Note: Descriptions are shown in the official language in which they were submitted.
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NATRIURETIC PEPTIDE MODIFIED TRANSFERRIN FUSION PROTEINS
INVENTORS: Homayoun Sadeghi, Andrew J. Turner, Christopher P. Prior and
David J. Ballance
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application
60/726,198, filed
October 14, 2005, which is herein incorporated by reference in its entirety.
[0002] This application is related to, but does not claim the benefit of, U.S.
Provisional
Application 60/658,140, filed March 4, 2005, U.S. Provisional Application
60/663,757, filed,
March 22, 2005, and to International Application PCT/US03/26818, filed August
28, 2003,
which claims the benefit of U.S. Application 10/378,094, filed March 4, 2003,
and U.S.
Application 10/231,494, filed August 30, 2002, whicli claiins the benefit of
U.S. Provisional
Application 60/315,745, filed August 30, 2001 and U.S. Provisional Application
60/334,059,
filed November 30, 2001, all of which are herein incorporated by reference in
their entirety.
This application is also related to U.S. Provisional Application 60/406,977,
filed August 30,
2002, which is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The preserit 'invention relates to tllerapeutic proteins or peptides
with extended seruin
stability or in vivo circulatory half-life fused to or inserted into a
transferrin molecule
modified to reduce or inhibit glycosylation, iron binding and/or transferrin
receptor binding.
Specifically, the present invention includes natriuretic peptides fused to or
inserted into a
transferrin molecule or a modified transferrin molecule.
BACKGROUND OF THE INVENTION
[0004] Therapeutic proteins or peptides in their native state, or when
recoinbinantly
produced, are typically labile molecules exhibiting short periods of serum
stability or short in
vivo circulatory half-lives. In addition, these molecules are often extremely
labile when
foilllulated, particularly wllen forinulated in aqueous solutions for
diagnostic and therapeutic
purposes.
[0005] Few practical solutions exist to extend or promote the stability in
vivo or in vitro of
proteinaceous therapeutic inolecules. Polyethylene glycol (PEG) is a substance
that can be
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2
attached to a protein, resulting in longer-acting, sustained activity of the
protein. If the
activity of a protein is prolonged by the attachment to PEG, the frequency
that the protein
needs to be administered may be decreased. PEG attachment, however, often
decreases or
destroys the protein's therapeutic activity. While in some instance PEG
attachment can
reduce immunogenicity of the protein, in other instances it may actually
increase
immunogenicity.
[0006] Therapeutic proteins or peptides have also been stabilized by fusion to
a protein
capable of extending the in vivo circulatory half-life of the therapeutic
protein. For instance,
therapeutic proteins fused to albumin or to antibody fragments may exhibit
extended in vivo
circulatory half-life when compared to the therapeutic protein in the unfused
state. See U.S.
Patents 5,876,969 and 5,766,883.
[0007] Another serum protein, glycosylated huinan transferrin (Tf) has also
been used to
make fusions with therapeutic proteins to target delivery to the interior of
cells or to carry
agents across the blood-brain barrier. These fusion proteins comprising
glycosylated human
Tf have been used to target nerve growth factor (NGF) or ciliary neurotrophic
factor (CNTF)
across the blood-brain barrier by fusing full-length Tf to the agent. See U.S.
Patents
5,672,683 and 5,977,307. In these fusion proteins, the Tf portion of the
molecule is
glycosylated and binds to two atoms of iron, which is required for Tf binding
to its receptor
on a cell and, according to the inventors of these patents, to target delivery
of the NGF or
CNTF moiety across the blood-brain barrier. Transferrin fusion proteins have
also been
produced by inserting an HIV-1 protease target sequence into surface exposed
loops of
glycosylated transferrin to investigate the ability to produce another fonn of
Tf fusion for
targeted delivery to the inside of a cell via the Tf receptor (Ali et al.
(1999) J. Biol. Chem.
274(34):24066-24073).
[0008] Serum transferrin (Tf) is a monomeric glycoprotein with a molecular
weight of
80,000 daltons that binds iron in the circulation and transports it to various
tissues via the
transferrin receptor (TfR) (Aisen et al. (1980) Aiul. Rev. Biochein. 49: 357-
393;
MacGillivray et al. (1981) J. Biol. Chein. 258: 3543-3553, U.S. Patent
5,026,651). Tf is one
of the most coininon seruin molecules, comprising up to about 5-10% of total
sei-uin proteins.
Carbohydrate deficient transferrin occurs in elevated levels in the blood of
alcoholic
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3
individuals and exhibits a longer half life (approximately 14-17 days) than
that of
glycosylated transferrin (approximately 7-10 days). See van Eijk et al. (1983)
Clin. Chim.
Acta 132:167-171, Stibler (1991) Clin. Chem. 37:2029-2037 (1991), Arndt (2001)
Clin.
Chem. 47(1):13-27 and Stibler et al. in "Carbohydrate-deficient consumption",
Advances in
the Biosciences, (Ed Nordmann et al.), Pergamon, 1988, Vol. 71, pages 353-
357).
[0009] The structure of Tf has been well characterized and the mechanism of
receptor
binding, iron binding and release and of carbonate ion binding have been
elucidated (U.S.
Patents 5,026,651, 5,986,067 and MacGillivray et al. (1983) J. Biol. Chem.
258(6):3543-
3546).
[0010] Transferrin and antibodies that bind the transferrin receptor have also
been used to
deliver or carry toxic agents to tumor cells as cancer therapy (Baselga and
Mendelsohn,
1994), and transferrin has been used as a non-viral gene therapy vector to
deliver DNA to
cells (Frank et al., 1994; Wagner et al., 1992). The ability to deliver
proteins to the central
nervous system (CNS) using the transferrin receptor as the entry point has
been demonstrated
with several proteins and peptides including CD4 (Walus et al., 1996), brain
derived
neurotrophic factor (Pardridge et al., 1994), glial derived neurotrophic
factor (Albeck et al.),
a vasointestinal peptide analogue (Bickel et al., 1993), a beta-amyloid
peptide (Saito et al.,
1995), and an antisense oligonucleotide (Pardridge et al., 1995).
[0011] Transferrin fusion proteins have not, however, been modified or
engineered to extend
the in vivo circulatory half-life of a therapeutic protein or peptide or to
increase
bioavailability by reducing or iiihibiting glycosylation of the Tf moiety nor
to reduce or
prevent iron and/or Tf receptor binding.
SUMMARY OF THE INVENTION
[0012] As described in more detail below, the present invention includes
modified Tf fusion
proteins comprising at least one natriuretic protein, polypeptide or peptide
entity, wllerein the
Tf portion is engineered to extend the in vivo circulatory half-life or
bioavailability of the
molecule. The invention also includes phannaceutical formulations and
compositions
comprising the fitsion proteins, methods of extending the serum stability, in
vivo circulatory
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4
half-life and bioavailability of a therapeutic protein by fusion to modified
transferrin, nucleic
acid molecules encoding the modified Tf fusion proteins, and the like. Another
aspect of the
present invention relates to methods of treating a patient with a modified Tf
fusion protein.
Preferably, the modified Tf fusion proteins comprise a human transferrin Tf
moiety that has
been modified to reduce or prevent glycosylation and/or iron and receptor
binding.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 shows an alignment of the N and C Domains of Human (Hu)
transferrin (Tf)
(SEQ ID NO: 3) wit11 similarities and identities highlighted.
[0014] Figures 2A-2B show an alignment of transferrin sequences from different
species.
Light shading: Similarity; Dark shading: Identity.
[0015] Figure 3 shows the location of a number of Tf surface exposed insertion
sites for
therapeutic proteins, polypeptides or peptides.
[0016] Figure 4 shows vector pREX0730.
[0017] Figure 5 shows vector pREX0731.
[0018] Figure 6 shows vector pREX0722
[0019] Figure 7 shows vector pREX0723.
[0020] Figure 8 shows vector pREX0549.
[0021] Figure 9 shows vector pREX0584.
[0022] Figure 10 shows vector pREX1 140.
[0023] Figure 11 shows vector pREX1146.
[0024] Figure 12 shows vector pREX0826.
[0025] Figure 13 sliows vector pREX0827.
[0026] Figure 14 shows vector pREX0828.
[0027] Figure 15 shows vector pREX0829.
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DETAILED DESCRIPTION
General Description
[0028] The present invention is based in part on the finding by the inventors
that therapeutic
proteins can be stabilized to extend their seruin half-life and/or activity in
vivo by genetically
fusing the therapeutic proteins to transferrin, modified transferrin, or a
portion of transferrin
or modified transferrin sufficient to extend the half-life of the therapeutic
protein in seruin.
The modified transferrin fusion proteins include a transferrin protein or
domain covalently
linked to a therapeutic protein or peptide, wherein the transferrin portion is
modified to
contain one or more amino acid substitutions, insertions or deletions compared
to a wild-type
transferrin sequence. In one embodiment, Tf fusion proteins are engineered to
reduce or
prevent glycosylation within the Tf or a Tf doinain as compared to fully
glycosylated Tf, for
instance fully N-linked glycosylated Tf. In other einbodiments, the Tf protein
or Tf
domain(s) is modified to exhibit reduced or no binding to iron or carbonate
ion, or to have a
reduced affinity or not bind to a Tf receptor (TfR).
[0029] The therapeutic proteins contemplated by the present invention include,
but are not
limited to polypeptides, antibodies, peptides, or fragments or variants
thereof. Preferably, the
therapeutic proteins of the present invention include natriuretic peptides and
their analogs,
derivatives, chimeric natriuretic peptides, and peptides or proteins that act
as natriuretic
receptor agonists or antagonists.
[0030] The present invention therefore includes transferrin fusion proteins,
therapeutic
compositions coinprising the fusion proteins, and methods of treating,
preventing, or
ameliorating diseases or disorders by adininistering the fusion proteins. A
transferrin fusion
protein of the invention includes at least a fraginent or variant of a
therapeutic protein and at
least a fraginent or variant of inodified transferrin, which are associated
with one another,
preferably by genetic fusion (i. e., the transferrin fusion protein is
generated by translation of a
nucleic acid in which a polynucleotide encoding all or a portion of a
therapeutic protein is
joined in-fraine with a polynucleotide encoding all or a portion of modified
transferrin) or
cheinical conjugation to one another. The therapeutic protein and transferrin
protein, once
part of the transferrin fusion protein, inay be referred to as a'iportion",
"region" ? or "inoiety"
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of the transferrin fusion protein (e.g., a "therapeutic protein portion' or a
"transferrin protein
portion").
[0031] In one embodiment, the invention provides a transfeiTin fusion protein
coinprising, or
alternatively consisting of, a therapeutic protein and a modified serum
transferrin protein. In
other embodiments, the invention provides a transferrin fusion protein
coinprising, or
alternatively consisting of, a biologically active and/or therapeutically
active fragment of a
therapeutic protein and a modified transferrin protein. In other embodiments,
the invention
provides a transferrin fasion protein comprising, or alternatively consisting
of, a biologically
active and/or therapeutically active variant of a therapeutic protein and
modified transferrin
protein. In further embodiments, the invention provides a transferrin fusion
protein
coinprising a therapeutic protein, and a biologically active and/or
therapeutically active
fragment of modified transferrin. In another embodiment, the therapeutic
protein portion of
the transferrin fusion protein is the active fonn of the therapeutic protein.
[0032] Unless defined otherwise, all technical and scientific terms us-ed
herein have the same
meaning as cominonly understood by one of ordinary skill in the art to which
this invention
belongs. Altliough any methods and materials similar or equivalent to those
described herein
can be used in the practice or testing of the present invention, the preferred
methods and
materials are described.
Definitions
[0033] As used herein, an "ainino acid corresponding to" or an "equivalent
ainino acid" in a
transferrin sequence is identified by aligmnent to maximize the identity or
similarity between
a first transferrin sequence and at least a second transfeiTin sequence. The
nuinber used to
identify an equivalent ainino acid in a second transferrin sequence is based
on the number
used to identify the corresponding amino acid in the first transferrin
sequence. In certain
cases, these phrases may be used to describe the amino acid residues in human
transferrin
coinpared to certain residues in rabbit serum transferrin.
[0034] As used herein, the term "biological activity" refers to a finiction or
set of activities
performed by a therapeutic molecule, protein or peptide in a biological
context (i.e., in an
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organism or an in vitro facsimile thereof). Biological activities may include
but are not
limited to the functions of the therapeutic molecule portion of the claimed
fusion proteins,
such as, but not limited to, the induction of extracellular matrix secretion
from responsive cell
lines, the induction of hormone secretion, the induction of chemotaxis, the
induction of
mitogenesis, the induction of differentiation, or the inhibition of cell
division of responsive
cells. A fusion protein or peptide of the invention is considered to be
biologically active if it
exhibits one or more biological activities of its therapeutic protein's native
counterpart.
[0035] As used herein, "binders" are agents used to impart cohesive qualities
to the powdered
material. Binders, or "granulators" as they are sometimes known, iinpart
cohesiveness to the
tablet formulation, which insures the tablet remaining intact after
compression, as well as
improving the free-flowing qualities by the fonnulation of granules of desired
hardness and
size. Materials commonly used as binders include starch; gelatin; sugars, such
as sucrose,
glucose, dextrose, molasses, and lactose; natural and synthetic gums, such as
acacia, sodium
alginate, extract of Irish inoss, panwar gum, ghatti guin, mucilage of isapol
husks,
carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, Veegum,
microcrystalline
cellulose, microcrystalline dextrose, ainylose, and larch arabogalactan, an.d
the like.
[0036] As used herein, the term "carrier" refers to a diluent, adjuvant,
excipient, or vehicle
with which a composition is administered. Such pharmaceutical carriers can be
sterile
liquids, such as water and oils, including those of petroleum, animal,
vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesaine oil and the
like.
[0037] As used herein, "coloring agents" are agents that give tablets a more
pleasing
appearance, and in addition help the manufacturer to control the product
during its
preparation and help the user to identify the product. Any of the approved
certified water-
soluble FD&C dyes, mixtures thereof, or their corresponding lakes may be used
to color
tablets. A color lake is the combination by adsorption of a water-soluble dye
to a hydrous
oxide of a heavy metal, resulting in an insoluble foi7n of the dye.
[0038] As used herein, "diluents" are inert substances added to increase the
bulk of the
fonnulation to malce the tablet a practical size for coinpression. Connnonly
used diluents
include calcium phosphate, calcium sulfate, lactose, lcaolin, inamiitol,
sodium cllloride, dry
starch, powdered sugar, silica, and the like.
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[0039] As used herein, "disintegrators" or "disintegrants" are substances that
facilitate the
breakup or disintegration of tablets after adininistration. Materials serving
as disintegrants
have been chemically classified as starches, clays, celluloses, algins, or
gums. Other
disintegrators include Veeguin HV, methylcellulose, agar, bentonite, cellulose
and wood
products, natural sponge, cation-exchange resins, alginic acid, guar gum,
citrus pulp, cross-
linlced polyvinylpyrrolidone, carboxyinethylcellulose, and the like.
[0040] The term "dispersibility" or "dispersible" means a dry powder having a
moisture
content of less than about 10% by weight (%w) water, usually below about 5%w
and
preferably less than about 3%w; a particle size of about 1.0-5.0 :m mass
median diameter
(MMD), usually 1.0-4.0 :m MMD, and preferably 1.0-3.0 :in MMD; a delivered
dose of
about >30%, usually >40%, preferably >50%, and most preferred >60%; and an
aerosol
particle size distribution of 1.0-5.0 :m mass median aerodynainic diameter
(MMAD), usually
1.5-4.5 :in MMAD, and preferably 1.5-4.0 :m MMAD.
[0041] The term "dry" means that the composition has a moisture content such
that the
particles are readily dispersible in an inhalation device to form an aerosol.
This moisture
content is generally below about 10% by weight (%w) water, usually below about
5%w and
preferably less than about 3%w.
[0042] As used herein, "effective amount" means an amount of a drug or
phannacologically
active agent that is sufficient to provide the desired local or systemic
effect and performance
at a reasonable benefit/risk ratio attending any medical treatment.
[0043] As used herein, "flavoring agents" vary considerably in their chemical
structure,
ranging from simple esters, alcohols, and aldehydes to carbohydrates and
coinplex volatile
oils. Synthetic flavors of almost any desired type are now available.
[0044] As used herein, the terms "fragment of a Tf protein" or "Tf protein,"
or "portion of a
Tf protein" refer to an ainino acid sequence coinprising at least about 5%,
10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of a naturally
occurring Tf protein or mutant thereof.
[0045] As used herein, the tenn "gene" refers to any seginent of DNA
associated with a
biological fiinction. Thus, genes include, but are not limited to, coding
sequences and/or the
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regulatory sequences required for their expression. Genes can also include non-
expressed
DNA segments that, for example, form recognition sequences for other proteins.
Genes can
be obtained from a variety of sources, including cloning from a source of
interest or
synthesizing from known or predicted sequence infonnation, and may include
sequences
designed to have desired parameters.
[0046] As used herein, a "heterologous polynucleotide" or a "heterologous
nucleic acid" or a
"heterologous gene" or a "heterologous sequence" or an "exogenous DNA segment"
refers to
a polynucleotide, nucleic acid or DNA seginent that originates from a source
foreign to the
particular host cell, or, if from the same source, is modified from its
original form. A
heterologous gene in a host cell includes a gene that is endogenous to the
particular host cell,
but has been modified. Thus, the terins refer to a DNA segment which is
foreign or
heterologous to the cell, or homologous to the cell but in a position within
the host cell
nucleic acid in whicll the element is not ordinarily found. As an example, a
signal sequence
native to a yeast cell but attached to a human Tf sequence is heterologous.
[0047] As used herein, an "isolated" nucleic acid sequence refers to a nucleic
acid sequence
which is essentially free of other nucleic acid sequences, e.g., at least
about 20% pure,
preferably at least about 40% pure, inore preferably about 60% pure, even more
preferably
about 80% pure, most preferably about 90% pure, and even most preferably about
95% pure,
as determined by agarose gel electrophoresis. For example, an isolated nucleic
acid sequence
can be obtained by standard cloning procedures used in genetic engineering to
relocate the
nucleic acid sequence from its natural location to a different site where it
will be reproduced.
The cloning procedures may involve excision and isolation of a desired nucleic
acid fraginent
comprising the nucleic acid sequence encoding the polypeptide, insertion of
the fragment into
a vector molecule, and incorporation of the recoinbinant vector into a host
cell where
multiple copies or clones of the nucleic acid sequence will be replicated. The
nucleic acid
sequence may be of genomic, eDNA, RNA, semi-synthetic, synthetic origin, or
any
combinations thereof.
[0048] As used herein, two or more DNA coding sequences are said to be
"joined" or "fused"
when, as a result of in-fraine fusions between the DNA coding sequences, the
DNA coding
sequences are translated into a fusion polypeptide. The tertn "fusion" in
reference to Tf
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fusions includes, but is not limited to, attachment of at least one
therapeutic protein,
polypeptide or peptide to the N-terminal end of Tf, attaclunent to the C-
terminal end of Tf,
and/or insertion between any two amino acids within Tf. As used herein
"joined" or "fused"
also includes a construct wherein the DNA sequences encoding two or more
moieties are
separated by an intron, the precise splicing of which (at the mRNA level)
would result in a
fusion protein.
[0049] As used herein, "lubricants" are materials that perform a number of
functions in tablet
manufacture, such as improving the rate of flow of the tablet granulation,
preventing
adhesion of the tablet material to the surface of the dies and punches,
reducing interparticle
friction, and facilitating the ejection of the tablets from the die cavity.
Commonly used
lubricants include talc, magnesium stearate, calcium stearate, stearic acid,
and hydrogenated
vegetable oils. Typical amounts of lubricants range from about 0.1% by weight
to about 5%
by weight.
[0050] As used herein, "Modified transferrin" (mTf) includes a transferrin
molecule that
exhibits at least one modification of its ainino acid sequence, coinpared to
wild-type
transferrin. Such modifications may include, but not liinited to,
modifications that reduce
glycosylations compared to fully glycosylated Tf protein. Modified Tf may also
include Tf
that has reduced glycosylation via enzyinatic removal of carbohydrate
residues.
[0051] As used herein, "Modified transferrin fusion protein" refers to a
protein forined by the
fusion of at least one molecule of modified transferrin (or a fraginent or
variant thereof) to at
least one molecule of a therapeutic protein (or fraginent or variant thereof).
[0052] As used herein, the tenns "nucleic acid" or "polynucleotide" refer to
deoxyribonucleotides or ribonucleotides and polymers thereof in either single-
or double-
stranded form. Unless specifically limited, the terms encompass nucleic acids
containing
analogues of natural nucleotides that have similar binding propei-ties as the
reference nucleic
acid and are metabolized in a manner similar to naturally occuiring
nucleotides. Unless
otherwise indicated, a particular nucleic acid sequence also implicitly
encompasses
conservatively modified variants tllereof (e.g. degenerate codon
substitutions) and
coinplementary sequences as well as the sequence explicitly indicated.
Specifically,
degenerate codon substitutions may be achieved by generating sequences in
which the third
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11
position of one or more selected (or all) codons is substituted with mixed-
base and/or
deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka
et al. (1985)
J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994)
Mol. Cell. Probes
8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, and
mRNA
encoded by a gene.
[0053] As used herein, a DNA seginent is refened to as "operably linked" when
it is placed
into a fiulctional relationship with another DNA segment. For exanple, DNA for
a signal
sequence is operably linked to DNA encoding a fusion protein of the invention
if it is
expressed as a preprotein that participates in the secretion of the fusion
protein; a promoter or
enhancer is operably linked to a coding sequence if it stimulates the
transcription of the
sequence. Generally, DNA sequences that are operably linked are contiguous,
and in the case
of a signal sequence or fusion protein both contiguous and in reading phase.
However,
enhancers need not be contiguous with the coding sequences whose transcription
they
control. Alternatively, DNA sequences that are operably linked may be
separated by one or,
more intron sequences wherein splicing of the intron sequences results in the
sequences being
contiguous in the resulting mature mRNA. Linking, in this context, is
accomplished by
ligation at convenient restriction sites or at adapters or linlcers inserted
in lieu thereof.
[0054] As used herein, "pharmaceutically acceptable" refers to materials and
compositions
that are physiologically tolerable and do not typically produce an allergic or
similar untoward
reaction, such as gastric upset, dizziness and the like, when administered to
a huinan.
Typically, as used herein, the term "phar-rnaceutically acceptable" ineans
approvable by a
regulatory agency of the Federal or a state govermnent or listed in the U.S.
Pharinacopeia or
other generally recognized phai7nacopeia for use in animals, and more
particularly in
humans.
[0055] As used herein, "physiologically effective ainount" is that ainount
delivered to a
subject to give the desired palliative or curative effect. This amount is
specific for each drug
and its ultimate approved dosage level.
[00561 As used herein, the terin "powder" means a coinposition that consists
of finely
dispersed solid particles that are free flowing and capable of being readily
dispersed in an
inhalation device and subsequently inhaled by a subject so that the particles
reach the lungs to
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12
permit penetration into the alveoli. Thus, the powder is said to be
"respirable". Preferably the
average particle size is less than about 10 microns (:m) in diameter with a
relatively uniform
spheroidal shape distribution. More preferably, the diaineter is less than
about 7.5 :m and
most preferably less than about 5.0 :m. Usually the particle size distribution
is between about
0.1 :m and about 5:in in diameter, particularly about 0.3 :m to about 5 :m.
[0057] As used herein, the term "promoter" refers to a region of DNA involved
in binding
RNA polymerase to initiate transcription.
[0058] As used herein, the term "recombinant" refers to a cell, tissue or
organism that has
undergone transformation with a new combination of genes or DNA.
[0059] As used herein, the term "subject" can be a human, a maminal, or an
animal. The
subject being treated is a patient in need of treatment.
[0060] As used herein, a targeting entity, protein, polypeptide or peptide
refers to a molecule
_ that binds specifically to a particular cell type (normal (e.g.,
lymphocytes) or abnorinal e.g.,
(cancer cell)) and tlierefore may be used to target a Tf fusion protein or
compound (drug, or
cytotoxic agent) to that cell type specifically.
[0061] As used herein, "tablets" are solid pharinaceutical dosage fonns
containing drug
substances with or without suitable diluents and prepared either by
coinpression or molding
methods well known in the art. Tablets have been in widespread use since the
latter part of
the 19tt' century and their popularity continues. Tablets remain popular as a
dosage form
because of the advantages afforded botli to the inanufacturer (e.g.,
simplicity and economy of
preparation, stability, and convenience in packaging, shipping, and
dispensing) and the
patient (e.g., accuracy of dosage, coinpactness, portability, blandness of
taste, and ease of
administration). Although tablets are most frequently discoid in shape, they
may also be
round, oval, oblong, cylindrical, or triangular. They may differ greatly in
size and weight
depending on the ainount of drug substance present and the intended method of
adininistration. They are divided into two general classes, (1) compressed
tablets, and (2)
molded tablets or tablet triturates. In addition to the active or therapeutic
ingredient or
ingredients, tablets contain a nuinber or inert materials or additives. A
first group of such
additives includes those materials that help to impart satisfactory
coinpression characteristics
to the fonnulation, including diluents, binders, and lubricants. A second
group of such
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13
additives helps to give additional desirable physical characteristics to the
finished tablet, such
as disintegrators, colors, flavors, and sweetening agents.
[0062] As used herein, the term "therapeutically effective amount" refers to
that amount of
the transferrin fusion protein comprising a therapeutic inolecule which, when
administered to
a subject in need thereof, is sufficient to effect treatment. The amount of
transferrin fusion
protein which constitutes a"therapeutically effective amount" will vary
depending on the
therapeutic protein used, the severity of the condition or disease, and the
age and body weight
of the subject to be treated, but can be determined routinely by one of
ordinary skill in the art
having regard to his/her own knowledge and to this disclosure.
[0063] As used herein, "therapeutic protein" refers to proteins, polypeptides,
peptides or
fragments or variants thereof, having one or more therapeutic, prophylactic
and/or biological
activities. Therapeutic proteins encompassed by the invention include but are
not limited to
proteins, polypeptides, peptides, antibodies, and biologics. The terzns
peptides, proteins, and
polypeptides are used interchangeably herein. Additionally, the term
"therapeutic peptide"
may refer to the endogenous or naturally occun-ing correlate of a therapeutic
protein. By a
polypeptide displaying a "therapeutic activity" or a protein that is
"therapeutically active" is
meant a polypeptide that possesses one or more known biological and/or
therapeutic activities
associated with a therapeutic protein such as one or more of the therapeutic
proteins
described herein or otherwise known in the art. As a non-limiting exalnple, a
"therapeutic
protein" is a protein that is useful to treat, prevent or aineliorate a
disease, condition or
disorder. Such a disease, condition or disorder may be in humans or in a non-
human animal,
e.g., veterinary use.
[0064] As used herein, the term "transformation" refers to the transfer of
nucleic acid (i.e., a
nucleotide polymer) into a cell. As used herein, the tenn "genetic
transfonnation" refers to
the transfer and incorporation of DNA, especially recombinant DNA, into a
cell.
[0065] As used herein, the term "transformant" refers to a cell, tissue or
organism that has
undergone transforination.
[0066] As used herein, the terln "transgene" refers to a nucleic acid that is
inserted into an
organism, host cell or vector in a mamler that ensures its fitnction.
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14
[0067] As used herein, the term "transgenic" refers to cells, cell cultures,
organisms, bacteria,
fungi, animals, plants, and progeny of any of the preceding, which have
received a foreign or
modified gene and in particular a gene encoding a modified Tf fusion protein
by one of the
various methods of transformation, wherein the foreign or modified gene is
from the same or
different species than the species of the organism receiving the foreign or
modified gene.
[0068] "Variants or variant" refers to a polynucleotide or nucleic acid
differing from a
reference nucleic acid or polypeptide, but retaining essential properties
thereof. Generally,
variants are overall closely similar, and, in many regions, identical to the
reference nucleic
acid or polypeptide. As used herein, "variant" refers to a therapeutic protein
portion of a
transferrin fusion protein of the invention, differing in sequence from a
native therapeutic
protein but retaining at least one functional and/or therapeutic property
thereof as described
elsewhere herein or otherwise known in the art.
[0069] As used herein, the term "vector" refers broadly to any plasmid,
phagemid or virus
encoding an exogenous nucleic acid. The term is also be construed to include
non-plasmid,
non-phageinid and non-viral compounds which facilitate the transfer of nucleic
acid into
virions or cells, such as, for example, polylysine compounds and the like. The
vector inay be
a viral vector that is suitable as a delivery vehicle for delivery of the
nucleic acid, or mutant
thereof, to a cell, or the vector may be a non-viral vector which is suitable
for the same
purpose. Exainples of viral and non-viral vectors for delivery of DNA to cells
and tissues are
well known in the art and are described, for example, in Ma et al. (1997,
Proc. Natl. Acad.
