Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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KAY- A NOVEL IMMUNE SYSTEM PROTEIN
BACKGROUND OF THE INVENTION
The present invention relates to a novel ligand, Kay, which is a member of the
Tumor Necrosis Factor Family. This protein or its receptor may have anti-
cancer
and/or immunoregulatory applications. Furthermore, cells transfected with the
genes for
this novel ligand may be used in gene therapy to treat tumors, autoimmune and
inflammatory diseases or inherited genetic disorders, and blocking antibodies
to these
proteins can have immunoregulatory applications.
BACKGROUND OF THE INVENTION
The tumor-necrosis factor (TNF)-related cytokines are mediators of host
defense
and immune regulation. Members of this family exist in membrane-anchored
forms,
acting locally through cell-to-cell contact, or as secreted proteins capable
of diffusing to
more distant targets. A parallel family of receptors signals the presence of
these
molecules leading to the initiation of cell death or cellular proliferation
and
differentiation in the target tissue. Presently, the TNF family of ligands and
receptors
has at least 11 recognized receptor-ligand pairs, including: TNF:TNF-R; LT-
a:TNF-R;
LT-a/~3:LT-(3-R; FasL:Fas; CD40L:CD40; CD30L:CD30; CD27L:CD27;
OX40L:OX40 and 4-IBBL:4-1BB. The DNA sequences encoding these ligands have
only about 25% to about 30% identity in even the most related cases, although
the
amino acid relatedness is about SO%.
The defining feature of this family of cytokine receptors is found in the
cysteine
rich extracellular domain initially revealed by the molecular cloning of two
distinct
TNF receptors.' This family of genes encodes glycoproteins characteristic of
Type I
transmembrane proteins with an extracellular ligand binding domain, a single
membrane spanning region and a cytoplasmic region involved in activating
cellular
functions. The cysteine-rich ligand binding region exhibits a tightly knit
disulfide
linked core domain, which, depending upon the particular family member, is
repeated
multiple times. Most receptors have four domains, although there may be as few
as
three, or as many as six.
Proteins in the TNF family of ligands are characterized by a short N-terminal
stretch of normally short hydrophilic amino acids, often containing several
lysine or
arginine residues thought to serve as stop transfer sequences. Next follows a
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transmembrane region and an extracellular region of variable length, that
separates the
C-terminal receptor binding domain from the membrane. This region is sometimes
referred to as the "stalk". The C-terminal binding region comprises the bulk
of the
protein, and often, but not always, contains glycosylation sites. These genes
lack the
classic signal sequences characteristic of type I membrane proteins, type II
membrane
proteins with the C terminus lying outside the cell, and a short N-terminal
domain
residing in the cytoplasm. In some cases, e.g., TNF and LT-a, cleavage in the
stalk
region can occur early during protein processing and the ligand is then found
primarily
in secreted form. Most ligands, however, exist in a membrane form, mediating
localized signaling.
The structure of these ligands has been well-defined by crystallographic
analyses of TNF, LT-a, and CD40L. TNF and lymphotoxin-a (LT-a) are both
structured into a sandwich of two anti-parallel ~i-pleated sheets with the
"jelly roll" or
Greek key topology." The rms deviation between the Ca and ~i residues is 0.61
C,
suggesting a high degree of similarity in their molecular topography. A
structural
feature emerging from molecular studies of CD40L, TNF and LT-a is the
propensity to
assemble into oligomeric complexes. Intrinsic to the oligomeric structure is
the
formation of the receptor binding site at the junction between the neighboring
subunits
creating a multivalent ligand. The quaternary structures of TNF, CD40L and LT-
a have
been shown to exist as trimers by analysis of their crystal structures. Many
of the
amino acids conserved between the different ligands are in stretches of the
scaffold (3-
sheet. It is likely that the basic sandwich structure is preserved in all of
these
molecules, since portions of these scaffold sequences are conserved across the
various
family members. The quaternary structure may also be maintained since the
subunit
conformation is likely to remain similar.
TNF family members can best be described as master switches in the immune
system controlling both cell survival and differentiation. Only TNF and LTa
are
currently recognized as secreted cytokines contrasting with the other
predominantly
membrane anchored members of the TNF family. While a membrane form of TNF has
been well-characterized and is likely to have unique biological roles,
secreted TNF
functions as a general alarm signaling to cells more distant from the site of
the
triggering event. Thus TNF secretion can amplify an event leading to the
well-described changes in the vasculature lining and the inflammatory state of
cells. In
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contrast, the membrane bound members of the family send signals though the TNF
type
receptors only to cells in direct contact. For example T cells provide CD40
mediated
"help" only to those B cells brought into direct contact via cognate TCR
interactions.
Similar cell-cell contact limitations on the ability to induce cell death
apply to the
well-studied Fas system.
It appears that one can segregate the TNF ligands into three groups based on
their ability to induce cell death (Table III). First, TNF, Fas ligand and
TRAIL can
efficiently induce cell death in many lines and their receptors mostly likely
have good
canonical death domains. Presumably the ligand to DR-3 (TRAMP/W5L-1) would
also
all into this category. Next there are those ligands which trigger a weaker
death signal
limited to few cell types and TWEAK, CD30 ligand and LTalb2 are examples of
this
class . How this group can trigger cell death in the absence of a canonical
death domain
is an interesting question and suggests that a separate weaker death signaling
mechanism exists. Lastly, there are those members that cannot efficiently
deliver a
death signal. Probably all groups can have antiproliferative effects on some
cell types
consequent to inducing cell differentiation e.g. CD40 (Funakoshi et al., 1994)
The TNF family has grown dramatically in recent years to encompass at least 11
different signaling pathways involving regulation of the immune system. The
widespread expression patterns of TWEAK and TRAIL indicate that there is still
more
functional variety to be uncovered in this family. This aspect has been
especially
highlighted recently in the discovery of two receptors that affect the ability
of rous
sacroma and herpes simplex virus to replicate as well as the historical
observations that
TNF has anti-viral activity and pox viruses encode for decoy TNF receptors
(Brojatsch
et al., 1996; Montgomery et al., 1996; Smith, 1994; Vassalli, 1992).
TNF is a mediator of septic shock and cachexia"', and is involved in the
regulation of hematopoietic cell development.'" It appears to play a major
role as a
mediator of inflammation and defense against bacterial, viral and parasitic
infections"
as well as having antitumor activity."' TNF is also involved in different
autoimmune
diseases."" TNF may be produced by several types of cells, including
macrophages,
fibroblasts, T cells and natural killer cells.""' TNF binds to two different
receptors, each
acting through specific intracellular signaling molecules, thus resulting in
different
effects of TNF."' TNF can exist either as a membrane bound form or as a
soluble
secreted cytokine.X
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LT-a shares many activities with TNF, i.e. binding to the TNF receptors,"' but
unlike TNF, appears to be secreted primarily by activated T cells and some (3-
lymphoblastoid tumors."" The heteromeric complex of LT-a and LT-(3 is a
membrane
bound complex which binds to the LT-/3 receptor.'°" The LT system (LTs
and LT-R)
appears to be involved in the development of peripheral lymphoid organs since
genetic
disruption of LT-(3 leads to disorganization of T and B cells in the spleen
and an
absence of lymph nodes.'°" The LT-(3 system is also involved in cell
death of some
adenocarcinoma cell lines.""
