Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
POLYMERIC IMMUNOGLOBULIN RECEPTOR (PIGR)-BINDING
DOMAINS AND METHODS OF USE THEREFOR
BACKGROUND OF THE INVENTION
This application claims priority to United States provisional patent
application
60/119,932, filed on February 12, 1999, which is specifically incorporated by
reference in its entirety herein without disclaimer. The United States
government
owns rights in the present invention pursuant to grant number AI44206-OIAl
from
the National Institutes of Health.
1. Field of the Invention
The present invention relates generally to the fields of diagnostic,
preventative,
and therapeutic treatment compositions and methods. More particularly, it
concerns a
system for delivering agents to mucosal epithelia using a binding domain that
recognizes and binds a polymeric immunoglobulin receptor.
2. Description of Related Art
The largest area of the body that is exposed to external pathogens is the
mucosal surfaces, which constitutes 400 square meters of surface area, as
compared to
1.8 square meters of skin coverage (Childers et al., 1989). Not surprisingly,
infections
frequently involve the mucosal surfaces. IgA is the antibody class primarily
found in
mucosal secretions; thus, IgA antibodies serve as a first line of immune
defense.
To be released in mucosal secretions, IgA, which is produced by plasma cells,
requires translocation across mucosal epithelium from the basal membrane side
of the
cells to the apical membrane side. Endocytosis and transcytosis of IgA is
mediated
through the polymeric immunoglobulin receptor (pIgR), which is located at the
basal
membrane of mucosal epithelium (Mostov, 1994; Mostov et al., 1982). IgA in
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mucosal secretions is then able to mediate an active or passive immune
response.
Two different mechanisms have been described to account for IgA's
antipathogenic
activities: active immunity involves generating a molecular and cellular
immune
response against the pathogen through Fc receptor binding or complement
activation;
passive immunity does not involve stimulation of the immune system and instead
occurs, for example, through IgA's ability to block viral attachment to host
cells or to
inhibit bacterial motility.
A number of studies have demonstrated the association between strong
mucosal IgA responses and protection against viral infection with rotavirus
(Underdown and Schiff, 1986, Feng et al., 1994), influenza virus (Taylor and
Dimmock, 1985, Liew et al., 1984), poliovirus (Ogra and Karzon, 1970),
respiratory
syncytial virus (Kaul et al., 1981 ), cytomegalovirus (Tamura et al., 1980)
and
Epstein-Barr virus (Yao et al., 1991). Secretory IgA is therefore, successful
in
preventing these viruses from gaining access to the body by blocking infection
at the
site of entry, namely the mucosal surface. Passive immunotherapy with
intranasal
IgG Fabs was protective against respiratory syncytial virus (Crowe et al.,
1994),
showing that the mere presence of neutralizing anti-viral antibodies, without
any
effector function, at the mucosal surface can prevent viral infection.
Furthermore,
HIV-specific IgA, which is transported to mucosal secretions, may be used as a
vaccine to elicit protective antibody-mediated immunity (U.S. Serial No.
08/779,597,
hereby incorporated by reference).
Despite this information, there remain a number of questions regarding how
IgA functions and, in particular, how IgA is transported. It would be of great
benefit
to know the nature and. identity of the structures, including IgA structures,
that are
responsible for targeting and transport of IgA to mucosal epithelium via pIgR.
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SUMMARY OF THE INVENTION
Therefore, it is a goal of the present invention to provide structures that
confers targeting and transport to the mucosal epithelium. It also is a goal
of the
present invention to provide a variety of compositions and uses for this
structure.
In accomplishing these and other goals, there is provided an isolated pIgR-
binding domain. A "pIgR-binding domain" refers to an amino acid motif or
structure
that is capable of binding a polymeric immunoglobin receptor (pIgR). This
domain
confers, upon molecules to which it is attached, the ability to be targeted to
mucosal
epithelium. In particular embodiments, the invention relates to an isolated
peptide of
between 10 and about 50 residues comprising a pIgR-binding domain. Additional
embodiments recite a peptide containing a pIgR-binding domain that is 10
residues in
length, about 15 residues in length, about 20 residues in length, about 25
residues in
length, about 30 residues in length, about 35 residues in length, about 40
residues in
length, about 45 residues in length, or about 50 residues in length. The
invention
further comprises a peptide that comprises the sequence SEQ ID NO:1, as well
as a
peptide that comprises a sequence selected from the group consisting of SEQ ID
NOS:2-33. Moreover, in other embodiments, the pIgR-binding domain comprises
the
Ca3 domain of IgA and fragments thereof. Amino acids flanking a pIgR-binding
domain that includes the Ca3 domain of IgA may also be included in
compositions of
the present invention. Flanking regions could include 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15,
20, 25, 30, 35, 50, or more amino acids on one or both sides of a pIgR-binding
domain. It is contemplated that the limitations and embodiments related to a
peptide
that contains one pIgR-binding domain could also be employed with respect to a
peptide containing more than one pIgR-binding domain. Such a multimeric
peptide
could contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more pIgR-binding domains.
Other embodiments of the present invention include a peptide containing a
pIgR-binding domain, where the peptide is attached to a linking moiety such as
SMTP, SPDP, LC-SPDP, Sulpho-LC-SDPD, SMCC, Sulfo-SMCC, MBS, Sulfo-
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MBS, SIAB, Sulfo-SIAB, SMPB, Sulfo-SMPB, EDC/Sulfo-NHS, and ABH.
Generally, the term "linking moiety" refers to a structure having the chemical
or
pharmacological property of linking or being able to link other compounds. In
a
multimeric peptide containing more than one pIgR-binding domain, a linking
moiety
may connect two or more pIgR-binding domains with each other.
The peptide can also be attached via the linking moiety to a selected agent,
which in some embodiments is a therapeutic or preventative compound. The
selected
agent can be, for example, a peptide, a polypeptide, an oligonucleotide, a
polynucleotide, a detectable label, or a drug. Moreover, the present invention
further
comprises a polypeptide that is an enzyme, antibody region, region involved in
protein-protein interactions or ligand-receptor interactions, cytokine, growth
factor,
hormone, toxin, tumor suppressor, transcription factor, or inducer of
apoptosis. In
additional embodiments, the selected agent is a polynucleotide that encodes a
polypeptide, a single chain antibody, an antisense construct, or a ribozyme.
Other
examples of the invention disclose a detectable label that is rhodamine or
fluorescein,
or is a radiolabel. In still further embodiments, the peptide containing the
pIgR-
binding domain is linked to a drug, such as an antibiotic, a DNA damaging
agent, an
enzyme inhibitor, or a metabolite.
The present invention also describes in some embodiments a pIgR-binding
domain within a peptide that is linked to a non-pIgR targeting agent. A "non-
pIgR
targeting agent" or "non-pIgR moiety" refers to a structure that allows the
targeting of
a molecule that is not pIgR. In some cases, the non-pIgR targeting agent is an
antigen
binding domain of an antibody, while in other cases it is a receptor ligand or
a ligand
binding domain. Thus, in these examples, the targeted molecule is either an
antigen, a
receptor, or a ligand, respectively.
In yet further embodiments, the invention covers a fusion protein containing a
pIgR-binding domain covalently linked to a non-antibody peptide or
polypeptide.
Some examples of the present invention involve a pIgR-binding domain that
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comprises a Ca3 domain of IgA. The non-antibody peptide or polypeptide can be
selected from the group consisting of an enzyme, region that mediates protein-
protein
or ligand-receptor interaction, cytokine, growth factor, hormone, toxin, tumor
suppressor, transcription factor, and inducer of apoptosis.
5
The present invention additionally encompasses a polynucleotide that encodes
a fusion protein containing a pIgR-binding domain covalently linked to a non-
antibody peptide or polypeptide sequence. In some embodiments, the non-
antibody
peptide or polypeptide is selected from the group consisting of an enzyme,
region that
mediates protein-protein or ligand-receptor interaction, cytokine, growth
factor,
hormone, toxin, tumor suppressor, transcription factor, and inducer of
apoptosis.
The invention also includes a method for targeting a selected agent to mucosal
epithelium by at least providing a complex containing the selected agent and
an
isolated peptide of between 10 and about 50 residues that comprises a pIgR-
binding
domain; and administering the targeting complex to a mammal, such that the
complex
binds to cells expressing pIgR, is taken up by those cells, and is transported
to the
mucosal epithelium. Embodiments further describe this method, which is
administered via oral, inhalation, ocular, nasal, vaginal, rectal,
intravenous,
subcutaneous, intramuscular, or intraarterial routes. In some examples, this
method
also includes a second, non-pIgR targeting moiety.
Another method of the present invention for targeting a non-antibody peptide
or polypeptide to mucosal epithelium includes: providing a fusion protein
containing a
pIgR-binding domain covalently linked to a non-antibody peptide or
polypeptide; and
administering said targeting complex to a mammal, whereby the targeting
complex
binds to cells expressing pIgR, is taken up by said cells, and is transported
to the
mucosal epithelium. In yet further embodiments, this method can be
administered via
oral, ocular, nasal, vaginal, rectal, intravenous, or intraarterial routes.
More
embodiments disclose a method where the fusion protein also includes a second
targeting moiety such as a non-pIgR targeting agent.
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The invention described herein also encompasses a method of delivering a
selected agent to a cell comprising: providing a complex containing the
selected
agent and an isolated peptide of between 10 and about 50 residues comprising a
pIgR-
binding domain; and contacting the targeting complex with a cell expressing
pIgR.
Additional examples include transforming the cell with an expression construct
encoding pIgR under the control of a promoter operable in said cell before
providing
the complex. Still further embodiments disclose a complex that also contains a
second targeting moiety such as a non-pIgR targeting agent.
Another method of delivering a non-antibody peptide or polypeptide to a cell
comprises: providing a fusion protein comprising a pIgR-binding domain
covalently
linked to a non-antibody peptide or polypeptide; and contacting that fusion
protein
with a cell expressing pIgR. This method also embraces the step of first
transforming
the cell with an expression construct encoding pIgR under the control of a
promoter
operable in the cell before providing the fusion protein. Other embodiments of
this
method include a fusion protein that also contains a second targeting moiety
such as a
non-pIgR targeting agent.
The use of the word "a" or "an" when used in conjunction with the term
"comprising" in the claims and/or the specification may mean "one," but it is
also
consistent with the meaning of "one or more," "at least one," and "one or more
than
one."
Other objects, features and advantages of the present invention will become
apparent from the following detailed description. It should be understood,
however,
that the detailed description and the specific examples, while indicating
preferred
embodiments of the invention, are given by way of illustration only, since
various
changes and modifications within the spirit and scope of the invention will
become
apparent to those skilled in the art from this detailed description.
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BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included
to further demonstrate certain aspects of the present invention. The invention
may be
better understood by reference to one or more of these drawings in combination
with
the detailed description of specific embodiments presented herein.
FIG. lA-D. Binding of monomeric and dimeric IgA/IgG domain swap mutant
antibodies to pIgR expressed on MDCK cells. FIG. lA. Staining of MDCK cells
with sheep anti-pIgR (heavy line) antiserum or normal sheep serum (broken
line)
followed by anti-sheep IgG FITC conjugate. FIG. 1B. Binding of wild-type IgA
monomer (thin line) or dimer (heavy line) to pIgR on MDCK cells. FIG. 1C.
Binding of VGAA mutant expressed as monomer (thin line) or dimer (heavy line)
to
pIgR on MDCK cells. FIG. 1D. Binding of VGGA mutant expressed as monomer
(thin line) or dimer (heavy line) to pIgR on MDCK cells. Bound IgA or IgA/G
chimeric antibodies were detected by rabbit anti-human kappa chain-FITC
conjugate.
Negative controls are shown as broken lines.
FIG. 2. Alignment of deduced peptide sequences from selection of phage
display peptide library against pIgR receptor-expressing cells with the human
Ca3
domain amino acid sequence (SEQ ID NOS:2-33). Peptides designated A or M are
from the acid-eluted and cell-associated fractions respectively. Numbering of
IgAl is
according to reference (Putnam et al., 1979).
FIG. 3A-C. Comparison of IgG 1 and IgA 1 CH3 sequences and IgG 1
structure in the area homologous to several phage-derived peptides. FIG. 3A.
The
A12 peptide alignment with both human IgAI and IgGI. IgGSTR indicates
structural
features of IgGI where < denotes a p-strand running in a descending
orientation (i.e.
hinge to CH3 direction), > denotes a a-strand running in an ascending
direction (i. e.
CH3 to hinge direction) and - denotes a loop or open structure (Deisenhofer et
al.,
1981 ). FIG. 3B. Comparison of several mammalian IgA sequences with the four
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human IgG subclasses showing the additional IgA-specific amino acids present
in the
loop at positions 402-410 in the IgA sequence. hu=human, gr=gorilla,
mur=murine,
rab=rabbit. FIG. 3C. IgAl Ca3 mutants L1, L2 and L3 aligned with the Ca3 and
Cy3 wild-type sequences and Cy3 structure (IgGSTR). = denotes sequence
identity in
the mutants, - denotes a space introduced in the IgG sequence to maximize
homology
and IgGSTR is labeled according to FIG 3a above. Numbering of IgAl and IgGI is
according to Putnam et al., 1979 and Deisenhofer et al., 1981, respectively.
FIG. 4. Binding of IgA mutants L1, L2 and L3 to purified human pIgR by
ELISA. The extracellular domain of human pIgR was purified following
expression in
baculovirus and coated onto ELISA plates at 10 ~g/ml. Chimeric IgAI and IgAI
Ca3
mutants L1, L2 and L3 were expressed as both monomeric (m) and dimeric (d)
forms
along with chimeric IgGl, purified and incubated on the pIgR-coated plates to
compare their abilities to bind to pIgR. Bound antibodies were detected with
anti
human-x light chain alkaline phosphatase conjugate.
FIG. 5. Alignment of phage peptides selected by transcytosis with human IgA
using the program LALIGN.
FIG. 6A-C. Basolateral to apical transport of phage peptides measured in the
MDCK transcytosis system. 5 x 10'° phage were added to the basolateral
medium of
wells containing 1.0 p,m pore inserts confluent with either polarized MDCK or
pIgR-
transfected MDCK cells. Transcytosis was allowed to occur for 4 hours. The
apical
supernatant fluids were collected and phage titers determined. A. SAM, IPS,
and
RSR peptides were evaluated. Note the very low phage titer level with the SAM
peptide in the absence of pIgR (bar graph is slightly above y-axis=0). B. MFV,
VDD, and QRN peptides were evaluated. C. LVL and WQA peptides were
evaluated.
FIG. 7. Procedure and kinetics determination of blood phage peptides
transported into hepatic bile.
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FIG. 8. Transcytosis of thioredoxin fusion proteins through non-transfected
and pIgR-transfected MDCK cells.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention is directed to compositions and methods employing
sequences, including IgA sequences, that target pIgR for the delivery of
therapeutic
compounds to the mucosal surfaces. The identification of a pIgR binding motif
of
IgA or of pIgR-binding peptide sequences reduces the complexity of using a
whole
IgA monomer or polymer to provide a targeted delivery system to the mucosal
epithelium. Delivery of therapeutic compounds to this area would bestow
preventative and therapeutic benefits through the body's enhanced ability to
prevent,
inhibit, or reduce the incidence of infections, diseases, or conditions. The
present
invention encompasses both general targeting of compounds to the mucosal
epithelium using the pIgR-binding domain, and additional specific targeting of
compounds to particular sites of action within the mucosal epithelium.
Specific
targeting is accomplished through the use of other targeting mechanisms that
utilize
sequences involved in specific protein-protein interactions, such as antigen-
antibody
interactions or ligand-receptor associations.
I. Treatment Uses
Mucosal surfaces of the body serve as a boundary with the environment. They
constitute the largest exposed area of the body to external pathogens
(Childers et al.,
1989), and consequently, infections commonly involve these surfaces. The
principal
mucosal antibody is IgA, which is considered to form a first line of immune
defense,
particularly against microbes, toxins, and other antigens.
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A. IgA Antibodies
The present invention encompasses sequences from IgA antibodies that
mediate IgA's binding to the pIgR, such as sequences within the Ca3 domain of
IgA;
in further embodiments of the present invention, other sequences from IgA
antibodies
5 are used such as those involved required for eliciting an immune response,
which can
include sequences that mediate antigen specificity. These IgA sequences can be
utilized in additional embodiments in conjunction with a preventative or
therapeutic
compound, which gets directed to mucosal epithelia via the pIgR-binding domain
of
IgA.
IgA antibodies generally are produced in the greatest quantities, and they are
manufactured locally by plasma cells within mucosal membranes before they are
released in mucosal secretions. They are located in respiratory,
nasopharyngeal,
ocular, gastrointestinal, urinary, and genital tracts, as well as being
present in saliva,
serum, tears, and colostrum.
IgA, like IgG, contains three constant domains (Cal, Ca2, Ca3) with a hinge
region joining the Cal and Ca2 domains. Unlike IgG, however, IgA isotypes have
an 18 residue "tailpiece" that is located at the C-terminal end of CH3, which
allows
IgA polymers to form (Putnam et al., 1979). Additionally, a small glycoprotein
called
the "J chain" forms disulfide bonds with two tailpiece cysteine residues,
while the
tailpieces each form a direct disulfide bond with each other (Garcia-Pardo et
al., 1981;
Bastian, et al., 1992). The J chain mediates serum IgA dimer formation (Zikan
et al.,
1986) by making polymerization more efficient, but it is not absolutely
required
(Hendrickson, et al., 1995).
IgA is secreted from plasma cells that underlie the mucosal epithelial
primarily
as dimers joined by an intersubunit J chain, with each IgA composed of two
identical
light chains and two identical heavy chains that confer its specificity. The
IgA dimers
bind to the polymeric immunoglobulin receptor (pIgR) on the basolateral
surface of
mucosal epithelia. Through its external IgA-binding domain, a pIgR-IgA complex
is
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formed through high-affinity non-covalent binding of pIgR domain I (Frutiger
et al.,
1986; Bakos et al., 1991 ), and this complex is subsequently endocytosed. The
pIgR
cytoplasmic domains contains sorting signals that cause the complex the be
transcytosed via vesicles to the apical surface, where proteolytic cleavage
separates
the external domain of pIgR (referred as "secretory component") from its
membrane
spanning domain (Lindh et al., 1974; Fallgrenn-Gebauer et al., 1993; Underdown
et
al., 1977). Consequently, the dimeric IgA-secretory component complex (termed
"secretory IgA" or sIgA) is released into the mucosal secretions (Underdown et
al.,
1977).
The polymeric immunoglobulin receptor (pIgR) is a type I transmembrane
protein with five immunoglobulin superfamily homology domains (I-V)
constituting
the extracellular region (Mostov et al., 1984). Polymeric immunoglobulins, IgA
and
IgM, are bound by pIgR at the basolateral surface of mucosal epithelial cells,
transported through these cells, and then secreted at the mucosae (Mostov et
al.,
1982).
While much is known about the macromolecular interaction and activity of
IgA and pIgR, relatively little is understood about the requirements of that
interaction
on a molecular scale. According to the present invention, a region of IgA that
mediates pIgR binding has been identified. The major binding motif of IgA to
pIgR
has been localized to the Ca3 domain, particularly to amino acids 402-410,
which
form a portion of a predicted exposed loop of the Ca3 domain. A pIgR-binding
domain may comprise any portion of amino acids 402-410 by itself or in
combination
with other pIgR-binding sequences, including IgA sequences, and can be
utilized as a
targeting peptide to recruit compounds to the mucosal epithelium; moreover,
sequences that mediate pIgR binding that are not derived from IgA are also
considered
to be a pIgR binding domain, and thus, part of the present invention. Thus,
complexes
comprising a pIgR-binding domain and a selected agent are contemplated by the
present invention. The pIgR-binding domain and the therapeutic/preventative
compound will bind either as a monomer or as a polymer (including concatemers)
to a
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pIgR located at the basal membrane of mucosal epithelium and be collectively
endocytosed. This complex will then be transcytosed until it reaches the
apical
membrane where the secretory component of pIgR is cleaved with the targeting
peptide and the therapeutic/preventative compound. Once secreted, the
therapeutic
compound can be utilized for various functions as will be discussed below,
including
prevention of diseases or conditions caused by pathogens; reduction or
amelioration
of disease states, conditions, or disorders, including cancer; and, mucosal
immunity
via vaccinations.
The mucosal surfaces of the body offer an important advantage over serum as
a site of immunological prevention or inhibition of disease, in that rather
than
responding to an infection that has already occurred, an immunological
response at
the mucosal surface prevents the infective agent from entering the body. Such
a
preventative method, as provided by the present disclosure, would be of
dramatic
benefit, not only in the prevention of sexually transmitted infections and
maternal
transmission of those diseases during birth, but also in the prevention of
other
infections that enter the body through mucosal surfaces such as the
genitourinary
tract, mouth, nasal passages, lungs, eyes, etc., in man and in domestic or non-
domestic
animals. Furthermore, access to the mucosal epithelia provides a method of
treating
many diseases, conditions, or disorders that are proximal to the mucosal
epithelia by
providing a passageway for preventative or therapeutic compounds to reach
sites
where they are needed.
This targeting is accomplished through the use of targeting sequences of IgA
that are involved in mediating its interaction with the polymeric
immunoglobulin
receptor (pIgR). The pIgR serves to shuttle IgA from the basal mucosal
epithelium
for its release in mucosal secretions. The present invention seeks to exploit
the
inventors' identification of a domain within IgA that is responsible for this
targeting
function. Therefore, IgA targeting sequences can be utilized to shuttle
preventative
and therapeutic compounds to mucosal epithelia for the treatment of diseases,
conditions, or disorders. Additionally, it is contemplated that the present
invention
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includes the use of peptide sequences that mimic the binding activity of IgA
to pIgR
such that these sequences can be used as the previously described delivery
shuttle
system.
As will be understood by those of skill in the art, small modification and
changes may be made in the structure of a domain that binds pIgR, including
those
changes that confer greating binding affinity for pIgR than a sequence from
IgA.
Furthermore, certain amino acids may be substituted for other amino acids in a
protein
structure without appreciable loss of interactive binding capacity with the
pIgR. Since
it is the interactive capacity and nature of a protein that defines that
protein's
biological functional activity, certain amino acid sequence substitutions can
be made
in a protein sequence (or, of course, its underlying DNA coding sequence) and
nevertheless obtain a protein with like (agonistic) properties. It is thus
contemplated
by the inventors that various changes may be made in the pIgR binding sequence
of
IgA or therapeutic or preventative compound polypeptides or peptides (or
underlying
DNA) without appreciable loss of their biological utility or activity.
In the present invention, residues shown to be necessary for a pIgR binding
generally should be substituted with conservative amino acids or not changed
at all,
such as the group of residues located in the region constituting a loop
between two
flanking (3-strand sequences of IgA. Introduction of alanine substitutions
into the L1
and L3 regions of the exposed loop abrogated pIgR binding, as they comprise a
major
binding domain of IgA.
Amino acid substitutions are generally based on the relative similarity of the
amino acid side-chain substituents, for example, their hydrophobicity,
hydrophilicity,
charge, size, and the like. An analysis of the size, shape, and type of the
amino acid
side-chain substituents reveals that arginine, lysine, and histidine are all
positively
charged residues; that alanine, glycine, and serine are all a similar size;
and that
phenylalanine, tryptophan, and tyrosine all have a generally similar shape.
Therefore,
based upon these considerations, the following subsets are defined herein as
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biologically functional equivalents: arginine, lysine, and histidine; alanine,
glycine,
and serine; and phenylalanine, tryptophan, and tyrosine.
