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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to the prevention and treatment of an
allergic disorder. In particular, the invention relates to the
administration of polypeptides of the unique domain of the tyrosine
kinase, Lyn, to the cells of a subject having, or at risk of having, an
allergic disorder. The polypeptides act within the cells to bind the
cytoplasmic domain of the high affinity receptor of IgE (Fc.epsilon.RI)
and inhibit signaling through the receptor which would result in release
of histamines and other substances associated with an allergic reaction,
thereby preventing or treating an allergic disorder.
2. Background Art
The family of proteins known as the "multichain immune recognition
receptors" includes the antigen receptors on B and T-lymphocytes and Fc
receptors including the receptor with high affinity for IgE (Fc.epsilon.RI
) (1). Highly homologous in structure, all these receptors utilize, at
least in part, a common mechanism to initiate cellular responses:
multi-valent interactions with antigen leads to aggregation of the
receptors and is followed by enhanced phosphorylation of tyrosines, in the
Immune-Receptor Tyrosine-based Activation Motifs (ITAMs) within the
cytoplasmic domains of the receptor itself, by a receptor-associated
Src-family kinase (2).
Aggregation of the Fc.epsilon.RI on mast cells initiates a cascade of
events leading to degranulation and release of mediators responsible for
the symptoms of atopy. Among the earliest events in the Fc.epsilon.RI
cascade is the phosphorylation of tyrosines in the ITAMs on the .beta. and
.gamma. chains of the receptor by the Src-family kinase, Lyn.
Several groups have studied the interaction between Fc.epsilon.RI and Lyn
kinase by a variety of techniques and have demonstrated (7-10) a direct
interaction between the kinase and the C-terminal cytoplasmic extension of
the receptor's .beta. chain. Previous studies have also demonstrated that
two forms of Lyn, designated Lyn A and Lyn B, are produced by differential
mRNA splicing, both of which behave equivalently (5) and become
equivalently attached to the receptor after chemical cross-linking (4).
For Fc.epsilon.RI, a "transphosphorylation" mechanism has been demonstrated
that accounts for the earliest cascade events (3,4). Data from these
earlier studies showed that a small percentage of resting
(unphosphorylated) receptors are constitutively associated with Lyn and
this constitutive association with the kinase is an absolute requirement
for the initial phosphorylation of the receptor.
The present invention provides polypeptides comprising the unique region of
the Lyn kinase which bind the C-terminal cytoplasmic domain of the .beta.
chain of the Fc.epsilon.RI and inhibit activation of the
Fc.epsilon.RI-mediated cascade of events that induce an allergic response
and methods for the use of these polypeptides in treating or preventing
allergic disorders.
SUMMARY OF THE INVENTION
The present invention provides an isolated polypeptide, in a
pharmaceutically acceptable carrier, comprising a polypeptide having an
amino acid sequence encoded by a nucleic acid which is at least 95%
identical to a nucleic acid selected from the group consisting of: a) a
nucleic acid encoding a polypeptide having the amino acid sequence of
amino acids 1-66 of the human tyrosine kinase, Lyn A; b) a nucleic acid
encoding a polypeptide having the amino acid sequence of SEQ ID NO:1; c) a
nucleic acid encoding a polypeptide having amino acids 1-10 of the human
tyrosine kinase, Lyn A; d) a nucleic acid encoding a polypeptide having
amino acids 1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acid
encoding a polypeptide having amino acids 27-66 of the human tyrosine
kinase, Lyn A; and f) a nucleic acid encoding a polypeptide having any
five or more contiguous amino acids of amino acids 1-66 of the human
tyrosine kinase, Lyn A, wherein the polypeptide has substantially the same
biologically functional activity of the polypeptide encoded by the nucleic
acid sequence as set forth in (a), (b), (c), (d), (e) or (f).
Also provided is an isolated polypeptide, in a pharmaceutically acceptable
carrier, comprising a polypeptide having an amino acid sequence encoded by
a nucleic acid which is at least 95% identical to a nucleic acid selected
from the group consisting of: a) a nucleic acid encoding a polypeptide
having the amino acid sequence of amino acids 1-45 of the human tyrosine
kinase, Lyn B; b) a nucleic acid encoding a polypeptide having the amino
acid sequence of SEQ ID NO:2; and c) a nucleic acid encoding a polypeptide
having any five or more amino acids of amino acids 1-45 of the human
tyrosine kinase, Lyn B, wherein the polypeptide has substantially the same
biologically functional activity of the polypeptide encoded by the nucleic
acid sequence as set forth in (a), (b) or (c).