Sci. U.S.A. 94:12744-12746). Examples of viral vectors include, but are not
limited to, a
recombinant vaccinia virus, a recoinbinant adenovirus, a recoznbinant
retrovirus, a
recombinant adeno-associated virus, a recoinbinant avian pox virus, and the
like (Cranage et
al., 1986, EMBO J. 5:3057-3063; International Patent Application No. WO
94/17810,
published August 18, 1994; International Patent Application No. WO 94/23744,
published
October 27, 1994). Examples of non-viral vectors include, but are not limited
to, liposomes,
polyainine derivatives of DNA, and the like.
[0070] As used herein, the ter7n "wild type" refers to a polynucleotide or
polypeptide
sequence that is naturally occuiTing.
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Transferrin and Transferrin Modifications
[0071] The present invention provides fusion proteins comprising a therapeutic
protein and
transferrin or modified transferrin. Preferably, the therapeutic protein
provided by the present
invention is a natriuretic peptide, analog, derivative, or chimeric
natriuretic molecule.
[0072] Any transferrin may be used to make modified Tf fusion proteins of the
invention. As
an example, the wild-type human Tf (Tf) is a 679 ainino acid protein of
approximately 75kDa
(not accounting for glycosylation), with two main domains (or lobes), N (about
330 amino
acids) and C (about 340 amino acids), which appear to originate from a gene
duplication. See
GenBank accession numbers NM 001063, XM 002793, M12530, XM 039845,
XM039847 and S95936 (www.ncbi.nhn.nih.gov/), all of which are herein
incorporated by
reference in their entirety, as well as SEQ ID NOS: 1, 2 and 3. The two
domains have
diverged over time but retain a large degree of identity/similarity (Fig. 1).
[0073] Each of the N and C domains is further divided into two subdomains, N1
and N2, C1
and C2. The function of Tf is to transport iron to the cells of the body. This
process is
mediated by the Tf receptor (TfR), which is expressed on all cells,
particularly actively
growing cells. TfR recognizes the iron bound forin of Tf (two molecules of
which are bound
per receptor), endocytosis then occurs whereby the TfR/Tf complex is
transported to the
endosome, at which point the localized drop in pH results in release of bound
iron and the
recycling of the TfR/Tf complex to the cell surface and release of Tf (known
as apoTf in its
iron-unbound forin). Receptor binding is through the C domain of Tf. The two
glycosylation
sites in the C domain do not appear to be involved in receptor binding as
unglycosylated iron
bound Tf does bind the receptor.
[0074] Each Tf molecule can carry two iron ions (Fe3+). These are coznplexed
in the space
between the N1 and N2, C1 and C2 sub domains resulting in a confonnational
change in the
molecule. Tf crosses the blood brain barrier (BBB) via the Tf receptor.
[0075] In human transferrin, the iron binding sites comprise at least ainino
acids Asp 63 (Asp
82 of SEQ ID NO: 2 which includes the native Tf signal sequence), Asp 392 (Asp
411 of
SEQ ID NO: 2), Tyr 95 (Tyr 114 of SEQ ID NO: 2), Tyr 426 (Tyr 445 of SEQ ID
NO: 2),
Tyr 188 (Tyr 207 of SEQ ID NO: 2), Tyr 514 or 517 (Tyr 533 or Tyr 536 SEQ ID
NO: 2),
His 249 (His 268 of SEQ ID NO: 2), a.nd His 585 (His 604 of SEQ ID NO: 2) of
SEQ ID NO:
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16
3. The hinge regions comprise at least N domain amino acid residues 94-96, 245-
247 and/or
316-318 as well as C domain amino acid residues 425-427, 581-582 and/or 652-
658 of SEQ
ID NO: 3. The carbonate binding sites comprise at least amino acids Thr 120
(Thr 139 of
SEQ ID NO: 2), Thr 452 (Thr 471 of SEQ ID NO: 2), Arg 124 (Arg 143 of SEQ ID
NO: 2),
Arg 456 (Arg 475 of SEQ ID NO: 2), Ala 126 (Ala 145 of SEQ ID NO: 2), Ala 458
(Ala 477
of SEQ ID NO: 2), Gly 127 (Gly 146 of SEQ ID NO: 2), and Gly 459 (Gly 478 of
SEQ ID
NO: 2) of SEQ ID NO: 3.
[0076] In one embodiment of the invention, the modified transferrin fusion
protein includes a
modified human transferrin, although any animal Tf molecule may be used to
produce the
fusion proteins of the invention, including human Tf variants, cow, pig,
sheep, dog, rabbit,
rat, mouse, hamster, echnida, platypus, chicken, frog, hornworm, monkey, as
well as other
bovine, canine and avian species. All of these Tf sequences are readily
available in GenBank
and other public databases. The human Tf nucleotide sequence is available (see
SEQ ID
NOS 1, 2 and 3 and the accession numbers described above and available at
www.ncbi.nlm.nih.gov/) and can be used to make genetic fusions between Tf or a
domain of
Tf and the therapeutic molecule of choice. Fusions may also be made from
related molecules
such as lacto transferrin (lactoferrin) GenBank Acc: NM 002343) or
melanotransferrin
(GenBank Acc. NM 013900, inurine melanotransferrin).
[0077] Melanotransferrin is a glycosylated protein found at high levels in
malignant
melanoma cells and was originally named huinan melanoma antigen p97 (Brown et
al., 1982,
Nature, 296: 171-173). It possesses high sequence homology with human seruin
transferrin,
human lactoferrin, and chicken transferrin (Brown et al., 1982, Nature, 296:
171-173; Rose et
al., Proc. Natl. Acad. Sci. USA, 1986, 83: 1261-1265). However, unlike these
receptors, no
cellular receptor has been identified for melanotransferrin. Melanotransferrin
reversibly
binds iron and it exists in two forins, one of which is bound to cell
ineinbrales by a glycosyl
phosphatidylinositol anchor wliile the other forin is both soluble and
actively secreted (Baker
et al., 1992, FEBS Lett, 298: 215-218; Alemany et al., 1993, J. Cell Sci.,
104: 1155-1162;
Food et al., 1994, J. Biol. Chem. 274: 7011-7017).
[0078] Lactofen-in (Lf), a natural defense iron-binding protein, has been
found to possess
antibacterial, antimycotic, antiviral, antineoplastic and anti-inflaminatory
activity. The
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17
protein is present in exocrine secretions that are cominonly exposed to normal
flora: milk,
tears, nasal exudate, saliva, bronchial mucus, gastrointestinal fluids,
cervico-vaginal inucus
and seminal fluid. Additionally, Lf is a major constituent of the secondary
specific granules
of circulating polymorphonuclear neutrophils (PMNs). The apoprotein is
released on
degranulation of the PMNs in septic areas. A principal function of Lf is that
of scavenging
free iron in fluids and inflamed areas so as to suppress free radical-mediated
damage and
decrease the availability of the metal to invading microbial and neoplastic
cells. In a study
that examined the turnover rate of 125I Lf in adults, it was shown that Lf is
rapidly taken up by
the liver and spleen, and the radioactivity persisted for several weeks in the
liver and spleen
(Bennett et al. (1979), Clin. Sci. (Lond.) 57: 453-460).
[0079] In one einbodiment, the traiisferrin portion of the transferrin fusion
protein of the
invention includes a transferrin splice variant. In one example, a transferrin
splice variant can
be a splice variant of human transferrin. In one specific einbodiinent, the
human transferrin
splice variant can be that of Genbank Accession AAA61140.
[0080] In another embodiment, the transferrin portion of the transferrin
fusion protein of the
invention includes a lactoferrin splice variant. In one example, a human serum
lactoferrin
splice variant can be a novel splice variant of a neutrophil lactofen-in. In
one specific
embodiment, the neutrophil lactoferrin splice variant can be that of Genbank
Accession
AAA59479. In another specific einbodiment, the neutrophil lactoferrin splice
variant can
comprise the following amino acid sequence EDCIALK GEADA (SEQ ID NO: 129),
which
includes the novel region of splice-variance.
[0081] In another embodiment, the transferrin portion of the transferrin
fusion protein of the
invention includes a melanotransferrin variant.
[0082] Modified Tf fusions may be made with any Tf protein, fraginent, domain,
or
engineered domain. For instance, fusion proteins may be produced using the
full-length Tf
sequence, with or without the native Tf signal sequence. Tf fusion proteins
may also be made
using a single Tf domain, such as an individual N or C domain or a modified
fonn of Tf
comprising 2N or 2C domains (see U.S. Provisional Application 60/406,977,
filed August 30,
2002, which is herein incorporated by reference in its entirety). In some
embodiinents,
fusions of a therapeutic protein to a single C domain may be produced, wherein
the C domain
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18
is altered to reduce, inhibit or prevent glycosylation. In other embodiments,
the use of a
single N domain is advantageous as the Tf glycosylation sites reside in the C
domain. A
preferred embodiment is the Tf fusion protein having a single N domain which
is expressed
at a high level.
[0083] As used herein, a C tenninal domain or lobe modified to function as an
N-like domain
is modified to exhibit glycosylation patterns or iron binding properties
substantially like that
of a native or wild-type N domain or lobe. In a preferred embodiment, the C
domain or lobe
is modified so that it is not glycosylated and does not bind iron by
substitution of the relevant
C domain regions or amino acids to those present in the corresponding regions
or sites of a
native or wild-type N domain.
[0084] As used herein, a Tf moiety comprising "two N domains or lobes"
includes a Tf
molecule that is modified to replace the native C domain or lobe with a native
or wild-type N
domain or lobe or a modified N domain or lobe or contains a C domain that has
been
modified to function substantially like a wild-type or modified N domain. -
[0085] Analysis of the two domains by overlay of the two domains (Swiss PDB
Viewer
3.7b2, Iterative Magic Fit) and by direct amino acid aligninent (ClustalW
inultiple aligmnent)
reveals that the two domains have diverged over time. Amino acid aligrunent
shows 42%
identity and 59% similarity between the two domains. However, approximately
80% of the
N domain matches the C domain for structural equivalence. The C domain also
has several
extra disulfide bonds compared to the N domain.
[0086] Aligmnent of molecular models for the N and C domain reveals the
following
structural equivalents:
36- 178- 283-
N
4-24 72 94- 138- 149- 168- 198 219- 259- 263- 271- 279- 288 309-
domain 139 164 173 255 260 268 275 280 327
(1-330) 75- 200- 290-
88 214 304
c 425-
domain 340- 365- 437 470- 475- 492- 507- 555- 593- 597- 605- 614- 620- 645-
(340- 361 415 439 471 490 497 542 591 594 602 609 615 640 663
-
679) 468
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19
The disulfide bonds for the two domains align as follows:
N C
T C339-C596
C9-C48 C345-C377
C19-C39 C355-C368
Bold aligned disulfide bonds
C402-C674 Italics bridging peptide
C418-C637
C118-C194 C450-C523
C137-C331
C474-C665
C158-C174 C484-C498
C161-C179
C171-C177 C495-C506
C227-C241 C563-C577
C615-C620
[0087] In one einbodiment, the transfeiTin portion of the transferrin fusion
protein includes at
least two N tenninal lobes of transferrin. In further einbodiments, the
transferrin portion of
the transferrin fusion protein includes at least two N tenninal lobes of
transferrin derived
from human seruin transfelTin.
[0088] In another einbodiment, the transferrin portion of the transferrin
fusion protein
includes, comprises, or consists of at least two N terininal lobes of
transferrin having a
inutation in at least one ainino acid residue selected from the group
consisting of Asp63,
G1y65, Tyr95, Tyr188, and His249 of SEQ ID NO: 3.
[0089] In another einbodiment, the transferrin portion of the modified
transferrin fusion
protein includes a recombinant huinan serum transferrin N-tenninal lobe mutant
having a
inutation at Lys206 or His207 of SEQ ID NO: 3.
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[0090] In another einbodiinent, the transferrin portion of the transferrin
fusion protein
includes, comprises, or consists of at least two C terminal lobes of
transferrin. In further
embodiments, the transferrin portion of the transferrin fusion protein
includes at least two C
terminal lobes of transferrin derived from human serum transferrin.
[0091] In a further embodiment, the C terminal lobe mutant further includes a
mutation of
one amino acid corresponding to at least one of Asn413 and Asn611 of SEQ ID
NO: 3 which
does not allow glycosylation.
[0092] In another embodiment, the transferrin portion of the transferrin
fusion protein
includes at least two C terminal lobes of transferrin having a mutation in at
least one amino
acid residue selected from the group consisting of Asp392, Tyr426, Tyr514,
Tyr517 and
His585 of SEQ ID NO: 3, wherein the mutant retains the ability to bind metal.
In an alternate
embodiment, the transferrin portion of the transferrin fusion protein includes
at least two C
terminal lobes of transferrin having a mutation in at least one amino acid
residue selected
from the -group consisting of Tyr426, Tyr514, Tyr517 and His5 85 of SEQ ID NO:
3, wherein
the mutant has a reduced ability to bind metal. In another embodiment, the
transferrin
portion of the transferrin fusion protein includes at least two C tenninal
lobes of transferrin
having a inutation in at least one amino acid residue selected from the group
consisting of
Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO:3, wherein the mutant does not
retain
the ability to bind metal and functions substantially like an N domain.
[0093] In some einbodiments, the Tf or Tf portion will be of sufficient length
to increase the
in vivo circulatory half-life, serum stability, in vitf-o solution stability
or bioavailability of the
therapeutic protein or peptide coinpared to the in vivo circulatory half-life,
seruin stability, in
vitro solution stability or bioavailability of the therapeutic protein or
peptide in an unfused
state. Such an increase in stability, serum half-life or bioavailability inay
be about a 30%,
50%, 70%, 80%, 90% or more increase over the unfused therapeutic protein. In
some cases,
the transferrin fusion proteins comprising modified transferrin exhibit a
seruin half-life of
about 10-20 or more days, about 12-18 days or about 14-17 days.
[0094] When the C domain of Tf is part of the fusion protein, the two N-lii-
Aced glycosylation
sites, ainino acid residues corresponding to N413 and N611 of SEQ ID NO: 3 may
be
inutated for expression in a yeast system to prevent glycosylation or
hypeimannosylatioml
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21
and extend the serum half-life of the fusion protein and/or therapeutic
protein ( to produce
asialo-, or in some instances, monosialo-Tf or disialo-Tf). In addition to Tf
amino acids
corresponding to N413 and N611, mutations may be to the adjacent residues
within the N-X-
S/T glycosylation site to prevent or substantially reduce glycosylation. See
U.S. Patent
5,986,067 of Funk et al. It has also been reported that the N domain of Tf
expressed in
Pichia pastoris becomes 0-linked glycosylated with a single hexose at S32
which also may
be mutated or modified to prevent such glycosylation.
[0095] Accordingly, in one embodiment of the invention, the transferrin fusion
protein
includes a modified transferrin molecule wherein the transferrin exhibits
reduced
glycosylation, including but not limited to asialo- monosialo- and disialo-
forms of Tf. In
another einbodiment, the transferrin portion of the transferrin fusion protein
includes a
recombinant transferrin mutant that is inutated to prevent glycosylation. In
another
embodiment, the transferrin portion of the transferrin fusion protein includes
a recombinant
transferrin mutant that is fully glycosylated. In a further embodiment, the
transferrin portion
of the transferrin fusion protein includes a recombinant huinan serum
transferrin inutant that
is mutated to prevent or reduce glycosylation, wherein at least one of Asn413
and Asn611 of
SEQ ID NO: 3 are mutated to an amino acid which does not allow or reduce
glycosylation as
compared to fully glycosylated Tf. In anotller embodiment, the transferrin
portion of the
transferrin fusion protein includes a recombinant human serum transferrin
mutant that is
inutated to prevent or substantially reduce glycosylation, wherein inutations
may be to the
adjacent residues within the N-X-S/T glycosylation site. Moreover,
glycosylation may be
reduced or prevented by inutating the serine or threonine residue. In one
einbodiment, the
modified transferrin protein contains inutations at S415 and T613 of SEQ ID
NO.: 3. For
instance, the invention includes fusion proteins comprising a modified Tf
protein with the
mutations S415A and T613A. Further, changing the X to proline is lalown to
inhibit
glycosylation.
[0096] As discussed below in more detail, modified Tf fusion proteins of the
invention may
also be engineered to not bind iron and/or bind the Tf receptor. In other
einbodiments of the
invention, the iron binding is retained and the iron binding ability of Tf may
be used to
deliver a therapeutic protein or peptide(s) to the inside of a cell, across an
epithelial or
endothelial cell meinbrane and/or across the BBB. These embodiments that bind
iron and/or
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the Tf receptor will often be engineered to reduce or prevent glycosylation to
extend the
serum half-life of the therapeutic protein. The N domain alone will not bind
to TfR when
loaded with iron, and the iron bound C domain will bind TfR but not with the
same affinity as
the whole molecule.
[0097] In another embodiment, the transferrin portion of the transferrin
fusion protein
includes a recoinbinant transferrin mutant having a mutation wherein the
mutant does not
retain the ability to bind metal ions. In an alternate embodiment, the
transferrin portion of the
transferrin fusion protein includes a recombinant transferrin mutant having a
mutation
wherein the mutant has a weaker binding avidity for metal ions than wild-type
serum
transferrin. In an alternate embodiment, the transferrin portion of the
transferrin fusion
protein includes a recombinant transferrin mutant having a mutation wlierein
the mutant has a
stronger binding avidity for metal ions than wild-type seruin transferrin.
[0098] In another embodiment, the transferrin portion of the transferrin
fusion protein
includes a recombinant transferrin mutant having a inutation wherein the
mutant does not
retain the ability to bind to the transferrin receptor. In an alternate
einbodiment, the
transferrin portion of the transferrin fusion protein includes a recombinant
transferrin mutant
having a mutation wherein the mutant has a weaker binding avidity for the
transferrin
receptor than wild-type serum transferrin. In an al.ternate embodiinent, the
transferrin portion
of the transferrin fusion protein includes a recombinant transferrin mutant
having a inutation
wherein the mutant has a stronger binding avidity for the transferrin receptor
than wild-type
seruin transferrin.
[0099] In another einbodiment, the transferrin portion of the transferrin
fusion protein
includes a recombinant transferrin mutant having a mutation wherein the mutant
does not
retain the ability to bind to carbonate ions. In an alternate einbodiinent,
the transferrin
portion of the transferrin fusion protein includes a recombinant transferrin
mutant having a
mutation wherein the inutant has a wealcer binding avidity for carbonate ions
than wild-type
serum transferrin. In an alternate embodiment, the transferrin portion of the
transferrin fusion
protein includes a recombinant transferrin inutant having a inutation wherein
the mutant has a
stronger binding avidity for carbonate ions than wild-type serum transferrin.
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23
[00100] In another embodiment, the transferrin portion of the transferrin
fusion protein
includes a recombinant human serum transferrin mutant having a mutation in at
least one
amino acid residue selected from the group consisting of Asp63, G1y65, Tyr95,
Tyrl88,
His249, Asp392, Tyr426, Tyr514, Tyr517 and His585 of SEQ ID NO: 3, wherein the
mutant
retains the ability to bind metal ions. In an alternate einbodiinent, a
recombinant huinan
serum transferrin mutant having a mutation in at least one ainino acid residue
selected fiom
the group consisting of Asp63, Gly65, Tyr95, Tyrl 88, His249, Asp392, Tyr426,
Tyr514,
Tyr517 and His585 of SEQ ID NO: 3, wherein the mutant has a reduced ability to
bind metal
ions. In another embodiment, a recombinant human serum transferrin mutant
having a
mutation in at least one amino acid residue selected from the group consisting
of Asp63,
G1y65, Tyr95, Tyrl88, His249, Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO:
3,
wherein the mutant does riot retain the ability to bind metal ions.
[00101] In another einbodiinent, the transferrin portion of the transferrin
fusion protein
includes a recombinant human seruin transferrin. mutant having a mutation at
Lys206 or
His207 of SEQ ID NO:3, wherein the mutant has a stronger binding avidity for
metal ions
than wild-type human serum transferrin (see U.S. Patent 5,986,067, which is
herein
incorporated by reference in its entirety). In an alternate embodiment, the
transferrin portion
of the transferrin fusion protein includes a recombinant human serum
transferrin mutant
having a mutation at Lys206 or His207 of SEQ ID NO:3, wherein the inutant has
a weaker
binding avidity for metal ions than wild-type huinan serum transferrin. In a
further
einbodiinent, the transferrin portion of the transferrin fusion protein
includes a recombinant
huinan serum transferrin inutant having a inutation at Lys206 or His207 of SEQ
ID NO:3,
wherein the mutant does not bind metal ions.
[00102] Any available technique may be used to produce the transferrin fusion
proteins of
the invention, including but not limited to molecular tecluliques coininonly
available, for
instance, those disclosed in Sainbrook et al. Molecular Cloning: A Laboratory
Manual, 2nd
Ed., Cold Spring Harbor Laboratory Press, 1989. When caiTying out nucleotide
substitutions
using techniques for accoinplishing site-specific inutagenesis that are well
known in the art,
the encoded amino acid changes are preferably of a minor nature, that is,
conservative ainino
acid substitutions, although other, non-conseivative, substitutions are
contemplated as well,
particularly wlien producing a modified transferrin portion of a Tf fusion
protein, e.g., a
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24
modified Tf protein exhibiting reduced glycosylation, reduced iron binding and
the like.
Specifically conteinplated are amino acid substitutions, small deletions or
insertions, typically
of one to about 30 amino acids; insertions between transferrin domains; small
amino- or
carboxyl-terminal extensions, such as an amino-terminal methionine residue, or
small linker
peptides of less than 50, 40, 30, 20 or 10 residues between transferrin
domains or linking a
transferrin protein and therapeutic protein or peptide or a small extension
that facilitates
purification, such as a poly-histidine tract, an antigenic epitope or a
binding domain.
[00103] Examples of conservative amino acid substitutions are substitutions
made within the
same group such as within the group of basic amino acids (such as arginine,
lysine, histidine),
acidic amino acids (such as glutamic acid and aspartic acid), polar amino
acids (such as
glutamine and asparagine), hydrophobic amino acids (such as leucine,
isoleucine, valine),
aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small
amino acids
(such as glycine, alanine, serine, threonine, inethionine).
[00104] Non-conservative substitutions encompass substitutions of amino acids
in one group
by ainino acids in another group. For example, a non-conservative substitution
would
include the substitution of a polar amino acid for a hydrophobic amino acid.
For a general
description of nucleotide substitution, see e.g. Ford et al. (1991), Pf ot.
Exp. Pur. 2: 95-107.
Non-conservative substitutions, deletions and insertions are particularly
useful to produce Tf
fusion proteins of the invention that exhibit no or reduced binding of iron,
no or reduced
binding of the fusion protein to the Tf receptor and/or no or reduced
glycosylation.
[00105] Iron binding and/or receptor binding may be reduced or disrupted by
mutation,
including deletion, substitution or insertion into, ainino acid residues
corresponding to one or
more of Tf N domain residues Asp63, Tyr95, Tyr188, His249 and/or C domain
residues Asp
392, Tyr 426, Tyr 514 and/or His 585 of SEQ ID NO: 3. Iron binding may also be
affected
by inutation to ainino acids Lys206, His207 or Arg632 of SEQ ID NO: 3.
Carbonate binding
may be reduced or disrupted by mutation, including deletion, substitution or
insertion into,
ainino acid residues corresponding to one or more of Tf N domain residues
Thr120, Arg 124,
Ala126, Gly 127 and/or C domain residues Thr 452, Arg 456, Ala 458 and/or Gly
459 of
SEQ ID NO: 3. A reduction or disruption of carbonate binding may adversely
affect iron
and/or receptor binding.
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[00106] Binding to the Tf receptor may be reduced or disrupted by mutation,
including
deletion, substitution or insertion into, amino acid residues corresponding to
one or more of
Tf N domain residues described above for iron binding.
[00107] As discussed above, glycosylation may be reduced or prevented by
mutation,
including deletion, substitution or insertion into, amino acid residues
corresponding to one or
more of Tf C domain residues around the N-X-S/T sites corresponding to C
domain residues
N413 and/or N611 (See U.S. Patent No. 5,986,067). For instance, the N413
and/or N6l 1
may be mutated to Glu residues.
[00108] In instances where the Tf fusion proteins of the invention are not
modified to
prevent glycosylation, iron binding, carbonate binding and/or receptor
binding, glycosylation,
iron and/or carbonate ions may be stripped from or cleaved off of the fiision
protein. For
instance, available deglycosylases may be used to cleave glycosylation
residues from the
fusion protein, in particular the sugar residues attached to the Tf portion,
yeast deficient in
glycosylation enzymes may be used to prevent glycosylation and/or recombinant
cells may be
grown in the presence of an agent that prevents glycosylation, e.g.,
tunicamycin.
[00109] The carbohydrates on the fusion protein may also be reduced or
completely
removed enzymatically by treating the fusion protein with deglycosylases.
Deglycosylases
are well known in the art. Examples of deglycosylases include but are not
limited to
galactosidase, PNGase A, PNGase F, glucosidase, mannosidase, fucosidase, and
Endo H
deglycosylase.
[00110] Nevertheless, in certain circuinstances, it inay be preferable for
oral delivery that the
Tf portion of the fusion protein be fully glycosylated.
[00111] Additional inutations may be made with Tf to alter the tliree
dimensional structure
of Tf, such as modifications to the hinge region to prevent the confonnational
cllange needed
for iron biding and Tf receptor recognition. For instance, inutations may be
made in or
around N domain amino acid residues 94-96, 245-247 and/or 316-318 as well as C
domain
amino acid residues 425-427, 581-582 and/or 652-658. In addition, mutations
may be made
in or around the flaillcing regions of these sites to alter Tf structure and
function.
[00112] In one aspect of the invention, the transfernn fusion protein can
function as a carrier
protein to extend the half life or bioavailability of the therapeutic protein
as well as, in some
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26
instances, delivering the therapeutic protein inside a cell and/or across the
blood brain barrier.
In an alternate embodiment, the transferrin fusion protein includes a modified
transferrin
molecule wherein the transferrin does not retain the ability to cross the
blood brain barrier.
[00113] In another embodiment, the transferrin fusion protein includes a
modified transferrin
molecule wherein the transferrin molecule retains the ability to bind to the
transferrin
receptor and transport the therapeutic peptide inside cells. In an alternate
einbodiinent, the
transferrin fusion protein includes a modified transferrin molecule wherein
the transferrin
molecule does not retain the ability to bind to the transferrin receptor and
transport the
therapeutic peptide inside cells.
[00114] In further embodiments, the transferrin fusion protein includes a
modified
transferrin molecule wherein the transferrin molecule retains the ability to
bind to the
transferrin receptor and transport the therapeutic peptide inside cells and
retains the ability to
cross the blood brain barrier. In an alternate embodiment, the transferrin
fusion protein
includes a modified transferrin molecule wherein the transferrin molecule
retains the ability
to cross the blood brain barrier, but does not retain the ability to bind to
the transferrin
receptor and transport the therapeutic peptide inside cells.
Modified Transferrin Fusion Proteins
[00115] The fusion of proteins of the invention may contain one or more copies
of the
therapeutic protein or polypeptide attached to the N-terininus and/or the C-
terininus of the Tf
protein. In some embodiments, the therapeutic protein or polypeptide is
attached to both the
N- and C-terininus of the Tf protein and the fusion protein may contain one or
more
equivalents of the therapeutic protein or polypeptide or one or more different
therapeutic
proteins or polypeptides on either or both ends of Tf. In other einbodiinents,
the therapeutic
protein or polypeptide is inserted into lalown domains of the Tf protein, for
instance, into one
or more of the surface loops of Tf (see Ali et al. (1999) J. Biol. Chein.
274(34):24066-
24073). Insertion may be made into inultiple loops of transferrin to create a
pentavalent
molecule with increased avidity for the antigen, receptor, or targeting
molecule, which the
therapeutic protein binds. In other einbodiinents, the tlierapeutic protein or
polypeptide is
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27
inserted between the N and C domains of Tf. Alternatively, the therapeutic
protein or
polypeptide is inserted anywhere in the transferrin molecule.
[00116] Generally, the transferrin fusion protein of the invention may have
one modified
transferrin-derived region and one therapeutic protein region. Multiple
regions of each
protein, however, may be used to malce a transferrin fusion protein of the
invention.
Similarly, more than one therapeutic protein may be used to make a transferrin
fusion protein
of the invention, thereby producing a multi-functional modified Tf fusion
protein.