Fas-L, another member of the TNF family, is expressed predominantly on
activated T cells.""' It induces the death of cells bearing its receptor,
including tumor
cells and HIV-infected cells, by a mechanism known as programmed cell death or
apoptosis.""" Furthermore, deficiencies in either Fas or Fas-L may lead to
lymphoproliferative disorders, confirming the role of the Fas system in the
regulation of
immune responses.x""' The Fas system is also involved in liver damage
resulting from
hepatitis chronic infection""' and in autoimmunity in HIV-infected patients.X"
The Fas
system is also involved in T-cell destruction in HIV
patients.'°° TRAIL, another
member of this family, also seems to be involved in the death of a wide
variety of
transformed cell lines of diverse origin."""
CD40-L, another member of the TNF family, is expressed on T cells and
induces the regulation of CD40-bearing B cells.'°°" Furthermore,
alterations in the
CD40-L gene result in a disease known as X-linked hyper-IgM
syndrome.'°"" The
CD40 system is also involved in different autoimmune diseases'°'" and
CD40-L is
known to have antiviral properties.'°'"' Although the CD40 system is
involved in the
rescue of apoptotic B cells,'°'"" in non-immune cells it induces
apoptosis'°'""'. Many
additional lymphocyte members of the TNF family are also involved in
costimulation.'°'"'
Generally, the members of the TNF family have fundamental regulatory roles in
controlling the immune system and activating acute host defense systems. Given
the
current progress in manipulating members of the TNF family for therapeutic
benefit, it
is likely that members of this family may provide unique means to control
disease.
Some of the ligands of this family can directly induce the apoptotic death of
many
transformed cells e.g. LT, TNF, Fas ligand and TRAIL (Nagata, 1997). Fas and
possibly
TNF and CD30 receptor activation can induce cell death in nontransformed
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lymphocytes which may play an immunoregulatory function (Amakawa et al., 1996;
Nagata, 1997; Sytwu et al., 1996; Zheng et al., 1995). In general, death is
triggered
following the aggregation of death domains which reside on the cytoplasmic
side of the
TNF receptors. The death domain orchestrates the assembly of various signal
S transduction components which result in the activation of the caspase
cascade (Nagata,
1997). Some receptors lack canonical death domains, e.g. LTb receptor and CD30
(Browning et al., 1996; Lee et al., 1996) yet can induce cell death, albeit
more weakly.
It is likely that these receptors function primarily to induce cell
differentiation and the
death is an aberrant consequence in some transformed cell lines, although this
picture is
unclear as studies on the CD30 null mouse suggest a death role in negative
selection in
the thymus (Amakawa et al., 1996). Conversely, signaling through other
pathways such
as CD40 is required to maintain cell survival. Thus, there is a need to
identify and
characterize additional molecules which are members of the TNF family thereby
providing additional means of controlling disease and manipulating the immune
system.
SUMMARY OF THE INVENTION
Accordingly, the present invention is directed to a novel polypeptide referred
to
as Kay-ligand, which substantially obviates one or more of the problems due to
the
limitations and disadvantages of the related art. The inventors have
discovered new
members of the TNF family of cytokines, and defined both the human amino acid
sequence of the protein, as well as the DNA sequences encoding these proteins.
The
claimed invention may be used to identify new diagnostics and therapeutics for
numerous diseases and conditions as discussed in more detail below, as well as
to
obtain information about, and manipulate, the immune system and its processes.
Additionally, the invention may be involved in the induction of cell death in
carcinomas.
Additional features and advantages of the invention will be set forth in the
description which follows, and in part will be apparent from the description,
or may be
learned by practice of the invention. The objectives and other advantages of
the
invention will be realized and attained by the compositions and methods
particularly
pointed out in the written description and claims hereof, as well as in the
appended
drawings.
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Thus, to achieve these and other advantages, and in accordance with the
purpose
of the invention, as embodied and broadly described herein, the invention
includes
DNA sequences encoding Kay-ligand. Specifically, the invention relates to DNA
sequences which encode the human Kay-ligand , SEQ. ID. NO.: 1. Additionally,
the
claimed invention relates to the amino acid sequence of this novel ligand. The
amino
acid sequence of human Kay-ligand is set forth in SEQ. m. NO.: 2. Applicants
have
additionally provided in part the DNA sequence for murine Kay-ligand, SEQ. m.
NO.:
3, and the protein encoded by SEQ. )D. NO. 3 is provided in SEQ. >17. NO.: 4.
In other
embodiments, the invention relates to sequences that have at least 50%
homology with
DNA sequences encoding the C terminal receptor binding domain of the ligand
and
hybridize to the claimed DNA sequences or fragments thereof, and which encode
the
Kay- ligand having the sequences identified in SEQ. )D. NO. 1 or SEQ. )D. NO.
4.
The invention in certain embodiments furthermore relates to DNA sequences
encoding Kay-ligand where the sequences are operatively linked to an
expression
I S control sequence. Any suitable expression control sequences are useful in
the claimed
invention, and can easily be selected by one skilled in the art.
The invention also contemplates recombinant DNAs comprising a sequence
encoding Kay-ligand or fragments thereof, as well as hosts with stably
integrated Kay-
ligand sequences introduced into their genome, or possessing episomal
elements. Any
suitable host may be used in the invention, and can easily be selected by one
skilled in
the art without undue experimentation.
In other embodiments, the invention relates to methods of producing
substantially pure Kay-ligands comprising the step of culturing transformed
hosts. In
yet other embodiments, the invention relates to the Kay ligand essentially
free of
normally associated animal proteins.
The invention encompasses Kay-ligand having the amino acid sequence
identified in SEQ. m. NO. 2. as well as fragments or homologs thereof. In
various
embodiments, the amino acid and/or the DNA sequences may comprise conservative
insertions, deletions and substitutions, as further defined below or may
comprise
fragments of said sequences.
The invention relates in other embodiments to soluble constructs comprising
Kay-ligand which may be used to directly trigger Kay-ligand mediated
pharmacological events. Such events may have useful therapeutic benefits in
the
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treatment of cancer, tumors or the manipulation of the immune system to treat
immunologic diseases. Soluble forms of the claimed ligands could be
genetically
reengineered to incorporate an easily recognizable tag, thereby facilitating
the
identification of the receptors for these ligands.
Additionally, in other embodiments the claimed invention relates to antibodies
directed against the Kay-Iigand, which can be used, for example, for the
treatment of
cancers, and manipulation of the immune system to treat immunologic disease.
In yet other embodiments the invention relates to methods of gene therapy
using
the genes for Kay-ligand, as disclosed and claimed herein.
The pharmaceutical preparations of the invention may, optionally, include
pharmaceutically acceptable carriers, adjuvants, fillers, or other
pharmaceutical
compositions, and may be administered in any of the numerous forms or routes
known
in the art.
It is to be understood that both the foregoing general description and the
I S following detailed description are exemplary and explanatory, and are
intended to
provide further explanation of the invention as claimed.
The accompanying drawings are included to provide a further understanding of
the invention, and are incorporated in, and constitute a part of this
specification,
illustrate several embodiments of the invention, and together with the
description serve
to explain the principles of the invention.
DESCRIPTION OF THE DRAWINGS
Figure 1: An alignment of the amino acid sequences of murine and human Kay
Ligand.
The murine sequence in the upper line was obtained by direct cloning of the
cDNA.
The human sequence reflects a composite of a partial cDNA sequence and 5' RACE
determination. The third, bottom sequence lines shows the consensus sequence.
