U.S. patent application number 11/463227 was filed with the patent office on 2006-12-21 for modulators of tissue regeneration.
Invention is credited to Joseph V. Bonventre, Richard L. Cate, Catherine Hession, Takaharu Ichimura, Michele Sanicola-Nadel, Henry Wei.
Application Number | 20060286031 11/463227 |
Document ID | / |
Family ID | 26690881 |
Filed Date | 2006-12-21 |
United States Patent
Application |
20060286031 |
Kind Code |
A1 |
Sanicola-Nadel; Michele ; et
al. |
December 21, 2006 |
Modulators of tissue regeneration
Abstract
Proteins which are upregulated in injured or regenerating
tissues, as well as the DNA encoding these proteins, are disclosed,
as well as therapeutic compositions and methods of treatment
encompassing these compounds.
Inventors: |
Sanicola-Nadel; Michele;
(Winchester, MA) ; Bonventre; Joseph V.; (Wayland,
MA) ; Hession; Catherine; (Hingham, MA) ;
Ichimura; Takaharu; (South Burlington, VT) ; Wei;
Henry; (Ossining, NY) ; Cate; Richard L.;
(Weston, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
26690881 |
Appl. No.: |
11/463227 |
Filed: |
August 8, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10655506 |
Sep 4, 2003 |
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11463227 |
Aug 8, 2006 |
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09197970 |
Nov 23, 1998 |
6664385 |
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10655506 |
Sep 4, 2003 |
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PCT/US97/09303 |
May 23, 1997 |
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09197970 |
Nov 23, 1998 |
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60018228 |
May 24, 1996 |
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60023442 |
Aug 23, 1996 |
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Current U.S.
Class: |
424/1.49 ;
424/178.1; 435/320.1; 435/325; 435/328; 435/69.1; 435/7.2; 530/350;
530/388.22; 530/391.1; 536/23.4 |
Current CPC
Class: |
A61K 47/6849 20170801;
C07K 2319/30 20130101; A61P 13/12 20180101; A61K 38/00 20130101;
A61K 51/1027 20130101; C07K 2319/00 20130101; C07K 16/2803
20130101; G01N 33/5082 20130101; C07K 14/705 20130101; A61K 48/00
20130101; C07K 14/47 20130101 |
Class at
Publication: |
424/001.49 ;
536/023.4; 435/069.1; 435/320.1; 435/325; 530/350; 530/388.22;
530/391.1; 435/328; 435/007.2; 424/178.1 |
International
Class: |
A61K 51/00 20060101
A61K051/00; G01N 33/567 20060101 G01N033/567; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; A61K 39/395 20060101
A61K039/395; C07K 14/575 20060101 C07K014/575 |
Claims
1-20. (canceled)
21. An antibody or antigen-binding fragment thereof that (i) binds
specifically to the extracellular domain of a polypeptide whose
amino acid sequence consists of SEQ ID NO:7, and (ii) is conjugated
or fused to a toxin or radionuclide.
22-36. (canceled)
37. The antibody or antigen-binding fragment thereof of claim 21,
wherein the antibody is a humanized antibody.
38. The antibody or antigen-binding fragment thereof of claim 21,
wherein the antibody is a human antibody.
39. The antibody or antigen-binding fragment thereof of claim 21,
wherein the antibody is a monoclonal antibody.
40. The antibody or antigen-binding fragment thereof of claim 21,
wherein the antibody is a single chain antibody.
41. The antibody or antigen-binding fragment thereof of claim 21,
wherein the antibody or antigen-binding fragment thereof is a
chimeric antibody, an F.sub.ab fragment, an F.sub.(ab')2 fragment,
an F.sub.ab' fragment, or an F.sub.v fragment.
42. A method of targeting a toxin or radionuclide to a KIM-1
expressing tumor cell, the method comprising contacting a KIM-1
expressing tumor cell with the antibody or antigen-binding fragment
thereof of claim 21.
43. The method of claim 42, wherein the tumor cell is disposed in
vivo in a subject and the antibody or antigen-binding fragment
thereof is administered to the subject.
44. The method of claim 43, wherein the tumor cell is a renal tumor
cell.
45. A method of targeting a toxin or radionuclide to a KIM-1
expressing tumor cell, the method comprising contacting a KIM-1
expressing tumor cell with the antibody or antigen-binding fragment
thereof of claim 37.
46. The method of claim 45, wherein the tumor cell is disposed in
vivo in a subject and the antibody or antigen-binding fragment
thereof is administered to the subject.
47. The method of claim 46, wherein the tumor cell is a renal tumor
cell.
48. A method of targeting a toxin or radionuclide to a KIM-1
expressing tumor cell, the method comprising contacting a KIM-1
expressing tumor cell with the antibody or antigen-binding fragment
thereof of claim 38.
49. The method of claim 48, wherein the tumor cell is disposed in
vivo in a subject and the antibody or antigen-binding fragment
thereof is administered to the subject.
50. The method of claim 49, wherein the tumor cell is a renal tumor
cell.
51. A method of targeting a toxin or radionuclide to a KIM-1
expressing tumor cell, the method comprising contacting a KIM-1
expressing tumor cell with the antibody or antigen-binding fragment
thereof of claim 39.
52. The method of claim 51, wherein the tumor cell is disposed in
vivo in a subject and the antibody or antigen-binding fragment
thereof is administered to the subject.
53. The method of claim 52, wherein the tumor cell is a renal tumor
cell.
54. A method of targeting a toxin or radionuclide to a KIM-1
expressing tumor cell, the method comprising contacting a KIM-1
expressing tumor cell with the antibody or antigen-binding fragment
thereof of claim 40.
55. The method of claim 54, wherein the tumor cell is disposed in
vivo in a subject and the antibody or antigen-binding fragment
thereof is administered to the subject.
56. The method of claim 55, wherein the tumor cell is a renal tumor
cell.
57. A method of targeting a toxin or radionuclide to a KIM-1
expressing tumor cell, the method comprising contacting a KIM-1
expressing tumor cell with the antibody or antigen-binding fragment
thereof of claim 41.
58. The method of claim 57, wherein the tumor cell is disposed in
vivo in a subject and the antibody or antigen-binding fragment
thereof is administered to the subject.
59. The method of claim 58, wherein the tumor cell is a renal tumor
cell.
Description
RELATED APPLICATIONS
[0001] This is a continuation-in-part of PCT/US97/09393, filed on
May 23, 1997 as a continuation-in-part of U.S. Ser. No. 60/018,228,
filed on May 24, 1996 and of U.S. Ser. No. 60/023,442, filed on
Aug. 23, 1996. The entire disclosure of each of the aforesaid
patent applications are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to proteins which are upregulated in
injured or regenerating tissues, as well as to the DNA encoding
these proteins. The invention further relates to therapeutic
compositions and methods of treatment encompassing these
proteins.
BACKGROUND OF THE INVENTION
[0003] A dynamic remodeling of tissue architecture occurs during
development and during tissue repair after injury. Herein are
presented results of studies of this process, using a model of
kidney injury caused by an ischemia-reperfusion insult.
[0004] The kidney is able to repair damage to the proximal tubule
epithelium through a complex series of events involving cell death,
proliferation of surviving proximal tubule epithelial cells,
formation of poorly differentiated regenerative epithelium over the
denuded basement membrane, and differentiation of the regenerative
epithelium to form fully functional proximal tubule epithelial
cells (Wallin et al., Lab. Invest. 66:474-484, 1992; Witzgall et
al., Mol. Cell. Biol. 13:1933-1942, 1994; Ichimura et al., Am. J.
Physiol. 269:F653-662, 1995; Thadhani et al., N. Engl. J. Med.
334:1448-1460, 1996). Growth factors such as IGF, EGF, and HGF have
been implicated in this process of repair, as has the endothelial
cell adhesion molecule ICAM-1. However, the mechanisms by which the
tubular epithelial cells are restored are still not understood.
[0005] To identify molecules involved in processes of injury and
repair of the tubular epithelium, differences were analyzed in the
mRNA populations between injured/regenerating and normal kidneys
using representational difference analysis (RDA). RDA is a
PCR-based method for subtraction which yields target tissue or cell
specific cDNA fragments by repetitive subtraction and amplification
(Hubank and Schutz, Nucl. Acids Res. 22:5640-5648, 1994).
SUMMARY OF THE INVENTION
[0006] The invention generally provides Kidney Injury-related
Molecules (each of which is henceforth called a "KIM") which are
upregulated in renal tissue after injury to the kidney. The KIM
proteins and peptides of the invention, as well as their agonists
and antagonists, and their corresponding nucleic acids are useful
in a variety of therapeutic interventions.
[0007] The invention provides a purified and isolated DNA molecule
having a nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:2,
SEQ ID NO:4 or SEQ ID NO:6. The invention also includes the
complementary strands of these sequences, DNA molecules which
hybridize under stringent conditions to the aforementioned DNA
molecules, and DNA molecules which, but for the degeneracy of the
genetic code, would hybridize to any of the DNA molecules defined
above. These DNA molecules may be recombinant, and may be operably
linked to an expression control sequence.
[0008] The invention further provides a vector comprising a
purified and isolated DNA molecule having a nucleotide sequence set
forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, or
one of the other DNA molecules defined above. This vector may be a
biologically functional plasmid or viral DNA vector. One embodiment
of the invention provides a prokaryotic or eukaryotic host cell
stably transformed or transfected by a vector comprising a DNA
molecule of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.
In another embodiment of the invention, a process is provided for
the production of a KIM polypeptide product encoded by a DNA
molecule as described above; the process involves growing, under
suitable culture conditions, prokaryotic or eukaryotic host cells
transformed or transfected with the DNA molecule in a manner
allowing expression of the DNA molecule, and recovering the
polypeptide product of said expression.
[0009] A purified and isolated human KIM protein substantially free
of other human proteins is specifically within the invention, as is
a process for the production of a polypeptide product having part
or all of the primary structural conformation and the biological
activity of a KIM protein. KIM proteins of the invention may have
an amino acid sequence which comprises SEQ ID NO:3,SEQ ID NO:5, or
SEQ ID NO:7, or may be a variant of SEQ ID NO:3, SEQ ID NO:5 or SEQ
ID NO:7, or a purified and isolated protein encoded by the DNA of
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6. These
proteins can be provided substantially free of other human
proteins. The invention further includes variants of these
proteins, such as soluble variants or fusion proteins. KIM fusion
proteins of the invention may comprise an immunoglobulin, a toxin,
an imageable compound or a radionuclide.
