 1) and
reducing (lane 2) conditions in a 10 % acrylamide gel.
The gel was stained with Coomassie Blue.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
0.20
0.14
0.18
0.16
E 0.12
GCSF-Tf
0.14
I 0.10
O I
m 0.08
0.12
G -CSF
0.10
GCSF-Tf
A570
0.08
0.06
0
eo
01 0.06
0.04
0.04
J Q
0.02
0.02
0.00 0.00
T -
CO CO
^i ^ o c o c o c r j c MLOo o
T - T - T ” T - CM o a CM
Fraction
Figure 8. Chromatographic analysis of G-CSF-Tf. G-CSF-Tf
was analyzed by S-200 gel chromatography (40 mL column).
The purity of the synthesized G-CSF-Tf was estimated by
comparing the position of eluted G-CSF-Tf to that of G-CSF and
Tf standards applied to the same column by measuring the
absorbance of the eluted samples at 280nm. The bioactivity of
the eluted samples was determined by the ability to stimulate
proliferation of NFS-60 cells as measured by MTT assay (Abs at
570nm).
( Q
m
E
c
o
lO
m
70
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3.2 The Effect of Wortmannin on TfR-Mediated Transcytosis in Caco-2
Monolayers
In light of previous findings that have shown that GTPase inhibitors are able
to elicit an enhancement of TfR-mediated transcytosis of protein drugs in vitro and in
vivo (113; 312), efforts were undertaken to examine whether or not inhibiting the
functions of downstream effectors of Rabs would result in a similar enhancement of
TfR-mediated transcytosis. Towards this end, PD kinases were identified as a
potential target for inhibition in order to alter sorting events to favor an increase in
net apical-to-basolateral transport. When the natural PDK inhibitor, wortmannin,
was applied to the apical compartment of Caco-2 monolayers at the beginning of a 6-
hour transport study in concentrations of either 1 or 10 |iM, a significant
enhancement of TfR-mediated transcytosis was observed (Figure 9). Specifically,
the TfR-specific apical-to-basolateral transport rate of ^^^I-Tf was 0.58
ng/hr/monolayer for the 10 pM wortmannin treatment group and 0.49
ng/hr/monolayer for the 1 jiM treatment group while the control monolayers
demonstrated a basal level TfR-mediated ^^^I-Tf transport rate of 0.28
ng/hr/monolayer. The monolayer integrity as determined by TEER measurement,
remained unaffected during the time course of the experiment, with both control and
10 jiM wortmannin treatment groups exhibiting nearly identical resistances over the
course of the experiment (Figure 10).
71
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3.5
Wortmannin 10 uM
Wortmannin luM
2.5
Control
05
0.5
0 1 2 3 4 6 5
Time (h)
Figure 9. Wortmannin induced enhancement of TfR-mediated
transcytosis in Caco-2 monolayers. Two-week old Caco-2 monolayers
were dosed with 1.5 |xg/mL of in the apical compartment.
Nonspecific transport was determined parallel by the addition of 100-fold
excess unlabelled Tf. Wortmannin was spiked into the apical bathing
fluid at the beginning of the transport experiment. Samples were
recovered from the basolateral compartment at regular intervals and the
radioactivity of the TCA precipitatable fraction was determined by
gamma counter (n=3).
7 2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
500
450
s r ^ o o
o
g 300
o 2 5 0
- 200
O J 150
UJ
H 100
Wortmannin 10 uM
Control
6 0 2 4
Time (h)
Figure 10. Wortmannin’s effect on TEER of Caco-2 monolayers.
Two-week Caco-2 monolayers were spiked with wortmannin to a final
concentration of 10 fiM in the apical compartment. The TEER was
measured over a 6-hour time course with a voltohmmeter (n=3).
73
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.3 LY294002 Mediated Enhancement of TfR-mediated Transcytosis in Caco-2
Monolayers
Since wortmannin is a non-specific phosphatidyl inositol kinase inhibitor,
LY294002 was chosen as a agent to be used for the exploration of a specific inhibitor
of PB-K. When LY294002 was applied to the apical compartment of the beginning
of a 6-hour transport study to a final concentration of 100 |iM, a significant
enhancement of TfR-mediated transcytosis was observed (Figure 11 A). Specifically,
the steady state TfR-specific apical-to-basolateral transport rate of ^^^I-Tf was 0.55
ng/hr/monolayer for the 100 jiM LY294002 treatment group while the control
monolayers demonstrated a roughly 5-fold lower basal level TfR-mediated *^^I-Tf
transport rate of 0.11 ng/hr/monolayer. The monolayer integrity as determined by
TEER measurement, remained unaffected during the time course of the experiment,
with both control and 100 jiM LY294002 treatment groups exhibiting nearly
identical resistances over the course of the experiment (Figure 12). In addition, as
can be seen in Figure 1 IB, the increase in ^^^I-Tf transport was due to an
enhancement of TfR-mediated processes, with the nonspecific ^^^I-Tf transport
remaining unchanged in the presence of 100 jiM LY294002 (Figure 5). The rate of
non-TfR-specific *^^I-Tf transport was 0.12 ± 0.015 ng ’ ■^^I-Tf/well/hr for the
LY94002 treatment group and 0.11 ± 0.016 ng ^^^I-Tf/well/hr for the control group.
7 4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 11. LY294002 induced enhancement of TfR-mediated
transcytosis in Caco-2 monolayers.
Two-week old Caco-2 monolayers were dosed with 1.5 jig/mL of
in the apical compartment. Nonspecific transport was determined
parallel by the addition of 100-fold excess non-radioabelled Tf.
LY294002 was spiked into the apical bathing fluid at the beginning of the
transport experiment. Samples were recovered from the basolateral
compartment at regular intervals and the radioactivity of the TCA
precipitatable fraction was determined by gamma counter. The time
course of TfR-mediated transport for the LY294002 and control groups is
shown in figure A. The average transport rate observed for the total,
nonspecific, TfR-mediated portions of the LY294002 and control groups
over the full six-hour period is shown in figure B (n=3)
75
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1.75-
L\»M }Q2100uM
c
Cortro! 1.25 4
r
I
la
CM
"0
^ 0 .7 5 j
Q.
( f >
S 0.5-
c o
h“
0 .2 5 4
1 2 6 0 3
Time(h)
4 5
0.80 - 1
0.70 -
0.60
0.50
h-
DS
S. 0.40
B
1 0.30
■ c
§. 0.20
O T
C
2 0.10
0.00
B
■ LY294002 100 u M
■ Control
Total Non-specific TfR-mediated
7 6
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
700 n
600 i:
£ 500 "-
o
E 400 -
300 -
♦ — LY294002 100 uM
200 -
■ ■ — Control
100 H
0 1 2 3 4 5 6
Time (h)
Figure 12. LY294002’s effect on TEER of Caco-2 monolayers. Two-
week old Caco-2 monolayers were spiked with LY294002 to a final
concentration of 100 jxM in the apical compartment. The TEER was
measured over a 6-hour time course with a voltohmmeter (n=3).
77
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.4 Effect of BFA, AG-10, and LY294002 on TfR-Speclfic Binding in
Caco-2 Monolayers
In order to determine the effect that LY294002 has on TfR distribution, two-
week old Caco-2 monolayers were dosed with 100 mM LY294002 in the apical
compartment, incubated at 37° C for 1 h and then radiolabeled Tf was applied to the
apical or basolateral compartment of the monolayers and the monolayers were
incubated at 4° C for two hours. Likewise, separate Caco-2 monolayers were also
simultaneously dosed with BFA and AG-10 (1.6 pg/mL and 500 mM, respectively)
and ^^^I-Tf was incubated at 4° C for two hours. Non-specific binding of Tf was
determined in parallel. As can be seen in Figure ISA, LY294002 treatment appeared
to result in a significant increase in TfR-specific binding at the apical surface
compared to the BFA and control treatment groups, with 119 ± 11 fmol TfR/
monolayer for the LY294002 treatment group and 68 ± 7 fmol TfR/monolayer and
84 ± 6 fmol TfR/monolayer detected for the BFA and control treatment groups,
respectively.
In contrast, LY294002 treatment resulted in a slight but significant decrease
in TfR levels present at the basolateral surface of Caco-2 monolayers compared to
control with 1920 ± 80 fmol TfR/monolayer and 2100 ± 85 fmol TfR/monolayer for
the LY294002 and control treatment groups, respectively (Figure 13B). In likewise
fashion, AG-10 and BFA treatment resulted in a significant decrease in TfR levels
7 8
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 13. The effect of BFA, AG-10, and LY294002 on the
surface distribution of TfR in Caco-2 monolayers.
TfR levels were determined through TfR specific binding of ^^^I-Tf
(1.5 |ig/mL) to the apical (A) and basolateral (B) surfaces of two-
week old Caco-2 cultures grown on 24mm Trans well filters. Non
specific binding was determined in parallel by the addition of 100-
fold molar excess of non-radiolabeled Tf to the respective donor
fluid. BFA (1.6 |Xg/mL), AG-10 (500 pM), and LY294002 (100
pM) were included in the in apical bathing fluid in all cases and
allowed to incubate for two hours at 37°C prior to replacing the
media with fresh media that had been stabilized at 4°C and which
contained the radiolabeled Tf. The surface binding experiments
were conducted at 4°C for two hours. TfR-specific binding was
determined as the difference between total and non-specific ^^® I-T f
binding. The monolayers were excised after incubation and the
level of radioactivity was determined on a Packard gamma counter.
79
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
> 100
LY 294002 AG-10 BFA Control
0)
>
m
o
c
o
E
P
o
E
2000
1500
1000
500
LY 294002 AG-10 BFA Control
B
8 0
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
at the basolateral surface of the Caco-2 monolayers, with 1900 ± 90 fmol
TfR/monolayer and 1750 ± 95 fmol TfR/monolayer for the AG-10 and BFA
treatment groups, respectively.
3.5 Effect of BFA, AG-10, and LY294002 on TfR-specific ®^Fe-Tf uptake in
Caco-2 monolayers
In order to explore the dynamic effects of the transcytosis enhancers on TfR
cycling events, the TfR-specific uptake of ^^Fe-loaded Tf was investigated in the
presence of transcytosis enhancers. In contrast to uptake of ^^^I-Tf, uptake of ^^Fe-Tf
allows for the recording of cumulative endocytosis events since upon entry into the
acidic environment of the endosome the ferric ion will be released from the
internalized Tf, whereas intemalized ^^^I-Tf will be merely recycled back outside of
the cell. As shown in Figure 14A, the apical TfR-specific ^^Fe-Tf uptake over a 6
hour time-course in Caco-2 monolayers is significantly enhanced by BFA treatment,
with 233 ± 44 fmol Tf/cm^ and 40 ± 33 fmol TFcm^ absorbed for the BFA and
control treatment groups, respectively. In contrast, treatment with AG-10 or
LY294002 did not have a significant effect on TfR-specific ^^Fe-Tf uptake in Caco-2
monolayers, with 5 ± 35 fmol Tf/cm^ and 4 ± 61 fmol Tf/cm^ absorbed for the AG
IO and LY294002 treatment groups, respectively. Basolateral uptake of ^^Fe-Tf in
Caco-2 monolayers displayed a different pattern in response to transcytosis enhancer
treatment compared to the effects that were recorded for the uptake at the apical
81
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 14. The effect of BFA, AG-10, and LY294002 on the uptake
of ®^Fe-Tf in Caco-2 monolayers.
TfR internalization was determined through TfR specific uptake of ^Ve-
Tf (1.5 |xg/mL) from the apical (A) and basolateral (B) surfaces of two-
week old Caco-2 cultures grown on 24mm Transwell filters. Non
specific uptake was determined in parallel by the addition of 100-fold
molar excess of non-radiolabeled Tf to the respective donor fluid. BFA
(1.6 |ig/mL), AG-10 (500 pM), and LY294002 (100 pM) were included
in the in either the apical or basolateral bathing fluid at and allowed to
incubate for five hours at 37°C. TfR-specific uptake was determined as
the difference between total and non-specific ® ® Fe-Tf uptake. The
monolayers were excised after incubation and the level of radioactivity
was determined on a Packard gamma counter.
8 2
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
300-
250-
^ 200-
o
P 150-
o
E 100-
50-
0 -
L i j _
C ontrd AG-10 LY
5000
4500
4000
3500
C M
E
3000
u
2500
o
i
2000
1500
1000
500
0
Control BFA AG-1 0 LY
B
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
surface. For example, all three of the transcytosis enhancers significantly reduced
the basolateral TfR-specific uptake of ^^Fe-Tf. BFA treatment caused the largest
percentage decrease in TfR-specific uptake of ^^Fe-Tf at the basolateral surface of
Caco-2 monolayers, with 41% decrease compared to control (2749 ± 70 fmol
Tf/monolayer and 4684 ± 20 fmol Tf/monolayer absorbed over 6 hours for the BFA
and control treatment groups, respectively). LY294002 and AG-10 treatments
caused a 14% and 32% respective decrease in TfR-specific uptake of ^^Fe-Tf at the
basolateral membrane with 4018 ± 40 fmol Tf/monolayer and 3162 ±73 fmol
Tf/monolayer absorbed for the LY294002 and AG-10 treatment groups, respectively,
during a 6 hour uptake experiment.
3.6 Comparison of Transcytosis Enhancers’ Effect on TfR-Mediated Transport
in Caco-2 Monolayers
In order to make a direct comparison between the effects of the transcytosis
enhancers on TfR-mediated transcytosis, two week old Caco-2 Monolayers were
dosed with 1.6 |ig/mL of BFA, 100 fiM LY294002, and 500 pM AG-10 in the apical
compartments at the beginning of a 6-hour transport study. Non-specific transport
was determined in a similar fashion as previously mentioned. As can be seen in
Figure 15, the steady state TfR-specific transport appeared to be enhanced to a
similar degree in the presence of LY294002 and AG-10, with transport rates of 2.00
± 0.16 ng Tf/hr/well and 2.08 ± 0.44 ng Tf/hr/well observed for the LY294002 and
84
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 15. Comparison of transcytosis enhancer effect on TfR-
mediated transcytosis in Caco-2 monolayers.
