Title: Antibodies to human
United States Patent: 7,097,985
Issued: August 29, 2006
Inventors: Li; Haodong
(Gaithersburg, MD), Adams; Mark D. (Rockville, MD)
Assignee: Human Genome
Sciences, Inc. (Rockville, MD)
Filed: December 27, 2002
Executive MBA in Pharmaceutical Management, U. Colorado
Human Ck.beta.-9 polypeptides and DNA
(RNA) encoding such chemokine polypeptides and a procedure for producing
such polypeptides by recombinant techniques is disclosed. Also disclosed
are methods for utilizing such Ck.beta.-9 polypeptides for the treatment
of leukemia, tumors, chronic infections, autoimmune disease, fibrotic
disorders, wound healing and psoriasis. Antagonists against such
polypeptides and their use as a therapeutic to treat rheumatoid arthritis,
autoimmune and chronic inflammatory and infective diseases, allergic
reactions, prostaglandin-independent fever and bone marrow failure are
also disclosed. Diagnostic assays are also disclosed which detect the
presence of mutations in the Ck.beta.-9 coding sequence and
over-expression of the Ck.beta.-9 protein.
OF THE INVENTION
In accordance with an aspect of the
present invention, there are provided isolated nucleic acids (polynucleotides)
which encode for the mature polypeptides having the deduced amino acid
sequences of FIG. 1 (SEQ ID NO:2) or for the mature polypeptide encoded by
the cDNA of the clones deposited with the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 (present
address) as ATCC Deposit No. 75803 on Jun. 7, 1994.
The polynucleotide encoding Ck.beta.-9 was discovered in a cDNA library
derived from a human breast lymph node. Ck.beta.-9 is structurally related
to the chemokine family. It contains an open reading frame encoding a
protein of 134 amino acid residues of which approximately the first 23
amino acids residues are the putative leader sequence such that the mature
protein comprises 111 amino acids. The protein exhibits the highest degree
of homology to eotaxin with 32% identity and 69% similarity over a stretch
of 75 amino acid residues. It is also important that the four spatially
conserved cysteine residues in chemokines are found in the polypeptides of
the present invention.
The polynucleotides of the present invention may be in the form of RNA or
in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic
DNA. The DNA may be double-stranded or single-stranded, and if single
stranded may be the coding strand or non-coding (anti-sense) strand. The
coding sequence which encodes the mature polypeptides may be identical to
the coding sequence shown in FIG. 1 (SEQ ID NO:1) or that of the deposited
clones or may be a different coding sequence which coding sequence, as a
result of the redundancy or degeneracy of the genetic code, encodes the
same mature polypeptides as the DNA of FIG. 1 (SEQ ID NO:1) or the
The polynucleotides which encodes for the mature polypeptides of FIG. 1 (SEQ
ID NO:2) or for the mature polypeptides encoded by the deposited cDNA may
include: only the coding sequence for the mature polypeptide; the coding
sequence for the mature polypeptide and additional coding sequence such as
a leader or secretory sequence or a proprotein sequence; the coding
sequence for the mature polypeptide (and optionally additional coding
sequence) and non-coding sequence, such as introns or non-coding sequence
5' and/or 3' of the coding sequence for the mature polypeptides.
Thus, the term "polynucleotide encoding a polypeptide" encompasses a
polynucleotide which includes only coding sequence for the polypeptide as
well as a polynucleotide which includes additional coding and/or
The present invention further relates to variants of the hereinabove
described polynucleotides which encode for fragments, analogs and
derivatives of the polypeptide having the deduced amino acid sequence of
FIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the
deposited clones. The variant of the polynucleotides may be a naturally
occurring allelic variant of the polynucleotides or a non-naturally
occurring variant of the polynucleotides.
Thus, the present invention includes polynucleotides encoding the same
mature polypeptides as shown in FIG. 1 (SEQ ID NO:2) or the same mature
polypeptides encoded by the cDNA of the deposited clones as well as
variants of such polynucleotides which variants encode for a fragment,
derivative or analog of the polypeptides of FIG. 1 (SEQ ID NO:2) or the
polypeptides encoded by the cDNA of the deposited clones. Such nucleotide
variants include deletion variants, substitution variants and addition or
As hereinabove indicated, the polynucleotides may have a coding sequence
which is a naturally occurring allelic variant of the coding sequence
shown in FIG. 1 (SEQ ID NO:2) or of the coding sequence of the deposited
clones. As known in the art, an allelic variant is an alternate form of a
polynucleotide sequence which may have a substitution, deletion or
addition of one or more nucleotides, which does not substantially alter
the function of the encoded polypeptide.
The present invention also includes polynucleotides, wherein the coding
sequence for the mature polypeptides may be fused in the same reading
frame to a polynucleotide sequence which aids in expression and secretion
of a polypeptide from a host cell, for example, a leader sequence which
functions as a secretory sequence for controlling transport of a
polypeptide from the cell. The polypeptide having a leader sequence is a
preprotein and may have the leader sequence cleaved by the host cell to
form the mature form of the polypeptide. The polynucleotides may also
encode for a proprotein which is the mature protein plus additional 5'
amino acid residues. A mature protein having a prosequence is a proprotein
and is an inactive form of the protein. Once the prosequence is cleaved an
active mature protein remains.