Furthermore, the present invention provides an isolated polypeptide, which
can be in a pharmaceutically acceptable carrier, produced from a cell
transformed with a nucleic acid which is at least 95% identical to a
nucleic acid selected from the group consisting of: a) a nucleic acid
encoding a polypeptide having the amino acid sequence of amino acids 1-66
of the human tyrosine kinase, Lyn A; b) a nucleic acid encoding a
polypeptide having the amino acid sequence of SEQ ID NO:1; c) a nucleic
acid encoding a polypeptide having amino acids 1-10 of the human tyrosine
kinase, Lyn A; d) a nucleic acid encoding a polypeptide having amino acids
1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acid encoding a
polypeptide having amino acids 27-66 of the human tyrosine kinase, Lyn A;
and f) a nucleic acid encoding a polypeptide having any five or more
contiguous amino acids of amino acids 1-66 of the human tyrosine kinase,
Lyn A, wherein the polypeptide has substantially the same biologically
functional activity of the polypeptide encoded by the nucleic acid
sequence as set forth in (a), (b), (c), (d), (e) or (f).
In addition, the present invention provides an isolated polypeptide, which
can be in a pharmaceutically acceptable carrier, produced from a cell
transformed with a nucleic acid which is at least 95% identical to a
nucleic acid selected from the group consisting of: a) a nucleic acid
encoding a polypeptide having the amino acid sequence of amino acids 1-45
of the human tyrosine kinase, Lyn B; b) a nucleic acid encoding a
polypeptide having the amino acid sequence of SEQ ID NO:2; and c) a
nucleic acid encoding a polypeptide having any five or more amino acids of
amino acids 1-45 of the human tyrosine kinase, Lyn B, wherein the
polypeptide has substantially the same biologically functional activity of
the polypeptide encoded by the nucleic acid sequence as set forth in (a),
(b) or (c).
A method of treating or preventing an allergic disorder in a subject is
also provided, comprising administering the nucleic acid of the present
invention to a cell of the subject under conditions whereby the nucleic
acid is expressed in the subject's cells, thereby treating the allergic
disorder.
The present invention also provides a fusion protein, comprising a
polypeptide having an amino acid sequence encoded by a nucleic acid which
is at least 95% identical to a nucleic acid selected from the group
consisting of: a) a nucleic acid encoding a polypeptide having the amino
acid sequence of amino acids 1-66 of the human tyrosine kinase, Lyn A; b)
a nucleic acid encoding a polypeptide having the amino acid sequence of
SEQ ID NO:1; c) a nucleic acid encoding a polypeptide having amino acids
1-10 of the human tyrosine kinase, Lyn A; d) a nucleic acid encoding a
polypeptide having amino acids 1-27 of the human tyrosine kinase, Lyn A;
e) a nucleic acid encoding a polypeptide having amino acids 27-66 of the
human tyrosine kinase, Lyn A; and f) a nucleic acid encoding a polypeptide
having any five or more contiguous amino acids of amino acids 1-66 of the
human tyrosine kinase, Lyn A, wherein the polypeptide has substantially
the same biologically functional activity of the polypeptide encoded by
the nucleic acid sequence as set forth in (a), (b), (c), (d), (e) or (f),
and a ligand which binds to and is internalized by cells which express a
high affinity receptor for IgE on the surface.
The present invention additionally provides a fusion protein, comprising a
polypeptide having an amino acid sequence encoded by a nucleic acid which
is at least 95% identical to a nucleic acid selected from the group
consisting of: a) a nucleic acid encoding a polypeptide having the amino
acid sequence of amino acids 1-45 of the human tyrosine kinase, Lyn B; b)
a nucleic acid encoding a polypeptide having the amino acid sequence of
SEQ ID NO:2; and c) a nucleic acid encoding a polypeptide having any five
or more amino acids of amino acids 1-45 of the human tyrosine kinase, Lyn
B, wherein the polypeptide has substantially the same biologically
functional activity of the polypeptide encoded by the nucleic acid
sequence as set forth in (a), (b) or (c) and a ligand which binds to and
is internalized by cells which express a high affinity receptor for IgE on
the surface.
Further provided is a fusion protein, produced from a cell transformed with
a nucleic acid which is at least 95% identical to a nucleic acid selected
from the group consisting of: a) a nucleic acid encoding a polypeptide
having the amino acid sequence of amino acids 1-66 of the human tyrosine
kinase, Lyn A; b) a nucleic acid encoding a polypeptide having the amino
acid sequence of SEQ ID NO:1; c) a nucleic acid encoding a polypeptide
having amino acids 1-10 of the human tyrosine kinase, Lyn A; d) a nucleic
acid encoding a polypeptide having amino acids 1-27 of the human tyrosine
kinase, Lyn A; e) a nucleic acid encoding a polypeptide having amino acids
27-66 of the human tyrosine kinase, Lyn A; and f) a nucleic acid encoding
a polypeptide having any five or more contiguous amino acids of amino
acids 1-66 of the human tyrosine kinase, Lyn A, wherein the polypeptide
has substantially the same biologically functional activity of the
polypeptide encoded by the nucleic acid sequence as set forth in (a), (b),
(c), (d), (e) or (f), and encoding a ligand which binds to and is
internalized by cells which express a high affinity receptor for IgE on
the surface.
