Note: Descriptions are shown in the official language in which they were submitted.
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POLYNUCLEOTDDESFROM HU~LANADULTPBMCENCODnNGSECRETEDPROTEnNS
This application is a continuation-in-part of application Ser. No. 08/659,224, filed
June 7, 1996.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by such
polynucleotides, along with therapeutic, diagnostic and research utilities for these
polynucleotides and proteins.
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g., cytokines, such
as lymphokines, inl~,fe~vns, CSFs and interleukins) has matured rapidly over the past
decade. The now routine hybridization cloning and expression cloning techniques clone
novel polynucleotides "directly" in the sense that they rely on information directly related
to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case
of hybridization cloning; activity of the protein in the case of expression cloning). More
2 0 recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA
sequences based on the presence of a now well-recognized secretory leader sequence motif,
as well as various PCR-based or low stringency hybridization cloning techniques, have
advanced the state of the art by making available large numbers of DNA/amino acid
sequences for proteins that are known to have biological activity by virtue of their secreted
2 5 nature in the case of leader sequence cloning, or by virtue of the cell or hssue source in the
case of PCR-based techniques. It is to these proteins and the polynucleotides encoding them
that the present invention is directed.
SUMMAl~Y OF THE INVENTION
3 0 In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2
from nucleotide 90 to nucleotide 564;
3 5 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2
from nucleotide 333 to nucleotide 564;
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(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone D147_17 deposited under accession numberATCC 98076;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone D147_17 deposited under accession number ATCC 98076;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone D147_17 deposited under accession number ATCC
98076;
(g) a polynucleotide encoding the mature protein encoded by the cDNA
1 0 insert of clone D147_17 deposited under accession number ATCC 98076;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:3;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:3 having biological activity;
1 5 (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-
(g) above; and
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above .
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:2from nucleotide 90 to nucleotide 564; the nucleotide sequence of SEQ ID NO:2 from
nucleotide 333 to nucleotide 564; the nucleotide sequence of the full-length protein coding
sequence of clone D147_17 deposited under accession number ATCC 98076; or the
nucleotide sequence of the mature protein coding sequence of clone D147_17 deposited
under accession number ATCC 98076. In other preferred embodiments, the polynucleotide
encodes the full-length or mature protein encoded by the cDNA insert of clone D147_17
deposited under accession number ATCC 98076. In yet other preferred embodiments, the
present invention provides a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:3 from amino acid 1 to amino acid 121.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
3 0 ID NO:2, SEQ ID NO:1 or SEQ ID NO:4 .
In other embodiments, the present invention provides a composition comprising a
protein, wherein said protein comprises an amino acid sequence selected from the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:3;
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(b) the amino acid sequence of SEQ ID NO:3 from amino acid 1 to amino
acid 121;
(c) fragments of the amino acid sequence of SEQ ID NO:3; and
(d) the amino acid sequence encoded by the cDNA insert of clone
D147_17 deposited under accession number ATCC 98076;
the protein being substantially free from other mammalian proteins. Preferably such protein
comprises the amino acid sequence of SEQ ID NO:3 or the amino acid sequence of SEQ ID
NO:3 from amino acid 1 to amino acid 121.
In certain p,e~ d embodiments, the polynucleotide is operably linked to an
10 expression control sequence. The invention also provides a host cell, including bacterial,
yeast, insect and mammalian cells, transformed with such polynucleotide compositions.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present invention.
Preferred embodiments include those in which the protein produced by such process is a
mature form of the protein.
Protein compositions of the present invention may further comprise a
2 0 pharmaceutically acceptable carrier. Compositions comprising an antibody which
specifically reacts with such protein are also provided by the present invention.
Methods are also provided for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a therapeutically effective
amount of a composition comprising a protein of the present invention and a
2 5 pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF FIGURES
Fig. 1 is a schematic representation of the pED6 and pNotS vectors used for deposit
of clones disclosed herein.
Fig. 2 is an autoradiograph evidencing the expression of clone D147_17 in COS cells.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported belowfor each clone and protein disclosed in the present application. The nucleotide sequence of
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each clone can readily be determined by sequencing of the deposited clone in accordance
with known methods. The predicted amino acid sequence (both full-length and mature) can
then be determined from such nucleotide sequence. The amino acid sequence of the protein
encoded by a particular clone can also be determined by expression of the clone in a suitable
5 host cell, collecting the protein and determining its sequence. For each disclosed protein
applicants have identified what they have determined to be the reading frame best
identifiable with sequence information available at the time of filing.
As used herein a "secreted" protein is one which, when expressed in a suitable host
cell, is transported across or through a membrane, including transport as a result of signal
10 sequences in its amino acid sequence. "Secreted" proteins include without limitation
proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in
which they are expressed. "Secreted" proteins also include without limitation proteins which
are transported across the membrane of the endoplpasmic reticulum.
Clone"D147 17"
A polynucleotide of the present invention has been identified as clone "D147_17".
D147_17 was isolated from a human adult blood (peripheral blood mononuclear cells treated
with concanavalin A and phorbol meristate acetate) cDNA library using methods which are
selective for cDNAs encoding secreted proteins. D147_17 is a full-length clone, including
the entire coding sequence of a secreted protein (also referred to herein as "D147_17
protein").
The nucleotide sequence of the 5' portion of D147_17 as presently determined is
reported in SEQ ID NO:1. An additional internal nucleotide sequence from D147 17 as
presently determined is reported in SEQ ID NO:2. What applicants believe is the proper
2 5 reading frame and the predicted amino acid sequence encoded by such internal sequence
is reported in SEQ ID NO:3. Amino acids 69 to 81 of SEQ ID NO:3 are a predicted
leader/signal sequence, with the predicted mature amino acid sequence beginning at amino
acid 82, or are a transmembrane domain. Additional nucleotide sequence from the 3'
portion of D147_17, including the polyA tail, is reported in SEQ ID NO:4.
3 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing clone
D147_17 should be approximately 2500 bp.
The nucleotide sequence disclosed herein for D147_17 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA
search protocols. D147_17 demonstrated at least some identity with sequences identified
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as AA011178 (ze22cO1.sl Soares fetal heart NbHH19W Homo sapiens cDNA clone 359712
3'), T24634 (Human gene signature HUMGS06694), H14129 (ym65bO4.rl Homo sapiens
cDNA clone 163759 5'), and AA181797 (zp55gO5.rl Stratagene NT2 neuronal precursor
937230 Homo sapiens cDNA clone 613400 5' similar to WP:C09G4.1 CE03978). The
predicted amino acid sequence disclosed herein for D147_17 was searched against the GenPept
~ and GeneSeq amino acid sequence ~ h~ccs using the BLASTX search protocol. The predicted
D147_17 protein demonstrated at least some identity with sequences identified as U42438 (similar
to S. cerevisiae longevit,v-assurance protein 1 (SP P38703) [Caenorhabditis elegans]), R20230
(hUOG-1: DNA segment encoding a mammalian GDF-1 protein - for diagnosis of tumours,
birth defects and genetic diseases). Based upon identity, D147_17 proteins and each
identical protein or peptide may share at least some activity. The TopPredII computer program
predicts three potential transmembrane domains within the D147_17 protein sequence, centered
around amino acids 40, 70, and 120 of SEQ ID NO:3.]