Thus, for example, the polynucleotide of the present invention may encode
for a mature protein, or for a protein having a prosequence or for a
protein having both a prosequence and a presequence (leader sequence).
The polynucleotides of the present invention may also have the coding
sequence fused in frame to a marker sequence which allows for purification
of the polypeptides of the present invention. The marker sequence may be a
hexa-histidine tag supplied by a pQE-9 vector to provide for purification
of the mature polypeptides fused to the marker in the case of a bacterial
host, or, for example, the marker sequence may be a hemagglutinin (HA) tag
when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds
to an epitope derived from the influenza hemagglutinin protein (Wilson,
I., et al., Cell, 37:767 (1984)).
The term "gene" means the segment of DNA involved in producing a
polypeptide chain; it includes regions preceding and following the coding
region (leader and trailer) as well as intervening sequences (introns)
between individual coding segments (exons).
Fragments of the full length gene of the present invention may be used as
a hybridization probe for a cDNA library to isolate the full length cDNA
and to isolate other cDNAs which have a high sequence similarity to the
gene or similar biological activity. Probes of this type preferably have
at least 30 bases and may contain, for example, 50 or more bases. The
probe may also be used to identify a cDNA clone corresponding to a full
length transcript and a genomic clone or clones that contain the complete
gene including regulatory and promoter regions, exons, and introns. An
example of a screen comprises isolating the coding region of the gene by
using the known DNA sequence to synthesize an oligonucleotide probe.
Labeled oligonucleotides having a sequence complementary to that of the
gene of the present invention are used to screen a library of human cDNA,
genomic DNA or mRNA to determine which members of the library the probe
The present invention further relates to polynucleotides which hybridize
to the hereinabove-described sequences if there is at least 70%,
preferably at least 90%, and more preferably at least 95% identity between
the sequences. The present invention particularly relates to
polynucleotides which hybridize under stringent conditions to the
hereinabove-described polynucleotides. As herein used, the term "stringent
conditions" means hybridization will occur only if there is at least 95%
and preferably at least 97% identity between the sequences. The
polynucleotides which hybridize to the hereinabove described
polynucleotides in a preferred embodiment encode polypeptides which either
retain substantially the same biological function or activity as the
mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1) or the
Alternatively, the polynucleotide may have at least 20 bases, preferably
30 bases, and more preferably at least 50 bases which hybridize to a
polynucleotide of the present invention and which has an identity thereto,
as hereinabove described, and which may or may not retain activity. For
example, such polynucleotides may be employed as probes for the
polynucleotide of SEQ ID NO:1, for example, for recovery of the
polynucleotide or as a diagnostic probe or as a PCR primer.
Thus, the present invention is directed to polynucleotides having at least
a 70% identity, preferably at least 90% and more preferably at least a 95%
identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2
as well as fragments thereof, which fragments have at least 30 bases and
preferably at least 50 bases and to polypeptides encoded by such
The deposit(s) referred to herein will be maintained under the terms of
the Budapest Treaty on the International Recognition of the Deposit of
Micro-organisms for purposes of Patent Procedure. These deposits are
provided merely as convenience to those of skill in the art and are not an
admission that a deposit is required under 35 U.S.C. .sctn.112. The
sequence of the polynucleotides contained in the deposited materials, as
well as the amino acid sequence of the polypeptides encoded thereby, are
incorporated herein by reference and are controlling in the event of any
conflict with any description of sequences herein. A license may be
required to make, use or sell the deposited materials, and no such license
is hereby granted.
The present invention further relates to chemokine polypeptides which have
the deduced amino acid sequences of FIG. 1 (SEQ ID NO:2) or which has the
amino acid sequence encoded by the deposited cDNA, as well as fragments,
analogs and derivatives of such polypeptides.
The terms "fragment," "derivative" and "analog" when referring to the
polypeptides of FIG. 1 (SEQ ID NO:2) or that encoded by the deposited cDNA,
means polypeptides which retain essentially the same biological function
or activity as such polypeptides. Thus, an analog includes a proprotein
which can be activated by cleavage of the proprotein portion to produce an
active mature polypeptide.
The chemokine polypeptides of the present invention may be recombinant
polypeptides, natural polypeptides or a synthetic polypeptides, preferably
The fragment, derivative or analog of the polypeptides of FIG. 1 (SEQ ID
NO:2) or that encoded by the deposited cDNA may be (i) one in which one or
more of the amino acid residues are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may not be one
encoded by the genetic code, or (ii) one in which one or more of the amino
acid residues includes a substituent group, or (iii) one in which the
mature polypeptide is fused with another compound, such as a compound to
increase the half-life of the polypeptide (for example, polyethylene
glycol), or (iv) one in which the additional amino acids are fused to the
mature polypeptide, such as a leader or secretory sequence or a sequence
which is employed for purification of the mature polypeptide or a
proprotein sequence. Such fragments, derivatives and analogs are deemed to
be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are
preferably provided in an isolated form, and preferably are purified to
The term "isolated" means that the material is removed from its original
environment (e.g., the natural environment if it is naturally occurring).