In addition, the present invention provides a fusion protein, produced from
a cell transformed with a nucleic acid which is at least 95% identical to
a nucleic acid selected from the group consisting of: a) a nucleic acid
encoding a polypeptide having the amino acid sequence of amino acids 1-45
of the human tyrosine kinase, Lyn B; b) a nucleic acid encoding a
polypeptide having the amino acid sequence of SEQ ID NO:2; and c) a
nucleic acid encoding a polypeptide having any five or more amino acids of
amino acids 1-45 of the human tyrosine kinase, Lyn B, wherein the
polypeptide has substantially the same biologically functional activity of
the polypeptide encoded by the nucleic acid sequence as set forth in (a),
(b) or (c), and encoding a ligand which binds to and is internalized by
cells which express a high affinity receptor for IgE on the surface.
Finally, the present invention provides a method of treating or preventing
an allergic disorder in a subject, comprising administering the fusion
protein of the present invention to a cell of the subject, whereby the
fusion protein treats or prevents the subject's allergic disorder.
Various other objectives and advantages of the present invention will
become apparent from the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-D. Receptor and kinase proteins and the constructs used for the
yeast two hybrid experiments. 1A: The .beta. chain and the Gal 4 binding
domain (BD)-receptor subunit fusion proteins based on the cytoplasmic
domains of the subunit. The four transmembrane domains of the subunit are
shown shaded. 1B: The .gamma. chain and the Gal 4 binding domain-receptor
subunit fusion proteins based on the cytoplasmic domain of the subunit.
The transmembrane domain is shown shaded. 1C: Lyn B and the Gal
4-activation domain(ACT)-kinase fusion proteins based on the complete
kinase or its unique domain. 1D: Lyn A and the Gal 4-activation
domain-kinase fusion proteins based on the complete kinase or its unique
domain.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, "a" can include multiples.
The present invention is based on the surprising discovery that binding of
a polypeptide consisting of only the unique domain of the Lyn kinase, or
portions thereof, to the C-terminal cytoplasmic domain of the .beta. chain
of the Fc.epsilon.RI results in inhibition of the transduction signaling
activity of the Fc.epsilon.RI. The signaling activity of the Fc.epsilon.RI
is triggered by binding of ligand to the extracellular domain of the
receptor and aggregation of ligand-bound receptors on the cell surface of
mast cells and basophils. Upon ligand binding and aggregation, the
Fc.epsilon.RI transduces a signal across the cell membrane which initiates
a cascade of events leading to release of chemical substances such as
histamine, serotonin, prostaglandins and cytokines, resulting in the
production of an allergic reaction in a subject. By inhibiting the
initiation of this cascade of events, the release of these substances is
blocked. In this manner, an allergic reaction can be treated or prevented
at the level of intracellular inhibition of the production and release of
allergy-inducing substances from the cells, as opposed to conventional
treatment methods which attempt to block the effects of the
allergy-inducing substances after they are released from activated cells.
Thus, by inhibiting an allergic response at an earlier stage, the present
invention provides a more efficient and effective way to treat and prevent
allergic reactions than has been previously achieved.
Thus, the present invention provides an isolated polypeptide consisting of
amino acids 1-66 of the tyrosine kinase, Lyn A, which can be the human Lyn
A kinase and an isolated polypeptide having the amino acid sequence of SEQ
ID NO:1 (human Lyn A kinase). Also provided is an isolated polypeptide
consisting of amino acids 1-45 of the tyrosine kinase, Lyn B, which can be
the human Lyn B kinase and an isolated polypeptide having the amino acid
sequence of SEQ ID NO:2 (human Lyn B kinase).
Also provided is an isolated polypeptide, comprising a polypeptide having
an amino acid sequence encoded by a nucleic acid which is at least 95%
identical to a nucleic acid selected from the group consisting of: a) a
nucleic acid encoding a polypeptide having the amino acid sequence of
amino acids 1-66 of the human tyrosine kinase, Lyn A; b) a nucleic acid
encoding a polypeptide having the amino acid sequence of SEQ ID NO:1; c) a
nucleic acid encoding a polypeptide having amino acids 1-10 of the human
tyrosine kinase, Lyn A; d) a nucleic acid encoding a polypeptide having
amino acids 1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acid
encoding a polypeptide having amino acids 27-66 of the human tyrosine
kinase, Lyn A; and f) a nucleic acid encoding a polypeptide having any
five or more contiguous amino acids of amino acids 1-66 of the human
tyrosine kinase, Lyn A, wherein the polypeptide has substantially the same
biologically functional activity of the polypeptide encoded by the nucleic
acid sequence as set forth in (a), (b), (c), (d), (e) or (f).
Thus, the polypeptide of this invention can comprise the amino acid
sequence of 1) amino acids 1-66 of the human tyrosine kinase, Lyn A, 2)
SEQ ID NO:1, 3) amino acids 1-10 of the human tyrosine kinase, Lyn A, 4)
amino acids 1-27 of the human tyrosine kinase, Lyn A, 5) amino acids 27-66
of the human tyrosine kinase, Lyn A, or 6) any five or more contiguous
amino acids of amino acids 1-66 of the human tyrosine kinase, Lyn A.