[0010] The invention also provides a specific antibody, such as a
monoclonal antibody (MAb), to the KIM proteins described above. The
present antibody binds to any epitope that is unique to a KIM
protein disclosed herein. In some embodiments, the epitope is
displayed on the surface of a cell expressing KIM. Antigen-binding
fragments of the antibody are also provided herein, preferably Fab,
Fab2, Fab' and Fv fragments, whether produced by chemical or
enzymatic cleavage, or by molecular engineering techniques.
Engineered versions of the present antibody include chimeric,
humanized and human antibodies, and antibody fusion proteins.
Monoclonal antibodies (MAbs) of the present invention can be of
mouse, rat, hamster or human origin. An exemplary MAb of the
invention is the murine AKG7 MAb disclosed herein, which binds
specifically to the human KIM polypeptide disclosed herein.
Anti-KIM antibodies of the present invention may be conjugated or
fused to a therapeutic agent, toxin, imageable compound or
radionuclide. Exemplary therapeutic agents include cytokines,
lymphokines, trophic factors, survival factors, chemokines and
chemoattractants. Exemplary toxins include ricin and diphtheria
toxin. Exemplary imageable compounds include luminescent proteins
(e.g., luciferin), fluorescent proteins (e.g., green fluorescent
protein), haptens (e.g., biotin), and radioactively labeled
proteins. Exemplary radionuclides include any radionuclide used for
medical imaging purposes. The invention further encompasses all
hybridoma cell lines and engineered host cells which produce
antibodies of the invention.
[0011] Pharmaceutical compositions are also within the scope of the
invention. A pharmaceutical composition of the invention may
comprise a therapeutically effective amount of a KIM protein or
anti-KIM antibody of the invention, along with a pharmacologically
acceptable carrier.
[0012] Diagnostic methods are within the invention, such as
assessing the presence or course of resolution of renal injury by
measuring the concentration of KIM in urine, serum, or urine
sediment of patients who have or who are at risk of developing
renal disease. Other diagnostic methods that are within the
invention include assessing KIM expression level in kidney tissue
(e.g., in kidney biopsy tissue) of patients who have, are suspected
of having, or are at risk of developing renal cancer (e.g., renal
carcinoma). The present methods involve contacting an appropriate
tissue or fluid sample derived from the patient being diagnosed,
with a KIM antibody or a KIM probe (as the case may be), under
binding conditions. Complexes formed by the binding of antibody or
probe to KIM protein or nucleic acid (e.g., RNA) in the sample are
detected by standard techniques. The presence or abnormal elevation
of KIM protein in urine or serum is expected to correlate with
renal failure or renal disease. The presence or abnormal elevation
of KIM gene expression in renal cells or tissue is expected to
correlate with disease processes, particularly carcinogenesis. Such
correlations are expected to be useful in the prognostication,
staging and clinical management of diseases or other conditions
deleterious to renal tissue and/or renal function.
[0013] Methods of treatment of the invention include treating
patients with therapeutically effective amounts of KIM, KIM
variants, KIM analogs, KIM fusion proteins, KIM agonists, and
antibodies to KIM or to KIM ligands. Other therapeutic compounds of
the invention include KIM ligands, anti-KIM antibodies, and fusions
proteins of KIM ligands. These compounds can be useful in
therapeutic methods which either stimulate or inhibit cellular
responses that are dependent on KIM function.
[0014] Further methods of the invention inhibit the growth of
KIM-expressing tumor cells by contacting the cells with a fusion
protein of a KIM ligand and either a toxin or radionuclide, or with
an anti-KIM antibody conjugated to a toxin or to a radionuclide.
Likewise, growth of tumor cells which express KIM ligand may be
inhibited by contacting the cells with a fusion protein of a KIM
and either a toxin or radionuclide, or with an anti-KIM ligand
antibody conjugated to a toxin or to a radionuclide.
[0015] The invention also encompasses methods of gene therapy.
These include a method of treating a subject with a renal disorder,
a method of promoting growth of new tissue in a subject, and a
method of promoting survival of damaged tissue in a subject,
wherein each method comprises administering to the subject a vector
which includes DNA comprising the nucleotide sequence of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.
[0016] The compounds of the invention are also useful for imaging
tissues, either in vitro or in vivo. One such method involves
targeting an imageable compound to a cell expressing a protein of
SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, comprising contacting the
cell with either a monoclonal antibody of the invention or a fusion
protein comprising a protein as described above, fused to an
imageable compound. For in vivo methods, the cell is within a
subject, and the protein or the monoclonal antibody is administered
to the subject.
[0017] The invention also includes diagnostic methods, such as a
method of identifying damage or regeneration of renal cells in a
subject, comprising comparing the level of expression of either SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 in renal cells of
the subject to a control level of expression of the sequence in
control renal cells. Another method of the invention includes
identifying upregulation of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4
or SEQ ID NO:6 in cells comprising contacting the cells with an
antisense probe and measuring hybridization to RNA within the
cell.
[0018] A further embodiment of the diagnostic methods of the
invention includes assessing the presence or concentration of a
molecule of the invention either in urine, serum, or other body
fluids, or in urine sediment or tissue samples. The measured
injury-related molecule can be correlated with the presence, extent
or course of a pathologic process. This correlation can also be
used to assess the efficacy of a therapeutic regime.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a text depiction of the nucleotide sequence of rat
clone cDNA 3-2, with putative protein reading frame of 615 to
1535.
[0020] FIG. 2 is a text depiction of the cDNA sequence of rat clone
1-7, with putative protein reading frame of 145 to 1065.
[0021] FIG. 3 is a text depiction of the cDNA sequence of rat clone
4-7, with putative protein reading frame of 107 to 1822.
[0022] FIG. 4 is a text depiction of the cDNA and deduced amino
acid sequences of human clone HI3-10-85, with putative protein
reading frame of 1 to 1002. The upper line of the listing is the
cDNA sequence (SEQ ID NO:6), and the lower line is the deduced
amino acid sequence (SEQ ID NO:7).
[0023] FIG. 5 is a text depiction of a BESTFIT comparison of the
nucleotide-sequence of human clone HI3-10-85 with that of rat clone
3-2.
[0024] FIG. 6 is a digitized image of a Northern blot of human
renal tissue samples isolated from renal carcinomas and from normal
renal tissue samples.
DETAILED DESCRIPTION OF THE INVENTION
[0025] KIM genes were identified by analyzing differences in mRNA
expression between regenerating and normal kidneys using
representational difference analysis (RDA). RDA is a PCR-based
method for subtraction which yields target tissue or cell-specific
cDNA fragments by repetitive subtraction and amplification. The
cDNA representation from 48 hr postischemic adult rat kidney RNA is
subtracted with the sample from normal (sham-operated) adult rat
kidney. In this procedure, sequences which are common to both
postischemic and to normal kidney samples are removed, leaving
those sequences which are significantly expressed only in the
injured kidney tissue. Such genes encode proteins that may be
therapeutically beneficial for renal disorders or involved in the
injury process. Several clones have been obtained, sequenced and
characterized. The clones are then investigated for their
expression patterns during kidney repair, development and tissue
distribution by northern analysis and RNA in situ
hybridization.
[0026] Sequence Identification Numbers
[0027] Nucleotide and amino acid sequences referred to in the
specification have been given the following sequence identification
numbers: [0028] SEQ ID NO:1--nucleotide sequence of rat 3-2 cDNA
insert [0029] SEQ ID NO:2--nucleotide sequence of rat 1-7 cDNA
insert [0030] SEQ ID NO:3--amino acid sequence of rat KIM-1,
encoded by rat 3-2 and 1-7 cDNAs [0031] SEQ ID NO:4--nucleotide
sequence of rat 4-7 cDNA insert [0032] SEQ ID NO:5--amino acid
sequence encoded by 4-7 cDNA insert [0033] SEQ ID NO:6--nucleotide
sequence of human cDNA clone H13-10-85 [0034] SEQ ID NO:7--amino
acid sequence encoded by human cDNA clone H13-10-85
[0035] Definitions of Terms
[0036] A "KIM protein", herein used synonymously with "KIM", is a
protein encoded by mRNA which is selectively upregulated following
injury to a kidney. One group of KIM proteins of interest includes
those coded for by mRNA which is selectively upregulated at any
time within one week following any insult which results in injury
to renal tissue. Examples of times at which such upregulation might
be identified include 10 hours, 24 hours, 48 hours or 96 hours
following an insult. Examples of types of insults include those
resulting in ischemic, toxic or other types of injury.
[0037] A "KIM agonist" is a molecule which can specifically trigger
a cellular response normally triggered by the interaction of KIM
with a KIM ligand. A KIM agonist can be a KIM variant, or a
specific antibody to KIM, or a soluble form of the KIM ligand.
[0038] A "KIM antagonist" is a molecule which can specifically
associate with a KIM ligand or KIM, thereby blocking or otherwise
inhibiting KIM binding to the KIM ligand. The antagonist binding
blocks or inhibits cellular responses which would otherwise be
triggered by ligation of the KIM ligand with KIM or with a KIM
agonist. Examples of KIM antagonists include certain KIM variants,
KIM fusion proteins and specific antibodies to a KIM ligand or
KIM.
[0039] A "KIM ligand" is any molecule which noncovalently and
specifically binds to a KIM protein. Such a ligand can be a
protein, peptide, steroid, antibody, amino acid derivative, or
other type molecule, in any form, including naturally-occurring,
recombinantly produced, or otherwise synthetic. A KIM ligand can be
in any form, including soluble, membrane-bound, or part of a fusion
construct with immunoglobulin, fatty acid, or other moieties. The
KIM ligand may be an integrin. A membrane-bound KIM ligand can act
as a receptor which, when bound to or associated with KIM, triggers
a cellular response. In some interactions, KIM may associate with
more than a single KIM ligand, or may associate with a KIM ligand
as part of a complex with one or more other molecules or cofactors.
In a situation where both the KIM and the KIM ligand are bound to
cell membranes, the KIM may associate and react with KIM ligand
which is bound to the same cell as the KIM, or it may associate and
react with KIM ligand be bound to a second cell. Where the KIM
ligation occurs between molecules bound to different cells, the two
cells may be the same or different with respect to cellular type or
origin, phenotypic or metabolic condition, or type or degree of
cellular response (e.g., growth, differentiation or apoptosis) to a
given stimulus. "KIM ligation" refers to the contact and binding of
KIM with a KIM ligand.