To effect more quantitative changes, the hydropathic index of amino acids
may be considered. Each amino acid has been assigned a hydropathic index on
the
basis of their hydrophobicity and charge characteristics, these are:
isoleucine (+4.5);
valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-
0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and
arginine (-4.5).
The importance of the hydropathic amino acid index in conferring interactive
biological function on a protein is generally understood in the art (Kyte &
Doolittle,
1982, incorporated herein by reference). It is known that certain amino acids
may be
substituted for other amino acids having a similar hydropathic index or score
and still
retain a similar biological activity. In making changes based upon the
hydropathic
index, the substitution of amino acids whose hydropathic indices are within ~2
is
preferred, those which are within ~l are particularly preferred, and those
within +0.5
are even more particularly preferred.
It also is understood in the art that the substitution of like amino acids can
be
made effectively on the basis of hydrophilicity, particularly where the
biological
functional equivalent protein or peptide thereby created is intended for use
in
immunological embodiments, as in the present case. U.S. Patent 4,554,101,
incorporated herein by reference, states that the greatest local average
hydrophilicity
of a protein, as governed by the hydrophilicity of its adjacent amino acids,
correlates
with its immunogenicity and antigenicity, i. e. with a biological property of
the
protein.
As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have
been assigned to amino acid residues: arginine (+3.0); lysine (+3.0);
aspartate (+3.0 ~
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1 ); glutamate (+3.0 ~ 1 ); serine (+0.3); asparagine (+0.2); glutamine
(+0.2); glycine
(0); threonine (-0.4); proline (-0.5 ~ 1); alanine (-0.5); histidine (-0.5);
cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine
(-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
5
In making changes based upon similar hydrophilicity values, the substitution
of amino acids whose hydrophilicity values are within ~2 is preferred, those
which are
within ~l are particularly preferred, and those within X0.5 are even more
particularly
preferred.
It also is conceivable that non-peptide structures such as "peptide mimetics"
may be used to duplicate the structure and contact points within the pIgR-
binding
domain structure, thereby also duplicating its ability to bind to, and hence
target, the
pIgR.
B. Treatment Uses
The present invention can be used to prevent or combat any diseases, ailments,
or conditions that involve mucosal epithelium, which includes any disease,
ailment or
condition that is accessible to mucosal epithelia. The effects described
herein can be
achieved within mucosal epithelia cells as well as in mucosal secretions or in
the
lumen after compounds of the present invention cross the mucosal epithelia.
The
present invention encompasses treatment of mucosal epithelia, which includes,
but is
not limited to, epithelia within respiratory, nasopharyngeal, ocular,
gastrointestinal,
urinary, and genital tracts.
Within the respiratory tract, the present invention includes, but is not
limited
to, treatment of the following: asthma; bronchitis; emphysema; cystic
fibrosis;
bronchiectasis; bronchiolitis; pulmonary edema; viral tracheobronchitis; sleep
apnea
syndrome; infectious diseases such as bacterial pneumonia, Mycoplasma
pneumonia,
influenza, tuberculosis, endemic fungal pneumonias, and invasive
aspergillosis;
neoplastic conditions such as lung cancer, carcinomas and lymphomas;
noninfectious,
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nonneoplastic diseases such as Loffler's syndrome, tropical pulmonary
eosinophilia,
sarcoidosis, pulomonary fibrosis, Caplan's sydrome, Wagner's granulomatosis,
bronchogenic cysts; disorders of the chest wall such as kyphoscoliosis,
neuromuscular
syndrome, diffuse parenchymal lung disease; as well as other chronic diffuse
infiltrative lung diseases.
The present invention is directed also to treatment of nasopharyngeal and
gastrointestinal disorders including, but not limited to, the following:
dysphagia;
peptic ulcers; diarrheal diseases; inflammatory bowel diseases such as
ulcerative
colitis and Chrohn's disease; acute pancreatitisgallstones and biliary tract
disease;
acute hepatits; chronic hepatitis; cirrhosis; gastrointestinal bleeding caused
by ulcers,
mucosal erosive diseases, malignancies, colonic diverticulosis, colitis, and
hemorrhoids among other causes; malabsorption and maldigestion diseases;
gastrointestinal motility disorders; and diverticulosis, diverticulitis, and
appendicitis.
In further embodiments, the present invention also covers the treatment of
urogenital tract disorders and conditions including renal disorders of water
and
sodium imbalance; disorders of acid-base and potassium balance; glomerular
diseases;
systemic vasculitis; tubulointerstitial diseases; polycistic kidney disease;
Alport's
syndrome; medullary cystic disease; nephrolithiasis; hyperproliferative
diseases such
as neoplasias and malignancies; sexually transmitted diseases such as AIDS,
hepatitis,
genital warts, herpes, gonorrhea, syphillis, and chlamydia.
Also contemplated by the present invention is the treatment of diseases and
disorders involving cell hyperproliferation including the treatment or
prevention of
malignancies, premalignant conditions (hyperplasia, metaplasia, dysplasia),
benign
tumors, hyperproliferative disorders, and benign dysproliferative disorders,
for
example. The present invention further encompasses the treatment of cancer or
precancer cells and tumors derived from bladder, blood, bone, bone marrow,
brain,
breast, colon, esophagus, gastrointestine, head, kidney, liver, lung,
nasopharynx, neck,
ovary, prostate, skin, stomach, and uterus.
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C. Pathogenic Protection
Another embodiment of the invention is the use of the target agent to deliver
a
therapeutic/preventative compound to mucosal epithelia to prevent or inhibit
pathogenic infection. The present invention contemplates the generation of
passive
and active immunity, in addition to therapeutic compounds and methods. One
mode
of operation includes a dIgA specific to a particular pathogen to prevent or
inhibit
infection against that particular pathogen.
The compositions of the invention are administered to a subject, such as an
animal or a human and are subsequently, after a latent period of up to 24
hours,
transported across a mucosal barrier. After active or passive transportation
into the
mucosa, the antibodies of the invention are available to inhibit an infection
at that site.
Because this is a method of passive immunity, it is understood based on the
serum
half lives of antibodies, that the protection may last for a period of several
weeks and
that the compositions may then be re-administered if the need persists. The
invention
provides, therefore, a method of inhibiting an infection prior to entry into
the body,
offering a first line of defense prior to exposure to the particular pathogen.
Pathogens
that may be inhibited from infecting a subject include, but are not limited
to, viruses,
bacteria, and macroscopic parasites such as protozoans.
The present invention would have applications therefore in the prevention and
treatment of viral diseases that may enter or exit the body through the
mucosal
surfaces such as the following pathogenic viruses which are mentioned by way
of
example, influenza A, B and C, parainfluenza, paramyxoviruses, Newcastle
disease
virus, respiratory syncytial virus, measles, mumps, adenoviruses,
adenoassociated
viruses, parvoviruses, Epstein-Barr virus, rhinoviruses, coxsackieviruses,
echoviruses,
reoviruses, rhabdoviruses, lymphocytic choriomeningitis, coronavirus,
polioviruses,
herpes simplex, human immunodeficiency viruses, cytomegaloviruses,
papillomaviruses, virus B, varicella-zoster, poxviruses, rubella, rabies,
picornaviruses,
rotavirus and Kaposi associated herpes virus.
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In addition to the viral diseases mentioned above, the present invention is
also
useful in the prevention, inhibition, or treatment of bacterial infections,
including, but
not limited to, the 83 or more distinct serotypes of pneumococci, streptococci
such as
S S. pyogenes, S. agalactiae, S. equi, S. canes, S. bovis, S. equinus, S.
anginosus, S.
sanguis, S. salivarius, S. mites, S. mutans, other viridans streptococci,
peptostreptococci, other related species of streptococci, enterococci such as
Enterococcus faecalis, Enterococcus faecium, Staphylococci, such as
Staphylococcus
epidermidis, Staphylococcus aureus, particularly in the nasopharynx,
Hemophilus
influenzae, pseudomonas species such as Pseudomonas aeruginosa, Pseudomonas
pseudomallei, Pseudomonas mallei, brucellas such as Brucella melitensis,
Brucella
sues, Brucella abortus, Bordetella pertussis, Neisseria meningitides,
Neisseria
gonorrhoeae, Moraxella catarrhalis, Corynebacterium diphtheriae,
Corynebacterium
ulcerans, Corynebacterium pseudotuberculosis, Corynebacterium
pseudodiphtheriticum, Corynebacterium urealyticum, Corynebacterium
hemolyticum,
Corynebacterium equi, etc. Listeria monocytogenes, Nocordia asteroides,
Bacteroides
species, Actinomycetes species, Treponema pallidum, Leptospirosa species and
related organisms. The invention may also be useful against gram negative
bacteria
such as Klebsiella pneumoniae, Escherichia coli, Proteus, Serratia species,
Acinetobacter, Yersinia pestis, Francisella tularensis, Enterobacter species,
Bacteriodes and Legionella species and the like. In addition, the invention
may prove
useful in controlling protozoan or macroscopic infections by organisms such as
Cryptosporidium, Isospora belle, Toxoplasma gondii, Trichomonas vaginalis,
Cyclospora species, for example, and for Chlamydia trachomatis and other
Chlamydia infections such as Chlamydia psittaci, or Chlamydia pneumoniae, for
example. Of course it is understood that the invention may be used on any
pathogen
against which an effective antibody can be made. In light of the present
disclosure,
one of skill in the art would be able to produce a composition of dimeric IgA
antibodies immunoreactive with any such pathogen and would further be able to
administer such a composition to a subject.
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Several groups have reported the use of the IgA isotype as protective
antibodies against a wide range of human pathogens. These include: viruses,
such as
HIV (Burnett et al., 1994), influenza A (Liew et al., 1984), Sendai (Manzanec
et al.,
1987), and respiratory syncytial virus (Weltzin et al., 1994); bacteria, such
as
Salmonella typhimurium (Michetti, 1992), Vibrio cholerae (Apter et al., 1993,
Winner
et al., 1991), Chlamydia trachomatis (Cotter et al., 1995); bacterial toxins,
and
macroscopic parasites (Grzych et al., 1993). Moreover, a recombinant dimeric
IgA
with antigen specificity directed against transmissible gastroenteritis
coronavirus
(TGEV) reduced TGEV production significantly when transfected into a cell line
(Castilla et al., 1997).
D. Therapeutic Compounds
The targeting agent of the present invention may be operatively linked or
attached to a selective agent or compound. Different and varied therapeutic
compounds are illustrated. These include enzymes, drugs (e.g., antibacterial,
antifungal, anti-viral), antibody regions, regions that mediate protein-
protein or
ligand-receptor interactions, cytokines, growth factors, hormones, toxins,
polynucleotides coding for proteins, antisense sequences, radiotherapeutics,
chemotherapeutics, ribozymes, tumor suppressors, transcription factors,
inducers of
apoptosis, or liposomes containing any of the foregoing. In addition to
encompassing
the delivery of purified compounds, the present invention further contemplates
the
delivery of nucleic acids that encode cognate compounds such as polypeptides.
Therefore, according to the present invention, both purified compounds and
nucleic
acid sequences encoding that compound, e.g., a cytokine, may be delivered in
conjunction with the pIgR-binding domain.
1. Enzymes
Various enzymes are of interest according to the present invention. Enzymes
that could be attached to the pIgR-binding domain include cytosine deaminase,
adenosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, galactose-
1-
phosphate uridyltransferase, phenylalanine hydroxylase, glucose-6-phosphate
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dehydrogenase, HSV thymidine kinase, and human thymidine kinase and
extracellular
proteins such as collagenase and matrix metalloprotease, lysosomal glucosidase
(Pompe's disease), muscle phosphorylase (McArdle's syndrome),
glucocerebosidase
(Gaucher's disease), a-L-iduronidase (Hurler syndrome), L-iduronate sulfatase
5 (Hunter syndrome), sphingomyelinase (Niemann-Pick disease) and
hexosaminidase
(Tay-Sachs disease).
2. Drugs
According to the present invention, a drug may be operatively linked to a
10 pIgR-binding domain to deliver the drug to the mucosal epithelia. It is
contemplated
that drugs such as antimetabolites (e.g., purine analogs, pyrimidine analogs,
folinic
acid analogs), enzyme inhibitors, metabolites, or antibiotics (e.g.,
mitomycin) are
useful in the present invention.
15 3. Antibody Regions
Regions from the various members of the immunoglobulin family are also
encompassed by the present invention. Both variable regions from specific
antibodies
are covered within the present invention, including complementarity
determining
regions (CDRs), as are antibody neutralizing regions, including those that
bind
20 effector molecules such as Fc regions. Antigen specific-encoding regions
from
antibodies, such as variable regions from IgGs, IgMs, or IgAs, can be employed
with
the pIgR-binding domain in combination with an antibody neutralization region
or
with one of the therapeutic compounds described above.
In yet another embodiment, one gene may comprise a single-chain antibody.
Methods for the production of single-chain antibodies are well known to those
of skill
in the art. The skilled artisan is referred to U.S. Patent No. 5,359,046,
(incorporated
herein by reference) for such methods. A single chain antibody is created by
fusing
together the variable domains of the heavy and light chains using a short
peptide
linker, thereby reconstituting an antigen binding site on a single molecule.
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Single-chain antibody variable fragments (scFvs) in which the C-terminus of
one variable domain is tethered to the N-terminus of the other via a 1 S to 25
amino
acid peptide or linker, have been developed without significantly disrupting
antigen
binding or specificity of the binding (Bedzyk et al., 1990; Chaudhary et al.,
1990).
These Fvs lack the constant regions (Fc) present in the heavy and light chains
of the
native antibody.
Antibodies to a wide variety of molecules are contemplated, such as
oncogenes, cytokines, growth factors, hormones, enzymes, transcription factors
or
receptors. Also contemplated are secreted antibodies targeted against serum,
angiogenic factors (VEGF/VPF; (3FGF; aFGF; and others), coagulation factors,
and
endothelial antigens necessary for angiogenesis (i.e., V3 integrin).
Specifically
contemplated are growth factors such as transforming growth factor, fibroblast
growth
factor, and platelet derived growth factor (PDGF) and PDGF family members.
The present invention further embodies composition targeting specific
pathogens through the use of antigen-specific sequences or targeting specific
cell
types, such as those expressing cell surface markers to identify the cell.
Examples of
such cell surface markers would include tumor-associated antigens or cell-type
specific markers such as CD4 or CDB.
4. Regions Mediating Protein-Protein or Ligand Receptor
Interaction
The use of a region of a protein that mediates protein-protein interactions,
including ligand-receptor interactions, also is contemplated by the present
invention.
This region could be used as an inhibitor or competitor of a protein-protein
interaction
or as a specific targeting motif. Consequently, the invention covers using the
pIgR-
binding domain to recruit a protein region that mediates a protein-protein
interaction
to mucosal epithelium. Once the compositions of the present invention reach
the
mucosal epithelia, more specific targeting of the composition is contemplated
through
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the use of a region that mediates protein-protein interactions including
ligand-receptor
interactions.
Protein-protein interactions include interactions between and among proteins
such as receptors and ligands; receptors and receptors; polymeric complexes;
transcription factors; kinases and downstream targets; enzymes and substrates;
etc.
For example, a ligand binding domain mediates the protein:protein interaction
between a ligand and its cognate receptor. Consequently, this domain could be
used
either to inhibit or compete with endogenous ligand binding or to target more
specifically cell types that express a receptor that recognizes the ligand
binding
domain operatively attached to the pIgR-binding domain.
Examples of ligand binding domains include ligands such as VEGF/VPF;
(3FGF; aFGF; coagulation factors, and endothelial antigens necessary for
angiogenesis (i.e., V3 integrin); growth factors such as transforming growth
factor,
fibroblast growth factor, colony stimulating factor, Kit ligand (KL), flk-
2/flt-3, and
platelet derived growth factor (PDGF) and PDGF family members; ligands that
bind
to cell surface receptors such as MHC molecules, among other.
The most extensively characterized ligands are asialoorosomucoid (ASOR)
(Wu and Wu, 1987) and transferrin (Wagner et al., 1990). Recently, a synthetic
neoglycoprotein, which recognizes the same receptor as ASOR, has been used as
a
gene delivery vehicle (Ferkol et al., 1993; Perales et al., 1994) and
epidermal growth
factor (EGF) has also been used to deliver genes to squamous carcinoma cells
(Myers,
EPO 0273085).
In other embodiments, Nicolau et al. (1987) employed lactosyl-ceramide, a
galactose-terminal asialganglioside, incorporated into liposomes and observed
an
increase in the uptake of the insulin gene by hepatocytes. Also, the human
prostate-
specific antigen (Watt et al., 1986) may be used as the receptor for mediated
delivery
to prostate tissue.
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5. Cytokines
Another class of compounds that is contemplated to be operatively linked to
the pIgR-binding domain of the present invention includes interleukins and
cytokines,
such as interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
IL-10, IL-
11, IL-12, IL-13, IL-14, IL-15, (3-interferon, a-interferon, y-interferon,
angiostatin,
thrombospondin, endostatin, METH-1, METH-2, Flk2/Flt3 ligand, GM-CSF, G-CSF,
M-CSF, and tumor necrosis factor (TNF)
6. Growth Factors
In other embodiments of the present invention, growth factors or ligands will
be encompassed by the therapeutic agent. Examples include VEGF/VPF, FGF,
TGF~3, ligands that bind to a TIE, tumor-associated fibronectin isoforms,
scatter
factor, hepatocyte growth factor, fibroblast growth factor, platelet factor
(PF4), PDGF,
KIT ligand (KL), colony stimulating factors (CSFs), LIF, and TIMP.
7. Hormones
Additional embodiments embrace the use of a hormone as a selective agent.
For example, the following hormones or steroids can be implemented in the
present
invention: prednisone, progesterone, estrogen, androgen, gonadotropin, ACTH,
CGH,
or gastrointestinal hormones such as secretin.
8. Toxins
In certain embodiments of the present invention, therapeutic agents will
include generally a plant-, fungus-, or bacteria-derived toxin such as ricin A-
chain
(Burbage, 1997), a ribosome inactivating protein, a-sarcin, aspergillin,
restrictocin, a
ribonuclease, diphtheria toxin A (Masuda et al., 1997; Lidor, 1997), pertussis
toxin A
subunit, E. coli enterotoxin toxin A subunit, cholera toxin A subunit, and
pseudomonas toxin c-terminal. Recently, it was demonstrated that transfection
of a
plasmid containing a fusion protein regulatable diphtheria toxin A chain gene
was
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cytotoxic for cancer cells. Thus, gene transfer of regulated toxin genes might
also be
applied to the treatment of diseases (Masuda et al., 1997).
9. Antisense Constructs
Antisense methodology takes advantage of the fact that nucleic acids tend to
pair with "complementary" sequences. By complementary, it is meant that
polynucleotides are those which are capable of base-pairing according to the
standard
Watson-Crick complementarity rules. That is, the larger purines will base pair
with
the smaller pyrimidines to form combinations of guanine paired with cytosine
(G:C)
and adenine paired with either thymine (A:T) in the case of DNA, or adenine
paired
with uracil (A:U) in the case of RNA. Inclusion of less common bases such as
inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in
hybridizing
sequences does not interfere with pairing.
Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix
formation; targeting RNA will lead to double-helix formation. Antisense
polynucleotides, when introduced into a target cell, specifically bind to
their target
polynucleotide and interfere with transcription, RNA processing, transport,
translation
and/or stability. Antisense RNA constructs, or DNA encoding such antisense
RNA's,
may be employed to inhibit gene transcription or translation or both within a
host cell,
either in vitro or in vivo, such as within a host animal, including a human
subject.
Antisense constructs may be designed to bind to the promoter and other
control regions, exons, introns or even exon-intron boundaries of a gene. It
is
contemplated that the most effective antisense constructs will include regions
complementary to intron/exon splice junctions. Thus, it is proposed that a
preferred
embodiment includes an antisense construct with complementarity to regions
within
50-200 bases of an intron-exon splice junction. It has been observed that some
exon
sequences can be included in the construct without seriously affecting the
target
selectivity thereof. The amount of exonic material included will vary
depending on
the particular exon and intron sequences used. One can readily test whether
too much
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exon DNA is included simply by testing the constructs in vitro to determine
whether
normal cellular function is affected or whether the expression of related
genes having
complementary sequences is altered.
5 As stated above, "complementary" or "antisense" means polynucleotide
sequences that are substantially complementary over their entire length and
have very
few base mismatches. For example, sequences of fifteen bases in length may be
termed complementary when they have complementary nucleotides at thirteen or
fourteen positions. Naturally, sequences which are completely complementary
will be
10 sequences which are entirely complementary throughout their entire length
and have
no base mismatches. Other sequences with lower degrees of homology also are
contemplated. For example, an antisense construct that has limited regions of
high
homology, but also contains a non-homologous region (e.g., ribozyme; see
below)
could be designed. These molecules, though having less than 50% homology,
would
15 bind to target sequences under appropriate conditions.
It may be advantageous to combine portions of genomic DNA with cDNA or
synthetic sequences to generate specific constructs. For example, where an
intron is
desired in the ultimate construct, a genomic clone will need to be used. The
cDNA or
20 a synthesized polynucleotide may provide more convenient restriction sites
for the
remaining portion of the construct and, therefore, would be used for the rest
of the
sequence.
Particular oncogenes that are targets for antisense constructs are ras, myc,
neu,
25 raf, erb, src, fms, jun, trk, ret, hst, gsp, bcl-2, and abl. Also
contemplated to be useful
are anti-apoptotic genes and angiogenesis promoters. Other antisense
constructs can
be directed at genes encoding viral or microbial genes to reduce or eliminate
pathogenicity. Specific constructs target genes such as viral env, pol, gag,
rev, tat or
coat or capsid genes, or microbial endotoxin, recombination, replication, or
transcription genes.
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10. Ribozymes
Although proteins traditionally have been used for catalysis of nucleic acids,
another class of macromolecules has emerged as useful in this endeavor.
Ribozymes
are RNA-protein complexes that cleave nucleic acids in a site-specific
fashion.
Ribozymes have specific catalytic domains that possess endonuclease activity
(Kim
and Cook; 1987; Gerlach et al., 1987; Forster and Symons, 1987). For example,
a
large number of ribozymes accelerate phosphoester transfer reactions with a
high
degree of specificity, often cleaving only one of several phosphoesters in an
oligonucleotide substrate (Michel and Westhof, 1990; Reinhold-Hurek and Shub,
1992). This specificity has been attributed to the requirement that the
substrate bind
via specific base-pairing interactions to the internal guide sequence ("IGS")
of the
ribozyme prior to chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific
cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al.,
1981).
For example, U.S. Patent No. 5,354,855 reports that certain ribozymes can act
as
endonucleases with a sequence specificity greater than that of known
ribonucleases
and approaching that of the DNA restriction enzymes. Thus, sequence-specific
ribozyme-mediated inhibition of gene expression may be particularly suited to
therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990).
Recently, it was
reported that ribozymes elicited genetic changes in some cells lines to which
they
were applied; the altered genes included the oncogenes H-ras, c-fos and genes
of HIV.
Most of this work involved the modification of a target mRNA, based on a
specific
mutant codon that is cleaved by a specific ribozyme. Targets for this
embodiment
will include angiogenic genes such as VEGFs and angiopoeiteins as well as the
oncogenes (e.g., ras, myc, neu, raf, erb, src, fms, jun, trk, ret, hst, gsp,
bcl-2, EGFR,
grb2 and ably. Other constructs will include overexpression of anti-apoptotic
genes
such as bcl-2, as well as microbial genes directed to viral or bacterial
genes.