In addition, the present invention provides an isolated polypeptide,
comprising a polypeptide having an amino acid sequence encoded by a
nucleic acid which is at least 95% identical to a nucleic acid selected
from the group consisting of: a) a nucleic acid encoding a polypeptide
having the amino acid sequence of amino acids 1-45 of the human tyrosine
kinase, Lyn B; b) a nucleic acid encoding a polypeptide having the amino
acid sequence of SEQ ID NO:2; and c) a nucleic acid encoding a polypeptide
having any five or more amino acids of amino acids 1-45 of the human
tyrosine kinase, Lyn B, wherein the polypeptide has substantially the same
biologically functional activity of the polypeptide encoded by the nucleic
acid sequence as set forth in (a), (b) or (c).
Thus, the polypeptide of this invention can comprise the amino acid
sequence of 1) amino acids 1-45 of the human tyrosine kinase, Lyn B, 2)
SEQ ID NO:2, or 3) any five or more amino acids of amino acids 1-45 of the
human tyrosine kinase, Lyn B.
Also provided is an isolated nucleic acid encoding the amino acid sequence
of the polypeptides described above, a vector comprising the nucleic acid
and cell comprising the vector. Furthermore, the polypeptides of this
invention and the nucleic acids encoding them can be in a pharmaceutically
acceptable carrier.
Furthermore, the present invention provides an isolated polypeptide, in a
pharmaceutically acceptable carrier, produced from a cell transformed with
a nucleic acid which is at least 95% identical to a nucleic acid selected
from the group consisting of: a) a nucleic acid encoding a polypeptide
having the amino acid sequence of amino acids 1-66 of the human tyrosine
kinase, Lyn A; b) a nucleic acid encoding a polypeptide having the amino
acid sequence of SEQ ID NO:1; c) a nucleic acid encoding a polypeptide
having amino acids 1-10 of the human tyrosine kinase, Lyn A; d) a nucleic
acid encoding a polypeptide having amino acids 1-27 of the human tyrosine
kinase, Lyn A; e) a nucleic acid encoding a polypeptide having amino acids
27-66 of the human tyrosine kinase, Lyn A; and f) a nucleic acid encoding
a polypeptide having any five or more contiguous amino acids of amino
acids 1-66 of the human tyrosine kinase, Lyn A, wherein the polypeptide
has substantially the same biologically functional activity of the
polypeptide encoded by the nucleic acid sequence as set forth in (a), (b),
(c), (d), (e) or (f). As used herein, "transformed" means a cell into
which exogenous nucleic acid has been introduced either as naked DNA or as
part of a vector.
The polypeptide of this invention can have from 80 to 100% identity with
the nucleic acid sequences set forth herein and still have substantially
the same biologically functional activity of the polypeptides encoded by
these nucleic acids. Such variation in nucleic acid sequence among
polypeptides having the same activity can be due to natural variation
within a species or among species, as well as a result of modification of
the amino acid sequence, as described below. The biologically functional
activity of a polypeptide having between 80 and 100% identity can be
determined according to the protocols for binding and inhibiting
phosphorylation as described herein.
Thus, the present invention also provides a polypeptide produced from a
cell transformed with a nucleic acid encoding the amino acid sequence of:
1) amino acids 1-66 of the human tyrosine kinase, Lyn A; 2). SEQ ID NO:1;
3) amino acids 1-10 of the human tyrosine kinase, Lyn A; 4) amino acids
1-27 of the human tyrosine kinase, Lyn A; 5) amino acids 27-66 of the
human tyrosine kinase, Lyn A; or 6) any five or more contiguous amino
acids of amino acids 1-66 of the human tyrosine kinase, Lyn A.
Also provided is an isolated polypeptide, in a pharmaceutically acceptable
carrier, produced from a cell transformed with a nucleic acid which is at
least 95% identical to a nucleic acid selected from the group consisting
of: a) a nucleic acid encoding a polypeptide having the amino acid
sequence of amino acids 1-45 of the human tyrosine kinase, Lyn B; b) a
nucleic acid encoding a polypeptide having the amino acid sequence of SEQ
ID NO:2; and c) a nucleic acid encoding a polypeptide having any five or
more amino acids of amino acids 1-45 of the human tyrosine kinase, Lyn B,
wherein the polypeptide has substantially the same biologically functional
activity of the polypeptide encoded by the nucleic acid sequence as set
forth in (a), (b) or (c).
Thus, the present invention provides a polypeptide produced from a cell
transformed with a nucleic acid encoding the amino acid sequence of: 1)
amino acids 1-45 of the human tyrosine kinase, Lyn B; 2) SEQ ID NO:2; or
3) any five or more contiguous amino acids of amino acids 1-45 of the
human tyrosine kinase, Lyn B. As used herein, the polypeptide of this
invention is the unique domain of the tyrosine kinase, Lyn A (amino acids
1-66) or Lyn B (amino acids 1-45)or any portion thereof.
Thus, the polypeptide of this invention can consist of any portion of the
amino acid sequence of amino acids 1-66 of Lyn A or of the amino acid
sequence of SEQ ID NO:1, as well as any portion of the amino acid sequence
of amino acids 1-45 of Lyn B or of the amino acid sequence of SEQ ID NO:2.