For example, a naturally-occurring polynucleotide or polypeptide present
in a living animal is not isolated, but the same polynucleotide or
polypeptide, separated from some or all of the coexisting materials in the
natural system, is isolated. Such polynucleotides could be part of a
vector and/or such polynucleotides or polypeptides could be part of a
composition, and still be isolated in that such vector or composition is
not part of its natural environment.
The polypeptides of the present invention include the polypeptide of SEQ
ID NO:2 (in particular the mature polypeptide) as well as polypeptides
which have at least 70% similarity (preferably at least 70% identity) to
the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity
(more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2
and still more preferably at least 95% similarity (still more preferably
at least 90% identity) to the polypeptide of SEQ ID NO:2 and also include
portions of such polypeptides with such portion of the polypeptide
generally containing at least 30 amino acids and more preferably at least
50 amino acids.
As known in the art "similarity" between two polypeptides is determined by
comparing the amino acid sequence and its conserved amino acid substitutes
of one polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be
employed for producing the corresponding full-length polypeptide by
peptide synthesis; therefore, the fragments may be employed as
intermediates for producing the full-length polypeptides. Fragments or
portions of the polynucleotides of the present invention may be used to
synthesize full-length polynucleotides of the present invention.
The present invention also relates to vectors which include
polynucleotides of the present invention, host cells which are genetically
engineered with vectors of the invention and the production of
polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or transformed or
transfected) with the vectors of this invention which may be, for example,
a cloning vector or an expression vector. The vector may be, for example,
in the form of a plasmid, a viral particle, a phage, etc. The engineered
host cells can be cultured in conventional nutrient media modified as
appropriate for activating promoters, selecting transformants or
amplifying the Ck.beta.-9 genes. The culture conditions, such as
temperature, pH and the like, are those previously used with the host cell
selected for expression, and will be apparent to the ordinarily skilled
The polynucleotides of the present invention may be employed for producing
polypeptides by recombinant techniques. Thus, for example, the
polynucleotide may be included in any one of a variety of expression
vectors for expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;
bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors
derived from combinations of plasmids and phage DNA, viral DNA such as
vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other
vector may be used as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety
of procedures. In general, the DNA sequence is inserted into an
appropriate restriction endonuclease site(s) by procedures known in the
art. Such procedures and others are deemed to be within the scope of those
skilled in the art.
The DNA sequence in the expression vector is operatively linked to an
appropriate expression control sequence(s) (promoter) to direct mRNA
synthesis. As representative examples of such promoters, there may be
mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda
P.sub.L promoter and other promoters known to control expression of genes
in prokaryotic or eukaryotic cells or their viruses. The expression vector
also contains a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more
selectable marker genes to provide a phenotypic trait for selection of
transformed host cells such as dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture, or such as tetracycline or
ampicillin resistance in E. coli.
The vector containing the appropriate DNA sequence as hereinabove
described, as well as an appropriate promoter or control sequence, may be
employed to transform an appropriate host to permit the host to express
As representative examples of appropriate hosts, there may be mentioned:
bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;
fungal cells, such as yeast; insect cells such as Drosophila S2 and
Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;
adenoviruses; plant cells, etc. The selection of an appropriate host is
deemed to be within the scope of those skilled in the art from the
More particularly, the present invention also includes recombinant
constructs comprising one or more of the sequences as broadly described
above. The constructs comprise a vector, such as a plasmid or viral
vector, into which a sequence of the invention has been inserted, in a
forward or reverse orientation. In a preferred aspect of this embodiment,
the construct further comprises regulatory sequences, including, for
example, a promoter, operably linked to the sequence. Large numbers of
suitable vectors and promoters are known to those of skill in the art, and
are commercially available. The following vectors are provided by way of
example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript,
psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene);
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO,
pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL
(Pharmacia). However, any other plasmid or vector may be used as long as
they are replicable and viable in the host.
Promoter regions can be selected from any desired gene using CAT (chloramphenicol
transferase) vectors or other vectors with selectable markers. Two
appropriate vectors are pKK232-8 and pCM7. Particular named bacterial
promoters include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L and trp.
Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,
early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.
Selection of the appropriate vector and promoter is well within the level
of ordinary skill in the art.
In a further embodiment, the present invention relates to host cells
containing the above-described constructs. The host cell can be a higher
eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,
such as a yeast cell, or the host cell can be a prokaryotic cell, such as
a bacterial cell. Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-Dextran mediated
transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic
Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional manner to
produce the gene product encoded by the recombinant sequence.