Fig. 2 is an autoradiograph evidencing expression of clone D147_17 of the present
invention. The D147_17 clone was expressed in COS cells.
Deposit of Clones
Clone D147_17 was deposited on June 6, 1996 with the American Type Culture
2 0 Collection under accession number ATCC 98076, from which the D147_17 clone comprising
a particular polynucleotide is obtainable. Each clone has been transfected into separate
bacterial cells (E. coli) in this composite deposit.
The D147_17 clone can be removed from the vector in which it was deposited by
performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, Notl) to produce the appropriate
fragment for such clone. The D147_17 clone was deposited in either the pED6 or pNotS
vector depicted in Fig. 1. In some instances, the deposited clone can become "flipped" (i.e.,
in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can
still be isolated by digestion with EcoRI and NotI. However, NotI will then produce the 5'
site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for
3 0 expression in a suitable vector. The cDNA may also be expressed from the vectors in which
they were deposited.
Bacterial cells containing a particular clone can be obtained from the compositedeposit as follows:
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An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone. This sequence can be derived from the sequences provided
herein, or from a combination of those sequences. The sequence of the oligonucleotide probe
that was used to isolate each full-length clone is identified below, and should be most
reliable in isolating the clone of interest.
Clone Probe Sequence
D147_17 SEQ ID NO:5
In the sequences listed above which include an N at position 2, that position is occupied in
l,refe.l~d probes/primers by a biotinylated phosphoaramidite residue rather than a
nucleotide (such as, for example, that produced by use of biotin phosphoramidite (1-
dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
The design of the oligonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ~ C (assuming 2~ for each A2 0 or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g 32p ATP (specific activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling
oligonucleotides. Other labeling techniques can also be used. Unincorporated label should
preferably be removed by gel filtration chromatography or other established methods. The
2 5 amount of radioactivity incorporated into the probe should be quantitated by measurement
in a scintillation counter. Preferably, specific activity of the resulting probe should be
approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should preferably be
thawed and 100 ,ul of the stock used to inoculate a sterile culture flask containing 25 ml of
3 0 sterile L-broth containing ampicillin at 100 ,ug/ml. The culture should preferably be grown
to saturation at 37~C, and the saturated culture should preferably be diluted in fresh L-broth.
Aliquots of these dilutions should preferably be plated to determine the dilution and volume
which will yield approximately 5000 distinct and well-separated colonies on solid
bacteriological media containing L-broth containing ampicillin at 100 ,ug/ml and agar at
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1.5% in a 150 mm petri dish when grown overnight at 37~C. Other known methods ofobtaining distinct, well-separated colonies can also be employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65~C for 1 hour with gentle agitation in 6X
SSC (20X stock is 175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH)
containing 0.5% SDS,100 ,ug/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per
150 mm filter). Preferably, the probe is then added to the hybridization mix at a
concentration greater than or equal to le+6 dpm/mL. The filter is then preferably incubated
1 0 at 65~C with gentle agitation overnight. The filter is then preferably washed in 500 mL of
2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL
of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash
with 0.1X SSC/0.5% SDS at 65~C for 30 minutes to 1 hour is optional. The filter is then
preferably dried and subjected to autoradiography for sufficient time to visualize the
1 5 positives on the X-ray film. Other known hybridization methods can also be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated using
standard procedures. The clones can then be verified by restriction analysis, hybridization
analysis, or DNA sequencing.
Fragments of the ~loLeins of the present invention which are capable of exhibiting
biological activity are also encompassed by the present invention. Fragments of the protein
may be in linear form or they may be cyclized using known methods, for example, as
described in H.U. Saragovi, et al., Bio/Technology ~g 773-778 (1992) and in R.S. McDowell,
2 5 et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by
reference. Such fragments may be fused to carrier molecules such as immunoglobulins for
many purposes, including increasing the valency of protein binding sites. For example,
fragments of the protein may be fused through "linker" sequences to the Fc portion of an
immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion
3 0 of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such
fusions. For example, a protein - IgM fusion would generate a decavalent form of the
protein of the invention.
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in the sequence
. .
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listing by translation of the nucleotide sequence of each disclosed clone. The mature form
of such protein may be obtained by expression of the disclosed full-length polynucleotide
(preferably those deposited with ATCC) in a suitable mammalian cell or other host cell. The
sequence of the mature form of the protein may also be determinable from the amino acid
5 sequence of the full-length form.
The present invention also provides genes corresponding to the cDNA sequences
disclosed herein. The corresponding genes can be isolated in accordance with known
methods using the sequence information disclosed herein. Such methods include the
preparation of probes or primers from the disclosed sequence information for identification
10 and/or amplification of genes in a~plo~l;ate genomic libraries or other sources of genomic
materials.
Where the protein of the present invention is membrane-bound (e.g., is a receptor),
the present invention also provides for soluble forms of such protein. In such forms part or
all of the intr~c~ ]l~r and transmembrane domains of the protein are deleted such that the
15 protein is fully secreted from the cell in which it is expressed. The intracellular and
transmembrane domains of proteins of the invention can be identified in accordance with
known techniques for determination of such domains from sequence information.
Proteins and protein fragments of the present invention include proteins with amino
acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably
2 0 at least 75%) of the length of a disclosed protein and have at least 60% sequence identity
(more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with
that disclosed protein, where sequence identity is determined by comparing the amino acid
sequences of the proteins when aligned so as to maximize overlap and identity while
minimizing sequence gaps. Also included in the present invention are proteins and protein
2 5 fragments that contain a segment preferably comprising 8 or more (more preferably 20 or
more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence
identity (more preferably, at least 85% identity; most preferably at least 95% identity) with
any such segment of any of the disclosed proteins.
Species homologs of the disclosed polynucleotides and proteins are also provided3 0 by the present invention. Species homologs may be isolated and identified by making
suitable probes or primers from the sequences provided herein and screening a suitable
nucleic acid source from the desired species.
The invention also encompasses allelic variants of the disclosed polynucleotides or
proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which
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also encode proteins which are identical, homologous or related to that encoded by the
polynucleotides .
The invention also includes polynucleotides with sequences complementary to those
of the polynucleotides disclosed herein.
The present invention also includes polynucleotides capable of hybridizing
under reduced stringency conditions, more preferably stringent conditions, and most
preferably highly stringent conditions, to polynucleotides described herein. Examples of
stringency conditions are shown in the table below: highly stringent conditions are those
that are at least as stringent as, for example, conditions A-F; stringent conditions are at least
as stringent as, for example, conditions G-L; and reduced stringency conditions are at least
as stringent as, for example, conditions M-R.