[00117] In one embodiment, the transferrin fusion protein of the invention
contains a
therapeutic protein or polypeptide or portion thereof is fused to a
transferrin molecule or
portion thereof. In another einbodiment, the transferrin fusion protein of the
inventions
contains a therapeutic protein or polypeptide fused to the N terininus of a
transferrin
molecule. In an alternate embodiment, the transferrin fusion protein of the
invention contains
a therapeutic protein or polypeptide fused to the C terminus of a transferrin
molecule. In a
further embodiment, the transferrin fusion-protein of the invention contains a
transferrin
molecule fused to the N terminus of a therapeutic protein or polypeptide. In
an alternate
embodiment, the transferrin fusion protein of the invention contains a
transferrin molecule
fused to the C tenninus of a therapeutic protein or polypeptide.
[00118] In otller embodiments, the transferrin fusion protein of the
inventions contains a
therapeutic protein fused to both the N-tenninus and the C-tenninus of
modified transferrin.
In another embodiment, the therapeutic proteins fused at the N- and C-
terinini bind the saine
therapeutic proteins. In an alternate einbodiinent, the therapeutic proteins
fused at the N- and
C- tennini are different therapeutic proteins. In another alternate
embodiment, the
therapeutic proteins fused to the N- and C- tennini bind different therapeutic
proteins which
may be used to treat or prevent the saine disease, disorder, or condition. In
another
embodiment, the therapeutic proteins fused at the N- and C- terinini are
different therapeutic
proteins which may be used to treat or prevent diseases or disorders wliich
are luzown in the
art to coinmonly occur in patients simultaneously.
[00119] In addition to modified transferrin fusion protein of the invention in
which the
modified transferrin portion is fused to the N tenninal and/or C-tenninal of
the therapeutic
protein portion, transferrin fusion protein of the invention may also be
produced by inserting
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28
the therapeutic protein or peptide of interest (e.g., a therapeutic protein or
peptide as
disclosed herein, or a fragment or variant thereof) into an internal region of
the modified
transferrin. Internal regions of inodified transfeiTin include, but are not
liinited to, the iron
binding sites, the hinge regions, the bicarbonate binding sites, or the
receptor binding domain.
[00120] Within the protein sequence of the modified transferrin inolecule a
nuinber of loops
or turns exist, which are stabilized by disulfide bonds. These loops are
useful for the
insertion, or internal fusion, of therapeutically active peptides particularly
those requiring a
secondary structure to be functional, or therapeutic proteins to generate a
modified transferrin
molecule with specific biological activity.
[00121] When therapeutic proteins are inserted into or replace at least one
loop of a Tf
molecule, insertions may be made within any of the surface exposed loop
regions, in addition
to other areas of Tf. For instance, insertions may be made within the loops
comprising Tf
amino acids 32-33, 74-75, 256-257, 279-280 and 288-289 (Ali et al., supr-a)
(See Figure 3).
As previously described, insertions may also be made within other regions of
Tf such as the
sites for iron and bicarbonate binding, hinge regions, and the receptor
binding domain as
described in more detail below. The loops in the Tf protein sequence that are
alnenable to
modification/replacement for the insertion of proteins or peptides may also be
used for the
development of a screenable library of random peptide inserts. Any procedures
may be used
to produce nucleic acid inserts for the generation of peptide libraries,
including available
phage and bacterial display systems, prior to cloning into a Tf domain and/or
fusion to the
ends of Tf.
[00122] The N-terminus of Tf is free and points away from the body of the
molecule.
Fusions of proteins or peptides on the N-terminus may therefore be a preferred
embodiment.
Such fusions may include a linker region, such as but not limited to a poly-
glycine stretch, to
separate the therapeutic protein from Tf. Attention to the junction between
the leader
sequence, the choice of leader sequence, and the structure of the mRNA by
codon
manipulation/optimization (no major stein loops to inhibit ribosome progress)
will increase
secretion and can be readily accoinplished using standard recoinbinant DNA
techniques.
[00123] The C-teianiiius of Tf appears to be more buried and secured by a
disulfide bond 6
amino acids fi=om the C-terminus. In human Tf, the C-tenninal ainino acid is a
proline which,
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29
depending on the way that it is orientated, will either point a fusion away or
into the body of
the molecule. A linker or spacer moiety at the C-terminus may be used in some
embodiments
of the invention. There is also a proline near the N-terminus. In one aspect
of the invention,
the proline at the N- and/or the C- termini may be changed. In another aspect
of the
invention, the C-terminal disulfide bond inay be eliminated to untether the C-
terminus.
[00124] In yet other einbodiments, small molecule therapeutics may be
coinplexed with iron
and loaded on a modified Tf protein fusion for delivery to the inside of cells
and across the
BBB. The addition of a targeting peptide or, for example, a single chain
antibody (SCA) can
be used to target the payload to a particular cell type, e.g., a cancer cell.
[00125] A modified transferrin protein may be used with any of the fusion
proteins, methods
and various other aspects of the invention. In one embodiment, the modified
transferrin
protein contains modifications within or adj acent to one or two N-linked
glycosylation sites
(e.g., N-X-S/T). For instance, the invention includes fusion proteins wherein
the Tf moiety
contains mutations at serine and/or threonine amino acids within the N-linked
glycosylation
site. In one embodiment, the modified transferrin protein contains mutations
at S415 and
T613 (SEQ ID NO.: 3). For instance, the invention includes fusion proteins
comprising a
modified Tf protein with the niutations S415A and T613A.
Therapeutic Proteins and Peptides
[00126] Any therapeutic molecule may be used as the fusion partner to Tf
according to the
methods and compositions of the present invention. As used herein, a
therapeutic molecule is
typically a protein or peptide capable of exerting a beneficial biological
effect in vitro or in
vivo and includes proteins or peptides that exert a beneficial effect in
relation to norinal
homeostasis, physiology or a disease state. Therapeutic molecules do not
include fusion
partners coinmonly used as inarlcers or protein purification aids, sucli as
bacterial
galactosidases (see for exainple, U.S. Patent 5, 986, 067 and Aldred et al.
(1984) Biochem.
Bioplzys. Res. Conznaun. 122: 960-965). For instance, a beneficial effect as
related to a
disease state includes any effect that is advantageous to the treated subject,
including disease
prevention, disease stabilization, the lessening or alleviation of disease
symptoms or a
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modulation, alleviation or cure of the underlying defect to produce an effect
beneficial to the
treated subject.
[00127] A transferrin fusion protein of the invention includes at least a
fragment or variant of
a therapeutic protein and at least a fragment or variant of serum transferrin,
for instance,
modified serum transferrin, which are associated with one another, preferably
by genetic
fusion.
[00128] In one embodiment, the transferrin fusion protein includes a modified
transferrin
molecule linked to a natriuretic peptide. In another embodiment, the modified
transferrin
fusion protein includes transferrin at the carboxyl terminus linked to a
natriuretic peptide at
the ainino terininus. In an alternate embodiment, the modified transferrin
fusion protein
includes transferrin at the amino terminus linked to a natriuretic peptide at
the carboxy
terminus.
[00129] In further embodiments, a transferrin fusion protein of the invention
may contain at
least a fragment or variant of a therapeutic protein such as a fragment or
variant of a
natriuretic peptide. In a further embodiment, the transferrin fusion proteins
can contain
peptide fragments or peptide variants of a natriuretic peptide wherein the
variant or fragment
retains at least one biological or therapeutic activity. The transferrin
fusion proteins can
contain therapeutic proteins that can be peptide fragments or peptide variants
at least about 3,
at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 11, at least
12, at least 13, at least 14, at least 15, at least 20, at least 25, at least
30, at least 35, or at least
about 40, at least about 50, at least about 55, at least about 60 or at least
about 70 or more
ainino acids in length fused to the N and/or C tennini, inserted within, or
inserted into a loop
of a transferrin or modified transferrin.
[00130] The transferrin fusion proteins of the present invention may contain
one or more
peptides. Increasing the nuinber of peptides enhances the function of the
peptides fused to
transferrin and the function of the entire transferrin fusion protein. The
peptides may be used
to make a bi- or inulti-functional fusion protein by including peptide or
protein domains witll
inultiple functions. For instance, a multi-functional fusion protein can be
made with a
therapeutic protein and a second protein to target the fusion protein to a
specific target. Other
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31
peptides may be used to induce the immune response of a cellular system, or
induce an
antiviral, antibacterial, or anti-pathogenic response.
[00131] In another embodiment, the transferrin fusion molecules contain a
therapeutic
protein portion that can be fragments of a therapeutic protein that include
the full length
protein as well as polypeptides having one or more residues deleted from the
ainino tenninus
of the amino acid sequence.
[00132] In another embodiment, the transferrin fusion molecules contain a
therapeutic
protein portion that can be fragments of a therapeutic protein that include
the full length
protein as well as polypeptides having one or more residues deleted from the
carboxy
tenninus of the amino acid sequence.
[00133] In another embodiment, the transferrin fusion molecules contain a
therapeutic
protein portion that can have one or more ainino acids deleted from both the
amino and the
carboxy termini.
[00134] In another embodiment, the transferrin fusion molecules contain a
therapeutic
protein portion that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99% identical
to a reference therapeutic protein set forth herein, or fraginents thereof. In
further
embodiinents, the transferrin fusion molecules contain a therapeutic protein
portion that is at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to reference
polypeptides
having the amino acid sequence of N- and C-terininal deletions as described
above.
[00135] In another einbodiment, the transferrin fusion molecules contain the
therapeutic
protein portion that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%
or 100%,
identical to, for exainple, the native or wild-type ainino acid sequence of a
therapeutic
protein. Fraginents, of these polypeptides are also provided.
[00136] The therapeutic proteins corresponding to a therapeutic protein
portion of a
transferrin fusion protein of the invention, such as cell surface and
secretory proteins, can be
modified by the attaclunent of one or more oligosaccharide groups. The
modification
referred to as glycosylation can significantly affect the physical properties
of proteins and can
be important in protein stability, secretion, and localization. Glycosylation
occurs at specific
locations along the polypeptide baclcbone. There are usually two major types
of
glycosylation: glycosylation characterized by 0-linked oligosaccharides, which
are attached
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to serine or threonine residues; and glycosylation characterized by N-linked
oligosaccharides,
which are attached to asparagine residues in an Asn-X-Ser/Thr sequence, where
X can be an
amino acid except proline.
[00137] Therapeutic proteins corresponding to a therapeutic protein portion of
a transferrin
fusion protein of the invention, as well as analogs and variants thereof, may
be modified so
that glycosylation at one or more sites is altered as a result of
manipulation(s) of their nucleic
acid sequence by the host cell in which they are expressed, or due to other
conditions of their
expression. For example, glycosylation isomers may be produced by abolishing
or
introducing glycosylation sites, e.g., by substitution or deletion of amino
acid residues, such
as substitution of glutamine for asparagine, or unglycosylated recombinant
proteins may be
produced by expressing the proteins in host cells that will not glycosylate
them, e.g. in
glycosylation-deficient yeast. These approaches are known in the art.
[00138] Therapeutic proteins and their nucleic acid sequences are well known
in the art and
availabte in public databases such as Chemical Abstracts Services Databases
(e.g., the CAS
Registry), GenBank, and GenSeq. The Accession Numbers and sequences referred
to herein
are incorporated by reference in their entirety.
[00139] In other embodiments, the transferrin fusion proteins of the invention
are capable of
a therapeutic activity and/or biologic activity, corresponding to the
therapeutic activity and/or
biologic activity of the therapeutic protein described elsewhere in this
application. In further
einbodiinents, the therapeutically active protein portions of the transferrin
fusion proteins of
the invention are fraginents or variants of the reference sequences cited
herein.
[00140] The present invention is further directed to Tf fusion proteins
coinprising fraginents
of the therapeutic proteins herein described. Even if deletion of one or more
ainino acids
from the N-tenninus of a protein results in modification or loss of one or
more biological
functions of the therapeutic protein portion, other therapeutic activities
and/or functional
activities (e.g., biological activities, ability to inultiinerize, ability to
bind a ligand) may still
be retained. For exainple, the ability of polypeptides with N-terminal
deletions to induce
and/or bind to antibodies which recognize the complete or inature foiins of
the polypeptides
generally will be retained with less than the majority of the residues of the
coinplete
polypeptide reinoved from the N-terininus. Whether a particular polypeptide
laclcing N-
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33
terminal residues of a complete polypeptide retains such immunologic
activities can be
assayed by routine methods described herein and otherwise known in the art. It
is not
unlikely that a mutant with a large nuinber of deleted N-terminal amino acid
residues may
retain some biological or immunogenic activities. In fact, peptides composed
of as few as six
amino acid residues may often evoke an immune response.
[00141] Also as mentioned above, even if deletion of one or more amino acids
from the N-
terminus or C-terminus of a therapeutic protein results in modification or
loss of one or more
biological functions of the protein, other functional activities (e.g.,
biological activities,
ability to multimerize, ability to bind a ligand) and/or therapeutic
activities may still be
retained. For example the ability of polypeptides with C-terminal deletions to
induce and/or
bind to antibodies which recognize the complete or mature forms of the
polypeptide generally
will be retained when less than the majority of the residues of the complete
or mature
polypeptide are removed from the C-tenninus. Whether a pai-ticular polypeptide
lacking the
N-terminal and/or, C-tenninal residues of a reference polypeptide retains
therapeutic activity
can readily be determined by routine methods described herein and/or otherwise
known in the
art.
[00142] Peptide fragments of the therapeutic proteins can be fragments
comprising, or
alternatively, consisting of, an amino acid sequence that displays a
therapeutic activity and/or
functional activity (e.g. biological activity) of the polypeptide sequence of
the therapeutic
protein of which the amino acid sequence is a fraginent.
[00143] The peptide fragments of the therapeutic protein may comprise only the
N- and C-
termini of the protein, i.e., the central portion of the therapeutic protein
has been deleted.
Alternatively, the peptide fragtnents may comprise non-adjacent and/or
adjacent portions of
the central part of the therapeutic protein.
[00144] Other polypeptide fragments are biologically active fraginents.
Biologically active
fraginents are those exhibiting activity similar, but not necessarily
identical, to an activity of a
therapeutic protein used in the present invention. The biological activity of
the fraginents
may include an iinproved desired activity, or a decreased undesirable
activity.
[00145] Generally, variants of proteins are overall very similar, and, in many
regions,
identical to the amino acid sequence of the therapeutic protein corresponding
to a therapeutic
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34
protein portion of a transferrin fusion protein of the invention. Nucleic
acids encoding these
variants are also encompassed by the invention.
[00146] Further therapeutic polypeptides that may be used in the invention are
polypeptides
encoded by polynucleotides which hybridize to the complement of a nucleic acid
molecule
encoding an amino acid sequence of a therapeutic protein under stringent
hybridization
conditions which are known to those of skill in the art. (see, for exainple,
Ausubel, F.M. et
al., eds., 1989 Current protocol in Molecular Biology, Green Publishing
Associates, Inc., and
John Wiley & Sons Inc., New. York). Polynucleotides encoding these
polypeptides are also
encompassed by the invention.
[00147] By a polypeptide-having an amino acid sequence at least, for example,
95%
"identical" to a query amino acid sequence of the present invention, it is
intended that the
amino acid sequence of the subject polypeptide is identical to the query
sequence except that
the subject polypeptide sequence may include up to five amino acid alterations
per each 100
amino acids of the query amino acid sequence. In other words, to obtain a
polypeptide
having an amino acid sequence at least 95% identical to a query amino acid
sequence, up to
5% of the amino acid residues in the subject sequence may be inserted,
deleted, or substituted
with another ainino acid. These alterations of the reference sequence may
occur at the
amino- or carboxy-terminal positions of the reference amino acid sequence or
anywhere
between those terminal positions, interspersed either individually ainong
residues in the
reference sequence, or in one or more contiguous groups within the reference
sequence.
[00148] As a practical matter, whether any particular polypeptide is at least
about 80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the ainino acid
sequence of a
transferrin fusion protein of the invention or a fraginent thereof (such, as
the therapeutic
protein portion of the transferrin fusion protein or the transferrin portion
of the transferrin
fusion protein), can be detennined conventionally using laiown coinputer
prograins. A
preferred method for detennining the best overall match between a query
sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence aligiunent, can be detennined using the FASTDB coinputer program
based on the
algorithin of Brufiag et al. (Coinp. App. Biosci 245 (1990)).
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[00149] The polynucleotide variants of the invention may contain alterations
in the coding
regions, non-coding regions, or both. Polynucleotide variants containing
alterations which
produce silent substitutions, additions, or deletions, but do not alter the
properties or activities
of the encoded polypeptide may be used to produce modified Tf fusion proteins.
Nucleotide
variants produced by silent substitutions due to the degeneracy of the genetic
code can be
utilized. Moreover, polypeptide variants in which less than about 50, less
than 40, less than
30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids
are substituted,
deleted, or added in any coinbination can also be utilized. Polynucleotide
variants can be
produced for a variety of reasons, e.g., to optimize codon expression for a
particular host
(change codons in the human mRNA to those preferred by a host, such as, yeast
or E. coli as
described above).
[00150] In other embodiments, the therapeutic protein moiety has conservative
substitutions
compared to the wild-type sequence. By "conservative substitutions" is
intended swaps
within groups such as replacement of the aliphatic or hydrophobic amino acids
Ala, Val, Leu
and Ile; replacement of the hydroxyl residues Ser and Thr; replaceinent of the
acidic residues
Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the
basic
residues Lys, Arg, and His; replaceinent of the aromatic residues Phe, Tyr,
and Trp, and
replaceinent of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
Guidance
concerning how to make phenotypically silent ainino acid substitutions is
provided, for
example, in Bowie et al., "Deciphering the Message in Protein Sequences:
Tolerance to
Ainino Acid Substitutions," Science 247:1306-1310 (1990). In specific
embodiments, the
polypeptides of the invention comprise, or alternatively, consist of,
fiaginents or variants of
the ainino acid sequence of a therapeutic protein described herein and/or
seruin transferrin,
and/ modified transferrin protein of the invention, wherein the fraginents or
variants have 1-5,
5-10, 5-25, 5-50, 10-50 or 50-150 amino acid residue additions, substitutions,
and/or
deletions when compared to the reference ainino acid sequence. In further
einbodiments, the
amino acid substitutions are conservative. Nucleic acids encoding these
polypeptides are also
encompassed by the invention.
[00151] The fusion proteins of the present invention can be coinposed of
ainino-acids joined
to each other by peptide bonds or modified peptide bonds and may contain amino
acids other
than the 20 gene-encoded amino acids. The polypeptides may be modified by
either natural
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36
processes, such as post-translational processing, or by chemical modification
techniques
which are well known in the art. Such modifications are well described in
basic texts and in
more detailed monographs, as well as in a voluminous research literature.
[00152] Modifications can occur anywhere in a polypeptide, including the
peptide backbone,
the amino acid side-chains and the ainino or carboxy termini. It will be
appreciated that the
same type of modification may be present in the same or varying degrees at
several sites in a
given polypeptide. Also, a given polypeptide may contain many types of
modifications.
Polypeptides may be branched, for example, as a result of ubiquitination, and
they may be
cyclic, with or without branching. Cyclic, branched, and branched cyclic
polypeptides may
result from posttranslation natural processes or may be made by syntlietic
methods.
Modifications include acetylation, acylation, ADP-ribosylation, amidation,
covalent
attachment of flavin, covalent attachment of a heme moiety, covalent
attachment of a
nucleotide or nucleotide derivative, covalent attachinent of a lipid or lipid
derivative, covalent
attachmeilt of phosphotidylinositol, cross-linking, cyclization, disulfide
bond formation,
demethylation, formation of covalent cross-links, formation of cysteine,
glycosylation, GPI
anchor formation, hydroxylation, iodination, methylation, myristylation,
oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
raceinization, sulfation,
transfer-RNA mediated addition of amino acids to proteins such as
arginylation, and
ubiquitination. (See, for instance, PROTEINS - STRUCTURE AND MOLECULAR
PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POST-
TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,
Academic Press, New York, pgs. 1-12 (1983); Seifter et ccl. (1990) Meth.
Enzyinol. 182:626-
646; Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62.
[00153] The therapeutic proteins of the present invention include, but are not
limited to
polypeptide, peptide, antibody, or fraginents and variants thereof.
Preferably, the therapeutic
proteins of the present invention include natriuretic peptides and their
analogs, derivatives,
and chimeric molecules. Other therapeutic peptide fusions for treatment of
cardiovascular
disease include fusions to adrenomedullin Shimosawa, T. et al. (2002)
Adrenomedullin, an
Endogenous Peptide, Counteracts Cardiovascular Dainage. Circulation 105,106-
111) and
fusions to urocortin (Donaldson, C.J. et al. (1996) Cloning and
Characterization of Huinan
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37
Urocortin. Endocrinology 137, 2167-2170).
Natriuretic Peptides
[00154] The present invention provides fusion proteins comprising one or more
copies of a
natriuretic peptide fused to a transferrin molecule. Preferably, the
transferrin molecule is a
modified transferrin molecule exhibiting reduced glycosylation as compared to
the native
transferrin molecule. The natriuretic peptide may be an endogenous natriuretic
peptide or an
exogenous peptide, such as an analog or derivative of an endogenous
natriuretic peptide, a
chimeric natriuretic peptide, or a peptide able to act as an agonist or
antagonist of a
natriuretic peptide receptor. The fusion protein may contain only one type of
natriuretic
peptide or a combination of different types of natriuretic peptides.
[00155] Natriuretic peptides are hormones involved in the regulation of fluid
and electrolyte
homeostasis. There are three major types of inaininalian natriuretic factors.
The first type,
atrial natriuretic peptide or factor (ANP or ANF), was discovered by DeBold
and coworkers
in 1981 when they found that granule-enriched atrial extracts contained a
substance which
caused natriuresis and vasodilatation (DeBold et al., Life Sci. (1981) 28:89-
94). Two years
later, Flynn et al. purified and sequenced ANP from maininalian atria (Flynn
et al., Biochem.
Biophys. Res. Commun. (1983) 117:859-865). Shortly tlzereafter, the second
type, brain
natriuretic peptide (BNP), and the third type, C-type natriuretic peptide
(CNP) were found.
BNP was initially found in porcine brain (Sudoh et al., Biochem Biophys Res
Comm (1988)
155:726-732), but the main source of BNP is the cardiac ventricle. CNP was
first identified
in the nervous system (Sudoh et al. Biochem Biophys Res Cominun (1990) 168(2):
863-
870), but later found to be produced by the endothelial cells (Suga et al. J
Clin Invest (1992)
90(3): 1145-1149). In addition to these maininalian natriuretic peptides, two
other peptides
have been isolated. Tervonen (1998) reported the isolation of a salmon
natriuretic factor
(Salmon cardiac peptide) witll similar structure and properties (Tervonen et
al.,
Endocrinology (1988) 139:4021-4025), and Schweitz et al. discovered
Dendroaspis
Natriuretic Peptide (DNP) in the venom of the green mamba (Schweitz et al., J.
Biol. Chein.,
(1992) 267: 13928-13932).
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38
[00156] Natriuretic peptides are synthesized by three different genes and then
stored as three
different prohormones (i.e., 126 ainino acid atrial natriuretic peptide (ANP),
108 a.a. brain
natriuretic peptide (BNP), and 126 amino acids C-natriuretic peptide (CNP)
prohormones).
Structurally, natriuretic peptides are homologous peptide hormones. Although
they are
peptides of varying lengths, they share the saine 17 amino acid ring
containing two cysteines
which provide the disulfide bond.
[00157] There also exist many nonendogenous or exogenous natriuretic peptides,
such as the
chimeric natriuretic peptides that contain the ainino acid sequence of one or
more natriuretic
peptides fused to another peptide, and mutant natriuretic peptides derived
from the
endogenous or wild-type natriuretic peptides. The inutant natriuretic peptides
may be
obtained by substituting and/or deleting one or more amino acids from the wild-
type peptides.
The mutant natriuretic peptides do not have the saine amino acid sequence as
the
corresponding endogenous natriuretic peptides but they share similar binding
specificity as
the corresponding endogenous peptides. The chimeric natriuretic peptides may
be obtained
by adding a portion of another natriuretic peptide or replacing a portion of
the natriuretic
peptide with another natriuretic peptide or therapeutic peptide.
[00158] Natriuretic peptides share common receptors and stimulate the
intracellular
production of cGMP as a second messenger. The functional activities of the
maminalian
natriuretic peptides are mediated through the binding of the natriuretic
peptides to three
distinct maminalian receptors, natriuretic peptide receptors A, B, and C
(NPRA, NPRB, and
NPRC). NPRA and NPRB are linked to guanyl cyclases (GC). Thus, ligand binding
to the
extracellular doznan activates the cytoplasnlic GC domain. On the other hand,
NPRC is not
coupled to cGMP production and may fiuiction in the clearance of ANP.
[00159] The existence of these specific receptors on mammalian ineinbranes has
been
demonstrated in a variety of kidney, adrenal cortex and vascular tissue
(Schenlc et al. (1985)
J. Biol. Chein. 260:14887-14890; Vandlen et al., (1985) J. Biol. Chern.
260:10889-10892;
Misono et al., (1985) Biochem. Biophys. Res. Coininun. 130:994-1001; Hirose et
al., (1985)
Biochem. Biophys. Res. Coininun. 130:574-579; Yip et al., (1985) J. Biol.
Chem. 260:8229-
8232; Schei-Ac et al. (II) (1985) Biochem. Biophys. Res. Coininun. 127:433-
442; Hirata et al.,
(1985) Biochern. Biophys. Res. Coininun. 128:538-546; Winquist et al., (1984)
Proc. Natl.
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39
Acad. Sci. USA 81:7661-7664; Napier et al., (1984) Proc. Natl. Acad. Sci. USA
81:5946-
5950; Hirata et al., (1984) Biochem. Biophys. Res. Commun. 125:562-568; De
Lean et al.,
Endocrinology 115:1636-1638; De Lean et al., (1984) Life Sci. 35:2311-2318).
[00160] Similar to all peptides, the natriuretic peptides have very short half-
lives after in vivo
delivery. Thus, there is an interest in obtaining natriuretic peptides with
extended serum
stability or in vivo circulatory half-live and with enhanced functional
activity.
Atrial Natriuretic Peptides (ANPs)
[00161] ANP has been given a variety of names including ANF, cardionatrin,
atrionatriuretic
factor, pronatriodilatin (PND), atriopeptin, but are now collectively known as
ANPs. The
main source of ANP is the atria of the heart, though its synthetic equivalent
is commercially
available in the fonn of a-H-ANP.
-[00162] ANP is synthesized in the atria of the h.eart as a prehormone and is
cleaved to a
prohormone. ANP is part of a hormonal system in which one gene synthesizes
four peptide
honnones. The ANP gene synthesizes a 151 amino acid preprohonnone (SEQ ID NO:
4)
which is processed within the endoplasmic reticulum to fonn a 126 amino acid
prohormone
(i.e., the storage form of the following peptide horinones) after removal of a
25 amino acid
signal peptide fiom its N-tenninal end.
MSSFSTTTVSFLLLLAFQLLGQTRANPMYNAVSNADLMDFKNLLDHLEEKMPLEDEVVPPQVLSEPNEEA
GAALSPLPEVPPWTGEVSPAQRDGGALGRGPWDSSDRSALLKSKLRALLTAPRSLRRSSCFGGRMDRIGA
QSGLGCNSFRY(SEQ ID NO: 4)
These four peptide honnones within the 126 ainino acid ANP prohonnone (SEQ ID
NO: 5,
after removal of the 25 ainino acid signal peptide) consist of: (1) the first
30 ainino acids
from the N-tenninal end of the prohormone (i.e., proANP 1-30 of SEQ ID NO: 5;
long acting
natriuretic peptide, LANP); (2) ainino acid 31-67 of SEQ ID NO: 5 (i.e.,
proANP 31-67;
Vessel Dilator); (3) ainino acid 79-98 of SEQ ID NO: 5 (proANP 79-98;
Kaliuretic Peptide);
and (4) ainino acid 99-126 of SEQ ID NO: 5 of this prohonnone (ANP:
S LRRS S CF GGRMDRIGAQ S GLGCN S FRY).
[00163] Each of these four peptide hoi7nones circulate witllin the blood
streain with LANP
and Vessel Dilator's concentrations in plasma being 15- to 20-fold higher than
ANP. Each of
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these peptide hormones has biologic effects, e.g., blood pressure lowering,
natriuretic and/or
diuretic effects in both animals and humans. ANP (amino acids 99-126 of SEQ ID
NO: 5) is
the principal circulating fonn of the peptide.
[00164] As used herein, the term "atrial natriuretic peptide (ANP)" means any
ANP from
various species, analogs, and derivatives thereof, and chimeric ANP peptides.