Alternatively, the polypeptides of the invention can be synthetically
produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or
other cells under the control of appropriate promoters. Cell-free
translation systems can also be employed to produce such proteins using
RNAs derived from the DNA constructs of the present invention. Appropriate
cloning and expression vectors for use with prokaryotic and eukaryotic
hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure
of which is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of the present
invention by higher eukaryotes is increased by inserting an enhancer
sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about from 10 to 300 bp that act on a promoter to increase its
transcription. Examples including the SV40 enhancer on the late side of
the replication origin bp 100 to 270, a cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication origin,
and adenovirus enhancers.
Generally, recombinant expression vectors will include origins of
replication and selectable markers permitting transformation of the host
cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae
TRP1 gene, and a promoter derived from a highly-expressed gene to direct
transcription of a downstream structural sequence. Such promoters can be
derived from operons encoding glycolytic enzymes such as
3-phosphoglycerate kinase (PGK), .alpha.-factor, acid phosphatase, or heat
shock proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and termination
sequences, and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence can encode a
fusion protein including an N-terminal identification peptide imparting
desired characteristics, e.g., stabilization or simplified purification of
expressed recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a
structural DNA sequence encoding a desired protein together with suitable
translation initiation and termination signals in operable reading phase
with a functional promoter. The vector will comprise one or more
phenotypic selectable markers and an origin of replication to ensure
maintenance of the vector and to, if desirable, provide amplification
within the host. Suitable prokaryotic hosts for transformation include E.
coli, Bacillus subtilis, Salmonella typhimurium and various species within
the genera Pseudomonas, Streptomyces, and Staphylococcus, although others
may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for
bacterial use can comprise a selectable marker and bacterial origin of
replication derived from commercially available plasmids comprising
genetic elements of the well known cloning vector pBR322 (ATCC 37017).
Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine
Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,
USA). These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host
strain to an appropriate cell density, the selected promoter is induced by
appropriate means (e.g., temperature shift or chemical induction) and
cells are cultured for an additional period.
Cells are typically harvested by centrifugation, disrupted by physical or
chemical means, and the resulting crude extract retained for further
Microbial cells employed in expression of proteins can be disrupted by any
convenient method, including freeze-thaw cycling, sonication, mechanical
disruption, or use of cell lysing agents, such methods are well know to
those skilled in the art.
Various mammalian cell culture systems can also be employed to express
recombinant protein. Examples of mammalian expression systems include the
COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell,
23:175 (1981), and other cell lines capable of expressing a compatible
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
Mammalian expression vectors will comprise an origin of replication, a
suitable promoter and enhancer, and also any necessary ribosome binding
sites, polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking nontranscribed
sequences. DNA sequences derived from the SV40 splice, and polyadenylation
sites may be used to provide the required nontranscribed genetic elements.
The Ck.beta.-9 polypeptides can be recovered and purified from recombinant
cell cultures by methods including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity chromatography, hydroxylapatite chromatography and lectin
chromatography. Protein refolding steps can be used, as necessary, in
completing configuration of the mature protein. Finally, high performance
liquid chromatography (HPLC) can be employed for final purification steps.
The polypeptides of the present invention may be a naturally purified
product, or a product of chemical synthetic procedures, or produced by
recombinant techniques from a prokaryotic or eukaryotic host (for example,
by bacterial, yeast, higher plant, insect and mammalian cells in culture).
Depending upon the host employed in a recombinant production procedure,
the polypeptides of the present invention may be glycosylated or may be
non-glycosylated. Polypeptides of the invention may also include an
initial methionine amino acid residue.
The Ck.beta.-9 polypeptides may be employed to inhibit bone marrow stem
cell colony formation as adjunct protective treatment during cancer
chemotherapy and for leukemia.
The chemokine polypeptides may also be used to inhibit epidermal
keratinocyte proliferation for treatment of psoriasis, which is
characterized by keratinocyte hyper-proliferation.
The chemokine polypeptides may also be used to treat solid tumors by
stimulating the invasion and activation of host defense cells, e.g.,
cytotoxic T cells and macrophages. They may also be used to enhance host
defenses against resistant chronic infections, for example, mycobacterial
infections via the attraction and activation of microbicidal leukocytes.
The chemokine polypeptides may also be used to treat auto-immune disease
and lymphocytic leukemia by inhibiting T cell proliferation by the
inhibition of IL2 biosynthesis.
Ck.beta.-9 may also be used in wound healing, both via the recruitment of
debris clearing and connective tissue promoting inflammatory cells and
also via its control of excessive TGF-mediated fibrosis. In this same
manner, Ck.beta.-9 may also be used to treat other fibrotic disorders,
including liver cirrhosis, osteoarthritis and pulmonary fibrosis.
The chemokine polypeptides also increase the presence of eosinophils which
have the distinctive function of killing the larvae of parasites that
invade tissues, as in schistosomiasis, trichinosis and ascariasis.