Two-week old Caco-2 monolayers were dosed with 1.5 jig/mL of
in the apical compartment. Nonspecific transport was determined in
parallel by the addition of 100-fold excess unlabelled Tf. TfR-specific
transport was determined to be the difference between the total and non
specific transport (A). AG-10 (500 fiM ), LY294002 (100 jiM ), or BFA
(1.6 jXg/mL) were spiked into the apical bathing fluid at the beginning of
the transport experiment. Samples were recovered from the basolateral
compartment at regular intervals and the radioactivity of the TCA
precipitatable fraction was determined by gamma counter. The transport
rates were determined for the total, non-specific, and TfR-mediated
components by linear regression for the steady state transport data (B).
In all cases, the error represents the standard error of the mean as
determined by ANOVA (n=3).
85
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
10
CD
I)
c
H
■e
o
a.
w
c
2
H
0 1 2 4 5 6
^ 4 5 -
§ 4 0 -
|a5-
gao-
ffl 2.5 -
| 2 0 -
r i . 5
«" 1-0
1 0.5^
0.0-
i / ^ 1 0
iO 2 9 « 0 2
■ BFA
B
Trtal Non-qDedfic TfFkmedatecI
86
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
AG-10 treatment groups, respectively. The steady state transport rate observed for
the BFA treatment group was significantly less than LY294002 or AG-10, with an
observed rate of 0.8 ± 0.10 ng Tf/well/hr.
3.7 The effect of LY294002 on TfR-mediated transport of a G-CSF-Tf
conjugate across Caco-2 monolayers
Two-week old Caco-2 monolayers, exhibiting TEER levels of approximately
500 Qcm^, were dosed with 1.5 pg/mL of ^^^I-G-CSF-Tf in the apical compartments
of 6-well Transwells. As shown in Figure 16, monolayers that received ^^^I-G-CSF-
Tf in combination with 100 jiM LY294002 exhibited significantly higher TfR-
mediated transcytosis compared to the monolayers that received only ^^^I-G-CSF-Tf.
For example, after six hours, the amount of transported ^^^I-G-CSF-Tf in the
presence of LY294002 was 4-fold higher than the amount of '^^I-G-CSF-Tf
transported in the absence of LY294002 (8.0 ± 0.9 ng/well ^^^I-G-CSF-Tf with 100
pM LY294002, 1.2 ± 0.7 fmol/well ^^^I-G-CSF). In addition, the transport rate was
also significantly higher, with ^^^I-G-CSF-Tf transported at 1.4 fmol/well-hr'^ and
^^^I-G-CSF transported at 0.3 fmol/well-hr ^ Non-specific ^^^I-G-CSF-Tf in the
presence or absence of LY294002 was significantly lower than TfR-mediated
transcytosis, with nonspecific transport composing 25% and 51% of the total
transport for ^^^I-G-CSF-Tf in the presence and absence of LY294002, respectively
(Figure 16 B).
87
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 16. LY294002 induced enhancement of TfR-mediated
transcytosis of ^^®I-G-CSF-Tf across Caco-2 monolayers.
Two-week old Caco-2 monolayers were dosed with 1.5 |ig/mL of
G-CSF-Tf in the apical compartment. Nonspecific transport was
determined in parallel by the addition of 100-fold excess non
radiolabelled Tf. TfR-specific transport was determined as the
difference between the total and non-specific transport (A). LY294002
(100 |i.M), was spiked into the apical bathing fluid at the beginning of
the transport experiment. Samples were recovered from the basolateral
compartment at regular intervals and the radioactivity of the TCA
precipitatable fraction was determined by gamma counter. The
transport rates were determined for the total, non-specific, and TfR-
mediated components by linear regression for the steady state transport
data (B). In all cases, the error represents the standard error of the
mean as determined by ANOVA (n=3).
88
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1 0 1
Q )
i
c
LY»ia)21(X3uM
u.
m
o
?
■ c
a
m
c
c e
Control
H -
1 0 2 3
Time (h)
4 5 6
3.0
2.5
i 2.0
0)
« 1-5
D C
? 1.0
a.
w
2
0.0
B
OLY294002
■ Control
Total Non-specific TfR-mediated
8 9
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
3.8 Comparison of the effect of BFA, AG-10, or LY294002 treatment on TfR-
mediated Transcytosis in MDCK monolayers
MDCK was chosen as another model cell line that exhibits a high degree of
polarization of TfR distribution. Previous studies have demonstrated that MDCK
and Caco-2 cells demonstrate differing responses to AG-10 treatment, which has
been postulated to be the result of dissimilar TfR recycling pathways within the cell.
In order to make a direct comparison between the effects of the transcytosis
enhancers on TfR-mediated transcytosis, MDCK monolayers exhibiting TEER
values > 3500 0»cm^ were dosed with 1.6 jig/mL of BFA, 100 pM LY294002, and
500 jiM AG-10 in the apical compartments at the beginning of a 6-hour transport
study. Non-specific transport was determined in a similar fashion as previously
mentioned. As can be seen in Figure 17, the steady state TfR-specific transport
appeared to be enhanced to a similar degree in the presence of LY294002 and AG
IO, with transport rates of 2.00 ± 0.16 ng Tf/hr/well and 2.08 ± 0.44 ng Tf/hr/well
observed for the LY294002 and AG-10 treatment groups, respectively. The steady
state transport rate observed for the BFA treatment group was significantly higher
than LY294002 or AG-10, with an observed rate of 0.8 ± 0.10 ng Tf/well/hr. The
relative degree of transcytosis enhancement, when comparing the effects of BFA
treatment to AG-10 and LY294002, was opposite of that seen for the Caco-2
monolayers. The MDCK monolayer integrity as determined by TEER measurement
(Figure 18), was examined over a 6 hour time period in response to BFA (1.6
90
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
|ig/mL), AG-10 (500 |iM), and LY294(X)2 (100 jiM) treatment in the apical
compartment.
Figure 17. Comparison of transcytosis enhancer effect on TfR-
mediated transcytosis in MDCK monolayers.
MDCK monolayers displaying TEER levels > 3500 0«cm^ were dosed
with 1.5 pg/mL of in the apical compartment. Nonspecific
transport was determined in parallel by the addition of 100-fold excess
unlabelled Tf. TfR-specific transport was determined to be the difference
between the total and non-specific transport (A). AG-10 (500 pM),
LY294002 (100 pM), or BFA (1.6 pg/mL) were spiked into the apical
bathing fluid at the beginning of the transport experiment. Samples were
recovered from the basolateral compartment at regular intervals and the
radioactivity of the TCA precipitatable fraction was determined by
gamma counter. The transport rates were determined for the total, non
specific, and TfR-mediated components by linear regression for the
steady state transport data (B). In all cases, the error represents the
standard error of the mean as determined by ANOVA (n=3).
91
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1 2
1 0 -
BFA
LY294002
T-8
Q .
0 1 2 3 4 5 6
Time(lh)
I 3 .0
5 2.0
t 1.5
e 0.5
■AG-10
□ LY294002
□ BFA
B
Total Non-specific TfR-mediated
92
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
4000
3000 -
*E
g 2000 "
o
Control
BFA 1.6 ug/mL
AG-10 500 uM
LY294002 lOOuM
1000 H
0 1 2 3 4 5 6
Time (h)
Figure 18. Effect of BFA, AG-10, and LY294002 treatment on
TEER of MDCK monolayers, monolayers were spiked with
BFA, AG-10, and LY294002 to a final concentrations of
l.Opg/mL, 500 }iM, and 100 pM, respectively, in the apical
compartment of MDCK transwells. The TEER was measured over
a 6-hour time course with a voltohmmeter (n=3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Monolayers that received AG-10 and LY294002 exhibited similar reduction in
TEER over the six hour time course, with both treatment groups exhibiting TEER
values near 1200 Q»cm^ at the end of the 6-hour time course. In contrast, the BFA
treatment group exhibited a significantly different TEER at the end of the six hour
time course, with an average TEER of 2200 ± 240 0®cm^. The control monolayers’
TEER did not decrease over the six hour time period.
3.9 Effect of BFA, AG-10, and LY294002 on TfR-Spedfic ^^® I-Tf Binding in
MDCK Monolayers
MDCK monolayers displaying TEER values near 3500 Q.»c t s ^ were treated
with BFA, AG-10, and LY294002 as described above for the Caco-2 monolayers. As
can be seen in Figure 19, BFA treatment resulted in a significant increase in TfR-
specific binding at the apical surface compared to the AG-10, LY294002, and
control treatment groups, with 210 ± 19 fmol TfR/ monolayer for the BFA
treatment group and 150 ± 10, 146 ± 12, and 145 ± 15 fmol TfR/monolayer detected
for the LY294002, AG-10, and control treatment groups, respectively. In contrast,
BFA treatment resulted in a slight but significant decrease in TfR levels present at
the basolateral surface of MDCK monolayers compared to control with 2400 ± 100
fmol TfR/monolayer and 3050 ±110 fmol TfR/monolayer for the BFA and control
treatment groups, respectively. In likewise fashion, LY294002
9 4
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 19. The effect of BFA, AG-10, and LY294002 on the surface
distribution of TfR in MDCK monolayers.
TfR levels were determined through TfR specific binding of *^^I-Tf (1.5
jig/mL) to the apical (A) and basolateral (B) surfaces of two-week old
MDCK cultures grown on 24mm Transwell filters. Non-specific binding
was determined in parallel by the addition of 100-fold molar excess of non
radiolabeled Tf to the respective donor fluid. BFA (1.6 fig/mL), AG-10
(500 joM), and LY294002 (100 |iM) were included in the in apical bathing
fluid in all cases and allowed to incubate for two hours at 37°C prior to
replacing the media with fresh media that had been stabilized at 4°C and
which contained the radiolabeled Tf. The surface binding experiments
were conducted at 4°C for two hours. TfR-specific binding was
determined as the difference between total and non-specific ^^I-Tf binding.
The monolayers were excised after incubation and the level of radioactivity
was determined on a Packard gamma counter.
95
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
200 -
0 )
>.
£ 150
100
E 50
LY 294002 AG-10 BFA Control
3500 1
3000
0 )
« 2500
O
c
o
E
p
■ g 1000
E
500
2000
1500
i 1
t "
LY 294002 AG-10 BFA Control
B
96
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
and AG-10 treatment did not significantly change TfR levels at the basolateral
surface of the MDCK monolayers, with 3050 ± 120 fmol TfR/monolayer and 3100 ±
97 fmol TfR/monolayer for LY294002 and AG-10 treatment groups, respectively.
3.10 Effect of BFA, AG-10, and LY294002 on TfR-specific ®*Fe-Tf uptake in
MDCK monolayers
TfR-specific uptake of ^^Fe-loaded Tf was investigated in MDCK monolayers in the
presence of transcytosis enhancers as described above for Caco-2 cell monolayers.
As shown in Figure 20, the apical TfR-specific ^®Fe-Tf uptake over a 6 hour time-
course in MDCK monolayers is significantly enhanced by BFA treatment, with 350
± 54 fmol Tf/cm^ and 45 ± 30 fmol Tf/cm^ absorbed for the BFA and control
treatment groups, respectively. In contrast, treatment with AG-10 or LY294002 did
not have a significant effect on TfR-specific ^®Fe-Tf uptake in MDCK monolayers,
with 50 ± 35 fmol Tf/cm^ and 39 ± 61 fmol Tf/cm^ absorbed for the AG-10 and
LY294002 treatment groups, respectively. The basolateral uptake of ^^Fe-Tf in
MDCK monolayers displayed a different pattern in response to transcytosis enhancer
treatment compared to the effects that were recorded for the uptake at the apical
surface. For example, BFA significantly reduced ^^Fe-Tf uptake, while AG-10 and
LY294002 did not significantly affect uptake, as seen at the apical surface. BFA
treatment caused a large decrease in TfR-specific uptake of ^^Fe-Tf at the basolateral
surface of MDCK monolayers, with 35% decrease compared to control (2496 ± 100
97
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 20. The effect of BFA, AG-10, and LY294002 on the uptake of
*®Fe-Tf in MDCK monolayers.
TfR intemalization was determined through TfR specific uptake of ^^Fe-
Tf (1.5 jig/mL) from the apical and basolateral surfaces of two-week old
MDCK cultures grown on 24mm Trans well filters. Non-specific uptake
was determined in parallel by the addition of 100-fold molar excess of
non-radiolabeled Tf to the respective donor fluid. BFA (1.6 pg/mL), AG
IO (500 pM), and LY294002 (100 pM) were included in the in either the
apical or basolateral bathing fluid at and allowed to incubate for five
hours at 37°C. TfR-specific uptake was determined as the difference
between total and non-specific ^^Fe-Tf uptake. The monolayers were
excised after incubation and the level of radioactivity was determined on a
Packard gamma counter.
98
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
300
C4
i 250
o
o
E
200
150
100
L i
C o r tr d B F A A G - 1 0 L Y
4500
4000
3500
3000
o 2500
o 2000
E
1 500
1 000
C ontrol
B
B F A AG-1 0 LY
9 9
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
fmol Tf/monolayer and 4100 ±40 fmol Tf/monolayer absorbed over 6 hours for the
BFA and control treatment groups, respectively).
3.11 Measurement of total surface transferrin receptor in primary cultures of
rat alveolar epithelial cell monolayers
Rat AEC monolayers were dosed with media containing 1.5 jig/mL ^^^I-Tf and
incubated at 4°C for two hours to determine total surface receptor binding. TfR-
specific ^^^I-Tf binding was determined as the difference between total and
nonspecifically bound ^^^I-Tf. The level of TfR at the plasma membrane, as
measured by specific binding of ^^^I-labeled Tf to total cell-surface, decreased from a
maximum of 10.7 (± 2.2) fmol/cm^ on day 1 to 1.49 (± 1.3) (n =6) fmol/cm^ on day 7
in culture (Figure 21). The decrease in total cell-surface TfR coincided with
transdifferentiation from the type II to type I cell-like phenotype, which normally
occurs by day 3-4 in culture (329-332).