[0040] By "alignment of sequences" is meant the positioning of one
sequence, either nucleotide or amino acid, with that of another, to
allow a comparison of the sequence of relevant portions of one with
that of the other. An example of one method of this procedure is
given in Needleman et al. (J. Mol. Biol. 48:443-453, 1970). The
method may be implemented conveniently by computer programs such as
the Align program (DNAstar, Inc.). As will be understood by those
skilled in the art, homologous or functionally equivalent sequences
include functionally equivalent arrangements of the cysteine
residues within the conserved cysteine skeleton, including amino
acid insertions or deletions which alter the linear arrangement of
these cysteines, but do not materially impair their relationship in
the folded structure of the protein. Therefore, internal gaps and
amino acid insertions in the candidate sequence are ignored for
purposes of calculating the level of amino acid sequence similarity
or identity between the candidate and reference sequences. One
characteristic frequently used in establishing the similarity of
proteins is the similarity of the number and location of the
cysteine residues between one protein and another.
[0041] "Antisense DNA" refers to the sequence of chromosomal DNA
that is transcribed.
[0042] An "antisense probe" is a probe which comprises at least a
portion of the antisense DNA for a nucleic acid portion of
interest.
[0043] By "cloning" is meant the use of in vitro recombination
techniques to insert a particular gene or other DNA sequence into a
vector molecule. In order to successfully clone a desired gene, it
is necessary to employ methods for generating DNA fragments, for
joining the fragments to vector molecules, for introducing the
composite DNA molecule into a host cell in which it can replicate,
and for selecting the clone having the target gene from amongst the
recipient host cells.
[0044] By "cDNA" is meant complementary or copy DNA produced from
an RNA template by the action of RNA-dependent DNA polymerase
(reverse transcriptase). Thus a "cDNA clone" means a duplex DNA
sequence complementary to an RNA molecule of interest, carried in a
cloning vector.
[0045] By "cDNA library" is meant a collection of recombinant DNA
molecules containing cDNA inserts which together comprise a
representation of the mRNA molecules present in an entire organism
or tissue, depending on the source of the RNA templates. Such a
cDNA library may be prepared by methods known to those of skill,
and described, for example, in Maniatis et al., Molecular Cloning:
A Laboratory Manual, supra. Generally, RNA is first isolated from
the cells of an organism from whose genome it is desired to clone a
particular gene. Preferred for the purposes of the present
invention are mammalian, and particularly human, cell lines.
Alternatively, RNA may be isolated from a tumor cell, derived from
an animal tumor, and preferably from a human tumor. Thus, a library
may be prepared from, for example, a human adrenal tumor, but any
tumor may be used.
[0046] As used herein, the term "DNA polymorphism" refers to the
condition in which two or more different nucleotide sequences can
exist at a particular site in DNA.
[0047] "Expression vector" includes vectors which are capable of
expressing DNA sequences contained therein, i.e., the coding
sequences are operably linked to other sequences capable of
effecting their expression. It is implied, although not always
explicitly stated, that these expression vectors must be replicable
in the host organisms either as episomes or as an integral part of
the chromosomal DNA. A useful, but not a necessary, element of an
effective expression vector is a marker encoding sequence, which is
a sequence encoding a protein which results in a phenotypic
property (such as tetracycline resistance) of the cells containing
the protein which permits those cells to be readily identified. In
sum, "expression vector" is given a functional definition, and any
DNA sequence which is capable of effecting expression of a
specified contained DNA code is included in this term, as it is
applied to the specified sequence. As at present, such vectors are
frequently in the form of plasmids, thus "plasmid" and "expression
vector" are often used interchangeably. However, the invention is
intended to include such other forms of expression vectors which
serve equivalent functions and which may, from time to time become
known in the art.
[0048] By "functional derivative" is meant the "fragments",
"variants", "analogs", or "chemical derivatives" of a molecule. A
"fragment" of a molecule, such as any of the antigens of the
present invention is meant to refer to any polypeptide subset of
the molecule. A "variant" of such molecules is meant to refer to a
naturally occurring molecule substantially similar to either the
entire molecule, or a fragment thereof. An "analog" of a molecule
is meant to refer to a non-natural molecule substantially similar
to either the entire molecule or a fragment thereof.
[0049] The term "gene" means a polynucleotide sequence encoding a
peptide.
[0050] By "homogeneous" is meant, when referring to a peptide or
DNA sequence, that the primary molecular structure (i.e., the
sequence of amino acids or nucleotides) of substantially all
molecules present in the composition under consideration is
identical.
[0051] "Isolated" refers to a protein of the present invention, or
any gene encoding any such protein, which is essentially free of
other proteins or genes, respectively, or of other contaminants
with which it might normally be found in nature, and as such exists
in a form not found in nature.
[0052] The term "label" refers to a molecular moiety capable of
detection including, by way of example, without limitation,
radioactive isotopes, enzymes, luminescent agents, and dyes.
[0053] The term "probe" refers to a ligand of known qualities
capable of selectively binding to a target antiligand. As applied
to nucleic acids, the term "probe" refers to a strand of nucleic
acid having a base sequence complementary to a target strand.
[0054] "Recombinant host cells" refers to cells which have been
transformed with vectors constructed using recombinant DNA
techniques. As defined herein, the antibody or modification thereof
produced by a recombinant host cell is by virtue of this
transformation, rather than in such lesser amounts, or more
commonly, in such less than detectable amounts, as would be
produced by the untransformed host.
[0055] By "substantially pure" is meant any protein of the present
invention, or any gene encoding any such protein, which is
essentially free of other proteins or genes, respectively, or of
other contaminants with which it might normally be found in nature,
and as such exists in a form not found in nature.
[0056] A molecule is said to be "substantially similar" to another
molecule if the sequence of amino acids in both molecules is
substantially the same, and if both molecules possess a similar
biological activity. Thus, provided that two molecules possess a
similar activity, they are considered variants as that term is used
herein even if one of the molecules contains additional amino acid
residues not found in the other, or if the sequence of amino acid
residues is not identical. As used herein, a molecule is said to be
a "chemical derivative" of another molecule when it contains
additional chemical moieties not normally a part of the molecule.
Such moieties may improve the molecule's solubility, absorption,
biological half life, etc. The moieties may alternatively decrease
the toxicity of the molecule, eliminate or attenuate any
undesirable side effect of the molecule, etc. Moieties capable of
mediating such effects are disclosed, for example, in Remington's
Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa.
(1980).
[0057] By "vector" is meant a DNA molecule, derived from a plasmid
or bacteriophage, into which fragments of DNA may be inserted or
cloned. A vector will contain one or more unique restriction sites,
and may be capable of autonomous replication in a defined host or
vehicle organism such that the cloned sequence is reproducible.
[0058] Compounds of the Invention
[0059] The invention includes the cDNA of SEQ ID NO: 1, SEQ ID
NO:2, SEQ ID NO:4 or SEQ ID NO:6, as well as sequences which
include the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, or
SEQ ID NO:6, and derivatives of these sequences. The invention also
includes vectors, liposomes and other carrier vehicles which
encompass these sequence or derivatives of them. The invention
further includes proteins transcribed from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:4, or SEQ ID NO:6, including but not limited to SEQ
ID NO:3, SEQ ID NO:5 or SEQ ID NO:7, and their derivatives and
variants.
[0060] One embodiment of the invention includes soluble variants of
a KIM protein that is usually synthesized as a membrane associated
protein, and which is upregulated after injury. Soluble variants
lack at least a portion of the transmembrane or intra-membrane
section of a native KIM protein. In some examples, the soluble
variant lacks the entire transmembrane or intra-membrane section of
a native KIM protein. The present soluble variants can be derived
from intact or native KIM proteins by any suitable means, including
chemical or enzymatic cleavage, or through molecular engineering
techniques such by expression of a truncated KIM nucleic acid.
Thus, the present soluble variants should be understood to be
derivatives of an intact KIM. Soluble variants include fusion
proteins which encompass derivatives of KIM proteins that lack at
least a portion of the transmembrane or intra-membrane section of a
native KIM protein. All types of KIM fusion proteins are included,
particularly those which incorporate his-tag, Ig-tag, and myc-tag
forms of the molecule. These KIM fusions may have characteristics
which are therapeutically advantageous, such as the increased
half-life conferred by the Ig-tag. Also included are fusion
proteins which incorporate portions of selected domains of the KIM
protein.
[0061] Variants can differ from naturally occurring KIM protein in
amino acid sequence or in ways that do not involve sequence, or
both. Variants in amino acid sequence are produced when one or more
amino acids in naturally occurring KIM protein is substituted with
a different natural amino acid, an amino acid derivative or
non-native amino acid. Particularly preferred variants include
naturally occurring KIM protein, or biologically active fragments
of naturally occurring KIM protein, whose sequences differ from the
wild type sequence by one or more conservative amino acid
substitutions, which typically have minimal influence on the
secondary structure and hydrophobic nature of the protein or
peptide. Variants may also have sequences which differ by one or
more non-conservative amino acid substitutions, deletions or
insertions which do not abolish the KIM protein biological
activity. Conservative substitutions typically include the
substitution of one amino acid for another with similar
characteristics such as substitutions within the following groups:
valine, glycine; glycine, alanine; valine, isoleucine; aspartic
acid, glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine. The non-polar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan and methionine. The
polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine, asparagine and glutamine. The positively
charged (basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic acid
and glutamic acid.
[0062] Other conservative substitutions can be taken from the table
below, and yet others are those which meet the criteria for an
accepted point mutation in Dayhoff et al. (1978), 5 Atlas of
Protein Sequence and Structure, Suppl. 3, ch. 22, Natl. Biomed.
Res. Found., Washington, D.C. 20007, the teachings of which are
incorporated by reference herein. TABLE-US-00001 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, homo-Arg, D-homo-Arg, Met, D-Met, Ile, 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, Beta-Ala,
Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine
L D-Leu, Val, D-Val, Met, D-Met 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, Ile, D-Ile, Leu, D-Leu, Val, D-Val, Norleu
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 5 phenylproline Proline P
D-Pro, L-I-thioazolidine-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), Val, D-Val 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
[0063] Other variants within the invention are those with
modifications which increase peptide stability. Such variants may
contain, for example, one or more non-peptide bonds (which replace
the peptide bonds) in the peptide sequence. Also included are:
variants that include residues other than naturally occurring
L-amino acids, such as D-amino acids or non-naturally occurring or
synthetic amino acids such as beta or gamma amino acids and cyclic
variants. Incorporation of D- instead of L-amino acids into the
polypeptide may increase its resistance to proteases. See, e.g.,
U.S. Pat. No. 5,219,990.