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11. Chemo- and Radiotherapeutics
According to the invention, chemotherapeutic and radiotherapeutic compounds
can be operatively attached to a pIgR targeting motif. Chemotherapeuticagents
contemplatedto be of use include, e.g., adriamycin, bleomycin, 5-fluorouracil
(SFU),
etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP),
podophyllotoxin, verapamil, and even hydrogen peroxide.
12. Transcription Factors and Regulators
Another class of genes that can be applied in an advantageous combination are
transcription factors, both negative and positive regulators. Examples include
C/EBPa, IKB, NFKB, AP-1, YY-l, Spl, CREB, VP16, and Par-4.
13. Cell Cycle Regulators
Cell cycle regulators provide possible advantages, when combined with other
genes. Such cell cycle regulators include p27, p16, p21, p57, p18, p73, p19,
p15,
E2F-1, E2F-2, E2F-3, p107, p130, and E2F-4. Other cell cycle regulators
include
anti-angiogenic proteins, such as soluble Flkl (dominant negative soluble VEGF
receptor), soluble Wnt receptors, soluble Tie2/Tek receptor, soluble hemopexin
domain of matrix metalloprotease 2, and soluble receptors of other angiogenic
cytokines (e.g., VEGFR1, VEGFR2/KDR, VEGFR3/Flt4, and neutropilin-1 and -2
coreceptors).
14. Chemokines
Chemokines also may be used in the present invention. Chemokines generally
act as chemoattractants to recruit immune effector cells to the site of
chemokine
expression. It may be advantageous to express a particular chemokine gene in
combination with, for example, a cytokine gene, to enhance the recruitment of
other
immune system components to the site of treatment. Such chemokines include
RANTES, MCAF, MIPl-alpha, MIP1-beta, and IP-10. The skilled artisan will
recognize that certain cytokines are also known to have chemoattractant
effects and
could also be classified under the term chemokines.
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15. Tumor Suppressors
A number of proteins have been characterized as tumor suppressors, which
define a class of proteins that are involved with regulated cell
proliferation. The loss
of wild-type tumor suppressor activity is associated with neoplastic or
unregulated
cell growth. It has been shown by several groups that the neoplastic growth of
cells
lacking a wild-type copy of a particular tumor suppressor can be halted by the
addition of a wild-type version of that tumor suppressor. This has been
observed, for
example, with p53 (Diller et al., 1990). The present invention contemplates
the use of
a pIgR-binding domain to target mucosal epithelia for the delivery of a tumor
suppressor, such as p53. Other tumor suppressors that may be employed
according to
the present invention include p21, p15, BRCA1, BRCA2, IRF-1, PTEN (MMAC1),
Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zacl, p73, VHL, FCC, and
MCC.
16. Inducers of Apoptosis
Inducers of apoptosis, such as Bax, Bak, Bcl-XS, Bad, Bim, Bik, Bid, Harakiri,
Ad ElB, and ICE-CED3 proteases, similarly could be of use according to the
present
invention.
17. Liposomes as Carriers of Selected Agents
In another embodiment of the invention, the selected agent may be entrapped in
a liposome. Liposomes are vesicular structures characterized by a phospholipid
bilayer
membrane and an inner aqueous medium. Multilamellar liposomes have multiple
lipid
layers separated by aqueous medium. They form spontaneously when phospholipids
are
suspended in an excess of aqueous solution. The lipid components undergo
self rearrangement before the formation of closed structures and entrap water
and
dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also
contemplated are cationic lipid-nucleic acid complexes, such as lipofectamine-
nucleic
acid complexes.
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"Liposome" is a generic term encompassing a variety of single and
multilamellar lipid vehicles formed by the generation of enclosed lipid
bilayers.
Phospholipids are used for preparing the liposomes according to the present
invention
and can carry a net positive charge, a net negative charge or are neutral.
Dicetyl
phosphate can be employed to confer a negative charge on the liposomes, and
stearylamine can be used to confer a positive charge on the liposomes.
Lipids suitable for use according to the present invention can be obtained
from
commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can
be
obtained from Sigma Chemical Co., dicetyl phosphate ("DCP") is obtained from K
& K
Laboratories (Plainview, NY); cholesterol ("Chol") is obtained from
Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids
may
be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions
of
lipids in chloroform, chloroform/methanol or t-butanol can be stored at about -
20°C.
Preferably, chloroform is used as the only solvent since it is more readily
evaporated
than methanol.
Phospholipids from natural sources, such as egg or soybean
phosphatidylcholine, brain phosphatidic acid, brain or plant
phosphatidylinositol, heart
cardiolipin and plant or bacterial phosphatidylethanolamine are preferably not
used as
the primary phosphatide, i.e., constituting 50% or more of the total
phosphatide
composition, because of the instability and leakiness of the resulting
liposomes.
Liposomes used according to the present invention can be made by different
methods. The size of the liposomes varies depending on the method of
synthesis. A
liposome suspended in an aqueous solution is generally in the shape of a
spherical
vesicle, having one or more concentric layers of lipid bilayer molecules. Each
layer
consists of a parallel array of molecules represented by the formula XY,
wherein X is a
hydrophilic moiety and Y is a hydrophobic moiety. In aqueous suspension, the
concentric layers are arranged such that the hydrophilic moieties tend to
remain in
contact with an aqueous phase and the hydrophobic regions tend to self
associate. For
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example, when aqueous phases are present both within and without the liposome,
the
lipid molecules will form a bilayer, known as a lamella, of the arrangement XY-
YX.
Liposomes within the scope of the present invention can be prepared in
5 accordance with known laboratory techniques. In one preferred embodiment,
liposomes
are prepared by mixing liposomal lipids, in a solvent in a container, e.g., a
glass,
pear-shaped flask. The container should have a volume ten-times greater than
the
volume of the expected suspension of liposomes. Using a rotary evaporator, the
solvent
is removed at approximately 40°C under negative pressure. The solvent
normally is
10 removed within about 5 min to 2 hours, depending on the desired volume of
the
liposomes. The composition can be dried further in a desiccator under vacuum.
The
dried lipids generally are discarded after about 1 week because of a tendency
to
deteriorate with time.
15 Dried lipids can be hydrated at approximately 25-50 mM phospholipid in
sterile,
pyrogen-free water by shaking until all the lipid film is resuspended. The
aqueous
liposomes can be then separated into aliquots, each placed in a vial,
lyophilized and
sealed under vacuum.
20 In the alternative, liposomes can be prepared in accordance with other
known
laboratory procedures: the method of Bangham et al. (1965), the contents of
which are
incorporated herein by reference; the method of Gregoriadis, as described in
DRUG
CARRIERSIN BIOLOGYAND MEDICINE, G. Gregoriadis ed. ( 1979) pp. 287-341, the
contents of which are incorporated herein by reference; the method of Deamer
and Uster
25 (1983), the contents of which are incorporated by reference; and the
reverse-phase
evaporation method as described by Szoka and Papahadjopoulos (1978). The
aforementioned methods differ in their respective abilities to entrap aqueous
material
and their respective aqueous space-to-lipid ratios.
30 The dried lipids or lyophilized liposomes prepared as described above may
be
reconstituted in a solution of nucleic acid and diluted to an appropriate
concentration
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31
with an suitable solvent, e.g., DPBS. The mixture is then vigorously shaken in
a vortex
mixer. Unencapsulated nucleic acid is removed by centrifugation at 29,000 x g
and the
liposomal pellets washed. The washed liposomes are resuspended at an
appropriate
total phospholipid concentration, e.g., about 50-200 mM. The amount of nucleic
acid
encapsulated can be determined in accordance with standard methods. After
determination of the amount of nucleic acid encapsulated in the liposome
preparation,
the liposomes may be diluted to appropriate concentration and stored at
4°C until use.
In another embodiment, the lipid dioleoylphosphatidylchoine is employed.
Nuclease-resistant oligonucleotides were mixed with lipids in the presence of
excess
t-butanol. The mixture was vortexed before being frozen in an acetone/dry ice
bath.
The frozen mixture was lyophilized and hydrated with Hepes-buffered saline ( 1
mM
Hepes, 10 mM NaCI, pH 7.5) overnight, and then the liposomes were sonicated in
a
bath type sonicator for 10 to 15 min. The size of the liposomal-
oligonucleotides
typically ranged between 200-300 nm in diameter as determined by the submicron
particle sizer autodilute model 370 (Nicomp, Santa Barbara, CA).
E. Diagnostic Compounds
Certain examples of protein conjugates are those conjugates in which a
protein sequence such as a peptide containing a pIgR-binding domain is linked
to a
detectable label. "Detectable labels" are compounds or elements that can be
detected due to their specific functional properties, or chemical
characteristics, the
use of which allows the peptide or protein to which they are attached to be
detected,
and further quantified if desired.
Many appropriate imaging agents are known in the art, as are methods for
their attachment to proteins (see, e.g., U.S. patents 5,021,236 and 4,472,509,
both
incorporated herein by reference). Certain attachment methods involve the use
of a
metal chelate complex employing, for example, an organic chelating agent such
a
DTPA attached to the antibody (U.S. Patent 4,472,509). Protein sequences may
also
be reacted with an enzyme in the presence of a coupling agent such as
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32
glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared
in
the presence of these coupling agents or by reaction with an isothiocyanate.
Rhodamine markers can also be prepared.
In the case of paramagnetic ions, one might mention by way of example ions
such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II),
nickel (II),
copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium
(III),
vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium
(III), with
gadolinium being particularly preferred.
Ions useful in other contexts, such as X-ray imaging, include but are not
limited to lanthanum (III), gold (III), lead (II), and especially bismuth
(III).
In the case of radioactive isotopes for therapeutic and/or diagnostic
application, one might mention astatinez", '4carbon, 5'chromium, 36chlorine,
5'cobalt, SBcobalt, copperb', 'szEu, galliumb', 3hydrogen, iodine'z3,
iodine'zs, iodine'3',
indium"', s9iron, 3zphosphorus, rhenium'86, rhenium'88, 'Sselenium, 35sulphur,
technicium~''"' and yttrium~°. 'zSIodine is often being preferred for
use in certain
embodiments, and technicium9~"' and indium"' are also often preferred due to
their
low energy and suitability for long range detection.
F. Combined therapy with immunotherapy, traditional
chemotherapy, radiotherapy or other anti-cancer agents
In many therapies, it will be advantageous to provide more than one functional
therapeutic. Such "combined" therapies may have particular import in treating
aspects
of multidrug resistant (MDR) cancers and in antibiotic resistant bacterial
infections.
Thus, one aspect of the present invention utilizes a pIgR-binding domain to
deliver
therapeutic compounds to mucosal epithelia for treatment of diseases, while a
second
therapy, either targeted or non-targeted, also is provided.
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33
The non-targeted treatment may precede or follow the targeted agent treatment
by intervals ranging from minutes to weeks. In embodiments where the other
agent and
expression construct are applied separately to the cell, one would generally
ensure that a
significant period of time did not expire between the time of each delivery,
such that the
agent and expression construct would still be able to exert an advantageously
combined
effect on the cell. In such instances, it is contemplated that one would
contact the cell
with both modalities within about 12-24 hours of each other and, more
preferably,
within about 6-12 hours of each other, with a delay time of only about 12
hours being
most preferred. In some situations, it may be desirable to extend the time
period for
treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to
several weeks
(1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
It also is conceivable that more than one administration of either agent will
be
desired. Various combinations may be employed, where the targeted agent is "A"
and
the non-targeted agent is "B", as exemplified below:
AB/A B/A/B B/B/A A/AB B/A/A A/B/B B/B/B/A B/B/AB
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/AB/A B/A/A/B BBBlA
A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/BB B/A/B/B BB/A/B
Other combinations are contemplated. For example, in the context of the
present
invention, it is contemplated that mucosal-epithelia-targeted therapy of the
present
invention could be used in conjunction with non-targeted anti-cancer agents,
including
chemo- or radiotherapeutic intervention. To kill cells, inhibit cell growth,
inhibit
metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant
phenotype
of tumor cells, using the methods and compositions of the present invention,
one would
generally contact a "target" cell with a targeting agent/therapeutic agent and
at least one
other agent; these compositions would be provided in a combined amount
effective
achieve these goals. This process may involve contacting the cells with the
expression
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34
construct and the agents) or factors) at the same time. This may be achieved
by
contacting the cell with a single composition or pharmacological formulation
that
includes both agents, or by contacting the cell with two distinct compositions
or
formulations, at the same time, wherein one composition includes the
expression
construct and the other includes the agent. Alternatively, a gene therapy
treatment
involving a tumor suppressor gene, an antisense oncogene or oncogene-specific
ribozyme may be used.
Agents or factors suitable for use in a combined cancer therapy are any
chemical
compound or treatment method with anticancer activity; therefore, the term
"anticancer
agent" that is used throughout this application refers to an agent with
anticancer activity.
These compounds or methods include alkylating agents, topisomerase I
inhibitors,
topoisomerase II inhibitors, RNA/DNA antimetabolites, DNA antimetabolites,
antimitotic agents, as well as DNA damaging agents, which induce DNA damage
when
applied to a cell.
Examples of alkylating agents include, inter alia, chloroambucil, cis-
platinum,
cyclodisone, flurodopan, methyl CCNU, piperazinedione, teroxirone.
Topisomerase I
inhibitors encompass compounds such as camptothecin and camptothecin
derivatives, as
well as morpholinodoxorubicin. Doxorubicin, pyrazoloacridine, mitoxantrone,
and
rubidazone are illustrations of topoisomerase II inhibitors. RNA/DNA
antimetabolites
include L-alanosine, 5-fluoraouracil, aminopterin derivatives, methotrexate,
and
pyrazofurin; while the DNA antimetabolite group encompasses, for example, ara-
C,
guanozole, hydroxyurea, thiopurine. Typical antimitotic agents are colchicine,
rhizoxin,
taxol, and vinblastine sulfate. Other agents and factors include radiation and
waves that
induce DNA damage such as, y-irradiation, X-rays, UV-irradiation, microwaves,
electronic emissions, and the like. A variety of anti-cancer agents, also
described as
"chemotherapeutic agents," function to induce DNA damage, all of which are
intended
to be of use in the combined treatment methods disclosed herein.
Chemotherapeutic
agents contemplated to be of use, include, e.g., adriamycin, bleomycin, 5-
fluorouracil
(SFU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin
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(CDDP), podophyllotoxin, verapamil, and even hydrogen peroxide. The invention
also
encompasses the use of a combination of one or more DNA damaging agents,
whether
radiation-based or actual compounds, such as the use of X-rays with cisplatin
or the use
of cisplatin with etoposide.
5
The skilled artisan is directed to "Remington's Pharmaceutical Sciences" 15th
Edition, chapter 33, in particular pages 624-652. Some variation in dosage
will
necessarily occur depending on the condition of the subject being treated. The
person
responsible for administration will, in any event, determine the appropriate
dose for the
10 individual subject. Moreover, for human administration, preparations should
meet
sterility, pyrogenicity, general safety and purity standards as required by
FDA Office of
Biologics standards.
The inventors propose that local, regional delivery of a
therapeutic/preventative
1 S agent targeted to the mucosal epithelium in patients with cancers,
precancers, or
hyperproliferative conditions that can be reached via mucosal epithelia will
be a very
efficient method for delivering a therapeutically effective compound to
counteract the
clinical disease. Similarly, the chemo- or radiotherapy may be directed to a
particular,
affected region of the subjects body. Alternatively, systemic delivery of
compounds
20 and/or the agents may be appropriate in certain circumstances, for example,
where
extensive metastasis has occurred.
In addition to combining mucosal epithelia-targeted therapies with chemo- and
radiotherapies, it also is contemplated that combination with gene therapies
will be
25 advantageous. For example, targeting of mucosal epithelia using a
combination of p53,
p16, p21, Rb, APC, DCC, NF-l, NF-2, BCRA2, p16, FHIT, WT-1, MEN-I, MEN-II,
BRCA1, VHL, FCC, or MCC, or antisense versions of the oncogenes ras, myc, neu,
raf,
erb, src, fms, jun, trk, ret, gsp, hst, bcl or abl are included within the
scope of the
invention.
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36
Another example of combined therapies is in the treatment of bacterial
infections. Likely combinations would be (a) two drugs or (b) a drug and an
antibody.
Various antibiotics include the fluoroquinolones (pefloxacin, norfloxacin,
ciprofloxacin, ofloxacin, levofloxacin, enoxacin, fleroxacin, lomefloxacin,
temofloxacin, amifloxacin, tosufloxacin, flumequine, rufloxacin,
clinafloxacin), the
penicillins (penicilln G or V, methicillin, oxacillin, nafcilling,
cloxacillin,
dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin,
azlocillin, mexlocillin,
piperacillin), the sulfonamides (silfanilamide, sulfamethoxazole,
sulfadiazine,
sulfisoxazole, sulfacetamide), the cephalosporins (cephalothin, cephapirin,
cefazolin,
cephalexin, cephradine, cefadroxil, cefamanadole, cefoxitin, cefaclor,
cefuroxime,
cefonicid, cefotetan, ceforanide, cefotaxime, ceftizoxime, ceftriaxone,
cefoperazone,
ceftazidime), the aminoglycosides (gentamicin, tobramycin, netilmicin,
amikacin,
kanamycin, neomycin), tetracyclines, erythromycin, lycomycin, clindamycin,
spectinomycin, vancomycin, and streptomycin. Anti-fungal agents (amphotericin
B,
flycytosine, ketoconazole, fluconazole, griseofulvin, clotrimazole, econazole,
miconazole, ciclopirox olamine, halprogein, tolnaftate, naftifine, natamycin
and
nystatin) and anti-virals (AZT, acyclovir, ganciclovir, vidarabine,
idoxuridine,
trifluiridine, foscarnet, alpha interferon, amatidine, ribavirin, and
rimantidine) also
will be useful in therapy of these diseases.
It should be reiterated that any of the agents listed here also can be used
individually to treat the related condition in conjunction with targeting to
the mucosal
epithelium by pIgR (see above).
II. Preparation Methods
The compounds of the present invention include a targeting agent and in some
embodiments, a therapeutic agent or diagnostic agent. The targeting agent of
the
invention may be linked, or operatively attached, to the therapeutic or
diagnostic agent
by either chemical conjugation (e.g., crosslinking) or through recombinant DNA
techniques to produce the compound.
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A. Protein Purification
To prepare a composition comprising the pIgR-binding domain and a selective
agent, it may be desirable to purify the components or variants thereof.
According to
one embodiment of the present invention, purification of a peptide comprising
the
pIgR-binding domain can be utilized ultimately to operatively link this domain
with a
selective agent. Protein purification techniques are well known to those of
skill in the
art. These techniques involve, at one level, the crude fractionation of the
cellular
milieu to polypeptide and non-polypeptide fractions. Having separated the
polypeptide from other proteins, the polypeptide of interest may be further
purified
using chromatographic and electrophoretic techniques to achieve partial or
complete
purification (or purification to homogeneity). Analytical methods particularly
suited
to the preparation of a pure peptide are ion-exchange chromatography,
exclusion
chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A
particularly efficient method of purifying peptides is fast protein liquid
chromatography or even HPLC.
Certain aspects of the present invention concern the purification, and in
particular embodiments, the substantial purification, of an encoded protein or
peptide,
such as IgA or pIgR-binding domain. The term "purified protein or peptide" as
used
herein, is intended to refer to a composition, isolatable from other
components,
wherein the protein or peptide is purified to any degree relative to its
naturally-
obtainable state. A purified protein or peptide therefore also refers to a
protein or
peptide, free from the environment in which it may naturally occur.
Generally, "purified" will refer to a protein or peptide composition, such as
the
pIgR-binding domain, that has been subjected to fractionation to remove
various other
components, and which composition substantially retains its expressed
biological
activity. Where the term "substantially purified" is used, this designation
will refer to
a composition in which the protein or peptide forms the major component of the
composition, such as constituting about 50%, about 60%, about 70%, about 80%,
about 90%, about 95% or more of the proteins in the composition.
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38
Various methods for quantifying the degree of purification of the protein or
peptide will be known to those of skill in the art in light of the present
disclosure.
These include, for example, determining the specific activity of an active
fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A
preferred method for assessing the purity of a fraction is to calculate the
specific
activity of the fraction, to compare it to the specific activity of the
initial extract, and
to thus calculate the degree of purity, herein assessed by a "-fold
purification
number." The actual units used to represent the amount of activity will, of
course, be
dependent upon the particular assay technique chosen to follow the
purification and
whether or not the expressed protein or peptide exhibits a detectable
activity.
Various techniques suitable for use in protein purification will be well known
to those of skill in the art. These include, for example, precipitation with
ammonium
sulphate, PEG, antibodies and the like or by heat denaturation, followed by
centrifugation; chromatography steps such as ion exchange, gel filtration,
reverse
phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel
electrophoresis; and combinations of such and other techniques. As is
generally
known in the art, it is believed that the order of conducting the various
purification
steps may be changed, or that certain steps may be omitted, and still result
in a
suitable method for the preparation of a substantially purified protein or
peptide.
There is no general requirement that the protein or peptide always be provided
in their most purified state. Indeed, it is contemplated that less
substantially purified
products will have utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or by utilizing
different forms of the same general purification scheme. For example, it is
appreciated that a cation-exchange column chromatography performed utilizing
an
HPLC apparatus will generally result in a greater "-fold" purification than
the same
technique utilizing a low pressure chromatography system. Methods exhibiting a
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39
lower degree of relative purification may have advantages in total recovery of
protein
product, or in maintaining the activity of an expressed protein.
It is known that the migration of a polypeptide can vary, sometimes
significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977).
It will
therefore be appreciated that under differing electrophoresis conditions, the
apparent
molecular weights of purified or partially purified expression products may
vary.
High Performance Liquid Chromatography (HPLC) is characterized by a very
rapid separation with extraordinary resolution of peaks. This is, achieved by
the use of
very fine particles and high pressure to maintain an adequate flow rate.
Separation
can be accomplished in a matter of minutes, or at most an hour. Moreover, only
a
very small volume of the sample is needed because the particles are so small
and
close-packed that the void volume is a very small fraction of the bed volume.
Also,
the concentration of the sample need not be very great because the bands are
so
narrow that there is very little dilution of the sample.
Gel chromatography, or molecular sieve chromatography, is a special type of
partition chromatography that is based on molecular size. The theory behind
gel
chromatography is that the column, which is prepared with tiny particles of an
inert
substance that contain small pores, separates larger molecules from smaller
molecules
as they pass through or around the pores, depending on their size. As long as
the
material of which the particles are made does not adsorb the molecules, the
sole factor
determining rate of flow is the size. Hence, molecules are eluted from the
column in
decreasing size, so long as the shape is relatively constant. Gel
chromatography is
unsurpassed for separating molecules of different size because separation is
independent of all other factors such as pH, ionic strength, temperature, etc.
There
also is virtually no adsorption, less zone spreading and the elution volume is
related in
a simple matter to molecular weight.
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Affinity Chromatography is a chromatographic procedure that relies on the
specific affinity between a substance to be isolated and a molecule that it
can
specifically bind to. This is a receptor-ligand type interaction. The column
material is
synthesized by covalently coupling one of the binding partners to an insoluble
matrix.
5 The column material is then able to specifically adsorb the substance from
the
solution. Elution occurs by changing the conditions to those in which binding
will not
occur (e.g., alter pH, ionic strength, and temperature.).
A particular type of affinity chromatography useful in the purification of
10 carbohydrate containing compounds is lectin affinity chromatography.
Lectins are a
class of substances that bind to a variety of polysaccharides and
glycoproteins.
Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A
coupled to Sepharose was the first material of this sort to be used and has
been widely
used in the isolation of polysaccharides and glycoproteins other lectins that
have been
15 include lentil lectin, wheat germ agglutinin which has been useful in the
purification
of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves
are
purified using affinity chromatography with carbohydrate ligands. Lactose has
been
used to purify lectins from castor bean and peanuts; maltose has been useful
in
extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is
used for
20 purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from
wheat
germ; D-galactosamine has been used in obtaining lectins from clams and L-
fucose
will bind to lectins from lotus.
The matrix should be a substance that itself does not adsorb molecules to any
25 significant extent and that has a broad range of chemical, physical and
thermal
stability. The ligand should be coupled in such a way as to not affect its
binding
properties. The ligand also should provide relatively tight binding. And it
should be
possible to elute the substance without destroying the sample or the ligand.
One of
the most common forms of affinity chromatography is immunoaffinity
30 chromatography. The generation of antibodies that would be suitable for use
in
accord with the present invention is discussed below.
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1. Synthetic Peptides
The present invention also describes a pIgR-binding domain, including an IgA
Ca,3 peptide, for use in various embodiments of the present invention.
Encompassed
within the invention is a peptide that demonstrates greater binding affinity
for pIgR
than even the corresponding region of IgA. The peptides of the invention can
be
synthesized in solution or on a solid support in accordance with conventional
techniques. Various automatic synthesizers are commercially available and can
be
used in accordance with known protocols. See, for example, Stewart and Young,
(1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield
(1979), each
incorporated herein by reference. Short peptide sequences, or libraries of
overlapping
peptides, usually from about 6 up to about 35 to 50 amino acids, which
correspond to
the selected regions described herein, can be readily synthesized and then
screened in
screening assays designed to identify reactive peptides. Peptides with at
least about
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95
or up to about 100 amino acid residues are contemplated by the present
invention.
The compositions of the invention may include a peptide comprising a pIgR-
binding domain that has been modified to enhance its pIgR-binding capability
or to
render it biologically protected. Biologically protected peptides have certain
advantages over unprotected peptides when administered to human subjects and,
as
disclosed in U.S. patent 5,028,592, incorporated herein by reference,
protected
peptides often exhibit increased pharmacological activity.
Compositions for use in the present invention may also comprise peptides that
include all L-amino acids, all D-amino acids, or a mixture thereof. The use of
D-
amino acids may confer additional resistance to proteases naturally found
within the
human body and are less immunogenic and can therefore be expected to have
longer
biological half lives.
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2. LinkerslCoupling Agents
If desired, dimers or multimers of the pIgR-binding domain, such as dimeric
IgA polypeptides, and the therapeutic or preventative compound may be joined
via a
biologically-releasable bond, such as a selectively-cleavable linker or amino
acid
sequence. For example, peptide linkers that include a cleavage site for an
enzyme
preferentially located or active within a tumor environment are contemplated.
Exemplary forms of such peptide linkers are those that are cleaved by
urokinase,
plasmin, thrombin, Factor IXa, Factor Xa, or a metallaproteinase, such as
collagenase,
gelatinase, or stromelysin.
It is also contemplated that a peptide containing multimers of pIgR-binding
domains may be comprised of heteromeric sequences, in which the pIgR-binding
sequences are not identical to each other, or homomeric sequences, in which a
pIgR-
binding domain sequence is repeated at least once. Amino acids such as
selectively-
cleavable linkers, synthetic linkers, or other amino acid sequences may be
used to
separate a pIgR-binding domain from another pIgR-binding domain.
Alternatively,
linker sequences may be employed both between at least once set of pIgR-
binding
domains, as well as between a pIgR-binding domain and a selective agent or
compound.
Additionally, while numerous types of disulfide-bond containing linkers are
known which can successfully be employed to conjugate the toxin moiety with
the
targeting agent, certain linkers will generally be preferred over other
linkers, based on
differing pharmacologic characteristics and capabilities. For example, linkers
that
contain a disulfide bond that is sterically "hindered" are to be preferred,
due to their
greater stability in vivo, thus preventing release of the toxin moiety prior
to binding at
the site of action. Furthermore, while certain advantages in accordance with
the
invention will be realized through the use of any of a number of toxin
moieties, the
inventors have found that the use of ricin A chain, and even more preferably
deglycosylated A chain, will provide particular benefits.
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43
a. Biochemical cross-linkers
The joining of any of the above components, to targeting peptide will
generally employ the same technology as developed for the preparation of
immunotoxins. It can be considered as a general guideline that any biochemical
cross-linker that is appropriate for use in an immunotoxin will also be of use
in the
present context, and additional linkers may also be considered.
Cross-linking reagents are used to form molecular bridges that tie together
functional groups of two different molecules, e.g., a stablizing and
coagulating agent.
To link two different proteins in a step-wise manner, hetero-bifunctional
cross-linkers
can be used that eliminate unwanted homopolymer formation.
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44
TABLE 1
HETERO-BIFUNCTIONAL CROSS-LINKERS
Spacer Arm
Length\after
cross-
v
linker Reactive TowardAdvantages and Applicationslinking
SMPT Primary amines~ Greater stability 11.2 A
Sulfhydryls
SPDP Primary amines~ Thiolation 6.8 A
Sulfhydryls ~ Cleavable cross-linking
LC-SPDP Primary amines~ Extended spacer arm 15.6 A
Sulfhydryls
Sulfo-LC-SPDPPrimary amines~ Extended spacer arm 15.6 A
Sulfhydryls ~ Water-soluble
SMCC Primary amines~ Stable maleimide reactive11.6 A
group
Sulfhydryls - Enzyme-antibody conjugation
Hapten-carrier protein
conjugation
Sulfo-SMCC Primary amines~ Stable maleimide reactive11.6 A
group
Sulfhydryls ~ Water-soluble
Enzyme-antibody conjugation
MBS Primary amines~ Enzyme-antibody conjugation9.9 A
Sulfhydryls ~ Hapten-carrier protein
conjugation
Sulfo-MBS Primary amines~ Water-soluble 9.9 A
Sulfhydryls
SIAB Primary amines~ Enzyme-antibody conjugation10.6 A
Sulfhydryls
Sulfo-SIAB Primary amines~ Water-soluble 10.6 A
Sulfhydryls
SMPB Primary amines~ Extended spacer arm 14.5 A
Sulfhydryls ~ Enzyme-antibody conjugation
Sulfo-SMPB Primary amines~ Extended spacer arm 14.5 A
Sulfhydryls ~ Water-soluble
EDC/Sulfo-NHSPrimary amines~ Hapten-Carrier conjugation0
Carboxyl groups
ABH Carbohydrates~ Reacts with sugar groups11.9 A
Nonselective
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An exemplary hetero-bifunctional cross-linker contains two reactive groups:
one reacting with primary amine group (e.g., N-hydroxy succinimide) and the
other
reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens,
etc.).
Through the primary amine reactive group, the cross-linker may react with the
lysine
5 residues) of one protein (e.g., the selected antibody or fragment) and
through the thiol
reactive group, the cross-linker, already tied up to the first protein, reacts
with the
cysteine residue (free sulfhydryl group) of the other protein (e.g., the
selective agent).
It can therefore be seen that a targeted peptide composition will generally
10 have, or be derivatized to have, a functional group available for cross-
linking
purposes. This requirement is not considered to be limiting in that a wide
variety of
groups can be used in this manner. For example, primary or secondary amine
groups,
hydrazide or hydrazine groups, carboxyl alcohol, phosphate, or alkylating
groups may
be used for binding or cross-linking. For a general overview of linking
technology,
15 one may wish to refer to Ghose & Blair (1987).
The spacer arm between the two reactive groups of a cross-linkers may have
various length and chemical compositions. A longer spacer arm allows a better
flexibility of the conjugate components while some particular components in
the
20 bridge (e.g., benzene group) may lend extra stability to the reactive group
or an
increased resistance of the chemical link to the action of various aspects
(e.g.,
disulfide bond resistant to reducing agents). The use of peptide spacers, such
as
L-Leu-L-Ala-L-Leu-L-Ala, is also contemplated.
25 It is preferred that a cross-linker having reasonable stability in blood
will be
employed. Numerous types of disulfide-bond containing linkers are known that
can
be successfully employed to conjugate targeting and therapeutic/preventative
agents.
Linkers that contain a disulfide bond that is sterically hindered may prove to
give
greater stability in vivo, preventing release of the targeting peptide prior
to reaching
30 the site of action. These linkers are thus one group of linking agents.
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Another cross-linking reagents for use in immunotoxins is SMPT, which is a
bifunctional cross-linker containing a disulfide bond that is "sterically
hindered" by an
adjacent benzene ring and methyl groups. It is believed that stearic hindrance
of the
disulfide bond serves a function of protecting the bond from attack by
thiolate anions
such as glutathione which can be present in tissues and blood, and thereby
help in
preventing decoupling of the conjugate prior to the delivery of the attached
agent to
the tumor site. It is contemplated that the SMPT agent may also be used in
connection with the bispecific coagulating ligands of this invention.
The SMPT cross-linking reagent, as with many other known cross-linking
reagents, lends the ability to cross-link functional groups such as the SH of
cysteine or
primary amines (e.g., the epsilon amino group of lysine). Another possible
type of
cross-linker includes the hetero-bifunctional photoreactive phenylazides
containing a
cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido)
ethyl-
1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary
amino
groups and the phenylazide (upon photolysis) reacts non-selectively with any
amino
acid residue.
In addition to hindered cross-linkers, non-hindered linkers also can be
employed in accordance herewith. Other useful cross-linkers, not considered to
contain or generate a protected disulfide, include SATA, SPDP and 2-
iminothiolane
(Wawrzynczak & Thorpe, 1987). The use of such cross-linkers is well understood
in
the art.
Once conjugated, the targeting peptide generally will be purified to separate
the conjugate from unconjugated targeting agents or coagulants and from other
contaminants. A large a number of purification techniques are available for
use in
providing conjugates of a sufficient degree of purity to render them
clinically useful.
Purification methods based upon size separation, such as gel filtration, gel
permeation
or high performance liquid chromatography, will generally be of most use.
Other
chromatographic techniques, such as Blue-Sepharose separation, may also be
used.
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In addition to chemical conjugation, a purified IgA protein or peptide may be
modified at the protein level. Included within the scope of the invention are
IgA
protein fragments or other derivatives or analogs that are differentially
modified
during or after translation, for example by glycosylation, acetylation,
phosphorylation,
amidation, derivatization by known protecting/blocking groups, and proteolytic
cleavage. Any number of chemical modifications may be carried out by known
techniques, including but not limited to specific chemical cleavage by
cyanogen
bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation,
formylation, farnesylation, oxidation, reduction; metabolic synthesis in the
presence
of tunicamycin.
B. Assays
Other aspects of the present invention involve the generation of an complex
that comprises a pIgR-binding domain and a selected agent, with the intent to
target
the complex to mucosal epithelium. Such compounds may be tested both in vitro,
for
pIgR binding, and in vivo, for targeting. The various assays for use in
determining
such changes in function are routine and easily practiced by those of ordinary
skill in
the art.
In vitro assays involve the use of isolated pIgR or cells bearing pIgR. A
convenient way to monitor binding is by use of a detetable label, and assess
the
binding of the label to the receptor which, for example, may be fixed to a
support
(column, plate, well, dipstick). Alternatively, a functional read out may be
preferred,
for example, the ability to affect (kill, promote growth of) a target cells.
In vivo assays, such as an MDCK transcytosis system assay, also can be easily
conducted (Mostov et al., 1986). In these systems, it again is generally
preferred to
label the test candidate IgA constructs with a detectable marker and to follow
the
presence of the marker after administration to the animal, preferably via the
route
intended in the ultimate therapeutic treatment strategy. As part of this
process, one
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would take samples of body fluids, particularly mucosal epithelial samples,
and one
would analyze the samples for the presence of the marker associated with the
IgA
construct.
C. Recombinant DNA Techniques
Alternatively, recombinant DNA technology may be employed as a way of
providing a polypeptide or peptide wherein a nucleotide sequence that encodes
a
polypeptide or peptide of the invention is inserted into an expression vector,
transformed or transfected into an appropriate host cell and cultivated under
conditions suitable for expression. While discussion has focused on
functionally
equivalent polypeptides arising from amino acid changes, it will be
appreciated that
these changes may be effected by alteration of the encoding DNA, taking into
consideration also that the genetic code is degenerate and that two or more
codons
may encode the same amino acid. A table of amino acids and their codons is
presented hereinabove for use in such embodiments, as well as for other uses,
such as
in the design of probes and primers and the like.
As with the synthetic peptides previously discussed, it is contemplated that
multimers of pIgR-binding domains may be comprised within a peptide, such that
the
peptide contains more than pIgR-binding domain. It is further contemplated
that the
pIgR-binding domains of a peptide may be identical (i.e., homomeric) or not
identical
(i. e., "heteromeric") to one another. Thus, the multimeric peptides of the
present
invention can be encoded by nucleic acid sequences. Linkers composed of amino
acids may be employed to separate pIgR-binding domains from each other or from
a
selective compound or agent. Such linkers and/or selective agents, if composed
of
amino acids, may be provided to a cell or organism by providing a nucleic acid
sequence encoding them such that the cell can express the encoded amino acid
sequences.
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1. IgA or PreventativelTherapeutic Compound Polypeptides and
Fragments Thereof
Aspects of the present invention concern the use of isolated DNA segments
and recombinant vectors encoding wild-type, polymorphic, or mutant IgA
polypeptides or preventative/therapeutic compound polypeptides, and fragments
thereof, and the use of recombinant host cells through the application of DNA
technology that express those wild-type, polymorphic or mutant polypeptides.
Alternatively, non-IgA peptides or polypeptides may be used in the present
invention
as a pIgR-binding domain. A mutant IgA polypeptide can be a sequence that is
not
identical to the wild-type sequence, but still retains pIgR binding activity.
Embodiments of the claimed methods include the use of amino acids from the Ca3
region of IgA, possibly including residues from the region encompassing amino
acids
402-410 of IgA. Moreover, the present invention encompasses the use of DNA
segments and recombinant vectors encoding residues that mediate pIgR binding
and
that are not derived from IgA. The present invention encompasses recombinant
DNA
techniques to produce a pIgR-binding domain by itself or in combination with
other
IgA regions, or other antibodies, or with other selective agents. A number of
these
techniques are well known to those of skill in the art.
a. Amplification and PCR
The pIgR-binding domain or other regions may be produced using recombinant
DNA techniques such as nucleic acid amplification methods. Nucleic acid used
as a
template for amplification of IgA sequences is isolated from cells contained
in the
biological sample, according to standard methodologies (Sambrook et al.,
1989). The
nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA
is used, it may be desired to convert the RNA to a complementary DNA. In one
embodiment, the RNA is whole cell RNA and is used directly as the template for
amplification.
A number of template dependent processes are available to amplify the marker
sequences present in a given template sample. One of the best known
amplification
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methods is the polymerase chain reaction (referred to as PCR) which is
described in
detail in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, and each
incorporated
herein by reference in entirety.
5 A reverse transcriptase PCR amplification procedure may be performed in
order to quantify the amount of mRNA amplified. Methods of reverse
transcribing
RNA into cDNA are well known and described in Sambrook et al., 1989.
Alternative
methods for reverse transcription utilize thermostable, RNA-dependent DNA
polymerases. These methods are described in WO 90/07641, filed December 21,
10 1990, incorporated herein by reference. Polymerase chain reaction
methodologies are
well known in the art. Another method for amplification is the ligase chain
reaction
("LCR"), disclosed in EPA No. 320 308, incorporated herein by reference in its
entirety. U.S. Patent 4,883,750 describes a method similar to LCR for binding
probe
pairs to a target sequence.
An isothermal amplification method, in which restriction endonucleases and
ligases are used to achieve the amplification of target molecules that contain
nucleotide 5'-[alpha-thio]-triphosphates in one strand of a restriction site
may also be
useful in the amplification of nucleic acids in the present invention. Strand
Displacement Amplification (SDA) is another method of carrying out isothermal
amplification of nucleic acids which involves multiple rounds of strand
displacement
and synthesis, i.e., nick translation. A similar method, called Repair Chain
Reaction
(RCR), involves annealing several probes throughout a region targeted for
amplification, followed by a repair reaction in which only two of the four
bases are
present. The other two bases can be added as biotinylated derivatives for easy
detection. A similar approach is used in SDA. Target specific sequences can
also be
detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5'
sequences of non-specific DNA and a middle sequence of specific RNA is
hybridized
to DNA that is present in a sample. Upon hybridization, the reaction is
treated with
RNase H, and the products of the probe identified as distinctive products that
are
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51
released after digestion. The original template is annealed to another cycling
probe
and the reaction is repeated.
Other nucleic acid amplification procedures include transcription-based
amplification systems (TAS), including nucleic acid sequence based
amplification
(NASBA) and 3SR Gingeras et al., PCT Application WO 88/10315, incorporated
herein by reference. Davey et al., EPA No. 329 822 (incorporated herein by
reference
in its entirety) disclose a nucleic acid amplification process involving
cyclically
synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA
(dsDNA), which may be used in accordance with the present invention. Miller et
al.,
PCT Application WO 89/06700 (incorporated herein by reference in its entirety)
disclose a nucleic acid sequence amplification scheme based on the
hybridization of a
promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by
transcription of many RNA copies of the sequence. This scheme is not cyclic,
i.e.,
new templates are not produced from the resultant RNA transcripts. Other
amplification methods include "RACE" and "one-sided PCR" (Frohman, M.A., In:
PCR PROTOCOLS.' A GUIDE TO METHODS AND APPLICATIONS, Academic
Press, N.Y., 1990 incorporated by reference).
b. Recombinant DNA cloning
The present invention also concerns DNA segments, isolatable from
mammalian and human cells, that are free from total genomic DNA and that are
capable of expressing a protein or polypeptide that binds to pIgR, including
one that is
derived from IgA, particularly the Ca3 domain.
As used herein, the term "DNA segment" refers to a DNA molecule that has
been isolated free of total genomic DNA of a particular species. Therefore, a
DNA
segment encoding an IgA polypeptide refers to a DNA segment that contains
wild-type, polymorphic, or mutant IgA polypeptide coding sequences yet is
isolated
away from, or purified free from, total mammalian or human genomic DNA.
Included within the term "DNA segment," are DNA segments and smaller fragments
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of such segments, and also recombinant vectors, including, for example,
plasmids,
cosmids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified wild-type,
polymorphic, or mutant IgA polypeptide gene or pIgR-binding domain refers to a
DNA segment including wild-type, polymorphic, or mutant IgA polypeptide or
pIgR-
binding domain coding sequences and, in certain aspects, regulatory sequences,
isolated substantially away from other naturally occurring genes or protein
encoding
sequences. In this respect, the term "gene" is used for simplicity to refer to
a
functional protein, polypeptide, or peptide-encoding unit. As will be
understood by
those in the art, this functional term includes genomic sequences, cDNA
sequences,
and smaller engineered gene segments that express, or may be adapted to
express,
proteins, polypeptides, domains, peptides, fusion proteins, and mutants.
"Isolated substantially away from other coding sequences" means that the gene
of interest, in this case the wild-type, polymorphic, or mutant IgA
polypeptide gene,
forms the significant part of the coding region of the DNA segment, and that
the DNA
segment does not contain large portions of naturally-occurring coding DNA,
such as
large chromosomal fragments or other functional genes or cDNA coding regions.
Of
course, this refers to the DNA segment as originally isolated, and does not
exclude
genes or coding regions later added to the segment by human manipulation.
In particular embodiments, the invention concerns isolated DNA segments and
recombinant vectors incorporating DNA sequences that encode a wild-type,
polymorphic, or mutant IgA polypeptide or peptide that includes within its
amino acid
sequence a contiguous amino acid sequence in accordance with, or essentially
corresponding to wild-type, polymorphic, or mutant IgA polypeptides.
In other embodiments, the invention concerns isolated DNA segments and
recombinant vectors incorporating DNA sequences that encode a IgA polypeptide
or
peptide that includes within its amino acid sequence a contiguous amino acid
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53
sequence in accordance with, or essentially corresponding to IgA's pIgR-
binding
domain.
The term "biologically functional equivalent" is well understood in the art
and
is further defined in detail herein. Accordingly, sequences that have between
about
70% and about 80%; or more preferably, between about 81 % and about 90%; or
even
more preferably, between about 91 % and about 99%; of amino acids that are
identical
or functionally equivalent to the amino acids of IgA polypeptide sequences
provided
the targeting activity of the protein is maintained.
The term "functionally equivalent codon" is used herein to refer to codons
that
encode the same amino acid, such as the six codons for arginine or serine, and
also
refers to codons that encode biologically equivalent amino acids (see Table 2,
below).
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TABLE 2
CODON TABLE
Amino Acids Codons
Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic Asp D GAC GAU
acid
Glutamic Glu E GAA GAG
acid
PhenylalaninePhe F UUC UUU
Glycine Gly G GGA GGC GGG GGU
Histidine His H CAC CAU
Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG
CUU
Methionine Met M AUG
Asparagine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glutamine Gln Q CAA CAG
Arginine Arg R AGA AGG CGA CGC CGG
CGU
Serine Ser S AGC AGU UCA UCC UCG
UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GUU
Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU
It will also be understood that amino acid and nucleic acid sequences may
include additional residues, such as additional N- or C-terminal amino acids
or 5' or 3'
sequences, and yet still be essentially as set forth in one of the sequences
disclosed
herein, so long as the sequence meets the criteria set forth above, including
the
maintenance of biological protein activity where protein expression is
concerned. The
addition of terminal sequences particularly applies to nucleic acid sequences
that may,
for example, include various non-coding sequences flanking either of the 5' or
3'
portions of the coding region or may include various internal sequences, i.e.,
introns,
which are known to occur within genes.
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The nucleic acid segments used in the present invention, regardless of the
length of the coding sequence itself, may be combined with other DNA
sequences,
such as promoters, polyadenylation signals, additional restriction enzyme
sites,
multiple cloning sites, other coding segments, and the like, such that their
overall
5 length may vary considerably. It is therefore contemplated that a nucleic
acid
fragment of almost any length may be employed, with the total length
preferably
being limited by the ease of preparation and use in the intended recombinant
DNA
protocol.
10 The DNA segments used in the present invention encompass biologically
functional equivalent IgA proteins and peptides. Such sequences may arise as a
consequence of codon redundancy and functional equivalency that are known to
occur
naturally within nucleic acid sequences and the proteins thus encoded.
Alternatively,
functionally equivalent proteins or peptides may be created via the
application of
15 recombinant DNA technology, in which changes in the protein structure may
be
engineered, based on considerations of the properties of the amino acids being
exchanged. Changes designed by man may be introduced through the application
of
site-directed mutagenesis techniques, e.g., to introduce improvements to the
antigenicity of the protein or to test mutants in order to examine DNA binding
activity
20 at the molecular level.
Encompassed by the invention are DNA segments encoding relatively small
peptides, such as, for example, peptides of from about 10, 1 l, 12, 13, 14,
15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40,
25 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95
or up to about
100 amino acids in length; and also larger polypeptides up to and including
proteins
corresponding to the full-length sequences of the IgA polypeptide.