100
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
I
o
E
14 1
12 -
10 H
T T 1
0 1 2 3 4 5 6 7
Culture day
Figure 21. Total surface TfR in rat alveolar epithelial cells (AEC)
as a function of day in culture. TfR levels were determined through
TfR specific binding of ^^^I-Tf (1.5 pg/mL) to the apical and basolateral
surfaces of AEC cultures grown on 12mm Trans well filters. Non
specific binding was determined in parallel by the addition of 100-fold
molar excess of non-radiolabeled Tf to the respective donor fluid. The
surface binding experiments were conducted at 4°C for two hours.
Data represent the difference between total and non-specific ^^^I-Tf
binding. In all cases, error bars represent standard error of the mean
(SEM) for n = 6. * indicates p<0.05 and ** indicates p<0.01 compared
to that observed for day 1 AEC as determined by one-way ANOVA.
101
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3,12 Total cellular TfR in rat alveolar epithelial cell monolayers as a function of
culture day
Total cellular TfR (soluble receptor fraction) was next assayed to determine
whether the decrease in surface TfR levels was due to a reduction in TfR protein
synthesis or the sequestration of cellular TfR into an intracellular compartment away
from the cell surface (see Table 1). Total soluble TfR on day 1 was 23.2 (± 1.09)
fmol/cm^ (69.5 (± 3.0) pg TfR/pg protein) and decreased to 0.94 (± 0.98) fmol/cm^
(5.3 (± 2.8) pg TfR/pg protein) by day 7 in culture.
Table 1
Total Cellular TfR (0.1% Triton X-100 Soluble Fraction) in Rat AEC.
pg TfR/ug protein fmol TfR/cm^
Day 1 69.5 (± 3.0) 23.2 (± 1.09)
Day 7 5.3 (±2.8)* 0.94 (±0.98)*
Note: Data are presented as mean (+ SEM) for n=3.
* indicates p<0.01 compared to respective Day 1 values as
determined by ANOVA.
102
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3.13 Rat alveolar epithelial cell monolayers exposed to keratinocyte growth
factor during culture retain TfR expression
Alveolar epithelial cell monolayers were grown continuously in the presence
of KGF (10 ng/mL) from day 0 through day 7 in culture. Monolayers that were
exposed to KGF exhibited retention of TfR levels at the cell-surface through day 7 in
culture (9.67 (± 1.12) fmol/cm^), while monolayers without KGF exposure
demonstrated a greatly reduced level of TfR by day 7 in culture (1.23 (± 1.31)
fmol/cm^), as compared to that of day 1 AEC cultures (9.81 (± 1.34) fmol/cm^)
without KGF (Figure 22). Exposure of alveolar epithelial cell monolayers to KGF
did not appreciably affect TEER, with both groups exhibiting resistances of about
2,500 flScm ^ on day 7 in culture (data not shown).
3.14 Confocal microscopy of keratinocyte growth factor treated rat alveolar
epithelial cell monolayers
The effect of KGF exposure on TfR levels in rat AEC monolayers was also
investigated by confocal microscopy. Day 1 monolayers (-KGF) and day 7
monolayers {+!- KGF) were stained with monoclonal antibody OX-26, which is
specific for rat TfR (352). Monolayers were subsequently labeled with FTTC-labeled
secondary antibody and visualized via confocal microscopy. Day 7 monolayers that
had been exposed to KGF retained significant levels of TfR staining, similar to that
seen for day 1 cells (Figure 23). This is in contrast to day 7 monolayers that were not
exposed to KGF, which exhibited very
103
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T
L _ M
H H l
illiiililM
Day7(»KGF) Day 7(+KGF)
Culture Day
Figure 22. Effect of KGF treatment on TfR expression in
rat alveolar epithelial cell monolayers. Total surface TfR was
determined by the specific binding of *^^I-Tf (1.5 |Xg/mL) with
and without KGF exposure (10 ng/mL) of rat AEC grown on 12
mm Trans well filters for seven days. In all cases, KGF was
added to culture media beginning on day 0 and replaced with
each feeding on days 3 and 6. Error bars represent SEM for n =
6. * indicates p<0.01 as compared to that observed in day 7
(+KGF) AEC as determined by one-way ANOVA.
104
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Figure 23. KGF treatment retains TfR expression in rat
alveolar epithelial cell monolayers.
Rat AEC monolayers examined by confocal microscopy for TfR on
days 1 (-KGF) and 7 (±KGF). Monoclonal antibody OX-26 was
used in conjunction with FTTC-labeled secondary antibody to
specifically localize TfR. TfR staining is more pronounced along
the cell-cell borders (i.e. lateral intercellular regions). Bar equals
10 pm.
105
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Day 1 (-KGF)
Day 7 (-
■ M i l
■ ■ ■ ■ ■ ■
illllli llllil M M w M
Day 7 (+KGF)
jr
106
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little staining for TfR, equivalent to background levels. The confocal images
obtained from the midsection of the monolayers also revealed that TfR staining in
KGF-exposed monolayers was predominantly localized to the basolateral cellular
plasma membrane.
3.15 Type II cell-like rat alveolar epithelial cell monolayers exhibit polarized
distribution of TfR at the cell surface
Previous studies have determined that many other highly polarized cell lines
that form tight monolayers in culture, such as MDCK and Caco-2 cells, localize
expressed TfR almost exclusively to the basolateral surface (353; 354). When KGF-
treated AEC monolayers were selectively exposed to *^^I-Tf, at either the apical or
basolateral surface on day 7 in culture, *^^I-Tf was preferentially bound to the
basolateral surface (Figure 24). The basolateral surface of these monolayers
exhibited 11.8 (± 0.5) fmol TfR/cm^, while the apical surface exhibited only 1.74 (±
2.4) fmol TfR/cm^ (Fig. 3). TfR levels observed for the apical surface were not
significantly different from zero. The basolateral surface of the monolayers not
exposed to KGF exhibited only 1.4 (± 0.5) fmol TfR/cm^, and the apical surface was
again essentially devoid of TfR, exhibiting only 0.19 (± 2.3) fmol TfR/cm^.
107
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3.16 Type II cell-like rat alveolar epithelial cell monolayers exhibit polarized
TfR-specific ® ’Fe-Tf uptake
o
E
12
10
8
2
0
Basolateral
(-KGF)
Apical
(-KGF)
Basolateral Apical
(+KGF) {+KGF)
Figure 24. Rat AEC monolayers tested on day 7 for
polarized distribution of TfR. TfR levels were deteraiined
by TfR-specific binding of ^^^I-Tf (1.5 pg/mL) on either the
apical or basolateral cell membranes of AEC monolayers
(+/- KGF). Error bars represent SEM for n = 6. * indicates
p<0.01 compared to basolateral (-KGF) and ** indicates
p<0.05 compared to basolateral (+KGF), as determined by
one-way ANOVA.
108
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To gain further insight into the functionality of TfR in alveolar epithelial cell
monolayers, TfR-dependent uptake of ^^Fe was determined. ^^Fe-Tf was prepared
and six hour TfR-dependent uptake experiments were performed, at either the apical
or basolateral surface, using day 7 monolayers (+/- KGF). The KGF-exposed
monolayers demonstrated highly polarized ^^Fe-Tf uptake, with a six hour uptake of
229 (±41) fmol ^^e/cm^ from the basolateral surface (Figure 25). In contrast, KGF-
exposed AEG monolayers showed essentially no TfR-dependent ^® Fe uptake [21 (±
33) fmol ^^Fe/cm^] from the apical surface. Monolayers unexposed to KGF
exhibited no significant ^^Fe uptake activity from either the apical or basolateral
surfaces, with respective six hour TfR-dependent ^^Fe uptakes of 0.90 (± 25.0) fmol
^^Fe/cm^ and 0.94 (± 30.9) fmol ^^Fe/cml
3.17 Effect of BFA, AG-10, and LY294002 on TEER of type II cell-like rat
alveolar epithelial cell monolayers
Treatment of rat AEG monolayers with AG-10 (500|iM) and LY294002 (100
|iM) resulted in a rapid decrease in TEER (Figure 26). By the fourth hour of
treatment with AG-10 and LY294002, the TEER had fallen to the point where the
barrier function of the monolayers had begun to fail. In contrast, treatment of AEG
monolayers with BFA resulted in a decrease in TEER to ~1 kOcm^ by the sixth hour.
While the TEER was significantly decreased upon BFA treatment, barrier function
of the monolayers was maintained for the duration of the experiment. BFA was thus
109
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250 -
E
200 -
o
a >
JF-
w
150 -
O
li
100 -
50 -
0 -
Basolateral
(-KGF)
Apical Basolateral Apical
(»KGF) (+KGF) (+KGF)
Figure 25. TfR-specific uptake of ^Ve in rat AEC monolayers
on day 7. ^®Fe-Tf (2)i.g/mL) was applied to either the apical or
basolateral cell surfaces of AEC monolayers (+/- KGF) and
incubated at 37°C for six hours. Nonspecific ^^Fe uptake was
determined in parallel by the addition of 100-fold molar excess of
unlabeled holo-Tf. TfR-specific uptake was determined as the
difference between total and non-specific ^^Fe uptake. Error bars
represent SEM for n = 3. * indicates p<0.01 compared to apical
(+KGF) and ** indicates p<0.01 compared to basolateral (+KGF)
as determined by one-way ANOVA.
110
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3.50
3.00
Control
BFA
^ 2.50
0
1 2.00
1.50
1.00 -
0.50
0.00
0 2 4 6
T im e (h)
Figure 26. Effect of BFA, AG-10, and LY294002
treatment on TEER of rat type II cell-like alveolar
epithelial cell monolayers. Monolayers were spiked with
BFA, AG-10, and LY294002 to a final concentrations of
1.6pg/mL, 500 pM, and 100 pM, respectively, in the apical
compartment of MDCK transwells. The TEER was measured
over a 6-hour time course with a voltohmmeter (n=6).
I l l
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further evaluated for its potential to be used as a TfR-dependent transcytosis
enhancer in AEC monolayers of the type-II cell-like phenotype.
3.18 Effects of BFA on distribution and endocytosis of TfR in rat alveolar
epithelial cell monolayers
Day 6 KGF-treated type II cell-like monolayers and non-KGF-treated type-I cell-like
monolayers were treated with BFA (1 pg/mL) for two hours. The cell-surface
distribution of TfR was subsequently detected by ^^^I-Tf binding (1 pg/ml) at 4°C for
two hours. TfR-specific binding was determined by subtracting the non-specific
component as measured by the separate inclusion of 100-fold excess non
radiolabeled Tf.
Non-KGF-treated AEC monolayers, or monolayers displaying the type I cell
like phenotype, did not exhibit significant differences in TfR expression at the apical
or basolateral membranes, compared to control AEC monolayers, in response to
BFA treatment (Figure 27A). TfR-specific ^^^I-Tf binding at the basolateral surface
was 1.3 ± 0.3 and 1.5 ± 0.3 fmol TfR/cm^, with and without BFA treatment,
respectively. TfR-specific *^^I-Tf binding at the apical surface was 0.20 ± 1.9 and
0.19 ± 2.0 fmol TfR/cm^, with and without BFA treatment respectively.
As shown in Figure 27B, KGF-treated type II cell-like monolayers exhibited
markedly increased TfR, although the inclusion of BFA (1 pg/mL) only slightly
altered the static distribution of TfR in KGF-treated monolayers. Specifically, KGF-
112
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treated monolayers showed a small significant reduction in TfR at the basolateral
membrane upon BFA treatment (10.5 ± 0.5 fmol TfR/cm^) as compared to KGF-
treated monolayers that did not receive BFA (11.8 ± 0.4 fmol TfR/cm^). The apical
membrane of KGF-treated AEC monolayers did not show significant differences in
TfR expression upon BFA treatment, with BFA treated monolayers exhibiting 1.2 ±
2.0 fmol TfR/cm^ and control monolayers exhibiting 1.74 ± 1.9 fmol TfR/cm^.
Since *^^I-Tf binding at 4°C presents only a static view of receptor
distribution, efforts were next undertaken to examine what effect BFA might have on
endocytosis of TfR at the apical and basolateral membranes of type I and tj^e II cell
like monolayers. ^^Fe-loaded Tf was applied to either the apical or basolateral
compartment of day 6 monolayers (± KGF), with or without BFA (1.0 ^ig/mL), and
incubated at 37° C for 5 hours. The use of ^^Fe-Tf enabled endocytosis events to be
measured, unlike the use of ^^^I-Tf, since upon endocytosis of the ^^Fe-Tf-TfR
complex, ^^Fe is known to disassociate from the complex in the acidic endosome and
accumulate inside the cell (292). BFA had no effect on ^^Fe-Tf uptake in non-KGF-
treated monolayers (Figure 27 C). However, BFA significantly altered the extent of
endocytosis in KGF-treated monolayers at the apical and basolateral membranes as
shown in Figure 27D. For example, upon BFA treatment, ^^e accumulation at the
basolateral membrane decreased from 225 ± 25 fmol ^®Fe/cm^ to 175 ± 23 fmol
^^Fe/cm^. In contrast, ^^Fe accumulation at the apical membrane increased upon
113
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Figure 27. The effect BFA on surface TfR expression and ®^Fe-Tf
uptake in rat alveolar epithelial cells (AEC).
TfR levels were determined through TfR specific binding of ^^^I-Tf (1.5
pg/mL) to the apical and basolateral surfaces of AEC cultures grown on
12mm Transwell filters in the presence (A) or absence (B) of KGF (10
ng/mL). Non-specific binding was determined in parallel by the addition
of 100-fold molar excess of non-radiolabeled Tf to the respective donor
fluid. The surface binding experiments were conducted at 4°C for two
hours. TfR-specific binding was determined as the difference between
total and non-specific ^^^I-Tf binding. BFA’s effect on TfR-mediated
uptake was determined via ^^Fe-Tf endocytosis. ^^Fe-Tf (2 pg/mL) was
added to either the apical or basolateral compartment of day 6 AEC
monlayers in the presence (C) or absence (D) of KGF (10 ng/mL). Non
specific uptake was determined in parallel by the addition of 100-fold
molar excess of non-radiolabeled Tf to the respective donor fluid. The
monolayers were excised after a six hour incubation and the level of
radioactivity was determined on a Packard gamma counter. BFA was
added to the apical compartment in all cases (1.0 pg/mL). TfR-specific
uptake was determined as the difference between total and non-specific
^Te uptake. In all cases, error bars represent standard error of the mean
(SEM) for n = 6. * indicates p<0.05 compared to respective non-BFA
treated membrane surfaces as determined by one-way ANOVA.