They may also be used to regulate hematopoiesis, by regulating the
activation and differentiation of various hematopoietic progenitor cells,
for example, to release mature leukocytes from the bone marrow following
The polynucleotides and polypeptides encoded by such polynucleotides may
also be utilized for in vitro purposes related to scientific research,
synthesis of DNA and manufacture of DNA vectors and for designing
therapeutics and diagnostics for the treatment of human disease.
Fragments of the full length Ck.beta.-9 genes may be used as a
hybridization probe for a cDNA library to isolate the full length gene and
to isolate other genes which have a high sequence similarity to the gene
or similar biological activity. Probes of this type can be, for example,
between 20 and 2000 bases. Preferably, however, the probes have between 30
and 50 base pairs. The probe may also be used to identify a cDNA clone
corresponding to a full length transcript and a genomic clone or clones
that contain the complete genes including regulatory and promoter regions,
exons, and introns. An example of a screen comprises isolating the coding
region of the genes by using the known DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary to that of the genes of the present invention are used to
screen a library of human cDNA, genomic DNA or mRNA to determine which
members of the library the probe hybridizes to.
This invention is also related to the use of the Ck.beta.-9 gene as part
of a diagnostic assay for detecting diseases or susceptibility to diseases
related to the presence of mutations in the Ck.beta.-9 nucleic acid
sequences. Such diseases are related to under-expression of the chemokine
polypeptides, for example, tumors and cancers.
Individuals carrying mutations in the Ck.beta.-9 gene may be detected at
the DNA level by a variety of techniques. Nucleic acids for diagnosis may
be obtained from a patient's cells, such as from blood, urine, saliva,
tissue biopsy and autopsy material. The genomic DNA may be used directly
for detection or may be amplified enzymatically by using PCR (Saiki et
al., Nature, 324:163 166 (1986)) prior to analysis. RNA or cDNA may also
be used for the same purpose. As an example, PCR primers complementary to
the nucleic acid encoding Ck.beta.-9 can be used to identify and analyze
Ck.beta.-9 mutations. For example, deletions and insertions can be
detected by a change in size of the amplified product in comparison to the
normal genotype. Point mutations can be identified by hybridizing
amplified DNA to radiolabeled Ck.beta.-9 RNA or alternatively,
radiolabeled Ck.beta.-9 antisense DNA sequences. Perfectly matched
sequences can be distinguished from mismatched duplexes by RNase A
digestion or by differences in melting temperatures.
Genetic testing based on DNA sequence differences may be achieved by
detection of alteration in electrophoretic mobility of DNA fragments in
gels with or without denaturing agents. Small sequence deletions and
insertions can be visualized by high resolution gel electrophoresis. DNA
fragments of different sequences may be distinguished on denaturing
formamide gradient gels in which the mobilities of different DNA fragments
are retarded in the gel at different positions according to their specific
melting or partial melting temperatures (see, e.g., Myers et al., Science,
Sequence changes at specific locations may also be revealed by nuclease
protection assays, such as RNase and S1 protection or the chemical
cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397 4401 (1985)).
Thus, the detection of a specific DNA sequence may be achieved by methods
such as hybridization, RNase protection, chemical cleavage, direct DNA
sequencing or the use of restriction enzymes, (e.g., Restriction Fragment
Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA sequencing,
mutations can also be detected by in situ analysis.
The present invention also relates to a diagnostic assay for detecting
altered levels of Ck.beta.-9 protein in various tissues since an
over-expression of the proteins compared to normal control tissue samples
may detect the presence of a disease or susceptibility to a disease, for
example, a tumor. Assays used to detect levels of Ck.beta.-9 protein in a
sample derived from a host are well-known to those of skill in the art and
include radioimmunoassays, competitive-binding assays, Western Blot
analysis, ELISA assays and "sandwich" assay. An ELISA assay (Coligan, et
al., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initially
comprises preparing an antibody specific to the Ck.beta.-9 antigen,
preferably a monoclonal antibody. In addition a reporter antibody is
prepared against the monoclonal antibody. To the reporter antibody is
attached a detectable reagent such as radioactivity, fluorescence or, in
this example, a horseradish peroxidase enzyme. A sample is removed from a
host and incubated on a solid support, e.g. a polystyrene dish, that binds
the proteins in the sample. Any free protein binding sites on the dish are
then covered by incubating with a non-specific protein like bovine serum
albumin. Next, the monoclonal antibody is incubated in the dish during
which time the monoclonal antibodies attach to any Ck.beta.-9 proteins
attached to the polystyrene dish. All unbound monoclonal antibody is
washed out with buffer. The reporter antibody linked to horseradish
peroxidase is now placed in the dish resulting in binding of the reporter
antibody to any monoclonal antibody bound to Ck.beta.-9. Unattached
reporter antibody is then washed out. Peroxidase substrates are then added
to the dish and the amount of color developed in a given time period is a
measurement of the amount of Ck.beta.-9 protein present in a given volume
of patient sample when compared against a standard curve.