Figure 2: A fragment of human KayL cDNA was used to probe a northern blot of
RNA's from various human tissues. It can be seen that a roughly 2.4 kb KayL
RNA is
expressed primarily in the spleen and peripheral blood lymphocytes, i.e. in
the
secondary-lymphoid organs.
DETAILED DESCRIPTION
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Reference will now be made in detail to the present preferred embodiments of
the invention. This invention relates to DNA sequences that code for human or
mouse
Kay-ligands, fragments and homologs thereof, and expression of those DNA
sequences
in hosts transformed with them. The invention relates to uses of these DNA
sequences
and the peptides encoded by them. Additionally, the invention encompasses both
human and mouse amino acid sequences for Kay-ligand or fragments thereof, as
well as
pharmaceutical compositions comprising or derived from them.
A. DEFINITIONS
"Homologous", as used herein, refers to the sequence similarity between
sequences of molecules being compared. When a position in both of the two
compared
sequences is occupied by the same base or amino acid monomer subunit, e.g., if
a
position in each of two DNA molecules is occupied by adenine, then the
molecules are
homologous at that position. The percent of homology between two sequences is
a
function of the number of matching or homologous positions shared by the two
sequences divided by the number of positions compared x 100. For example, if 6
of 10
of the positions in two sequences are matched or homologous then the two
sequences
are 60% homologous. By way of example, the DNA sequences ATTGCC and
TATGGC share 50% homology. Generally, a comparison is made when two sequences
are aligned to give maximum homology.
A "purified preparation" or a "substantially pure preparation" of a
polypeptide,
as used herein, means a polypeptide that has been separated from other
proteins, lipids,
and nucleic acids with which it naturally occurs. Preferably, the polypeptide
is also
separated from other substances, e.g., antibodies, matrices, etc., which are
used to
purify it.
"Transformed host" as used herein is meant to encompass any host with stably
integrated sequence, i.e. Kay-ligand sequence, introduced into its genome or a
host
possessing sequence, i.e. Ligand encoding episomal elements.
A "treatment", as used herein, includes any therapeutic treatment, e.g., the
administration of a therapeutic agent or substance, e.g., a drug.
A "substantially pure nucleic acid", e.g., a substantially pure DNA, is a
nucleic
acid which is one or both of: (1) not immediately contiguous with either one
or both of
the sequences, e.g., coding sequences, with which it is immediately contiguous
(i.e.,
one at the 5' end and one at the 3' end) in the naturally-occurring genome of
the
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organism from which the nucleic acid is derived; or (2) which is substantially
free of a
nucleic acid sequence with which it occurs in the organism from which the
nucleic acid
is derived. The term includes, for example, a recombinant DNA which is
incorporated
into a vector, e.g., into an autonomously replicating plasmid or virus, or
into the
genomic DNA of a prokaryote or eukaryote, or which exists as a separate
molecule
(e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction
endonuclease treatment) independent of other DNA sequences. Substantially pure
DNA also includes a recombinant DNA which is part of a hybrid gene encoding
Kay-
ligand.
The terms "peptides", "proteins", and "polypeptides" are used interchangeably
herein.
"Biologically active" as used herein, means having an in vivo or in vitro
activity
which may be performed directly or indirectly. Biologically active fragments
of Kay
ligand may have, for example, 70% amino acid homology with the active site of
the
Ligands, more preferably at least 80%, and most preferably, at least 90%
homology.
Identity or homology with respect to the Ligands is defined herein as the
percentage of
amino acid residues in the candidate sequence which are identical to the Kay-
ligand
residues in SEQ. ID. NO. 2.
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of cell biology, cell culture, molecular biology,
transgenic
biology, microbiology, recombinant DNA, and immunology, which are within the
skill
of the art. Such techniques are described in the literature.
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B. DNA SEC,~UENCES OF THE INVENTION
As described herein, one aspect of the invention features a substantially pure
(or
recombinant) nucleic acid which includes a nucleotide sequence encoding a Kay-
ligand,
such as the DNA described in SEQ. >D. NO. 1 and/or equivalents of such nucleic
acids.
The term nucleic acid as used herein can include fragments and equivalents,
such as, for
example, sequences encoding functionally equivalent peptides. Equivalent
nucleotide
sequences may include sequences that differ by one or more nucleotide
substitutions,
additions or deletions, such as allelic variants, mutations, etc. and include
sequences
that differ from the nucleotide sequence encoding Kay-ligand shown in SEQ. ID
NO: 1,
due to the degeneracy of the genetic code.
The inventor describes herein the human and sequences; the invention will be
described generally by reference to the human sequences, although one skilled
in the art
will understand that the mouse sequences are encompassed herein. The human
proteins
appear to have all of the characteristics of the TNF family, i.e., a type II
membrane
protein organization and conservation of the sequence motifs involved in the
folding of
the protein into the TNF anti-parallel (3-sheet structure.
The nucleotide sequence for Kay-ligand is set forth in SEQ. ID. NO. 1; the
amino acid sequence for Kay-ligand is described in SEQ. ID. NO. 2.
The sequences of the invention can be used to prepare a series of DNA probes
that are useful in screening various collections of natural and synthetic DNAs
for the
presence of DNA sequences that are closely related to Kay-ligand, or fragments
or
derivatives thereof. One skilled in the art will recognize that reference to
Kay-ligand as
used herein, refers also to biologically active derivatives, fragments or
homologs
thereof.
The DNA sequences encoding the Kay-Ligand of the invention can be employed
to produce the claimed peptides on expression in various prokaryotic and
eukaryotic
hosts transformed with them. These peptides may be used in anti-cancer, and
immunoregulatory applications. In general, this comprises the steps of
culturing a host
transformed with a DNA molecule containing the sequence encoding Kay-ligand,
operatively-linked to an expression control sequence.
The DNA sequences and recombinant DNA molecules of the present invention
can be expressed using a wide variety of hostlvector combinations. For
example, useful
vectors may consist of segments of chromosomal, non-chromosomal or synthetic
DNA
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sequences. The expression vectors of the invention are characterized by at
least one
expression control sequence that may be operatively linked to the Kay-Ligand
DNA
sequence inserted in the vector, in order to control and to regulate the
expression of the
DNA sequence.
S Furthermore, within each expression vector, various sites may be selected
for
insertion of the Kay-Ligand sequence of the invention. The sites are usually
designated
by a restriction endonuclease which cuts them, and these sites and
endonucleases are
well recognized by those skilled in the art. It is of course to be understood
that an
expression vector useful in this invention need not have a restriction
endonuclease site
for insertion of the desired DNA fragment. Instead, the vector may be cloned
to the
fragment by alternate means. The expression vector, and in particular the site
chosen
therein for insertion of a selected DNA fragment, and its operative linking
therein to an
expression control sequence, is determined by a variety of factors. These
factors
include, but are not limited to, the size of the protein to be expressed, the
susceptibility
of the desired protein to proteolytic degradation by host cell enzymes, number
of sites
susceptible to a particular restriction enzyme, contamination or binding of
the protein to
be expressed by host cell proteins which may prove difficult to remove during
purification. Additional factors which may be considered include expression
characteristics such as the location of start and stop codons relative to the
vector
sequences, and other factors which will be recognized by those skilled in the
art. The
choice of a vector and insertion site for the claimed DNA sequences is
determined by a
balancing of these factors, not all selections being equally effective for a
desired
application. However, it is routine for one skilled in the art to analyze
these parameters
and choose an appropriate system depending on the particular application.