114
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8
i F A
0 + 8 F A
Basolateral
Basolateral
115
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Figure 27 continued
2i0
200
M
I -
too
50
. 0
2 9 0 -
2 0 0 -
1
m 1 1 0 "
o
2:
m i l -
1 _ J L
Basolateral
BFA
+ BFA
Basolateral
116
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BFA treatment to 70 ± 10 fmol ^^Fe/cm^ as compared to a negligible uptake of 5.1 ±
8.2 fmol ^^Fe/cm^ without BFA treatment.
3.19 TfR-mediated apical-to-basolateral Tf transport in BFA-treated type II
cell-like rat alveolar epithelial cell monolayers
Transport studies were performed to determine if BFA treatment was able to
elicit TfR-specific apical-to basolateral transcytosis in type II cell-like AEC
monolayers (i.e., KGF-treated) and type I cell-like AEC monolayers (i.e., treated
with control vehicle). Day 7 AEC monolayers (with or without KGF treatment)
were dosed with 1.5 pg/mL of ’^^I-Tf in the apical compartment. The apical
compartments of the monolayers were subsequently spiked with BFA and incubated
at 37° C. Samples were taken from the basolateral compartments at regular intervals,
subjected to 15% TCA precipitation, and radioactivity was counted on a Packard
gamma counter. Non-specific '^^I-Tf transport was determined in parallel by
inclusion of 150 pg/mL of unlabelled Tf. As shown in Figure 2, KGF-treated
monolayers demonstrated a significant increase in TfR-specific transcytosis in
response to BFA treatment. For example, after six hours, the amount of transported
i^Sf-Tf was 4.4-fold higher in the presence of BFA (0.31 ± 0.3 ng/well ^^^I-Tf with
BFA, 0.07 ± 0.02 ng/well ^^^I-Tf without BFA) (Fig. 28A). BFA treatment did not
result in significant change in non-specific transport (0.06 ± 0.3 ng/well '^^I-Tf with
BFA, 0.05 ± 0.02 ng/well ^^^I-Tf without BFA) (data not shown). In contrast, the
117
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Figure 28. The effects of BFA on transport in rat alveolar
epithelial cell monolayers.
Day 7 AEC monolayers, with (A) or without (B) KGF-treatment, were
dosed with 1.5 |Xg/mL of in the apical compartment. The apical
compartments of the monolayers were subsequently spiked with BFA and
incubated at 37° C. Samples were taken from the basolateral
compartments at regular intervals, subjected to 15% TCA precipitation, and
radioactivity counted on a Packard gamma counter. Non-specific ^^^I-Tf
transport was determined in parallel by the inclusion of 10-fold molar
excess of non-radiolabeled Tf. BFA (1.0 (Xg/mL) significantly enhanced
TfR-mediated apical-to-basal transport in KGF-treated (10 ng/mL) AEC
monolayers, yet had no effect on monolayers not treated with KGF. KGF-
treated cells exhibited a small amount of transport in the absence of BFA.
BFA treatment did not result in significant change in non-specific transport
(0.06 ± 0.3 ng/well ^^^I-Tf with BFA, 0.05 ± 0.02 ng/well ^^^I-Tf without
BFA) (data not shown), n = 6.
118
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0.35 1
0.30 -
+ BFA
&
0.25 -
-BFA
S 0.20 •
i 0.15 ■
0.10 -
0.05 -
0.00
0 1 2 3
Time (h)
4 5 6
0.35 1
KGF
+ BFA
j .
I
-BFA
•g 0.15 -
^ 0.10 -
c
I
^ 0.05 -
0.00
0 2 3
Tim e (h)
4 5 1 6
119
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type I cell-like AEC monolayers (no KGF) did not demonstrate significant TfR-
mediated ^^^I-Tf transcytosis in the absence or presence of BFA (Figure 28B).
3.20 TfR-mediated apical-to-basolateral transcytosis of In-Tf conjugate across
type II cell-like rat alveolar epithelial cell monolayers
An insulin-transferrin conjugate was synthesized as previously described
(113). Day 7 AEC monolayers (with or without KGF treatment) were dosed with 1.5
|Xg/mL of ^^^I-In-Tf in the apical compartment. The apical compartments of the
monolayers were subsequently spiked with BFA (1.6 pg/mL) and incubated at 37° C.
Samples were taken from the basolateral compartments at regular intervals, subjected
to 15% TCA precipitation, and radioactivity was counted on a Packard gamma
counter. Non-specific *^I-Tf transport was determined in parallel by inclusion of
150 pg/mL of unlabelled Tf. As shown in Figure 29, KGF-treated monolayers
demonstrated a significant increase in TfR-specific transcytosis in response to BFA
treatment. For example, after six hours, the amount of transported ^^^I-ln-Tf was
4.3-fold higher in the presence of BFA (0.30 ± 0.4 ng/well ^^^I-In-Tf with BFA, 0.07
± 0.02 ng/well ’^^I-In-Tf without BFA) (Fig. 2A). BFA treatment did not result in
significant change in non-specific transport (0.08 ± 0.2 ng/well *^^I-In-Tf with BFA,
0.05 ± 0.03 ng/well ^^^I-In-Tf without BFA) (data not shown).
1 2 0
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0.35
+ BFA
lir— BFA
™ 0.20
X3
©
I 0.15
I 0.10
0.05
0.00
0 2 4 6
Time (h)
Figure 29. ^^®I-In-Tf transport in rat alveolar epithelial cell
monolayers. Day 7 AEC monolayers were dosed with 1.5 |ig/mL
of ^^I-Tf in the apical compartment. The apical compartments of
the monolayers were subsequently spiked with BFA and incubated
at 37° C. Samples were taken from the basolateral compartments at
regular intervals, subjected to 15% TCA precipitation, and
radioactivity counted on a Packard gamma counter. Non-specific
i25i_Tf transport was determined in parallel by the inclusion of 10-
fold molar excess of non-radiolabeled Tf. BFA (1.0 |Xg/mL)
significantly enhanced TfR-mediated apical-to-basal transport in
KGF-treated (10 ng/mL) AEC monolayers, yet had no effect on
monolayers not treated with KGF. KGF-treated cells exhibited a
small amount of transport in the absence of BFA. n = 6.
.21
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3.21 Analysis of transcytosed across type II cell-like rat alveolar
epithelial cell monolayers
Apical compartments of BFA-treated (1.0 pg/mL) type II cell-like AEC monolayers
were dosed with 1.5 pg/mL ^^^I-In-Tf and the basolateral media collected after six
hour incubation at 37° C. Samples were subjected to size exclusion chromatography
analysis
When samples recovered from the basolateral compartments were applied to
a 10 ml Sephadex G-50 column, the major recorded peak coincided with the ^^I-In-
Tf column standard at fraction 4, indicating that the molecular weight of the ^^^I-In-
Tf (recovered post-TfR-mediated transcytosis) was identical to the molecular weight
of the *^^I-In-Tf standard. The extent of degradation appeared to be minor, with
relatively little small-molecule products appearing around fraction 10, accounting for
13% of total radioactivity.
3.22 TfR-Mediated ^^®I-GCSF-Tf transcytosis in type II cell-like rat alveolar
epithelial cell monolayers
*^^I-GCSF-Tf transport studies were performed in KGF-treated day 7 AEC
monolayers. The transport studies were performed in an analogous manner to the
i^Sf-Tf studies, with the apical compartment dosed with 1.5 pg/mL *^^I-GCSF-Tf and
122
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Figure 30. Specific TfR-mediated transport of ^^®I-GCSF-transferrin
conjugate determined on day 7 and comparison to ^^®I-GCSF.
The apical compartments of day 7 type II cell-like monolayers were dosed
with 1.5 p,g/mL of ‘^^I-GCSF-Tf (A) or ^^^I-GCSF (B) and spiked with
BFA (1.0 |Xg/mL) at the beginning of the transport study. Samples were
taken from the basolateral compartments at regular intervals, subjected to
15% TCA precipitation, and radioactivity counted on a Packard gamma
counter. Non-specific ^^^I-Tf transport was determined in parallel by the
inclusion of 10-fold molar excess of non-radiolabeled Tf. BFA (1.0
lig/mL) significantly enhanced TfR-mediated apical-to-basolateral
transport in KGF-treated (10 ng/mL) AEC monolayers. Monolayers that
were not exposed to BFA displayed negligible TfR-mediated transcytosis.
BFA treatment did not significantly affect non-specific ^^^I-GCSF-Tf
transport (0.67 ± 0.3 fmol/well ‘^^I-GCSF-Tf with BFA, 0.61 ± 0.3 ng/well
^^^I-GCSF-Tf without BFA) (data not shown). The apical-to-basolateral
transport of '^^I-GCSF was not affected by the presence of BFA, exhibiting
nearly identical transport profiles with or without BFA. n = 6.
123
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4 .5
% BFA
3.5
- BFA 3.0
li.
2.0
-II-------
o.o;
1 2 0 4 5 3
Time (h)
6
4.5
4.0
I 3.5
+/BFA
, - BFA
1
■c
a
m
c
0.5
0.0
0 2 5 1 3
Tim e (h)
4 6
124
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then spiked with BFA (1.0 fig/ml). As shown in Figure 30A, BFA significantly
enhanced TfR-specific apical-to-basolateral transcytosis of ^^^I-GCSF-Tf (3.7 ± 0.68
fmol/well '^^I-GCSF-Tf after six hours), while those monolayers that were not
exposed to BFA displayed negligible TfR-specific ^^^I-GCSF-Tf transcytosis (0.21 ±
0.45 fmol/well ^^^I-GCSF-Tf after six hours). BFA treatment did not significantly
affect the degree of non-specific ^^^I-GCSF-Tf transport (0.67 ± 0.3 fmol/well
GCSF-Tf with BFA, 0.61 ± 0.3 ng/well ^^^I-GCSF-Tf without BFA) (data not
shown).
3.23 Comparison of ^^^I-GCSF-Tf and ^^®I-GCSF apical-to-basolateral
transport in AEC monolayers.
The efficacy of BFA-induced enhancement of ^^^I-GCSF-Tf apical-to-basolateral
transcytosis was compared to the apical-to-basolateral transport of ^^^I-GCSF.
Transport studies were performed as described above, with the apical compartments
of day 7 type II cell-like monolayers dosed with 1.5 |ig/mL of ^^^I-GCSF and spiked
with BFA (1.0 p-g/mL) at the beginning of the transport study. TfR-specific
transport of ^^^I-GCSF-Tf (+ BFA) (Figure 3GB) was significantly greater than that
of the similarly treated ^^^I-GCSF group (+ BFA) (Fig. 3B), with respective
cumulative 6-hour apical-to-basolateral transports of 3.7 ± 0.68 fmol/well ^^I-
GCSF-Tf and 1.2 ±0.11 fmol/well ^^^I-GCSF. The apical-to-basolateral transport of
125
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^^^I-GCSF was not affected by the presence of BFA, exhibiting nearly identical
transport profiles with or without BFA.
3.24 Analysis of transcytosed ^^®I-GCSF-Tf
Apical compartments of BFA-treated (1.0 jig/mL) type II cell-like AEC monolayers
were dosed with 1.5 |Xg/mL ^^®I-GCSF-Tf and the basolateral media collected after
six hour incubation at 37° C. Samples were subjected to size exclusion
chromatography analysis and assayed for bioactivity by measuring the ability to
stimulate NFS-60 cell proliferation (349).
When samples recovered from the basolateral compartments were applied to
a 40 ml Sephacryl-200 column, the major recorded peak coincided with the ^^^I-
GCSF-Tf column standard at fraction 19, indicating that the molecular weight of the
'^^I-GCSF-Tf (recovered post-TfR-mediated transcytosis) was identical to the
molecular weight of the ^^^I-GCSF-Tf standard (Figure 31). The extent of
degradation appeared to be minor, with relatively little small-molecule products
appearing around fraction 40, accounting for 14% of total radioactivity.
The biological activity of transcytosed conjugate was next determined
through a NFS-60 MTT proliferation assay. Basolateral media was collected as
described above. The samples were then sterile filtered, normalized for GCSF
content, and used as assay substrate. The recovered conjugate displayed relatively
minor proliferative ability, with 0.1 ng/mL GCSF equivalents displaying an
126
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absorbance (570 nm) of only 0.23 ± 0.5 (Figure 32). However, when the sample is
subjected to reducing conditions prior to the assay (25 mM DTT, 15 min), we find
TfR Specific
GCSF-Tf
Transport
-o-G C SF-T f Standard
Fraction #
Figure 31. Gel filtration chromatogram of TfR-mediated
transcytosed ^^®I-GCSF-Tf. Apical compartments of BFA-treated (1.0
|ig/mL) type II cell-like AEC monolayers were dosed with 1.5 |ig/mL
1-GCSF-Tf and the basolateral media collected after six hour
incubation at 37° C. The basolateral samples were applied to a 40 mL
Sephacryl 200 column, eluted with PBS (pH 7.4), and compared to the
standard ^^^I-GCSF-Tf solution. The fractions (1 mL) were subsequently
counted on a Packard gamma counter.
127
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- ^ ► - gcsf standard
HD- Reduced Conjugate
Non-reduced
Conjugate
0.000 0.006 0.012 0.025 0.050 0.100 0.200 0.400
GCSF Equivalent (ng/mL)
Figure 32. Evaluation of biological activity of transcytosed
GCSF-transferrin. Apical compartments of BFA-treated (1.0
pg/mL) type II cell-like AEC monolayers were dosed with 1.5
pg/mL ^^^I-GCSF or ^^^I-GCSF-Tf and the basolateral media
collected after six hour incubation at 37° C. Biological activity
assays were conducted for transcytosed ^^^I-GCSF-Tf or reduced
^^^I-GSCF-Tf (25mM DTT treatment for 15 min) by measuring
proliferation of the murine myeloblastic cell line NFS-60 via MTT
assay. Results are also shown for GCSF control (Neupogen).