The term also
refers to synthetically produced ANP having the same amino acid sequence as an
endogenous
ANP peptide. For example, the term "ANP" may include, ANPs having a sequence
derived
from mainmals, such as, but not limited to, human, rat, mouse, equine, or
porcine sources.
[00165] ANP is a potent natriuretic and vasorelaxant polypeptide. One of its
main biologic
functions is to enhance sodium excretion (natriuresis). ANP has been shown to
play a
significant role in blood-pressure homeostasis, regulation of extracellular
fluid volume, and
as an antagonist to the hypertensive effects of the reiiin-angiotensin system
and other
hormonal and neurotransmitter systems. ANP has been detected in the blood by
radioimmunoassay (Gutkowska et -al., (1984) Biochem. Biophys. Res. Coinmon.
125:315-
323; Tanaka et al., (1984) Biochein. Biophys. Res. Cominun. 124:663-668).
[00166] Because of the potent biological activity of ANP, regulation of its
levels in the blood
would be a therapeutic approach to the treatment of sucll disorders as
hypertension, shock,
and the like. While current native and synthetic ANP, as well as analogs
thereof, would
allow for the modulation of fluid volume and vascular function by increasing
ANP levels,
effective therapies may also require ANP levels to be reduced in order to
achieve the desired
extracellular fluid voluine and electrolytic homeostasis.
[00167] ANP has been infused intravenously in treating hypertension, heart
disease, acute
renal failure and edeina. ANP, when infused intravenously, has been shown to
increase the
glomerular filtration rate (GFR) and filtration fraction. ANP has also been
shown to reduce
proximal tubule sodium ion concentration and water reabsorption. Further, ANP
has been
shown to inhibit net sodiuin ion reabsorption and water reabsorption in the
collecting duct,
lower plasma renin concentration and iifllibit aldosterone secretion. Use of
ANP
intravenously has also resulted in mean arterial pressure reduction and has
led to natriuresis
and diuresis.
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41
[00168] Vessel Dilator has been shown to have significant beneficial diuretic,
natriuretic and
heinodynamic properties in humans with congestive heart failure (Vesely, D.L.
et al. (1998)
Circulation. 98: 323-329).
[00169] Dialysis provides a method for supplementing or replacing renal
function in certain
patients. Principally, hemodialysis and peritoneal dialysis are the two
methods that are
currently utilized.
[00170] In hemodialysis, the patient's blood is passed through an artificial
kidney dialysis
machine. A membrane in the machine acts as an artificial kidney for cleansing
the blood.
Because it is an extracorporeal treatment that requires special machinery,
heinodialysis is
fraught with certain inllerent disadvantages such as the availability of
dialysis machines and
the possibility of infection and containination.
[00171] To overcome the disadvantages associated with hemodialysis, peritoneal
dialysis
was developed. Peritoneal dialysis utilizes the patient's own peritoneum as a
semi-penneable
ineinbrane. The peritoneum is a meinbranous lining of the abdominopelvic walls
of the body.
The peritoneum is capable of acting as a natural semi-permeable membrane
because of its
large number of blood vessels and capillaries.
[00172] In operation, a peritoneal dialysis solution is introduced into the
peritoneal cavity
utilizing a catlleter. After a sufficient period of time, an exchange of
solutes between the
dialysate and blood is achieved. Fluid removal is achieved by providing a
suitable osmotic
gradient from the dialysate to the blood to perinit water outflow from the
blood. This allows
the proper acid-base, electrolyte and fluid balance to be achieved in the
blood. After an
appropriate dwell period, the dialysis solution or dialysate is drained from
the body through a
catheter.
[00173] While peritoneal dialysis provides some advantages over heinodialysis,
primary
disadvantages of peritoneal dialysis include an insufficient net
ultrafiltration and insufficient
clearances of urea nitrogen and sodium. As a result, overall peritoneal
dialysis adequacy can
be insufficient. Therefore, there is a need for an iinproved peritoneal
dialysis solution which
provides a greater net ultrafiltration and increased clearances of components
such as urea
nitrogen.
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42
[00174] U.S. Patent 5,965,533 provides a peritoneal dialysis solution that
contains atrial
natriuretic peptide (ANP), a derivative of ANP, an analogue of ANP, a
substance that binds
ANP to clearance receptors or a substance that promotes ANP synthesis, which
results in an
increased net ultrafiltration and increased sodiuin clearance experienced in
peritoneal dialysis
patients.
[00175] A ineans to obtain ANPs with extended serum stability or in vivo
circulatory half-
life is to fuse the ANP to a transferrin or modified transferrin. Fusing the
ANP to modified
transferrin improves the stability of the ANP during delivery and enhances its
therapeutic
effects at the target site. The present invention provides fusion proteins
comprising ANPs
fused to modified transferrins exhibiting reduced glycosylation as compared to
a native
transferrin. The ANPs in the fusion proteins may be endogenous peptides or
exogenous
peptides, i.e., analogs, derivatives, and chimeric molecules. The exogenous
ANPs have, if
not enhanced, at least the same functional activity and stability as the
endogenous ANPs.
The ANP sequence may be fused to the N-terminus of Tf, the C-terminus of Tf,
to botlh the
N- and C-termini, or inserted into one or more of the surface exposed loops of
Tf.
Brain Natriuretic Peptides (BNPs)
[00176] Although brain natriuretic peptides were first discovered in porcine
brain (Sudoh, P.
(1988) Nature 332:78-81), BNPs have been localized to the cardiac ventricle.
As used
herein, the term "brain natriuretic peptide (BNP)" refers to any naturally
occurring BNPs
from various species, analogs and derivatives thereof, and chimeric BNPs. The
term also
refers to synthetically produced BNPs having the saine ainino acid sequence as
an
endogenous BNP. For exainple, the tenn "BNP" will include, BNPs having a
sequence
derived from mammals, such as, but not limited to, human, rat, mouse, equine,
or porcine
sources.
[00177] As mentioned above, Sudoh, P. (Nature (1988) 332:78-81) was the first
to isolate
and sequence BNP. BNP is a 26-amino acid peptide syntllesized in porcine brain
and atrial
tissue at about 1/100 of the concentration of analyzed atrial natriuretic
peptide (ANP) activity.
The spectruin of activity of this porcine brain natriuretic peptide, or pBNP,
is similar to that
of the porcine ANP.
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43
[00178] Like the ANPs, BNPs are derived from a larger precursor molecule.
Subsequent
papers from Sudoh et al. further characterized these proteins. Sudoh et al.
(Biochem Biophys
Res Comm (1988) 155:726-732) reported the isolation of a 32-ainino acid
natriuretic peptide
("BNP-32") from porcine brain which contains the 26 amino acids of the porcine
BNP
described above at its C-terminus and an additional N-terininal 6-amino acid
extended
portion of the sequence Ser-Pro-Lys-Thr-Met-Arg- (SEQ ID NO: 6). In papers
following on
subsequent pages, levels of various natriuretic peptides in tissues were
reported. Ueda et al.
(Biochem Biophys Res Comin (1988) 155: 733-739) utilized a radioimmunoassay to
localize
and messure the levels of porcine BNP and porcine BNP-32 in the brain and
spinal cord. The
results showed that both BNP and BNP-32 were major forms of immunoreactive BNP
in the
porcine brain, and that the highest concentrations were found in the medulla-
pons, striatuin,
and spinal cord. The porcine form of atrial natriuretic peptide (pANP) was
also found in the
porcine brain but at a level approximately 13 times lower than that
characteristic of BNP.
Minamino et al. (Biochem Biophys Res Comin (1988) 155:740-746) reported the
results of
radioiinmunoassay for porcine BNP and ANP in peripheral tissue. The
concentration of BNP
was highest in cardiac atrium of the tissues assayed. The iinmunoreactive foim
of this
protein was characterized as mostly a 12 kDa high molecular weight forin; less
than 15% of
the total immunoreactive BNP in atrial tissue is of the lower molecular weight
forms pBNP
or pBNP-32.
[00179] In a subsequent publication, Minamino et al. (Biochein Biophys Res
Coinm (1988)
157:402-409) reported the isolation and characterization of this higher
molecular weight form
of BNP from porcine heart. The coinplete amino acid sequence of this protein
was obtained
and shown to contain the 26-ainino acid pBNP (aiid 32-ainino acid pBNP-32) at
its carboxy
terminus. The full-length protein contains 106 amino acids. Finally, Maelcawa
et al.
(Biochem Biophys Res Comin (1988) 155:410-416) report the cloning and sequence
analysis
of a eDNA encoding a precursor protein for porcine BNP. A cDNA library was
obtained
from porcine cardiac atriuin and the relevant BNP-encoding gene was isolated
and sequenced.
The gene was found to include a 25-residue putative signal peptide at the N-
terininus
followed by the codons corresponding to the 106 amino acids of the reported
protein. These
results are consistent with the inforination available from studies of the
atrial-derived
natriuretic peptides which are generally also associated with longer
precursors.
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44
[00180] Kambayashi et al. (FEBS Lett. (1990) 259(2):341-5) isolated human
brain
natriuretic peptide (human BNP) from the human atrium. SEQ ID NO: 7 discloses
the human
BNP sequences including its signal peptide. The first 26 ainino acid in SEQ ID
NO: 7 is the
signal peptide.
MDPQTAPSRALLLLLFLHLAFLGGRSHPLGSPGSASDLETSGLQEQRNHLQGKLSELQVEQTSLEPLQES
PRPTGVWKSREVATEGIRGHRKMVLYTLRAPRSPKMVQGSGCFGRKNIDRISSSSGLGCKVLRRH (SEQ ID
NO: 7)
[00181] Sequence analysis has revealed that it is a 32-amino-acid peptide with
the sequence
S-P-K-M-V-Q-G-S-G-C-F-G-R-K-M-D-R-I-S-S-S-S-G-L-G-C-K-V-L-R-R-H, which is
identical to the C-terininal sequence (103-134 of SEQ ID NO: 8) of the human
BNP
precursor deduced fiom the cDNA sequence. The sequence of huinan BNP (103-134)
is
preceded by Pro101-Arg102 in the human BNP precursor, which is the saine
processing
signal as Pro97-Arg98 of the precursor of atrial natriuretic peptide (ANP, SEQ
ID NO: 4).
The processing of the BNP precursor occurs in the cardiocyte, although that of
the ANP
precursor in the cardiocyte is unclear at present.
[00182] U.S. Patent 5,948,761 discloses recombinant canine BNPs useful in
treating
conditions characterized by high levels of extracellular fluid. The patent
discloses various
peptides, such as, R1-Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly-
Leu-
Gly-Cys-R2 wherein Rl is selected from the group consisting of: (H); Gly-; Ser-
Gly-; Lys-
Ser-Gly-; His-Lys-Ser-Gly-; Met-His-Lys-Ser-Gly-; Thr-Met-His-Lys-Ser-Gly-;
Lys-Thr-
Met-His-Lys-Ser-Gly-; Pro-Lys-Thr-Met-His-Lys-Ser-Gly-; and Ser-Pro-Lys-Thr-
Met-His-
Lys-Ser-Gly; and R2 is (OH), NH2, or NR2 wherein each R is independently H or
lower alkyl
(1-4C) or is Asn; Asn-Val; Asn-Val-Leu; Asn-Val-Leu-Arg; Asn-Val-Leu-Arg-Lys;
Asn-
Val-Leu-Arg-Lys-Tyr (SEQ ID NO: 130).
[00183] Similar to the ANPs, BNPs have short half-lives after in vivo
delivery. One way to
extend the seruin stability or in vivo circulatory half-life of the BNPs after
in vivo delivery is
to fuse them to transferrin or modified transferrin. The present invention
provides fusion
proteins coinprising BNPs fused to modified transferrins exhibiting reduced
glycosylation as
coinpared to a native transfeiTin. The BNPs in the fusion proteins may be
endogenous
peptides or exogenous peptides, i.e. analogs, derivatives, and chimeric
molecules. The
exogenous BNPs have, if not enhanced, at least the same functional activity
and stability as
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the endogenous BNPs. The BNP sequence may be fused to the N-terminus of Tf,
the C-
terminus of Tf, to both the N- and C-termini, or inserted into one or more of
the surface
exposed loops of Tf.
C-Type Natriuretic Peptides (CNPs)
[00184] Like the BNP, C-type natriuretic peptide (CNP) was isolated from
porcine brain
extracts on the basis of their potent relaxant effects on chick rectum (Sudoh
et al. Biochem
Biophys Res Commun (1990) 168(2): 863-870); Sudoh et al. Biochem Biophys Res
Commun (1990) 168(2): 863-870). CNP is of endothelial cell origin and
functions as a
vasodilating and growth-inhibiting peptide (Suga et al. J Clin Invest (1992)
90(3): 1145-
1149).
[00185] As used herein, the term "C-type Natriuretic Peptide (CNP)" refers to
any naturally
occurring CNPs from various species, analogs and derivatives thereof, and
chimeric CNPs.
The term also refers to synthetically produced CNPs having the same amino acid
sequence as
an endogenous CNP. For example, the term "CNP" will include, CNPs having a
sequence
derived from mammals, such as, but notlimited to, liuman, camel, rat, mouse,
equine, or
porcine sources.
[00186] Similar to the ANP and BNP, CNP is synthesized from large precursor
proteins, and
the mature, active peptides have a 17 amino acid loop forined by an
intramolecular disulfide
linkage. In the human peptides, eleven of these ainino acids are identical in
ANP, BNP, and
CNP, whereas the C-terminal tails vary in both length and coinposition
(Kambayashi et al.
FEBS Lett. (1990) 259(2):341-5). However, CNP has no C-terminal tail, and
studies of the
structure of the gene for CNP deinonstrated that translation is tenninated by
a stop codon
iininediately after the final cysteine codon in the inRNA. The amino acid
sequence of CNP
precursoris
MHLSQLLACALLLTLLSLRPSEAKPGAPPKVPRTPPAEELAEPQAAGGGQKKGDKAPGGGGANLKGDRSRLLRDL
RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 8)
[00187] Ainong species, the ainino acid sequence of both ANP and CNP are
highly
conserved, wliereas the structure of BNP varies greatly. For exainple, the
mature 28 ainino
acid huinan and porcine ANPs are identical, and there is only one substitution
in the rat
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46
peptide. The existence of this structural variation, coupled with the presence
of at least three
types of receptors specific for the natriuretic peptides, suggests that the
physiological control
of body fluid hoineostasis is complex. ANP and CNP both decrease cardiac
preload.
However, unlike ANP, CNP is not natriuretic (Stingo et al., Am. J. Physiol.
(1992) 262(1 Pt
2):H308-12).
[00188) The present invention provides CNP with extended serum stability and
in vivo
circulatory half-life. The present invention provides fusion proteins
comprising a CNP fused
to transferrin or modified transferrin. Preferably, the transferrin molecule
is modifed to
exhibit reduced glycosylation as compared to the wild-type transferrin. The
CNP may be an
endogenous peptide or an exogenous peptide such as an analog, derivative, or
chimeric
peptide. The analogs, derivatives, or chimeric peptide have, if not enhanced,
at least the saine
functional activity and stability as the endogenous CNP. Moreover, the fusion
protein may
contain a combination of endogenous and exogenous CNP peptides. The CNP
sequence may
be fused to the N-terminus of Tf, the C-terininus of Tf, to both the N- and C-
termini, or -
inserted into one or more of the surface exposed loops of Tf.
Variants of Natriuretic Peptides
[00189] The present invention also provides variants of the endogenous
natriuretic peptides
that function as agonists, mimetics or antagonists. Variants of eildogenous
natriuretic
peptides include analogs, derivatives and chiineric peptides, that can be
generated by
mutagenesis, e.g., discrete point inutation, ainino acid additions,
substitutions, or deletions.
A variant of a parent natriuretic peptide can retain substantially the saine,
or a subset of, the
biological activities of the naturally occurring forin of the parent peptide.
Thus, specific
biological effects can be elicited by treatinent with a variant with a limited
function. In one
einbodiment, treatinent of a subject with a variant having a subset of the
biological activities
of the naturally occurring form of the peptide has fewer side effects in a
subject relative to
treatment with the naturally occurring form of the parent peptide.
[00190] The variant natriuretic peptides are functionally active. As utilized
herein, the terin
"functionally active" refers to species displaying one or more 1c1own
functional attributes of a
full-length peptide. "Variant" refers to a polynucleotide or polypeptide
differing from the
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47
polynucleotide or polypeptide of the present invention, but retaining
essential properties
thereof. Generally, variants are overall closely similar, and in many regions,
identical to the
endogenous polynucleotide or polypeptide.
[00191] Variants of the natriuretic peptides that function as either agonists
or mimetics can
be identified by screening combinatorial libraries of mutants of the
endogenous peptide for
peptide agonist. In one embodiment, a library of variants is generated by
combinatorial
nlutagenesis at the nucleic acid level and is encoded by a gene library. A
library of variants
can be produced by, for example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of potential
sequences is
expressible as individual peptides, or alternatively, as a set of larger
fusion proteins (e.g., for
phage or mTf display) containing the set of sequences therein. There are a
variety of
methods whicll can be used to produce libraries of potential variants fiom a
degenerate
oligonucleotide sequence.
[00192] The present invention also encompasses libraries coinprising peptides
of agonists
and antagonists of natriuretic receptors. These peptides include those that
are not related in
by sequence to known natriuretic peptides.
[00193] Variants of endogenous natriuretic peptides include a sequence of at
least 6
(contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length
sufficient to allow
for specific hybridization in the case of nucleic acids or for specific
recognition of an epitope
in the case of amino acids, respectively. Variants may be full length or other
than full length,
if said variant contains a modified nucleic acid or amino acid. Variants
include, but are not
limited to, molecules comprising regions that are substantially homologous in
various
einbodiinents, of at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or preferably 95%
ainino
acid identity when: (i) compared to an ainino acid sequence of identical size;
(ii) compared to
an aligned sequence in that the alignment is done by a computer homology
prograin lcnown
within the art (e.g., Wisconsin GCG software) or (iii) the encoding nucleic
acid is capable of
hybridizing to a sequence encoding the aforementioned peptides under
stringent, moderately
stringent, or non-stringent conditions (Ausubel et al., CuiTent Protocols in
Molecular
Biology, Jolul Wiley and Sons, New York, N.Y., 1993).
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48
[00194] Variant may be produced by alteration of their sequences by
substitutions, additions
or deletions that result in functionally-equivalent molecules. Thus, the
invention includes
DNA sequences that encode substantially the saine ainino acid sequence. In
another
embodiment, one or more amino acid residues within the sequence of interest
may be
substituted by another ainino acid of a similar polarity and net charge, thus
resulting in a
silent alteration. Substitutes for an amino acid within the sequence may be
selected from
other members of the class to which the amino acid belongs. For example,
nonpolar
(hydrophobic) ainino acids include alanine, leucine, isoleucine, valine,
proline,
phenylalanine, tryptophan and methionine. Polar neutral ainino acids include
glycine, serine,
tlzreonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged
(basic) amino
acids include arginine, lysine and histidine. Negatively charged (acidic)
ainino acids include
aspartic acid and glutamic acid.
[00195] In particular einbodiments, variants, are related to animals (e.g.,
mouse, rat, pig,
cow, dog, monkey, frog), or human natriuretics. Homologs (i.e., nucleic acids
encoding
peptides derived from species other than human) or other related sequences
(e.g., paralogs)
can also be obtained by low, moderate or high stringency hybridization with
all or a portion
of the particular huinan sequence as a probe using methods well laiown in the -
art for nucleic
acid hybridization and cloning (Ausubel et al., (eds.), 1993, CuiTent
Protocols in Molecular
Biology, John Wiley and Sons, NY; and Kriegler, 1990, Gene Transfer and
Expression, A
Laboratory Manual, Stoclcton Press, NY).
[00196] The variant NP (natriuretic peptide) sequence may be fused to the N-
terininus of Tf,
the C-terininus of Tf, to both the N- and C-terinini, or inserted into one or
more of the surface
exposed loops of Tf.
Chimeric Natriuretic Peptides
[00197] The present invention also provides fusion proteins coinprising one or
more copies
of a chimeric natriuretic peptide fused to a Tf or mTf molecule. The chimeric
natriuretic
peptides may contain sequences derived from two or more different natriuretic
peptides or a
natriuretic peptide and another peptide.
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49
[00198] U.S. Patent 6,818,619 (which is herein incorporated by reference in
its entirety)
provides an isolated and purified peptide compound having natriuretic, renin-
suppressing,
diuretic and/or vasodilator activity in maminals. The peptide comprises a
compound of
formula (I):
XO-Pro-Xl-A5-Al-A3-Pro-Al-Pro-Al-A5-Pro-X1-Xl-Xl-A4 (SEQ ID NO:9)
wherein Al is Leu, Lys, Arg, His, Orn, Asn or Gln; A3 is Asp or Glu; A4 is
Lys, Arg, Orn,
Ala, Thr, Asn, or Gln; A5 is Gly, Ala, Val, Met, Leu, Norleucine or Ile; X0 is
absent or is a
peptide of from 1 to 35 amino acid residues, preferably from 1 to 25 amino
acid residues,
which peptide has a Cys residue at the C-terininus, and more preferably
residues from the N-
terininus of BNP or CNP; and X1 is Ser or Thr. Alternatively, the peptide
coinprises a
compound of fonnula (II):
X0-Pro-X1-A5 -Al-A3-Pro-Al-Pro-Al-A5 -Pro-Xl-X1-Xl-A4 -X2 (SEQ ID NO:10)
wherein A 1 is Leu, Lys, Arg, His, Orn, Asn or Gln; A3 is Asp or Glu; A4 is
Lys, Arg, Om,
Ala, Thr, Asn, or Gln; A5 is Gly, Ala, Val, Met, Leu, Norleucine or Ile; X2 is
absent or is a
peptide of from 1 to 35 amino acid residues, preferably of from 1 to 25 ainino
acid residues;
X0 is absent or is a peptide of from 1 to 35 ainino acid residues, preferably
of from 1 to 25
amino acid residues, which peptide has a Cys residue at the C-terininus, and
more preferably
residues residues from the N-tenninus of BNP or CNP; and X1 is Ser or Thr.
[00199] A preferred peptide of U.S. Patent 6,818,619 includes a chimeric
peptide which is a
41 amino acid peptide coinbining the core ring structure of BNP wit11 the C-
tenninus of DNP
(dendroaspis natriuretic peptide). Thus, a preferred coinpound of fonnula (I)
is a chimeric
peptide comprising Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-
Asp-
Arg-Ile-Se r-Ser-Ser-Ser-Gly-Leu-Gly-Cys-Pro-Ser-Leu-Arg-Asp-Pro-Arg-Pro-Asn-
Ala-Pro-
S er-Thr-Ser-Ala (SEQ ID NO: 11), or a biologically active variant or
fiaginent thereof.
Preferably, the chiineric peptide has a disulfide bridge between Cys 10 and
Cys 26. Other
preferred peptides of the invention include a 37 amino acid peptide combining
the core ring
sti-ucture of CNP with the C-tenninus of DNP. Thus, anotlzer preferred
coinpound of formula
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(I) is a chimeric peptide comprising Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-
Leu-Asp-
Arg-Ile-Gly-Ser-Met-Ser-GI y-Leu-Gly-Cys-Pro-Ser-Leu-Arg-Asp-Pro-Arg-Pro-Asn-
Ala-
Pro-Ser-Thr-Ser-Ala (SEQ ID NO: 12), or a biologically active variant or
fraginent thereof.
Preferably, the cliimeric peptide has a disulfide bridge between Cys 6 and Cys
22. Thus, XO,
if present, is preferably the N-terminus of human BNP, i.e., Ser-Pro-Lys-Met-
Val-Gln-Glu-
Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Se r-Ser-Ser-Ser-Gly-Leu-Gly-Cys
(SEQ
ID NO: 13), or the N-terminus of human CNP, i.e., Gly-Leu-Ser-Lys-Gly-Cys-Phe-
Gly-Leu-
Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gl y-Leu-Gly-Cys (SEQ ID NO: 14). Yet
another
preferred peptide includes a portion of the carboxy-terminus of DNP,
preferably which
includes the carboxy-terminal 15 ainino acids or a biologically active variant
or fragment
thereof.
[00200] As used herein, the term "biologically active" means that a peptide
has at least one
of the activities of a native natriuretic peptide.
[00201] Preferably, the fusion proteins of the present invention comprise a
chimeric -
natriuretic peptide fused to a modified transferrin exhibiting reduced
glycosylation as
coinpared to a wild-type transferrin fusion protein.
_ Methods of Using Natriuretic Peptide/mTf Fusion Proteins
[00202] Atrial natriuretic peptide is synthesized, stored, and released by
atrial myocytes in
response to atrial distension, angiotensin II stimulation, endothelin, and
sympathetic
stiinulation (beta-adrenoceptor mediated). Once it is in the circulation, its
effects are
primarily on the kidney, vascular tissue, and adrenal gland, in which its
actions lead to the
excretion of sodium and water by the kidneys and a decrease in intravascular
volume and
blood pressure. Elevated levels of ANP are found during hypervoleinic states
(elevated blood
voluine) and congestive heart failure.
[00203] ANP is involved in the long-terin regulation of sodiuin and water
balance, blood
voluine and arterial pressure. This honnone decreases aldosterone release by
the adrenal
cortex, increases glomerular filtration rate (GFR), produces natriuresis and
diuresis
(potassium sparing), and decreases renin release thereby decreasing
angiotensin II. These
actions contribute to reductions in blood volume and therefore central venous
pressure
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51
(CVP), cardiac output, and arterial blood pressure. Chronic elevations of ANP
appear to
decrease arterial blood pressure primarily by decreasing systemic vascular
resistance. The
mechanism of systemic vasodilation may involve ANP receptor-mediated
elevations in
vascular smooth muscle cGMP as well as by attenuating sympathetic vascular
tone. This
latter mechanism may involve ANP acting upon sites within the central nervous
systein as
well as through inhibition of norepinephrine release by sympathetic nerve
tenninals.
[00204] Therefore, ANP is a counter-regulatory system for the renin-
angiotensin-aldosterone
system. A class of drugs that are neutral endopeptidase (NEP) inhibitors have
been shown to
be efficacious in animal models of heart failure. These drugs inhibit neutral
endopeptidase,
the enzyme responsible for the degradation of ANP, and thereby elevate plasma
levels of
ANP. NEP inhibition is particularly effective in heart failure when the drug
is combined with
an ACE iiihibitor.
[00205] BNP is of inyocardial cell origin, and like ANP circulates in huinan
plasma (de Bold
et al., Life Sci., 28, 89 (1981); Burnett et al., Am. J. Physiol. (1984) 247;
F863). BNP is
natriuretic, renin inhibiting, vasodilating, and lusitropic (Mulcoyaina et
al., J. Clin.
Invest.(1991) 87, 1402; Yamamoto et al., Am. J. Physiol. (1996) 271, R1529;
Grantham et
al., in Natriuretic Peptides in Health and Disease, Samson W. K., Levin E. R.,
eds, Huinana
Press, pp. 309-326 (1997)).
[00206] ANP and BNP are increased in the plasma and heart during congestive
heart failure
(CHF) in huinans, and they exert important cardiorenal protective actions in
addition to
serving as serum markers for ventricular dysfunction.
[00207] The diverse actions of ANP, BNP and CNP on both the cardiovascular
system and
the kidney, as well as their roles in pathophysiological states such as heart
failure,
hypertension, and renal disease, have made the native peptides and their
analog molecules of
great interest to both clinical and basic scientists as therapeutic agents.
See, for example,
Lewicki et al. (U.S. Pat. Nos. 5,114,923, 4,804,650 and 4,757,048), Joluison
et al. (U.S. Pat.
No. 5,047,397) and Jolulson et al. (U.S. Pat. No. 4,935,492), and Wei et al.
(U.S. Pat. No.
5,583,108). U.S. Pat. No. 5,583,108 relates to a chimera of ANP and CNP,
termed vasonatrin
peptide (VNP). VNP, which includes 22 amino acids of CNP and the 5 amino acids
at the
carboxy-terminus of ANP, has arterial and venous vasodilating and natriuretic
effects.
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52
[00208] The present invention provides methods of using natriuretic peptide/Tf
or mTf
fusion proteins for the reduction of blood pressure; inhibition of cardiac
hypertrophy;
treatment of cardiovascular diseases, such as congestive heart failure and
decompensated
heart failure; enhanceinent in post surgical repair for CVD; inhibition of
aldosterone
production and release; diuresis; modulating salt excretion; treatment of
various renal
diseases which cause renal hypertrophy, such as chronic kidney disease;
inhibition of
pulmonary diseases, such as pulmonary hypertension and reduction of
coinplications
associated with pulmonary diseases; inhibit vascular cell growth and regulate
vessel tone in
the eye for various diseases sucli as diabetic retinopathy and glaucoma;
increasing the rate of
lipolysis in fat cells; and reduction of inflammation and inflammatory
mediators comprising
administering said fusion protein at a therapeutically effective dosage to a
patient in need
thereof. The dosage may be a single administration or may comprise inultiple
administrations for a time frame that results in a desired outcome. The
present invention
further provides methods of using the disclosed the natriuretic peptide/mTf
fusion proteins
for treating various other diseases and conditions.