One skilled in the art can readily make appropriate modifications to the
expression control sequences to obtain higher levels of protein expression,
i.e. by
substitution of codons, or selecting codons for particular amino acids that
are
preferentially used by particular organisms, to minimize proteolysis or to
alter
glycosylation composition. Likewise, cysteines may be changed to other amino
acids to
simplify production, refolding or stability problems.
Thus, not all host/expression vector combinations function with equal
efficiency
in expressing the DNA sequences of this invention. However, a particular
selection of
a host/expression vector combination may be made by those of skill in the art.
Factors
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one may consider include, for example, the compatibility of the host and
vector, toxicity
to the host of the proteins encoded by the DNA sequence, ease of recovery of
the
desired protein, expression characteristics of the DNA sequences and
expression
control sequences operatively linked to them, biosafety, costs and the
folding, form or
S other necessary post-expression modifications of the desired protein.
The Kay-ligand and homologs thereof produced by hosts transformed with the
sequences of the invention, as well as native Kay-ligand purified by the
processes of
this invention, or produced from the claimed amino acid sequences, are useful
in a
variety of compositions and methods for anticancer, antitumor and
immunoregulatory
applications. They are also useful in therapy and methods directed to other
diseases.
This invention also relates to the use of the DNA sequences disclosed herein
to
express this ligand under abnormal conditions, i.e. in a gene therapy setting.
Kay-
ligand may be expressed in tumor cells under the direction of promoters
appropriate for
such applications. Such expression could enhance anti-tumor immune responses
or
directly affect the survival of the tumor. The claimed ligand can also affect
the survival
of an organ graft by altering the local immune response. In this case, the
graft itself or
the surrounding cells would be modified with an engineered gene encoding Kay-
ligand.
Another aspect of the invention relates to the use of the isolated nucleic
acid
encoding the Kay-ligand in "antisense" therapy. As used herein, "antisense"
therapy
refers to administration or in situ generation of oligonucleotides or their
derivatives
which specifically hybridize under cellular conditions with the cellular mRNA
and/or
DNA encoding the ligand of interest, so as to inhibit expression of the
encoded protein,
i.e. by inhibiting transcription and/or translation. The binding may be by
conventional
base pair complementarity, or, for example, in the case of binding to DNA
duplexes,
through specific interactions in the major groove of the double helix. In
general,
"antisense" therapy refers to a range of techniques generally employed in the
art, and
includes any therapy which relies on specific binding to oligonucleotide
sequences.
An antisense construct of the present invention can be delivered, for example,
as
an expression plasmid, which, when transcribed in the cell, produces RNA which
is
complementary to at least a portion of the cellular mRNA which encodes Kay-
ligand.
Alternatively, the antisense construct can be an oligonucleotide probe which
is
generated ex vivo. Such oligonucleotide probes are preferably modified
oligonucleotides which are resistant to endogenous nucleases, and are therefor
stable in
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vivo. Exemplary nucleic acids molecules for use as antisense oligonucleotides
are
phosphoramidates, phosphothioate and methylphosphonate analogs of DNA (See,
e.g.,
5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to
constructing
oligomers useful in antisense therapy have been reviewed, for example, by Van
Der
Krol et al., ( 1988) Biotechniques 6:958-976; and Stein et al. ( 1988) Cancer
Res 48:
2659-2668, specifically incorporated herein by reference.
C. KAY-LIGAND AND AMINO ACID SE(~UENCES THEREFOR
The Kay-ligand of the invention, as discussed above, is a member of the TNF
family. The protein, fragments or homologs thereof may have wide therapeutic
and
diagnostic applications.
The Kay-ligand is present primarily in the spleen and in peripheral blood
lymphocytes, strongly indicating a regulatory role in the immune system.
Comparison
of the claimed Kay-ligand sequences with other members of the human TNF family
reveals considerable structural similarity. All the proteins share several
regions of
sequence conservation in the extracellular domain.
Although the precise three-dimensional structure of the claimed ligand is not
known, it is predicted that, as a member of the TNF family, it may share
certain
structural characteristics with other members of the family.
The novel polypeptides of the invention specifically interact with a receptor,
which has not yet been identified. However, the peptides and methods disclosed
herein
enable the identification of receptors which specifically interact with the
claimed Kay-
ligand or fragments thereof.
The claimed invention in certain embodiments includes peptides derived from
Kay-ligand which have the ability to bind to their receptors. Fragments of the
Kay-
ligands can be produced in several ways, e.g., recombinantly, by PCR,
proteolytic
digestion or by chemical synthesis. Internal or terminal fragments of a
polypeptide can
be generated by removing one or more nucleotides from one end or both ends of
a
nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA
produces polypeptide fragments.
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Polypeptide fragments can also be chemically synthesized using techniques
known in the art such as conventional Merrifield solid phase f- moc or t-boc
chemistry.
For example, peptides and DNA sequences of the present invention may be
arbitrarily
divided into fragments of desired length with no overlap of the fragment, or
divided
into overlapping fragments of a desired length. Methods such as these are
described in
more detail below.
D. Generation of Soluble Forms of Kay-ligand and Tumor-li~and
Soluble forms of the Kay-ligand can often signal effectively and hence can be
administered as a drug which now mimics the natural membrane form. It is
possible
that the Kay-ligand claimed herein are naturally secreted as soluble
cytokines, however,
if not, one can reengineer the gene to force secretion. To create a soluble
secreted form
of Kay-ligand, one would remove at the DNA level the N-terminus transmembrane
regions, and some portion of the stalk region, and replace them with a type I
leader or
alternatively a type II leader sequence that will allow efficient proteolytic
cleavage in
the chosen expression system. A skilled artisan could vary the amount of the
stalk
region retained in the secretion expression construct to optimize both
receptor binding
properties and secretion efficiency. For example, the constructs containing
all possible
stalk lengths, i.e. N-terminal truncations, could be prepared such that
proteins starting at
amino acids 81 to 139 would result. The optimal length stalk sequence would
result
from this type of analysis.
E. Generation of Antibodies Reactive with the Kay-ligand
The invention also includes antibodies specifically reactive with the claimed
Kay-ligand or its receptors. Anti-protein/anti-peptide antisera or monoclonal
antibodies
can be made by standard protocols (See, for example, Antibodies: A Laboratory
Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal such
as a mouse, a hamster or rabbit can be immunized with an immunogenic form of
the
peptide. Techniques for conferring immunogenicity on a protein or peptide
include
conjugation to carriers, or other techniques, well known in the art.
An immunogenic portion of the claimed Kay-ligand or its receptors can be
administered in the presence of an adjuvant. The progress of immunization can
be
monitored by detection of antibody titers in plasma or serum. Standard ELISA
or other
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immunoassays can be used with the immunogen as antigen to assess the levels of
antibodies.
In a preferred embodiment, the subject antibodies are immunospecific for
antigenic determinants of Kay-ligand or its receptors, e.g. antigenic
determinants of a
polypeptide of SEQ. ID. NO.: 2, or a closely related human or non-human
mammalian
homolog (e.g. 70, 80 or 90 percent homologous, more preferably at least 95
percent
homologous). In yet a further preferred embodiment of the present invention,
the anti-
Kay-ligand or anti-Kay-ligand-receptor antibodies do not substantially cross
react (i.e.
react specifically) with a protein which is e.g., less than 80 percent
homologous to SEQ.
ll~. NO. 2 or 6; preferably less than 90 percent homologous with SEQ. >D. NO.:
2; and,
most preferably less than 95 percent homologous with SEQ.1D. N0.:2. By "not
substantially cross react", it is meant that the antibody has a binding
affinity for a non-
homologous protein which is less than 10 percent, more preferably less than 5
percent,
and even more preferably less than 1 percent, of the binding affinity for a
protein of
SEQ.1D. NO. 2.