2. Recombinant Fusion Proteins
30 The pIgR targeted compositions of the invention may also be fusion proteins
prepared by molecular biological techniques, whereby a pIgR-binding domain,
such
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56
as Ca3 domain from IgA including the pIgR targeting motif, is fused with a
preventative or therapeutic compound. Alternatively, it is contemplated that a
pIgR-
binding domain can be expressed in the context of a non-IgA antibody, such as
IgG or
IgGM, to target that antibody to the mucosal epithelia. Furthermore, it is
contemplated that multiple pIgR-binding domains may be contained on a single
molecule, such as dimers and trimers of pIgR-binding domains. The use of
recombinant DNA techniques to achieve such ends is now standard practice to
those
of skill in the art. These methods include, for example, in vitro recombinant
DNA
techniques, synthetic techniques and in vivo recombination/genetic
recombination.
DNA and RNA synthesis may, additionally, be performed using an automated
synthesizers (see, for example, the techniques described in Sambrook et al.,
1989; and
Ausubel et al., 1989).
The preparation of such a fusion protein generally entails the preparation of
a
first and second DNA coding region and the functional ligation or joining of
said
regions, in frame, to prepare a single coding region that encodes the desired
fusion
protein. In the present context, the pIgR-binding domain DNA sequence will
generally be joined in frame with a DNA sequence encoding a preventative or
therapeutic compund and/or inert protein carrier, immunoglobulin, Fc region,
or such
like. The invention encompasses constructs where either the IgA portion of the
fusion
protein or the non-IgA portion is prepared as the N-terminal region or as the
C-
terminal region.
Once the coding region desired has been produced, an expression vector is
created. Expression vectors contain one or more promoters upstream of the
inserted
DNA regions that act to promote transcription of the DNA and to thus promote
expression of the encoded recombinant protein. This is the meaning of
"recombinant
expression" and has been discussed elsewhere in the specification.
In addition to the many known vectors that are commercially available, other
useful vectors include pIN vectors (Inouye et al., 1985); and pGEX vectors,
for use in
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generating glutathione S-transferase (GST) soluble fusion proteins for later
purification
and separation or cleavage. Other suitable fusion proteins are those with
13-galactosidase, ubiquitin, hexahistidine, or the like.
3. Vectors for Cloning, Gene Transfer, and Expression
Within certain embodiments, expression vectors are employed to express the
pIgR-binding domain polypeptide product, which can then be purified and, for
example, be used to vaccinate animals to generate antisera or monoclonal
antibody with
which further studies may be conducted. In other embodiments, the expression
vectors
are used to express a protein of interest in mucosal epithelia. In either
case, expression
requires that appropriate signals be provided in the vectors, and which
include various
regulatory elements, such as enhancers/promoters from both viral and mammalian
sources that drive expression of the genes of interest in host cells. Elements
designed
to optimize messenger RNA stability and translatability in host cells also are
defined.
The conditions for the use of a number of dominant drug selection markers for
establishing permanent, stable cell clones expressing the products also are
provided,
as is an element that links expression of the drug selection markers to
expression of
the polypeptide.
The construction and use of expression vectors and plasmids is well known to
those of skill in the art. Virtually any mammalian cell expression vector may
thus be
used connection with the humanized genes disclosed herein. Preferred vectors
and
plasmids will be constructed with at least one multiple cloning site. In
certain
embodiments, the expression vector will comprise a multiple cloning site that
is
operatively positioned between a promoter and sequences from IgA. Such vectors
may be used, in addition to their uses in other embodiments, to create N-
terminal
fusion proteins by cloning a second protein-encoding DNA segment into the
multiple
cloning site so that it is contiguous and in-frame with the pIgR-binding
domain
sequence, including the pIgR-binding domain sequence from IgA.
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In other embodiments, expression vectors may comprise a multiple cloning
site that is operatively positioned downstream from expressible IgA nucleic
acid
sequences. These vectors are useful, in addition to their uses, in creating C-
terminal
fusion proteins by cloning a second protein-encoding DNA segment into the
multiple
cloning site so that it is contiguous and in-frame with the IgA sequence.
Vectors and plasmids in which a second protein- or RNA-encoding nucleic
acid segment is also present are, of course, also encompassed by the
invention,
irrespective of the nature of the nucleic acid segment itself. A second
reporter gene
may be included within an expression vector of the present invention. The
second
reporter gene may be comprised within a second transcriptional unit. Suitable
second
reporter genes include those that confer resistance to agents such as
neomycin,
hygromycin, puromycin, zeocin, mycophenolic acid, histidinol and methotrexate.
Expression vectors may also contain other nucleic acid sequences, such as
IRES elements, polyadenylation signals, splice donor/splice acceptor signals,
and the
like.
a. Viral vectors
The methods and compositions described herein include multigene adenoviral
constructs; the methods and compositions described may be applicable to the
construction of multigene constructs using other viral vectors including but
not
limited to retroviruses, herpes viruses, adeno-associated viruses, vaccinia
viruses. The
discussion below provides details regarding the characteristics of each of
these viruses
in relation to their application in therapeutic compositions.
i) Adenovirus
One of the preferred methods for in vivo delivery involves the use of an
adenovirus expression vector. "Adenovirus expression vector" is meant to
include
those constructs containing adenovirus sequences sufficient to (a) support
packaging
of the construct and (b) to express an antisense polynucleotide, a protein, a
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polynucleotide (e.g., ribozyme, or an mRNA) that has been cloned therein. In
this
context, expression does not require that the gene product be synthesized.
The expression vector comprises a genetically engineered form of adenovirus.
Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-
stranded
DNA virus, allows substitution of large pieces of adenoviral DNA with foreign
sequences up to 7 kb (Grunhaus and Horwitz, 1992). In contrast to
retroviruses, the
adenoviral infection of host cells does not result in chromosomal integration
because
adenoviral DNA can replicate in an episomal manner without potential
genotoxicity.
As used herein, the term "genotoxicity" refers to permanent inheritable host
cell
genetic alteration. Also, adenoviruses are structurally stable, and no genome
rearrangement has been detected after extensive amplification of normal
derivatives.
Adenovirus can infect virtually all epithelial cells regardless of their cell
cycle stage.
Adenovirus is particularly suitable for use as a gene transfer vector because
of
its mid-sized genome, ease of manipulation, high titer, wide target cell range
and high
infectivity. Both ends of the viral genome contain 100-200 base pair inverted
repeats
(ITRs), which are cis elements necessary for viral DNA replication and
packaging.
The early (E) and late (L) regions of the genome contain different
transcription units
that are divided by the onset of viral DNA replication. The El region (ElA and
ElB)
encodes proteins responsible for the regulation of transcription of the viral
genome
and a few cellular genes. The expression of the E2 region (E2A and E2B)
results in
the synthesis of the proteins for viral DNA replication. These proteins are
involved in
DNA replication, late gene expression and host cell shut-off (Renan, 1990).
The
products of the late genes, including the majority of the viral capsid
proteins, are
expressed only after significant processing of a single primary transcript
issued by the
major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly
efficient
during the late phase of infection, and all the mRNA's issued from this
promoter
possess a 5'-tripartite leader (TPL) sequence which makes them preferred
mRNA's for
translation.
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The E3 region encodes proteins that appears to be necessary for efficient
lysis
of Ad infected cells as well as preventing TNF-mediated cytolysis and CTL
mediated
lysis of infected cells. In general, the E4 region encodes is believed to
encode seven
proteins, some of which activate the E2 promoter. It has been shown to block
host
5 mRNA transport and enhance transport of viral RNA to cytoplasm. Further the
E4
product is in part responsible for the decrease in early gene expression seen
late in
infection. E4 also inhibits ElA and E4 (but not E1B) expression during lytic
growth.
Some E4 proteins are necessary for efficient DNA replication however the
mechanism
for this involvement is unknown. E4 is also involved in post-transcriptional
events in
10 viral late gene expression; i.e., alternative splicing of the tripartite
leader in lytic
growth. Nevertheless, E4 functions are not absolutely required for DNA
replication
but their lack will delay replication. Other functions include negative
regulation of
viral DNA synthesis, induction of sub-nuclear reorganization normally seen
during
adenovirus infection, and other functions that are necessary for viral
replication, late
15 viral mRNA accumulation, and host cell transcriptional shut off.
In a current system, recombinant adenovirus is generated from homologous
recombination between shuttle vector and provirus vector. Possible
recombination
between the proviral vector and Ad sequences in 293 cells, or in the case of
pJMl7
20 plasmid spontaneous deletion of the inserted pBR322 sequences, may generate
full
length wild-type Ad5 adenovirus. Therefore, it is critical to isolate a single
clone of
virus from an individual plaque and examine its genomic structure.
Generation and propagation of the current adenovirus vectors, which are
25 replication deficient, depend on a unique helper cell line, designated 293,
which was
transformed from human embryonic kidney cells by Ad5 DNA fragments and
constitutively expresses El proteins (Graham et al., 1977). Since the E3
region is
dispensable from the adenovirus genome (Jones and Shenk, 1978), the current
adenovirus vectors, with the help of 293 cells, carry foreign DNA in either
the El, the
30 E3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can
package
approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987),
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providing capacity for about 2 extra kb of DNA. Combined with the
approximately
5.5 kb of DNA that is replaceable in the E1 and E3 regions, the maximum
capacity of
the current adenovirus vector is under 7.5 kb, or about 15% of the total
length of the
vector.
Helper cell lines may be derived from human cells such as human embryonic
kidney cells, muscle cells, hematopoietic cells or other human embryonic
mesenchymal or epithelial cells. Alternatively, the helper cells may be
derived from
the cells of other mammalian species that are permissive for human adenovirus.
Such
cells include, e.g., Vero cells or other monkey embryonic mesenchymal or
epithelial
cells. As stated above, the preferred helper cell line is 293.
Recently, Racher et al. (1995) disclosed improved methods for culturing 293
cells and propagating adenovirus. In one format, natural cell aggregates are
grown by
inoculating individual cells into 1 liter siliconized spinner flasks (Techne,
Cambridge,
UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell
viability is estimated with trypan blue. In another format, Fibra-Cel
microcarriers
(Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum,
resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml
Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h.
The
medium is then replaced with 50 ml of fresh medium and shaking is initiated.
For
virus production, cells are allowed to grow to about 80% confluence, after
which time
the medium is replaced (to 25% of the final volume) and adenovirus added at an
MOI
of 0.05. Cultures are left stationary overnight, following which the volume is
increased to 100% and shaking commenced for another 72 h.
Other than the requirement that the adenovirus vector be replication
defective,
or at least conditionally defective, the nature of the adenovirus vector is
not believed
to be crucial to the successful practice of the invention. The adenovirus may
be of any
of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of
subgroup C is the preferred starting material in order to obtain the
conditional
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replication-defective adenovirus vector for use in the present invention. This
is
because Adenovirus type 5 is a human adenovirus about which a great deal of
biochemical, medical and genetic information is known, and it has historically
been
used for most constructions employing adenovirus as a vector.
As stated above, the typical vector according to the present invention is
replication defective and will not have an adenovirus E1 region. Thus, it will
be most
convenient to introduce the polynucleotide encoding the gene of interest at
the
position from which the E1-coding sequences have been removed. However, the
position of insertion of the construct within the adenovirus sequences is not
critical to
the invention. The polynucleotide encoding the gene of interest may also be
inserted
in lieu of the deleted E3 region in E3 replacement vectors as described by
Karlsson et
al. ( 1986), or in the E4 region where a helper cell line or helper virus
complements the
E4 defect.
Adenovirus is easy to grow and manipulate and exhibits broad host range in
vitro and in vivo. This group of viruses can be obtained in high titers, e.g.,
109-10"
plaque-forming units per ml, and they are highly infective. The life cycle of
adenovirus does not require integration into the host cell genome. The foreign
genes
delivered by adenovirus vectors are episomal and, therefore, have low
genotoxicity to
host cells. No side effects have been reported in studies of vaccination with
wild-type
adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating their safety
and
therapeutic potential as in vivo gene transfer vectors.
Adenovirus vectors have been used in eukaryotic gene expression
investigations (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine
development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently,
animal studies suggested that recombinant adenovirus could be used for gene
therapy
(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al.,
1990; Rich
et al., 1993). Studies in administering recombinant adenovirus to different
tissues
include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992),
muscle
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injection (Ragot et al., 1993), peripheral intravenous injections (Herz and
Gerard,
1993), intranasal inoculation (Ginsberg et al., 1991), aerosol administration
to lung
(Bellon, 1996) intra-peritoneal administration (Song et al., 1997), Intra-
pleural
injection (Elshami et al., 1996) administration to the bladder using intra-
vesicular
administration (Werthman, et al., 1996), Subcutaneous injection including
intraperitoneal, intrapleural, intramuscular or subcutaneously) (Ogawa, 1989)
ventricular injection into myocardium (heart, French et al., 1994), liver
perfusion
(hepatic artery or portal vein, Shiraishi et al., 1997) and stereotactic
inoculation into
the brain (Le Gal La Salle et al., 1993).
ii) Retrovirus
The retroviruses are a group of single-stranded RNA viruses characterized by
an ability to convert their RNA to double-stranded DNA in infected cells by a
process
of reverse-transcription (Coffin, 1990). The resulting DNA then stably
integrates into
cellular chromosomes as a provirus and directs synthesis of viral proteins.
The
integration results in the retention of the viral gene sequences in the
recipient cell and
its descendants. The retroviral genome contains three genes, gag, pol, and env
that
code for capsid proteins, polymerase enzyme, and envelope components,
respectively.
A sequence found upstream from the gag gene contains a signal for packaging of
the
genome into virions. Two long terminal repeat (LTR) sequences are present at
the 5'
and 3' ends of the viral genome. These contain strong promoter and enhancer
sequences and are also required for integration in the host cell genome
(Coffin, 1990).
In order to construct a retroviral vector, a nucleic acid encoding a gene of
interest is inserted into the viral genome in the place of certain viral
sequences to
produce a virus that is replication-defective. In order to produce virions, a
packaging
cell line containing the gag, pol, and env genes but without the LTR and
packaging
components is constructed (Mann et al., 1983). When a recombinant plasmid
containing a cDNA, together with the retroviral LTR and packaging sequences is
introduced into this cell line (by calcium phosphate precipitation for
example), the
packaging sequence allows the RNA transcript of the recombinant plasmid to be
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packaged into viral particles, which are then secreted into the culture media
(Nicolas
and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing
the
recombinant retroviruses is then collected, optionally concentrated, and used
for gene
transfer. Retroviral vectors are able to infect a broad variety of cell types.
However,
S integration and stable expression require the division of host cells
(Paskind et al.;
1975).
A novel approach designed to allow specific targeting of retrovirus vectors
was recently developed based on the chemical modification of a retrovirus by
the
chemical addition of lactose residues to the viral envelope. This modification
could
permit the specific infection of hepatocytes via sialoglycoprotein receptors:
A different approach to targeting of recombinant retroviruses was designed in
which biotinylated antibodies against a retroviral envelope protein and
against a
specific cell receptor were used. The antibodies were coupled via the biotin
components by using streptavidin (Roux et al., 1989). Using antibodies against
major
histocompatibility complex class I and class II antigens, they demonstrated
the
infection of a variety of human cells that bore those surface antigens with an
ecotropic
virus in vitro (Roux et al., 1989).
There are certain limitations to the use of retrovirus vectors in all aspects
of
the present invention. For example, retrovirus vectors usually integrate into
random
sites in the cell genome. This can lead to insertional mutagenesis through the
interruption of host genes or through the insertion of viral regulatory
sequences that
can interfere with the function of flanking genes (Varmus et al., 1981).
Another
concern with the use of defective retrovirus vectors is the potential
appearance of
wild-type replication-competent virus in the packaging cells. This can result
from
recombination events in which the intact- sequence from the recombinant virus
inserts
upstream from the gag, pol, env sequence integrated in the host cell genome.
However, new packaging cell lines are now available that should greatly
decrease the
likelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,
1990).
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iii) Herpesvirus
Because herpes simplex virus (HSV) is neurotropic, it has generated
considerable interest in treating nervous system disorders. Moreover, the
ability of
5 HSV to establish latent infections in non-dividing neuronal cells without
integrating
in to the host cell chromosome or otherwise altering the host cell's
metabolism, along
with the existence of a promoter that is active during latency makes HSV an
attractive
vector. And though much attention has focused on the neurotropic applications
of
HSV, this vector also can be exploited for other tissues given its wide host
range.
Another factor that makes HSV an attractive vector is the size and
organization of the genome. Because HSV is large, incorporation of multiple
genes or
expression cassettes is less problematic than in other smaller viral systems.
In
addition, the availability of different viral control sequences with varying
performance
(temporal, strength, etc.) makes it possible to control expression to a
greater extent
than in other systems. It also is an advantage that the virus has relatively
few spliced
messages, further easing genetic manipulations.
HSV also is relatively easy to manipulate and can be grown to high titers.
Thus, delivery is less of a problem, both in terms of volumes needed to attain
sufficient MOI and in a lessened need for repeat dosings. For a review of HSV
as a
gene therapy vector, see Glorioso et al. (1995).
HSV, designated with subtypes 1 and 2, are enveloped viruses that are among
the most common infectious agents encountered by humans, infecting millions of
human subjects worldwide. The large, complex, double-stranded DNA genome
encodes for dozens of different gene products, some of which derive from
spliced
transcripts. In addition to virion and envelope structural components, the
virus
encodes numerous~other proteins including a protease, a ribonucleotides
reductase, a
DNA polymerase, a ssDNA binding protein, a helicase/primase, a DNA dependent
ATPase, a dUTPase and others.
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HSV genes form several groups whose expression is coordinately regulated
and sequentially ordered in a cascade fashion (Honess and Roizman, 1974;
Honess
and Roizman 1975; Roizman and Sears, 1995). The expression of a genes, the
first
set of genes to be expressed after infection, is enhanced by the virion
protein number
16, or a-transinducing factor (Post et al., 1981; Batterson and Roizman, 1983;
Campbell, et al., 1983). The expression of (3 genes requires functional a gene
products, most notably ICP4, which is encoded by the a4 gene (DeLuca et al.,
1985).
y genes, a heterogeneous group of genes encoding largely virion structural
proteins,
require the onset of viral DNA synthesis for optimal expression (Holland et
al., 1980).
In line with the complexity of the genome, the life cycle of HSV is quite
involved. In addition to the lytic cycle, which results in synthesis of virus
particles
and, eventually, cell death, the virus has the capability to enter a latent
state in which
the genome is maintained in neural ganglia until some as of yet undefined
signal
triggers a recurrence of the lytic cycle. Avirulent variants of HSV have been
developed and are readily available for use in gene therapy contexts (U.S.
Patent No.
5,672,344).
iv) Adeno-Associated Virus
Recently, adeno-associated virus (AAV) has emerged as a potential alternative
to the more commonly used retroviral and adenoviral vectors. While studies
with
retroviral and adenoviral mediated gene transfer raise concerns over potential
oncogenic properties of the former, and immunogenic problems associated with
the
latter, AAV has not been associated with any such pathological indications.
In addition, AAV possesses several unique features that make it more
desirable than the other vectors. Unlike retroviruses, AAV can infect non-
dividing
cells; wild-type AAV has been characterized by integration, in a site-specific
manner,
into chromosome 19 of human cells (Kotin and Berns, 1989; Kotin et al., 1990;
Kotin
et al., 1991; Samulski et al., 1991); and AAV also possesses anti-oncogenic
properties
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(Ostrove et al., 1981; Berns and Giraud, 1996). Recombinant AAV genomes are
constructed by molecularly cloning DNA sequences of interest between the AAV
ITRs, eliminating the entire coding sequences of the wild-type AAV genome. The
AAV vectors thus produced lack any of the coding sequences of wild-type AAV,
yet
retain the property of stable chromosomal integration and expression of the
recombinant genes upon transduction both in vitro and in vivo (Berns, 1990;
Berns
and Bohensky, 1987; Bertran et al., 1996; Kearns et al., 1996; Ponnazhagan et
al.,
1997a). Until recently, AAV was believed to infect almost all cell types, and
even
cross species barriers. However, it now has been determined that AAV infection
is
receptor-mediated (Ponnazhagan et al., 1996; Mizukami et al., 1996).
AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted
terminal repeats flank the genome. Two genes are present within the genome,
giving
rise to a number of distinct gene products. The first, the cap gene, produces
three
different virion proteins (VP), designated VP-l, VP-2 and VP-3. The second,
the rep
gene, encodes four non-structural proteins (NS). One or more of these rep gene
products is responsible for transactivating AAV transcription. The sequence of
AAV
is provided by Srivastava et al., (1983) and in U.S. Patent 5,252,479 (entire
text of
which is specifically incorporated herein by reference).
The three promoters in AAV are designated by their location, in map units, in
the genome. These are, from left to right, p5, pl9 and p40. Transcription
gives rise to
six transcripts, two initiated at each of three promoters, with one of each
pair being
spliced. The splice site, derived from map units 42-46, is the same for each
transcript.
The four non-structural proteins apparently are derived from the longer of the
transcripts, and three virion proteins all arise from the smallest transcript.
AAV is not associated with any pathologic state in humans. Interestingly, for
efficient replication, AAV requires "helping" functions from viruses such as
herpes
simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course,
adenovirus. The best characterized of the helpers is adenovirus, and many
"early"
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functions for this virus have been shown to assist with AAV replication. Low
level
expression of AAV rep proteins is believed to hold AAV structural expression
in
check, and helper virus infection is thought to remove this block.
v) Vaccinia Virus
Vaccinia virus vectors have been used extensively because of the ease of their
construction, relatively high levels of expression obtained, wide host range
and large
capacity for carrying DNA. Vaccinia contains a linear, double-stranded DNA
genome
of about 186 kb that exhibits a marked "A-T" preference. Inverted terminal
repeats of
about 10.5 kb flank the genome. The majority of essential genes appear to map
within
the central region, which is most highly conserved among poxviruses: Estimated
open reading frames in vaccinia virus number from 150 to 200. Although both
strands are coding, extensive overlap of reading frames is not common.
At least 25 kb can be inserted into the vaccinia virus genome (Smith and
Moss, 1983). Prototypical vaccinia vectors contain transgenes inserted into
the viral
thymidine kinase gene via homologous recombination. Vectors are selected on
the
basis of a tk-phenotype. Inclusion of the untranslated leader sequence of
encephalomyocarditis virus, the level of expression is higher than that of
conventional
vectors, with the transgenes accumulating at 10% or more of the infected
cell's
protein in 24 h (Elroy-Stein et al., 1989).
b. Non-viral transfer
Several non-viral methods for the transfer of expression constructs into
cultured mammalian cells also are contemplated by the present invention. These
include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and
Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation
(Tur-Kaspa et al., 1986; Potter et al., 1984), direct microinjection (Harland
and
Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al.,
1979), cell sonication (Fechheimer et al., 1987), gene bombardment using high
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velocity microprojectiles (Yang et al., 1990), and receptor-mediated
transfection (Wu
and Wu, 1987; Wu and Wu, 1988).
For therapeutic endeavors, once the construct has been delivered into the
cell,
the gene may be positioned and expressed at different sites. In certain
embodiments,
the nucleic acid encoding the therapeutic gene may be stably integrated into
the
genome of the cell. This integration may be in the cognate location and
orientation
via homologous recombination (gene replacement) or it may be integrated in a
random, non-specific location (gene augmentation). In yet further embodiments,
the
nucleic acid may be stably maintained in the cell as a separate, episomal
segment of
DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to
permit maintenance and replication independent of or in synchronization with
the host
cell cycle. How the expression construct is delivered to a cell and where in
the cell
the nucleic acid remains is dependent on the type of expression construct
employed.
In a particular embodiment of the invention, the expression construct may be
entrapped in a liposome. Liposomes are vesicular structures characterized by a
phospholipid bilayer membrane and an inner aqueous medium. Multilamellar
liposomes have multiple lipid layers separated by aqueous medium. They form
spontaneously when phospholipids are suspended in an excess of aqueous
solution.