[00209] In a further embodiment of the invention, the natriuretic peptide/Tf
or mTf fusion is
administered witll an inhibitor of proteases or peptidases that may inactivate
the natriuretic
peptide, e.g. an NEP inhibitor. The inhibitor may be adininistered at the
saine time as the
fusion protein of the invention or at a dose and frequency appropriate to
providing adequate
inhibition, e.g. the fusion protein may be adininistered once per week and the
inhibitor
adininistered daily.
Nucleic Acids
[00210] The present invention also provides nucleic acid molecules encoding
transferrin
fusion proteins comprising a transferrin protein or a portion of a transferrin
protein covalently
lii-Aced or joined to a therapeutic protein, preferably a therapeutic protein.
As discussed in
more detail below, any therapeutic protein may be used. The fusion protein may
f-urther
coinprise a linlcer region, for instance a lii-~lcer less than about 50, 40,
30, 20, or 10 ainino acid
residues. The linker can be covalently linlced to and between the transferrin
protein or
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53
portion thereof and the therapeutic protein, preferably the therapeutic
protein. Nucleic acid
molecules of the invention may be purified or not.
[00211] Host cells and vectors for replicating the nucleic acid molecules and
for expressing
the encoded fusion proteins are also provided. Any vectors or host cells may
be used,
whether prokaryotic or eukaryotic, but eukaryotic expression systems, in
particular yeast
expression systeins, may be preferred. Many vectors and host cells are known
in the art for
such purposes. It is well within the skill of the art to select an appropriate
set for the desired
application.
[00212] DNA sequences encoding transferrin, portions of transferrin and
therapeutic
proteins of interest may be cloned from a variety of genomic or cDNA libraries
known in the
art. The techniques for isolating such DNA sequences using probe-based methods
are
conventional techniques and are well known to those skilled in the art. Probes
for isolating
such DNA sequences may be based on published DNA or protein sequences (see,
for
example, Baldwin, G.S. (1993) Comparison of Transferrin Sequences from
Different Species.
Comp. Biochem. Physiol. 106B/1:203-218 and all references cited tllerein,
which are hereby
incorporated by reference in their entirety). Alternatively, the polymerase
clzain reaction
(PCR) method disclosed by Mullis et al. (U.S. Pat. No. 4,683,195) and Mullis
(U.S. Pat. No.
4,683,202), incorporated herein by reference may be used. The choice of
library and
selection of probes for the isolation of such DNA sequences is within the
level of ordinary
skill in the art.
[00213] As known in the art "similarity" between two polynucleotides or
polypeptides is
detennined by comparing the nucleotide or amino acid sequence and its
conserved nucleotide
or amino acid substitutes of one polynucleotide or polypeptide to the sequence
of a second
polynucleotide or polypeptide. Also known in the art is "identity" which means
the degree of
sequence relatedness between two polypeptide or two polynucleotide sequences
as
detennined by the identity of the match between two strings of suc11
sequences. Both identity
and similarity can be readily calculated (Computational Molecular Biology,
Lesk, A. M., ed.,
Oxford University Press, New Yorlc, 1988; Biocomputing: Infonnatics and Genome
Projects,
Smith, D. W., ed., Academic Press, New Yorlc, 1993; Computer Analysis of
Sequence Data,
Part I, Griffin, A. M., and Griffin, H. G., eds., Huinana Press, New Jersey,
1994; Sequence
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54
Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and
Sequence
Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stoclcton Press, New
York, 1991).
[00214] While there exist a number of methods to measure identity and
similarity between
two polynucleotide or polypeptide sequences, the terms "identity" and
"similarity" are well
known to skilled artisans (Sequence Analysis in Molecular Biology, von Heinje,
G.,
Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M
Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J.
Applied Math.,
48: 1073 (1988). Methods commonly employed to determine identity or similarity
between
two sequences include, but are not limited to those disclosed in Guide to Huge
Computers,
Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and
Lipman, D.,
SIAM J. Applied Math. 48:1073 (1988).
[00215] Preferred methods to determine identity are designed to give the
largest match
between the two sequences tested. Methods to deterinine identity and
similarity are codified
in computer programs. Preferred computer program methods to determine identity
and -
similarity between two sequences include, but are not limited to, GCG program
package
(Devereux, et al., Nucl. Acid Res. 12(l):387 (1984)), BLASTP, BLASTN, FASTA
(Atschul,
et al., J. Mol. Biol. 215:403 (1990)). The degree of similarity or identity
referred to above is
deterinined as the degree of identity between the two sequences, often
indicating a derivation
of the first sequence from the second. The degree of identity between two
nucleic acid
sequences may be determined by means of coinputer programs lcnown in the art
such as GAP
provided in the GCG program package (Needleinan and Wunsch J. Mol. Biol.
48:443-453
(1970)). For purposes of detennining the degree of identity between two
nucleic acid
sequences for the present invention, GAP is used with the following settings:
GAP creation
penalty of 5.0 and GAP extension penalty of 0.3.
Codon Optimization
[00216] The degeneracy of the genetic code pennits variations of the
nucleotide sequence of
a transferrin protein and/or therapeutic protein of interest, while still
producing a polypeptide
having the identical amino acid sequence as the polypeptide encoded by the
native DNA
sequence. The procedure, known as "codon optimization" (described in U.S.
Patent
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5,547,871 which is incorporated herein by reference in its entirety) provides
one with a
means of designing such an altered DNA sequence. The design of codon optimized
genes
should take into account a variety of factors, including the frequency of
codon usage in an
organism, nearest neighbor frequencies, RNA stability, the potential for
secondary structure
forination, the route of synthesis and the intended future DNA manipulations
of that gene. In
particular, available methods may be used to alter the codons encoding a given
fusion protein
with those most readily recognized by yeast when yeast expression systems are
used.
[00217] The degeneracy of the genetic code peinlits the same amino acid
sequence to be
encoded and translated in many different ways. For example, leucine, serine
and arginine are
each encoded by six different codons, while valine, proline, threonine,
alanine and glycine
are each encoded by four different codons. However, the frequency of use of
such
synonymous codons varies from genome to genome ainong eukaryotes and
prokaryotes. For
example, synonymous codon-choice patterns ainong mammals are very similar,
while
evolutionarily distant organisms such as yeast (such as S. cerevisiae),
bacteria (such as E.
coli) and insects (such as D. inelanogaster) reveal a clearly different
pattern of genomic
codon use frequencies (Grantham, R., et al., Nucl. Acid Res., 8, 49-62 (1980);
Grantham, R.,
et al., Nucl. Acid Res., 9, 43-74 (1981); Maroyaina, T., et al., Nucl. Acid
Res., 14, 151-197
(1986); Aota, S., et al., Nucl. Acid Res., 16, 315-402 (1988); Wada, K., et
al., Nucl. Acid
Res., 19 Supp., 1981-1985 (1991); Eurland, C. G., FEBS Lett., 285, 165-169
(1991)). These
differences in codon-choice patterns appear to contribute to the overall
expression levels of
individual genes by modulating peptide elongation rates. (Kurland, C. G., FEBS
Lett., 285,
165-169 (1991); Pedersen, S., EMBO J., 3, 2895-2898 (1984); Sorensen, M. A.,
J. Mol. Biol.,
207, 365-377 (1989); Randall, L. L., et al., Eur. J. Biochein., 107, 375-379
(1980); Curran, J.
F., and Yaras, M., J. Mol. Biol., 209, 65-77 (1989); Varemie, S., et al., J.
Mol. Biol., 180,
549-576 (1984), Varenne, S., et al., J. Mol, Biol., 180, 549-576 (1984);
Garel, J.-P., J. Theor.
Biol., 43, 211-225 (1974); Ilcemura, T., J. Mol. Biol., 146, 1-21 (1981);
Ikeinura, T., J. Mol.
Biol., 151, 389-409 (1981)).
The preferred codon usage frequencies for a synthetic gene should reflect the
codon usages of
nuclear genes derived froin the exact (or as closely related as possible)
genoine of the
cell/organism that is intended to be used for recombinant protein expression,
particularly that
of yeast species. As discussed above, in one preferred einbodiinent the
1luinan Tf sequence is
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56
codon optimized, before or after modification as herein described for yeast
expression as may
be the therapeutic protein nucleotide sequence(s).
Vectors
[00218] Expression units for use in the present invention will generally
comprise the
following elements, operably linked in a 5' to 3' orientation: a
transcriptional promoter, a
secretory signal sequence, a DNA sequence encoding a modified. Tf fusion
protein
comprising transferrin protein or a portion of a transferrin protein joined to
a DNA sequence
encoding a therapeutic protein or peptide of interest and a transcriptional
terminator. As
discussed above, any arrangement of the therapeutic protein or peptide fused
to or within the
Tf portion may be used in the vectors of the invention. The selection of
suitable promoters,
signal sequences and terminators will be deterinined by the selected host cell
and will be
evident to one skilled in the art and are discussed more specifically below.
[00219] Suitable yeast vectors for use in the present invention are described
in U.S. Patent
6,291,212 and include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-
1039, 1978),
YEp13 (Broach et al., Gene 8: 121-133, 1979), pJDB249 and pJDB219 (Beggs,
Nature
275:104-108, 1978), pPPC0005, pSeCHSA, pScNHSA, pC4 and derivatives thereof.
Useful
yeast plasmid vectors also include pRS403-406, pRS413-416 and the Pichia
vectors available
from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Plasmids pRS403,
pRS404,
pRS405 and pRS406 are Yeast Integrating plasmids (Ylps) and incorporate the
yeast
selectable marlcers HIS3, TRPI, LEU2 and URA3. Plasmids pRS413-41.6 are Yeast
Centromere plasmids (YCps).
[00220] Such vectors will generally include a selectable marker, which may be
one of any
number of genes that exhibit a dominant phenotype for which a phenotypic assay
exists to
enable transfonnants to be selected. Preferred selectable markers are those
that compleinent
host cell auxotrophy, provide antibiotic resistance or enable a cell to
utilize specific carbon
sources, and include LEU2 (Broach et al. ibid.), URA3 (Botstein et al., Gene
8: 17, 1979),
HIS3 (Struhl et al., ibid.) or POT] (Kawasalci and Bell, EP 171,142). Other
suitable
selectable markers include the CAT gene, which confers chlorainphenicol
resistance on yeast
cells. Preferred promoters for use in yeast include promoters from yeast
glycolytic genes
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57
(Hitzeinan et al., J Biol. Chem. 225: 12073-12080, 1980; Alber and Kawasaki,
J. Mol. Appl.
Genet. 1: 419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or alcohol
dehydrogenase
genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals,
Hollaender et
al., (eds.), p. 355, Plenum, N.Y., 1982; Ammerer, Meth. Enzymol. 101: 192-201,
1983). In
this regard, particularly preferred promoters are the TPII promoter
(Kawasalci, U.S. Pat. No.
4,599,311) and the ADH2-4C (see U.S. Patent 6,291,212 promoter (Russell et
al., Nature 304:
652-654, 1983). The expression units may also include a transcriptional
terminator. A
preferred transcriptional tei7ninator is the TPII terininator (Alber and
Kawasaki, ibid.). Other
preferred vectors and preferred components such as promoters and terminators
of a yeast
expression system are disclosed in European Patents EP 0258067, EP 0286424,
EP0317254,
EP 0387319, EP 0386222, EP 0424117, EP 0431880, and EP 1002095; European
Patent
Publications EP 0828759, EP 0764209, EP 0749478, and EP 0889949; PCT
Publication WO
00/44772 and WO 94/04687; and U.S. Patents 5,739,007; 5,637,504; 5,302,697;
5,260,202;
5,667,986; 5,728,553; 5,783,423; 5,965,386; 6150,133; 6,379,924; and
5,714,377; which are
herein incorporated by reference in their entirety.
[00221] In addition to yeast, modified fusion proteins of the present
invention can be
expressed in filamentous fungi, for exainple, strains of the fungi
Aspergillus. Examples of
useful promoters include those derived from Aspergillus nidulans glycolytic
genes, such as
the adh3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1985) and the tpiA
promoter.
An example of a suitable tenninator is the adh3 terminator (McIs'-light et
al., ibid.). The
expression units utilizing such components may be cloned into vectors that are
capable of
insertion into the chromosomal DNA ofAspergillus, for exainple.
[00222] Mainmalian expression vectors for use in carrying out the present
invention will
include a promoter capable of directing the transcription of the modified Tf
fusion protein.
Preferred promoters include viral promoters and cellular promoters. Preferred
viral
promoters include the major late promoter from adenovirus 2(Kaufinan and
Sharp, Mol.
Cell. Biol. 2: 1304-13199, 1982) and the SV40 promoter (Subrainani et al.,
Mol. Cell. Biol.
1: 854-864, 1981). Preferred cellular promoters include the mouse
metallothionein 1
promoter (Palmiter et al., Science 222: 809-814, 1983) and a mouse V6 (see
U.S. Patent
6,291,212) promoter (Grant et al., Nuc. Acids Res. 15: 5496, 1987). A
particularly preferred
promoter is a mouse VH (see U.S. Patent 6,291,212) promoter (Loh et al.,
ibid.). Such
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58
expression vectors may also contain a set of RNA splice sites located
downstream from the
promoter and upstream from the DNA sequence encoding the transferrin fusion
protein.
Preferred RNA splice sites may be obtained from adenovirus and/or
iminunoglobulin genes.
[00223] Also contained in the expression vectors is a polyadenylation signal
located
downstream of the coding sequence of interest. Polyadenylation signals include
the early or
late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the
polyadenylation
signal from the adenovirus 5 E1B region and the human growth hormone gene
terininator
(DeNoto et al., Nucl. Acid Res. 9: 3719-3730, 1981). A particularly preferred
polyadenylation signal is the VH (see U.S. Patent 6,291,212) gene terminator
(Loh et al.,
ibid.). The expression vectors may include a noncoding viral leader sequence,
such as the
adenovirus 2 tripartite leader, located between the promoter and the RNA
splice sites.
Preferred vectors may also include enhancer sequences, such as the SV40
enhancer and the
mouse :(see U.S. Patent 6,291,212) enhancer (Gillies, Cel133: 717-728, 1983).
Expression
vectors may also include sequences encoding the adenovirus VA RNAs.
Transformation
[00224] Techniques for transforming fungi are well known in the literature,
and have been
described, for instance, by Beggs (ibid.), Hinnen et al. (Proc. Natl. Acad.
Sci. USA 75: 1929-
1933, 1978), Yelton et al., (Proc. Natl. Acad. Sci. USA 81: 1740-1747, 1984),
and Russell
(Nature 301: 167-169, 1983). Other techniques for introducing cloned DNA
sequences into
fungal cells, such as electroporation (Becker and Guarente, Methods in
Enzyinol. 194: 182-
187, 1991) may be used. The genotype of the host cell will generally contain a
genetic defect
that is coinplemented by the selectable marker present on the expression
vector. Choice of a
particular host and selectable marker is well within the level of ordinary
skill in the art.
[00225] Cloned DNA sequences comprising modified Tf fusion proteins of the
invention
may be introduced into cultured mainmalian cells by, for example, calcium
phosphate-
mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson,
Somatic Cell
Genetics 7: 603, 1981; Grahain and Van der Eb, Virology 52: 456, 1973.) Other
tecluliques
for introducing cloned DNA sequences into mammalian cells, sucll as
electroporation
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59
(Neumaim et al., EMBO J. 1: 841-845, 1982), or lipofection may also be used.
In order to
identify cells that have integrated the cloned DNA, a selectable marker is
generally
introduced into the cells along with the gene or cDNA of interest. Preferred
selectable
markers for use in cultured mammalian cells include genes that confer
resistance to drugs,
such as neomycin, hygromycin, and methotrexate. The selectable marker may be
an
amplifiable selectable marlcer. A preferred amplifiable selectable inarlcer is
the DHFR gene.
A particularly preferred amplifiable marlcer is the DHFR' (see U.S. Patent
6,291,212) cDNA
(Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: 2495-2499, 1983).
Selectable
markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth
Publishers,
Stoneham, Mass.) and the choice of selectable markers is well within the level
of ordinary
skill in the art.
Host Cells
[00226] The present invention also includes a cell, preferably a yeast cell
transformed to
express a modified transferrin fusion protein of the invention. In addition to
the transformed
host cells themselves, the present invention also includes a culture of those
cells, preferably a
monoclonal (clonally homogeneous) culture, or a culture derived from a
monoclonal culture,
in a nutrient medium. If the polypeptide is secreted, the medium will contain
the polypeptide,
with the cells, or without the cells if they have been filtered or centrifuged
away.
[00227] Host cells for use in practicing the present invention include
eukaryotic cells, and in
some cases prokaryotic cells, capable of being transfonned or transfected with
exogenous
DNA and grown in culture, such as cultured mammalian, insect, fungal, plant
and bacterial
cells.
[00228] Fungal cells, including species of yeast (e.g., Saccharoinyces spp.,
Schizosaccharonayces spp., Pichia spp.) may be used as host cells within the
present
invention. Exalnples of fungi including yeasts contemplated to be useful in
the practice, of
the present invention as hosts for expressing the, transferrin fusion protein
of the inventions
are Pichia (some species of which were fonnerly classified as Hansenula),
Saccharofnyces,
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Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora,
Schizosaccharomyces,
Citeromyces, Pachysolen, Zygosaccharomyces, Debaroinyces, Tirichodef-ma,
Ceplzalosporium, Hun2icola, Mucor, Neurospora, Yarrowia, Metschunikowia,
Rhodosporidiuzn, Leucosporidium, Botyyoascus, Sporidiobolus, Endomycopsis, and
the lilce.
Examples of Sacchaz-omyces spp. are S. cez~evisiae, S. italicus and S. youxii.
Examples of
Kluyves=oynyces spp. are K. fragilis, K. lactis and K. marxianus. A suitable
Torulaspora
species is T. delbrueckii. Examples of Pichia spp. are P. angusta (formerly H.
polymorph.a),
P. an.oinala (fonnerly H. anomala) and P. pastoris.
[00229] Particularly useful host cells to produce the Tf fusion proteins of
the invention are
the methylotrophic Pichia pastoris (Steinlein et al. (1995) PYotein Express.
Purif. 6:619-
624). Pichia pastoris has been developed to be an outstanding host for the
production of
foreign proteins since its alcohol oxidase promoter was isolated and cloned;
its
transformation was first reported in 1985. P. pastof=is can utilize methanol
as a carbon source
in the absence of glucose. The P. pastoris expression system can use the
methanol-induced
alcohol oxidase (AOX7) promoter, which controls the gene that codes for the
expression of
alcohol oxidase, the enzyme which catalyzes the first step in the metabolism
of methanol.
This promoter has been characterized and incorporated into a series of P.
pastoris expression
vectors. Since the proteins produced in P. pastoris are typically folded
colTectly and secreted
into the mediuin, the fennentation of genetically engineered P. pastoris
provides an excellent
alternative to E. coli expression systems. A ilumber of proteins have been
produced using
this system, including tetanus toxin fraginent, Boi datella peytussis
pertactin, huinan serum
albumin and lysozyine.
[002301 Strains of the yeast Saccharomyces ceyevisiae are another preferred
host. In a
preferred einbodiment, a yeast cell, or more specifically, a Sacchaf ornyces
cerevisiae host
cell that contains a genetic deficiency in a gene required for asparagine-
linked glycosylation
of glycoproteins is used. S. cer=evisiae host cells having sucli defects may
be prepared using
standard tecluliques of mutation and selection, although many available yeast
strains have
been modified to prevent or reduce glycosylation or hypennamiosylation. Ballou
et al. (J.
Biol. Chem. 255: 5986-5991, 1980) have described the isolation of
rnarmoprotein
biosyntliesis inutants that are defective in genes wliich affect asparagine-
linked glycosylation.
Gentzsch and Tanner (Glycobiology 7:481-486, 1997) have described a family of
at least six
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61
genes (PMTI-6) encoding enzymes responsible for the first step in 0-
glycosylation of
proteins in yeast. Mutants defective in one or more of these genes show
reduced 0-linked
glycosylation and/or altered specificity of 0-glycosylation.
[00231] To optiinize production of the heterologous proteins, it is also
preferred that the host
strain carries a mutation, such as the S. cef-evisiae pep4 mutation (Jones,
Genetics 85: 23-33,
1977), which results in reduced proteolytic activity. Host strains containing
mutations in
other protease encoding regions are particularly useful to produce large
quantities of the Tf
fusion proteins of the invention.
[00232] Host cells containing DNA constructs of the present invention are
grown in an
appropriate growth medium. As used herein, the term "appropriate growth
medium" means a
medium containing nutrients required for the growth of cells. Nutrients
required for cell
growth may include a carbon source, a nitrogen source, essential amino acids,
vitainins,
minerals and growth factors. The growth medium will generally select for cells
containing
the DNA construct by, for example, drug selection or deficiency in an
essential nutrient
which is coinplemented by the selectable inarker on the DNA construct or co-
transfected with
the DNA construct. Yeast cells, for example, are preferably grown in a
chemically defined
medium, comprising a carbon source, e.g. sucrose, a non-amino acid nitrogen
source,
inorganic salts, vitamins and essential ainino acid suppleinents. The pH of
the medium is
preferably maintained at a pH greater than 2 and less than 8, preferably at pH
5.5-6.5.
Methods for maintaining a stable pH include buffering and constant pH control.
Preferred
buffering agents include succinic acid and Bis-Tris (Sigina Chemical Co., St.
Louis, Mo.).
Yeast cells having a defect in a gene required for asparagine-linked
glycosylation are
preferably grown in a medium containing an osmotic stabilizer. A preferred
osmotic
stabilizer is sorbitol suppleinented into the medium at a concentration
between 0.1 M and 1.5
M., preferably at 0.5 M or 1.0 M.
[00233] Cultured maininalian cells are generally grown in commercially
available seruin-
containing or seruin-free media. Selection of a medium appropriate for the
particular cell line
used is within the level of ordinary skill in the art. Transfected maminalian
cells are allowed
to grow for a period of time, typically 1-2 days, to begin expressing the DNA
sequence(s) of
interest. Drug selection is then applied to select for growth of cells that
are expressing the
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62
selectable marker in a stable fashion. For cells that have been transfected
with an amplifiable
selectable marker the drug concentration may be increased in a stepwise manner
to select for
increased copy nuinber of the cloned sequences, thereby increasing expression
levels.
[00234] Baculovirus/insect cell expression systeins may also be used to
produce the modified
Tf fusion proteins of the invention. The BacPAI,'-TM Baculovirus Expression
System (BD
Biosciences (Clontech)) expresses recoinbinant proteins at high levels in
insect host cells.
The target gene is inserted into a transfer vector, which is cotransfected
into insect host cells
with the linearized BacPAK6 viral DNA. The BacPAK6 DNA is inissing an
essential portion
of the baculovirus genome. When the DNA recombines with the vector, the
essential eleinent
is restored and the target gene is transferred to the baculovirus genome.
Following
recombination, a few viral plaques are picked and purified, and the
recoinbinant phenotype is
verified. The newly isolated recombinant virus can then be amplified and used
to infect
insect cell cultures to produce large amounts of the desired protein.
[00235] Tf fusion proteins of the present invention may also be produced using
transgenic
plants and animals. For example, sheep and goats can make the therapeutic
protein in their
milk. Or tobacco plants can include the protein in their leaves. Both
transgenic plant and
animal production of proteins comprises adding a new gene coding the fusion
protein into the
genome of the organism. Not only can the transgenic organism produce a new
protein, but it
can also pass this ability onto its offspring.
Secretory Signal Sequences
[00236] The terms "secretory signal sequence" or "signal sequence" or
"secretion leader
sequence" are used interchangeably and are described, for example in U.S. Pat.
6,291,212
and U.S. Pat 5,547,871, both of which are herein incorporated by reference in
their entirety.
Secretory signal sequences or signal sequences or secretion leader sequences
encode
secretory peptides. A secretory peptide is an amino acid sequence that acts to
direct the
secretion of a mature polypeptide or protein from a cell. Secretory peptides
are generally
characterized by a core of hydrophobic amino acids and are typically (but not
exclusively)
found at the amino tennini of newly syntliesized proteins. Very often the
secretory peptide is
cleaved from the mature protein during secretion. Secretory peptides may
contain processing
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sites that allow cleavage of the signal peptide from the mature protein as it
passes through the
secretory pathway. Processing sites may be encoded within the signal peptide
or may be
added to the signal peptide by, for example, in vitro inutagenesis.
[00237] Secretory peptides may be used to direct the secretion of modified Tf
fusion
proteins of the invention. One such secretory peptide that may be used in
coinbination with
other secretory peptides is the alpha mating factor leader sequence. Secretory
signal
sequences or signal sequences or secretion leader sequences are required for a
complex series
of post-translational processing steps which result in secretion of a protein.
If an intact signal
sequence is present, the protein being expressed enters the lumen of the rough
endoplasmic
reticulum and is then transported through the Golgi apparatus to secretory
vesicles and is
finally transported out of the cell. Generally, the signal sequence
iminediately follows the
initiation codon and encodes a signal peptide at the ainino-terminal end of
the protein to be
secreted. In most cases, the signal sequence is cleaved off by a specific
protease, called a
signal peptidase. Preferred signal sequences iinprove. the processing and
export efficiency of
recombinant protein expression using viral, mammalian or yeast expression
vectors.
[00238] In one embodiment, the native Tf signal sequence may be used to
express and
secrete fusion proteins of the present invention. Since transferrin molecules
exist in various
types of secretions such as blood, tears, and milk, there are many different
transferrin signal
peptides. For exainple, the transferrin signal peptide could be from seruin
transferrin,
lactotransferrin, or melanotransferrin. The native transferrin signal peptide
also could be
from various species such as insects, maminals, fish, frog, duck, chicken, or
other species.
Preferably, the signal peptide is from a mammalian transferrin molecule. More
preferably,
the signal peptide is from human serum transferrin. The table below
suinmarizes the signal
peptide sequences from various mainmalian transferrin molecules
(http://www. chatham.
edu/undergraduate/bio/lainbert/transfeiTiii/signal.Iltm).
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Signal Peptide Sequences (from GenBank entries)
Species Type Sequence Location in immature
protein, SEQ ID NO:
Mammals
Bos taurus (cow) serum MRPAVRALLA CAVLGLCLA 1-19, SEQ ID NO: 107
E uus caballus (horse) serum MRLAIRALLA CAVLGLCLA 1-19, SEQ ID NO: 108
Homo sapiens (human) serum MRLAVGALLV CAVLGLCLA amino acids 1-19 of SEQ ID
NO:2
Mus musculus (mouse) serum MRLTVGALLA CAALGLCLA 1-19, SEQ ID NO: 109
Or ctola us cuniculus serum MRLAAGALLA CAALGLCLA 1-19, SEQ ID NO: 110
(rabbit)
Rattus norve icus (rat) serum MRFAVGALLA CAALGLCLA 1-19, SEQ ID NO: 111
Bos taurus (cow) lacto MKLFVPALLS LGALGLCLA 1-19; SEQ ID NO: 112
Bubalus bubalis (buffalo) lacto MKLFVPALLS LGALGLCLA 1-19, SEQ ID NO: 113
Camelus dromedaries lacto MKLFFPALLS LGALGLCLA 1-19, SEQ ID NO: 1-14
(camel)
Ca ra hircus (goat) lacto MKLFVPALLS LGALGLCLA 1-19, SEQ ID NO: 115
E uus caballus (horse) lacto LGLCLA 1-6, SEQ ID NO: 116
(partial sequence)
Mus musculus (mouse) lacto MRLLIPSLIF LEALGLCLA 1-19, SEQ ID NO: 117
Sus scrofa (pig) lacto MKLFIPALLF LGTLGLCLA 1-19, SEQ ID NO: 118
Sus scrofa (pig) ica MRLAFCVLLC AGSLGLCLA 1-19, SEQ ID NO: 119
Homo sapiens (human) melano MRGPSGALWL LLALRTVLG 1-19, SEQ ID NO: 120
Mus musculus (mouse) melano MRLLSVTFWL LLSLRTVVC 1-19, SEQ ID NO: 121
Or ctola us cuniculus melano MRCRSAAMWI FLALRTALG 1-19, SEQ ID NO: 122
(rabbit) (by inference) Not available in GenBank
description
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[00239] In another einbodiment, the signal peptides are from variant or
modified transferrin
molecules that have functionally active signal peptides. Additionally, the
signal peptides are
variant or modified forms of transferrin signal peptides that retain the
ability to transport a
transferrin fusion protein of the present invention across the cell membrane
and then to
process the fusion protein.