[0064] Generally, substitutions that may be expected to induce
changes in the functional properties of KIM polypeptides are those
in which: (I) a hydrophilic residue, e.g., serine or threonine, is
substituted by a hydrophobic residue, e.g., leucine, isoleucine,
phenylalanine, or alanine; (ii) a cysteine residue is substituted
for (or by) any other residue; (iii) a residue having an
electropositive side chain, e.g., lysine, arginine or histidine, is
substituted for (or by) a residue having an electronegative charge,
e.g., glutamic acid or aspartic acid; or (iv) a residue having a
bulky side chain, e.g., phenylalanine, is substituted for (or by)
one not having such a side chain, e.g., glycine.
[0065] The peptides of this invention may also be modified by
various changes such as insertions, deletions and substitutions,
either conservative or nonconservative where such changes might
provide for certain advantages in their use. Splice variants are
specifically included in the invention.
[0066] In other embodiments, variants with amino acid substitutions
which are less conservative may also result in desired derivatives,
e.g., by causing changes in charge, conformation and other
biological properties. Such substitutions would include for
example, substitution of hydrophilic residue for a hydrophobic
residue, substitution of a cysteine or proline for another residue,
substitution of a residue having a small side chain for a residue
having a bulky side chain or substitution of a residue having a net
positive charge for a residue having a net negative charge. When
the result of a given substitution cannot be predicted with
certainty, the derivatives may be readily assayed according to the
methods disclosed herein to determine the presence or absence of
the desired characteristics.
[0067] In accordance with the foregoing, the scope of the invention
includes proteins and peptides with variant amino acid sequences
(sequence variants) having at least eighty percent similarity with
a KIM protein. More preferably the sequence similarity is at least
ninety percent, or at least ninety-five percent. For example, a
sequence variant within the scope of the invention is any which
shares at least eighty percent similarity with a KIM selected from
rat KIM1 (SEQ ID NO:3) and human KIM1(SEQ ID NO:7). The percentage
of similarity between a variant sequence and a reference sequence
(such as SEQ ID NO:3 or SEQ ID NO:7) is determined after the
variant is aligned with the reference, preferably according to the
method of Needelman et al. (1970), 48 J. Mol. Biol. 443-454, or
according to an appropriate sequence alignment program (discussed
more fully below). The percentage of sequence "identity" of the
variant to the reference is the percentage of amino acid or nucleic
acid residues of the variant sequence that are identical to the
aligned (corresponding) residues of the reference. The percentage
of sequence "similarity" is the sum of the percentage of identical
corresponding residues and the percentage of variant residues that
are conservative substitutions for the aligned (corresponding)
reference residues.
[0068] A number of algorithms and computer software programs can be
utilized to determine whether a sequence of interest is a KIM
sequence variant within the scope of this invention. One such is
the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci.
USA 87:2264-68, as modified in Karlin and Altschul (1993), Proc.
Natl. Acad. Sci. USA 90:5873-77. This algorithm is incorporated
into the NBLAST and XBLAST programs of Altschul et al. (1990) J.
Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed
with NBLAST. BLAST protein searches can be performed with XBLAST.
To obtain gapped alignments, gapped BLAST can be utilized as
described in Altschul et al. (1997) Nucl. Acids Res.
25(17):3389-3402. All of the foregoing are available through
http:\\www.ncbi.nlm.nih.gov. Another suitable algorithm is that of
Myers and Miller, CABIOS (1989), which is incorporated into the
ALIGN program (version 2.0), which is a part of the Wisconsin
Sequence Analysis package of the Genetics Computing Group (GCG).
Thus, variants within the invention include any polypeptide or
nucleic acid sequence which is identified through BLAST or CABIOS
analysis as being related to a KIM nucleic acid or polypeptide
sequence disclosed herein (e.g., rat KIM1, SEQ ID NO:3 or human
KIM1, SEQ ID NO:7). "Related" sequences are those for which, in the
judgement of an ordinarily skilled practitioner, the results of
sequence analysis indicate a biologically significant relationship
with the reference sequence (e.g., rat KIM1 or human KIM1).
[0069] Preferably, sequence variants of the KIM proteins disclosed
herein are those having an amino acid sequence which is at least
forty (40) percent identical overall (i.e., when aligned) to a
disclosed KIM polypeptide sequence, and sharing at least 80% of the
aligned KIM cysteine residues. Particularly preferred sequence
variants are those which meet the foregoing analysis criteria when
analyzed, relative to a KIM reference sequence disclosed herein
(e.g., rat KIM1, SEQ ID NO:3 or human KIM1, SEQ ID NO:7), using the
program GAP, which is a part of the Wisconsin analysis package of
the GCG. To run a GAP analysis, the following program parameters
should be used: the gap creation penalty (gap weight) should be set
to 3.0, and the gap extension penalty (gap length) should be set to
0.1. GAP uses the algorithm of Needelman et al. (1970), 48 J. Mol.
Biol. 443453, to create an alignment with the largest number of
matched (aligned identical) residues and the fewest number of
sequence gaps.
[0070] Just as it is possible to replace substituents of the
scaffold, it is also possible to substitute functional groups which
are bound to the scaffold with groups characterized by similar
features. These substitutions will initially be conservative, i.e.,
the replacement group will have approximately the same size, shape,
hydrophobicity and charge as the original group. Non-sequence
modifications may include, for example, in vivo or in vitro
chemical derivatization of portions of naturally occurring KIM
protein, as well as changes in acetylation, methylation,
phosphorylation, carboxylation or glycosylation.
[0071] Also included within the invention are agents which
specifically bind to the protein, or a fragment of the protein (SEQ
ID NO:3, 5 or 7). These agents include ligands and antibodies
(including monoclonal, single chain, double chain, Fab fragments,
and others, whether native, human, humanized, primatized, or
chimeric). Additional descriptions of these categories of agents
are in PCT application 95/16709, the specification of which is
herein incorporated by reference.
[0072] Experimental Procedures
[0073] 1. Generation of RNA from Ischemic and Normal Rat Adult
Kidneys
[0074] Ischemic injured rat kidneys are generated as described by
Witzgall et al. (J. Clin Invest. 93: 2175-2188, 1994). Briefly, the
renal artery and vein from one kidney of an adult Sprague-Dawley
rat are clamped for 40 minutes and then reperfused. Injured kidneys
are harvested from the rats at 24 hours and at 48 hours after
reperfusion. Kidneys from sham-operated, normal adult
Sprague-Dawley rats are also harvested.
[0075] Total RNA is prepared from the organs based on the protocol
by Glisin et al. (Biochemistry 13: 2633, 1974). Briefly, the
harvested organs are placed immediately into GNC buffer (4M
guanidine thiocyanate, 0.5% SDS, 25 mM sodium. citrate, 0.1% Sigma
anti foam) and disrupted on ice with a polytron. Cell debris is
removed with a low speed spin in a clinical centrifuge and the
supernatant fluid is placed on a 5.7 M CsCl, 25 mM sodium acetate,
1 mM EDTA cushion. RNA is pelleted through the cushion in a SW40Ti
rotor at 22K for 15 hrs. RNA is resuspended in sterile DEPC-treated
water, precipitated twice with 1/10 volume 3M sodium acetate and
2.5 volumes of EtOH. Poly A+ RNA is isolated using an mRNA
purification kit (Pharmacia, catalog No.27-9258-02).
[0076] 2. Representational Difference Analysis (RDA) Method to
Isolate 1-7, 3-2 and 4-7 RDA Fragments
[0077] Double stranded cDNA is synthesized from sham-operated and
from 48 hr post-ischemic kidney poly A+ RNA using Gibco BRL
"Superscript Choice.TM. System cDNA Synthesis Kit", catalog No.
18090. First strand is synthesized by priming with oligo dT and
using Superscript II.TM. reverse transcriptase. Second strand is
generated using E. coli DNA polymerase I and RNase H followed by T4
DNA polymerase using BRL recommended conditions.
[0078] RDA analysis is performed essentially as described by Hubank
and Schatz (Nucleic Acid Research 22: 5640-48, 1994). Briefly, 48
hr post-ischemic kidney cDNA is digested with the restriction
enzyme Dpn II, and ligated to R-Bgl-12/24 oligonucleotides (see
reference for exact sequence). PCR amplification (performed with
Perkin-Elmer Taq polymerase and their corresponding PCR buffer) of
the linker ligated cDNA is used to generate the initial
representation. This PCR product is designated "tester amplicon."
The same procedure is used to generate "driver amplicon" from
sham-operated rat kidney cDNA.
[0079] Hybridization of tester and driver amplicons followed by
selective amplification are performed three times to generate
Differential Product One (DP1), Two (DP2) and Three (DP3).
Generation of the DP1 product is performed as described by Hubank
and Schatz (Nucleic Acid Research 22: 56400-48, 1994). The DP2 and
DP3 products are also generated as described by Hubank and Schatz
(id.), except that the driver:tester ratios are changed to 5,333:1
for DP2 and to 40,000:1 or 4,000:1. for DP3.
[0080] Three RDA products are cloned from DP3 into the cloning
vector pUC 18: RDA product 1-7 (252 bp) when the DP3 was generated
using a ratio of 40,000:1, and product RDA 3-2 (445 bp) and 4-7
(483 bp) when the DP3 was generated using a ratio of 4,000:1. The
DNA fragments;are subcloned using the Pharmacia Sureclone.TM. kit
(catalog No. 27-9300-01) to repair the ends of the PCR fragments
with Klenow enzyme and to facilitate blunt end ligation of the
fragments into the pUC18 vector.
[0081] 3. Northern Analysis
[0082] Poly A+ RNA (2.5 .mu.g) from rat normal adult kidney (sham
operated), from 48 hr post-ischemic injured adult kidney, and from
day 18 embryonic kidney is electrophoresed and Northern blotted
(Cate, Cell 45:685, 1986) to a GeneScreen.TM. membrane (Dupont).
Hybridization in PSB buffer (50 mM Tris 7.5, 1M NaCl, 0.1% Na
pyrophosphate, 0.2% PVP, 0.2% Ficoll, 0.2% BSA, 1% SDS), containing
10% dextran sulphate and 100 .mu.g/ml tRNA, is performed at 65 C
using three different probes: 1-7 RDA product, 3-2 RDA product and
4-7 RDA product. All are radiolabeled using Pharmacia's "Ready to
GoT.TM." random priming labeling kit (catalog No.27-9251-01). RDA
products 1-7, 3-2 and 4-7 hybridize to mRNAs present in all three
samples, but most intensely to mRNAs in the 48 hr post-ischemic
kidney RNA samples.