For example, the polypeptide of this invention can be amino acids 1-10
(SEQ ID NO:3), amino acids 1-27 (SEQ ID NO:4) or amino acids 27-66 (SEQ ID
NO:5) of Lyn A, all of which have been demonstrated to associate with the
Fc.epsilon.RI .beta.c at levels greater than the negative control
(consisting of Lyn A residues 27-66 out of frame (OOF).
It would be well understood by one of skill in the art that any portion of
the amino acid sequence of amino acids 1-66 of Lyn A or any portion of the
amino acid sequence of amino acids 1-45 of Lyn B can be identified based
on the known sequence of Lyn A and Lyn B (22, 24) and produced according
to methods well known in the art (e.g., peptide synthesis; expression of
synthesized oligonucleotides). The portion of the amino acid sequence of
Lyn A or of Lyn B can then be identified as effective in inhibiting the
signaling activity of Fc.epsilon.RI according to the methods described in
the Examples herein. Thus, the portion of the amino acid sequence of Lyn A
or Lyn B which can be produced and tested for inhibitory activity
according the methods of this invention can be any five or more contiguous
amino acids of amino acids 1-66 of Lyn A or 1-45 of Lyn B. For example,
the portion of this invention can be amino acids 1-5, 15-30, 22-45, etc.,
of Lyn A or Lyn B.
As used herein, "isolated" and/or "purified" means a polypeptide which is
substantially free from the naturally occurring materials with which the
polypeptide is normally associated in nature. Also as used herein,
"polypeptide" refers to a molecule comprised of amino acids which
correspond to those encoded by a nucleic acid. The polypeptides of this
invention can consist of the entire amino acid sequence of amino acids
1-66 of Lyn A or of amino acids 1-45 of Lyn B or portions thereof, as set
forth above. The polypeptides or portions thereof of the present invention
can be obtained by isolation and purification from cells where they are
produced naturally or by expression of DNA encoding amino acids 1-66 of
Lyn A or amino acids 1-45 of Lyn B or portions thereof. The polypeptides
of the present invention or portions thereof can be obtained by chemical
synthesis of peptides, by proteolytic cleavage of the polypeptides and by
synthesis from nucleic acids (either naturally occurring or synthesized)
encoding the amino acid sequence of interest. The polypeptide may include
conservative substitutions where a naturally occurring amino acid is
replaced by one having similar properties. Such conservative substitutions
do not alter the Fc.epsilon.RI signal transduction-inhibiting activity of
the polypeptide and would be understood to include at least those listed
in Table 1 (35).
Thus, it is understood that, where desired, modifications and changes may
be made in the nucleic acid and/or amino acid sequence of the polypeptides
of the present invention and still obtain a polypeptide having like or
otherwise desirable characteristics. Such changes may occur in natural
isolates or may be synthetically introduced using site-specific
mutagenesis, the procedures for which, such as mis-match polymerase chain
reaction (PCR), are well known in the art.
For example, certain amino acids may be substituted for other amino acids
in a Lyn A or Lyn B polypeptide of this invention without appreciable loss
of Fc.epsilon.Ri signal transduction-inhibiting activity. 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 the Lyn A or Lyn B amino acid sequence (or, of course, the
underlying nucleic acid sequence) and nevertheless obtain a Lyn A or Lyn B
polypeptide of this invention with like properties. It is thus
contemplated that various changes may be made in the amino acid sequence
of the Lyn A or Lyn B polypeptide (or underlying nucleic acid sequence) of
this invention without appreciable loss of biological utility or activity
and possibly with an increase in such utility or activity.
The present invention also provides an isolated nucleic acid encoding a
polypeptide consisting of amino acids 1-66 of the human tyrosine kinase,
Lyn A and an isolated nucleic acid encoding a polypeptide having the amino
acid sequence of SEQ ID NO:1. Also provided is an isolated nucleic acid
encoding a polypeptide consisting of amino acids 1-45 of the human
tyrosine kinase, Lyn B and an isolated nucleic acid encoding a polypeptide
having the amino acid sequence of SEQ ID NO:2. The nucleic acids of this
invention can be in a pharmaceutically acceptable carrier.
"Nucleic acid" as used herein refers to single- or double-stranded
molecules which may be DNA, comprised of the nucleotide bases A, T, C and
G, or RNA, comprised of the bases A, U (substitute for T), C and G. The
nucleic acid may represent a coding strand or its complement. Nucleic
acids may be identical in sequence to the sequence which is naturally
occurring or may include alternative codons which encode the same amino
acid as that which is found in the naturally occurring sequence (34).
Furthermore, nucleic acids may include codons which represent conservative
substitutions of amino acids as described in Table 1.
As used herein, the term "isolated" means a nucleic acid separated or
substantially free from at least some of the other components of the
naturally occurring organism, for example, the cell structural components
commonly found associated with nucleic acids in a cellular environment
and/or other nucleic acids. The isolation of nucleic acids can therefore
be accomplished by techniques such as cell lysis followed by phenol plus
chloroform extraction, followed by ethanol precipitation of the nucleic
acids (33). The nucleic acids of this invention can be isolated from cells
according to methods well known in the art. Alternatively, the nucleic
acids of the present invention can be synthesized according to standard
protocols well described in the literature.