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Stringency Polynucleotide Hybrid Hybridization T~ p~:ldlu-~ and Wash
Condition Hybrid Length Buffert Temperature
(bp)~ and Buffert
A DNA:DNA 2 50 65~C; IxSSC -or- 65DC; 0 3xSSC
42~C; lxSSC, 50% formamide
B DNA:DNA <50 TB~;1XSSC TB~;1XSSC
C DNA:RNA 2 50 67~C; lxSSC-or- 67~C;0.3xSSC
45~C; lxSSC, 50% formamide
D DNA:RNA <50 TD~;1XSSC TD+;1XSSC
E RNA:RNA 2 50 70~C; 1xSSC -or- 70~C; 0.3xSSC
50~C; 1xSSC, 50% formamide
F RNA:RNA c50 TF+;1XSSC TF*;1XSSC
G DNA:DNA 2 50 65~C; 4xSSC -or- 65~C; 1xSSC
42~C; 4xSSC, 50% formamide
1 0H DNA:DNA <50 TH~; 4xSSC TH~; 4xSSC
DNA:RNA 2 50 67~C; 4xSSC -or- 67~C; 1xSSC
45~C; 4xSSC, 50% formamide
J DNA:RNA c50 Tl~; 4xSSC TJ~; 4xSSC
K RNA:RNA 2 50 70~C;4xSSC -or- 67~C; 1xSSC
50~C; 4xSSC, 50% formamide
L RNA:RNA <50 T,~; 2xSSC T,"; 2xSSC
15 M DNA:DNA 2 50 50~C;4xSSC-or- 50~C;2xSSC
40~C; 6xSSC, 50% formamide
N DNA:DNA <50 TN~; 6xSSC TN~; 6xSSC
O DNA:RNA 2 50 55~C; 4xSSC -or- 55~C; 2xSSC
42~C; 6xSSC, 50% formamide
P DNA:RNA <50 Trl'; 6xSSC Tr'l; 6xSSC
Q RNA:RNA 2 50 60~C; 4xSSC -or- 60~C; 2xSSC
45~C; 6xSSC, 50% formamide
2 0R RNA:RNA <50 TR"; 4xSSC TR+; 4xSSC
~: The hybrid length is that anticipated for the hybridized region(s) of the hybridizing polynucleotides. When
hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region
or regions of optimal sequence complementarity.
t: SSPE (lxSSPE is 0.15M NaCl, lOmM NaH2PO~, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (lxSSC
is 0.15M NaCI and 15mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15
minutes after hybridization is complete.
~TB-TR The hybridization l~ll",~ t,lre for hybrids anticipated to be less than 50 base pairs in length should be
5-10~C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following
equations. For hybrids less than 18 base pairs in length, Tm(~C) = 2(# of A + T bases) + 4(# of G + C bases). For
hybrids between 18 and 49 base pairs in length, Tm(~C) = 81.5 + 16.6(1Og,0[Na~]) + 0.41(%G+C) - (600/N), where
N is the number of bases in the hybrid, and [Na~] is the concentration of sodium ions in the hybridizahon buffer
3 5 ([Na+] for 1xSSC = 0.165 M).
.
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Additional examples of stringency conditions for polynucleotide hybridization are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11,
and Current Protocols in Molecular Biology, 1995, F.M. Ausubel et al., eds., John Wiley & Sons,
Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the length of the
polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity)
with the polynucleotide of the present invention to which it hybridizes, where sequence
identity is determined by comparing the sequences of the hybridizing polynucleotides when
aligned so as to maximize overlap and identity while minimizing sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an expression
control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al.,
Nucleic Acids Res. ~, 4485-4490 (1991), in order to produce the protein recombinantly.
Many suitable expression control sequences are known in the art. General methods of
expressing recombinant proteins are also known and are exemplified in R. Kaufman,
Methods in Enzymology ~, 537-566 (1990). As defined herein "operably linked" means
that the isolated polynucleotide of the invention and an expression control sequence are
2 0 situated within a vector or cell in such a way that the protein is expressed by a host cell
which has been transformed (transfected) with the ligated polynucleotide/expression
control sequence.
A number of types of cells may act as suitable host cells for expression of the protein.
Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary
2 5 (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells,3T3
cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains
derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells,
BHK, HL-60, U937, HaK or Jurkat cells.
Alternatively, it may be possible to produce the protein in lower eukaryotes such as
yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimt~rium, or any bacterial strain
capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it
11
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may be necessary to modify the protein produced therein, for example by phosphorylation
or glycosylation of the appropriate sites, in order to obtain the functional protein. Such
covalent attachments may be accomplished using known chemical or enzymatic methods.
The protein may also be produced by operably linking the isolated polynucleotide5 of the invention to suitable control sequences in one or more insect expression vectors, and
employing an insect expression system. Materials and methods for baculovirus/insect cell
expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego,
California, U.S.A. (the MaxBac(13) kit), and such methods are well known in the art, as
described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555
10 ~1987), incorporated herein by reference. As used herein, an insect cell capable of expressing
a polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cellsunder culture conditions suitable to express the recombinant protein. The resulting
expressed protein may then be purified from such culture (i.e., from culture medium or cell
15 extracts) using known purification processes, such as gel filtration and ion exchange
chromatography. The purification of the protein may also include an affinity column
containing agents which will bind to the protein; one or more column steps over such
affinity resins as concanavalin A-agarose, heparin-toyopearl(~) or Cibacrom blue 3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography using
2 0 such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
Alternatively, the protein of the invention may also be expressed in a form which will
facilitate purification. For example, it may be expressed as a fusion protein, such as those
of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX).
Kits for expression and purification of such fusion proteins are commercially available from
2 5 New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen,respectively. The protein can also be tagged with an epitope and subsequently purified by
using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially
available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP-
3 0 HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant
methyl or other aliphatic groups, can be employed to further purify the protein. Some or
all of the foregoing purification steps, in various combinations, can also be employed to
provide a substantially homogeneous isolated recombinant protein. The protein thus
. .
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purified is substantially free of other mammalian proteins and is defined in accordance with
the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are
5 characterized by somatic or germ cells containing a nucleotide sequence encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic means are
known to those skilled in the art. The synthetically-constructed protein sequences, by virtue
10 of sharing primary, secondary or tertiary structural and/or conformational characteristics
with proteins may possess biological properties in common therewith, including protein
activity. Thus, they may be employed as biologically active or immunological substitutes
for natural, purified proteins in screening of therapeutic compounds and in immunological
processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification are naturally
provided or deliberately engineered. For example, modifications in the peptide or DNA
sequences can be made by those skilled in the art using known techniques. Modifications
of interest in the protein sequences may include the alteration, substitution, replacement,
2 0 insertion or deletion of a selected amino acid residue in the coding sequence. For example,
one or more of the cysteine residues may be deleted or replaced with another amino acid to
alter the conformation of the molecule. Techniques for such alteration, substitution,
replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S.
Patent No. 4,518,~84). Preferably, such alteration, substitution, replacement, insertion or
2 5 deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be
expected to retain protein activity in whole or in part and may thus be useful for screening
or other immunological methodologies may also be easily made by those skilled in the art
given the disclosures herein. Such modifications are believed to be encompassed by the
3 0 present invention.