The lipid components undergo self rearrangement before the formation of closed
structures and entrap water and dissolved solutes between the lipid bilayers
(Ghosh
and Bachhawat, i 991 ). The addition of DNA to cationic liposomes causes a
topological transition from liposomes to optically birefringent liquid-
crystalline
condensed globules (Radler et al., 1997). These DNA-lipid complexes are
potential
non-viral vectors for use in gene therapy.
Liposome-mediated nucleic acid delivery and expression of foreign DNA in
vitro has been very successful. Using the (3-lactamase gene, Wong et al.
(1980)
demonstrated the feasibility of liposome-mediated delivery and expression of
foreign
DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et al. (1987)
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accomplished successful liposome-mediated gene transfer in rats after
intravenous
injection. Also included are various commercial approaches involving
"lipofection"
technology.
5 In certain embodiments of the invention, the liposome may be complexed with
a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with
the cell
membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al.,
1989). In other embodiments, the liposome may be complexed or employed in
conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al.,
10 1991 ). In yet further embodiments, the liposome may be complexed or
employed in
conjunction with both HVJ and HMG-1. In that such expression constructs have
been
successfully employed in transfer and expression of nucleic acid in vitro and
in vivo,
then they are applicable for the present invention.
15 Other vector delivery systems that can be employed to deliver a nucleic
acid
encoding a therapeutic gene into cells are receptor-mediated delivery
vehicles. These
take advantage of the selective uptake of macromolecules by receptor-mediated
endocytosis in almost all eukaryotic cells. Because of the cell type-specific
distribution of various receptors, the delivery can be highly specific (Wu and
Wu,
20 1993).
In another embodiment of the invention, the expression construct may simply
consist of naked recombinant DNA or plasmids. Transfer of the construct may be
performed by any of the methods mentioned above which physically or chemically
25 permeabilize the cell membrane. This is applicable particularly for
transfer in vitro,
however, it may be applied for in vivo use as well. Dubensky et al. ( 1984)
successfully injected polyomavirus DNA in the form of CaP04 precipitates into
liver
and spleen of adult and newborn mice demonstrating active viral replication
and acute
infection. Benvenisty and Neshif (1986) also demonstrated that direct
intraperitoneal
30 injection of CaP04 precipitated plasmids results in expression of the
transfected
genes.
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4. Regulatory Elements
The recombinant DNA techniques encompassed by the present invention to
prepare and produce pIgR-targeted compositions including compositions
comprising
IgA sequences may utilize recombinant vectors or expression constructs
containing
regulatory elements. These regulatory elements can include promoters (tissue-
specific, non-tissue-specific, and inducible) and enhancers, polyadenylation
sequences, and internal ribosomal entry sites (IRES).
a. Promoters
Throughout this application, the term "expression construct" is meant to
include any type of genetic construct containing a nucleic acid coding for
gene
products in which part or all of the nucleic acid encoding sequence is capable
of being
transcribed. The transcript may be translated into a protein, but it need not
be. In
certain embodiments, expression includes both transcription of a gene and
translation
of mRNA into a gene product. In other embodiments, expression only includes
transcription of the nucleic acid encoding genes of interest.
The nucleic acid encoding a gene product is under transcriptional control of a
promoter. A "promoter" refers to a DNA sequence recognized by the synthetic
machinery of the cell, or introduced synthetic machinery, required to initiate
the
specific transcription of a gene. The phrase "under transcriptional control"
means that
the promoter is in the correct location and orientation in relation to the
nucleic acid to
control RNA polymerase initiation and expression of the gene.
The term promoter will be used here to refer to a group of transcriptional
control modules that are clustered around the initiation site for RNA
polymerase II.
Much of the thinking about how promoters are organized derives from analyses
of
several viral promoters, including those for the HSV thymidine kinase (tk) and
SV40
early transcription units. These studies, augmented by more recent work, have
shown
that promoters are composed of discrete functional modules, each consisting of
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approximately 7-20 by of DNA, and containing one or more recognition sites for
transcriptional activator or repressor proteins.
At least one module in each promoter functions to position the start site for
S RNA synthesis. The best known example of this is the TATA box, but in some
promoters lacking a TATA box, such as the promoter for the mammalian terminal
deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a
discrete
element overlying the start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional
initiation. Typically, these are located in the region 30-110 by upstream of
the start
site, although a number of promoters have recently been shown to contain
functional
elements downstream of the start site as well. The spacing between promoter
elements frequently is flexible, so that promoter function is preserved when
elements
are inverted or moved relative to one another. In the tk promoter, the spacing
between
promoter elements can be increased to 50 by apart before activity begins to
decline.
Depending on the promoter, it appears that individual elements can function
either co-
operatively or independently to activate transcription.
The particular promoter employed to control the expression of a nucleic acid
sequence of interest is not believed to be important, so long as it is capable
of
directing the expression of the nucleic acid in the targeted cell. Thus, where
a human
cell is targeted, it is preferable to position the nucleic acid coding region
adjacent to
and under the control of a promoter that is capable of being expressed in a
human cell.
Generally speaking, such a promoter might include either a human or viral
promoter.
In various embodiments, the human cytomegalovirus (CMV) immediate early
gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal
repeat,
(3-actin, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase
can be
used to obtain high-level expression of the coding sequence of interest. The
use of
other viral or mammalian cellular or bacterial phage promoters which are well-
known
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in the art to achieve expression of a coding sequence of interest is
contemplated as
well, provided that the levels of expression are sufficient for a given
purpose. By
employing a promoter with well-known properties, the level and pattern of
expression
of the protein of interest following transfection or transformation can be
optimized:
Selection of a promoter that is regulated in response to specific physiologic
or
synthetic signals can permit inducible expression of the gene product. For
example in
the case where expression of a transgene, or transgenes when a multicistronic
vector is
utilized, is toxic to the cells in which the vector is produced in, it may be
desirable to
prohibit or reduce expression of one or more of the transgenes. Examples of
transgenes that may be toxic to the producer cell line are pro-apoptotic and
cytokine
genes. Several inducible promoter systems are available for production of
viral
vectors where the transgene product may be toxic.
The ecdysone system (Invitrogen, Carlsbad, CA) is one such system. This
system is designed to allow regulated expression of a gene of interest in
mammalian
cells. It consists of a tightly regulated expression mechanism that allows
virtually no
basal level expression of the transgene, but over 200-fold inducibility. The
system is
based on the heterodimeric ecdysone receptor of Drosophila, and when ecdysone
or
an analog such as muristerone A binds to the receptor, the receptor activates
a
promoter to turn on expression of the downstream transgene high levels of mRNA
transcripts are attained. In this system, both monomers of the heterodimeric
receptor
are constitutively expressed from one vector, whereas the ecdysone-responsive
promoter that drives expression of the gene of interest is on another plasmid.
Engineering of this type of system into the gene transfer vector of interest
would
therefore be useful. Cotransfection of plasmids containing the gene of
interest and the
receptor monomers in the producer cell line would then allow for the
production of
the gene transfer vector without expression of a potentially toxic transgene.
At the
appropriate time, expression of the transgene could be activated with ecdysone
or
muristeron A.
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Another inducible system that would be useful is the Tet-OffrM or Tet-OnTM
system (Clontech, Palo Alto, CA) originally developed by Gossen and Bujard
(Gossen
and Bujard, 1992; Gossen et al., 1995). This system also allows high levels of
gene
expression to be regulated in response to tetracycline or tetracycline
derivatives such
as doxycycline. In the Tet-OnTM system, gene expression is turned on in the
presence
of doxycycline, whereas in the Tet-OffrM system, gene expression is turned on
in the
absence of doxycycline. These systems are based on two regulatory elements
derived
from the tetracycline resistance operon of E. coli. The tetracycline operator
sequence
to which the tetracycline repressor binds, and the tetracycline repressor
protein. The
gene of interest is cloned into a plasmid behind a promoter, that has
tetracycline-
responsive elements present in it. A second plasmid contains a regulatory
element
called the tetracycline-controlled transactivator, which is composed, in the
Tet-OffrM
system, of the VP16 domain from the herpes simplex virus and the wild-type
tertracycline repressor. Thus in the absence of doxycycline, transcription is
constituitively on. In the Tet-OnTM system, the tetracycline repressor is not
wild type
and in the presence of doxycycline activates transcription. For gene therapy
vector
production, the Tet-OffrM system would be preferable so that the producer
cells could
be grown in the presence of tetracycline or doxycycline and prevent expression
of a
potentially toxic transgene, but when the vector is introduced to the patient,
the gene
expression would be constituitively on.
In some circumstances, it may be desirable to regulate expression of a
transgene in a gene therapy vector. For example, different viral promoters
with
varying strengths of activity may be utilized depending on the level of
expression
desired. In mammalian cells, the CMV immediate early promoter if often used to
provide strong transcriptional activation. Modified versions of the CMV
promoter
that are less potent have also been used when reduced levels of expression of
the
transgene are desired. When expression of a transgene in hematopoetic cells is
desired, retroviral promoters such as the LTRs from MLV or MMTV are often
used.
Other viral promoters that may be used depending on the desired effect include
SV40,
RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as from the ElA, E2A,
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or MLP region, AAV LTR, cauliflower mosaic virus, HSV-TK, and avian sarcoma
virus.
Similarly tissue specific promoters may be used to effect transcription in
5 specific tissues or cells so as to reduce potential toxicity or undesirable
effects to non-
targeted tissues. In the present invention, embodiments cover promoters that
direct
expression in epithelium cells, particularly mucosal epithelium. Endothelial-
specific
promoters direct the regulation of genes such as E-selectin, von Willebrand
factor,
TIE (Korhonen et al., 1995) and KDR/flk-1.
In certain indications, it may be desirable to activate transcription at
specific
times after administration of the gene therapy vector. This may be done with
such
promoters as those that are hormone or cytokine regulatable. For example in
gene
therapy applications where the indication is a gonadal tissue where specific
steroids
are produced or routed to, use of androgen or estrogen regulated promoters may
be
advantageous. Such promoters that are hormone regulatable include MMTV, MT-1,
ecdysone and RuBisco. Other hormone regulated promoters such as those
responsive
to thyroid, pituitary and adrenal hormones are expected to be useful in the
present
invention. Cytokine and inflammatory protein responsive promoters that could
be
used include K and T Kininogen (Kageyama et al., 1987), c-fos, TNF-alpha, C-
reactive protein (Arcone et al., 1988), haptoglobin (Oliviero et al., 1987),
serum
amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3
(Wilson et al., 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann,
1988),
alpha-1 antitypsin, lipoprotein lipase (Zechner et al., 1988), angiotensinogen
(Ron et
al., 1991), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV
radiation,
retinoic acid, and hydrogen peroxide), collagenase (induced by phorbol esters
and
retinoic acid), metallothionein (heavy metal and glucocorticoid inducible),
Stromelysin (inducible by phorbol ester, interleukin-l and EGF), alpha-2
macroglobulin and alpha-1 antichymotrypsin.
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It is envisioned that cell cycle regulatable promoters may be useful in the
present invention. For example, in a bi-cistronic gene therapy vector, use of
a strong
CMV promoter to drive expression of a first gene such as pl6 that arrests
cells in the
G1 phase could be followed by expression of a second gene such as p53 under
the
control of a promoter that is active in the G l phase of the cell cycle, thus
providing a
"second hit" that would push the cell into apoptosis. Other promoters such as
those of
various cyclins, PCNA, galectin-3, E2F1, p53 and BRCAI could be used.
Tumor specific promoters such as osteocalcin, hypoxia-responsive element
(HRE), MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may also be
used to regulate gene expression in tumor cells. Other promoters that could be
used
according to the present invention include Lac-regulatable, chemotherapy
inducible
(e.g. MDR), and heat (hyperthermia) inducible promoters, radiation-inducible
(e.g.,
EGR (Joki et al., 1995)), Alpha-inhibin, RNA pol III tRNA met and other amino
acid
promoters, U1 snRNA (Bartlett et al., 1996), MC-l, PGK, (3-actin and a-globin.
Many other promoters that may be useful are listed in Walther and Stein
(1996).
It is envisioned that any of the above promoters alone or in combination with
another may be useful according to the present invention depending on the
action
desired. In addition, this list of promoters is should not be construed to be
exhaustive
or limiting, those of skill in the art will know of other promoters that may
be used in
conjunction with the promoters and methods disclosed herein.
b. Enhancers
Enhancers are genetic elements that increase transcription from a promoter
located at a distant position on the same molecule of DNA. Enhancers are
organized
much like promoters. That is, they are composed of many individual elements,
each
of which binds to one or more transcriptional proteins. The basic distinction
between
enhancers and promoters is operational. An enhancer region as a whole must be
able
to stimulate transcription at a distance; this need not be true of a promoter
region or its
component elements. On the other hand, a promoter must have one or more
elements
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that direct initiation of RNA synthesis at a particular site and in a
particular
orientation, whereas enhancers lack these specificities. Promoters and
enhancers are
often overlapping and contiguous, often seeming to have a very similar modular
organization.
In some embodiments of the invention, the expression construct comprises a
virus or engineered construct derived from a viral genome. The ability of
certain
viruses to enter cells via receptor-mediated endocytosis and to integrate into
host cell
genome and express viral genes stably and efficiently have made them
attractive
candidates for the transfer of foreign genes into mammalian cells (Ridgeway,
1988;
Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The
first
viruses used as gene vectors were DNA viruses including the papovaviruses
(simian
virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and
Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).
These have a relatively low capacity for foreign DNA sequences and have a
restricted
host spectrum. Furthermore, their oncogenic potential and cytopathic effects
in
permissive cells raise safety concerns. They can accommodate only up to 8 kB
of
foreign genetic material but can be readily introduced in a variety of cell
lines and
laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).
c. Polyadenylation signals
Where a cDNA insert is employed, one will typically desire to include a
polyadenylation signal to effect proper polyadenylation of the gene
transcript. The
nature of the polyadenylation signal is not believed to be crucial to the
successful
practice of the invention, and any such sequence may be employed such as human
or
bovine growth hormone and SV40 polyadenylation signals. Also contemplated as
an
element of the expression cassette is a terminator. These elements can serve
to
enhance message levels and to minimize read through from the cassette into
other
sequences.
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d. IRES
In certain embodiments of the invention, the use of internal ribosome entry
site
(IRES) elements is contemplated to create multigene, or polycistronic,
messages.
IRES elements are able to bypass the ribosome scanning model of 5' methylated
Cap
dependent translation and begin translation at internal sites (Pelletier and
Sonenberg,
1988). IRES elements from two members of the picornavirus family (poliovirus
and
encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as
well
an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements
can be linked to heterologous open reading frames. Multiple open reading
frames can
be transcribed together, each separated by an IRES, creating polycistronic
messages.
By virtue of the IRES element, each open reading frame is accessible to
ribosomes for
efficient translation. Multiple genes can be efficiently expressed using a
single
promoter/enhancer to transcribe a single message.
Any heterologous open reading frame can be linked to IRES elements. This
includes genes for secreted proteins, mufti-subunit proteins, encoded by
independent
genes, intracellular or membrane-bound proteins and selectable markers. In
this way,
expression of several proteins can be simultaneously engineered into a cell
with a
single construct and a single selectable marker.
5. In vitro protein production
Following transduction with an expression construct or vector according to
some
embodiments of the present invention, primary mammalian cell cultures may be
prepared in various ways. In order for the cells to be kept viable while in
vitro and in
contact with the expression construct, it is necessary to ensure that the
cells maintain
contact with the correct ratio of oxygen and carbon dioxide and nutrients but
are
protected from microbial contamination. Cell culture techniques are well
documented
and are disclosed herein by reference (Freshner,1992).
One embodiment of the foregoing involves the use of gene transfer to
immortalize cells for the production and/or presentation of proteins. The gene
for the
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protein of interest may be transferred as described above into appropriate
host cells
followed by culture of cells under the appropriate conditions. The gene for
virtually
any polypeptide may be employed in this manner. The generation of recombinant
expression vectors, and the elements included therein, are discussed above.
Alternatively, the protein to be produced may be an endogenous protein
normally
synthesized by the cell in question.
Another embodiment of the present invention uses cell lines, which are
transfected with an expression construct or vector that expresses IgA protein.
Examples of mammalian host cell lines include Vero and HeLa cells, other B-
and T-
cell lines, such as CEM, 721.221, H9, Jurkat, Raji, etc., as well as cell
lines of
Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK
cells. In addition, a host cell strain may be chosen that modulates the
expression of
the inserted sequences, or that modifies and processes the gene product in the
manner
desired. Such modifications (e.g., glycosylation) and processing (e.g.,
cleavage) of
protein products may be important for the function of the protein. Different
host cells
have characteristic and specific mechanisms for the post-translational
processing and
modification of proteins. Appropriate cell lines or host systems can be chosen
to
insure the correct modification and processing of the foreign protein
expressed.
A number of selection systems may be used including, but not limited to, HSV
thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine
phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively.
Also, anti-
metabolite resistance can be used as the basis of selection: for dhfi°,
which confers
resistance to; gpt, which confers resistance to mycophenolic acid; neo, which
confers
resistance to the aminoglycoside 6418; and hygro, which confers resistance to
hygromycin.
Animal cells can be propagated in vitro in two modes: as non-anchorage-
dependent cells growing in suspension throughout the bulk of the culture or as
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anchorage-dependent cells requiring attachment to a solid substrate for their
propagation (i. e., a monolayer type of cell growth).
Non-anchorage dependent or suspension cultures from continuous established
5 cell lines are the most widely used means of large-scale production of cells
and cell
products. However, suspension cultured cells have limitations, such as
tumorigenic
potential and lower protein production than adherent cells.
III. Therapeutic Formulations and Routes of Administration
10 The present invention discloses the compositions and methods involving a
pIgR-binding domain that provides a targeting system for delivering selective
agents
to the mucosal epithelia. While systemic administration of formulations can
provide a
treatment method, frequently this delivery method fails to reach a location
where it
can confer a therapeutic benefit or it does so with reduced efficacy. The
present
15 invention, however, provides in some embodiments the ease of systemic
formulation
while providing a targeting method that allows the delivery of formulations
specifically to the mucosal epithelia.
Where clinical applications are contemplated, it will be necessary to prepare
20 the compositions of the present invention as pharmaceutical compositions,
i.e., in a
form appropriate for in vivo applications. Generally, this will entail
preparing
compositions that are essentially free of pyrogens, as well as other
impurities that
could be harmful to humans or animals.
25 A. Formulations
One will generally desire to employ appropriate salts and buffers to render
delivery vectors stable and allow for uptake by target cells. Buffers also
will be
employed when recombinant cells are introduced into a patient. Aqueous
compositions of the present invention comprise an effective amount of the
vector to
30 cells, dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous
medium. Such compositions also are referred to as inocula. The phrase
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"pharmaceutically or pharmacologically acceptable" refer to molecular entities
and
compositions that do not produce adverse, allergic, or other untoward
reactions when
administered to an animal or a human. As used herein, "pharmaceutically
acceptable
carrier" includes any and all solvents, dispersion media, coatings,
antibacterial and
antifungal agents, isotonic and absorption delaying agents and the like. The
use of
such media and agents for pharmaceutically active substances is well know in
the art.
Except insofar as any conventional media or agent is incompatible with the
vectors or
cells of the present invention, its use in therapeutic compositions is
contemplated.
Supplementary active ingredients also can be incorporated into the
compositions.
The active compositions of the present invention include classic
pharmaceutical preparations. Administration of these compositions according to
the
present invention will be via any common route so long as the target tissue is
available via that route. This includes oral, nasal, buccal, rectal, vaginal
or topical.
Alternatively, administration may be by orthotopic, intradermal, subcutaneous,
intramuscular, intraperitoneal or intravenous injection. Such compositions
would
normally be administered as pharmaceutically acceptable compositions,
described
supra.
The active compounds may be administered via any suitable route, including
parenterally or by injection or inhalation. Solutions of the active compounds
as free
base or pharmacologically acceptable salts can be prepared in water suitably
mixed
with a surfactant, such as hydroxypropylcellulose. Dispersions also can be
prepared
in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
Under
ordinary conditions of storage and use, these preparations contain a
preservative to
prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of
sterile injectable solutions or dispersions. In all cases the form must be
sterile and
must be fluid to the extent that easy syringability exists. It must be stable
under the
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conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and fungi. The
carrier can
be a solvent or dispersion medium containing, for example, water, ethanol,
polyol (for
example, glycerol, propylene glycol, and liquid polyethylene glycol, and the
like),
suitable mixtures thereof, and vegetable oils. The proper fluidity can be
maintained,
for example, by the use of a coating, such as lecithin, by the maintenance of
the
required particle size in the case of dispersion and by the use of
surfactants. The
prevention of the action of microorganisms can be brought about by various
antibacterial an antifungal agents, for example, parabens, chlorobutanol,
phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be preferable to
include
isotonic agents, for example, sugars or sodium chloride. Prolonged absorption
of the
injectable compositions can be brought about by the use in the compositions of
agents
delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active
compounds in the required amount in the appropriate solvent with various of
the other
ingredients enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the various sterilized
active
ingredients into a sterile vehicle which contains the basic dispersion medium
and the
required other ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the preferred
methods of
preparation are vacuum-drying and freeze-drying techniques which yield a
powder of
the active ingredient plus any additional desired ingredient from a previously
sterile-
filtered solution thereof.
As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and
absorption delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active ingredient, its
use in the
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therapeutic compositions is contemplated. Supplementary active ingredients
also can
be incorporated into the compositions.
For oral administration the polypeptides and expression constructs of the
present invention may be incorporated with excipients and used in the form of
non-
ingestible mouthwashes and dentifrices. A mouthwash may be prepared
incorporating
the active ingredient in the required amount in an appropriate solvent, such
as a
sodium borate solution (Dobell's Solution). Alternatively, the active
ingredient may
be incorporated into an antiseptic wash containing sodium borate, glycerin and
potassium bicarbonate. The active ingredient may also be dispersed in
dentifrices,
including: gels, pastes, powders and slurries. The active ingredient may be
added in a
therapeutically effective amount to a paste dentifrice that may include water,
binders,
abrasives, flavoring agents, foaming agents, and humectants.
The compositions of the present invention may be formulated in a neutral or
salt form. Pharmaceutically-acceptable salts include the acid addition salts
(formed
with the free amino groups of the protein) and which are formed with inorganic
acids
such as, for example, hydrochloric or phosphoric acids, or such organic acids
as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free
carboxyl
groups also can be derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a manner compatible with
the dosage formulation and in such amount as is therapeutically effective. The
formulations are easily administered in a variety of dosage forms such as
injectable
solutions, drug release capsules and the like. For parenteral administration
in an
aqueous solution, for example, the solution should be suitably buffered if
necessary
and the liquid diluent first rendered isotonic with sufficient saline or
glucose. These
particular aqueous solutions are especially suitable for intravenous,
intramuscular,
subcutaneous and intraperitoneal administration.
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Compositions may be conventionally administered parenterally, by inj ection,
for
example, either subcutaneously or intramuscularly. Additional formulations
which are
suitable for other modes of administration include suppositories and, in some
cases, oral
formulations. For suppositories, traditional binders and carriers may include,
for
example, polyalkalene glycols or triglycerides: such suppositories may be
formed from
mixtures containing the active ingredient in the range of about 0.5% to about
10%,
preferably about 1 to about 2%. Oral formulations include such normally
employed
excipients as, for example, pharmaceutical grades of mannitol, lactose,
starch,
magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the
like.