The term antibody as used herein is intended to include fragments thereof
which
are also specifically reactive with Kay-ligand, or its receptors. Antibodies
can be
fragmented using conventional techniques and the fragments screened for
utility in the
same manner as described above for whole antibodies. For example, F(ab~~
fragments
can be generated by treating antibody with pepsin. The resulting F(ab~2
fragment can
be treated to reduce disulfide bridges to produce Fab' fragments. The
antibodies of the
present invention are further intended to include biospecific and chimeric
molecules
having anti-Kay-ligand or anti-Kay-ligand -receptor activity. Thus, both
monoclonal
and polyclonal antibodies {Ab) directed against Kay-ligand, Tumor-ligand and
their
receptors, and antibody fragments such as Fab' and F(ab')2, can be used to
block the
action of the Ligand and their respective receptor.
Various forms of antibodies can also be made using standard recombinant DNA
techniques. (Winter and Milstein, Nature 349: 293-299 ( 1991 ) specifically
incorporated by reference herein.) For example, chimeric antibodies can be
constructed
in which the antigen binding domain from an animal antibody is linked to a
human
constant domain (e.g. Cabilly et al., U.S. 4,816,567, incorporated herein by
reference).
Chimeric antibodies may reduce the observed immunogenic responses elicited by
animal antibodies when used in human clinical treatments.
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In addition, recombinant "humanized antibodies" which recognize Kay-Iigand
or its receptors can be synthesized. Humanized antibodies are chimeras
comprising
mostly human IgG sequences into which the regions responsible for specific
antigen-
binding have been inserted. Animals are immunized with the desired antigen,
the
corresponding antibodies are isolated, and the portion of the variable region
sequences
responsible for specific antigen binding are removed. The animal-derived
antigen
binding regions are then cloned into the appropriate position of human
antibody genes
in which the antigen binding regions have been deleted. Humanized antibodies
minimize the use of heterologous (i.e. inter species) sequences in human
antibodies,
and thus are less likely to elicit immune responses in the treated subject.
Construction of different classes of recombinant antibodies can also be
accomplished by making chimeric or humanized antibodies comprising variable
domains and human constant domains (CHl, CH2, CH3) isolated from different
classes
of immunoglobulins. For example, antibodies with increased antigen binding
site
valencies can be recombinantly produced by cloning the antigen binding site
into
vectors carrying the human : chain constant regions. (Arulanandam et al., J.
Exp. Med.,
177: 1439-1450 (1993), incorporated herein by reference.)
In addition, standard recombinant DNA techniques can be used to alter the
binding affinities of recombinant antibodies with their antigens by altering
amino acid
residues in the vicinity of the antigen binding sites. The antigen binding
affinity of a
humanized antibody can be increased by mutagenesis based on molecular
modeling.
(Queen et al., Proc. Natl. Acad. Sci. 86: 10029-33 ( 1989) incorporated herein
by
reference.
F. Generation of Analogs: Production of Altered DNA and Peptide Sequences
, Analogs of the claimed Kay-ligand can differ from the naturally occurring
Kay-
Iigand in amino acid sequence, or in ways that do not involve sequence, or
both. Non-
sequence modifications include in vivo or in vitro chemical derivatization of
the Kay-
ligand. Non-sequence modifications include, but are not limited to, changes in
acetylation, methylation, phosphorylation, carboxylation or glycosylation.
Preferred analogs include Kay-ligand biologically active fragments thereof,
whose sequences differ from the sequence given in SEQ. m NO. 2, by one or more
conservative amino acid substitutions, or by one or more non-conservative
amino acid
substitutions, deletions or insertions which do not abolish the activity of
Kay-ligand.
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Conservative substitutions typically include the substitution of one amino
acid for
another with similar characteristics, e.g. substitutions within the following
groups:
valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid,
glutamic
acid; asparagine, glutamine; serine, threonine; lysine, arginine; and,
phenylalanine,
tyrosine.
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TABLE 1
CONSERVATIVE AMINO ACID REPLACEMENTS
for amino Acid code replace with any
of:
Alanine A D-Ala, Gly, Beta-Ala,
L-
Cys, D-Cys
Arginine R D-Arg, Lys, D-Lys,
homo-
Arg, D-homo-Arg,
Met,
Ile, D-Met, D-Ile,
Orn, D-
Orn
Asparagine N D-Asn, Asp, D-Asp,
Glu,
D-Glu, Gln, D-Gln
Aspartic Acid D D-Asp, D-Asn, Asn,
Glu,
D-Glu, Gln, D-Gln
Cysteine C D-Cys, S-Me-Cys,
Met, D-
Met, Thr, D-Thr
Glutamine Q D-Gln, Asn, D-Asn,
Glu,
D-Glu, Asp, D-Asp
Glutamic Acid E D-Glu, D-Asp, Asp,
Asn,
D-Asn, Gln, D-Gln
Glycine G Ala, D-Ala, Pro,
D-Pro, -
Ala, Acp
Isoleucine I D-Ile, Val, D-Val,
Leu, D-
Leu, Met, D-Met
Leucine L D-Leu, Val, D-Val,
Leu,
D-Leu, Met, D-Met
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Lysine K D-Lys, Arg, D-Arg,
Homo-
arg, D-homo-Arg, Met,
D-
Met, Ile, D-Ile, Orn,
D-Orn
Methionine M D-Met, S-Me-Cys, lle,
D-
Ile, Leu, D-Leu, Val,
D-
Val
Phenylalanine F D-Phe, Tyr, D-Thr,
L-
Dopa, His, D-His,
Trp, D-
Trp, Trans-3, 4 or
5-
phenylproline, cis-3,
4, or
S-phenylproline
Proline P D-Pro, L-I-thoazolidine-4-
carboxylic acid, D-or
L-1-
oxazolidine-4-carboxylic
acid
Serine S D-Ser, Thr, D-Thr,
allo-
Thr, Met, D-Met, Met(O),
D-Met(O), L-Cys, D-Cys
Threonine T D-Thr, Ser, D-Ser,
allo-
Thr, Met, D-Met, Met(O),
D-Met(O), Val, D-Val
Tyrosine Y D-Tyr, Phe, D-Phe,
L-
Dopa, His, D-His
Valine V D-Val, Leu, D-Leu,
Ile, D-.
Ile, Met, D-Met
Useful methods for mutagenesis include PCR mutagenesis and saturation
mutagenesis as discussed in more detail below. A library of random amino acid
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sequence variants can also be generated by the synthesis of a set of
degenerate
oligonucleotide sequences.
-PCR Mutagenesis
In PCR mutagenesis, reduced Taq polymerase fidelity can be used to introduce
random mutations into a cloned fragment of DNA (Leung et al., 1989, Technigue
1:11-
15). This is a very powerful and relatively rapid method of introducing random
mutations. The DNA region to be mutagenized can be amplified using the
polymerase
chain reaction (PCR) under conditions that reduce the fidelity of DNA
synthesis by Taq
DNA polymerase, e.g., by using a dGTP/dATP ratio of five and adding Mn''+ to
the
PCR reaction. The pool of amplified DNA fragments can be inserted into
appropriate
cloning vectors to provide random mutant libraries.