128
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that biological activity is recovered and fairly closely matches activity of the GCSF
standard, with 0.1 ng/mL GCSF equivalents displaying an absorbance (570 nm) of
0.95 ± 0.07 and 0.80 ± 0.05 for the GCSF standard and the reduced ^^^I-GCSF-Tf
conjugate, respectively.
3.25 Apical-to-basolateral Transcytosis of G-CSF-Tf and TF across Caco-2
monolayers
Two-week old Caco-2 monolayers, exhibiting TEER levels of approximately 500
Ocm^, were dosed with 1.5 pg/mL of ^^^I-G-CSF-Tf or ^^^I-G-CSF in the apical
compartments of 6-well Transwells. As shown in Figure 1, monolayers that received
G-CSF-Tf exhibited significantly higher TfR-mediated transcytosis compared to the
monolayers that received ^^^I-G-CSF. For example, after six, hours, the amount of
transported ’^^I-G-CSF-Tf was 7.8-fold higher than ^^^I-G-CSF (9.3 ± 0.8 fmol/well
*^^I-G-CSF-Tf, 1.2 ± 0.7 fmol/well ^^^I-G-CSF) (Figure 33). In addition, the
transport rate was also significantly higher, with ^^^I-G-CSF-Tf transported at 1.7
fmol/well-hr'^ and ^^^I-G-CSF transported at 0.3 fmol/well h r'\ Non-specific ^^^I-G-
CSF-Tf and ^^^I-G-CSF transport was significantly lower than TfR-mediated
transcytosis, with nonspecific transport composing 25% and 27% of the total
transport for ^^^I-G-CSF-Tf and '^^I-G-CSF, respectively (data not shown).
129
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10 1
o
G-CSF-Tf
c
I
e
a.
xs
«
1 C
o
a .
(0
c
s
H
0 1 2 3 4 5 6
Tlme(h|
Figure 33. Specific TfR-mediated transport of ^^®I-GCSF-
transferrin conjugate determined in Caco-2 monolayers and
comparison to ^ I-GCSF . The apical compartments of two
week-old Caco-2 monolayers were dosed with 1.5 pg/mL of
G-CSF-Tf or ^^^I-G-CSF. Samples were taken from the
basolateral compartments at regular intervals, subjected to 15%
TCA precipitation, and radioactivity counted on a Packard
gamma counter. Non-specific ^^^I-Tf transport was determined
in parallel by the inclusion of 10-fold molar excess of non-
radiolabeled Tf. The apical-to-basolateral transport of ^^^I-GCSF
was not affected by the presence of excess Tf while ^^^I-G-CSF-
Tf exhibited an 80% reduction in transport (data not shown) n=3.
130
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3.26 Chromatographic analysis of transcytosed G-CSF-Tf
Apical compartments of Caco-2 monolayers were dosed with 1.5 )Xg/mL ^^^I-G-CSF-
Tf and the basolateral media collected after six hour incubation at 37° C. Samples
were subjected to size exclusion chromatography analysis. When samples recovered
from the basolateral compartments were applied to a 40 ml Sephacryl-200 column,
the major recorded peak coincided with the ^^^I-G-CSF-Tf column standard at
fraction 19, indicating that the molecular weight of the ^^^I-G-CSF-Tf (recovered
post-T fR-mediated transcytosis) was identical to the molecular weight of the ^^^I-G-
CSF-Tf standard (Figure 34). The extent of degradation appeared to be minor, with
relatively little small-molecule products appearing around fraction 40, accounting for
13% of total radioactivity.
3.27 Analysis of biological activity of transcytosed ^^I-G-CSF-Tf in Caco-2
monolayers
The biological activity of transcytosed conjugate was next determined through a
NFS-60 MTT proliferation assay (Figure 35). Basolateral media was collected as
described above after a six-hour transport experiment across Caco-2 monolayers.
The samples were then sterile filtered, normalized for G-CSF content, and used as
assay substrate. The sample was subjected to reducing conditions prior to the assay
131
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(25 mM DTT, 15 min) in order to mimic reducing conditions that would encountered
by the conjugate in vivo. The biological activity of the recovered ^^^I-G-CSF-TF
400
350 -i
300 -
-■-TfR Specific G-CSF-Tf
Transport
-o-G -C SF-Tf Standdard
250 i
1 200 -
a.
u
150 -
100
o
Fraction #
Figure 34. Gel filtration chromatogram of TfR-mediated
transcytosed ^^®I-GCSF-Tf. Apical compartments of Caco-2
monolayers were dosed with 1.5 |ig/mL ^^^I-GCSF-Tf and the
basolateral media collected after six hour incubation at 37° C.
The basolateral samples were applied to a 40 mL Sephacryl 200
column, eluted with PBS (pH 7.4), and compared to the standard
'^^I-GCSF-Tf solution. The fractions (1 mL) were subsequently
counted on a Packard gamma counter.
132
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1.2
G-CSF Startdard
0.8
E
c -o-G-CSF-Tf
e
w 0.6
0.4
0.2
0.0
0.000 0.006 0.012 0.025 0.050 0.100 0.200 0.400
G-CSF Equivalent (ng/mL)
Figure 35. Evaluation of biological activity of transcytosed
G-CSF-Tf. Apical compartments of Caco-2 monolayers were
dosed with 1.5 |Xg/mL ^^^I-G-CSF or ^^^I-G-CSF-Tf and the
basolateral media collected after six hour incubation at 37° C.
Biological activity assays were conducted for transcytosed ^^I-
GSCF-Tf after reduction of the disulfide linkage (25mM DTT
treatment for 15 min) by measuring proliferation of the murine
myeloblastic cell line NFS-60 via MTT assay. Results are also
shown for GCSF control (filgrastim).
133
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conjugate closely matches the activity of the G-CSF standard, with 0.1 ng/mL G-
CSF equivalents displaying an absorbance (570 nm) of 0.87 ± 0.08 and 0.78 ± 0.06
for the G-CSF standard and the reduced ^^^I-G-CSF-Tf conjugate, respectively.
3.28 Neutrophil proliferation in BDFl mice dosed with subcutaneous and oral
G-CSF-Tf
BDFl mice were given 1 mg/kg filgrastim, 5 mg/kg G-CSF-Tf, or control vehicle
subcutaneously. The day of dosage administration was denoted as day 0. By day 1,
both the G-CSF-Tf and filgrastim treatment groups exhibited an increase in absolute
neutrophil counts (2420 ± 450 cells/|jL for the G-CSF-Tf treatment group and 2375
± 400 cell/|iL for the filgrastim treatment group) (Figure 36). However, by day 3
there was a marked significant difference between the two treatment groups, with
4100 ± 510 cells/fiL for the G-CSF-Tf treatment group and 1200 ± 385 cells/jiL for
the filgrastim treatment groups. The neutrophil levels remained elevated for the G-
CSF-Tf treatment group, relative to control and filgrastim groups, until day 3 and
then returned to normal levels by day 4.
For the oral dosing experiments, BDFl mice were given 10 mg/kg filgrastim,
50 mg/kg G-CSF-Tf, or control vehicle by gavage needle. The mice that received G-
CSF-Tf demonstrated a statistically significant elevation in absolute neutrophil
counts by day 1 with 2350 ± 225 cells/|iL (Figure 37). In contrast, oral
administration of filgrastim did not result in a statistically significant change in
134
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neutrophil levels compared to control (1080 ± 280 cells/|iL and 990 ± 95 cells/jxL for
filgrastim and control groups on day 1, respectively). Absolute neutrophil counts
continued to be significantly elevated for the G-CSF-Tf treatment compared to
control until day 3, when they retumed to normal levels.
A dose response experiment was next performed on orally administered G-
CSF-Tf. BDFl mice were given either 50 mg/kg, 25 mg/kg, or 12.5 mg/kg G-CSF-
Tf orally by gavage needle. The group that received 50 mg/kg demonstrated a
similar response as seen in the previous experiment, with 2300 ± 295 ceUs/|iL
recorded for absolute neutrophil counts on day 1 (Fig. 6), which was significantly
different from the other two treatment groups. There was an indication that the
neutrophil counts were elevated for the 25 mg/kg group compared to the 12.5 mg/kg
group on day 1 (1400 ± 200 cells/pL and 1030 ± 168 cells/jiL for 25 mg/kg and 12.5
mg/kg groups, respectively, p < 0.10).
135
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5000
_ 4500
"i
1 4000
"5
— 3500
I
I sooo
0
1 2500
a.
0
Z
2000
1500
1 1000
XI
<
500
* G-CSF-Tf
-♦-Filgrastim
Control
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5
Figure 36. Myelopoietic effect of subcutaneously
administered G-CSF-Tf. G-CSF-Tf (5 mg/kg), filgrastim
(1 mg/mL), or control vehicle was administered
subcutaneously to 8 week old male BDFl mice. Absolute
neutrophil counts were determined daily. * indicates p <
0.01 compared to control.
136
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2500
i
§ 2000
O 1500
Ic
a
z
I
o
< n
XI
<
1000
500
G-CSF"Tf
Filgrastim
Control
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5
Figure 37. Myelopoietic effect of orally administered G-
CSF-Tf. G-CSF-Tf (50 mg/kg), filgrastim (10 mg/kg), or
control vehicle was administered orally by gavage needle to
8 week old male BDFl mice. Absolute neutrophil counts
were determined daily. * indicates p < 0.01 compared to
control.
137
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2500
2000
■ 4
tn
c
O 1500
a.
8
S 1000
I
0
©
m
§
500
-♦"G-CSF-Tf ( » n ^ )
-■-G-CSF-Tf (25ni/kg)
G^SF-Tf (125 nr^g)
DayO Dayl E :te y 2
Figure 38. Dose response effect of orally administered
G-CSF-Tf. G-CSF-Tf was given orally to 8 week old
BDFl mice by gavage needle at doses of 50 mg/kg, 25
mg/kg, and 12.5 mg/kg. Myelopoietic effect was
determined by daily absolute neutrophil counts. * indicates
p < 0.01 compared to 12.5 mg/kg group.
138
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DISCUSSION
As shown in Figures 6-8, the G-CSF that was obtained through the GST-G-
CSF fusion construct closely matched the commercially obtained G-CSF (filgrastim)
in the ability to stimulate the proliferation of the G-CSF-dependent murine cell-line
NFS-60. Synthesis of the G-CSF-Tf conjugate was initially attempted using the
heterobifunctional linker SPDP to create a sulfhydryl-reactive modified-Tf that
would be targeted to the free sulfhydryl group of Cysl7 in G-CSF. Even though the
free sulfhydryl group of G-CSF demonstrated reactivity when incubated with
Ellman’s reagent, initial attempts at reacting Tf-PDP with G-CSF-Cysl7 proved
unsuccessful (data not shown). In order to understand why the reaction was not
occurring, the crystal structure of G-CSF was examined using the molecular
visualization computer program, Rasmol. From the crystal structure (Figure 39), it
was apparent that the reaction of SPD-Tf with G-CSF-Cysl7 may have been
impeded due to the fact that Cysl7 of G-CSF resides within a pocket on the surface
of the molecule, thereby suggesting the possibility of steric hindrance to the reaction.
In light of this possibility, a longer -SH reactive linking agent was first used to
modify the free sulfhydryl group so that the -SH reactive species of DPDPB may
reach beyond the borders of the pocket to freely interact with the free -SH groups of
the DTT-reduced Tf-PDP. This modification allowed the reaction to proceed and the
release of pyridine-2-thione was followed by measuring the absorbance at 343nm.
139
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Figure 39. Spacefill model of G-CSF x-ray crystal structure. PDB
coordinates were obtained from the Brookhaven protein data bank and the
image was rendered with Rasmol. The arrow denotes the position of the free
sulfhydryl group of Cysl7, shown in the lighter shaded atom, which resides in
a pocket on the surface of G-CSF.
140
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G-CSF was conjugated to Tf by disulfide linkage chemistry. Disulfide
linkage chemistry provides the advantage of allowing cleavage of the conjugate to
liberate the free protein-dmg into the circulation (355). Densitometric scanning
analysis of the conjugate revealed that the ratio of Tf to G-CSF in the conjugate was
approximately 1:1 (Figure 7). However, when the non-reduced conjugate was
observed under non-reducing conditions, it appeared to be composed of a
heterogeneous mixture of higher ordered conjugate species (100 kDA to 300 kDa).
It is possible that crosslinking of Tf molecules may occur, as there is multiple
reactive free sulfhydryl groups on the surface of Tf after Tf-PDP has been reduced
with DTT.
Even though the limitations of the current disulfide linkage prevents the
formation of a defined product, the fact that cross-linking occurs between Tf
molecules allows the conjugate to be conveniently separated from unreacted
constituent proteins (i.e. G-CSF and Tf) merely by using size exclusion
chromatography of limited resolution power (i.e. Sephacryl 200) (Figure 8). In
addition, this purification process results in the NFS-60 cell proliferative ability to be
mostly restricted to the protein fraction that contains the conjugate, indicating that
the conjugate is well isolated from its constituent proteins.
Previous findings have demonstrated that the GTPase inhibitor, AG-10, was
able to elicit a dose-dependent TfR-specific enhancement of Tf and In-Tf
transcytosis across Caco-2 monolayers, as well as enhancing the hypoglycemic effect
of orally administered In-Tf in streptozocin-induced diabetic rat (113; 312). Even
141
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though evidence exists of in vitro and in vivo TfR-specific transport across epithelial
barriers in the absence of enhancers, investigation of agents such as AG-10 is
interesting because uptake of Tf-protein drug conjugates from the GI epithelium is
still relatively inefficient when compared to invasive delivery methods. The rate of
apical-to-basolateral TfR transcytosis can be thought of as relatively low when one
considers that most of the TfR that is present on the surface of polarized intestinal
epithelial cells exists at the basolateral surface of the cell (356). Because of this
polarity, most of the TfR that enters the cell will be from basolaterally derived
endocytosis (357). Alteration of these events, such that the equilibrium of TfR
endocytosis is shifted to some extent toward the apical membrane, could conceivably
result in significant improvement of net apical-to-basolateral flux of Tf-protein-drug
conjugates across epithelial barriers.