[00240] In another embodiment, the transferrin derived signal sequence may be
used to
secrete a heterologous protein, for instance, any protein of interest that is
heterologous to the
Tf signal sequence may be expressed and secreted using a Tf signal. In
particular, a Tf signal
sequence may be used to secrete proteins from recombinant yeast. Preferably,
the signal
peptide is from human serum transferrin (nL, amino acids 1-19 of SEQ ID NO:
2).
[00241] In order to ensure efficient removal of the signal sequence, in some
cases it may be
preferable to include a short pro-peptide sequence between the signal sequence
and the
mature protein in which the C-terminal portion of the pro-peptide comprises a
recognition
site for a protease, such as the yeast Kex2p protease. Preferably, the pro-
peptide sequence is
about 2-12 amino acids in length, more preferably about 4-8 amino acids in
length.
Examples of such pro-peptides are Arg-Ser-Leu-Asp-Lys-Arg (SEQ ID NO: 125, Arg-
Ser-
Leu-Asp-Arg-Arg (SEQ ID NO: 126), Arg-Ser-Leu-Glu-Lys-Arg (SEQ ID NO: 127),
and
Arg-Ser-Leu-Glu-Arg-Arg (SEQ ID NO: 128).
Linkers
[00242] The Tf moiety and the therapeutic protein of the modified transferrin
fusion proteins
of the invention can be fused directly or using a linker peptide of various
lengths to provide
greater physical separation and allow more spatial mobility between the fused
proteins and
thus maxiinize the accessibility of the therapeutic protein, for instance, for
binding to its
cognate receptor. The linker peptide may consist of amino acids that are
flexible or more
rigid. For example, a linker such as but not limited to a poly-glycine stretch
may be used.
The liiilcer can be less than about 50, 40, 30, 20, 10, or 5 ainino acid
residues. The linker can
be covalently lii-dced to and between the transferrin protein or portion
thereof and the
therapeutic protein, such as the natriuretic peptide.
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[00243] The present invention provides long flexible linkers, short flexible
linkers, and rigid
linkers. Exainples of long flexible linkers include glucagon-like peptide 2
(GLP-2),
(SGGG)2(SEQ ID NO: 131), (SGGG)3 (SEQ ID NO: 132), and (SGGG)n (SEQ ID NO:
133), (GGGS)2 (SEQ ID NO: 134), (GGGS)3 (SEQ ID NO: 135), and (GGGS)n (SEQ ID
NO: 136), (SSSG)2 (SEQ ID NO: 137) (SSSG)3 (SEQ ID NO: 138) and (SSSG)n (SEQ
ID
NO: 139) wllerein n is an integer greater than 3. Short flexible linkers
include S, SS, and
SSG. Examples of short linkers include one Ser residue, two Ser residues, or
the peptide Ser-
Ser-Gly, or alteinatively one Gly residue, two Gly residues, three Gly
residues or the peptide
Gly-Gly-Gly-Ser (SEQ ID NO: 140). Examples of rigid linkers include PE, PEA,
PEAPTD
(SEQ ID NO: 141), (PEAPTD)2 (SEQ ID NO: 142), (PEAPTD)3 (SEQ ID NO: 143), or
(PEAPTD)õ (SEQ ID NO: 144), wherein n is an integer. The present invention
also provides
the IgG hinge linker (SEQ ID NO: 145-147), the CEx linker (SSGAPPPS (C-
terminal
extension to Exendin-4) (SEQ ID NO: 148)), the IgG hinge linker in conjunction
with the
PEAPTD linker (SEQ ID NOS: 149-158) and the IgG hinge linker in conjunction
with the
CEx linker (SEQ ID NOS: 159-164).
Detection of Tf Fusion Proteins
[00244] Assays for detection of biologically active modified transferrin-
fusion protein may
include Western transfer, protein blot or colony filter as well as activity
based assays that
detect the fusion protein coinprising transferrin and therapeutic protein. A
Western transfer
filter may be prepared using the method described by Towbin et al. (Proc.
Natl. Acad. Sci.
USA 76: 4350-4354, 1979). Briefly, sainples are electrophoresed in a sodiuin
dodecylsulfate
polyacrylamide gel. The proteins in the gel are electrophoretically
transferred to
nitrocellulose paper. Protein blot filters may be prepared by filtering
supernatant sainples or
concentrates through nitrocellulose filters using, for example, a Minifold
(Schleicher &
Schuell, Keene, N.H.). Colony filters may be prepared by growing colonies on a
nitrocellulose filter that has been laid across an appropriate growtll medium.
In this method,
a solid medium is preferred. The cells are allowed to grow on the filters for
at least 12 llours.
The cells are removed from the filters by washing witlz an appropriate buffer
that does not
remove the proteins bound to the filters. A prefeiTed buffer coinprises 25 mM
Tris-base, 19
mM glycine, pH 8.3, 20% methanol.
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[00245] Transferrin fusion proteins of the present invention may be labeled
with a
radioisotope or other imaging agent and used for in vivo diagnostic purposes.
Preferred
radioisotope imaging agents include iodine-125 and technetiuin-99, with
technetiuin-99 being
particularly preferred. Methods for producing protein-isotope conjugates are
well known in
the art, and are described by, for example, Eclcelman et al. (U.S. Pat. No.
4,652,440), Parker
et al. (WO 87/05030) and Wilber et al. (EP 203,764). Alternatively, the
transferrin fusion
proteins may be bound to spin label enhancers and used for magnetic resonance
(MR)
imaging. Suitable spin label enhancers include stable, sterically hindered,
free radical
coinpounds such as nitroxides. Methods for labeling ligands for MR imaging are
disclosed
by, for exainple, Coffinan et al. (U.S. Pat. No. 4,656,026).
[00246] Detection of a transferrin fusion protein of the present invention can
be facilitated
by coupling (i.e., physically linking) the therapeutic protein to a detectable
substance.
Examples of detectable substances include various enzyines, prosthetic groups,
fluorescent
materials, luminescent materials, bioluminescent materials, aild radioactive
materials.
Examples of suitable enzymes include horseradish peroxidase, alkaline
phosphatase, 0-
galactosidase, or acetylcholinesterase; examples of suitable prosthetic group
complexes
include streptavidin/biotin and avidin/biotin; exainples of suitable
fluorescent materials
include uinbelliferone, fluorescein, fluorescein isothiocyanate, rhodainine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luininescent material includes luminol; examples of bioluininescent materials
include
luciferase, luciferin, and aequorin, and exainples of suitable radioactive
material include 125I1131I, 35S or 3H.
[00247] In one einbodiment where one is assaying for the ability of a
transferrin fusion
protein of the invention to bind or coinpete with an antigen for binding to an
antibody,
various iininunoassays known in the art can be used, including but not limited
to, conipetitive
and non-competitive assay systeins using techniques such as
radioiminunoassays, ELISA
(enzyine linlced iininunosorbent assay), sandwich immunoassays,
iinmunoradiometric assays,
gel diffusion precipitation reactions, iminunodiff-usion assays, in situ
iminunoassays (using
colloidal gold, enzyine or radioisotope labels, for example), western blots,
precipitation
reactions, agglutination assays (e.g., gel agglutination assays), coinpleinent
fixation assays,
inununofluorescence assays, protein A assays, and immunoelectrophoresis
assays, etc. In
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one embodiment, the binding of the transferrin fusion protein is detected by
detecting a label
on the transferrin fusion protein. In another embodiment, the transferrin
fusion protein is
detected by detecting binding of a secondary antibody or reagent that
interacts with the
transferrin fusion protein. In a further embodiment, the secondary antibody or
reagent is
labeled. Many means are known in the art for detecting binding in an
iimnunoassay and are
within the scope of the present invention.
[00248] Fusion proteins of the invention may also be detected by assaying for
the activity of
the tlierapeutic protein moiety. Specifically, transferrin fusion proteins of
the invention may
be assayed for functional activity (e.g., biological activity or therapeutic
activity) using
assays known to one of ordinary skill in the art. Additionally, one of skill
in the art may
routinely assay fragments of a therapeutic protein corresponding to a
therapeutic protein
portion of a fusion protein of the invention, for activity using well-known
assays. Further,
one of slcill in the art may routinely assay fragments of a modified
transferrin protein for
activity using assays known in the art.
[00249] For example, in one einbodiment where one is assaying for the ability
of a
transferrin fusion protein of the invention to bind or coinpete with a
therapeutic protein for
binding to an anti-therapeutic polypeptide antibody and/or anti-transferrin
antibody, various
immunoassays known in the art can be used, including but not limited to,
coinpetitive and
non-coinpetitive assay systems using techniques such as radioimmunoassays,
ELISA
(enzyine linked iinmunosorbent assay), sandwich iininunoassays,
iininunoradiometric assays,
gel diffusion precipitation reactions, immunodiffusion assays, in sitat
immunoassays (using
colloidal gold, enzyine or radioisotope labels, for exainple), western blots,
precipitation
reactions, agglutination assays (e.g., gel agglutination assays), compleinent
fixation assays,
immunofluorescence assays, protein A assays, and iinmunoelectrophoresis
assays, etc. In
one einbodiment, antibody binding is detected by detecting a label on the
primary antibody.
In another einbodiment, the primary antibody is detected by detecting binding
of a secondary
antibody or reagent to the primary antibody. In a f-urther einbodiment, the
secondary
antibody is labeled. Many means are known in the art for detecting binding in
an
iminunoassay and are within the scope of the present invention.
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[00250] In a further embodiment, where a binding partner (e.g., a receptor or
a ligand) of a
therapeutic protein is identified, binding to that binding partner by a
transferrin fusion protein
containing that therapeutic protein as the therapeutic protein portion of the
fusion can be
assayed, e.g., by means well-known in the art, such as, for example, reducing
and non-
reducing gel chromatography, protein affinity chromatography, and affinity
blotting. Other
methods will be known to the skilled artisan and are within the scope of the
invention.
Production of Fusion Proteins
[00251] The present invention further provides methods for producing a
modified fusion
protein of the invention using nucleic acid molecules herein described. In
general terms, the
production of a recombinant form of a protein typically involves the following
steps.
[00252] A nucleic acid molecule is first obtained that encodes a transferrin
fusion protein of
the invention. The nucleic acid molecule is then preferably placed in operable
linkage with
suitable control sequences, as described above, to form an expression unit
containing the
protein open reading fraine. The expression unit is used to transform a
suitable host and the
transformed host is cultured under conditions that allow the production of the
recoinbinant
protein. Optionally the recombinant protein is isolated from the medium or
from the cells;
recovery and purification of the protein may not be-necessary in some
instances where some
impurities may be tolerated.
[00253] Eacli of the foregoing steps can be accomplished in a variety of ways.
For exainple,
the construction of expression vectors that are operable in a variety of hosts
is accomplished
using appropriate replicons and control sequences, as set forth above. The
control sequences,
expression vectors, and transformation metllods are dependent on the type of
host cell used to
express the gene and were discussed in detail earlier and are otherwise known
to persons
skilled in the art. Suitable restriction sites can, if not norinally
available, be added to the ends
of the coding sequence so as to provide an excisable gene to insert into these
vectors. A
skilled artisan can readily adapt any host/expression system lcliown in the
art for use with the
nucleic acid molecules of the invention to produce a desired recombinant
protein.
[00254] As discussed above, any expression system may be used, including
yeast, bacterial,
aniinal, plant, eukaryotic and prokaryotic systems. In some embodiinents,
yeast, mainmalian
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cell culture and transgenic animal or plant production systems are preferred.
In other
embodiments, yeast systems that have been modified to reduce native yeast
glycosylation,
hyper-glycosylation or proteolytic activity may be used.
Isolation/Purification of Transferrin Fusion Proteins
[00255] Secreted, biologically active transferrin fusion proteins may be
isolated from the
medium of host cells grown under conditions that allow the secretion of the
biologically
active fusion proteins. The cell material is removed from the culture mediuin,
and the
biologically active fusion proteins are isolated using isolation techniques
known in the art.
Suitable isolation techniques include precipitation and fiactionation by a
variety of
chromatographic methods, including gel filtration, ion exchange chromatography
and affinity
chromatography.
[00256] A particularly preferred purification method is affinity
chromatography on an iron
binding or metal chelating column or an immunoaffinity chromatography using an
antibody
directed against the transferrin or therapeutic protein of the polypeptide
fusion. The antibody
is preferably irrimobilized or attached to a solid support or substrate. A
particularly preferred
substrate is CNBr-activated Sepharose (Pharmacia LKB Technologies, Inc.,
Piscataway,
N.J.). By this inethod, the medium is combined with the antibody/substrate
under conditions
that will allow binding to occur. The complex may be washed to remove unbound
material,
and the transferrin fusion protein is released or eluted through the use of
conditions
unfavorable to complex forination. Particularly useful methods of elution
include changes in
pH, wherein the immobilized antibody has a high affinity for the transferrin
fusion protein at
a first pH and a reduced affinity at a second (higher or lower) pH; changes in
concentration of
certain chaotropic agents; or through the use of imidazole.
Delivery of a Drug or Therapeutic Protein to the inside of a Cell and/or
across
the Blood Brain Barrier (BBB)
[00257] Within the scope of the invention, the transfei-l-in fusion proteins
may be used as a
carrier to deliver a molecule or small molecule therapeutic coinplexed to the
ferric ion of
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transferrin to the inside of a cell or across the blood brain barrier or other
barriers including
across the cell membrane of any cell type that naturally or engineered to
express a Tf
receptor. In these embodiments, the Tf fusion protein will typically be
engineered or
modified to inhibit, prevent or remove glycosylation to extend the seruin half-
life of the
fusion protein and/or therapeutic protein portion. The addition of a targeting
peptide is
specifically contemplated to further target the Tf fusion protein to a
particular cell type, e.g.,
a cancer cell.
[00258] In one embodiment, the iron-containing, anti-anemic drug, ferric-
sorbitol-citrate
coinplex is loaded onto a modified Tf fusion protein of the invention. Ferric-
sorbitol-citrate
(FSC) has been shown to inhibit proliferation of various murine cancer cells
in vitf-o and
cause tumor regression in vivo, while not having any effect on proliferation
of non-inalignant
cells (Poljak-Blazi et al. (June 2000) Cancer Biotherapy and
Radiopharmaceuticals (United
States), 15/3:285-293).
[00259] In another embodiment, the antineoplastic drug Adriamycin RO
(doxorubicin) and/or
the chemotherapeutic drug bleomycin, both of which are known to form
coinplexes with
ferric ion, is loaded onto a Tf fusion protein of the invention. In other
embodiments, a salt of
a drug, for instance, a citrate or carbonate salt, may be prepared and
complexed witll the
ferric iron that is then bound to Tf. As tumor cells often display a higher
turnover rate for
iron; transferrin modified to carry at least one anti-tuinor agent, may
provide a means of
increasing agent exposure or load to the tumor cells. (Demant, E.J., (1983)
Eur. J. Biochem.
137(1-2):113-118; Padbury et al. (1985) J. Biol. Chein. 260:7820-7823).
Pharmaceutical Formulations and Treatment Methods
[00260] The fusion proteins of the invention coinprising transferrin, for
instance, modified
transferrin, may be administered to a patient in need thereof using standard
administration
protocols. For instance, the Tf fusion proteins of the present invention can
be provided alone,
or in coinbination, or in sequential combination with other agents that
modulate a particular
pathological process. As used herein, two agents are said to be administered
in combination
when the two agents are administered simultaneously or are adininistered
independently in a
fashion such that the agents will act at the saine or near the saine time.
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[00261] The fusion proteins of the present invention can be administered via
parenteral,
subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal and
buccal routes. For
example, an agent may be administered locally to a site of injury via
inicroinfusion.
Alternatively, or concurrently, administration may be noninvasive by either
the oral,
inhalation, nasal, or pulmonary route. The dosage administered will be
dependent upon the
age, health, and weight of the recipient, kind of concurrent treatinent, if
any, frequency of
treatment, and the nature of the effect desired.
[00262] While any method of administration may be used to deliver the Tf
fusion proteins of
the invention, administration or delivery orally may be a preferred embodiment
for certain
classes of fusion proteins or to treat certain conditions.
[00263] The present invention further provides coinpositions containing one or
more fusion
proteins of the invention. While individual needs vary, determination of
optimal ranges of
effective amounts of each coinponent is within the skill of the art. Typical
dosages comprise
about 1 pg/kg to about 100 mg/kg body weight. The preferred dosages for
systemic
administration comprise about 100 ng/kg to about 100 mg/kg body weight. The
preferred
dosages for direct administration to a site via microinfusion comprise about 1
ng/kg to about
1 mg/kg body weight. When administered via direct injection or microinfusion,
modified
fusion proteins of the invention may be engineered to exhibit reduced or no
binding of iron to
prevent, in part, localized iron toxicity.
[00264] In addition to the phannacologically active fusion protein, the
compositions of the
present invention may contain suitable phannaceutically acceptable carriers
comprising
excipients and auxiliaries that facilitate processing of the active coinpounds
into preparations
which can be used pharinaceutically for delivery to the site of action.
Suitable forinulations
for parenteral administration include aqueous solutions of the active
compounds in water-
soluble forin, for exainple, water-soluble salts. In addition, suspensions of
the active
coinpounds as appropriate oily injection suspensions may be administered.
Suitable
lipophilic solvents or vehicles include fatty oils, for exainple, sesaine oil,
or synthetic fatty
acid esters, for example, ethyl oleate or triglycerides. Aqueous injection
suspensions may
contain substances which increase the viscosity of the suspension and include,
for exainple,
sodiuin carboxylnethyl cellulose, sorbitol and dextran. Optionally, the
suspension may also
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73
contain stabilizers. Liposomes can also be used to encapsulate the agent for
delivery into the
cell.
[00265] The pharmaceutical formulation for systemic adininistration according
to the
invention may be formulated for enteral, parenteral or topical administration.
Indeed, all
three types of formulations inay be used siinultaneously to achieve systemic
administration of
the active ingredient. Suitable formulations for oral administration include
hard or soft
gelatin capsules, pills, tablets, including coated tablets, elixirs,
suspensions, syrups or
inhalations and controlled release forms thereof.
[00266] The pharmaceutical composition of the present invention can be in unit
dosage
form, e.g. as tablets or capsules. In such form, the composition is sub-
divided in unit dose
containing appropriate quantities of the active ingredient; the unit dosage
forms can be
packaged coinpositions, for example, packeted powders, vials, ampoules,
prefilled syringes
or sachets containing liquids. The unit dosage form can be, for example, a
capsule or tablet
itself, or it- can be the appropriate number of any such compositions in
package form. The
dosage to be used in the treatment must be subjectively determined by the
physician.
[00267] In practicing the metllods of this invention, the fusion proteins of
this invention may
be used alone or in combination, or in combination with other therapeutic or
diagnostic
agents. In certain preferred embodiments, the compounds of this invention may
be co-
administered along witll other compounds typically prescribed for these
conditions according
to generally accepted medical practice. The compounds of this invention can be
utilized iti
vivo, ordinarily in maminals, such as humans, sheep, horses, cattle, pigs,
dogs, cats, rats and
mice, or in vitro.
Oral Pharmaceutical Compositions and Delivery Methods
[00268] In the present invention, Tf fusion proteins, including but not
limited to modified Tf
fusion proteins, may be forinulated for oral delivery. In particular, certain
fusion proteins of
the invention that are used to treat certain classes of diseases or medical
conditions may be
particularly amenable for oral formulation and delivery. Such classes of
diseases or
conditions include, but are not limited to, acute, chronic and recurrent
diseases. Chronic or
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74
recurrent diseases include, but are not limited to, viral disease or
infections, cancer, a
metabolic diseases, obesity, autoimmune diseases, inflammatory diseases,
allergy, graft-vs.-
host disease, systemic microbial infection, anemia, cardiovascular disease,
psychosis, genetic
diseases, neurodegenerative diseases, disorders of hematopoietic cells,
diseases of the
endocrine system or reproductive systems, gastrointestinal diseases. Examples
of these
classes of disease include diabetes, inultiple sclerosis, astluna, HCV or HIV
infections,
llypertension, hypercholesterolemia, arterial scllerosis, arthritis, and
Alzheimer's disease. In
many chronic diseases, oral formulations of Tf fusion proteins of the
invention and methods
of administration are particularly useful because they allow long-tenn patient
care and
therapy via home oral administration without reliance on injectable treatment
or drug
protocols.
[00269] Oral formulations and delivery methods coinprising Tf fusion proteins
of the
invention take advantage of, in part, transferrin receptor mediated
transcytosis across the
gastrointestinal (GI) epithelium. The Tf receptor is found at a very high
density in the human
GI epithelium, transferrin is highly resistant to tryptic and chymotryptic
digestion and Tf
chemical conjugates have been used to successfully deliver proteins and
peptides across the
GI epitheliuin (Xia et al., (2000) J. Pharmacol. Experiment. Therap., 295:594-
600; Xia et al.
(2001) Pharmaceutical Res., 18(2):191-195; and Shah et al. (1996) J.
Pharmaceutical Sci.,
85(12):1306-1311, all of which are herein incorporated by reference in their
entirety). Once
transported across the GI epithelium, Tf fusion proteins of the invention
exhibit extended
half-life in serum, that is, the tllerapeutic protein or peptide(s) attaclled
or inserted into Tf
exhibit an extended serum half-life coinpared to the protein or peptide in its
non-fused state.
[00270] Oral fonnulations of Tf fusion proteins of the invention may be
prepared so that they
are suitable for transport to the GI epithelium and protection of the Tf
fusion protein
component and other active coinponents in the stomach. Such fonnulations may
include
carrier and dispersant components and may be in any suitable forin, including
aerosols (for
oral or pulmonary delivery), syrups, elixirs, tablets, including cllewable
tablets, hard or soft
capsules, troches, lozenges, aqueous or oily suspensions, einulsions, cacllets
or pellets
granulates, and dispersible powders. Preferably, Tf fasion protein
fonnulations are
employed in solid dosage fornns suitable for simple, and preferably oral,
adininistration of
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precise dosages. Solid dosage forms for oral administration are preferably
tablets, capsules,
or the like.
[00271] For oral adininistration in the form of a tablet or capsule, care
should be talcen to
ensure that the composition enables sufficient active ingredient to be
absorbed by the host to
produce an effective response. Thus, for example, the amount of Tf fusion
protein may be
increased over that theoretically required or other known measures such as
coating or
encapsulation may be taken to protect the polypeptides from enzyinatic action
in the stomach.
[00272] Traditionally, peptide and protein drugs have been adininistered by
injection because
of the poor bioavailability when administered by other means, and in
particular orally. These
drugs are prone to chemical and confonnational instability and are often
degraded by the
acidic conditions in the stomach, as well as by enzymes in the stomach and
gastrointestinal
tract. In response to these delivery problems, certain technologies for oral
delivery have been
developed, such as encapsulation in nanoparticles composed of polyiners with a
hydrophobic
backbone and hydrophilic branches as drug carriers, encapsulatioiT in-
microparticles, insertion
into liposomes in emulsions, and conjugation to other molecules. All of which
may be used
with the Tf fusion molecules of the present invention.
[00273] Examples of nanoparticles include inucoadhesive nanoparticles coated
with chitosan
and Carbopol (Takeuchi et al., Adv. Drug Deliv. Rev. 47(l):39-54, 2001) and
nanopai-ticles
containing charged coinbination polyesters, poly(2-sulfobutyl-vinyl alcohol)
and poly(D,L-
lactic-co-glycolic acid) (Jung et al., Eur. J. Phann. Biopharm. 50(1):147-160,
2000).
Nanoparticles containing surface polyiners witli poly-N-isopropylacrylainide
regions and
cationic poly-vinylainine groups showed iinproved absorption of salmon
calcitonin when
administered orally to rats.
[00274] Drug delivery particles composed of alginate and pectin, strengthened
with
polylysine, are relatively acid and base resistant and can be used as a
carrier for drugs. These
particles coinbine the advantages of bioadhesion, enhanced absoiption and
sustained release
(Liu et al., J. Pharm. Phannacol. 51(2):141-149, 1999).
[00275] Additionally, lipoainino acid groups and liposaccharide groups
conjugated to the N-
and C-termini of peptides sucll as synthetic somatostatin, creating an
amphipathic surfactant,
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76
were shown to produce a composition that retained biological activity (Toth et
al., J. Med.
Chem. 42(19):4010-4013, 1999).
100276] Examples of other peptide delivery technologies include carbopol-
coated
mucoadhesive emulsions containing the peptide of interest and either nitroso-N-
acetyl-D,L-
penicillainine and carbolpol or taurocholate and carbopol. These were shown to
be effective
when orally administered to rats to reduce serum calciuin concentrations
(Ogiso et al., Biol.
Pharm. Bull. 24(6):656-661, 2001). Phosphatidylethanol, derived froin
phosphatidylcholine,
was used to prepare liposomes containing phosphatidylethanol as a carrier of
insulin. These
liposomes, when administered orally to rats, were shown to be active (Kisel et
al., Int. J.
Phann. 216(1-2):105-114, 2001).
[00277] Insulin has also been formulated in poly(vinyl alcohol)-gel spheres
containing
insulin and a protease inhibitor, such as aprotinin or bacitracin. The glucose-
lowering
properties of these gel spheres have been deinonstrated in rats, where insulin
is released
largely in the lower intestine f Ifimura et al:, Biol. Pharm. Bull. 19(6):897-
900, 1996.
[00278] Oral delivery of insulin has also been studied using nanoparticles
made of poly(alkyl
cyanoacrylate) that were dispersed with a surfactant in an oily phase (Dainge
et al., J. Pharm.
Sci. 86(12):1403-1409, 1997) and using calcium alginate beads coated with
chitosan (Onal et
al., Artif. Cells Blood Substit. Im.mobil. Biotechnol. 30(3):229-237, 2002). -
[00279] In other methods, the N- and C-termini of a peptide are linlced to
polyethylene glycol
and then to allyl chains to form conjugates with improved resistance to
enzyinatic
degradation and improved diffusion through the GI wall (www.nobexcorp.com).
[00280] BioPORTEROO is a cationic lipid mixture, wllich interacts non-
covalently with
peptides to create a protective coating or layer. The peptide-lipid complex
can fuse to the
plasma membrane of cells, and the peptides are intenlalized into the cells
(www. genetherapysystems . com).
[00281] In a process using liposoines as a starting inaterial, cochleate-
shaped particles have
been developed as a phannaceutical vehicle. A peptide is added to a suspension
of liposoines
containing mainly negatively charged lipids. The addition of calciurn causes
the collapse and
fusion of the liposarnes into large sheets coinposed of lipid bilayers, which
then
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77
spontaneously roll up or stack into cochleates (U.S. Patent 5,840,707;
http://www.biodeliveiysciences.com).
[00282] Compositions comprising Tf fusion protein intended for oral use may be
prepared
according to aiiy method lcnown to the art for the inanufacture of
pharmaceutical
coinpositions and such compositions may contain one or more agents selected
from the group
consisting of sweetening agents in order to provide a phannaceutically elegant
and palatable
preparation. For example, to prepare orally deliverable tablets, Tf fusion
protein is mixed
with at least one pharmaceutical excipient, and the solid formulation is
compressed to form a
tablet according to known methods, for delivery to the gastrointestinal tract.
The tablet
composition is typically formulated with additives, e.g. a saccharide or
cellulose carrier, a
binder such as starch paste or methyl cellulose, a filler, a disintegrator, or
other additives
typically usually used in the manufacture of medical preparations. To prepare
orally
deliverable capsules, DHEA is mixed with at least one pharmaceutical
excipient, and the
solid formulation is placed in a capsular container suitable for delivery to
the gastrointestinal
tract. Compositions comprising Tf fusion protein may be prepared as described
generally in
Remington's Pharinaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co.
Easton Pa.
18042) at Chapter 89, which is herein incorporated by reference.
[00283] As described above, many of the oral fonnulations of the invention may
contain
inert ingredients which allow for protection against the stomach environment,
and release of
the biologically active material in the intestine. Such formulations, or
enteric coatings, are
well known in the art. For example, tablets containing Tf fusion protein in
adinixture with
non-toxic phannaceutically acceptable excipients which are suitable for
manufacture of
tablets may be used. These excipients may be inert diluents, such as calcium
carbonate,
sodiuin carbonate, lactose, calciuin phosphate or sodiuin phosphate;
granulating and
disintegrating agents, for exainple, maize starch, gelatin or acacia, and
lubricating agents, for
example, inagnesiuin stearate, stearic acid, or talc.