-Saturation Mutagenesis
Saturation mutagenesis allows for the rapid introduction of a large number of
single base substitutions into cloned DNA fragments (Mayers et al., 1985,
Science
229:242). This technique includes generation of mutations, e.g., by chemical
treatment
or irradiation of single-stranded DNA in vitro, and synthesis of a
complimentary DNA
strand. The mutation frequency can be modulated by modulating the severity of
the
treatment, and essentially all possible base substitutions can be obtained.
Because this
procedure does not involve a genetic selection for mutant fragments both
neutral
substitutions, as well as of a protein can be prepared by random mutagenesis
of DNA
which those that alter function, can be obtained. The distribution of point
mutations is
not biased toward conserved sequence elements.
-Degenerate Oligonucleotides
A library of homologs can also be generated from a set of degenerate
oligonucleotide sequences. Chemical synthesis of degenerate sequences can be
carried
out in an automatic DNA synthesizer, and the synthetic genes then ligated into
an
appropriate expression vector. The synthesis of degenerate oligonucleotides is
known
in the art'°°' Such techniques have been employed in the
directed evolution of other
proteins'°°".
Non-random or directed, mutagenesis techniques can be used to provide specific
sequences or mutations in specific regions. These techniques can be used to
create
variants which include, e.g., deletions, insertions, or substitutions, of
residues of the
known amino acid sequence of a protein. The sites for mutation can be modified
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individually or in series, e.g., by (1) substituting first with conserved
amino acids and
then with more radical choices depending upon results achieved, (2) deleting
the target
residue, or (3) inserting residues of the same or a different class adjacent
to the located
site, or combinations of options 1-3.
-Alanine Scanning Mutagenesis
Alanine scanning mutagenesis is a useful method for identification of certain
residues or regions of the desired protein that are preferred locations or
domains for
mutagenesis, Cunningham and Wells (Science 244.:1081-1085, 1989) specifically
incorporated by reference. In alanine scanning, a residue or group of target
residues are
identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and
replaced by a
neutral or negatively charged amino acid (most preferably alanine or
polyalanine).
Replacement of an amino acid can affect the interaction of the amino acids
with the
surrounding aqueous environment in or outside the cell. Those domains
demonstrating
functional sensitivity to the substitutions can then be refined by introducing
further or
other variants at or for the sites of substitution. Thus, while the site for
introducing an
amino acid sequence variation is predetermined, the nature of the mutation per
se need
not be predetermined. For example, to optimize the performance of a mutation
at a
given site, alanine scanning or random mutagenesis may be conducted at the
target
codon or region and the expressed desired protein subunit variants are
screened for the
optimal combination of desired activity.
-Oligonucleotide-Mediated Mutagenesis
Oligonucleotide-mediated mutagenesis is a useful method for preparing
substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et
al., (DNA
2:183, 1983) incorporated herein by reference. Briefly, the desired DNA can be
altered
by hybridizing an oligonucleotide encoding a mutation to a DNA template, where
the
template is the single-stranded form of a plasmid or bacteriophage containing
the
unaltered or native DNA sequence of the desired protein. After hybridization,
a DNA
polymerase is used to synthesize an entire second complementary strand of the
template
that will thus incorporate the oligonucleotide primer, and will code for the
selected
alteration in the desired protein DNA. Generally, oligonucleotides of at least
25
nucleotides in length are used. An optimal oligonucleotide will have 12 to 15
nucleotides that are completely complementary to the template on either side
of the
nucleotides) coding for the mutation. This ensures that the oligonucleotide
will
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hybridize properly to the single-stranded DNA template molecule. The
oligonucleotides are readily synthesized using techniques known in the art
such as that
described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978])
incorporated
herein by reference.
-Cassette Mutagenesis
Another method for preparing variants, cassette mutagenesis, is based on the
technique described by Wells et al. (Gene, 34:315[1985]) incorporated herein
by
reference. The starting material can be a plasmid (or other vector) which
includes the
protein subunit DNA to be mutated. The codon(s) in the protein subunit DNA to
be
mutated are identified. There must be a unique restriction endonuclease site
on each
side of the identified mutation site(s). If no such restriction sites exist,
they may be
generated using the above-described oligonucleotide-mediated mutagenesis
method to
introduce them at appropriate locations in the desired protein subunit DNA.
After the
restriction sites have been introduced into the plasmid, the plasmid is cut at
these sites
to linearize it. A double-stranded oligonucleotide encoding the sequence of
the DNA
between the restriction sites but containing the desired mutations) is
synthesized using
standard procedures. The two strands are synthesized separately and then
hybridized
together using standard techniques. This double-stranded oligonucleotide is
referred to
as the cassette. This cassette is designed to have 3' and 5' ends that are
comparable with
the ends of the linearized plasmid, such that it can be directly ligated to
the plasmid.
This plasmid now contains the mutated desired protein subunit DNA sequence.
-Combinatorial Mutagenesis
Combinatorial mutagenesis can also be used to generate mutants. E.g., the
amino acid sequences for a group of homologs or other related proteins are
aligned,
preferably to promote the highest homology possible. All of the amino acids
which
appear at a given position of the aligned sequences can be selected to create
a
degenerate set of combinatorial sequences. The variegated library of variants
is
generated by combinatorial mutagenesis at the nucleic acid level, and is
encoded by a
variegated gene library. For example, a mixture of synthetic oligonucleotides
can be
enzymatically ligated into gene sequences such that the degenerate set of
potential
sequences are expressible as individual peptides, or alternatively, as a set
of larger
fusion proteins containing the set of degenerate sequences.
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Various techniques are known in the art for screening generated mutant gene
products. Techniques for screening large gene libraries often include cloning
the gene
library into replicable expression vectors, transforming appropriate cells
with the
resulting library of vectors, and expressing the genes under conditions in
which
detection of a desired activity, e.g., in this case, binding to Kay-ligand or
its receptor,
facilitates relatively easy isolation of the vector encoding the gene whose
product was
detected. Each of the techniques described below is amenable to high through-
put
analysis for screening large numbers of sequences created, e.g., by random
mutagenesis
techniques.
The invention also provides for reduction of the protein binding domains of
the
claimed polypeptides or their receptors, to generate mimetics, e.g. peptide or
non-
peptide agents. The peptide mimetics are able to disrupt binding of Kay-ligand
with its
receptor. The critical residues of the Kay-ligand involved in molecular
recognition of a
receptor polypeptide or of a downstream intracellular protein, can be
determined and
used to generate the Kay-ligand or its receptor-derived peptidomimetics which
competitively or noncompetitively inhibit binding of the Kay-ligand with a
receptor.
(see, for example, "Peptide inhibitors of human papilloma virus protein
binding to
retinoblastoma gene protein" European patent applications EP-412,762A and EP-
B31,080A), specifically incorporated herein by reference.
G. PHARMACEUTICAL COMPOSTTIONS
By making available purified and recombinant- Kay-ligands, the present
invention provides assays which can be used to screen for drug candidates
which are
either agonists or antagonists of the normal cellular function, in this case,
of Kay-
ligand, or its receptor. In one embodiment, the assay evaluates the ability of
a .
compound to modulate binding between the Kay-ligand and their receptors. A
variety
of assay formats will suffice and, in light of the present inventions, will be
comprehended by the skilled artisan.