The nucleic acid encoding the polypeptide of Lyn A or of Lyn B or portion
thereof of this invention can be part of a recombinant nucleic acid
comprising any combination of restriction sites and/or functional elements
as are well known in the art which facilitate molecular cloning,
expression and other recombinant DNA manipulations. Thus, the present
invention further provides a recombinant nucleic acid comprising the
nucleic acid encoding the polypeptide of Lyn A or Lyn B, or portion
thereof of the present invention. In particular, the isolated nucleic acid
encoding the polypeptide of Lyn A or of Lyn B or portion thereof can be
present in a vector and the vector can be present in a cell, which can be
a cell cultured in vitro or a cell in a transgenic animal.
Thus, the present invention further provides a vector comprising a nucleic
acid encoding the polypeptide of Lyn A or of Lyn B or a portion thereof
(e.g., peptides consisting of amino acids 1-10, 1-27 or 27-66 of Lyn A) of
this invention. The vector can be in a pharmaceutically acceptable
carrier. The vector can be an expression vector which contains all of the
genetic components required for expression of the nucleic acid encoding
the polypeptide of Lyn A or of Lyn B or portion thereof in cells into
which the vector has been introduced, as are well known in the art. The
expression vector can be a commercial expression vector or it can be
constructed in the laboratory according to standard molecular biology
protocols. The expression vector can comprise viral nucleic acid
including, but not limited to, adenovirus, retrovirus and or
adeno-associated virus nucleic acid. The nucleic acid or vector of this
invention can also be in a liposome or a delivery vehicle which can be
taken up by a cell via receptor-mediated or other type of endocytosis.
A polypeptide of Lyn A or of Lyn B or portion thereof of the present
invention which is identified to inhibit the signaling activity of
Fc.epsilon.RI according to the methods provided herein can be administered
to a subject to treat or prevent an allergic disorder. Thus, the present
invention further provides a method for treating or preventing an allergic
disorder in a subject, comprising administering the nucleic acid of this
invention, which encodes a polypeptide of Lyn A or of Lyn B or a portion
thereof, to a cell of the subject under conditions whereby the nucleic
acid is expressed in the subject's cells, thereby treating the allergic
disorder.
The subject can be any animal in which it is desirable to inhibit the
signal transducing activity of an immunoglobulin receptor which mediates
an allergic reaction. In a preferred embodiment, the animal of the present
invention is a human. In addition, non-human animals which can be treated
by the method of this invention can include, but are not limited to, cats,
dogs, birds, horses, cows, goats, sheep, guinea pigs, hamsters, gerbils
and rabbits, as well as any other animal in which the polypeptide of the
present invention can inhibit the signal transducing activity of an
immunoglobulin receptor, thereby treating or preventing an allergic
response.
As recited herein, an allergic disorder describes a disease state or
syndrome whereby the body produces a dysfunctional immune response to
environmental antigens comprising immunoglobulin E (IgE) antibodies which
evoke allergic symptoms such as itching, sneezing, coughing, respiratory
congestion, rhinorrhea, skin eruptions and the like, as well as severe
reactions, such as asthma attacks and systemic anaphylaxis. Examples of
allergic diseases and disorders which can be treated or prevented by the
methods of this invention include, but are not limited to, drug
hypersensitivity, allergic rhinitis, bronchial asthma, ragweed pollen
hayfever, anaphylactic syndrome, urticaria, angioedema, atopic dermatitis,
erythema nodosum, erythema multiforme, Stevens-Johnson Syndrome, cutaneous
necrotizing venulitis, bullous skin diseases, allergy to food substances
and insect venom-induced allergic reactions (36-39), as well as any other
allergic disease or disorder now known or identified in the future.
As described above, the nucleic acid of the present invention can be
administered in a pharmaceutically acceptable carrier and can be delivered
to the subject's cells in vivo and/or ex vivo by a variety of mechanisms
well known in the art (e.g., uptake of naked DNA, viral infection,
liposome fusion, intramuscular injection of DNA via a gene gun,
endocytosis and the like).
If ex vivo methods are employed, cells or tissues can be removed and
maintained outside the body according to standard protocols well known in
the art. The nucleic acids of this invention can be introduced into the
cells via any gene transfer mechanism, such as, for example,
virus-mediated gene delivery, calcium phosphate mediated gene delivery,
electroporation, microinjection or proteoliposomes. The transduced cells
can then be infused (e.g., in a pharmaceutically acceptable carrier) or
homotopically transplanted back into the subject per standard methods for
the cell or tissue type. Standard methods are known for transplantation or
infusion of various cells into a subject.