A competition assay may be employed wherein antibodies specific to Ck.beta.-9
are attached to a solid support and labeled Ck.beta.-9 and a sample
derived from the host are passed over the solid support and the amount of
label detected, for example by liquid scintillation chromatography, can be
correlated to a quantity of Ck.beta.-9 in the sample.
A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay
Ck.beta.-9 is passed over a solid support and binds to antibody attached
to a solid support. A second antibody is then bound to the Ck.beta.-9. A
third antibody which is labeled and specific to the second antibody is
then passed over the solid support and binds to the second antibody and an
amount can then be quantified.
This invention provides a method for identification of the receptors for
Ck.beta.-9 polypeptides. The gene encoding the receptor can be identified
by numerous methods known to those of skill in the art, for example,
ligand panning and FACS sorting (Coligan, et al., Current Protocols in
Immun., 1(2), Chapter 5, (1991)). Preferably, expression cloning is
employed wherein polyadenylated RNA is prepared from a cell responsive to
the polypeptides, and a cDNA library created from this RNA is divided into
pools and used to transfect COS cells or other cells that are not
responsive to the polypeptides. Transfected cells which are grown on glass
slides are exposed to the labeled polypeptides. The polypeptides can be
labeled by a variety of means including iodination or inclusion of a
recognition site for a site-specific protein kinase. Following fixation
and incubation, the slides are subjected to autoradiographic analysis.
Positive pools are identified and sub-pools are prepared and re-transfected
using an iterative sub-pooling and re-screening process, eventually
yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled
polypeptides can be photo-affinity linked with cell membrane or extract
preparations that express the receptor molecule. Cross-linked material is
resolved by PAGE analysis and exposed to x-ray film. The labeled complex
containing the receptors of the polypeptides can be excised, resolved into
peptide fragments, and subjected to protein micro-sequencing. The amino
acid sequence obtained from micro-sequencing would be used to design a set
of degenerate oligonucleotide probes to screen a cDNA library to identify
the genes encoding the putative receptors.
This invention provides a method of screening compounds to identify
agonists and antagonists to the Ck.beta.-9 polypeptides of the present
invention. An agonist is a compound which has similar biological functions
of the polypeptides, while antagonists block such functions. Chemotaxis
may be assayed by placing cells, which are chemo-attracted by either of
the polypeptides of the present invention, on top of a filter with pores
of sufficient diameter to admit the cells (about 5 m). Solutions of
potential agonists are placed in the bottom of the chamber with an
appropriate control medium in the upper compartment, and thus a
concentration gradient of the agonist is measured by counting cells that
migrate into or through the porous membrane over time.
When assaying for antagonists, the polypeptides of the present invention
are placed in the bottom chamber and the potential antagonist is added to
determine if chemotaxis of the cells is prevented.
Alternatively, a mammalian cell or membrane preparation expressing the
receptors of the polypeptides would be incubated with a labeled Ck.beta.-9
polypeptide, e.g. radioactivity, in the presence of the compound. The
ability of the compound to block this interaction could then be measured.
When assaying for agonists in this fashion, the chemokines would be absent
and the ability of the agonist itself to interact with the receptor could
Examples of potential Ck.beta.-9 antagonists include antibodies, or in
some cases, oligonucleotides, which bind to the polypeptides. Another
example of a potential antagonist is a negative dominant mutant of the
polypeptides. Negative dominant mutants are polypeptides which bind to the
receptor of the wild-type polypeptide, but fail to retain biological
Antisense constructs prepared using antisense technology are also
potential antagonists. Antisense technology can be used to control gene
expression through triple-helix formation or antisense DNA or RNA, both of
which methods are based on binding of a polynucleotide to DNA or RNA. For
example, the 5' coding portion of the polynucleotide sequence, which
encodes for the mature polypeptides of the present invention, is used to
design an antisense RNA oligonucleotide of from about 10 to 40 base pairs
in length. A DNA oligonucleotide is designed to be complementary to a
region of the gene involved in transcription (triple-helix, see Lee et
al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456
(1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing
transcription and the production of the chemokine polypeptides. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into the polypeptides (antisense--Okano,
J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). The
oligonucleotides described above can also be delivered to cells such that
the antisense RNA or DNA may be expressed in vivo to inhibit production of
Another potential Ck.beta.-9 antagonist is a peptide derivative of the
polypeptides which are naturally or synthetically modified analogs of the
polypeptides that have lost biological function yet still recognize and
bind to the receptors of the polypeptides to thereby effectively block the
receptors. Examples of peptide derivatives include, but are not limited
to, small peptides or peptide-like molecules.
The antagonists may be employed to inhibit the chemotaxis and activation
of macrophages and their precursors, and of neutrophils, basophils, B
lymphocytes and some T cell subsets, e.g., activated and CD8 cytotoxic T
cells and natural killer cells, in auto-immune and chronic inflammatory
and infective diseases. Examples of auto-immune diseases include
rheumatoid arthritis, multiple sclerosis, and insulin-dependent diabetes.