As mentioned previously, PD kinases are thought to be downstream effectors
of Rab GTPases. It was postulated that inhibition of PDK could enable a more
specific enhancement of TfR-mediated transcytosis compared to AG-10 treatment.
Initial efforts focused on the effects of wortmannin on TfR-mediated transcytosis of
i^Sf-Tf across Caco-2 monolayers. As was shown in Figure 9, treatment with
wortmannin exhibited a dose dependent and TfR-specific enhancement of *^^I-Tf
apical-to-basolateral transport. In addition, use of wortmannin did not adversely
affect TEER of the monolayers over the course of the experiment (Figure 10),
indicating that integrity of the monolayer’s barrier function was maintained. Though
it is very potent PIK inhibitor, wortmannin is a natural product and it is not very
142
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specific, affecting a broad range of PIK subtypes (224-227). Because of this,
LY294002 was chosen as a specific inhibitor of PDK to investigate whether
inhibition of events that are downstream of Rab5 functions will result in an
enhancement of TfR-mediated transcytosis that is similar in magnitude or perhaps
even better than that seen for AG-10. As was shown in figure 11-A, LY294002
treatment results in a significant enhancement of total ^^^I-Tf apical-to-basolateral
transport across Caco-2 monolayers. When the ^^^I-Tf transport is examined by
comparing non-specific and TfR-mediated processes (Figure 1 IB), it can be seen
that the enhancement of ^^^I-Tf transport in the presence of LY294002 (100 |iM) is
due to an increase in only the TfR-mediated ^^^I-Tf apical-to-basolateral transport.
LY294002 treatment had no effect on the non-specific transport of ^^^I-Tf across the
Caco-2 monolayers, with both the LY294002 and control groups exhibiting identical
non-specific ^^^I-Tf transport rates. From this data and the fact that LY294002 did
not adversely affect the TEER of Caco-2 monolayers (Figure 12), it is apparent that
treatment with LY294002 does not result in loss of monolayer integrity.
Once it had been established that a PDK inhibitor (i.e. LY294002) was able
to elicit specific enhancement of TfR-mediated transcytosis, efforts were undertaken
next to compare the effect of LY294002 on TfR surface distribution and TfR specific
uptake of ^^e-Tf with that of the previously investigated transcytosis enhancers,
BFA and AG-10. As previously discussed, AG-10 and LY294002 treatment resulted
in a significant increase in TfR at the apical surface of the monolayer (Figure 13). In
contrast, BFA treatment resulted in a significant decrease in TfR at the apical
143
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surface. When the basolateral membrane was examined, the amount of TfR present
at the surface was not significantly different from control for the LY294002 and AG
IO treatment groups, while the BFA group exhibited a slight but significant decrease
in basolateral surface TfR compared to control. Since BFA treatment has been
shown to result in intemalization and localization of TfR to the trans-Golgi network
(307), one might expect to see a reduction in total surface TfR.
The results from the surface binding studies in Caco-2 cell monolayers in the
presence of transcytosis enhancers would suggest that BFA is acting in a distinct
fashion when compared to AG-10 and LY294002. This difference becomes even
more striking when one looks at intemalization events as opposed to static binding of
^25i-Tf at the cell surface. Uptake studies with ^^Fe-Tf allow for the recording of TfR
intemalization since upon endocytosis, ^Ve will be released from Tf and
accumulated in the cytoplasm as the endosome is acidified. As shown in Figure 14,
BFA treatment had a dramatic and significant effect on Tf uptake from the apical
surface, with an approximately 5-fold increase in ^^Fe-Tf uptake. However, AG-10
and LY294002 apparently did not affect the uptake of ^^Fe from the apical surface,
with results that were not significantly different than control. This result seemingly
contrasts with the ^^^I-Tf transport data across Caco-2 monolayers in the presence
the transcytosis enhancers (Figure 15). As was shown, AG-10 and LY294002 had a
similar and significantly greater enhancement effect on Tf A-to-B transport
compared to BFA. Thus LY294002 would seem to affect a similar TfR recycling
pathway as AG-10.
144
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This trend is also seen when one examines the effect of LY294002 on TfR-
mediated transcytosis across MDCK monolayers. As shown in Figure 17, BFA had
a significantly greater effect on TfR-mediated transcytosis compared to those
monolayers that received AG-10 or LY294002. In addition, LY294G02 had an
almost identical effect on MDCK TEER as compared to AG-10 over the course of
the transport experiment (Figure 18). The similarity of LY294002 and AG-10
effects on TfR mediated processes is not limited to transport phenomenon.
LY294002 also demonstrates a lack of effect on TfR surface distribution in MDCK
monolayers (Figure 19), as well as ^^Fe-Tf uptake (Figure 20).
Variability in sorting mechanisms for apically destined TfR in Caco-2 and
MDCK cultures forms the basis for understanding the functional differences between
the transcytosis enhancers examined in this study. The predominant mechanisms
whereby cells maintain membrane TfR polarity lies in the ability to sort apical or
basolateral proteins and target them to their respective membranes. There exists two
types of mechanisms by which epithelial cells separate apically destined surface
proteins from those proteins that are meant to be sorted to the basolateral membrane,
namely indirect or direct sorting. This sorting is primarily initiated at two distinct
cellular domains, the trans-Golgi network and the basolateral endosome (358).
Direct sorting, which is used by MDCK cells, utilizes the trans-Golgi network to
fully separate apical and basolateral proteins from each other and deliver these
proteins in a vectorial fashion directly to their respective cellular surface (359). In
addition to direct sorting, Caco-2 cells may utilize indirect sorting of apically
145
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destined plasma membrane proteins (360; 361). For example, proteins that are
targeting to the apical membrane may initially be directed to the basolateral surface
from the trans-Golgi network. These proteins are then sorted only after they are
endocytosed into the basolateral endosomal system. Once in the basolateral
endosome, the apical proteins are separated from proteins that are destined for
recycling back to the basolateral membrane or for degradation in the lysosomal
compartments, and then transcytosed to the apical surface.
From the data presented in this report, it is postulated that LY294002
dependent enhancement of TfR mediated transcytosis is derived from inhibition of
PD-K dependent processes in the indirect basolateral endosomal sorting of TfR that
is distinct from enhancement by modification of direct sorting mechanisms, as with
BFA (Figures 40 and 41). For example, whereas BFA caused an enhancement of
TfR-mediated transcytosis in both MDCK and Caco-2 cultures, LY294002 treatment
was only able to elicit an enhancement of TfR-mediated transcytosis in Caco-2
cultures (Figure 14). In addition, similarly to previous observations made in human
hepatoma Hep 0 2 cells (362), BFA treatment was shown to decrease TfR expression
at the apical and basolateral surfaces of the Caco-2 cultures and MDCK cultures
(Figures 13 and 19, respectively).
146
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/Y y yYVYYYVYYYYYYVYYVY|
Direct Sorting
+BFA
Indirect
Sorting
+AG-10
+LY294002
Newly Synthesized
TfR
Figure 40. Possible mechanisms of transcytosis enhancer’s effect
on TfR distribution in polarized epithelia. BFA is known to cause
fusion of TGN and endosomal system and may disrupt the direct
sorting of TfR in MDCK and Caco-2 cells. AG-10, Wortmannin, and
LY2940Q2 may affect Rab and PDK dependent processes in
basolaterally derived TfR sorting. This would result in enhancement of
indirect TfR sorting in Caco-2 cells, but not MDCK cells.
147
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Apical.membrane
Recycling
/
Late ^
endosomes
'X .
C > Recycling
i-L & A iy ^
\
\
/
Basolateral
endocytosis
i
1 FYVL , ___
Rab5
-------- ^
PI-3K
t
AG-10 LY294002
W ortm annin
Figure 41. PI3K Inhibitors as Potential TfR-Mediated
Transcytosis Enhancers. A possible method of action for wortmannin
and LY294002 on TfR distribution in caco-2 cells that may involve
effects downstream from AG-10 activity. LY294002 and wortmannin
may inhibit TfR recycling to the basolateral membrane and result in a
transient shift to the apical plasma membrane .
148
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X). In contrast, both AG-10 and LY294002 caused an increase in apical surface
expression of TfR and a possible decrease in basolateral expression of TfR in Caco-2
monolayers, while causing no statistically significant change in TfR expression in
MDCK monolayers.
LY294002 treatment of Caco-2 monolayers resulted in an enhancement of
TfR-mediated transcytosis in the apical to basolateral direction, on the other hand the
uptake of ^®Fe-Tf was not enhanced in the presence of LY294002 nor AG-10, while
BFA treatment resulted in a significant increase in ^^Fe-Tf uptake from the apical
membrane. It is possible that since BFA treatment results in the retrograde
movement of TfR to the TGN, that this indirect transcytotic movement of TfR would
allow sufficient time for the acidification of the intravesicular milieu such that ^^Fe
would be released from the TfTfR complex. LY294002 and AG-10 may enhance
the movement of rapid transcellular vesicle trafficking where the vesicles may not be
sufficiently acidified to release ^^e before the vesicle is merges with the plasma
membrane for exocytosis. Or alternatively, the presence of AG-10 and LY294002 in
these cells types may potentially cause a lack of divalent metal ion transporters at the
apical surface, resulting in retention of ^^Fe as the vesicle is transcytosed across the
cell.
Investigations were also performed in primary culture system using rat
alveolar epithelial cell monolayers. The level of TfR at the plasma membrane, as
measured by specific binding of ^^I-labeled Tf to total cell-surface, decreased from a
maximum on day 1 to a minimum on day 7 in culture (Figure 21). The decrease in
149
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total cell-surface TfR coincided with transdifferentiation from the type II to type I
cell-like phenotype, which normally occurs by day 3-4 in culture (329-332). This
suggests that TfR synthesis may be down-regulated in rat alveolar epithelial cells
upon transdifferentiation to the type I cell-like phenotype and that TfR synthesis
might be limited to alveolar epithelial cells of the type II phenotype.
Total cellular TfR (soluble receptor fraction) was also assayed to determine
whether the decrease in surface TfR levels was due to a reduction in TfR protein
synthesis or the sequestration of cellular TfR into an intracellular compartment away
from the cell surface (see Table 1). The fact that the total soluble receptor also
decreased confirms the decreased synthesis of TfR after ceUs transdifferentiated
towards the alveolar type I cell-like phenotype.
The high level of TfR in cells of the type II cell-like phenotype early in
culture presents a viable target for TfR-mediated transcytosis. However, the lack of
substantial TEER early in culture (e.g., days 0-2) precludes conducting transport
studies using type II cell-like monolayers. Exposure of rat alveolar cell monolayers
to KGF has previously been implicated in prevention of the transition of alveolar cell
monolayers from the type II cell-like to the type I cell-like phenotype. In particular,
KGF exposure of AEG monolayers leads to retention of expression of lung surfactant
proteins and lamellar bodies, which are identifying characteristics of the type H cell
phenotype (333; 336), and KGF prevention and reversal of expression of the type I
cell-specific marker proteins, T la and aquaporin-5 (333; 334).
150
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Alveolar epithelial cell monolayers were grown continuously in the presence
of KGF (10 ng/mL) from day 0 through day 7 in culture. Monolayers that were
exposed to KGF exhibited retention of TfR levels at the cell-surface through day 7 in
culture, while monolayers without KGF exposure demonstrated a greatly reduced
level of TfR by day 7 in culture.
Previous studies have determined that many other highly polarized cell lines that
form tight monolayers in culture, such as MDCK and Caco-2 cells, localize
expressed TfR almost exclusively to the basolateral surface (353; 354). When KGF-
treated AEC monolayers were selectively exposed to ^^^I-Tf, at either the apical or
basolateral surface on day 7 in culture, ^^^I-Tf was preferentially bound to the
basolateral surface. TfR levels observed for the apical surface were not significantly
different from zero, allowing us to conclude that the apical surface can be considered
to be predominantly lacking in TfR. The binding studies for day 7 monolayers not
exposed to KGF support the findings noted above that the amount of TfR present at
the cell surface is greatly reduced in type I cell-like monolayers.
The confocal images obtained from staining with the monoclonal anti-body
OX-26 supported the binding studies that were performed with radiolabeled Tf
(Figure 23). For example, monolayers of the type II cell-like phenotype retained TfR
expression while monolayers of the type I cell-like phenotype did not demonstrate
any TfR-specific staining. The confocal images obtained from the midsection of the
monolayers also revealed that TfR staining in KGF-exposed monolayers was
predominantly localized to the basolateral cellular plasma membrane. This is
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consistent with the normal cellular routing of internalized TfR, where endocytosed
TfR is promptly recycled back to the plasma membrane after the release of ferric
ions in acidic endosomes, thus localizing the potential staining to the basolateral
plasma membrane of the cell at steady state (324).
TfR-dependent uptake of ^^Fe was determined in order to gain insight into the
effect apparent cellular phenotype, i.e. type I or type It cell-like monolayers, had on
TfR endocytosis function (Figure 25). To gain further insight into the functionality
of TfR in alveolar epithelial cell monolayers. The KGF-exposed monolayers
demonstrated highly polarized uptake with most of the TfR-dependent uptake
occurring from the basolateral surface. In contrast, KGF-exposed AEC monolayers
showed essentially no TfR-dependent ^^Fe uptake from the apical surface.
Monolayers unexposed to KGF exhibited no significant ® ^F e uptake activity from
either the apical or basolateral surfaces. The amount of ^^Fe uptake is directly
proportional to the amount of ^^Fe-Tf that has been endocytosed by the monolayers.