[0083] A Northern blot analysis of adult rat tissues indicates that
the 1-7 gene is expressed at very low levels in normal adult
kidney, testis, spleen and lung. The 3-2 gene is expressed in
liver, kidney, spleen, and brain. The 4-7 gene is expressed in
spleen, kidney, lung, testis, heart, brain, liver, and skeletal
muscle. The presence of different sized mRNAs in some tissues in
the 1-7 and 3-2 blot indicates that the primary transcription
product of the 1-7 gene and of the 3-2 gene may undergo alternate
splicing and/or polyadenylation.
[0084] 4. Isolation of 3-2 and 4-7 cDNA Clones
[0085] A cDNA library is generated from 4 .mu.g of polyA+ RNA from
48 hr post-ischemic injured kidney using reagents from BRL
Superscript Choice.TM. System for cDNA synthesis, and
Stratagene.TM. Lambda ZapII cloning kit (catalog No. 236201),
according to protocols recommended by the manufacturers.
[0086] 10.sup.5 clones are screened with the 3-2 RDA product as a
probe (random primed labeled as described above). Eight positive
clones are selected and four are randomly chosen for secondary
analysis to obtain pure phage plaques. After tertiary screening,
four pure phage clones are isolated. Cloned inserts from the phage
are isolated by in vivo excision procedure according to
Stratagene.TM. Lambda Zap II kit. The largest insert, of
approximately 2.6 kb (referred to as cDNA clone 3-2), is subjected
to DNA sequencing. The sequence of the insert (SEQ ID NO:1) is
shown in FIG. 1. cDNA clone 3-2 (E. coli K-12, SOLR/p3-2#5-1) has
been deposited as ATCC No.98061. The sequence of cDNA clone 3-2 is
identical to that of clone 1-7 cDNA (SEQ ID NO:2), except that
nucleotides 136-605 of SEQ ID NO:1 represent an insertion. Thus,
SEQ ID NO:2 represents a splice variant form of SEQ ID NO:1. The
clone for 1-7 (E. coli K-12, SOLR/p1-7#3-1) has been deposited as
ATCC No. 98060.
[0087] 10.sup.5 clones are screened with the 1-7 RDA product as a
probe (random primed radiolabeled as described above). Eight
positive clones are selected and four are randomly chosen for
secondary analysis to obtain pure phage plaques. After tertiary
screening, four pure phage clones are isolated. Cloned inserts from
the phage are isolated by in vivo excision procedure according to
Stratagene.TM. Lambda Zap II kit. The largest insert of
approximately 2.0 kb (referred to as cDNA clone 1-7) is subjected
to DNA sequencing; the sequence of the insert (SEQ ID NO: 2) is
shown in FIG. 2.
[0088] 10.sup.5 clones are screened with the 4-7 RDA product as a
probe (random primed labeled as described above and hybridized in
PSB at 65 C). Eight positive clones are selected and four are
randomly chosen for secondary analysis to obtain pure phage
plaques. After secondary screening, two pure phage clones are
isolated. Cloned inserts from the phage are isolated by in vivo
excision procedure according to Stratagene.TM. Lambda Zap II kit.
The largest insert, approximately 2.4 kb (referred to as cDNA clone
4-7), is subjected to DNA sequencing. The sequence of the insert,
SEQ ID NO: 4, is shown in FIG. 3. The cDNA clone 4-7 (E. coli K-12,
SOLR/p4-7#1-1) has been deposited as ATCC No. 98062.
[0089] 5. Characterization of the 1-7, 3-2 and 4-7 cDNA Clones
[0090] A.) DNA and Protein Sequences:
[0091] The sequence of 3-2 cDNA (FIG. 1;.SEQ ID NO:1) contains an
open reading frame of 307 amino acids (FIG. 1; SEQ ID NO:3). A
sequence of 21 amino acids is inferred from Von Heijne analysis
(Von Heijne et al., Nucl. Acid Res. 14:14683 (1986)), and a
transmembrane region spanning approximately aa 235-257 indicates
that the 3-2 product is a cell surface protein. The sequence of 1-7
cDNA (FIG. 2; SEQ ID NO:2) contains an open reading frame of 307
amino acids, which is identical to the open reading frame contained
in the 3-2 cDNA (SEQ ID NO: 3). This 307 amino acid polypeptide is
herein designated rat KIM1. The sequence of 4-7 cDNA (FIG. 3; SEQ
ID NO:4) contains an open reading frame of 572 amino acids (SEQ ID
NO:5). A transmembrane region is located at approximately amino
acids. 501-521. This 572 amino acid polypeptide is related to the
polypeptide known as nmb. Weterman et al. (1995) 60 Int. J. Cancer
73-81.
[0092] B.) In Situ Analysis of 1-7, 3-2 and 4-7 mRNAs in
Contralateral and in Post-Ischemic Adult Rat Kidneys:
[0093] In situ hybridization is carried out according to the method
described by Finch et al., Dev. Dynamics 203: 223-240, 1995.
Briefly, both ischemic and contralateral kidneys are perfusion
fixed with 4% paraformaldehyde in PBS. Kidneys are further fixed
overnight at 4 C and processed. Paraffin sections are
deparaffinized and rehydrated, fixed with 4% paraformaldehyde in
PBS, digested with proteinase K, refixed, then acetylated with
acetic anhydride in triethanolamine buffer. Sections are then
dehydrated and hybridized with .sup.32P-labeled riboprobes at
55.degree. C. overnight, with 33 P-labeled riboprobes generated
from 3-2 RDA or 1-7 RDA products subcloned into BamH1 site of
pGEM-11Z. After hybridization, sections were washed under high
stringency conditions (2.times.SSC, 50% formamide at 65.degree.
C.). Sections are finally dehydrated, emulsion (NBT-2) coated for
autoradiography, and exposed for at least a week. Silver grains are
developed and sections are counterstained with toluidine blue and
microphotographed.
[0094] Analysis of 1-7 and 3-2 mRNA expression by in situ
hybridization indicates that these genes are greatly upregulated in
damaged kidney cells compared to their expression in normal kidney
sections. The expression seen is in regenerative cells of the
cortex and outer medulla, most of which appear to be proximal
tubule cells.
[0095] Analysis of the 4-7 in situ RNA expression pattern also
reveals abundant expression of this gene in the injured ischemic
kidney compared to the normal adult kidney. The site of expression
appears to be infiltrating cells.
[0096] 6.) Isolation of a Human cDNA Clone Which Cross Hybridizes
to the Rat 3-2 cDNA
[0097] A .sup.32P-labeled DNA probe comprising nucleotides 546-969
of the insert of clone 3-2 shown in FIG. 1 is generated and used to
screen a human embryonic liver lambda gt10 cDNA library (Clontech
Catalog #HL5003a). 1.times.10.sup.6 plaques are screened in
duplicate using standard conditions as described above but
temperature for screening was 55 C. For the high stringency wash,
the filters are washed in 2.times.SSC at 55 C. Fifty positive phage
are identified and plaque purified, and DNA is prepared. The phage
DNAs are subjected to Southern analysis using the same probe as
above. The Southern blot filter is subjected to a final wash with
0.5.times.SSC at 55 C. Two clones are identified as positive. The
insert of clone H13-10-85 is sequenced and a region is found that
encodes a protein with a high level of identity to the 3-2 protein
shown in FIG. 3.
[0098] The nucleotide sequence (SEQ ID NO:6) and predicted amino
acid sequence (SEQ ID NO:7) of the human 3-2 related protein are
shown in FIG. 4. As shown by the bestfit analysis depicted in FIG.
5, the human 3-2 related protein is 43.8% identical and 59.1%
similar to the rat 3-2 protein. Both contain IgG, mucin,
transmembrane, and cytoplasmic domains. The six cysteines within
the IgG domains of both proteins are conserved. Accordingly, the
polypeptide of SEQ ID NO:7 is designated human KIM1 (or KIM-1)
herein. It is believed that the mammalian genome (e.g., the human
genome) includes genes encoding a family of KIM-1 related proteins.
Thus, the human KIM-1 disclosed herein is more precisely designated
human KIM-1a.
[0099] 7) Production of KIM-1 Ig Fusion Protein
[0100] A fusion protein of the extracellular domain of KIM and the
Fc region of immunoglobulin (Ig) is a useful tool for the study of
the molecular and cellular biology of the injured/regenerating
kidney and as a therapeutic molecule. To produce KIM Ig fusion
protein with the extracellular domain of human and rat KIM-1
protein, a fragment of the extracellular domain of KIM-1 cDNA was
amplified by PCR and cloned in the Biogen expression vector,
pCA125, for transient expression in COS cells. The expression
vector pCA125 produces a fusion protein which has a structure from
gene cloned at N-terminus and a human Ig Fc region at the
C-terminus. COS cells were transfected with the plasmids SJR 103 or
104; these plasmids express a fusion protein which contains the
human KIM sequences 263-1147 (SEQ ID NO:6; SJR 103) or rat KIM
sequences 599-1319 (SEQ ID NO:1; SJR 104) of the extracellular
domain fused to human Ig Fc region. The cells were grown in 10% FBS
in DMEM in the cell factory (Nunc, Naperville, Ill.). Two to three
days post-transfection, medium was harvested, concentrated using
Amicon concentrator, and fusion protein was purified using
Protein-A Sepharose column. After purification, purity of fusion
protein was evaluated by SDS-PAGE.
[0101] 8) Production of Antibodies, Including Monoclonal Antibody
(MAb) AKG7, with Binding Specificity for Human KIM Protein
[0102] Antibody production involves administration of one or more
immunogenic doses of a KIM polypeptide preparation (whether
isolated, or as part of a cell suspension, fraction or extract) to
an appropriate non-human animal, such as a mouse, rat, rabbit,
gunea pig, turkey, goat, sheep, pig or horse. To enhance
immunogenicity, the preparation is emulsified with a conventional
adjuvant (e.g., Complete Freund's Adjuvant, CFA). Serum
immunoglobulins (Ig), using peripheral blood samples withdrawn at
intervals (e.g., weekly) after initial or a subsequent
immunization, are monitored to detect the onset and/or maturation
of a humoral immune response to KIM. Any conventional technique,
e.g., ELISA, RIA, Western blotting, or the like, can be used to
detect and/or quantitate (titer) the KIM-specific Ig. High titer
immune sera are preferred for use as anti-KIM polyclonal antibodies
herein. High titer sera (e.g., having a titer of at least 1:1000)
can be harvested in bulk for purification of anti-KIM Ig,
using-conventional immunoaffinity chromatography (e.g., using a
Protein A resin).