In many drug screening programs which test libraries of compounds and natural
extracts, high throughput assays are desirable in order to maximize the number
of
compounds surveyed in a given period of time. Assays which are performed in
cell-free
systems, such as may be derived with purified or semi-purified proteins, are
often
preferred as "primary" screens in that they can be generated to permit rapid
development and relatively easy detection of an alteration in a molecular
target which is
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mediated by a test compound. Moreover, the effects of cellular toxicity and/or
bioavailability of the test compound can be generally ignored in the in vitro
system, the
assay instead being focused primarily on the effect of the drug on the
molecular target
as may be manifest in an alteration of binding affinity with other proteins or
change in
enzymatic properties of the molecular target.
Pharmaceutical compositions of the invention may comprise a therapeutically
effective amount of Kay-ligand, or its receptor, or fragments or mimetics
thereof, and,
optionally may include pharmaceutically acceptable carriers. Accordingly, this
invention provides methods for treatment of cancer, and methods of
stimulating, or in
certain instances, inhibiting the immune system, or parts thereof by
administering a
pharmaceutically effective amount of a compound of the invention or its
pharmaceutically acceptable salts or derivatives. It should of course by
understood that
the compositions and methods of this invention can be used in combination with
other
therapies for various treatments.
The compositions can be formulated for a variety of routes of administration,
including systemic, topical or localized administration. For systemic
administration,
injection is preferred, including intramuscular, intravenous, intraperitoneal,
and
subcutaneous for injection, the compositions of the invention can be
formulated in
liquid solutions, preferably in physiologically compatible buffers such as
Hank's
solution or Ringer's solution. In addition, the compositions may be formulated
in solid
form and, optionally, redissolved or suspended immediately prior to use.
Lyophilized
forms are also included in the invention.
The compositions can be administered orally, or by transmucosal or transdermal
means. For transmucosal or transdermal administration, penetrants appropriate
to the
barrier to be permeated are used in the formulation. Such penetrants are known
in the
art, and include, for example, for transmucosal administration, bile salts,
fusidic acid
derivatives, and detergents. Transmucosal administration may be through nasal
sprays
or using suppositories. For oral administration, the compositions are
formulated into
conventional oral administration forms such as capsules, tablets, and tonics.
For topical
administration, the compositions of the invention are formulated into
ointments, salves,
gels, or creams as known in the art.
Preferably the compositions of the invention will be in the form of a unit
dose
and will be administered one or more times a day. The amount of active
compound
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administered at one time or over the course of treatment will depend on many
factors.
For example, the age and size of the subject, the severity and course of the
disease
being treated, the manner and form of administration, and the judgments of the
treating
physician. However, an effective dose may be in the range of from about 0.005
to
about 5 mg/kg/day, preferably about 0.05 to about 0.5 mg/kg/day. One skilled
in the art
will recognize that lower and higher doses may also be useful.
Gene constructs according to the invention can also be used as a part of a
gene
therapy protocol to deliver nucleic acids encoding either an agonistic or
antagonistic
form of a Kay-ligand polypeptide.
Expression constructs of the claimed Kay-ligand can be administered in any
biologically effective carrier, e.g., any formulation or composition capable
of
effectively delivering the gene for the claimed Kay-ligand to cells in vivo.
Approaches
include insertion of the gene in viral vectors which can transfect cells
directly, or
delivering plasmid DNA with the help of, for example, liposomes, or
intracellular
carriers, as well as direct injection of the gene construct. Viral vector
transfer methods
are preferred.
A pharmaceutical preparation of the gene therapy construct can consist
essentially of the gene delivery system in an acceptable diluent, or can
comprise a slow
release matrix in which the gene delivery vehicle is imbedded. Alternatively,
where the
complete gene delivery system can be produced intact from recombinant cells,
e.g.
retroviral vectors, the pharmaceutical preparation can comprise one or more
cells which
produce the gene delivery system.
In addition to use in therapy, the oligomers of the invention may be used as
diagnostic reagents to detect the presence or absence of the target DNA, RNA
or amino
acid sequences to which they specifically bind. In other aspects, the claimed
invention
may be used to evaluate a chemical entity for its ability to interact with,
e.g., bind or
physically associate with the claimed Kay-ligand, or fragment thereof. The
method
includes contacting the chemical entity with the Kay-ligand, and evaluating
the ability
of the entity to interact with the Kay-ligand. Additionally, the Kay-ligand of
the
invention can be used in methods of evaluating naturally occurring Kay-ligand
or
receptors of the Kay-ligand, as well as to evaluate chemical entities which
associate or
bind with receptors of the Kay-ligand.
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In certain aspects, the claimed invention features a method for evaluating a
chemical entity for the ability to modulate the interaction between Kay-ligand
and its
receptor. The method includes combining a Kay-ligand receptor, and the Kay-
Iigand
under conditions wherein the pair is capable of interacting, adding the
chemical entity
to be evaluated and detecting the formation or dissolution of complexes. These
modulating agents may be further evaluated in vitro, e.g. by testing its
activity in a cell
free system, and then, optionally administering the compound to a cell or
animal, and
evaluating the effect.
H. EXAMPLES
c) Isolation of a receptor binding to the claimed Kay-ligand.
Ligands of the TNF family can be used to identify and clone receptors. With
the
described Kay-ligand sequences, one could fuse the 5' end of the extracellular
domain
of the Kay-ligand which constitutes the receptor binding sequence to a marker
or
tagging sequence and then add a leader sequence that will force secretion of
the Kay-
ligand in any of a number of expression systems. One example of this
technology is
described by Browning et al., (1996) (JBC 271, 8618-8626) where the LT-(3
ligand was
secreted in such a form. The VCAM leader sequence was coupled to a short myc
peptide tag followed by the extracellular domain of the LT-(3. The VCAM
sequence is
used to force secretion of the normally membrane bound LT-(3 molecule. The
secreted
protein retains a myc tag on the N-terminus which does not impair the ability
to bind to
a receptor. Such a secreted protein can be expressed in either transiently
transfected
Cos cells or a similar system, e.g., EBNA derived vectors, insect
cell/baculovirus,
picchia etc. The unpurified cell supernatant can be used as a source of the
tagged
ligand.
Cells expressing the receptor can be identified by exposing them to the tagged
ligand. Cells with bound ligand are identified in a FACS experiment by
labeling the
myc tag with an anti-myc peptide antibody (9E10) followed by phycoerythrin (or
a
similar label) labeled anti-mouse immunoglobulin. FACS positive cells can be
readily
identified and would serve as a source of RNA encoding for the receptor. An
expression library would then be prepared from this RNA via standard
techniques and
separated into pools. Pools of clones would be transfected into a suitable
host cell and
binding of the tagged ligand to receptor positive transfected cells determined
via
microscopic examination, following labeling of bound myc peptide tag with an
enzyme
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labeled anti-mouse Ig reagent, i.e. galactosidase, alkaline phosphatase or
luciferase
labeled antibody. Once a positive pool has been identified, the pool size
would be
reduced until the receptor encoding cDNA is identified. This procedure could
be
carried out with either the mouse of human Kay-ligand as one may more readily
lead to
a receptor.
It will be apparent to those skilled in the art that various modifications and
variations
can be made in the novel Kay-ligand, compositions and methods of the present
invention without departing from the spirit or scope of the invention. Thus,
it is
intended that the present invention cover the modifications and variations of
this
invention provided that they come within the scope of the appended claims and
their
equivalents.