The cells of the subject to which the nucleic acid of this invention can be
administered can include any cell which can take up and express exogenous
DNA and which expresses an Fc.epsilon.RI whose C-terminal .beta. chain
cytoplasmic domain can be bound by the polypeptide of this invention, the
binding of which results in inhibition of the signal transducing activity
of the Fc.epsilon.RI. For example, the cells can be mast cells, basophils
and/or eosinophils, as well as any other cell type which expresses an
Fc.epsilon.RI and in which it would be desirable to inhibit the signal
transducing activity of the Fc.epsilon.RI.
In the methods described above which include the administration and uptake
of exogenous DNA into the cells of a subject (i.e., gene transduction or
transfection), the nucleic acids of the present invention can be in the
form of naked DNA or the nucleic acids can be in a vector for delivering
the nucleic acids to the cells for expression of the nucleic acid encoding
Lyn A or Lyn B polypeptide or portion thereof inside the cell. The vector
can be a commercially available preparation, such as an adenovirus vector
(Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the
nucleic acid or vector to cells can be via a variety of mechanisms. As one
example, delivery can be via a liposome, using commercially available
liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc.,
Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) and
TRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as other
liposomes developed according to procedures standard in the art. In
addition, the nucleic acid or vector of this invention can be delivered in
vivo by electroporation, the technology for which is available from
Genetronics, Inc. (San Diego, Calif.) as well as by means of a
SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).
As one example, vector delivery can be via a viral system, such as a
retroviral vector system which can package a recombinant retroviral genome
(40, 41). The recombinant retrovirus can then be used to infect and
thereby deliver to the infected cells nucleic acid encoding the
polypeptide of Lyn A or of Lyn B or a portion thereof. The exact method of
introducing the nucleic acid into mammalian cells is, of course, not
limited to the use of retroviral vectors. Other techniques are widely
available for this procedure including the use of adenoviral vectors (42),
adeno-associated viral (AAV) vectors (43), lentiviral vectors (44),
pseudotyped retroviral vectors (45). Physical transduction techniques can
also be used, such as liposome delivery and receptor-mediated and other
endocytosis mechanisms (see, for example, 46). This invention can be used
in conjunction with any of these or other commonly used gene transfer
methods.
As described above, the nucleic acid or vector of the present invention can
also be administered in vivo in a pharmaceutically acceptable carrier. By
"pharmaceutically acceptable" is meant a material that is not biologically
or otherwise undesirable, i.e., the material may be administered to a
subject, along with the nucleic acid or vector, without causing any
undesirable biological effects or interacting in a deleterious manner with
any of the other components of the pharmaceutical composition in which it
is contained. The carrier would naturally be selected to minimize any
degradation of the active ingredient and to minimize any adverse side
effects in the subject, as would be well known to one of skill in the art.
The nucleic acid or vector may be administered orally, parenterally (e.g.,
intravenously), by intramuscular injection, by intraperitoneal injection,
transdermally, extracorporeally, topically or the like, although topical
intranasal administration or administration by inhalant is typically
preferred. As used herein, "topical intranasal administration" means
delivery of the nucleic acid or vector into the nose and nasal passages
through one or both of the nares and can comprise delivery by a spraying
mechanism or droplet mechanism, or through aerosolization of the nucleic
acid or vector. The latter may be effective when a large number of animals
is to be treated simultaneously. Administration of the nucleic acid or
vector by inhalant can be through the nose or mouth via delivery by a
spraying or droplet mechanism. Delivery can also be directly to any area
of the respiratory system (e.g., lungs) via intubation. The exact amount
of the nucleic acid or vector required will vary from subject to subject,
depending on the species, age, weight and general condition of the
subject, the severity of the allergic disorder being treated, the
particular nucleic acid or vector used, its mode of administration and the
like. Thus, it is not possible to specify an exact amount for every
nucleic acid or vector. However, an appropriate amount can be determined
by one of ordinary skill in the art using only routine experimentation
given the teachings herein (see, e.g., 47).
As one example, if the nucleic acid of this invention is delivered to the
cells of a subject in an adenovirus vector, the dosage for administration
of adenovirus to humans can range from about 10.sup.7 to 10.sup.9 plaque
forming unit (pfu) per injection, but can be as high as 10.sup.12 pfu per
injection (48, 49).
Parenteral administration of the nucleic acid or vector of the present
invention, if used, is generally characterized by injection. Injectables
can be prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for solution of suspension in liquid
prior to injection, or as emulsions. A more recently revised approach for
parenteral administration involves use of a slow release or sustained
release system such that a constant dosage is maintained. See, e.g., U.S.
Pat. No. 3,610,795, which is incorporated by reference herein.
The present invention additionally provides a fusion protein comprising a
polypeptide consisting of amino acids 1-66, or a portion thereof, of the
human tyrosine kinase, Lyn A or a polypeptide having the amino acid
sequence of SEQ ID NO:1, or a portion thereof and a ligand which binds to
and is internalized by cells which express a high affinity receptor for
IgE on the surface.
Also provided is a fusion protein comprising a polypeptide consisting of
amino acids 1-45, or a portion thereof, of the human tyrosine kinase, Lyn
B or a polypeptide having the amino acid sequence of SEQ ID NO:2, or a
portion thereof and a ligand which binds to and is internalized by cells
which express a high affinity receptor for IgE on the surface.