These compositions take the form of solutions, suspensions, tablets, pills,
capsules,
sustained release formulations or powders and contain about 10 to about 95% of
active
ingredient, preferably about 25 to about 70%.
1. Liposomes
Liposomes are formed from phospholipids that are dispersed in an aqueous
medium and spontaneously form multilamellar concentric bilayer vesicles.
Liposomes
bear many resemblances to cellular membranes and are contemplated for use in
connection with the present invention as drug delivery agents.
The formation and use of liposomes is generally known to those of skill in the
art. For example, several U. S. Patents concern the preparation and use of
liposomes that
encapsulate biologically active materials, e.g., U.S. Patent 4,485,054;
4,089,801;
4,234,871; and 4,016,100; each incorporated herein by reference. Mostly, it is
contemplated that intravenous injection of liposomal preparations would be
used, but
pessaries are also possible.
2. Nasal Administration
One may also use nasal solutions or sprays, aerosols or inhalants in the
present
invention. Nasal solutions are usually aqueous solutions designed to be
administered to
the nasal passages in drops or sprays. Nasal solutions are prepared so that
they are
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similar in many respects to nasal secretions, so that normal ciliary action is
maintained.
Thus, the aqueous nasal solutions usually are isotonic and slightly buffered
to maintain a
pH of 5.5 to 6.5.
5 In addition, antimicrobial preservatives, similar to those used in
ophthalmic
preparations, and appropriate drug stabilizers, if required, may be included
in the
formulation. Various commercial nasal preparations are known and include, for
example, antibiotics and antihistamines and are used for asthma prophylaxis.
10 3. Oral Administration
In certain embodiments, active compounds may be administered orally. This is
contemplated to be useful as many substances contained in tablets designed for
oral use
are absorbed by mucosal epithelia along the gastrointestinal tract.
15 Also, if desired, the peptides, antibodies and other agents may be rendered
resistant, or partially resistant, to proteolysis by digestive enzymes. Such
compounds
are contemplated to include chemically designed or modified agents;
dextrorotatory
peptides; and peptide and liposomal formulations in time release capsules to
avoid
peptidase and lipase degradation.
For oral administration, the active compounds may be administered, for
example, with an inert diluent or with an assimilable edible carrier, or they
may be
enclosed in hard or soft shell gelatin capsule, or compressed into tablets, or
incorporated
directly with the food of the diet. For oral therapeutic administration, the
active
compounds may be incorporated with excipients and used in the form of
ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups,
wafers, and the like.
The oral compositions and preparations should contain at least 0.1 % of active
compound. The percentage of the compositions and preparations may, of course,
be
varied and may conveniently be between about 2 to about 60% of the weight of
the unit.
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The amount of active compounds in such therapeutically useful compositions is
such
that a suitable dosage will be obtained.
The tablets, troches, pills, capsules and the like may also contain the
following:
a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such
as dicalcium
phosphate; a disintegrating agent, such as corn starch, potato starch, alginic
acid and the
like; a lubricant, such as magnesium stearate; and a sweetening agent, such as
sucrose,
lactose or saccharin may be added or a flavoring agent, such as peppermint,
oil of
wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it
may
contain, in addition to materials of the above type, a liquid carrier.
Various other materials may be present as coatings or to otherwise modify the
physical form of the dosage unit. For instance, tablets, pills, or capsules
may be coated
with shellac, sugar or both. A syrup of elixir may contain the active
compounds sucrose
as a sweetening agent methyl and propylparabens as preservatives, a dye and
flavoring,
such as cherry or orange flavor. Of course, any material used in preparing any
dosage
unit form should be pharmaceutically pure and substantially non-toxic in the
amounts
employed. In addition, the active compounds may be incorporated into sustained-
release preparation and formulations.
Upon formulation, the compounds will be administered in a manner compatible
with the dosage formulation and in such amount as is therapeutically
effective. The
formulations are easily administered in a variety of dosage forms, as
described herein.
4. Pessaries
Additional formulations which are suitable for other modes of administration
include vaginal suppositories and pessaries. A rectal pessary or suppository
may also be
used.
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Suppositories are solid dosage forms of various weights and shapes, usually
medicated, for insertion into the rectum, vagina or the urethra. After
insertion,
suppositories soften, melt or dissolve in the cavity fluids.
In general, for suppositories, traditional binders and carriers may include,
for
example, polyalkylene glycols or triglycerides; such suppositories may be
formed from
mixtures containing the active ingredient in the range of 0.5% to 10%,
preferably
1 %-2%.
Vaginal suppositories or pessaries are usually globular or oviform and
weighing
about 5 g each. Vaginal medications are available in a variety of physical
forms, e.g.,
creams, gels or liquids, which depart from the classical concept of
suppositories.
Vaginal tablets, however, do meet the definition, and represent convenience
both of
administrationand manufacture.
B. Vaccines
The present invention contemplates vaccines for use in both active and passive
immunization embodiments. Immunogenic compositions, proposed to be suitable
for
use as a vaccine, may be prepared most readily directly from immunogenic
calcium
binding peptides prepared in a manner disclosed herein. Preferably the
antigenic
material is extensively dialyzed to remove undesired small molecular weight
molecules
and/or lyophilized for more ready formulation into a desired vehicle.
The preparation of vaccines that contain IgR-binding domain sequences as
active ingredients is generally well understood in the art, as exemplified by
U.S. Patents
4,608,251; 4,601,903; 4,599,231; and 4,599,230, all incorporated herein by
reference.
Typically, such vaccines are prepared as injectables. Either as liquid
solutions or
suspensions: solid forms suitable for solution in, or suspension in, liquid
prior to
injection may also be prepared. The preparation may also be emulsified. The
active
immunogenic ingredient is often mixed with excipients which are
pharmaceutically
acceptable and compatible with the active ingredient. Suitable excipients are,
for
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example, water, saline, dextrose, glycerol, ethanol, or the like and
combinations thereof.
In addition, if desired, the vaccine may contain minor amounts of auxiliary
substances
such as wetting or emulsifying agents, pH buffering agents, or adjuvants which
enhance
the effectiveness of the vaccines. Additionally, iscom, a supramolecular
spherical
structure, may be used for parenteral and mucosal vaccination (Morein et al.,
1998).
Vaccines may be conventionally administered parenterally, by injection, for
example, either subcutaneously or intramuscularly. Additional formulations
which are
suitable for other modes of administration include suppositories and, in some
cases, oral
formulations. For suppositories, traditional binders and carriers may include,
for
example, polyalkalene glycols or triglycerides: such suppositories may be
formed from
mixtures containing the active ingredient in the range of about 0.5% to about
10%,
preferably about 1 to about 2%. Oral formulations include such normally
employed
excipients as, for example, pharmaceutical grades of mannitol, lactose,
starch,
1 S magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and
the like.
These compositions take the form of solutions, suspensions, tablets, pills,
capsules,
sustained release formulations or powders and contain about 10 to about 95% of
active
ingredient, preferably about 25 to about 70%.
The calcium binding protein-derived peptides of the present invention may be
formulated into the vaccine as neutral or salt forms. Pharmaceutically-
acceptablesalts,
include the acid addition salts (formed with the free amino groups of the
peptide) and
those which axe formed with inorganic acids such as, for example, hydrochloric
or
phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic,
and the like.
Salts formed with the free carboxyl groups may also be derived from inorganic
bases
such as, for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and
such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,
histidine,
procaine, and the like.
The vaccines axe administered in a manner compatible with the dosage
formulation, and in such amount as will be therapeutically effective and
immunogenic.
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The quantity to be administered depends on the subject to be treated,
including, e.g., the
capacity of the individual's immune system to synthesize antibodies, and the
degree of
protection desired. Precise amounts of active ingredient required to be
administered
depend on the judgment of the practitioner. However, suitable dosage ranges
are of the
order of several hundred micrograms active ingredient per vaccination.
Suitable
regimes for initial administration and booster shots are also variable, but
are typified by
an initial administration followed by subsequent inoculations or other
administrations.
The manner of application may be varied widely. Any of the conventional
methods for administration of a vaccine are applicable. These are believed to
include
oral application on a solid physiologically acceptable base or in a
physiologically
acceptable dispersion, parenterally, by injection or the like. The dosage of
the vaccine
will depend on the route of administration and will vary according to the size
of the
host.
Various methods of achieving adjuvant effect for the vaccine includes use of
agents such as aluminum hydroxide or phosphate (alum), commonly used as about
0.05
to about 0.1 % solution in phosphate buffered saline, admixture with synthetic
polymers
of sugars (Carbopol~) used as an about 0.25% solution, aggregation of the
protein in the
vaccine by heat treatment with temperatures ranging between about 70°
to about 1 O 1 °C
for a 30-second to 2-minute period, respectively. Aggregation by reactivating
with
pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such
as C.
parvum or endotoxins or lipopolysaccharide components of Gram-negative
bacteria,
emulsion in physiologically acceptable oil vehicles such as mannide mono-
oleate
(Aracel A) or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA~)
used
as a block substitute may also be employed.
In many instances, it will be desirable to have multiple administrations of
the
vaccine, usually not exceeding six vaccinations, more usually not exceeding
four
vaccinations and preferably one or more, usually at least about three
vaccinations. The
vaccinations will normally be at from two to twelve week intervals, more
usually from
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three to five week intervals. Periodic boosters at intervals of 1-5 years,
usually three
years, will be desirable to maintain protective levels of the antibodies. The
course of the
immunization may be followed by assays for antibodies for the supernatant
antigens.
The assays may be performed by labeling with conventional labels, such as
5 radionuclides, enzymes, fluorescents, and the like. These techniques are
well known
and may be found in a wide variety of patents, such as U.S. Patent Nos.
3,791,932;
4,174,384 and 3,949,064, as illustrative of these types of assays.
"Unit dose" is defined as a discrete amount of a therapeutic composition
10 dispersed in a suitable carrier. For example, in accordance with the
present methods,
viral doses include a particular number of virus particles or plaque forming
units
(pfu). For embodiments involving adenovirus, particular unit doses include
103, 104,
105, 106, 10', 108, 109, 10'°, 10", 10'2, 10'3 or 10'4 pfu. Particle
doses may be
somewhat higher (10 to 100-fold) due to the presence of infection defective
particles.
In this connection, sterile aqueous media which can be employed will be
known to those of skill in the art in light of the present disclosure. For
example, a unit
dose could be dissolved in 1 ml of isotonic NaCI solution and either added to
1000 ml
of hypodermoclysis fluid or injected at the proposed site of infusion, (see
for example,
"Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-
1580). Some variation in dosage will necessarily occur depending on the
condition of
the subject being treated. The person responsible for administration will, in
any event,
determine the appropriate dose for the individual subject. Moreover, for human
administration, preparations should meet sterility, pyrogenicity, general
safety and
purity standards as required by FDA Office of Biologics standards.
In a preferred embodiment, the present invention is directed at the treatment
of
human malignancies. A variety of different routes of administration are
contemplated. For example, a classic and typical therapy will involve direct,
intratumoral injection of a discrete tumor mass. The injections may be single
or
multiple; where multiple, injections are made at about 1 cm spacings across
the
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accessible surface of the tumor. Alternatively, targeting the tumor
vasculature by
direct, local or regional intra-arterial injection are contemplated. The
lymphatic
systems, including regional lymph nodes, present another likely target given
the
potential for metastasis along this route. Further, systemic injection may be
preferred
when specifically targeting secondary (i.e., metastatic) tumors.
In another embodiment, the viral gene therapy may precede or following
resection of the tumor. Where prior, the gene therapy may, in fact, permit
tumor
resection where not possible before. Alternatively, a particularly
advantageous
embodiment involves the prior resection of a tumor (with or without prior
viral gene
therapy), followed by treatment of the resected tumor bed. This subsequent
treatment
is effective at eliminating microscopic residual disease which, if left
untreated, could
result in regrowth of the tumor. This may be accomplished, quite simply, by
bathing
the tumor bed with a viral preparation containing a unit dose of viral vector.
Another
preferred method for achieving the subsequent treatment is via catheterization
of the
resected tumor bed, thereby permitting continuous perfusion of the bed with
virus
over extended post-operative periods.
C. Kits
All the essential materials and reagents required for delivering agents to the
mucosal epithelia using a pIgR-binding domain may be assembled together in a
kit.
This generally will comprise selected expression constructs. Also included may
be
various media for replication of the expression constructs and host cells for
such
replication. Such kits will comprise distinct containers for each individual
reagent.
When the components of the kit are provided in one or more liquid solutions,
the
liquid solution preferably is an aqueous solution, with a sterile aqueous
solution being
particularly preferred. For in vivo use, the expression construct may be
formulated into
a pharmaceutically acceptable syringeable composition. In this case, the
container
means may itself be an inhalent, syringe, pipette, eye dropper, or other such
like
apparatus, from which the formulation may be applied to an infected area of
the body,
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such as the lungs, injected into an animal, or even applied to and mixed with
the other
components of the kit.
The components of the kit may also be provided in dried or lyophilized forms.
When reagents or components are provided as a dried form, reconstitution
generally is
by the addition of a suitable solvent. It is envisioned that the solvent also
may be
provided in another container means.
The kits of the present invention also will typically include a means for
containing the vials in close confinement for commercial sale such as, e.g.,
injection or
blow-moldedplastic containers into which the desired vials are retained.
Irrespective of the number or type of containers, the kits of the invention
also
may comprise, or be packaged with, an instrument for assisting with the
injection/administrationor placement of the ultimate complex composition
within the
body of an animal. Such an instrument may be an inhalant, syringe, pipette,
forceps,
measured spoon, eye dropper or any such medically approved delivery vehicle.
The following examples are included to demonstrate preferred embodiments
of the invention. It should be appreciated by those of skill in the art that
the
techniques disclosed in the examples which follow represent techniques
discovered by
the inventor to function well in the practice of the invention, and thus can
be
considered to constitute preferred modes for its practice. However, those of
skill in
the art should, in light of the present disclosure, appreciate that many
changes can be
made in the specific embodiments which are disclosed and still obtain a like
or similar
result without departing from the spirit and scope of the invention.
EXAMPLE l: Materials and Methods
A. Baculovirus Expression
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Arsonate hapten-specific chimeric IgAI and IgAI/IgGI domain swap
mutants were expressed as described (Carayannopoulos et al., 1994; O'Reilly et
al.,
1992). Dimeric IgA was generated by coexpression of IgA with J chain. Affinity
purification was carried out on arsonate-sepharose. Antibodies were eluted
with 200
S mM arsanillic acid (Sigma, St. Louis, MO), 200 mM Tris-HCl pH8.0, which was
removed by extensive dialysis against PBS. Monomeric and dimeric IgA was
confirmed by 4% non-reducing SDS-PAGE analysis. The hexahistidine tagged
human pIgR extracellular domain was expressed in a similar manner and purified
on
a Ni-NTA Agarose (Qiagen, Chatsworth, CA) column (Rindisbacher et al., 1995).
B. Construction of Mutant IgA Antibodies for Baculovirus
Expression
The Ca3 loop mutants L1, L2 and L3 were constructed by PCR SOEing
(Splicing by Overlap Extension, Horton et al., 1990) using the following
complementary pairs of sense (S) and antisense (AS) primers:
L1S 5'-GAGC CCAGCGCGGGCGCCGCCGCCTTCGCTGTG-3',
L1AS 5'-CTCGGGTCGCGCCCGCGGCGGCGGAAGCGACAC-3';
L2S 5'TACCTGACTGCGGCAGCCGCGCAGGAGCCC-3',
L2AS 5'-ATGGACTGACGCCGTCGGCGCGT CCTCGGG-3';
L3S 5'-CGGCAGGAGGCCGCCGCGGCCACCACCACC-3',
L3AS 5'-GCCGTCCTCCGGCGG CGCCGGTGGTGGTGG-3'
The outer primers B1-2 (S'-CCTATAACCATGGGATGGAGCTTCATC-3'), SpeClflC
for the 5' leader of the VH region of this chimeric IgA heavy chain, and Ca3-
3' (5' -
CCCTCTAGATTAGTAGCAGGTGCCGTCCAC-3' ) , SpeClflC fOr the 3' tailpiece-encoding
sequence of the IgA 1 gene, were used with the above primer pairs L 1-L3 to
generate
pairs of 5' and 3' fragments with complementary overlaps. These fragments were
gel
purified then spliced in a further PCR reaction using the outer primers B1-2
and Ca3-
3'. Modified IgAI genes were cloned into the baculovirus transfer vector using
Xba
I and Nco I digestion and the insert sequences verified. Recombinant
baculovirus
were produced using the BacPAK system (Clontech, Palo Alto, CA).
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C. FACS Analysis
MDCK cells were placed in serum-free MEM plus Earle's salts (MediaTech,
Inc., Herndon, VA) 16 hr before the experiment. Cells were harvested in 10 mM
EDTA in PBS and washed in PBS 0.1% BSA. pIgR binding of human IgAI
antibodies and mutants was assessed by incubation of 100 ~1 antibody in PBSBSA
with approximately 106 cells for 1 hr. Cells were washed three times in
PBS/BSA
and bound antibody was detected with 100 ~1 of an anti-human kappa FITC
conjugate (Sigma, St. Louis, MO) diluted 1/100. Cells were washed as above and
resuspended in 1 ml PBS/BSA. FACS analysis was carried out on a Becton
Dickinson FACSCAN instrument. Data collection and analysis were performed with
the LYSYSII (Becton Dickinson, San Jose, CA) and WinMIDI
(http//facs.scripps.edu) or with the CELLQUEST programs (Becton Dickinson, San
Jose, CA).
D. Phage Display Peptide Library Selection
The random 40mer peptide library was constructed in the pCANTABSe
vector and has an actual total diversity of 1.55x10'° (Ravera et al.,
1998). The random
40mer is flanked by two peptide tag sequences, preceded by a leader peptide
and
fused to the membrane-proximal domain of the M 13 phage coat protein III. 1-2x
1 O6
MDCK cells were harvested in 5 ml PBS + 10 mM EDTA at 37°C, washed
twice in
15 ml PBS and resuspended in 1.8 ml PBS at 4 °C. 100 ~l phagemid
library stock
(4.5x10" cfu) was added and incubated for one hour at 37°C or
4°C. The cells were
then washed five times with 15 ml PBS at 4 °C. Bound phage were eluted
with 2 ml
of 0.1 M HCl/glycine pH 2.2 containing 0.1% BSA for 10 min and neutralized
immediately with 400 ~1 of 2 M Tris base. Phage rescue and amplification were
carried out in E. coli strain TG1 (Pharmacia, Piscataway, NJ) according to
standard
procedures (Hexham, 1998).
E. DNA Sequencing and Analysis
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DNA sequencing was carried out on double-stranded plasmid or phagemid
DNA using an ABI 377 Prism automated sequencer. Alignments of deduced peptide
sequences and immunoglobulin constant regions were carried out using the MAP
(Huang, 1994) and PIMA (Smith et al., 1992) software.
5
EXAMPLE 2: Domain Mapping of dIGA
Chimeric human IgA 1 (Caryannopoulos et al., 1994) and a panel of
IgAI/IgGI constant region domain swap mutants (Caryannopoulos et al., 1996)
with
10 murine-encoded arsonate-specificity were expressed in baculovirus as both
monomer
and dimer, affinity purified, and then used to define the pIgR binding site.
Dimeric
IgA (dIgA) was operationally defined as an IgA preparation generated by co-
expression of IgA with J chain. MDCK cells, transfected with rabbit pIgR
(Mostov et
al., 1986), were used to measure binding of recombinant IgAI mutants to the
receptor
15 by FACS analysis (FIG. lA). Specific binding was observed with dIgA and not
with
monomeric IgA (FIG. 1 B), a medium control (FIG. 1 b) or IgG. Mutant VGAA, in
which the Cal domain was substituted with the Cyl domain, bound to the pIgR in
a
similar manner to wild-type IgAl (FIG. 1C). The dimeric molecule (heavy line)
bound to the receptor, while the monomer (light line) did not. Similarly, the
VGGA
20 mutant, in which both Cal and Ca2 including the hinge of IgA were replaced
with the
analogous domains from IgG, bound as a dimer but not as a monomer (FIG. 1 D).
Thus, the Cal and Ca2 domains of dIgA are not necessary for pIgR binding
suggesting that the presence of the Ca3 domain is required.
25 EXAMPLE 3: IgA Minimum Peptide Binding Unit
dIgA contains four Ca3 domains and the covalently bound J chain which,
together with the IgA tailpiece, are responsible for IgA polymerization. To
reduce the
complexity of this problem, a library of random 40mer peptides, expressed as a
phage
30 display library (Ravera et al., 1998), was selected against pIgR-expressing
MDCK
cells. The goal was to identify putative pIgR binding sites within IgA by
reducing
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them to a minimum peptide binding unit, a proven approach for several receptor-
ligand interactions (Cwirla et al., 1990; Parmley et al., 1988; Devlin et al.,
1990).
Selection was carried out on live pIgR-expressing MDCK cells in suspension
with negative selection on non-receptor expressing cells. Bound phage were
eluted
with acid or by cell lysis. Recovery of both acid-eluted and cell-associated
phage
increased gradually from approximately 6x104 to 5x10' c.f.u. over 4-6
successive
rounds, indicating enrichment for specific binding clones. Individual clones
were
randomly selected from the final panning from the acid-eluted and membrane-
associated fractions and sequenced. Binding of the enriched , phage
populations to
recombinant human pIgR, as measured by ELISA, increased with successive rounds
of panning and was inhibited by polymeric IgM.
Sequencing of phagemid DNA showed that 20 out of 32 acid-eluted clones
and 12 out of 32 cell-associated clones had open reading frames (FIG. 2).
There is
little clonality among these two groups of sequences, although the A22 peptide
was
recovered three times. These peptides were aligned for maximum homology with
the
human IgAI Ca3 region amino acid sequence (FIG. 2) using the PIMA program
(Smith et al., 1992). Many of the peptides, particularly A12 (9/30 identical
amino
acids) (FIG. 3A), show homology with human IgAI Ca3 domain, prompting a
further
examination of the amino acid sequence and structure in this area.
EXAMPLE 4: Mutational Analysis
The human Ca3 domain is 40% identical and 62% homologous to the
corresponding region of human IgGI at the amino acid level. In addition, all
the
sequence hallmarks of the immunoglobulin superfamily fold are conserved.
Accordingly, the human IgGI crystal structure (Deisenhofer et al., 1981) was
used to
predict the likely positions of the major structural motifs (~3-strands and
loops) within
the IgAl sequence, an approach used previously to map the FcaR (CD89) binding
site
on IgAl (Caryannopoulos et al., 1996). FIG. 3A shows the alignment of the
peptide
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A 12 with the IgA 1 sequence and the corresponding IgG 1 sequence with its
secondary
structural features. The A12 peptide is homologous to a region that in the IgG
structure forms an exposed 6 amino acid loop between two p-strands. However,
in
IgAI, this area contains a 3 amino acid insertion to expand the loop to 9
amino acids.
The flanking p-strand sequences and part of the loop are conserved between IgA
and
IgG, which suggests that gross structural features are also conserved. FIG. 3B
shows
alignment of this region in the CH3 domain of five mammalian IgA molecules
aligned
with the four human IgG subclasses. Despite sequence differences in the loop,
all IgA
sequences have the additional 3 amino acids whereas the IgG sequences do not.
Similar to IgA, the sequence of IgM contains a two amino acid insertion at
this site.
On the basis of these observations, three mutant IgAI molecules were
constructed and expressed in baculovirus to examine the effect of amino acid
changes
in this area on pIgR binding (FIG. 3C). Mutations were made in the loop itself
(L1
and L3) and in the p-strand N-terminal to the loop (L2) as a negative control.