[0103] Animals (e.g., mice, rats, guinea pigs, hamsters) having a
high serum titer of anti-KIM Ig also can be used for the
conventional production of monoclonal antibodies (MAb) with binding
specificity for KIM. Splenocytes are isolated from such animals and
are fused with a myeloma cell line according to standard techniques
to produce hybridomas. Such hybridomas are cloned by limiting
dilution, fluorescence-activated cell sorting, or any other
suitable cell isolation technique, to produce monoclonal hybridoma
cell lines of the present invention. Hybridoma lines of the present
invention are useful for the production of anti-KIM MAbs, and also
as sources from which nucleic acids encoding the present anti-KIM
MAbs can be routinely isolated. Such nucleic acids, encoding heavy
and/or light chain polypeptides of the present MAbs, can be used to
produce recombinant, engineered versions of the present MAbs,
including truncated MAb polypeptides, chimeric or humanized MAbs,
and MAb fusion proteins.
[0104] By way of illustration, mice have been immunized with a
human KIM1-Ig fusion protein as disclosed herein, which was
immobilized on Protein A Sepharose beads and emulsified in CFA. The
fusion protein included the extracellular domain of human KIM1
(amino acids 1 to 291) fused to a truncated human IgG1 polypeptide
comprising the hinge-C2-C3 domains of the IgG1 heavy chain
polypeptide. The onset and maturation of a humoral immune response
was monitored using a conventional ELISA, in which another Ig
fusion protein was used as a blocking agent to subtract serum Ig's
having undesired specificity for the IgG1 portion of the KIM1
fusion protein. Following conventional isolation of immune
splenocytes, fusion, ELISA screening, and cloning by limiting
dilution, ten MAbs were obtained with desirable binding specificity
for human KIM1. Among these, one, designated AKG7, is preferred
herein. The AKG7 antibody is useful in ELISA, Western blot and
immunohistochemistry analysis of human KIM1 polypeptides. The
present technique can be repeated and/or adapted routinely to
produce any number of MAbs to domains (e.g., the extracellular
domain) of KIM proteins, that have the KIM-specific binding
properties of MAb AKG7 and are useful in the analytical, diagnostic
and therapeutic methods described herein.
[0105] MAbs of the present invention, such as AKG7, are obtained
from cell culture media conditioned by the present hybridoma lines,
or from ascites fluid, or from media conditioned by an engineered
host cell expressing a heavy and/or light chain nucleic acid
construct encoding an anti-KIM MAb of the present invention. Such
MAbs are purified using conventional immunoaffinity chromatography,
and used in the various diagnostic, analytical and therapeutic
methods described herein.
[0106] Diagnostic Uses of the Compounds of the Invention
[0107] Anti-KIM antibodies of the invention, which specifically
bind to the protein of SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 or a
fragment thereof, are useful in several diagnostic methods. These
agents may be labeled with detectable markers, such as
fluoroscopically or radiographically opaque substances, and
administered to a subject to allow imaging of tissues which express
KIM protein. The agents may also be bound to substances, such as
horseradish peroxidase, which can be used as immunocytochemical
stains to allow visualization of areas of KIM protein-positive
cells on histological sections. A specific antibody could be used
alone in this manner, and sites where it is bound can be visualized
in a sandwich assay using an anti-immunoglobulin antibody which is
itself bound to a detectable marker.
[0108] For example and not by way of limitation, the AKG7 MAb can
be used to visualize cells producing KIM-1 protein in
immunohistochemistry analysis of human renal tissue samples.
Immunohistochemistry is carried out on paraformaldehyde-fixed
paraffin sections by deparaffinizing the sections, then ablating
endogenous peroxidase activity by incubation in 2% hydrogen
peroxide in methanol for 20 min. Sections are then heated in a
microwave oven in 0.1 M citrate buffer, pH 6.0, for 10 min., then
blocked with diluted goat serum (1:67) overnight at 4.degree. C.
Thereafter, sections are incubated with affinity purified AKG7 or
control IgG at a concentration of 5 .mu.g/mL. After 1 hr, the
sections are washed in PBS and incubated with biotinylated goat
anti-mouse IgG for 30 min. After further washes with PBS, the
sections are incubated with avidin-biotinylated horseradish
peroxidase complex (Vectastain Elite ABC kit, Vector Laboratories)
for 1 hr. Sections are washed in PBS and developed by the addition
of 50 mM sodium phosphate buffer, pH 7.6, containing 0.12%
3,3-diaminobenzidine tetrahydrochloride(Sigma), 0.0075% nickel
chloride, 0.0075%.cobalt chloride, and 0.0075% hydrogenperoxide for
2-5 min., producing a dark brown stain indicating immunoreactivity.
Sections are then counterstained with 0.01% toluidine blue. This
technique has been used to visualize KIM-1 immunoreactivity in
paraffin sections from two different human patients with renal cell
carcinoma. Sections comprising tumor tissue and adjacent normal
renal tissue were assessed. In patients, strong KIM-1
immunoreactivity was noted in tumor tissue. By contract,
immunoreactivity was virtually absent in adjacent normal tissue (as
judged by pathology). Immunoreactivity was also virtually absent in
normal control renal tissue sections. These data are consistent
with the utility of antibodies to KIM-1 as reagents for the
diagnosis, prognostication and/or staging of renal cancers.
[0109] Specific antibodies to KIM protein are also useful in
immunoassays to measure KIM presence or concentration in samples of
body tissues and fluids. Such concentrations may be correlated with
different disease states. As an embodiment of particular interest,
the invention includes a method of diagnosing renal injury, or of
monitoring a process of renal repair, by measuring the
concentration of KIM or of KIM fragments in the urine, plasma or
serum of a patient. Similarly, KIM can be measured in urine
sediment, in particular in cellular debris in the urine sediment.
Casts of renal tubule cells, which may be present in urine sediment
from patients with ongoing renal disease, may contain elevated
levels of KIM protein and mRNA.
[0110] Specific antibodies to KIM protein may also be bound to
solid supports, such as beads or dishes, and used to remove the
ligand from a solution, either for measurement, or for purification
and characterization of the protein or its attributes (such as
posttranslational modifications). Such characterization of a
patient's KIM protein might be useful in identifying deleterious
mutants or processing defects which interfere with KIM function and
are associated with abnormal patient phenotypes. Each of these
techniques is routine to those of skill in the immunological
arts.
[0111] Additional imaging methods utilize KIM or KIM fragments,
fused to imageable moieties, for diagnostic imaging of tissues that
express KIM ligands, particularly tumors.
[0112] Further diagnostic techniques are based on demonstration of
upregulated KIM mRNA in tissues, as an indication of injury-related
processes. This technique has been tested and found workable in a
model of isohemic injury in rats, as follows.
[0113] To determine if the amount of KIM-1 protein is increased
after injury, kidney homogenates of contralateral and postischemic
kidneys were examined 24 and 48 hours following a 40 minute
clamping of the renal artery and vein of a single kidney for each
rat. The kidney homogenate was assessed for the presence of KIM-1
protein. Western blot analysis identifies three proteins detected
by two different antibodies after ischemic injury, which are not
detectable in homogenates from contralateral-kidneys which were not
exposed to ischemic injury. The apparent molecular weights of the
bands are approximately 40 kDa, 50 kDa and 70-80 kDa. The three
protein species detected by western blotting could represent
glycosylated forms of the same protein given the presence of
potential N and O linked glycosylation sites. The fact that each of
these proteins react with two different sets of polyclonal
antibodies supports the idea that they are related to KIM-1 and are
not cross-reacting bands. Confirmation of this prediction came from
the results of partial CNBr cleavage of the three proteins which
revealed they shared common CNBr cleavage fragments. Since the
cytoplasmic domain of the KIM-1 protein is not predicted to contain
any major post-translational modifications, the two smallest
products of the digest (4.7 kDa and 7.4 kDa) detected with
antibodies directed against the cytoplasmic domain of KIM-1 should
be the same size for the three different KIM-1 protein bands if
they originate from the same protein. The KIM1 40 kDa and 70-80 kDa
proteins indeed produced fragments migrating at the predicted size.
Digest of the 50 kDa protein band gave also the same C-terminal
signature band peptide.
[0114] The KIM-1 sequence presents two putative sites for
N-glycosylation and a mucin domain where O-glycosylation could
cover-the polypeptide chain. The three KIM-1 bands detected in
postischemic kidney could correspond to glycosylation variants of
the same core protein. De-N-glycosylation with PNGase F resulted in
a shift of all three bands to a lower molecular weight,
corresponding to a loss of about 3 kDa, indicating that all three
proteins are N-glycosylated. Differences in O-glycosylation might
explain the differences in sizes of these three bands.
[0115] Further studies are expected to corroborate the correlation
between KIM-1 expression and disease status of renal tissue. For
example and not by way of limitation, Northern blot analysis can be
carried out to establish levels of KIM-1 expression in normal and
cancerous renal tissue samples. Poly(A)+ RNA is obtained from
tissue samples of interest (e.g., kidney biopsy or necropsy
samples, or control samples), size fractionated by electrophoresis,
and transferred to a GeneScreen membrane (NEN Life Science
Products). Hybridization with a KIM-1 probe corresponding to part
or all of the open reading frame of the KIM-1 gene of interest
(e.g., human KIM-1, SEQ ID NO:6) is carried out in plaque screening
buffer (PSB, 50 mM Tris, pH 7.5, 1 M NaCl, 0.1% sodium
pyrophosphate, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine
serum albumin, 1% SDS) containing 10% dextran sulfate and 100
.mu.g/mL tRNA at 65.degree. C. The KIM-1 probe is preferably
detectably labeled, e.g., radiolabelled. The membrane is then
washed to remove unbound probe, and KIM-1 mRNA is visualized using
standard membrane development techniques. Thereafter, the KIM-1
probe is stripped from the blot, and the blot is rehybridized with
a control probe, such as a .beta.-actin probe.