Some infectious diseases include silicosis, sarcoidosis, idiopathic
pulmonary fibrosis by preventing the recruitment and activation of
mononuclear phagocytes, idiopathic hypereosinophilic syndrome by
preventing eosinophil production and migration, endotoxic shock by
preventing the migration of macrophages and their production of the
chemokine polypeptides of the present invention.
The antagonists may also be employed for treating atherosclerosis, by
preventing monocyte infiltration in the artery wall.
The antagonists may also be employed to treat histamine-mediated allergic
reactions and immunological disorders including late phase allergic
reactions, chronic urticaria, and atopic dermatitis by inhibiting
chemokine-induced mast cell and basophil degranulation and release of
histamine. IgE-mediated allergic reactions such as allergic asthma,
rhinitis, and eczema may also be treated.
The antagonists may also be employed to treat chronic and acute
inflammation by preventing the attraction of monocytes to a wound area.
They may also be employed to regulate normal pulmonary macrophage
populations, since acute and chronic inflammatory pulmonary diseases are
associated with sequestration of mononuclear phagocytes in the lung.
Antagonists may also be employed to treat rheumatoid arthritis by
preventing the attraction of monocytes into synovial fluid in the joints
of patients. Monocyte influx and activation plays a significant role in
the pathogenesis of both degenerative and inflammatory arthropathies.
The antagonists may be employed to interfere with the deleterious cascades
attributed primarily to IL-1 and TNF, which prevents the biosynthesis of
other inflammatory cytokines. In this way, the antagonists may be employed
to prevent inflammation. The antagonists may also be employed to inhibit
prostaglandin-independent fever induced by chemokines.
The antagonists may also be employed to treat cases of bone marrow
failure, for example, aplastic anemia and myelodysplastic syndrome.
The antagonists may also be used to treat asthma and allergy by preventing
eosinophil accumulation in the lung. The antagonists may also be employed
to treat subepithelial basement membrane fibrosis which is a prominent
feature of the asthmatic lung.
The antagonists may be employed in a composition with a pharmaceutically
acceptable carrier, e.g., as hereinafter described.
The Ck.beta.-9 polypeptides and agonists and antagonists may be employed
in combination with a suitable pharmaceutical carrier. Such compositions
comprise a therapeutically effective amount of the polypeptide, and a
pharmaceutically acceptable carrier or excipient. Such a carrier includes
but is not limited to saline, buffered saline, dextrose, water, glycerol,
ethanol, and combinations thereof. The formulation should suit the mode of
The invention also provides a pharmaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients of the
pharmaceutical compositions of the invention. Associated with such
container(s) can be a notice in the form prescribed by a governmental
agency regulating the manufacture, use or sale of pharmaceuticals or
biological products, which notice reflects approval by the agency of
manufacture, use or sale for human administration. In addition, the
polypeptides and agonists and antagonists may be employed in conjunction
with other therapeutic compounds.
The pharmaceutical compositions may be administered in a convenient manner
such as by the topical, intravenous, intraperitoneal, intramuscular,
intratumor, subcutaneous, intranasal or intradermal routes. The
pharmaceutical compositions are administered in an amount which is
effective for treating and/or prophylaxis of the specific indication. In
general, the polypeptides will be administered in an amount of at least
about 10 .mu.g/kg body weight and in most cases they will be administered
in an amount not in excess of about 8 mg/Kg body weight per day. In most
cases, the dosage is from about 10 .mu.g/kg to about 1 mg/kg body weight
daily, taking into account the routes of administration, symptoms, etc.
The Ck.beta.-9 polypeptides, and agonists or antagonists which are
polypeptides, may be employed in accordance with the present invention by
expression of such polypeptides in vivo, which is often referred to as
Thus, for example, cells from a patient may be engineered with a
polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the
engineered cells then being provided to a patient to be treated with the
polypeptide. Such methods are well-known in the art. For example, cells
may be engineered by procedures known in the art by use of a retroviral
particle containing RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide
in vivo by, for example, procedures known in the art. As known in the art,
a producer cell for producing a retroviral particle containing RNA
encoding the polypeptide of the present invention may be administered to a
patient for engineering cells in vivo and expression of the polypeptide in
vivo. These and other methods for administering a polypeptide of the
present invention by such method should be apparent to those skilled in
the art from the teachings of the present invention. For example, the
expression vehicle for engineering cells may be other than a retrovirus,
for example, an adenovirus which may be used to engineer cells in vivo
after combination with a suitable delivery vehicle.