Binding
was then measured to the physiologically relevant human receptor by ELISA
using
the purified recombinant extracellular domain of human pIgR expressed in
baculovirus as described (Rindisbacher et al., 1995). FIG. 4 shows the binding
of
IgA 1 monomer and dimer compared to the monomeric and dimeric forms of the L
1,
L2 and L3 mutants to purified human pIgR. Only dimeric wild-type IgAI and
dimeric L2 mutant, in which the mutations are in the li-strand N-terminal to
the loop,
show binding. Mutations within the loop itself, namely L 1 and L3, abrogate
the
binding of the dimeric IgAl mutant molecules to the pIgR. Similar binding
patterns
were obtained with the loop mutants and rabbit pIgR-expressing cells as
measured by
FACS. These results indicate that this Ca3 loop is the major binding motif for
the
pIgR on dIgA, and based on this observation and on the selection of random
peptides
and comparison between them and the IgA sequence, amino acids 402-410 of IgA
(SEQ ID NO: l ) constitute a binding site to pIgR.
IgA is, in functional terms, closely related to IgM, sharing its ability to
polymerize and be secreted. However, the overall IgA domain organization
resembles
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that of IgG. The presence of amino acid sequence insertions in all the
polymeric
immunoglobulins that are ligands for this receptor and the absence of
insertions from
non-pIgR-binding immunoglobulins (FIG. 3b) supports its role in immunoglobulin
secretion. The variation in the insertion size and the actual IgA and IgM
sequences
may reflect differences in fine structure of these polymeric antibodies or in
their
affinity for pIgR binding.
The fact that monomeric IgA is not secreted suggests that either a
conformational change induced by polymerization is required for dIgA binding
to the
receptor or that the binding requires a polyvalent interaction of these Ca3
sites with
the receptor. The presence of J chain is required for optimal IgA (or IgM)
polymerization but its precise role in Ig secretion remains to be elucidated.
The
increase in binding observed with dimeric L3 when compared to monomeric L3
(and
to a lesser extent with the L 1 mutants) suggests that J chain and/or
polymerization
may play a role in binding (FIG. 4). Although amino acids 402-410 in the Ca3
domain of dIgA define a major pIgR-binding site, other dIgA structures may be
involved. J chain deficient mice express lower levels of polymeric IgA, have
impaired
hepatic transport of IgA (which humans lack) but normal levels of IgA at
mucosal
epithelial sites, compared to wild type mice (Hendrickson et al., 1995;
Hendrickson et
al., 1996). J chain may thus not be necessary for secretion of IgA but
required for
stable binding to the secretory component in the mucosal environment, however
alternative secretory mechanisms may also be involved.
EXAMPLE 5: Protective Immunity
Protective immunity against various pathogenic microorganisms will be
delivered with the pIgR-binding domain linked to single-chain Fv (scFv)
antibodies
with anti-microbial specificity. The prevention of HIV infection may be
accomplished in the SHIV/baboon model for heterosexual AIDS transmission using
anti-gp120 scFv linked to the pIgR-binding domain.
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Single-chain Fv fragments linked to the pIgR-binding domain will be
generated from a human anti-gp 120 IgG antibody employing oligonucleotide
primers
in a series of polymerise chain reactions (PCR) using "splice by overlap
extension"
(SOE). The resulting fragment will consist of the VH gene, a linker sequence,
the VL
gene segment, and the pIgR-binding domain. The 5' primer will contain a
restriction
site, Xho I, and the 3' primer will contain a second site, Spe I, for cloning
into the
phage display vector, pCOMB3 (Barbas, 1991 ). The resulting product will be
cloned
into the Xho I and Spe I sites contained within the pCOMB3 vector.
Consequently,
this vector will contain the bacterial periplasmic signal peptide, pelB,
cloned in-frame
and upstream from a single-chain Fv and the carboxy-terminus of the
filamentous
phage coat protein III. Phage particles will be generated using helper phage,
VCSM13, as described previously (Barbas, 1991). The resulting phage will be
used
in a panning technique to select for antigen-specificity. The techniques for
panning
with HIV antigens to select to high affinity antibodies are straightforward
and have
been published (Hexham, 1996). Briefly, gp120 antigen will be coated onto the
wells
of ELISA plates. Varying amounts of specific- and non-specific phage will be
added
to the wells and allowed to bind to the antigens. Unbound phage will be
removed by
washing and stored. Bound phage will be eluted from the wells using low pH,
followed by re-infection of fresh bacterial cultures, and further rounds of
selection.
DNA from selected phage will then be modified by restriction digestion with
Spe I and Nhe I to facilitate excision of the gene III fragment. The vector
will be re-
legated (Spe I and Nhe I have compatible ends) to form a vector that will
allow for
production of soluble scFv. The fragment is under the control of the lacZ
promoter,
inducible with IPTG, for maximal protein production. In this system, a
hexahistidine
sequence has been inserted into the vector between the Nhe I site and the stop
codons,
resulting in a protein with a hexahistidine tag. This permits the purification
of any
protein using Ni++ affinity chromatography. To determine if the recombinant
scFv
antibody binds comparably to the native molecule, the affinities of both will
be
compared using BIAcore and ELISA determinations.
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After purification of the scFV-pIgR-binding domain, the ability of this
molecule to prevent HIV infection in a baboon SHIV model for heterosexual AIDS
transmission will be assessed.
Using the same approach, other anti-pathogen scFV-pIgR binding-domain
combinations can be utilized to mediate immunity against influenza,
respiratory
syncytial virus, and gonorrhoeae.
EXAMPLE 6: Identify Other Peptide Motifs with High Binding Affinity for
pIgR
Phage display technology was used to identify peptide motifs that would bind
to the pIgR with high affinity. More specifically, a modification of the MDCK
(Madin-Darby canine kidney) cell transcytosis system was employed for
screening
random 40-mer and 20-mer peptide phage libraries. Three separate rounds of
experiments were conducted: the first two involved the 40-mer peptide library
while
the third involved the 20-mer peptide library. The MDCK cell line, transfected
with
the pIgR, is used as an in vitro transcytosis assay (Mostov et al., 1995).
MDCK is a
polarized cell line, capable of forming monolayers with tight junctions,
which, when
grown on a semipermeable support, will transport dIgA from the lower
(basolateral)
to the upper (apical) chamber of a tissue culture well. MDCK cells expressing
pIgR
and non-transfected MDCK cells were seeded on tissue culture inserts with 1.0
pm
pore size polycarbonate membranes to allow pore sizes to which the phage may
traverse. The phage library is introduced into the lower or basolateral
chamber of the
wells and incubated for 3-4 hours at 37°C. Phage that display peptides
capable of
transcytosis are then collected by harvesting the apical supernatant fluid.
The
collected phage are then amplified and subjected to further rounds of
selection by
transcytosis. After successive rounds of panning (assessed by phage titer but
typically
about 6), random phage clones were chosen and sequenced.
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Twenty clones from the first round of experiments (40-mer library) and 18
clones from the second (40-mer library) and third rounds (20-mer library) of
experiments were chosen at random and sequenced. Table 3 shows the eight phage
peptides selected from the three experiments and the frequency of selection as
indicated by sequences obtained (in parentheses):
TABLE 3
EXP 1
SAM FVPFDIAVGVRDGQQGLGGSRRKGARLREAISSYAE 9/20
IPS VTRMTVGGTLRKEFQDWLGVIFGLVLVINRCSFL 3/20
LVL RGNQVFAFCRSDNNRQQAPAGCCYVGFSLFVTRGGYE 1/20
EXP 2
WQA YPVQYLFVVATGYGGKVINHLRGKVRRESADQVPGYF 4/18
MFV CVDAKQCLLGAAGGLRLIFA 3/18
VDD LTLQSRSPPSQLNSQHLLLSQLCGYWMFRVRSRSCCG 1/18
RSR MFVLGVLEVDSGLLNCLCWVGVSVDGRKSSCRWTAY 1/18
EXP 3
QRN PRLRLIRRHPTLRIPPI 11/18
There were no apparent sequence motifs among the peptides as assessed by
peptide alignment computer programs. However, when these selected phage
peptides
were aligned to the sequence of IgA, three peptides (VDD, SAM, and IPS) show
significant identity in and around the 402-410 region that has previously been
shown
by the inventors to be important for pIgR binding (Hexham et al., 1999) (FIG.
5).
Phage peptides are referred to by the first three amino acids of their
sequence.
The eight phage peptides shown above were selected by successive rounds of
transcytosis through pIgR-transfected cells, a positive selection method.
Therefore,
the ability of the individual phage-displayed peptides to be transcytosed
specifically
by the pIgR in the MDCK transcytosis system was assessed using both pIgR-
transfected and non-transfected MDCK cells as a negative control (FIG. 6).
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These results clearly indicate that the SAM, IPS, RSR, MFV, VDD, and QRN
peptides displayed on the surface of the phage allow the phage to be
specifically
transported by the pIgR through MDCK cells (FIGS. 6A & 6B). In contrast, the
LVL
and WQA phage peptides direct apical transport of phage by an alternate
transcytosis
pathway (FIG. 6C).
Further peptides are being selected using different phage display libraries,
i.e.,
constrained libraries. The molecules that result from these and other
experiments
described in the original submission will be tested in the rat model.
Furthermore,
mutagenic libraries will be derived from the peptides and derivatives selected
by
transcytosis. These libraries will produce more candidate transport molecules
that
will ultimately be tested in both the in vitro MDCK transcytosis system and
the rat in
vivo model described below.
EXAMPLE 7: in vivo Transcytosis Model
The ability of the phage peptides to transport in an in vivo model of
transcytosis was then evaluated. In the rat, polymeric immunoglobulins are
selectively transported from the blood to the bile by pIgR expressed by the
liver. The
rat hepatic bile transcytosis system was therefore chosen as a means to assess
the
transport of the phage peptides in vivo. For this experiment, a rat was fasted
overnight
before bile duct cannulation. The rat was anesthetized with ketamine
hydrocloride (44
mg/kg body weight), an intravenous (i.v.) saline line was established, and
then the bile
duct was cannulated with the outlet of the cannula resting approximately 10 cm
below
the rat. Bile collection began after i.v. injection of test phage and negative
control
phage particles (in 0.2 ml sterile saline) and was continued for 30 minute
time points
in separate tubes. Duplicate serum samples may also be obtained to assess
peripheral
clearance of the phage particles. After bile and serum collection was
complete, the
animal was sacrificed. FIG. 7 shows the procedure used and results from one
rat
injected with the IPS test phage. One hundred percent of the phage recovered
from all
time points displayed the IPS peptide. No nonspecific phage was recovered in
this
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103
experiment. In addition to hepatic bile transport, the rat model also allows
sampling
of various other mucosal sites for the presence of a targeting molecule.
Mutagenesis studies can be employed to further optimize the binding and
transcytosis of the selected phage peptides as well as the native region from
dIgA.
These reagents will first be tested for transcytosis in the in vitro MDCK
transcytosis
system and then will need to be assessed in an in vivo model of secretion.
EXAMPLE 8: Multimeric Peptides
Other experiments were aimed at localizing the smallest possible peptides)
required for the pIgR- dIgA interaction. To this end, the native region from
IgA
(amino acids 402-410) that is involved in pIgR-binding was synthesized in both
monomeric and dimeric form (with a small linker) as thioredoxin or Green
1 S Fluorescent Protein (GFP) fusion proteins. Flanking amino acids (five
residues) on
each side of the pIgR-binding domain were included. With both the monomer and
dimer, a GP(G)6PG non-cleavable linker moiety was placed between a pIgR-
binding
domain and the selected agent (SA), either thioredoxin or GFP. An additional
GP(G)6PG linker was placed between the pIgR-binding domains as well.
MONOMERIC PEPTIDE:
TWASRQEPSQGTTTFAVTSGP(G)EPG~ [SA]
402-410 lirzke~~
DIMERIC PEPTIDE:
TWASRQEPSQGTTTFAVTSGP(G)EPGTWASRQEPS GTTTFAVTSGP(G)6PG
[SA]
402-410 lirrkc~r 402-410 linket~
The peptides were constructed by standard PCR (for the monomeric peptide)
and PCR SOEing reactions (for the dimeric peptide) (Horton et al., 1990) using
human IgA cDNA and primers which added the GP(G)6PG linker as well as
convenient restriction sites to the ends of the PCR products. The monomeric
and
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104
dimeric peptide constructs were then cloned into thioredoxin and GFP fusion
protein
vectors, expressed in E. coli, and purified by affinity chromatography
methods.
The ability of thioredoxin alone (negative control) and the thioredoxin fusion
proteins to be specifically transported by the pIgR in the MDCK transcytosis
system
has been assessed (FIG. 8). Data indicate that a peptide derived from the
native
region from the CH3 region of dIgA can indeed direct transport of a fusion
protein
through pIgR-transfected MDCK cells. There appears to be a significant amount
of
polymerization or aggregation in the preparations of thioredoxin fusion
proteins.
Clearance/transport analyses will be performed using intact dimeric IgA, Fc
fragments of dIgA, recombinant peptides with and without a selected agent,
synthetic
peptides, and monomeric IgA and IgG as controls. In these experiments, rats
will be
injected intravenously with the various proteins, the clearance of the
molecules
measured from serum samples, and the mucosal transport of the molecules
assessed
from bile samples. In addition, other mucosal sites may be sampled to provide
information concerning the quantitative limits of detection of these molecules
in this
system.
All of the compositions and methods disclosed and claimed herein can be
made and executed without undue experimentation in light of the present
disclosure.
While the compositions and methods of this invention have been described in
terms of
preferred embodiments, it will be apparent to those of skill in the art that
variations
may be applied to the compositions and methods and in the steps or in the
sequence of
steps of the method described herein without departing from the concept,
spirit and
scope of the invention. More specifically, it will be apparent that certain
agents that
are both chemically and physiologically related may be substituted for the
agents
described herein while the same or similar results would be achieved. All such
similar substitutes and modifications apparent to those skilled in the art are
deemed to
be within the spirit, scope and concept of the invention as defined by the
appended
claims.
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105
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The following references, to the extent that they provide exemplary procedural
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HEXHAM, MARK J.
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<400> 17
Ala Glu Tyr Phe Val Ala Gln Cys Val Ala Glu Glu Asp Phe Ala Gly
CA 02362373 2001-08-13
WO 00/47611 PCT/US00/03650
6
1 5 10 15
Val Thr Ser Leu Asp Leu Asn Asn Leu Gly Ala Met Leu Phe Leu Phe
20 25 30
Asn Arg Tyr Leu Gly Trp Leu Ile
35 40
<210> 18
<211> 26
<212> PRT
<213> Homo Sapiens
<400> 18
Ala Gly Arg Phe Asp Pro Pro Val Leu Leu Ser Val Phe Asp Phe Gly
1 5 10 15
Ser Phe Phe GlyThrSer SerGln Lys
Arg
20 25
<210> 19
<211> 40
<212> PRT
<213> Homo
Sapiens
<400> 19
Arg Arg Glu ValSerGln GlyGlu Gly Leu Asp Val Thr
Arg Ala Leu
1 5 10 15
Glu Asp Pro ValPheIle ValGlu Trp Leu Asp Lys Thr
Ile Phe Gly
20 25 30
Leu His Leu IleValTrp Leu
Pro
35 40
<210> 20
<211> 40
<212> PRT
<213> Homo sapiens
<400> 20
Arg Ala Asp Asp Trp Ser Gly Arg Gly Glu Gly Asp Val Phe Trp Tyr
1 5 10 15
Trp Gly Pro Phe Ala Phe Tyr Pro Ser Phe Ser Ala Ala Phe Leu Gly
20 25 30
Gly Met Phe Gly Gln Lys Trp His
35 40
<210> 21
<211> 40
CA 02362373 2001-08-13
WO 00/47611 PCT/US00/03650
7
<212> PRT
<213> Homo sapiens
<400> 21
His Gly Leu Glu Ser Leu Tyr Asp Pro Asp Leu Gly Gly Gln Arg Asp
1 5 10 15
Cys Cys Glu Ser Ile Phe Thr Val Val Ala Val Gly Met Gly Phe Met
20 25 30
Thr Phe Phe Leu Pro Trp Trp Met
35 40
<210> 22
<211> 38
<212> PRT
<213> Homo Sapiens
<400> 22
Trp Trp Met Gly Val Ala Gly Trp Met Phe Ser Phe Trp Thr Met Arg
1 5 10 15
Asp Cys Tyr Asn Asp Arg Asp Gly Gly Asp Val Val Cys Ser Leu Gly
20 25 30
Glu Ala Pro Leu Gly Leu
<210> 23
<211> 40
<212> PRT
<213> Homo Sapiens
<400> 23
Glu Gly Leu Arg Asp Arg Ala Arg Ala Cys Ser Met Ser Asp Cys Asp
1 5 10 15
Glu Gly Leu Asp Ser Met Gly Leu Trp Ser Trp Ala Gly Leu Thr Leu
20 25 30
Phe Gly Gly Val Gly Gln Leu Ile
35 40
<210> 24
<211> 40
<212> PRT
<213> Homo Sapiens
<400> 24
Gly Met Gln Asn Val Gly Ser Asp Arg Gly Pro Asn Gly Leu Ala Leu
1 5 10 15
Gly Glu Ala Val Phe Ser Phe Trp Asp Ile Phe Gly Ala Gly Ala Gly
CA 02362373 2001-08-13
WO 00/47611 PCT/US00/03650
8
20 25 30
Gly Val Ala Ala Asp Asn Gly Trp
35 40
<210> 25
<211> 39
<212> PRT
<213> Homo Sapiens
<400> 25
Cys Gly Leu Met Gly Leu Ser Gly Leu Phe Val Gly Cys Asn Asp Val
1 5 10 15
Trp Glu Pro Met Gly Val Asn Gly Tyr Ala Met Leu Tyr Arg Asn Ala
20 25 30
Trp Phe Ser Arg Pro Arg Thr
<210> 26
<211> 29
<212> PRT
<213> Homo Sapiens
<400> 26
Gly Pro Ala Ala Ile Arg Gln Val His Ala Trp Trp Ser Val Pro Trp
1 5 10 15
Phe Gly Leu Ala Gly Arg Glu Ser Ala Gly Gly Leu Ser
20 25
<210> 27
<211> 44
<212> PRT
<213> Homo sapiens
<400> 27
Leu Asn Ser Gly Ala Cys Ser Ser Glu Cys Ile Trp Phe Leu Ser Gln
1 5 10 15
Ser Gly Ile Leu Trp Pro His Ile Pro Cys Ala Val Gly Cys Leu Gly
20 25 30
Met Lys Ser Trp Trp Ser Glu Leu Thr Ser Gly Leu
35 40
<210> 28
<211> 24
<212> PRT
<213> Homo Sapiens
CA 02362373 2001-08-13
WO 00/47611 PCT/US00/03650
9
<400> 28
Pro Ser Leu Arg Arg Leu Gly Phe Phe Gly Phe Gly Ser Glu Arg Gly
1 5 10 15
Ser Leu Leu His Leu Trp Asp Arg
<210> 29
<211> 25
<212> PRT
<213> Homo Sapiens
<400> 29
Arg Gly Gly Asn Gly Ala Leu Ser Trp Arg Gly Phe Gly Trp Ala His
1 5 10 15
Asp Ser Trp Phe Pro Trp Phe Gly Gly
20 25
<210> 30
<211> 11
<212> PRT
<213> Homo Sapiens
<400> 30
Glu Gly Trp Trp Ser Trp Leu Phe Pro Arg Glu
1 5 10
<210> 31
<211> 11
<212> PRT
<213> Homo Sapiens
<400> 31
Gly Trp Leu Gly Glu Gly Trp Trp Glu Leu Leu
1 5 10
<210> 32
<211> 40
<212> PRT
<213> Homo sapiens
<400> 32
Gly Asn Leu Ala Val Ser Glu Leu Ala Met Thr Gly Ser Ser Ala Leu
1 5 10 15
Pro Thr Arg Met Arg Ser Gly Thr Gly Ser Ala Ala Arg Glu Trp Trp
20 25 30
Glu Gly Leu Ile Arg Leu Arg Pro
35 40
CA 02362373 2001-08-13
WO 00/47611 PCT/US00/03650
<210> 33
<211> 11
<212> PRT
<213> Homo Sapiens
<400> 33
Gly Trp Leu Gly Glu Gly Trp Trp Glu Leu Leu
1 5 10
<210> 34
<211> 39
<212> PRT
<213> Synthetic Peptide
<400> 34
Ser Ala Met Phe Val Pro Phe Asp Ile Ala Val Gly Val Arg Asp Gly
1 5 10 1'5
Gln Gln Gly Leu Gly Gly Ser Arg Arg Lys Gly Ala Arg Leu Arg Glu
25 30
Ala Ile Ser Ser Tyr Ala Glu
<210> 35
<211> 38
<212> PRT
<213> Synthetic Peptide
<400> 35
Ile Pro Ser Val Thr Arg Met Thr Val Gly Gly Thr Leu Arg Lys Glu
1 5 10 15
Phe Gln Asp Val Val Leu Gly Val Ile Phe Gly Leu Val Leu Val Ile
20 25 30
Asn Arg Cys Ser Phe Leu
<210> 36
<211> 40
<212> PRT
<213> Synthetic Peptide
<400> 36
Leu Val Leu Arg Gly Asn Gln Val Phe Ala Phe Cys Arg Ser Asp Asn
5 10 15
Asn Arg Gln Gln Ala Pro Ala Gly Cys Cys Tyr Val Gly Phe Ser Leu
20 25 30
Phe Val Thr Arg Gly Gly Tyr Glu
35 40
CA 02362373 2001-08-13
WO 00/47611 PCT/US00/03650
11
<210> 37
<211> 40
<212> PRT
<213> Synthetic Peptide
<400> 37
Trp Gln Ala Tyr Pro Val Gln Tyr Leu Phe Val Val Ala Thr Gly Tyr
1 5 10 15
Gly Gly Lys Val Ile Asn His Leu Arg Gly Lys Val Arg Arg Glu Ser
20 25 30
Ala Asp Gln Val Pro Gly Tyr Phe
35 40
<210> 38
<211> 23
<212> PRT
<213> Synthetic Peptide
<400> 38
Met Phe Val Cys Val Asp Ala Lys Gln Cys Leu Leu Gly Ala Ala Gly
1 5 10 15
Gly Leu Arg Leu Ile Phe Ala
<210> 39
<211> 40
<212> PRT
<213> Synthetic Peptide
<400> 39
Val Asp Asp Leu Thr Leu Gln Ser Arg Ser Pro Pro Ser Gln Leu Asn
1 5 10 15
Ser Gln His Leu Leu Leu Ser Gln Leu Cys Gly Tyr Trp Met Phe Arg
20 25 30
Val Arg Ser Arg Ser Cys Cys Gly
35 40
<210> 40
<211> 39
<212> PRT
<213> Synthetic Peptide
<400> 40
Arg Ser Arg Met Phe Val Leu Gly Val Leu Glu Val Asp Ser Gly Leu
1 5 10 15
CA 02362373 2001-08-13
WO 00/47611 PCT/US00/03650
12
Leu Asn Cys Leu Cys Trp Val Gly Val Ser Val Asp Gly Arg Lys Ser
20 25 30
Ser Cys Arg Trp Thr Ala Tyr
<210> 41
<211> 20
<212> PRT
<213> Synthetic Peptide
<400> 41
Gln Arg Asn Pro Arg Leu Arg Leu Ile Arg Arg His Pro Thr Leu Arg
1 5 10 15
Ile Pro Pro Ile