[0116] The results of an exemplary Northern blot analysis are set
forth in FIG. 6: biopsy samples containing histologically normal
renal tissue were compared to renal tumor tissues (renal cell
carcinoma) obtained from four different patients afflicted with
renal cancers. KIM-1 expression was virtually undetectable in three
of the four normal samples and was weakly detectable in the fourth.
In contract, two of the four renal cancer samples displayed strong
KIM-1 expression. These results are consistent with the utility of
KIM-1 probes as reagents for the diagnosis, prognostication or
staging of renal cancers. Without being limited hereby, it is
believed that aberrant KIM-1 gene expression may correlate with the
degree of neoplastic transformation, agressiveness or metastatic
propensity of a given renal cell carcinoma. Thus, assessment of
KIM-1 gene expression is believed to provide information useful in
conjunction with other, conventional diagnostic and/or clinical
criteria for characterizing renal cancers.
[0117] Therapeutic Uses of the Compounds of the Invention
[0118] The therapeutic methods of the invention involve selectively
promoting or inhibiting cellular responses that are dependent on
KIM ligation. Where the KIM and the KIM ligand are both membrane
bound, and expressed by different cells, the signal transduction
may occur in the KIM-expressing cell, in the KIM ligand-expressing
cell, or in both.
[0119] KIM ligation-triggered response in a KIM ligand-expressing
cell may be generated by contacting the cell with exogenous KIM,
KIM fusion proteins or activating antibodies against KIM ligand,
either in vitro or in vivo. Further, responses of the KIM
ligand-expressing cell that would otherwise be triggered by
endogenous KIM could be blocked by contacting the KIM
ligand-expressing cell with a KIM ligand antagonist (e.g., an
antagonist antibody that binds to KIM ligand), or by contacting the
endogenous KIM with an anti-KIM antibody or other KIM-binding
molecule which prevents the effective ligation of KIM with a KIM
ligand.
[0120] Similarly, the responses triggered by KIM ligation in the
KIM-expressing cell may be promoted or inhibited with exogenous
compounds. For example, KIM ligation-triggered response in a
KIM-expressing cell may be generated by contacting the cell with a
soluble KIM ligand, or certain anti-KIM activating antibodies.
Further, responses of the KIM-expressing cell that would otherwise
be triggered by interaction with endogenous KIM ligand could be
blocked by contacting the KIM-expressing cell with an antagonist to
KIM (e.g., a blocking antibody that binds to KIM in a manner that
prevents effective, signal-generating KIM ligation), or by
contacting the endogenous KIM ligand with an anti-KIM ligand
antibody or other KIM ligand-binding molecule which prevents the
effective ligation of KIM with the KIM ligand.
[0121] Which of the interventions described above are useful for
particular therapeutic uses depend on the relevant etiologic
mechanism of either the pathologic process to be inhibited, or of
the medically desirable process to be promoted, as is apparent to
those of skill in the medical arts. For example, where KIM ligation
results in desirable cellular growth, maintenance of differentiated
phenotype, resistance to apoptosis induced by various insults, or
other medically advantageous responses, one of the above-described
interventions that promote ligation-triggered response may be
employed. In the alternative, one of the inhibitory interventions
may be useful where KIM ligation invokes undesirable consequences,
such as neoplastic growth, deleterious loss of cellular function,
susceptibility to apoptosis, or promotion of inflammation
events.
[0122] Following are examples of the previously described
therapeutic methods of the invention. One therapeutic use of the
KIM-related compounds of the invention is for treating a subject
with renal disease, promoting growth of new tissue in a subject, or
promoting survival of damaged tissue in a subject, and includes the
step of administering to the subject a therapeutically effective
amount of a KIM protein of the invention, or of a pharmaceutical
composition which includes a protein of the invention. The protein
used in these methods may be a fragment of a full-length KIM
protein, a soluble KIM ligand protein or fusion fragment, or a KIM
agonist. These methods may also be practiced by administering to
the subject a therapeutically effective amount of an agonist
antibody of the invention, or a pharmaceutical composition which
includes an agonist antibody of the invention. A KIM protein may be
administered concurrently with a therapeutically effective amount
of a second compound which exerts a medically desirable adjunct
effect. While tissues of interest for these methods may include any
tissue, preferred tissues include renal tissue, liver, neural
tissue, heart, stomach, small intestine, spinal cord, or lung.
Particular renal conditions which may be beneficially treated with
the compounds of the invention include acute renal failure, acute
nephritis, chronic renal failure, nephrotic syndrome, renal tubule
defects, kidney transplants, toxic injury, hypoxic injury, and
trauma. Renal tubule defects include those of either hereditary or
acquired nature, such as polycystic renal disease, medullary cystic
disease, and medullary sponge kidney. This list is not limited, and
may include many other renal disorders (see, e.g., Harrison's
Principles of Internal Medicine, 13th ed., 1994, which is herein
incorporated by reference.) The subject of the methods may be
human.
[0123] A therapeutic intervention for inhibiting growth of
undesirable, KIM ligand-expressing tissue in a subject includes the
step of administering to the subject a therapeutically effective
amount of a KIM antagonist (e.g., an antagonist antibody that binds
to KIM ligand), or by administering a therapeutically effective
amount of an anti-KIM antibody or other KIM-binding molecule which
blocks KIM binding to the KIM ligand-expressing tissue. In an
embodiment of interest, the KIM antagonist or anti-KIM antibody may
be used therapeutically to inhibit or block growth of tumors which
depend on KIM protein for growth.
[0124] Other methods of the invention include killing KIM
ligand-expressing tumor cells, or inhibiting their growth, by
contacting the cells with a fusion protein of a KIM and a toxin or
radionuclide, or an anti-KIM ligand antibody conjugated to a toxin
or radionuclide. The cell may be within a subject, and the protein
or the conjugated antibody is administered to the subject.
[0125] Also encompassed within the invention is a method for
targeting a toxin or radionuclide to a cell expressing a KIM,
comprising contacting the cell with a fusion protein comprising a
KIM ligand and a toxin or radionuclide, or an anti-KIM antibody
conjugated to a toxin or radionuclide. Another embodiment includes
the method of suppressing growth of a tumor cell which expresses
KIM, comprising contacting the cell with a fusion protein of KIM
ligand and a toxin or radionuclide or with an anti-KIM antibody
conjugated to a toxin or radionuclide; the cell may be within a
subject, and the protein administered to the subject.
[0126] The term "subject" used herein is taken to mean any mammal
to which KIM may be administered. Subjects specifically intended
for treatment with the method of the invention include humans, as
well as nonhuman primates, sheep, horses, cattle, goats, pigs,
dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice
as well as the organs, tumors, and cells derived or originating
from these hosts.
[0127] Use of Compounds of the Invention in Gene Therapy
[0128] The KIM genes of the invention are introduced into damaged
tissue, or into tissue where stimulated growth is desirable. Such
gene therapy stimulates production of KIM protein by the
transfected cells, promoting cell growth and/or survival of cells
that express the KIM protein.
[0129] In a specific embodiment of a gene therapy method, a gene
coding for a KIM protein may be introduced into a renal target
tissue. The KIM protein would be stably expressed and stimulate
tissue growth, division, or differentiation, or could potentiate
cell survival. Furthermore, a KIM gene may be introduced into a
target cell using a variety of well-known methods that use either
viral or non-viral based strategies.
[0130] Non-viral methods include electroporation, membrane fusion
with liposomes, high velocity bombardment with DNA-coated
microprojectiles, incubation with calcium-phosphate-DNA
precipitate, DEAE-dextran mediated transfection, and direct
micro-injection into single cells. For instance, a KIM gene may be
introduced into a cell by calcium phosphate coprecipitation
(Pillicer et al., Science, 209: 1414-1422 (1980); mechanical
microinjection and/or particle acceleration (Anderson et al., Proc.
Nat. Acad. Sci. USA, 77: 5399-5403 (1980); liposome based DNA
transfer (e.g., LIPOFECTIN-mediated transfection-Fefgner et al.,
Proc. Nat. Acad. Sci., USA, 84: 471-477, 1987; Gao and Huang,
Biochim. Biophys. Res. Comm., 179: 280-285,1991; DEAE
Dextran-mediated transfection; electroporation (U.S. Pat. No.
4,956,288); or polylysine-based methods in which DNA is conjugated
to deliver DNA preferentially to liver hepatocytes (Wolff et al.,
Science, 247: 465-468, 1990; Curiel et al., Human Gene Therapy 3:
147-154, 1992).
[0131] Target cells may be transfected with the genes of the
invention by direct gene transfer. See, e.g., Wolff et al., "Direct
Gene Transfer Into Moose Muscle In Vivo", Science 247:1465-68,
1990. In many cases, vector-mediated transfection will be
desirable. Any of the methods known in the art for the insertion of
polynucleotide sequences into a vector may be used. (See, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; and
Ausubel et al., Current Protocols in Molecular Biology, J. Wiley
& Sons, NY, 1992, both of which are incorporated herein by
reference.) Promoter activation may be tissue specific or inducible
by a metabolic product or administered substance. Such
promoters/enhancers include, but are not limited to, the native
c-ret ligand protein promoter, the cytomegalovirus immediate-early
promoter/enhancer (Karasuyama et al., J. Exp. Med., 169: 13, 1989);
the human beta-actin promoter (Gunning et al., Proc. Nat. Acad.
Sci. USA, 84: 4831, 1987; the glucocorticoid-inducible promoter
present in the mouse mammary tumor virus long terminal repeat (MMTV
LTR) (Klessig et al., Mol. Cell. Biol., 4: 1354, 1984); the long
terminal repeat sequences of Moloney murine leukemia virus (MuLV
LTR) (Weiss et al., RNA Tumor Viruses, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1985); the SV40 early region
promoter (Bernoist and Chambon, Nature, 290:304, 981); the promoter
of the Rous sarcoma virus (RSV) (Yamamoto et al., Cell, 22:787,
1980); the herpes simplex virus (HSV) thymidine kinase promoter
(Wagner et al., Proc. Nat. Acad. Sci. USA, 78: 1441, 1981); the
adenovirus promoter (Yamada et al., Proc. Nat. Acad. Sci. USA, 82:
3567, 1985).
[0132] The KIM genes may also be introduced by specific viral
vectors for use in gene transfer systems which are now well
established. See for example: Madzak et al., J. Gen. Virol., 73:
1533-36, 1992 (papovavirus SV40); Berkner et al., Curr. Top.