The sequences of the present invention are also valuable for chromosome
identification. The sequence is specifically targeted to and can hybridize
with a particular location on an individual human chromosome. Moreover,
there is a current need for identifying particular sites on the
chromosome. Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking chromosomal
location. The mapping of DNAs to chromosomes according to the present
invention is an important first step in correlating those sequences with
genes associated with disease.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15 25 bp) from the cDNA. Computer analysis of the 3'
untranslated region is used to rapidly select primers that do not span
more than one exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic cell
hybrids containing individual human chromosomes. Only those hybrids
containing the human gene corresponding to the primer will yield an
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular DNA to a particular chromosome. Using the present invention
with the same oligonucleotide primers, sublocalization can be achieved
with panels of fragments from specific chromosomes or pools of large
genomic clones in an analogous manner. Other mapping strategies that can
similarly be used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection by
hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clones to a metaphase
chromosomal spread can be used to provide a precise chromosomal location
in one step. This technique can be used with cDNA as short as 50 or 60
bases. For a review of this technique, see Verma et al., Human
Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York
Once a sequence has been mapped to a precise chromosomal location, the
physical position of the sequence on the chromosome can be correlated with
genetic map data. Such data are found, for example, in V. McKusick,
Mendelian Inheritance in Man (available on line through Johns Hopkins
University Welch Medical Library). The relationship between genes and
diseases that have been mapped to the same chromosomal region are then
identified through linkage analysis (coinheritance of physically adjacent
Next, it is necessary to determine the differences in the cDNA or genomic
sequence between affected and unaffected individuals. If a mutation is
observed in some or all of the affected individuals but not in any normal
individuals, then the mutation is likely to be the causative agent of the
With current resolution of physical mapping and genetic mapping
techniques, a cDNA precisely localized to a chromosomal region associated
with the disease could be one of between 50 and 500 potential causative
genes. (This assumes 1 megabase mapping resolution and one gene per 20
The polypeptides, their fragments or other derivatives, or analogs
thereof, or cells expressing them can be used as an immunogen to produce
antibodies thereto. These antibodies can be, for example, polyclonal or
monoclonal antibodies. The present invention also includes chimeric,
single chain, and humanized antibodies, as well as Fab fragments, or the
product of an Fab expression library. Various procedures known in the art
may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence
of the present invention can be obtained by direct injection of the
polypeptides into an animal or by administering the polypeptides to an
animal, preferably a nonhuman. The antibody so obtained will then bind the
polypeptides itself. In this manner, even a sequence encoding only a
fragment of the polypeptides can be used to generate antibodies binding
the whole native polypeptides. Such antibodies can then be used to isolate
the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides
antibodies produced by continuous cell line cultures can be used. Examples
include the hybridoma technique (Kohler and Milstein, 1975, Nature,
256:495 497), the trioma technique, the human B-cell hybridoma technique (Kozbor
et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to
produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96).
Techniques described for the production of single chain antibodies (U.S.
Pat. No. 4,946,778) can be adapted to produce single chain antibodies to
immunogenic polypeptide products of this invention. Also, transgenic mice
may be used to express humanized antibodies to immunogenic polypeptide
products of this invention.
The present invention will be further described with reference to the
following examples; however, it is to be understood that the present
invention is not limited to such examples. All parts or amounts, unless
otherwise specified, are by weight.
In order to facilitate understanding of the following examples certain
frequently occurring methods and/or terms will be described.
"Plasmids" are designated by a lower case p preceded and/or followed by
capital letters and/or numbers. The starting plasmids herein are either
commercially available, publicly available on an unrestricted basis, or
can be constructed from available plasmids in accord with published
procedures. In addition, equivalent plasmids to those described are known
in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a
restriction enzyme that acts only at certain sequences in the DNA. The
various restriction enzymes used herein are commercially available and
their reaction conditions, cofactors and other requirements were used as
would be known to the ordinarily skilled artisan. For analytical purposes,
typically 1 .mu.g of plasmid or DNA fragment is used with about 2 units of
enzyme in about 20 .mu.l of buffer solution. For the purpose of isolating
DNA fragments for plasmid construction, typically 5 to 50 .mu.g of DNA are
digested with 20 to 250 units of enzyme in a larger volume. Appropriate
buffers and substrate amounts for particular restriction enzymes are
specified by the manufacturer. Incubation times of about 1 hour at 37 C.
are ordinarily used, but may vary in accordance with the supplier's
instructions. After digestion the reaction is electrophoresed directly on
a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent
polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide
or two complementary polydeoxynucleotide strands which may be chemically
synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus
will not ligate to another oligonucleotide without adding a phosphate with
an ATP in the presence of a kinase. A synthetic oligonucleotide will
ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between
two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p.
146). Unless otherwise provided, ligation may be accomplished using known
buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 .mu.g
of approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as described in the
method of Graham, F. and Van der Eb, A., Virology, 52:456 457 (1973).
Claim 1 of 23 Claims
1. An isolated antibody or
fragment thereof that specifically binds to a protein selected from the
group consisting of: (a) a protein whose amino acid sequence consists of
amino acid residues 1 to 134 of SEQ ID NO:2; (b) a protein whose amino acid
sequence consists of amino acid residues 24 to 134 of SEQ ID NO:2; (c) a
protein whose amino acid sequence consists of a portion of SEQ ID NO:2,
wherein said portion is at least 30 contiguous amino acid residues in
length; and (d) a protein whose amino acid sequence consists of a portion of
SEQ ID NO:2, wherein said portion is at least 50 contiguous amino acid
residues in length.
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