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to exhibit one
or more of the uses or biological activities (including those associated with assays cited
CA 022~7243 1998-11-30
W 097/46682 PCTAUS97/09865
herein) identified below. Uses or activities described for proteins of the present invention
may be provided by administration or use of such proteins or by administration or use of
polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors
suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the research
community for various purposes. The polynucleotides can be used to express recombinant
protein for analysis, characterization or therapeutic use; as markers for tissues in which the
10 corresponding protein is preferentially expressed (either constitutively or at a particular
stage of tissue differentiation or development or in disease states); as molecular weight
markers on Southern gels; as chromosome markers or tags (when labeled) to identify
chromosomes or to map related gene positions; to compare with endogenous DNA
sequences in patients to identify potential genetic disorders; as probes to hybridize and thus
15 discover novel, related DNA sequences; as a source of information to derive PCR primers
for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of
discovering other novel polynucleotides; for selecting and making oligomers for attachrnent
to a "gene chip" or other support, including for examination of expression patterns; to raise
anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-
2 0 DNA antibodies or elicit another immune response. Where the polynucleotide encodes aprotein which binds or potentially binds to another protein (such as, for example, in a
recep~ -ligand interaction), the polynucleotide can also be used in interaction trap assays
(such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify
polynucleotides encoding the other protein with which binding occurs or to identify
2 5 inhibitors of the binding interaction.
The proteins provided by the present invention can similarly be used in assay todetermine biological activity, including in a panel of multiple proteins for high-throughput
screening; to raise antibodies or to elicit another immune response; as a reagent (including
the labeled reagent) in assays designed to quantitatively determine levels of the protein (or
3 0 its receptor) in biological fluids; as markers for tissues in which the corresponding protein
is preferentially expressed (either constitutively or at a particular stage of tissue
differentiation or development or in a disease state); and, of course, to isolate correlative
receptors or ligands. Where the protein binds or potentially binds to another protein (such
as, for example, in a receptor-ligand interaction), the protein can be used to identify the other
14
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W O 97/46682 PCTrUS97/09865
protein with which binding occurs or to identify inhibitors of the binding interaction.
Proteins involved in these binding interactions can also be used to screen for peptide or
small molecule inhibitors or agonists of the binding interaction.
Any or all of these research utilities are capable of being developed into reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled in the
art. References disclosing such methods include without limitation "Molecular Cloning: A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch
and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning
Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as nutritional
sources or supplements. Such uses include without limitation use as a protein or amino acid
supplement, use as a carbon source, use as a nitrogen source and use as a source of
carbohydrate. In such cases the protein or polynucleotide of the invention can be added to
the feed of a particular organism or can be administered as a separate solid or liquid
preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the
case of microorganisms, the protein or polynucleotide of the invention can be added to the
2 0 medium in or on which the microorganism is cultured.
Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation (either
inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may
2 5 induce production of other cytokines in certain cell populations. Many protein factors
discovered to date, including all known cytokines, have exhibited activity in one or more
factor dependent cell proliferation assays, and hence the assays serve as a convenient
confirmation of cytokine activity. The activit,v of a protein of the present invention is
evidenced by any one of a number of routine factor dependent cell proliferation assays for
3 0 cell lines including, without limitation, 32D, DA2, DAlG, T10, B9, B9/11, BaF3, MC9/G, M+
(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured by
the following methods:
.
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Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986;
Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology
133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph node cells
1 0 or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation,
Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immllnology. J.E.e.a. Coligan eds.
Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse
and human I~ on ~, Schreiber, R.D. In Current Protocols in ~mmunology. J.E.e.a. Coligan
eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
1 5 Assays for proliferation and .lifr~ liation of hematopoietic and lymphopoietic cells
include, without limitation, those described in: Measurement of Human and MurineInterleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols
in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto.
1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692,
2 0 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of
mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc.
Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11 - Bennett,
F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a.
2 5 Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse
and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In C~rrent
Protocols in Imml~nology. J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.
1991.
Assays for T-cell clone responses to antigens (which will identify, among others,
3 0 proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring
proliferation and cytokine production) include, without limitation, those described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their
16
.
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cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc.
Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981;
Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
Immune Stimulating or Suppressing Activity
A protein of the present invention may also exhibit immune stimulating or immunesuppressing activity, including without limitation the activities for which assays are
described herein. A protein may be useful in the treatment of various immune deficiencies
and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up
or down) growth and proliferation of T and/or B Iymphocytes, as well as effecting the
cytolytic activity of NK cells and other cell populations. These immune deficiencies may be
genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may
result from autoimmune disorders. More specifically, infectious diseases causes by viral,
bacterial, fungal or other infection may be treatable using a protein of the present invention,
1 5 including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp.,
malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a
protein of the present invention may also be useful where a boost to the immune system
generally may be desirable, i.e., in the treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present
2 0 invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus
erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre
syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis,
graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the
present invention may also to be useful in the treatment of allergic reactions and conditions,
such as asthma (particularly allergic asthma) or other respiratory problems. Other
conditions, in which immune suppression is desired (including, for example, organ
transplantation), may also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to immune responses, in
a number of ways. Down regulation may be in the form of inhibiting or blocking an immune
response already in progress or may involve preventing the induction of an immune
response. The functions of activated T cells may be inhibited by suppressing T cell
responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell
responses is generally an active, non-antigen-specific, process which requires continuous
exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-
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responsiveness or anergy in T cells, is disffnguishable from immunosuppression in that it
is generally antigen-specific and persists after exposure to the tolerizing agent has ceased.
Operationally, tolerance can be demonstrated by the lack of a T cell response upon
reexposure to specific antigen in the absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without
limitation B Iymphocyte antigen functions (such as, for example, B7)), e.g., preventing high
level Iymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and
organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of
T cell function should result in reduced tissue destruction in tissue transplantation.
1 0 Typically, in tissue transplants, rejection of the transplant is initiated through its recognition
as foreign by T cells, followed by an immune reaction that destroys the transplant. The
administration of a mol~clllP which inhibits or blocks interaction of a B7 lymphocyte antigen
with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide
having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an
activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody), prior to
transplantation can lead to the binding of the molecule to the natural ligand(s) on the
immune cells without transmitting the corresponding costimulatory signal. Blocking B
lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells,
such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation
2 0 may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the
necessity of repeated administration of these blocking reagents. To achieve sufficient
immunosuppression or tolerance in a subject, it may also be necessary to bloclc the function
of a combination of B lymphocyte antigens.
2 5 The efficacy of particular blocking reagents in preventing organ transplant rejection
or GVHD can be assessed using animal models that are predictive of efficacy in humans.
Examples of a~r~liate systems which can be used include allogeneic cardiac grafts in rats
and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine
the immunosu~"e:jsive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow
3 0 et al., Science 2~7:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105
(1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology,
Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking
B lymphocyte antigen function in vivo on the development of that disease.
18
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W 097/46682 PCTrUS97/09865
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activaffon of T cells that are reactive against self tissue and which promote the production
of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the
activation of autoreactive T cells may reduce or eliminate disease ~y~ loms.
Administration of reagents which block costimulation of T cells by disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation
and prevent production of autoantibodies or T cell-derived cytokines which may be
involved in the disease process. Additionally, blocking reagents may induce antigen-specific
tolerance of autoreactive T cells which could lead to long-term relief from the ~ A~e The
efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be
determined using a number of well-characterized animal models of human autoimmune
diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus
erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen
arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia
gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen function),
as a means of up regulating immune responses, may also be useful in therapy. Upregulation
of immune responses may be in the form of enhancing an existing immune response or
2 0 eliciting an initial immune response. For example, enhancing an immune response through
stimulating B lymphocyte antigen function may be useful in cases of viral infection. In
addition, systemic viral diseases such as influenza, the common cold, and encephalitis might
be alleviated by the administration of stimulatory forms of B lymphocyte antigens
systemically.