Microbiol. Immunol., 158: 39-61, 1992 (adenovirus); Hofmann et al.,
Proc. Natl. Acad. Sci. 92: 10099-10103, 1995 (baculovirus); Moss et
al., Curr. Top. Microbiol. Immunol., 158: 25-38, 1992 (vaccinia
virus); Muzyczka, Curr. Top. Microbiol. Immunol., 158: 97-123, 1992
(adeno-associated virus); Margulskee, Curr. Top. Microbiol.
Immunol., 158: 67-93, 1992 (herpes simplex virus (HSV) and
Epstein-Barr virus (HBV)); Miller, Curr. Top. Microbiol. Immunol.,
158: 1-24, 1992 (retrovirus); Brandyopadhyay et al., Mol. Cell.
Biol., 4, 749-754, 1984 (retrovirus); Miller et al., Nature, 357:
455-450, 1992 (retrovirus); Anderson, Science, 256: 808-813, 1992
(retrovirus), Current Protocols in Molecular Biology: Sections
9.10-9.14 (Ausubel et al., Eds.), Greene Publishing Associcates,
1989, all of which are incorporated herein by reference.
[0133] Preferred vectors are DNA viruses that include adenoviruses
(preferably Ad-2 or Ad-5 based vectors), baculovirus, herpes
viruses (preferably herpes simplex virus based vectors), and
parvoviruses (preferably "defective" or non-autonomous parvovirus
based vectors, more preferably adeno-associated virus based
vectors, most preferably AAV-2 based vectors). See, e.g., Ali et
al., Gene Therapy 1: 367-384, 1994; U.S. Pat. Nos. 4,797,368 and
5,399,346 and discussion below.
[0134] The choice of a particular vector system for transferring,
for instance, a KIM sequence will depend on a variety of factors.
One important factor is the nature of the target cell population.
Although retroviral vectors have been extensively studied and used
in a number of gene therapy applications, they are generally
unsuited for infecting cells that are not dividing but may be
useful in cancer therapy since they only integrate and express
their genes in replicating cells. They are useful for ex vivo
approaches and are attractive in this regard due to their stable
integration into the target cell genome.
[0135] Adenoviruses are eukaryotic DNA viruses that can be modified
to efficiently deliver a therapeutic or reporter transgene to a
variety of cell types. The general adenoviruses types 2 and S (Ad2
and Ad5, respectively), which cause respiratory disease in humans,
are currently being developed for gene therapy of Duchenne Muscular
Dystrophy (DMD)and Cystic Fibrosis (CF). Both Ad2 and Ad5 belong to
a subclass of adenovirus that are not associated with human
malignancies. Adenovirus vectors are capable of providing extremely
high levels of transgene delivery to virtually all cell types,
regardless of the mitotic state. High titers (10.sup.13 plaque
forming units/ml) of recombinant virus can be easily generated in
293 cells (an adenovirus-transformed, complementation human
embryonic kidney cell line: ATCC CRL1573) and cryo-stored for
extended periods without appreciable losses. The efficacy of this
system in delivering a therapeutic transgene in vivo that
complements a genetic imbalance has been demonstrated in animal
models of various disorders. See Watanabe, Atherosclerosis, 36:
261-268, 1986; Tanzawa et al., FEBS Letters 118(1):81-84, 1980;
Golasten et al., New Engl. J. Med. 309:288-296, 1983; Ishibashi et
al., J. Clin. Invest. 92: 883-893, 1993; and Ishibashi et al., J.
Clin. Invest. 93: 1889-1893, 1994, all of which are incorporated
herein by reference. Indeed, recombinant replication defective
adenovirus encoding a cDNA for the cystic fibrosis transmembrane
regulator (CFTR) has been approved for use in at least two human CF
clinical trials. See, e.g., Wilson, Nature 365:691-692, 1993.
Further support of the safety of recombinant adenoviruses for gene
therapy is the extensive experience of live adenovirus vaccines in
human populations.
[0136] The first-generation recombinant, replication-deficient
adenoviruses which have been developed for gene therapy of DMD and
other inherited disorders contain deletions of the entire E1a and
part of the E1b regions. This replication-defective virus is grown
in 293 cells containing a functional adenovirus E1a gene which
provides a transacting E1a protein. E1-deleted viruses are capable
of replicating and producing infectious virus in the 293 cells,
which provide E1a and E1b region gene products in trans. The
resulting virus is capable of infecting many cell types and can
express the introduced gene (providing it carries its own
promoter), but cannot replicate in a cell that does not carry the
E1 region DNA unless the cell is infected at a very high
multiplicity of infection. Adenoviruses have the advantage that
they have a broad host range, can infect quiescent or terminally
differentiated cells such as neurons, and appear essentially
non-oncogenic. Adenoviruses do not appear to integrate into the
host genome. Because they exist extrachromasomally, the risk of
insertional mutagenesis is greatly reduced. Ali et al., supra, at
373. Recombinant adenoviruses (rAdV) produce very high titers, the
viral particles are moderately stable, expression levels are high,
and a wide range of cells can be infected. Their natural host cells
are airway epithelium, so they are useful for therapy of lung
cancers.
[0137] Baculovirus-mediated transfer has several advantages.
Baculoviral gene transfer can occur in replicating and
nonreplicating cells, and can occur in renal cells, as well as in
hepatocytes, neural cells, spleen, skin, and muscle. Baculovirus is
non-replicating and nonpathogenic in mammalian cells. Humans lack
pre-existing antibodies to recombinant baculovirus which could
block infection. In addition, baculovirus is capable of
incorporating and transducing very large DNA inserts.
[0138] Adeno-associated viruses (AAV) have also been employed as
vectors for somatic gene therapy. AAV is a small, single-stranded
(ss) DNA virus with a simple genomic organization (4-7 kb) that
makes it an ideal substrate for genetic engineering. Two open
reading frames encode a series of rep and cap polypeptides. Rep
polypeptides (rep78, rep68, rep 62 and rep 40) are involved in
replication, rescue and integration of the AAV genome. The cap
proteins (VP1, VP2 and VP3) form the virion capsid. Flanking the
rep and cap open reading frames at the 5' and 3' ends are 145 bp
inverted terminal repeats (ITRs), the first 125 bp of which are
capable of forming Y- or T-shaped duplex structures. Of importance
for the development of AAV vectors, the entire rep and cap domains
can be excised and replaced with a therapeutic or reporter
transgene. See B. J. Carter, in Handbook of Parvoviruses, ed., P.
Tijsser, CRC Press, pp. 155-168 (1990). It has been shown that the
ITRs represent the minimal sequence required for replication,
rescue, packaging, and integration of the AAV genome.
[0139] Adeno-associated viruses (AAV) have significant potential in
gene therapy. The viral particles are very stable and recombinant
AAVs (rAAV)have "drug-like" characteristics in that rAAV can be
purified by pelleting or by CsCl gradient banding. They are heat
stable and can be lyophilized to a powder and rehydrated to full
activity. Their DNA stably integrates into host chromosomes so
expression is long-term. Their host range is broad and AAV causes
no known disease so that the recombinant vectors are non-toxic.
[0140] Once introduced into a target cell, sequences of interest
can be identified by conventional methods such as nucleic acid
hybridization using probes comprising sequences that are
homologous/complementary to the inserted gene sequences of the
vector. In another approach, the sequence(s) may be identified by
the presence or absence of a "marker" gene function (e.g, thymidine
kinase activity, antibiotic resistance, and the like) caused by
introduction of the expression vector into the target cell.
[0141] Formulations and Administration
[0142] The compounds of the invention are formulated according to
standard practice, such as prepared in a carrier vehicle. The term
"pharmacologically acceptable carrier" means one or more organic or
inorganic ingredients, natural or synthetic, with which the mutant
proto-oncogene or mutant onco protein is combined to facilitate its
application. A suitable carrier includes sterile saline although
other aqueous and non-aqueous isotonic sterile solutions and
sterile suspensions known to be pharmaceutically acceptable are
known to those of ordinary skill in the art. In this regard, the
term "carrier" encompasses liposomes and the HIV-1 tat protein (See
Chen et al., Anal. Biochem. 227: 168-175, 1995) as well as any
plasmid and viral expression vectors.
[0143] Any of the novel polypeptides of this invention may be used
in the form of a pharmaceutically acceptable salt. Suitable acids
and bases which are capable of forming salts with the polypeptides
of the present invention are well known to those of skill in the
art, and include inorganic and organic acids and bases.
[0144] A compound of the invention is administered to a subject in
a therapeutically-effective amount, which means an amount of the
compound which produces a medically desirable result or exerts an
influence on the particular condition being treated. An effective
amount of a compound of the invention is capable of ameliorating or
delaying progression of the diseased, degenerative or damaged
condition. The effective amount can be determined on an individual
basis and will be based, in part, on consideration of the physical
attributes of the subject, symptoms to be treated and results
sought. An effective amount can be determined by one of ordinary
skill in the art employing such factors and using no more than
routine experimentation.
[0145] A liposome delivery system for a compound of the invention
may be any of a variety of unilamellar vesicles, multilamellar
vesicles, or stable plurilamellar vesicles, and may be prepared and
administered according to methods well known to those of skill in
the art, for example in accordance with the teachings of U.S. Pat.
Nos. 5,169,637, 4,762,915, 5,000,958 or 5,185,154. In addition, it
may be desirable to express the novel polypeptides of this
invention, as well as other selected polypeptides, as lipoproteins,
in order to enhance their binding to liposomes. As an example,
treatment of human acute renal failure with liposome-encapsulated
KIM protein may be performed in vivo by introducing a KIM protein
into cells in need of such treatment using liposomes. The liposomes
can be delivered via catheter to the renal artery. The recombinant
KIM protein is purified, for example, from CHO cells y
immunoaffinity chromatography or any other convenient method, then
mixed with liposomes and incorporated into them at high efficiency.
The encapsulated protein may be tested in vitro for any effect on
stimulating cell growth.
[0146] The compounds of the invention may be administered in any
manner which is medically acceptable. This may include injections,
by parenteral routes such as intravenous, intravascular,
intraarterial, subcutaneous, intramuscular, intratumor,
intraperitoneal, intraventricular, intraepidural, or others as well
as oral, nasal, ophthalmic, rectal, or topical. Sustained release
administration is also specifically included in the invention, by
such means as depot injections or erodible implants. Localized
delivery is particularly contemplated, by such means as delivery
via a catheter to one or more arteries, such as the renal artery or
a vessel supplying a localized tumor.
[0147] Equivalents
[0148] Those skilled in the arts will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. All such equivalents are embraced by the following claims.
Sequence CWU 1
1
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