The present invention further provides a fusion protein, comprising a
polypeptide having an amino acid sequence encoded by a nucleic acid which
is at least 95% identical to a nucleic acid selected from the group
consisting of: a) a nucleic acid encoding a polypeptide having the amino
acid sequence of amino acids 1-66 of the human tyrosine kinase, Lyn A; b)
a nucleic acid encoding a polypeptide having the amino acid sequence of
SEQ ID NO:1; c) a nucleic acid encoding a polypeptide having amino acids
1-10 of the human tyrosine kinase, Lyn A; d) a nucleic acid encoding a
polypeptide having amino acids 1-27 of the human tyrosine kinase, Lyn A;
e) a nucleic acid encoding a polypeptide having amino acids 27-66 of the
human tyrosine kinase, Lyn A; and f) a nucleic acid encoding a polypeptide
having any five or more contiguous amino acids of amino acids 1-66 of the
human tyrosine kinase, Lyn A, wherein the polypeptide has substantially
the same biologically functional activity of the polypeptide encoded by
the nucleic acid sequence as set forth in (a), (b), (c), (d), (e) or (f),
and a ligand which binds to and is internalized by cells which express a
high affinity receptor for IgE on the surface.
The fusion protein of the present invention can comprise a polypeptide
comprising the amino acid sequence of: 1) amino acids 1-66 of the human
tyrosine kinase, Lyn A, 2) SEQ ID NO:1, 3) amino acids 1-10 of the human
tyrosine kinase, Lyn A, 4) amino acids 1-27 of the human tyrosine kinase,
Lyn A, 5) amino acids 27-66 of the human tyrosine kinase, Lyn A, or 6) any
five or more contiguous amino acids of amino acids 1-66 of the human
tyrosine kinase, Lyn A, and a ligand which binds to and is internalized by
cells which express a high affinity receptor for IgE on the surface.
Further provided is a fusion protein, comprising a polypeptide having an
amino acid sequence encoded by a nucleic acid which is at least 95%
identical to a nucleic acid selected from the group consisting of: a) a
nucleic acid encoding a polypeptide having the amino acid sequence of
amino acids 1-45 of the human tyrosine kinase, Lyn B; b) a nucleic acid
encoding a polypeptide having the amino acid sequence of SEQ ID NO:2; and
c) a nucleic acid encoding a polypeptide having any five or more amino
acids of amino acids 1-45 of the human tyrosine kinase, Lyn B, wherein the
polypeptide has substantially the same biologically functional activity of
the polypeptide encoded by the nucleic acid sequence as set forth in (a),
(b) or (c) and a ligand which binds to and is internalized by cells which
express a high affinity receptor for IgE on the surface.
The fusion protein of the present invention can comprise a polypeptide
comprising the amino acid sequence of: 1) amino acids 1-45 of the human
tyrosine kinase, Lyn B, 2) SEQ ID NO:2, or any five or more contiguous
amino acids of amino acids 1-45 of the human tyrosine kinase, Lyn B, and a
ligand which binds to and is internalized by cells which express a high
affinity receptor for IgE on the surface.
Additionally, the present invention provides a fusion protein, produced
from a cell transformed with a nucleic acid which is at least 95%
identical to a nucleic acid selected from the group consisting of: a) a
nucleic acid encoding a polypeptide having the amino acid sequence of
amino acids 1-66 of the human tyrosine kinase, Lyn A; b) a nucleic acid
encoding a polypeptide having the amino acid sequence of SEQ ID NO:1; c) a
nucleic acid encoding a polypeptide having amino acids 1-10 of the human
tyrosine kinase, Lyn A; d) a nucleic acid encoding a polypeptide having
amino acids 1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acid
encoding a polypeptide having amino acids 27-66 of the human tyrosine
kinase, Lyn A; and f) a nucleic acid encoding a polypeptide having any
five or more contiguous amino acids of amino acids 1-66 of the human
tyrosine kinase, Lyn A, wherein the polypeptide has substantially the same
biologically functional activity of the polypeptide encoded by the nucleic
acid sequence as set forth in (a), (b), (c), (d), (e) or (f), and encoding
a ligand which binds to and is internalized by cells which express a high
affinity receptor for IgE on the surface.
The fusion protein of the present invention can comprise a polypeptide
produced by a cell transformed with a nucleic acid encoding the amino acid
sequence of: 1) amino acids 1-66 of the human tyrosine kinase, Lyn A, 2)
SEQ ID NO:1, 3) amino acids 1-10 of the human tyrosine kinase, Lyn A, 4)
amino acids 1-27 of the human tyrosine kinase, Lyn A, 5) amino acids 27-66
of the human tyrosine kinase, Lyn A, or 6) any five or more contiguous
amino acids of amino acids 1-66 of the human tyrosine kinase, Lyn A, and a
ligand which binds to and is internalized by cells which express a high
affinity receptor for IgE on the surface.
In addition, the present invention provides a fusion protein, produced from
a cell transformed with a nucleic acid which is at least 95% identical to
a nuc | | |