2 5 Alternatively, anti-viral immune responses may be enhanced in an infected patient
by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-
pulsed APCs either expressing a peptide of the present invention or together with a
stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro
activated T cells into the patient. Another method of enhancing anti-viral immune responses
3 0 would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding
a protein of the present invention as described herein such that the cells express all or a
portion of the protein on their surface, and reintroduce the transfected cells into the patient.
The infected cells would now be capable of delivering a costimulatory signal to, and thereby
activate, T cells in vivo.
19
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W 097146682 PCTrUS97/09865
In another application, up regulation or enhancement of antigen function (preferably
B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor
cells (e.g., sarcoma, melanoma, Iymphoma, leukemia, neuroblastoma, carcinoma) transfected
with a nucleic acid encoding at least one peptide of the present invention can be
administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the
tumor cell can be transfected to express a combination of peptides. For example, tumor cells
obtained from a patient can be transfected ex vivo with an expression vector directing the
expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide
having B7-1-like activity and/or B7-3-like activity. The transfected tumor cells are returned
to the patient to result in expression of the peptides on the surface of the transfected cell.
Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in
V2VO.
The presence of the peptide of the present invention having the activity of a B
lymphocyte antigen(s) on the surface of the tumor cell provides the necessary costimulation
signal to T cells to induce a T cell mediated immune response against the transfected tumor
cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which
fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be
transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain
truncated portion) of an MHC class I a chain protein and ,B2 microglobulin protein or an
2 0 MHC class II a chain protein and an MHC class II ~ chain protein to thereby express MHC
class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or
class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen
(e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected
tumor cell. C~ptionally, a gene encoding an antisense construct which blocks expression of
2 5 an MHC class II associated protein, such as the invariant chain, can also be cotransfected
with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote
presentation of tumor associated antigens and induce tumor specific immunity. Thus, the
induction of a T cell mediated immune response in a human subject may be sufficient to
overcome tumor-specific tolerance in the subject.
3 0 The activity of a protein of the invention may, among other means, be measured by
the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
CA 022~7243 1998-11-30
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Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter
7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J.
Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492,1981;
Herrmann et al., J. Immunol. 128:1968-1974,1982; Handa et al., J. Immunol. 135:1564-1572,
1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998;
Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., Cellular Immunology 133:327-
341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent antibody
responses and that affect Thl /Th2 profiles) include, without limitation, those described in:
Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro
antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl and CTL responses) include, without limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
2 0 Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et
al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without limitation, those
2 5 described in: Guery et al., J. Immunol.134:536-544,1995; Inaba et al., Journal of Experimental
Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995;
Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of
Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al.,
Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical
3 0 Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-
640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that regulate
lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et
CA 022~7243 1998-11-30
WO 97146682 PCTrUS97109865
al., Cytometry 13:795-808, 1992; Gorczyca et al., ~eukemia 7:659-670, 1993; Gorczyca et al.,
Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal
of Immunology 145:4037~045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorc~yca et
al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence ear}y steps of T-cell commitment and development
include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et
al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,
Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Regulating Activity
A protein of the present invention may be useful in regulation of hematopoiesis and,
consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal
biological activity in support of colony forming cells or of factor-dependent cell lines
indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and
proliferation of erythroid progenitor cells alone or in combination with other cytokines,
thereby indicating utility, for example, in treating various anemias or for use in conjunction
with irradiation/chemotherapy to stimulate the production of ~ uid precursors and/or
erythroid cells; in supporting the growth and proliferation of myeloid cells such as
granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for
example, in conjunction with chemotherapy to prevent or treat consequent myelo-
suppression; in supporting the growth and proliferation of megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various platelet
disorders such as thrombocytopenia, and generally for use in place of or complimentary to
platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic
stem cells which are capable of maturing to any and all of the above-mentioned
hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such
as those usually treated with transplantation, including, without limitation, aplastic anemia
and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell
compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction
with bone marrow transplantation or with peripheral progenitor cell transplantation
(homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured by
the following methods:
CA 022~7243 1998-11-30
WO 97/46682 PCT/US97/09865
Suitable assays for proliferation and difrelcntiation of various hematopoietic lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among others,
proteins that influence embryonic diL[~.~.,Iiation hematopoiesis) include, without limitation,
those described in: Johansson et al. Cellular Biology 15:141-151,1995; Keller et al., Molecular
and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
Assays for stem cell survival and di~rerellLiation (which will identify, among others,
proteins that regulate lympho-hematopoiesis) include, without limitation, those described
in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells.
1 0 R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama
et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming
cells with high proliferative potential, McNiece, I.K. and Briddell, R.A. In Culh~re of
Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY.
1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming
1 5 cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp.
1-21, Wiley-Liss, Inc.., New York, NY. 1994; Long term bone marrow cultures in the presence
of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I.
Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term
culture initiating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney,
2 0 et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activit,v
A protein of the present invention also may have utility in compositions used for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for
2 5 wound healing and tissue repair and replacement, and in the treatment of burns, incisions
and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth in
circumstances where bone is not normally formed, has application in the healing of bone
fractures and cartilage damage or defects in humans and other animals. Such a preparation
3 0 employing a protein of the invention may have prophylactic use in closed as well as open
fracture reduction and also in the improved fixation of artificial joints. De novo bone
formation induced by an osteogenic agent contributes to the repair of congenital, trauma
induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic
plastic surgery.
23
CA 022~7243 1998-11-30
W O g7/46682 PCT~US97/09865
A protein of this invention may also be used in the treatment of periodontal disease,
and in other tooth repair processes. Such agents may provide an environment to attract
bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of
progenitors of bone-forming cells. A protein of the invention may also be useful in the
5 treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or
cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase
activity, osteoclast activity, etc.) mediated by inflammatory processes.
Another category of tissue regeneration activity that may be attributable to theprotein of the present invention is tendon/ligament formation. A protein of the present
10 invention, which induces tendon/ligament-like tissue or other tissue formation in
circumstances where such tissue is not normally formed, has application in the healing of
tendon or ligament tears, deformities and other tendon or ligament defects in humans and
other animals. Such a preparation employing a tendon/ligament-like tissue inducing
protein may have prophylactic use in preventing damage to tendon or ligament tissue, as
15 well as use in the improved fixation of tendon or ligament to bone or other tissues, and in
repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue
formation induced by a composition of the present invention contributes to the repair of
congenital, trauma induced, or other tendon or ligament defects of other origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The
2 0 compositions of the present invention may provide an environment to attract tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of
tendon/ligament cells or progenitors ex vivo for return i~l vivo to effect tissue repair. The
compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel
2 5 syndrome and other tendon or ligament defects. The compositions may also include an
appropriate matrix and/or se~uestering agent as a carrier as is well known in the art.
The protein of the present invention may also be useful for proliferation of neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and
peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic
3 0 disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More
specifically, a protein may be used in the treatment of diseases of the peripheral nervous
system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies,
and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's
disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which
24
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may be treated in accordance with the present invention include mechanical and traumatic
disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as
stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies
may also be treatable using a protein of the invention.