^^Fe-free apo-Tf is subsequently recycled to the basolateral surface and exocytosed
by the monolayers (363). Since the molar ratio of ^^Fe:Tf is 2:1, it can be estimated
that the turnover of Tf by AEC monolayers in a six hour period is about 115 fmol
Tf/cm^, which is approximately 10 fold higher than the molar amount of TfR that is
resident on the basolateral surface of KGF-exposed AEC monolayers as determined
by the ^^^I-Tf binding studies (11.8 (± 0.5) fmol TfR/ cm^). This indicates an
apparent half-life for TfR residence at the basolateral membrane of approximately 18
min. These data provide evidence that the elevated synthesis of TfR, as a result of
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KGF exposure, leads to avid ^^Fe uptake activity, and that the activity is restricted to
the basolateral surface.
This part of the study demonstrated that alveolar type Tl-like epithelial cells
express significant TfR, and that synthesis of TfR is down regulated upon
transdifferentiation from the type II to the type I cell-like phenotype. Exposure of
the AEC cultures to KGF results in retention of TfR, indicating that maintenance of
type n cell-like phenotype results in retention of TfR synthesis. In addition, the
synthesized TfR is limited exclusively to the basolateral surface of AEC monolayers
and TfR activity, as determined by TfR-specific ^^Fe-Tf uptake, is restricted
predominantly to the basolateral surface of KGF-exposed AEC monolayers. The
presence of TfR exclusively at the basolateral surface of AEC monolayers exhibiting
type n cell-like characteristics indicates that type II cells are likely involved in the
transcytotic transport of Tf between plasma and alveolar lining fluid. These findings
may provide insight into how Tf, an important constituent of the surface fluid lining
the alveoli of the lung, is present at relatively high concentrations in alveolar fluid.
Elucidating the magnitude, directionality, and regulation of these putative processes
presents an attractive area for future research.
We previously reported that Tf-protein drug conjugates have been
successfully used to facilitate the transcytosis of the protein drugs across epithelial
barriers in vitro and in vivo (112) (312). While past efforts focused on the
development of oral protein drug delivery methods, the current study explores the
possibility of non-invasive protein drug delivery facilitated by TfR transcytosis
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across the pulmonary epithelium. One of the limitations of using Tf as a carrier
molecule for pulmonary delivery of protein-based therapeutics is the predominant
localization of TfR to the basolateral membrane of alveolar epithelium (114). There
is very little transcytosis of TfR from the basolateral membrane to the apical
membrane under normal conditions. Indeed, epithelial tissues will in general have
TfR predominantly localized to the basolateral (serosal) membrane in order to
facilitate iron uptake from the blood stream. In addition, basolateral TfR is
predisposed to enter into a recycling pathway upon endocytosis, precluding the
transfer of receptors to the apical membrane. Transcytosis enhancers have
previously been utilized to overcome this dilemma to enable oral in vitro and in vivo
protein drug delivery (112; 292; 312). Transcytosis enhancers were thus considered
a means to enable Tf carrier-mediated drug delivery at the pulmonary epithelium.
BFA and T- 8 have previously been used as transcytosis enhancers. In contrast to
previously studied systems (312), T- 8 proved to rapidly destabilize the AEC
monolayer through unknown mechanisms, precluding the study of TfR-mediated
transcytosis (data not shown), while cellular tight junctions were maintained
throughout the experiment in the presence of BFA. Our efforts therefore focused on
the use of BFA as an enhancer of TfR-mediated transcytosis across alveolar
epithelium.
BFA was found not to significantly alter the polar distribution of TfR in type
n cell-like AEC monolayers (i.e., KGF-treated), as measured by binding of '^I-Tf at
4° C (Figure 27). However, BFA treatment did enhance uptake of ^®Fe-Tf from the
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apical membrane and concomitantly decreased uptake of ^^Fe-Tf from the basolateral
membrane of type II cell-like AEC monolayers (Figure 27D). This apparent
discrepancy can be resolved through the following hypothesis. BFA treatment is
postulated to induce missorting of TfR, derived from the basolateral endocytic-
recycling pool or from nascently synthesized receptor, such that a portion of the TfR
is misrouted to the apical membrane where the receptor is only fleetingly situated
prior to being resorted back to the Golgi complex. In support of this hypothesis, it is
useful to note that BFA has previously been shown to reversibly alter the cis- and
trans-Golgi complexes and to disrupt normal endoplasmic reticulum-Golgi
interactions (364-366), which would tend to support the possibility that TfR could be
missorted to the apical membrane. In addition, we have previously demonstrated a
similar result when Madin-Darby canine kidney (MDCK) cell monolayers are treated
with BFA (292). The MDCK cells also exhibited similar behavior in response to
BFA treatment, with rate of uptake increased at the apical membrane without any
change in the average number of receptors present at the apical membrane (292).
The MDCK cell line used exhibits high TEER (~2500 Qcm^), characteristics similar
to the primary AEC monolayers used in the current study. Lastly, the fact that the
amount of TfR residing at the basolateral membrane was slightly diminished upon
BFA treatment (Figure 27B) in type II ceU-like monolayers, and that the level of TfR
endocytosis from the basolateral membrane was also diminished (Fig. ID), suggests
a possible receptor source-pool for the increase in TfR endocytosis from the apical
membrane. It is likely that a portion of internalized basolateral TfR would be subject
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to misrouting to the apical membrane. Even though the 4° C binding studies
were unable to demonstrate a change in static apical TfR distribution in response to
BFA treatment, the ability of BFA to increase uptake of ^^Fe-Tf from the apical
surface suggested a possible means to facilitate apical-to-basolateral transcytosis in
type n cell-like AFC monolayers.
Our results indicate that BFA is able to specifically enhance apical-to-
basolateral TfR-mediated Tf transcytosis in AFC monolayers of the type II cell-like
phenotype (-f-KGF, Figure 28A), but not in AFC monolayers of the type I cell-like
phenotype (-KGF, Figure 28B). This result contrasts with previously published
reports that indicated enhancement of TfR-mediated transcytosis in AFC monolayers
presumed to be of the type I cell-like phenotype (367). AFC monolayers used in our
study had much higher TFFR values than those in the previous report (-2500 Ocm^
compared to -1300 Ocm^), suggesting a possible difference in monolayer
composition. Our results are not surprising when one considers that type II cells are
postulated to be much more metabolically active, playing a critical role in
synthesizing and secreting lung surfactant and surfactant proteins, as compared to the
relatively quiescent type I cells, whose major biological roles include formation of
the air-interface barrier and fluid clearance from the alveolar spaces, and as such
would be more likely to have a much lower iron requirement as compared to type II
cells.
Our previous results demonstrated that apical-to-basolateral transcytosis of an
insulin-Tf conjugate across Caco-2 monolayers was enhanced by BFA treatment
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(112). In addition, BFA enhanced the in vivo hypoglycemic effect of the insulin-Tf
conjugate when administered to streptozotocin-induced diabetic rats (312). Our
current results extend these findings to show that the TfR-mediated transcytosis
pathway is not limited to Tf conjugates covalently linked to small protein drugs like
insulin, but that it can also be utilized with much larger protein drugs like G-CSF.
Since the molecular weight of GCSF (19 kDa) is rather ubiquitous for cytokines in
general, this pathway could hold promise for many more protein drugs of interest.
We observed that a covalently linked GCSF-Tf conjugate is able to be
transcytosed across type II cell-like AEC monolayers in a manner almost identical to
that observed for Tf itself (Figure 30A). This not unexpected, since BFA-derived
enhancement of transcytosis is specific for TfR and does not influence the non
specific transport of other proteins. In support of this statement. Figure 3B
demonstrated that transport of ^^^I-GCSF in type II cell-like AEC monolayers was
not influenced by the presence of BFA. Indeed, GCSF had a substantially higher
transport across the AEC monolayer when covalently linked to Tf and subjected to
BFA exposure.
In addition to enhancement of transport, TfR-mediated transcjdosis of the
conjugate appears to deliver the protein drug to the receiver compartment in an
unaltered native state. We demonstrated that the molecular weight of the conjugate
was not altered when transcytosed via TfR-specific processes (Figure 31). A minor
fraction of degradation products were noted in the chromatographic analyses, most
likely due to the routing of a small percentage of the TfR enriched endosomes to the
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lysosomal pathway. In addition, the protein drug retained biological activity when
released from the conjugate via reductive chemistry (Figure 32). Our previous
studies indicated that free insulin is released from an insulin-Tf conjugate both ex
vivo (when incubated with rat liver) and in vivo (as measured by human insulin
radioimmunoassay) (113). The GCSF-Tf conjugate utilized in this study uses
similar disulfide linker chemistry, and it is postulated that GCSF-Tf will be reduced
in a likewise fashion in vivo. In addition, the lack of substantial biological activity
for the conjugate does not necessarily obviate the potential for the monomer
conjugate to retain biological activity. The current disulfide linker chemistry results
in formation of higher-ordered cross-linked products that might potentially be
responsible for the lack of biological activity seen for the conjugate in this study.
Optimization of the cross-linking process could result in formation of conjugate
products that retain substantial biological activity. These data indicate that the
conjugate is not degraded as a result of TfR-mediated transcytosis and that the GCSF
retains biological activity. Reduction of the labile disulfide bonds within the linker
moiety liberates free active protein drug from the conjugate. This reduction
phenomenon could be predictive of in vivo conjugate reduction characteristics.
We have thus demonstrated that TfR is expressed in a polarized fashion in
monolayers of the AEC type n cell-like phenotype. In addition, it is proposed that
BFA exposure results in transient localization of TfR to the apical membrane with
subsequent rapid intemalization of these missorted receptors. This was evidenced by
the substantial increase in ^^Fe-Tf uptake at the apical surface of these monolayers in
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response to BFA treatment, with the net number of receptors remaining unchanged at
the apical surface (4° C, ^^^I-Tf binding studies). BFA also enhanced TfR-mediated
transcytosis of ^^^I-Tf and the *^^I-GCSF-Tf conjugate in the apical-to-basolateral
direction across the monolayers. The conjugate was not degraded by this transport
process, and the conjugated protein retained biological activity when recovered from
the receiver compartment. This study suggests the possibility of using TfR-mediated
transcytosis for systemic delivery of therapeutic proteins via the alveolar epithelium.
Caco-2 cell culture model has come to serve as a leading qualitative indicator
in screening GI permeability and absorption of therapeutic agents (368-371). In this
report, we have investigated the amount of ^^^I-G-CSF-Tf transported in the apical-
to-basolateral direction, relative to ^^^I-G-CSF, in two-week old Caco-2 monolayers
(Figure 1). Our data demonstrate that the rate of apical-to-basolateral ^^^I-G-CSF-Tf
transport is significantly higher than that of *^^I-G-CSF (1.70 fmol/well/hr and 0.25
fmol/well/hr for ^^^I-G-CSF-Tf and ^^^I-G-CSF, respectively). The enhanced
transport of the G-CSF-Tf conjugate, compared to G-CSF, was presumed to be as a
result of TfR-mediated transcytosis processes. This was suggested by competitive
inhibition studies that demonstrated 80% reduction in ^^^I-G-CSF-Tf apical-to-
basolateral transport in the presence of 100-fold molar excess unlabeled Tf (data not
shown). We have previously demonstrated that an insulin-Tf conjugate is able to be
transcytosed across Caco-2 monolayers by TfR-specific processes, while the
unconjugated insulin was unable to be transported across Caco-2 monolayers (112).
The fact that we have demonstrated that G-CSF, a much larger protein-drug than
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insulin, is also able to transported across Caco-2 monolayers as a Tf-conjugate
suggests that TfR-mediated transcytosis may serve as a multi-platform vehicle to
deliver proteins of varying sizes.
Efforts were next undertaken to examine the protein-drug downstream after
the transcytosis process. Our results indicate that G-CSF only suffered from a minor
degree of degradation as a result of the TfR-mediated transcytosis process. For
example, as shown in Figure 34, when G-CSF-Tf is recovered from the basolateral
compartment, post-TfR-mediated transport, and applied to a 40 mL S-200 column,
the major recorded peak coincides with the peak for the G-CSF-Tf standard at
fraction number 19. A low level of small molecular weight degradation products can
also be seen at fraction number 40, accounting for only 13% of the total applied
radioactivity. One can infer from this data that the molecular weight of the G-CSF-
Tf conjugate is not altered by the transcytosis process in Caco-2 monolayers and
relatively little conjugate is degraded. This result is supported by previous studies
that have shown similar results in monolayers of various cell types and for different
cargo-proteins (112; 292; 310).
In addition to determination of molecular weight, the biological activity of
the transcytosed conjugate was also determined. The conjugate was recovered after
a transcytosis experiment from the receiver compartment of Caco-2 monolayers,
concentrated ten-fold, and then subjected to a brief mild DTT reduction (25 mM
DTT, 15 min) in order to liberate free G-CSF from the conjugate. The DTT
reduction also serves to mimic the natural reduction of di-sulfide linked Tf protein-
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drag conjugates that one would observe in vivo (113). As shown in Figure 35, the G-
CSF that has undergone TfR-mediated transcytosis across Caco-2 monolayers retains
almost all of its biological activity relative to control, as measured by the ability to
stimulate the proliferation of NFS-60 cells (349) (ED50 values of 0.06 ng/mL and
0.07 ng/mL for the G-CSF standard and the transcytosed-G-CSF, respectively).
Taken together, these experiments suggest that TfR-mediated transport might be able
to deliver protein-based therapeutics that are much larger than insulin (312), across
GI epithelial barriers while retaining biological function of the cargo protein.