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SEQ. ID. N0. 1
1 TGCCAAGCCCTGCCATGTAGTGCACGCAGGACATCAACAA
ACACAGATAA
51 CAGGAAATGATCCATTCCCTGTGGTCACTTATTCTAAAGGCCCCAACCTT
S 101 CAAAGTTCAAGTAGTGATATGGATGACTCCACAGAAAGGGAGCAGTCACG
151 CCTTACTTCTTGCCTTAAGAAAAGAGAAGAAATGAAACTGAAGGAGTGTG
201 TTTCCATCCTCCCACGGAAGGAAAGCCCCTCTGTCCGATCCTCCAAAGAC
251 GGAAAGCTGCTGGCTGCAACCTTGCTGCTGGCACTGCTGTCTTGCTGCCT
301 CACGGTGGTGTCTTTCTACCAGGTGGCCGCCCTGCAAGGGGACCTGGCCA
IO 351 GCCTCCGGGCAGAGCTGCAGGGCCACCACGCGGAGAAGCTGCCAGCAGGA
401 GCAGGAGCCCCCAAGGCCGGCCTGGAGGAAGCTCCAGCTGTCACCGCGGG
451 ACTGAAAATCTTTGAACCACCAGCTCCAGGAGAAGGCAACTCCAGTCAGA
501 ACAGCAGAAATAAGCGTGCCGTTCAGGGTCCAGAAGAAACAGTCACTCAA
551 GACTGCTTGCAACTGATTGCAGACAGTGAAACACCAACTATACAAAAAGG
IS 601 ATCTTACACATTTGTTCCATGGCTTCTCAGCTTTAAAAGGGGAAGTGCCC
651 TAGAAGAAAAAGAGAATAAAATATTGGTCAAAGAAACTGGTTACTTTTTT
701 ATATATGGTCAGGTTTTATATACTGATAAGACCTACGCCATGGGACATCT
751 AATTCAGAGGAAGAAGGTCCATGTCTTTGGGGATGAATTGAGTCTGGTGA
801 CTTTGTTTCGATGTATTCAAAATATGCCTGAAACACTACCCAATAATTCC
2O 851 TGCTATTCAGCTGGCATTGCAAAACTGGAAGAAGGAGATGAACTCCAACT
901 TGCAATACCAAGAGAAAATGCACAAATATCACTGGATGGAGATGTCACAT
951 TTTTTGGTGCATTGAAACTGCTGTGACCTACTTACACCATGTCTGTAGCT
1001 ATTTTCCTCCCTTTCTCTGTACCTCTAAGAAGAAAGAATCTAACTGAAAA
1051 TA
2S
SEQ. ID
NO. 2
1 MDDSTEREQSRLTSCLKKREEMKLKECVSILPRKESPSVRSSKDGKLLAA
51 TLLLALLSCCLTWSFYQVAALQGDLASLRAELQGHHAEKLPAGAGAPKA
101 GLEEAPAVTAGLKIFEPPAPGEGNSSQNSRNKRAVQGPEETVTQDCLQLI
3O 151 ADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVL
201 YTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSCYSAGI
251 AKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL
SEQ. ID
NO. 3
3S 1 GTGGTCACTTACTCCAAAGGCCTAGACCTTCAAAGTGCTCCTCGTGGAAT
51 GGATGAGTCTGCAAAGACCCTGCCACCACCGTGCCTCTGTTTTTGCTCCG
101 AGAAAGGAGAAGATATGAAAGTGGGATATGATCCCATCACTCCGCAGAAG
151 GAGGAGGGTGCCTGGTTTGGGATCTGCAGGGATGGAAGGCTGCTGGCTGC
201 TACCCTCCTGCTGGCCCTGTTGTCCAGCAGTTTCACAGCGATGTCCTTGT
4O 251 ACCAGTTGGCTGCCTTGCAAGCAGACCTGATGAACCTGCGCATGGAGCTG
301 CAGAGCTACCGAGGTTCAGCAACACCAGCCGCCGCGGGTGCTCCAGAGTT
351 GACCGCTGGAGTCAAACTCCTGACACCGGCAGCTCCTCGACCCCACAACT
401 CCAGCCGCGGCCACAGGAACAGACGCGCTTTCCAGGGACCAGAGGAAACA
451 GAACAAGATGTAGACCTCTCAGCTCCTCCTGCACCATGCCTGCCTGGATG
4S 501 CCGCCATTCTCAACATGATGATAATGGAATGAACCTCAGAAACAGAACTT
551 ACACATTTGTTCCATGGCTTCTCAGCTTTAAAAGAGGAAATGCCTTGGAG
601 GAGAAAGAGAACAAAATAGTGGTGAGGCAAACAGGCTATTTCTTCATCTA
651 CAGCCAGGTTCTATACACGGACCCCATCTTTGCTATGGGTCATGTCATCC
701 AGAGGAAGAAAGTACACGTCTTTGGGGACGAGCTGAGCCTGGTGACCCTG
SO 751 TTCCGATGTATTCAGAATATGCCCAAAACACTGCCCAACAATTCCTGCTA
801 CTCGGCTGGCATCGCGAGGCTGGAAGAAGGAGATGAGATTCAGCTTGCAA
851 TTCCTCGGGAGAATGCACAGATTTCACGCAACGGAGACGACACCTTCTTT
901 GGTGCCCTAAAACTGCTGTAACTCACTTGCTGGAGTGCGTGATCCCCTTC
951 CCTCGTCTTCTCTGTACCTCCGAGGGAGAAACAGACGACTGGAAAAACTA
SS 1001 AAAGATGGGGAAAGCCGTCAGCGAAAGTTTTCTCGTGACCCGTTGAATCT
1051 GATCCAAACC AGGAAATATA ACAGACAGCC ACAACCGAAG TGTGCCATGT
1101 GAGTTATGAG AAACGGAGCC CGCGCTCAGA AAGACCGGAT GAGGAAGACC
1151 GTTTTCTCCA GTCCTTTGCC AACACGCACC GCAACCTTGC TTTTTGCCTT
CA 02303424 2000-03-10
WO - - PCT/US98/19037
99/12964
1201 GGGTGACACATGTTCAGAATGCAGGGAGATTTCCTTGTTTTGCGATTTGC
1251 CATGAGAAGAGGGCCCACAACTGCAGGTCACTGAAGCATTCACGCTAAGT
1301 CTCAGGATTTACTCTCCCTTCTCATGCTAAGTACACACACGCTCTTTTCC
1351 AGGTAACTACTATGGGATACTATGGAAAGGTTGTTTGTTTTTAAATCTAG
S 1401 AAGTCTTGAACTGGCAATAGACAAAAATCCTTATAAATTCAAGTGTAAAA
1451 TAAACTTAATTAAAAAGGTTTAAGTGTG
SEQ. ID
N0. 4
1 MDESAKTLPPPCLCFCSEKGEDMKVGYDPITPQKEEGAWFGICRDGRLLA
lO51 ATLLLALLSSSFTAMSLYQLAALQADLMNLRMELQSYRGSATPAAAGAPE
101 LTAGVKLLTPAAPRPHNSSRGHRNRRAFQGPEETEQDVDLSAPPAPCLPG
151 CRHSQHDDNGMNLRNRTYTFVPWLLSFKRGNALEEKENKIVVRQTGYFFI
201 YSQVLYTDPIFAMGHVIQRKKVHVFGDELSLVTLFRCIQNMPKTLPNNSC
251 YSAGIARLEEGDEIQLAIPRENAQISRNGDDTFFGALKLL
15
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