Proteins of the invention may also be useful to promote better or faster closure of
non-healing wounds, including without limitation pressure ulcers, ulcers associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit activity for
generation or regeneration of other tissues, such as organs (including, for example, pancreas,
l 0 liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular
(including vascular endothelium) tissue, or for promoting the growth of cells comprising
such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic
scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit
angiogenic activity.
A protein of the present invention may also be useful for gut protection or
regeneration and treatrnent of lung or liver fibrosis, reperfusion injury in various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or inhibiting
differentiation of tissues described above from precursor tissues or cells; or for inhibiting the
2 0 growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured by
the following methods:
Assays for ffssue generation activity include, without limitation, those described in:
International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International
2 5 Patent Publication No. W095/05846 (nerve, neuronal); International Patent Publication No.
WO91 /07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and l~ovee, DT, eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest.
3 0 Dermatol 71:382-84 (1978).
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-related
activities. Inhibins are characterized by their ability to irlhibit the release of follicle
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stimulating hormone (FSH), while activins and are characterized by their ability to stimulate
the release of follicle stimulating hormone (FSH). Thus, a protein of the present invention,
alone or in heterodimers with a member of the inhibin a family, may be useful as a
contraceptive based on the ability of inhibins to decrease fertility in female mammals and
decrease spermatogenesis in male mammals. Administration of sufficient amounts of other
inhibins can induce infertility in these mammals. Alternatively, the protein of the invention,
as a homodimer or as a heterodimer with other protein subunits of the inhibin-~ group, may
be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in
stimulating FSH release from cells of the anterior pituitary. See, for example, United States
1 0 Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset
of fertility in sexually immature mammals, so as to increase the lifetime reproductive
performance of domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by
the following methods:
Assays for activin/inhibin activity include, without limitation, those described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al.,
Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl.
Acad. Sci. USA 83:3091-3095, 1986.
2 0 Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic activity(e.g., act as a chemokine) for mammalian cells, including, for example, monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins provide
particular advantages in treatment of wounds and other trauma to tissues, as well as in
treatrnent of localized infections. For example, attraction of lymphocytes, monocytes or
neutrophils to tumors or sites of infection may result in improved immune responses against
the tumor or infecting agent.
3 0 A protein or peptide has chemotactic activity for a particular cell population if it can
stimulate, directly or indirectly, the directed orientation or movement of such cell
population. Preferably, the protein or peptide has the ability to directly stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a population
26
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of cells can be readily determined by employing such protein or peptide in any known assay
for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured by
the following methods:
Assays for chemotactic activity (which will identify proteins that induce or prevent
chemotaxis) consist of assays that measure the ability of a protein to induce the migration
of cells across a membrane as well as the ability of a protein to induce the adhesion of one
cell population to another cell population. Suitable assays for movement and adhesion
include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E.
1 0 Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS
103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol.
152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994.
Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic activity. As
a result, such a protein is expected to be useful in treatment of various coagulation disorders
(including hereditary disorders, such as hemophilias) or to enhance coagulation and other
2 0 hemostatic events in treating wounds resulting from trauma, surgery or other causes. A
protein of the invention may also be useful for dissolving or inhibiting formation of
thromboses and for treatment and prevention of conditions resulting therefrom (such as, for
example, infarction of cardiac and central nervous system vessels (e.g., stroke).
The activity of a protein of the invention may, among other means, be measured by
2 5 the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res.
45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-
474, 1988.
Receptor/Ligand Activity
A protein of the present invention may also demonstrate activity as receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions. Examples of such
receptors and ligands include, without limitation, cytokine receptors and their ligands,
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receptor kinases and their ligands, receptor phosphatases and their ligands, receptors
involved in cell-cell interactions and their ligands (including without limitation, cellular
adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs
involved in antigen presentation, antigen recognition and development of cellular and
humoral immune responses). Receptors and ligands are also ~lseful for screening of
potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
A protein of the present invention (including, without limitation, fragments of receptors and
ligands) may themselves be useful as inhibitors of receptor/ligand interactions.The activity of a protein of the invention may, among other means, be measured by
1 0 the following methods:
Suitable assays for receptor-ligand activity include without limitation those described
in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies,
E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience
(Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22),
Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med.
168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al.,
J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.
Anti-Inflammatory Activity
2 0 Proteins of the present invention may also exhibit anti-inflammatory activity. The
anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the
inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or
suppressing production of other factors which more directly inhibit or promote an
inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory
conditions including chronic or acute conditions), including without limitation inflammation
associated with infection (such as septic shock, sepsis or systemic inflammatory response
syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-
mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury,
inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines
such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and
hypersensitivity to an antigenic substance or material.
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Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major roles
during development, particularly in defining specific cell types. Loss or alteration of normal
cadherin expression can lead to changes in cell adhesion properties linked to tumor growth
and metastasis. Cadherin malfunction is also implicated in other human diseases, such as
pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases),
Crohn's disease, and some developmental abnormalities.
The cadherin superfamily includes well over forty members, each with a distinct
pattern of expression. All members of the superfamily have in common conserved
extracellular repeats (cadherin domains), but structural differences are found in other parts
of the molecule. The cadherin domains bind calcium to form their tertiary structure and
thus calcium is required to mediate their adhesion. Only a few amino acids in the first
cadherin domain provide the basis for homophilic adhesion; modification of this recognition
site can change the specificity of a cadherin so that instead of recognizing only itself, the
mutant molecule can now also bind to a different cadherin. In addition, some cadherins
engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become
invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides
expressing E-cadherin has reversed cancer-associated changes by returning altered cell
shapes to normal, restoring cells' adhesiveness to each other and to their substrate,
decreasing the cell growth rate, and drastically reducing anchorage-independent cell
growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced
stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas
2 5 derived from other tissue types. Therefore, proteins of the present invention with cadherin
activity, and polynucleotides of the present invention encoding such proteins, can be used
to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or
eliminate the cancerous changes observed in these cells by providing normal cadherin
expression.
3 0 Cancer cells have also been shown to express cadherins of a different tissue type than
their origin, thus allowing these cells to invade and metastasize in a different tissue in the
body. Proteins of the present invention with cadherin activity, and polynucleotides of the
present invention encoding such proteins, can be substituted in these cells for the
29
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W O 97/46682 PCTrUS97/09865
inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing
or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
polynucleotides of the present invention encoding such proteins, can used to generate
antibodies recognizing and binding to cadherins. Such antibodies can be used to block the
adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the
cancer, the less cadherin expression there will be, and this decrease in cadherin expression
1 0 can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity, preferably a
polypeptide comprising a decapeptide of the cadherin recognition site, and poly-nucleotides
of the present invention encoding such protein fragments, can also be used to block cadherin
function by binding to cadherins and preve~ g them from binding in ways that produce
undesirable effects. Additionally, fragments of proteins of the present invention with
cadherin activity, preferably truncated soluble cadherin fragments which have been found
to be stable in the circulation of cancer patients, and polynucleotides encoding such protein
fragments, can be used to disturb proper cell-cell adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
2 0 limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki
et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
2 5 prevention of tumors, a protein of the invention may exhibit other anti-tumor activities. A
protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC).