The in vivo efficacy of G-CSF-Tf was investigated next. Acute doses of G-
CSF-Tf, filgrastim, or control vehicle were administered subcutaneously to 6 - 8 week
old BDFl mice (n=5). Absolute neutrophil counts were performed daily. As can be
seen in figure 4, the G-CSF-Tf treatment group demonstrated an increased duration
of action and significantly higher neutrophil counts (for days 2 and 3) relative to the
group receiving filgrastim. G-CSF is known to foUow a non-linear pharmacokinetic
clearance profile, which is most likely due in major part to receptor-mediated
endocytosis (372). The clearance rate of G-CSF, when covalently conjugated to Tf,
may be significantly reduced relative to filgrastim. This phenomenon could arise
due to several factors. The first is that the conjugation of G-CSF to Tf may reduce or
eliminate a predominant means of G-CSF clearance, which is dependent upon the
amount of circulating neutrophils (372). In addition, the large molecular weight of
the conjugate, relative to G-CSF, should result in reduced renal clearance, which is
the other major route of G-CSF elimination. This hypothesis is supported by
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previous studies that have demonstrated a large decrease in clearance rate when G-
CSF has been genetically fused to albumin (373) or when G-CSF has been modified
by pegylation (374). In addition, the serum half-life of Tf in mice (40 h) (375) is
much longer than the terminal half-life of G-CSF (~2.5 h) (376). It is possible that
the G-CSF-Tf conjugate may have a clearance rate that is more similar to Tf than G-
CSF, as Tf is predominantly recycled after binding to its receptor. G-CSF may also
be slowly released from the conjugate as the disulfide linkage is reduced, as has been
seen for other disulfide linked protein-drug conjugates (113; 377), enabling sustained
neutrophilic leukocytosis relative to filgrastim. Another possibility is that
subcutaneous administration of G-CSF-Tf may result in the binding of the conjugate
to TfR in the interstitial tissues, creating a depot effect. G-CSF would also be slowly
released from the site of administration in this situation, resulting in a sustained
therapeutic effect.
In light of our findings that G-CSF-Tf can be actively transported across
Caco-2 monolayers (Figures 33-35), the feasibility of using G-CSF-Tf in an oral
route of administration was investigated. BDFl mice (6 - 8 week old) were given G-
CSF-Tf (50 mg/kg) or filgrastim (10 mg/kg) by gavage needle. Absolute neutrophil
counts were performed as in the subcutaneous administration studies. Oral G-CSF-
Tf was able to elicit a significant increase in neutrophil counts (for days 1 and 2)
while filgrastim had no effect when administered orally (Figure 37). In addition, G-
CSF-Tf appears to have a dose-specific response when administered orally,
providing evidence that G-CSF-Tf has a true pharmacological effect (Figure 38).
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Since G-CSF will only have a myelopoietic effect if it is absorbed into the
bloodstream, we presume from this data that G-CSF-Tf has a statistically significant
oral bioavailability. In addition, when comparing the area under the curve of
subcutaneously and orally administered G-CSF-Tf (Figs. 36 and 37), the oral
bioequivalence of G-CSF-Tf based only on biological activity in vivo is about 4%.
TfR is known to be highly expressed in the small intestine and Tf is absorbed by the
GI epithelium as a part of normal physiological processes (324; 378), G-CSF-Tf may
be taken up by similar processes. In addition, this result is supported by our previous
report where we demonstrated that an orally administered insulin-Tf conjugate is
able to elicit a hypoglycemic effect in diabetic rats while free insulin had no effect
(113). G-CSF-Tf may therefore be able to be orally absorbed by TfR-specific
transcellular transport processes.
We have described the creation a G-CSF-Tf conjugate that is transported
across Caco-2 monolayers by TfR-mediated transcytosis at a rate that is several-fold
higher than non-specific apical-to-basolateral G-CSF flux. G-CSF-Tf is also
recovered from the receiver compartment of Caco-2 monolayers with the molecular
weight intact and retains full biological function. The conjugate also exhibits a
prolonged myelopoietic effect in mice compared to filgrastim. This result was
observed in both subcutaneous and oral administration. The mechanism whereby G-
CSF-Tf exhibits a prolonged therapeutic effect as well as the in vivo kinetics of G-
CSF release from the conjugate remain to be determined. However, previous studies
with PEG (373) and albumin (374) have indicated that conjugation to the relatively
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large molecular weight Tf may reduce clearance rate of G-CSF. In addition to the
improved pharmacokinetic characteristics of G-CSF-Tf, the development of an orally
bioavailable G-CSF has the potential to provide great benefit for patients that are
indicated for a chronic G-CSF dosing regime.
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CONCLUSIONS
This report focused on the development of G-CSF-Tf conjugate as a means to
enable the transepithelial systemic delivery of G-CSF for therapeutic purposes. The
ability of the conjugate to pass through various biological barriers hinged upon the
utilization and manipulation of TfR-specific transcytosis. The transport properties of
this conjugate were investigated in in vivo systems using established cells lines (i.e.
Caco-2 and MDCK) as well as primary cultures of alveolar epithelial cells. The
effects of novel and established transcytosis enhancers on altering the polarity,
uptake, and transcytosis of TfR in these epithelial systems were also investigated. In
vivo efficacy was investigated by measuring the ability of subcutaneously and orally
delivered GCSF-Tf to stimulate the proliferation of neutrophils in BDFl mice.
The major findings of this investigation are summarized as follows:
1) G-CSF-Tf conjugate was synthesized at a 1:1 ratio using SPDP-modified Tf
coupled to DPDP-modified G-CSF. The use of DPDP enabled the free sulfhydryl
group of G-CSF’s Cysl7 to be targeted to create a defined site of linkage to Tf.
2) The G-CSF-Tf conjugate was able to be well separated from its constituent
reactant proteins using size exclusion chromatography.
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3) Wortmannin, a non-specific PI-K inhibitor, treatment resulted in a significant
increase in TfR mediated transcytosis in Caco-2 monolayers. Wortmannin treatment
did not affect TEER of the Caco-2 monolayers, indicating that monolayer integrity
was maintained..
4) LY294002, a PI-K inhibitor that is specific for PD-K, treatment resulted in a
significant increase in TfR-mediated transcytosis in Caco-2 monolayers. The TEER
of the Caco-2 monolayers was not affected by LY294002 treatment.
5) When LY294002 was investigated for the ability to alter TfR distribution in
Caco-2 monolayers it was found to exhibit similar effects as the GTPase inhibitor,
AG-10, and dissimilar effects compared to BFA. For example, the amount of ^^^I-Tf
bound to the apical surface of Caco-2 monolayers at 4°C, was increased after pre
treatment with LY294002 and AG-10 (compared to non-treated monolayers), while
monolayers that received BFA pre-treatment exhibited a reduction in ^^I-Tf bound
to the apical surface.
6 ) LY294002 was found to not significantly affect TfR-mediated uptake of ^^Fe
(present in bathing fluid as ^^Fe-Tf) from the apical surface of Caco-2 monolayers
while it significantly decreased the uptake of ^^Fe-Tf from the basolateral surface of
Caco-2 monolayers. In comparison, AG-10 treatment also demonstrated no
significant alterations in TfR-mediated ^^Fe uptake from the apical surface of Caco-2
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monolayers, while significantly decreasing uptake from the basolateral surface. In
contrast BFA treatment resulted in a significant increase in TfR-mediated ^^Fe
uptake from the apical surface of Caco-2 monolayers, while significantly decreasing
TfR-mediated ^^e uptake from the basolateral membrane to a greater extent than
either AG-10 or LY294002.
7) LY294002 enhanced the apical-to-basolateral transport of Tf and G-CSF-Tf
conjugate across Caco-2 monolayers more effectively than BFA and in a similar
magnitude as AG-10. The increased transport was due to an elevation of TfR
specific processes, with non-specific transport being not affected by the presence of
LY294002.
8 ) LY294002 treatment did not result in an enhancement of TfR-mediated transport
in MDCK monolayers. MDCK cells differ from Caco-2 cells in that they are thought
to only use a direct sorting mechanism from the TGN to localize TfR to the apical
membrane of the cells, while Caco-2 cells are thought to be able to also utilize an
indirect sorting mechanism that first directs TfR to the basolateral membrane before
direction to the apical surface. LY294002 was also found to have no effect on the
distribution of TfR at the apical or basolateral membrane and it did not affect the
uptake of ^^Fe-Tf from the apical or basolateral surfaces of MDCK monolayers.
LY294002 was thus theorized to affect indirect TfR sorting that is downstream of
AG-10 sensitive sorting processes.
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9) The transport characteristics of G-CSF-Tf were also investigated in the
pulmonary system using primary cultured rat alveolar type n epithelial monolayers.
The primary cultured type II cell system is interesting to use because they exhibit
many in vivo like characteristics when grown on semi-permeable membranes,
including the trans-differentiation to the type I eell-like phenotype. This
investigation established that TfR is a characteristic of type II cell-like monolayers
and that TfR expression is greatly attenuated, both in cell-surface and total cell-
surface expression, after the transdifferentiation to the type I phenotype.
10) When KGF was included in the culture medium of type II monolayers, TfR
expression was maintained through culture day 8 as determined by ^^^I-Tf binding
studies and eonfocal microseopy. TfR was also expressed in a polarized fashion,
with most of the TfR being expressed at the basolateral surface and ^^Fe-Tf uptake
occurring primarily at the basolateral surface.. Since KGF has been previously
shown to maintain type II cell-like characteristics in vitro, it is assumed that these
observations are due to maintenance of type II cell-like phenotype.
11) BFA treatment was found to significantly enhance the uptake of ^®Fe-Tf from
the apical surface of rat alveolar epithelial cell monolayers. TEER was maintained
through the experiment with BFA, while AG-10 and LY294002 treatment resulted in
a rapid decrease in TEER such that monolayer barrier function was compromised.
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BFA also significantly enhanced the TfR-specific transport of Tf, In-Tf, and G-CSF-
Tf across type II cell-like monolayers. BFA treatment did not affect the transport of
G-CSF alone, which was lower than G-CSF-Tf. G-CSF-Tf that was recovered from
the basolateral compartment, post-transcytosis, maintained full biological activity
after a short reduction reaction.
12) The feasibility of using G-CSF-Tf in an oral route of administration was
investigated using Caco-2 monolayers and oral administration to BDFl mice.
Conjugation of G-CSF to Tf significantly enhanced the apical-to basolateral
transport of G-CSF across Caco-2 monolayers, with biological activity of G-CSF
being retained. G-CSF-Tf administration (subcutaneous and oral) to BDFl mice
resulted in a prolonged myelopoietic effect compared to control (filgrastim).
This report has demonstrated the development of G-CSF-Tf as an efficient means to
specifically deliver G-CSF across biological barriers. The transport process is
specific to TfR-mediated transcytosis ensuring that only Tf-conjugated proteins will
pass through the biological barrier of interest. Conjugation of G-CSF to Tf may also
improve the pharmacological and pharmacokinetic properties of G-CSF. The release
of G-CSF from the conjugate complex by reduction of the disulfide linkage bonds
may result in a sustained release of G-CSF and thereby an enhanced drug half-life.
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FUTURE DIRECTIONS
The future direction of this project hinges upon the ability to produce large
quantities of structurally defined product, whether with chemical or genetic linkages,
as a feasible pharmaceutical product for oral protein drug delivery. However, in the
current conjugation methodology, the Tf molecule is modified by the cross-linking
reagent, SPDP, on multiple lysine residues of variable position. It is estimated that at
least 3-5 Lys residues in Tf are equally accessible to modification by SPDP. By
using current techniques, with carefully controlled conditions, SPDP modified Tf is
usually obtained with 2-3 covalently attached SPDP groups per Tf molecule. The
end result is that Tf-protein conjugates are obtained with linkages at undetermined
lysines, creating an undefined molecular structure. In addition, even though the G-
CSF-Tf conjugate was formed with a stoichiometry of 1:1, of G-CSF to Tf, the
additional SPDP modification sites on Tf result in the formation of cross-linked
products, creating a heterogeneous mixture of variable molecular weight. This
dilemma may be solved by allowing for the formation of defined molecular species.
The success of this approach may lie in the creation of a series of fusion proteins that
will represent the next generation of Tf delivered protein drugs (figure X). The
creation of fusion proteins, consisting of defined molecular structures, will also solve
the problems presented by the current conjugation methodology, as they will be free
of the potential to form covalently linked aggregates and will be of a defined
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Tf
t .
GGGGGG
IgG3 hinge
G-CSF
] GGGGGG
IgG3 hinge
G-CSF
T f
Tf-G-GCSF
Tf-lgG-GCSF
GCSF-G-Tf
GGSF-lgG-Tf
Figure 42. Possible G-CSF-Tf fusion constructs. The order of the
genetic linkage is shown along with the intergenic linkage. The
smaller box within the N-terminal domain represents the respective
leader peptide signaling sequences for G-CSF and Tf.
171
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structure. In addition, fusion protein constructs will provide a means to quickly and
efficiently produce large amounts of the orally available protein drugs. These two
themes, the creation of a defined product and rapid and efficient production, are
needed for TfR mediated delivery of protein drugs to become a reality for the
treatment of humans. The ability to non-invasively deliver these drugs will provide a
great benefit for humanity, through increased compliance to treatment regimes and
patient pain reduction.
The G-CSF-Tf fusion protein will be created by using a baculovirus
expression system. The baculovirus system has been chosen to express the fusion
protein because it provides a rapid and efficient means to produce the recombinant
protein. Nevertheless, E. coli expression is the system of choice for the production of
recombinant G-CSF because no post-translational modifications are needed for the
production of biologically active protein. However, E. coli expression for the
production of the G-CSF-Tf fusion proteins will most likely result in a functionally
inactive Tf. Bacterial expression systems for Tf have been reported, however the Tf
was biologically inactive due to improper disulfide-bond formation (379). In
contrast, the high-level production of functionally active Tf by baculovirus
expression has been previously described (380). The proteins that are produced by
the insect cells undergo post-translational processing, which is necessary for
preserving functionality in many mammalian proteins (381). In addition, the insect
cells recognize many mammalian leader signaling sequences that often greatly
increases the ease of isolation of the protein. For example, in High Five insect cells
172
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(Life Invitrogen) the leader sequence of Tf is cleaved from the protein, to form the
mature Tf molecule, prior to the secretion into the cell culture media (382).
Therefore, it is expected that the use of baculovirus expression for the production of
G-CSF and Tf fusion constructs will result in biologically active domains for both G-
CSF andTf.
173
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Creator Widera, Adam Scott (author)

Core Title Trans-epithelial transport of G -CSF -transferrin conjugate

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,biology, molecular,Health Sciences, Pharmacology,OAI-PMH Harvest

Language English

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-531455

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