[00284] The tablets may be uncoated or they may be coated with known
techniques to delay
disintegration and absorption in the gastrointestinal track and thereby
provide a sustained
action over a longer period of time. For exainple, a time delay material such
as glyceryl
monostearate or glyceryl distearate alone or witll a wax may be employed.
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[00285] Formulations for oral use may also be presented as hard gelatin
capsules wherein the
active ingredient is mixed with an inert solid diluent, for example, calcium
carbonate,
calciuin phosphate, or kaolin or as soft gelatin capsules wherein the active
ingredient is
mixed with an aqueous or an oil medium, for example, arachis oil, peanut oil,
liquid paraffin
or olive oil.
[00286] Aqueous suspensions may contain Tf fusion protein in the adinixture
with excipients
suitable for the manufacture of aqueous suspensions. Such excipients are
suspending agents,
for exainple, sodium carboxymethylcellulose, inethylcellulose,
hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum
tragacanth and
gum acacia; dispersing or wetting agents may be a naturally occurring
phosphatide, for
example, lecithin, or condensation products of an alkylene oxide with fatty
acids, for
example, polyoxyethylene stearate, or condensation products of ethylene oxide
with long
chain aliphatic alcohols, for example, heptadecylethyloxycetanol, or
condensation products of
ethylene oxide with partial esters derived from fatty acids and a hexitol such
as
polyoxyethylene sorbitol monooleate, or condensation products of ethylene
oxide with partial
esters derived from fatty acids and hexitol anliydrides, for example
polyoxyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more preservatives
for
example, ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one
or more
flavoring agents and one or more sweetening agents such as sucrose or
saccharin.
[00287] Oily suspensions may be formulated by suspending the active ingredient
in a
vegetable oil, for exarnple, arachis oil, olive oil, sesame oil or coconut
oil, or in a mineral oil
such as liquid paraffin. The oil suspensions may contain a thickening agent,
for example,
beeswax, hard paraffin or cetyl alcoliol. Sweetening agents, such as those set
forth above,
and flavoring agents may be added to provide a palatable oral preparation.
These
coinpositions may be preserved by the addition of an antioxidant such as
ascorbic acid.
[002881 Dispersible powders and granules suitable for preparation of an
aqueous suspension
by the addition of water provide the active ingredient and admixture with
dispersing or
wetting agent, suspending agent and one or more preservatives. Suitable
dispersing or
wetting agents and suspending ageiits are exemplified by those already
mentioned above.
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Additional excipients, for example, sweetening, flavoring and coloring agents,
may also be
present.
[00289] The pharmaceutical coinpositions containing Tf fusion protein may also
be in the
form of oil-in-water emulsions. The oil phase may be a vegetable oil, for
example, olive oil
or arachis oil, or a mineral oil for exainple, gum acacia or gum tragacanth,
naturally-
occurring phosphotides, for example soybean lecithin, and esters or partial
esters derived
from fatty acids and hexitol anhydrides, for exainple, sorbitan monooleate,
and condensation
products of the same partial esters with etliylene oxide, for example,
polyoxyethylene
sorbitan monooleate. The emulsions may also contain sweetening and flavoring
agents.
[00290] Syrups and elixirs containing Tf fusion protein may be formulated with
sweetening
agents, for example, glycerol, sorbitol or sucrose. Such fonnulations may also
contain a
demulcent, a preservative and flavoring and coloring agents. The
pharmaceutical
compositions may be in the forni of a sterile injectable preparation, for
example, as a sterile
injectable aqueous or oleaginous suspension. This suspension may be formulated
according
to the known art using those suitable dispersing or wetting agents and
suspending agents
which have been mentioned above. The sterile injectable preparations may also
be a sterile
injectable solution or suspension in a non-toxic parenterally-acceptable
diluent or solvate, for
example as a solution in 1, 3-butanediol. Among the acceptable vehicles and
solvents that
may be employed are water, Ringer's solution and isotonic sodium chloride
solution. In
addition, sterile, fixed oils are conventionally einployed as a solvent or
suspending mediuin.
For this period any bland fixed oil may be employed including synthetic mono-
or
diglycerides. In addition, fatty acids such as oleic acid find use in the
preparation of
injectables.
[00291] Phannaceutical compositions may also be formulated for oral delivery
using
polyester microspheres, zein microspheres, proteinoid microspheres,
polycyanoacrylate
microspheres, and lipid-based systems (see, for example, DiBase and MoiTel,
Oral Delivery
ofMicf=oencapszalcctecl Proteins, in Protein Delivery: Physical Systeins,
Sanders and Hendren
(eds.), pages 255-288 (Plenuin Press 1997)).
[00292] The proportion of phannaceutically active Tf fusion protein to carrier
and/or other
substances may vary from about 0.5 to about 100 wt. %(weight percent). For
oral use, the
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pharmaceutical formulation will generally contain from about 5 to about 100%
by weight of
the active material. For other uses, the formulation will generally have from
about 0.5 to
about 50 wt. % of the active material.
[00293] Tf fusion protein formulations employed in the invention provide an
effective
amount of Tf fusion protein upon adininistration to an individual. As used in
this context, an
"effective amount" of Tf fusion is an ainount that is effective to ameliorate
a symptom of a
disease.
[00294] The Tf fusion protein composition of the present invention may be,
though not
necessarily, administered daily, in an effective amount to ameliorate a
symptom. Generally,
the total daily dosage will be at least about 50 mg, preferably at least about
100 mg, and more
preferably at least about 200 mg, and preferably not more than 500 mg per day,
administered
orally, e.g., in 4 capsules or tablets, each containing 50 mg Tf fusion
protein. Capsules or
tablets for oral delivery can conveniently contain up to a fiill daily oral
dose, e.g., 200 mg or
more. -
[00295] In a particularly preferred embodiment, oral pharinaceutical
compositions
coinprising Tf fusion protein are fonnulated in buffered liquid form which is
then
encapsulated into soft or hard-coated gelatin capsules which are then coated
with an
appropriate enteric coating. For the oral pharmaceutical compositions of the
invention, the
location of release may be anywhere in the GI system, including the small
intestine (the
duodenuin, the jejunum, or the ileuin), or the large intestine.
[00296] In other embodiments, oral compositions of the invention are
formulated to slowly
release the active ingredients, including the Tf fusion proteins of the
invention, in the GI
system using known delayed release forinulations.
[00297] Tf fusion proteins of the invention for oral delivery are capable of
binding the Tf
receptor found in the GI epithelium. To facilitate this binding and receptor
mediated
transport, Tf fusion proteins of the invention are typically produced with
iron and in some
instances carbonate, bound to the Tf moiety. Processes and methods to load the
Tf moiety of
the fusion protein compositions of the invention with iron and carbonate are
lcnown in the art
[00298] In some phannaceutical fonnulations of the invention, the Tf moiety of
the Tf fusion
protein may be modified to increase the affinity or avidity of the Tf moiety
to iron. Such
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methods are lenown in the art. For instance, mutagenesis can be used to
produce mutant
transferrin moieties that bind iron more avidly than natural transferrin. In
human serum
transferrin, the amino acids which are ligands for metal ion chelation
include, but are not
limited to N lobe amino acids Asp63, Tyr 95, Tyr188, Lys206, His207 and
His249; and C
lobe amino acids Asp392, Tyr426, Tyr517 and His585 of SEQ ID NO: 3 (the
nuinber beside
the amino acid indicates the position of the ainino acid residue in the
primary ainino acid
sequence where the valine of the mature protein is designated position 1). See
U.S. Patent
5,986,067, which is herein incorporated be reference. In one einbodiment, the
Lys206 and
His207 residues witliin the N lobe are replaced with Gln and Glu,
respectively.
[00299] In some pharmaceutical formulations of the invention, the Tf fusion
protein is
engineered to contain a cleavage site between the therapeutic protein or
peptide and the Tf
moiety. Such cleavable sites or linkers are known in the art.
[00300] Pharmaceutical compositions of the invention and methods of the
invention may
include the addition of a transcytosis enhancer to facilitate transfer of the
fusion protein
across the GI epithelium. Such enhancers are known in the art. See Xia et al.,
(2000) J.
Pharriiacol. ExpeJ iment. Therap., 295:594-600; and Xia et al. (2001)
Pharmaceutical Res.,
18(2):191-195.
[00301] In preferred embodiments of the invention, oral pharmaceutical
formulations include
Tf fusion proteins comprising a modified Tf moiety exhibiting reduced or no
glycosylation
fused at the N tenninal end to a natriuretic peptide as described above. Such
pharmaceutical
coinpositions may be used to treat glucose imbalance disorders such as
diabetes by oral
adm.inistration of the phannaceutical composition comprising an effective dose
of fusion
protein.
100302] The effective dose of fusion protein may be measured in a numbers of
ways,
including dosages calculated to alleviate syinptoms associated with a specific
disease state in
a patient, such as the syinptoins of diabetes. In other foiinulations, dosages
are calculated to
coniprise an effective amount of fusion protein to induce a detectable cllange
in blood
glucose levels in the patient. Sucli detectable changes in blood glucose may
include a
decrease in blood glucose levels of between about 1% and 90%, or between about
5% and
about 80%. These decreases in blood glucose levels will be dependent on the
disease
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condition being treated and pharmaceutical compositions or methods of
administration may
be modified to achieve the desired result for each patient. In other
instances, the
pharmaceutical compositions are fonnulated and methods of administration
modified to
detect an increase in the activity level of the therapeutic protein or peptide
in the patient, for
instance, detectable increases in the activities of a natriuretic peptide.
Such fonnulations and
methods may deliver between about 1 pg to about 100 mg /kg body weight of
fusion protein,
about 100 ng to about 100 :g/kg body weight of fusion protein, about 100 :g/
to about 100
mg/kg body weight of fusion protein, about 1:g to about 1 g of fusion protein,
about 10 :g to
about 100 mg of fusion protein or about 10 mg to about 50 mg of fusion
protein.
Formulations may also be calculated using a unit measurement of therapeutic
protein activity,
such as about 5 to about 500 units of human insulin or about 10 to about 100
units of human
insulin. The measurements by weight or activity can be calculated using known
standards for
each therapeutic protein or peptide fused to Tf.
[00303] The invention also includes methods of orally administering the
pharmaceutical
compositions of the invention. Such methods may include, but are not liinited
to, steps of
orally administering the compositions by the patient or a caregiver. Such
administration steps
may include adminisfration on intervals such as once or twice per day
depending on the Tf
fusion protein, disease or patient condition or individual patient. Such
methods also include
the adininistration of various dosages of the individual Tf fusion protein.
For instance, the
initial dosage of a pharinaceutical composition may be at a higher level to
induce a desired
effect, such as reduction in blood pressure. Subsequent dosages may then be
decreased once
a desired effect is achieved. These clianges or modifications to
adininistration protocols may
be done by the attending physician or health care worker. In some instances,
the changes in
the adininistration protocol may be done by the individual patient, such as
when a patient is
monitoring blood pressure and administering a mTf-natriuretic peptide oral
composition of
the invention.
[00304] The invention also includes methods of producing oral compositions or
inedicant
coinpositions of the invention comprising fonnulating a Tf fusion protein of
the invention
into an orally administerable form. In other instances, the invention includes
methods of
producing compositions or medicant compositions of the invention coinprising
foi-tnulating a
Tf fusion protein of the invention into a form suitable for oral
administration.
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[00305] Moreover, the present invention includes pulmonary delivery of the Tf
fusion
protein formulations. Pulmonary delivery is particularly promising for the
delivery of
macromolecules which are difficult to deliver by other routes of
administration. Such
pulmonary delivery can be effective both for systemic delivery and for
localized delivery to
treat diseases of the lungs, since drugs delivered to the lung are readily
absorbed through the
alveolar region directly into the blood circulation.
[00306] The present invention provides compositions suitable for forming a
drug dispersion
for oral inhalation (pulmonary delivery) to treat various conditions or
diseases. The Tf fusion
protein formulation could be delivered by different approaches such as liquid
nebulizers,
aerosol-based metered dose inlialers (MDI's), and dry powder dispersion
devices. In
forinulating compositions for pulmonary delivery, pharmaceutically acceptable
carriers
including surface active agents or surfactants and bulk carriers are
coininonly added to
provide stability, dispersibility, consistency, and/or bulking characteristics
to enliance
uniform pulmonary delivery of the composition to the subject.
[00307] Surface active agents or surfactants promote absorption of polypeptide
through
inucosal membrane or lining. Useful surface active agents or surfactants
include fatty acids
and salts thereof, bile salts, phospholipid, or an alkyl saccharide. Exainples
of fatty acids and
salts thereof include sodium, potassium and lysine salts of caprylate (C8),
caprate (Clo),
laurate (C12) and myristate (C14). Examples of bile salts include cholic acid,
chenodeoxycholic acid, glycocholic acid, taurocholic acid,
glycochenodeoxycholic acid,
taurochenodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid,
taurodeoxycholic
acid, lithocholic acid, and ursodeoxycholic acid.
Exainples of phospholipids include single-chain phospholipids, such as
lysophosphatidylclzoline, lysophosphatidylglycerol,
lysophosphatidylethanolamine,
lysophosphatidylinositol and lysophosphatidylserine; or double-chain
phospholipids, such as
diacylphosphatidylcholines, diacylphosphatidylglycerols,
diacylphosphatidylethanolainines,
diacylphosphatidylinositols and diacylphosphatidylserines. Exainples of alkyl
saccharides
include alkyl glucosides or alkyl maltosides, such as decyl glucoside and
dodecyl maltoside.
[00308] Pharinaceutical excipients that are useful as cai-liers include
stabilizers suc11 as
human serum albumin (HSA); bulking agents sucli as carbohydrates, amino acids
and
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84
polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the
like. These
carriers may be in a crystalline or amorphous form or may be a mixture of the
two.
[00309] Exanples of carbohydrates for use as bulking agents include
monosaccharides such
as galactose, D-mannose, sorbose, and the like; disaccharides, such as
lactose, trehalose, and
the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and
polysaccharides,
such as raffinose, maltodextrins, dextrans, and the like; alditols, sucli as
mannitol, xylitol, and
the like. Examples of polypeptides for use as bulking agents include
aspartame. Amino acids
include alanine and glycine, with glycine being preferred.
[00310] Additives, which are minor components of the composition, may be
included for
confonnational stability during spray drying and for improving dispersibility
of the powder.
These additives include hydrophobic amino acids such as tryptophan, tyrosine,
leucine,
phenylalanine, and the like.
[00311] Suitable pH adjusters or buffers include organic salts prepared from
organic acids
and bases, such as sodiuin citrate, sodium ascorbate, and the like; sodium
citrate is preferred.
[00312] The Tf fusion compositions for pulmonary delivery may be packaged as
unit doses
where a therapeutically effective amount of the composition is present in a
unit dose
receptacle, such as a blister pack, gelatin capsule, or the like. The
manufacture of blister
packs or gelatin capsules is typically carried out by inethods that are
generally well known in
the packaging art.
[00313] U.S. Patent 6,524,557 discloses a pharinaceutical aerosol forinulation
comprising
(a) a HFA propellant; (b) a pharinaceutically active polypeptide dispersible
in the propellant;
and (c) a surfactant which is a C8 -C16 fatty acid or salt thereof, a bile
salt, a phospholipid, or
an alkyl saccharide, which surfactant enllances the systemic absoiption of the
polypeptide in
the lower respiratory tract. The invention also provides methods of
manufacturing such
fonnulations and the use of such formulations in treating patients.
[00314] One approach for the pulmonary delivery of dry powder drugs utilizes a
hand-held
device with a hand puinp for providing a source of pressurized gas. The
pressurized gas is
abruptly released through a powder dispersion device, such as a venturi
nozzle, and the
dispersed powder made available for patient inhalation.
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[00315] Dry powder dispersion devices are described in several patents. U.S.
Pat. No.
3,921,637 describes a manual pump with needles for piercing through a single
capsule of
powdered medicine. The use of multiple receptacle disks or strips of
medication is described
in European Patent Application No. EP 0 467 172; International Patent
Publication Nos. WO
91/02558; and WO 93/09832; U.S. Pat. Nos. 4,627,432; 4,811,731; 5,035,237;
5,048,514;
4,446,862; 5,048,514, and 4,446,862.
[00316] The aerosolization of protein therapeutic agents is disclosed in
European Patent
Application No. EP 0 289 336. T1lerapeutic aerosol formulations are disclosed
in
International Patent Publication No. WO 90/09781.
[00317] The present invention provides formulating Tf fusion protein for oral
inhalation.
The formulation comprises Tf fusion protein and suitable pharmaceutical
excipients for
pulmonary delivery. The present invention also provides administering the Tf
fusion protein
composition via oral inhalation to subjects in need thereof.
Transgenic Animals
[00318] The production of transgenic non-human animals that contain a
transferrin fusion
construct with increased serum half-life increased seruin stability or
increased bioavailability
of the instant invention is contemplated in one einbodiinent of the present
invention. In some
einbodiinents, lactoferrin may be used as the Tf portion of the fusion protein
so that the
fusion protein is produced and secreted in milk.
[00319] The successful production of transgenic, non-human animals has been
described in
a number of patents and publications, such as, for example U.S. Patent
6,291,740 (issued
Septeinber 18, 2001); U.S. Patent 6,281,408 (issued August 28, 2001); and U.S.
Patent
6,271,436 (issued August 7, 2001) the contents of wliich are hereby
incorporated by reference
in their entireties.
[00320] The ability to alter the genetic make-up of animals, sucli as
domesticated mammals
including cows, pigs, goats, horses, cattle, and sheep, allows a nuinber of
coimnercial
applications. These applications include the production of animals which
express large
quantities of exogenous proteins in an easily harvested form (e.g., expression
into the milk or
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86
blood), the production of animals with increased weight gain, feed efficiency,
carcass
composition, milk production or content, disease resistance and resistance to
infection by
specific microorganisms and the production of animals having enhanced growth
rates or
reproductive perforinance. Animals which contain exogenous DNA sequences in
their
genome are referred to as transgenic animals.
[00321] The most widely used method for the production of transgenic animals
is the
microinjection of DNA into the pronuclei of fertilized embiyos (Wall et al.,
J. Cell. Biochem.
49:113 [1992]). Other methods for the production of transgenic animals include
the infection
of embryos with retroviruses or with retroviral vectors. Infection of both pre-
and post-
implantation mouse embryos with either wild-type or recombinant retroviruses
has been
reported (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 (1976); Janenich et
al., Cell 24:519
(1981); Stuhlmann et al., Proc. Natl. Acad. Sci. USA 81:7151 (1984); Jalmer et
al., Proc.
Natl. Acad Sci. USA 82:6927 (1985); Van der Putten et al., Proc. Natl. Acad
Sci. USA
82:6148-6152 (1985); Stewart et al., EMBO J. 6:383-388 (1987)).
[00322] An alternative means for infecting embryos with retroviruses is the
injection of
virus or virus-producing cells into the blastocoele of mouse embryos (Jahner,
D. et al., Nature
298:623 [1982]). The introduction of transgenes into the gennline of mice has
been reported
using intrauterine retroviral infection of the midgestation mouse embryo
(Jahner et al., supra
[1982]). Infection of bovine and ovine embryos with retroviruses or retroviral
vectors to
create transgenic animals has been reported. These protocols involve the
inicro-injection of
retroviral particles or growth aiTested (i.e., initomycin C-treated) cells
which shed retroviral
particles into the perivitelline space of fertilized eggs or early einbryos
(PCT International
Application WO 90/08832 [1990]; and Haskell and Bowen, Mol. Reprod. Dev.,
40:386
[1995]. PCT International Application WO 90/08832 describes the injection of
wild-type
feline leukemia virus B into the perivitelline space of sheep einbryos at the
2 to 8 cell stage.
Fetuses derived from injected einbryos were shown to contain multiple sites of
integration.
[00323] U.S. Patent 6,291,740 (issued Septeinber 18, 2001) describes the
production of
transgenic animals by the introduction of exogenous DNA into pre-maturation
oocytes and
mature, unfertilized oocytes (i.e., pre-fertilization oocytes) using
retroviral vectors wliich
transduce dividing cells (e.g., vectors derived from inurine leulceinia virus
(MLV)). This
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patent also describes methods and compositions for cytomegalovirus promoter-
driven, as
well as mouse mammary tumor LTR expression of various recombinant proteins.
[00324] U.S. Patent 6,281,408 (issued August 28, 2001) describes methods for
producing
transgenic aniinals using embryonic stem cells. Briefly, the embryonic stein
cells are used in
a mixed cell co-culture with a morula to generate transgenic animals. Foreign
genetic
material is introduced into the embryonic stem cells prior to co-culturing by,
for exainple,
electroporation, microinjection or retroviral delivery. ES cells transfected
in this manner are
selected for integrations of the gene via a selection marker such as neomycin.
[00325] U.S. Patent 6,271,436 (issued August 7, 2001) describes the production
of
transgenic animals using metllods including isolation of primordial germ
cells, culturing these
cells to produce primordial germ cell-derived cell lines, transforming both
the primordial
germ cells and the cultured cell lines, and using these transforined cells and
cell lines to
generate transgenic animals. The efficiency at wlzich transgenic animals are
generated is
greatly increased, thereby allowing the use of homologous recoinbinati<7n in
producing
transgenic non-rodent animal species.
Gene Therapy
[00326] The use of transferrin fusion constructs for gene therapy wherein a
transferrin or
modified transferrin protein or domain is joined to a therapeutic protein or
peptide is
contemplated in one einbodiment of this invention. The modified transferrin
fusion
constructs with increased serum half-life or serum stability of the instant
invention are ideally
suited to gene therapy treatinents.
[00327] The successful use of gene therapy to express a soluble fusion protein
has been
described. Briefly, gene therapy via injection of an adenovirus vector
containing a gene
encoding a soluble fusion protein consisting of cytotoxic lyinphocyte antigen
4 (CTLA4) and
the Fc portion of human iininunoglubulin G1 was recently shown in Ijima et al.
(June 10,
2001) Human Gene Therapy (United States) 12/9:1063-77. In this application of
gene
therapy, a murine model of type II collagen-induced artluitis was successfully
treated via
intraarticular injection of the vector.
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[00328] Gene therapy is also described in a number of U.S. patents including
U.S. Pat.
6,225,290 (issued May 1, 2001); U.S. Pat. 6,187,305 (issued February 13,
2001); and U.S.
Pat. 6,140,111 (issued October 31, 2000).
[00329] U.S. Patent 6,225,290 provides methods and constructs whereby
intestinal epithelial
cells of a maininalian subject are genetically altered to operatively
incorporate a gene which
expresses a protein which has a desired therapeutic effect. Intestinal cell
transforination is
accomplished by administration of a formulation composed primarily of naked
DNA, and the
DNA may be administered orally. Oral or other intragastrointestinal routes of
administration
provide a simple method of administration, while the use of naked nucleic acid
avoids the
coinplications associated with use of viral vectors to accomplish gene
therapy. The expressed
protein is secreted directly into the gastrointestinal tract and/or blood
stream to obtain
therapeutic blood levels of the protein thereby treating the patient in need
of the protein. The
transformed intestinal epithelial cells provide short or long tenn therapeutic
cures for diseases
associated with a deficiency in a particular protein or which are amenable to
treatment by
overexpression of a protein.
[00330] U.S. Pat. 6,187,305 provides methods of gene or DNA targeting in cells
of
vertebrate, particularly maimnalian, origin. Briefly, DNA is introduced into
primary or
secondary cells of vertebrate origin througli homologous recombination or
targeting of the
DNA, which is introduced into genomic DNA of the primary or secondary cells at
a
preselected site.
[00331] U.S. Pat. 6,140,111 (issued October 31, 2000) describes retroviral
gene therapy
vectors. The disclosed retroviral vectors include an insertion site for genes
of interest and are
capable of expressing high levels of the protein derived fioin the genes of
interest in a wide
variety of transfected cell types. Also disclosed are retroviral vectors
lacking a selectable
marker, thus rendering thein suitable for huinan gene therapy in the treatment
of a variety of
disease states without the co-expression of a marker product, suc11 as an
antibiotic. These
retroviral vectors are especially suited for use in certain packaging cell
lines. The ability of
retroviral vectors to insert into the genome of mammalian cells has made them
particularly
promising candidates for use in the genetic therapy of genetic diseases in
huinans and
animals. Genetic therapy typically involves (1) adding new genetic material to
patient cells in
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vivo, or (2) removing patient cells from the body, adding new genetic material
to the cells and
reintroducing them into the body, i.e., in vitro gene therapy. Discussions of
how to perform
gene therapy in a variety of cells using retroviral vectors can be found, for
example, in U.S.
Pat. Nos. 4,868,116, issued Sep. 19, 1989, and 4,980,286, issued Dec. 25, 1990
(epithelial
cells), WO 89/07136 published Aug. 10, 1989 (liepatocyte cells), EP 378,576
published Jul.
25, 1990 (fibroblast cells), and WO 89/05345 published Jun. 15, 1989 and
WO/90/06997,
published Jun. 28, 1990 (endothelial cells), the disclosures of which are
incorporated herein
by reference.
Kits Containing Transferrin Fusion Proteins
[00332] In a further embodiment, the present invention provides kits
containing transferrin
fusion proteins, which can be used, for instance, for the therapeutic or non-
therapeutic
applications. The kit coinprises a container with a label. Suitable containers
include, for
exainple, bottles, vials, and test tubes. The containers may be forined from a
variety of
materials such as glass or plastic. The container holds a coinposition which
includes a
transferrin fusion protein that is effective for therapeutic or no.n-
therapeutic applications, such
as described above. The active agent in the coinposition is the therapeutic
protein. The label
on the container indicates that the composition is used for a specific therapy
or non-
therapeutic application, and may also indicate directions for either in vivo
or in vitro use, such
as those described above.
[00333] The kit of the invention will typically comprise the container
described above and
one or more other containers coinprising materials desirable from a
coininercial and user
standpoint, including buffers, diluents, filters, needles, syringes, and
package inserts with
instructions for use.
[00334] Without further description, it is believed that a person of ordinary
skill in the art
can, using the preceding description and the following illustrative examples,
make and utilize
the present invention and practice the claimed methods. For example, a skilled
artisan would
readily be able to detennine the biological activity, both in vitro and in
vivo, for the fusion
protein constructs of the present invention as coinpared with the coinparable
activity of the
therapeutic moiety in its unfused state. Similarly, a person skilled in the
art could readily
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determine the serum half life and serum stability of constructs according to
the present
invention. The following working examples therefore, specifically point out
the preferred
einbodiments of the present invention, and are not to be construed as limiting
in any way the
remainder of the disclosure.
EXAMPLES
Example 1: BNP/mTf Fusion Proteins
[00335] Natriuretic peptides are hormones involved in the regulation of fluid
and electrolyte
homeostasis. Brain natriuretic peptide (BNP) was initially found in porcine
brain (Sudoh et
al. (1988) Biochem Biophys Res Comm 155:726-732), but the main source of BNP
is the
cardiac ventricle. Like other peptides, BNP has a short plasma half-life in
humans. The
present invention provides fusion proteins with BNP fused to mTf (S415A,
T613A) with
extended seruin stability and in vivo circulatory half-life and phannaceutical
coinpositions of
such fusion proteins for treating patients in need thereof. The patient may be
suffering from,
for exainple, from congestive heart failure or renal disease.
[00336] In this example, the steps for producing a BNP/mTf fusion protein are
described.
The same steps may be used to generate transferrin fusion proteins with other
natriuretic
peptides, BNP analogs or derivatives, etc.
[00337] The human BNP protein sequence was obtained from PubMed (Accession no.
NM002521), back translated into DNA codon optimized for yeast.
TCTCCAAAAATGGTTCAAGGTTCTGGTTGTTTTGGTAGAAAAATGGATAGAATTT
CTTCTTCTTCTGGTTTGGGTTGTAAAGTTTTGAGAAGACAT (SEQ ID NO: 165)
N-terminal fusion: nL BNP (1-32) (PEAPTD)2 mTf (pREX0730/731)
[00338] The human BNP sequence (SPIeIVIVQGSGCFGRKIVIDRISSSSGLGCKVLRRH )
(SEQ ID NO: 166) was inserted at the N-terininus of inTf (S415A, T613A) using
overlapping
primer sequences encoding the adjoining mTf sequence. Primers P1228 (reverse
primer) and
P 1229 (forward primer) were designed to introduce the BNP sequence and
(PEAPTD)2 linlcer
(SEQ ID NO: 142) at the 5' of the mTf sequence and 3' of the nL leader
sequence.