A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor
precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such
as, for example, by inhibiting angiogenesis), by causing production of other factors, agents
3 0 or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting
factors, agents or cell types which promote tumor growth.
Other Activities
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A protein of the invention may also exhibit one or more of the following additional
activities or effects: inhibiting the growth, infection or function of, or killing, infectious
agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without limitation, height,
5 weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or
body part size or shape (such as, for example, breast augmentation or diminution, change
in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the
fertility of male or female subjects; effecting the metabolism, catabolism, anabolism,
processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate,
10 vitamins, minerals, cofactors or other nutritional factors or component(s); effecting
behavioral characteristics, including, without limitation, appetite, libido, stress, cognition
(including cognitive disorders), depression (including depressive disorders) and violent
behaviors; providing analgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages other than hematopoietic
15 lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of
the enzyme and treating deficiency-related diseases; treatment of hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for
example, the ability to bind antigens or complement); and the ability to act as an antigen in
a vaccine composition to raise an immune response against such protein or another material
2 0 or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including without
2 5 limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical
composition when combined with a pharmaceutically acceptable carrier. Such a
composition may also contain (in addition to protein and a carrier) diluents, fillers, salts,
buffers, stabilizers, solubilizers, and other materials well known in the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not interfere with the
3 0 effectiveness of the biological activity of the active ingredient(s). The characteristics of the
carrier will depend on the route of administration. The pharmaceutical composition of the
invention may also contain cytokines, Iymphokines, or other hematopoietic factors such as
M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-
13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor,
.... . .. . ...
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and erythropoietin. The pharmaceutical composition may further contain other agents
which either enhance the activity of the protein or compliment its activity or use in
treatment. Such additional factors and/or agents may be included in the pharmaceutical
composition to produce a synergistic effect with protein of the invention, or to minimize side
5 effects. Conversely, protein of the present invention may be included in formulations of the
particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic
factor, or anti-infl~mm~tory agent to minimize side effects of the cytokine, Iymphokine,
other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-infl~mm~tory
agent.
1 0 A protein of the present invention may be active in multimers (e.g., heterodimers or
homodimers) or complexes with itself or other proteins. As a result, pharmaceutical
compositions of the invention may comprise a protein of the invention in such multimeric
or complexed form.
The pharmaceutical composition of the invention may be in the form of a complex
1 5 of the protein(s) of present invention along with protein or peptide antigens. The protein
and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR) following
presentation of the antigen by MHC proteins. MHC and structurally related proteins
2 0 including those encoded by class I and class II MHC genes on host cells will serve to present
the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied
as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly
signal T cells. Alternatively antibodies able to bind surface immunolgobulin and other
molecules on B cells as well as antibodies able to bind the TCR and other molecules on T
2 5 cells can be combined with the pharmaceutical composition of the invention.
The pharmaceutical composition of the invention may be in the form of a liposomein which protein of the present invention is combined, in addition to other pharmaceutically
acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form
as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
30 Suitable lipids for liposomal formulation include, without limitation, monoglycerides,
diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.
Preparation of such liposomal formulations is within the level of skill in the art, as disclosed,
for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No.
4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference.
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As used herein, the term "therapeutically effective amount" means the total amount
of each active component of the pharmaceutical composition or method that is sufficient to
show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the
relevant medical condition, or an increase in rate of treatment, healing, prevention or
5 amelioration of such conditions. When applied to an individual active ingredient,
administered alone, the term refers to that ingredient alone. When applied to a combination,
the term refers to combined amounts of the active ingredients that result in the therapeutic
effect, whether administered in combination, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a
10 therapeutically effective amount of protein of the present invention is administered to a
mammal having a condition to be treated. Protein of the present invention may beadministered in accordance with the method of the invention either alone or in combination
with other therapies such as treatments employing cytokines, lymphokines or other
hematopoietic factors. When co-administered with one or more cytokines, lymphokines or
15 other hematopoietic factors, protein of the present invention may be administered either
simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s),
thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the
attending physician will decide on the appropriate sequence of administering protein of the
present invention in combination with cytokine(s), Iymphokine(s), other hematopoietic
2 0 factor(s), thrombolytic or anti-thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried out in a variety
of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous,
subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous
2 5 administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a tablet, capsule,
powder, solution or elixir. When administered in tablet form, the pharmaceuticalcomposition of the invention may additionally contain a solid carrier such as a gelatin or an
3 0 adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the
present invention, and preferably from about 25 to 90% protein of the present invention.
When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils
may be added. The liquid form of the pharmaceutical composition may further contain
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W 097/46682 PCTA~S97/09865
physiological saline solution, dextrose or other saccharide solution, or glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form,
the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the
present invention, and preferably from about 1 to 50% protein of the present invention.
When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of the present
invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
The preparation of such pd~ dlly acceptable protein solutions, having due regard to pH,
isotonicity, stability, and the like, is within the skill in the art. A IJrer~,L~d pharmaceutical
composition for intravenous, cutaneous, or subcutaneous injection should contain, in
addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride
Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection,
Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical
composition of the present invention may also contain stabilizers, preservatives, buffers,
antioxidants, or other additives known to those of skill in the art.
The amount of protein of the present invention in the pharmaceutical compositionof the present invention will depend upon the nature and severity of the condition being
treated, and on the nature of prior treatments which the patient has undergone. Ultimately,
the attending physician will decide the amount of protein of the present invention with
2 0 which to treat each individual patient. Initially, the attending physician will administer low
doses of protein of the present invention and observe the patient's response. Larger doses
of protein of the present invention may be administered until the optimal therapeutic effect
is obtained for the patient, and at that point the dosage is not increased further. It is
contemplated that the various pharmaceutical compositions used to practice the method of
2 5 the present invention should contain about 0.01 ,ug to about 100 mg (preferably about 0.1ng
to about 10 mg, more preferably about 0.1 ,ug to about 1 mg) of protein of the present
invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of the
present invention will vary, depending on the severity of the disease being treated and the
3 0 condition and potential idiosyncratic response of each individual patient. It is contemplated
that the duration of each application of the protein of the present invention will be in the
range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending
physician will decide on the appropriate duration of intravenous therapy using the
pharmaceutical composition of the present invention.
34
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W O 97/46682 PCTrUS97/09865
Protein of the invention may also be used to immunize animals to obtain polyclonal
and monoclonal antibodies which specifically react with the protein. Such antibodies may
be obtained using either the entire protein or fragments thereof as an imm~mogen. The
peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus,
5 and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for
synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J.
Amer.Chem.Soc. ~, 2149-2154 (1963); J.L. Krstenansky, e~ al., FEBS Lett. ~, 10 (1987).
Monoclonal antibodies binding to the protein of the invention may be useful diagnostic
agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding
10 to the protein may also be useful therapeutics for both conditions associated with the protein
and also in the treatment of some forms of cancer where abnormal expression of the protein
is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal
antibodies against the protein may be useful in detecting and preventing the metastatic
spread of the cancerous cells, which may be mediated by the protein.