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91
P 1228 reverse primer:
AAACTTTACAACCCAAACCAGAAGAAGAAGAAATTCTATCCATTTTTCTACCAAA
ACAACCAGAACCTTGAACCATTTTTGGAGACGCCAGACACAGCCC (SEQ ID NO:
167)
P 1229 forward primer:
CTTCTTCTTCTGGTTTGGGTTGTAAAGTTTTGAGAAGACATCCAGAAGCTCCAACT
GATCCAGAAGCTCCAACTGATGTACCTGATAAAACTGTGAGATG (SEQ ID NO:
168
[00339] In the first round of PCR, two products were generated using
P1228+P0025 or
P1229+P0012 with pREX0197. The products from these reactions were then joined
in a
second round of PCR using the outer primers P0012 and P0025.
Outer primers
P0012: CATGATCTTGGCGATGCAGTC (SEQ ID NO: 169)
P0025: AGCGGATAACAATTTCACACAGGA (SEQ ID NO: 170)
The product of this reaction was cut with AflII and EcoRI and subcloned into
pREX0052 cut
with AflII and EcoRl to create pREX0730 (Figure 4).
[00340] The plasmid was cut with the restriction enzyines NotI and Pvzal and
ligated into
pSAC35 (Sleep et al. (1991) Biotecluiology 9:183-187) cut with NotI to create
the yeast
expression vector pREX0731 (Figure 5).
C terminal fusion of BNP: nL mTf (PEAPTD)2 BNP(1-32) (pREX0722/723)
[00341] The BNP protein was inserted at the C-tenninus of mTf using
overlapping primer
sequences encoding the adjoining mTf sequence. Priiners P1230 (reverse primer)
and P1231
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92
(forward primer) were designed to introduce the BNP sequence and (PEAPTD)2
(SEQ ID
NO: 142) linker at the 3' of the mTf sequence.
P1230 reverse primer:
TTTTCTACCAAAACAACCAGAACCTTGAACCATTTTTGGAGAATCAGTTGGAGCT
TCTGGATCAGTTGGAGCTTCTGGAGGTCGACGGAAAGTGCAGGC (SEQ ID NO:
171)
P 1231 forward primer:
CAAGGTTCTGGTTGTTTTGGTAGAAAAATGGATAGAATTTCTTCTTCTTCTGGTTT
GGGTTGTAAAGTTTTGAGAAGACATTAATAAGCTTAATTCTTA (SEQ ID NO: 172)
[00342] In the first round of PCR, two products were generated using
P1230+P0007 or
P1231+P0026 with pREX0197 as the template.
Outer primers:
P0007: TGCGTCCACAAGATATTACG (SEQ ID NO: 173)
P0026: GTTTTCCCAGTCACGAC (SEQ ID NO: 174)
The products from these reactions were then joined in a second round of PCR
using outer
primers P0007 and P0026. The product of this reaction was cut with HindI1I and
SaII and
subcloned into pREX0197 cut with the HiiadIII and Sall to create pREX0722
(Figure 6).
[00343] This plasmid was cut with the restriction enzynnes NotI and Pvut and
ligated into
pSAC35 cut with Notl to create the yeast expression vector pREX0723 (Figure
7).
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+1 A P U U A b) A
4-) E +-) ro
~ ~
d = 'u b ,
-P c = ,.... ~ ,.. U
E cd N x ro N 41
E (d M U U C] P, +J = .~
E (d W P-i '... U U E N =
C7 m U U 41 W
N U ro U Z7
N 41 = U) C7 b) W m =
w ~ ~ A ro ~ ~ ~ ro ~ ~
v U A ~
) U U A b U = 4-I
U. b) = rl U U A 4-)
t~ ,: FC .I-) . ,.. E 4-i A ,LI lJ =
"0 = n
~ tm
~ ~ . ~ ~ ~
0 . b+
Z U~ bl ; H A ~ r0 = S; U =
Ll G U = rn '... C7 tT = U = 0
H . C) b) = b~ N 'd [r
C) tn N C7 tn ,-I .t1
~ E +~ tõ x
c.
a ) ~
W a
u] tn = rl H 41 = ~d =
U = 5 ~ c~ tn U 4-)
x rtl
J-) = H rG ro
tn = H' FFCG rd = U = a1
U ; ro ~ m E ~!-) = U b~
UU = U U
(y' E+
tJ) = FC . E 4j = tT tT 4-I
i~ = U O 0 b) = t~ H -ci
U Z C? b) = ro b~ ~ ~ = H b, = N
b) = ~-I H H 41 = ro
=
U = U Oa 0 0) = ~ C) = cn
tT = i-1 'U W U' tT = U =
4-1 = FC cn . H1~ = m 4-1
=
U U' U = _. tn = D
U d E +1 V
b : Ft7C' v ~ ~ ro ~ ro
0) ~U U . U7 t
T ; U
4-1
b~ = ~ E 41 cV _ tT =
b~ = t7'.... .. CE,> . U M U) b, . 3
a,
u 4-1
OZ) ro fA rl ', ~ ~ : FI
~ = rl N
.!J = N H t6 = }.l r~y ctl = _
UU = N W H ro = EU-, ~ tn
0) FC +3 d c7 ~ = c-
f(i
O) = v U tr) = U U = ~ H
b~ A r-F~6 F~ ~ ~ = x Z
~C
ro H,.. ro x E~ ~ H
U H ro = H U' 0) . O~
U)
rtl H N~ = A U U = Q
ro r-C +~ = tT rn U U =
N
b U ~ ~ E 4-1 A al
ro .u = 4-4 w C7 a) A
U
A
U ~.. ~ = FC N A
41 ~~~CCC N
4-1 U b) = U H ~ = 4J
=r1 d-) U b) = ~ ~ rd N p~ ,.~
N = Q
w ro 9 h U
C = N H .u 9 rt1 'Z~
N rotl r~L -~ = b~ CUr~7 b~ W C
+-I U J ~ = r~ b U =i
~ b EH-H co ro p,
~ r~ C7 U A U U A
z r-I ~-1 rl o
0 0 o N
PW 00 m C cm
z ~ o
an
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x
n H ~ m A
b = E b
tn = rl I H.''~.. N =
tD E , ~d . S-I
U H ~ ..,. H 4-) b,
11 U U' bi
U = ul U A 0 0 =
rd = ~ H 4-) w
U = .-I
m E 4-)
N . ul I ~N-I H ~ = U
u U w 0 U)
U ; ~V ,..~.. ~ ~~,.~..~..~,..~.. H ~ = tT
U = ...~.. U ...~~~.. 0 b) =
N = u~ ', U' C7 b, =
U .N U)
U C7 N U U N
}1
U r-C ~ E a-~
b~ = FUC... .~....,, ~ w ~
ro : x E 0 m azi rr
~ Q v ~
ro oI : ~.
~ = ~--i ,. U .., cWi) tn = H
41 = A .~-I ro = E
U H rd A" H
b . .. E.y
N ts O H ~d
tT 2 CE7 U ~ H I H
b~ = ',~ C1 C'0 C) = H I U
41 . H '.. FG 41 = m Tf 1 U'
-P = ~ I
tn b a ~ .~ A x +
4-J = m ~ .u A ~ El
ro x o ~ b A 10 = P m ~. H cd = FC
~ w ~ ~
tn F~ a
v 0 .u
~ u
~ ~ ~ U ~
ca = E (e = a ~C
ca E m ~
b) = 4) U. t71 = ' E
r6 = C7 U H Q H
tD = b~ C7 U () H
(a = x
bI ~ H r41d ~riI ~ A U
b~ = r-I U' tn rl ~--i a;
~ N FHG
. ,-.. ~ U = -I~
dJ >r E ' ttl = 1-1 C7
U H ro S~, W H
cd C9 U H
A-1 = X CD U = C9
rd = ~ .I-J = (0 U"
rt
C7 U C7
lo ro ~ ; U '.....H
ro = H ro = E
tn = ~ U tn = C7
rU = f.7.~' C) E
rt . ~ .l~ A P, E
Q U = C7~, U A H
CU-+ rUtl : FI H
ro = u ro i ~ ~ = v
ro , en +
b) = 1 ~ U~~, b) S-1 E
- Cl
U ~ ~ U
U va H
~ H
N b) = .~ ~,FC 41
~.,.. U .N
N w ~ ... I
H
~ rl ~ ~
ro n
H U H
N r O
oC) o 0)
o z 00
'-' (rI N
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tDn
ro
ro
41
ro
ro
ro
-P
rt
-P
a~
ro
~
~
4J
~p
.u
rt
rn
-P
ro
~
-P
-P
ro
-P
ro
-P
-P
U
~
ro
ro
4J
+1
U
ro -x
(d
-P
ro -x
ro
+1
A 4
ro A
U
ro s4
ro =
ro =
ts =
ro =
tn =
.u
_u b
ro = ~,
ro =
(Ti
1J U _
4-) = r
tm
ZT N rl O
.IJ M
4-) 00
-F1 b Ca
w H -I
4-) U) O
u ~ z
4-)
4-) N
U H
4-) a)
N rn ~ Ol
U
4-) a) cf)
4-) N l7
U v
~
4-J U
rti
ro S-I
41 v)
ro z
tD)
ro A
O
O N CO
M O O
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96
Example 2: ANP/mTf Fusion Proteins.
[00344] ANP is part of a hormonal system in which one gene synthesizes four
peptide
hormones. Regulation of ANP levels in the blood would be a therapeutic
approach to the
treatment of such disorders as hypertension, shoclc, and the like. While
current native and
synthetic ANP, as well as analogs thereof, would allow for the modulation of
fluid volume
and vascular function by increasing ANP levels, effective therapies may also
require ANP
levels to be reduced in order to achieve the desired extracellular fluid
volume and electrolytic
homeostasis.
[00345] The present invention provides fusion proteins with ANP fused to mTf
(S415A,
T613A) in order to extend stability in vivo and also provides pharmaceutical
compositions for
treating a patient in need thereof. In this exainple, the steps for producing
an ANP/mTf
fusion protein are described. The same steps may be used to generate
transferrin fusion
proteins with other natriuretic peptides such as ANP analogs or derivatives,
etc.
[00346] The human ANP protein sequence (SLRRSSCFGGRMDRIGAQSGLGCNSFRY)
(SEQ ID NO: 179) was obtained from PubMed (Accession no. NM_006172), back
translated
into DNA codon optimized for yeast:
TCTTTGAGAAGATCTTCTTGTTTTGGTGGTAGAATGGATAGAATTGGTGCTCAAT
CTGGTTTGGGTTGTAATTCTTTTAGATAT (SEQ ID NO: 180)
N-terminal fusion of ANP: nL ANP(1-28) (PEAPTD)2 mTf (pREX0826/827)
[00347] The ANP protein was inserted at the N-tenninus of mTf using
overlapping primer
sequences encoding the adjoining mTf sequence. Primers P1552 (reverse primer)
and P1553
(forward primer) were designed to introduce the ANP sequence and (PEAPTD)2
linker at the
5' of the mTf sequence, 3' of the nL leader sequence.
P1552 reverse primer:
CAACCCAAACCAGATTGAGCACCAATTCTATCCATTCTACCACCAAAACAAGATG
ATCTCCTCAAAGACGCCAGACACAGC (SEQ ID NO: 181)
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P1553 forward primer:
TGCTCAATCTGGTTTGGGTTGTAATTCTTTTCGTTATCCAGAAGCTCCAACTGATC
CAGAAGCTCCAACTGATGTACCTGATAAAACT (SEQ ID NO: 182)
[00348] In the first round of PCR, two products were generated using
P1552+P0025 or
P1553+P0012 with pREX0197 as the template.
Outer primers
P0012 : CATGATCTTGGCGATGCAGTC (SEQ ID NO: 169)
P0025 : AGCGGATAACAATTTCACACAGGA (SEQ ID NO: 170)
The products from these reactions were then joined in a second round of PCR
using outer
primers P0025 and P0012. The product of this reaction was cut with the
restriction enzyines
AfiIl and EcoRI and subcloned into pREX0052 cut with AflII and EcoRI to create
pREX0826
(Figure 12).
[00349] This plasmid was cut with the restriction enzymes Notl and Pvul and
ligated into
pSAC35 cut with the restriction enzymes Notl to create the yeast expression
vector
pREX0827 (Figure 13).
C-terminal fusion of ANP: nL mTf(PEAPTD)2 ANP(1-28) (pREX0828/829)
[00350] The ANP protein was inserted at the C-terininus of mTf using
overlapping primer
sequences encoding the adjoining mTf sequence. Primers P1554 (reverse primer)
and P1555
(forward primer) were designed to introduce the ANP sequence and (PEAPTD)2
(SEQ ID
NO: 142) linlcer at the 3' of the inTf sequence.
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98
P1554 reverse primer:
ATTCTACCACCAAAACAAGATGATCTCCTCAAAGAATCAGTTGGAGCTTCTGGAT
CAGTTGGAGCTTCTGGAGGTCGACGGAAAGTGCAGGC (SEQ ID NO: 183)
P1555 forward primer:
CATCTTGTTTTGGTGGTAGAATGGATAGAATTGGTGCTCAATCTGGTTTGGGTTGT
AATTCTTTTCGTTATTAATAAGCTTAATTCTTATG (SEQ ID NO: 184)
[00351] In a first round of PCR, two products were generated using P
1554+P0007 or
P1231+P1668 with pREX0197 as the template. The products of these reactions
were then
joined in a second round of PCR using the outer primers P0007 and P 1668.
Outer primers
P0007: TGCGTCCACAAGATATTACG (SEQ ID'NO: 173)
P1668: GAGCGACCTCATGCTATACC (SEQ ID NO: 185)
[00352] The product of this reaction was cut with was cut with the restriction
enzyines
HindI11 and SaII and cloned into pREX0197 cut with HindIIl and SaII to create
pREX0828
(Figure 14).
[00353] This plasmid was cut with the restriction enzymes Not1 and PvuI and
ligated into
pSAC35 cut with the restriction enzymes Notl to create the yeast expression
vector
pREX0829 (Figure 15).
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99
U' U A Ul ~ A 'U A
ro S-I U U = U =
U tn oo H, ro U
'. H ~d N U U = 4-) =
.'. U b) 1 44 C7 b) = 4J =
=
... U tn rl .,-...,. N = N ro
U b) a, rl C7 b~ ~
4-) ~ v ~ : 0 ~
u
~ ch u n ~ H .u '~ ro
P4 ~ A ro 0 ~ Q ro
v U b~ A U U ~ ~ m
t U bI = rl (d W d-) =
'.,Fz~ ... 41 = cd w ui co
=
~ u : U u
~ H ~
+
~ u -) ro ~
1-4 U ~ = =-I tU'J U) = N
C~J U n1 ~ UtDi
Z U ~ = ~ ,~' ~ = F14 ~ =
b) = A U U U
q A
H 0) = =-I M U U A 41 =
O U tn H rt A > ~
W U =U ' 0) =
cn 41 = w H 4J = ~I ro =
ts = C7 t3~ b~
U ro C) U (d
H +J 4-1 U
b) H 4-) = U
U U H 4-J = b~
N
b, = m H .t~ = U) =p
41 = ,-I U U = .. b~ =
.t-U) Fl -P = ~ 1
b~ C =-I rtl U
41 H =1-1 U (tl
U U L U' b) = U 4-
~ = H ~ H H ~ = b~ t7~ ~
O,
U . U W U' tT _ b
U m U m c7 b ro
U t)' ~ 0 ts = rl b~
b H +-) = 4-)
b 0) ~ H i . b) U
[-
is U (7 bl = b)
t:
41 y) : ~> ~ H ~
b', Ur U U
U r0 FC H +J U
~ U N 23' ~
.u = ~C H .u co tn
=
U = C.7 U U 1-1 co
9 [ 0 ~ - tT b'
tl U U) w d-~
b~ = ~ C-> tT ~ ~'
-P A N ~ 4-) = =,q 4-)
b~ A ~n H c~a = b~
rtl -i H co = ~4 4-1 =
ra w U tn = ro
b, v E
ro U ~ ~ tr~
U r-C a-) = H 4u
co H (d = U U
ro H rtS = N ro
U H (0 R3
d U b) = ~ H +1
u ~ 0)
o ro ~ .~ A
~I +J U b) = m H 4-)
Ln
~H U U ~ = 4-4 =N-1 C) U
ro ~ 4J w < co
N ro .~ : U C H 7 b~ A
~ 0 .~ ro A
b) .u u) t7 b
ai ~ FC .~ A U U A
-P
Zi -I r-I
p p O
a co rn o
- ~
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100
P4 6)
4J . ~ ro
~ u m ~
ro : u) H ro ~ s4
U A
U t7' m
u) U) ~ ~ H ~
U = U u') U U~
-14 4J 4-) U r-I 0 m
U U P4 CH7 b~ ts
F.j m
U CJ E~' 4-4
U = ~', H =U
.P m
~ ~ ~ ~ a U
r~6 x H -4pJ ~ ro
r6 = C7 O D U
~4 ~ = ~ ~
H z ~ tl m
b~ = CJ q CU. U
4-1 (tl = rl KG' H .. . 4J =
~
H a ra u
4-) = ~r w ~
m U = q H ~ nf
U U, ~ b 54
N = U b~
b' rd tT H N
.P U b~ rl
U b) b -P
n
4-) = ~~CC 4-)
4-3
.14 4-) A -0
U H co A
u ~ ~ 4-J
rt U' U
co U = ~ , ~ ................ ~ = 04 H
4-) 4-I <n C7 U = H
N 4-) 4-) = ~ '0
ro = W C7 U = -rl
.p = N tJ b) = ~
rd = V [-i (d = N
rt = H ro
4) b~ = N UtT H
N = CH'J U q ~
m b = ts t7 U H
ro = ~=, a
co FG +J ~C Cf
0) - H m w
~
~ u ~ w
ro .u = ~, z H ro
U = H (d = S~
i = q C7' U
U 4j = .~ H (7 U =
m = < 4-J = b
(d a
W
(d U, U
3 a~ u~ U tn
c6 = -- H ,......... rd = N
r6 . H .... R9 =
S-I b) = ~ U' b)
rn -I j H R1 G~+
rl qf = (.7 U A
-Fj = .U U' .. 0 A
O U = C~~j U A ~
z
4-i v) cz = C I U' .. U =
q rn U = H I H b ' ~4
H .-I rd = -I I C7 b~ =
0 ~ ; s~ ~ + ~ ~ ~
z c ~ ~ = w
q 0 (d 4-) 4-I
Q U H N ~ 4-3 1~
4-3 = 4-J
FC4 w U = ~ N ro
a~i ro ~ : ~
'0 cn (D ~ U = ?,'' r-C; .;. 4-J
CD7 C U ro
a) U A ~ U fd A N
rt b' .1J U
N I
0 ~n U H
C -I
~ z 7j m N
q F4 H
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101
ro
m
ro
ro
ro
ro
ro
ro
ro
-P
~
ro
ro
-P
ro
ro
m
~
-P
ro
-
,N
ro
~
-P
~p
-P
4J
ro
~
-P
ro
~
~
-P
ro
H -P
~ ro
H -P
H -P
U U
H -P
H -P
~ ro
ro
H .P
H 41
U U
U' tr
+ FU r6
FC rt
~ ~ ~
(Z
H d-)
E. 41 A >1
(0 A
H .IJ
H -1-J ~4
t7 0)
U U
H 4-) 4-a
A H 4-)
H 41
.n E-H 4J m
Un U U
N 4-)
ri
Pa E- 1-) : A
a~GG ro
aS
H +J U
C~ b) = ~
H 41 rn
H a-3 b~ rl
CD 0) co
C7' b~ N_
w7 z
4J
H rn
H .I-) = b~ H -1
(D 0)
(D o)
H 4J U) W O
U U = u) Z
H' ,N
~ ro ; ~ == q
d 4) H
= U
U U = q 01
H +) = (tl 4) W
U U ~ uo
C7 O~ = U'
H .F- b~ U)
(7 tT = cq N
U D~ U
H .1-) =~ 'U q
H d-) =r-I a)
~ rd A
ro b
a)
I 0 a)
O C
O ~ Z
G1
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Example 3: Vessel Dilator (1-48) (PEAPTD)2 mTf
[00354] Fusion of the vessel dilator sequence to the N-tenninus of mTf (S415A,
T613A)
was achieved in a three step process. Two PCR reactions were performed
initially to add the
5' and 3' ends of the vessel dilator DNA sequence to the ends of the leader
sequence and
linker peptide respectively. Using the plasmid pREX0549 (Figure 8) as a
template with
primers P0669 and P2280 added the 5' sequence of vessel dilator to the leader
sequence
(PCR1). Using the template pREX0584 (Figure 9) and the primers P2278 and P0478
added
the 3' sequence of vessel dilator to the linker peptide (PCR2).
[00355] The products of these two reactions were then extended to add
sufficient sequence
of vessel dilator, PCR1 with P0669 and P2281 (PCR3) or PCR2 with P2279 and
P0478
(PCR4), that the 3' of PCR3 overlapped with the 5' of PCR4. The final reaction
was to use
the outer primers P0669 and P0478 to join PCR3 and PCR 4 together.
[00356] The product of this final reaction was digested with the restriction
enzymes AflII
and EcoRI and ligated into pREX0549 cut with the same enzyines to give
pREX1140 (Figure
10). Clones were checked by restriction digest and DNA sequenced to confinn
correct
insertion free of any PCR induced errors.
[00357] The expression cassette was recovered from pREX1140 by digestion with
the
restriction enzyine NotI, with the addition of P>>ut to cut the vector
backbone. This was then
ligated into the yeast expression vector pSAC3 5 cut with NotI and
dephosphatased with
Antarctic phosphatase to give pREX1146 (Figure 11).
P2278
GAAGTCAGCCCAGCCCAGAGAGATGGAGGTGCCCTCGGTAGAGGTCCATGGGAC
CCAGAAGCTCCAACTGATCCAG (SEQ ID NO: 186)
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103
P2279
CCCTGAGGTGCCTCCCTGGACCGGTGAAGTCAGCCCAGCCCAGAGAGATGGAGG
TGCCCTCGGTAG (SEQ ID NO: 187)
P2280
GAGCAGCACCCGCTTCTTCATTCGGCTCACTGAGCACTTGTGGTGGCACGACCTC
CGCCAGACACAGCCCCAGGACG (SEQ ID NO: 188)
P2281
GTCCAGGGAGGCACCTCAGGGAGTGGGCTGAGAGCAGCACCCGCTTCTTCATTC
GG (SEQ ID NO: 189)
P0669 CCGCGATAAAGAGCGCGATG (SEQ ID NO: 190)
P0478 GTACATCTTGGCATCCATCC (SEQ ID NO: 191)
PstT
-----+-
601 tgttgaaaaa gcaaaatttg ggctcagtaa tgccactgca gtggcttatc acgccaggac
(SEQ ID NO: 190) P0669 >
CCGC GATAAAGAGC GCGATG
661 tgcgggagtg gcgggggcaa acacacccgc gataaagagc gcgatgaata taaaaggggg
Af1II
721 ccaatgttac gtcccgttat attggagttc ttcccataca aacttaagag tccaattagc
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781 ttcatcgcca ataaaaaaac aagctaaacc taattctaac aagcaaagat gaggctcgcc
...nL..... >
m r 1 a
841 gtgggagccc tgctggtctg cgccgtcctg gggctgtgtc tggcggaggt cgtgccacca
GCAGGAC CCCGACACAG ACCGCCTCCA GCACGGTGGT
(SEQ ID NO: 188) < P2280
> ...................... nL ...................... >>
v g a 1 1 v c a v 1 g l c 1 a e v v p p
vessel dilator >> ............. >
(SEQ ID NO: 187) P2279
CCCTGAGGTG
901 caagtgctca gtgagccgaa tgaagaagcg ggtgctgctc tcagcccact ccctgaggtg
GTTCACGAGT CACTCGGCTT ACTTCTTCGC CCACGACGAG
P2280
GGCTT ACTTCTTCGC CCACGACGAG AGTCGGGTGA GGGACTCCAC
(SEQ ID NO: 189) < P2281
> ........................ vessel dilator ......................... >
q v 1 s e p n e e a g a a 1 s p 1 p e v
P2279 >
CCTCCCTGGA CCGGTGAAGT CAGCCCAGCC CAGAGAGATG GAGGTGCCCT CGGTAG
P2278
GAAGT CAGCCCAGCC CAGAGAGATG GAGGTGCCCT CGGTAGAGGT
961 cctccctgga ccggtgaagt cagcccagcc cagagagatg gaggtgccct cggtagaggt
GGAGGGACCT G
P2281
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105
> ........................ vessel dilator......................... >
p p w t g e v s p a q r d g g a 1 g r g
P2278 > (SEQ ID NO: 186)
CCATGGGACC CAGAAGCTCC AACTGATCCA G
1021 ccatgggacc cagaagctcc aactgatcca gaagctccaa ctgatgtacc tgataaaact
>...... vessel dilator
p w d p e a p t d p e a p t d v p d k t
>> ............. (PEAPTD)2 .............. ....mTf.. ....
1081 gtgagatggt gtgcagtgtc ggagcatgag gccactaagt gccagagttt ccgcgaccat
> .............................. mTf.............................. >
v r w c a v s e h e a t k c q s f r d h
1141 atgaaaagcg tcattccatc cgatggtccc agtgttgctt gtgtgaagaa agcctcctac
> .............................. mTf............. ................. >
m k s v i p s d g p s v a c v k k a s y
1201 cttgattgca tcagggccat tgcggcaaac gaagcggatg ctgtgacact ggatgcaggt
> .............................. mTf .............................. >
1 d c i r a i a a n e a d a v t 1 d a g
1261 ttggtgtatg atgcttacct ggctcccaat aacctgaagc ctgtggtggc agagttctat
> .............................. mTf.............................. >
1 v y d a y 1 a p n n 1 k p v v a e f y
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BamHI
-+----
1321 gggtcaaaag aggatccaca gactttctat tatgctgttg ctgtggtgaa gaaggatagt
> .............................. mTf.............................. >
g s k e d p q t f y y a v a v v k k d s
1381 ggcttccaga tgaaccagct tcgaggcaag aagtcctgcc acacgggtct aggcaggtcc
> .............................. mTf .............................. >
g f q m n q 1 r g k k s c h t g 1 g r s
1441 gctgggtgga acatccccat aggcttactt tactgtgact tacctgagcc acgtaaacct
> .............................. mTf.............................. >
a g w n i p i g 1 1 y c d 1 p e p r k p
1501 cttgagaaag cagtggccaa tttcttctcg ggcagctgtg ccccttgtgc ggatgggacg
>
> .............................. mTf.........
1 e k a v a n f f s g s c a p c a d g t
1561 gacttccccc agctgtgtca actgtgtcca gggtgtggct gctccaccct taaccaatac
> .............................. mTf.............................. >
d f p q 1 c q 1 c p g c g c s t 1 n q y
1621 ttcggctact cgggagcctt caagtgtctg aaggatggtg ctggggatgt ggcctttgtc
> .............................. mTf.............................. >
f g y s g a f k c 1 k d g a g d v a f v
1681 aagcactcga ctatatttga gaacttggca aacaaggctg acagggacca gtatgagctg
> .............................. mTf.............................. >
k h s t i f e n 1 a n k a d r d q y e 1
CA 02625600 2008-04-10
WO 2007/047504 PCT/US2006/040207
107
1741 ctttgcctgg acaacacccg gaagccggta gatgaataca aggactgcca cttggcccag
> .............................. mTf .............................. >
1 c 1 d n t r k p v d e y k d c h 1 a q
1801 gtcccttctc ataccgtcgt ggcccgaagt atgggcggca aggaggactt gatctgggag
> .............................. mTf .............................. >
v p s h t v v a r s m g g k e d 1 i w e
EcoRI
--}------
1861 cttctcaacc aggcccagga acattttggc aaagacaaat caaaagaatt ccaactattc
> .............................. mTf .............................. >
1 1 n q a q e h f g k d k s k e f q 1 f
1921 agctctcctc atgggaagga cctgctgttt aaggactctg cccacgggtt tttaaaagtc
> .............................. mTf .............................. >
s s p-h g k d 1 1 f k d s a h g f 1 k v
1981 ccccccagga tggatgccaa gatgtacctg ggctatgagt atgtcactgc catccggaat
CCT ACCTACGGTT CTACATG
< P0478 (SEQ ID NO: 191)
> .............................. mTf.............................. >
p p r m d a k m y 1 g y e y v t a i r n
[00358] Amino Acid Sequence: SEQ ID NO: 192
[00359] DNA Sequence: SEQ ID NO: 193
CA 02625600 2008-04-10
WO 2007/047504 PCT/US2006/040207
108
[00360] Although the present invention has been described in detail with
reference to
examples above, it is understood that various modifications can be made
without departing
from the spirit of the invention. Accordingly, the invention is liinited only
by the following
claims. All cited patents, patent applications and publications referred to in
this application
are herein incorporated by reference in their entirety.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 108
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