For compositions of the present invention which are useful for bone, cartilage,
tendon or ligament regeneration, the therapeutic method includes administering the
composition topically, systematically, or locally as an implant or device. When
administered, the therapeutic composition for use in this invention is, of course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may desirably be
2 0 encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue
damage. Topical administration may be suitable for wound healing and tissue repair.
Therapeutically useful agents other than a protein of the invention which may also
optionally be included in the composition as described above, may alternatively or
additionally, be administered simultaneously or sequentially with the composition in the
2 5 methods of the invention. Preferably for bone and/or cartilage formation, the composition
would include a matrix capable of delivering the protein-containing composition to the site
of bone and/or cartilage damage, providing a structure for the developing bone and
cartilage and optimally capable of being resorbed into the body. Such matrices may be
formed of materials presently in use for other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The particular
application of the compositions will define the appropriate formulation. Potential matrices
for the compositions may be biodegradable and chemically defined calcium sulfate,
tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides.
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Other potential materials are biodegradable and biologically well-defined, such as bone or
dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix
components. Other potential matrices are nonbiodegradable and chemically defined, such
as sintered hydroxapatite, bioglass, aluminates, or other ceramics. Matrices may be
5 comprised of combinations of any of the above mentioned types of material, such as
polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics
may be altered in composition, such as in calcium-aluminate-phosphate and processing to
alter pore size, particle size, particle shape, and biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid
in the form of porous particles having diameters ranging from 150 to 800 microns. In some
applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose
or autologous blood clot, to prevent the protein compositions from disassociating from the
matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and
carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose
(CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate,
poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol).
2 0 The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based
on total formulation weight, which represents the amount necessary to prevent desorbtion
of the protein from the polymer matrix and to provide appropriate handling of the
composition, yet not so much that the progenitor cells are prevented from infiltrating the
matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the
2 5 progenitor cells.
In further compositions, proteins of the invention may be combined with other
agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in
question. These agents include various growth factors such as epidermal growth factor
(EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-a and TGF-
3 0 ~), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary applications.
Particularly domestic animals and thoroughbred horses, in addition to humans, are desired
patients for such treatment with proteins of the present invention.
36
,
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The dosage regimen of a protein-containing pharmaceutical composition to be usedin tissue regeneration will be determined by the attending physician considering various
factors which modify the action of the proteins, e.g., amount of tissue weight desired to be
formed, the site of damage, the condition of the damaged tissue, the size of a wound, type
5 of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection,
time of administration and other clinical factors. The dosage may vary with the type of
matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical
composition. For example, the addition of other known growth factors, such as IGF I
(insulin like growth factor I), to the final composition, may also effect the dosage. Progress
10 can be monitored by periodic assessment of tissue/bone growth and /or repair, for example,
X-rays, histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy. Suchpolynucleotides can be introduced either in vivo or ex vivo into cells for expression in a
mammalian subject. Polynucleotides of the invention may also be administered by other
15 known methods for introduction of nucleic acid into a cell or organism (including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present invention
in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells
can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as if fully
set forth.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Racie, Lisa A.
Merberg, David
Treacy, Maurice
Evans, Cheryl
Bowman, Michael
Spaulding, Vikki
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 5
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: MA
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFT~ARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger, Suzanne A.
(B) REGISTRATION NUMBER: P-41,323
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 498-8284
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
38
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W 097/46682 PCTAUS97/09865
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
NGCTGGGCTC GGGCTCANCT CGACTGGGCT CGGCGGGCGG CGGCGGCGGC GCCCGCGGCT 60
GGCGGAAGAA GGAGGGCGAG GGCGGGCGCG GGCCGGCGGG CGGGCGGAAA AAGGAGGAAA 120
GGCGCGGGGA GCCAGGCCTC GGGGCCTCGG ANCAACCACC CGAGCAGACG GAGTACACGG 180
AGCAGCGGCC CCGGCCCCGC CAACGCTGCC GCCGGGATGC TCCAA 225
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 605 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CCAAAGAGGC CGTGACGCCC AGTCTCCTCA AGAAGTTCCG AGGAGCCAGC TGGAGATTCA 60
CATTTTACCT GATTGCCTTC ATTGCCGGCA TGGCCGTCAT TGTGGATAAA CCCTGGTTCT 120
ATGACATGAA GAAAGTTTGG GAGGGATATC CCATACAGAG CACTATCCCT TCCCCGTATT 180
GGTACTACAT GATTGAACTT TCCTTCTACT GGTCCCTGCT CTTCAGCATT GCCTCTGATG 240
TCAAGCGAAA GGATTTCAAG GAACAGATCA TCCACCATGT GGCCACCATC ATTCTCATCA 300
GCTTTTCCTG GTTTGCCAAT TACATCCGAG CTGGGACTCT AATCATGGCT CTGCATGACT 360
CTTCCGATTA CCTGCTGGAG TCAGCCAAGA TGTTTAACTA CGCGGGATGG AAGAACACCT 420
GCAACAACAT CTTCATCGTC TTCGCCATTG TTTTTATCAT CACCCGACTG GTCATCCTGC 480
CCTTCTGGAT CCTGCATTGC ACCCTGGTGT ACCCACTGGA GCTCTATCCT GCCTTCTTTG 540
GCTATTACTT CTTCAATCCA TGATGGGAGT TCTACAGCTG CTGCATATCT CCTGGGCCTA 600
CCTCA 605
(2) INFORMATION FO~ SEQ ID No:3:
(i) SEQUENCE CHARACTERISTICS:
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~A) LENGTH: 157 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Ala Val Ile Val Asp Lys Pro Trp Phe Tyr Asp Met Lys Lys Val
1 5 10 15
Trp Glu Gly Tyr Pro Ile Gln Ser Thr Ile Pro Ser Pro Tyr Trp Tyr
Tyr Met Ile Glu Leu Ser Phe Tyr Trp Ser Leu Leu Phe Ser Ile Ala
Ser Asp Val Lys Arg Lys Asp Phe Lys Glu Gln Ile Ile His His Val
Ala Thr Ile Ile Leu Ile Ser Phe Ser Trp Phe Ala Asn Tyr Ile Arg
Ala Gly Thr Leu Ile Met Ala Leu His Asp Ser Ser Asp Tyr Leu Leu
g0 95
Glu Ser Ala Lys Met Phe Asn Tyr Ala Gly Trp Lys Asn Thr Cys Asn
100 105 110
Asn Ile Phe Ile Val Phe Ala Ile Val Phe Ile Ile Thr Arg Leu Val
115 120 125
Ile Leu Pro Phe Trp Ile Leu His Cys Thr Leu Val Tyr Pro Leu Glu
130 135 140
Leu Tyr Pro Ala Phe Phe Gly Tyr Tyr Phe Phe Asn Pro
145 150 155
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 145 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
. .
CA 02257243 l998-ll-30
W 097/46682 PCTrUS97/09865
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GGTTTTAAAG CCAGAATTAC GGNTAGCACC TAGCATTTCA GCAGAGGGAC CATTTTAGAC 60
CAAAATGTAC TGTTAANGGG ~ l'A AAATTAAAAG ATTAAATAAA AAATATTAAA 120
TAAAACANGA AAAAAAAAAA AAAAA 145
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = ~oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ACCTGCTGGA GTCAGCCAAG ATGTTTA 27
41
